WO2014085801A1 - Cryo-treatment in a microfluidic device - Google Patents

Abstract

The present invention generally relates to methods and compositions for cryo preservation and/or cryolysis.

Description

CRYO-TREATMENT IN A MICROFLUIDIC DEVICE

RELATED APPLICATIONS AND/OR INCORPORATION BY REFERENCE

[0001] This application claims priority to and benefit of US provisional patent application 61/732,179 entitled MULTIPURPOSE MICROFLUIDIC DEVICE FOR CRYOLYSIS AND CRYOPRESERVATION filed on November 30, 2012.

[0002] All documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated herein by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

FIELD OF THE INVENTION

[0003] The present invention generally relates to methods and compositions for cryolysis and/or cryopreservation.

BACKGROUND OF THE INVENTION

[0004] The ability to lyse cells is a key step in sample preparation to provide quantitative access to the cell contents. Current cell lysis methods can be classified into mechanical, chemical, acoustic, thermal, and pressure based methods. Each of these methods varies, with respect to, e.g., device complexity, their ability to lyse <20 cells, and flexibility to lyse a range of microbial pathogens with minimal changes. Mechanical methods require significantly greater sample volumes than may be available, and are thus not suitable. Chemical methods are not a universal solution in that they require customization, of e.g., reagent concentration and mix, for each pathogen type to ensure cell wall breakdown while avoiding RNA damage. Although methods employing pressure, e.g., "French press" methods, are reliable, complete, and the gold standard in microbiology, the requirement for cumbersome vessel pressurization, high pressure (>9,000 psi), and larger sample volumes (>40 ml), eliminates it as an option for the present application. Acoustic methods such as sonication or cavitation, while able to lyse even microbes with very strong cell walls, impart significant energy to the sample and thus can degrade RNA. This leaves thermal methods, where freeze/thaw is preferred due to the gentleness of the approach on RNA, ability to halt cell expression in the frozen state, and simplicity of the device compared to acoustic methods.

[0005] Microfluidics involves micro-scale devices that handle small volumes of fluids. Because microfluidics may accurately and reproducibly control and dispense small fluid volumes, in particular volumes less than 1 μΐ, the application of microfluidics provides significant cost-savings. The use of microfluidics technology reduces cycle times, shortens tirae- to-results, and increases throughput. Furthermore, incorporation of microfluidics technology enhances system integration and automation. Microfluidic reactions are often conducted in microdroplets. The ability to conduct reactions in microdroplets depends on being able to merge different sample fluids and different microdroplets. See, e.g., US Patent Publication No. 20120219947. Droplet microfluidics offer significant advantages for performing high-throughput screens and sensitive assays. Droplets allow sample volumes to be significantly reduced, leading to concomitant reductions in cost. Manipulation and measurement at kilohertz speeds enable up to 10⁸ samples to be screened in a single day. Compartmentalization in droplets increases assay sensitivity by increasing the effective concentration of rare species and decreasing the time required to reach detection thresholds. Droplet microfluidics combines these powerful features to enable currently inaccessible high-throughput screening applications, including single-cell and single-molecule assays. See, e.g., Guo et al., Lab Chip, 2012,12, 2146-2155. Although there are many advantages for studying biological processes via droplet microfluidics, there remain problems in the art.

[0006] Many lysis methods rely on the use of harsh chemicals that must be neutralized or eliminated prior to downstream processing. The neutralization/elimination of such chemicals does not lend itself well to a microfluidic environment, particularly an emulsion droplet based system.

[0007] Furthermore, in cell lysis rapid freeze/thaw cycling is most often performed as a "bulk" process (i.e., cells are processed in multiwell plates or centrifuge tubes).

[0008] Accordingly, problems with cell lysis, especially in a microfluidic system, include: harsh chemicals or bulk freeze thawing techniques, and heretofore unavailable apparatus and methods for cryolysis of a specific volume of a cell culture or even a controlled number of cells. [0009] There are also problems with the cryopreservation of cells, especially in a microfluidic system.

[0010] For example, for accessing a specific volume of a cell culture or even a controlled numbers of cells of a cell culture that has been cryopreserved, heretofore there was a need to thaw the entire culture, including because apparatus and methods for ciyopreserving a specific volume of a cell culture or even a controlled number of cells was heretofore unavailable.

[0011] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

[0012] Applicants have developed freeze/thaw lysis methods for use with all cell types and particularly to be able to recover useful RNA sample of microbial pathogens. The techniques developed are suitable as freeze/thaw lysis or cryopreservation methods with microfluidic systems and devices enabling efficient sample preparation and evaluation on these systems.

[0013] The invention particularly relates to devices for cryolysis and/or cryopreservation, particularly microfluidic devices and methods for microfluidic cryolysis or cryopreservation.

[0014] The invention provides a device for receiving a cell in a droplet comprising an inlet and an outlet and a channel in between for a cell to travel from inlet to outlet (cell channel). The cell channel passes near or through one or more blocks. The one or more blocks may be chilling blocks, whereby the device may be for cryopreservation. Alternatively, the first block may be a chiller block and second block may be a warming block, whereby the device may be for cryolysis. Alternatively, multiple blocks, alternately for chilling and warming, may be put in series for multiple freeze/thaw cycles for cryolysis. Alternatively, the cell channel may run back and forth (in a zigzag, serpentine, sinusoidal or other pattern) between a chiller block and a warming block to achieve the same cyclic freezing and thawing. Alternatively, the cell channel may include a substantially straight line configuration including, for example, zones along the aforementioned straight line configuration such that, via temperature sensing and regulation means cells passing through the cell channel experience temperatures (e.g., temperature gradients, differences) as if the cell channel were serpentine or sinusoidal.

[0015] Associated with the cell channel is means for temperature regulation. The means for temperature regulation may include one or more temperature regulation conducting pathways (e.g. copper rods) or one or more temperature regulation channels for warming, cooling, or warming and cooling to vary the temperature in the cell channel and/or chiller/warming blocks. Accordingly, the temperature regulation conducting pathway(s) or channel(s) is/are in proximity to or associated with the cell channel and the chiller/warming blocks. Means for removal/delivery of heat from/to the temperature regulation conducting pathways is provided so that heat may be removed/delivered via the temperature regulation conductive pathways. Means for introducing fluid into or out of the temperature regulation channel(s) is provided so that warming or cooling fluid may be added into and/or removed from the temperature regulation channels. The temperature regulation conducting pathway(s) and channel(s) and the means for adding/removing heat or fluid to/from the same are thus temperature regulation means.

[0016] Temperature sensing means is also associated with the temperature regulation conducting pathway(s) and channel(s), chiller/warming blocks, and/or the cell channel for sensing the temperature along the path from inlet to outlet. The temperature sensing means is in electrical communication with a microprocessor means which is also in association with the temperature regulation means, and indeed in some embodiments the temperature sensing means may be part of the temperature regulation means. Accordingly, temperature along the path from inlet to outlet is sensed at various points and if it is not a desired temperature, the temperature regulation means may adjust temperature, e.g., by adding or removing warming heat or cooling fluid into the temperature regulation conducting pathway(s) or channel(s).

[0017] Thus, in accordance with one disclosed exemplary embodiment, an apparatus for cryo-treatment of a microfluidic sample provided that in some embodiments comprises a channel having an inlet for receiving a freeze-resistant carrier fluid containing at least one microfluidic droplet. The freeze-resistant carrier medium is immiscible with the droplet fluid. The surface of the droplet, which is also the interface between the freeze-resistant carrier fluid and the droplet solution, may be stabilized by surfactants. The apparatus may include a means for adding surfactant to the droplet surface either before or after cryo-treatment. The apparatus may also include a means for controlling the trajectory of the microfluidic droplet through the channel and at least one temperature regulation means in thermal communication with the channel. A controller may also be provided for controlling temperature of the at least one temperature regulation means. As used herein, cryo-treatment may refer to either cryolysis or cryopreservation or any other methodology involving similar conditions. [0018] In accordance with another disclosed exemplary embodiment, a method of performing cryo-treatment of a microfiuidic sample is provided that in some embodiments comprises preparing a microfiuidic droplet including a freeze-resistant carrier medium, introducing the microfiuidic droplet into a channel, and passing the microfiuidic droplet through at least one temperature zone at a predetermined, continuous flow rate via a channel.

[0019] In yet another advantageous embodiment, the present invention relates to a pipette- friendly lysis device design which may comprise a well plate format that may be compatible with centrifuges, PCR plates, and/or plate liquid handlers. The top of the plate may be covered with a substance for heat transfer, such as foil.

[0020] The present invention also compasses methods of cryolysis of a sample utilizing a pipette-friendly lysis device which may comprise filling a well in a well plate with a sample, capping and centrifuging of the sample to redistribute it into a shallow configuration with small thermal inertia enabling the sample to be rapidly frozen and thawed, freeze/thaw, and either centrifuging or removing cap and pressing, thereby resulting in a lysate in the well.

[0021] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.

[0022] It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", 'included", ''including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. [0023] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

DESCRIPTION OF THE DRAWINGS

[0024] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[002S] FIG. 1A illustrates a cryolysis embodiment of the invention. One or more droplets 100 each containing one or more cells 110 and buffer solution 120 are suspended in a carrier solution 130. The carrier solution does not freeze or boil at all temperatures present in the device. Moreover, the carrier solution 130 flows at all temperatures present in the device. These droplets 100 suspended in the carrier solution 130 enter the device through an inlet 210 to a channel 175. Channel 17S passes alternately between a chiller block 260 and a wanning block 240. Thus, each cell travels through the chiller block 260 set to a temperature or temperatures to promote the formation of ice crystals within the cell 110, or ice crystals within the buffer solution 120 outside the cell and protruding into the cell 110, but not within the carrier solution 1130 transporting the droplet 100 with cell 110. Each cell 110 is passed to the wanning block 240 to promote the melting of the intracellular ice crystals. The droplet 100 and cell 110 thus pass through channel segments in the chiller block 260 and channel segments in the warming block 240, as many times as desired, typically anywhere from 2 to 100 times. The duration and temperature of each cycle is also adjustable. The repeated freeze/thaw cycling leads to membrane instability and cell lysis. Emerging from outlet 290 is a droplet containing a lysed cell 1300 in emulsion 320. Sensors 215, 225, 235, 245, 255, 265, and 295 are in communication with the chiller block 260 and/or warming block 240 and provide feedback in order to advantageously adjust the temperatures of the chiller block 260 and warming block 240, either locally or globally.

[0026] FIG IB illustrates a preferred embodiment of FIG 1 A in which warming block 240 is separated from chiller block 260 to avoid direct heat transfer between the two. Temperature sensor 225 is now placed in the region between blocks and measures the contents of channel 175 halfway between the chiller and warming block. Temperature sensor 235 may be positioned near where channel 175 enters the chiller block 260, and sensor 265 at points where channel 175 is well inside the chiller block 260. Similarly, sensor 255 may be positioned near where channel 175 enters the warming block 240, and sensor 245 at points where channel 175 is well inside the wanning block 240.

[0027] FIG. 2A illustrates a cryopreservation embodiment of the invention. One or more droplets 1000 each containing one or more cells 1110 and cryopreservant buffer solution 1120 that readily permeabilizes the cell (e.g., a solution containing a DMSO-containing composition or glycerol-containing composition) are suspended in a carrier solution 1130. The carrier solution 1130 does not freeze or boil at all temperatures present in the device. Moreover, the carrier solution 1130 flows at all temperatures present in the device. These droplets 1000 suspended in the carrier solution 1130 enter the device through an inlet 1210 to a channel 1175. Channel 1175 passes through a module 1200 that includes a user controlled cooling element (a chiller block 1260). Thus, each cell travels through the chiller block 1260 set to a temperature or temperatures to promote the controlled freezing of the cell 1110, in the cryopreservant buffer solution 1120 contained in the emulsion droplet 1000. A uniform cooling rate of 1°C per minute from ambient temperature is effective for a wide variety of cells and organisms. In this manner, the duration, rate, and temperature of the freezing process may be precisely controlled so as to promote maximum viability upon subsequent thawing. Emerging from outlet 1290 is a droplet containing a cryopreserved cell 1300 in emulsion 1320. Since the cryopreservant 1120 may comprise or consist essentially of an emulsion oil that freezes at significantly lower temperatures than the cell 1110, specific volumes (or numbers of cells) may be harvested without the need to thaw an entire culture of frozen cells. Sensors 1215, 1225, 1235, 1245, 1255, 1265, and 1295 are in communication with the cooling element (chiller block 260) and provide feedback in order to advantageously adjust the temperatures of the chiller block 260 either locally or globally.

[0028] FIG. 2B illustrates a preferred cryopreservation embodiment of the invention, with alternate shape of channel 1175 and alternate placement of temperature sensors 1215, 1225, 1235, 1245, 1255, 1265, and 1295.

[0029] FIG. 3 illustrates a cryolysis embodiment of the invention. Each piece of the FIG. 3 system that is analogous to a piece of the FIG. 1 system is analogously numbered, using a prime (') after the number. Thus, for example, the channel is 175', the module is 200', etc. The channel 175' is not serpentine or sinusoidal as in the embodiments of FIG. 1, and hence these sensors 215', 225', 235', 265', 255', 245' and 295' are analogous to sensors 215, 225, 235, 265, 255, 245 and 295 and extend for regions along channel 175'. The repeated freeze/thaw cycling leads to membrane instability and cell lysis as in the sinusoidal or serpentine channel 175 of the embodiments of FIG. 1. Thus, just as there are temperature differences or gradients at various points along channel 175, e.g., between sensors 215 and 295 etc. (enumerated above as items 1- 11), there are those temperature difference or gradients along channel 175' at analogous points (e.g., between sensors 215' and 295').

[0030] FIG. 4 illustrates a cryopreservation embodiment of the invention. Each piece of the FIG. 4 system that is analogous to a piece of the FIG. 2 system is analogously numbered, using a prime (') after the number. Thus, for example, the channel is 1175', the module is 1200', etc. The channel 1175' is not serpentine or sinusoidal as in the embodiments of FIG. 2, and hence these sensors 1215', 1225', 1235', 1265', 1255', 1245' and 1295' are analogous to sensors 1215, 1225, 1235, 1265, 1255, 1245 and 1295 and extend for regions along channel 1175'. The freeze cycling leads to cryopreservation as in the sinusoidal or serpentine channel 1175 of the embodiments of FIG. 2. Thus, just as there are temperature differences or gradients at various points along channel 1175, e.g., between sensors 1215 and 1295 etc. (enumerated above as items 1-11), mere are those temperature difference or gradients along channel 1175' at analogous points (e.g., 1. between sensors 1215' and 1295' etc.).

[0031] FIG. SAi illustrates temperature regulation channels 575 and 675 alongside or adjoining channel 175 or 1175. In embodiments of cryolysis, channel 675 may contain water or fluid that warms channel 175, and in embodiments of cryopreservation, two channels 575 are present, one in place of channel 675, with channel 575 providing fluid that cools channel 175 or 1175.

[0032] FIG. 5Aii illustrates a single channel 175 or 1175 and a single channel 575 that provides cooling in embodiments of cryopreservation of FIG. 2 to channel 1175. However, if regions of channel 575 may receive warm water or fluid and cold fluid at other regions, e.g., coordinating with sensors, then one temperature regulation channel may run alongside or adjoin channel 175 for cryolysis embodiments of FIG. 1.

[0033] FIG. 5A111 illustrates an embodiment wherein temperature regulation channel 575 or 675 rotates about, wraps around, or zigzags along channel 175, 175', 1175, or 1175'.

[0034] FIG. 5Bi illustrates a preferred embodiment of FIG. 5Ai in which a serpentine channel 175 or 1175 comes into communication with the temperature regulation channels 575 and/or 675. The temperature regulation channels are separated by insulation to reduce cross-talk. The temperature sensors 225, 235, 245, 255, and 265 are placed advantageously near channel 175 or 1175. One set of sensors is shown in the drawing; however, multiple sets may be advantageously placed along channel 175 or 1175. In embodiments of cryolysis, channel 675 may contain water or fluid that warms channel 175, and in embodiments of cryopreservation, two channels 575 are present, one in place of channel 675, with channel 575 providing fluid that cools channel 175 or 1175.

[0035] FIG. 5Bii illustrates a preferred embodiment of FIG. 5Aii in which a single channel 575 provides cooling to channel 175 or 1175 for embodiments of cryopreservation of FIG. 2. However, if warm and cold fluid may be alternately circulated through channel 575, e.g., coordinating with sensors, then one temperature regulation channel may run alongside or adjoin channel 175 for cryolysis embodiments of FIG. 1.

[0036] FIG. 5Biii illustrates a preferred embodiment of FIG. 5Aiii wherein temperature regulation channel 575 or 675 rotates about, wraps around, or zigzags along channel 175, 175', 1175, or 1175'.

[0037] FIG.5Biv illustrates an embodiment wherein two temperature regulation channel 575 and 675 rotate about, wrap around, or zigzag along channel 175, 175', 1175, or 1175' to produce cold and hot regions alternately positioned in space along channel 175, 175', 1175, or 1175'. Sets of temperature sensors 215, 225, 235, 245, 255, 265, and 295 are placed advantageously near channel 175 or 1175 to provide feedback for controlling the flow and fluid temperature in the temperature regulation channels 575 and 675. One set of sensors is shown in the drawing; however, multiple sets may be advantageously placed along channel 175 or 1175.

[0038] FIG.5C illustrates embodiments where channel 175, 175', 1175 or 1175' is generally adjacent to temperature regulation channel 575 or 675.

[0039] FIG.5D illustrates embodiments where channel 175, 175', 1175 or 1175' is generally coaxial with temperature regulation channel 575 or 675.

[0040] FIG. 5E-1 illustrates sensor 215, 215', 1215, 1215', 225, 225', 1225, 1225', 235, 235', 1235, 1235', 265, 265' 1265, 1265', 255, 255', 1255, 1255', 245 245', 1245, 1245' 295, 295', 1295, and/or 1295' associated with temperature regulation channel 575 or 675, e.g., temperature of the temperature regulation channel and/or fluid therein is being measured to control temperature in the channel 175, 175' 1175, or 1175'. [0041] FIG. 5E-2 illustrates sensor 215, 215', 1215, 1215', 225, 225', 1225, 1225', 235, 235', 1235, 1235', 265, 265' 1265, 1265', 255, 255', 1255, 1255', 245 245', 1245, 1245' 295, 295', 1295, and/or 1295' associated with channel 175, 175' 1175, or 1175', e.g., temperature of the channel 175, 175' 1175, or 1175' and/or contents therein is being measured to control temperature in the channel 175, 175' 1175, or 1175'.

[0042] FIGS. 5E-3 and 5E-4 illustrate means for controlling or regulating temperature, with temperature regulation unit 475. Specifically, in each of these figures there is unit 475 including a removing and/or adding channel 775 in communication with temperature regulation channel 575 or 675 and optional removing and/or adding channel 875 in communication with temperature regulation channel 575 or 675, with the junction thereof being control means 975 which is in electronic communication with microprocessor means whereby at select times or temperatures or when desired fluid from channel 575 or 675 may be removed or added to by one of channels 775 or 875. For example, in a one removal / addition channel system, i.e., where there is no channel 875, at select times or temperatures or when desired, cooling or warming fluid may be added to channel 575 or 675 by controller 975 opening valve means between channels 775 and 575 or 675 to allow the flow into channel 575 or 675 from channel 775, and the system may also provide for removal of fluid by means of channel 775, e.g., controller 975 may open valve means between channels 775 and 575 or 675 whereby fluid flows from channel 575 or 675 into channel 775. Assuming a plurality of these controllers and channels, an upstream channel 775 may have warming or cooling fluid flowing into the system, and a downstream channel 775 may provide for removal to equilibrate or control the amount of fluid in channel 575 or 675. Alternatively, each controller 975 may be associated with two channels, 775 and 875, and valve means therefor, whereby the means for removing fluid that may be one of channels 775 and 875 and the other of channels 775 and 875 may be means for adding fluid, whereby at select times or temperatures or when desired, channel 775 (or 875) may remove fluid from channel 675 or 575 and at select times or temperatures or when desired, channel 875 (or 775) may add fluid into channel 675 or 575. In FIG. 5E-3, the controller 975 also includes sensor 215, 215', and 1215. 1215', 225, 225', 1225, 1225', 235, 235', 1235, 1235', 265, 265' 1265, 1265', 255, 255', 1255, 1255', 245 245', 1245, 1245' 295, 295', 1295, and/or 1295', i.e., the sensor is associated with temperature regulation channel 575 or 675, e.g., temperature of the temperature regulation channel and/or fluid therein is being measured to control temperature in the channel 175, 175' 1175, or 1175'. FIG. 5E-4 is analogous to FIG. 5E-2 insofar as sensor 215, 215', 1215, 1215', 225, 225', 1225, 1225', 235, 235', 1235, 1235', 265, 265' 1265, 1265', 255, 255', 1255, 1255', 245 245', 1245, 1245' 295, 295', 1295, and/or 1295' is associated with channel 175, 175' 1175, or 1175', e.g., temperature of the channel 175, 175' 1175, or 1175' and/or contents therein is being measured to control temperature in the channel 175, 175' 1175, or 1175'. The channels 575, 675, 775 and 875 may be of a material as used for channel 175, 175' 1175, or 1175'.

[0043] While the sensor 215, 215', 1215, 1215', 225, 225', 1225, 1225', 235, 235', 1235, 1235', 265, 265' 1265, 1265', 255, 255', 1255, 1255', 245 245', 1245, 1245' 295, 295', 1295, and/or 1295' in FIGS. 5E-1 and 5E-2 and the unit 475 of FIGS. 5E-3 and 5E-4 are illustrated with respect to coaxial arrangement of channels 575 or 675 and channel 175, 175' 1175, or 1175', the sensors may be on or associated with the channels 575 or 675 or channel 175, 175' 1175, or 1175' and the unit 475 may be on or associated with channel 575 or 675 in any of the other arrangements of channels 575 or 675 and channel 175, 175' 1175, or 1175' of FIGS. 5A, 5B, 5C, 5D, and 5F.

[0044] FIG 5F illustrates a cryolysis device consisting of interdigitated conducting fingers extending from a chiller block 260 or 5006 and a warming block 240 or 5004 which create cold zones 5010 or warm zones 5008 in a straight sample channel design 175, 175', 1175, or 1175'. Droplets containing intact cells are carried in the carrier solution that enters f om the inlet 210, 1210, 210', or 1210' and first passes a chiller finger 5002 to freeze the cells. The chiller fingers 5002 are connected to a chiller block 260 or 5006. The cell droplets then pass a warming finger 5000 to thaw the cell. The warming fingers 5000 are connected to a warming block 240 or 5004. The cell droplet then passes another chiller finger 5002, then a wanning finger 5000, and so forth until reaching the end 290, 1290, 290', or 1290' of the cell channel 175, 1175, 175', 1175', at which point sufficiently many freeze-thaw cycles have been accomplished to lyse the cells in each droplet. Temperature regulation means 215, 1215, 215', and/or 1215' are present at the inlet, means 295, 1295, 295', and/or 1295' at the outlet, and means 225, 235, 245, 255, 265, 1225, 1235, 1245, 1255, 1265, 225', 235', 245', 255', 265', 1225', 1235', 1245', 1255', and/or 1265' at advantageous points along the channel 175, 1175, 175', or 1175'.

[0045] FIG. 5G illustrates a cryolysis device with a circulation loop and sensor and temperature regulation means. Cryolysis is performed by circulating sample between a chiller block 260 or 5006 and a warming block 240 or 5004. With outlet valve 351 closed and input valve 350 open, sample flows into inlet 210, 1210, 210', or 1210' and fills channel loop 175, 1175, 175', or 1175^*. Inlet valve 350 is then closed, and pump 360 circulates the fluid around the channel loop 175, 1175, 175', or 1175'. On each pass around the channel loop, the sample passes through the chiller block 260 or 5006 to freeze the cell inside the droplets and then the wanning block 240 or 5004 to thaw the droplets. Temperature regulation means (numbered) are present at the inlet and at advantageous points along the channel loop 175, 1175, 175', or 1175'. Once the sample has circulated the desired number of times around the loop, the pump 360 is stopped, inlet valve 350 and outlet valve 360 are opened, and the lysed sample is flowed out of the loop. Additional valves could be added at advantageous points on the loop, for example before or after the pump 360, to improve control of sample loading/unloading.

[0046] FIGS. 6A and 6B illustrate disclosed droplets subjected to experimental processes according to an exemplary embodiment of the invention.

[0047] FIG. 7 schematically illustrates a process according to an exemplary embodiment of the invention.

[0048] FIGS. 8A-8E provide graphical analysis of the results from the process of FIG. 7 according to an exemplary embodiment of the invention.

[0049] FIG. 9A depicts the microfluidic lysis chamber 380. Channel inlet 385 accepts intact cells 110 in carrier solution 120 and delivers these to lysis chamber 380. Lysis chamber 380 has small height 381 so fluidic contents of chamber freeze and thaw rapidly. To handle 10-100 uL volumes, the chamber 380 may have a wide plan area 383. The chamber 380 is mounted on a base 382 with low thermal resistance. The chamber base 382 contacts the surface of an external rapid chiller/warming module 390, which may rapidly change temperature from a chiller temperature for freezing to a warming temperature for thawing. Temperature of the chamber contents may be monitored by advantageously placed sensors 225, 225', 1225, 1225', 235, 235', 1235, 1235', 265, 265', 1265, 1265', 255, 255', 1255, 1255', 245, 245', 1245 and/or 1245' on the top of the chamber, and at the inlet, sensors 215, 1215, 215', and/or 1215', and at the outlet, sensors 295, 1295, 295', and/or 1295'; output from these sensors may be fed back into an electronic or computer controller for the external rapid chiller/warming module 390. Once the sample is flowed into the chamber, the desired freeze-thaw cycles are accomplished to lyse the sample, after which the sample is pumped out of the chamber 380 via outlet channel 386. Cells 300 are now lysed, i.e. their walls are rendered permeable or disintegrated 310 sufficiently to allow the genomic material to exit cell 300.

[0050] FIG. 9B depicts pre-lysis and post-lysis concentration means added to microfluidic lysis chamber 380. Concentration of sample allows a more concentrated lysate to be sent to downstream components, enhancing detection capability. The lysis chamber 380 is connected to a nanofilter 370, which is connected to valve 3S7, which is connected to outlet 360. The lysis chamber 380 is also connected to an ultrafiltration (UF) membrane 371, which is connected to valve 3S8, which is connected to outlet 361. The UF membrane 371 has a specified molecular weight (MW) cutoff (e.g. 50 kDa), i.e. molecules above the cutoff are retained in the lysis chamber 380. For pre-concentration prior to lysis, pusher fluid, reagent, or additional sample enters through inlet channel 385, while valves 353 and 358 are closed and valve 357 open, forcing excess fluid through nanofilter 370 and waste outlet 360, but retaining intact cells within the lysis chamber 380. For post-concentration following lysis, pusher fluid or reagent enters through inlet channel 385, while valves 353 and 357 are closed and valve 358 open, forcing excess fluid through UF membrane 371 and waste outlet 361, but retaining debris and genomic material down to the specified MW cutoff, e.g. 50 kDa. An additional use of the nanofilter 370 and associated waste outlet 360 is to allow perfusion of reagents from the inlet channel 385 into the lysis chamber 380 without increasing the overall volume of the treated sample.

[0051] FIG. 9C depicts a means for the addition of drugs, RNA protectant(s), and pusher fluid to lysis chamber 380. Valved inlets for reagents 356, 357 (such as drugs, RNA protectant), and valved inlet 358 for pusher fluid are connected via a channel 387 to the main inlet channel 385, downstream of inlet valve 385. Pre-lysis, closing valves 352, 354, and 355 and opening valve 354, reagents and additional sample may be added without increasing the overall volume of the sample in the lysis chamber 380, and without losing intact cells. Pre-lysis, the sample may be pre-concentrated by closing valves 352, 353, and 355, and opening valves 354 and 358 to add pusher fluid to reduce the sample volume within the lysis chamber 380, without losing intact cells. Post-lysis, closing valves 352, 353, and 354 and opening valves 356 and 355 to add reagents without increasing overall sample volume in lysis chamber 380, and without losing genomic material of MW greater than the cutoff of the UF membrane 371. Post-lysis concentration may be achieved by closing valves 352, 353, and 354, and opening valves 355 and 358 to add pusher fluid to reduce the sample volume within the lysis chamber 380, without losing genomic material of MW greater than the cutoff of the UF membrane 371.

[0052] FIG. 9D depicts a means for sample separation and distribution in parallel lysis chambers. To enable sub-sample isolation and high-throughput treatment by different reagent cocktails prior to lysis, and then subsequent isolated sub-sample lysis and analysis, a bifurcation channel network 388 is added between the sample inlet 1385 and downstream sub-sample lysis chambers 1380, 2380, 3380, 4380 and deliver lysed samples to outlets 1386, 2386, 3386, and 4386. Additional or fewer channel bifurcations could be added or removed as needed to divide the sample into any number of sub-samples, and output said sub-samples into individual lysis chambers and following lysis to individual outlets.

[0053] FIG. 9E depicts a means for sample preparation and treatment prior to sample division and/or lysis. Valved inputs 1356, 1357, and 1358 allow for reagents, sample, and pusher fluid to be added and flowed through inlet channel 1385 into the sample preparation chamber 401. To handle 10-1000 uL volumes, chamber 401 may have a wide plan area 1383. Chamber 401 is mounted on a base 1382 with low thermal resistance. The chamber base 1382 contacts the surface of an external warming module 1390, which may maintain or vary the warming temperature between 10 °C and 40 ° C, or as desired. Temperature of the chamber contents may be monitored by advantageously placed sensors 225, 225', 1225, 1225', 235, 235', 1235, 1235', 265, 265', 1265, 1265', 255, 255', 1255, 1255', 245, 245', 1245 and/or 1245' on the top of the chamber; output from these sensors may be fed back into an electronic or computer controller for the external warming module 1390. A nanofilter 1370 may be connected between chamber 401 and valve 1357, and waste outlet channel 1360, to allow reagents or additional sample to be added without increasing volume of sample in chamber, or to allow pusher fluid to be added while retaining intact cells inside the chamber 401, to accomplish sample concentration.

[0054] FIG. 10 illustrates finite element heat transfer simulations in Comsol of the freezing of 30 uL of water in different geometries (thin rods, thick blocks, and thin discs). The geometries are axisymmetric (2D simulation) and are symmetric about their mid-planes. Thus, only half the domains are simulated. The phase change is calculated using the popular Apparent Heat Capacity method. Sections of each geometry are shown in the top row at different times, indicating the ice-water phase boundary. The plots along the bottom row show the temperature of the centroid of each shape. By symmetry, the centroid of each shape is the position of highest temperature. [00SS] FIG. 11A depicts a user-friendly system for freeze-thaw lysis. Design includes a disposable (consumable) base unit 500 with an inlet chamber 504 for sample input, connected via narrow aperture 503 to shallow region 502. Shallow region 502 bounded by foil layer 510 on one side. Foil layer held in place by collar 511, which forms a mechanical seal. Base unit 500 maybe sealed with cap 512, for example a Mi era Amp tube caps, which fits 513 over the top 509 of the base unit 500. In brief, the device is operated as follows. Sample is input into inlet chamber 504, then moved via tapping or centrifuge through narrow aperture 504 and redistributed into shallow region 502. Once the fluid is in the shallow region, it is sandwiched between the base unit wall and foil layer boundaries, limiting the maximum thickness 553 of the sample to a specified amount, in this case 0.5 mm, which allows the sample to be frozen within 1 s and thawed within 4 s. The foil layer makes contact with an external thermal module 1590, which comprises either a single module whose temperature cycles between chilling and wanning temperatures, like 590, or the device would be moved robotically or manually between chiller 260 or 5006 and wanning 240 or 5004 blocks or modules. Other designs are as follows. (1) The base unit 500, cap 512, collar 511, and foil layer 510 are rotationally symmetric about the axis 506. (2) The collar 511 shape mates with the outer edge 505 of the base unit 500, to form a mechanical seal to keep the sample in the shallow region 502. (3) The volume of the shallow region must be greater than the sample volume, so that the entire sample may fit inside the shallow region. The radius 554 of the shallow region may be increased to allow for larger sample volumes, but keeping the thickness 553 of the shallow region less than or equal to 0.5 mm to maintain rapid freeze and thaw times. (4) The diameter of the aperture 503 must be much less than the capillary length of the sample solution (e.g. 2.7 mm for pure water) so that the sample will not bulge into the aperture 503, increasing the sample thickness and therefore the times required for freezing and thawing. If the shallow region is full of sample, the sample contact line must be pinned on the lower edge 555 of the aperture 503. To achieve this, the edge must be sufficiently sharp and non-wetting, either by using a non-wetting plastic or coating the region with hydrophobic coating. Alternatively, a sample volume lower than the shallow region 502 volume could be used, so that when redistributed within the shallow region 503 the sample stays away from the aperture 503. (5) Device 500 may be disposable (i.e. a consumable) or reusable.

[0056] FIG. 11B depicts further views of base unit 500 of user-friendly device for freeze- thaw lysis. Inlet region 504 is connected to narrow aperture 503 which is connected to shallow region 502. Shape of outer edge SOS of base unit S00 allows it to mate with collar Sll (not shown). Inlet region 504 has outer surface structures, including structure S09 that fits inside Micro Amp tube caps, structure S08 that fits inside PCR plate wells, structure S07 that sits outside and on top of PCR plate well and prevents structure S08 from getting stuck inside PCR plate well. The design of base unit S00 is rotationally symmetric about the axis S06.

[00S7] FIG. llC depicts the assembly of base unit S00, foil layer S10, and collar Sll to produce user-friendly device for freeze-thaw lysis. Shape of outer edge SOS of base unit S00 mates with the shape of the inner side of collar Sll so that the collar Sll snaps over foil layer S10 and base unit S00 as shown to form a mechanical tight seal to keep fluid in shallow region S02. A non-wetting coating may be applied to edge SOS and foil layer Sll to discourage fluid incursion between the foil layer and base unit S10, when assembled, to prevent sample loss, and maintain sample isolation and containment within shallow region. All surfaces of device in potential contact with sample may also be coated with non-fouling materials to prevent sample loss and attachment to device surfaces.

[00S8] FIG. 11D depicts views of assembled user-friendly device 1S00 for freeze-thaw lysis. Shown are mated base layer S00, foil layer S10, and collar Sll. Tight mechanical fit between outer edge SOS of base unit S00, foil layer S10, and collar Sll enables sample to be isolated within shallow region.

[00S9] FIG. HE depicts example protocol for use of user-friendly freeze-thaw lysis device 1S00. (a) Sample 518 is pipetted or otherwise inserted into inlet chamber 504 of base unit 500. Sample 518 may include may include cells of different types and a cocktail of reagents to aid lysis and protect RNA. (b) MicroAmp tube cap or similar closure means is secured on base unit 500, mated with structure 509 on base unit, making a tight mechanical seal 513. (c) Device 1500 is tapped or centrifuged or other means to move sample solution 518 from inlet chamber 504 through narrow aperture 503 and into shallow region 502 to redistribute the sample solution into a thin shape that may rapidly freeze and thaw, (d) Foil layer 510 on device 1500 is then brought in contact 519 with surface of external thermal module 1590, which may be the same or similar to module 590, which rapidly changes temperature from a chiller temperature for freezing to a wanning temperature for thawing. Alternately, device 1500 is robotically or manually set on a chiller block 260 or 5006 until sample 518 is frozen, and then moved to a warming block 240 or 5004 until sample 518 is thawed. A sufficient number of freeze-thaw cycles are carried out until sample 518 is lysed, becoming lysate 2518. (e) Cap 512 and base unit 500 are then removed 520, exposing lysate 2518 on foil layer 510, optionally still connected to collar 511. Lysate 2518 is then removed by pipette 521 or other means. Wetting properties of foil layer may be adjusted to enable facile lysate 2518 removal.

[0060] FIG. 11F depicts arrayed user-friendly freeze-thaw lysis device 516. Design enables the high-throughput freeze-thaw lysis of multiple samples simultaneously, (a) Collar 510 in arrayed configuration 515. (b) Steps to assemble device 516. Foil layers 510 are clamped between base units 500 and collar array 515. (c) Views (top, bottom, isometric, from top to bottom) of device 516.

[0061] FIG. 11G depicts optional mating of user-friendly lysis device 1500 with PCR plate well 514. Device base unit 500 has structure 508 that fits inside PCR plate well 514, structure 507 that sits outside and on top of PCR plate well and prevents structure 508 from getting stuck inside PCR plate well 514. The design of base unit 500 and assembled device 1500 are rotationally symmetric about the axis 506.

[0062] FIG. 11H depicts the mating of arrayed freeze-thaw lysis device 516 with 96-well PCR plate 517. (a) Device 516 mated in arrayed manner similar to mating of single device 1500 to PCR plate well 514 in FIG. 11G. Collars in array 515 are spaced at 18 mm, equal to twice the center-to-center distance of PCR plate wells. Thus, the necks of the devices 1500 fit into every other well in a line of PCR plate wells, as shown, (b) Side and top-down views of arrayed device 516 mated with PCR plate 517.

[0063] FIG. 11I depicts an alternate protocol using centrifuge to remove sample 518 or lysate 2518 from a single lysis device 1500 or arrayed lysis device 516. (a) Following freeze- thaw lysis, all caps removed from device 1500 or 516. (b) Device 1500 or 516 is inverted and mated with PCR plate wells 514 or 517 as in FIGS. 11G or 11H, respectively, (c) Sample 518 or lysate 2518 is moved 522 via centrifugation from shallow region 502 through narrow aperture 503 and inlet chamber 504 and into the PCR plate well(s) 514. (d) Device 1500 or 516 is then removed from PCR plate well(s) 514.

[0064] FIG. 11 J illustrates a 3D printed prototype of user-friendly freeze-thaw lysis device, (i) Components: collar 511; foil 510; base unit 500. Foil 510 is sandwiched between base unit 500 and collar 511, which snaps in place, (ii) Assembled device, ready for use. Collar 511 is present, but hidden by foil 510. Sample is pipetted into inlet 504, which is then capped 512. By manual tapping, sample is moved into thin (shallow) region, which changes the sample geometry into a thin disc-like shape, ideal for rapid freeze/thaw cycles, (iii) Capped device ready for freeze-thaw lysis, currently done manually by moving device (containing sample) between metal plate at -78 °C (not shown) and metal plate at room temperature (not shown). Foil layer 511 enables rapid heat transfer between plates and sample. Freezing of water is completed within 1 s, and thawing within 4 s. (iv) Device is disassembled and lysate is removed from foil 510, for easy sample recovery.

[006S] FIG. 12A depicts the design of a thin tube device 600 and corresponding system 601 for freeze-thaw lysis. Thin tube geometry allows sample to be frozen and thawed rapidly and repeatedly, (a) Mating of thin tube 600 to sample input/recovery receptacle 601 through connection 602. (b) Thin tube 600 mated with sample input/recovery receptacle 601. (c) Fill tube 600 with buffer or pusher fluid 603. (d) Inject sample 604. (e) Add additional buffer or pusher fluid 605 to position sample 604 in bottom portion of tube 600. (f,g) Perform freeze/thaw lysis by alternately (609) dipping tube 600 with sample 604 into (f) cryo-liquid 607 and (g) warming liquid 608. Repeat (609) for as many freeze-thaw cycles as necessary so that sample 604 is fully lysed 606. (h) Elute lysed sample 606 from tube for downstream processing. Design allows disposable thin-tube to be used for freeze-thaw, or a permanent thin tube that may be washed after step (h) and then re-used.

[0066] FIG. 12B depicts the assembly of an arrayed thin tube device 1600 and corresponding system 1601 for freeze-thaw lysis. Arrayed design allows separate samples to be lysed via freeze-thaw in parallel to increase throughput, (a) Mating of thin tube array 1600 to sample input/recovery receptacle array 1601 through connection 1602. (b) Thin tube array 1600 mated with sample input/recovery receptacle array 1601.

[0067] FIG. 12C depicts a protocol for multiplexed thin tube device for freeze-thaw lysis, (a) Fill arrayed tubes 1600 with parallel streams of buffer or pusher fluid 1603. (b) Inject different samples in parallel 1604. (c) Add additional buffer or pusher fluid 1605 in parallel streams to position samples 1604 in bottom portion of tubes in array 1600. (f,g) Perform freeze/thaw lysis by alternately (1609) dipping tube array 1600 with samples 1604 into (f) cryo- liquid 607 and (g) warming liquid 608. Repeat (1609) for as many freeze-thaw cycles as necessary so that the samples 1604 are sufficiently lysed 1606. (h) Elute lysed samples 1606 from tube for downstream processing. [0068] FIG. 13A depicts an alternate design of a thin tube device for freeze-thaw lysis: the loop device. A loop of microtubing 640 containing sample 641 passes through a guide tube 642. The guide tube passes into an insulated cooling chamber 647, within which resides a cryo-liquid 644, for example ethanol in dry ice. The cryo-liquid circulates through holes 643 in the guide tube 642 to cool the loop 640 and hence the sample 641. Room temperature or warm air surrounds the loop outside of the guide tube 642 and cooling chamber 647. The loop 640 is rotated 650 through the guide tube 642, passing through the inlet 648, into the portion of the guide tube 651 immersed in the cryo-liquid, thereby freezing the sample. The loop is continuously rotated, so that the frozen portion exits the guide tube outlet 649 and thaws in the room temperature or warm air 646. The loop 640 is continuously rotated 650 to achieve the desired number of freeze-thaw cycles. Advantage of loop design is that external thermal module is static; freeze-thaw cycles are provided by rotating loop.

[0069] FIG. 13B depicts a protocol for loading microtubing with sample for use with loop device, (a) Sample 641 contained in syringe 652. Syringe tip 653 is inserted into microtubing 654. (b) Sample 641 in syringe 652 injected into microtubing 654. Syringe 652 then removed from microtubing 654.

[0070] FIG. 13C depicts a protocol for threading and connecting microtubing for loop device, (a) Connector plug 655 inserted into one end of microtubing 654. (b) Microtubing 654 threaded 656 through guide tube 642, and pulled through. Connector plug 655 is inserted into open end of microtubing 654 to form a microtubing loop 640 containing sample 641.

[0071] FIG. 13D depicts a fabricated loop device, (a) Loop device set up and ready for freeze-thaw cycles, (b) Inside of cooling chamber 647, showing guide tube 642, loop 640, sample 641, cryo-liquid 644, and cooling means 657 (in this case an ethanol and dry ice mixture), (c) Operating loop lysis device. Loop 640 is rotated through guide tube 642, passing through the cooling chamber 647 and cryo-liquid 644 to achieve the desired number of freeze- thaw cycles, (d) Closeup of cooling chamber 647 and guide 642 tube outlet 649 , showing loop 640, sample 641, connector plug 655 and join 645 in microtubing.

[0072] FIG. 13Έ depicts data from laboratory experiments with loop device demonstrating freeze-thaw lysis. Fluorescent images of BL-1 microbial strains stained by LIVE (green) / DEAD (red) after 10 freeze/thaw cycles with loop device at 20 s per cycle. [0073] FIG. 14A depicts the design of dunk device 700 for freeze-thaw lysis, another alternate embodiment of a thin tube device. The advantages of dunk device design are: ease of use; static external thermal module (freeze-thaw cycles enabled by moving dunk device); rapid freeze-thaw lysis due to thin tube geometry, (a) Isometric and front views of dunk device design. Segments of microtubing 701 are clamped 702 in place and spaced along a rod 706, which is insulated 703 and connected to an insulated handle 704. The clamped tubing segments 701 are spaced 705 sufficiently to allow ample cryo-fluid circulation to expedite heat transfer, (b) Closeup of array of parallel clamped microtubing segments 701.

[0074] FIG. 14B depicts a protocol for freeze-thaw lysis with dunk device 700. Microtubing segments are filled with sample according to protocol in FIG. 13B, and then clamped in place on dunk device, as shown in FIG. 14A. Clamping mechanically seals sample inside tubing. Dunk device with microtubing segments 1701 containing sample is then alternately dipped ("dunked") 707 in cryo-liquid 607 to freeze the samples and warming fluid (liquid or air) 608 to thaw.

[0075] FIG. 14C depicts a fabricated dunk device 700 for freeze-thaw lysis.

[0076] FIG. 15 depicts utility of adding heat conductive baffles or barriers to a tube to expedite freeze-thaw lysis. (A) An empty tube 800. (B) A tube 801 with heat conductive baffles 802. Sample containing cells or emulsion with cells is placed in tube. Baffles 802 separate sample to reduce maximum distance between heat conductive surfaces, thereby reducing thermal mass of sample. (C) Top view of tube 801 with baffles 802. (D) Section CC view (see (C) for reference) of tube 801 with baffles 802.

DETAILED DESCRIPTION OF THE INVENTION

[0077] The invention will now be described with reference to the drawing figures, in which like reference numerals refer to like parts throughout. The following detailed description consists of example embodiments of the presently claimed invention with references to the accompanying drawings. Such description is intended to be illustrative and not limiting with respect to the scope of the present invention. Such embodiments are described in sufficient detail to enable one of ordinary skill in the art to practice the subject invention, and it will be understood that other embodiments may be practiced with some variations without departing from the spirit or scope of the subject invention. Reference is made to all of the Figures.

[0078] A novel, nonobvious and inventive feature of the invention is the integration of elements into a microfluidic device so that the microfluidic device is a multi-purpose - either cryolysis or cryopreservation - microfluidic device. In the practice of the invention, a cell may pass through up to 1000 to 10,000 freeze-thaw or cycles per second in the microfluidic device, when the cell channel is in either the straight or serpentine or sinusoidal configuration.

[0079] Cryolysis embodiments are illustrated in FIGS. 1A, 1B, 3, 5F and 5G. Referring to FIG. 1A or IB, one or more droplets 100 each containing one or more cells 110 and buffer solution 120 are suspended in a carrier solution 130. The carrier solution does not freeze or boil at all temperatures present in the device. Moreover, the carrier solution 130 flows at all temperatures present in the device. These droplets 100 suspended in the carrier solution 130 enter the device through an inlet 210 to a channel 175. Channel 175 passes alternately between a chiller block 260 and a wanning block 240. Thus, each cell travels through the chiller block 260 set to a temperature or temperatures to promote the formation of ice crystals within the cell 110, or ice crystals within the buffer solution 120 outside the cell and protruding into the cell 110, but not within the carrier solution 1130 transporting the droplet 100 with cell 110. Each cell 110 is passed to the wanning block 240 to promote the melting of the intracellular ice crystals. The droplet 100 and cell 110 thus pass through channel segments in the chiller block 260 and channel segments in the warming block 240, as many times as desired, typically anywhere from 2 to 100 times. The time and temperature of each cycle is also adjustable. The repeated freeze/thaw cycling leads to membrane instability and cell lysis. Emerging from outlet 290 is a droplet containing a lysed cell 1300 in emulsion 320.

[0080] Sensors 215, 225, 235, 245, 255, 265, and 295 are in communication with the chiller block 260 and/or warming block 240 and provide feedback in order to advantageously adjust the temperatures of the chiller block 260 and warming block 240, either locally or globally. Additional details on sensors and temperature regulation channels are illustrated in FIGS. 5A-E and discussed herein. Alternate disclosed embodiments may provide multiple independent thermal zones (i.e. hot and cold regions) that may contain multiple independently controlled elements such that each cycle may be tuned progressively according to a predetermined sequence. An alternate embodiment involving interdigitated thermally conductive fingers connected to chiller and warming blocks is illustrated in FIG. 5F and discussed herein. An alternate embodiment involving a circulation loop is illustrated in FIG. 5G and discussed herein.

[0081] In the embodiment illustrated in FIG. 1A, the warming block is positioned adjacent to the chiller block. In the preferred embodiment illustrated in FIG. IB, the warming block and chiller block are separated by an insulator, to prevent heat transfer between the two. While depicted in a top to bottom configuration, blocks 240 and 260 may also be in a side-by-side configuration, e.g., module 200 may be rotated 90 degrees, whereby the chiller block 260 is to the left of the wanning block 240 as one faces module 200, or the chiller block 260 may be to the right of the warming block 240 as one faces module 200.

[0082] The main consideration for lysis is the full freezing of the cell such that ice crystals may form disrupting the cell wall. Furthermore, there is sufficient water content to form ice crystals in sufficient number and appropriate size to disrupt a cell wall. There is also one or more sufficient additional reagents (such as a surfactant) to isolate and stabilize cell walls. As such the temperature must be sufficiently below freezing for the cell to freeze within the short time it spends flowing through the cold section and may be significantly less than the freezing point of pure water. Temperatures may range below 0 degrees C down to the freezing point of the carrier oil. Low-temperature heat transfer oils may have freezing points lower than -80°C. Typical temperatures of the chiller block and nearby portions of the cell channels 175 range from to - 40°C to -20°C to avoid significant changes in the viscosity of the oil. The thaw aspect of course considers thawing the cell at suitably higher temperatures. Accordingly, between the cold temperature of the chiller block and cell channel segment within and the warm temperature of the warming block and the cell channel segment within there is the possibility for temperature gradients along the system as herein discussed.

[0083] The current invention thus enables rapid freeze/thawing of a continuous stream of individual cells 110 in droplets 100 in a micro fluidic device. In this device, the freeze/thaw cycling occurs very rapidly given the reaction volumes (nano- to picoliter volumes). The advantages to a device and methods involving or arising from it include : optimal preservation of RNA/protein expression due to the rapid time scale (i.e., minimal nucleic acid and/or protein degradation), no need for neutralization or buffer exchange prior to downstream processes, ease of modular integration with other microfluidic devices and efficient recovery of intracellular milieu since the cell is "trapped" in an emulsion droplet 300.

[0084] Cryopreservation embodiments are illustrated in FIGS. 2A, 2B, and 4. Referring to FIG. 2A or 2B, one or more droplets 1000 each containing one or more cells 1110 and cryopreservant buffer solution 1120 that readily permeabilizes the cell (e.g., a solution containing a DMSO-containing composition or glycerol-containing composition) are suspended in a carrier solution 1130. The carrier solution 1130 does not freeze or boil at all temperatures present in the device. Moreover, the carrier solution 1130 flows at all temperatures present in the device. These droplets 1000 suspended in the carrier solution 1130 enter the device through an inlet 1210 to a channel 1175. Channel 1175 passes through a module 1200 that includes a user controlled cooling element (a chiller block 1260). Thus, each cell travels through the chiller block 1260 set to a temperature or temperatures to promote the controlled freezing of the cell 1110, in the cryopreservant buffer solution 1120 contained in the emulsion droplet 1000. A uniform cooling rate of 1°C per minute from ambient temperature is effective for a wide variety of cells and organisms. In this manner, the duration, rate, and temperature of the freezing process may be precisely controlled so as to promote maximum viability upon subsequent thawing. Emerging from outlet 1290 is a droplet containing a cryopreserved cell 1300 in emulsion 1320.

[0085] Since the cryopreservant 1120 may comprise or consist essentially of an emulsion oil that freezes at significantly lower temperatures than the cell 1110, specific volumes (or numbers of cells) may be harvested without the need to thaw an entire culture of frozen cells.

[0086] Sensors 1215, 1225, 1235, 1245, 1255, 1265, and 1295 are in communication with the cooling element (chiller block 260) and provide feedback in order to advantageously adjust the temperatures of the chiller block 260 either locally or globally. Additional details on sensors and temperature regulation channels are illustrated in FIGS. 5A-E and discussed herein. Alternate disclosed embodiments may provide multiple independent thermal zones that may contain multiple independently controlled elements such that each cycle may be tuned progressively according to a predetermined sequence.

[0087] While depicted in a top to bottom configuration in FIG 2A, the sinusoidal or serpentine channel 1175 of chiller block 1260 may also be in a side-by-side configuration, e.g., module 1200 may be rotated 90 degrees, whereby region 1202 is to the left of region 1201 as one faces module 1200, or the chiller block 1260 may such that an initial curve of the serpentine or sinusoidal path of channel 1175 is to the right as one faces module 1200.

[0088] As may be readily apparent from FIGS. 1-4, 5F, 5G, 9, 10, 11, 12, 13, and 14 by making simple adjustments to user controlled parameters on the device of FIGS. 1, 3, 5F, 5G, 9, 10, 11, 12, 13, and 14, it may be used for the cryopreservation of cells of the device of FIGS. 2 and 4. For example, if in the embodiments of FIG. 1, cooling is applied in that which is designated as warmer block 240, i.e., if warmer block 240 were instead a second chiller block 260, the device of FIG. 1 may be for cryopreservation. The invention thus allows for the utilization of a microfluidic device to process cells, each contained in a buffer-in-oil emulsion. Depending on the buffer solution used, and whether one of the blocks is a warming block, the device may be utilized to promote either cryopreservation or cryolysis of the cells in the emulsion.

[0089] For cryolysis embodiments, referring to FIG. 1, droplet 100 contains cell 110 suspended in a buffer solution 120 that may have a different freezing point than the interior of the cell; for instance a hypotonic solution in order to promote cell swelling and lysis. The freeze- resistant carrier solution is outside the droplet. The carrier solution remains in the liquid state throughout the operation to allow the droplets (or pellets/beads when frozen) to keep moving through the system. The cell freezes during the freeze portion of the cycle for either cryopreservation or cryolysis. The solution inside the drop but outside the cell does not necessarily freeze, but in general it may, and likely before the cell (since usually this solution will be hypotonic, i.e. have a higher water concentration than the cell, to maintain a high water concentration within the cell to promote freezing and ice crystal growth). The carrier solution has a lower freezing temperature than the cell, droplet, and chiller block. The freezing point of the cell is higher than the temperature of the chiller block. The carrier solution also has a boiling point well above the temperature of the warming block, so that it stays in the liquid state and ideally no gas bubbles nucleate during wanning. The droplet may comprise water, the cell, and dissolved chemicals including reagents to protect from RNA degradation, lysis chemicals, etc.

[0090] The buffer solution may also contain cryopreservants, RNA and DNA degradation inhibitors, and other ingredients to promote lysis such as zwitterioinic detergents such as 3-(N,N- Dimethylmyristylammonio)propanesulfonate, nonionic detergents such as TWEEN® from Sigma Aldrich, anionic detergents such as sodium deoxycholate, or alkaline buffer solutions such as EDTA with sodium dodecyl sulfate with sodium hydroxide followed by potassium acetate.

[0091] Some examples of cryolysis agents that could be put in the droplets: For difficult to lyse cells including prokaryotes such as bacteria, and others such as fungi, yeast, protozoa, algae, and plant cells, a variety of detergents and lysis agents may be used such as Lysozyme, Lysostaphin, zwitterioinic detergents such as 3-(N,N-Dimemylmyristylammonio) propanesulfonate, nonionic detergents such as TWEEN® from Sigma Aldrich, anionic detergents such as sodium deoxycholate, or alkaline buffer solutions such as EDTA with sodium dodecyl sulfate with sodium hydroxide followed by potassium acetate. For animal cells, hybridomas, stem cells, embryos, blood, and other eukaryotic cells the same variety of lysis agents are available but may require gentler conditions such as a hypotonic solution to promote swelling and lysis. The agent is in the droplet containing typically a single cell, but sometimes one to three cells, e.g., two cells may be in droplet.

[0092] For cryopreservation embodiments, referring to FIG. 2, droplet 1000 contains a cell 1110 suspended in cryopreservant buffer 1120. The cryopreservant is advantageously a type that readily permeabilizes the cell (e.g., a solution containing DMSO, or glycerol where DMSO is not suitable; for example, formulated complete cryopreservation medium such as Recovery™ Cell Culture Freezing Medium or Synth-a-Freeze® Cryopreservation Medium of Invitrogen or Life Technologies; other media may also be used such as about 50% to about 80%, e.g., 70% basal medium, about 10% to about 30%, e.g. about 20% FBS (fetal bovine serum), and about 5% to about 15%, e.g., about 10% DMSO (dimethyl sulfoxide), w/v. See, e.g., Polge et al, "Revival of spermatozoa after vitrification and dehydration at low temperatures." Nature 1949;164(4172):666; Lovelock et al, "Prevention of freezing damage to living cells by dimethyl sulphoxide," Nature 1959; 183(4672): 1394-5; Matsumura et al, "Polyampholytes as low toxic efficient cryoprotective agents with antifreeze protein properties," Biomaterials 30 (2009) 4842- 4849; EP1181865 Al ("Cryoprotective Solutions"); US Patent Application 20100311036 ("Methods for Augmentation of Cell Cryopreservation"; and US Patents Nos. 6,673,607 and 7,094,601, each of which is incorporated herein by reference.

[0093] Exemplary cryoprotective agents by cell type that may be in the droplets:

[0094] Cell /Organism Type Cryoprotective Agent (% w/v)

[0095] Bacteria Glycerol (about 9% to about 11%, e.g. about 10%)

[0096] Bacteriophage Glycerol (about 9% to about 11%, e.g. about 10%)

[0097] Fungi Glycerol (about 9% to about 11%, e.g. about 10%)

[0098] Yeast Glycerol (about 9% to about 11%, e.g. about 10%)

[0099] Protozoa DMSO (about 3% to about 12%, e.g., about 5%- about 10%)

[00100] or

[00101] Glycerol (about 8% to about 22%, e.g. about 10%-about 20%)

[00102] Algae Methanol (about 3% to about 12%, e.g., about 5%- about 10%)

[00103] or [00104; DMSO (about 3% to about 12%, e.g., about 5%- about 10%)

[00105 Plant Cells DMSO (about 3% to about 12%, e.g., about 5%- about 10%)

[00106: + (plus)

[00107: Glycerol (about 3% to about 12%, e.g., about 5%- about 10%) [00108 Animal Cells DMSO (about 3% to about 12%, e.g., about 5%- about 10%)

[00109: or

[00110 Glycerol (about 3% to about 12%, e.g., about 5%- about 10%) [00111 Hybridomas DMSO (about 3% to about 12%, e.g., about 5%- about 10%)

[00112 + (plus)

[00113 Serum (e.g., FBS) (about 15%-about 25%, e.g., about 20%)

[00114 Stem Cells DMSO (about 3% to about 12%, e.g., about 5%- about 10%)

[00115 + (plus)

[00116: Serum (FBS) (about 15%-about 95%. e.g.. about 20%-about 90%)

[00117: For Multicellular Embryos, a cryopreservant may be 1,2-propanediol, glycerol or ethylene glycol, and for Blood, a cryopreservant Glycerol. See also "Thermo Scientific Nalgene and Nunc Cryopreservation Guide", and references cited therein (see, e.g., www.atcc.0rg/~/media/PDFs/Cryopreservation_Tech11ical_M The agent is in the droplet containing typically a single cell, but sometimes one to three cells, e.g., two cells may be in droplet.

[00118] Referring to FIG 5F, thermally conductive fingers may be attached to chiller blocks 240 or 5004 to create cold zones 5010 and warming blocks 260 or 5006 to create warm zones 5008 in a straight sample channel design 175 or 175' or 1175 or 1175'. Wanning block 240 or 5004 and chiller block 260 or 5006 have interdigitated metal elements 5000, 5002, respectively, or interdigitated thermally conductive elements corresponding to each heating element. The heating elements may be driven by chilled or heated water or other suitable heat exchange medium. More particularly, wanning block 240 or 5004 and chiller block 260 or 5006 may be driven by Peltier electronic control systems, exothermic or endothermic reactions, or thermodynamic processes such as melting point solutions such as an ice-water bath. For example, warming block may maintain a constant temperature heat source such as with boiling water. Chiller block 260 or 5006 may achieve a constant temperature heat sink such as with a dry ice alcohol bath. A suitable feedback loop may be utilized to regulate temperature based on parameters including, for example, inlet temperature, flow rate, outlet temperature and heat sink temperatures. The disclosed embodiment may include any appropriate number of sensors, 235', 1235, 1235', 265, 265' 1265, 1265', 255, 255', 1255, 1255', 245 245', 1245, 1245' 295, 295', 1295, and/or 1295' appropriately disposed at suitable locations, for example, on straight sample channel 175 or 175' or 1175 or 1175' or within suitable temperature zones thereof in order to facilitate measurement of the temperature regulation channel and/or fluid therein is to control temperature in channel 175 or 175' or 1175 or 1175'. While a straight sample channel design is described herein, it is understood that the described channel design is exemplary and that the invention may utilize alternate geometric channel designs and should not be considered as limited to only a straight channel design configurations.

[00119] Channel 175 or 175' or 1175 or 1175' may include a recirculating or spiral configuration. In one embodiment shown in FIG 5G, recirculating channel loop 175 or 175' or 1175 or 1175' may include a valve 350 at the inlet 210, 1210, 210' or 1210' and a valve 351 at the outlet 290, 1290, 290' or 1290' and a pump 360 that enable the fluid within the loop 175 or 175' or 1175 or 1175' to be recirculated an arbitrary number of times. A portion, for example, the bottom portion of recirculating channel 175 or 175' or 1175 or 1175' may encounter the chiller block 260 or 5006. Likewise another portion, for example, the top portion of recirculating channel 175 or 175 ' or 1175 or 1175 ' may encounter a wanning block 240 or 5004. Chiller block 260 or 5006 and warming block 240 or 5004 may be driven by Peltier electronic control systems, exothermic or endothermic reactions, or thermodynamic processes such as melting point solutions such as an ice-water bath. For example, warming block 240 or 5004 may maintain a constant temperature heat source such as with boiling water. Chiller block 260 or 5006 may achieve a constant temperature heat sink such as with a dry ice alcohol bath. A suitable feedback loop may be utilized to regulate temperature based on parameters including, for example, inlet temperature, flow rate, outlet temperature and heat sink temperatures. The disclosed embodiment may include any appropriate number of temperature sensors, 235', 1235, 1235', 265, 265', 1265, 1265', 255, 255', 1255, 1255', 245 245', 1245, 1245', 295, 295', 1295, and/or 1295' appropriately located at suitable positions, for example, on the recirculating channel 175 or 175' or 1175 or 1175' or within suitable temperature zones thereof in order to measure and help control the temperature of the fluid in channel 175 or 175' or 1175 or 1175'. Again, the design configuration of the present embodiment is for illustrative purposes and should not be considered as limiting.

[00120] Individual cells, in both the cryolysis and cryopreservation embodiments, are captured in emulsion droplets using flow-focusing techniques for droplet formation. For example the creation of droplets are disclosed in U.S. Patent Application Serial No. 111024,228, filed December 28, 2004, entitled "Method and Apparatus for Fluid Dispersion," by Stone, et al., published as U.S. Patent Application Publication No. 2005/0172476 on August 11, 2005; US Patent Application Serial No. 111246,911, filed October 7, 2005, entitled "Formation and Control of Fluidic Species," by Link, et al., published as U.S. Patent Application Publication No. 2006/0163385 on July 27, 2006; US Patent Application Serial No. 111360,845, filed February 23, 2006, entitled "Electronic Control of Fluidic Species," by Link, et al., published as U.S. Patent Application Publication No. 2007/0003442 on January 4, 2007, International Patent Application No. PCT/US2008/007941, filed June 26, 2008, entitled "Methods and Apparatus for Manipulation of Fluidic Species," published as WO 2009/005680 on 5 January 8, 2009; Miller et al, "Manipulation of Microfluidic Droplets," United States Patent Application 20110000560, published January 6, 2011; Yurkovetsky et al, "Methods for Forming Mixed Droplets," United States Patent Application 20120219947, published August 30, 2012; Samuels et al, "Sandwich Assays in Droplets," United States Patent Application 20120122714, published May 17, 2012; Weitz et al, "Droplet Creation Techniques," WO 2011/056546, published May 12, 2011, each incorporated herein by reference. As is evident from the foregoing, there are many methods to make droplets, and the "Droplet Creation Techniques" of Weitz and of the documents cited and incorporated therein and herein are considered advantageous methods for making droplets.

[00121] With respect to prior methods for making droplets, the invention encompasses generating droplets in bulk using rapid stirring methods and running the resultant droplets through a device (cryopreservation or cryolysis or cryolysis and/or cryopreservation device) of the invention.

[00122] Thus, droplet 100 or 1000 of FIGS. 1 or 2 in either the cryolysis or cryopreservation aspects of the invention may originate from any of the foregoing including an apparatus of any of the foregoing connected or in fluid communication with channel 175 or 1175 or 175' or 1175' of any of FIGS. 1-4. [00123] The microfluidics of the systems of FIGS. 1-4 may be continuous flow (e.g., activation of liquid flow is implemented by either external pressure or external mechanical pump(s) or integrated mechanical micropump(s) or a combination of capillary forces and electrokinetic mechanisms), for instance, closed channel systems or open structures involving digital microfluidics, e.g., using electro-wetting, see, e.g., US Patent No. 7,816,121 entitled, "Droplet actuation system and method"; US Patent No. 7,815,871, entitled, "Droplet microactuator system"; and US Patent No. 7,763,471, entitled, "Method of electrowetting droplet operations for protein crystallization"; each incorporated herein by reference; or surface acoustic waves, optoelectrowetting, or mechanical actuation. Standard high pressure pumps (e.g., Mitos Pressure Pumps, Ultrafast High-pressure AC Electro-osmotic Pumps, Elveflow AF1 pressure pumps, etc.) may be used to push the cells 110 or 1110 through the serpentine or sinusoidal channel 175 or 1175 that, for the cryolysis embodiment, passes back and forth between the warming block 240 and chiller block 260 or, for the cryopreservation embodiment, through a single chiller block 1260.

[00124] With regard to all of FIGS. 1-4, 5F and 5G, channel 175 or 1175 or 175' or 1175' may be a chip, a plurality of chips connected, a capillary tube, a microchannel, and the like, composed of silicone, glass, plastic and the like. The channel width and height are each somewhat larger than the maximum diameter of a droplet containing a cell, which is to pass through the channel, typically on the range of 25 to 100 microns wide and high. The channel may however be designed to be much larger than the droplet, at least 2 times or more to regulate temperature gradients or enable slowing of the droplet flow rate upon channel expansion to control exposure times of the droplet to varying temperatures.

[00125] The entire region through which the droplet containing the cell travels may fit on a microfluidic chip that would be on average about the size of most credit cards, e.g., about 74 to about 94 mm x about 48 to about 60 mm, e.g., 80 to about 90 mm x about 50 to about 58 mm, as the average size of most credit cards is about 85-about 86 mm x about 53-about 55 mm or more precisely about 85.60 x about 53.98 mm. However, the size of the device is not limited to these dimensions and the principle works at smaller scales as well as for larger systems. Furthermore, with reference to FIG. 1, channel 175 runs through a chiller block 260 and a wanning block 240 and in FIG. 3, channel 175' is running between chiller block 260' and wanning block 240'. Similarly, in FIG. 2, channel 1175 runs through chiller block 1260 and in FIG. 4, channel 1175' is running through chiller block 1260*.

[00126] Channel 175 and 1175 of FIGS. 1 and 2 are illustrated as being serpentine, and channels 175' and 1175' are illustrated as being generally straight, but with zones along the course thereof that allow for temperature changes in the serpentine channels. A serpentine layout is useful to change the shape of the domain occupied by the channel, from linear (linear channel) to square or rectangular (serpentine or zigzag channel). Accordingly, considering how one describes a sine wave, there are cycles, where each cycle has a peak and a trough. In either the configuration of FIGS. 1 and 2 or 3 and 4, the droplet may be put through anywhere from two (2) to more than one hundred (100) cycles, and the channel from inlet 210, 1210, 210' or 1210' to outlet 290, 1290, 290' or 1290' may vary in dimensions from less than 40 microns to over 200 microns in height, from less than 100 microns to more than 1 mm in width, and from less than 1 mm to over 10 cm in length.

[00127] In another embodiment, the emulsified sample in the carrier oil is contained in straight channel with alternating hot and cold regions (e.g., see FIGS. 3 and 5F). This design avoids channel corners and thus has the benefit of lower pressure drop along the channel length.

[00128] Embodiments of the invention may provide channel 175, 175' 1175, or 1175' fabricated from a variety of materials, including, but not limited to, polydimethylsiloxane (PDMS) using photolithography. As an alternative, other materials may be employed such as those including polycarbonate. The apparatus of the present invention may typically be made in two pieces with the channels etched using photolithography and then utilizing a cover slip joined to seal the device. The heating and cooling sections of the disclosed embodiments may be liquid filled channels connected in continuous flow to a heating or cooling block or other heat sink. Alternatively, metal may be deposited on the surface of the device or on the cover plate acting as a heat conductor to the heating or cooling blocks, which, in turn, may act as Peltier systems or simply chambers filled with dry ice, hot water, or other appropriate constant temperature system.

[00129] Accordingly there are temperature gradients in various aspects of embodiments illustrated in FIG. 1. for instance:

[00130] 1. considering input to output, between inlet 210 (sensor 215) and outlet 290 (sensor 295),

[00131] 2. considering segment of channel 175 between sensors 215 and 225, [00132] 3. considering segment of channel 175 between sensors 225 and 235,

[00133] 4. considering segment of channel 175 between sensors 235 and 265,

[00134] 5. considering segment of channel 175 between sensors 265 and 235,

[00135] 6. considering segment of channel 175 between sensors 235 and 225,

[00136] 7. considering segment of channel 175 between sensors 225 and 255,

[00137] 8. considering segment of channel 175 between sensors 255 and 245,

[00138] 9. considering segment of channel 175 between sensors 245 and 255,

[00139] 10. considering segment of channel 175 between sensors 255 and 225, and

[00140] 11. considering additional cycles of items analogous to items 2-10, but downstream or closer to outlet 290 and sensor 295 than the first cycle, e.g., in a second temperature regulation region spanning 203 and 204, downstream from that spanning regions 201 and 202, as herein discussed.

[00141] Accordingly there may be temperature gradients in various aspects of embodiments illustrated in FIG.2. for instance:

[00142] 1. considering input to output, between inlet 1210 (sensor 1215) and outlet 1290 (sensor 1295),

[00143] 2. considering segment of channel 1175 between sensors 1215 and 1225,

[00144] 3. considering segment of channel 1175 between sensors 1225 and 1235,

[00145] 4. considering segment of channel 1175 between sensors 1235 and 1265,

[00146] 5. considering segment of channel 1175 between sensors 1265 and 1235,

[00147] 6. considering segment of channel 1175 between sensors 1235 and 1225,

[00148] 7. considering segment of channel 1175 between sensors 1225 and 1255,

[00149] 8. considering segment of channel 1175 between sensors 1255 and 1245,

[00150] 9. considering segment of channel 1175 between sensors 1245 and 1255,

[00151] 10. considering segment of channel 1175 between sensors 1255 and 1225, and

[00152] 11. considering additional cycles of items analogous to items 2-10, but downstream or closer to outlet 1290 and sensor 1295 than the first cycle, e.g., in a second temperature regulation region spanning 1203 and 1204, downstream from that spanning regions 1201 and 1202, as herein discussed.

[00153] Such temperature gradients may be a uniform or non-uniform cooling rate of 1°C per minute from ambient temperature to about minus 80 degrees C. [00154] The chiller and wanning blocks in the cryolysis and cryopreservation inventions may be divided into thermal regions, each with a different or independent temperature regulation system. For example, each of FIGS. 1 to 4 show imaginary line 205, 1205, 205' and 1205', perpendicular to generally straight embodiments illustrated in FIGS. 3 and 4 and the horizontal axis of FIGS. 1 and 2. That imaginary line is provided to illustrate that each block of FIGS. 1-4 may be divided into regions. With reference to FIG. 1, those regions are 201, 202, 203 and 204; with reference to FIG. 2, those regions are 1201, 1202, 1203, and 1204; with reference to FIG. 3 those regions are 201', 202', 203' and 204'; and with reference to FIG. 4, those regions are 1201', 1202', 1203', and 1204'. Each of those regions may have a different or independent temperature regulation system.

[00155] For example, considering FIG. 1, there are temperature gradients along the channel 175 from inlet 210 through to outlet 290, and temperature gradients) from the warming block 240 to the chiller block 260, including across the length from inlet 210 to outlet 290. Region 201 may thus be different from region 202. Region 203 may be the same or different than 201 and region 204 may be the same or different than region 202. Further region 203 may be the same as or similar to region 201 but independently controlled, and region 204 may be the same or similar to region 202 but independently controlled. In this way, the droplet 100 may be subjected to chilling and warming across a temperature gradient from inlet 210 to outlet 290. For instance, region 203 may warm either to about the same as or to a greater or lesser degree than that of region 201 and region 204 may cool or lower temperature to about the same as or to either a greater or lesser extent than region 202, to optimize freezing and thawing for lysis. While illustrated as four regions, one may readily envision one or more different or independent temperature regulation systems, e.g., one warming block region and one chiller block region, two warming block regions and two chiller block regions (as illustrated), three warming block regions, etc.

[00156] Similarly, considering FIG. 2, regions 1201 and 1202 may be one or as illustrated two or more independent or different temperature regulation systems and regions 1203 and 1204 may be one or as illustrated two or more independent or temperature regulations systems. Thus, chiller block 1260 may be one or more different or independent temperature regulation systems. For instance, regions 1201 and 1202 may be independent, but of a same or similar type of temperature regulation system to gradually decrease the temperature of the droplet 1100. Regions 1203 and 1204 may be independent but of a same or similar type of temperature regulation system (as each other and/or regions 1201 and 1202), and cooler than that of regions 1201 and 1202, to thereby result in cryopreserved cell 1300 in droplet 1310.

[001S7] The provision of regions, designated 201 ', 202', 203' and 204', for example about an imaginary axis defined by imaginary line 205' and a line following the general direction of channel 175' is provided for in FIG. 3. The provision of regions, designated 1201', 1202', 1203' and 1204', for example about an imaginary axis defined by imaginary line 1205' and a line following the general direction of channel 1175' is provided for in FIG. 4. The regions of the FIGS. 3 and 4 are for temperature regulation system(s) and are analogous to those in FIGS. 1 and 2. Channel 175' thus adjoins region(s) of temperature regulation system(s) and channel 1175' thus adjoins region(s) of temperature regulation system(s). Referring to FIG. 1, droplet 100 containing intact cell 110 in buffered solution 120 after formation of the droplet as discussed above are passed inlet 210 of channel 175.

[001S8] Fast and precise temperature regulation and controlled variation may be obtained in these blocks of the invention. See, e.g., Casquillas et al, "Fast microfluidic temperature control for high resolution live cell imaging," Lab Chip, 2011,11, 484-489, incorporated herein by reference; and products of ELVEBIO™ for temperature control, e.g., those that allow for temperature from minus 20°C to positive 100°C that allows 5°C temperature changes, and the ELVEBIO™ Cell-Cius product(s) / technology, manuals and product information which are incorporated herein by reference. Temperature regulation of the channel 175 or 1175 or 175' or 1175' in the instant invention may come from various temperature regulation systems. For instance, water or other suitable fluid circulated over a thin glass slide in contact with the exterior of the channel to either increase temperature (add energy) or decrease temperature (remove energy), or one or more sister channels alongside channel 175 or 1175 or 175' or 1175' (e.g. parallel and advantageously having an exterior surface contacting the exterior surface of channel 175 or 1175 or 175' or 1175'), and/or coiling channel 175 or 1175 or 175' or 1175' (wrapping around and contacting channel 175 or 1175 or 175' or 1175'). Through the one or more sister channels, water or other suitable fluid is circulated to either increase temperature (add energy) or decrease temperature (remove energy) to allow either cycling for fast temperature change between minus 20°C to an appropriate warming temperature in the range 20°C to 100°C for suitable freeze-thaw and hence cryolysis while the cell remains in the droplet, or gradual freezing of the cell in the droplet and hence cryopreservation. The switching of the temperature of the droplet in the channel may be within or less than 5 seconds, for instance, by either manual programming therefor or by a programmed sequence, controlled by microprocessors or processors that introduce and control the flow of water or other suitable fluid in the temperature regulation system(s). Likewise, as also discussed herein, the invention comprehends the use of chiller and wanning blocks (e.g., see FIGS. 5F and SG) such as Peltier blocks or en do thermic or exothermic reactions to create cold or hot zones.

[001S9] An array of temperature sensors may be distributed at advantageous positions across the device to provide feedback to the temperature regulation system(s). Referring to FIGS. 1A and IB, at or just after inlet 210 is sensor 21S, which may be within the channel or on the exterior surface of the channel. There are sensors 225 at or near the region adjoining the warming and chiller blocks 240, 260, advantageously at each transition from the chiller block 260 to the wa ming block 240. There are sensors 235 approximately a quarter and three quarters the distance along each channel segment within the chiller block 260. There are also sensors 265 approximately halfway along each channel segment within the chiller block 260. Similarly there are sensors 255 approximately one quarter and three quarters the distance along each channel segment within the wanning block 240. And there are sensors 245 at or near the halfway point along each channel segment within the warming block 240. There is also a sensor 295 at or near the outlet 290. Referring to FIGS. 5Ai and 5Bi, similar sensor arrangements may be used in conjunction with temperature regulation channel 575 and 675.

[00160] Similarly, the cryopreservation embodiments have advantageously positioned temperature sensors. Sensor 1215 is positioned at or just after inlet 1210 and sensor 1295 is positioned at or just after outlet 1290 of the user controlled module 1200 that has chiller block 1260. Sensors 1225, 1235, 1245, 1255, 1265 and 1295 are placed at advantageous positions along or near channel 1175 within the cooling module 1200 that has chiller block 1260. An example sensor positioning is illustrated in FIG. 5Bi when both temperature regulation channels are cooling channels 575, i.e. applicable to cryopreservation applications.

[00161] Each of these sensors 215, 225, 235, 245, 255, 265, 295, 1215, 1225, 1235, 1245, 1255, 1265 and 1295 are in electrical communication with a microprocessor (not illustrated) and may include means for temperature regulation as herein discussed. Thus, if the temperature at any of these sensors is less than a particular desired temperature, the temperature of the chiller block or warming block may be adjusted upward, either globally or locally. Likewise, if the temperature at the sensor is greater than a particular desired inlet temperature, the temperature of the chiller block or wanning block or may be adjusted downward, either globally or locally.

[00162] While disclosed embodiments describe and illustrate a selected number of sensors, it is readily appreciated that less or more sensors may be utilized by the present invention, for example, to facilitate measurement and/or regulation of temperature within the disclosed system. This may include measuring temperature gradients at or within temperature zones and/or measuring discrete temperature points disposed within the disclosed system. Additional embodiments may include utilizing the disclosed sensors to measure flow rates through prescribed components of the system (such as the cell channel, wanning block, and chiller block). Further embodiments of the invention may provide altering the composition of the different solutions to change the freezing or thawing points of the aforementioned solutions as measured by the disclosed sensors.

[00163] Temperature regulation may be provided by fluidic channels transporting warm or cold heat transfer fluids (liquids or gases) to adjust the temperature warming blocks 240 or chiller blocks 260 or 1260. If the temperature at a sensor is less than a particular desired temperature, warm water or other fluid may flow through a particular temperature regulation channel to increase temperature near the sensor. Likewise, if the temperature at the sensor is greater than a particular desired inlet temperature, chilled fluid may flow via a temperature regulation channel to decrease the temperature near the sensor. Each sensor is therefore a temperature sensing means and/or a temperature adjusting means and/or means for sensing and/or adjusting temperature.

[00164] While FIG. 1 illustrates sensors 225, 235, 245, 255, and 265 sequentially placed along a segment of channel 175, these sensors or sets of these sensors may be positioned advantageously at other segments of channel 175. There may be segments of channel 175 without any temperature sensing and/or regulating means associated therewith. While FIG. 2 illustrates sensors 1225, 1235, 1245, 1255, and 1265 sequentially placed along a segment of channel 1175, these sensors or sets of these sensors may be positioned advantageously at other segments of channel 1175. There may be segments of channel 1175 without any temperature sensing and/or regulating means associated therewith. While FIGS. 5A and 5B are illustrated with sensors 215, 225, 235, 245, 255, 265, 295 or 1215, 1225, 1235, 1245, 1255, 1265, 1295 sequentially placed along a segment of channel 175 or 1175, these sensors or sets of these sensors may be placed along other segments of channel 175 or 1175. There may also be segments of channel 175 or 1175 without any temperature sensing and/or regulating means associated therewith.

[00165] As may be seen from FIGS. 5Ai and 5Aii, the temperature regulation channels may run on the outside or the inside of the curved shape of channel 175 or 1175. This is also illustrated in FIG. 5C. Thus, in any of the systems of FIGS. 1-4, the temperature regulation channels may be adjacent to or adjoining or next to or in close proximity to the channel 175, 175', 1175, or 1175'.

[00166] Potential uses of the invention include cryolysis and cryopreservation. Each cryolysed cell in a droplet and cryopreserved cell in a droplet are useful tools in cell biological or microbiological research. The lysed cell in a droplet 300 or frozen cell in a droplet 1300 from output 290, 1290, 290' or 1290' of FIGS. 1-4 are thus useful, and that output may be directly linked to other devices utilizing such frozen cells or lysed cells and/or for storage of such frozen cells or lysed cells. See also Choi, J.W., Rosset, S., Niklaus, M., Adleman, J.R., Shea, H., Psaltis, D. "3-dimensional electrode patterning within a microfluidic channel using a metal ion implantation," Lab on a Chip 10, 738-788, 2010, incorporated herein by reference with respect to the utility of microfluidic devices for confining cells.

[00167] One application of this invention is for the lysis of mammalian and bacterial cells in a microfluidic environment. It may also be used for the lysis of other cell types (e.g., fungal and plant). The contents of the lysed cells include RNA, DNA and proteins which may then be used for a number of downstream applications including cDNA synthesis, PCR, immunoassays, etc.

[00168] Many aspects and advantages of the cryolysis aspects of the invention as herein discussed, e.g., no need to rely on electrodes or sharp or pointed edges, various sensors throughout the device, means for sensing and/or regulating and/or controlling temperature, e.g., involving adding or removing cooling or warming fluid, positioning of cooling / warming channel, and the ability of the device to be for both cryopreservation and cryolysis, are not taught or suggested by Tai, US Patent No. 6,543 5 and/or Yang, US Patent No. 7,521,246, both of which are incorporated herein by reference (as aspects therein that may be common may be employed in the practice of the invention). [00169] Another application for this invention is for the cryopreservation of cells. Typically, cells are frozen in bulk cultures and the entire volume must be thawed in order to reactivate the cells for downstream assays. With this device, a discrete volume of cells may be harvested from a frozen culture without the need to thaw the entire volume. The invention offers a unique advantage for the cryopreservation and storage of precious cell samples. Such samples include, stem cells, primary explants, non-transformed cell lines and any other cell types were it is desirable to keep passage number at an absolute minimum The invention also has potential applications in the storage of samples to be used for in vitro fertilization. The cryopreservation aspect takes advantage of the fact that the emulsion oil freezes at significantly lower temperatures than the cell. This means that specific volumes of culture (or even controlled numbers of cells) may be harvested without the need to thaw the entire culture.

[00170] Embodiments of the present invention provide distinct advantages of convention systems by providing a continuous flow or flow-through device. For example, since convention systems do not provide a carrier fluid, emulsion samples would fail due to clogging within the cold region. In contrast, the present invention provides a carrier fluid to facilitate continuous flow. This allows not only allows the disclosed invention to provide high and continuous flow rates through the hot section, but as importantly, the chambers maintain high and continuous flow throughout the cold sections as well.

[00171] The present invention provides the use of emulsions which means individual (single) cells may be lysed, and post lysis, the contents of the emulsion droplet may be kept together and isolated from the contents of other droplets - a novel advantage which is not possible in the prior art. This is an essential part of single cell or single group analysis, but as important, the capability of the disclosed system allows for the manipulation of the single cell downstream through various droplet methods. Careful control is required to reform droplets in emulsions after freezing, since the surfactant may precipitate out of the oil, may become dissociated from the frozen droplet, and needs to return to the droplet meniscus (surface) upon thawing. This requires careful temperature and fluidic control, as disclosed by the present invention, and in some disclosed embodiments is accomplished using a thermoelectric device to control hot and cold zones and flow rate along the channel 175, 1175, 175', or 1175'. This provides significant impact in industrial systems. [00172] The use of emulsions also allows for the introduction of cell lysis agents such as hypotonic solution or detergents according to the cell type which is not readily available in the prior art. Advantages of the disclosed invention include the high throughput nature (due to the use of a non-freezing carrier medium, e.g., the oil), the encapsulation of lysis contents, and the ability to modify lysis conditions in numerous ways by adding reagents to individual droplets (i.e. detergents). In addition, useful for this and for cryo-preservation the invention provides that the cells may also be encapsulated entirely within the oil without the need for an "emulsion," as the cells themselves would serve as the droplet.

[00173] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations may be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[00174] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

Examples

Example 1: Cryofysis

[00175] Freshly cultured cells were washed twice in cold IX PBS buffer in a conical bottom SO mL centrifuge tube.

[00176] The washed cells were suspended in 0.5X PBS buffer at room temperature (approximately 20 degrees C), with a cell density of approximately lxl0⁶/mL. (for RNA- Sequence / amplification applications, cells were suspended in Reverse Transcription (PCR) reaction mix).

[00177] Using a 40 urn Nylon cell strainer clumps in the cell suspension were filtered out at room temperature.

[00178] The filtered cell suspension was transferred into an injection vial that was driven by pure fluorocarbon oil at injection rate 100 μΙΛ. with the carrier oil flowing at 200 μΙΑι (all at room temperature).

[00179] From the injection vial, cells in oil were injected into a flow-focusing droplet generator chip to encapsulate cells in droplets (generally single cells, but sometimes up to three, e.g., two cells per droplet) with a droplet size approximately 50 picoliters at room temperature. [00180] A first portion of the cell-encapsulated droplets (with Poisson distribution) was fed into Polyethylene (PE) tubing with diameter 400 urn that flowed into an region of rapid cooling and a region of wanning. Specifically, a central section of the PE tubing (3 cm in length) was immersed into a dry ice-ethanol bath having a temperature of about minus 40 degrees C, and from that the cells passed to room temperature to thereby rapidly thaw. Typical flow rates were about 60mm/second requiring heating and cooling regions on the order of about 2mm. The tubing was in a generally straight path (e.g., see FIGS. 3, 4 and/or 5F).

[00181] As a second portion of the cell-encapsulated droplets (with Poisson distribution) was NOT put through the foregoing freeze-thaw technique that the first portion of cell-encapsulated droplets was subjected to. But rather, these cells after forming into droplets at room temperature were kept at room temperature.

[00182] A sample of droplets from each of the first portion and second portion were examined under an optical microscope to check the effectiveness of cryo-lysis. FIGS. 6A and 6B show the droplets subjected to freeze-thaw and those not subjected freeze-thaw, respectively, under the optical microscope. As is clear from comparing FIGS. 6A and 6B, in FIG. 6 A, lysed cells were readily seen in the droplets as cell contents were clearly seen outside of the cell membrane and within the droplet, whereas cells not subject to freeze-thaw were intact in the droplets. Arrows are added to FIG. 6A to highlight where in each one may easily see where cell contents have penetrated the cell wall and hence cells are lysed and to FIG. 6B where cells are intact.

[00183] The first portion of rapidly frozen and thawed droplets from the PE tubing output were collected into a 0.2 uL PCR (polymerase chain reaction) vial. An equal amount of the second portion droplets were collected in a separate 0.2 uL PCR vial.

[00184] Then RT PCR (16 cycles) was carried out using the SMARTer RT system of Clontech on the droplets of the first portion that was subjected to freeze-thaw according to the invention, and the second portion droplets that were not subjected to freeze-thaw according to the invention. A control of simply the contents of the droplets without either cells or cell contents, and the reagents of the SMARTer RT system was also subject to 16 cycles of what would be RT PCR if RNA was present in the control. Because the droplets subject to freeze-thaw have clearly been lysed as shown in FIGS. 6A and 6B, it appears that subjecting equal amounts of the first (freeze-thaw) and second (no freeze-thaw) portions of droplets to PCR results in a higher input of freeze-thaw plus RNasel cells (as indicated in FIG. 8E, discussed below). [00185] FIG. 7 shows schematically the process undertaken in this Example.

[00186] FIGS. 8A-E provide the results from this Example. FIG. 8A titled FT_+R aseI graphically shows RNA amplification from carrying out RT PCR ("+RNaseI") on the cells that were subject to freeze-thaw ("FT") according to the invention. FIG. 8B titled NF_+RNaseI shows little RNA amplification from carrying out RT PCR ("+RNaseI") on cells that were not subject to freeze-thaw according to the invention ("NF"). FIG. 8C titled SMRT_+Control also shows little RNA amplification from carrying out RT PCR conditions in the absence of cells or lysed cells. The products of the RT PCR of the three samples FT RNasel (cells in droplets subject to freeze-thaw according to the invention), NF_+RNaseI (cells in droplets not subject to freeze-thaw), and the control (SMRT_+Control) were subject to electrophoresis and FIG. 8D is the image of the gel from that electrophoresis showing the respective amplified RNA in comparison to a base pair (bp) ladder. FIG. 8E provides a table of the results in FIGS. 8A-D. As seen on the gel of FIG. 8D and as discussed in the Table of FIG. 8E, there was essentially no RNA amplification from the cells that were not subject to freeze-thaw (500-3000 bp length, concentration ng/μΐ, 3.73 with molarity nmol/L of 6.0), and the "background noise" amplification from the control (400-3000 bp length, concentration ng/uL 7.27 with molarity nmol/L of 7.9) was approximately twice that of RNA amplification from the cells not subject to freeze thaw. However, the RNA amplification from the cells subjected to freeze-thaw according to the invention had an order of magnitude greater RNA amplification (150-3000 bp length, concentration ng/uL 35.76 with molarity nmol/L of 65.8) than the cells not subjected to freeze thaw (compare concentration ng/uL 35.76 with molarity nmol/L of 65.8 from RT PCR of droplets subjected to freeze thaw according to the invention with concentration ng/^L 3.73 with molarity nmol/L of 6.0 from RT PCR of droplets not subject to freeze thaw according to the invention).

[00187] This Example clearly shows that the present invention may provide droplets, each of which contains a cell (or two or most three cells) that have been lysed and that contain reagents for running a reaction, whereby the reaction is run successfully. With the technology of this invention, cells in a medium for a downstream application may be readily lysed in a microfluidic environment, and the downstream application, e.g., PCR, may be readily performed, successfully and accurately.

Example 2: For Freeze/Thaw Lysis or for Cryopreservation [00188] A typical implementation of a cooling section, either for freeze / thaw lysis or for cryo preservation would have the following parameters. Typical sample PDMS channel size cross section may be SO micro-meters by 25 micro-meters at a flow rate of 300 micro liters per hour. For a sample inlet temperature of 20 degrees C and a desired -20 degree C droplet temperature during exposure to the cold region the typical region length for the droplet to travel along the channel may be 174 micro meters and a cooling block temperature of -26.2 degrees C for the center line of the channel containing oil plus droplets to reach -20 degrees C freezing the cell and droplet contents subject to the buffer or additives selected for the cell type or experiment Many variations of these parameters are possible to control the cooling rates, freezing temperature, and flow rates of droplets through the region, and oil types with different heat diffusivities, and device materials. Hearing for thawing or heat lysis may be accomplished in the same manner through a heating region raising the temperature of the inlet sample containing emulsified cells in droplets along with the carrier oil. For the case of an identical flow rate of 300 micro liters per hour through the same 50 micro-meters by 25 micro-meter channel, an inlet temperature of 20 degrees C and a desired outlet temperature of 60 degrees C may require 174 micro meters along the channel and a wall temperature from the heating block of 66.2 degrees C. Further travel along the heating or cooling blocks may result in an asymptotic approach to the region temperature. These heating and cooling parameters are representative of the regions in all of the regions described in the devices in this application.

Example 3: DeviceModule for Rapid Cryolysis or Cryopreservation

[00189] Applicants developed an efficient, rapid, ultra-low volume, and R A-safe cryolysis device able to handle the full range of organisms (with varying degrees of cell wall strength) to enable novel research in microbial expression, single cell analysis, and accurate unbiased analyses of microbial populations in environmental and clinical samples. The system uses rapid repeated freeze/thaw cycles in low thermal mass micro-chambers able to handle a wide range of lysis conditions, from easy to difficult The method also enables the use of additives to stabilize RNA, add detergents, remove proteins, or extract viral RNA, and Applicants aim to complete lysis of a 10 μΐ sample in < 20 min with SO freeze/thaw cycles, each < 20 s long.

[00190] The rapid cryolysis system is an innovative combination of effective lysis techniques, including cryolysis, with advanced micro systems engineering. The cryolysis system provides a rapid and universal method of lysing cells without harsh chemicals that could reduce the yield of intra-cellular analytes such as RNA or proteins or interfere with downstream processes. This cr olysis system may be used as a standalone tool in a number of other processes in proteomics and genomics, where either lysis speed, analyte yield, or completeness of lysis across a population are essential.

[00191] Additional pre- and post-lysis functionalities may be included, including sample division, sample concentration, reagent delivery for cell pre-treatment, and detection. As shown in FIG. 9, such functionalities may be carried out in separate modules, but with common specifications and controls or, where appropriate, the modules may be integrated in series or into a single device. Where possible, the same chamber is used for multiple steps, in order to reduce sample loss and simplify fabrication and operation. Reducing sample loss is critical to increasing sensitivity for certain clinical samples such as those with low pathogen burden. Minimizing transfers between processing steps or eliminating steps entirely may be advantageous to maximize the yield of RNA into a detection system. Specific modules may be integrated and tailored for particular applications.

[00192] Applicants have designed a rapid cryolysis system consisting of a millimetric scale fluidic channel with a metal base to expedite freezing and thawing (FIG. 9A). An important feature of the lysis device or module is that the PDMS channel layer is bonded to a metal base (such as copper) for rapid heat transfer between the sample and an external heat source/sink. Applicants use published methods including that by Cai et al. (Cai D, Neyer A. Cost-effective and reliable sealing method for PDMS (PolyDiM ethylSiloxane)-based microfluidic devices with various substrates. Microfluid Nanofluid.2010;9(4):855-64) to bond PDMS to copper substrates.

[00193] The small sample chamber height and base thickness keep the overall thermal mass of the system small so that the external heat transfer module may accomplish up to 100 to 1000 freeze/thaw cycles at under 20 s per cycle with the chamber filled with water.

[00194] An advantageous step before and/or after is sample concentration. For cases where cells of interest are in low abundance, to improve detection accuracy, an advantageous concentration strategy may involve concentration pre- and post-lysis: pre-concentration of intact cells prior to lysis against a nanofilter, and following lysis, concentration of the lysate against an ultrafiltration membrane (FIG. 9B). A post-lysis concentration strategy may be used separately from and/or in combination with a pre-lysis concentration strategy. [00195] Prior to lysis, the intact cell solution is pre-concentrated by pushing it against a nanofilter with 0.1 urn pore size (e.g. illipore filter VCWP) (FIG. 9B). Since the cells are intact, they retain the molecular sized genomic material. A biocompatible oil-based pusher fluid upstream of the sample solution applies the necessary pressure to drain excess fluid from the sample through the filter and to waste. The oil does not mix with water and a single meniscus separates the two, provided the flow is sufficiently slow and smooth.

[00196] Post lysis, an advantageous concentration strategy may be to use pusher fluid to push the lysate against an ultrafiltration (UF) membrane (FIG. 9B). Excess fluid from the lysate is therefore removed and output to a waste channel (FIG. 9B). Suitable semi-permeable membranes include Millipore UF membranes or Thermo Scientific SnakeSkin* Dialysis Tubing with a range of molecular weight cut offs (MWCOs) as low as 1 kDa. An advantageous design calculation includes the MWCO of the UF membrane: to retain R A transcripts longer than SO base pairs (bp), which have MWs of -50 bp * (330 g/mole/bp) = 33 kDa, a UF membrane with a MWCO lower than 33 kDa must be employed.

[00197] In the concentration strategies outlined herein, the nanofilters and/or UF membranes may be advantageously coated to prevent sample loss, as described herein.

[00198] Drugs, RNA protectants), and pusher fluid may be fed to lysis chamber 380 (FIG. 9C). These additional fluids or additional sample may be added without increasing the overall volume of the sample in the lysis chamber 380, and without losing intact cells, by eluting through either the nanofilter (pre-lysis) or the UF membrane (post-lysis) and sending excess liquid to the appropriate waste output. Computer controlled syringe pumps drive these fluids as needed into and out of the ports to the lysis chamber 380. A bio-compatible oil-based pusher fluid may be advantageously used to fill the void behind the sample and drain excess fluid through the membrane and into the waste channel.

[00199] FIG. 9D illustrates a means for sample separation and distribution into parallel lysis chambers to enable sub-sample isolation and high-throughput treatment by different reagent cocktails. Additional or fewer channel bifurcations could be added or removed as needed to divide the sample into any number of sub-samples, and output said sub-samples into individual lysis chambers and following lysis to individual outlets.

[00200] The sample may be treated pre and post lysis both within the device and before or after the lysis module to pre-condition, perturb and/or dilute or concentrate the sample. Perturbation includes exposing microbial pathogens to antibiotics for a desired exposure time (seconds, minutes, and hours). An example perturbation embodiment is illustrated in FIG. 9E. Following controlled perturbation, Applicants may preserve the transcriptional state of the cells by mixing R AProtect (Qiagen) with the sub-samples in the equilibration/perturbation chambers. Note that the ability to add reagents for cell perturbation may also be used to process viral particles to release their UNA such as TRIzol and SDS buffers with EDTA. Drug solutions may be advantageously pumped into the appropriate inlets and transported to the appropriate chambers (FIGS. 9C, 9D, 9E). Sample may advantageously be concentrated before or after perturbation to reduce sample size and/or to improve downstream processes such as detection (FIG. 9E).

[00201] Biological material loss including proteins, nucleic acids, metabolites and cells is a significant problem on most surfaces and degrades the sensitivity of detection of these components in a diagnostic application. To mitigate such loss, Applicants work with collaborators who have outlined a surface modification protocol for the prevention of biofouling. Applicants' collaborators recently reported a method for coating surfaces to minimize loss of biological materials. Their work describes the first synthesis of zwitterionic thin films via initiated chemical vapor deposition (iCVD) (Y ang R, Xu J, Ozaydin-Ince G, Wong SY, Gleason KK. Surface-tethered zwitterionic ultrathin antifouling coatings on reverse osmosis membranes by initiated chemical vapor deposition. Chem Mater. 2011;23(5):1263-72). Proceeding from vapor phase monomers, polymer synthesis, and film deposition occur simultaneously at modest vacuum and low surface temperatures. The iCVD method achieves 100% retention of the pendent organic functional groups present from the monomelic reactants. The iCVD provides for the facile integration of polymer coating on virtually any substrates, including PDMS and other plastic materials commonly used in microfiuidics.

[00202] The external thermal module may house two stages, hot and cold. A direct contact switching mechanism may alternately connect the cold or hot stage via a highly thermally conductive pathway to the lysis module. An example switching mechanism includes a solenoid- actuated metal contact between the hot and cold stages and a highly conductive plate on the thermal module, which is in turn in direct contact with the lysis module. The cold stage could consist of a metal conducting pathway partially immersed in a cold bath of dry ice and either ethanol or a mixture of ethanol and glycerol (Jensen CM, Lee DW. Dry-Ice Bath Based on Ethylene Glycol Mixtures. J Chem Educ.2000;77(5):629). The hot stage could consist of a metal conducting pathway partially immersed in a hot bath or room temperature air or water. The hot and cold stages could also be commercial chillers, Peltier blocks, and hot plates. An example of a suitable low temperature chiller is the Cole-Parmer Polystat® model EW- 14575-41 with a cold- finger with sustained temperatures between -100 °C and -60 °C. As for Peltier systems, they are widely used in research (Stan CA, Schneider GF, Shevkoplyas SS, Hashimoto M, Ibanescu M, Wiley BJ, et al. A microfluidic apparatus for the study of ice nucleation in supercooled water drops. Lab Chip. 2009;9(16):2293-305) and may be purchased (e.g. LTS120, Linkam Scientific Instruments Ltd.).

Model-based engineering: heat transfer model and freeze/thaw times.

[00203] FIG. 10 illustrates finite element heat transfer simulations in Comsol of the cooling and freezing of 30 uL of water in different geometries (thin rods, thick blocks, and thin discs). The water is initially at a temperature of 20 degrees C throughout. The phase change is calculated using the popular Apparent Heat Capacity method. Though all geometries have the same volume, the thin rods and discs cool and freeze that fastest. Thus, the sample geometry is an important feature to consider in the design of a device that rapidly freezes and thaws a sample.

[00204] The proposed lysis module design consists of a 0.5 mm high * 2 mm wide * 1 cm long PDMS channel bonded to a copper plate (FIG. 9). To estimate the cooling and freezing times of water in the chamber of height L, we neglect the sidewalls and approximate the water in the chamber as an infinitely wide slab of thickness L. The time to change the temperature of the slab of water from T_x to T₂ is t = (4L²/πα)log(π(T_l-T_m)/4(T₂-T_m)) when the metal base is at a temperature T_m, where a = k_w/ρ_wC_pw for liquid water and a = ki/ρ_iC_pi for ice (Eq. 3.44 in Mills AF. Heat Transfer. Boston, MA: Irwin; 1992). The time to freeze or thaw the sample is t_f = L²ρ_ih_fs/2k_i(T_f-T_m) (p. 184-185 in Mills AF. Heat Transfer. Boston, MA: Irwin; 1992), where T_f is the freezing temperature of the material. Water at 20 °C has a density of ρ_w = 1000 kg/m³ and freezes at T_f = 0 °C. The thermal conductivity and density of ice are ki = 2.1 W/mK and ρ_i = 881 kg/m³. The latent heat of fusion of water is h_fs = 334 J/g. If the metal base plate is held at T_m = - 20 °C, the sample takes 0.66 s to cool to freezing and 0.84 s to freeze. Similar calculations may be used to calculate the wanning and thawing times, which may be longer due to the slower heat transfer in liquid water than ice. The presence of chemicals or biological material may lower the freezing temperature of the water. [00205] The key factors influencing the freezing and thawing times are the height of the channel L (i.e. thickness of the liquid layer) and the thermal conductivities of the sample in the channel (in liquid and solid (frozen) phases) and the metal base. The freeze/thaw and cooling/warming times are all proportional to L², and thus may be shortened by decreasing the channel height L, at the tradeoff of lengthening the channel and adding real estate on chip. In addition, freeze/thaw cycles may be expedited by adding more metal channel walls or baffles. The duration of the freeze/thaw cycle also depends on the speed at which the metal base may be heated and cooled.

[00206] A diagnostic system that is rapid, sensitive, cost-effective is achieved by integrating the various modules in terms of physical workflow by matching input and output volumes and by synchronizing processing times into an overall process. This addresses three (3) distinct sample workflows and associated pathogens: urine microbial, blood microbial, and blood viral. Each of these workflows may be run on the same system infrastructure (e.g. computer, pumps, detection) but may have distinct microfluidic chips and consumables to match the particular assay. The modular design approach enables subsets of modules to be integrated into each workflow, and advantageously makes use of programmable logic controllers (PLCs), precision mechanical attachment points, power supplies, air sources for pneumatic controls, dispensing systems for reagents, chillers, and any other "services" required across the modules.

Example 4: User-friendly disposable or reusable devices for freeze-thaw lysis

[00207] Another advantageous embodiment of the present cryolysis / cryopreservation invention is a user- friendly cryolysis / cryopreservation device design that may be operated with pipettes and is compatible with centrifuges, plate covers, and/or plate liquid handlers (FIG. 11). The "user-friendly" nature of the design means that it may be operated without specialized equipment such as pumps or syringes. Referring to FIGS. 11 A, 11B, and 11D, the device consists of a disposable (consumable) base unit 500 with an inlet chamber 504 for sample input, connected via narrow aperture 503 to shallow region 502. The shallow region 502 bounded by a foil layer 510 on one side. The foil layer is held in place by collar 511, which forms a mechanical seal. The base unit 500 may be sealed with cap 512, for example a Micro Amp tube caps, which fits 513 over the top 509 of the base unit 500. The device is simple to assemble (see FIG. 11C).

[00208] Referring to FIG. 1 IE, the device is operated for cryolysis as follows. Sample is input into inlet chamber 504, then moved via tapping or centrifuge through narrow aperture 504 and redistributed into shallow region 502. Once the fluid is in the shallow region, it is sandwiched between the base unit wall and foil layer boundaries, limiting the maximum thickness 553 of the sample to a specified amount, for example, 0.5 mm. The shape of the sample in the shallow region is that of a thin disc, which is advantageous for rapid cooling, freezing, wanning, and thawing as discussed in FIG. 10. The foil layer 510 on device 1500 is then brought in contact 519 with surface of external thermal module 1590, which may be the same or similar to module 590 described herein, which rapidly changes temperature from a cold temperature for f eezing to a warm temperature for thawing. Alternately, device 1500 is robotically or manually set on a chiller block 260 or 5006 until sample 518 is frozen, and then moved to a warming block 240 or 5004 until sample 518 is thawed. A sufficient number of freeze-thaw cycles are carried out until sample 518 is lysed, becoming lysate 2518. The cap 512 and base unit 500 are then removed 520, exposing lysate 2518 on foil layer 510, optionally still connected to collar 511. Lysate 2518 is then removed by pipette 521 or other means.

[00209] The wetting properties of the foil layer 510 may be adjusted to enable facile lysate 2518 removal.

[00210] The foil layer may be aluminum, copper, gold, iron, silver, steel, tin, titanium or zinc or a mixture thereof, as long as it allows rapid heat transfer. The foil layer may be replaced by any thin layer of glass or thermally conductive plastic or other thermally conductive material, as long as it allows rapid heat transfer.

[00211] The duration and speed of the freeze/thaw cycle depends on the speed at which the metal base may be heated and cooled between cold (e.g. -20 °C) and warm (e.g. 20 °C) temperatures, or how quickly the device may be moved from a cold plate to a warm plate. For example, if the device containing water in the shallow region is initially at room temperature (20 °C) and is placed on a cold plate at less than -20 °C, the water in the shallow region will freeze in under 1 s. If the device is then placed on a warm plate at 20 °C, the water in the shallow region will thaw in about 4 s.

[00212] The duration and speed of the freeze/thaw cycle also depends on the temperatures of the cold and warm plates or on the extreme cold and warm temperatures of a single plate whose temperature varies between extremes.

[00213] Freeze/thaw cycles may be expedited by adding more metal channel walls or baffles. [00214] Referring to FIG. HE, the device may be operated as a cryopreservation means by using only a cold plate to rapidly freeze the sample. Later, following storage, the frozen samples may be thawed prior to use by placing the sample, still in the device, on the warm plate or a warming surface of an external thermal module 1S90 in a similar manner illustrated in FIG. 1 IE.

[00215] The volume of the shallow region must be greater than the sample volume, so that the entire sample may fit inside the shallow region. The radius 554 of the shallow region may be increased to allow for larger sample volumes, but keeping the thickness 553 of the shallow region less than or equal to 0.5 mm to maintain rapid freeze and thaw times. Typical samples volumes range between 1 -500 uL, but others could be readily considered.

[00216] The inlet well may be sufficiently large to allow pre- and post- processing of sample, including: reagent addition, removal; sample mixing; etc. The inlet well may have the capacity of the sample volume plus an additional volume ranging from 1-500 μL.

[00217] The diameter of the aperture 503 must be sufficiently less than the capillary length of the sample solution (e.g. 2.7 mm for pure water) so that the sample will not bulge into the aperture 503, increasing the sample thickness and therefore the times required for freezing and thawing. If the shallow region is full of sample, the sample contact line must be pinned on the lower edge 555 of the aperture 503. To achieve this, the edge must be sufficiently sharp and non- wetting, either by using a non-wetting plastic or coating the region with hydrophobic coating. Alternatively, a sample volume lower than the shallow region 502 volume could be used, so that when redistributed within the shallow region 503 the sample stays away from the aperture 503.

[00218] Device 500 may be disposable (i.e. a consumable) or reusable.

[00219] The thickness of plastic under shallow region is kept thin to avoid adding excessive thermal resistance, which is a key feature enabling rapid heat transfer, cryo lysis, and cryopreservation.

[00220] Other embodiments may employ ultrasonic or heat or other sealing method to seal the foil 510 onto the base unit 500. Such methods may be more suitable for industrial mass production, and may provide better seals.

[00221] FIG. 11F illustrates an arrayed embodiment 516 of the user-friendly cryolysis/cryopreservation device, enabling the high-throughput cryolysis/cryopreservation of multiple samples simultaneously. In addition, the well spacing is compatible with 96-well plates and multichannel pipettes (for example, by using every other pipette tip connection on the multichannel pipettes).

[00222] FIG. 11G illustrates an example of mating cryolysis/cryopreservation device 1500 with a PCR plate well 514. The device base unit 500 has advantageous structure 508 that fits inside PCR plate well 514, structure 507 that sits outside and on top of PCR plate well and prevents structure 508 from getting stuck inside PCR plate well 514.

[00223] F G. 11H illustrates an example of mating cryolysis/cryopreservation device 516 with 96-well PCR plate 517. Collars in array 515 are spaced at 18 mm, equal to twice the center-to- center distance of PCR plate wells. Thus, the necks of the devices 1500 fit into every other well in a line of PCR plate wells. The device 1500 may therefore be made compatible with plate sealers, readers, centrifuge plate holders, and other equipment designed for 96-well plates. The device 516 may be modified to be compatible with any multiwell plate design, such as a Corning Costar 96-well multiwell plate or 384 well or 1536 well multiwell plates.

[00224] FIG. 111 depicts an alternate protocol using centrifuge to remove thawed sample 518 (for cryopreservation applications) or lysate 2518 (for cryolysis applications) from a single lysis device 1500 or arrayed lysis device 516. This enables the sample or lysate to be removed simultaneously from multiple devices and moved to appropriate wells in a multiwall PCR plate, in a single centrifuge step.

[00225] FIG. 11 J illustrates a 3D printed prototype of user-friendly cryolysis/cryopreservation device.

[00226] Alternate embodiments of the user-friendly disposable or reusable devices for cryolysis or cryopreservation include thin tube devices illustrated in FIGS. 12-14. In this case, thin tube-like structures or channels within the device force the samples into thin rod geometries, advantageous for rapid cooling, freezing, warming, and thawing as discussed in FIG. 10. General design features are considered in FIG. 12, and particular fabricated designs are presented in FIG. 13 (loop device) and FIG. 14 (dunk device).

[00227] A particular device for cryolysis/cryopreservation having a thin tube geometry is illustrated in FIG. 12. A thin tube is connected to a sample input/recovery receptacle. Loading is accomplished by pushing sample with pusher fluid or other sample. Cryolysis or is accomplished by alternately dipping thin tube into cryo-liquid and warming liquid, and repeating with sufficiently many cycles to achieve lysis. Thawed lysate is eluted for downstream processing. [00228] Design allows disposable thin-tube to be used for freeze-thaw, or a permanent thin tube that may be washed after step (h) and then re-used.

[00229] An arrayed embodiment of the thin tube device is illustrated in FIG. 12B. Arrayed design allows separate samples to be cryolysed or cryopreserved in parallel to increase throughput. An example cryolysis protocol for arrayed thin tube device illustrated in FIG. 12C.

[00230] In an alternate embodiment, cryopreservation is accomplished by dipping a thin tube or array of thin tubes into cryoliquid, freezing the sample(s). The thin tube(s) may then be disconnected from the receptacle and stored. Later, following cold storage, the thin tube(s) with frozen sample(s) may be reconnected to the receptacle, dipped in warming liquid to thaw sample(s), which may then be optionally eluted for downstream processing.

[00231] An alternate embodiment of the thin tube cryolysis/cryopreservation device is illustrated in FIG. 13. A loop of microtubing 640 containing sample 641 passes through a guide tube 642. The guide tube passes into an insulated cooling chamber 647, within which resides a cryo-liquid 644, for example ethanol in dry ice. The cryo-liquid circulates through holes 643 in the guide tube 642 to cool the loop 640 and hence the sample 641. Room temperature or warm air surrounds the loop outside of the guide tube 642 and cooling chamber 647. The loop 640 is rotated 650 through the guide tube 642, passing through the inlet 648, into the portion of the guide tube 651 immersed in the cryo-liquid, thereby freezing the sample. The loop is continuously rotated, so that the frozen portion exits the guide tube outlet 649 and thaws in the room temperature or warm air 646. The loop 640 is continuously rotated 650 to achieve the desired number of freeze-thaw cycles. The advantage of loop design is that external thermal module is static; freeze-thaw cycles are provided by rotating loop. The protocol for loading the microtubing with sample is illustrated in FIG. 13B. The protocol for threading and connecting microtubing is illustrated in FIG. 13C.

[00232] A fabricated loop device is presented in FIG. 13D, showing the inside of cooling chamber 647, guide tube 642, loop 640, sample 641, cryo-liquid 644, and cooling means 657 (in this case an ethanol and dry ice mixture), and operation.

[00233] Data from laboratory experiments is presented in FIG. 13E demonstrating cryolysis. Fluorescent images of BL-1 microbial strains stained by LIVE (green) / DEAD (red) after 10 freeze/thaw cycles with loop device at 20 s per cycle. [00234] To accomplish cryopreservation, the loop of microtubing could be run through the cryoliquid once, freezing the sample. The frozen sample could then be stored. Later, following storage, the frozen sample could be retrieved and dipped in wanning liquid or thawed in air.

[0023S] An additional alternate embodiment of the thin tube cryolysis/cryopreservation device is illustrated in FIG. 14. In this case, segments of microtubing 701 are clamped 702 in place and spaced along a rod 706, which is insulated 703 and connected to an insulated handle 704 (FIG. 14A). The clamped tubing segments 701 are spaced 705 sufficiently to allow ample cryo-fluid circulation to expedite heat transfer. Microtubing segments are filled with sample according to protocol in FIG. 13B, and then clamped in place on the dunk device (FIG. 14A). Clamping mechanically seals sample inside tubing.

[00236] To accomplish cryolysis, the dunk device with microtubing segments 1701 containing sample is then alternately dipped ("dunked") 707 in cryo-liquid 607 to freeze the samples and warming fluid (liquid or air) 608 to thaw (FIG. 14B).

[00237] To accomplish cryopreservation, the dunk device with microtubing segments 1701 containing sample is dipped ("dunked") 707 in cryo-liquid 607 to freeze the samples. The samples may then be removed from the device and stored, or the entire device with clamped microtubing segments 1701 may be stored at cold temperatures. Later, following storage, the frozen samples may be retrieved, and, if not clamped to the device, re-clamped. The device would then be dipped ("dunked") in wanning fluid to thaw the samples. Alternately, the frozen samples inside the microtubing segments could be thawed in room temperature air.

[00238] A fabricated dunk device 700 is shown in FIG. 14C.

[00239] Alternate embodiments of the user-friendly disposable or reusable devices for cryolysis or cryopreservation include devices consisting of tubes, wells, or other containers with heat conducting baffles or barriers illustrated in FIG. 15. Such baffles or barriers divide the fluid into thin regions with low thermal mass which can freeze and thaw quickly. Tubes, wells, or containers may be singleton devices or arrayed in a micro we 11 plate format or other preferred arrangements.

_{* *} ·

[00240] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. An apparatus for cryo-treatment of a microfluidic sample, comprising:

a channel having an inlet for receiving therein at least one microfluidic droplet, wherein the at least one microfluidic droplet is in a freeze-resistant carrier medium;

means for controlling flow of the microfluidic droplet through the channel;

at least one temperature regulation means in thermal communication with the channel; and

a controller for controlling temperature of the at least one temperature regulation means.

2. The apparatus for cryo-treatment of a microfluidic sample of claim 1, comprising a plurality of temperature regulation means, wherein each of the temperature regulation means corresponds to one of a plurality of thermal control regions.

3. The apparatus for cryo-treatment of a microfluidic sample of claim 2, wherein the channel passes through the plurality of thermal control regions.

4. The apparatus for cryo-treatment of a microfluidic sample of claim 3, wherein the channel has a sinusoidal shape.

5. The apparatus for cryo-treatment of a microfluidic sample of claim 3, wherein the channel is substantially straight.

6. The apparatus for cryo-treatment of a microfluidic sample of claim 2, wherein at least one of the thermal control regions is a warming region.

7. The apparatus for cryo-treatment of a microfluidic sample of claim 2, wherein at least one of the thermal control regions is a chiller region.

8. The apparatus for cryo-treatment of a microfluidic sample of claim 1, wherein the microfluidic droplet comprises cellular material and resides in freeze-resistant carrier medium.

9. The apparatus for cryo-treatment of a microfluidic sample of claim 1, wherein the microfluidic droplet comprises cellular material and a reagent and resides in freeze- resistant carrier medium

10. The apparatus for cryo-treatment of a microfluidic sample of claim 1, wherein the freeze-resistant carrier medium is oil.

11. A method of performing cryo-treatment of a microfluidic sample, comprising: preparing a microfluidic droplet in a freeze-resistant carrier medium; introducing the microfluidic droplet into a channel; and

passing the microfluidic droplet through at least one temperature zone at a predetermined, continuous flow rate via a channel.

12. The method of performing cryo-treatment of a microfluidic sample of claim 11, wherein passing the microfluidic droplet through the at least one temperatures zone comprise passing the microfluidic droplet through a warming zone and a chiller zone.

13. The method of performing cryo-treatment of a microfluidic sample of claim 11, wherein the channel is sinusoidal.

14. The method of performing cryo-treatment of a microfluidic sample of claim 11, wherein the channel is straight.

15. The method of performing cryo-treatment of a microfluidic sample of claim 11, wherein microfluidic droplet comprises cellular material and resides in freeze-resistant carrier.

16. The method of performing cryo-treatment of a microfluidic sample of claim 11, wherein the microfluidic droplet comprises cellular material and a reagent and resides in freeze-resistant carrier medium.

17. The method of performing cryo-treatment of a microfluidic sample of claim 11, wherein the freeze-resistant carrier medium is oil.

18. The method of performing cryo-treatment of a microfluidic sample of claim 11, wherein there is a first microfluidic droplet and a second microfluidic droplet.

19. The method of performing cryo-treatment of a microfluidic sample of claim 18, wherein the first microfluidic droplet comprises a biological sample and the second microfluidic droplet comprises a reagent for cryolysis or cryopreservation.

20. The method of performing cryo-treatment of a microfluidic sample of claim 18, wherein there is a first and second microfluidic droplet, and wherein the first microfluidic droplet comprises a bacteria, bacteriophage, fungi, yeast, protozoa, algae, plant cell, animal cell, hybridoma or stem cell and wherein the second microfluidic droplet comprises glycerol, DMSO, methanol or serum.

21. An apparatus for cryo-treatment of a microfluidic or millifluidic sample, comprising the rapid cryo-treatment designs of FIG. 11.

22. The apparatus of claim 21 further comprising a highly thermally conductive layer such as foil, copper, tin, gold, silver, glass, thermally conductive plastic, etc.

23. The apparatus of claim 21 or 22 further comprising a channel network to divide sample into sub-samples and deliver sub-samples to individual cryo-treatment chambers.

24. The apparatus of claim 21, 22, or 23 further comprising a nanofilter for sample concentration prior to cryolysis or cryopreservation, and/or an ultrafiltration membrane with desired molecular weight cutoff for sample concentration following cryolysis.

25. The apparatus of claim 21, 22, 23, or 24 further comprising a perturbation chamber or means for perturbation of an existing chamber, where reagents, drugs, R A protectants, cryolysis or cryopreservation reagents may be added to sample.

26. A method of performing cryo-treatment of a sample, comprising filling the cryo- treatment chambers) of the apparatus of claim 21, 22, 23, 24, or 25 with sample, freezing the sample(s), optionally thawing the sample(s), optionally re-freezing and re-thawing the sample(s) as many times as desired, then either storing the frozen samples) or eluting the thawed cryo-treated sample(s) for downstream processing.

27. The method of claim 26, wherein the freezing and thawing of the sample is performed with an external thermal module comprising two stages of hot and cold.

28. The method of claim 27, wherein the external thermal module is connected with a cryolysis module via a thermally conductive pathway.

29. The method of claim 28, wherein the thermally conductive pathway is a solenoid- actuated metal contact between the stages and the bottom plate.

30. The method of any one of claims 27 to 29 wherein stages of hot and cold comprises a commercial chiller, a Peltier block, a hot plate, or any combination thereof, that alternately make contact with the device's metal base to freeze and thaw the sample.

31. The method of any one of claims 26 to 30, wherein the sample is a biological sample.

32. The method of claim 31, wherein the biological sample is a bacteria, bacteriophage, fungi, yeast, protozoa, algae, plant cell, animal cell, hybridoma or stem cell.

33. An apparatus for cryo-treatment of a sample, comprising the user-friendly cryo- treatment designs of FIGS. 12-15.

34. The apparatus of claim 33 further comprising a highly thermally conductive layer such as foil, copper, tin, gold, silver, glass, thermally conductive plastic, etc.

35. The apparatus of claim 33 or 34 further comprising an arrayed format compatible with multiwell plates.

36. A method of performing cryo-treatment of a sample, comprising filling the inlet well(s) of the apparatus of claim 33, 34, or 35 with sample, capping the inlet well(s), and tapping or centrifuging to move the sample from the inlet well(s) into shallow regions, thereby redistributing the sample(s) into a thin disc-like shape, with a narrow aperture between inlet well and shallow region restricting flow of sample out of shallow region, freezing the sample, thawing the sample, optionally re-freezing and re-thawing the sample as many times as desired, removing the base unit from the thin conductive layer to allow sample to be removed.

37. The method of claim 36, wherein the freezing and thawing of the sample is performed with an external thermal module comprising two stages of hot and cold.

38. The method of claim 37, wherein the external thermal module is connected with a cryolysis module via a thermally conductive pathway.

39. The method of claim 38, wherein the thermally conductive pathway is a solenoid- actuated metal contact between the stages and the bottom plate.

40. The method of any one of claims 37 to 39 wherein stages of hot and cold comprises a commercial chiller, a Peltier block, a hot plate, or any combination thereof, that alternately make contact with the device's metal base to freeze and thaw the sample.

41. The method of any one of claims 36 to 40, wherein the sample is a biological sample.

42. The method of claim 41, wherein the biological sample is a bacteria, bacteriophage, fungi, yeast, protozoa, algae, plant cell, animal cell, hybridoma or stem cell.