Journal of Bone Research

Journal of Bone Research
Open Access

ISSN: 2572-4916

+44 7868 792050


3-D Perfusion Bioreactor Process Optimization for CD34+ Hematopoietic Stem Cell Culture and Differentiation towards Red Blood Cell Lineage

Greggory J. Housler, Christopher Pekor, Toshio Miki, Eva Schmelzer, Katrin Zeilinger and Jörg C. Gerlach

Process optimization for in vitro cellular engineering of CD34+ hematopoietic stem cell (HSC) culture expansion and differentiation towards red blood cell (RBC) lineages continues to remain a multifaceted challenging operation. This work focuses on three process aspect experiments with the goal of providing improved conditions for the culture of HCSs towards RBC lineages in four-compartment hollow fiber based bioreactors. In a first set of experiments, ideal conditions for the expansion and differentiation of CD34+ HSCs into RBCs were determined by testing the impact of initial cell plating density (3,000 cells/mL versus 20,000 cells/mL), the frequency of replenishing medium, and the transfer to new wells for expansion using 2D transwell plate cultures over 28 days. Results show that a lower density of 3,000 cells/mL and more frequent media changes promote higher levels of cell expansion. In a second independent set of experiments, hollow fiber bioreactor cultures were used to assess if cell inoculation and harvest from such a bioreactor technology platform are potentially damaging HSCs, yielding unfavorable outcomes. Four 8-mL cell chamber volume laboratory scale bioreactors were inoculated with an initial HSC seeding density of 20,000 cells/mL each, perfused for 4 hours, and then harvested to determine the percent recovery. Cells were effectively recovered from the bioreactors, and in follow-up 2D conventional plate cultures the recovered cells expanded as well as the control cultures, indicating that inoculation and harvest procedures are not a source of mechanical injury or cell loss during bioreactor culture. Finally, a third independent set of experiments used multiple 8-mL laboratory scale bioreactors with an initial HSC seeding density of 20,000 cells/mL. Cells were cultured at three time intervals for 8 to 11 days (n=10), 12 to 14 days (n=15), or 15 to 22 days (n=3) with fold-expansion results of 106.0 ± 94.0, 999.5 ± 589.6, and 456.3 ± 33.6, respectively. Although additional studies are necessary for complete large scale-up RBC optimization, the results of these studies have led to a methodical understanding of improved conditions for HSC culture in hollow fiber perfusion bioreactor systems.