System for freezing and / or vitrifying biological sample

Abstract

The present invention relates to a system for freezing and / or vitrifying a biological sample. The system comprises an enclosure (40) for containing a cryogenic liquid (C); a support (4) for receiving the container (16); and an upper reservoir (2) optionally provided with a first overflow edge (5) which allows cryogenic liquid to flow from the upper reservoir into a lower reservoir (12) with a second overflow edge (6). The support may comprise one or more cavities (10) adapted to receive an aperture or bottle for receiving a container. The support may include a material having a wicking effect on cryogenic liquids, or be disposed on the spacer. The system may also include a pipetting device. The present disclosure also relates to a receiving container; a vent plate for thawing a biological sample; and methods for freezing and / or vitrifying, storing and thawing a biological sample.

Description

Technical Field

[0001] The present application relates to systems and methods for freezing and / or vitrifying biological specimens. Background Art

[0002] Biological specimens play an important role in biological sciences and applications. As living materials, they have a limited lifespan and therefore often require proper preservation.

[0003] Flash freezing is the process of rapidly cooling a substance for preservation. Widely used in scientific research, this technique involves rapidly cooling a sample to ultra-low temperatures, such as below -70°C, typically by immersing the sample in liquid nitrogen or a mixture of dry ice and ethanol. This process is particularly useful for freezing purified biomolecules, such as proteins, to prevent ice crystals from forming and damaging the protein.

[0004] For other biological samples, such as cells or microtissues, slow freezing is often used. This involves cooling the sample in an aqueous medium below freezing. At a certain stage, ice forms, containing pure water of crystallization. This confines the biological material and any solutes, increasing the concentration of substances such as sugars, salts, and / or cryoprotectants, while the volume of the unfrozen portion decreases. This increase in osmotic pressure causes water to escape from the cells. Slow cooling is necessary to ensure sufficient water escape and minimize the formation of ice crystals within the cells. As cooling continues, the viscosity of the unfrozen portion eventually becomes too high to allow further crystallization. The remaining unfrozen portion transforms into an amorphous solid free of ice crystals.

[0005] To avoid the above problems, it is desirable to also perform quick freezing on biological samples such as cells or microtissues. However, since the containers in which biological samples are usually stored or transported have a high heat capacity, the high freezing and heating rates required for quick freezing and thawing cannot be achieved.

[0006] Therefore, it is an object of the present invention to provide improved methods and systems for rapid freezing and / or vitrification of biological specimens.

[0007] This and other objects are achieved by the features of the independent claims. The dependent claims disclose preferred embodiments of the invention in certain cases. Likewise, the description discloses further preferred embodiments of the invention in certain cases. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1Figure A shows, in simplified schematic form, a transverse cross-sectional view of a system for freezing and / or vitrifying biological specimens according to the present invention. In the context of this specification, this system, as well as the other systems described herein, is also referred to as a "cryogenic chamber" or "cryogenic chamber." The system includes a housing 40 for containing a cryogenic liquid C. As an optional feature, the housing 40 includes a cover 14 with an opening 15 for filling with the cryogenic liquid, and is provided with a support 4 for receiving a receptacle 16 suitable for and / or configured to hold one or more biological specimens.

[0009] Figure 1 FIG. 2B shows, in simplified schematic form, a transverse cross-sectional view of another system for freezing and / or vitrifying biological specimens according to the present invention. The system includes a housing 40 for containing a cryogenic liquid C. As an optional feature, the housing 40 includes a cover 14 with an opening 15 for filling the cryogenic liquid and a support 4 for receiving a receptacle 16 suitable for and / or configured to hold one or more biological specimens.

[0010] The support member 4 of the receiving vessel 16 is comprised of a material having an open-pore, sponge-like structure that retains elastic deformation at low temperatures (e.g., -70°C or lower). This creates a wicking effect for the cryogenic liquid. Consequently, the cryogenic liquid C in the cladding 40 and / or lower reservoir 12 penetrates the support member 4, cooling the receiving vessel and its contents thereon from the sides and bottom.

[0011] As an optional feature, the system also comprises a positioning grid 11 which allows a safe and repeatable positioning of the support 4 receiving the container.

[0012] As an optional feature, the system includes a reservoir 12 with an overflow edge 6, which forms an overflow compartment 7. The overflow edge 6 is arranged to allow cryogenic liquid to flow from the reservoir 12 into the overflow compartment 7 if the liquid level of the cryogenic liquid in the reservoir 12 exceeds the height of the overflow edge 6. To this end, the height of the overflow edge 6 is aligned with the height of the support 4 of the receiving container.

[0013] As an optional feature, the system further comprises a compartment 8 disposed below the reservoir 12. The compartment 8 is in fluid communication with the overflow compartment 7 and the support 4 and is adapted and / or arranged to contain a cryogenic liquid.

[0014] Compartment 8 may comprise a material having an open-pore, sponge-like structure that retains elastic deformation at low temperatures. This creates a wicking effect on the cryogenic liquid. Consequently, the cryogenic liquid C in the overflow compartment penetrates compartment 8 and support member 4, providing cooling from the sides and below to the receiving container and its contents positioned thereon.

[0015] Figure 1FIG. 1 shows, in simplified schematic form, a transverse cross-sectional view of another system for freezing and / or vitrifying biological specimens 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 adapted and / or configured to hold a cryogenic liquid C. The second compartment 3 is disposed below the first compartment 1. The second compartment comprises a support 4 for a receiving container. The support 4 for the receiving container includes a cavity 10 for accommodating a hole or bottle of the receiving container. The first compartment comprises an overflow edge 5 that allows cryogenic liquid C (see arrow) to flow from the upper reservoir 2 into the lower reservoir 12 of the second compartment 3. The cryogenic liquid C in the second compartment 3 can evaporate, thereby forming a cold air zone that cools the support 4, the receiving container disposed thereon, and its contents from above. The cryogenic liquid C in the lower reservoir 12 of the second compartment 3 laterally infiltrates the support 4, the receiving container 16 disposed thereon, and its contents.

[0016] Figure 1 D shows a transverse cross-sectional view of another system for freezing and / or vitrifying biological samples according to the present invention, the system Figure 1 In contrast, the support 4 of the receiving container is arranged on the spacer 24 so that the cryogenic liquid C in the lower reservoir 12 of the second compartment 3 wets the support 4 and the receiving container and its contents thereon from the sides and below.

[0017] Figure 2 A transverse cross-sectional view of another system for freezing and / or vitrifying biological samples according to the present invention is shown. Figure 1 The system shown in FIG. 1 is similar to, but more complex than, the system shown in FIG. 2 . In contrast, the receiving container's support member 4 is not necessarily mounted on a spacer. Instead, it comprises a material with an open-pore, sponge-like structure that maintains elastic deformation at low temperatures (e.g., -70°C or lower). This creates a wicking effect on the cryogenic liquid. Accordingly, the cryogenic liquid C in the lower reservoir 12 of the second compartment 3 penetrates the support member 4, cooling the receiving container and its contents mounted thereon from the sides and below.

[0018] As an optional feature, the system further comprises a cover plate 14 comprising an opening 15 adapted and / or arranged for filling the upper reservoir 2 of the first compartment 1 with cryogenic liquid.

[0019] As an optional feature, the system further comprises a side portion 13 of the upper reservoir 2 , shown in dotted lines, forming a U-shaped or fully circumferential channel of the upper reservoir 2 .

[0020] As an optional feature, the system further comprises a groove 28 on the overflow edge 5 of the first compartment, which allows cryogenic liquid C (see arrow) to flow from the upper reservoir 2 into the lower reservoir 12 of the second compartment 3 .

[0021] As an optional feature, the system also comprises a positioning grid 11 which allows a safe and repeatable positioning of the support 4 receiving the container.

[0022] As an optional feature, the second compartment 3 includes a second overflow edge 6, which forms an overflow compartment 7 within the second compartment. The second overflow edge 6 is configured to allow cryogenic liquid to flow from the lower reservoir 12 within the second compartment into the overflow compartment 7 if the liquid level of the cryogenic liquid in the lower reservoir 12 exceeds the height of the second overflow edge 6. To this end, the height of the second overflow edge 6 is aligned with the height of the support 4 of the receiving container.

[0023] As an optional feature, the system further comprises a third compartment 8 disposed 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 adapted and / or arranged to contain a cryogenic liquid.

[0024] The third compartment 8 may comprise a material with an open-pore, sponge-like structure that retains elastic deformation at low temperatures. This creates a wicking effect on the cryogenic liquid. Accordingly, the cryogenic liquid C in the lower reservoir 12 of the second compartment 3 penetrates the third compartment 8 and the support member 4, cooling the receiving container and its contents from the sides and below.

[0025] As an optional feature, the system further comprises a cryogenic liquid filling nozzle 25 , which is adapted and / or arranged to fill the lower reservoir 12 of the second compartment 2 with cryogenic liquid.

[0026] Figure 3 A front cross-sectional view of a system for freezing and / or vitrifying biological samples according to the present invention is shown. Figure 2 It can be seen that the side 13 of the upper reservoir 2 forms a U-shaped channel of the upper reservoir 2.

[0027] Figure 4 A perspective view of a system for freezing and / or vitrifying biological samples according to the present invention is shown. Figure 2 The system shown is similar. The opening 15 adapted and / or configured to fill the upper reservoir 2 of the first compartment 1 with cryogenic liquid is clearly visible. It can be seen that the side 13 of the upper reservoir 2 forms a U-shaped channel (which could also be a fully circumferential channel) for the upper reservoir 2. The 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 the positioning grid 11 that allows for secure and repeatable positioning of the support 4 of the receiving container.

[0028] Figure 5 A perspective view of the support member 4 and the receiving container 16 disposed thereon is shown. The receiving container 16 is in the form of a microporous plate. The support member 4 comprises a material having an open-pore, sponge-like structure and maintaining elastic deformation at low temperatures. This creates a wicking effect on the cryogenic liquid. Accordingly, the cryogenic liquid C in the lower reservoir 12 of the second compartment 3 penetrates the support member 4, providing cooling from the side and bottom to the receiving container disposed thereon and its contents. The cavity of the support member 4 is configured to substantially lock with at least a portion of the holes of the receiving container. This allows the holes to come into direct contact with the cryogenic liquid.

[0029] Figure 6 Figure A shows a cross-sectional view of a support member 4 and a receiving container 16 mounted thereon. A biological sample 17 is placed within the well 9 of the receiving container 16. The support member 4 comprises a material with an open-pore, sponge-like structure that retains elastic deformation at low temperatures. This creates a wicking effect on the cryogenic liquid. The cavity 10 of the support member 4 is configured to substantially form-lock with at least a portion of the well of the receiving container, thereby achieving direct contact between the well and the cryogenic liquid.

[0030] Figure 6 B shows a cross-sectional view of a receiving container 16 with a hole 9, in which a frozen and / or vitrified biological sample 17 is placed. The receiving container is placed on a ventilation plate 18 for thawing the biological sample contained in the receiving container.

[0031] The upper side of the vent plate 18 is provided with a cavity 19, which is adapted and / or configured to accommodate the hole 9 of the receiving container 16. The vent plate further includes at least one supply channel, which is adapted and / or configured to supply thawing medium to the outer wall of the hole 9 of the receiving container 16. The vent plate further includes at least one exhaust channel 21, which is adapted and / or configured to discharge the thawing medium from the hole of the receiving container or the outer wall of the bottle body.

[0032] Figure 7 A detailed cross-sectional view of a system for thawing a biological sample 17 contained in a well 9 of a receiving container 16 is shown. The system comprises Figure 6 Shown is an aeration plate 18 and a pipetting device 22 adapted and / or arranged to dispense a thawing medium 23 into the well 9 of the receiving container 16 .

[0033] Figure 8 A and B show Figure 6 B shows a perspective view of the ventilation plate.

[0034] Figure 9An embodiment of a system for freezing and / or vitrifying biological samples according to the present invention is shown, illustrating three different steps of the freezing process. A pusher 26 is used to pump cryogenic liquid into the system, causing it to laterally infiltrate the support 4 and the receiving container disposed thereon and its contents. Simultaneously, the cryogenic liquid is injected into the first compartment 1, which includes an overflow edge 5 that allows the cryogenic liquid to flow from the upper reservoir 2 into the lower reservoir of the second compartment 3. As can be seen, the upper reservoir 2 includes a side portion 13 that forms a U-shaped channel for the upper reservoir 2.

[0035] Figure 10 A shows the results of measuring intracellular ATP in HepG2 microtissues shortly after freezing / thawing and 2 days later, and compared with control microtissues that were not frozen / thawed. Figure 10 B shows the immunofluorescence images and the corresponding GFP signal quantitative data ( Figure 10 C) shows the results of HepG2 microtissues expressing GFP compared to unfrozen control tissue 2 days after freezing / thawing (vitrification). The data show that the vitrification and thawing process did not negatively affect cell viability, morphology, and growth rate.

[0036] Figure 11 The measured temperature gradient is shown, starting above the liquid nitrogen level in the second compartment and extending into the first compartment, with the distance above the liquid nitrogen level measured in millimeters (mm). The red dashed line represents the temperature gradient achieved when the upper reservoir is installed and filled with liquid nitrogen, while the yellow line represents the temperature gradient when the upper reservoir is not installed. The installation of the upper reservoir filled with liquid nitrogen maintains a stable temperature in the second compartment at a height of 30 to 45 mm above the liquid nitrogen level and ensures a temperature drop of 70 to 80°C at a height of 105 mm above the liquid nitrogen level.

[0037] Figure 12 A transverse cross-sectional view of a system for freezing and / or vitrifying biological samples according to the present invention is shown. Figure 2 The system is similar to the one shown in FIG. This system also includes a control nozzle 27 having an opening aligned with the height of the second overflow edge of the second compartment. Thus, if the level of cryogenic liquid in the overflow compartment exceeds the height of the second overflow edge, cryogenic liquid is allowed to flow from the overflow compartment into the control nozzle and out of the control nozzle. Line AA' indicates the height of the second overflow edge, the height of the support member of the receiving container, and the height of the opening of the control nozzle.

[0038] Figure 13 The shapes of microplate wells used for 3D cell culture are shown, including conical or tapered cross-sections ( Figure 13 A, InSphero provides "AkuraTM 3D microplate") and hemispherical cross-section ( Figure 13 B, "Spherical Microplate" provided by the company). Figure 13 C shows other possible shapes of the microplate wells. In any case, those skilled in the art will appreciate that by properly designing the cavity shape of the support member, a form-locking connection between one or more cavities of the support member and the wells or bottle body of the receiving container can be achieved.

[0039] Figure 14 Another embodiment is shown, in which a receiving container 16 in the form of a microplate is provided with wells 9 but has a flat bottom 29. In this case, the support body 4, which optionally comprises a material having an open-pore sponge-like structure and retains elastic deformation capability at low temperatures, is not provided with any cavities 10 but is floated so as to form-lock with the flat bottom 29 of the receiving container. As a result, the wells 9 are in direct contact with the cryogenic liquid.

[0040] Figure 15 Shown are hematoxylin-eosin (H&E) staining and immunostaining results for albumin (hepatocytes), BSEP (tubular structures), and CD68 (Kupffer cells) of microtissues frozen / thawed according to the present invention versus unfrozen microtissue (control).

[0041] Figure 16 ATP content and albumin secretion of microtissues frozen / thawed according to the present invention relative to unfrozen microtissues are shown.

[0042] Figure 17 The CYP450 enzyme activities (1A2, 2B6, 2C9, 3A4) of microtissues frozen / thawed according to the present invention relative to unfrozen microtissues (control) are shown. CYP450 enzyme activities were assessed by exposure to prototype substrates for 24 hours and quantification of the corresponding metabolites using LC-MS.

[0043] Figure 18 Shown are dose response toxicity studies (ATP) for chlorpromazine, troglitazone, and tolcapone of microtissues frozen / thawed according to the present invention relative to unfrozen microtissues (control).

[0044] Figure 19 A schematically simplified cross-sectional view of another system for freezing and / or vitrifying biological samples according to the present invention is shown. The system comprises a pipette array 33 with pipette tips 22 for dispensing the biological sample into one or more wells or vials 9 of a receiving container 16 arranged on a support 4.

[0045] The system also includes a ventilation system to prevent the liquid sample in the pipette tip 22 from freezing rapidly when the liquid sample containing the biological specimen is dispensed into the well or bottle 9 of the receiving container 16. The ventilation system includes

[0046] The outer shell 31 forms a cavity 42 between the cladding 40 and the outer shell 32;

[0047] an air intake 32 for drawing cool air from the empty chamber 42 via a corresponding pump (not shown), and

[0048] Flow guide plates 30 , which enable a non-turbulent gas flow, such as air, to be formed, which only wets the pipette tips without generating any turbulence that could affect the overall temperature conditions in the system, including a “cold air lake”.

[0049] Figure 20 A schematically simplified cross-sectional view of another system for freezing and / or vitrifying biological samples according to the present invention is shown. The system comprises a pipette array 33 with pipette tips 22 for dispensing the biological sample into one or more wells or vials 9 of a receiving container 16 arranged on a support 4.

[0050] The system also includes a ventilation system to prevent the liquid sample in the pipette tip 22 from freezing rapidly when the liquid sample containing the biological specimen is dispensed into the well or bottle 9 of the receiving container 16. The ventilation system includes

[0051] Suction port 32, for passing hot air source (39, see Figure 23 ) extracts cold gas (e.g., cold air) from the system, and

[0052] An air inlet (34) for receiving hot gas (eg, hot air) provided by a hot air source (39).

[0053] Figure 21 A shows a schematically simplified transverse cross-sectional view of another system for freezing and / or vitrifying biological samples according to the present invention. The system comprises a pipette array 33 with pipette tips 22 for dispensing biological samples into one or more wells or vials 9 of a receiving container 16 arranged on a support 4.

[0054] The system also includes a ventilation system to prevent the liquid sample in the pipette tip 22 from freezing rapidly when the liquid sample containing the biological specimen is dispensed into the well or bottle 9 of the receiving container 16. The ventilation system includes a tube heating device 37 and an air pump 38 (both as shown in FIG. Figure 23 A flat air nozzle 36 is connected to the nozzle 36 (shown).

[0055] Such flat air nozzles 36 are able to generate a laminar flow 41 of hot gas (e.g. hot air), thereby ensuring that the hot gas only wets the pipette tips 22 of the pipette array without generating any turbulence, including a "cold air lake", which could affect the overall temperature conditions within the system.

[0056] Figure 21 B shows an example of such a flat air nozzle 36 and the laminar flow 41 of hot gas (eg hot air) it generates, which wets only the pipette tips 22 of the pipette array 33 .

[0057] Figure 22 The vertical temperature gradient of the first and second compartments is shown, with the upper reservoir empty (■) or filled with liquid nitrogen (●), as measured by a temperature probe.

[0058] Figure 23 Shown schematically and exemplarily

[0059] Hot air source 39, consisting of a warm water wash bottle and an air tube connected to a pump, allowing cool air to flow from Figure 20 The air intake 32 of the system is drawn in, heated and then supplied to the air inlet 34 of the system, and

[0060] Tube heating device 37 and air pump 38, connected to flat air nozzle 36, as Figure 21 shown.

[0061] Figure 24 A shows a receiving container 16 comprising wells or vials 9, each of which contains a frozen or vitrified biological sample 17. The inner volume V of the well or vial is i At least the volume V of the biological sample 17 s 100 times the volume of the sample, including surrounding vitrified or frozen storage or culture medium (if any).

[0062] Figure 24 B shows Figure 24 The hole or bottle body 9 is tapered or conical. Its volume can be calculated by the formula V = (1 / 3)·π·h·(r1 2 +r2 2 +(r1·r2)) approximate calculation, where h is the height of the hole or bottle, r1 is the lower radius of the bottle, and r2 is the upper radius of the bottle.

[0063] Figure 24 C shows an example of a frozen or vitrified biological sample 17 including a surrounding vitrified or frozen storage or culture medium 43. Volume V s By the formula V = 4 / 3·π·r 3Approximate calculation, where r is the radius of the biological sample 17, including the surrounding vitrified or frozen storage or culture medium 43. DETAILED DESCRIPTION

[0064] According to a first aspect of the present invention, a system for freezing and / or vitrifying biological samples is provided, comprising a housing (40) for containing a cryogenic liquid (C), and a support (4) for a receiving container (16) adapted and / or arranged to receive one or more biological samples.

[0065] According to an embodiment of the system, the support (4) is a support as described below.

[0066] According to one embodiment, the system comprises a first compartment (1) and a second compartment (3). The first compartment comprises an upper reservoir (2), and the second compartment comprises a lower reservoir (12). Each reservoir is adapted and / or configured to hold a cryogenic liquid (C). The second compartment is disposed below the first compartment and comprises a support (4) adapted and / or configured to hold a receiving container for one or more biological samples.

[0067] The system enables rapid freezing and / or vitrification of biological samples without the formation of ice crystals that could damage or compromise the sample. Furthermore, the system avoids the need to apply a cryogenic liquid (C) directly to the biological sample. Such direct contact is often hindered by the difficulty of accurately metering the cryogenic liquid (C) and by the splashing caused by the Leidenfrost effect when the cryogenic liquid (C) contacts a warmer surface. Furthermore, the system avoids the need to pipette the cryogenic liquid (C) directly into a vial containing the biological sample, a process that is difficult to control due to evaporation of the cryogenic liquid in the pipette and the extrusion of the cryogenic liquid from the pipette tip.

[0068] With respect to embodiments of supports, systems and / or methods described elsewhere herein, the features, characteristics and advantages associated with said embodiments, as well as the embodiments themselves, should be understood to apply to the systems and embodiments thereof described above and below, even if not reiterated.

[0069] According to an embodiment of the system according to the present invention, the system further comprises a receiving container (16) adapted and / or arranged to receive one or more biological samples.

[0070] According to one embodiment of the system according to the present invention, the upper liquid reservoir (2) comprises a first overflow edge (5) which is arranged to allow the cryogenic liquid to flow into the lower liquid reservoir (12) if the liquid level of the cryogenic liquid in the upper liquid reservoir (2) exceeds the height of the first overflow edge (5).

[0071] This arrangement ensures that the support of the receiving vessel is always completely immersed in the cryogenic liquid and forms a cold vapor zone ("cold air lake (Kaltluftsee)") above the cryogenic liquid, which can still cool all components below it, including the support and the receiving vessel and its contents arranged thereon.

[0072] According to one embodiment of the system according to the present invention, the second compartment (3) includes a second overflow edge (6) which is arranged to form an overflow compartment (7) within the second compartment (3), wherein the second overflow edge (6) is arranged to allow cryogenic liquid to flow from the lower liquid reservoir (12) within the second compartment into the overflow compartment (7) if the liquid level of the cryogenic liquid in the lower liquid reservoir (12) exceeds the height of the second overflow edge (6).

[0073] According to one embodiment of the system according to the invention, the height of the second overflow edge (6) is aligned with the height of the support (4) of the receiving container (16), thereby ensuring that the hole or the bottle in the receiving container is not immersed in the cryogenic liquid when placed on the support.

[0074] According to one embodiment of the system according to the present invention, the system further comprises a third compartment (8) arranged below the second compartment (3), the third compartment (8) being fluidically connected to the second compartment (3) and / or the overflow compartment (7), and being suitable for and / or arranged to accommodate a cryogenic liquid.

[0075] According to one embodiment of the system according to the present invention, the system further comprises a cover plate (14) comprising an opening (15) adapted and / or arranged for filling the upper reservoir (2) of the first compartment (1) with cryogenic liquid (C).

[0076] According to one embodiment of the system according to the present invention, the system further comprises a cryogenic liquid filling nozzle (25), which is adapted and / or arranged to fill the cryogenic liquid directly into the second compartment (3) and / or the overflow compartment (7) and / or the third compartment (8).

[0077] According to an embodiment of the system according to the invention, the system further comprises a control nozzle (27) with an opening, wherein the height of the opening of the control nozzle is aligned with the height of the second overflow edge (6) of the second compartment (3).

[0078] Thus, if the liquid level of the cryogenic liquid in the overflow compartment (7) exceeds the height of the second overflow edge (6), the cryogenic liquid is allowed to flow from the overflow compartment (7) into the control nozzle (27) and out of the control nozzle (27).

[0079] According to one embodiment of the system according to the present invention, the upper side of the support (4) of the receiving container (16) is provided with one or more cavities (10), which are suitable and / or arranged to accommodate one or more holes or bottles (9) of the receiving container (16), which are suitable and / or arranged to accommodate biological samples.

[0080] According to one embodiment of the system according to the invention, the one or more cavities (10) are arranged to be substantially form-locked with one or more holes of the receiving container (16) or at least part of the body (9).

[0081] According to an embodiment of the system according to the present invention, the support (4) of the receiving container comprises a material having a wicking effect on the cryogenic liquid.

[0082] In this way, it is ensured that the cryogenic liquid is introduced quickly and directly into the hole of the receiving container (16) or the bottle (9), which enables the biological sample contained therein to be cooled quickly and continuously.

[0083] According to an embodiment of the system according to the invention, the support (4) of the receiving container comprises a material having an open-pore sponge-like structure.

[0084] According to one embodiment of the system according to the invention, the support (4) of the receiving container comprises a material that retains elastic deformation capability at temperatures below -70°C and / or in the presence of a cryogenic liquid (C).

[0085] In this way, even under low temperature conditions, it is possible to ensure that the one or more cavities (10) of the support (4) and the hole or bottle (9) of the receiving container (16) are in permanent close contact, which allows the biological sample contained therein to be cooled quickly and continuously.

[0086] According to one embodiment of the system according to the invention, the absolute height of the support (4) of the receiving container is ≥5 and ≤50 mm.

[0087] In various embodiments, the absolute height of the support (4) of the receiving container is ≥10 and ≤40 mm, ≥15 and ≤30 mm, or 20 mm ± 3 mm.

[0088] According to one embodiment of the system according to the present invention, the support (4) of the receiving container comprises a material selected from the group consisting of:

[0089] Melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin,

[0090] Polyurethane, and / or

[0091] Cross-linked polyolefin foam.

[0092] The inventors unexpectedly discovered that these materials

[0093] a) has a wicking effect on cryogenic liquids as described above, and

[0094] b) Maintaining elastic deformability at temperatures below -70°C and / or in the presence of cryogenic liquids.

[0095] As mentioned above, these two features enable rapid and uninterrupted cooling of the contained biological specimen.

[0096] GB2377985A discloses a disposable container for refrigerating animal / human tissue or fluids. The device comprises a liquid refrigerant (liquid nitrogen) absorbing material and is provided with one or more recesses for receiving biological sample containers so that the refrigerant is in close proximity to the container.

[0097] The liquid absorbing material is preferably a foam material or a sponge, preferably a material commonly used by florists as a base for flower arrangements. Foam material. GB2377985A mentions that this material has a particular advantage because the grooves retain their shape once formed. This means that, unlike the preferred materials of the present invention, the foam material described in GB2377985A has the ability to plastically deform (i.e., retain its shape after deformation) and does not have the ability to elastically deform.

[0098] The inventors of the present invention have confirmed through experiments that the preferred The material lacks elastic deformation capability, either at room temperature or at low temperatures (e.g., -196°C). This means that, contrary to the corresponding embodiments of the present invention, such materials cannot ensure permanent and intimate contact between the cavity ("recess") of the support and the well or bottle ("container") of the receiving container, thereby failing to achieve rapid and uninterrupted cooling of the contained biological sample.

[0099] The inventors of the present invention have also confirmed through experiments that, for example, melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin does

[0100] a) has a wicking effect on cryogenic liquids, and

[0101] b) Maintaining elastic deformability at temperatures below -70°C and / or in the presence of cryogenic liquids.

[0102] According to one embodiment of the system according to the invention, the third compartment (8) comprises a material having at least one of the following properties:

[0103] a) has a wicking effect on cryogenic liquids,

[0104] b) open-pore sponge-like structure,

[0105] c) maintains elastic deformability at temperatures below -70°C and / or in the presence of cryogenic liquids, and / or

[0106] d) comprising a material selected from the group consisting of:

[0107] Melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin,

[0108] Polyurethane, and / or

[0109] Cross-linked polyolefin foam.

[0110] According to one embodiment of the system according to the present invention, the cryogenic liquid is selected from the group consisting of:

[0111] Liquid nitrogen,

[0112] Liquid helium,

[0113] Liquid argon,

[0114] Liquid oxygen,

[0115] A mixture of dry ice (CO2) and an organic solvent.

[0116] Preferably, the organic solvent mixed with the dry ice is acetone and / or ethanol.

[0117] According to one embodiment of the system according to the invention, the support (4) of the receiving container is arranged on one or more spacers (24), so that the cryogenic liquid can wet the support from below.

[0118] According to one embodiment of the system according to the present invention, the receiving container (16) comprises one or more wells or bottles (9) adapted and / or configured to receive a biological sample.

[0119] According to one embodiment of the system according to the present invention, the receiving container (16) is a microplate or an array of bottles corresponding to a 6-well, 24-well, 96-well or 384-well microplate layout.

[0120] As described herein, the one or more cavities (10) of the support (4) are preferably configured to substantially positively engage with one or more apertures of the receiving container (16) or at least part of the body (9).

[0121] The present invention can be used with different types of microplates, including conventional microplates comprising tapered, conical, cylindrical or hemispherical wells, as well as plates designed specifically for microsphere culture as described in Larsen B (2015), the contents of which are incorporated herein by reference for practical purposes.

[0122] Such plates may include holes of more complex shapes, such as the "Akura" offered by InSphero.TM 3D microplates” with a tapered cross section (see Figure 13 A), or The company provides "spherical microplate" (see Figure 13 B).

[0123] The principle of form-locking between one or more cavities (10) of the support (4) and the hole of the receiving container (16) or the bottle body (9) is as follows Figure 6 A of FIG. Those skilled in the art can extend this principle to other receiving containers with holes or bottles of different shapes (see FIG. Figure 13 ), even in embodiments where the receiving container has a flat bottom (see Figure 14 ).

[0124] According to another embodiment of the present invention, the system further comprises a pipetting device (22, 33) for dispensing the biological sample (17) into one or more wells or bottles (9) of the receiving container (16).

[0125] According to another embodiment of the present invention, the system further comprises ventilation means (30, 32, 34, 36, 37, 38, 39) for generating a warm air flow along the pipette tip (22) of the pipetting device (33).

[0126] Such ventilation device optionally comprises at least one element selected from the group consisting of:

[0127] ·Deflector(30)

[0128] Air intake (32)

[0129] Air Inlet (34)

[0130] Flat air nozzle (36)

[0131] Tube heating device (37)

[0132] Air pump (38)

[0133] Hot air source (39)

[0134] Such a venting device prevents rapid freezing of the liquid sample in the pipette tip (22) when dispensing the liquid sample containing the biological specimen (17) into the well or bottle (9) of the receiving container (16). It is important to ensure that this effect remains locally confined, as shown in the figure.

[0135] Such flat air nozzles (36) are able to generate a laminar flow (41) of hot gas (e.g. hot air), thereby ensuring that the hot gas only wets the pipette tips without generating any turbulence that could affect the overall temperature conditions within the system, including a "cold air lake".

[0136] A similar effect can be achieved by the baffles (30), which produce a non-turbulent flow of gas (41), such as air.

[0137] Furthermore, the preferred embodiments and features described elsewhere herein apply mutatis mutandis to this aspect of the invention, i.e. the system.

[0138] According to another aspect of the present invention, there is provided a support member (4) for freezing or vitrifying a biological specimen contained in a receiving container (16). The support member comprises a material having a wicking effect on cryogenic liquids.

[0139] According to an embodiment of the support member of the present invention, the cryogenic liquid is selected from the group consisting of:

[0140] Liquid nitrogen,

[0141] Liquid helium,

[0142] Liquid argon,

[0143] Liquid oxygen,

[0144] A mixture of dry ice (CO2) and an organic solvent.

[0145] The following table lists the boiling points of suitable cryogenic liquids.

[0146] type The boiling point at normal pressure is approximately liquid nitrogen -196℃ Liquid helium -269℃ Liquid argon -186℃ Liquid oxygen -183℃ <![CDATA[Mixture of dry ice (CO2) and organic solvent]]> -72℃(when mixed with ethanol)

[0147] Preferably, the organic solvent mixed with the dry ice is acetone and / or ethanol.

[0148] According to an embodiment of the support element according to the present invention, the receiving container is adapted and / or configured to receive one or more biological samples.

[0149] According to an embodiment of the support member of the present invention, the support member comprises a material having an open-pore sponge-like structure. In one embodiment, the support member comprises an open-pore three-dimensional fiber network structure.

[0150] In one embodiment, the support member comprises an open-cell three-dimensional fiber network structure.

[0151] According to one embodiment of the support element according to the invention, the support element comprises a material which retains elastic deformation capability at temperatures below -70°C and / or in the presence of cryogenic liquids. This allows for a suitable form-locking connection with the receiving container over a wide temperature range.

[0152] According to an embodiment of the support member of the present invention, the absolute height of the support member is ≥5 and ≤50 mm.

[0153] In various embodiments, the absolute height of the support is > 10 and < 40 mm, > 15 and < 30 mm, or 20 mm ± 3 mm.

[0154] This height range has proven to be practical because it meets two constraints: a) it is not too high to negatively impact the wicking effect that draws the cryogenic liquid into the support; and b) it is high enough to allow a sufficient level of cryogenic liquid to be provided in the compartment housing the support while avoiding immersion of the receiving vessel.

[0155] According to an embodiment of the support member according to the present invention, the support member comprises a material selected from the group consisting of:

[0156] Melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin,

[0157] Polyurethane, and / or

[0158] Cross-linked polyolefin foam.

[0159] These materials meet the above-mentioned requirements regarding wicking effect and elastic deformation capacity under low temperature conditions.

[0160] Melamine and its resins are most commonly used in tableware, laminate flooring, and whiteboards. Melamine foam is used as thermal and sound insulation, as well as in polymer cleaning products such as so-called "magic erasers."

[0161] The inventors unexpectedly discovered that melamine and melamine resin, as materials used for the support member of the present invention, have excellent properties because the material

[0162] Maintains elastic deformation capability at temperatures below -70°C and / or in the presence of cryogenic liquids

[0163] Can be configured to have an open-pore sponge-like structure

[0164] It can also be configured to have a wicking effect on cryogenic liquids.

[0165] Cross-linked polyolefin foams have been found to be suitable materials for cryogenic containers. Several properties of polyolefin foams make them particularly well-suited for use as cryogenic containers: they can be easily formed into suitable shapes by molding, casting, or other methods of foaming from monomers or polymers, or by machining finished blocks; their cell structure is typically fine-grained enough to contain, for example, liquid nitrogen without leaking; they are durable enough to withstand repeated exposure to low temperatures; they are essentially inert; and they typically have both low thermal conductivity and low volumetric heat capacity. As used herein, the term "polyolefin foam" refers to polyethylene foam, polypropylene foam, polyethylene-polypropylene blends and copolymers, and foams containing blends or copolymers of olefin monomers with other monomers, provided that the blended foam possesses at least some of these desirable properties.

[0166] According to one embodiment of the support according to the present invention, the upper side of the support is provided with one or more cavities (10), which are suitable and / or arranged to accommodate one or more holes or bottles (9) of a receiving container (16), which are suitable and / or arranged to accommodate a biological sample.

[0167] According to one embodiment of the support member of the present invention, one or more cavities (10) are configured to be substantially form-locked with at least a portion of one or more holes or bottle bodies (9). This form-locked relationship between the one or more cavities (10) of the support member (4) and the hole or bottle body (9) of the receiving container (16) is shown in FIG6A. A person skilled in the art will be able to extend this principle to other receiving containers having holes or bottle bodies of different shapes.

[0168] According to one embodiment of the support member of the present invention, the receiving container (16) is a microplate or a bottle array corresponding to a 6-well, 24-well, 96-well or 384-well microplate layout.

[0169] According to another embodiment, the receiving container (16) is provided with a hole or a bottle (9) but has a flat bottom (29). In this case, the support body (4), which optionally comprises a material having an open-pore sponge-like structure and retaining elastic deformation ability at low temperatures, is not provided with any cavity (10) but is in a floating state so as to be form-locked with the flat bottom (29) of the receiving container (16). As a result, the hole (9) is in direct contact with the cryogenic liquid.

[0170] According to another aspect of the present invention, a vent plate (18) is provided for thawing a biological sample contained in a receiving container (16). The vent plate has one or more cavities (19) on its upper side, the cavities being adapted and / or configured to receive one or more wells or vials (9) of a receiving container adapted and / or configured to receive the biological sample.

[0171] The vent panel also includes

[0172] one or more supply channels (20) adapted and / or arranged to supply thawing medium to the hole of the receiving container or the outer wall of the bottle body (9), and

[0173] One or more exhaust channels (21) adapted and / or arranged to exhaust the thawing medium from the hole of the receiving container or the outer wall of the bottle body (9).

[0174] According to one embodiment of the aeration plate (18) according to the present invention, the thawing medium (23) is a fluid, a liquid or a gas.

[0175] According to one embodiment, the thawing medium (23) is air.

[0176] According to one embodiment of the aeration plate according to the present invention, the temperature of the thawing medium (23) is ≥-150°C and ≤+100°C.

[0177] According to embodiments, the temperature of the thawing medium is ≥4°C and ≤+100°C, ≥15°C and ≤+90°C, preferably ≥20°C and ≤+37°C.

[0178] According to another aspect of the present invention, a system for thawing a biological sample contained in a receiving container (16) is provided.

[0179] The system includes

[0180] a) a vent plate (18) as described above, and

[0181] b) A pipetting device (22) adapted and / or arranged to dispense a thawing medium (23) into a well or bottle (9) of a receiving container (16).

[0182] According to an embodiment of the thawing system of the present invention, the thawing medium is a fluid, liquid or gas.

[0183] According to one embodiment of the thawing system of the present invention, the thawing medium used in the aeration plate is gas, preferably air, as described above, while involving an appropriate temperature.

[0184] According to one embodiment of the thawing system of the present invention, the thawing medium used in the pipetting system is a liquid, preferably an aqueous liquid. According to various embodiments, the temperature of the thawing medium used in the pipetting system is ≥4°C and ≤+80°C, ≥15°C and ≤+70°C, preferably ≥20°C and ≤+37°C.

[0185] According to other embodiments of the system, the ventilation plate (18) or the support (4) according to the present invention, the biological sample is at least one selected from the group consisting of:

[0186] One or more tissue samples,

[0187] One or more gametocytes (oocytes or spermatocytes),

[0188] One or more blastocysts,

[0189] One or more single cells,

[0190] One or more cell aggregates (e.g., microtissues, spheroids), and / or

[0191] One or more organoids formed through the self-organization and differentiation of stem cells.

[0192] In one embodiment, the biological sample is contained in a liquid, such as a storage or culture medium. In this case, the biological sample is described as being contained in a liquid sample.

[0193] According to one embodiment, the biological sample does not involve a biological artificial scaffold, that is, is a scaffold-free structure. In one embodiment, the biological sample is a scaffold-free microtissue, spheroid or organoid.

[0194] Such scaffold materials are disclosed, for example, in Carletti et al. (2011) and include natural polymers (e.g., collagen, chitosan, glycosaminoglycans, silk fibroin, agarose, alginate, and starch) and synthetic polymers (e.g., polyglycolic acid, polylactic acid, and copolymers thereof, poly-ε-caprolactone, polyurethane, polyorthoesters, and polyanhydrides). Such scaffold materials are commonly used for the formation of microtissues but may negatively impact the results of assays using the microtissues.

[0195] According to another aspect of the present invention, a method for freezing and / or vitrifying a biological sample is provided. The method comprises the following steps:

[0196] a) placing a receiving container (16) suitable for and / or arranged to receive one or more biological samples on a support (4) as described above, the support being arranged in a system as described above;

[0197] b) before or after step a), dispensing one or more biological samples (17) into one or more wells or vials (9) of a receiving container; and

[0198] c) Before or after step a) or b), the system as described above is filled with cryogenic liquid.

[0199] According to one embodiment, the method further comprises the step of adding one or more cryoprotectants (CPA) to the biological sample prior to step b).

[0200] According to one embodiment, the method comprises equilibrating the biological sample sequentially with two or three different increasing concentrations of CPA.

[0201] In various embodiments, the cryoprotectant comprises at least one of the following: dimethyl sulfoxide (DMSO) and / or one or more selected from alcohols, sugars, amides or imides and / or macromolecular compounds, wherein the alcohol is selected from methanol, glycerol, sorbitol, ethylene glycol, propylene glycol, butylene glycol, inositol, xylitol, mannitol, adonitol, arabitol, ribitol, erythritol, avocado, threitol, galactitol, pinitol, xylitol and combinations thereof; the sugar is selected from sucrose, Trehalose, maltose, arabinose, lactose, mannose, xylose, galactose, fructose, glucose, dextran, melezitose, raffinose, nigerose, maltotriose, maltotriketose, kestose, cellobiose, chitobiose, lactulose and combinations thereof; the amides and imides are selected from formamide, acetamide, propionamide, lactamide, butanamide, malonamide and combinations thereof; the macromolecular compound is selected from polysucrose, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, hyaluronic acid and combinations thereof. For this aspect, see Whaley et al. (2021), the contents of which are incorporated herein by reference for practicable purposes.

[0202] According to one embodiment, dispensing one or more biological samples (17) to one or more wells or vials (9) of a receiving container comprises the following steps:

[0203] i) using one or more pipettes or a pipette array (33) comprising one or more pipette tips (22) to aspirate the one or more biological samples from a plate containing the biological samples;

[0204] ii) allowing the one or more biological samples to settle to the bottom of the one or more pipette tips (22); and

[0205] iii) dispensing the one or more biological samples from a pipette tip (22) into one or more wells or vials (9) of the receiving container by releasing a droplet containing the biological sample.

[0206] Even after sedimentation, the biological sample can still be selectively surrounded by the liquid medium.

[0207] According to one embodiment of the method, only one biological sample (eg only one microtissue) is dispensed into each well or bottle (9) of the receiving container.

[0208] According to an embodiment of the method, the volume of the droplet containing the biological sample is ≥ 0.05 μL and ≤ 8 μL. According to other embodiments of the method, the volume of the droplet containing the biological sample is about 0.1 μL, about 0.2 μL, about 0.3 μL, about 0.4 μL, about 0.5 μL, about 0.6 μL, about 0.7 μL, about 0.8 μL, about 0.9 μL, about 1 μL, about 1.2 μL, about 1.4 μL, about 1.5 μL, about 1.6 μL, about 1.8 μL, about 2 μL, about 2.2 μL, about 2.4 μL, about 2.6 μL, about 2.8 μL, about 3 μL, about 3.2 μL, about 3.4 μL, about 3.6 μL, about 3.8 μL, or about 4 μL.

[0209] Various embodiments of the present invention ensure that during the dispensing step, the sample containing the biological specimen does not freeze in the pipette tip (22), even though the pipette tip is close to one or more wells or vials (9) of the receiving container, all of which are cooled due to the presence of the cryogenic liquid. Thus, freezing / vitrification begins immediately after the sample is dispensed into its well or vial.

[0210] According to another aspect of the present invention, a method for storing biological samples frozen and / or vitrified by the method described above is provided. The method comprises the following steps:

[0211] a) maintaining a receiving container (16) containing one or more frozen and / or vitrified biological specimens within a system as described above;

[0212] b) optionally, positioning a cover plate (14) on top of the system; and

[0213] c) Optionally, supplementing the system as described above with cryogenic liquid.

[0214] According to another aspect of the present invention, a method for thawing a biological sample frozen and / or vitrified by the method described above is provided. The method comprises the following steps:

[0215] a) removing the receiving container (16) from the support (4);

[0216] b) placing the receiving container (16) on the aeration plate (18) as described above;

[0217] c) supplying a thawing medium to the ventilation plate (18);

[0218] d) Optionally, simultaneously with step c) or before or after step c), a thawing medium is dispensed from a pipetting device (22) arranged above the receiving container (16).

[0219] According to one embodiment of the method according to the present invention, the thawing medium is a fluid, a liquid or a gas.

[0220] According to one embodiment of the method according to the invention, the thawing medium used in the aeration plate is a gas, preferably air, as described above, while involving an appropriate temperature.

[0221] According to one embodiment of the method according to the present invention, the thawing medium used in the pipetting system is a liquid, preferably an aqueous liquid. According to various embodiments, the temperature of the thawing medium used in the pipetting system is ≥4°C and ≤+80°C, ≥15°C and ≤+70°C, preferably ≥20°C and ≤+37°C.

[0222] According to one embodiment, the method further comprises e) replacing the preheated thawing medium with room temperature medium once or twice.

[0223] According to another aspect of the present invention, a receiving container (16) is provided, comprising one or more wells or vials (9) containing one or more frozen or vitrified biological samples (17). The inner volume V of at least one well or vial is i At least the volume V of the biological sample (17) s 50 times the inner volume V including the surrounding vitrified or frozen storage or culture medium (if present). According to an embodiment, the inner volume V i At least the volume V of the biological sample (17) s 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 105 times, 110 times, 115 times, 120 times, 125 times, 150 times or 200 times.

[0224] This ensures rapid freezing or vitrification of the sample, for example, avoiding the formation of harmful ice crystals. It further ensures that when thawing medium is dispensed into a receiving container containing a frozen biological sample, the storage medium surrounding the biological sample will be sufficiently diluted. This may be beneficial in mitigating any potential harmful effects of the medium components on the biological sample, such as those caused by certain cryoprotectants (present in the medium), such as DMSO.

[0225] Typically, biological specimens (e.g., cells, cell aggregates or clusters, or microtissues) are more or less spherical, including the surrounding vitrified or frozen medium (if present). In this case, their volume can be calculated by the formula V = 4 / 3·π·r 3 Approximate calculation, where r is the radius of the biological sample (see Figure 24 C).

[0226] If the hole or bottle is cylindrical, its volume can be calculated by the formula V = π·r 2h is calculated approximately, where r is the radius of the circular cross section and h is the height of the hole or bottle.

[0227] If the hole or bottle body is conical or conical, its volume can be calculated by the formula V = (1 / 3)·π·h·(r1 2 +r2 2 +(r1·r2)) approximate calculation, where h is the height of the hole or bottle, r1 is the lower radius of the bottle, and r2 is the upper radius of the bottle.

[0228] If the hole or bottle is hemispherical, its volume can be calculated by the formula V = 2 / 3·π·r 3 Approximate calculation, where r is the radius of the hole or bottle.

[0229] With respect to the embodiments of receiving containers, bottles or wells or biological samples described elsewhere herein, the features, characteristics and advantages associated with said embodiments, as well as the embodiments themselves, should be understood to apply to the receiving containers and their embodiments described above and below, even if not repeated again.

[0230] In one embodiment, the receiving container is a 96-well plate, such as an Akura TM 96-well plate (InSphero). In such embodiments, the volume of the well is typically ≥100 μL and ≤400 μL. In such embodiments, if V i At least V s If the volume of the biological sample is 100 times that of the sample, the maximum volume of the biological sample is, for example, ≤1 μL to ≤4 μL (including surrounding vitrified or frozen storage or culture medium, if any).

[0231] In one embodiment, the receiving container is a 384-well plate. In such embodiments, the volume of the wells is typically ≥80 μL and ≤200 μL. In such embodiments, if V i At least V s If the volume of the biological sample is 100 times that of the sample, the maximum volume of the biological sample is, for example, ≤ 0.8 μL to ≤ 2 μL (including surrounding vitrified or frozen storage or culture medium, if any).

[0232] In one embodiment, the receiving container is a 1536-well plate. In such embodiments, the volume of the wells is typically ≥10 μL and ≤200 μL. In such embodiments, if V i At least V s If the volume of the biological sample is 100 times that of the sample, the maximum volume of the biological sample is, for example, ≤ 0.1 μL to ≤ 2 μL (including surrounding vitrified or frozen storage or culture medium, if any).

[0233] The following table lists additional non-limiting examples:

[0234] Orifice Plate 96 384 <![CDATA[Typical pore volume range (V i )(μL)]]> ≥100 to ≤400 ≥80 to ≤200 <![CDATA[Volume of InSphero Akura V i (μL)]]> 280 130 <![CDATA[When V i V when it is at least 50 times s (μL)]]> ≤5.6 ≤2.6 <![CDATA[When V i V when it is at least 80 times s (μL)]]> ≤3.5 ≤1.65 <![CDATA[When V i is at least 100 times the V s (μL)]]> ≤2.8 ≤1.3 <![CDATA[When V i V when it is at least 120 times s (μL)]]> ≤2.3 ≤1.83 <![CDATA[When V i is at least 150 times the V s (μL)]]> ≤1.87 ≤0.87 <![CDATA[When V i is at least 200 times the V s (μL)]]> ≤1.4 ≤0.65

[0235] *V s Includes surrounding vitrified or frozen storage or culture medium (if present)

[0236] According to one embodiment, the receiving container has been prepared or is obtainable by the method as described above.For this purpose, one or more biological samples (17) are dispensed into one or more wells or vials (9) of the receiving container (16) as described above.

[0237] According to one embodiment of the method, only one biological sample (eg only one microtissue) is present in each well or bottle (9) of the receiving container.

[0238] Example

[0239] Although the invention has been described and illustrated in detail in the drawings and the foregoing description, such description and illustration should be regarded as illustrative or exemplary rather than restrictive; the invention is not limited to the disclosed embodiments. In implementing the claimed invention, other variations of the disclosed embodiments may be understood and implemented by a person skilled in the art by studying the drawings, the disclosure and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain technical features are recited in mutually different dependent claims does not indicate that the combination of these technical features cannot be fully utilized. Any figure marks in the claims should not be construed as limiting the scope of protection.

[0240] Materials and Methods

[0241] 1. The method itself

[0242] 1.1. Vitrification method

[0243] In Akura TM Human tumor microtissues (HepG2 cells) expressing eGFP formed and cultured in 96-well plates (InSphero) were removed from the incubator (37°C, 5% CO2) and placed in designated slots on the workbench of a Hamilton Vantage liquid handling system (Hamilton AG, Bonaduz, Switzerland). A column of cell culture medium was aspirated from the well plate and replaced with a culture medium containing a specific cryoprotectant (CPA) mixture selected from a range of cell permeable and non-permeable components (see Swain & Smith 2010, the contents of which are incorporated herein by reference for practicable purposes). This process was repeated twice while gradually increasing the CPA concentration. Each culture medium composition was maintained on the microtissue for 3 minutes to allow the microtissue to reach equilibrium with the culture medium. After the third equilibrium step, eight Hamilton An array of pipetting tips picks up the microtissue and settles it to the bottom of the tip.

[0244] Another Akura TM A 96-well plate is pre-cooled to below -190°C in a cryogenic chamber according to the present invention and cooled with liquid nitrogen. After the microtissues have equilibrated and settled, the cryogenic chamber lid is briefly opened, and the microtissues are spotted into the wells of the plate in a minimal volume of microliter or submicroliter culture medium. This minimal volume of culture medium is dispensed onto the pre-cooled culture plate, resulting in instantaneous rapid freezing of the culture medium and vitrification of the microtissues. The cryogenic chamber is then closed again to maintain the low temperature, and the process is repeated for the remaining columns of the same source plate.

[0245] 1.2. Thawing method

[0246] When thawing the culture plates, the medium array is rebalanced using three decreasing concentrations of CPA (see Swain & Smith 2010) and subsequently replaced with standard cell culture medium. The first medium used is preheated to above ambient temperature. The frozen plates are automatically removed from the cryogenic chamber and placed on a vented plate holder (vented plate) according to the present invention, which directs pressurized air to the bottom of the plate to dissipate the cold air around the plate and its wells. A 96-channel pipetting head (Hamilton) is used to simultaneously add preheated medium to all 96 wells of the plate, resulting in immediate thawing of the plate and microtissue while minimizing damage caused by potential ice crystal formation when the plate, medium and microtissue are warmed to temperatures above freezing.

[0247] After 1 minute, remove the prewarmed medium from the plate and replace it with the same medium at room temperature. Repeat the wash step 1 minute later. Subsequently, add re-equilibrated medium 2 and 3, incubate for 2 minutes each, and replenish with fresh medium after 1 minute of equilibration. Finally, replace the medium with standard tissue culture medium and place the plate in a 37°C, 5% CO2 incubator.

[0248] 2. Experiment

[0249] Preformed, unsupported microtissues are equilibrated in a mixture of a specific cryoprotectant (CPA), for example, during an equilibration process with increasing CPA concentrations, while the receiving container is precooled to -196°C in liquid nitrogen in a cryogenic chamber. The microtissues are then spotted as small droplets (0.3 to 1.0 μL or 0.5 to 1.0 μL volume) onto a receiving container (e.g., a 96-well microtissue plate; one microtissue per well) using Hamilton's MagPip pipetting technology. The droplets containing the microtissue freeze instantly upon contact with the cold surface. This process enables the aqueous sample to be cooled at the high rate required to achieve a non-crystalline, amorphous glass state (vitrification). Vitrification transforms the aqueous solution into an amorphous glass state, avoiding the cellular damage associated with ice crystal formation typically observed using traditional freezing methods. The tissue can then be stored in the liquid nitrogen vapor phase at temperatures below 150°C for long-term storage.

[0250] During thawing, preheated thawing medium is added to the receiving container from the top. Simultaneously, bottom heating is achieved using a vent plate, allowing the tissue to transition rapidly from a vitrified state to a liquid state, minimizing the potential for harmful ice crystal formation.

[0251] This process has been tested using preformed liver microtissues constructed from primary human hepatocytes and non-parenchymal hepatocytes (see Proctor et al. (2017), which is incorporated herein by reference for applicable purposes). The microtissues were frozen in standard 96-well microplates using the cryostat described above. After thawing using a vented plate, the microtissues were compared to unfrozen control microtissues. Morphology showed that the thawed liver microtissues retained a dense spherical shape, similar to unfrozen liver microtissues. H&E staining and immunostaining for albumin (hepatocytes), BSEP (tubule structure), and CD68 (Kupffer cells) showed a staining pattern similar to that of unfrozen microtissues (see Figure 15 ).

[0252] On days 2 and 7 after thawing, the ATP content of individual microtissues was 85% to 75% of that of unfrozen control tissues (see Figure 16 A). Thawed microtissues and control microtissues showed significant differences in albumin secretion (see Figure 16 B), CYP450 enzyme activity (1A2, 2B6, 2C9, 3A4, see Figure 17 ) and reactivity to three known hepatotoxic compounds, as shown by a 7-day repeated dose-response toxicity study (ATP) with chlorpromazine, troglitazone, and tolcapone, where the IC50 values of thawed microtissues were similar to those of control microtissues ( Figure 18 , see also Messner et al. (2013), the contents of which are incorporated herein by reference for all applicable purposes).

[0253] These results demonstrate the feasibility of cryopreserving highly differentiated spheroids based on primary cells in standard microplates for long-term storage without loss of functionality, and that these spheroids can be used directly for drug safety testing once thawed.

[0254] Reference numerals

[0255] 1. First compartment

[0256] 2. Upper reservoir

[0257] 3. Second compartment

[0258] 4. Support for receiving container

[0259] 5. Overflow edge

[0260] 6. (Second) overflow edge

[0261] 7. Overflow compartment

[0262] 8. (Third) compartment

[0263] 9. One or more holes or bottles

[0264] 10. One or more cavities

[0265] 11. Positioning grid

[0266] 12. Lower reservoir

[0267] 13. Side of upper reservoir

[0268] 14. Cover

[0269] 15. Opening for filling with cryogenic liquid

[0270] 16. Receiving container

[0271] 17. Biological samples

[0272] 18. Ventilation panel

[0273] 19. Cavity in the vent plate

[0274] 20. Thawing medium supply channel

[0275] 21. Exhaust duct

[0276] 22. Pipette Tips

[0277] 23. Thawing Medium

[0278] 24. Spacers

[0279] 25. Cryogenic liquid filling nozzle

[0280] 26. Thruster

[0281] 27. Control nozzle

[0282] 28. Overflow edge groove

[0283] 29. Flat bottom of microplate

[0284] 30. Deflector

[0285] 31. Housing

[0286] 32. Air intake (cold air)

[0287] 33. Pipette Array

[0288] 34. Air intake (hot air)

[0289] 35. Plug for sealing opening 15

[0290] 36. Flat air nozzle

[0291] 37. Tube heating device

[0292] 38. Air Pump

[0293] 39. Hot air source

[0294] 40. Cladding

[0295] 41. Laminar / Non-Turbulent Gas Flow

[0296] 42. The space formed between the cladding 40 and the outer shell 31

[0297] 43. Vitrified or frozen storage or culture medium surrounding biological specimens 17

[0298] V i The inner volume of the hole or bottle 9

[0299] V s The volume of the biological sample 17, including the surrounding vitrified or frozen storage or culture medium 43 (if present)

[0300] C Cryogenic liquid

[0301] evC Evaporated cryogenic liquid

[0302] A-A' This line marks the height of the second overflow edge (6), the height of the support (4) of the receiving container and the height of the opening of the control nozzle (27)

[0303] References

[0304] Jason E.Swain and Gary D.Smith,4-Cryoprotectants from Cryobiology,in:Ri-Cheng Chian and Patrick Quinn(eds)Fertility Cryopreservation,pp.24-38Cambridge University Press(2010).

[0305] Whaley D,Damyar K,Witek RP,Mendoza A,Alexander M,LakeyJR.Cryopreservation:An Overview of Principles and Cell-SpecificConsiderations.Cell Transplant.2021Jan-Dec;30.

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Claims

1. A system for freezing and / or vitrifying a biological sample (17), the system comprising a cladding (40) configured to contain a cryogenic liquid (C), and A support (4) for a receiving container (16), the receiving container (16) being suitable and / or arranged to receive one or more biological samples (17).

2. The system according to claim 1, further comprising a first compartment (1) and a second compartment (3), wherein the first compartment (1) comprises an upper liquid reservoir (2), wherein the second compartment (3) comprises a lower liquid reservoir (12), wherein each liquid reservoir (2, 12) is adapted and / or configured to contain a cryogenic liquid (C), The second compartment (3) is arranged below the first compartment (1), and The second compartment comprises a support (4) for a receiving container (16), wherein the receiving container (16) is suitable for and / or arranged to receive one or more biological samples (17).

3. The system according to any of the preceding claims, wherein the system further comprises a receiving container (16) adapted and / or arranged to receive one or more biological samples (17).

4. A system according to any one of the preceding claims, wherein the upper liquid reservoir (2) comprises a first overflow edge (5), which is arranged to allow the cryogenic liquid (C) to flow into the lower liquid reservoir (12) if the liquid level of the cryogenic liquid (C) in the upper liquid reservoir (2) exceeds the height of the first overflow edge (5).

5. A system according to any one of the preceding claims, wherein the second compartment (3) includes a second overflow edge (6), the second overflow edge (6) being configured to form an overflow compartment (7) within the second compartment (3), wherein the second overflow edge (6) is configured to allow the cryogenic liquid (C) to flow from the lower liquid reservoir (12) within the second compartment into the overflow compartment (7) if the liquid level of the cryogenic liquid (C) in the lower liquid reservoir (12) exceeds the height of the second overflow edge (6).

6. System according to any of the preceding claims, wherein the height of the second overflow edge (6) is aligned with the height of the support (4) of the receiving container (16).

7. A system according to any one of the preceding claims, wherein the system further comprises a third compartment (8) arranged below the second compartment (3), the third compartment (8) being fluidically connected to the second compartment (3) and / or the overflow compartment (7), and being suitable for and / or arranged to accommodate a cryogenic liquid (C).

8. A system according to any of the preceding claims, wherein the system further comprises a cover plate (14), the cover plate (14) comprising a first opening (15) suitable for and / or arranged to fill the upper liquid reservoir (2) of the first compartment (1) with a cryogenic liquid (C).

9. A system according to any of the preceding claims, wherein the system further comprises a cryogenic liquid (C) filling nozzle (25) adapted for and / or arranged to fill the cryogenic liquid (C) directly into the second compartment (3) and / or the overflow compartment (7) and / or the third compartment (8).

10. System according to any of the preceding claims, wherein the system further comprises a control nozzle (27) with an opening, wherein the height of the opening of the control nozzle is aligned with the height of the second overflow edge (6) of the second compartment (3).

11. A system according to any of the preceding claims, wherein the support (4) of the receiving container (16) comprises one or more cavities (10) on its upper side, suitable and / or arranged to accommodate one or more holes or bottles (9) of the receiving container (16), and the receiving container (16) is suitable and / or arranged to accommodate a biological sample.

12. System according to any of the preceding claims, wherein the one or more cavities (10) are arranged to be substantially form-locked with at least part of the body (9) or one or more holes of the receiving container (16).

13. System according to any of the preceding claims, wherein the support (4) of the receiving container comprises a material having a wicking effect on the cryogenic liquid.

14. System according to any of the preceding claims, wherein the support (4) of the receiving container comprises a material having an open-pore sponge-like structure.

15. System according to any of the preceding claims, wherein the support (4) of the receiving container comprises a material that retains elastic deformation capability at temperatures below -70°C and / or in the presence of a cryogenic liquid (C).

16. System according to any of the preceding claims, wherein the absolute height of the support (4) of the receiving container is ≥ 5 and ≤ 50 mm.

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

18. The system according to any one of the preceding claims, wherein the third compartment (8) comprises a material having at least one of the following properties: a) has a wicking effect on cryogenic liquids, b) open-pore sponge-like structure, c) maintains elastic deformability at temperatures below -70°C and / or in the presence of cryogenic liquids, and / or d) comprising a material selected from the group consisting of: Melamine (1,3,5-triazine-2,4,6-triamine) or melamine resin, Polyurethane, and / or Cross-linked polyolefin foam.

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

20. System according to any of the preceding claims, wherein the support (4) is a support according to any of claims 13 to 18.

21. The system according to any of the preceding claims, further comprising a pipetting device (22, 33) for dispensing a biological sample to one or more wells or vials (9) of the receiving container (16).

22. The system according to claim 23, further comprising ventilation means (30, 32, 34, 36, 37, 38, 39) for generating a flow of warm air along the pipette tips (22) of the pipetting device (33).

23. The system of claim 24, wherein the ventilation device comprises at least one element selected from the group consisting of: ·Deflector(30) Air intake (32) Air Inlet (34) Flat air nozzle (36) Tube heating device (37) Air pump (38) Warm water bath (39).

24. System according to any of the preceding claims, wherein the support (4) of the receiving container is arranged on one or more spacers (24).

25. The system according to any of the preceding claims, wherein the receiving container (16) comprises one or more wells or bottles (9) adapted and / or arranged to receive a biological sample.

26. The system according to any of the preceding claims, wherein the receiving container (16) is a microplate or an array of vials corresponding to a 6-well, 24-well, 96-well or 384-well microplate layout.

27. A support (4) for freezing or vitrifying a biological specimen contained in a receiving container (16), the support comprising a material having a wicking effect for cryogenic liquids.

28. The support according to claim 27, wherein the cryogenic liquid (C) is selected from the group consisting of: Liquid nitrogen, Liquid helium, Liquid argon, Liquid oxygen, A mixture of dry ice (CO2) and an organic solvent.

29. A support according to any one of the preceding claims, wherein the support comprises a material having an open-cell sponge-like structure.

30. A support according to any preceding claim, wherein the support comprises a material that retains the ability to deform elastically at temperatures below -70°C and / or in the presence of cryogenic liquids.

31. A support according to any one of the preceding claims, wherein the absolute height of the support is > 5 and < 50 mm.

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

33. A support according to any of the preceding claims, wherein the support comprises on its upper side one or more cavities (10), suitable and / or arranged to receive one or more holes or bottles (9) of a receiving container (16), the receiving container (16) being suitable and / or arranged to receive a biological sample.

34. Support according to claim 33, wherein said one or more cavities (10) are arranged to be substantially form-locked with at least part of said one or more holes or bottle bodies (9).

35. The support according to any one of the preceding claims, wherein the receiving container (16) is a microplate or an array of vials corresponding to a 6-well, 24-well, 96-well or 384-well microplate layout.

36. A vent plate (18) for thawing a biological sample (17) contained in a receiving container (16), the vent plate comprising one or more cavities (19) on its upper side adapted and / or arranged to receive one or more wells or bottles (9) of a receiving container (16), the receiving container (16) being adapted and / or arranged to receive the biological sample (17), the vent plate (18) further comprising one or more supply channels (20) adapted and / or arranged to supply thawing medium to the hole of the receiving container (16) or the outer wall of the bottle body (9), and One or more exhaust channels (21) adapted and / or arranged to exhaust the thawing medium from the hole of the receiving container or the outer wall of the bottle body (9).

37. The aeration panel (18) of claim 36, wherein the thawing medium (23) is a fluid, liquid or gas.

38. The aeration plate according to any one of claims 36 to 37, wherein the temperature of the thawing medium (23) is ≥ -150°C and ≤ +100°C.

39. A system for thawing a biological sample contained in a receiving container (16), the system comprising a) A vent plate (18) according to any one of claims 36 to 38, and b) a pipetting device (22) adapted and / or arranged to dispense a thawing medium (23) into the well or bottle (9) of the receiving container (16).

40. The system, aeration panel (18) or support (4) according to any one of the preceding claims, wherein the biological sample (17) is at least one selected from the group consisting of: One or more tissue samples, One or more gametocytes (oocytes or spermatocytes), One or more blastocysts, One or more single cells, One or more cell aggregates (e.g., microtissues, spheroids), and / or One or more organoids formed through the self-organization and differentiation of stem cells.

41. A method for freezing and / or vitrifying a biological sample (17), the method comprising the steps of: a) placing a receiving container (16) suitable for and / or arranged to receive one or more biological samples (17) on a support (4) according to any one of claims 13 to 18, said support (4) being arranged in a system according to any one of claims 27 to 35; b) before or after step a), dispensing one or more biological samples (17) into one or more wells or bottles (9) of the receiving container (16); and c) before or after step a) or b), filling the system according to any one of claims 1 to 26 with a cryogenic liquid (C).

42. The method of claim 34, further comprising the step of adding one or more cryoprotectants to the biological sample prior to step b).

43. A method for storing a biological sample frozen and / or vitrified by the method according to claim 34 or 35, the method comprising the following steps: a) maintaining a receiving container (16) containing one or more frozen and / or vitrified biological samples (17) in a system according to any one of claims 1 to 26; b) optionally, positioning a cover plate (14) on top of the system according to any one of claims 1 to 26; and c) Optionally, the system according to any one of claims 1 to 26 is supplemented with cryogenic liquid (C).

44. A method for thawing a biological sample (17) frozen and / or vitrified by the method according to claim 39 or 40 and / or stored by the method according to claim 43, the method comprising the following steps: a) removing the receiving container (16) from the support (4); b) placing the receiving container (16) on the aeration plate (18) according to any one of claims 36 to 38; c) supplying a thawing medium (23) to the ventilation plate (18); Optionally, the thawing medium (23) is dispensed from a pipetting device (22, 33) arranged above the receiving container (16) simultaneously with step c) or before or after step c).

45. A receiving container (16) comprising one or more wells or vials (9) containing one or more frozen or vitrified biological samples (17), wherein the inner volume V of at least one well or vial (9) is i At least the volume V of the biological sample (17) s 100-fold, including surrounding vitrified or frozen storage or culture medium, if any.

46. The receiving container according to claim 45, which is prepared or obtained by the method according to any one of claims 41 to 42.

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