Methods for large-scale banking of human pluripotent stem cells and products derived therefrom - Patents.com

Abstract

The present invention relates to a method for establishing a cell bank, comprising the steps of providing a batch of cell suspension comprising stem cells or stem cell-derived cells and a cryopreservation medium, maintaining the batch of cell suspension at a temperature below 15° C., and transferring a quantity of the cell suspension from the batch of cell suspension to one or more storage containers, wherein establishing the cell bank takes at least (30) minutes.

Description

[Technical field]

[0001] The present invention relates generally to the field of stem cells, such as human pluripotent stem cells, and cell products derived from stem cells.Specifically, a method is provided for establishing cell banks from human pluripotent stem cells (hPSCs), such as undifferentiated hPSC banks and hPSC-derived drug substances and drug products. [Background technology]

[0002] The prospects of using hPSCs to treat a variety of disease conditions seem very promising. As efforts are made to bring live cell-based therapies closer to the market, it is becoming increasingly relevant to consider manufacturing at a large scale and in a production environment. For some indications, an estimated 10 8 cells are required. The production of stem cell-based products typically uses differentiation of hPSCs as a starting point. Cell lines may be derived from early stage human embryos or from induced pluripotency of somatic cells. The resulting cell lines are then cultured for expansion and establishment of cell banks, in which the cells are cryopreserved for storage until they undergo further expansion and / or differentiation into a specific cell type product.

[0003] Over the years, there have been significant improvements in the cryopreservation of hPSCs. The basic cryopreservation principle has its origins in other mammalian cell types, and the procedure relies on chemicals in the cryopreservation medium, such as the disaccharides sucrose, maltose, trehalose, and others with or without DMSO.

[0004] The main cause of cell death is not the long-term storage at low temperature per se, but the glass transition temperature (T g ) are events that occur in cells at ultra-high temperatures. Intracellular crystallization during freezing and recrystallization upon warming are known to be the most damaging to cells, aside from other known causes such as toxicity, cell shrinkage, and osmotic imbalance. To combat these harmful events to cells, especially hPSCs, different cryopreservation methods (vitrification, slow cooling, rapid thawing, etc.) and cryopreservation media have been developed.

[0005] For therapeutic cell production, long-term cell stability and methodologies that allow for the generation of large cell banks are essential for success. It is therefore an object of the present invention to improve current methods for establishing cell banks with high cell viability, while complying with the requirements of therapeutic drug products, including good cell culture practice (GCCP) and current good manufacturing practice (cGMP), and in particular addressing the problems that arise from the establishment of cell banks, which take a long time due to preparation and / or large-scale banking. Summary of the Invention

[0006] The above outlined objectives are achieved by the aspects of the present invention, which may also solve further problems that will become apparent from the disclosure of the exemplary embodiments.

[0007] In a first aspect of the present invention, a method for establishing a cell bank is provided, comprising the steps of providing a batch of cell suspension comprising stem cells or stem cell-derived cells and a cryopreservation medium, maintaining the batch of cell suspension at a temperature below 15° C., and transferring an amount of cell suspension from the batch of cell suspension to one or more storage containers to establish a cell bank, where establishing the cell bank takes at least 30 minutes. The inventors have realized that establishing a large cell bank by filling numerous storage containers, such as vials, with cells requires a long time, during which the viability of cells in the cryopreservation medium is significantly reduced. The same is true during the establishment of a cell bank, where the batch of cell suspension requires additional preparation time. Maintaining the batch of cell suspension at a temperature below 15° C. throughout the process of establishing a cell bank will improve the quality and cell number of each vial.

[0008] In a further aspect, a cooling device for cooling a batch of cell suspension during preparation of a cell bank is provided, the cooling device comprising a housing including an outer wall, a first compartment configured to receive a refrigerant, and a second compartment configured to hold a container with the batch of cell suspension, the first and second compartments being separated by a heat conducting element such that the refrigerant is capable of cooling the batch of cell suspension when the refrigerant is loaded into the first compartment and the container with the batch of cell suspension is loaded into the second compartment. The inventors have designed a cooling device suitable for maintaining the batch of cell suspension at a temperature below 15° C. while establishing a cell bank, and the use of the cooling device facilitates compliance with the stringent requirements of GCCP and cGMP. The cooling equipment is designed not to generate any particles during operation and therefore the equipment can be handled within a Grade A (or ISO designation 5) cleanroom area classification that complies with the requirements of EU GMP Guidance Annex 1: Manufacturing of Sterile Medicinal Products and US Food and Drug Administration's (FDA's) Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing Current Good Manufacturing Practice.

[0009] Thus, another aspect of the present invention relates to the use of a cooling device for preparing a cell bank from a batch of cell suspension comprising maintaining the temperature of the batch of cell suspension below 15°C, preferably between 0 and 12°C, more preferably between 0 and 10°C, more preferably between 0 and 8°C, more preferably between 2 and 8°C, more preferably between 2 and 6°C, more preferably between 3 and 5°C, even more preferably at about 4°C.

[0010] In a final aspect, there is provided a method of cooling a batch of cell suspension comprising the steps of providing a cooling apparatus as described above, loading a refrigerant into a first compartment of the cooling apparatus, the temperature of the refrigerant being suitable for maintaining a temperature of the batch of cell suspension below 15°C, loading a flask containing the batch of cell suspension into a second compartment of the cooling apparatus, and maintaining a temperature of the batch of cell suspension below 15°C. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows a housing of a cooling device having a temperature conducting element separating a first compartment from a second compartment. [Diagram 2] FIG. 2 shows a top view of a cooling device having a heat conducting element separating a first compartment and a second compartment. [Diagram 3] FIG. 3 illustrates a partially disassembled cooling device with a lid, a heat conducting element, and a housing with one heat conducting element attached to separate a first compartment and a second compartment. [Figure 4] FIG. 4 shows the trypan blue exclusion viability of hESCs over different exposure times to cryopreservation media (specifically STEM-CELLBANKER) at room temperature. [Diagram 5] FIG. 5 shows a trypan blue exclusion study of hESC viability over increasing exposure times to cryopreservation media (specifically STEM-CELLBANKER) when cell suspensions were maintained at approximately 4° C. [Figure 6] FIG. 6 shows the temperature measurement curve of the medium in the container of the cooling device. [Figure 7]Figure 7 shows the % viability of hESC-derived beta-like cells measured by NC-202 for different holding times in cryopreservation media (e.g., StemcellBanker) at room temperature and approximately 4 degrees Celsius. The average % viability drops from 82 to 66 at room temperature and from 89 to 85 at 4°C (5 min to 240 min). Six to eight vials were thawed for each time point and were thawed by three different operators. Two-way ANOVA Sidak's multiple comparison test was performed to compare room temperature and 4°C. [Figure 8] Figure 8 shows flow cytometry dot plots comparing the percentage of cells expressing the NKX6.1 and Islet-1 markers, with 49.5% co-expressing the two markers indicative of beta cells. [Figure 9] Figure 9 shows the % viability of hESC-derived RPE cells measured by NC-202 for different holding times in cryopreservation media (e.g., StemcellBanker) at room temperature and approximately 4 degrees Celsius. The % viability drops from 95 to 81 at room temperature and from 95 to 89 at 4°C (0 min to 240 min). Eight to ten vials were thawed for each time point and were thawed by three different operators. Two-way ANOVA Sidak's multiple comparison test was performed to compare room temperature and 4°C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology, and pharmacology known to those skilled in the art.

[0013] Please note that all headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[0014] The use of any and all examples or exemplary phrases (e.g., "such as") presented herein is intended merely to make the invention more clear and does not limit the scope of the invention unless otherwise claimed.

[0015] General definition Throughout this application, the terms "method" and "protocol" when referring to a process for differentiating cells may be used interchangeably. As used herein, "a" or "an" or "the" may mean one or more than one. Unless otherwise indicated herein, terms presented in the singular also include plural situations. Also, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or"). Furthermore, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features described herein may be excluded or omitted.

[0016] In the following, the method according to the invention is described in more detail by non-limiting embodiments and examples. A method is provided for establishing a large stem cell bank. The method is therefore offset in the use of stem cells.

[0017] "Stem cells" should be understood as cells that have differentiation and proliferation potential (especially self-renewal potential) but maintain differentiation potential. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, unipotent stem cells, and the like according to their differentiation potential. As used herein, the term "pluripotent stem cells" (PSCs) refers to stem cells that can be cultured in vitro and have the ability to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm) and / or extraembryonic tissues (pluripotency). As used herein, the term "pluripotent stem cells" refers to stem cells that have the ability to differentiate into multiple, but not all, types of tissues or cells. As used herein, the term "unipotent stem cells" refers to stem cells that have the ability to differentiate into specific tissues or cells. Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germ stem cells, stem cells in tissues, somatic cells, and the like. Examples of pluripotent stem cells (PSCs) include embryonic stem cells (ESCs), EG cells (embryonic germ cells), induced pluripotent stem cells (iPSCs), and the like. Muse cells (multilineage differentiation stress-resistant cells) obtained from mesenchymal stem cells (MSCs) and GS cells produced from germ cells (e.g., testes) are also included in pluripotent stem cells. As used herein, the term "induced pluripotent stem cells" (also known as iPS cells or iPSCs) refers to a type of pluripotent stem cell that can be generated directly from adult cells. As used herein, the term "embryonic stem cells" (also known as hES cells or hESCs) refers to a type of pluripotent stem cell that is derived from either a single blastomere or the inner cell mass of a blastocyst, or from a parthenogenetic organism (e.g., as described in International Publication No. WO2003 / 046141). Embryonic stem cells are available from a given tissue and are also commercially available. Preferably, the methods and products of the invention are based on hPSCs, ie stem cells derived from either induced pluripotent stem cells or embryonic stem cells, including parthenogenetic organisms.

[0018] "NC-202" viability measurements refer to The NucleoCounter® NC-202™ Automated Cell Counter (NucleoCounter® NC-202™ Leading Automated Cell Counter System (chemometec.com)).

[0019] As used herein, the term "RPE cells" means retinal pigment epithelial cells.

[0020] Methods for establishing cell banks In a first aspect of the present application, there is provided a method for establishing a cell bank, comprising the steps of: a) providing a batch of cell suspension comprising stem cells or stem cell-derived cells and a cryopreservation medium; b) maintaining the batch of cell suspension at a temperature below 15° C.; and c) transferring an amount of cell suspension from the batch of cell suspension to one or more storage containers to establish the cell bank, wherein establishing the cell bank takes at least 30 minutes.

[0021] As used herein, the term "cell bank" refers to aliquots of a single pool of cells, typically prepared from an individual source derived under defined conditions, dispensed into multiple containers, and stored under defined conditions, which may further be pools of different cell types.

[0022] As used herein, the term "cell suspension" means that cells are freely suspended in a liquid medium, thus allowing for a substantially uniform concentration of cells to be maintained, in which an aliquot of the liquid medium containing the cells can be transferred. The cells in the cell suspension may be single cells or cell aggregates / spheroids, with or without associated biomaterial.

[0023] As used herein, the term batch of cell suspension refers to a cell suspension that is to be transferred to one or more storage vessels.

[0024] As used herein, the term "stem cell-derived cell" refers to a stem cell that has been differentiated. The term "differentiation" with respect to pluripotent stem cells refers to the process by which a cell progresses from an undifferentiated state to a specific differentiated state, i.e., from an immature state to a less immature or mature state. Changes in cell interactions and maturation occur when a cell loses markers of an undifferentiated cell or acquires markers of a differentiated cell. Loss or acquisition of a single marker can indicate that a cell has become "mature or fully differentiated".

[0025] "Providing a batch of cell suspension" in step a) means obtaining a batch of cell suspension by any suitable means. In one embodiment, the batch of cell suspension is provided by compounding stem cells or stem cell-derived cells with a cryopreservation medium. In one embodiment, the concentration of cells in the batch of cell suspension is about 1×10 5 cells / ml ~ approx. 1×10 8 cells / ml. In one embodiment, the stem cells are PSCs. In a further embodiment, the stem cells are hPSCs. The stem cells may be provided by any suitable method as described above, for example, the stem cells may be provided by deriving them from human embryonic stem cells. A person skilled in the art will recognize suitable methods for obtaining or providing stem cells, which may include deriving stem cells from blastomeres or the inner cell mass of blastocysts. As used herein, "cryopreservation medium" refers to a liquid medium composition suitable for preserving cells during freezing. In one embodiment, the cryopreservation medium comprises dimethyl sulfoxide (DMSO). In a preferred embodiment, the cryopreservation medium is chemically defined, non-xenogeneic, and GMP grade. Suitable cryopreservation media are commercially available, for example, STEM-CELLBANKER.

[0026] In step b) the batch of cell suspension is maintained at a temperature below 15°C. In one embodiment the temperature is maintained at 0-12°C, preferably 0-11°C, preferably 0-10°C, preferably 0-8°C, more preferably 2-8°C, more preferably 2-6°C, more preferably 3-5°C, even more preferably about 4°C. In one embodiment the temperature is maintained below 15°C, 14°C, 13°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C or 4°C, preferably below 8°C. However, the temperature of the batch of cell suspension should not be below the freezing point of the cell suspension. The person skilled in the art can easily establish the freezing point of the cell suspension. In one embodiment the temperature is maintained above the freezing point of the cell suspension. In a further embodiment the temperature is maintained above 0°C, 1°C, 2°C or 3°C. The temperature may be maintained at said temperature by any suitable means, such as by cooling the container with the batch of cell suspension or by cooling the surrounding environment in which the cell bank is established. In one embodiment, the batch of cell suspension in step b) is subjected to agitation, such as shaking or stirring. The batch of cell suspension may be stirred by any suitable means, such as by magnetic stirring. Agitation may facilitate a homogenous cell suspension, ensuring a substantially uniform concentration of cells transferred to each vial in step c).

[0027] In step c), a quantity of cell suspension from the batch of cell suspension is transferred to one or more storage containers. In one embodiment, a quantity of cell suspension is transferred to at least 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 storage containers. As used herein, the term "storage container" refers to a container suitable for storing cells. Any container suitable for storing cells can be used, such as glass or plastic containers or bottles or bags. In one embodiment, the storage container is a vial or a cryopreservation bag. As used herein, the terms "amount" and "aliquot" are used interchangeably and may refer to a volume smaller than the initial volume of the cell suspension. Any process or method for transferring a quantity of cell suspension can be used. The transferring is typically performed manually by a pipette, but may be automated by robotic assistance. In one embodiment, the amount of cell suspension transferred to each vial is 0.25 to 1000 ml. In one embodiment, about 1 ml of the volume is transferred to a 2 ml cryovial. In one embodiment, about 20 ml of the volume is transferred to a 50 ml cryovial. In one embodiment, the volume of the batch of cell suspension is at least 25 ml, 50 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, or 1 L.

[0028] In one embodiment, it takes at least 1 hour, at least 2 hours, or at least 3 hours to establish a cell bank. The time that the cell suspension is maintained at the defined temperature depends on the time that it takes to establish a cell bank, i.e., the time that it takes to prepare and / or transfer a certain amount of cell suspension to each of a defined number of vials. It is easily understood that the step of transferring an aliquot of the cell suspension to a storage container is performed while the remaining batch of cell suspension is maintained at the temperature. Once a certain amount of cell suspension is transferred to a storage container, it is no longer necessary to maintain it at the temperature.

[0029] In one embodiment, the method further comprises a step d) of freezing each of the storage containers immediately after transferring the amount of cell suspension. In addition to the discovery by the inventors, it is easily understood that the individual storage containers after transferring the aliquot of cell suspension should not be left at room temperature, for example, while establishing the remaining cell bank. The term "immediately" means that the cell suspension transferred to the vial is subjected to freezing within less than 10 minutes after transfer, preferably within less than 8, 6, 4, or 2 minutes after transfer. Those skilled in the art will recognize suitable methods for freezing, as well as recommended freezing temperatures for storage.

[0030] In one embodiment, the method is for increasing the viability of cells in a cell suspension.

[0031] In one embodiment, the stem cells or stem cell-derived cells are single cells or aggregates, hi another embodiment, the batch of cell suspension further comprises a biological material.

[0032] In one embodiment, the batch of cell suspension of step a) is compounded in a cell culture spinner bottle, which is placed in a cooling device according to the present invention.

[0033] cooling device In another aspect, there is provided a cooling device 100 for cooling a batch of cell suspension during preparation of a cell bank, the cooling device 100 comprising a housing 101 including an outer wall 102, a first compartment 104, 105 configured to receive a refrigerant, and a second compartment 103 configured to hold a container having a batch of cell suspension, the first compartment 104, 105 and the second compartment 103 being separated by heat conducting elements 106, 107 such that the refrigerant is capable of cooling the batch of cell suspension when the refrigerant is loaded into the first compartment 104, 105 and the container having the batch of cell suspension is loaded into the second compartment 103.

[0034] In one embodiment, the housing 101 is made from nylon. In one embodiment, the housing 101 is 3D printed using SLS printing, which results in a nylon material that is an inert and robust material that can withstand frequent sanitization with EtOH / IPA.

[0035] In one embodiment, the cooling device 100 comprises two first compartments 104, 105 on either side of the second compartment 103. As a result, in this embodiment, both first compartments 104, 105 are separated by heat conducting elements 106, 107. This results in a more even distribution of cooling of the batch of cell suspension. The heat conducting elements 106, 107 can be any suitable material for conducting heat. In one embodiment, the heat conducting elements 106, 107 are metal blocks, preferably stainless steel blocks. In one embodiment, the heat conducting elements 106, 107 are curved to closely fit the container with the batch of cell suspension.

[0036] In one embodiment, the cooling apparatus further comprises a lid 200 to cover the one or more first compartments 104,105.

[0037] As used herein, the term "refrigerant" refers to a substance suitable for cooling. In one embodiment, the refrigerant is in a sealed container. In a preferred embodiment, the refrigerant is in a bag, such as a gel pack. In one embodiment, the refrigerant is pre-cooled to a temperature below -20°C.

[0038] As used herein, the term "container" in relation to the cooling device refers to a container containing a batch of cell suspension that is to be dispensed into smaller storage containers such as vials. In one embodiment, the container with the batch of cell suspension has a volume of 50 ml to 10 L, preferably 1 L. In one embodiment, the container with the batch of cell suspension is a cell culture spinner bottle.

[0039] In a preferred embodiment, the cooling system is GMP compliant, specifically GMP compliant for a clean room Class A environment.

[0040] How to cool a cell suspension Consequently, another aspect of the present invention relates to a method for cooling a batch of cell suspension comprising the steps of: i) providing a cooling device 100 as described above; ii) loading a refrigerant into a first compartment 104, 105 of the cooling device 100, the temperature of the refrigerant being suitable for maintaining a temperature of the batch of cell suspension below 15°C; iii) loading a container containing the batch of cell suspension into a second compartment 103 of the cooling device 100; and iv) maintaining a temperature of the batch of cell suspension below 15°C.

[0041] In one embodiment, the refrigerant is a cold pack. As used herein, the term "cold pack" refers to a container, such as a plastic bag, filled with a refrigerant. In one embodiment, the temperature of the refrigerant is -20 to -80°C upon loading into the first compartment 104, 105. In one embodiment, the temperature of the cell suspension is maintained at 0 to 15°C, preferably 0 to 12°C, preferably 0 to 10°C, preferably 0 to 8°C, more preferably 2 to 8°C, more preferably 2 to 6°C, more preferably 3 to 5°C, and even more preferably about 4°C.

[0042] In a preferred embodiment, the method is GMP compliant.

[0043] Use of cooling device Another aspect of the present invention relates to the use of a cooling device as described hereinbefore for preparing a cell bank from a batch of cell suspension, comprising maintaining the temperature of the batch of cell suspension below 15° C., preferably between 0 and 12° C., preferably between 0 and 10° C., more preferably between 0 and 8° C., more preferably between 2 and 8° C., more preferably between 2 and 6° C., more preferably between 3 and 5° C., even more preferably at about 4° C. In a preferred embodiment, the cells are stem cells or are derived from stem cells. In an even more preferred embodiment, the stem cells are human pluripotent stem cells (hPSCs).

[0044] In one embodiment, the refrigerant is exchanged as needed to maintain a low temperature for the batch of cell suspension.

[0045] Specific Embodiments Aspects of the present invention will now be further illustrated by the following non-limiting embodiments. 1. A cooling device (100) for cooling a batch of cell suspension during preparation of a large cell bank, the cooling device (100) comprising a housing (101) including an outer wall (102), a first compartment (104) configured to receive a refrigerant, and a second compartment (103) configured to hold a container with the batch of cell suspension, the first compartment (104) and the second compartment (103) being separated by a heat conducting element (106) such that the refrigerant is capable of cooling the batch of cell suspension when the refrigerant is loaded into the first compartment (104) and the container with the batch of cell suspension is loaded into the second compartment (103). 2. A cooling device according to embodiment 1, wherein the housing (101) is made of nylon. 3. The cooling device according to embodiment 1 or 2, wherein the heat conducting element (106) is a metal block, preferably a stainless steel block. 4. A cooling device according to any one of embodiments 1 to 3, wherein the refrigerant is in a sealed bag, such as a gel pack. 5. A cooling device according to any one of embodiments 1 to 4, wherein the container with the batch of cell suspension has a volume of 50 ml to 10 L, preferably 1 L. 6. The cooling device according to any one of embodiments 1 to 5, wherein the container with the batch of cell suspension is a cell culture spinner. 7. A cooling device according to any one of embodiments 1-6, wherein the cooling device complies with GMP. 8. A cooling device according to any one of the preceding embodiments, wherein the cooling device comprises two first compartments (104, 105) on either side of a second compartment (103). 9. A cooling device according to embodiment 8, wherein each of the two first compartments (104, 105) is separated from the second compartment (103) by a heat conducting element (106, 107). 10. A cooling device according to any one of embodiments 1 to 9, further comprising a lid (200) for covering the one or more first compartments (104, 105). 11. A method for establishing a cell bank, comprising: a. providing a batch of cell suspension comprising stem cells or stem cell-derived cells and a cryopreservation medium; b. maintaining the batch of cell suspension at a temperature below 15° C.; c. transferring a quantity of the cell suspension from the batch of cell suspension to one or more storage containers to establish a cell bank; Establishing a cell bank takes at least 30 minutes. 12. d. The method according to embodiment 11, comprising the additional step of freezing each of the storage containers immediately after transferring said amount of cell suspension. 13. The method according to any one of embodiments 11 and 12, wherein a batch of a volume of cell suspension is transferred to at least 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 storage containers. 14. The method according to any one of embodiments 11-13, wherein the temperature is maintained at 0-15°C, preferably 0-12°C, preferably 0-11°C, preferably 0-10°C, preferably 0-8°C, more preferably 2-8°C, more preferably 2-6°C, more preferably 3-5°C, even more preferably about 4°C. 15. The method according to any one of embodiments 11-14, wherein the temperature is maintained below 15°C, 14°C, 13°C, 12°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, or 4°C, preferably below 12°C, more preferably below 8°C. 16. The method according to any one of embodiments 11-15, wherein the temperature is maintained above the freezing point of the cell suspension. 17. The method according to any one of embodiments 11-16, wherein the temperature is maintained above 0°C, 1°C, 2°C, or 3°C. 18. The method according to any one of embodiments 11 to 17, wherein establishing the cell bank takes at least 1 hour, at least 2 hours, or at least 3 hours. 19. The method according to any one of embodiments 11 to 18, wherein the storage container is a vial and / or a cryopreservation bag. 20. The method according to any one of embodiments 11 to 19, wherein the method is for increasing the viability of cells. 21. The concentration of cells in a batch of cell suspension is approximately 1 x 10 5 cells / ml ~ approx. 1×10 8 The method according to any one of embodiments 11 to 20, wherein the amount of the cell per ml is 1000 cells / ml. 22. The method according to any one of embodiments 11 to 21, wherein the cryopreservation medium comprises dimethyl sulfoxide (DMSO). 23. The method according to any one of embodiments 11 to 22, wherein the cryopreservation medium is STEM-CELLBANKER. 24. The method according to any one of embodiments 11 to 23, wherein the stem cells are pluripotent stem cells (PSCs). 25. The method according to embodiment 24, wherein the stem cells are human pluripotent stem cells (hPSCs). 26. The method according to any one of embodiments 11 to 25, wherein the stem cells or stem cell-derived cells are single cells or aggregates. 27. The method according to any one of embodiments 11 to 26, wherein the stem cell-derived cells are beta-like cells, cardiomyocytes, or RPE cells. 28. The method according to any one of embodiments 11 to 27, wherein the cell suspension further comprises a biological material. 29. The method according to any one of embodiments 11-28, wherein the volume of the batch of cell suspension is at least 25ml, 50ml, 100ml, 200ml, 300ml, 400ml, 500ml, 600ml, 700ml, 800ml, 900ml, or 1L. 30. The method according to any one of embodiments 11 to 29, wherein the amount of cell suspension transferred to each storage container is 0.25 to 1000 ml. 31. The method according to any one of embodiments 11 to 30, wherein a volume of about 1 ml is transferred to a 2 ml cryovial. 32. The method according to any one of embodiments 11-30, wherein a volume of about 20 ml is transferred to a 50 ml cryopreservation bag. 33. The method according to any one of embodiments 11 to 32, wherein a batch of cell suspension of step a) is compounded in a cell culture spinner bottle, and the cell culture spinner bottle is placed in a cooling device according to any one of embodiments 1 to 10. 34. The method according to any one of embodiments 11 to 33, wherein the batch of cell suspension of step b) is subjected to agitation, such as shaking or stirring. 35. The method according to embodiment 34, wherein the means for stirring is within the batch of cell suspension, such as a magnetic stirrer. 36. The method according to any one of embodiments 11-35, wherein at least a portion of the cell suspension remains within the batch of cell suspension maintained at said temperature for the time it takes to establish a cell bank. 37. The method according to any one of embodiments 11 to 36, wherein the method is carried out in a laminar flow workstation or a clean room. 38. Use of a cooling device according to any one of embodiments 1-10 for preparing a cell bank from a batch of cell suspension, comprising maintaining the temperature of the batch of cell suspension at less than 15°C, preferably less than 12°C, preferably between 0 and 12°C, preferably between 0 and 11°C, preferably between 0 and 10°C, more preferably between 0 and 8°C, more preferably between 2 and 8°C, more preferably between 2 and 6°C, more preferably between 3 and 5°C, even more preferably at about 4°C. 39. The use according to embodiment 38, wherein the batch of cell suspension comprises or is derived from stem cells. 40. The method according to embodiment 39, wherein the stem cells are human pluripotent stem cells (hPSCs). 41. A method for cooling a batch of a cell suspension, comprising the steps of: i. Providing a cooling device according to any one of embodiments 1 to 10; ii. charging a refrigerant into a first compartment of the cooling device, the temperature of the refrigerant being suitable to maintain the temperature of the batch of cell suspension below 15° C.; iii. loading a flask containing a batch of cell suspension into a second compartment of the cooling device; iv. maintaining the temperature of the batch of cell suspension below 15°C. 42. The method according to embodiment 41, wherein the method is GMP compliant. 43. The method according to any one of embodiments 41 and 42, wherein the refrigerant is a cooling pack. 44. The method according to any one of embodiments 41 to 43, wherein the temperature of the refrigerant is −20 to −80 °C when it is loaded into the first compartment. 45. The method according to any one of embodiments 41 to 44, wherein the batch of cell suspension is maintained at said temperature for at least 1 hour. 46. ​​The method according to any one of embodiments 41 to 45, wherein the temperature of the batch of cell suspension is maintained at 0 to 15°C, preferably 0 to 12°C, preferably 0 to 10°C, preferably 0 to 8°C, more preferably 2 to 8°C, more preferably 2 to 6°C, more preferably 3 to 5°C, even more preferably about 4°C. EXAMPLES

[0046] Below are non-limiting examples for carrying out the present invention.

[0047] Example 1: Protocol for establishing a large cell bank During preparation, pre-cool the vials at 2-8 °C. Freeze the gel packs and metal supports for the cooling device for at least 4 h and overnight at -20 °C prior to cryopreservation.

[0048] First, hPSCs are formulated in STEM-CELLBANKER (SCB) cryopreservation medium. Cells at appropriate confluency are then photographed, and cells are harvested and counted. Based on the total viable cell count obtained, for example, 1 x 10 per vial. 6Determine the number of vials that can be filled with 1000 total viable cells. Then transfer the calculated volume of cell suspension into an appropriate sterile conical tube and keep cold. Pellet the cells by centrifugation at 300 g for 3 and 5 minutes for small and large cell suspension volumes depending on the culture format. In a biosafety cabinet (BSC), aspirate the supernatant and then tap the tube to loosen the cell pellet. Gently resuspend the cell pellet in 1-5 mL of SCB, e.g., 1 x 10 6 Additional SCB is added to the cell suspension until a volume is reached that gives a concentration of cells / ml.

[0049] The cell suspension is transferred to a 1 L spinner flask and placed in a refrigerator according to the present invention. The pre-frozen cold pack and metal support are inserted into the refrigerator. The refrigerator is placed on the magnetic stirrer with the cell suspension, ensuring that the refrigerator is in the center of the stir plate. Mixing is set to the required speed. The mixing speed may be changed accordingly while filling as the volume is reduced.

[0050] A benchtop automated cryovial processing system, the Fill-it System (Sartorius Stedim Biotech), is used to automatically uncap, fill, and recapp the cryovials, and connect 8-way single-use sterile tubing set up to fill a 48 tube rack to the spinner flask liquid transfer port tubing. The required number of cryovials are then filled using the system's peristaltic pump with a fill volume of 1 mL. Alternatively, cryovials can be filled manually, for example, using a serological pipette.

[0051] The cryovials are then transferred to a rate-controlled freezer for cryopreservation. Once the target temperature is reached, the cryovials are placed on dry ice or directly onto a pre-chilled rack and transferred to the vapor phase of liquid nitrogen for long-term storage. Alternatively, use a Mr. Frosty or BioCision CoolCell cryovial. The cryovials with the filled cryovials are then placed in a -80°C freezer for 4-48 hours. The cryovials are then transferred to the vapor phase of liquid nitrogen for long-term storage.

[0052] Example 2: Comparison of cell viability at different temperatures We investigated the effect of longer exposure times to cryoprotectants on post-thaw hPSCs (% viability, plating, and recovery). Samples were taken at specific time points, immediately cryopreserved, and then thawed at a later time and cell samples were assessed for viability. Trypan blue viability assessment is fully described, for example, in Strober W. Trypan Blue Exclusion Test of Cell Viability. Curr Protoc Immunol. 2015;111:A3.B.1-.A3.B.3. Published 2015 Nov 2. doi:10.1002 / 0471142735.ima03bs111.

[0053] A comparison was made between hPSCs subjected to the method described in Example 1 and a similar method in which the cell suspension was not maintained at a temperature below 12° C. Cryopreserved cell samples were thawed and tested for viability using the trypan blue exclusion method.

[0054] Figure 4 shows the trypan blue exclusion viability of hESCs over different exposure times to cryopreservation media (specifically STEM-CELLBANKER) at room temperature (approximately 20°C) in two separate experiments. Clearly, viability decreases the longer the hPSCs are in cell suspension maintained at room temperature (RT). The data are shown in Table 1. [Table 1]

[0055] Figure 5 shows the results when the cell suspension was kept at approximately 4°C, along with comparative samples kept at room temperature (RT). The data are shown in Table 2, and the statistics for comparison with samples taken at 4°C and RT, respectively, at 240 minutes are shown in Table 3. [Table 2] [Table 3]

[0056] Example 3: Measurement of temperature of cryopreservation medium In one experiment, we measured the temperature of the cryopreservation medium in a flask when it was placed in a cooling device according to the present invention. Two temperature probes were used at the top and bottom of the flask, respectively. STEM-CELLBANKER was used as the cryopreservation medium in a 1 L spinner flask with magnetic stirring, with a medium volume of 850 ml. The refrigerant and metal plate were frozen prior to the experiment at -20°C.

[0057] Temperature measurements over time are shown in Figure 6. Just before 0 minutes, STEM-CELLBANKER was added at a temperature of approximately 6°C. No coolant exchange was performed during the experiment.

[0058] Example 4: Comparison of cell viability of differentiated cells (hESC-derived beta-like cells) at different temperatures The following experiment was set up to test the retention time of immature beta cells, single cells, at 100E6 VC / mL in the StemCell Banker: After harvesting BC03 from a 1L DASgip bioreactor using the standard SC2BC differentiation protocol. Figure 8 shows a flow cytometry dot plot comparing the percentage of cells expressing the NKX6.1 and Islet-1 markers. 49.5% co-express the two markers indicative of beta cells. Q0720882.1.8mL vials containing 1mL of 100E6 VC / mL were stored under the following conditions before being transferred to -80°C. [Table 4] [Table 5]

[0059] The next day, all vials are transferred to liquid nitrogen. Upon thawing, the following protocol was followed: [Table 6] [Table 7]

[0060] The results are shown in Figure 7, which shows the % viability measured by NC-202 for different holding times in cryopreservation media (such as StemcellBanker) at room temperature and approximately 4 degrees Celsius. The average % viability drops from 82 to 66 at room temperature and from 89 to 85 at 4°C (5 min to 240 min). Six to eight vials were thawed for each time point and were thawed by three different operators. Two-way ANOVA Sidak's multiple comparison test was performed to compare room temperature and 4°C (Table 6). [Table 8]

[0061] Example 5: Cryopreservation of hESC-derived RPE cells E1C3 (NN GMP0050E1C3) cultured on iMatrix-511 (0.25 mg / cm2, Nippi) were differentiated into RPE cells as described in Petrus-Reurer, Sandra et al. "Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation." Stem cell reports vol. 17, 6 (2022): 1458-1475. doi: 10.1016 / j.stemcr.2022.05.005.

[0062] To test the duration of cryopreservation media at different temperatures on RPE and how it affects RPE viability, cells were first kept in the Stem Cell Banker (at room temperature and 4°C for the following periods described below) before being transferred to a -80°C freezer. Room temperature: 60 minutes, 120 minutes, and 240 minutes 4C: 10 minutes, 30 minutes, 60 minutes, 120 minutes, and 240 minutes.

[0063] A positive control was prepared, which (0 min) consisted of cells transferred directly to -80°C.

[0064] The results are shown in Figure 9, which shows the % viability measured by NC-202 for different holding times in cryopreservation media (such as StemcellBanker) at room temperature and approximately 4 degrees Celsius. The % viability drops from 95 to 81 at room temperature and from 95 to 89 at 4°C (0 to 240 minutes). Eight to ten vials were thawed for each time point and were thawed by three different operators. A two-way ANOVA Sidak's multiple comparison test was performed to compare room temperature and 4°C, as shown in Table 7. [Table 9]

[0065] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art, and it is therefore understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

1. A method for establishing a cell bank, comprising: a. providing a batch of cell suspension comprising stem cells or stem cell-derived cells and a cryopreservation medium; b. maintaining the batch of cell suspension at a temperature below 15°C; c. transferring the cell suspension from the batch of cell suspension into one or more storage containers to establish the cell bank, wherein establishing the cell bank takes at least 30 minutes.

2. The method according to claim 1, wherein the batch of cell suspension is transferred into at least 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 storage containers.

3. The method according to claim 1, wherein the temperature is maintained below 15°C, 14°C, 13°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, or 4°C, preferably below 12°C, more preferably below 8°C.

4. The method according to claim 1, wherein the temperature is maintained at 0 to 15°C, preferably 0 to 12°C, more preferably 0 to 10°C, more preferably 0 to 8°C, more preferably 2 to 8°C, more preferably 2 to 6°C, more preferably 3 to 5°C, and even more preferably about 4°C.

5. The method according to claim 1, wherein the temperature is maintained higher than the freezing point of the cell suspension.

6. The method according to claim 1, wherein the temperature is maintained higher than 0°C, 1°C, 2°C, or 3°C.

7. The method according to claim 1, wherein establishing the cell bank takes at least 1 hour, at least 2 hours, or at least 3 hours.

8. The method according to claim 1, further comprising d. immediately after transferring the cell suspension from the batch of cell suspension, freezing each of the storage containers.

9. The method according to claim 1, wherein the amount of the cell suspension transferred to each storage container is 0.25 to 1000 ml.

10. The method according to claim 1, wherein the volume of the batch of cell suspension is at least 25 ml, 50 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, or 1 L.

11. The method according to claim 1, wherein the stem cells are pluripotent stem cells (PSCs) and / or the stem cell-derived cells are beta-like cells, cardiomyocytes, or RPE cells.

12. A cooling device (100) for cooling a batch of cell suspension during the preparation of a cell bank, comprising a housing (101) including an outer wall (102), a first compartment (104, 105) configured to receive a refrigerant, and a second compartment (103) configured to hold a container having the batch of the cell suspension, wherein the first compartment (104, 105) and the second compartment (103) are separated by heat conduction elements (106, 107), so that when the refrigerant is loaded into the first compartment (104, 105) and the container having the batch of the cell suspension is loaded into the second compartment (103), the refrigerant can cool the batch of the cell suspension, the cooling device (100).

13. A method for cooling a batch of cell suspension, comprising: i. providing the cooling device according to claim 12; ii. loading a refrigerant into the first compartment of the cooling device, wherein the temperature of the refrigerant is suitable for maintaining the temperature of the batch of the cell suspension below 15°C; iii. loading a flask containing the batch of the cell suspension into the second compartment of the cooling device; iv. maintaining the temperature of the batch of the cell suspension below 15°C.

14. The method according to claim 13, wherein the temperature of the batch of the cell suspension is maintained at 0 to 15°C, preferably 0 to 12°C, preferably 0 to 10°C, preferably 0 to 8°C, more preferably 2 to 8°C, more preferably 2 to 6°C, more preferably 3 to 5°C, and even more preferably about 4°C.

15. Use of the cooling device according to claim 12 for preparing a cell bank from a batch of cell suspension, comprising maintaining the temperature of the batch of the cell suspension below 15°C, preferably 0 to 12°C, preferably 0 to 10°C, more preferably 0 to 8°C, more preferably 2 to 8°C, more preferably 2 to 6°C, more preferably 3 to 5°C, and even more preferably about 4°C.

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