Spheroid cryopreservation

Abstract

The disclosure relates to a cryopreservation medium comprising a diol, sugar, or both a diol and sugar. The disclosure also provides a use of the cryopreservation medium to cryopreserve a cell, a method of preparing a cell for preservation, and a method of cryopreserving a cell.

Description

Attorney Docket No. 124324-5003-WOSPHEROID CRYOPRESERVATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U. S. Provisional Application Serial No. 63 / 721,058 filed on November 15, 2024, the entire disclosure of which is incorporated herein by reference.BACKGROUND

[0002] Traditional slow cooling cryopreservation techniques used for cultured cells tend to not work well for multicellular models such as spheroids and organoids because cryoprotectants (typically 10% dimethylsulfoxide, DMSO) do not penetrate reliably to the core of such three- dimensional cellular structures. As a result, the cryoprotection at the center of the spheroid is poor, which results in a large necrotic core of spheroids or organoids after cry opreservation and thawing. If DMSO equilibration time is increased or if the starting concentration of DMSO is increased to improve final DMSO at the core of the spheroid upon cryopreservation, DMSO toxicity' is increased at the outer layer.SUMMARY

[0003] The disclosure relates to a cry opreservation medium comprising a diol, sugar, or both a diol and sugar. The disclosure also provides a use of the cryopreservation medium to cry opreserve a cell, a method of preparing a cell for preservation, and a method of cryopreservmg a cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 shows cryopreserved HepG2 spheroids having a diameter of 52.83 ± 4.06 pm (top left), 78.87 ± 6.73 pm (top right), 106.71 ± 7.92 pm (bottom left), and 410.83 ± 3.92 pm (bottom right).

[0005] Figure 2 shows cell count per spheroid versus spheroid size (pm). The starting cell density per well is 12.5 × 104Total cell count, red solid line; viable cell count, blue dashed line.

[0006] Figure 3 shows HepG2 spheroids growing in the well plate.1DBl / 164222938.1Attorney Docket No. 124324-5003-WO

[0007] Figure 4 shows fluorescence images of the HepG2 spheroids grown in 3D plate well post-thaw. Live-dead fluorescent staining was performed where the bright red spots indicate the cells with compromised membrane integrity possibly due to necrosis triggered by the diffusion limitations at the core.

[0008] Figure 5 shows HepG2 spheroids before cry opreservation, just after thawing, and after growing in media for 3-4 days. The successful survival of the spheroids were demonstrated by the bioactivity post thaw in culture conditions as indicated by merging of the individual spheroids into a bigger structure. This demonstrates successful cryopreservation of spheroids after thawing.

[0009] Figure 6A shows an example of successful cry opreservation of spheroids with smaller necrotic core (colored red using propidium iodide).

[0010] Figure 6B shows an example of a failed cry opreservation of spheroids. The failed condition is a result of using only 5% DMSO as a negative control.

[0011] Figure 7 shows characterization of the growth of spheroids in a successful example.

[0012] Figure 8 show’s characterization of the growth of spheroids in a successful example.

[0013] Figure 9 shows characterization of the growth of spheroids in an unsuccessful example.

[0014] Figure 10 shows fusion assays demonstrating the superior performance of post¬ cry opr eservati on functionality and viability’ of the spheroids cryopreserved with thecry opreservation medium (Experimental Condition) compared to cryopreservation in cell culture media (Negative Control 1) and 5% DMSO (Negative Control 2).DETAILED DESCRIPTION

[0015] In one aspect, the disclosure provides a cry opreservation medium.

[0016] In some embodiments, the cry opreservation medium provided herein comprises an alcohol, e.g., an alcohol, a diol, a triol, or mixtures thereof. In some embodiments, the alcohol comprises methanol, ethanol, 1 -propanol, isopropyl alcohol, ethanediol, propane- 1,2-diol, 1,3- propanediol, 1,4- butanediol, 1,3-butanediol, 2,3-butanediol, glycerol, or mixtures thereof. In some embodiments, the cryopreservation medium comprises a diol, e.g., ethanediol, propane- 2DBl / 164222938.1Attorney Docket No. 124324-5003-WO1,2-diol, 1,3-propanediol, 1,4-butanediol, 1,3 -butanediol, 2, 3-butanediol, or mixtures thereof. In some embodiments, the diol comprises propanediol, e.g., propane- 1,2-diol, 1,3-propanediol, or mixtures thereof. In some embodiments, the diol comprises propane- 1,2-diol. In some embodiments, the diol comprises 1,3 -propanediol.

[0017] In some embodiments, a concentration of alcohol (e.g., an alcohol, a diol, a triol, or mixtures thereof) in the cry opreservation medium is at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 wt%. In some embodiments, a concentration of alcohol (e.g., an alcohol, a diol, a triol, or mixtures thereof) in the cry opreservation medium is less than or equal to about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 wt%. In some embodiments, a concentration of alcohol (e.g., an alcohol, a diol, a triol, or mixtures thereof) in the cryopreservation medium is from about 0.1 to 20 wt%, from about 0.5 to 20 wt%, from about 1 to 20 wt%, from about 2 to 20 wt%, from about 3 to 20 wt%, from about 4 to 20 wt%, from about 5 to 20 wt%, from about 6 to 20 wt%, from about 7 to 20 wt%, from about 8 to 20 wt%, from about 9 to 20 wt%, from about 10 to 20 wt%, from about 15 to 20 wt%, from about 0.1 to 15 wt%, from about 0.5 to 15 wt%, from about 1 to 15 wt%, from about 2 to 15 wt%, from about 3 to 15 wt%, from about 4 to 15 wt%, from about 5 to 15 wt%, from about 6 to 15 wt%, from about 7 to 15 wt%, from about 8 to 15 wt%, from about 9 to 15 wt%, from about 10 to 15 wt%, from about 0.1 to 10 wt%, from about 0.5 to 10 wt%, from about 1 to 10 wt%, from about 2 to 10 wt%, from about 3 to 10 wt%, from about 4 to 10 wt%, from about 5 to 10 wt%, from about 6 to 10 wt%, from about 7 to 10 wt%, from about 8 to 10 wt%, from about 0.1 to 5 wt%, from about 0.5 to 5 wt%, from about 1 to 5 wt%, from about 2 to 5 wt%, from about 3 to 5 wt%, from about 4 to 5 wt%, from about 4 to 6 wt%, from about 3 to 7 wt%, from about 2 to 8 wt%, or from about 1 to 9 wt%. In some embodiments, a concentration of alcohol (e.g., an alcohol, a diol, a triol, or mixtures thereof) in the cryopreservation medium is from about 1 to 10 wt%. In some embodiments, a concentration of diol in the cry opreservation medium is from about 1 to 10 wt%. In some embodiments, a concentration of propanediol in the cryopreservation medium is from about 1 to 10 wt%.

[0018] As used herein, the term “about” means modifying, for example, lengths of nucleotide sequences, degrees of errors, dimensions, the quantity of an ingredient in a composition, concentrations, volumes, densities, diameters, sizes, process temperature, process time, yields, flow rates, pressures, and like values, and ranges thereof, refers to variation in the numerical 3DBl / 164222938.1Attorney Docket No. 124324-5003-WOquantity that may occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term “about” also encompasses amounts that differ due to aging of, for example, a composition, formulation, or cell culture with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture. Whether modified by the term “about” the claims appended hereto include equivalents to these quantities. The term “about” further may refer to a range of values that are similar to the stated reference value. In certain embodiments, the term “about” refers to a range of values that fall within 50, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 percent or less of the stated reference value. In certain embodiments, the term “about,” when used in connection with pH values of a solution, refers to a range of ±0.5, 0.4, 0.3, 0.2, or 0.1.

[0019] In some embodiments, the cryopreservation medium provided herein comprises a sugar. Any suitable sugar can be used in the cryopreservation medium provided herein. The sugar may comprise a sugar alcohol, a sugar acid, a carbohydrate, a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide, or derivatives thereof. In some embodiments, the sugar comprises dextrose, dextrin, fructose, galactose, glucose, lactose, trehalose, maltose, sucrose, erythritol, maltitol, sorbitol, mannitol, xylitol, lactitol, isomalt, N-acetylglucosamine, hydrogenated starch hydrolysates, or mixtures thereof. In some embodiments, the sugar comprises trehalose.

[0020] In some embodiments, a concentration of sugar in the cryopreservation medium is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM. In some embodiments, a concentration of sugar in the cry opreservation medium is less than or equal to about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 mM. In some embodiments, a concentration of sugar in the cry opreservation medium is from about 1 to 100 mM, from about 1 to 90 mM, from about 1 to 80 mM, from about 1 to 70 mM, from about 1 to 60 mM, from about 1 to 50 mM, from about 1 to 40 mM, from about 1 to 30 mM, from about 1 to 20 mM, from about 1 to 10 mM, from about 5 to 100 mM, from about 5 to 90 mM, from about 5 to 80 mM, from about 5 to 70 mM, from about 5 to 60 mM, from about 5 to 50 mM, from about 5 to 404DBl / 164222938.1Attorney Docket No. 124324-5003-WOmM, from about 5 to 30 mM, from about 5 to 20 mM, from about 5 to 10 mM, from about 10 to 100 mM, from about 10 to 90 mM, from about 10 to 80 mM, from about 10 to 70 mM, from about 10 to 60 mM, from about 10 to 50 mM, from about 10 to 40 mM, from about 10 to 30 mM, from about 10 to 20 mM, from about 20 to 100 mM, from about 20 to 90 mM, from about 20 to 80 mM, from about 20 to 70 mM, from about 20 to 60 mM, from about 20 to 50 mM, from about 20 to 40 mM, from about 20 to 30 mM, from about 30 to 100 mM, from about 30 to 90 mM, from about 30 to 80 mM, from about 30 to 70 mM, from about 30 to 60 mM, from about 30 to 50 mM, from about 30 to 40 mM, from about 40 to 100 mM, from about 40 to 90 mM, from about 40 to 80 mM, from about 40 to 70 mM, from about 40 to 60 mM, from about 40 to 50 mM, from about 50 to 100 mM, from about 50 to 90 mM, from about 50 to 80 mM, from about 50 to 70 mM, or from about 50 to 60 mM, In some embodiments, a concentration of sugar in the cryopreservation medium is from about 5 to 50 mM. In some embodiments, a concentration of trehalose in the cry opreservation medium is from about 5 to 50 mM.

[0021] In some embodiments, the cryopreservation medium provided herein comprises an alcohol and a sugar. In some embodiments, the cryopreservation medium provided herein comprises a diol and a sugar. In some embodiments, the cry opreservation medium provided herein comprises propanediol and a sugar. In some embodiments, the cry opreservation medium provided herein comprises an alcohol and trehalose. In some embodiments, the cry opreservation medium provided herein comprises a diol and trehalose. In some embodiments, thecry opreservation medium provided herein comprises propanediol and trehalose.

[0022] In some embodiments, the cryopreservation medium provided herein comprises an organic solvent. Any suitable sugar can be used in the cryopreservation medium provided herein. In some embodiments, the organic solvent comprises dimethyl sulfoxide ( DMSO), acetone, acetonitrile, methoxy ethanol, butanone, dimethyl formamide (DMF), polyethylene glycol (PEG), dichlorom ethane, tetrahydrofuran, ethyl acetate, hexamethylphosphoric triamide, or mixtures thereof. In some embodiments, the cry opreservation medium comprises DMSO.

[0023] In some embodiments, a concentration of organic solvent in the cryopreservation medium is at least about 0.1, 0.5, I, 2, 3, 4, 5, 6, 7, 8, 9, or 10 wt%. In some embodiments, a concentration of organic solvent in the cryopreservation medium is less than or equal to about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 wt%. In some embodiments, a 5DBl / 164222938.1Attorney Docket No. 124324-5003-WOconcentration of organic solvent in the cryopreservation medium is from about 0.1 to 20 wt%, from about 0.5 to 20 wt%, from about 1 to 20 wt%, from about 2 to 20 wt%, from about 3 to 20 wt%, from about 4 to 20 wt%, from about 5 to 20 wt%, from about 6 to 20 wt%, from about 7 to 20 wt%, from about 8 to 20 wt%, from about 9 to 20 wt%, from about 10 to 20 wt%, from about 15 to 20 wt%, from about 0.1 to 15 wt%, from about 0.5 to 15 wt%, from about 1 to 15 wt%, from about 2 to 15 wt%, from about 3 to 15 wt%, from about 4 to 15 wt%, from about 5 to 15 wt%, from about 6 to 15 wt%, from about 7 to 15 wt%, from about 8 to 15 wt%, from about 9 to 15 wt%, from about 10 to 15 wt%, from about 0.1 to 10 wt%, from about 0.5 to 10 wt%, from about 1 to 10 wt%, from about 2 to 10 wt%, from about 3 to 10 wt%, from about 4 to 10 wt%, from about 5 to 10 wt%, from about 6 to 10 wt%, from about 7 to 10 wt%, from about 8 to 10 wt%, from about 0.1 to 5 wt%, from about 0.5 to 5 wt%, from about 1 to 5 wt%, from about 2 to 5 wt%, from about 3 to 5 wt%, from about 4 to 5 wt%, from about 4 to 6 wt%, from about 3 to 7 wt%, from about 2 to 8 wt%, or from about 1 to 9 wt%. In some embodiments, a concentration of organic solvent in the cryopreservation medium is from about 1 to 10 wt%. In some embodiments, a concentration of DMSO in the cryopreservation medium is from about 1 to 10 wt%.

[0024] In some embodiments, the cryopreservation medium provided herein comprises a cell culture medium. In some embodiments, the cell culture medium comprises Eagle’s Minimum Essential Medium, Dulbecco’s Modified Eagle Medium (DMEM), DMEM / F-12, Roswell Park Memorial Institute (RPMI) 1640 Medium, human embryonic stem cell medium (hESC medium), Ham's F12 Medium, KnockOut™ Serum Replacer (KOSR), Minimum Essential Medium Eagle¬ alpha modification (Alpha MEM), Knockout DMEM (KO-DMEM), N-2, MS-5 stromal cell culture medium, StemSpan™ Serum-Free Expansion Medium (SFEM), Basal Medium Eagle (BME), Glasgow's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco’s Medium, or the like. In some embodiments, the cry opreservation medium provided herein comprises Eagle’s Minimum Essential Medium.

[0025] In some embodiments, the cryopreservation medium provided herein comprises a growth supplement. In some embodiments, the growth supplement is fetal bovine serum (FBS), bovine calf serum, newborn calf serum, adult bovine serum, donor bovine serum, horse serum, rabbit serum, chicken serum, goat serum, sheep serum, lamp serum, porcine serum, bovine ocular fluid,6DBl / 164222938.1Attorney Docket No. 124324-5003-WOsericin protein, human platelet lysate, or earthworm heat inactivated coelomic fluid. In some embodiments, the cry opreservation medium provided herein comprises fetal bovine serum.

[0026] In some embodiments, a concentration of the growth supplement in the cell culture medium or cryopreservation medium is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 v / v%. In some embodiments, a concentration of the growth supplement in the cell culture medium or cry opreservation medium is less than or equal to about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 v / v%. In some embodiments, a concentration of the growth supplement in the cell culture medium or cryopreservation medium is from about 1 to 20 v / v%, from about 2 to 20 v / v%, from about 3 to 20 v / v%, from about 4 to 20 v / v%, from about 5 to 20 v / v%, from about 6 to 20 v / v%, from about 7 to 20 v / v%, from about 8 to 20 v / v%, from about 9 to 20 v / v%, from about 10 to 20 v / v%, from about 15 to 20 v / v%, from about 1 to 15 v / v%, from about 2 to 15 v / v%, from about 3 to 15 v / 'v%, from about 4 to 15 v / v%, from about 5 to 15 v / v%, from about 6 to 15 v / v%, from about 7 to 15 v / v%, from about 8 to 15 v / v%, from about 9 to 15 v / v%, from about 10 to 15 v / v%, from about 1 to 10 v / v%, from about 2 to 10 v / v%, from about 3 to 10 v / v%, from about 4 to 10 v / v%, from about 5 to 10 v / v%, from about 6 to 10 v / v%, from about 7 to 10 v / v%, from about 8 to 10 v / v%, or from about 1 to 5 v / v%. In some embodiments, a concentration of the growth supplement (e.g., FBS) in the cell culture medium (e.g., Eagle’s Minimum Essential Medium) is from about 1 to 20 v / v%. In some embodiments, a concentration of the growth supplement (e.g., FBS) in the cell culture medium (e.g.. Eagle’s Minimum Essential Medium) is from about 5 to 15 v / v%. In some embodiments, a concentration of the growth supplement (e.g., FBS) in the cry opreservation medium is from about 1 to 20 v / v%. In some embodiments, a concentration of the growth supplement (e.g., FBS) in the cry opreservation medium is from about 5 to 15 v / v%.

[0027] In some embodiments, the cryopreservation medium provided herein comprises at least one of an alcohol, a sugar, an organic solvent, a growth supplement, and a cell culture medium. In some embodiments, the cry opreservation medium provided herein comprises at least one of propanediol, trehalose, DMSO, FBS, and Eagle’s Minimum Essential Medium. In some embodiments, the cry opreservation medium provided herein comprises an alcohol, a sugar, an organic solvent, a growth supplement, and a cell culture medium. In some embodiments, the7DBl / 164222938.1Attorney Docket No. 124324-5003-WOcryopreservation medium provided herein comprises propanediol, trehalose, DMSO, FBS, and Eagle’s Minimum Essential Medium.

[0028] In one aspect, the cryopreservation medium provided herein is used to cryopreserve a cell. As used herein, "a cell" refers to a single cell, multiple cells, or a cell cluster.

[0029] In some embodiments, the cell comprises a mammalian cell. In some embodiments, the cell comprises a human cell. In some embodiments, the cell comprises an animal or non-human cell.

[0030] Exemplary cell types include, but are not limited to, a germ cell such as spermatozoa or ova; a somatic cell; a stem cell (e.g., pluripotent stem cell, etc.); a precursor cell; a cancer cell; an immortalized cell (cell line); a cell separated from a living body and artificially gene-modified; a cell separated from a living body and the nucleus of which is artificially exchanged, etc.

[0031] In some embodiments, the cell comprises a somatic cell. Exemplary somatic cells include, but are not limited to, fibroblast, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, macrophages, monocytes, bone cells, bone marrow cells, perithelial cells, dendritic cells, keratinocytes, fat cells, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatic parenchymal cells, cartilage cells, cumulus cells, neural cells, glial cells, neurons, oligodendrocyte, microglia, astroglial cells, heart cells, esophagus cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic precursor cells (e.g., CD34 positive cells derived from cord blood), and mononuclear cells, etc. In some embodiments, the somatic cells include cells collected from a tissue such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including cord blood), bone marrow, heart, eye, brain, or nerve tissue, etc,

[0032] In some embodiments, the cell comprises a stem cell. Exemplary stem cells include, but are not limited to, embryonic stem cells, embryonic tumor cells, embryonic germline stem cells, induced pluripotent stem cells (iPS cell), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, etc.8DBl / 164222938.1Attorney Docket No. 124324-5003-WO

[0033] In some embodiments, the cell comprises a precursor cell. Exemplary precursor cells are cells in a halfway stage of differentiating from the above- referenced stem cells to specific somatic cells or germ cells.

[0034] In some embodiments, the cell comprises a cancer cell. Exemplary cancer cells comprise cells from cell lines include, but are not limited to, human breast cancer cell lines such as HBC- 4, BSY-1, BSY-2, MCF-7, MCF-7 / ADR RES, HS578T, MDA-MB-231, MDA-MB-435, MDA- N, BT-549 and T47D; human cervical cancer cell lines such as HeLa; human lung cancer cell lines such as A549, EKVX, HOP-62, HOP-92, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, DMS273 and DMS114; human colorectal cancer cell lines such as Caco-2, COLO- 205, DLD-1, HCC-2998, HCT-15, HCT-116, HT-29, KM- 12, SW-620 and WiDr; human prostate cancer cell lines such as DU- 145, PC-3 and LNCaP; human central nervous system cancer cell lines such as U251, SF-295, SF-539, SF-268, SNB-75, SNB-78 and SNB-19; human ovarian cancer cell lines such as OVCAR-3, OVCAR-4, O VC AR- 5, OVCAR-8, SK-OV-3 and IGROV-1; human kidney cancer cell lines such as RXF-631L, ACHN, UO- 1, SN-12C, A498, CAKI-1, RXF-393L, 786-0 and TK-10; human stomach cancer cell lines such as MKN45, MKN28, St-4, MKN-1, MKN-7 and MKN-74; skin cancer cell lines such as LOX-IMVI, LOX, MALME-3M, SK-MEL-2, SK- MEL-5, SK-MEL-28, U ACC-62, UACC-257 and Ml 4; leukemia cell lines such as CCRF-CRM, K562, MOLT-4, HL-60 TB, RPMI8226, SR, UT7 / TPO and Jurkat; etc.

[0035] In some embodiments, the cell comprises a liver cell. Exemplary cells from liver cell lines include, but are not limited, HepG2, Hep3B, HepaRG™, JHH7, HLF, HLE, PLC / PRF / 5, WRL68, HB611, SK-HEP-1, Huh-4, Huh-7, etc.

[0036] Other exemplary cells from cell lines include, but are not limited to, HEK293 (human embryonal kidney cells), MDCK, MDBK, BHK, C-33A, AE-1, 3D9, Ns0 / l, NIH3T3, PC12, S2, SD, S 19 and Sf21.

[0037] In some embodiments, the cell comprises an immortalized human hepatoma cell (e.g., HepG2 cell). In some embodiments, the cell comprises an immortalized human embryonic kidney cell (e.g., HEK293 cell). In some embodiments, the cell comprises a colorectal adenocarcinoma cell (e.g., DLD-1 cell). In some embodiments, the cell comprises an9DBl / 164222938.1Attorney Docket No. 124324-5003-WOimmortalized human colorectal carcinoma cell (e g., HCT116 cell). In some embodiments, the cell is selected from the group consisting of a HepG2 cell, a HEK293 cell, a DLD-1 cell, and an HCT116 cell.

[0038] In some embodiments, the cell comprises a spheroid. As used herein, “spheroid” refers to a three-dimensional sphere-like aggregate of cells (i.e., at least about 40 cells) having proper compaction and cell-cell junctions developed, and having dimensions that exceed the diameter of any individual cell in each of the three dimensions of the aggregate.

[0039] In some embodiments, the cell comprises a spheroid having a diameter of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 pm. In some embodiments, the cell comprises a spheroid having a diameter of at least about 50 um. In some embodiments, the cell comprises a spheroid having a diameter of at least about 80 pm.

[0040] In some embodiments, the cell comprises a spheroid having a diameter of less than or equal to about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, or 120 pm. In some embodiments, the cell comprises a spheroid having a diameter of less than or equal to about 200 pm. In some embodiments, the cell comprises a spheroid having a diameter of less than or equal to about 120 pm.

[0041] In some embodiments, the cell comprises a spheroid having a diameter from about 10 to 1000 pm, from about 10 to 500 pm, from about 10 to 400 pm, from about 10 to 300 pm, from about 10 to 200 pm, from about 10 to 100 pm, from about 50 to 1000 pm, from about 50 to 500 pm, from about 50 to 400 pm, from about 50 to 300 pm, from about 50 to 200 pm, from about 50 to 120 pm, from about 50 to 100 pm, from about 80 to 1000 pm, from about 80 to 500 pm, from about 80 to 400 pm, from about 80 to 300 pm, from about 80 to 200 pm, from about 80 to 120 pm, from about 80 to 100 pm, from about 100 to 1000 pm, from about 100 to 500 pm, from about 100 to 400 pm, from about 100 to 300 pm, from about 100 to 200 pm, or from about 100 to 120 pm. In some embodiments, the cell comprises a spheroid having a diameter from about 10 to 1000 pm. In some embodiments, the cell comprises a spheroid having a diameter from about 80 to 200 pm. In some embodiments, the cell comprises a spheroid having a diameter from about 80 to 120 pm.10DBl / 164222938.1Attorney Docket No. 124324-5003-WO

[0042] In one aspect, the disclosure provides a method of preparing a cell (e.g., spheroid) for preservation. As used herein, definition of the term “preservation” or “preserving” as it relates to the disclosure is understood by one of skill in the art. In some embodiments, preserving relates to a process of maintaining the activity, integrity, and / or viability of a cell (e.g., a spheroid).

[0043] In some embodiments, the preservation is cryopreservation. As used herein, definition of the term “cryopreservation” or “cryopreserving” is understood by one of skill in the art. In some embodiments, cryopreservation relates to preserving a cell (e.g., spheroid) at thecry opreservation temperature and / or under vapor phase liquid nitrogen.

[0044] In some embodiments, the method of preparing a cell (e.g., spheroid) for preservation comprises incubating the cell at a temperature above about 0 °C in the cry opreservation medium described herein.

[0045] In some embodiments, the method of preparing a cell (e.g., spheroid) for preservation comprises incubating the cell at a temperature of at least about 1, 5, 10, 15, 20, 25, 30, 35, 36, 37 38, 39, or 40 °C. In some embodiments, the method of preparing a cell (e.g., spheroid) for preservation comprises incubating the cell at a temperature of less than or equal to about 50, 40, 39, 38, or 37 °C. In some embodiments, the temperature is from about 5 to 40 °C, from about 5 to 39 °C, from about 5 to 38 °C, from about 5 to 37 °C, from about 5 to 36 °C, from about 5 to 35 °C. from about 5 to 30 °C. from about 5 to 25 °C. from about 5 to 20 °C. from about 5 to 15 °C, from about 5 to 10 °C, from about 10 to 40 °C, from about 10 to 39 °C, from about 10 to 38 °C, from about 10 to 37 °C, from about 10 to 36 °C, from about 10 to 35 °C, from about 10 to 30 °C, from about 10 to 25 °C, from about 10 to 20 °C, from about 15 to 40 °C, from about 15 to 39 °C, from about 15 to 38 °C, from about 15 to 37 °C, from about 15 to 36 °C, from about 15 to 35 °C, from about 15 to 30 °C, from about 15 to 25 °C, from about 15 to 20 °C, from about 20 to 40 °C, from about 20 to 39 °C, from about 20 to 38 °C, from about 20 to 37 °C, from about 20 to 36 °C, from about 20 to 35 °C, from about 20 to 30 °C, from about 20 to 25 °C, from about 25 to 40 °C, from about 25 to 39 °C, from about 25 to 38 °C, from about 25 to 37 °C, from about 25 to 36 °C, from about 25 to 35 °C, from about 25 to 30 °C, from about 30 to 40 °C, from about 30 to 39 °C, from about 30 to 38 °C, from about 30 to 37 °C, from about 30 to 36 °C, from about 30 to 35 °C, from about 35 to 40 °C, from about 35 to 39 °C, from about 35 to 38 °C, from about 35 to 37DBl / 164222938.1Attorney Docket No. 124324-5003-WO°C, from about 35 to 36 °C, from about 36 to 40 °C, from about 36 to 39 °C, from about 36 to 38 °C, or from about 36 to 37 °C.

[0046] In some embodiments, the method provided herein further comprises changing the temperature to a cryogenic temperature. In some embodiments, the cryogenic temperature is less than or equal to about 0, -10, -20, -30, -40, -50, -60, -70, -80, or -90 °C. In some embodiments, the cryogenic temperature is greater than or equal to about -200, -150 or -100 °C. In some embodiments, the cryogenic temperature is from about 0 to -200 °C, from about -10 to -200 °C, from about -20 to -200 °C, from about -30 to -200 °C, from about -40 to -200 °C, from about -50 to -200 °C, from about -60 to -200 °C, from about -70 to -200 °C, from about -80 to -200 °C, from about -90 to -200 °C, from about -100 to -200 °C, from about 0 to -150 °C, from about -10 to -150 °C, from about -20 to -150 °C, from about -30 to -150 °C, from about -40 to -150 °C, from about -50 to -150 °C, from about -60 to -150 °C, from about -70 to -150 °C, from about -80 to -150 °C, from about -90 to -150 °C, from about -150 to -200 °C, from about 0 to -100 °C, from about -10 to -100 °C, from about -20 to -100 °C, from about -30 to -100 °C, from about -40 to -100 °C, from about -50 to -100 °C, from about -60 to -100 °C, from about -70 to -100 °C, from about -80 to -100 °C, or from about -90 to -100 °C. In some embodiments, the cryogenic temperature is lower than -50 °C.

[0047] In some embodiments, the temperature is changed at a rate of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, or 2.5 °C / minute. In some embodiments, the temperature is changed at a rate of less than or equal to about 5, 4.5, 4, 3.5, 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1 °C / minute. In some embodiments, the temperature is changed at a rate from about 0.1 to 5 °C / minute, from about 0.2 to 5 °C / minute, from about 0.3 to 5 °C / minute, from about 0.4 to 5 °C / minute, from about 0.5 to 5 °C / minute, from about 0.6 to 5 °C / minute, from about 0.7 to 5 °C / minute, from about 0.8 to 5 °C / minute, from about 0.9 to 5 °C / minute, from about 1 to 5 °C / minute, from about 0.1 to 2.5 °C / minute, from about 0.2 to 2.5 °C / minute, from about 0.3 to 2.5 °C / minute, from about 0.4 to 2.5 °C / minute, from about 0.5 to 2.5 °C / minute, from about 0.6 to 2.5 °C / minute, from about 0.7 to 2.5 °C / minute, from about 0.8 to 2.5 °C / minute, from about 0.9 to 2.5 °C / minute, from about 1 to 2.5 °C / minute, from about 0.1 to 2 °C / minute, from about 0.2 to 2 °C / minute, from about 0.3 to 2 °C / minute, from about 0.4 to 2 °C / minute, from about 0.512DBl / 164222938.1Attorney Docket No. 124324-5003-WOto 2 °C / minute, from about 0.6 to 2 °C / minute, from about 0.7 to 2 °C / minute, from about 0.8 to 2 °C / minute, from about 0.9 to 2 °C / minute, from about 1 to 2 °C / minute, from about 0.1 to 1.5 °C / minute, from about 0.2 to 1.5 °C / minute, from about 0.3 to 1.5 °C / minute, from about 0.4 to 1.5 °C / minute, from about 0.5 to 1.5 °C / minute, from about 0.6 to 1.5 °C / minute, from about 0.7 to 1.5 °C / minute, from about 0.8 to 1.5 °C / minute, from about 0.9 to 1.5 °C / minute, from about 1 to 1.5 °C / minute, from about 0.1 to 1 °C / minute, from about 0.2 to 1 °C / minute, from about 0.3 to 1 °C / minute, from about 0.4 to 1 °C / minute, from about 0.5 to 1 °C / minute, from about 0.6 to 1 °C / minute, from about 0.7 to 1 °C / minute, from about 0.8 to 1 °C / minute, or from about 0.9 to 1 °C / minute. In some embodiments, the temperature is changed at a rate from 0.2 °C / minute to 2.5 °C / minute.

[0048] In some embodiments, the cell (e.g., spheroid) is incubated in a well plate. In some embodiments, the well plate comprises a multi-well plate (e.g., a 4-well plate, a 6-well plate, an 8-well plate, a 12-well plate, a 16-well plate, a 24-well plate, a 36-well plate, a 48-well plate, a 60-well plate, a 96-well plate, or a 384-well plate). The multi-well plates may be comprised of a polymer such as polycarbonate, polyethylene, polyester, polypropylene, polystyrene, a cycloolefin polymer, a vinyl polymer, or any blends, derivatives, or copolymers thereof. In some embodiments, the well plate comprises a 96-well plate.

[0049] In some embodiments, the cell (e.g., spheroid) is incubated on a bottom of a well (e.g., a well in a 96-well plate) having a flat shape. In some embodiments, the cell (e.g., spheroid) is incubated on a botom of a well (e.g., a well in a 96-well plate) having a cone shape. In some embodiments, the cell (e.g., spheroid) is incubated on a bottom of a tube.

[0050] In one aspect, the disclosure provides a method of cryopreservmg a cell (e.g., spheroid). In some embodiments, the method comprises preparing the cell according to the method as disclosed herein, and preserving the cell in the cryogenic temperature.

[0051] In some embodiments, the method comprises preserving the cell for at least about 1 week, at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least13DBl / 164222938.1Attorney Docket No. 124324-5003-WOabout 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 25 years, at least about 50 years, at least about 75 years, at least about 100 years, at least about 500 years, or at least about 1,000 years.

[0052] In some embodiments, the method comprises thawing the cells.

[0053] In one aspect, the disclosure provides a cell prepared according to the method as provided herein. In one aspect, the disclosure provides a spheroid prepared according to the method as provided herein.

[0054] In one aspect, the use of the product or method is characterized by one or more elements disclosed in the application.

[0055] The term “and / or” used herein is defined to indicate any combination of the components. Moreover, the singular forms “a,” “an,” and “the” may further include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a sample” refers to one, more than one, or mixtures of such samples, and reference to “a method” may include reference to equivalent steps and methods known to those skilled in the art, and so forth.EXAMPLESExample 1: Spheroid Cryopreservation

[0056] Cell line derived HepG2 (HB-8065) spheroids (80-200 pm diameter) were cryopreserved using plate freezing with a cry opreservation medium comprising: Eagle’s Minimum Essential Medium (EMEM) + 10% fetal bovine serum (FBS), 5% propane- 1,2-diol, 5% DMSO, and 50 mM trehalose.

[0057] Generally, in the plate freezing method 96 well plates containing the cryopreservation medium were directly placed in controlled rate freezers to cool at approximately 1 °C / minute until the cryogenic temperature was reached, then the plates were maintained in vapor phase liquid nitrogen. This method offers the convenience of direct plating and culture on tliaw’ed plates.

[0058] Specifically, HepG2 spheroids were created in 96 well plates in which the bottom of each well has a cone shape to facilitate the creation of the spheroids (defined herein as “3D 96- well plates”). The HepG2 spheroids were grown to about 100 μm diameter, then cryopreserved in the 14DBl / 164222938.1Attorney Docket No. 124324-5003-WOcryopreservation medium. The cryopreserved spheroids were then thawed and incubated in fresh media for 3 to 4 days.

[0059] Figure 1 show’s cryopreserved HepG2 spheroids having a diameter of 52.83 ± 4.06 pm (top left), 78.87 ± 6.73 pm (top right), 106.71 ± 7.92 pm (bottom left), and 410.83 ± 3.92 pm (bottom right). Figure 2 shows cell count per spheroid versus spheroid size (pm). The starting cell density’ per well is 12.5 104.

[0060] It was found that the spheroids were intact following preservation. Figure 3 shows HepG2 spheroids growing in the well plate. Similarly, HEK293 cells were also grown in a well plate (data not shown). Figure 4 shows fluorescence images of the HepG2 spheroids grown in 3D plate well post-thaw. Live-dead fluorescent staining was performed where the bright red spots indicate the cells with compromised membrane integrity possibly due to necrosis triggered by the diffusion limitations at the core.

[0061] Figure 5 shows HepG2 spheroids before cryopreservation, just after thawing, and after growing in media for 3-4 days. The successful survival of the spheroids were demonstrated by the bioactivity post thaw in culture conditions as indicated by merging of the individual spheroids into a bigger structure. This demonstrates successful cryopreservation of spheroids after thawing.

[0062] Figure 6A shows an example of successful cryopreservation of spheroids with smaller necrotic core (colored red using propidium iodide), and Figure 6B shows an example of a failed cryopreservation of spheroids. The failed condition is a result of using only 5% DMSO as a negative control (“Negative Control 2”). Figures 7 and 8 show characterization of the growth of spheroids in a successful example. Figure 9 shows characterization of the growth of spheroids in an unsuccessful example, due to use of only culture media or 5% DMSO with culture media as the cryopreservation medium.

[0063] Successful cryopreservation was also demonstrated using HEK293, DLD-1, and HCT116 cell types (data not shown).

[0064] In summary, it was shown that the cryopreservation medium performed better than 10% DMSO as the standard cryopreservation media. Importantly, the cryopreservation medium15DBl / 164222938.1Attorney Docket No. 124324-5003-WOperformed better than DMSO due to reduced toxicity associated with DMSO which is an essential consideration for fertility preservation applications.Example 2: Fusion Assays of Cryopreserved Spheroids

[0065] A fusing spheroid assay was done to demonstrate the retention of functionality, viability and overall function of spheroids derived from HepG2 liver carcinoma cells after cryopreserving with the cryopreservation medium (Figure 10). Under normal physiological conditions, cell-line derived spheroids fuse together when in close proximity to create a larger cellular aggregate structure, provides lower injury of surface proteins, cytoskeletal rearrangement ability, ability to create gap junction, as well as overall compaction. This data demonstrates the ability of cry opreservation using the cry opreservation medium to create ‘tissue-like morphology widely accepted as a functional model (Kosheleva et al., Sei. Rep., 2020, 10, 12614; Zamani et al., Front. Cardiovasc. Med., 2018, 5, 147; Bao et al., Nat. Commun., 2017, 8, 1962). Thus, using the cry opreservation medium maintains superior physiological performance upon cryopreservation using the cryopreservation medium. This observation was also verified using HEK293 cell derived spheroids (data not shown).16DBl / 164222938.1

Claims

1. Attorney Docket No. 124324-5003-WO2.CLAIMS1. A cry opreservation medium comprising a diol, sugar, or both a diol and sugar.

2. The cry opreservation medium according to claim 1, further comprising DMSO.

3. The cryopreservation medium according to claim 1 or 2, wherein a concentration of DMSO in the cry opreservation medium is from 1 wt% to 10 wt%.

4. The cryopreservation medium according to any one of the preceding claims, wherein the cryopreservation medium comprises a diol and sugar.

5. The cryopreservation medium according to any one of the preceding claims, wherein the diol comprises propanediol.

6. The cryopreservation medium according to any one of the preceding claims, wherein the diol comprises propane- 1,2-diol.

7. The cryopreservation medium according to any one of the preceding claims, wherein the diol comprises 1,3-propanediol.

8. The cryopreservation medium according to any one of the preceding claims, wherein a concentration of propanediol in the cryopreservation medium is from 1 wt% to 10 wt%.

9. The cryopreservation medium according to any one of the preceding claims, wherein the sugar comprises trehalose.

10. The cryopreservation medium according to any one of the preceding claims, wherein a concentration of trehalose in the cryopreservation medium is from 5 mM to 50 mM.

11. The cry opreservation medium according to any one of the preceding claims, wherein the cry opreservation medium comprises Eagle’s minimum essential medium.

12. The cry opreservation medium according to any one of the preceding claims, wherein the cryopreservation medium comprises fetal bovine serum.

13. Use of the cry opreservation medium according to any one of the preceding claims to cryopreserve a cell.

14. The use according to claim 13, wherein the cell comprises a spheroid.

15. The use according to claim 13 or 14, wherein the cell comprises a human cell.18.1719.DBl / 164222938.1 Attorney Docket No. 124324-5003-WO16. The use according to any one of claims 13-15, wherein the cell is selected from the group consisting of a HepG2 cell, a HEK293 cell, a DLD-1 cell, and an HCT116 cell.

17. A method of preparing a cell for preservation, comprising:22.incubating the cell at a temperature above 0 °C in the cryopreservation medium of any one of claims 1-12.

18. The method according to claim 17, wherein the cell comprises a spheroid.

19. The method according to claim 17 or 18, wherein the cell comprises a spheroid having a diameter from 10 pm to 1000 pm.

20. The method according to any one of claims 17-19, wherein the cell comprises a spheroid having a diameter from 80 pm to 200 pm.

21. The method according to any one of claims 17-20, wherein the cell comprises a human cell.

22. The method according to any one of claims 17-21, wherein the cell is selected from the group consisting of a HepG2 cell, a HEK293 cell, a DLD-1 cell, and an HCT116 cell.

23. The method according to any one of claims 17-22, further comprising:29.changing the temperature to a cryogenic temperature.

24. The method according to claim 23, wherein the cryogenic temperature is lower than -50 °C.

25. The method according to claim 23 or 24, wherein the temperature is changed at a rate from 0.2 °C / minute to 2.5 °C / minute.

26. The method according to any one of claims 17-25, wherein the cell is incubated in a well plate.

27. The method according to any one of claims 17-26, wherein the cell is incubated on a bottom of a well having a cone shape.

28. The method according to any one of claims 17-27, wherein the cell is incubated on a bottom of a tube.35.1836.DBl / 164222938.1 Attorney Docket No. 124324-5003-WO29. A method of cryopreserving a cell, the method comprising preparing the cell according to the method of any one of claims 17-28, and preserving the cell in the cryogenic temperature.

30. The method according to claim 29, further comprising preserving the cell for at least one week.

31. The method according to claim 29 or 30, further comprising thawing the cells.

32. A cell prepared according to the method of any one of claims 17-31.40.1941.DBl / 164222938.1

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