Cell cryopreservation formulations and methods of use

Abstract

Some aspects of the present disclosure provide improved cryopreservation formulations as well as improved methods of cryopreserving and thawing cells. These formulations facilitate high density, low volume cell cryopreservation strategies that overcome the need for cleaning cells after thawing and thus increase cell recovery and survival. Also provided are pharmaceutical compositions comprising a population of cells and a cryopreserved formulation as provided herein. Also provided are methods of treating a subject having a disease or condition that can benefit from transplantation cryopreserved and thawed cell populations.

Description

Related Applications

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 430,976 (filed on December 7, 2022, entitled “CELL CRYOPRESERVATIVE FORMULATIONS AND METHODS OF USE”) and U.S. Provisional Application Serial No. 63 / 400,382 (filed on August 23, 2022, entitled “CELL CRYOPRESERVATIVE FORMULATIONS AND METHODS OF USE”) under 35 U.S.C. §119(e), the entire contents of each of which are incorporated herein by reference. Background Art

[0002] Effective cell cryopreservation is an important aspect of many cell therapy strategies. The ability to freeze, store for different time periods, and optionally transport cell-based therapies in a frozen state expands their clinical utility. Improper cryopreservation can lead to cell damage, including changes in cell properties, function, metabolic activity, and cell death. Summary of the invention

[0003] The ability to cryopreserve cell-based therapeutic agents decouples the manufacture and clinical use of cell therapy in time and place. Designing and selecting suitable cryoprotectant preparations is important for ensuring cell survival and functional maintenance. The present disclosure provides novel cryoprotectant preparations, which can be used for cryopreservation and optionally transporting cell populations, including cell populations used as therapeutic agents. Importantly, the present disclosure contemplates the use of such preparations to cryopreserve cell populations at high density and low volume, so that thawing is subsequently achieved by adding a diluent, and then applied to a subject, without the need to wash the cells after thawing. In this way, the method can achieve freezing and thawing of cell-based therapeutic agents with minimal processing and operation. This reduces the possibility of cell loss (e.g., cell death) that may result from operating cells during the post-thawing period.

[0004] Therefore, the present disclosure provides cryoprotectant preparations and methods of use thereof. Cryoprotectant preparations can be used for a variety of cell types. Compared with the methods of freezing cells of the prior art and commercially available cryoprotectant preparations, cryoprotectant preparations provided herein obtain higher survival rates and cell growth after thawing. Although this paper is exemplified by ophthalmic applications, the present disclosure also contemplates and encompasses other applications.

[0005] Some aspects of the present disclosure provide a formulation comprising about 4-10% (v / v) cryoprotectant, about 2-8% (w / v) albumin, about 0-1.5% (w / v) glucose, and a buffer.

[0006] In some embodiments, the cryoprotectant is selected from DMSO, glycerol, and ethylene glycol.

[0007] In some embodiments, the cryoprotectant is DMSO.

[0008] In some embodiments, the formulation comprises about 4-6% (v / v) cryoprotectant.

[0009] In some embodiments, the formulation comprises about 0.08-0.10% (w / v) glucose.

[0010] In some embodiments, the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and a buffer.

[0011] In some embodiments, the formulation comprises about 0.6% (w / v) glucose.

[0012] In some embodiments, the formulation comprises about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and a buffer.

[0013] In some embodiments, the albumin is human albumin.

[0014] In some embodiments, the albumin is recombinant human albumin.

[0015] In some embodiments, the buffer is buffered saline.

[0016] In some embodiments, the buffer is phosphate buffered saline (PBS).

[0017] In some embodiments, the buffered saline comprises Ca2+ and Mg2+.

[0018] In some embodiments, the buffered saline does not contain Ca2+ and Mg2+.

[0019] In some embodiments, the preparation further comprises a cell population or tissue.

[0020] In some embodiments, the cell population is a dissociated cell population.

[0021] In some embodiments, the cell population comprises a cell monolayer, a cell sheet, a cell cluster, a cell spheroid, or a cell aggregate.

[0022] In some embodiments, the cell population includes retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells.

[0023] In some embodiments, the formulation is stored frozen.

[0024] In some embodiments, the formulation does not include a polymer excipient.

[0025] In some embodiments, the formulation does not comprise dextran.

[0026] Some aspects of the present disclosure provide a cell formulation comprising (a) a formulation described herein and (b) a cell population.

[0027] In some embodiments, the cell population includes retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells.

[0028] In some embodiments, the cell population concentration in the cell preparation is about 300 cells-1000 cells / μL. In some embodiments, the cell population concentration in the cell preparation is about 300 cells-10,000 cells / μL. In some embodiments, the cell population concentration in the cell preparation is about 1000 cells-10,000 cells / μL.

[0029] In some embodiments, the cell population in the cell formulation has a concentration of about 3,000 cells to 5,000 cells / μL or a concentration of about 3,000 cells to 4,000 cells / μL.

[0030] In some embodiments, the concentration of the cell population in the cell preparation is about 10,000 cells-100,000 cells / μL.

[0031] In some embodiments, the concentration of the cell population in the cell formulation is about 15,000 cells-150,000 cells / μL.

[0032] In some embodiments, the concentration of the cell population in the cell formulation is about 15,000-50,000 cells / μL.

[0033] In some embodiments, the cell formulations described herein are in cryopreservation vials, syringes, ampoules, bottles, or freezer packaging.

[0034] In some embodiments, about 10 to about 500 μL of the formulation is in a vial, syringe, ampoule, bottle, or freezer packaging.

[0035] In some embodiments, about 10 5 to about 10 7 In some embodiments, there are about 4x10 5 cells to about 5x10 5 In some embodiments, there are about 4.5 x 10 5In some embodiments, there are about 1x10 6 cells to about 2x10 6 In some embodiments, there are about 1.5 x 10 6 In some embodiments, the cells are RPE cells. A similar number of cells may be present in a syringe, ampoule, bottle, or freezer packaging.

[0036] In some embodiments, there are about 0.5 x 10 6 cells to about 1.5x10 6 In some embodiments, there are about 1x10 6 In some embodiments, there are about 5x10 6 cells to about 7x10 6 In some embodiments, there are about 6 x 10 6 In some embodiments, the cells are PRC cells. A similar number of cells may be present in a syringe, ampoule, bottle, or freezer packaging.

[0037] In some embodiments, about 10 5 Up to 5x10 6 cells.

[0038] In some embodiments, about 10 5 Up to 2x10 6 cells.

[0039] In some embodiments, the cell preparation is cryopreserved.

[0040] Some aspects of the present disclosure provide methods of preparing a cell formulation comprising contacting a formulation described herein with a cell population.

[0041] In some embodiments, the methods described herein further comprise cryopreserving the cell preparation.

[0042] In some embodiments, the cell population includes retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells.

[0043] Some aspects of the present disclosure provide methods of treating a subject having a disorder or condition comprising thawing a cell preparation described herein and administering the cell preparation to the subject.

[0044] Some aspects of the present disclosure provide methods of treating a subject having a disorder or condition, comprising thawing a cell preparation described herein, diluting the cell preparation with a diluent at a ratio ranging from 1:2 to 1:20 (including a ratio selected from 1:2, 1:3, 1:4, and 1:5) or a ratio of 1:10 to 1:15 (including 1:10, 1:11, 1:12, 1:13, 1:14, and 1:15), and administering the diluted cell preparation to the subject.

[0045] In some embodiments, the disorder or condition is selected from retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration, or retinal atrophy, or is associated with a variety of vision-altering diseases that lead to photoreceptor damage and blindness, such as choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's Disease.

[0046] In some embodiments, the diluent is GS2, GS2 Plus, BSS, BSS Dextran and HSA solution, PBS, DMEM, MEM, or albumin.

[0047] Some aspects of the present disclosure provide methods of treating a subject having a disorder or condition, comprising thawing a cell preparation described herein, diluting the cell preparation with a diluent at a ratio ranging from 1:2 to 1:20, including a ratio selected from 1:15, 1:16, 1:17, 1:18, and 1:19, and administering the diluted cell preparation to the subject.

[0048] In some embodiments, the disorder or condition is selected from retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration, or retinal atrophy, or is associated with a variety of vision-altering diseases that lead to photoreceptor damage and blindness, such as choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (yellow spots on the fundus).

[0049] In some embodiments, the diluent is GS2 or GS2 Plus, BSS, BSS Dextran and HSA solution, PBS, DMEM, MEM, or albumin.

[0050] Some aspects of the present disclosure provide kits comprising (a) a cryopreserved cell preparation described herein, (b) a diluent, and (c) instructions for diluting the cell preparation prior to administration to a subject.

[0051] In some embodiments, the instructions further include instructions for thawing the cryopreserved cell preparation.

[0052] Some aspects of the present disclosure provide a combination comprising a formulation described herein and a cell population.

[0053] In some embodiments, the cell population includes retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells.

[0054] In some embodiments, the cell population includes neural cells, neural stem cells, or neural precursor cells.

[0055] Some aspects of the present disclosure provide pharmaceutical compositions comprising the formulations described herein, wherein the pharmaceutical composition is suitable for administration to a subject.

[0056] Some aspects of the present disclosure provide pharmaceutical compositions comprising a formulation described herein and a diluent, wherein the ratio of formulation to diluent is in the range of 1:2 to 1:100, including 1:2 to 1:20, including 1:2 to 1:5, including 1:2, 1:3, 1:4, 1:5, or a ratio of 1:10 to 1:15, including 1:10, 1:11, 1:12, 1:13, 1:14, and 1:15, wherein the pharmaceutical composition is suitable for administration to a subject.

[0057] In some embodiments, the pharmaceutical composition is suitable for administration to the eye.

[0058] In some embodiments, the pharmaceutical composition is suitable for subretinal administration.

[0059] Some aspects of the present disclosure provide pharmaceutical compositions comprising the cell formulations described herein, wherein the pharmaceutical composition is suitable for administration to a subject.

[0060] Some aspects of the present disclosure provide pharmaceutical compositions comprising a cell preparation described herein and a diluent, wherein the ratio of the cell preparation to the diluent is in the range of 1:2 to 1:100, including 1:2 to 1:20, including 1:2 to 1:5, including 1:2, 1:3, 1:4, 1:5, or a ratio of 1:10 to 1:15, including 1:10, 1:11, 1:12, 1:13, 1:14, and 1:15, wherein the pharmaceutical composition is suitable for administration to a subject.

[0061] In some embodiments, the pharmaceutical composition is suitable for administration to the eye.

[0062] In some embodiments, the pharmaceutical composition is suitable for subretinal administration.

[0063] Some aspects of the present disclosure provide a cell preparation comprising a formulation described herein, wherein the preparation comprises a population of cells, wherein the cell preparation is suitable for administration to a subject.

[0064] Some aspects of the present disclosure provide a cell formulation comprising a formulation described herein and a diluent, wherein the ratio of formulation to diluent is in the range of 1:2 to 1:100, including 1:2 to 1:20, including 1:2 to 1:5, including 1:2, 1:3, 1:4, 1:5, or a ratio of 1:10 to 1:15, including 1:10, 1:11, 1:12, 1:13, 1:14, and 1:15, wherein the formulation comprises a population of cells, wherein the cell formulation is suitable for administration to a subject.

[0065] In some embodiments, the cell formulation is suitable for administration to the eye.

[0066] In some embodiments, the cell formulation is suitable for administration subretinaly.

[0067] In some embodiments, the pharmaceutical composition or cell preparation is not washed before or after dilution with a diluent.

[0068] In some embodiments, the pharmaceutical composition or cell formulation is not centrifuged before or after dilution with the diluent.

[0069] In some embodiments, the pharmaceutical composition or cell formulation is undiluted (eg, not diluted prior to administration to a subject).

[0070] In some embodiments, the formulation or cell preparation is stored frozen prior to dilution with a diluent.

[0071] In some embodiments, DMSO is present in a detectable amount at a level equal to or less than about 1% (v / v).

[0072] In some embodiments, DMSO is present in a detectable amount at a level equal to or less than about 0.037 mg / dose.

[0073] Some aspects of the present disclosure provide a cell preparation comprising a formulation and a cell population described herein, wherein the cell preparation is thawed by dilution with a diluent and is not washed.

[0074] In some embodiments, the cell preparation is diluted with a diluent at a ratio selected from the group consisting of: 1:2 to 1:100, including 1:2 to 1:20, including 1:2 to 1:5, including 1:2, The cell preparation may be diluted 1:3, 1:4, 1:5, or at a ratio of 1:10 to 1:15, including 1:10, 1:11, 1:12, 1:13, 1:14 and 1:15.

[0075] In some embodiments, DMSO is present in a detectable amount at a level equal to or less than about 0.037 mg / dose.

[0076] In some embodiments, the diluent is GS2 or GS2 Plus.

[0077] The above summary is intended to illustrate 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

[0078] Figure 1 Shown are pre-freezing cell viability of retinal pigment epithelial (RPE) cells prepared in formulations containing different concentrations of dimethyl sulfoxide (DMSO), albumin, and glucose as shown in Table 1. Cryopreserved Formulation #1 and Cryopreserved Formulation #1 (Second) refer to different preparations of the same formulation.

[0079] Figure 2 Shown in the preparation (as shown in Table 1) containing different concentrations of DMSO, albumin and glucose, prepared in the RPE cell thawing cell viability.The first bar in each pair of columns represents manual (Trypan blue) viability measurement, and the second bar in each pair of columns represents the automatic viability measurement performed using the NC-200 device.

[0080] Figure 3 Shows the use The thawing cell growth of the RPE cells prepared in the preparation (as shown in Table 1) containing different concentrations of DMSO, albumin and glucose of cell proliferation assay evaluation.The first bar in each pair of columns represents the measurement carried out on the 2nd day after thawing, and the second bar in each pair of columns represents the measurement carried out on the 4th day after thawing.

[0081] Figure 4A The images are of cryopreserved RPE cells (including multinucleated cells) under a microscope after thawing, plating and culturing in growth medium for 3 days. The arrows indicate multinucleated cells. Figure 4B is a graph showing the quantitative analysis of multinucleated cells present in post-thaw cultures in formulations AE. The first bar of each group represents binucleated cells, the second bar represents trinucleated cells, the third bar represents tetranucleated cells, and the fourth bar represents cells with five or more nuclei.

[0082] Figure 5AShown are the cell viability (circles above the bar graph) and cell growth on the 2nd day (the first bar in each pair of bars) and 3rd day (the second bar in each pair of bars) after thawing of RPE cells prepared in formulations (as shown in Table 3) containing 2.5% or 5% glycerol ("G"), 7.5% or 10% dextran 40 ("D") and 2.5% rHA in DPBS (with or without Ca and Mg, expressed as "PBS-" and "PBS+", respectively), as assessed by DNA concentration. Figure 5B is a graph showing quantitative analysis of multinucleated cells present in post-thaw culture in the preparations. The first bar of each group represents binucleated cells, the second bar represents trinucleated cells, the third bar represents tetranucleated cells, and the fourth bar represents cells with five or more nuclei.

[0083] Fig. 6A Shown are cell viability (circles above histogram) and cell growth on day 2 (first bar in each pair of bars) and day 3 (second bar in each pair of bars) after thawing of RPE cells prepared in formulations containing 5% DMSO (D5) or 5% glycerol+7.5% dextran 40 (G5) or 7.5% dextran 40 (D0) or 2.5% glycerol+7.5% dextran 40 (G2.5) or 2.5% propylene glycol (P2.5)+7.5% dextran 40 (P2.5), as assessed by DNA concentration, each formulation also containing 0.09% glucose, 2.5% rHA in PBS (+Ca, +Mg), as shown in Table 4. Figure 6B is a graph showing quantitative analysis of multinucleated cells present in post-thaw culture in the preparations. The first bar of each group represents binucleated cells, the second bar represents trinucleated cells, the third bar represents tetranucleated cells, and the fourth bar represents cells with five or more nuclei.

[0084] Fig. 7A Figure 3 shows the post-thaw cell viability of photoreceptor rescue cell (PRC) cells prepared in formulations containing 2.5% rHA, DPBS with Ca and Mg, 0.6% glucose, and different concentrations of DMSO (5% DMSO or 10% DMSO), as well as commercially available PRC cells prepared in formulations containing 10% DMSO. Post-thaw cell viability of PRC cells formulated in CS10 cell cryopreparation for comparison. Figure 7B Shown are post-thaw cell viability of photoreceptor rescue cell (PRC) cells prepared in formulations containing 2.5% rHA, DPBS with Ca and Mg, 0.6% glucose, and varying concentrations of DMSO (5%, 7.5% DMSO, or 10% DMSO).

[0085] Figure 8Shown is the post-thaw cell viability of corneal endothelial cell (CEC) cells prepared at a density of 30,000 cells / μL and a volume of 50 μL in formulations containing 2.5% rHA, 0.09% glucose, DPBS with Ca and Mg, and varying concentrations of DMSO (5% or 10%), compared to the post-thaw cell viability of CEC cells cryopreserved at a lower cell density (3 million cells / mL) and a standard volume (1 mL / vial) in 2.5% rHA, 0.09% glucose, DPBS with Ca and Mg, and 5% DMSO concentrations. DETAILED DESCRIPTION

[0086] Cell-based therapies, including those involving cell transplantation, such as in the context of regenerative medicine approaches, often require the formulation, storage, transport and / or injection of delicate or fragile cells that may be damaged or lose their repopulation capacity upon improper handling, extensive manipulation and / or processing steps, or exposure to non-physiological conditions.

[0087] The present disclosure provides preparations and methods for cell cryopreservation, cell storage, cell transportation, and final cell administration to subjects, wherein the treatment and operation of cells are minimal, and the survival rate increases after thawing. The preparations provided herein have several advantages over currently available preparations. For example, quite different from currently available cryopreservation preparations, the preparations provided herein require minimal post-thaw processing, and can be administered to subjects together with therapeutic agents, with almost no side effects (e.g., toxicity). The preparations provided herein allow cryopreservation at high density in a small volume, which provides many advantages, including a thawing scheme of a single step in which only a diluent needs to be added. Since the preparation does not need to be cleaned after thawing, the possibility of cell loss and cell damage is small, thereby improving the therapeutic effect of the cell preparations of cryopreservation. Currently available cryoprotectants and cryoprotectant preparations require a large number of cell processing procedures, which may be costly, time-consuming, prone to human errors, and may eventually reduce cell survival rate, re-seeding efficiency, and the re-proliferation ability of cells contained therein. In addition, it is worth noting that it is observed that compared with the cryopreservation preparations and methods of the prior art, the survival rate of cell groups cryopreserved using the preparations provided herein is improved. Improved cell growth after thawing was also observed when using the formulations provided herein. The formulations provided herein can improve the functionality, viability and stability of cell populations.

[0088] In addition to the above advantages, cryopreservation preparations disclosed herein are also surprisingly and unexpectedly relevant to the reduction in the frequency (or ubiquity) of multinucleated cells in culture, particularly in RPE cell cultures. Multinucleated cells, such as multinucleated RPE cells, have been observed using other preparations. However, surprisingly, cryopreservation preparations provided herein seem to reduce the frequency of such cells.

[0089] The present disclosure provides a novel cryoprotectant formulation comprising at least a cryoprotectant and albumin. In certain embodiments, the cryoprotectant formulation comprises at least a cryoprotectant, albumin and optional sugar. The cryoprotectant may be DMSO, glycerol, ethylene glycol, trehalose or taurine. In some embodiments, the cryoprotectant is DMSO. The albumin may be human albumin. The sugar may be glucose. The formulation may also include a buffer or a buffered saline, such as, but not limited to, phosphate buffered saline (PBS). The pH of the formulation may be close to physiological pH (e.g., in the range of 6-8). The presence of the cryoprotectant may range from about 1% to about 10% volume / volume (v / v). The presence of the cryoprotectant may range from about 2% to about 10% volume / volume (v / v). The presence of the cryoprotectant may range from about 3% to about 10% volume / volume (v / v). The presence of the cryoprotectant may range from about 4% to about 10% volume / volume (v / v), such as 4%, 5%, 6%, 7%, 8%, 9% or 10%. Albumin may be present in a range of about 2% to about 3% (w / v). Albumin may be present in a range of about 2% to about 10% (w / v). Albumin may be present in a range of about 2% to about 8% (w / v). Albumin may be present in a range of about 2% to about 7.5% (w / v). Albumin may be present in a range of about 3.5% to about 7.5% (w / v). Albumin may be present in a range of about 4% to about 7.5% (w / v). Albumin may be present in a range of about 4% to about 8% (w / v). Albumin may be present in a range of about 4% to about 8.5% (w / v). Albumin may be present in a range of about 4% to about 9% (w / v). Albumin may be present in a range of about 4% to about 10% (w / v). Sugar may be present in a range of about 0–about 1.5% (w / v).

[0090] In some embodiments, the cryopreservation formulation comprises about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0-1.5% glucose (w / v) and a buffer or buffered saline. The formulation may be substantially composed of about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0-1.5% glucose (w / v) and a buffer or buffered saline. The formulation may be composed of about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0-1.5% glucose (w / v) and a buffer or buffered saline. The albumin may be human albumin, including recombinant human albumin. The buffered saline may be phosphate buffered saline (PBS).

[0091] In some embodiments, the cryopreservation formulation comprises about 4% to about 6% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0.08% to about 0.1% glucose (w / v), and a buffer or buffered saline. In some embodiments, the cryopreservation formulation consists essentially of about 4% to about 6% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0.08% to about 0.1% glucose (w / v), and a buffer or buffered saline. In some embodiments, the cryopreservation formulation consists of about 4% to about 6% DMSO (v / v), about 2% to about 3% albumin (w / v), about 0.08% to about 0.1% glucose (w / v), and a buffer or buffered saline. The albumin can be human albumin, including recombinant human albumin. The buffered saline can be phosphate buffered saline (PBS). Table 1 and Figure 1 Explanation, RPE cell is placed in the preparation that comprises about 4% to about 6%DMSO (v / v), about 2% to about 3% recombinant albumin (w / v) and about 0.08% to about 0.1% glucose (w / v) in the PBS containing calcium and magnesium, and its survival rate exceeds 96%.When cell is frozen in these preparations and then thawed, its survival rate is within the scope of 94-98%.The cell growth in these preparations after thawing is also relatively uniform.

[0092] In some embodiments, the cryopreservation formulation comprises about 4% to about 10% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0-1.5% glucose (w / v) and a buffer or buffered saline. The formulation may be substantially composed of about 4% to about 10% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0-1.5% glucose (w / v) and a buffer or buffered saline. The formulation may be composed of about 4% to about 10% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0-1.5% glucose (w / v) and a buffer or buffered saline. The albumin may be human albumin, including recombinant human albumin. The buffered saline may be phosphate buffered saline (PBS).

[0093] In some embodiments, the cryopreservation formulation comprises about 4% to about 6% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0.08% to about 0.1% glucose (w / v), and a buffer or buffered saline. In some embodiments, the cryopreservation formulation consists essentially of about 4% to about 6% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0.08% to about 0.1% glucose (w / v), and a buffer or buffered saline. In some embodiments, the cryopreservation formulation consists of about 4% to about 6% DMSO (v / v), about 2% to about 7.5% albumin (w / v), about 0.08% to about 0.1% glucose (w / v), and a buffer or buffered saline. The albumin can be human albumin, including recombinant human albumin. The buffered saline can be phosphate buffered saline (PBS). Table 1 and Figure 1 Explanation, RPE cell is placed in the preparation that comprises about 4% to about 6%DMSO (v / v), about 2% to about 3% recombinant albumin (w / v) and about 0.08% to about 0.1% glucose (w / v) in the PBS containing calcium and magnesium, and its survival rate exceeds 96%.When cell is frozen in these preparations and then thawed, its survival rate is within the scope of 94-98%.The cell growth in these preparations after thawing is also relatively uniform.

[0094] The preparation comprising about 2.5% (v / v) DMSO, about 50mM trehalose, about 7.5% (w / v) dextran-40, about 2.5% albumin (w / v) and buffered saline is relevant with freezing and thawing after each cell has the incidence rate reduction of multinuclear RPE cells more than 4 nuclei.After freezing and thawing, the DMSO of 2.5%v / v level is compared with the DMSO of 1%v / v level, and can more effectively reduce the incidence rate of multinuclear RPE cells more than 4 nuclei of each cell.The preparation comprising even higher levels of DMSO (for example, 5%) and optionally also comprising ethylene glycol (for example, 5%) is relevant with each cell having the incidence rate reduction of multinuclear cells more than 3 nuclei.The preparation containing 5% glycerol can not produce the cell that each cell has more than 4 nuclei.

[0095] In some embodiments, the cryoprotectant is a cell-penetrating cryoprotectant, such as DMSO, glycerol, or ethylene glycol, all of which have been found to be associated with multinucleation of target cells, including but not limited to RPE cells, photoreceptor cells, photoreceptor progenitor cells, photoreceptor rescue cells, corneal endothelial cells, corneal endothelial progenitor cells, mesenchymal cells, mesenchymal stem cells, hematopoietic stem cells, neural or neuronal stem or progenitor cells, glial stem or progenitor cells, pancreatic stem or progenitor cells, β cells, keratinocytes, chondrocytes, osteoblasts, osteoclasts, or cells in a group or tissue (e.g., a monolayer or sheet or aggregate or cluster of RPE cells, corneal endothelial cells, photoreceptor rescue cells, islets of Langerhans, or skin grafts).

[0096] In some embodiments, the cryoprotectant is a cell-permeable cryoprotectant, such as DMSO, glycerol, or ethylene glycol, all of which have been found to be associated with a reduction in the incidence of multinucleated RPE cells.

[0097] Other formulations include about 5% DMSO (v / v), about 2.5% albumin (w / v), about 0.09% glucose (w / v), and buffer or buffered saline. Other formulations include about 2.5% albumin (w / v), about 0.09% glucose (w / v), about 5% (v / v) glycerol, about 200mM taurine, and buffer or buffered saline. Other formulations include 2.5% albumin (w / v), about 0.09% glucose (w / v), about 5% (v / v) glycerol, about 100mM trehalose, and buffer or buffered saline.

[0098] Other formulations include about 5% DMSO (v / v), about 2.5% albumin (w / v), about 0.6% glucose (w / v), and buffer or buffered saline. Other formulations include about 2.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerol, about 200mM taurine, and buffer or buffered saline. Other formulations include 2.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerol, about 100mM trehalose, and buffer or buffered saline.

[0099] Other formulations include about 5% DMSO (v / v), about 7.5% albumin (w / v), about 0.6% glucose (w / v), and buffer or buffered saline. Other formulations include about 7.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerol, about 200 mM taurine, and buffer or buffered saline. Other formulations include 7.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerol, about 100 mM trehalose, and buffer or buffered saline.

[0100] The present disclosure further contemplates that, in any embodiment provided herein, "glycerol" may also encompass "glycerin", and conversely, in any embodiment provided herein, "glycerin" may also encompass "glycerol".

[0101] In some embodiments, the cryopreserved cell preparation may include target cells (e.g., RPE cells, photoreceptor rescue cells, photoreceptor cells, corneal endothelial cells, corneal endothelial progenitor cells, neural cells or neural stem cells or neural progenitor cells or other cell types disclosed herein), 5% or 10% DMSO, 27 μg glucose, 750 μg recombinant human albumin, 110 μg sodium chloride, 26.6 μg sodium phosphate, 2.10 μg calcium chloride, 4.19 μg potassium chloride, 4.19 μg potassium dihydrogen phosphate, 2.10 μg magnesium chloride-6H 2 O, 168 μg sodium chloride and 24.1 μg sodium phosphate dibasic. In some embodiments, the cryopreserved cell preparation may include target cells (e.g., RPE cells), 5% or 10% DMSO, 0.9 μg / ml glucose, 259.5 μg / ml Recombumin Elite (recombinant human albumin), and Dulbecco's phosphate-buffered saline as a buffer.

[0102] After thawing and diluting in a diluent provided herein, in some embodiments, the final cell formulation to be administered to a subject can include target cells (e.g., RPE cells), sodium hyaluronate, sodium chloride, potassium chloride, sodium hydrogen phosphate dodecahydrate, dextrose (anhydrous), calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, and sodium citrate dihydrate.

[0103] Compared to standard prior art techniques, cells can be cryopreserved at a higher concentration (or density). For example, cells can be cryopreserved at a concentration (or density) of about 3x10 2 Cryopreserved at 10 cells / μL or more, including about 10 3 cells / μL, about 2x10 3 cells / μL, about 3x10 3 cells / μL, 10 4 cells / μL or higher, including approximately 2x10 4 cells / μL, about 3x10 4 cells / μL, about 4x10 4 cells / μL, about 5x10 4 cells / μL, about 6x10 4 cells / μL, about 7x10 4 cells / μL, about 8x10 4cells / μL, about 9x10 4 cells / μL or about 10 5 cells / μL or more. In some embodiments, the cell density is about 3x10 2 In some embodiments, the cell density is about 3.3 x 10 3 In some embodiments, the cell density is about 5x10 4 cells / μL.

[0104] In some embodiments, the cell density is about 10 3 cells / μL to about 6x10 4 cells / μL, including about 3x10 3 cells / μL to about 5x10 4 In some embodiments, the cell density is about 10 4 cells / μL to about 6x10 4 cells / μL, including 1.5x10 4 cells / μL to about 5x10 4 cells / μL, including approximately 2x10 4 cells / μL to about 5x10 4 cells / μL, and about 3x10 4 cells / μL to about 5x10 4 In some embodiments, the cell density is about 3x10 2 In some embodiments, the cell density is about 3.3 x 10 3 In some embodiments, the cell density is about 5x10 4 cells / μL.

[0105] In some embodiments, the number of cells per vial is about 10 5 Up to 10 7 In some embodiments, the number of cells per vial is about 450,000 to 1,500,000 cells per vial. In some embodiments, the number of cells per vial is about 1,750,000 to about 3,500,000 cells per vial.

[0106] In addition, the volume of the cryopreserved cell preparation can be reduced compared to standard prior art. For example, cells can be cryopreserved in a volume range of about 10 μL to about 100 μL, in some embodiments, including a volume of about 10 μL to about 50 μL, including a volume of about 10 μL, about 20 μL, about 30 μL, about 40 μL, about 50 μL, about 60 μL, about 70 μL, about 80 μL, about 90 μL, or about 100 μL. In some embodiments, cells can be cryopreserved in a volume range of about 10 μL to about 500 μL, in some embodiments, including 10 μL, about 20 μL, about 30 μL, about 40 μL, about 50 μL, about 60 μL, about 70 μL, about 80 μL, about 90 μL, about 100 μL, about 150 μL, about 200 μL, about 250 μL, about 300 μL, about 350 μL, about 400 μL, about 450 μL or about 500 μL. Since cells can be frozen in large quantities and small volumes, the frozen preparation can be diluted with a suitable diluent to reach the required volume and concentration, and then directly applied to the subject without performing a washing step. Standard prior art usually freezes cells of lower concentration in a larger volume (e.g., the order of 1 mL), and then the thawed preparation needs to be cleaned, and sometimes it needs to be cleaned multiple times to remove the cryoprotectant before applying to the subject.

[0107] In some embodiments, cryoprotectant preparation comprises the following composition, is substantially composed of them or is composed of them: about 4% to about 10% DMSO (w / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v) and buffer or buffered saline, and is used for freezing and preserving RPE cells. The preparation causes the survival rate of RPE cells after thawing to be at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95%. This also causes the frequency of multinucleated cells to be lower. In some embodiments, cryoprotectant preparation comprises the following composition, is substantially composed of them or is composed of them: about 10% DMSO (v / v), about 2.4% albumin (w / v), 0-1.5% glucose (w / v) and buffer.

[0108] In some embodiments, the cryoprotectant formulation comprises, consists essentially of, or consists of about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v), and a buffer, and is used to cryopreserve photoreceptor rescue cells, photoreceptor cells, or photoreceptor progenitor cells. The amount of DMSO may be about 5% or about 10%. The cells may be stored at about 50 x 10 3 The cells were frozen at a concentration of 10 cells / μL. Compared with the commercially available cryoprotectant CS10, this preparation can improve the cell survival rate after thawing.

[0109] In some embodiments, the cryoprotectant formulation comprises, consists essentially of, or consists of about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v), and a buffer or buffered saline, and is used to cryopreserve corneal endothelial progenitor cells or corneal endothelial cells. DMSO may be present in an amount of about 5% or about 10% (v / v). The cells may be stored at about 30x10 3 Compared with the traditional cryoprotectant formulation of 90% FBS (v / v) and 10% DMSO (v / v), this formulation can improve the survival rate of cells after thawing.

[0110] In some embodiments, the cryoprotectant formulation comprises, consists essentially of, or consists of the following ingredients: about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v), and a buffer or buffered saline. DMSO may be present in an amount of about 4% (v / v), about 5% (v / v), and about 6% (v / v). The albumin may be human albumin, including recombinant human albumin, and may be present at about 2% (w / v), or about 2.5% (w / v), or about 3% (w / v). Glucose may be absent, or may be present at about 0.08% (w / v), or about 0.09% (w / v), or about 0.1% (w / v). The buffered saline may be phosphate buffered saline.

[0111] In some embodiments, the cryoprotectant formulation comprises, consists essentially of, or consists of about 4% to about 6% DMSO (v / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v), and a buffer or buffered saline. The albumin may be present in an amount of about 2% (w / v), about 2.5% (w / v), or about 3% (w / v). The glucose may be present in an amount of about 0.08% (w / v), or about 0.09% (w / v), or about 0.10% (w / v).

[0112] In some embodiments, the cryoprotectant formulation comprises, consists essentially of, or consists of about 4% to about 10% DMSO (v / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v), and a buffer or buffered saline. DMSO may be present in an amount of about 4% (v / v), about 5% (v / v), and about 6% (v / v). The albumin may be human albumin, including recombinant human albumin, and may be present at about 2% (w / v), or about 2.5% (w / v), or about 3% (w / v), or about 4% (w / v), or about 5% (w / v), or about 6% (w / v), or about 7% (w / v), or about 7.5% (w / v), or about 8% (w / v), or about 9% (w / v), or about 10% (w / v). Glucose may be absent, or may be present at about 0.08% (w / v), or about 0.09% (w / v), or about 0.1% (w / v).The buffered saline may be phosphate buffered saline.

[0113] In some embodiments, the cryoprotectant formulation comprises, consists essentially of, or consists of the following ingredients: about 4% to about 6% DMSO (v / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v), and a buffer or buffered saline. The albumin may be present in an amount of about 2% (w / v), about 2.5% (w / v), or about 3% (w / v). In some embodiments, the cryoprotectant formulation comprises, consists essentially of, or consists of the following ingredients: about 4% to about 6% DMSO (v / v), about 2% to about 7.5% albumin (w / v), 0-1.5% glucose (w / v), and a buffer or buffered saline. The albumin may be present in an amount of about 2% (w / v), about 5% (w / v), or about 7.5% (w / v). The glucose may be present in an amount of about 0.08% (w / v), or about 0.09% (w / v), or about 0.10% (w / v). Cryopreserved preparations

[0114] In some embodiments, provided herein is a formulation (formulation) or preparation (preparation) showing physiological pH and physiological osmotic pressure, also referred to as physiological osmotic pressure.Physiological pH refers to a pH without cytotoxicity and similar to the pH of a cell or tissue in its natural environment.For most cells and tissues, physiological pH is a pH of about 6.8 to about 7.8, such as pH is about 7 to about 7.7, pH is about 7.2 to about 7.6, pH is about 7.2 to about 7.4, or pH is about 7.4 to about 7.5.Therefore, in some embodiments, provided herein is a formulation or preparation 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 refers to an osmotic pressure without cytotoxicity and similar to the osmotic pressure of a cell or tissue in its natural environment. For some cells and tissues, the physiological osmotic pressure is about 270 to about 345mOsm / l, for example, about 280 to about 330mOsm / l, about 290 to about 325mOsm / l, about 300 to about 315mOsm / l. In some embodiments, the physiological osmotic pressure is about 300, about 305, about 310, about 315, about 320, or about 325mOsm / l. In some embodiments, the formulations or preparations provided herein exhibit a physiological pH and an osmotic pressure greater than the physiological osmotic pressure, for example, about 350 to about 450mOsm / l, for example, about 360 to about 440mOsm / l, about 370 to about 430mOsm / l, about 380 to about 420mOsm / l, or about 390 to about 410mOsm / l. In some embodiments, the osmotic pressure greater than the physiological osmotic pressure is about 350, about 355, about 360, about 365, about 370, about 375, about 380, about 385, about 390, about 395, about 400, about 405, about 410, about 415, about 420, about 425, about 430, about 435, about 440, about 445, or about 450 mOsm / l. In some embodiments, the osmotic pressure of the formulations or preparations provided herein is about 1250mOsm / kg, for example, about 1250mOsm / kg, about 1000mOsm / kg, about 900mOsm / kg, about 800mOsm / kg, about 700mOsm / kg, about 600mOsm / kg, about 500mOsm / kg, about 400mOsm / kg or about 300mOsm / kg. In some embodiments, the osmotic pressure of the formulations or preparations provided herein is about 300-450mOsm / kg, about 300-400mOsm / kg, about 300-450mOsm / kg, about 350-450mOsm / kg or about 350-400mOsm / kg.

[0115] In some embodiments, the cryopreservation formulations provided herein comprise (a) a cryoprotectant; (b) albumin; (c) a buffer that maintains the solution at physiological pH; and (d) glucose.

[0116] In some embodiments, the preparation includes a cryoprotectant. Cryoprotectants are used to protect cells from damage during freezing. In some embodiments, the preparation includes a cryoprotectant selected from dimethyl sulfoxide (DMSO), glycerol and ethylene glycol. In some embodiments, the cryoprotectant is DMSO. In some embodiments, the preparation includes about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5% or about 10% (volume / volume, v / v) of DMSO. In some embodiments, the solution comprises about 4%-10%, about 4%-9.5%, about 4%-9%, about 4%-8.5%, about 4%-8%, about 4%-7.5%, about 4%-7%, about 4%-6.5%, about 4%-6%, about 4%-5.5%, about 4%-5%, about 4%-4.5%, about 4.5%-10%, about 4.5%-9.5%, about 4.5%-9%, about 4.5%-8.5%, about 4.5%-8%, about 4.5%-7.5%, about 4.5%-7%. , about 4.5%-6.5%, about 4.5%-6%, about 4.5%-5.5%, about 4.5%-5%, about 5%-10%, about 5%-9.5%, about 5%-9%, about 5%-8.5%, about 5%-8%, about 5%-7.5%, about 5%-7%, about 5%-6.5%, about 5%-6%, about 5%-5.5%, about 5.5%-10%, about 5.5%-9.5%, about 5.5%-9%, about 5.5%-8.5%, about 5.5%-8%, about 5.5%-7.5% , about 5.5%-7%, about 5.5%-6.5%, about 5.5%-6%, about 6%-10%, about 6%-9.5%, about 6%-9%, about 6%-8.5%, about 6%-8%, about 6%-7.5%, about 6%-7%, about 6%-6.5%, about 6.5%-10%, about 6.5%-9.5%, about 6.5%-9%, about 6.5%-8.5%, about 6.5%-8%, about 6.5%-7.5%, about 6.5%-7%, about 7%-10%, about 7%-9.5%, About 7%-9%, about 7%-8.5%, about 7%-8%, about 7%-7.5%, about 7.5%-10%, about 7.5%-9.5%, about 7.5%-9%, about 7.5%-8.5%, about 7.5%-8%, about 8%-10%, about 8%-9.5%, about 8%-9%, about 8%-8.5%, about 8.5%-10%, about 8.5%-9.5%, about 8.5%-10%, about 8.5%-9.5%, about 8.5%-9%, about 9%-10%, about 9%-9.5%, or about 9.5%-10% (v / v) DMSO.In some embodiments, the formulation comprises about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9%, about 6%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7%. %, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9% or about 10% (volume / volume, v / v) DMSO.

[0117] In some embodiments, the formulation comprises albumin. In some embodiments, the albumin is recombinant albumin. In some embodiments, the albumin is recombinant human (rh) albumin. The term "recombinant albumin" can be used interchangeably with the term "rA" or "r albumin". The term "recombinant human albumin" can be used interchangeably with the term "rHA", "rHSA", "rh albumin". In some embodiments, the cryopreservation formulation comprises about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about 5.8%, about 5.9 %, about 6.0%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about 7.0%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8.0%, About 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9.0%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9% or about 10.0% (w / v) albumin.

[0118] In some embodiments, the cryopreservation formulation comprises about 2.0%-10.0%, about 2.0%-9.5%, about 2.0%-9.0%, about 2.0%-8.5%, about 2.0%-8.0%, about 2.0%-7.5%, about 2.0%-7.0%, about 2.0%-6.5%, about 2.0%-6.0%, about 2.0%-5.5%, about 2.0%-5.0%, about 2.0%-4.5%, about 2.0%-4.0%, about 2.0%-3.5%, about 2.0%-3.0%, about 2.0%-2.5%, about 2.5%-10.0%, about 2.5%-9.5%, about 2.5%-9.0%, about 2.5%-8.5%, about 2.5%-8. 0%, about 2.5%-7.5%, about 2.5%-7.0%, about 2.5%-6.5%, about 2.5%-6.0%, about 2.5%-5.5%, about 2.5%-5.0%, about 2.5%-4.5%, about 2.5%-4.0%, about 2.5%-3.5%, about 2.5%-3.0%, about 3.0%-10.0% , about 3.0%-9.5%, about 3.0%-9.0%, about 3.0%-8.5%, about 3.0%-8.0%, about 3.0%-7.5%, about 3.0%-7.0%, about 3.0%-6.5%, about 3.0%-6.0%, about 3.0%-5.5%, about 3.0%-5.0%, about 3.0%-4.5%, about 3. 0%-4.0%, about 3.0%-3.5%, about 3.5%-10.0%, about 3.5%-9.5%, about 3.5%-9.0%, about 3.5%-8.5%, about 3.5%-8.0%, about 3.5%-7.5%, about 3.5%-7.0%, about 3.5%-6.5%, about 3.5%-6.0%, about 3.5%-5.5%, about 3.5%-5.0%, about 3.5%-4.5%, about 3.5%-4.0%, about 4.0%-10.0%, about 4.0%-9.5%, about 4.0%-9.0%, about 4.0%-8.5%, about 4.0%-8.0%, about 4.0%-7.5%, about 4.0%-7.0%, about 4.0%-6. 5%, about 4.0%-6.0%, about 4.0%-5.5%, about 4.0%-5.0%, about 4.0%-4.5%, about 4.5%-10.0%, about 4.5%-9.5%, about 4.5%-9.0%, about 4.5%-8.5%, about 4.5%-8.0%, about 4.5%-7.5%, about 4.5%-7.0%, About 4.5%-6.5%, about 4.5%-6.0%, about 4.5%-5.5%, about 4.5%-5.0%, about 5.0%-10.0%, about 5.0%-9.5%, about 5.0%-9.0%, about 5.0%-8.5%, about 5.0%-8.0%, about 5.0%-7.5%, about 5.0%-7.0%, about 5.0%-6.5%, about 5.0%-6.0%, about 5.0%-5.5%, about 5.5%-10.0%, about 5.5%-9.5%, about 5.5%-9.0%, about 5.5%-8.5%, about 5.5%-8.0%, about 5.5%-7.5%, about 5.5%-7.0%, about 5.5%-6.5%, about 5.5%-6.0%, about 6.0%-10.0%, about 6.0%-9.5%, about 6.0%-9.0%, about 6.0%-8.5%, about 6.0%-8.0%, about 6.0%-7.5%, about 6.0%-7.0%, about 6.0%-6.5%, about 6.5%-10.0%, about 6.5%- %, about 9.5%, about 6.5%-9.0%, about 6.5%-8.5%, about 6.5%-8.0%, about 6.5%-7.5%, about 6.5%-7.0%, about 7.0%-10.0%, about 7.0%-9.5%, about 7.0%-9.0%, about 7.0%-8.5%, about 7.0%-8.0%, about 7.0%-7.5%, about 8.0%-10.0%, about 8.0%-9.5%, about 8.0%-9.0%, about 8.0%-8.5%, about 8.5%-10.0%, about 8.5%-9.5%, about 8.5%-9.0%, about 9.0%-10.0%, or about 9.5%-10.0% (w / v) albumin.

[0119] In some embodiments, the cryopreservation formulation comprises about 2.0%-3.0%, about 2.0%-2.9%, about 2.0%-2.8%, about 2.0%-2.7%, about 2.0%-2.6%, about 2.0%-2.5%, about 2.0%-2.4%, about 2.0%-2.3%, about 2.0%-2.2%, about 2.0%-2.1%, about 2.1%-3.0%, about 2.1%-2.9%, about 2.1%-2. .8%, about 2.1%-2.7%, about 2.1%-2.6%, about 2.1%-2.5%, about 2.1%-2.4%, about 2.1%-2.3%, about 2.1%-2.2%, about 2.2%-3.0%, about 2.2%-2.9%, about 2.2%-2.8%, about 2.2%-2.7%, about 2.2%-2.6%, about 2.2%-2.5%, about 2.2%-2.4%, about 2.2%-2.3%, about 2.3%-3.0%, about 2.3%-2.9%, about 2.3%-2.8%, about 2.3%-2.7%, about 2.3%-2.6%, about 2.3%-2.5%, about 2.3%-2.4%, about 2.4%-3.0%, about 2.4%-2.9%, about 2.4%-2.8%, about 2.4%-2.7%, about 2.4%-2.6%, about 2.4%-2.5%, about 2.5%-3.0%, about 2.5 %-2.9%, about 2.5%-2.8%, about 2.5%-2.7%, about 2.5%-2.6%, about 2.6%-3.0%, about 2.6%-2.9%, about 2.6%-2.8%, about 2.6%-2.7%, about 2.7%-3.0%, about 2.7%-2.9%, about 2.7%-2.8%, about 2.8%-3.0%, about 2.8%-2.9% or about 2.9%-3.0% (w / v) albumin.

[0120] In some embodiments, the cryopreservation formulation comprises a buffer. A buffer refers to a reagent that can maintain the pH of a solution, formulation, or formulation relatively stable by neutralizing an added acid or base. Typically, the buffer comprises a weak conjugate acid-base pair, i.e., a weak acid and its conjugate base, or a weak base and its conjugate acid. In some embodiments, the buffer included in the cryopreservation formulation provided herein is a buffer comprising disodium hydrogen phosphate (Na 2 HPO 4 ) and sodium chloride (NaCl) in an aqueous salt solution. In some embodiments, the buffer included in the formulations provided herein also includes potassium chloride (KCl) and potassium dihydrogen phosphate (KH 2 PO 4). In some embodiments, the formulation comprises a buffered saline, such as phosphate buffered saline (PBS). In some embodiments, the buffer is Dulbecco's phosphate buffered saline (DPBS). In some embodiments, the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). In some embodiments, the buffer is 3-(N-morpholino)propanesulfonic acid (MOPS). In some embodiments, the buffer is N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES). In some embodiments, the buffer is 3-morpholino-2-hydroxypropanesulfonic acid sodium salt (MOPSO). In some embodiments, the buffer is N-tris(hydroxymethyl)methylglycine (Tricine). In some embodiments, the buffer is 2-[tris(hydroxymethyl)methylamino]-1-ethanesulfonic acid (TES). In some embodiments, the buffer is 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS or HEPPS).

[0121] In some embodiments, the buffer comprises 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 some embodiments, the divalent cation comprises calcium. In some embodiments, the divalent cation comprises magnesium. In some embodiments, the divalent cation comprises two or more different divalent cations, such as calcium and magnesium. In some embodiments, the buffer comprises calcium. In some embodiments, the buffer comprises a pharmaceutically acceptable calcium salt. In some embodiments, the buffer comprises magnesium. In some embodiments, the buffer comprises a pharmaceutically acceptable magnesium salt. In some embodiments of the solutions provided herein, the buffer comprises calcium chloride. In some embodiments, the buffer comprises calcium chloride dihydrate. In some embodiments, the buffer comprises magnesium chloride. In some embodiments, the buffer comprises magnesium chloride hexahydrate.

[0122] In some embodiments, the cryopreservation formulations provided herein include glucose. In some embodiments, the solution includes about 0%-5.0% (w / v) glucose. For example, in some embodiments, the cryopreservation formulations include about 0%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6 %, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9% or about 5% (w / v) glucose. The amount of glucose can be expressed as a range. The disclosure contemplates and provides herein any range of glucose selected from any of the following lower limits and any of the following upper limits. Any combination of these is disclosed herein.The lower limit of the range includes any of the following: 0%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5% , 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8% and 4.9%, and the The range includes any of the following upper limits: 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4. %, 4.8%, 4.9% or 5% (w / v) glucose. The present disclosure contemplates any range of glucose with any combination of a disclosed lower limit and a disclosed upper limit.For example, ranges provided herein are, for example, about 0-0.5%, about 0-1%, about 0-1.5%, about 0-2%, about 0-2.5%, about 0-3%, about 0-3.5%, about 0-4%, about 0-4.5%, about 0-5%, about 0.5-1%, about 0.5-1.5%, about 0.5-2%, about 0.5-2.5%, about 0.5-3%, about 0.5-3.5%, about 0.5-4%, about 0.5-4.5%, about 0.5-5%, about 1-1.5%, about 1-2%, about 1-2.5%, about 1-3%, about 1-3.5%, about 1-4%, about 1-4.5%, about 1-5%, about 1.5-2. %, about 1.5-2.5%, about 1.5-3%, about 1.5-3.5%, about 1.5-4%, about 1.5-4.5%, about 1.5-5%, about 2-2.5%, about 2-3%, about 2-3.5%, about 2-4%, about 2-4.5%, about 2-5%, about 2.5-3%, about 2.5-3.5%, about 2.5-4%, about 2.5-4.5%, about 2.5-5%, about 3-3.5%, about 3-4%, about 3-4.5%, about 3-5%, about 3.5-4%, about 3.5-4.5%, about 3.5-5%, about 4-4.5%, about 4-5%, and about 4.5-5% (w / v) glucose. In some embodiments, the solution comprises about 0-1.5% (w / v) glucose. It should be understood that, as an example, a range of about 1-1.5% means about 1% to about 1.5%. Similar meanings are given to each and every range described herein.

[0123] In some embodiments, the formulation comprises about 4-10% (v / v) of a cell cryoprotectant, about 2-3% (w / v) of albumin, about 0-1.5% (w / v) of glucose, and a buffer (optionally buffered saline). In some embodiments, the formulation comprises about 4-10% (v / v) of a cell cryoprotectant, about 2-3% (w / v) of albumin, about 0.08-0.10% (w / v) of glucose, and a buffer (optionally buffered saline). In some embodiments, the formulation comprises about 4-6% (v / v) of a cell cryoprotectant, about 2-3% (w / v) of albumin, about 0.08-0.10% (w / v) of glucose, and a buffer (optionally buffered saline).

[0124] In some embodiments, the formulation comprises about 4-10% (v / v) DMSO, about 2-3% (w / v) albumin, about 0-1.5% (w / v) glucose, and a buffer. In some embodiments, the formulation comprises about 4-10% (v / v) DMSO, about 2-3% (w / v) albumin, about 0.08-0.10% (w / v) glucose, and a buffer. In some embodiments, the formulation comprises about 4-6% (v / v) DMSO, about 2-3% (w / v) albumin, about 0.08-0.10% (w / v) glucose, and a buffer. In some embodiments, the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose and buffer. In some embodiments, the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.6% (w / v) glucose and buffer. In some embodiments, the formulation comprises about 4-10% (v / v) DMSO, about 2-8% (w / v) albumin, about 0-1.5% (w / v) glucose and buffer. In some embodiments, the formulation comprises about 4-6% (v / v) DMSO, about 2-8% (w / v) albumin, about 0-1.5% (w / v) glucose and buffer. In some embodiments, the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.6% (w / v) glucose and buffer. In some embodiments, the formulation comprises about 5% (v / v) DMSO, about 7.5% (w / v) albumin, about 0.6% (w / v) glucose, and a buffer.

[0125] The present disclosure further contemplates any of the foregoing formulations comprising about 0%-5% (w / v) glucose, or comprising any one of the ranges specified herein (eg, in the preceding paragraphs).

[0126] In some embodiments, the formulations provided herein further comprise at least one additional excipient. In some embodiments, the formulation comprises about 1 mM-500 mM, about 5 mM-400 mM, about 10 mM-300 mM, about 25 mM-250 mM, about 50 mM-200 mM of at least one additional excipient. In some embodiments, the formulation comprises about 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61mM, 62mM, 63mM, 64mM, 65mM , 66mM, 67mM, 68mM, 69mM, 70mM, 71mM, 72mM, 73mM, 74mM, 75mM, 76mM, 77mM, 78mM, 79mM, 80mM, 81mM, 82mM, 83mM, 84mM, 85mM, 86mM, 87mM , 88mM, 89mM, 90mM, 91mM, 92mM, 93mM, 94mM, 95mM, 96mM, 97mM, 98mM, 99mM, 100mM, 101mM, 102mM, 103mM, 104mM, 105mM, 106mM, 107mM, 10 8mM, 109mM, 110mM, 111mM, 112mM, 113mM, 114mM, 115mM, 116mM, 117mM, 118mM, 119mM, 120mM, 121mM, 122mM, 123mM, 124mM, 125mM, 126m M, 127mM, 128mM, 129mM, 130mM, 131mM, 132mM, 133mM, 134mM, 135mM, 136mM, 137mM, 138mM, 139mM, 140mM, 141mM, 142mM, 143mM, 144mM, 1 45mM, 146mM, 147mM, 148mM, 149mM, 150mM, 151mM, 152mM, 153mM, 154mM, 155mM, 156mM, 157mM, 158mM, 159mM, 160mM, 161mM, 162mM, 163 mM, 164mM, 165mM, 166mM, 167mM, 168mM, 169mM, 170mM, 171mM, 172mM, 173mM, 174mM, 175mM, 176mM, 177mM, 178mM, 179mM, 180mM, 181mM,182mM, 183mM, 184mM, 185mM, 186mM, 187mM, 188mM, 189mM, 190mM, 191mM, 192mM, 193mM, 194mM, 195mM, 196mM, 197mM, 198mM, 199m M, 200mM, 201mM, 202mM, 203mM, 204mM, 205mM, 206mM, 207mM, 208mM, 209mM, 210mM, 211mM, 212mM, 213mM, 214mM, 215mM, 216mM, 21 244mM, 245mM, 246mM, 247mM, 248mM, 249mM or 250mM of at least one additional excipient. In some embodiments, the at least one additional excipient is selected from taurine, trehalose, sucrose, lactose, propylene glycol, mannitol, sorbitol, proline, glutathione, glycine, alanine, or lysine. In some embodiments, the at least one additional excipient is taurine. In some embodiments, the at least one additional excipient is trehalose. In some embodiments, the at least one additional excipient is 1,2-propylene glycol or 1,3-propylene glycol. In some embodiments, the at least one additional excipient is propylene glycol.

[0127] In some embodiments, the formulation comprises a polymeric excipient. In some embodiments, the formulation comprises about 1%-20% of a polymeric excipient. In some embodiments, the formulation comprises about 3%-18%, about 5%-15%, or about 7.5%-12.5% ​​of a polymeric excipient. In some embodiments, the formulation comprises about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, about 13%, about 13.5%, about 14%, about 14.5%, about 15%, about 15.5%, about 16%, about 16.5%, about 17%, about 17.5%, about 18%, about 18.5%, about 19%, about 19.5%, or about 20% of a polymer excipient. In some embodiments, the polymer excipient is selected from polyethylene glycol (PEG), dextran and polyvinyl pyrrolidone (PVP). In some embodiments, the polymer excipient is dextran. In some embodiments, the formulation does not include a polymer excipient. In some embodiments, the formulation does not include dextran. Cell preparations Some aspects of the present disclosure provide cell preparations, which are included in the cell populations or tissues in the cryopreservation preparations provided herein. In some embodiments, the cell preparations are cryopreserved. The cell preparations provided herein can be cryopreserved by cryogenic freezing at any appropriate temperature known in the art. For example, in some embodiments, the cell preparations provided herein are cryopreserved at a temperature between -100 ° C and -200 ° C. In some embodiments, the cell preparations provided herein are cryopreserved at a temperature lower than -125 ° C. In some embodiments, the cell preparations provided herein are cryopreserved at or below a temperature of -135 ° C. In some embodiments, the cell preparations provided herein are cryopreserved at a temperature lower than -150 ° C.

[0129] Cryopreservation preparations and cell preparations provided herein can be used in combination with different cell types and different application sites. Although ophthalmic applications are preferred, the disclosure also contemplates and encompasses other applications. In some embodiments, cell populations are selected from retinal pigment epithelial (RPE) cells, photoreceptor cells and / or its progenitor cells, photoreceptor rescue cells and corneal endothelial cells and / or its progenitor cells.

[0130] In some embodiments, the cell preparation is suitable for transplantation into a subject once thawed, optionally without dilution. In some embodiments, the cell preparation is suitable for transplantation into a subject once thawed and diluted with a diluent. In some embodiments, the diluent is GS2, GS2 Plus, BSS, BSS Dextran and HSA solution, PBS, DMEM, MEM, albumin or any other suitable diluent. In some embodiments, the diluent is GS2 or GS2 Plus. In some embodiments, the diluent is a solution comprising the following ingredients: (a) a buffer solution maintaining a solution at physiological pH; (b) at least 2mM of glucose; and (c) an osmotic agent maintaining a solution at a physiological osmotic pressure. In some embodiments, the diluent is GS2 or GS2 Plus. In some embodiments, the diluent is a solution comprising the following ingredients: (a) a buffer solution maintaining a solution at physiological pH; (b) at least 2mM of glucose; and (c) an osmotic agent maintaining a solution at an osmotic pressure (e.g., in the range of 350-450mOsm / kg) or an osmotic pressure in the range of 300-1250mOsm / kg higher than the physiological osmotic pressure. In some embodiments, the diluent further comprises divalent cations. In some embodiments, divalent cations include calcium and / or magnesium. In some embodiments, the diluent comprises calcium. In some embodiments, the diluent further comprises magnesium. In some embodiments, diluent includes acetate buffer and / or citrate buffer.In some embodiments, diluent is a solution combining two or more or any number of standards (for example, pH, osmotic pressure, solute (buffer, glucose, osmotic active agent, magnesium, calcium, potassium, polymer), concentration, etc.).In some embodiments, diluent is a solution comprising buffer, glucose and osmotic active agent (adding or not adding polymer), a solution comprising potassium and a solution not comprising potassium and a solution comprising any solute combination under any concentration provided for corresponding solute.It should also be understood that the present disclosure encompasses solutions comprising listed solutes and solutions consisting of or consisting of listed solutes and solvents (for example water) substantially.For brevity, these alternatives are not described in detail here.

[0131] For example, in some embodiments, the diluent is calcium chloride, magnesium chloride, sodium citrate, sodium chloride and glucose (e.g., D-glucose) contained in water or a solution consisting essentially of them. In some embodiments, the diluent is calcium chloride, magnesium chloride, sodium citrate, sodium chloride, glucose (e.g., D-glucose) and potassium chloride contained in water or a solution consisting essentially of them. In some embodiments, the diluent is GS2 or GS2Plus, as described in WO2017 / 031312A1, the contents of which are incorporated herein by reference.

[0132] In some embodiments, the diluent is a diluent comprising about 0.1 to about 1.2 mM CaCl 2 , about 0.05 to about 5 mM MgCl 2 , about 1 to about 2.5 mM KCl, about 0.5 to about 2 mM sodium citrate, about 14 to about 17 mM dextrose, and about 125 to about 175 mM NaCl. In some embodiments, the diluent is a solution comprising about 0.9 mM CaCl 2 , about 0.3 mM MgCl 2 The diluent may also include a solution of about 2 mM KCl, about 1.2 mM sodium citrate, about 15 mM dextrose and about 145 mM NaCl. The diluent may also include sodium acetate. The diluent may also include a polymer such as hyaluronic acid or a solvate thereof such as sodium hyaluronate, optionally at a concentration of about 0.01 to about 0.05% (w / v), including about 0.05% (w / v). It may also include a buffer component, such as disodium hydrogen phosphate heptahydrate and / or sodium dihydrogen phosphate monohydrate and / or sodium dihydrogen phosphate dihydrate.

[0133] In some embodiments, the diluent is a diluent comprising about 0.008% to about 0.012% CaCl 2 Dihydrate, about 0.0048% to about 0.0072% MgCl 2 In some embodiments, the diluent is a solution comprising about 0.01% CaCl 2 Dihydrate, about 0.006% MgCl 2 The diluent may also include a solution of about 0.25% dextrose and about 0.85% NaCl. The diluent may also include sodium acetate. The diluent may also include a polymer such as hyaluronic acid or a solvate thereof such as sodium hyaluronate, optionally at a concentration of about 0.01 to about 0.05% (w / v), including about 0.05% (w / v). It may also include a buffer component such as disodium hydrogen phosphate heptahydrate and / or sodium dihydrogen phosphate monohydrate and / or sodium dihydrogen phosphate dihydrate.

[0134] In some embodiments, the diluent comprises 5% dextrose, 0.9% NaCl, Sterile flushing solution, 0.1 N NaOH and 0.9% NaCl flushing solution. In some embodiments, the diluent includes 3% sodium hyaluronate, 5% anhydrous dextrose, disodium hydrogen phosphate dodecahydrate, Sterile irrigation solution (e.g., 0.64% NaCl, 0.075% KCl, 0.048% CaCl 2 Dihydrate, 0.03% MgCl 2 hexahydrate, 0.39% sodium acetate trihydrate and 0.17% sodium citrate dihydrate).

[0135] In some embodiments, the diluent comprises about 0.27% dextrose, about 0.84% ​​sodium chloride, about 0.016% potassium chloride, about 0.01% calcium chloride, about 0.006% magnesium chloride, about 0.036% sodium citrate, and optionally sodium acetate (e.g., about 0.08%), optionally sodium hyaluronate (e.g., about 0.049%), and optionally sodium phosphate dibasic heptahydrate (e.g., about 0.0007%) and sodium phosphate monobasic monohydrate (e.g., about 0.0001%). In some embodiments, the diluent comprises about 15 mM glucose, about 144 mM sodium chloride, about 2.1 mM potassium chloride, about 0.9 mM calcium chloride, about 0.3 mM magnesium chloride, about 1.2 mM sodium citrate, optional sodium acetate (e.g., about 6 mM), optional sodium hyaluronate, and optional sodium dihydrogen phosphate heptahydrate (e.g., about 0.027 mM) and sodium dihydrogen phosphate monohydrate (e.g., about 0.007 mM). Such diluents include GS2.

[0136] In some embodiments, the diluent comprises about 0.27% glucose, about 0.84% ​​sodium chloride, about 0.016% potassium chloride, about 0.01% calcium chloride, about 0.006% magnesium chloride, about 0.036% sodium citrate, and optionally sodium acetate (e.g., about 0.08%), optionally sodium hyaluronate (e.g., about 0.049%), and optionally sodium dihydrogen phosphate dihydrate (e.g., about 0.047%). In some embodiments, the diluent comprises about 15mM glucose, about 144mM sodium chloride, about 2.1mM potassium chloride, about 0.9mM calcium chloride, about 0.3mM magnesium chloride, about 1.2mM sodium citrate, optionally sodium acetate (e.g., about 6mM), optionally sodium hyaluronate, and optionally sodium dihydrogen phosphate dihydrate (e.g., about 3mM). Such diluents include GS2 Plus.

[0137] In some embodiments, provided herein is a cell preparation, once thawed and diluted, formulated for application to a subject, such as by injection. Provided herein is a cell preparation that can include a cell population, such as RPE cells and / or its progenitor cells, photoreceptor cells and / or its progenitor cells and corneal endothelial cells and / or its progenitor cells and photoreceptor rescue cells.

[0138] Exemplary cell or tissue preparations, after thawing, 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 osmotic pressure. In some embodiments, the preparations provided herein are formulated for injection into a specific site, e.g., in the case of an ophthalmic preparation for treating a retinal disease or disorder, injection into the vitreous humor for delivery to a site of retinal or choroidal damage.

[0139] The cell formulations provided by the present disclosure may include additional therapeutic agents, such as immunosuppressants, pro-angiogenic agents, or nutrients or growth factors that support the survival and / or engraftment of the cells in the formulation.

[0140] The volume and cell number of the cell preparation to be administered will depend on specific application. Generally, for cell transplantation applications, it is desirable to reduce the volume of administration as much as possible. Therefore, the cell preparation can be prepared so that the minimum volume can be delivered. The cell concentration of injection can be any effective and nontoxic concentration. In some embodiments, the volume of the cell preparation to be administered is 1 to 1000 μL. In some embodiments, the volume of the cell preparation to be administered is 1 to 50 μL, or 10 to 50 μL, or 25 to 50 μL, or 50 to 100 μL, or 50 to 200 μL, or 50 to 300 μL, or 50 to 400 μL, or 50 to 500 μL, or 100 to 500 μL, or 100 to 400 μL, or 100 to 300 μL, or 100 to 300 μL, or about 200 μL or about 150 μL. In some embodiments, the volume of the cell preparation to be administered is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, 950, or 1000 μl.

[0141] In some embodiments, the cell number and / or cell concentration in the cell preparations provided herein can be determined by counting live cells and excluding non-living cells. For example, non-living cells can be detected by failing to exclude vital dyes (e.g., trypan blue) or using functional assays (e.g., ability to adhere to a culture matrix, phagocytosis, etc.). In addition, the cell number or cell concentration of the desired cell type can be determined by counting cells expressing one or more cell markers characteristic of the cell type and / or excluding cells expressing one or more markers indicating a cell type other than the desired cell type.

[0142] In some embodiments, the cell preparation to be administered comprises about or at least 1×10 3 , 2x103 、3x10 3 、4x10 3 、5x10 3 、6x10 3 、7x10 3 、8x10 3 、9x10 3 、1x10 4 、2x10 4 、3x10 4 、4x10 4 、5x10 4 、6x10 4 、7x10 4 、8x10 4 、9x10 4 、1x10 5 、2x10 5 、3x10 5 、4x10 5 、5x10 5 、6x10 5 、7x10 5 、8x10 5 、9x10 5 、1x10 6 、2x10 6 、3x10 6 、4x10 6 、5x10 6 、6x10 6 、7x10 6 、8x10 6 、9x10 6 、1x10 7 、2x10 7 、3x10 7 、4x10 7 、5x10 7 、6x10 7 、7x10 7 、8x10 7 、9x10 7 、1x10 8 、2x10 8 、3x10 8 、4x10 8 、5x10 8 、6x10 8 、7x10 8 、8x10 8 、9x10 8 、1x10 9 、2x10 9 、3x10 9, 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 or 9x10 10 cells or more.

[0143] In some embodiments, the number of cells per vial prior to cryopreservation is about 10 5 Up to 10 7 In some embodiments, the number of cells per vial prior to cryopreservation is about 450,000 to 1,500,000 cells per vial. In some embodiments, the number of cells per vial prior to cryopreservation is about 1,750,000 to about 3,500,000 cells per vial. In some embodiments, the number of cells per vial after cryopreservation is about 10 5 Up to 10 7 In some embodiments, the number of cells per vial is about 450,000 to 1,500,000 cells per vial after cryopreservation, thawing, and dilution. In some embodiments, the number of cells per vial is about 1,750,000 to about 3,500,000 cells per vial before and after cryopreservation, thawing, and dilution.

[0144] The above-described number of cells may be present in a cryopreserved cell preparation in a single cryovial. The cells may be present in about 10 to about 500 μL of cryoprotectant formulation, including about 10 to about 200 μL of cryoprotectant formulation, or about 10 to about 100 μL of cryoprotectant formulation, or about 10 to about 50 μL of cryoprotectant formulation, or about 20 to about 50 μL of cryoprotectant formulation.

[0145] In some embodiments, cell mass is suitable for transplanting into the eyes of experimenter.In some embodiments, the cell mass suitable for transplanting into the eyes of experimenter includes RPE cell, RPE progenitor cell, iris pigment epithelium (IPE) cell and other visual related nerve cells, such as contact neuron (such as " relay " neuron of inner nuclear layer (INL)) and amacrine cell, retinal cell, rod cell, cone cell and keratocyte (such as corneal endothelial cell), nerve cell, neural progenitor cell, neural stem cell, photoreceptor cell (such as photoreceptor progenitor cell), photoreceptor rescue cell and mesenchymal cell, such as mesenchymal stem cell (MSC).The cell so prepared can be produced by the directed differentiation of pluripotent (pluripotent) or pluripotent (multipotent) stem cell, including inducible human pluripotent stem cell (hiPSC), human embryonic stem cell (hESC) and somatic cell (including transdifferentiation cell and stem cell).

[0146] In some embodiments, cell preparation includes the RPE cell group in the cryopreservation preparation provided herein.Suitable RPE cell can be differentiated from pluripotent stem cells (such as human embryonic stem (ES) cells or induced pluripotent stem cells (iPS cells)), and is molecularly different from embryonic stem cells, iPS cells, adult-derived RPE cells and fetus-derived RPE cells.In some embodiments, adult-derived RPE cells and fetus-derived RPE cells are used.

[0147] When using ES cell-derived RPE cells, in some embodiments, cell preparations do not include the residual ES cells of detectable amount, so that the cell preparations provided herein will not cause unacceptable risks to the recipients of such preparations. In the case of using iPS cell-derived RPE cells, in some embodiments, cell preparations do not include the residual iPS cells of detectable amount, so that the cell preparations provided herein will not bring unacceptable risks to the recipients of such preparations.

[0148] In some embodiments, the cell formulation includes a population of photoreceptor rescue cells in a cryopreservation preparation provided herein. Suitable photoreceptor rescue cells can be differentiated from pluripotent stem cells (e.g., human embryonic stem cells or iPS cells) and are molecularly distinct from embryonic stem cells, iPS cells, adult-derived photoreceptor rescue cells, and fetal-derived photoreceptor rescue cells. In some embodiments, adult-derived photoreceptor rescue cells and fetal-derived photoreceptor rescue cells are used.

[0149] When ES cell-derived photoreceptor rescue cells are used, in some embodiments, the cell preparations do not contain detectable amounts of residual ES cells, such that the cell preparations provided herein do not pose an unacceptable risk to the recipient of such preparations. When iPS cell-derived photoreceptor rescue cells are used, in some embodiments, the cell preparations do not contain detectable amounts of residual iPS cells, such that the cell preparations provided herein do not pose an unacceptable risk to the recipient of such preparations.

[0150] In some embodiments, cell preparations include a group of corneal endothelial cells in a cryopreservation preparation provided herein. Suitable corneal endothelial cells can be differentiated from pluripotent stem cells (e.g., human embryonic stem cells or iPS cells) and are molecularly different from embryonic stem cells, iPS cells, corneal endothelial cells derived from adults and corneal endothelial cells derived from fetuses. In some embodiments, corneal endothelial cells derived from adults and corneal endothelial cells derived from fetuses are used.

[0151] When ES cell-derived corneal endothelial cells are used, in some embodiments, the cell preparations do not contain detectable amounts of residual ES cells, such that the cell preparations provided herein do not pose an unacceptable risk to the recipient of such preparations. When iPS cell-derived corneal endothelial cells are used, in some embodiments, the cell preparations do not contain detectable amounts of residual iPS cells, such that the cell preparations provided herein do not pose an unacceptable risk to the recipient of such preparations.

[0152] In some embodiments, a cell preparation comprising a cell population suitable for transplantation into an eye of a subject is suitable for injection into an eye of a subject. In some embodiments, such a cell preparation can be used to treat retinal degenerative diseases or conditions, including but not limited to retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration, or retinal atrophy, or associated with a variety of vision-altering diseases that lead to photoreceptor damage and blindness (e.g., choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (yellow spot disease of the fundus)).

[0153] RPE cells can be stable, terminally differentiated RPE cells that do not dedifferentiate into non-RPE cell types. RPE cells as described herein can be functional RPE cells, characterized in that they can be integrated into the retina when being administered to humans or non-human animals in the cornea, under the retina or otherwise.

[0154] RPE cells 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) may be equal to the expression level in naturally occurring RPE cells.The maturity level of RPE cells can be assessed by measuring at least one expression in PAX2, PAX6 and tyrosinase or their expression level separately.

[0155] In some embodiments, the RPE cell included in the pharmaceutical composition provided herein can be identified and characterized based on the pigmentation degree of the cell.The change of pigment can be controlled by the density and the duration of maintaining RPE in the culture that RPE cells are cultured and maintained.The pigmentation of the differentiated RPE cells of rapid division is lighter.On the contrary, the RPE that divides slower or does not divide will present its characteristic polygonal or hexagonal shape, and increase the pigmentation level by accumulating melanin and lipofuscin.For example, static RPE culture (for example, due to fusion) will usually increase its pigmentation level over time.Therefore, the accumulation of pigmentation serves as an index of RPE differentiation, and the increase of pigmentation related to cell density serves as an index of RPE maturation.For example, mature RPE cells can be subcultured at a lower density, so that pigmentation is reduced.In this case, mature RPE cells can be cultivated to produce less mature RPE cells.This type of RPE cell is still the RPE cell of differentiation, and it expresses the marker of RPE differentiation.

[0156] For example, in some embodiments, a preparation is provided, comprising RPE cells, 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 -2pg / cell, between 0.1-1pg / cell, between 1-8pg / cell, between 1-7pg / cell, between 1-6pg / cell, between 1-5pg / cell, between 1-4pg / cell, between 1-3pg / cell, between 1-2pg / cell, between 2-6pg / cell, between 3-5pg / cell, or between 4-5pg / cell, for example, between 4.2-4.8pg / cell, or between 0.1-5pg / cell. In further examples, the average melanin content can be less than 5pg / cell, for example, between 0.1-5pg / cell, between 0.2-5pg / cell, between 0.5-5pg / cell, between 1-5pg / cell, between 2-5pg / cell, between 3-5pg / cell, between 4-5pg / cell or between 4.5-5pg / cell.

[0157] In some embodiments, provide the preparation comprising the RPE cell in the cell cryopreservation preparation described herein, wherein after RPE cell is transplanted into experimenter (for example, after preparation is injected into experimenter's eyes), RPE cell maintains its phenotype.After transplantation, RPE cell may maintain its phenotype in recipient's lifetime.For example, RPE cell 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, RPE cell can maintain their phenotype at least about 1,2,3,4,5,6,7,8,9 or 10 weeks after transplantation.RPE cell can maintain their phenotype at least about 1,2,3,4,5,6,7,8,9,10,11 or 12 months after transplantation. 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.

[0158] The cells can be formulated in the formulations provided herein for delivery in a pharmaceutically acceptable ophthalmic vehicle such that the formulation remains in contact with the ocular surface for a sufficient time to allow the cells to penetrate the affected area of ​​the eye, such as the anterior chamber, posterior chamber, vitreous, aqueous humor, vitreous humor, cornea, iris / ciliary body, lens, choroid, retina, sclera, suprachoroidal space, conjunctiva, subconjunctival space, episcleral space, intracorneal space, extracorneal space, pars plana, surgically induced avascular area, or macula. The cells may have several possible arrangements, such as single cells, clusters, spheres, aggregates, sheets, or any combination thereof, which may be contained in an aqueous vehicle, gel, matrix, polymer, etc.

[0159] In some embodiments, provided herein is a formulation wherein the cells are contained in a cell sheet. For example, RPE cells or corneal endothelial cells or photoreceptor cells or photoreceptor rescue cells can be contained in the sheet. For example, a cell sheet comprising RPE cells can be prepared by culturing RPE cells on a substrate that releases the intact cell sheet (e.g., a thermoresponsive polymer, such as a surface grafted with thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm), the cells adhere and proliferate thereon at the culture temperature, and then upon a change in temperature, the surface properties change, resulting in 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 December; 25(12):577-83; Hsiue et al., Transplantation. 2006 February 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 may be adhered to a matrix suitable for transplantation, such as a matrix that dissolves in vivo when the sheet is transplanted into a host organism, such as prepared by culturing cells on a matrix suitable for transplantation, or releasing cells from another matrix (e.g., a thermoresponsive polymer) onto a matrix suitable for transplantation. Exemplary matrices that may be suitable for transplantation may include gelatin (see Hsiue et al., supra). Alternative matrices that may be suitable for transplantation include fibrin-based matrices, etc. The cell sheet may be used to manufacture a drug for preventing or treating retinal degenerative diseases.

[0160] In some embodiments, provided herein is a formulation wherein the cells are in the form of clusters, spheres, or aggregates. In some embodiments, provided herein are cells administered in combination with a biomaterial (eg, a polymerizable biomaterial).

[0161] In some embodiments, provided herein is cell (for example, RPE cell) as implant or device administration.In certain embodiments, the device is the bioerodible implant for the medical condition of the treatment eye, it comprises the activating agent dispersed in the biodegradable polymer matrix, wherein the diameter of at least about 75% of the activating agent particles is less than about 10pm.The bioerodible implant has the size for implanting eye region.The eye region can be any one or more in the anterior chamber, posterior chamber, vitreous cavity, choroid, suprachoroidal cavity, conjunctiva, subconjunctival cavity, sclera outer space, corneal inner space, corneal outer space, sclera, ciliary body flat part, the avascular area caused by surgery, macula and retina.Biodegradable polymer can be for example poly (lactic acid-co-glycolic acid) (PLGA) copolymer.In certain embodiments, the ratio of lactic acid to glycolic acid monomer in the polymer is about 25 / 75,40 / 60,50 / 50,60 / 40,75 / 25 weight percentage, more preferably about 50 / 50. In addition, PLGA copolymer can account for about 20%, 30%, 40%, 50%, 60%, 70%, 80% to about 90% of the weight of bioerodible implant.In some preferred embodiments, PLGA copolymer can account for about 30 to about 50% of the weight of bioerodible implant, preferably about 40%.Cell provided herein (for example, RPE cell, photoreceptor cell, photoreceptor rescue cell and corneal endothelial cell) can be with biocompatible polymer (for example, polylactic acid, poly (lactic acid-glycolic acid), PDLGA 50:50, Pd Lg A 85:15 and biodegradable film INION (biocompatible polymer mixture)) combined with implantation. See U.S. Patent Nos. 6,331,313, 7,462,471 and 7,625,582. See also Hutala et al. (2007) "In vitro biocompatibility of degradable biopolymers in cell line cultures from various ocular tissues: Direct contact studies". Journal of Biomedical Materials Research 83A(2):407-413; Lu et al. (1998) J Biomater Sci Polym Ed 9:1187 205; and Tomita et al. (2005) Stem Cells 23:1579-88. In some cases, the biodegradable material can be polymeric.

[0162] The volume of the pharmaceutical composition provided in some embodiments of the present disclosure depends on factors such as mode of administration, number of cells to be delivered, age and weight of the patient, and type and severity of the disease treated. For example, if administered by injection, the volume of the pharmaceutical composition of the cell disclosed herein can be about 1-1000 μL. In some embodiments, the volume can be about 1-200 μL. For example, the volume of the composition disclosed herein can be about 10-50, 20-50, 25-50 or 1-200 μL. The volume of the composition disclosed herein can be about 10, 20, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400 or 450 μL, or higher.

[0163] In some embodiments, the cell concentration in the cryopreserved preparation (or cryopreserved cell preparation) is about 300, 500, 1 x 10 3 , 1.5x10 3 , 2x10 3 , 2.5x10 3 , 3x10 3 ,3.333x10 3 , 3.5x10 3 , 4x10 3 , 4.4x10 3 , 5x10 3 , 5.5x10 3 , 7.5x10 3 , 1x10 4 , 1.5x10 4, 2x10 4 , 2.5x10 4 , 3x10 4 , 3.5x10 4 , 4x10 4 , 4.5x10 4 , 5x10 4 , 5.5x10 4 , 6x10 4 , 6.5x10 4 , 7x10 4 , 7.5x10 4 , 8x10 4 , 8.5x10 4 , 9x10 4 , 9.5x10 4 , 1x10 5 , 1.05x10 5 , 1.1x10 5 , 1.15x10 5 , 1.2x10 5 , 1.25x10 5 , 1.3x10 5 , 1.35x10 5 , 1.4x10 5 , 1.45x10 5 or 1.5x10 5In some embodiments, the cell concentration in the cryopreserved preparation (or cryopreserved cell preparation) is about 2,000-80,000 cells / μL, 2,000-70,000 cells / μL, 3,000-70,000 cells / μL, 3,000-60,000 cells / μL, 3,000-50,000 cells / μL, 3,000-10,000 cells / μL, 3,000-5,000 cells / μL, 3,000-4,000 cells / μL, 3,500 cells / μL, 10,000-100,000 cells / μL, 10,000-90,000 cells / μL, 10,000-80 ,000 cells / μL, 10,000-70,000 cells / μL, 10,000-60,000 cells / μL, 10,000-50,000 cells / μL, 10,000-40,000 cells / μL, 10,000-30,000 cells / μL, 10,000-20,000 cells / μL, 20,000-100,000 cells / μL, 20,000-90,000 cells / μL, 20,000-80,000 cells / μL, 20,000-70,000 cells / μL, 20,000-60,000 cells / μL, 20,000-50,000 cells / μL, 20,000-40,000 cells / μL, 20,000-30,000 cells / μL, 30,000-100,000 cells / μL, 30,000-90,000 cells / μL, 30,000-80,000 cells / μL, 30,000-70,000 cells / μL, 30,000-60,000 cells / μL, 30,000-50,000 cells / μL, 30,000-40,000 cells / μL, 40,000-100,000 cells / μL, 40,000-90,000 cells / μL, 40,000-80,000 cells / μL, 40,0 00-70,000 cells / μL, 40,000-60,000 cells / μL, 40,000-50,000 cells / μL, 50,000-100,000 cells / μL, 50,000-90,000 cells / μL, 50,000-80,000 cells / μL, 50,000-70,000 cells / μL, 50,000-60,000 cells / μL, 60,000-100,000 cells / μL, 60,000-90,000 cells / μL, 60,000-80,000 cells / μL, 60,000-70,000 cells / μL, 70,000-100,000 cells / μL000 cells / μL, 70,000-90,000 cells / μL, 70,000-80,000 cells / μL, 80,000-100,000 cells / μL, 80,000-90,000 cells / μL, 90,000-100,000 cells / μL, 10,000-150,000 cells / μL, 20,000-150,000 cells / μL, 30,000-150,000 cells / μL, 40,000-150,000 cells / μL L, 50,000-150,000 cells / μL, 60,000-150,000 cells / μL, 70,000-150,000 cells / μL, 80,000-150,000 cells / μL, 90,000-140,000 cells / μL, 100,000-150,000 cells / μL, 100,000-130,000 cells / μL, 120,000-150,000 cells / μL, or 120,000-125,000 cells / μL. ,

[0164] In some embodiments, the formulation supports the survival of cells in the cell population during storage of the formulation. In some embodiments, the formulation supports the survival of cells during storage for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years or more. In some embodiments, the formulation supports the survival of cells during storage for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 1 week to about 8 weeks, about 1 week to about 4 weeks, about 1 week to about 2 weeks. In some embodiments, the formulation supports cell survival during storage for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days, or about 1 day to about 7 days, about 1 day to about 5 days, about 1 day to about 3 days, or about 1 day to about 2 days. The formulation can be stored in a temperature range of about -100 to about -200°C, or about -135°C to about -200°C, but is not limited thereto.

[0165] In some embodiments, at least 30% of the cells in the cell population are viable after storage of the formulation at -100 to -200°C, preferably at or below -135°C, for about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years, about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years, or about 1-2 years or less. In some embodiments, at least 40% of the cells in the cell population are viable after storage of the formulation at -100 to -200°C, preferably at or below -135°C, for about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years, about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years, or about 1-2 years or less. In some embodiments, at least 50% of the cells in the cell population are viable after storage of the formulation at -100 to -200°C, preferably at or below -135°C, for about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years, about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years, or about 1-2 years or less. In some embodiments, at least 55% of the cells in the cell population are viable after storage of the formulation at -100 to -200°C, preferably at or below -135°C, for about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years, about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years, or about 1-2 years or less. In some embodiments, at least 60% of the cells in the cell population are viable after storage of the formulation at -100 to -200°C, preferably at or below -135°C, for about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years, about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years, or about 1-2 years or less. In some embodiments, at least 65% of the cells in the cell population are viable after storage of the formulation at -100 to -200°C, preferably at or below -135°C, for about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years, about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years, or about 1-2 years or less. In some embodiments, at least 70% of the cells in the cell population are viable after storage of the formulation at -100 to -200°C, preferably at or below -135°C, for about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years, about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years, or about 1-2 years or less. In some embodiments, at least 80% of the cells in the cell population are viable after storage of the formulation at -100 to -200°C, preferably at or below -135°C, for about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years, about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years, or about 1-2 years or less.In some embodiments, at least 90% of the cells in the cell population are viable after storage of the formulation for about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years, about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years, or about 1-2 years or less at -100 to -200° C., preferably at or less than -135° C. In some embodiments, the formulation supports maintenance of seeding efficiency of the cell population during storage of the formulation. In some embodiments, after storing the formulation for about 1-10 years, about 1-9 years, about 1-8 years, about 1-7 years, about 1-6 years, about 1-5 years, about 1-4 years, about 1-3 years, or about 1-2 years or less at -100 to -200°C, preferably at -135°C or less, the cell population exhibits at least 50%, at least 55%, at least 60%, at least 65%, 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 seeding efficiency, wherein the original seeding efficiency refers to the seeding efficiency of the cell population at the beginning of the storage period. In some embodiments, the formulation is in a storage container. In some embodiments, the storage container is a vial, an ampoule, a bottle, a syringe, a freezer package, or other suitable storage container. In some embodiments, the formulation is in a vial. In some embodiments, the formulation is in a syringe.

[0166] The cell preparation provided herein is suitable for being applied to a subject after minimal processing (e.g., dilution) after thawing. In some embodiments, the preparation is substantially composed of cells, cell groups or tissues and cryopreservation preparations provided herein. In some embodiments, the preparation comprises one or more pharmaceutically active ingredients, such as preservatives, antioxidants, free radical scavengers, immunosuppressants, angiogenic factors, anti-angiogenic factors, hormones (e.g., growth hormones) or cell nutrients or matrix that support cell growth, survival and implantation.

[0167] Preservatives may include, but are not limited to, benzalkonium chloride (BAK), benzethonium chloride, chlorobutanol, acetic acid or phenylmercuric nitrate, thimerosal, polyquaternium-1 Stabilized oxychlorine complex or methyl or propyl paraben. Antioxidants and / or free radical scavengers may include, but are not limited to, vitamin C, vitamin E, β-carotene, coenzyme Q-10, selenium, thioredoxin reductase, glutathione, α-tocopherol, L-cysteine ​​and N-acetylcysteine ​​(NAC), ascorbic acid-2-phosphate (AAP), zinc (e.g., zinc oxide), copper (e.g., copper oxide), dimethyl sulfoxide (DMSO), glycerol, mannitol, and polyphenolic compounds. Immunosuppressants may 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, (anti-CD20 antibody), sirolimus and tacrolimus, mesenchymal stem cells and mycophenolate mofetil. Pro-angiogenic factors may include, but are not limited to, VEGF, HGF, KGF, bFGF, TIMP1, ANG2, PDGF-bb, TPO, HB-EGF, TGF-β1, MMP-9 and MMP-2. Anti-angiogenic factors may include, but are not limited to, somatostatin, angiostatin and endostatin, thrombospondin (TSP), pigment epithelium-derived factor (PEDF), transforming growth factor β (TGF-β), ranibizumab or bevacizumab Tissue inhibitors of metalloproteinases (TiMPs), plasminogen activator inhibitors (PAI), prolactin, interferon, and angiopoietin 2. Hormones and hormone antagonists may include 17B-estradiol, adrenocorticotropic hormone, adrenomedullin, alpha-melanocyte stimulating hormone, chorionic gonadotropin, corticosteroid binding globulin, corticosterone, dexamethasone, estriol, follicle stimulating hormone, gastrin 1, glucagon, gonadotropin, hydrocortisone, insulin, insulin-like growth factor binding protein, L-3,3′,5′-triiodothyronine, L-3,3′,5′-triiodothyronine, leptin, luteinizing hormone, L-thyroxine, melatonin, MZ4, oxytocin, parathyroid hormone, PEC-60, pituitary growth hormone, progesterone, prolactin, secretin, sex hormone binding globulin, thyroid stimulating hormone, thyrotropin releasing factor, thyroxine binding globulin, and vasopressin. Cell nutrients may include, but are not limited to, albumin, B-27 supplement, fetal bovine serum (FBS), ethanolamine, fetuin, glutamine, insulin, peptone, purified lipoprotein material, sodium selenite, transferrin, vitamin A, vitamin B9, vitamin B12, vitamin C or vitamin E, glucose, galactose, riboflavin, thiamine, biotin, cholesterol, iron, zinc, copper, selenium, magnesium, and tricarboxylic acid. The matrix that supports cell growth, survival, and implantation may include, but are not limited to, extracellular matrix from bovine corneal epithelium, Matrigel TM (basement membrane matrix) or gelatin.

[0168] The present disclosure also encompasses pharmaceutical packages and / or kits. The pharmaceutical packages and / or kits provided may include a pharmaceutical composition provided herein, a diluent, and instructions for thawing the pharmaceutical composition by diluting the pharmaceutical composition prior to administration to a subject, preferably without washing the cells prior to adding the diluent. In some embodiments, the diluent is GS2, GS2 Plus, BSS, BSS Dextran and HSA solution, PBS, DMEM, MEM, albumin or any other suitable diluent. In some embodiments, the diluent is GS2 or GS2 Plus. In some embodiments, the pharmaceutical composition is cryopreserved. In some embodiments, the instructions also include instructions for thawing the cryopreserved pharmaceutical composition, and optionally instructions for combining the composition with a diluent and then applying the resulting composition to a subject. In some embodiments, the pharmaceutical composition is not cryopreserved. In some embodiments, the instructions also include instructions for mixing the composition with a diluent and then applying the resulting composition to a subject.

[0169] Some aspects of the present disclosure provide methods including contacting a cell group with a cell cryopreservation preparation provided herein.Cell group can be a RPE cell group, a mesenchymal stem cell group, a mesenchymal cell group, a photoreceptor cell group, a photoreceptor progenitor cell group, a photoreceptor rescue cell group, a retinal ganglion cell group, a retinal ganglion progenitor cell group, a corneal endothelial cell group or a corneal endothelial progenitor cell group.Cell group can be produced by the in vitro differentiation of pluripotent stem cells (such as embryonic stem cells or induced pluripotent stem cells).Cell group may be a human cell or a non-human cell.

[0170] Some aspects of the present disclosure provide methods, including contacting a cell population with a cell cryopreservation preparation provided herein to form a pharmaceutical composition, and then cryopreserving the pharmaceutical composition. Any suitable method can be used to cryopreserve the pharmaceutical composition, including at any appropriate temperature. For example, the pharmaceutical composition can be cryopreserved at a temperature between -100 ° C and -200 ° C. In some embodiments, the pharmaceutical composition is cryopreserved at a temperature below -125 ° C. In some embodiments, the pharmaceutical composition is cryopreserved at a temperature equal to or below 135 ° C. In some embodiments, the pharmaceutical composition is cryopreserved at a temperature below -150 ° C. The pharmaceutical composition can be cryopreserved in liquid nitrogen or in the gas phase of liquid nitrogen.

[0171] Some aspects of the present disclosure provide methods of treating a disease or condition comprising (a) administering a diluent in a ratio ranging from about 1:1 to about 1:20, or about 1:2 to about 1:20, or about 1:3 to about 1:20, or about 1:4 to about 1:20, or about 1:5 to about 1:20, or about 1:4 to about 1:19, or about 1:4 to about 1:18, or about 1:4 to about 1:17 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:115, 1:120, 1:13, 1:14, 1:15 In some embodiments, the cell preparation is diluted with a warm diluent. In some embodiments, the cell preparation is diluted with a cold diluent. In some embodiments, the cell preparation is gently pipetted after dilution. In some embodiments, the cell preparation is vigorously pipetted after dilution. As provided in the examples, there is no difference between the embodiment of diluting the cell preparation with a warm diluent and the embodiment of diluting the cell preparation with a cold diluent. There was also no difference in cell viability between embodiments in which the cell preparation was gently pipetted after dilution and embodiments in which the cell preparation was vigorously pipetted after dilution (e.g., into a storage container or into any container in which the cell preparation was transferred for dilution). Exemplary Uses and Methods

[0172] The cell preparations disclosed herein (including cryopreserved preparations once thawed) can be used for various clinical applications. As described herein, the cryopreserved preparations can be thawed by simply adding a diluent (such as GS2 or GS2 Plus), after which they can be applied to a subject without washing or other operations. In some embodiments, the cryopreserved preparations can be thawed without adding a diluent, after which they can be applied to a subject without washing or other operations.

[0173] The preparation of cryopreservation can comprise dissociated cells or cell monolayer or cell aggregate or cluster or sheet.The cryopreservation preparation provided herein is compatible with various cell types, cell groups and tissues, including but not limited to adult stem cells and progenitor cells, differentiated cells and groups and tissues comprising such cells.In some embodiments, the cell, cell group or tissue so prepared are intended to be used for the clinical use of regenerative medicine method.For example, cell, cell group or tissue can be cells, cell groups or tissues that can replace the cell of loss or degeneration in the subject or repair or replace the tissue that has been damaged or dysfunctional in the subject.For example, in some embodiments, cell, cell group or tissue can include pluripotent stem cells, such as iPS cells or ES cells, pluripotent stem cells or progenitor cells or functional differentiated cells or groups or tissues comprising such cells or the combination of such cells.

[0174] Cells, cell populations, or tissues to be cryopreserved using the formulations and methods disclosed herein include, but are not limited to, RPE cells, photoreceptor cells, photoreceptor progenitor cells, photoreceptor rescue cells, corneal endothelial cells, corneal endothelial progenitor cells, mesenchymal cells, mesenchymal stem cells, hematopoietic stem cells, neural or neuronal stem or progenitor cells, glial stem or progenitor cells, pancreatic stem or progenitor cells, β cells, keratinocytes, chondrocytes, osteoblasts, osteoclasts, or a population or tissue comprising or substantially consisting of such cells, such as a monolayer or sheet or aggregate or cluster of RPE cells, corneal endothelial cells, photoreceptor rescue cells, pancreatic islets, or skin grafts.

[0175] In some embodiments, the cell preparation of cryopreservation can be stored indefinitely, or can store the time period of regulation, and optionally can be transported to clinic or other end user location.It is worth noting that it can be thawed by simply adding diluent, and then can be applied to the subject without further operation.The preparation of cryopreservation is generally a small volume preparation.For example, its volume can be less than 1mL, less than 500 μ L, less than 250 μ L, less than 100 μ L or less than or about 50 μ L or any volume disclosed herein. It can be diluted at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, or more to achieve a volume and cell concentration suitable for the intended clinical use. The ratio of the volume of the cryoprotectant formulation to the volume of the diluent can be expressed as a ratio, and can include, but is not limited to, about 1:1 to about 1:20, or about 1:2 to about 1:20, or about 1:3 to about 1:20, or about 1:4 to about 1:20, or about 1:5 to about 1:20, or about 1:4 to about 1:19, or about 1:4 to about 1:18, or about 1:4 to about 1:17 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:115, 1:120, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:116, 1:18, 1:19, 1:117, 1:12 :9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100 or higher).

[0176] Cryopreserved cell preparations typically contain cells at a high density, such as but not limited to at least or about 3×10 2 / μL, or at least or about 10 3 / μL, at least or about 2x10 3 / μL, at least or about 3x10 3 / μL, at least or about 3.5x10 3 / μL, at least or about 5x10 3 / μL, at least or about 10 4 / μL, at least or about 5x10 4 / μL, at least or about 105 / μL, or at least or about 5x10 5 One skilled in the art will appreciate that the cell concentration and intended use will guide the degree of dilution required prior to administration to a subject.

[0177] Cryopreserved formulations can be stored in a frozen state, e.g., in the vapor phase of a liquid nitrogen tank, for extended periods of time, e.g., the periods described herein, including but not limited to at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, or longer, with minimal effect on cell viability or cell function, as measured, e.g., by dye exclusion, replating efficiency, or repopulation capacity. Cells or cell populations cryopreserved and thawed according to the present disclosure may have a viability, reseeding efficiency and / or repopulation capacity of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, %, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% cell viability, reseeding efficiency and / or repopulation capacity.

[0178] In some embodiments, the cryopreserved cell formulations provided herein are stored at a temperature between -100° C. and -200° C., including a temperature below -125° C., a temperature below -135° C., or a temperature below -150° C. In some embodiments, the cryopreserved cell formulations provided herein are stored in liquid nitrogen or in the vapor phase of liquid nitrogen.

[0179] Cryopreserved cell preparations are produced by combining target cells, cell groups or tissues into cryoprotectant preparations provided herein, optionally dispersing cells, cell groups, tissues in preparations, and then slowly freezing the mixture. Slow freezing can be done manually or by a device, as described in the examples. In one embodiment, the preparation is frozen at a rate of -1°C per minute until the desired temperature, optional intermediate temperature, such as -80°C is reached. Alternatively, the freezing rate can be -1.5°C per minute or -2°C per minute, etc. Cryopreserved cell preparations can be stored at intermediate temperatures for hours, days, weeks or longer. It can then be stored at a lower temperature. For example, a slow freezing process can be used to reduce the preparation to -80°C, after which it can be stored overnight (or up to about 12 or about 24 hours) at -80°C, and then it can be stored in the vapor phase of a liquid nitrogen tank (for example, at about -125°C). Once at a lower temperature, it may be stored there for days, weeks, months or even years. If necessary, it can even be transported to, for example, a clinical site in the form of cryopreservation at a lower temperature. The temperature of the cryoprotectant formulation can be, for example, between 0°C and 45°C, such as about 1°C, about 2°C, about 3°C, about 4°C, about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36%, about 37°C and about 40°C.

[0180] Some aspects of the present disclosure provide methods for treating a subject with a disorder or condition that can benefit from cell transplantation. The method may include thawing a cryopreserved cell preparation comprising a cryopreserved formulation provided herein, and then administering the thawed cell preparation to a subject in need, optionally without diluting with a diluent. In some embodiments, the method may include thawing a cryopreserved cell preparation comprising a cryopreserved formulation provided herein by adding a diluent, and then administering the thawed and diluted cell preparation to a subject in need. In some embodiments, the diluent is GS2, GS2Plus, BSS, BSS Dextran and HSA solution, PBS, DMEM, MEM or albumin. In some embodiments, the diluent is GS2 or GS2 Plus. In some embodiments, the diluent is GS2 or GS2 Plus. The subject may suffer from or may have been diagnosed with a disease or condition that can be treated by administering cells, cell groups or tissues. The cell preparation is administered at a cell dose that effectively treats the subject's disease or condition. In some embodiments, the disease or condition includes but is not limited to retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration or retinal atrophy, or is related to a variety of vision-changing diseases (e.g., choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), dry or wet age-related macular degeneration, retinitis pigmentosa and Stargardt's disease (fundus yellow spot disease)) that cause photoreceptor damage and blindness. In some embodiments, the method also includes monitoring at least one symptom of the subject's disease or condition, optionally to determine whether the severity or frequency of such symptoms is alleviated after administration.

[0181] The term "treatment" refers to a clinical intervention intended to reverse, alleviate, delay the onset of a disease or condition or to inhibit its progress. In some cases, the effect of the intervention can be assessed by monitoring the symptoms associated with the disease or condition. In some embodiments, the cell preparation can be administered after developing one or more symptoms and / or after diagnosing a disease or condition. In other embodiments, the cell preparation can be administered in the absence of symptoms, for example, to prevent or delay the appearance of symptoms or to inhibit the appearance or progress of a disease or condition. For example, the cell preparation can be administered to susceptible individuals (for example, in view of symptom history and / or in view of genetic or other susceptibility factors) before the onset of symptoms. The cell preparation can be administered once or more than once, including at regular and prescribed time points or as needed.

[0182] The subject can be a human or a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, cat, or dog. In some embodiments, the subject is a research animal. In some embodiments, the subject is a genetically modified, such as a genetically modified non-human subject. The subject can be of any gender and can be at any stage of development.

[0183] The term "effective amount" as used herein refers to the amount of a bioactive agent sufficient to cause a desired biological response. For example, in some embodiments, the effective amount of a cell preparation may refer to the amount of a preparation comprising a cell number or tissue amount sufficient to provide a therapeutic benefit to a subject suffering from a disease or condition (e.g., sufficient to improve the vision of a subject suffering from a retinal disease or condition). It will be appreciated by those skilled in the art that an effective amount may vary according to various factors, for example, according to a desired biological response, for example, according to a specific disease being treated, a specific symptom to be alleviated, or a cell or tissue targeted, and according to the age, sex, and general health of the subject.

[0184] Some aspects of the present disclosure provide methods for treating retinal diseases, wherein the method includes applying an effective amount of cell preparations provided herein to the eye of a subject suffering from retinal diseases. In some embodiments, the subject suffers from or is diagnosed with retinal diseases. In some embodiments, retinal diseases are rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy macular degeneration, such as wet or dry age-related macular degeneration, Leber congenital amaurosis or Stargardt disease. In some embodiments, the cells administered include RPE cells, photoreceptor cells, photoreceptor progenitor cells, photoreceptor rescue cells, corneal endothelial cells, corneal endothelial progenitor cells or retinal ganglion cells or retinal ganglion progenitor cells, any of which can be produced by the in vitro differentiation of pluripotent stem cells (such as embryonic stem cells or induced pluripotent stem cells). Reagent test kit

[0185] Some aspects of the present disclosure provide kits that include (a) a cryopreserved pharmaceutical composition comprising target cells provided herein; (b) a diluent, such as but not limited to GS2 or GS2 Plus, and (c) instructions for adding the diluent to the composition to thaw and dilute the composition and make it suitable for administration to a subject. The instructions may also provide that the cryopreserved components do not need to be cleaned or otherwise manipulated after thawing. The kit may also include a device for administering the composition, such as a syringe and a needle.

[0186] Therefore, the present disclosure provides, inter alia, the following: Item 1. A formulation comprising about 4-10% (v / v) cryoprotectant, about 2-8% (w / v) albumin, about 0-1.5% (w / v) glucose, and a buffer. Clause 2. The formulation of Clause 1, wherein the cryoprotectant is selected from the group consisting of DMSO, glycerol and ethylene glycol. Clause 3. The formulation of Clause 1, wherein the cryoprotectant is DMSO. Clause 4. The formulation of any one of the preceding clauses, wherein the formulation comprises about 4-6% (v / v) cryoprotectant. Clause 5. The formulation of any one of the preceding clauses, wherein the formulation comprises about 0.08-0.10% (w / v) glucose. Clause 6. The formulation of any of the preceding clauses, wherein the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and a buffer. Clause 7. The formulation of any of clauses 1-4, wherein the formulation comprises about 0.6% (w / v) glucose. Clause 8. The formulation of any one of Clauses 1-4 and 7, wherein the formulation comprises about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and a buffer. Clause 9. The formulation of any one of the preceding clauses, wherein the albumin is human albumin. Clause 10. The formulation of any one of the preceding clauses, wherein the albumin is recombinant human albumin. Clause 11. The formulation of any of the preceding clauses, wherein the buffer is buffered saline. Clause 12. The formulation of any of the preceding clauses, wherein the buffer is phosphate buffered saline (PBS). Clause 13. The formulation of Clause 11 or 12, wherein the buffered saline comprises Ca2+ and Mg2+. Clause 14. The formulation of Clause 11 or 12, wherein the buffered saline does not contain Ca2+ and Mg2+. Clause 15. The preparation of any of the preceding clauses, further comprising a cell population or tissue. Item 16. The preparation of Item 15, wherein the cell population is a dissociated cell population. Clause 17. The preparation of Clause 15, wherein the cell population comprises a cell monolayer, a cell sheet, a cell cluster, a cell sphere or a cell aggregate. Clause 18. The preparation of Clause 15, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells. Clause 19. The formulation of any of the preceding clauses, wherein the formulation is stored frozen. Clause 20. The formulation of any of the preceding clauses, wherein the formulation does not comprise a polymer excipient. Clause 21. The formulation of any of the preceding clauses, wherein the formulation does not comprise dextran. Clause 22. A cell formulation comprising (a) the formulation of any of clauses 1-14 and (b) a cell population. Clause 23. The cell preparation of clause 22, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells. Clause 24. The cell preparation of clause 22 or 23, wherein the cell population in the cell preparation has a concentration of about 10,000 cells to 100,000 cells / μL. Clause 25. The cell preparation of Clause 22 or 23, wherein the cell population concentration in the cell preparation is about 15,000-50,000 cells / μL. Clause 26. The cell formulation of any one of clauses 22-25, in a cryopreservation vial, syringe, ampoule, bottle or freezer packaging. Clause 27. The cell formulation of Clause 26, wherein about 10 to about 500 μL of the formulation is in a vial, syringe, ampoule, bottle, or freezer packaging. Clause 28. The cell preparation of clause 26 or 27, wherein about 10 5 Up to 10 7 cells. Clause 29. The cell preparation of clause 24 or 25, wherein about 10 5 Up to 5x10 6 cells. Clause 30. The cell preparation of clause 24 or 25, wherein about 10 5 Up to 2x10 6 cells. Clause 31. The cell preparation of any one of clauses 22-30, wherein the cell preparation is cryopreserved. Clause 32. A method of preparing a cell preparation comprising contacting the formulation of any one of clauses 1-14 with a cell population. Clause 33. The method of Clause 29, further comprising cryopreserving the cell preparation. Clause 34. The method of Clause 32 or 33, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells. Clause 35. A method of treating a subject having a disorder or condition, comprising thawing the cell preparation of Clause 31, diluting the cell preparation with a diluent at a ratio selected from 1:2, 1:3, 1:4 and 1:5, and The diluted cell preparation is administered to the subject. Clause 36. The method of clause 35, wherein the disorder or condition is selected from retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration or retinal atrophy, or is associated with a variety of vision-altering diseases that lead to photoreceptor damage and blindness (e.g., choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (yellow spots on the eye)). Clause 37. The method of clause 35, wherein the diluent is GS2, GS2 Plus, BSS, BSS Dextran and HSA solution, PBS, DMEM, MEM, or albumin. Clause 38. A method of treating a subject having a disorder or condition, comprising thawing the cell preparation of Clause 31, diluting the cell preparation with a diluent at a ratio selected from 1:15, 1:16, 1:17, 1:18 and 1:19, and The diluted cell preparation is administered to the subject. Item 39. The method of Item 37, wherein the disorder or condition is selected from retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration or retinal atrophy, or is associated with a variety of vision-altering diseases that lead to photoreceptor damage and blindness (e.g., choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (yellow spots on the eye)). Clause 40. The method of clause 38, wherein the diluent is GS2 or GS2 Plus, BSS, BSS Dextran and HSA solution, PBS, DMEM, MEM, or albumin. Clause 41. A kit comprising (a) a cryopreserved cell preparation as defined in clause 31, (b) a diluent, and (c) instructions for diluting the cell preparation prior to administration to a subject. Clause 42. The kit of clause 41, wherein the instructions further comprise instructions for thawing the cryopreserved cell preparation. Clause 43. A pharmaceutical composition comprising the preparation of any one of clauses 1-14 in combination with a cell population. Clause 44. The pharmaceutical composition of Clause 43, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells. Clause 45. The pharmaceutical composition of Clause 43, wherein the cell population comprises neural cells, neural stem cells, or neural precursor cells. Clause 46. A pharmaceutical composition comprising the formulation of any one of clauses 15-21 and a diluent, wherein the ratio of formulation to diluent is in the range of 1:2 to 1:20, wherein the pharmaceutical composition is suitable for administration to a subject, optionally to the eye, further optionally to the subretina. Clause 47. A pharmaceutical composition comprising the cell preparation of any one of clauses 22-25 and 31 and a diluent, wherein the ratio of the cell preparation to the diluent is in the range of 1:2 to 1:20, wherein the pharmaceutical composition is suitable for administration to a subject, optionally to the eye, further optionally to the subretina. Clause 48. A cell preparation comprising the formulation of any one of clauses 1-14, 19, 20 and 21 and a diluent, wherein the ratio of formulation to diluent is in the range of 1:2 to 1:20, wherein the formulation comprises a population of cells, wherein the cell preparation is suitable for administration to a subject, optionally to the eye, further optionally to the subretina. Clause 49. The pharmaceutical composition of Clause 46 or 47 or the cell preparation of Clause 48, wherein the pharmaceutical composition or the cell preparation is not washed before or after dilution with the diluent. Clause 50. The pharmaceutical composition of Clause 46 or 47 or the cell preparation of Clause 48, wherein the pharmaceutical composition or the cell preparation is not centrifuged before or after dilution with the diluent. Clause 51. The pharmaceutical composition of Clause 46 or 47 or the cell preparation of Clause 48, wherein the formulation or cell preparation is stored frozen prior to dilution with the diluent. Clause 52. The pharmaceutical composition or cell formulation of any one of clauses 48-51, wherein DMSO is present in a detectable amount at a level equal to or less than about 1% (v / v). Clause 53. The pharmaceutical composition or cell formulation of any one of Clauses 48-51, wherein DMSO is present in a detectable amount at a level equal to or less than about 0.037 mg / dose. Clause 54. A cell preparation comprising the preparation of any one of clauses 1-14 and a cell population, wherein the cell preparation is thawed by dilution with a diluent and is not washed. Clause 55. The cell preparation of clause 54, wherein the cell preparation is diluted with a diluent at a ratio selected from 1:2 to 1:20. Clause 56. The cell formulation of Clause 54 or 55, wherein DMSO is present in a detectable amount at a level equal to or less than about 0.037 mg / dose. Clause 57. The cell formulation of any one of clauses 54-56, wherein the diluent is GS2 or GS2Plus. Example Having now generally described the invention, the invention will be more readily understood by reference to the following examples, which are included merely to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention. Example 1 - Cryoprotectant formulations maintain post-thaw cell viability and cell growth

[0188] Human embryonic stem cell-derived RPE were pooled in a 225 mL bottle and washed with 200 mL 0.5% rHA / DPBS by centrifugation at 160 x g for 5 minutes. Washing was repeated a total of 3 times.

[0189] RPE cells were resuspended in 0.09% glucose / 2.5% rHA / DPBS and then mixed with a buffer containing DMSO, rHA, and glucose at a volume ratio of 1: 1. As shown in Table 1, nine (9) formulations containing different concentrations of DMSO, albumin, and glucose were prepared. Table 1

[0190] Thirty (30) μL of the prepared RPE cells were loaded into the Crystal (CZ) vials (West Pharmaceutical Services, Inc. Exton, PA) and used The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0191] Trypan blue cell counting was used to evaluate cell viability before freezing. No significant differences were observed among the nine preparations (see Figure 1 ). The pre-freezing survival rates of all preparations were higher than 95%.

[0192] Manual trypan blue cell counting and use NC-200 TM Post-thaw cell viability was assessed using an automated method using a ChemoMetec Inc. device. No significant differences were observed among the nine preparations using either method (see Figure 2 )(n=3). Post-thaw viability was higher than 90% for all preparations, ranging from 94% to 98%.

[0193] use Post-thaw cell growth was assessed using a cell proliferation assay (Thermo Fisher Scientific, Waltham, MA). Cryopreserved RPE vials were thawed, seeded, and cultured in growth medium in 96-well plates. Plates were frozen on days 2 and 4 to assess their cell growth index, which was defined by the concentration of DNA extracted from cells after cell lysis. No significant differences were observed among the nine preparations (see Figure 3 ). Example 2 - Cryoprotectants reduce the production of multinucleated cells

[0194] Human embryonic stem cell-derived RPE cells were pooled into a 50 mL tube and washed with 30 mL 0.5% rHA / DPBS by centrifugation at 160 x g for 5 minutes. The wash was repeated 3 times in total.

[0195] RPE cells were resuspended in 2.5% rHA / DPBS at a density of 50K cells / μl and then mixed with a buffer containing a cryoprotectant at a volume ratio of 1:1. As shown in Table 2, five (5) formulations containing 1% DMSO, 2.5% DMSO, 5% DMSO, 5% glycerol, or 5% ethylene glycol were prepared, and each of the 5 formulations contained 50 mM trehalose, 7.5% dextran 40, and 2.5% rHA in DPBS. Table 2

[0196] Ten (10) μL of the prepared RPE cells were added to the cryovial and the cells were frozen using Mr. Frosty TM The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0197] The cryopreserved RPE cells were thawed, inoculated and cultured in growth medium for 3 days, and the presence of multinucleated cells was evaluated under a microscope. The RPE cells in formulation A (1% DMSO) showed the presence of some multinucleated cells in the field of view of hundreds of RPE cells, which had more than 4 nuclei in the cells (see Figure 4A ), while formulation B (2.5% DMSO) showed one multinucleated cell with more than 4 nuclei in the field of view among hundreds of RPE cells.

[0198] RPE cells in formulation C (5% DMSO) and formulation E (5% ethylene glycol) did not show multinucleated cells with more than three nuclei in the cell in the field of view of hundreds of RPE cells, while RPE cells in formulation D (5% glycerol) did not show any multinucleated cells with more than four nuclei in the cell in the field of view of hundreds of RPE cells ( Figure 4B ).

[0199] These results suggest that cell-permeable cryoprotectants (such as DMSO, glycerol, and ethylene glycol) reduce the generation of multinucleated cells. Example 3 - Effects of other excipients on multinucleation, cell growth and viability

[0200] Human embryonic stem cell-derived RPE cells were pooled in a 50 mL tube and washed with 30 mL 0.5% rHA / DPBS by centrifugation at 160 x g for 5 minutes. The wash was repeated 3 times in total.

[0201] RPE cells were resuspended in 2.5% rHA / DPBS and then mixed with a cryoprotectant buffer at a volume ratio of 1:1. Eight (8) formulations containing different amounts of glycerol and dextran 40 were prepared, each containing 0.09% glucose and 2.5% rHA in DPBS (with or without Ca, Mg), as shown in Table 3. Table 3

[0202] Ten (10) μL of the prepared RPE cells were added to the cryovial and the cells were frozen using Mr. Frosty TM The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0203] Trypan blue cell counting was used to evaluate cell viability after thawing. Post-thaw cell growth was assessed using a cell proliferation assay (Thermo Fisher Scientific, Waltham, MA). Cryopreserved RPE vials were thawed, seeded, and cultured in growth medium in 96-well plates. Plates were frozen on days 2 and 3 to assess DNA concentration as an index of cell growth. No significant differences were observed among the eight preparations (see Table 1). Figure 5A ) (survival rate: n=1, cell growth: n=3). Multinucleated cells were observed under all 2.5% glycerol conditions, but not under 5% glycerol ( Figure 5B ).

[0204] RPE cells were pooled into a 50 mL tube and washed with 30 mL 0.5% rHA / DPBS by centrifugation at 160 x g for 5 minutes. Washing was repeated 3 times in total.

[0205] RPE cells were resuspended in 2.5% rHA / DPBS and then mixed with a buffer containing a cryoprotectant at a volume ratio of 1:1. As shown in Table 4, five (5) formulations were prepared. Table 4

[0206] Ten (10) μL of the prepared RPE cells were added to the cryovial and the cells were frozen using Mr. Frosty TM The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0207] Trypan blue cell counting was used to evaluate cell viability after thawing. Post-thaw cell growth was assessed using a cell proliferation assay (Thermo Fisher Scientific, Waltham, MA). Cryopreserved RPE vials were thawed, seeded, and cultured in growth medium in 96-well plates. Plates were frozen on days 2 and 3 to assess DNA concentration as an index of cell growth. No significant differences were observed among the five preparations (see Table 1). Fig. 6A ) (survival rate: n=1, cell growth: n=3). Multinucleated cells were observed using 2.5% glycerol and 2.5% propylene glycol (eg, 1,2-propylene glycol), but not using 5% DMSO or 5% glycerol (see Figure 6B ). Example 4 - Effect of albumin on adsorption and cryoprotection

[0208] RPE cells were pooled in 225 mL bottles and washed with 200 mL 0.5% rHA / DPBS by centrifugation at 500 x g for 5 minutes. Washing was repeated a total of 4 times.

[0209] RPE cells were resuspended in 2.5% rHA / 0.09% glucose / DPBS and then mixed with DMSO-containing buffer at a volume ratio of 1:1. As shown in Table 5, four (9) formulations containing different cell densities and albumin concentrations were prepared. Table 5

[0210] Thirty (30) μL of the prepared RPE cells were added to the Crystal (CZ) vials (West Pharmaceutical Services, Inc. Exton, PA) and cryopreserved using a controlled rate freezer under different cryopreservation conditions (e.g., different types of storage containers, different pre-freezing storage temperatures, and different pre-freezing durations) as shown in Table 6. The vials were then transferred and stored in the vapor phase of liquid nitrogen. Table 6

[0211] Post-thaw cell viability was assessed using the trypan blue method using a Vi-CELL BLU cell viability analyzer (Beckman Coulter, Inc., Indianapolis, IN). Formulations containing albumin exhibited higher post-thaw viability than formulations without albumin, especially when stored for up to 6 hours before freezing, as shown in Table 7. Table 7

[0212] Based on this result, albumin showed a stabilizing effect during the formulation process. Example 5 - Cellular Drug Product Formulation Exhibits No Toxicity

[0213] The following four (4) test vehicles or articles as well as GS2 and GS2 Plus were prepared as shown in Table 8 and subjected to non-GLP acute toxicity studies. Table 8

[0214] Each of the six formulations was placed in a plastic vial and stored at 2 to 8°C or -10 to -30°C. The frozen formulations were thawed and diluted prior to administration and administered as a single unilateral dose by subretinal injection into the NIH-Lyst bg-J Foxn1 nu Btk xid (NIH III) immunocompromised mice. Animals were evaluated after 1 and 14 day observation periods. Toxicity assessment was based on clinical observations, body weight and food consumption measurements, ophthalmological examinations, and clinical and anatomical pathology evaluations.

[0215] No toxicity was observed in any formulation. Example 6 - Applicability to different cell types

[0216] Photoreceptor rescue cells (PRCs) were pooled into 50 mL tubes and prepared using OptiPrep TM(STEMCELL Technologies, Inc., Vancouver, Canada) to remove dead cells and debris from the cell suspension, followed by washing with growth medium at 300 x g for 10 min and then with 0.5% rHA / DPBS at 300 x g for 10 min.

[0217] Photoreceptor rescue cells were resuspended in 0.5% rHA / DPBS and then mixed with a cryoprotectant buffer at a volume ratio of 1:2. PRC cells were prepared in formulations containing 2.5% rHA, DPBS containing Ca / Mg, 0.6% glucose, and different concentrations of DMSO (10% DMSO and 5% DMSO). For comparison, PRC cells were also formulated in commercially available CS10 cell freezing medium (STEMCELL Technologies, Inc., Vancouver, Canada) containing 10% DMSO.

[0218] Add cells to Crystal Biosystems at 70,000 cells / μl in 50 μL (3.5 million cells total per vial). (CZ) vials (West Pharmaceutical Services, Inc. Exton, PA) and cryopreserved at -1°C / min using a controlled rate freezer. The vials were then transferred and stored in the vapor phase of liquid nitrogen. By using 70,000 cells / μl in 50 μL, the following target doses can be achieved: 1,000,000 cells in 150 μl when cells are diluted 1:4 in a buffer (e.g., GS2 Plus buffer) (accounting for approximately 50% cell loss due to cell death and recovery loss); 300,000 cells in 150 μl when cells are diluted 1:17 in GS2 Plus buffer (accounting for approximately 50% cell loss due to cell death and recovery loss).

[0219] The cell viability after thawing was evaluated using the trypan blue cell counting method. The formulation of the present invention containing 5% DMSO, 2.5% rHA, DPBS containing Ca / Mg and 0.6% glucose showed similar viability to the formulation of the present invention containing 10% DMSO, 2.5% rHA, DPBS containing Ca / Mg and 0.6% glucose (see Fig. 7A )(n=3). Both preparations of the present invention are better than the commercially available CS10 showed a higher survival rate. The survival rate of PRC after freeze-thaw was also determined using the formulations of the present invention containing 5% DMSO, 7.5% DMSO and 10% DMSO (see Figure 7B ). Example 7 - Applicability to different cell types (2)

[0220] Human corneal endothelial cells (CECs) derived from human induced pluripotent stem (iPS) cells were centrifuged to remove media components and any dissociation enzymes, and then washed with 0.5% rHA / DPBS by centrifugation at 200 x g for 5 minutes.

[0221] The CEC cell pellet was then resuspended with a formulation containing 2.5% rHA, 0.09% glucose, DPBS with Ca and Mg, and either 5% DMSO or 10% DMSO. The target cell density was 30,000 cells / μL.

[0222] Fifty (50) μL of the prepared CEC cells were added to the cryovial and The vials were stored frozen in 1% CO2-freezing containers (Corning Life Sciences, Tewksbury, MA) at a freezing rate of approximately -1°C / min. After storage at -80°C overnight, the vials were transferred and stored in the vapor phase of liquid nitrogen.

[0223] The cell viability after thawing was evaluated using the trypan blue cell counting method. The vials were thawed in a 37°C water bath and diluted with DPBS and then mixed with the trypan blue reagent. For the freezing buffer containing 5% and 10% DMSO, the cell viability was 89.3% and 88.2%, respectively.

[0224] For comparison, CEC cells were also cryopreserved at a lower cell density (3 million cells / mL) and a standard volume (1 mL / vial) in 2.5% rHA, 0.09% glucose, DPBS with calcium and magnesium, and 5% DMSO. Under these conditions, post-thaw cell viability was 86.2% when measured by trypan blue cell counting (see Figure 8 ). Example 8 - Additional Candidates for Cellular Drug Product Formulation

[0225] Human embryonic stem cell-derived RPE cells were pooled in a 50 mL tube and washed with 30 mL 0.5% rHA / DPBS by centrifugation at 160 x g for 5 minutes. The wash was repeated 3 times in total.

[0226] RPE cells were resuspended in 2.5% rHA / 0.09% glucose / DPBS and then mixed with a buffer containing a cryoprotectant at a volume ratio of 1: 1. As shown in Table 9, preparations containing 5% glycerol, 200 mM taurine or 100 mM trehalose, 0.09% glucose and 2.5% rHA in DPBS were prepared (cryopreservation preparation #5 and cryopreservation preparation #6). Table 9

[0227] Ten (10) μL of the prepared RPE cells were added to the cryovial and The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0228] Trypan blue cell counting was used to evaluate cell viability after thawing. Cell proliferation assay (Thermo Fisher Scientific, Waltham, MA) evaluates the cell growth after thawing. Cryopreserved RPE bottles are thawed, inoculated and cultivated in the growth medium in 96 well plates. Plates were frozen at the 2nd and 3rd days to evaluate DNA concentration as cell growth index. As shown in Table 10, no significant differences were observed between cryopreserved preparation #1, cryopreserved preparation #5 and cryopreserved preparation #6, and multinucleated cells with more than four nuclei were not observed in cells in the hundreds of RPE cells using cryopreserved preparation #1, cryopreserved preparation #5 and cryopreserved preparation #6. Table 10

[0229] Cryopreservation preparation #2 does not contain any cell penetrating cryoprotectant, therefore may potentially cause toxicity to cell, and due to dextran 40, also shows high viscosity.In addition, multinucleated cells are observed in the RPE cells cryopreserved in cryopreservation preparation #2.Although cryopreservation preparation #3 and cryopreservation preparation #4 demonstrate less multinucleated cells, they also demonstrate high viscosity because of the dextran 40 in the preparation.Cryopreservation preparation #5 and cryopreservation preparation #6 lack multinucleated cells, and show low viscosity owing to lacking polymer excipient. EQUIVALENTS AND SCOPE, INCORPORATED BY REFERENCE

[0230] 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 is instead set forth by the claims appended hereto.

[0231] Unless the context clearly dictates otherwise, articles such as "a," "an," and "the" include singular and plural referents. Thus, for example, reference to "an agent" includes a single agent and a plurality of such agents.

[0232] For claims or descriptions that include an "or" between two or more members of a group, if one, more than one, or all of the group members are present, then this is deemed satisfied unless otherwise stated or obvious from the context. Disclosures of groups that include an "or" between two or more 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 of the group members are present. For the sake of brevity, these embodiments are not individually described in detail herein, but it is understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0233] It should be understood that the present invention covers 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 basic claim. In addition, when a composition is mentioned in the claims, it should be understood that any method of making or using the composition disclosed herein or according to methods known in the art (if any) is included, unless otherwise stated or unless a contradiction or inconsistency is obvious to a person of ordinary skill in the art.

[0234] When the elements are presented in a list form, for example, in a Markush group format, it should be understood that each possible subgroup of the elements is also disclosed, and any element or subgroup of the elements can be removed from the group. It should also be noted that the term "comprising" is intended to be open, allowing the inclusion of additional elements or steps. It should be understood that, in general, when an embodiment, product or method is referred to as including a specific element, feature or step, an embodiment, product or method consisting of or substantially consisting of such elements, features or steps is also provided. For the sake of brevity, these embodiments are not described in detail separately herein, but it should be understood that each of these embodiments is provided herein, and protection can be specifically claimed or abandoned.

[0235] When a range is given, endpoints are also included. In addition, it should be understood that, unless otherwise stated or apparent from the context and / or understanding of a person of ordinary skill in the art, the value represented by a range can take any specific value within the range in some embodiments, up to one-tenth of the unit of the lower limit of the range, unless the context clearly specifies otherwise. For the sake of brevity, the values ​​within each range are not described in detail herein, but it should be understood that each of these values ​​is provided herein and can be specifically stated or abandoned. It should also be understood that, unless otherwise stated or apparent from the context and / or understanding of a person of ordinary skill in the art, the value represented by a range can take any sub-range within a given range, wherein the endpoint of the sub-range is represented with the same precision as one-tenth of the unit of the lower limit of the range.

[0236] In addition, it should be understood that any specific embodiment of the present invention may be expressly excluded from any one or more claims. When a range is given, any value within the range may be expressly 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 sake of brevity, all embodiments in which one or more elements, features, objects or aspects are excluded are not expressly set forth herein.

[0237] All publications, patents, patent applications, publications, and database entries (e.g., sequence database entries) mentioned herein, for example, in the Background, Summary, Detailed Description, Examples, and / or References sections, are hereby incorporated by reference in their entirety, just as if each individual publication, patent, patent application, publication, and database entry were specifically and individually incorporated herein by reference. In the event of a conflict, the present application (including any definitions herein) will control.

Claims

1. A formulation comprising about 4-10% (v / v) cryoprotectant, about 2-8% (w / v) albumin, about 0-1.5% (w / v) glucose and a buffer.

2. The formulation of claim 1, wherein the cryoprotectant is selected from the group consisting of DMSO, glycerol and ethylene glycol.

3. The formulation of claim 1, wherein the cryoprotectant is DMSO.

4. The formulation of any one of the preceding claims, wherein the formulation comprises about 4-6% (v / v) cryoprotectant.

5. The formulation of any one of the preceding claims, wherein the formulation comprises about 0.08-0.10% (w / v) glucose.

6. The formulation of any one of the preceding claims, wherein the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and buffer.

7. The formulation of any one of claims 1-4, wherein the formulation comprises about 0.6% (w / v) glucose.

8. The formulation of any one of claims 1-4 and 7, wherein the formulation comprises about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and a buffer.

9. The formulation of any one of the preceding claims, wherein the albumin is human albumin.

10. The formulation of any one of the preceding claims, wherein the albumin is recombinant human albumin.

11. The formulation of any one of the preceding claims, wherein the buffer is buffered saline.

12. The formulation of any one of the preceding claims, wherein the buffer is phosphate buffered saline (PBS).

13. The formulation of claim 11 or 12, wherein the buffered saline comprises Ca2+ and Mg2+.

14. The formulation of claim 11 or 12, wherein the buffered saline does not contain Ca2+ and Mg2+.

15. The preparation of any one of the preceding claims, further comprising a cell population or tissue.

16. The preparation of claim 15, wherein the cell population is a dissociated cell population.

17. The preparation of claim 15, wherein the cell population comprises a cell monolayer, a cell sheet, a cell cluster, a cell sphere, or a cell aggregate.

18. The preparation of claim 15, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells.

19. The formulation of any one of the preceding claims, wherein the formulation is stored frozen.

20. The formulation of any one of the preceding claims, wherein the formulation does not comprise a polymeric excipient.

21. The formulation of any one of the preceding claims, wherein the formulation does not comprise dextran.

22. A cell preparation comprising (a) a preparation according to any one of claims 1 to 14 and (b) Cell population.

23. The cell preparation of claim 22, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells.

24. The cell preparation of claim 22 or 23, wherein the cell population concentration in the cell preparation is about 10,000 cells to 100,000 cells / μL.

25. The cell preparation of claim 22 or 23, wherein the cell population concentration in the cell preparation is about 15,000-50,000 cells / μL.

26. The cell formulation of any one of claims 22-25 in a cryopreservation vial, syringe, ampoule, bottle or freezer packaging.

27. The cell formulation of claim 26, wherein about 10 to about 500 μL of the formulation is in a vial, syringe, ampoule, bottle, or freezer packaging.

28. The cell formulation of claim 26 or 27, wherein about 10 5 Up to 10 7 cells.

29. The cell formulation of claim 24 or 25, wherein about 10 5 Up to 5x10 6 cells.

30. The cell formulation of claim 24 or 25, wherein about 10 5 Up to 2x10 6 cells.

31. The cell preparation of any one of claims 22-30, wherein the cell preparation is cryopreserved.

32. A method of preparing a cell formulation comprising contacting a cell population with the formulation of any one of claims 1-14.

33. The method of claim 32, further comprising cryopreserving the cell preparation.

34. The method of claim 32 or 33, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells.

35. A method of treating a subject having a disorder or condition comprising thawing the cell preparation of claim 31, diluting the cell preparation with a diluent at a ratio selected from 1:2, 1:3, 1:4 and 1:5, and The diluted cell preparation is administered to the subject.

36. The method of claim 35, wherein the disorder or condition is selected from retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration, or retinal atrophy, or is associated with a variety of vision-altering diseases that lead to photoreceptor damage and blindness, such as choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (yellow spots on the eye).

37. The method of claim 35, wherein the diluent is GS2, GS2 Plus, BSS, BSS Dextran and HSA solution, PBS, DMEM, MEM, or albumin.

38. A method of treating a subject suffering from a disorder or condition comprising thawing the cell preparation of claim 31, diluting with a diluent selected from the group consisting of 1:15, 1:16, diluting the cell preparation at ratios of 1:17, 1:18 and 1:19, and The diluted cell preparation is administered to the subject.

39. The method of claim 38, wherein the disorder or condition is selected from retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration, or retinal atrophy, or is associated with a variety of vision-altering diseases that lead to photoreceptor damage and blindness, such as choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (yellow spots on the eye).

40. The method of claim 38, wherein the diluent is GS2 or GS2 Plus, BSS, BSS Dextran and HSA solution, PBS, DMEM, MEM, or albumin.

41. A kit comprising (a) the cryopreserved cell preparation of claim 31, (b) a diluent, and (c) instructions for diluting the cell preparation prior to administration to a subject.

42. The kit of claim 41, wherein the instructions further comprise instructions for thawing the cryopreserved cell preparation.

43. A pharmaceutical composition comprising a combination of the formulation of any one of claims 1-14 and a cell population.

44. The pharmaceutical composition of claim 43, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells.

45. The pharmaceutical composition of claim 43, wherein the cell population comprises neural cells, neural stem cells, or neural precursor cells.

46. ​​A pharmaceutical composition comprising the formulation of any one of claims 15-21 and a diluent, wherein the ratio of formulation to diluent is in the range of 1:2 to 1:20, wherein the pharmaceutical composition is suitable for administration to a subject, optionally to the eye, further optionally to the subretina.

47. A pharmaceutical composition comprising the cell preparation of any one of claims 22-25 and 31 and a diluent, wherein the ratio of the cell preparation to the diluent is in the range of 1:2 to 1:20, wherein the pharmaceutical composition is suitable for administration to a subject, optionally to the eye, further optionally to the subretina.

48. A cell preparation comprising the formulation of any one of claims 1-14, 19, 20 and 21 and a diluent, wherein the ratio of formulation to diluent is in the range of 1:2 to 1:20, wherein the formulation comprises a population of cells, wherein the cell preparation is suitable for administration to a subject, optionally to the eye, further optionally to the subretina.

49. The pharmaceutical composition of claim 46 or 47 or the cell preparation of claim 48, wherein the pharmaceutical composition or cell preparation is not washed before or after dilution with a diluent.

50. The pharmaceutical composition of claim 46 or 47 or the cell preparation of claim 48, wherein the pharmaceutical composition or cell preparation is not centrifuged before or after dilution with the diluent.

51. The pharmaceutical composition of claim 46 or 47 or the cell preparation of claim 48, wherein the pharmaceutical composition or the cell preparation is stored frozen prior to dilution with a diluent.

52. The pharmaceutical composition or cell preparation of any one of claims 48-51, wherein DMSO is present in a detectable amount at a level equal to or less than about 1% (v / v).

53. The pharmaceutical composition or cell formulation of any one of claims 48-51, wherein DMSO is present in a detectable amount at a level equal to or less than about 0.037 mg / dose.

54. A cell preparation comprising the preparation of any one of claims 1-14, 19, 20 and 21 and a cell population, wherein the cell preparation is thawed by dilution with a diluent and is not washed.

55. The cell preparation of claim 54, wherein the cell preparation is diluted with a diluent at a ratio selected from 1:2 to 1:

20.

56. The cell preparation of claim 54 or 55, wherein DMSO is present in a detectable amount at a level equal to or less than about 0.037 mg / dose.

57. The cell formulation of any one of claims 54-56, wherein the diluent is GS2 or GS2 Plus.

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