Spheroid co-culture of HSPC and monolayer expanded MSC in PDMS microwells modestly improves in vitro HSPC expansion

While 2-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSC) have been shown to enhance haematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long-term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel 3-dimensional (3D) co-culture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular co-culture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated co-culture spheroids, each containing 25-to-400 MSC and 10 umbilical cord blood (CB)-derived CD34 + progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC co-culture modestly improved overall haematopoietic cell and CD34 + cell expansion outcomes. By contrast, a substantial increase in CD34 + CD38 - cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSC. This outcome indicated that CD34 + CD38 - cell culture yield could be increased using the microwell platform alone, even without MSC co-culture support. We found that the increase in CD34 + CD38 - cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NSG mice, or a modification in the relative human haematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34 + cell yield from 2D or 3D co-cultures, and MSC co-culture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in co-culture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.