Cell delivery medium and method of using the same

Abstract

The present disclosure is directed to various compositions comprising one or more energy source components; one or more pH buffers; one or more salts; and one or more stabilizers as well as one or more cell types.

Description

Background Art

[0001] Various biological cells and / or tissues are stored in various materials such as cryoprotective substances at freezing temperatures for various times. These cryoprotective substances used during storage should be removed or substantially removed from the biological cells and / or tissues after or during the thawing process so that the biological cells and / or tissues can be utilized.

[0002] Generally, a container containing biological cells and / or tissues is taken out of a freezing temperature storage, thawed, and the contents of the container are "washed" or diluted with a cell washing solution so that some, most, or all of the cryoprotective substance is removed. Typical cell "washing" solutions are: one or more amino acids such as glycine, L-alanine, L-arginine hydrochloride, L-asparagine-H 2 O, L-glutamine, L-cysteine, L-histidine hydrochloride-H 2 O, 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 such as albumin, e.g., human serum albumin; one or more vitamins such as ascorbic acid, choline chloride, calcium D-pantothenate, folic acid, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and i-inositol; and calcium chloride (CaCl 2 ), ferric nitrate (Fe(NO 3 )3-9H 2 O), magnesium chloride (MgCl), potassium chloride (KCl), sodium bicarbonate (NaHCO 3 ), sodium chloride (NaCl), sodium dihydrogen phosphate (NaH 2 PO 4 -H 2(O) and zinc sulfate (ZnSO 4 -7H 2 O) and has many components including but not limited to one or more salts such as

[0003] After dilution with the cell washing solution, the container can undergo one or more precipitation procedures such as centrifugation or precipitation over time. The supernatant can be completely or partially removed from the container such that all or most of the cells remain. The process can then be repeated one or more times with the precipitation procedure after adding the cell washing solution to the container again.

[0004] After the cells have been "washed", the remaining biological cells and / or tissues, which can be in the same storage container or may have been transferred to a different container, are resuspended in a cell delivery solution (transplantation medium), whereby the biological cells and / or tissues can be used for any required procedure. Generally, this cell delivery solution has some similarities in composition to the cell washing solution, and the cell delivery solution has components at the same or different concentrations or additional components compared to the cell washing solution.

[0005] Desirable are cell washing solutions and / or cell delivery solutions that have an appropriate shelf life at standard storage temperatures, contain little or no components of human or mammalian origin, have a substantially stable pH value for days, weeks or longer, have a substantially physiological osmolarity concentration, and maintain cell health better than commercially available solutions. In addition, the cell delivery solution composition can be designed to sufficiently prevent cell precipitation and make it possible to guarantee an accurate dosage during preparation and administration. This guarantee and delivery of an accurate dosage can be the result of the ability of the cell delivery solution to maintain the cells in a substantially suspended state for 8 hours or longer.

[0006] Embodiments of the present disclosure provide solutions and methods that address the above needs.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Document

[0008]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0009] The present disclosure is directed to various formulations or compositions comprising one or more energy source components; one or more pH buffers; one or more salts; and one or more stabilizers. The present disclosure is also directed to cell wash solutions (CWS), cell delivery solutions (CDS), and cell suspensions for injection (CSI). The cell suspension for injection provides cells in a suspended state for several hours while maintaining the health of the cells at a sufficient level.

[0010] This patent or application file contains at least one drawing created in color. Copies of this patent or patent application publication, including the color drawings, will be provided by the Office upon request and payment of the necessary fee.

[0011] The present disclosure will be better understood by reference to the following drawings, which are provided as examples of specific embodiments of the subject application, and are not meant to limit the scope of the present disclosure.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5A

Figure 5B

Figure 5C

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11A

Figure 11B

Figure 12

Figure 13A

Figure 13B

Figure 14A

Figure 14B

Figure 15

Best Mode for Carrying Out the Invention

[0013] In the discussion and claims of this specification, the term "about" indicates that the recited value may be changed somewhat, provided that no incompatibility of the process results from the change. For example, for some elements, the term "about" can refer to a variation of ±0.1%, and for other elements, the term "about" can refer to a variation of ±1% or ±10%, or any point therebetween.

[0014] In this specification, the terms "substantially" or "substantial" are equally applicable when used with a negative connotation to refer to a complete or almost complete absence of an action, feature, characteristic, state, structure, item, or result. For example, a "substantially" flat surface would be either completely flat or almost flat, such that its effect would be the same as if it were completely flat.

[0015] In this specification, terms such as "a", "an", and "the" do not refer only to single entities, but also include general classes exemplified by specific instances.

[0016] In this specification, terms defined in the singular are intended to include terms defined in the plural, and vice versa.

[0017] References to "one embodiment", "certain embodiments", "some embodiments" or "embodiments" in this specification indicate that the described embodiments may include certain features or characteristics, but not every embodiment necessarily includes the specific features, structures or characteristics. Further, such phrases do not necessarily refer to the same embodiment. Further, when a specific feature, structure or characteristic is described in relation to an embodiment, it is considered within the knowledge of those skilled in the art that such feature, structure or characteristic may affect other embodiments, whether or not explicitly described. For the purposes of the following description, the terms "upper", "lower", "right", "left", "vertical", "horizontal", "uppermost", "lowermost" and their derivatives are to be taken in relation to the orientation of the drawings as relevant to the present invention. The terms "lying on", "on top of", "located on" or "positioned on top of" mean that a first element is present above a second element, with intervening elements continuous between the first and second elements. The terms "in direct contact" or "attached to" mean that a first element and a second element are connected without any intermediate element at the interface of the two elements.

[0018] Any reference to a numerical range in this specification explicitly includes each numerical value (including fractions and integers) encompassed by that range. By way of example, a reference in this specification to a range of "at least 50" or "at least about 50" includes the integers 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and the fractions 50.1, 50.2, 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc. In a further example, a reference in this specification to a range of "less than 50" or "less than about 50" includes the integers 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, etc. and the fractions 49.9, 49.8, 49.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1, 49.0, etc.

[0019] As used herein, the term "stabilizer" refers to any component that may act to reduce or prevent the decomposition of other solution components.

[0020] As used herein, the term "energy source" refers to any component capable of providing chemical energy to one or more cells.

[0021] As used herein, the term "pH indicator" refers to any component or substance that changes its properties in response to a change in pH. Such changes in properties include changes in optical properties such as color change.

[0022] As used herein, the term "vitamin" refers to various fat-soluble or water-soluble organic substances that can be obtained naturally from plant and animal foods or produced synthetically [non-limiting examples include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or nicotinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid), and vitamin B12 (various cobalamins; generally 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 any of its provitamins, derivatives, and / or analogs.

[0023] As used herein, the term "cell" can refer to any one or more types of cells noted in this or any other paragraph of the present disclosure, or alternatives and / or equivalents thereof. In some embodiments, the cell is a nerve cell. The cell can be derived from embryonic stem cells or induced pluripotent stem cells. In some embodiments, the cell can be 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.

[0024] In some embodiments, "cell" can refer to floor plate-derived DA neurons from floor plate midbrain progenitor cells. As used herein, the terms "floor plate-derived DA neurons" or "authentic 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" refer to a population of transplantable midbrain DA neurons obtained by any suitable method.

[0025] As used herein, the 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 hESCs and non-embryonic hiPSCs, somatic stem cells, disease stem cells, i.e., isolated pluripotent cells, and engineered stem cells isolated from patients with Parkinson's disease, cancer stem cells, human or mammalian pluripotent cells, etc. The floor plate midbrain progenitor cells and midbrain fate FOXA2 / LMX1A+ dopamine (DA) neurons, or these cells for obtaining differentiated midbrain dopamine-activating neurons, "DMD" cells, can also be the "cells" of the present disclosure.

[0026] As used herein, the term "stem cell" refers to a cell that has the ability to divide indefinitely in culture and give rise to specialized cells. Stem cells can be obtained from patients including animals and humans; for example, human stem cells refer to stem cells that are human. Stem cells can be obtained from a variety of sources including embryonic as well as non-embryonic sources such as umbilical cord cells, child-derived cells, and adult-derived cells. In the context of the present invention, adult stem cells generally refer to cells that are not originally obtained from a fetus, in other words, infant-derived cells, discarded umbilical cords, discarded placenta cells, pediatric-derived cells, adult-derived cells, etc.

[0027] As used herein, the term "umbilical cord blood stem cell" refers to stem cells collected from the umbilical cord at birth and having the ability to give rise to at least all blood cells in the body (hematopoiesis).

[0028] As used herein, the term "somatic (adult) stem cell" refers to relatively rare undifferentiated cells found in many organs and differentiated tissues and having limited capabilities for both self-renewal (in the laboratory) and differentiation. Such cells have different differentiation capabilities, but those capabilities are usually limited to cell types in the original organ. As used herein, the term "somatic cell" refers to any cell of the body except sperm and eggs. Somatic cells are diploid, which means they contain two sets of chromosomes, one 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.

[0029] As used herein, the term "allogeneic cell" or "allogeneic stem cell" refers to cells that belong to the same species but are obtained from genetically different individuals. The allogeneic stem cells of the present disclosure are derived from either a person other than the patient, a compatible related donor, or an unrelated donor.

[0030] As used herein, the term "neural lineage cell" refers to cells that contribute to the fate of the nervous system (both the central and peripheral nervous systems) or neural crest cells during development or in the adult. The nervous system includes the brain, spinal cord, and peripheral nervous system. The fate of neural crest cells includes the head, trunk, vagal, sacral, and cardiac, and gives rise to ectodermal mesenchyme, head cartilage, skull bone, thymus, teeth, melanocytes, iridial pigment cells, cranial ganglia, dorsal root ganglia, sympathetic / parasympathetic ganglia, endocrine cells, enteric nervous system, and parts of the heart.

[0031] As used herein, the term "adult stem cell" refers to somatic stem cells, and in one example, "hematopoietic stem cells" that give rise to all red blood cells, white blood cells, and platelets in infants, children, and adults.

[0032] As used herein, the term "embryonic stem cell" refers to a primitive (undifferentiated) cell, and is obtained from one of several sources including, but not limited to, pre-implantation embryos, artificial embryos created by in vitro fertilization, etc., and is capable of dividing in culture without differentiating for a long period of time and is known to have the ability to develop into cells and / or tissues of the three primary germ layers, the ectoderm, mesoderm, and endoderm.

[0033] As used herein, the term "endoderm" refers to the layer of cells obtained from the inner cell mass of a blastocyst; it has the ability to give rise to various cell types in vivo in the lungs, other respiratory structures, and the digestive tract or generally the "gut" and in vitro.

[0034] As used herein, the term "embryonic stem cell line" refers to a population of embryonic stem cells cultured for several days, months, or years under in vitro conditions that can proliferate without differentiating, such as the cells of the human WA-09 cell line.

[0035] As used herein, the term "human embryonic stem cell" or "hESC" refers to a type of pluripotent stem cell obtained from an early human embryo, including up to the blastocyst stage, and is capable of dividing in culture without differentiating for a long period of time and is known to be able to develop into cells and tissues of the three primary germ layers, the ectoderm, mesoderm, and endoderm.

[0036] As used herein, the term "pluripotent stem cell" or "PSC" has its ordinary meaning to those skilled in the art, i.e., a self-renewing cell having the ability to develop into endoderm, ectoderm, and mesoderm cells. As used herein, PSC includes "genetically edited or modified PSC". In some embodiments, the PSC is a human PSC. PSCs include embryonic stem cells (ESCs) and induced pluripotent stem cells ("iPS cells" or "iPSCs" or "hiPSCs"). The terms ES cells and iPS cells have their ordinary meaning to those skilled in the art. As used herein, "genetically edited or modified PSC" includes cells in which the aEF1a-IDUA overexpression cassette has been engineered within the AAVS1 safe harbor locus in human induced pluripotent stem cells (hiPSCs).

[0037] As used herein, the term "non-pluripotent stem cell" has its ordinary meaning to those skilled in the art, i.e., the cell does not have the potential to differentiate into all three germ layers (i.e., endoderm, ectoderm, and mesoderm). Examples of such cells include umbilical cord blood stem cells and epidermal stem cells.

[0038] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a type of pluripotent stem cell similar to embryonic stem cells, which is reprogrammed so that somatic cells (adults) are forced to express factors important for maintaining the "stemness" of embryonic stem cells (ESCs) and enter an embryonic stem cell-like state. Mouse iPSCs were reported in 2006 (Takahashi and Yamanaka), and human iPSCs were reported in late 2007 (Takahashi et al. and Yu et al.). Mouse iPSCs demonstrate important characteristics 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 a very early stage of development. Human iPSCs also express stem cell markers and can generate cells characteristic of all three germ layers. Different from embryonic stem cells, iPSCs are artificially formed by introducing specific embryonic genes (such as introduced genes of OCT4, SOX2, and KLF4) [see, for example, Takahashi and Yamanaka, Cell 126, pp. 663-676 (2006), which is incorporated herein by reference] into somatic cells of cell lines derived from, for example, inducible cells C14, C72, etc. Another example of iPSCs is adult human skin cells or fibroblasts transformed by using genes (OCT4, SOX2, NANOG, LIN28, and KLF4) cloned into plasmids [see, for example, Yu et al., Science DOI: 10.1126 / science.1172482, which is incorporated herein by reference].

[0039] As used herein, the term "totipotency" refers to the ability to give rise to all cell types of the body + all cell types that make up extra-embryonic tissues such as the placenta.

[0040] As used herein, the term "plurality" refers to the ability to develop into two or more cell types of the body.

[0041] As used herein, the term "pluripotent" refers to cells that have the ability to give rise to at least two, but often many different cell types of the body. Pluripotent cells often form teratomas after injection into immunosuppressed mice.

[0042] As used herein, the term "specialized cell" refers to a type of cell that performs a specific function in a multicellular organism. For example, groups of specialized cells such as neurons act together to form systems such as the nervous system.

[0043] As used herein, the term "neuroectoderm" refers to cells or cell fates that are seen during early development or during the differentiation of pluripotent stem cells that can give rise to cells of the nervous system.

[0044] However, it should be recognized that the composition may contain, alone or in combination, other types of "cells" such as mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells or induced pluripotent stem cells, red blood cells, 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, heart tissue, chondrocytes, osteocytes, glial restricted progenitors, astrocytes, oligodendrocytes, neuroblasts, megakaryoblasts, megakaryocytes, monoblasts, monocytes, macrophages, myeloid cells, spinal cord dendritic cells, microglial cells, differentiated microglial cells, microglial progenitor cells, proerythroblasts, erythroblasts, normoblasts, reticulocytes, platelets, myeloblasts, promyelocytes, neutrophilic myelocytes, neutrophilic band cells, neutrophils, eosinophilic myelocytes, eosinophilic band cells, eosinophils, basophilic myelocytes, basophilic band cells, basophils, lymphoid progenitors, pre-NK cells, NK lymphoblasts, NK cells, thymocytes, T-lymphoblasts, T cells, plasmacytoid dendritic cells, pre-B cells, B lymphoblasts, B cells, plasma cells, osteoblasts, chondrocytes, myoblasts, myotubes, fibroblasts, adipocytes, mesoderm, ectoderm, primordial germ cells, sperm, eggs, embryonic endoderm, heart cells or any other suitable type of cells, etc. Heart 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 lymphoid), adipocytes, mesothelial cells and neuron cells.

[0045] As used herein, the term "microglial progenitor cells" refers to PSC-derived myeloid cells.

[0046] As used herein, the term "microglia" refers to PSC-derived myeloid cells.

[0047] Furthermore, "cell" can refer to myeloid cells concentrated in microglial cells obtained from unedited human induced pluripotent stem cells (hiPSC). As used herein, the term "myeloid cell" refers to PSC-derived cells and / or non-PSC-derived cells. PSC-derived cells are differentiated after hematopoietic differentiation and express spinal cord markers (including but not limited to CD45, CD11b, CD33, CD14, CX3CR1) and can perform normal spinal cord functions including but not limited to phagocytosis, response to external stimuli, cytokine secretion, and polarization into pro-inflammatory or anti-inflammatory states. These myeloid cells will become tissue-resident macrophages upon delivery to different organs of a living organism. For example, when delivered to the brain of a living animal, they will become microglia and express standard microglial markers including but not limited to TMEM119, IBA1, CD163, CX3CR1, CD45, CD206. The payload of these cells can be filled by supplementing the missing lysosomal enzymes and can have no genetic modification for containing any additional payload.

[0048] In addition to any of the cells noted above as "cells" of the present disclosure, "cell" can refer to non-living biologic agents including but not limited to endosomes and lipid-based vesicles. Further in addition to any of the cells noted above as "cells" of the present disclosure, "cell" can refer to any cells and / or tissues disclosed in U.S. Patent No. 10,280,398, U.S. Patent No. 10,711,243, and International Publication No. 2013 / 067362, the respective contents of which are incorporated herein by reference in their entireties.

[0049] As used herein, the term "cell wash solution" or "CWS" is any solution that is added to a container containing cells and cryoprotectant after the container has been 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 freeze-thaw process, which necessarily involves ice crystal formation and imbalances in ions and osmotic pressure when cells and / or tissues are stored at temperatures below 0°C. Cryoprotectants are not limited to specific substances as long as they can reduce cell damage during storage below 0°C. Examples thereof include permeating cryoprotectants such as dimethyl sulfoxide (DMSO), glycerin, propylene glycol, ethylene glycol, etc., or non-permeating cryoprotectants such as sucrose, carboxymethylcellulose salts, carboxymethylcellulose (CMC), monosaccharides, disaccharides, etc., but are not limited thereto.

[0050] As used herein, the term "cell delivery solution" or "CDS" is any solution that is added to a container containing non-washed cells (directly to thawed cells) or washed cells so that the cells can be administered to a subject. The cell delivery solution may minimally or trace contain a cryoprotectant and / or a cell wash solution, and the cryoprotectant and / or the cell wash solution are those that the cells have been stored and / or washed with before contacting the cell delivery solution and have not been completely removed from the container after the supernatant discard process, which may occur after any centrifugation step of the container that may optionally form a cell pellet or concentrated cell solution. This supernatant discard process reduces and / or removes the concentration of components of the storage sample that are not desirable for injection, such as cryoprotectants. The cell delivery solution can be used to reconstitute an injectable cell solution (CSI) after thawing the cells before administration for clinical use.

[0051] As used herein, the term "cell suspension for injection" or "CSI" refers to either or both of a cell delivery solution (CDS) and / or a cell wash solution (CWS) combined with one or more cells and one or more optional additional components. The cell suspension for injection can contain additional components such as, for example, a composition containing phosphate buffered saline (PBS), a composition containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Bio-Plex Pro™ Cell Signaling Wash Buffer, and Cultrex™ 3-D Cell Wash Buffer, such as a cell wash buffer. The cell suspension for injection may contain trace amounts of cryoprotective substances and / or cell wash solutions that were not completely removed from the container after the supernatant discard process. However, the possible trace amounts of cryoprotective substances and / or cell wash solutions do not modify the effectiveness of the cell suspension for injection.

[0052] The cell delivery solution and / or the cell wash solution can be used at a manufacturing site that creates a CSI, which is a composition or product, for example, to be administered to a subject, such as by injection. The manufacturing site can receive a plurality of cells, "wash" the cells, mix the cell delivery solution and / or the cell wash solution with the cells, and then prepare the cells for administration, such as by injection, to the subject as a CSI. Injectable types contemplated by the present disclosure include, but are not limited to, intracerebroventricular (ICV), intravenous (IV), intramuscular (IM), and intrathecal (IT). The manufacturing site can perform these steps under a sterile environment and appropriate environmental conditions. Further, the manufacturing site can perform quality control tests on the cells at any point in their preparation prior to leaving the manufacturing site to determine whether the cells are sufficiently viable.

[0053] The present disclosure provides a “Cell Wash Solution (CWS)” and a “Cell Delivery Solution (CDS)”, either or both of which may be referred to as a “Cell Solution”. Both the cell wash solution and the cell delivery solution are formulated to have 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 concentration of about 100 to about 700 mOsm / L, or an osmolarity concentration of about 150 to about 500 mOsm / L, or an osmolarity concentration of about 200 to about 500 mOsm / L, or an osmolarity concentration of about 225 to about 400 mOsm / L, or an osmolarity concentration of about 250 to about 350 mOsm / L, or an osmolarity concentration of about 270 to about 325 mOsm / L, or an osmolarity concentration of about 280 to about 300 mOsm / L, and (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, 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, relatively low viscosity, and good cell compatibility such that both the cell wash solution and the cell delivery solution are substantially non-cytotoxic. Both the cell wash solution and the cell delivery solution are configured to have a sufficient shelf life under typical or standard storage conditions and to be immediately available for clinical applications.

[0054] These qualities can potentially affect the ability of the cell delivery solution to maintain the cells in a suspended state. As used herein, the term "suspension" refers to cells dispersed within a liquid. In the present disclosure, cells dispersed within a liquid can continue to be dispersed for 0 to 104 hours or longer. The shelf life of cells dispersed or suspended within 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 maintains cells dispersed within the cell delivery solution 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 longer, either without agitation (mixing) and / or homogenization or after agitation (mixing) and / or homogenization.

[0055] 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. Some components of both the cell washing solution and the cell delivery solution can be combined by any suitable method, such as the methods discussed in the following examples, to reach either the cell washing solution or the cell delivery solution. 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) prior to contact with any cells.

[0056] Any one or more energy source components of the cell washing solution or the cell delivery solution can contain any suitable sugar such as dextrose, fructose, galactose, glucose, lactose, maltose, sucrose, etc. Any of the cell washing solution or the cell delivery solution can contain an energy source in any amount that achieves the desired effect, such as about 24.5 mM to about 24.8 mM, about 24.4 mM to about 24.9 mM, about 24.3 mM to about 25.0 mM, about 24.1 mM to about 25.2 mM, about 23.9 mM to about 25.4 mM, about 23.7 mM to about 25.6 mM, or about 23.5 mM, or about 23 mM, or about 22 mM, or about 21 mM, or less than about 20 mM, or about 26 mM, or about 27 mM, or about 28 mM, or about 29 mM, or about 30 mM or more, but not limited thereto.

[0057] Any one or more pH buffers of the cell washing solution or the cell delivery solution can contain any suitable buffer such as zwitterionic organic chemical buffers. Examples thereof include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium bicarbonate, 4-morpholinepropanesulfonic acid, 3-propanesulfonic acid (MOPS), and 2-(N-morpholino)ethanesulfonic acid (MES), but not limited thereto. Any of the cell washing solution or the cell delivery solution can contain a pH buffer in any amount that achieves the desired effect, such as about 10.6 mM to about 10.9 mM, about 10.5 mM to about 11.0 mM, about 10.4 mM to about 11.1 mM, about 10.2 mM to about 11.3 mM, about 10.0 mM to about 11.5 mM, about 9.8 mM to about 11.7 mM, or about 9.5 mM, or about 9 mM, or about 8 mM, or about 7 mM, or less than about 6 mM, or higher than about 12 mM, or about 13 mM, or about 14 mM, or about 15 mM, or about 16 mM, but not limited thereto.

[0058] Any one or more salts of the cell washing solution or the cell delivery solution can include any suitable salts, such as one or more salts selected from the group consisting of calcium chloride, magnesium chloride, potassium chloride, monosodium phosphate, and sodium chloride.

[0059] Either the cell washing solution or the cell delivery solution can contain any amount of calcium chloride, not limited to this, for example, 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 less than about 0.1 mM, or about 3 mM, or about 3.5 mM, or about 4 mM, or 5 mM or more, to achieve the desired effect.

[0060] Either the cell washing solution or the cell delivery solution can contain any amount of magnesium chloride, not limited to this, for example, 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 less than about 0.05 mM, or about 2.5 mM, or about 3.0 mM or more, to achieve the desired effect.

[0061] Either the cell washing solution or the cell delivery solution can contain any amount of potassium chloride, which is not limited to, but can be, for example, from about 5.1 mM to about 5.4 mM, or from about 5.0 mM to about 5.5 mM, or from about 4.9 mM to about 5.7 mM, or from about 4.8 mM to about 5.8 mM, or from about 4.6 mM to about 6.0 mM, or from about 4.4 mM to about 6.2 mM, or from about 4.2 mM to about 6.4 mM, or about 4.0 mM, or about 3.5 mM, or less than about 3.0 mM, or about 6.5 mM, or about 7.0 mM, or about 7.5 mM, or about 8.0 mM or more, to achieve the desired effect.

[0062] Either the cell washing solution or the cell delivery solution can contain any amount of monosodium phosphate, which is not limited to, but can be, for example, from about 0.88 mM to about 0.91 mM, or from about 0.87 mM to about 0.92 mM, or from about 0.86 mM to about 0.93 mM, or from about 0.85 mM to about 0.94 mM, or from about 0.83 mM to about 0.96 mM, or from about 0.81 mM to about 0.98 mM, or about 0.8 mM, or about 0.75 mM, or less than about 0.7 mM, or about 1.0 mM, or about 1.05 mM, or about 1.1 mM or more, to achieve the desired effect.

[0063] In one embodiment, either the cell washing solution or the cell delivery solution can contain any amount of sodium chloride, which is not limited to, but can be, for example, from about 119 mM to about 122 mM, or from about 118 mM to about 123 mM, or from about 117 mM to about 124 mM, or from about 115 mM to about 126 mM, or from about 113 mM to about 128 mM, or about 110 mM, or about 105 mM, or less than about 100 mM, or about 130 mM, or about 135 mM, or about 140 mM or more, to achieve the desired effect.

[0064] In another embodiment, either the cell washing solution or the cell delivery solution can contain any amount of sodium chloride, not limited to, but including, for example, about 74 mM to about 77 mM, or about 73 mM to about 78 mM, or about 72 mM to about 79 mM, or about 70 mM to about 81 mM, or about 68 mM to about 83 mM, or about 66 mM to about 85 mM, or about 65 mM, or about 60 mM, or less than about 55 mM, or about 90 mM, or about 95 mM, or about 100 mM or more, to achieve the desired effect.

[0065] In another embodiment, either the cell washing solution or the cell delivery solution can contain any amount of sodium chloride, not limited to, but including, for example, about 93 mM to about 96 mM, or about 92 mM to about 97 mM, or about 91 mM to about 98 mM, or about 90 mM to about 99 mM, or about 88 mM to about 101 mM, or about 85 mM to about 103 mM, or about 83 mM to about 105 mM, or about 80 mM, or about 75 mM, or less than about 70 mM, or about 105 mM, or about 110 mM, or about 115 mM or more, to achieve the desired effect.

[0066] One or more stabilizers of either the cell washing solution or the cell delivery solution can include any suitable stabilizer such as one or more proteins like one or more albumins, recombinant albumin (rHSA), dextran (including dextran 40 as an example), poloxamer (including poloxamer 188 as an example), etc. Further, one or more stabilizers can include the following: polyethylene glycol, carboxymethyl cellulose, hyaluronic acid, starch, acrylate, methacrylate, polyvinyl alcohol, polyethylene oxide, polypropylene oxide, polyacrylate, polyvinyl pyrrolidone, polymethacrylate, poly lactic - co - glycolic acids, polyacrylamide, polylactic acid, chitosan, rubber, guar gum, xanthan gum, carrageenan, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, cyclodextrin derivatives, beta - cyclodextrin derivatives, alginates, calcium alginate, and stearates, and they can also be excipients of either the cell washing solution or the cell delivery solution.

[0067] In one embodiment, either the cell washing solution or the cell delivery solution can contain a protein such as recombinant albumin (rHSA) or one or more albumins in any amount, for example, from about 0.07 mass / mass% to about 0.09 mass / mass%, or from about 0.06 mass / mass% to about 0.1 mass / mass%, or from about 0.05 mass / mass% to about 0.11 mass / mass%, or from about 0.04 mass / mass% to about 0.12 mass / mass%, or from about 0.03 mass / mass% to about 0.13 mass / mass%, or from about 0.02 mass / mass% to about 0.15 mass / mass%, or from about 0.01 mass / mass% to about 0.17 mass / mass%, or less than about 0.005 mass / mass%, or about 0.2 mass / mass%, or about 0.25 mass / mass% or more, but not limited to these amounts, to achieve the desired effect.

[0068] In another embodiment, either the cell washing solution or the cell delivery solution may contain one or more proteins such as recombinant albumin (rHSA) in an amount of about 0.08 mass / mass% to about 0.11 mass / mass%, or about 0.07 mass / mass% to about 0.12 mass / mass%, or about 0.06 mass / mass% to about 0.13 mass / mass%, or about 0.05 mass / mass% to about 0.14 mass / mass%, or about 0.04 mass / mass% to about 0.16 mass / mass%, or about 0.03 mass / mass% to about 0.18 mass / mass%, or about 0.02 mass / mass% to about 0.2 mass / mass%, or about 0.01 mass / mass% to about 0.22 mass / mass%, or less than about 0.05 mass / mass%, or about 0.25 mass / mass%, or about 0.3 mass / mass%, or about 0.35 mass / mass% or more, but can include any amount not limited thereto to achieve the desired effect.

[0069] In another embodiment, either the cell washing solution or the cell delivery solution may contain one or more proteins such as recombinant albumin (rHSA) in an amount of about 6.50 mass / mass% to about 6.8 mass / mass%, or about 6.4 mass / mass% to about 6.9 mass / mass%, or about 6.3 mass / mass% to about 7.0 mass / mass%, or about 6.1 mass / mass% to about 7.2 mass / mass%, or about 5.9 mass / mass% to about 7.4 mass / mass%, or about 5.7 mass / mass% to about 7.6 mass / mass%, or about 5.5 mass / mass%, or about 5.0 mass / mass%, or less than about 4.5 mass / mass%, or about 8.0 mass / mass%, or about 8.5 mass / mass%, or about 9.0 mass / mass% or more, but can include any amount not limited thereto to achieve the desired effect.

[0070] In one embodiment, either the cell washing solution or the cell delivery solution can contain any amount of dextran, not limited thereto, to achieve the desired effect, such as from about 17.27 mass / mass% to about 17.30 mass / mass%, or from about 17.26 mass / mass% to about 17.31 mass / mass%, or from about 17.24 mass / mass% to about 17.33 mass / mass%, or from about 17.2 mass / mass% to about 17.35 mass / mass%, or from about 17.1 mass / mass% to about 17.4 mass / mass%, or from about 17.0 mass / mass% to about 17.5 mass / mass%, or about 16.5 mass / mass%, or about 16.0 mass / mass%, or less than about 15.0 mass / mass%, or about 18.0 mass / mass%, or about 18.5 mass / mass%, or about 19.0 mass / mass% or more.

[0071] In one embodiment, either the cell washing solution or the cell delivery solution can contain any amount of dextran, not limited thereto, to achieve the desired effect, such as from about 13.02 mass / mass% to about 13.05 mass / mass%, or from about 13.0 mass / mass% to about 13.1 mass / mass%, or from about 12.9 mass / mass% to about 13.2 mass / mass%, or from about 12.7 mass / mass% to about 13.4 mass / mass%, or from about 12.5 mass / mass% to about 13.5 mass / mass%, or from about 12.2 mass / mass% to about 13.8 mass / mass%, or about 12.0 mass / mass%, or about 11.5 mass / mass%, or less than about 11.0 mass / mass%, or about 14.0 mass / mass%, or about 14.5 mass / mass%, or about 15.0 mass / mass% or more.

[0072] In one embodiment, either the cell washing solution or the cell delivery solution can contain any amount of poloxamer, for example, from about 0.07 mass / mass% to about 0.09 mass / mass%, or from about 0.06 mass / mass% to about 0.1 mass / mass%, or from about 0.05 mass / mass% to about 0.11 mass / mass%, or from about 0.04 mass / mass% to about 0.12 mass / mass%, or from about 0.03 mass / mass% to about 0.13 mass / mass%, or from about 0.02 mass / mass% to about 0.15 mass / mass%, or from about 0.01 mass / mass% to about 0.17 mass / mass%, or less than about 0.005 mass / mass%, or about 0.2 mass / mass%, or about 0.25 mass / mass% or more, but not limited thereto, to achieve the desired effect.

[0073] There are some components that are not included in either the cell washing solution or the cell delivery solution and can be completely excluded or excluded to below the detectable limit. Some examples of components that can be excluded are: certain components of animal origin; 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; glycine, L-alanine, L-arginine hydrochloride, L-asparagine-H 2 O, L-glutamine, L-cysteine, L-histidine hydrochloride-H 2 O, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, etc.; and certain vitamins such as ascorbic acid, choline chloride, calcium D-pantothenate, folic acid, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and i-inositol.

[0074] The present disclosure also targets a method of treating a subject. The method can include administering a single dose of a cell suspension for injection (CSI) comprising 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 can include any of the cells disclosed herein and can be cells of the subject being treated or cells from another source or subject. The subject to be treated by the method can be an adult or a pediatric patient. The subject to be treated by the method of the present disclosure can be a mammal, preferably a human. The subject can also be an animal such as a primate, dog, cat, livestock, or horse.

[0075] The number of cells administered can vary based on the particular treatment. The number of cells can include any range having as an endpoint any of the following values: less than about 10 cells, 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, but can be any suitable and effective number, not limited thereto. The number of these cells can be from about 25×10 6 cells / mL to about 150×10 6 cells / mL, or from about 50×10 6 cells / mL to about 100×10 6 cells / mL, or from about 60×10 6 cells / mL to about 90×10 6 cells / mL, or from about 65×10 6 cells / mL to about 85×10 6 cells / mL, or from 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 6cells / mL, or about 125×10 6 cells / mL, or about 150×10 6 cells / mL or cells at any suitable concentration such as these can be administered in any suitable volume. In one embodiment, the number of cells administered is 120×10 6 cells / mL, 130×10 6 cells / mL or 140×10 6 cells / mL.

[0076] The method can be directed to a single administration of a single dose of CSI, or the method can include a further step of waiting a predetermined time after a previous administration and then administering another single dose of CSI, the dose of which can be the same dose, such as a dose containing the same or similar components, or a different dose of CSI. The method can be directed to unilateral or bilateral administration, for example, an injection such as an ICV injection. The amount of the predetermined time can vary based on the treatment and can include any suitable time, including any range having any of the following values as endpoints, 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 longer, etc.

[0077] These additional steps of waiting a predetermined time after a previous administration and then administering another dose of CSI can be repeated based on the treatment and can be any suitable number of repetitions, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more.

[0078] The treatment method can also include a step prior to the administration of CSI, or a step simultaneous with the administration of CSI, or a step subsequent to the administration of CSI. This optional simultaneous or subsequent step can be the administration of one or more treatments, and the treatment is selected from the group consisting of immunosuppressive agents, chemotherapeutic agents, radiation treatments, bone marrow treatments, hormonal treatments, surgical treatments, or any treatment intended to improve the subject's response to CSI administration. Each of these treatments can vary based on the treatment required.

[0079] The present disclosure also targets a method of administering a dosing volume to a subject. This method of administering a dosing volume includes loading a dosing volume of an injectable cell suspension (CSI) into a dosing delivery device such as a syringe or any other device capable of delivering a solution to the subject. CSI can 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, and the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer. The plurality of cells can be differentiated midbrain dopaminergic neurons, about 10 0 cells to about 10 10 cells, or can include a range of about 10 8 cells to 10 10 cells.

[0080] After loading the dosing volume of the solution into the delivery device, the method then includes the step of injecting the dosing volume into a part of the subject. The subject can be a human, and this injection step can occur directly into a part of the subject and / or into a venous line within the subject's blood vessels.

[0081] This administration method can further include a simultaneous or subsequent step of administering a treatment, and the treatment is selected from the group consisting of chemotherapeutic agents, radiation treatments, bone marrow treatments, hormonal treatments, and surgical treatments.

[0082] The methods and models of the present disclosure will be well understood by referring to the following examples, which are provided as typical examples of the present disclosure and are not limiting in any form.

Example

[0083] An example of forming the cell washing solution of the present disclosure is disclosed in this example.

[0084] This cell washing solution can be used to wash any cells, including cells in cryoprotectant after thawing. The procedure is described for a 1 kg scale but can be scaled up or down as appropriate.

[0085]

Table 1

[0086] Using these starting materials, the following procedure was followed: 1. Weighed the tare of a 1 L container with a stir bar and filled it with 500 g of water for injection (WFI) using a serological pipette. 2. Using a balance, weighed the tare of separate weighing dishes and weighed each reagent. While stirring on a magnetic stirrer plate, quantitatively transferred it to a 1 L bottle. 3. Considering the volume of water already added in the previous step, the total mass including WFI was 980.0 g. 4. Adjusted the pH of the bulk solution to about 6.95 to about 7.05 at about 17 °C to about 25 °C using 1 M NaOH and 1 M HCl solutions. Recorded the added volume (about 3.500 mL of 1 M NaOH). 5. Considering the volumes of acid and base used to adjust the pH of the solution in step 3, added rHSA and up to 20.0 mL of WFI (so that the total mass becomes 1 kg). Placed it on a magnetic stirrer plate for about 20 to about 30 minutes until well mixed. 6. Took an aliquot to measure the osmolarity. a. This ongoing measurement was about 295 mOsm / kg H 2It is expected to become O. 7. Filter the solution using a filtration sterilization device in a sterile environment. 8. Take aliquots in a sterile environment to confirm that the pH, weight molar osmotic concentration, and density of the final formulation are sufficient. 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. b. The weight molar osmotic concentration should be about 282 to about 302 mOsm / kgH 2 O. 1 . c. The density should be about 1.0075 to about 1.0105 g / mL. 9. Store at about 4 °C (or as desired / required). ( 1 The specification of the weight molar osmotic concentration is based on the conversion from the specification of the volumetric molar osmotic concentration of 280 - 300 mOsm / L (in accordance with USP <785>), assuming a density of about 1.0090 g / mL and a solute concentration of about 0.0167617 g / mL.)

Examples

[0087] An example of forming the cell delivery solution of the present disclosure is provided in this example.

[0088] This solution can be used to resuspend cells during the preparation of a cell suspension for injection (CSI). The procedure is described for a 1 kg scale but can be scaled up or down as appropriate.

[0089]

Table 2

[0090] Using these starting materials, the following procedure was followed: 1. Fill a 1 L container with 500 g of water for injection (WFI). 2. Use an appropriate balance to weigh the tare of separate weighing dishes and weigh each reagent. Quantitatively transfer to a 1 L flask while stirring on a magnetic stirrer plate. 3. Considering the amount of water already added in the previous step, the total mass including WFI will be approximately 750.0 g. Solubilize for about 30 to about 45 minutes. 4. Adjust the bulk solution pH to about 7.00 to about 7.05 at about 17 to about 25 °C using 1M NaOH and 1M HCl solutions. Record the addition volumes (NaOH approximately 3.000 to 3.125 mL). 5. Add rHSA under constant stirring. Dissolve for about 30 to about 45 minutes. 6. Add Dextran 40 under constant stirring. Dissolve at medium speed for about 30 to about 45 minutes. 7. Considering the volumes of acid and base used to adjust the pH of the solution in step 4, make up the bulk to 1 kg with WFI. Place on a magnetic stirrer plate for about 5 to about 10 minutes until well mixed. 8. Take an aliquot to measure the osmolarity. · This ongoing measurement is expected to reach about 344 mOsm / kgH 2 2O. 9. Filter the solution using a sterile filtration device in a sterile environment. 10. Take an aliquot in a sterile environment to confirm that the pH, osmolarity and density of the final formulation are satisfactory. · The pH should be about 6.9 to about 7.1 at about 17 to about 25 °C. a. The osmolarity should be about 341 to about 366 mOsm / kgH 2 2O. · This will correspond to an osmolarity of about 280 to about 300 mOsm / L based on a density of about 1.081 g / mL and a solute concentration of about 0.249 g / mL at about 5 °C. b. The density should be from about 1.0800 to about 1.0820 g / mL at about 5 °C. However, the final density of the CSI can include one or more additional components that contain substantially little or substantially trace amounts of cell wash solution and / or buffer. The presence of these one or more additional components can 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). 11. Store at 2 - 8 °C (or as desired / required).

Examples

[0091] The following examples of the present disclosure are intended to be illustrative of one embodiment of the cell wash solution and cell delivery solution when used with microglial cells.

[0092] All of the data shown in Figures 1 - 4 were for cryopreserved microglial cells. After thawing the frozen cells, the cryoprotectant was washed from the cells by addition of one embodiment of the cell wash solution. The mixture containing the cells was then centrifuged to pellet the cells, and thus the cell wash solution supernatant could be removed. After removing the supernatant, the cells were resuspended in one embodiment of the cell delivery solution to form an injectable cell suspension (CSI).

[0093] The CSI was then stored for 0, 8 or 24 hours and characterized by several assays. The control groups used were commercial solutions containing a number of excipients or several excipients that had to be added prior to use, and thus this solution is not ideal for late stage clinical and commercial use.

[0094] As shown in Figure 1, the cell viability of cells in a typical preparation of injectable cells was evaluated at different time points. After storage in a container on ice for 6 hours, the microglial cells stored in the cell washing solution of the present disclosure had almost the same cell viability (92.0%) compared to the cell viability (92.7%) of the cells in the commercially available solution.

[0095] Apoptosis is the process by which cells enter a state of cell death. As shown in Figure 2, the percentage of apoptotic cells in cells stored in a commercially available solution compared to cells stored in the cell washing solution of the present disclosure had almost the same percentage (approximately 7.9%) of apoptotic cells compared to the percentage of apoptotic cells (approximately 6.4%) in the commercially available solution after storage in a container on ice for 6 hours.

[0096] As shown in Figure 3, after storage in a container on ice for 24 hours, the microglial cells stored in the cell delivery solution of the present disclosure had almost the same cell viability (approximately 90%) compared to the cell viability (approximately 80%) of the cells in the commercially available solution.

[0097] As shown in Figure 4, when the cells were stored in the cell delivery solution of the present disclosure, the dose of microglial cells (evaluated by viable cell concentration) was substantially stable after 24 hours of storage and did not significantly differ from the stability of the cells stored in the commercially available solution.

Example

[0098] The following examples of the present disclosure are intended to illustrate an embodiment of the cell washing solution and the cell delivery solution when used with dopamine-producing cells.

[0099] All of the data shown in FIGS. 5 - 12 are for cryopreserved dopamine - producing cells. After thawing the frozen cells, the cryoprotectant was washed from the cells by addition of one embodiment of a cell wash solution. The solution containing the cells was then centrifuged to remove the supernatant, and the cells were resuspended in one embodiment of a cell delivery solution to form an injectable cell suspension (CSI). The CSI was stored for 0, 8, 24, or 48 hours and then stored. The control used was a commercially available solution containing components of non - human origin.

[0100] As seen in FIGS. 5A - 5C, cell viability and cell dose were substantially stable after 96 hours of storage. The grey lines and data points in FIGS. 5A - 5C represent the average of 11 samples of cells stored in a commercially available solution. The blue lines and data points in FIGS. 5A - 5C represent the average of 12 samples of cells stored in one embodiment of a cell delivery solution.

[0101] As seen in FIG. 5B, the loss of cell viability (- 2.5%) of cells stored for more than 24 hours in the cell delivery solution was minimal and lower than the loss (- 5%) in the viability of cells stored in a commercially available solution.

[0102] As seen in FIG. 5C, in all of the formulations tested, the viable cell dose remained within the clinical target dose range of about 100E6 ± 10 cells / mL up to 96 hours.

[0103] Apoptosis is the process that describes early cell death. Further data on apoptotic cells are shown in FIG. 6. The grey data points in FIG. 6 represent the average of 5 samples of cells stored in a commercially available solution. The blue data points in FIG. 6 represent the average of 5 samples of cells stored in one embodiment of a cell delivery solution. Both cells suspended in the commercially available solution and cells suspended in the cell delivery solution had low apoptosis levels (< 10%) after 24 hours of storage.

[0104] The effect of the formulation on the morphology of dopamine-producing cells after storage when the cells were formulated in a commercial solution or an embodiment of a cell delivery solution was evaluated. Figure 7 shows representative images of cells cultured after storage in a commercial solution (left) or a cell delivery solution (right). As seen in Figure 5, no visual differences in cell morphology were observed after 5 days of culture, and the morphology was as expected.

[0105] An advantage of one embodiment of the cell delivery solution is its ability to maintain the cells in a suspended state and improve dose assurance. In this example, the cells were suspended in a cell delivery solution or a commercial solution. As seen in Figure 8, and as demonstrated by sequential photographs of the container over time, one embodiment of the cell delivery solution maintained the cells in a suspended state for at least 72 hours, in contrast to a commercial solution in which the cells settled within minutes. As seen in Figure 8, the visual non-uniformity of the cells in the embodiment of the cell delivery solution began approximately 96 hours later, significantly longer than that of the cells in the commercial solution.

[0106] Figure 9 is a graph of the visualized results from Figure 8. At each time point, the container was visually observed and scored according to the scoring system shown in Figure 10, with a score of 3 being the initial score after mixing and the cells being uniformly distributed in the solution. As seen in the results graphed over time in Figure 9, the cells resuspended in the commercial solution quickly settled to a score of 1. As also seen in the results, the embodiment of the cell delivery solution maintained the cells in a suspended state at a score of 3 for a considerably long time (72 hours).

[0107] Figures 11A and 11B are graphs of the quantitative determination of cell precipitation. The cell suspension was sampled three times at the upper, middle, and lower parts of the container, and the variation between the three measurements was calculated as the coefficient of variation (%CV). In this scenario, the greater the %CV, the more the cells have settled.

[0108] The gray data points in FIGS. 11A and 11B represent the variability in dose sampling for cells stored in a commercial solution. The blue dots in FIGS. 11A and 11B represent the variability in dose sampling for cells stored in two embodiments of the cell delivery solution: "A" and "B". In particular, FIGS. 11A and 11B demonstrate that two different cell delivery solutions, A and B, reduce cell precipitation up to 24 hours compared to the commercial solution.

[0109] The comparison can be made between FIG. 11A, which demonstrates the variability in dose sampling after 8 hours of storage, and FIG. 11B, which demonstrates the variability in dose sampling after 24 hours of storage. This comparison shows that cell delivery solutions A and B maintain the cells in suspension for at least 24 hours (%CV < 25%), and are significantly better than cells stored in a commercial solution, where the cell suspension is non-uniform after at most 8 hours of storage (%CV approximately 150 - 175).

[0110] As seen in FIG. 12, dopamine-producing cells were able to produce dopamine above a certain threshold regardless of whether the cells were stored for 0, 8, or 24 hours in a commercial solution or in one embodiment of the cell delivery solution.

[0111] Overall, the data presented here suggest that dopamine-producing cells remain healthy and functional regardless of whether they are stored in a commercial solution or in one embodiment of the cell delivery solution. The advantages of using the cell delivery solution include: excellent dose assurance due to the cells remaining in suspension for an extended period; higher manufacturing and commercial viability due to the formulation containing minimal pharmaceutical-grade components and no components of human or animal origin; and ease of use due to the formulation being storable under standard storage conditions and ready for use.

Example

[0112] In this example, the cell washing solution (CWS) of the present disclosure was evaluated as a component of the cell solution for injection (CSI). In this example, the CSI containing CWS and a commercially available solution were evaluated for the delivery of cells at two different concentrations of approximately 75,000 cells / μL and approximately 20,000 cells / μL. The tests were performed in triplicate, and the data shown in this example are the average of these triplicate data.

[0113] Each of the CSI and the commercially available solution of this example was prepared at both a cell concentration of approximately 75,000 cells / μL and a cell concentration of approximately 20,000 cells / μL, and the cell viability / viable cell concentration and apoptosis of the cells were measured at approximately 0 hours and approximately 6 hours of storage at 2°C to 8°C.

[0114] Figure 13A shows the results at approximately 0 hours and approximately 6 hours for approximately 75,000 cells / μL stored in both a commercially available solution and one embodiment of the CWS. Figure 13B shows the results at approximately 0 hours and approximately 6 hours for approximately 20,000 cells / μL stored in both a commercially available solution and one embodiment of the CWS. As seen in Figures 13A and 13B, at both cell concentrations, the CWS of the present disclosure had less of a decrease in viable cell concentration compared to the commercially available solution.

[0115] Specifically, in Figure 13A, at a cell concentration of approximately 75,000 cells / μL, after a storage time of approximately 6 hours, there was a decrease in viable cell concentration of approximately 10.2% for the cells stored in the CWS of the present disclosure compared to a decrease in viable cell concentration of approximately 16.2% for the cells stored in the commercially available solution. In Figure 13B, at a cell concentration of approximately 20,000 cells / μL, after a storage time of approximately 6 hours, there was a decrease in viable cell concentration of approximately 3.8% for the cells stored in the CWS of the present disclosure compared to a decrease in viable cell concentration of approximately 18.7% for the cells stored in the commercially available solution.

[0116] In the same experiment, as shown in Figures 14A and 14B, the cell viability of the cells stored in the CWS of the present disclosure was determined in comparison to the commercially available solution.

[0117] Figure 14A shows the results of cells after thawing and cells after storing the cells for approximately 0 hours and approximately 6 hours for both approximately 75,000 cells / μL and approximately 20,000 cells / μL stored in both a commercially available solution and one embodiment of the CWS. As can be seen from Figure 14A, at both cell concentrations, the CWS of the present disclosure has a cell viability percentage comparable to that of the commercially available solution after approximately 6 hours and maintains a cell viability well above 70% after approximately 6 hours of storage.

[0118] Figure 14B is a diagram of the total cell concentration of cells with an initial concentration of approximately 75,000 cells / μL stored for approximately 0 hours and approximately 6 hours in both a commercially available solution and an embodiment of the CWS. As can be seen from Figure 14B, the CWS of the present disclosure has less reduction in total cell concentration (approximately 3.4%) compared to the cells stored in the commercially available solution (approximately 17%).

[0119] Also, measurements of morphology were performed on cells stored at 2°C to 8°C for approximately 0 hours and approximately 6 hours for both approximately 75,000 cells / μL and approximately 25,000 cells / μL stored in both a commercially available solution and an embodiment of the CWS. This test was performed in triplicate by seeding approximately 100,000 cells per 96-well plate.

[0120] Representative images were taken and the results are shown in a series of photographs in Figure 15. As shown in Figure 15, in both the CWS of the present disclosure and the commercially available solution, at both cell concentrations, no visual differences in cell morphology were observed after approximately 6 hours of storage, and the morphology was as expected.

[0121] The embodiments and examples described in this disclosure are intended to be illustrative rather than restrictive, and are not intended to represent every embodiment or example of the disclosure. While the basic novel features of the disclosure are shown, described, and pointed out as applied to various specific embodiments thereof, it will also be understood that various omissions, substitutions, and modifications in the form and detail of the exemplified compositions and in the operations thereof may be made by those skilled in the art without departing from the spirit of the disclosure. For example, all combinations of those elements and / or method steps that perform substantially the same function in substantially the same way to achieve the same result are expressly intended to be within the scope of the disclosure. Further, it should be recognized that structures and / or elements and / or method steps shown and / or described in connection with any disclosed form or embodiment of the disclosure may be incorporated into any other disclosed or described or suggested form or embodiment as a general matter of design choice. Moreover, various modifications and changes may be made both literally and in equivalents legally recognized without departing from the spirit or scope of the disclosure as set forth in the following claims.

Claims

1. One or more energy source components; One or more pH buffers; One or more salts; and The present invention comprises one or more stabilizers, wherein the one or more stabilizers are selected from the group consisting of recombinant albumin (rHSA), dextran, and poloxamer. Cell delivery solution.

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

3. The solution according to claim 2, wherein the sugar is dextrose.

4. The solution according to claim 1, wherein the one or more pH buffers comprise one or more amphoteric organic chemical buffers.

5. The solution according to claim 4, wherein the one or more amphoteric organic chemical buffers include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

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

7. The solution according to claim 1, wherein the solution does not contain human serum albumin (HSA).

8. The solution according to claim 1, wherein the solution contains no animal-derived components.

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

10. The solution according to claim 1, wherein the solution does not contain a pH indicator.

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

12. The solution according to claim 1, wherein the solution does not contain sodium pyruvate.

13. The solution according to claim 1, wherein the solution does not contain amino acids.

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

15. The solution according to claim 1, further comprising one or more excipients selected from the group consisting of polyethylene glycol, carboxymethylcellulose, hyaluronic acid, starch, acrylate, methacrylate, polyvinyl alcohol, polyethylene oxide, polypropylene oxide, polyacrylate, polyvinylpyrrolidone, polymethacrylate, lactic acid-glycolic acid copolymer, polyacrylamide, polylactic acid, chitosan, rubber, guar gum, xanthan gum, carrageenan, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, cyclodextrin derivatives, beta-cyclodextrin derivatives, alginate, calcium alginate, and stearate.

Images