Abstract
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.
Highlights
Significant advances have been made towards the in vitro culture of human CD34+ hematopoietic stem cell (HSC) populations, bioreactor culture processes and differentiation into red blood cells (RBCs) in vitro have yet to be fully optimized for a therapeutic production of RBCs [1]
We previously demonstrated a proof of principle expansion and differentiation of human CD34+ HSCs towards RBC lineages using a four-compartment hollow fiber based 3D perfusion bioreactor that provides integral oxygenation and high performance mass exchange with low gradients, making it suitable for larger scale cell production that approaches the cell densities under which RBC production is performed in vivo
The following conditions were compared as shown in Figure 3: 1) 3,000 cells per mL density (3000)/Celgene Cellular Therapeutics (CCT) Protocol (CG)/Expansion (Exp), 2) 20,000 cells per mL density (20000)/CG/Exp, 3) 3000/Bioreactor Simulation (BR)/No Expansion, 4) 20000/Bioreactor volumetric media simulation (BR)/no expansion (NoExp), 5) 3000/BR/Exp, and 6) 20000/BR/Exp
Summary
Significant advances have been made towards the in vitro culture of human CD34+ hematopoietic stem cell (HSC) populations, bioreactor culture processes and differentiation into red blood cells (RBCs) in vitro have yet to be fully optimized for a therapeutic production of RBCs [1]. Substrate interactions with the bone marrow niche hydroxyapatite provide a mechanical stabilization and protection of these components Recreating such a niche in vitro, in order to enable cell production behaviors similar to the human body, is challenging (Table 1). Plate cultures with static medium conditions, bioreactor systems that can provide a dynamic three-dimensional (3D) perfusion more closely mimic the bone marrow stem cell niche and allow for spatial cell-tocell interaction. We worked on three objectives in order to optimize bioreactor culture conditions: (1) We sought to determine how cultures are affected by higher and lower seeding densities and more frequent medium changes. This was performed simulating general bioreactor conditions in 2D transwell cultures that are independent of the platform we promote. (2) We proceeded to determine if the cell inoculation or harvest procedures in the specific bioreactors are mechanically stressing HSCs, and if cells can be effectively recovered from the bioreactor after short periods of culture using standard perfusion protocols when employing an 8-mL culture volume bioreactor scale system. (3) Increasing the cell expansion within one bioreactor while extending the culture time was investigated to enhance differentiation towards RBC lineage, using an initial cell seeding density of 20,000 cells/mL in 8-mL volume bioreactor systems
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