Cell delivery vehicle and methods of use thereof

Abstract

The present disclosure relates to various compositions comprising one or more energy source components; one or more pH buffering agents; one or more salts; and one or more stabilizers and one or more cell types.

Description

Background Art

[0001] Various biological cells and / or tissues are stored at freezing temperatures for different amounts of time and in various materials such as cryoprotectants. These cryoprotectants used during storage will be removed or substantially removed from the biological cells and / or tissues after or during the thawing process so that those biological cells and / or tissues can be utilized.

[0002] Typically, the container containing the biological cells and / or tissue is removed from freezing temperature storage, thawed, and the contents of the container are "washed" or diluted with a cell washing solution to remove some, most, or all of the cryoprotectant. A typical cell "wash" solution has many, many components, including, but not limited to: one or more amino acids such as glycine, L-alanine, L-arginine hydrochloride, L-asparagine-H2O, L-glutamine, L-cysteine, L-histidine hydrochloride-H2O, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; one or more glycolytic components such as dextrose and sodium pyruvate; one or more pH control components and / or pH buffers such as 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and phenol red; one or more proteins, for example, albumins, such as human serum albumin; one or more vitamins, such as ascorbic acid, choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12 and i-inositol; and one or more salts such as calcium chloride (CaCl2), ferric nitrate (Fe(NO3)3-9H2O), magnesium chloride (MgCl), potassium chloride (KCl), sodium bicarbonate (NaHCO3), sodium chloride (NaCl), sodium dihydrogen phosphate (NaH2PO4-H2O) and zinc sulfate (ZnSO4-7H2O).

[0003] After dilution with the cell washing solution, the container can undergo one or more sedimentation procedures, such as centrifugation or sedimentation over time. The supernatant can then be removed from the container in whole or in part so that all or most of the cells are retained. The process can then be repeated, wherein the cell washing solution is added to the container again, and then the sedimentation procedure is performed one or more times.

[0004] After "washing" the cells, the remaining biological cells and / or tissue (which may be in the same storage container or may have been transferred to a different container) are resuspended with a cell delivery solution (transplantation medium) so that the biological cells and / or tissue can be used for any desired procedure. Typically, the cell delivery solution has some similarities in composition to the cell washing solution, wherein the cell delivery solution has the same or different concentrations of components or has additional components compared to the cell washing solution.

[0005] What is desired is a cell washing solution and / or cell delivery solution that has an appropriate shelf life at standard storage temperatures, has few or no components of human or mammalian origin, has a substantially stable pH over days, weeks, or longer, substantially physiological osmolarity, and maintains better cell health than commercially available solutions. In addition, the cell delivery solution composition can be designed to sufficiently prevent cell sedimentation to allow accurate dose assurance during preparation and administration. Such accurate dose assurance and delivery can be a result of the ability of the cell delivery solution to substantially maintain cells in suspension for up to 8 hours or more.

[0006] Embodiments of the present disclosure provide solutions and methods to address the above-mentioned needs. Summary of the invention

[0007] The present disclosure relates to various formulations or compositions, which include one or more energy source components; one or more pH buffers; one or more salts; and one or more stabilizers. The present disclosure also relates to cell washing solutions (CWS), cell delivery solutions (CDS) and cell suspensions for injection (CSI). Cell suspensions for injection provide cells suspended for several hours while maintaining adequate levels of cell health. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0010] The present disclosure will be better understood by referring to the following drawings, which are provided as illustrations of certain embodiments of the present application and are not intended to limit the scope of the present disclosure.

[0011] Figure 1 is a graphical representation of cell viability over time.

[0012] Figure 2 is a graphical representation of the percentage of apoptotic cells over time.

[0013] Figure 3is a graphic representation of cell health over time.

[0014] Figure 4 is a graphical representation of cell dose over time.

[0015] Figures 5A-5C is a graphical representation of cell viability and dose over time.

[0016] Figure 6 is a graphical representation of the percentage of apoptotic cells over time.

[0017] Figure 7 This is a photograph of the cultured cells after storage for 8 hours.

[0018] Figure 8 is a table of photographs of cells in suspension or out of suspension when stored in different culture media.

[0019] Fig. 9 is a graphical representation of cell sedimentation over time using the scoring system.

[0020] Fig.10 is a visual illustration of the cell sedimentation scoring system.

[0021] Figures 11A-11B is a graphical representation of the variability of dose sampling over time.

[0022] Fig.12 is a graphical representation of cell potency over time.

[0023] Fig.13A and 13B is a graphical representation of the viable cell concentration over time for cells stored in different solutions.

[0024] Fig.14A is a graphical representation of cell viability over time for cells stored in different solutions.

[0025] Fig. 14B is a graphical representation of the total cell concentration over time for cells stored in different solutions.

[0026] Fig.15 are multiple representative images of cells stored in different solutions over time. DETAILED DESCRIPTION

[0027] In the discussion herein and in the claims, the term "about" means that the listed value may vary as long as the variation does not result in an inconsistency in the method. For example, for some elements, the term "about" may refer to a variation of ±0.1%, and for other elements, the term "about" may refer to a variation of ±1% or ±10% or any point therein.

[0028] As used herein, the terms "substantially" or "substantially" apply equally when used in a negative sense to refer to the complete or near complete lack of an action, feature, characteristic, state, structure, matter, or result. For example, a "substantially" flat surface would be completely flat, or so nearly flat that the effect would be the same as if it were completely flat.

[0029] As used herein, terms such as "a," "an," and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration.

[0030] As used herein, terms defined in the singular are intended to include those terms defined in the plural and vice versa.

[0031] References in the specification to "one embodiment", "certain embodiments", "some embodiments" or "embodiments" indicate that the embodiments may include specific features or characteristics, but each embodiment does not necessarily include specific features, structures or characteristics. In addition, such phrases do not necessarily refer to the same embodiment. In addition, when a specific feature, structure or feature is described in conjunction with an embodiment, it is considered that, whether or not explicitly described, it is within the knowledge of those skilled in the art to affect such features, structures or features in conjunction with other embodiments. For the purposes of the following description, the terms "upper", "lower", "right", "left", "vertical", "horizontal", "top", "bottom" and their derivatives should be related to the present invention as it is oriented in the accompanying drawings. The terms "covering", "above", "located on..." or "located on..." mean that the first element is present on the second element, wherein the intermediate element forms an interface between the first element and the second element. The term "directly in contact" or "attached to" means that the first element and the second element are connected without any intermediate element at the interface of the two elements.

[0032] Reference to any numerical range herein explicitly includes each numerical value (including fractions and integers) encompassed by the range. For illustration, the range of "at least 50" or "at least about 50" mentioned herein includes integers 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and fractions 50.1, 50.2, 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc. In further illustration, the range of "less than 50" or "less than about 50" mentioned herein includes integers 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, etc., and fractions 49.9, 49.8, 49.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1, 49.0, etc.

[0033] As used herein, the term "stabilizer" refers to any component that can be used to reduce or prevent the degradation of other solution components.

[0034] As used herein, the term "energy source" refers to any component that can provide chemical energy to one or more cells.

[0035] As used herein, the term "pH indicator" refers to any component or substance that changes its properties in response to changes in pH. Changes in such properties include changes in optical properties, such as color changes.

[0036] As used herein, the term "vitamin" includes various fat-soluble or water-soluble organic substances obtained naturally from plant and animal foods or synthesized (non-limiting examples include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal or pyridoxamine or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid) and vitamin B12 (various cobalamines; usually cyanocobalamin in vitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, K1 and K2 (i.e., MK-4, MK-7), folic acid and biotin), as well as provitamins, derivatives and / or analogs thereof.

[0037] As used herein, the term "cell" may refer to any one or more types of cells mentioned in this paragraph or any other paragraph of the present disclosure, or a substitute and / or equivalent thereof. In some embodiments, the cell is a neural cell. The cell may be derived from embryonic stem cells or induced pluripotent stem cells. In some embodiments, the cell is a dopaminergic neuron cell, a transplantable midbrain dopaminergic neuron, a midbrain dopaminergic neuron, an authentic midbrain dopamine (DA) neuron, a midbrain dopaminergic neuron progenitor cell, a dopaminergic neuron progenitor cell, a dopaminergic neuron precursor cell, and in some embodiments, an iPSC-derived dopaminergic neuron cell.

[0038] In some embodiments, "cells" may refer to floor plate midbrain progenitors floor plate derived DA neurons. As used herein, the term "floor plate derived DA neurons" or "true midbrain DA neurons" or "midbrain fate FOXA2+LMX1A+ dopamine (DA) neurons" or "floor plate midbrain dopamine (DA) neurons" or "transplantable midbrain DA neurons" or "mDA neurons" or "FOXA2+LMX1A+TH+" or "FOXA2 / LMX1A / TH" or "FOXA2+LMX1A+NURR1+TH+" or "FOXA2 / LMX1A / NURR1 / TH" refers to a transplantable midbrain DA neuron population obtained in any suitable manner.

[0039] As used herein, cells used to obtain floor plate midbrain progenitor cells and midbrain fate FOXA2 / LMX1A+ dopamine (DA) neurons are obtained from a variety of sources, including embryonic and non-embryonic sources, such as hESC and non-embryonic hiPSC, somatic stem cells, disease stem cells, i.e., isolated pluripotent cells and engineered derived stem cells isolated from Parkinson's disease patients, cancer stem cells, human or mammalian pluripotent cells, etc. These cells used to obtain floor plate midbrain progenitor cells and midbrain fate FOXA2 / LMX1A+ dopamine (DA) neurons, or differentiated midbrain dopaminergic neuron cells ("DMD" cells) can also be "cells" of the present disclosure.

[0040] As used herein, the term "stem cell" refers to a cell that has the ability to divide indefinitely in culture and produce specialized cells. Stem cells can be obtained from animals and patients, including humans; for example, human stem cells refer to human stem cells. Stem cells can be obtained from a variety of sources, including embryos and non-embryos, such as umbilical cord cells, cells from children, and cells from adults. For the purposes of the present invention, adult stem cells generally refer to cells that were not originally obtained from a fetus, in other words, cells from infants, shed umbilical cords, shed placental cells, cells from children, cells from adults, etc.

[0041] As used herein, the term "cord blood stem cells" refers to stem cells collected from the umbilical cord at birth that have the ability to generate at least all blood cells (hematopoietic cells) in the body.

[0042] As used herein, the term "somatic (adult) stem cell" refers to relatively rare undifferentiated cells found in many organs and differentiated tissues, which have limited self-renewal (in the laboratory) and differentiation capabilities. Such cells differ in their differentiation capabilities, but are generally limited to the cell type in the organ of origin. As used herein, the term "somatic cell" refers to any cell of the body except sperm and egg cells. Somatic cells are diploid, which means that they contain two sets of chromosomes, one set inherited from each parent. Somatic cells include brain cells. Brain cells include, but are not limited to, neurons, oligodendrocytes, astrocytes, microglia, perivascular macrophages, meningeal macrophages, endothelial cells, pericytes, ependymal cells, and blood cells.

[0043] As used herein, the term "allogeneic cells" or "allogeneic stem cells" refers to cells obtained from an individual belonging to the same species but genetically different. The allogeneic stem cells of the present disclosure are from a person other than the patient (a matched relative or an unrelated donor).

[0044] As used herein, the term "neural lineage cell" refers to a cell that contributes to the nervous system (both central and peripheral) or neural crest cell fate during development or in an adult. The nervous system includes the brain, spinal cord, and peripheral nervous system. Neural crest cell fate includes those of the cranium, trunk, vagus nerve, sacrum, and heart, producing mesectoderm, cranial cartilage, skull, thymus, teeth, melanocytes, iris pigment cells, cranial ganglia, dorsal root ganglia, sympathetic / parasympathetic ganglia, endocrine cells, parts of the enteric nervous system and heart.

[0045] As used herein, the term "adult stem cell" refers to a somatic stem cell, for example, a "hematopoietic stem cell," which refers to the stem cell that gives rise to all red and white blood cells and platelets in infants, children, and adults.

[0046] As used herein, the term "embryonic stem cell" refers to a primitive (undifferentiated) cell derived from one of several sources, including but not limited to pre-implantation stage embryos, artificially created embryos (i.e., by in vitro fertilization), etc., which is capable of dividing for a long time in culture under non-differentiating conditions and is known to have the ability to develop into cells and / or tissues of the three primary germ layers (ectoderm, mesoderm, and endoderm).

[0047] As used herein, the term "endoderm" refers to the cell layer derived from the inner cell mass of the blastocyst; which has the ability to give rise to the lungs, other respiratory structures and digestive organs, or generally the "gut" "in vivo" and to a variety of cell types in vitro.

[0048] As used herein, the term "embryonic stem cell line" refers to a population of embryonic stem cells that have been cultured under in vitro conditions that allow proliferation without differentiation for days, months to years, such as cells in the human WA-09 cell line.

[0049] As used herein, the term "human embryonic stem cell" or "hESC" refers to a type of pluripotent stem cell derived from an early human embryo, up to and including the blastocyst stage, which is capable of dividing for long periods of time in culture under non-differentiating conditions and is known to develop into cells and tissues of the three primary germ layers (ectoderm, mesoderm, and endoderm).

[0050] As used herein, the term "pluripotent stem cell" or "PSC" has its usual meaning in the art, i.e., a self-replicating cell with the ability to develop into endoderm, ectoderm, and mesoderm cells. As used herein, PSC includes "genetically edited or modified PSC". In some embodiments, PSC is a human PSC. PSC includes embryonic stem cells (ESC) and induced pluripotent stem cells ("iPS cells" or "iPSC" or "hiPSC"). The terms ES cells and iPS cells have their usual meanings in the art. As used herein, "genetically edited or modified PSC" includes cells in which the aEF1a-IDUA overexpression cassette is engineered into the AAVS1 safe harbor locus in human induced pluripotent stem cells (hiPSC).

[0051] As used herein, the term "non-pluripotent stem cells" has its usual meaning in the art, i.e., cells that do not have the potential to differentiate into all three germ layers (i.e., endoderm, ectoderm, and mesoderm). Examples of such cells include cord blood stem cells and epidermal stem cells.

[0052] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to the type of pluripotent stem cell, which is similar to embryonic stem cells, whereby somatic cells (adults) are reprogrammed to enter an embryonic stem cell-like state by being forced to express factors important for maintaining the "stemness" of embryonic stem cells (ESC). Mouse iPSC was reported in 2006 (Takahashi and Yamanaka), and human iPSC was reported in late 2007 (Takahashi et al. and Yu et al.). Mouse iPSC shows the important features of pluripotent stem cells, including the expression of stem cell markers, the formation of tumors containing cells from all three germ layers, and the ability to contribute to many different tissues when injected into mouse embryos at the very early stages of development. Human iPSC also expresses stem cell markers, and can generate cells characteristic of all three germ layers. Unlike embryonic stem cells, iPSCs are artificially formed by introducing certain embryonic genes (such as OCT4, SOX2 and KLF4 transgenes) (see, e.g., Takahashi and Yamanaka Cell 126, 663-676 (2006), incorporated herein by reference) into somatic cells, such as cell lines from induced cells C14, C72, etc. Another example of iPSCs is adult human skin cells or fibroblasts transformed with genes (OCT4, SOX2, NANOG, LIN28 and KLF4) cloned into plasmids, e.g., see Yu et al., Science DOI: 10.1126 / science.1172482, incorporated herein by reference.

[0053] As used herein, the term "totipotent" refers to the ability to give rise to all cell types of the body plus all cell types that make up extra-embryonic tissues such as the placenta.

[0054] As used herein, the term "multipotent" refers to the ability to develop into more than one cell type of the body.

[0055] As used herein, the term "pluripotent" refers to a cell having the ability to give rise to at least two, but usually many, different cell types of the body. Pluripotent cells typically generate teratomas after injection into immunosuppressed mice.

[0056] As used herein, the term "specialized cell" refers to a cell type that performs a specific function in a multicellular organism. For example, a group of specialized cells (such as neurons) work together to form a system (such as a nervous system).

[0057] As used herein, the term "neurectoderm" refers to cells or cell fates found early in development or during differentiation of pluripotent stem cells that can give rise to cells of the neural lineage.

[0058] However, it should be understood that the composition can include other types of "cells", such as mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells or induced pluripotent stem cells, erythrocytes, platelets, chondrocytes, skin cells, immune cells (e.g., tumor infiltrating lymphocytes, virus-reconstituted T cells, dendritic cells, regulatory T cells, macrophages), neural crest stem cells, neurons, glia, smooth muscle, cardiac tissue, chondrocytes, osteocytes, glial-restricted progenitor cells, astrocytes, oligodendrocytes, neuroblasts, promegakaryocytes, megakaryocytes, promonocytes, monocytes, macrophages, myeloid cells, myeloid dendritic cells, microglia, differentiated microglia, microglial progenitor cells, proerythroblasts, erythroblasts, normoblasts, reticulocytes, thrombocytes, myeloblasts, promyelocytes, neutrophilic myeloid cells, neutrophilic myeloid cells, promyelocytes ...

[00136] The present invention also includes, but is not limited to, a myelocyte, a neutrophilic band cell, a neutrophil, an eosinophilic myelocyte, an eosinophilic band cell, an eosinophil, a basophilic myelocyte, a basophilic band cell, a basophil, a committed lymphoid progenitor cell, a pre-NK cell, a NK lymphoblast, a NK cell, a thymocyte, a T lymphoblast, a T cell, a plasmacytoid dendritic cell, a pre-B cell, a B lymphoblast, a B cell, a plasma cell, an osteoblast, a chondrocyte, a myoblast, a myotube, a fibroblast, an adipocyte, a mesoderm, an ectoderm, a primordial germ cell, a sperm, an ovum, a definitive endoderm, a cardiac cell, or any other suitable type of cell. Cardiac cells may include, but are not limited to, cardiomyocytes, atrial cardiomyocytes, ventricular cardiomyocytes, fibroblasts (FB), endothelial cells (EC), pericytes, smooth muscle cells (SMC), immune cells (myeloid and lymphoid), adipocytes, mesothelial cells, and neuronal cells.

[0059] As used herein, the term "microglial progenitor cells" refers to PSC-derived myeloid cells.

[0060] As used herein, the term "microglia" refers to PSC-derived myeloid cells.

[0061] In addition, "cell" may refer to myeloid cells, which are rich in microglia derived from unedited human induced pluripotent stem cells (hiPSC). As used herein, the term "myeloid cells" refers to PSC-derived cells and / or non-PSC-derived. PSC-derived cells differentiate after hematopoietic differentiation, express myeloid markers (including but not limited to: CD45, CD11b, CD33, CD14, CX3CR1), and can perform conventional myeloid functions, including but not limited to phagocytosis, responding to external stimuli, secreting cytokines and polarizing to pro-inflammatory or anti-inflammatory states. These myeloid cells will become tissue-resident macrophages after being delivered to different organs in living organisms, for example, when delivered to the brain of living animals, they will become microglia, because they will express typical microglial markers, including but not limited to TMEM119, IBA1, CD163, CX3CR1, CD45, CD206. The payload of these cells may be full of the complement of the missing lysosomal enzymes and may not have any genetic modifications to include any additional payload.

[0062] In addition to any cell mentioned above as a "cell" of the present disclosure, "cell" may also refer to a non-living biological product, including but not limited to endosomes and lipid-based vesicles. Further, in addition to any cell mentioned above as a "cell" of the present disclosure, "cell" may also refer to any cell and / or tissue disclosed in U.S. Pat. No. 10,280,398, U.S. Pat. No. 10,711,243, and International Application WO2013 / 067362, the contents of each of which are incorporated herein in their entirety.

[0063] As used herein, the term "cell washing solution" or "CWS" is any solution added to a container containing cells and a cryoprotectant after the container is removed from an environment below 0°C. As used herein, the term "cryoprotectant" refers to a substance used to reduce or eliminate cell damage caused by the freezing and thawing process, which is inevitably accompanied by ice crystal formation and ionic and osmotic imbalances when cells and / or tissues are stored at temperatures below 0°C. Cryoprotectants are not limited to certain substances as long as they are able to reduce cell damage during storage below 0°C. Examples thereof may include permeable cryoprotectants such as dimethyl sulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, etc., or non-permeable cryoprotectants such as sucrose, carboxymethylcellulose salts, carboxymethylcellulose (CMC), monosaccharides, disaccharides, etc., but are not limited thereto.

[0064] As used herein, the term "cell delivery solution" or "CDS" is any solution that is added to a container containing unwashed cells (directly on thawed cells) or washed cells so that the cells can be administered to a subject. The cell delivery solution may be free of cryoprotectants and / or cell washing solutions, containing a minimum or trace amount of cryoprotectants and / or cell washing solutions, which are stored and / or washed cells before contacting the cell delivery solution, and are not completely removed from the container after the supernatant discarding process, which may occur after the optional centrifugation step of the container, and which may optionally form a cell precipitate or a concentrated cell solution. This supernatant discarding process reduces the concentration of components such as cryoprotectants that are undesirable for injection in the stored sample and / or removes the component. The cell delivery solution may be used for reconstructing a cell solution for injection (CSI) after the cell is thawed before being administered for clinical use.

[0065] As used herein, the term "cell suspension for injection" or "CSI" refers to one or both of a cell delivery solution (CDS) and / or a cell washing solution (CWS) in combination with one or more cells, and one or more optional additional components. The cell suspension for injection may include additional components such as a cell washing buffer, such as, for example, a composition comprising phosphate buffered saline (PBS), a composition comprising 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), a Bio-Plex Pro TM Signaling Wash Buffer and Cultrex TM 3-D cell washing buffer. The cell suspension for injection may also contain trace amounts of cryoprotectants and / or cell washing solutions that are not completely removed from the container after the supernatant discarding process. However, possible trace amounts of cryoprotectants and / or cell washing solutions do not change the efficacy of the cell suspension for injection.

[0066] The cell delivery solution and / or cell washing solution can be used at a preparation site to create a CSI, which is a composition or product that is administered to a subject, such as by injection into a subject. The preparation site can receive a plurality of cells, "wash" those cells, mix those cells with the cell delivery solution and / or cell washing solution, and then prepare those cells for administration to a subject, such as by injection into a subject, with a CSI. The types of injections contemplated by the present disclosure include, but are not limited to, intracerebroventricular (ICV), intravenous (IV), intramuscular (IM), intrathecal (IT). The preparation site can perform these steps in a sterile environment and under appropriate environmental conditions. In addition, the preparation site can perform quality control testing on the cells during any point in their preparation to determine whether the cells are sufficiently viable before leaving the preparation site.

[0067] The present disclosure provides a "cell washing solution (CWS)" and a "cell delivery solution (CDS)", one or both of which may be referred to as a "cell solution". Both the cell washing solution and the cell delivery solution may be formulated to constitute various qualities, such as (A) a pH level of about 5.5 to about 9.0, or a pH level of about 6.0 to about 8.0, or a pH level of about 6.4 to about 7.8, or a pH level of about 6.8 to about 7.6, or a pH level of about 7.0 to about 7.5, or a pH level of about 7.2 to about 7.4, (B) an osmolarity of about 100 to about 700 mOsm / L, or an osmolarity of about 150 to about 500 mOsm / L, or an osmolarity of about 200 to about 500 mOsm / L, or an osmolarity of about 225 to about 400 mOsm / L. (C) a density of about 1.00 to about 1.30 g / mL, or a density of about 1.02 to about 1.20 g / mL, or a density of about 1.04 to about 1.15 g / mL, or a density of about 1.05 to about 1.11 g / mL, or a density of about 1.07 to about 1.09 g / mL, or a density of about 1.08 g / mL, a relatively low viscosity and good cell compatibility, so that both the cell washing solution and the cell delivery solution are substantially free of cytotoxicity. Both the cell washing solution and the cell delivery solution are configured to have sufficient shelf life under typical or standard storage conditions so that they are ready for clinical use.

[0068] These qualities can affect the ability of the cell delivery solution to maintain cells in suspension. As used herein, the term "suspension" refers to cells dispersed in a liquid. In the present disclosure, cells dispersed in a liquid can remain dispersed for 0 to 104 hours or longer. The shelf life of cells dispersed or suspended in a liquid according to the present disclosure is up to about 104 hours. More specifically, in the present disclosure, the liquid can be a cell delivery solution that can maintain cells dispersed therein for up to about 15 minutes, up to about 30 minutes, up to about 45 minutes, up to about 1 hour, up to about 90 minutes, up to about 2 hours, up to about 4 hours, up to about 6 hours, up to about 8 hours, up to about 12 hours, up to about 16 hours, up to about 20 hours, up to about 24 hours, up to about 30 hours, up to about 36 hours, up to about 42 hours, up to about 48 hours, up to about 56 hours, up to about 64 hours, up to about 72 hours, up to about 80 hours, up to about 88 hours, up to about 96 hours, up to about 104 hours or more, without or after agitation (mixing) and / or homogenization.

[0069] Both the cell washing solution and the cell delivery solution can include several components, including but not limited to one or more energy source components; one or more pH buffers; one or more salts; and one or more stabilizers. Several components of both the cell washing solution and the cell delivery solution can be combined in any suitable manner to reach any one of the cell washing solution and the cell delivery solution, such as by the method discussed in the following embodiments. Before contacting with any cell, both the cell washing solution and the cell delivery solution can be warmed or cooled to any suitable temperature (e.g., about room temperature, or about 37°C, or about 4°C, or about 0°C, or about 2°C to about 8°C, or about 1°C to about 10°C, or about 0°C to about 12°C).

[0070] One or more energy source components of the cell washing solution or cell delivery solution can include any suitable sugar, such as dextrose, fructose, galactose, glucose, lactose, maltose, sucrose. The cell washing solution or cell delivery solution can include any amount of energy source to achieve the desired effect, including but not limited to about 24.5mM to about 24.8mM, about 24.4mM to about 24.9mM, about 24.3mM to about 25.0mM, about 24.1mM to about 25.2mM, about 23.9mM to about 25.4mM, about 23.7mM to about 25.6mM, or about 23.5mM, or about 23mM, or about 22mM, or about 21mM, or about 20mM or less, or about 26mM, or about 27mM, or about 28mM, or about 29mM, or about 30mM or more.

[0071] The one or more pH buffers of the cell wash solution or the cell delivery solution may include any suitable buffer, such as a zwitterionic organic chemical buffer, examples of which include, but are not limited to, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium bicarbonate, 4-morpholinepropanesulfonic acid, 3-propanesulfonic acid (MOPS), and 2-(N-morpholino)ethanesulfonic acid (MES). The cell washing solution or the cell delivery solution can contain any amount of pH buffer to achieve the desired effect, including but not limited to about 10.6mM to about 10.9mM, about 10.5mM to about 11.0mM, about 10.4mM to about 11.1mM, about 10.2mM to about 11.3mM, about 10.0mM to about 11.5mM, about 9.8mM to about 11.7mM, or about 9.5mM, or about 9mM, or about 8mM, or about 7mM, or about 6mM or less, or about 12mM, or about 13mM, or about 14mM, or about 15mM, or about 16mM or more pH buffer.

[0072] The one or more salts of the cell wash solution or the cell delivery solution may include any suitable salts, such as one or more salts selected from the group consisting of calcium chloride, magnesium chloride, potassium chloride, sodium dihydrogen phosphate, and sodium chloride.

[0073] The cell washing solution or cell delivery solution can contain any amount of calcium chloride to achieve the desired effect, including but not limited to about 1.6 mM to about 1.9 mM, or about 1.5 mM to about 2.0 mM, or about 1.4 mM to about 2.1 mM, or about 1.3 mM to about 2.2 mM, or about 1.1 mM to about 2.4 mM, or about 0.9 mM to about 2.6 mM, or about 0.7 mM to about 2.8 mM, or about 0.5 mM, or about 0.3 mM, or about 0.1 mM or less, or about 3 mM, or about 3.5 mM, or about 4 mM, or about 5 mM or more.

[0074] The cell washing solution or cell delivery solution can contain any amount of magnesium chloride to achieve the desired effect, including but not limited to about 0.7 mM to about 1.0 mM, or about 0.6 mM to about 1.1 mM, or about 0.5 mM to about 1.2 mM, or about 0.4 mM to about 1.4 mM, or about 0.3 mM to about 1.6 mM, or about 0.2 mM to about 1.9 mM, or about 0.1 mM to about 2.2 mM, or about 0.05 mM or less, or about 2.5 mM, or about 3.0 mM or more.

[0075] The cell washing solution or the cell delivery solution can contain any amount of potassium chloride to achieve the desired effect, including but not limited to about 5.1 mM to about 5.4 mM, or about 5.0 mM to about 5.5 mM, or about 4.9 mM to about 5.7 mM, or about 4.8 mM to about 5.8 mM, or about 4.6 mM to about 6.0 mM, or about 4.4 mM to about 6.2 mM, or about 4.2 mM to about 6.4 mM, or about 4.0 mM, or about 3.5 mM, or about 3.0 mM or less, or about 6.5 mM, or about 7.0 mM, or about 7.5 mM, or about 8.0 mM or more.

[0076] The cell washing solution or cell delivery solution can contain any amount of sodium phosphate monobasic to achieve the desired effect, including but not limited to about 0.88mM to about 0.91mM, or about 0.87mM to about 0.92mM, or about 0.86mM to about 0.93mM, or about 0.85mM to about 0.94mM, or about 0.83mM to about 0.96mM, or about 0.81mM to about 0.98mM, or about 0.8mM, or about 0.75mM, or about 0.7mM or less, or about 1.0mM, or about 1.05mM, or about 1.1mM or more.

[0077] In one embodiment, the cell washing solution or cell delivery solution can contain any amount of sodium chloride to achieve the desired effect, including but not limited to about 119mM to about 122mM, or about 118mM to about 123mM, or about 117mM to about 124mM, or about 115mM to about 126mM, or about 113mM to about 128mM, or about 110mM, or about 105mM, or about 100mM or less, or about 130mM, or about 135mM, or about 140mM or more.

[0078] In another embodiment, the cell washing solution or cell delivery solution can contain any amount of sodium chloride to achieve the desired effect, including but not limited to about 74mM to about 77mM, or about 73mM to about 78mM, or about 72mM to about 79mM, or about 70mM to about 81mM, or about 68mM to about 83mM, or about 66mM to about 85mM, or about 65mM, or about 60mM, or about 55mM or less, or about 90mM, or about 95mM, or about 100mM or more.

[0079] In another embodiment, the cell washing solution or cell delivery solution can contain any amount of sodium chloride to achieve the desired effect, including but not limited to about 93mM to about 96mM, or about 92mM to about 97mM, or about 91mM to about 98mM, or about 90mM to about 99mM, or about 88mM to about 101mM, or about 85mM to about 103mM, or about 83mM to about 105mM, or about 80mM, or about 75mM, or about 70mM or less, or about 105mM, or about 110mM, or about 115mM or more.

[0080] The one or more stabilizers of the cell washing solution or the cell delivery solution may include any suitable stabilizer, such as one or more of proteins, such as one or more albumins, such as recombinant albumin (rHSA), dextran (e.g., including dextran 40), poloxamer (e.g., including poloxamer 188). In addition, the one or more stabilizers may include one or more of the following, which may also be excipients of the cell washing solution or the cell delivery solution: polyethylene glycol, carboxymethylcellulose, hyaluronic acid, starch, acrylates, methacrylates, polyvinyl alcohol, polyethylene oxide, polypropylene oxide, polyacrylates, polyvinyl pyrrolidone, polymethacrylates, polylactic acid-co-glycolic acid, polyacrylamide, polylactic acid, chitosan, gum, guar gum, xanthan gum, carrageenan, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, cyclodextrin derivatives, β-cyclodextrin derivatives, alginate esters, calcium alginate and stearate.

[0081] In one embodiment, the cell washing solution or cell delivery solution can contain any amount of protein, such as one or more albumins, such as recombinant albumin (rHSA) to achieve the desired effect, including but not limited to about 0.07 w / w% to about 0.09 w / w%, or about 0.06 w / w% to about 0.1 w / w%, or about 0.05 w / w% to about 0.11 w / w%, or about 0.04 w / w% to about 0.12 w / w%, or about 0.03 w / w% to about 0.13 w / w%, or about 0.02 w / w% to about 0.15 w / w%, or about 0.01 w / w% to about 0.17 w / w%, or about 0.005 w / w% or less, or about 0.2 w / w%, or about 0.25 w / w% or more.

[0082] In another embodiment, the cell washing solution or cell delivery solution can contain any amount of protein, such as one or more albumins, such as recombinant albumin (rHSA) to achieve the desired effect, including but not limited to about 0.08w / w% to about 0.11w / w%, or about 0.07w / w% to about 0.12w / w%, or about 0.06w / w% to about 0.13w / w%, or about 0.05w / w% to about 0.14w / w%, or about 0.04w / w% to about 0.16w / w%, or about 0.03w / w% to about 0.18w / w%, or about 0.02w / w% to about 0.2w / w%, or about 0.01w / w% to about 0.22w / w%, or about 0.05w / w% or less, or about 0.25w / w%, or about 0.3w / w%, or about 0.35w / w% or more.

[0083] In one embodiment, the cell washing solution or cell delivery solution can contain any amount of protein, such as one or more albumins, such as recombinant albumin (rHSA) to achieve the desired effect, including but not limited to about 6.50 w / w% to about 6.8 w / w%, or about 6.4 w / w% to about 6.9 w / w%, or about 6.3 w / w% to about 7.0 w / w%, or about 6.1 w / w% to about 7.2 w / w%, or about 5.9 w / w% to about 7.4 w / w%, or about 5.7 w / w% to about 7.6 w / w%, or about 5.5 w / w%, or about 5.0 w / w%, or about 4.5 w / w% or less, or about 8.0 w / w%, or about 8.5 w / w%, or about 9.0 w / w% or more.

[0084] In one embodiment, the cell washing solution or cell delivery solution can contain any amount of dextran to achieve the desired effect, including but not limited to about 17.27 w / w% to about 17.30 w / w%, or about 17.26 w / w% to about 17.31 w / w%, or about 17.24 w / w% to about 17.33 w / w%, or about 17.2 w / w% to about 17.35 w / w%, or about 17.1 w / w% to about 17.4 w / w%, or about 17.0 w / w% to about 17.5 w / w%, or about 16.5 w / w%, or about 16.0 w / w%, or about 15.0 w / w% or less, or about 18.0 w / w%, or about 18.5 w / w%, or about 19.0 w / w% or more.

[0085] In one embodiment, the cell washing solution or cell delivery solution can contain any amount of dextran to achieve the desired effect, including but not limited to about 13.02 w / w% to about 13.05 w / w%, or about 13.0 w / w% to about 13.1 w / w%, or about 12.9 w / w% to about 13.2 w / w%, or about 12.7 w / w% to about 13.4 w / w%, or about 12.5 w / w% to about 13.5 w / w%, or about 12.2 w / w% to about 13.8 w / w%, or about 12.0 w / w%, or about 11.5 w / w%, or about 11.0 w / w% or less, or about 14.0 w / w%, or about 14.5 w / w%, or about 15.0 w / w% or more.

[0086] In one embodiment, the cell washing solution or cell delivery solution can contain any amount of poloxamer to achieve the desired effect, including but not limited to about 0.07 w / w% to about 0.09 w / w%, or about 0.06 w / w% to about 0.1 w / w%, or about 0.05 w / w% to about 0.11 w / w%, or about 0.04 w / w% to about 0.12 w / w%, or about 0.03 w / w% to about 0.13 w / w%, or about 0.02 w / w% to about 0.15 w / w%, or about 0.01 w / w% to about 0.17 w / w%, or about 0.005 w / w% or less, or about 0.2 w / w%, or about 0.25 w / w% or more.

[0087] There are several components that are not included in the cell wash solution or the cell delivery solution and can be completely excluded or below detectable limits. Some examples of components that may be excluded are: certain animal-derived components; certain stabilizers such as human serum albumin (HSA); certain salts such as zinc sulfate, sodium bicarbonate, and ferric nitrate; certain pH indicators such as phenol red; certain energy sources such as sodium pyruvate; certain amino acids such as glycine, L-alanine, L-arginine hydrochloride, L-asparagine-H2O, L-glutamine, L-cysteine, L-histidine hydrochloride-H2O, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; and certain vitamins such as ascorbic acid, choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and i-inositol.

[0088] The present disclosure also relates to a method for treating a subject. The method may include the step of administering a dose of a cell suspension for injection (CSI), wherein the CSI comprises a plurality of cells; one or more energy source components; one or more pH buffers; one or more salts; and one or more stabilizers. The plurality of cells may include any cell disclosed herein, and may be cells of a subject undergoing treatment, or cells from another source or subject. The subject treated with the method may be an adult or a child. The subject treated with the method of the present disclosure may be a mammal and preferably a human. The subject may also be an animal, such as a primate, a dog, a cat, a livestock animal, or a horse.

[0089] The number of cells administered can vary depending on the particular treatment. The number of cells can be any suitable and effective number, including but not limited to about 10 cells or less, about 100 cells, about 1,000 cells, about 10,000 cells, about 100,000 cells, about 10 6 cells, about 10 7 cells, about 10 8 cells, about 10 9 cells, about 10 10 cells, about 10 11 cells, about 10 15 cells, about 10 20 cells or more, including any range with any of these values ​​as endpoints. These numbers of cells can be administered in any suitable volume, at any suitable cell concentration, such as about 25×10 6 cells / mL to about 150×10 6 cells / mL, or about 50×10 6 cells / mL to about 100×106 cells / mL, or about 60×10 6 cells / mL to about 90×10 6 cells / mL, or about 65×10 6 cells / mL to about 85×10 6 cells / mL, or about 70×10 6 cells / mL to about 80×10 6 cells / mL, or about 50×10 6 cells / mL, or about 75×10 6 cells / mL, or about 100×10 6 cells / mL, or about 125×10 6 cells / mL, or about 150×10 6 In one embodiment, the number of cells administered can be 120×10 6 cells / mL, 130×10 6 cells / mL or 140×10 6 cells / mL.

[0090] The method may involve a single administration of a dose of CSI, or the method may include the further step of waiting a predetermined time after the previous administration and then administering another dose of CSI, which may be a dose of the same dose type with the same or similar components or a different dose of CSI. The method may involve unilateral or bilateral administration, for example, injection such as ICV injection. The predetermined amount of time may vary with the treatment and may be any suitable amount of time, such as about 1 minute, about 1 hour, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 2 days, about 4 days, about 7 days, about 10 days, about 14 days, about 21 days, about 1 month, about 6 weeks, about 2 months, about 4 months, about 6 months, about 9 months, about 12 months, about 18 months, about 2 years or more, including any range with any of these values ​​as endpoints.

[0091] These additional steps of waiting a predetermined time after a previous administration and then administering another dose of the CSI can be repeated based on the treatment, and can be repeated any suitable amount, such as two, three, four, five, six, seven, eight, nine, ten or more times.

[0092] The treatment method may also include a step prior to the administration of the CSI, or a step concurrent with the administration of the CSI, or a step after the administration of the CSI. This optional concurrent step or subsequent step may be one or more of the administration of a treatment selected from the group consisting of an immunosuppressant, a chemotherapeutic agent, radiation therapy, bone marrow therapy, hormone therapy, surgical therapy, or any treatment intended to improve the subject's response to the administration of the CSI. Each of these treatments may vary depending on the treatment desired.

[0093] The present disclosure also relates to a method of administering a dose volume to a subject. This method of administering a dose volume includes loading a dose delivery device, such as a syringe or any other device capable of delivering a solution to a subject, with a dose volume of a cell suspension for injection (CSI). The CSI may include a plurality of cells; one or more energy source components; one or more pH buffers; one or more salts; and one or more stabilizers, wherein the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer. The plurality of cells may be differentiated midbrain dopaminergic neuronal cells, and may include about 10 0 cells to about 10 10 range of cells, or about 10 8 cells to about 10 10 The range of cells.

[0094] After loading the solution delivery device with the dose volume, the method then includes the step of injecting the dose volume into a portion of the subject. The subject can be a human, and this injection step can occur directly into the portion of the subject, and / or into an intravenous line in a blood vessel of the subject.

[0095] This method of administration may further comprise a concurrent or subsequent step of administering a treatment selected from the group consisting of chemotherapeutic agents, radiation therapy, bone marrow therapy, hormone therapy, and surgical therapy.

[0096] The methods and models of the present disclosure will be better understood by reference to the following examples, which are provided as illustrations of the present disclosure and not limiting in any way.

[0097] Example 1

[0098] Examples of forming cell wash solutions of the present disclosure are disclosed in this Example.

[0099] This cell wash solution can be used to wash any cells after thawing, including cells in cryoprotectants. The procedure is described for a 1 kg scale, but can be scaled up or down accordingly.

[0100]

[0101]

[0102] For these starting materials, the following procedure was followed:

[0103] 1. Taree a 1 L container with a stir bar and fill with 500 g of water for injection (WFI) using a serological pipette.

[0104] 2. Using a balance, tare a separate weigh boat and weigh out each reagent. Transfer quantitatively to a 1 L bottle while stirring on a magnetic stir plate.

[0105] 3. Taking into account the volume of water added in the previous step, make up the weight to 980.0 g with WFI.

[0106] 4. Adjust the bulk solution pH to about 6.95 to about 7.05 with 1 M NaOH and 1 M HCl solutions at about 17° C. to about 25° C. Record the volume added (about 3.500 mL of 1 M NaOH).

[0107] 5. Add rHSA and up to 20.0 mL of WFI (to bring total weight to 1 kg), taking into account the volumes of acid and base used to adjust the pH of the solution in step 3. Place on a magnetic stir plate until well mixed, about 20 minutes - about 30 minutes.

[0108] 6. Take an aliquot to measure osmolarity.

[0109] a. Expected to be about 295 mOsm / kg H2O measured during this process.

[0110] 7. Filter the solution using a sterile filtration device in a sterile environment.

[0111] 8. Take an aliquot in a sterile environment to confirm that the final formulation pH, osmolarity and density are adequate.

[0112] a. The pH should be about 6.9 to about 7.1 at about 17 to about 25°C and about 7.2 to about 7.4 at about 0 to about 5°C.

[0113] b. The osmotic pressure should be between about 282 mOsm / kg·H2O and about 302 mOsm / kg·H2O. 1

[0114] c. The density should be from about 1.0075 g / mL to about 1.0105 g / mL.

[0115] 9. Store at about 4°C (or as desired / as needed).

[0116] 1 Osmolarity specifications are based on osmolarity specifications from 280-300mOsm / L (according to USP <785> ) conversion, considering the density to be about 1.0090 g / mL and the solute concentration to be about 0.0167617 g / mL.

[0117] Example 2

[0118] Examples of forming cell delivery solutions of the present disclosure are provided in this Example.

[0119] This solution can be used to resuspend cells during preparation of cell suspension for injection (CSI).The procedure is described for a 1 kg scale but can be scaled up or down accordingly.

[0120] Material Chemical formula / acronym HEPES <![CDATA[C8H 18 N2O4S]]> Calcium chloride, dihydrate <![CDATA[CaCl2·2H2O]]> Magnesium chloride hexahydrate <![CDATA[MgCl2·6H2O]]> Anhydrous potassium chloride KCl Anhydrous Sodium Chloride NaCl Sodium dihydrogen phosphate, dihydrate <![CDATA[NaH2PO4·2H2O]]> Dextrose (D-glucose), monohydrate <![CDATA[C6H 12 O6·H2O]]> Recombinant Albumin <![CDATA[rHSA (s) ]]> Dextran 40 Dex40 Poloxamer P188 P188 Water for Injection (WFI) <![CDATA[H2O]]>

[0121] For these starting materials, the following procedure was followed:

[0122] 1. Fill a 1 L container with 500 g of water for injection (WFI).

[0123] 2. Using an appropriate balance, tare a separate weigh boat and weigh out each reagent. Transfer quantitatively to a 1 L bottle while stirring on a magnetic stir plate.

[0124] 3. Complete the mass to about 750.0 g with WFI, taking into account the amount of water already added in the previous step. Allow to dissolve for about 30 minutes to about 45 minutes.

[0125] 4. Adjust the bulk solution pH to about 7.00 to about 7.05 with 1 M NaOH and 1 M HCl solution at about 17 to about 25° C. Record the volume added (about 3.000-3.125 mL of NaOH).

[0126] 5. Add rHSA with continuous stirring. Allow to dissolve for about 30 minutes to about 45 minutes.

[0127] 6. Add Dextran 40 with continuous stirring. Allow to dissolve at medium speed for about 30 minutes to about 45 minutes.

[0128] 7. Top up the bulk to 1 kg with WFI, taking into account the volumes of acid and base used to adjust the pH of the solution in step 4. Place on a magnetic stir plate until well mixed, about 5 minutes - about 10 minutes.

[0129] 8. Take an aliquot to measure the osmotic pressure.

[0130] • The in-process measurement was estimated to be approximately 344 mOsm / kg H2O.

[0131] 9. Filter the solution using a sterile filtration device in a sterile environment.

[0132] 10. Take an aliquot in a sterile environment to confirm that the final formulation pH, osmolarity and density are adequate.

[0133] .The pH should be between about 6.9 and about 7.1 at about 17 to about 25°C.

[0134] a. The osmolarity should be between about 341 and about 366 mOsm / kg·H2O.

[0135] Based on a density of about 1.081 g / mL at about 5°C and a solute concentration of about 0.249 g / mL, this would correspond to an osmolarity of about 280 to about 300 mOsm / L.

[0136] b. The density should be about 1.0800-about 1.0820 g / mL at about 5°C. However, the final density of the CSI may contain one or more additional components, including a substantially small amount or substantially trace amount of a cell washing solution and / or a buffer. The presence of the one or more additional components may have substantially no effect on the density of the cell delivery solution, have a substantially small effect on the density of the cell delivery solution (e.g., about 1% change, about 2% change, about 3% change, about 4% change, about 5% change, or greater), or have a substantially large effect on the density of the cell delivery solution (e.g., about 10% change, about 11% change, about 12% change, about 13% change, about 14% change, about 15% change, or greater).

[0137] 11. Store at 2-8°C (or as desired / as needed).

[0138] Example 3

[0139] The following examples of the present disclosure are intended to illustrate embodiments of cell wash solutions and cell delivery solutions when used with microglial cells.

[0140] Figure 1-4 All data presented in relate to microglia that have been cryopreserved. After thawing the frozen cells, the cryoprotectant is washed from the cells by adding an embodiment of a cell washing solution. The mixture containing the cells is then centrifuged to precipitate the cells so that the cell washing solution supernatant can be removed. After removing the supernatant, the cells are resuspended in an embodiment of a cell delivery solution to form a cell suspension for injection (CSI).

[0141] The CSIs were then stored for 0, 8, or 24 hours and characterized by several assays. The control group used was a commercial solution containing large amounts of excipients or some excipients that had to be added before use, making the solution suboptimal for later clinical and commercial use.

[0142] like Figure 1 As can be seen in the Figures, the cell viability of cells was evaluated at different points in a typical cell preparation for injection. After six hours of storage in a container on ice, microglial cells stored in the cell washing solution of the present disclosure had almost the same cell viability (92.0%) as compared to the cell viability of cells in the commercial solution (92.7%).

[0143] Apoptosis is the process by which cells enter a state of cell death. Figure 2 As can be seen, after six hours of storage in a container on ice, the percentage of apoptotic cells in the cells stored in the commercial solution was almost the same as the percentage of apoptotic cells in the commercial solution (about 7.9%) compared to the cells stored in the cell washing solution of the present disclosure (about 6.4%).

[0144] like Figure 3 As can be seen, after being stored in a container on ice for 24 hours, microglial cells stored in the cell delivery solution of the present disclosure had almost the same cell viability (around 90%) as that of cells in the commercial solution (around 80%).

[0145] like Figure 4 As can be seen, when cells were stored in the cell delivery solution of the present disclosure, the microglial cell dose (assessed by viable cell concentration) was essentially stable after 24 hours of storage and was not significantly different from the stability of cells stored in the commercial solution.

[0146] Example 4

[0147] The following examples of the present disclosure are intended to illustrate embodiments of cell wash solutions and cell delivery solutions when used with dopamine-producing cells.

[0148] All data presented in Figures 5-12 relate to cells that have been cryopreserved to produce dopamine. After thawing the frozen cells, the cryoprotectant is washed from the cells by adding an embodiment of a cell washing solution. Then, the solution containing the cells is centrifuged to remove the supernatant, and the cells are resuspended in an embodiment cell delivery solution to form a cell suspension for injection (CSI). Then, the CSI is stored for 0, 8, 24 or 48 hours. The control used is a commercially available solution containing a non-human origin component.

[0149] like Figure 5A As can be seen in Figure 5C, cell viability and cell dose were basically stable after 96 hours of storage. Figures 5A-5C The grey line and data points in represent the average of 11 samples of cells stored in commercially available solution. Figures 5A-5CThe blue line and data points in represent the average of 12 samples of cells stored in an embodiment of the cell delivery solution.

[0150] like Figure 5B As can be seen, there was minimal loss in viability of cells stored in the cell delivery solution for more than 24 hours (-2.5%), which was less than the loss of viability of cells stored in the commercial solution (-5%).

[0151] like Figure 5C As can be seen in Figure 5, in all formulations tested, the viable cell dose remained within the clinical target dose of approximately 100E6 ± 10 cells / mL for up to 96 hours.

[0152] Apoptosis is a process that describes early cell death. Further data on apoptotic cells such as Figure 6 shown. Figure 6 The grey data points in represent the average of five samples of cells stored in the commercial solution. Figure 6 The blue data points in represent the average of five samples of cells stored in an embodiment of the cell delivery solution. Both cells suspended in the commercial solution and cells suspended in the cell delivery solution had low levels of apoptosis (<10%) after storage for 24 hours.

[0153] When cells are formulated in either the commercial solution or the embodiments of the cell delivery solution, the effect of the formulation on the morphology of the dopamine producing cells after storage is evaluated. Figure 7 Representative pictures of cells cultured after storage in commercial solution (left) or cell delivery solution (right) are shown. As can be seen in Figure 5, no visual differences were observed in cell morphology, and after 5 days in culture the morphology was as expected.

[0154] One advantage of the cell delivery solution embodiment is the ability to maintain cells in suspension to improve dose assurance. In this example, cells are suspended in a cell delivery solution or a commercially available solution. Figure 8 As can be seen in the Figure 1 and demonstrated by a series of photographs of the container over time, embodiments of the cell delivery solution maintain cells in suspension for at least 72 hours, in contrast to the commercial solution, where the cells settle within minutes. Figure 8 As can be seen in the Figure 1 , visual heterogeneity of cells in the embodiment of the cell delivery solution begins around 96 hours, which is significantly longer than cells in the commercial solution.

[0155] Fig. 9 is from Figure 8 Visualize the results in a graphical representation. At each time point, visually inspect the container and Fig.10The scores were scored using the scoring system shown in , where a score of 3 is the initial score upon mixing when the cells are uniformly distributed in the solution. Fig. 9 As can be seen in the results plotted over time in FIG. 1 , cells resuspended in the commercial solution quickly settled to a score of 1. It can also be seen from the results that an embodiment of the cell delivery solution maintained cells in suspension at a score of 3 for a considerable amount of time (72 h).

[0156] Figures 11A-11B is a graphic representation of the quantitative assessment of cell sedimentation. The cell suspension was sampled three times: in the top, middle and lower part of the container, and the variability between the three measurements was calculated as the coefficient of variation (%CV). In this case, the greater the %CV, the more cells that precipitated.

[0157] Figures 11A-11B The grey data points in represent the variability in dose sampling for cells stored in commercially available solutions. Figures 11A-11B The blue dots in the graph represent the variability in the dose sampling of cells stored in two embodiments of the cell delivery solution: “A” and “B”. Specifically, Figures 11A-11B It was shown that two different cell delivery solutions, A and B, reduced cell sedimentation for up to 24 hours compared to commercial solutions.

[0158] Can be Fig.11A and Fig. 11B For comparison, Fig.11A Showing the variability in dose sampling after 8 hours of storage, Fig. 11B Variability in dose sampling after 24 hours of storage is shown. This comparison shows that cell delivery solutions A and B maintained cells in suspension for at least 24 hours (%CV < 25%), which is significantly better than cells stored in commercial solutions, where cell suspensions were heterogeneous after up to 8 hours of storage (%CV approximately 150-175).

[0159] like Fig.12 As can be seen in the Figures 2 and 3, dopamine-producing cells are able to produce dopamine above a certain threshold, regardless of whether the cells have been stored for 0, 8, or 24 h in the commercial solution or in the embodiments of the cell delivery solution.

[0160] In summary, the data presented herein demonstrate that dopamine-producing cells remain healthy and functional, whether they have been stored in commercial solutions or in embodiments of cell delivery solutions. Advantages of using cell delivery solutions include: excellent dose assurance, since the cells remain in suspension for extended periods of time; greater manufacturing and commercial relevance, since the formulation contains minimal amounts of pharmaceutical grade components and contains no components of human or animal origin; and ease of use, since the formulation can be stored under standard storage conditions and ready for use.

[0161] Example 5

[0162] In this example, a cell washing solution (CWS) of the present disclosure was evaluated as a component of a cell solution for injection (CSI). In this example, CWS and commercial solutions containing CSI were evaluated for delivery of two different concentrations of cells (about 75,000 cells / μL and about 20,000 cells / μL. The tests were performed in triplicate, and the data presented in this example are the average of these triplicate data.

[0163] Each of the CSI of this example and the commercially available solution was prepared at both a cell concentration of about 75,000 cells / μL and a cell concentration of about 20,000 cells / μL, where cell viability / live cell concentration and apoptosis were measured at about 0 hours and at 2°C-8°C after storage for about 6 hours.

[0164] Fig.13A Results at about 75,000 cells / μL stored in both the commercial solution and an embodiment of CWS are shown at about 0 hours and about 6 hours. Fig. 13B Results are shown for about 20,000 cells / μL stored in both the commercial solution and an embodiment of CWS at about 0 hours and about 6 hours. Fig.13A and 13B It can be seen that at both cell concentrations, the CWS of the present disclosure had a smaller decrease in viable cell concentration compared to the commercial solution.

[0165] Specifically, in Fig.13A In the present invention, at a cell concentration of about 75,000 cells / μL, after about 6 hours of storage time, there was about a 10.2% reduction in viable cell concentration of cells stored in the CWS of the present disclosure compared to about a 16.2% reduction in viable cell concentration of cells stored in the commercial solution. Fig. 13B In the present invention, at a cell concentration of about 20,000 cells / μL, after about 6 hours of storage time, there was about a 3.8% reduction in the viable cell concentration of cells stored in the CWS of the present disclosure compared to about an 18.7% reduction in the viable cell concentration of cells stored in the commercial solution.

[0166] In the same experiment, the cell viability of cells stored in CWS of the present disclosure compared to commercial solutions was determined, as Fig.14A and 14B shown.

[0167] Fig.14AResults are shown for cells stored at about 75,000 cells / μL and about 20,000 cells / μL in both commercial solutions and embodiments of CWS after thawing, and for cells stored for about 0 hours and about 6 hours. Fig.14A It can be seen that at both cell concentrations, the CWS of the present disclosure had comparable cell viability percentages to the commercial solution after about 6 hours, and maintained well over 70% cell viability after about 6 hours of storage.

[0168] Fig. 14B is a graphical representation of the total cell concentration of cells at an initial concentration of about 75,000 cells / μL stored for about 0 hours and about 6 hours in both the commercial solution and an embodiment of CWS. Fig. 14B It can be seen that the CWS of the present disclosure has a smaller reduction in total cell concentration (about 3.4%) compared to cells stored in the commercial solution (about 17%).

[0169] Morphological measurements were also performed for cells stored at about 0 hours and about 6 hours at 2° C.-8° C. for both about 75,000 cells / μL and about 25,000 cells / μL stored in both the commercial solution and the embodiment of CWS. This test was performed in triplicate with about 100,000 cells dispensed per 96-well plate.

[0170] Representative images were taken and the results are shown in Fig.15 In a series of photos. Fig.15 As shown, no visual differences were observed in cell morphology, and the morphology was as expected after storage for about 6 hours in both the CWS of the present disclosure and the commercial solution at both cell concentrations.

[0171] The embodiments and examples of the present disclosure described are intended to be illustrative rather than restrictive, and are not intended to show each embodiment or example of the present disclosure. Although the basic novel features of various specific embodiments applied to the present disclosure have been shown, described and pointed out, it will also be understood that, without departing from the spirit of the present disclosure, those skilled in the art may perform various omissions, substitutions and changes to the form and details of the composition shown and its operation. For example, it is explicitly considered that all combinations of those elements and / or method steps that perform substantially the same function in substantially the same manner to achieve the same result are within the scope of the present disclosure. In addition, it should be recognized that the structure and / or element and / or method step shown and / or described in conjunction with any disclosed form or embodiment of the present disclosure can be incorporated into any other disclosed or described or suggested form or embodiment as a general matter of design selection. In addition, without departing from the spirit or scope of the present disclosure as set forth in the appended claims, various modifications and changes may be made.

Claims

1. A cell delivery solution, comprising: one or more energy source components; one or more pH buffers; one or more salts; and One or more stabilizers, wherein the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer.

2. The solution of claim 1, wherein the one or more energy source components include sugar.

3. The method of claim 2, wherein the sugar is dextrose.

4. The solution of claim 1, wherein the one or more pH buffers comprise one or more zwitterionic organic chemical buffers.

5. The solution of claim 4, wherein the one or more zwitterionic organic chemical buffers comprise 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

6. The solution of claim 1, wherein the one or more salts are selected from the group consisting of calcium chloride, magnesium chloride, potassium chloride, sodium dihydrogen phosphate, and sodium chloride.

7. The solution of claim 1, wherein the solution does not comprise human serum albumin (HSA).

8. The solution of claim 1, wherein the solution does not contain any components of animal origin.

9. The solution of claim 1, wherein the solution does not contain one or more of zinc sulfate, sodium bicarbonate, and ferric nitrate.

10. The solution of claim 1, wherein the solution does not comprise a pH indicator.

11. The solution of claim 10, wherein the pH indicator is phenol red.

12. The solution of claim 1, wherein the solution does not comprise sodium pyruvate.

13. The solution of claim 1, wherein the solution does not comprise amino acids.

14. The solution of claim 1, wherein the solution does not contain vitamins.

15. The solution of claim 1, further comprising one or more excipients selected from the group consisting of polyethylene glycol, carboxymethyl cellulose, hyaluronic acid, starch, acrylates, methacrylates, polyvinyl alcohol, polyethylene oxide, polypropylene oxide, polyacrylates, polyvinyl pyrrolidone, polymethacrylates, polylactic-co-glycolic acid, polyacrylamide, polylactic acid, chitosan, gum, guar gum, xanthan gum, carrageenan, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, cyclodextrin derivatives, beta-cyclodextrin derivatives, alginates, calcium alginate, and stearates.

16. A cell suspension for injection, comprising: Multiple cells; one or more energy source components; one or more pH buffers; one or more salts; and One or more stabilizers, wherein the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer.

17. The suspension of claim 16, wherein the cells are differentiated midbrain dopaminergic neuronal cells.

18. The suspension of claim 17, wherein the differentiated midbrain dopaminergic neuron cells are differentiated midbrain dopaminergic neuron progenitor cells.

19. The suspension of claim 16, further comprising one or both of a cell washing buffer and a cryoprotectant.

20. The suspension of claim 19, wherein the cell washing buffer is selected from the group consisting of phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Bio-Plex Pro TM Cell Signaling Wash Buffer and Cultrex TM 3-D Cell Wash Buffer.

21. The suspension of claim 19, wherein the cryoprotectant is selected from the group consisting of: a permeating cryoprotectant and a non-permeating cryoprotectant.

22. The suspension of claim 21, wherein the permeating cryoprotectant is selected from the group consisting of dimethyl sulfoxide (DMSO), glycerol, propylene glycol, and ethylene glycol.

23. The suspension of claim 21, wherein the non-permeating cryoprotectant is selected from the group consisting of sucrose, carboxymethylcellulose salts, carboxymethylcellulose (CMC), monosaccharides, and disaccharides.

24. The suspension of claim 16, wherein the one or more energy source components comprise sugar.

25. The suspension of claim 24, wherein the sugar is dextrose.

26. The suspension of claim 16, wherein the one or more pH buffers comprise one or more zwitterionic organic chemical buffers.

27. The suspension of claim 26, wherein the one or more zwitterionic organic chemical buffers comprise 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

28. The suspension of claim 16, wherein the one or more salts are selected from the group consisting of calcium chloride, magnesium chloride, potassium chloride, sodium dihydrogen phosphate, and sodium chloride.

29. The suspension of claim 16, wherein the suspension does not comprise human serum albumin (HSA).

30. The suspension of claim 16, wherein the suspension does not contain any components of animal origin.

31. The suspension of claim 16, wherein the suspension does not contain one or more of zinc sulfate, sodium bicarbonate, and ferric nitrate.

32. The suspension of claim 16, wherein the solution does not comprise a pH indicator.

33. The suspension of claim 32, wherein the pH indicator is phenol red.

34. The suspension of claim 16, wherein the suspension does not comprise sodium pyruvate.

35. The suspension of claim 16, wherein the suspension does not comprise amino acids.

36. The suspension of claim 16, wherein the suspension does not contain vitamins.

37. The suspension of claim 16, further comprising one or more excipients selected from the group consisting of polyethylene glycol, carboxymethyl cellulose, hyaluronic acid, starch, acrylates, methacrylates, polyvinyl alcohol, polyethylene oxide, polypropylene oxide, polyacrylates, polyvinyl pyrrolidone, polymethacrylates, polylactic-co-glycolic acid, polyacrylamide, polylactic acid, chitosan, gums, guar gum, xanthan gum, carrageenan, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, cyclodextrin derivatives, beta-cyclodextrin derivatives, alginates, calcium alginate, and stearates.

38. A cell washing solution, the solution comprising: one or more energy source components; one or more pH buffers; one or more salts; and One or more stabilizers, wherein the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer.

39. The solution of claim 38, wherein the one or more energy source components include sugar.

40. The method of claim 39, wherein the sugar is dextrose.

41. The solution of claim 38, wherein the one or more pH buffers comprise one or more zwitterionic organic chemical buffers.

42. The solution of claim 41, wherein the one or more zwitterionic organic chemical buffers comprise 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

43. The solution of claim 38, wherein the one or more salts are selected from the group consisting of calcium chloride, magnesium chloride, potassium chloride, sodium dihydrogen phosphate, and sodium chloride.

44. The solution of claim 38, wherein the solution does not comprise human serum albumin (HSA).

45. The solution of claim 38, wherein the solution does not contain any components of animal origin.

46. ​​The solution of claim 38, wherein the solution does not comprise one or more of zinc sulfate, sodium bicarbonate, and ferric nitrate.

47. The solution of claim 38, wherein the solution does not comprise a pH indicator.

48. The solution of claim 47, wherein the pH indicator is phenol red.

49. The solution of claim 38, wherein the solution does not comprise sodium pyruvate.

50. The solution of claim 38, wherein the solution does not comprise amino acids.

51. The solution of claim 38, wherein the solution does not comprise vitamins.

52. The solution of claim 38, further comprising one or more excipients selected from the group consisting of polyethylene glycol, carboxymethyl cellulose, hyaluronic acid, starch, acrylates, methacrylates, polyvinyl alcohol, polyethylene oxide, polypropylene oxide, polyacrylates, polyvinyl pyrrolidone, polymethacrylates, polylactic-co-glycolic acid, polyacrylamide, polylactic acid, chitosan, gums, guar gum, xanthan gum, carrageenan, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, cyclodextrin derivatives, beta-cyclodextrin derivatives, alginates, calcium alginate, and stearates.

53. A cell delivery solution, the solution comprising: one or more energy source components, wherein the one or more energy source components include dextrose; one or more pH buffers, wherein the one or more pH buffers include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); one or more salts, wherein the one or more salts are selected from the group consisting of calcium chloride, magnesium chloride, potassium chloride, sodium dihydrogen phosphate, and sodium chloride; One or more stabilizers, wherein the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer.

54. The solution of claim 53, wherein the solution does not comprise one or more of the following components: components of animal origin; human serum albumin (HSA); zinc sulfate, sodium bicarbonate, and ferric nitrate; pH indicators; sodium pyruvate; amino acids; and vitamins.

55. The solution of claim 53, wherein the solution does not contain any of the following components: any components of animal origin, human serum albumin (HSA); zinc sulfate, sodium bicarbonate, and ferric nitrate; pH indicators; sodium pyruvate; amino acids; and vitamins.

56. The solution of claim 53, further comprising one or more excipients selected from the group consisting of polyethylene glycol, carboxymethyl cellulose, hyaluronic acid, starch, acrylates, methacrylates, polyvinyl alcohol, polyethylene oxide, polypropylene oxide, polyacrylates, polyvinyl pyrrolidone, polymethacrylates, polylactic-co-glycolic acid, polyacrylamide, polylactic acid, chitosan, gums, guar gum, xanthan gum, carrageenan, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, cyclodextrin derivatives, beta-cyclodextrin derivatives, alginates, calcium alginate, and stearates.

57. A cell suspension for injection, the suspension comprising: Multiple cells; one or more energy source components, wherein the one or more energy source components include dextrose; one or more pH buffers, wherein the one or more pH buffers include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); one or more salts, wherein the one or more salts are selected from the group consisting of calcium chloride, magnesium chloride, potassium chloride, sodium dihydrogen phosphate, and sodium chloride; One or more stabilizers, wherein the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer.

58. The suspension of claim 57, wherein the cells are differentiated midbrain dopaminergic neuronal cells.

59. The suspension of claim 58, wherein the differentiated midbrain dopaminergic neuronal cells are differentiated midbrain dopaminergic neuronal progenitor cells.

60. The suspension of claim 57, further comprising one or both of a cell washing buffer and a cryoprotectant.

61. The suspension of claim 60, wherein the cell washing buffer is selected from the group consisting of phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Bio-Plex Pro TM Cell Signaling Wash Buffer and Cultrex TM 3-D Cell Wash Buffer.

62. The suspension of claim 60, wherein the cryoprotectant is selected from the group consisting of: a permeating cryoprotectant and a non-permeating cryoprotectant.

63. The suspension of claim 62, wherein the permeating cryoprotectant is selected from the group consisting of dimethyl sulfoxide (DMSO), glycerol, propylene glycol, and ethylene glycol.

64. The suspension of claim 62, wherein the non-permeating cryoprotectant is selected from the group consisting of sucrose, carboxymethylcellulose salts, carboxymethylcellulose (CMC), monosaccharides, and disaccharides.

65. The suspension of claim 57, wherein the suspension does not comprise one or more of the following components: components of animal origin; human serum albumin (HSA); zinc sulfate, sodium bicarbonate, and ferric nitrate; pH indicators; sodium pyruvate; amino acids; and vitamins.

66. The suspension of claim 57, further comprising one or more excipients selected from the group consisting of polyethylene glycol, carboxymethyl cellulose, hyaluronic acid, starch, acrylates, methacrylates, polyvinyl alcohol, polyethylene oxide, polypropylene oxide, polyacrylates, polyvinyl pyrrolidone, polymethacrylates, polylactic-co-glycolic acid, polyacrylamide, polylactic acid, chitosan, gums, guar gum, xanthan gum, carrageenan, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, cyclodextrin derivatives, beta-cyclodextrin derivatives, alginates, calcium alginate, and stearates.

67. A cell washing solution, the solution comprising: one or more energy source components, wherein the one or more energy source components include dextrose; one or more pH buffers, wherein the one or more pH buffers include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); one or more salts, wherein the one or more salts are selected from the group consisting of calcium chloride, magnesium chloride, potassium chloride, sodium dihydrogen phosphate, and sodium chloride; One or more stabilizers, wherein the one or more stabilizers include recombinant albumin (rHSA).

68. The solution of claim 67, wherein the solution does not comprise one or more of the following components: components of animal origin; human serum albumin (HSA); zinc sulfate, sodium bicarbonate, and ferric nitrate; pH indicators; sodium pyruvate; amino acids; and vitamins.

69. The solution of claim 67, further comprising one or more excipients selected from the group consisting of polyethylene glycol, carboxymethyl cellulose, hyaluronic acid, starch, acrylates, methacrylates, polyvinyl alcohol, polyethylene oxide, polypropylene oxide, polyacrylates, polyvinyl pyrrolidone, polymethacrylates, polylactic-co-glycolic acid, polyacrylamide, polylactic acid, chitosan, gums, guar gum, xanthan gum, carrageenan, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, cyclodextrin derivatives, beta-cyclodextrin derivatives, alginates, calcium alginate, and stearates.

70. A method of suspending a plurality of cells in a cell solution, the method comprising: adding the cell solution to the container of the plurality of cells, wherein the cell solution comprises: one or more energy source components; one or more pH buffers; one or more salts; and one or more stabilizers, wherein the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer; mixing the plurality of cells and the cell solution to form a cell suspension for injection; and Wait for the scheduled time.

71. The method of claim 70, further comprising removing a portion of the cell suspension for injection from the container.

72. The method of claim 70, wherein the predetermined time is from about 8 hours to about 96 hours.

73. The method of claim 70, wherein the predetermined time is about 24 hours, about 36 hours, about 48 hours, or about 96 hours.

74. The method of claim 70, wherein the plurality of cells has a dose sampling variability of less than about 25% in the container.

75. The method of claim 70, wherein the cell is a differentiated midbrain dopaminergic neuronal cell.

76. A method of treating a subject, the method comprising: A dose of a cell suspension for injection (CSI) is administered, the CSI comprising: Multiple cells one or more energy source components; one or more pH buffers; one or more salts; and One or more stabilizers, wherein the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer.

77. The method of claim 76, wherein the cell is a differentiated midbrain dopaminergic neuronal cell.

78. The method of claim 76, wherein the plurality of cells comprises about 10 0 cells to about 10 10 The range of cells.

79. The method of claim 76, wherein the plurality of cells comprises about 10 8 cells to about 10 10 The range of cells.

80. The method of claim 76, further comprising the steps of: a. Wait for the scheduled time; and b. Administering another dose of the CSI.

81. The method of claim 80, further comprising repeating steps a. and b. two, three, four or more times.

82. The method of claim 76, further comprising a concurrent or subsequent step of administering a therapy selected from the group consisting of a chemotherapeutic agent, radiation therapy, bone marrow therapy, hormone therapy, and surgical therapy.

83. A method of administering a dose volume, the method comprising: The dose delivery device is loaded with the dose volume of a cell suspension for injection (CSI), the CSI comprising: Multiple cells; one or more energy source components; one or more pH buffers; one or more salts; and one or more stabilizers, wherein the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer; and The dose volume is injected into a portion of the subject.

84. The method of claim 83, wherein the cell is a differentiated midbrain dopaminergic neuronal cell.

85. The method of claim 83, wherein the plurality of cells comprises about 10 0 cells to about 10 10 The range of cells.

86. The method of claim 83, wherein the plurality of cells comprises about 10 8 cells to about 10 10 The range of cells.

87. The method of claim 83, further comprising a concurrent or subsequent step of administering a therapy selected from the group consisting of a chemotherapeutic agent, radiation therapy, bone marrow therapy, hormone therapy, and surgical therapy.

88. The method of claim 83, wherein the subject is a human.

89. The method of claim 83, wherein the injecting step occurs directly into a portion of the subject.

90. The method of claim 83, wherein the injecting step occurs in an intravenous line, wherein the intravenous line is in a blood vessel of the subject.

91. The method of claim 83, wherein the dose delivery device is a syringe.

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