MEDIA AND PROCESSES FOR THE EX VIVO PRODUCTION OF MEGAKARYOCYTES FROM HUMAN CD34+ CELLS

• US 20100227401A1
• Filed: 04/05/2010
• Published: 09/09/2010
• Est. Priority Date: 08/06/2003
• Status: Abandoned Application

• Current Assignee: Food Industry Research & Development Institute
• Original Assignee: Food Industry Research & Development Institute
• Inventors: HWANG, Shiaw-Min, CHEN, Te-Wei, YAO, Chao-Ling
• Application Number:
  US12/754,247
• Publication Number:
  US 20100227401A1
• Time in Patent Office: Days
• US Class Current: 435/372
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/10 Growth factors
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/22 Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 5/0634 Cells from the blood or the immune system

Abstract

Disclosed herein are media and processes for the ex vivo production of megakaryocytes from human CD34⁺ cells, in which human CD34⁺ cells, either being freshly isolated from a newborn'"'"'s cord blood or having been subcultured in an expansion medium after isolation from a newborn'"'"'s cord blood, are subjected to cultivation in a cultivating medium consisting essentially of a basal medium, a serum substitute and a cytokine cocktail, wherein the serum substitute consists of human serum albumin, insulin, and transferrin; and the cytokine cocktail consists of thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (FL), interleukin-3 (IL-3), IL-6, IL-9, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

Claims

1. A process for the ex vivo production of megakaryocytes from human CD34⁺ cells, comprising:

cultivating a population of human CD34⁺ cells in a cultivating medium consisting essentially of a basal medium, a serum substitute and a cytokine cocktail, wherein;

the serum substitute consists essentially of human serum albumin, insulin, and transferrin; and

the cytokine cocktail consists essentially of thrombopoietin, stem cell factor, Flt-3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor; and

harvesting a population of megakaryocytes thus formed from the cultivation of the population of human CD34⁺ cells.

2. The process according to claim 1, wherein the basal medium is selected from the group consisting of an Iscove'"'"'s modified Dulbecco'"'"'s medium, a Dulbecco'"'"'s modified Eagle'"'"'s medium, a RPMI 1640 medium, a minimum essential medium alpha medium, a basal medium Eagle medium, an F-12K nutrient mixture medium, and a Medium 199.

3. The process according to claim 2, wherein the basal medium is an Iscove'"'"'s modified Dulbecco'"'"'s medium.

4. The process according to claim 1, wherein based on the volume of the cultivating medium, the human serum albumin in the serum substitute is present at a concentration ranging from 4.0 to 32.0 g/L.

5. The process according to claim 1, wherein based on the volume of the cultivating medium, the insulin in the serum substitute is present at a concentration ranging from 0.9 to 7.2 μ

g/mL.

6. The process according to claim 1, wherein based on the volume of the cultivating medium, the transferrin in the serum substitute is present at a concentration ranging from 25.3 to 202.0 μ

g/mL.

7. The process according to claim 1, wherein based on the volume of the cultivating medium, the thrombopoietin in the cytokine cocktail is present at a concentration ranging from 1.8 to 13.2 ng/mL.

8. The process according to claim 1, wherein based on the volume of the cultivating medium, the stem cell factor in the cytokine cocktail is present at a concentration ranging from 7.5 to 55.0 ng/mL.

9. The process according to claim 1, wherein based on the volume of the cultivating medium, the Flt-3 ligand in the cytokine cocktail is present at a concentration ranging from 0.8 to 6.1 ng/mL.

10. The process according to claim 1, wherein based on the volume of the cultivating medium, the interleukin-3 in the cytokine cocktail is present at a concentration ranging from 1.7 to 12.7 ng/mL.

11. The process according to claim 1, wherein based on the volume of the cultivating medium, the interleukin-6 in the cytokine cocktail is present at a concentration ranging from 0.3 to 2.1 ng/mL.

12. The process according to claim 1, wherein based on the volume of the cultivating medium, the interleukin-9 in the cytokine cocktail is present at a concentration ranging from 1.0 to 7.5 ng/mL.

13. The process according to claim 1, wherein based on the volume of the cultivating medium, the granulocyte-macrophage colony-stimulating factor in the cytokine cocktail is present at a concentration ranging from 4.4 to 32.1 ng/mL.

14. The process according to claim 1, wherein the basal medium is an Iscove'"'"'s modified Dulbecco'"'"'s medium;

and based on the volume of the cultivating medium, the serum substitute consists essentially of 8 g/L human serum albumin, 1.8 μ

g/mL insulin and 50.5 μ

g/mL transferrin, and the cytokine cocktail consists essentially of 3.0 ng/mL thrombopoietin, 12.5 ng/mL stem cell factor, 1.4 ng/mL Flt-3 ligand, 2.9 ng/mL interleukin-3, 0.5 ng/mL interleukin-6, 1.7 ng/mL interleukin-9 and 7.3 ng/mL granulocyte-macrophage colony-stimulating factor.

15. The process according to claim 1, wherein the population of human CD34⁺ cells is any one of the following:

(i) a population of human CD34⁺ cells freshly isolated from a newborn'"'"'s cord blood; and

(ii) a population of human CD34⁺ cells that have been subcultured in an expansion medium after isolation from a newborn'"'"'s cord blood.

16. A cultivating medium for the ex vivo production of megakaryocytes from human CD34⁺ cells, the medium consisting essentially of a basal medium, a serum substitute and a cytokine cocktail, wherein:

the serum substitute consists essentially of human serum albumin, insulin, and transferrin; and

the cytokine cocktail consists essentially of thrombopoietin, stem cell factor, Flt-3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor.

17. The cultivating medium according to claim 16, wherein the basal medium is selected from the group consisting of an Iscove'"'"'s modified Dulbecco'"'"'s medium, a Dulbecco'"'"'s modified Eagle'"'"'s medium, a RPMI 1640 medium, a minimum essential medium alpha medium, a basal medium Eagle medium, an F-12K nutrient mixture medium, and a Medium 199.

18. The cultivating medium according to claim 17, wherein the basal medium is an Iscove'"'"'s modified Dulbecco'"'"'s medium.

19. The cultivating medium according to claim 16, wherein based on the volume of the cultivating medium, the human serum albumin in the serum substitute is present at a concentration ranging from 4.0 to 32.0 g/L.

20. The cultivating medium according to claim 16, wherein based on the volume of the cultivating medium, the insulin in the serum substitute is present at a concentration ranging from 0.9 to 7.2 μ

g/mL.

21. The cultivating medium according to claim 16, wherein based on the volume of the cultivating medium, the transferrin in the serum substitute is present at a concentration ranging from 25.3 to 202.0 μ

g/mL.

22. The cultivating medium according to claim 16, wherein based on the volume of the cultivating medium, the thrombopoietin in the cytokine cocktail is present at a concentration ranging from 1.8 to 13.2 ng/mL.

23. The cultivating medium according to claim 16, wherein based on the volume of the cultivating medium, the stem cell factor in the cytokine cocktail is present at a concentration ranging from 7.5 to 55.0 ng/mL.

24. The cultivating medium according to claim 16, wherein based on the volume of the cultivating medium, the Flt-3 ligand in the cytokine cocktail is present at a concentration ranging from 0.8 to 6.1 ng/mL.

25. The cultivating medium according to claim 16, wherein based on the volume of the cultivating medium, the interleukin-3 in the cytokine cocktail is present at a concentration ranging from 1.7 to 12.7 ng/mL.

26. The cultivating medium according to claim 16, wherein based on the volume of the cultivating medium, the interleukin-6 in the cytokine cocktail is present at a concentration ranging from 0.3 to 2.1 ng/mL.

27. The cultivating medium according to claim 16, wherein based on the volume of the cultivating medium, the interleukin-9 in the cytokine cocktail is present at a concentration ranging from 1.0 to 7.5 ng/mL.

28. The cultivating medium according to claim 16, wherein based on the volume of the cultivating medium, the granulocyte-macrophage colony-stimulating factor in the cytokine cocktail is present at a concentration ranging from 4.4 to 32.1 ng/mL.

29. The cultivating medium according to claim 16, wherein the basal medium is an Iscove'"'"'s modified Dulbecco'"'"'s medium;

and based on the volume of the cultivating medium, the serum substitute consists essentially of 8 g/L human serum albumin, 1.8 μ

g/mL insulin and 50.5 μ

g/mL transferrin, and the cytokine cocktail consists essentially of 3.0 ng/mL thrombopoietin, 12.5 ng/mL stem cell factor, 1.4 ng/mL Flt-3 ligand, 2.9 ng/mL interleukin-3, 0.5 ng/mL interleukin-6, 1.7 ng/mL interleukin-9 and 7.3 ng/mL granulocyte-macrophage colony-stimulating factor.

30. The cultivating medium according to claim 16, wherein the cultivating medium is used to cultivate human CD34⁺ cells freshly isolated from a newborn'"'"'s cord blood or human CD34⁺ cells having been subcultured in an expansion medium after isolation from a newborn'"'"'s cord blood.

31. A process for the ex vivo production of megakaryocytes from human CD34⁺ cells, comprising:

cultivating a population of human CD34⁺ cells in a cultivating medium consisting essentially of a basal medium, a serum substitute and a cytokine cocktail, wherein;

the basal medium is an Iscove'"'"'s modified Dulbecco'"'"'s medium;

the serum substitute consists essentially of human serum albumin, insulin, and transferrin, wherein based on the volume of the cultivating medium, the human serum albumin is present at a concentration ranging from 4.0 to 32.0 g/L, the insulin is present at a concentration ranging from 0.9 to 7.2 μ

g/mL, and the transferrin is present at a concentration ranging from 25.3 to 202.0 μ

g/mL; and

the cytokine cocktail consists essentially of thrombopoietin, stem cell factor, Flt-3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor, wherein based on the volume of the cultivating medium, the thrombopoietin is present at a concentration ranging from 1.8 to 13.2 ng/mL, the stem cell factor is present at a concentration ranging from 7.5 to 55.0 ng/mL, the Flt-3 ligand is present at a concentration ranging from 0.8 to 6.1 ng/mL, the interleukin-3 is present at a concentration ranging from 1.7 to 12.7 ng/mL, the interleukin-6 is present at a concentration ranging from 0.3 to 2.1 ng/mL, the interleukin-9 is present at a concentration ranging from 1.0 to 7.5 ng/mL, and the granulocyte-macrophage colony-stimulating factor is present at a concentration ranging from 4.4 to 32.1 ng/mL; and

harvesting a population of megakaryocytes thus formed from the cultivated population of human CD34⁺ cells.

32. A cultivating medium for the ex vivo production of megakaryocytes from human CD34⁺ cells, the medium consisting essentially of a basal medium, a serum substitute and a cytokine cocktail, wherein:

the basal medium is an Iscove'"'"'s modified Dulbecco'"'"'s medium;

the serum substitute consists essentially of human serum albumin, insulin, and transferrin, wherein based on the volume of the cultivating medium, the human serum albumin is present at a concentration ranging from 4.0 to 32.0 g/L, the insulin is present at a concentration ranging from 0.9 to 7.2 μ

g/mL, and the transferrin is present at a concentration ranging from 25.3 to 202.0 μ

g/mL; and

the cytokine cocktail consists essentially of thrombopoietin, stem cell factor, Flt-3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor, wherein based on the volume of the cultivating medium, the thrombopoietin is present at a concentration ranging from 1.8 to 13.2 ng/mL, the stem cell factor is present at a concentration ranging from 7.5 to 55.0 ng/mL, the Flt-3 ligand is present at a concentration ranging from 0.8 to 6.1 ng/mL, the interleukin-3 is present at a concentration ranging from 1.7 to 12.7 ng/mL, the interleukin-6 is present at a concentration ranging from 0.3 to 2.1 ng/mL, the interleukin-9 is present at a concentration ranging from 1.0 to 7.5 ng/mL, and the granulocyte-macrophage colony-stimulating factor is present at a concentration ranging from 4.4 to 32.1 ng/mL.