Granted Patent B2

US 12,433,916 · App. 15/769,873 · Granted Oct 7, 2025

Inventors: Jens Kastrup (Birkerød, DK); Annette Ekblond (Birkerød, DK); Mandana Haack-Sørensen (Vanløse, DK)

Assignee: Rigshospitalet

A61K35/28A61K9/0019A61K38/19A61P29/00A61P37/06C12N5/0667A61P3/10A61P19/02A61P19/08C12N2500/34C12N2500/38C12N2500/40C12N2500/44C12N2500/60C12N2500/62C12N2500/84

Abstract

Claims (72)

1. 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 1.5×10 7 cells per mL to 5×10 7 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.

2. The cell bank of claim 1 , wherein at least about 80% of the ASC population express CD90, CD73, CD13, CD105, CD29, CD166, CD10, CD140b, CD160, CD204, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTβR and at most about 15% of the ASC population express CD45, CD19, CD14, CD106, CD31 and CD36.

3. The cell bank of claim 1 wherein, of the ASC population,

at least 90% express CD90, CD73, CD13, CD29 and CD166; at most 5% express CD45, CD19, CD14 and CD31; at most 10% express CD106; between 2 and 15% express CD36; at least 10% express CD146; at least 80% express CD105 and at most 40% express CD34; and/or

at least 90% express CD10, CD140b, CD160, CD204, CD272, CD44, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTβR; at least 80% express CD49a; at least 60% express CD258 and CD270 and at least 5% express CD200; at most 15% express CD15, CD152, CD163, CD18, CD274, CD39, CD40, CD62L, CD80 and CD86; and at most 30% express CXCR4.

4. The cell bank of claim 1 wherein, after thawing of a vial, the ASC population is such that upon interferon-gamma stimulation comprising cultivation for 3 days in the presence of 50 ng/ml interferon-gamma, the percentage of the ASC population expressing CD274 is increased to at least 80% and the expression level of CD54 on CD54-positive cells is increased at least 25-fold as compared to a control ASC population cultivated for 3 days in the absence of interferon-gamma.

5. The cell bank of claim 1 , wherein, immediately after thawing, the ASC population in a vial of the pharmaceutical composition is capable of a population doubling of at least 1 when cultured for 48 hours.

6. The cell bank of claim 1 , wherein each vial comprises about 5 mL of the pharmaceutical composition and wherein the cell concentration is in the range of about 2.0 ×10 7 to about 2.5×10 7 cells per mL.

7. 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 1×10 6 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 1.5×10 7 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.

8. The process of claim 7 , wherein

a) at least one surface of the bioreactor is pre-treated with a composition comprising or consisting of cryoprecipitate;

b) the culture medium comprises from about 2% to about 15% human platelet lysate;

c) in step (v), thawing the frozen cells and repeating steps (ii) and (iii) is carried out once; or

d) a combination of any two or more of (a) to (c).

9. 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 1.5×10 7 cells per mL to 5×10 7 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 1×10 6 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 1.5×10 7 cells/mL to 5×10 7 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.

10. The cell bank of claim 1 , wherein the ASC concentration is between 2×10 7 cells and 5×10 7 cells per mL.

11. The pharmaceutical composition according to claim 9 , wherein

a) at least one surface of the bioreactor is pre-treated with a composition comprising or consisting of cryoprecipitate;

b) the culture medium comprises from about 2% to about 15% human platelet lysate;

c) in step (v), thawing the frozen cells and repeating steps (ii) and (iii) is carried out once; or

d) a combination of any two or more of (a) to (c).

12. The cell bank of claim 10 , wherein the ASC concentration is from 2.0×10 7 to 2.4×10 7 cells per mL.

13. The cell bank of claim 10 , wherein the ASC concentration is 5×10 7 cells per mL.

Assignments (1)

Priority Claims (1)

Continuity (1)

References Cited (61)