The hollow fiber bioreactor as a stroma‐supported, serum‐free ex vivo expansion platform for human umbilical cord blood cells

Abstract

The bone marrow microenvironment plays an integral role in the regulation of hematopoiesis. Residing stromal cells and the extracellular matrix in the bone marrow microenvironment provide biological signals that control hematopoietic stem cell (HSC) function. In this study, we developed a bio-mimetic co-culture platform using the hollow fiber bioreactor (HFBR) for ex vivo expansion of HSCs. We evaluated the efficacy of such a platform in comparison to standard cultures performed on tissue culture polystyrene (TCP), using a human stromal cell line (HS-5) as stromal support, co-cultured with lineage-depleted human cord blood cells in serum-free medium supplemented with a cytokine cocktail. Our results showed that the performance of the HFBR in supporting total cell and CD34(+) progenitor cell expansion was comparable to that of cultures on TCP. Cells harvested from the HFBR had a higher clonogenic ability. The performance of ex vivo-expanded cells from the HFBR in hematopoietic reconstitution in humanized mice was comparable to that of the TCP control. Scanning electron microscopy revealed that stroma cell growth inside the HFBR created a three-dimensional cell matrix architecture. These findings demonstrate the feasibility of utilizing the HFBR for creating a complex cell matrix architecture, which may provide good in vitro mimicry of the bone marrow, supporting large-scale expansion of HSCs.

In Vitro Modeling of Tissue-Specific 3D Microenvironments and Possibile Application to Pediatric Cancer Research

“…Moreover, in the oncological field, these 3D models can be useful for in vitro production of human hematopoietic cells. Indeed, the HSCs transplantation, a widely used therapy for several hematological malignancies, presents two important limitations: an insufficient number of matching donors and the difficulty of obtaining enough cells [174]. Thus, expanding HSCs, obtained from donors, can be a suitable strategy to overcome these obstacles.…”

3D models of the hematopoietic stem cell niche under steady-state and active conditions

“…Cultures in rotating wall vessel bioreactors and orbital shake flasks with intermittent shaking both resulted in an elevated multiplication of cells expressing the surface marker CD34 + —that labels hematopoietic stem and progenitor cells (HSPCs)—compared to static cultures 51, 52 . Studies on more complex dynamic 3D setups including a co-culture of lineage-negative UCB cells with bone marrow stroma cells in a hollow fibre continuous perfusion reactor, however, reported expansion rates similar to static 2D co-culture setups 53 or an enrichment of myeloid progenitors rather than of HSCs 54 . The bone marrow-on-a-chip by Torisawa et al .…”

Stromalized microreactor supports murine hematopoietic progenitor enrichment

“…Both these cell types lie in very close proximity to quiescent HSCs in vivo suggesting the potential involvement for flow on HSC development and function (Kumar and Geiger 2017). Only a handful of previous studies have investigated the effects of flow on hematopoietic-associated co-cultures within hollow fiber reactors (Davis et al 1996; Xue et al 2014). Both these studies showed success in expanding sub-purified Lineage negative and purified CD34 + cells from human umbilical cord blood in hollow fiber bioreactors directly co-cultured with MSCs and endothelial cells respectively, and supplemented with cytokines.…”

“…Both these studies showed success in expanding sub-purified Lineage negative and purified CD34 + cells from human umbilical cord blood in hollow fiber bioreactors directly co-cultured with MSCs and endothelial cells respectively, and supplemented with cytokines. Xue et al was able to demonstrate a 60-fold increase in CD34 + cell numbers and unaltered homing capacities through mice reconstitution studies (Xue et al 2014).…”

“…This report describes a scalable hollow-fiber bioreactor system whereby a mouse embryonic fibroblast cell line, known to provide hematopoietic support (Roberts et al 1987), was indirectly cocultured with mouse HSCs in a continuous, concentrated, and recycling flow to stabilize and enrich HSCs for short-term bioprocessing. This study builds on prior reports of direct coculture of HSCs with stromal cells whereby purification of HSCs is more difficult as cells were seeded on the extraluminal side of a hollow fiber membrane and subject to lower yields with additional downstream processing (Davis et al 1996; Xue et al 2014). Since feeder cells were separated from HSCs by the hollow fiber membrane in our system, the isolation of HSCs was simplified.…”