Superiority of 3-D Hematopoietic Stem Cell (HSC) & Stroma Cell Co-Cultures in Supporting the Proliferation of Transplantable HSC.

Stem cells are intimately associated in-vivo with discrete niches within the 3-dimensional (3-D) bone marrow micro-environment collectively provide molecular signals that mediate their differentiation and self-renewal. A recently developed Plug-Flow Bioreactor System (PluriX^TM), which closely mimics the physiological bone marrow environment by combining 3-D stromal cultures with a continuous flow system, is capable of supporting long term maintenance/expansion of transplantable human HSC.

The stroma cells, growing inside the PluriX^TM bioreactor on porrosive carriers enable the propagation of high density (>100-fold input) 3-D stroma cell cultures for prolonged periods of time. Within 40 days, the number of primary human stroma cells seeded in the bioreactor at a density of 5*10⁴ cells/ml reached 1.5*10⁶ cells/ml and the stroma cell lines reached a density of 5*10⁷–1*10⁸ cells/ml. Three-dimensional static stroma HSC co-cultures were found to be superior to 2-D co-cultures in supporting the maintenance of CB HSC and early hematopoietic progenitors.

When cord blood CD34+ cells were seeded on 2-D & 3-D stroma cell static cultures, the 3-D stroma cultures proved superior in supporting the growth of CD34+, CD34+38− and CD34+38−CXCR4+ cells. The 3-D primary human stroma static cultures supported these cells 4.7, 22, 7.4 fold better than the 2-D cultures, respectively.

The CD34+ cells seeded on the high-density 3-D stroma cell cultures within the PluriX^TM bioreactor demonstrated significant superiority to 2-D and 3-D stroma cell static cultures and 2-D stroma cultures in supporting the growth and expansion of HSC and earlier hematopoietic progenitors.

These findings verify that a system based on 3-D stroma co-cultures is preferable for ex-vivo expansion of human HSC’s. The PluriX^TM system offers wide-range potential for expansion of stem cells from cord blood and production of specific blood products.