Methods: First passage rat ASCs were used in monolayer or microencapsulated. To examine the effect of osmolytes during microencapsulation, cells were incorporated into low viscosity, high mannuronate alginate in 75mM Ca++containing glucose, mannitol, or trehalose and microbead diameters and cell viability were determined. In addition, microbeads were fabricated with alginate-lyase and the number of rASCs per microbead and the number of microbeads per unit volume were varied to study effects on time of cell release and local factor production determined. Degradation was followed by measuring DNA content of released cells. Statistical significance was determined by multi-way ANOVA with Bonferroni post test (n=6, per variable).
Results: Smallest microbead diameters were achieved using glucose. After a week in culture, viability of rASCs encapsulated with organic osmolytes was significantly higher than with NaCl. Local factor production was not osmolyte dependent. Total factor production over the last 24 hours of culture was independent of the number microbeads per well for TGF-β2 and TGF-β3 both in the media and retained within microbead. There was an inverse correlation between the number of beads/unit volume or number of cells/bead and the level of IGF1, VEGF, TGF-β2 and TGF-β3. The time of cell release increased as the number of cells/bead decreased.
Conclusion: Only a small number of alginate microbeads and a low cell density may be required to achieve the levels of local factor production for clinical results.