Systems, methods, and devices for rapid warming of cells and other biological materials

Abstract

Presented herein are systems, methods, and devices for handling small biological samples, e.g., for heating the samples at predefined warming rates during sample thawing. A method of immersing a sample in a fluid solution includes attaching the sample onto a sample support surface of a sample support, and moving the sample support to thereby align the sample support surface and sample with an open end of a well containing the fluid solution. The sample support surface and sample are then inserted into the fluid solution. The method also includes generating a relative speed between the sample and the fluid solution until the sample reaches a predefined temperature by: moving the sample support surface continuously along a predefined distance within the fluid solution; rotating and / or vibrating the sample support surface within the fluid solution; and / or agitating the fluid solution while the sample support surface is within the fluid solution.

Description

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

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

[0001] This disclosure relates generally to the field of biotechnology. More particularly, aspects of this disclosure relate to systems for processing biological samples, including the rapid warming of millimeter-sized and smaller cell samples and other biological materials.BACKGROUND

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

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

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

[0005] For warming / thawing, samples used in assisted reproduction may be removed from a cryogenic storage Dewar, transferred through air, and then plunged into a well of a multiwell plate (e.g., CRYOTEC™ plate from REPROLIFE™) or a straw containing a warming solution at room temperature or biological temperature (e.g., 37 °C), with all steps performed by hand. Other wells in the plate may contain solutions to promote cell re-expansion and postthaw growth / development. The wells of the plates used typically range from 5 millimeters (mm) to 10 mm deep, which severely limits the vertical plunge distance into the warming solution. The small well volume reduces consumption of thawing media and makes it easier to find and retrieve samples after thawing. Separate wells and warming solutions may be used for each sample to prevent cross-contamination. Similar manual protocols are used for thawing of many other types of cryopreserved samples. In the future, warming may be performed automatically using robots or other dedicated systems.

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

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

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

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

[0010] Efforts to improve warming rates have focused primarily on methods that involve inputting energy from laser beams or alternating-current (AC) electromagnetic fields. To achieve warming rates substantially faster than can be achieved in warm liquids, light absorbing or electrically conducting nano / microparticles may be introduced throughout the sample. This approach is a serious obstacle to most applications, especially those connected with human health. The time interval during which the laser light / AC field may be applied to heat the sample above ice’s melting temperature without overheating the sample depends on sample size, shape, and composition, among other factors. Uncertainties in determining this time interval for each sample and the possibility of under or overheating is another obstacle to routine application.

[0011] Recent direct measurements of bovine oocyte warming, e.g., using X-ray diffraction to measure the temperature-dependent unit cell of ice within oocytes, yielded warming rates of around 150,000 °C / min. As noted above, this is a factor of four larger than has been reported when using a CRYOTOP® vitrification container - a popular support for oocyte and embryo cry opreservation. This is surprising, since the 150,000 °C / min was achieved not by plunging in a 37 °C warming solution, but instead using a room temperature N2 gas stream flowing at a few meters per second. Comparison of fluid parameters relevant to heat transfer suggests that plunging in a warm warming solution at a few meters per second should give warming rates 20-30 times larger than in the N2 gas stream, of order 3,000,000 °C / min. The fact that observed warming rates in warming solutions are more than 60 times smaller suggests that substantial improvements in plunge warming methods are possible.SUMMARY

[0012] Aspects of the present disclosure relate to the design, function, and use of sample supports, methods, and systems for warming / thawing small biological samples that have been cooled to low temperatures. As used herein in relation to biological samples, the term “small” may be defined to include a sample having a diameter or minimum dimension that is smaller than about 2 millimeters or, in some applications, smaller than about 500 micrometers, and a volume that is smaller than about 10 microliters (pL) or, in some applications, smaller than about 1 pL. An example criterion for determining smallness is that the internal sample temperature gradients that develop within the sample during warming in a warm liquid (typically an aqueous solution) be small, e.g., less than about 20-40 °C or, in some applications,less than about 10 °C, so that thawing is reasonably uniform throughout the sample volume and so that no large stresses develop.

[0013] As used herein, the term “low temperature” may be defined to include any temperature that leads to full or partial vitrification of a solvent within a sample, which may depend on the solvent composition and solutes present. For example, a typical low temperature in this application is the temperature of boiling liquid nitrogen (77 K = -196 °C). Temperatures less than about 150 K (-123 °C) may completely inhibit ice crystal formation in samples held at those temperatures. Temperatures as high as about -80 °C (the temperature of standard laboratory freezers) may also be used. While not per se limited, eggs, embryos, and blastocysts having up to approximately 100 cells are primary targets, although stem cells, sperm cells, and any other “small” biological (human or nonhuman) sample containing cells, larvae, antibodies, proteins, etc., may benefit from warming using features of the present disclosure.

[0014] Aspects of this disclosure include structures, apparatuses, and approaches to facilitate the handling of small cryopreserved biological samples so as to achieve the largest possible warming rates during thawing.

[0015] According to aspects of this disclosure, a small biological sample having a volume of less than about 10 microliters or, in some applications, less than about 1 microliter and a thickness of less than about 5 mm or, in some applications, less than 500 micrometers may be held on a sample support having low thermal mass in a region or regions adjacent the sample.

[0016] According to aspects of this disclosure, the sample support may be comprised of a thin film of polymer, glass, semiconductor, metal, or composite with a thickness of between about 2 μm and about 100 μm or, in some applications, between about 10 and 25 micrometers. The thin film of the sample support may be attached by a rod or frame to a base.

[0017] According to aspects of this disclosure, the thin film may have at least one or, in some configurations, an array of through-holes beneath the sample position, e.g., to maximize direct contact of the warming liquid with the sample.

[0018] According to aspects of this disclosure, the portion of the thin film that supports thereon the sample may have lateral dimensions of between about 100 micrometers to about 2 millimeters, e.g., that are comparable to the size of the sample so as to minimally perturb fluid flow around the sample as it is plunged into a warming solution.

[0019] According to aspects of this disclosure, the thin film may be unframed and may be unsupported except along one edge. That edge may be curved along an axis perpendicular to the supported edge to impart rigidity.

[0020] According to aspects of this disclosure, the thin film may be attached to a thin rigid frame (e.g., less than about 1 millimeter thick or, in some applications, about 100-250 micrometers thick) containing an aperture so that the film spans the aperture in the frame.

[0021] According to aspects of this disclosure, the frame may have the shape of a “U” so that one edge of the thin sample-supporting film is unobstructed and, thus, open. During plunging in a liquid cryogen for cryogenic storage, for example, the frame may be oriented with the open edge of the frame leading / pointing along the plunge direction, e.g., so that the cryogenic liquid flows freely and without impediment over the thin film and sample so as to produce the fastest and most uniform cooling of the sample. During warming by plunging into a warm solution, the open end of the frame may again be oriented along the plunge direction, e.g., so that the warming liquid flows freely and without impediment over the thin film and sample so as to produce the fastest most uniform warming of the sample.

[0022] According to aspects of this disclosure, the sample support film may have a portion that extends beyond the open end of the “U” of the frame and forms a shovel that may be pressed into contact with the bottom of a well and slid underneath a sample to facilitate the sample’s retrieval.

[0023] According to aspects of this disclosure, the base of the sample support may include a magnet or be of a magnetic steel for magnetically attaching the support to an actuator or a base.

[0024] According to aspects of this disclosure, the sample support may be attached to an actuator that allows the axis of the sample support to be oriented perpendicular to the surface of a warming liquid, parallel to the surface of the warming liquid, and at oblique angles to the surface.

[0025] According to aspects of this disclosure, the sample and sample support may be retrieved from a cold / cryogenic temperature sample storage system using a sample handling system.

[0026] According to aspects of this disclosure, the sample handling system may include a portion that holds the sample support and a shield that surrounds at least the sample support holding portion of the sample handling system.

[0027] According to aspects of this disclosure, the shield that surrounds the sample holding portion may be cooled to near the sample’s storage temperature (e.g., below about 180 K or, in some applications, near T=77 K, the boiling temperature of liquid nitrogen) by contact with a liquid cryogen or with cryogenic / low temperature gas within a storage Dewar or refrigerator, as may occur when the sample holding portion captures the sample support from a storage Dewar.

[0028] According to aspects of this disclosure, the shield may be configured as a radiation shield, e.g., to help maintain the sample at a low temperature and trap / generate cold nitrogen gas within it so as to prevent ambient moisture-laden air from coming into contact with the sample and frost forming on the sample from this moisture.

[0029] According to aspects of this disclosure, the shield may include a reservoir and / or a lining of absorbent material that holds liquid nitrogen when the shield and sample are withdrawn from the liquid nitrogen. The liquid nitrogen held within the reservoir / absorbent material lining may then boil away, generating cold nitrogen gas that keeps moist ambient air out of the shield’s interior and away from the sample.

[0030] According to aspects of this disclosure, the absorbent material may include glass or metal wool, sintered metal powder, perlite, activated carbon, silica gel, and / or vermiculite.

[0031] According to aspects of this disclosure, the shield may be fabricated from metal, glass, ceramic, and / or a cryogenic-compatible polymer or polymer composite. Together with any internal absorbent material and trapped liquid nitrogen, the shield may have a much larger thermal mass than the sample support, e.g., to help ensure that the shield remains cold as the sample is transferred from the storage Dewar to the warming solution.

[0032] According to aspects of this disclosure, the shield may have a length that is sufficient to extend across and cover a length of the sample support and to extend at least a distance beyond the sample support equal to at least the shield’s diameter or, in some applications, between 2 and 5 times the diameter, e.g., to help ensure that the sample is properly shielded from ambient radiation and air.

[0033] According to aspects of this disclosure, a shield diameter of the shield may be between about 2 mm and about 2 centimeters (cm) or, in some applications, is about 1 cm.

[0034] According to aspects of this disclosure, the sample handling system may include a small cup or funnel (e.g., a pipette tip) with a volume of between about 0.1 milliliters (mL) andabout 10 mL. The cup or funnel is configured to capture (or can be loaded with) a small volume of liquid nitrogen when the sample is removed from a storage Dewar.

[0035] According to aspects of this disclosure, the small cup or funnel may have an outlet through which liquid nitrogen contained therein flows under gravity. The cup or funnel may be configured such that this transient liquid nitrogen flow is directed at the sample, e.g., to keep the sample cold and frost-free while it is being transferred from the Dewar to the warming solution.

[0036] According to aspects of this disclosure, after retrieval from the storage Dewar, the sample holding system may translate the sample, sample support, and shield (e.g., as a single unit) into position above the warming solution.

[0037] According to aspects of this disclosure, after translating the sample and shield into position above the warming solution, the bottom of the shield may be positioned within about 2 cm or, in some applications, about 3 mm of a top surface of the plate holding the warming solution, but does not contact the top surface of the plate or exchange significant heat with it.

[0038] According to aspects of this disclosure, after being positioned above the warming solution, the sample may be plunged, translated, or otherwise moved at a high speed out of the shield (if present) and into and through the warming solution. As used herein, a “high speed” may be in the range of about 0.1 meters per second (m / s) to about 5 m / s or, in some applications, about 2 m / s.

[0039] According to aspects of this disclosure, the sample plunging / translation / movement may be performed by a pneumatic linear actuator, a cam mechanism, a motor driven linear translation stage, a gravity drop linear motion stage, a solenoid, and / or by a multi-axis robot arm.

[0040] According to aspects of this disclosure, a distance over which the cold sample is accelerated from rest to its peak high speed, which is in the range of about 0.1 m / s to about 5 m / s or, in some applications, about 2 m / s, may be less than about 10 cm or, in some applications, less than about 5 cm or, in some applications, less than about 2 cm. This may help to minimize the vertical extent of the plunging mechanism above the warming solution, leaving more room for manipulation and microscope observation.

[0041] According to aspects of this disclosure, the sample handling system may plunge / translate / move the sample into and through the warming solution along a vertical axis or along an inclined axis angled to the vertical at up to about 75 degrees.Plunging / translating / moving the sample along an inclined axis may allow a total distance traveled through the solution to be increased while keeping the height of the wells and plate fixed.

[0042] According to aspects of this disclosure, the sample may be rotated around the plunge axis so as to maintain a total sample speed relative to the warming solution of at least about 0.1 m / s until the sample has reached a target temperature above 0 °C and completely thawed, for example, as well as to help maintain effective convective heat transfer and prevent freezing of the warming solution on the sample surface.

[0043] According to aspects of this disclosure, the sample handling system may have both a linear translation mechanism, which is operable to translate the sample, e.g., in a plunge direction on a plunge axis, into a warming solution, and a sample rotation mechanism, which is operable to rotate the sample on a rotational axis parallel to or coaxial with the plunge axis.

[0044] According to aspects of this disclosure, the sample rotation mechanism may include an electric motor that is attached to the bottom of a plunge arm of the sample handling system.

[0045] According to aspects of this disclosure, the sample may be placed off the rotational axis via a member extending perpendicular from the drive shaft.

[0046] According to aspects of this disclosure, a sample support surface plane of the sample film may be oriented perpendicular to a radial direction of the off-axis motion and tangential to the circular motion, e.g., so as to reduce stress on the sample and film during rotation.

[0047] According to aspects of this disclosure, the sample rotation mechanism may rotate the sample support about the rotational axis at an angular speed of at least about 10 revolutions per second during the plunge until the sample has fully thawed and reached a target temperature above 0 °C, for example.

[0048] According to aspects of this disclosure, the sample handling system may have an off-axis blade attached to a rotation shaft whose axis is along the sample plunge / translation / movement direction, such that the off-axis blade dips into the warming solution and spins the warming solution up to a speed of greater than about 0.1 m / s, e.g., immediately before the sample is plunged into the warming solution.

[0049] According to aspects of this disclosure, the blade may have the form of a fan or a plate configured to push into the liquid and set it into motion through openings in the fan or plate.

[0050] According to aspects of this disclosure, the sample handling system may include an actuator, a motor, and / or a linear transducer operable to vibrate the sample as it is plunged / translated / moved, e.g., so as to maintain an average speed relative to the initially stationary warming solution of at least about 0.1 m / s even once the linear motion has ended.

[0051] According to aspects of this disclosure, the sample’s vibrational motion may be along an axis or arc that lies in the plane of the sample support film so as to reduce mechanical stress on the sample during the vibrational motion.

[0052] According to an aspect of this disclosure, a warming solution and / or other solutions may be held in a deep well block, such as a Society for Biomolecular Screening (SBS) standard format deep well block, with multiple wells each having a well depth of at least about 4 cm.

[0053] According to an aspect of this disclosure, the warming solution and / or other solutions are held in a cell culture plate, such as a standard height SBS format cell culture plate, with multiple wells each having a well depth of at least about 1.6 cm.

[0054] According to aspects of this disclosure, the cell culture plate may have multiple cells each including a discrete circular well with a flat bottom or an arcuate (concave-upward) bottom.

[0055] According to aspects of this disclosure, some of the wells may be fluidly connected to a sloped channel that runs from a point at the top of the plate at a distance d from the center of the well and into the well, with the channel bottom at an angle theta, allowing a sample to be plunged, translated or otherwise moved through a warming solution contained in the channel and into the well, and increasing a maximum plunge / translation / movement distance through the warming solution without increasing the plate height.

[0056] According to an aspect of this disclosure, the plate may have a single well or multiple wells (e.g., twenty -four distinct wells).

[0057] According to an aspect of this disclosure, each of the wells may have a diameter of between about 1 cm and about 3 cm, and may have an internal volume sufficiently sized to hold a volume of solution of between about 0.1 ml and about 100 ml.

[0058] According to an aspect of this disclosure, a distance between the sloped channel at the top of the plate and the center of the well may range from about 5 mm to about 10 cm.

[0059] According to an aspect of this disclosure, the angle theta between the sloped channel and the channel bottom may range from about 0 degrees (e.g., in which case the channel bottom is horizontal) to about 75 degrees to the horizontal.

[0060] According to an aspect of this disclosure, the system may include a pipette to retrieve a thawed sample from the warming solution in the well.

[0061] According to aspects of this disclosure, the system may include a flow-through gripper to retrieve a thawed sample from the warming solution. The flow-through gripper may include two jaws each having a depression containing an array of through-holes. Each hole may have a hole size (diameter / width) that is smaller than the size of the cell or sample, e.g., in the range of about 20 μm to about 100 μm, such that when the jaws are slowly closed down on the sample, liquid flows through the through-holes until the jaws have fully enclosed the sample. The sample and j aws may then be translated from solution to solution for equilibration.

[0062] According to aspects of this disclosure, each jaw may include a frame, which has a frame thickness of about 200 micrometers to about 2 millimeters and defines a large aperture, and a film, which has a film thickness of between about 10 μm and about 100 μm, defines an array of through holes, and is adhered to the frame.

[0063] According to aspects of this disclosure, the jaws may be opened and closed manually or via a controller-automated actuator using a variety of mechanisms.

[0064] The above summary does not represent every embodiment or every aspect of the present disclosure. Rather, the foregoing summary merely provides a synopsis of some of the novel concepts and features set forth herein. The above features and advantages, and other features and attendant advantages of this disclosure, will be readily apparent from the following Detailed Description of illustrated examples and representative modes for carrying out the disclosure when taken in connection with the accompanying drawings and appended claims. Moreover, this disclosure expressly includes any and all combinations and subcombinations of the elements and features presented above and below.BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIGS. 1A and IB illustrate representative temperature-vs-time measurements obtained when plunging a dry 125-micrometer junction thermocouple (FIG. 1A) and an iced125-micrometer junction thermocouple with a drop of frozen water (FIG. 1B) into a warming solution in accordance with aspects of the present disclosure.

[0066] FIG. 2 is a perspective-view illustration of a representative sample support according to aspects of the present disclosure.

[0067] FIGS. 3A and 3B are perspective-view illustrations of other representative sample supports according to aspects of the present disclosure.

[0068] FIGS. 4A and 4B are perspective-view illustrations of yet other representative sample supports according to aspects of the present disclosure.

[0069] FIGS. 5A and 5B are perspective-view illustrations of representative shields that surround sample supports according to aspects of the present disclosure, with FIG. 5A illustrating a shield with a reduced-diameter bottom opening through which a sample can be projected, and FIG. 5B illustrating a shield that is lined with a porous, liquid-nitrogen- absorbing material.

[0070] FIG. 6 is a perspective-view illustration of a representative sample holder gripper, motion arm with shield, and linear motion shaft according to aspects of the present disclosure.

[0071] FIGS. 7A and 7B are perspective-view illustrations of representative sample rotation stages, each of which is attached to an end of a linear motion shaft or motion arm and includes a shield according to aspects of the present disclosure, with FIG. 7A illustrating a sample rotation stage configured for on-axis rotation of a sample support, and FIG. 7B illustrating another sample rotation stage configured for off-axis rotation, e.g., before, during, and after translation into a warming solution.

[0072] FIG. 8 is a perspective-view illustration of a representative fluid excitation / agitation mechanism for generating rotational motion of a warming solution, e.g., before, during, and after a sample is plunged into the warming solution, according to aspects of the present disclosure.

[0073] FIGS. 9A and 9B are perspective-view illustrations of a representative sample support system transferring a sample into a warming solution in a well plate according to aspects of the present disclosure, with FIG. 9A illustrating the sample and shield positioned above a well and FIG. 9B illustrating the sample being plunged out from the shield and into the well.

[0074] FIG. 10 illustrates a representative well plate having multiple wells with some including angled channels leading to a well body according to aspects of the present disclosure.

[0075] FIGS. 11A and 11B are partially cutaway perspective-view illustrations of a representative well plate with an array of wells for storing a solution and receiving a sample according to aspects of the present disclosure.

[0076] The present disclosure is amenable to various modifications and alternative forms, and some representative embodiments of the disclosure are shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the novel aspects of this disclosure are not limited to the particular forms illustrated in the aboveenumerated drawings. Rather, this disclosure covers all modifications, equivalents, combinations, permutations, groupings, and alternatives falling within the scope of this disclosure as encompassed, for example, by the appended claims.DETAILED DESCRIPTION

[0077] This disclosure is susceptible of embodiment in many different forms. Representative embodiments of the disclosure are shown in the drawings and will herein be described in detail with the understanding that these embodiments are provided as an exemplification of the disclosed principles, not limitations of the broad aspects of the disclosure. To that extent, elements and limitations that are described, for example, in the Abstract, Introduction, Summary, Description of the Drawings, and Detailed Description sections, but not explicitly set forth in the claims, should not be incorporated into the claims, singly or collectively, by implication, inference or otherwise. Moreover, recitation of “first”, “second”, “third”, etc., in the specification or claims is not per se used to establish a serial or numerical limitation; unless specifically stated otherwise, these designations may be used for ease of reference to similar features in the specification and drawings and to demarcate between similar elements in the claims.

[0078] For purposes of this Detailed Description, unless specifically disclaimed: the singular includes the plural and vice versa (e.g., indefinite articles “a” and “an” are to be construed as meaning “one or more” unless expressly disclaimed); the words “and” and “or” shall be both conjunctive and disjunctive; the words “any” and “all” shall both mean “any and all”; and the words “including,” “containing,” “comprising,” “having,” and the like, shall each mean “including without limitation.” Moreover, words of approximation, such as “about,”“almost,” “substantially,” “generally,” “approximately,” and the like, may each be used herein to denote “at, near, or nearly at,” or “within 0-5% of,” or “within acceptable manufacturing tolerances,” or any logical combination thereof, for example.

[0079] Features of the present disclosure aim to address the design criteria and challenges listed below to achieve the fastest possible warming of small biological samples that have been cryopreserved, and to do so using standard warming solutions with the goal of minimizing sample damage resulting from the freeze-thaw cycle and of maximizing post-thaw survival and development of cellular systems. As discussed above, some key principles that have been identified for maximizing warming rates of small biological samples may include:(1) minimizing a thermal mass of sample + surrounding liquid + sample support in contact with a sample, and maximizing direct contact of the sample with a warming solution;(2) protecting a sample during transfer from a cold / cryogenic liquid to a thawing liquid using a partial enclosure that has a large thermal mass and surrounds the sample to maintain the sample at or near the original cold temperature so as to maintain the sample at a nearly constant temperature until it enters a warming solution and thereby prevent partial thawing during the transfer through air;(3) protecting a sample from frosting due to moisture in ambient air / gas during transfer from a cryogenic liquid to a thawing liquid, as frost adds thermal mass, reduces convective heat transfer at the sample surface, and adds latent heat that slows warming through 0 °C;(4) maximizing a velocity of the sample relative to a warming solution to maximize convective heat transfer rates;(5) maintaining a large sample velocity relative to a warming solution for a sufficient time / distance until the sample has warmed above 0 °C or, in some applications, above 5 °C; and(6) automating sample motions and handling as much as possible to maximize warming rates, maximize sample throughput, and minimize opportunities for operator error and sample damage or mis-labeling.

[0080] Detailed experiments described below show that principle (5), above, may be particularly important for maximizing warming rates of small biological samples. In currentinvitro fertilization (IVF) practices, for example, a sample and its support are manually inserted into a roughly 5 millimeter (mm) deep well containing a warming solution. This limits the maximum sample speed relative to the warming solution when it is plunged into the warming solution because of the very short stopping distance afforded by the shallow well, both for routine hand plunging and also if automated plunging is implemented. A shallow well also reduces the maximum time that the sample maintains high speed motion relative to the warming solution. When using a sample speed on entry into the warming liquid of 1 m / s, for example, the average speed before stopping will be roughly 0.5 m / s, and to travel 5 mm will take about 10 milliseconds. With a warming rate of 105°C / minute - the largest sample warming rate that is likely achievable using standard sample supports used in IVF, such as CRYOTOPS™ - the sample temperature will rise less than 20 °C before it stops. The sample deceleration needed to stop in 5 mm-deep well at an initial speed of 1 m / s is approximately 100 m / s2or 10 g, which may cause large sample oscillations after braking and possible damage to the sample support.

[0081] Sample warming may be primarily via convective heat transfer, which increases as the sample speed relative to the warming liquid increases. Experimental data has shown that if a sample speed relative to a warming solution becomes too small before a sample has warmed above 0 °C, the warming solution may solidify (e.g., crystallize or vitrify) onto the sample and dramatically slow the sample’s warming rate. Cooled warming liquid next to the sample may concomitantly sink, which may cause the sample to descend to the bottom of the warming solution and thereby reduce a warming rate of the sample. Some of these effects may be reduced by manually “stirring” the sample in the warming solution. However, relative speeds between the sample and the warming solution that can be safely achieved by manual stirring are small, typically no greater than about 0.1 m / s. To achieve extremely large warming rates may necessitate innovation and integration of all six principle components listed above.

[0082] An additional goal to optimize sample warming may include reducing the concentrations of ice-inhibiting cryoprotectants, especially of potentially toxic dimethyl sulfoxide (DMSO), that is oftentimes used in soaking the sample prior to cooling to prevent ice formation during both cooling and warming. This may necessitate maximizing both cooling and warming rates. Minimizing cryoprotectant concentrations may be desirable to: (1) help reduce the time needed to soak samples to predefined final concentrations prior to cooling; (2) help reduce any osmotic shock and associated damage during the soak and also during thawingand re-equilibration to the native state; and (3) help reduce the thermal contraction of the solutions on cooling, reducing mechanical damage and fracturing.

[0083] Turning to FIG. 1, there are shown temperature-time measurements obtained when plunging two thermocouples into a warming solution comprised of pure water having an initial temperature of 30-32 °C. FIG. 1A shows data obtained using a dry (first) thermocouple that is a dry 125-micrometer junction thermocouple (e.g., having a thermal mass similar to that of a human or bovine oocyte or blastocyst). In contrast, FIG. IB shows data obtained using a wet (second) thermocouple that is a 25-micrometer junction thermocouple that is embedded in an approximately 150-micrometer diameter drop of water on a 10-micrometer thick polymer mesh support. The two thermocouple samples are initially cooled in liquid nitrogen with a temperature T=77 K (-196 °C), transferred within a cold shield into position above the warming solution, and then plunged into the warming solution.

[0084] Continuing with the discussion of the foregoing temperature-time measurement examples, maximum warming rates (given by the maximum slope of each curve, measured in the first part of warming) are 3.6 x 106°C / min for the dry (first) thermocouple and 6.8 x 106°C / min for the wet (second) thermocouple. Crystalline ice formation in an initially vitrified sample can begin at temperatures near 150 K (-123 °C) but the rate of ice crystal growth is too slow to yield appreciable ice below 180 K (-93 °C). Average warming rates from 77 K = -196 °C to 5 °C are 1.0 x 106°C / min for the first thermocouple and 1.1 x 106°C / min for the second thermocouple sample, and corresponding warming times are ~11 milliseconds in both cases. Warming rates reported by IVF sample support manufacturers are less than 50,000 °C / min, and the largest warming rate reported in the literature for a thermocouple attached to a CRYOTOP™ is 117,000 °C / min, corresponding to warming times of 240 and 100 milliseconds, respectively. The plateau in temperature near 0 °C in FIG. IB is due to absorption of heat at constant temperature (the latent heat of fusion) as the ice surrounding the thermocouple junction converts to liquid water. Measurements at plunge speeds from 0.2 to 2 m / s (not shown) indicate that the maximum warming rate increases approximately as the square root of the plunge speed.

[0085] One key to understanding why warming rates in current practice are so small is to consider the distance the sample travels at each plunge speed from the time it enters the warming liquid and begins warming to when its temperature reaches 5 °C. For the plunge speed of 1 m / s in FIG. 1A the distance is ~1.1 cm, and for the plunge speed of 2 m / s in FIG.IB the distance is 2.2 cm. These distances are larger than the 3-5 mm warming solution depth typical in current IVF practice. Biological samples that have larger volumes or that are held on thicker, larger thermal mass supports (such as a CRYOTOP™) will have smaller warming rates, longer warming times, and require larger plunge distances to warm to 5 °C.

[0086] Turning next to FIG. 2, there is shown an example of a sample support 20 according to aspects of the present disclosure. This sample support 20 design may help to minimize a thermal mass of the sample + the surrounding liquid + the sample support in contact with the sample, while also maximizing direct contact of the sample with a warming solution. A biological sample 30 is supported on a thin polymer film 40, which is attached to and projects upwards from a distal end of an elongated support rod 60. A proximal end of the support rod 60, opposite that of the film 40, is inserted into an open end of a cylindrical support rod sleeve 50 that projects upward from a sample support base 80 that enables easy handling of the sample support 20. Although differing in appearance, it is envisioned that any of the features and options described herein with reference to the sample support configurations of FIG. 2 may be incorporated, singly or in any combination, into the sample support configurations of FIGS. 3- 11, and vice versa.

[0087] It may be desirable that the polymer film 40 has a film thickness that is less than 100 micrometers (μm) or, in some applications, between about 5 μm and about 25 μm, e.g., to minimize thermal mass and maximize heat transfer rates. As seen in the inset view of FIG. 2, the polymer film 40 may be fabricated with a pattern of through holes, such as larger single holes or an array of holes 110 forming a mesh region 100. These through holes 110 allow the warming solution to contact both sides of the sample. The sample is placed on a region 100 with the through holes 110 or mesh if present. The polymer film 40 may be fabricated, in whole or in part, from a polyimide material, an epoxy -based, negative-tone photoresist material (SU-8), a mylar material, and / or any suitable polymer material that can be processed into a desired size and shape. A film width of the polymer film 40 may be approximately equal to or, if desired, somewhat larger than a sample width of the sample. In the case of oocytes and embryos, the sample width is approximately 100-200 micrometers; for this example, the film width may be between 100-500 micrometers with a maximum width of 2 millimeters. The polymer film 40 of FIG. 2 may have one mesh region 100 or several mesh regions 100 and, as such, can support thereon one or several samples. For multi-region configurations, the mesh regions 100 may be arranged in a rectilinear line and spaced equidistant from each other.

[0088] Polymer film 40 of FIG. 2 may be curved, e.g., by wrapping one end of the film 40 around a curved distal end of the elongated support rod 60. Doing so will impart curvature to the polymer film 40 and, thus, make the film 40 substantially rigid against bending when it enters a warming solution. An overall film length of the polymer film 40 extending beyond the rod 60 may be less than about 2 mm when using thinner films (e.g., 5-25 micrometers thick) so that there is adequate rigidity to prevent excessive bending as the film 40 travels through a warming solution. The support rod 60 can be a single-piece, cylindrical structure formed with a polymeric material, a metallic material, or other suitable rigid or semi-rigid materials. The sample support base 80 may also be made from a rigid material; in crystallography, for example, the base 80 is made of magnetic steel to allow handling using magnets. A detailed shape of the base 80 can be customized for use with the specific tools used for sample handling; the shape shown may be particularly useful in crystallography.

[0089] FIG. 3A shows another example of a sample support 120 for supporting thereon and transferring a sample (not labelled) in accord with aspects of the present disclosure. The sample support 120 includes a two-tined sample support frame 140 with an elongated stem that inserts into the support sleeve 50 of the base 80. Projecting from a distal end of the flat frame 140 are parallel tines 145 that define therebetween a U-shaped opening. Attached to the parallel tines 145 and spanning across this U-shaped opening is a thin polymer film 160 that supports thereon one or more samples. Similar to polymer film 40 of FIG. 2, the thin polymer film 160 of FIG. 3A may have one or more through holes, which may be typified by a single aperture, a single array of apertures, or multiple mesh regions 170 (as shown), on which one or more samples may be placed. Moreover, the thin film 160 may be fabricated from a polymeric material, including those described above with respect to the film 40 of FIG. 2, but may be of any metallic or other material that can be fabricated in a desired size and shape. A patterned film may be fabricated by photolithography, by laser cutting, by micro-embossing, etc.

[0090] A distal-most (top) open end of the U-shaped opening and a distal-most (top) edge of the film 60 may be perpendicular to a plunge direction Dp (FIG. 8) during sample thawing, e.g., so that the frame 140 does not obstruct or inhibit flow of warming solution over the film 160 and sample. The orientation of the “U” can be tilted with respect to the sample support axis to align with the axis of motion during thawing. The frame 140 may have a frame thickness of between about 50 μm and about 1 mm or, in some applications, about 100 μm to about 250 μm, e.g., to ensure rigidity. Moreover, the frame 140 may be fabricated (e.g., by laser cutting) from any material in thin sheet form that is rigid and not prone to breaking understress, including a polymer (e.g., polycarbonate), a composite (e.g., a glass-filled epoxy such as G-10), and a metal (e.g., brass or stainless steel). The sample support frame 140 may be made of a material that has low thermal mass and a low thermal conductivity so that ice does not form on it when plunged into the warming solution.

[0091] The exposed, uppermost edge of the film 160 may be set back from the uppermost edge of the sample support frame 140 so that the film 160 is protected by the frame 140, e.g., should the sample support 120 impact the bottom of the well holding the warming solution. Alternatively, FIG. 3B shows an alternative example of a sample support 120’ in which a thin polymer film 180 is mounted to and extends between the tines 145. The exposed, uppermost edge of the film 180 projects beyond the uppermost edge of the frame 140 to form a flexible “scoop” projection 185 that can be pushed into the bottom of a well to slide underneath a sample and the lift the sample up and onto the framed portion of the film. The projection 185 may project up to 2 millimeters from the terminal edges of the frame tines 145 or, for at least some applications, project between about 200 μm and about 800 μm, e.g., so that the projection 185 does not flutter excessively during plunging in liquids.

[0092] FIGS. 4A and 4B show two more examples of sample support 120” and 120’” configurations for supporting thereon and transferring a sample 30 in accord with aspects of the present disclosure. To help maximize a sample’s warming rate and minimize ice formation within the sample during warming, the sample should remain in motion relative to the warming solution at least until its entire volume has warmed to at least a few degrees Celsius above 0 °C. It is possible that the portion of the sample in contact with the sample support film 160 may melt first, in which case the sample may be released from the support before it has fully thawed. The sample may then have a smaller speed relative to the warming solution than it would had it remained in contact with the sample support, and thus may warm at a smaller rate.

[0093] FIG. 4A and 4B show sample supports 120 and 120’”, respectively, that include structure for capturing the sample and keeping it moving through the warming solution in the event that it becomes detached from the sample support film. In FIG. 4A, a second covering film 190 spans the opposite side of the frame 140. The covering film 190 is thin and has an array of through holes forming a mesh with a large open area fraction to maximize contact of the warming solution with the sample, and where the holes are sized to prevent passage of the sample through them. The covering film 190 may leave a portion of the sample support film exposed to allow the sample to be easily deposited on the sample support film, and may cover portions of the film that are in the direction the sample will move should it become detachedduring plunging. For example, when the sample support is plunged vertically into a warming solution, corresponding to the direction DP(FIG. 8), on detaching from the sample support film the sample will tend to move downwards and thus be captured by the covering film 190. If the sample support undergoes rotational motion through the fluid, as discussed later, with the plane of the support 120 along a tangent to the motion and the vertical axis of the support perpendicular to the plane of the motion, the sample will displace sideways relative to the support after detaching, and will be captured by the covering film.

[0094] After the sample has completed thawing it may be desirable to keep the sample on / in the support for subsequent processing steps, which may include soaks in solutions having different compositions. Once the subsequent steps are complete, it is desirable to make it as easy as possible to extract the sample from the holder. The stage that holds the sample support may include at the sample holding end an actuator / effector that allows the axis of the sample support to be rotated through at least 90 degrees while it is in the warming solution within a well. The sample support may then be oriented with its axis perpendicular to the surface of a warming liquid, parallel to the surface, or at an angle in between. Once the sample has thawed and detached from the supporting film, rotating the support to the horizontal will keep the sample on the support and prevent it from falling out the open end of frame. Alternatively, the “U” frame may be modified, e.g., by adding inward projecting members extending horizontally from the top of each arm of the “U”, by additionally adding vertical members at the end of each horizontal member that projects toward the closed end of the “U” (thereby forming “pockets”) on either side of the opening of the frame. The thawed thawed sample can then be displaced into these pockets by relative motion of the sample holder and warming solution and / or by tilting the sample support,, and where the sample can then be held during subsequent soaking steps. Alternatively, pockets can be formed in the sides of the “U” to hold a sample. In either case, structures are provided that can hold the sample within the sample holder after it thaws and during subsequent manipulations.

[0095] FIG. 4B shows an alternative structure for capturing the sample 30 should it detach from the support film during thawing. After loading the sample onto the support film, a sample cover 195 is positioned and held over the sample support film, leaving a gap between the film and cover large enough to accommodate the sample. The cover 195 may be comprised of a frame spanned by a thin film that has an array of through holes forming a mesh with a large open area fraction to maximize contact of the warming solution with the sample, and where the holes are sized to prevent passage of the sample through them. The cover 195 may be attachedto the sample support film’s frame by a hinge-like member, which may be held closed, for example, by a clip or finger that can be extended from the base. Alternatively, the cover 195 could be structured to slide onto and over the sample support frame, for example by having a “U” channel in the cover’s frame that can capture the sample support frame (not shown). In both FIG. 4A and FIG. 4B, there may be an open gap between the sample support film along the edge that leads during plunging, to facilitate flow of warming solution across the sample.

[0096] FIGS. 5A and 5B show two examples of sample shields 200 and 240, respectively, that surround a sample support 20 and a sample (no shown), for example, as they are transferred from cold storage to a position just above a warming solution. For small samples (e.g., with volumes below roughly 5 microliters and thicknesses below 1 millimeter) on thin, low-thermal mass films, appreciable sample warming may occur when the sample and holder are transferred from cold storage through ambient temperature air to a position above the warming solution, depending on the amount of time needed for that transfer. Sample shields 200, 240 are structurally configured to cool to near the initial temperature of the sample when it is removed from cold storage and have a much larger thermal mass than the sample / sample support such that its temperature does not rise appreciably during the transfer. The shields 200, 240 act as thermal jackets that maintain the sample temperature at or near its initial cold temperature until the sample is plunged into the warming solution, so that nearly all sample warming occurs in the warming solution at a (large) rate determined by heat transfer to the warming solution, and not in the ambient air through which the sample is transferred.

[0097] In FIG. 5A, the sample shield 200 is an elongated and hollow cylinder that slidably receives therein and surrounds the sample holder 20 and, thus, the sample(s) borne by the sample holder 20. A distal (top) end of the shield 200 has a large hole sufficiently sized to receive therethrough the sample holder 20. Comparatively, a proximal (bottom) end of the shield 200 has a small hole 220 with a hole diameter that is approximately 1 / 5 to 1 / 10 of a shield diameter of an outer-diameter (OD) surface of the shield 200. The hole 220 in the sample shield 200 is sized to pass therethrough the support rod 60 such that the sample may be ejected from the sample shield 200 and plunged into a warming solution. If desired, a modified cryovial, such as those used in cryocrystallography, with a central hole in its cap and a magnetic ring in its base that captures the sample support could be used. The sample support 20 and shield 200 could then be held in “pucks,” similar to those that are used in cryocrystallography, and retrieved as a unit.

[0098] In FIG. 5B, the sample shield 240 is also an elongated and hollow cylinder through which the sample support 20 and sample(s) can be pushed into the warming solution. In this case, the shield 240 may be attached to the sample-holding arm that is used to retrieve the sample from cold storage (as shown in FIG. 6). Both of the illustrated sample shields 200, 240 may be formed, in whole or in part, from materials such as a polymer or a metal (such as stainless steel), and may have a wall thickness of at least about 1 mm so as to have adequate thermal mass. Optionally, the inner surface of the shield 200, 240 may be coated, textured, or fabricated with grooves / recesses (e.g., by threading) that, when the sample is stored in liquid nitrogen, help to hold liquid nitrogen when the shield 200, 240 is removed from cold storage.

[0099] Whereas shield 200 of FIG. 5A may be fabricated as a one-piece, unitary structure, the shield 240 of FIG. 5B may be a bipartite construction generally composed of an outer shell 260 portion, e.g., made with a metal or a polymer, and an inner lining 280 portion, e.g., made with a material that absorbs liquid nitrogen, such as metal or glass wool, sintered metal powder, perlite, activated carbon, vermiculite, and / or silica gel. By trapping liquid nitrogen inside the outer shell 260 portion of the shield 240, the inner lining 280 helps to reduce the warming rate of the shield 240. Boiling / evaporation of liquid nitrogen within the shield 240 may generate dry nitrogen gas that will prevent warm moist ambient air from entering the shield 240 and inadvertently warming and frosting the sample. Either of the shield 200, 240 configurations shown may include or may be thermally connected to a small heater (not shown) that heats the shield 200, 240, e.g., to just above the temperature of liquid nitrogen to ensure that cold dry nitrogen gas is generated at an adequate rate to prevent warm room temperature gas from impinging on the sample.

[0100] In addition to a sample shield 200, 240, any of the herein described sample support systems may include a small cup or funnel (like a pipette tip) with a volume between about 0.1 milliliter (mL) and about 10 lOmL (not shown). This cup / funnel can be loaded with or can capture a small volume of liquid nitrogen when the sample is removed from its storage Dewar. The cup / funnel has a small-diameter outlet port through which liquid nitrogen contained therein flows under the force of gravity. This transient liquid nitrogen flow is directed at the sample to keep it cold and frost-free while it is being transferred from the Dewar to the warming solution.

[0101] FIG. 6 shows select parts of a representative sample holding system 300 for translating a sample support 20 contained within a sample shield 200, 240 and, when desired, plunging the sample borne by the sample support 20 into a warming solution. In accord withthe illustrated example, the sample holding system 300 includes a sample handling end effector (sample handling member) 310, which may be attached to a multi-axis robot arm or to a linear or multi-axis translation mechanism that translates the support 20 and shield 200, 240 from cold storage to a a warming solution station. Alternatively, the sample support 20 may be retrieved from cold storage using a wand that incorporates the shield 200, 240; this wand may be loaded on a stage for necessary translations during warming.

[0102] Sample shield 200, 240 of FIG. 6, which protects the sample(s) from warming in ambient air, is securely mounted, e.g., via threads, camlock connection, or keylock connection, proximate a bottom -most end of the sample handling member 310. Sample support 20 of FIG. 6 is securely mounted e.g., via threads, camlock, or quick connector, to a bottom-most end of a slidable plunge shaft (sliding member) 320. This sliding member 320 slides within the sample handling member 310, e.g., driven by a motor or linear transducer, to thereby linearly translate the sample support 20 through the shield 200, 240 and out an open end thereof into a warming solution.

[0103] To achieve fast (or the fastest) warming rates, the plunge speed should be as fast (or fastest) as feasible. The plunge speed may be a mathematical sum of the respective speeds of the sample handling member 310 and of the sliding member 320 relative to sample handling member 310. Sliding member 320 may be translated by mechanisms including pneumatics, a solenoid, and a bidirectional direct-current (DC) induction motor. The sample holding system 300 should accelerate the sample support 20 to a speed of between about 0.1 m / s and about 5 m / s or, in some applications, at least 1-2 m / s in as short a distance as is feasible from the initial position of the sliding member 320. The system 300 should then maintain adequate speed of the sample(s) to maintain a large warming rate until each sample has fully thawed and its temperature has risen above roughly 5 °C; this speed should be maintained for at least a predefined sample travel time and sample travel distance, e.g., as discussed above. The predefined sample travel distance may be at least about 1 cm or, in some applications, about 2 to 4 cm; for larger samples that warm more slowly, a larger travel distance may be necessary, perhaps 10 cm or greater.

[0104] For at least some applications, the sample speed relative to the warming fluid and the warming rate during the initial phase of sample warming from near the temperature of boiling liquid nitrogen (T=77 K or -196 °C) to about T= 180 K (or -93 °C) is less critical, as the rate of ice formation in this temperature range is very small. In this instance, the warming rate from about T=180 K to about 5 °C is more critical, as this will primarily determine theamount of ice (ideally, zero) that forms. Consequently, the peak translational speed of the sample relative to the warming liquid can occur after the sample has entered the warming solution and before it has warmed to roughly 180 K. This allows the distance over which the sample is accelerated to be larger and the peak sample acceleration to be smaller. Since the sample is initially cold and solid and rigidly attached to the support, it can withstand very large accelerations without falling off the support or being damaged in any way.

[0105] The sample support 20 can be gradually decelerated to a stop or can be stopped abruptly (e.g., by hitting a stop) after each sample has fully melted so that the sample is no longer in rigid contact with the support; if the sample has not fully melted, it may experience large stresses during deceleration. Use of thick sample supports made from materials with relatively low thermal conductivity (e.g., polymers), such as the CRYOTOPS™ used in current practice, results in sample thawing occurring most slowly at the boundary between the support and sample, and thus increases the likelihood that the sample will experience damaging stresses during deceleration. By using very thin supports with holes that allow direct contact of warming solution with both sides of the sample may give more uniform warming and release of sample from the support when the entire sample has thawed. Alternatively, the thin sample support films can be fabricated from a good thermal conductor, including metals like copper, nickel, tungsten, and brass, and perhaps coated with a very thin film for biocompatibility, to ensure that the sample first melts adjacent to the sample support and releases. A possible risk here is that the sample releases before its entire volume has warmed above its melting temperature, in which case it may subsequently move more slowly and thaw more gradually.

[0106] Motion of sample handling member 310 can be effected by a pneumatic linear actuator, a cam mechanism, a motor driven linear translation stage, a gravity drop linear motion stage, a solenoid, or by a multiaxis robot arm. The multiaxis robot arm option may be preferred if the motion of sample handling member 310 is not simply linear, e.g., as is oftentimes needed to retrieve sample holders from storage and deliver them to warming solutions. Motion of member 320 relative to member 310 can be produced, for example, by a pneumatic linear actuator, a cam mechanism, a motor driven linear translation stage, a gravity drop linear motion stage or a solenoid.

[0107] Depending upon the size, volume, and / or thickness of a sample, the time for warming the sample to at least about 5 °C and complete thawing may be long, and the total distance the sample travels relative to the warming solution may be large. If the warming rate of the sample is maximized by linearly translating the sample at a high speed through thewarming solution, the corresponding distance traveled through the warming solution before complete thawing to about 5 °C could be several centimeters to tens of centimeters.

[0108] To help ensure that the sample has fully thawed and warmed to at least about 5-10 °C, the sample may need to be translated relative to the warming solution for a minimum distance that depends on: (1) the sample size, (2) the plunge speed on entering the warming solution, and (3) the braking profile once the sample is in the warming solution. At average speeds over the plunge distance of 0.1 m / s and 1 m / s and for sample sizes of about 100 micrometers, and assuming a maximum warming rate as obtained in current practice of 5 x 104K / min and an exponential approach to the final sample temperature, minimum sample travel distances may be in the range of about 5 centimeters (cm) to about 50 cm. These distances are much larger than the ~5 millimeter depth of warming solution wells provided in current IVF practices. Assuming a warming rate of 1 x106K / min, the minimum sample travel distances drop to about 2.4 millimeters to about 2.4 centimeters. If the sample comes to rest relative to the surrounding fluid while the sample is still cold, the warming solution may freeze onto the sample and slow its warming. Alternatively, a layer of cold liquid may form around the sample that, once the sample comes to rest near the bottom of the well, may remain around the sample and slow its warming.

[0109] For some samples, it may be practical to use deep well blocks, e.g., with well depths of at least about 4 cm, or custom wells that are much deeper to hold the warming solution. However, for many samples, manual retrieval becomes more difficult as the depth of the warming well increases. The volume of warming solution needed for each sample may also increase as the depth of the well increases. To facilitate sample retrieval and minimize warming solution consumption, it may be desirable to minimize the depth and total volume of the warming well. In that case, some other mechanism besides unidirectional linear translation through the warming solution is needed to maintain an adequate constant sample speed relative to the warming solution until the sample is warmed and released from the sample support.

[0110] FIGS. 7A and 7B show two approaches for rotating a sample relative to a warming solution to maintain a constant relative sample speed during sample warming. In FIG. 7A, for example, a sample support system 350 includes an electric motor 340 that is attached to a proximal, bottom-most end of the rectilinearly translatable plunge shaft 320 (FIG.6). In this example, a sample support 20 is securely mounted to a proximal, bottom-most end of the connecting arm 360, both of which are circumscribed by and slidable within a sample shield 380 (e.g., sample shields 200, 240). The electric motor 340 is selectively activated, e.g., via asystem controller, to rotate both the cylindrical connecting arm 360 and the sample support 20 about a common rotational axis Al-Al, e.g., upon plunging of the sample support 20 into a warming solution.[0111 In FIG. 7B, a sample support system 350’ includes an electric motor 340 that is attached to the bottom-most end of the plunge shaft 320 (FIG.6), and a mounting plate member 390 that is securely mounted onto a terminal end of the output shaft of the motor 340 to rotate in unison therewith. A sample support 20 is securely mounted onto a bottom face of the mounting plate member 390, spaced radially outward from a central rotational axis Al-Al. Electric motor 340 is selectively activated, e.g., via a system controller, to rotate the mounting plate member 390 and, thus, the sample support 20 such that the sample support 20 orbits about the axis Al-Al so that the sample(s) borne by the support 20 execute a circular motion.

[0112] In both FIGS. 7A and 7B, rotational motion of the sample support 20 via the motor 360 can be initiated immediately before the sample is plunged into the warming solution, e.g., to help ensure the maximum possible sample speed relative to the warming solution, and the rotation can be maintained until the sample has fully warmed. The sample support system 350 in FIG. 7A will produce less mixing of the warming solution relative to the sample than the system 350’ in FIG. 7B and may not be as effective. A maximum radial displacement of the sample support 20 and sample from the rotational axis Al-Al may be limited by the diameter of the warming well (e.g., about 1 cm). For a rotation radius of 0.5 centimeters and a rotational speed of 1 m / s, the sample may need to be rotated at about 30 or more revolutions per second (RPS). Suitable rotation rates may range from about 5 RPS to about 200 RPS, e.g., depending on the available radius of the motion.

[0113] A compact commercial vibration motor has a rotation speed of 160 revolutions per second and rotates a circular mass roughly 3 mm off its axis. The speed of a sample completing 160 revolutions per second in a 3 millimeter radius circle is roughly 1.6 meters per second, comparable to what can be achieved in linear motion plunging. In FIG. 7B, to reduce forces on the sample support and sample once they enter the warming solution, the plane of the sample support film can be oriented perpendicular to a radius of the motion. This could be achieved, for example, using a keyed sample support design to ensure that the support is held to the platform 390 in the correct orientation. If the linear sample motion is pneumatically driven, rotational motion can also be pneumatically driven using an air motor. Suitably compact motors with rotation speeds below 200 revolutions per second are available.

[0114] Presented in FIG. 8 is a sample support system 400 that generates rotational motion within a deep well 415 of a well plate 425 to agitate a liquid warming solution 435 immediately prior to plunging a sample on a sample support 20. Rather than rotating the sample and sample support 20, one or more helical blades 410 are mounted via a mounting disc 430 to motor shaft 345 of an electric motor 340 to rotate in unison therewith. These blades 410 wrap around the sample support 20 and project downwards from the mounting disc 430, extending beyond the sample support 20 and into the warming solution 435. Similar to the support system configurations of FIGS. 7A, and 7B, the motor 340 of FIG. 8 is attached to the bottom-most end of a plunge shaft 320 (FIG.6) to effect rectilinear “plunging” motion of the motor 340, sample support 20, and blades 410.

[0115] A system control module (shown schematically in FIG. 8 as electronic control unit (ECU) 450) selectively activates the motor 340, e.g., immediately prior to the sample plunge; motor activation effects rotation of the blades 410 and not the sample support 20. At high rotational speeds, the liquid warming solution 435 may rise to the upper edge of the well 415 and may eventually overflow. Instead of the “stir-type” helical blades 410 shown in FIG. 8, the sample support system 400 may employ other blade form factors, such as fan blades, that are shaped to direct the solution 435 downwards and, thus, keep it from rising up on the sides of the well 415. As another option, a disk with a central hole may be used to force liquid solution 435 to flow up through the hole as it descends. After the blades begin stirring the solution 435, the sample support 20 and sample are plunged into the solution 435. It is also envisioned that the blades need not generate circular motion of the fluid; any rapid organized or disorganized fluid motion relative to the sample should be effective in increasing warming rates.

[0116] Instead of rotational motion, the sample support 20 and any samples supported thereon can be vibrated up and down, side to side, torsionally, or in a random orbit. A fluidagitating up-down motion may be generated using any suitable actuator mechanism, including a voice-coil type actuator, a piezoelectric actuator, and a linear motor. A disadvantage of using an up-down linear motion is that the sample may be pulled in and out of the warming liquid as it enters the liquid, depending on the vibration amplitude and frequency and the liquid surface tension. Side-to-side fluid-agitating motion and other horizonal plane vibrations can be generated using a vibration motor with an asymmetric rotating mass (e.g., similar to FIG. 7B) and an ultrasonic vibrating motor, e.g., where vibration is generated electromagnetically or electromechanically.

[0117] Turning next to FIGS. 9A and 9B, the sample holding system 300 of FIG. 6 is shown plunging the sample support 20 and any samples supported thereon into a well 420 of a microplate 440 that contains a warming solution 460 according to aspects of the present disclosure. First, a sample support motion mechanism, such as robot arm wrist 445, positions the sample handling member 310 along with the sample support 20 and sample shield 200, 240 attached thereto immediately above the well 420 of the microplate 440 containing the warming solution 460. As shown in FIG. 9A, the bottommost end of the shield 200, 240 is located approximately within about 5 mm of the top surface of the microplate 440 with an air gap therebetween, e.g., so that heat does not flow from the well 420 into the shield 200, 240. This helps to minimize the time during which the sample support 20 is exposed to ambient air that can warm or generate condensation on the sample(s) prior to entering the warming solution 460

[0118] After locating the sample support 20 above the well 420, the linear motion sliding member 320 plunges the sample support 20 through the hole 220 / open bottom end of the shield 200, 240 and into the warming solution 460 to a predefined depth close to bottom of the well 420. It may be desirable that the average plunge speed of the sample support 20 be as large as is feasible given the available acceleration and deceleration distances, e.g., with a maximum plunge speed of about 2 m / s. The plunge depth through the solution may be between about 4 mm and about centimeters, e.g., for 100-200 micrometer size samples; plunge depth, speed, and / or acceleration / decel eration may be varied for smaller and larger samples that warm more quickly / slowly. This plunge stage may include sample rotation / vibration and / or solution agitation, such as the options described above with respect to FIGS. 6-8, so that the relative speed between the sample and the warming fluid maintains a significant average even after linear translation motion has stopped.

[0119] FIG. 10 shows an example of a multi -well microplate 500 device with a primary (first) set of thawing wells 560 that may hold a warming solution and from which will be retrieved thawed samples, and s secondary (second) set of post-treatment wells 520 that may hold additional solutions used for post-thaw treatment. If rotational and vibrational motion of the sample and sample support prove problematic, the microplate thawing wells 560 provide a longer plunge path through the warming solution to help ensure that the samples have fully warmed before their motion relative to the warming solution stops. In particular, multi-well microplate 500 of FIG. 10 includes angled well extension channels 540 that are fluidly connected to the thawing well 560 so that the sample may travel laterally as well as verticallythrough the wanning solution. Each well extension channel 540 has a ramped bottom surface that slopes, e.g., at an oblique angle, from the top surface of the microplate 500 toward the base of a respective well 560. With this well configuration, a sample can be easily moved down the slope to the main body of the well 560. If the sample falls off the sample support before reaching the main body of the well 560, it can easily be retrieved.

[0120] Each well extension channel 540 may be narrow - about 2 mm to about 10 mm or, in some applications, about 4 mm - to help minimize the amount of additional warming solution needed. Moreover, each well extension channel 540 can span at least about 30-40% a plate width of the microplate 500, e.g., having a channel length and corresponding plunge distance of at least about 7 cm to about 10 cm. Unlike traditional SBS well blocks, the individual wells 520, 560 in the microplate 500 may have a depth of at least about 12-18 millimeters; laterally extending the wells 520, 560 in with the channels 540 provides a large increase in plunge distance. To avoid cross contamination of samples, a single plate may only have a single set of wells needed to thaw and prepare a single sample. The bottom of each well 520, 560 may be substantially flat or arcuate (concave upward) so that a dislodged sample settles to the center of the well for simplified retrieval. In the illustrated example, a biological sample may be translated at a high speed along a predefined trajectory through a warming solution and toward the bottom of the main well compartment. The angled well extension channels 540 increase a total available travel distance over which the sample can be linearly translated during warming.

[0121] FIGS. 11A and 11B show how the sample handling end effector 310 may translate a sample on a sample support 20 through the sloped well extension channels 540 and into the well 560. In this example, warming solution 580 is contained in both the channels 540 and the main body of the wells 560. As shown, the member 310 may be moved by a robotic arm, a gantry system, etc. Alternatively, sample handling member 310 may be attached to a linear motion stage whose motion axis is parallel to the desired sample trajectory. If a shield is present, the sample may be ejected through the shield and into the well as it moves laterally using the mechanism shown in FIG. 6.

[0122] Aspects of the present disclosure have been described in detail with reference to the illustrated embodiments; those skilled in the art will recognize, however, that many modifications may be made thereto without departing from the scope of the present disclosure. The present disclosure is not limited to the precise construction and compositions disclosed herein; any and all modifications, changes, and variations apparent from the foregoingdescriptions are within the scope of the disclosure as defined by the appended claims. Moreover, the present concepts expressly include any and all combinations and subcombinations of the preceding elements and features.

[0123] Additional features and options of this disclosure may be reflected in the following clauses:

[0124] Clause 1 : a method of warming a sample in a fluid solution, the method comprising: attaching the sample onto a sample support surface of a sample support; moving the sample support to thereby align the sample support surface and the sample with an open end of a well containing the fluid solution; inserting the sample support surface with the sample into the well containing the fluid solution; and generating a relative speed between the sample and the fluid solution by: moving the sample support surface continuously along a predefined distance within the fluid solution until the sample reaches a predefined temperature; rotating and / or vibrating the sample support surface within the fluid solution until the sample reaches the predefined temperature; and / or agitating the fluid solution via a fluid agitating device while the sample support surface is within the fluid solution.

[0125] Clause 2: the method of clause 1, further comprising locating the sample support with the sample support surface inside a sample shield configured to thermally insulate the sample on the sample support surface.

[0126] Clause 3: the method of clause 2, wherein the sample shield includes a hollow cylindrical shield body, and wherein the sample support is slidable within the hollow cylindrical shield body.

[0127] Clause 4: the method of clause 3, wherein a longitudinal end of the hollow cylindrical shield body is open or defines therethrough a sample hole such that the longitudinal end is configured to pass therethrough sample support surface with the sample.

[0128] Clause 5: the method of clause 3, wherein hollow cylindrical shield body includes an outer shell and an inner lining covering some or all of an interior surface of the outer shell.

[0129] Clause 6: the method of clause 5, wherein the inner lining is formed with an absorbent material configured to hold liquid nitrogen.

[0130] Clause 7: the method of clause 3, further comprising cooling the sample shield prior to or contemporaneous with locating the sample support with the sample support surface inside the sample shield.

[0131] Clause 8: the method of any one of clauses 1 through 7, wherein inserting the sample support surface with the sample into the well includes a sample handling member mounting thereto and translating the sample support in a first direction at a first speed along a substantially linear path.

[0132] Clause 9: the method of clause 8, wherein moving the sample support surface continuously along the predefined distance within the fluid solution includes the sample handling member translating the sample support in the first direction at a second speed, greater than the first speed, along the substantially linear path.

[0133] Clause 10: the method of clause 8, wherein rotating and / or vibrating the sample support surface within the fluid solution includes a rotational actuator or a linear actuator mounting thereto and rotating or vibrating the sample support.

[0134] Clause 11 : the method of clause 8, wherein the fluid agitating device includes one or more blades configured to rotate within and thereby agitate the fluid solution.

[0135] Clause 12: the method of any one of clauses 1 through 11, wherein the sample support includes a thin film attached to a sample support frame, and wherein the sample support surface is a surface of the thin film.

[0136] Clause 13: the method of clause 12, wherein the thin film has a film thickness of between about 2 micrometers (μm) to about 100 μm.

[0137] Clause 14: the method of clause 12, wherein the thin film is curved and projects from one end of the sample support frame.

[0138] Clause 15: the method of clause 12, wherein the sample support frame includes first and second tines defining therebetween an opening, and wherein first and second edges of the thin film are attached to the first and second tines, respectively, such that the thin film spans across the opening.

[0139] Clause 16: the method of clause 15, wherein the thin film includes a third edge extending between and connecting the first and second edges of the thin film, the third edge projecting past terminal ends of the first and second tines.

[0140] Clause 17: the method of clause 15, wherein the thin film includes a third edge extending between and connecting the first and second edges of the thin film, the third edge aligned with terminal ends of the first and second tines.

[0141] Clause 18: the method of clause 12, wherein the thin film defines at least one hole extending through the thin film.

[0142] Clause 19: the method of clause 18, wherein the at least one hole includes an array of holes and / or a mesh region with multiple mesh holes.

[0143] Clause 20: the method of clause 18, wherein the at least one hole includes a plurality of mesh regions each containing multiple mesh holes.

[0144] Clause 21 : the method of clause 18, wherein the mesh regions are arranged in a line and each spaced substantially equidistant from neighboring ones of the mesh regions.

[0145] Clause 22: the method of any one of clauses 1 through 21, wherein the fluid solution is a liquid warming solution, and wherein the predefined temperature is at least about 0 degrees centigrade.

[0146] Clause 23: the method of any one of clauses 1 through 22, wherein the sample is a biological sample with a sample diameter or minimum dimension of less than about 2 millimeters.

[0147] Clause 24: the method of any one of clauses 1 through 23, the sample is a biological sample with a sample diameter or minimum dimension of less than about 500 micrometers.

[0148] Clause 25: the method of any one of clauses 1 through 23, wherein the sample is a biological sample with a sample volume of less than about 10 microliters.

[0149] Clause 26: the method of any one of clauses 1 through 23, wherein the sample is a biological sample with a sample volume of less than about 1 microliter.

[0150] Clause 27: the method of any one of clauses 1 through 29, wherein the relative speed between the sample and the fluid solution is between about 0.1 m / s and about 5 m / s, and wherein the relative speed is maintained until the sample reaches a predefined temperature.

[0151] Clause 28: the method of any one of clauses 1 through 26, wherein the predefined temperature is between about 0° and about 5° C.

[0152] Clause 29: a system for warming a cold biological sample on a sample support in a fluid solution, the system comprising: a sample support with a sample support surface configured to attach thereto the sample; a sample handling member and system controller configured to move the sample support to thereby align the sample support surface and the sample with an open end of a well containing the fluid solution and then to translate the samplesupport into and through the fluid solution at some speed relative to the fluid solution, wherein the motion of the sample support relative to the fluid solution may be obtained by translating the sample support continuously along a predefined distance within the fluid solution until the sample reaches a predefined temperature; rotating and / or vibrating the sample support within the fluid solution until the sample reaches the predefined temperature; and / or agitating the fluid solution via a fluid agitating device while the sample support surface is within the fluid solution.

[0153] Clause 30: a system for warming a sample in a fluid solution, the system comprising: a sample support with a sample support surface configured to attach thereto the sample; a sample handling member configured to move the sample support to thereby align the sample support surface and the sample with an open end of a well containing the fluid solution; a sample sliding member configured to insert the sample support surface with the sample into the well containing the fluid solution; and a system controller configured to generate a relative speed between the sample and the fluid solution by: moving the sample support surface continuously along a predefined distance within the fluid solution until the sample reaches a predefined temperature; rotating and / or vibrating the sample support surface within the fluid solution until the sample reaches the predefined temperature; and / or agitating the fluid solution via a fluid agitating device while the sample support surface is within the fluid solution.

[0154] Clause 31 : the system clause 29 or clause 30, comprising any of the elements and limitations presented in any one or more of clauses 1 through 28.

Claims

AMENDED CLAIMS received by the International Bureau on 22 April 2025 (22.04.2025)

1. A method of warming a sample in a fluid solution, the method comprising: attaching the sample onto a sample support surface of a sample support; moving the sample support to thereby align the sample support surface and the sample with an open end of a well containing the fluid solution; inserting the sample support surface with the sample into the well containing the fluid solution; and generating a relative speed between the sample and the fluid solution by: moving the sample support surface continuously along a predefined distance within the fluid solution until the sample reaches a predefined temperature; rotating and / or vibrating the sample support surface within the fluid solution until the sample reaches the predefined temperature; and / or agitating the fluid solution via a fluid agitating device while the sample support surface is within the fluid solution.

2. The method of claim 1 , further comprising locating the sample support with the sample support surface inside a sample shield configured to thermally insulate the sample on the sample support surface.

3. The method of claim 2, wherein the sample shield includes a hollow cylindrical shield body, and wherein the sample support is slidable within the hollow cylindrical shield body.

4. The method of claim 3, wherein a longitudinal end of the hollow cylindrical shield body is open or defines therethrough a sample hole such that the longitudinal end is configured to pass therethrough the sample support surface with the sample.

5. The method of claim 3, wherein the hollow cylindrical shield body includes an outer shell and an inner lining covering some or all of an interior surface of the outer shell.

6. The method of claim 5, wherein the inner lining is formed with an absorbent material configured to hold liquid nitrogen.

7. The method of claim 3, further comprising cooling the sample shield prior to or contemporaneous with locating the sample support with the sample support surface inside the sample shield.

8. The method of claim 1 , wherein inserting the sample support surface with the sample into the well includes a sample handling member mounting thereto and translating the sample support in a first direction at a first speed along a substantially linear path.

9. The method of claim 8, wherein moving the sample support surface continuously along the predefined distance within the fluid solution includes the sample handling member translating the sample support in the first direction at a second speed, greater than the first speed, along the substantially linear path.

10. The method of claim 8, wherein rotating and / or vibrating the sample support surface within the fluid solution includes a rotational actuator or a linear actuator mounting thereto and rotating or vibrating the sample support.

11. The method of claim 8, wherein the fluid agitating device includes one or more blades configured to rotate within and thereby agitate the fluid solution.

12. The method of claim 1 , wherein the sample support includes a thin film attached to a sample support frame, and wherein the sample support surface is a surface of the thin film.

13. The method of claim 12, wherein the thin film has a film thickness of between about 2 micrometers (μm) to about 100 μm.

14. The method of claim 12, wherein the thin film is curved and projects from one end of the sample support frame.

15. The method of claim 12, wherein the sample support frame includes first and second tines defining therebetween an opening, and wherein first and second edges of the thin film are attached to the first and second tines, respectively, such that the thin film spans across the opening.

16. The method of claim 15, wherein the thin film includes a third edge extending between and connecting the first and second edges of the thin film, the third edge projecting past terminal ends of the first and second tines.

17. The method of claim 15, wherein the thin film includes a third edge extending between and connecting the first and second edges of the thin film, the third edge aligned with terminal ends of the first and second tines.

18. The method of claim 12, wherein the thin film defines at least one hole extending through the thin film.

19. The method of claim 18, wherein the at least one hole includes an array of holes and / or a mesh region with multiple mesh holes.

20. The method of claim 18, wherein the at least one hole includes a plurality of mesh regions each containing multiple mesh holes.

21. The method of claim 20, wherein the mesh regions are arranged in a line and each spaced substantially equidistant from neighboring ones of the mesh regions.

22. The method of claim 1 , wherein the fluid solution is a liquid warming solution, and wherein the predefined temperature is at least about 0 degrees centigrade.

23. The method of claim 1 , wherein the sample is a biological sample with a sample diameter or minimum dimension of less than about 2 millimeters.

24. The method of claim 1 , wherein the sample is a biological sample with a sample diameter or minimum dimension of less than about 500 micrometers.

25. The method of claim 1 , wherein the sample is a biological sample with a sample volume of less than about 10 microliters.

26. The method of claim 1 , wherein the sample is a biological sample with a sample volume of less than about 1 microliter.

27. The method of claim 1 , wherein the relative speed between the sample and the fluid solution is between about 0.1 meters per second (m / s) and about 5 m / s, and wherein the relative speed is maintained until the sample reaches the predefined temperature.

28. The method of claim 1 , wherein the predefined temperature is between about 0 degrees and about 5 degrees centigrade.

29. A system for warming a cold biological sample in a fluid solution, the system comprising:a sample support with a sample support surface configured to attach thereto the sample; a sample handling member configured to move the sample support to thereby align the sample support surface and the sample with an open end of a well containing the fluid solution and then to translate the sample support into and through the fluid solution at a predefined speed relative to the fluid solution, wherein the motion of the sample support relative to the fluid solution is obtained by: translating the sample support continuously along a predefined distance within the fluid solution until the sample reaches a predefined temperature; rotating and / or vibrating the sample support within the fluid solution until the sample reaches the predefined temperature; and / or agitating the fluid solution via a fluid agitating device while the sample support surface is within the fluid solution.

30. A system for warming a sample in a fluid solution, the system comprising: a sample support with a sample support surface configured to attach thereto the sample; a sample handling member configured to move the sample support to thereby align the sample support surface and the sample with an open end of a well containing the fluid solution; a sample sliding member configured to insert the sample support surface with the sample into the well containing the fluid solution; and a system controller configured to generate a relative speed between the sample and the fluid solution by: moving the sample support surface continuously along a predefined distance within the fluid solution until the sample reaches a predefined temperature; rotating and / or vibrating the sample support surface within the fluid solution until the sample reaches the predefined temperature; and / or agitating the fluid solution via a fluid agitating device while the sample support surface is within the fluid solution.

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