System for freezing and / or vitrifying biological samples

Abstract

The present invention relates to a support for freezing or vitrifying a biological sample contained in a receptor, comprising a material having an absorption effect on low-temperature liquids, as well as a system and method comprising such a support.

Description

[Technical Field]

[0001] This application relates to a system and method for freezing and / or vitrifying biological samples. [Background technology]

[0002] Biological specimens play a vital role in biology and its applications. As living materials, they have a limited lifespan and therefore often require proper conservation.

[0003] Snap freezing is a process of rapidly cooling a substance for preservation purposes. It is widely used in the scientific industry when a sample needs to be cooled very rapidly to extremely low temperatures, such as below -70°C. This is usually achieved by immersing the sample in liquid nitrogen or a mixture of dry ice and ethanol. This method is particularly useful for freezing biomolecules such as purified proteins, as it prevents ice crystals from forming and damaging them.

[0004] For other biological samples such as cells or microtissues, slow freezing is often used, in which the sample contained in an aqueous medium is cooled below its freezing point. At some point, ice blocks containing pure crystalline water are formed. Thus, the biological material and all solutes are trapped, and the concentrations of, for example, sugars, salts, and / or cryoprotectants increase, while the volume of the unfrozen fraction decreases. The increased osmotic intensity causes water to leach out of the cells. Slow cooling is required to allow sufficient water leaching and minimize the opportunity for intracellular ice formation. As cooling continues, the viscosity of the unfrozen fraction eventually becomes too high for further crystallization. The remaining unfrozen fraction becomes an amorphous solid without ice crystals.

[0005] To avoid the problems mentioned above, it is desirable to snap-freeze biological samples such as cells or microtissues. However, due to the high heat capacity of the containers in which biological samples are typically stored or transported, the high freezing and heating rates required for snap-freezing and thawing cannot be achieved.

[0006] Therefore, one object of the present invention is to provide an improved method and system for rapid freezing and / or vitrification of biological samples.

[0007] These and other objectives are addressed by the features of the independent claims. Dependent claims disclose embodiments of the invention that may be preferred under certain circumstances. Similarly, this specification discloses further embodiments of the invention that may be preferred under certain circumstances. [Brief explanation of the drawing]

[0008] Figure 1A shows a schematic and simplified cross-section of a system for freezing and / or vitrifying biological samples according to the present invention. In the context of this specification, this system and other systems described herein are also referred to as “cryogenic boxes” or “cryogenic chambers.” The system includes a housing 40 arranged to contain a cryogenic liquid C. The housing 40 optionally includes a cover lid 14 having an opening 15 for filling with the cryogenic liquid and a support 4 for a receiver 16 suitable for and / or arranged to contain one or more biological samples.

[0009] Figure 1B shows a schematic and simplified cross-section of another system for freezing and / or vitrifying biological samples according to the present invention. The system includes a housing 40 arranged to contain a cryogenic liquid C. The housing 40 optionally includes a cover lid 14 having an opening 15 for filling with the cryogenic liquid and a support 4 for a receiver 16 suitable for and / or arranged to contain one or more biological samples.

[0010] The support 4 for the receptor 16 has an open-cell sponge-like structure and contains a material that maintains elastic deformability at low temperatures (e.g., below -70°C). In this way, it exerts a wicking effect on the cryogenic liquid. Therefore, the cryogenic liquid C contained in the housing 40 and / or the lower reservoir 12 permeates the support 4, and the receptor and its contents placed on it are cooled from the sides and from below.

[0011] The system optionally further includes a positioning grid 11 that enables reliable and reproducible positioning of the support 4 for the receptor.

[0012] Optionally, the system includes a reservoir 12 having an overflow rim 6 that forms an overflow compartment 7. The overflow rim 6 is positioned to allow cryogenic liquid to flow from the reservoir 12 into the overflow compartment 7 when the liquid level of the cryogenic liquid in the reservoir 12 exceeds the height of the overflow rim 6. For this purpose, the height of the overflow rim 6 is aligned with the height of the support 4 for the receiver.

[0013] The system optionally further includes a compartment 8 located below the reservoir 12. Compartment 8 is in fluid communication with the overflow compartment 7 and the support 4 and is suitable for and / or configured to contain cryogenic liquids.

[0014] Compartment 8 may contain a material having an open-cell sponge-like structure that maintains elastic deformability at low temperatures. In this way, it exerts an suction effect on the cryogenic liquid. Thus, the cryogenic liquid C in the overflow compartment permeates into compartment 8 and the support 4, and the receiver and its contents placed on it are cooled from the sides and from below.

[0015] Figure 1C shows a schematic and simplified cross-section of another system for freezing and / or vitrifying biological samples according to the present invention. The system comprises a first compartment 1 and a second compartment 3. The first compartment comprises an upper reservoir 2, and the second compartment 3 comprises a lower reservoir 12. Each reservoir is suitable for and / or configured to contain cryogenic liquid C. The second compartment 3 is located below the first compartment 1. The second compartment comprises a support 4 for receptors. The support 4 for receptors comprises a cavity 10 for housing the receptor well or vial. The first compartment comprises an overflow rim 5 that allows cryogenic liquid C (see arrow) to flow from the upper reservoir 2 to the lower reservoir 12 of the second compartment 3. The cryogenic liquid C in the second compartment 3 can be evaporated (evC), thus establishing a region of cold air that cools the support 4 and the receptor and its contents placed on top of it from above. The cryogenic liquid C in the lower reservoir 12 of the second compartment 3 immerses the support 4, the receiver, and its contents placed on top of it from the side.

[0016] Figure 1D shows a cross-section of another system according to the present invention for freezing and / or vitrifying biological samples, similar to the system shown in Figure 1A. In contrast, the support 4 for the receptor is placed on a spacer 24, so that the cryogenic liquid C in the lower reservoir 12 of the second compartment 3 immerses the support 4, the receptor and its contents placed on it, from the sides and below.

[0017] Figure 2 shows a cross-section of another system for freezing and / or vitrifying biological samples according to the present invention, which is similar to the system shown in Figure 1B but has greater complexity. In contrast, the support 4 for the receptor does not necessarily need to be placed on a spacer and has an open-cell sponge-like structure and contains a material that maintains elastic deformability at low temperatures (e.g., below -70°C). In this way, it exerts an suction effect on the cryogenic liquid. Thus, the cryogenic liquid C in the lower reservoir 12 of the second compartment 3 permeates the support 4, and the receptor and its contents placed on it are cooled from the sides and below.

[0018] As an optional feature, the system further includes a cover lid 14 having an opening 15 suitable for filling and / or arranged to fill the upper reservoir 2 of the first compartment 1 with a cryogenic liquid.

[0019] As an optional feature, the system further includes a side portion 13 of the upper reservoir 2 shown by a dotted line, thus forming a U-shaped or fully surrounding corridor of the upper reservoir 2.

[0020] As an optional feature, this system further includes a recess 28 in the overflow rim 5 of the first compartment, whereby a cryogenic liquid C (see arrow) can flow from the upper reservoir 2 into the lower reservoir 12 of the second compartment 3.

[0021] As an optional feature, the system further includes a positioning grid 11 that enables reliable and reproducible positioning of the support 4 for the receptacle.

[0022] As an optional feature, the second compartment 3 includes a second overflow rim 6 that forms an overflow compartment 7 within the second compartment 3. The second overflow rim 6 is arranged such that when the height of the cryogenic liquid in the lower reservoir 12 exceeds the height of the second overflow rim 6, the cryogenic liquid can flow from the lower reservoir 12 in the second compartment into the overflow compartment 7. For this purpose, the height of the second overflow rim 6 is aligned with the height of the support 4 for the receptacle.

[0023] As an optional feature, the system further includes a third compartment 8 arranged below the second compartment 3. The third compartment 8 is in fluid communication with the second compartment 3 and / or the overflow compartment 7 and is suitable for containing and / or arranged to contain a cryogenic liquid.

[0024] The third compartment 8 may contain a material having an open-cell sponge-like structure that maintains elastic deformability at low temperatures. In this way, it exerts an suction effect on the cryogenic liquid. Therefore, the cryogenic liquid C in the lower reservoir 12 of the second compartment 3 permeates into the third compartment 8 and the support 4, and the receiver and its contents placed on it are cooled from the sides and below.

[0025] The system optionally further includes a filling nozzle 25 for cryogenic liquids that is suitable for and / or positioned to fill the lower reservoir 12 of the second compartment 2 with cryogenic liquid.

[0026] Figure 3 shows a front cross-section of a system for freezing and / or vitrifying biological samples according to the present invention, similar to the system shown in Figure 2. It can be seen that the sides 13 of the upper reservoir 2 form a U-shaped corridor of the upper reservoir 2.

[0027] Figure 4 shows a perspective view of a system for freezing and / or vitrifying biological samples according to the present invention, similar to the system shown in Figure 2. An opening 15 suitable for and / or positioned to fill the upper reservoir 2 of the first compartment 1 with cryogenic liquid is clearly visible. The sides 13 of the upper reservoir 2 can be seen to form a U-shaped corridor of the upper reservoir 2 (the corridor may be entirely along the perimeter). A third compartment 8, which is in fluid communication with the second compartment 3 and / or the overflow compartment 7, is also clearly visible, as is a positioning grid 11 that allows for reliable and reproducible positioning of the support 4 for the receptor.

[0028] Figure 5 shows a perspective view of the support 4 and the receptor 16 placed on it. The receptor 16 takes the form of a microtiter plate. The support 4 has an open-cell sponge-like structure and contains a material that maintains elastic deformability at low temperatures. In this way, it exerts an suction effect on the cryogenic liquid. Thus, the cryogenic liquid C in the lower reservoir 12 of the second compartment 3 permeates the support 4, and the receptor and its contents placed on it are cooled from the sides and below. The cavities of the support 4 are positioned to be essentially fixed in shape with at least a portion of the wells of the receptor. In this way, direct contact between the wells and the cryogenic liquid is established.

[0029] Figure 6A shows a cross-section of the support 4 and the receptor 16 placed thereon. The biological sample 17 is provided in the well 9 of the receptor 16. The support 4 contains a material having an open-cell sponge-like structure that maintains elastic deformability at low temperatures. In this way, it exerts an aspiration effect on the cryogenic liquid. The cavities 10 of the support 4 are positioned to be essentially shape-fixed with at least a portion of the well of the receptor. In this way, direct contact between the well and the cryogenic liquid is established.

[0030] Figure 6B shows a cross-section of a receptor 16 having a well 9 into which a frozen and / or vitrified biological sample 17 is provided. The receptor is placed on a vent plate 18 for thawing the biological sample contained within the receptor.

[0031] The ventilation plate 18 includes a cavity 19 on its upper side that is suitable for and / or disposed to accommodate the well 9 of the receiver 16. The ventilation plate further includes at least one supply passage suitable for and / or disposed to supply the thawing medium to the outer wall of the well 9 of the receiver 16. The ventilation plate further includes at least one exhaust passage 21 suitable for and / or disposed to discharge the thawing medium from the well or the outer wall of the vial of the receiver.

[0032] Figure 7 shows a detailed cross-sectional view of a system for thawing a biological sample 17 contained in the wells 9 of a receptor 16. The apparatus includes a vent plate 18 as described in Figure 6 and pipetting means 22 suitable for and / or positioned to dispense a thawing medium 23 into the wells 9 of the receptor 16.

[0033] Figures 8A and 8B show perspective views of the ventilation plate shown in Figure 6B.

[0034] Figure 9 shows an embodiment of a system for freezing and / or vitrifying a biological sample according to the present invention in three different steps of the freezing process. A pusher 26 is used to pressurize the cryogenic liquid into the system, which then immerses the support 4 and the receptor and its contents placed thereon from the sides. The cryogenic liquid is also filled into a first compartment 1, which includes an overflow rim 5 that allows the cryogenic liquid to flow from the upper reservoir 2 to the lower reservoir of the second compartment 3. As can be seen in the figure, the upper reservoir 2 includes sides 13 that form a U-shaped corridor of the upper reservoir 2.

[0035] Figure 10(A) shows intracellular ATP measurements in HepG2 microtissue immediately after and 2 days after freezing / thawing, compared with unfreezed control microtissue. Figure 10(B) shows immunofluorescence images identifying GFP-expressing HepG2 microtissue 2 days after freezing / thawing (vitrification), and corresponding GFP signal quantification data (Figure 10(C)), compared with unfreezed control tissue. The data demonstrate that viability, morphology, and growth rate were not adversely affected by the vitrification and thawing processes.

[0036] Figure 11 shows the measured temperature gradient starting above the liquid nitrogen surface in the second compartment and reaching the first compartment, with the distance above the liquid nitrogen surface indicated in millimeters (mm). The red dotted line shows the temperature gradient achieved in the presence of the upper reservoir filled with liquid nitrogen, while the yellow line shows the temperature gradient in the absence of the upper reservoir. The placement of the upper reservoir filled with liquid nitrogen stabilizes the temperature in the second compartment up to a height of 30-45 mm above the liquid nitrogen surface and ensures a temperature 70-80°C lower up to a height of 105 mm.

[0037] Figure 12 shows a cross-section of a system for freezing and / or vitrifying biological samples according to the present invention, similar to the system shown in Figure 2. The system further includes a control nozzle 27 having an opening, the height of which is aligned with the height of the second overflow rim of the second compartment. In this way, if the liquid level of the cryogenic liquid in the overflow compartment exceeds the height of the second overflow rim, the cryogenic liquid can enter and exit the control nozzle from the overflow compartment. The line A-A' indicates the height of the second overflow rim, the height of the support for the receptor, and the height of the opening of the control nozzle.

[0038] Figure 13 shows the shape of the wells in a microtiter plate used for culturing 3D cell cultures, with a conical or tapered cross-section (Figure 13A, Akura from Insfero). TM Examples include 3D microplates and hemispherical cross-sections (Figure 13B, Corning® "Spheroid Microplate"). Figure 13C shows other possible shapes of wells in a microtiter plate. In all cases, those skilled in the art will recognize how fixation between one or more cavities in the support and the receptor wells or vials can be formed by appropriately shaping the cavities in the support.

[0039] Figure 14 shows another embodiment in which the receptor 16, provided in the form of a microtiter plate, includes a well 9 but has a flat bottom 29. In this case, the support 4 optionally includes a material having an open-cell sponge-like structure and maintaining elastic deformability at low temperatures, but does not have a cavity 10 and floats to fix the flat bottom 29 of the receptor. In this way, direct contact between the well 9 and the cryogenic liquid is established.

[0040] Figure 15 shows the results for hematoxylin and eosin (H&E) staining, as well as immunostaining, albumin (hepatocytes), BSEP (tubule structure), and CD68 (Kupffer cells) in microtissues frozen / thawed according to the present invention, compared with unfrozen microtissues (control).

[0041] Figure 16 shows the ATP content and albumin secretion of microtissues frozen / thawed according to the present invention, compared to unfrozen microtissues.

[0042] Figure 17 shows the CYP450 activity (1A2, 2B6, 2C9, 3A4) of microtissues frozen / thawed according to the present invention, compared to unfrozen microtissues (control). CYP450 activity was evaluated by 24-hour exposure to a prototype substrate and quantification of each metabolite by LC-MS.

[0043] Figure 18 shows dose-response toxicity (ATP) studies of chlorpromazine, troglitazone, and tolcapone in microtissues frozen / thawed according to the present invention, compared to unfrozen microtissues (control).

[0044] Figure 19 shows a schematic and simplified cross-section of another system for freezing and / or vitrifying biological samples according to the present invention. The system includes a pipette array 33 having tips 22 for dispensing biological samples into one or more wells of a receptor 16 or into vials 9 placed on a support 4.

[0045] The system further includes a venting system to prevent rapid freezing of the liquid sample, including the biological sample in the pipette tip 22, at the time of dispensing into the wells of the receiver 16 or vial 9. The venting system is • An outer shell 31 such that a hollow chamber 42 is formed between the housing 40 and the outer shell 31, • An intake port 32 draws in cold air from the hollow chamber 42 using an individual pump (not shown), The system includes a deflection plate 30 that can establish non-turbulent flow of a gas, such as air, immersing only the pipette tip, and that does not generate turbulence that could affect the overall temperature control within the system, including a "cold air lake (Kaltluftsee)".

[0046] Figure 20 shows a schematic and simplified cross-section of another system for freezing and / or vitrifying biological samples according to the present invention. The system includes a pipette array 33 having tips 22 for dispensing biological samples into one or more wells of a receptor 16 or into vials 9 placed on a support 4.

[0047] The system further includes a venting system to prevent rapid freezing of the liquid sample, including the biological sample in the pipette tip 22, at the time of dispensing into the wells of the receiver 16 or vial 9. The venting system is • A suction port 32 draws in cold gas (e.g., cold air) from the system via a hot air source (39, see Figure 23), The system includes an input port (34) for warm gas (e.g., warm air) provided by a warm air source (39).

[0048] Figure 21A shows a schematic and simplified cross-section of another system for freezing and / or vitrifying biological samples according to the present invention. The system includes a pipette array 33 having tips 22 for dispensing biological samples into one or more wells of a receptor 16 or into vials 9 placed on a support 4.

[0049] The system further includes a ventilation system to prevent rapid freezing of the liquid sample, including the biological sample in the pipette tip 22, at the time of dispensing into the wells of the receiver 16 or the vial 9. The ventilation system includes a flat air nozzle 36 connected to a tube heater 37 and an air pump 38 (both shown in Figure 23).

[0050] Such a flat air nozzle 36 can generate a laminar flow 41 of a warm gas, such as air, and thus ensure that the latter immerses only the pipette tips 22 of the pipette array but does not generate turbulence that could affect the overall temperature control in the system, including a "cold air lake".

[0051] Figure 21B shows an example of such a flat air nozzle 36, in which a laminar flow 41 of a warm gas, such as air, immerses only the pipette tips 22 of the pipette array 33.

[0052] Figure 22 shows the vertical temperature gradient across the first and second compartments, as measured by a temperature probe, where the upper reservoir is either empty (■) or filled with liquid N2 (●).

[0053] Figure 23 is a schematic and illustrative example. • A hot air source 39, including a hot water washing bottle and gas piping connected to a pump, is provided so that cold air is drawn in from the intake port 32 of the system shown in Figure 20, heated, and resupplied to the input port 34 of the system. As shown in Figure 21, the system includes a pipe heating device 37 and an air pump 38 connected to a flat air nozzle 36.

[0054] Figure 24A shows a receptor 16 containing wells or vials 9, each containing a frozen or vitrified biological sample 17. The wells or vials contain the volume V of the biological sample 17. s Internal volume V is at least 100 times larger than the surrounding vitrified or frozen storage medium or culture medium (if present). i Includes.

[0055] Figure 24B shows an enlarged detail of Figure 24A. The well or vial 9 has a tapered or conical shape. Its volume is given by the formula V = (1 / 3)·π·h·(r1 2 +r2 2 It can be approximated by +(r1·r2)), where h is the height of the well or vial, r1 is the lower radius of the vial, and r2 is the upper radius of the vial.

[0056] Figure 24C shows an example of a frozen or vitrified biological sample 17, including the surrounding vitrified or frozen storage medium or culture medium 43. Volume V s The formula is V = 4 / 3·π·r 3 This is approximated by the formula, where r is the radius of the biological sample 17 including the surrounding vitrified or frozen preservation medium or culture medium 43. [Modes for carrying out the invention]

[0057] Detailed explanation According to a first aspect of the present invention, a system for freezing and / or vitrifying biological samples is provided. The system includes a housing (40) arranged to contain a cryogenic liquid (C) and a support (4) for a receptor (16) suitable for and / or arranged to contain one or more biological samples.

[0058] According to this embodiment of the system, the support (4) is the support as described below.

[0059] According to one embodiment, the system includes a first compartment (1) and a second compartment (3). The first compartment includes an upper reservoir (2), and the second compartment includes a lower reservoir (12). Each reservoir is suitable for and / or configured to contain a cryogenic liquid (C), the second compartment is located below the first compartment, and the second compartment includes a support (4) for receptors suitable for and / or configured to contain one or more biological samples.

[0060] Such a system allows for the rapid freezing and / or vitrification of biological samples without forming ice crystals that could damage or affect the sample. Furthermore, it avoids the direct application of cryogenic liquid (C) to the biological sample. Such direct contact is generally difficult because it is hard to accurately dispense the cryogenic liquid (C), and when the cryogenic liquid (C) comes into contact with a warmer surface, it is hindered by splashing caused by the Leidenfrost effect. In addition, pipetting cryogenic liquid (C) directly into a vial containing a biological sample is difficult to control due to the effect of the cryogenic liquid being pushed out of the pipette tip by evaporation within the pipette, but such a system can avoid this.

[0061] With respect to embodiments of supports, systems, and / or methods described elsewhere in this specification, it will be understood that the features, characteristics, and advantages described in relation to the embodiments, as well as such embodiments, apply to the systems and embodiments described above and below, even if they are not repeated.

[0062] According to one embodiment of the system according to the present invention, the system further includes a receptor (16) suitable for and / or arranged to contain one or more biological samples.

[0063] According to one embodiment of the system according to the present invention, the upper reservoir (2) includes a first overflow rim (5) which is arranged to allow the cryogenic liquid to flow into the lower reservoir (12) when the liquid level of the cryogenic liquid in the upper reservoir (2) exceeds the height of the first overflow rim (5).

[0064] This configuration ensures that the support for the receptor is always completely immersed in the cryogenic liquid, and that a zone of cold vapor ("cold lake") is established on the cryogenic liquid, still cooling everything beneath it, including the support, the receptor and its contents placed on top of it.

[0065] According to one embodiment of the system according to the present invention, the second compartment (3) includes a second overflow rim (6) positioned to form an overflow compartment (7) within the second compartment (3), wherein the second overflow rim (6) is positioned to allow the cryogenic liquid to flow from the lower reservoir (12) into the overflow compartment (7) within the second compartment when the liquid level of the cryogenic liquid in the lower reservoir (12) exceeds the height of the second overflow rim (6).

[0066] According to one embodiment of the system according to the present invention, the height of the second overflow rim (6) is aligned with the height of the support (4) for the receptor (16). In this way, it is ensured that the well or vial in the receptor is not filled with cryogenic liquid when placed on the support.

[0067] According to one embodiment of the system according to the present invention, the system further includes a third compartment (8) located below a second compartment (3), the third compartment (8) being in fluid communication with the second compartment (3) and / or the overflow compartment (7), and being suitable for and / or arranged to contain a cryogenic liquid.

[0068] According to one embodiment of the system according to the present invention, the system further includes a cover lid (14) which includes an opening (15) suitable for and / or arranged to fill with a cryogenic liquid (C) into an upper reservoir (2) of a first compartment (1).

[0069] According to one embodiment of the system according to the present invention, the system further includes a filling nozzle (25) for cryogenic liquid that is suitable for and / or arranged to directly fill a second compartment (3) and / or an overflow compartment (7) and / or a third compartment (8) with cryogenic liquid.

[0070] According to one embodiment of the system according to the present invention, the system further includes a control nozzle (27) having an opening, the height of which is aligned with the height of the second overflow rim (6) of the second compartment (3).

[0071] In this way, if the liquid level of the cryogenic liquid in the overflow compartment (7) exceeds the height of the second overflow rim (6), the cryogenic liquid can flow from the overflow compartment (7) into and out of the control nozzle (27).

[0072] According to one embodiment of the system according to the present invention, the support (4) for the receptor (16) includes, on its upper side, one or more cavities (10) suitable for and / or arranged to accommodate one or more wells or vials (9) of the receptor (16) that are suitable for and / or arranged to accommodate a biological sample.

[0073] According to one embodiment of the system according to the present invention, one or more cavities (10) are arranged to be essentially fixed in shape with one or more wells of a receptor (16) or at least a portion of a vial (9).

[0074] According to one embodiment of the system according to the present invention, the support (4) for the receptor includes a material that has an absorption effect on low-temperature liquids.

[0075] In this way, the cryogenic liquid is directed rapidly and directly to the well or vial (9) of the receptor (16). This allows for rapid and uninterrupted cooling of the biological sample provided therein.

[0076] According to one embodiment of the system according to the present invention, the support (4) for the receptor includes a material having an open-cell sponge-like structure.

[0077] According to one embodiment of the system according to the present invention, the support (4) for the receptor comprises a material that maintains elastic deformability in the presence of a temperature below -70°C and / or a low-temperature liquid (C).

[0078] In this way, permanent close contact between one or more cavities (10) of the support (4) and the well or vial (9) of the receptor (16) is provided even under low-temperature conditions. This allows for rapid and uninterrupted cooling of the biological sample provided therein.

[0079] According to one embodiment of the system according to the present invention, the support (4) for the receptor has an absolute height of 5 to 50 mm.

[0080] In some embodiments, the support (4) for the receptor has an absolute height of 10-40 mm, 15-30 mm, or 20 mm ± 3 mm.

[0081] According to one embodiment of the system according to the present invention, the support (4) for the receptor includes a material selected from the group consisting of the following: • Melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin, • Polyurethane, and / or • Cross-linked polyolefin foam.

[0082] The inventors have discovered that, surprisingly, these materials are a) As described above, it has an absorption effect on low-temperature liquids, b) It was demonstrated that the elastic deformability is maintained at temperatures below -70°C and / or in the presence of low-temperature liquids.

[0083] As mentioned above, both features allow for rapid and uninterrupted cooling of the biological sample provided within.

[0084] The UK Patent Application Publication No. 2377985A discloses a disposable container for refrigerated animal / human tissue or fluid. The device includes a liquid refrigerant (liquid nitrogen) absorbent material having one or more pockets for receiving a container for a biological sample, such that the refrigerant is in close proximity to the container.

[0085] The liquid absorbent material is preferably a foamed material or sponge, preferably a foam commonly known as Oasis®, which is typically used by florists as a base for flower arrangements. English Patent Application Publication No. 2377985A states that this material is particularly advantageous because, once a pocket is formed, the pocket retains its shape. This then means that, unlike the preferred material in the present invention, the foamed material of English Patent Application Publication No. 2377985A is plastically deformable (i.e., once deformed, retains its shape) and not elastically deformable.

[0086] The inventors of the present invention have experimentally confirmed that the Oasis® material, disclosed as preferred in UK Patent Application Publication No. 2377985A, is not elastically deformable at room temperature or under low-temperature conditions (e.g., -196°C). This means that, unlike each embodiment of the present invention, such material does not provide permanent adhesion between the cavity ("pocket") of the support and the well or vial ("container") of the receptor, and therefore does not allow for rapid and uninterrupted cooling of the biological sample provided therein.

[0087] The inventors further suggest that, for example, melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin may be used. a) Has an suction effect on low-temperature liquids, b) We experimentally confirmed that the elastic deformability is maintained at temperatures below -70°C and / or in the presence of low-temperature liquids.

[0088] According to one embodiment of the system according to the present invention, the third compartment (8) includes a material having at least one of the following properties: a) Absorption effect on low-temperature liquids, b) Open-cell sponge-like structure, c) Maintaining elastic deformability in the presence of a temperature below -70°C and / or a low-temperature liquid, and / or d) Includes a material selected from the group consisting of the following: • Melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin, • Polyurethane, and / or • Cross-linked polyolefin foam.

[0089] According to one embodiment of the system according to the present invention, the cryogenic liquid is selected from the group consisting of the following: • Liquid nitrogen, • Liquid helium, • Liquid argon, • Liquid oxygen, A mixture of dry ice (CO2) and an organic solvent.

[0090] Preferably, the organic solvent to be mixed with dry ice is acetone and / or ethanol.

[0091] According to one embodiment of the system according to the present invention, the support (4) for the receptor is placed on one or more spacers (24). In this way, the cryogenic liquid can also immerse the support from below.

[0092] According to one embodiment of the system according to the present invention, the receptor (16) includes one or more wells or vials (9) suitable for and / or arranged to contain a biological sample.

[0093] According to one embodiment of the system according to the present invention, the receptor (16) is an array of microtiter plates or vials corresponding to a 6, 24, 96, or 384 microtiter plate arrangement.

[0094] As discussed herein, one or more cavities (10) of the support (4) are preferably positioned to be essentially shape-fixed with one or more wells of the receptor (16) or at least a portion of the vial (9).

[0095] In the context of the present invention, different types of microtiter plates can be used, including conventional microtiter plates containing tapered, conical, cylindrical, or hemispherical wells, as well as plates specifically made for culturing microspheres, as described in Larsen B (2015), the contents of which are incorporated herein by reference for the purpose of enabling the invention.

[0096] Such plates include, for example, the "Akura" manufactured by Insfero, which has a tapered cross-section. TM It may include wells with more sophisticated shapes, such as "3D microplates" (see Figure 13A) or Corning®'s "spheroid microplates" (see Figure 13B).

[0097] Figure 6A shows the principle of shape fixation between one or more cavities (10) of the support (4) and the well or vial (9) of the receptor (16). A person skilled in the art can adapt this principle to other receptors having different shapes of their wells or vials (see Figure 13), or even embodiments in which the receptor has a flat bottom (see Figure 14).

[0098] According to another embodiment of the present invention, the system further includes pipetting means (22, 33) for dispensing a biological sample (17) into one or more wells or vials (9) of a receptor (16).

[0099] According to another embodiment of the present invention, the system further includes aeration means (30, 32, 34, 36, 37, 38, 39) for generating a warm gas flow along the tip (22) of the pipette means (33).

[0100] Such ventilation means may optionally include at least one element selected from the group consisting of the following: · Deflection plate (30), • Air intake port (32), • Air intake port (34), • Flat air nozzle (36), · Tube heating device (37), • Air pump (38) · Hot air source (39).

[0101] Such aeration means prevent the liquid sample containing the biological sample (17) in the pipette tip (22) from freezing rapidly when it is dispensed into the well of the receptor (16) or the vial (9). The important thing is to ensure that such an effect is contained and maintained locally, as shown in the figure, for example.

[0102] Such a flat air nozzle (36) can generate a laminar flow (41) of a warm gas, such as air, and thus ensure that the latter only immerses the pipette tip but does not generate turbulence that could affect the overall temperature plan in the system, including a "cold air lake".

[0103] A similar effect can be achieved by a deflection plate (30) that generates non-turbulent flow of a gas (41), such as air.

[0104] Furthermore, this aspect of the present invention, i.e., the system, is subject to the preferred embodiments and their characteristics discussed elsewhere in this specification, with necessary modifications.

[0105] According to another aspect of the present invention, a support (4) for freezing or vitrifying a biological sample contained in a receptor (16) is provided. The support comprises a material having an absorption effect on the cryogenic liquid.

[0106] According to one embodiment of the support according to the present invention, the cryogenic liquid is selected from the group consisting of the following: • Liquid nitrogen, • Liquid helium, • Liquid argon, • Liquid oxygen, A mixture of dry ice (CO2) and an organic solvent.

[0107] The following table shows the boiling points of appropriate low-temperature liquids.

[0108] [Table 1]

[0109] Preferably, the organic solvent to be mixed with dry ice is acetone and / or ethanol.

[0110] According to one embodiment of the support according to the present invention, the receptor is suitable for and / or arranged to contain one or more biological samples.

[0111] According to one embodiment of the support according to the present invention, the support comprises a material having an open-cell sponge-like structure. In one embodiment, the support comprises an open-cell three-dimensional fiber network structure.

[0112] In one embodiment, the support includes an open-cell three-dimensional fiber network structure.

[0113] According to one embodiment of the support according to the present invention, the support comprises a material that maintains elastic deformability at temperatures below -70°C and / or in the presence of a low-temperature liquid. In this way, proper shape fixation with the receptor is achieved over a wide temperature range.

[0114] According to one embodiment of the support according to the present invention, the support has an absolute height of 5 to 50 mm.

[0115] In some embodiments, the support has an absolute height of 10-40 mm, 15-30 mm, or 20 mm ± 3 mm.

[0116] This height range has proven feasible because it satisfies two constraints: a) it is not so high as to adversely affect the suction effect that draws the cryogenic liquid into the support, and b) it is high enough to allow a sufficiently high liquid level of the cryogenic liquid provided within the compartment containing the support without causing the receptor to float.

[0117] According to one embodiment of the support according to the present invention, the support comprises a material selected from the group consisting of the following: • Melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin, • Polyurethane, and / or • Cross-linked polyolefin foam.

[0118] These materials satisfy the above-mentioned requirements regarding suction effect and elastic deformability at low temperatures.

[0119] Melamine and its resins are most commonly known for their use in tableware, laminate flooring, and whiteboards. Melamine foam is used as an insulating and soundproofing material, and in polymer cleaning products such as the so-called "Magic Eraser."

[0120] To our surprise, the inventors have found that melamine and melamine resin have the following desirable properties as materials used in the support according to the present invention: • Maintaining elastic deformability at temperatures below -70°C and / or in the presence of low-temperature liquids. • It can be provided to have an open-cell sponge-like structure. • And, it can be provided in a manner that has an absorption effect on low-temperature liquids.

[0121] Cross-linked polyolefin foams have been found to be suitable materials for cryogenic containers. Several characteristics of polyolefin foams are advantageous for use as cryogenic containers: they can be easily manufactured into suitable shapes by molding, casting, or otherwise forming foams from monomers or polymers, or by machining finished blocks; their cellular structure is typically fine enough to contain, for example, liquid nitrogen without leakage; they are durable enough to withstand repeated exposure to low temperatures; they are largely non-reactive; and they typically possess both low thermal conductivity and low volumetric heat capacity. As used herein, the term “polyolefin foam” refers to polyethylene foams, polypropylene foams, polyethylene-polypropylene mixtures and copolymers, as well as foams containing olefin monomers and mixtures or copolymers of other monomers, where mixed foams possess at least some of the preferred properties described above.

[0122] According to one embodiment of the support according to the present invention, the support includes, on its upper side, one or more cavities (10) suitable for and / or arranged to accommodate one or more wells or vials (9) of a receptor (16) suitable for and / or arranged to accommodate a biological sample.

[0123] According to one embodiment of the support according to the present invention, one or more cavities (10) are arranged to be essentially shape-fixed with at least a portion of one or more wells or vials (9). Such shape-fixing between one or more cavities (10) of the support (4) and the wells or vials (9) of the receptor (16) is shown in Figure 6A. Those skilled in the art can adapt this principle to other receptors having wells or vials of different shapes.

[0124] According to one embodiment of the support according to the present invention, the receptor (16) is an array of microtiter plates or vials corresponding to a 6, 24, 96, or 384 microtiter plate arrangement.

[0125] In another embodiment, the receptor (16) comprises a well or vial (9) and further has a flat bottom (29). In this case, the support (4) optionally comprises a material having an open-cell sponge-like structure and maintaining elastic deformability at low temperatures, but without a cavity (10), and floats to fix the flat bottom (29) of the receptor (16). In this way, direct contact between the well (9) and the cryogenic liquid is established.

[0126] According to another aspect of the present invention, a venting plate (18) for thawing a biological sample contained in a receptor (16) is provided. The venting plate includes, on its upper side, one or more cavities (19) suitable for and / or arranged to accommodate one or more wells or vials (9) of a receptor suitable for and / or arranged to accommodate a biological sample.

[0127] The ventilation plate further, • One or more supply channels (20) suitable for and / or arranged to supply a thawing medium to the outer wall of the receptor well or vial (9), and • Includes one or more exhaust passages (21) suitable for and / or configured to discharge the thawing medium from the well or outer wall of the receptor or vial (9).

[0128] According to one embodiment of the ventilation plate (18) of the present invention, the thawing medium (23) is a fluid, liquid, or gas.

[0129] According to one embodiment, the thawing medium (23) is air.

[0130] According to one embodiment of the ventilation plate according to the present invention, the thawing medium (23) has a temperature of -150°C to +100°C.

[0131] According to some embodiments, the thawing medium has a temperature of 4°C to +100°C, 15°C to +90°C, preferably 20°C to +37°C.

[0132] According to another aspect of the present invention, a system for thawing a biological sample contained in a receptor (16) is provided. This system is a) The ventilation plate (18) described above, and b) Includes a pipette means (22) suitable for and / or configured to dispense a thawing medium (23) into the wells of a receptor (16) or a vial (9).

[0133] According to one embodiment of the thawing system of the present invention, the thawing medium is a fluid, liquid, or gas.

[0134] According to one embodiment of the thawing system of the present invention, the thawing medium used in the ventilated plate is a gas, preferably air, in terms of the suitable temperature, as described above.

[0135] According to one embodiment of the thawing system of the present invention, the thawing medium used in the pipette system is a liquid, preferably an aqueous liquid. According to some embodiments, the thawing medium used in the pipette system has a temperature of 4°C to +80°C, 15°C to +70°C, preferably 20°C to +37°C.

[0136] According to further embodiments of the system, ventilation plate (18), or support (4) of the present invention, the biological sample is at least one selected from the group consisting of the following: • One or more tissue samples, • One or more types of germ cells (oocytes or spermatocytes), • One or more types of blastocysts, • One or more individual cells, • One or more types of cell aggregates (e.g., microtissues, spheroids), and / or • One or more organoids derived from stem cells through self-organization and differentiation.

[0137] In one embodiment, the biological sample is contained in a liquid, such as a storage medium or culture medium. In such a case, the biological sample is described as being contained in a liquid sample.

[0138] According to one embodiment, the biological sample does not include a bio-artificial scaffold, i.e., it is scaffold-free. In one embodiment, the biological sample is a scaffold-free microtissue, spheroid, or organoid.

[0139] Such scaffolding materials are disclosed, for example, by Carletti et al. (2011) and include natural polymers (e.g., collagen, chitosan, glycosaminoglycans, silk fibroin, agarose, alginates, and starches) and synthetic polymers (e.g., poly(glycolic acid), poly(lactic acid), and copolymers, poly(e-caprolactone), polyurethane, poly(orthoesters), and poly(anhydrides)). Such scaffolding materials are often used to form microtissues, but they can adversely affect the results of assays in which the microtissues are used.

[0140] According to another aspect of the present invention, a method for freezing and / or vitrifying a biological sample is provided. This method includes the following steps: a) Installing a receptor (16) on the support (4) as described above, which is suitable for and / or arranged to contain one or more biological samples, wherein the support is arranged within the system as described above. b) Before or after step a), dispense one or more biological samples (17) into one or more wells or vials (9) of the receptor. c) Fill the system with cryogenic liquid as described above, either before or after step a) or b).

[0141] According to one embodiment, the method further includes the step of adding one or more cryoprotective agents (CPAs) to the biological sample prior to step b).

[0142] According to one embodiment, the method includes sequentially equilibrating a biological sample with two or three different increasing concentrations of CPA.

[0143] In some embodiments, the cryoprotectant comprises at least one of the following: dimethyl sulfoxide (DMSO), and / or one or more alcohols, sugars, amides or imides, and / or polymeric compounds. Here, the alcohol is selected from methanol, glycerol, sorbitol, ethylene glycol, propylene glycol, butylene glycol, inositol, xylitol, mannitol, adonitol, arabitol, ribitol, erythritol, perseitol, treitol, galactitol, pinitol, xylitol, and combinations thereof, and the sugar is sucrose, trehalose, maltose, arabinose, lactose, mannose, xylose, galactose, fructose, glucose, dextran, melegitose, raffinose, nigerotriose, maltotriose The polymers are selected from maltotriose, maltotriulose, kestose, cellobiose, chitobiose, lactulose, and combinations thereof; the amides and imides are selected from formamide, acetamide, propionamide, lactamide, butylamide, malonamide, and combinations thereof; and the polymers are selected from Ficoll, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, hyaluronic acid, and combinations thereof. For further information, see Whaley et al. (2021), the contents of which are incorporated herein by reference for the purpose of enabling implementation.

[0144] According to one embodiment, dispensing one or more biological samples (17) into one or more wells or vials (9) of a receptor is performed. i) Using a pipette array (33) containing one or more pipettes or one or more pipette tips (22), to remove one or more biological samples from a plate containing biological samples. ii) Allowing one or more biological samples to settle at the bottom of one or more pipette tips (22), and iii) Dispensing one or more biological samples from a pipette tip (22) into one or more wells or vials (9) of a receptor by releasing droplets containing the biological samples.

[0145] Biological samples, even if they precipitate, can be arbitrarily surrounded by a liquid medium.

[0146] According to one embodiment of the method, only one biological sample (e.g., only one microtissue) is dispensed into each receptor well or vial (9).

[0147] According to one embodiment of the method, the droplet containing the biological sample has a volume of 0.05 ml to 8 ml. According to a further embodiment of the method, the droplet containing the biological sample has a volume of about 0.1 ml, about 0.2 ml, about 0.3 ml, about 0.4 ml, about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about 0.9 ml, about 1 ml, about 1.2 ml, about 1.4 ml, about 1.5 ml, about 1.6 ml, about 1.8 ml, about 2 ml, about 2.2 ml, about 2.4 ml, about 2.6 ml, about 2.8 ml, about 3 ml, about 3.2 ml, about 3.4 ml, about 3.6 ml, about 3.8 ml, or about 4 ml.

[0148] Some embodiments of the present invention ensure that during the dispensing step, the sample containing the biological sample does not freeze in the pipette tip (22), but the latter is brought close to one or more wells or vials (9) of a receptor, all of which are cooled due to the presence of the cryogenic liquid. Thus, freezing / vitrification begins immediately only after the sample has been dispensed into its well or vial.

[0149] According to another aspect of the present invention, a method for storing a biological sample frozen and / or vitrified in the manner described above is provided. This method includes the following steps: a) A receptor (16) containing one or more frozen and / or vitrified biological samples is kept within the system as described above. b) Optionally, place a cover (14) on top of the system. c) Optionally, replenish the system with cryogenic liquid as described above.

[0150] According to another aspect of the present invention, there is provided a method for thawing a biological sample frozen and / or vitrified by the above-described method. This method includes the following steps: a) Removing the receptacle (16) from the support (4); b) Placing the receptacle (16) on the ventilation plate (18) as described above; c) Supplying a thawing medium to the ventilation plate (18); d) Optionally, simultaneously with step c), or continuously before or after step c), dispensing the thawing medium from pipetting means (22) disposed on the receptacle (16).

[0151] According to one embodiment of the method according to the present invention, the thawing medium is a fluid, liquid or gas.

[0152] According to one embodiment of the method according to the present invention, the thawing medium used for the ventilation plate is also a gas with respect to a suitable temperature as described above, preferably air.

[0153] According to one embodiment of the method according to the present invention, the thawing medium used in the pipette system is a liquid, preferably an aqueous liquid. According to some embodiments, the thawing medium used in the pipette system has a temperature of 4°C to +80°C, 15°C to +70°C, preferably 20°C to +37°C.

[0154] According to one embodiment, the method further includes e) replacing the preheated thawing medium one or two times with a medium at room temperature.

[0155] According to another aspect of the present invention, there is provided a receptacle (16) containing one or more wells or vials (9) containing one or more biological samples (17) frozen or vitrified. At least one of the wells or vials has a volume V of the biological sample (17) sInternal volume V (including surrounding vitrified or frozen storage medium or culture medium, if present) is at least 50 times larger than the surrounding vitrified or frozen storage medium or culture medium. i Includes. According to the embodiment, internal volume V i This is the volume V of the biological sample (17). s It is at least 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 150, or 200 times larger than the given value.

[0156] In this way, rapid freezing or vitrification of the sample is ensured, for example, without the formation of harmful ice crystals. Dispensing the thawing medium into the receptor containing the frozen biological sample further ensures that the preservation medium surrounding the biological sample is sufficiently diluted. This can be beneficial in mitigating potentially harmful effects of medium components on the biological sample, such as those provided by certain cryoprotective agents (present in the medium), such as DMSO.

[0157] In many cases, biological samples, such as cells, cell aggregates or clusters, or microtissues, take on a more or less spheroidal form (including the surrounding vitrified or frozen culture medium, if present). In this case, the volume is V = 4 / 3·π·r 3 This can be approximated by r, where r is the radius of the biological sample (see Figure 24C).

[0158] If the well or vial is cylindrical, its volume is given by the formula V = π·r 2 It can be approximated by h, where r is the diameter of the circular cross-section and h is the height of the well or vial.

[0159] If the well or vial is tapered or conical, its volume is given by the formula V = (1 / 3)·π·h·(r¹ 2 +r2 2 It can be approximated by +(r1·r2)), where h is the height of the well or vial, r1 is the lower radius of the vial, and r2 is the upper radius of the vial.

[0160] If the well or vial is hemispherical, its volume is V = 2 / 3·π·r 3It can be approximated by the following equation, where r is the radius of the well or vial.

[0161] With regard to embodiments of receptors, vials or wells, or biological specimens described elsewhere in this specification, it is understood that the features, properties and advantages discussed in connection with such embodiments, as well as such embodiments, apply to the receptors and embodiments discussed above and below, even if they are not repeated.

[0162] In one embodiment, the receptor is, for example, Akura TM This is a 96-well plate, such as a 96-well plate (manufactured by Insfero). In such embodiments, the wells typically have a volume of 100 μl to 400 μl. In such embodiments, V i ga V s If it is at least 100 times larger than the specified size, the biological sample has a maximum volume of, for example, 1 μl to 4 μl (including the surrounding vitrified or frozen storage medium or culture medium, if present).

[0163] In one embodiment, the receptor is a 384-well plate. In such an embodiment, the wells typically have a volume of 80 μl to 200 μl. In such an embodiment, V i ga V s If at least 100 times larger than the specified size, the biological sample has a maximum volume of 0.8 μl to 2 μl (including the surrounding vitrified or frozen storage medium or culture medium, if present).

[0164] In one embodiment, the receptor is a 1536-well plate. In such an embodiment, the wells typically have a volume of 10 μl to 200 μl. In such an embodiment, V i ga V s If it is at least 100 times larger than the specified value, the biological sample has a maximum volume of 0.1 μl to 2 μl (including the surrounding vitrified or frozen storage medium or culture medium, if present).

[0165] The following table shows some further non-limiting examples:

[0166] [Table 2]

[0167] According to one embodiment, the receptor is manufactured or available by the method described above. For such purposes, one or more biological samples (17) are dispensed into one or more wells or vials (9) of the receptor (16) as described above.

[0168] According to one embodiment of the method, each receptor well or vial (9) contains only one biological sample (e.g., only one microtissue). [Examples]

[0169] Although the present invention is illustrated and described in detail in the drawings and the foregoing description, such illustrations and descriptions should be considered illustrative or exemplary and not limiting, and the present invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and achieved by those skilled in the art in carrying out the claimed invention, from a consideration of the drawings, disclosure and appended claims. In the claims, the term “including” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude plurals. The mere fact that certain means are described in different dependent claims does not imply that combinations of these means cannot be used advantageously. No reference numeral in the claims should be construed as limiting.

[0170] material and method 1. The method itself 1.1. Vitrification Method Akura TMeGFP-expressing human tumor microtissue (HepG2 cells) formed and cultured in a 96-well plate (Insfero) was removed from an incubator (37°C, 5% CO2) and placed in designated grooves on the deck of a Hamilton Vantage Solution Apparatus (Hamilton, Bonadus, Switzerland). The cell culture medium was aspirated from one column of the plate and replaced with a medium containing a specified mixture of cryoprotective agents (CPAs) selected from a list of cell-permeable and cell-impermeable components (see Swain and Smith 2010, the contents of which are incorporated herein by reference for the purpose of enabling the procedure). This process was repeated twice, increasing the concentration of CPA. Each medium composition was allowed to stand on the microtissue for 3 minutes to equilibrate the microtissue and medium. After the third equilibration step, the microtissue was collected using an array of eight Hamilton MagPip® pipette heads and settled at the bottom of the tips.

[0171] Another Akura TM A 96-well plate was pre-cooled to below -190°C in a cryogenic chamber according to the present invention and then cooled with liquid nitrogen. After equilibration and precipitation of microtissues, the lid of the cryogenic chamber was briefly opened to allow placement of the microtissues onto the plate in the smallest medium volume in the range of low microliters or submicroliters. Dispensing the smallest medium volume onto the pre-cooled plate resulted in immediate and rapid snap freezing of the medium and vitrification of the microtissues. To maintain the low temperature, the cryogenic chamber was closed again, and the process was repeated using additional columns from the same source plate.

[0172] 1.2. How to unzip To thaw the plates, an array of three re-equilibriumated media with decreasing CPA concentrations (see Swain and Smith 2010) was used before replacing the medium with standard cell culture medium. The first medium used was preheated to a temperature above ambient level. The frozen plates were automatically lifted from the cryogenic chamber and placed on a vented plate holder (vented plate) according to the present invention, which directed pressurized air towards the bottom of the plate to dissipate the cold air around the plate and its wells. The preheated medium was added synchronously to all 96 wells of the plate using a 96-tip pipette head (Hamilton), resulting in immediate thawing of the plate and microtissues, minimizing damage from potential ice crystal formation while the plate, medium, and microtissues were heated to a temperature above their freezing point.

[0173] After 1 minute, the preheated medium was removed from the plate and replaced with the same medium at room temperature, and this washing step was repeated after another minute. Subsequently, re-equilibrium mediums 2 and 3 were added for 2 minutes each, and the medium was further refreshed after the first minute of equilibration. Finally, the medium was replaced with standard tissue culture medium and placed in an incubator at 37°C and 25% CO2.

[0174] 2. Experiment Microtissues without pre-formed scaffolds are equilibrated in a specified mixture of cryoprotectant (CPA) in an equilibration process involving increasing CPA concentration, for example, while the receptor is pre-cooled to -196°C in liquid nitrogen in a cryogenic chamber. The microtissue is then placed in droplets (0.3–1.0 ml or 0.5–1.0 ml volume) onto the receptor (e.g., a 96-well microtissue plate; one microtissue per well) using Hamilton's MagPip pipetting technique. The droplets containing the microtissue freeze immediately upon contact with the cold surface. This method allows for the rapid cooling required for aqueous samples to obtain an amorphous, glassy state (vitrification). Vitrification transforms the aqueous solution into an amorphous, glassy state, preventing cell damage associated with ice crystal formation, as typically observed with conventional freezing methods. The tissue can then be stored in a liquid N2 gas phase below 150°C for long-term preservation.

[0175] For thawing, a preheated thawing medium is applied to the receiver from the top. In parallel, the use of a ventilated plate allows heating from the bottom, enabling a rapid transition from the vitrified state to the liquid state and minimizing the opportunity for harmful ice crystals to form again.

[0176] This method has been tested using pre-formed liver microtissues produced from primary human hepatocytes and non-parenchymal hepatocytes (see Proctor et al. (2017), the contents of which are incorporated herein by reference for the purpose of enabling the procedure). Microtissues were frozen in standard 96-well microwell plates using the cryo chamber described above. After thawing using a vented plate, the microtissues were compared with unfrozen control microtissues. Morphologically, they showed compact, round liver microtissues similar to their unfrozen counterparts. H&E staining and immunostaining for albumin (hepatocytes), BSEP (tubule structures), and CD68 (Kupffer cells) showed patterns similar to those of the unfrozen microtissues (see Figure 15).

[0177] The ATP content of a single microtissue was 85%–75% of that of the unfrozen control tissue on days 2 and 7 after thawing (see Figure 16A). The thawed and control microtissues were very similar in their albumin secretion (see Figure 16B), CYP450 activity (see 1A2, 2B6, 2C9, 3A4, Figure 17), and responsiveness to the application of three known hepatotoxic compounds (see Figure 18), as shown in the 7-day repeated dose-response toxicity study (ATP) for chlorpromazine, troglitazone, and tolcapone. Similar IC50 values ​​were observed for both the thawed and control microtissues (see also Messner et al. (2013), whose contents are incorporated herein by reference for the purpose of enabling the practice).

[0178] These results demonstrate the feasibility of cryopreserving highly differentiated spheroids based on primary cells in standard microplates, enabling long-term storage without loss of function, and demonstrating that these thawed spheroids can be directly used in drug safety testing. [Explanation of Symbols]

[0179] 1. First section 2. Upper reservoir 3. Second section 4. Support for receptors 5. Overflow rim 6. (Second) Overflow Rim 7. Overflow compartment 8. (Third) section 9. One or more wells or vials 10. One or more cavities 11. Positioning grid 12. Lower Reservoir 13. Side of the upper reservoir 14. Cover Lid 15. Opening for cryogenic liquid filling 16. Receptors 17. Biological samples 18. Ventilation plate 19. Cavities in the ventilation plate 20. Supply route for thawing medium 21. Exhaust passage 22. Pipette tips 23. Decompression medium 24. Spacer 25. Filling nozzle for cryogenic liquids 26. Pusher 27. Control nozzle 28. Recess of the overflow rim 29. Flat bottom of the microtiter plate 30. Deflection plate 31. Outer shell 32. Intake port (cold air) 33. Pipette array 34. Input port (warm air) 35. Plug to seal opening 15 36. Flat air nozzle 37. Tube heating device 38. Air pump 39. Hot air source 40. Enclosure 41. Laminar / Non-turbulent flow of gases 42. Hollow chamber formed between the housing 40 and the outer shell 31 43. Vitrified or frozen storage medium or culture medium surrounding the biological sample 17 V i Internal volume of well or vial 9 V s Surrounding vitrified or frozen storage medium or culture medium 43 Volume of biological sample 17 including (if present) C Cryogenic liquid evC: Evaporated cryogenic liquid A-A' Lines indicating the height of the second overflow rim (6), the height of the support for the receptor (4), and the height of the opening of the control nozzle (27).

[0180] References Jason E. Swain, Gary D. Smith, Ri-Cheng Chian, and Patrick Quinn (eds.), "4. Cryoprotective Agents from Cryobiology" in *Cryopreservation in Infertility Treatment*, pp. 24-38, Cambridge University Press (2010). Whaley D, Damyar K, Witek RP, Mendoza A, Alexander M, Lakey JR. Cryopreservation: An Overview of Principles and Cell-Specific Considerations. Cell Transplantation. January-December 2021; 30 Larsen B, 3D Cell Culture: A Review of Current Techniques. Agilent White Paper, November 12, 2015. https: / / www.biotek.com / resources / white-papers / 3d-cell-culture-a-review-of-current-techniques Carletti E, Motta A, Migliaresi C. Scaffolds for tissue engineering and 3D cell culture. Methods Mol Biol. 2011;695:17-39. William R. Proctor, Alison J. Foster, Jennifer Vogt, Claire Summers, Brian Middleton, Mark A. Pilling, Daniel Shienson, Monika Kijanska, Simon Stroebel, Jens M. Kelm, Paul Morgan, Simon Messner, Dominic Williams. Usefulness of globular human liver microtissue for predicting clinical drug-induced liver injury. Arch Toxicol (2017) 91:2849-2863 S Messner, I Agarkova, W Moritz, JM Kelm. Multicellular human liver microtissue for hepatotoxicity studies. Arch Toxicol. 2013;87:209-13.

Claims

1. A system for freezing and / or vitrifying a biological sample (17), comprising a housing (40) arranged to contain a cryogenic liquid (C), and a support (4) for a receptor (16) suitable for and / or arranged to contain one or more biological samples (17).

2. The system according to claim 1, further comprising a first compartment (1) and a second compartment (3), The first section (1) includes the upper reservoir (2), The second section (3) includes the lower reservoir (12), Each reservoir (2, 12) is suitable for and / or arranged to contain a cryogenic liquid (C), The second section (3) is located below the first section (1), and The second compartment is a system comprising a support (4) for a receptor (16) suitable for and / or arranged to contain one or more biological samples (17).

3. The system according to claim 1 or 2, further comprising a receptor (16) suitable for and / or arranged to contain one or more biological samples (17).

4. The system according to any one of claims 1 to 3, wherein the upper reservoir (2) includes a first overflow rim (5), the first overflow rim (5) is arranged to allow the cryogenic liquid (C) to flow into the lower reservoir (12) when the height of the cryogenic liquid (C) in the upper reservoir (2) exceeds the height of the first overflow rim (5).

5. The system according to any one of claims 1 to 4, wherein the second compartment (3) includes a second overflow rim (6) positioned to form an overflow compartment (7) within the second compartment (3), the second overflow rim (6) being positioned to allow cryogenic liquid (C) to flow from the lower reservoir (12) into the overflow compartment (7) within the second compartment when the height of cryogenic liquid (C) in the lower reservoir (12) exceeds the height of the second overflow rim (6).

6. The system according to any one of claims 1 to 5, wherein the height of the second overflow rim (6) is aligned with the height of the support (4) for the receptor (16).

7. The system according to any one of claims 1 to 6, further comprising a third compartment (8) located below the second compartment (3), the third compartment (8) being in fluid communication with the second compartment (3) and / or the overflow compartment (7), and being suitable for and / or arranged to contain a cryogenic liquid (C).

8. The system according to any one of claims 1 to 7, further comprising a cover lid (14) having a first opening (15) suitable for and / or arranged to fill with a cryogenic liquid (C) into the upper reservoir (2) of the first compartment (1).

9. The system according to any one of claims 1 to 8, further comprising a filling nozzle (25) for the cryogenic liquid (C) that is suitable for and / or positioned to fill the second compartment (3) and / or the overflow compartment (7) and / or the third compartment (8) with the cryogenic liquid (C).

10. The system further includes a control nozzle (27) having an opening, wherein the height of the opening of the control nozzle is aligned with the height of the second overflow rim (6) of the second compartment (3), according to any one of claims 1 to 9.

11. The system according to any one of claims 1 to 10, wherein the support (4) for the receptor (16) includes, on its upper side, one or more cavities (10) suitable for and / or arranged to accommodate one or more wells or vials (9) of the receptor (16) that are suitable for and / or arranged to accommodate a biological sample.

12. The system according to any one of claims 1 to 11, wherein one or more cavities (10) are arranged to be essentially shape-fixed with one or more wells of a receptor (16) or at least a portion of a vial (9).

13. The system according to any one of claims 1 to 12, wherein the support for the receptor (4) includes a material that has an absorption effect on low-temperature liquids.

14. The system according to any one of claims 1 to 13, wherein the support for the receptor (4) comprises a material having an open-cell sponge-like structure.

15. The system according to any one of claims 1 to 14, wherein the support (4) for the receptor comprises a material that maintains elastic deformability in the presence of a temperature below -70°C and / or a low-temperature liquid (C).

16. The system according to any one of claims 1 to 15, wherein the support for the receptor (4) has an absolute height of 5 to 50 mm.

17. The system according to any one of claims 1 to 16, wherein the support for the receptor (4) comprises a material selected from the group consisting of the following: Melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin, • Polyurethane, and / or Cross-linked polyolefin foam.

18. The third compartment (8) is the system according to any one of claims 1 to 17, comprising a material having at least one of the following properties: a) Absorption effect on low-temperature liquids, b) Open-cell sponge-like structure, c) Maintaining elastic deformability in the presence of temperatures below -70°C and / or low-temperature liquids, and / or d) Includes a material selected from the group consisting of the following: Melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin, • Polyurethane, and / or Cross-linked polyolefin foam.

19. The cryogenic liquid (C) is selected from the group consisting of the following, in the system according to any one of claims 1 to 18: • Liquid nitrogen, • Liquid helium, • Liquid argon, • Liquid oxygen, A mixture of dry ice and organic solvent.

20. The system according to any one of claims 1 to 19, wherein the support (4) is the support according to any one of claims 13 to 18.

21. The system according to any one of claims 1 to 20, further comprising pipetting means (22, 33) for dispensing a biological sample into one or more wells of a receptor (16) or vials (9).

22. The system according to claim 23, further comprising aeration means (30, 32, 34, 36, 37, 38, 39) for generating a flow of warm gas along the pipette tip (22) of the pipette means (33).

23. The ventilation means comprises at least one element selected from the group consisting of the following, according to claim 24. ・ Deflection plate (30), - Air intake port (32), - Air input port (34), - Flat air nozzle (36), ・Pipe heating device (37), - Air pump (38) ・ Hot water tank (39).

24. The system according to any one of claims 1 to 23, wherein the support for the receptor (4) is arranged on one or more spacers (24).

25. The system according to any one of claims 1 to 24, wherein the receptor (16) comprises one or more wells or vials (9) suitable for and / or arranged to contain a biological sample.

26. The system according to any one of claims 1 to 25, wherein the receptor (16) is an array of microtiter plates or vials corresponding to a 6, 24, 96, or 384 microtiter plate arrangement.

27. A support (4) for freezing or vitrifying a biological sample contained in a receptor (16), the support comprising a material that has an absorption effect on low-temperature liquids.

28. The cryogenic liquid (C) is selected from the group consisting of the following, for the support according to claim 27: • Liquid nitrogen, • Liquid helium, • Liquid argon, • Liquid oxygen, Dry ice (CO ２ ) and a mixture of organic solvents.

29. The support according to claim 27 or 28, comprising a material having an open-cell sponge-like structure.

30. A support according to any one of claims 27 to 29, comprising a material that maintains elastic deformability at temperatures below -70°C and / or in the presence of a low-temperature liquid.

31. A support according to any one of claims 27 to 30, having an absolute height of 5 to 50 mm.

32. A support according to any one of claims 27 to 31, comprising a material selected from the group consisting of the following: Melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin, • Polyurethane, and / or Cross-linked polyolefin foam.

33. The support according to any one of claims 27 to 32, comprising on its upper side one or more cavities (10) suitable for and / or arranged to accommodate one or more wells or vials (9) of a receptor (16) suitable for and / or arranged to accommodate a biological sample.

34. The support according to claim 33, wherein one or more cavities (10) are arranged to be essentially shaped-fixed with at least a portion of one or more wells or vials (9).

35. The support according to any one of claims 27 to 34, wherein the receptor (16) is an array of microtiter plates or vials corresponding to a 6, 24, 96, or 384 microtiter plate arrangement.

36. A ventilation plate (18) for thawing a biological sample (17) contained within a receptor (16), wherein the plate includes one or more cavities (19) on its upper side that are suitable for and / or arranged to accommodate one or more wells or vials (9) of a receptor (16) suitable for and / or arranged to accommodate the biological sample (17), The ventilation plate (18) further, - One or more supply channels (20) suitable for supplying and / or configured to supply a thawing medium to the wells of the receptor (16) or the outer wall of the vial (9), and A ventilation plate including one or more exhaust passages (21) suitable for and / or configured to discharge the thawing medium from the well or outer wall of the receptor or vial (9).

37. The thawing medium (23) is a fluid, liquid, or gas, as described in claim 36, for the vent plate (18).

38. The thawing medium (23) has a temperature of -150°C to +100°C, as described in claim 36 or 37.

39. A system for thawing a biological sample contained in a receptor (16), a) A ventilation plate (18) according to any one of claims 36 to 38, and b) A system comprising a pipette (22) suitable for and / or arranged to dispense a thawing medium (23) into a well (16) of a receptor or a vial (9).

40. The biological sample (17) is at least one selected from the group consisting of the following: the system, ventilation plate (18), or support (4) according to any one of claims 1 to 39: - One or more tissue samples, - One or more types of germ cells (oocytes or spermatocytes), - One or more types of blastocysts, - One or more individual cells, - One or more types of cell aggregates (e.g., microtissues, spheroids), and / or - One or more organoids derived from stem cells through self-organization and differentiation.

41. A method for freezing and / or vitrifying a biological sample (17), comprising the following steps: a) A receptor (16) suitable for and / or arranged to contain one or more biological samples (17) is placed on a support (4) according to any one of claims 13 to 18, wherein the support (4) is arranged in the system as described in any one of claims 27 to 35. b) Before or after step a), dispense one or more biological samples (17) into one or more wells or vials (9) of the receptor (16). c) Filling the system according to any one of claims 1 to 26 with cryogenic liquid (C) before or after step a) or b).

42. The method according to claim 34, further comprising the step of adding one or more cryoprotective agents to the biological sample before step b).

43. A method for preserving a frozen and / or vitrified biological sample by the method described in claim 34 or 35, comprising the following steps: a) A receptor (16) containing one or more frozen and / or vitrified biological samples (17) is held within the system according to any one of claims 1 to 26. b) Optionally, a lid (14) is placed on top of the system according to any one of claims 1 to 26. c) Optionally, replenishing the system according to any one of claims 1 to 26 with cryogenic liquid (C).

44. A method for thawing a biological sample (17) that has been frozen and / or vitrified by the method of claim 39 or 40 and / or preserved by the method of claim 43, comprising the following steps: a) Removing the receptor (16) from the support (4), b) Placing the receiver (16) on the ventilation plate (18) according to any one of claims 36 to 38, c) Supply the thawing medium (23) to the ventilation plate (18), Optionally, simultaneously with step c) or immediately before or after step c) the thawing medium (23) is dispensed from pipetting means (22, 33) positioned on the receptor (16).

45. A receptor (16) comprising one or more wells or vials (9) containing one or more frozen or vitrified biological samples (17), wherein at least one of the wells or vials (9) contains the volume V of the biological sample (17). ｓ Internal volume V is at least 100 times larger than ｉ A receptor, including the surrounding vitrified or frozen storage medium or culture medium (if present).

46. The receptor according to claim 45, manufactured or available by the method described in claim 41 or 42.

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