Hydrogels as model scaffolds to induce 3-dimensional adipocyte culture from stem cells

Abstract

Event Abstract Back to Event Hydrogels as model scaffolds to induce 3-dimensional adipocyte culture from stem cells Kristen Newman1, Kendra Clark1 and Amol Janorkar1 1 University of Mississippi Medical Center, Biomedical Materials Science, School of Dentistry, United States Introduction: The need to treat obesity is becoming ever more apparent as the rate of obesity related ailments, such as heart disease and diabetes, continues to rise[1],[2]. Better understanding of the mechanisms at play on a cellular scale will help develop such treatments. Cell culture in hydrogel matrices has been shown to more readily mimic the 3-dimensional in vivo structure of an adipose tissue making it more advantageous to use than a 2-dimensional monolayer[3]. However, how the properties such as hydrogel scaffold density and composition may affect the ultimate function of the encapsulated adipocytes has not been systematically investigated. This study aimed to probe the effect of these factors on the viability of human adipose derived stem cells (hASCs) and their differentiation along the adipogenic lineage in composite hydrogels prepared using collagen and elastin-like polypeptide (ELP). Methods: Elastin-like polypeptide (ELP) was produced as before[4],[5]. Composite hydrogels were produced with the varied conditions being collagen concentration (2mg/mL or 6 mg/mL; rat tail tendon), addition of ELP (in 1:3 mass ratio with respect to collagen), and crosslinking (Table 1). hASCs isolated from elective liposuction aspirates under an IRB-approved protocol were seeded in hydrogels (40,000 cells/hydrogel) and differentiated for 3 days in 50:50 DMEM and F12 media with 1 μM dexamethasone, 1 μM indomethacin, 0.5 μM IBMX, and 1U/mL insulin. Subsequently, the differentiated cells were given adipocyte maturation media (50:50 DMEM and F12 media with 10% v/v FBS and 1U/mL insulin) for up to 15 days. Following these time blocks Live/Dead, DNA content, and Oil Red O assays were preformed according to manufacturers’ protocols and the results were analyzed using ANOVA and Games-Howell post hoc test . Results: hASCs cultured in 2mg/mL collagen and collagen-ELP (2:6 mg/mL) hydrogels showed a spread morphology and high viability. hASCs cultured in all other hydrogels did not spread, but instead formed spheroid aggregates and showed lower viability (Figure 1a). In general, the Live/Dead fluorescence assay showed evidence of a higher number of living cells than dead cells (>70% viability) in all hydrogels. The DNA content was highest for the 2mg/mL collagen hydrogel attesting the high viability as well as higher proliferation of cells within these hydrogels. Crosslinking reduced the DNA content by 2-fold in 2mg/mL collagen hydrogel, but did not show detrimental results in collagen-ELP (2:6 mg/mL) and 6mg/mL collagen hydrogels (Figure 1b). Oil Red O staining showed maturing adipocytes by day 11 but not at day 5 (Figure 2), a result consistent with Ramirez-Zacarias et al[6]. Quantitative measurement of the extracted Oil Red O stain showed that the hydrogels where hASCs had formed spheroidal aggregates possessed higher adipocytic maturation than the 2mg/mL collagen and collagen-ELP (2:6 mg/mL) hydrogels that showed the spread morphology (Figure 2b). Such higher differentiation in the spheroidal aggregates was consistent with our previous work showing enhanced adipogenesis in 3-dimensional spheroids[5]. Conclusion: These results indicate that hASC viability and differentiation are dependent on their morphology, which in turn is governed by the composition and density of the hydrogel scaffolds. Supported by NIH/NIDCR R03DE024257