KeywordsAIM 2: Utilization of the high throughput microwell platform to manufacture HSPC/MSC spheroids to improve the efficacy of direct BM transplantation.AIM 3: Development of an improved microwell platform that retains spheroids within discrete microwells throughout culture.
AIM 4:Characterization of HSPC surface marker change in response to microwell material.
DEVICE AND APPLICATION DEVELOPMENT FOR HSPC AND MSC 3D SPHEROID CULTURES iiiChapter 2 describes the development of a high throughput HSPC/MSC 3D spheroid co--culture embodied in AIM 1. I developed a high throughput polydimethylsiloxane (PDMS) microwell platform that enabled the manufacture of thousands of uniform 3D multicellular co--culture spheroids. Spheroids were assembled such that each contained 25--to--400 BM--derived MSC and 10 CB--derived CD34 + cells (a cell population enriched for HSPC). The outcomes from this Chapter guided the design of the subsequent three chapters. The specific outcomes and subsequent study results were as follows:Chapter 3: As described in Chapter 2, only modest improvements in expansion could be achieved by co--culturing HSPC in 3D MSC spheroids using a microwell platform. We reasoned that the 3D spheroids could, however, function as a biologically active anchor to improve direct BM transplantation outcomes. In this Chapter, we contrasted transplantation methods, including the use of MSC suspensions and 3D multicellular spheroids to modulate human CB--derived CD34 + cell retention within the BM cavity of NOD/SCID gamma (NSG) mice. Analysis revealed that the BM of injected femurs in some animals was saturated with human cells, while distal haematopoietic tissue engraftment was poor. These data show, for the first time, that retention of human haematopoietic cells within the BM enhances local engraftment at the expense of systemic engraftment.Chapter 4: As identified in Chapter 2, improvements in 3D spheroid co--cultures will require that the microwell platform is compatible with repeated medium exchange or dilution feeding. Multiple partial or complete medium exchanges can displace spheroids from discrete microwells, and this can result in either the loss of spheroids from culture, or spheroid amalgamation when displaced microtissues fall into common microwells. In this Chapter, I describe the first microwell platform that incorporates a mesh to retain microtissues within discrete microwells. We called this platform the microwell--mesh. We show that bonding a nylon mesh with an appropriate pore size over the microwell openings allows single cells to pass through the mesh into the microwells during the seeding process, but subsequently retains assembled microtissues within discrete microwells.Chapter 5: As identified in Chapter 2, the presence of PDMS, the material used to fabricate the microwell platform, altered CD38 surface expression on CB--derived CD34 + cells. Previous reports have shown that inhibition of retinoic acid signaling down--regulated CD38 iv DEVICE AND APPLICATION DEVELOPMENT FOR HSPC AND MSC 3D SPHE...