Methods for cryoprotection and / or cryoprotection of cells

Abstract

Compositions are provided that include a population of cells that are suitable for freezing or lyophilization while maintaining viable cells. The compositions include a simple sugar, urea, and in some embodiments, a bulking agent. Also provided are methods for preparing and storing the cells by freezing or lyophilization in a manner that allows the cells to remain viable during storage.

Description

[Technical field]

[0001] The present disclosure provides methods for providing cryoprotection and / or lyoprotection of cells preserved by freezing or lyophilization. [Background technology]

[0002] Human mesenchymal stem cells (hMSCs) are important pluripotent stem cells that have the ability to differentiate (in vitro) into different tissues such as fat, muscle, cartilage, bone, or neural cells [1-3]. The use of hMSCs in regenerative medicine and cell therapy is expanding [4, 5]. hMSCs have the potential to be harvested from adult donors, expanded, modified, and subsequently used in allogeneic therapies [5], as they exhibit immunosuppressive capabilities [6]. To make this possible, hMSCs need to be stored for long periods of time. The current protocol for long-term storage of hMSCs is by cryopreservation in liquid nitrogen [7]. However, storing cells in liquid nitrogen is cumbersome and expensive, especially for distribution and transportation, as a regular supply of liquid nitrogen is required to avoid uncontrolled thawing and damage to the cells [9], and thus, the cost of storage is high, especially for the sake of storage and transportation [8].

[0003] Liquid nitrogen storage of hMSCs also requires the addition of fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) to preserve the cells [7, 10]. DMSO is toxic [11-14] and may alter transcription factor expression and gene expression in hMSCs [15, 16]. Furthermore, FBS is highly immunogenic in humans and may transmit pathogens [7]. Therefore, DMSO and FBS are usually removed before administration to humans, a process that is labor-intensive and requires specially trained personnel and special facilities. Therefore, there is a high risk that the administered product will be substandard and of inconsistent quality, with safety implications.

[0004] It would also be beneficial to store cell therapy products, like many other biological products, at refrigerated temperatures to make them easier to handle and facilitate economical transportation. One possible way to achieve this is to lyophilize (freeze-dry) the cells [18, 19]. Lyophilization is routinely applied to (drug) products that are unstable in liquid formulations but are stabilized in solid / dried forms, such as vaccines, proteins, peptides, or antibiotics

[20] .

[0005] EP3403502A1 discloses cryoprotectants and / or cryopreservation compositions, methods and uses thereof in the preservation and / or protection of cells, organisms and / or tissues.

[0006] US2009 / 123436A1 discloses compositions and related methods for the cryopreservation of biological materials.

[0007] WO2021 / 050896A1 discloses a method for producing a freeze-dried cell aggregate, the method comprising: (a) freezing a composition comprising a cell aggregate, an aqueous component, a polyol, a sugar, and a polysaccharide; and (b) removing at least about 90% of the aqueous component from the frozen composition to produce the freeze-dried cell aggregate.

[0008] One object of the present invention is to provide a method for cryopreservation and freeze-drying of cells that allows the cells to remain viable during storage. Summary of the Invention

[0009] The present disclosure relates to a composition comprising a population of viable cells, an aqueous component, urea, and a simple sugar.

[0010] In embodiments, the composition further comprises a bulking agent. In embodiments, the composition further comprises hyaluronan gel.

[0011] In an embodiment of the composition, the concentration of the monosaccharide is from about 0.2M to about 1.25M, or from about 0.3M to about 0.8M, or from about 0.4M to about 0.6M.

[0012] In embodiments of the composition, the concentration of urea is from about 0.2M to about 1.25M, or from about 0.2M to about 0.8M, or from about 0.4M to about 0.6M.

[0013] In an embodiment of the composition, the concentration of the monosaccharide is from about 0.6M to about 2.0M, or from about 0.7M to about 1.7M, or from about 0.8M to about 1.4M.

[0014] In embodiments of the composition, the concentration of urea is from about 0.6M to about 2.0M, or from about 0.7M to about 1.7M, or from about 0.8M to about 1.4M.

[0015] In embodiments of the composition, the molar ratio of urea to monosaccharide in the composition is from about 5:1 to about 1:5, or from about 1:3 to about 3:1, or from about 1:2 to about 2:1, or is about 1:1.

[0016] In an embodiment of the composition, the monosaccharide is glucose, fructose, or galactose.

[0017] In an embodiment of the composition, the bulking agent comprises a) a disaccharide, where the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, lactulose, isomaltose, melibiose, or gentiobiose; or b) a sugar alcohol, where the sugar alcohol is mannitol, sorbitol, galactitol, fucitol, iditol, or inositol; or c) both a disaccharide and a sugar alcohol; or d) both sucrose and mannitol.

[0018] In embodiments of the composition, the composition does not include DMSO, or the composition includes DMSO at a concentration of about 1% to about 10% prior to freezing, or the composition includes DMSO at a concentration of about 1% to about 5% prior to freezing.

[0019] In an embodiment of the composition, the population of cells comprises mammalian cells.

[0020] In embodiments, the composition is in a frozen state at a temperature of about -10°C to about -100°C, or about -20°C to about -90°C, or about -40°C to about -60°C.

[0021] In an embodiment, the composition is a lyophilized composition.

[0022] The composition of any one of claims 1 to 14, wherein the composition contains less than about 90% (volume / volume) water.

[0023] In an embodiment, the present disclosure also relates to a method for producing a frozen cell aggregate, comprising the step (a) of freezing the composition defined above to produce a frozen cell aggregate.

[0024] In an embodiment, the present disclosure also relates to a method for producing a reconstituted population of viable cells, comprising the steps of (a) freezing a composition defined above to produce a population of frozen cells, and (b) resuspending the population of frozen cells in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable.

[0025] In an embodiment, the present disclosure also relates to a method for producing a freeze-dried cellular aggregate, comprising the steps of (a) freezing a composition as defined above, and (b) removing at least about 10% (volume / volume) water from the frozen composition to produce a freeze-dried cellular aggregate.

[0026] In an embodiment, the present disclosure also relates to a method for producing a reconstituted viable cell population, comprising the steps of (a) freezing a composition as defined above, (b) removing at least about 10% (volume / volume) of water from the frozen composition to produce a lyophilized cell population, and (c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable.

[0027] In an embodiment of the above method, prior to freezing in step (a), the cell population is isolated and contacted with a poration solution. In an embodiment, the poration solution comprises trehalose. In an embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 1.0 M, or about 0.1 M to about 0.6 M.

[0028] The present disclosure also relates to a method of producing a frozen cell aggregate, comprising freezing a composition comprising a cell aggregate, an aqueous component, urea, a simple sugar, and optionally a bulking agent to produce the frozen cell aggregate.

[0029] The present disclosure also relates to a method of producing a reconstituted viable cell population, the method comprising: a) freezing a composition comprising a cell population, an aqueous component, urea, a simple sugar, and optionally a bulking agent to produce a frozen cell population; and b) resuspending the frozen cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable.

[0030] In an embodiment of the method, the composition further comprises a hydrogel. In an embodiment, the hydrogel is a biocompatible hydrogel. In an embodiment, the hydrogel is a hyaluronan gel, an alginate gel, an agarose gel, a collagen gel, or a combination thereof.

[0031] In an embodiment, the cell population is suspended in a hydrogel.

[0032] In embodiments of the method, the concentration of the monosaccharide before freezing is from about 0.6M to about 2M, or from about 0.7M to about 1.7M, or from about 0.8M to about 1.4M.

[0033] In an embodiment of the method, the monosaccharide is glucose, fructose, or galactose.

[0034] In an embodiment of the method, the monosaccharide is glucose, and the concentration of glucose before freezing is about 0.2M to about 1.0M, or about 0.2M to about 0.8M, or about 0.4M to about 0.6M.

[0035] In embodiments of the method, the concentration of urea before freezing is from about 0.2M to about 1.0M, or from about 0.2M to about 0.8M, or from about 0.4M to about 0.6M.

[0036] In embodiments of the method, the molar ratio of urea to monosaccharides prior to freezing is from about 1:3 to about 3:1, or from about 1:2 to about 2:1, or is about 1:1.

[0037] In an embodiment of the method, the monosaccharide is glucose and the molar ratio of urea to glucose before freezing is from about 1:3 to about 3:1, or from about 1:2 to about 2:1, or is about 1:1.

[0038] In embodiments of the method, freezing is performed with cells in suspension or attached to a surface of a container, or with a collection of cells suspended in a hydrogel, and / or in a container with or without a collagen coating, optionally the container is a glass or plastic container with or without a membrane for cell attachment or growth.

[0039] The disclosure also relates to a method of producing freeze-dried cell aggregates, the method comprising: a) freezing a composition comprising cell aggregates, hyaluronan gel, an aqueous component, urea, a monosaccharide, and a bulking agent, wherein the concentration of the monosaccharide before freezing is at least about 0.6 M and the concentration of the urea before freezing is at least about 0.6 M; and b) removing at least about 90% of the aqueous component from the frozen composition to produce the freeze-dried cell aggregates.

[0040] The disclosure also relates to a method of producing a reconstituted viable cell aggregate, comprising: a) freezing a composition comprising a cell aggregate, hyaluronan gel, an aqueous component, urea, a monosaccharide, and a bulking agent, wherein the concentration of the monosaccharide before freezing is at least about 0.6M and the concentration of the urea before freezing is at least about 0.6M; b) removing at least about 90% of the aqueous component from the frozen composition to produce a lyophilized cell aggregate; and c) resuspending the lyophilized cell aggregate in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable.

[0041] In an embodiment of the method, the cell aggregates are suspended in a hyaluronan gel.

[0042] In embodiments of the method, the concentration of the monosaccharide before freezing is from about 0.6M to about 2.0M, or from about 0.7M to about 1.7M, or from about 0.8M to about 1.4M.

[0043] In an embodiment of the method, the monosaccharide is glucose, fructose, or galactose.

[0044] In an embodiment of the method, the monosaccharide is glucose, and the concentration of glucose before freezing is about 0.6M to about 2.0M, or about 0.7M to about 1.7M, or about 0.8M to about 1.4M.

[0045] In embodiments of the method, the concentration of urea before freezing is from about 0.6M to about 2.0M, or from about 0.7M to about 1.7M, or from about 0.8M to about 1.4M.

[0046] In embodiments of the method, the molar ratio of urea to monosaccharides prior to freezing is from about 1:3 to about 3:1, or from about 1:2 to about 2:1, or is about 1:1.

[0047] In an embodiment of the method, the monosaccharide is glucose and the molar ratio of urea to glucose before freezing is from about 1:3 to about 3:1, or from about 1:2 to about 2:1, or is about 1:1.

[0048] In an embodiment of the method, the cell population is about 1×10 per mL. 4 ~1 x 10 cells per mL 7 In an embodiment, the cell population is about 1 x 10 cells per mL. 5 ~4 x 10 cells per mL 5 In an embodiment, the cell population is about 2×10 cells per mL. 5 ~2.5 x 10 cells per mL 5 Each cell is a single cell.

[0049] In an embodiment of the method, the bulking agent comprises a disaccharide. In an embodiment, the concentration of the disaccharide before freezing is about 0.1 M to about 1.0 M. In an embodiment, the concentration of the disaccharide before freezing is about 0.1 M to about 0.5 M. In an embodiment, the concentration of the disaccharide before freezing is about 0.2 M to about 0.4 M. In an embodiment, the bulking agent is sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, lactulose, isomaltose, melibiose, or gentiobiose.

[0050] In an embodiment, the disaccharide is sucrose. In an embodiment, the concentration of sucrose before freezing is about 0.1 M to about 1.0 M. In an embodiment, the concentration of sucrose before freezing is about 0.1 M to about 0.5 M. In an embodiment, the concentration of sucrose before freezing is about 0.2 M to about 0.4 M.

[0051] In an embodiment of the method, the bulking agent comprises a sugar alcohol. In an embodiment, the concentration of the sugar alcohol before freezing is about 0.1 M to about 1.0 M. In an embodiment, the concentration of the sugar alcohol before freezing is about 0.3 M to about 0.8 M. In an embodiment, the concentration of the sugar alcohol before freezing is about 0.4 M to about 0.6 M.

[0052] In an embodiment, the sugar alcohol is mannitol, sorbitol, galactitol, fucitol, iditol, or inositol. In an embodiment, the sugar alcohol is mannitol. In an embodiment, the concentration of mannitol before freezing is about 0.1 M to about 1.0 M. In an embodiment, the concentration of mannitol before freezing is about 0.3 M to about 0.8 M. In an embodiment, the concentration of mannitol before freezing is about 0.4 M to about 0.6 M.

[0053] In an embodiment of the method, the bulking agent comprises both a disaccharide and a sugar alcohol, hi an embodiment, the bulking agent comprises both sucrose and mannitol.

[0054] In an embodiment of the method, the composition does not include DMSO. In an embodiment, the composition includes DMSO at a concentration of about 1% to about 10% prior to freezing. In an embodiment, the composition includes DMSO at a concentration of about 1% to about 5% prior to freezing.

[0055] In an embodiment of the method, prior to freezing in (a), the cell population is isolated and contacted with a poration solution. In an embodiment, the poration solution comprises trehalose. The poration solution may optionally further comprise a cell culture medium. In an embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 1.0 M. In an embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 0.6 M.

[0056] In embodiments of the methods involving reconstitution of cells, the reconstitution agent comprises cell culture medium. In embodiments, the reconstitution agent comprises a phosphate buffer solution.

[0057] In an embodiment of the method, the composition is an isotonic solution. In an embodiment, the composition is a hypertonic solution.

[0058] In an embodiment of the method, the population of cells comprises mammalian cells. In an embodiment, the population of cells comprises stem cells. In an embodiment, the population of cells comprises pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or hematopoietic stem cells. In an embodiment, the population of cells comprises mesenchymal stem cells. In an embodiment, the population of cells comprises induced pluripotent stem cells. In an embodiment, the population of cells comprises neuroblastoma cells. In an embodiment, the neuroblastoma cells are SK-N-AS cells.

[0059] In the context of the present invention, the aqueous component is water, optionally comprising further components such as buffers or cell culture media.

[0060] In embodiments of the method, the aqueous component comprises a buffer. In embodiments, the buffer comprises a phosphate buffer, a Tris buffer, an acetate buffer, a bicarbonate buffer, a histidine buffer, a citrate buffer, or a combination thereof. In embodiments, the aqueous component comprises a cell culture medium. In embodiments, the cell culture medium is serum-free.

[0061] In embodiments of the method, removing the aqueous component comprises reducing pressure, applying heat, or both to the frozen composition to remove the aqueous component. In embodiments, removing the aqueous component comprises a primary drying step and a secondary drying step. In embodiments, removing the aqueous component comprises only a primary drying step. In embodiments, the primary drying step comprises reducing pressure to remove the aqueous component. In embodiments, the secondary drying step comprises applying heat to remove the aqueous component.

[0062] In an embodiment of the method, the freezing is performed at -10°C to about -100°C. In an embodiment, the freezing is performed at -20°C to about -90°C. In an embodiment, the freezing is performed at -40°C to about -60°C. In an embodiment, the freezing reduces the temperature of the composition to about -80°C.

[0063] In an embodiment of the method, freezing reduces the temperature of the composition to −40° C., and the aqueous components are removed at a chamber pressure of about 60 mTorr to about 80 mTorr.

[0064] In embodiments of the method, resuspending the frozen cells occurs more than 2 hours after freezing. In embodiments, resuspending the frozen cells occurs more than 1 day after freezing. In embodiments, resuspending the frozen cells occurs more than 1 week after freezing. In embodiments, resuspending the frozen cells occurs more than 1 month after freezing.

[0065] In embodiments where the cells are freeze-dried, resuspending the freeze-dried cells occurs more than 2 hours after removal of the aqueous component. In embodiments, resuspending the freeze-dried cells occurs more than 1 day after removal of the aqueous component. In embodiments, resuspending the freeze-dried cells occurs more than 1 week after removal of the aqueous component. In embodiments, resuspending the freeze-dried cells occurs more than 1 month after removal of the aqueous component.

[0066] In embodiments of the methods, the frozen or lyophilized cells are stored below about -20°C prior to resuspension. In embodiments, the frozen or lyophilized cells are stored at about -20°C to about 30°C prior to resuspension. In embodiments, the frozen or lyophilized cells are stored at about 4°C to about 28°C prior to resuspension. In embodiments, the frozen or lyophilized cells are stored at about 10°C to about 27°C prior to resuspension. In embodiments, the frozen or lyophilized cells are stored at about 2°C to about 8°C prior to resuspension.

[0067] In embodiments of the methods, the frozen or lyophilized cells are stored above about -20°C for more than 2 days before resuspension. In embodiments, the lyophilized cells are stored at about 20°C to about 25°C for more than 1 week before resuspension. In embodiments, the lyophilized cells are stored at about 2°C to about 8°C for more than 1 week before resuspension.

[0068] In embodiments of the methods, at least about 20% of the cells in the reconstituted composition are viable, for example, as measured by the ALAMARBLUE® test. In embodiments, at least about 30% of the cells in the reconstituted composition are viable, as measured by the ALAMARBLUE® test. In embodiments, at least about 40% of the cells in the reconstituted composition are viable, as measured by the ALAMARBLUE® test. In embodiments, at least about 50% of the cells in the reconstituted composition are viable, as measured by the ALAMARBLUE® test.

[0069] In embodiments, the present disclosure also relates to a composition comprising a population of viable cells, hyaluronan gel, an aqueous component, urea, a monosaccharide, and a bulking agent, wherein the urea and the monosaccharide are present in the composition in a molar ratio of about 1:3 to about 3:1, and the composition contains less than about 90% (volume / volume) water.

[0070] In an embodiment of the composition, the monosaccharide is glucose, and urea and glucose are included in the composition in a molar ratio of about 1:3 to about 3:1. In an embodiment of the composition, urea and glucose are included in the composition in a molar ratio of about 1:2 to about 2:1. In an embodiment of the composition, urea and glucose are included in the composition in a molar ratio of about 1:1 to about 1:1.

[0071] In embodiments, the present disclosure also relates to a composition comprising a population of viable cells, an aqueous component, urea, a monosaccharide, and optionally a bulking agent, wherein the molar ratio of the monosaccharide to urea in the composition is about 5:1 to about 1:5. In embodiments, the molar ratio of the monosaccharide to urea in the composition is about 3:1 to about 1:3. In embodiments, the molar ratio of the monosaccharide to urea in the composition is about 2:1 to about 1:2. In embodiments, the molar ratio of the monosaccharide to urea in the composition is about 1:1 to about 1:1.

[0072] In an embodiment of the composition, the monosaccharide is glucose. In an embodiment, the molar ratio of glucose to urea in the composition is about 3:1 to about 1:3. In an embodiment, the molar ratio of glucose to urea in the composition is about 2:1 to about 1:2. In an embodiment, the molar ratio of glucose to urea in the composition is about 1:1 to about 1:1.

[0073] In embodiments, the present disclosure also relates to a composition comprising a population of viable cells, an aqueous component, about 0.2 M to about 1.0 M urea, about 0.2 M to about 1.0 M glucose, and a bulking agent. In embodiments, the composition comprises about 0.3 M to about 0.8 M glucose. In embodiments, the composition comprises about 0.4 M to about 0.6 M glucose.

[0074] In an embodiment, the composition comprises about 0.3 M to about 0.8 M urea. In an embodiment, the composition comprises about 0.4 M to about 0.6 M urea.

[0075] In an embodiment, the composition further comprises sucrose. In an embodiment, the composition further comprises about 0.1 M to about 0.5 M sucrose.

[0076] In an embodiment, the composition further comprises mannitol. In an embodiment, the composition further comprises about 0.1 M to about 0.8 M mannitol.

[0077] In an embodiment of the composition, the population of viable cells comprises mammalian cells. In an embodiment, the population of viable cells comprises stem cells. In an embodiment, the population of viable cells comprises pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or hematopoietic stem cells. In an embodiment, the population of viable cells comprises mesenchymal stem cells. In an embodiment, the population of viable cells comprises induced pluripotent stem cells. In an embodiment, the population of viable cells comprises neuroblastoma cells. In an embodiment, the neuroblastoma cells are SK-N-AS cells.

[0078] In an embodiment of the composition, the composition does not include DMSO. In an embodiment of the composition, the composition includes DMSO at a concentration of about 1% to about 10%. In an embodiment of the composition, the composition includes DMSO at a concentration of about 1% to about 5%.

[0079] In embodiments, the present disclosure also relates to a method of freezing a collection of cells, comprising freezing a composition comprising the collection of cells, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.1 M to about 0.5 M sucrose, wherein the freezing is performed at less than about -30°C.

[0080] In embodiments, the present disclosure also relates to a method of freeze-drying a cell aggregate, the method comprising: a) freezing a composition comprising a cell aggregate, hyaluronan gel, an aqueous component, about 0.6 M to about 2.0 M urea, about 0.6 M to about 2.0 M glucose, and about 0.1 M to about 1.0 M sucrose, wherein the freezing occurs at less than about -30°C; and b) removing the aqueous component from the frozen composition to produce a freeze-dried cell aggregate, wherein the aqueous component in the freeze-dried cell aggregate is less than about 10% w / w.

[0081] In embodiments, the present disclosure also relates to a method of producing a viable aggregate of cells, comprising: a) freezing a composition comprising an aggregate of cells, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.1 M to about 0.5 M sucrose, wherein the freezing is performed at less than about -30°C; and b) resuspending the lyophilized aggregate of cells in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0082] In embodiments, the present disclosure also relates to a method of producing a viable aggregate of cells comprising: a) freezing a composition comprising an aggregate of cells, hyaluronan gel, an aqueous component, about 0.6 M to about 2.0 M urea, about 0.6 M to about 2.0 M glucose, and about 0.1 M to about 1.0 M sucrose, wherein the freezing occurs at less than about −30° C.; b) removing the aqueous component from the frozen composition to produce a freeze-dried aggregate of cells, wherein the aqueous component in the freeze-dried aggregate of cells is less than about 10% w / w; and c) resuspending the freeze-dried aggregate of cells in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0083] In embodiments, the present disclosure also relates to a method of freezing a collection of cells, comprising freezing a composition comprising the collection of cells, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.1 M to about 0.8 M mannitol, wherein the freezing occurs at less than about -30°C.

[0084] In embodiments, the present disclosure also relates to a method of freeze-drying a cell aggregate, the method comprising: a) freezing a composition comprising the cell aggregate, hyaluronan gel, an aqueous component, about 0.6 M to about 2.0 M urea, about 0.6 M to about 2.0 M glucose, and about 0.1 M to about 0.8 M mannitol, wherein the freezing occurs at less than about -30°C; and b) removing the aqueous component from the frozen composition to produce a freeze-dried cell aggregate, wherein the aqueous component in the freeze-dried cell aggregate is less than about 10% w / w.

[0085] In embodiments, the present disclosure also relates to a method of producing a viable aggregate of cells, comprising: a) freezing a composition comprising an aggregate of cells, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.1 M to about 0.8 M mannitol, wherein the freezing occurs at less than about -30°C; and b) resuspending the lyophilized aggregate of cells in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0086] In embodiments, the present disclosure also relates to a method of producing a viable aggregate of cells comprising: a) freezing a composition comprising an aggregate of cells, hyaluronan gel, an aqueous component, about 0.6 M to about 2.0 M urea, about 0.6 M to about 2.0 M glucose, and about 0.1 M to about 0.8 M mannitol, wherein the freezing occurs at less than about −30° C.; b) removing the aqueous component from the frozen composition to produce a freeze-dried aggregate of cells, wherein the aqueous component in the freeze-dried aggregate of cells is less than about 10% w / w; and c) resuspending the freeze-dried aggregate of cells in a reconstituting agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0087] In an embodiment of the method, the composition further comprises about 1% to about 8% DMSO. In an embodiment of the method, the composition further comprises a hydrogel. In an embodiment, the hydrogel is a hyaluronan gel, an alginate gel, or a collagen gel. In an embodiment, the cell aggregates are suspended in the hydrogel. In an embodiment, the composition does not comprise DMSO.

[0088] In embodiments of the method in which the composition is reconstituted, at least about 20% of the cells in the reconstituted composition are viable, for example, as measured by the ALAMARBLUE® test. In embodiments, at least about 30% of the cells in the reconstituted composition are viable, as measured by the ALAMARBLUE® test. In embodiments, at least about 40% of the cells in the reconstituted composition are viable, as measured by the ALAMARBLUE® test. In embodiments, at least about 50% of the cells in the reconstituted composition are viable, as measured by the ALAMARBLUE® test.

[0089] The following drawings form part of the present specification and are included to further demonstrate illustrative embodiments of certain aspects of the present invention. [Brief description of the drawings]

[0090] [Figure 1]1A-1C are plots showing the viability of hMSCs after freeze-thaw in different cryoprotectant (CPA) formulations, as described in Example 1. Each experiment was repeated five times, the bars show the mean, and the error bars show the standard deviation. Viability was assessed by metabolic conversion of a fluorescent dye. U=urea, G=glucose, DMSO=dimethylsulfoxide, M=mannitol, S=sucrose. Cell viability is plotted as the percentage of viable cells. (FIG. 1A) Urea alone does not readily cryoprotect, whereas glucose does. The two CPAs act synergistically, yielding viability comparable to the DMSO control. (FIG. 1B) Evaluation of the ratio of urea to glucose (added to 1M CPA in total), with a 1:1 molar ratio being preferred. (FIG. 1C) Addition of bulking agents to the U / G formulation also provided viable cells despite increased osmotic stress. Furthermore, prior to CPA application and freeze-thawing, hMSCs were incubated overnight in medium supplemented with 0.2 M trehalose (Figure 1C "With Tre Inc"). Pretreatment slightly increased viability in the U / G / S formulation. Addition of 2% DMSO further improves viability. (Figure 1D1-Figure 1D3) Shown are microscopic images of hMSCs subjected to trehalose incubation and subsequent FT. Cells convert fluorescent dye, morphology remains unchanged, and they attach to the plastic surface, indicating viability. Scale bar is 200 μm. [Diagram 2] FIG. 2 shows microscopic images of hMSCs growing after freeze-drying in different formulations with high concentrations of CPA, demonstrating that hMSCs remain viable and can withstand the harsh conditions of the freeze-drying process described in Example 1. U=urea, G=glucose, DMSO=dimethylsulfoxide, M=mannitol, S=sucrose, Tre=trehalose. All cells were pre-incubated in trehalose before freeze-drying. Scale bar is 400 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0091] The present disclosure provides methods for preparing and storing cells by freezing or lyophilization in a manner that allows the cells to remain viable during storage. The present disclosure also provides compositions comprising a collection of cells that are suitable for freezing or lyophilization while maintaining viable cells.

[0092] As used herein, "a" or "an" can mean one or more. As used herein, the words "a" or "an" when used in conjunction with the word "comprising" can mean one or more than one. As used herein, "another" or "further" can mean at least a second or more.

[0093] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device used to determine the value or the variation that exists between study subjects. Typically, the term "about" is meant to encompass a variation of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or less, depending on the context. In embodiments, a person skilled in the art will understand the level of variation indicated by the term "about" depending on the context in which it is used herein. It should also be understood that the use of the term "about" includes the specifically recited value.

[0094] Although use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0095] As used herein, the terms "comprising" (and any variant or form of comprising, such as "comprise" and "comprises"), "having" (and any variant or form of having, such as "have" and "has"), "including" (and any variant or form of including, such as "includes" and "include"), or "containing" (and any variant or form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0096] The use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) means that the particular term recited is representative of examples and embodiments of the present disclosure that are not intended to be limited to the specific example referenced or cited, unless expressly stated otherwise.

[0097] As used herein, "between" is a range that includes the ends of the range. For example, a number between x and y explicitly includes the numbers x and y, as well as any number that falls within the range between x and y.

[0098] Method for producing cell aggregates In embodiments, the present disclosure provides a method of producing a frozen cell aggregate, comprising freezing a composition comprising a cell aggregate, an aqueous component (e.g., water), urea, a simple sugar, and optionally a bulking agent to produce the frozen cell aggregate.

[0099] In embodiments, the present disclosure provides a method of producing a reconstituted viable cell population, the method comprising: a) freezing a composition comprising a cell population, an aqueous component (e.g., water), urea, a simple sugar, and optionally a bulking agent to produce a frozen cell population; and b) resuspending the frozen cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable.

[0100] In embodiments, the present disclosure provides a method of producing a freeze-dried cell aggregate, the method comprising: a) freezing a composition comprising a cell aggregate, an aqueous component, urea, a simple sugar, and a bulking agent; and b) removing at least about 90% of the water from the frozen composition to produce the freeze-dried cell aggregate.

[0101] In embodiments, the present disclosure provides a method of producing a reconstituted viable cell population, the method comprising: a) freezing a composition comprising a cell population, an aqueous component, urea, a simple sugar, and a bulking agent; b) removing at least about 90% of the water from the frozen composition to produce a lyophilized cell population; and c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable.

[0102] Freezing and freeze-drying In embodiments, freezing is the transition of a substance (e.g., a composition containing one or more components) from a liquid state to a solid state. "Lyophilization" (also called "freeze-drying" and "low-temperature drying") and variations thereof refer broadly to freezing a substance and then reducing the concentration of one of the solutes, typically water, by digestion and dissociation (i.e., "drying"). In embodiments, drying includes applying a vacuum to a frozen substance, e.g., a frozen composition. In embodiments, drying includes reducing pressure, applying heat, or both to a frozen composition.

[0103] Method embodiments provided herein involve freezing the composition as part of the lyophilization process. Method embodiments provided herein involve freezing the composition but do not involve a drying step. Any of the devices and methods described herein for freezing a composition can be used in either the freezing only methods provided herein or the lyophilization methods provided herein.

[0104] Freeze drying can be used to dry thermally unstable compounds such as microorganisms and proteins. Instead of using the evaporation mechanism of solvent removal common to small molecules, sublimation is used. Freeze drying may consist of three process steps: freezing, primary drying, and secondary drying. Freeze drying can be achieved by placing the composition to be freeze-dried in a freeze drying chamber, which is followed by freezing, drying, and finally quenching under an inert atmosphere. During drying, water vapor may enter the condenser through a connecting duct. In some embodiments, the temperature in the condenser is lower than that of the freeze drying chamber, so that the water vapor turns into ice.

[0105] frozen Freezing can have a significant effect on the rate and uniformity of drying. Solvent freezing can be subdivided into cooling, phase change, and solidification. In some embodiments, the formulation does not freeze at the equilibrium freezing temperature, but freezes at a lower temperature. This is because ice crystal nuclei have not yet formed in the solution or there is a significant temperature difference in the system. The degree to which a phase change occurs, starting with primary nucleation and then secondary nucleation, is called the degree of supercooling. Primary nucleation is defined as the appearance of the first ice nuclei, while secondary nucleation is characterized by the formation of additional nucleation sites. After the phase change stage, solidification is initiated. Here, ice crystals start to grow, the amount of liquid water phase decreases, and the solute concentration increases. Above a critical concentration, the concentrated solution undergoes either eutectic freezing or vitrification. The temperature at which this occurs is called the eutectic temperature (T) for crystalline systems. eu ), and for amorphous systems, the glass transition temperature (T gBelow these temperatures, the system is considered to be fully solidified. Depending on the degree of supercooling, different crystal sizes and morphologies are formed, which, upon sublimation during the drying step, will affect the pore size in the dried product. Smaller ice crystals will form smaller pores, which will cause a higher resistance to mass transfer and therefore slower primary drying. However, due to the larger surface area, secondary drying in this case can proceed at a higher rate. For larger ice crystals, the opposite can be true. In some embodiments, more rapid crystallization will result in smaller crystals, slower primary drying but faster secondary drying.

[0106] Primary drying In an embodiment of the freeze-drying method, the method includes a primary drying step. After the formulation is in its frozen state, the chamber is evacuated to near vacuum pressure and the shelf temperature is increased to the collapse temperature (T c ) slowly until the temperature is close to, but below, T c is usually T g 'A few degrees above the temperature at which the cake loses its structure. The correlation between a collapsed cake and product stability is still controversial. However, visual collapse may lead to the rejection of a freeze-dried cell product after visual inspection by the manufacturer.

[0107] The driving force for primary drying is the gradient between the vapor pressure of the ice and the chamber pressure. The vapor pressure of the ice depends on the temperature of the product, which is the most important parameter in the process. The product temperature is controlled by varying both the shelf temperature and the chamber pressure. The higher the shelf temperature, the higher the product temperature and therefore the more rapid the sublimation. On the other hand, since sublimation is an endothermic process, a low chamber pressure cools the product and slows down the rate of sublimation.

[0108] In addition to bulking agents, it may be important to use a protectant against water loss and low temperatures when drying cells. Protectants can include sorbitol, sucrose, trehalose, mannitol, polyvinylpyrrolidone (PVP), dextrose, and glycine. Most of these compounds are used in the formulation T. g ' has the added positive effect of increasing the crystallinity of the lyophilized cell product. This is desirable because it allows the process to be run at higher temperatures, which makes the process faster and therefore more efficient. Bulking agents provide a good appearance and structure to the freeze-dried cell product, and some also act as protectants. Mannitol is one example. Crystalline bulking agents are desirable because they tend to provide a beautiful cake structure.

[0109] Secondary drying In embodiments of the freeze-drying process, the method includes a secondary drying step. In other embodiments, no secondary drying step is performed. The free ice is sublimated during primary drying, which is the longest step of the freeze-drying steps. However, some of the water remains bound to the product and therefore needs to be removed with more aggressive treatment. This is accomplished during secondary drying, where the chamber pressure remains the same as in primary drying and the shelf temperature is raised, typically to 30-50°C. At this point, it is possible to raise the temperature to such a level because there is no more free ice in the vial and therefore no risk of collapse of the freeze-dried cake.

[0110] In an embodiment, freezing the composition comprises placing a container containing the composition in liquid nitrogen. In an embodiment, freezing the composition comprises placing a container containing the composition in a -80°C freezer. In an embodiment, freezing the composition comprises placing a container containing the composition in a freezing container, such as a MR.FROSTY Freezing Container from THEFRMO SCIENTIFIC. In an embodiment, freezing the composition comprises placing a container containing the composition on a pre-cooled shelf in a freezer. In an embodiment, the container comprises a cryogenic vial or an injection glass vial, such as a 2R glass vial. In an embodiment, freezing the composition does not use liquid nitrogen.

[0111] In an embodiment, the rate at which the temperature of the composition containing the cell aggregates is reduced may affect the viability of the cells. The cooling rate depends on many factors, including the cooling device, the container, the composition, and the volume of the composition. In an embodiment, the temperature of the composition is reduced by about 1°C to about 50°C / sec, about 2°C to about 40°C / sec, about 3°C ​​to about 30°C / sec, about 4°C to about 20°C / sec, or about 5°C to about 10°C / sec. In an embodiment, the temperature of the composition is reduced by about 1°C to about 50°C / min, about 2°C to about 40°C / min, about 3°C ​​to about 30°C / min, about 4°C to about 20°C / min, or about 5°C to about 10°C / min. In an embodiment, the temperature of the composition containing the cell aggregates ... g not exceed.

[0112] In some embodiments, removing the aqueous components (particularly water) in the method includes reducing pressure, applying heat, or both to the frozen composition to remove the aqueous components. In embodiments, reducing the pressure includes applying a vacuum to the frozen composition. In embodiments, reducing the pressure includes reducing the pressure to less than 10 torr, e.g., less than about 8 torr, less than about 5 torr, less than about 2 torr, or less than about 1 torr. In embodiments, reducing the pressure includes reducing the pressure to less than about 0.8 torr. In embodiments, reducing the pressure includes reducing the pressure to less than about 0.4 torr.

[0113] In some embodiments, removing the aqueous components (especially water) comprises a primary drying step and a secondary drying step. In embodiments, the primary drying step comprises reducing the pressure to remove the water. In embodiments, the secondary drying step comprises applying heat to remove the aqueous components. In embodiments, the primary drying step comprises sublimation (i.e., transition from a solid phase to a gas phase) of the water of the composition. In embodiments, the secondary drying step comprises dissociation of the water of the composition. When used in the context of freeze-drying, "dissociation" refers to the disruption of physiochemical interactions between the aqueous components (e.g., water molecules) and one or more components of the frozen composition. In embodiments, the pressure and temperature are selected to allow the water of the composition to sublime and / or dissociate.

[0114] In some embodiments, applying heat in the secondary drying step comprises heating the composition to above −20° C. In embodiments, applying heat comprises heating the composition to above −10° C. In embodiments, applying heat comprises heating the composition to above 0° C.

[0115] In some embodiments, lyophilization removes solutes (e.g., water or aqueous components) from the composition. One of skill in the art will understand that "lyophilized cells" may still contain some amount of water. In embodiments, the amount of solutes remaining in the freeze-dried cell aggregates may be intracellular factors that maintain viability when the cells are reconstituted. In embodiments, the freeze-dried cell aggregates contain less than 10% w / w water. In embodiments, the freeze-dried cell aggregates contain less than 5% w / w water. In embodiments, the freeze-dried cell aggregates contain less than 10% w / w, less than 9% w / w, less than 8% w / w, less than 7% w / w, less than 6% w / w, less than 5% w / w, less than 4% w / w, less than 3% w / w, less than 2% w / w, or less than 1% w / w water. In embodiments, a composition comprising a freeze-dried mixture of cells and a freeze-drying agent selected from glycerol, propylene glycol, or a combination thereof contains less than 10% water. In embodiments, the composition comprising a lyophilized mixture of cells and a lyophilizing agent selected from glycerol, propylene glycol, or a combination thereof comprises less than 5% water, In embodiments, the composition comprising a lyophilized mixture of cells and a lyophilizing agent selected from glycerol, propylene glycol, or a combination thereof comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% water.

[0116] In other embodiments, the compositions contain less than about 90% (v / v), sometimes less than about 80% (v / v), sometimes less than about 70% (v / v), sometimes less than about 60% (v / v), sometimes less than about 50% (v / v), sometimes less than about 40% (v / v), sometimes less than about 30% (v / v), sometimes less than about 20% (v / v), sometimes less than about 10% (v / v), sometimes less than about 9% (v / v), sometimes less than about 8% (v / v), sometimes less than about 7% (v / v), sometimes less than about 6% (v / v), sometimes less than about 5% (v / v), sometimes less than about 4% (v / v), sometimes less than about 3% (v / v), sometimes less than about 2% (v / v), or sometimes less than about 1% (v / v) water after lyophilization.

[0117] In some embodiments, lyophilization is performed in a device capable of both freezing the composition and removing water, such as the FREEZONE freeze dryer from LABCONCO, the FREEZEMOBILE, VIRTIS, and HULL freeze dryers from SP SCIENTIFIC, and the STELLAR, REVO, MAGNUM, and EPIC freeze dryers from MILLROCK TECHNOLOGY. In embodiments, the freezing of the composition and removal of the aqueous components are performed in separate devices (e.g., a freezer, e.g., a -80°C freezer) or with liquid nitrogen to freeze the composition and a vacuum system to remove the water.

[0118] In some embodiments, cells are freeze-dried on a two-dimensional surface, for example, on the surface of a plate or container. In embodiments, freeze-drying is performed on a surface (e.g., in a container) with or without a collagen coating. In embodiments, the surface (e.g., container) is a glass or plastic surface / container with or without a membrane for cell attachment or growth.

[0119] In some embodiments, the cells are freeze-dried onto the three-dimensional matrix. In embodiments, freeze-drying is performed on the 3D matrix in a container, for example, with or without a collagen coating.

[0120] In some embodiments, the composition comprising cells, an aqueous component, and a lyophilizing agent further comprises a matrix. Non-limiting examples of matrices include, for example, collagen (e.g., type I, type II, or type IV collagen), elastin, fibronectin, laminin, vitronectin, cadherin, tenascin-C, and other matrix-derived peptides. In embodiments, such cell aggregates are suspended in a matrix. In embodiments, the matrix is ​​a hydrogel.

[0121] In an embodiment of any of the above methods, the composition further comprises a hydrogel. A hydrogel is a network of polymer chains that are hydrophilic and may be viewed as a colloidal gel in which water is the dispersion medium. In an embodiment, the hydrogel is a biocompatible hydrogel. In an embodiment, the hydrogel is a hyaluronan gel, an alginate gel, an agarose gel, a collagen gel, or a combination thereof. In an embodiment, the hydrogel is a combination of a hyaluronan gel, an alginate gel, an agarose gel, or a collagen gel. In an embodiment, the cell aggregates are suspended in the hydrogel. In an embodiment, the freezing of the cell aggregates is performed in suspension or attached to a container. In an embodiment, the freezing of the cell aggregates is performed with the cell aggregates suspended in the hydrogel. In an embodiment, the hydrogel is a hyaluronan gel.

[0122] In some embodiments, the hydrogel is HYSTEM™ Gel available from Sigma Aldrich. HYSTEM™ may include HYSTEM™, which is composed of thiol-modified HA (GLYCOSIL®) and a thiol-reactive crosslinker (EXTRALINK®). See, e.g., Chen D,et al.,Eye(Lond),2017 Jun;31(6):962-971, Devarasetty M,,et al.,Biofabrication,2017 Jun 7;9(2):021002, Mannino RG,et al.,Lab Chip,2017 Jan 31;17(3):407-414, Chen X,et al.,J Tissue Eng Regen Med,2016 May;10(5):437-46, and Engel BJ,,et al.,Adv Healthc Mater,2015 Aug 5;4(11):1664-74. The crosslinker enables the gelation process (without the crosslinker, the HA solution remains liquid). HYSTEM™ may also include HYSTEM™, which may include GLYCOSIL™ and EXTRALINK™, as well as thiol-modified modified collagen fibers called GELIN-S™ to accommodate some cell attachment needs (e.g., stem cells). HYSTEM™ may also include HYSTEM™ HP (HYSHP020-1KT, sigmaaldrich.com / catalog / product / sigma / hyshp020?lang=en®ion=US), which includes both HYSTEM™ C and heparin sulfate to ensure release of growth factors in close proximity to the cells. In an embodiment, the hydrogel includes more than one HYSTEM™ gel. In an embodiment, the hydrogel includes a HYSTEM™ gel that has been further processed (e.g., chemically modified). In an embodiment, the hydrogel includes a HYSTEM gel and an alginate gel, an agarose gel, a collagen gel, and / or a different hyaluronan gel.

[0123] In embodiments of the methods provided herein, the cell aggregates are suspended in a hydrogel. In embodiments, freezing of the cell aggregates is performed in suspension or attached to a container. In embodiments, freezing of the cell aggregates is performed on the cell aggregates suspended in a hydrogel.

[0124] In an embodiment of the method provided herein, the concentration of the monosaccharide in the composition is adjusted before freezing. As used herein, the concentration before freezing is the concentration of the component in the composition before application of the method provided herein.

[0125] In an embodiment of the method provided herein, the concentration of the monosaccharide before freezing is about 0.6 M to about 2.0 M, or about 0.7 M to about 1.7 M, or about 0.8 M to about 1.4 M, or about 0.2 M to about 1.0 M. In an embodiment, the concentration of the monosaccharide before freezing is about 0.3 M to about 0.8 M. In an embodiment, the concentration of the monosaccharide before freezing is about 0.4 M to about 0.6 M. In an embodiment, the concentration of the monosaccharide before freezing is about 0.1 M to about 1.0 M. In an embodiment, the concentration of the monosaccharide before freezing is about 0.25 M to about 0.75 M. In embodiments, the concentration of the monosaccharide before freezing is about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, or 1.0M.

[0126] In embodiments, the monosaccharides may include both D- and L-forms of sugars. In some embodiments, the monosaccharides are glucose, fructose, galactose, mannose, ribose, or deoxyribose. In embodiments, the sugars may include non-naturally occurring or semi-artificial monosaccharides. In embodiments, the sugars may include (i) hexoses (containing six carbons), including D- and L-allose, D- and L-altrose, D- and L-fucose, D- and L-gulose, D-sorbose, and D-tagatose; (ii) pentoses (containing five carbons), such as D- and L-arabinose, D- and L-lyxose, rhamnose, D-ribose, ribulose and its synthetic form, sucroribulose, and D-xylose or wood sugar.

[0127] In embodiments of the methods provided herein, the monosaccharide is glucose, fructose, or galactose. In embodiments, the monosaccharide is glucose.

[0128] In an embodiment of the method where the monosaccharide is glucose, the concentration of glucose before freezing is about 0.2 M to about 1.0 M. In an embodiment, the concentration of glucose before freezing is about 0.3 M to about 0.8 M. In an embodiment, the concentration of glucose before freezing is about 0.4 M to about 0.6 M. In an embodiment, the concentration of glucose before freezing is about 0.1 M to about 1.0 M. In an embodiment, the concentration of glucose before freezing is about 0.25 M to about 0.75 M. In an embodiment, the concentration of glucose before freezing is about 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, 0.5 M, 0.55 M, 0.6 M, 0.65 M, 0.7 M, 0.75 M, 0.8 M, 0.85 M, 0.9 M, 0.95 M, or 1.0 M.

[0129] In embodiments of the methods provided herein, the concentration of urea before freezing is about 0.6 M to about 2.0 M, or about 0.7 M to about 1.7 M, or about 0.8 M to about 1.4 M, or about 0.2 M to about 1.0 M. In embodiments, the concentration of urea before freezing is about 0.3 M to about 0.8 M. In embodiments, the concentration of urea before freezing is about 0.4 M to about 0.6 M. In embodiments, the concentration of urea before freezing is about 0.1 M to about 1.0 M. In embodiments, the concentration of urea before freezing is about 0.25 M to about 0.75 M. In embodiments, the concentration of urea before freezing is about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, or 1.0M.

[0130] In embodiments of the methods provided herein, the molar ratio of urea to monosaccharides can be adjusted by varying the concentration of either component. In embodiments, the molar ratio of urea to monosaccharides before freezing is about 1:3 to about 3:1. In embodiments, the molar ratio of urea to monosaccharides before freezing is about 1:2 to about 2:1. In embodiments, the molar ratio of urea to monosaccharides before freezing is about 1:1. In embodiments, the molar ratio of urea to monosaccharides before freezing is about 1:5 to about 5:1. In embodiments, the molar ratio of urea to monosaccharides before freezing is about 1:4 to about 4:1. In embodiments, the molar ratio of urea to monosaccharides before freezing is about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.

[0131] In embodiments of the method where the monosaccharide is glucose, the molar ratio of urea to glucose before freezing is about 1:3 to about 3:1. In embodiments, the molar ratio of urea to glucose before freezing is about 1:2 to about 2:1. In embodiments, the molar ratio of urea to glucose before freezing is about 1:1. In embodiments, the molar ratio of urea to glucose before freezing is about 1:5 to about 5:1. In embodiments, the molar ratio of urea to glucose before freezing is about 1:4 to about 4:1. In embodiments, the molar ratio of urea to glucose before freezing is about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.

[0132] In embodiments of the methods provided herein, freezing may occur on cells in suspension or attached to a surface of a container. In embodiments, freezing may occur with some cells in suspension and some cells attached to a surface of a container, e.g., both suspended and attached cells. In embodiments, freezing occurs on a collection of cells suspended in a hydrogel. Suitable hydrogels are described herein.

[0133] In embodiments, freezing is performed in a container with or without a collagen coating. In embodiments, the container is a glass or plastic container with or without a membrane for cell attachment or growth. In embodiments, freezing is performed on a surface with or without a collagen coating. In embodiments, the surface is glass or plastic.

[0134] In embodiments of the methods provided herein, the cell population is about 1×10 cells / mL. 4 ~1 x 10 cells per mL 7 In an embodiment, the cell population is about 1 x 10 cells per mL. 5 ~4 x 10 cells per mL 5 In an embodiment, the cell population is about 2×10 cells per mL. 5 ~2.5 x 10 cells per mL 5In an embodiment, the cell population is about 1 x 10 cells per mL. 5 1.5 x 10 cells per mL 5 2 x 10 cells per mL 5 2.5 x 10 cells per mL 5 3 x 10 cells per mL 5 3.5 x 10 cells per mL 5 Cells, 4 x 10 per mL 5 4.5 x 10 cells per mL 5 cells or 5 x 10 per mL 5 Each cell is a single cell.

[0135] In embodiments of the methods provided herein, the bulking agent comprises a disaccharide. In embodiments, the concentration of the disaccharide before freezing is about 0.1 M to about 1.0 M. In embodiments, the concentration of the disaccharide before freezing is about 0.1 M to about 0.5 M. In embodiments, the concentration of the disaccharide before freezing is about 0.2 M to about 0.4 M. In embodiments, the concentration of the disaccharide before freezing is about 0.1 M to about 0.4 M. In embodiments, the concentration of the disaccharide before freezing is about 0.1 M to about 0.3 M. In embodiments, the concentration of the disaccharide before freezing is about 0.25 M to about 0.45 M. In embodiments, the concentration of the disaccharide before freezing is about 0.25 M to about 0.75 M. In embodiments, the concentration of the disaccharide before freezing is about 0.3 M to about 0.8 M. In embodiments, the concentration of the disaccharide prior to freezing is about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, or 1.0M.

[0136] In embodiments, the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, lactulose, isomaltose, melibiose, or gentiobiose. In embodiments, the disaccharide has a C1-C1 glycosidic bond. In embodiments, the disaccharide may include, for example, an α(1→2)β, β(1→4), or α(1→4) bond. In embodiments, the disaccharide is sucrose.

[0137] In an embodiment of the method in which sucrose is used, the concentration of sucrose before freezing is about 0.1 M to about 1.0 M. In an embodiment, the concentration of sucrose before freezing is about 0.1 M to about 0.5 M. In an embodiment, the concentration of sucrose before freezing is about 0.2 M to about 0.4 M. In an embodiment, the concentration of sucrose before freezing is about 0.1 M to about 0.4 M. In an embodiment, the concentration of sucrose before freezing is about 0.1 M to about 0.3 M. In an embodiment, the concentration of sucrose before freezing is about 0.25 M to about 0.45 M. In an embodiment, the concentration of sucrose before freezing is about 0.25 M to about 0.75 M. In an embodiment, the concentration of sucrose before freezing is about 0.3 M to about 0.8 M. In embodiments, the concentration of sucrose before freezing is about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, or 1.0M.

[0138] In embodiments of the methods provided herein, the bulking agent comprises a sugar alcohol. In embodiments, the concentration of the sugar alcohol before freezing is about 0.1 M to about 1.0 M. In embodiments, the concentration of the sugar alcohol before freezing is about 0.3 M to about 0.8 M. In embodiments, the concentration of the sugar alcohol before freezing is about 0.4 M to about 0.6 M. In embodiments, the concentration of the sugar alcohol before freezing is about 0.1 M to about 1.0 M. In embodiments, the concentration of the sugar alcohol before freezing is about 0.25 M to about 0.75 M. In embodiments, the concentration of the sugar alcohol prior to freezing is about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, or 1.0M.

[0139] In an embodiment, the sugar alcohol is mannitol, sorbitol, galactitol, fucitol, iditol, or inositol. In an embodiment, the sugar alcohol is mannitol. In an embodiment, the concentration of mannitol before freezing is about 0.6 M to about 2.0 M, or about 0.7 M to about 1.7 M, or about 0.8 M to about 1.4 M, or about 0.1 M to about 1.0 M. In an embodiment, the concentration of mannitol before freezing is about 0.3 M to about 0.8 M. In an embodiment, the concentration of mannitol before freezing is about 0.4 M to about 0.6 M. In an embodiment, the concentration of mannitol before freezing is about 0.1 M to about 1.0 M. In an embodiment, the concentration of mannitol before freezing is about 0.25 M to about 0.75 M. In embodiments, the concentration of mannitol prior to freezing is about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, or 1.0M.

[0140] In embodiments of the methods provided herein, the bulking agent comprises both a disaccharide and a sugar alcohol. In embodiments, the bulking agent comprises only a disaccharide. In embodiments, the bulking agent comprises only a sugar alcohol. In embodiments, the bulking agent comprises both sucrose and mannitol. In embodiments, the bulking agent comprises only sucrose. In embodiments, the bulking agent comprises only mannitol.

[0141] In embodiments, the methods provided herein do not use DMSO for the preservation of cells. In embodiments, the methods provided herein allow for the use of DMSO at a lower concentration than that required for the preservation of cells using conventional methods. In embodiments of the methods provided herein, the composition does not include DMSO. In embodiments, the composition includes DMSO at a concentration of about 1% to about 10% prior to freezing. In embodiments, the composition includes DMSO at a concentration of about 1% to about 10%, or about 1.5% to about 8%, or about 2% to about 5% prior to freezing. In embodiments, the composition includes DMSO at a concentration of about 3% to about 8% prior to freezing. In embodiments, the composition includes DMSO at a concentration of about 1% to about 5% prior to freezing. In embodiments, the composition includes DMSO at a concentration of about 1% to about 4.5%, or about 1% to about 4%, or about 1% to about 3% prior to freezing. In embodiments, the composition comprises DMSO at a concentration of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% prior to freezing.

[0142] In an embodiment of the method provided herein, prior to freezing in (a), the cell aggregates are isolated and contacted with a poration solution. The term "poration solution" as used herein typically refers to a solution composed of cell culture medium and sugar. There is no purpose to perforate the cell membrane. Rather, the poration solution is used as a pre-incubation solution to introduce sugar, e.g., trehalose, contained therein to the cells and then facilitate cryoprotection. In an embodiment, the method includes a step in which, prior to freezing, the cell aggregates are isolated (e.g., by centrifugation or filtration, or by simple removal of the culture medium from the cells, usually attached to the wall or bottom of a container such as a culture flask) and resuspended in the poration solution. The poration solution includes a sugar (e.g., trehalose) in an aqueous solution. In an embodiment, the poration solution includes a sugar and water. In an embodiment, the poration solution includes trehalose and water. In an embodiment, the poration solution includes trehalose and cell culture medium. In an embodiment, the cell aggregates are placed in the poration solution and then subjected to heat treatment. In an embodiment, the heat treatment is carried out as outlined in He et al., (He, X., et al., Cell Preservation Technology 4, 178-187 (2006)). In an embodiment, the heat treatment includes, for example, placing a cell aggregate in a poration solution, then cooling the composition for 1-60 minutes, e.g., 2-50 minutes, 3-40 minutes, 4-30 minutes, 5-20 minutes, 5-15 minutes, or about 10 minutes, and then heating the composition for 1-60 minutes, e.g., 2-50 minutes, 3-40 minutes, 4-30 minutes, 5-20 minutes, 5-15 minutes. The cooling and heating can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In an embodiment, the cooling and heating can be repeated 1, 2, or 3 times. In an embodiment, the cooling was -20° C. to 10° C., or -10° C. to 10° C., or 0° C. to 10° C., or 2° C. to 8° C. In an embodiment, the heating was 0° C. to 50° C., or 4° C. to 40° C., 8° C. to 40° C., or 20° C. to 40° C.In embodiments, the difference between the cooling temperature and the heating temperature is greater than 4°C, greater than 6°C, greater than 8°C, greater than 10°C, greater than 15°C, or greater than 20°C.

[0143] In an embodiment in which the poration solution contains trehalose, the concentration of trehalose in the poration solution is about 0.1 M to about 1.0 M. In an embodiment, the concentration of trehalose in the poration solution is about 0.2 M to about 0.6 M. In an embodiment, the concentration of trehalose in the poration solution is about 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, 0.5 M, 0.55 M, 0.6 M, 0.65 M, 0.7 M, 0.75 M, 0.8 M, 0.85 M, 0.9 M, 0.95 M, or 1.0 M.

[0144] In embodiments, the tonicity of a composition can affect cell viability and attachment. "Tonicity" refers to the ability of an extracellular solution to move water into or out of cells by osmosis. Tonicity is related to the osmotic pressure of a solution, i.e., the total concentration of all solutes in the solution. An "isotonic" solution refers to a solution that has the same osmotic pressure as the cells, and therefore there is no net movement of water into or out of the cells. A "hypertonic" solution refers to a solution that has a higher osmotic pressure than the cells, and therefore water moves from the cells to the extracellular solution. A "hypotonic" solution refers to a solution that has a lower osmotic pressure than the cells, and therefore water moves from the extracellular solution into the cells. In embodiments, a composition that includes a collection of cells is an isotonic solution. In embodiments, a composition that includes cells is a hypertonic solution. In some embodiments, the tonicity of a composition facilitates the freeze-drying process, protects the cells from damage or lysis, and / or increases cell viability.

[0145] In some embodiments, the aqueous component of the composition comprises a buffer. Non-limiting examples of buffers include phosphate buffer, Tris buffer, HEPES buffer, acetate buffer, bicarbonate buffer, citrate buffer, Tricine buffer, TES buffer, etc. In embodiments, the aqueous component of the composition comprises a phosphate buffer, a Tris buffer, an acetate buffer, a bicarbonate buffer, a histidine buffer, a citrate buffer, or a combination thereof.

[0146] In some embodiments, the aqueous component of the composition comprises a cell culture medium. Non-limiting examples of cell culture media include DMEM, MEM, RPMI 1640, EXPI293, OPTI-MEM, and STEMPRO from GIBCO, HYQ-RS from HYCLONE, X-VIVO and Hybridoma Serum-Free Culture Media from LONZA BIOWHITTAKER, etc. In embodiments, the medium is serum-free. In embodiments, the medium is fetal bovine serum, bovine serum, or human serum-free. In embodiments, the medium is a chemically defined medium. Exemplary chemically defined media may include basal media (e.g., DMEM, F12, or RPMI 1640), sugars such as dextrose and glucose, amino acids, vitamins, inorganic salts, buffers, antioxidants, growth factors and energy sources, recombinant serum albumin, chemically defined lipids, recombinant insulin and / or zinc, recombinant transferrin or iron, selenium, and / or antioxidant thiols such as 2-mercaptoethanol or 1-thioglycerol. In embodiments, the cell culture medium includes dextrose, glucose, amino acids, recombinant serum albumin, growth factors, or combinations thereof.

[0147] In embodiments of the methods provided herein, the cells are reconstituted in a reconstitution agent, e.g., any liquid that can be used to suspend frozen or lyophilized cells. In embodiments, the reconstitution agent comprises a cell culture medium. In embodiments, the reconstitution agent comprises a cell culture medium as described above.

[0148] In some embodiments, the freeze-drying process does not lyse or damage the cells, at least not all of the cells. In embodiments, freeze-dried cells are capable of being reconstituted and are viable. In the context of freeze-drying, "reconstitution" refers to the process of rehydrating freeze-dried materials, e.g., freeze-dried cells. In embodiments, cells reconstituted after freeze-drying are viable. In embodiments, cells reconstituted after freeze-drying are capable of performing the same cellular functions as cells that are not subjected to freeze-drying. In embodiments, cells reconstituted after freeze-drying are viable. In embodiments, cells reconstituted after freeze-drying retain the ability to adhere or attach to other cells. In embodiments, cells reconstituted after freeze-drying retain the ability to proliferate. In embodiments, stem cells reconstituted after freeze-drying retain the ability to differentiate. In embodiments, cells reconstituted after freeze-drying are suitable for use in cell therapy, e.g., introduction into humans or animals. In embodiments, cells reconstituted after freeze-drying attach to culture flasks, proliferate, and are of regular morphology, thus indicating viability and therefore regular cellular functions.

[0149] In some embodiments, reconstitution of lyophilized cells comprises resuspending the lyophilized cells in a reconstitution agent to form a reconstituted composition. In the context of lyophilization, "reconstitution agent" refers to a substance that resuspends and rehydrates the lyophilized product. In embodiments, the reconstitution agent comprises water. In embodiments, the reconstitution agent comprises a buffer. In embodiments, the reconstitution agent comprises cell culture medium. In embodiments, the reconstitution agent comprises polyvinylpyrrolidone (PVP). In embodiments, the reconstitution agent comprises trehalose. In embodiments, the reconstitution agent comprises PVP, trehalose, or a combination thereof. In embodiments, the reconstitution agent comprises phosphate buffered solution (PBS). In some embodiments, the reconstitution agent can be a solution known to maintain cell viability.

[0150] The methods provided herein may be used for freezing or lyophilizing various types of cells and cells from various sources. In some embodiments, the cells are obtained from a cell culture, i.e., cultured cells. In embodiments, the population of cells comprises mammalian cells. In embodiments, the population of cells comprises stem cells. In embodiments, the population of cells comprises pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or hematopoietic stem cells. In embodiments, the population of cells comprises mesenchymal stem cells. In embodiments, the population of cells comprises induced pluripotent stem cells. In embodiments, the population of cells comprises neuroblastoma cells. In embodiments, the neuroblastoma cells are SK-N-AS cells. In embodiments, any of the cells listed above are human cells.

[0151] In embodiments, the population of cells comprises bacterial cells. Examples of such bacterial cells include, but are not limited to, E. coli, S. aureus, V. cholerae, S. pneumoniae, B. subtilis, C. crescentus, M. genitalium, A. fischeri, Synechocystis, P. fluorescens, A. vinelandii, S. coelicolor. In embodiments, the bacterial cells are of bacteria used in the preparation of foods and / or beverages. Non-limiting exemplary genera of such cells include, but are not limited to, Acetobacter, Arthrobacter, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium. rium, Corynebacterium, Enterococcus, Gluconacetobacter, Hafnia, Halomonas, Kocuria, Lactobacillus (L.acetotolerans, L.acidipiscis, L. .acidophilus, L.alimentarius, L.brevis, L.bucheri, L.casei, L.curvatus, L.fermentum, L.hilgardii, L.jensenii, L.kimchii, L.lactis, L. paracasei, L. plantarum, and L. sakei), Leuconostoc, Microbacterium, Pediococcus, Propionibacterium, Weissella, and Zymomonas.

[0152] In an embodiment, the collection of cells comprises eukaryotic cells. In an embodiment, the eukaryotic cells are animal or human cells. In an embodiment, the eukaryotic cells are human, rodent or bovine cell lines or cell lines. Examples of such cells, cell lines or cell lines include, but are not limited to, mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cell, COS, e.g., COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLA, EBl, EB2, EB3, oncolytic or hybridoma cell lines. In an embodiment, the eukaryotic cells are CHO cell lines. In an embodiment, the eukaryotic cells are CHO cells. In embodiments, the cell is a CHO-K1 cell, a CHO-K1 SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS cell, a CHO GS knockout cell, a CHO FUT8 GS knockout cell, a CHOZN cell, or a CHO-derived cell. The CHO GS knockout cell (e.g., a GSKO cell) is, for example, a CHO-K1 SV GS knockout cell. The eukaryotic cell may also be an avian cell, cell line, or cell lineage, such as, for example, an EBX cell, EB14, EB24, EB26, EB66, or Ebvl3 cell.

[0153] In some embodiments, the eukaryotic cells are human cells. In embodiments, the human cells are stem cells. In embodiments, the methods provided herein are advantageous for producing freeze-dried stem cells that are viable upon reconstitution, for example, for use in cell therapy. The stem cells may be pluripotent stem cells, including, for example, embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells, neural stem cells, epithelial stem cells, skin stem cells, etc.), and mesenchymal stem cells (MSCs). In embodiments, the stem cells are mesenchymal stem cells (MSCs). In embodiments, the MSCs are obtained from bone marrow, umbilical cord blood, peripheral blood, fallopian tubes, liver, and / or lungs of a human subject. In embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In embodiments, the iPSCs have at least one vector capable of expressing one or more pluripotency factors. In embodiments, the iPSCs are derived from fibroblasts, keratinocytes, peripheral blood mononuclear cells (PBMCs), hepatocytes, neural cells, B cells, muscle cells, adrenal cells, and / or renal epithelial cells of a human subject, or any other type of cell known to be suitable for becoming an induced pluripotent stem cell. In embodiments, the human cells are any differentiated form of the cells described herein. In embodiments, the cells comprise at least one vector capable of expressing one or more pluripotency factors, but the pluripotency factors have not yet been expressed prior to lyophilization, and therefore the cells have not yet been induced to pluripotency. In embodiments, the eukaryotic cells are cells derived from any primary cell in culture.

[0154] In some embodiments, the eukaryotic cells are hepatocytes, such as human hepatocytes, animal hepatocytes, or non-parenchymal cells. For example, the eukaryotic cells can be seedable metabolically competent human hepatocytes, seedable induction competent human hepatocytes, seedable human hepatocytes, suspension competent human hepatocytes (including pooled hepatocytes of 10 donors and 20 donors), human hepatic Kupffer cells, human hepatic stellate cells, dog hepatocytes (including single and pooled beagle hepatocytes), mouse hepatocytes (including CD-1 and C57BI / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including cynomolgus or rhesus hepatocytes), feline hepatocytes (including domestic shorthair hepatocytes), and rabbit hepatocytes (including New Zealand white hepatocytes). In embodiments, the cells are derived from the eye. In embodiments, the cells are retinal cells, scleral cells, choroidal epithelial cells, macrophage cells, or immune cells.

[0155] In some embodiments, the cells are obtained from a cell line. Non-limiting examples of cell lines include MOLT-4 (differentiated or undifferentiated), Jurkat, HL60 (differentiated or undifferentiated), U-937 (differentiated or undifferentiated), HDLM-2, THP-1 (differentiated or undifferentiated), GA10, Ramos, HUVEC, PANC-1, Expi293, HaCat, HCT-15, H-2228, peripheral blood mononuclear cells (PBMC), KU-812, MC-04, HT-1376, TT, HCT-1116, MCF-7, Calu-3, and the like. Exemplary monocytic cell lines include THP-1, differentiated THP-1, HL60, and differentiated HL60. An exemplary NK cell line is NK92. Exemplary T cell lines include Jurkat and Molt-4. An exemplary B cell line is GA-10. Exemplary endothelial cell lines include HUVEC and differentiated HUVEC. Exemplary hepatic cell lines include HepG2 and differentiated HepG2. Exemplary epithelial cell lines include A549, A431, Caco-2, HT29, LNCap, SKOV3, SW480, PC3, MDMB-468, MDMB-231, MCF7, HT-1376, PANC-1, HCT15, Calu-3, Skov3, Bewo, K562, and HeLa. Further additional cell lines include, for example, HT-29 sARPE-19, SH-SY5Y, and U87-MG.

[0156] Further examples of cells include T cells, B cells, dendritic cells, NK cells, monocytes, macrophages, granulocytes, platelets, red blood cells, endothelial cells (e.g., aortic endothelial cells), epithelial cells, stem cell precursors, mesenchymal stem cells, hematopoietic stem cells, leukocytes, senescent cells, adipocytes, hepatocytes, muscle cells, or skeletal muscle cells. T cells include, for example, helper T cells, such as subtypes Th1, Th2, Th9, Th17, Th22, and Tfh; regulatory T cells; killer T cells; γδTCR+ T cells; and natural killer T cells. Adipocytes include, for example, normal adipocytes, diabetic adipocytes, omental adipocytes, MSC-derived adipocytes, preadipocytes, and omental preadipocytes.

[0157] In some embodiments, the eukaryotic cell is a plant cell. For example, the plant cell can be from a crop plant, such as cassava, corn, sorghum, wheat, or rice. The plant cell can be from an algae, a tree, or a plant. The plant cell can be from a monocotyledonous or dicotyledonous plant, or from a crop or cereal plant, a productive plant, a fruit, or a vegetable. For example, the plant cell can be from a tree, such as a citrus tree, such as an orange, grapefruit, or lemon tree; a peach or nectarine tree; an apple or pear tree; a nut tree, such as an almond, walnut, or pistachio tree; a plant of the Solanaceae family, such as potato; a plant of the Brassica genus, a plant of the Lactuca genus; a plant of the Spinacia genus; a plant of the Capsicum genus; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, and the like.

[0158] In some embodiments, the cells include cells in a microbiota. In embodiments, the cells are a combination of cells that comprise a microbiota. In embodiments, the cells include a large number of intact microorganisms in a microbiota that live on and in an organism, such as a human, livestock (e.g., cows, pigs, chickens, horses, etc.), or zoo animals. In embodiments, the microbiota includes bacteria, archaea (primitive single-celled organisms), fungi, and even some protozoa and non-living viruses. In embodiments, the microbiota is a gastrointestinal microbiota (e.g., esophageal, stomach, and / or intestinal microbiota), oral microbiota, urinary tract microbiota, nasal microbiota, respiratory microbiota, skin microbiota, vaginal microbiota, rectal microbiota, or a combination thereof. In embodiments, the microbiota is an infant's microbiota. In embodiments, the microbiota is an adult's microbiota.

[0159] In the freeze-drying methods provided herein, removing water includes reducing pressure, applying heat, or both to the frozen composition to remove water. Freeze-drying methods are described herein. In embodiments, removing water includes a primary drying step and a secondary drying step. Primary and secondary drying steps are described herein. In embodiments, removing water includes only a primary drying step. In embodiments, the primary drying step includes reducing pressure to remove aqueous components. In embodiments, the secondary drying step includes applying heat to remove aqueous components.

[0160] In some embodiments, the freezing is performed at about -30°C to about -100°C. In embodiments, the freezing is performed at about -60°C to about -90°C. In embodiments, the freezing is performed at about -70°C to about -80°C. In embodiments, the freezing is performed at about -60°C, about -65°C, about -70°C, about -75°C, about -80°C, about -85°C, or about -90°C. In embodiments, the freezing is performed at about -10°C to about -100°C. In embodiments, the freezing is performed at about -20°C to about -90°C. In embodiments, the freezing is performed at about -40°C to about -60°C. In embodiments, the freezing reduces the temperature of the composition to about -50°C. In embodiments, the freezing reduces the temperature of the composition to about -80°C. In embodiments, freezing reduces the temperature of the composition to about -40°C, -50°C, -60°C, -70°C, -80°C, or -90°C.

[0161] In some embodiments, freezing reduces the temperature of the composition to −50° C. and the water is removed at a pressure of about 20 mTorr to about 40 mTorr chamber pressure. In embodiments, freezing reduces the temperature of the composition to −40° C. and the water is removed at a pressure of about 60 mTorr to about 80 mTorr chamber pressure.

[0162] Once processed using the methods provided herein, the cells can be stored for extended periods of time. In an embodiment of the method of freezing cells, resuspending the frozen cells occurs more than 2 hours after freezing. In an embodiment, resuspending the frozen cells occurs more than 1 day after freezing. In an embodiment, resuspending the frozen cells occurs more than 1 week after freezing. In an embodiment, resuspending the frozen cells occurs more than 1 month after freezing.

[0163] In an embodiment of the method of freeze-drying cells, resuspending the freeze-dried cells occurs more than 2 hours after removal of water. In an embodiment, resuspending the freeze-dried cells occurs more than 1 day after removal of water. In an embodiment, resuspending the freeze-dried cells occurs more than 1 week after removal of water. In an embodiment, resuspending the freeze-dried cells occurs more than 1 month after removal of water.

[0164] In embodiments of the methods, the frozen or lyophilized cells are stored below about -20°C prior to resuspension. In embodiments, the frozen or lyophilized cells are stored at about -20°C to about 30°C prior to resuspension. In embodiments, the frozen or lyophilized cells are stored at about 4°C to about 28°C prior to resuspension. In embodiments, the frozen or lyophilized cells are stored at about 10°C to about 27°C prior to resuspension. In embodiments, the frozen or lyophilized cells are stored at about 2°C to about 8°C prior to resuspension.

[0165] In embodiments, the frozen or lyophilized cells are stored below about -20°C for more than 2 days before resuspension, e.g., more than 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months before resuspension. In embodiments, the frozen or lyophilized cells are stored above about -20°C for more than 2 days before resuspension, e.g., more than 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months before resuspension. In embodiments, the lyophilized cells are stored at about 20°C to about 25°C for more than 1 week before resuspension, e.g., more than 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months before resuspension. In embodiments, the lyophilized cells are stored at about 2°C to about 8°C for more than 1 week before resuspension, e.g., more than 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months before resuspension.

[0166] As discussed herein, the method provides viable cells after freezing or lyophilization. A "viable" cell refers to a cell that is alive and functioning, e.g., a cell that is alive and capable of performing normal cell functions. In embodiments, the normal cell function of a cell is adhesion or attachment to other cells. "Cell adhesion" or "cell attachment" refers to the process by which cells interact and attach to adjacent cells and / or the surface of their growth vessel through surface protein interactions. Cells that are unable to attach may not be able to proliferate or perform normal cell functions. For example, stem cells that are unable to attach may not be able to differentiate. In another example, cells that are introduced into a patient for cell therapy but are unable to attach may not provide therapeutic benefit because the cells are unable to perform normal functions in the patient. Thus, in embodiments, a viable cell is a cell that is capable of attachment. In embodiments, a viable cell is a cell that is capable of proliferation. In embodiments, a viable stem cell is a stem cell that is capable of differentiation.

[0167] In some embodiments, the viability of cells is determined, for example, by measuring the redox potential and / or metabolic activity of the cell aggregates. The redox potential and / or metabolic activity of the cell aggregates can be measured by reagents such as resazurin, a component of the ALAMARBLUE® and PRESTOBLUE® reagents, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) used in the VYBRANT MTT Cell Viability Assay, or XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) used in the CYQUANT XTT Cell Viability Assay. In embodiments, the viability of cells is determined by measuring or examining the integrity of the cell membrane, for example, using microscopy or flow cytometry. When used to determine viability, microscopy allows for observing, for example, whether the morphology of the cells appears similar to cells known to be viable, and / or whether the cells grow, and / or whether the cells are attached to the walls or bottom of a culture flask. In an embodiment, the viability of the cells is measured after the cells are frozen and then thawed or resuspended. In an embodiment, the viability of the cells is measured after the cells are lyophilized and then reconstituted.

[0168] Additional examples of cell viability assays are described, for example, in Riss et al., “Cell Viability Assays,” 2013 May 1 [Updated 2016 Jul 1]. In:Sittampalam GS, Coussens NP, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004. Available at www.ncbi.nlm.nih.gov / books / NBK144065.

[0169] One of skill in the art can understand that when discussing the viability of a cell (i.e., a collection of cells), not 100% of the cells are viable. In embodiments, a viable cell collection comprises at least 1% viable cells. In embodiments, a viable cell collection comprises at least 5% viable cells. In embodiments, a viable cell collection comprises at least 10% viable cells. In embodiments, a viable cell collection comprises at least 15% viable cells. In embodiments, a viable cell collection comprises at least 20% viable cells. In embodiments, a viable cell collection comprises at least 30% viable cells. In embodiments, a viable cell collection comprises at least 40% viable cells. In embodiments, a viable cell collection comprises at least 50% viable cells. In embodiments, a viable cell collection comprises at least 60% viable cells. In embodiments, a viable cell collection comprises at least 70% viable cells. In embodiments, a viable cell collection comprises at least 80% viable cells. In embodiments, the viable cell population comprises at least 90% viable cells. In embodiments, the viable cell population comprises about 1% viable cells to about 99% viable cells, 5% viable cells to about 99% viable cells, 10% viable cells to about 99% viable cells, 20% viable cells to about 99% viable cells, about 30% viable cells to about 99% viable cells, about 40% viable cells to about 99% viable cells, about 50% viable cells to about 99% viable cells, about 60% viable cells to about 99% viable cells, about 70% viable cells to about 99% viable cells, about 80% viable cells to about 99% viable cells, or about 90% viable cells to about 99% viable cells.

[0170] In some embodiments, the viable cell population comprises at least 1% adherent cells. In embodiments, the viable cell population comprises at least 5% adherent cells. In embodiments, the viable cell population comprises at least 10% adherent cells. In embodiments, the viable cell population comprises at least 20% adherent cells. In embodiments, the viable cell population comprises at least 30% adherent cells. In embodiments, the viable cell population comprises at least 40% adherent cells. In embodiments, the viable cell population comprises at least 50% adherent cells. In embodiments, the viable cell population comprises at least 60% adherent cells. In embodiments, the viable cell population comprises at least 70% adherent cells. In embodiments, the viable cell population comprises at least 80% adherent cells. In embodiments, the viable cell population comprises at least 90% adherent cells. In an embodiment, the viable cell population comprises about 1% to about 99% adherent cells, 5% to about 99% adherent cells, 10% to about 99% adherent cells, 20% to about 99% adherent cells, about 30% to about 99% adherent cells, about 40% to about 99% adherent cells, about 50% to about 99% adherent cells, about 60% to about 99% adherent cells, about 70% to about 99% adherent cells, about 80% to about 99% adherent cells, or about 90% to about 99% adherent cells.

[0171] In some embodiments, the reconstituted cells are viable. Methods for measuring cell viability are described herein. In embodiments, cell viability is measured by staining and detecting intact cell membranes. In embodiments, cell viability is measured by assessing metabolic activity, for example, by using the ALAMARBLUE®, MTT, or XTT test. In embodiments, at least about 1% of the cells in the reconstituted composition are viable. In embodiments, at least about 5% of the cells in the reconstituted composition are viable. In embodiments, at least about 10% of the cells in the reconstituted composition are viable. In embodiments, at least about 20% of the cells in the reconstituted composition are viable. In embodiments, at least about 30% of the cells in the reconstituted composition are viable. In embodiments, at least about 40% of the cells in the reconstituted composition are viable. In embodiments, at least about 50% of the cells in the reconstituted composition are viable. In embodiments, at least about 60% of the cells in the reconstituted composition are viable. In embodiments, at least about 70% of the cells in the reconstituted composition are viable. In embodiments, at least about 80% of the cells in the reconstituted composition are viable. In embodiments, at least about 90% of the cells in the reconstituted composition are viable.

[0172] In some embodiments, the reconstituted cells are capable of attachment as measured by the ALAMARBLUE® test. As described herein, the ALAMARBLUE® test assesses the metabolic activity of cells, which may be an indicator of cell attachment. In embodiments, at least 20% of the cells in the reconstituted composition are attached as measured by the ALAMARBLUE® test. In embodiments, at least 30% of the cells in the reconstituted composition are attached as measured by the ALAMARBLUE® test. In embodiments, at least 40% of the cells in the reconstituted composition are attached as measured by the ALAMARBLUE® test. In embodiments, at least 50% of the cells in the reconstituted composition are attached as measured by the ALAMARBLUE® test. In embodiments, at least 60% of the cells in the reconstituted composition are attached as measured by the ALAMARBLUE® test. In embodiments, at least 70% of the cells in the reconstituted composition are attached as measured by the ALAMARBLUE® test. In embodiments, at least 80% of the cells in the reconstituted composition are attached as measured by the ALAMARBLUE® test. In embodiments, at least 90% of the cells in the reconstituted composition are attached as measured by the ALAMARBLUE® test.

[0173] Compositions Comprising a Population of Viable Cells In embodiments, the present disclosure provides a composition comprising a population of viable cells, an aqueous component, urea, and a simple sugar. In embodiments, the composition further comprises a bulking agent. In embodiments, the composition further comprises hyaluronan gel.

[0174] In an embodiment of the composition, the concentration of the monosaccharide is from about 0.2M to about 1.25M, or from about 0.3M to about 0.8M, or from about 0.4M to about 0.6M.

[0175] In embodiments of the composition, the concentration of urea is from about 0.2M to about 1.25M, or from about 0.2M to about 0.8M, or from about 0.4M to about 0.6M.

[0176] In an embodiment of the composition, the concentration of the monosaccharide is from about 0.6M to about 2.0M, or from about 0.7M to about 1.7M, or from about 0.8M to about 1.4M.

[0177] In embodiments of the composition, the concentration of urea is from about 0.6M to about 2.0M, or from about 0.7M to about 1.7M, or from about 0.8M to about 1.4M.

[0178] In embodiments of the composition, the molar ratio of urea to monosaccharide in the composition is from about 5:1 to about 1:5, or from about 1:3 to about 3:1, or from about 1:2 to about 2:1, or is about 1:1.

[0179] In an embodiment of the composition, the bulking agent comprises a) a disaccharide, where the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, lactulose, isomaltose, melibiose, or gentiobiose, or b) a sugar alcohol, where the sugar alcohol is mannitol, sorbitol, galactitol, fucitol, iditol, or inositol, or c) both a disaccharide and a sugar alcohol, or d) both sucrose and mannitol. In an embodiment of the composition, the concentration of the disaccharide before freezing is about 0.1M to about 1.0M, or about 0.1M to about 0.5M, or about 0.2M to about 0.4M, and / or the concentration of the sugar alcohol before freezing is about 0.1M to about 1.0M, or about 0.3M to about 0.8M, or about 0.4M to about 0.6M.

[0180] In an embodiment of the composition, the composition comprises about 0.2M to about 1.25M urea and about 0.2M to about 1.25M glucose.

[0181] In embodiments of the composition, the composition does not include DMSO, or the composition includes DMSO at a concentration of about 1% to about 10% prior to freezing, or the composition includes DMSO at a concentration of about 1% to about 5% prior to freezing.

[0182] In an embodiment, the composition is in a frozen state at a temperature of about -10°C to about -100°C, or about -20°C to about -90°C, or about -40°C to about -60°C. In an embodiment, the composition is a lyophilized composition. In an embodiment, the composition contains less than about 90% (volume / volume) water. In an embodiment, the composition contains less than about 80% (volume / volume) water. In an embodiment, the composition contains less than about 70% (volume / volume) water. In an embodiment, the composition contains less than about 60% (volume / volume) water. In an embodiment, the composition contains less than about 50% (volume / volume) water. In an embodiment, the composition contains less than about 40% (volume / volume) water. In an embodiment, the composition contains less than about 30% (volume / volume) water. In an embodiment, the composition contains less than about 20% (volume / volume) water. In embodiments, the composition contains less than about 10% (v / v), less than about 9% (v / v), less than about 8% (v / v), less than about 7% (v / v), less than about 6% (v / v), less than about 5% (v / v), less than about 4% (v / v), less than about 3% (v / v), less than about 2% (v / v), or less than about 1% (v / v) water.

[0183] In an embodiment, the composition is in a frozen state at a temperature of about −10° C. to about −100° C., or about −20° C. to about −90° C., or about −40° C. to about −60° C. In an embodiment, the composition is a freeze-dried composition. In embodiments, the compositions described above contain less than about 90% (v / v), or less than about 80% (v / v), or less than about 70% (v / v), or less than about 60% (v / v), or less than about 50% (v / v), or less than about 40% (v / v), or less than about 30% (v / v), or less than about 20% (v / v), or less than about 10% (v / v), or less than about 9% (v / v), or less than about 8% (v / v), or less than about 7% (v / v), or less than about 6% (v / v), or less than about 5% (v / v), or less than about 4% (v / v), or less than about 3% (v / v), or less than about 2% (v / v), or less than about 1% (v / v) water.

[0184] In an embodiment, the present disclosure provides a composition comprising a population of viable cells, an aqueous component, urea, a simple sugar, and optionally a bulking agent, wherein said composition contains less than about 90% (v / v), or less than about 80% (v / v), or less than about 70% (v / v), or less than about 60% (v / v), or less than about 50% (v / v), or less than about 40% (v / v), or less than about 30% (v / v), or less than about 20% (v / v), or less than about 10% (v / v), or less than about 9% (v / v), or less than about 8% (v / v), or less than about 7% (v / v), or less than about 6% (v / v), or less than about 5% (v / v), or less than about 4% (v / v), or less than about 3% (v / v), or less than about 2% (v / v), or less than about 1% (v / v) water.

[0185] In embodiments, the present disclosure provides a composition comprising a population of viable cells, an aqueous component, urea, a simple sugar, and a bulking agent, wherein the composition contains less than about 10% (volume / volume) water. In embodiments, the composition contains less than about 5% (volume / volume) water. In embodiments, the composition contains less than about 3% (volume / volume) water. In embodiments, the composition contains less than about 1% (volume / volume) water.

[0186] In embodiments of the composition, the molar ratio of glucose to urea (i.e., the (M / M) ratio) in the composition is from about 30:1 to about 1:3. In embodiments, the molar ratio of glucose to urea in the composition is from about 15:1 to about 1:2. In embodiments, the molar ratio of glucose to urea in the composition is from about 3:1 to about 1:1. In embodiments, the molar ratio of glucose to urea in the composition is about 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.

[0187] In embodiments, the present disclosure also provides a composition comprising a population of viable cells, an aqueous component, urea, a monosaccharide, and optionally a bulking agent, wherein the molar ratio of glucose to urea in the composition is about 30:1 to about 1:3. In embodiments, the molar ratio of glucose to urea in the composition is about 15:1 to about 1:2. In embodiments, the molar ratio of glucose to urea in the composition is about 3:1 to about 1:1. In embodiments, the molar ratio of glucose to urea in the composition is about 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.

[0188] In embodiments of the compositions provided herein, the monosaccharide is glucose, fructose, galactose, mannose, ribose, or deoxyribose. In embodiments, the monosaccharide is glucose, fructose, or galactose. In embodiments, the monosaccharide is glucose.

[0189] In embodiments, the present disclosure also provides a composition comprising a population of viable cells, an aqueous component, about 0.2 M to about 1.0 M urea, about 0.2 M to about 1.0 M glucose, and optionally a bulking agent.

[0190] In an embodiment, the composition comprises about 0.3 M to about 0.8 M glucose. In an embodiment, the composition comprises about 0.4 M to about 0.6 M glucose. In an embodiment, the composition comprises about 0.5 M glucose.

[0191] In an embodiment, the composition comprises about 0.3 M to about 0.8 M urea. In an embodiment, the composition comprises about 0.4 M to about 0.6 M urea. In an embodiment, the composition comprises about 0.5 M urea.

[0192] In an embodiment, the composition further comprises sucrose. In an embodiment, the composition comprises from about 0.1 M to about 0.5 M sucrose. In an embodiment, the composition comprises from about 0.25 M to about 0.5 M sucrose. In an embodiment, the composition comprises about 0.25 M sucrose. In an embodiment, the composition comprises about 0.5 M sucrose.

[0193] In an embodiment, the composition further comprises mannitol. In an embodiment, the composition comprises about 0.3 M to about 0.8 M mannitol. In an embodiment, the composition comprises about 0.1 M to about 0.5 M mannitol. In an embodiment, the composition comprises about 0.25 M to about 0.5 M mannitol. In an embodiment, the composition comprises about 0.25 M mannitol. In an embodiment, the composition comprises about 0.5 M mannitol. In an embodiment, the composition comprises about 0.4 M mannitol.

[0194] In an embodiment of the composition, the population of viable cells comprises eukaryotic or prokaryotic cells as described herein. In an embodiment, the population of viable cells comprises mammalian cells. In an embodiment, the population of viable cells comprises stem cells. In an embodiment, the population of viable cells comprises pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or hematopoietic stem cells. In an embodiment, the population of viable cells comprises mesenchymal stem cells. In an embodiment, the population of viable cells comprises induced pluripotent stem cells. In an embodiment, the population of viable cells comprises neuroblastoma cells. In an embodiment, the neuroblastoma cells are SK-N-AS cells.

[0195] In embodiments, the compositions provided herein do not include DMSO. In embodiments, the compositions provided herein include DMSO at a concentration lower than that required for compositions for cell preservation using conventional methods. In embodiments, the compositions do not include DMSO. In embodiments, the compositions include DMSO at a concentration of about 1% to about 10%. In embodiments, the compositions include DMSO at a concentration of about 2% to about 5%. In embodiments, the compositions include DMSO at a concentration of about 3% to about 8%. In embodiments, the compositions include DMSO at a concentration of about 1% to about 5%. In embodiments, the compositions include DMSO at a concentration of about 1% to about 3%. In embodiments, the compositions include DMSO at a concentration of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In embodiments, the compositions include DMSO at the concentrations listed above on a volume percent (vol%) basis. In embodiments, the compositions include DMSO at the concentrations listed above on a weight percent (wt%) basis. In some embodiments, the concentration of DMSO is less than 1% (by weight), less than 0.5% (by weight), less than 0.2% (by weight), or less than 0.1% (by weight) of the composition.

[0196] Further methods In an embodiment, the present disclosure also relates to a method for producing a frozen cell aggregate, comprising the step (a) of freezing the composition defined above to produce a frozen cell aggregate.

[0197] In embodiments, the present disclosure provides a method of freezing a cell population, the method comprising freezing a composition comprising the cell population, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.1 M to about 0.5 M sucrose, wherein the freezing occurs at less than about -30°C.

[0198] In an embodiment, the present disclosure provides a method for freeze-drying a cell aggregate, the method comprising: a) freezing a composition comprising the cell aggregate, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.1 M to about 0.5 M sucrose, where the freezing occurs at less than about -30°C; and b) removing the aqueous component (particularly water) from the frozen composition to produce a freeze-dried cell aggregate, where the aqueous component (particularly water) in the freeze-dried cell aggregate is less than about 10% w / w.

[0199] In embodiments, the present disclosure also relates to a method of producing a freeze-dried cellular aggregate, comprising the steps of (a) freezing a composition as defined herein and (b) removing at least about 10% (volume / volume) water from the frozen composition to produce a freeze-dried cellular aggregate, in embodiments, step (b) removing at least about 20% (volume / volume), at least about 30% (volume / volume), at least about 40% (volume / volume), at least about 50% (volume / volume), at least about 60% (volume / volume) water from the frozen composition to produce a freeze-dried cellular aggregate. In some embodiments, the method includes removing at least about 90% (vol / vol), at least about 70% (vol / vol), at least about 80% (vol / vol), at least about 90% (vol / vol), at least about 91% (vol / vol), at least about 92% (vol / vol), at least about 93% (vol / vol), at least about 94% (vol / vol), at least about 95% (vol / vol), at least about 96% (vol / vol), at least about 97% (vol / vol), at least about 98% (vol / vol), or at least about 99% (vol / vol) of the water.

[0200] In an embodiment, the present disclosure also relates to a method for producing freeze-dried cell aggregates, comprising the steps of (a) freezing the composition defined above, and (b) removing at least about 10% (volume / volume) water from the frozen composition to produce freeze-dried cell aggregates. In an embodiment, step (b) comprises removing at least about 20% (volume / volume) water. In an embodiment, step (b) comprises removing at least about 30% (volume / volume) water. In an embodiment, step (b) comprises removing at least about 40% (volume / volume) water. In an embodiment, step (b) comprises removing at least about 50% (volume / volume) water. In an embodiment, step (b) comprises removing at least about 60% (volume / volume) water. In an embodiment, step (b) comprises removing at least about 70% (volume / volume) water. In an embodiment, step (b) comprises removing at least about 80% (volume / volume) water. In embodiments, step (b) comprises removing at least about 90% (v / v), about 91% (v / v), about 92% (v / v), about 93% (v / v), about 94% (v / v), about 95% (v / v), about 96% (v / v), about 97% (v / v), about 98% (v / v), or about 99% (v / v) of the water.

[0201] In embodiments, the disclosure provides a method of producing a viable aggregate of cells comprising: a) freezing a composition comprising an aggregate of cells, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.1 M to about 0.5 M sucrose, wherein the freezing occurs at less than about -30°C; and b) resuspending the lyophilized aggregate of cells in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0202] In an embodiment, the present disclosure provides a method of producing a viable aggregate of cells comprising: a) freezing a composition comprising an aggregate of cells, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.1 M to about 0.5 M sucrose, wherein the freezing is performed at less than about -30°C; b) removing the aqueous component (particularly water) from the frozen composition to produce a freeze-dried aggregate of cells, wherein the aqueous component (particularly water) in the freeze-dried aggregate of cells is less than about 10% w / w; and c) resuspending the freeze-dried aggregate of cells in a reconstituting agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0203] In an embodiment, the present disclosure also relates to a method for producing a reconstituted population of viable cells, comprising the steps of (a) freezing a composition defined above to produce a population of frozen cells, and (b) resuspending the population of frozen cells in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable.

[0204] In embodiments, the present disclosure also relates to a method of producing a reconstituted viable cell population, comprising the steps of (a) freezing a composition as defined above, (b) removing at least about 10% (volume / volume) water from the frozen composition to produce a lyophilized cell population, and (c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable, and in embodiments, step (b) removes at least about 20% (volume / volume), at least about 30% (volume / volume), at least about 40% (volume / volume), at least about 50% (volume / volume), at least about 60% (volume / volume), at least about 70% (volume / volume), at least about 80% (volume / volume), at least about 90% (volume / volume), at least about 10% (volume / volume), at least about 15% (volume / volume), at least about 20% (volume / volume), at least about 30% (volume / volume), at least about 40% (volume / volume), at least about 50% (volume / volume), at least about 10% (volume / volume), at least about 15% (volume / volume), at least about 20% (volume / volume), at least about 3 ...40% (volume / volume), at least about 15% (volume / volume), at least about 15% (volume / volume), at least about 20% (volume / volume), at least about 20% (volume / volume), at least about 30% (volume / volume), at least about 15% (vol In some embodiments, the method includes removing at least about 90% (v / v), at least about 91% (v / v), at least about 92% (v / v), at least about 93% (v / v), at least about 94% (v / v), at least about 95% (v / v), at least about 96% (v / v), at least about 97% (v / v), at least about 98% (v / v), or at least about 99% (v / v) of the water.

[0205] In embodiments, the present disclosure provides a method of freezing a cell population, the method comprising freezing a composition comprising the cell population, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.2 M to about 0.8 M mannitol, wherein the freezing occurs at less than about -30°C.

[0206] In an embodiment, the present disclosure provides a method for freeze-drying a cell aggregate, the method comprising: a) freezing a composition comprising the cell aggregate, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.2 M to about 0.8 M mannitol, where the freezing occurs at less than about -30°C; and b) removing the aqueous component (particularly water) from the frozen composition to produce a freeze-dried cell aggregate, where the aqueous component (particularly water) in the freeze-dried cell aggregate is less than about 10% w / w.

[0207] In embodiments, the disclosure provides a method of producing a viable aggregate of cells, the method comprising: a) freezing a composition comprising an aggregate of cells, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.2 M to about 0.8 M mannitol, wherein the freezing occurs at less than about -30°C; and b) resuspending the lyophilized aggregate of cells in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0208] In an embodiment, the present disclosure provides a method of producing a viable aggregate of cells comprising: a) freezing a composition comprising an aggregate of cells, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.2 M to about 0.8 M mannitol, wherein the freezing is performed at less than about -30°C; b) removing the aqueous component (particularly water) from the frozen composition to produce a freeze-dried aggregate of cells, wherein the aqueous component (particularly water) in the freeze-dried aggregate of cells is less than about 10% w / w; and c) resuspending the freeze-dried aggregate of cells in a reconstituting agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0209] In an embodiment of any of the above methods, the composition further comprises about 1% to about 8% DMSO.

[0210] In embodiments of any of the above methods, the composition further comprises a hydrogel. In embodiments, the hydrogel is a hyaluronan gel, an alginate gel, or a collagen gel. In embodiments, the cell aggregates are suspended in the hydrogel.

[0211] In an embodiment of any of the above methods, the composition does not include DMSO.

[0212] In an embodiment of any of the above methods, prior to freezing in step (a), the cell population is isolated and contacted with a poration solution. In an embodiment, the poration solution comprises trehalose. In an embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 1.0 M, or about 0.1 M to about 0.6 M.

[0213] In embodiments of any of the above methods, at least about 20% of the cells in the reconstituted composition are viable as measured by the ALAMARBLUE® test. In embodiments, at least about 30% of the cells in the reconstituted composition are viable as measured by the ALAMARBLUE® test. In embodiments, at least about 40% of the cells in the reconstituted composition are viable as measured by the ALAMARBLUE® test.

[0214] In an embodiment of any of the above methods, at least about 50% of the cells in the reconstituted composition are viable as measured by the ALAMARBLUE® test.

[0215] The present invention is further illustrated by the following embodiments.

[0216] method In one embodiment, the present invention provides a method for producing a population of frozen cells, comprising the steps of: The method includes freezing a composition comprising a cell aggregate, an aqueous component, urea, a simple sugar, and optionally a bulking agent to produce a frozen cell aggregate.

[0217] In a further embodiment, the invention provides a method for producing a population of reconstituted viable cells, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, urea, a simple sugar, and optionally a bulking agent to produce a frozen cell mass; (b) resuspending the frozen cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable.

[0218] In a further embodiment, the present invention provides a method for producing a freeze-dried cell population, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, urea, a simple sugar, and a bulking agent; (b) removing at least about 90% of the aqueous components from the frozen composition to produce a freeze-dried cell aggregate.

[0219] In a further embodiment, the invention provides a method for producing a population of reconstituted viable cells, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, urea, a simple sugar, and a bulking agent; (b) removing at least about 90% of the aqueous components from the frozen composition to produce a freeze-dried cell aggregate; (c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable.

[0220] In any one of the above embodiments, the composition may further comprise a hydrogel.

[0221] In one embodiment, the hydrogel is a biocompatible hydrogel.

[0222] In one embodiment, the hydrogel is a hyaluronan gel, an alginate gel, an agarose gel, a collagen gel, or a combination thereof.

[0223] In any one of the above embodiments, the cell aggregates may be suspended in the hydrogel.

[0224] In any one of the above embodiments, the concentration of the monosaccharide before freezing may be from about 0.2M to about 1.0M.

[0225] In any one of the above embodiments, the concentration of the monosaccharide before freezing may be from about 0.3M to about 0.8M.

[0226] In any one of the above embodiments, the concentration of the monosaccharide before freezing may be from about 0.4M to about 0.6M.

[0227] In any one of the above embodiments, the monosaccharide may be glucose, fructose, or galactose.

[0228] In one embodiment, the monosaccharide is glucose.

[0229] In one embodiment, the glucose concentration before freezing is about 0.2M to about 1.0M.

[0230] In one embodiment, the glucose concentration before freezing is about 0.3M to about 0.8M.

[0231] In one embodiment, the glucose concentration before freezing is about 0.4M to about 0.6M.

[0232] In any one of the above embodiments, the concentration of urea before freezing may be from about 0.2M to about 1.0M.

[0233] In any one of the above embodiments, the concentration of urea before freezing may be from about 0.3M to about 0.8M.

[0234] In any one of the above embodiments, the concentration of urea before freezing may be from about 0.4M to about 0.6M.

[0235] In any one of the above embodiments, the molar ratio of urea to monosaccharides before freezing may be from about 1:3 to about 3:1.

[0236] In any one of the above embodiments, the molar ratio of urea to monosaccharides before freezing may be from about 1:2 to about 2:1.

[0237] In any one of the above embodiments, the molar ratio of urea to monosaccharides before freezing may be about 1:1.

[0238] In any one of the above embodiments, the molar ratio of urea to glucose before freezing may be from about 1:3 to about 3:1.

[0239] In any one of the above embodiments, the molar ratio of urea to glucose before freezing may be from about 1:2 to about 2:1.

[0240] In any one of the above embodiments, the molar ratio of urea to glucose before freezing may be about 1:1.

[0241] In any one of the above embodiments, freezing may be performed with the cells in suspension or attached to the surface of the container.

[0242] In any one of the above embodiments, freezing may be performed on a collection of cells suspended in a hydrogel.

[0243] In any one of the above embodiments, the freezing may be performed in a container with or without a collagen coating.

[0244] In any one of the above embodiments, the container may be a glass or plastic container with or without a membrane for cell attachment or growth.

[0245] In any one of the above embodiments, the cell population is about 1 x 10 cells per mL. 4 ~1 x 10 cells per mL 6 It may be a single cell.

[0246] In any one of the above embodiments, the cell population is about 1 x 10 cells per mL. 5 ~4 x 10 cells per mL 5 It may be a single cell.

[0247] In any one of the above embodiments, the cell population is about 2×10 per mL.5 ~2.5 x 10 cells per mL 5 It may be a single cell.

[0248] In any one of the above embodiments, the bulking agent may include a disaccharide.

[0249] In one embodiment, the concentration of the disaccharide before freezing is about 0.1M to about 1.0M.

[0250] In one embodiment, the concentration of the disaccharide before freezing is about 0.1M to about 0.5M.

[0251] In one embodiment, the concentration of the disaccharide before freezing is about 0.2M to about 0.4M.

[0252] In one embodiment, the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, lactulose, isomaltose, melibiose, or gentiobiose.

[0253] In one embodiment, the disaccharide is sucrose.

[0254] In one embodiment, the concentration of sucrose before freezing is about 0.1M to about 1.0M.

[0255] In one embodiment, the concentration of sucrose before freezing is about 0.1M to about 0.5M.

[0256] In one embodiment, the concentration of sucrose before freezing is about 0.2M to about 0.4M.

[0257] In any one of the above embodiments, the bulking agent may include a sugar alcohol.

[0258] In one embodiment, the sugar alcohol concentration before freezing is about 0.1M to about 1.0M.

[0259] In one embodiment, the sugar alcohol concentration before freezing is about 0.3M to about 0.8M.

[0260] In one embodiment, the sugar alcohol concentration before freezing is about 0.4M to about 0.6M.

[0261] In one embodiment, the sugar alcohol is mannitol, sorbitol, galactitol, fucitol, iditol, or inositol.

[0262] In one embodiment, the sugar alcohol is mannitol.

[0263] In one embodiment, the concentration of mannitol before freezing is about 0.1M to about 1.0M.

[0264] In one embodiment, the concentration of mannitol before freezing is about 0.3M to about 0.8M.

[0265] In one embodiment, the concentration of mannitol before freezing is about 0.4M to about 0.6M.

[0266] In any one of the above embodiments, the bulking agent may include both a disaccharide and a sugar alcohol.

[0267] In any one of the above embodiments, the bulking agent may include both sucrose and mannitol.

[0268] In any one of the above embodiments, the composition may be free of DMSO.

[0269] In any one of the above embodiments, the composition may contain DMSO at a concentration of about 1% to about 10% prior to freezing.

[0270] In any one of the above embodiments, the composition may contain DMSO at a concentration of about 2% to about 5% prior to freezing.

[0271] In any one of the above embodiments, the cell population may be isolated and contacted with a poration solution prior to freezing in (a).

[0272] In one embodiment, the poration solution comprises trehalose and optionally cell culture medium.

[0273] In one embodiment, the concentration of trehalose in the poration solution is about 0.1M to about 1.0M.

[0274] In one embodiment, the concentration of trehalose in the poration solution is about 0.2M to about 0.6M.

[0275] In any one of the above embodiments, the reconstitution agent may comprise cell culture medium.

[0276] In any one of the above embodiments, the reconstitution agent may comprise a phosphate buffer solution.

[0277] In any one of the above embodiments, the composition may be an isotonic solution.

[0278] In any one of the above embodiments, the composition may be a hypertonic solution.

[0279] In any one of the above embodiments, the population of cells may comprise mammalian cells.

[0280] In any one of the above embodiments, the population of cells may include stem cells.

[0281] In any one of the above embodiments, the population of cells may include pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or hematopoietic stem cells.

[0282] In any one of the above embodiments, the population of cells may include mesenchymal stem cells.

[0283] In any one of the above embodiments, the population of cells may include induced pluripotent stem cells.

[0284] In any one of the above embodiments, the population of cells may comprise neuroblastoma cells.

[0285] In one embodiment, the neuroblastoma cells are SK-N-AS cells.

[0286] In any one of the above embodiments, the aqueous component may include a buffer.

[0287] In one embodiment, the buffer comprises a phosphate buffer, a Tris buffer, an acetate buffer, a bicarbonate buffer, a histidine buffer, a citrate buffer, or a combination thereof.

[0288] In any one of the above embodiments, the aqueous component may include cell culture medium.

[0289] In one embodiment, the cell culture medium is serum-free.

[0290] In any one of the above embodiments, removing the aqueous component may include reducing pressure, applying heat, or both to the frozen composition to remove the aqueous component.

[0291] In any one of the above embodiments, removing the aqueous components may include a primary drying step and a secondary drying step.

[0292] In any one of the above embodiments, removing the aqueous component may include only a primary drying step.

[0293] In any one of the above embodiments, the primary drying step may include reducing the pressure to remove the aqueous components.

[0294] In any one of the above embodiments, the secondary drying step may include the application of heat to remove the aqueous components.

[0295] In any one of the above embodiments, the freezing may be performed at about -10°C to about -100°C.

[0296] In any one of the above embodiments, the freezing may be performed at about -20°C to about -90°C.

[0297] In any one of the above embodiments, the freezing may be performed at about -40°C to about -60°C.

[0298] In any one of the above embodiments, freezing reduces the temperature of the composition to about -80°C.

[0299] In any one of the above embodiments, freezing may reduce the temperature of the composition to -40°C, and the aqueous components may be removed at a pressure of about 60 mTorr to about 80 mTorr chamber pressure.

[0300] In any one of the above embodiments, resuspending the frozen cells may occur more than two hours after freezing.

[0301] In any one of the above embodiments, resuspending the frozen cells may occur more than one day after freezing.

[0302] In any one of the above embodiments, resuspending the frozen cells may occur more than one week after freezing.

[0303] In any one of the above embodiments, resuspending the frozen cells may occur more than one month after freezing.

[0304] In any one of the above embodiments, resuspending the freeze-dried cells may occur more than two hours after removal of the aqueous component.

[0305] In any one of the above embodiments, resuspending the freeze-dried cells may occur more than one day after removal of the aqueous components.

[0306] In any one of the above embodiments, resuspending the freeze-dried cells may occur more than one week after removal of the aqueous components.

[0307] In any one of the above embodiments, resuspending the freeze-dried cells may occur more than one month after removal of the aqueous components.

[0308] In any one of the above embodiments, the frozen or lyophilized cells may be stored at below about -20°C prior to resuspension.

[0309] In any one of the above embodiments, the frozen or lyophilized cells may be stored at about -20°C to about 30°C prior to resuspension.

[0310] In any one of the above embodiments, the frozen or lyophilized cells may be stored at about 4° C. to about 28° C. prior to resuspension.

[0311] In any one of the above embodiments, the frozen or lyophilized cells may be stored at about 10° C. to about 27° C. prior to resuspension.

[0312] In any one of the above embodiments, the frozen or lyophilized cells may be stored at about 2° C. to about 8° C. prior to resuspension.

[0313] In any one of the above embodiments, the frozen or lyophilized cells may be stored above about -20°C for more than two days before being resuspended.

[0314] In any one of the above embodiments, the lyophilized cells may be stored at about 20° C. to about 25° C. for more than one week before being resuspended.

[0315] In any one of the above embodiments, the lyophilized cells may be stored at about 2° C. to about 8° C. for more than one week before being resuspended.

[0316] In any one of the above embodiments, at least about 20% of the cells in the reconstituted composition may be viable as measured by the ALAMARBLUE® test.

[0317] In any one of the above embodiments, at least about 30% of the cells in the reconstituted composition may be viable as measured by the ALAMARBLUE® test.

[0318] In any one of the above embodiments, at least about 40% of the cells in the reconstituted composition may be viable as measured by the ALAMARBLUE® test.

[0319] In any one of the above embodiments, at least about 50% of the cells in the reconstituted composition may be viable as measured by the ALAMARBLUE® test.

[0320] composition The present invention further relates to a composition comprising a population of viable cells, an aqueous component, urea, a simple sugar, and optionally a bulking agent, wherein the composition contains less than about 10% (volume / volume) water.

[0321] In one embodiment, the composition contains less than about 5% (volume / volume) water.

[0322] In one embodiment, the molar ratio of glucose to urea in the composition is from about 30:1 to about 1:3.

[0323] In one embodiment, the molar ratio of glucose to urea in the composition is from about 15:1 to about 1:2.

[0324] In one embodiment, the molar ratio of glucose to urea in the composition is from about 3:1 to about 1:1.

[0325] The present invention further relates to a composition comprising a population of viable cells, an aqueous component, urea, a monosaccharide, and optionally a bulking agent, wherein the molar ratio of glucose to urea in the composition is from about 30:1 to about 1:3.

[0326] In one embodiment, the molar ratio of glucose to urea in the composition is from about 15:1 to about 1:2.

[0327] In one embodiment, the molar ratio of glucose to urea in the composition is from about 3:1 to about 1:1.

[0328] In one embodiment of any of the compositions, the monosaccharide may be glucose.

[0329] The present invention further relates to a composition comprising a population of viable cells, an aqueous component, about 0.2 M to about 1.0 M urea, about 0.2 M to about 1.0 M glucose, and optionally a bulking agent.

[0330] In one embodiment, the composition comprises about 0.3M to about 0.8M glucose.

[0331] In one embodiment, the composition comprises about 0.4M to about 0.6M glucose.

[0332] In one embodiment, the composition comprises about 0.3M to about 0.8M urea.

[0333] In one embodiment, the composition comprises about 0.4M to about 0.6M urea.

[0334] In one embodiment of any of the compositions, the composition may further comprise sucrose.

[0335] In one embodiment, the composition comprises about 0.1 M to about 0.5 M sucrose.

[0336] In one embodiment of any of the compositions, the composition may further comprise mannitol.

[0337] In one embodiment, the composition comprises about 0.3M to about 0.8M mannitol.

[0338] In one embodiment of any of the compositions, the population of viable cells may comprise mammalian cells.

[0339] In one embodiment of any of the compositions, the population of viable cells may comprise stem cells.

[0340] In one embodiment of any of the compositions, the population of viable cells may comprise pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or hematopoietic stem cells.

[0341] In one embodiment of any of the compositions, the population of viable cells may comprise mesenchymal stem cells.

[0342] In one embodiment of any of the compositions, the population of viable cells may comprise induced pluripotent stem cells.

[0343] In one embodiment of any of the compositions, the population of viable cells may comprise neuroblastoma cells.

[0344] In one embodiment, the neuroblastoma cells are SK-N-AS cells.

[0345] In one embodiment of any of the compositions, the composition may be free of DMSO.

[0346] method The present invention further relates to a method of freezing a cell mass, comprising freezing a composition comprising the cell mass, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.1 M to about 0.5 M sucrose, wherein the freezing is performed at less than about -30°C.

[0347] The present invention further provides a method for freeze-drying a collection of cells, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.1M to about 0.5M sucrose, wherein the freezing is performed at less than about -30°C; (b) removing the aqueous component from the frozen composition to produce a freeze-dried cell mass, wherein the aqueous component in the freeze-dried cell mass is less than about 10% w / w.

[0348] The present invention further provides a method for producing a viable aggregate of cells, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.1M to about 0.5M sucrose, wherein the freezing is performed at less than about -30°C; (b) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0349] The present invention further provides a method for producing a viable aggregate of cells, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.1M to about 0.5M sucrose, wherein the freezing is performed at less than about -30°C; (b) removing aqueous components from the frozen composition to produce a freeze-dried cell mass, wherein the aqueous components in the freeze-dried cell mass are less than about 10% w / w; (c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0350] The present invention further relates to a method of freezing a cell aggregate, comprising freezing a composition comprising the cell aggregate, an aqueous component, about 0.2 M to about 1 M urea, about 0.2 M to about 1 M glucose, and about 0.2 M to about 0.8 M mannitol, wherein the freezing is performed at less than about -30°C.

[0351] The present invention further provides a method for freeze-drying a collection of cells, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.2M to about 0.8M mannitol, wherein the freezing is performed at less than about -30°C; (b) removing the aqueous component from the frozen composition to produce a freeze-dried cell mass, wherein the aqueous component in the freeze-dried cell mass is less than about 10% w / w.

[0352] The present invention further provides a method for producing a viable aggregate of cells, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.2M to about 0.8M mannitol, wherein the freezing is performed at less than about -30°C; (b) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0353] The present invention further provides a method for producing a viable aggregate of cells, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.2M to about 0.8M mannitol, wherein the freezing is performed at less than about -30°C; (b) removing aqueous components from the frozen composition to produce a freeze-dried cell mass, wherein the aqueous components in the freeze-dried cell mass are less than about 10% w / w; (c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable.

[0354] In one embodiment of any of the methods, the composition may further comprise about 1% to about 8% DMSO.

[0355] In one embodiment of any of the methods, the composition may further comprise a hydrogel.

[0356] In one embodiment, the hydrogel is a hyaluronan gel, an alginate gel, or a collagen gel.

[0357] In one embodiment, the cell population is suspended in a hydrogel.

[0358] In one embodiment of any of the methods, the composition may be free of DMSO.

[0359] In one embodiment of any of the methods, at least about 20% of the cells in the reconstituted composition may be viable as measured by the ALAMARBLUE® test.

[0360] In one embodiment of any of the methods, at least about 30% of the cells in the reconstituted composition may be viable as measured by the ALAMARBLUE® test.

[0361] In one embodiment of any of the methods, at least about 40% of the cells in the reconstituted composition may be viable as measured by the ALAMARBLUE® test.

[0362] In one embodiment of any of the methods, at least about 50% of the cells in the reconstituted composition may be viable as measured by the ALAMARBLUE® test.

[0363] item The items are as follows: 1. A method for producing a frozen cell population, comprising: A method comprising freezing a composition comprising a cell aggregate, an aqueous component, urea, a simple sugar, and optionally a bulking agent to produce a frozen cell aggregate. 2. A method for producing a population of reconstituted viable cells, comprising: (a) freezing a composition comprising a cell mass, an aqueous component, urea, a simple sugar, and optionally a bulking agent to produce a frozen cell mass; (b) resuspending the frozen cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable. 3. A method for producing a freeze-dried cell aggregate, comprising: (a) freezing a composition comprising a cell mass, an aqueous component, urea, a simple sugar, and a bulking agent; (b) removing at least about 90% of the aqueous components from the frozen composition to produce a freeze-dried cell aggregate. 4. A method for producing a population of reconstituted viable cells, comprising: (a) freezing a composition comprising a cell mass, an aqueous component, urea, a simple sugar, and a bulking agent; (b) removing at least about 90% of the aqueous components from the frozen composition to produce a freeze-dried cell aggregate; (c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable. 5. The method of any one of items 1 to 4, wherein the composition further comprises a hydrogel. 6. The method of item 5, wherein the hydrogel is a biocompatible hydrogel. 7. The method of item 5, wherein the hydrogel is a hyaluronan gel, an alginate gel, an agarose gel, a collagen gel, or a combination thereof. 8. The method of any one of items 1 to 7, wherein the cell aggregates are suspended in a hydrogel. 9. The method of any one of items 1 to 8, wherein the concentration of the monosaccharide before freezing is about 0.2 M to about 1.0 M. 10. The method of any one of items 1 to 8, wherein the concentration of the monosaccharide before freezing is about 0.3 M to about 0.8 M. 11. The method of any one of items 1 to 8, wherein the concentration of the monosaccharide before freezing is about 0.4 M to about 0.6 M. 12. The method of any one of items 1 to 11, wherein the monosaccharide is glucose, fructose, or galactose. 13. The method of item 12, wherein the monosaccharide is glucose. 14. The method of item 13, wherein the concentration of glucose before freezing is about 0.2 M to about 1.0 M. 15. The method of item 13, wherein the concentration of glucose before freezing is about 0.3 M to about 0.8 M. 16. The method of item 13, wherein the concentration of glucose before freezing is about 0.4 M to about 0.6 M. 17. The method of any one of items 1 to 16, wherein the concentration of urea before freezing is about 0.2 M to about 1.0 M. 18. The method of any one of items 1 to 16, wherein the concentration of urea before freezing is about 0.3 M to about 0.8 M. 19. The method of any one of items 1 to 16, wherein the concentration of urea before freezing is about 0.4 M to about 0.6 M. 20. The method of any one of items 1 to 19, wherein the ratio of urea to monosaccharides before freezing is about 1:3 to about 3:1. 21. The method of any one of items 1 to 19, wherein the ratio of urea to monosaccharides before freezing is about 1:2 to about 2:1. 22. The method of any one of items 1 to 19, wherein the ratio of urea to monosaccharides before freezing is about 1:1. 23. The method of any one of items 13 to 19, wherein the ratio of urea to glucose before freezing is about 1:3 to about 3:1. 24. The method of any one of items 1 to 19, wherein the ratio of urea to glucose before freezing is about 1:2 to about 2:1. 25. The method of any one of items 1-19, wherein the ratio of urea to glucose before freezing is about 1:1. 26. The method of any one of items 1 to 25, wherein freezing is performed on cells in suspension or attached to a container surface. 27. The method of any one of items 1 to 25, wherein freezing is performed on a collection of cells suspended in a hydrogel. 28. The method of any one of items 1 to 27, wherein freezing is performed in a container with or without a collagen coating. 29. The method of any one of items 1 to 28, wherein the container is a glass or plastic container with or without a membrane for cell attachment or growth. 30. The cell population is approximately 1 x 10 per mL. 4 ~1 x 10 cells per mL 7 30. The method of any one of items 1 to 29, wherein the cells are 31. The cell population is approximately 1 x 10 per mL. 5 ~4 x 10 cells per mL 5 30. The method of any one of items 1 to 29, wherein the cells are 32. The cell population is approximately 2 x 10 per mL. 5 ~2.5 x 10 cells per mL 5 30. The method of any one of items 1 to 29, wherein the cells are 33. The method of any one of items 1 to 32, wherein the bulking agent comprises a disaccharide. 34. The method according to item 33, wherein the concentration of the disaccharide before freezing is about 0.1 M to about 1.0 M. 35. The method according to item 33, wherein the concentration of the disaccharide before freezing is about 0.1 M to about 0.5 M. 36. The method according to item 33, wherein the concentration of the disaccharide before freezing is about 0.2 M to about 0.4 M. 37. The method according to any one of items 33 to 36, wherein the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, lactulose, isomaltose, melibiose or gentiobiose. 38. The method of item 33, wherein the disaccharide is sucrose. 39. The method of item 35, wherein the concentration of sucrose before freezing is about 0.1 M to about 1.0 M. 40. The method of item 35, wherein the concentration of sucrose before freezing is about 0.1 M to about 0.5 M. 41. The method of item 35, wherein the concentration of sucrose before freezing is about 0.2 M to about 0.4 M. 42. The method of any one of items 1 to 32, wherein the bulking agent comprises a sugar alcohol. 43. The method according to item 42, wherein the concentration of the sugar alcohol before freezing is about 0.1 M to about 1.0 M. 44. The method according to item 42, wherein the concentration of the sugar alcohol before freezing is about 0.3 M to about 0.8 M. 45. The method according to item 42, wherein the concentration of the sugar alcohol before freezing is about 0.4 M to about 0.6 M. 46. ​​The method of any one of items 42 to 45, wherein the sugar alcohol is mannitol, sorbitol, galactitol, fucitol, iditol, or inositol. 47. The method of item 46, wherein the sugar alcohol is mannitol. 48. The method according to item 47, wherein the concentration of mannitol before freezing is about 0.1 M to about 1.0 M. 49. The method of item 47, wherein the concentration of mannitol before freezing is about 0.3 M to about 0.8 M. 50. The method of item 47, wherein the concentration of mannitol before freezing is about 0.4 M to about 0.6 M. 51. The method of any one of items 1 to 50, wherein the bulking agent includes both a disaccharide and a sugar alcohol. 52. The method of any one of items 1 to 50, wherein the bulking agent includes both sucrose and mannitol. 53. The method of any one of items 1 to 52, wherein the composition does not contain DMSO. 54. The method of any one of items 1 to 52, wherein the composition comprises DMSO at a concentration of about 1% to about 10% prior to freezing. 55. The method of any one of items 1 to 52, wherein the composition comprises DMSO at a concentration of about 2% to about 5% prior to freezing. 56. The method of any one of items 1 to 55, wherein prior to freezing in (a), the cell aggregates are isolated and contacted with a poration solution. 57. The method of item 56, wherein the poration solution comprises trehalose and a cell culture medium. 58. The method of item 57, wherein the concentration of trehalose in the poration solution is about 0.1 M to about 1.0 M. 59. The method of item 57, wherein the concentration of trehalose in the poration solution is about 0.2 M to about 0.6 M. 60. The method of any one of items 2 or 4-59, wherein the reconstitution agent comprises cell culture medium. 61. The method of any one of items 2 or 4 to 62, wherein the reconstitution agent comprises a phosphate buffer solution. 62. The method of any one of items 1 to 62, wherein the composition is an isotonic solution. 63. The method of any one of items 1 to 63, wherein the composition is a hypertonic solution. 64. The method of any one of items 1 to 64, wherein the population of cells comprises mammalian cells. 65. The method of any one of items 1 to 65, wherein the cell population comprises stem cells. 66. The method of any one of items 1 to 66, wherein the population of cells comprises pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or hematopoietic stem cells. 67. The method of any one of items 1 to 67, wherein the cell population comprises mesenchymal stem cells. 68. The method of any one of items 1 to 68, wherein the population of cells comprises induced pluripotent stem cells. 69. The method of any one of items 1 to 67, wherein the population of cells comprises neuroblastoma cells. 70. The method of item 70, wherein the neuroblastoma cells are SK-N-AS cells. 71. The method of any one of items 1 to 71, wherein the aqueous component comprises a buffer. 72. The method of item 72, wherein the buffer comprises a phosphate buffer, a Tris buffer, an acetate buffer, a bicarbonate buffer, a histidine buffer, a citrate buffer, or a combination thereof. 73. The method of any one of items 1 to 73, wherein the aqueous component comprises a cell culture medium. 74. The method of item 74, wherein the cell culture medium is serum-free. 75. The method of any one of items 3 to 75, wherein removing the aqueous component includes reducing pressure, applying heat, or both to the frozen composition to remove the aqueous component. 76. The method of any one of items 3 to 76, wherein removing the aqueous component comprises a primary drying step and a secondary drying step. 77. The method of any one of items 3 to 77, wherein removing the aqueous component includes only a primary drying step. 78. The method of any one of items 3 to 78, wherein the primary drying step includes reducing the pressure to remove the aqueous components. 79. The method of any one of items 3 to 79, wherein the secondary drying step includes applying heat to remove aqueous components. 80. The method of any one of items 1 to 80, wherein the freezing is carried out at about -10°C to about -100°C. 81. The method of any one of items 1 to 80, wherein the freezing is carried out at about -20°C to about -90°C. 82. The method of any one of items 1 to 80, wherein the freezing is carried out at about -40°C to about -60°C. 83. The method of any one of items 1 to 80, wherein freezing reduces the temperature of the composition to about -80°C. 84. The method of any one of items 3 to 84, wherein freezing reduces the temperature of the composition to -40°C and the aqueous components are removed at a chamber pressure of about 60 mTorr to about 80 mTorr. 85. Any one of the methods of items 2 or 5 to 85, wherein resuspending the frozen cells is performed more than 2 hours after freezing. 86. Any one of the methods of items 2 or 5 to 85, wherein resuspending the frozen cells is performed more than one day after freezing. 87. Any one of the methods of items 2 or 5 to 85, wherein resuspending the frozen cells is performed more than one week after freezing. 88. Any one of the methods of items 2 or 5 to 85, wherein resuspending the frozen cells is performed more than one month after freezing. 89. The method of any one of items 4 to 85, wherein resuspending the freeze-dried cells is performed more than 2 hours after removal of the aqueous components. 90. The method of any one of items 4 to 85, wherein resuspending the freeze-dried cells is performed more than one day after removal of the aqueous components. 91. The method of any one of items 4 to 85, wherein resuspending the freeze-dried cells is performed more than one week after removal of the aqueous components. 92. The method of any one of items 4 to 85, wherein resuspending the freeze-dried cells is performed more than one month after removal of the aqueous components. 93. The method of any one of items 2 or 4 to 93, wherein the frozen or lyophilized cells are stored at less than about -20°C prior to resuspension. 94. The method of any one of items 2 or 4 to 93, wherein the frozen or lyophilized cells are stored at about -20°C to about 30°C before resuspension. 95. The method of any one of items 2 or 4 to 93, wherein the frozen or lyophilized cells are stored at about 4°C to about 28°C before resuspension. 96. The method of any one of items 2 or 4 to 93, wherein the frozen or lyophilized cells are stored at about 10°C to about 27°C before resuspension. 97. The method of any one of items 2 or 4 to 93, wherein the frozen or lyophilized cells are stored at about 2°C to about 8°C before resuspension. 98. The method of any one of items 2 or 4 to 93, wherein the frozen or lyophilized cells are stored above about -20°C for more than 2 days before being resuspended. 99. The method of any one of items 4 to 98, wherein the lyophilized cells are stored at about 20°C to about 25°C for more than one week before being resuspended. 100. The method of any one of items 4 to 98, wherein the lyophilized cells are stored at about 2°C to about 8°C for more than one week before being resuspended. 101. The method of any one of items 1 to 101, wherein at least about 20% of the cells in the reconstituted composition are viable. 102. The method of any one of items 1 to 101, wherein at least about 30% of the cells in the reconstituted composition are viable. 103. The method of any one of items 1 to 101, wherein at least about 40% of the cells in the reconstituted composition are viable. 104. The method of any one of items 1 to 101, wherein at least about 50% of the cells in the reconstituted composition are viable as measured by the ALAMARBLUE test. 105. A composition comprising a population of viable cells, an aqueous component, urea, a simple sugar, and a bulking agent, wherein the composition contains less than about 10% (volume / volume) water. 106. The composition of item 106, wherein the composition contains less than about 5% (volume / volume) water. 107. The composition of item 107, wherein the weight ratio of glucose to urea in the composition is from about 30:1 to about 1:3. 108. The composition of item 107, wherein the weight ratio of glucose to urea in the composition is from about 15:1 to about 1:2. 109. The composition of item 107, wherein the weight ratio of glucose to urea in the composition is from about 3:1 to about 1:1. 110. A composition comprising a population of viable cells, an aqueous component, urea, a monosaccharide, and optionally a bulking agent, wherein the weight ratio of glucose to urea in the composition is from about 30:1 to about 1:3. 111. The composition of item 111, wherein the weight ratio of glucose to urea in the composition is from about 15:1 to about 1:2. 112. The composition of item 111, wherein the weight ratio of glucose to urea in the composition is from about 3:1 to about 1:1. 113. The composition of any one of items 107 to 113, wherein the monosaccharide is glucose. 114. A composition comprising a population of viable cells, an aqueous component, about 0.2M to about 1.0M urea, about 0.2M to about 1.0M glucose, and optionally a bulking agent. 115. The composition of item 115, comprising about 0.3M to about 0.8M glucose. 116. The composition of item 115, comprising about 0.4M to about 0.6M glucose. 117. The composition of any one of items 115 to 117, comprising about 0.3M to about 0.8M urea. 118. The composition of any one of items 115 to 117, comprising about 0.4M to about 0.6M urea. 119. The composition of any one of items 115 to 117, further comprising sucrose. 120. The composition of item 120, comprising about 0.1 M to about 0.5 M sucrose. 121. The composition of any one of items 115 to 117, further comprising mannitol. 122. The composition of item 122, comprising about 0.3M to about 0.8M mannitol. 123. The composition of any one of items 107 to 123, wherein the population of viable cells comprises mammalian cells. 124. The composition of any one of items 107 to 123, wherein the population of viable cells comprises stem cells. 125. The composition of any one of items 107 to 123, wherein the population of viable cells comprises pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or hematopoietic stem cells. 126. The composition of any one of items 107 to 123, wherein the population of viable cells comprises mesenchymal stem cells. 127. The composition of any one of items 107 to 123, wherein the population of viable cells comprises induced pluripotent stem cells. 128. The composition of any one of items 107 to 123, wherein the population of viable cells comprises neuroblastoma cells. 129. The composition of item 129, wherein the neuroblastoma cells are SK-N-AS cells. 130. The composition of any one of items 107 to 130, wherein the composition does not contain DMSO. 131. A method for freezing a cell mass, comprising freezing a composition comprising the cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.1M to about 0.5M sucrose, wherein the freezing is performed at less than about -30°C. 132. A method for freeze-drying a cell aggregate, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.1M to about 0.5M sucrose, wherein the freezing is performed at less than about -30°C; (b) removing aqueous components from the frozen composition to produce a freeze-dried cell mass, wherein the aqueous components in the freeze-dried cell mass are less than about 10% w / w. 133. A method for producing a viable aggregate of cells, comprising: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.1M to about 0.5M sucrose, wherein the freezing is performed at less than about -30°C; (b) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable. 134. A method for producing a viable aggregate of cells, comprising: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.1M to about 0.5M sucrose, wherein the freezing is performed at less than about -30°C; (b) removing aqueous components from the frozen composition to produce a freeze-dried cell mass, wherein the aqueous components in the freeze-dried cell mass are less than about 10% w / w; (c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable. 135. A method for freezing a cell mass, comprising freezing a composition comprising the cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.2M to about 0.8M mannitol, wherein the freezing is performed at less than about -30°C. 136. A method for freeze-drying a cell aggregate, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.2M to about 0.8M mannitol, wherein the freezing is performed at less than about -30°C; (b) removing aqueous components from the frozen composition to produce a freeze-dried cell mass, wherein the aqueous components in the freeze-dried cell mass are less than about 10% w / w. 137. A method for producing a viable aggregate of cells, comprising: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.2M to about 0.8M mannitol, wherein the freezing is performed at less than about -30°C; (b) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable. 138. A method for producing a viable aggregate of cells, comprising: (a) freezing a composition comprising a cell mass, an aqueous component, about 0.2M to about 1M urea, about 0.2M to about 1M glucose, and about 0.2M to about 0.8M mannitol, wherein the freezing is performed at less than about -30°C; (b) removing aqueous components from the frozen composition to produce a freeze-dried cell mass, wherein the aqueous components in the freeze-dried cell mass are less than about 10% w / w; (c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells in the reconstituted composition are viable. 139. The method of any one of items 132 to 138, wherein the composition further comprises about 1% to about 8% DMSO. 140. The method of any one of items 132 to 139, wherein the composition further comprises a hydrogel. 141. The method of item 140, wherein the hydrogel is a hyaluronan gel, an alginate gel, or a collagen gel. 142. The method of item 141, wherein the cell aggregates are suspended in a hydrogel. 143. The method of any one of items 132 to 142, wherein the composition does not contain DMSO. 144. The method of any one of items 133, 134, 137, or 138-143, wherein at least about 20% of the cells in the reconstituted composition are viable. 145. The method of any one of items 133, 134, 137, or 138-143, wherein at least about 30% of the cells in the reconstituted composition are viable. 146. The method of any one of items 133, 134, 137, or 138-143, wherein at least about 40% of the cells in the reconstituted composition are viable. 147. The method of any one of items 133, 134, 137, or 138-143, wherein at least about 50% of the cells in the reconstituted composition are viable as measured by the ALAMARBLUE test.

[0364] The invention further relates to the following numbered embodiments: 1. A method for producing a frozen cell population, comprising: A method comprising freezing a composition comprising a cell aggregate, an aqueous component, urea, a simple sugar, and optionally a bulking agent to produce a frozen cell aggregate. 2. A method for producing a population of reconstituted viable cells, comprising: (a) freezing a composition comprising a cell mass, an aqueous component, urea, a simple sugar, and optionally a bulking agent to produce a frozen cell mass; (b) resuspending the frozen cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable. 3. A method for producing a freeze-dried cell aggregate, comprising: (a) freezing a composition comprising a cell mass, an aqueous component, urea, a simple sugar, and a bulking agent; (b) removing at least about 90% of the aqueous components from the frozen composition to produce a freeze-dried cell aggregate. 4. A method for producing a population of reconstituted viable cells, comprising: (a) freezing a composition comprising a cell mass, an aqueous component, urea, a simple sugar, and a bulking agent; (b) removing at least about 90% of the aqueous components from the frozen composition to produce a freeze-dried cell aggregate; (c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable. 5. The method of any one of embodiments 1 to 4, wherein the composition further comprises a hydrogel, preferably, the hydrogel is a biocompatible hydrogel, or a hyaluronan gel, an alginate gel, an agarose gel, a collagen gel, or a combination thereof. 6. The method of any one of embodiments 1-5, wherein the cell aggregates are suspended in a hydrogel. 7. The method of any one of embodiments 1 to 6, wherein the concentration of the monosaccharide before freezing is from about 0.2M to about 1.0M, or from about 0.3M to about 0.8M, or from about 0.4M to about 0.6M. 8. The method of any one of embodiments 1 to 7, wherein the monosaccharide is glucose, fructose, or galactose, preferably, the monosaccharide is glucose, and optionally, the concentration of glucose before freezing is about 0.2M to about 1.0M, or about 0.3M to about 0.8M, or about 0.4M to about 0.6M. 9. The method of any one of embodiments 1 to 8, wherein the concentration of urea before freezing is about 0.2M to about 1.0M, preferably about 0.3M to about 0.8M, or about 0.4M to about 0.6M, and / or the ratio of urea to monosaccharides before freezing is about 1:3 to about 3:1, preferably about 1:2 to about 2:1, or about 1:1. 10. The method of embodiment 8, wherein the ratio of urea to glucose before freezing is from about 1:3 to about 3:1, preferably from about 1:2 to about 2:1, or is about 1:1. 11. Freezing with cells in suspension or attached to a vessel surface, or using a collection of cells suspended in a hydrogel, and / or The method of any one of embodiments 1-10, performed in a container with or without a collagen coating, optionally the container is a glass or plastic container with or without a membrane for cell attachment or growth. 12. The cell population is approximately 1 x 10 per mL. 4 ~1 x 10 cells per mL 7 cells, or Approximately 1 x 10 per mL 5 ~4 x 10 cells per mL 5 cells, or Approximately 2 x 10 per mL 5 ~2.5 x 10 cells per mL 5 13. The method of any one of the preceding embodiments, wherein the cell is a cell. 13. Bulking agents are a) a disaccharide, Optionally, the concentration of the disaccharide before freezing is from about 0.1 M to about 1.0 M, or from about 0.1 M to about 0.5 M, or from about 0.2 M to about 0.4 M; Optionally, the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, lactulose, isomaltose, melibiose, or gentiobiose, preferably, the disaccharide is sucrose, optionally, the concentration of sucrose before freezing is about 0.1 M to about 1.0, or about 0.1 M to about 0.5 M, or about 0.2 M to about 0.4 M; or b) a sugar alcohol, Optionally, the concentration of the sugar alcohol before freezing is about 0.1 M to about 1.0 M, or about 0.3 M to about 0.8 M, or about 0.4 M to about 0.6 M; Optionally, the sugar alcohol is mannitol, sorbitol, galactitol, fucitol, iditol, or inositol, preferably the sugar alcohol is mannitol, and optionally the concentration of mannitol before freezing is about 0.1 M to about 1.0 M, or about 0.3 M to about 0.8 M, or about 0.4 M to about 0.6 M; or c) both a disaccharide and a sugar alcohol, or d) The method of any one of the preceding embodiments, comprising both sucrose and mannitol. 14. The composition does not contain DMSO, or The method of any one of the preceding embodiments, wherein the composition comprises DMSO at a concentration of about 1% to about 10% prior to freezing, or wherein the composition comprises DMSO at a concentration of about 2% to about 5% prior to freezing. 15. (a) Prior to freezing, a population of cells is isolated and contacted with a poration solution; Preferably, the poration solution comprises trehalose and a cell culture medium; Optionally, the method of any one of the preceding embodiments, wherein the concentration of trehalose in the poration solution is from about 0.1 M to about 1.0 M, or from about 0.2 M to about 0.6 M. 16. The method of any one of embodiments 2 or 4-15, wherein the reconstitution agent comprises cell culture medium or the reconstitution agent comprises phosphate buffer solution. 17. The method of any one of the preceding embodiments, wherein the composition is an isotonic solution or wherein the composition is a hypertonic solution. 18. The method of any one of the preceding embodiments, wherein the population of cells comprises mammalian cells, preferably the population of cells comprises stem cells, optionally the population of cells comprises pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or hematopoietic stem cells, and / or the population of cells comprises mesenchymal stem cells, and / or the population of cells comprises induced pluripotent stem cells, and / or the population of cells comprises neuroblastoma cells, preferably the neuroblastoma cells are SK-N-AS cells. 19. The aqueous component is a) a buffer solution, preferably comprising a phosphate buffer, a Tris buffer, an acetate buffer, a bicarbonate buffer, a histidine buffer, a citrate buffer, or a combination thereof; and / or b) The method of any one of the preceding embodiments, comprising a cell culture medium, preferably wherein the cell culture medium is serum-free. 20. Removing the aqueous component includes reducing pressure, applying heat, or both to the frozen composition to remove the aqueous component; and / or Removing the aqueous component comprises a primary drying step and a secondary drying step; and / or removing the aqueous components comprises only a primary drying step, preferably the primary drying step comprises reducing the pressure to remove the aqueous components; and / or 20. The method of any one of embodiments 3-19, wherein the secondary drying step comprises applying heat to remove the aqueous components. 21. The method of any one of the preceding embodiments, wherein the freezing is performed at about -10°C to about -100°C, preferably about -20°C to about -90°C, or about -40°C to about -60°C, or wherein the freezing reduces the temperature of the composition to about -80°C, preferably wherein the freezing reduces the temperature of the composition to about -40°C and the aqueous component is removed at a pressure of about 60mTorr to about 80mTorr chamber pressure. 22. The method of any one of embodiments 2 or 5-21, wherein resuspending the frozen cells is performed more than 2 hours after freezing, preferably more than 1 day after freezing, or more than 1 week after freezing, or more than 1 month after freezing. 23. The method of any one of embodiments 4-22, wherein resuspending the freeze-dried cells is performed more than 2 hours after removal of the aqueous component, preferably more than 1 day after removal of the aqueous component, or more than 1 week after removal of the aqueous component, or more than 1 month after removal of the aqueous component. 24. The method of any one of embodiments 2 or 4-23, wherein the frozen or lyophilized cells are stored below about -20°C prior to resuspension, preferably at about -20°C to about 30°C prior to resuspension, or at about 4°C to about 28°C prior to resuspension, or at about 10°C to about 27°C prior to resuspension, or at about 2°C to about 8°C prior to resuspension, or above about -20°C for more than 2 days prior to resuspension. 25. The method of any one of embodiments 4 to 24, wherein the lyophilized cells are stored at about 20°C to about 25°C for more than one week before resuspension, preferably at about 2°C to about 8°C for more than one week before resuspension. 26. The method of any one of the preceding embodiments, wherein at least about 20% of the cells in the reconstituted composition are viable, preferably at least about 30% of the cells in the reconstituted composition are viable, or at least about 40% of the cells in the reconstituted composition are viable, or at least about 50% of the cells in the reconstituted composition are viable, as measured by the ALAMARBLUE test. 27. A composition comprising a population of viable cells, an aqueous component, urea, a simple sugar, and a bulking agent, the composition containing less than about 10% (volume / volume) water, preferably less than about 5% (volume / volume) water, and optionally A composition, wherein the weight ratio of glucose to urea in the composition is from about 30:1 to about 1:3, or from about 15:1 to about 1:2, or from about 3:1 to about 1:1. 28. A composition comprising a population of viable cells, an aqueous component, urea, a monosaccharide, and optionally a bulking agent, wherein the weight ratio of glucose to urea in the composition is from about 30:1 to about 1:3, and optionally, the weight ratio of glucose to urea in the composition is from about 15:1 to about 1:2, or from about 3:1 to about 1:1. 29. The composition of any of embodiments 27-28, wherein the monosaccharide is glucose. 30. A composition comprising a population of viable cells, an aqueous component, about 0.2M to about 1.0M urea, about 0.2M to about 1.0M glucose, and optionally a bulking agent, and optionally further comprising about 0.3M to about 0.8M glucose, or about 0.4M to about 0.6M glucose, and / or about 0.3M to about 0.8M urea, or about 0.4M to about 0.6M urea, and / or sucrose, preferably about 0.1M to about 0.5M sucrose.

[0365] The present invention also relates to the following numbered embodiments: 1. A method for producing a frozen cell population, comprising: A method comprising freezing a composition comprising a cell aggregate, an aqueous component, urea, a simple sugar, and optionally a bulking agent to produce a frozen cell aggregate. 2. A method for producing a population of reconstituted viable cells, comprising: (a) freezing a composition comprising a cell mass, an aqueous component, urea, a simple sugar, and optionally a bulking agent to produce a frozen cell mass; (b) resuspending the frozen cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable. 3. The method of embodiment 1 or 2, wherein the concentration of the monosaccharide before freezing is from about 0.2 M to about 1.25 M, or from about 0.3 M to about 0.8 M, or from about 0.4 M to about 0.6 M. 4. The method of any one of embodiments 1-3, wherein the monosaccharide is glucose, fructose, or galactose. 5. The method of any one of embodiments 1 to 4, wherein the monosaccharide is glucose and the concentration of glucose before freezing is from about 0.2 M to about 1.25 M, or from about 0.2 M to about 0.8 M, or from about 0.4 M to about 0.6 M. 6. The method of any one of embodiments 1-5, wherein the concentration of urea before freezing is from about 0.2M to about 1.25M, or from about 0.2M to about 0.8M, or from about 0.4M to about 0.6M. 7. The method of any one of embodiments 1-6, wherein the molar ratio of urea to monosaccharides before freezing is from about 1:3 to about 3:1, or from about 1:2 to about 2:1, or is about 1:1. 8. The method of any one of embodiments 1-7, wherein the monosaccharide is glucose and the molar ratio of urea to glucose before freezing is about 1:3 to about 3:1, or about 1:2 to about 2:1, or about 1:1. 9. A method for producing a freeze-dried cell aggregate, comprising: (a) freezing a composition comprising a cell aggregate, hyaluronan gel, an aqueous component, urea, a monosaccharide, and a bulking agent, wherein the concentration of the monosaccharide before freezing is at least about 0.6 M and the concentration of the urea before freezing is at least about 0.6 M; (b) removing at least about 90% of the aqueous components from the frozen composition to produce a freeze-dried cell aggregate. 10. A method for producing a population of reconstituted viable cells, comprising: (a) freezing a composition comprising a cell aggregate, hyaluronan gel, an aqueous component, urea, a monosaccharide, and a bulking agent, wherein the concentration of the monosaccharide before freezing is at least about 0.6 M and the concentration of the urea before freezing is at least about 0.6 M; (b) removing at least about 90% of the aqueous components from the frozen composition to produce a freeze-dried cell aggregate; (c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable. 11. The method of embodiment 9 or 10, wherein the concentration of the monosaccharide before freezing is from about 0.6 M to about 2.0 M, or from about 0.7 M to about 1.7 M, or from about 0.8 M to about 1.4 M. 12. The method of any one of embodiments 9-11, wherein the monosaccharide is glucose, fructose, or galactose. 13. The method of any one of embodiments 9 to 12, wherein the monosaccharide is glucose and the concentration of glucose before freezing is from about 0.6 M to about 2.0 M, or from about 0.7 M to about 1.7 M, or from about 0.8 M to about 1.4 M. 14. The method of any one of embodiments 9 to 13, wherein the concentration of urea before freezing is from about 0.6M to about 2.0M, or from about 0.7M to about 1.7M, or from about 0.8M to about 1.4M. 15. The method of any one of embodiments 9-14, wherein the molar ratio of urea to monosaccharides before freezing is from about 1:3 to about 3:1, or from about 1:2 to about 2:1, or is about 1:1. 16. The method of any one of embodiments 9 to 15, wherein the monosaccharide is glucose and the molar ratio of urea to glucose before freezing is about 1:3 to about 3:1, or about 1:2 to about 2:1, or about 1:1. 17. Bulking agents are a) a disaccharide, the concentration of the disaccharide before freezing is about 0.1 M to about 1.0 M, or about 0.1 M to about 0.5 M, or about 0.2 M to about 0.4 M; A disaccharide, wherein the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, lactulose, isomaltose, melibiose or gentiobiose; or b) a sugar alcohol, The concentration of the sugar alcohol before freezing is about 0.1 M to about 1.0 M, about 0.3 M to about 0.8 M, or about 0.4 M to about 0.6 M; A sugar alcohol, wherein the sugar alcohol is mannitol, sorbitol, galactitol, fucitol, iditol, or inositol; or c) both a disaccharide and a sugar alcohol, or d) The method of any one of the preceding embodiments, comprising both sucrose and mannitol. 18. The composition does not contain DMSO, or The method of any one of the preceding embodiments, wherein the composition comprises DMSO at a concentration of about 1% to about 10% prior to freezing, or wherein the composition comprises DMSO at a concentration of about 1% to about 5% prior to freezing. 19. The method of any one of the preceding embodiments, wherein the aggregates of cells are isolated and contacted with a poration solution prior to freezing in (a). 20. The method of Example 19, wherein the poration solution contains trehalose, and the concentration of trehalose in the poration solution is from about 0.1 M to about 1.0 M, or from about 0.1 M to about 0.6 M. 21. The method of any one of the preceding embodiments, wherein the population of cells comprises mammalian cells.

[0366] Cryoprotection of cells stored by freezing: The present invention relates to a method for producing a frozen cell aggregate, the method comprising freezing a composition comprising a cell aggregate, an aqueous component, urea, and a simple sugar to produce the frozen cell aggregate.

[0367] The present invention further provides a method for producing a population of reconstituted viable cells, comprising the steps of: (a) freezing a composition comprising a cell mass, an aqueous component, urea, a simple sugar, and optionally a bulking agent to produce a frozen cell mass; (b) resuspending the frozen cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable.

[0368] In one embodiment, the concentration of the monosaccharide before freezing is about 0.1 M to about 1.5 M. In one embodiment, the concentration of the monosaccharide before freezing is about 0.2 M to about 1.2 M. Preferably, the concentration of the monosaccharide before freezing is about 0.2 M to about 1.0 M. More preferably, the concentration of the monosaccharide before freezing is about 0.2 M to about 0.8 M. Even more preferably, the concentration of the monosaccharide before freezing is about 0.3 M to about 0.7 M. Even more preferably, the concentration of the monosaccharide before freezing is about 0.4 M to about 0.6 M. Most preferably, the concentration of the monosaccharide before freezing is about 0.5 M.

[0369] In one embodiment, the concentration of the monosaccharide prior to freezing is about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, 1.0M, 1.05M, 1.10M, 1.15M, 1.20M, 1.25M, 1.30M, 1.35M, 1.40M, 1.45M, or 1.50M.

[0370] In one embodiment, the monosaccharide is glucose, fructose, or galactose.

[0371] In one embodiment, the glucose concentration before freezing is about 0.1 M to about 1.5 M. In one embodiment, the glucose concentration before freezing is about 0.2 M to about 1.2 M. Preferably, the glucose concentration before freezing is about 0.2 M to about 1.0 M. More preferably, the glucose concentration before freezing is about 0.2 M to about 0.8 M. Even more preferably, the glucose concentration before freezing is about 0.3 M to about 0.7 M. Even more preferably, the glucose concentration before freezing is about 0.4 M to about 0.6 M. Most preferably, the glucose concentration before freezing is about 0.5 M.

[0372] In one embodiment, the concentration of glucose prior to freezing is about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, 1.0M, 1.05M, 1.10M, 1.15M, 1.20M, 1.25M, 1.30M, 1.35M, 1.40M, 1.45M, or 1.50M.

[0373] In one embodiment, the concentration of urea before freezing is about 0.1 M to about 1.5 M. In one embodiment, the concentration of urea before freezing is about 0.2 M to about 1.2 M. Preferably, the concentration of urea before freezing is about 0.2 M to about 1.0 M. More preferably, the concentration of urea before freezing is about 0.2 M to about 0.8 M. Even more preferably, the concentration of urea before freezing is about 0.2 M to about 0.8 M. Even more preferably, the concentration of urea before freezing is about 0.4 M to about 0.6 M. Most preferably, the concentration of urea before freezing is about 0.5 M.

[0374] In one embodiment, the concentration of urea prior to freezing is about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, 1.0M, 1.05M, 1.10M, 1.15M, 1.20M, 1.25M, 1.30M, 1.35M, 1.40M, 1.45M, or 1.50M.

[0375] In one embodiment, the molar ratio of urea to monosaccharides before freezing is about 1:5 to about 5:1. In one embodiment, the molar ratio of urea to monosaccharides before freezing is about 1:4 to about 4:1. In one embodiment, the molar ratio of urea to monosaccharides before freezing is about 1:3 to about 3:1. In one embodiment, the molar ratio of urea to monosaccharides before freezing is about 1:2 to about 2:1. Preferably, the molar ratio of urea to monosaccharides before freezing is about 1:1.

[0376] In one embodiment, the molar ratio of urea to glucose before freezing is about 1:5 to about 5:1. In one embodiment, the molar ratio of urea to glucose before freezing is about 1:4 to about 4:1. In one embodiment, the molar ratio of urea to glucose before freezing is about 1:3 to about 3:1. In one embodiment, the molar ratio of urea to glucose before freezing is about 1:2 to about 2:1. Preferably, the molar ratio of urea to glucose before freezing is about 1:1.

[0377] In one embodiment, the concentration of glucose before freezing is about 0.2M to about 1.2M, and the concentration of urea before freezing is about 0.2M to about 1.2M. Preferably, the concentration of glucose before freezing is about 0.2M to about 1.0M, and the concentration of urea before freezing is about 0.2M to about 1.0M. More preferably, the concentration of glucose before freezing is about 0.2M to about 0.8M, and the concentration of urea before freezing is about 0.2M to about 0.8M. Even more preferably, the concentration of glucose before freezing is about 0.4M to about 0.6M, and the concentration of urea before freezing is about 0.4M to about 0.6M. Most preferably, the concentration of glucose before freezing is about 0.5M, and the concentration of urea before freezing is about 0.5M.

[0378] In one embodiment, the composition further comprises a bulking agent.

[0379] In one embodiment, the bulking agent is mannitol. In one embodiment, the concentration of mannitol before freezing is about 0.1 M to about 1.0 M. In one embodiment, the concentration of mannitol before freezing is about 0.1 M to about 0.8 M. Preferably, the concentration of mannitol before freezing is about 0.1 M to about 0.6 M. More preferably, the concentration of mannitol before freezing is about 0.15 M to about 0.55 M.

[0380] In one embodiment, the concentration of mannitol before freezing is about 0.1 M to about 0.3 M. In one embodiment, the concentration of mannitol before freezing is about 0.4 M to about 0.6 M.

[0381] In one embodiment, the bulking agent is sucrose. In one embodiment, the concentration of sucrose before freezing is about 0.1 M to about 1.0 M. In one embodiment, the concentration of sucrose before freezing is about 0.1 M to about 0.5 M. Preferably, the concentration of sucrose before freezing is about 0.1 M to about 0.4 M. More preferably, the concentration of sucrose before freezing is about 0.2 M to about 0.3 M.

[0382] In one embodiment, the composition comprises DMSO at a concentration of about 1% to about 10% prior to freezing. In one embodiment, the composition comprises DMSO at a concentration of about 1% to about 5% prior to freezing. In one embodiment, the composition comprises DMSO at a concentration of about 1% to about 3% prior to freezing.

[0383] In one embodiment, prior to freezing in (a), the cell aggregates are isolated and contacted with a poration solution. In one embodiment, the poration solution comprises trehalose and a cell culture medium. In one embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 1.0 M. In one embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 0.8 M. In one embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 0.6 M. In one embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 0.3 M.

[0384] In one embodiment, the cells are contacted with the poration solution for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, or at least 12 hours. In one embodiment, the cells are contacted with the poration solution for 1 hour to 24 hours, 1 hour to 18 hours, or 1 hour to 12 hours.

[0385] In one embodiment, the composition comprises urea, glucose, and mannitol, wherein the concentration of glucose before freezing is about 0.4 M to about 0.6 M, the concentration of urea before freezing is about 0.4 M to about 0.6 M, and the concentration of mannitol before freezing is about 0.1 M to about 0.6 M.

[0386] In one embodiment, the composition comprises urea, glucose, and sucrose, wherein the concentration of glucose before freezing is about 0.4 M to about 0.6 M, the concentration of urea before freezing is about 0.4 M to about 0.6 M, and the concentration of sucrose before freezing is about 0.2 M to about 0.3 M.

[0387] In one embodiment, the composition comprises urea, glucose, sucrose, and DMSO, wherein the concentration of glucose before freezing is about 0.4 M to about 0.6 M, the concentration of urea before freezing is about 0.4 M to about 0.6 M, the concentration of sucrose before freezing is about 0.2 M to about 0.3 M, and the concentration of DMSO before freezing is about 1% to about 3%.

[0388] The present invention further relates to a composition comprising a population of viable cells, an aqueous component, about 0.2 M to about 1.0 M urea, about 0.2 M to about 1.0 M glucose, and optionally a bulking agent.

[0389] In one embodiment, the composition comprises about 0.3M to about 0.8M glucose, preferably about 0.4M to about 0.6M glucose.

[0390] In one embodiment, the composition comprises about 0.3M to about 0.8M urea, preferably about 0.4M to about 0.6M urea.

[0391] In one embodiment, the bulking agent is sucrose. In one embodiment, the composition comprises from about 0.1 M to about 0.5 M. Preferably, the composition comprises from about 0.1 M to about 0.4 M. More preferably, the composition comprises from about 0.2 M to about 0.3 M.

[0392] In one embodiment, the bulking agent is mannitol. In one embodiment, the composition comprises from about 0.1 M to about 0.8 M. Preferably, the composition comprises from about 0.1 M to about 0.6 M. More preferably, the composition comprises from about 0.15 M to about 0.55 M.

[0393] In one embodiment, the composition further comprises DMSO. In one embodiment, the composition comprises about 1% to about 10% DMSO. In one embodiment, the composition comprises about 1% to about 5% DMSO. In one embodiment, the composition comprises about 1% to about 3% DMSO.

[0394] Cryoprotection of cells stored by lyophilization: The present invention also provides a method for producing a freeze-dried cell population, comprising the steps of: (a) freezing a composition comprising a cell aggregate, a hyaluronan gel, an aqueous component, urea, a simple sugar, and a bulking agent, freezing, wherein the concentration of monosaccharides before freezing is at least about 0.6M and the concentration of urea before freezing is at least about 0.6M; (b) removing at least about 90% of the water from the frozen composition to produce a freeze-dried cell aggregate.

[0395] The present invention further provides a method for producing a population of reconstituted viable cells, comprising the steps of: (a) freezing a composition comprising a cell aggregate, a hyaluronan gel, an aqueous component, urea, a simple sugar, and a bulking agent, freezing, wherein the concentration of monosaccharides before freezing is at least about 0.6M and the concentration of urea before freezing is at least about 0.6M; (b) removing at least about 90% of the water from the frozen composition to produce a freeze-dried cell aggregate; (c) resuspending the lyophilized cell population in a reconstitution agent to form a reconstituted composition, wherein at least about 1% of the cells are viable.

[0396] Hyaluronan gels are known to those skilled in the art. In one embodiment, the hyaluronan gel is derived from hyaluronic acid or hyaluronic acid sodium salt, preferably hyaluronic acid sodium salt. In one embodiment, the hyaluronan gel is based on PBS (phosphate buffered saline) or water and hyaluronic acid sodium salt. Preferably, the hyaluronan gel is based on PBS (phosphate buffered saline) and hyaluronic acid sodium salt.

[0397] In one embodiment, hyaluronic acid or sodium hyaluronate is contained in the composition at a concentration of 5 mg / mL to about 50 mg / mL before freezing, for example, about 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL.Preferably, hyaluronic acid or sodium hyaluronate is contained in the composition at a concentration of about 5 mg / mL to about 35 mg / mL before freezing, for example, about 5 mg / mL to about 15 mg / mL, about 15 mg / mL to about 25 mg / mL, or about 25 mg / mL to about 35 mg / mL.

[0398] In one embodiment, the concentration of the monosaccharide before freezing is about 0.6 M to about 2.0 M. In one embodiment, the concentration of the monosaccharide before freezing is about 0.7 M to about 1.7 M. Preferably, the concentration of the monosaccharide before freezing is about 0.8 M to about 1.4 M. More preferably, the concentration of the monosaccharide before freezing is about 0.9 M to about 1.2 M. Most preferably, the concentration of the monosaccharide before freezing is about 1.0 M.

[0399] In one embodiment, the concentration of the monosaccharide prior to freezing is about 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, 1.0M, 1.05M, 1.1M, 1.15M, 1.2M, 1.25M, 1.3M, 1.35M, 1.4M, 1.45M, 1.5M, 1.55M, 1.60M, 1.65M, 1.70M, 1.75M, 1.80M, 1.85M, 1.90M, 1.95M, or 2.0M.

[0400] In one embodiment, the monosaccharide is glucose, fructose, or galactose, preferably glucose.

[0401] In one embodiment, the glucose concentration before freezing is about 0.6 M to about 2.0 M. In one embodiment, the glucose concentration before freezing is about 0.7 M to about 1.7 M. Preferably, the glucose concentration before freezing is about 0.8 M to about 1.4 M. More preferably, the glucose concentration before freezing is about 0.9 M to about 1.2 M. Most preferably, the glucose concentration before freezing is about 1.0 M.

[0402] In one embodiment, the concentration of glucose prior to freezing is about 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, 1.0M, 1.05M, 1.1M, 1.15M, 1.2M, 1.25M, 1.3M, 1.35M, 1.4M, 1.45M, 1.5M, 1.55M, 1.60M, 1.65M, 1.70M, 1.75M, 1.80M, 1.85M, 1.90M, 1.95M, or 2.0M.

[0403] In one embodiment, the concentration of urea before freezing is about 0.6 M to about 2.0 M. In one embodiment, the concentration of urea before freezing is about 0.7 M to about 1.7 M. Preferably, the concentration of urea before freezing is about 0.8 M to about 1.4 M. More preferably, the concentration of urea before freezing is about 0.9 M to about 1.2 M. Most preferably, the concentration of urea before freezing is about 1.0 M.

[0404] In one embodiment, the concentration of urea prior to freezing is about 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, 1.0M, 1.05M, 1.1M, 1.15M, 1.2M, 1.25M, 1.3M, 1.35M, 1.4M, 1.45M, 1.5M, 1.55M, 1.60M, 1.65M, 1.70M, 1.75M, 1.80M, 1.85M, 1.90M, 1.95M, or 2.0M.

[0405] In one embodiment, the molar ratio of urea to monosaccharides before freezing is about 1:3 to about 3:1. In one embodiment, the molar ratio of urea to monosaccharides before freezing is about 1:2 to about 2:1. Preferably, the molar ratio of urea to monosaccharides before freezing is about 1:1.

[0406] In one embodiment, the molar ratio of urea to glucose before freezing is about 1:3 to about 3:1. In one embodiment, the molar ratio of urea to glucose before freezing is about 1:2 to about 2:1. Preferably, the molar ratio of urea to glucose before freezing is about 1:1.

[0407] In one embodiment, the concentration of glucose before freezing is about 0.6M to about 2.0M, and the concentration of urea before freezing is about 0.6M to about 2.0M. Preferably, the concentration of glucose before freezing is about 0.7M to about 1.7M, and the concentration of urea before freezing is about 0.7M to about 1.7M. More preferably, the concentration of glucose before freezing is about 0.8M to about 1.4M, and the concentration of urea before freezing is about 0.8M to about 1.4M. Even more preferably, the concentration of glucose before freezing is about 0.9M to about 1.2M, and the concentration of urea before freezing is about 0.9M to about 1.2M. Most preferably, the concentration of glucose before freezing is about 1.0M, and the concentration of urea before freezing is about 1.0M.

[0408] In one embodiment, the bulking agent is mannitol. In one embodiment, the concentration of mannitol before freezing is about 0.1 M to about 1.0 M. In one embodiment, the concentration of mannitol before freezing is about 0.1 M to about 0.8 M. Preferably, the concentration of mannitol before freezing is about 0.1 M to about 0.6 M. More preferably, the concentration of mannitol before freezing is about 0.1 M to about 0.4 M. In one embodiment, the concentration of mannitol before freezing is at least about 0.2 M.

[0409] In one embodiment, the bulking agent is sucrose. In one embodiment, the concentration of sucrose before freezing is about 0.1 M to about 1.0 M. In one embodiment, the concentration of sucrose before freezing is about 0.2 M to about 0.8 M. Preferably, the concentration of sucrose before freezing is about 0.3 M to about 0.7 M. More preferably, the concentration of sucrose before freezing is about 0.4 M to about 0.6 M. In one embodiment, the concentration of sucrose before freezing is at least about 0.4 M.

[0410] In one embodiment, the composition comprises DMSO at a concentration of about 1% to about 10% prior to freezing. In one embodiment, the composition comprises DMSO at a concentration of about 1% to about 8% prior to freezing. In one embodiment, the composition comprises DMSO at a concentration of about 2% to about 6% prior to freezing. In one embodiment, the composition comprises DMSO at a concentration of about 3% to about 5% prior to freezing.

[0411] In one embodiment, prior to freezing in (a), the cell aggregates are isolated and contacted with a poration solution. In one embodiment, the poration solution comprises trehalose and a cell culture medium. In one embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 1.0 M. In one embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 0.8 M. In one embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 0.6 M. In one embodiment, the concentration of trehalose in the poration solution is about 0.1 M to about 0.3 M.

[0412] In one embodiment, the cells are contacted with the poration solution for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, or at least 12 hours. In one embodiment, the cells are contacted with the poration solution for 1 hour to 24 hours, 1 hour to 18 hours, or 1 hour to 12 hours.

[0413] In one embodiment, the composition comprises urea, glucose, sucrose, and mannitol, wherein the concentration of glucose before freezing is about 0.8 M to about 1.2 M, the concentration of urea before freezing is about 0.8 M to about 1.2 M, the concentration of sucrose before freezing is about 0.4 M to about 0.6 M, and the concentration of mannitol before freezing is about 0.1 M to about 0.4 M.

[0414] In one embodiment, the composition comprises urea, glucose, sucrose, and DMSO, wherein the concentration of glucose before freezing is about 0.8M to about 1.2M, the concentration of urea before freezing is about 0.8M to about 1.2M, the concentration of sucrose before freezing is about 0.4M to about 0.6M, and the concentration of DMSO before freezing is about 3% to about 5%.

[0415] In one embodiment, the composition comprises urea, glucose, sucrose, mannitol, and DMSO, wherein the concentration of glucose before freezing is about 0.8M to about 1.2M, the concentration of urea before freezing is about 0.8M to about 1.2M, the concentration of sucrose before freezing is about 0.4M to about 0.6M, the concentration of mannitol before freezing is about 0.1M to about 0.4M, and the concentration of DMSO before freezing is about 3% to about 5%.

[0416] In one embodiment, the composition comprises urea, glucose, mannitol, and DMSO, wherein the concentration of glucose before freezing is about 0.8 M to about 1.2 M, the concentration of urea before freezing is about 0.8 M to about 1.2 M, the concentration of mannitol before freezing is about 0.1 M to about 0.4 M, and the concentration of DMSO before freezing is about 3% to about 5%.

[0417] The present invention further relates to a composition comprising a cell aggregate, hyaluronan gel, an aqueous component, urea, a monosaccharide, and optionally a bulking agent, wherein the urea and the monosaccharide are present in the composition in a molar ratio of about 1:3 to about 3:1, and the composition contains less than about 10% (volume / volume) water.

[0418] In one embodiment, the molar ratio of urea to monosaccharides is about 1:2 to about 2:1. Preferably, the molar ratio of urea to monosaccharides is about 1:1.

[0419] In one embodiment, the monosaccharide is glucose. In one embodiment, the molar ratio of urea to glucose before freezing is about 1:2 to about 2:1. Preferably, the molar ratio of urea to glucose before freezing is about 1:1.

[0420] In one embodiment, the composition contains less than about 5% (volume / volume) water. EXAMPLES

[0421] Example 1 - Cryoprotection and cryoprotection of human mesenchymal stem cells overview Human mesenchymal stem cells (hMSCs) can be differentiated in vitro into various types of tissues, such as bone, fat, cartilage, or muscle, making them suitable for use in research and cell therapy products. However, the current method of storage of hMSCs in liquid nitrogen has drawbacks. Storage and distribution are expensive and labor intensive, and standard freezing protocols commonly use reagents such as fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO), which can cause aversive immune responses or be toxic, respectively. Therefore, these agents need to be removed before products containing frozen hMSCs can be applied to humans. Lyophilization of cells in a non-toxic formulation could be a solution, but currently no satisfactory non-toxic formulations with cryoprotective and cryoprotective properties have been reported.

[0422] The work presented herein provides the first known evaluation of the cryoprotective capacity of the non-toxic and synergistically acting cryoprotectants (CPAs), urea and glucose, with hMSCs. Different freeze-drying strategies using urea / glucose as CPAs are qualitatively evaluated herein. Freezing hMSCs at -80°C in 0.5 M urea and 0.5 M glucose yielded viability comparable to DMSO controls. Also, hMSCs in formulations with high amounts of CPAs were able to withstand the harsh conditions of freeze-drying and proliferated upon thawing.

[0423] Introduction Human mesenchymal stem cells (hMSCs) are important pluripotent stem cells that have the ability to differentiate (in vitro) into different tissues such as fat, muscle, cartilage, bone, or nerve cells. The use of hMSCs in regenerative medicine and cell therapy is expanding. hMSCs have the potential to be harvested from adult donors, expanded, modified, and then used in allogeneic therapy, as they exhibit immunosuppressive capabilities. To make this possible, hMSCs need to be stored for long periods of time. The current protocol for long-term storage of hMSCs is by cryopreservation in liquid nitrogen. However, storing cells in liquid nitrogen is cumbersome and expensive, especially for distribution and transportation, since a regular supply of liquid nitrogen is required to avoid uncontrolled thawing and damage of the cells.

[0424] Liquid nitrogen storage of hMSCs also requires the addition of fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) to preserve the cells. DMSO is toxic and can alter transcription factor expression and gene expression in hMSCs. Furthermore, FBS is highly immunogenic in humans and can transmit pathogens. Therefore, DMSO and FBS are usually removed before administration to humans, a process that is labor intensive and requires specially trained personnel and special facilities. Therefore, there is a high risk that the administered product will be substandard and of inconsistent quality, with safety implications.

[0425] It would also be beneficial to store cell therapy products (CTPs) at refrigerated temperatures, as with many other biological products, to make them easier to handle and facilitate economical transport. One possible way to achieve this is to lyophilize (freeze-dry) the cells. Lyophilization is routinely applied to (drug) products that are unstable in liquid formulations but are stabilized in solid / dried form, such as vaccines, proteins, peptides, or antibiotics. Applying the principles of lyophilization to hMSCs would greatly support their utility as CTPs.

[0426] Various alternatives to DMSO have been explored for cryopreserving hMSCs, primarily sugars or sugar alcohols such as sucrose, trehalose, glucose, mannitol, glycerol, or ethylene glycol, but also intracellular or extracellular incorporation of polyvinylpyrrolidone, pentaisomaltose, or ectoine have been evaluated.

[0427] In some species of hibernating frogs, accumulation of urea has been reported to have osmoprotective and cryoprotective effects. These effects were further strengthened by further increase in somatic glucose levels, and the glucose / urea combination improved resistance to freezing stress. Because these agents are relatively safe in humans, inexpensive, and commercially available, the potential of the urea / glucose combination for cryoprotection and lyophilization in hMSCs was explored by advanced formulations, hydrogels, and stabilizers.

[0428] Materials and Methods cell culture Adherent bone marrow-derived human mesenchymal stem cells (hMSCs) (ATCC) were expanded to generate cell banks and stored at passage 3 in the vapor phase of liquid nitrogen in growth medium supplemented with 50% FBS and 7.5% DMSO. Aliquots of these samples were warmed in a 37°C water bath and cultured in MSCGM™ Mesenchymal Stem Cell Growth Medium BulletKit™ (containing FBS, Lonza), a commercially available medium that facilitates undifferentiated growth of hMSCs. Cells were kept in a humidified incubator at 37°C, 5% CO2, and growth medium was changed every 3-4 days. When hMSCs reached approximately 90% confluence, they were split and cultured at approximately 5000 cells / cm. 2 To promote further proliferation, cells at passages 4 to 8 were used for the experiments.

[0429] Freezing and thawing experiments Confluent or nearly confluent hMSCs were cultured by aspirating the medium and washing with pre-warmed phosphate buffered saline pH 7.4 (PBS, Gibco) at approximately 0.02 ml / cm 2 The cells were harvested by dissociating them by applying 100 mL of trypsin solution (Lonza). The trypsin reaction was then stopped by adding growth medium. An aliquot was taken for cell concentration determination (using a NucleCounter® NC-200™). The cells were then pelleted by centrifugation and subsequently resuspended in PBS to a concentration of 2×10 per mL. 6 A volume of 0.5 mL of the cell suspension was added to a 6R vial (Schott) containing 0.5 ml of a 2x concentrated solution of cryoprotectant (CPA) (urea: GE Healthcare, glucose: Aldrich, sucrose and trehalose: Pfanstiehl, mannitol: Avantor, DMSO: Arcos) diluted in PBS to achieve a target concentration of 1 × 10 cells. 6A total volume of 1 ml was obtained containing 1000 cells and 1x the target concentration of cryoprotectant. The vials were then stopped with stoppers (West) and transferred to a -80 °C freezer where they were stored for 2-3 days until viability measurements were performed. Each experiment was repeated five times.

[0430] Freeze-drying experiment hMSCs were cultured on different hydrogels (HyStem™, HyStem™ C, or agar (Sigma) or suspended in hyaluronic acid (HA) (Alfa Aesar) to provide a gel matrix that can be used for culture and / or as a scaffold for the frozen cake.

[0431] Samples for the freeze-drying experiments were prepared in the following manner.

[0432] Agarose gels were prepared by dissolving 2% agar in PBS with heating. The gels were cooled and allowed to gel for approximately 20 hours. Cells were then seeded on the surface of the gels at a concentration of approximately 20,000 cells per square centimeter of gel and incubated with cell culture medium for approximately 20 hours, allowing the cells to reattach to the gel surface. Prior to lyophilization, the medium was removed and a layer of a viscous solution containing HA and 1× CPA (the so-called viscous CPA / HA formulation as seen in Table 1, prepared by dissolving approximately 1× CPA in PBS and then adding hyaluronic acid sodium salt, stirring the solution for approximately 20 minutes to ensure complete solution of HA (once HA is dissolved, the solution becomes viscous) to provide a viscous CPA / HA formulation) was added on top of the agar gel / cell layer, and the samples were frozen at −80° C. for approximately 20 hours before lyophilization.

[0433] HyStem™ gels were prepared according to the manufacturer's instructions. On top of these HyStem™ gels, cells were seeded onto the gel surface and then layered with a viscous CPA / HA formulation as described for the preparation of agarose gels.

[0434] A viscous CPA / HA formulation was prepared by dissolving approximately 1x CPA in PBS, then adding hyaluronic acid sodium salt to reach a target concentration of 10mg / ml HA salt (=1% w / v). The solution was stirred for approximately 20 minutes to ensure complete solution of HA (once the HA is dissolved, the solution becomes viscous) to provide the viscous CPA / HA formulation.

[0435] For cells suspended in HA, a viscous CPA / HA formulation was prepared as described. Cells were prepared similar to the cell preparation described for the freeze-thaw experiments. After stopping the trypsin reaction by adding growth medium, cells were pelleted by centrifugation and subsequently resuspended in PBS to give 2×10 cells per ml. 6 A target concentration of 1000 cells was achieved. A volume of 0.5 mL of the cell suspension was added to a 6R vial (Schott), and instead of layering the cells on top of the gel, they were allowed to settle within the 6R vial and then resuspended in 1 ml of the viscous CPA / HA formulation.

[0436] After adding the CPA solution or viscous CPA / HA formulation, all vials were immediately transferred to a -80°C freezer and frozen overnight. For lyophilization, samples were placed in a pilot-scale LyoStar™ 3 freeze dryer pre-cooled to -65°C. Samples were lyophilized at -40°C, below 75 mTorr for either 1'000 (cycle 1) or 1'800 (cycle 2) or 3'640 (cycle 3) minutes. The secondary drying step was omitted to avoid possible loss of viable cells. After lyophilization, samples were reconstituted in 6 ml of warm growth medium and transferred to flasks to allow cells to reattach to the plastic surface.

[0437] After lyophilization, the composition still contains some water, typically less than about 90% (vol / vol) water.

[0438] Survival assessment For frozen samples, i.e., samples after freezing and thawing, viability was measured by performing the alamarBlue™ assay (Invitrogen) according to the manufacturer's instructions. The assay is based on the reduction of a non-fluorescent dye to a fluorescent dye. This reaction occurs only in cells that are metabolically active (viable), thus correlating measured fluorescence with viable cells. Frozen cells were thawed in a 37°C water bath and incubated for 1 cm. 2 The cells were placed in 48-well plates at a density of 20,000 cells (dead and viable) per well and kept in an incubator overnight to allow time for the viable cells to attach to the surface and regain their metabolic activity. The assay reagent was then applied and after 4 hours of incubation, the lambda ex = 550 nm and λ em Fluorescence was measured using a SpectraMax® iD3 with a wavelength of 590 nm. Background fluorescence values ​​were subtracted from measurements and compared to fluorescence values ​​of untreated cells.

[0439] For lyophilized samples, hMSCs were qualitatively assessed for viability by reconstituting the sample in growth medium and reseeding the entire contents of the vial in a flask. After overnight incubation, cells were examined microscopically to see if they had reattached to the flask surface and if their morphology showed any irregularities compared to the known morphology of viable cells. Cells were then cultured as previously described and examined for proliferation after several days. For all experiments, the sample size was one per condition.

[0440] Results and Discussion Urea and glucose as cryoprotectants for hMSCs during freezing and thawing Different concentrations of urea and glucose were evaluated for cryoprotection of hMSCs (Figure 1A). Urea at concentrations of 0.05, 0.1 (these two are not shown in Figure 1A), 0.2, and 0.5M had virtually no viable cells, indicating that urea alone is not sufficient to protect hMSCs during cryopreservation. Interestingly, about 26% cell viability was achieved with 0.5M glucose alone, which slightly decreased to about 20% when the glucose concentration was reduced to 0.2M. The combination of urea and glucose (0.5M glucose and 0.5M urea) further increased the number of viable cells to a level similar to the 5% DMSO control (58%). When urea / glucose was further increased to 1M each, the viability of hMSCs decreased to about 50%. These results suggest that urea and glucose act synergistically in hMSCs and their combined cryoprotective capacity is greater than that of each agent alone. As shown in Figure 1A, the combination of 0.5 M urea and 0.5 M glucose was optimal for protecting hMSCs from freeze / thaw (FT) stress, with cell viability comparable to the control. Further evaluation of different ratios of urea to glucose to potentially increase FT viability (Figure 1B) confirmed that a 1:1 molar ratio of urea / glucose at 0.5 M provided the greatest cryoprotection. The following cell viabilities were observed: urea:glucose molar ratios: 1:2 ratio: 45%, 1:3 ratio: 40%; 2:1 ratio: 45%, 3:1 ratio: 30%. None of the ratios tested had greater viability than the optimal 1:1 molar ratio.

[0441] Formulation for freeze-drying tested by freeze-thaw Since cryopreservation was successfully achieved using a 1:1 molar ratio of urea / glucose at a concentration of 0.5 M, we sought to improve the formulation for the primary drying step of the freeze-drying process. Sucrose and mannitol were selected as bulking agents for their cake-forming ability with favorable cryoprotective properties, as sucrose can improve post-FT survival in stem cells.

[0442] The addition of 0.2M and 0.5M mannitol provided 45% and 40% viable cells, respectively, and 0.25M sucrose provided 39% viable cells (Figure 1C). Although cell viability is lower than the previous FT control or formulations containing 0.5M urea / glucose, viability was further improved by adding a small amount of DMSO (2%) to the urea / glucose / sucrose formulation. The decrease in viability with increasing sugar concentration is likely due to increased osmotic stress. Sugar concentrations with high viability appear to provide the advantage of high molar amounts of extracellular sugar as CPAs without causing an excessive increase in osmolarity. Internalization and intracellular accumulation of sugars may be beneficial for FT survival of hMSCs.

[0443] Internalization of trehalose as a cryoprotectant and cryoprotectant. Trehalose is known to function as a cryoprotectant and cryoprotectant, but this is only possible when it is present both intracellularly and extracellularly. However, this molecule is not membrane permeable. Thus, several methods for intracellular trehalose uptake use genetic modification of cell pores, cell-penetrating peptides, liposomes, or electroporation, but these methods are complex, expensive, time-consuming, and may cause significant changes in the genetic makeup of hMSCs. An alternative, simple internalization method involves trehalose uptake by endocytosis from trehalose-supplemented medium, which has been reported to act as a CPA

[32] .

[0444] Incubation of hMSCs with 0.2 M trehalose dissolved in cell culture medium for about 20 h followed by FT in the optimal formulation of 0.5 M urea / 0.5 M glucose / 0.25 M sucrose showed good viability without morphological changes such as shrinkage, suggesting that hMSCs were able to withstand the applied osmotic stress (Figure 1C and Figure 1D1-1D3). Further incubation in trehalose slightly increased the viability of hMSCs in the urea / glucose / sucrose formulation to about 65%, and the addition of 2% DMSO could further increase it to about 72%, suggesting that a combination of different CPAs is superior to a single CPA for improving cell viability for FT. In the 0.2 M mannitol formulation, the cell viability was about 70%. These results demonstrate that hMSCs can tolerate short-term hypertonic conditions in growth medium supplemented with 0.2 M trehalose, further helping to improve the cryopreservation and potential cryopreservation of hMSCs.

[0445] Freeze-drying of hMSCs Optimizing the freeze-drying cycle Freezing cycles and residual moisture can have a significant effect on hMSC viability. Two different drying times, 1'000 - 1'800 - 3'640 min, were evaluated at -40 °C. Also, steps were taken to further optimize the loading and unloading temperatures as well as the reconstitution procedure.

[0446] Freeze-drying using 3D gel matrix Different freeze-drying strategies were evaluated for hMSCs using urea and glucose as primary CPAs as well as sucrose, mannitol, and trehalose. hMSCs were grown on different gel matrices, namely HyStem™, HyStem™ C, and 2% agarose gels. HyStem™ and HyStem™ C are commercially available for 3D cell culture based on thiol-modified hyaluronic acid. HyStem™ C further contains collagen fibers that act as attachment sites, thus improving cell attachment to the gel

[33] . Alternatively, hMSCs were suspended and then freeze-dried in hyaluronic acid (HA) and CPA.

[0447] hMSCs cultured on HyStem™ had no viable cells after freeze-drying, regardless of which CPA was used (Table 1). The cells also could not attach to the hydrogel surface and form a cell layer. Cells cultured on HyStem™ C matrix were able to attach to the gel but did not show significant cell viability after freeze-drying. Also, cell viability remained very low in agarose / HA dual matrix. However, hMSCs suspended in a viscous formulation of HA at higher concentrations and different amounts of CPA could obtain viable cells that attached and proliferated in the flask after freeze-drying (Figure 2), indicating that hMSCs are viable and can withstand the harsh conditions of the freeze-drying process. To the best of the inventors' knowledge, this is the first case to report freeze-drying of unmodified hMSCs with unchanged morphology and utilizing a combination of urea and glucose in such a process. hMSCs also remained viable in HA gels with optimal CPA amounts but without intracellular trehalose incubation or addition of DMSO (Table 1). Cells survived the freeze-drying process, but cake formation was substandard with many visible droplets, suggesting partial melting. Cake formation and quality did not increase with further increases in the amount of HA and / or addition of mannitol, but cells remained viable. [Table 1-1] [Table 1-2] [Table 1-3]

[0448] conclusion Human mesenchymal stem cell CTPs still use toxic and / or immune response eliciting agents (DMSO and FBS) for cell storage. CPA, a safe and effective alternative to DMSO, has been identified for cryopreservation. Urea and glucose showed synergistic CPA activity at equimolar concentrations in cryopreservation of hMSCs. Further addition of sugars and optimization of the formulation helped improve cryopreservation. Attempts were made to improve cell viability by optimizing the formulation and freeze-drying process. Early attempts at freeze-drying showed survival and proliferation capacity of hMSCs. For freeze-drying, examples were also found that provided viable cells after freeze-drying without DMSO.

[0449] References 1. Barry, FP and JMMurphy, Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol, 2004.36(4):p.568-84. 2.Ullah, I., RBSubbarao, and GJRho, Human mesenchymal stem cells-current trends and future prospects. Biosci Rep, 2015.35(2). 3.Pittenger, MF, et al., Multilineage potential of adult human mesenchymal stem cells.Science, 1999.284(5411):p.143-7. 4. Nakamizo, A., et al., Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res, 2005. 65(8): p. 3307-18. 5. Yong, K.W., et al., Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges. Biopreserv Biobank, 2015. 13(4): p. 231-9. 6. De Miguel, M.P., et al., Immunosuppressive properties of mesenchymal stem cells: advances and applications. Curr Mol Med, 2012. 12(5): p. 574-91. 7. Yong, K.W., J.R. Choi, and W.K.Z. Wan Safwani, Biobanking of Human Mesenchymal Stem Cells: Future Strategy to Facilitate Clinical Applications, in Biobanking and Cryopreservation of Stem Cells, F. Karimi-Busheri及びM. Weinfeld, Editors. 2016, Springer International Publishing: Cham. p. 99-110. 8. Arav, A.及びJ. Saragusty, Directional freezing of sperm and associated derived technologies. Anim Reprod Sci, 2016. 169: p. 6-13. 9. Germann, A., et al., Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function. Cryobiology, 2013. 67(2): p. 193-200. 10. Stewart, S. and X. He, Intracellular Delivery of Trehalose for Cell Banking. Langmuir, 2019. 35(23): p. 7414-7422. 11. Galvao, J., et al., Unexpected low-dose toxicity of the universal solvent DMSO. FASEB J, 2014. 28(3): p. 1317-30. 12. Hanslick, J.L., et al., Dimethyl sulfoxide (DMSO) produces widespread apoptosis in the developing central nervous system. Neurobiol Dis, 2009. 34(1): p. 1-10. 13. Martino, M., et al., Fractionated infusions of cryopreserved stem cells may prevent DMSO-induced major cardiac complications in graft recipients. Haematologica, 1996. 81(1): p. 59-61. 14. Ruiz-Delgado, G.J., et al., Dimethyl sulfoxide-induced toxicity in cord blood stem cell transplantation: report of three cases and review of the literature. Acta Haematol, 2009. 122(1): p. 1-5. 15.Davies,O.G.,et al.,The effects of cryopreservation on cells isolated from adipose,bone marrow and dental pulp tissues.Cryobiology,2014.69(2):p.342-7. 16.Woodbury,D.,et al.,Adult rat and human bone marrow stromal cells differentiate into neurons.J Neurosci Res,2000.61(4):p.364-70. 17.Thirumala,S.,et al.,Evaluation of polyvinylpyrrolidone as a cryoprotectant for adipose tissue-derived adult stem cells.Tissue Eng Part C Methods,2010.16(4):p.783-92. 18.Oliver,A.E.,Dry state preservation of nucleated cells:progress and challenges.Biopreserv Biobank,2012.10(4):p.376-85. 19.Weng,L.,Technologies and Applications Toward Preservation of Cells in a Dry State for Therapies.Biopreserv Biobank,2021. 20. Izutsu, K.-i., Applications of Freezing and Freeze-Drying in Pharmaceutical Formulations, in Survival Strategies in Extreme Cold and Desiccation: Adaptation Mechanisms and Their Applications, M. Iwaya-Inoue, M. Sakurai, and M. Uemura, Editors. 2018, Springer Singapore: Singapore. p. 371-383. 21. Weng, L. and P. R. Beauchesne, Dimethyl sulfoxide-free cryopreservation for cell therapy: A review. Cryobiology, 2020. 94: p. 9-17. 22. Gomez-Fernandez, J., et al., Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation. Anim Reprod Sci, 2012. 133(1-2): p. 109-16. 23. Pollock, K., et al., Combinations of Osmolytes, Including Monosaccharides, Disaccharides, and Sugar Alcohols Act in Concert During Cryopreservation to Improve Mesenchymal Stromal Cell Survival. Tissue Eng Part C Methods, 2016. 22(11): p. 999-1008. 24. Freimark, D., et al., Systematic parameter optimization of a Me(2)SO- and serum-free cryopreservation protocol for human mesenchymal stem cells. Cryobiology, 2011. 63(2): p. 67-75. 25. Svalgaard, J.D., et al., Low-molecular-weight carbohydrate Pentaisomaltose may replace dimethyl sulfoxide as a safer cryoprotectant for cryopreservation of peripheral blood stem cells. Transfusion, 2016. 56(5): p. 1088-95. 26. Costanzo, J.P. and R.E. Lee, Jr., Urea loading enhances freezing survival and postfreeze recovery in a terrestrially hibernating frog. J Exp Biol, 2008. 211(Pt 18): p. 2969-75. 27. Costanzo, J.P. and R.E. Lee, Jr., Cryoprotection by urea in a terrestrially hibernating frog. J Exp Biol, 2005. 208(Pt 21): p. 4079-89. 28. Pan, M., et al., Urea: a comprehensive review of the clinical literature. Dermatol Online J, 2013. 19(11): p. 20392. 29.Janz Fde,L.,et al.,Evaluation of distinct freezing methods and cryoprotectants for human amniotic fluid stem cells cryopreservation.J Biomed Biotechnol,2012.2012:p.649353. 30.Bissoyi,A.,et al.,Recent Advances and Future Direction in Lyophilisation and Desiccation of Mesenchymal Stem Cells.Stem Cells Int,2016.2016:p.3604203. 31.Oliver,A.E.,et al.,Loading Human Mesenchymal Stem Cells with Trehalose by Fluid-Phase Endocytosis.Cell Preservation Technology,2004.2:p.35-49. 32.Zhang,S.Z.,et al.,Preliminary study on the freeze-drying of human bone marrow-derived mesenchymal stem cells.J Zhejiang Univ Sci B,2010.11(11):p.889-94. 33.HyStemC Product Information.2021[cited 2021 22.Apr.2021];Available from:https: / / www.sigmaaldrich.com / content / dam / sigma-aldrich / docs / Sigma / Bulletin / hysc020bul.pdf.

Claims

1. A composition comprising an aggregate of viable cells, an aqueous component, urea, and a monosaccharide, wherein the concentration of the monosaccharide is from about 0.4 M to about 2.0 M.

2. The composition further comprises a bulking agent and / or The composition further comprises a hyaluronic acid gel, the composition according to claim 1.

3. The concentration of the monosaccharide is from about 0.6 M to about 2.0 M, or from about 0.7 M to about 1.7 M, or from about 0.8 M to about 1.4 M, or from about 0.4 M to about 1.25 M, or from about 0.4 M to about 0.8 M, or from about 0.4 M to about 0.6 M, and / or The concentration of the urea is from about 0.7 M to about 1.7 M, or from about 0.8 M to about 1.4 M, or from about 0.2 M to about 1.25 M, or from about 0.2 M to about 0.8 M, or from about 0.4 M to about 0.6 M, or from about 0.6 M to about 2.0 M, and / or The molar ratio of the urea to the monosaccharide in the composition is from about 5:1 to about 1:5, or from about 1:3 to about 3:1, or from about 1:2 to about 2:1, or about 1:1, the composition according to claim 1.

4. The monosaccharide is glucose, fructose, or galactose, and / or The bulking agent is a) a disaccharide, wherein the disaccharide is sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, lactulose, isomaltose melibiose or gentiobiose, the disaccharide, or b) a sugar alcohol, wherein the sugar alcohol is mannitol, sorbitol, galactitol, fucitol, iditol, or inositol, the sugar alcohol, or c) both a disaccharide and a sugar alcohol, or d) both sucrose and mannitol, the composition according to claim 1.

5. The monosaccharide is glucose, the bulking agent is sucrose and optionally mannitol and / or the concentration of the urea is from about 0.7 M to about 1.7 M, the concentration of the monosaccharide, preferably glucose, is from about 0.6 M to about 2.0 M, and the concentration of the sucrose is from about 0.1 M to about 0.5 M, the composition according to claim 2.

6. The composition further comprises DMSO at a concentration of from about 1% to about 5% before freezing, the concentration of the urea is from about 0.7 M to about 1.7 M, the concentration of the glucose is from about 0.6 M to about 2.0 M, the concentration of the sucrose is from about 0.1 M to about 0.5 M; or the composition further comprises mannitol, The concentration of the urea is from about 0.7 M to about 1.7 M, the concentration of the glucose is from about 0.6 M to about 2.0 M, the concentration of the sucrose is from about 0.1 M to about 0.5 M, the concentration of the mannitol before freezing is from about 0.3 M to about 0.8 M, the composition according to claim 5.

7. the composition further comprises mannitol, the concentration of the urea is from about 0.7 M to about 1.7 M, the concentration of the glucose is from about 0.6 M to about 2.0 M, the concentration of the sucrose is from about 0.1 M to about 0.5 M, the concentration of the mannitol before freezing is from about 0.1 M to about 1.0 M, the composition according to claim 5.

8. the composition does not contain DMSO, or the composition contains DMSO at a concentration of about 1% to about 10% before freezing, or the composition contains DMSO at a concentration of about 1% to about 5% before freezing, the composition according to claim 1.

9. the aggregate of cells contains mammalian cells, the composition according to claim 1.

10. the composition is in a frozen state at a temperature of about -10°C to about -100°C, or about -20°C to about -90°C, or about -40°C to about -60°C, and / or the composition is a lyophilized composition, and / or the composition contains less than about 90% (volume / volume) of water, the composition according to claim 1.

11. A method for producing an aggregate of frozen cells, comprising: (a) freezing the composition according to any one of claims 1 to 9 to produce the aggregate of frozen cells, step (a).

12. A method for producing an aggregate of reconstituted viable cells, comprising: (a) freezing the composition according to any one of claims 1 to 9 to produce an aggregate of frozen cells, step (a); and (b) resuspending the aggregate of frozen cells in a reconstituting agent to form a reconstituted composition, step (b), wherein at least about 1% of the cells are viable.

13. A method for producing an aggregate of lyophilized cells, comprising: (a) freezing the composition according to any one of claims 1 to 9, step (a); and (b) removing at least about 10% (volume / volume) of water from the frozen composition to produce the aggregate of lyophilized cells, step (b).

14. A method for producing an aggregate of reconstituted viable cells, Step (a) of freezing the composition according to any one of claims 1 to 9; Step (b) of removing at least about 10% (volume / volume) of water from the frozen composition to produce an aggregate of lyophilized cells; Step (c) of resuspending the aggregate of lyophilized cells in a reconstituting agent to form a reconstituted composition, wherein at least about 1% of the cells are viable, method.

15. The method according to claim 11, wherein prior to said freezing in step (a), the aggregate of cells is isolated and contacted with a poration solution.

16. The method according to claim 15, wherein the poration solution contains trehalose, preferably the concentration of trehalose in the poration solution is from about 0.1 M to about 1.0 M, or from about 0.1 M to about 0.6 M.

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