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

Abstract

Systems, methods, and apparatus for handling biological samples during cryopreservation and thawing. One method includes loading a sample into a sample carrier, immersing the sample in a solution to prepare for cryogenic cooling, removing excess liquid from the sample and carrier, imaging the sample, cooling the sample to a cryogenic temperature, imaging and storing the sample at the cryogenic temperature, retrieving and warming the cryogenically cooled sample, immersing and culturing the warmed sample, and then removing the sample from the carrier. Automated systems may use robots to perform sample movement between and within a series of modules or stations to perform each of the preceding steps. The sample may be held within and sealed by a porous sample carrier that allows high-speed fluid to pass through and move around the sample while containing it, as the sample carrier is translated through a fluid.

Description

[0001] Cross-referencing of priority claims and related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 573,809, filed April 3, 2024, and U.S. Provisional Patent Application No. 63 / 601,937, filed November 22, 2023. Technical Field

[0003] This disclosure generally relates to the field of biotechnology. More specifically, aspects of this disclosure relate to automated systems and methods for handling and processing samples, including cryopreservation and thawing of biological samples of millimeters and smaller, and sample processing steps for cryopreservation and thawing. Background Technology

[0004] A wide variety of small biological samples are cryopreserved for storage and later use. For example, eggs, embryos, blastocysts, and sperm from humans and many other livestock and wild animals are typically frozen, stored at low temperatures, and then thawed for use in assisted reproduction. Small organisms (e.g., coral symbiont larvae, mosquito larvae, etc.), small-volume cells, and solutions of antibodies, proteins, and other biomolecules are also stored as samples for scientific and commercial purposes. Human eggs and embryos typically have a size of about 100 micrometers (μm) to about 120 μm, and blastocysts used for human assisted reproduction can have a size of about 180 μm to about 200 μm.

[0005] Samples used for assisted reproduction can be immersed in a cryoprotectant / vitrification solution, 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 can be slowly cooled (e.g., over twenty minutes to two hours) to an intermediate temperature (e.g., -40 degrees Celsius (°C)) and then dripped into liquid nitrogen for long-term storage. During the slow cooling step, ice can nucleate extracellularly; this ice growth draws water out of the cells, increasing the concentration of intracellular proteins and other solutes. As cooling and dehydration continue, the intracellular solvent eventually vitrifies.

[0006] Alternatively, samples at room temperature can be immersed in liquid nitrogen or other liquid cryoprotectants to rapidly cool them, resulting in little or no ice formation inside and significant vitrification of the solvent within the sample. To eliminate intracellular ice formation, much higher cryoprotectant concentrations can be used in pre-cooling immersions than in slow cooling methods. These high concentrations can be toxic and may cause damage due to osmotic stress if not introduced gradually. This latter rapid cooling method now dominates most small-sample cryopreservation practices, including in assisted reproduction.

[0007] For warming / thawing, samples intended for assisted reproduction can be removed from the cryogenic storage Dewar flask, transferred with air, and then immersed in a multi-well plate (e.g., from Reprolife) at room temperature or biological temperature (e.g., 37°C). TM Cryotec TM All steps are performed manually, either in the wells of a plate or in pipettes containing warming solutions. Other wells in the plate may contain solutions to promote cell re-expansion and post-thawing growth / development. The wells of the plates used are typically in the range of 3 mm to 10 mm deep, which severely limits the vertical immersion distance into the warming solutions. The small well volume reduces the consumption of thawing medium and makes it easier to locate and retrieve samples after thawing. Separate wells and warming solutions can be used for each sample to prevent cross-contamination. Similar manual procedures are used to thaw many other types of cryopreserved samples. In the future, warming can be automated using robots or other specialized systems.

[0008] During cooling, samples can be damaged by several mechanisms. First, ice can nucleate and grow inside cells; this ice can pierce cell membranes and disrupt internal cell tissues. Second, solutes may be repelled by growing ice crystals and thus concentrate in the remaining uncrystallized solution, potentially leading to protein aggregation. Third, the intracellular solution may expand or contract more than other cellular components, causing cell damage through mechanical stress. In cell clusters or other samples, inhomogeneous composition and thermal gradients during heating can generate mechanical stress and breakage. Fourth, proteins can change their conformation due to the temperature dependence of hydrophobic interactions driving folding, as well as the temperature dependence of side chain pH (acidity), pKas (the negative logarithm of the acid dissociation constant base 10), and many other physicochemical properties, and typically unfold upon cooling—a process known as “cold denaturation.” Their solubility may also be temperature-dependent. As a result, proteins may aggregate, and other potentially irreversible biomolecular changes may occur during cooling.

[0009] Cryoprotectants can be added to reduce ice formation. At the cooling rates used in current assisted reproductive technology (ART) practices (e.g., approximately 30,000 degrees Celsius per minute (°C / min)), high concentrations of cryoprotectants—approximately 20-30% by weight / volume (w / v)—and 0.5 M to 1 M of sugar are typically required to prevent ice formation. These cryoprotectants are present in commercial vitrification solutions. To prevent cell damage from osmotic shock, cells / embryos can be immersed in a series of solutions with increased concentrations of cryoprotectants, which can be a very time-consuming process. Vitrification solutions with these high concentrations of sugar and cryoprotectants can shrink dramatically when cooled from room temperature to 77 Kelvin (K)—by about 5%, which can cause mechanical stress and breakage.

[0010] It is generally desirable to cool the sample to a low temperature to bring the internal solvent to a vitrified, glassy, ​​or amorphous state. During sample heating / thawing, the glassy solvent may develop a certain degree of molecular mobility above its glass transition temperature (typically between 140 and 180 K). Ice can then nucleate and grow, and the growth rate increases with temperature as solvent molecules become increasingly mobile, becoming significant above 180–200 K and peaking slightly below the melting temperature. Therefore, samples often exhibit a "momentary whitening" during heating because ice crystals form rapidly and then melt around 273 K.

[0011] The heating rate required to prevent the formation of a significant ice fraction (e.g., 5% or more) within an initially ice-free sample is typically much larger—one to three orders of magnitude—than the cooling rate required to prevent significant ice formation during cooling. For example, recent X-ray diffraction experiments on bovine oocytes have shown that while the cryoprotectant concentrations and cooling rates currently used in assisted reproduction are sufficient to prevent ice formation during cooling, a large portion of the internal solvent crystallizes during heating, even at heating rates of 150,000°C / min, four times the typical values ​​in current practice (<40,000°C / min). Using the same cryoprotectant concentrations as in current practice, a 20-fold increase in cooling rate (e.g., 600,000°C / min or higher) combined with a several-fold increase in heating rate (e.g., 150,000°C / min or higher) allows for almost complete elimination of ice formation during both cooling and heating. These results suggest that increasing the heating rate should be a primary focus in efforts to improve post-thawing survival, development, and other outcomes in cryopreservation.

[0012] In current practice, the vast majority of cryopreservation and thawing of small biological samples are performed manually. Manual sample handling is time-consuming, error-prone, and cannot achieve the performance of key steps (such as cooling and heating) that could be accomplished using automated systems. Automated systems have been developed for some steps, such as sample immersion in cryoprotectant solutions and sample cooling in liquid nitrogen. However, none of these systems are designed to maximize the rate of sample cooling and heating, which is crucial for maximizing survival and developmental outcomes after heating. Automated systems have been developed for cooling and storing samples used in crystallography, but these systems cannot be used for heating or for multiple steps of sample immersion before, during, and after cryopreservation and thawing. Summary of the Invention

[0013] This disclosure relates to the design, function, and use of automated systems for the cryopreservation and thawing of small biological samples, including oocytes, embryos, cells, and tissues. As used herein with respect to biological samples, the term "small" can be defined as a sample having the following characteristics: a diameter or minimum size less than about 2 millimeters, or in some applications less than about 500 micrometers, and a volume less than about 10 microliters (μL), or in some applications less than about 1 μL, or in some cases less than 0.1 μL or less than about 0.01 μL. For example, mammalian oocytes have a diameter of 200 micrometers or less and a volume less than 0.01 μL. Exemplary criteria for determining smallness may include a small internal sample temperature gradient formed within the sample during heating in a heating solution (typically an aqueous solution), for example, less than about 20–40°C, or in some applications less than about 10°C, such that thawing is fairly uniform throughout the sample volume and does not generate significant stress.

[0014] As used herein, the term “low temperature” can be defined as any temperature that causes complete or partial vitrification of the solvent within a sample, which can depend on the solvent composition and the solute present. For example, a typical low temperature in this application is the temperature at which liquid nitrogen boils (77 K = -196°C). Temperatures below approximately 150 K (-123°C) can completely inhibit ice crystal formation in samples held at these temperatures. Temperatures up to approximately -80°C (the temperature of a standard laboratory freezer) can also be used. While not inherently limited, eggs, embryos, and blastocysts with up to approximately 100 cells are primary targets, although stem cells, sperm cells, and any other “small” biological (human or non-human) samples containing cells, larvae, antibodies, proteins, etc., can benefit from the features of this disclosure.

[0015] This disclosure includes structures, apparatus, and methods for facilitating the handling of small cryopreserved biological samples in order to achieve the maximum possible cooling rate and the maximum possible heating rate.

[0016] This disclosure includes structures and methods for holding small biological samples within a carrier while maximizing convective liquid flow through and around the small biological sample as the sample and the sample carrier holding the sample translate through the liquid at speeds of at least about 0.25 m / s and up to about 5 m / s.

[0017] This disclosure includes structures and methods for removing excess liquid from around a sample.

[0018] The aspects of this disclosure include structures and methods that enable samples to be easily loaded into a sample carrier prior to a cryopreservation step and easily retrieved from the sample carrier after warming and thawing.

[0019] This disclosure includes structures and methods that enable samples to be automatically immersed in various solutions at different temperatures before cooling and after heating, and to be cultured after heating to allow them to grow and develop.

[0020] This disclosure includes structures and methods for facilitating automated optical imaging of small biological samples during or after one or more steps in cryopreservation and thawing.

[0021] According to aspects of this disclosure, small biological samples—e.g., samples with a volume of less than about 10 microliters, or less than about 1 microliter in some applications, or less than about 0.1 microliters in some applications, and a thickness of less than about 2 mm, or less than about 500 micrometers in some applications, or less than about 200 micrometers in applications involving mammalian oocytes and embryos—can be held within a sample carrier having a low thermal mass in the region adjacent to the sample. The sample carrier may be porous and may be structurally configured to allow liquid to flow largely unimpeded from the outside of the carrier to the sample during high-speed immersion, translation, or other movement of the sample carrier into the liquid.

[0022] According to aspects of this disclosure, multiple stations can be used to process small biological samples contained within sample carriers for cryopreservation and thawing, with each station performing one function or a set of functions for the sample. In this configuration, a robot can move the sample between stations and / or within each station.

[0023] According to aspects of this disclosure, samples can be handled by an automated system using a robot.

[0024] According to aspects of this disclosure, a robot may have an end effector that is structurally configured to grasp, hold, and manipulate one or more sample carriers.

[0025] According to this disclosure, the robot can transfer samples between several stations within the system that perform different processing steps.

[0026] According to aspects of this disclosure, a robot can provide, for example, multi-axis translational motion in the x, y, and z directions, and single-axis or multi-axis rotational motion, for example, along the z-axis.

[0027] According to this disclosure, the robot can be a pick-and-place robot.

[0028] According to aspects of this disclosure, the robot may be a selectively compliant articulated robotic arm (SCARA) robot or a Cartesian gantry robot.

[0029] According to aspects of this disclosure, the robot's vertical and horizontal speeds may each have a maximum permissible speed of at least about 1 m / s, or in some applications about 2 m / s, or in some applications not exceeding about 5 m / s.

[0030] According to aspects of this disclosure, the end effector of a robot may include a motorized stage for high-speed on-axis or off-axis rotational and / or vibratory motion of a sample.

[0031] According to aspects of this disclosure, the maximum speed of the sample in its rotational motion can reach at least about 0.25 m / s, or about 1-2 m / s in some applications, for example, generated by the combined rotation of the robot's z-axis and the end effector.

[0032] According to aspects of this disclosure, the end effector may have hardware to superimpose the vertical velocity of the sample carrier on the robot's z-axis velocity.

[0033] According to aspects of this disclosure, the end effector may include hardware for picking up and holding a sample carrier and ejecting or releasing the sample carrier.

[0034] According to aspects of this disclosure, the robot can be partially or substantially enclosed in a shell with transparent portions to protect the user from harm during robot operation.

[0035] According to aspects of this disclosure, the robot housing may have an emergency stop button on its outer surface.

[0036] According to aspects of this disclosure, the robot housing may have hinged or sliding doors, which enable the loading of plates containing solutions, blocks containing cryogenically cooled samples, liquid nitrogen, and / or other components into stations within the housing.

[0037] According to aspects of this disclosure, the door may have an interlocking device that prevents the operation of a robot when the door is open.

[0038] According to aspects of this disclosure, a sample transfer station (also referred to herein as a “sample loading station”) can translate and / or rotate a sample (including at room temperature or biological temperature) from a location outside the housing to a location inside the housing, where a robot can access the sample.

[0039] According to aspects of this disclosure, a sample transfer station may be a motor-driven turntable having one or more containers for holding one or more sample carriers.

[0040] According to aspects of this disclosure, the motor-driven turntable can hold a single sample carrier or can hold multiple sample carriers at a time.

[0041] According to aspects of this disclosure, the motor-driven turntable may include a heater and a temperature control system to maintain the sample temperature of one or more samples at a temperature above room temperature and / or close to biological temperature.

[0042] According to aspects of this disclosure, the system may include a pre-soaking station, wherein the sample may be soaked in one or more solutions prior to cryogenic cooling.

[0043] According to aspects of this disclosure, the pre-soaking station can enable the solution temperature to vary from about 0°C to about 50°C (e.g., between about 4°C and 50°C or between about 20°C and 40°C) and be maintained near biological temperature.

[0044] According to aspects of this disclosure, the pre-soaking station can accept multiple heterogeneous porous plates, for example, in thin, standard and / or deep-pore formats, for containing the pre-soaking solution.

[0045] According to aspects of this disclosure, the pre-soaking station can accept a custom porous plate configured to receive and hold the sample carrier when it is released by a robot.

[0046] According to aspects of this disclosure, the pre-soaking station may include a commercially available orifice plate temperature control system with a custom orifice plate carrier.

[0047] According to aspects of this disclosure, the system may include a liquid removal and sample inspection station capable of removing excess liquid from the sample, for example, to minimize total thermal mass and maximize cooling and heating rates, and capable of optically imaging the sample after liquid removal, for example, to record its state before cryopreservation.

[0048] According to aspects of this disclosure, liquid removal can be performed by a suction device that selectively applies suction to selected portions or portions of a sample container adjacent to the sample, the selected portions or portions being in fluid communication with the sample.

[0049] According to aspects of this disclosure, liquid removal can be achieved, for example, by imprinting a portion of a sample carrier adjacent to and in fluid communication with the sample using a hydrophilic absorbent pad or similar suitable medium.

[0050] According to aspects of this disclosure, a liquid removal station may include a backlighting device and a digital imaging device, the backlighting device providing illumination through a suction channel to illuminate the sample, and the digital imaging device having forward illumination near the sample carrier to image the sample, for example, after the liquid has been removed and before the sample is immersed / translated / moved into liquid nitrogen.

[0051] According to aspects of this disclosure, the system may include a sample cryogenic cooling station that rapidly cools the sample and sample carrier to a cryogenic temperature, for example, to minimize ice nucleation and growth within the sample and any surrounding liquid.

[0052] According to aspects of this disclosure, a sample cryogenic cooling station may include an insulated container or Dewar flask configured to receive and contain a first volume of liquid nitrogen, and an insulated container / Dewar flask cap that isolates and insulates the contents of the container / Dewar flask from the warm, humid surrounding air.

[0053] According to aspects of this disclosure, the container / Dewar may include a second inner cavity configured to contain a second volume of liquid nitrogen and to be in “good” geothermal communication with a first volume of liquid nitrogen in the Dewar volume between the inner wall of the Dewar and the second inner cavity.

[0054] According to aspects of this disclosure, the inner chamber may be made wholly or partially of a metallic material such as stainless steel or aluminum.

[0055] According to aspects of this disclosure, by periodically filling and overflowing the cavity, and if necessary, by keeping the liquid nitrogen level in the Dewar region outside the cavity lower than the liquid nitrogen level inside the cavity, the liquid nitrogen level in the cavity can be maintained near a predetermined level, for example, at the top of the cavity wall or the bottom of the orifice within the wall.

[0056] According to aspects of this disclosure, thermocouples, resistance temperature detectors (RTDs), diodes, and / or laser level sensors can be used to monitor the liquid nitrogen level in the inner chamber and in the volume between the inner chamber and the insulating outer shell.

[0057] According to aspects of this disclosure, the heat-insulating cap may include a manifold within a portion of the cap, the manifold including an orifice or through-hole through which a sample and sample carrier may be immersed / translated / moved into liquid nitrogen within the inner chamber. The manifold within the heat-insulating cap may also include a set of gas channels intersecting the orifice to apply suction and / or deliver dry, ambient-temperature gas into the orifice, thereby removing cold gas near the liquid nitrogen surface within the orifice and replacing it with dry, ambient-temperature gas. These gas channels may also fill the orifice with excess dry gas to prevent the infiltration of moist ambient air. The manifold within the heat-insulating cap may also include one or more heaters lining the orifice to prevent frost formation on or around the orifice or other manifold surfaces.

[0058] According to aspects of this disclosure, the heat-insulating cover may also include a second opening or through-hole in a region adjacent to the manifold. The shape and size of this second opening / hole may be designed to receive a box or disc having one or more containers for holding one or more samples. A hinged, sliding, or rotating cover may extend over and cover the opening and may be positioned over an area entirely contained within the interior chamber.

[0059] According to an aspect of this disclosure, the manifold orifice and the second opening of the cap can be in communication, such that a sample immersed, translated, or moved by a robotic arm through the orifice and into liquid nitrogen can subsequently be translated out of the orifice and into the region below the second opening.

[0060] According to an aspect of this disclosure, the manifold orifice may include a hinged door defining one side of the orifice, the hinged door opening to allow the lateral transfer of a cryogenically cooled sample from the orifice to an area near the orifice below the second cover opening and above the cassette.

[0061] According to aspects of this disclosure, the system may include one or more hydraulic, pneumatic and / or electrically activated actuators for automating the opening and closing of the aforementioned covers, hoods, doors, etc.

[0062] According to aspects of this disclosure, the bottom of the inner cavity located below the lid opening may include a cavity structure whose dimensions and shape are adapted to receive and hold a cassette or disk in a defined orientation, for example, allowing a robot to place a sample into the cassette or disk after cooling, or to retrieve a sample from the cassette or disk for subsequent processing. The cavity structure may be integrally formed or machined into the inner cavity; alternatively, the cavity structure may be a different structure fixed to the bottom of the inner cavity.

[0063] According to aspects of this disclosure, tools can be used to load or remove a box or disc into or from a chamber structure within the cavity.

[0064] According to aspects of this disclosure, a cryogenically cooled box or disk containing a previously cryogenically cooled sample can be loaded through a second opening in the lid of an insulated container and placed into a receiving box or disk structure within the insulated container, for example, to allow a robot to approach the sample and transfer it to a heating station.

[0065] According to aspects of this disclosure, a sample cryogenic cooling station may include a camera, a lens, and one or more light sources and / or other optical devices to image a cryogenically cooled sample in or directly above liquid nitrogen as needed, and then transfer the sample to a cassette or disk.

[0066] According to aspects of this disclosure, the sample cooling rate achieved by the cryogenic cooling system with a robot and an end effector may be approximately 25,000. oC / min (for samples of approximately 500 micrometers) and approximately 3,000,000 o Between C / min (for a sample of approximately 25 micrometers).

[0067] According to aspects of this disclosure, a robot can immerse, translate, or otherwise move a sample through a hole and into liquid nitrogen at a speed of at least about 1 m / s or, in some applications, at a speed of at least about 2 m / s but not more than 5 m / s, for example, in order to maximize convective heat transfer without causing excessive splashing of liquid nitrogen or requiring excessive travel distance through the liquid nitrogen.

[0068] According to aspects of this disclosure, the Dewar flask depth and liquid nitrogen filling level can be predetermined to allow the sample to travel a distance between about 2 cm and about 20 cm before stopping, or at least about 4 cm in some applications, for example, to ensure that the sample continues to travel at high speed and that the convection velocity of the liquid nitrogen relative to the sample remains large until the sample cools to a sample temperature below a predetermined target temperature (e.g., about 150 K).

[0069] According to aspects of this disclosure, the robotic arm itself can realize or may have an end effector that enables high-speed, on-axis or off-axis rotation of the sample carrier, for example, so as to maintain a high velocity and a high convective heat transfer rate of the sample carrier relative to the liquid even when the translational motion of the robotic arm has stopped.

[0070] According to aspects of this disclosure, the rotational motion provided by the robot can result in a rotational speed of at least about 0.25 m / s relative to the frame of the insulating container, or in some applications, about 0.5 m / s to about 2 m / s.

[0071] According to aspects of this disclosure, a robotic arm and / or end effector can provide high-speed vibrational motion of a sample, wherein the peak sample velocity 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 wherein the inter-peak vibration amplitude is at least about 2 mm.

[0072] According to aspects of this disclosure, the system may include a sample warming or thawing station, wherein a sample previously cooled to a cryogenic temperature may be rapidly warmed in the sample warming station to a predefined room temperature or a predefined biological temperature between about 20°C and about 60°C. As used herein, the term “rapid warming” and its substitutions may include a sample warming rate between about 50,000°C / min (e.g., for a 500-micron sample) and about 6,000,000°C / min (e.g., for a 25-micron sample).

[0073] According to aspects of this disclosure, the heating station may include a temperature-controlled plate or block heater, a high-conductivity stage for receiving a plate attached to the plate / block heater, a container (e.g., vial, test tube, cuvette, etc.) for holding the solution used in thawing, and an insulator wrapped around the outer surface of the heat-conducting stage to reduce heat loss.

[0074] According to aspects of this disclosure, the heat-conducting stage can be shaped to provide close thermal contact with any liquid-sealed surface of a plate, vial, test tube, etc., for example, to ensure maximum heat transfer rate and temperature uniformity.

[0075] According to aspects of this disclosure, a robot can immerse, translate, or otherwise move a sample into, translate, or otherwise move it into a heated solution contained in a well in a plate, vial, test tube, or cuvette at a speed between about 0.25 m / s and about 5 m / s, or about 2 m / s in some applications, for example, to maximize the rate of convective heat transfer while minimizing liquid splashing when the sample carrier impacts the liquid.

[0076] According to aspects of this disclosure, plates, vials, test tubes, cuvettes, etc., may contain liquid containers with a depth between about 1 cm and about 10 cm, or about 4 cm in some applications, for example, such that the sample can be kept in high-speed motion for a sufficiently long time to ensure that it has substantially heated to a predetermined sample temperature, such as at least about -10°C, or about 0°C in some applications, such that any and all ice nucleation and growth within the sample has ceased before the translational motion stops.

[0077] According to aspects of this disclosure, in addition to vertical translation, a robotic arm or an end effector on the robotic arm can be used to rotate the sample, for example, such that the sample movement relative to the heated liquid can maintain a large convective heat transfer rate until the sample has been heated to at least about -10°C, or in some applications, about 0°C.

[0078] According to aspects of this disclosure, the rotational motion provided by the robot can result in a rotational speed of at least about 0.25 m / s relative to the frame of the insulating container, or in some applications about 0.5 m / s to about 2 m / s.

[0079] According to aspects of this disclosure, the robotic arm and / or end effector can allow high-speed vibrational motion of the sample, for example, having a peak sample vibration velocity of at least about 0.25 m / s, or in some applications, between about 0.5 m / s and about 2 m / s, wherein the interpeak amplitude of the motion is at least about 2 millimeters.

[0080] According to aspects of this disclosure, the temperature of the heated liquid can be a biological temperature (e.g., close to 37°C for mammalian samples), including heated biological temperatures close to the upper limit that the sample can tolerate (up to about 60°C for at least some eukaryotic samples), lasting for several seconds without adverse effects.

[0081] According to aspects of this disclosure, before entering a heated liquid at a biological temperature (e.g., close to 37°C), a cold sample may be initially immersed, translated, or otherwise moved through a heated liquid at a temperature substantially above the biological temperature (between 50°C and 100°C), thereby increasing the average heating rate of the sample without raising the sample temperature to a non-biological temperature.

[0082] According to this disclosure, the robot can quickly remove the sample from the heating solution before the sample temperature reaches the temperature of the heating solution, thus protecting the sample from excessively high temperatures.

[0083] According to aspects of this disclosure, the heating solution can be contained in two separate compartments, each heated to a different temperature. For example, one compartment may maintain the heating solution at biological temperature (e.g., close to 37°C), while the other compartment may maintain it at a temperature well above biological temperature (e.g., 50 to 100°C) and below the solution's boiling point. The heating solution compartments may be connected to allow the sample to be transferred from one compartment to the other during heating. The sample may initially travel through the first high-temperature chamber before being transferred to and through the second low-temperature chamber, for example, to maximize the sample heating rate while preventing sample damage that could occur if its temperature rises well above biological temperature.

[0084] According to aspects of this disclosure, the two heated solution compartments can be arranged vertically, with the high-temperature compartment located above the low-temperature compartment, for example, such that the sample can be continuously moved through the high-temperature compartment and into the low-temperature compartment.

[0085] According to aspects of this disclosure, the two heated solution compartments can be arranged side by side.

[0086] According to an aspect of this disclosure, the two heated solution compartments can be in direct fluid communication via pores in the walls separating the compartments. The pores can be large enough to allow the sample-containing portion of the sample carrier to pass through them.

[0087] According to an aspect of this disclosure, the opening in the wall separating the two heated solution compartments can be covered by a door or flexible flap that can be opened before or during sample transfer between the compartments, for example, thereby keeping the heated solutions in the two compartments separated until the transfer occurs.

[0088] According to aspects of this disclosure, the aperture cover door can be opened by direct contact with a sample carrier, a robot end effector, or a robotic arm.

[0089] According to aspects of this disclosure, the door may be spring-loaded or driven by an actuator.

[0090] According to aspects of this disclosure, the movement of the door can be within the plane of the door, for example, to minimize fluid resistance to opening the door and to facilitate opening the door within a short time range compared to the heating time of the sample.

[0091] According to aspects of this disclosure, the heating solution can be contained in a channel that slopes downward from near the top surface of the microplate / solution container to the bottom of the microplate / solution container, for example, such that the sample can be “immersed” in the heating solution along a trajectory parallel to the bottom of the channel.

[0092] According to aspects of this disclosure, a first heating block adjacent to the upper portion of the channel can be used to heat the upper portion of the channel to a first temperature significantly higher than the normal biological temperature, such as 50°C to 100°C, and a second heating block adjacent to the lower portion of the channel can be used to heat the lower portion of the channel below the upper portion to a second temperature different from the desired final sample temperature, such as 37°C.

[0093] According to aspects of this disclosure, the width of the channel can be between about 1 mm and about 10 mm, or in some applications, between about 2 mm and about 5 mm, for example, in order to minimize the convective mixing of the heated solution in the lower and upper parts of the channel.

[0094] According to aspects of this disclosure, the channels may have a maximum depth of no more than about 13 mm, for example, to be compatible with SBS / SLAS standard height microplates (14.35 mm high), or no more than about 27.3 mm to be compatible with SBS / SLAS dual height microplates (28.7 mm high), or no more than about 41.7 mm to be compatible with SBS / SLAS deep well plates (43.05 mm high).

[0095] According to aspects of this disclosure, the system may include a post-heating soaking and incubation station, wherein the sample may be soaked in one or more solutions before cryogenic cooling.

[0096] According to aspects of this disclosure, the station can allow the solution temperature to vary between about 20°C and about 50°C after heating.

[0097] According to aspects of this disclosure, the post-heating station can accept standard multi-well plates in thin-hole, standard-hole, or deep-hole format for containing the post-heated solution.

[0098] According to aspects of this disclosure, the heated station can use a commercially available temperature-controlled orifice plate heater with a custom orifice plate carrier, as needed.

[0099] According to aspects of this disclosure, a custom porous plate can be used at the post-heating station, the custom porous plate being designed to receive and hold sample carriers placed therein by a robot.

[0100] According to aspects of this disclosure, the system may include a rinsing and drying station for rinsing and drying a robotic end effector to remove heated liquid from the end effector in the event of liquid splashing during heated immersion, and to heat and dry the end effector after immersion in liquid nitrogen or a heated solution.

[0101] According to aspects of this disclosure, the station's rinsing components may involve one or more containers containing pure water, water plus detergent, or isopropanol.

[0102] According to aspects of this disclosure, the robot can translate within a rinsing and drying station and then immerse the end effector in one or more rinsing liquids.

[0103] According to aspects of this disclosure, the robot can rotate the end effector and cause it to oscillate up and down during rinsing within the rinsing and drying station.

[0104] According to aspects of this disclosure, the heating and drying components of the station may include an air amplifier supplied with compressed drying gas, into which an end effector is inserted and moves up and down until dry.

[0105] According to aspects of this disclosure, the heating and drying components of the station may include a single heater or multiple heaters arranged concentrically near the air amplifier and having the same central axis as the air amplifier, for example, to allow the end effector to move in and out of the heater and air amplifier along the line.

[0106] According to aspects of this disclosure, any or all of the robot, motor, actuator attached to the robot and forming an end effector, temperature-controlled heater / cooler of the pre-soaking station, heating station and post-soaking station, sensor, actuator, motor, valve, etc., can be electronically controlled within any other system component and, if desired, can be automated via a computer, microcontroller, programmable logic circuit, integrated circuit (IC) device, control module or network of computer / microcontroller / logic circuit / IC device / etc.

[0107] According to aspects of this disclosure, multiple sample carriers can be processed simultaneously by sequencing robot operations, allowing samples to be released for immersion while the robot performs movements required for other processing steps on another sample.

[0108] According to aspects of this disclosure, the sample carrier may contain one or more samples to prevent their loss during processing. The sample carrier may have a pore array that allows the liquid to flow through and over the sample at high speeds while the sample carrier moves relative to the liquid at high speeds, with minimal flow resistance, thereby maximizing the convective heat transfer rate between the sample and the liquid.

[0109] According to aspects of this disclosure, the sample can be supported on a thin film of a transparent polymer, glass, or semiconductor. The thickness of the film can be from about 2 μm to about 100 μm, or in some applications, from about 5 μm to about 50 μm. Small thickness can help minimize flow resistance, maximize heat transfer through the film to the sample, and maximize optical transparency.

[0110] According to aspects of this disclosure, the membrane can have regions of varying thicknesses, such as a first “thin” region, for example having pores on which the sample resides, with a thickness between about 5 μm and about 15 μm, and a second “thick” region, for example for mechanical strength and for bonding to other portions of the sample carrier, with a thickness between about 10 μm and about 50 μm, or, for some applications, up to about 100 micrometers. Thick regions forming markers, pillars, or other features may be present within the thin regions of the membrane, which can be used to position the sample on the membrane.

[0111] According to aspects of this disclosure, portions of the thin film receiving the sample may have an open area fraction between about 50% and about 95%, or in some applications, between about 70% and about 95%. The open regions may be formed by a dense array of through-holes, wherein the pores are as large as possible but small enough to prevent the sample from passing through them, thereby minimizing resistance to fluid flow through the membrane in those regions.

[0112] According to aspects of this disclosure, the pores in the film can have a diameter between about 40% and about 80% of the sample diameter. For mammalian oocytes and embryos, for example, the pore diameter can be between about 40 micrometers and 150 micrometers.

[0113] According to this disclosure, thin films can be micro-patterned using photolithography and microfabrication processes, micro-embossing, stamping, etc.

[0114] According to aspects of this disclosure, the film can be made of an optically transparent polymer, such as polyimide, cyclic olefin copolymer, polyester film, or SU-8, for example, to facilitate imaging of a sample placed on the film.

[0115] According to aspects of this disclosure, the film can be attached to a thin rigid frame, wherein the frame can be marked with information (e.g., laser marking) to allow sample identification and tracking.

[0116] According to aspects of this disclosure, the frame can be attached to a base configured to be manipulated by a robotic end effector. The base can be made of rigid, dimensionally stable materials, including magnetic stainless steel, and can be marked or may contain RFID tags for sample identification and tracking.

[0117] According to aspects of this disclosure, the frame may include a rigid sheet having a hole disposed adjacent to one end of the sheet. The sheet may be made of a rigid metal (e.g., brass), a polymer (e.g., polycarbonate), or a polymer-glass composite (e.g., G10). The sheet thickness may be comparable to or slightly greater than the corresponding sample size (between 80% and 150%), for example, to ensure that the sample is close to the sample carrier surface and close to the liquid flowing over these surfaces to maximize the heat transfer rate between the liquid and the sample. For mammalian oocytes and embryos, this thickness may be between about 80 μm and about 250 μm, or about 150 μm in some applications.

[0118] According to aspects of this disclosure, the width of the frame can be between about 3 mm and about 10 mm, or for some applications, it can be as large as about 30 mm (e.g., if the carrier is to hold a very large number of samples).

[0119] According to aspects of this disclosure, the length of the frame—including the portion inserted / attached to the base—can be between about 1 cm and about 10 cm, or in some applications about 2 cm.

[0120] According to aspects of this disclosure, the frame may have at least one hole disposed near the end furthest from the base, the width of which is equal to the width of the frame, less than about 2 mm (e.g., between about 1 and about 8 mm, up to about 28 mm, depending on the frame width), or, for frames with a width of 10 mm or greater, less than about 4 mm, and the length of the hole is between about 2 mm and about 10 mm, up to about 30 mm, such that the hole in the frame extends to a range not exceeding about 0.5 mm, or, in some applications, not exceeding about 1 mm of the frame edge, to allow sufficient surface area to bond the film to the frame.

[0121] According to aspects of this disclosure, the pores in the frame can be spanned and sealed by a membrane on one side (e.g., the "bottom" side of the frame), as discussed above. The portion of the membrane filling the pores can be located within the pores, while the portion without a dense array of pores can be predominantly located over the solid region of the frame defining the pores. The membrane can be bonded to the frame using methods including adhesives, waxes, ultrasonic bonding, or thermal bonding.

[0122] According to aspects of this disclosure, the membrane may have a thickness (e.g., 5-15 micrometers) in the region of the pores and a second larger thickness (e.g., 10-50 micrometers) in the region of the contact frame.

[0123] According to aspects of this disclosure, one or more samples are placed onto a porous membrane within a pore.

[0124] According to aspects of this disclosure, after the sample has been placed on the first film, a second film (which may be substantially similar to the first film, containing a dense array of pores of similar size and density) may be applied to the “top” side of the frame, for example, to seal the sample within the pores and between the two films.

[0125] According to aspects of this disclosure, the first “bottom” membrane and / or the second “top” membrane may include perforations or tabs within the opening surrounding the respective inner edge of the opening to facilitate removal of the membrane portion within the opening and access to the sample within the opening.

[0126] According to an aspect of this disclosure, the holes in the frame can be replaced by U-shaped cuts at the frame ends furthest from the base.

[0127] According to aspects of this disclosure, the “top” and “bottom” sides of the frame and the open ends of the frame can be sealed with one or two perforated films, such that the top, bottom and end openings of the U-shaped cut are partially sealed by a film portion having a dense array of pores, for example, to allow liquid to flow into the interior of the U-shaped cut from all three open sides with minimal flow resistance, while retaining one or more samples inside the cut.

[0128] According to aspects of this disclosure, the sample carrier may include a basket and a lid, or, if desired, may consist substantially of a basket and a lid. The basket may receive and hold one or more samples, and the lid may be configured to seal the basket and be grasped or handled by a robotic end effector.

[0129] According to aspects of this disclosure, the diameter of the bottom of the basket can be between about 2 mm and about 20 mm, or in some applications, between about 3 mm and about 10 mm.

[0130] According to aspects of this disclosure, the basket may include a thin, rigid frame with low thermal mass, a substantially open bottom, and an opening extending upward from the bottom in the side of the frame by a distance d. The distance d may be equivalent to or greater than the diameter of the basket at its base, and may be about 2 to 4 times the diameter of the bottom, or in some applications, between about 6 and about 40 mm.

[0131] According to aspects of this disclosure, the open bottom and sides may be spanned by a porous membrane or a mesh formed of filaments. The pores of the membrane or mesh may be as large as possible to prevent sample passage, typically in the size range of about 40 μm to 120 μm for mammalian oocytes and embryos, and more typically between 40% and 80% of the sample diameter.

[0132] According to aspects of this disclosure, the bottom opening of the basket can be spanned and sealed by a porous film of a transparent polymer (such as polyimide, SU-8, and cyclic olefin copolymers), for example, to allow optical inspection of the sample on the film. The film may have a thickness between 5 μm and about 15 μm in the region within the bottom opening, and a thickness between about 10 μm and about 50 μm in the region where it is bonded to the frame.

[0133] According to aspects of this disclosure, the film portion within the opening in the bottom of the basket may have an opening area fraction of about 40% to about 95%, or in some applications, an opening area fraction of about 70% to about 95%.

[0134] According to aspects of this disclosure, the openings on the sides of the basket frame can be sealed using a perforated membrane, wherein the perforated area of ​​the membrane is located within the frame opening, or sealed by a fine mesh of metal or polymer (e.g., nylon) with a mesh size between about 60 and about 400 meshes, or for mammalian oocytes and embryos, between about 100 and about 240 meshes.

[0135] According to aspects of this disclosure, one or more samples may be placed on the inner bottom of a basket, on a porous membrane with an opening spanning the bottom of the basket.

[0136] According to aspects of this disclosure, a sample inside the basket can be sealed by inserting a lid into the top opening of the basket to form a seal between the lid and the basket.

[0137] According to this disclosure, the cover can be engaged with the basket in a pressing or twisting motion.

[0138] According to aspects of this disclosure, the upper part of the basket frame can extend beyond the outer diameter of the cover, thereby allowing the basket to be removed from the cover by pressing down on the frame extension while holding the cover in place.

[0139] According to aspects of this disclosure, the cover may have a structure that facilitates gripping by a robotic arm. The cover structure may include a torsion locking structure, a press-lock locking structure, or a magnet.

[0140] According to aspects of this disclosure, the cover may include a tapered feature that is axially positioned and projects downward into the basket. This tapered feature can be used to guide liquid flowing upward through the bottom of the basket outward to the side of the basket and out through a porous membrane or mesh covering portion on the side of the basket, thereby generating a liquid flow closer to laminar flow when the sample carrier is immersed in cold or hot liquid.

[0141] According to aspects of this disclosure, the cover may include openings spanned by a thin porous membrane or mesh to allow liquid to pass through during cooling and heating.

[0142] According to aspects of this disclosure, a series of baskets and caps can be held within a container carried by the baskets and caps. Samples can be loaded into the baskets using a pipette or other tools, and then the caps are positioned on the baskets and sealed to them.

[0143] According to aspects of this disclosure, the bottom of the basket may be dome-shaped, with most of the dome spanned by a thin porous membrane.

[0144] According to aspects of this disclosure, the basket frame or lid may be marked or patterned to allow identification of each sample, or the lid may contain an RFID tag for sample identification.

[0145] The foregoing summary does not represent every embodiment or aspect of this disclosure. Rather, it provides only a summary of some novel concepts and features set forth herein. The foregoing features and advantages, as well as other features and accompanying advantages, will become apparent from the following detailed description of illustrated examples and representative modes for carrying out this disclosure, when taken in conjunction with the accompanying drawings and appended claims. Furthermore, this disclosure expressly includes any and all combinations and sub-combinations of the elements and features presented above and below. Attached Figure Description

[0146] Figure 1 This is a perspective view of a representative automated sample system for cryopreservation and resuscitation of small biological samples according to aspects of this disclosure.

[0147] Figure 2 It is based on the aspects of this disclosure that have the same Figure 1 A perspective view of a representative robotic system that uses key structures and functions in conjunction with an automated sample system.

[0148] Figure 3 A to Figure 3 C is a perspective view of different representative robot systems having end effectors suitable for use with the disclosed system, according to aspects of this disclosure.

[0149] Figure 4 This is a partial cross-sectional perspective view of a representative turntable used for transferring samples into and out of the system housing, according to aspects of this disclosure.

[0150] Figure 5 A and 5B are perspective views and partially exploded perspective views of representative stations where samples are immersed in solution prior to cryopreservation, according to aspects of this disclosure.

[0151] Figure 6 A and 6B are, respectively, partial exploded and partial cross-sectional perspective views of representative stations used for removing excess liquid from a sample and imaging the sample prior to immersion cooling, according to aspects of this disclosure.

[0152] Figure 7 A and Figure 7 B are partial cross-sectional perspective views and elevation perspective views of representative stations for rapid cooling of samples in liquid nitrogen and for cryogenic sample storage, respectively, according to various aspects of this disclosure, showing the hinged door of the housing in the open position.

[0153] Figure 8 A and 8B are respectively Figure 7 Partial sectional perspective and elevation perspective views of representative rapid cooling stations A and 7B, showing the hinged doors in the closed position.

[0154] Figure 9 A and 9B are schematic diagrams and partial cross-sectional perspective views, respectively, of another representative station used for rapid cooling of samples in liquid nitrogen and for cryogenic sample storage, according to aspects of this disclosure.

[0155] Figure 10 A and 10B are perspective views and partially exploded perspective views, respectively, of a representative station for rapidly heating a cold sample according to aspects of this disclosure.

[0156] Figure 11 A and 11B are schematic diagrams of two representative dual-chamber units and heating blocks used to maintain the solution during rapid heating of a cold sample, according to aspects of this disclosure.

[0157] Figure 12 A and 12B are schematic diagrams of another representative unit and heating block for maintaining the solution during rapid heating of a cold sample, according to aspects of this disclosure.

[0158] Figure 13 This is a side perspective view of a representative microplate and a matching heating block used to contain a solution during sample heating, according to an aspect of this disclosure.

[0159] Figure 14 This is a partial exploded perspective view of a representative station for a heating and drying robot end effector according to aspects of this disclosure.

[0160] Figure 15 A to Figure 15C is a perspective view of a representative first sample carrier suitable for use with the disclosed system, according to various aspects of this disclosure.

[0161] Figure 16 A and Figure 16 B is a perspective view of a representative second sample carrier suitable for use with the systems described in various aspects of this disclosure.

[0162] Figure 17 A to 17C are perspective views of the “basket” portion of a representative third sample carrier applicable to the disclosed system according to aspects of this disclosure.

[0163] Figure 18 This is a perspective view of the alternative "basket" portion of a representative third sample carrier applicable to the disclosed system, in accordance with aspects of this disclosure.

[0164] Figure 19 A and Figure 19 B is a perspective view of a basket and lid of a representative sample carrier according to various aspects of this disclosure, and a representative tool suitable for use with the disclosed system.

[0165] Figure 20 This is a perspective view of a representative tray used for holding, loading, and assembling sample carriers according to various aspects of this disclosure and suitable for use with the disclosed system.

[0166] Figure 21 A to Figure 21 C is a perspective view of a representative basket-based sample carrier according to various aspects of this disclosure.

[0167] Figure 22 It is a side perspective view of a representative sample carrier that can be opened with a tool to allow sample loading and then closed to seal the sample for processing, in accordance with aspects of this disclosure.

[0168] This disclosure is adaptable to various modifications and alternatives, and some representative embodiments of this disclosure are illustrated by way of example in the accompanying drawings and will be described in detail herein. However, it should be understood that the novel aspects of this disclosure are not limited to the specific forms illustrated in the foregoing drawings. Rather, this disclosure covers all modifications, equivalents, combinations, substitutions, groupings, and alternatives that fall within the scope of this disclosure, for example, as covered by the appended claims. Detailed Implementation

[0169] This disclosure allows for numerous different forms of embodiments. Representative embodiments of this disclosure are shown in the accompanying drawings and will be described in detail herein. It should be understood that these embodiments are provided as examples of the principles disclosed and not as limitations on the broad aspects of this disclosure. To this extent, elements and limitations described, for example, in the abstract, introduction, summary of the invention, description of the drawings, and detailed description but not expressly set forth in the claims should not be incorporated into the claims individually or collectively by implication, inference, or otherwise. Furthermore, the use of terms such as “first,” “second,” “third,” etc., in the specification or claims is not in itself intended to establish a sequence or numerical limitation; unless expressly stated otherwise, these names may be used to facilitate reference to similar features in the specification and drawings and to delineate similar elements in the claims.

[0170] For the purposes of this detailed description, unless otherwise stated: the singular includes the plural, and vice versa (e.g., unless explicitly stated, the indefinite articles “a” and “one” should be interpreted as meaning “one or more”); the words “and” and “or” should be both connected and separate; the words “any” and “all” should both mean “any and all”; and the words “including,” “containing,” “comprising,” “having,” etc., should each mean “including but not limited to.” Furthermore, for example, approximate words such as “approximately,” “almost,” “substantially,” “generally,” “approximately,” etc., may each be used herein to mean “in, near, or almost in,” or “within 0-5%,” or “within acceptable manufacturing tolerances,” or any logical combination thereof.

[0171] The features of this disclosure are intended to address the design criteria and challenges listed below to optimize the cryopreservation and thawing of small biological samples, including minimizing sample damage caused by handling, osmotic shock, cryoprotectant toxicity, freezing, rupture, and other factors, thereby maximizing the survival and development of cellular systems. Additional information relating to this disclosure can be found, for example, in U.S. Patent No. 9,417,166B2 to Thorne et al., U.S. Patent No. 11,473,826B2 to Closs et al., and U.S. Patent No. 11,653,644B2 to Thorne et al., the entire contents of which are incorporated herein by reference and used for all purposes.

[0172] The key physical and design principles of cryopreservation and revival systems are as follows:

[0173] - The sample is enclosed in a support that allows liquid to flow freely and easily, and at high speeds (1 m / s to 5 m / s), around the sample, with minimal thermal mass in the region adjacent to the sample, and maximizes convective and conductive heat transfer to / from the sample when moving relative to cold and warm liquids, while minimizing the chance of sample loss during all processing steps. This support concept is key to enabling automated sample handling.

[0174] - The use of a single, inexpensive robotic platform and sample carrier to automate all steps, the type of which is described in (1) and disclosed, for example, in U.S. Provisional Patent Application No. 63 / 601,937, filed November 22, 2023, which is incorporated herein by reference in its entirety for all purposes.

[0175] - Using a modular design, where each module performs a specific set of functions for the samples and carriers delivered to it by the robot, allowing for easy modification to improve functionality and easy addition of new functions.

[0176] - The sample is automatically immersed in a dehydrating solution containing cryoprotectant before cryogenic cooling to improve reproducibility and facilitate multiple immersion steps at increased concentrations to minimize osmotic shock without the risk of other sample loss or damage.

[0177] - Automatically remove excess liquid around the sample before cryogenic cooling to minimize thermal mass and maximize heat transfer rate and cooling / heating rate.

[0178] - The state of the sample is automatically recorded using optical imaging just before it is cryogenically cooled.

[0179] - The sample is cooled at the maximum feasible rate by high-speed immersion in liquid nitrogen, by maintaining a high relative velocity between the sample and the liquid nitrogen until the sample vitrifies, and by eliminating sample pre-cooling in the cold gas that is usually present above the liquid nitrogen.

[0180] - The state of the cryogenically cooled sample is automatically recorded using optical imaging before sample storage.

[0181] - Automatically transfer cryogenically cooled samples to a multi-sample storage box, which can be removed for short-term or long-term cryogenic storage.

[0182] - To minimize the preheating of samples before they enter the heating solution, the cryogenically cooled samples are automatically transferred from the storage container in liquid nitrogen to the heating solution.

[0183] - The sample is heated at the maximum feasible rate by high-speed immersion in the heating solution, by maintaining a high relative velocity between the sample and the heating solution until the sample is fully heated (or reaches at least 0°C), and also by first immersing the sample in a heating solution at a temperature significantly higher than the final target temperature, and then rapidly transferring the sample to a second solution at the target temperature.

[0184] - Automatically transfer samples from the heated solution to a reservoir containing additional solutions for soaking and recovery, enabling multiple soaking steps to be performed at increased concentrations to minimize osmotic shock without the risk of further sample loss or damage.

[0185] - Automatically transfer samples that have been cryogenically cooled, thawed, and revived to a location where they can be retrieved.

[0186] -Automatic cleaning, heating, and drying of samples by robotic end effectors to minimize frost buildup and remove soaking and immersion contaminants.

[0187] - Offers full programmability for most functions, allowing easy modification of soaking / cooling / thawing parameters for optimization.

[0188] Figure 1 A perspective view is provided of a representative example of an automated system 10 for cryopreservation and resuscitation of small biological samples based on the above principles. System 10 includes: a pick-and-place robot 20 having a customized end effector 40 for holding a sample carrier 60; a housing 80 having a door 100 that allows materials (including liquid microplates, liquid nitrogen, etc.) to be loaded into the housing and an externally accessible emergency stop button 120 for the robot; a sample loading turntable 140 into which samples can be loaded using, for example, a rod 160, and into which samples can be unloaded to the outside of the housing; a pre-cooled sample immersion station 180; a sample liquid removal and imaging station 200; a sample cryogenic cooling and storage station 220; a robot end effector heating and drying station 240; a sample heating station 260; and a post-heating sample immersion and incubation station 280. The advantageous feature of the system 10 shown is its great flexibility: a single robot with a properly designed end effector and sample carrier can perform all the required functions of cryopreservation and resuscitation; stations for each function can be swapped out and replaced when maintenance or improved versions are available; and additional stations can be added for new functions, all of which require only modest reprogramming of the robot and control system.

[0189] Figure 2 A perspective view of a commercial pick-and-place robot (e.g., the EPSON T3 desktop SCARA robot) is provided, showing its application in... Figure 1The system 10 contains the key structures and functions required for use. Key features may include: (1) high-speed (e.g., at least about 1 m / s and up to about 5 m / s) lateral movement for rapid sample transfer between stations; (2) high-speed vertical translation (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 immersing samples in liquid nitrogen and heated solutions and for rapid sample transfer between stations; (3) a sample-holding z-axis shaft of the robot that rotates about its axis at at least 2 revolutions per second and (if an end effector is used) up to 30 revolutions per second. Figure 2 The SCARA robot 300 in this design provides vertical "z" motion via a driven axis 320, which can also rotate about its axis. Horizontal motion is provided by two rotary stages 340 and 360. The robotic arm should allow for the addition of an end effector 40 to hold the sample carrier 60 and may also generate additional motion of the sample. Cartesian robots and spherical robots are also suitable for this application.

[0190] Figure 3 Three examples of robotic end effectors are shown, which may include any relevant features presented in U.S. Provisional Patent Application No. 63 / 601,937, applicable to the illustrated system 10. The end effector allows for the "grabbing," holding, and releasing of a sample carrier. Grabbing can utilize, for example, magnets, press fits, torsion fits, threaded fits, or snap-fit ​​fits. Release can utilize electromagnets or mechanical release. The end effector may also provide additional degrees of freedom for sample movement. Figure 3 In A, the end effector has a vertical translation mechanism comprising an outer cylinder 400 and an inner cylinder 410, the inner cylinder 410 holding a sample carrier 60, which can be translated in and out (e.g., by a motor and screw mechanism or pneumatically). Attached to the outer fixed cylinder may be a shield 420, which, for example, prevents the sample from significantly heating during transfer from liquid nitrogen to the heated solution before the sample enters the heated solution. The end effector allows the sample to translate in and out of the shield. Figure 3 In B, the end effector includes a motor 430 that generates axial rotational motion of the sample. Figure 3 In C, the end effector includes a motor 440 and a stage 450 to generate off-axis rotational motion of the sample. Rotational motion helps ensure uniform and reproducible immersion during sample soaking. It also helps generate and maintain a high velocity of the liquid nitrogen / heating solution relative to the sample during the cooling / heating process, minimizing the required cooling and heating times. As an example, with a rotation radius of 0.5 cm and a rotational speed of 7200° / s (20 rpm), the sample's velocity in its circular motion is 0.63 m / s.

[0191] Figure 4An example of a turntable-type sample transfer mechanism 500 for transferring samples from the outside of a housing to the inside of the housing and vice versa is shown. A rotating sample holding section 520 is driven by a motor 540 and has one or more containers 560 for holding the sample carrier. The containers can form a tight seal with the sample carrier to reduce sample evaporation and dehydration while the sample is inside the container. The sample holding section may include means for maintaining the temperature of the sample held therein at a desired temperature (e.g., 37°C), and may include a strip heater 580. The sample in the carrier is loaded into a container outside the housing, and then the turntable rotates the sample to the appropriate position inside the housing for retrieval by a robot. Similarly, when sample processing is complete, the robot can place the sample into the turntable, which then rotates it outside the housing. Depending on the number of samples to be processed per unit time, other sample transfer mechanisms (such as sliding stages that slide in and out of the housing, a single rotating arm, etc.) may be suitable.

[0192] Figure 5 A possible implementation of a station for immersing samples in solution prior to cryogenic cooling is shown. A typical protocol involves immersion in two solutions – an equilibrium solution and a vitrification solution, the latter having approximately twice the cryoprotectant concentration of the first. This reduces osmotic shock, but such immersion negatively impacts the subsequent survival and development of the sample (without cooling and heating). Breaking the immersion into more steps, with gradually increasing concentrations, may be helpful. Figure 5 The station 600 includes a commercial microplate heater 620 (or a thermoelectric microplate stage capable of simultaneous heating and cooling), a high thermal conductivity metal block 640 shaped to match the underside of the microplate for good heat transfer and uniform temperature, and a commercial or custom microplate 660 containing wells for holding the immersion solution. The volume of each well depends in part on the sample size and the size of the portion of the sample carrier immersed in the liquid in each well. The volume of each well, the number of wells on each plate, and the required number of immersion steps / solutions determine the total solution consumption and the number of samples that can be processed before a new plate must be loaded. The well volume should be as small as possible to minimize solution consumption.

[0193] In some applications, it may be desirable to perform cryogenic immersion at reduced temperatures to limit toxicity or other degradation effects. In this case, a temperature-controlled station containing, for example, a thermoelectric heater / cooler can be used. To achieve a more uniform and reproducible equilibrium in each solution, a robotic arm can oscillate the sample up and down, left and right, and / or in a circular motion, and an end effector can be designed to perform similar movements (with much less inertia than a robot).

[0194] and Figure 5 The same basic configuration can be used for sample soaking and incubation station 280 after heating.

[0195] The microplates used can be custom-manufactured to fit the sample carrier and allow the sample carrier to be placed and held in the well by a robot, thus allowing the robot to perform other functions while the sample is immersed.

[0196] Figure 6 A possible implementation of a station for removing excess liquid from a sample and imaging the sample before cooling is illustrated. Station 700 includes a body 710 with a closed, hollow interior; a port 720 to which suction can be applied (e.g., generated by a pump or compressed air vacuum generator), which may include a connector / adapter for connecting the body to the pump via a hose; a sample suction port 730 having an opening connected to the hollow interior of the body and sized and shaped to provide a seal to the sample holding portion of the sample carrier; a gasket 740 of a compliant material (e.g., rubber) for sealing the sample carrier to the suction port; a backlight source 750 (e.g., an LED lamp and lens) for illuminating the sample when it is in place at the suction port; a transparent or translucent window 760 in the station body for transmitting light from the source to the sample; and a compact digital microscope / endoscope / pipe endoscope 770, which may include additional LEDs for sample illumination and for imaging the sample.

[0197] Figures 7 to 9 A possible implementation of a station for rapid cooling and cryogenic storage of samples in liquid nitrogen is illustrated. Experiments show that cooling rates up to approximately 25,000°C / min can be achieved when cooling 500 μm samples, and up to approximately 3,000,000°C / min when cooling 25 μm samples. Station 800 includes an insulated housing / Dewar flask 810 and a reservoir 820 within the housing. A volume 830 enclosed between the housing and the reservoir holds a first volume of liquid nitrogen, and a 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 the reservoir 840 can be good thermal conductors, such as steel, aluminum, or copper, and the walls of the housing 810 can be good thermal insulators, such as polyurethane foam.

[0198] The liquid nitrogen volume in reservoir 820 is higher than that in volume 830, and is in fluid communication with that volume, so that if the liquid nitrogen in reservoir 820 rises above a preset level, excess liquid nitrogen will flow into volume 830. The strong thermal contact between the first and second volumes of liquid nitrogen inhibits boiling of the liquid nitrogen within the reservoir. Together with the overflow mechanism, this helps maintain a defined and stable liquid nitrogen level within the reservoir.

[0199] The outer casing 810 is covered by an insulated cover 850, which has a hinged / sliding / rotating door 860 providing access to a portion of the reservoir. The bottom of the reservoir beneath the door includes a container 870 for holding a box or disc 880, which in turn has a container 890 for one or more sample carriers. The box is made of a cryogenically compatible and dimensionally stable material, such as aluminum or steel. The box can be placed in a Dewar flask and removed from the Dewar flask using tools. When the door is open, dry gas can be supplied to the space in the reservoir above the liquid nitrogen to minimize the infiltration of humid ambient air and frost formation.

[0200] The heat-insulating cover 850 includes an opening adjacent to the door that accommodates a gas management manifold 900, which includes an immersion port 910 extending from the top surface of the manifold to its bottom surface, thereby allowing direct access to liquid nitrogen within the reservoir. The immersion port may be defined on one side by a hinged door 920 that extends below the surface of the liquid nitrogen within the reservoir, and its function will be described later.

[0201] The key function of the manifold is to remove all cold gas above the liquid nitrogen in the orifice and replace it with dry gas at ambient temperature before the sample is immersed, and also to prevent warm, humid ambient air from causing frost on cold surfaces and in liquid nitrogen.

[0202] Now go to Figure 8 Figure A shows a cross-sectional view of manifold 900, which has a port 930 for drying ambient temperature gas and a port 940 for suction. These ports connect to opposing channels whose openings 950 and 960 at the immersion orifice form a rectangle. The liquid nitrogen level within reservoir 820 is set by the aforementioned filling and overflow mechanism, described in more detail below, such that the gas-liquid interface rises approximately in the middle of the channel openings, making it easier for cold gas to be suctioned and for supplemental drying gas to be scavenged, and ensuring that the airflow is largely laminar, so that the thickness of any cold gas layer is compressed to below 100 micrometers. The manifold may include an additional opening located above the suction and supplemental gas channels, leading to the immersion orifice, through which additional drying gas can be supplied to further suppress the infiltration of moist air into the immersion orifice and frost formation. The manifold may also include a heater, particularly on the orifice wall, to minimize frost buildup.

[0203] like Figure 8 As shown in B, the door 860 covering the container may be transparent and made of glass or polymer to allow visual inspection of the cassette and the samples inside.

[0204] Before immersion in the cooled sample, cold gas is removed from above the liquid nitrogen within the orifice 910, and the sample is immersed in the liquid nitrogen by a robot through the orifice 910, and then transferred from the orifice to container 890 within the cassette / disc 880. To facilitate transfer without removing the sample from the liquid nitrogen, one or more doors 920 may be provided on one side of the orifice, which are pushed open by the robot, allowing the sample to be dragged through the orifice across the liquid nitrogen and into a position above the container in the cassette. The doors (which may be spring-loaded or magnetically retained) then automatically close after the transfer. Once the cassette has been filled, it can be removed and transferred to a storage Dewar flask for storage. The door 860 covering the portion of the storage flask that holds the cassette may be optically transparent to allow visualization of the transfer and detection of errors during the transfer.

[0205] The correct operation of the cooling station 800 requires that the liquid nitrogen level in the orifice 910 of the manifold 900 be precisely maintained near the midpoint of the vacuum at the orifice and the openings of the supplementary gas channels 950 and 960. Figure 9 A illustrates a possible configuration for implementing control of liquid nitrogen levels within a cooling station. For example... Figure 7 and Figure 8 As shown, a reservoir 1000, formed of a good thermal conductor (such as steel, aluminum, or copper), is located within a second, larger insulating housing 1010. Thermal insulation can be provided by a solid thermal insulator 1020 (e.g., polyurethane foam) or by maintaining a vacuum within the sealed chamber. The inner surface of the insulating chamber can be constructed of a metallic (e.g., steel) "barrel" 1030 to contain liquid nitrogen, preventing it from penetrating the insulation in the event of insulation degradation or failure, and also providing a more uniform temperature to the inner surface. The top of the wall of the reservoir 1000 is located below the top of the wall of the housing 1010.

[0206] Liquid nitrogen supply lines 1040 and 1050 connect to the interior of reservoir 1000 and to the space 1060 between reservoir 1000 and the inner surface 1030 of insulating housing 1010. These can be connected to a pressurized liquid nitrogen storage Dewar flask or to a liquid nitrogen supply source via valves. Alternatively, they can be connected via valves 1070 and 1080 to an insulating liquid nitrogen supply container 1090 (which can be formed of insulating foam lined with a metal (steel) "bucket"). Liquid nitrogen supply container 1090 can be raised relative to reservoir 1000 and insulating housing 1010, such that the flow of liquid nitrogen between the supply container and the reservoir and housing is driven by gravity. The supply lines can be connected near the bottom of the reservoir and near the bottom inner surface of the insulating housing to reduce vaporization during filling. The supply lines should be well insulated or cooled by contact with cold gas or liquid nitrogen.

[0207] The liquid nitrogen level within the reservoir 1000 and the space 1060 between the reservoir and the insulating housing can be measured using level sensors 1100 and 1110, such as heated RTD sensors, thermocouples, diodes, laser level sensors, or float-type sensors.

[0208] By maintaining the liquid nitrogen level within the reservoir 1000 at or very close to the top of the reservoir wall, the liquid nitrogen level within the immersion port 900 can be kept nearly constant. When the liquid level in the reservoir drops below the target value (typically about 2-3 mm below the top of the reservoir), liquid nitrogen flows into the reservoir to fill and then overflows, with the overflow collected in volume 1060 between the reservoir and the insulating housing. Experiments show that almost all boiling of the liquid nitrogen occurs in volume 1060 between the reservoir and the housing; the liquid nitrogen level within the reservoir 1000 drops much more slowly, and the surface of the liquid nitrogen within the reservoir is largely still, indicating minimal boiling. This allows for long operating times between liquid nitrogen filling and reservoir replenishment. In this way, the liquid nitrogen level within the orifice of the manifold 900 is maintained near the midpoint of the openings of the vacuum and replenishment gas channels 950 and 960, as required for effective removal of the cold gas layer.

[0209] Instead of being set by the top of the reservoir wall, the liquid nitrogen level inside the reservoir 1000 can be set by a hole located on one of its side walls (below the top of the side wall), through which liquid nitrogen will overflow into the space between the reservoir and the outer casing.

[0210] An alternative method for filling reservoir 1000 is to pump liquid nitrogen from volume 1060 into reservoir 1000 until the reservoir overflows, wherein pumping is turned on and off based on readings from level sensor 1100. Any pump compatible with liquid nitrogen can be used. Impeller pumps or centrifugal pumps are particularly suitable because they do not require valves or seals and there is no need for contact between moving and non-moving parts. The pump can be driven from above by a shaft that protrudes downwards into the liquid nitrogen to the impeller.

[0211] In operation, the liquid nitrogen supply container 1090 can be filled with liquid nitrogen first. During or after filling the supply container, valve 1070, which controls the flow to volume 1060 between the reservoir and the housing, can be opened to cool and then fill the volume to a preset level measured by 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 level sensor 1100. When the liquid level in reservoir 1000 or volume 1060 drops below the target value, the corresponding volume will be filled with liquid nitrogen.

[0212] Functionally similar to Figure 9 As shown in A and Figure 9In the prototype system shown in B, the storage container is cylindrical with a diameter of 15 cm, a height of 10 cm, and a volume of approximately 1.8 liters; the external isolation chamber has internal dimensions of 25 cm long × 25 cm wide × 15 cm high and a volume of approximately 10 liters; and the insulated supply container has internal dimensions of 10 cm wide × 30 cm long × 4 cm high and a volume of approximately 3 liters.

[0213] Cooling stations can include similar Figure 6 The optical imaging system shown is used to image a sample after cryogenic cooling to assess whether the water in the sample has vitrified (in which case the sample will appear clear and its internal structure will remain visible) or whether some parts have crystallized (in which case the sample will appear milky white). The imaging system may include an LED light source (with a lens or translucent plate in front and possibly optical fibers or mirrors to guide light to the sample), which provides illumination from behind the sample for transmitted light illumination; a compact digital microscope / endoscope / pipe endoscope, which may include an imaging chip, a series of lenses, and possibly optical fibers, and may also include additional LEDs for incident illumination. These components may be mounted in a 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 sources of drying ambient temperature gas used to prevent moisture ingress shut off when the door is open, cold gas will accumulate above the surface of the liquid nitrogen. The sample can then be briefly raised into this cold gas above the liquid nitrogen surface for imaging. Preliminary experiments using a $30 endoscope camera show that it works well at low temperatures as long as it is kept dry. Alternatively, a fiber optic endoscope could be used, where optical fibers are used to transmit light to the sensors at room temperature.

[0214] To warm and revive cold samples, sample cartridge 880 can be loaded into storage unit 820 of the cooling station, and the samples can be retrieved directly by a robot through door 860 above the storage unit, or by first translating them from the cartridge through door 920 and into the immersion port. The latter is likely desirable to minimize frost formation on cold surfaces. Alternatively, sample cartridge 880 can be placed in a separate insulating Dewar flask filled with liquid nitrogen, with a container at the bottom for the cartridge (similar to 870) to ensure accurate and repeatable cartridge placement, thus enabling reliable robotic retrieval.

[0215] Figure 10A possible implementation of a station for rapidly heating cold samples is shown. For the fastest heating, the sample must travel at high speed (1-5 m / s is the practical range) relative to the heating solution until it is heated to a temperature well above the sample's melting point (typically close to -10°C if it has been immersed in a cryoprotectant). If the relative motion is merely vertical / translational, this may require a large immersion depth for larger samples that heat more slowly – much greater than that used in current practice. Alternatively, if the sample is rotated and translated at high speeds, the immersion depth can be reduced. Figure 10 The heating station implementation 1200 includes a commercially available microplate temperature-controlled block heater 1210, a high thermal conductivity plate housing 1220, and a deep-hole block plate 1230. The plate housing can be constructed of a high thermal conductivity metal (e.g., aluminum) in close thermal contact with the block heater, and its sides can be covered with an insulating material (e.g., foam). Standard deep-hole block plates have a hole depth of approximately 4 cm, which should be sufficient to heat samples with a diameter less than approximately 150 micrometers, including most mammalian oocytes and embryos, when using immersion velocities up to approximately 2 m / s. For larger samples or higher immersion velocities, significantly deeper holes may be required – potentially up to 10 cm or even 20 cm – to ensure a high convective fluid velocity relative to the sample until the sample is fully heated. This would require a custom-made deep-hole block plate or an array of test tubes, cuvettes, or similar containers. In this case, combining vertical and off-axis rotational motion of the sample may be more ideal to minimize solution consumption.

[0216] The heating rate of a sample at any given time is partly determined by the temperature difference ΔT between the surrounding liquid and the sample. Therefore, the heating rate is highest immediately after the sample enters the heating solution, when this temperature difference is greatest, and decreases as the sample temperature approaches the temperature of the heating solution. For a heating solution temperature of 37°C, the "driving force" for heating decreases from ΔT = 37°C + 196°C = 233°C when the sample first enters the heating solution to ΔT = 37°C + 10°C = 47°C when the sample temperature reaches -10°C, a decrease of about one-quarter of the original value. The specific heat of the sample increases with increasing temperature, further reducing the heating rate. Once the sample temperature rises above approximately -70°C, ice rapidly nucleates and grows, peaking at around -40°C, at which point the heating rate decreases significantly. Therefore, it is necessary to increase the heating rate within the temperature range between approximately -70°C and the typical sample melting temperature (approximately -10°C). Eukaryotic cells typically perform poorly at temperatures above 45°C, although some can survive at temperatures up to 60°C. This sets an upper limit on the temperature of the final solution in which the sample can reside.

[0217] One way to increase the heating rate is to increase the temperature of the heating solution. For example, when the sample temperature reaches 10°C to ΔT = 100°C + 10°C = 110°C, raising the solution temperature to near 100°C increases the driving force by 2.3 times. However, most biological samples cannot survive at temperatures far above biologically viable levels. This suggests that after heating beyond the “danger zone” for ice nucleation and growth, the heating solution temperature should be reduced to a biologically appropriate temperature, such as 37°C.

[0218] Figure 11 A possible implementation of a heated solution unit suitable for generating a temperature change in the heated solution along the sample's falling path is shown. Figure 11 A shows a side cross-sectional view of the heating unit 1300. The heating unit 1300 is divided into an upper chamber 1310 and a lower chamber 1320. A heating block 1330 heats the heating solution in the upper chamber to an elevated temperature T1 in the range of 50-100°C. A second heating block 1340 heats the heating solution in the lower chamber to a biological temperature T2 (e.g., 37°C) that the biological sample can tolerate, or other lower (or higher) temperatures. Because the hotter solution has a lower density, this configuration should be stable for buoyancy-driven convection. The opening between the upper and lower chambers can be covered by a door 1350 or a flexible fin to isolate the two chambers. During heating, the sample is vertically immersed, first through the heating solution in the first upper chamber, then through the heating solution in the second lower chamber and remaining there. The immersion rate, chamber size, chamber temperature, and sample size can be adjusted to provide the desired sample temperature upon exiting the upper chamber. The door or flap can be pushed open by contact with the sample carrier or the robot's end effector. The travel time / distance / speed required to pass through the solution at the first higher temperature can be determined in calibration experiments using actual (live or dead) samples. Figure 11 B shows a top view of the alternative heating unit 1360. "Hot" chamber 1370 and "cold" chamber 1380 are arranged side-by-side and surrounded by heating blocks 1390 and 1400. The chambers are separated by doors or flexible members 1410. The sample is first immersed in the heating solution in the hot chamber and then moved through the heating solution in the hot chamber, and then moved into and through the heating solution in the cold chamber. Figure 11 The overall vertical cell height in A will depend on the sample size and heating rate, but can range from 2 cm to 10 cm, and may be as large as 20 cm, for example closer to 4 cm, to minimize heating solution consumption.

[0219] Figure 12The diagram shows top and side cross-sectional views of the alternative sample heating unit 1420. This unit has a long channel 1430 intersecting with a larger orifice 1440. The bottom of channel 1430 descends from the top of the unit to the bottom of orifice 1440. The upper part of the unit and channel is adjacent to a block heater 1450, which heats the heating solution in the channel to a high temperature T1 in the range of 40-100°C. A second heating block 1460 heats the heating solution in the lower part of the channel and orifice 1440 to a biological temperature T2 (e.g., 37°C) that the biological sample can tolerate, or other lower (or higher) temperatures. The heating solution heated in this manner should be stable against buoyancy-induced convection, which is further reduced by ensuring that channel 1430 is not wider than the width required to ensure good flow of the heating solution through the sample, and by having a much larger volume of solution within orifice 1440. During heating, the sample travels down the channel at a high speed (1-5 m / s) at an angle and enters the orifice. This configuration is convenient because it allows for long sample travel distances while reducing the total volume of the solution required for heating.

[0220] Figure 13 A top view of a sample heating microplate 1460 with a long channel 1470 and a matching metal block 1480 for block heating is shown. This microplate is designed for use with a sample carrier that completely encapsulates the sample, eliminating the chance of sample loss during heating. As a result, it eliminates the need for... Figure 12 The orifice at the end of the heating channel for sample retrieval. Such as... Figure 15 and Figure 16 The sample support shown is well-suited for heating in this configuration. For supports with a thickness of less than about 250 micrometers, the channel width can be between about 1 and about 10 mm, or for some applications, between about 2 and about 5 mm, to reduce the consumption of heating solution.

[0221] Figure 14A representative example of a station 1500 for washing, heating, and drying a robotic end effector 40 after cooling and heating is shown. Station 1500 includes a cylindrical orifice tube 1510 lined with resistive or radiant heaters (or a group of heaters arranged generally in a cylindrical manner) and a cylindrical air amplifier 1520 (e.g., manufactured by Exair), which, when connected to a compressed gas source, generates a strong airflow through its central orifice. An electrically actuated valve can be used to open and close the compressed gas flow to the air amplifier. In use, the robot's z-axis motion arm 320 translates the end effector 40 through the heater 1510 and air amplifier 1520 once or multiple times until the actuator is warm and all moisture has evaporated. The station may also include a sprayer for spraying water, detergent solution, isopropanol, or other solvents, as well as cleaning and rinsing solutions, or a series of containers containing different rinsing solutions.

[0222] Figure 15-21 Examples of sample carriers suitable for use with this system are shown. Desired characteristics of these carriers may include, for example:

[0223] - After loading the sample, seal or close the carrier to prevent the sample from escaping during processing / treatment. Retrieve the sample from the carrier after treatment.

[0224] - The carrier has an open grid design that allows the immersion, cooling and heating fluids to be in full contact with the sample, and allows the cooling and heating fluids to flow through and around the sample at high speed with very little resistance, while having sufficiently small grid openings to prevent sample escape / loss.

[0225] The thickness of the portion of the sample residing on the screen is less than approximately 100 micrometers, or, for some applications, less than approximately 25 micrometers, ideally between 5 and 15 micrometers. This minimizes thermal mass and maximizes heat transfer through the solid portion of the screen to the sample. This helps ensure that the sample residing on the screen cools and freezes onto the screen rapidly during immersion in liquid nitrogen, rather than detaching from it. These characteristics help maximize cooling and heating rates.

[0226] - The portion of the mesh in contact with the sample should be flexible so that differential shrinkage between the mesh and the sample during cooling does not cause the sample to break. This suggests using a thin polymer instead of metal for the mesh portion supporting the sample.

[0227] - The mesh openings should be as large as possible, yet small enough that no sample can pass through them. For a 100-micron sample, openings of 50-80 microns might be suitable. For mammalian oocytes and embryos, openings of 40-120 microns might be appropriate. The ratio of sample size to mesh size will vary depending on the sample size and deformability.

[0228] - Considering the limitations of the mechanical strength required to withstand high-speed impacts, the solid area fraction of the mesh should be as small as possible to minimize flow resistance.

[0229] The sample carrier includes a rigid section that holds the mesh section and maintains its overall shape and rigidity during high-speed immersion. The rigid section should be easily grasped by a robot and should have precisely defined dimensions for automated handling.

[0230] - The sample carrier may include components made of magnets or magnetic materials to facilitate handling with magnets.

[0231] Figure 15 A representative sample carrier 1600 is shown, which may include any of the options and features disclosed in U.S. Provisional Patent Application No. 63 / 601,937. The carrier 1600 includes a thin, rigid frame 1610 attached to a base 1620, wherein the base is designed to engage with and be reliably manipulated by a robotic end effector. The frame 1610 may be made of a metallic material, a polymeric material, a polymer-glass composite material, or a combination thereof, and should be thin and very rigid so that it does not significantly bend when impacted during high-speed immersion and into liquid nitrogen and heated solutions. The frame thickness should be comparable to or slightly larger than the size of the sample to be held (e.g., 80% to 150%), so that the sample can be sealed within the frame without excessive compression, and that fluid flow in and around the sample during immersion is optimal for heat transfer. For human and bovine oocytes with a size of 110–120 micrometers, the frame may be approximately 100 to 150 micrometers thick. The frame width may vary depending on the size and number of samples to be held and processed on each sample carrier. Frames may be required to be from approximately 3 mm to approximately 10 mm wide, and can be as wide as 30 mm; as the frame width decreases, attaching the film to the frame becomes difficult, and frames that are too wide may not be suitable for large-capacity storage. The length of the frame can range from approximately 2 to 4 times its width to a maximum of 10 cm. During high-speed immersion in liquid, the greatest liquid disturbance and splashing will occur if the frame is held at the base and impacts the liquid. To eliminate this, the frame length extending beyond the base can be equivalent to or slightly longer than the immersion depth, which can vary between 1 and 20 cm, depending on the sample size and immersion speed.

[0232] The base 1620 can be any rigid, dimensionally stable material. Magnetic stainless steel is a good choice if the end effector is to use a magnet. The base can be marked or can contain RFID tags for sample identification and tracking.

[0233] The 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 membrane 1640, wherein the membrane portion within the aperture is provided with openings 1650, which are too small to allow the sample to pass through, but are as large as possible in other respects and occupy as large an area fraction as possible within the aperture, taking into account limitations on mechanical robustness during processing.

[0234] The membrane 1640 can be a polymer, metal, or semiconductor, but polymers are likely preferred because they are compliant and should therefore reduce stress on the sample during cooling and heating, and also because they are easy to handle. The membrane “mesh” can have two different thicknesses. For example, the thickness within the pores can be 5–15 micrometers to maximize heat transfer rate and compliance, while the thickness in the area in contact with the frame can be 10–50 micrometers for ease of handling and assembly. The membrane mesh can have special patterns of through-holes or thicker regions in specific areas of the membrane to indicate where the sample should be loaded and to create texture to help hold the sample on the membrane. Ideally, the sample should be loaded away from the edges of the frame apertures to maximize the heat transfer rate, as fluid flow is most disturbed near the inner edges.

[0235] To seal the sample within the frame, a second, similar film 1660 can be applied to the top surface of the frame to cover and seal the openings. Again, this film should have a large open area fraction and openings just small enough to prevent sample escape. The film can be applied with a permanent adhesive, a removable adhesive, a material with a relatively low melting temperature (close to biological temperatures) (e.g., wax), or a material that is easily mechanically removed after application (e.g., wax). One or both films may include features such as perforations 1670 or tabs 1680, which facilitate easy removal of the mesh and extraction of the sample.

[0236] If the frame thickness is less than the sample thickness, the sample will be compressed when the second membrane is applied. Slight compression is well tolerated by most biological samples and ensures optimal sample holding to maximize cooling and heating rates. However, unless the membranes are highly flexible, contact with both membranes can cause sample stress during cooling and heating. Contact with both membranes can also make retrieved samples after processing more difficult.

[0237] During high-speed immersion, liquid nitrogen and the heating solution immediately flow through the mesh and wet the sample, ensuring rapid cooling. In typical use, the frame can be immersed with the perforated end pointing downwards and the frame's axis perpendicular to the liquid surface to minimize the cross-section affecting the liquid surface and the forces on the frame and film. Alternatively, the frame can be immersed at an angle to the liquid surface to force the liquid through the mesh. The sample carrier can also be rotated at high speed during immersion to force the liquid through the mesh. This rotation can be around the carrier's central axis or around an axis parallel to the carrier's axis.

[0238] Figure 16 It shows the relationship with Figure 14 The design shown relates to a second sample carrier design 1700. In this case, the frame 1710 has a U-shaped opening 1720 at its end (in... Figure 16 In this design, the top opening of the "U" is located at the bottom of the frame, and the mesh membrane 1730 covers the opening on the first side of the frame and is attached to the frame, then wrapped around the ends of the frame. After the sample is loaded onto the mesh, the sample can be sealed in the frame by pressing down the "free" end of the mesh membrane 1730 (which may have adhesive gaskets) if the membrane extends to cover the opening on the second side of the frame, or by placing and pressing down the second mesh membrane. The advantage of this design is that liquid nitrogen or heated solutions can enter the front edge of the support through the mesh and flow over the mesh on the front edge and through the sample, instead of having to... Figure 15 It flows around the leading edge of the frame in that way.

[0239] for Figure 15 and 16 In one of the two designs, the second membrane can be attached to a separate frame and then held in alignment with the first frame using clips or a similar mechanism.

[0240] Figure 17-20 The design of the third sample carrier is shown. In this case, the carrier consists of two parts: a "basket" 1800 ( Figure 17 ) and Gai 1900 ( Figure 19 ).basket( Figure 17 The basket is formed using a rigid frame 1810 (e.g., polymer), with openings at the bottom and sides such as... Figure 15 and 16The design incorporates a thin, open mesh and / or a thin mesh cover made of polymer monofilaments or metal wires. The lid 1900 is designed to press, twist, or snap onto the top of the basket, sealing the sample inside and allowing for easy removal from the basket at the end of sample processing. The lid is designed to be gripped / held by a robotic end effector and may include a magnet or a piece of magnetic stainless steel. The lid can be formed from any rigid, dimensionally stable material, but its thermal expansion should match well with that of the basket frame. Ideally, it should have low thermal conductivity and relatively low thermal mass so that minimal heat transfer is required for cooling and heating. This suggests that the same polymer used for the basket frame should be used for the portion of the lid that inserts into the basket.

[0241] The advantages of this design are (1) when the sample carrier basket is immersed side down in the liquid, the liquid is forced to flow directly through the basket and around the sample, which can increase the heat transfer rate; and (2) loading the samples into the carrier and then sealing them into the carrier is simplified, without the need for adhesives or the use of membranes.

[0242] The diameter of the basket will be determined by the number of samples to be cooled together, the size of each sample, the size of the tools used to place and remove samples from the basket, and the minimum dimensions that allow for easy manufacture and assembly. For samples typically 50–200 micrometers in size, such as mammalian oocytes and embryos, the diameter at the bottom of the basket can range from about 2 mm to 10 mm, or possibly up to 20 mm. The height of the basket is determined by the following requirements: that the liquid can flow freely through the basket with minimal resistance, that the samples are far enough from the lid that their cooling and heating are unaffected by the lid, and that it is easy to retrieve the samples placed in the basket. The holes in the sides of the basket through which the liquid can exit during immersion should extend upwards from the base by 2 to 4 times the diameter of the base (e.g., from about 6 mm to about 40 mm). If good flow is required, the lid may also include a mesh section to allow liquid to flow through and out of the mesh.

[0243] The bottom of the basket can be covered with a thin (5-50 μm) polymer film of a mechanically robust and low-temperature compatible polymer (such as polyimide or cyclic olefin copolymer), having the features described above for use... Figure 17 and 18The sample carrier's perforation pattern is shown. Since the sample will be placed at the bottom of the basket, these films provide optimal cooling and heating performance, both in terms of cooling / heating rates and in minimizing stress caused by differential shrinkage of the sample. Both polyimide and COC are optically transparent, thus allowing visualization of the sample placed on the film within the carrier. The film can be fabricated, for example, via microfabrication. The film can then be bonded or ultrasonically welded / 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 bonded during the frame's injection molding. Individual films 1830 fabricated as flat can be wound around the frame and bonded to it.

[0244] The bottom and side openings of the basket can alternatively be covered by a mesh of fine metal wires or polymer monofilaments. Woven mesh will generate greater flow resistance than micropatterned films and also has poorer thermal conductivity.

[0245] Since only the sample support bottom of the basket needs to provide good transparency for imaging and compliance to minimize sample stress during cooling, the bottom of basket 1840 can be covered with a thin polymer film, while the side openings in basket 1850 (and any openings in the lid) can more easily be covered with nylon monofilaments or mesh, which will be more mechanically robust and easier to handle than a thin microfabricated membrane. A sieve size in the range of 60 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 that of the bottom opening, the flow rate through the sides will be lower, thus offsetting the greater flow resistance when using a mesh there.

[0246] like Figure 18 As shown, basket 1860 may have a hemispherical bottom 1870 instead of a flat bottom, wherein the hemispherical portion is covered with a porous membrane or mesh and supported by a suitable frame.

[0247] like Figure 19 As shown, the cap 1900 can be press-fitted into the basket 1800 and can be held by a hand tool 1910 or a robotic end effector that magnetically grips the cap via a torsion fit or other standard mechanism. The cap can be designed to facilitate laminar flow of liquid away from the sides of the basket at the sample holding base. This can involve having a downward-pointing tapered surface 1920, where the cone deflects the fluid away from the central axis of the basket and toward the side of the mesh cover. The tool or end effector can have a mechanism 1530 that allows the basket to be pushed away from the cap to allow the sample to be retrieved after processing. The cap can have a notch or other feature that engages with and holds it in place by a rigid support to facilitate the installation of the cap 1900 after the sample is loaded into the basket and the release of the cap from the tool 1930 or end effector after sample processing.

[0248] Figure 20 An example is shown of a tray 1950 with a series of containers 1960 holding a basket 1800 (and possibly a lid) for initial sample loading and basket + lid assembly. Samples can be placed into the basket, for example, using a pipette (manually or robotically). The lid can then be picked up using tool 1910, pressed into the basket, and then the lid and basket are picked up and transferred to a container in a sample transfer or loading station. Alternatively, the sample + basket + lid tray can be rotated or translated into a robot housing, and the robot can assemble the basket + lid and then begin the movements required for sample processing.

[0249] Figure 21 Another representative sample carrier design is shown. Here, the initially flat film 2000 is patterned with slits 2010, allowing a portion of the film to deform into a basket 2020 (as known in paper-cutting techniques), in which a sample can be placed. The slit openings in the deformable basket must be smaller than the minimum size of the sample to prevent sample loss. Prototypes made using polyimide work very well and require only mechanical force to permanently deform into a basket. Other materials may require thermoforming. The advantage of this method of creating 3D baskets is that it can produce pores of 100 micrometers and smaller within the basket, necessary to retain many biological samples of interest. Figure 2 C illustrates how two such baskets can be assembled face-to-face to create a carrier that completely surrounds the sample.

[0250] Figure 22 An example of a sample carrier that can be opened and closed using a tool that mates with the base of the carrier is shown. In this case, the porous film 2100 (which can be as follows) Figure 15 The flat or formed shown Figure 21 A basket (of the type shown) is attached to one end of a semi-rigid member 2120 such that it spans and seals a hole in the member, or alternatively, it protrudes from the end of the semi-rigid member. The semi-rigid members 2120 are mounted on opposite sides of a base 2140 and pressed together, causing the membrane to form a closed volume. The base has a notch 2160 to allow a tool to be attached to the base in a specific orientation about the base axis. The tool can be magnetically attached. The tool can have a central rod connected at one end to a button and protruding through a hole 2180 in the base toward the semi-rigid member 2120. When the button is pressed, the rod pushes the two semi-rigid members apart, opening the "jaws" and allowing access to each membrane. Other mechanisms can be used to open and close the carrier. For example, two sample-receiving "jaws" can be hinged together and have a naturally opening configuration. Pulling the hinge into the container can close the jaws like the structure of a sponge mop. Alternatively, a sliding member can slide and push one of the jaws to close it, as in some kettle lids.

[0251] Various aspects of this disclosure have been described in detail with reference to the illustrated embodiments; however, those skilled in the art will recognize that many modifications can be made thereto without departing from the scope of this disclosure. This disclosure is not limited to the precise construction and composition disclosed herein; any and all modifications, alterations, and variations that are apparent from the foregoing description are within the scope of this disclosure as defined by the appended claims. Furthermore, this concept expressly includes any and all combinations and sub-combinations of the foregoing elements and features.

[0252] Additional features and options of this disclosure may be reflected in the following terms:

[0253] Clause 1: A robotic system for cryopreservation and resuscitation of small biological samples, the system comprising a sample carrier that encloses and holds at least one sample, a series of sample processing stations, and a robot and a robot end effector that perform the required sample movements between and within stations.

[0254] Clause 2: The system as described in Clause 1, wherein the volume of the small biological sample is less than about 10 microliters, or less than about 1 microliter in some applications, or less than about 0.1 microliters in some applications, or less than about 10 nanoliters in applications involving mammalian oocytes and embryos, and the thickness is less than about 2 mm, and less than 500 micrometers in some applications, or less than about 200 micrometers in applications involving mammalian oocytes and embryos.

[0255] Clause 3: The system of Clause 1, wherein a series of stations for sample handling includes a station for immersing the sample in a solution (e.g., an equilibration and vitrification solution) prior to cryogenic cooling, a station for removing excess liquid from the sample and carrier and imaging the sample prior to cryogenic cooling, a station for rapidly cooling the sample by high-speed immersion in a liquid cryogenic agent (e.g., liquid nitrogen), a station for storing the cryogenically cooled sample in liquid nitrogen and loading the cryogenically cooled sample for heating and revival, a station for heating the cryogenically cooled sample by immersing the sample in a heating solution, a station for immersing and culturing the sample after heating, and a station for heating and drying the robotic end effector.

[0256] Clause 4: A system as described in Clause 1, wherein the robot and station are located within a housing, wherein at least a portion of the housing is transparent to allow observation and monitoring, and wherein the housing has at least one door that allows the transfer of materials and equipment between the interior and exterior of the housing.

[0257] Clause 5: A system as described in Clause 4, wherein the system includes a sample transfer station for transferring a sample between the exterior and interior of the housing and transferring the sample to a location where it can be retrieved or placed by the robot.

[0258] Robots and end effectors

[0259] Clause 6: A system as described in Clause 1, wherein the robotic end effector is configured to grip and release the sample carrier and to provide lateral and vertical translation of the sample carrier, wherein the vertical translation speed of the sample carrier is at least about 1 m / s and up to about 5 m / s, or for some applications, about 2 m / s, and the sample carrier rotates about its axis.

[0260] Clause 7: A system as described in Clause 6, wherein the robotic end effector provides high-speed rotational motion of the sample carrier about an axis, such that the maximum speed of the sample carrier in its rotational motion can reach at least about 0.25 m / s, or for some applications, about 0.5-2 m / s, and wherein the robotic end effector can also allow vibrational motion of the sample carrier.

[0261] Clause 8: A system as described in Clause 1, wherein the robot is a pick-and-place robot.

[0262] Clause 9: The system described in Clause 1, wherein the robot is a SCARA robot.

[0263] Sample transfer station

[0264] Clause 10: A system as described in Clause 5, wherein the sample transfer station rotates or translates the sample between locations outside and inside the housing at room temperature or biological temperature, wherein the location inside the housing is accessible by a robot.

[0265] Clause 11: The system as described in Clause 10, wherein the sample transfer station is a motor-driven turntable having one or more receivers for a sample carrier, and wherein the sample transfer station may include a heater and a temperature control system to maintain the sample at a temperature above room temperature and, for example, close to biological temperature.

[0266] Precooling and heat dissipation station

[0267] Clause 12: The system of Clause 3, wherein the station for immersing the sample in a solution (e.g., an equilibration and vitrification solution) prior to cryogenic cooling includes a temperature control system that allows the temperature of the solution and sample therein to vary within a range of 0°C and 50°C (e.g., between 4°C and 50°C or between 20°C and 40°C) and to be kept near biological temperature.

[0268] Clause 13: The system as described in Clause 12, wherein the station for immersing the sample in the solution prior to cryogenic cooling may accept standard or custom porous microplates in thin, standard and deep-pore form for containing the pre-cooled immersion solution.

[0269] Clause 14: The system as described in Clause 13, wherein the microplate is held on / within a commercial orifice plate heater / orifice plate temperature control system.

[0270] Clause 15: The system as described in Clause 13, wherein multiple different solutions may be present for immersing each sample carrier, each sample carrier is held in different wells in a microplate, and wherein each sample carrier may be immersed in different groups of solutions held in different wells.

[0271] Clause 16: A system as described in Clause 12, wherein the robotic end effector translates the sample carrier to immerse its sample in each solution in each well, and wherein it may oscillate or rotate the sample to ensure uniform and reproducible immersion.

[0272] Clause 17: A system as described in Clause 12, wherein the microplate and sample carrier are designed to cooperate in a manner that allows the robot to release the first sample carrier and perform other tasks with other sample carriers while the first sample is immersed, and then return to the first sample carrier and locate it in the same position and orientation for retrieval.

[0273] Immersion and incubation station after heating

[0274] Clause 18: The system of Clause 3, wherein the station for soaking and culturing samples after heating comprises the same or similar components as the station for soaking samples before cooling, but the solution is different.

[0275] Clause 19: The system of Clause 18, wherein the station for immersing and culturing samples after heating may further include means for controlling the atmosphere (including CO2 content) above the immersion liquid during culturing.

[0276] Liquid removal and sample imaging station

[0277] Clause 20: A system of Clause 3, wherein a liquid removal and sample inspection station allows the removal of excess liquid from samples and carriers to minimize total thermal mass and maximize cooling and heating rates, and the station allows optical imaging of the sample after liquid removal to record its state prior to cryopreservation.

[0278] Clause 21: The system of Clause 20, wherein liquid is removed from the sample and carrier by bringing the sample into contact with or adjacent to the station and briefly applying suction to a portion of the sample carrier in fluid communication with the sample, wherein the suction may be generated by a pump or a compressed air vacuum generator and the suction may be opened and closed by an electric valve.

[0279] Clause 22: A system as described in Clause 20, wherein liquid is removed by blowing compressed gas onto the sample and the carrier.

[0280] Clause 23: A system as described in Clause 20, wherein liquid can be additionally removed by contacting the sample carrier with an absorbent material such as filter paper.

[0281] Clause 24: The system as described in Clause 20 further includes an LED or fiber optic light source for transmitted light imaging of the sample, an LED or fiber optic light source for incident illumination of the sample, and a sample backlight for a compact digital camera / microscope / endoscope for imaging the sample within the carrier.

[0282] Sample Low Temperature Cooling Station

[0283] Clause 25: The system as described in Clause 3, wherein the sample cryogenic cooling station allows the sample and carrier to be rapidly cooled to a cryogenic temperature in order to minimize ice nucleation and growth in and around the sample, and wherein, depending on the sample size, the rapid cooling rate is approximately 25,000°C / min for a 500-micron sample and 3,000,000°C / min for a 25-micron sample.

[0284] Clause 26: The system according to Clause 25, wherein the sample cryogenic cooling station comprises: an insulated container or Dewar flask configured to receive and hold a first volume of liquid nitrogen; an insulating cap for isolating and insulating the contents of the Dewar flask from the surrounding humid and hot air; and an immersion port through which the sample and carrier can be immersed in the liquid nitrogen.

[0285] Clause 27: The system according to Clause 26, wherein the Dewar flask may include a second inner chamber containing a second volume of liquid nitrogen, wherein the liquid nitrogen in the second chamber is in good thermal communication with the first volume of liquid nitrogen, and wherein the inner chamber may be made of a material such as a metal having high thermal conductivity.

[0286] Clause 28: The system of Clause 26, wherein the surface level of the second volume of liquid nitrogen is higher than the surface level of the first volume of liquid nitrogen, and liquid nitrogen is added to the first chamber to first fill the chamber and then overflow, and the overflow is added to the first volume of liquid nitrogen outside the inner chamber.

[0287] Clause 29: The system of Clause 26, wherein a level sensor is used to monitor the liquid nitrogen level inside and outside the cavity, including a laser level sensor, thermocouple, heated RTD or float, and when the liquid level in the cavity drops below a target value, a valve connected to the liquid nitrogen supply source (e.g., a third volume of liquid nitrogen contained in an additional insulating cavity located above the fill level of a second cavity) can open and fill the cavity to overflow.

[0288] Clause 30: The system as described in Clause 26, wherein the heat-insulating cap includes a manifold within a portion of the cap, the manifold including the orifice through which the sample and carrier can be immersed in the second volume of liquid nitrogen within the inner chamber; a set of gas channels within the manifold intersecting the orifice and capable of applying suction or delivery of dry ambient temperature gas to the orifice at and near the liquid nitrogen level within the orifice to remove cold gas present near the surface of the liquid nitrogen within the orifice and replace it with dry ambient temperature gas, and also to fill the orifice with excess dry gas to prevent the infiltration of moist ambient air; and a heater lining the orifice to prevent frost formation on the orifice surface.

[0289] Clause 31: The system according to Clause 26, wherein the heat-insulating cover further includes a second opening or through-hole in a region adjacent to the manifold, the second opening or through-hole being positioned above a region contained within the inner cavity, wherein the through-hole is sized to receive one or more boxes or disks, each box or disk having a plurality of receivers for holding a sample, and wherein the through-hole is covered by a sliding, rotating, or hinged cover that opens to allow access to the box and the sample carrier within the box.

[0290] Clause 32: The system of Clause 31, wherein the orifice of the manifold and the second opening within the cap are in communication, such that a sample carried by a robotic arm and immersed in liquid nitrogen through the orifice can then be translated out of the orifice and into the area below the second opening without being lifted out of the liquid nitrogen.

[0291] Clause 33: The system of Clause 31, wherein the lid is opened and closed, slid, or rotated using an electronically or pneumatically controlled actuator, and a dry gas supply is connected to the inner cavity and opens when the lid is opened to ensure overpressure and prevent humid ambient gases from entering the inner cavity and frosting. Clause 34: The system of Clause 31, wherein the bottom of the inner cavity below the opening in the lid includes a structure whose size and shape are configured to receive and hold a cassette or disk in a clearly defined orientation, thereby allowing the robot to place or retrieve samples into or from the cassette or disk after cooling for subsequent processing.

[0292] Clause 35: A system of Clause 31 in which a tool is used to load or remove a box or disc into or from a structure containing a cavity.

[0293] Clause 36: A system as described in Clause 31, wherein a box or disk containing a previously cryogenically cooled sample is loaded through an opening in the cap of a Dewar flask and enters a receiving box or disk within the Dewar flask to allow a robot to approach the sample and transfer it to a heating station.

[0294] Clause 37: The system of Clause 31, wherein the sample cryogenic cooling station includes a digital camera / microscope / endoscope and lens and other optics as required, and an LED illuminator that provides reflected and transmitted light illumination to allow imaging of the cryogenically cooled sample in or directly above liquid nitrogen and before transferring the sample to a cassette or disk.

[0295] Details of low-temperature cooling

[0296] Clause 38: A system of Clause 26, wherein a robotic end effector immerses a sample and carrier in a second volume of liquid nitrogen within an inner cavity at a speed between about 0.25 m / s and about 5 m / s, or for certain applications, at a speed of about 2 m / s, thereby maximizing convective heat transfer without causing excessive splashing of liquid nitrogen or requiring an excessively long travel distance through the liquid nitrogen before cooling is complete.

[0297] Clause 39: The system as described in Clause 26, wherein the depth of the inner chamber and the liquid nitrogen level within the chamber allow the sample to travel a distance between about 2 cm and about 20 cm before stopping, or, for some applications, at least about 4 cm, to ensure that the sample continues to travel at high speed until the sample is cooled to below T~140 K.

[0298] Clause 40: A system as described in Clause 26, wherein the robotic arm may have an end effector that allows high-speed coaxial or off-axis rotation of the sample carrier in order to maintain a high velocity and a high rate of convective heat transfer of the sample carrier relative to the liquid nitrogen, even when the translational motion of the robotic arm has stopped, as may be required to maximize the average cooling rate when immersing large (approximately millimeters and larger) samples that are being slowly cooled.

[0299] Heating Station

[0300] Clause 41: A system as described in Clause 3, wherein a sample previously cooled to a cryogenic temperature can be rapidly heated to room temperature or biological temperature between 20°C and 60°C in the sample heating station, wherein the rapid heating includes a heating rate between approximately 50,000°C / min and 6,000,000°C / min.

[0301] Clause 42: The system according to Clause 41, wherein the heating station includes a temperature control plate / block heater, a high thermal conductivity stage attached to the block heater, the high thermal conductivity stage receiving a plate, vial, test tube, cuvette or other container for holding the solution used in the heating process, and may also include an insulator surrounding the outer surface of the stage to reduce heat loss.

[0302] Clause 43: The system as described in Clause 41, wherein the heat-conducting stage can be shaped to provide close thermal contact with any liquid-sealed surface of a plate, vial, test tube, etc., in order to ensure maximum heat transfer rate and temperature uniformity.

[0303] Clause 44: A system as described in Clause 41, wherein the robotic end effector immerses the sample into a heated solution contained in a well of a plate, vial, test tube, cuvette, or other container at a speed between about 0.25 and about 5 m / s, or about 2 m / s for some applications, to maximize the rate of convective heat transfer while minimizing liquid splashing when the sample carrier impacts the liquid and minimizing potential damage to the sample carrier and the sample.

[0304] Clause 45: A system as described in Clause 41, wherein the plate, vial, test tube, etc., has a container for liquid therein, the depth of which is between about 1 cm and about 10 cm, with a maximum depth of about 20 cm, or for some applications, about 4 cm, the distance depending on the sample size and the time required for its heating (increasing with the sample size and the time required for its heating), such that the sample can be kept in linear translational motion for a sufficient time to ensure that it has substantially heated to at least -10°C, or for some applications, 0°C, such that any and all ice nucleation and growth within the sample has ceased before the translational motion stops.

[0305] Clause 46: The system of Clause 41, wherein, in addition to linear vertical motion, the robot end effector can also cause the sample to rotate at a certain angular velocity ω at a non-zero vertical distance r from the axis of rotation, such that after the translational motion of the robotic arm and the end effector stops, the sample can maintain motion relative to the heated liquid, maintain a large heat transfer rate until heated to a temperature and / or a target temperature at which ice nucleation and growth no longer occur, and such that the required depth of heated solution can be reduced compared to the depth required when the sample motion is purely vertical.

[0306] Clause 47: The system of Clause 41, wherein, in addition to linear vertical motion, the robot end effector can also cause the sample to vibrate at a certain angular velocity and a certain amplitude A, such that after the translational motion of the robotic arm and the end effector stops, the sample can maintain its motion relative to the heated liquid, maintain a large heat transfer rate until it is heated to a temperature and / or a target temperature at which ice nucleation and growth no longer occur, and such that the required depth of heated solution can be reduced from the depth required when the sample motion is purely vertical.

[0307] Clause 48: A system as described in Clause 46 or Clause 47, wherein the sample is maintained at a net velocity of about 0.5 m / s to about 5 m / s relative to the surrounding liquid by a combination of translational and rotational and / or vibratory motions, or for some applications, at about 2 m / s, until the sample is heated to at least about -10°C, or for some applications, to about 0°C.

[0308] Clause 49: A system as described in Clause 46 or Clause 47, wherein the net velocity of the sample relative to the surrounding liquid is maintained at at least about 0.25 m / s by a combination of translational and rotational and / or vibratory motions until it is heated to within 10°C of its final temperature.

[0309] Clause 50: Systems described in Clause 41, wherein the temperature of the heating liquid can be significantly higher than the normal biological temperature of the sample, for example at 50°C. o C and possible 100 o Between C, in order to maximize the rate of temperature rise of the sample in the temperature range with the greatest ice growth rate, thereby minimizing the amount of ice formed in the sample.

[0310] Clause 51: A system of Clause 50, wherein a robot can rapidly remove a sample from a heating solution before the sample temperature reaches the temperature of the heating solution, thereby protecting the sample from damage caused by prolonged exposure to high temperatures.

[0311] Clause 52: The system of Clause 50, wherein each immersion port in the heating station may be vertically divided into two chambers connected by a sufficiently large channel to allow the sample-containing portion of the sample carrier to pass through, wherein the liquid in the upper chamber is maintained at a higher temperature than the liquid in the lower chamber, such that the sample can first move in the upper chamber to be heated at a higher rate than in the liquid at the same temperature in the lower chamber, and then be transferred to the lower chamber before its temperature becomes too high to protect the sample from damage due to excessive temperature.

[0312] Robot end effector flushing and drying station

[0313] Clause 53: The system according to Clause 3, wherein the system includes a heating and drying station for the robot end effector, the heating and drying station being capable of heating and drying the end effector after immersion in a liquid.

[0314] Clause 54: The system according to Clause 53, wherein the end effector is heated by a cylindrical arrangement passing through a heater.

[0315] Clause 55: The system according to Clause 53, wherein the end effector is dried by passing over or through a compressed air-driven air amplifier or through a high-speed gas jet or sheet-like airflow.

[0316] Clause 56: The system according to Clause 53, wherein the heater and the air dryer are arranged such that they are on the same axis and the end effector can pass through them via linear motion oscillation.

[0317] Clause 57: The system according to Clause 53, wherein the heating and drying station includes means for rinsing the end effector to remove residues from any liquid it has come into contact with.

[0318] Clause 58: The system according to Clause 57, wherein the rinsing components of the station may include one or more containers containing pure water, water + detergent, or isopropanol, and wherein the robot delivers the end effector to one or more rinsing solutions and between one or more rinsing solutions.

[0319] Clause 59: The system according to Clause 57, wherein the rinsing is accomplished by spraying water or other suitable solvent onto the end effector.

[0320] Clause 60: A system as described in Clause 57, wherein the robot is capable of rotating and oscillating the end effector during the rinsing process.

[0321] System control and monitoring

[0322] Clause 61: In the system described in Clause 3, any one or all of the robot, the motors and actuators attached to the robot to form the end effector, the temperature control block, the sensors, actuators, motors, valves of various other system components, and the digital camera and illumination source of the imaging component can be electronically controlled via a computer or microcontroller.

[0323] Clause 62: A system as described in Clause 61, wherein the system controller can sequence robotic and other operations so that two or more sample carriers can undergo processing simultaneously.

[0324] Clause 63: A system as described in Clause 1, wherein two or more robots may be used to increase the number of samples that can be processed by a given set of processing stations.

[0325] Sample holder

[0326] Clause 64: A system of Clause 1, wherein a sample carrier comprising one or more samples has a thin material covering most of its surface area near the sample, the thin material containing a dense array of pores that provides little resistance to liquid flow and allows the liquid to flow at high speed through and across the sample when the sample carrier moves at high speed relative to the liquid, thereby maximizing convective and conductive heat transfer between the sample and the liquid.

[0327] Clause 65: A system as described in Clause 64, wherein the sample is supported on a thin film of polymer, glass, semiconductor, metal or composite having a thickness between about 2 μm and about 100 μm, or in some applications between about 5 and 50 micrometers.

[0328] Clause 66: A system of Clause 65, wherein the film has regions of two thicknesses, a thin region having a through-hole on which the sample is placed, having a thickness of about 5 to 15 micrometers, and a thick region for mechanical strength and for adhesion to other parts of the sample carrier, having a thickness of between 10 and 50 micrometers, and wherein within the thin region of the film there may be a thick region forming markings or pillars or other features that may help to position the sample on the film.

[0329] Clause 67: A system as described in Clause 65, wherein the thin film is micro-patterned using photolithography and standard microfabrication processes, micro-embossing, or stamping.

[0330] Clause 68: A system as described in Clause 65, wherein the film has a micropatterned polymer, such as polyimide, cyclic olefin copolymer or SU-8, the micropatterned polymer being optically transparent to facilitate imaging of a sample placed thereon.

[0331] Clause 69: A system as described in Clause 65, wherein the film comprises a large area patterned with through-holes, wherein the opening area fraction of the film is as large as possible given mechanical strength requirements and is between about 50% and about 95%, or for some applications, between about 70% and about 95%, and wherein the pores have as large a diameter as possible, but not large enough to allow the sample to pass through; for a 100-micrometer oocyte, the diameter may be on the order of 50-80 micrometers, and more typically between 40% and 80% of the diameter of the sample to be cryopreserved.

[0332] Clause 70: The system as described in Clause 65, wherein the thin film of the sample carrier can be added to a thin rigid frame.

[0333] Clause 71: In a system pursuant to Clause 69 or Clause 70, the rod or frame may be permanently or removably attached to a base configured to be handled by the robot end effector.

[0334] Clause 72: In the system described in Clause 70, the base may be a rigid, dimensionally stable material, including magnetic stainless steel, and the base may be marked or may contain RFID tags for sample identification and tracking.

[0335] Clause 73: A system as described in Clause 70, wherein the frame may be marked or may be combined with RFID tags for sample identification and tracking.

[0336] Clause 74: The system of Clause 70, wherein the frame is composed of a rigid sheet having a hole disposed near one end, wherein the sheet may be a rigid metal or polymer or polymer-glass composite or semiconductor, and the thickness of the sheet may be comparable to or slightly greater than the corresponding sample size (approximately 80% to 150%). For mammalian oocytes and embryos, this thickness may be between 80 and 250 micrometers.

[0337] Clause 75: The system according to Clause 70, wherein the width of the frame is between about 3 mm and 10 mm and can be up to 30 mm, wherein the length of the frame is between about 1 cm and 10 cm, or about 2 cm for some applications.

[0338] Clause 76: A system as described in Clause 70, wherein the frame has at least one hole disposed near the end furthest from the base, and wherein the hole is spanned and sealed on one side (the “bottom” side of the frame) by a thin film attached using an adhesive, ultrasonic bonding, or other method, and wherein the thin porous membrane may have a thickness (e.g., 5-15 micrometers) in the region of the hole and a second greater thickness (e.g., 10-50 micrometers) in the region in contact with the frame, and wherein the portion of the membrane within the hole is filled with a dense array of pores, the pores being as large as possible to prevent the sample from passing through, and such that the opening area fraction of the portion of the membrane within the hole is as large as possible, consistent with mechanical strength requirements, and between about 50% and about 95%, or for some applications, between about 70% and about 95%.

[0339] Clause 77: A system as described in Clause 70, wherein one or more samples are placed onto a porous membrane, and then a second porous membrane is applied to the top surface of a frame to seal the samples within the pores.

[0340] Clause 78: A system as described in Clause 70, wherein the membrane may have perforations, tabs or other patterns to facilitate their removal within the perforations, thereby facilitating access to the sample after processing.

[0341] Clause 79: The system as described in any one of Clauses 75 to 78, wherein the holes in the frame extend to within about 0.5 mm of the frame edge, or for some applications, not more than about 1 mm, to allow sufficient surface area for the membrane to bond to the frame.

[0342] Clause 80: A system as described in Clause 70, wherein the carrier is immersed along the axis of the frame during cooling and heating.

[0343] Clause 81: The system according to Clause 70, wherein during cooling and heating, the carrier is immersed along and rotates about the frame axis.

[0344] Clause 82: The system according to Clause 70, wherein, during cooling and heating, the carrier is immersed along its axis and rotated about different axes.

[0345] Clause 83: The system according to Clause 70, wherein the frame has a U-shaped cut at the end furthest from the base.

[0346] Clause 84: The system of Clause 83, wherein a thin porous membrane attached to one side of the frame covers and seals a U-shaped cutout on one side of the frame, and after the sample is loaded, covers and seals the open end of the 'U' shape, and then seals the cutout on the other side of the frame to retain the sample inside, thereby forming a series of holes with a large opening area ratio at the leading edge of the sample carrier to allow cooling and heating liquids to flow directly to the sample therein with minimal resistance.

[0347] Clause 85: A system as described in Clause 63, wherein the sample carrier comprises two parts, namely a basket and a lid, wherein the basket receives and holds one or more samples, and the lid is configured to seal the basket and be grasped or handled by the robotic end effector.

[0348] Clause 86: The system according to Clause 85, wherein the basket has a diameter at its bottom between about 2 mm and about 20 mm, or for some applications, a diameter of about 3-10 mm.

[0349] Clause 87: The system of Clause 86, wherein the basket comprises a thin, low-thermal-mass frame having a substantially open bottom and substantially open sides, the opening extending upward from the bottom at a distance d from the bottom, wherein d is equal to or greater than the diameter of the bottom of the basket, for example, about 2 to 4 times the diameter of the bottom or between about 6 and 40 millimeters.

[0350] Clause 88: A system of Clause 85, wherein the open bottom and sides are spanned by a porous membrane or a mesh formed of filaments, wherein the pores of the membrane or mesh are as large as possible to prevent the sample from passing through, and for mammalian oocytes and embryos, the size range is typically between about 40 and 120 micrometers, more generally between 40% and 80% of the cell diameter.

[0351] Clause 89: A system of Clause 88, wherein the porous film is made wholly or partially of a metallic material or a transparent polymer (e.g., polyimide, SU-8, and cyclic olefin copolymers) to allow optical inspection of a sample on the film.

[0352] Clause 90: The system according to Clause 88, wherein the film has a thickness of 5 to 15 micrometers in the region spanning the opening and a thickness of 10 to 50 micrometers in the region in which it is bonded to the frame.

[0353] Clause 91: A system as described in Clause 88, wherein the membrane portion within the opening in the frame has a large opening area fraction ranging from about 50% to about 95%, or for some applications between about 70% and about 95%.

[0354] Clause 92: The system according to Clause 88, wherein the mesh is formed of filaments of metal or polymers such as nylon.

[0355] Clause 93: A system as described in Clause 88, wherein the mesh size is between about 60 and about 400 meshes, or between about 100 and about 240 meshes for mammalian oocytes and embryos.

[0356] Clause 94: A system as described in Clause 88, wherein the bottom of the basket in which the sample is placed is made of a thin, porous, transparent polymer film to provide the fastest cooling and heating as well as optical transparency, and wherein the openings on the sides of the basket are covered and sealed with a mesh formed of filaments.

[0357] Clause 95: The system according to Clause 85, wherein the cover is press-fitted into the basket to form a seal, and wherein the basket can be removed by pushing its upper edge downward while firmly holding the cover.

[0358] Clause 96: A system according to Clause 85, wherein the cover includes a tapered feature that is axially positioned and projectes downward into the basket, the tapered feature being used to guide liquid upward through the bottom of the basket to the sides and out through a porous membrane or mesh covering portion on the sides of the basket, thereby creating a near-laminar liquid flow when the sample carrier is immersed in a cold or hot liquid.

[0359] Clause 97: The system according to Clause 85, wherein the cover includes an opening spanned by a thin porous membrane or mesh to allow liquid to pass through during cooling and heating.

[0360] Clause 98: A system as described in Clause 85, wherein a series of baskets and caps can be held in a receiver within a base, and wherein a sample can be loaded into the baskets using a pipette or other tool, and then the caps are positioned and sealed to the base.

[0361] Clause 99: The system described in Clause 85, wherein the basket has a dome shape rather than a flat bottom.

[0362] Clause 100: A system as described in Clause 46, wherein the basket frame or cover may be marked or patterned to allow identification of each sample, or wherein the cover is incorporated with an RFID tag for sample identification.

Claims

1. A method for processing biological samples, the method comprising: The biological sample is loaded into a sample carrier; The biological sample is immersed in a solution configured to prepare the biological sample for cryogenic cooling; Remove liquid from the biological sample and the sample carrier; The biological sample is cooled to a predetermined low temperature to produce a cooled sample; Store the cooled sample at a storage temperature that is at least at a predetermined low temperature; The cooled sample is heated to produce a heated sample; Incubate the sample by raising the temperature; and Remove the sample from the sample carrier.

2. The method of claim 1, wherein the sample carrier completely encloses the sample during sample processing, such that the sample remains within the sample carrier from loading until removal at the end of processing.

3. The method according to claim 1, wherein, The sample carrier is porous, allowing fluid to flow into the interior of the sample carrier and contact the sample.

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

5. The method according to claim 1, wherein, The sample carrier is translated and / or rotated at a minimum speed relative to the initially stationary heating fluid until the sample has been heated to above 0°C.

6. The method of claim 1, wherein the sample carrier comprises a thin film, and wherein the sample is placed on the thin film.

7. A robotic system for cryopreserving biological samples, the robotic system comprising: A sample carrier configured to enclose and retain the biological sample; A series of sample processing stations; and A robot with a robotic end effector configured to hold the biological sample carrier containing the biological sample, wherein the robot is configured to perform multiple predefined sample movements between and within the series of sample processing stations.

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

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

10. The robotic system of claim 7, wherein the biological sample has a volume of 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 robot system according to claim 7, wherein, The biological sample has a thickness of less than approximately 500 micrometers.

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

14. The robot system according to 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 handling comprises: The first step involves immersing biological samples in a equilibration and vitrification solution before cryogenic cooling. The second station is configured to remove liquid from around the biological sample; The third station is configured to image the biological sample before cryogenic cooling and after heating. The fourth station is configured to cool the biological sample to a cryogenic temperature; The fifth station is configured to store the biological samples at low temperatures; The sixth station is configured to heat the sample to biological temperature; The seventh station is configured to soak and culture the biological sample; and / or The eighth station is configured to heat and dry the robot's end effector.

16. The robot system of claim 15, wherein the robot and the first station, the second station, the third station, the fourth station, the fifth station, the sixth station, the seventh station, and the eighth station are located within a housing.

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

18. The robot system of claim 17, wherein the housing includes at least one door that allows materials and equipment to be transferred between the interior and exterior of the housing.

19. The robotic system of claim 18, further comprising a sample transfer station configured to transfer a sample between the exterior and interior of the housing and to transfer the sample to a location within the interior of the housing, where the sample can be retrieved or placed by the robot.

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

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

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

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

24. The robot system of claim 7, wherein the robot actuator is configured to provide rotational motion of the sample carrier about a rotation axis.

25. The robot system according to claim 24, wherein, The rotation axis is offset from the moving axis of the sample carrier by a predetermined distance.

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

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

28. The robot system of claim 7, wherein the robot actuator is configured to provide vibrational motion of the sample carrier.

29. The robot system according to 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 includes a sample transfer station configured to transfer samples, wherein the sample transfer station is a motor-driven turntable having one or more containers, each container being configured to hold a corresponding sample carrier.

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

32. The robotic system of claim 7 further includes an immersion station configured to immerse the biological sample in a solution prior to cryogenic cooling, wherein the immersion station includes a temperature control system configured to vary the temperature of the solution and the sample immersed in the solution within a range of 0°C and 50°C, and to maintain it near a biological temperature, wherein the biological temperature may be approximately 37°C.

33. The robotic system of claim 34, wherein the soaking station is configured to accept thin, standard, and deep-well format Biomolecular Screening Society (SBS) porous microplates and / or Laboratory Automation and Screening Society (SLAS) porous microplates for containing the pre-cooled soaking solution.

34. The robot system according to claim 35, wherein, The soaking station also includes a temperature-controlled perforated plate heater configured to hold the microporous plate thereon.

35. The robot system according to claim 35, wherein, Multiple different solutions may be present, each solution being held in different wells of the microplate, each sample carrier being immersed in said different wells, and each sample carrier may be immersed in different groups of solutions held in different wells.

36. The robot system according to claim 35, wherein, The microplate and the sample carrier are designed to cooperate in a manner that allows the robot to release the first sample carrier and perform other tasks with other sample carriers while the first sample is immersed, and then return to the first sample carrier and locate it in the same position and orientation for retrieval.

37. The robotic system of claim 34, wherein the robotic end effector is configured to translate the sample carrier to immerse its sample in each of a plurality of solutions.

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

39. The robot system according to claim 34, wherein, The station for soaking and culturing samples after heating includes the same or similar components as the station for soaking samples before cooling, but with a different solution.

40. The robot system according to claim 7, wherein, The station for immersing and culturing samples after heating includes means for controlling the atmosphere above the immersion liquid during culturing, the atmosphere including a CO2 content.

41. The robotic system of claim 14, wherein the station for soaking and culturing samples includes a temperature control system that allows the temperature of the solution and sample therein to vary within a range between 0°C and 50°C and to be maintained near biological temperature.

42. The robot system according to claim 14, wherein, The station for soaking and culturing samples is configured to accept thin, standard, and deep-well SBS and / or SLAS porous microplates for containing soaking and culturing solutions.

43. The robot system according to claim 44, wherein, The microplate is held on a temperature-controlled orifice plate heater.

44. The robot system according to claim 44, wherein, The microplate may include multiple wells, each well containing one of a variety of different solutions, each sample carrier being immersed in the solution, and wherein each sample carrier may be immersed in a different group of solutions held in different wells.

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

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

47. The robot system according to claim 48, wherein, The suction can be generated using a pump or a compressed air vacuum generator, and the suction can be opened and closed using an electrically actuated valve.

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

49. The robot system according to claim 48, wherein, Liquid can be removed by contacting the sample carrier with an absorbent material such as filter paper.

50. The robot system according to claim 14, wherein, The station for imaging the sample before cryogenic cooling and after heating consists of a camera with sufficient resolution, a digital microscope or digital endoscope, and optics that provide sufficient magnification to resolve fine details within the sample.

51. The robot system according to claim 52, wherein, The station for imaging the sample includes a backlight using an LED or fiber optic light source for transmitted light imaging of the sample and / or an LED or fiber optic light source for incident illumination of the sample.

52. The robot system according to claim 14, wherein, The sample cryogenic cooling station allows the sample and carrier to be rapidly cooled to a cryogenic temperature in order to minimize ice nucleation and growth in the sample and surrounding solution.

53. The robot system according to claim 54, wherein, For a sample with a diameter of 500 micrometers, the cooling rate, depending on the sample size, is approximately 25,000°C / min, and for a sample with a diameter of 25 micrometers, the cooling rate, depending on the sample size, is approximately 3,000,000°C / min.

54. The robot system according to claim 14, wherein, The sample cryogenic cooling station includes an insulated container or Dewar flask defining a first chamber.

55. The robot system of claim 56, wherein a second inner chamber exists within the first chamber.

56. The robotic system of claim 57, wherein the space between the first chamber and the second chamber comprises a first volume of liquid nitrogen.

57. The robot system of claim 57, wherein the second chamber contains a second volume of liquid nitrogen, the second volume of liquid nitrogen not being in direct contact with the first volume of liquid nitrogen.

58. The robotic system of claim 59, wherein the surface level of the second volume of liquid nitrogen is higher than the surface level of the first volume of liquid nitrogen.

59. The robotic system of claim 59, wherein adding liquid nitrogen to the second chamber will first fill the chamber and then overflow, and wherein the overflow will be added to the first volume of liquid nitrogen.

60. The robotic system of claim 59, wherein liquid nitrogen can be pumped from the first volume between the first chamber and the second chamber to fill the second inner chamber to overflow, thereby maintaining a nearly constant liquid nitrogen level in the second chamber.

61. The robot system of claim 59, wherein a third volume of liquid nitrogen can be maintained in a second insulated container in fluid communication with the second inner chamber, and wherein the filling of the inner chamber with the third volume of liquid nitrogen is controlled by a valve.

62. The robot system according to claim 59, wherein, The liquid nitrogen levels in the first and second volumes are monitored using level sensors.

63. The robot system according to claim 64, wherein, The liquid level sensor can be a laser liquid level sensor, a thermocouple, a heated RTD, or a float.

64. The robot system according to claim 64, wherein, The sensor readings are used to control the valve, which controls the flow of liquid nitrogen from the third volume to the second volume, and to pump liquid nitrogen from the first volume to the second volume.

65. The robot system according to claim 59, wherein, The second volume of liquid nitrogen in the internal chamber is in thermal communication with the first volume of liquid nitrogen in the first insulated container between the first chamber and the second chamber.

66. The robot system according to claim 59, wherein, The inner chamber can be made of a material such as a metal with high thermal conductivity.

67. The robot system according to claim 14, wherein, The sample cryogenic cooling station has an insulated cover that isolates and insulates the contents of the Dewar flask from the warm, humid surrounding air.

68. The robot system according to claim 69, wherein, The heat-insulating cover includes a manifold within a portion of the cover.

69. The robot system according to claim 70, wherein, The manifold includes: The sample and carrier can be immersed in the second volume of liquid nitrogen within the inner chamber through the pore; A set of gas channels within the manifold intersects the orifice and can apply suction or delivery of dry, ambient-temperature gas to the orifice at and near the liquid nitrogen level within the orifice, in order to remove the cold gas present near the liquid nitrogen surface within the orifice and replace it with dry, ambient-temperature gas; and A heater lined inside a hole to prevent frost from forming on the hole surface.

70. The robotic system of claim 69, wherein the heat-insulating cover further includes a second opening or through-hole in a region adjacent to the manifold, the second opening or through-hole being positioned above a region contained within the second inner chamber, wherein the through-hole is sized to receive one or more boxes or disks, each box or disk having a plurality of receivers for holding samples.

71. The robotic system of claim 72, wherein the through-hole is covered by a sliding, rotating, or hinged cover that opens to allow access to the cassette and the sample carrier within the cassette.

72. The robot system according to claim 73, wherein, The cover is opened and closed, slid or rotated using an electronically or pneumatically controlled actuator.

73. The robot system according to claim 59, wherein, The bottom of the second inner chamber includes a structure sized and shaped to receive and hold a box or disc in a defined location and a defined orientation.

74. The robot system of claim 75, wherein a tool is used to load or remove a box or disk into or from the structure in the second inner chamber.

75. The robot system according to claim 75, wherein, A box or disk containing a previously cryogenically cooled sample, at a low temperature, can be loaded into the receiving box or disk structure inside the Dewar flask.

76. The robotic system of claim 72, wherein the orifice of the manifold and the second opening within the cap communicate, such that a sample immersed in liquid nitrogen passing through the orifice and entering the second volume can subsequently be translated out of the orifice and into a box or disk without being lifted out of the liquid nitrogen.

77. The robot system according to claim 59, wherein, The sample cryogenic cooling station includes a digital camera, digital microscope or digital endoscope and lenses, as well as other optics as needed, and LED illuminators to allow imaging of the cryogenically cooled sample in liquid nitrogen or in a cold gas present directly above the liquid nitrogen and before transferring the sample to a cassette or disk.

78. The robot system according to claim 59, wherein, The robotic end effector immerses the sample and carrier into the second volume of liquid nitrogen within the second inner chamber at a speed between approximately 0.25 m / s and approximately 5 m / s.

79. The robot system according to claim 59, wherein, The robot end effector immerses the sample and carrier into the second volume of liquid nitrogen within the internal chamber at a speed of approximately 2 m / s.

80. The robot system according to claim 59, wherein, The depth of the second inner chamber and the liquid nitrogen level within the chamber allow the sample to travel a distance between approximately 2 cm and approximately 20 cm before stopping, ensuring that the sample continues to move at high speed until it cools to below T~140 K.

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

82. The robotic system of claim 7, wherein the sample heating station heats a sample previously cooled to a cryogenic temperature to room temperature or biological temperature between 20°C and 60°C by contacting a heating solution.

83. The robot system according to claim 84, wherein, The sample size-related heating rate is between approximately 50,000°C / min and 6,000,000°C / min.

84. The robot system according to claim 14, wherein, The heating station includes a temperature control block heater.

85. The robotic system of claim 86, wherein the heating station includes a high thermal conductivity block attached to the block heater, the high thermal conductivity block receiving a plate, vial, test tube, cuvette, or other container for holding a solution used in the heating process.

86. The robotic system of claim 87, wherein the heat-conducting block can be shaped to provide close thermal contact with any liquid-sealed surface of a plate, vial, test tube, etc., in order to ensure maximum heat transfer rate and temperature uniformity.

87. The robot system according to claim 14, wherein, The robotic end effector transfers the sample into a heated solution contained in a hole in the plate, vial, test tube, cuvette, or other container held on the block and heater block.

88. The robotic system of claim 89, wherein the robotic end effector translates the sample into and through the heated solution at a speed of about 0.25 to about 5 m / s.

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

90. The robotic system of claim 90, wherein the sample translation may be vertical, horizontal, or at different points in the movement of the sample through the heated solution at an angle.

91. The robot system according to claim 90, wherein, Maintain the sample heating rate relative to the static heating solution until the sample has been heated above the predetermined temperature.

92. The robotic system of claim 89, wherein the depth of the plate, vial, test tube, cuvette or other container is between about 1 cm and about 20 cm.

93. The robot system of claim 89, wherein the length of the hole in the plate is from about 1 cm to about 20 cm.

94. The robot system of claim 89, wherein, in addition to translational motion, the robot end effector can also cause the sample to undergo rotational motion at a desired angular velocity at a non-zero vertical distance r from the axis of rotation.

95. The system according to claim 96, wherein, In addition to translational and / or rotational motions, the robot end effector can subject the sample to vibrational motion of a certain amplitude.

96. The robotic system of claim 97, wherein the net velocity of the sample relative to the initially stationary heated solution is maintained at about 0.5 m / s to about 5 m / s due to the translational, rotational and / or vibrational motion of the sample until the sample has been heated above 0°C and any ice in the sample has melted.

97. The robot system according to claim 97, wherein, Due to the translational, rotational, and / or vibrational motion of the sample, the net velocity of the sample relative to the initially stationary heated solution is maintained at at least about 0.25 m / s until the sample is heated to within 10°C of the temperature of the heated solution.

98. The robot system according to claim 89, wherein, The temperature of the heated solution can be substantially higher than the normal biological temperature of the sample.

99. The robot system of claim 89, wherein the temperature of the heating solution can be between 50°C and 100°C.

100. The robot system according to claim 100, wherein, The robot is able to remove the sample from the heating solution before the sample temperature reaches the temperature of the heating solution, in order to protect the sample from damage caused by prolonged exposure to elevated temperatures.

101. The robot system according to claim 100, wherein, Each orifice in the heating station can be vertically divided into two chambers connected by a channel large enough to allow the sample-containing portion of the sample carrier to pass through, wherein the liquid in the upper chamber is maintained at a higher temperature than the liquid in the lower chamber, such that the sample can first move in the upper chamber to heat at a higher rate than possible in the liquid at the temperature of the lower chamber, and then be transferred to the lower chamber before its temperature becomes too high, in order to protect the sample from damage due to excessive heat.

102. The robot system according to claim 100, wherein, Each hole in the heating station can be horizontally divided into two chambers connected by a sufficiently large channel to allow the sample-containing portion of the sample carrier to pass through, and the liquid in one chamber is maintained at a higher temperature than the heating solution in the other chamber, so that the sample can first move within the liquid in the high-temperature chamber to heat at a higher rate than possible in the heating solution at the final sample temperature, and then be transferred to the low-temperature chamber before the sample temperature becomes high enough to cause sample damage.

103. The robot system according to claim 14, wherein, The system includes a heating and drying station for the robot end effector, which is capable of heating and drying the end effector after immersion in a liquid.

104. The robot system according to claim 105, wherein, The end effector is heated by a cylindrical arrangement passing through the heater.

105. The robot system of claim 105, wherein the robot end effector dries by passing over or through a compressed air-driven air amplifier or through a high-speed gas jet or sheet-like airflow.

106. The robot system according to claim 105, wherein, The heater and the dryer are arranged such that they are on the same axis, and the end effector is able to pass through them via linear motion oscillation.

107. The robot system according to claim 105, wherein, The heating and drying station includes means for rinsing the end effector to remove residues from any liquid it has come into contact with.

108. The robot system according to claim 109, wherein, The station's rinsing components may involve one or more containers containing pure water, water + detergent, or isopropanol, and wherein the robot transfers the end effector into one or more rinsing solutions.

109. The robot system according to claim 109, wherein, The rinsing is accomplished by spraying water or other suitable solvent onto the end effector.

110. The robot system according to claim 109, wherein, The robot can rotate the end effector and make it swing up and down during rinsing.

111. The robot system according to claim 14, wherein, The robot, the motors and actuators attached to the robot forming the end effector, the temperature control block, the sensors, actuators, motors, valves of various other system components, and any or all of the digital camera and illumination source of the imaging component can be electronically controlled via a computer or microcontroller.

112. The robot system of claim 113, wherein the system controller can sequence the robot and other operations so that two or more sample carriers can undergo processing simultaneously.

113. The robot system according to claim 7, wherein, Two or more robots can be used to increase the number of samples that can be processed with a given set of processing stations.

114. The robot system according to claim 7, wherein, The sample carrier, which contains and surrounds one or more samples, has a surface containing a plurality of pores, which are sufficient to allow the liquid to flow at high speed through the sample carrier and over the sample when the sample carrier moves at high speed relative to an initially stationary liquid.

115. The sample carrier of claim 116, wherein the hole is sized to prevent the sample from passing through.

116. The sample carrier of claim 116, wherein the surface of the sample carrier may be composed of a membrane having a region with an array of pores, and wherein the sample may be in direct contact with the membrane.

117. The sample carrier according to claim 118, wherein the membrane may be a polymer, glass, semiconductor, metal or composite material.

118. The sample carrier according to claim 118, wherein the thickness of the membrane is between about 2 μm and about 100 μm.

119. The sample carrier according to claim 118, wherein the membrane has a thickness between about 5 micrometers and 50 micrometers.

120. The sample carrier of claim 118, wherein the membrane has two regions of different thicknesses, a region of first thickness with through holes for the sample to be placed thereon, and a region of second thickness for providing mechanical strength and bonding to other parts of the sample carrier.

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

122. The sample carrier according to claim 122, wherein the second thickness is between about 10 micrometers and 50 micrometers.

123. The sample carrier according to claim 118, wherein the film is micropatterned using photolithography and standard microfabrication processes, microembossing or stamping.

124. The sample carrier of claim 118, wherein the membrane is an optically transparent micropatterned polymer, such as polyimide, cyclic olefin copolymer or SU-8, to facilitate imaging of a sample placed thereon or within it.

125. The sample carrier of claim 118, wherein the opening area fraction of the porous portion of the membrane is between about 50% and 95%.

126. The sample carrier according to claim 118, wherein the diameter of the pore is between 40% and 80% of the minimum size of the sample.

127. The sample carrier of claim 118, wherein the pore has a diameter between 50 micrometers and 80 micrometers.

128. The sample carrier of claim 118, wherein the membrane is attached to a rigid frame.

129. The sample carrier of claim 130, wherein the frame may be made of metal, polymer, polymer-glass composite or semiconductor.

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

131. The sample carrier according to claim 132, wherein the base may be a rigid, dimensionally stable and cryogenically compatible material.

132. The sample carrier according to claim 132, wherein the base material may be magnetic stainless steel or glass-filled polymer.

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

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

135. The sample carrier of claim 130, wherein the frame is made of a rigid sheet having a hole disposed adjacent to one end, and wherein the membrane is applied across the hole.

136. The sample carrier according to claim 137, wherein the thickness of the sheet is between 80% and 150% of the corresponding sample thickness.

137. The sample carrier of claim 137, wherein the thickness of the sheet is between 80 micrometers and 250 micrometers.

138. The sample carrier according to claim 137, wherein the width of the frame is between about 3 mm and 10 mm.

139. The sample carrier according to claim 137, wherein the length of the frame is between about 1 cm and 10 cm.

140. The sample carrier of claim 137, wherein the pores in the frame are spanned by a first membrane and sealed on a first side, and one or more samples are placed on the first membrane.

141. The sample carrier according to claim 142, wherein the sample is sealed into the sample carrier by attaching a second membrane to a second side of the frame.

142. The sample carrier of claim 142, wherein the membrane may be attached to the frame using an adhesive, adhesive gasket, or ultrasonic bonding.

143. The sample carrier according to claim 142, wherein each membrane has a porous region of a first thickness and a solid region of a second greater thickness, wherein the membrane region in contact with the frame is a solid region.

144. The sample carrier of claim 145, wherein the first thickness is between 5 micrometers and 15 micrometers.

145. The sample carrier of claim 145, wherein the second thickness is between about 10 micrometers and 50 micrometers.

146. The sample carrier of claim 145, wherein one or both membranes have perforations, tabs or other patterns to facilitate tearing and removal of portions within the perforations, thereby facilitating access to and release of the sample after processing.

147. The sample carrier of claim 130, wherein the frame is made of a rigid sheet and wherein the frame has a cut at one end such that the frame has a "U" shape at the end.

148. The sample carrier of claim 149, wherein a membrane having a porous portion is attached to a first side of the frame to cover and seal the U-shaped portion and wrap around the open end of the "U".

149. The sample carrier of claim 149, wherein after the sample is loaded onto the membrane, the free portion of the membrane is bonded to the second side of the frame, thereby sealing the sample within the frame.

150. The sample carrier according to claim 116, wherein the sample carrier comprises two parts, namely a basket and a lid.

151. The sample carrier of claim 152, wherein the basket receives and holds one or more samples.

152. The sample carrier of claim 152, wherein the cover is configured to seal the basket and is grasped or handled by the robotic end effector.

153. The sample carrier according to claim 152, wherein the basket has a diameter at its bottom between about 2 mm and about 20 mm.

154. The sample carrier of claim 152, wherein the basket comprises a thin, low-thermal-mass frame having a substantially open bottom and substantially open sides.

155. The sample carrier of claim 156, wherein the open bottom and sides of the frame are spanned and sealed by a mesh of porous membrane or filaments.

156. The sample carrier according to claim 157, wherein the pores in the membrane or mesh are smaller than the minimum size of the sample to be held within the carrier.

157. The sample carrier of claim 157, wherein the membrane is formed wholly or partially of a transparent polymer comprising polyimide, SU-8 and cyclic olefin copolymers to allow optical inspection of the sample within the basket.

158. The sample carrier of claim 157, wherein the membrane has a thickness between 5 micrometers and 15 micrometers in the region spanning the opening in the frame, and a thickness between 10 micrometers and 50 micrometers in the region to which it is bonded to the frame.

159. The sample carrier of claim 157, wherein the membrane portion within the opening in the frame has a large opening area fraction ranging from about 50% to about 95%.

160. The sample carrier of claim 157, wherein the mesh is formed of filaments of metal or polymer such as nylon.

161. The sample carrier according to claim 157, wherein the mesh size is between about 60 and 400 meshes.

162. The sample carrier of claim 152, wherein the cover is fitted into the basket to form a seal.

163. The sample carrier of claim 152, wherein the cover includes a conical feature axially positioned and projecting downward into the basket, the conical feature being used to guide liquid flowing through the bottom of the basket to its sides.

164. The sample carrier of claim 152, wherein a series of baskets can be held in a container within the carrier, wherein a sample can be loaded into the baskets using a pipette or other tool, and then a cap is positioned and sealed to each basket.

165. The sample carrier of claim 152, wherein the cover may be marked or incorporated with an RFID tag for sample identification.

166. The robot system according to claim 1, wherein, The sample carrier may comprise a basket formed by deforming a suitably patterned flat membrane.

167. The sample carrier of claim 168, wherein the membrane contains a slit pattern that facilitates the deformation of the basket and defines the opening in the basket.

168. The sample carrier of claim 169, wherein the size of the opening in the basket is set between 50% and 80% of the minimum size of the sample.

169. The sample carrier of claim 168, wherein the membrane is attached to a rigid sheet at one end, and wherein the rigid sheet is inserted into a base.

170. The sample carrier of claim 171, wherein the sheet is semi-rigid, and wherein similar or identical films and semi-rigid sheets are attached to the same base such that the sheets are pressed together and the two baskets form a closed volume.

171. The sample carrier of claim 168, wherein the membrane is attached to a first side of a rigid sheet comprising a hole to extend across the hole and such that the basket protrudes from the hole, and wherein the rigid sheet is attached to a base.

172. The sample carrier of claim 173, wherein the sheet is semi-rigid, and wherein similar or identical films and semi-rigid sheets are attached to the same base such that the sheets are pressed together and the two baskets form a closed volume.

173. The sample carrier of claim 137, wherein the sheet is semi-rigid, and wherein similar or identical films and semi-rigid sheets are attached to the same base such that the sheets are pressed together.

174. The sample carrier of claim 175, wherein when the sheets are pressed together, the membrane is applied to the outer surface of the sheets to form a closed volume between the membranes when the sheets are pressed together. The sample carrier according to claim 172, 174 or 176, wherein the semi-rigid sheet can be pushed open using a button tool that holds the base.

175. The sample carrier of claim 176, wherein the base has a central through hole, and wherein when the button is pressed, a rod connected to the button and passing through the hole in the base can push the sheet open.

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