ALTERED ADHERENT STROMAL CELLS AND METHODS OF PRODUCING AND USING SAME

• US 20200131481A1
• Filed: 06/06/2016
• Published: 04/30/2020
• Est. Priority Date: 06/06/2016
• Status: Active Grant

• Current Assignee: Pluri Biotech Limited
• Original Assignee: Pluristem Ltd. (Pluri Inc.)
• Inventors: ABRAHAM, Eytan, RAVIV, Lior, WADAMANY, Maya, ESHKOL, Nadav
• Granted Patent: US 10,851,347 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/50 Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
• CPC Class Codes: C12M 21/00 Bioreactors or fermenters specially adapted for specific uses digesters for manure A01C3/023; apparatus for PCR B01L7/52; destroying or transforming solid waste B09B3/00; methods for genetic engineering C12N15/00, C12Q1/68; nucleic acid amplification reactions C12Q1/6844
• CPC Class Codes: C12M 23/14 Bags
• CPC Class Codes: C12N 2501/052 Lipopolysaccharides [LPS]
• CPC Class Codes: C12N 2501/24 Interferons [IFN]
• CPC Class Codes: C12N 2501/25 Tumour necrosing factors [TNF]
• CPC Class Codes: C12N 2513/00 3D culture
• CPC Class Codes: C12N 2533/30 Synthetic polymers
• CPC Class Codes: C12N 5/0668 Mesenchymal stem cells from other natural sources

Abstract

Disclosed herein are altered adherent stromal cells and methods of producing and utilizing same.

Claims

1. A method of producing modified adherent stromal cells (ASC), said method comprising the step of incubating ASC from placental or adipose tissue in a three-dimensional (3D) culture in a cytokine-containing growth medium, wherein one or more pro-inflammatory cytokines have been added to said cytokine-containing growth medium, thereby producing modified ASC.

2. The method of claim 1, said method comprising the steps of:

(a) incubating said ASC from placental or adipose tissue in a 3D culture in a first growth medium, wherein no pro-inflammatory cytokines have been added to said first growth medium; and

(b) subsequently performing the step of incubating said ASC in said cytokine-containing growth medium.

3. The method of claim 1, further comprising the subsequent step of harvesting said ASC by removing said ASC from said 3D culture.

4. The method of claim 1, wherein said 3D culture is performed in a bioreactor.

5. The method of claim 1, wherein said 3D culture comprises a synthetic adherent material.

12. The method of claim 1, wherein the step of incubating said ASC in said cytokine-containing growth medium is commenced during exponential phase.

13. The method of claim 4, wherein the step of incubating said ASC in said cytokine-containing growth medium comprises:

(i) adding a bolus of said one or more pro-inflammatory cytokines to a medium in said bioreactor, thereby generating said cytokine-containing growth medium; and

(ii) operably connecting said cytokine-containing growth medium with an external reservoir comprising an additional amount of said cytokine-containing growth medium.

14. The method of claim 1, wherein said one or more pro-inflammatory cytokines comprises Tumor Necrosis Factor alpha (TNF-alpha).

16. The method of claim 1, wherein said one or more pro-inflammatory cytokines comprises Interferon-Gamma (IFN-gamma).

17. The method of claim 14, wherein each of said one or more pro-inflammatory cytokines is present at a concentration between 1-12 nanograms/milliliter.

21. The method of claim 14, wherein said one or more pro-inflammatory cytokines further comprises Interferon-Gamma (IFN-gamma), wherein concentrations of said TNF-alpha and said IFN-gamma are both between 1-12 nanograms/milliliter;

and the ratio of said concentrations is between 1;

5 &

5;

1.

24. The method of claim 1, wherein said ASC originate from placenta tissue.

25. The method of claim 1, wherein said ASC originate from adipose tissue.

26. Modified ASC produced by the method of claim 1.

35. A pharmaceutical composition comprising the modified ASC of claim 26.

36. Conditioned medium derived from the method of claim 1.

46. A bioreactor comprising the modified ASC of claim 26.

6-11. -11. (canceled)

15. (canceled)

18-20. -20. (canceled)

22-23. -23. (canceled)

27. A population of cells, wherein the majority of said population express, on a population level, each of CD10, CD29, CD38, and CD40.

33. The population of cells of claim 27, wherein said population is at least 40% positive for at least one of a marker selected from:

CD42a, CD45Ra, CD77, CD243, and CD275.

28-29. -29. (canceled)

30. A population of cells, wherein the majority of said population express, on a population level, each of CD10, CD29, HLA-DR, and CD74.

31-32. -32. (canceled)

34. (canceled)

37-45. -45. (canceled)

47. (canceled)