WO2018195107A1 - Systèmes, dispositifs et procédés de décongélation automatique d'échantillon de gros calibre - Google Patents

Systèmes, dispositifs et procédés de décongélation automatique d'échantillon de gros calibre Download PDF

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Publication number
WO2018195107A1
WO2018195107A1 PCT/US2018/028012 US2018028012W WO2018195107A1 WO 2018195107 A1 WO2018195107 A1 WO 2018195107A1 US 2018028012 W US2018028012 W US 2018028012W WO 2018195107 A1 WO2018195107 A1 WO 2018195107A1
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WO
WIPO (PCT)
Prior art keywords
sample
vial
temperature
heater block
thawing
Prior art date
Application number
PCT/US2018/028012
Other languages
English (en)
Inventor
Brian Schryver
Scott COMISO
Thomas MCPHERSON
Original Assignee
MedCision, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MedCision, Inc. filed Critical MedCision, Inc.
Publication of WO2018195107A1 publication Critical patent/WO2018195107A1/fr

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Classifications

    • 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
    • G01N1/44—Sample treatment involving radiation, e.g. heat
    • 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
    • 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
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • 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

Definitions

  • the present disclosure is generally related to systems and methods for thawing a frozen sample in a vial, and in particular relatively large gauge sample vials.
  • the present disclosure relates to the cryogenic preservation of cells, and to systems, devices, and methods for the recovery of cryogenically-preserved cells and tissue.
  • Cryogenic preservation of cells in suspension is a well-established and accepted technique for long term archival storage and recovery of live cells.
  • cells are suspended in a cryopreservation media typically including salt solutions, buffers, nutrients, growth factors, proteins, and cryopreservatives.
  • the cells are then distributed to archival storage containers of the desired size and volume, and the containers are then reduced in temperature until the container contents are frozen.
  • Typical long-term archival conditions include liquid nitrogen vapor storage where temperatures are typically between -196°C and -150°C.
  • intracellular ice crystal growth may be reduced by adding a cryoprotectant compound to the tissues or cell suspension solution that inhibits ice crystal nucleation and growth both extracellularly and intracellularly.
  • the growth of intracellular ice can be controlled through management of the rate of sample temperature reduction. During the freezing process extracellular ice crystal formation will exclude solutes and cells from the developing ice crystal structure thereby concentrating the solutes and cells in the remaining liquid phase.
  • solute concentration will establish an osmotic potential that will promote the dehydration of the cells while allowing time for cell membrane-permeable cryoprotectants to equilibrate in concentration within the intracellular volume.
  • a temperature will be reached at which the high solute concentration will solidify in a glass state with minimal size of ice crystal nuclei within the intracellular volume.
  • the solid-state cell suspension is then further reduced in temperature until the cryogenic storage temperature is reached. At this temperature molecular activity is sufficiently reduced that the cells may be stored indefinitely.
  • the rate of temperature reduction during the freezing process must fall within a range of values.
  • the temperature reduction rate during the freezing process is typically controlled.
  • one method of controlling the rate of temperature reduction includes surrounding the sample with an insulating material and placing the assembly in a static temperature environment, while another method includes placing the exposed sample container into an isolation chamber in which the interior temperature is reduced at a controlled rate.
  • thermodynamically favored extension of the small ice nuclei within the cells As the growth of the intracellular ice crystals have an associated potential for cell damage, and as the degree of crystal growth is a time-dependent the phenomenon, minimizing the time interval of the transition through the phase change is desirable.
  • a rapid slew rate in the sample vessel temperature is typically achieved by partial submersion of the vessel in a water bath set to a temperature of approximately 37°C.
  • thawing protocols typically include a rapid thaw phase that is terminated when a small amount of solid material still remains in the sample container. Following removal from the water bath, the sample temperature will quickly equilibrate to a temperature that is near to the phase change temperature. Thawing protocols typically seek to minimize the duration at which the thawed sample is held in a state where the cryoprotectant is concentrated, and subsequent steps to dilute the sample or exchange the cryopreservation media for culture media are commonly applied in as short of an interval as possible.
  • thawing cells For thawing cells, conventional practice is to warm the cells quickly in a warm water bath (e.g., 37 °C) to just about the point at which the last bit of ice is about to melt and then to dilute the cells slowly into growth media. If the sample is allowed to get too warm, the cells may start to metabolize, and be poisoned by the DMSO (dimethyl sulfoxide) that is used in the freezing process.
  • DMSO dimethyl sulfoxide
  • the thawing of cryogenically preserved cells and tissue is performed by lab technicians and the applied protocol can not only vary between each lab technician, but may also be technique dependent.
  • sample thaw is generally subjectively judged by each individual technician and may result in variation in the thaw rate or samples which have been allowed to become too warm.
  • a repeatable thawing profile is theoretically possible to achieve using a bath and manual control of the vial insertion, expected variance in both technique and degree of protocol compliance, particularly combined with the requirement to frequently remove the vial from bath to monitor the thaw status, makes deviation from the standard profile a near certainty.
  • the removal of the vial from the bath interrupts the thermal energy transfer from the bath water to the vial and visual assessment of the thaw status is often difficult and may be complicated by the presence of vial labels and printed writing surfaces that are provided as integrated features of the vial product. Further water baths are also a source of contamination and inadvertent submersion of the vial body-cap junction can result in the introduction of bath liquid into the vial contents during removal of the vial cap.
  • Systems, devices, and methods that provide simplified, automated, and/or more consistent sample thawing may be advantageous and may increase cell recovery. While some thawing devices and methods have been proposed, further improvements may be desirable. For example, in some instances, it may be advantageous to increase a heat transfer rate, especially when thawing larger cells and/or multicellular organisms, or when thawing samples held within a sample vial having a volume of, for example, 6 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, or greater than 50 mL. Embodiments of the disclosure may address one or more of these issues.
  • the disclosed sample thawing device includes: a housing;
  • a heater block housed within the housing, the heater block being configured to split into three segments and wherein each of the three heater block segments is arranged to actuate between an outward position and an inward position; a pliable lining mechanically coupled to inner surfaces of the three segments of the heater block, wherein the pliable lining and the heater block, with the three heather block segments each located at the inward position, form a vessel receptacle;
  • each of the three heater block segments are mounted on a track and are coupled to move linearly between the outward position and the inward position.
  • each of the three heater block segments are mounted on a hinge and are coupled to move in concert with each other between the outward position and the inward position.
  • the agitation element comprises a motor and a plate configured to oscillate the vessel receptacle at a rate of from 1 cycle per second to 100 cycles per second.
  • the agitation element can include a counterweight configured to balance the oscillation motion of the motor.
  • the vessel receptacle is further configured to receive a collapsible sleeve in combination with a vessel.
  • the sample thawing device can include one or more infrared sensors, one or more temperature thermsistors, thermocouple sensors, or a combination thereof, configured to sense temperature of the contents of a sample vial via readings on the exterior surface of that sample vial.
  • one of the infrared sensors, one of the temperature thermsistors, thermocouple sensors, or combinations thereof are positioned proximate to the bottom of the vessel receptacle.
  • one of the infrared sensors, one of the temperature thermsistors, thermocouple sensors, or combinations thereof are accommodated within the heater block and the pliable lining and positioned proximate to a vertical side of the vessel receptacle.
  • the vessel receptacle is configured to receive vessels of various sizes, and can have a modular configuration, configured to hold a sample vessel with a 10 mL volume, a 50 mL volume, or sample vials that hold volumes within that range.
  • the disclosed sample thawing system for thawing a frozen sample in a vial includes: a heater block, configured to split into three or more segments, and wherein each of the heater block segments is arranged to move between a first orientation and a second orientation; a thermally-conductive pliable material mechanically coupled to form a lining along inner surfaces of the heater block segments, wherein the thermally-conductive pliable material and the heater block, with the heater block segments each located at the second position, form a vial receptacle; an agitation motor configured to vibrate the vial receptacle; a heating element thermally coupled with the heater block and being configured to heat the heating block; and a communication module, configured to collect and transmit sample data relating to a sample vial held within the vial receptacle.
  • the sample system can further include a collapsible sleeve, configured to fit around the sample vial, and further configured to substantially reduce in diameter; and an analysis device, comprising a microprocessor, configured to receive sample data from the communications module, to process the sample data, to provide the sample data to a display interface, and to send instructions to the temperature control device for a duration of thaw for a sample vail inserted into the vial receptacle.
  • a collapsible sleeve configured to fit around the sample vial, and further configured to substantially reduce in diameter
  • an analysis device comprising a microprocessor, configured to receive sample data from the communications module, to process the sample data, to provide the sample data to a display interface, and to send instructions to the temperature control device for a duration of thaw for a sample vail inserted into the vial receptacle.
  • FIG. 1 illustrates an perspective view of the exterior of a sample thawing apparatus, according to various embodiments.
  • FlG. 2 illustrates a partial interior view of the apparatus shown in FlG. 1, according to various embodiments.
  • FIG. 3 illustrates a partial front-plane cross-sectional view of the apparatus shown in FlG. 1 , according to various embodiments.
  • FIG. 4 illustrates an isolated view of an orbital module used within the sample thawing apparatus, according to various embodiments.
  • FlG. 5 illustrates a further isolated view of the orbital assembly illustrated in
  • FIG. 4 according to various embodiments.
  • FlG. 6 illustrates the underside of the isolated orbital assembly as shown in
  • FIG. 5, according to various embodiments.
  • FIG. 7 illustrates is a cross-sectional view of the orbital module assembly, according to various embodiments.
  • FlGS. 8A-8D illustrate views of a three-part heater block assembly, according to various embodiments.
  • FlGS. 9A-9C illustrate views of a four-part heater block assembly, according to various embodiments.
  • FlG. lOA shows a side schematic view of a model thawing system for a typical cryogenic storage vial that is used in describing the thermal energy flow partem and vial temperature detection methods, according to various embodiments of this technology.
  • FlG. l OB shows a top schematic view of the model thawing system for a typical cryogenic storage vial shown in FlG. 10A.
  • FlG. 1 1 shows a graphic representation of the heat conduction pathways and heat flow rates into a storage vial at two different levels of sample loading.
  • FlG. 12 shows an embodiment of a vial temperature equilibration device with dry ice refrigerant that equilibrates a vial temperature to approximately -77°C.
  • FlG. 13 shows a dimensioned drawing of the device described in FlG. 12.
  • FlG. 14 shows a second embodiment of a vial temperature adjustment device.
  • FlG. 15 shows two dimensioned drawings of the device described in FlG. 14.
  • FlG. 16 shows two graphs, with graph A showing a graphic display of the cooling and temperature-holding duration of the embodiment described in FlGS. 12 and 13, and with graph B showing the uniformity in the temperature transition of sample vial contents when transferred from liquid nitrogen to the temperature-equilibrated device described in FIGS. 12 and 13.
  • FlGS. 17A and 17B show a collapsible sleeve in an initial/open orientation and in an final/griping orientation, according to aspects of the disclosure.
  • the present disclosure describes various embodiments for the thermal control and/or thawing of organic samples, along with associated devices, systems, and methods. Certain details are set forth in the following description and in the Figures to provide a thorough understanding of various embodiments of the present technology. Other details describing known structures and systems often associated with heating and/or cooling processes, motors, etc., however, are not set forth below to avoid unnecessarily obscuring the description of the various embodiments of the present technology.
  • the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result.
  • an object that is “substantially” uniform in height to another object would mean that the objects are either completely or nearly completely uniform in height.
  • the exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context, however, generally speaking, the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained.
  • the term "about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be greater than or less than the indicated value.
  • the given value modified by about may be at or within ⁇ 5% from that value.
  • direct liquid contact with the sample vial exterior can be eliminated, as opposed to the partial submersion of the sample in a water bath.
  • the exterior surface of the sample vial (which can include the sample vial exterior plus laminations such as labels or shrink-wrap sleeves) will be in contact only with solid materials.
  • the solid material in contact with the vial exterior can be a homogenous solid, while in other cases, the solid material can be a complex material.
  • the solid material can have a thermal conductivity of greater than 0.2 Watts per meter-Kelvin (0.2 W/(m-K)).
  • the solid material can be formed from aluminum, copper, zinc, magnesium, titanium, iron, chromium, nickel, carbon, or alloys of the same elements.
  • the solid material can be formed from a synthetic material, such as a polymer or ceramic.
  • the solid material can be formed from a synthetic thermally-conductive pliable material such as a silicone polymer foam or putty.
  • the solid material can be formed from a combination of materials, for example and without limitation, a conductive pliable material and a metal alloy.
  • the solid material in contact with the sample vial exterior can be a polymer shell or tank, the shell or tank containing a liquid within the shell or tank volume.
  • such a polymer shell or tank holding a liquid can further include a thermally conductive lining of a pliable material that contacts the sample vial.
  • the liquid in the polymer shell can be water or aqueous solutions, while in other embodiments the liquid can be an oil or a liquid organic material.
  • the polymer shell can be filled with a wax that is in a liquid phase at some temperatures while in a solid phase at other
  • the sample vial or a portion of the sample vial is in continuous contact with the solid material on the circumference of the vial while in other embodiments, the solid material contacts the vial intermittently.
  • the solid material comprises a recess or cavity which closely matches the outer surface of the sample vial for the purpose of receiving the sample vial such that the sample vial is partially contained within the solid material in direct and close contact.
  • the container cavity comprises one or more sides and a floor, while in other embodiments, the container comprises only one or more sides such that a sample vial with a foot or base portion can fit within the container cavity.
  • the solid material containing the sample vial is segmented to facilitate the insertion and removal of the sample vial from the material and to interrupt the thermal conduction pathway between the solid material and the sample vial.
  • the solid material segments of the container are relationally constrained such that when separated to facilitate insertion or removal of the sample vial or to interrupt the thermal conduction between the solid material segments and the sample vial, the segments can be easily reassembled into a closely joined configuration.
  • the segments are joined by slide mechanisms, hinge mechanisms, track mechanisms, hydraulic or pneumatic pistons, rails, kinematic linkages, pin and groove linkages, electro-magnetic, or magnetic interface.
  • the segments are, without limitations, actively separated or joined by electric motors, solenoid activators, pneumatic or hydraulic actuators, linear actuators, either directly acting on the segments or by gear systems, kinematic linkages, cam systems, pushrods, cable system, and screw mechanisms.
  • the solid material container for the sample vial comprises one or more heater elements for the purpose of increasing the temperature of the solid material such that when a sample vial is placed into the receiving cavity, thermal energy will migrate into the sample vial from the solid material (alternatively referred as “heater blocks", “heating block”, or “warming blocks”).
  • the heater elements are electrically resistive heaters while in other warming blocks, the heater elements are thermoelectric element heaters.
  • the warming block may be alternatively heated and cooled by a thermoelectric element.
  • the warming block comprises one or more temperature sensors that can detect the temperature of the block and provide an analog or digital signal to a microcontroller that is configured to interpret the thermometric signal and thereby regulate the power level or duty cycle supplied to the heater element in order to maintain the temperature of the warming block at the desired temperature.
  • the heater block includes one or more temperature sensors that are thermally isolated from the heater block material, but are positioned to contact with the exterior surface of a sample vial received within the heater block, such that the temperature of the sample vial at the surface can be ascertained and tracked over time.
  • the vial sensor can be a thermocouple, a thermistor, an resistance temperature detector (RTD), or a non-contact infrared sensor.
  • cryogenic temperatures in the range of -150°C to -196°C is a commonly applied method for preserving live cell suspensions indefinitely. Within this temperature range, single cells and even multicellular organisms may be safely preserved, and later returned to a biologically active state by the application of proper thawing techniques. Subject to the damage imposed on cellular membranes and organelles by the formation of intracellular ice crystals, a transition through the frozen state would be catastrophic for most eukaryotic cell types. While some specialized organisms have evolved the means of preparative conditioning and are adapted to withstand a transition through a frozen state, mammalian cells are notably susceptible to freezing injury with cellular death being the universal outcome.
  • the thawing rate can directly influence the viability of cryogenically preserved cells.
  • a range of temperature roughly from -50°C to 0°C, wherein molecular movement is suppressed, but nevertheless extant, and within this temperature range, minute ice crystals may migrate and reform into larger molecular structures that have an increased potential for cellular damage. It is therefore desirable that the transit time through this temperature range be as brief as possible such that the opportunity for ice crystal reformation is minimized.
  • a typical target for the applied rate for the return of frozen cells to the thawed state is "as fast as possible".
  • the thaw rate is typically a prime consideration.
  • cylindrical vessels are commonly selected. Cylindrical storage vessels have multiple advantages over alternative storage vessel formats in that the cylindrical vessels are quite robust with regards to structural integrity over the full temperature range of cryogenic temperatures to environmental temperatures, and can withstand substantial mechanical load and shock.
  • the cylindrical format integrates well with a variety of filling options, both manual and automated.
  • thermal energy redistribution will commence that will eventually bring the combined masses to a common temperature. If the warming block temperature is actively maintained, for example at 45°C, then the temperature of the combined masses will in time equilibrate at a temperature of 45°C.
  • the thermal energy redistribution pattern may be considered to be a migration or flow of thermal energy in a radial partem toward the central axis of the combined mass.
  • Cylindrical storage vessels present some challenges in that the mechanical stability comes at a cost of material wall thickness, and being typically constructed from synthetic materials that have relatively poor thermal conductivity, present a resistive impediment to rapid thermal energy influx as is desirable for high thawing rates.
  • the thermal resistance of the vessel materials can be compensated for by increasing the temperature differential across the material, in other words by increasing the temperature of the heat source in contact with the vessel exterior, however there lies an additional source of resistance in the thermal pathway of such a system in the form of the heat source-vessel interface. Because a gap of any size, however small, introduces a thermal resistance to the pathway, the elimination or reduction of any gaps will improve the efficiency of the thermal energy transfer.
  • a common solution is to fill the gaps using a compliant or pliable material.
  • a thermal grease material is an effective solution, however for thawing instruments that will receive and subsequently disengage with sample vessels, this is not an acceptable solution.
  • a favored solution is to fill the gap with a compliant putty-like material, preferably one that has a high degree of shape recovery such that the material may be re-used indefinitely without the negative effects of material extrusion that requires the gap-filler to be frequently reset into position.
  • the materials for this application include synthetic sheet materials that may be adhesively bonded to the heat source surface in a manner such that the gap filler sheet will be interposed between the heat source and the vessel surface when the surfaces are mated. Even with the incorporation of gap filler materials into the heat-source-vessel interface, gaps will remain if some amount of force is not applied to induce the compliant material to completely fill the interface region. Without adequate application of force, the thermal conductivity of the heat source-vessel interface will not be optimized and therefore the rate of thermal energy transfer for a given temperature differential between the heat source and vessel will not be accurately calculated.
  • the application of force to the interface of a heat source and cylindrical sample vessel would be to introduce a cylindrical well into the heat source that has a diameter slightly larger than the vessel diameter, such that the interstitial space can be occupied by the gap-filling material and bisecting the heat source on a plane that is coincident with the axis of the cylinder.
  • the two parts With a small reduction of the self-mating faces of the bisected heat source, the two parts can be pressed together so that there is a force applied to the vessel interface.
  • Such a configuration would apply the greatest compressive force along a line where the direction of the force vector is normal to the vessel surface, and the least compressive force at the line where the force vector is nearly tangential to the surface of the vessel, at the location of the heat source bisection plane.
  • This configuration does provide for excellent heat transfer characteristics, as discussed in the Applicant's co-owned U. S. Patent Application No. 14/712, 120.
  • the heat source in order to reduce the area of the heat source-vessel interface where the force vector is near to tangential, the heat source can be further segmented into three or more parts.
  • the number of heat source segments increases, so does the complexity for articulation of the parts.
  • an optimal number of segments for several kinematic mechanisms may be heater blocks separated into to three or four parts.
  • Combinations of various actuation mechanisms, variation in vessel sizes, or other such alternative configurations of the assembly can provide for the advantageous use of two heater block segments, or five or more heater block segments.
  • thermal redistribution may not equilibrate the volume of fluid at a rate sufficient or ideal for maintaining the integrity of cells within the sample.
  • physical agitation can be applied to the sample vial via an internal module to ensure that the complete volume of sample is thawed and reaches a target temperature within a target time range.
  • the target time range for thawing any given sample can depend on the size of the sample vial, the starting temperature of the sample, the volume of sample fluid within the sample vial, and the characteristics of the contents within the sample vial (e.g., the type of cells, the amount of cryopreservatives added to the sample fluid, the density of cells within the volume of fluid, etc.).
  • the sample vial can be labeled with at barcode, a QR code, or the like, to identify the contents of the sample held within a sample vial.
  • the thawing system can include a visual sensor to read such codes and thereby identify the contents of the sample vial, with that data then being used to calculate a corresponding thawing time for the sample vial.
  • FIG. 1 illustrates an elevated view of the exterior of a sample thawing apparatus.
  • the instrument shell can be formed of a lower case 101, with an upper deck 102 that forms an inner horizontal surface 103 (alternatively referred to as the inner deck) in which a removable access ring 104 can be placed or received.
  • lower case 101 can be cast of a synthetic or polymer material.
  • the access ring 104 has a center hole 105 through which sample vials 106 can be inserted.
  • the upper deck 102 on the right side of the instrument is contiguous with the upper surface 107 surrounding a touchscreen panel 108.
  • the upper deck panel connection to the lower case 101 can be masked or covered by a rim piece 109 which can attached to the lower case by means of frictional or snapping features.
  • the rim piece 109 can be formed of a synthetic plastic, and can further have cosmetic features such as a specific color, texture, or other such ornamentation.
  • a lid piece covers the inner deck on the right side of the instrument, and is attached by a hinge 1 14 at the inner back wall of the instrument.
  • the lid piece 1 10 can be a clear panel 1 11 through which a user may view the action of the mechanism and progress of the sample vial thawing process.
  • the clear panel can be made of a synthetic material such as a polymer or clear plastic, or of glass or another clear material.
  • an LED light source can be placed in the in the vertical wall of the inner deck (not visible in this figure).
  • the lid piece 1 10 can be closed by a push-lock / push-release fastener 112 that captures an extension 1 13 fixed to the underside of the lid piece 110.
  • features of the instrument can be positioned on the opposite sides of the lower case 101.
  • the hinged lid, inner deck, and aperture for receiving a sample vial can be on the left side of the apparatus, while the touchscreen panel and upper deck can be on the right side of the apparatus.
  • the size of the center hole in the access ring for receiving a sample vial can have different diameters, in order to accommodate different sized vials.
  • the instrument can be formed without a touchscreen panel.
  • FlG. 2 illustrates a partial interior view of the apparatus shown in FlG. 1.
  • FlG. 2 shows the instrument the upper deck 107, lid piece 110 and clear panel 1 11 , and the lower case 101 removed.
  • the access ring 104 with its center hole 105 can be more clearly seen, as well as an exemplary sample vial 106 received within the center hole 105 and resting in the instrument.
  • light source 201 is shown, which in some embodiments is an LED.
  • FIG. 3 illustrates a partial front-plane cross-sectional view of the apparatus shown in FlG. 1 , with the inner horizontal surface 103, access ring 104, and touchscreen panel 108 removed from view to further expose the internal mechanisms and circuit boards that control and power the mechanics of the instrument.
  • This exemplary embodiment of the apparatus includes three separate circuit boards: a touch-screen circuit board 301 , a power circuit board 302, and a driver circuit board 303.
  • the power circuit board 302 is connected to a high voltage AC receptacle 304, and is likewise connected to both the touchscreen circuit board 301 and the driver circuit board 303 through a combination of power and data bus connection cords (not shown).
  • the driver circuit board 303 is attached to the underside of a heating, sensing, and mixing module which includes a base plate 305 that supports the entire module and is attached to four pylon projections (not seen in this view) that are cast and are contiguous with the lower case 101.
  • the base plate 305 supports an orbital module 306 through three offset crank shafts 307.
  • the orbital module 306 is moved relative to the base plate 305 by an orbit drive gear motor 308 that is connected to one of the offset crank shafts 307 through a flex drive 309.
  • the orbit drive gear motor 308 is attached to the base plate 305 by a mount bracket 310.
  • FIG. 4 illustrates a detail view of an isolated orbital module used within the sample thawing apparatus, particularly showing an exemplary version of the isolated orbital module 308 that includes a horizontal bearing race plate 401 and a vertical race plate 402 that are fastened through six (6) screw mounts 403.
  • the horizontal bearing race plate 401 and vertical race plate 402 capture the tractor assemblies 404 (described in detail below in relation to FIG. 5).
  • the tractor assemblies 404 articulate the three heater-block assemblies 405 along a path that is radial from and perpendicular to the axis of a sample vial 406, shown here as a cylindrical sample vial.
  • the vertical race plate 402 is formed in part from six (6) binding posts 407 that are arranged in a hexagonal partem around the axis of the cylindrical sample vial 406, with two (2) of the binding posts 407 flanking each of the three tractor assemblies 404.
  • Each tractor assembly 404 can include a single vertical binding post 408 that is connected to each of the flanking vertical race plate 402 binding posts 407 through a pair of coil extension springs 409.
  • the respective pairs of coil extension springs 409 provide an inward vector force on each tractor assembly 404 such that each of the three heater-block assemblies 405 apply an inward-vector force on the sample vial 406.
  • the three heater-block assemblies are configured to apply pressure onto the longitudinal exterior surface of a sample vial 406 to hold the sample vial securely within the instrument.
  • the shaft of each of the vertical binding posts 408 are surrounded by a synthetic-material flange bearing 410 that reduces friction between the binding post shaft and the vertical bearing races 411 in the vertical bearing race plate 402.
  • the orbital assembly is configured to rotate at a frequency from less than one (1) cycle per minute to one hundred (100) cycles per second, and at increments and gradients within that range.
  • the orbital assembly can be configured to rotate at about three (3) rotations per minute
  • FIG. 5 illustrates a further detailed view of the orbital assembly illustrated in
  • the internal articulation mechanism has a circular cam ring 501 that is captured by three (3) outer and two (2) internal flange bearings 502 that are centered shafts 503 that are held in position between the horizontal bearing race plate 401 and vertical bearing race plate 402.
  • the circular cam ring 501 is further formed from three (3) cam lobes 504 that interface with the vertical flange bearings 410.
  • the circular cam ring 501 is rotated relative to the horizontal bearing plate 402 through the action of an inside-tooth gear segment 505 that interfaces with a spur gear 506.
  • the spur gear 506 is mounted on the shaft of the gear motor assembly 507 so that when the gear motor is active, the spur gear 506 rotates the cam ring 501, thereby advancing or retracting the three tractor assemblies 404 relative to the cylindrical axis of the sample vial 406.
  • the tractor assemblies 404 are attached to the three heater-block assemblies 405, the action of the gear motor assembly 507 can advance and retract the heater-block assemblies 405 relative to the axis of the sample vial 406.
  • the curve of the cam lobes 504 is such that when the cam ring 501 is positioned at the limit of clockwise motion (as seen from above), the heater-block assemblies 405 are in full contact with the surface of the sample vial 406, and the flange bearing 410 surface is no longer in contact with the face of the cam lobe 504. Accordingly, the heater block assemblies are held in contact with the sample vial surface exclusively by the force provided by the coil springs 409.
  • the pressure of the heater block assemblies 405 on the surface of any sample vial 406 is constant, predictable, and adjustable by altering the force constant of the individual coil springs 409.
  • the three heater block assemblies 405, when located at their innermost position along an inward vector, can be in contact with each other and can function as a single heater block assembly.
  • the disengagement of the cam lobe 504 surface from the flange bearing 410 surface (which thereby allows the uniform coil spring force to be applied to the interface between the heater block assemblies 405 and the sample vial 406 surface) provides for the control of the rate of thermal energy transfer from the heater block assemblies 405 to the sample vial 406.
  • the precise control of the thermal energy transfer from the heater block assemblies 405 to the sample vial 406 resulting from the uniform force applied to the interface of the heater block assemblies 405 with the sample vial 406 surface allows the rate of thermal energy transfer, and therefore the thawing end-point time, to be a calculable value.
  • the release of the sample vial 406 from the heater block assemblies 405 can be achieved by rotating the cam ring 501 counter-clockwise relative to the horizontal bearing race plate 401 (as observed from above), during which motion the cam lobes 504 re-engage with the flange bearings 410 and retract the tractor assemblies 404 and heater block assemblies 405 in an outward vector from the axis of the sample vial 406. Once the heater block assemblies 405 (now again separated in three segments) have disengaged from the sample vial 406, the sample vial 406 may be removed. FiG.
  • FIG. 5 additionally shows the temperature sensor retraction solenoid 508, and the solenoid mounting bracket 509, which connects the sensor retraction solenoid 508 to the horizontal bearing race plate 401.
  • FlG. 6 illustrates the underside of the orbital assembly as shown in FlG. 5, with the solenoid mounting bracket 509 removed, allowing a better view of the heater block assemblies 405.
  • Each of the three heater block assemblies 405 include an outer insulation jacket 601 that, in some aspects, is constructed from a synthetic material and is connected to an inner heater block 602 that is the inner portion of the heater block assembly 405, and is typically constructed from a thermally conductive material in the range of 10 to 300 Watts per meter-Kelvin (0.2 W/(m-K) by means of multiple screw fasteners 603.
  • Each insulation jacket 601 can have a flange feature 604 by which the respective heater block is fastened to the corresponding tractor assembly 404, being mechanically coupled via a single pin 605.
  • the inner heater block 602 in some embodiments, can be constructed from a material with a high thermal conductivity, such as aluminum, copper, and the like, or alloys thereof. Attached to the underside of each heater block is a temperature sensor screw tab 608 by which the temperature of the heater block can be monitored.
  • Each heater block 602 comprises an access boring 609 into which is inserted and fastened a resistance cartridge heater (not visible) each of which is connected to a regulated power source by two wire conductors 610.
  • FlG. 6 further shows the sample vial underside temperature sensor mount 61 1 that is attached to the shaft 612 of the solenoid actuator 508.
  • the sensor mount 611 is terminated on the upper side by a thin layer 613 of highly conductive material, such as copper foil, to which is bonded a temperature sensor such as a thermocouple, a thermistor sensor, or an RTD sensor.
  • a temperature sensor such as a thermocouple, a thermistor sensor, or an RTD sensor.
  • a layer of insulating material 614 Interposed between the conductive layer and the sensor mount 611 is a layer of insulating material 614, positioned such so that the temperature sensor thermally couples with the sample vial 406 and not the sensor mount 611.
  • the sensor mount assembly is pressed to the underside of the sample vial by an upward force applied by the spring element 615.
  • the solenoid shaft 612 and sensor mount 61 1 are retracted from the under-surface of the sample vial.
  • the sensor assembly and solenoid shaft 612 are held in a retracted state until a sample vial is inserted.
  • the solenoid is de-energized, the sensor assembly is pressed against the underside of the vial by the spring force of spring element 615, thereby assuring optimal contact and thermal coupling of the sensor with the vial undersurface.
  • FlG. 6 also provides a better view of the mounting of the tractor assemblies
  • Each tractor assembly 404 is formed with three (3) pairs of laterally opposed bearings 616, two (2) pairs that interact with the upper surface of the horizontal bearing race plate 401, and one (1) pair that interacts with the underside of the horizontal bearing race plate 401.
  • the laterally opposed bearings 616 are shown as flange sleeve bearings that guide the tractor in the bearing race slot 617. Also visible in FlG. 6 are flange bearings 618 by which the offset crank shafts 307 are mounted to the base plate 305.
  • the offset crank shaft 307 attached to the flex drive 309 shaft is rotated, and by inducing a similar rotation in the remaining two offset crank shafts 307, thereby inducing a horizontal circular motion in the entire orbital assembly.
  • the orbital motion will introduce a mixing oscillation in the vessel payload, thereby increasing thermal convection and reducing thermal gradients within the sample fluid, having a net effect of (1) decreasing the overall thaw time and (2) suppression of regions of higher temperature that may, in some situations, be deleterious to cell viability.
  • the fluid mixing is also helpful in homogenizing and diluting regions of concentrated salt solution that are imposed by the freezing process.
  • FIG. 7 illustrates is a cross-sectional view of the orbital module assembly.
  • the cross-section reveals the heater cartridge 701 mounted inside the inner heater block 602 and held in place by a set screw 702.
  • the tractor assembly 404 which comprises the three bearing shafts 703 on which the three bearing pairs 616 of the assembly are mounted.
  • Further aspects of the overall sample thawing apparatus can include a communications module, formed of a non-transitory computer-readable medium, and configured to transmit to other devices sample data, including thermal data about a sample vial held by the sample thawing apparatus.
  • the communications module can be directly coupled with temperature sensors of the sample thawing apparatus.
  • the communications module can be further configured to communicate with a remote microprocessor (e.g. a cloud-based server or computer) in order to sort and display data.
  • the communications module is also configured to receive instruction data or sample vial identification data, and to control the heating of a sample vial accordingly.
  • FIGS. 8A-8D illustrate an exemplary three-part heater block (alternatively referred to as a three-jaw or three-segment heater/heating block) in further detail.
  • FIG. 8 A shows the module of the three-part heater block assembly, and particularly shows a counterweight that is arranged to be 180° out of phase with the orbital platform holding the heater jaws. The counterweight can mitigate excess vibration of the overall apparatus during phases of operation when the sample vial is being agitated or oscillating.
  • FIGS. 8A-8C all show the three-part heater blocks in an inward position (which can be alternatively referred to as an operating position or a contact position), such that the heater block segments effectively form a single heater block and vessel receptacle to contact and conduct heat into a sample vial.
  • FIGS. 8B and 8C illustrate the module with certain plates removed from view to better see specific components of the three-part heater block assembly, such as the bearings and tractor assemblies.
  • FIG. 8D shows the three-part heater blocks in an outward position (which can be alternatively referred to as an initial position or a non-contact position), which gives an indication of the distance the heater block segments can move along their respective vectors toward and away from a normal centerline of the module. With the three-part heater blocks in their outward positions, a sample vial can be easily (without disadvantageous friction) inserted into or removed from the apparatus.
  • the three-part heater block configuration has the three heat source segments articulated by a radially-arranged tractor bearing system.
  • a circular cam is arranged to move the heater block segments in a centrifugal direction (in other words, outwardly from the longitudinal axis of a sample vial held by the heater block, or away from a centerline in the vessel receiving space equidistant from the inner surfaces of the heater block segments and perpendicular to the direction of movement for all of the heater block segments), while a spring force is applied to move the heater block segments in the opposite direction.
  • the cam surfaces are structured such that when the cam is rotated in one direction, the springs forces will bring the heater source inner surfaces (alternatively referred to as "well surfaces") into contact with the vessel surface (e.g. a sample vial). Further, it can be understood that all three of the heater block segments move in concert with each other as based on the motion of the circular cam, moving inward or outward at the same rate and at the same relative distances from the sample vial or centerline of the module.
  • the cam surface lifts away from the cam follower thereby allowing the springs to apply their respective full forces to the interface of the heater blocks and the sample vessel.
  • the spring force is a function of the spring constant and the extension of the spring at the position where contact made by the heater well wall with the vessel surface, a known and repeatable amount of force per unit area of the vessel can be generated.
  • the rate of heat transfer can be accurately controlled by the regulation of the temperature differential between the heat source and the vessel temperature.
  • the desired end-point of a thawing process can be accurately calculated and predicted if the mass, the heat capacity, and the starting temperature of the vessel and contents are known. As such, the correct termination of a thawing process may be regulated even without the application of thermal metrology to the vessel contents.
  • a radially-arranged tractor bearing system as discussed herein can be implemented with a two-part heater block configuration, a three-part heater block configuration, a four-part heater block configuration, a five-part heater block configuration, or heater block arrangements with six or more segments.
  • a subset of heater blocks can be used to contact a vessel received within the a radially-arranged tractor bearing system.
  • the heater blocks can be configured to move independently from each other, or sequentially, between inward and outward positions.
  • the force applied by the heater block segments is calibrated to securely hold a sample vessel without damaging or crushing the vessel. Accordingly, in combination or individually, the force applied by each spring of the system can be about 15 Newtons.
  • the a standard warming block is shown.
  • the warming block is segmented by three right-angle planar interfaces to create independent block segments.
  • the vertical segmentation planes are aligned with the centerline of a cylindrical sample vial receiving well coincident with the cylindrical axis of the receiving well.
  • the receiving well can have a taper that is configured to match the taper of a standard screw-capped cryovial such as those available commercially from multiple vendors and being formed of a material.
  • cryovials can be made of materials having a thermal conductivity of less than 0.2 Watts per meter-Kelvin (0.2 W/(m-K)), for example but not limited to, polypropylene, polyethylene, or blends of polypropylene, polyethylene and additional plastic materials, plastic resins and resin blends, and glass.
  • the receiving well walls form a 0.5 mm-thick layer of thermally conductive material such as, but not limited to, thermally conductive foam, with the innermost surface matched to the surface of the standard screw-capped cryovial such that when the cryovial is placed into the well and the two receiver blocks close to near contact at the surface, the cryovial surface and the conductive foam are in close and complete contact at all points.
  • the movable sliding segments can have a linear horizontal motion driven by a tractor assembly.
  • the tractor assembly can include two push-rods that are secured at the end by a push-bar. By horizontal actuation of such a push-bar, the segments of the heater block may be separated to allow insertion or removal of the cryovial.
  • the thawing apparatus does not require a thermally conductive compliant materal, although because precisely and closely matching the taper of the cryovial is a difficult achievement, particularly when considering manufacturing variability in the angle of the taper and the diameter of the vial.
  • the aqueous contents of the sample vial will expand with the potential to distort the exterior surface of the cryovial. Further complications may arise in mating the receiver well surface to the vial exterior surface in that the vials may be unpredictably laminated with an identification label. Therefore a compliant surface in the receiver well is helpful to ensure uniform, complete, and repeatable contact of the heater block and sample vial surfaces, as any disruption in the physical contact will alter the thermal transfer by imposing additional thermal resistance at the location of the disruption.
  • the warming block can be heated by an electric resistance heater that is embedded in the undersurface and powered by an electrical current.
  • the temperature of the warming block may be determined by a thermocouple sensor that is inserted into the warming block segment.
  • the block segments can be further joined by embedded magnet pairs both on the vertical interface and the horizontal interface, thereby assuring close thermal conductive contact of the two adjacent segments in addition to supplying clamping pressure to the inserted vial.
  • the thermally conductive foam lining can be constructed from a thermally- conductive silicone composition, or other such comparable materials.
  • FlGS. 9A-9C illustrate views of an exemplary four-part heater block assembly in further detail.
  • the four-part heater block configuration has the four heat source segments articulated on individual hinges mounted to a surrounding frame.
  • the four respective hinges are connected to a vessel support plate, such that when a sample vial or other such vessel is placed within the module and presses downward on the vessel support plate, the four hinges all move in concert with each other, and the four heater blocks all tilt and shift from an open position (alternatively referred to as an outward or receive/release position) to a closed position (alternatively referred to as an inward or contact position).
  • all four of the heater block segments move in concert with each other as based on the position of the vessel support plate, tilting angles and shifting inward or outward at the same rate and at the same relative distances from the sample vial or centerline of the module. Coordination and axial orientation and axial travel of the hinged elements are controlled by an underlying Sarrus linkage system controlling the sample vial stage into which a second pivot axis for each heater segment is embedded.
  • the inclusion of spring elements in the upper hinge assemblies can be configured such that when in the closed position, an inward force will be applied to the heater blocks that contact the vessel such that the heater-block / vessel interface is under compression.
  • a hinge articulating heater block and support plate system as discussed herein can be implemented with a two-part heater block configuration, a three-part heater block configuration, a four-part heater block configuration, a five-part heater block configuration, or heater block arrangements with six or more segments.
  • a subset of heater blocks can be used to contact a vessel received within the a hinge articulating system.
  • the heater blocks can be configured to tilt and shift independently from each other, or sequentially, between inward and outward positions.
  • a thawing apparatus configured to hold a sample vial using either a radially-arranged tractor bearing system or a hinge articulating heater block and support plate system can be adapted to have a variable diameter. Because such three-jaw or four-jaw heater blocks can change in diameter in order to receive and release sample vials, the distance that each of the sets of heater blocks can move or adjust can be selected or configured to adjust for different-sized vessels. Accordingly, a thawing apparatus can be used for multiple sample vial types, with multiple access rings having correspondingly sized center holes to accommodate the sample vials.
  • a cylindrical container 1020 forms a central cavity in which a lining of thermally conductive pliable material 1025 surrounds the vertical wall of the cavity, except where an opening allows one or more side wall sensors 1030 to protrude into the cavity.
  • the cylindrical container can be formed of various metals or alloys, such as aluminum or alloys thereof.
  • a side wall sensor 1030 can be a thermistor temperature sensor, an infrared sensor, a pressure sensor, a humidity sensor, or combinations thereof.
  • the cavity is occupied by a sample vial tube 1010 that is sealed with a screw cap 1015, the combination of which isolates the sample contents of a liquid phase 1040 and a solid phase 1035.
  • the sample vial tube 1010 further has a foot structure 101 1 which has an exterior surface that can also be used to sense characteristics of the fluid held in the volume.
  • a floor sensor 1032 can be positioned underneath the sample vial tube 1010 extending through, or between segments of, the cylindrical container 1020.
  • a floor sensor 1032 can be a thermistor temperature sensor, an infrared sensor, a pressure sensor, a humidity sensor, or combinations thereof.
  • the vial interior also contains a gas phase volume 1045.
  • the floor sensor 1032 shown in FiG. 1 OA is an infrared sensor proximate to the exterior bottom surface of the sample vial 1010 foot structure 1011.
  • the cylindrical container 1020 can have and use both side wall sensors 1030 and floor sensors 1032. Segmentation lines 150 section the cylindrical container 1020 and the thermally conductive pliable material 1025.
  • the aggregate of the components shown in FlGS. 10A and 10B can be considered collectively to represent a system in reference to the thermodynamic illustrations herein.
  • the boundaries can include: a region formed of the cylindrical container 1020 material (e.g. an aluminum alloy); a region formed of thermally conductive pliable material; a region formed of sample vial material such as polypropylene, polyethylene, or blends of polypropylene, polyethylene and additional plastic materials; a region of a liquid sample phase; and a region of solid sample phase.
  • a model simulating the temperature transitions will show both liquid and solid phases, where the state of the sample shown is during the melting or phase transition process.
  • the amount of thermal energy required to raise the temperature of a sample vial and the contents from one temperature to a second temperature is dependent only on the heat capacity of the sample vial and the sample mass contained therein. Therefore if the material masses and the hence the amount of heat required to achieve the temperature transition do not change, and the start temperature of the warmer block and the start temperature of the sample vial are consistent, the same time temperature profile may be expected upon repeated freeze-thaw cycles of the same sample. If the sample vial dimensions, vial materials and mass, and sample payload mass and composition are uniform from sample to sample, the time-temperature profiles obtained should be identical regardless of whether the same sample is repeatedly cycled through a freeze-thaw or another sample is subjected to the same process. Therefore, the inclusion of a step or device for equilibrating all samples to a uniform starting temperature, and an accurate and uniform warming block temperature will allow the prediction of the thawing process duration to be made exclusively on the basis of prior experience.
  • Cylindrical vessels while structurally favorable, present a challenge that lies in the surface area to volume ratio.
  • the surface area increases linearly with the length, however the volume also increases by the square of half the diametric dimension, and as a result an increase in the diameter of the vessel not only reduces the surface to volume ratio, but increases the maximum path length that thermal energy must travel to reach the axis of the internal frozen payload of the vessel.
  • the reduced surface area relative to the vessel volume, and the increased internal thermal energy pathway may be countered by an increased temperature differential between the vessel and the heat source and an increased interval of thermal energy transfer. Increasing the heat source temperature, however will increase the temperature gradient between the vessel interior wall and the core of the frozen mass.
  • thermal energy will transfer at a greater rate through solid material as opposed to the liquid phase of that material, as the sample begins to melt, the thermal gradient becomes even greater, thereby increasing the potential for a decrease in viability for cells located at regions of elevated temperature.
  • a gentle mixing of the contents will be beneficial by both suppressing regions of elevated temperature and while at the same time increasing the rate of thermal energy transfer between the vessel wall and the remainder of the solid payload.
  • FlG. 11 shows a cross-section diagram of two sample vials illustrating the load-volume independence of a thawing vial temperature trace for a cylindrical sample vial.
  • vial A where the contents of the vial is greater, an identical thermal conduction pathway exists at the position of both of the arrows in that the inner wall of the vial is in direct contact with the sample.
  • vial B the sample volume is reduced and therefore at the position of the upper arrow, the interior wall of the vial does not contact the sample, therefore thermal energy entering the vial at this location must either migrate downward through the thermally resistant polymer of the vial wall or migrate through the gas above the vial which has a thermal conductivity approximately one-tenth that of the polymer vessel wall. Therefore, the amount of heat entering the sample contained within the vial is proportional to the amount of sample in the vial.
  • FIG. 12 shows a device is shown that may be used to equilibrate sample vials to a reference temperature (or intermediate temperature).
  • a sample vial receiver forms a rectangular upper block of solid material 1215 and a horizontal flange 1230 that is mated to upper block forming a sample receiver block.
  • the upper block 1215 forms one or more recesses 1220 that are sufficient diameter and depth to receive and surround the sample vial such that the top of the sample contained within the vial is below the top surface of the block.
  • the receiver block forms one or more recesses on the sidewall of the upper block 1240 to assist in the grip security of the invention.
  • the flange 1230 and the upper block 1215 interface as an uninterrupted continuum of the material from which the parts are made, while in other embodiment the upper block 1215 and the flange 1230 are separate pieces that are joined (for example and without limitation), by mechanical fasteners, adhesive bonds, magnetic fasteners, or weldments.
  • the upper block forms a hole (not shown) into which a thermometric sensor may be inserted and secured.
  • the sample receiver block is constructed from a metal.
  • the metal forms aluminum, copper, magnesium, zinc, titanium, iron, chromium, nickel or alloys of these metal elements.
  • the receiver block is surrounded on the sides and bottom by an insulating container 1210 that has a cavity 1245 with an interior height that is greater than the height of the receiver block plus one inch (1 in.).
  • the insulating container forms an insulating foam material.
  • the insulating foam material forms polyethylene, polyurethane, or polystyrene, while in other embodiments, the insulating material forms a blend of materials such as a polyethylene polymer blend.
  • the insulating container forms a cover (not shown). The receiver block is positioned in the insulating container such that as layer of solid carbon dioxide or dry ice 1225 is positioned under and above the lateral surface of the flange 1230.
  • the receiver block forms a solid material has a thermal conductivity greater than 16 W/(m-K) such as aluminum alloy.
  • the receiver block shown in FIG. 12 will maintain a steady temperature of -77°C in an open-top configuration.
  • the amount of dry ice beneath the receiver block may be limited such that the entirety of a sample vial placed into a receiver well will be positioned below the top surface of the insulating container, thereby holding the sample in a well of cold gas, and insulating the upper portion of the vial from the environmental temperature.
  • vial temperatures as measured with an internal thermocouple can be equilibrated to and held at a reference temperature of -77°C.
  • the reference temperature device shown in FIG. 12 can be used to provide a standard starting temperature for the sample thawing process that will allow the prediction of the thaw process status based exclusively on the duration of the thaw process.
  • the embodiment has an outside width of approximately 7 inches, a width of approximately 5.5 inches and a depth of approximately 3.5 inches.
  • the internal cavity has a length of approximately 5.25 inches, a width of approximately 3.6 inches, and a depth of
  • the receiver block has a length of approximately 5 inches, a width of approximately 3.4 inches and a height of approximately 1.35 inches.
  • the sample vial receiver wells of the receiver block have a diameter of approximately 0.55 inches and a depth of approximately 1.1 inches.
  • the wells of the receiver block form a passage in the floor of the wells that extends to the undersurface of the receiver block so that the receiver block may be used efficiently with a liquid refrigerant such as liquid nitrogen.
  • the passage way has a diameter of approximately 0.2 inches.
  • the diameter and depth of the wells may be adjusted to provide a go/no-go gauge for the sample vial by which a user may determine if the vial intended for the thawing process is too large for the thawing apparatus or is too small to be used properly with the same.
  • a second embodiment of a temperature equilibration device is shown.
  • a circular receiver block 1430 is shown comprising a radial distribution of sample vial receiver wells 1440.
  • the receiver block may form a central well 1450 that may be provide an additional vial receiver well, be used as a through hole by which to assess the presence of dry ice beneath the receiver block, or provide a gauge for the purpose of confirming the appropriate vial dimensions that are compatible with the thawing device.
  • the receiver block 1430 is situated within the internal cavity of an insulating container 1410.
  • the receiver block 1430 forms a hole into which a temperature sensor may be inserted and secured (not shown).
  • the insulating container 1410 forms internal extensions of the cavity wall 1420 that support or limit the movement of the receiver block 1430, while in other embodiments, the internal wall of the insulation housing 1410 is a cylindrical shape without extensions.
  • the receiver block and the insulating container form the same materials described for the embodiments presented in FlGS. 2 and 3.
  • the receiver block 1430 forms a disc-shaped flange attached at the bottom surface of the receiver block (not shown) while in other embodiments the receiver block forms the upper block only.
  • the insulating container has an outside diameter of approximately 5.5 inches and a height of approximately 3.5 inches with an internal cavity diameter of approximately 2 inches and a depth of approximately 2.8 inches.
  • the receiver block has an outside diameter of approximately 2.5 inches and a height of approximately 1.4 inches.
  • the vial receiver wells of the receiver block have a diameter of approximately 0.51 inches and a depth of
  • the central cavity has a diameter of approximately 0.7 inches and in the embodiment shown extends through to the undersurface of the block.
  • the vial receiver wells of the receiver block form a passage 1470 extending through the floor of the well to allow flooding of the well when the receiver rack is used with a liquid refrigerant such as liquid nitrogen.
  • the passage has a diameter of approximately 0.2 inches.
  • the sample vial receiver well floor is solid and does not form a passageway.
  • FlG. 16 shows two graphs of temperature tracking.
  • Graph A in FlG. 16 is a data graph of the temperature of a receiver block as described in FIGS. 2 and 3.
  • the temperature measurements were collected by a thermocouple sensor that was positioned into a receiver hole drilled into the vial receiver block to a depth of 0.5 inches.
  • the receiver block was placed upon an approximately 0.75 inch thick layer of dry ice, and additional dry ice was placed over the flange portion of the receiver block to a level equal to the top of the receiver.
  • the receiver was allowed to temperature equilibrate. As seen in the graph, the receiver block reached a temperature of -77°C in approximately 5 minutes and held the temperature for over seven (7) hours until the dry ice was exhausted.
  • a sample vial containing 90% buffered saline and 10% dimethyl sulfoxide in a volume of 1 ml was configured with a thermocouple temperature sensor held in an axial orientation with the thermocouple sensor positioned mid-height in the sample liquid.
  • the sample vial was then equilibrated to a temperature of -194°C in liquid nitrogen, then transferred to the -77°C equilibration block.
  • Graph B in FIG. 16 shows the temperature of the vial contents equilibrated to the -77°C temperature within an interval of approximately ten minutes (10 min.). Following repeated cycles of thawing, re-equilibration in liquid nitrogen and transfer to the -77°C receiver block, the temperature profiles of the sample contents are highly repeatable.
  • a sample stored at cryogenic temperature may be retrieved from archival storage and be rapidly equilibrated to a steady temperature of -77°C. The sample may then be stored for an extended period of up to seven (7) hours or longer if the dry ice refrigerant is replenished.
  • the receiver block at -77°C provides a highly reproducible temperature start point for a thawing process, allowing the thaw time of a sample to be accurately predicted following placement into a warming block that has been equilibrated to the appropriate temperature.
  • the receiver block prevents direct contact of the sample with the dry ice refrigerant.
  • some vial designs form a skirt extension on the undersurface, direct insertion of these vial into dry ice will capture dry ice in the underside recesses and if subsequently inserted into a warming block will experience a significant change in the thaw time due to the additional heat influx required to change the dry ice to the gas phase. Therefore the use of a receiver block that isolates the sample vials from direct contact with the dry ice is preferable for the standardization of the thawing process.
  • FlG. 17A shows a collapsible sleeve in an initial/open orientation, with a generally circular perimeter interrupted with foldable wings positioned around the circumference of the sleeve cylinder.
  • FIG. 17B shows the collapsible sleeve in an final/griping orientation, where the generally circular perimeter is relatively smaller than when in the initial orientation, and where the foldable wings have been folded, thereby extending out ward further, but allowing the arc portions of the cylindrical shape to move closer together.
  • the collapsible sleeve is configured to receive a sample vial and to thereby provide for a layer of protection to the sample thawing apparatus.
  • the sample thawing apparatus is protected from such flaws affecting the instrument because the collapsible sleeve is present to contain such leaks or disadvantageous material.
  • the collapsible sleeve Similar to the multi-part blocks, the collapsible sleeve has an initial position in which it is easy to insert or remove a sample vial, but can reduce in diameter in order for the heater block to come into contact with the sample vial; this is shown by the vector lines in FIG. 17B.
  • the collapsible sleeves are disposable, generally intended for one use only, in order to avoid cross contamination between samples.
  • the collapsible sleeve can be made from any one of a number of materials, the only requirements being that a temperature sensor can still accurately read the temperature of the vessel receiver and sample vial through the material of the collapsible sleeve.
  • materials can be polymers having a specific hardness and transparency.
  • the collapsible sleeve can also have an aperture in its bottom surface, to allow for certain sensors to directly contact the base of a sample vial held by the sleeve.
  • thermally pliant conductive material linings are laminated onto the inside walls of the three segments of the heater block that thereby form the vial receiver well.
  • the block segments can be warmed by a resistance heater that is embedded into the underside of the joined heater block in a cavity, and the heater element is held in close thermal contact with the cavity walls by pressure from a segment, the pressure on which may be adjusted by a force of a screw impinging on the back side of the wedge.
  • the heater is powered by an electrical current that is conducted through power wires.
  • the temperature of the warming block may be monitored by a thermocouple sensor inserted into the warming block at the sensor receiver hole.
  • the multi-part heater blocks considered herein can be articulated and automated by active propulsion machinery including (but not limited to): motors, solenoid actuators, hydraulic and pneumatic actuators, and electromagnets.
  • the propulsion machinery may be linked to the block segments by hardware including (but not limited to): screw machines, kinematic linkages, hinges, cables, belts, chains, pin and slot, tracks, rails, slide bearings, linear bearings, rotational bearings, cams, gears, and combinations thereof.
  • the system may include more than one temperature sensors by which the temperature of the warming block may be monitored.
  • the heater block allows for one or more temperature sensors that are thermally isolated from the heater block, but are in contact with the surface of the sample vial.
  • the temperature sensors can be thermocouples, thermistors, or RTD sensors.
  • the warming block can also include a microprocessor circuit board which receives warming block temperature feedback signals from the block sensors, and which regulates the power supplied to the heaters to maintain the desired block temperature.
  • the microprocessor board can receive position sensor data from proximity sensors on the warming block to determine when the block is open or closed. Further, the microprocessor board actively opens and closes the heater block according to a state algorithm that conducts the thawing process.
  • the microprocessor can also be configured to receive thermometric signal data from sensors in contact with the surface of the sample vial received into the warming block, infrared signal data from sensors proximate to the surface of the sample vial, or combinations thereof.
  • the microprocessor makes determinations of the thawing status of the vial by algorithmic interpretation of the thermometric data.
  • the microprocessor board controls a user interface that displays the status of the thawing process, alerts users to fault conditions, and signals the readiness of the warming block to receive a sample vial and initiate the thawing sequence.
  • the size of the magnet pairs and/or the field strength of the magnet pairs may be used to adjust the clamping pressure of the conductive pliant material linings on the vessel, thereby changing the thermal conduction between linings and the vessel.
  • the clamping pressure is provided by, without limitations, magnetic, electromagnetic, spring, pneumatic, hydraulic, or mechanical force, or any combination thereof.
  • the time-temperature traces collected using an intra-vial temperature sensor or an exterior surface temperature detector may be divided into three regions.
  • the first region coincides with the time interval in which the contents of the vial are in the solid phase
  • the second coincides with the time interval where the vial contents are mixed solid and liquid phase
  • the third time interval coincides with the region where the vial contents are liquid phase exclusively.
  • the first and third regions where the vial contents are one of two homogenous phases, the combined mass of the vial and the vial contents behave as a lumped capacity system, and the temperature transition behavior may be described by a linear time invariant equation:
  • T(t) the temperature of the system at time t
  • Th the bath temperature
  • T c the starting temperature of the vial
  • tpc the time offset (required to mathematically match the calculated values to the actual data plot)
  • x v the effective thermal time constant of the vile and contents.
  • the formula describes the warming of a mass subject to a fixed temperature at its outer boundary.
  • the warming of the solid phase content of the vial between the point where the sensor reading reaches a minimum at approximately 11 seconds after the insertion of the vial into the warming block until a time of approximately 60 seconds can be closely approximated using the Equation 1 above where the value of Th is the warm block temperature (39°C), T c is the temperature selected at the beginning of the solid phase warming curve at a time after the vial surface sensor and the vial have reached thermal equilibrium, and at the time point where the value of the effective thermal time constant reaches a minimum value (23.8°C), at 31 seconds past the time of insertion of the vial into the warm block.
  • the value of the time constant x v may be calculated from the value of Th and the values of T(t) by the following derivation:
  • the denominator of equation 4 may be obtained.
  • the numerator of equation 4 may be obtained.
  • the x v value may then be obtained by taking the negative value of the division result.
  • the x v value obtained by this treatment of the data will only relate to the linear time- invariant equation that describes the lumped capacity system of the vial and the solid sample during the portion of the curve prior to the commencement of the phase change, and therefore a deviation from the a constant x v value in excess of a pre-set limit may be used to identify the beginning of the phase change.
  • a data processing algorithm is embedded into the software of the instant invention to determine the beginning of the phase change of the contents of a vial.
  • the time of the phase change completion may be estimated.
  • the duration of the thaw (Tthaw) may be calculated where AH f is the specific heat of fusion of the sample, m so in is the mass of the sample, R v is the absolute thermal resistance of the sample vial wall, T v iai is the temperature of the vial exterior wall, and T m is the melting temperature of the sample.
  • the termination of the phase change is determined by a software algorithm that combines the time value calculated for the start of the phase change as described above with an experimentally derived phase change duration value for a given sample vial to determine the phase change completion time.
  • the reduction of the determined phase change completion time value by a constant will be used to terminate the thawing process while some solid phase is still remaining in the vial.
  • multiple algorithms may be provided for determining a thaw end time.
  • each of the multiple algorithms may be concurrently run to provide separate estimates for the thaw end time.
  • the system may be configured to end the thawing based on the algorithm which first provides an estimated thaw end time.
  • the system may be configured to allow each of the algorithms to complete their estimations and may utilize the shortest thawing interval calculated.
  • a system may be configured to average the estimated thawing intervals and utilize the averaged thawing interval to determine the thaw end time.
  • One or more computing devices may be adapted to provide desired functionality by accessing software instructions rendered in a computer-readable form.
  • software any suitable programming, scripting, or other type of language or combinations of languages may be used to implement the teachings contained herein.
  • Embodiments of the methods disclosed herein may be executed by one or more suitable computing devices.
  • Such system(s) may comprise one or more computing devices adapted to perform one or more embodiments of the methods disclosed herein.
  • such devices may access one or more computer -readable media that embody computer-readable instructions which, when executed by at least one computer, cause the at least one computer to implement one or more embodiments of the methods of the present subject matter.
  • the computing device(s) may comprise circuitry that renders the device(s) operative to implement one or more of the methods of the present subject matter.
  • Any suitable computer-readable medium or media may be used to implement or practice the presently-disclosed subject matter, including but not limited to, diskettes, drives, and other magnetic-based storage media, optical storage media, including disks (e.g., CD-ROMS, DVD-ROMS, variants thereof, etc.), flash, RAM, ROM, and other memory devices, and the like. It can be understood that, as used herein, a "microprocessor" includes a suitable computer-readable medium.

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Abstract

La présente invention concerne des dispositifs, des systèmes et des procédés de décongélation d'échantillon, conçus de façon à prendre des flacons d'échantillonnage individuels de 10 mL à 50 mL et à décongeler le contenu desdits flacons. Les contenus de tels flacons de relativement gros calibre sont des échantillons comprenant des cellules, et la décongélation de tels échantillons est basée sur la température, le volume et la masse de l'échantillon de manière à répartir uniformément la chaleur et éviter des dégâts de cristallisation de la glace sur les cellules pendant la décongélation. Un bloc chauffant en plusieurs parties revêtu d'un matériau souple thermoconducteur peut être moulé afin de prendre la forme d'un flacon de manière à s'apparier de manière ajustée aux tailles de flacons connues. Le bloc chauffant peut se séparer en trois ou quatre segments, ce qui permet d'insérer et de retirer facilement des échantillons. Les segments de bloc chauffant peuvent être déplacés linéairement le long d'un dispositif de traction, ou s'incliner le long d'une charnière, de manière à se déplacer entre des positions vers l'intérieur et vers l'extérieur.
PCT/US2018/028012 2017-04-17 2018-04-17 Systèmes, dispositifs et procédés de décongélation automatique d'échantillon de gros calibre WO2018195107A1 (fr)

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CN114740921A (zh) * 2022-04-13 2022-07-12 上海冠赛生物科技有限公司 一种用于生物样品的解冻控制***及其控制方法

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