Cryopreserved cell preparations and methods of use

Abstract

Some aspects of the present disclosure provide improved cryopreservation formulations and methods for cryopreserving and thawing cells. These formulations overcome the need for washing cells after thawing, thus facilitating high-density, low-volume cell cryopreservation strategies, resulting in increased cell recovery and viability. Pharmaceutical compositions comprising the cell populations and cryopreservation formulations provided herein are also provided. Methods for treating subjects with diseases or conditions that would benefit from the transplantation of cryopreserved and thawed cell populations are also provided.

Description

[Technical Field]

[0001] Related Applications

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

[0002]

[0002] Effective cell cryopreservation is a critical aspect of many cell therapy strategies. The ability to freeze cell-based therapeutics, store them for various periods of time, and optionally transport them in a frozen state expands their clinical use. Improper cryopreservation can result in cellular damage, including alterations in cellular characteristics, function, metabolic activity, and cell death. Summary of the Invention

[0003]

[0003] The ability to cryopreserve cell-based therapeutics separates the production and clinical use of the cell therapy in time and space. The design and selection of an appropriate cryoprotective formulation is important to ensure the maintenance of cell viability and function. The present disclosure presents novel cryoprotective formulations that can be used to cryopreserve and, optionally, transport cell populations, including those used as therapeutics. Importantly, the present disclosure contemplates using such formulations to cryopreserve cell populations at high densities and low volumes, with subsequent thawing achieved by the addition of a diluent, followed by administration to a subject without the need for post-thaw cell washing. In this manner, the method allows for the freezing and thawing of cell-based therapeutics with minimal processing and manipulation. This reduces the potential for cell loss (e.g., cell death) that can result from manipulation of the cells in the post-thaw period.

[0004]

[0004] Accordingly, the present disclosure provides cryoprotective formulations and methods of use thereof. The cryoprotective formulations may be used with a variety of cell types. The cryoprotective formulations provided herein result in increased cell viability and proliferation after thawing compared to prior art methods for freezing cells and commercially available cryoprotective formulations. While ophthalmic applications are exemplified herein, other applications are also contemplated and encompassed by the present disclosure.

[0005]

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

[0006]

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

[0007]

[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]

[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]

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

[0012]

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

[0013]

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

[0014]

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

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

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

[0017]

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

[0018]

[0018] In some embodiments, the buffered saline solution is free of Ca2+ and Mg2+.

[0019]

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

[0020]

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

[0021]

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

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

[0023]

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

[0024]

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

[0025]

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

[0026]

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

[0027]

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

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

[0029]

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

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

[0031] In some embodiments, the cell population is at a concentration of about 15,000 to 150,000 cells / μL in the cell preparation.

[0032] In some embodiments, the cell population is at a concentration of about 15,000 to 50,000 cells / μL in the cell preparation.

[0033]

[0033] In some embodiments, the cell preparations described herein are in vials, syringes, ampoules, bottles, or refrigerated packages for cryopreservation.

[0034] In some embodiments, about 10 to about 500 μL of the preparation is in a vial, syringe, ampoule, bottle, or refrigerated package.

[0035] In some embodiments, about 10 cells 5 ~about 10 7 In some embodiments, the cells are present in a vial, syringe, ampoule, bottle, or refrigerated package. 5 pieces~cells approx. 5×10 5 In some embodiments, about 4.5 x 10 cells are present in the vial. 5 In some embodiments, about 1 x 10 cells are present in the vial. 6 pieces~cells approx. 2×10 6 In some embodiments, about 1.5 x 10 cells are present in the vial. 6 In these embodiments, the cells are RPE cells. Similar numbers of cells may be present in a syringe, ampoule, bottle, or refrigerated package.

[0036] In some embodiments, about 0.5 x 10 cells 6 pieces~cells approx. 1.5×106 In some embodiments, about 1 x 10 cells are present in the vial. 6 In some embodiments, about 5 x 10 cells are present in the vial. 6 pieces~cells approx. 7×10 6 In some embodiments, about 6 x 10 cells are present in the vial. 6 In these embodiments, the cells are PRC cells. Similar numbers of cells may be present in a syringe, ampoule, bottle, or refrigerated package.

[0037] In some embodiments, about 10 cells 5 ~5×10 6 The individual may be in a vial, syringe, ampoule, bottle, or refrigerated package.

[0038] In some embodiments, about 10 cells 5 ~2×10 6 The individual may be in a vial, syringe, ampoule, bottle, or refrigerated package.

[0039]

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

[0040]

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

[0041]

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

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

[0043]

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

[0044]

[0044] Some aspects of the present disclosure provide a method of treating a subject having a disorder or condition, comprising thawing a cell preparation described herein and diluting the cell preparation with a diluent at a ratio ranging from 1:2 to 1:20, including ratios selected from the group consisting of 1:2, 1:3, 1:4, and 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, and administering the diluted cell preparation to the subject.

[0045]

[0045] In some embodiments, the disorder or condition is selected from the group consisting of retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration, or retinal atrophy, or is associated with several vision-altering diseases that result in photoreceptor damage and blindness, such as, for example, congenital choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (fundus flava).

[0046]

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

[0047]

[0047] Some aspects of the present disclosure provide a method 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 ratios selected from the group consisting of 1:15, 1:16, 1:17, 1:18, and 1:19, and administering the diluted cell preparation to the subject.

[0048]

[0048] In some embodiments, the disorder or condition is selected from the group consisting of retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration, or retinal atrophy, or is associated with several vision-altering diseases that result in photoreceptor damage and blindness, such as, for example, congenital choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (fundus flava).

[0049]

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

[0050]

[0050] Some aspects of the present disclosure provide a kit 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]

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

[0052]

[0052] Some aspects of the present disclosure provide the formulations described herein in combination with a cell population.

[0053]

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

[0054]

[0054] In some embodiments, the cell population comprises neural cells, neural stem cells, or neural progenitor cells.

[0055]

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

[0056]

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

[0057]

[0057] In some embodiments, the pharmaceutical compositions are suitable for administration to the eye.

[0058]

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

[0059]

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

[0060]

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

[0061]

[0061] In some embodiments, the pharmaceutical compositions are suitable for administration to the eye.

[0062]

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

[0063]

[0063] Some aspects of the present disclosure include the formulations described herein, which formulations comprise a cell population and present a cell preparation suitable for administration to a subject.

[0064]

[0064] Some aspects of the present disclosure include the formulations and diluents described herein, 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, 1:3, 1:4, 1:5, or 1:10 to 1:15, including 1:10, 1:11, 1:12, 1:13, 1:14, and 1:15, and wherein the formulation comprises a cell population and provides a cell preparation suitable for administration to a subject.

[0065]

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

[0066]

[0066] In some embodiments, the cell preparation is suitable for subretinal administration.

[0067]

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

[0068]

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

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

[0070]

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

[0071]

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

[0072]

[0072] In some embodiments, DMSO is present in detectable amounts at a level of about 0.037 mg or less per dose.

[0073]

[0073] Some aspects of the present disclosure provide cell preparations that include the formulations and cell populations described herein and that are thawed via dilution with a diluent and are not washed.

[0074]

[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, 1:3, 1:4, 1:5, including 1:2 to 1:5, including 1:2 to 1:20, or 1:10 to 1:15, including 1:10, 1:11, 1:12, 1:13, 1:14, and 1:15.

[0075]

[0075] In some embodiments, DMSO is present in detectable amounts at a level of about 0.037 mg or less per dose.

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

[0077]

[0077] The above summary is intended to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the detailed description, brief description of the drawings, examples, and claims. [Brief explanation of the drawings]

[0078] [Figure 1]

[0078] Figure 1 shows the 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 (part 2) refer to different preparations of the same formulation. [Figure 2]

[0079] Figure 1 shows post-thaw cell viability of RPE cells prepared in formulations containing different concentrations of DMSO, albumin, and glucose, as shown in Table 1. The first bar of each doublet represents manual (Trypan Blue) viability measurements, and the second bar of each doublet represents automated viability measurements using the NC-200 device. [Figure 3]

[0080] Figure 1 shows post-thaw cell proliferation of RPE cells prepared in formulations containing different concentrations of DMSO, albumin, and glucose, as assessed using the CyQUANT® cell proliferation assay, as shown in Table 1. The first bar of each doublet represents measurements performed on day 2 post-thaw, and the second bar of each doublet represents measurements performed on day 4 post-thaw. [Figure 4]

[0081] Figure 4A is a microscopic image of cryopreserved RPE cells containing multinucleated cells after thawing, plating, and culturing in growth medium for three days. Arrows indicate multinucleated cells. Figure 4B is a graph showing quantitative analysis of multinucleated cells present in post-thaw cultures in Formulations A-E. The first bar in each grouping 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. [Figure 5]

[0082] Figure 5A shows post-thaw cell viability (circles above the bars) and post-thaw cell proliferation, as assessed by DNA concentration, at 2 days (first bar of each doublet) and 3 days (second bar of each doublet) after thawing for RPE cells prepared in formulations 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, labeled "PBS-" and "PBS+," respectively), as shown in Table 3. Figure 5B is a graph showing quantitative analysis of multinucleated cells present in post-thaw cultures in the formulations. The first bar in each grouping 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. [Figure 6]

[0083] Figure 6A shows post-thaw cell viability (circles above the bars) and post-thaw cell proliferation, as assessed by DNA concentration, on days 2 (first bar of each doublet) and 3 (second bar of each doublet) of RPE cells prepared in PBS (+Ca, +Mg) containing 5% DMSO (D5), 5% glycerol + 7.5% dextran 40 (G5), 7.5% dextran 40 (D0), 2.5% glycerol + 7.5% dextran 40 (G2.5), or 2.5% propanediol (P2.5) + 7.5% dextran 40 (P2.5), each formulation also containing 0.09% glucose and 2.5% rHA, as shown in Table 4. Figure 6B is a graph showing quantitative analysis of multinucleated cells present in post-thaw cultures in the formulations. The first bar in each grouping 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. [Figure 7A]

[0084] FIG. 1 shows post-thaw cell viability of photoreceptor rescue cells (PRC) cells prepared in a formulation containing 2.5% rHA, DPBS with Ca and Mg, 0.6% glucose, and different concentrations of DMSO (5% DMSO or 10% DMSO), and for comparison, PRC cells formulated in the commercially available CryoStor® CS10 cell freezing formulation containing 10% DMSO. [Figure 7B] FIG. 1 shows post-thaw cell viability of photoreceptor rescue cell (PRC) cells prepared in a formulation containing 2.5% rHA, DPBS with Ca and Mg, 0.6% glucose, and different concentrations of DMSO (5% DMSO, 7.5% DMSO, or 10% DMSO). [Figure 8]

[0085] FIG. 10 shows post-thaw cell viability of corneal endothelial cells (CEC) cells prepared in a formulation containing 2.5% rHA, 0.09% glucose, DPBS with Ca and Mg, and different concentrations of DMSO (5% or 10%) at a density of 30,000 cells / μL and in a volume of 50 μL, compared with the post-thaw cell viability of CEC cells cryopreserved in 2.5% rHA, 0.09% glucose, DPBS with Ca and Mg, and DMSO at a concentration of 5% at a low cell density (3 million cells per mL) and in a standard volume (1 mL / vial). DETAILED DESCRIPTION OF THE INVENTION

[0079]

[0086] For example, in the context of regenerative medicine, cell-based therapies, including those involving cell transplantation, often require formulating, storing, transporting, and / or injecting delicate or fragile cells that may be damaged or lose their ability to repopulate if subjected to improper handling, extensive manipulation and / or processing steps, or exposure to non-physiological conditions.

[0080]

[0087] The present disclosure provides formulations and methods for cryopreserving cells, storing cells, transporting cells, and ultimately administering cells to a subject with minimal cell processing and manipulation and increased post-thaw viability. The formulations provided herein have several advantages over currently available formulations. For example, in contrast to currently available cryopreserved formulations, the formulations provided herein require minimal post-thaw processing, exhibit few, if any, adverse effects, such as toxicity, and can be administered to a subject with a therapeutic agent. The formulations provided herein allow for high-density cryopreservation in a low volume, which offers several advantages, including a thawing regimen requiring only a single step, the addition of a diluent. Because there is no need to wash out the formulation after thawing, there is less chance of cell loss and cell damage, thereby increasing the therapeutic efficacy of cryopreserved cell preparations. Currently available cryoprotectants and cryoprotective formulations require extensive cell processing procedures that are costly, time-consuming, and prone to human error, and ultimately can reduce the cell viability, reseeding efficiency, and repopulation ability of the cells contained therein.It is also worth noting that cell populations cryopreserved using the formulations provided herein have been observed to have increased viability compared to prior art cryopreservation formulations and cryopreservation methods.Using the formulations provided herein, improved cell growth after thawing has also been observed.The formulations provided herein can result in improved functionality, survival, and stability of cell populations.

[0081]

[0088] In addition to the above-mentioned advantages, the cryopreservation preparation of the present disclosure is also surprisingly and unexpectedly associated with the frequency (or occurrence rate) of multinucleated cells in culture, particularly in RPE cell culture.When using other preparations, multinucleated cells such as multinucleated RPE cells are observed.But surprisingly, the cryopreservation preparation presented herein is thought to reduce the frequency of such cells.

[0082]

[0089] The present disclosure provides novel cryoprotective formulations comprising, at a minimum, a cryoprotectant and albumin. In certain embodiments, the cryoprotective formulation comprises, at a minimum, a cryoprotectant, albumin, and, optionally, a 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 further comprise a buffer or buffered saline solution, such as, but not limited to, phosphate-buffered saline (PBS). The formulation may have a pH that is approximately physiological pH (e.g., a pH in the range of 6-8). The cryoprotectant may be present in a range of about 1% to about 10% (v / v) volume / volume. The cryoprotectant may be present in a range of about 2% to about 10% (v / v) volume / volume. The cryoprotectant may be present in a range of about 3% to about 10% (v / v) volume / volume. The cryoprotectant may be present in a range of about 4% to about 10% (v / v) volume / volume, e.g., 4%, 5%, 6%, 7%, 8%, 9%, or 10% volume / volume. 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 the range of about 4% to about 8% (w / v). Albumin may be present in the range of about 4% to about 8.5% (w / v). Albumin may be present in the range of about 4% to about 9% (w / v). Albumin may be present in the range of about 4% to about 10% (w / v). Sugar may be present in the range of about 0 to about 1.5% (w / v).

[0083]

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

[0084]

[0091] In some embodiments, the cryopreserved 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 solution. In some embodiments, the cryopreserved 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 solution. In some embodiments, the cryopreserved 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 solution. The albumin can be human albumin, including recombinant human albumin. The buffered saline solution can be phosphate-buffered saline (PBS). Table 1 and Figure 1 illustrate that RPE cells placed in a formulation containing 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 PBS with calcium and magnesium had a viability of greater than 96%. When cells were frozen in these formulations and then thawed, their viability ranged from 94 to 98%. Post-thaw cell growth was also relatively uniform across these formulations.

[0085]

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

[0086]

[0093] In some embodiments, the cryopreserved 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 solution. In some embodiments, the cryopreserved 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 solution. In some embodiments, the cryopreserved 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 solution. The albumin can be human albumin, including recombinant human albumin. The buffered saline solution can be phosphate-buffered saline (PBS). Table 1 and Figure 1 illustrate that RPE cells placed in a formulation containing 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 PBS with calcium and magnesium had a viability of greater than 96%. When cells were frozen in these formulations and then thawed, their viability ranged from 94 to 98%. Post-thaw cell growth was also relatively uniform across these formulations.

[0087]

[0094] A formulation containing about 2.5% (v / v) DMSO, about 50 mM trehalose, about 7.5% (w / v) dextran 40, about 2.5% (w / v) albumin, and buffered saline was associated with a reduction in the occurrence of multinucleated RPE cells with more than four nuclei per cell after freezing and thawing. DMSO at a level of 2.5% v / v was more effective than DMSO at a level of 1% v / v in reducing the occurrence of multinucleated RPE cells with more than four nuclei per cell after freezing and thawing. Formulations containing even higher levels of DMSO (e.g., 5%), optionally also containing ethylene glycol (e.g., 5%), were associated with a reduction in the occurrence of multinucleated cells with more than three nuclei per cell. Formulations containing 5% glycerol did not result in cells with more than four nuclei per cell.

[0088]

[0095] 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 multinucleation of cells of interest, 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 stem or progenitor cells, glial stem or progenitor cells, pancreatic stem or progenitor cells, beta cells, keratinocytes, chondrocytes, osteoblasts, osteoclasts, or cells within a population or tissue, e.g., in a monolayer or sheet, or in aggregates or clusters, of RPE cells, corneal endothelial cells, photoreceptor rescue cells, pancreatic islets, or skin grafts.

[0089]

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

[0090]

[0097] Still other formulations include about 5% DMSO (v / v), about 2.5% albumin (w / v), about 0.09% glucose (w / v), and a buffer or buffered saline solution. Another formulation includes about 2.5% albumin (w / v), about 0.09% glucose (w / v), about 5% (v / v) glycerin, about 200 mM taurine, and a buffer or buffered saline solution. Another formulation includes 2.5% albumin (w / v), about 0.09% glucose (w / v), about 5% (v / v) glycerin, about 100 mM trehalose, and a buffer or buffered saline solution.

[0091]

[0098] Still other formulations include about 5% DMSO (v / v), about 2.5% albumin (w / v), about 0.6% glucose (w / v), and a buffer or buffered saline solution. Another formulation includes about 2.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerin, about 200 mM taurine, and a buffer or buffered saline solution. Another formulation includes 2.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerin, about 100 mM trehalose, and a buffer or buffered saline solution.

[0092]

[0099] Still other formulations include about 5% DMSO (v / v), about 7.5% albumin (w / v), about 0.6% glucose (w / v), and a buffer or buffered saline solution. Another formulation includes about 7.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerin, about 200 mM taurine, and a buffer or buffered saline solution. Another formulation includes 7.5% albumin (w / v), about 0.6% glucose (w / v), about 5% (v / v) glycerin, about 100 mM trehalose, and a buffer or buffered saline solution.

[0093]

[0100] The present disclosure further contemplates that in any of the embodiments presented herein, "glycerol" may also encompass "glycerin," and conversely, in any of the embodiments presented herein, "glycerin" may also encompass "glycerol."

[0094]

[0101] In some embodiments, the cryopreserved cell preparation may include cells of interest (e.g., RPE cells, photoreceptor rescue cells, photoreceptor cells, corneal endothelial cells, corneal endothelial progenitor cells, neuronal cells or neural stem or progenitor cells, or other cell type 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 phosphate monobasic, 2.10 μg magnesium chloride 6H2O, 168 μg sodium chloride, and 24.1 μg sodium phosphate dibasic. In some embodiments, the cryopreserved cell preparation may include cells of interest (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.

[0095]

[0102] In some embodiments, upon thawing and dilution in the diluents provided herein, the final cell preparation administered to a subject may include cells of interest (e.g., RPE cells), sodium hyaluronate, sodium chloride, potassium chloride, dibasic sodium phosphate dodecahydrate, dextrose (anhydrous), calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, and sodium citrate dihydrate.

[0096]

[0103] Cells can be cryopreserved at higher concentrations (or densities) compared to standard prior art techniques. For example, cells can be cryopreserved at approximately 2 x 10 cells per μL. 4 Approximately 3 x 10 cells per μL 4Approximately 4 x 10 cells per μL 4 Approximately 5 x 10 cells per μL 4 Approximately 6 x 10 cells per μL 4 Approximately 7 x 10 cells per μL 4 Approximately 8 x 10 cells per μL 4 Approximately 9 x 10 cells per μL 4 or approximately 10 cells per μL 5 Concentrations (or densities) of approximately 10 cells per μL, or greater 3 Approximately 2 x 10 cells per μL 3 Approximately 3 x 10 cells per μL 3 10 cells per μL 4 Containing a concentration (or density) of approximately 3 x 10 cells per µL, or greater 2 In some embodiments, the cell density is about 3 x 10 cells per μL. 2 In some embodiments, the cell density is about 3.3 x 10 cells per μL. 3 In some embodiments, the cell density is about 5 x 10 cells per μL. 4 There are individuals.

[0097]

[0104] In some embodiments, the cell density is about 3 x 10 cells per μL. 3 Approximately 5 x 10 cells per 1 µL 4 Contains approximately 10 cells per μL 3 Approximately 6 x 10 cells per 1 µL 4 In some embodiments, the cell density is in the range of about 2 x 10 cells per μL. 4 Approximately 5 x 10 cells per 1 µL 4 and approximately 3 x 10 cells per µL 4 Approximately 5 x 10 cells per 1 µL 4 Contains 1.5 x 10 cells per µL 4 Approximately 5 x 10 cells per 1 µL 4 Contains approximately 10 cells per μL 4 Approximately 6 x 10 cells per 1 µL 4In some embodiments, the cell density is in the range of about 3 x 10 cells per μL. 2 In some embodiments, the cell density is about 3.3 x 10 cells per μL. 3 In some embodiments, the cell density is about 5 x 10 cells per μL. 4 There are individuals.

[0098]

[0105] In some embodiments, the number of cells per vial is about 10 cells per vial. 5 ~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.

[0099]

[0106] Additionally, the volume of the cryopreserved cell preparation can be reduced compared to standard prior art techniques. For example, cells can be cryopreserved in volumes ranging from about 10 μL to about 100 μL, including volumes 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, and in some embodiments, including volumes of about 10 μL to about 50 μL. In some embodiments, cells may be cryopreserved in volumes ranging from about 10 μL to about 500 μL, including volumes of 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. Because cells can be frozen in large numbers in low volumes, the frozen preparation can then be diluted with an appropriate diluent to reach the desired volume and concentration, and then administered directly to a subject without the need to perform a washing step. Standard prior art techniques typically require freezing lower concentrations of cells in large volumes (e.g., volumes on the order of 1 mL) and then washing the thawed preparation, sometimes multiple times, to remove the cryoprotectant prior to administration to a subject.

[0100]

[0107] In some embodiments, a cryoprotective formulation used to cryopreserve RPE cells comprises, consists essentially of, or consists of about 4% to about 10% DMSO (w / v), about 2% to about 3% albumin (w / v), 0-1.5% glucose (w / v), and a buffer or buffered saline solution. This formulation results in a viability of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% of RPE cells after thawing. The cryoprotective formulation also results in a lower frequency of multinucleated cells. In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of about 10% DMSO (v / v), about 2.4% albumin (w / v), 0-1.5% glucose (w / v), and a buffer.

[0101]

[0108] In some embodiments, a cryoprotective 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 buffer, and is used to cryopreserve photoreceptor rescue cells, photoreceptor cells, or photoreceptor progenitor cells. DMSO may be used at about 5% or about 10%. Cells are cryopreserved at about 50 x 10 cells per μL. 3 The formulation results in improved cell viability after thawing compared to the commercially available cryoprotectant formulation, CS10.

[0102]

[0109] In some embodiments, the cryoprotective 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% to 1.5% glucose (w / v), and a buffer or buffered saline solution, and is used to cryopreserve corneal endothelial progenitor cells or corneal endothelial cells. DMSO may be present at about 5% or about 10% (v / v). Cells are cryopreserved at a concentration of about 30 x 10 cells per μL. 3 The formulation results in improved cell viability after thawing compared to conventional cryoprotective formulations of 90% FBS (v / v) and 10% DMSO (v / v).

[0103]

[0110] In some embodiments, the cryoprotective 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 solution. DMSO can be present at about 4% (v / v), about 5% (v / v), and about 6% (v / v). The albumin can be human albumin, including recombinant human albumin, and can be present at about 2% (w / v), about 2.5% (w / v), or about 3% (w / v). Glucose can be absent or present at about 0.08% (w / v), about 0.09% (w / v), or about 0.1% (w / v). The buffered saline solution can be phosphate buffered saline.

[0104]

[0111] In some embodiments, the cryoprotective 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 solution. The albumin may be present at about 2% (w / v), about 2.5% (w / v), or about 3% (w / v). The glucose may be present at about 0.08% (w / v), about 0.09% (w / v), or about 0.10% (w / v).

[0105]

[0112] In some embodiments, the cryoprotective 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 solution. DMSO may be present at about 4% (v / v), about 5% (v / v), and about 6% (v / v). The albumin can be human albumin, including recombinant human albumin, and can 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 can be absent or present at about 0.08% (w / v), or about 0.09% (w / v), or about 0.1% (w / v). The buffered saline solution can be phosphate buffered saline.

[0113] In some embodiments, the cryoprotective 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 solution. The albumin may be present at about 2% (w / v), about 2.5% (w / v), or about 3% (w / v). In some embodiments, the cryoprotective formulation comprises, consists essentially of, or consists of 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 solution. The albumin may be present at about 2% (w / v), about 5% (w / v), or about 7.5% (w / v). Glucose may be present at about 0.08% (w / v), or about 0.09% (w / v), or about 0.10% (w / v).

[0106] Cryopreserved preparations

[0114] In some embodiments, the formulations or preparations provided herein exhibit physiological pH and physiological osmolality, also referred to as physiological osmolality. Physiological pH refers to a pH that is not cytotoxic and approximates the pH of a cell or tissue in its natural environment. For most cells and tissues, physiological pH is about 6.8 to about 7.8, e.g., about 7 to about 7.7, about 7.2 to about 7.6, about 7.2 to about 7.4, or about 7.4 to about 7.5. Thus, in some embodiments, the formulations or preparations provided herein exhibit 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 osmolality refers to an osmotic pressure that is not cytotoxic and approximates the osmotic pressure of a cell or tissue in its natural environment. For some cells and tissues, physiological osmolality is about 270 to about 345 mOsm / L, e.g., about 280 to about 330 mOsm / L, about 290 to about 325 mOsm / L, or about 300 to about 315 mOsm / L. In some embodiments, physiological osmolality is about 300, about 305, about 310, about 315, about 320, or about 325 mOsm / L. In some embodiments, the formulations or preparations provided herein exhibit physiological pH and osmolality exceeding physiological osmolality, e.g., about 350 to about 450 mOsm / L, e.g., about 360 to about 440 mOsm / L, about 370 to about 430 mOsm / L, about 380 to about 420 mOsm / L, or about 390 to about 410 mOsm / L. In some embodiments, the supra-physiological osmolality 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 formulations or preparations provided herein have an osmolality of about 1250 mOsm / kg, e.g., about 1250 mOsm / kg, about 1000 mOsm / kg, about 900 mOsm / kg, about 800 mOsm / kg, about 700 mOsm / kg, about 600 mOsm / kg, about 500 mOsm / kg, about 400 mOsm / kg, or about 300 mOsm / kg.In some embodiments, the formulations or preparations provided herein have an osmolality of about 300-450 mOsm / kg, about 300-400 mOsm / kg, about 300-450 mOsm / kg, about 350-450 mOsm / kg, or about 350-400 mOsm / kg.

[0107]

[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.

[0108]

[0116] In some embodiments, the formulation includes a cryoprotectant. Cryoprotectants are used to protect cells from damage during the freezing process. In some embodiments, the formulation includes a cryoprotectant selected from dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol. In some embodiments, the cryoprotectant is DMSO. In some embodiments, the formulation 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) DMSO. In some embodiments, the solution is about 4% to 10%, about 4% to 9.5%, about 4% to 9%, about 4% to 8.5%, about 4% to 8%, about 4% to 7.5%, about 4% to 7%, about 4% to 6.5%, about 4% to 6%, about 4% to 5.5%, about 4% to 5%, about 4% to 4.5%, about 4.5% to 10%, about 4.5% to 9.5%, about 4.5% to 9%, about 4.5% to 8.5%, about 4.5% to 8%, about 4.5% to 7.5%, about 4.5% to 7%, about 4% to 5.5 ...5.5%, about 4% to 4.5%, about 4.5% to 10%. .5%~6.5%, approximately 4.5%~6%, approximately 4.5%~5.5%, approximately 4.5%~5%, approximately 5%~10%, approximately 5%~9.5%, approximately 5%~9%, approximately 5%~8.5%, approximately 5%~8%, approximately 5%~7.5%, approximately 5%~7%, approximately 5%~6.5%, approximately 5%~6%, approximately 5%~5.5%, approximately 5.5%~10%, approximately 5.5%~9.5%, approximately 5.5%~9%, approximately 5.5%~8.5%, approximately 5.5%~8%, approximately 5.5%~7.5%, approximately 5. 5% to 7%, approximately 5.5% to 6.5%, approximately 5.5% to 6%, approximately 6% to 10%, approximately 6% to 9.5%, approximately 6% to 9%, approximately 6% to 8.5%, approximately 6% to 8%, approximately 6% to 7.5%, approximately 6% to 7%, approximately 6% to 6.5%, approximately 6.5% to 10%, approximately 6.5% to 9.5%, approximately 6.5% to 9%, approximately 6.5% to 8.5%, approximately 6.5% to 8%, approximately 6.5% to 7.5%, approximately 6.5% to 7%, approximately 7% to 10%, approximately 7% to 9.5%, approximately 7% to 9 %, about 7% to 8.5%, about 7% to 8%, about 7% to 7.5%, about 7.5% to 10%, about 7.5% to 9.5%, about 7.5% to 9%, about 7.5% to 8.5%, about 7.5% to 8%, about 8% to 10%, about 8% to 9.5%, about 8% to 9%, about 8% to 8.5%, about 8.5% to 10%, about 8.5% to 9.5%, about 8.5% to 9%, about 9% to 10%, about 9% to 9.5%, or about 9.5% to 10% (v / v) DMSO.In some embodiments, the formulation may comprise 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%, or about 7%. 0.1%, 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.

[0109]

[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" is used interchangeably with the terms "rA" or "rAlbumin." The term "recombinant human albumin" is used interchangeably with the terms "rHA," "rHSA," and "rhAlbumin." In some embodiments, the cryopreserved formulation contains 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 5.10%, about 5.11%, about 5.12%, about 5.13%, about 5.14%, about 5.15%, about 5.16%, about 5.17%, about 5.18%, about 5.19%, about 5.20%, about 5.21%, about 5.22%, about 5.23%, about 5.24%, about 5.25%, about 5.26%, about 5.27%, about 5.28%, about 5.29%, about 5.30%, about 5.31%, about 5.3 %, approximately 3.9%, approximately 4.0%, approximately 4.1%, approximately 4.2%, approximately 4.3%, approximately 4.4%, approximately 4.5%, approximately 4.6%, approximately 4.7%, approximately 4.8%, approximately 4.9%, approximately 5.0%, approximately 5.1%, approximately 5.2%, approximately 5.3%, approximately 5.4%, approximately 5.5%, approximately 5.6%, approximately 5.7%, approximately 5.8%, approximately 5.9%, approximately 6. 0%, approx. 6.1%, approx. 6.2%, approx. 6.3%, approx. 6.4%, approx. 6.5%, approx. 6.6%, approx. 6.7%, approx. 6.8%, approx. 6.9%, approx. 7.0%, approx. 7.1%, approx. 7.2%, approx. 7.3%, approx. 7.4%, approx. 7.5%, approx. 7.6%, approx. 7.7%, approx. 7.8%, approx. 7.9%, approx. 8.0%, approx. 8.1%, approx. Contains 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 cryopreserved formulation is about 2.0% to 10.0%, about 2.0% to 9.5%, about 2.0% to 9.0%, about 2.0% to 8.5%, about 2.0% to 8.0%, about 2.0% to 7.5%, about 2.0% to 7.0%, about 2.0% to 6.5%, about 2.0% to 6.0%, about 2.0% to 5.5%, about 2.0% to 5.0%, about 2.0% to 4.5%, about 2.0% to 4.0%, about 2.0% to 3.5%, about 2.0% to 3.0%, about 2.0% to 2.5%, about 2.5% to 10.0%, about 2.5% to 9.5%, about 2.5% to 9.0%, about 2.5% to 8.5%, or about 2.5% to 8.0%. , approximately 2.5% to 7.5%, approximately 2.5% to 7.0%, approximately 2.5% to 6.5%, approximately 2.5% to 6.0%, approximately 2.5% to 5.5%, approximately 2.5% to 5.0%, approximately 2.5% to 4.5%, approximately 2.5% to 4.0%, approximately 2.5% to 3.5%, approximately 2.5% to 3.0%, approximately 3.0% to 10.0%, approximately 3 .0%~9.5%, approximately 3.0%~9.0%, approximately 3.0%~8.5%, approximately 3.0%~8.0%, approximately 3.0%~7.5%, approximately 3.0%~7.0%, approximately 3.0%~6.5%, approximately 3.0%~6.0%, approximately 3.0%~5.5%, approximately 3.0%~5.0%, approximately 3.0%~4.5%, approximately 3.0%~ 4.0%, approximately 3.0% to 3.5%, approximately 3.5% to 10.0%, approximately 3.5% to 9.5%, approximately 3.5% to 9.0%, approximately 3.5% to 8.5%, approximately 3.5% to 8.0%, approximately 3.5% to 7.5%, approximately 3.5% to 7.0%, approximately 3.5% to 6.5%, approximately 3.5% to 6.0%, approximately 3.5% to 5.5 %, approximately 3.5% to 5.0%, approximately 3.5% to 4.5%, approximately 3.5% to 4.0%, approximately 4.0% to 10.0%, approximately 4.0% to 9.5%, approximately 4.0% to 9.0%, approximately 4.0% to 8.5%, approximately 4.0% to 8.0%, approximately 4.0% to 7.5%, approximately 4.0% to 7.0%, approximately 4.0% to 6.5%, approximately 4.0%~6.0%, approximately 4.0%~5.5%, approximately 4.0%~5.0%, approximately 4.0%~4.5%, approximately 4.5%~10.0%, approximately 4.5%~9.5%, approximately 4.5%~9.0%, approximately 4.5%~8.5%, approximately 4.5%~8.0%, approximately 4.5%~7.5%, approximately 4.5%~7.0%, approximately 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%, approximately 5.0% to 6.0%, approximately 5.0% to 5.5%, approximately 5.5% to 10.0%, approximately 5.5% to 9.5%, approximately 5.5% to 9.0%, approximately 5.5% to 8.5%, approximately 5.5% to 8.0%, approximately 5.5% to 7.5%, approximately 5.5% to 7.0%, approximately 5.5% to 6.5%, approximately 5.5% to 6.0%, approximately 6.0% to 10.0%, approximately 6.0% to 9.5%, approximately 6.0% to 9.0%, approximately 6.0% to 8.5%, approximately 6.0% to 8.0%, approximately 6.0% to 7.5%, approximately 6.0% to 7.0%, approximately 6.0% to 6.5%, approximately 6.5% to 10.0%, approximately 6.5% to 9.5%, approximately 6 The composition contains about 0.5% to 9.0%, about 6.5% to 8.5%, about 6.5% to 8.0%, about 6.5% to 7.5%, about 6.5% to 7.0%, about 7.0% to 10.0%, about 7.0% to 9.5%, about 7.0% to 9.0%, about 7.0% to 8.5%, about 7.0% to 8.0%, about 7.0% to 7.5%, about 8.0% to 10.0%, about 8.0% to 9.5%, about 8.0% to 9.0%, about 8.0% to 8.5%, about 8.5% to 10.0%, about 8.5% to 9.5%, about 8.5% to 9.0%, about 9.0% to 10.0%, or about 9.5% to 10.0% (w / v) albumin.

[0110]

[0119] In some embodiments, the cryopreserved formulation is about 2.0% to 3.0%, about 2.0% to 2.9%, about 2.0% to 2.8%, about 2.0% to 2.7%, about 2.0% to 2.6%, about 2.0% to 2.5%, about 2.0% to 2.4%, about 2.0% to 2.3%, about 2.0% to 2.2%, about 2.0% to 2.1%, about 2.1% to 3.0%, about 2.1% to 2.9%, about 2.1% to 2.8% , approximately 2.1% to 2.7%, approximately 2.1% to 2.6%, approximately 2.1% to 2.5%, approximately 2.1% to 2.4%, approximately 2.1% to 2.3%, approximately 2.1% to 2.2%, approximately 2.2% to 3.0%, approximately 2.2% to 2.9%, approximately 2.2% to 2.8%, approximately 2.2% to 2.7%, approximately 2.2% to 2.6%, approximately 2.2% to 2.5%, approximately 2.2% to 2.4%, approximately 2.2% to 2.3%, approximately 2.3% ~3.0%, approximately 2.3%~2.9%, approximately 2.3%~2.8%, approximately 2.3%~2.7%, approximately 2.3%~2.6%, approximately 2.3%~2.5%, approximately 2.3%~2.4%, approximately 2.4%~3.0%, approximately 2.4%~2.9%, approximately 2.4%~2.8%, approximately 2.4%~2.7%, approximately 2.4%~2.6%, approximately 2.4%~2.5%, approximately 2.5%~3.0%, approximately 2.5%~2.9%, Contains about 2.5% to 2.8%, about 2.5% to 2.7%, about 2.5% to 2.6%, about 2.6% to 3.0%, about 2.6% to 2.9%, about 2.6% to 2.8%, about 2.6% to 2.7%, about 2.7% to 3.0%, about 2.7% to 2.9%, about 2.7% to 2.8%, about 2.8% to 3.0%, about 2.8% to 2.9%, or about 2.9% to 3.0% (w / v) albumin.

[0111]

[0120] In some embodiments, the cryopreserved formulation contains a buffer. A buffer refers to an agent that can maintain the pH of a solution, preparation, or formulation relatively stable by neutralizing added acids or bases. Typically, a buffer contains 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 contained in the cryopreserved formulation provided herein is a water-based salt solution containing disodium hydrogen phosphate (NaHPO) and sodium chloride (NaCl). In some embodiments, the buffer contained in the formulation provided herein further contains potassium chloride (KCl) and potassium dihydrogen phosphate (KHPO). In some embodiments, the formulation contains a buffered saline solution, 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).

[0112]

[0121] In some embodiments, the buffer comprises a divalent cation. Suitable divalent cations include, but are not limited to, Ca 2+ , Mg 2+ , Zn 2+ , Fe 2+ , Mn 2+ , Cr 2+ , Cu 2+ , Ba2+ , 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, for example, 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 presented 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.

[0113]

[0122] In some embodiments, the cryopreservation formulations provided herein contain glucose. In some embodiments, the solution contains about 0% to 5.0% (w / v) glucose. For example, in some embodiments, the cryopreservation formulation contains 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 2.0%, 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 2.1%. The glucose concentration may be about 0.7%, 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 may be expressed as a range. The present disclosure contemplates, and these ranges are presented herein, any range for glucose selected from any one of the following lower limits and any one of the following upper limits. Any combination of these is disclosed herein.The lower limits of the ranges are: 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%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 2.10%, 2.11%, 2.12%, 2.13%, 2.14%, 2.15%, 2.16%, 2.17%, 2.18%, 2.19%, 2.20%, 2.21%, 2.22%, 2.23%, 2.24%, 2.25%, 2.26%, 2.27%, 2.28%, 2.29%, 2.30%, 2.31%, 2.32%, 2.33%, 2.34%, 2.35%, 2.36%, 2.37%, 2.38%, 2.39%, 2.40%, 2.41%, 2.42%, 2.43%, 2.44%, 2.45%, 2.46%, 2.47%, 2.48%, 2.49%, 2.50%, 2.51%, 2.52%, 2.53%, 2.54%, 2.55%, 2.56%, 2.57%, 2.58%, 2.59%, The range includes any one of the following: 0.8%, 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%. The following: 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 ... %, 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%, 4.9%, or 5% (w / v) glucose. The present disclosure contemplates any range for glucose having any combination of a disclosed lower limit and a disclosed upper limit.For example, in the present specification, the terms "amount of oxidized or unoxidized amine" include about 0 to 0.5%, about 0 to 1%, about 0 to 1.5%, about 0 to 2%, about 0 to 2.5%, about 0 to 3%, about 0 to 3.5%, about 0 to 4%, about 0 to 4.5%, about 0 to 5%, about 0.5 to 1%, about 0.5 to 1.5%, about 0.5 to 2%, about 0.5 to 2.5%, about 0.5 to 3%, about 0.5 to 3.5%, about 0.5 to 4%, about 0.5 to 4.5%, about 0.5 to 5%, about 1 to 1.5%, about 1 to 2%, about 1 to 2.5%, about 1 to 3%, about 1 to 3.5%, about 1 to 4%, about 1 to 4.5%, about 1 to 5%, about 1.5 to 2%, about 1.5 to 2.5%, about 1 to 3%. Ranges such as 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 are presented. In some embodiments, the solution contains about 0-1.5% (w / v) glucose. By way of example, a range of about 1 to 1.5% shall be understood to mean about 1% to about 1.5%, with the same meaning being assigned to each and every range recited herein.

[0114]

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

[0115]

[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 solution. 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 solution. 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 solution. 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 solution. In some embodiments, the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.6% (w / v) glucose, and a buffer solution. 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 a buffer solution. 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 a buffer solution. In some embodiments, the formulation comprises about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.6% (w / v) glucose, and a buffer solution. 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.

[0116]

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

[0117]

[0126] In some embodiments, the formulations provided herein further comprise at least one additional excipient, hi some embodiments, the formulations comprise about 1 mM to 500 mM, about 5 mM to 400 mM, about 10 mM to 300 mM, about 25 mM to 250 mM, or about 50 mM to 200 mM of at least one additional excipient. In some embodiments, the formulation comprises a concentration of about 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM , 65mM, 66mM, 67mM, 68mM, 69mM, 70mM, 71mM, 72mM, 73mM, 74mM, 75mM, 76mM, 77mM, 78mM, 79mM, 80mM, 81mM, 82mM, 83mM, 84mM, 85mM, 8 6mM, 87mM, 88mM, 89mM, 90mM, 91mM, 92mM, 93mM, 94mM, 95mM, 96mM, 97mM, 98mM, 99mM, 100mM, 101mM, 102mM, 103mM, 104mM, 105mM, 106 mM, 107mM, 108mM, 109mM, 110mM, 111mM, 112mM, 113mM, 114mM, 115mM, 116mM, 117mM, 118mM, 119mM, 120mM, 121mM, 122mM, 123mM, 12 4mM, 125mM, 126mM, 127mM, 128mM, 129mM, 130mM, 131mM, 132mM, 133mM, 134mM, 135mM, 136mM, 137mM, 138mM, 139mM, 140mM, 141mM, 1 42mM, 143mM, 144mM, 145mM, 146mM, 147mM, 148mM, 149mM, 150mM, 151mM, 152mM, 153mM, 154mM, 155mM, 156mM, 157mM, 158mM, 159mM, 160mM, 161mM, 162mM, 163mM, 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, 1 97mM, 198mM, 199mM, 200mM, 201mM, 202mM, 203mM, 204mM, 205mM, 206mM, 207mM, 208mM, 209mM, 210mM, 211mM, 212mM, 213mM, 214mM, 215mM, 21 6mM, 217mM, 218mM, 219mM, 220mM, 221mM, 222mM, 223mM, 224mM, 225mM, 226mM, 227mM, 228mM, 229mM, 230mM, 231mM, 232mM, 233mM, 234mM, 235mM, 236mM, 237mM, 238mM, 239mM, 240mM, 241mM, 242mM, 243mM, 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 the group consisting of taurine, trehalose, sucrose, lactose, propanediol, 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 propanediol or 1,3 propanediol. In some embodiments, the at least one additional excipient is propylene glycol.

[0118]

[0127] In some embodiments, the formulation comprises a polymeric excipient. In some embodiments, the formulation comprises about 1% to 20% polymeric excipient. In some embodiments, the formulation comprises about 3% to 18%, about 5% to 15%, or about 7.5% to 12.5% ​​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% polymeric excipient. In some embodiments, the polymeric excipient is selected from the group consisting of poly(ethylene glycol) (PEG), dextran, and poly(vinylpyrrolidone) (PVP). In some embodiments, the polymeric excipient is dextran. In some embodiments, the formulation does not comprise a polymeric excipient. In some embodiments, the formulation does not comprise dextran.

[0119] cell preparation

[0128] Some aspects of the present disclosure provide cell preparations comprising cell populations or tissues in the cryopreserved formulations provided herein. In some embodiments, the cell preparations are cryopreserved. The cell preparations provided herein can be cryopreserved by deep-freezing at any suitable temperature known in the art. For example, in some embodiments, the cell preparations provided herein are cryopreserved at temperatures between -100°C and -200°C. In some embodiments, the cell preparations provided herein are cryopreserved at temperatures below -125°C. In some embodiments, the cell preparations provided herein are cryopreserved at temperatures at or below -135°C. In some embodiments, the cell preparations provided herein are cryopreserved at temperatures below -150°C.

[0120]

[0129] The cryopreserved formulations and cell preparations presented herein can be used in conjunction with different cell types and different administration sites. Although ophthalmic applications are preferred, other applications are also envisioned and encompassed by the present disclosure. In some embodiments, the cell population is selected from retinal pigment epithelial (RPE) cells, photoreceptor cells and / or their progenitor cells, photoreceptor rescue cells, and corneal endothelial cells and / or their progenitor cells.

[0121]

[0130] In some embodiments, the cell preparation, optionally undiluted, is suitable for transplantation into a subject upon thawing. In some embodiments, the cell preparation, optionally undiluted, is suitable for transplantation into a subject upon thawing and dilution with a diluent. In some embodiments, the diluent is GS2, GS2 Plus, BSS, BSS Plus®, 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 (a) a buffer that maintains the solution at physiological pH, (b) at least 2 mM glucose, and (c) an osmotically active agent that maintains the solution at physiological osmolality. In some embodiments, the diluent is GS2 or GS2 Plus. In some embodiments, the diluent is a solution comprising (a) a buffer that maintains the solution at physiological pH; (b) at least 2 mM glucose; and (c) an osmotically active agent that maintains the solution at a higher than physiological osmolality, e.g., an osmolality in the range of 350-450 mOsm / kg, or an osmolality in the range of 300-1250 mOsm / kg. In some embodiments, the diluent further comprises a divalent cation. In some embodiments, the divalent cation comprises calcium and / or magnesium. In some embodiments, the diluent comprises calcium. In some embodiments, the diluent further comprises magnesium. In some embodiments, the diluent comprises an acetate buffer and / or a citrate buffer. In some embodiments, the diluent is a solution that combines two or more, or any number of criteria (e.g., pH, osmolality, solutes (buffer, glucose, osmotically active agent, magnesium, calcium, potassium, polymer), concentration, etc.). In some embodiments, the diluent is a solution containing a buffer, glucose, and an osmotically active agent, with or without the addition of a polymer, a solution with potassium, and a solution without potassium, as well as a solution containing any combination of solutes at any concentration listed for each solute.It will also be understood that the present disclosure encompasses solutions containing the recited solutes, as well as solutions consisting essentially of or consisting of the recited solutes and a solvent, such as water, and for purposes of brevity, these alternatives will not be detailed herein.

[0122]

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

[0123]

[0132] In some embodiments, the diluent is a solution containing about 0.1 to about 1.2 mM CaCl, about 0.05 to about 5 mM MgCl, 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 containing about 0.9 mM CaCl, about 0.3 mM MgCl, about 2 mM KCl, about 1.2 mM sodium citrate, about 15 mM dextrose, and about 145 mM NaCl. The diluent may further contain sodium acetate. The diluent may further comprise 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). The diluent may further comprise a buffer component such as dibasic sodium phosphate heptahydrate, and / or monobasic sodium phosphate monohydrate, and / or monobasic sodium phosphate dihydrate.

[0124]

[0133] In some embodiments, the diluent is a solution containing about 0.008% to about 0.012% CaCl2 dihydrate, about 0.0048% to about 0.0072% MgCl2 hexahydrate, about 0.012% to about 0.018% KCl, about 0.028% to about 0.042% sodium citrate dihydrate, about 0.23% to about 0.35% dextrose, and about 0.68% to about 1.02% NaCl. In some embodiments, the diluent is a solution containing about 0.01% CaCl2 dihydrate, about 0.006% MgCl2 hexahydrate, about 0.015% KCl, about 0.035% sodium citrate dihydrate, at least 0.25% dextrose, and about 0.85% NaCl. The diluent may further contain sodium acetate. The diluent may further comprise 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). The diluent may further comprise a buffer component such as dibasic sodium phosphate heptahydrate, and / or monobasic sodium phosphate monohydrate, and / or monobasic sodium phosphate dihydrate.

[0125]

[0134] In some embodiments, the diluent comprises 5% dextrose, 0.9% NaCl, BSS® sterile irrigation solution, 0.1 N NaOH, and 0.9% NaCl irrigation solution. In some embodiments, the diluent comprises 3% sodium hyaluronate, 5% dextrose dehydrate, dibasic sodium phosphate dodecahydrate, BSS® sterile irrigation solution (e.g., 0.64% NaCl, 0.075% KCl, 0.048% CaCl2 dihydrate). 、 0.03% MgCl hexahydrate, 0.39% sodium acetate trihydrate, and 0.17% sodium citrate dihydrate).

[0126]

[0135] 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, optionally sodium acetate (e.g., at about 0.08%), optionally sodium hyaluronate (e.g., at about 0.049%), and optionally dibasic sodium phosphate heptahydrate (e.g., at about 0.0007%) and monobasic sodium phosphate monohydrate (e.g., at 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, optionally sodium acetate (e.g., at about 6 mM), optionally sodium hyaluronate, and optionally sodium phosphate dibasic heptahydrate (e.g., at about 0.027 mM) and sodium phosphate monobasic monohydrate (e.g., at about 0.007 mM). Such diluents include GS2.

[0127]

[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, optionally sodium acetate (e.g., at about 0.08%), optionally sodium hyaluronate (e.g., at about 0.049%), and optionally monobasic sodium phosphate dihydrate (e.g., at about 0.047%). 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, optionally sodium acetate (e.g., at about 6 mM), optionally sodium hyaluronate, and optionally monobasic sodium phosphate dihydrate (e.g., at about 3 mM). Such diluents include GS2 Plus.

[0128]

[0137] In some embodiments, the cell preparations provided herein, once thawed and diluted, are formulated for administration to a subject, for example, via injection. The cell preparations provided herein may include cell populations such as RPE cells and / or their progenitors, photoreceptor cells and / or their progenitors, and corneal endothelial cells and / or their progenitors, and photoreceptor rescue cells.

[0129]

[0138] Exemplary cell or tissue preparations can be formulated such that, after thawing, they are suitable for use in treating human patients, e.g., pyrogen-free or essentially pyrogen-free, pathogen-free, sterile, and at physiological pH and osmolality. In some embodiments, the preparations provided herein are formulated for injection into the vitreous humor for delivery to a specific site, e.g., in the case of ophthalmic preparations for treating retinal diseases or disorders, to the site of retinal or choroidal damage.

[0130]

[0139] The cell preparations provided by the present disclosure may additionally contain therapeutic agents, such as immunosuppressants, pro-angiogenic agents, or trophic substances or growth factors that support the survival and / or engraftment of the cells in the preparation.

[0131]

[0140] The volume and number of cells in the administered cell preparation will depend on the specific application. Typically, for cell transplant applications, it is desirable to reduce the administered volume as much as possible. Therefore, the cell preparation can be formulated so that a minimized volume can be delivered. The cell concentration for injection can be any concentration that is effective and non-toxic. In some embodiments, the volume of the administered cell preparation is between 1 and 1000 μL. In some embodiments, the volume of the cell preparation administered is between 1 and 50 μl, or between 10 and 50 μl, or between 25 and 50 μl, or between 50 and 100 μl, or between 50 and 200 μl, or between 50 and 300 μl, or between 50 and 400 μl, or between 50 and 500 μl, or between 100 and 500 μl, or between 100 and 400 μl, or between 100 and 300 μl, or between 100 and 300 μl, or about 200 μl, or about 150 μl. In some embodiments, the volume of the cell preparation 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.

[0132]

[0141] In some embodiments, cell number and / or cell concentration in the cell preparations provided herein can be determined by counting viable cells and excluding nonviable cells. For example, nonviable cells can be detected by their inability to exclude a vital dye (such as Trypan Blue) or by using functional assays (such as the ability to adhere to a culture substrate, phagocytosis, etc.). Additionally, cell number or cell concentration of a desired cell type can be determined by counting cells expressing one or more cell markers characteristic of that cell type and / or excluding cells expressing one or more markers indicative of a cell type other than the desired cell type.

[0133]

[0142] In some embodiments, the cell preparation administered comprises about 1 x 10 cells. 3 , 2 × 103 、3×10 3 、4×10 3 、5×10 3 、6×10 3 、7×10 3 、8×10 3 、9×10 3 、1×10 4 、2×10 4 、3×10 4 、4×10 4 、5×10 4 、6×10 4 、7×10 4 、8×10 4 、9×10 4 、1×10 5 、2×10 5 、3×10 5 、4×10 5 、5×10 5 、6×10 5 、7×10 5 、8×10 5 、9×10 5 、1×10 6 、2×10 6 、3×10 6 、4×10 6 、5×10 6 、6×10 6 、7×10 6 、8×10 6 、9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9, 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9×10 10 The invention may include at least these number of cells or more than these number of cells.

[0134]

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

[0135]

[0144] The aforementioned number of cells can be present in a cryopreserved cell preparation in a single cryovial. The cells can be present in about 10 to about 200 μL of cryoprotected formulation, or about 10 to about 100 μL of cryoprotected formulation, or about 10 to about 50 μL of cryoprotected formulation, including about 20 to about 50 μL of cryoprotected formulation.

[0136]

[0145] In some embodiments, the cell population is suitable for transplantation into the eye of a target. In some embodiments, the cell population suitable for transplantation into the eye of a target comprises RPE cells, RPE progenitor cells, iris pigment epithelial (IPE) cells, and other vision-related neural cells, such as interneurons (e.g., "relay" neurons of the inner nuclear layer (INL)) and amacrine cells, retinal cells, retinal rod cells, retinal cone cells, and corneal cells (e.g., corneal endothelial cells), neural cells, neural progenitor cells, neural stem cells, photoreceptor cells (e.g., photoreceptor progenitor cells), photoreceptor rescue cells, and mesenchymal cells, such as mesenchymal stem cells (MSCs). Such formulated cells can be produced by directed differentiation of pluripotent or multipotent stem cells, including human induced pluripotent stem cells (hiPSCs), human embryonic stem cells (hESCs), and somatic cells (including transformed cells and stem cells).

[0137]

[0146] In some embodiments, cell preparation comprises a population of RPE cells in the cryopreserved preparation presented herein.Suitable RPE cells can be differentiated from pluripotent stem cells such as human embryonic stem (ES) cells or induced pluripotent stem cells (iPS cells), and are molecularly distinct 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.

[0138]

[0147] When ES cell-derived RPE cells are used, in some embodiments, the cell preparations provided herein do not contain detectable amounts of residual ES cells, such that the cell preparations provided herein do not pose an unacceptable risk to recipients of such preparations. When iPS cell-derived RPE cells are used, in some embodiments, the cell preparations provided herein do not contain detectable amounts of residual iPS cells, such that the cell preparations provided herein do not pose an unacceptable risk to recipients of such preparations.

[0139]

[0148] In some embodiments, the cell preparation comprises a population of photoreceptor rescue cells in a cryopreserved formulation as provided herein. Suitable photoreceptor rescue cells may be differentiated from pluripotent stem cells, such as human embryonic stem cells or iPS cells, and are significantly 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.

[0140]

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

[0141]

[0150] In some embodiments, the cell preparation comprises a population of corneal endothelial cells in a cryopreserved formulation as presented herein.Suitable corneal endothelial cells may be differentiated from pluripotent stem cells such as human embryonic stem cells or iPS cells, and are molecularly significantly different from embryonic stem cells, iPS cells, adult-derived corneal endothelial cells, and fetal-derived corneal endothelial cells.In some embodiments, adult-derived corneal endothelial cells and fetal-derived corneal endothelial cells are used.

[0142]

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

[0143]

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

[0144]

[0153] The RPE cells can be stable, terminally differentiated RPE cells that do not dedifferentiate into non-RPE cell types. The RPE cells described herein can be functional RPE cells characterized by their ability to integrate into the retina upon intracorneal, subretinal, or other administration to a human or non-human animal.

[0145]

[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 can be comparable to the expression level in naturally occurring RPE cells.The maturity level of RPE cells can be evaluated by measuring the expression of at least one of PAX2, PAX6 and tyrosinase, or their respective expression levels.

[0146]

[0155] In some embodiments, RPE cells contained in the pharmaceutical compositions provided herein can be identified and characterized based on the degree of cell pigmentation. Pigment changes can be controlled by the density at which RPE cells are cultured and maintained, and the duration for which RPE cells are maintained in culture. Rapidly dividing, differentiated RPE cells are pigmented more lightly. In contrast, RPE cells that divide more slowly or do not divide adopt their characteristic polygonal or hexagonal shape and increase their pigmentation level by accumulating melanin and lipofuscin. For example, dormant RPE cultures (e.g., due to confluence) typically increase their pigmentation level over time. Thus, pigmentation accumulation is used as an indicator of RPE differentiation, and increased pigmentation relative to cell density is used as an indicator of RPE maturity. For example, mature RPE cells can be subcultured at low density to reduce pigmentation. In this context, mature RPE cells can also be cultured to generate less mature RPE cells, which are also differentiated RPE cells that express RPE differentiation markers.

[0147]

[0156] For example, in some embodiments, their average melanin content is less than 8 pg per cell, less than 7 pg per cell, less than 6 pg per cell, or less than 5 pg per cell, such as between 4.2 and 4.8 pg per cell, or between 0.1 and 5 pg per cell, e.g., between 0.1 and 8 pg per cell, between 0.1 and 7 pg per cell, between 0.1 and 6 pg per cell, between 0.1 and 5 pg per cell, between 0.1 and 4 pg per cell, Preparations are provided that include RPE cells that are between 0.1-3 pg per cell, between 0.1-2 pg per cell, between 0.1-1 pg per cell, between 1-8 pg per cell, between 1-7 pg per cell, between 1-6 pg per cell, between 1-5 pg per cell, between 1-4 pg per cell, between 1-3 pg per cell, between 1-2 pg per cell, between 2-6 pg per cell, between 3-5 pg per cell, or between 4-5 pg per cell. In further examples, the average melanin content can be less than 5 pg per cell, e.g., between 0.1 and 5 pg per cell, between 0.2 and 5 pg per cell, between 0.5 and 5 pg per cell, between 1 and 5 pg per cell, between 2 and 5 pg per cell, between 3 and 5 pg per cell, between 4 and 5 pg per cell, or between 4.5 and 5 pg per cell.

[0148]

[0157] In some embodiments, a preparation comprising RPE cells is provided in the cell cryopreservation formulation described herein, wherein the RPE cells maintain their phenotype after transplantation into a subject, for example, after injection of the preparation into the subject's eye. The RPE cells may maintain their phenotype for the recipient's lifetime after transplantation. For example, the RPE cells may 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, or 20 days after transplantation. Furthermore, the RPE cells may maintain their phenotype for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after transplantation. The RPE cells may maintain their phenotype for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after transplantation. RPE cells may 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.

[0149]

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

[0150]

[0159] In some embodiments, the present invention provides a preparation in which cells are contained within a cell sheet. For example, RPE cells, corneal endothelial cells, photoreceptor cells, or photoreceptor rescue cells can be contained within the sheet. For example, cell sheets containing RPE cells can be prepared by culturing RPE cells on a substrate from which the intact cell sheet can be released, e.g., a thermoresponsive polymer, such as a thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surface, onto which the cells attach and grow at culture temperatures, and then upon a temperature shift (e.g., by cooling to a temperature below the lower critical solution temperature (LCST)), which alters the surface characteristics and causes the release of the cultured cell sheet (e.g., by cooling to a temperature below the lower critical solution temperature (LCST)). Each of these is incorporated herein by reference in its entirety; see da Silva et al., Trends Biotechnol., December 2007, 25(12):577-83; Hsiue et al., Transplantation, February 15, 2006, 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). Cell sheets can be adherent to a substrate suitable for transplantation, such as a substrate that can dissolve in vivo when the sheet is transplanted into a host organism. For example, cells can be cultured on a substrate suitable for transplantation or can be prepared by releasing cells from another substrate (such as a thermoresponsive polymer) onto a substrate suitable for transplantation. An exemplary substrate potentially suitable for transplantation can include gelatin (see Hsiue et al., supra). Alternative substrates that may be suitable for transplantation include fibrin-based matrices, etc. Cell sheets can be used in the manufacture of medicaments for the prevention or treatment of retinal degenerative diseases.

[0151]

[0160] In some embodiments, provided herein are preparations in which the cells are in clusters, spheres, or aggregates, hi some embodiments, the cells provided herein are administered in combination with a biomaterial, for example, a biomaterial that can be polymerized.

[0152]

[0161] In some embodiments, the cells (e.g., RPE cells) provided herein are administered as an implant or device. In certain embodiments, the device is a biodegradable implant for treating an ocular medical condition, comprising an active agent dispersed within a biodegradable polymer matrix, wherein at least about 75% of the particles of the active agent have a diameter of less than about 10 pm. The biodegradable implant is sized for implantation within an ocular region. The ocular region may be any one or more of the anterior chamber, posterior chamber, vitreous cavity, choroid, suprachoroidal space, conjunctiva, subconjunctival space, extrascleral space, intracorneal space, extracorneal space, sclera, pars plana, surgically induced avascular region, macula, and retina. The biodegradable polymer may be, for example, poly(lactic-co-glycolic acid) (PLGA) copolymer. In certain embodiments, the ratio of lactic acid monomer to glycolic acid monomer in the polymer is about 25 / 75, 40 / 60, 50 / 50, 60 / 40, 75 / 25, or more preferably about 50 / 50 by weight. Additionally, the PLGA copolymer can be about 20, 30, 40, 50, 60, 70, 80, or about 90 percent by weight of the bioerodible implant. In certain preferred embodiments, the PLGA copolymer can be about 30 to about 50 percent by weight of the bioerodible implant, preferably about 40 percent by weight of the bioerodible implant. The cells provided herein (e.g., RPE cells, photoreceptor cells, photoreceptor rescue cells, and corneal endothelial cells) can be implanted with biocompatible polymers such as polylactic acid, poly(lactic-co-glycolic acid), PDLGA 50:50, Pd Lg A 85:15, and the biodegradable membrane INION GTR® (a mixture of biocompatible polymers). 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, biodegradable materials can be polymerized.

[0153]

[0162] The volume of a pharmaceutical composition provided by some embodiments of the present disclosure depends on factors such as the mode of administration, the number of cells to be delivered, the age and weight of the patient, and the type and severity of the disease being treated. For example, when administered by injection, the volume of a pharmaceutical composition comprising cells of the present disclosure may be about 1-1000 μL. In some embodiments, the volume may be about 1-200 μL. For example, the volume of a composition of the present disclosure may be about 10-50, 20-50, 25-50, or 1-200 μL. The volume of a composition of the present disclosure may 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 more.

[0154]

[0163] In some embodiments, the cells have a concentration in a cryopreserved formulation (or cryopreserved cell preparation) of about 300, 500, 1 x 10 cells, or about 1 x 10 cells. 3 , 1.5×10 3 , 2 × 10 3 , 2.5×10 3 , 3×10 3 , 3.333×10 3 , 3.5×10 3 , 4×10 3 , 4.4×10 3 , 5×10 3 , 5.5×10 3, 7.5×10 3 , 1×10 4 , 1.5×10 4 , 2 × 10 4 , 2.5×10 4 , 3×10 4 , 3.5×10 4 , 4×10 4 , 4.5×10 4 , 5×10 4 , 5.5×10 4 , 6×10 4 , 6.5×10 4 , 7×10 4 , 7.5×10 4 , 8×10 4 , 8.5×10 4 , 9×10 4 , 9.5×10 4 , 1×10 5 , 1.05×10 5 , 1.1×10 5 , 1.15×10 5 , 1.2 × 10 5 , 1.25×10 5 , 1.3 × 10 5 , 1.35×10 5 , 1.4×10 5 , 1.45×10 5 , or 1.5 × 10 5cells / μL. In some embodiments, the concentration of cells in the cryopreservation formulation (or cryopreserved cell preparation) is approximately 2,000 to 80,000 cells / μL, 2,000 to 70,000 cells / μL, 3,000 to 70,000 cells / μL, 3,000 to 60,000 cells / μL, 3,000 to 50,000 cells / μL, 3,000 to 10,000 cells / μL, 3,000 to 5,000 cells / μL, 3,000 to 4,000 cells / μL, 3,500 cells / μL, 10,000 to 100,000 cells / μL, 10,000 to 90,000 cells / μL, 10,000 to 80,000 cells / μL, 10,000 to 70,000 cells / μL, 10,000 to 60,000 cells / μL, 10,000 to 50,000 cells / μL, 10,000 to 40,000 cells / μL, 10,000 to 30,000 cells / μL, 10,000 to 20,000 cells / μL, 20,000 to 100,000 cells / μL, 20,000 to 90,000 cells / μL, 20,000 to 80,000 cells / μL, 20,000 to 70,000 cells / μL, 20,000 to 60,000 cells / μL, 20,000 to 50,000 cells / μL, 20,000 to 40,000 cells / μL, 20,000 to 30,000 cells / μL, 30,000 to 100,000 cells / μL, 30,000 to 90,000 cells / μL, 30,000 to 80,000 cells / μL, 30,000 to 70,000 cells / μL, 30,000 to 60,000 cells / μL, 30,000 to 50,000 cells / μL, 30,000 to 40,000 cells / μL, 40,000 to 100,000 cells / μL, 40,000 to 90,000 cells / μL, 40,000 to 80,000 cells / μL, 40,000 to 70,000 cells / μL, 40,000 to 60,000 cells / μL, 40,000 to 50,000 cells / μL, 50,000 to 100,000 cells / μL, 50,000 to 90,000 cells / μL, 50,000 to 80,000 cells / μL, 50,000 to 70,000 cells / μL, 50,000 to 60,000 cells / μL, 60,000 to 100,000 cells / μL, 60,000 to 90,000 cells / μL, 60,000 to 80,0 cells / μL, 70,000 - 100,000 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, 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 preparation supports survival of cells within the cell population during storage of the preparation. In some embodiments, the preparation supports 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 preparation supports 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, or about 1 week to about 2 weeks. In some embodiments, the preparation 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 between about 1 day and about 7 days, between about 1 day and about 5 days, between about 1 day and about 3 days, or between about 1 day and about 2 days. The preparation may be stored at a temperature ranging, without limitation, from about -100 to about -200°C, or from about -135°C to about -200°C.

[0155]

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

[0156]

[0166] The cell preparations provided herein are suitable for administration to a subject after thawing and minimal processing (e.g., dilution). In some embodiments, the preparations consist essentially of cells, cell populations, or tissues and the cryopreserved formulations provided herein. In some embodiments, the preparations include one or more active pharmaceutical ingredients, such as preservatives, antioxidants, radical scavengers, immunosuppressants, pro-angiogenic factors, anti-angiogenic factors, hormones (e.g., growth hormones), or cell nutrients or substrates that support cell growth, survival, and engraftment.

[0157]

[0167] Preservatives may include, but are not limited to, benzalkonium chloride (BAK), benzethonium chloride, chlorobutanol, phenylmercuric acetate or nitrate, thimerosal, polyquaternium 1 (POLYQUAD®), stabilized oxychloro complex (SOC) (PURITE®), or methylparaben or propylparaben. Antioxidants and / or radical scavengers may include, but are not limited to, vitamin C, vitamin E, beta-carotene, coenzyme Q-10, selenium, thioredoxin reductase, glutathione, alpha-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 antibody, anti-thymocyte globulin (ATG) polyclonal antibody, azathioprine, BASILIXIMAB® (anti-IL-2Rα receptor antibody), cyclosporine (cyclosporine A), DACLIZUMAB® (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, RITUXIMAB® (anti-CD20 antibody), sirolimus, and tacrolimus, mesenchymal stem cells, and mycophenolate mofetil. Angiogenesis-promoting 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 beta (TGF beta), ranibizumab (LUCENTIS®) or bevacizumab (AVASTIN®), tissue inhibitors of metalloproteinases (TiMPs), plasminogen activator inhibitors (PAIs), prolactin, interferons, and angiopoietin 2. Hormones and hormone antagonists can include 17B-estradiol, adrenocorticotropic hormone, adrenomedullin, alpha-melanocyte-stimulating hormone, chorionic gonadotropin, corticosteroid-binding globulin, corticosterone, dexamethasone, estriol, follicle-stimulating hormone, gastrin 1, glucagon, gonadotropins, 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. Cellular 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 acids. Substrates that support cell growth, survival, and implantation may include, but are not limited to, extracellular matrix derived from bovine corneal epithelium, Matrigel™ (basement membrane substrate), or gelatin.

[0158]

[0168] Pharmaceutical packs and / or kits are also encompassed by the present disclosure. The provided pharmaceutical packs and / or kits 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 before adding the diluent. In some embodiments, the diluent is GS2, GS2 Plus, BSS, BSS Plus®, 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 further include instructions for thawing the cryopreserved pharmaceutical composition, optionally combining the composition with a diluent, and then administering the resulting composition to a subject. In some embodiments, the pharmaceutical composition is not cryopreserved. In some embodiments, the instructions further include instructions for combining the composition with a diluent and then administering the resulting composition to a subject.

[0159]

[0169] Some embodiments of the present disclosure provide a method comprising contacting a cell population with the cell cryopreservation formulation provided herein. The cell population can be a population of RPE cells, a population of mesenchymal stem cells, a population of mesenchymal cells, a population of photoreceptor cells, a population of photoreceptor precursor cells, a population of photoreceptor rescue cells, a population of retinal ganglion cells, a population of retinal ganglion precursor cells, a population of corneal endothelial cells, or a population of corneal endothelial precursor cells. The cell population can be generated by in vitro differentiation of pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells. The cell population can be human or non-human cells.

[0160]

[0170] Some aspects of the present disclosure provide methods that include contacting a cell population with a cell cryopreservation formulation provided herein to form a pharmaceutical composition, and then cryopreserving the pharmaceutical composition. The pharmaceutical composition can be cryopreserved using any suitable process, including any suitable process at any suitable 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 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 vapor phase of liquid nitrogen.

[0161]

[0171] Some embodiments of the present disclosure relate to a method for treating a disease or condition, comprising: (a) diluting a cell preparation with a diluent in a ratio ranging from about 1:1 to about 1:20, or from about 1:2 to about 1:20, or from about 1:3 to about 1:20, or from about 1:4 to about 1:20, or from about 1:5 to about 1:20, or from about 1:4 to about 1:19, or from about 1:4 to about 1:18, or from 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, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, and 1:20 or greater, e.g., 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, to form a diluted cell preparation; and (b) administering the diluted cell preparation to a subject. In some embodiments, the diluent is GS2 or GS2 Plus. 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 presented in the examples, no difference was observed between embodiments in which the cell preparation was diluted with a warm diluent and embodiments in which the cell preparation was diluted with a cold diluent. For example, no difference in cell viability was observed between embodiments in which the cell preparation was gently pipetted after dilution and embodiments in which the cell preparation was vigorously pipetted after dilution into a storage container or any container to which the cell preparation is transferred for dilution.

[0162] Exemplary Uses and Methods

[0172] The cell preparations disclosed herein, including cryopreserved preparations, can be used in a variety of clinical applications once thawed. As mentioned herein, cryopreserved preparations can be thawed by simply adding a diluent, such as GS2 or GS2 Plus, and then administered to a subject without the need for washing or other manipulations. In some embodiments, cryopreserved preparations can be thawed without adding a diluent, and then administered to a subject without the need for washing or other manipulations.

[0163]

[0173] Cryopreserved preparations may contain dissociated cells, cell monolayers, cell aggregates, cell clusters, or cell sheets. The cryopreserved preparations provided herein are compatible with a variety of cell types, cell populations, and tissues, including, but not limited to, adult stem cells and progenitor cells, differentiated cells, and populations and tissues containing such cells. In some embodiments, the cells, cell populations, or tissues thus formulated are intended for clinical use in regenerative medicine. For example, the cells, cell populations, or tissues may be cells, cell populations, or tissues that can replace lost or degenerated cells in a subject, or cells, cell populations, or tissues that can repair or replace damaged or dysfunctional tissues in a subject. For example, in some embodiments, the cells, cell populations, or tissues may include pluripotent stem cells, such as iPS cells or ES cells, multipotent stem cells, or multipotent progenitor cells, or functionally differentiated cells, or populations or tissues containing such cells or combinations of such cells.

[0164]

[0174] Cells, cell populations, or tissues that may 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 stem or progenitor cells or neuronal stem or progenitor cells, glial stem or progenitor cells, pancreatic stem or progenitor cells, beta cells, keratinocytes, chondrocytes, osteoblasts, osteoclasts, or populations or tissues comprising or consisting essentially of such cells, such as monolayers or sheets, or aggregates or clusters of RPE cells, corneal endothelial cells, photoreceptor rescue cells, pancreatic islets, or skin grafts.

[0165]

[0175] In some embodiments, cryopreserved cell preparations can be stored indefinitely or for a specified period of time, and optionally, cryopreserved cell preparations can be transported to clinical sites or other end-user sites.In particular, cryopreserved cell preparations can be thawed by simply adding diluent, and then administered to subjects without further manipulation.Cryopreserved formulations are typically low-volume formulations.For example, cryopreserved formulations can have a volume of less than 1 mL, less than 500 μL, less than 250 μL, less than 100 μL, or less than or about 50 μL, or any volume disclosed herein. The cryopreserved formulation may 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 reach a volume and cell concentration suitable for intended clinical use. The ratio of cryoprotectant formulation volume to diluent volume may be expressed as a ratio, and may be 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, 1:14, 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 greater.

[0166]

[0176] Cryopreserved cell preparations typically contain at least 3 x 10 cells per μL. 2 pieces or approximately 3 x 10 2 or at least 10 per μL 3 pieces or about 10 3 At least 2 x 10 cells per µL 3 or approximately 2 x 10 3 At least 3 x 10 cells per µL 3 or approximately 3 x 10 3 At least 3.5 x 10 cells per µL 3 pieces or approximately 3.5 x 10 3 At least 5 x 10 cells per µL 3 pieces or approximately 5 x 10 3 At least 10 per μL 4 pieces or about 10 4 At least 5 x 10 cells per µL 4 pieces or approximately 5 x 10 4 At least 10 per μL 5 pieces or about 10 5 or at least 5 x 10 per µL 5 pieces or approximately 5 x 10 5 As will be appreciated by those skilled in the art, the cell concentration and intended use will guide the degree of dilution required prior to administration to a subject.

[0167]

[0177] Cryopreserved formulations may be stored under frozen conditions, such as in the vapor phase of a liquid nitrogen tank, with minimal impact on cell viability or cell function, e.g., as measured by dye exclusion, replating efficiency, or repopulation ability, for extended periods of time, such as those enumerated 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 more. Cells or cell populations cryopreserved and thawed according to the present disclosure may have 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 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 110% of their pre-cryopreservation viability, replating efficiency, and / or repopulation potential. 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, replating efficiency, and / or repopulation ability.

[0168]

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

[0169]

[0179] Cryopreserved cell preparations are made by combining the desired cells, cell populations, or tissues in the cryoprotective formulations presented herein, optionally dispersing the cells, cell populations, or tissues in the formulation, and then slowly freezing the mixture. Slow freezing can be achieved 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 it reaches a desired temperature, optionally an intermediate temperature, such as -80°C. Alternatively, the freezing rate can be -1.5°C per minute or -2°C per minute. Cryopreserved cell preparations can be kept at intermediate temperatures for several hours, days, or weeks, or longer. The cryopreserved cell preparations can then be kept at low temperatures. By way of example, the preparation can be brought to -80°C using a slow freezing process, then stored at -80°C overnight (or for up to about 12 hours or about 24 hours), and then stored in the vapor phase of a liquid nitrogen tank (e.g., at about -125°C). Once at the low temperature, the preparation can be stored in the vapor phase of a liquid nitrogen tank for days, weeks, months, or even years. The preparation can even be transported, for example, to a clinical facility while in cryogenically frozen storage, if so desired. The temperature of the cryoprotected formulation can be, for example, between 0°C and 45°C, e.g., 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°C, about 37°C, and about 40°C.

[0170]

[0180] Some aspects of the present disclosure provide methods for treating a subject with a disorder or condition that would 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, optionally without dilution with a diluent, to a subject in need thereof. 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, diluted cell preparation to a subject in need thereof. In some embodiments, the diluent is GS2, GS2 Plus, BSS, BSS Plus®, 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 have or be diagnosed with a disease or disorder that can be treated by administering cells, cell populations, or tissues. The cell preparation is administered at a cell dose effective to treat the disorder or condition in the subject. In some embodiments, the disorder or condition includes, but is not limited to, retinal detachment, retinal dysplasia, retinal pigmentary streaks, myopic macular degeneration, or retinal atrophy, or is associated with some vision-altering diseases that result in photoreceptor damage and blindness, such as congenital 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 flava). In some embodiments, the method optionally further comprises monitoring at least one symptom of the disorder or condition in the subject to determine whether such symptom is reduced in severity or frequency after administration.

[0171]

[0181] The terms "treatment," "treating," and "treating" refer to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of a disorder or condition. In some cases, the effectiveness of the intervention may be assessed by monitoring symptoms associated with the disorder or condition. In some embodiments, the cell preparation may be administered after one or more symptoms have developed and / or after the disorder or condition has been diagnosed. In other embodiments, the cell preparation may be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms, or inhibit the onset or progression of a disorder or condition. For example, the cell preparation may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). The cell preparation may be administered once or more than once, including administration regularly and at defined time points, or on an as-needed basis.

[0172]

[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 genetically engineered, e.g., a genetically engineered non-human subject. The subject can be of either gender and at any stage of development.

[0173]

[0183] As used herein, the term "effective amount" refers to the amount of a bioactive agent that is sufficient to induce a desired biological response.For example, in some embodiments, an effective amount of a cell preparation may refer to the amount of preparation containing some cells, or the amount of tissue sufficient to provide a therapeutic benefit to a subject with a disorder or condition, for example, the amount of tissue sufficient to improve vision in a subject with a retinal disease or disorder.As will be appreciated by those skilled in the art, the effective amount may vary depending on a variety of factors, such as the desired biological response, for example, the specific disorder to be treated, the specific symptoms to be alleviated, or the cells or tissue to be targeted, as well as the age, sex, and general health of the subject.

[0174]

[0184] Some aspects of the present disclosure provide a method for treating a retinal disease, comprising administering an effective amount of a cell preparation provided herein to the eye of a subject with the retinal disease. In some embodiments, the subject has or has been diagnosed with a retinal disease. In some embodiments, the retinal disease is rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration (e.g., wet age-related macular degeneration or dry age-related macular degeneration), Leber's congenital amaurosis, or Stargardt's disease. In some embodiments, the administered cells 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 generated by in vitro differentiation of pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells.

[0175] kit

[0185] Some embodiments of the present disclosure provide kits that include (a) a cryopreserved pharmaceutical composition comprising a cell of interest as 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, thereby thawing and diluting the composition and rendering it suitable for administration to a subject. These instructions may also provide that no washing or other manipulation of the cryopreserved composition is necessary after thawing. The kit may further include a device for administering the composition, such as a syringe and needle.

[0176]

[0186] Thus, the present disclosure provides, among other things: Appendix 1: A formulation containing approximately 4-10% (v / v) cryoprotectant, approximately 2-8% (w / v) albumin, approximately 0-1.5% (w / v) glucose, and a buffer solution. Appendix 2: The formulation of Appendix 1, wherein the cryoprotectant is selected from DMSO, glycerol, and ethylene glycol. Appendix 3: The formulation of Appendix 1, wherein the cryoprotectant is DMSO. Appendix 4: The formulation of any one of Appendixes 1 to 3, comprising about 4-6% (v / v) cryoprotectant. Appendix 5: The formulation of any one of Appendixes 1 to 4, comprising about 0.08 to 0.10% (w / v) glucose. Appendix 6: The formulation of any one of Appendixes 1-5, comprising about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and a buffer. Appendix 7: The formulation of any one of Appendixes 1 to 4, comprising about 0.6% (w / v) glucose. Appendix 8: The formulation of any one of Appendixes 1-4 and 7, comprising about 5% DMSO, about 2.5% albumin, about 0.6% glucose, and a buffer. Appendix 9: The formulation of any one of Appendixes 1 to 8, wherein the albumin is human albumin. Appendix 10: The formulation of any one of Appendixes 1 to 9, wherein the albumin is recombinant human albumin. Appendix 11: The formulation of any one of Appendixes 1 to 10, wherein the buffer is a buffered saline solution. Appendix 12: The formulation of any one of Appendixes 1 to 11, wherein the buffer is phosphate buffered saline (PBS). Appendix 13: The formulation of Appendix 11 or 12, wherein the buffered saline solution comprises Ca2+ and Mg2+. Appendix 14: The formulation of Appendix 11 or 12, wherein the buffered saline solution is free of Ca2+ and Mg2+. Appendix 15: The formulation of any one of appendices 1 to 14, further comprising a cell population or tissue. Appendix 16: The formulation of Appendix 15, wherein the cell population is a dissociated cell population. Appendix 17: The formulation of Appendix 15, wherein the cell population comprises a cell monolayer, a cell sheet, a cell cluster, a cell sphere, or a cell aggregate. Appendix 18: The formulation of Appendix 15, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells. Appendix 19: The formulation of any one of Appendixes 1 to 18, which is cryopreserved. Appendix 20: The formulation of any one of Appendixes 1 to 19, which does not contain a polymeric excipient. Appendix 21: The formulation of any one of Appendixes 1 to 20, which does not contain dextran. Appendix 22: (a) a formulation according to any one of Appendixes 1 to 14, and (b) Cell population A cell preparation comprising: Appendix 23: The cell preparation of Appendix 22, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells. Appendix 24: The cell preparation of Appendix 22 or 23, wherein the cell population has a concentration of about 10,000 to 100,000 cells / μL in the cell preparation. Appendix 25: The cell preparation of Appendix 22 or 23, wherein the cell population has a concentration of about 15,000 to 50,000 cells / μL in the cell preparation. Appendix 26: The cell preparation of any one of Appendixes 22 to 25 in a vial, syringe, ampoule, bottle, or cooler package for cryopreservation. Appendix 27: The cell preparation of Appendix 26, wherein about 10 to about 500 μL of the preparation is in a vial, syringe, ampoule, bottle, or refrigerated package. Appendix 28: Approximately 10 cells 5 ~10 7 28. The cell preparation of claim 26 or 27, wherein the cells are present in a vial, syringe, ampoule, bottle, or refrigerated package. Appendix 29: Cells approximately 10 5 ~5×10 6 26. The cell preparation of claim 24 or 25, wherein the cells are present in a vial, syringe, ampoule, bottle, or refrigerated package. Appendix 30: Approximately 10 cells 5 ~2×10 6 26. The cell preparation of claim 24 or 25, wherein the cells are present in a vial, syringe, ampoule, bottle, or refrigerated package. Appendix 31: The cell preparation of any one of Appendixes 22 to 30, which is cryopreserved. Appendix 32: A method for preparing a cell preparation, the method comprising contacting a population of cells with a formulation described in any one of appendices 1 to 14. 33. The method of claim 29, further comprising cryopreserving the cell preparation. Appendix 34: The method of appendix 32 or 33, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells. Appendix 35: A method of treating a subject having a disorder or condition, comprising: thawing the cell preparation of claim 31 and diluting the cell preparation with a diluent at a ratio selected from the group consisting of 1:2, 1:3, 1:4, and 1:5; administering the diluted cell preparation to a subject; A method comprising: Appendix 36: The method of Appendix 35, wherein the disorder or condition is selected from the group consisting of retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration, or retinal atrophy, or is associated with several vision-altering diseases that result in photoreceptor damage and blindness, such as, for example, congenital choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (fundus flava). Appendix 37: The method of Appendix 35, wherein the diluent is GS2, GS2 Plus, BSS, BSS Plus®, dextran, and HSA solution, PBS, DMEM, MEM, or albumin. Appendix 38: A method of treating a subject having a disorder or condition, comprising: thawing the cell preparation of claim 31 and diluting the cell preparation with a diluent at a ratio selected from the group consisting of 1:15, 1:16, 1:17, 1:18, and 1:19; administering the diluted cell preparation to a subject; A method comprising: Appendix 39: The method of Appendix 37, wherein the disorder or condition is selected from the group consisting of retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration, or retinal atrophy, or is associated with several vision-altering diseases that result in photoreceptor damage and blindness, such as, for example, congenital choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (fundus flava). Appendix 40: The method of Appendix 38, wherein the diluent is GS2 or GS2 Plus, BSS, BSS Plus®, dextran, and HSA solution, PBS, DMEM, MEM, or albumin. Appendix 41: (a) a cryopreserved cell preparation according to Appendix 31; (b) a diluent; (c) instructions for diluting the cell preparation prior to administration to a subject; Kit including: Appendix 42: The kit of Appendix 41, wherein the instructions further comprise instructions for thawing the cryopreserved cell preparation. Appendix 43: A pharmaceutical composition comprising the formulation of any one of Appendixes 1 to 14 in combination with a cell population. Appendix 44: The pharmaceutical composition of Appendix 43, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells. Appendix 45: The pharmaceutical composition of Appendix 43, wherein the cell population comprises neural cells, neural stem cells, or neural progenitor cells. Appendix 46: A pharmaceutical composition comprising the formulation of any one of appendices 15 to 21 and a diluent, wherein the ratio of formulation to diluent is in the range of 1:2 to 1:20, and suitable for administration to a subject, optionally ocularly, and further optionally subretinally. Appendix 47: A pharmaceutical composition comprising a cell preparation and a diluent according to appendices 22-25 and 31, wherein the cell preparation to diluent ratio is in the range of 1:2 to 1:20, and suitable for administration to a subject, optionally ocularly, and further optionally subretinally. Appendix 48: A cell preparation comprising the formulation of any one of appendices 1-14, 19, 20, and 21 and a diluent, wherein the ratio of the formulation to the diluent is in the range of 1:2 to 1:20, and wherein the formulation comprises a population of cells, the cell preparation being suitable for administration to a subject, optionally ocular administration, and further optionally subretinal administration. Appendix 49: The pharmaceutical composition of Appendix 46 or 47, or the cell preparation of Appendix 48, which is not washed before or after dilution with a diluent. Appendix 50: The pharmaceutical composition of Appendix 46 or 47, or the cell preparation of Appendix 48, which is not centrifuged prior to or after dilution with a diluent. Appendix 51: The pharmaceutical composition of Appendix 46 or 47, or the cell preparation of Appendix 48, wherein the formulation or cell preparation is cryopreserved prior to dilution with a diluent. Appendix 52: The pharmaceutical composition or cell preparation of any one of Appendixes 48 to 51, wherein DMSO is present in a detectable amount at a level of about 1% (v / v) or less. Appendix 53: The pharmaceutical composition or cell preparation of any one of Appendixes 48 to 51, wherein DMSO is present in a detectable amount at a level of about 0.037 mg or less per dose. Appendix 54: A cell preparation comprising the formulation and cell population of any one of appendices 1 to 14, wherein the cell preparation is thawed via dilution with a diluent and is not washed. Appendix 55: The cell preparation of Appendix 54, diluted with a diluent at a ratio selected from the group consisting of 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 of about 0.037 mg or less per dose. Appendix 57: The cell preparation of any one of Appendixes 54 to 56, wherein the diluent is GS2 or GS2 Plus. [Example]

[0177]

[0187] Having now generally described the invention, it will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0178] Example 1 Cryoprotectants preserve post-thaw cell viability and proliferation

[0179]

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

[0180]

[0189] RPE cells were resuspended with 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. Nine formulations containing different concentrations of DMSO, albumin, and glucose were prepared as shown in Table 1.

[0181] [Table 1]

[0182]

[0190] 30 μL of formulated RPE cells were filled into Crystal Zenith® (CZ) vials (West Pharmaceutical Services, Inc., Exton, PA) and cryopreserved using Corning® CoolCell® containers (Corning Life Sciences, Tewksbury, MA) for slow cryopreservation. The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0183]

[0191] Pre-freeze cell viability was assessed using the Trypan Blue cell counting method. As a result, no significant differences were observed among the nine formulations (see Figure 1). Pre-freeze viability was greater than 95% for all formulations.

[0184]

[0192] Post-thaw cell viability was assessed using a manual Trypan Blue cell counting method and an automated method using a NucleoCounter® NC-200™ (ChemoMetec Inc., Bohemia, NY) instrument. No significant differences were observed among the nine formulations using either method (see Figure 2) (n=3). Post-thaw viability was greater than 90% for all formulations, ranging from 94% to 98%.

[0185]

[0193] Post-thaw cell proliferation was assessed using the CyQUANT® Cell Proliferation Assay (Thermo Fisher Scientific, Waltham, MA). Cryopreserved RPE vials were thawed, seeded in growth medium in 96-well plates, and cultured. Plates were frozen on days 2 and 4 to assess their cell proliferation index, defined by the DNA concentration extracted from the cells after cell lysis. No significant differences were observed among the nine formulations (see Figure 3).

[0186] Example 2 Cryoprotectants reduce the occurrence of multinucleated cells

[0187]

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

[0188]

[0195] RPE cells were resuspended in 2.5% rHA / DPBS at a density of 50,000 cells / μL and then mixed with a cryoprotectant-containing buffer at a volume ratio of 1:1. Five formulations, each containing 50 mM trehalose, 7.5% dextran 40, and 2.5% rHA in DPBS, and five formulations containing 1% DMSO, 2.5% DMSO, 5% DMSO, 5% glycerol, or 5% ethylene glycol, were prepared as shown in Table 2.

[0189] [Table 2]

[0190]

[0196] 10 μL of formulated RPE cells were added to a cryovial and cryopreserved using a Mr. Frosty™ Freezing Container (Thermo Fisher Scientific, Inc., Waltham, MA) for slow cryopreservation. The vial was then transferred and stored in the vapor phase of liquid nitrogen.

[0191]

[0197] Cryopreserved RPE cells were thawed, seeded, and cultured in growth medium for 3 days and assessed under a microscope for the presence of multinucleated cells. RPE cells in Formulation A (1% DMSO) showed some multinucleated cells with more than four nuclei per cell within a field of several hundred RPE cells (see Figure 4A), while Formulation B (2.5% DMSO) showed one multinucleated cell with more than four nuclei per cell within a field of several hundred RPE cells.

[0192]

[0198] RPE cells in formulations C (5% DMSO) and E (5% ethylene glycol) did not show any multinucleated cells with more than three nuclei within a field of several hundred RPE cells, and RPE cells in formulation D (5% glycerol) did not show any multinucleated cells with more than four nuclei within a field of several hundred RPE cells (Figure 4B).

[0193]

[0199] These results indicate that cell-permeant cryoprotectants such as DMSO, glycerol, and ethylene glycol reduce the occurrence of multinucleated cells.

[0194] Example 3 Effects of other excipients on multinucleation, cell proliferation, and viability

[0195]

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

[0196]

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

[0197] [Table 3]

[0198]

[0202] 10 μL of formulated RPE cells were added to a cryovial and cryopreserved using a Mr. Frosty™ Freezing Container (Thermo Fisher Scientific, Inc., Waltham, MA) for slow cryopreservation. The vial was then transferred and stored in the vapor phase of liquid nitrogen.

[0199]

[0203] Post-thaw cell viability was assessed using the Trypan Blue cell counting method. Post-thaw cell proliferation was also assessed using the CyQUANT® Cell Proliferation Assay (Thermo Fisher Scientific, Waltham, MA). Cryopreserved RPE vials were thawed, seeded in growth medium in 96-well plates, and cultured. Plates were frozen on days 2 and 3 to assess DNA concentration as a cell proliferation index. No significant differences were observed among the eight formulations (see Figure 5A) (viability: n=1, cell proliferation: n=3). Multinucleated cells were observed for all 2.5% glycerol conditions, but not for 5% glycerol (Figure 5B).

[0200]

[0204] The RPE cells were pooled in a 50 mL tube and washed with 30 mL of 0.5% rHA / DPBS via centrifugation at 160×g for 5 minutes. The wash was repeated a total of three times.

[0201]

[0205] RPE cells were resuspended with 2.5% rHA / DPBS and then mixed with the cryoprotectant-containing buffer at a volume ratio of 1:1. Five formulations were prepared as shown in Table 4.

[0202] [Table 4]

[0203]

[0206] 10 μL of formulated RPE cells were added to a cryovial and cryopreserved using a Mr. Frosty™ Freezing Container (Thermo Fisher Scientific, Inc., Waltham, MA) for slow cryopreservation. The vial was then transferred and stored in the vapor phase of liquid nitrogen.

[0204]

[0207] Post-thaw cell viability was assessed using trypan blue cell counting. Post-thaw cell proliferation was also assessed using the CyQUANT® Cell Proliferation Assay (Thermo Fisher Scientific, Waltham, MA). Cryopreserved RPE vials were thawed, seeded in growth medium in 96-well plates, and cultured. Plates were frozen on days 2 and 3 to assess DNA concentration as a cell proliferation index. No significant differences were observed among the five formulations (see Figure 6A) (viability: n = 1, cell proliferation: n = 3). Multinucleated cells were observed with 2.5% glycerol and 2.5% propanediol (e.g., 1,2-propanediol), but not with 5% DMSO or 5% glycerol (see Figure 6B).

[0205] Example 4 Effect of albumin on adsorption and cryoprotection

[0206]

[0208] The RPE cells were pooled into a 225 mL bottle and washed with 200 mL of 0.5% rHA / DPBS via centrifugation at 500×g for 5 minutes. The wash was repeated a total of four times.

[0207]

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

[0208] [Table 5]

[0209]

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

[0210] [Table 6]

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

[0212] [Table 7]

[0213]

[0212] Based on the results, albumin exhibits a stabilizing effect in the formulation process.

[0214] Example 5 The cell therapy formulation did not demonstrate toxicity

[0215]

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

[0216] [Table 8]

[0217] 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, diluted, and stored at NIH-Lyst before administration. bg-J Foxn1 nu Btk xid (NIH III) immunocompromised mice were administered a single unilateral dose via subretinal injection. Animals were evaluated after 1-day and 14-day observation periods. Toxicity assessment was based on clinical observations, body weight and food intake measurements, ophthalmologic examinations, and clinical and anatomic pathology assessments.

[0218]

[0215] No toxicity was observed in any of the formulations.

[0219] Example 6 Applicability to different cell types

[0220]

[0216] Photoreceptor rescue cells (PRCs) were pooled into 50 mL tubes and centrifuged using OptiPrep™ (STEMCELL Technologies, Inc., Vancouver, Canada) to remove dead cells and debris from the cell suspension. Following this, the cells were washed first with growth medium at 300 x g for 10 minutes and then with 0.5% rHA / DPBS via centrifugation at 300 x g for 10 minutes.

[0221] Photoreceptor rescue cells were resuspended with 0.5% rHA / DPBS and then mixed with a cryoprotectant-containing buffer solution at a volume ratio of 1:2. PRC cells were prepared in a formulation containing 2.5% rHA, DPBS with 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 CryoStor® CS10 cell freezing medium (STEMCELL Technologies, Inc., Vancouver, Canada) containing 10% DMSO.

[0222] Cells were added to Crystal Zenith® (CZ) vials (West Pharmaceutical Services, Inc., Exton, PA) at 70,000 cells / μL in 50 μL (3.5 million cells total per vial) 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 if the cells are diluted 1:4 in buffer (e.g., GS2 Plus buffer) (leading to a loss of approximately 50% of the cells due to cell death and loss upon harvesting); 300,000 cells in 150 μL if the cells are diluted 1:17 in GS2 Plus buffer (leading to a loss of approximately 50% of the cells due to cell death and loss upon harvesting).

[0223] Post-thaw cell viability was assessed using the Trypan Blue cell counting method. The formulation of the present invention containing 5% DMSO, 2.5% rHA, DPBS with Ca / Mg, and 0.6% glucose showed similar viability to the formulation of the present invention containing 10% DMSO, 2.5% rHA, DPBS with Ca / Mg, and 0.6% glucose (see Figure 7A) (n=3). All formulations of the present invention showed higher viability than the commercially available CryoStor® CS10. Post-freeze-thaw PRC viability was also determined using formulations of the present invention containing 5% DMSO, 7.5% DMSO, and 10% DMSO (see Figure 7B).

[0224] Example 7 Applicability to different cell types (2)

[0225]

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

[0226] The CEC cell pellet was then resuspended with a formulation containing 2.5% rHA, 0.09% glucose, DPBS with Ca and Mg, and 5% or 10% DMSO. The target cell density was 30,000 cells / μL.

[0227] 50 μL of formulated CEC cells were added to a cryovial and stored frozen in a Corning® CoolCell® container (Corning Life Sciences, Tewksbury, MA) at a freezing rate of approximately −1° C. / min. After overnight storage at −80° C., the vial was transferred and stored in the vapor phase of liquid nitrogen.

[0228] Post-thaw cell viability was assessed using the trypan blue cell count method. Vials were thawed in a 37°C water bath and diluted with DPBS before mixing with Trypan Blue reagent. Cell viability was 89.3% and 88.2% for the 5% and 10% DMSO-containing freeze buffers, respectively.

[0229] For comparison, CEC cells were also cryopreserved at low cell density (3 million cells per mL) in 2.5% rHA, 0.09% glucose, DPBS with Ca and Mg, and 5% DMSO in a standard volume (1 mL / vial). Post-thaw cell viability under these conditions was 86.2%, as determined by the Trypan Blue cell counting method (see FIG. 8).

[0230] Example 8 Further candidates for cell therapy formulations

[0231] Human embryonic stem cell-derived RPE cells were pooled into a 50 mL tube and washed with 30 mL of 0.5% rHA / DPBS via centrifugation at 160 × g for 5 minutes. The wash was repeated a total of three times.

[0232] RPE cells were resuspended with 2.5% rHA / 0.09% glucose / DPBS and then mixed with a cryoprotectant-containing buffer at a volume ratio of 1:1. Formulations containing 5% glycerol, 200 mM taurine, or 100 mM trehalose, 0.09% glucose, and 2.5% rHA in DPBS were prepared as shown in Table 9 (Cryopreservation Formulation 5 and Cryopreservation Formulation 6).

[0233] [Table 9]

[0234]

[0227] 10 μL of formulated RPE cells were added to a cryovial and cryopreserved using Corning® CoolCell® containers (Corning Life Sciences, Tewksbury, MA) for slow cryopreservation. The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0235] Post-thaw cell viability was assessed using the Trypan Blue cell counting method. Post-thaw cell proliferation was also assessed using the CyQUANT® Cell Proliferation Assay (Thermo Fisher Scientific, Waltham, MA). Cryopreserved RPE vials were thawed, seeded in growth medium in 96-well plates, and cultured. Plates were frozen on days 2 and 3 to assess DNA concentration as a cell proliferation index. As shown in Table 10, no significant differences were observed between cryopreserved formulations 1, 5, and 6, and no multinucleated cells with more than four nuclei were observed among hundreds of RPE cells for cryopreserved formulations 1, 5, and 6.

[0236] [Table 10]

[0237] Cryopreserved Formulation 2 did not contain a cell-permeable cryoprotectant, potentially causing toxicity to cells, and also exhibited high viscosity due to dextran 40. Furthermore, multinucleated cells were observed in RPE cells cryopreserved in Cryopreserved Formulation 2. Cryopreserved Formulations 3 and 4 showed a small number of multinucleated cells, but also exhibited high viscosity due to dextran 40 in the formulation. Cryopreserved Formulations 5 and 6 lacked multinucleated cells and exhibited low viscosity due to the absence of polymeric excipients. Equivalence and Scope, Incorporation by Reference

[0238]

[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 set forth in the appended claims.

[0239]

[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 as well as a plurality of such agents.

[0240]

[0232] Unless indicated to the contrary or otherwise clear from the context, a claim or description containing "or" between two or more members of a group is considered to be satisfied if one, more than one, or all of the group members are present. A disclosure of a group containing "or" between two or more group members presents 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 purposes of brevity, these embodiments have not been individually detailed herein, but it will be understood that each of these embodiments may or may not be presented and specifically claimed herein.

[0241] It is also to be understood that the present invention encompasses all variations, combinations, and permutations of one or more limitations, elements, clauses, or descriptive terms introduced into another claim from one or more of the claims or from one or more relevant portions of the specification. For example, a claim that is dependent on another claim may be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, if a claim recites a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included unless otherwise indicated or unless a contradiction or inconsistency would arise apparent to one of ordinary skill in the art.

[0242]

[0234] When elements are presented as a list, for example, in Markush group format, it is to be understood that all possible subgroups of the elements are also disclosed, and that any element or subgroup of elements is excluded from the group. It should also be noted that the term "comprising" is intended to be an open term, allowing for the inclusion of additional elements or steps. Generally, when referring to an embodiment, product, or method that includes a particular element, feature, or step, it should be understood that an embodiment, product, or method that consists of or consists essentially of such element, feature, or step is also presented. For purposes of brevity, these embodiments have not been individually detailed herein, but it will be understood that each of these embodiments is presented herein and may or may not be specifically claimed.

[0243]

[0235] When ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or apparent from the context and / or the understanding of one of ordinary skill in the art, it is understood that values ​​expressed as ranges can assume any specific value within the stated range, and in some embodiments, to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, although the values ​​within each range have not been individually detailed herein, it will be understood that each of these values ​​may or may not be presented and specifically claimed herein. It is also understood that, unless otherwise indicated or apparent from the context and / or the understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any subranges within the given range, in which case the endpoints of the subranges are expressed to the same degree of precision as one-tenth of the unit of the lower limit of the range.

[0244]

[0236] Additionally, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may be explicitly excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the present invention may be excluded from any one or more of the claims. For purposes of brevity, this specification does not specify all of the embodiments in which one or more elements, features, purposes, or aspects are excluded.

[0245] All publications, patents, patent applications, publications, and database data entries (e.g., sequence database data entries) mentioned herein, e.g., in the "Background" section, "Summary" section, "Description of the Invention" section, "Examples" section, and / or "References" section, are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, patent application, publication, and database data entry was specifically and individually incorporated herein by reference. In the case of conflict, the present application, including any definitions therein, will control.

Claims

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

2. The formulation according to claim 1, wherein the cryoprotectant is selected from DMSO, glycerol, and ethylene glycol.

3. The formulation according to claim 2, wherein the cryoprotectant is DMSO.

4. The formulation according to claim 1, comprising approximately 4-6% (v / v) of a cryoprotective agent.

5. The formulation according to claim 1, comprising approximately 0.08 to 0.10% (w / v) glucose.

6. The formulation according to claim 1, comprising approximately 0.6% (w / v) glucose.

7. (i) comprising about 5% (v / v) DMSO, about 2.5% (w / v) albumin, about 0.09% (w / v) glucose, and a buffer, or (ii) A solution comprising approximately 5% (v / v) DMSO, approximately 2.5% (w / v) albumin, approximately 0.6% (w / v) glucose, and a buffer, The formulation according to claim 1.

8. The formulation according to claim 1, wherein the albumin is human albumin.

9. The formulation according to claim 1, wherein the buffer solution is buffered physiological saline.

10. The preparation according to claim 1, which is stored by freezing.

11. The formulation according to claim 1, which does not contain a polymeric excipient or dextran.

12. (a) A formulation according to any one of claims 1 to 11, (b) Cell population Cell preparations containing the above.

13. A group of cells (i) A population of dissociated cells, (ii) including cell monolayers, cell sheets, cell clusters, cell spheres, or cell aggregates, (iii) Retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells, and / or (iv) The cell preparation contains approximately 10,000 to 100,000 cells / μL. The cell preparation according to claim 12.

14. The cell preparation according to claim 12, wherein the preparation is contained in a vial, syringe, ampoule, bottle, or in a refrigerated package in an amount of approximately 10 to 500 μL.

15. The cell preparation according to claim 12, which is cryopreserved.

16. The cell preparation according to claim 15, which has been melted by dilution with a diluent and has not been washed.

17. The cell preparation according to claim 16, wherein the diluent is GS2 or GS2 Plus.

18. A pharmaceutical composition for treating a subject having a disorder or condition, comprising the cell preparation described in Claim 16, wherein the ratio of the cell preparation to a diluent is selected from the group consisting of 1:2, 1:3, 1:4, 1:5, 1:15, 1:16, 1:17, 1:18 and 1:

19.

19. (a) A cryopreserved cell preparation according to claim 15, (b) Diluent and (c) Instructions for diluting the cell preparation before administration to the subject, A kit that includes this.

20. A method for preparing a cell preparation, comprising the step of bringing a preparation according to any one of claims 1 to 11 into contact with a cell population.

21. The method according to claim 20, wherein the cell population comprises retinal pigment epithelial (RPE) cells, photoreceptor rescue cells, photoreceptor progenitor cells, or corneal endothelial cells.

22. A pharmaceutical composition comprising a cryopreserved cell preparation and a diluent according to claim 15, wherein the ratio of the cell preparation to the diluent is in the range of 1:2 to 1:20, and the pharmaceutical composition is suitable for administration to a subject.

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