Method of producing erythrocytes without feeder cells

• US 8,586,360 B2
• Filed: 07/01/2010
• Issued: 11/19/2013
• Est. Priority Date: 07/02/2009
• Status: Active Grant

• Current Assignee: Celularity, Inc.
• Original Assignee: Anthrogenesis Corporation (Bristol-Myers Squibb Company)
• Inventors: Abbot, Stewart, Kang, Lin, Voskinarian-Berse, Vanessa, Zhang, Xiaokui
• Primary Examiner(s): Ketter, Jim
• Assistant Examiner(s): Ghafoorian, Reza
• Application Number:
  US12/829,326
• Publication Number:
  US 20110003387A1
• Time in Patent Office: 1,237 Days
• Field of Search: None
• US Class Current: 435/377
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/14 Erythropoietin [EPO]
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/2303 Interleukin-3 (IL-3)
• CPC Class Codes: C12N 2501/2311 Interleukin-11 (IL-11)
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2501/90 Polysaccharides
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0641 Erythrocytes

Abstract

Provided herein are methods of producing erythrocytes from hematopoietic cells, particularly hematopoietic cells from placental perfusate in combination with hematopoietic cells from umbilical cord blood, wherein the method results in accelerated expansion and differentiation of the hematopoietic cells to more efficiently produce administrable erythrocytes. Further provided herein is a bioreactor in which hematopoietic cell expansion and differentiation takes place.

Claims

1. A method of producing erythrocytes, comprising expanding a population of human hematopoietic cells in a medium in the absence of feeder cells and in contact with one or more factors, wherein a plurality of human hematopoietic cells within said population of human hematopoietic cells differentiate into erythrocytes during said expanding;

and isolating said erythrocytes from said medium,

wherein said factors comprise SCF at a concentration of about 10 to about 10,000 ng/mL, IL-3 at a concentration of about 0.01 to about 500 ng/mL, and Epo at a concentration of about 0.1 to about 10 IU/mL,

wherein said medium comprises insulin-like growth factor 1 (IGF-1) at a concentration of about 1 to about 1000 ng/mL,

wherein said medium does not comprise one or more of Flt-3L, IL-11, thrombopoietin (Tpo), homeobox-B4 (HoxB4), or methylcellulose, and

wherein said SCF, IL-3 and Epo are not comprised within an undefined component of said medium.

2. The method of claim 1, wherein said SCF is present at a concentration of about 100 ng/mL.

3. The method of claim 1, wherein said IL-3 is present at a concentration of about 5 ng/mL.

4. The method of claim 1, wherein said EPO is present at a concentration of about 2 to about 3 IU/mL.

5. The method of claim 1, wherein said hematopoietic cells are CD34⁺.

6. The method of claim 1, wherein said medium further comprises lipids at a concentration of about 1 to about 1000 μ

g/mL, wherein said lipids comprise a mixture of protein and cholesterol; and

wherein said medium comprises hydrocortisone at a concentration of about 0.01 to about 100 μ

M, or dexamethasone at a concentration of about 0.01 μ

M to about 100 μ

M.

7. The method of claim 6, wherein said IGF-1 is present at a concentration of about 20 to about 100 ng/mL.

8. The method of claim 6, wherein said lipids are present at a concentration of about 20 to about 100 μ

g/mL.

9. The method of claim 6, wherein said dexamethasone is present at a concentration of about 0.1 to about 10 μ

M.

10. The method of claim 6 wherein said medium comprises about 100 ng/mL SCF, about 3 IU/mL Epo, about 40 ng/mL IGF-1, about 1 μ

M Dexamethasone, and 40 μ

g/ml lipids.

11. The method of claim 6 wherein said medium comprises about 100 ng/mL SCF, about 2 IU/mL Epo, about 40 ng/mL IGF-1, about 1 μ

M hydrocortisone, and 50 μ

g/mL lipids, wherein said lipids comprise a mixture of protein and cholesterol.

12. A method of producing erythrocytes, comprising expanding a population of human hematopoietic cells in a medium in the absence of feeder cells, wherein a plurality of human hematopoietic cells within said population of human hematopoietic cells differentiate into erythrocytes during said expanding;

and isolating said erythrocytes from said medium,

wherein said medium comprises about 100 ng/mL SCF, about 3 IU/mL Epo, about 40 ng/mL IGF-1, about 1 μ

M dexamethasone, about 40 μ

g/ml lipids, and about 5 ng/mL IL-3,

wherein said medium does not comprise one or more of Flt-3L, IL-11, thrombopoietin (Tpo), homeobox-B4 (HoxB4), or methylcellulose, and

wherein said SCF, IL-3 and Epo are not comprised within an undefined component of said medium.

13. A method of producing erythrocytes, comprising expanding a population of human hematopoietic cells in a medium in the absence of feeder cells, wherein a plurality of human hematopoietic cells within said population of human hematopoietic cells differentiate into erythrocytes during said expanding;

and isolating said erythrocytes from said medium,

wherein said medium comprises about 100 ng/mL SCF, about 3 IU/mL Epo, about 40 ng/mL IGF-1, about 1 μ

M hydrocortisone, about 40 μ

g/mL lipids, and about 5 ng/mL IL-3,

wherein said medium does not comprise one or more of Flt-3L, IL-11, thrombopoietin (Tpo), homeobox-B4 (HoxB4), or methylcellulose, and

wherein said SCF, IL-3 and Epo are not comprised within an undefined component of said medium.

14. A method of producing erythrocytes, comprising expanding a population of human hematopoietic cells in a medium in the absence of feeder cells, wherein a plurality of human hematopoietic cells within said population of human hematopoietic cells differentiate into erythrocytes during said expanding;

and isolating said erythrocytes from said medium,

wherein said medium comprises about 10 to about 10,000 ng/mL SCF, about 0.01 to about 500 ng/mL IL-3, about 0.1 to about 10 IU/mL Epo, about 40 ng/mL IGF-1, about 1 μ

M dexamethasone, and about 40 μ

g/mL lipids,

wherein said medium does not comprise one or more of Flt-3L, IL-11, thrombopoietin (Tpo), homeobox-B4 (HoxB4), or methylcellulose, and

wherein said SCF, IL-3 and Epo are not comprised within an undefined component of said medium.