(355a) 3D Spheroid Stability and Morphology on Elastin-Based Coatings

Objective: Current in vitro adipose cell culture models do not replicate progressive adiposity due to limitations in facilitating the gradual intracellular fat buildup in 3D clusters of adipose-derived stem cells (ASCs) over extended periods. Previously, we cultured human ASCs as 3D spheroids on elastin-like polypeptide-polyethyleneimine (ELP-PEI) coatings and ELP-PEI coatings containing Arginine-Glycine-Aspartic Acid (RGD) residues. We observed that adding three RGD residues to the C-terminus of ELP showed superior spheroid retention. In this study, we qualitatively and quantitatively assessed the impact of the RGD-containing ELP-PEI coating on adipocyte morphology and differentiation over an eight-week culture period.

Materials and Methods: ELP conjugated with RGD was produced from genetically modified Escherichia coli bacteria via suspension culture. These ELP-RGD molecules were conjugated with PEI (MW= 800 Da) via carbodiimide chemistry. 5 mg/mL of 5 mol% ELP-(RGD)₃-PEI conjugates were adsorbed onto 24-well tissue culture polystyrene (TCPS) plates by placing 200 µL solution in each well. 50,000 human ASCs per well were cultured for 3 days on coated plates to form 3D spheroids. The spheroids were differentiated along the adipogenic lineage for 3 days and matured for 8 weeks.

Results: We observed several dynamic changes in cell morphology over the cell culture period. Initially, on Day 3, the cells aggregated to form spheroids and maintained the spheroid morphology after 3 days of differentiation. After 7 days of maturation in adipogenic media, it was observed that the spheroids were surrounded by a monolayer of 2D cells. By day 14, there was a shift toward a predominantly 2D morphology, although with scattered cell clusters. Interestingly, by day 28, there was a reformation of spheroids, indicating a cyclic pattern of spheroid formation and dispersal within the culture period. The spheroids then continued to increase in size till day 56.

Conclusion: This observation underscored the dynamic nature of cell-surface interactions of the human stem cells with the elastin-based coatings containing RGD. We will further probe these results by measuring the adipocyte gene expression and integrin binding.