Bioreactor for isolation of rare cells and methods of use
View Patent ↗The present invention relates to a bioreactor apparatus, and methods of use, for the isolation of rare blood cells, including hematopoietic stem cells and megakaryocytes. The apparatus includes a soft substrate and an anti-contractility agent, thereby providing a soft microenvironment to cultured cells. The apparatus of the invention is permissive for the survival of non-dividing cells while dividing cells are eliminated. This unique property allows for the simple isolation of rare blood cells without the use of costly equipment and antibodies.
1. An apparatus for the isolation of rare blood cells from a heterogeneous population of hematopoietic cells, the apparatus comprising:
a hollow tube housed within a syringe, wherein the hollow tube comprises: two opposing openings and an inner surface to form a passage therethrough, a sterile tip attached to one opening of the hollow tube, and a plunger unit inserted into and through the opposing opening of the hollow tube;
rare blood cells; and
a substrate layer on at least a portion of the inner surface of the hollow tube, wherein the rare blood cells are in contact with at least a portion of the substrate layer;
wherein the substrate layer comprises a composition comprising at least one anti-contractility agent selected from the group consisting of blebbistatin, ML-7, and Y-27632;
wherein the substrate layer comprises cross-linked polyacrylamide gel having stiffness of about 0.3 kPa to 2 kPa; and
wherein the rare blood cells comprise polyploid megakaryocytes, polyploid non-megakaryocytes, granulocyte-macrophage progenitors, and erythroid progenitors.
2. The apparatus of claim 1 , wherein the composition is embedded within the substrate layer.
3. The apparatus of claim 1 , wherein the substrate layer is coated with at least one protein selected from the group consisting of collagen, fibronectin, laminin, and vitronectin.
4. A method of isolating rare blood cells from a heterogeneous population of hematopoietic cells, the method comprising the steps of:
(a) culturing a heterogeneous population of hematopoietic cells obtained from a subject within an apparatus having a substrate layer, wherein the heterogeneous population of hematopoietic cells is in contact with a least a portion of the substrate layer,
wherein the apparatus comprises a hollow tube housed within a syringe, wherein the hollow tube comprises: two opposing openings and an inner surface to form a passage therethrough, a sterile tip attached to one opening of the hollow tube, and a plunger unit inserted into and through the opposing opening of the hollow tube;
wherein the substrate layer is on at least a portion of the inner surface of the tube;
wherein the substrate layer comprises cross-linked polyacrylamide gel having a stiffness of about 0.3 kPa to 2 kPa;
wherein the substrate layer comprises a composition comprising at least one anti-contractility agent selected from the group consisting of blebbistatin, ML-7, and Y-27632; and
wherein the rare blood cells comprise polyploid megakaryocytes, polyploid non-megakaryocytes, granulocyte-macrophage progenitors, and erythroid progenitors; and
(b) removing non-viable cells from the population of cells, thereby isolating rare blood cells from a heterogeneous population of hematopoietic cells.
5. The method of claim 4 , wherein the at least one composition is embedded within the substrate layer.
6. The method of claim 4 , wherein the surface of the substrate layer is coated with at least one protein selected from the group consisting of collagen, fibronectin, laminin, and vitronectin.
7. The method of claim 4 , wherein the rare blood cells are used to program the outcome of blood cell transplantation in a subject.
8. The method of claim 7 , wherein the rare blood cells are used to perform at least one function selected from the group consisting of early recovery of platelet count, clearance of granulocyte-macrophage progenitors, maintenance of long term hematopoietic stem cell activity and red blood cell generation.
9. The apparatus of claim 1 , wherein the composition further comprises an aryl-hydrocarbon receptor antagonist selected from the group consisting of StemRegenin-1, CH-223191 and resveratrol.
10. The apparatus of claim 9 , wherein the composition further comprises a growth factor.
11. The apparatus of claim 10 , wherein the growth factor is selected from the group consisting of stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), GDNF-family receptor (including GFRα1), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), insulin-like growth factor, keratinocyte growth factor (KGF), nerve growth factor (NGF), transforming growth factor beta (TGF-β), vascular endothelial cell growth factor (VEGF), platelet-derived growth factor (PDGF), FMS-like tyrosine kinase 3 ligand (Flt3L or Flt3LG), transforming growth factor, interleukins, colony-stimulating factors, Sonic hedgehog, notch, leptin, hormones, and interferons.