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US 12,433,916 B2Granted
Cell bank of high concentration adipose-derived stem cell composition and related methods
AssigneeRigshospitalet
InventorsJens Kastrup, Annette Ekblond, Mandana Haack-Sørensen
Life of this patent
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Abstract
The present invention relates to adipose-derived stem cells (ASCs) and compositions, as well as methods for preparing and using such ASCs and compositions for therapy.
Claims13
A cell bank comprising a plurality of vials stored under freezing conditions, each vial comprising a pharmaceutical composition comprising a suspension of an immunosuppressive adult human adipose-tissue derived stem cell (ASC) population in a protein-free cryoprotectant at a concentration in the range of 1.5×107 cells per mL to 5×107 cells per mL,
- wherein at least 80% of the ASC population express CD90, CD105, CD13, CD73, CD166, and CD29, and at most 15% of the ASC population express CD45, CD31, CD14, and CD19,
- wherein immediately after thawing a vial at least 80% of the stem cell population are viable cells, and
- wherein the protein-free cryoprotectant comprises a 1:10 to 1:20 mixture of dimethyl sulfoxide (DMSO) and an aqueous solution comprising:
- (a) one or more electrolytes selected from the group consisting of potassium ions at a concentration ranging from 35-45 mM, sodium ions ranging from 80-120 mM, magnesium ions ranging from 2-10 mM, and calcium ions ranging from 0.01-0.1 mM;
- (b) a macromolecular oncotic agent selected from the group consisting of a polysaccharide and colloidal starch;
- (c) a biological pH buffer;
- (d) at least one of sucrose and glucose;
- (e) mannitol;
- (f) at least one impermeant anion selected from the group consisting of lactobionate, gluconate, citrate and glycerophosphate;
- (g) at least one member selected from the group consisting of adenosine, fructose, ribose and adenine;
- and
- (h) glutathione.
9 more elements
A process for preparing a pharmaceutical composition comprising an adult human stem cell population,
- wherein at least 80% of the stem cell population express CD90, CD105, CD13, CD73, CD166, and CD29, and at most 15% of the stem cell population express CD45, CD31, CD14, and CD19,
- said process comprising the steps of
- (i) adding the stromal vascular fraction (SVF) of a lipoaspirate collected from a donor to a bioreactor wherein at least one surface is pre-treated to promote adhesion of adult human stem cells;
- (ii) in the bioreactor, cultivating adherent cells to confluence in a serum-free culture medium supplemented with human platelet lysate;
- (iii) detaching the adherent cells;
- (iv) freezing the detached cells in a protein-free cryoprotectant at a concentration of at least 1×106 cells/mL;
- (v) thawing the frozen cells and repeating steps (ii) and (iii), and optionally (iv), once, twice, or three times;
- (vi) freezing the detached cells at a concentration of at least 1.5×107 cells/mL;
- and
- (vii) prior to clinical use, thawing the frozen composition
- thereby obtaining the pharmaceutical composition, wherein immediately after thawing a vial at least 80% of the stem cell population are viable cells,
- wherein the protein-free cryoprotectant comprises a 1:10 to 1:20 mixture of DMSO and an aqueous solution comprising:
- (a) one or more electrolytes selected from the group consisting of potassium ions at a concentration ranging from 35-45 mM, sodium ions ranging from 80-120 mM, magnesium ions ranging from 2-10 mM, and calcium ions ranging from 0.01-0.1 mM;
- (b) a macromolecular oncotic agent selected from the group consisting of a polysaccharide and colloidal starch;
- (c) a biological pH buffer;
- (d) at least one of sucrose and glucose;
- (e) mannitol;
- (f) at least one impermeant anion selected from the group consisting of lactobionate, gluconate, citrate and glycerophosphate;
- (g) at least one member selected from the group consisting of adenosine, fructose, ribose and adenine;
- and
- (h) glutathione.
18 more elements
A pharmaceutical composition comprising a suspension of an adult human adipose-tissue derived stem cell (ASC) population in a protein-free cryoprotectant, at a concentration in the range of 1.5×107 cells per mL to 5×107 cells per mL,
- wherein at least 80% of the ASC population express CD90, CD105, CD13, CD73, CD166, and CD29, and at most 15% of the ASC population express CD45, CD31, CD14, and CD19, and
- wherein the composition is obtained or obtainable according to a process comprising the steps of
- (i) adding the stromal vascular fraction (SVF) of a lipoaspirate collected from a donor to a bioreactor wherein at least one surface is pre-treated to promote adhesion of adult human stem cells;
- (ii) in the bioreactor, cultivating adherent cells to confluence in a serum-free culture medium supplemented with human platelet lysate;
- (iii) detaching the adherent cells;
- (iv) freezing the detached cells in a protein-free cryoprotectant at a concentration of at least 1×106 cells/mL;
- (v) thawing the frozen cells and repeating steps (ii) and (iii), and optionally (iv), once, twice, or three times,
- (vi) freezing the detached cells at a concentration in the range of 1.5×107 cells/mL to 5×107 cells/mL;
- and
- (vii) prior to clinical use, thawing the frozen composition,
- thereby obtaining the pharmaceutical composition, wherein immediately after thawing a vial at least 80% of the stem cell population are viable cells,
- wherein the protein-free cryoprotectant comprises a 1:10 to 1:20 mixture of DMSO and an aqueous solution comprising:
- (a) one or more electrolytes selected from the group consisting of potassium ions at a concentration ranging from 35-45 mM, sodium ions ranging from 80-120 mM, magnesium ions ranging from 2-10 mM, and calcium ions ranging from 0.01-0.1 mM;
- (b) a macromolecular oncotic agent selected from the group consisting of a polysaccharide and colloidal starch;
- (c) a biological pH buffer;
- (d) at least one of sucrose and glucose;
- (e) mannitol;
- (f) at least one impermeant anion selected from the group consisting of lactobionate, gluconate, citrate and glycerophosphate;
- (g) at least one member selected from the group consisting of adenosine, fructose, ribose and adenine;
- and
- (h) glutathione.
18 more elements
Dependencies are inferred from the claim text.