Methods of preparing hematopoietic progenitor cells in vitro
AssigneeThe United States of America, as represented by the Secretary, Department of Health and Human Services
InventorsRaul E. Vizcardo, Naritaka Tamaoki, Meghan L. Good, Nicholas P. Restifo
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Abstract
Disclosed are methods of preparing CD34+CD43+ hematopoietic progenitor cells (HPC) in vitro according to embodiments of the invention. Also disclosed are methods of differentiating CD34+CD43+ hematopoietic progenitor cells to hematopoietic lineage cells according to embodiments of the invention. Also disclosed are methods of treating or preventing a condition in a mammal, e.g., cancer, according to embodiments of the invention.
Claims23
A method of preparing CD34+CD43+ hematopoietic progenitor cells (HPC) in vitro, the method comprising:
- (i) culturing source cells in a vessel;
- (ii) collecting the source cells from the vessel and preparing a single cell suspension of source cells;
- (iii) culturing the single cell suspension in a first three-dimensional (3D) culture and forming embryoid bodies (EBs) from the source cells in the first 3D culture;
- (iv) collecting the EBs, mixing the EBs with mesoderm lineage cells (MLC), and preparing a single cell suspension comprising EBs and MLC;
- (v) co-culturing the EBs and MLC in the single cell suspension in a second 3D culture and forming hematopoietic spheroids in the second 3D culture;
- (vi) collecting the hematopoietic spheroids from the second 3D culture and culturing the hematopoietic spheroids in a third 3D culture;
- and
- (vii) harvesting CD34+CD43+ hematopoietic progenitor cells from the hematopoietic spheroids.
A method of preparing CD34+CD43+ hematopoietic progenitor cells (HPC) in vitro, the method comprising:
- (i) forming source cells into spheroids in a bioreactor by culturing the source cells in suspension in the bioreactor;
- (ii) collecting the spheroids from the bioreactor and mixing the spheroids with mesoderm lineage cells (MLC);
- (iii) co-culturing the spheroids with the MLC in a first 3D culture and forming hematopoietic spheroids in the first 3D culture;
- (iv) collecting the hematopoietic spheroids from the first 3D culture and culturing the hematopoietic spheroids in a second 3D culture;
- and
- (v) harvesting CD34+CD43+ hematopoietic progenitor cells from the hematopoietic spheroids.
Dependencies are inferred from the claim text.