Jornal de Pesquisa Óssea
Acesso livre

Abstrato

Otimização do processo de biorreator de perfusão 3-D para cultura de células estaminais hematopoiéticas CD34+ e diferenciação para linhagem de glóbulos vermelhos

Greggory J. Housler, Christopher Pekor, Toshio Miki, Eva Schmelzer, Katrin Zeilinger e 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.