Patent Description:
Cryopreservation techniques at temperatures at or below <NUM> are routinely used for long-time preservation of biological materials such as cells and tissues of animals (including human cells and tissues) and plants. Effective long-term storage of mammalian cells is critical to the successful application of such cells as clinical and research tools. For example, stem cells can be used for cell transplantation, tissue engineering, and regenerative medicine. Cryopreserved oocytes, sperm, and embryos can be used in assisted reproductive technologies. In transplantation medicine, living tissues such as the skin, cornea, pancreatic islets and heart valves need to be cryopreserved.

Cells can be stored at subzero temperatures (e.g., below -<NUM>) for months or years. However, cells are not stable when and after they are thawed. The stability, mainly indicated by the viability, of the cells, varies with the environment with which the cells contact during and after the thawing process. It has been shown that the thawing rate, osmotic stress, and cryoprotectant toxicity would damage cells after thawing. If the cells will be injected into a subject (e.g., through cell therapy, blood transfusion, or bone marrow transplantation etc.), it is very important to obtain a high cell viability after thawing, as it can be futile to reinject dead or damaged cells. Likewise, when cells have to be re-cultured, it is equally important that the cell viability is high.

How to maintain cell viability as they are transitioned from a cryogenic state to a metabolically active state is a challenging task. Thus, there is a need for improved methods for maintaining cell viability after thawing. In <CIT> and in <NPL>, gelatin is used in cryopreservation for improving the post-thaw viability, but in these cases gelatin was added before the freezing step.

The present invention is defined by the granted claims. The present invention refers to method for maintaining post-thaw cell viability of at least <NUM>%, the method comprising the steps of:.

In certain embodiments, the one or more cells are in a cell suspension before the mixing step.

In certain embodiments, for the mixing step, the volume ratio of the cell stabilizing medium to the cell suspension ranges from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>.

In certain embodiments, the cells are in the cell suspension at a concentration ranging from about <NUM> x <NUM><NUM> cells/ml to about <NUM> x <NUM><NUM> cells/ml.

In certain embodiments, the method further comprises placing the cell stabilizing medium at a temperature ranging from about <NUM> to about <NUM> before the mixing step.

In certain embodiments, before the mixing step, the one or more cells are in a cryopreservation composition which has been thawed from a cryopreserved state. In certain embodiments, the cryopreserved state is at a temperature ranging from about - <NUM> and -<NUM>.

In certain embodiments, the cells have a post-thaw viability of at least <NUM>%.

In certain embodiments, the cryopreservation composition comprises glycerol, dimethyl sulfoxide (DMSO), and/or polyethylene glycol (PEG).

In certain embodiments, after the mixing step, the cells are present in the mixture at a concentration ranging from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml.

In certain embodiments, gelatin has a weight average molecular mass (or molecular weight, or average molecular weight) ranging from about <NUM> kilodalton (kD) to about <NUM> kD. In certain embodiments, gelatin comprises denatured collagen.

In certain embodiments, the cell stabilizing medium is a thermoreversible hydrogel having a bloom value ranging from about <NUM> to about <NUM>.

In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human, porcine, canine, equine or bovine cells. In certain embodiments, the cells comprise tumor cells. In certain embodiments, the cells comprise fibroblasts. In certain embodiments, the cells comprise stem cells.

In certain embodiments, the cell stabilizing medium further comprises an amino acid, a cytokine, a lipid, a growth factor, an antibiotic, an antimycotic, a steroid hormone, a protein hormone, or a combination thereof.

The present specification describes a cell stabilizing medium which comprises, e.g., gelatin. The cell stabilizing medium help maintain cell viability, e.g., after thawing of a biological material post-cryopreservation. By mixing with the present cell stabilizing medium, the viability of the cells can be maintained for a desirable time.

The biological material (e.g., cells) can then be used in a variety of research and clinical settings, for example, for cell-based therapeutics, in assisted reproductive technology, or for patients undergoing chemotherapy or radiation therapy. In one embodiment, the biological material may be administered to a subject.

In certain embodiments, the biological material (e.g., cells, tissues, organs, or viral particles) is thawed (or has been thawed) from cryopreservation (from a cryopreserved state). In certain embodiments, the biological material has been under cryopreservation. In certain embodiments, while being mixed with the cell stabilizing medium, the biological material is in the process of being thawed from, or has been thawed from, cryopreservation. In certain embodiments, after being combined/mixed with the present cell stabilizing medium, the cells have a post-thaw viability of at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or at least about <NUM>%.

In certain embodiments, when the biological material (e.g., cells, tissues, organs, or viral particles) is thawed (or has been thawed) from cryopreservation, the biological material is in a cryopreservation composition which comprises one or more permeating cryoprotectants, and/or one or more non-permeating cryoprotectants. Non-limiting examples of permeating cryoprotectants include glycerol, DMSO, polyethylene glycol, ethylene glycol, and propylene glycol (<NUM>,<NUM>-propanediol, propane-<NUM>,<NUM>-diol). Non-limiting examples of non-permeating cryoprotectants include high molecular weight molecules, such as saccharides (e.g., sucrose, trehalose, maltose), sugars, starches (e.g., hydroxyethyl starch), protein (e.g., albumin such as serum albumin), percoll, ficol, polyethylene glycol, dextran, polyvinyl pyrrolidone, polyvinylalcohol (PVA), serum, plasma and other macromolecules. In certain embodiments, the cryopreservation composition comprises one or more cryoprotectants including, but not limited to, glycerol, dimethyl sulfoxide (DMSO), and/or polyethylene glycol (PEG).

As used herein, the term "cryopreserved state" means a state of being at a cryopreserved temperature.

In specific embodiments, a cryopreservation temperature comprises a temperature of at or below about <NUM>, at or below about -<NUM>, at or below about - <NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, from about -<NUM> to about -<NUM>, or in liquid nitrogen.

In certain embodiments, the biological material (e.g., cells, tissues, organs, or viral particles) is in a hypothermic preserved state before being combined/mixed with the cell stabilizing medium. In certain embodiments, the biological material (cells, tissues, organs) was in a lyophilized state before being combined/mixed with the cell stabilizing medium.

In certain embodiments, the cells are under cell culture before being combined/mixed with the cell stabilizing medium. Cells may be harvested at sub-confluence, at the exponential growth phase, at confluence, or post-confluence.

In certain embodiments, the cells are harvested from a subject (e.g., a patient) before being combined/mixed with the cell stabilizing medium.

In certain embodiments, after being combined/mixed with the present cell stabilizing medium, the cells have a viability of at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or at least about <NUM>%.

In certain embodiments, the cells comprise tumor cells. In certain embodiments, the cells comprise fibroblasts. In certain embodiments, the cells comprise stem cells.

In certain embodiments, the cells comprise mammalian cells, including, but not limited to, human, porcine, canine, equine or bovine cells.

In certain embodiments, the biological material treated with the present cell stabilizing medium may be administered to a subject (e.g., a patient).

In certain embodiments, the cell stabilizing medium comprises about <NUM> wt% to about <NUM> wt% of gelatin (based on the total weight of the cell stabilizing medium). In certain embodiments, the cell stabilizing medium comprises about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, or about <NUM> wt%, of gelatin based on the total weight of the cell stabilizing medium.

In certain embodiments, the cell stabilizing medium comprises (or consists of, or consists essentially of) about <NUM> wt% to about <NUM> wt% of gelatin, and a solvent (e.g., a culture medium such as DMEM, water, a buffer, a saline solution etc.). In certain embodiments, the cell stabilizing medium comprises (or consists of, or consists essentially of) an aqueous gelatin solution where the concentration of gelatin ranges from about <NUM> wt% to about <NUM> wt%.

In certain embodiments, the cell stabilizing medium comprises (or consists of, or consists essentially of) about <NUM> wt% to about <NUM> wt% of gelatin, and a buffer system (e.g., a physiological buffer). In certain embodiments, the cell stabilizing medium comprises (or consists of, or consists essentially of) about <NUM> wt% to about <NUM> wt% of gelatin, and a salt solution, and/or any physiological solution.

In certain embodiments, the cell stabilizing medium comprises (or consists of, or consists essentially of) about <NUM> wt% to about <NUM> wt% of gelatin, and a culture medium (e.g., a cell culture medium).

In certain embodiments, in the present cell stabilizing medium, gelatin is mixed with a liquid composition. In certain embodiments, the cell stabilizing medium comprises (or consists of, or consists essentially of) about <NUM> wt% to about <NUM> wt% of gelatin, and a liquid composition. Non-limiting examples of liquid compositions include water, a culture medium, a mixture of culture media, a buffer system (e.g., a physiological buffer), a salt solution, and/or any physiological solution. In certain embodiments, the liquid composition further comprises supplements, additives, additional amounts of some medium components, etc..

In certain embodiments, the cell stabilizing medium comprises (or consists of, or consists essentially of) two constituents: the first constituent being gelatin which is present in the cell stabilizing medium at a concentration ranging from about <NUM> wt% to about <NUM> wt%; the second constituent being a saline solution (e.g., an isotonic saline solution), a buffer system (e.g., a physiological buffer), water, and/or a culture medium (e.g., a cell culture medium).

The present disclosure provides for a method for maintaining the viability of a biological material (e.g., cell viability). The method may comprise the step of mixing a biological material (e.g., cells, tissues, organs, or viral particles) with a cell stabilizing medium to form a mixture. In certain embodiments, the cell stabilizing medium comprises about <NUM> wt% to about <NUM> wt% of gelatin based on the total weight of the cell stabilizing medium.

The biological material (e.g., cells, tissues, organs, or viral particles) can be combined with the present cell stabilizing medium by any suitable method. In certain embodiments, the cell stabilizing medium is added to the biological material. In certain embodiments, the biological material is added to the cell stabilizing medium. In one embodiment, the method comprises providing cells in a cell suspension, and adding the cell stabilizing medium to the cell suspension, optionally with mixing. In another embodiment, the method comprises providing cells in a cell suspension, and adding the cell suspension to the cell stabilizing medium, optionally with mixing.

In certain embodiments, cells are freed of their culture medium or preservation medium (e.g., centrifuged, harvested, and optionally washed in a buffer solution), before being combined/mixed with the present cell stabilizing medium.

In certain embodiments, before mixing the cells and the present cell stabilizing medium, the cells are in a cell suspension. In certain embodiments, the cells are suspended in a culture medium (e.g., a cell culture medium), a buffer system (e.g., a physiological buffer), a salt solution, and/or a physiological solution.

In certain embodiments, the cells are present in the cell suspension at a concentration ranging from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, from about <NUM> x <NUM><NUM> cells/ml to about <NUM> x <NUM><NUM> cells/ml, from about <NUM> x <NUM><NUM> cells/ml to about <NUM> x <NUM><NUM> cells/ml, from about <NUM> x <NUM><NUM> cells/ml to about <NUM> x <NUM><NUM> cells/ml, from about <NUM> x <NUM><NUM> cells/ml to about <NUM> x <NUM><NUM> cells/ml, from about <NUM><NUM> cells/ml to about <NUM> x <NUM><NUM> cells/ml, from about <NUM> x <NUM><NUM> cells/ml to about <NUM> x <NUM><NUM> cells/ml, from about <NUM> x <NUM><NUM> cells/ml to about <NUM> x <NUM><NUM> cells/ml, from about <NUM> x <NUM><NUM> cells/ml to about <NUM> x <NUM><NUM> cells/ml, from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, about <NUM> x <NUM><NUM> cells/ml, about <NUM> x <NUM><NUM> cells/ml, or about <NUM> x <NUM><NUM> cells/ml, about <NUM><NUM> cells/ml, about <NUM><NUM> cells/ml, or about <NUM><NUM> cells/ml. In certain embodiments, the cells are present in the cell suspension at a concentration ranging from about <NUM> x <NUM><NUM> cells/ml to about <NUM> x <NUM><NUM> cells/ml. In certain embodiments, the cells are present in the cell suspension at a concentration of about <NUM> x <NUM><NUM> cells/ml. The concentration of the cells in the cell suspension may be higher than <NUM><NUM> cells/ml or lower than <NUM><NUM> cells/ml.

In certain embodiments, the cells and the present cell stabilizing medium are mixed to form a mixture.

The present disclosure provides for a method for maintaining cell viability. The method may comprise the step of mixing one or more cells with a cell stabilizing medium to form a mixture. In certain embodiments, the mixture comprises about <NUM> wt% to about <NUM> wt% of gelatin.

In certain embodiments, the mixture of cells (which may or may not be in a cell suspension) and the present cell stabilizing medium, (or the combination of a biological material and the cell stabilizing medium) comprises about <NUM> wt% to about <NUM> wt% of gelatin (based on the total weight of the mixture). In certain embodiments, the mixture (or the combination) comprises about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, or about <NUM> wt%, of gelatin based on the total weight of the mixture (or the combination).

In certain embodiments, the cells and the present cell stabilizing medium are mixed to form a mixture, where the cells are present in the mixture at a concentration ranging from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml, about <NUM><NUM> cells/ml, about <NUM><NUM> cells/ml, or about <NUM><NUM> cells/ml. In certain embodiments, the cells are present in the mixture at a concentration ranging from about <NUM><NUM> cells/ml to about <NUM><NUM> cells/ml. In certain embodiments, the cells are present in the mixture at a concentration of about <NUM><NUM> cells/ml. The concentration of the cells in the mixture may be higher than <NUM><NUM> cells/ml or lower than <NUM><NUM> cells/ml.

In certain embodiments, for the mixing step, the volume ratio of the cell stabilizing medium to the cell suspension (or a composition containing the biological material) ranges from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>.

In certain embodiments, the viability of the cells in the mixture of a biological material (such as cells which may or may not be in a cell suspension) and the present cell stabilizing medium is reduced less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, or less than about <NUM>%, after the mixture is incubated at a temperature of about <NUM>, about <NUM>, about <NUM>, or about <NUM>, for a time period ranging from about <NUM> hour to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hour to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hour to about <NUM> hours, from about <NUM> hour to about <NUM> hours, from about <NUM> hour to about <NUM> hours, from about <NUM> hour to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> minutes to about <NUM> minutes, from about <NUM> minutes to about <NUM> minutes, from about <NUM> minutes to about <NUM> hour, from about <NUM> minutes to about <NUM> hour, about <NUM> minutes, about <NUM> hour, about <NUM> hours, about <NUM> hours, about <NUM> hours, about <NUM> hours, about <NUM> hours, about <NUM> hours, or about <NUM> hours.

In certain embodiments, the viability of the cells in the mixture of a biological material (such as cells which may or may not be in a cell suspension) and the present cell stabilizing medium is at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or at least about <NUM>%, after the mixture is incubated at a temperature of about <NUM>, about <NUM>, about <NUM>, or about <NUM>, for a time period ranging from about <NUM> hour to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hour to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hour to about <NUM> hours, from about <NUM> hour to about <NUM> hours, from about <NUM> hour to about <NUM> hours, from about <NUM> hour to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> hours to about <NUM> hours, from about <NUM> minutes to about <NUM> minutes, from about <NUM> minutes to about <NUM> minutes, from about <NUM> minutes to about <NUM> hour, from about <NUM> minutes to about <NUM> hour, about <NUM> minutes, about <NUM> hour, about <NUM> hours, about <NUM> hours, about <NUM> hours, about <NUM> hours, about <NUM> hours, about <NUM> hours, or about <NUM> hours.

The present specification describes a composition comprising a biological material (e.g., one or more cells, tissues, organs) and about <NUM> wt% to about <NUM> wt% of gelatin. In certain embodiments, the composition comprises about <NUM> wt% to about <NUM> wt% of gelatin (based on the total weight of the composition). The composition comprises for example about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, or about <NUM> wt%, of gelatin based on the total weight of the composition.

In certain embodiments, the biological material (e.g., cells, tissues, organs, or viral particles) is thawed (or has been thawed) from cryopreservation (a cryopreserved state). In certain embodiments, the biological material has been under cryopreservation. In certain embodiments, the biological material is in the process of being thawed from, or has been thawed from, cryopreservation.

The present cell stabilizing medium may be a liquid or a solid. The present cell stabilizing medium is for example a concentrate composition, such as, in a dry form (e.g., powder, tablet, granular or any other suitable physical form) or in liquid form as, e.g., 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X etc. stock solutions. The stock solutions can be diluted 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X etc. by, e.g., a culture medium, a physiologic solution, a buffer, water etc. the dry form of the cell stabilizing medium may be converted to a liquid form by adding, e.g., a culture medium, a physiologic solution, a buffer, water etc. (e.g., dissolved in, e.g., a culture medium, a physiologic solution, a buffer, water etc.).

The concentrations of the components discussed herein are for example the concentrations of the components in a stock solution of the present cell stabilizing medium. The concentrations of the components discussed herein are for example the concentrations of the components in a working solution of the present cell stabilizing medium.

The present cell stabilizing medium may be a solution. The cell stabilizing medium is for example an aqueous solution of the components discussed herein.

When preparing the present cell stabilizing medium, the components discussed herein (e.g., gelatin, a gelatin derivative, and/or albumin) are for example dissolved in a balanced electrolyte solution (e.g., a saline solution, a culture medium such as a cell culture medium). The cell stabilizing medium has for example an appropriate concentration of electrolytes (such as sodium, potassium, and/or chloride ions) to maintain a normal osmolality. The saline solution is for example a phosphate-buffered saline solution (PBS). The saline solution comprises for example one or more of the following: Sodium Chloride, Potassium Chloride, Magnesium Sulfate, Potassium Phosphate, Calcium Chloride, and Sodium Bicarbonate. In one embodiment, the saline solution is an isotonic saline solution (e.g., isotonic with the plasma, or a body fluid).

The present cell stabilizing medium may comprise a buffer system (e.g., a physiological buffer). The present cell stabilizing medium may comprise a balanced salt solution or any physiological solution.

Non-limiting examples of the buffer systems include phosphoric acid buffers (for example, phosphate buffered saline (PBS)), BES, TES, acetamidoglycine, glycine amides, glycylglycine, TRICINE, TALP, tris-ethanolamine, veronal, and HEPES.

The concentration of the buffer in the present cell stabilizing medium ranges for example from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>.

Non-limiting examples of culture media include, Dulbecco's Modified Eagle Media (DMEM), Minimal Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimal Essential Medium (G-MEM), Basal Medium Eagle (BME), DMEM/Ham's F12, Advanced DMEM/Ham's F12, Iscove's Modified Dulbecco's Media and Minimal Essential Media (MEM), Ham's F-<NUM>, Ham's F-<NUM>, Medium <NUM>, RPMI <NUM> Media, and combinations thereof and/or modifications thereof. The cell culture is preferably DMEM.

The present cell stabilizing medium has for example a pH ranging from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, or from about <NUM> to about <NUM> at room temperature or ambient temperature (for example, at <NUM>).

The cell stabilizing medium is packaged for example in unit forms. The cell stabilizing medium is for example packaged in a volume of <NUM>, <NUM>, <NUM>, <NUM> or <NUM>. The cell stabilizing medium is for example packaged as a 1X, 5X, 10X, or 20X solution.

The present cell stabilizing medium can be obtained in a solid form by mixing the components discussed herein, or as an aqueous solution by dissolving the components in water, a buffer, a solution, a culture medium, etc..

As used herein, the percentage "% (w/v)" is percent weight to volume (w in gram and v in milliliter); the percentage "% (v/v)" is percent volume to volume; the percentage "% (w/w)" or "wt%" is percent weight to weight.

The term "about" in reference to a numeric value refers to ±<NUM>% of the stated numeric value. In other words, the numeric value can be in a range of <NUM>% of the stated value to <NUM>% of the stated value.

The present cell stabilizing medium may comprise a gelatin and/or a gelatin derivative. As used herein, the term "gelatin" may refer to gelatin or a gelatin derivative. Any gelatin or a gelatin derivative may be used in the present cell stabilizing medium.

In certain embodiments, gelatin in the present cell stabilizing medium has a molecular weight (or weight average molecular mass, or average molecular mass) ranging from about <NUM> kilodalton (kD) to about <NUM> kD, from about <NUM> kD to about <NUM> kD, from about <NUM> kD to about <NUM> kD, from about <NUM> kD to about <NUM> kD, from about <NUM> kD to about <NUM> kD, from about <NUM> kD to about <NUM> kD, from about <NUM> kD to about <NUM> kD, from about <NUM> kD dalton to about <NUM> kD, from about <NUM> kD to about <NUM> kD, from about <NUM> kD to about <NUM> kD, from about <NUM> kD to about <NUM> kD, from about <NUM> kD to about <NUM> kD, from about <NUM> kD to about <NUM> kD, or from about <NUM> kD to about <NUM> kD.

In certain embodiments, gelatin in the present cell stabilizing medium has an isoelectric point (pI) ranging from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>.

In certain embodiments, gelatin is derived from mammalian tissue. In certain embodiments, gelatin is obtained from animal collagen. In certain embodiments, gelatin is derived from raw materials including, but not limited to, the skin, bones, connective tissues, tendons, ligaments, etc. of animals such as cattle, chicken, pigs, and fish. In one embodiment, gelatin is of bovine source, porcine source, or a combination thereof. In certain embodiments, gelatin is sourced from bovine bones and porcine skin, bovine skin, pork, bovine hides, and/or fish skin. In one embodiment, gelatin is skin-derived gelatin or bone-derived gelatin.

In certain embodiments, gelatin is a mixture of peptides and proteins produced by partial hydrolysis of collagen. In certain embodiments, gelatin is a hydrolyzed form of collagen. In certain embodiments, gelatin is a form of denatured collagen. In certain embodiments, gelatin comprises denatured collagen.

In certain embodiments, gelatin may be type A gelatin or type B gelatin. As used herein, type A gelatin is the gelatin obtained from acid-treated raw material; type B gelatin is the gelatin obtained from alkali-treated raw material.

In certain embodiments, to produce gelatin, collagen hydrolysis is performed by chemical hydrolysis, and/or thermal hydrolysis. In one embodiment, collagen is boiled (e.g., in water) or heated (extensively) to produce gelatin.

In certain embodiments, to produce gelatin, collagen hydrolysis is performed by acid-hydrolysis, alkali-hydrolysis, and/or enzymatic hydrolysis.

In certain embodiments, the manufacturing processes of gelatin contain three main stages: the pretreatment, the main extraction step, and the refining and recovering treatments. Pretreatments make the raw materials ready for the main extraction step and remove impurities that may have negative effects on physiochemical properties of the final gelatin product. The main extraction step may be done with hot water or dilute acid solutions as a multistage extraction to hydrolyze collagen into gelatin. The refining and recovering treatments include filtration, clarification, evaporation, sterilization, drying, rutting, grinding, and/or sifting to remove the water from the gelatin solution, to blend the gelatin extracted, and/or to obtain dried, blended and ground final product.

In certain embodiments, the present cell stabilizing medium comprises fractionated gelatin which is obtained from conventional gelatin by special preparation techniques, such as ultrafiltration. In certain embodiments, fractionated gelatin is obtained by removal of a selected portion(s) of peptides/polypeptides, or by mixtures of individual fractions of peptides/polypeptides.

Gelatin derivatives are chemically modified gelatins, including, but not limited to, succinylated gelatin, thiolated gelatin, acetylated gelatin, phthalated gelatin, succinyl gelatin, oxypolygelatin, or urea cross-linked gelatin. In one embodiment, succinylated gelatin is a gelatin cross-linked by succinic acid or its salt, or succinic anhydride. In certain embodiments, a gelatin derivative is obtained by by reacting gelatin with an anhydride, such as succinic, citraconic, itaconic, aconitic or maleic anhydride. <CIT> and <CIT>.

The present cell stabilizing medium may contain any suitable polypeptide. The cell stabilizing medium comprises for example a polypeptide component and a liquid component.

A polypeptide, as used herein, is intended to encompass any tissue-derived or synthetically produced polypeptide, such as collagen-derived components (such as gelatin). A polypeptide can comprise (or consists of) from about <NUM> amino acid residues to about <NUM>,<NUM> amino acid residues, preferably about <NUM> amino acid residues to about <NUM>,<NUM> amino acid residues, more preferably about <NUM> amino acid residues to about <NUM>,<NUM> amino acid residues, still more preferably about <NUM> amino acid residues to about <NUM>,<NUM> amino acid residues, and most preferably about <NUM> amino acid residues to about <NUM>,<NUM> amino acid residues.

The polypeptide comprises for example gelatin, albumin, or a combination thereof.

The polypeptide is for example gelatin or a gelatin derivative (e.g., succinylated gelatin). The polypeptide is for example other gelatin-like components, such as keratin, decorin, aggrecan, elastin, laminin, nidogen, fibulin, fibrillin, collagen, fractionated gelatin, collagen hydrolyzates, plant proteins, plant protein hydrolyzates, elastin hydrolyzates, glycoproteins (including proteoglycans), and mixtures thereof.

Polypeptides derived from other types of tissue could also be used. Examples include, but are not limited to, tissue extracts from arteries, vocal chords, pleura, trachea, bronchi, pulmonary alveolar septa, ligaments, auricular cartilage or abdominal fascia; the reticular network of the liver; the basement membrane of the kidney; or the neurilemma, arachnoid, dura mater or pia mater of the nervous system.

The polypeptide may comprise natural components, and/or synthetic components. Examples of natural components include, but are not limited to, naturally occurring proteins and polypeptides.

The cell stabilizing medium comprises for example about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, about <NUM> wt%, or about <NUM> wt%, of one or more polypeptides (as discussed herein) based on the total weight of the cell stabilizing medium.

Any albumin or an albumin derivative may be used in the present cell stabilizing medium.

Non-limiting examples of albumin include serum albumin (e.g., human serum albumin or HSA), plasma albumin (e.g., human plasma albumin), bovine serum albumin, and/or synthetic serum albumin), ovalbumin, plant albumin, or combinations thereof. Non-limiting examples of albumin also include fetal bovine serum.

Albumin may be either of natural origin (e.g., purified from a natural source) or of recombinant origin (recombinant albumin). Albumin is for example produced by purification from biological material of human origin. It may be obtained by conventional techniques for fractionation of plasma obtained from blood (<NPL>), or by extraction from the human placenta, according to the technique described by <NPL>). Recombinant albumin is for example produced in a eukaryotic host.

The term "albumin" comprises any natural variant of human albumin, resulting from the polymorphism of this protein.

The present cell stabilizing medium is a thermoreversible hydrogel, which undergoes a transition from a flowable state (a liquid state) to a gel state in response to a change in temperature. The present cell stabilizing medium is for example in a free flowing or liquid phase at or above a phase transition temperature, and is in a gel phase (a solid phase, a non-flowable phase) below a phase transition temperature. <CIT> and <CIT>.

The present cell stabilizing medium has for example a fluid phase (e.g., a gelatin solution) above a phase transition temperature and has a gel phase (e.g., a gelatin hydrogel) at or below the phase transition temperature. The conversion between the fluid phase and the gel phase is for example a continuous process. In the gel phase, the extent of the gelation of the cell stabilizing medium provides for example an operable hydrogel. The phase transition temperature can also be a critical temperature at which the viscosity of the cell stabilizing medium ensures that the cell stabilizing medium is an operable hydrogel. The phase transition temperature is for example the melting point.

The cell stabilizing medium has for example a phase transition temperature ranging from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, above about <NUM>, or about <NUM>.

The cell stabilizing medium in its liquid phase is for example combined/mixed with a biological material (e.g., cells, tissues, organs, viral particles etc.) to form a mixture.

The cell stabilizing medium is placed for example at a temperature ranging from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, above about <NUM>, or about <NUM>, to liquefy the cell stabilizing medium (or to maintain the cell stabilizing medium in a liquid state) before being mixed with a biological material (e.g., cells, tissues, organs, viral particles etc.) to form a mixture.

The cell stabilizing medium has for example a gelation time ranging from about <NUM> minutes to about <NUM> hour, from about <NUM> minutes to about <NUM> minutes, from about <NUM> minutes to about <NUM> minutes, from about <NUM> minutes to about <NUM> minutes, from about <NUM> minutes to about <NUM> minutes, from about <NUM> minutes to about <NUM> minutes, from about <NUM> minutes to about <NUM> minutes, from about <NUM> minutes to about <NUM> minutes, or from about <NUM> minutes to about <NUM> minutes. As used herein, the term "gelation time" refers to the time required for the present cell stabilizing medium to convert from its fluid phase into its gel phase.

The cell stabilizing medium (the gel phase of the cell stabilizing medium) has for example a bloom value ranging from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, at least or equal to about <NUM>, at least or equal to about <NUM>, at least or equal to about <NUM>, about <NUM>, or about <NUM>.

The cell stabilizing medium further comprises for example a saccharide, an amino acid, a cytokine, a lipid, a growth factor, an antibiotic (e.g., penicillin, streptomycin, etc.), an antimycotic, a steroid hormone, a protein hormone, serum, amino acid analogues, amino acid derivatives, and divalent cation chelators, such as ethylenediaminetetraacetic acid (EDTA) or salts thereof, proteins, salts, formamide, methoxylated compounds, and/or polymers (e.g., polyvinyl pyrrolidone and polyvinyl alcohol), or a combination thereof. In certain embodiments, the cell stabilizing medium further comprises glycine, glycerol, sucrose, glucose, or combinations thereof.

The cell stabilizing medium comprises for example from about <NUM>/L to about <NUM>,<NUM>/L, from about <NUM>/L to about <NUM>,<NUM>/L, from about <NUM>/L to about <NUM>/L, from about <NUM>/L to about <NUM>/L, from about <NUM>/L to about <NUM>/L, about <NUM>,<NUM>/L, about <NUM>,<NUM>/L, about <NUM>/L, or about <NUM>,<NUM>/L, of glucose.

Saccharides include oligosaccharides such as monosaccharides and disaccharides, polysaccharides, and the like. Saccharides include sugars.

Non-limiting examples of saccharides include sucrose, sorbitol, glucose, fructose, galactose, trehalose, mannose, raffinose, stachyose, dextran, xylose, arabinose, mannitol, xylitol, myo-inositol, lactose, maltose, cellobiose, lactitol, maltitol, methyl cellulose, carboxymethyl cellulose, glycogen, amylose, amylopectin, inulin, sodium alginate, ethyl cellulose, hydroxyethyl cellulose, xanthan gum, glucosamine, galactosamine, and combinations thereof. <CIT> and <CIT>.

The present cell stabilizing medium may or may not comprise one or more amino acids.

Amino acids include optical isomers, namely both D-isomers and L-isomers. Amino acids include alpha-amino acids, as well as beta-amino acids, gamma-amino acids, delta-amino acids, and unnatural amino acids. Non-limiting examples of amino acids include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, glutamine, asparagine, tyrosine, lysine, arginine, aspartic acid, glutamic acid, and combinations thereof.

Amino acid derivatives may also be used in the present methods. Non-limiting examples of amino acid derivatives include amino acid salts and amino acid solvates. Non-limiting examples of the amino acid salts include alkaline metal salts or alkaline earth metal salts such as sodium salts, potassium salts, and calcium salts; halogen acid salts such as hydrofluoric acid salts, hydrochloric acid salts, hydrobromic acid salts, and hydroiodic acid salts; inorganic acid salts such as nitrate salts, perchlorate salts, sulfate salts, and phosphate salts; and organic acid salts such as fumarate salts, succinate salts, citrate salts, oxalate salts, maleate salts, acetate salts, lactate salts, and ascorbate salts. Non-limiting examples of the amino acid solvates include hydrates, alcoholates (for example, methanolates, ethanolates), and etherates (for example, diethyl etherates).

In certain embodiments, the amino acid concentration in the present cell stabilizing medium is <NUM>-<NUM>% by weight, or <NUM>-<NUM>% by weight.

The cell stabilizing medium further comprises for example one or more vitamins. Non-limiting examples of vitamins include D-calcium pantothenate, choline chloride, folic acid, niacinamide, pyridoxine HCl, thiamine HCl, and riboflavin.

The present cell stabilizing medium further comprises for example one or more salts, including inorganic salts, and/or organic salts. Non-limiting examples of inorganic salts include, potassium chloride, sodium bicarbonate, sodium chloride, and sodium phosphate monobasic, potassium phosphate monobasic, potassium phosphate dibasic, sodium bicarbonate, calcium chloride, magnesium chloride, potassium bicarbonate, potassium monophosphate, and combinations thereof.

The cell stabilizing medium or the composition does for example not comprise serum. The cell stabilizing medium or the composition does for example not comprise any raw materials of direct human or animal origin, or materials that have been produced using materials of human or animal origin.

The cell stabilizing medium or the composition may comprise other optional components, including, but not limited to, peptides, other proteins, sugar alcohols, amino saccharides, glycoproteins, and alcohols, pH controlling agents, moisturizing agents, preservatives, viscosity controlling agents, or combinations thereof.

The biological material (cells, tissues, organs) is thawed (or has been thawed) from cryopreservation (a cryopreserved state), before or during mixing with the present cell stabilizing medium.

The appropriate storage conditions for preserving a biological material may comprise any such conditions that maintain the biological material viable. Such conditions can include a cryopreservation temperature of at or below about <NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, at or below about -<NUM>, or in liquid nitrogen. For hypothermic preservation, the temperature can be between <NUM> and <NUM>. In the case of lyophilized samples, the temperature may be any temperature above <NUM> (e.g., room temperature, an ambient temperature, etc.) or below <NUM>, as long as, the material is kept away from humidity.

The biological material can remain in a preserved state (e.g., a cryopreserved state) for periods of days, weeks, months or years, until the biological material is required. When required, the cryopreserved biological material is retrieved and thawed.

In certain embodiments, the biological material in the cryopreservation composition is thawed in a water bath (e.g., by placing the cryotube or cryovial in a water bath), at a temperature at or below about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, room temperature, or about <NUM>.

In one embodiment, the biological material in the cryopreservation composition is thawed in a water bath at about <NUM>. Optionally, it would be then moved to a lower temperature such as <NUM> or on ice.

In certain embodiments, a "step up" thawing process having a step up heating rate (or a temperature ramp-up heating rate) is used. For example, the cryovial may be placed in sequential storage environments with increasing temperatures before being transferred to a temperature that is around body temperature, for example a water bath having a temperature of around <NUM>, or any other suitable temperature.

In certain embodiments, the cryopreserved biological material in the cryopreservation composition is thawed at a warming rate ranging from about <NUM>/min to about <NUM>/min, from about <NUM>/min to about <NUM>/min, from about <NUM>/min to about <NUM>/min, from about <NUM>/min to about <NUM>/min, from about <NUM>/min to about <NUM>/min, from about <NUM>/min to about <NUM>/min, about <NUM>/min to about <NUM>/min, from about <NUM>/min to about <NUM>/min, greater than about <NUM>/min, greater than about <NUM>/min, greater than about <NUM>/min, greater than about <NUM>/min, greater than about <NUM>/min, or about <NUM>/min.

In certain embodiments, after thawing, before or after being treated with the present cell stabilizing medium, the biological material is washed, suspended in the appropriate media and treated as needed for use in research or clinical applications.

In certain embodiments, after thawing and after being treated with the present cell stabilizing medium, the cells are transferred to a culture dish for re-culturing. The cells may be cultured under appropriate conditions for a period of about <NUM> minutes, about <NUM> hour, about <NUM> hours, about <NUM> hours, about <NUM> hours, about <NUM> hours, about <NUM> hours, about <NUM> hours, about <NUM> hours, about <NUM> week, about <NUM> weeks, or more than <NUM> weeks prior to research or clinical applications.

In certain embodiments, resuscitation of adherent cells or semi-adherent cells are re-cultured immediately upon thawing and after treatment with the present cell stabilizing medium.

In certain embodiments, after thawing and after treatment with the present cell stabilizing medium, the biological material is used in vivo without an intervening culturing step.

In certain embodiments, after thawing and after treatment with the present cell stabilizing medium, the cells may be re-suspended in a fluid or other medium suitable for the intended use. For example, the cells can be re-suspended in any osmotically supportive solution. In certain embodiments, the cells can be re-suspended in a physiologically compatible buffer, such as the buffer solutions described herein. Preferably, any physiologically compatible material providing a composition for convenient delivery in vivo can be used to re-suspend the cells.

The present compositions and methods maintain cell viability.

As used herein, the term "viability" refers to the percentage of viable biological material (such as cells, e.g., based on the presence of DNA and/or an intact cell membrane system, or viable viruses). In certain embodiments, viable biological material refers to a biological material comprising some viable cells or fractions of cells that are metabolically active or would become metabolically active after their release from the preservation state.

In certain embodiments, the viability of the biological material (e.g., cells or viruses) is at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, at least about <NUM>%, or at least about <NUM>%.

In certain embodiments, the present compositions and methods ensure that the cells display a limited amount of, or minimal, necrosis and apoptosis. In certain embodiments, necrosis and/or apoptosis is observed in less than about <NUM>%, less than about <NUM>%, less than about <NUM>%, less than about10%, less than about <NUM>%, or less than about <NUM>% of the cells.

The viability can be measured by any methods known in the art. In certain embodiments, the viability is measured using a Trypan blue internalization test or by measuring propidium iodide uptake. In certain embodiments, the viability is measured by assaying the ability of cells to attach efficiently (e.g., the attachment assays). In certain embodiments, proliferation assays can be used to determine if the attached cells can proliferate as expected after cryopreservation. Attachment and proliferation efficiency can be compared to control cells which have not undergone cryopreservation.

There are various tests known in the art to determine the viability and function of the cells. In certain embodiments, these tests are dependent on the cell type and the desired use of the cell.

For stem cells or progenitor cells, the methods described herein may further ensure that the cells maintain their pluripotency. This can be established by the determination of expression of lineage-specific markers. For instance, functional characterization of the mesenchymal stem cells may include induction of adipogenic, osteogenic and chondrogenic differentiation in vitro using commercially available differentiation kits and RT-PCR to detect lineage specific expression of mRNA, indicative for adipogenic, osteogenic and chondrogenic differentiation potential. Similarly, the quality of the undifferentiated stem cells can be tested by isolation of mRNA and testing on cell-specific markers. In particular embodiments, the ability to differentiate into a cell of the specified lineage is maintained, i.e., does not significantly differ from unprocessed cells. The pluripotency of the embryonic stem (ES) cells can be tested using art known methods, including, for example, Oct4-GFP expression, elevated alkaline phosphatase expression, and SSEA-<NUM> surface glycoprotein expression. Several in vitro methods can be applied to assess stem cell recovery after experimental treatment. These assessments may include, but are not limited to, membrane integrity, metabolic and other functional assays and/or colony growth in culture, and fluorescent assays, such as SYTO/EB. In certain embodiments, differentiation tests, immunophenotype characterization, and/or an inspection of the morphology may be used to assay stem cells and/or progenitor cells.

For zygotes, cleavage rates can be determined and compared to control groups to determine if there has been any cellular damage. The viability of oocytes can be determined by examination of the morphological characteristics of the cells following cryopreservation. Morphologically viable oocytes exhibit intact zona pellucida and plasma membrane and refractive cytoplasm, while non-viable oocytes appear degenerated when visualized under a light microscope. The ultimate criterion for oocyte viability and function is their capability to be fertilized by healthy sperm in vitro and in vivo, followed by cleavage, blastocyst, and/or hatching or development of the fetus.

In certain embodiments, the present preservation compositions and methods, as well as the biological material, can be used for research and/or clinical application (e.g., cell-based therapies, transplantation, regenerative medicine, diagnostics and genetic testing, cell/tissue banking for surveillance, toxicity testing and for in vitro fertilization).

The term "biological material" denotes cells, cell aggregates, tissue, organs, biological fluids, viral particles, and any other membranous entity such as liposomes (natural or synthetic).

Any type of cells or tissues may be treated with the present compositions (e.g., the cell stabilizing medium) and methods.

In certain embodiments, the cells are mammalian cells, including, but not limited to, human cells, murine cells, porcine cells, canine cells, equine cells and bovine cells. The cells may be from a mammal that is of an endangered or threatened species. The cells may be from a human or non-human mammal, for example Cercopithecoidea family, Hominoidea superfamily, Canis familiaris, Felis catus, Cricetidae spp. , Equus spp. (e.g., Equus caballus, Equus assinus), Equidae family, Bos taurus, Bos indicus, Bovidae family, Camelidae family, Bubalus bubalis, Capra aegagrus hircus, Cervidae family, Cervinae family, Ovis aries, Ovis canadensis, Capra hircus, Sus scrofa domestica, Mesocricetus spp. , Mustela vison, Cavia porcellus, Meriones unguiculatus, Chinchilla laniger, Rattus norvegicus, Rattus spp. , Mus musculus, Leporidae family, Oryctolagus cuniculus, Kobus spp. , Gallus spp. , Meleagria gallopavo, Anatidae spp. , Mustela putorius, Columba domestica, Columba livia, Numida meleagris, Ornithorhynchus anatinus, Pavo cristatus, Bison spp. , Struthio spp. , Lama glama, Rhea spp. , Dromiceius spp. , Lama pacos, Rangifer tarandus, Bos grunniens, Camelus bactrianus, Camelus dromedarius), and any endangered or threatened species.

The present compositions and methods may be used to treat microorganisms, bacteria, non-mammalian animal cells (e.g., insect cells, avian cells, fish cells, etc.), or plant cells.

Non-limiting examples of the cell include stem cells, progenitor cells, embryos, sperm, oocytes, gametocytes, and zygotes.

The cells may be tumor cells or non-tumor cells. In one embodiment, the cells are fibroblasts.

Biological materials may comprise, without limitation, any of the following: fibroblasts, stem cells, progenitor cells, whole blood or fractions thereof, red blood cells, white blood cells, umbilical cord blood or fractions thereof, umbilical cord blood cells, bone marrow, oocytes, sperm, ova, embryos, cartilage, ovary, heart, skin, kidney, liver, lung. In addition, such biological material may comprise cellular organisms, which may be eukaryotes or prokaryotes, including bacteria, and yeast, etc. Additionally, biological material may also comprise whole multi-cellular organisms that are capable of surviving cryopreservation such as nematodes. Fractions of blood may comprise any fraction of blood comprising blood cells (white and/or red), plasma and/or solutes and/or sub-cellular components (e.g. fractions of cells, such as platelets, components of degraded cells, etc.), proteins, lipids, antibodies, etc..

The present methods may be used to treat any types of cells, including but not limited to, cellular materials derived from tissues and organs, including, but not limited to, pancreatic islet cells, chondrocytes, cells of neural origin, cells of hepatic origin, cells of opthalmolic origin, cells of orthopedic origin, cells from connective tissues, and cells of reproductive origin, and cells of cardiac and cardiovascular origin.

Stem cells include adult stem cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), peripheral blood stem cells, umbilical cord blood stem cells, mesenchymal stem cells, stem cells derived from tissues and organs or other sources, including fetal and/or embryonic sources, as well as mixtures of stem cells with other cells and from different sources. Adult stem cells include bone marrow stem cells, hematopoietic stem cells, skin stem cells, ocular stem cells, neural stem cells, cardiac stem cells, etc..

In certain embodiments, the stem cells of endodermal origin are pulmonary epithelial stem cells, gastrointestinal tract stem cells, pancreatic stem cells or hepatic oval cells and/or progenitor cells thereof. In particular embodiments, the cells of urogenital origin are either categorized as mammary and prostatic gland stem cells or ovarian and testicular stem cells and/or progenitor cells thereof. In particular embodiments, the cells of mesodermal origin are bone marrow cells, hematopoietic stem cells, stromal stem cells or cardiac stem cells and/or progenitor cells thereof. In particular embodiments, the cells of ectodermal origin are neural stem cells, skin stem cells or ocular stem cells and/or progenitor cells thereof.

Cell types that may be treated using the methods of the present disclosure include, for example, differentiated cells, such as fibroblasts, epithelial cells, cardiomyocytes, hepatocytes, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-cells, T-cells, erythrocytes, macrophages, monocytes, or muscle cells; and undifferentiated cells, such as embryonic, mesenchymal, or adult stem cells. The cells can be haploid, diploid, or tetraploid. Other cells include cells from the bladder, brain, esophagus, fallopian tube, heart, intestines, gallbladder, kidney, liver, lung, ovaries, pancreas, prostate, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, or uterus.

In further particular embodiments, the cells are obtained from adult brain, bone marrow, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, or other adult tissues. In particular embodiments, the cells are selected from the group consisting of endodermal, urogenital, mesodermal or ectodermal origin.

Tissues include cornea, cartilage, bone, skin, heart valves, Islets of Langerhans, embryos from humans, animals, fish, shellfish and plants, and ovarian tissues from humans and animals. The present compositions and methods may also treat engineered tissues and tissue constructs.

In certain embodiments, the present methods can be used to treat oocytes or sperm in assisted reproductive technology, or for patients undergoing chemotherapy or radiation therapy. The method can also be used for the treatment of stem cells, which can then be used as the basis of stem cell-based therapies, cell transplantation, tissue engineering, and regenerative medicine. The method can also be used to treat oocytes or sperm from an animal that is rare or at risk of becoming extinct for future use in assisted reproductive technologies for the preservation of the species. The method can further be used for animal husbandry purposes (e.g., the breeding and raising of animals), for example, for treating embryonic stem cells, gametocytes, oocytes, or sperm from animals such as cows, pigs, and sheep.

The biological material may be used in the treatment of a variety of diseases. For example, ocular cells are used to treat ocular diseases including, but not limited to age related macular degeneration (wet or dry), diabetic macular edema, idiopathic choroidal neovascularization, or high myopia macular degeneration. In some ocular embodiments, RPE cells are used. In several embodiments, cardiac stem cells are used to treat cardiovascular disorders such as myocardial infarction, ischemic cardiac tissue damage, congestive heart failure, aneurysm, atherosclerosis-induced events, cerebrovascular accident (stroke), and coronary artery disease. In several embodiments, liver stem cells are used to treat liver disease such as hepatitis, cirrhosis, cancer, and the like. Diseases in other tissues, such as the kidney, lung, pancreas, intestine, bone and/or cartilage, and neural tissues, among others, may be treated with the methods and devices disclosed herein. In some embodiments, harvested bone marrow stem cells may be used to repopulate hematopoietic cells that are reduced due to leukemias, cancers, or therapies that reduce blood cell counts.

Cellular therapy, or cell therapy, can generally encompass transplantation of human or animal cells to replace or repair damaged tissue and/or cells. Cell therapy has been used to rebuild damaged cartilage in joints, repair spinal cord injuries, strengthen a weakened immune system, treat autoimmune diseases, and help patients with neurological disorders such as Alzheimer's disease, Parkinson's disease, and epilepsy. Further uses have included treatment of a wide range of chronic conditions such as arteriosclerosis, congenital defects, and sexual dysfunction.

Cell therapy typically involves the injection of either whole cells or cell extracts that are xenogenic, allogenic (from another human donor), or autologous (wherein the cells are extracted from and transplanted back into the same patient).

Viruses or viral particles can be any viruses. The viruses or viral particles comprises adenoviruses, adeno-associated viruses, retroviruses, herpes viruses and the like. The viruses or viral particles are for example those which may be used in gene therapy.

The present specification also describes a kit comprising the present cell stabilizing medium (in solid or liquid form as described herein). Such kits may include one or more containers comprising the cell stabilizing medium. The kit comprises for example the cell stabilizing medium. The kit comprises for example the biological material for treating with the present cell stabilizing medium.

The kit can comprise instructions for use in any of the methods described herein. The kit comprises instructions for treating a biological material using the cell stabilizing medium and method. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. The instructions comprise a description of the biological material treated with the present cell stabilizing medium to be administered to a subject who is in need of the treatment. Instructions supplied in the kits are for example written instructions on a label or package insert. The label or package insert may also indicate clinical and/or research applications of the biological material.

Parts of a kit may be used simultaneously or chronologically staggered, i.e., at different points in time and with equal or different time intervals for any component of a kit. Time intervals can be selected to obtain the desired effect.

The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, a vial (e.g., a cryovial), a bottle, an ampoule, a tube (e.g., a cryotube), a bag, a flask, a jar, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as a freezing container, a cryovial and/or a cryotube.

Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. The disclosure provides articles of manufacture comprising contents of the kits described above.

Gelatin was obtained from Gelita (gelatin was prepared from bovine hides; Batch No. L600217). A cell stabilizing medium containing <NUM> wt% gelatin was prepared by dissolving gelatin in DMEM.

FE002-SK2 cells, which are fetal skin fibroblast cells, in a cryopreservation composition comprising glycerol were thawed from cryopreservation. This cell suspension was then mixed with the cell stabilizing medium to form a mixture.

As a control sample, cells in a cryopreservation composition comprising glycerol were thawed from cryopreservation, and then the cell suspension was mixed with DMEM to form a mixture.

The volume ratio of the cell stabilizing medium (or DMEM for the control samples) to the cell suspension is <NUM>. The cells (in the cell suspension) and the cell stabilizing medium (or DMEM for the control samples) were mixed by aspiration using <NUM>µL PIPETMAN® and tips.

The mixture was incubated at <NUM> or <NUM> for <NUM> hour (Time <NUM>, no incubation), <NUM> hours, or <NUM> hours. Samples for each condition were duplicated. The total number of the cells, the number of the live cells and the number of the dead cells were assayed by using ADAM-MC Automatic Cell Counter (Digital Bio). Viability of the cells was calculated by the following formula: [(live cell number)/(total cell number)] × <NUM>%.

As shown in Table <NUM>, cells in the cell stabilizing medium comprising gelatin maintained their viability after being incubated at <NUM> or <NUM> for <NUM> hours or <NUM> hours. In other words, after being incubated at <NUM> or <NUM> for <NUM> hours or <NUM> hours, the viability of the cells was similar to their viability at Time <NUM>. In contrast, the viability of the cells mixed with DMEM reduced about <NUM>% (<NUM> hours) or <NUM>% (<NUM> hours) after being incubated at <NUM> or <NUM> for <NUM> hours or <NUM> hours. This experiment confirms the protective effect of the cell stabilizing medium containing gelatin.

Gelatin was obtained from GELITA (Lot. A cell stabilizing medium containing <NUM> wt% gelatin was prepared by dissolving gelatin in water.

Cells in a cryopreservation composition comprising glycerol were thawed from cryopreservation. This cell suspension was then mixed with the cell stabilizing medium to form a mixture. The volume ratio of the cell stabilizing medium to the cell suspension is <NUM>. The cells (in the cell suspension) and the cell stabilizing medium were mixed by aspiration using <NUM>µL PIPETMAN® and tips.

The mixture was incubated at <NUM> for <NUM> hour (Time <NUM>, no incubation), <NUM> hour, <NUM> hours, <NUM> hours, <NUM> hours, or <NUM> hours. Samples for each condition were duplicated. The total number of the cells, the number of the live cells and the number of the dead cells were assayed by using ADAM-MC Automatic Cell Counter (Digital Bio). Viability of the cells was calculated as described in herein.

As shown in Table <NUM>, cells in the cell stabilizing medium comprising gelatin maintained their viability after being incubated at <NUM> up to <NUM> hours (for <NUM> hour, <NUM> hours or <NUM> hours). In other words, after being incubated at <NUM> for up to <NUM> hours, the viability of the cells was similar to their viability at Time <NUM>. After <NUM> hours of incubation at <NUM>, the cell viability can still be maintained to be above <NUM>%. This experiment confirms the protective effect of the cell stabilizing medium containing gelatin.

Cells in a cryopreservation composition comprising DMSO were thawed from cryopreservation. This cell suspension was then mixed with the cell stabilizing medium to form a mixture. As a control sample, cells in a cryopreservation composition comprising DMSO were thawed from cryopreservation, and then the cell suspension was mixed with DMEM or <NUM>% FBS/DMEM to form a mixture.

The volume ratio of the cell stabilizing medium (or DMEM for the control samples) to the cell suspension is <NUM>. The cells (in the cell suspension) and the cell stabilizing medium (or DMEM, or <NUM>% FBS/DMEM, for the control samples) were mixed by aspiration using <NUM>µL PIPETMAN® and tips.

The mixture was incubated at <NUM> for <NUM> hour (Time <NUM>, no incubation), <NUM> hours, <NUM> hours, or <NUM> hours. Samples for each condition were duplicated. The total number of the cells, the number of the live cells and the number of the dead cells were assayed by using ADAM-MC Automatic Cell Counter (Digital Bio). Viability of the cells was calculated.

As shown in Table <NUM>, cells in the cell stabilizing medium comprising gelatin maintained their viability after being incubated at <NUM> for <NUM> hours or <NUM> hours. In other words, after being incubated at <NUM> for <NUM> hours or <NUM> hours, the viability of the cells was similar to their viability at Time <NUM>. After <NUM> hours of incubation at <NUM>, the cell viability can still be maintained to be above <NUM>%. In contrast, the viability of the cells mixed with DMEM reduced about <NUM>% (<NUM> hours), <NUM>% (<NUM> hours), or <NUM>% (<NUM> hours) after being incubated at <NUM> for up to <NUM> hours. Similarly, the viability of the cells mixed with <NUM>% FBS/DMEM reduced about <NUM>% (<NUM> hours) or <NUM>% (<NUM> hours or <NUM> hours) after being incubated at <NUM> for up to <NUM> hours. This experiment confirms the protective effect of the cell stabilizing medium containing gelatin.

Gelatin was obtained from Nippi (gelatin was prepared from bovine, swine and/or fish, etc. source; Lot No. S150806). A cell stabilizing medium containing <NUM> wt% gelatin was prepared by dissolving gelatin in water.

Cells in a cryopreservation composition comprising glycerol were thawed from cryopreservation. This cell suspension was then mixed with the cell stabilizing medium to form a mixture. The volume ratio of the cell stabilizing medium to the cell suspension is <NUM>.

Differently from Experiment Nos. <NUM>-<NUM>, in this experiment, the cells (in the cell suspension) and the cell stabilizing medium were mixed by aspiration using a syringe with a needle. In a clinical setting, an <NUM> needle connected to a syringe will be used to aspirate and mix cells and the cell stabilizing medium.

The mixture was incubated at <NUM> for <NUM> hour (Time <NUM>, no incubation), <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, or <NUM> hours. Samples for each condition were either triplicated or duplicated. The total number of the cells, the number of the live cells and the number of the dead cells were assayed by using ADAM-MC Automatic Cell Counter (Digital Bio). Viability of the cells was calculated as described in herein.

As shown in Table <NUM>, cells in the cell stabilizing medium comprising gelatin maintained their viability after being incubated at <NUM> up to <NUM> hours (for <NUM> hours, <NUM> hours, or <NUM> hours). In other words, after being incubated at <NUM> for up to <NUM> hours, the viability of the cells was similar to their viability at Time <NUM>. After <NUM> hours of incubation at <NUM>, the cell viability slightly decreased. After <NUM> hours of incubation at <NUM>, the cell viability decreased to about <NUM>%. This experiment confirms the protective effect of the cell stabilizing medium containing gelatin.

Gelatin was obtained from Nippi (Lot. A cell stabilizing medium containing <NUM> wt% gelatin was prepared by dissolving gelatin in water.

The cells (in the cell suspension) and the cell stabilizing medium were mixed by aspiration using a syringe with a needle. In a clinical setting, an <NUM> needle connected to a syringe will be used to aspirate and mix cells and the cell stabilizing medium.

The mixture was incubated at <NUM> or <NUM> for <NUM> hour (Time <NUM>, no incubation), <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, or <NUM> hours, or <NUM> hours. Samples for each condition were either triplicated or duplicated. The total number of the cells, the number of the live cells and the number of the dead cells were assayed by using ADAM-MC Automatic Cell Counter (Digital Bio). Viability of the cells was calculated as described in herein.

As shown in Table <NUM>, cells in the cell stabilizing medium comprising gelatin maintained their viability after being incubated at <NUM> for up to <NUM> hours (for <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, or <NUM> hours). In other words, after being incubated at <NUM> for up to <NUM> hours, the viability of the cells was similar to their viability at Time <NUM>. Cells in the cell stabilizing medium comprising gelatin maintained their viability after being incubated at <NUM> for up to <NUM> hours (for <NUM> hours, <NUM> hours, <NUM> hours, or <NUM> hours). In other words, after being incubated at <NUM> for up to <NUM> hours, the viability of the cells was similar to their viability at Time <NUM>. After <NUM> hours of incubation at <NUM>, the cell viability can still be maintained to be above <NUM>%.

Cells in a cryopreservation composition comprising glycerol were thawed from cryopreservation. This cell suspension was then mixed with the cell stabilizing medium to form a mixture.

The mixture was incubated at <NUM> for <NUM> hour (Time <NUM>, no incubation), or <NUM> hours. The total number of the cells, the number of the live cells and the number of the dead cells were assayed by using ADAM-MC Automatic Cell Counter (Digital Bio). Viability of the cells was calculated.

As shown in Table <NUM>, after being incubated at <NUM> for <NUM> hours, the viability of the cells mixed with the cell stabilizing medium containing gelatin was higher than the viability of the cells mixed with DMEM.

The volume ratio of the cell stabilizing medium to the cell suspension is <NUM>. The cells (in the cell suspension) and the cell stabilizing medium were mixed by aspiration using <NUM>µL PIPETMAN® and tips.

The mixture was incubated at <NUM> or <NUM> for <NUM> hour (Time <NUM>, no incubation), <NUM> hours, or <NUM> hours. Samples for each condition were duplicated. The total number of the cells, the number of the live cells and the number of the dead cells were assayed by using ADAM-MC Automatic Cell Counter (Digital Bio). Viability of the cells was calculated.

As shown in Table <NUM>, cells in the cell stabilizing medium comprising gelatin maintained their viability after being incubated at <NUM> or <NUM> for <NUM> hours or <NUM> hours. In other words, after being incubated at <NUM> or <NUM> for <NUM> hours or <NUM> hours, the viability of the cells was similar to their viability at Time <NUM>.

The mixture was incubated at <NUM>, <NUM>, or <NUM> for <NUM> hour (Time <NUM>, no incubation), <NUM> hour, or <NUM> hours. Samples for each condition were duplicated. The total number of the cells, the number of the live cells and the number of the dead cells were assayed by using ADAM-MC Automatic Cell Counter (Digital Bio). Viability of the cells was calculated as described in herein (Table <NUM>).

The experimental conditions are shown in Table <NUM>.

Cell stabilizing media containing <NUM> wt%, <NUM> wt%, or <NUM> wt% gelatin was prepared. The cell stabilizing medium was incubated in a <NUM> water bath to ensure conversion to fluid phase before being mixed with the cells.

Cells were expanded, harvested and cryopreserved. Before the experiment, <NUM> - <NUM> cryotubes of FE002-SK2 cells (passage <NUM>; P12), which are fetal skin fibroblast cells, were thawed. Every cryotube contained <NUM> x <NUM><NUM> cells in <NUM>. The contents of all of the <NUM> - <NUM> cryotubes were combined and mixed.

For the cell stabilizing medium (comprising gelatin) group:.

Similarly, cell viabilities were also tested for the <NUM>-fold dilution and <NUM>-fold dilution at <NUM>.

The results show that the post-thaw cell viability was higher when incubated with the cell stabilizing medium comprising <NUM> wt%, <NUM> wt% or <NUM> wt% of gelatin, compared to DMEM. At <NUM> or <NUM>, the cells diluted <NUM>-fold with the cell stabilizing medium exhibited a higher and prolonged viability compared to the cells diluted <NUM>-fold with the cell stabilizing medium.

Table <NUM> and <FIG> show the effects of the working concentrations of gelatin on the cell viabilities, based on the data of Experiment Nos. <NUM>-<NUM> ("Exp. <NUM>" to "Exp. <NUM>") in Example <NUM> and Example <NUM>.

ADAM Accuchip and ADAM Solution were from NanoEnTek. DMEM and fetal bovine serum (FBS) were from Gibco. Bovine serum albumin was from Sigma.

Testing solutions (cell stabilizing medium, DMEM, or FBS) include:.

Fibroblast cells (FE002-SK2) were frozen in a cryopreservation composition comprising glycerol at the concentration of <NUM> × <NUM><NUM>cells/mL.

<NUM>µL of each of testing solutions A-F were dispensed into <NUM>-mL Eppendorf tubes, with <NUM> tubes for each solution.

The cryopreserved fibroblast cells were thawed in a <NUM> water bath. All cell suspensions were pooled into one tube. <NUM>µL thawed cell suspension was added to each of the Eppendorf tubes containing <NUM>µL of the testing solution to form a mixture. The volume ratio of the testing solutions (cell stabilizing medium, or DMEM, or FBS) to the cell suspension is <NUM>.

The mixture was incubated at <NUM> for <NUM> hours. Samples for each condition were triplicated. The total number of the cells, the number of the live cells and the number of the dead cells were assayed by using ADAM-MC Automatic Cell Counter (Digital Bio). Viability of the cells was calculated by the following formula: [(live cell number)/(total cell number)] × <NUM>%.

As shown in Table <NUM>, after being incubated at <NUM> for <NUM> hours, the viability of the cells in the cell stabilizing medium comprising gelatin was about <NUM>%. The viabilities of the cells in the cell stabilizing medium comprising <NUM> wt%, <NUM> wt%, or <NUM> wt% albumin were about <NUM>%, <NUM>% and <NUM>%, respectively. Gelatin-based solution was able to maintain thawed cell viability better than other tested solutions.

Claim 1:
A method for maintaining post-thaw cell viability of at least <NUM>%, the method comprising the steps of:
providing a cell suspension comprising one or more cells which have been thawed from a cryopreserved state; and
mixing the cell suspension with a cell stabilizing medium to form a mixture,
wherein the cell stabilizing medium is a thermoreversible hydrogel in a liquid state when mixed with the cell suspension that has been thawed and comprises <NUM> wt% to <NUM> wt%, <NUM> wt% to <NUM> wt%, or <NUM> wt% to <NUM> wt% of gelatin based on the total weight of the cell stabilizing medium,
and
wherein the volume ratio of the cell stabilizing medium to the cell suspension ranges from <NUM> to <NUM>, <NUM> to <NUM>, or <NUM> to <NUM>.