Systems, methods, and devices for cryopreservation and recovery of cells and other biological materials

Abstract

Presented are systems, methods, and devices for handling biological samples during cryopreservation and recovery. A method includes loading samples into sample holders, soaking samples in solutions to prepare for cryocooling, removing excess liquid from the sample and holder, imaging the sample, cooling the sample to cryogenic temperature, imaging and storing the sample at cryogenic temperature, retrieving and warming a cryogenically cooled sample, soaking and incubating a warmed sample, and then removing the sample of its holder. An automated system may use a robot to perform sample motions between and within a series of modules or stations to perform each of the foregoing steps. The sample may be held within and enclosed by a porous sample holder that allows high-speed fluid motion past and around the sample while it is held within the holder when the sample holder is translated through a fluid, while containing the sample within the holder.

Description

SYSTEMS, METHODS, AND DEVICES FOR CRYOPRESERVATION ANDRECOVERY OF CELLS AND OTHER BIOLOGICAL MATERIALSCLAIM OF PRIORITY AND CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 573,809, which was filed on April 3, 2024, and U.S. Provisional Patent Application No. 63 / 601,937, which was filed on November 22, 2023.TECHNICAL FIELD

[0001] This disclosure relates generally to the field of biotechnology. More particularly, aspects of this disclosure relate to automated systems and methods for the handling and processing of samples, including cryopreservation and recovery of millimeter and smaller biological samples and for sample processing steps before cry opreservation and after recovery.BACKGROUND

[0002] A wide variety of small biological samples are cryopreserved for storage and later use. Eggs, embryos, blastocysts, and sperm of humans and many other domestic and wild animals, for example, are routinely frozen, stored at cryogenic temperature, and then thawed for use in assisted reproduction. Small organisms (e.g., coral symbiont larvae, mosquito larvae, etc.), small volumes of cells, and solutions of antibodies, proteins, and other biological molecules are also stored as samples for scientific and commercial purposes. Human eggs and embryos have typical sizes of about 100 micrometers (pm) to about 120 pm, and blastocysts used in human assisted reproduction may have sizes of about 180 pm to about 200 pm.

[0003] Samples used in assisted reproduction may be soaked in cryoprotectant / vitrification solutions, which may contain dimethyl sulfoxide (DMSO), ethylene glycol, sucrose, trehalose, and other sugars to dehydrate cells and inhibit ice formation during cooling and thawing. Samples may be slowly cooled (e.g., over twenty minutes to two hours) to an intermediate temperature (e.g., -40 degrees Centigrade (oC)), and then dropped into liquid nitrogen for long term storage. During the slow cooling step, ice may nucleate outside the cells; growth of this ice may draw water out of the cells, increasing the concentration of protein and other solutes within the cells. As cooling and water removal continues, eventually the solvent inside the cells vitrifies.

[0004] Alternatively, samples at room temperature may be plunged into liquid nitrogen or another liquid cryogen with the goal of cooling the sample so fast that little or no ice forms inside and the solvent within the sample is largely vitrified. To eliminate ice formation within cells, much larger cryoprotectant concentrations may be used in pre-cooling soaks than in slow cooling approaches. These large concentrations may be toxic and may cause damage due to osmotic stress if not introduced gradually. This latter, fast-cooling approach is now overwhelmingly dominant in most small-sample cryopreservation practices, including in human-assisted reproduction.

[0005] For warming / thawing, samples used in assisted reproduction may be removed from a cryogenic storage Dewar, transferred through air, and then plunged into a well of a multiwell plate (e.g., CRYOTEC™ plate from REPROLIFE™) or a straw containing a warming solution at room temperature or biological temperature (e.g., 37 oC), with all steps performed by hand. Other wells in the plate may contain solutions to promote cell re-expansion and postthaw growth / development. The wells of the plates used typically range from 3 millimeters (mm) to 10 mm deep, which severely limits the vertical plunge distance into the warming solution. The small well volume reduces consumption of thawing media and makes it easier to find and retrieve samples after thawing. Separate wells and warming solutions may be used for each sample to prevent cross-contamination. Similar manual protocols are used for thawing of many other types of cryopreserved samples. In the future, warming may be performed automatically using robots or other dedicated systems.

[0006] During cooling, samples may be damaged by several mechanisms. First, ice may nucleate and grow inside the cells; this ice can puncture cell membranes and disrupt a cell’s internal organization. Second, solutes may be rejected from growing ice crystals and, as such, may become concentrated in the remaining uncrystallized solution, which can lead to protein aggregation. Third, the solution inside a cell may expand or contract more than the other cell components, causing cell damage by mechanical stresses. In cell clusters or other samples, inhomogeneous composition and thermal gradients during warming may generate mechanical stress and fracture. Fourth, proteins may change their conformation and often unfold as they are cooled - a process called “cold denaturation” - due to the temperature dependence of the hydrophobic interaction that drives folding, as well as of pH (acidity), pKas (negative base- 10 logarithm of acid dissociation constant) of side chains, and many other physico-chemical properties. Their solubility may also be temperature dependent. As a result, proteins may aggregate and other biomolecular changes that may be irreversible may occur during cooling.

[0007] Cryoprotectants may be added to reduce ice formation. At the cooling rates used in current assisted reproductive technology (ART) practices (e.g., -30,000 degrees Centigrade per minute (oC / min)), large concentrations of cryoprotectants - on the order of 20-30% weight per volume (w / v) - as well as 0.5 M to 1 M of sugars are often needed to prevent ice formation. These cryoprotectants are present in commercial vitrification solutions. To prevent cell damage by osmotic shock, the cells / embryos may be soaked in a series of solutions with increasing cryoprotectant concentrations, which can be a very time-consuming process. Vitrification solutions with these large sugar and cryoprotectant concentrations may contract drastically - by perhaps 5% - on cooling from room temperature to 77 Kelvin (K), which can cause mechanical stress and fracturing.

[0008] It is oftentimes desirable that cooling a sample to cryogenic temperature leaves the solvent inside in a vitrified or glassy or amorphous state. During sample warming / thawing, the vitrified solvent may develop some molecular mobility above the solvent’s glass transition temperature - typically between 140 and 180 K. Ice can then nucleate and grow, and the growth rate increases with temperature as the solvent molecules become more and more mobile, becoming significant at temperatures above 180-200 K and peaking at temperatures somewhat below the melting temperature. As a result, samples often “flash white” during warming, as ice crystals form rapidly during warming before melting near 273 K.

[0009] Warming rates needed to prevent the formation of a significant ice fraction (e.g., 5% or more) within an initially crystalline-ice-free sample are generally much larger - by one to three orders of magnitude - than cooling rates needed to prevent significant ice formation during cooling. Recent X-ray diffraction experiments on bovine oocytes, for example, show that while cryoprotectant concentrations and cooling rates currently used in assisted reproduction are adequate to prevent ice formation during cooling, a large fraction of the internal solvent crystallizes during warming even at a warming rate of 150,000 oC / min, four times larger than is typical (<40,000 oC / min) in current practice. Using the same cryoprotectant concentrations as in current practices, an increase of cooling rates by a factor of 20 over current best practices (e.g., 600,000 oC / min or higher) combined with a multifold increase in warming rates (e.g., 150,000 oC / min or higher) allows almost complete elimination of ice formation during cooling and warming. These results may suggest that increasing warming rates should be a primary focus of efforts to improve post-thaw survival, development, and other outcomes in cryopreservation.

[0010] In current practice, the overwhelming majority of cry opreservation and recovery of small biological samples is done manually. Manual handling of samples is time consuming, prone to error, and cannot achieve the performance in key steps, such as cooling and warming, that is possible using automated systems. Automated systems have been developed for some steps, such as sample soaking in cryoprotectant solutions and sample cooling in liquid nitrogen. However, none of these systems is designed to maximize sample cooling and warming rates, which is critical to maximizing post-warming survival and developmental outcomes. Automated systems have been developed for cooling and storage of samples used in crystallography, but these systems cannot be used for warming or for the multiple steps of sample soaking preceding, during, and following cryopreservation and recovery.SUMMARY

[0011] Aspects of the present disclosure relate to the design, function, and use of automated systems for cryopreservation and recovery of small biological samples, including oocytes, embryos, cells, and tissues. As used herein in relation to biological samples, the term “small” may be defined to include a sample having a diameter or minimum dimension that is smaller than about 2 millimeters or, in some applications, smaller than about 500 micrometers, and a volume that is smaller than about 10 microliters (pL) or, in some applications, smaller than about 1 pL or, in some instances, smaller than 0.1 pL or less than about 0.01 pL For example, mammalian oocytes have diameters of 200 micrometers or less, and volumes of less than 0.01 pL. An example criterion for determining smallness may include the internal sample temperature gradients that develop within a sample during warming in a warm liquid (typically an aqueous solution) be small, e.g., less than about 20-40 oC or, in some applications, less than about 10 oC, so that thawing is reasonably uniform throughout the sample volume and so that no large stresses develop.

[0012] As used herein, the term “low temperature” may be defined to include any temperature that leads to full or partial vitrification of a solvent within a sample, which may depend on the solvent composition and solutes present. For example, a typical low temperature in this application is the temperature of boiling liquid nitrogen (77 K = -196 oC). Temperatures less than about 150 K (-123 oC) may completely inhibit ice crystal formation in samples held at those temperatures. Temperatures as high as about -80 oC (the temperature of standard laboratory freezers) may also be used. While not per se limited, eggs, embryos, and blastocysts having up to approximately 100 cells are primary targets, although stem cells, sperm cells, andany other “small” biological (human or nonhuman) sample-containing cells, larvae, antibodies, proteins, etc., may benefit from using features of the present disclosure.

[0013] Aspects of this disclosure include structures, apparatuses, and approaches to facilitate the handling of small cryopreserved biological samples so as to achieve the largest possible cooling rates and the largest possible warming rates.

[0014] Aspects of this disclosure include structures and approaches that retain small biological samples within a holder while maximizing convective liquid flow past and around the small biological sample when the sample and a sample holder holding the sample are translated through liquids at speeds of at least about 0.25 m / s and up to about 5 m / s.

[0015] Aspects of this disclosure include structures and approaches that enable removal of excess liquid from around a sample.

[0016] Aspects of this disclosure include structures and approaches that enable samples to be easily loaded into sample holders prior to the steps of cryopreservation, and to be easily retrieved from sample holders after warming and recovery.

[0017] Aspects of this disclosure include structures and approaches that enable samples to be automatically soaked in various solutions at different temperatures before cooling and after warming, and to be incubated to enable growth and development after warming.

[0018] Aspects of this disclosure include structures and approaches to facilitate automated optical imaging of small biological samples during or following one or more steps in cryopreservation and recovery.

[0019] According to aspects of this disclosure, a small biological sample - such as a sample having a volume of less than about 10 microliters or, in some applications, less than about 1 microliter or, in some applications, less than about 0.1 microliter, and having a thickness of less than about 2 mm or, in some applications, less than about 500 micrometers or, in applications involving mammalian oocytes and embryos, less than about 200 micrometers - may be held within a sample holder having a low thermal mass in a region adjacent the sample. This sample holder may be porous and may be structurally configured to enable largely unimpeded liquid flow from outside the holder to the sample during high-speed plunging, translation, or other movement of the sample holder into liquids.

[0020] According to aspects of this disclosure, a small biological sample contained within a sample holder may be processed for cryopreservation and recovery using multiple stationsthat each performs a function or a set of functions on the sample. In this instance, a robot may move the sample between stations and / or within each station.

[0021] According to aspects of this disclosure, samples may be handled by the automated system using a robot.

[0022] According to aspects of this disclosure, the robot may have an end effector that is structurally configured to grab, hold, and manipulate one or more sample holders.

[0023] According to aspects of this disclosure, the robot may transfer samples between several stations within the system where different processing steps are performed.

[0024] According to aspects of this disclosure, the robot may provide multiaxis translational motion, e.g., in the x, y and z directions, as well as single-axis or multiaxis rotational motion, e.g., of a z-motion shaft.

[0025] According to aspects of this disclosure, the robot may be a pick-and-place robot.

[0026] According to aspects of this disclosure, the robot may be a Selective ComplianceArticulated Robot Arm (SCARA) robot or a cartesian-type gantry robot.

[0027] According to aspects of this disclosure, a vertical speed and a horizontal speed of the robot may each have a maximum allowable speed of at least about 1 m / s or, in some applications, about 2 m / s or, in some applications, not more than about 5 m / s.

[0028] According to aspects of this disclosure, the end effector of the robot may include a motorized stage for high speed on-axis or off-axis rotational and / or vibrational motion of the sample.

[0029] According to aspects of this disclosure, the maximum speed of the sample in its rotational motion may reach at least about 0.25 m / s or, in some applications, about 1-2 m / s, e.g., as produced by the combined rotations of the robot’s z-motion shaft and the end effector.

[0030] According to aspects of this disclosure, the end effector may have hardware to increase the vertical speed of the sample holder additively to the speed of the robot’s z-motion shaft.

[0031] According to aspects of this disclosure, the end effector may include hardware to pick up and hold a sample holder and to eject or release a sample holder.

[0032] According to aspects of this disclosure, the robot may be partially or substantially enclosed in an enclosure with a transparent portion to protect users from the robot during its operation.

[0033] According to aspects of this disclosure, the robot enclosure may have an emergency stop button on its exterior surface.

[0034] According to aspects of this disclosure, the robot enclosure may have a hinged or sliding door that enables loading of plates containing solutions, blocks containing cryocooled samples, liquid nitrogen, and / or other components into stations within the enclosure.

[0035] According to aspects of this disclosure, the door may have an interlock that prevents operation of the robot when the door is open.

[0036] According to aspects of this disclosure, a sample transfer station (also referred to herein as “sample loading station”) may translate and / or rotate samples, including at room temperature or biological temperature, from a position outside the enclosure to a position within the enclosure, where the samples may be accessed by the robot.

[0037] According to aspects of this disclosure, the sample transfer station may be a motor- driven carousel with one or more receptacles for retaining one or more sample holders.

[0038] According to aspects of this disclosure, the motor-driven carousel may hold a single sample holder or may hold multiple sample holders at a time.

[0039] According to aspects of this disclosure, the motor-driven carousel may include a heater and a temperature control system to maintain a sample temperature of the sample or samples at a temperature above room temperature and / or near a biological temperature.

[0040] According to aspects of this disclosure, the system may include a presoaking station where samples can be soaked in one or more solutions prior to cryocooling.

[0041] According to aspects of this disclosure, the presoaking station may enable a solution temperature of a solution to be varied from about 0 oC to about 50 oC (e.g., between about 4oC and 50oC or between about 20oC and 40 oC) and to be held near biological temperature.

[0042] According to aspects of this disclosure, the presoaking station may accept multiple heterogeneous multiwell plates, e.g., in thin, standard, and / or deep well formats, for containing the presoaking solutions.

[0043] According to aspects of this disclosure, the presoaking station may accept custom multiwell plates configured to accept and hold sample holders when they are released by the robot.

[0044] According to aspects of this disclosure, the presoaking station may include a commercial well plate temperature control system with custom well plate holders.

[0045] According to aspects of this disclosure, the system may include a liquid removal and sample inspection station that enables removal of excess liquid from the sample, e.g., to minimize total thermal mass and maximize cooling and warming rates, and that enables optical imaging of the sample(s) after liquid removal, e.g., to document its pre-cry opreservation state.

[0046] According to aspects of this disclosure, the liquid removal may be performed by a suction device that selectively applies suction to a select portion or portions of the sample holder adjacent to the sample that is in fluid communication with the sample.

[0047] According to aspects of this disclosure, the liquid removal may be performed by blotting a portion of the sample holder adjacent to the sample and in fluid communication with the sample, e.g., with a hydrophilic blotting pad or similarly suitable medium.

[0048] According to aspects of this disclosure, the liquid removal station may include a backlighting device that provisions lighting through the suction channel to illuminate the sample, and a digital imaging device with forward lighting in proximity to the sample holder to image the sample, e.g., after liquid has been removed and before the sample is plunged / translated / moved into liquid nitrogen.

[0049] According to aspects of this disclosure, the system may include a sample cryocooling station that rapidly cools the sample and the sample holder to a cryogenic temperature, e.g., so as to minimize ice nucleation and growth within the sample and within any surrounding liquid.

[0050] According to aspects of this disclosure, the sample cryocooling station may include a thermally insulated container or Dewar that is configured to accept and hold a first volume of liquid nitrogen, and an insulated container / Dewar cover that isolates and insulates the container / Dewar’s contents from warm moist surrounding air.

[0051] According to aspects of this disclosure, the container / Dewar may contain a second, inner chamber that is configured to hold a second volume of liquid nitrogen and is in“excellent” thermal communication with the first volume of liquid nitrogen in the Dewar volume between the inner Dewar walls and the second inner chamber.

[0052] According to aspects of this disclosure, the inner chamber may be fabricated, in whole or in part, using a metallic material, such as stainless steel or aluminum.

[0053] According to aspects of this disclosure, the level of liquid nitrogen within the inner chamber may be maintained near a predefined level, e.g., either at the top of the walls of the inner chamber or at the bottom of an aperture within the walls, by periodically filling and overfilling the inner chamber and, if desired, by maintaining a liquid nitrogen level in the Dewar region outside the inner chamber that is below that in the inner chamber.

[0054] According to aspects of this disclosure, the level of liquid nitrogen within the inner chamber and within the volume between the inner chamber and insulated enclosure may be monitored using a thermocouple, a resistance temperature detector (RTD), a diode, and / or a laser level sensor.

[0055] According to aspects of this disclosure, the insulated cover may include a manifold within a portion of the cover that includes a bore or a through-hole through which a sample and a sample holder may be plunged / translated / moved into liquid nitrogen within the inner chamber. The manifold within the insulated cover may also include a set of gas channels that intersects the bore to apply suction and / or deliver dry ambient-temperature gas to the bore so as to remove cold gas present near the surface of the liquid nitrogen within the bore and replace it with ambient temperature dry gas. These gas channels may also flood the bore with excess dry gas to prevent infiltration of moist surrounding air. The manifold within the insulated cover may also include one or more heaters lining the bore to prevent frost formation in or around the bore or other manifold surfaces.

[0056] According to aspects of this disclosure, the insulated cover may also include a second opening or through-hole in a region adjacent the manifold. This second opening / hole may be shaped and sized to receive a cassette or puck with one or more receptacles for holding one or more samples. A hinged, sliding, or rotating lid may extend over and cover the opening, and may be positioned above a region contained entirely within the inner chamber.

[0057] According to aspects of this disclosure, the bore of the manifold and the second opening of the cover may be in communication such that a sample plunged, translated, or moved by the robot arm through the bore and into liquid nitrogen can be translated latterly out of the bore and into the region below the second opening.

[0058] According to aspects of this disclosure, the bore of the manifold may include a hinged door defining one side of the bore that opens and allows lateral transfer of cryocooled samples from the bore to the area adjacent the bore beneath the second cover opening and above a cassette.

[0059] According to aspects of this disclosure, the system may include one or more hydraulically, pneumatically, and / or electrically activated actuators for automating the opening and closing of the aforementioned lids, covers, doors, etc.

[0060] According to aspects of this disclosure, the bottom of the inner chamber beneath the opening in the cover may contain a chamber structure that is sized and shaped to receive and hold a cassette or puck in a well-defined orientation, e.g., allowing the robot to deposit samples after cooling into the cassette or puck or to retrieve samples from the cassette or puck for subsequent processing. The chamber structure may be integrally formed or machined into the inner chamber; alternatively, the chamber structure may be a distinct structure that is secured to the bottom of the inner chamber.

[0061] According to aspects of this disclosure, a cassette or puck may be loaded into the chamber structure in the inner chamber or removed from it using a tool.

[0062] According to aspects of this disclosure, a cryogenically cooled cassette or puck containing previously cryocooled samples may be loaded through the second opening in the cover of the insulated container and into the structure within the insulated container that accepts a cassette or puck, e.g., to allow the robot to access the samples and transfer them to the warming station.

[0063] According to aspects of this disclosure, the sample cryocooling station may include a camera, a lens, and one or more light sources and / or other optics as needed to image a cryocooled sample in liquid nitrogen or in cold gas present immediately above the liquid nitrogen and before the sample is transferred into a cassette or puck.

[0064] According to aspects of this disclosure, the sample cooling rates achieved by the cryocooling system with robot and end effector may be between about 25,000 oC / min for roughly 500 micrometer samples and about 3,000,000 oC / min for roughly 25 micrometer samples.

[0065] According to aspects of this disclosure, the robot may plunge, translate, or otherwise move the sample through the bore and into the liquid nitrogen at a speed of at least about 1 m / s or, in some applications, at least about 2 m / s but not greater than 5 m / s, e.g., so as to maximizeconvective heat transfer without causing excessive splashing of liquid nitrogen or requiring excessive travel distances through the liquid nitrogen.

[0066] According to aspects of this disclosure, the Dewar depth and nitrogen fill level may be predetermined to allow the sample to travel a distance of between about 2 centimeters (cm) and about 20 cm or, in some applications, at least about 4 cm before coming to a stop, e.g., to ensure that the sample continues to travel at high speed and to ensure that the convective velocity of the liquid nitrogen relative to the sample remains large until the sample has cooled to a sample temperature that is below a predefined target temperature (e.g., -150 K).

[0067] According to aspects of this disclosure, the robot arm may by itself enable or may have an end effector that enables high-speed, on-axis or off-axis rotation of the sample holder, e.g., so as to maintain a large speed of the sample holder relative to liquid and a large rate of convective heat transfer even when translational motion of the robot arm has ceased.

[0068] According to aspects of this disclosure, the rotational motion provided by the robot may result in a sample holder rotational speed relative to the frame of the insulated container of at least about 0.25 m / s or, in some applications, between about 0.5 m / s and about 2 m / s.

[0069] According to aspects of this disclosure, the robot arm and / or end effector may provide high-speed vibrational motion of the sample, where the peak sample speed in its vibrational motion is at least about 0.25 m / s or, in some applications, about 0.5 m / s to about 2 m / s, and where the peak-to-peak vibration amplitude is at least about 2 mm.

[0070] According to aspects of this disclosure, the system may include a sample warming or thawing station where samples previously cooled to cryogenic temperature may be rapidly warmed to a predefined room temperature or a predefined biological temperature of between about 20 oC and about 60 oC in the sample warming station. As used herein, the term “rapid warm” and permutations thereof may include sample warming rates of between about 50,000 oC / min (e.g., for 500 micrometer samples) and about 6,000,000 oC / min (e.g., for 25 micrometer samples).

[0071] According to aspects of this disclosure, the warming station may include a temperature-controlled plate or block heater, a highly thermally conductive stage attached to the plate / block heater that accepts plates, receptacles for holding solutions used in thawing (e.g., vials, test tubes, cuvettes, etc.), and insulation wrapped around outer faces of the thermally conductive stage to reduce heat loss.

[0072] According to aspects of this disclosure, the thermally conductive stage may be shaped to provide close thermal contact with any liquid-enclosing surfaces of a plate, vial, test tube, etc., e.g., so as to ensure maximum rates of heat transfer and temperature uniformity.

[0073] According to aspects of this disclosure, the robot plunges, translates, or otherwise moves samples into warm liquid contained with the well of a plate, a vial, a test tube, a cuvette at a speed of between about 0.25 m / s and about 5 m / s or, in some applications, about 2 m / s, e.g., to maximize the rate of convective heat transfer while minimizing splashing of liquid when the sample holder impacts the liquid.

[0074] According to aspects of this disclosure, the plates, vials, test tubes, cuvettes, etc., may contain liquid receptacles with a depth of between about 1 cm and about 10 cm or, in some applications, about 4 cm, e.g., so that the sample may remain in high-speed motion long enough to ensure that it has substantially warmed to a predefined sample temperature, for example at least about -10 oC or, in some applications, about 0 oC so that any and all ice nucleation and growth within the sample has ceased before translational motion is ceased.

[0075] According to aspects of this disclosure, the sample may be rotated using the robot arm or using an end effector on the robot arm in addition to being vertically translated, e.g., so that sample motion relative to the warming liquid can maintain large convective heat transfer rates until the sample has warmed to at least about -10 oC or, in some applications, about 0 oC.

[0076] According to aspects of this disclosure, the rotational motion provided by the robot may result in a sample holder rotational speed relative to the frame of the insulated container of at least about 0.25 m / s or, in some applications, about 0.5 m / s to about 2 m / s.

[0077] According to aspects of this disclosure, the robot arm and / or end effector may allow high-speed vibrational motion of the sample, e.g., with a peak sample vibrational speed of at least about 0.25 m / s or, in some applications, between about 0.5 m / s and about 2 m / s, where the peak-to-peak amplitude of the motion is at least about 2 millimeters.

[0078] According to aspects of this disclosure, a liquid temperature of a warming liquid may be a biological temperature (e.g., near 37 oC for mammalian samples), including a warm biological temperature near the upper bound of what a sample can tolerate (up to about 60 oC for at least some eukaryotic samples) for periods of seconds without adverse effects.

[0079] According to aspects of this disclosure, a cold sample may initially be plunged, translated, or otherwise moved through a warming liquid at a temperature substantially above biological temperature - between 50oC and 100 oC - before entering warming liquid at abiological temperature (e.g., near 37 oC), thereby increasing the sample’s average warming rate without the sample temperature rising to non-biological temperatures.

[0080] According to aspects of this disclosure, the robot may quickly remove the sample from the warming solution before the sample temperature has reached the temperature of the warming solution so as to protect the sample from excessive temperatures.

[0081] According to aspects of this disclosure, warming solution may be contained within two separate compartments that are separately heated to different temperatures. For example, one compartment may maintain a warming solution at a biological temperature (e.g., near 37 oC), and another compartment may maintain a warming solution at a temperature well above a biological temperature (e.g., 50 to 100 oC) and below the solution’s boiling point. The warming solution compartments may be connected to allow transfer of a sample from one compartment to the other during warming. The sample may initially travel within a first high- temperature compartment before being translated into and through a second low-temperature compartment, e.g., so as to maximize sample warming rates while preventing sample damage that may result should its temperature rise well above a biological temperature.

[0082] According to aspects of this disclosure, the two warming solution compartments may be arranged vertically, with the high-temperature compartment located above the low- temperature compartment, e.g., so that the sample may be plunged in a continuous motion through the high-temperature compartment and into the low-temperature compartment.

[0083] According to aspects of this disclosure, the two warming solution compartments may be arranged side-by-side.

[0084] According to aspects of this disclosure, the two warming solution compartments may be in direct fluid communication through an aperture in a wall separating the compartments. The aperture may be large enough for a sample-containing portion of a sample holder to pass through the aperture.

[0085] According to aspects of this disclosure, the aperture in the wall separating the two warming solution compartments may be covered by a door or a flexible flap that may be opened immediately prior to or during sample transfer between the compartments, e.g., thereby keeping warming solution within the two compartments separate until the transfer occurs.

[0086] According to aspects of this disclosure, the aperture-covering door may be opened via direct contact with the sample holder, robot end effector, or robot arm.

[0087] According to aspects of this disclosure, the door may be spring loaded or driven by an actuator.

[0088] According to aspects of this disclosure, the door’s motion may be within the plane of the door, e.g., to minimize fluid resistance to the door’s opening and to facilitate the door’s opening within a timeframe that is short compared with the warming time of the sample.

[0089] According to aspects of this disclosure, the warming solution may be contained in a channel that slopes downward from near a top surface of the micropl ate / soluti on container toward a bottom of the microplate / solution container, e.g., such that the sample may be “plunged” through the warming solution along a trajectory parallel to the bottom of the channel.

[0090] According to aspects of this disclosure, an upper portion of the channel may be heated to a first temperature substantially above normal biological temperature, for example, 50oC to 100 oC, using a first heated block adjacent to the upper portion of the channel, and a lower portion of the channel, which is below the upper portion, may be heated to a distinct, second temperature equal to a desired final sample temperature, for example, 37 oC, using a second heated block adjacent to the lower portion of the channel.

[0091] According to aspects of this disclosure, the channel may have a width of between about 1 mm and about 10 mm or, in some applications, between about 2 mm and about 5 mm, e.g., so as to minimize convective mixing of warming solution in the lower and upper portions of the channel.

[0092] According to aspects of this disclosure, the channel may have a maximum depth of no more than about 13 mm, e.g., so as to be compatible with Society for Biomolecular Screening (SBS) / Society for Laboratory Automation and Screening (SLAS) standard height microplates (14.35 mm height), or no more than about 27.3 mm so as to be compatible with SBS / SLAS double height microplates (28.7 mm height), or no more than about 41.7 mm to be compatible with an SBS / SLAS deep well plates (43.05 mm height).

[0093] According to aspects of this disclosure, the system may include a post-warming soaking and incubation station where samples can be soaked in one or more solutions prior to cryocooling.

[0094] According to aspects of this disclosure, the post-warming station may allow the solution temperature to be varied from roughly about 20 oC to about 50 oC.

[0095] According to aspects of this disclosure, the post-warming station may accept standard multiwell plates in thin, standard, or deep well formats for containing the postwarming solutions.

[0096] According to aspects of this disclosure, the post-warming station may use commercial temperature-controlled well plate heaters with custom well plate holders as needed.

[0097] According to aspects of this disclosure, the post-warming station may use custom multiwell plates designed to accept and hold sample holders deposited into them by the robot.

[0098] According to aspects of this disclosure, the system may include a rinsing and drying station for rinsing and drying a robot end effector to remove warming liquid from the end effector in the event of liquid splashing during the warming plunge, and to warm and dry the end effector after plunges into liquid nitrogen or warming solution.

[0099] According to aspects of this disclosure, the rinsing component of the station may involve one or more receptacles containing pure water, water + detergent, or isopropyl alcohol.

[0100] According to aspects of this disclosure, the robot may translate within the rinsing and drying station and then immerse the end effector into one or more rinsing liquids.

[0101] According to aspects of this disclosure, the robot may rotate the end effector and oscillate it up and down during the rinsing within the rinsing and drying station.

[0102] According to aspects of this disclosure, the warming and drying component of the station may include an air amplifier fed by compressed dry gas, into which the end effector is inserted and moved up and down until dry.

[0103] According to aspects of this disclosure, the warming and drying component of the station may include a single heater or multiple heaters arranged concentrically adjacent to the air amplifier and having the same central axis as the air amplifier, e.g., to allow the end effector to be moved along a line in and out of the heater and air amplifier.

[0104] According to aspects of this disclosure, any one or all of the robots, motors, actuators attached to the robot and forming the end effector, the temperature-controlled heaters / coolers of the pre-soak, warming, and post-soak stations, the sensors, actuators, motors, valves etc., within any of the other system components may be controlled electronically and, if desired, automated via a computer, microcontroller, programmable logic circuit, integratedcircuit (IC) device, control module, or network of computers / microcontrollers / logic circuits / IC devices / etc.

[0105] According to aspects of this disclosure, multiple sample holders may be processed concurrently by sequencing robot operations so that samples can be released for soaking while the robot executes motions required for other processing steps on another sample.

[0106] According to aspects of this disclosure, the sample holder may contain one or more samples so as to prevent their loss during handling. The sample holder may have an array of holes that allows liquids to flow through and past the sample(s) at high speed and with minimal flow resistance when the sample holder is moving at high speed relative to the liquids so as to maximize convective heat transfer rates between the sample and liquid.

[0107] According to aspects of this disclosure, the sample may be supported on a thin film of a transparent polymer or glass or semiconductor. The thin film may have a thickness of between about 2 pm and about 100 pm or, in some applications, between about 5 pm and about 50 pm. A small thickness may help to minimize flow resistance, maximize heat transfer through the film to the sample, and maximize optical transparency.

[0108] According to aspects of this disclosure, the film may have regions of with different thicknesses, such as a first “thin” region, e.g., with through holes on which the sample resides, with a thickness of between about 5 pm and about 15 pm, and a second “thick” region, e.g., for mechanical strength and for bonding to other parts of the sample holder, with a thickness of between about 10 pm and about 50 pm or, for some applications, up to about 100 micrometers. There may be thick regions forming markings or pillars or other features within the thin region of the film that may be useful in positioning samples on the film.

[0109] According to aspects of this disclosure, the portion of the thin film that receives samples may have an open area fraction of between about 50% and about 95% or, in some applications, between about 70% and about 95%. The open area may be formed by a dense array of through-holes, where the holes are sized to be as large as possible but small enough that the sample cannot pass through the holes, so as to minimize resistance to fluid flow through the film in those regions.

[0110] According to aspects of this disclosure, the through-holes in the thin film may have a diameter that is between about 40% and about 80% of a sample diameter of a sample. For mammalian oocytes and embryos, for example, the through-hole diameter may be between about 40 micrometers and 150 micrometers.

[0111] According to aspects of this disclosure, the thin film may be micropattemed using photolithography and microfabrication processes, micro-embossing, stamping, etc.

[0112] According to aspects of this disclosure, the thin film may be fabricated with an optically transparent polymer, such as polyimide, cyclic olefin copolymer, mylar, or SU8, e.g., to facilitate imaging of a sample placed upon the film.

[0113] According to aspects of this disclosure, the thin film may be attached to a thin rigid frame, where the frame may be marked (e.g., laser marked) with information to allow sample identification and tracking.

[0114] According to aspects of this disclosure, the frame may be attached to a base configured to be handled by the robot end effector. The base may be fabricated with a hard, dimensionally stable material, including magnetic stainless steel, and may be marked or may incorporate an RFID tag for sample identification and tracking.

[0115] According to aspects of this disclosure, the frame may include a rigid sheet with an aperture disposed adjacent to one end of the sheet. The sheet may be made with a rigid metal (e.g., brass), polymer (e.g., polycarbonate), or polymer-glass composite (e.g., G10). A sheet thickness of the sheet may be comparable to or somewhat larger than (between 80% and 150% of) the corresponding sample dimension, e.g., so as to ensure that the sample is near the sample holder surfaces and near the liquid flowing past those surfaces to maximize the rate of heat transfer between the liquid and the sample. For mammalian oocytes and embryos, this thickness may be between about 80 pm and about 250 pm or, in some applications, about 150 micrometers.

[0116] According to aspects of this disclosure, the width of the frame may be between about 3 mm and about 10 mm or, for some applications, may be as large as about 30 mm (e.g., if the holder is to hold a very large number of samples).

[0117] According to aspects of this disclosure, the length of the frame - including the portion that inserts into / attaches to a base - may be between about 1 cm and about 10 cm or, in some applications, about 2 cm.

[0118] According to aspects of this disclosure, the frame may have at least one aperture disposed near the end furthest from the base, with a width equal to the width of the frame less about 2 millimeters (e.g., between about 1 and about 8 millimeters and as large as about 28 millimeters, depending on frame width) or, for frames of width 10 mm and greater, less about 4 millimeters and where the length of the aperture is between about 2 millimeters andabout 10 millimeters and as large as about 30 millimeters, in such a way that the aperture in the frame extends to within no more than about 0.5 mm or, in some applications, no more than about 1 mm of the frame edges to allow sufficient surface for film bonding to the frame.

[0119] According to aspects of this disclosure, the aperture in the frame may be spanned and sealed on one side (e.g., a “bottom” side of the frame) by a thin film, such as those discussed above. The portion of the film that is filled with holes may be located within the aperture, whereas portions without a dense array of holes may be largely located over solid areas of the frame bounding the aperture. The film may be bonded to the frame using methods including adhesives, waxes, ultrasonic bonding, or thermal bonding.

[0120] According to aspects of this disclosure, the film may have one thickness in the area of the aperture (for example, 5-15 micrometers) and a second, larger thickness (for example 10-50 micrometers) in the area contacting the frame.

[0121] According to aspects of this disclosure, one or more samples are deposited onto the hole-filled film within the aperture.

[0122] According to aspects of this disclosure, after samples have been deposited on the a first thin film, a second thin film, which may be substantially similar to the first flm, containing a dense array of holes of similar size and density, may be applied to a “top” side of the frame, e.g., to seal the samples within the aperture and between the two films.

[0123] According to aspects of this disclosure, the first “bottom” film and / or the second “top” film may include perforations around a respective inside edge of the aperture or tabs within the aperture to facilitate removal of the portion of the film within the aperture and access to the samples within the aperture.

[0124] According to aspects of this disclosure, the aperture in the frame may be replaced by a U-shaped cutout at the frame end furthest from the base.

[0125] According to aspects of this disclosure, the “top” and “bottom” sides of the frame, as well as the open end of the frame, may be sealed with one or two hole-filled thin films so that the top, bottom, and end openings of the U-shaped cutout are sealed by portions of the film(s) having a dense array of holes, e.g., so as to allow liquid to flow into the interior of the U-shaped cutout from all three open sides with minimal flow resistance while retaining one or more samples held within the interior of the cutout.

[0126] According to aspects of this disclosure, a sample holder may include or, if desired, may consist essentially of a basket and a cap. The basket may receive and hold one or more samples, and the cap may be configured to seal the basket and to be grabbed or handled by a robot end effector.

[0127] According to aspects of this disclosure, a basket bottom of the basket may have a diameter of between about 2 mm and about 20 mm or, in some applications, between about 3 mm and about 10 mm.

[0128] According to aspects of this disclosure, the basket may include a thin, rigid frame with a low thermal mass, a substantially open bottom, and openings in the sides of theframe extending from the bottom up a distance d from the bottom. The distance d may be comparable to or larger than the basket diameter at its base, and may be about 2-4 times the bottom diameter or, in some applications, between about 6 and about 40 millimeters.

[0129] According to aspects of this disclosure, the open bottom and sides may be spanned by a hole-filled thin film or a mesh formed of fine filaments. The holes of the film or mesh may be as large as possible without allowing a sample to pass through, e.g., typically in a size range of between about 40 pm and 120 pm for mammalian oocytes and embryos and more generally between 40% and 80% of the sample diameter.

[0130] According to aspects of this disclosure, the open bottom of the basket may be spanned and sealed by a hole-filled thin film of a transparent polymer, such as polyimide, SU- 8, and cyclic olefin copolymer, e.g., to allow optical inspection of samples on the film. The film may have a thickness of between 5 pm and about 15 pm in the area within the bottom opening and a thickness of between about 10 pm and about 50 pm in the areas where it is bonded to the frame.

[0131] According to aspects of this disclosure, the thin film portion within the opening in the bottom of the basket may have an open area fraction of between about 40% and about 95% or, in some applications, between about 70% and about 95%.

[0132] According to aspects of this disclosure, the openings in the side of the basket frame may be sealed using a hole-filled thin film in which the hole-filled regions of the film are located within the frame openings, or by a mesh of fine filaments of a metal or of a polymer such as nylon with a mesh size between about 60 and about 400 mesh or, for mammalian oocytes and embryos, may be between about 100 and about 240 mesh.

[0133] According to aspects of this enclosure, one or more samples may be deposited on the inside bottom of the basket, on the thin, hole-filled film spanning the opening in the bottom of the basket.

[0134] According to aspects of this disclosure, samples within the basket can be sealed within by inserting the cap into the top opening of the basket, forming a seal between the cap and basket.

[0135] According to aspects of this disclosure, the cap may press or twist fit with the basket.

[0136] According to aspects of this disclosure, an upper portion of the basket frame may extend beyond an outer diameter of the cap, allowing the basket to be removed from the cap by pressing down on the frame extension while holding the cap.

[0137] According to aspects of this disclosure, the cap may have structure that facilitate gripping by the robot arm. The cap structure may include a twist-and-lock structure, a press-and-snap lock structure, or a magnet.

[0138] According to aspects of this disclosure, the cap may include a conical feature located axially and projecting down into the basket. This conical feature may serve to direct liquid flowing upward through basket bottom outward toward the basket sides and out through a holey film or mesh-covered portion of the basket side so as to produce more nearly laminar liquid flow when the sample holder is plunged in cold or warm liquid.

[0139] According to aspects of this disclosure, the cap may include openings spanned by thin holey films or mesh to allow passage of liquid during cooling and warming.

[0140] According to aspects of this disclosure, a series of baskets and caps may be held in receptacles within a basket and cap holder. Samples may be loaded into the baskets using a pipette or other tool, and then the cap positioned on and sealed to the basket.

[0141] According to aspects of this disclosure, the bottom of the basket may be dome shaped with a large fraction of the area of the dome spanned by a thin holey film.

[0142] According to aspects of this disclosure, the basket frame or cap may be marked or patterned to allow identification of each sample, or the cap may incorporate an RFID tag for sample identification.

[0143] The above summary does not represent every embodiment or every aspect of the present disclosure. Rather, the foregoing summary merely provides a synopsis of some ofthe novel concepts and features set forth herein. The above features and advantages, and other features and attendant advantages of this disclosure, will be readily apparent from the following Detailed Description of illustrated examples and representative modes for carrying out the disclosure when taken in connection with the accompanying drawings and appended claims. Moreover, this disclosure expressly includes any and all combinations and subcombinations of the elements and features presented above and below.BRIEF DESCRIPTION OF THE DRAWINGS

[0144] FIG. 1 is a perspective-view illustration of a representative automated sample system for cryopreservation and recovery of small biological samples in accord with aspects of this disclosure.

[0145] FIG. 2 is a perspective-view illustration of a representative robotic system with key structures and functions for use with the automated sample system of FIG. 1 in accord with aspects of this disclosure.

[0146] FIGS. 3A to 3C are perspective-view illustrations of different representative robot systems with end effectors suitable for use with disclosed systems in accord with aspects of this disclosure.

[0147] FIG. 4 is a partially cut away, perspective-view illustration of a representative carousel for transferring samples in and out of a system enclosure in accord with aspects of this disclosure.

[0148] FIGS. 5 A and 5B are perspective-view and partially exploded perspective-view illustrations, respectively, of a representative station for soaking samples in solutions prior to cry opreservation in accord with aspects of this disclosure.

[0149] FIGS. 6 A and 6B are partially exploded and partially cutaway perspective-view illustrations, respectively, of a representative station for removing excess liquid from samples and imaging samples prior to plunge cooling in accord with aspects of this disclosure.

[0150] FIGS. 7 A and 7B are partially cutaway perspective-view and elevated perspectiveview illustrations, respectively, of a representative station for rapid cooling of samples in liquid nitrogen and for cryogenic sample storage, showing a hinged door of an enclosure in an open position, in accord with aspects of this disclosure.

[0151] FIGS. 8 A and 8B are partially cutaway perspective-view and elevated perspectiveview illustrations, respectively, of the representative rapid cooling station of FIGS. 7 A and 7B showing the hinged door in a closed position.

[0152] FIGS. 9 A and 9B are schematic and partially cutaway perspective-view illustrations, respectively, of yet another representative station for rapid cooling of samples in liquid nitrogen and for cryogenic sample storage in accord with aspects of this disclosure.

[0153] FIGS. 10A and 10B are perspective-view and partially exploded perspective-view illustrations, respectively, of a representative station for rapid warming of cold samples in accord with aspects of this disclosure.

[0154] FIGS. 11A and 11B are schematic illustrations of two representative two- compartment cells and heating blocks for holding solution during rapid warming of cold samples in accord with aspects of this disclosure.

[0155] FIGS. 12A and 12B are schematic illustrations of another representative cell and heating block for holding solution during rapid warming of cold samples in accord with aspects of this disclosure.

[0156] FIG. 13 is a side perspective-view illustration of a representative microwell plate and matching heating block for holding a solution during warming of a sample in accord with aspects of this disclosure.

[0157] FIG. 14 is a partially exploded, perspective-view illustration of a representative station for warming and drying a robot end effector in accord with aspects of this disclosure.

[0158] FIGS. 15Ato 15C are perspective-view illustrations of a representative first sample holder suitable for use with disclosed systems in accord with aspects of this disclosure.

[0159] FIGS. 16A and 16B are perspective-view illustrations of a representative second sample holder suitable for use with disclosed systems in accord with aspects of this disclosure.

[0160] FIGS. 17A to 17C are perspective-view illustrations of a “basket” portion of a representative third sample holder suitable for use with disclosed systems in accord with aspects of this disclosure.

[0161] FIG. 18 is a perspective-view illustration of an alternative “basket” portion of a representative third sample holder suitable for use with disclosed systems in accord with aspects of this disclosure.

[0162] FIGS. 19A and 19B are perspective-view illustrations of a basket and cap of a representative sample holder and a representative tool suitable for use with disclosed systems in accord with aspects of this disclosure.

[0163] FIG. 20 is a perspective-view illustration of a representative tray for holding, loading, and assembling sample holders and suitable for use with disclosed systems in accord with aspects of this disclosure.

[0164] FIGS. 21 A to 21C are perspective view illustrations of representative basket-based sample holders in accord with aspects of this disclosure.

[0165] FIG. 22 is a side, perspective-view illustration of a representative sample holder that may be opened using a tool to allow sample loading and then closed to seal in the sample for processing in accord with aspects of this disclosure.

[0166] The present disclosure is amenable to various modifications and alternative forms, and some representative embodiments of the disclosure are shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the novel aspects of this disclosure are not limited to the particular forms illustrated in the aboveenumerated drawings. Rather, this disclosure covers all modifications, equivalents, combinations, permutations, groupings, and alternatives falling within the scope of this disclosure as encompassed, for example, by the appended claims.DETAILED DESCRIPTION

[0167] This disclosure is susceptible of embodiment in many different forms. Representative embodiments of the disclosure are shown in the drawings and will herein be described in detail with the understanding that these embodiments are provided as an exemplification of the disclosed principles, not limitations of the broad aspects of this disclosure. To that extent, elements and limitations that are described, for example, in the Abstract, Introduction, Summary, Description of the Drawings, and Detailed Description sections, but not explicitly set forth in the claims, should not be incorporated into the claims, singly or collectively, by implication, inference or otherwise. Moreover, recitation of “first”, “second”, “third”, etc., in the specification or claims is not per se used to establish a serial or numerical limitation; unless specifically stated otherwise, these designations may be used for ease of reference to similar features in the specification and drawings and to demarcate between similar elements in the claims.

[0168] For purposes of this Detailed Description, unless specifically disclaimed: the singular includes the plural and vice versa (e.g., indefinite articles “a” and “an” are to be construed as meaning “one or more” unless expressly disclaimed); the words “and” and “or” shall be both conjunctive and disjunctive; the words “any” and “all” shall both mean “any and all”; and the words “including,” “containing,” “comprising,” “having,” and the like, shall each mean “including without limitation.” Moreover, words of approximation, such as “about,” “almost,” “substantially,” “generally,” “approximately,” and the like, may each be used herein to denote “at, near, or nearly at,” or “within 0-5% of,” or “within acceptable manufacturing tolerances,” or any logical combination thereof, for example.

[0169] Features of the present disclosure aim to address the design criteria and challenges listed below to optimize cryopreservation and recovery of small biological samples, including minimizing sample damage resulting from handling, osmotic shock, cryoprotectant toxicity, ice formation, fracturing, and other factors, so as to maximize survival and development of cellular systems. Additional information pertinent to this disclosure may be found, for example, in U.S. Patent Nos. 9,417,166 B2, to Thorne et al., 11,473,826 B2, to Closs et al., and 11,653,644 B2 to Thome et al., all of which are incorporated herein by reference in their respective entireties and for all purposes.

[0170] Key physical and design principles of the cryopreservation and recovery system are as follows:

[0171] - Enclosing the sample in a holder that allows liquids to flow freely and easily and at high speed (1 to 5 m / s) past and around the sample, has minimal thermal mass in regions adjacent to the sample, and maximizes convective and conductive heat transfer to / from the sample when moved relative to cold and warming liquids, while minimizing the chance of sample loss during all handling steps. This holder concept is key to allowing automated sample processing.

[0172] - Automating all steps using a single inexpensive robotic platform and sample holders of the type described in (1) and disclosed, for example, in U.S. Provisional Patent Application No. 63 / 601,937, which was filed on November 22, 2023, and is incorporated herein by reference in its entirety and for all purposes.

[0173] - Using a modular design where each module performs a specific set of functions on samples and holders delivered to it by the robot, allowing easy modifications to improve function and easy addition of new functions.

[0174] - Automating soaking of samples in dehydrating and cryoprotectant-containing solutions prior to cryocooling to improve reproducibility, and to facilitate execution of multiple soaking steps at increasing concentration to minimize osmotic shock, without risk of other sample loss or damage.

[0175] - Automating removal of excess liquid surrounding samples prior to cryocooling to minimize thermal mass and maximize heat transfer rates and cooling / warming rates.

[0176] - Automatically documenting the state of the sample using optical imaging immediately prior to cryocooling.

[0177] - Cooling samples at the largest feasible rates by plunging at high speed in liquid nitrogen, by maintaining high relative speeds of sample and liquid nitrogen until the sample has vitrified, and by eliminating sample precooling in cold gas normally present above the liquid nitrogen.

[0178] - Automatically documenting the state of the cryocooled sample using optical imaging prior to sample storage.

[0179] - Automatically transferring the cryocooled sample into a multiple-sample storage cassette, that can be removed for short or long-term cryogenic storage.

[0180] - Automatically transferring cryocooled samples from a storage cassette in liquid nitrogen to a warming solution in a way that minimizes sample prewarming prior to entering the warming solution.

[0181] - Warming samples at the largest feasible rates by plunging at high speed into warming solution, by maintaining high relative speeds of sample and warming solution until the sample has fully warmed (or to at least 0 oC), and also by first plunging in a warming solution at a temperature substantially above the final target temperature and then quickly transferring the sample to a second solution at the target temperature.

[0182] - Automatically transferring samples from the warming solution to reservoirs containing additional solutions for soaking and recovery, to facilitate execution of multiple soaking steps at increasing concentration to minimize osmotic shock, without risk of other sample loss or damage.

[0183] - Automatic transfer of cryocooled, thawed, and recovered samples to a location where they can be retrieved.

[0184] - Automated washing, warming and drying of the sample holding robot end effector to minimize frost accumulation and remove contamination from soaks and plunges.

[0185] - Providing full programmability of most functions, allowing easy modification of soaking / cooling / thawing parameters for optimization.

[0186] Figure 1 gives a perspective-view illustration of a representative example of an automated system 10 for cry opreservation and recovery of small biological samples based on the principles outlined above. The system 10 includes a pick-and-place robot 20 with a custom end effector 40 holding a sample holder 60; an enclosure 80 with a door 100 to allow loading of materials (liquid-containing microplates, liquid nitrogen, etc.) in the enclosure and an externally accessible emergency stop button 120 for the robot, a sample loading carousel 140 into which samples may be loaded and unloaded outside the enclosure using, e.g., a wand 160, a pre-cooling sample soaking station 180, a sample liquid removal and imaging station 200, a sample cryocooling and storage station 220, a robot end effector warming and drying station 240, a sample warming station 260, and a post-warming sample soaking and incubation station 280. An advantageous feature of the illustrated system 10 is its tremendous flexibility: a single robot with a properly designed end effector and sample holder can perform all required functions in cryopreservation and recovery; stations for each function can be swapped out and replaced when they need maintenance or when improved versions become available; and additional stations can be added for new functions, all requiring only modest reprogramming of the robot and control system.

[0187] Figure 2 gives a perspective view of a commercial pick-and-place robot (e.g., an EPSON T3 table-top SCARA robot) illustrating key structures and functions desired for use within the system 10 of Figure 1. The key features may include: (1) high-speed (e.g., at least about 1 m / s and up to about 5 m / s) lateral motion, for rapid transfers of samples between stations; (2) high-speed vertical translations (e.g., at least about 1 m / s and up to about 5 m / s or, in some applications, about 2-3 m / s) for plunging samples into liquid nitrogen and warming solution and rapid transfers of samples between stations; (3) rotation of the sample holding z- axis shaft of the robot about its axis at at least 2 revolutions per second and (with an end effector as needed) up to 30 revolutions / s. For the SCARA robot 300 in Figure 2, the vertical “z” motion is provided by the driven shaft 320, which can also rotate about its axis. The horizontal motion is provided by two rotation stages 340 and 360. The robot arm should allow addition of an end effector 40 to hold the sample holder 60 and possibly also to generate additionalmotion of the sample. Cartesian robots and spherical robots are also suitable for this application.

[0188] Figure 3 shows three examples of robot end effectors, which may include any of the related features presented in U.S. Provisional Pat. App. No. 63 / 601,937, suitable for use in the illustrated system 10. The end effectors allow “grabbing”, holding, and release of a sample holder. Grabbing may use, for example, a magnet, a press fit, a twist fit, a screw fit or a snap fit. Release may use an electromagnet or a mechanical release. The end effector may also provide additional degrees of freedom of sample motion. In Figure 3A, the end effector has a vertical translation mechanism comprised of an outer cylinder 400 and an inner cylinder 410 that holds the sample holder 60 that may be translated in and out (e.g. via a motor and screw mechanism or pneumatically). Attached to the outer, fixed cylinder may be a shield 420 that, e.g., prevents the sample from warming appreciably as it is being transferred from liquid nitrogen to a warming solution, before the sample enters the warming solution. The effector allows translation of the sample in and out of the shield. In Figure 3B, the end effector includes a motor 430 that generates on-axis rotational motion of the sample. In Figure 3C, the end effector includes a motor 440 and stage 450 to generate off-axis rotational motion of the sample. Rotational motions can aid during sample soaking to ensure uniform and reproducible soaking. They can also help in generating and maintaining large velocities of liquid nitrogen / warming solution relative to the sample during cooling / warming required to minimize cooling and warming times. As an example, with a rotation radius of 0.5 cm and a rotation speed of 7200° / second (20 revolutions / s), the sample speed in its circular motion is 0.63 m / s.

[0189] Figure 4 shows an example of a carousel-type sample transfer mechanism 500 for transferring samples from outside the enclosure to inside the enclosure, and vice versa. The rotating sample holding portion 520 is driven by a motor 540 and has one or more receptacles 560 for holding a sample holder. The receptacles may form a tight seal with the sample holder to reduce evaporation from the sample and sample dehydration while it is within the receptacle. The sample holding portion may include a means for maintaining the temperature of samples held within at some desired temperature (e.g., 37 oC), which could include strip heaters 580. Samples in holders are loaded into a receptacle on the outside of the enclosure, and then the carousel rotates the sample into the enclosure in position for retrieval by the robot. Similarly, when sample processing is complete, the robot may deposit the sample into the carousel, which then rotates it outside the enclosure. Other sample transfer mechanisms, such as a sliding stageT1that slides in and out of the enclosure, a single rotating arm, etc., could be suitable, depending upon the number of samples to be processed per unit time.

[0190] Figure 5 shows a possible realization of a station for soaking samples in solutions prior to cryocooling. Typical protocols involve soaks in two solutions - an equilibration solution and a vitrification solution, with the latter having roughly twice the concentration of cryoprotective agents as the first. This reduces osmotic shock, but this soaking can have negative impacts (without cooling and warming) on subsequent survival and development of the sample. Breaking the soaking up into more steps with a more gradual increase in concentration can help. The station 600 in Figure 5 includes a commercial microplate heater 620 (or a thermoelectric microplate stage capable of both heating and cooling), a highly thermally conductive metal block 640 machined to match the shape of the underside of the microplate to provide good heat transfer and uniform temperature, and a commercial or custom microplate 660 containing wells holding soaking solutions. The volume of each well in part depends on the sample size and the size of the portion of the sample holder that is immersed in the liquid in each well. The volume of each well, the number of wells on each plate, and the number of soaking steps / solutions required determine the total consumption of solutions and also the number of samples that can be processed before a new plate must be loaded. The well volume should be as small as is feasible to minimize solution consumption.

[0191] In some applications, it may be desirable to perform the pre-cryocooling soaks at reduced temperatures to limit toxicity or other degrading effects. In that case, a temperature- controlled station incorporating, e.g., a thermoelectric heater / cooler could be used. To obtain more uniform and reproducible equilibration in each solution, the robot arm can oscillate the sample up and down, side to side, and / or in a circle, and the end effector can be designed to execute similar motions (with much less inertia than the robot.)

[0192] The same basic configuration as in Figure 5 can be used for the post-warming sample soaking and incubation station 280.

[0193] The microplates used can be custom fabricated as needed to mate with the sample holders and allow sample holders to be deposited and held in a well by the robot, allowing the robot to perform other functions while a sample is soaking.

[0194] Figure 6 shows a possible realization of a station for removing excess liquid from samples and imaging samples prior to cooling. The station 700 includes of a body 710 enclosing a hollow interior; a port 720 to which suction (e.g., produced by a pump or acompressed air vacuum generator) can be applied, which may include a connector / adapter for connecting the body to the pump via a hose; a sample suction port 730 with an aperture that connects to the hollow interior of the body, and that is sized and shaped so as to provide a seal to a sample holding portion of the sample holder, a gasket 740 of a compliant material such as rubber to seal the sample holder to the suction port; a back illumination source 750 (e.g., an LED light and lens) for illuminating the sample when it is in position on the suction port; a transparent or translucent window 760 in the station body that transmits light from the source to the sample; and a compact digital microscope / endoscope / borescope 770, which may including additional LEDs for sample illumination, for imaging the sample.

[0195] Figures 7 to 9 show a possible realization of a station for rapid cooling and cold storage of samples in liquid nitrogen. Experiments indicate cooling rates of up to about 25,000 °C / min can be achieved when cooling 500 pm samples and up to about 3,000,000 °C / min can be achieved when cooling 25 pm samples. The station 800 includes of a thermally insulated enclosure / Dewar 810 and a reservoir 820 within the enclosure. The volume 830 enclosed between the enclosure and the reservoir holds a first volume of liquid nitrogen, and the volume enclosed by the reservoir holds a second volume of liquid nitrogen. The liquid nitrogen within the reservoir is in good thermal communication with the first volume of liquid nitrogen within volume 830. The walls and bottom of reservoir 840 may be of a good thermal conductor like steel, aluminum or copper, and walls of enclosure 810 may be of a good thermal insulator such as urethane foam.

[0196] The volume of liquid nitrogen within the reservoir 820 is at a level above the level of the liquid nitrogen in the volume 830 and is in fluid communication with that volume such that if the liquid nitrogen within the reservoir 820 rises above a preset level, the excess liquid nitrogen will flow into the volume 830. The strong thermal contact between the first and second volumes of liquid nitrogen suppresses boiling of liquid nitrogen within the reservoir. Together with the overflow mechanism, this helps maintain a well-defined and steady level of liquid nitrogen within the reservoir.

[0197] Enclosure 810 is covered by an insulated cover 850 with a hinged / sliding / rotating door 860 providing access to a portion of the reservoir. The bottom of the reservoir below the door includes a receptacle 870 for holding a cassette or puck 880, which in turn has receptacles 890 for one or more sample holders. The cassette is of a cryogenically compatible and dimensionally stable material like aluminum or steel. The cassette may be placed in the dewar and removed from it using a tool. Dry gas may be supplied to the space in the reservoir abovethe liquid nitrogen when the door is open to minimize infiltration of moist ambient temperature air and frosting.

[0198] The insulated cover 850 includes an aperture adjacent to the door that holds a gas management manifold 900 containing a plunge bore 910 extending from the top surface of the manifold through its bottom surface, allowing direct access to liquid nitrogen within the reservoir. The plunge bore may be bounded on one side by hinged doors 920 that extend below the surface of the liquid nitrogen within the reservoir and whose function will be described later.

[0199] A key function of the manifold is to remove all cold gas above the liquid nitrogen within the bore and replace it with ambient temperature dry gas prior to the sample plunge, and also to prevent warm moist ambient air from causing frost build up on cold surfaces and in the liquid nitrogen.

[0200] Turning now to Figure 8A which shows a cross section view of the manifold 900, the manifold 900 has ports for dry ambient temperature gas 930 and for suction 940. These ports connect to opposing channels whose openings 950 and 960 at the plunge bore form rectangles. The liquid nitrogen level within the reservoir 820 is set by the fill and overflow mechanism mentioned above and described in more detail below, so that the gas-liquid interface rises roughly midway up the channel openings, so that cold gas is more readily swept away by suction and make-up dry gas and so that gas flows are largely laminar to collapse the thickness of any cold gas layer below 100 micrometers. The manifold may contain additional openings to the bore above the vacuum and make-up gas channels through which additional dry gas may be supplied to further inhibit moist air infiltration into the bore and frosting. The manifold may also include heaters, particularly on the bore walls, to minimize frost accumulation.

[0201] As shown in Figure 8 B, the door 860 covering the reservoir may be transparent and made of a glass or polymer to allow visual inspection of cassettes and samples within the cassette.

[0202] Before plunge cooling a sample, the cold gas will be removed from above the liquid nitrogen within the bore 910, and a sample will be plunged by the robot through the bore 910 into liquid nitrogen, and then transferred from the bore and into a receptacle 890 in the cassette / puck 880. To facilitate that transfer without requiring that the sample be removed from liquid nitrogen, one side of the bore may have a door or doors 920 that are pushed open by the robot, allowing the sample to be dragged from the bore through liquid nitrogen and into position above a receptacle in the cassette. The door(s) (which may be spring loaded or heldmagnetically then automatically close after the transfer. Once a cassette has been filled, the cassette may be removed and transferred to a storage dewar for storage. The door 860 covering the portion of the reservoir holding the cassette may be optically transparent to allow visualization of transfers and to detect errors in transfers.

[0203] Proper operation of the cooling station 800 requires that the level of liquid nitrogen within bore 910 of manifold 900 be precisely maintained near the middle of the openings of vacuum and make-up gas channels 950 and 960 at the bore. Figure 9A shows a possible configuration for implementing control of liquid nitrogen levels within the cooling station. As in Figures 7 and 8, a reservoir 1000 formed of a good thermal conductor like steel, aluminum, or copper is located within a second, larger, thermally insulated enclosure 1010. Thermal insulation may be provided by a solid thermal insulator 1020 (e.g., urethane foam) or by vacuum held within a sealed chamber. The inner surface of the insulated chamber may be comprised of a metal (e.g., steel) “tub” 1030 to contain the liquid nitrogen, prevent its infiltration into the insulation should the insulation degrade or fail, and also to provide a more uniform temperature for the internal surface. The top of the walls of reservoir 1000 is located below the top of the walls of enclosure 1010.

[0204] Liquid nitrogen supply lines 1040 and 1050 connect to the interior of the reservoir 1000 and to the space 1060 between the reservoir 1000 and inner surface 1030 of the insulated enclosure 1010. These may be connected to a pressurized liquid nitrogen storage dewar or to house liquid nitrogen supply via valves. Alternatively, they may be connected via valves 1070 and 1080 to an insulated liquid nitrogen supply container 1090 (which may be formed of insulating foam lined with a metal (steel) “tub”). The liquid nitrogen supply container 1090 may be elevated relative to the reservoir 1000 and insulated enclosure 1010 so that flow of liquid nitrogen between the supply container and reservoir and enclosure is driven by gravity. The supply lines may connect near the bottom of the reservoir and near the bottom inner surface of the insulated enclosure, to reduce boil-off during filling. The supply lines should be well insulated or else cooled by contact with cold gas or liquid nitrogen.

[0205] Liquid nitrogen levels within reservoir 1000 and within the space 1060 between the reservoir and the insulated enclosure may be measured with level sensors 1100 and 1110, such as a heated RTD sensor, a thermocouple, a diode, a laser level sensor, or a float-type sensor.

[0206] A nearly constant level of liquid nitrogen within the plunge bore 900 is maintained by maintaining the liquid nitrogen level within the reservoir 1000 at or very near the top of thewalls of the reservoir. When the level in the reservoir drops below a target value (typically about 2-3 mm below the top of the reservoir), liquid nitrogen flows into the reservoir to fill and then overfill it, with the overflow being collected in the volume 1060 between the reservoir and insulated enclosure. Experiment shows that nearly all boiling of liquid nitrogen occurs in the volume 1060 between reservoir and enclosure; the level of liquid nitrogen within the reservoir 1000 drops much more slowly, and the surface of the liquid nitrogen within the reservoir is largely quiescent, indicating a near absence of boiling. This allows long operating time between liquid nitrogen filling / top-up of the reservoir. In this way, the liquid nitrogen level within the bore of the manifold 900 is maintained near the middle of the openings of the vacuum and make-up gas channels 950 and 960, as required for effective cold gas layer removal.

[0207] Rather than being set by the top of the walls of the reservoir, the liquid nitrogen level within the reservoir 1000 could be set by an aperture in one of its walls located below the top of its walls, through which liquid nitrogen will (over) flow into the space between the reservoir and enclosure.

[0208] An alternative approach to filling the reservoir 1000 is to pump liquid nitrogen from the volume 1060 into the reservoir 1000 until the reservoir overflows, with pumping turned on and off depending on the reading of the level sensor 1100. Any liquid-nitrogen compatible pump can be used. Impeller or centrifugal pumps are particularly well suited as they require no valves or seals and no contact between moving and non-moving parts. The pump may be driven from above by a shaft projecting down into the liquid nitrogen to the impeller.

[0209] In operation, liquid nitrogen supply container 1090 may first be filled with liquid nitrogen. During or after filling of the supply container, the valve 1070 controlling flow to the volume 1060 between the reservoir and enclosure may be opened, cooling and then filling that volume to a preset level as measured by the level sensor 1100. Valve 1070 is then closed, and valve 1080 to the reservoir will open, filling the reservoir until it overflows, as detected by the level sensor 1100. When the level in either the reservoir 1000 or the volume 1060 drops below a target value, the corresponding volume will be topped up with liquid nitrogen.

[0210] In a prototype system functionally similar to that shown in Figure 9A and illustrated in Figure 9B, the reservoir was cylindrical with a diameter of 15 cm, a height of 10 cm, and a volume of about 1.8 liters; the outer insulated chamber had inner dimensions of 25 cm long x25 cm wide x 15 cm tall, and a volume of about 10 liters; and the insulated supply container had inner dimensions of 10 cm wide x 30 cm long x 4 cm tall and a volume of about 3 liters.

[0211] The cooling station may include an optical imaging system similar to that shown in Figure 6 for imaging the sample after cryocooling to evaluate whether the water in the sample has vitrified (in which case the sample will appear clear and internal structures within the sample will remain visible) or if some fraction has crystallized (in which case the sample will appear milky.) The imaging system may be comprised of an LED light source (with a lens or translucent plate in front and possibly a fiber optic or mirror to direct the light at the sample) providing illumination from the behind the sample for transmitted light illumination, a compact digital microscope / endoscope / borescope, which may include an imaging chip, a series of lenses, and possibly also a fiber optic, and possibly also additional LEDs for epi illumination. These components can be mounted in reservoir 820 (or 1000), above the sample cassette and, if desired, immediately above the surface of the liquid nitrogen. With the door to the reservoir closed and any source of dry ambient temperature gas used to prevent moisture infiltration when the door is opened turned off, cold gas will accumulate above the liquid nitrogen surface. A sample may then be briefly raised into this cold gas above the liquid nitrogen surface for imaging. Preliminary experiments using a $30 endoscope camera show that such cameras function well at cryogenic temperature provided they are kept dry. Alternatively, a fiber endoscope in which optical fibers are used to transmit light to a sensor at room temperature may be used.

[0212] For warming and recovery of cold samples, a sample cassette 880 can be loaded into the reservoir 820 of the cooling station, and samples retrieved by the robot directly, through the door 860 over the reservoir, or by first translating them from the cassette, through the doors 920 and into the plunge bore. The latter may be desirable to minimize frosting of cold surfaces. Alternatively, the sample cassette 880 could be loaded into a separate insulated Dewar filled with liquid nitrogen and having a receptacle for the cassette in its bottom (similar to 870) so ensure accurate and reproducible cassette placement for reliable robotic retrieval.

[0213] Figure 10 shows a possible realization of a station for rapid warming of cold samples. For the fastest warming, the sample must travel at high speed (1-5 m / s is a practical range) relative to the warming solution until it has warmed well above the melting point of the sample (typically near -10 oC if it has been soaked in cryoprotectants). If the relative motion is solely vertical / translational, then for larger samples that warm more slowly, this may require a large plunge depth - much larger than is used in current practice. Alternatively, if the sample is rotated at high speed as well as translated, the plunge depth can be reduced. The warmingstation realization 1200 in Figure 10 includes a commercial microplate temperature-controlled block heater 1210, a highly thermally conductive plate enclosure 1220, and a deep well block plate 1230. The plate enclosure can be comprised of a high thermal conductivity metal (e.g., aluminum) in intimate thermal contact with the block heater, whose sides may be covered by a thermal insulator (e.g., foam.) Standard deep well block plates have well depths of roughly 4 cm, which should be sufficient for warming of samples with diameters less than roughly 150 micrometers when using plunge speeds up to about 2 m / s, which includes most mammalian oocytes and embryos. For larger samples or larger plunge speeds, significantly deeper wells - perhaps up to 10 cm or even 20 cm - may be required to ensure that large convective fluid velocities relative to the sample are maintained until the sample is fully warmed. This would require either custom deep well block plates or use of arrays of test tubes, cuvettes, or similar containers. Combining vertical and off-axis rotational motion of the sample is likely to be more desirable in that case to minimize consumption of solutions.

[0214] The warming rate of the sample at any given time is determined in part by the temperature difference AT between the surrounding liquid and the sample. Sample warming rates are thus largest right after the sample enters the warming solution, when this temperature difference is largest, and get smaller as the sample’s temperature approaches that of the warming solution. For a warming solution temperature of 37 oC, the “driving force” for warming decreases from AT =37 oC + 196 oC = 233 oC when the sample first enters the warming solution to AT =37 oC + 10 oC = 47 oC when the sample temperature has reached -10 oC, a decrease by a factor of 4. The specific heat of the sample increases as temperature increases, further decreasing the warming rate. Ice nucleates and grows rapidly once the sample temperature rises above about -70 oC and nucleation peaks around -40 oC, where the warming rate has decreased substantially. Consequently, there is a need to increase warming rates in the temperature range between about -70 oC and typical sample melting temperatures (about -10 oC). Eukaryotic cells generally do not fare well at temperatures above 45 oC although some can survive at temperatures as high as 60 oC. This sets an upper bound for the temperature of the final solution in which sample may reside.

[0215] One way to increase warming rates is to increase the temperature of the warming solution. For example, increasing the solution temperature to near 100 oC increases the driving force when the sample temperature has reached -10 oC to AT =100 oC + 10 oC = 110 oC, a factor of 2.3 increase. However, most biological samples cannot survive heating to temperatures well above biological temperatures. This suggests that, after warming beyond theice nucleation and growth “danger zone”, the warming solution temperature should be reduced to a biologically suitable temperature, e.g., 37 oC.

[0216] Figure 11 shows possible realizations of warming solution cells suitable for producing changes in warming solution temperature along a sample’s plunge path. Figure 11A shows a side section view of a warming cell 1300. The warming cell 1300 is divided into an upper chamber 1310 and a lower chamber 1320. A heated block 1330 warms the warming solution in the upper chamber to an elevated temperature Ti in the range of 50-100 oC. A second heated block 1340 warms the warming solution in the lower chamber to a biological temperature T2 (e.g., 37 oC) or other lower (or higher) temperature that is tolerated by the biological sample. Because warmer solutions are less dense, this configuration should be stable against buoyancy driven convection. The opening between the upper and lower chamber may be covered by a door 1350 or flexible flap to isolate the two chambers. During warming, the sample is plunged vertically, first traveling through the warming solution in the first, upper chamber, and then traveling through and coming to rest in the warming solution in the second, lower chamber. The plunge speed, chamber sizes, chamber temperatures and sample size can be adjusted to give a desired sample temperature upon leaving the upper chamber. The door or flap may be pushed open by contact with the sample holder or with the robot’s end effector. The required travel time / distance / speed through the solution at the first, higher temperature can be determined in calibration experiments using actual (live or dead) samples. Figure 11B shows a top view of an alternative warming cell 1360. The “hot” and “cold” chambers 1370 and 1380 are arranged side by side, and are surrounded by heated blocks 1390 and 1400. The chambers are separated by a door or flexible member 1410. The sample is first immersed in and translated through the warming solution in the hot chamber and then translated into and through the warming solution in the cold chamber. The overall vertical cell height in Figure 11A will depend on sample size and warming rate but may be in the range of 2 cm to 10 cm and possibly as large as 20 cm, e.g., closer to 4 cm to minimize warming solution consumption.

[0217] Figure 12 shows top and side section views of an alternative sample warming cell 1420. The cell has a long channel 1430 that intersects a larger well 1440. The bottom of channel 1430 may descend from the top of the cell to the bottom of the well 1440. The upper portion of the cell and channel are adjacent to a block heater 1450 that heats the warming solution in the channel to an elevated temperature Ti in the range of 40-100 oC. A second heated block 1460 warms the warming solution in the lower portion of the channel and in the well 1440 to a biological temperature T2 (e.g., 37 oC) or other lower (or higher) temperature that is tolerated bythe biological sample. The wanning solution heated in this way should be stable against buoyancy-induced convection, which can be further reduced by making the channel 1430 no wider than is necessary to ensure good warming solution flow past the sample, and by having a much larger volume of solution within the well 1440. During warming, the sample travels at high speed (1-5 m / s) at an angle down the channel and into the well. This configuration is convenient in that it allows a long sample travel distance while reducing the total volume of warming solution required.

[0218] Figure 13 shows top views of a sample warming microplate 1460 with long channels 1470 and a matching metal block 1480 for block heating. This microplate is designed for use with sample holders that fully enclose the sample and where there is no chance of sample loss during warming. As a result, there is no need for a well for sample retrieval at the end of the warming channel as in Figure 12. Sample holders such as that shown in Figures 15 and 16 are well suited to warming in this configuration. For those holders, which have a thickness of less than about 250 micrometers, the channel may be between about 1 and about 10 mm wide or, for some applications, between about 2 and about 5 mm wide to reduce warming solution consumption.

[0219] Figure 14 shows a representative example of a station 1500 for washing, warming, and drying the robot end effector 40 after cooling and warming. The station 1500 includes a cylindrical bore tube 1510 lined with resistive or radiant heaters (or a set of heaters roughly arranged in a cylindrical manner) and a cylindrical air amplifier 1520 (e.g., as manufactured by Exair) that generates a strong air flow through its center bore when connected to a source of compressed gas. An electrically actuated valve can be used to turn the compressed gas flow to the air amplifier on and off. In use, the robot’s z axis motion arm 320 translates the end effector 40 through the heaters 1510 and air amplifier 1520 one or more times until the effector has warmed and all moisture has evaporated. The station could also include a sprayer that sprays water, detergent solution, isopropanol, or other solvents and cleaning and rinsing solutions, or a series of containers filled with different rinsing solutions.

[0220] Figures 15-21 show examples of sample holders suitable for use with the present system. Desirable features of these holders may include, for example:After the sample is loaded, the holder is sealed or closed so that the sample cannot escape from the holder during handling / processing. The sample is retrieved from the holder after processing.The holders have an open mesh design that allows full contact of soaking, cooling and warming fluids with the sample, and that allows cooling and warming fluids to flow with little resistance at high-speed past and around a sample, while having mesh openings small enough to prevent sample escape / loss.The thickness of the portion of the mesh on which the sample(s) resides is less than about 100 micrometers or, for some applications, is less than about 25 micrometers and ideally between 5 and 15 micrometers. This will minimize thermal mass and maximize heat transfer through solid portions of the mesh to the sample. This will help ensure that the sample residing on the mesh quickly cools and becomes frozen to the mesh during plunging in liquid nitrogen, rather than becoming detached from the mesh. These features will help maximize cooling and warming rates.The portion of the mesh in contact with the sample should be compliant so that differential contraction of the mesh and sample during cooling does not cause sample fracturing. This suggests use of thin polymers rather than metals for the mesh portion supporting the sample.The mesh openings are as large as possible while being small enough that there is no chance of samples passing through them. For 100 micrometer samples, 50-80 micrometer openings may be suitable. For mammalian oocytes and embryos, 40 to 120 micrometer openings may be suitable. The ratio of sample dimensions to mesh opening dimensions will vary depending on the sample size and deformability.The solid area fraction of the mesh should be as small as is feasible given constraints on mechanical strength required to withstand high speed plunges, to minimize flow resistance.The sample holder includes a rigid portion that holds the mesh portion and maintains overall shape and rigidity during high-speed plunges. The rigid portion should be easily grabbed by the robot and should have precisely defined dimensions to facilitate automated handling.The sample holder may include components made of magnetic steel or that are magnetic to facilitate handling using magnets.

[0221] Figure 15 shows a representative sample holder 1600 which may include any of the options and features disclosed in U.S. Provisional Pat. App. No. 63 / 601,937. Holder 1600 includes a thin rigid frame 1610 attached to a base 1620, where the base is designed to mate withthe robot end effector and be reliably handled by the robot. The frame 1610 may be fabricated from metallic materials, polymeric materials, polymer-glass composite materials, or combinations thereof, and should be thin and very rigid, so that it does not bend appreciably when it impacts and enters liquid nitrogen and warming solution during high-speed plunges. The frame thickness should be comparable to or somewhat larger (e.g., 80% to 150% of) the dimension of the sample to be held, so that the sample can be sealed within the frame without excessive compression and so that fluid flows adjacent to and around the sample during plunging are optimal for heat transfer. For human and bovine oocytes, which have a size of 110-120 micrometers, the frame may be roughly 100 to 150 micrometers thick. The frame width can vary depending on the size and number of samples to be held and processed on each sample holder. Frames from about 3 mm to about 10 mm in width may be desired and may be up to 30 mm; attaching the thin film to the frame becomes difficult as the frame width becomes smaller, and too wide a frame may be inconvenient for high volume storage. The length of the frame can range from roughly 2 to 4 times the width to a maximum of 10 cm. During high-speed plunges into liquids, the greatest liquid disturbance and splashing will occur if and when the frameholding base 1620 impacts the liquid. To eliminate this, the frame length extending beyond the base can be comparable to or somewhat longer than the plunge depth, which may vary between 1 and 20 cm, depending on sample size and plunge speed.

[0222] The base 1620 may be of any hard, dimensionally stable material. Magnetic stainless steel is a good choice if the effector is to use magnets. The base can be marked or can incorporate an RFID tag for sample identification and tracking.

[0223] Frame 1610 has at least one aperture 1630, at the end furthest from the base. The aperture is sealed on one side (the “bottom” side) by a thin film 1640, where the film portion within the aperture is filled with openings 1650 that are too small to pass the sample but otherwise are as large as possible, and that occupy as large an area fraction within the aperture as is feasible given constraints on mechanical robustness during handling.

[0224] The film 1640 may be of polymers, metals, and semiconductors, but polymers may be desired because they are compliant and so should reduce stress on the sample during cooling and warming, and also because they can be easily processed. The thin film “mesh” may have two different thicknesses. For example, the thickness within the aperture may be 5-15 micrometers to maximize heat transfer rates and compliance, and 10-50 micrometers in the region in contact with the frame to facilitate handling and assembly. The thin film mesh may have special patterns either of through holes or of thicker regions in particular areas of the filmto indicate where samples should be loaded and also to create textures to help keep samples in place on the film. Ideally, the sample should be loaded away from the edges of the frame’s aperture to maximize heat transfer rates, as fluid flows are most perturbed near the interior edges.

[0225] To seal the sample within the frame, a second, similar thin film 1660 may be applied to the top surface of the frame to cover and seal the aperture. Again, this film should have a large open area fraction and openings that are just small enough to prevent the sample from escaping. The films may be applied with a permanent adhesive, a removable adhesive, a material that has a relatively low melting temperature (near biological temperature) such as a wax, or a material like a wax that is easy to remove mechanically after the film is applied. One or both films may include features such as perforations 1670 or tabs 1680 that make it easy to remove the mesh and extract the sample.

[0226] If the frame thickness is less than the sample thickness, the sample will be compressed when the second film is applied. Slight compression is well tolerated by most biological samples and ensures that the sample is optimally held to maximize cooling and warming rates. However, contact with both films may lead to sample stresses during cooling and warming unless the films are highly compliant, contact with both films may also make sample retrieval after processing more difficult.

[0227] During high-speed plunges, liquid nitrogen and warming solution will immediately flow through the mesh and wet the sample, ensuring rapid cooling. In typical use, the frame may be plunged with the aperture-containing end pointing downward and the frame’s axis oriented perpendicular to the liquid surface, to minimize the cross-section impacting the liquid surface and the force on the frame and films. Alternatively, the frame could be plunged at an angle to the liquid surface to force liquid flow through the mesh. The sample holder could also be rotated at high speed during plunging to force liquid through the mesh. This rotation can be about the central axis of the holder, or about an axis parallel to the holder’s axis.

[0228] Figure 16 shows a second sample holder design 1700 related to the design shown in Figure 14. In this case, the frame 1710 has a U-shaped opening 1720 at its end (with the top, open portion of the “U” at the bottom of the frame in Figure 16), and the mesh film 1730 covers the opening on a first side of the frame and is attached to the frame, and then wraps around the frame’s end. After sample loading onto the mesh, the sample may be sealed in the frame by either pressing down the “free” end of the mesh film 1730 (which may have anadhesive gasket) if the film extends to cover the aperture on the second side of the frame, or by placing and pressing down a second mesh film. The advantage of this design is that liquid nitrogen or warming solution can enter the leading edge of the holder through the mesh and flow through the mesh on the leading edge and past the sample, rather than having to flow around the leading edge of the frame as in Figure 15.

[0229] For both the designs of Figures 15 and 16, the second film could be attached to a separate frame and then held in place aligned with the first frame using a clip or similar mechanism.

[0230] Figures 17-20 show a third sample holder design. In this case, the holder includes two parts: a “basket” 1800 (Figure 17) and a cap 1900 (Figure 19). The basket (Figure 17) is formed using a rigid frame 1810 (e.g., of a polymer) whose openings at the bottom and sides of the basket are covered by a thin open mesh film as in the design of Figures 15 and 16 and / or a thin mesh made of polymer monofilament or metal wire. Cap 1900 is designed to press fit, twist-fit, or snap fit into the top of the basket, sealing the samples inside, and to be easily removed from the basket at the end of sample processing. The cap is designed to be grabbed / held by a robot end effector and may include a magnet or a piece of magnetic stainless steel. The cap can be formed of any hard, dimensionally stable material, but its thermal expansion should be well matched to that of the basket’s frame. It should ideally have low thermal conductivity and relatively low thermal mass so as not to require excessive heat transfer for its cooling and warming. This suggests the use of the same polymer as is used for the basket frame for the portion of the cap that inserts into the basket.

[0231] The advantages of this design are (1) liquids are forced to flow directly through the basket and around the sample as the sample holder is plunged basket-side down into liquid, which may increase rates of heat transfer; and (2) loading samples into the holder and then sealing them into the holder is simplified, requiring no adhesives or playing with films.

[0232] The diameter of the basket will be set by the number of samples to be cooled together, by the size of each sample, by the size of the tool used to deposit and remove samples from the basket, and by minimum dimensions that can be conveniently manufactured and assembled. For 50-200 micrometer size samples, typical of mammalian oocytes and embryos, the diameter at the bottom end of the basket may range from ~2 mm to 10 mm or, perhaps, as large as 20 mm. The height of the basket is set by the requirement that liquids be able to flow freely with minimal resistance through the basket, that the samples be far enough away from the cap that their coolingand warming is not impacted by it, and by the ease of retrieving samples placed within the basket. The apertures in the side of the basket through which liquid can exit during a plunge should extend between 2 and 4 times the base diameter upward from the base (e.g., between about 6 mm and about 40 mm). If needed to ensure good flow, the cap may also include a mesh portion to allow liquid to flow through it and out of the mesh.

[0233] The bottom of the basket may be covered by a thin (5-50 um) polymer film of a mechanically robust and cryogenic compatible polymer like polyimide or cyclic olefin copolymer, with a pattern of holes as described above for the sample holders shown in Figures 17 and 18. Since samples will be deposited onto the bottom of the basket, these thin films may provide the best cooling and warming performance, both in terms of cooling / warming rates and in terms of minimizing stress due to differential contraction with the sample. Polyimide and COC are both optically transparent and so will allow visualization of the samples placed on the film within the holder. The film can be manufactured, for example, by microfabrication. The film may then be glued or ultrasonically bonded to the basket frame, either to the inside of the frame (as shown) or, perhaps more conveniently, to the outside of the frame, or else incorporated during injection molding of the frame. A single film 1830, manufactured flat, can be wrapped around and bonded to the frame.

[0234] The basket bottom and side openings can instead be covered by a mesh of fine metal wire or polymer monofilament. Woven meshes will give larger flow resistance than the micropattemed thin films and also have worse thermal conduction.

[0235] Since only the sample supporting bottom of the basket need provide good transparency for imaging and be compliant to minimize sample stress during cooling, the bottom of the basket 1840 can be covered with the thin polymer film, while the side openings in the basket 1850 (and any openings in the cap) may more conveniently be covered with nylon monofilament or wire mesh, which will be more mechanically robust and easier to handle than thin microfabricated films. Mesh sizes in the range of 60 mesh to 200 mesh should be suitable for mammalian oocytes and embryos. Because the area of the side openings in the basket is much larger than the area of the bottom opening, the flow velocities through the sides will be smaller, offsetting the greater flow resistance when using a mesh there.

[0236] As shown in Figure 18, the basket 1860 may have a hemispherical bottom 1870 rather than a flat bottom, with the hemispherical portion covered with a holey film or mesh and supported by a suitable frame.

[0237] As shown in Figure 19, the cap 1900 may be press fit into the basket 1800 and may be held by hand tool 1910 or a robot end effector that grabs the cap magnetically, via a twist fit, or other standard mechanism. The cap may be designed to encourage laminar flow of liquid out the sides of the basket away from the sample-holding base of the basket. This could involve having a downward-pointing conical surface 1920 where the cone steers fluid away from the central axis of the basket and toward the mesh covered sides. The tool or end effector may have a mechanism 1530 that allows the basket to be pushed off the cap to allow samples to be retrieved after processing. The cap may have indentations or other features that mate with a rigid support and keep it fixed to facilitate installation of the cap 1900 after sample loading in the basket and release of the cap from the tool 1930 or end effector after sample processing.

[0238] Figure 20 shows an example of a tray 1950 with a series of receptacles 1960 that hold baskets 1800 (and possibly also caps) for initial sample loading and basket+cap assembly. The sample may be deposited into the basket, e.g., using a pipette (either manually or robotically). A tool 1910 can then be used to pick up the cap, press it into the basket, and then pick up the cap and basket and transfer to a receptacle in the sample transfer or loading station. Alternatively, a tray of samples + baskets + caps could be rotated or translated into the robot enclosure and the robot could assemble the basket + cap and then begin motions required for sample processing.

[0239] Figure 21 shows another representative sample holder design. Here, an initially flat thin film 2000 is patterned with slits 2010 so that a portion of the film can be deformed to form a basket 2020 (as is known in kirigami) into which a sample may be deposited. The slit openings in the deformed basket must be smaller than the minimum dimension of the sample to prevent sample loss. Prototypes fabricated using polyimide work very well and require only mechanical force for permanent deformation into a basket. Other materials may require thermoforming. The advantage of this approach to creating a 3D basket is that it can produce 100 micron and smaller holes in the basket, required to retain many biological samples of interest. Figure 2 C shows how two such baskets 2020 can be assembled face-to-face to produce a holder that fully encloses a sample.

[0240] Figure 22 shows an example of a sample holder that can be opened and closed using a tool that mates to the base of the holder. In this case, the hole-filled thin films 2100 (which may be flat as in Figure 15 or form a basket of the type shown in Figure 21) are attached to one end of a semi-rigid member 2120 so that they span and seal an aperture in the member or, alternatively so that they project from the end of the semirigid member. The semi-rigid members 2120 are mounted on the opposite sides of the base 2140 and presstogether, so that the films form an enclosed volume. The base has a notch 2160 to allow a tool to be attached to the base with a particular orientation about the base axis. The tool may attach magnetically. The tool may have a central rod connected at one end to a push button and that projects through the hole 2180 in the base toward the semi-rigid members 2120. When the push button is pressed, the rod pushes the two semi-rigid members apart, opening the “jaws” and allowing access to each film. Other mechanisms can be used to open and close the holder. For example, the two sample accepting “jaws” may be hinged together and have a naturally open configuration. Pulling the hinge into a receptacle can close the jaws, as with a sponge mop. Alternatively, a sliding member can slide and push on one of the jaws, closing it, as in some jug lids.

[0241] Aspects of the present disclosure have been described in detail with reference to the illustrated embodiments; those skilled in the art will recognize, however, that many modifications may be made thereto without departing from the scope of the present disclosure. The present disclosure is not limited to the precise construction and compositions disclosed herein; any and all modifications, changes, and variations apparent from the foregoing descriptions are within the scope of the disclosure as defined by the appended claims. Moreover, the present concepts expressly include any and all combinations and subcombinations of the preceding elements and features.

[0242] Additional features and options of this disclosure may be reflected in the following clauses:

[0243] Clause 1 : A robotic system for cryopreservation and recovery of small biological samples, the system comprising a sample holder that encloses and retains at least one sample, a series of sample processing stations, and a robot and robot end effector that executes required sample motions between and within stations.

[0244] Clause 2: the system of clause 1, where the small biological sample has a volume less than about 10 microliters or, in some applications, less than about 1 microliter or, some applications, less than about 0.1 microliters, or in applications involving mammalian oocytes and embryos, less than about 10 nanoliters, a thickness of less than about 2 mm and in some applications, less than 500 micrometers or, in applications involving mammalian oocytes and embryos, less than about 200 micrometers.

[0245] Clause 3: the system of clause 1, where the series of stations for sample processing includes a station for soaking the sample in solutions (for example, equilibration andvitrification solutions) prior to cryocooling, a station for removing excess liquid from the sample and holder and imaging the sample prior to cryocooling, a station for rapid cooling of the sample by plunging at high speed into a liquid cryogen such as liquid nitrogen, a station for storing cryocooled samples in liquid nitrogen and for loading cryocooled samples to be warmed and recovered, a station for warming cryocooled samples by plunging samples into a warm solution, a station for soaking and incubating samples after warming, and a station for warming and drying the robot end effector.

[0246] Clause 4: the system of clause 1, where the robot and stations are located within an enclosure, where at least a portion of the enclosure is transparent to allow viewing and monitoring, and where the enclosure has at least one door allowing transfer of materials and equipment between the interior and exterior of the enclosure.

[0247] Clause 5: the system of clause 4, where the system includes a sample transfer station for transferring samples between the exterior and interior of the enclosure, and to a position where they may be retrieved by or placed by the robot.Robot and end effector

[0248] Clause 6: the system of clause 1, where the robot end effector is configured to grip and release the sample holder, and to provision lateral and vertical translation of the sample holder, wherein the vertical translation of the sample holder is at speeds of at least about 1 m / s and up to about 5 m / s or, for some applications, about 2 m / s, and rotation of the sample holder about its axis.

[0249] Clause 7: the system of clause 6, where the robot end effector provision highspeed rotational motion of the sample holder about an axis, so that the maximum speed of the sample holder in its rotational motion may reach at least about 0.25 m / s or, for some applications, about 0.5-2 m / s, and where the robot end effector may also allow vibrational motion of the sample holder.

[0250] Clause 8: the system of clause 1, where the robot is a pick and place robot.

[0251] Clause 9: the system of clause 1, where the robot is a SC ARA robot.Sample transfer station

[0252] Clause 10: the system of clause 5, where the sample transfer station rotates or translates samples at room or biological temperature between positions outside and inside the enclosure, where the location inside the enclosure is accessible by the robot.

[0253] Clause 11 : the system of clause 10, where the sample transfer station is a motor driven carousel with one or more receptacles for sample holders, and where the sample transfer station may including a heater and temperature control system to maintain the samples at a temperature above room temperature and, for example, near a biological temperature.Pre-cooling soaking station

[0254] Clause 12: the system of clause 3, where the station for soaking the samples in solutions (for example, equilibration and vitrification solutions) prior to cryocooling includes a temperature control system allowing the temperature of solutions and samples held within to be varied within a range between OoC and 50oC (for example, between 4oC and 50oC or 20oC and 40 oC) , and to be held near a biological temperature.

[0255] Clause 13: the system of clause 12, where the station for soaking the samples in solutions prior to cryocooling may accept standard or custom multiwell microplates in thin, standard, and deep well formats for containing the pre-cooling soaking solutions.

[0256] Clause 14: the system of clause 13, where microplates are held on / within a commercial well plate heater / well plate temperature control system.

[0257] Clause 15: the system of clause 13, where there may be multiple different solutions in which each sample holder is soaked, each held within a different well in the microplate, and where each sample holder may be soaked in a different set of solutions held in different wells.

[0258] Clause 16: the system of clause 12, where the robot end effector translate the sample holder to immerse its samples in each solution in each well, and where they may oscillate or rotate the sample to ensure uniform and reproducible soaking.

[0259] Clause 17: the system of clause 12, where the microplates and sample holders are designed to mate in a way that allows the robot to release a first sample holder and perform other tasks with other sample holders while the first sample is soaking, and then to return to the first sample holder and find it in the same position and orientation for retrieval.Post-warming soaking and incubation station

[0260] Clause 18: the system of clause 3, where the station for soaking and incubation of samples after warming includes the same or similar components but with different solutions from those of the station for soaking samples prior to cooling.

[0261] Clause 19: the system of clause 18, where the station for soaking and incubation of samples after warming may further include means for controlling the atmosphere above the soaking liquids including the CO2 content during incubation.Liquid removal and sample imaging station

[0262] Clause 20: the system of clause 3, where the liquid removal and sample inspection station allows excess liquid to be removed from the sample and holder to minimize total thermal mass and maximize cooling and warming rates, and where the station allows optical imaging of the sample after liquid removal to document its pre-cry opreservation state.

[0263] Clause 21 : the system of clause 20, where liquid is removed from the sample and holder by bringing the sample in contact with or closely adjacent to the station and briefly applying suction to a portion of the sample holder in fluid communication with the sample, where the suction may be generated using a pump or compressed air vacuum generator, and where it may be turned on an off using an electrically actuated valve.

[0264] Clause 22: the system of clause 20, where liquid is removed by blowing compressed gas onto the sample and holder.

[0265] Clause 23 : the system of clause 20, where liquid may be additionally removed by bringing the sample holder in contact with an absorbent material such a filter paper.

[0266] Clause 24: the system of clause 20, further including sample backlighting using an LED or fiber light source for transmitted light imaging of the sample, an LED or fiber light source for epi illumination of the sample, and a compact digital camera / microscope / endoscope for imaging the sample within the holder.Sample Cryocooling station

[0267] Clause 25: the system of clause 3, where the sample cryocooling station allows the sample and holder to be rapidly cooled to cryogenic temperature so as to minimize ice nucleation and growth within the sample and in surrounding solution, and where rapid cooling is at rates of about 25,000 oC / min for 500 micrometer samples and 3,000,000 oC / min for 25 micrometer samples, depending on sample size.

[0268] Clause 26: the system of clause 25, where the sample cryocooling station is comprised of an insulated container or Dewar configured to accept and hold a first volume of liquid nitrogen; an insulated cover that isolates and insulates the Dewar’s contents from warmmoist surrounding air, and a plunge bore through which the sample and holder may be plunged into liquid nitrogen.

[0269] Clause 27: the system of clause 26, where the Dewar may contain a second, inner chamber containing a second volume of liquid nitrogen, where the liquid nitrogen in the second chamber is in excellent thermal communication with the first amount of liquid nitrogen, and where the inner chamber may be made of a material such as a metal with high thermal conductivity.

[0270] Clause 28: the system of clause 26, where the level of the surface of the second amount of liquid nitrogen is above the level of the surface of the first amount of liquid nitrogen, and where addition of liquid nitrogen to the first chamber first fills the chamber and then overflow, and where the overflow will add to the first volume of liquid nitrogen outside the inner chamber.

[0271] Clause 29: the system of clause 26, where the levels of liquid nitrogen within the inner chamber and outside the inner chamber are monitored using level sensors, including a laser level sensor, a thermocouple, a heated RTD, or a float and where a valve connected to a supply of liquid nitrogen (for example, a third volume of liquid nitrogen held within an additional insulated chamber located above the fill level of the second chamber) may open and fill the inner chamber to overflowing when the level in that chamber drops below a target value.

[0272] Clause 30: the system of clause 26, where the insulated cover includes a manifold within a portion of the cover that includes the bore through which a sample and holder may be plunged into the second volume of liquid nitrogen within the inner chamber; a set of gas channels within the manifold that intersect the bore and that can apply suction or deliver dry ambient temperature gas to the bore at and near the level of the liquid nitrogen within the bore so as to remove cold gas present near the surface of the liquid nitrogen within the bore and replace it with ambient temperature dry gas, and also to flood the bore with excess dry gas to prevent infiltration of moist surrounding air; as well as heaters lining the bore to prevent frost formation on bore surfaces.

[0273] Clause 31 : the system of clause 26, where the insulating cover is further comprised, in a region adjacent the manifold, of a second opening or through-hole positioned above a region contained within the inner chamber, where the through-hole is sized to accept one or more cassettes or pucks, each with multiple receptacles for holding samples, and where thethrough-hole is covered by a sliding, rotating or hinged lid that opens to allow access to the cassettes and sample holders within them.

[0274] Clause 32: the system of clause 31, where the bore of the manifold and the second opening within the cover are in communication such that a sample plunged on the robot arm through the bore and into liquid nitrogen can be translated latterly out of the bore and into the region below the second opening using the robot arm without being lifted out of the liquid nitrogen.

[0275] Clause 33: the system of clause 31, where the lid is opened and closed, slid, or rotated using an electronically or pneumatically controlled actuator, and where a supply of dry gas is connected to the inner chamber and is turned on when the lid is opened to ensure an overpressure and to prevent moist ambient gas from entering inner chamber and forming frost.

[0276] Clause 34: the system of clause 31, where the bottom of the inner chamber beneath the opening in the cover contains a structure sized and shaped to accept and hold a cassette or puck in a well-defined orientation, allowing the robot to deposit samples after cooling into the cassette or puck, or to retrieve samples from the cassette or puck for subsequent processing.

[0277] Clause 35: the system of clause 31, where a cassette or puck is loaded into the structure in the inner chamber or removed from it using a tool.

[0278] Clause 36: the system of clause 31, where a cassette or puck at cryogenic temperature containing previously cryocooled samples can be loaded through the opening in the cover of the Dewar and into the structure within the Dewar that accepts a cassette or puck, to allow the robot to access the samples and transfer them to the warming station.

[0279] Clause 37: the system of clause 31, where the sample cryocooling station includes a digital camera / microscope / endoscope and lens and other optics as needed as well as LED illuminators providing reflected and transmitted light illumination to allow imaging of the cryocooled sample in liquid nitrogen or in cold gas present immediately above the liquid nitrogen and before it is transferred into a cassette or puck.Details of cryocooling

[0280] Clause 38: the system of clause 26, where the robot end effector plunge the sample and holder through the bore and into the second volume of liquid nitrogen within the inner chamber at a speed of between about 0.25 m / s and about 5 m / s or, for some applications, about 2 m / s, so as to maximize convective heat transfer without causing excessive splashing of liquidnitrogen or requiring excessive travel distances through liquid nitrogen before cooling is complete.

[0281] Clause 39: the system of clause 26, where the depth of the inner chamber and the nitrogen fill level within the chamber are such as to allow the sample to travel a distance of between about 2 cm and about 20 cm or, for some applications, at least about 4 cm before coming to a stop, to ensure that the sample continues to travel at high speed until sample cooling below T-140 K has completed.

[0282] Clause 40: the system of clause 26, where the robot arm may have an end effector that allows high speed on- or -off axis rotation of the sample holder, so as to maintain a large speed of the sample holder relative to the liquid nitrogen and a large rate of convective heat transfer even when translational motion of the robot arm has ceased, as may be needed to maximize average cooling rate when plunging large (roughly millimeter and larger) samples that cool slowly.Warming station

[0283] Clause 41 : the system of clause 3, where samples previously cooled to cryogenic temperature may be rapidly warmed to room or biological temperatures between 20 oC and 60 oC in the sample warming station, where rapid warming includes warming rates between about 50,000 oC and 6,000,000 oC / minute.

[0284] Clause 42: the system of clause 41, where the warming station is comprised of a temperature-controlled plate / block heater, a highly thermally conductive stage attached to the block heater that accepts plates, vials, test tubes, cuvettes or other receptacles for holding solutions used in warming, and possibly also insulation around the outer faces of the stage to reduce heat loss.

[0285] Clause 43: the system of clause 41, where the thermally conductive stage may be shaped to provide close thermal contact with any liquid-enclosing surfaces of a plate, vial, test tube, etc. so as to ensure maximum rates of heat transfer and temperature uniformity.

[0286] Clause 44: the system of clause 41, where the robot end effector plunges samples into warm liquid contained with the well of a plate, a vial, a test tube, a cuvette or other receptacle at a speed of between about 0.25 and about 5 m / s or, for some applications, about 2 m / s, to maximize the rate of convective heat transfer while minimizing splashing of liquid when the sample holder impacts the liquid and minimizing possible damage to the sample holder and sample.

[0287] Clause 45: the system of clause 41, where the plates, vials, test tubes etc. have receptacles for liquid within them with a depth of between about 1 cm and about 10 cm with a maximum depth of about 20 cm or, for some applications, about 4 cm, a distance depending upon (increasing with) the sample size and the time required for it to warm, so that the sample can remain in linear translational motion long enough to ensure that it has substantially warmed to at least -10 oC or, for some applications, 0 oC, so that any and all ice nucleation and growth within the sample has ceased before translational motion is ceased.

[0288] Clause 46: the system of clause 41, where, in addition to linear vertical motion, the robot end effector may subject the sample to rotational motion at some nonzero perpendicular distance r from the rotation axis at some angular speed o so that sample motion relative to the warming liquid can be maintained after translational motion of the robot arm and end effector has ceased, maintaining large heat transfer rates until warming to temperatures where ice nucleation and growth no long occur and / or to a target temperature, and so that the required depth of warming solution can be reduced from that required when sample motion is purely vertical.

[0289] Clause 47: the system of clause 41, where, in addition to linear vertical motion, the robot end effector may subject the sample to vibrational motion of some amplitude A at some angular speed o so that sample motion relative to the warming liquid can be maintained after translational motion of the robot arm and end effector has ceased, maintaining large heat transfer rates until warming to temperatures where ice nucleation and growth no long occur and / or to a target temperature, and so that the required depth of warming solution can be reduced from that required when sample motion is purely vertical.

[0290] Clause 48: the system of clause 46 or clause 47, where the net speed of the sample relative to the surrounding liquid through a combination of translational motion and rotational motion and / or vibrational motion is maintained at about 0.5 m / s to about 5 m / s or, for some applications, about 2 m / s until the sample has warmed to at least about -10 oC or, for some applications, about 0 oC.

[0291] Clause 49: the system of clause 46 or clause 47, where the net speed of the sample relative to the surrounding liquid through a combination of translational and rotational and / or vibrational motion is maintained at a speed of at least about 0.25 m / s until it has warmed to within 10 oC of its final temperature.

[0292] Clause 50: the system of clause 41, where the temperature of the warming liquid may be substantially above the sample’s normal biological temperature, for example between 50 oC and possibly 100 oC, so as to maximize the rate at which the sample warms through the temperature range where ice growth rates are largest and thus minimize the amount of ice formed in the sample.

[0293] Clause 51 : the system of clause 50, where the robot may quickly remove the sample from the warming solution before the sample temperature has reached the temperature of the warming solution so as to protect the sample from damage due to prolonged exposure to elevated temperatures.

[0294] Clause 52: the system of clause 50, where each plunge well in the warming station may be vertically divided into two chambers connected by a passage sufficiently large to allow the sample-containing portion of the sample holder to pass through, where the liquid in the upper chamber is held at a higher temperature than the liquid in the lower chamber, so that the sample may first be moved within the upper chamber so as to warm at higher rate than is possible in liquid at a temperature equal to that of the lower chamber, and then translated to the lower chamber before its temperature becomes too large so as to protect the sample from damage due to excessive temperatures.Robot end effector rinsing and drying station

[0295] Clause 53 : the system of clause 3, where the system includes a warming and drying station for the robot end effector, that can warm and dry the end effector after plunging in liquids.

[0296] Clause 54: the system of clause 53, where the end effector is warmed by passage through a cylindrical arrangement of heaters.

[0297] Clause 55 : the system of clause 53, where the end effector is dried by passage above or through a compressed air driven air amplifier or through a high-speed gas jet or sheet.

[0298] Clause 56: the system of clause 53, where the heaters and air dryers are arranged so that they are on the same axis and so that the end effector may be oscillated through them via a linear motion.

[0299] Clause 57: the system of clause 53, where the warming and drying station includes means for rinsing the end effector to remove residues from any liquids it has been in contact with.

[0300] Clause 58: the system of clause 57, where the rinsing component of the station may involve one or more receptacles containing pure water, water + detergent, or isopropyl alcohol, and where the robot transfers the end effector into and between one or more rinsing solutions.

[0301] Clause 59: the system of clause 57, where the rinsing is accomplished by spraying water or other suitable solvent on the end effector.

[0302] Clause 60: the system of clause 57, where the robot may rotate the end effector and oscillate it up and down during the rinsing.System control and monitoring

[0303] Clause 61 : the system of clause 3, where any one or all of the robot, the motors and actuators attached to the robot forming the end effector, the temperature-controlled blocks, the sensors, actuators, motors, valves of various other system components, and the digital cameras and illumination sources of the imaging components may be controlled electronically via computer or microcontroller.

[0304] Clause 62: the system of clause 61, where the system controller may sequence robot and other operations so that two or more sample holders may undergo processing at the same time.

[0305] Clause 63 : the system of clause 1, where two or more robots may be used to increase the number of samples that can be processed with a given set of processing stations.Sample holders

[0306] Clause 64: the system of clause 1, where the sample holder that contains one or more samples has a substantial portion of its surface area in the vicinity of the sample covered with a thin material containing a dense array of holes that presents little resistance to liquid flow, that allows liquids to flow through and past the sample at high speed when the sample holder is moving at high speed relative to the liquids, and thereby maximizes convective and conductive heat transfer between the sample and the liquid.

[0307] Clause 65: the system of clause 64, where the sample is supported on a thin film of polymer, glass, semiconductor, metal, or composite with a thickness of between about 2 pm and about 100 pm or, in some applications, between about 5 and 50 micrometers.

[0308] Clause 66: the system of clause 65, where the thin film has regions of two thicknesses, a thin region with through holes on which the sample resides with a thickness of about 5 to 15 micrometers, and a thick region for mechanical strength and for bonding to otherparts of the sample holder with a thickness between 10 and 50 micrometers, and where there may be thick regions forming markings or pillars or other features within the thin region of the film that may be useful in positioning samples on the film.

[0309] Clause 67: the system of clause 65, where the thin film is micropatterned using photolithography and standard microfabrication processes, micro-embossing, or stamping.

[0310] Clause 68: the system of clause 65, where the thin film is of a micropatterned polymer such as polyimide, cyclic olefin copolymer or SU8 that is optically transparent to facilitate imaging of a sample placed upon it.

[0311] Clause 69: the system of clause 65, where the thin film includes substantial areas patterned with through holes, where the open area fraction of the film is as large as feasible given mechanical strength requirements and is between about 50% and about 95% or, for some applications, between about 70% and about 95%, and where the holes have a diameter that is as large as possible without being so large as to allow the sample to pass through, which, for a 100 micrometer oocyte, may be of order 50-80 micrometers, and more generally between 40% and 80% of the diameter of the samples to be cryopreserved.

[0312] Clause 70: the system of clause 65, where the thin film of the sample holder may be added to a thin rigid frame.

[0313] Clause 71 : the system of clause 69 or clause 70, where the rod or frame may be attached either permanently or removably to a base configured to be handled by the robot end effector.

[0314] Clause 72: the system of clause 70, where the base may be of a hard, dimensionally stable material, including magnetic stainless steel, and where the base may be marked or may incorporate an RFID tag for sample identification and tracking.

[0315] Clause 73: the system of clause 70, where the frame may be marked or may incorporate an RFID tag for sample identification and tracking.

[0316] Clause 74: the system of clause 70, where the frame is comprised of a rigid sheet with an aperture disposed adjacent to one end, where the sheet may be of a rigid metal or polymer or polymer glass composite or semiconductor, and where the thickness of the sheet may be comparable to or somewhat larger than (from about 80% to 150%) the corresponding sample dimension. For mammalian oocytes and embryos, this thickness may be between 80 and 250 micrometers.

[0317] Clause 75: the system of clause 70, where the width of the frame is between about 3 and 10 millimeters and may be as wide as 30 millimeters, where the length of the frame is between about 1 and 10 centimeters or, for some applications about 2 centimeters.

[0318] Clause 76: the system of clause 70, where the frame has at least one aperture disposed near the end furthest from the base, and where the aperture is spanned and sealed on one side (the “bottom” side of the frame) by a thin film, attached using adhesive, ultrasonic bonding, or other methods, and where the thin, hole-filled film may have one thickness in the area of the aperture (for example, 5-15 micrometers) and a second, larger thickness (for example 10-50 micrometers) in the area contacting the frame, and where the portion of the film within the aperture is filled with a dense array of holes that are as large as possible without allowing passage of the samples and so that the open area fraction of the portion of the film within the aperture is as large as possible consistent with mechanical strength requirements and is between about 50% and about 95% or, for some applications, between about 70% and about 95%.

[0319] Clause 77: the system of clause 70, where one or more samples are deposited onto the hole-filled film and then a second, hole-filled film is applied to the top face of the frame to seal the sample within the aperture.

[0320] Clause 78: the system of clause 70, where the films may have perforations, tabs or other patterns to facilitate their removal within the aperture to facilitate access to the sample after processing.

[0321] Clause 79: The system of any one of clauses 75 to 78, where the aperture in the frame extends to within no more than about 0.5 mm or, for some applications, no more than about 1 mm of the frame edges to allow sufficient surface for film bonding to the frame.

[0322] Clause 80: the system of clause 70, where the holder is plunged along the frame axis during cooling and warming.

[0323] Clause 81: the system of clause 70, where the holder is plunged along and rotated about the frame axis during cooling and warming.

[0324] Clause 82: the system of clause 70, where the holder is plunged along its axis and rotated about a different axis during cooling and warming.

[0325] Clause 83 : the system of clause 70, where the frame has a U shaped cutout at the end furthest from the base.

[0326] Clause 84: the system of clause 83, where the thin, hole-filled film attached to one side of the frame covers and seals the U-shaped cutout on one side of the frame, and, after a sample is loaded, wraps around the open end of the “U”, sealing it, and then seals the cutout on the other side of the frame so as to retain the sample, thus providing a series of holes comprising a large open area fraction on the leading edge of the sample holder to allow cooling and warming liquids to flow directly to the samples within with minimal resistance.

[0327] Clause 85: the system of clause 63, where the sample holder includes two parts, a basket and a cap, where the basket receives and holds one or more samples and the cap is configured to seal the basket and to be grabbed or handled by the robot end effector.

[0328] Clause 86: the system of clause 85, where the basket has a diameter at its bottom of between about 2 mm and about 20 mm or, for some applications, about 3-10 mm.

[0329] Clause 87: the system of clause 86, where the basket includes a thin, low thermal mass frame with a substantially open bottom and substantially open sides with the openings extending from the bottom up a distance d from the bottom, where d is comparable to or larger than the basket diameter at its base, e.g., about 2-4 times the bottom diameter or between about 6 and 40 millimeters.

[0330] Clause 88: the system of clause 85, where the open bottom and sides are spanned by a hole-filled thin film or a mesh formed of fine filaments, where the holes of the film or mesh are as large as possible without allowing a sample to pass through, and typically in a size range of between about 40 and 120 micrometers for mammalian oocytes and embryos and more generally between 40% and 80% of the cell diameter.

[0331] Clause 89: the system of clause 88, where the hole-filled thin film is fabricated, in whole or in part, from a metallic material or a transparent polymer, such as polyimide, SU-8, and cyclic olefin copolymer to allow optical inspection of samples on the film.

[0332] Clause 90: the system of clause 88, where the thin film has a thickness of between 5 and 15 micrometers in the areas where it spans openings and between 10 and 50 micrometers in the areas where it is bonded to the frame.

[0333] Clause 91: the system of clause 88, where the film portion within openings in the frame has a large open area fraction in the range of about 50% to about 95% or, for some applications, between about 70% and about 95%.

[0334] Clause 92: the system of clause 88, where the mesh is formed of fine filaments of metal or of a polymer such as nylon.

[0335] Clause 93: the system of clause 88, where the mesh size is between about 60 and about 400 mesh or, for mammalian oocytes and embryos, between about 100 and about 240 mesh.

[0336] Clause 94: the system of clause 88, where the bottom of the basket on which samples are placed is made of a thin hole-filled, transparent polymer film so as to provide the fastest cooling and warming and optical transparency, and where the openings in the side of the basket are covered and sealed using a mesh formed of fine filaments.

[0337] Clause 95: the system of clause 85, where the cap press fits into the basket to form a seal, and where the basket may be removed by pushing down on its upper edge while holding the cap securely.

[0338] Clause 96: the system of clause 85, where the cap includes a conical feature located axially and projecting down into the basket that serves to direct liquid flowing upward through basket bottom to the sides and out through a holey film or mesh-covered portion of the basket side so as to produce near laminar liquid flow when the sample holder is plunged in cold or warm liquid.

[0339] Clause 97: the system of clause 85, where the cap includes openings spanned by thin holey films or mesh to allow passage of liquid during cooling and warming.

[0340] Clause 98: the system of clause 85, where a series of baskets and caps may be held in receptacles within in base, and where samples may be loaded into the baskets using a pipette or other tool, and then the cap positioned and sealed on the base.

[0341] Clause 99: the system of clause 85, where the basket has a dome-shaped rather than flat bottom.

[0342] Clause 100: the system of clause 46, where the basket frame or cap may be marked or patterned to allow identification of each sample, or where the cap incorporates an RFID tag for sample identification.

Claims

CLAIMSWhat is claimed:

1. A method for handling a biological sample, the method comprising: loading the biological sample into a sample holder; soaking the biological sample in a solution configured to prepare the biological sample for cryocooling; removing liquid from the biological sample and the sample holder; cooling the biological sample to a predefined cryogenic temperature to create a cooled sample; storing the cooled sample at a storage temperature of at least the predefined cryogenic temperature; warming the cooled sample to create a warmed sample; incubating the warmed sample; and removing the sample from the sample holder.

2. The method of claim 1, wherein the sample holder fully encloses the sample during sample handling so that the sample is retained within the sample holder from loading to removal at the end of processing.

3. The method of claim 1, wherein the sample holder is porous, allowing fluids to flow to the interior of the sample holder and contact the sample.

4. The method of claim 1, wherein the sample holder is translated and / or rotated at a minimum speed with respect to the initially stationary cooling fluid until the sample has substantially cooled toward the temperature of the cooling fluid.

5. The method of claim 1, wherein the sample holder is translated and / or rotated at a minimum speed with respect to the initially stationary warming fluid until the sample has warmed above OoC.

6. The method of claim 1, wherein the sample holder contains a thin film, and where the sample rests on the thin film.

7. A robotic system for cryopreservation of a biological sample, the robotic system comprising: a sample holder configured to enclose and retain the biological sample; a series of sample processing stations; and a robot with a robot end effector configured to retain the biological sample holder with the biological sample, wherein the robot is configured to execute a plurality of predefined sample motions between and within the series of sample processing stations.

8. The robotic system of claim 7, wherein the biological sample has a volume less than about 10 microliters.

9. The robotic system of claim 7, wherein the biological sample has a volume less than about 1 microliter.

10. The robotic system of claim 7, wherein the biological sample has a volume less than about 0.1 microliters.

11. The robotic system of claim 7, wherein the biological sample has a thickness of less than about 2 mm.

12. The robotic system of claim 7, wherein the biological sample has a thickness less than about 500 micrometers.

13. The robotic system of claim 7, wherein the biological sample has a thickness less than about 200 micrometers.

14. The robotic system of claim 7, wherein the biological sample is a mammalian oocyte or embryo.

15. The robotic system of claim 7, wherein the series of stations for sample processing includes: a first station configured to soak a biological sample in equilibration and vitrification solutions prior to cryocooling; a second station configured to remove liquid from around the biological sample;a third station configured to image the biological sample prior to cryocooling and after warming; a fourth station configured to cool the biological sample to a cryogenic temperature; a fifth station configured to store the biological sample at cryogenic temperature; a sixth station configured to warm the sample to a biological temperature; a seventh station configured to soak and incubate the biological sample; and / or an eighth station configured to warm and dry the robot end effector.

16. The robotic system of claim 15, wherein the robot and the first, second, third, fourth, fifth, sixth, seventh, and eighth stations are located within an enclosure.

17. The robotic system of claim 16, wherein at least a portion of the enclosure is sufficiently transparent to allow viewing and monitoring.

18. The robotic system of claim 17, wherein the enclosure includes at least one door allowing transfer of materials and equipment between the interior and exterior of the enclosure.

19. The robotic system of claim 18, further including a sample transfer station configured to transfer samples between the exterior and interior of the enclosure, and to a position in the interior of the enclosure where they may be retrieved by or placed by the robot.

20. The robotic system of claim 7, wherein the robot end effector is configured to grip and release the sample holder.

21. The robotic system of claim 7, wherein the robot end effector is configured to provision lateral translation and vertical translation of the sample holder.

22. The robotic system of claim 7, wherein the robot end effector is configured to translate the sample holder at a vertical speed of between about 1 m / s and about 5 m / s.

23. The robotic system of claim 7, wherein the robot end effector is configured to translate the sample holder at a horizontal speed of between about 1 m / s and about 5 m / s.

24. The robotic system of claim 7, wherein the robot effector is configured to provision rotational motion of the sample holder about an axis of rotation.

25. The robotic system of claim 24, wherein the axis of rotation is offset a predefined distance from an axis of movement of the sample holder.

26. The robotic system of claim 23, wherein the robot effector is configured to rotate the sample holder at a rotational speed of at least about 0.25 m / s.

27. The robotic system of claim 23, wherein the robot effector is configured to rotate the sample holder at a rotational speed of between about 0.5 m / s and about 2 m / s.

28. The robotic system of claim 7, wherein the robot effector is configured to provision vibrational motion of the sample holder.

29. The robotic system of claim 7, wherein the robot is a pick-and-place robot, a SCARA robot, a spherical robot, or a cartesian robot.

30. The robotic system of claim 7, further including a sample transfer station configured to transfer samples, wherein the sample transfer station is a motor driven carousel with one or more receptacles each configured to hold a respective sample holder.

31. The robotic system of claim 30, wherein the sample transfer station includes a heater and a temperature control system configured to maintain the samples at a temperature above room temperature and near a biological temperature.

34. The robotic system of claim 7, further comprising a soaking station configured to soak the biological sample in a solution prior to cryocooling, wherein the soaking station includes a temperature control system configured to vary a temperature of solutions and samples immersed in the solutions to be varied within a range between 0 °C and 50 °C, and to be held near a biological temperature, where a biological temperature may be about 37 °C35. The robotic system of claim 34, wherein the soaking station is configured to accept a Society for Biomolecular Screening (SBS) multiwell microplate and / or a Society forLaboratory Automation and Screening (SLAS) multiwell microplate in thin, standard, and deep well formats for containing the pre-cooling soaking solutions.

36. The robotic system of claim 35, wherein the soaking station further includes a temperature-controlled well-plate heater configured to hold thereon the microplates.

37. The robotic system of claim 35, wherein there may be multiple different solutions, each held within different wells in the microplate, in which each sample holder is soaked, and where each sample holder may be soaked in a different set of solutions held in different wells.

38. The robotic system of claim 35, wherein the microplates and the sample holders are designed to mate in a way that allows the robot to release a first sample holder and perform other tasks with other sample holders while the first sample is soaking, and then to return to the first sample holder and find it in the same position and orientation for retrieval.

39. The robotic system of claim 34, wherein the robot end effector is configured to translate the sample holder to immerse its sample in each of multiple solutions.

40. The robotic system of claim 34, wherein the robot end effector is configured to oscillate and / or rotate the sample to ensure uniform and reproducible soaking.

41. The robotic system of claim 34, wherein the station for soaking and incubation of samples after warming includes the same or similar components but with different solutions from those of the station for soaking samples prior to cooling.

42. The robotic system of claim 7, wherein the station for soaking and incubation of samples after warming includes a device for controlling the atmosphere above the soaking liquids including the CO2 content during incubation.

43. The robotic system of claim 14, wherein the station for soaking and incubation of samples includes a temperature control system allowing the temperature of solutions and samples within them to be varied within a range between OoC and 50oC, and to be held near a biological temperature.

44. The robotic system of claim 14, wherein the station for soaking and incubation of samples is configured to accept an SBS and / or SLAS multiwell microplate in thin, standard, and deep well formats for containing the soaking and incubation solutions.

45. The robotic system of claim 44, wherein the microplates are held on a temperature-controlled well-plate heater.

46. The robotic system of claim 44, wherein the microplate may include multiple wells each containing therein one of multiple different solutions, in which each sample holder is soaked, and where each sample holder may be soaked in a different set of solutions held in different wells.

47. The robotic system of claim 14, wherein the liquid removal station allows excess liquid to be removed from the sample and sample holder to minimize total thermal mass and maximize cooling and warming rates.

48. The robotic system of claim 47, wherein liquid is removed from the sample and holder by bringing the sample in contact with or closely adjacent to the station and briefly applying suction to a portion of the sample holder in fluid communication with the sample.

49. The robotic system of claim 48, wherein the suction may be generated using a pump or a compressed air vacuum generator, and where it may be turned on an off using an electrically actuated valve.

50. The robotic system of claim 48, wherein liquid is removed by blowing compressed gas onto the sample and the sample holder.

51. The robotic system of claim 48, wherein liquid may be removed by bringing the sample holder in contact with an absorbent material such a filter paper.

52. The robotic system of claim 14, wherein the station for imaging the sample prior to cryocooling and after warming consists of a camera or digital microscope or digital endoscope with sufficient resolution and optics providing sufficient magnification to resolve fine details within the sample.

53. The robotic system of claim 52, wherein the station for imaging the sample includes backlighting using an LED or fiber light source for transmitted light imaging of the sample, and / or an LED or fiber light source for epi illumination of the sample.

54. The robotic system of claim 14, wherein the sample cryocooling station allows the sample and holder to be rapidly cooled to cryogenic temperature so as to minimize ice nucleation and growth within the sample and in surrounding solution.

55. The robotic system of claim 54, wherein the sample-size-dependent cooling rate is about 25,000 oC / minute for a 500 micrometer diameter sample and about 3,000,000 oC / minute for 25 micrometer diameter sample.

56. The robotic system of claim 14, wherein the sample cryocooling station is comprised of an insulated container or Dewar defining a first chamber.

57. The robotic system of claim 56, wherein there is a second, inner chamber within the first chamber.

58. The robotic system of claim 57, wherein the space between the first and second chambers contains a first volume of liquid nitrogen.

59. The robotic system of claim 57, wherein the second chamber contains a second volume of liquid nitrogen that is not in direct contact with the first volume of liquid nitrogen.

60. The robotic system of claim 59, wherein the level of the surface of the second amount of liquid nitrogen is above the level of the surface of the first amount of liquid nitrogen.

61. The robotic system of claim 59, wherein the addition of liquid nitrogen to the second chamber will first fill the chamber and then overflow, and where the overflow will add to the first volume of liquid nitrogen.

62. The robotic system of claim 59, wherein liquid nitrogen may be pumped from the first volume between the first and second chambers so as to fill the second, inner chamber to overflow, thereby maintaining a near constant level of liquid nitrogen within the second chamber.

63. The robotic system of claim 59, wherein a third volume of liquid nitrogen may be held in a second insulated container in fluid communication with the second, inner chamber, and where filling of the inner chamber using the third volume of liquid nitrogen is controlled by a valve.

64. The robotic system of claim 59, wherein the levels of liquid nitrogen within the first volume and the second volume are monitored using level sensors.

65. The robotic system of claim 64, wherein the level sensor may be a laser level sensor, a thermocouple, a heated RTD, or a float.

66. The robotic system of claim 64, wherein the sensor readings are used to control the valve controlling flow from the third volume of liquid nitrogen to the second volume, and pumping of liquid nitrogen from the first volume to the second volume.

67. The robotic system of claim 59, wherein the second volume of liquid nitrogen within in the inner chamber is in thermal communication with the first amount of liquid nitrogen within the first insulated container between the first and second chambers.

68. The robotic system of claim 59, wherein the inner chamber may be made of a material such as a metal with high thermal conductivity.

69. The robotic system of claim 14, wherein the sample cryocooling station has an insulated cover that isolates and insulates the Dewar’s contents from warm moist surrounding air.

70. The robotic system of claim 69, wherein the insulated cover includes a manifold within a portion of the cover.

71. The robotic system of claim 70, wherein the manifold includes: a bore through which a sample and holder may be plunged into the second volume of liquid nitrogen within the inner chamber; a set of gas channels within the manifold that intersect the bore and that can apply suction or deliver dry ambient temperature gas to the bore at and near the level of the liquid nitrogen within the bore so as to remove cold gas present near the surface of the liquid nitrogen within the bore and replace it with ambient temperature dry gas; and heaters lining the bore to prevent frost formation on bore surfaces.

72. The robotic system of claim 69, wherein the insulating cover is further comprised, in a region adjacent the manifold, of a second opening or through-hole positioned above a region contained within the second, inner chamber, where the through-hole is sized to accept one or more cassettes or pucks, each with multiple receptacles for holding samples.

73. The robotic system of claim 72, wherein the through-hole is covered by a sliding, rotating or hinged lid that opens to allow access to the cassettes and sample holders within them.

74. The robotic system of claim 73, wherein the lid is opened and closed, slid, or rotated using an electronically or pneumatically controlled actuator.

75. The robotic system of claim 59, wherein the bottom of the second, inner chamber contains a structure sized and shaped to accept and hold a cassette or puck at a well- defined position and in a well-defined orientation.

76. The robotic system of claim 75, wherein a cassette or puck is loaded into the structure in the second, inner chamber or removed from it using a tool.

77. The robotic system of claim 75, wherein a cassette or puck at cryogenic temperature containing previously cryocooled samples can be loaded into the structure within the Dewar that accepts a cassette or puck.

78. The robotic system of claim 72, wherein the bore of the manifold and the second opening within the cover are in communication such that a sample plunged through the bore and into the second volume of liquid nitrogen can be translated latterly out of the bore and into a cassette or puck without being lifted out of the liquid nitrogen.

79. The robotic system of claim 59, wherein the sample cryocooling station includes a digital camera, digital microscope, or digital endoscope and lens and other optics as needed as well as LED illuminators to allow imaging of the cryocooled sample in liquid nitrogen or in cold gas present immediately above the liquid nitrogen and before it is transferred into a cassette or puck.

80. The robotic system of claim 59, wherein the robot end effector plunge the sample and holder into the second volume of liquid nitrogen within the second, inner chamber at a speed of between about 0.25 m / s and about 5 m / s.

81. The robotic system of claim 59, wherein the robot end effector plunge the sample and holder into the second volume of liquid nitrogen within the inner chamber at a speed of about 2 m / s.

82. The robotic system of claim 59, wherein the depth of the second, inner chamber and the nitrogen fill level within the chamber are such as to allow the sample to travel a distance of between about 2 cm and about 20 cm before coming to a stop, to ensure that the sample continues to travel at high speed until sample cooling below T-140 K has completed.

83. The robotic system of claim 59, wherein the robot arm may have an end effector that allows high speed on- or -off axis rotation of the sample holder, so as to maintain a large speed of the sample holder relative to the liquid nitrogen and a large rate of convective heat transfer even when translational motion of the robot arm has ceased.

84. The robotic system of claim 7, wherein the sample warming station warms samples previously cooled to cryogenic temperature to room or biological temperatures between 20 oC and 60 oC by contact with a warming solution.

85. The robotic system of claim 84, wherein the sample-size-dependent warming rates are between about 50,000 oC / minute and 6,000,000 oC / minute.

86. The robotic system of claim 14, wherein the warming station includes a temperature-controlled block heater.

87. The robotic system of claim 86, wherein the warming station includes a highly thermally conductive block attached to the block heater that accepts plates, vials, test tubes, cuvettes or other receptacles for holding solutions used in warming.

88. The robotic system of claim 87, wherein the thermally conductive block may be shaped to provide close thermal contact with any liquid-enclosing surfaces of a plate, vial, test tube, etc. so as to ensure maximum rates of heat transfer and temperature uniformity.

89. The robotic system of claim 14, wherein the robot end effector translate sample into warm liquid contained with the well of a plate, a vial, a test tube, a cuvette or other receptacle held on the block and heater block.

90. The robotic system of claim 89, wherein the robot end effector translate the sample into and through the warming solution at a speed of between about 0.25 and about 5 m / s.

91. The robotic system of claim 89, wherein the robot end effector translate the sample into and through the warming solution at a speed of about 2 m / s.

92. The robotic system of claim 90, wherein the sample translation may be vertical, horizontal or at an angle at different points in the sample’s motion through the warming solution.

93. The robotic system of claim 90, wherein the sample speed relative to the stationary warming solution is maintained until the sample has warmed above a predetermined temperature.

94. The robotic system of claim 89, wherein the plate, vial, test tube, cuvette or other receptacle has a depth of between about 1 cm and about 20 cm.

95. The robotic system of claim 89, wherein the wells in the plate have a length between about 1 cm and about 20 cm.

96. The robotic system of claim 89, wherein, in addition to translational motion, the robot end effector may subject the sample to rotational motion at some nonzero perpendicular distance r from the rotation axis at a desired angular speed.

97. The system of claims 96, wherein, in addition to translational and / or rotational motion, the robot end effector may subject the sample to vibrational motion of some amplitude.

98. The robotic system of claim 97, wherein the net speed of the sample relative to the initially stationary warming solution due to its translational, rotational and / or vibrational motion is maintained at about 0.5 m / s to about 5 m / s until the sample has warmed above 0 oC and any ice within the sample has melted.

99. The robotic system of claim 97, wherein the net speed of the sample relative to the initially stationary warming solution due to its translational, rotational and / or vibrational motion is maintained at at least about 0.25 m / s until it has warmed to within 10 oC of the temperature of the warming solution.

100. The robotic system of claim 89, wherein the temperature of the warming solution may be substantially above the sample’s normal biological temperature.

101. The robotic system of claim 89, wherein the temperature of the warming solution may be between 50 oC and 100 °C.

102. The robotic system of claim 100, wherein the robot may remove the sample from the warming solution before the sample temperature has reached the temperature of the warming solution so as to protect the sample from damage due to prolonged exposure to elevated temperatures.

103. The robotic system of claim 100, wherein each well in the warming station may be vertically divided into two chambers connected by a passage sufficiently large to allow the sample-containing portion of the sample holder to pass through, and where the liquid in the upper chamber is held at a higher temperature than the liquid in the lower chamber, so that the sample may first be moved within the upper chamber so as to warm at higher rate than is possible in liquid at a temperature equal to that of the lower chamber, and then translated to the lower chamber before its temperature becomes too large so as to protect the sample from damage due to excessive temperatures.

104. The robotic system of claim 100, wherein each well in the warming station may be horizontally divided into two chambers connected by a passage sufficiently large to allow the sample-containing portion of the sample holder to pass through, and where the liquid in one chamber is held at a higher temperature than the warming solution in the other chamber, so that the sample may first be moved within the liquid in the high temperature chamber so as to warm at higher rate than is possible in warming solution at the final sample temperature, and then translated to the low temperature chamber before the sample temperature becomes large enough to cause sample damage.

105. The robotic system of claim 14, wherein the system includes a warming and drying station for the robot end effector, that can warm and dry the end effector after plunging in liquids.

106. The robotic system of claim 105, wherein the end effector is warmed by passage through a cylindrical arrangement of heaters.

107. The robotic system of claim 105, wherein the robot end effector is dried by passage above or through a compressed air driven air amplifier or through a high-speed gas jet or sheet.

108. The robotic system of claim 105, wherein the heaters and dryers are arranged so that they are on the same axis and so that the end effector may be oscillated through them via a linear motion.

109. The robotic system of claim 105, wherein the warming and drying station includes means for rinsing the end effector to remove residues from any liquids it has been in contact with.

110. The robotic system of claim 109, wherein the rinsing component of the station may involve one or more receptacles containing pure water, water + detergent, or isopropyl alcohol, and where the robot transfers the end effector into and between one or more rinsing solutions.

111. The robotic system of claim 109, wherein the rinsing is accomplished by spraying water or other suitable solvent on the end effector.

112. The robotic system of claim 109, wherein the robot may rotate the end effector and oscillate it up and down during the rinsing.

113. The robotic system of claim 14, wherein any one or all of the robot, the motors and actuators attached to the robot forming the end effector, the temperature-controlled blocks, the sensors, actuators, motors, valves of various other system components, and the digital cameras and illumination sources of the imaging components may be controlled electronically via computer or microcontroller.

114. The robotic system of claim 113, wherein the system controller may sequence robot and other operations so that two or more sample holders may undergo processing at the same time.

115. This robotic system of claim 7, wherein two or more robots may be used to increase the number of samples that can be processed with a given set of processing stations.

116. The robotic system of claim 7, wherein the sample holder that contains and encloses one or more samples has surfaces containing multiple holes sufficient to allow liquids to flow through the sample holder and past the sample at high speed when the sample holder is moving at high speed relative to the initially stationary liquid.

117. The sample holder of claim 116, wherein the holes are sized so as to not allow passage of sample.

118. The sample holder of claim 116, wherein sample holder surfaces may be comprised of films having regions with arrays of holes, and where the sample may make direct contact with the films.

119. The sample holder of claim 118, wherein the films may be of polymer, glass, semiconductor, metal, or composite.

120. The sample holder of claim 118, wherein the film has a thickness of between about 2 pm and about 100 pm.

121. The sample holder of claim 118, wherein the film has a thickness of between about 5 and 50 micrometers.

122. The sample holder of claim 118, wherein the film has regions of two different thicknesses, a first thickness with through holes on which the sample resides, and a second thickness for mechanical strength and for bonding to other parts of the sample holder.

123. The sample holder of claim 122, wherein the first thickness is between about 5 and 15 micrometers.

124. The sample holder of claim 122, wherein the second thickness is between about10 and 50 micrometers.

125. The sample holder of claim 118, wherein the film is micropattemed using photolithography and standard microfabrication processes, micro-embossing, or stamping.

126. The sample holder of claim 118, wherein the film is of a micropatterned polymer such as polyimide, cyclic olefin copolymer or SU8 that is optically transparent to facilitate imaging of a sample placed upon or within it.

127. The sample holder of claim 118, wherein the open area fraction of the holecontaining portion of the film is between about 50% and 95%.

128. The sample holder of claim 118, wherein the holes have a diameter of between 40% and 80% of the minimum dimension of the samples.

129. The sample holder of claim 118, wherein the holes have a diameter of between 50 and 80 micrometers.

130. The sample holder of claim 118, wherein the films are attached to a rigid frame.

131. The sample holder of claim 130, wherein the frame may be composed of a metal, a polymer, a polymer-glass composite, or a semiconductor.

132. The sample holder of claim 130, wherein the frame is attached to a base configured to be handled by the robot end effector.

133. The sample holder of claim 132, wherein the base may be of a hard, dimensionally stable and cryogenic compatible material.

134. The sample holder of claim 132, wherein the material of the base may be a magnetic stainless steel or a glass filled polymer.

135. The sample holder of claim 132, wherein the base may be marked or may incorporate an RFID tag for sample identification and tracking.

136. The sample holder of claim 130, wherein the frame may be marked or may incorporate an RFID tag for sample identification and tracking.

137. The sample holder of claim 130, wherein the frame is comprised of a rigid sheet with an aperture disposed adjacent to one end, and where the film is applied to span the aperture.

138. The sample holder of claim 137, wherein the sheet has a thickness between 80% and 150% of the corresponding sample thickness.

139. The sample holder of claim 137, wherein the sheet has a thickness between 80 micrometers and 250 micrometers.

140. The sample holder of claim 137, where the width of the frame is between about 3 and 10 millimeters.

141. The sample holder of claim 137, where the length of the frame is between about 1 and 10 centimeters.

142. The sample holder of claim 137, where the aperture in the frame is spanned and sealed on a first side by a first film, onto which one or more samples are deposited.

143. The sample holder of claim 142, where the sample is sealed into the sample holder by attaching a second film to a second side of the frame.

144. The sample holder of claim 142, where the films may be attached to the frame using an adhesive, an adhesive gasket, or ultrasonic bonding.

145. The sample holder of claim 142, where each film has hole-filled region of a first thickness, and solid regions of a second, larger thickness, where the film regions contacting the frame are solid regions.

146. The sample holder of claim 145, where the first thickness is between 5 and 15 micrometers.

147. The sample holder of claim 145, where the second thickness is between about 10 micrometers and 50 micrometers.

148. The sample holder of claim 145, where one or both films have perforations, tabs or other patterns to facilitate tearing and removal of the portion within the aperture to facilitate access to and release of the sample after processing.

149. The sample holder of claim 130, where the frame is comprised of a rigid sheet, and where the frame has a cut-out at one end so that it has a “U” shape at that end.

150. The sample holder of claim 149, where a film with a hole-filled portion attaches to a first side of the frame, covering and sealing the U-shaped portion, and wraps around the open end of the “U”.

151. The sample holder of claim 149, where, after a sample is loaded on the film, the free portion of the film is bonded to the second side of the frame, sealing the sample within the frame.

152. The sample holder of claim 116, where the sample holder consists of two parts, a basket and a cap.

153. The sample holder of claim 152, where the basket receives and holds one or more samples.

154. The sample holder of claim 152, where the cap is configured to seal the basket and to be grabbed or handled by the robot end effector.

155. The sample holder of claim 152, where the basket has a diameter at its bottom of between about 2 mm and about 20 mm.

156. The sample holder of claim 152, where the basket includes a thin, low thermal mass frame with a substantially open bottom and substantially open sides.

157. The sample holder of claim 156, where the open bottom and sides of the frame are spanned and sealed by a hole-filled film or a mesh of fine filaments.

158. The sample holder of claim 157, where the holes in the film or mesh are smaller than the minimum dimension of the samples to be held within the holder.

159. The sample holder of claim 157, where the film is formed, in whole or in part, from a transparent polymer including polyimide, SU-8, and cyclic olefin copolymer to allow optical inspection of samples within the basket.

160. The sample holder of claim 157, where the film has a thickness of between 5 and 15 micrometers in the areas where it spans openings in the frame and between 10 and 50 micrometers in the areas where it is bonded to the frame.

161. The sample holder of claim 157, where the film portion within openings in the frame has a large open area fraction in the range of about 50% to about 95%.

162. The sample holder of claim 157, where the mesh is formed of fine filaments of metal or of a polymer such as nylon.

163. The sample holder of claim 157, where the mesh size is between about 60 and 400 mesh.

164. The sample holder of claim 152, where the cap press fits into the basket to form a seal.

165. The sample holder of claim 152, where the cap includes a conical feature located axially and projecting down into the basket that serves to direct liquid flowing through the basket bottom to its sides.

166. The sample holder of claim 152, where a series of baskets may be held in receptacles within a holder, where samples may be loaded into the baskets using a pipette or other tool and then a cap positioned and sealed to each basket.

167. The sample holder of claim 152, where the cap may be marked or incorporate an RFID tag for sample identification.

168. The robotic system of claim 1, where the sample holder may contain a basket formed by deformation of a suitably patterned flat film.

169. The sample holder of claim 168, where film contains a pattern of slits that facilitate deformation into a basket and that define the openings in the basket.

170. The sample holder of claim 169, where the openings in the basket are sized to be between 50% and 80% of the minimum dimension of the sample.

171. The sample holder of claim 168, where the film is attached at one end to a rigid sheet, and where the rigid sheet inserts into a base.

172. The sample holder of claim 171, where the sheet is semi-rigid, and where an similar or identical film and semirigid sheet are attached to the same base so that the sheets press together and the two baskets form an enclosed volume.

173. The sample holder of claim 168, where the film is attached to a first face of a rigid sheet containing an aperture so as to span the aperture and so that the basket projects from the aperture, and where the rigid sheet is attached to a base.

174. The sample holder of claim 173, where the sheet is semi-rigid, and where a similar or identical film and semi-rigid sheet are attached to the same base so that the sheets press together and the two baskets form an enclosed volume.

175. The sample holder of claim 137, where the sheet is semi-rigid, and where a similar or identical film and semi-rigid sheet are attached to the same base so that the sheets press together.

176. The sample holder of claim 175, the films are applied to outer faces of the sheets when they are pressed together, so as to form an enclosed volume between the films when the sheets are pressed together.

176. The sample holder of claims 172, 174, or 176, where the semi-rigid sheets may be pushed apart using a push-button tool that holds the base.

177. The sample holder of claim 176, where the base has a central through-hole, and where a rod connected to the push button and passing through the hole in the base can push apart the sheets when the button is depressed.

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