Cryopreserving composition comprising stem cells

Abstract

The present invention provides a cry opreserving composition comprising a low concentration of DMSO for cry opreserving human stem cells and uses thereof for cryopreserving the cells or for preparing therapeutic cell suspensions for parenteral administration useful for treating neurodegenerative diseases or disorders.

Description

[0001] CRYOPRESERVING COMPOSITION COMPRISING STEM CELLS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method of preserving and using human stem cells derived from oral mucosa.

[0004] BACKGROUND OF THE INVENTION

[0005] Cryopreservation of cell-based products for therapeutic purposes involves the utilization of cryopreservative agents (CA). The most commonly used CA for cell cry opreservation is dimethyl sulfoxide (DMSO). DMSO is an amphipathic agent that crosses the plasma membrane and stabilizes the intracellular organelles during the freezing and thawing processes. However, DMSO is toxic to a variety of organs and systems as hepatic, cardiovascular, and central nervous systems (Shu et al., Bone Marrow Transplant. 2014). This toxicity is of particular importance when cell suspensions containing DMSO are delivered directly to the central nervous system for example intrathecal administration, intraventricular or administration into the brain and spinal cord parenchyma. DMSO freezing solutions are hyperosmotic, a property that may cause cell disruption and loss during the thawing and administration processes or may cause damage to the recipient tissue.

[0006] Typically, DMSO is used for cryopreservation of stem cells in high concentrations, 5% and higher. There is a substantial amount of research showing that low concentrations of DMSO are less effective in cry opreserving cells. For example, Akkok et al (Cytotherapy, 2009, Vol. 11, issue 6, 749-760) concluded that higher viability of neutrophils and lymphocytes was detected with 4% and 5% DMSO, whereas decreased viability was observed with 2% and 10% DMSO. A similar conclusion was made by Liseth et al (Cytotherapy, 2005, Vol. 7, issue 4, 328-333). They observed that PBPC cryopreserved at 150xl06cells / mL with 2% DMSO yielded significantly inferior CD34+ cell recovery (P < 0.001) and survival (P < 0.001) compared with cryopreservation with 4% and 5% DMSO. Smagur et al., (The International Journal of Transfusion Medicine, 2013, Vol. 104, issue 3, 240-247) observed that reduction in DMSO concentrations of 10% to 7.5% may have the best recovery and clonogenic potential, respectively, when compared to lower DMSO concentration (2.5% and 5%).

[0007] There is an unmet need for developing compositions of cells that may be used immediately after thawing without further manipulations. SUMMARY OF THE INVENTION

[0008] In some clinical cases, there is a requirement to administer a low volume of highly concentrated cells. One such example is the intrathecal administration of stem cells during which, only a limited volume of cells, typically up to 20 ml may be administered. Usually, cryopreserved cells are used and it is important to avoid administering a high concentration of a cryopreservative agent, which is commonly DMSO. For this aim, cell manipulations, such as washing, require the use of laboratory facilities and may cause a reduction of both the number of cells and their viability. The present invention relates to new methods and compositions for cryopreservation of a high concentration of human oral mucosa stem cells at low DMSO concentrations. As described above, this is a non-trivial task considering that DMSO at low concentration is considered and was shown to have lower cryopreserving potency for stem cells. It was unexpectedly found that increasing the concentration of cells while using a low concentration of DMSO improves the stability of cells during storage and thawing. Such compositions may be thawed and diluted at the bedside and immediately administered into the central nervous system of a patient to treat neural disorders caused by disease or trauma. Due to the low concentration of DMSO in the cryopreservation composition, the final diluted cell suspension has a non-toxic concentration of DMSO and may be immediately used without additional steps, e.g. without further steps aimed at removing DMSO. Therefore, the composition cancels the need for any further manipulations of cells which are time-consuming, require special equipment and facilities and increase the risk of cell contamination, cell loss and reduced efficacy. To further clarify, increasing cell concentration at freezing enables the delivery of a large number of cells in a non-toxic concentration of DMSO.

[0009] According to one aspect, the present invention provides a cryopreserving composition comprising stem cells, wherein the composition comprises less than 4% w / v of dimethyl sulfoxide (DMSO) and at least 5xl06cells / ml stem cells, wherein the cells are human stem cells derived from the lamina propria of the oral mucosa (hOMSC). In some examples, the composition comprises less than 3% v / v of DMSO, from 1 to 4%, or from 1.5% to 2.5% v / v of DMSO. In some examples, the composition comprises from IxlO6to 5xl08cells / ml or from 10xl06to lOOxlO6cells / ml. According to some embodiments, the cryopreserving composition is a frozen composition.

[0010] In some examples, the composition comprises less than 10% or less than 3% Human Serum Albumin (HSA). In some examples, the composition is devoid of HSA. According to another aspect, the present invention provides a therapeutic cell suspension for injection, comprising the cryopreserving composition of any one of the above aspects, examples or embodiments, and a solution for injection. In some examples, the solution for injection is selected from PlasmaLyte, Saline solution, and Ringer’s Lactate. In some examples, therapeutic cell suspension comprises less than 0.5% or less than 0.1% v / v of DMSO. In some examples, the therapeutic cell suspension comprises less than 1% or less than 0.5% of HSA (w / v). In some examples, the therapeutic cell suspension is devoid of HSA. In some examples, the therapeutic cell suspension comprises from IxlO5to IxlO7cells / ml. In some examples, the therapeutic cell suspension is for use in the treatment of a neurodegenerative disease or disorder, preferably for use in treating Multiple System Atrophy.

[0011] According to a further aspect, the present invention provides a method for preparing a therapeutic cell suspension for injection, comprising (i) thawing the frozen cryopreserving composition comprising stem cells as described in any one of the aspects and embodiments of the present application into a solution for injection either comprising HSA or not and (ii) dissolving the composition obtained in step (i) in a solution for injection. In some examples, the cry opreserving composition of step (i) comprises from 10xl06to 500xl06cells / ml. In some examples, the solution for injection is selected from PlasmaLyte, Saline solution, Ringer’s Lactate, or a combination thereof. In some examples, no HSA is further added. In other examples, the method comprises adding a solution for injection comprising HSA during thawing wherein the volume of the added solution is equal to or less than the volume of the cry opreserving composition provided in step (i). In some examples, the concentration of HSA in the added solution is from 1 to 20 % w / v or from 7 to 12% w / v. In some examples, the final concentration of HSA after the dilution in step (ii) is less than 0.5% w / v. In some examples, the final concentration of DMSO after the dilution in step (ii) is less than 0.5% v / v.

[0012] In some examples, the method further comprises washing the cells with a solution for injection before step (ii).

[0013] According to a further aspect, the present invention provides a method of treatment of a neurodegenerative disease or disorder in a subject, comprising the steps:

[0014] (i) providing a cryopreserving composition of any one of the above aspects, examples and embodiments;

[0015] (ii) thawing the composition of step (i) into a solution for injection optionally further comprising HSA, optionally from 5 to 20 % w / v; (iii) diluting the composition of step (ii) in a solution for injection to achieve a therapeutic cell suspension for injection comprising stem cells at a concentration ranging from IxlO6to 50xl06cells / ml, DMSO less than 0.5%, and HSA, if present, less than 0.5%, and

[0016] (iv) intrathecally administering the composition obtained in step (iv) to the subject.

[0017] According to another aspect, the present invention provides a method for cry opreserving human stem cells derived from the lamina propria of the oral mucosa, the method comprises dissolving the stem cells in a cry opreserving composition comprising from 1 to 4% w / v of dimethyl sulfoxide (DMSO) and freezing the cells at a temperature below -70°C wherein the final concentration of the cells is at least IxlO7cells / ml. In some examples, the cryopreserving composition comprises less than 3 % w / v or from 1.5% to 2.5% v / v of DMSO (w / v). In some examples, the final concentration of the cells is from 2xl07to 1.5xl08cells / ml. In some examples, the cry opreserving composition comprises less than 10%, or less than 3%, from 0.5 to 8% HSA or substantially devoid of Human Serum Albumin (HSA). In some examples, the cry opreserving composition is free of non-human animal components.

[0018] According to another aspect, the present invention provides a therapeutic cell suspension for injection, comprising from O.lxlO5to IxlO7cells / ml, from 0.01 to 0.5% v / v of DMSO, optionally from 0.01 to 0.5% w / v of HSA, and solution for injection, wherein the cells are human stem cells derived from the lamina propria of the oral mucosa (hOMSC).

[0019] According to any one of the above aspects, examples and embodiments, the cells are derived from the lining and masticatory oral mucosa. In some examples, the cells are characterized by simultaneously expressing the following markers: Oct-4, SSEA4, Nanog, Sox2, KLF4, c-MYC, nestin, p-III tubulin, p75, CD29, CD 73, CD90, CD105, and CD166. In some examples, the cells are characterized by simultaneously expressing the following markers: KLF4, c-MYC, nestin, P-III tubulin and p75, and wherein the cells are negative for CD45 and CD31.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In case of conflict, the patent specification, including definitions, will control.

[0022] According to one aspect, the present invention provides a cryopreserving composition comprising stem cells, wherein the composition comprises less than 4% v / v of dimethyl sulfoxide (DMSO) and at least 106cells / ml stem cells. According to some embodiments, the present invention provides a cry opreserving composition comprising stem cells, wherein the composition comprises less than 4% v / v of dimethyl sulfoxide (DMSO) and at least 5xl06cells / ml stem cells. According to some embodiments, the stem cells are human stem cells derived from the lamina propria of the oral mucosa (hOMSC). Thus, according to some embodiments, the present invention provides a cryopreserving composition comprising stem cells, wherein the composition comprises less than 4% v / v of dimethyl sulfoxide (DMSO) and at least 5xl06cells / ml stem cells and wherein the stem cells are human stem cells derived from the lamina propria of the oral mucosa (hOMSC).

[0023] As used herein, the term "cry opreserving composition" refers to a composition that is suitable for the storage of a biological material (e.g., cells, tissues, organs and biological molecules) at temperatures below 4°C. In some embodiments, the cry opreserving composition of the invention is in a frozen state, e.g., at a temperature of less than 0°C less than -5°C, less than -20°C, less than -60°C, less than -70°C, less than -80°C, less than -90°C, less than -170°C.

[0024] According to some embodiments, the cryopreserving composition comprises less than 3.5 % v / v DMSO, less than 3% v / v DMSO, less than 2.5% v / v DMSO, less than 2% v / v DMSO, less than 1.5% v / v DMSO or less than 1% v / v DMSO. The term "less than" as used herein with respect to DMSO in cryopreserving composition only does not include zero. Therefore, the term "less than 4% v / v DMSO" with respect to cryopreserving composition has the meaning of less than 4% but above 0%. In other embodiments, the term "less than" has its common meaning.

[0025] According to some embodiments, the cryopreserving composition comprises from 0.5 to 4% v / v DMSO. According to some embodiments, the cry opreserving composition comprises from 1 to 3.5% v / v DMSO. According to some embodiments, the cryopreserving composition comprises from 1 to 3% v / v DMSO. According to some embodiments, the cryopreserving composition comprises from 1.5 to 2.8% v / v DMSO. According to some embodiments, the cryopreserving composition comprises from 1.5 to 2.5% v / v DMSO. According to some embodiments, the cry opreserving composition comprises from 1.6 to 2.3% v / v DMSO. According to some embodiments, the cryopreserving composition comprises from 1.8 to 2.3% v / v DMSO. According to some embodiments, the cryopreserving composition comprises from 1.9 to 2.2% v / v DMSO. According to some embodiments, the cry opreserving composition comprises about 2% v / v DMSO. According to some embodiments, DMSO is the main cry opreserving agent in the cryopreserving composition. According to some embodiments, the content of DMSO is at least 50% of all cryopreservative agents. According to some embodiments, the content of DMSO is at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of all cryopreservative agents. According to some embodiments, the content of DMSO is from 50 to 99%, from 55 to 95%, from 60 to 90%, from 65 to 85%, from 70 to 80% of all cryopreservative agents. According to some embodiments, DMSO is the only cry opreserving agent in the cryopreserving composition. The terms “cry opreserving agents”, “cryopreservative agents” and "cryoprotectant" may be used herein interchangeably and refer to an agent used to preserve cell viability when cooling to sub-zero centigrade temperatures.

[0026] According to some embodiments, the cryopreserving composition comprises at least IxlO6, at least 1.5xl06, at least 2xl06, at least 2.5xl06, at least 5xl06, at least IxlO7, at least 2xl07, or at least 3xl07cells / ml. According to some embodiments, the cryopreserving composition comprises at least 5xl06cells / ml. According to some embodiments, the cryopreserving composition comprises at least IxlO7cells / ml. According to some embodiments, the cry opreserving composition comprises at least 4xl07cells / ml. According to some embodiments, the cryopreserving composition comprises at least 5xl07cells / ml. According to some embodiments, the cry opreserving composition comprises at least 6xl07cells / ml. According to some embodiments, the cry opreserving composition comprises at least 7xl07cells / ml. According to some embodiments, the cry opreserving composition comprises at least 8xl07cells / ml. According to some embodiments, the cryopreserving composition comprises at least 9xl07cells / ml. According to some embodiments, the cryopreserving composition comprises at least IxlO8cells / ml. According to some embodiments, the cry opreserving composition comprises at least 1.2xl08cells / ml. According to some embodiments, the cryopreserving composition comprises at least 1.5xl08cells / ml. According to some embodiments, the cry opreserving composition comprises at least 2xl08cells / ml. According to some embodiments, the cry opreserving composition comprises from 5xl06to 5xl08cells / ml. According to some embodiments, the cryopreserving composition comprises from IxlO7to 1.2xl08cells / ml. According to some embodiments, the cry opreserving composition comprises from 1.5xl07to IxlO8cells / ml. According to some embodiments, the cryopreserving composition comprises from 10xl06to 200xl06cells / ml. According to some embodiments, the cryopreserving composition comprises from 10xl06to lOOxlO6cells / ml. According to some embodiments, the cryopreserving composition comprises from 50xl06to lOOxlO6cells / ml. According to some embodiments, the cry opreserving composition comprises from 30xl06to 80xl06cells / ml.

[0027] According to any one of the above embodiments, the cryopreserving composition comprises less than 10% Human Serum Albumin (HSA). According to some embodiments, the cryopreserving composition comprises less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% HSA. According to some embodiments, the cry opreserving composition comprises from 0.5% to 10%, from 1 to 10%, from 2 to 9%, from 3 to 8%, from 4 to 7%, or from 4 to 6% or about 5% of HSA. According to some embodiments, the cry opreserving composition comprises from 0.5% to 8% HSA. According to some embodiments, the cry opreserving composition comprises from 1% to 6% HSA. According to some embodiments, the cry opreserving composition comprises from 1.5% to 5% HSA. According to some embodiments, the cry opreserving composition comprises from 1.5% to 4% HSA. According to some embodiments, the cryopreserving composition comprises about 1%, about 2%, about 35, about 4%, about 5%, or about 6% of HSA. According to some embodiments, the cryopreserving composition is devoid of HSA. The terms “substantially devoid”, “essentially devoid”, “devoid”, “does not include” and “does not comprise” may be used interchangeably and refer to a composition that does not include, contain or comprise a particular component, e.g. said composition comprises less than 0.1 wt%, less than 0.01 wt%, or less than 0.001 wt% of the component. In some embodiments, the term devoid contemplates a composition comprising traces of the devoid component such as traces of a component used in the purification process.

[0028] According to some embodiments, the stem cells are human stem cells. According to some embodiments, the human stem cells are derived from the lining and masticatory oral mucosa. According to some embodiments, the cells are characterized by simultaneously expressing the following markers: Oct-4, SSEA4, Nanog, Sox2, KLF4, c-MYC, nestin, 0- III tubulin, p75, CD29, CD 73, CD90, CD105, and CD166. According to some embodiments, the cells are characterized by simultaneously expressing the following markers KLF4, c-MYC, nestin, 0 -III tubulin, and p75, and being negative for CD45 and CD31. According to some embodiments, the cells are characterized by simultaneously expressing the following markers: OCT-4, SSEA4, NANOG, SOX2, KLF4, c-MYC, nestin, 0 -III tubulin, p75, CD29, CD 73, CD90, CD105, and CD166 and the cells are negative for CD45 and CD31. According to some embodiments, the cells are isolated cells. According to some embodiments, the cells are pluripotent or multipotent stem cells. According to any one of the above embodiments, the cry opreserving composition is free of non-human animal components.

[0029] According to any one of the above embodiments, the cryopreserving composition is sterile.

[0030] According to any one of the above embodiments, the cryopreserving composition is frozen.

[0031] According to another aspect, the present invention provides a therapeutic cell suspension for injection, comprising the composition of the present invention as described in any one of the above aspects and embodiments, and a solution for injection. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

[0032] The term "therapeutic cell suspension” refers to a suspension comprising cells and is intended for therapeutic purposes, typically administered through injection or infusion into a patient's body. This suspension contains a specific population of cells that possess therapeutic properties, such as stem cells, or other specialized cell types with therapeutic potential. These cells are suspended in a suitable solution or medium to maintain their viability and functionality. The therapeutic cell suspension of the present invention comprises the human stem cells as defined in the application, e.g., hOMSC. The terms "therapeutic cell suspension" and "pharmaceutical composition comprising cells" may be used interchangeably. The term “pharmaceutical composition” as used herein refers to a composition comprising at least one active agent as disclosed herein such as hOMSC optionally formulated together with one or more pharmaceutically acceptable carriers. Formulation of the pharmaceutical composition may be adjusted according to applications. In particular, the pharmaceutical composition may be formulated using a method known in the art so as to provide rapid, continuous or delayed release of the active ingredient after administration to mammals. For example, the formulation may be any one selected from among liquids and solutions, emulsions, suspensions, infusions, and injections. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffering agents, enhancers, wetting agents, solubilizing agents, surfactants, antioxidants the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well-known in the art. Pharmaceutical compositions adapted for parenteral administration include, but are not limited to, aqueous and non-aqueous sterile injectable solutions or suspensions, which can contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of an intended recipient. Such compositions can also comprise water, alcohols, polyols, glycerin and vegetable oils. The term “parenteral” refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, intraperitoneal and intracranial injection, as well as various infusion techniques.

[0033] The term "solution for injection" refers to any isotonic solution suitable for injection into a human body. According to some embodiments, the solution for injection is selected from PlasmaLyte 148, PBS, Saline solution, and Ringer’s Lactate or a combination of thereof. According to some embodiments, the solution for injection is PlasmaLyte 148.

[0034] In some embodiments, the therapeutic cell suspension comprises from IxlO5to IxlO7cells / ml. In some embodiments, the therapeutic cell suspension comprises from 5xl05to 5xl06cells / ml. In some embodiments, the therapeutic cell suspension comprises from IxlO6to IxlO7cells / ml. In some embodiments, the therapeutic cell suspension comprises from 2xl06to 8xl06cells / ml. In some embodiments, the therapeutic cell suspension comprises from 3xl06to 7xl06cells / ml. According to some embodiment, the cells are hOMSC.

[0035] According to some embodiments, therapeutic cell suspension comprises less than 0.5% v / v of DMSO. According to some embodiments, therapeutic cell suspension comprises less than 0.4 %N / N, less than 0.3% v / v, less than 0.2% v / v, or less than 0.1% v / v DMSO. According to some embodiments, therapeutic cell suspension comprises less than 0.4% v / v of DMSO. According to some embodiments, therapeutic cell suspension comprises from 0.01 to 1 % v / v of DMSO. According to some embodiments, therapeutic cell suspension comprises from 0.01 to 0.5 % v / v of DMSO. According to some embodiments, therapeutic cell suspension comprises from 0.03 to 0.5 % v / v, from 0.05 to 0.4 % v / v of DMSO, from 0.1 to 0.4 % v / v of DMSO, from 0.2 to 0.4 % v / v of DMSO. According to some embodiments, therapeutic cell suspension comprises from 0.11 to 0.3 % v / v of DMSO.

[0036] According to some embodiments, therapeutic cell suspension comprises less than 1% of HSA (w / v). According to some embodiments, therapeutic cell suspension comprises less than 0.8% of HSA (w / v). According to some embodiments, therapeutic cell suspension comprises less than 0.5% of HSA (w / v). According to some embodiments, therapeutic cell suspension comprises less than 0.4% of HSA (w / v). According to some embodiments, therapeutic cell suspension comprises less than 0.3% of HSA (w / v). According to some embodiments, therapeutic cell suspension comprises from 0.01 to 0.5 % w / v of HSA. According to some embodiments, therapeutic cell suspension comprises from 0.03 to 0.5 % w / v, from 0.05 to 0.4 % w / v, from 0.1 to 0.4 % w / v, from 0.2 to 0.4 % w / v of HSA. According to some embodiments, therapeutic cell suspension comprises from 0.1 to 0.3 % w / v of HSA. According to some embodiments, therapeutic cell suspension is devoid of HSA.

[0037] According to any one of the above embodiments, the therapeutic cell suspension is sterile.

[0038] According to some embodiments, the therapeutic cell suspension of the present invention is for use in the treatment of a neurodegenerative disease or disorder. In some embodiments, neurodegenerative disease is a disease of basal ganglia and brain stem. In some embodiments, the neurodegenerative disease of basal ganglia and brain stem is selected from a Multiple System Atrophy, Parkinsonism, Idiopathic Parkinson Disease, Progressive Supranuclear Palsy, Corticobasal Degeneration, Striatonigral Degeneration, Shy-Drager Syndrome, Olivopontocerebellar Atrophy, Huntington Disease and ALS. According to some embodiments, the neurodegenerative disease is Multiple System Atrophy.

[0039] The term “treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, ameliorating, abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating or alleviating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and / or (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s). The term "administering” or “administration of’ a composition to a subject can be carried out using one of a variety of methods known to those skilled in the art suitable for the administration of cells. According to some embodiments, the administration is a systemic administration. According to some embodiments, the administration is intravenous (IV) According to some embodiments, the administration is into CSF. In some embodiments, the therapeutic cell suspension is administered intrathecally. The term “intrathecal administration” or “intrathecal injection” or "IT" refers to an injection into the spinal canal (intrathecal space surrounding the spinal cord). Various techniques may be used including, without limitation, lumbar puncture or lateral cerebro ventricular injection through a burrhole or cisternal or the like. In some embodiments, “intrathecal administration” or “intrathecal delivery” according to the present invention refers to IT administration or delivery via the lumbar area or region, i.e., lumbar IT administration or delivery.

[0040] According to another aspect, the present invention provides a therapeutic cell suspension for injection, comprising from IxlO5to 5xl07cells / ml, from 0.01 to 0.5% v / v of DMSO, optionally from 0.01 to 0.5% w / v of HSA, and a solution for injection. According to some embodiments, the stem cells are human stem cells derived from the lamina propria of the oral mucosa (hOMSC). In some embodiments, the therapeutic cell suspension comprises from 5xl05to 50xl06cells / ml. In some embodiments, the therapeutic cell suspension comprises from 5xl05to 10xl06cells / ml. In some embodiments, the therapeutic cell suspension comprises from IxlO6to 10xl06cells / ml. In some embodiments, the therapeutic cell suspension comprises from 5xl05to 7xl06cells / ml. In some embodiments, the therapeutic cell suspension comprises from IxlO6to IxlO7cells / ml. In some embodiments, the therapeutic cell suspension comprises from IxlO6to 5xl07cells / ml. In some embodiments, the therapeutic cell suspension comprises from IxlO6to 8xl06cells / ml. In some embodiments, the therapeutic cell suspension comprises from 0.01 to 0.5% w / v of HSA. In some embodiments, the therapeutic cell suspension comprises from 0.01 to 0.4% w / v of HSA. In some embodiments, the therapeutic cell suspension comprises from 0.01 to 0.3% w / v of HSA. In some embodiments, the therapeutic cell suspension comprises from 0.01 to 0.2% w / v of HSA. In some embodiments, the therapeutic cell suspension comprises from 0.05 to 0.3% w / v of HSA. In some embodiments, the therapeutic cell suspension comprises from 0.1 to 0.3% w / v of HSA.

[0041] All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

[0042] According to some embodiments, therapeutic cell suspension comprises from 0.01 to 0.5 % v / v of DMSO. According to some embodiments, therapeutic cell suspension comprises from 0.03 to 0.5 % w / v, from 0.05 to 0.4 % v / v of DMSO, from 0.1 to 0.4 % v / v of DMSO, from 0.2 to 0.4 % v / v of DMSO. According to some embodiments, therapeutic cell suspension comprises from 0.03 to 0.5 % w / v, from 0.05 to 0.4 % w / v, from 0.1 to 0.4 % w / v, from 0.2 to 0.4 % v / v of DMSO. According to some embodiments, therapeutic cell suspension comprises from 0.01 to 0.4 % w / v, from 0.01 to 0.35 % v / v of DMSO, from 0.01 to 0.3 % v / v of DMSO, from 0.01 to 0.2 % v / v of DMSO.

[0043] According to any one of the above embodiments, the therapeutic cell suspension is sterile.

[0044] According to another aspect, the present invention provides a method for preparing a therapeutic cell suspension for injection comprising stem cells, the method comprising dissolving the cryopreserving composition according to any one of the above aspects and embodiments comprising stem cells in a solution for injection to obtain a therapeutic cell suspension for injection comprising less than 0.4% v / v of DMSO.

[0045] According to some embodiments, the present invention provides a method for preparing a therapeutic cell suspension for injection, the method comprising (i) thawing a cryopreserving composition according to any one of the above aspects and embodiments comprising stem cells, and (ii) dissolving the composition of step (i) in a solution for injection to obtain a therapeutic cell suspension for injection comprising less than 0.4% v / v of DMSO.

[0046] According to some embodiments, the present invention provides a method for preparing a therapeutic cell suspension for injection, the method comprising the steps: (i) thawing a cry opreserving composition according to any one of the above aspects and embodiments comprising stem cells in a solution for injection comprising from 1 to 20 % of has, and (ii) dissolving the composition of step (i) in a solution for injection to obtain a therapeutic cell suspension for injection comprising less than 0.4% v / v of DMSO and less than 0.4% w / v of HSA. According to some embodiments, the volume ratio between the cryopreserving composition and the solution for injection comprising HSA in step (i) is from 8:1 to 1:3. According to some embodiments, the ratio between the cry opreserving composition and the solution for injection comprising HSA in step (i) is from 6:1 to 1:2. According to some embodiments, the ratio between the cryopreserving composition and the solution for injection comprising HSA in step (i) is from 5:1 to 1:2. According to some embodiments, the ratio between the cry opreserving composition and the solution for injection comprising HSA in step (i) is from 4:1 to 1:2. According to some embodiments, the ratio between the cryopreserving composition and the solution for injection comprising HSA in step (i) is from 3:1 to 1:2. According to some embodiments, the ratio between the cryopreserving composition and the solution for injection comprising HSA in step (i) is from 2:1 to 1:2. According to some embodiments, the ratio between the cry opreserving composition and the solution for injection comprising HSA in step (i) is from 1.5:1 to 1:1.5. According to some embodiments, the ratio between the cryopreserving composition and the solution for injection comprising HSA in step (i) is from 1.3:1 to 1:1.3, or about 1.2:1. According to some embodiments, the volume ratio between the cry opreserving composition and the solution for injection comprising HSA in step (i) is from about 1.2:1. The term "volume ratio" refers to ratio between the volumes of the two compositions. According to some embodiments, the concentration of HSA in the added solution for injection in step (i) is from 1 to 20 % w / v. In some embodiments of the invention, HSA may be replaced by any other equivalent protein or compound. According to some embodiments, the concentration of HSA in the added solution for injection in step (i) is from 1 to 15 % w / v. According to some embodiments, the concentration of HSA in the added solution injection in step (i) is from 1 to 10 % w / v. According to some embodiments, the concentration of HSA in the added solution injection in step (i) is from 2 to 9 % w / v, from 3 to 8% w / v, from 4 to 7 % w / v, from 4 to 6% w / v of HSA. According to some embodiments, the concentration of HSA in the added solution is from 5 to 15%, from 7 to 12%, from 8 to 12% or about 10%. According to some embodiments, the stem cells are human stem cells derived from the lamina propria of the oral mucosa (hOMSC).

[0047] According to some embodiments, dissolving may be performed from 1 to 60 min after step (i). According to some embodiments, dissolving may be performed from 5 to 60 min after step (i). According to some embodiments, dissolving may be performed from 10 to 50 min after step (i).

[0048] According to some embodiments, the present invention provides a method for preparing a therapeutic cell suspension for injection, the method comprising: (i) thawing a cryopreserving composition according to any one of the above aspects and embodiments comprising step cells in a solution for injection wherein the ratio between the cryopreserving composition and the solution for injection is from 8:1 to 1:3, and (ii) dissolving the composition of step (i) in a solution for injection to obtain a therapeutic cell suspension for injection comprising less than 0.4% of DMSO. According to some embodiments, the ratio between the cry opreserving composition and the solution for injection in step (i) is from 5:1 to 1:2. According to some embodiments, the ratio between the cry opreserving composition and the solution for injection in step (i) is from 2:1 to 1:2. According to some embodiments, the ratio between the cryopreserving composition and the solution for injection in step (i) is from 1.5: 1 to 1:1.5. According to some embodiments, the ratio between the cry opreserving composition and the solution for injection in step (i) is from 1: 1 to 1:1, or about 1.2:1. According to some embodiments, the stem cells are human stem cells derived from the lamina propria of the oral mucosa (hOMSC).

[0049] All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

[0050] According to some embodiments, the cryopreserving composition comprises less than 3% v / v DMSO, less than 2.5% v / v DMSO, from 1 to 3% v / v DMSO, or from 1.5 to 2.5% v / v DMSO. According to some embodiments, the cry opreserving composition comprises from 1.8 to 2.3% v / v DMSO or about 2% v / v DMSO. According to some embodiments, the cryopreserving composition comprises from 5xl06to 5xl08cells / ml / ml. According to some embodiments, the cry opreserving composition comprises from IxlO7to 1.2xl08cells / ml. According to some embodiments, the cry opreserving composition comprises from 1.5xl07to IxlO8cells / ml. According to some embodiments, the cry opreserving composition comprises from 10xl06to 200xl06cells / ml. According to some embodiments, the cryopreserving composition comprises from 10xl06to lOOxlO6cells / ml.

[0051] According to some embodiments, the solution for injection is selected from PlasmaLyte, Saline solution, PBS, and Ringer’s Lactate. According to some embodiments, the solution for injection is PlasmaLyte 148.

[0052] According to any embodiment of the present invention, the solution for injection, as well as cry opreserving composition and therapeutic cell suspension, are of a pharmaceutically acceptable grade. The terms "pharmaceutically acceptable" and "pharmacologically acceptable" include molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, or human, as appropriate. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by a government drug regulatory agency, e.g., the United States Food and Drug Administration (FDA) Office of Biologies standards.

[0053] According to some embodiments, no HSA is further added.

[0054] According to some embodiments, the present invention provides a method for preparing a therapeutic cell suspension for injection, the method comprising (i) providing a cryopreserving composition according to any one of the above aspects and embodiments comprising stem cells and (ii) dissolving the composition of step (i) in a solution for injection to obtain a therapeutic cell suspension for injection comprising less than 0.4% of DMSO. According to some embodiments, the method further comprises thawing the cryopreserving composition before step (i). According to some embodiments, no HSA is further added. According to other embodiments, the method further comprises adding a solution for injection comprising HSA before, during or immediately after thawing the cryopreserving composition. According to some embodiments, the volume of the added solution comprising HSA is equal to or less than the volume of the cryopreserving composition provided in step (i). According to some embodiments, the ratio between the volume of cry opreserving composition and solution for injection comprising HSA is from 10:1 to 1:3. According to some embodiments, the ratio between the volume of cry opreserving composition and solution for injection comprising HSA is from 8:1 to 1:3. According to some embodiments, the ratio between the volume of cry opreserving composition and solution for injection comprising HSA is from 5:1 to 1:2. According to some embodiments, the ratio between the volume of cryopreserving composition and solution for injection comprising HSA is from 3:1 to 1:2. According to some embodiments, the ratio between the volume of cry opreserving composition and solution for injection comprising HSA is from 2:1 to 1:2. According to some embodiments, concentration of HSA in the added solution is from 1 to 15 % w / v. According to some embodiments, the concentration of HSA in the added solution is from 1 to 10 % w / v. According to some embodiments, the concentration of HSA in the added solution is from 2 to 9 % w / v, from 3 to 8% w / v, from 4 to 7 % w / v, from 4 to 6% w / v of HSA. According to some embodiments, the concentration of HSA in the added solution is from 1 to 20%, from 5 to 15%, from 7 to 12%, from 8 to 12% or about 10%.

[0055] According to any one of the above embodiments, the final concentration of HSA after the dilution in step (ii) is less than 0.5% w / v. According to some embodiments, the final concentration of HSA after the dilution in step (ii) is less than 0.4% w / v. According to some embodiments, the final concentration of HSA after the dilution in step (ii) is less than 0.35% w / v. According to some embodiments, the final concentration of HSA is from 0.01 to 0.5 % w / v of HSA. According to some embodiments, the final concentration of HSA is from 0.03 to 0.5 % w / v, from 0.05 to 0.4 % w / v, from 0.1 to 0.4 % w / v, from 0.2 to 0.4 % w / v of HSA. With respect to HAS, the units w / v and v / v are equivalent and may be used interchangeably.

[0056] According to some embodiments, therapeutic cell suspension is devoid of HSA.

[0057] According to any one of the above embodiments, resulting therapeutic cell suspension comprises less than 0.4 % v / v of DMSO, less than 0.3 % w / v of HSA, devoid of HSA, devoid of DMSO or any reasonable combination of the above. According to any one of the above embodiments, the resulting therapeutic cell suspension comprises less than 0.4 % v / v of DMSO and less than 0.3 % w / v of HSA. According to any one of the above embodiments, the resulting therapeutic cell suspension comprises from 0% to 0.4 % v / v of DMSO and from 0% to 0.3 % w / v of HSA.

[0058] In some embodiments, the resulting therapeutic cell suspension comprises from IxlO5to IxlO7cells / ml. In some embodiments, the resulting therapeutic cell suspension comprises from 5xl05to 5xl06cells / ml. In some embodiments, the therapeutic cell suspension comprises from IxlO6to IxlO7cells / ml. In some embodiments, the therapeutic cell suspension comprises from IxlO6to 10xl06cells / ml. In some embodiments, the therapeutic cell suspension comprises from 3xl06to 8xl06cells / ml or about 5xl06cells / ml.

[0059] According to some embodiments, the volume of the resulting therapeutic cell suspension is from 10 to 40 ml. According to some embodiments, the volume of the resulting therapeutic cell suspension is from 10 to 30 ml. According to some embodiments, the volume of the resulting therapeutic cell suspension is from 10 to 20 ml. According to some embodiments, the volume of the resulting therapeutic cell suspension is from 15 to 25 ml. According to some embodiments, the volume of the resulting therapeutic cell suspension is about 20 ml.

[0060] According to some embodiments, the volume of the resulting therapeutic cell suspension comprises from 10xl06cells to lOOxlO6cells. According to some embodiments, the volume of the resulting therapeutic cell suspension comprises from 30xl06cells to 90xl06cells. According to some embodiments, the volume of the resulting therapeutic cell suspension comprises from 50xl06cells to 90xl06cells. According to some embodiments, the volume of the resulting therapeutic cell suspension comprises from 60xl06cells to 80xl06cells or about 75xl06cells.

[0061] According to some embodiments, the method further comprises washing the cells with a solution for injection before step (ii). According to any one of the above embodiments, the method further comprises washing the cells with a solution for injection after the addition of a solution for injection comprising HSA and before step (ii). According to some embodiments, the washing comprises centrifuging the cells, discarding the supernatant and diluting the cells in a solution for injection. The step of washing may be repeated 1, 2, 3 or more times.

[0062] According to some embodiments, the present invention provides a therapeutic cell suspension obtained or obtainable by a method according to any one of the above embodiments.

[0063] As discussed above, the method of the present invention using 2-steps, i.e. thawing into small amount of solution for injection and further dilution provides a much higher rate of viable and recovered cells. Even more than that, using HSA in the solution for injection in the first step increases the viability of the cells after thawing even more. According to any one of the above embodiments, at least 85% of the cells in the resulting therapeutic cell suspension are viable cells. According to any one of the above embodiments, at least 90% of the cells in the resulting therapeutic cell suspension are viable cells. According to any one of the above embodiments, at least 92% of the cells in the resulting therapeutic cell suspension are viable cells. According to some embodiments, the cells are viable after 2, 3, or 4 hours when maintained at a temperature of 2-8°C. According to some embodiments, the cells are viable after 5 or 6 hours when maintained at a temperature of 2- 8 °C.

[0064] According to another aspect, the present invention provides a method of treatment of a disease or disorder in a subject, comprising the steps of:

[0065] (i) providing a frozen cry opreserving composition according to any one of the above aspects and embodiments comprising stem cells;

[0066] (ii) thawing the composition of step (i) into a solution for injection, optionally comprising HSA;

[0067] (iii) diluting the composition of step (ii) with a solution for injection to achieve a therapeutic cell suspension for injection comprising stem cells with a concentration of from IxlO6to lOOxlO6cells / ml; and

[0068] (iv) administering the composition obtained in step (iii) to the subject.

[0069] According to some embodiments, aspect, the present invention provides a method of treatment of a neurodegenerative disease or disorder in a subject, comprising the steps of:

[0070] (i) providing a frozen cryopreserving composition according to any one of the above aspects and embodiments comprising stem cells;

[0071] (ii) thawing the composition of step (i) into a solution for injection;

[0072] (iii) diluting the composition of step (ii) with a solution for injection to achieve a therapeutic cell suspension for injection comprising stem cells with a concentration of from IxlO6to lOOxlO6cells / ml; and

[0073] (iv) intrathecally administering the composition obtained in step (iv) to the subject.

[0074] All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

[0075] According to some embodiments, the cryopreserving composition comprises less than 3% v / v DMSO, less than 2.5% v / v DMSO, from 1 to 3% v / v DMSO, or from 0.5 to 2.5% v / v DMSO. According to some embodiments, the cry opreserving composition comprises from 1.8 to 2.3% v / v DMSO or about 2% v / v DMSO. According to some embodiments, the cryopreserving composition comprises from IxlO6to 5xl08cells / ml. According to some embodiments, the cry opreserving composition comprises from IxlO7to 1.2xl08cells / ml. According to some embodiments, the cry opreserving composition comprises from 1.5xl07to IxlO8cells / ml. According to some embodiments, the cryopreserving composition comprises from 10xl06to lOOxlO6cells / ml.

[0076] According to some embodiments, the solution for injection is selected from PlasmaLyte, PBS, Saline solution, and Ringer’s Lactate. According to some embodiments, the solution for injection is PlasmaLyte 148.

[0077] According to some embodiments, the method comprises thawing the cryopreserving composition in step (ii). The thawing is performed as described in any one of the above aspects and embodiments. According to some embodiments, thawing comprises adding a solution for injection comprising from 1 to 20% w / v of HSA to the composition of step (ii). Therefore, according to some embodiments, the solution for injection in step (ii) comprises from 1 to 15% of HSA. According to some embodiments, the concentration of HSA in the added solution is from 5 to 15%, from 7 to 12%, from 8 to 12% or about 10%. According to some embodiments, the ratio between the cry opreserving composition and the solution for injection in step (ii) is from 8:1 to 1:3. According to some embodiments, the ratio between the cry opreserving composition and the solution for injection in step (ii) is from 5:1 to 1:2. According to some embodiments, the ratio between the cryopreserving composition and the solution for injection in step (ii) is from 3:1 to 1:2. According to some embodiments, the ratio between the cryopreserving composition and the solution for injection in step (ii) is from 2:1 to 1:2, or about 1.2:1 According to some embodiments, the ratio between the volume of cryopreserving composition and solution for injection comprising HSA is from 10:1 to 1:2. According to some embodiments, the ratio between the volume of cryopreserving composition and solution for injection comprising HSA is from 10: 1 to 1 : 10. According to some embodiments, the concentration of HSA in the added solution is from 1 to 10 % w / v. According to some embodiments, the concentration of HSA in the added solution is from 1 to 10, from 2 to 9 % w / v, from 3 to 8% w / v, from 4 to 7 % w / v, from 4 to 6% w / v of HSA. According to some embodiments, the concentration of HSA in the added solution is from 3 to 7%. According to some embodiments, the concentration of HSA in the added solution is from 4 to 6% or about 5%.

[0078] According to some embodiments, aspect, the present invention provides a method of treatment of a neurodegenerative disease or disorder in a subject, comprising the steps of: (i) thawing a frozen cryopreserving composition according to any one of the above aspects and embodiments comprising stem cells into a solution for injection;

[0079] (ii) diluting the composition of step (i) with a solution for injection to achieve a therapeutic cell suspension for injection comprising stem cells with a concentration of from IxlO6to lOOxlO6cells / ml; and

[0080] (iii) intrathecally administering the composition obtained in step (ii) to the subject. According to some embodiments, thawing in step (i) comprises adding a solution for injection comprising from 1 to 20% w / v of HSA, as described above.

[0081] In some embodiments, the resulting therapeutic cell suspension comprises from IxlO5to 5xl07cells / ml. In some embodiments, the resulting therapeutic cell suspension comprises from 5xl05to 50xl06cells / ml. In some embodiments, the therapeutic cell suspension comprises from IxlO6to IxlO7cells / ml. In some embodiments, the therapeutic cell suspension comprises from IxlO6to 10xl06cells / ml.

[0082] In some embodiments, the therapeutic cell suspension for injection comprises less than 0.5% v / v of DMSO as defined herein above. In some embodiments, the therapeutic cell suspension for injection comprises less than 0.5% w / v of HSA as defined herein above. According to some embodiments, the stem cells are human stem cells derived from the lamina propria of the oral mucosa as defined herein above.

[0083] In some embodiments, the neurodegenerative disease is a disease of basal ganglia and brain stem. In some embodiments, the neurodegenerative disease of basal ganglia and brain stem is selected from Multiple System Atrophy, Parkinsonism, Idiopathic Parkinson's Disease, Progressive Supranuclear Palsy, Corticobasal Degeneration, Striatonigral Degeneration, Shy-Drager Syndrome, Olivopontocerebellar Atrophy, ALS, and Huntington Disease. According to some embodiments, the neurodegenerative disease is Multiple System Atrophy.

[0084] According to yet another aspect, the present invention provides a method for cryopreserving human stem cells, the method comprises dissolving the stem cells in a cryopreserving composition comprising less than 4% w / v of dimethyl sulfoxide (DMSO) and freezing the cells at a temperature below -70°C wherein the final concentration of the cells is at least IxlO7cells / ml. According to some embodiments, the human stem cells derived from the lamina propria of the oral mucosa (hOMSC)

[0085] All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well. According to some embodiments, human stem cells are derived from the lining and masticatory oral mucosa. According to some embodiments, the human stem cells are characterized by simultaneously expressing the following markers: Oct-4, SSEA4, Nanog, Sox2, KLF4, c-MYC, nestin, p-III tubulin, p75, CD29, CD 73, CD90, CD105, and CD166. According to some embodiments, the cells are characterized by simultaneously expressing the following markers KLF4, c-MYC, nestin, P -III tubulin, and p75, and being negative for CD45 and CD31. According to some embodiments, the cells are characterized by simultaneously expressing the following markers: OCT-4, SSEA4, NANOG, SOX2, KEF4, c-MYC, nestin, p-III tubulin, p75, CD29, CD 73, CD90, CD105, and CD166; the cells are negative for CD45 and CD31. According to some embodiments, the cells are isolated cells. According to some embodiments, the cells are pluripotent or multipotent stem cells.

[0086] According to some embodiments, the final concentration of DMSO in the cryopreserving composition comprising the hOMSC is less than 4 % v / v or less than 3 % v / v. According to some embodiments, the final concentration of DMSO in the cryopreserving composition comprising the hOMSC is from 2% to 3% v / v of DMSO. According to some embodiments, the final concentration of DMSO in the cryopreserving composition comprising the hOMSC is from 1 to 4% v / v. According to some embodiments, the final concentration of DMSO in the cryopreserving composition comprising the hOMSC is from 1 to 3% v / v. According to some embodiments, the final concentration of DMSO in the cry opreserving composition comprising the hOMSC is from 1.5% to 2.5% v / v of DMSO (v / v). According to some embodiments, the final concentration of DMSO in the cryopreserving composition comprising the hOMSC is about 2%v / v of DMSO (v / v).

[0087] According to some embodiments, the final concentration of the cells is from 2xl07to 1.5xl08cells / ml. According to some embodiments, the final concentration of the cells is from 2.5xl07to IxlO8cells / ml. According to some embodiments, the final concentration of the cells is from 3xl07to 9xl07cells / ml. According to some embodiments, the final concentration of the cells is from 4xl07to 7xl07cells / ml.

[0088] According to some embodiments, the method comprises adding up to 10% w / v, up to 9% w / v, up to 8% w / v, up to 7% w / v, up to 6% w / v, up to 5% w / v, up to 4% w / v, up to 3% w / v, up to 2% w / v, or up to 1% w / v HSA up to 9%. According to some embodiments, the method is substantially devoid of adding HSA.

[0089] According to some embodiments, the cells cryopreserved according to the methods of the present invention are stable in a frozen state for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. According to some embodiments, the cells cryopreserved according to the methods of the present invention are stable in the frozen state for 6 months. According to some embodiments, the cells cryopreserved according to the methods of the present invention are stable in the frozen state for 12 months. According to some embodiments, the cells cryopreserved according to the methods of the present invention are stable in the frozen state for 18 months. According to some embodiments, the cells cryopreserved according to the methods of the present invention are stable in the frozen state for 24 months. According to some embodiments, the cells cryopreserved according to the methods of the present invention are stable in the frozen state for 36 months. According to some embodiments, the cells cryopreserved according to the methods of the present invention are stable in the frozen state for 1, 2, 3, 4 or 5 years. The term "stable" when referring to frozen stem cells contemplates that the cells at least 85% of the cells after thawing according to the methods of the present invention are viable cells. According to any one of the above embodiments, at least 90% of the cells after thawing according to the methods of the present invention are viable cells. According to any one of the above embodiments, at least 92% of the cells after thawing according to the methods of the present invention are viable cells. According to some embodiments, the cells are viable after 2, 3, or 4 hours when maintained at a temperature of 2-8°C. According to some embodiments, the cells are viable after 5 or 6 hours when maintained at a temperature of 2-8°C.

[0090] Clause 1. A cryopreserving composition comprising stem cells, wherein the composition comprises from 0.5 to 4% v / v of dimethyl sulfoxide (DMSO) and at least 5xl06cells / ml of stem cells, wherein the cells are human stem cells derived from the lamina propria of the oral mucosa (hOMSC).

[0091] Clause 2. The composition of any clauses herein, particularly clause 1, comprising less than 3% v / v of DMSO.

[0092] Clause 3. The composition of any clauses herein, particularly clause 2, comprising from 1.5% to 2.5% v / v of DMSO.

[0093] Clause 4. The composition of any clauses herein, particularly clause 3, comprising less than or about 2% v / v of DMSO.

[0094] Clause 5. The composition of any clauses herein, particularly clauses 1 to 4, comprising from IxlO6to 5xl08cells / ml.

[0095] Clause 6. The composition of any clauses herein, particularly clause 5, comprising from 10xl06to lOOxlO6cells / ml.

[0096] Clause 7. The composition of any clauses herein, particularly clauses 1 to 6, wherein the composition comprises less than 10% Human Serum Albumin (HSA). Clause 8. The composition of any clauses herein, particularly clause 7, wherein the composition comprises from 0.5 to 8% HSA.

[0097] Clause 9. The composition of any clauses herein, particularly clauses 1 to 8, wherein the composition is devoid of HSA.

[0098] Clause 10. The composition of any clauses herein, particularly clauses 1 to 9, wherein the composition is free of non-human animal components.

[0099] Clause 11. The composition of any clauses herein, particularly clauses 1 to 10, wherein DMSO is the only cryopreservative agent.

[0100] Clause 12. A therapeutic cell suspension for injection, comprising the cryopreserving composition of any clauses herein, particularly clauses 1 to 11, and a solution for injection. Clause 13. The therapeutic cell suspension of any clauses herein, particularly clause 12, wherein the solution for injection is selected from PlasmaLyte, Saline solution, PBS, and Ringer’s Lactate.

[0101] Clause 14. The therapeutic cell suspension of any clauses herein, particularly clauses 12 or 13, comprising less than 0.5% v / v of DMSO.

[0102] Clause 15. The therapeutic cell suspension of any clauses herein, particularly clause 14, comprising less than 0.1% v / v of DMSO.

[0103] Clause 16. The therapeutic cell suspension of any clauses herein, particularly clauses 11 to 14, comprising from 0.01 to 0.4 % v / v of DMSO.

[0104] Clause 17. The therapeutic cell suspension of any clauses herein, particularly clauses 11 to 16, comprising less than 1% of HSA (w / v).

[0105] Clause 18. The therapeutic cell suspension of any clauses herein, particularly clause 17, comprising less than 0.3% of HSA (w / v).

[0106] Clause 19. The therapeutic cell suspension of any clauses herein, particularly clauses 11 to 16, wherein the composition is devoid of HSA.

[0107] Clause 20. The therapeutic cell suspension of any clauses herein, particularly clauses 11 to 19, comprising from IxlO5to IxlO7cells / ml.

[0108] Clause 21. The therapeutic cell suspension of any clauses herein, particularly clauses 11 to 20, for use in the treatment of a neurodegenerative disease or disorder, preferably for use in treating Multiple System Atrophy.

[0109] Clause 22. A method for preparing a therapeutic cell suspension for injection comprising stem cells, wherein the method comprising the steps: (i) thawing the cryopreserving composition of any clauses herein, particularly clauses 1 to 11 into a solution for injection, wherein the ratio between the cryopreserving composition and the solution for injection is from 8:1 to 1:3, and (ii) diluting the solution obtained in step (i) with a solution for injection to obtain the therapeutic cell suspension for injection comprising less than 0.5% DMSO.

[0110] Clause 23. The method of any clauses herein, particularly clause 22, wherein the cryopreserving composition comprises from 10xl06to lOOxlO6cells / ml.

[0111] Clause 24. The method of any clauses herein, particularly clauses 22 to 23, wherein the solution for injection is selected from PlasmaLyte, Saline solution, PBS, and Ringer’s Lactate or a combination of thereof.

[0112] Clause 25. The method of any clauses herein, particularly clauses 22 to 24, wherein no HSA is added.

[0113] Clause 26. The method of any clauses herein, particularly clauses 22 or 24, comprises adding a solution for injection in step (i) comprising from 1 to 20% HSA.

[0114] Clause 27. The method of any clauses herein, particularly clause 26, wherein the concentration of HSA in the solution for injection in step (i) is from 5 to 15 % w / v.

[0115] Clause 28. The method of any clauses herein, particularly clauses 22 to 27, wherein the final concentration of HSA in the resulting therapeutic cell suspension for injection is less than 0.5% w / v.

[0116] Clause 29. The method of any clauses herein, particularly clauses 22 to 28, wherein the final volume of the therapeutic cell suspension for injection comprising stem cells is from 5 to 40 ml or from 5 to 20 ml.

[0117] Clause 30. A method of treatment of a neurodegenerative disease or disorder in a subject, comprising the steps of:

[0118] (i) providing a cryopreserving composition of any clauses herein, particularly clauses 1 to 11;

[0119] (ii) thawing the composition of step (i), optionally, into a solution for injection comprising from 1 to 20% w / v of HSA;

[0120] (iii) diluting the composition of step (ii) with a solution for injection to achieve the concentration of stem cells of from IxlO6to 50xl06cells / ml, DMSO from 0 to 0.4% v / v and HSA, if added in step (ii) less than 0.3 % w / v, and

[0121] (iv) intrathecally administering the composition obtained in step (iii ) to the subject, optionally, wherein the ratio between the cryopreserving composition and the solution for injection in step (ii) is from 8:1 to 1:3.

[0122] Clause 31. A method for cryopreserving human stem cells derived from the lamina propria of the oral mucosa, the method comprises dissolving the stem cells in a cryopreserving composition comprising from 0.5 w / v to 4% w / v of dimethyl sulfoxide (DMSO) and freezing the cells at a temperature below -70°C wherein the final concentration of the cells is at least IxlO7cells / ml.

[0123] Clause 32. The method of any clauses herein, particularly clause 31, wherein the cryopreserving composition comprises less than 3 % w / v or from 1.5% to 2.5% v / v of DMSO (w / v).

[0124] Clause 33. The method of any clauses herein, particularly clauses 31 to 32, wherein the final concentration of the cells is from 2xl07to 1.5xl08cells / ml.

[0125] Clause 34. The method of any clauses herein, particularly clauses 31 to 33, wherein the cryopreserving composition comprises less than 10%, or less than 3% Human Serum Albumin (HSA) or substantially devoid of HSA.

[0126] Clause 35. The method of any clauses herein, particularly clauses 31 to 34, wherein the cryopreserving composition is free of non-human animal components.

[0127] Clause 36. The method of any clauses herein, particularly clauses 31 to 35, wherein DMSO is the only cryopreserving agent.

[0128] Clause 37. A therapeutic cell suspension for injection, comprising from O.lxlO5to IxlO7cells / ml, from 0.01 to 0.5% v / v of DMSO, optionally from 0.01 to 0.5% w / v of HSA, and a solution for injection, wherein the cells are human stem cells derived from the lamina propria of the oral mucosa (hOMSC).

[0129] Clause 38. The composition, the cell suspension or the method according to any one of claims 1 to 37, wherein the cells are derived from the lining and masticatory oral mucosa.

[0130] Clause 39. The composition, the cells suspension or the method of any clauses herein, particularly clause 38, wherein said cells are characterized by simultaneously expressing the following markers: Oct-4, SSEA4, Nanog, Sox2, KLF4, c-MYC, nestin, P-III tubulin, p75, CD29, CD 73, CD90, CD105, and CD166.

[0131] Clause 40. The method of any clauses herein, particularly clause 39 or 40, wherein said cells are characterized by simultaneously expressing the following markers: KLF4, c-MYC, nestin, P-III tubulin, and p75, and wherein the cells are negative for CD45 and CD31.

[0132] According to any one of the above embodiments, the cry opreserving composition is free of non-human animal components.

[0133] The terms “a,” “an,” and “the” are used herein interchangeably and mean one or more. The term “and / or” is used to indicate one or both stated cases may occur, for example A and / or B includes, (A and B) and (A or B). The term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination if the combination is not mutually exclusive.

[0134] The terms “comprising”, "comprise(s)", "include(s)", "having", "has" and "contain(s)," are used herein interchangeably and have the meaning of “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. The terms “have”, “has”, having” and “comprising” may also encompass the meaning of “consisting of’ and “consisting essentially of’, and may be substituted by these terms. The term “consisting of’ excludes any component, step or procedure not specifically delineated or listed. The term “consisting essentially of’ means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.

[0135] As used herein, the term “about”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + / -10%, or + / -5%, + / -1%, or even + / -0.1% from the specified value.

[0136] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting to the present invention.

[0137] EXAMPLES

[0138] Example 1. Viability test for cells frozen at high concentrations

[0139] To spare the necessity of specialized preparation of cryopreserved stem cells, prior to administration thereof into a subject, an attempt was made to reduce the concentration of cryoprotectant, which is potentially harmful to human health, in the cryopreservation composition, while retaining the necessary cell quantity required for treatment of the subject.

[0140] To this end, cryopreservation compositions were prepared using CryoStor® CS10 and CryoStor® CSB at a ratio of 1 :4, thereby achieving 2% of dimethyl sulfoxide (DMSO) (w / v). Escalating concentrations of human Oral Mucosa Stem Cells (hOMSC) were suspended in the compositions, as shown in Table 1 below, and were stored at -190°C for 7 days. No albumin was used in the cryopreservation compositions. On the 8th day, the cells were thawed and diluted in PlasmaLyte (PL) 148. Following the thawing of the cells, the cells were quantified and their viability was tested using NucleoCounter® NC-200™. Samples of the cells were then stained with Trypan blue and tested for viability The cells were then seeded on well plates in a growth medium including 10% fetal bovine serum (FBS). 24 hours following seeding, the cells were recovered and were again counted and tested for viability. As a positive control, hOMSC at a concentration of 6.6xl06cells / ml were cryopreserved in a composition containing DMSO at a concentration of 5% (v / v). The results of are set out in Table 1 below.

[0141] Table 1. Quantification and viability tests of high-concentration cryopreserved cells As can be seen in Table 1 above, unexpectedly, the number and viability of cells frozen and cryopreserved in the low concentration of DMSO (2%) were equivalent to those of the Control cells that were frozen and cryopreserved in 5% DMSO. Surprisingly, these high recovery and viability results persevered also at high concentrations of 75 million cells per milliliter of the cryopreservation composition. To further test the potency of the thawed cells, ELISA assay was performed for VEGF,

[0142] HGF, and CXCL12 proteins, which are indicative of the healthy functionality of the cells. The results of the tests, as compared to the positive control, are shown in Table 2, below.

[0143] Table 2: ELISA assay for testing cell potency

[0144] As can be seen in Table 2 above, the protein expression which was detected from hOMSC frozen in 2% DMSO, at the high concentrations of 2.73xl07, 4.40xl07, and 7.55xl07cells / mL, was of the same order of magnitude as the positive control in the case of HGF, and in the case of VEGF and CSCL12 was even higher than the positive control, indicating high potency of the concentrated thawed cells.

[0145] The high cell concentrations in 2% DMSO were substantially as good and in some criteria even better than the control.

[0146] In our further experiments, we showed that increasing the concentration of cells, e.g. above 106improves the rate of viable cells upon thawing.

[0147] It was further concluded that in this study the expression of VEGF from cells frozen in higher concentration is superior to cells frozen in low cell concentration.

[0148] Example 2. Viability test for cells frozen at different HSA concentrations

[0149] To further assess the potency of concentrated cells frozen in a cryopreserving composition containing low levels of DMSO, cells with similar (high) concentrations were examined under different thawing regimens. hOMSC were cryopreserved at a concentration of 30xl06cells / ml in a composition containing 2% DMSO (w / v) and were then divided into 3 groups. The cells were then thawed by dilution in PlasmaLyte (PL) 148, including different concentrations of Human Serum Albumin (HSA), at different dilution ratios. As a positive control, hOMSC at a concentration of 20xl06cells / ml were cryopreserved in a composition containing 5% DMSO (w / v) and thawed in PL including 5% HSA. The thawing regimens of each of the groups are set out in Table 3 below.

[0150] Table 3: Thawing regimens of high-concentration cryopreserved cells

[0151] Following thawing and dilution in PL (with or without HSA), each of the cell groups was tested for cell recovery (designated "TO" and calculated as percent of cells recovered from thawing) at 3-time frames, namely, immediately following thawing, 20 minutes following thawing, and 40 minutes following thawing. The recovered cells were further tested for viability. The cell counting and viability tests were performed as detailed in Example 1 above. The respective cell recovery and viability results of each group are set out in Table 4 below. It is noted that all tested cell groups (including each of the time frames) were seeded for 24hr from each time point, and all displayed 100% recovery from seeded cells.

[0152] Table 4: Cell recovery and viability following thawing of high-concentration cryopreserved cells

[0153] As can be seen in the results above, the cell viability was very high in all cell groups. Method 4 (i.e., thawing in the presence of 5% HSA followed by a dilution such that the final concentration of HSA is less than 0.5%) seems to be most effective as it does not require manipulation of cells such as centrifugation.

[0154] Example 3. Protocol for cell cryopreservation at high cell concentration and low DMSO / HSA concentration.

[0155] This experiment provides a protocol for freezing cells at a high concentration with a low concentration of DMSO and thawing them by dilution. The aim is to provide a viable drug product containing a low residual amount of HSA and DMSO.

[0156] The experiment was performed with two new specific cryo-cell banks and one bank which was frozen for ~1 year before thawing and testing.

[0157] The cells were cultured in a growth medium until reaching the desired number of cells and then cryopreserved in a freezing solution containing DMSO. The thawing was carried out while maintaining the following parameters:

[0158] 1. Final cell concentration: 5xl06cells per ml.

[0159] 2. The concentration of HSA in the final product should not exceed 0.3%.

[0160] 3. The concentration of DMSO in the final product should not exceed 0.4%.

[0161] Cell thawing is done in duplicates.

[0162] Two thawing protocols are tested and compared:

[0163] Protocol- 1 -

[0164] Control protocol for cell thawing.

[0165] Cell bank were frozen in freezing solution consisting of 5%DMSO, at high concentration of 25xl06cells / ml and low concentration of 10xl06cells / ml

[0166] Step A - Add the thawed cells to a tube containing PlasmaLyte (PL)+5%HSA.

[0167] Step B - Centrifuge the tube containing the cells.

[0168] Step C - Resuspension of the cell pellet with PL. Protocol-2: A Protocol for cells frozen with 2%DMS0

[0169] Cell banks were frozen in freezing solution consisting of 2%DMS0 at 3 escalating cell concentrations; 30xl06cells / ml, 50xl06cells / ml and 70xl06cells / ml

[0170] Cell thawing was done in two steps (with HSA in the first step).

[0171] Step A - Add PL with 5%HSA to the thawed test tube at a volume ratio of final injective volume / HSA total dilution factor. Mix gently.

[0172] Step B - Dilute the contents of the test tube to a final volume with PL to reach cell concentration of 5x106cells / ml.

[0173] The experimental matrix is shown in the following Table 5.

[0174] Table 5. Experimental procedure.

[0175] The experiment is designed to test the stability of the cryopreserved cells over time; Time 0 after freezing, 6 months and 12 months.

[0176] At each stability time point, two time points of stability after thawing were tested: T=0, T=40 min.

[0177] Each thawed test tube was tested under the hOMSC300 Drug Product release quality control tests (except safety tests) in two formulation endpoints, immediately after formulation end (TO), and 40 minutes after formulation (incubation at room temperature for 40 minutes). The QC results are presented in Table 6.

[0178] Table 6. Results of stability and cell viability at T=0 and T=40 min. In an additional experiment, an additional protocol is tested.

[0179] This protocol is performed with the escalating cell concentration (30xl06cells / ml, 50xl06cells / ml and 70xl06cells / ml), all banks are frozen with 2%DMS0, and thawing is performed in PL without HSA in one step.

[0180] Step A - Add PL without HSA to the thawed test tube at a volume ratio of cryopreserved cells:PL of about 1.5: 1 to 1:1.5.

[0181] Step B - Dilute the contents of the test tube to a final volume with PL, till the final dilution of 1 to 15.

[0182] Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims

Claims

CLAIMS1. A cry opreserving composition comprising stem cells, wherein the composition comprises from 0.5 to 4% v / v of dimethyl sulfoxide (DMSO) and at least 5xl06cells / ml of stem cells, wherein the stem cells are human stem cells derived from the lamina propria of the oral mucosa (hOMSC).

2. The composition according to claim 1, comprising less than 3% v / v of DMSO.

3. The composition according to claim 2, comprising from 1.5% to 2.5% v / v of DMSO.

4. The composition according to claim 3, comprising less than or about 2% v / v of DMSO.

5. The composition according to any one of claims 1 to 4, comprising from 5xl06to 5xl08cells / ml.

6. The composition according to claim 5, comprising from 10xl06to lOOxlO6cells / ml.

7. The composition according to any one of claims 1 to 6, wherein the composition comprises less than 10% Human Serum Albumin (HSA).

8. The composition according to claim 7, wherein the composition comprises from 0.5 to 8% HSA.

9. The composition according to any one of claims 1 to 6, wherein the composition is devoid of HSA.

10. The composition according to any one of claims 1 to 9, wherein the composition is free of non-human animal components.

11. The composition according to any one of claims 1 to 10, wherein DMSO is the only cryopreservative agent.

12. A therapeutic cell suspension for injection, comprising the cryopreserving composition of any one of claims 1 to 11, and a solution for injection.

13. The therapeutic cell suspension according to claim 12, wherein the solution for injection is selected from PlasmaLyte, Saline solution, PBS, and Ringer’s Lactate.

14. The therapeutic cell suspension according to claim 12 or 13, comprising less than 0.5% v / v of DMSO.

15. The therapeutic cell suspension according to claim 14, comprising less than 0.1% v / v of DMSO.

16. The therapeutic cell suspension according to any one of claims 11 to 14, comprising from 0.01 to 0.4 % v / v of DMSO.

17. The therapeutic cell suspension according to any one of claims 11 to 16, comprising less than 1% of HSA (w / v).

18. The therapeutic cell suspension according to claim 17, comprising less than 0.3% of HSA (w / v).

19. The therapeutic cell suspension according to any one of claims 11 to 16, wherein the composition is devoid of HSA.

20. The therapeutic cell suspension according to any one of claims 11 to 19, comprising from IxlO5to IxlO7cells / ml.

21. The therapeutic cell suspension according to any one of claims 11 to 20, for use in the treatment of a neurodegenerative disease or disorder, preferably for use in treating Multiple System Atrophy.

22. A method for preparing a therapeutic cell suspension for injection comprising stem cells, wherein the method comprising the steps: (i) thawing the cryopreserving composition according to any one of claims 1 to 11 into a solution for injection, wherein the ratio between the cryopreserving composition and the solution for injection is from 8:1 to 1:3, and (ii) diluting the solution obtained in step (i) with a solution for injection to obtain the therapeutic cell suspension for injection comprising less than 0.5% DMSO.

23. The method according to claim 22, wherein the cry opreserving composition comprises from 10xl06to lOOxlO6cells / ml.

24. The method according to any one of claims 22 to 23, wherein the solution for injection is selected from PlasmaLyte, Saline solution, PBS, and Ringer’s Lactate or a combination of thereof.

25. The method according to any one of claims 22 to 24, wherein no HSA is added.

26. The method according to claim 22 or 24, comprises adding a solution for injection in step (i) comprising from 1 to 20% HSA.

27. The method according to claim 26, wherein the concentration of HSA in the solution for injection in step (i) is from 5 to 15 % w / v.

28. The method according to any one of claims 22 to 27, wherein the final concentration of HSA in the resulting therapeutic cell suspension for injection is less than 0.5% w / v.

29. The method according to any one of claims 22 to 27, wherein the final volume of the therapeutic cell suspension for injection comprising stem cells is from 5 to 40 ml or from 5 to 20 ml.

30. A method of treatment of a neurodegenerative disease or disorder in a subject, comprising the steps of:(i) providing a cry opreserving composition to any one of claims 1 to 11;(ii) thawing the composition of step (i), optionally, into a solution for injection comprising from 1 to 20% w / v of HSA;(iii) diluting the composition of step (ii) with a solution for injection to achieve the concentration of stem cells of from IxlO6to 50xl06cells / ml, DMSO from 0 to 0.4% v / v and HSA, if added in step (ii) less than 0.3 % w / v, and(iv) intrathecally administering the composition obtained in step (iii ) to the subject, optionally, wherein the ratio between the cryopreserving composition and the solution for injection in step (ii) is from 8:1 to 1:3.

31. A method for cry opreserving human stem cells derived from the lamina propria of the oral mucosa, the method comprises dissolving the stem cells in a cryopreserving composition comprising from 0.5 w / v to 4% w / v of dimethyl sulfoxide (DMSO) and freezing the cells at a temperature below -70°C wherein the final concentration of the cells is at least IxlO7cells / ml.

32. The method according to claim 31, wherein the cry opreserving composition comprises less than 3 % w / v or from 1.5% to 2.5% v / v of DMSO (w / v).

33. The method according to any one of claims 31 to 32, wherein the final concentration of the cells is from 2xl07to 1.5xl08cells / ml.

34. The method according to any one of claims 31 to 33, wherein the cry opreserving composition comprises less than 10%, or less than 3% of Human Serum Albumin (HSA) or substantially devoid of HSA.

35. The method according to any one of claims 31 to 34, wherein the cryopreserving composition is free of non-human animal components.

36. The method according to any one of claims 31 to 35, wherein DMSO is the only cryopreserving agent.

37. A therapeutic cell suspension for injection, comprising from 0. IxlO5to IxlO7cells / ml, from 0.01 to 0.5% v / v of DMSO, optionally from 0.01 to 0.5% w / v of HSA, and a solution for injection, wherein the cells are human stem cells derived from the lamina propria of the oral mucosa (hOMSC).

38. The composition, the cells suspension or the method according to any one of claims 1 to 37, wherein the cells are derived from the lining and masticatory oral mucosa.

39. The composition, the cells suspension or the method according to claim 38, wherein said cells are characterized by simultaneously expressing the following markers: Oct-4, SSEA4, Nanog, Sox2, KLF4, c-MYC, nestin, p-III tubulin, p75, CD29, CD 73, CD90, CD105, and CD166.

40. The method according to claim 39 or 40, wherein said cells are characterized by simultaneously expressing the following markers: KLF4, c-MYC, nestin, P-III tubulin, p75 and wherein the cells are negative for CD45 and CD31.

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