Cell cryopreservation formulations and methods of use thereof

Abstract

Some aspects of the present disclosure provide improved cryopreservation formulations and improved methods for cryopreserving and thawing cells. Also provided are cell preparations comprising a population of cells and a cryopreservation formulation as provided herein. Also provided are methods for treating a subject having a disease or condition that benefits from transplantation of a population of cryopreserved and thawed cells.

Description

[0001] Related applications This application claims the benefit of U.S. Provisional Application Serial No. 63 / 559,174, filed February 28, 2024, entitled “Cell cryopreservation formulation and method of use,” pursuant to 35 USC § 119(e), the entire contents of which are incorporated herein by reference.

[0002] Reference to electronic sequence listing The contents of the electronic serial number (A102570066WO00-SEQ-CBD.xml; size: 59,322 bytes; and creation date: February 24, 2025) are incorporated herein by reference in their entirety. Background Technology

[0003] Effective cell cryopreservation is an important aspect of many cell therapy strategies. The ability to cryopreserve cell-based therapies, store them for different time periods, and optionally transport them in a frozen state expands their clinical use. Dimethyl sulfoxide (DMSO) is a commonly used, permeable type of cryoprotectant for cell products, used to maintain cell quality during cryopreservation and thawing. However, some cryoprotectants, such as DMSO, can cause cytotoxicity when used in vitro at high concentrations and may also be harmful to patients when administered at high concentrations.

[0004] Existing methods for minimizing the negative effects of certain cryoprotectants (such as DMSO) involve diluting and / or washing cells with physiological buffer to remove the cryoprotectant before administration to the patient, followed by reconstitution with physiological buffer. However, these methods present several challenges, including the need for additional processing steps, which can be costly, and shorten the shelf life of cell preparations. There is a need for cryopreservation formulations that overcome these difficulties and limitations. Summary of the Invention

[0005] The ability to cryopreserve cell-based therapeutics separates the production and clinical use of cell therapies in time and space. The design and selection of suitable cryoprotectant formulations are crucial to ensuring cell survival and functional maintenance. This disclosure provides novel cryopreservation formulations that can be used to cryopreserve cell populations, optionally transport cell populations, and ultimately administer cell populations to subjects in need. Importantly, this disclosure envisions using such formulations to cryopreserve cell populations so that subsequent thawing and administration to subjects can occur without the need for washing or diluting the cells. In this way, the method allows for the freezing and thawing of cell-based therapeutics with virtually no handling steps and manipulations. This reduces the likelihood of cell loss that may result from manipulating the cells during post-thawing.

[0006] This disclosure therefore provides specific cryopreservation formulations and methods for preparing such formulations, as well as methods of using them. The cryopreservation formulations can be used with various cell types. The cryopreservation formulations provided herein result in robust post-thawing viability and cell growth. Furthermore, such formulations are capable of preserving cell populations for extended periods at room temperature and at 4ºC before cryopreservation (e.g., as may occur during the cryopreservation of cell populations) or after thawing. It has also been shown that, as described in the examples, cell populations cryopreserved in such formulations are resistant to temperature fluctuations during cryopreservation (e.g., as may occur during the transport of cell populations).

[0007] The formulations described herein can be used for cryopreservation of various cell types, including but not limited to retinal pigment epithelial (RPE) cells. Therefore, although many examples in this disclosure refer to RPE cells, it should be understood that other cell types may also be used. For example, the formulations provided herein can be used for photoreceptor repair cells (PRCs) (as described in WO2024044134, which is incorporated herein by reference in its entirety), mesenchymal cell populations (e.g., angiogenic mesenchymal cells (HMCs)), vascular progenitor cells (VPCs), photoreceptor cell populations, photoreceptor progenitor cell populations, corneal epithelial cell (CEC) populations, macrophage populations, natural killer cell populations, T-cell populations, vascular cell populations, hepatocyte populations, neural cell populations, and pancreatic cell populations.

[0008] One aspect of this disclosure provides a formulation comprising albumin, glucose, buffered saline, and one or more, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch (HES) with a hydroxyethyl substitution degree ranging from up to 100%, and ethylene glycol. In some embodiments, the formulation may be free of dimethyl sulfoxide (DMSO). In other embodiments, it may contain less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% DMSO. In some embodiments, the formulation lacks albumin and may be referred to as albumin-free. In some embodiments, the formulation lacks glucose and may be referred to as glucose-free. In some embodiments, the formulation lacks both glucose and albumin and may be referred to as glucose-free and albumin-free. In some embodiments, the formulation contains at least (or only) hydroxyethyl starch (HES) as a cryoprotectant, such HES having a hydroxyethyl substitution degree ranging from up to 100%.

[0009] One aspect of this disclosure provides a formulation comprising albumin, glucose, buffered saline, and one or more cryoprotectants, optionally selected from propylene glycol, dextran-40, proline, trehalose, glycerol, pentathione, and ethylene glycol. In some embodiments, the formulation may be free of dimethyl sulfoxide (DMSO). In other embodiments, it may contain less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% DMSO. In some embodiments, the formulation lacks albumin and may be referred to as albumin-free. In some embodiments, the formulation lacks glucose and may be referred to as glucose-free. In some embodiments, the formulation lacks both glucose and albumin and may be referred to as glucose-free and albumin-free. In some embodiments, pentathione may be replaced with one or more other HES with a hydroxyethyl substitution degree ranging from up to 100%, including but not limited to 2-hydroxyethyl starch (2-HES).

[0010] Another aspect of this disclosure provides a cryopreservation formulation substantially composed of albumin, glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch (HES) with a hydroxyethyl substitution degree ranging from up to 100%, and ethylene glycol. In some embodiments, the formulation lacks albumin and may be referred to as albumin-free. In some embodiments, the formulation lacks glucose and may be referred to as glucose-free. In some embodiments, the formulation lacks both glucose and albumin and may be referred to as glucose-free and albumin-free. In some embodiments, the formulation contains at least (or only) hydroxyethyl starch as a cryoprotectant, such hydroxyethyl starch having a hydroxyethyl substitution degree ranging from up to 100%.

[0011] Another aspect of this disclosure provides a cryopreservation formulation substantially composed of albumin, glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, pentathione, and ethylene glycol. In some embodiments, the formulation lacks albumin and may be referred to as albumin-free. In some embodiments, the formulation lacks glucose and may be referred to as glucose-free. In some embodiments, the formulation lacks both glucose and albumin and may be referred to as glucose-free and albumin-free. In some embodiments, pentathione may be replaced with one or more other HES with a hydroxyethyl substitution degree ranging from up to 100%, including but not limited to 2-hydroxyethyl starch (2-HES).

[0012] This disclosure provides various embodiments of the aforementioned formulation, which are described below.

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

[0014] In some embodiments, albumin is present in the range of about 2% to about 8% (w / v). In some embodiments, albumin is present in the range of about 1% to about 4% (w / v). In some embodiments, albumin is present in the range of about 2.5% (w / v).

[0015] In some implementations, albumin is not present in the formulation.

[0016] In some embodiments, glucose is present in the range of about 0.01% to about 1.5% (w / v). In some embodiments, glucose is present in the range of about 0.01% to about 0.5% (w / v). In some embodiments, glucose is present in the range of about 0.09% (w / v).

[0017] In some implementations, glucose is not present in the formulation.

[0018] In some implementations, albumin and glucose are not present in the formulation.

[0019] In some implementations, the buffered saline is phosphate-buffered saline (PBS).

[0020] In some embodiments, the one or two, or optionally more, cryoprotectants comprise propylene glycol.

[0021] In some embodiments, propylene glycol is present in the range of about 0.1% to about 20% (v / v). In some embodiments, propylene glycol is present in the range of about 0.2% to about 15% (v / v). In some embodiments, propylene glycol is present in the range of about 0.2%, about 2%, about 6%, about 10%, or about 15% (v / v).

[0022] In some embodiments, the one or two, or optionally more, cryoprotectants comprise dextran-40.

[0023] In some embodiments, dextran-40 is present in the range of about 1% to about 20% (w / v). In some embodiments, dextran-40 is present in the range of about 2% to about 20% (w / v). In some embodiments, dextran-40 is present in the range of about 4% to about 20% (w / v). In some embodiments, dextran-40 is present in the range of about 5% to about 15% (w / v). In some embodiments, dextran-40 is present in the range of about 5%, about 6%, about 10%, or about 15% (w / v).

[0024] In some embodiments, the one or two, or optionally more, cryoprotectants comprise proline.

[0025] In some embodiments, the one or two, or optionally more, cryoprotectants comprise trehalose.

[0026] In some implementations, the one or two, or optionally more, cryoprotectants comprise glycerin.

[0027] In some embodiments, the one or two, or optionally more, cryoprotectants comprise hydroxyethyl starch (HES) with a hydroxyethyl substitution range of up to 100%, the substitution including, for example, substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 11 hydroxyl groups of the HES. Some of these hydroxyethyl starches (HES) may be referred to as 2-HES, tetrastarch, pentastarch, and heptastarch. The cryoprotectant may comprise two or more hydroxyethyl starches with different degrees of hydroxyethyl substitution.

[0028] In some embodiments, hydroxyethyl-substituted hydroxyethyl starch (HES) of up to 100% is present in the range of about 4% to about 20% (w / v). In some embodiments, hydroxyethyl-substituted hydroxyethyl starch (HES) of up to 100% is present in the range of about 5% to about 15% (w / v). In some embodiments, hydroxyethyl-substituted hydroxyethyl starch (HES) of up to 100% is present in the range of about 5%, about 10%, or about 15% (w / v). Such hydroxyethyl starch includes, but is not limited to, 2-HES.

[0029] In some implementations, the one or two, or optionally more, cryoprotectants comprise penta-starch.

[0030] In some embodiments, the pentathione is present in the range of about 4% to about 20% (w / v). In some embodiments, the pentathione is present in the range of about 5% to about 15% (w / v). In some embodiments, the pentathione is present in the range of about 5%, about 10%, or about 15% (w / v).

[0031] In some embodiments, the one or two, or optionally more, cryoprotectants comprise ethylene glycol.

[0032] In some embodiments, ethylene glycol is present in the range of about 4% to about 6% (v / v). In some embodiments, ethylene glycol is present in the range of about 5% (v / v).

[0033] In some embodiments, the one or two, or optionally more, cryoprotectants are propylene glycol and dextran-40.

[0034] In some embodiments, propylene glycol is present in the range of about 0.1% to about 20% (v / v) and dextran-40 is present in the range of about 0.2% to about 20% (w / v). In some embodiments, propylene glycol is present in the range of about 0.15% to about 20% (v / v) and dextran-40 is present in the range of about 4% to about 20% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2% to about 15% (v / v) and dextran-40 is present in the range of about 5% to about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2%, about 2%, about 6%, about 10%, or about 15% (v / v) and dextran-40 is present in the range of about 5%, about 6%, about 10%, or about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2% to about 15% (v / v) and dextran-40 is present in the range of about 6%, about 10%, or about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2%, about 2%, about 6%, about 10%, or about 15% (v / v) and dextran-40 is present in the range of about 10% (w / v). In some embodiments, propylene glycol is present in the range of about 10% (v / v) and dextran-40 is present in the range of about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 10% (v / v) and dextran-40 is present in the range of about 10% (w / v). In some embodiments, propylene glycol is present in the range of about 6% (v / v) and dextran-40 is present in the range of about 20% (w / v). In some embodiments, propylene glycol is present at about 6% (v / v) and dextran-40 at about 15% (w / v). In some embodiments, propylene glycol is present at about 6% (v / v) and dextran-40 at about 10% (w / v). In some embodiments, propylene glycol is present at about 6% (v / v) and dextran-40 at about 6% (w / v). In some embodiments, propylene glycol is present at about 6% (v / v) and dextran-40 at about 2% (w / v).

[0035] In some embodiments, the one or two, or optionally more, cryoprotectants are propylene glycol and proline.

[0036] In some embodiments, propylene glycol is present in the range of about 0.1% to about 20% (v / v) and proline is present in the range of about 2% to about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2% to about 15% (v / v) and proline is present in the range of about 5% to about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2% to about 15% (v / v) and proline is present in the range of about 5% to about 10% (w / v). In some embodiments, propylene glycol is present in the range of about 6% (v / v) and proline is present in the range of about 5% or about 10% (w / v).

[0037] In some embodiments, the one or two, or optionally more, cryoprotectants are propylene glycol and trehalose.

[0038] In some embodiments, propylene glycol is present in the range of about 0.1% to about 20% (v / v) and trehalose is present in the range of about 0.1% to about 20% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2% to about 15% (v / v) and trehalose is present in the range of about 0.2% to about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 10% (v / v) and trehalose is present in the range of about 0.2% to about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 10% (v / v) and trehalose is present in the range of about 7% (w / v). In some embodiments, propylene glycol is present in the range of about 10% (v / v) and trehalose is present in the range of about 2% (w / v).

[0039] In some embodiments, the one or two, or optionally more, cryoprotectants are glycerol and trehalose.

[0040] In some embodiments, glycerol is present in the range of about 4% to about 16% (v / v) and trehalose is present in the range of about 2% to about 12% (w / v). In some embodiments, glycerol is present in the range of about 8% to about 12% (v / v) and trehalose is present in the range of about 4% to about 10% (w / v). In some embodiments, glycerol is present in the range of about 10% (v / v) and trehalose is present in the range of about 7% (w / v).

[0041] In some embodiments, the formulation comprises about 2.5% (w / v) albumin (optionally recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 5% (v / v) propylene glycol, and about 7.5% (w / v) dextran-40.

[0042] In some embodiments, the formulation comprises about 2.5% (w / v) albumin (optionally recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol, and about 5% (w / v) dextran-40.

[0043] In some embodiments, the formulation comprises about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol and about 5% (w / v) dextran-40, and is albumin-free.

[0044] In some embodiments, the formulation comprises about 2.5% (w / v) albumin (optionally recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 5% (w / v) dextran-40.

[0045] In some embodiments, the formulation comprises about 2.5% (w / v) albumin (optionally recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 7.5% (w / v) pentasyl starch.

[0046] Another aspect of this disclosure provides a cell preparation comprising a cell population and any of the aforementioned formulations.

[0047] Another aspect of this disclosure provides a cell preparation comprising a cell population and any of the aforementioned formulations, wherein the concentration of one or more, or optionally more, cryoprotectants in the preparation is halved compared to their concentration in the formulation.

[0048] Another aspect of this disclosure provides a cell preparation comprising (i) a cell population in a 1:1 (v / v) mixture of (i) any of the aforementioned formulations and (ii) a suspension comprising albumin, glucose, and buffered saline, each in the same concentration as the formulation, wherein the suspension does not contain a (non-albumin) cryoprotectant.

[0049] Another aspect of this disclosure provides a cell preparation comprising a cell population in a formulation comprising albumin, glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch (e.g., but not limited to 2-hydroxyethyl starch and / or pentastarch) and ethylene glycol, optionally wherein the formulation is free of dimethyl sulfoxide (DMSO-free).

[0050] Other aspects of this disclosure provide a cell preparation comprising a cell population in a formulation, said formulation comprising: (a) Albumin, glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol, optionally wherein the formulation is free of dimethyl sulfoxide (DMSO). (b) Glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol, optionally wherein the formulation is free of dimethyl sulfoxide (DMSO). (c) Albumin, buffered saline, and one or more cryoprotectants, said cryoprotectants being selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol, optionally said formulation being free of dimethyl sulfoxide (DMSO). (d) A buffered saline solution and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol, optionally wherein the formulation is free of dimethyl sulfoxide (DMSO). (e) Albumin, glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, optionally wherein the formulation is free of dimethyl sulfoxide (DMSO). (f) Glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, optionally wherein the formulation is free of dimethyl sulfoxide (DMSO). (g) Albumin, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, optionally wherein the formulation is free of dimethyl sulfoxide (DMSO), or (h) Buffered saline and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch and ethylene glycol, optionally wherein the formulation is free of dimethyl sulfoxide (DMSO).

[0051] Another aspect of this disclosure provides a cell preparation comprising a cell population in a cryopreservation formulation, the formulation being substantially composed of albumin, glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch (e.g., but not limited to 2-hydroxyethyl starch and / or pentastarch), and ethylene glycol.

[0052] Other aspects of this disclosure provide a cell preparation comprising a cell population in a cryopreservation formulation, said formulation being substantially composed of: (a) Albumin, glucose, buffered saline, and one or more cryoprotectants, said cryoprotectants being selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol. (b) Glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol. (c) Albumin, buffered saline, and one or more cryoprotectants, said cryoprotectants being selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol. (d) a buffered saline solution and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol. (e) Albumin, glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol. (f) Glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol. (g) Albumin, buffered saline, and one or more cryoprotectants, said cryoprotectants being selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, or (h) a buffered saline solution and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol.

[0053] This disclosure provides various embodiments of the aforementioned cell preparations, which are described below and may also be shared with the aforementioned formulations (therefore, reference may also be made to the previously described embodiments).

[0054] In some embodiments, the albumin is human albumin (optionally recombinant human albumin). In some embodiments, albumin is present in the range of about 2% to about 8% (w / v), optionally in the range of about 1% to about 4% (w / v), and further optionally in the range of about 2.5% (w / v). In some embodiments, albumin is absent.

[0055] In some embodiments, glucose is present in the range of about 0.01% to about 1.5% (w / v), optionally in the range of about 0.01% to about 0.5% (w / v), and more optionally in the range of about 0.09% (w / v). In some embodiments, glucose is absent.

[0056] In some implementations, the buffer brine is a phosphate-buffered brine.

[0057] In some embodiments, the one or two, or optionally more, cryoprotectants comprise propylene glycol.

[0058] In some embodiments, propylene glycol is present in the range of about 0.05% to about 10%, optionally in the range of about 0.1% to about 7.5%, and further optionally in the range of about 0.1%, about 1%, about 3%, about 5% or about 7.5% (v / v).

[0059] In some embodiments, the one or two, or optionally more, cryoprotectants comprise dextran-40.

[0060] In some embodiments, dextran-40 is present in the range of about 0.5% to about 10%, optionally in the range of about 1% to about 10%, optionally in the range of about 2% to about 10%, optionally in the range of about 2.5% to about 7.5%, and further optionally in the range of about 2.5%, about 3%, about 5%, or about 7.5% (w / v).

[0061] In some embodiments, the one or two, or optionally more, cryoprotectants comprise proline, trehalose, and / or glycerol.

[0062] In some embodiments, the one or two, or optionally more, cryoprotectants comprise hydroxyethyl-substituted starch (HES) up to 100%, including but not limited to 2-HES.

[0063] In some embodiments, hydroxyethyl starch, such as, but not limited to, 2-HES, is present in the range of about 2% to about 10%, optionally in the range of about 2.5% to about 7.5%, and further optionally in the range of about 2.5%, about 5%, or about 7.5% (w / v).

[0064] In some embodiments, the one or two, or optionally more, cryoprotectants comprise pentastarch. In some embodiments, the one or two, or optionally more, cryoprotectants comprise tetrastarch. In some embodiments, the one or two, or optionally more, cryoprotectants comprise heptastarch. In some embodiments, the one or two, or optionally more, cryoprotectants comprise two or more hydroxyethyl starches with different degrees of hydroxyethyl substitution. As an example, the one or two, or optionally more, cryoprotectants may comprise 2-HES and pentastarch.

[0065] In some embodiments, individual hydroxyethyl starches, such as, but not limited to, tetra-, penta-, or hepta-starch, are present in the range of about 2% to about 10%, optionally in the range of about 2.5% to about 7.5%, and further optionally in the range of about 2.5%, about 5%, or about 7.5% (w / v). In some embodiments, all hydroxyethyl starches are present in the range of about 2% to about 10%, optionally in the range of about 2.5% to about 7.5%, and further optionally in the range of about 2.5%, about 5%, or about 7.5% (w / v).

[0066] In some embodiments, the one or two, or optionally more, cryoprotectants comprise ethylene glycol.

[0067] In some embodiments, ethylene glycol is present in the range of about 2% to about 3%, and optionally in the range of about 2.5% (v / v).

[0068] In some embodiments, the one or two, or optionally more, cryoprotectants are propylene glycol and dextran-40.

[0069] In some embodiments, propylene glycol is present in the range of about 0.05% to about 10% (v / v) and dextran-40 is present in the range of about 0.1% to about 10% (w / v). Optionally, propylene glycol is present in the range of about 0.05% to about 10% (v / v) and dextran-40 is present in the range of about 2% to about 10% (w / v). Optionally, propylene glycol is present in the range of about 0.1% to about 7.5% (v / v) and dextran-40 is present in the range of about 2.5% to about 7.5% (w / v). Optionally, propylene glycol is present in the range of about 0.1%, about 1%, about 3%, about 5%, or about 7.5% (v / v) and dextran-40 is present in the range of about 2.5%, about 3%, about 5%, or about 7.5% (w / v). Optionally, propylene glycol is present in the range of about 0.1% to about 7.5%. The range of (v / v) is present and dextran-40 is present at about 3%, about 5% or about 7.5% (w / v), and further optionally, propylene glycol is present at about 0.1%, about 1%, about 3%, about 5% or about 7.5% and dextran-40 is present at about 5% (w / v).

[0070] In some embodiments, propylene glycol is present at about 5% (v / v) and dextran-40 at about 7.5% (w / v), or propylene glycol is present at about 5% (v / v) and dextran-40 at about 5% (w / v), or propylene glycol is present at about 3% (v / v) and dextran-40 at about 10% (w / v), or propylene glycol is present at about 3% (v / v) and dextran-40 at about 7.5% (w / v), or propylene glycol is present at about 3% (v / v) and dextran-40 at about 5% (w / v), or propylene glycol is present at about 3% (v / v) and dextran-40 at about 3% (w / v), or propylene glycol is present at about 3% (v / v) and dextran-40 at about 1% (w / v).

[0071] In some embodiments, the one or two, or optionally more, cryoprotectants are propylene glycol and proline.

[0072] In some embodiments, propylene glycol is present in the range of about 0.05% to about 10% (v / v) and proline is present in the range of about 1% to about 7.5% (w / v), optionally wherein propylene glycol is present in the range of about 0.1% to about 7.5% (v / v) and proline is present in the range of about 2.5% to about 7.5% (w / v), optionally wherein propylene glycol is present in the range of about 0.1% to about 7.5% (v / v) and proline is present in the range of about 2.5% to about 5% (w / v), and further optionally wherein propylene glycol is present in the range of about 3% (v / v) and proline is present in the range of about 2.5% or about 5% (w / v).

[0073] In some embodiments, the one or two, or optionally more, cryoprotectants are propylene glycol and trehalose.

[0074] In some embodiments, propylene glycol is present in the range of about 0.05% to about 10% (v / v) and trehalose is present in the range of about 0.05% to about 10% (w / v), optionally wherein propylene glycol is present in the range of about 0.1% to about 7.5% (v / v) and trehalose is present in the range of about 0.1% to about 7.5% (w / v), optionally wherein propylene glycol is present in the range of about 5% (v / v) and trehalose is present in the range of about 0.1% to about 7.5% (w / v), optionally wherein propylene glycol is present in the range of about 5% (v / v) and trehalose is present in the range of about 3.5% (w / v), and further optionally wherein propylene glycol is present in the range of about 5% (v / v) and trehalose is present in the range of about 1% (w / v).

[0075] In some embodiments, the one or two, or optionally more, cryoprotectants are glycerol and trehalose.

[0076] In some embodiments, glycerol is present in the range of about 2% to about 8% (v / v) and trehalose is present in the range of about 1% to about 6% (w / v), optionally wherein glycerol is present in the range of about 4% to about 6% (v / v) and trehalose is present in the range of about 2% to about 5% (w / v), and further optionally wherein glycerol is present in the range of about 5% (v / v) and trehalose is present in the range of about 3.5% (w / v).

[0077] In some embodiments, the cell population is a retinal pigment epithelial (RPE) cell population, a photoreceptor cell population, a photoreceptor progenitor cell population, a photoreceptor repair cell (PRC) population, a corneal epithelial cell (CEC) population, a mesenchymal cell population (e.g., angiogenic mesenchymal cell (HMC) population), a macrophage population, a natural killer cell population, a T-cell population, a vascular cell population, a vascular progenitor cell population, a hepatocyte population, a nerve cell population, and a pancreatic cell population.

[0078] In some embodiments, the cell population is a retinal pigment epithelium (RPE) cell population. In some embodiments, the cell population is a hemangioblast-derived mesenchymal cell (HMC) population. In some embodiments, the cell population is a vascular progenitor cell population.

[0079] In some embodiments, the cell population is a cell population in which the cells express at least one polypeptide selected from human leukocyte antigen E (HLA-E), human leukocyte antigen F (HLA-F), and human leukocyte antigen G (HLA-G); wherein the cells contain a genetically modified disruption of one or more copies of endogenous β-2 microglobulin (B2M); and wherein the cells contain a genetically modified disruption of one or more copies of human leukocyte antigen (HLA) class II related genes selected from regulatory factor X-associated ankyrin repeat sequence protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-associated protein (RFXAP), and class II transactivator (CIITA).

[0080] In some embodiments, the cell further comprises a nucleic acid molecule encoding a suicide gene product.

[0081] In some implementations, the cell contains the herpes simplex virus thymidine kinase (TK) suicide gene.

[0082] In some implementations, the cells are derived from pluripotent stem cells.

[0083] In some implementations, the pluripotent stem cells are embryonic stem cells.

[0084] In some implementations, the pluripotent stem cells are induced pluripotent stem cells.

[0085] In some implementations, the cells contain genetically modified disruptions of all copies of endogenous B2M.

[0086] In some embodiments, the cells contain genetically modified disruptions of all copies of HLA class II-related genes selected from RFXANK, RFX5, RFXAP, and CIITA.

[0087] In some implementations, the cell population is a dissociated cell population, a cell sheet, a cell monolayer, or a cell aggregate.

[0088] In some implementations, the cell population is a human cell population.

[0089] In some implementations, the cell population is generated from in vitro differentiated pluripotent stem cells, optionally embryonic stem cells, or induced pluripotent stem cells.

[0090] In some embodiments, the cell preparation has a cell density of about 300 cells to about 10,000 cells / µL. In some embodiments, the cell preparation has a cell density of about 300 cells to about 7,000 cells / µL. In some embodiments, the cell preparation has a cell density of about 350 cells to about 7,000 cells / µL. In some embodiments, the cell preparation has a cell density of about 350 cells / µL, or about 3,000 cells / µL, or about 3,500 cells / µL, or about 6,500 cells / µL, or about 7,000 cells / µL. In some embodiments, the cell preparation has a cell density of about 3,333 cells / µL.

[0091] In some embodiments, the cell preparation is in a volume of about 400-500 microliters, and more optionally in a volume of about 450 microliters.

[0092] In some implementations, the cell preparation is cryopreserved.

[0093] In some implementations, the cell preparations have been cryopreserved and thawed.

[0094] In some embodiments, the cell preparation is provided at room temperature (about 20°C to about 25°C), at about 4°C, at about -20°C, at about -70°C, or at about -180°C before cryopreservation or after thawing.

[0095] In some embodiments, the cell preparations are held at room temperature (about 20°C to about 25°C) for about 1 to 2 hours and at about 4°C for about 4 to 7 hours prior to cryopreservation, optionally at -180°C.

[0096] In some embodiments, the cell preparation is a DMSO-free cell preparation.

[0097] Another aspect of this disclosure provides a cell preparation comprising a cell population in a formulation comprising albumin, glucose, buffered saline, and one or more cryoprotectants, and having, after an event, a cell viability equal to or greater than about 80% and / or a cell growth index equal to or less than 110 hours:3, optionally having a cell viability equal to or greater than about 85% and / or a cell growth index equal to or less than 100 hours:3, and further optionally having a cell viability equal to or greater than about 90% and / or a cell growth index equal to or less than 90 hours:3: a. Store at room temperature (about 20°C to about 25°C) for about 1 to 2 hours, then at about 4°C for about 4 to 7 hours, optionally at room temperature for about 1.5 hours, then at 4°C for about 4.5 hours or 6.5 hours. b. Store frozen at -180°C for approximately 1 to 2 years, optionally for approximately 1 month to 1 year. c. Freeze at -180°C, then thaw and store at approximately 4°C for approximately 3 to 6 hours, optionally approximately 4 hours. d. Freeze at -180°C, then thaw and allow to cool at room temperature (approximately 20°C to approximately 25°C) for approximately 3 to 6 hours, optionally approximately 4 hours. e. Freeze at -180°C, then freeze at about -20°C for about 0.5 to about 1 hour, then freeze at -180°C, or optionally freeze at about -20°C for about 0.5 to about 1 hour, then freeze at -180°C. f. Freeze at -180°C, then at about -70°C for about 2 to about 4 weeks, then freeze again at -180°C.

[0098] In some embodiments, the cell preparation is a DMSO-free cell preparation. In some embodiments, the formulation or cell preparation is an albumin-free formulation or cell preparation (meaning albumin is absent). In some embodiments, the formulation or cell preparation is a glucose-free cell preparation or cell preparation (meaning glucose is absent). In some embodiments, the formulation or cell preparation is both albumin-free and glucose-free (meaning albumin and glucose are absent). In some embodiments, after any one of events (a) to (f), the cell preparation has a viability equal to or greater than about 85% and / or a cell growth index equal to or less than 100 hours: 3. In some embodiments, after any one of events (a) to (f), the cell preparation has a viability equal to or greater than about 90% and / or a cell growth index equal to or less than 90 hours: 3.

[0099] Another aspect of this disclosure provides a method for preparing a cell preparation comprising contacting a cell population with (i) any of the aforementioned preparations and (ii) a suspension comprising albumin, glucose, and buffered saline, each at the same concentration as the preparation, wherein the suspension does not contain a (non-albumin) cryoprotectant.

[0100] In some embodiments, the method further includes cryopreserving the cell preparation. In some embodiments, the method further includes thawing the cell preparation. In some embodiments, the method further includes administering the cell preparation to a subject in need. In some embodiments, the cell preparation is kept at about 4°C for about 4 hours prior to administration to the subject. In some embodiments, the cell preparation is kept at room temperature (about 20°C to about 25°C) for about 4 hours prior to administration to the subject. In some embodiments, the cell preparation is administered to the subject after thawing without washing or dilution.

[0101] Another aspect of this disclosure provides a method for treating a subject with a disease or condition, comprising administering an effective amount of any of the aforementioned cell preparations to the subject in need after cryopreservation and thawing without washing or dilution.

[0102] Another aspect of this disclosure provides treatment for subjects with a condition or illness, including administering an effective amount of a cell preparation prepared according to any of the aforementioned methods for cryopreservation and thawing of cell preparations to a subject in need without washing or dilution after thawing.

[0103] In some embodiments, the cell preparation is kept at about 4°C for about 4 hours prior to administration to the subject. In some embodiments, the cell preparation is kept at room temperature (about 20°C to about 25°C) for about 4 hours prior to administration to the subject. In some embodiments, the condition or ailment is selected from retinal detachment, retinal tear, retinal dysplasia, vascular streaks, myopic macular degeneration, or retinal atrophy, or is associated with a variety of visual impairments leading to photoreceptor damage and blindness, such as choroidal agenesis, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration, AMD), retinitis pigmentosa, Staggart's disease (yellow macular degeneration), cancer, liver disease, Crohn's disease, arthritis, pulmonary hypertension, severe limb ischemia, Fuchs' keratodystrophy, corneal injury, retinal tear, and geographic atrophy (e.g., geographic atrophy secondary to age-related macular degeneration (AMD)). In some embodiments, the cell preparation is in a vial. In some embodiments, the cell preparation is in a syringe.

[0104] The foregoing summary of the invention is intended to illustrate, in a non-limiting manner, some embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will become apparent from the detailed description, drawings, examples, and claims. Attached Figure Description

[0105] Figure 1A-1B This demonstrates cell viability using RPE cells in the initial formulation screening. Figure 1A ) and cell growth ( Figure 1B Results. 5% (v / v) DMSO was used as a control. Target cell density: 3,333 cells / µL.

[0106] Figure 2A-2B The cell viability of RPE cells screened using dextran-40 was shown. Figure 2A ) and cell growth ( Figure 2B Results. 5% (v / v) DMSO was used as a control. Target cell density: 3,333 cells / µL.

[0107] Figures 3A-3B This demonstrates the cell viability of UC-RPE cells during propylene glycol screening. Figure 3A ) and cell growth ( Figure 3B Results. 5% (v / v) DMSO was used as a control. Target cell density: 3,333 cells / µL.

[0108] Figures 4A-4B This demonstrates the cell viability of RPE cells in trehalose screening. Figure 4A ) and cell growth ( Figure 4B Results. Target cell density: 3,333 cells / µL.

[0109] Figures 5A-5B This demonstrates cell viability using RPE cells in process time assessment. Figure 5A ) and cell growth ( Figure 5B Results. Process time indicates the duration from the addition of cryoprotectant to the initiation of rate-controlled freezing. The process times used corresponded to 6 h (1.5 h RT + 4.5 h 4ºC) and 8 h (1.5 h RT + 6.5 h 4ºC). 5% (v / v) DMSO was used as a control. Target cell density: 3,333 cells / µL.

[0110] Figures 6A-6B The stability after thawing is shown after 4 hours at 4ºC. RPE cell viability was measured after thawing in frozen vials and storing at 4ºC for 4 hours. Figure 6A ) and cell growth ( Figure 6B 5% (v / v) DMSO was used as a control. Target cell density: 3,333 cells / µL.

[0111] Figures 7A-7B This indicates stability after thawing at room temperature for 4 hours. RPE cell viability was measured after thawing the frozen vials and storing them at room temperature for 4 hours. Figure 7A ) and cell growth ( Figure 7B 5% (v / v) DMSO was used as a control. Target cell density: 3,333 cells / µL.

[0112] Figures 8A-8B Stability was demonstrated at -20ºC. RPE cell viability was measured after temperature deviation from -20ºC. Figure 8A ) and cell growth ( Figure 8B Transfer the frozen vials to a -20ºC chamber and hold for 30 or 60 minutes, then transfer them back to the liquid nitrogen tank; repeat the process twice, then evaluate. Target cell density: 3,333 cells / µL.

[0113] Figures 9A-9B Stability was demonstrated at -70ºC. RPE cell viability was measured after a temperature deviation from -70ºC. Figure 9A ) and cell growth ( Figure 9B The frozen vials were transferred to a -70ºC chamber and held for 4 weeks, then transferred back to the liquid nitrogen tank for evaluation. 5% (v / v) DMSO was used as a control. Target cell density: 3,333 cells / µL.

[0114] Figure 10 The results of the multinucleation assay are displayed. Thawed cells were seeded and cultured for 4 hours, then fixed. The stained cells were counted, and the ratio of multinucleated cells was assessed.

[0115] Figure 11A-11B This demonstrates cell viability using UC-RPE cells during time-lapse evaluation. Figure 11A ) and cell growth ( Figure 11B Results. Process time indicates the duration from the addition of cryoprotectant to the initiation of rate-controlled freezing. The process times used correspond to 6 h and 8 h. 5% (v / v) DMSO was used as a control. Target cell density: 3,333 cells / µL. Detailed Implementation

[0116] Cell-based therapies, including those involving cell transplantation, often require the formulation, storage, transportation, and / or injection of fragile or vulnerable cells that may be damaged or lose their regenerative capacity due to improper handling, extensive manipulation and / or processing steps, or exposure to non-physiological conditions, in the context of regenerative medicine protocols.

[0117] Dimethyl sulfoxide (DMSO) is a common cryoprotectant used to protect cells from damage during the cryopreservation and thawing of cell products. However, DMSO can be harmful to patients if applied in high concentrations. DMSO can also cause cytotoxicity when used in high concentrations in cell preparations. Existing methods for minimizing this negative effect of DMSO include diluting and / or washing cells with physiological buffer to remove DMSO and reconstituteing them with physiological buffer before administration to patients.

[0118] However, these methods present several challenges. One such challenge is that even after the cells are diluted with another solution, some cryoprotectant residue may still remain, which could be administered to the subject. This can increase the risk of side effects, thus limiting the use of such cell populations. Furthermore, the development of suitable physiological buffers can be expensive and labor-intensive, ultimately increasing the cost of the therapeutic agent. Another challenge is that the process of diluting and / or washing cells before administration is complex for clinical pharmacists and can increase the risk of contamination, leading to loss of sterility and potentially fatal effects on patients.

[0119] When the washing step is used to remove DMSO, the shelf life of cell products is typically very short (e.g., usually 48–96 hours), which requires robust and challenging planning for product administration and limits the geographical location from which the product can be distributed and used. Regarding dilution strategies, appropriate reconstitution buffers and additional processes may be necessary, increasing costs and the risk of contamination. Furthermore, many clinical sites are not prepared for additional processing steps prior to administration.

[0120] Finally, because the reconfiguration process will be performed by various clinicians and pharmacists, a degree of variability is introduced. This variability may lead to inconsistent dosing and consequently inconsistent therapeutic effects.

[0121] This disclosure provides formulations and methods for cryopreservation, storage, transport, and final administration of cells to subjects with minimal handling and manipulation of the cells and increased viability after thawing. The formulations provided herein possess several properties that make them suitable for cryopreserving cell-based therapeutics. For example, the formulations provided herein enable the cryopreservation of cell-based therapeutics without the use of DMSO as a cryoprotectant, thereby avoiding significant reductions in cell quality after freezing and thawing and increasing cell stability. Furthermore, the formulations can be used directly after freezing and thawing, eliminating the need for dilution and remodeling with another solution before administration (or transplantation) to a subject. The formulations also enable the preservation of frozen cell populations for extended periods, such as one year or longer. Moreover, cells stored in these formulations exhibit tolerance to temperature changes at room temperature or 4°C, either in the frozen state or before cryopreservation or after thawing. In contrast, various currently available cryopreservation formulations require extensive cell handling procedures, which can be expensive, time-consuming, prone to human error, and ultimately potentially reduce cell viability, plating efficiency, and the reproliferative capacity of the cells contained therein. Furthermore, it is noteworthy that increased cell viability has been observed in cell populations cryopreserved using the formulations provided herein, compared to existing cryopreservation formulations and methods. Improved cell growth after thawing has also been observed when using the formulations provided herein. The formulations provided herein offer improved cell population functionality, viability, and stability.

[0122] In addition to the aforementioned advantages, the cryopreservation formulations of this disclosure are also surprisingly and unexpectedly associated with a reduction in the frequency (or proportion) of multinucleated cells in cultures (particularly RPE cell cultures). Multinucleated cells, such as multinucleated RPE cells, have been observed using other formulations.

[0123] Cryopreservation preparations This disclosure provides novel cryopreservation formulations. Some of these formulations are DMSO-free. If DMSO is present in the formulations provided herein, it is present in amounts of less than 10% (v / v), less than 5% (v / v), less than 1% (v / v), less than 0.1% (v / v), or less than 0.01% (v / v). Some cryopreservation formulations provided herein comprise albumin, glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol (1,3-propanediol), dextran-40, proline, trehalose, glycerol, hydroxyethyl starch (e.g., but not limited to 2-HES and / or pentamethane), and ethylene glycol. In some embodiments, the cryopreservation formulations consist essentially of albumin, glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, pentamethane, and ethylene glycol. In other embodiments, the cryopreservation preparation comprises albumin, glucose, buffered saline, and one or two, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol.

[0124] Other formulations also lack albumin and / or glucose.

[0125] These formulations may be referred to as cell-free, meaning they contain no cells or contain cells. However, as discussed in more detail herein, such formulations may be combined with cell populations, and optionally suspensions, to form cell preparations.

[0126] albumin Some cryopreservation formulations provided herein contain albumin. In some embodiments, the albumin is human albumin. In some embodiments, the albumin is recombinant human albumin. The term "recombinant human albumin" is used interchangeably with the terms "rHA," "rHSA," and "rh albumin." In some embodiments, albumin is present in the range of about 1% to about 10% (w / v). In some embodiments, albumin is present in the range of about 2% to about 8% (w / v). In some embodiments, albumin is present in the range of about 2% to about 7.5% (w / v). In some embodiments, albumin is present in the range of about 1% to about 4% (w / v). In some embodiments, albumin is present in the range of about 2% to about 3% (w / v). In some embodiments, albumin is present at 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%, or about 10% (w / v). In some embodiments, albumin is present at 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%, or about 3% (w / v). In some embodiments, albumin is present at about 2.5% (w / v). Albumin may have cryoprotectant properties, but it should be understood that the cryoprotectants discussed herein do not involve albumin. Some formulations provided herein lack albumin and may be referred to as albumin-free.

[0127] glucose Some of the cryopreservation formulations provided herein contain glucose. In some embodiments, glucose is present in the range of about 0.01% to about 3% (w / v). In some embodiments, glucose is present in the range of about 0.01% to about 1.5% (w / v). In some embodiments, glucose is present in the range of about 0.01% to about 0.5% (w / v). In some embodiments, glucose is present in the range of about 0.02% to about 1.5% (w / v). In some embodiments, glucose is present in the range of about 0.05% to about 1.2% (w / v). In some embodiments, glucose is present at 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%, about 1.1%, or about 1.2% (w / v). In some embodiments, glucose is present at about 0.09% (w / v). Some formulations provided herein lack glucose and may be referred to as glucose-free.

[0128] Buffered saline The cryopreservation formulations provided herein contain buffered saline. In some embodiments, the buffered saline is phosphate-buffered saline (PBS). In some embodiments, the buffered saline is Duchenne phosphate-buffered saline (DPBS).

[0129] In some embodiments, the buffer brine contains a divalent cation. Suitable divalent cations include, but are not limited to, Ca2+. 2+ Mg 2+ Zn 2+ Fe 2+ Mn 2+ Cr 2+ Cu 2+ Ba 2+ and Sr 2+ In some embodiments, the divalent cation comprises calcium. In some embodiments, the divalent cation comprises magnesium. In some embodiments, the divalent cation comprises two or more different divalent cations (e.g., calcium and magnesium). In some embodiments, the buffer brine does not contain calcium and / or magnesium (Ca). 2+ and Mg 2+ In some embodiments, the buffer saline contains one or more pharmaceutically acceptable salts, such as, but not limited to, pharmaceutically acceptable magnesium salts and / or pharmaceutically acceptable calcium salts.

[0130] In some embodiments, buffered saline may be excluded from any formulation provided herein. In some embodiments, any formulation provided herein may contain a buffer and / or a saline solution (or electrolyte or electrolyte solution) instead of buffered saline. In some cases, the formulation may lack buffered saline but may contain a buffer and may subsequently be combined with a saline solution (or electrolyte or electrolyte solution). In some cases, the formulation may lack buffered saline but may contain a saline solution (or electrolyte or electrolyte solution) and may subsequently be combined with a buffer. Examples of buffers that can be used in the various formulations disclosed herein include alkali metal (sodium and potassium) or alkaline earth metal (calcium and magnesium) carbonates, phosphates, bicarbonates, citrates, borates, acetates, phthalates, tartrates, succinates, etc., such as sodium or potassium phosphate, sodium or potassium citrate, sodium or potassium borate, sodium or potassium acetate, sodium or potassium bicarbonate, and sodium or potassium carbonate; and salts of Group IA metals, including, for example, bicarbonates of Group IA metals and carbonates of Group IA metals; alkaline earth metal buffers, amino acids, basic salts of amino acids, aluminum buffers, calcium buffers, sodium buffers, or magnesium buffers. Other buffers are provided in published PCT application number 2008 / 057802, and examples of buffers provided in that application are incorporated herein by reference. Examples of electrolytes that may be used in the various formulations disclosed herein include calcium chloride; sodium chloride; magnesium chloride; sodium acetate; potassium chloride; potassium acetate; sodium or potassium dihydrogen phosphate, disodium or potassium hydrogen phosphate, or sodium or potassium phosphate; sodium or potassium citrate; sodium or potassium tartrate; sodium benzoate; sodium or potassium sorbate; sodium or potassium phthalate; sodium or potassium metabisulfite; or other similar salts commonly used in pharmaceutical products.

[0131] One or two, or optionally more, cryoprotectants The cryopreservation formulations provided herein contain one or two, or optionally more (non-albumin) cryoprotectants. In some embodiments, the one or two, or optionally more cryoprotectants do not include DMSO in certain cases. Some formulations provided herein contain one (non-albumin) cryoprotectant. Some formulations provided herein contain two (non-albumin) cryoprotectants. Some formulations provided herein contain more than two (non-albumin) cryoprotectants. The one or two, or optionally more cryoprotectants are selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch (e.g., but not limited to pentathione), and ethylene glycol.

[0132] A. Propylene glycol (1,3-propanediol) In some embodiments, the one or two, or optionally more, cryoprotectants comprise propylene glycol. In some embodiments, propylene glycol is present in the range of 0.1% to about 20% (v / v). In some embodiments, propylene glycol is present in the range of 0.2% to about 15% (v / v). In some embodiments, propylene glycol is present in the range of 0.2% to about 6% (v / v). In some embodiments, propylene glycol is present in the range of 2% to about 15% (v / v). In some embodiments, propylene glycol is present in the range of 6% to about 15% (v / v). In some embodiments, propylene glycol is present in the range of about 0.2%, about 1%, about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 14%, or about 15% (v / v). In some embodiments, propylene glycol is present in the range of about 0.2%, about 2%, about 6%, about 10%, or about 15% (v / v). In some embodiments, propylene glycol is present in the range of about 0.2% (v / v). In some embodiments, propylene glycol is present at about 2% (v / v). In some embodiments, propylene glycol is present at about 6% (v / v). In some embodiments, propylene glycol is present at about 10% (v / v). In some embodiments, propylene glycol is present at about 15% (v / v). The concentration of the propylene glycol stock solution is 99% + propylene glycol.

[0133] B. Glucan-40 In some embodiments, the one or two, or optionally more, cryoprotectants comprise dextran-40. In some embodiments, dextran-40 is present in the range of about 1% to about 20% (w / v). In some embodiments, dextran-40 is present in the range of about 2% to about 20% (w / v). In some embodiments, dextran-40 is present in the range of about 4% to about 20% (w / v). In some embodiments, dextran-40 is present in the range of about 5% to about 15% (w / v). In some embodiments, dextran-40 is present in the range of about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 7.5%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% (w / v). In some embodiments, dextran-40 is present at about 5%, about 6%, about 10%, or about 15% (w / v). In some embodiments, dextran-40 is present at about 2% (w / v). In some embodiments, dextran-40 is present at about 5% (w / v). In some embodiments, dextran-40 is present at about 6% (w / v). In some embodiments, dextran-40 is present at about 10% (w / v). In some embodiments, dextran-40 is present at about 15% (w / v). In some embodiments, dextran-40 is present at about 20% (w / v).

[0134] C. Proline In some embodiments, the one or two, or optionally more, cryoprotectants comprise proline. In some embodiments, proline is present in the range of about 2% to about 15% (w / v). In some embodiments, proline is present in the range of about 5% to about 15% (w / v). In some embodiments, proline is present in the range of about 5% to about 10% (w / v). In some embodiments, proline is present in the range of about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% (w / v). In some embodiments, proline is present in the range of about 5% or 10% (w / v). In some embodiments, proline is present in the range of about 5% (w / v). In some embodiments, proline is present in the range of about 10% (w / v).

[0135] D. Trehalose In some embodiments, the one or two, or optionally more, cryoprotectants comprise trehalose. In some embodiments, trehalose is present in the range of about 0.1% to about 20% (w / v). In some embodiments, trehalose is present in the range of about 0.2% to about 15% (w / v). In some embodiments, trehalose is present in the range of about 0.2% to about 7% (w / v). In some embodiments, trehalose is present in the range of about 2% to about 12% (w / v). In some embodiments, trehalose is present in the range of about 2% to about 7% (w / v). In some embodiments, trehalose is present in the range of about 4% to about 10% (w / v). In some embodiments, trehalose is present in the range of about 6% to about 15% (w / v). In some embodiments, trehalose is present in the range of about 2%, about 3%, about 4%, about 5%, about 6%, or about 7% (w / v). In some embodiments, trehalose is present in the range of about 2% or about 7% (w / v). In some embodiments, trehalose is present at about 2% (w / v). In some embodiments, trehalose is present at about 7% (w / v).

[0136] E. glycerin In some embodiments, the one or two, or optionally more, cryoprotectants comprise glycerol. In some embodiments, glycerol is present in the range of about 4% to about 16% (v / v). In some embodiments, glycerol is present in the range of about 8% to about 12% (v / v). In some embodiments, glycerol is present in the range of about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or about 16% (v / v). In some embodiments, glycerol is present in the range of about 10% (v / v). The initial stock solution of glycerol has a concentration of 99% glycerol.

[0137] This disclosure further envisions that in any of the embodiments provided herein, "glycerol" may also include "glycerol".

[0138] F. Hydroxyethyl starch In some embodiments, the one or two, or optionally more, cryoprotectants comprise hydroxyethyl starch (HES) (e.g., but not limited to 2-hydroxyethyl starch). Examples of such hydroxyethyl starches include tetrastarch (approximately 40% hydroxyethylated HES), pentastarch (approximately 50% hydroxyethyl substituted), and heptastarch (approximately 70% hydroxyethyl substituted). The degree of hydroxyethyl substitution may involve a single HES molecule, or it may involve a group of HES. In some embodiments, the one or two, or optionally more, cryoprotectants comprise pentastarch. Pentastarch is a subgroup of hydroxyethyl starch that has five hydroxyethyl groups out of every 11 hydroxyl groups, resulting in approximately 50% hydroxyethyl substitution. Pentastarch is commercially available as Pentaspan (Bristol-Myers Squibb). The examples used a stock solution of pentasyl starch comprising: 20% (w / v) pentasyl starch (200 g / L), 0.18% (w / v) dextrose (1.8 g / L), and an electrolyte solution comprising: 526 mg NaCl, 502 mg sodium gluconate, 368 mg sodium acetate 3H₂O, 37 mg KCl, and 30 mg MgCl₂·6H₂O (per 100 mL volume). In some embodiments, the pentasyl starch is present in the range of about 4% to about 20% (w / v). In some embodiments, the pentasyl starch is present in the range of about 5% to about 15% (w / v). In some embodiments, the pentasyl starch is present in the range of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, or about 20% (w / v). In some embodiments, the pentathione is present at about 5%, about 10%, or about 15% (w / v). In some embodiments, the pentathione is present at about 5% (w / v). In some embodiments, the pentathione is present at about 10% (w / v). In some embodiments, the pentathione is present at about 15% (w / v). Similar ranges apply when other HES are used instead of pentathione.

[0139] G. Ethylene glycol In some embodiments, the one or two, or optionally more, cryoprotectants comprise ethylene glycol. In some embodiments, ethylene glycol is present in the range of about 2% to about 8% (v / v). In some embodiments, ethylene glycol is present in the range of about 4% to about 6% (v / v). In some embodiments, ethylene glycol is present in the range of about 4%, about 4.2%, about 4.4%, about 4.6%, about 4.8%, about 5%, about 5.2%, about 5.4%, about 5.6%, about 5.8%, or about 6% (v / v). In some embodiments, ethylene glycol is present in the range of about 5% (v / v). The initial stock solution of ethylene glycol has a concentration of 99% ethylene glycol.

[0140] Combination of cryoprotectants The cryopreservation formulations described herein may contain two (non-albumin) cryoprotectants. Various combinations of cryoprotectants are provided below.

[0141] Propylene glycol and dextran-40 In some embodiments, the formulation comprises propylene glycol and dextran-40. In some embodiments, propylene glycol is present in the range of about 0.1% to about 20% (v / v) and dextran-40 is present in the range of about 0.2% to about 20% (w / v). In some embodiments, propylene glycol is present in the range of about 0.1% to about 20% (v / v) and dextran-40 is present in the range of about 4% to about 20% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2% to about 15% (v / v) and dextran-40 is present in the range of about 5% to about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2%, about 2%, about 6%, about 10%, or about 15% (v / v) and dextran-40 is present in the range of about 5%, about 6%, about 10%, or about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2% to about 15% (v / v) and dextran-40 is present in the range of about 6%, about 10%, or about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2%, about 2%, about 6%, about 10%, or about 15% (v / v) and dextran-40 is present in the range of about 10% (w / v). In some embodiments, propylene glycol is present in the range of about 10% (v / v) and dextran-40 is present in the range of about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 10% (v / v) and dextran-40 is present in the range of about 10% (w / v). In some embodiments, propylene glycol is present in the range of about 6% (v / v) and dextran-40 is present in the range of about 20% (w / v). In some embodiments, propylene glycol is present at about 6% (v / v) and dextran-40 at about 15% (w / v). In some embodiments, propylene glycol is present at about 6% (v / v) and dextran-40 at about 10% (w / v). In some embodiments, propylene glycol is present at about 6% and dextran-40 at about 6% (w / v). In some embodiments, propylene glycol is present at about 6% (v / v) and dextran-40 at about 2% (w / v).

[0142] Propylene glycol and trehalose In some embodiments, the formulation comprises propylene glycol and trehalose. In some embodiments, propylene glycol is present in the range of about 0.1% to about 20% (v / v) and trehalose is present in the range of about 0.1% to about 20% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2% to about 15% (v / v) and trehalose is present in the range of about 0.2% to about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 10% (v / v) and trehalose is present in the range of about 0.2% to about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 10% (v / v) and trehalose is present in the range of about 7% (w / v). In some embodiments, propylene glycol is present in the range of about 10% (v / v) and trehalose is present in the range of about 2% (w / v).

[0143] Propylene glycol and proline In some embodiments, the formulation comprises propylene glycol and proline. In some embodiments, propylene glycol is present in the range of about 0.1% to about 20% (v / v) and proline is present in the range of about 2% to about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2% to about 15% (v / v) and proline is present in the range of about 5% to about 15% (w / v). In some embodiments, propylene glycol is present in the range of about 0.2% to about 15% (v / v) and proline is present in the range of about 5% to about 10% (w / v). In some embodiments, propylene glycol is present in the range of about 6% (v / v) and proline is present in the range of about 5% or about 10% (w / v).

[0144] Glycerin and Trehalose In some embodiments, the formulation comprises glycerol and trehalose. In some embodiments, glycerol is present in the range of about 4% to about 16% (v / v) and trehalose is present in the range of about 2% to about 12% (w / v). In some embodiments, glycerol is present in the range of about 8% to about 12% (v / v) and trehalose is present in the range of about 4% to about 10% (w / v). In some embodiments, glycerol is present in the range of about 10% (v / v) and trehalose is present in the range of about 7% (w / v).

[0145] Examples of cryopreserved preparations In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin); about 0.09% (w / v) glucose; buffered saline (e.g., PBS); about 0.2%, about 2%, about 6%, about 10%, or about 15% (w / v) propylene glycol; and about 5%, about 6%, about 10%, or about 15% (w / v) dextran-40.

[0146] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin); about 0.09% (w / v) glucose; buffered saline (e.g., PBS); about 0.2% to about 15% (v / v) propylene glycol; and about 6%, about 10%, or about 15% (w / v) dextran-40.

[0147] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin); about 0.09% (w / v) glucose; buffered saline (e.g., PBS); about 0.2%, about 2%, about 6%, about 10% or about 15% (v / v) propylene glycol; and about 10% (w / v) dextran-40.

[0148] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 10% (v / v) propylene glycol and about 15% (w / v) dextran-40.

[0149] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 10% (v / v) propylene glycol and about 10% (w / v) dextran-40.

[0150] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 6% (v / v) propylene glycol, and about 20% (w / v) dextran-40.

[0151] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 6% (v / v) propylene glycol, and about 15% (w / v) dextran-40.

[0152] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 6% (v / v) propylene glycol, and about 10% (w / v) dextran-40.

[0153] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 6% (v / v) propylene glycol, and about 6% (w / v) dextran-40.

[0154] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 6% (v / v) propylene glycol and about 2% (w / v) dextran-40.

[0155] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 5% (v / v) propylene glycol and about 7.5% (w / v) dextran-40, and optionally contains cell populations, such as, but not limited to, RPE cell populations, HMC populations and VPC populations.

[0156] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol and about 5% (w / v) dextran-40, and optionally contains cell populations, such as, but not limited to, RPE cell populations, HMC populations and VPC populations.

[0157] In some embodiments, the cryopreservation formulation comprises about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol and about 5% (w / v) dextran-40, and optionally includes cell populations, such as, but not limited to, RPE cell populations, HMC populations and VPC populations.

[0158] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 5% (w / v) dextran-40, and optionally contains cell populations, such as, but not limited to, RPE cell populations, HMC populations, and VPC populations.

[0159] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 7.5% (w / v) pentasyl starch, and optionally contains cell populations, such as, but not limited to, RPE cell populations, HMC populations, and VPC populations.

[0160] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 6% (v / v) propylene glycol and about 5% (w / v) proline.

[0161] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 6% (v / v) propylene glycol and about 10% (w / v) proline.

[0162] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 0.02% to about 1% (w / v) glucose, buffered saline (e.g., PBS), about 10% (v / v) propylene glycol and about 0.2% to about 15% (w / v) trehalose.

[0163] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 10% (v / v) propylene glycol and about 7% (w / v) trehalose.

[0164] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 10% (v / v) propylene glycol and about 2% (w / v) trehalose.

[0165] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 10% glycerol and about 7% (w / v) trehalose.

[0166] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 5% (w / v) hydroxyethyl starch.

[0167] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 10% (w / v) hydroxyethyl starch.

[0168] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 15% (w / v) hydroxyethyl starch.

[0169] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 5% (w / v) starch.

[0170] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 10% (w / v) pentasyl starch.

[0171] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 15% (w / v) pentasyl starch.

[0172] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 15% (w / v) dextran-40.

[0173] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 10% (w / v) dextran-40.

[0174] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 5% (w / v) dextran-40.

[0175] In some embodiments, the cryopreservation formulation comprises about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 5% (v / v) ethylene glycol.

[0176] In some embodiments, the cryopreservation formulation may be any of the aforementioned formulations in an albumin-free form (i.e., it does not contain albumin). In some embodiments, the cryopreservation formulation may be any of the aforementioned formulations in a glucose-free form (i.e., it does not contain glucose). In some embodiments, the cryopreservation formulation may be any of the aforementioned formulations in both an albumin-free and glucose-free form (i.e., it does not contain albumin and it does not contain glucose).

[0177] In some embodiments, the cryopreservation formulation comprises glucose, buffered saline (e.g., PBS) (or optionally buffer and / or saline or electrolyte) and hydroxyethyl starch, and is albumin-free.

[0178] In some embodiments, the cryopreservation formulation comprises albumin (e.g., human albumin, such as recombinant human albumin), buffered saline (e.g., PBS) (or optionally buffer and / or saline or electrolyte) and hydroxyethyl starch, and is glucose-free.

[0179] In some embodiments, the cryopreservation formulation comprises buffered saline (e.g., PBS) (or optionally buffer and / or saline or electrolyte) and hydroxyethyl starch, and is albumin-free and glucose-free.

[0180] This disclosure envisions and provides formulations that "comprise," "are substantially composed of," or "are composed of" the components provided herein. For the sake of brevity, most formulations are described herein as "comprise" the various components they specify; however, it should be understood that this disclosure provides formulations that are "substantially composed of" or "are composed of" the same components.

[0181] Cell preparations Some aspects of this disclosure provide cell preparations that include cell populations and cryopreservation formulations as provided herein.

[0182] In some embodiments, the cell preparation exhibits at least 80% (e.g., 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%, and at least 99%) cell viability. In some embodiments, the cell preparation exhibits a robust cell growth index. In some embodiments, the cell preparation exhibits a cell growth index of 3 for at least 110 hours, at least 109 hours, at least 108 hours, at least 107 hours, at least 106 hours, at least 105 hours, at least 104 hours, at least 103 hours, at least 102 hours, at least 101 hours, at least 100 hours, at least 99 hours, at least 98 hours, at least 97 hours, at least 96 hours, at least 95 hours, at least 94 hours, at least 93 hours, at least 92 hours, at least 91 hours, at least 90 hours, at least 89 hours, at least 88 hours, at least 87 hours, at least 86 hours, at least 85 hours, at least 84 hours, at least 83 hours, at least 82 hours, at least 81 hours, or at least 80 hours. In some embodiments, the cell preparation is resistant to changes in storage temperature while maintaining robust viability and cell growth index.

[0183] This document provides cell preparations comprising (i) a cryopreservation formulation provided herein and (ii) a 1:1 (v / v) mixture of cell populations comprising a suspension of albumin, glucose, and buffered saline, each at the same concentration as said formulation, wherein said suspension does not contain a (non-albumin) cryoprotectant. In some embodiments, the cell preparations are DMSO-free. If DMSO is present in the cell preparations provided herein, it is present in amounts of less than 10% (v / v), less than 5% (v / v), less than 1% (v / v), less than 0.1% (v / v), or less than 0.01% (v / v).

[0184] This document provides cell preparations comprising cell populations in formulations provided herein, the formulations comprising glucose, albumin, buffered saline, and one or more, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch (e.g., but not limited to pentamylon), ethylene glycol, and poloxamer-188. In some embodiments, the cell preparations are DMSO-free. If DMSO is present in the cell preparations provided herein, it is present in amounts of less than 10% (v / v), less than 5% (v / v), less than 1% (v / v), less than 0.1% (v / v), or less than 0.01% (v / v).

[0185] This article provides cell preparations comprising cell populations in a cryopreservation formulation, the cryopreservation formulation being substantially composed of albumin, glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch (e.g., but not limited to pentamylon), and ethylene glycol.

[0186] In some embodiments, the cell preparation comprises a cell population in a formulation provided herein, the formulation comprising glucose, albumin, buffered saline, and one or two, or optionally more, cryoprotectants. In some embodiments, the components in the cell preparation are the same as those in the cryopreservation formulation provided herein, except that the concentrations of the one or two, or optionally more, cryoprotectants are halved.

[0187] In some embodiments, albumin is present in the range of about 1% to about 10% (w / v). In some embodiments, albumin is present in the range of about 2% to about 8% (w / v). In some embodiments, albumin is present in the range of about 2% to about 7.5% (w / v). In some embodiments, albumin is present in the range of about 1% to about 4% (w / v). In some embodiments, albumin is present in the range of about 2% to about 3% (w / v). In some embodiments, albumin is present in the range of 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%, or about 10% (w / v). In some embodiments, albumin is present at 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%, or about 3% (w / v). In some embodiments, albumin is present at about 2.5% (w / v). In some embodiments, albumin is absent (i.e., the formulation is albumin-free).

[0188] In some embodiments, glucose is present in the range of about 0.01% to about 3% (w / v). In some embodiments, glucose is present in the range of about 0.01% to about 1.5% (w / v). In some embodiments, glucose is present in the range of about 0.01% to about 0.5% (w / v). In some embodiments, glucose is present in the range of about 0.02% to about 1.5% (w / v). In some embodiments, glucose is present in the range of about 0.05% to about 1.2% (w / v). In some embodiments, glucose is present in the range of 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 1%, about 1.1%, or about 1.2% (w / v). In some embodiments, glucose is present in the range of about 0.09% (w / v). In some embodiments, glucose is absent (i.e., the formulation is glucose-free).

[0189] In some embodiments, the cell preparations provided herein comprise a cell population in a formulation containing one or two, or optionally more, (non-albumin) cryoprotectants. In some embodiments, the one or two, or optionally more, cryoprotectants do not include DMSO in certain cases. Some formulations provided herein contain one (non-albumin) cryoprotectant. Some formulations provided herein contain two (non-albumin) cryoprotectants. Some formulations provided herein contain more than two (non-albumin) cryoprotectants. The one or two, or optionally more, cryoprotectants are selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch (e.g., but not limited to pentamylon), and ethylene glycol.

[0190] In some embodiments, the one or two, or optionally more, cryoprotectants comprise propylene glycol. In some embodiments, propylene glycol is present in the range of 0.05% to about 10% (v / v). In some embodiments, propylene glycol is present in the range of 0.1% to about 7.5% (v / v). In some embodiments, propylene glycol is present in the range of 0.1% to about 3% (v / v). In some embodiments, propylene glycol is present in the range of 1% to about 7.5% (v / v). In some embodiments, propylene glycol is present in the range of 3% to about 7.5% (v / v). In some embodiments, propylene glycol is present in the range of about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, or about 7.5% (v / v). In some embodiments, propylene glycol is present in the range of about 0.1%, about 1%, about 3%, about 5%, or about 7.5% (v / v). In some embodiments, propylene glycol is present at about 0.1% (v / v). In some embodiments, propylene glycol is present at about 1% (v / v). In some embodiments, propylene glycol is present at about 3% (v / v). In some embodiments, propylene glycol is present at about 5% (v / v). In some embodiments, propylene glycol is present at about 7.5% (v / v).

[0191] In some embodiments, the one or two, or optionally more, cryoprotectants comprise dextran-40. In some embodiments, dextran-40 is present in the range of about 0.5% to about 10% (w / v). In some embodiments, dextran-40 is present in the range of about 1% to about 10% (w / v). In some embodiments, dextran-40 is present in the range of about 2% to about 10% (w / v). In some embodiments, dextran-40 is present in the range of about 2.5% to about 7.5% (w / v). In some embodiments, dextran-40 is present in the range of 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%, or about 10% (w / v). In some embodiments, dextran-40 is present at about 2.5%, about 3%, about 5%, or about 7.5% (w / v). In some embodiments, dextran-40 is present at about 1% (w / v). In some embodiments, dextran-40 is present at about 2.5% (w / v). In some embodiments, dextran-40 is present at about 3% (w / v). In some embodiments, dextran-40 is present at about 5% (w / v). In some embodiments, dextran-40 is present at about 7.5% (w / v). In some embodiments, dextran-40 is present at about 10% (w / v).

[0192] In some embodiments, the one or two, or optionally more, cryoprotectants comprise proline. In some embodiments, proline is present in the range of about 1% to about 7.5% (w / v). In some embodiments, proline is present in the range of about 2.5% to about 7.5% (w / v). In some embodiments, proline is present in the range of about 2.5% to about 5% (w / v). In some embodiments, proline is present in the range of about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% (w / v). In some embodiments, proline is present in the range of about 2.5% or 5% (w / v). In some embodiments, proline is present in the range of about 2.5% (w / v). In some embodiments, proline is present in the range of about 5% (w / v).

[0193] In some embodiments, the one or two, or optionally more, cryoprotectants comprise trehalose. In some embodiments, trehalose is present in the range of about 0.05% to about 10% (w / v). In some embodiments, trehalose is present in the range of about 0.1% to about 7.5% (w / v). In some embodiments, trehalose is present in the range of about 0.1% to about 3.5% (w / v). In some embodiments, trehalose is present in the range of about 1% to about 6% (w / v). In some embodiments, trehalose is present in the range of about 1% to about 3.5% (w / v). In some embodiments, trehalose is present in the range of about 2% to about 5% (w / v). In some embodiments, trehalose is present in the range of about 3% to about 7.5% (w / v). In some embodiments, trehalose is present in the range of about 1%, about 1.5%, about 2%, about 2.5%, about 3%, or about 3.5% (w / v). In some embodiments, trehalose is present at about 1% or about 3.5% (w / v). In some embodiments, trehalose is present at about 1% (w / v). In some embodiments, trehalose is present at about 3.5% (w / v).

[0194] In some embodiments, the one or two, or optionally more, cryoprotectants comprise glycerol. In some embodiments, glycerol is present in the range of about 2% to about 8% (v / v). In some embodiments, glycerol is present in the range of about 4% to about 6% (v / v). In some embodiments, glycerol is present in the range of 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%, or about 8% (v / v). In some embodiments, glycerol is present in the range of about 5% (v / v).

[0195] In some embodiments, the one or two, or optionally more, cryoprotectants comprise hydroxyethyl starch, such as, but not limited to, tetra-starch, penta-starch, or hepta-starch. In some embodiments, the one or two, or optionally more, cryoprotectants comprise penta-starch. Penta-starch is a subgroup of hydroxyethyl starch, having five hydroxyethyl groups out of every 11 hydroxyl groups, resulting in approximately 50% hydroxyethyl substitution. Penta-starch is commercially available as Pentaspan (Bristol-Myers Squibb).

[0196] In some embodiments, hydroxyethyl starch, such as pentamyl starch, is present in the range of about 2% to about 10% (w / v). In some embodiments, hydroxyethyl starch, such as pentamyl starch, is present in the range of about 2.5% to about 7.5% (w / v). In some embodiments, hydroxyethyl starch, such as pentamyl starch, is present in the range of 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%, or about 10% (w / v). In some embodiments, hydroxyethyl starch, such as pentamyl starch, is present in the range of about 2.5%, about 5%, or about 7.5% (w / v). In some embodiments, hydroxyethyl starch, such as pentamyl starch, is present in the range of about 2.5% (w / v). In some embodiments, hydroxyethyl starch, such as pentamyl starch, is present in the range of about 5% (w / v). In some implementations, hydroxyethyl starch, such as pentastarch, is present at about 7.5% (w / v).

[0197] In some embodiments, the one or two, or optionally more, cryoprotectants comprise ethylene glycol. In some embodiments, ethylene glycol is present in the range of about 1% to about 4% (v / v). In some embodiments, ethylene glycol is present in the range of about 2% to about 3% (v / v). In some embodiments, ethylene glycol is present in the range of 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%, or about 3% (v / v). In some embodiments, ethylene glycol is present in the range of about 2.5% (v / v).

[0198] Combination of cryoprotectants In some embodiments, the cell preparations provided herein comprise cell populations in formulations containing two (non-albumin) cryoprotectants. Various combinations of cryoprotectants are provided below.

[0199] Propylene glycol and dextran-40 In some embodiments, the cell preparation comprises propylene glycol and dextran-40. In some embodiments, propylene glycol is present in the range of about 0.05% to about 10% (v / v) and dextran-40 is present in the range of about 0.1% to about 10% (w / v). In some embodiments, propylene glycol is present in the range of about 0.05% to about 10% (v / v) and dextran-40 is present in the range of about 2% to about 10% (w / v). In some embodiments, propylene glycol is present in the range of about 0.1% to about 7.5% (v / v) and dextran-40 is present in the range of about 2.5% to about 7.5% (w / v). In some embodiments, propylene glycol is present in the range of about 0.1% (v / v), about 1%, about 3%, about 5%, or about 7.5% and dextran-40 is present in the range of about 2.5%, about 3%, about 5%, or about 7.5%. In some embodiments, propylene glycol is present in the range of about 0.1% to about 7.5% (v / v) and dextran-40 is present in the range of about 3%, about 5%, or about 7.5%. In some embodiments, propylene glycol is present in the range of about 0.1%, about 1%, about 3%, about 5%, or about 7.5% and dextran-40 is present in the range of about 5%. In some embodiments, propylene glycol is present in the range of about 5% (v / v) and dextran-40 is present in the range of about 5%. In some embodiments, propylene glycol is present in the range of about 3% (v / v) and dextran-40 is present in the range of about 10%. In some embodiments, propylene glycol is present in the range of about 3% (v / v) and dextran-40 is present in the range of about 7.5% (w / v). In some embodiments, propylene glycol is present at about 3% (v / v) and dextran-40 is present at about 5% (w / v). In some embodiments, propylene glycol is present at about 3% and dextran-40 is present at about 3% (w / v). In some embodiments, propylene glycol is present at about 3% (v / v) and dextran-40 is present at about 1% (w / v).

[0200] Propylene glycol and trehalose In some embodiments, the cell preparation comprises propylene glycol (1,3-propanediol) and trehalose. In some embodiments, propylene glycol is present in the range of about 0.05% to about 10% (v / v) and trehalose in the range of about 0.05% to about 10% (w / v). In some embodiments, propylene glycol is present in the range of about 0.1% to about 7.5% and trehalose in the range of about 0.1% to about 7.5% (w / v). In some embodiments, propylene glycol is present in the range of about 5% and trehalose in the range of about 0.1% to about 7.5% (w / v). In some embodiments, propylene glycol is present in the range of about 5% (v / v) and trehalose in the range of about 3.5% (w / v). In some embodiments, propylene glycol is present in the range of about 5% (v / v) and trehalose in the range of about 1% (w / v).

[0201] Propylene glycol and proline In some embodiments, the cell preparation comprises propylene glycol (1,3-propanediol) and proline. In some embodiments, propylene glycol is present in the range of about 0.05% to about 10% (v / v) and proline in the range of about 1% to about 7.5% (w / v). In some embodiments, propylene glycol is present in the range of about 0.1% to about 7.5% (v / v) and proline in the range of about 2.5% to about 7.5% (w / v). In some embodiments, propylene glycol is present in the range of about 0.1% to about 7.5% and proline in the range of about 2.5% to about 5% (w / v). In some embodiments, propylene glycol is present in the range of about 3% (v / v) and proline in the range of about 2.5% or about 5% (w / v).

[0202] Glycerin and Trehalose In some embodiments, the cell preparation comprises glycerol and trehalose. In some embodiments, glycerol is present in the range of about 2% to about 8% (v / v) and trehalose is present in the range of about 1% to about 6% (w / v). In some embodiments, glycerol is present in the range of about 4% to about 6% (v / v) and trehalose is present in the range of about 2% to about 5% (w / v). In some embodiments, glycerol is present in the range of about 5% (v / v) and trehalose is present in the range of about 3.5% (w / v).

[0203] Examples of cell preparations In some embodiments, the cell preparations provided herein comprise a cell population in a formulation containing about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin); about 0.09% (w / v) glucose; buffered saline (e.g., PBS); about 0.1%, about 1%, about 3%, about 5%, or about 7.5% (w / v) propylene glycol; and about 2.5%, about 3%, about 5%, or about 7.5% (w / v) dextran-40.

[0204] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin); about 0.09% (w / v) glucose; buffered saline (e.g., PBS); about 0.1% to about 7.5% (v / v) propylene glycol; and about 3%, about 5%, or about 7.5% (w / v) dextran-40.

[0205] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin); about 0.09% (w / v) glucose; buffered saline (e.g., PBS); about 0.1%, about 1%, about 3%, about 5%, or about 7.5% (v / v) propylene glycol; and about 5% (w / v) dextran-40.

[0206] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 5% (v / v) propylene glycol, and about 7.5% (w / v) dextran-40.

[0207] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 5% (v / v) propylene glycol, and about 5% (w / v) dextran-40.

[0208] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol, and about 10% (w / v) dextran-40.

[0209] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol, and about 7.5% (w / v) dextran-40.

[0210] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol, and about 5% (w / v) dextran-40.

[0211] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol, and about 3% (w / v) dextran-40.

[0212] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol, and about 1% (w / v) dextran-40.

[0213] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol and about 2.5% (w / v) proline.

[0214] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol and about 5% (w / v) proline.

[0215] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 0.01% to about 0.5% (w / v) glucose, buffered saline (e.g., PBS), about 5% (v / v) propylene glycol, and about 0.1% to about 7.5% (w / v) trehalose.

[0216] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 5% (v / v) propylene glycol and about 3.5% (w / v) trehalose.

[0217] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 5% (v / v) propylene glycol and about 1% (w / v) trehalose.

[0218] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 5% glycerol, and about 3.5% (w / v) trehalose.

[0219] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 2.5% (w / v) hydroxyethyl starch with a hydroxyethyl substitution range of up to 100%.

[0220] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 5% (w / v) hydroxyethyl-substituted starch ranging from up to 100%.

[0221] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 7.5% (w / v) hydroxyethyl-substituted starch ranging from up to 100%.

[0222] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 2.5% (w / v) pentasyl starch.

[0223] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 5% (w / v) pentasyl starch.

[0224] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 7.5% (w / v) pentasyl starch.

[0225] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 7.5% (w / v) dextran-40.

[0226] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 5% (w / v) dextran-40.

[0227] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 2.5% (w / v) dextran-40.

[0228] In some embodiments, the cell preparations provided herein contain a cell population in a formulation comprising about 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), about 0.09% (w / v) glucose, buffered saline (e.g., PBS), and about 2.5% (v / v) ethylene glycol.

[0229] This disclosure also contemplates any of the aforementioned cell preparations in an albumin-free form. This disclosure also contemplates any of the aforementioned cell preparations in a glucose-free form. This disclosure also contemplates any of the aforementioned cell preparations in both albumin-free and glucose-free forms.

[0230] This disclosure envisions and provides cell preparations that “contain,” “are substantially composed of,” or “are composed of.” For the sake of brevity, most cell preparations are described herein as “containing” the various components they specify; however, it should be understood that this disclosure provides cell preparations that “are substantially composed of” or “are composed of” the same components.

[0231] In some embodiments, the cell preparation has a cell density of about 100 cells to about 10,000 cells / µL. In some embodiments, the cell preparation has a cell density of about 300 cells to about 7,000 cells / µL. In some embodiments, the cell preparation has a cell density of about 350 cells to about 7,000 cells / µL. In some embodiments, the cell preparation has a cell density of about 350 cells / µL, or about 3,000 cells / µL, or about 3,500 cells / µL, or about 6,500 cells / µL, or about 7,000 cells / µL. In some embodiments, the cell preparation has a cell density of about 2,000 cells to about 4,000 cells / µL, or about 3,000 cells to about 3,500 cells / µL. In some embodiments, the cell preparation has a cell density of about 3,333 cells / µL. In some embodiments, the cell preparation is in a volume of about 400-500 microliters. In some embodiments, the cell preparation is in a volume of about 450 microliters.

[0232] cell population Cell types The cryopreservation formulations and cell preparations provided herein can be used in combination with different cell types and different sites and routes of application. While ophthalmic applications are preferred, other applications are also contemplated and included in this disclosure. In some embodiments, the cell population is selected from retinal pigment epithelial (RPE) cells, photoreceptor cells, their photoreceptor progenitor cells, photoreceptor repair cells (PRCs), corneal endothelial cells, corneal endothelial progenitor cells, corneal epithelial cells, or mesenchymal cells, such as angiogenic mesenchymal cells. In some embodiments, the cell population is a retinal pigment epithelial (RPE) cell population. In some embodiments, the cell population is an angiogenic mesenchymal cell (HMC) population. In other embodiments, the cell population is selected from macrophages, natural killer cells, T cells, vascular cells, vascular progenitor cells, hepatocytes, nerve cells, and pancreatic cells. In some embodiments, the cell population is a vascular progenitor cell population.

[0233] Cell source In some embodiments, the cell population in the cell preparations provided herein may be derived from primary cells. In some embodiments, the cell population in the cell preparations provided herein may be derived from freshly harvested cells.

[0234] In some embodiments, the cell population in the cell preparations provided herein can be generated from differentiated pluripotent stem cells, including human induced pluripotent stem cells (hiPSCs), human embryonic stem cells (hESCs), and somatic cells (including transdifferentiated cells and stem cells). Pluripotent stem cells can be derived from universal donor cells. The differentiation protocol can be directed or spontaneous. In some embodiments, the cell population is generated by in vitro differentiation of pluripotent stem cells. In some embodiments, the cell population is generated by in vitro differentiation of embryonic stem (ES) cells. Embryonic stem cells can be derived from a single blastomeres without embryonic destruction. In some embodiments, the cell population is generated by in vitro differentiation of induced pluripotent stem cell (iPSC) cells.

[0235] In some embodiments, cell preparations containing cell populations suitable for transplantation into a subject's eye are suitable for injection into the subject's eye. In some embodiments, such cell preparations can be used to treat retinal degenerative diseases or conditions, including but not limited to retinal detachment, retinal tears, retinal dysplasia, vascular streaks, myopic macular degeneration, or retinal atrophy, or those associated with a variety of visual impairments leading to photoreceptor damage and blindness, such as choroidal atrophy, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration (AMD)), retinitis pigmentosa, Staggart's disease (yellow macular degeneration), and geographic atrophy (including geographic atrophy secondary to AMD).

[0236] In some embodiments, the cell preparation comprises a population of RPE cells and the cryopreservation formulation provided herein. The RPE cells may be stable terminally differentiated RPE cells that do not dedifferentiate into non-RPE cell types. The RPE cells described herein may be functional RPE cells characterized by their ability to integrate into the retina after administration to the cornea, subretina, or other means to humans or non-human animals.

[0237] RPE cells can express RPE cell markers. For example, the expression levels of markers such as RPE65, PAX2, PAX6, tyrosinase, bestrophin, PEDF, PMEL17, CRALBP, Otx2, and / or MITF can be equivalent to those in naturally occurring RPE cells. The maturity level of RPE cells can be assessed by measuring the expression of at least one of PAX2, PAX6, PMEL17, and tyrosinase, or their respective expression levels.

[0238] The cells may be of human or animal origin.

[0239] Engineered non-immunogenic cells In some embodiments, the cell populations provided herein are engineered cell populations (universal donor cells or cells with low immunogenicity or derived therefrom), wherein said cells express at least one polypeptide selected from human leukocyte antigen E (HLA-E), human leukocyte antigen F (HLA-F), and human leukocyte antigen G (HLA-G). The cell populations also contain genetically modified disruptions of one or more copies (e.g., all copies) of endogenous β-2 microglobulin (B2M) and / or genetically modified disruptions (UC-RPEs) of one or more copies (e.g., all copies) of human leukocyte antigen (HLA) class II related genes selected from regulatory factor X-associated ankyrin repeat sequence protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-associated protein (RFXAP), and class II transactivator (CIITA). For example, said cells contain genetically modified disruptions of endogenous B2M and RFXANK and express HLA-E. In another example, the cells contain genetically modified disruptions of all copies of endogenous B2M and all copies of RFXANK, and express HLA-E. In various respects, the cells further contain nucleic acid molecules encoding suicide gene products (e.g., the herpes simplex virus thymidine kinase (TK) suicide gene). This disruption allows the cells to, for example, reduce or evade immune detection, and allows for the generation of cell populations that do not require HLA matching for therapeutic applications, as well as providing homogeneous cell populations with reduced unwanted cell appearance, improved safety profiles, strong in vivo retention, enhanced colonization capacity in subjects, and / or increased tumor burden reduction (see, for example, WO2012145384 and WO2013158292, which are incorporated herein by reference in their entirety).

[0240] The cells, such as RPE cells (or PSCs derived from them, such as RPE cells, or intermediates thereof), are genetically modified to disrupt one or more copies (e.g., all copies) of the endogenous B2M gene in the cells. B2M is a key component of the HLA class I (HLA-I) complex. HLA is a cell surface complex that mediates leukocyte interactions or interactions between leukocytes and other cells. The HLA-I molecule is a cell surface complex that presents antigens to CD8+ cytotoxic T cells, thereby mediating cellular immunity. The HLA-I molecule contains the HLA-I heavy chain and B2M. Disruption of one or more (e.g., all) copies of the B2M gene reduces or eliminates the production of the B2M protein (SEQ ID NO: 5), thereby reducing or eliminating the display of the HLA-I complex on the cell. A representative nucleic acid sequence encoding B2M is provided in SEQ ID NO: 1.

[0241] Cells, such as RPE cells (or PSCs or intermediates thereof from which said cells are derived), are also envisioned that are genetically modified to disrupt one or more copies (e.g., all copies) of a subset of HLA-I. Six HLA class I alpha (α) chains have been identified, including three canonical (HLA-A, HLA-B, and HLA-C) and three non-canonical (HLA-E, HLA-F, and HLA-G) HLA class I α chains, which are responsible for the specificity of peptide binding at the HLA-I binding groove. In this regard, this disclosure envisions cells genetically modified to disrupt one or more copies (e.g., all copies) of the HLA-I gene selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, and combinations thereof. For example, said cells (or PSCs or intermediates thereof from which said cells are derived) could be genetically modified to disrupt one or more copies (e.g., all copies) of HLA-A, HLA-B, and / or HLA-C. HLA molecules are further described, for example, in Choo, Yonsei Med J., 48(1): 11–23 (2007); www.ebi.ac.uk / ipd / imgt / hla / ; and GenBank accession numbers NG_029217 (HLA-A), XP_041536701 (HLA-A), NG_023187 (HLA-B), NP_005505 (HLA-B), NG_029422 (HLA-C), NP_001229971 (HLA-C), NM_005516 (HLA-E), NP_005507 (HLA-E), NG_012009 (HLA-F), NP_001091949 (HLA-F), NG_029039 (HLA-G) and NP_001371219 (HLA-G).

[0242] Cells disclosed herein, such as RPE cells (or PSCs from which said cells are derived, or intermediates thereof), are genetically engineered to disrupt one or more (e.g., all) copies of HLA class II (HLA-II)-associated genes selected from RFXANK, RFX5, RFXAP, and CIITA. HLA-II molecules are transmembrane proteins found on antigen-presenting cells (APCs) and sometimes on solid organs. HLA-II molecules contain two homologous subunits, an alpha (α) subunit and a beta (β) subunit. HLA-II-associated genes encode HLA-II regulatory proteins that regulate the expression of HLA class II molecules. Disruption of one or more (e.g., all) copies of one or more of the HLA-II-associated genes RFXANK, RFX5, RFXAP, and / or CIITA reduces or eliminates the production of the encoded protein, thereby reducing or eliminating the display of the HLA-II complex on the cell. The cell (or a PSC or intermediate thereof from which the cell is derived) may contain disruption of one or more copies (e.g., all copies) of a gene encoding one of RFXANK, RFX5, RFXAP, and / or CIITA (e.g., the cell is engineered to disrupt one or more copies (e.g., all copies) of the RFXANK gene); disruption of one or more copies (e.g., all copies) of a gene encoding any two combinations of RFXANK, RFX5, RFXAP, and / or CIITA; disruption of one or more copies (e.g., all copies) of a gene encoding any three combinations of RFXANK, RFX5, RFXAP, and / or CIITA; or disruption of one or more copies (e.g., all copies) of a gene encoding all of RFXANK, RFX5, RFXAP, and CIITA. RFXANK is encoded by, for example, sequences of SEQ ID NO: 3 and 5; RFX5 is encoded by, for example, sequences of SEQ ID NO: 7 and 9; RFXAP is encoded by, for example, SEQ ID NO: 11; and CIITA is encoded by, for example, SEQ ID NO: 13. In all respects of this disclosure, the cells are engineered to destroy all copies of B2M and RFXANK.

[0243] Any suitable technique can be used to introduce disruption into a target gene (e.g., an HLA-II-related gene, a B2M gene, or any other target gene). Many techniques for disrupting endogenous coding sequences are known in the art, including the use of gene editing systems such as CRISPR / Cas (clustered regularly spaced short palindromic repeats and CRISPR-related proteins) systems, transcription activator-like effector nucleases (TALENs) and zinc finger nucleases, and targeting vectors that cleave target sites in the cellular genome and / or insert nucleic acid sequences into target sites in the cellular genome. Exemplary techniques utilize adeno-associated virus (AAV) vector-based editing, which optionally includes targeted insertion of the edited sequence (potentially a transgene) into the cellular genome to disrupt the endogenous coding sequence, thereby resulting in reduced or avoided production of endogenous proteins. In various respects, serotype 3B AAV vectors are used. The use of AAV vectors in engineered stem cells is further described, for example, in Riolobos et al., Molecular Therapy, 21(6), 1232-1241 (2013), which is incorporated herein by reference in its entirety, and particularly with respect to the disclosures regarding the use of AAV vectors and disruption of the HLA complex. Methods for confirming target gene disruption are known in the art and include, but are not limited to, PCR detection methods (e.g., real-time polymerase chain reaction (qRT-PCR)), RNA sequencing, next-generation sequencing methods, fluorescence-activated cell sorting (FACS), and immunostaining.

[0244] In various aspects, engineered cells, such as RPE cells, express at least one polypeptide selected from HLA-E, HLA-F, and HLA-G. In the context of this disclosure, the cells are engineered to express HLA-E, HLA-F, and / or HLA-G, i.e., nucleic acids encoding HLA-E, HLA-F, and / or HLA-G are introduced into the cells (or PSCs from which the cells are derived, or intermediates thereof), and the encoded proteins are produced and displayed on the cell surface. Optionally, the engineered cells express HLA-E. Optionally, the engineered cells express single-stranded fusion HLA-E, comprising at least a portion of B2M directly or via an adapter sequence linked to at least a portion of the HLA-I α chain, such as HLA-E. Optionally, the engineered cells express single-stranded fusion HLA-F or HLA-G, comprising at least a portion of B2M directly or via an adapter sequence linked to at least a portion of the HLA-I α chain, such as HLA-F or HLA-G. Engineered cells can overexpress proteins naturally produced by said cells, can express proteins encoded by exogenous nucleic acids after knocking out or disrupting the corresponding endogenous protein-coding sequence, or can express proteins not naturally encoded by the wild-type cell genome. Representative DNA and protein sequences of HLA-E are provided in SEQ ID NO: 15 and 16. Representative DNA and protein sequences of HLA-F are provided in SEQ ID NO: 17 and 18. Representative DNA and protein sequences of HLA-G are provided in SEQ ID NO: 19 and 20. It will be understood that variants of the sequences provided herein may be used, and therefore this disclosure contemplates the use of nucleic acids containing at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleic acid sequences disclosed herein. This disclosure further envisions the use of nucleic acids encoding peptides containing amino acid sequences having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any amino acid sequence described herein. Preferably, the variant retains HLA-I function, for example, by forming a functional peptide-binding groove to display the peptide and / or connect to inhibitory receptors on NK cells. Cells expressing HLA-E, HLA-F, and / or HLA-G minimize the risk of rejection when used as adoptive cell therapy. In various aspects of this disclosure, the cells are engineered to destroy all copies of B2M and RFXANK and are engineered to express HLA-E, for example, by single-chain fusion of HLA-E.

[0245] Optionally, the cells, such as RPE cells, further comprise nucleic acid molecules encoding suicide gene products. Combinations of nucleic acid molecules encoding different suicide gene products are also envisioned. Suicide genes can be used to selectively kill cells by inducing apoptosis or converting a non-toxic compound into a toxic compound, resulting in the death of unwanted cells. Examples of suicide gene products include, but are not limited to, caspases (e.g., caspase 9), thymidine kinase, cytosine deaminase, cytochrome P450, telomerase, and DNase. See, for example, U.S. Patent Publication No. 2022 / 0025001, which is incorporated herein by reference. Optionally, the nucleic acid encoding the suicide gene product is operatively linked to an inducible promoter. See, for example, Straathof et al. Blood, 105(11), 4247-4254 (2005), which is incorporated herein by reference. Optionally, the engineered cells comprise the herpes simplex virus thymidine kinase (TK) suicide gene. See, for example, Bonini et al., Science, 276, 1719-1724 (1997), which is incorporated herein by reference. In various respects, the cells contain nucleic acids encoding a hygromycin-thymidine kinase fusion protein. For example, the cells may contain nucleic acids encoding HSV TK and nucleic acids encoding a hygromycin-thymidine kinase fusion protein.

[0246] Methods for genetically modifying host cells to stably produce one or more target gene products are known in the art. Exemplary methods include the use of viral vectors. Viral vectors may include any suitable viral vector, including, for example, retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus), coronaviruses, orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), microRNA viruses, alphaviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., cowpox, fowlpox, and canarypox). Exogenous nucleic acids may also be introduced into host cells using gene-editing techniques, such as those described herein. Preferably, nucleic acids encoding HLA-E, HLA-F, or HLA-G are integrated into the cell genome, for example, into the B2M or HLA-II locus. Thus, in various aspects, the B2M locus is disrupted by inserting nucleic acids encoding different gene products into the B2M locus to replace the expression of the endogenous wild-type B2M protein. Alternatively or additionally, certain HLA-II loci may be disrupted by inserting nucleic acids encoding different gene products into the HLA-II loci to replace the expression of endogenous wild-type HLA-II proteins.

[0247] In all aspects of this disclosure, cells in the cell population, such as RPE cells, are derived from pluripotent stem cells. The term "pluripotent stem cell" includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells, regardless of the method by which pluripotent stem cells are obtained. Pluripotent stem cells are generally defined functionally as stem cells that: (a) induce teratomas when transplanted into immunodeficient (SCID) mice; (b) are capable of differentiating into cell types of all three germ layers (e.g., differentiateable into ectoderm, mesoderm, and endoderm cell types); and (c) express one or more markers of embryonic stem cells (e.g., expression of Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.). Pluripotent stem cells can be generated using methods known in the art, for example. Exemplary pluripotent stem cells include embryonic stem cells. "Embryonic stem cells" (ESCs) include, for example, cells derived from the inner cell mass of a human blastocyst or morula, including those cells that have been continuously passaged as cell lines. Embryonic stem cells, regardless of their origin or the specific method used to generate them, can be identified based on, for example, (i) their ability to differentiate into cells of all three germ layers, (ii) the expression of at least Oct-4 and alkaline phosphatase, and (iii) their ability to produce teratomas when transplanted into immunodeficient animals.

[0248] Another exemplary pluripotent stem cell is induced pluripotent stem cell (iPSC). iPSCs are generated, for example, by reprogramming somatic cells through the expression of factors (“reprogramming factors”) such as, but not limited to, combinations of Oct4, Sox2, c-Myc, Nanog, Lin28, and / or Klf4. Induced pluripotent stem cells can be generated using virtually any somatic cell at any developmental stage as a starting point. For example, the cells can be derived from embryonic, fetal, neonatal, juvenile, or adult donors. Exemplary somatic cells that can be used include fibroblasts, such as dermal fibroblasts obtained from skin samples or biopsies, synovial cells from synovial tissue, prepuce cells, buccal cells, or lung fibroblasts. In some embodiments, the somatic cells are not fibroblasts.

[0249] Pluripotent stem cells can be derived from any species. Embryonic stem cells have been successfully obtained from, for example, mice, various species of non-human primates, and humans. Therefore, those skilled in the art can generate embryonic stem cells from any species, including but not limited to humans, non-human primates, rodents (mice and rats), ungulates (cows, sheep, etc.), canines (domestic and wild canines), felines (domestic and wild felines, such as lions, tigers, and leopards), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, and marine mammals (dolphins, whales, etc.). Similarly, iPSCs can be derived from any species. iPSCs have been successfully generated using, for example, mouse and human cells. Therefore, iPSCs can be readily generated using donor cells from any species, such as any species described herein.

[0250] Cell state Cell populations can be formulated in cell preparations as provided herein for delivery in pharmaceutically acceptable ophthalmic media, such that the preparation remains in contact with the ocular surface for a sufficient time to allow the cells to penetrate into affected areas of the eye, such as the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous fluid, cornea, iris / ciliary body, lens, choroid, retina, sclera, suprachoroidal space, conjunctiva, subconjunctival space, suprascleral space, intracorneal space, supracorneal space, pars plana of the ciliary body, surgically induced avascular areas, macular or geographic atrophy areas.

[0251] The cell population can have several possible arrangements, such as single cells, cell clusters, spheroids, aggregates, sheets, or any combination thereof, and can be contained in an aqueous medium, gel, matrix, polymer, etc. In some embodiments, the cell population is combined with biomaterials. In some embodiments, the cell population is part of a 3D organoid. In some embodiments, the cell population in the cell preparations provided herein is a dissociated cell population, cell sheets, cell monolayers, or cell aggregates. In some embodiments, the cell population is a human cell population.

[0252] In some embodiments, this document provides cell preparations in which cell populations are contained in cell sheets. For example, RPE cells, corneal endothelial cells, photoreceptor cells, or photoreceptor repair cells may be contained in the sheets. For example, cell sheets containing RPE cells can be prepared by culturing RPE cells on a substrate from which intact cell sheets can be released. The substrate may be a thermosensitive polymer, such as a thermosensitive poly(N-isopropylacrylamide) (PNIPAAm) grafted surface, on which cells adhere and proliferate at culture temperatures, and whose surface characteristics change upon temperature change, resulting in the release of the cultured cell sheets (e.g., by cooling to below the lower critical dissolution temperature (LCST)). (See da Silva et al., Trends Biotechnol. 2007 Dec;25(12):577-83; Hsiue et al., Transplantation. 2006 Feb 15;81(3):473-6; Ide, T. et al. (2006), Biomaterials s27, 607–614; Sumide, T. et al. (2005), FASEB J. 20, 392–394; Nishida, K. et al. (2004), Transplantation 77, 379–385; and Nishida, K. et al. (2004), N. Engl. J. Med. 351, 1187–1196, each incorporated herein by reference in its entirety. Cell sheets can adhere to a suitable substrate for transplantation, such as a substrate that dissolves in vivo when the sheet is transplanted into a host organism, for example, by culturing cells on a suitable substrate, or by releasing cells from another substrate (e.g., a heat-sensitive polymer) onto the suitable substrate. Exemplary substrates that may be suitable for transplantation may comprise gelatin (see Hsiue et al., ibid.). Alternative substrates suitable for transplantation include fibrin-based matrices, etc. Cell sheets can be used to prepare drugs for the prevention or treatment of retinal degenerative diseases.

[0253] In some embodiments, preparations are provided herein in which cells are in the form of cell clusters, spheroids, or aggregates. In some embodiments, the cells provided herein are administered in combination with biological materials, such as polymerizable biological materials. In some embodiments, the cells are administered as organoids.

[0254] cell preparation form In some embodiments, the cell preparations are in cryopreserved form. The cell preparations provided herein can be cryopreserved by freezing at any suitable temperature known in the art. For example, in some embodiments, the cell preparations provided herein are cryopreserved at temperatures from -100ºC to -200ºC. In some embodiments, the cell preparations provided herein are cryopreserved at temperatures below -125ºC. In some embodiments, the cell preparations provided herein are cryopreserved at temperatures equal to or below -135ºC. In some embodiments, the cell preparations provided herein are cryopreserved at temperatures below -150ºC. In some embodiments, the cryopreserved cell preparations provided herein are stored in liquid nitrogen or in a vapor phase of liquid nitrogen.

[0255] Cryopreserved cell preparations are produced by combining cell populations with a cryopreservation formulation provided herein, optionally dispersing the cell populations in the formulation, and then slowly freezing (e.g., rate-controlled freezing) the mixture. Slow freezing can be performed manually or by a device (e.g., a rate-controlled freezer, as described in the examples). In one embodiment, the preparation is frozen at a rate of -2ºC / min until a desired (optionally intermediate) temperature, such as -80ºC, is reached. Alternatively, the rate-controlled freezing rate can be -1ºC / min or -1.5ºC / min, etc. Cryopreserved cell preparations can be held at intermediate temperatures for hours, days, weeks, or longer. It can then be held at even lower temperatures. As an example, the preparation can be reached to -80ºC using a slow freezing process, after which it can be stored at -80ºC overnight (or at most about 12 or about 24 hours), after which it can be stored in the vapor phase of a liquid nitrogen tank (e.g., at about -125ºC). Once at even lower temperatures, it can be stored therein for days, weeks, months, or even years. It can even be transported to, for example, clinical settings, while being cryopreserved at even lower temperatures (if desired). The cryopreservation temperature can be, for example, from 0ºC to 25ºC, such as about 1ºC, about 2ºC, about 3ºC, about 4ºC, about 10ºC, about 15ºC, about 20ºC, or about 25ºC. In some embodiments, the cell preparation has been cryopreserved and thawed. Once a cell preparation has been cryopreserved and thawed, it is considered a “thawed” cell preparation. The cell preparations provided herein can be thawed without dilution, washing, or other manipulation.

[0256] The following is a non-limiting procedure for preparing cells for cryopreservation using any of the cryopreservation formulations provided herein.

[0257] RPE cells are harvested from in vitro cultures (e.g., on day 11 or 12). They may be washed once or multiple times. A suitable wash buffer contains albumin (e.g., about 5% (w / v) albumin buffer). After washing, the cell pellet may be resuspended in a suitable resuspension buffer containing a minimum of albumin and glucose, and may also contain PBS, such as DPBS. Typically, the resuspension buffer will contain the same concentration of albumin and glucose as the cryopreservation formulation. As an example, both the resuspension buffer and the cryopreservation formulation may contain about 2.5% (w / v) albumin and about 0.09% (w / v) glucose in buffered saline. The resuspended cells may have a cell density ranging from, for example, about 200 cells / µL to about 20,000 cells / µL, including about 1,000 cells / µL to about 10,000 cells / µL, and about 3,000 cells / µL to about 10,000 cells / µL. In some specific instances, the cell density can be from about 6,000 cells / µL to about 7,000 cells / µL, including about 6,500 cells / µL, about 6,600 cells / µL, or about 6,700 cells / µL.

[0258] The resuspended cell mixture is then mixed 1:1 with one of the cryopreservation buffers provided herein. This 1:1 dilution is used to halve the cell density and the concentration of each cryoprotectant in the cryopreservation formulation. Thus, the cell density can range, for example, from about 100 cells / µL to about 10,000 cells / µL, including about 500 cells / µL to about 5,000 cells / µL, and from about 1,500 cells / µL to about 5,000 cells / µL. In some specific instances, the cell density can be from about 2,000 cells / µL to about 4,000 cells / µL, including about 3,000 cells / µL. The cell preparation can then be placed in cryopreservation vials, such as 2 mL CZ vials. Each vial can contain, for example, about 450 µL of the cell preparation containing about 1.5 million cells.

[0259] These vials can then be cryopreserved, for example using a rate-controlled freezer and a procedure. A non-limiting procedure may include the following steps: (a) placing the vial in a 4°C environment, (b) maintaining the vial at 4°C for 10 minutes, (c) gradually decreasing the temperature at a rate of 2°C / minute until -40°C is reached, (d) gradually decreasing the temperature at a rate of 10°C / minute until -80°C is reached, and optionally then placing it in liquid nitrogen (LN2) (-180°C).

[0260] It should be understood that the cell precipitate can be directly resuspended in a cryopreservation formulation diluted 1:1 with another solution, provided that the formulation and the solution contain the same concentrations of albumin, glucose, and buffer. Therefore, in some cases, the resuspension buffer or dilution solution is the same as the cryopreservation formulation, except that the cryopreservation formulation contains one or two, or optionally more, cryopreservatives, and the resuspension buffer and dilution solution do not.

[0261] When ready to thaw, the cryopreservation vial can be placed in a 37°C water bath for approximately 2–3 minutes. Afterward, the thawed cell preparation can be gently pipetted several times (e.g., 7–10 times) into the vial. The cell preparation can be used directly and immediately, or it can be kept at 4°C or room temperature for 0–4 hours.

[0262] Cell viability and cell growth potential (referred to herein as the cell growth index) of the cell preparation can also be assessed. Viability can be measured from several aliquots of the thawed cell preparation using any standard techniques or instruments known in the art. Typically, viability can be measured within approximately 5 to approximately 30 minutes after thawing.

[0263] Cell growth potential can be measured in many ways. In one approach, the cell density of the thawed cell preparation is adjusted to approximately 100-200 cells / µL (e.g., approximately 160 cells / µL), and approximately 80 cells / µL are seeded per well in a 96-well plate (e.g., an e-plate). These plates are then incubated at 37°C for 7 days, during which time cell growth can be monitored and evaluated. One metric that can be used is the time it takes for the cell number to triple. Cells are typically seeded within approximately 2 hours of thawing.

[0264] In some cases, suitable cell preparations are those demonstrating viability equal to or greater than about 75%, or equal to or greater than about 80%, or equal to or greater than about 85%, or equal to or greater than about 90%, or equal to or greater than about 95%. In some cases, suitable cell preparations are those demonstrating a cell growth index of 3 less than or equal to about 100 hours, less than or equal to about 90 hours, less than or equal to about 80 hours, or less than or equal to about 70 hours. In some cases, suitable cell preparations are those demonstrating viability equal to or greater than about 80% and a cell growth index of 3 less than or equal to about 100 hours. Other suitable cell preparations include those demonstrating viability equal to or greater than about 90% and a cell growth index of 3 less than or equal to about 80 hours.

[0265] In some embodiments, the cell preparation is maintained at room temperature (about 20°C to about 25°C). In some embodiments, the cell preparation is maintained at about 4°C. In some embodiments, the cell preparation is maintained at about -20°C. In some embodiments, the cell preparation is maintained at about -70°C. In some embodiments, the cell preparation is maintained at about -180°C.

[0266] In some embodiments, the cell preparation has been cryopreserved, thawed, and maintained at room temperature (about 20°C to about 25°C).

[0267] In some embodiments, the cell preparation has been cryopreserved, thawed, and maintained at approximately 4°C.

[0268] In some embodiments, the cell preparations have been held at room temperature (about 20°C to about 25°C) for about 1 to 2 hours prior to cryopreservation at -180°C. In some embodiments, the cell preparations have been held at about 4°C for about 4 to 7 hours prior to cryopreservation at -180°C. In some embodiments, the cell preparations have been held at room temperature (about 20°C to about 25°C) for about 1 to 2 hours and at about 4°C for about 4 to 7 hours prior to cryopreservation at -180°C.

[0269] This article also provides cell preparations comprising cell populations in a formulation containing albumin, glucose, buffered saline, and one or more, or optionally more, cryoprotectants, and having a cell viability of 80% or greater and / or a cell growth index of 110 hours or less after the following event: 3: a. Store at room temperature (about 20°C to about 25°C) for about 1 to 2 hours, then at about 4°C for about 4 to 7 hours, optionally at room temperature for about 1.5 hours, then at 4°C for about 4.5 hours or 6.5 hours. b. Store frozen at -180°C for approximately 1 to 2 years, optionally for approximately 1 month to 1 year. c. Freeze at -180°C, then thaw and store at approximately 4°C for approximately 3 to 6 hours, optionally approximately 4 hours. d. Freeze at -180°C, then thaw and allow to cool at room temperature (approximately 20°C to approximately 25°C) for approximately 3 to 6 hours, optionally approximately 4 hours. e. Freeze at -180°C, then freeze at about -20°C for about 0.5 to about 1 hour, then freeze at -180°C, or optionally freeze at about -20°C for about 0.5 to about 1 hour, then freeze at -180°C, or f. Freeze at -180°C, then at about -70°C for about 2 to about 4 weeks, then freeze again at -180°C.

[0270] In some embodiments, the cell preparation is dimethyl sulfoxide-free (DMSO-free). In some embodiments, after any one of events (a) to (f), the cell preparation has a cell viability of 85% or greater. In some embodiments, after any one of events (a) to (f), the cell preparation has a cell viability of 90% or greater. In some embodiments, after any one of events (a) to (f), the cell preparation has a cell viability of 95% or greater.

[0271] In some embodiments, the cell growth index is assessed by cell index 3 (as shown in the examples). In some embodiments, after any one of (a)-(f), the cell preparations provided herein have a cell growth index of 3 equal to or less than 100 hours, equal to or less than 90 hours, or equal to or less than 80 hours.

[0272] In some embodiments, after any of a, the cell preparations provided herein maintain a robust cell growth index. In some embodiments, after any of a, the cell preparations provided herein maintain robust viability and a robust cell growth index. In some embodiments, after any of (a)-(f), the cell preparations provided herein maintain robust viability (e.g., equal to or greater than 80% or greater than 85%) and a cell growth index of at least 110 hours (e.g., equal to or less than 110 hours, equal to or less than 100 hours, equal to or less than 90 hours, or equal to or less than 80 hours): 3.

[0273] Preparation method Some aspects of this disclosure provide methods for preparing cell preparations, including contacting a cell population with a cryopreservation formulation provided herein and a suspension comprising albumin, glucose, and buffered saline (e.g., about 2.5% (w / v) albumin (e.g., recombinant human albumin), about 0.09% (w / v) glucose, and buffered saline (e.g., PBS) at concentrations equal to those of the formulation), wherein the suspension does not contain a (non-albumin) cryoprotectant. In some embodiments, the cell preparation comprises a 1:1 (v / v) ratio of the cryopreservation formulation and the suspension. In some embodiments, the method further includes cryopreserving the cell preparation. In some embodiments, the method further includes thawing the cell preparation. In some embodiments, the method further includes administering the cell preparation to a subject in need. In some embodiments, the cell preparation is administered to the subject without washing or dilution after thawing. In some embodiments, the cell preparation is kept at about 4°C for about 4 hours prior to administration to the subject. In some embodiments, the cell preparation is kept at room temperature (about 20°C to about 25°C) for about 4 hours before administration to the subject. The cell population may be an RPE cell population, a mesenchymal stem cell population (e.g., an angiogenic mesenchymal stem cell population), a mesenchymal cell population (e.g., an angiogenic mesenchymal cell population), a photoreceptor cell population, a photoreceptor progenitor cell population, a photoreceptor repair cell population, a retinal ganglion cell population, a retinal ganglion progenitor cell population, a corneal endothelial cell population, a corneal endothelial progenitor cell population, or a vascular progenitor cell population, or a population of any cell type (e.g., as disclosed herein). The cell population may be generated by in vitro differentiation of pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells. The cell population may be human cells or non-human cells.

[0274] Some aspects of this disclosure provide methods for preparing cell preparations, including suspending cell populations in a cryopreservation formulation, said formulation comprising: (i) approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin); approximately 0.09% (w / v) glucose; buffered saline (e.g., PBS); approximately 0.1%, approximately 1%, approximately 3%, approximately 5%, or approximately 7.5% (v / v) propylene glycol; and approximately 5% (w / v) dextran-40, (ii) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 5% (v / v) propylene glycol, and approximately 7.5% (w / v) dextran-40. (iii) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 5% (v / v) propylene glycol, and approximately 5% (w / v) dextran-40. (iv) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 3% (v / v) propylene glycol, and approximately 10% (w / v) dextran-40. (v) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 3% (w / v) propylene glycol, and approximately 7.5% (w / v) dextran-40, (vi) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 3% (v / v) propylene glycol, and approximately 5% (w / v) dextran-40. (vii) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 3% (v / v) propylene glycol, and approximately 3% (w / v) dextran-40. (viii) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 3% (w / v) propylene glycol, and approximately 1% (w / v) dextran-40. (ix) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 3% (v / v) propylene glycol, and approximately 2.5% (w / v) proline. (x) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 3% (v / v) propylene glycol, and approximately 5% (w / v) proline. (xi) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 5% (v / v) propylene glycol, and approximately 3.5% (w / v) trehalose. (xii) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 5% (v / v) propylene glycol, and approximately 1% (w / v) trehalose. (xiii) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), approximately 5% glycerol, and approximately 3.5% (w / v) trehalose. (xiv) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), and approximately 2.5% (w / v) pentathione (or other hydroxyethyl starch with up to 100% hydroxyethyl substitution), (xv) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), and approximately 5% (w / v) pentathione (or other hydroxyethyl starch with up to 100% hydroxyethyl substitution), (xvi) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), and approximately 7.5% (w / v) pentathione (or other hydroxyethyl starch with up to 100% hydroxyethyl substitution), (xviii) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), and approximately 7.5% (w / v) dextran-40. (xix) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), and approximately 5% (w / v) dextran-40. (xx) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), and approximately 2.5% (w / v) dextran-40. (xxi) Approximately 2.5% (w / v) albumin (e.g., human albumin, such as recombinant human albumin), approximately 0.09% (w / v) glucose, buffered saline (e.g., PBS), and approximately 2.5% (v / v) ethylene glycol. (xxii) Buffered saline (e.g., PBS) and approximately 2.5% (w / v) hydroxyethyl starch, such as, but not limited to, tetra-, penta-, or hepta-starch. (xxiii) Buffered saline (e.g., PBS) and approximately 5% (w / v) hydroxyethyl starch, such as, but not limited to, tetra-, penta-, or hepta-starch, or (xxiv) buffered saline (e.g., PBS) and about 7.5% (w / v) hydroxyethyl starch, such as, but not limited to, tetra-starch, penta-starch or hepta-starch.

[0275] In some embodiments, the formulation may be a non-albumin form of the aforementioned formulation (i.e., the formulation may be as described above and herein, except that the formulation may lack albumin). As demonstrated in the examples, the cryoprotectant formulations provided herein result in higher post-thaw cell viability compared to formulations containing DMSO, even in the absence of albumin.

[0276] In some implementations, the cell population can be an RPE cell population, a mesenchymal stem cell population (e.g., angioblast-derived mesenchymal stem cell (HMC) population), a mesenchymal cell population (e.g., angioblast-derived mesenchymal cell (HMC) population), a photoreceptor cell population, a photoreceptor progenitor cell population, a photoreceptor repair cell population, a retinal ganglion cell population, a retinal ganglion progenitor cell population, a corneal endothelial cell population, a corneal endothelial progenitor cell population, or a vascular progenitor cell population. The cell population can be generated through in vitro differentiation of pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells. The cell population can be human cells or non-human cells.

[0277] How to use The cell preparations disclosed herein, including cryopreserved formulations, are available for various clinical applications once thawed. As described herein, cryopreserved formulations can be thawed without dilution, washing, or other procedures. In some embodiments, cryopreserved formulations can be thawed without the addition of a diluent, and subsequently administered to subjects without washing or other procedures.

[0278] In some embodiments, the cell populations thus formulated are intended for clinical use in regenerative medicine approaches. For example, the cell populations may be cell populations that can replace lost or degenerated cells in a subject, or repair or replace damaged or dysfunctional tissues in a subject. For example, in some embodiments, the cell populations may comprise pluripotent stem cells (e.g., iPS cells or ES cells), pluripotent stem cells or progenitor cells, or functionally differentiated cells, or populations or tissues containing such cells or combinations of such cells.

[0279] In some implementations, the cryopreserved cell preparation can be stored indefinitely, or it can be stored for a defined period of time, and optionally it can be transported to clinical or other end-user sites.

[0280] Cryopreservation formulations can be stored in a frozen state, such as in the vapor phase of a liquid nitrogen tank, for an extended period of time, such as the periods described herein, and including but not limited to at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40 weeks, at least 44 weeks, at least 48 weeks, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years or longer, while having minimal effect on cell viability or cell function, as measured by, for example, dye rejection, recoiling efficiency or reproliferative capacity. Cells or cell populations cryopreserved and thawed according to this disclosure may have at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cell viability, replate efficiency, and / or reproliferative capacity prior to cryopreservation.

[0281] In some embodiments, the cell preparations provided herein, once thawed, are formulated for administration to a subject without dilution, e.g., by injection. In some embodiments, the cell preparations are administered using a syringe. In some embodiments, the cell preparations are administered using a cannula. Exemplary cell preparations, after thawing, may be formulated to be suitable for treating human patients, e.g., pyrogen-free or substantially pyrogen-free, pathogen-free, sterile, and at physiological pH and osmotic pressure. In some embodiments, the preparations provided herein are formulated for administration by injection to a specific site, e.g., in the case of ophthalmic preparations for treating retinal diseases or conditions, injection into vitreous fluid to deliver to sites of retinal or choroidal damage, including, for example, vesicular or geographic atrophic areas. In some embodiments, the cell preparations are in a storage container. In some embodiments, the storage container is a vial, ampoule, bottle, syringe, refrigerated packaging, or other suitable storage container. In some embodiments, the cell preparations are in a vial. In some embodiments, the cell preparations are in a syringe.

[0282] The cell preparations provided in this disclosure may additionally include therapeutic agents, such as immunosuppressants, angiogenic agents, or nutrients or growth factors that support cell survival and / or are implanted in the preparation.

[0283] The volume and number of cells to be administered will depend on the specific application. Generally, for cell transplantation applications, it is ideal to minimize the administration volume as much as possible. Therefore, the cell preparation may be formulated to deliver the minimum possible volume. The cell concentration for injection may be any effective and non-toxic concentration. In some embodiments, the volume of the cell preparation to be administered is 1-1000 µL. In some embodiments, the volume of the cell preparation to be administered is 1-50 µL, or 10-50 µL, or 25-50 µL, or 50-100 µL, or 50-200 µL, or 50-300 µL, or 50-400 µL, or 50-500 µL, or 100-500 µL, or 100-400 µL, or 100-300 µL, or 100-300 µL, or about 200 µL, or about 150 µL. In some implementations, the volume of the cell preparation to be applied 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.

[0284] In some embodiments, the number and / or concentration of cells in the cell preparations provided herein can be determined by counting live cells and excluding non-live cells. For example, non-live cells can be detected by the inability to reject live dyes (e.g., trypan blue) or by using functional assays (e.g., ability to adhere to culture medium, phagocytosis, etc.). Additionally, the number or concentration of cells of a desired cell type can be determined by counting cells expressing one or more cell markers specific to that cell type and / or excluding cells expressing one or more markers indicating a cell type other than the desired cell type.

[0285] In some implementations, the cell preparation to be applied contains approximately 100 cells / µL to approximately 10,000 cells / µL.

[0286] In some embodiments, the cell count per vial is approximately 45,000 to approximately 4,500,000 cells / via. In some embodiments, the cell count per vial is approximately 1,500,000 cells / via.

[0287] The aforementioned cell quantity may be present in a single cryopreserved vial of the cryopreserved cell preparation. The cells may be present in approximately 10 to approximately 500 µL of cryopreservation formulation, including approximately 100 to approximately 500 µL of cryopreservation formulation, or approximately 200 to approximately 500 µL of cryoprotection formulation, or approximately 300 to approximately 500 µL of cryopreservation formulation, or approximately 400 to approximately 500 µL of cryopreservation formulation. In some embodiments, the cells are present in approximately 450 µL of cryopreservation formulation.

[0288] Some aspects of this disclosure provide methods for treating a subject with a condition or illness. In some embodiments, the subject has a condition or illness that would benefit from cell transplantation. In some embodiments, the method includes administering an effective amount of the cell preparation provided herein to the subject in need without washing or dilution after thawing. In some embodiments, the method includes administering an effective amount of a cell preparation prepared according to the methods provided herein to the subject in need without washing or dilution after thawing. In some embodiments, the cell preparation is kept at about 4°C for about 4 hours prior to administration to the subject. In some embodiments, the cell preparation is kept at room temperature (about 20°C to about 25°C) for about 4 hours prior to administration to the subject. In some embodiments, the cell preparation is administered at a cellular dose that is effective in treating the subject's condition or illness. In some embodiments, the condition or ailment includes, but is not limited to, retinal detachment, retinal dysplasia, vascular streaks, myopic macular degeneration, or retinal atrophy, or is associated with a variety of visual impairments leading to photoreceptor damage and blindness, such as choroidal agenesis, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration (AMD)), dry or wet age-related macular degeneration, retinitis pigmentosa, Fuchs dystrophy, Staggart's disease (yellow macular degeneration), and geographic atrophy (including geographic atrophy secondary to AMD). In some embodiments, the method further includes monitoring the subject for at least one symptom of the condition or ailment, optionally determining whether such symptom is reduced in severity or frequency after administration. In other embodiments, the condition or ailment includes, but is not limited to, cancer, liver disease (including advanced liver disease), Crohn's disease, arthritis (including osteoarthritis), pulmonary hypertension, and severe limb ischemia.

[0289] The term "treatment" refers to a clinical intervention designed to reverse, alleviate, delay the onset of a symptom or condition, or inhibit its progression. In some cases, the effectiveness of the intervention can be assessed by monitoring symptoms associated with the symptom or condition. In some embodiments, cell preparations may be administered after the development of one or more symptoms and / or after a diagnosis of the symptom or condition. In other embodiments, cell preparations may be administered in the absence of symptoms, for example, to prevent or delay the onset of symptoms or to inhibit the onset or progression of the symptom or condition. For example, cell preparations may be administered to susceptible individuals before the onset of symptoms (e.g., taking into account a history of symptoms and / or genetic or other susceptibility factors). Cell preparations may be administered once or more, including at regular and scheduled times or as needed.

[0290] Subjects can be human or non-human mammals. In some embodiments, subjects are non-human primates. In some embodiments, subjects are rodents. In some embodiments, subjects are sheep, goats, cattle, cats, or dogs. In some embodiments, subjects are research animals. In some embodiments, subjects are genetically modified, such as genetically modified non-human subjects. Subjects can be of any sex and at any developmental stage.

[0291] As used herein, the term "effective amount" refers to an amount of bioactive agent sufficient to elicit the desired biological response. For example, in some embodiments, an effective amount of a cell preparation may refer to an amount of preparation containing a number of cells or tissues sufficient to provide a therapeutic benefit to a subject with a condition or symptom (e.g., sufficient to improve the vision of a subject with a retinal disease or symptom). Those skilled in the art will understand that an effective amount may vary depending on various factors, such as the desired biological response, for example, the specific condition being treated, the specific symptoms to be alleviated, or the cells or tissues targeted, and the subject's age, sex, stage of disease, and general health condition.

[0292] Some aspects of this disclosure provide methods for treating retinal diseases, wherein the method includes administering an effective amount of the cell preparation provided herein to the eye of a subject suffering from a 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 or dry age-related macular degeneration (AMD)), Leber congenital amaurosis, Staggart's disease, or geographic atrophy (e.g., geographic atrophy secondary to AMD). In some embodiments, the cells administered include RPE cells, photoreceptor cells, photoreceptor progenitor cells, photoreceptor repair cells, corneal endothelial cells, corneal endothelial progenitor cells, or retinal ganglion cells or retinal ganglion progenitor cells, any of which may be generated through in vitro differentiation of pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells).

[0293] Reagent test kit Some aspects of this disclosure provide kits comprising (a) a cryopreserved pharmaceutical composition containing the target cells provided herein; and (b) instructions for thawing said composition and making it suitable for administration to a subject. These instructions may specify that no dilution, washing, or other manipulation of the cryopreserved composition is required. The kit may also include devices for administering the composition, such as syringes, cannulas, and needles. Example

[0294] The present invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are included only to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

[0295] Example 1 – First Screening of Cell Preparations RPE derived from human embryonic stem cells (as described in, for example, WO2021086911, which is incorporated herein by reference) were pooled in a 250 mL flask and washed with 200 mL of 0.5% rHA / DPBS by centrifugation at 500 x g for 5 min.

[0296] RPE cells were resuspended in 2.5% rHA / DPBS / 0.09% (w / v) glucose and then mixed with cryoprotectant buffer. As used herein, the cryopreservation preparation is sometimes referred to as "cryoprotectant buffer." The final composition of each cell preparation is shown in Table 1.

[0297] Table 1 450 µL of formulated RPE cells were loaded into CRYSTAL ZENITH® (CZ) vials (WestPharmaceutical Services, Inc. Exton, PA) and frozen at -2ºC / min using a rate-controlled freezer (THERMO SCIENTIFIC, Waltham, MA). Rate-controlled freezing was initiated within 3 hours of adding cryoprotectant buffer to the cells. The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0298] Thawed cell viability was assessed using a Vi-CELL BLU cell analyzer (Beckman Coulter Inc., Brea, CA). The cell index (CI) is a dimensionless parameter. It is obtained as a relative change in measured electrical impedance to represent cell state. The more cells attached to the electrode, the higher the CI value. In this experiment, cell proliferation was assessed by the time it took to reach a CI of 3, meaning that a shorter time indicated higher proliferative capacity. Cell growth was evaluated under the following conditions: E-Plates 96 (ACEA Bioscience, catalog number: 111902) were coated with 0.1% gelatin (Stemcell Tech, catalog number: 7903). RPE cells were seeded at 9,600 cells / well into the wells of an E-Plates 96 with 120 µL of DMEM-based cell culture medium containing 45% v / v high glucose DMEM (Gibco, catalog number: 10313-021), 42.6% v / v Knockout DMEM (Gibco, catalog number: 10829-018), 1% v / v GlutaMAX-1 (100X) (Gibco, catalog number: 35050-061), 0.5% v / v MEM non-essential amino acids (Gibco, catalog number: 11140-050), 5% v / v fetal bovine serum (irradiated) (Hyclone, catalog number: SH30406.02IR), and 6.4% v / v Knockout serum substitute (Gibco, catalog number: 10828-028). Plates containing cells were incubated at 37ºC in a CO2 incubator (Panasonic, Newark, NJ) at 5% CO2.

[0299] Except for 3% (v / v) propylene glycol / 2.5% (w / v) proline and 3% (v / v) propylene glycol / 10% (w / v) dextran-40, all cell preparations showed cell viability exceeding 90%, a value comparable to or better than the control 5% (v / v) DMSO cell preparation. (See also...) Figure 1A (Table 2).

[0300] Thawed cell growth was assessed using the xCelligence system (Agilent Technology, Santa Clara, CA). Frozen RPE vials were thawed, seeded and cultured in 96-well plates in growth medium, and cell growth was monitored in real time. Cell preparations 1A, 1B, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1M, and 1L showed a cell index 3 below 80 hours, indicating that cell growth rates were within the normal range, and cell preparations 1B, 1D, 1E, 1F, 1G, 1I, and 1J exhibited cell growth rates comparable to those of 5% (v / v) DMSO cell preparations. Cell preparations 1C and 1K showed a cell index 3 above 80 hours, indicating relatively low cell growth rates. (See also...) Figure 1B (Table 2).

[0301] Table 2 Example 2 – Screening of Dextran-40 Density with Propylene Glycol RPE derived from human embryonic stem cells (as described in, for example, WO2021086911, which is incorporated herein by reference) were pooled in a 250 mL flask and washed with 200 mL of 0.5% rHA / DPBS by centrifugation at 500 x g for 5 min.

[0302] RPE cells were resuspended in 2.5% rHA / DPBS / 0.09% (w / v) glucose and then mixed with cryoprotectant buffer. The final composition of each cell preparation is shown in Table 3.

[0303] Table 3 450 µL of formulated RPE cells were loaded into CRYSTAL ZENITH® (CZ) vials (WestPharmaceutical Services, Inc. Exton, PA) and frozen at -2ºC / min using a rate-controlled freezer (THERMO SCIENTIFIC, Waltham, MA). Rate-controlled freezing was initiated within 3 hours of adding cryoprotectant buffer to the cells. The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0304] Thawed cell viability was assessed using a Vi-CELL BLU cell analyzer (Beckman Coulter Inc., Brea, CA) as described in Example 1. Cell preparations 2A to 2C, comprising 3 to 7.5% (w / v) dextran-40 and 3% (v / v) propylene glycol, showed cell viability exceeding 90%. (See also...) Figure 2A (Table 4).

[0305] Thawed cell growth was assessed using the xCelligence system (Agilent Technology, Santa Clara, CA). Frozen RPE vials were thawed, seeded and cultured in 96-well plates in growth medium, and cell growth was monitored in real time. Cell preparations 2A to 2C, containing 3 to 7.5% (w / v) dextran-40 and 3% (v / v) propylene glycol, showed a cell index 3 below 70 hours, indicating that cell growth rates were within the normal range. Cell preparations 2D to 2E, containing 0.1 to 1% (w / v) dextran-40 and 3% (v / v) propylene glycol, showed a cell index 3 above 80 hours, indicating relatively low cell growth rates. (See also...) Figure 2B (Table 4).

[0306] These results show that dextran-40 exhibits significant cryoprotective properties at concentrations of 3% and higher.

[0307] Table 4 # Cell preparations Average viability (%) SD becomes vibrant Average cell growth (in hours) for Cell Index 3. SD of cell growth 2A 3% (v / v) Propylene Glycol / 7.5% (w / v) Glucan-40 96.3 0.3 58.0 0.0 2B 3% (v / v) Propylene Glycol / 5% (w / v) Dextran-40 95.6 0.5 63.0 2.8 2C 3% (v / v) Propylene Glycol / 3% (w / v) Dextran-40 93.6 0.4 68.5 2.1 2D 3% (v / v) Propylene Glycol / 1% (w / v) Dextran-40 89.2 0.9 84.5 2.1 2E 3% (v / v) propylene glycol / 0.1% (w / v) dextran-40 86.8 1.3 83.0 0.0 Example 3 – Screening Trehalose Density with Propylene Glycol RPE derived from human embryonic stem cells (as described in, for example, WO2021086911, which is incorporated herein by reference) were pooled in a 250 mL flask and washed with 200 mL of 0.5% rHA / DPBS by centrifugation at 500 x g for 5 min.

[0308] RPE cells were resuspended in 2.5% rHA / DPBS / 0.09% and then mixed with cryoprotectant buffer. The final composition of each cell preparation is shown in Table 5.

[0309] Table 5 450 µL of formulated RPE cells were loaded into CRYSTAL ZENITH® (CZ) vials (WestPharmaceutical Services, Inc. Exton, PA) and frozen at -2ºC / min using a rate-controlled freezer (THERMO SCIENTIFIC, Waltham, MA). Rate-controlled freezing was initiated within 3 hours of adding cryoprotectant buffer to the cells. The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0310] Thawed cell viability was assessed using a Vi-CELL BLU cell analyzer (Beckman Coulter Inc., Brea, CA) as described in Example 1. Cell preparations 3A to 3E showed cell viability exceeding 90%. (See Example 1) Figure 4A (Table 6)

[0311] Thawed cell growth was assessed using the xCelligence system (Agilent Technology, Santa Clara, CA). Frozen RPE vials were thawed, seeded and cultured in 96-well plates in growth medium, and cell growth was monitored in real time. All cell preparations were within acceptable limits, and there were no significant differences between cell preparations. (See [link to relevant documentation]) Figure 4B (Table 6)

[0312] Table 6 Example 4 – Robustness of cell preparations in terms of process time RPE derived from human embryonic stem cells (as described in, for example, WO2021086911, which is incorporated herein by reference) were pooled in a 250 mL flask and washed with 200 mL of 0.5% rHA / DPBS by centrifugation at 500 x g for 5 min.

[0313] RPE cells were resuspended in 2.5% rHA / DPBS / 0.09% (w / v) glucose and then mixed with cryoprotectant buffer. The final composition of each cell preparation is shown in Table 7.

[0314] Table 7 450 µL of prepared RPE cells were loaded into CRYSTAL ZENITH® (CZ) vials (WestPharmaceutical Services, Inc. Exton, PA) and cryopreserved at -2ºC / min using a rate-controlled freezer (THERMO SCIENTIFIC, Waltham, MA). For preparations 4A, 4C, 4E, 4G, 4I, 4K, and 4M, rate-controlled freezing began approximately 6 hours after the addition of cryoprotectant buffer (1.5 hours at room temperature plus 4.5 hours at 4ºC), and for preparations 4B, 4D, 4F, 4H, 4J, and 4L, rate-controlled freezing began approximately 8 hours after the addition of cryoprotectant buffer (1.5 hours at room temperature plus 6.5 hours at 4ºC). The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0315] Thawed cell viability was assessed using a Vi-CELL BLU cell analyzer (Beckman Coulter Inc., Brea, CA) as described in Example 1. All cell preparations showed cell viability exceeding 90%. Cell preparations 4A, 4B, 4C, 4D, 4G, 4H, and 4K exhibited higher cell viability than 5% (v / v) DMSO cell preparations (4L and 4M). Cell preparation 4D showed better viability than 4B. (See Example 1) Figure 5A (Table 8)

[0316] Thawed cell growth was assessed using the xCelligence system (Agilent Technology, Santa Clara, CA). Frozen RPE vials were thawed, seeded and cultured in 96-well plates in growth medium, and cell growth was monitored in real time. All cell preparations showed growth within 70 hours, indicating good growth was observed in all cell preparations. (See [link to documentation]) Figure 5B (Table 8)

[0317] Table 8 Example 5 – Post-thaw stability at 4ºC and room temperature RPE derived from human embryonic stem cells (as described in, for example, WO2021086911, which is incorporated herein by reference) were pooled in a 250 mL flask and washed with 200 mL of 0.5% rHA / DPBS by centrifugation at 500 x g for 5 min.

[0318] RPE cells were resuspended in 2.5% rHA / DPBS / 0.09% (w / v) glucose and then mixed with cryoprotectant buffer. The final composition of each cell preparation is shown in Table 9.

[0319] Table 9 Four hundred and fifty (450) µL of formulated RPE cells were loaded into CRYSTAL ZENITH® (CZ) vials (WestPharmaceutical Services, Inc. Exton, PA) and frozen at -2ºC / min using a rate-controlled freezer (THERMO SCIENTIFIC, Waltham, MA). Rate-controlled freezing was initiated approximately 8 hours after the addition of cryoprotectant buffer to the cells (as described in Example 4). The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0320] Thaw the frozen vials and store at 4ºC (cell preparations 5A to 5G) or room temperature (cell preparations 5H to 5N) for 4 hours, then evaluate cell viability using a Vi-CELL BLU cell analyzer (Beckman Coulter Inc., Brea, CA). All cell preparations (5A to 5F) showed higher viability than the 5% (v / v) DMSO cell preparation (5G). In particular, cell preparation 5B exhibited a significant cell viability of 95.1%. (See also...) Figure 6A (Table 10). Cell preparations 5H, 5I, 5K, 5L, and 5M showed higher viability than the 5% (v / v) DMSO cell preparation (5N). (See Table 10) Figure 7A (Table 11). Cell preparation 5J had a cell viability of 80.6%, which is slightly lower than that of 5% (v / v) DMSO cell preparation (5N).

[0321] Thaw frozen vials and store at 4ºC (cell preparations 5A to 5G) or room temperature (5H to 5N) for 4 hours, then evaluate cell growth using the xCelligence system (Agilent Technology, Santa Clara, CA). Thaw frozen RPE vials, seed and culture in 96-well plates in growth medium, and monitor cell growth in real time. Cell preparations 5A, 5B, 5C, 5E, and 5F showed higher cell growth rates than 5% (v / v) DMSO cell preparation (5G). (See...) Figure 6B(Table 10). Cell preparation 5D exhibited a cell growth rate comparable to that of 5% (v / v) DMSO cell preparation (5G). All cell preparations (5H to 5M) showed a cell index of less than 3 after 70 hours, indicating good growth was detected in all cell preparations. (See Table 10) Figure 7B (Table 11).

[0322] Table 10 (4ºC) # Cell preparations Average viability (%) SD becomes vibrant Average cell growth (in hours) for Cell Index 3. SD of cell growth 5A 5% (v / v) Propylene Glycol / 7.5% (w / v) Dextran-40 91.7 0.8 60.0 2.8 5B 3% (v / v) Propylene Glycol / 5% (w / v) Dextran-40 95.1 0.4 66.0 0.0 5C 5% (v / v) propylene glycol / 1% (w / v) trehalose 86.1 1.7 66.0 0.0 5D 5% (w / v) glucan-40 88.8 0.4 74.5 2.1 5E 5% (w / v) pentasyl starch 91.9 0.1 58.2 0.3 5F 7.5% (w / v) pentasyl starch 90.4 0.7 60.2 0.5 5G 5% (v / v) DMSO 85.6 1.6 75.5 3.5 Table 11 (RT) # Cell preparations Average viability (%) SD becomes vibrant Average cell growth (in hours) for Cell Index 3. SD of cell growth 5H 5% (v / v) Propylene Glycol / 7.5% (w / v) Dextran-40 93.3 1.1 61.5 0.7 5I 3% (v / v) Propylene Glycol / 5% (w / v) Dextran-40 92.7 0.6 67.5 0.7 5J 5% (v / v) propylene glycol / 1% (w / v) trehalose 80.6 5.5 69.5 3.5 5K 5% (w / v) glucan-40 90.2 0.8 67.0 0.0 5L 5% (w / v) pentasyl starch 88.6 0.7 56.2 0.4 5M 7.5% (w / v) pentasyl starch 86.6 0.5 67.7 0.4 5N 5% (v / v) DMSO 83.8 1.5 64.0 1.4 Example 6 – Robustness of cell preparations against temperature deviation at -70ºC RPE derived from human embryonic stem cells (as described in, for example, WO2021086911, which is incorporated herein by reference) were pooled in a 250 mL flask and washed with 200 mL of 0.5% rHA / DPBS by centrifugation at 500 x g for 5 min.

[0323] RPE cells were resuspended in 2.5% rHA / DPBS / 0.09% (w / v) glucose and then mixed with cryoprotectant buffer. The final composition of each cell preparation is shown in Table 12.

[0324] Table 12 Four hundred and fifty (450) µL of formulated RPE cells were loaded into CRYSTAL ZENITH® (CZ) vials (WestPharmaceutical Services, Inc. Exton, PA) and frozen at -2ºC / min using a rate-controlled freezer (THERMO SCIENTIFIC, Waltham, MA). Rate-controlled freezing was initiated approximately 8 hours after the addition of cryoprotectant buffer to the cells (as described in Example 4). The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0325] The frozen vials are transferred to a -70ºC chamber and kept for 2 or 4 weeks, and then transferred to a tank filled with liquid nitrogen vapor.

[0326] Thawed cell viability was assessed using a Vi-CELL BLU cell analyzer (Beckman Coulter Inc., Brea, CA) as described in Example 1. After 4 weeks, cell preparations 6A, 6B, 6D, 6E, and 6F showed cell viability exceeding 94%, and higher viability than the 5% (v / v) DMSO cell preparation (6G). (See Example 1) Figure 9A(Table 13). Cell preparation 7C showed 84.4% cell viability, which is comparable to 5% (v / v) DMSO cell preparation (6G).

[0327] Thawed cell growth was assessed using the xCelligence system (Agilent Technology, Santa Clara, CA). Frozen RPE vials were thawed, seeded and cultured in 96-well plates in growth medium, and cell growth was monitored in real time. All cell preparations (6A to 6G) showed a cell index of less than 3 after 80 hours, indicating good growth in all cell preparations. (See [link to documentation]) Figure 9B (Table 13).

[0328] Table 13 # Cell preparations Average viability (%) SD becomes vibrant Average cell growth (in hours) for Cell Index 3. SD of cell growth 6A 5% (v / v) Propylene Glycol / 7.5% (w / v) Dextran-40 97.7 0.4 55.5 2.1 6B 3% (v / v) Propylene Glycol / 5% (w / v) Dextran-40 95.7 1.2 57.0 0.0 6C 5% (v / v) propylene glycol / 1% (w / v) trehalose 84.4 1.6 68.5 3.5 6D 5% (w / v) glucan-40 94.1 0.6 58.0 4.2 6E 5% (w / v) pentasyl starch 94.3 1.1 54.1 0.4 6F 7.5% (w / v) pentasyl starch 96.8 1.6 76.6 10.3 6G 5% (v / v) DMSO 87.4 1.0 57.5 0.7 Example 7 – Robustness of cell preparations against temperature deviation at -20ºC RPE derived from human embryonic stem cells (as described in, for example, WO2021086911, which is incorporated herein by reference) were pooled in a 250 mL flask and washed with 200 mL of 0.5% rHA / DPBS by centrifugation at 500 x g for 5 min.

[0329] RPE cells were resuspended in 2.5% rHA / DPBS / 0.09% (w / v) glucose and then mixed with cryoprotectant buffer. The final composition of each cell preparation is shown in Table 14.

[0330] Table 14 Four hundred and fifty (450) µL of formulated RPE cells were loaded into CRYSTAL ZENITH® (CZ) vials (WestPharmaceutical Services, Inc. Exton, PA) and frozen at -2ºC / min using a rate-controlled freezer (THERMO SCIENTIFIC, Waltham, MA). Rate-controlled freezing was initiated approximately 8 hours after the addition of cryoprotectant buffer to the cells (as described in Example 4). The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0331] Transfer the frozen vials to a -20ºC chamber and hold for 30 minutes (7A, 7C, 7E, 7G, 7I, 7K, 7M) or 60 minutes (7B, 7D, 7F, 7H, 7J, 7L, 7N), then transfer these vials to a tank filled with liquid nitrogen vapor. This procedure is repeated a total of two times before evaluation.

[0332] Thawed cell viability was assessed using a Vi-CELL BLU cell analyzer (Beckman Coulter Inc., Brea, CA) as described in Example 1. Cell preparations 7A, 7C, 7G, 7I, and 7K showed viability exceeding 94%, and higher viability than the 5% (v / v) DMSO cell preparation (7M). (See Example 1) Figure 8A (Table 15). Cell preparation 7E showed 86.1% cell viability, which is lower than that of 5% (v / v) DMSO cell preparation (7M). Cell preparations 7B, 7D, 7H, 7J, and 7L showed significantly higher viability than 5% (v / v) DMSO cell preparation (7N). In particular, cell preparations 7B, 7D, and 7L showed viability exceeding 90% even after two exposures to -20ºC for 60 minutes.

[0333] Thawed cell growth was assessed using the xCelligence system (Agilent Technology, Santa Clara, CA). Frozen RPE vials were thawed, seeded and cultured in 96-well plates in growth medium, and cell growth was monitored in real time. Formulations 7A, 7C, 7E, 7G, 7I, and 7K showed a cell index of less than 3 after 80 hours, indicating good growth in all cell preparations. (See [link to documentation]) Figure 8B (Table 15). Cell preparations 7B and 7D showed a cell index 3 of less than 80 hours, and cell preparation 8L showed a cell index 3 of 80.5 hours. These values ​​are comparable to those of 5% (v / v) DMSO cell preparation (7N).

[0334] Table 15 Example 8 – Reduced production of multinucleated cells RPE derived from human embryonic stem cells (as described in, for example, WO2021086911, which is incorporated herein by reference) were pooled in a 250 mL flask and washed with 200 mL of 0.5% rHA / DPBS by centrifugation at 500 x g for 5 min.

[0335] RPE cells were resuspended in 2.5% rHA / DPBS / 0.09% (w / v) glucose and then mixed with cryoprotectant buffer. The final composition of each cell preparation is shown in Table 16.

[0336] Table 16 Four hundred and fifty (450) µL of formulated RPE cells were loaded into CRYSTAL ZENITH® (CZ) vials (WestPharmaceutical Services, Inc. Exton, PA) and frozen at -2ºC / min using a rate-controlled freezer (THERMO SCIENTIFIC, Waltham, MA). Rate-controlled freezing was initiated approximately 6 hours after the addition of cryoprotectant buffer to the cells (as described in Example 4). The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0337] Frozen RPE cells were thawed, seeded in growth medium, and cultured for 4 hours. The presence of multinucleated cells was assessed under a microscope. The control 5% (v / v) DMSO cell preparation (8I) resulted in 4.3% multinucleated cells, exceeding the threshold (below 10%). (See...) Figure 10 (Table 17). Cell preparations 8E, 8F, and 8G showed similar levels of multinucleated cells as cell preparation 8I. On the other hand, cell preparations 8A, 8B, 8C, 8D, and 8H resulted in a lower percentage of multinucleated cells. In particular, the combination of propylene glycol and dextran-40 strongly inhibited the production of multinucleated cells.

[0338] Table 17 # Cell preparations Average percentage of multi-core Multi-core percentage SD 8A 5% (v / v) Propylene Glycol / 7.5% (w / v) Dextran-40 1.3 0.3 8B 3% (v / v) Propylene Glycol / 5% (w / v) Dextran-40 2.3 0.1 8C 5% (v / v) propylene glycol / 1% (w / v) trehalose 3.0 0.0 8D 5% (w / v) glucan-40 2.7 0.9 8E 5% (v / v) Propylene Glycol 4.2 0.2 8F 1% (w / v) glucan-40 5.4 1.1 8G 5% (w / v) pentasyl starch 4.2 0.3 8H 7.5% (w / v) pentasyl starch 3.1 0.2 8I 5% (v / v) DMSO 4.3 0.0 Example 9 – Cell preparation robustness over process time (UC-RPE) RPE derived from human induced pluripotent stem cells (obtained from universal donor cells) (UC-RPE) were pooled in a 250 mL flask and washed with 200 mL of 0.5% rHA / DPBS after centrifugation at 500 x g for 5 minutes.

[0339] UC-RPE cells were resuspended in 2.5% rHA / DPBS / 0.09% (w / v) glucose and then mixed with cryoprotectant buffer. The final composition of each cell preparation is shown in Table 18.

[0340] Table 18 Four hundred and fifty (450) µL of formulated UC-RPE cells were loaded into CRYSTAL ZENITH® (CZ) vials (WestPharmaceutical Services, Inc. Exton, PA) and frozen at -2ºC / min using a rate-controlled freezer (THERMO SCIENTIFIC, Waltham, MA). For preparations 9A, 9C, 9E, 9G, 9I, 9K, and 9M, rate-controlled freezing began approximately 6 hours after the addition of cryoprotectant buffer to the cells, and for preparations 9B, 9D, 9F, 9H, 9J, 9L, and 9N, rate-controlled freezing began approximately 8 hours after the addition of cryoprotectant buffer to the cells. The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0341] Thawed cell viability was assessed using a NucleoCounter® NC-200™ instrument (ChemoMetec Inc., Bohemia, NY) as described in Example 1. At a 6-hour process time, all cell preparations showed cell viability exceeding 89% (see Table 19). Cell preparations 9A, 9C, 9E, and 9G resulted in higher cell viability than the 5% (v / v) DMSO cell preparation (9M). At an 8-hour process time, all cell preparations showed cell viability exceeding 87% (see Table 19). Figure 11A Cell preparations 9B, 9D, 9H, and 9L resulted in higher cell viability than 5% (v / v) DMSO cell preparation (9N).

[0342] Thawed cell growth was assessed using the xCelligence system (Agilent Technology, Santa Clara, CA). Frozen UC-RPE vials were thawed, seeded, and cultured in 96-well plates in DMEM-based growth medium, with cell growth monitored in real time. All cell preparations showed growth within 70 hours, indicating good growth was observed in all cell preparations. (See Table 19 and...) Figure 11B ).

[0343] Table 19 Example 10 – Screening of propylene glycol density (UC-RPE) using dextran-40 RPE derived from human induced pluripotent stem cells (obtained from universal donor cells) (UC-RPE) were pooled in a 250 mL flask and washed with 200 mL of 0.5% rHA / DPBS after centrifugation at 500x g for 5 minutes.

[0344] UC-RPE cells were resuspended in 2.5% rHA / DPBS / 0.09% glucose solution, followed by mixing with cryoprotectant buffer. The final composition of each cell preparation is shown in Table 20.

[0345] Table 20 Four hundred and fifty (450) µL of formulated UC-RPE cells were loaded into CRYSTAL ZENITH® (CZ) vials (WestPharmaceutical Services, Inc. Exton, PA) and frozen at -2ºC / min using a rate-controlled freezer (THERMO SCIENTIFIC, Waltham, MA). For preparations 10A, 10B, and 10C, rate-controlled freezing was initiated approximately 6 hours after the addition of cryoprotectant buffer to the cells (as described in Example 4). The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0346] Cell viability was restored after thawing using NucleoCounter. ® The NC-200™ (ChemoMetec Inc., Bohemia, NY) instrument was used for evaluation, as described in Example 1. At a process time of 6 hours, all cell preparations showed cell viability exceeding 89% (see Table 21). Cell preparations 10A to 10C showed cell viability exceeding 90%. (See...) Figure 3A (Table 21).

[0347] Thawed cell growth was assessed using the xCelligence system (Agilent Technology, Santa Clara, CA). Frozen RPE vials were thawed, seeded, and cultured in 96-well plates in DMEM-based growth medium, with cell growth monitored in real time. All cell preparations were within acceptable limits, and there were no significant differences between cell preparations. (See [link to relevant documentation]) Figure 3B (Table 21).

[0348] Table 21 # Cell preparations Average viability (%) SD becomes vibrant Average cell growth (in hours) for Cell Index 3. SD of cell growth 10A 5% (v / v) Propylene Glycol / 7.5% (w / v) Dextran-40 94.8 0.9 58.0 2.8 10B 3% (v / v) Propylene Glycol / 5% (w / v) Dextran-40 94.7 0.8 59.0 1.4 10C 1% (v / v) Propylene Glycol / 5% (w / v) Dextran-40 94.4 1.3 58.5 0.7 Example 11 – Albumin-free cell preparation RPE derived from human embryonic stem cells (as described in, for example, WO2021086911, which is incorporated herein by reference) were pooled in a 250 mL flask and washed with 200 mL of DPBS by centrifugation at 500 x g for 5 min.

[0349] RPE cells were resuspended in DPBS / 0.09% (w / v) glucose and then mixed with cryoprotectant buffer. The final composition of each cell preparation is shown in Table 22. In this example, albumin was replaced with DPBS in both the resuscitation buffer and the cryoprotectant buffer.

[0350] Table 22 Four hundred and fifty (450) µL of prepared RPE cells were placed in CZ vials and frozen at a rate of -2ºC / min using a rate-controlled freezer. Rate-controlled freezing was initiated within 3 hours of adding cryoprotectant buffer to the cells. The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0351] Cell viability after thawing was assessed using a Vi-CELL BLU cell analyzer. Cell preparations containing non-DMSO cryoprotectants (11A, 11B, 11C, 11D, 11E, 11F, 11G) showed higher viability than those containing DMSO (11H, 11I, 11J). Cell preparations containing 2-hydroxyethyl starch (11E, 11F, 11G) showed comparable or higher viability than those containing pentamyl starch (approximately 50% hydroxyethyl-substituted HES). These data are shown in Table 23. The finding that using 2-HES or pentamyl starch as cryoprotectants improves efficacy can be extrapolated to other HES, including but not limited to tetradyl starch (approximately 40% hydroxyethylated HES) and heptadyl starch (approximately 70% hydroxyethylated HES).

[0352] Table 23 # Cell preparations Average vitality (%) SD becomes vibrant 11A 3% (v / v) Propylene Glycol / 5% (w / v) Dextran-40 85.5 3.3 11B 5% (w / v) glucan-40 87.2 0.5 11C 5% (w / v) pentasyl starch 83.3 2.3 11D 10% (w / v) pentasyl starch 90.2 0.8 11E 5% (w / v) 2-Hydroxyethyl Starch 88.9 0.8 11F 7.5% (w / v) 2-Hydroxyethyl Starch 89.5 1.9 11G 10% (w / v) 2-Hydroxyethyl Starch 90.2 0.5 11H 5% (v / v) DMSO 84.1 1.1 11I 7.5% (v / v) DMSO 83.2 1.5 11J 10% (v / v) DMSO 83.4 0.8 Example 12 – Cell preparations using vascular progenitor cells Vascular progenitor cells (VPCs) are an immune-masked, allogeneic therapeutic cell product derived from class I and class II HLA double knockout iPSC lines (universal donor cells). VPCs were obtained by expanding the iPSC seed stock through two passages, followed by a 10-day cell culture period (maintained at -80°C / LN2 on day 6) to differentiate into VPCs, and then culturing for another 4 days (a total of 10 days of differentiation protocol). The cells were washed and resuspended in suitable cryopreservation buffer and cryopreserved. Vascular progenitor cells and their manufacture are described in WO2021041592, which is incorporated herein by reference in its entirety.

[0353] The cryopreserved vascular progenitor cells (VPCs) derived from human induced pluripotent stem cells (obtained from universal donor cells) were thawed, pooled in 50 mL tubes, and washed with 9 mL of 0.5% rHA / DPBS by centrifugation at 300 x g for 5 minutes at 4ºC.

[0354] VPCs were resuspended in 2.5% rHA / DPBS / 0.09% glucose and then mixed with cryoprotectant buffer. The final compositions of each cell preparation are shown in Table 24.

[0355] Table 24 Four hundred and fifty (450) µL of prepared VPC was placed in CZ vials and frozen at a rate of -2ºC / min using a rate-controlled freezer. Rate-controlled freezing was initiated within 3 hours after the addition of cryoprotectant buffer to the cells. The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0356] Cell viability after thawing was assessed using a Vi-CELL BLU cell analyzer. Cell preparations containing non-DMSO cryoprotectant (12A, 12B, 12C) showed higher viability than cell preparations containing DMSO (12D). These data are shown in Table 25.

[0357] Table 25 # Cell preparations Average vitality (%) SD becomes vibrant 12A 3% (v / v) Propylene Glycol / 5% (w / v) Dextran-40 83.9 1.4 12B 5% (w / v) glucan-40 79.0 0.9 12C 7.5% (w / v) pentasyl starch 87.2 0.8 12D 5% (v / v) DMSO 76.2 0.6 These data show that cryopreservation formulations containing propylene glycol / dextran-40, or dextran-40, or pentasyl starch as cryoprotectants, examples of which include 12A, 12B, and 12C, are comparable to or better than cryopreservation formulations containing DMSO when VPC is frozen.

[0358] Example 13 – Cell preparation using angiogenic mesenchymal cells (HMCs) The differentiation of angiogenic mesenchymal stem cells (HMCs) is a 33-day process starting from human embryonic stem cells (hESCs). One million cryopreserved vials of hESCs are expanded to form embryoid bodies. The first step in differentiation is the formation of embryoid bodies (EBs) from hESCs, followed by the differentiation of EBs into hematopoietic cells (HBs). HMCs are selected by platenating HBs onto fibronectin-coated tissue culture plates (PO). The selected HMCs are expanded for one generation and cryopreserved as intermediate material (P1CS) for reproducible large-scale expansion. The expansion of angiogenic mesenchymal stem cells (HMCs) is a 14-day process starting from HMC P1CSs. Cryopreserved vials of HMC P1CSs are expanded three times. The first generation is expanded in T225 vials. The second and third generations are expanded in XPN50 and XPN200 disposable bioreactors, respectively. HMCs harvested from the XPN200 are concentrated and washed in a UF Mini (a continuous flow centrifuge). HMCs were resuspended in a suitable cryopreservation buffer and cryopreserved. Angiogenic mesenchymal cells and their manufacture are described in WO2021041592 and WO2007120811, which are incorporated herein by reference in their entirety.

[0359] The cryopreserved angiogenic mesenchymal cells (HMCs) were thawed, pooled in 50 mL tubes, and washed with 8 mL of 1% rHA / DPBS by centrifugation at 500 x g for 5 minutes at 4ºC.

[0360] HMCs were resuspended in 2.5% rHA / DPBS / 0.09% glucose and then mixed with cryoprotectant buffer. The final composition of each cell preparation is shown in Table 26.

[0361] Table 26 Four hundred and fifty (450) µL of prepared HMCs were placed in CZ vials and frozen at a rate of -2ºC / min using a rate-controlled freezer. Rate-controlled freezing was initiated within 3 hours after the addition of cryoprotectant buffer to the cells. The vials were then transferred and stored in the vapor phase of liquid nitrogen.

[0362] Cell viability after thawing was assessed using a Vi-CELL BLU cell analyzer. Cell preparations containing non-DMSO cryoprotectant (13A, 13B, 13C) showed comparable viability to those containing DMSO (13D), as shown in Table 27.

[0363] Table 27 # Cell preparations Average vitality (%) SD becomes vibrant 13A 3% (v / v) Propylene Glycol / 5% (w / v) Dextran-40 82.4 2.4 13B 5% (w / v) glucan-40 80.4 1.7 13C 7.5% (w / v) pentasyl starch 83.1 2.3 13D 5% (v / v) DMSO 82.4 1.2 These data show that cryopreservation formulations containing propylene glycol / dextran-40, or dextran-40, or pentasyl starch as cryoprotectants, examples of which include 13A, 13B, and 13C, are comparable to or better than cryopreservation formulations containing DMSO when HMC is frozen.

[0364] Equivalents and scope, incorporated by reference Those skilled in the art will recognize or be able to determine many equivalents of the embodiments described herein using only conventional experiments. The scope of this disclosure is not intended to be limited to the foregoing description, but is set forth by the appended claims.

[0365] Unless the context clearly indicates otherwise, articles such as “a,” “an,” and “the” include both singular and plural references. Thus, for example, a reference to “a reagent” includes both a single reagent and multiple such reagents.

[0366] For any claim or description involving "or" between two or more members of a group, the presence of one, more than one, or all members of the group is deemed satisfied unless otherwise stated or clearly apparent from the context. The disclosure of groups involving "or" between two or more members provides 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 members of the group are present. For the sake of brevity, these embodiments are not described in detail separately herein, but it should be understood that each of these embodiments is provided herein and may be specifically claimed or waived.

[0367] It should be understood that this invention covers all variations, combinations, and arrangements in which one or more limitations, elements, clauses, or descriptive terms from one or more claims or from one or more related portions of the specification are incorporated into another claim. For example, a claim dependent on another claim may be modified to include one or more limitations seen in any other claim dependent on the same basic claim. Furthermore, when a composition is referred to in the claims, it should be understood that any method of preparation or use of the composition disclosed herein or any method known in the art (if any) is included, unless otherwise stated, or unless a contradiction or inconsistency would be apparent to a person skilled in the art.

[0368] When elements are presented in list form, such as in Markush group format, it should be understood that every possible subgroup of the elements is also disclosed, and any element or subgroup of elements can be removed from that group. It should also be noted that the word "comprise" is intended to be open, allowing for the inclusion of additional elements or steps. It should be understood that, in general, when an implementation, product, or method is referred to as including a particular element, feature, or step, an implementation, product, or method consisting of or substantially consisting of such element, feature, or step is also provided. For the sake of brevity, these implementations are not described in detail separately herein, but it should be understood that each of these implementations is provided herein, and protection may be specifically claimed or waived.

[0369] When a range is given, the endpoints are also included. Furthermore, it should be understood that, unless otherwise stated or clearly apparent from the context and / or understanding of someone skilled in the art, values ​​expressed as ranges in some embodiments may take any specific value within the range, up to one-tenth of a unit of the lower limit of the range, unless the context explicitly specifies otherwise. For the sake of brevity, values ​​within each range are not described in detail separately herein, but it should be understood that each of these values ​​is provided herein and may be specifically claimed or waived. It should also be understood that, unless otherwise stated or clearly apparent from the context and / or understanding of someone skilled in the art, values ​​expressed as ranges may take any subrange within a given range, where the endpoints of the subranges are represented with the same precision as one-tenth of a unit of the lower limit of the range.

[0370] Furthermore, it should be understood that any particular embodiment of the invention may be explicitly excluded from any one or more claims. When a scope is given, any value within that scope may be explicitly excluded from any one or more claims. Any embodiment, element, feature, application, or aspect of the composition and / or method of the invention may be excluded from any one or more claims. For the sake of brevity, not all embodiments in which one or more elements, features, objects, or aspects are explicitly excluded herein.

[0371] All publications, patents, patent applications, publications, and database entries (e.g., sequence database entries) mentioned herein, such as in the Background, Summary of the Invention, Detailed Description, Embodiments, and / or References sections, are hereby incorporated in their entirety by reference, just as each individual publication, patent, patent application, publication, and database entry is specifically and separately incorporated herein by reference. In case of conflict, this application (including any definitions herein) shall prevail.

Claims

1. A formulation comprising: (a) Albumin, glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (b) Glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (c) Albumin, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (d) A buffered saline solution and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, pentamylose, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (e) Albumin, glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (f) Glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (g) albumin, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, wherein said formulation is free of dimethyl sulfoxide (DMSO), or (h) Buffered saline and one or more cryoprotectants, optionally more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch and ethylene glycol, optionally wherein the formulation is free of dimethyl sulfoxide (DMSO).

2. A cryopreservation preparation, which is basically composed of the following: (a) Albumin, glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol. (b) Glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol. (c) Albumin, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol. (d) Buffered saline and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, pentamylose, and ethylene glycol. (e) Albumin, glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol. (f) Glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol. (g) Albumin, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, or (h) Buffered saline and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch and ethylene glycol.

3. The formulation according to claim 1 or 2, wherein the albumin is human albumin.

4. The formulation according to claim 1 or 2, wherein the albumin is recombinant human albumin.

5. The formulation according to any one of the preceding claims, wherein, if present, albumin is present in the range of about 2% to about 8% (w / v).

6. The formulation according to any one of the preceding claims, wherein, if present, albumin is present in the range of about 1% to about 4% (w / v).

7. The formulation according to any one of the preceding claims, wherein, if present, albumin is present at about 2.5% (w / v).

8. The formulation according to any one of the preceding claims, wherein, if present, glucose is present in the range of about 0.01% to about 1.5% (w / v).

9. The formulation according to any one of the preceding claims, wherein, if present, glucose is present in the range of about 0.01% to about 0.5% (w / v).

10. The formulation according to any one of the preceding claims, wherein glucose, if present, is present at about 0.09% (w / v).

11. The formulation according to any one of the preceding claims, wherein the buffered saline is phosphate-buffered saline (PBS).

12. The formulation according to any one of claims 1-11, wherein one or more cryoprotectants comprise propylene glycol.

13. The formulation of claim 12, wherein propylene glycol is present in the range of about 0.1% to about 20% (v / v).

14. The formulation of claim 12, wherein propylene glycol is present in the range of about 0.2% to about 15% (v / v).

15. The formulation according to claim 12, wherein propylene glycol is present at about 0.2%, about 2%, about 6%, about 10%, or about 15% (v / v).

16. The formulation according to any one of claims 1-11, wherein one or two, or optionally more, cryoprotectants comprise dextran-40.

17. The formulation of claim 16, wherein dextran-40 is present in the range of about 1% to about 20% (w / v).

18. The formulation of claim 16, wherein dextran-40 is present in the range of about 2% to about 20% (w / v).

19. The formulation of claim 16, wherein dextran-40 is present in the range of about 4% to about 20% (w / v).

20. The formulation of claim 16, wherein dextran-40 is present in the range of about 5% to about 15% (w / v).

21. The formulation according to claim 16, wherein dextran-40 is present at about 5%, about 6%, about 10%, or about 15% (w / v).

22. The formulation according to any one of claims 1-11, wherein one or two, or optionally more, cryoprotectants comprise proline.

23. The formulation according to any one of claims 1-11, wherein one or two, or optionally more, cryoprotectants comprise trehalose.

24. The formulation according to any one of claims 1-11, wherein one or two, or optionally more, cryoprotectants comprise glycerol.

25. The formulation according to any one of claims 1-11, wherein one or two, or optionally more, cryoprotectants comprise hydroxyethyl starch having up to 100% hydroxyethyl substitution, optionally penta-starch.

26. The formulation according to claim 25, wherein hydroxyethyl starch, optionally penta-starch, is present in the range of about 4% to about 20% (w / v).

27. The formulation according to claim 25, wherein hydroxyethyl starch, optionally penta-starch, is present in the range of about 5% to about 15% (w / v).

28. The formulation according to claim 25, wherein hydroxyethyl starch, optionally penta-starch, is present at about 5%, about 10%, or about 15% (w / v).

29. The formulation according to any one of claims 1-11, wherein one or two, or optionally more, cryoprotectants comprise ethylene glycol.

30. The formulation of claim 29, wherein ethylene glycol is present in the range of about 4% to about 6% (v / v).

31. The formulation according to claim 29, wherein ethylene glycol is present at about 5% (v / v).

32. The formulation according to any one of claims 1-11, wherein one or two, or optionally more, cryoprotectants are propylene glycol and dextran-40.

33. The formulation of claim 32, wherein propylene glycol is present in the range of about 0.1% to about 20% (v / v), and dextran-40 is present in the range of about 0.2% to about 20% (w / v).

34. The formulation of claim 32, wherein propylene glycol is present in the range of about 0.15% to about 20% (v / v), and dextran-40 is present in the range of about 4% to about 20% (w / v).

35. The formulation of claim 32, wherein propylene glycol is present in the range of about 0.2% to about 15% (v / v), and dextran-40 is present in the range of about 5% to about 15% (w / v).

36. The formulation of claim 32, wherein propylene glycol is present at about 0.2%, about 2%, about 6%, about 10% or about 15% (v / v), and dextran-40 is present at about 5%, about 6%, about 10% or about 15% (w / v).

37. The formulation of claim 32, wherein propylene glycol is present in the range of about 0.2% to about 15% (v / v), and dextran-40 is present in the range of about 6%, about 10%, or about 15% (w / v).

38. The formulation according to claim 32, wherein propylene glycol is present at about 0.2%, about 2%, about 6%, about 10% or about 15% (v / v), and dextran-40 is present at about 10% (w / v).

39. The formulation of claim 32, wherein propylene glycol is present at about 10% (v / v) and dextran-40 is present at about 15% (w / v).

40. The formulation of claim 32, wherein propylene glycol is present at about 10% (v / v) and dextran-40 is present at about 10% (w / v).

41. The formulation of claim 32, wherein propylene glycol is present at about 6% (v / v) and dextran-40 is present at about 20% (w / v).

42. The formulation of claim 32, wherein propylene glycol is present at about 6% (v / v) and dextran-40 is present at about 15% (w / v).

43. The formulation of claim 32, wherein propylene glycol is present at about 6% (v / v) and dextran-40 is present at about 10% (w / v).

44. The formulation of claim 32, wherein propylene glycol is present at about 6% (v / v) and dextran-40 is present at about 6% (w / v).

45. The formulation of claim 32, wherein propylene glycol is present at about 6% (v / v) and dextran-40 is present at about 2% (w / v).

46. ​​The formulation according to any one of claims 1-11, wherein one or two, or optionally more, cryoprotectants are propylene glycol and proline.

47. The formulation of claim 46, wherein propylene glycol is present in the range of about 0.1% to about 20% (v / v), and proline is present in the range of about 2% to about 15% (w / v).

48. The formulation of claim 46, wherein propylene glycol is present in the range of about 0.2% to about 15% (v / v), and proline is present in the range of about 5% to about 15% (w / v).

49. The formulation of claim 46, wherein propylene glycol is present in the range of about 0.2% to about 15% (v / v), and proline is present in the range of about 5% to about 10% (w / v).

50. The formulation of claim 46, wherein propylene glycol is present at about 6% (v / v) and proline is present at about 5% or about 10% (w / v).

51. The formulation according to any one of claims 1-11, wherein one or two, or optionally more, cryoprotectants are propylene glycol and trehalose.

52. The formulation of claim 51, wherein propylene glycol is present in the range of about 0.1% to about 20% (v / v), and trehalose is present in the range of about 0.1% to about 20% (w / v).

53. The formulation of claim 51, wherein propylene glycol is present in the range of about 0.2% to about 15% (v / v), and trehalose is present in the range of about 0.2% to about 15% (w / v).

54. The formulation of claim 51, wherein propylene glycol is present at about 10% (v / v) and trehalose is present at a range of about 0.2% to about 15% (w / v).

55. The formulation of claim 51, wherein propylene glycol is present at about 10% (v / v) and trehalose is present at about 7% (w / v).

56. The formulation according to claim 51, wherein propylene glycol is present at about 10% (v / v) and trehalose is present at about 2% (w / v).

57. The formulation according to any one of claims 1-11, wherein one or two, or optionally more, cryoprotectants are glycerol and trehalose.

58. The formulation of claim 57, wherein glycerol is present in the range of about 4% to about 16% (v / v), and trehalose is present in the range of about 2% to about 12% (w / v).

59. The formulation of claim 57, wherein glycerol is present in the range of about 8% to about 12% (v / v), and trehalose is present in the range of about 4% to about 10% (w / v).

60. The formulation of claim 57, wherein glycerol is present at about 10% (v / v) and trehalose is present at about 7% (w / v).

61. The formulation of claim 1, wherein the formulation comprises about 2.5% (w / v) albumin, optionally recombinant human albumin, about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 5% (v / v) propylene glycol and about 7.5% (w / v) dextran-40.

62. The formulation of claim 1, wherein the formulation comprises about 2.5% (w / v) albumin, optionally recombinant human albumin, about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol and about 5% (w / v) dextran-40.

63. The formulation of claim 1, wherein the formulation comprises about 0.09% (w / v) glucose, buffered saline (e.g., PBS), about 3% (v / v) propylene glycol and about 5% (w / v) dextran-40, and is free of albumin.

64. The formulation of claim 1, wherein the formulation comprises about 2.5% (w / v) albumin, optionally recombinant human albumin, about 0.09% (w / v) glucose, buffered saline (e.g., PBS) and about 5% (w / v) dextran-40.

65. The formulation of claim 1, wherein the formulation comprises about 2.5% (w / v) albumin, optionally recombinant human albumin, about 0.09% (w / v) glucose, buffered saline (e.g., PBS) and about 7.5% (w / v) pentasyl starch.

66. A cell preparation comprising a cell population and a formulation according to any one of claims 1-11, 12, 16, 22-25, 29, 32, 46, 51 and 57.

67. A cell preparation comprising a cell population and a formulation according to any one of claims 13-15, 17-21, 26-28, 30-31, 33-45, 47-50, 52-56 and 58-65, wherein the concentration of each of the one or two, or optionally more, cryoprotectants is halved.

68. A cell preparation comprising (i) a formulation according to any one of claims 1-65 and (ii) a 1:1 (v / v) mixture of a suspension comprising albumin, glucose and buffered saline, each in the same concentration as the formulation, wherein the suspension does not contain a (non-albumin) cryoprotectant.

69. A cell preparation comprising a cell population in a formulation, said formulation comprising: (a) Albumin, glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (b) Glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (c) Albumin, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (d) A buffered saline solution and one or more, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, pentamylose, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (e) Albumin, glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (f) Glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, wherein said formulation is optionally free of dimethyl sulfoxide (DMSO). (g) albumin, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, optionally wherein said formulation is free of dimethyl sulfoxide (DMSO), or (h) Buffered saline and one or more, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch and ethylene glycol, wherein the formulation is optionally free of dimethyl sulfoxide (DMSO-free).

70. A cell preparation comprising a cell population in a cryopreservation formulation, said cryopreservation formulation being substantially composed of: (a) Albumin, glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol. (b) Glucose, buffered saline, and one or more, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol. (c) Albumin, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, starch, and ethylene glycol. (d) Buffered brine and one or more, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, pentamylose, and ethylene glycol. (e) Albumin, glucose, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol. (f) Glucose, buffered saline, and one or more, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol. (g) albumin, buffered saline, and one or more cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch, and ethylene glycol, or (h) Buffered brine and one or more, or optionally more, cryoprotectants selected from propylene glycol, dextran-40, proline, trehalose, glycerol, hydroxyethyl starch and ethylene glycol.

71. The cell preparation according to any one of claims 66-70, wherein, if present, the albumin is human albumin, optionally recombinant human albumin.

72. The cell preparation according to any one of claims 66-71, wherein, if present, albumin is present in the range of about 2% to about 8% (w / v), optionally in the range of about 1% to about 4% (w / v), and further optionally in the range of about 2.5% (w / v).

73. The cell preparation according to any one of claims 66-72, wherein, if present, glucose is present in the range of about 0.01% to about 1.5% (w / v), optionally in the range of about 0.01% to about 0.5% (w / v), and more preferably in the range of about 0.09% (w / v).

74. The cell preparation according to any one of claims 66-72, wherein the buffered saline is a phosphate-buffered saline.

75. The cell preparation according to any one of claims 66-74, wherein one or two, or optionally more, cryoprotectants comprise propylene glycol.

76. The cell preparation of claim 75, wherein propylene glycol is present in the range of about 0.05% to about 10%, optionally in the range of about 0.1% to about 7.5%, and more optionally in the range of about 0.1%, about 1%, about 3%, about 5% or about 7.5% (v / v).

77. The cell preparation according to any one of claims 66-74, wherein one or two, or optionally more, cryoprotectants comprise dextran-40.

78. The cell preparation of claim 77, wherein dextran-40 is present in the range of about 0.5% to about 10%, optionally in the range of about 1% to about 10%, optionally in the range of about 2% to about 10%, optionally in the range of about 2.5% to about 7.5%, and further optionally in the range of about 2.5%, about 3%, about 5%, or about 7.5% (w / v).

79. The cell preparation according to any one of claims 66-74, wherein one or two, or optionally more, cryoprotectants comprise proline, trehalose, and / or glycerol.

80. The cell preparation according to any one of claims 66-74, wherein one or two, or optionally more, cryoprotectants comprise hydroxyethyl starch, optionally penta-starch.

81. The cell preparation of claim 80, wherein hydroxyethyl starch, optionally penta-starch, is present in the range of about 2% to about 10%, optionally in the range of about 2.5% to about 7.5%, and further optionally in the range of about 2.5%, about 5%, or about 7.5% (w / v).

82. The cell preparation according to any one of claims 66-74, wherein one or two, or optionally more, cryoprotectants comprise ethylene glycol.

83. The cell preparation according to claim 82, wherein ethylene glycol is present in the range of about 2% to about 3%, optionally in the range of about 2.5% (v / v).

84. The cell preparation according to any one of claims 66-74, wherein one or two, or optionally more, cryoprotectants are propylene glycol and dextran-40.

85. The cell preparation of claim 84, wherein propylene glycol is present in the range of about 0.05% to about 10% (v / v) and dextran-40 is present in the range of about 0.1% to about 10% (w / v), optionally wherein propylene glycol is present in the range of about 0.05% to about 10% (v / v) and dextran-40 is present in the range of about 2% to about 10% (w / v), optionally wherein propylene glycol is present in the range of about 0.1% to about 7.5% (v / v) and dextran-40 is present in the range of about 2.5% to about 7.5% (w / v), optionally wherein propylene glycol is present in the range of about 0.1%, about 1%, about 3%, about 5% or about 7.5% (v / v) and dextran-40 is present in the range of about 2.5%, about 3%, about 5% or about 7.5% (w / v), optionally wherein propylene glycol is present in the range of about 0.1% to about 7.5%. The range of (v / v) is present and dextran-40 is present at about 3%, about 5% or about 7.5% (w / v), and further optionally, propylene glycol is present at about 0.1%, about 1%, about 3%, about 5% or about 7.5% and dextran-40 is present at about 5% (w / v).

86. The cell preparation according to claim 84, wherein... a. Propylene glycol is present at about 5% (v / v) and dextran-40 is present at about 7.5% (w / v). b. Propylene glycol is present at about 5% (v / v) and dextran-40 is present at about 5% (w / v). c. Propylene glycol is present at approximately 3% (v / v) and dextran-40 at approximately 10% (w / v). d. Propylene glycol is present at about 3% (v / v) and dextran-40 at about 7.5% (w / v). e. Propylene glycol is present at about 3% (v / v) and dextran-40 is present at about 5% (w / v). f. Propylene glycol is present at about 3% (v / v) and dextran-40 is present at about 3% (w / v), or g. Propylene glycol is present at about 3% (v / v) and dextran-40 is present at about 1% (w / v).

87. The cell preparation according to any one of claims 66-74, wherein one or two, or optionally more, cryoprotectants are propylene glycol and proline.

88. The cell preparation of claim 87, wherein propylene glycol is present in the range of about 0.05% to about 10% (v / v) and proline is present in the range of about 1% to about 7.5% (w / v), optionally wherein propylene glycol is present in the range of about 0.1% to about 7.5% (v / v) and proline is present in the range of about 2.5% to about 7.5% (w / v), optionally wherein propylene glycol is present in the range of about 0.1% to about 7.5% (v / v) and proline is present in the range of about 2.5% to about 5% (w / v), and further optionally wherein propylene glycol is present in the range of about 3% (v / v) and proline is present in the range of about 2.5% or about 5% (w / v).

89. The cell preparation according to any one of claims 66-74, wherein one or two, or optionally more, cryoprotectants are propylene glycol and trehalose.

90. The cell preparation of claim 89, wherein propylene glycol is present in the range of about 0.05% to about 10% (v / v) and trehalose is present in the range of about 0.05% to about 10% (w / v), optionally wherein propylene glycol is present in the range of about 0.1% to about 7.5% (v / v) and trehalose is present in the range of about 0.1% to about 7.5% (w / v), optionally wherein propylene glycol is present in the range of about 5% (v / v) and trehalose is present in the range of about 0.1% to about 7.5% (w / v), optionally wherein propylene glycol is present in the range of about 5% (v / v) and trehalose is present in the range of about 3.5% (w / v), and further optionally wherein propylene glycol is present in the range of about 5% (v / v) and trehalose is present in the range of about 1% (w / v).

91. The cell preparation according to any one of claims 66-74, wherein one or two, or optionally more, cryoprotectants are glycerol and trehalose.

92. The cell preparation of claim 91, wherein glycerol is present in the range of about 2% to about 8% (v / v) and trehalose is present in the range of about 1% to about 6% (w / v), optionally wherein glycerol is present in the range of about 4% to about 6% (v / v) and trehalose is present in the range of about 2% to about 5% (w / v), and further optionally wherein glycerol is present in the range of about 5% (v / v) and trehalose is present in the range of about 3.5% (w / v).

93. The cell preparation according to any one of claims 66-92, wherein the cell population is a retinal pigment epithelial (RPE) cell population, a photoreceptor cell population, a photoreceptor progenitor cell population, a photoreceptor repair cell (PRC) population, a corneal epithelial cell (CEC) population, a mesenchymal cell population, angiogenic mesenchymal cells (HMC) population, a macrophage population, a natural killer cell population, a T-cell population, a vascular cell population, a vascular progenitor cell (VPC) population, a hepatocyte population, a nerve cell population, and a pancreatic cell population.

94. The cell preparation according to any one of claims 66-92, wherein the cell population is a retinal pigment epithelium (RPE) cell population.

95. The cell preparation according to any one of claims 66-92, wherein the cell population is a cell population expressing at least one polypeptide selected from human leukocyte antigen E (HLA-E), human leukocyte antigen F (HLA-F), and human leukocyte antigen G (HLA-G); wherein the cells contain a genetically modified disruption of one or more copies of endogenous β-2 microglobulin (B2M); and wherein the cells contain a genetically modified disruption of one or more copies of human leukocyte antigen (HLA) class II related genes selected from regulatory factor X-associated ankyrin repeat sequence protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-associated protein (RFXAP), and class II transactivator (CIITA).

96. The cell preparation of claim 95, wherein the cells further comprise a nucleic acid molecule encoding a suicide gene product.

97. The cell preparation of claim 96, wherein the cells comprise the herpes simplex virus thymidine kinase (TK) suicide gene.

98. The cell preparation according to any one of claims 95-97, wherein the cells are derived from pluripotent stem cells.

99. The cell preparation according to claim 98, wherein the pluripotent stem cells are embryonic stem cells.

100. The cell preparation according to claim 98, wherein the pluripotent stem cells are induced pluripotent stem cells.

101. The cell preparation according to any one of claims 95-100, wherein the cells comprise genetically modified destruction of all copies of endogenous B2M.

102. The cell preparation according to any one of claims 95-101, wherein the cells comprise genetically modified disruption of all copies of an HLA class II-related gene selected from RFXANK, RFX5, RFXAP, and CIITA.

103. The cell preparation according to any one of claims 66-102, wherein the cell population is a dissociated cell population, a cell sheet, a cell monolayer, or a cell aggregate.

104. The cell preparation according to any one of claims 66-103, wherein the cell population is a human cell population.

105. The cell preparation according to any one of claims 66-104, wherein the cell population is generated by in vitro differentiated pluripotent stem cells, optionally embryonic stem cells or induced pluripotent stem cells.

106. The cell preparation according to any one of claims 66-105, having a cell density of about 300 cells to about 10,000 cells / µL.

107. The cell preparation according to any one of claims 66-105, having a cell density of about 300 cells to about 7,000 cells / µL.

108. The cell preparation according to any one of claims 66-105, having a cell density of about 350 cells to about 7,000 cells / µL.

109. The cell preparation according to any one of claims 66-105, having a cell density of about 350 cells / µL, or about 3,000 cells / µL, or about 3,500 cells / µL, or about 6,500 cells / µL, or about 7,000 cells / µL.

110. The cell preparation according to any one of claims 66-105, having a cell density of about 3,333 cells / µL.

111. The cell preparation according to any one of claims 66-110, wherein it is in a volume of about 400-500 μL, more preferably in a volume of about 450 μL.

112. The cell preparation according to any one of claims 66-111, wherein it is in cryopreserved form.

113. The cell preparation according to any one of claims 66-111, wherein the preparation has been cryopreserved and thawed.

114. The cell preparation according to any one of claims 66-111, provided at room temperature (about 20°C to about 25°C), at about 4°C, at about -20°C, at about -70°C, or at about -180°C before cryopreservation or after thawing.

115. The cell preparation according to any one of claims 66-111, wherein the cell preparation has been kept at room temperature (about 20°C to about 25°C) for about 1 to 2 hours and at about 4°C for about 4 to 7 hours prior to cryopreservation, optionally at -180°C.

116. A cell preparation comprising a cell population in a formulation comprising albumin, glucose, buffered saline, and one or more cryoprotectants, and having, after an event, a cell viability equal to or greater than about 80% and / or a cell growth index equal to or less than 110 hours:3, optionally having a cell viability equal to or greater than about 85% and / or a cell growth index equal to or less than 100 hours:3, and further optionally having a cell viability equal to or greater than about 90% and / or a cell growth index equal to or less than 90 hours:3: a. Store at room temperature (about 20°C to about 25°C) for about 1 to 2 hours, then at about 4°C for about 4 to 7 hours, optionally at room temperature for about 1.5 hours, then at 4°C for about 4.5 hours or 6.5 hours. b. Store frozen at -180°C for approximately 1 to 2 years, optionally for approximately 1 month to 1 year. c. Freeze at -180°C, then thaw and store at approximately 4°C for approximately 3 to 6 hours, optionally approximately 4 hours. d. Freeze at -180°C, then thaw and allow to cool at room temperature (approximately 20°C to approximately 25°C) for approximately 3 to 6 hours, optionally approximately 4 hours. e. Freeze at -180°C, then freeze at about -20°C for about 0.5 to about 1 hour, then freeze at -180°C, or optionally freeze at about -20°C for about 0.5 to about 1 hour, then freeze at -180°C. f. Freeze at -180°C, then at about -70°C for about 2 to about 4 weeks, then freeze again at -180°C.

117. The cell preparation of claim 116, wherein the cell preparation is a DMSO-free cell preparation.

118. The cell preparation according to claim 116 or 117, wherein after any one of events (a) to (f), the cell preparation has a viability of about 85% and / or a cell growth index of 3 equal to or less than 100 hours.

119. The cell preparation according to any one of claims 116-118, wherein after any one of events (a) to (f), the cell preparation has a cell viability equal to or greater than about 90% and / or a cell growth index equal to or less than 90 hours:

3.

120. A method for preparing a cell preparation comprising contacting a cell population with (i) a formulation according to any one of claims 1-65 and (ii) a suspension comprising albumin, glucose and buffered saline, each in the same concentration as the formulation, wherein the suspension does not contain a (non-albumin) cryoprotectant.

121. The method of claim 120, further comprising cryopreserving the cell preparation.

122. The method of claim 121, further comprising thawing the cell preparation.

123. The method of claim 122, further comprising administering the cell preparation to a subject in need.

124. The method of claim 123, wherein the cell preparation is kept at about 4°C for about 4 hours prior to administration to the subject.

125. The method of claim 123, wherein the cell preparation is kept at room temperature (about 20°C to about 25°C) for about 4 hours before being administered to the subject.

126. The method according to any one of claims 123-125, wherein the cell preparation is administered to the subject without washing or dilution after thawing.

127. A method of treating a subject with a symptom or condition, comprising administering an effective amount of the cell preparation of claim 113 to the subject in need without washing or dilution after thawing.

128. A method of treating a subject with a symptom or condition, comprising administering to the subject in need an effective amount of a cell preparation prepared according to the method of claim 122 without washing or dilution after thawing.

129. The method of claim 127 or 128, wherein the cell preparation is kept at about 4°C for about 4 hours before being administered to the subject.

130. The method of claim 127 or 128, wherein the cell preparation is kept at room temperature (about 20°C to about 25°C) for about 4 hours before being administered to the subject.

131. The method according to any one of claims 127-130, wherein the condition or ailment is selected from retinal detachment, retinal tear, retinal dysplasia, vascular streaks, myopic macular degeneration, or retinal atrophy, or is associated with a variety of visual impairments leading to photoreceptor damage and blindness, such as choroidal agenesis, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, Staggart's disease (yellow macular degeneration), cancer, liver disease, Crohn's disease, arthritis, pulmonary hypertension, severe limb ischemia, Fuchs' dystrophy, corneal injury, retinal tear, and geographic atrophy (e.g., geographic atrophy secondary to age-related macular degeneration).

132. The cell preparation according to any one of claims 66-119, wherein the cell preparation is in a vial.

133. The cell preparation according to any one of claims 66-119, wherein the cell preparation is contained in a syringe.

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