Methods and apparatuses for umbilical cord blood collection and isolation of cells
The invention relates to method for the separation of cord blood endothelial cells from hematopoietic cells in a manner that preserves the hematopoietic cells' repopulating ability and primitivity, without significantly reducing the yield of hematopoietic cells. The said endothelial cells from cord blood have a utility in cell based therapeutics. The system described takes advantage of the endogenous adherence properties of the cells to facilitate the separation and separate storage of the cell types.
1. A method for cell separation, comprising:
plating a freshly harvested umbilical cord blood sample that has not been enriched, stored, thawed, or washed on a surface at a surface density of approximately 100 million cells per 100 cm 2 , the surface coated in a component configured to selectively bind with endothelial cells within the whole umbilical cord blood sample;
allowing adhesion of cells in said umbilical cord blood sample to said surface for about 5 minutes to about 60 minutes, to generate an adherent fraction and a non-adherent fraction of said whole umbilical cord blood sample;
separating said non-adherent fraction from said adherent fraction, wherein said non-adherent fraction comprises a hematogenic progenitor cell or a hematopoietic stem cell;
wherein said adherent fraction comprises at least one of an endothelial cell and an endothelial progenitor cell; and
processing and storing the non-adherent fraction.
2. The method of claim 1 , wherein separating said non-adherent fraction from said adherent fraction does not cause detachment of the majority of the cells in said adherent fraction from the surface.
3. The method of claim 1 , wherein about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of cells in said adherent fraction remain attached to the surface following separating said non-adherent fraction from said adherent fraction.
4. The method of claim 1 , wherein said umbilical cord blood sample is from a human.
5. The method of claim 1 , wherein said umbilical cord blood sample is frozen and thawed before the plating step.
6. The method of claim 1 , wherein the said umbilical cord blood sample is plated at a temperature between about 0° C. and about 37° C.
7. The method of claim 1 , wherein the surface is coated with an extracellular matrix component.
8. The method of claim 7 , wherein the extracellular matrix component is collagen I, gelatin, or a combination thereof.
9. The method of claim 1 , wherein the cell viability, primitivity, proliferation ability, differentiation ability, and/or repopulating ability of the hematogenic progenitor cell or hematopoietic stem cell is maintained.
10. The method of claim 1 , wherein the cell viability and/or primitivity of cells in the adherent fraction is maintained.
11. The method of claim 1 , further comprising isolating an adherent cell from the adherent fraction and reprogramming the isolated adherent cell to a stem cell or progenitor cell.
12. The method of claim 1 , further comprising expanding the hematogenic progenitor cell or hematopoietic stem cell.
13. The method of claim 12 , further comprising:
(1) isolating a more primitive cell from the expanded hematogenic progenitor cell or hematopoietic stem cell population, or
(2) reprogramming said hematogenic progenitor cell or hematopoietic stem cell to a more primitive cell.
14. The method of claim 1 , further comprising isolating an adherent cell from the adherent fraction, and:
(a) reprogramming the isolated adherent cell to a stem cell or progenitor cell, and differentiating said stem cell or progenitor cell to a somatic cell; or
(b) reprogramming the isolated adherent cell to a somatic cell directly.
15. The method of claim 1 , wherein a device is used for cell separation from the umbilical cord blood sample, the device comprising a first vessel, a second vessel, and a sterile tubing with stopcock flow regulation for the transfer of cells in suspension between the vessels, and
wherein one of the vessels is pre-coated for cell attachment of cells in the adherent fraction, and the other vessel is a collection vessel for cells in the non-adherent fraction.
16. The method of claim 15 , wherein the device is prefilled with a hypoxic, normoxic, or hyperoxic gas.
17. The method of claim 1 , wherein said adherent fraction comprises a mesenchymal stem cell.
18. The method of claim 17 , further comprising isolating said endothelial cell, endothelial progenitor cell, and/or mesenchymal stem cell from said adherent fraction.
19. The method of claim 1 , further comprising isolating said hematogenic progenitor cell or hematopoietic stem cell from said non-adherent fraction.
20. The method of claim 1 , further comprising using the non-adherent fraction for treatment of a hematologic disorder.