Viable cell compositions and associated methods

Description

BACKGROUND

[0001] Aspects of the present disclosure relate generally to viable cellular compositions and methods of their preparation, storage and use.

[0002] Administration of cellular compositions to humans and animals in the treatment of various pathologies or disorders has become increasingly prevalent and bears hope to improve a multitude of therapies. The ability to stably store viable cells and to then prepare them and deliver them to patients is important. Additives to the compositions for cryopreservation must be effective to preserve viability and also biologically acceptable when ultimately delivered to the patient. As well, to facilitate their broad distribution and use, it is highly beneficial if the compositions can be effectively stored through a range of conditions that are achievable without undue capital or other expense in the manufacturing and / or distribution chain. EP 2 721 930 relates to a solution for cryopreservation, a method for manufacturing the same and the use of such a solution. US 4 004 975 provides a method of salvaging and cryopreserving granulocytes, or white cells, form buffy coat of centrifuged whole blood. US 2012 / 149108 provides a method for cryopreservation of cells that is capable of maintaining a high viability rate after thawing, and a cryopreservation solution used in the cryopreservation method. Maruyama et al. Transplant Proc. (2004); 36(4):1133-1134 describes a method for cryopreservation of islets using hydroxyethyl starch (HES) and dimethyl sulfoxide (DMSO). The authors identified that the use of HES allowed a decrease in DMSO concentration and simplified the freeze-thawing procedure. Stiff et al. Blood (1987); 70(4): 974-8 describes changes that were made to the standard method of bone marrow cryopreservation in three areas: the cryoprotectant, the method of cell freezing and the storage temperature to develop a simplified cryopreservation method. Stolzing et al. Transfus Apher Sci. (2012); 46 (2): 137-47 discusses the use of hydroxyethyl starch (HES) as a cryoprotectant. Chen et al. Stem Cells Int. (2016); Article ID 1396783 identified that low concentration of DMSO with the addition of trehalose might improve the cryopreservation outcome. In view of the background in the area, there remain needs for improved and / or alternative methods and compositions related to storage-stable cell preparation, and their distribution, storage and ultimate use. Aspects of the present disclosure are addressed to these needs.SUMMARY

[0003] It has been discovered that highly robust, storable, viable cell preparations can be achieved when incorporating controlled concentrations of additives that help to preserve viability during cryopreservation, but which ultimately provide easy access to therapeutic, viable cell compositions for administration to patients. The present invention is defined in the independent claims and certain optional features thereof are defined in the dependent claims. In so far as the terms "invention", "aspect", "example" and "embodiment" are used herein, this will be interpreted in such a way that the only protection sought is for the invention as claimed. The present invention provides a method for preparing viable cells, comprising: thawing a cryopreserved composition that includes viable cells and an aqueous cryopreservation medium, wherein the aqueous cryopreservation medium of the cryopreserved composition consists of dimethylsulfoxide at a concentration of 2% by weight, hydroxyethyl starch at a concentration of 6% by weight and water, to provide a thawed viable cell composition having the viable cells suspended in an aqueous liquid; and combining an aqueous liquid diluting medium with the thawed viable cell composition, preferably in a volume ratio of at least 2:1 and more preferably at least 5:1, to provide a diluted viable cell composition containing hydroxyethyl starch and dimethylsulfoxide, wherein the dimethylsulfoxide is present in the diluted viable cell composition at a concentration of 0.5% by weight or less. The present invention also provides a method for maintaining viable cells, comprising: maintaining in a cryopreserved condition a composition that includes viable cells and an aqueous cryopreservation medium, wherein the aqueous cryopreservation medium consists of dimethylsulfoxide at a concentration of 2% by weight, hydroxyethyl starch at a concentration of 6% by weight and water, wherein said maintaining comprises maintaining the composition at a temperature in the range of -60°C to -100°C, and wherein said maintaining comprises cooling the composition in a mechanical freezer.DETAILED DESCRIPTION

[0004] The technical information set out below may in some respects go beyond the scope of the invention, which is defined exclusively by the appended claims. The additional technical information is provided to place the actual invention in a broader technical context and to illustrate possible related technical developments.

[0005] In addition, incidental references to methods for treatment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body are not to be construed as claiming protection for such methods as such, but are instead to be construed as referring to products, in particular substances or compositions, for use in any of these methods.

[0006] A wide variety of cell types may be used in embodiments of the present disclosure. For example, the cells can be skin cells, skeletal muscle cells, cardiac muscle cells, lung cells, mesentery cells, adipose cells, or stem cells such as mesenchymal stem cells. Adipose cells may be from omental fat, properitoneal fat, perirenal fat, pericardial fat, subcutaneous fat, breast fat, or epididymal fat. In certain embodiments, the cells comprise stromal cells, stem cells, or combinations thereof. As used herein, the term "stem cells" is used in a broad sense and includes traditional stem cells, adipose derived stem cells, progenitor cells, preprogenitor cells, reserve cells, and the like. Exemplary stem cells include embryonic stem cells, adult stem cells, pluripotent stem cells, neural stem cells, liver stem cells, muscle stem cells, muscle precursor stem cells, endothelial progenitor cells, bone marrow stem cells, chondrogenic stem cells, lymphoid stem cells, mesenchymal stem cells, hematopoietic stem cells, central nervous system stem cells, peripheral nervous system stem cells, and the like. Additional illustrative cells which can be used include hepatocytes, epithelial cells, Kupffer cells, fibroblasts, neurons, cardiomyocytes, myocytes, chondrocytes, pancreatic acinar cells, islets of Langerhans, osteocytes, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, biliary epithelial cells, and progentior cells of any of these cell types.

[0007] When used, mesenchymal stem cells (MSC) can be obtained from any suitable tissue. These include as examples MSCs derived from dental tissue (such as those harvested from dental pulp, periodontal ligaments, or other dental tissues), testicle tissue, bone marrow; peripheral blood, placental tissue, uterine tissue (including endometrial regenerative cells), umbilical cord blood, umbilical cord tissue, or skin tissue (including full thickness skin tissue). These or other MSCs can be used in aspects of the present disclosure. The MSCs can be generally an adherent cell population expressing markers CD90 and CD105 (>90%) and lacking expression of CD34 and CD45 and MHC class II (<5%) as detected by flow cytometry.

[0008] The cells used in the embodiments herein can be from any suitable species of animal, for example a mammal, such as a human, canine (e.g. dog), feline (e.g. cat), equine (e.g. horse), porcine, ovine, caprine, or bovine mammal.

[0009] The cells, in a viable state, will be combined with an aqueous cryopreservation medium that includes dimethylsulfoxide (DMSO) and a carbohydrate which in preferred embodiments is hydroxyethyl starch (HES). HES and DMSO are readily available commercially. The combination of the cells with the DMSO and HES can be conducted in any suitable fashion, including by combining the viable cells with a solution containing both DMSO and HES, or by combining the cells with separate solutions, one of which contains the DMSO and another of which contains the HES. These and other modes of providing the viable cells in the aqueous cryopreservation medium will be suitable. As well, it will be understood that the cryopreservation medium can contain agents other than the DMSO and HES, including other cryopreservation agents. However, in preferred forms, the DMSO and HES will constitute at least 50%, at least 70%, at least 80%, at least 90%, essentially all (98% or more) or all of the cryopreservation medium other than the water. In the invention, the cryopreservation medium includes DMSO at a concentration of 2% and HES at a concentration of 6%.

[0010] In preferred prepared viable cell compositions to be cryopreserved, the viable cells will be present at a concentration of at least about 200,000 cells / ml, at least about 500,000 cells / ml, at least about 1 million cells / ml, at least about 5 million cells / ml, or at least about 10 million cells / ml; and / or the viable cells will be present at a concentration not exceeding about 50 million cells / ml, not exceeding about 30 million cells / ml, or not exceeding about 20 million cells / ml. In certain variants, the viable cells will be present in the cell composition at a concentration in the range of about 500,000 to about 20 million cells / ml, or in the range of about 3 million to about 20 million cells / ml, or in the range of about 5 million to about 15 million cells / ml, or in the range of about 8 million to about 12 million cells / ml. In some forms, the viable cells will be present in the viable cell composition at a concentration of about 10 million cells / ml.

[0011] The viable cell composition including the viable cells and the above-disclosed cryopreservation medium, in unfrozen (liquid) form, can be prepared in, delivered into, or otherwise provided in a cryopreservation container such as a cryopreservation vial or cryopreservation bag. In preferred forms, the cell composition is provided in a cryopreservation vial having features as disclosed in U.S. Patent No. 8,709,797 issued April 29, 2014 .

[0012] Preferred cryopreservation vials will thus include an access port, preferably a needle septum, through which the viable cells can be sterilely accessed with a needle or other cannulated device after a period of storage (e.g. in cryopreserved state) in the vial. The preferred vials will also include two additional ports to the interior of the vial, which can be used as filling and vent ports (the vent port preferably containing a sterile filter). These two additional ports can be fitted with heat sealable tubing, which can be heat sealed to seal the vial after filling, and which can thereafter be cut prior to accessing the cells in the vial, for example to provide a vent for needle-based removal of the cell composition through the needle septum. In preferred forms, each cryopreservation container will contain about 1ml to about 10ml of the cell composition, more preferably about 1ml to about 5ml of the composition, and most preferably about 2ml to about 3ml of the composition. Preferred cryopreservation vials for use in embodiments herein are commercially available as CellSeal ®< Cryogenic Vials (Cook Regentec LLC, Bloomington, IN).

[0013] The cryopreservation container containing the viable cell composition can be subjected to cryopreservation conditions to cause the composition to be cryopreserved. For example, the cryopreservation conditions can include storing the cryopreservation container at a temperature at which the viable cell composition freezes. For example, the cell composition can be maintained in a cryopreserved state at a temperature in the range of about -60°C or lower, for example in the range of about -60°C to -150°C, more preferably about-60°C to -100°C, and even more preferably about -70°C to -90°C. In certain forms, the cell composition can be maintained in a cryopreserved state at a temperature of about -80°C ± 5°C. In most preferred forms, the cryopreservation container is stored in a mechanical freezer (a freezer, typically electrically-powered, that uses a re-circulating refrigerant within an air cooler that exchanges heat from air circulating within the freezer to reduce the temperature of contents within the freezer) to maintain the cell composition in a cryopreserved state at the stated temperatures. Advantageously, this avoids the need to use liquid nitrogen tanks for cryopreservation storage, at least during a storage phase when the mechanical freezer is used (e.g. a mechanical freezer in operation at a point of care location to which the viable cell composition is shipped, stored in the mechanical freezer, thawed and then used, e.g. to treat a patient). Thus, in some embodiments, the cryopreservation container containing the viable cell composition can be stored under cryopreservation conditions (e.g. immersed in liquid nitrogen and / or in a mechanical freezer as disclosed herein) at the manufacturing facility, a distribution facility, or another facility that is a distance from a point of care facility, the cryopreservation container containing the viable cells can be shipped to the point of care while maintaining cryopreservation conditions (e.g. in a shipping package containing a liquid nitrogen system and / or dry ice - frozen carbon dioxide), and the cryopreservation container containing the viable cells can be stored at the point of care (e.g. a veterinary clinic or hospital) under cryopreservation conditions until needed for administration, preferably within a mechanical freezer as disclosed herein.

[0014] Preferred viable cell compositions containing the cells, HES and DMSO exhibit good capacity to maintain the viability of the cells during storage at a temperature in the range of about -60°C to -100°C. For example, preferred compositions will lose no more than 20% of their initial viable cells, no more than 10% of their viable cells, or no more than 5% of their viable cells, when stored at a temperature in the range of -60°C to -100°C for a period of six months, and more preferably when stored at a temperature in the range of -60°C to -100°C for a period of one year. Such storage stabilities under the relatively moderate cooling conditions enable advantageous product distribution and use methods in which multiple point of care locations (e.g. 10 or more, or 20 or more locations) can maintain mechanical freezers in which a plurality of cryopreservation containers each containing a cell composition as described herein are stored. The stability of the viability of the cells under these storage conditions can provide an acceptable shelf life at the point of care. Immediately prior to administration (e.g. within 6 hours, or within 2 hours, or within 1 hour), a cryopreservation container can be removed from the freezer and the composition therein thawed for additional manipulation (e.g. dilution as described below) and / or administration.

[0015] In some methods herein, to thaw the cell composition, the cryopreservation container can be caused to warm to a temperature at which the frozen cell composition reverts to a liquid form. This can be achieved in any suitable manner. For example, the container, remaining sterilely sealed, can be exposed to a gaseous environment (e.g. the room air environment at the point of care) at room temperature (e.g. about 20°C to 25°C) to thaw the cell composition. In another form, the container can be incubated in a heated liquid bath to thaw the cell composition, for example heated to a temperature in the range of about 33°C to 37°C, preferably about 37°C. The cryopreservation and subsequent thawing of the cell composition will preferably result in high maintenance of cell viability, for example with the thawed composition retaining at least 80%, at least 90%, or at least 95% of the viable cells of the initial cell composition prior to cryopreservation.

[0016] After thawing, the viable cell composition can be removed from the cryopreservation container. For example, in some embodiments, the cryopreservation container has a septum through which a needle or other cannulated device may be passed to access the container contents, while maintaining a sterile environment within the container. The needle or other cannulated device can then be used to withdraw the cell composition from the container sterilely, for example into a syringe barrel or other vessel. It will be understood that other methods for accessing the cell composition within and removing the viable cell composition from the cryopreservation container may be used in other embodiments.

[0017] In some forms, the viable cell composition can be administered to a patient, or put to a research or other use, unmodified from its composition upon removal from the cryopreservation container. In some embodiments, the viable cell composition is modified for use, including for use in administration to a patient. The modification can include, for example, the addition of one or more substances to the viable cell composition.

[0018] In preferred methods, the viable cell composition is combined with an aqueous liquid diluting medium to dilute the cell concentration of the composition, and potentially also dilute the concentration of other components, such as HES and / or DMSO, of the viable cell composition. In beneficial aspects, the diluting aqueous liquid medium can be a physiologically acceptable aqueous liquid, such as a phosphate-buffered or otherwise buffered saline solution, potentially with other additives. Also, it will be understood that the diluting aqueous liquid medium can be combined with the viable cell composition as a single volume of a given composition, or as multiple volumes of the same composition or of differing compositions.

[0019] In preferred methods, to prepare a diluted viable cell composition for administration to a patient or other use, a diluting aqueous liquid medium will be combined with the cell composition in a volume ratio of at least 2:1, at least 3:1, at least 5:1, or at least 8:1. In more preferred forms, such volume ratio will be in the range of about 3:1 to about 20:1, or about 5:1 to about 15:1. It is contemplated that the cell concentration, DMSO concentration and HES concentration in the prepared diluted composition will be correspondingly reduced relative to the starting cell composition prior to dilution; however, in some forms the diluting aqueous liquid medium may itself contain some concentration of cells, HES and / or DMSO, albeit typically lower than the concentration in the starting cell composition so as to result in some dilution of cells, HES and DMSO in the diluted cell composition.

[0020] The combination of the thawed cell composition with the diluting aqueous liquid medium can be conducted in any suitable container or vessel. In beneficial modes, the combination is conducted in a second container (other than the cryopreservation or other container in which the cells were stored or held). This second container can include an input port or other input member, for example a septum, for sterile transfer of materials such as the cell composition and / or the diluting aqueous liquid medium into the second container. In some forms, combining the cell composition and diluting aqueous liquid medium can include delivering both of these into the second container, for example in either order or simultaneously. In other forms, the second container can be provided as a pre-manufactured container already containing the diluting aqueous liquid medium in sterile condition, and the cell composition can be added to the pre-manufactured container. In any of these embodiments, the second container can be a bag and / or can have an outlet port spaced from the input port or other member for delivery of the prepared diluted cell composition from the bag or other container, e.g. for delivery into a patient. The second container can be a bag having a septum for sterile input of materials and a valved port for outlet of materials, e.g. as occurs in common saline bags for patient treatment in medical care. The thawed cell composition, and potentially the trehalose-containing medium (if not pre-manufactured in the bag) can be sterilely delivered into the bag by needle through the septum, and the prepared diluted cell composition can be sterilely delivered to a patient through the valved port.

[0021] In certain embodiments, the diluting aqueous liquid medium will be combined with the viable cell composition, and the prepared diluted cell composition will have a DMSO concentration that has been reduced from its initial level (e.g. any of those levels indicated above for the cryopreserved cell composition) to a diluted concentration of about 0.5% or less, more preferably about 0.3% or less, and in some embodiments to a diluted concentration in the range of about 0.05% to about 0.5% or in the range of about 0.1% to about 0.3%. In addition or alternatively, the diluting aqueous liquid medium will be combined with the cell composition, and the prepared diluted cell composition will have an HES concentration that is greater than that of the DMSO and / or which is at least about 0.1%, more preferably at least about 0.3%, and in some embodiments a concentration in the range of about 0.1% to about 6% or in the range of about 0.3% to about 2%. HES (at 6% concentration) is a known volume extender for administration to the bloodstream, and thus it is contemplated that the diluting aqueous liquid medium can in some embodiments contain significant levels of HES while nonetheless preparing a diluted cell composition well suited for administration to the bloodstream of a human or other animal patient. Thus in some modes of operation, the diluted composition can have a DMSO concentration that is reduced to a greater extent than the reduction, if any, of the HES concentration, as compared to the starting cell composition prior to dilution. In regard to the cell concentration in the diluted cell composition, the diluting aqueous liquid medium will typically not contain any cells, and the cell concentration of the diluted viable cell composition will the lower than that of the starting cell composition, for example in the range of about 100,000 cells / ml to about 20 million cells / ml, or about 250,000 cells / ml to about 5 million cells / ml, or about 500,000 cells / ml to about 2 million cells / ml. In certain forms, the cell concentration of the prepared diluted cell composition will be about 1 million cells / ml.

[0022] In certain embodiments herein, the diluting aqueous liquid medium will contain trehalose. Trehalose, also known as mycose or tremalose, is an alpha-linked disaccharide formed by an α,α-1,1-glucoside bond between two α-glucose units. It has a chemical name of (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxane-3,4,5-triol (IUPAC naming convention). The diluting aqueous liquid medium can contain any suitable concentration of trehalose for these purposes. In certain aspects, the diluting aqueous liquid medium will contain trehalose at a concentration of about 1% to about 20% by weight, or about 1% to about 10% by weight, or about 2% to about 7% by weight, or about 2.5% to about 5% by weight, or about 3% to about 4% by weight. The diluting aqueous liquid medium, whether it contains trehalose as specified herein or not, can also include other components and / or have a specified osmolarity. For example, it can include sodium chloride at a physiologically acceptable level, for example at a level in the range of about 0.5% to about 1.5%, e.g. about 0.9% (isotonic). The diluting aqueous liquid medium can also include a buffer, for example phosphate buffer, and can have a pH in the range of about 6 to 8, or about 6.8 to 7.8, or about 7 to 7.5. The diluting aqueous liquid medium can also have an osmolarity in the range of 200 to 600 milliosmols per kilogram (mosm / kg), or 250 to 500 mosm / kg, or 250 to 400 mosm / kg.

[0023] A diluting aqueous liquid medium containing trehalose can be combined with the cell composition containing DMSO and HES, to prepare a trehalose-containing diluted cell composition having a suitable concentration of trehalose, along with HES and DMSO (e.g. with the HES and DMSO in the concentrations for the diluted cell composition specified above). This concentration of trehalose in the prepared diluted viable cell composition, in certain embodiments, is in the range of about 1% to about 10% by weight trehalose or about 2% to about 7% by weight, or about 2.5% to about 5% by weight, or about 3% to about 4% by weight. Additionally or alternatively, the concentration of trehalose can be effective to inhibit clumping of the cells as compared to a corresponding cell composition without the trehalose. Inhibition of clumping can be observed by the formation of fewer and / or smaller clumps of cells in the prepared cell composition, for example at a time point at least ten minutes after preparation of the cell composition, at least twenty minutes after preparation of the cell composition, or at least after 60 minutes after preparation of the cell composition. The capacity of the trehalose to inhibit clumping for significant periods of time following preparation of the cell composition can, for example, provide sufficient time to administer the prepared cell composition to a patient, for example by injecting or infusing the cell composition into the bloodstream of a patient by venous or arterial access and / or by local implantation of the cell composition. In therapeutic applications of cell compositions, the composition can be administered to the patient over a relatively prolonged period of time, for example at least 10 minutes, at least 20 minutes, or at least 60 minutes.

[0024] As disclosed herein, the prepared diluted viable cell composition can have specified concentrations of cells, DMSO, HES and / or trehalose. Where minimum or maximum concentrations, or ranges of concentrations, are stated, it will be understood that the conditions of the dilution of the starting viable cell composition can be controlled to achieve the stated amounts in the prepared diluted viable cell composition. These conditions include, for example, the concentrations of components in the starting viable cell composition prior to dilution, the volume ratio of the diluting aqueous liquid medium used relative to the starting viable cell composition, and the concentrations (if any) of the identified components in the diluting aqueous liquid medium.

[0025] The prepared diluted cell composition can be put to any suitable use, including for example research or therapeutic uses. For therapeutic use, the cell composition may as examples be administered to a human or animal patient to treat or prevent a disease or condition such as degenerative bone disease, osteoarthritis, rheumatoid arthritis, polyarthritis, systemic lupus erythematosus, inflammatory bowel disease, atopy, hepatitis, chronic steroid responsive meningitis-arteritis, beagle pain syndrome, degenerative myelopathy, chronic renal failure disease, dilated and mitral cardiomyopathy, keratoconjunctivitis sicca, immune mediated non-erosive arthritis, immune mediated hemolytic anemia, immune mediated thrombocytopenia, Evans syndrome, intervertebral disc disease, muscle fibrosis secondary to disease or trauma, refractory corneal ulcer, diabetes mellitus, spinal trauma, eosinophilic granuloma complex, hypertrophic cardiomyopathy, cholangitis, spinal injury, exercise induced pulmonary hemorrhage, rhabdomyolysis, corneal ulcer, eczema, multiple sclerosis, muscular dystrophy, spinal injury, diabetes mellitus, hepatitis, myocardial infarction, congestive heart failure, or muscle fibrosis.

[0026] The prepared diluted cell composition can be administered to a patient in any suitable manner. In certain forms, the cell composition is delivered systemically into the bloodstream of a patient, for example by delivery into a vein or artery. In other forms, the cell composition is delivered topically to the patient (e.g. in the treatment of atopy or other skin disorders). In still other forms, the cell composition is delivered to a local implant site in a patient. Any of these or any combination of these modes of administration may be used in the treatment of a patient. In certain combination treatments, a first amount of a prepared cell composition herein can be delivered systemically into the bloodstream of a patient, and a second amount of a prepared cell composition herein (e.g. prepared with or separately from the first amount and including the same type(s) or a different type(s) of cells) is implanted locally in or near one or more skeletal joints in a patient to treat an arthritic condition, e.g. any of those arthritic conditions identified herein. Also, in patient treatments herein, not covered by the claimed invention, a single administration of a prepared cell composition as described herein can be made in some instances, while in others multiple separate administrations of prepared cell compositions as described herein may be made over time (e.g. weekly or monthly administrations). In further embodiments, the prepared, diluted cell composition can be filtered prior to administration to the patient, e.g. to remove any clumps of cells that may be present. In certain forms, the cell composition can be passed through an in-line filter positioned in tubing through which the cell composition is passed into the bloodstream of the patient, e.g. into a vein or artery of the patient. Such a filter can, in certain variants, have a particle size cutoff of about 200 micrometers (i.e. exclude from passage particles having a maximum cross-sectional dimension of greater than about 200 micrometers) or lower, or a particle size cutoff of about 170 micrometers or lower, or a particle size cutoff of about 100 micrometers or lower, while allowing the passage of singly suspended cells through the filter. Additional instances herein, not covered by the claimed invention, include products useful in preparing diluted cell compositions as described herein. In one instance not covered by the claimed invention, provided is a diluting aqueous liquid medium useful for preparing a diluted cell composition. The diluting aqueous liquid medium can contain those components, and in amounts, as specified herein.

[0027] As well, the diluting aqueous liquid medium can be provided in sterile form in a container that is included in a kit. That container may be a vial, bag or other container. In certain forms, the container has the features of the "second container" discussed hereinabove in which the diluted cell composition can be prepared, including for example having an inlet port or other member (e.g. needle septum) and a separate outlet port as discussed above. Kits disclosed herein may include the container containing the diluting aqueous liquid medium along with one or more additional components, for example including but not limited to a liquid transfer device such as a syringe and attached or attachable needle, and potentially also a container containing the starting cell composition to be used to prepare the diluted cell composition. The container containing the starting cell composition can include the cell composition in a cryopreserved state (e.g. shipped frozen with the kit) or in a non-cryopreserved (e.g. thawed where the cells were previously cryopreserved) state, including instances, not covered by the claimed invention, in which the cryopreserved cell composition contains HES and DMSO in any of those amounts therefor specified above. Kits disclosed herein may also include at least one filter, for example a filter as described above, through which a prepared diluted cell composition can be passed prior to administration into a patient, and / or tubing through which the diluted cell composition can be passed during administration to a patient.

[0028] The following specific Experimental is provided to facilitate a further understanding of aspects of the present disclosure.Example 1 C-URCs Isolation and Culture

[0029] Fully intact uteri are obtained from a local low-cost spay-neuter clinic from female canines that have presented for ovariohysterectomy. Once the samples arrive at the laboratory, the ovaries are removed and discarded then the uterus separated into one gram, full thickness sections.

[0030] A one gram sample is then minced to ≤1mm 3< fragments using a sterile scalpel. The chopped tissue is placed into an enzymatic bath and digested for 30min at 37 °C. Once digestion is complete, the enzymes are neutralized with culture media (DMEM-HG with 10% fetal bovine serum and 0.25 mg / mL amphotericin B, 100 IU / mL penicillin-G, and100 mg / mL streptomycin), centrifuged at 300×g for 5min and re-suspended in fresh culture media. The contents are then strained through a 200 µm sterile membrane and plated in a 25 cm 2< flask. After 14 days of culture, the cells are split as Passage 0 (P0) using TrypZean ™< solution and cell counts and viability are assessed using a standard trypan blue dye exclusion assay and hematocytometer. The resulting cells are termed canine uterine regenerative cells (C-URCs).Example 2 C-URCs Cryopreservation, Thawing, and Dilution

[0031] C-URC's prepared as in Example 1, or prepared by expanding a culture prepared as in Example 1 in a bioreactor, are suspended at a concentration of 10 million cells / ml in a cryopreservation solution containing 2% DMSO and 6% HES, and this cell composition is sterilely filled in 3ml aliquots into respective cryopreservation vials (available as 5ml capacity CellSeal ®< Cryogenic Vials from Cook Regentec LLC, Bloomington, IN). The vials are sealed and then frozen and stored at -80°C in a mechanical freezer for one to twelve months.

[0032] After its storage period, a vial is removed from the freezer and thawed by immersing the vial in a 37°C water bath for several minutes. After thawing, the sterile filter-containing vent tube of the vial is cut to open the vent, and a sterile syringe needle is used to penetrate the needle septum of the vial and withdraw the cell composition into the syringe barrel. The needle is then removed from the vial's septum, and used to penetrate the septum input port of a sterile bag containing 27ml of a buffered physiologic saline solution containing 3.67% trehalose. The prepared, diluted cell composition has a cell concentration of 1 million cells / ml, and DMSO, HES and trehalose concentrations of 0.2%, 0.6% and 3.3%, respectively. The prepared diluted cell composition can be administered to a patient from a valved output port of the bag, for example through medical tubing having an in-line filter to remove clumped cells. The administration can be intravenous.

Claims

1. A method for preparing viable cells, comprising: thawing a cryopreserved composition that includes viable cells and an aqueous cryopreservation medium, wherein the aqueous cryopreservation medium of the cryopreserved composition consists of dimethylsulfoxide at a concentration of 2% by weight, hydroxyethyl starch at a concentration of 6% by weight and water, to provide a thawed viable cell composition having the viable cells suspended in an aqueous liquid; and combining an aqueous liquid diluting medium with the thawed viable cell composition, preferably in a volume ratio of at least 2:1 and more preferably at least 5:1, to provide a diluted viable cell composition containing hydroxyethyl starch and dimethylsulfoxide, wherein the dimethylsulfoxide is present in the diluted viable cell composition at a concentration of 0.5% by weight or less.

2. The method of claim 1, also comprising prior to the thawing: storing a cryopreservation container in a freezer at a temperature in the range of -60°C to - 100°C, the cryopreservation container containing the cryopreserved composition.

3. The method of any one of the preceding claims, wherein the thawed viable cell composition has a concentration of the viable cells in the range of 1 million cells / ml to 20 million cells / ml, and wherein the diluted viable cell composition has a concentration of the viable cells in the range of 200,000 cells / ml to 5 million cells / ml.

4. The method of any one of the preceding claims, wherein said volume ratio is at least 8:1, and wherein the dimethylsulfoxide is present in the diluted viable cell composition at a concentration of 0.3% by weight or less.

5. The method of any one of the preceding claims, wherein the viable cells comprise viable mesenchymal stem cells.

6. The method of any preceding claim, wherein the diluted viable cell composition contains the hydroxyethyl starch at a concentration of at least 0.1% by weight, and more preferably at least 0.3% by weight, and / or wherein the diluted viable cell composition contains the DMSO at a concentration in the range of 0.05% to 0.5% by weight and the hydroxyethyl starch at a concentration in the range of 0.1% to 6% by weight.

7. The method of any preceding claim, wherein the aqueous liquid diluting medium comprises trehalose.

8. A method for maintaining viable cells, comprising: maintaining in a cryopreserved condition a composition that includes viable cells and an aqueous cryopreservation medium, wherein the aqueous cryopreservation medium consists of dimethylsulfoxide at a concentration of 2% by weight, hydroxyethyl starch at a concentration of 6% by weight and water, wherein said maintaining comprises maintaining the composition at a temperature in the range of -60°C to -100°C, and wherein said maintaining comprises cooling the composition in a mechanical freezer.

9. The method of claim 8, wherein the composition has a concentration of the viable cells in the range of 1 million cells / ml to 20 million cells / ml.

10. The method of any one of claims 8 to 9, wherein the viable cells comprise viable mesenchymal stem cells.