Clinical formulations

Abstract

Some aspects of the present disclosure provide clinical media that support viability, re-planking efficiency, and re-proliferative capacity of cells and tissues during storage up to 48 hours or longer. The clinical media provided herein may also be used for clinical irrigation. Also provided are cell or tissue articles comprising a population of cells or tissues and a clinical medium provided herein, as well as methods for making such articles. Also disclosed are methods of using the clinical media and the cell and tissue preparations provided herein, for example, administering an effective amount of a cell or tissue to a subject in need thereof.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of August 18, 2016, application number 201680059201.6, and invention name “Clinical Preparations”.

[0002] Related Applications

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 206,821, filed on August 18, 2015, entitled “Clinical Formulations,” under 35 U.S.C. 119(e), the entire contents of which are incorporated herein by reference. background

[0005] Currently available clinical media for storage and transport of cells and tissues to be transplanted do not support cell viability and function over relatively short periods of time (e.g., 4-6 hours maximum), and even during those short storage times, significant loss of cells and cell function is often observed. In addition, current clinical media containing bicarbonate anions cannot withstand heat sterilization and often form carbon salt precipitation even during ambient temperature storage, resulting in a short shelf life. Summary of the invention

[0006] A variety of medical procedures rely on the use of clinical solutions, for example, for rinsing surgical areas, wound cleaning, post-surgical adhesion prevention, and removal of debris from surgical areas. In the context of cell or tissue implantation or transplantation, clinical solutions are used to formulate the cells or tissues, store the cells or tissues after formulation and prior to administration to a subject, and serve as a medium for carrying cell or tissue constructs during administration (e.g., during injection of cells or tissues). Solutions that come into contact with cells or tissues in a clinical setting (e.g., during rinsing, wound cleaning, injection, etc.) are typically sterile, pyrogen-free, buffered at physiological pH, and exhibit physiological osmolarity.

[0007] One problem associated with currently available surgical irrigation solutions for use during surgical procedures to prevent trauma to sensitive cells or tissues is the use of bicarbonate anions as buffers with salts that can form precipitates with bicarbonate anions (e.g., essentially all ionic salts of calcium and magnesium commonly used as pharmaceutical excipients). When solutions containing bicarbonate and calcium and / or magnesium are heat sterilized, carbonate formation and subsequent precipitation can occur rapidly, and also often occurs over time under ambient storage conditions.

[0008] One possible solution to the precipitation problem is to provide surgical irrigation solutions as a two-part kit, with one part containing a bicarbonate buffer and the other part containing a calcium salt and / or a magnesium salt. The parts are typically mixed together to form a single solution just before use. The use of such two-part solutions is associated with several disadvantages, including the need to prepare two separate solutions, an inconvenient mixing step that represents a risk of mixing errors, and a generally short half-life of the mixed solution. Therefore, a single-part irrigation solution would be advantageous and highly desirable.

[0009] Attempts have been made to prepare single-part rinse solutions. See, for example, European Patent Application EP 1067907B1 (Armitage). Such attempts generally rely on the use of zwitterionic organic buffers such as N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (commonly referred to as HEPES) to prevent the carbonate precipitation problem discussed above. However, zwitterionic organic buffers used in such single-part rinse solutions are generally incompatible with cell or tissue culture media and thus do not provide a broadly applicable solution to the problem of precipitate formation during cell storage.

[0010] In addition to the problems of precipitate formation and incompatibility with common cell culture components as noted above, commonly used surgical irrigation solutions also often contain combinations of components that cannot withstand steam sterilization, such as certain carbohydrates or glutathione disulfide (GSSG).

[0011] Unlike previously developed single-part rinse solutions, the present disclosure provides single-part solutions that do not require the use of zwitterionic organic buffers. The stabilized rinse solutions of the present invention solve the problems of precipitation and the associated short shelf life of current rinse solutions. The solutions provided herein have greatly improved shelf life compared to the shelf life of currently available solutions.

[0012] The solutions provided herein can be used for flushing, cell reconstitution (e.g., cell reconstitution of cryopreserved or precipitated cells), cell storage (e.g., for shipment or transplantation after formulation), transport and / or administration of cells to a subject (e.g., in the context of cell implantation or transplantation). The solutions provided herein can be used in conjunction with different cell types and different sites of administration. Although ophthalmic applications are preferred, other applications are also contemplated and are encompassed by the present disclosure.

[0013] Some aspects of the present disclosure provide solutions for cell reconstruction, storage, transportation and / or administration to a subject. In certain embodiments, the solution comprises (a) a buffer for maintaining the solution at physiological pH; and (b) at least 2 mM glucose; and (c) an osmotically active agent (osmotically active agent) for maintaining the solution at physiological molar osmotic pressure concentration. In certain embodiments, the solution comprises 2-150 mM glucose, for example, 5-150 mM, 10-150 mM, 15-150 mM, 2-100 mM, 2-50 mM, 5-30 mM, 10-100 mM, 10-50 mM, 10-30 mM, 10-20 mM, 12-18 mM, 14-17 mM, 15-17 or 16-17 mM. In certain embodiments, the solution comprises at least 2.5mM, at least 3mM, at least 5mM, at least 7.5mM, at least 10mM, at least 15mM, at least 20mM, at least 25mM or at least 30mM glucose. In certain embodiments, the glucose is made up of dextrose or comprises dextrose. In certain embodiments, the solution comprises at least 2.5mM, at least 3mM, at least 5mM, at least 6mM, at least 7.5mM, at least 10mM, at least 15mM, at least 20mM, at least 25mM or at least 30mM dextrose. In certain embodiments, the solution comprises at least 0.03% (w / v), at least 0.05% (w / v), at least 0.1% (w / v), at least 0.125% (w / v), at least 0.15% (w / v), at least 0.175% (w / v), at least 0.2% (w / v), at least 0.225% (w / v), at least 0.25% (w / v), at least 0.275% (w / v), at least 0.28% (w / v), at least 0.29% (w / v), at least 0.3% (w / v), at least 0.35% (w / v), at least 0.4% (w / v), at least 0.45 In some embodiments, the solution further comprises a source of divalent cations. In some embodiments, the divalent cations comprise calcium and / or magnesium cations. In some embodiments, the source of divalent cations comprises a calcium source and / or a magnesium source.In certain embodiments, the solution comprises a calcium source.In certain embodiments, the solution comprises a magnesium source.In certain embodiments, the buffer comprises an acetate buffer and / or a citrate buffer.

[0014] Some aspects of the present disclosure provide solutions for cell reconstitution, storage, transportation and / or administration to a subject, wherein the solution comprises (a) a buffer that maintains the solution at physiological pH; and (b) glucose; and (c) an osmotically active agent that maintains the solution at physiological osmotic pressure; and (d) a source of divalent cations. In certain embodiments, the source of divalent cations comprises a calcium source and / or a magnesium source. In certain embodiments, the buffer comprises an acetate and / or citrate buffer.

[0015] In certain embodiments of the solutions provided herein, the glucose is D-glucose (dextrose). In certain embodiments, the concentration of the glucose is 5-50mM. In certain embodiments, the concentration of the glucose is 10-25mM. In certain embodiments, the concentration of the glucose is 10-20mM. In certain embodiments, the concentration of the glucose is about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM or about 20mM.

[0016] In certain embodiments of the solutions provided herein, the source of the divalent cations comprises a pharmaceutically acceptable salt of a divalent cation. In certain embodiments, the source of the divalent cations comprises a pharmaceutically acceptable calcium salt. In certain embodiments, the source of the divalent cations comprises a pharmaceutically acceptable magnesium salt. In certain embodiments, the source of the divalent cations comprises a pharmaceutically acceptable calcium salt and / or a pharmaceutically acceptable magnesium salt, the salt being selected from a calcium salt and / or a magnesium salt formed with an acid selected from acetic acid, ascorbic acid, citric acid, hydrochloric acid, maleic acid, oxalic acid, phosphoric acid, stearic acid, succinic acid and sulfuric acid. In certain embodiments, the source of the divalent cations comprises a calcium source. In certain embodiments, the calcium source comprises calcium chloride. In certain embodiments, the calcium source comprises calcium chloride dihydrate. In certain embodiments, the source of the divalent cations comprises a magnesium source. In certain embodiments, the magnesium source comprises magnesium chloride. In certain embodiments, the magnesium source comprises magnesium chloride hexahydrate. In certain embodiments of the solutions provided herein, the concentration of the calcium source is 0.25-0.75 mM. In certain embodiments, the concentration of the calcium source is 0.4-0.65 mM. In certain embodiments, the concentration of the calcium source is 0.5-0.6 mM. In certain embodiments, the concentration of the calcium source is about 0.6 mM. In certain embodiments, the concentration of the calcium source is 0.5-0.9 mM. In certain embodiments, the concentration of the calcium source is 0.6-0.8 mM. In certain embodiments, the concentration of the calcium source is about 0.7 mM.

[0017] In certain embodiments of the solutions provided herein, the concentration of the magnesium source is 0.05-5 mM. In certain embodiments, the concentration of the magnesium source is 0.1-0.3 mM. In certain embodiments, the concentration of the magnesium source is about 0.3 mM.

[0018] In certain embodiments of the solutions provided herein, the citrate or acetate buffer is provided in the form of citrate or acetate. In certain embodiments of the solutions provided herein, the citrate buffer is provided as sodium citrate. In certain embodiments, the concentration of the citrate or acetate is 0.1-5mM. In certain embodiments, the concentration of the citrate or acetate is 0.5-2mM. In certain embodiments, the concentration of the citrate or acetate is about 1mM.

[0019] In certain embodiments of the solutions provided herein, the pH of the solution is 6.8-7.8. In certain embodiments of the solutions provided herein, the pH of the solution is 7.2-7.6. In certain embodiments, the pH of the solution is 7.4-7.5. In certain embodiments, the pH of the solution is about 7.5.

[0020] In certain embodiments of the solutions provided herein, the osmotic pressure active agent is a salt. In certain embodiments, the osmotic pressure active agent is a sodium salt. In certain embodiments, the osmotic pressure active agent is sodium chloride. In certain embodiments, the concentration of the osmotic pressure active agent is about 100-250mM. In certain embodiments, the concentration of the osmotic pressure active agent is about 125-175mM. In certain embodiments, the concentration of the osmotic pressure active agent is about 150mM.

[0021] In certain embodiments of the solutions provided herein, the solution is isotonic. In certain embodiments, the solution is hypertonic. In certain embodiments, the solution exhibits an osmotic pressure of about 270-345mOsm / l. In certain embodiments, the solution exhibits an osmotic pressure of about 300-330mOsm / l. In certain embodiments, the osmotic pressure of the solution is about 315mOsm / l.

[0022] In certain embodiments, the solution further comprises a potassium salt. In certain embodiments, the potassium salt is potassium chloride. In certain embodiments, the concentration of the KCl is 0.2-5 mM. In certain embodiments, the concentration of the KCl is 1-2.5 mM. In certain embodiments, the concentration of the KCl is about 2 mM.

[0023] In certain embodiments, the solution further comprises a viscoelastic polymer. In certain embodiments, the polymer is a synthetic polymer. In certain embodiments, the polymer is present at a concentration effective to reduce the exposure of cells in the solution to shear stress. In certain embodiments, the concentration of the polymer is 0.001-5% w / v. In certain embodiments, the concentration of the polymer is about 0.05% w / v. In certain embodiments, the polymer is hyaluronic acid or a salt or solvate thereof. In certain embodiments, the polymer is sodium hyaluronate. In certain embodiments, the hyaluronic acid or a salt or solvate thereof is Healon (Abbott), (Novozymes) or (Alcon). In certain embodiments, the concentration of the hyaluronic acid or a salt or solvate thereof is about 0.001%-0.05% w / v, e.g., 0.01%-0.05% w / v, about 0.02%-0.05% w / v, about 0.01%, about 0.02%, about 0.03%, about 0.04%, or about 0.05% w / v.

[0024] In some embodiments of solution provided herein, the solution is included in calcium chloride, magnesium chloride, sodium citrate, sodium chloride and glucose (for example, D-glucose) in the aqueous solution or is substantially composed of it. In certain embodiments, the solution is included in calcium chloride, magnesium chloride, sodium citrate, sodium chloride, glucose (for example, D-glucose) and potassium chloride in the aqueous solution or is substantially composed of it. In certain embodiments, the solution is included in about 0.7mM CaCl (calcium chloride), about 0.3mM MgCl (magnesium chloride), about 1mM sodium citrate, about 16mM dextrose and about 145mM NaCl in the aqueous solution or is substantially composed of it. In certain embodiments, the solution is included in about 0.5-0.9mM CaCl (calcium chloride), about 0.2-.4mM MgCl (magnesium chloride), about 0.8-1.2mM sodium citrate, about 13-19mM dextrose and about 116-174mM NaCl in the aqueous solution or is substantially composed of it.

[0025] In certain embodiments, the solution also includes about 2mM KCl. In certain embodiments, the solution is included in or is substantially composed of about 0.7mM CaCl (calcium chloride), about 0.3mM MgCl (magnesium chloride), about 1mM sodium citrate, about 16mM dextrose, about 145mM NaCl and about 2mM KCl in the aqueous solution. In certain embodiments, the solution is included in or is substantially composed of about 0.5-0.9mM CaCl (calcium chloride), about 0.2-.4mM MgCl (magnesium chloride), about 0.8-1.2mM sodium citrate, about 13-19mM dextrose, about 116-174mM NaCl and about 1.6-2.4mM KCl in the aqueous solution.

[0026] In some embodiments of the solution provided herein, the solution is included in calcium chloride, magnesium chloride, sodium citrate, sodium chloride, glucose (e.g., D-glucose) and viscoelastic polymers (e.g., hyaluronic acid or its salt or solvate) in an aqueous solution or is substantially composed of it. In certain embodiments, the solution is included in calcium chloride, magnesium chloride, sodium citrate, sodium chloride, glucose (e.g., D-glucose), potassium chloride and viscoelastic polymers (e.g., hyaluronic acid or its salt or solvate) in an aqueous solution or is substantially composed of it. In certain embodiments, the solution is included in about 0.7mM CaCl (calcium chloride), about 0.3mM MgCl (magnesium chloride), about 1mM sodium citrate, about 16mM dextrose, about 145mM NaCl and about 1-5% w / v of viscoelastic polymers (e.g., hyaluronic acid or its salt or solvate) in an aqueous solution or is substantially composed of it. In certain embodiments, the solution comprises or consists essentially of about 0.7 mM CaCl (calcium chloride), about 0.3 mM MgCl (magnesium chloride), about 1 mM sodium citrate, about 16 mM dextrose, about 145 mM NaCl, about 2 mM KCl, and about 0.01-5% w / v of a viscoelastic polymer (e.g., about 0.01-0.05% w / v hyaluronic acid or a salt or solvate thereof) in aqueous solution. In certain embodiments, the solution comprises or consists essentially of about 0.7 mM CaCl (calcium chloride), about 0.3 mM MgCl (magnesium chloride), about 2 mM KCl, about 1 mM sodium citrate, about 16 mM dextrose, about 145 mM NaCl, and about 0.05% hyaluronic acid. In certain embodiments, the solution comprises or consists essentially of about 0.5-0.8 mM CaCl (calcium chloride), about 0.2-.4 mM MgCl (magnesium chloride), about 1.6-2.4 mM KCl, about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, about 116-174 mM NaCl, and about 0.04-0.06% hyaluronic acid.

[0027] In certain embodiments, the solution is sterile.In certain embodiments, the solution is substantially pyrogen-free.

[0028] In certain embodiments, the solution does not comprise a carbonate buffer. In certain embodiments, the solution does not comprise glutathione or glutathione disulfide (GSSG). In certain embodiments, the solution does not comprise a zwitterionic organic buffer.

[0029] In certain embodiments, the solutions provided herein can be stored at 25°C for at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month without measurable solute precipitation and / or measurable loss of the ability of the solution to support the survival and viability of cells stored in the solution. In certain embodiments, the solutions provided herein can be stored at 2-8°C for at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month without measurable solute precipitation and / or measurable loss of the ability of the solution to support the survival and viability of cells stored in the solution.

[0030] Some aspects of the present disclosure provide preparations (preparation) of cell colonies included in the solutions provided herein. In certain embodiments, the cell colony is suitable for transplanting into a subject. In certain embodiments, the cell colony is suitable for transplanting into the eyes of a subject. In certain embodiments, the cell colony comprises RPE cells. In certain embodiments, the cell colony comprises photoreceptor cells. In certain embodiments, the cell colony comprises mesenchymal cells. In certain embodiments, the cell colony comprises retinal ganglion cells. In certain embodiments, the cell colony comprises retinal progenitor cells. In certain embodiments, the cell colony comprises hematopoietic stem cells or hematopoietic progenitor cells (hematopoietic stem or progenitor cell), neural stem cells or neural progenitor cells (neural stem or progenitor cell), neural cells, astrocytes or astrocyte progenitors (progenitor), glial cells or glial cell progenitors and / or pancreatic cells. In certain embodiments, the product is refrigerated. In certain embodiments, the product is refrigerated at about 2-8°C. In certain embodiments, during the storage of the product, the product supports the survival of the cells in the cell colony. In certain embodiments, at least 70% of the cells in the cell colony are alive after the product is stored at 2-8°C for 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, or 144 hours. In certain embodiments, at least 80% of the cells in the cell colony are alive after the product is stored at 2-8°C for 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, or 144 hours. In certain embodiments, at least 90% of the cells in the cell colony are alive after the product is stored at 2-8°C for 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, or 144 hours. In certain embodiments, during the storage of the product, the product supports the maintenance of the plating efficiency of the cell colony. In certain embodiments, after storing the article at 2-8°C for 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, or 144 hours, the cell population exhibits at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of its original plating efficiency, wherein the original plating efficiency represents the plating efficiency of the cell population at the beginning of the storage phase. In certain embodiments, the article is in a storage container. In certain embodiments, the article is in a syringe.

[0031] Some aspects of the present disclosure provide methods for preparing a product provided herein (e.g., a product of a cell colony contained in a solution provided herein), wherein the method includes contacting the cell colony with the solution. In certain embodiments, the method includes contacting a cryopreserved or precipitated cell colony with the solution, thereby reconstructing the cells.

[0032] Some aspects of the present disclosure provide pharmaceutical compositions suitable for administration to a subject, wherein the pharmaceutical preparation comprises a solution provided herein or a preparation of a cell population contained in a solution provided herein.

[0033] Some aspects of the present disclosure provide methods, including applying the solution or product provided herein to a subject in need thereof. In certain embodiments, the method includes applying the solution or product to the eyes of the subject. In certain embodiments, the method includes applying the product to the subject after storing the product for at least 4, at least 6, at least 12, at least 24, at least 36, at least 24, at least 48, at least 60, at least 72, at least 96, at least 120 or at least 144 hours. In certain embodiments, the subject has or is diagnosed with a retinal disease. In certain embodiments, the retinal disease is rod or cone dystrophy, retinal degeneration, pigmentary retinitis, diabetic retinopathy, macular degeneration, Leber congenital amaurosis, diseases related to retinal ganglion cells, glaucoma or Stargardt disease. In certain embodiments, the product includes a cell colony of the size of at least one symptom of a retinal disease in the subject that is effectively improved. In certain embodiments, the cell colony includes RPE cells, photoreceptor cells or mesenchymal stem cells. In certain embodiments, the method further comprises monitoring the subject for at least one symptom of a retinal disease.

[0034] Some aspects of the present disclosure provide methods, comprising: (a) contacting a cell population with a solution provided herein, thereby producing a cell product. In certain embodiments, the method further comprises (b) storing the cell product of (a) for at least 4, at least 6, at least 12, at least 18, at least 24, at least 36, at least 48, at least 60, at least 72, at least 96, at least 120 or at least 144 hours. In certain embodiments, the method further comprises (c) administering the cell product of (a) to a subject after the storage stage of (b). In certain embodiments, the administration of (c) comprises injecting the cells into the eyes of the subject. In certain embodiments, wherein the method further comprises determining the cell viability in the cell product of (a) after the storage stage of (b). In certain embodiments, the method comprises refrigerating the cell product of (a) during the storage stage of step (b). In certain embodiments, refrigeration comprises storing the cell product at a temperature of 2-8°C. In certain embodiments, the method further comprises transporting the product produced in (a) to a location different from the location where the product was produced during the storage stage of (b). In certain embodiments, the transporting comprises transporting the article to a clinic or operating room where the administration of (c) occurs.

[0035] Some aspects of the present disclosure provide a method for treating a retinal disease, wherein the method includes administering an effective amount of a cell preparation provided herein to the eyes of a subject with a retinal disease. In certain embodiments, the subject has or is diagnosed with a retinal disease. In certain embodiments, the retinal disease is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, pathological myopia, Leber congenital amaurosis, glaucoma or Stargardt disease. In certain embodiments, the product includes a cell colony of the size of at least one symptom of the retinal disease in the subject that is effectively improved. In certain embodiments, the cell colony includes RPE cells, photoreceptor cells, retinal ganglia or mesenchymal stem cells. In certain embodiments, the method also includes monitoring at least one symptom of the retinal disease in the subject.

[0036] Some aspects of the present disclosure provide a kit comprising: (a) a solution provided herein; and (b) instructions for contacting a cell population with the solution of (a) to produce a cell product; and (c) a container for contacting (b) and / or storing the cell product of (b). In certain embodiments, the solution of (a) and the container of (c) are suitable for the use of the cell product of (b) for transplantation to a subject.

[0037] The above summary is intended to explain some embodiments, advantages, features and uses of the technology disclosed herein in a non-limiting manner. Other embodiments, advantages, features and uses of the technology disclosed herein will be apparent from the detailed description, drawings, examples and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 RPE cells could be maintained in HypoThermosol for 24 hours (but not 48 hours) without significant loss of ability to be subsequently plated and grown in culture.

[0039] Figure 2 . RPE stability in GS2 (2-8°C). RPE cells can be maintained in GS2 for at least 48 hours without significant loss in viable cell number or ability to be subsequently plated and grown in culture.

[0040] Figure 3 . RPE stability in GS2 (2-8°C). RPE cells can be maintained in GS2 for 4-5 days with only a slight loss of cell viability and no significant decrease in viable cell density.

[0041] Figure 4 . RPE stability in GS2 (2-8°C) The ability of RPE cells to plate and grow in culture began to decline after 5 days of refrigerated storage in GS2.

[0042] Figure 5 . RPE stability in GS2 (2-8°C). GS2 is compatible with current injection systems.

[0043] Figure 6 . show the average viable cell density ± standard deviation from three test tubes.Use hemocytometer to determine human RPE cell density.Exclude obtaining cell viability by trypan blue.From the cell calculation mean value of three test tubes prepared for each condition, determine the viable cell concentration of each test tube from three counts.For each condition, show the percentage change (δ) of the cell number observed after the cell extrusion through MedOne#3233 cannula above each group of values.

[0044] Figure 7. Show the mean and standard deviation (± SD) of human RPE cells / well from 6 holes for each condition of test.For each condition, about 20,000 cells / well are inoculated on the 96-well plate of gelatin coating and cultured 3 days in RPE growth medium (EBM-2 containing EGM2 Single Quots, Stem Cell, Inc.) in 5%CO2, 37-degrees Celsius humidity controlled incubator.In order to determine the cell number after 3-day growth, the 1:1 mixture of cell trypsin (Sigma) and dissociation medium (Gibco.) based on HEPES is raised (lifted).Once the cell is raised, the medium containing 10% fetal bovine serum is neutralized by trypsin and the cell is counted using a hemocytometer.

[0045] Figure 8 Comparison of different media for storing MSCs. Human embryonic stem cell-derived MSCs were grown to 70% confluence, harvested with 0.05% trypsin, resuspended in aMEM + 15% FCS (MSC medium) and centrifuged at 200×g for 5 min. The cell pellet was resuspended in a small volume of MSC medium and viability was counted using trypan blue exclusion. Then, 5 million MSCs were placed in each of 4 Eppendorf tubes, centrifuged and resuspended in 1 ml of the indicated buffer. The tubes were placed in a refrigerator set at 4°C for the specified amount of time. CS: canine serum; FBS: fetal bovine serum.

[0046] Fig. 9 MSC viability was enhanced when stored at higher density in GS2, although the presence of FBS contributed little to the enhanced viability in GS2 over 24 hours.

[0047] Fig.10 MSC viability was maintained when stored and removed via 26G needle / syringe (in GS2 at 4°C).

[0048] Fig.11 Electroretinograms (ERGs) of a group of 16 rat eyes treated with RPE cells suspended in BSS-Plus or GS2 transport medium 60 days after treatment compared with eyes of animals treated with BSS-Plus or GS2 transport medium alone (without cells) or sham or no treatment.

[0049] Fig.12 .Flowchart of the steps of RPE Product Preparation-I (GS2 processing steps are underlined).

[0050] Fig.13 . Flowchart of the steps of RPE product formulation-II (GS2 processing steps are underlined).

[0051] Fig.14 .In BSS Flowchart of the packaging and shipping steps in.

[0052] Fig.15 .Packaging and delivery of RPE in GS2. Detailed Description

[0054] Preface

[0055] Although many advanced surgical procedures minimize damage to cells and tissues compared to older techniques, certain delicate surgeries are still very sensitive to the techniques and materials used. For example, eye surgeries such as cataract surgery and vitrectomy surgery involve very delicate cells and tissues (such as the corneal endothelium), and therefore have little room for error and great potential for damage to such visual tissues and the patient's vision. In addition, the transplantation of cells, for example, in the context of regenerative medicine protocols, often requires the preparation, storage, transportation and / or injection of delicate or fragile cells that can be damaged or lose repopulation capacity after improper manipulation or exposure to non-physiological conditions.

[0056] The disclosure provides for flushing, cell reconstruction, cell storage, cell transport and / or cell administration to experimenter's solution.Compared with currently available solution for flushing, cell reconstruction, preparation, storage and / or transplantation, solution provided herein has several advantages.For example, different from currently available medium, solution provided herein supports the survival of various cells and tissue types (including fragile cells and tissues), and even maintains the cell and tissue viability, re-plating efficiency and population recovery (repopulating) ability of improving level in the storage stage of extension.As explained in more detail elsewhere herein, currently available medium for flushing or surgical operation front cell preparation has short half-life (for example, based on carbon salt precipitation) and / or will not support the survival of the cell stored in long time period (for example, more than 4-6 hours), re-plating efficiency and repopulation ability. Because short half-life and the support of cell viability and function exceeding relatively short time period lack, the application of currently available medium needs to be very close to the clinical site of using (for example, transplanting) medium or cell preparation, for example, in the clinic inside or in the laboratory preparation medium and / or the cell and tissue in various media very close.Therefore, currently available solution limits the clinical application of the cell or tissue of preparation to those applications of administration that allow in short time span (during this period, cell viability, re-plating efficiency and / or population recovery ability are acceptable).The requirement of preparation very close to clinical site can produce the restriction of extra expense, risk and extra place outer (off-site) processing.

[0057] On the contrary, solution provided herein has a shelf life extended compared with currently available solution, and also supports cell function, viability, re-plating efficiency and population recovery ability of various cell types (including fragile cells, such as RPE and photoreceptor cells and mesenchymal stem cells), even in long storage stages (for example, up to 24 hours, up to 48 hours or longer storage stages). Solution provided herein is further biocompatible, and thus is suitable for being administered to a subject. Cells or tissues prepared in solution provided herein can thus be directly administered to a subject, without the need for medium replacement.

[0058] The enhanced features of the solution provided herein allow the solution, cells and tissues prepared to be transported to a clinical site away from the preparation site, which can achieve the central processing and preparation of the final product and eliminate the need for preparation very close to the clinical site. The improved storage and transportation capabilities of the solution provided herein further allow the use of clinical sites further away from the preparation site of the final product, and also increase the flexibility of arranging clinical applications (e.g., arranging for the surgical operation of cells or tissues prepared in the solution provided herein). The expanded storage time window further provides the additional quality control opportunity of the product prepared, for example, before the clinical operation begins, for example, before the surgical team begins surgical preparation or before the subject prepares for surgical operation, the presence of test pathogenic contaminants in the final preparation product.

[0059] Clinical Solutions

[0060] Some aspects of the present disclosure provide clinical solutions for flushing and for formulating, storing, shipping, and administering cells and tissues.

[0061] Unlike currently available media for flushing and cell formulation (eg, balanced salt solutions or saline), the clinical solutions described herein support long-term survival and function of sensitive cells or tissues, are easily prepared and sterilized, and have an extended shelf life.

[0062] Simple salt solutions (such as phosphate-buffered saline or 0.9% sodium chloride solutions) can be used for short-term storage of cells, but these solutions do not adequately support cell viability or cell function over longer-term storage, resulting in significant and often unacceptable decreases in cell viability, re-plating efficiency, and reproliferation capacity, even after only brief storage periods.

[0063] More complex balanced salt solutions may be used for clinical purposes such as clinical flushing or cell and tissue storage and typically contain agents for maintaining osmolarity, a calcium source, a magnesium source, and a buffer.

[0064] Sodium chloride is often used to maintain the osmolarity of a solution. Calcium ions play a role in maintaining cell-to-cell junctions (e.g., in the corneal endothelium). Like calcium ions, magnesium ions are present in the aqueous humor and are necessary for many cellular processes.

[0065] Bicarbonate anions are commonly used as buffers because they represent physiological buffers for many tissues and are widely compatible with other solutes. However, certain forms of calcium and magnesium can react with bicarbonate to form calcium carbonate or magnesium carbonate, which can precipitate out of solution in some cases. When solutions containing bicarbonate and calcium and / or magnesium are heat sterilized, the reaction and precipitation can occur rapidly and can occur over time under ambient storage conditions. For substantially all calcium and magnesium ion salts commonly used as pharmaceutical excipients, the reaction between calcium or magnesium and bicarbonate appears to occur.

[0066] One approach to avoid precipitation is to provide clinical solutions using bicarbonate buffer as two separate stock solutions that are mixed shortly before use. For example, a widely used clinical medium is BSS Sterile intraocular irrigation solution (Alcon Laboratories, Inc.). BSS Sterile intraocular irrigation solution is a two-part solution. The parts are mixed together to form a single solution immediately prior to surgery. This mixing step can be inconvenient and represents a risk of mixing errors in a busy operating room. In addition, preparing two separate solutions is more complicated and expensive than preparing a one-part formulation. Therefore, a one-part clinical solution can be advantageous and highly desirable.

[0067] BSS Part I of the sterile intraocular irrigation solution contains sodium chloride, potassium chloride, sodium bicarbonate, and disodium hydrogen phosphate dissolved in water for injection. The pH of Part I is close to neutral, and it has an osmolality that is close to isotonic with respect to physiological fluids. BSS Part II of the sterile intraocular irrigation solution contains calcium chloride, magnesium chloride, dextrose, and glutathione disulfide (GSSG) dissolved in water for injection. The pH of Part II is adjusted to between 3-5, and the solution has a hypotonic osmolality.

[0068] Reconstructed BSS Sterile intraocular irrigation solution has a neutral pH and isotonic osmolality. If combined with BSS The two parts of the sterile intraocular irrigation solution, divalent cations such as calcium and magnesium in part II will react with the bicarbonate and phosphate in part I to form a precipitate. This reaction occurs almost immediately if the combined solution is steam sterilized, and more slowly at room temperature, typically over a period of hours to days. To prevent this precipitation, reconstituted BSS The labeled shelf life of sterile intraocular irrigation solution is 6 hours, during which time the solution must be used.

[0069] Previous attempts have been made to prepare two-part BSS with comparable performance. A single-part clinical solution comparable to a sterile intraocular irrigation solution. European patent application EP 1067907 B1 (Armitage) teaches the use of zwitterionic organic buffers such as N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (commonly referred to as HEPES) to prevent precipitation as discussed above. The formulation disclosed by Armitage does not contain components such as dextrose and GSSG that are known to be unstable when autoclaved or incorporated into physiological pH solutions. The formulation disclosed by Armitage also does not contain components of the type commonly found in tissue culture media, such as amino acids. The teachings of Armitage therefore do not provide a solution to the problem of precipitate formation.

[0070] Different from the single-part solutions developed previously, some aspects of the present disclosure provide single-part clinical solutions that do not require the use of zwitterionic organic buffers such as HEPES, BES, MOPS, TES, EPPS and TRICINE to maintain the solution within the physiological pH range. The clinical solutions of the present invention solve the problem of short shelf life. The clinical solutions provided herein have greatly improved shelf life compared to currently available clinical media, and support the viability, re-plating efficiency and re-proliferation capacity of cells, even after long-term storage of 24, 48, 60, 72, 96, 120, 144 or 168 hours or longer.

[0071] The term "solution" as used herein refers to an aqueous medium comprising water as the primary solvent and one or more solutes dissolved in the solution, e.g., buffers, osmotically active agents, glucose, salts, polymers, etc. In certain embodiments, the solutions provided herein are for clinical use and are thus non-toxic, substantially non-pyrogenic, and sterile.

[0072] In certain embodiments, provided herein is a solution showing physiological pH and physiological osmotic pressure, also referred to as physiological osmotic pressure concentration. Physiological pH means such pH: it has no cytotoxicity and is similar to the pH of the cell or tissue applied by the solution or the pH of the cell or tissue prepared in the solution that encounters in its natural environment. For most cells and tissues, physiological pH is a pH of about 6.8-7.8, for example, a pH of 7-7.7, a pH of 7.2-7.6, a pH of 7.2-7.4, or a pH of 7.4-7.5. Therefore, in certain embodiments, provided herein is a solution showing a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7. or about 7.8. Physiological osmotic pressure means such osmotic pressure: it has no cytotoxicity and is similar to the osmotic pressure of the cell or tissue applied by the solution or the osmotic pressure of the cell or tissue prepared in the solution that encounters in its natural environment. For most cells and tissues, the physiological osmotic pressure is about 270-345mOsm / l, for example, 280-330mOsm / l, 290-325mOsm / l, 300-315mOsm / l. In certain embodiments, the physiological osmotic pressure is about 300, about 305, about 310, about 315, about 320 or about 325mOsm / l.

[0073] The term "osmotic pressure" or "osmotic pressure concentration" of a solution is the pressure required to stop the flow of a solvent through a semipermeable membrane to a solution, wherein the semipermeable membrane is isolated from the pure solvent on one side and the solution on the other side, wherein the semipermeable membrane is permeable to solvent molecules, but impermeable to solute molecules. The osmotic pressure of a solution is proportional to the molar concentration of the solute particles in the solution, and is measured in units of mOsm / l or mOsm / kg. In certain embodiments, provided herein is a solution that exhibits an osmotic pressure concentration between about 290mOsm / l and about 320mOsm / l or between about 300mOsm / l and 310mOsm / l or about 305mOsm / l. In certain embodiments, the solution exhibits an osmotic pressure concentration of about 300-330mOsm / l. In certain embodiments, the osmolarity of the solution is about 300, about 305, about 310, about 315, about 320, or about 325 mOsm / l.

[0074] In certain embodiments, the molar osmotic pressure concentration of the solution provided herein is mentioned in the form of its osmotic pressure (tonicity), wherein a hypertonic solution is a solution that causes cell contraction, a hypotonic solution is a solution that causes cell expansion, and an isotonic solution does not produce a change in cell volume. The terms "hypertonic", "hypotonic" and "isotonic" are generally used with respect to the cells, cell colonies or tissues that the solution contacts. For example, in an embodiment, in the case of providing a flushing solution, isotonicity represents the osmotic pressure of the change in the volume of the cell or tissue that will not cause contact with the solution during flushing. Similarly, in an embodiment, in the case of using the solution for the preparation of cells, cell colonies or tissues for clinical applications (for example, for transplantation to a subject), isotonicity represents the osmotic pressure of the change in the volume of the cell, cell colony or tissue that will not cause the cell when prepared in the solution. In some embodiments of the solution provided herein, the solution is isotonic. In certain embodiments, the solution is hypertonic.

[0075] In certain embodiments of the solutions provided herein, the osmotic pressure active agent is a salt. In certain embodiments, the salt is a pharmaceutically acceptable salt. In certain embodiments, the osmotic pressure active agent is a sodium salt. In certain embodiments, the osmotic pressure active agent is sodium chloride. In certain embodiments, the concentration of the osmotic pressure active agent is about 100-200mM, 125-175mM or 140-160mM. In certain embodiments, the concentration of the osmotic pressure active agent is about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195 or about 200mM.

[0076] As used herein, the term "physiological osmolarity" refers to an osmotic pressure that is not cytotoxic (e.g., it does not cause rupture or otherwise damage to a given cell or cell type) and is similar to the osmotic pressure of the tissue to which the solution is applied or the osmotic pressure encountered by a cell or tissue formulated in the solution in its natural environment. For most applications, the range of physiological osmolarity is between about 280 mOsm / l and about 325 mOsm / l, between about 290 mOsm / l and about 320 mOsm / l, or between about 300 mOsm / l and 310 mOsm / l, or about 305 mOsm / l.

[0077] In certain embodiments, provided herein are solutions for cell reconstruction, storage, transportation and / or administration to a subject comprising: (a) a buffer for maintaining the solution at physiological pH; and (b) at least 2 mM glucose; and (c) an osmotic pressure active agent for maintaining the solution at a physiological molar osmotic pressure concentration. In certain embodiments, the solution further comprises a source of divalent cations. In certain embodiments, the source of divalent cations comprises a calcium source and / or a magnesium source. In certain embodiments, the solution comprises a calcium source. In certain embodiments, the solution further comprises a magnesium source. In certain embodiments, the buffer comprises an acetate buffer and / or a citrate buffer. In certain embodiments, the solution comprises at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 7.5 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 40 mM or at least 50 mM glucose. In certain embodiments, the solution comprises at least 0.5mM, at least 1mM, at least 2mM, at least 2.5mM, at least 3mM, at least 5mM, at least 6mM, at least 7mM, at least 7.5mM, at least 8mM, at least 9mM, at least 10mM, at least 15mM, at least 16mM, at least 20mM, at least 25mM, at least 30mM, at least 40mM, or at least 50mM dextrose. In certain embodiments, the solution comprises no more than 3mM, no more than 4mM, no more than 5mM, no more than 6mM, no more than 7mM, no more than 7.5mM, no more than 8mM, no more than 9mM, no more than 10mM, no more than 15mM, no more than 17mM, no more than 20mM, no more than 25mM, no more than 30mM, no more than 40mM, or no more than 50mM glucose. In certain embodiments, the solution comprises no more than 0.5 mM, no more than 1 mM, no more than 2 mM, no more than 2.5 mM, no more than 3 mM, no more than 4 mM, no more than 5 mM, no more than 6 mM, no more than 7 mM, no more than 7.5 mM, no more than 8 mM, no more than 9 mM, no more than 10 mM, no more than 15 mM, no more than 20 mM, no more than 25 mM, no more than 30 mM, no more than 40 mM, or no more than 50 mM dextrose.

[0078] In certain embodiments, provided herein are solutions for cell reconstitution, storage, transportation and / or administration to a subject comprising: (a) a buffer for maintaining the solution at physiological pH; and (b) glucose; and (c) an osmotic pressure active agent for maintaining the solution at physiological molar osmotic pressure concentration; and (d) a calcium source; and (e) a magnesium source. In certain embodiments, the buffer comprises acetate and / or citrate buffer. In certain embodiments, the solution comprises at least 0.5 mM, at least 1 mM, at least 2 mM, at least 2.5 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 7.5 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 15 mM, at least 16 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 40 mM or at least 50 mM glucose. In certain embodiments, the solution comprises at least 0.5mM, at least 1mM, at least 2mM, at least 2.5mM, at least 3mM, at least 5mM, at least 6mM, at least 7mM, at least 7.5mM, at least 8mM, at least 9mM, at least 10mM, at least 15mM, at least 16mM, at least 20mM, at least 25mM, at least 30mM, at least 40mM, or at least 50mM dextrose. In certain embodiments, the solution comprises no more than 0.5mM, no more than 1mM, no more than 2mM, no more than 2.5mM, no more than 3mM, no more than 4mM, no more than 5mM, no more than 6mM, no more than 7mM, no more than 7.5mM, no more than 8mM, no more than 9mM, no more than 10mM, no more than 15mM, no more than 17mM, no more than 20mM, no more than 25mM, no more than 30mM, no more than 40mM, or no more than 50mM glucose. In certain embodiments, the solution comprises no more than 0.5 mM, no more than 1 mM, no more than 2 mM, no more than 2.5 mM, no more than 3 mM, no more than 4 mM, no more than 5 mM, no more than 6 mM, no more than 7 mM, no more than 7.5 mM, no more than 8 mM, no more than 9 mM, no more than 10 mM, no more than 15 mM, no more than 17 mM, no more than 20 mM, no more than 25 mM, no more than 30 mM, no more than 40 mM, or no more than 50 mM dextrose.

[0079] In certain embodiments of the solutions provided herein, the concentration of the glucose or the dextrose is 0.5-150 mM, 0.5-50 mM, 2.5-50 mM, 5-50 mM, 10-50 mM, 0.5-25 mM, 2.5-25 mM, 5-25 mM, 10-25 mM or 10-20 mM. In certain embodiments of the solutions provided herein, the concentration of the glucose or the dextrose is about 0.5 mM, about 1 mM, about 2 mM, about 2.5 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 7.5 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 12.5 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 22.5 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.

[0080] In certain embodiments, the solutions provided herein include a source of divalent cations. Suitable divalent cations include, but are not limited to, for example, Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , Mn 2+ Cr 2+ , Cu 2+ , Ba 2+ and Sr 2+ In certain embodiments, the source of divalent cations comprises a calcium source. In certain embodiments, the source of divalent cations comprises a magnesium source. In certain embodiments, the source of divalent cations comprises two or more different sources of divalent cations, e.g., a calcium source and a magnesium source.

[0081] In certain embodiments of the solutions provided herein, the solutions include a calcium source, e.g., a source of calcium ions. In certain embodiments, the calcium source includes a pharmaceutically acceptable calcium salt. In certain embodiments of the solutions provided herein, the solutions include a magnesium source, e.g., a source of magnesium ions. In certain embodiments, the magnesium source includes a pharmaceutically acceptable magnesium salt.

[0082] The term "pharmaceutically acceptable salt" as used herein means a salt that is considered suitable for administration to a human subject. In certain embodiments, a pharmaceutically acceptable salt is a salt prepared with an acid selected from acetic acid, ascorbic acid, citric acid, hydrochloric acid, maleic acid, oxalic acid, phosphoric acid, stearic acid, succinic acid and sulfuric acid. In certain embodiments, the source of the pharmaceutically acceptable divalent cation is selected from a calcium salt and / or a magnesium salt formed with an acid selected from acetic acid, ascorbic acid, citric acid, hydrochloric acid, maleic acid, oxalic acid, phosphoric acid, stearic acid, succinic acid and sulfuric acid. For example, the pharmaceutically acceptable calcium salt of this group of embodiments will include calcium acetate, calcium ascorbate, calcium citrate, calcium chloride, calcium maleate, calcium oxalate, calcium phosphate, calcium stearate, calcium succinate and calcium sulfate. It will be apparent to those skilled in the art that, in embodiments, where the solution comprises two or more pharmaceutically acceptable salts (e.g., calcium, magnesium, and potassium salts), some or all of the salts may be formed with the same acid (e.g., calcium chloride, magnesium chloride, and potassium chloride), or two or more salts may be formed with different acids (e.g., calcium chloride, magnesium chloride, and potassium acetate; calcium chloride, magnesium citrate, and potassium maleate, etc.).

[0083] In certain embodiments, the pharmaceutically acceptable salt, for example, the pharmaceutically acceptable calcium salt or magnesium salt, is a salt selected from the following acids: 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L), benzenesulfonic acid, benzoic acid, camphoric acid (+), camphor-10-sulfonic acid (+), capric acid (decanoic acid), caprylic acid (caproic acid), caprylic acid (caprylic acid). acid)(octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid (DL), lactobionic acid, lauric acid, maleic acid, malic acid (-L), malonic acid, mandelic acid (DL), methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyroglutamic acid (-L), salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid (+L), thiocyanic acid, toluenesulfonic acid (p), and undecylenic acid. Additional suitable pharmaceutically acceptable salts will be apparent to those skilled in the art, and it will be appreciated that the present disclosure is not limited in this regard.

[0084] In certain embodiments, the source of divalent cations comprises a total concentration of divalent cations of 0.1-20 mM (e.g., about 0.5-10 mM, 0.5-5 mM, 1-10 mM, or 2-10 mM). In certain embodiments, the concentration of the divalent cation source is about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. In certain embodiments, the source of divalent cations comprises a calcium source and / or a magnesium source.

[0085] In certain embodiments of the solutions provided herein, the calcium source comprises calcium chloride. In certain embodiments, the calcium source comprises calcium chloride dihydrate. In certain embodiments, the magnesium source comprises magnesium chloride. In certain embodiments, the magnesium source comprises magnesium chloride hexahydrate. In certain embodiments of the solutions provided herein, the concentration of the calcium source is 0.1-1.2mM, 0.25-0.75mM, 0.4-0.65mM or 0.5-0.7mM. In certain embodiments, the concentration of the calcium source is about 0.2mM, about 0.3mM, about 0.4mM, about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM or about 1.2mM. In certain embodiments of the solutions provided herein, the concentration of the magnesium source is 0.05-5mM, 0.1-0.5mM, 0.25-2.5mM, 0.1-1mM or 0.1-0.3mM. In certain embodiments, the concentration of the magnesium source is about 0.05mM, about 0.1mM, about 0.2mM, about 0.3mM, about 0.4mM, about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 2mM, about 3mM, about 4mM or about 5mM.

[0086] In some embodiments of the solution provided herein, the solution comprises a buffer. The term "buffering agent" is used interchangeably with the term "buffer" herein to represent a relatively stable agent that can maintain the pH of a solution by neutralizing an added acid or base. Typically, a buffer comprises a weak conjugate acid-base pair, that is, a weak acid and its conjugate base, or a weak base and its conjugate acid.

[0087] In certain embodiments, the buffer included in the solution provided herein is a citrate or acetate buffer, for example, provided in the form of a citrate or acetate. In certain embodiments of the solution provided herein, the citrate buffer is provided as sodium citrate. In certain embodiments, the concentration of the citrate or acetate is 0.1-5mM. In certain embodiments, the concentration of the citrate or acetate is 0.5-2mM. In certain embodiments, the concentration of the citrate or acetate is about 0.05mM, 0.06mM, 0.07mM, 0.09mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.9mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM or 10mM.

[0088] In certain embodiments, the solution further comprises a potassium salt, preferably a pharmaceutically acceptable potassium salt. In certain embodiments, the potassium salt is potassium chloride. In certain embodiments, the concentration of the KCl is 0.2-5mM or 1-2.5mM. In certain embodiments, the concentration of the KCl is about 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.9mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 4mM or 5mM.

[0089] In certain embodiments, the solution further comprises a viscoelastic polymer. Without wishing to be bound by theory, it is believed that the addition of a viscoelastic polymer will enhance cell and tissue viability, re-plating efficiency, and population recovery ability by protecting cells and tissues from shear stress after being stored in a solution provided herein and / or after being administered to a subject (e.g., by an administration route comprising cannula insertion). Viscoelastic polymers are well known to those skilled in the art, and exemplary suitable viscoelastic polymers include, but are not limited to: hyaluronic acid (e.g., Healon (Abbott), (Novozymes) and (Alcon)), alginates (including sodium alginate), poly(ethylene glycol) (PEG) (also known as poly(ethylene oxide) (PEO)), polyacrylamide, poly(vinyl alcohol) (PVA), hydroxyethylcellulose (HEC), poly(N-hydroxyethylacrylamide) (PHEA), hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose, carboxymethylcellulose, poly(2-hydroxyethylmethacrylate) (pHEMA), polymethacrylic acid (carbomer), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), dextran, chondroitin sulfate, poly(2-methacryloyloxyethylphosphorylcholine) (PMPC), and triblock copolymers, for example, poloxamer 188 ( F68), Poloxamer P108 ( F38), Poloxamer P184 ( L64), Poloxamer P401, Poloxamer P402, Poloxamer P407 ( F127) and Poloxamer P408 ( F108), hydroxypropyl guar gum (guar) polyvinyl pyrrolidone, polyoxyethylene polyoxypropylene copolymer (poloxamer) or its salt or mixture, including (but not limited to) mixture of hyaluronic acid and alginate or its salt. In certain embodiments, the polymer is a nonionic polymer. In certain embodiments, the polymer is a polyether. In certain embodiments, the polymer is a polyalkyl ether. In certain embodiments, the polymer is a copolymer of a polyalkyl ether and another polymer (e.g., a polyalkyl ether). In certain embodiments, the polymer is poloxamer (also known as poloxymer). Poloxymer is a nonionic triblock copolymer composed of a central hydrophobic chain of polyoxypropylene (POP) (also known as polypropylene glycol) flanked by two polyoxyethylene (POE) hydrophilic chains (also known as polyethylene glycol (PEG)). Those skilled in the art will appreciate additional suitable viscoelastic polymers for use in the solutions provided herein based on the present disclosure, and it should be understood that the present disclosure is not limited in this regard. Those skilled in the art will appreciate that the amount of viscoelastic polymer suitable for use in the solutions and articles provided herein will depend on the viscoelastic properties of the polymer, for example, in particular, on the molecular weight of the polymer used. In certain embodiments, the viscoelastic polymer is used in the solutions and articles provided herein at a concentration of 0.001% w / v-5% w / v. In certain embodiments, the viscoelastic polymer is used in a concentration that provides a solution or article provided herein with a viscosity that corresponds to the viscosity of the same solution or article containing 0.01%-0.05% hyaluronic acid, for example, corresponding to a solution or article containing 0.01%-0.05% hyaluronic acid. The viscosity of the same solution or preparation.

[0090] In certain embodiments, the transfer medium containing the viscoelastic polymer has a zero shear viscosity greater than 1,000, 10,000, 50,000 or even 100,000 Pas, and preferably has a zero shear viscosity in the range of 1,000-200,000 Pas, and more preferably in the range of 1,000-20,000 Pas. In certain embodiments, the viscoelastic polymer increases the zero shear viscosity of the resulting transport medium by 5%, 10%, 15%, 25% or even 40% relative to the transport medium without the viscoelastic polymer.

[0091] In embodiments, where the solution is administered to a subject (eg, in the form of a preparation of cells or tissue contained in the solution), the polymers utilized in the present invention are biocompatible and / or biodegradable.

[0092] In certain embodiments, the polymer is hyaluronic acid or a salt or solvate thereof. In certain embodiments, the polymer is sodium hyaluronate. In certain embodiments, the polymer is present at a concentration effective to reduce the exposure of cells in the solution to shear stress. In certain embodiments, the concentration of the polymer is 0.01-5% w / v. In certain embodiments, the concentration of the polymer is about 0.01%-0.05% w / v. In certain embodiments, the polymer is Healon

[0093] In certain embodiments, the solution does not comprise a carbonate buffer. In certain embodiments, the solution does not comprise glutathione or glutathione disulfide (GSSG). In certain embodiments, the solution does not comprise a zwitterionic organic buffer.

[0094] The present disclosure includes solutions that combine two or more or any number of standards (e.g., pH, osmolarity, solutes (buffers, glucose, osmotic pressure active agents, magnesium, calcium, potassium, polymers), concentrations, etc.). For example, the present disclosure includes solutions comprising buffers, glucose, and osmotic pressure active agents (with or without added polymers), solutions comprising potassium and solutions not comprising potassium, and solutions comprising any combination of solutes at any concentration provided for the various solutes. It should also be understood that the present disclosure includes solutions comprising the listed solutes, as well as solutions consisting essentially of or consisting of the listed solutes and a solvent (e.g., water). For the sake of brevity, these alternatives are not described in detail here.

[0095] For example, in some embodiments of solution provided herein, the solution is included in calcium chloride, magnesium chloride, sodium citrate, sodium chloride and glucose (for example, D-glucose) in water or is substantially composed of it.In certain embodiments, the solution is included in calcium chloride, magnesium chloride, sodium citrate, sodium chloride, glucose (for example, D-glucose) and potassium chloride in water or is substantially composed of it.In certain embodiments, the solution is included in about 0.7mM CaCl (calcium chloride), about 0.03mM MgCl (magnesium chloride), about 1mM sodium citrate, about 16mM dextrose and about 145mM NaCl in water or is substantially composed of it.In certain embodiments, the solution also includes about 2mM KCl.In certain embodiments, the solution is included in about 0.7mM CaCl (calcium chloride), about 0.03mM MgCl (magnesium chloride), about 1mM sodium citrate, about 16mM dextrose, about 145mM NaCl and about 2mM KCl in water or is substantially composed of it. In certain embodiments, the solution comprises about 0.7mM CaCl (calcium chloride), about 0.3mM MgCl (magnesium chloride), about 1mM sodium citrate, about 16mM dextrose, about 145mM NaCl and about 2mM KCl in water or consists essentially of it. In certain embodiments, the solution also comprises a viscoelastic polymer. In certain embodiments, the polymer is hyaluronic acid or a salt or solvate thereof. In certain embodiments, the polymer is sodium hyaluronate. In certain embodiments, the polymer is present in a concentration that effectively reduces the exposure of cells in the solution to shear stress. In certain embodiments, the concentration of the polymer is 0.01-5% w / v. In certain embodiments, the concentration of the polymer is about 0.01-0.05% w / v. In certain embodiments of the solution provided herein, the solution comprises calcium chloride, magnesium chloride, sodium citrate, sodium chloride, glucose (e.g., D-glucose) and a viscoelastic polymer (e.g., hyaluronic acid or a salt or solvate thereof) in water or consists essentially of it. In certain embodiments, the solution comprises or consists essentially of calcium chloride, magnesium chloride, sodium citrate, sodium chloride, glucose (e.g., D-glucose), potassium chloride, and a viscoelastic polymer (e.g., hyaluronic acid or a salt or solvate thereof) in water. In certain embodiments, the solution comprises or consists essentially of about 0.7 mM CaCl (calcium chloride), about 0.03 mM MgCl (magnesium chloride), about 1 mM sodium citrate, about 16 mM dextrose, about 145 mM NaCl, and about 0.005-5% w / v of a viscoelastic polymer (e.g., hyaluronic acid or a salt or solvate thereof) in water.In certain embodiments, the solution comprises or consists essentially of about 0.7 mM CaCl (calcium chloride), about 0.03 mM MgCl (magnesium chloride), about 1 mM sodium citrate, about 16 mM dextrose, about 145 mM NaCl, about 2 mM KCl, and about 0.005-5% w / v of a viscoelastic polymer (e.g., hyaluronic acid or a salt or solvate thereof) in water. In certain embodiments, the solution comprises or consists essentially of about 0.85% NaCl, about 0.015% KCl, about 0.01% CaCl dihydrate (calcium chloride dihydrate), about 0.006% MgCl hexahydrate (magnesium chloride hexahydrate), about 0.035% sodium citrate dihydrate, and about 0.29% dextrose in water, and optionally comprises about 0.01-5% w / v of a viscoelastic polymer (e.g., hyaluronic acid or a salt or solvate thereof), e.g., 0.01-0.05% Healon. In certain embodiments, the solution comprises or consists essentially of about 0.68-1.02% NaCl, about 0.008-0.012% CaCl dihydrate (calcium chloride dihydrate), about 0.0048-0.0072% MgCl hexahydrate (magnesium chloride hexahydrate), about 0.028-0.042% sodium citrate dihydrate, and about 0.23-0.35% dextrose in water, and optionally comprises about 0.01-5% w / v of a viscoelastic polymer (e.g., hyaluronic acid or a salt or solvate thereof), e.g., 0.01-0.05% Healon In certain embodiments, the solution comprises or consists essentially of about 0.68-1.02% NaCl, about 0.012-0.018% KCl, about 0.008-0.012% CaCl dihydrate (calcium chloride dihydrate), about 0.0048-0.0072% MgCl hexahydrate (magnesium chloride hexahydrate), about 0.028-0.042% sodium citrate dihydrate, and about 0.23-0.35% dextrose in water, and optionally comprises about 0.01-5% w / v of a viscoelastic polymer (e.g., hyaluronic acid or a salt or solvate thereof), e.g., 0.01-0.05% Healon

[0096] Cell and tissue products

[0097] Some aspects of the present disclosure provide preparations of cell colonies or tissues contained in solutions provided herein. In certain embodiments, the cell colonies are suitable for transplantation into a subject. In certain embodiments, the preparations provided herein are formulated for administration to a subject, e.g., for administration via injection or irrigation. The preparations provided herein may be contained in the cell colonies or tissues in solutions described herein (e.g., GS2), either alone or in combination with one or more additional compounds or agents, e.g., in combination with antioxidants, bacteriostatic agents, or pharmaceutically active agents. Exemplary pharmaceutical preparations include cells or tissues in GS2 as described in Example 1 of the present disclosure.

[0098] Exemplary cell or tissue preparations in solutions provided herein can be formulated to be suitable for use in treating human patients, e.g., pyrogen-free or substantially pyrogen-free, pathogen-free, sterile, and at physiological pH and osmolarity. In certain embodiments, the preparations provided herein are formulated for injection into a specific site, e.g., in the case of ophthalmic preparations for treating retinal diseases or disorders, injection into the vitreous humor for delivery to a site of retinal or choroidal damage.

[0099] The articles provided by the present disclosure may include additional therapeutic agents, for example, immunosuppressants, pro-angiogenic agents, or nutrients or growth factors that support the survival and / or engraftment of cells in the article.

[0100] The volume of the product and the number of cells in the product will depend on the specific application. Generally, for cell transplantation applications, it is desirable to reduce the volume of administration as much as possible. Therefore, the product can be formulated so that a minimized volume can be delivered. The cell concentration for injection can be any amount that is effective and non-toxic. For example, in certain embodiments, a cell product for transplantation is provided, which comprises at least about 10% of the solution provided herein (e.g., in GS2). 4 In some embodiments, at least about 10 3 , at least about 10 4 , at least about 10 5 , at least about 10 6 , at least about 10 7 , at least about 10 8 , at least about 10 9 or at least about 10 10 The cell product for transplantation is prepared at a dose of 10 cells / ml.

[0101] In certain embodiments, by counting live cells and excluding non-living cells, the number of cells and / or the concentration of cells in the products provided herein can be determined. For example, by not being able to exclude vital dyes (such as trypan blue), or using functional assays (such as the ability to adhere to a culture substrate, phagocytosis, etc.), non-living cells can be detected. In addition, the number of cells or the concentration of cells of the desired cell type can be determined as follows: cells expressing one or more cell markers specific to the cell type are counted, and / or cells expressing one or more markers indicating cell types other than the desired cell type are excluded.

[0102] In certain embodiments, provided herein is a cell preparation comprising at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, or 9,000 cells. In certain embodiments, the cell preparation may comprise at least about 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 ,7×10 4 , 8×10 4 ,9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 ,7×10 5 , 8×10 5 ,9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 ,7×10 6 , 8×10 6 ,9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 ,7×10 7 , 8×10 7 ,9×10 7 , 1×108 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 ,7×10 8 , 8×10 8 ,9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 ,7×10 9 , 8×10 9 ,9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 ,7×10 10 , 8×10 10 or 9×10 10 In certain embodiments, the cell preparation may contain at least about 1×10 2 Up to 1×10 3 , 1×10 2 Up to 1×10 4 , 1×10 4 Up to 1×10 5 or 1×10 3 Up to 1×10 6 In certain embodiments, the cell preparation may comprise at least about 10,000, 20,000, 25,000, 50,000, 75,000, 100,000, 125,000, 150,000, 175,000, 180,000, 185,000, 190,000, or 200,000 cells, for example, the cell preparation may comprise at least about 20,000-200,000 cells in a volume of about 50-200 μl of a solution (e.g., GS2) provided herein.

[0103] In certain embodiments, the cell colony is suitable for being transplanted into the eyes of experimenter.In certain embodiments, the cell colony suitable for being transplanted into the eyes of experimenter comprises RPE cell, RPE progenitor cell, iris pigment epithelium (IPE) cell and other visually relevant neural cells, such as contact neuron (for example, " relay " neuron of inner nuclear layer (INL)) and amacrine cell, retinal cell, rod, cone and keratocyte, neuron, photoreceptor cell and mesenchymal cell, for example, mesenchymal stem cell (MSC).

[0104] In certain embodiments, the goods provided are included in the RPE cell colony in the solution provided herein (for example, in the GS2 medium described in Example 1).Suitable RPE cell can be differentiated from pluripotent stem cells (such as human embryonic stem cells or iPS cells), and can be different from embryonic stem cells, adult-derived RPE cells and fetus-derived RPE cells in molecules.In certain embodiments, adult-derived RPE cells and fetus-derived RPE cells are used.

[0105] Where ES cell-derived RPE cells are used, in certain embodiments, the preparations do not contain detectable amounts of residual ES cells, such that the preparations provided herein do not create an unacceptable risk of contamination in RPE cell cultures and preparations.

[0106] In certain embodiments, a preparation comprising a cell population suitable for transplantation into a subject's eye is suitable for injection into the subject's eye. In certain embodiments, such preparations can be used to treat retinal degenerative diseases or disorders, including, but not limited to, retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration, or retinal atrophy or associated with a number of vision-altering conditions that lead to photoreceptor damage and blindness, e.g., choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt disease (yellow spot disease).

[0107] The RPE cells can be stable, terminally differentiated RPE cells that do not dedifferentiate into non-RPE cell types. The RPE cells described herein can be functional RPE cells, characterized in that they integrate into the ability of the retina after being administered into a human or non-human animal by corneal, subretinal or other means.

[0108] The RPE cell can express RPE cell markers.For example, the expression level of markers such as RPE65, PAX2, PAX6, tyrosinase, bestrophin, PEDF, CRALBP, Otx2 and / or MITF can be equivalent to the expression level in naturally occurring RPE cells.By measuring at least one expression in PAX2, PAX6 and tyrosinase, or their expression level separately, the maturation level of RPE cells can be assessed.

[0109] In certain embodiments, based on the pigmentation degree of cell, the RPE cell included in the goods provided herein described can be identified and characterized.By cultivating and maintaining the density of RPE cells and maintaining the duration of RPE in culture, the change of pigment can be controlled.The RPE cells of differentiation of rapid division have lighter pigmentation.On the contrary, the RPE that divides more slowly or does not divide presents their unique polygonal or hexagonal shape and increases the pigmentation level by accumulating melanin and lipofuscin.For example, static RPE culture (for example, due to converging) usually increases their pigmentation level over time.Like this, the accumulation of pigmentation serves as the indicator of RPE differentiation, and the pigmentation of the increase relevant to cell density serves as the indicator of RPE maturity.For example, the RPE cells that can be cultured mature at lower density passage culture so that pigmentation is reduced.Under this background, mature RPE cells can be cultivated to produce less mature RPE cells.Such RPE cells are still the RPE cells of differentiation expressing the marker of RPE differentiation.

[0110] For example, in certain embodiments, a product comprising RPE cells is provided, wherein the average melanin content of the RPE cells is less than 8 pg / cell, less than 7 pg / cell, less than 6 pg / cell, or less than 5 pg / cell, for example, between 0.1-8 pg / cell, between 0.1-7 pg / cell, between 0.1-6 pg / cell, between 0.1-5 pg / cell, between 0.1-4 pg / cell, between 0.1-3 pg / cell, between 0.1-2 In another embodiment, the average melanin content can be less than 5pg / cell, for example, between 0.1-5pg / cell, between 0.2-5pg / cell, 0.5-5pg / cell, 1-5pg / cell, 2-5pg / cell, 3-5pg / cell, or 4-5pg / cell, for example, between 4.2-4.8pg / cell or between 0.1-5pg / cell. In another embodiment, the average melanin content can be less than 5pg / cell, for example, between 0.1-5pg / cell, between 0.2-5pg / cell, 0.5-5pg / cell, 1-5pg / cell, 2-5pg / cell, 3-5pg / cell, 4-5pg / cell or 4.5-5pg / cell.

[0111] In certain embodiments, a kind of goods is provided, it is included in the RPE cells in the solution described herein (for example, GS2), wherein the RPE cells maintain their phenotype after the RPE cells are transplanted to the experimenter (for example, after the products are injected into the eyes of the experimenter). The RPE cells can maintain their phenotype in the life span of the recipient after transplantation. For example, the RPE cells can maintain their phenotype at least about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 days after transplantation. In addition, the RPE cells can maintain their phenotype at least about 1,2,3,4,5,6,7,8,9 or 10 weeks after transplantation. The RPE cells can maintain their phenotype at least about 1,2,3,4,5,6,7,8,9,10,11 or 12 months after transplantation. The RPE cells can maintain their phenotype for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 years or more after transplantation.

[0112] In certain embodiments, the disclosure provides a product of the RPE cells in the solution provided herein for injection into the eyes of a subject. In such an embodiment, the product is a pharmaceutically acceptable ophthalmic preparation for intraocular injection. When the product is administered by intravitreal injection, for example, the product can be formulated so that a minimized volume can be delivered. The injection concentration can be any amount that is effective and nontoxic. It can be at least about 10 4 The dosage of the solution provided herein is prepared for treating the RPE cells of a patient. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 The RPE cell product is formulated at a dose of 10 RPE cells / mL for treating a patient.

[0113] The preparation of RPE cells described herein can include at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, or 9,000 RPE cells in a solution described herein (e.g., in GS2 described in Example 1). The pharmaceutical preparation of RPE cells can include at least about 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 ,7×10 4 , 8×10 4 ,9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 ,7×10 5 , 8×10 5 ,9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 ,7×10 6 , 8×10 6 ,9×10 6 , 1×107 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 ,7×10 7 , 8×10 7 ,9×10 7 , 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 ,7×10 8 , 8×10 8 ,9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 ,7×10 9 , 8×10 9 ,9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 ,7×10 10 , 8×10 10 or 9×10 10 RPE cells. The pharmaceutical preparation of RPE cells may contain at least about 1×10 2 Up to 1×10 3 , 1×10 2 Up to 1×10 4 , 1×10 4 Up to 1×10 5 or 1×10 3 Up to 1×10 6In some embodiments, the pharmaceutical preparation of RPE cell can include at least about 10,000, 20,000, 25,000, 50,000, 75,000, 100,000, 125,000, 150,000, 175,000, 180,000, 185,000, 190,000 or 200,000 RPE cells. For example, the pharmaceutical preparation of RPE cell can include at least about 20,000-200,000 RPE cells in at least about 50-200 μ L volume. In addition, the pharmaceutical preparation of RPE cell can include about 50,000 RPE cells in 150 μ L volume, about 200,000 RPE cells in 150 μ L volume, or at least about 180,000 RPE cells in at least about 150 μ l volume.

[0114] RPE cells can be formulated in the preparations provided herein for delivery in a pharmaceutically acceptable ocular vehicle such that the preparations remain in contact with the ocular surface for a sufficient period of time to allow the cells to penetrate the affected ocular region, e.g., the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humour, cornea, iris / ciliary body, lens, choroid, retina, sclera, suprachoroidal space, conjunctiva, subconjunctival space, suprascleral space, intracorneal space, epicorneal space, par plana, surgically induced avascular area, or macula.

[0115] In certain embodiments, provided herein is a cell preparation, wherein RPE cells are contained in a cell sheet. For example, a cell sheet comprising RPE cells can be prepared as follows: RPE cells are cultured on a substrate from which a complete cell sheet can be released, e.g., a surface implanted with a thermoresponsive polymer such as thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm), to which the cells attach and propagate at the culture temperature, and then, after a temperature shift, the surface characteristics are altered to cause the release of the cultured cell sheet (e.g., by cooling to below the lower critical solution temperature (LCST) (see da Silva et al., Trends Biotechnol. 2007 Dec; 25(12):577-83; Hsiue et al., Transplantation. 2006 Feb 15; 81(3):473-6; Ide, T. et al. (2006); Biomaterials 27, 607-614, Sumide, T. et al. (2005), FASEB J. 20, 392-394; Nishida, K. et al. (2004), Transplantation 77, 379-385; and Nishida, K. et al. (2004), N. Engl. J. Med. 351, 1187-1196, each of which is incorporated herein by reference in its entirety). The cell sheet can be attached to a substrate suitable for transplantation, such as a substrate that can dissolve in vivo when the sheet is transplanted into a host organism, for example, by culturing the cells on a substrate suitable for transplantation or releasing the cells from another substrate (such as a thermoresponsive polymer) onto a substrate suitable for transplantation. An exemplary substrate that may be suitable for transplantation may include gelatin (see Hsiue et al., supra). The alternative substrate that can be suitable for transplanting comprises matrix based on fibrin and other.Described cell sheet can be used for the preparation of medicine, and described medicine is used for preventing or treating retinal degenerative diseases.Can be as follows RPE cell sheet be formulated in cell or tissue product for introducing the eyes of experimenter having this need: make it contact with solution (for example, GS2 solution) described herein.In certain embodiments, by subfoveal membrane resection (subfovealmembranectomy) and transplant RPE cell sheet, described cell sheet can be introduced into the eyes of experimenter having this need, or can be used for the preparation of medicine, and described medicine is used for transplanting after subfoveal membrane resection.

[0116] The volume of the goods provided by some embodiments of the present disclosure depends on multiple factors such as mode of administration, the number of cells to be delivered, age and weight of the patient and the type and severity of the disease being treated. For example, if used by injection, the volume of the drug product of the RPE cell of the present disclosure can be about 1,1.5,2,2.5,3,4 or 5ml. The volume can be about 1-2mL. For example, if administered by injection, the volume of the pharmaceutical preparation of RPE cells of the present disclosure can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 100 , 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156 6, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 μL (microliters). For example, the volume of the preparation of the present disclosure can be about 10-50, 20-50, 25-50, or 1-200 μL. The volume of an article of the present disclosure may be about 10, 20, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μL or more.

[0117] In certain embodiments, the articles provided herein may comprise about 1×10 3 , 2×10 3 , 3×10 3 , 4×10 3 , 5×10 3 , 6×10 3 ,7×10 3 , 8×10 3 ,9×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 ,7×10 4 , 8×10 4 or 9×10 4 For example, in certain embodiments, the preparation may comprise 2000 RPE cells / μL, for example, 100,000 RPE cells / 50 μL or 180,000 RPE cells / 90 μL.

[0118] In certain embodiments, the preparation is refrigerated. In certain embodiments, the preparation is refrigerated at about 2-8°C.

[0119] In certain embodiments, during the storage of the product, the product supports the survival of cells in the cell colony. In certain embodiments, at least 70% of the cells in the cell colony are alive after the product is stored at 2-8°C for 48 hours. In certain embodiments, at least 80% of the cells in the cell colony are alive after the product is stored at 2-8°C for 48 hours. In certain embodiments, at least 90% of the cells in the cell colony are alive after the product is stored at 2-8°C for 48 hours. In certain embodiments, during the storage of the product, the product supports the maintenance of the plating efficiency of the cell colony. In certain embodiments, after the product is stored at 2-8°C for 48 hours, the cell colony shows at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% of its original plating efficiency, wherein the original plating efficiency represents the plating efficiency of the cell colony at the beginning of the storage stage. In certain embodiments, the product is in a storage container. In certain embodiments, the article is in a syringe.

[0120] Some aspects of the present disclosure provide pharmaceutical preparations of cells and tissues in solutions provided herein.Such preparations are suitable for administration to subjects.In certain embodiments, the pharmaceutical preparations are substantially composed of cells, cell colonies or tissues and solutions provided herein.In certain embodiments, the pharmaceutical preparations include one or more pharmaceutically active ingredients, for example, preservatives, antioxidants, free radical scavengers, immunosuppressants, angiogenic factors, anti-angiogenic factors, growth hormones or cell nutrients or substrates that support cell growth, survival and implantation.

[0121] The disclosure also includes a drug package and / or a test kit. The drug package and / or test kit provided may include a cell or tissue product and a container (e.g., vial, ampoule, bottle, syringe and / or cooler bag or other suitable container) provided herein. In certain embodiments, the test kit provided may optionally also include a second container, which includes a solution for preparing the product, and the solution is used to dilute, wash and / or reconstruct the cell or tissue product. In certain embodiments, the contents of the preparation container and the solvent container provided are combined to form at least one unit dosage form.

[0122] Some aspects of the present disclosure provide methods for preparing a product provided herein (e.g., a product of a cell colony contained in a solution provided herein), wherein the method includes contacting the cell colony with the solution. In certain embodiments, the method includes contacting a cryopreserved or precipitated cell colony with the solution, thereby reconstructing the cells.

[0123] For example, some aspects of the present disclosure provide methods, comprising: (a) contacting a cell population with a solution provided herein, thereby producing a cell product. In certain embodiments, the method further comprises (b) storing the cell product of (a) for at least 4, at least 6, at least 12, at least 18, at least 24, at least 36, at least 48, at least 60, at least 72, or at least 96 hours. In certain embodiments, the method further comprises (c) administering the cell product of (a) to a subject after the storage stage of (b). In certain embodiments, the administration of (c) comprises injecting the cells into the eyes of the subject. In certain embodiments, wherein the method further comprises determining the cell viability in the cell product of (a) after the storage stage of (b). In certain embodiments, the method comprises refrigerating the cell product of (a) during the storage stage of step (b). In certain embodiments, refrigeration comprises storing the cell product at a temperature of 2-8°C. In certain embodiments, the method further comprises transporting the product produced in (a) to a location different from the location where the product was produced during the storage stage of (b). In certain embodiments, the transporting comprises transporting the article to a clinic or operating room where the administration of (c) occurs.

[0124] Exemplary Applications and Methods

[0125] The solutions disclosed herein can be used in a variety of clinical applications. Such applications include clinical flushing, reconstitution or formulation of cells (e.g., after precipitation or cryopreservation), and formulation of cells, e.g., for clinical applications, including, but not limited to, cell storage and transportation prior to administration to a subject, and / or as a carrier medium for administering cells, cell monolayers, or tissues to a subject.

[0126] For example, in certain embodiments, the solution disclosed herein can be used as a solution for cell reconstruction. The term "cell reconstruction" used herein means contacting a cell colony with a solution (e.g., a solution provided herein) to produce a process comprising a solution of the cell colony. In certain embodiments, the reconstruction of the cell colony includes producing a solution comprising a cell colony from a cell precipitate (e.g., a cell precipitate obtained after a centrifugation step by discarding the supernatant). In certain embodiments, reconstructing a cell colony with a solution provided herein includes diluting or replacing any medium in which the cell is initially suspended to obtain a cell colony in a medium consisting essentially of a solution provided herein or consisting of a solution provided herein. In certain such embodiments, the cell colony suspended in a medium other than a solution provided herein can be washed once or multiple times with a solution provided herein. In certain embodiments, a washing step can include contacting the cell with a solution provided herein, precipitating the cell (e.g., by centrifugation), discarding the supernatant, and reconstructing a cell precipitate with the solution. Depending on the volume of the solution used and the volume of the cell precipitate, the initial medium can be substantially replaced with the solution after a single wash-reconstruction cycle or after 2, 3, 4, 5, 6, 7, 8, 9 or 10 such cycles.

[0127] In certain embodiments, provided herein is a solution that can be used for cell and tissue preparation. In the context of cells and tissues, the term "preparation" used herein means contacting cells, cell colonies or tissues with a certain volume of solution provided herein to obtain cells or tissue products suitable for clinical applications (e.g., for administration to a subject). Provided herein is a solution that is widely compatible with a variety of cell types, cell colonies and tissues, including, but not limited to, adult stem cells and progenitor cells, differentiated cells and colonies and tissues comprising such cells. In certain embodiments, the cells, cell colonies or tissues so prepared in the solution provided herein are therapeutic cells, cell colonies or tissues, for example, for clinical applications in regenerative medicine schemes. For example, in certain embodiments, the cells, cell colonies or tissues prepared in the solution provided herein can include such cell colonies: it can replace cells lost or degenerated in a subject, or repair or replace tissues that have been damaged or dysfunctional in a subject. For example, in certain embodiments, the cells, cell colonies or tissues prepared in the solution provided herein can include pluripotent stem cells or progenitor cells, or functional differentiated cells, or colonies or tissues comprising such cells or a combination of such cells. In certain embodiments, solution provided herein is for preparing RPE cell, photoreceptor cell, mesenchymal stem cell, hematopoietic stem cell, nerve or neuronal stem cell or progenitor cell, glial stem cell or progenitor cell, pancreatic stem cell or progenitor cell, beta cell, keratinocyte, chondrocyte, osteoblast, osteoclast or comprise this type of cell or colony or tissue basically composed of this type of cell, for example, RPE cell monolayer, islet or skin graft.In certain embodiments, the cell preparation in solution provided herein can be stored and / or transported to clinical site before clinical application (for example, before being administered to experimenter), and to provide state or only through micro-processing (such as described preparation is diluted to the volume of expectation or the cell concentration of expectation) and be administered to experimenter.

[0128] In certain embodiments, solution provided herein can be used for cell or tissue storage.Under the background of cell and tissue, term " storage " used herein means the time period between the clinical application of preparation cell or tissue and another processing step or cell or tissue in solution provided herein.Being different from previously available solution, solution provided herein supports multiple cells and tissue types (including sensitive cells and tissues such as RPE cells, photoreceptors and MSC) to store the extended time period, for example, at least 4, at least 6, at least 8, at least 12, at least 18, at least 24, at least 30, at least 36, at least 48 hours, at least 60 hours or at least 72 hours, and cell viability, re-plating efficiency or population recovery ability only slightly decline. For example, in certain embodiments, cell, cell colony or tissue (for example, RPE cell, photoreceptor cell or MSC) are stored in solution provided herein for at least 4, at least 6, at least 8, at least 12, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60 or at least 72 hours. The cell viability produced by the time period, re-plating efficiency and / or the re-proliferation capacity are the viability when the storage stage starts, re-plating efficiency and / or the re-proliferation capacity at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%.In certain embodiments, the storage period, for example, the time period between the preparation of cell or tissue and their clinical application will not exceed 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 ​​hours, 60 hours or 72 hours.Usually, the cell or tissue refrigerated storage formulated in the solution provided herein, for example, to the temperature between 2-8 ℃. Thus, in certain embodiments, cells and tissues are stored in solutions provided herein at temperatures below ambient temperature, e.g., at temperatures between 2-8° C. However, in certain embodiments, storage at higher temperatures is contemplated, e.g., at temperatures between 8° C. and 16° C., at 16-22° C., or at ambient temperature (typically about 25° C.).

[0129] Solution provided herein is also used for transporting cells, cell colonies and tissues to clinical sites after they are prepared. Solution provided herein supports cell survival and minimizes metabolic stress and physical stress (including shear stress) during transportation. Cell or tissue transportation will usually be completed in the storage stage of the cell or tissue, and thus under the appropriate conditions of storage, as described above. In an embodiment, when the cell or tissue formulated in the solution provided herein is transported under refrigerated conditions (for example, at a temperature below ambient temperature), the application of a removable refrigeration device is preferred for transportation. Such equipment includes, but is not limited to, insulated transport or shipping containers, wet ice bags, cooling gels, cooling containers and removable freezing units. Some exemplary suitable transport methods, containers and devices for refrigerated transportation are described in more detail elsewhere herein, and those skilled in the art will understand other suitable methods, containers and devices in view of the present disclosure.

[0130] Some aspects of the present disclosure provide a method for treating a subject in need of the subject by administering an effective amount of a clinical solution as described herein or a cell or tissue product to the subject. In certain embodiments, the subject has or is diagnosed with a disease or disorder, which can be treated by administering cells, cell colonies or tissues (e.g., in the form of cells or tissue products described herein). In certain embodiments, the product administered to the subject comprises a cell colony of the size of at least one symptom of the disease or disorder of the subject that is effectively improved. In certain embodiments, the subject is undergoing surgery, and a solution or product described herein is used to rinse the surgical site. In certain embodiments, the method also includes monitoring at least one symptom of the disease in the subject.

[0131] The terms "treatment" and "treatment" represent clinical interventions, the purpose of which is to reverse, alleviate or inhibit the progression of a disease or disorder as described herein or one or more symptoms thereof, to reverse, alleviate, delay the onset of a disease or disorder as described herein or one or more symptoms thereof. In certain embodiments, treatment may be administered after one or more symptoms have been formed and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, for example, to prevent or delay the onset of symptoms or to inhibit the onset or progression of a disease. For example, treatment may be administered to susceptible individuals (e.g., in view of the history of symptoms and / or in view of genetic factors or other susceptibility factors) before the onset of symptoms. Treatment may also be continued after symptoms have subsided, for example, to prevent or delay their recurrence.

[0132] As used herein, the term "subject" refers to a single organism, e.g., a single mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a non-human primate. In certain embodiments, the subject is a rodent. In certain embodiments, the subject is a sheep, goat, cow, cat, or dog. In certain embodiments, the subject is a research animal. In certain embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject can be of either sex and at any stage of development.

[0133] The term "effective amount" as used herein means an amount of a bioactive agent sufficient to cause a desired biological response. For example, in certain embodiments, an effective amount of a cell or tissue preparation as described herein may mean an amount of a preparation comprising a plurality of cells or an amount of tissue sufficient to improve symptoms associated with a disease or disorder (e.g., sufficient to improve vision in a subject with a retinal disease or disorder). A skilled artisan will appreciate that the effective amount of a solution or preparation provided herein may vary with a variety of factors, such as a desired biological response, e.g., a specific disease being treated, a specific symptom to be alleviated, a cell or tissue being targeted, and the age, sex, and general health of the subject.

[0134] Some aspects of the present disclosure provide methods, including applying the solution or product provided herein to a subject in need thereof. In certain embodiments, the method includes applying the solution or product to the eyes of the subject. In certain embodiments, the method includes applying the product to the subject after storing the product for at least 4, at least 6, at least 12, at least 24, at least 36 or at least 48 hours. In certain embodiments, the subject has or is diagnosed with a retinal disease. In certain embodiments, the retinal disease is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, Leber congenital amaurosis or Stargardt disease. In certain embodiments, the product includes a cell colony of the size of at least one symptom of the retinal disease in the subject that is effectively improved. In certain embodiments, the cell colony includes RPE cells, photoreceptor cells or mesenchymal stem cells. In certain embodiments, the method also includes monitoring at least one symptom of the retinal disease in the subject.

[0135] Some aspects of the present disclosure provide methods for treating retinal diseases, wherein the methods include administering an effective amount of a cell preparation provided herein to the eyes of a subject with a retinal disease. In certain embodiments, the subject has or is diagnosed with a retinal disease. In certain embodiments, the retinal disease is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, Leber congenital amaurosis or Stargardt disease. In certain embodiments, the product includes a cell colony of the size of at least one symptom of the retinal disease in the subject that is effectively improved. In certain embodiments, the cell colony includes RPE cells, photoreceptor cells or mesenchymal stem cells. In certain embodiments, the method also includes monitoring at least one symptom of the retinal disease in the subject.

[0136] The term "monitoring the symptoms of a disease" as used herein means assessing the severity of the symptoms of a disease at multiple time points within a time period. For example, the severity of the symptoms can be assessed in a subject before the treatment of the subject begins, and then assessed again after a time period after the treatment. In certain embodiments, the monitoring may include assessing the severity of symptoms after a certain period of time, and it is known or expected that the time is sufficient to treat a measurable improvement in symptoms in similar subjects (e.g., subjects of the same species, sex, age, general health, etc.). In certain embodiments, the monitoring may include regularly assessing the severity of symptoms. For example, in a subject treating a retinal disorder, a preliminary assessment of the subject's vision may be performed. Those skilled in the art will appreciate that the assessment is exemplary, and other assessments may be performed outside or alternatively in the assessment of the subject's vision. Such an assessment may include retinal degeneration levels, retinal ablation, macular degeneration, and the like. By using cell preparation provided herein (for example, the product of the RPE cell of effective number included in the GS2 medium provided herein) treatment experimenter, experimenter's vision can be assessed again, ideally after surgery, past the time period allowing the use of cells to implant and realize their function, for example, after about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year later. The result of assessment after surgery can be recorded, and compared with the assessment before surgery to determine whether symptom has improved, for example, whether the visual level in the experimenter has recovered. Assessment can be repeated one or more times to determine whether the improvement of symptoms is still in progress or whether terminal has been reached. Depend on the result of monitoring after surgery, other surgery can be planned to (further) improve the symptom of assessment. If improvement is not observed after initial surgery, the dosage of cell or tissue product can be increased to promote the improvement of symptoms.

[0137] The therapeutic method of retinal disease can comprise the RPE cell product provided herein of the effective amount of using single dose, for example, the product of the RPE cell of the effective amount contained in the GS2 medium described herein.And, the therapeutic method described herein can comprise such a course for the treatment of: wherein RPE cell is used repeatedly in a certain stage, and wherein the cell of each dosage can effectively alleviate the symptom of disease, or wherein multiple dosages are cumulatively delivered effective amount.Exemplary course for the treatment of can comprise weekly, every 2 weeks, monthly, every season, every half a year or annual treatment.Alternately, treatment can be carried out in stages, wherein initially multiple doses (for example, the daily dose of the first week) are used, and subsequently less and less frequent dosages are needed.

[0138] If by intraocular injection use RPE cell products as described herein, for example, the product of the RPE cell included in the GS2 solution, can periodically deliver RPE cell products once or many times in the patient's life span.For example, can deliver RPE cell products once a year, once every 6-12 months, once every 3-6 months, once every 1-3 months or once every 1-4 weeks.Alternately, more frequent use can be desirable for some illness or obstacle.If by implant or device use, RPE cells can be used once, or if necessary for the specific patient being treated and obstacle or illness periodically deliver one or many times in the patient's life span.Similarly foresee the treatment scheme that changes with time.For example, may need more frequent treatment (for example, every day or weekly treatment) at the beginning.Over time, due to the improvement of patient's situation, may need less frequent treatment or even do not need further treatment.

[0139] The methods described herein may further comprise the step of monitoring the efficacy of treatment or prevention by measuring the electroretinogram response, visual acuity threshold or brightness threshold in the subject. The methods may also comprise monitoring the efficacy of treatment or prevention by monitoring the immunogenicity of the cells or the migration of the cells in the eye.

[0140] The RPE cell or the product comprising RPE cell and clinical solution provided herein (for example, GS2 solution) can be used for preparing medicine to treat retinal degeneration.The present disclosure also includes the purposes of the product comprising RPE cell disclosed herein in treating blindness.For example, the product comprising human RPE cell can be used for treating retinal degeneration relevant with many diseases (it causes photoreceptor damage and blindness) that change vision, for example, diabetic retinopathy, macular degeneration (comprising age-related macular degeneration, for example, wet age-related macular degeneration and dry age-related macular degeneration), retinitis pigmentosa and Stargardt disease (fundus yellow spot disease). The preparation can contain at least about 5,000-500,000 RPE cells (e.g., 100,000 RPE cells), which can be administered to the retina to treat retinal degeneration associated with a number of vision-altering conditions that lead to photoreceptor damage and blindness, such as diabetic retinopathy, macular degeneration (including age-related macular degeneration), retinitis pigmentosa, and Stargardt disease (yellow spots on the fundus).

[0141] The cell used in the product for treating a subject (for example, the RPE cell used in the RPE cell product provided herein) can be a human cell. Human cells can be used in human patients, and in animal models or animal patients. For example, human cells can be tested in mice, rats, cats, dogs or non-human primate models of retinal degeneration. In addition, human cells can be used for treating animals in need thereof, such as in veterinary medicine.

[0142] In certain embodiments, the method for treating retinal diseases may further comprise the administration of an immunosuppressant close in time to the administration of the clinical preparations provided herein. The immunosuppressants that can be used include, but are not limited to, anti-lymphocyte globulin (ALG) polyclonal antibodies, anti-thymocyte globulin (ATG) polyclonal antibodies, azathioprine, (anti-IL-2Rα receptor antibody), cyclosporine (cyclosporine A), (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, In some embodiments, the present invention relates to immunosuppressive agents for treating RPE cells. For example, immunosuppressive agents for treating RPE cells can be used to treat RPE cells. For example, immunosuppressive agents for treating RPE cells can be used to treat RPE cells. For example, immunosuppressive agents for treating RPE cells can be used to treat RPE cells. For example, immunosuppressive agents for treating RPE cells can be used to treat RPE cells. For example, immunosuppressive agents for treating RPE cells can be used to treat RPE cells. For example, immunosuppressive agents for treating RPE cells can be used to treat RPE cells. For example, immunosuppressive agents for treating RPE cells can be used to treat RPE cells. For example, immunosuppressive agents for treating RPE cells can be used to treat RPE cells. For example, immunosuppressive agents for treating RPE cells can be used to treat RPE cells.

[0143] In some aspects, provided herein is a solution that can be used as a clinical flushing solution. Such a solution can be used for clinical flushing, for example, for flushing a wound or a surgical site. The term "clinical flushing" used interchangeably with the term "flushing" herein generally means that a solution (usually an aqueous solution) is applied to a wound or a surgical site. As provided herein, a flushing solution can be applied for a variety of purposes, for example, for tissue hydration, cleaning, removal of debris or surface pathogens, lubrication, avoidance of tissue attachment or auxiliary visual inspection. In certain embodiments, flushing is included in a stable flow of applying a flushing solution on an open wound or a surgical site. In certain embodiments, the flushing solution is applied intermittently. The mode of application and the volume and flow rate of the flushing solution applied will depend on the specific circumstances, for example, the size of the wound, the tissue being flushed, and the state of the wound or surgical site (for example, the presence of debris, exposure to surface pathogens). Those skilled in the art will be able to determine the appropriate method and device suitable for application and suitable volume and flow rate.

[0144] Although in some cases, simple irrigation solutions may be sufficient to achieve some irrigation purposes, conventional irrigation solutions (such as saline, phosphate buffered saline (PBS), antiseptics or antibiotics) may not support the survival of sensitive cells or tissues or are cytotoxic to sensitive cells or tissues in certain surgical situations, thereby negatively affecting the outcome of the surgical procedure.

[0145] A commonly used clinical irrigation solution, normal saline (0.9% NaCl in water), is isotonic and often used for wound irrigation due to its low toxicity, physiological properties (pH and osmolarity), ease of preparation and sterilization (including steam sterilization), and long shelf life at ambient temperature. One disadvantage of normal saline is that it does not support the long-term survival of sensitive cells or tissues, and relatively high rates of wound infection have been reported after irrigation with normal saline compared to other irrigation solutions.

[0146] Many commercially available surgical irrigation solutions have been developed for the purpose of irrigating sensitive surgical sites or wounds, e.g., by irrigating sites or wounds that may be negatively affected, e.g., during ophthalmic surgery, with saline or other simple irrigation solutions. There are generally four key ingredients in currently available surgical irrigation solutions (which are intended for use in surgery to prevent trauma to sensitive cells or tissues, e.g., during ophthalmic surgery): an agent for maintaining osmolarity, a calcium source, a magnesium source, and a buffer.

[0147] Some embodiments of the present disclosure provide methods of irrigating a surgical site with a solution provided herein (e.g., with a GS2 solution). In certain embodiments, the surgical site is an eye of a subject.

[0148] Methods and devices for flushing surgical sites are well known to those of ordinary skill in the art. The skilled artisan will appreciate that the method of delivery, volume, and pressure used will depend on the nature and condition of the surgical site. Suitable devices for clinical flushing include, but are not limited to, bulb syringes, piston syringes, pressure tanks, vortex mixers, vortex hose sprayers, flushing fluids in plastic containers with pouring caps or nozzles, and pulse irrigation devices (e.g., jet irrigation, mechanical irrigation, pulsating irrigation, mechanical irrigation, high pressure irrigation devices).

[0149] In certain embodiments, irrigation is continuous, wherein a continuous stream of irrigation fluid is applied to the surgical site. In other embodiments, pulsed or intermittent irrigation is employed, wherein intermittent or discontinuous delivery of irrigation fluid is performed. The irrigation volume will depend on the characteristics of the surgical site and the purpose of the irrigation (wound cleaning, hydration, etc.).

[0150] Reagent test kit

[0151] Some aspects of the present disclosure provide a kit comprising (a) a solution provided herein; and (b) instructions for contacting a cell population with the solution of (a) to produce a cell product; and (c) a container for contacting (b) and / or storing the cell product of (b). In certain embodiments, the solution of (a) and the container of (c) are suitable for the cell product of (b) for transplantation to a subject.

[0152] Therefore, the present disclosure provides, inter alia, the following:

[0153] Item 1. A solution comprising

[0154] (a) a buffer to maintain the solution at physiological pH; and

[0155] (b) at least 2 mM or at least 0.05% (w / v) glucose; and

[0156] (c) an osmotically active agent that maintains the solution at physiological osmolarity.

[0157] Item 2. The solution of Item 1, wherein the solution comprises at least 5 mM or at least 0.1% (w / v) glucose.

[0158] Clause 3. The solution of Clause 1, wherein the solution comprises at least 7.5 mM or at least 0.14% (w / v) glucose.

[0159] Clause 4. The solution of Clause 1, wherein the solution comprises at least 10 mM or at least 0.2% (w / v) glucose.

[0160] Clause 5. The solution of Clause 1, wherein the solution comprises at least 15 mM or at least 0.25% (w / v) glucose.

[0161] Clause 6. The solution of Clause 1, wherein the solution comprises at least 20 mM or at least 0.4% (w / v) glucose.

[0162] Clause 7. The solution of Clause 1, wherein the solution comprises at least 25 mM or at least 0.5% (w / v) glucose.

[0163] Clause 8. The solution of any one of Clauses 1-7, wherein the solution further comprises a source of divalent cations.

[0164] Item 9. The solution of Item 8, wherein the source of divalent cations comprises a calcium source and / or a magnesium source.

[0165] Clause 10. The solution of any one of Clauses 1-9, wherein the buffer comprises acetate buffer and / or citrate buffer.

[0166] Item 11. A solution comprising

[0167] (a) a buffer that maintains the solution at physiological pH, wherein the buffer is not a bicarbonate buffer; and

[0168] (b) glucose; and

[0169] (c) an osmotically active agent that maintains the solution at physiological osmolarity; and

[0170] (d) Source of divalent cations.

[0171] Item 12. The solution of Item 11, wherein the source of divalent cations of (d) comprises a calcium source and / or a magnesium source.

[0172] Clause 13. The solution of any one of Clauses 11-12, wherein the buffer comprises acetate buffer and / or citrate buffer.

[0173] Clause 14. The solution of any one of Clauses 9-10 or 12-13, wherein the calcium source comprises a pharmaceutically acceptable calcium salt.

[0174] Clause 15. The solution of any one of Clauses 9-10 or 12-14, wherein the magnesium source comprises a pharmaceutically acceptable magnesium salt.

[0175] Clause 16. The solution of any one of Clauses 14-15, wherein the pharmaceutically acceptable calcium and / or the pharmaceutically acceptable magnesium salt is selected from calcium salts and / or magnesium salts formed with an acid selected from acetic acid, ascorbic acid, citric acid, hydrochloric acid, maleic acid, oxalic acid, phosphoric acid, stearic acid, succinic acid and sulfuric acid.

[0176] Clause 17. The solution of any of clauses 9-10 or 12-16, wherein the calcium source comprises calcium chloride.

[0177] Clause 18. The solution of any of clauses 9-10 or 12-17, wherein the calcium source comprises calcium chloride dihydrate.

[0178] Clause 19. The solution of any of clauses 9-10 or 12-18, wherein the magnesium source comprises magnesium chloride.

[0179] Clause 20. The solution of any one of clauses 9-10 or 12-18, wherein the magnesium source comprises magnesium chloride hexahydrate.

[0180] Clause 21. The solution of any one of clauses 10 or 13-20, wherein the citrate buffer is provided as sodium citrate.

[0181] Clause 22. The solution of any one of clauses 1-21, wherein the glucose is D-glucose (dextrose).

[0182] Clause 23. The solution of any one of Clauses 1-22, wherein the osmotically active agent is a salt.

[0183] Clause 24. The solution of any one of Clauses 1-23, wherein the osmotically active agent is a sodium salt.

[0184] Clause 25. The solution of any one of Clauses 1-24, wherein the osmotically active agent is sodium chloride.

[0185] Clause 26. The solution of any one of clauses 1-25, wherein the solution comprises calcium chloride, magnesium chloride, sodium citrate, sodium chloride, and glucose.

[0186] Clause 27. The solution of any one of clauses 1-26, wherein the pH of the solution is 6.8-7.8.

[0187] Clause 28. The solution of any one of clauses 1-27, wherein the pH of the solution is 7.4-7.5.

[0188] Clause 29. The solution of any one of Clauses 1-28, wherein the pH of the solution is about 7.5.

[0189] Clause 30. The solution of any one of Clauses 1-29, wherein the solution is isotonic.

[0190] Clause 31. The solution of any one of Clauses 1-29, wherein the solution is hypertonic.

[0191] Clause 32. The solution of any one of clauses 1-31, wherein the solution exhibits an osmolarity of about 270-345 mOsm / l.

[0192] Clause 33. The solution of any one of clauses 1-32, wherein the osmolarity of the solution is about 315 mOsm / l.

[0193] Clause 34. The solution of any one of clauses 9-10 or 12-33, wherein the concentration of the calcium source is 0.25-0.75 mM.

[0194] Clause 35. The solution of any one of clauses 9-10 or 12-34, wherein the concentration of the calcium source is 0.4-0.65 mM.

[0195] Clause 36. The solution of any one of clauses 9-10 or 12-35, wherein the concentration of the calcium source is 0.5-0.6 mM, or the solution of any one of clauses 9-10 or 12-35, wherein the concentration of the calcium source is 0.5-0.9 mM, or wherein the concentration of the calcium source is 0.6-0.8 mM.

[0196] Clause 37. The solution of any one of clauses 9-10 or 12-36, wherein the concentration of the calcium source is about 0.6 mM, or the solution of any one of clauses 9-10 or 12-36, wherein the concentration of the calcium source is about 0.7 mM.

[0197] Clause 38. The solution of any of clauses 9-10 or 12-37, wherein the concentration of the magnesium source is 0.05-5 mM.

[0198] Clause 39. The solution of any one of clauses 9-10 or 12-38, wherein the concentration of the magnesium source is 0.1-0.3 mM.

[0199] Clause 40. The solution of any of clauses 9-10 or 12-39, wherein the concentration of the magnesium source is about 0.3 mM.

[0200] Clause 41. The solution of any one of clauses 1-40, wherein the concentration of glucose is 5-50 mM.

[0201] Clause 42. The solution of any one of clauses 1-41, wherein the concentration of glucose is 10-25 mM.

[0202] Clause 43. The solution of any one of clauses 1-42, wherein the concentration of glucose is 10-20 mM.

[0203] Clause 44. The solution of any one of clauses 1-43, wherein the concentration of glucose is about 16 mM.

[0204] Clause 45. The solution of any one of clauses 1-44, wherein the concentration of the osmotically active agent is about 100-200 mM.

[0205] Clause 46. The solution of any one of Clauses 1-45, wherein the concentration of the osmotically active agent is about 125-175 mM.

[0206] Clause 47. The solution of any one of Clauses 1-46, wherein the concentration of the osmotically active agent is about 150 mM.

[0207] Clause 48. The solution of any one of clauses 10 or 13-47, wherein the concentration of citrate or acetate is 0.1-5 mM.

[0208] Clause 49. The solution of any one of clauses 10 or 13-48, wherein the concentration of citrate or acetate is 0.5-2 mM.

[0209] Clause 50. The solution of any one of clauses 10 or 13-39, wherein the concentration of citrate or acetate is about 1 mM.

[0210] Clause 51. The solution of any of Clauses 1-51, wherein the solution further comprises a potassium salt.

[0211] Item 52. The solution of Item 51, wherein the potassium salt is potassium chloride.

[0212] Item 53. The solution of Item 51 or 52, wherein the concentration of KCl is 0.2-5 mM.

[0213] Item 54. The solution of Item 53, wherein the concentration of KCl is 1-2.5 mM.

[0214] Clause 55. The solution of Clause 54, wherein the concentration of KCl is about 2 mM.

[0215] Item 56. The solution of any of items 1-55, wherein the solution comprises about 0.7 mM CaCl (calcium chloride), about 0.03 mM MgCl (magnesium chloride), about 1 mM sodium citrate, about 16 mM dextrose and about 145 mM NaCl, or the solution of any of items 1-55, wherein the solution comprises about 0.7 mM CaCl (calcium chloride), about 0.3 mM MgCl (magnesium chloride), about 1 mM sodium citrate, about 16 mM dextrose and about 145 mM NaCl, or the solution of any of items 1-55, wherein the solution comprises about 0.5-0.9 mM CaCl (calcium chloride), about 0.2-.4 mM MgCl (magnesium chloride), about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose and about 116-174 mM NaCl.

[0216] Clause 57. The solution of any of clauses 1-55, wherein the solution comprises about 0.85% NaCl, about 0.01% CaCl dihydrate (calcium chloride dihydrate), about 0.006% MgCl hexahydrate (magnesium chloride hexahydrate), about 0.035% sodium citrate dihydrate, and about 0.29% dextrose, or the solution of any of clauses 1-55, wherein the solution comprises about 0.68-1.02% NaCl, about 0.008-0.012% CaCl dihydrate (calcium chloride dihydrate), about 0.0048-0.0072% MgCl hexahydrate (magnesium chloride hexahydrate), about 0.028-0.042% sodium citrate dihydrate, and about 0.23-0.35% dextrose.

[0217] Clause 58. The solution of any one of clauses 1-57, wherein the solution further comprises about 2 mM KCl.

[0218] Clause 59. The solution of any one of Clauses 1-58, wherein the solution further comprises a viscoelastic polymer.

[0219] Item 60. The solution of Item 59, wherein the polymer is hyaluronic acid or a salt or solvate thereof.

[0220] Item 61. The solution of Item 59 or 60, wherein the polymer is sodium hyaluronate.

[0221] Clause 62. The solution of any one of clauses 59-61, wherein the polymer is present at a concentration effective to reduce exposure of cells in the solution to shear stress.

[0222] Clause 63. The solution of any one of clauses 59-62, wherein the concentration of the polymer is 0.005-5% w / v.

[0223] Clause 64. The solution of Clause 63, wherein the concentration of the polymer is about 0.05% w / v.

[0224] Clause 65. The solution of any one of clauses 1-64, wherein the solution comprises about 0.7 mM CaCl (calcium chloride), about 0.03 mM MgCl (magnesium chloride), about 2 mM KCl, about 1 mM sodium citrate, about 16 mM dextrose, about 145 mM NaCl and about 0.05% hyaluronic acid, or the solution of any one of clauses 1-64, wherein the solution comprises about 0.7 mM CaCl (calcium chloride), about 0.3 mM MgCl (magnesium chloride), about 2 mM KCl, about 1 mM sodium citrate, about 16 mM dextrose, about 145 mM NaCl and about 0.05% hyaluronic acid, or the solution of any one of clauses 1-64, wherein the solution comprises about 0.5-0.8 mM CaCl (calcium chloride), about 0.2-.4 mM MgCl (magnesium chloride), about 1.6-2.4 mM KCl, about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, about 116-174 mM NaCl, and about 0.04-0.06% hyaluronic acid.

[0225] Clause 66. The solution of any one of clauses 1-65, wherein the solution does not comprise a carbonate buffer.

[0226] Clause 67. The solution of any one of clauses 1-66, wherein the solution does not comprise glutathione or glutathione disulfide (GSSG).

[0227] Clause 68. The solution of any one of clauses 1-67, wherein the solution does not comprise a zwitterionic organic buffer.

[0228] Item 69. The solution of any of items 1-68, wherein the solution can be stored at 25°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month without measurable precipitation of solutes and / or measurable loss of the ability of the solution to support the survival and viability of cells stored in the solution.

[0229] Item 70. The solution of any of items 1-69, wherein the solution can be stored at 2-8°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks or at least 1 month without measurable precipitation of solutes and / or measurable loss of the ability of the solution to support the survival and viability of cells stored in the solution.

[0230] Clause 71. The solution of any one of Clauses 1-70, wherein the solution is suitable for administration to a subject, suitable for administration to an eye of a subject, and / or suitable for transplanting cells into an eye of a subject.

[0231] Clause 72. The solution of any one of clauses 1-71, wherein the solution is substantially pyrogen-free.

[0232] Clause 73. The solution of any one of clauses 1-72, wherein the solution is sterile.

[0233] Clause 74. The solution of any one of clauses 1-73, wherein the solution is for flushing, cell reconstitution, cell storage, cell transport and / or administration to a subject.

[0234] Clause 75. An article comprising a population of cells in the solution of any one of clauses 1-74.

[0235] Clause 76. The article of manufacture of Clause 75, wherein the cell population is suitable for transplantation into a subject.

[0236] Clause 77. The article of manufacture of Clause 76, wherein the cell population is suitable for transplantation into an eye of a subject.

[0237] Clause 78. The article of any one of clauses 75-77, wherein the cell population comprises RPE cells.

[0238] Clause 79. The article of any one of clauses 75-78, wherein the cell population comprises photoreceptor cells.

[0239] Clause 80. The article of any one of clauses 75-79, wherein the cell population comprises mesenchymal cells.

[0240] Clause 81. The product of any one of clauses 75-80, wherein the product is refrigerated.

[0241] Clause 82. The product of clause 81, wherein the product is refrigerated at about 2-8°C.

[0242] Clause 83. The preparation of any one of clauses 75-82, wherein the preparation supports survival of cells in the cell population during storage of the preparation, and wherein at least 70% of the cells in the cell population are alive after storing the preparation at 2-8°C for 48 hours.

[0243] Clause 84. The preparation of Clause 83, wherein at least 80% of the cells in the cell population are viable after storing the preparation at 2-8°C for 48 hours.

[0244] Clause 85. The preparation of Clause 83, wherein at least 90% of the cells in the cell population are viable after storing the preparation at 2-8°C for 48 hours.

[0245] Item 86. The product of any one of items 75-85, wherein the product supports the maintenance of the plating efficiency of the cell population during storage of the product, and wherein after storing the product at 2-8°C for 48 hours, the cell population exhibits at least 70% of its original plating efficiency, wherein the original plating efficiency is the plating efficiency of the cell population at the beginning of the storage period.

[0246] Clause 87. The preparation of Clause 86, wherein the cell population exhibits at least 80% of its original plating efficiency after storage of the preparation at 2-8°C for 48 hours.

[0247] Clause 88. The article of manufacture of Clause 86, wherein the cell population exhibits at least 90% of its original plating efficiency.

[0248] Clause 89. The article of any of clauses 75-88, wherein the article is in a storage container.

[0249] Clause 90. The article of any one of clauses 75-89, wherein the article is in a syringe.

[0250] Clause 91. A method for preparing the article of any one of clauses 75-90, wherein the method comprises contacting a population of cells with the solution of any one of clauses 1-74.

[0251] Clause 92. The method of clause 91, wherein the method comprises contacting a cryopreserved or pelleted population of cells with the solution of any one of clauses 1-74, thereby reconstituting the cells.

[0252] Clause 93. A pharmaceutical composition comprising the solution of any one of clauses 1-74 or the preparation of any one of clauses 75-90, wherein the pharmaceutical composition is suitable for administration to a subject.

[0253] Clause 94. A method comprising administering the solution of any one of clauses 1-74 or the preparation of any one of clauses 75-90 or the pharmaceutical composition of clause 88 to a subject in need thereof.

[0254] Clause 95. The method of Clause 94, wherein the method comprises applying the solution or the preparation to an eye of the subject.

[0255] Clause 96. The method of Clause 94 or 95, wherein the method comprises administering the preparation to the subject after storing the preparation for at least 4, at least 6, at least 12, at least 24, at least 36 or at least 48 hours.

[0256] Clause 97. The method of Clause 96, wherein the subject has or has been diagnosed with a retinal disease.

[0257] Clause 98. The method of Clause 97, wherein the retinal disease is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, Leber congenital amaurosis, or Stargardt's disease.

[0258] Clause 99. The method of clause 97 or 98, wherein the preparation comprises a population of cells of a size effective to improve at least one symptom of a retinal disease in the subject.

[0259] Clause 100. The method of any one of clauses 97-99, wherein the method further comprises monitoring at least one symptom of a retinal disease in the subject.

[0260] Clause 101. A method comprising:

[0261] (a) contacting a population of cells with a solution according to any one of clauses 1 to 74 to produce a cell preparation.

[0262] Clause 102. The method of clause 101, further comprising:

[0263] (b) storing the cell preparation of (a) for at least 4, at least 6, at least 12, at least 18, at least 24, at least 36, at least 48, at least 60 or at least 72 hours.

[0264] Clause 103. The method of clause 102, wherein the method further comprises

[0265] (c) administering the cell product of (a) to a subject after the storage period of (b).

[0266] Clause 104. The method of Clause 103, wherein administering of (c) comprises injecting the cells into the eye of the subject.

[0267] Clause 105. The method of any one of clauses 101-104, wherein the method further comprises determining the viability of cells in the cell preparation of (a) after the storage period of (b).

[0268] Clause 106. The method of any one of clauses 101-105, wherein the method comprises refrigerating the cell preparation of (a) during the storage phase of step (b).

[0269] Clause 107. The method of Clause 106, wherein refrigeration comprises storing the cell product at a temperature of 2-8°C.

[0270] Clause 108. A method for treating a retinal disease, the method comprising

[0271] An effective amount of the preparation of any one of clauses 75 to 90 or the pharmaceutical composition of clause 93 is administered to an eye of a subject having a retinal disease.

[0272] Clause 109. The method of Clause 108, wherein the retinal disease is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, Leber congenital amaurosis, or Stargardt's disease.

[0273] Clause 110. A kit comprising

[0274] (a) a solution according to any one of clauses 1 to 74;

[0275] (b) instructions for contacting the cell population with the solution of (a) to produce a cell product; and

[0276] (c) a container for contacting (b) and / or for storing the cell product of (b).

[0277] Clause 111. The kit of Clause 110, wherein the solution of (a) and the container of (c) are suitable for use of the cell preparation of (b) for transplantation into a subject. Example

[0278] Introduction

[0279] Using Alcon BSS As an RPE cell formulation, storage and transplantation medium, a Phase 1 clinical trial is being conducted on the administration of RPE cells to the eyes of subjects with SMD and AMD. BSS It is a physiologically compatible (osmolarity ~310 mOs, pH ~7.4) solution approved for use in intraocular surgery. When stored at 2-8°C prior to clinical administration (injection), it is The shelf life of the RPE cells prepared in is limited to about 4 hours. Due to this limited product shelf life, a satellite cell processing laboratory must be established in the immediate vicinity of each clinical site participating in the trial.

[0280] Developing a medium that significantly extends the shelf life of the final product (e.g., to 48 hours or more) will confer numerous advantages. The enhanced shelf life will allow the final product formulation to be consolidated in a single location, from which the final product can be shipped to all clinical sites in the U.S. In this way, the number of clinical sites can be expanded, which is currently limited to those located in the immediate vicinity of the cGMP processing site. The extended product shelf life will also eliminate the logical complexity associated with maintaining multiple material inventories, training personnel, and supervising multiple subsidiary processing sites. The extended shelf life will enhance the flexibility of arranging transplants, which currently must occur in a tight 4-hour window.

[0281] Extending the final product shelf life will allow ample time to notify any delay or cancellation just before the patient is prepared for surgery or enters the operating room (OR). In addition, if the final product fails in a mass release test, an additional final product formulation may be prepared that day without delaying or canceling the surgery. Extending the shelf life allows additional time for supplemental final product release testing such as DNA qPCR using pan primers to detect the presence of microbial contaminants set forth below.

[0282] Through MedOne REF 3233 Cannula 23 / 38 delivered at BSS The final RPE product is formulated in. Using the current BSS Preparation, an average loss of -23% viable cell density was observed. This loss was consistent across all cell densities tested and had no apparent effect on the remaining 77% of cells extruded through the cannula in terms of viability or ability to subsequently be seeded, propagated, and differentiated in culture. Although some cell loss due to attachment was expected, subsequent experiments were consistent with cell loss and possible cell lysis (attributable to shear forces generated during cannula extrusion).

[0283] To compensate for the expected loss, the cannula loading density is increased accordingly to ensure the required dose is delivered. In addition to increasing the shelf life, a final formulation medium with suitable viscoelastic properties will minimize cell loss during cannula delivery. Minimizing cell loss will reduce the delivery of cell debris, thereby alleviating potential immune responses to intracellular components.

[0284] Embodiment 1: medium composition

[0285] GS2 Media

[0286] A medium for cell reconstitution, storage, transport and / or administration to a subject is prepared. The medium designated "GS2" is prepared as follows:

[0287] combination

[0288] 48.75 ml of 0.9% NaCl solution in water;

[0289] 13.10 ml Alcon balanced salt solution 300mOsm in water; and

[0290] 3.75 ml 5% dextrose in 0.9% NaCl in water (560 mOsm)

[0291] This yielded 65.6 mL of medium with a final concentration of 0.29% dextrose and an osmolarity of 315 mOsm.

[0292] The basic GS2 medium thus contains

[0293] About 145 mM NaCl (about 0.85% NaCl),

[0294] About 2 mM KCl (about 0.015% KCl),

[0295] About 0.7 mM CaCl (calcium chloride) (about 0.01% CaCl dihydrate (calcium chloride dihydrate)),

[0296] about 0.3 mM MgCl (magnesium chloride) (about 0.006% MgCl hexahydrate (magnesium chloride hexahydrate)),

[0297] about 1 mM sodium citrate (about 0.035% sodium citrate dihydrate), and

[0298] About 16 mM glucose (about 0.29% dextrose).

[0299] Optionally, the GS2 medium may further comprise a viscoelastic polymer in an amount effective to reduce shear stress on cells (eg, at a final concentration of about 0.005-5% w / v). In certain embodiments, the viscoelastic polymer is hyaluronic acid or a salt or solvate thereof.

[0300] Alcon Balanced Salt Solution

[0301] Alcon Balanced Salt Solution ( Sterile Irrigation Solution) is a sterile balanced salt solution containing

[0302] 0.64% sodium chloride (NaCl),

[0303] 0.075% potassium chloride (KCl),

[0304] 0.048% calcium chloride dihydrate (CaCl2·2H2O),

[0305] 0.03% magnesium chloride hexahydrate (MgCl2·6H2O),

[0306] 0.39% sodium acetate trihydrate (C2H3NaO2·3H2O),

[0307] 0.17% sodium citrate dihydrate (C6H5Na3O7·2H2O),

[0308] Sodium hydroxide and / or hydrochloric acid (to adjust pH),

[0309] and water for injection.

[0310] The pH is about 7.5, and the osmolality is about 300 mOsm / Kg.

[0311] Alcon BSS thus includes

[0312] About 109mM NaCl,

[0313] About 10 mM KCl

[0314] About 3mM CaCl (Calcium Chloride)

[0315] About 0.1mM MgCl (magnesium chloride)

[0316] About 5mM sodium citrate

[0317] Alcon Balanced Salt Solution PLUS( PLUS)

[0318] Alcon Balanced Salt Solution PLUS (BSS ) is a sterile intraocular irrigation solution for use during intraocular surgical procedures. It is reconstituted prior to use from two parts designated "Part I" and "Part II".

[0319] Part I is a sterile 480 mL solution in a 500 mL single-dose bottle to which the Part II concentrate is added. BSS Part I contains

[0320] 7.440mg / ml sodium chloride,

[0321] 0.395mg / ml potassium chloride,

[0322] 0.433mg / ml disodium hydrogen phosphate,

[0323] 2.190mg / ml sodium bicarbonate,

[0324] hydrochloric acid and / or sodium hydroxide (to adjust pH), and

[0325] Water for injection.

[0326] Part II is a sterile concentrate in a 20 ml single-dose vial for addition to Part I. BSS Part II contains:

[0327] 3.85mg / ml calcium chloride dihydrate,

[0328] 5mg / ml magnesium chloride hexahydrate,

[0329] 23mg / ml dextrose,

[0330] 4.6 mg / ml glutathione disulfide (oxidized glutathione), and

[0331] Water for injection.

[0332] BSS After adding Part II to the Part I bottle, the reconstituted product contains in Water for Injection:

[0333] 7.14mg / ml sodium chloride,

[0334] 0.38mg / ml potassium chloride,

[0335] 0.154mg / ml calcium chloride dihydrate,

[0336] 0.2mg / ml magnesium chloride hexahydrate,

[0337] 0.42mg / ml disodium hydrogen phosphate,

[0338] 2.1mg / ml sodium bicarbonate,

[0339] 0.92mg / ml dextrose,

[0340] Glutathione disulfide (oxidized glutathione) 0.184 mg / ml,

[0341] Hydrochloric acid and / or sodium hydroxide (to adjust pH).

[0342] The reconstituted product had a pH of about 7.4 and an osmolality of about 305 mOsm.

[0343] Embodiment 2: Shelf life of the final RPE product formulated in

[0344] In BSS The RPE final product prepared in refrigeration (2-8 ℃) maintained its viability for 4 hours. A more comprehensive evaluation of the final product shelf life has been completed. In this study, RPE bulk product (Bulk Product) is melted and prepared at 2 viable cell densities supporting the final storage density of 2,000 viable cells / μl. For all dosages prepared, this storage density is constant. Perform BSS with pre-measured volume in the OR before loading the syringe. Final Dilution of Cells. This dilution step determines the final cell density delivered in the 150 μL injection volume (also constant for all doses).

[0345] Final cold storage was assessed at formulation (0 h) and after 4 and 6 h of cold storage (2-8°C). The cell viability, viable cell density, purity and potency of the RPE cell product prepared.Show that viable cell density and cell viability are constant for 6 hours in cold storage.In addition, the RPE cell inoculation of the preparation stored for 0,4 and 6 hours, and cultivate the purity and potency assessment subsequently.For each time point of inoculation (0,4 and 6 hours), purity is assessed by MITF and PAX6 immunostaining, and potency is assessed by measuring the FACS analysis of the granule engulfed.Data show that all product attributes of test keep constant in the 6 hour period of evaluation.

[0346] In BSS RPE cells stored in BSS-Plus for 12 hours typically exhibited less than 70% viability, and after 24 hours of storage in BSS-Plus, RPE cell viability was typically less than 20%.

[0347] Example 3: Development of Improved Cell Reconstitution, Storage, Delivery and / or Transplantation Media

[0348] Improved media for cell reconstitution (e.g., from a cryopreserved state, cell storage (e.g., cold storage between reconstitution or harvesting from cell culture and transport to a transplant facility or administration to a subject)), cell transport, and cell transplantation have been developed. The components of the resulting medium (GS2) are listed in Table 1 below. Exemplary vendors are provided. One skilled in the art will appreciate that there are additional sources for the listed components and will be able to determine suitable sources for these agents:

[0349]

[0350] The GS2 final formulation was physiological in terms of pH (7.2-7.6) and osmolarity (calculated osmolarity 315).

[0351] Prepare GS2 medium and distribute in the dedicated RPE cell final product Iso-7 clean room in Iso-5 BSC.The sterile components in Table 1 are added in the sterile reservoir (reservoir) and placed on a rotary shaker (30-40RPM).After minimum 3 hours, sample is taken out from the reservoir, and pH is measured and the pH is adjusted to 7.4 ± 0.2 with the incremental addition of 0.1N NaOH.The GS2 for filling does not come into contact with the pH probe.The solution is sterilized with a membrane filter and pH is rechecked.

[0352] 3 ml aliquots of GS2 are distributed into gamma-irradiated cryovials consisting of fresh polypropylene resin that meets USP Class VI limits. Samples are taken out from each bottle filled and the vials are capped. QC tests are performed on the samples merged as described below. Visual inspection is performed on each bottle under visible light and ultraviolet light to confirm the absence of microparticles. The vials are returned to sterile conditions for labeling and storage at 2-8°C. After refrigeration for a minimum of 1 day, several vials are taken out and the pH is re-tested using a pH meter calibrated with 2-8°C pH standards to confirm acceptability at the use temperature (2-8°C).

[0353] Example 4: Quality Control and Release Specifications of RPE Cell Final Product in GS2

[0354] The aliquots of the GS2 (which is composed of the sample taken out from all filled bottles) merged are subjected to 14 days USP sterility, pH, osmotic pressure concentration and endotoxin test. The performance of maintaining RPE cell viability and growth of each batch of GS2 after preparation and extruding through the injection cannula is also assessed. Exemplary quality control test and release specification are provided in Table 2 below: 4

[0356] It should be understood that the release criteria listed in Table 2 are exemplary, and any combination of any of the criteria listed in Table 2 may be combined and used alone or in combination with additional criteria for release testing and quality control.

[0357] In an exemplary embodiment of RPE cell growth assay, after the GS2 storage stage, cells are seeded in RPE GM or EGM2 / EBM2 culture medium (Lonza, for example, catalog number: CC-3156, CC-4176) in 96 well plates coated with gelatin with initial density (for example, at a density of 20,000 cells / well). Cells are grown for 2-3 days under suitable conditions, for example, at 5% CO2, at 37 ° C, and in a humidity controlled incubator. Cells are raised from holes, for example, by trypsinization, and then counted. If at the end of the culture phase, the cell count in each hole is 125% or more of the initial cell density (for example, for the initial density of 20,000 cells / well, > / =25,000 cells / well), then the GS2 quality release test standard for the test is met.

[0358] Example 5: RPE final product formulation, packaging and delivery

[0359] In the following process, BSS Used in the initial wash step of GS2 processing. Embodiments of the present disclosure also contemplate and include use in GS2 instead of BSS Wash in . Vials of MA09-hRPE cells cryopreserved for clinical use are recovered from liquid nitrogen storage. Depending on the dose, 2-4 vials of cells are required. The cryovials are transported to a clean room and thawed quickly in a 37°C water bath. The thawed contents of each bottle (1ml cryopreservation medium (90% FCS + 10% DMSO) containing 2 million cells during cryopreservation) are gently resuspended in warm DMEM, transferred to a 50ml conical tube and reached a final volume of 40ml with additional warm DMEM. The resuspended cells of each test tube are centrifuged (at room temperature 160 × g for 5 minutes), and each pellet is resuspended in 40ml room temperature BSS Each cell suspension was centrifuged again, and the pellets were combined and resuspended in 10 ml of room temperature BSS. The resuspended pooled cells were centrifuged (160×g for 5 minutes at room temperature) for a third time and the supernatant was aspirated.

[0360] A flow chart of the steps for the preparation of the above product (GS2 processing steps are underlined) is shown below Fig.12 shown.

[0361] After removing as much supernatant as possible, resuspend the pellet in a certain volume of cold BSS. (current treatment) or cold GS2 (GS2 treatment), for every 1,000,000 cells that melt, target a final volume of 50 μ l. From this point on, cells are maintained in a pre-cooled test tube holder so that cells are maintained at 2-8 ° C in the remaining method steps. The typical recovery of 15-25% target volume will produce a cell suspension of approximately 4,000 live RPE cells / μ l.

[0362] Remove a sample, perform a viable cell count, and determine the viable cell density and total number of cells recovered. Add additional cold BSS Or cold GS2 was added to the cell suspension to bring the final cell concentration to 2,300 viable RPE cells / μl (300 cells more than the target final preparation concentration of 2,000 viable RPE cells / μl). Perform a confirmatory cell count and add additional cold BSS or cold GS2 to a final concentration of 2,000 viable RPE cells / μl. Dispense the desired volume of cells into the final product closure (0.5 ml sterile microcentrifuge tubes; Fisher, catalog number 02-707-351). Attach the product label to the tube for BSS Each tube treated or Whirl-Pak containing a tube of product for GS2 treatment, specify BSS 4 hours expiration for RPE or 48 hours expiration for GS2RPE. Samples were removed from each product tube and these samples were combined for documentation and QC testing.

[0363] In addition, developed the preparation scheme of cultivated cells.In this scheme, the RPE cells of cryopreservation are thawed, and pre-cultured 3-7 days before the cell preparation in GS2 transplantation medium.Cultivated cells are raised from culture dish, and first washed in DMEM, then washed in BSS-Plus, and finally washed in BSS-Plus mixed with GS2 medium 1:1.After the final washing step, cells are transferred into cold (2-8 ℃) GS2 medium.The volume adjustment of sample taking out, test and final cell density is as mentioned above.

[0364] A flow chart of the steps for the preparation of the above product (GS2 processing steps are underlined) is shown below Fig.13 shown.

[0365] In BSS Packaging and delivery of RPE in clinical trials. For clinical trial transplants (SMD and AMD), each dose consists of a pair of tubes: one tube contains a pre-measured volume of RPE cells formulated at 2,000 viable RPE cells / μl and the other tube contains a pre-measured volume of BSS Each pair of tubes was placed in a Labtop tube cooling rack at 2-8° C. The cooling racks were bagged, placed in a Coleman Cooler containing -16 lbs of pre-cooled Insul-Ice, and hand delivered to the clinic by courier.

[0366] In the OR, immediately prior to implantation, BSS is placed in the Add cells. Mix cells and load into 1 ml syringe. Extrude product (150 μl for all doses) through injection cannula into subretinal space. Load cell density in syringe is set at 1.33 times delivery density to compensate for 25% expected loss during cannula extrusion.

[0367] In BSS A flowchart of the packaging and shipping steps is shown in Fig.14 shown.

[0368] The packaging and delivery of RPE in GS2.Each dosage is made up of 1 microcentrifuge tube containing 250 μ l RPE cells prepared with 2,300 live RPE cells / μ l.The single test tube of the final prepared RPE product containing 1 dosage is placed in 2 ounces of sterile Whirl-Pak bags, and the bag has attached the final product label.The bagged test tube is placed in a clean, pre-cooled (2-8 ℃), uniquely numbered portable test tube cooler.Except the labeled product test tube, the labeled attached test tube (not bagged) containing 30 μ l RPE product cells and the separate test tube containing 100 μ l 0.4% trypan blue are placed in each Chillette cooler.The final product label is also attached to each Chillette cooler.Each cooler containing product is placed in a sterile Whirl-Pak bag under aseptic conditions. The bagged coolers containing product, the accompanying clinical order form and two product labels are placed in the cleanroom pass-through leading to the final packaging and shipping area. To avoid mix-ups, each Chillette cooler is aseptically loaded with product tubes and placed into the cleanroom pass-through one at a time.

[0369] The packaging personnel retrieve each Chillette cooler and accompanying documentation and final product label from the cleanroom access and place the bagged Chillette coolers, foam packaging, blunt filling needles, syringes, injection cannulas, and hemacytometers into the shipping room of the pre-chilled Nano Cool cooling unit. The lid of the cooling unit is fastened and the outer shipping box is filled with documentation regarding receipt, inspection, storage, and post-shipment viability testing. The packaged product is placed in quarantine until all product release testing has been performed and the "Final Product Certificate for Release of RPE Cells for Transplantation" has been issued. The NanoCool shipping box with the product and documentation is sent to the clinic overnight. Upon receipt, the shipping box can be stored at room temperature or at 2-8°C until the product is used within 48 hours from the time of final filling. The final product label with the expiration date and time is attached to the outer shipping box, Chillette tube cooler, and the bag containing the final product tube. Packaging and Shipping of RPE in GS2 as Fig.15 shown.

[0370] RPE final product quality release testing. Current final product (at BSS) performed prior to transplantation (Medium) Quality release testing includes cell viability check and Gram stain. Samples are sent for USP 14-day sterility testing and results are reported after transplantation.

[0371]

[0372] qPCR for detecting contaminants. In the 4 hours of GS2, it is extended to at least 48 hours in GS2, and it is possible to perform a determination with a longer report time (turn-around time) before the product is released. For example, it is now possible to perform PCR determination and receive the result for quality control and release purpose to the RPE final product before the release and use of the product. A suitable determination that can be performed is the pan-primer DNA qPCR determination. Such a determination can be completed in 2 hours. The qPCR determination is extremely sensitive, and can be used to detect a wide range of pathogenic pollutants, such as environmental pollutants, microbial pollutants and viral pollutants and common skin commensals (commensules). The qPCR determination can be used as an auxiliary final product release determination for detecting microbial pollutants except Gram staining and USP sterility test. Known qPCR results before product shipment, so as to prevent the product from potentially contaminated from leaving the equipment. It should be understood that the qPCR determination described herein is exemplary, and other suitable determinations can be performed alternatively or additionally, including, but not limited to, other PCR determinations and other types of determinations that can be performed in the extended shelf life of the final RPE product.

[0373] Final Product Post-Shipping Inspection and Viability Check. Upon receipt at the clinical site, trained personnel will retrieve the external shipping box, verify the correct batch and lot information, and inspect the shipping box for signs of damage. If the clinical site has the capability to perform cell viability checks, remove the attached tubes containing cells and the tube containing Trypan Blue from the Chillette at this time. Transfer the tubes to the testing laboratory for post-shipping cell viability checks. If cell viability is less than 70%, the product is discarded.

[0374] Brief introduction about loading injection cannula.Before about to transplant in OR, cells are mixed and loaded into 1ml syringe using blunt filling needle.Product (150 μ l) is squeezed into subretinal space through injection cannula.Due to only nominal cell loss in cannula extrusion process in GS2, the loading cell density (2,000 live RPE cells / μ l) in syringe is the density delivered with 150 μ L (for 300K dosage injection).

[0375] Example 6: Cell stability in GS2

[0376] Figure 1 It was shown that RPE cells could be maintained in HypoThermosol (BioLife Solutions, Inc., Bothell, WA, USA) for 24 hours (but not 48 hours) without significant loss of ability to be subsequently plated and grown in culture.

[0377] Figure 2-5 The stability of RPE in GS2 at 2-8°C was explained. Figure 2 It was demonstrated that RPE cells could be maintained in GS2 for at least 48 hours without significant loss in viable cell number or ability to be subsequently plated and grown in culture. Figure 3 It was demonstrated that RPE cells could be maintained in GS2 for 4-5 days with only a slight loss of cell viability and no significant decrease in viable cell density. Figure 4 It was shown that after 5 days of refrigeration in GS2, the ability of RPE cells to plate and grow in culture began to decline. Figure 5 It was shown that GS2 is compatible with current injection systems.

[0378] Example 7: GS2 Implantation Medium - Effect of Viscoelastic Polymers

[0379] The effect of different concentrations of viscoelastic polymers on cell viability after cannula insertion was evaluated. Human retinal pigment epithelial cells (RPE) bulk lot NRPE-313 5C P2 were prepared and stored frozen according to cGMP regulations. On the day of the experiment, the vials were thawed and prepared. The cells were thawed into pre-warmed (37°C) DMEM (Gibco). The cells were then centrifuged (at 160×g for 5 minutes). Each pellet was resuspended in 40ml room temperature BSS. (Alcon) and centrifuged again. The pellets were then combined into a single centrifuge tube and resuspended in room temperature BSS. The cells were then divided into multiple (4) tubes and a final centrifugation step was performed with a volume of 10 ml per tube. The cell pellets were placed into different transplantation medium preparations (named GS2 TM).

[0380] Prepare transplant medium GS2 TM as follows: combine 5% dextrose in saline (0.9% NaOH) solution, Braun NDC#00264-7610-00 or Baxter NDC#0338-0089-04 in a sterile reservoir; saline (0.9% NaOH), Baxter NDC#0338-0049-11; Alcon BSS flushing solution, NDC#0065-0795-15; and hyaluronic acid or sodium hyaluronate (HA), such as Abbott Healon EndoCoat, NDC#05047-4547-06, and mix the components on an orbital shaker. Measure pH and adjust to a pH of 7.4±0.2 with incremental additions of 0.1N NaOH, then sterile filter. In this experiment, GS2 TM was prepared containing final concentrations of 0.15%, 0.1%, 0.05% or 0% hyaluronic acid (HA) from Healon EndoCoat (Abbott).

[0381] The cells were diluted incrementally to obtain a final storage cell density of approximately 2,000 cells / microliter. After reconstitution and dilution in different transplant media, three vials of cells were prepared for each condition. The vials of cells were stored in a refrigerator at 2-8°C for 2 days. Thereafter, a hemocytometer was used to determine the cell number. Cell viability was obtained by trypan blue exclusion. The mean value was calculated from the cells of the three test tubes prepared for each condition, and the viable cell concentration of each test tube was determined from the three counts. The difference in the number of cells before and after extrusion through the MedOne#3233 cannula, or δ, was calculated.

[0382] The results of this experiment are shown in Figure 6In. Shown are the average viable cell density ± standard deviation from three test tubes. Human RPE cell density is determined using a hemocytometer. Obtain cell viability by excluding trypan blue. From the cell calculation mean value of three test tubes prepared for each condition, the viable cell concentration of each test tube is determined from three counts. For each condition, the percentage change (δ) of the cell number observed after the cell extrusion through the MedOne#3233 cannula is presented above each group of values.

[0383] Although all concentrations of the HA of the test show the cell viability value that improves after cannula insertion, it is determined that the interpolation of 0.05% HA is preferred for transplantation medium preparation.Compared with the GS2 TM prepared without HA or with 0.1% or 0.15% HA, observe less cell loss after passing MedOne#3233 cannula to extrude RPE cells with the GS2 TM containing 0.05% HA.Most noteworthy is that, compared with the GS2 TM not adding HA, observe less 10% loss when comprising 0.05% HA.

[0384] Example 8: Effect of GS2 medium-glucose concentration on cell viability

[0385] The effect of viscoelastic polymers of different concentrations on cell viability after cannula insertion was assessed. For this purpose, human retinal pigment epithelial cells (RPE) were prepared in bulk (bulk lot) NRPE-313 5C P2 and cryopreserved according to cGMP regulations. On the day of the experiment, the vial was thawed and the cells were reconstructed in pre-warmed (37°C) DMEM (Gibco). The cells were then centrifuged (at 160 × g for 5 minutes). Each precipitation was resuspended in 40ml room temperature BSS. (Alcon) and centrifuged again. The pellets were then combined into a single centrifuge tube and resuspended in room temperature BSS. Then divide into multiple (6) test tubes, and then add 10ml BSS to each test tube. A final centrifugation step was performed and the cell pellet was placed into different transplantation medium preparations (GS2 TM).

[0386] Transplantation medium (GS2 TM) was prepared as follows: a 5% dextrose solution in saline (0.9% NaOH); saline (0.9% NaOH); and Alcon BSS flushing solution, NDC#0065-0795-15 were combined in a sterile reservoir and mixed on an orbital shaker. The pH was measured and adjusted to a pH of 7.4±0.2 with incremental additions of 0.1 N NaOH, then sterile filtered. GS2 TM medium for this experiment was prepared with different concentrations of glucose, as shown in Table 3 below. The volumes noted are in milliliters:

[0387] Table 3:

[0388]

[0389] After preparation in different GS2 transplantation media, cells are diluted incrementally to obtain a final storage cell density of about 2,000 cells / microliter. The cells are then stored in a sterile vial (Fischer) in a refrigerator at 2-8 ° C for 3-days. Subsequently, a hemocytometer is used to determine the number of viable cells, and approximately 20,000 viable cells / well are plated in a gelatin-coated (Stem Cell, Inc.) 96-well tissue culture plate (COSTAR). Cells are cultured in 5% CO2, 37 ° C humidity-controlled incubators in RPE growth medium (EBM-2, which contains EGM2 Single Quots, Stem Cell, Inc., for example, Lonza catalog number: CC-3156, CC-4176) for 3-days. Subsequently, 40 μl / well 1: 1 trypsin (Sigma) and HEPES-based dissociation medium (Gibco) are used to raise cells from flat boards. Trypsin action was neutralized with serum-containing medium (40 μl / well), and cells were titrated to lift and counted using a hemacytometer. Figure 7 Shown are the mean number of human RPE cells / well from 6 wells for each condition tested ± SD. Each glucose concentration produced enhanced results compared to the control without glucose.

[0390] Example 9: Enhanced viability of mesenchymal stem cells in GS2 medium

[0391] Figure 8 The cell viability of mesenchymal stem cells (MSC) in different formulated media is explained. Human embryonic stem cell-derived MSCs were grown to 70% confluence, harvested with 0.05% trypsin, resuspended in aMEM+15% FCS (MSC medium), and centrifuged at 200×g for 5 min. The cell pellet was resuspended in a small volume of MSC medium and counted for viability using trypan blue exclusion. 5 million MSCs were placed in each of 4 Eppendorf tubes, centrifuged, and resuspended in 1 ml of each indicated medium. The tubes were placed in a 4°C refrigerator for the specified time period. CS: canine serum; FBS: fetal bovine serum. Figure 8 It was demonstrated that formulation in GS2 enhanced MSC cell viability and prolonged MSC storage time in the presence and absence of serum.

[0392] Fig. 9It is shown that higher cell density (shown in millions of cells / ml) enhances MSC viability when stored in GS2. After 24 hours of storage in GS2, the presence of serum (here FBS) was found to have no effect on cell viability (arrows).

[0393] Fig.10 It was demonstrated that MSC viability was retained after storage in GS2 at 4°C and subsequent extrusion through a 26G needle / syringe.

[0394] Example 10: Exemplary Certificate of Analysis for GS2 Shipping Media

[0395] As part of quality control, a group of tests are carried out on the subject transport medium intended for human and / or veterinary patients, with the intention of maximizing the utility of the medium as a part of a drug or surgical procedure, and minimizing the potential of adverse reactions or events. For example, endotoxins are very effective, heat-stable, and pass through most sterilizing membrane filters, and are present in every place where bacteria are located or have existed. In the case where the transport medium is to be used for local delivery, such as in the subretinal space (as expected when used to prepare RPE cells for injection), endotoxins are most worried. Therefore, a group of tests have been carried out on GS2 transport preparations, and it has been confirmed that the following standards are met:

[0396]

[0397] Example 11: Testing the transport medium by light obscuration method

[0398] Also as part of quality control, particularly for use in cells formulated for use in human patients, the transport medium of the present invention generally does not contain unacceptable amounts of particulate matter. Particulate matter consists of mobile insoluble particles (i.e., it is not gas bubbles) that are generally not quantifiable by chemical analysis due to the small mass it represents and its heterogeneous composition. When tested using the light obstruction method (USP 788 test method) (i.e., it counts particulate matter with an electronic particle counter), it has been confirmed that the transport medium formulations of the present invention have no more than 25 particles of size ≥ 10 microns / mL of transport medium and no more than 3 particles of size ≥ 25 microns / mL of transport medium.

[0399] USP 788 provides procedures for determining particulate matter, Method 1 (Obstruction Particle Count Test) and Method 2 (Microscopic Particle Count Test). When inspecting a transport medium for particles that are visible only under a microscope, Method 1 is preferably applied. However, in certain cases of subject formulations, it may be necessary to test the article by the obstruction particle count test and subsequently by the microscopic particle count test to reach a conclusion regarding conformance with the requirements.

[0400] Not all parenteral products can be checked for particles that can only be seen under a microscope by one or two of these methods. When method 1 is not available, for example, in the case of products with reduced clarity or increased viscosity, the test should be carried out according to method 2. In the case where the transport medium includes colloids or liposomes as components, it is an embodiment of the latter. Similarly, for those embodiments of the transport medium that may produce air or bubbles when drawn into the sensor, microscopic particle counting tests may also be required. Although the current preferred embodiment of the subject transport is not very sticky, if the viscosity of the product to be tested is high enough to hinder its inspection by any test method, quantitative dilution with an appropriate diluent can be made to reduce the viscosity when necessary to allow the analysis to be performed.

[0401] USP method 1. light-blocking particle counting test. Samples of GS2 transport media (containing and not containing hyaluronic acid components) are tested in a suitable device based on the light-blocking principle, which allows the automatic determination of the size of particles and the number of particles according to the size. The device is calibrated using a dispersion of spherical particles of known size between 10 μm and 25 μm, USP ParticleCount Reference Standard. These standard particles are dispersed in particle-free water. Carefully avoid particle aggregation during the dispersion process. The particle test is carried out under conditions of limited particle matter (i.e., in a laminar flow cabinet). Glassware and filter equipment are washed and rinsed very carefully. Immediately before use, the equipment is rinsed from top to bottom with particle-free water outside and inside. Samples are analyzed for the number of particles equal to or greater than 10 microns and 25 microns using a number of test samples sufficient to provide a statistically reliable assessment of the transport media being analyzed. For each GS2 transport medium sample tested, the sample includes ≤25 particles / mL of ≥10 micron size transport medium and ≤3 particles / mL of ≥25 micron size transport medium.

[0402] Example 12: Demonstration of RPE cell viability in vivo and efficacy in GS2 medium

[0403] The purpose is to evaluate and compare the safety, engraftment and functionality of the following cells: 1) in BSS Human retinal pigment epithelial (hRPE) cells derived from the embryonic stem cell (ES) line MA09 were prepared in BSS and transplanted within 4 hours; 2) 3) hRPE cells prepared in GS2 transport medium and (a) transplanted in 22-28 hours and (b) in 44-52 hours, derived from J1 cell line. The research confirms that, compared with the currently used clinical preparation medium (BSS-PLUS), the GS2 medium can prolong the shelf life of the final product.

[0404] A total of 32 RCS (Royal College of Surgeon) adolescent rats (16 males and 16 females) were received for the study. At the start of dosing, the rats were 21-25 days old.

[0405] Compliance phase: minimum 7 days. Day 1 corresponds to the day of subretinal injection.

[0406] Experimental design: 8 RCS rats (4 / sex) were randomized to four dose groups of MA09-hRPE (Group 1) and J1-hRPE (Groups 2, 3, 4), each consisting of three subgroups (Table 1 below). All rats received subretinal injections of hRPE cells in the right eye (OD) via a transscleral administration route under anesthesia. Group 1 was administered with BSS. MA-09hRPE in BSS (within 4 hours) was injected into Group 2 J1 hRPE cells in GS2 (within 4 hours), J1 hRPE cells in GS2 (within 22-28 hours) were injected to group 3, and J1 hRPE cells in GS2 (within 44-52 hours) were injected to group 4. Subgroups were defined by administration of the left eye; the sham subgroup (1 / sex) only received needle puncture to the subretinal space of the left eye; no injection (NI or untreated) subgroup (1 / sex) did not receive injection in the left eye; and two vehicle subgroups (2 / sex) received subretinal injections of GS2 or BSS Plus vehicles. Rats were euthanized 70-80 days after injection.

[0407] Table 1 Experimental study design

[0408]

[0409] NI = no injection, sham = puncture of the subretinal space with an empty pipette.

[0410] Vehicle (cell-free) GS2 = 2 μL vehicle (cell-free)

[0411] hRPE = 100,000 cells in 2 μL BSS or in GS2 medium.

[0412] A summary of in-life measurements, necropsy and histopathology endpoints evaluated are presented in Table 2 below.

[0413] Table 2 Evaluation parameters and intervals

[0414]

[0415] Reconstitute as per label on day of dosing and stored refrigerated (2-8°C) for dilution of RPE cells during preparation, for use by surgeons to fill injection devices and for use as a vehicle reference. Contents of Section II transferred to BSS Part I was used to resuspend RPE cells within 6 hours of reconstitution. The time of reconstitution and injection was recorded and maintained in the study log.

[0416] The GS2 medium batches used in this study were formulated and stored at 2-8° C. GS2 was used to wash cells during formulation at room temperature and at 2-8° C. for the final formulation of RPE cells, as well as, used by surgeons to fill injection devices and as a vehicle reference to be injected.

[0417] BBS-Plus Preparation: Cryopreserved MA09-hRPE and J1-hRPE cells were stored in liquid nitrogen (LN2) to maintain the cells in the vapor phase at a temperature of ≤135°C. The cells were maintained in LN2 storage until the day of treatment and transplantation. On the day of transplantation, cryopreserved hRPE were thawed and plated at approximately 50,000 viable cells / μL. The concentrated hRPE cells were delivered to the surgeon in a Final Fill Tube (on wet ice in a LabTop Cooler) for use within 4 hours of preparation ( Fig.11 , see “MA-09 RPE < 4 h” and “J1 RPE < 4 h”). The time of reconstitution and injection was recorded and maintained in the study records.

[0418] GS2 preparations: Cryopreserved J1-hRPE is stored in dry liquid nitrogen (LN2) - in the vapor phase at a temperature of ≤135°C. Before the day of treatment, the cells are maintained in LN2 storage. 1 or 2 days before transplantation, the J1-hRPE cells are thawed and prepared in GS2 at approximately 1,500 viable cells / μL. The prepared RPE is then stored at 2-8°C, and the number of viable cells is assessed 2-6 hours after preparation. About 20 hours or 42 hours after preparation, the RPE cells are concentrated in GS2 to about 50,000 viable cells / μL. The concentrated hRPE cells are delivered to the surgeon in a Final Fill Tube (placed on wet ice in a LabTop Cooler). About 22 hours or 44 hours after preparation in GS2, transplanted RPE cells ( Fig.11 , see “J1 RPE <22” and “J1 RPE <44” respectively).

[0419] Dosage: Subretinal injection

[0420] Animals were anesthetized with a mixture of ketamine (75 mg / kg) and dexmedetomidine (0.25 mg / kg), followed by carprofen (5 mg / kg) via SQ for dosing. Prior to dosing, the eyes were flushed with 0.9% sodium chloride for injection (sterile NaCl) USP. The eyes were then cleaned with 2 drops of 0.3% Ocuflux ophthalmic solution USP and dilated with mydriatic drops (1% tropicamide) followed by 2.5% phenylephrine hydrochloride ophthalmic solution USP. The animals were placed in a BSS The test material MA09 and J1 hRPE cells in GS2 were injected in 0.25-4 hours of preparation (group 1 and 2, table 1). The test material J1 hRPE cells in GS2 were (group 3 and 4, table 1) in 22 or 44 hours of preparation. The test material of each group (accepting MA09-hRPE cells or J1-hRPE cells) was applied to the right eye of each group of 8 animals by subretinal injection, as indicated in the experimental design (and table 1) above. The left eye of four animals in each group of groups 1 and 2 was used for reference material BSS. The reference substance GS2 was administered to the left eyes of four animals in each of groups 3 and 4. The left eyes of two animals in each of the four groups were subjected to the injection procedure but without injection of substance (sham injection). The left eyes of the remaining two animals in each of the four groups did not receive an injection (untreated).

[0421] Briefly, subretinal injections were performed using a surgical microscope. The eye was stabilized using a purse-string loop around the eyeball using sutures (Ethicon 4-0 Perma-Hand Silk) behind the equator of the eyeball. Hydroxypropyl methylcellulose (or similar) solution was applied to the eye and held in place with the loop. A small area of ​​the conjunctiva was cut away using scissors, and a 30G x 1 / 2 "metal needle was applied to perform a sclerotomy at the upper dorsal temporal region of the eyeball. A drug delivery device consisting of a calibrated sterile glass pipette (World Precision Instruments, Item # 1B150-4) was connected to an approximately 0.8 mm hole Tygon TM Plastic tubing (Saint-Gobain Performance Plastics #R-3603) connected to an 18G blunt needle (Becton-Dickenson, Inc. Reference #305196) connected to a 25 μL Hamilton syringe (Model #702 LT Catalog No. 804010) prefilled with the appropriate vehicle (Table 1) as described in the study design. A small amount of air was introduced into the line to separate the injectate from the vehicle in the dosing device, after which the test or reference was drawn into a glass pipette to a volume of 2 μL. A new sterile glass pipette was used for each injection / eye.

[0422] The sclerotomy was closed with nonabsorbable surgical sutures (Ethicon Prolene 10-0). The sutures (Ethicon 4-0 Perma-Hand ) was removed and the eyelids were eventually restored to their normal position. After the injection procedure was completed, topical antibiotics (5 mg / g erythromycin eye ointment) were applied to the treated eye.

[0423] During surgery, careful intraoperative fundus mapping is performed to record the size and location of the bleb and any other ocular changes.

[0424] Animals were maintained on a warming board or heating blanket (~37°C) until fully recovered, after which they were returned to their home cages. Additional applications of 0.5% erythromycin ophthalmic ointment were applied to the surface of the eye to prevent dryness as needed until the animal was fully awake and could blink normally.

[0425] All animals were maintained on oral cyclosporin A (CsA) administered in the drinking water (210 g / L, resulting in a target blood concentration of approximately 300 g / L). Intraperitoneal injections of dexamethasone were also given once daily for 14 days following surgery (1.6 mg / kg / day).

[0426] Cage side observation and clinical observation of mortality / moribundity were performed at least once a day. There were no clinical signs of disease or response to treatment. Animals were weighed at least once during the compliance period, and weighed (terminal weight) approximately weekly and immediately before autopsy during the study. No exceptional changes in body weight were observed for the study animals. Eye examinations were performed again before administration, approximately 40 days after administration, and before autopsy. 1% tropicamide was used as 1 drop / eye instillation to dilate the animal's eyes for examination. No significant differences were revealed from eye examinations between different RPE preparation groups. Similarly, at the time described in Table 2, the photokinetic response (OKR) performed using moving bars of different spatial frequencies was measured for all animals. Visual acuity was measured by OKR in all eyes (experimental and control). The method to be used, the visual acuity test equipment (Prusky et al., 2000), is composed of a rotating cylinder covered by a vertical sine wave grid (grafting), and is presented on four computer monitors arranged in a square in a virtual three-dimensional (3-D) space. Rats are placed on the platform without restriction in the center of the square, where they track the grid with reflexive head movements. By repeatedly repositioning the center of the 'cylinder' on the head of the test subject, the spatial frequency of the grid is clamped in the viewing position. Visual acuity is quantified by increasing the spatial frequency of the grid using a psychophysical staircase progression until the optokinetic reflex is lost, thereby obtaining the maximum threshold. It is measured in units of c / d (cycle / degree). No significant differences were revealed from the OKR experiment between different RPE preparation groups. Spectral domain optical coherence tomography (SD-OCT) was also performed as above, and no significant differences were revealed from the eye examination between different RPE preparation groups. Rats are anesthetized by IP injection of anesthetics. For better imaging of the retina, tropicamide is applied to both eyes to dilate the pupil. Rats are placed in an imaging system, and eye drops of Genteal and Systane are applied to the cornea to keep the cornea moist.

[0427] Fig.11The results of the electroretinogram (ERG) of a group of 16 rats 60 days after treatment are shown. As above, no significant differences are revealed from the ERG examination between different RPE preparation groups. In short, animals are kept in complete darkness overnight (at least 12 hours) to reach the dark adaptation state of the retina. In order to record ERG, animals are anesthetized with IP injection of anesthetic and placed in a stereotaxic head holder. Under blurred red light, recording electrodes (two coaxial wire loops, wire diameter 50um, attached to neutral contact lenses) are placed on the animal eyes pretreated with lidocaine. One hind limb is trimmed with electric clippers, and the skin is prepared with betadyne, and then the cannula is inserted (the cannula is inserted into the hind limb muscle during the operation duration). The pupil is enlarged with tropicamide. Before starting recording, an additional 1-hour dark adaptation phase is used to recover the adaptation of the animal preparation later. For both eyes (eyes will be tested in the order of left-right or right to left), the whole ERG recording lasts for about 20 minutes. Eyes are stimulated with full-field flashes. Corneal potentials were recorded with an amplifier connected to the electrodes. Flash presentation was controlled by a computer program. Responses were averaged for 5-100 stimulus presentations, depending on the ERG intensity, which can be very low in animals with progressive retinal degeneration.

[0428] In a word, the result indication of these researches, GS2 transfer medium can provide the living and functional RPE cell (even suspending up to 44 hours later in GS2 transfer medium) of about same effective dose, when compared with the BSSPlus medium less than 4 hours later with suspension, described 4 hours are the storage life of the use of the BSS-Plus for RPE cell injection of current FDA and EMA approval.In each case, as a control, the false and untreated eyes of described animal do not show any improvement relative to the eyes of RPE cell treatment in BSS-Plus and GS2 transfer medium.

[0429] Equivalents and scope, incorporated by reference

[0430] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments described herein.The scope of the present disclosure is not intended to be limited to the above description, but rather is set forth in the claims that follow.

[0431] Articles such as "a", "an" and "the" include both the singular and the plural, unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a single agent and a plurality of such agents.

[0432] A claim or description including "or" between two or more members of a group is deemed to be satisfied if one, more than one, or all of the group members are present, unless otherwise indicated or otherwise obvious from the context. Disclosures of groups including "or" between two or more group members provide embodiments in which exactly one member of the group is present, embodiments in which more than one member of the group is present, and embodiments in which all group members are present. For the purpose of brevity, those embodiments are not individually set forth herein, but it is understood that each of these embodiments is provided herein and may be specifically claimed or waived.

[0433] It should be understood that the invention includes all variations, combinations and permutations in which one or more limitations, elements, clauses or descriptive terms from one or more claims or from one or more relevant parts of the specification are introduced into another claim. For example, a claim that is dependent on another claim can be amended to include one or more limitations found in any other claim that is dependent on the same base claim. In addition, when a claim recites a composition, it should be understood to include methods of making or using the composition according to any method disclosed herein or according to methods known in the art (if any), unless otherwise stated or unless it is obvious to one of ordinary skill in the art that it would cause a conflict or contradiction.

[0434] In the case where the elements are presented as a list, for example, in the form of Markush groups, it should be understood that each possible subgroup of the elements is also disclosed, and any element or element subgroup can be removed from the group. It is also noted that the term "comprising" is intended to be open and allows the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product or method is referred to as comprising a specific element, feature or step, an embodiment, product or method consisting of or substantially consisting of such an element, feature or step is also provided. For the purpose of brevity, those embodiments are not separately set forth herein, but it should be understood that each of these embodiments is provided herein and can be specifically stated or abandoned.

[0435] In the case of providing a range, endpoints are included. In addition, it should be understood that unless otherwise noted or otherwise apparent from the context and / or understanding of those of ordinary skill in the art, the value expressed as a range can adopt any specific value within the range, and in certain embodiments, it is accurate to one-tenth of the lower limit unit of the range, unless the context clearly indicates otherwise. For the purpose of brevity, the value within each range is not separately set forth herein, but it should be understood that each of these values ​​is provided herein, and it can be specifically stated or abandoned. It should also be understood that unless otherwise noted or otherwise apparent from the context and / or understanding of those of ordinary skill in the art, the value expressed as a range can adopt any sub-range within a given range, wherein the endpoint of the sub-range is expressed to the same accuracy as 1 / 10 of the lower limit unit of the range.

[0436] In addition, it should be understood that any specific embodiment of the present invention may be explicitly excluded from any one or more claims. Where a range is given, any value within the range may be explicitly excluded from any one or more claims. Any embodiment, element, feature, application or aspect of the composition and / or method of the present invention may be excluded from any one or more claims. For the purpose of brevity, all such embodiments are not explicitly set forth herein: wherein one or more elements, features, purposes or aspects are excluded.

[0437] All publications, patents, patent applications, disclosures, and database entries (e.g., sequence database entries) mentioned herein (e.g., in the Background, Summary, Detailed Description, Examples, and / or References sections) are hereby incorporated by reference in their entirety, to the same extent as if each individual publication, patent, patent application, disclosure, and database entry was specifically and individually incorporated by reference herein. In the event of a conflict, the present application (including any definitions herein) will control.

[0438] This application also includes the following specific implementation schemes:

[0439] 1. A solution comprising

[0440] (a) a buffer to maintain the solution at physiological pH; and

[0441] (b) at least 2 mM or at least 0.05% (w / v) glucose; and

[0442] (c) an osmotically active agent that maintains the solution at physiological osmolarity.

[0443] 2. The solution of embodiment 1, wherein the solution comprises at least 5 mM or at least 0.1% (w / v) glucose; or at least 7.5 mM or at least 0.14% (w / v) glucose; or at least 10 mM or at least 0.2% (w / v) glucose; or at least 15 mM or at least 0.25% (w / v) glucose; or at least 20 mM or at least 0.4% (w / v) glucose; or at least 25 mM or at least 0.5% (w / v) glucose.

[0444] 3. The solution of embodiment 1 or 2, wherein the solution further comprises

[0445] (a) a source of divalent cations, optionally wherein the source of divalent cations comprises a calcium source and / or a magnesium source, and / or

[0446] (b) acetate buffer and / or citrate buffer.

[0447] 4. A solution comprising

[0448] (a) a buffer that maintains the solution at physiological pH, wherein the buffer is not a bicarbonate buffer; and

[0449] (b) glucose; and

[0450] (c) an osmotically active agent that maintains the solution at physiological osmolarity; and

[0451] (d) the source of divalent cations,

[0452] Optionally wherein the source of divalent cations comprises a calcium source and / or a magnesium source, and / or wherein the buffer comprises an acetate buffer and / or a citrate buffer.

[0453] 5. The solution of any one of embodiments 3 or 4, wherein the calcium source comprises a pharmaceutically acceptable calcium salt, and / or the magnesium source comprises a pharmaceutically acceptable magnesium salt.

[0454] 6. The solution of embodiment 5, wherein the pharmaceutically acceptable calcium and / or the pharmaceutically acceptable magnesium salt is selected from calcium salts and / or magnesium salts formed with an acid selected from acetic acid, ascorbic acid, citric acid, hydrochloric acid, maleic acid, oxalic acid, phosphoric acid, stearic acid, succinic acid and sulfuric acid.

[0455] 7. The solution of any one of embodiments 3-6, wherein the calcium source comprises calcium chloride, optionally wherein the calcium source comprises calcium chloride dihydrate.

[0456] 8. The solution of any one of embodiments 3-7, wherein the magnesium source comprises magnesium chloride, optionally wherein the magnesium source comprises magnesium chloride hexahydrate.

[0457] 9. The solution of any one of embodiments 3-9, wherein the citrate buffer is provided as sodium citrate.

[0458] 10. The solution of any one of embodiments 1-9, wherein the glucose is D-glucose (dextrose).

[0459] 11. The solution of any one of embodiments 1-10, wherein the osmotically active agent is a salt, optionally wherein the osmotically active agent is a sodium salt, further optionally wherein the osmotically active agent is sodium chloride.

[0460] 12. The solution of any one of embodiments 1-11, wherein the solution comprises calcium chloride, magnesium chloride, sodium citrate, sodium chloride and glucose.

[0461] 13. The solution of any one of embodiments 1-12, wherein the pH of the solution is 6.8-7.8, or 7.4-7.5, or about 7.5.

[0462] 14. The solution of any one of embodiments 1-13, wherein the solution is isotonic or hypertonic.

[0463] 15. The solution of any one of embodiments 1-14, wherein the solution exhibits an osmolarity of about 270-345 mOsm / l or about 315 mOsm / l.

[0464] 16. The solution of any one of embodiments 3-15, wherein the concentration of the calcium source is

[0465] (a) 0.25-0.75 mM, or 0.4-0.65 mM, or 0.5-0.6 mM, or about 0.6 mM; or

[0466] (b) 0.5-0.9 mM, or 0.6-0.8 mM, or about 0.7 mM.

[0467] 17. The solution of any one of embodiments 3-16, wherein the concentration of the magnesium source is 0.05-5 mM, or 0.1-0.3 mM, or about 0.3 mM.

[0468] 18. The solution of any one of embodiments 1-40, wherein the concentration of glucose is 5-50 mM, or 10-25 mM, or 10-20 mM, or about 16 mM.

[0469] 19. The solution of any one of embodiments 1-18, wherein the concentration of the osmotically active agent is about 100-200 mM, or about 125-175 mM, or about 150 mM.

[0470] 20. The solution of any one of embodiments 3-19, wherein the concentration of citrate or acetate is 0.1-5 mM, or 0.5-2 mM, or about 1 mM.

[0471] 21. The solution of any one of embodiments 1-20, wherein the solution further comprises a potassium salt, optionally wherein the potassium salt is potassium chloride, further optionally wherein the concentration of KCl is 0.2-5 mM, or 1-2.5 mM, or about 2 mM.

[0472] 22. The solution of any one of embodiments 1-21, wherein the solution comprises

[0473] (a) about 0.7 mM CaCl (calcium chloride), about 0.3 mM MgCl (magnesium chloride), about 1 mM sodium citrate, about 16 mM dextrose, and about 145 mM NaCl, or

[0474] (b) about 0.5-0.9 mM CaCl (calcium chloride), about 0.2-.4 mM MgCl (magnesium chloride), about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, and about 116-174 mM NaCl, or

[0475] (c) about 0.85% NaCl, about 0.01% CaCl dihydrate (calcium chloride dihydrate), about 0.006% MgCl hexahydrate (magnesium chloride hexahydrate), about 0.035% sodium citrate dihydrate, and about 0.29% dextrose, or

[0476] (d) about 0.68-1.02% NaCl, about 0.008-0.012% CaCl dihydrate (calcium chloride dihydrate), about 0.0048-0.0072% MgCl hexahydrate (magnesium chloride hexahydrate), about 0.028-0.042% sodium citrate dihydrate, and about 0.23-0.35% dextrose.

[0477] 23. The solution of any one of embodiments 1-22, wherein the solution further comprises

[0478] (a) about 2 mM KCl, and / or

[0479] (b) a viscoelastic polymer, optionally wherein the polymer is hyaluronic acid or a salt or solvate thereof, further optionally wherein the polymer is sodium hyaluronate.

[0480] 24. The solution of embodiment 23, wherein the polymer is present in a concentration effective to reduce exposure of cells in the solution to shear stress, optionally wherein the concentration of the polymer is 0.005-5% w / v or about 0.05% w / v.

[0481] 25. The solution of any one of embodiments 1-24, wherein the solution comprises

[0482] (a) about 0.7 mM CaCl (calcium chloride), about 0.3 mM MgCl (magnesium chloride), about 2 mM KCl, about 1 mM sodium citrate, about 16 mM dextrose, about 145 mM NaCl and about 0.05% hyaluronic acid, or

[0483] (b) about 0.5-0.8 mM CaCl (calcium chloride), about 0.2-.4 mM MgCl (magnesium chloride), about 1.6-2.4 mM KCl, about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, about 116-174 mM NaCl, and about 0.04-0.06% hyaluronic acid.

[0484] 26. The solution of any one of embodiments 1-25, wherein the solution does not contain

[0485] (a) carbonate buffer, and / or

[0486] (b) glutathione or glutathione disulfide (GSSG), and / or

[0487] (c) Zwitterionic organic buffers.

[0488] 27. The solution of any one of embodiments 1-26, wherein the solution can

[0489] (a) stored at 25°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month without measurable precipitation of solutes and / or measurable loss of the ability of the solution to support the survival and viability of cells stored in the solution, and / or

[0490] (b) storage at 2-8°C for at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month without measurable precipitation of solutes and / or measurable loss of the ability of the solution to support the survival and viability of cells stored in the solution.

[0491] 28. The solution of any one of embodiments 1-27, wherein the solution is

[0492] (a) suitable for administration to a subject, suitable for administration to the eye of a subject, and / or suitable for transplanting cells into the eye of a subject, and / or

[0493] (b) substantially non-pyrogenic, and / or

[0494] (c) sterile, and / or

[0495] (d) for flushing, cell reconstitution, cell storage, cell delivery, and / or administration to a subject.

[0496] 29. A preparation comprising a cell population in a solution according to any one of embodiments 1-28, optionally wherein the cell population

[0497] (a) suitable for transplantation into a subject, further optionally suitable for transplantation into an eye of a subject, and / or

[0498] (b) comprises RPE cells, and / or

[0499] (c) contains photoreceptor cells, and / or

[0500] (d) Contains mesenchymal cells.

[0501] 30. The product of embodiment 29, wherein the product is refrigerated, optionally wherein the product is refrigerated at about 2-8°C.

[0502] 31. The article of manufacture of embodiment 29 or 30, wherein during storage of the article, the article supports survival of cells in the cell population, and wherein at least 70% of the cells in the cell population are viable after storing the article at 2-8° C. for 48 hours,

[0503] Optionally wherein at least 80% of the cells in the cell population are viable after storage of the preparation at 2-8°C for 48 hours, or

[0504] wherein at least 90% of the cells in the cell population are viable after storing the preparation at 2-8°C for 48 hours.

[0505] 32. The article of any one of embodiments 29-31, wherein the article supports maintenance of the plating efficiency of the cell population during storage of the article, and wherein the cell population exhibits at least 70% of its original plating efficiency after storage of the article at 2-8°C for 48 hours, wherein the original plating efficiency is the plating efficiency of the cell population at the beginning of the storage period,

[0506] Optionally wherein said cell population exhibits at least 80% of its original plating efficiency after storage of said preparation at 2-8°C for 48 hours, or

[0507] wherein the cell population exhibits at least 90% of its original plating efficiency.

[0508] 33. The article of any one of embodiments 29-32, wherein the article is

[0509] (a) in storage containers, and / or

[0510] (b) Inside the syringe.

[0511] 34. A method for preparing the product of any one of embodiments 29-33, wherein the method comprises contacting a cell population with a solution of any one of embodiments 1-28, optionally wherein the method comprises contacting a cryopreserved or precipitated cell population with a solution of any one of embodiments 1-28, thereby reconstructing the cells.

[0512] 35. A pharmaceutical composition comprising the solution of any one of embodiments 1-28 or the preparation of any one of embodiments 29-33, wherein the pharmaceutical composition is suitable for administration to a subject.

[0513] 36. A method comprising administering the solution of any one of embodiments 1-28 or the preparation of any one of embodiments 29-33 or the pharmaceutical composition of embodiment 35 to a subject in need thereof, optionally wherein the method comprises administering the solution or the preparation to the eye of the subject, further optionally wherein the method comprises administering the preparation to the subject after storing the preparation for at least 4, at least 6, at least 12, at least 24, at least 36 or at least 48 hours.

[0514] 37. The method of embodiment 36, wherein the subject has or is diagnosed with a retinal disease, optionally wherein the retinal disease is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, Leber congenital amaurosis, or Stargardt's disease.

[0515] 38. The method of embodiment 37, wherein the preparation comprises a cell population of a size effective to improve at least one symptom of a retinal disease in the subject, optionally wherein the method further comprises monitoring at least one symptom of a retinal disease in the subject.

[0516] 39. A method comprising

[0517] (a) contacting a cell population with a solution according to any one of embodiments 1 to 28 to produce a cell product, optionally further comprising

[0518] (b) storing the cell preparation of (a) for at least 4, at least 6, at least 12, at least 18, at least 24, at least 36, at least 48, at least 60, or at least 72 hours, optionally further comprising

[0519] (c) administering the cell preparation of (a) to a subject after the storage period of (b), optionally wherein administering comprises injecting the cells into an eye of the subject.

[0520] 40. The method of embodiment 39, wherein the method further comprises

[0521] (i) determining the viability of cells in the cell preparation of (a) after the storage period of (b), and / or

[0522] (ii) refrigerating the cell preparation of (a) during the storage phase of step (b), optionally wherein refrigerating comprises storing the cell preparation at a temperature of 2-8°C.

[0523] 41. A method for treating a retinal disease, the method comprising

[0524] An effective amount of the preparation of any one of Embodiments 29-33 or the pharmaceutical composition of Embodiment 35 is administered to an eye of a subject having a retinal disease, optionally wherein the retinal disease is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, Leber congenital amaurosis, or Stargardt's disease.

[0525] 42. A kit comprising

[0526] (a) the solution of any one of embodiments 1-28;

[0527] (b) instructions for contacting the cell population with the solution of (a) to produce a cell product; and

[0528] (c) a container for contacting (b) and / or for storing the cell product of (b), optionally wherein the solution of (a) and the container are suitable for use of the cell product of (b) for transplantation into a subject.

Claims

1. A solution for cell reconstruction, cell storage, cell transplantation and / or cell administration, wherein the solution comprises (a) about 0.5-0.9 mM CaCl2, about 0.2-0.4 mM MgCl2, about 1.6-2.4 mM KCl, about 0.8-1.2 mM sodium citrate, about 13-19 mM dextrose, and about 116-174 mM NaCl in aqueous solution, or (b) about 0.008-0.012% CaCl2 dihydrate, about 0.0048-0.0072% MgCl2 hexahydrate, about 0.012-0.018% KCl, about 0.028-0.042% sodium citrate dihydrate, about 0.23-0.35% dextrose, and about 0.68-1.02% NaCl.

2. goods, it is comprised in the cell colony in the solution of claim 1, optionally wherein the cell colony comprises and is suitable for being transplanted into experimenter, further optionally is suitable for being transplanted into the cell of experimenter's eye, multipotent stem cell or progenitor cell or functional differentiated cell, RPE cell, RPE progenitor cell, iris pigment epithelium (IPE) cell and other visual relevant neural cell, photoreceptor cell, mesenchymal cell, retinal ganglion cell, retinal progenitor cell, hematopoietic stem cell or hematopoietic progenitor cell, neural stem cell or neural progenitor cell, neural cell, astrocyte or astrocyte ancestor, glial cell, glial stem cell or progenitor cell, pancreatic cell, pancreatic stem cell or progenitor cell, beta cell, keratinocyte, chondrocyte, osteoblast and / or osteoclast.

3. A product comprising a cell colony in the solution of claim 1, wherein the cell colony comprises RPE cells.

4. An article comprising a cell population in the solution of claim 1, wherein the cell population comprises photoreceptor cells.

5. An article of manufacture comprising a cell population in the solution of claim 1, wherein the cell population comprises mesenchymal cells.

6. A method for preparing a product containing a cell population, wherein the method comprises contacting the cell population with a solution of claim 5, optionally wherein the method comprises contacting a cryopreserved or precipitated cell population with a solution, thereby reconstituting the cells.

7. A pharmaceutical composition comprising the preparation of any one of claims 2-5, wherein the pharmaceutical composition is suitable for administration to a subject.

8. Use of the solution of claim 1 or the preparation of any one of claims 2-5 or the pharmaceutical composition of claim 7 for the preparation of a medicament for use in a subject in need thereof, comprising administering the medicament to the eye of the subject.

9. A method comprising: (a) contacting a cell population with the solution of claim 1 to produce a cell product, optionally further comprising (b) storing the cell preparation of (a) for at least 4, at least 6, at least 12, at least 18, at least 24, at least 36, at least 48, at least 60 or at least 72 hours.

10. Use of the pharmaceutical composition of claim 7 in the preparation of a drug for treating retinal diseases, comprising An effective amount of the medicament is administered to an eye of a subject having a retinal disease, optionally wherein the retinal disease is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, Leber congenital amaurosis, or Stargardt's disease.

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