WO2014085801A1 - Traitement cryogénique dans un dispositif microfluidique - Google Patents

Traitement cryogénique dans un dispositif microfluidique Download PDF

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Publication number
WO2014085801A1
WO2014085801A1 PCT/US2013/072568 US2013072568W WO2014085801A1 WO 2014085801 A1 WO2014085801 A1 WO 2014085801A1 US 2013072568 W US2013072568 W US 2013072568W WO 2014085801 A1 WO2014085801 A1 WO 2014085801A1
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WIPO (PCT)
Prior art keywords
sample
channel
microfluidic
cryo
treatment
Prior art date
Application number
PCT/US2013/072568
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English (en)
Inventor
Matthew J. HANCOCK
Robert Nicol
Francesco PIRAINO
Scott Steelman
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The Broad Institute, Inc.
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Application filed by The Broad Institute, Inc. filed Critical The Broad Institute, Inc.
Publication of WO2014085801A1 publication Critical patent/WO2014085801A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
    • A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0278—Physical preservation processes
    • A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid

Definitions

  • the present invention generally relates to methods and compositions for cryolysis and/or cryopreservation.
  • 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.
  • 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 ⁇ , the application of microfluidics provides significant cost-savings.
  • the use of microfluidics technology reduces cycle times, shortens tirae- to-results, and increases throughput.
  • 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 10 8 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.
  • 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.
  • problems with the cryopreservation of cells especially in a microfluidic system.
  • 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.
  • the invention particularly relates to devices for cryolysis and/or cryopreservation, particularly microfluidic devices and methods for microfluidic cryolysis or cryopreservation.
  • 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.
  • the first block may be a chiller block and second block may be a warming block, whereby the device may be for cryolysis.
  • multiple blocks, alternately for chilling and warming may be put in series for multiple freeze/thaw cycles for cryolysis.
  • 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.
  • 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.
  • 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.
  • 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).
  • an apparatus for cryo-treatment of a microfluidic sample 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.
  • cryo-treatment may refer to either cryolysis or cryopreservation or any other methodology involving similar conditions.
  • a method of performing cryo-treatment of a microfiuidic sample 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.
  • 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.
  • 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.
  • 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.
  • 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 warmthing block 240.
  • 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 warmthing 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.
  • 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.
  • 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 warmthing block 240.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the channel is 175'
  • the module is 200'
  • 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'.
  • 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.
  • the channel is 1175'
  • the module is 1200'
  • 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.
  • 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.).
  • FIG. SAi illustrates temperature regulation channels 575 and 675 alongside or adjoining channel 175 or 1175.
  • 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.
  • 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.
  • 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.
  • 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'.
  • 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.
  • 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.
  • 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.
  • a single channel 575 provides cooling to channel 175 or 1175 for embodiments of cryopreservation of FIG. 2.
  • 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.
  • 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'.
  • 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.
  • FIG.5C illustrates embodiments where channel 175, 175', 1175 or 1175' is generally adjacent to temperature regulation channel 575 or 675.
  • FIG.5D illustrates embodiments where channel 175, 175', 1175 or 1175' is generally coaxial with temperature regulation channel 575 or 675.
  • 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'.
  • FIG. 1 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'.
  • 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'.
  • FIGS. 5E-3 and 5E-4 illustrate means for controlling or regulating temperature, with temperature regulation unit 475.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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'.
  • 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.
  • 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 warmthing 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'.
  • 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.
  • 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'.
  • the sample passes through the chiller block 260 or 5006 to freeze the cell inside the droplets and then the warmthing block 240 or 5004 to thaw the droplets.
  • Temperature regulation means 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.
  • FIGS. 6A and 6B illustrate disclosed droplets subjected to experimental processes according to an exemplary embodiment of the invention.
  • FIG. 7 schematically illustrates a process according to an exemplary embodiment of the invention.
  • FIGS. 8A-8E provide graphical analysis of the results from the process of FIG. 7 according to an exemplary embodiment of the invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • MW molecular weight
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 9D depicts a means for sample separation and distribution in parallel lysis chambers.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the foil layer makes contact with an external thermal module 1590, which comprises either a single module whose temperature cycles between chilling and warmthing temperatures, like 590, or the device would be moved robotically or manually between chiller 260 or 5006 and warmthing 240 or 5004 blocks or modules.
  • an external thermal module 1590 comprises either a single module whose temperature cycles between chilling and warmthing temperatures, like 590, or the device would be moved robotically or manually between chiller 260 or 5006 and warmthing 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.
  • 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.
  • 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.
  • 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.
  • Device 500 may be disposable (i.e. a consumable) or reusable.
  • 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.
  • 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.
  • 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.
  • FIG. HE depicts example protocol for use of user-friendly freeze-thaw lysis device 1S00.
  • 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.
  • 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.
  • 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,
  • 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 Warming temperature for thawing.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 11H depicts the mating of arrayed freeze-thaw lysis device 516 with 96-well PCR plate 517.
  • 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.
  • (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.
  • 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.
  • sample is moved into thin (shallow) region, which changes the sample geometry into a thin disc-like shape, ideal for rapid freeze/thaw cycles,
  • 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.
  • Device is disassembled and lysate is removed from foil 510, for easy sample recovery.
  • 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,
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 13 ⁇ 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.
  • 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.
  • 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.
  • FIG. 14C depicts a fabricated dunk device 700 for freeze-thaw lysis.
  • 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.
  • 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.
  • 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.
  • FIGS. 1A, 1B, 3, 5F and 5G Cryolysis embodiments are illustrated in FIGS. 1A, 1B, 3, 5F and 5G.
  • a carrier solution 130 The carrier solution does not freeze or boil at all temperatures present in the device.
  • 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 warmthing block 240.
  • 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 warmthing 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.
  • 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.
  • the warming block is positioned adjacent to the chiller block.
  • the warming block and chiller block are separated by an insulator, to prevent heat transfer between the two.
  • 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 warming 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.
  • 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.
  • the current invention thus enables rapid freeze/thawing of a continuous stream of individual cells 110 in droplets 100 in a micro fluidic 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.
  • FIGS. 2A, 2B, and 4 Cryopreservation embodiments are illustrated in FIGS. 2A, 2B, and 4.
  • one or more droplets 1000 each containing one or more cells 1110 and cryopreservant buffer solution 1120 that readily permeabilizes the cell are suspended in a carrier solution 1130.
  • the carrier solution 1130 does not freeze or boil at all temperatures present in the device.
  • 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).
  • a 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.
  • 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. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 warming.
  • the droplet may comprise water, the cell, and dissolved chemicals including reagents to protect from RNA degradation, lysis chemicals, etc.
  • 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.
  • zwitterioinic detergents such as 3-(N,N- Dimethylmyristylammonio)propanesulfonate
  • nonionic detergents such as TWEEN® from Sigma Aldrich
  • anionic detergents such as sodium deoxycholate
  • alkaline buffer solutions such as EDTA with sodium dodecyl sulfate with sodium hydroxide followed by potassium acetate.
  • 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.
  • detergents and lysis agents such as Lysozyme, Lysostaphin, zwitterioinic detergents such as 3-(N,N-Dimemylmyristylammonio) propanesulfonate, nonionic detergents such as TWEEN® from Sigma Aldrich,
  • lysis agents 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.
  • 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 RecoveryTM 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.
  • cryoprotective agents by cell type that may be in the droplets:
  • Bacteria Glycerol (about 9% to about 11%, e.g. about 10%)
  • Bacteriophage Glycerol (about 9% to about 11%, e.g. about 10%)
  • Fungi Glycerol (about 9% to about 11%, e.g. about 10%)
  • Yeast Glycerol (about 9% to about 11%, e.g. about 10%)
  • Protozoa DMSO (about 3% to about 12%, e.g., about 5%- about 10%)
  • Glycerol (about 8% to about 22%, e.g. about 10%-about 20%)
  • Algae Methanol (about 3% to about 12%, e.g., about 5%- about 10%)
  • Plant Cells DMSO (about 3% to about 12%, e.g., about 5%- about 10%)
  • Glycerol (about 3% to about 12%, e.g., about 5%- about 10%)
  • Animal Cells DMSO about 3% to about 12%, e.g., about 5%- about 10%
  • Glycerol (about 3% to about 12%, e.g., about 5%- about 10%)
  • Hybridomas DMSO (about 3% to about 12%, e.g., about 5%- about 10%)
  • Serum e.g., FBS (about 15%-about 25%, e.g., about 20%)
  • Serum (FBS) (about 15%-about 95%. e.g.. about 20%-about 90%)
  • 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.
  • 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, Warming 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.
  • 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.
  • Channel 175 or 175' or 1175 or 1175' may include a recirculating or spiral configuration.
  • 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.
  • 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.
  • 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'.
  • the design configuration of the present embodiment is for illustrative purposes and should not be considered as limiting.
  • 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.
  • 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.
  • 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.
  • Standard high pressure pumps e.g., Mitos Pressure Pumps, Ultrafast High-pressure AC Electro-osmotic Pumps, Elveflow AF1 pressure pumps, etc.
  • 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.
  • 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.
  • the size of the device is not limited to these dimensions and the principle works at smaller scales as well as for larger systems.
  • channel 175 runs through a chiller block 260 and a warmthing block 240 and in FIG. 3, channel 175' is running between chiller block 260' and warmthing block 240'.
  • channel 1175 runs through chiller block 1260 and in FIG. 4, channel 1175' is running through chiller block 1260*.
  • 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.
  • 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.
  • 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.
  • 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.
  • PDMS polydimethylsiloxane
  • 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.
  • 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.
  • 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.
  • the chiller and warmthing blocks in the cryolysis and cryopreservation inventions may be divided into thermal regions, each with a different or independent temperature regulation system.
  • 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.
  • 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.
  • 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.
  • 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.
  • one warming block region and one chiller block region e.g., two warming block regions and two chiller block regions (as illustrated), three warming block regions, etc.
  • 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.
  • chiller block 1260 may be one or more different or independent temperature regulation systems.
  • 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.
  • FIG. 3 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).
  • 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 ELVEBIOTM 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 ELVEBIOTM 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.
  • 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').
  • 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).
  • the invention comprehends the use of chiller and warmthing 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.
  • An array of temperature sensors may be distributed at advantageous positions across the device to provide feedback to the temperature regulation system(s).
  • sensor 21S at or just after inlet 210 is sensor 21S, which may be within the channel or on the exterior surface of the channel.
  • 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.
  • sensors 235 approximately a quarter and three quarters the distance along each channel segment within the chiller block 260.
  • sensors 265 approximately halfway along each channel segment within the chiller block 260.
  • sensors 255 approximately one quarter and three quarters the distance along each channel segment within the warmthing block 240.
  • sensors 245 at or near the halfway point along each channel segment within the warming block 240.
  • sensors 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.
  • 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.
  • 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 warmthing block or may be adjusted downward, either globally or locally.
  • 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, warmthing 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.
  • 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.
  • 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.
  • 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.
  • 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'.
  • 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.
  • 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.
  • 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).
  • Another application for this invention is for the cryopreservation of cells.
  • 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.
  • 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.
  • 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.
  • thermoelectric device to control hot and cold zones and flow rate along the channel 175, 1175, 175', or 1175'.
  • 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).
  • 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.
  • the washed cells were suspended in 0.5X PBS buffer at room temperature (approximately 20 degrees C), with a cell density of approximately lxl0 6 /mL. (for RNA- Sequence / amplification applications, cells were suspended in Reverse Transcription (PCR) reaction mix).
  • PCR Reverse Transcription
  • the filtered cell suspension was transferred into an injection vial that was driven by pure fluorocarbon oil at injection rate 100 ⁇ . with the carrier oil flowing at 200 ⁇ (all at room temperature).
  • FIGS. 6A and 6B show the droplets subjected to freeze-thaw and those not subjected freeze-thaw, respectively, under the optical microscope.
  • FIGS. 6A and 6B show the droplets subjected to freeze-thaw and those not subjected freeze-thaw, respectively, under the optical microscope.
  • 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.
  • 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.
  • 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.
  • FIG. 7 shows schematically the process undertaken in this Example.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • Example 3 DeviceModule for Rapid Cryolysis or Cryopreservation
  • 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 ⁇ sample in ⁇ 20 min with SO freeze/thaw cycles, each ⁇ 20 s long.
  • 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.
  • 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.
  • 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.
  • 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.
  • an advantageous step before and/or after is sample concentration.
  • 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.
  • the intact cell solution 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.
  • 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).
  • UF ultrafiltration
  • 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.
  • the nanofilters and/or UF membranes may be advantageously coated to prevent sample loss, as described herein.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • iCVD initiated chemical vapor deposition
  • 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.
  • 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.
  • 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.
  • the thin rods and discs cool and freeze that fastest.
  • the sample geometry is an important feature to consider in the design of a device that rapidly freezes and thaws a sample.
  • 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.
  • T f is the freezing temperature of the material.
  • the presence of chemicals or biological material may lower the freezing temperature of the water.
  • 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 L 2 , and thus may be shortened by decreasing the channel height L, at the tradeoff of lengthening the channel and adding real estate on chip.
  • 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.
  • 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.
  • 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.
  • PLCs programmable logic controllers
  • Example 4 User-friendly disposable or reusable devices for freeze-thaw lysis
  • 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).
  • 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.
  • 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, warmthing, 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.
  • 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.
  • the wetting properties of the foil layer 510 may be adjusted to enable facile lysate 2518 removal.
  • 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.
  • 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.
  • 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.
  • Freeze/thaw cycles may be expedited by adding more metal channel walls or baffles.
  • 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.
  • 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.
  • 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 ⁇ L.
  • 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.
  • Device 500 may be disposable (i.e. a consumable) or reusable.
  • 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.
  • 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.
  • FIG. 11F illustrates an arrayed embodiment 516 of the user-friendly cryolysis/cryopreservation device, enabling the high-throughput cryolysis/cryopreservation of multiple samples simultaneously.
  • 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).
  • 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.
  • 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.
  • 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.
  • FIG. 11 J illustrates a 3D printed prototype of user-friendly cryolysis/cryopreservation device.
  • Alternate embodiments of the user-friendly disposable or reusable devices for cryolysis or cryopreservation include thin tube devices illustrated in FIGS. 12-14.
  • 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).
  • FIG. 12 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.
  • 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.
  • FIG. 12B 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.
  • FIG. 12C An example cryolysis protocol for arrayed thin tube device illustrated in FIG. 12C.
  • 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.
  • FIG. 13 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.
  • FIG. 13D 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.
  • cooling means 657 in this case an ethanol and dry ice mixture
  • FIG. 13E 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 warmthing liquid or thawed in air.
  • FIG. 14 An additional alternate embodiment of the thin tube cryolysis/cryopreservation device is illustrated in FIG. 14.
  • 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.
  • 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).
  • 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 warmthing fluid to thaw the samples. Alternately, the frozen samples inside the microtubing segments could be thawed in room temperature air.
  • a fabricated dunk device 700 is shown in FIG. 14C.
  • 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.

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Abstract

La présente invention concerne d'une manière générale des procédés et des compositions pour la cryoconservation et/ou la cryolyse.
PCT/US2013/072568 2012-11-30 2013-12-02 Traitement cryogénique dans un dispositif microfluidique WO2014085801A1 (fr)

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DE102017210725A1 (de) * 2017-06-26 2018-12-27 Robert Bosch Gmbh Mikrofluidische Vorrichtung und Verfahren zur Prozessierung einer biologischen Probe mit einer Kühlkammer
CN110314715A (zh) * 2019-07-17 2019-10-11 西安交通大学 基于聚焦式表面声波和微液滴技术的粒子富集微流控芯片
CN111235085A (zh) * 2020-01-20 2020-06-05 丁林 基于静态液滴阵列分离单个细胞的方法
CN113210024A (zh) * 2021-06-03 2021-08-06 北京中科生仪科技有限公司 基于pcr的连续进液装置
US11122796B1 (en) 2016-02-19 2021-09-21 Regents Of The University Of Minnesota Cryoprotection compositions and methods
US11213824B2 (en) 2017-03-29 2022-01-04 The Research Foundation For The State University Of New York Microfluidic device and methods
US11311008B2 (en) 2016-04-19 2022-04-26 Regents Of The University Of Minnesota. Cryopreservation compositions and methods involving nanowarming
CN116375790A (zh) * 2023-06-05 2023-07-04 厦门胜泽泰医药科技有限公司 一种微流控芯片固相肽合成方法及装置
US11708556B2 (en) 2014-10-20 2023-07-25 University Of Utah Research Foundation Tissue sample processing system and associated methods
US11971377B2 (en) 2018-03-12 2024-04-30 The Penn State Research Foundation Method and apparatus for temperature gradient microfluidics
WO2024155998A1 (fr) * 2023-01-20 2024-07-25 Massachusetts Institute Of Technology Système microfluidique à base de gouttes suspendues, dispositif et procédé d'échange de liquide pour la vitrification d'un échantillon

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Cited By (14)

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Publication number Priority date Publication date Assignee Title
US11708556B2 (en) 2014-10-20 2023-07-25 University Of Utah Research Foundation Tissue sample processing system and associated methods
US11122796B1 (en) 2016-02-19 2021-09-21 Regents Of The University Of Minnesota Cryoprotection compositions and methods
US11311008B2 (en) 2016-04-19 2022-04-26 Regents Of The University Of Minnesota. Cryopreservation compositions and methods involving nanowarming
US11213824B2 (en) 2017-03-29 2022-01-04 The Research Foundation For The State University Of New York Microfluidic device and methods
US11911763B2 (en) 2017-03-29 2024-02-27 The Research Foundation For The State University Of New York Microfluidic device and methods
DE102017210725A1 (de) * 2017-06-26 2018-12-27 Robert Bosch Gmbh Mikrofluidische Vorrichtung und Verfahren zur Prozessierung einer biologischen Probe mit einer Kühlkammer
US11971377B2 (en) 2018-03-12 2024-04-30 The Penn State Research Foundation Method and apparatus for temperature gradient microfluidics
CN110314715A (zh) * 2019-07-17 2019-10-11 西安交通大学 基于聚焦式表面声波和微液滴技术的粒子富集微流控芯片
CN111235085A (zh) * 2020-01-20 2020-06-05 丁林 基于静态液滴阵列分离单个细胞的方法
CN113210024A (zh) * 2021-06-03 2021-08-06 北京中科生仪科技有限公司 基于pcr的连续进液装置
CN113210024B (zh) * 2021-06-03 2022-04-08 北京中科生仪科技有限公司 基于pcr的连续进液装置
WO2024155998A1 (fr) * 2023-01-20 2024-07-25 Massachusetts Institute Of Technology Système microfluidique à base de gouttes suspendues, dispositif et procédé d'échange de liquide pour la vitrification d'un échantillon
CN116375790B (zh) * 2023-06-05 2023-08-18 厦门胜泽泰医药科技有限公司 一种微流控芯片固相肽合成方法及装置
CN116375790A (zh) * 2023-06-05 2023-07-04 厦门胜泽泰医药科技有限公司 一种微流控芯片固相肽合成方法及装置

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