Abstract P1088: Biophysical Crowding Of In Vitro Culture Media Alters The Growth Rate And Collagen Production Of Cells Used For Seeding Tissue-Engineered Heart Valves

Introduction: Ideal tissue-engineered heart valves (TEHVs) for valve replacement in congenital heart disease patients would have durability and growth potential. Endothelial colony-forming cells (ECFCs) isolated from peripheral blood are an attractive source for pre-cellularizing TEHVs with a patient’s own cells, as they are isolated non-invasively and can transform into valve endothelial and interstitial phenotypes. ECFC limitations include slow proliferation and uncertain mechanisms to control extracellular matrix (ECM) production. In other cell types, culture media “crowded” with large macromolecules limits paracrine diffusion and augments both ECM production and cell growth. Hypothesis: As a novel study of biophysical crowding on ECFCs, we hypothesized that media crowded with macromolecules dextran sulfate or Ficoll-400 would enhance the growth rate and collagen production of ovine ECFCs. Methods: ECFC lines (CD31+, α-SMA+) from n=3 animals at passages 2, 3, or 7 were cultured in media with no crowder or either Ficoll-400 or dextran sulfate at four concentrations in three separate replicates (n=10, n=120, n=384). Deposited and suspended collagen was quantified. Immunocytochemical cell scoring was quantified for vWF, VEGFR-2, CD34, vimentin, and α-SMA markers. Results and Conclusions: Anionic dextran sulfate (DS) caused significant dose-dependent increases in media soluble collagen, despite significantly lowering cell counts. Crowding with DS significantly increased CD34 positivity, a marker for anti-angiogenic and quiescent ECFC populations. Conversely, neutral Ficoll-400 significantly increased cell counts but did not alter media collagen or CD34 positivity. Both crowder groups had altered cellular morphology but did not cause reproducible differences in positivity for endothelial markers vWF and VEGFR-2 or interstitial markers vimentin and α-SMA. In conclusion, biophysical crowding of ECFC media alters cell count and morphology, CD34 positivity, and collagen production, which may allow for fine-tuning of ECFC properties when seeding onto TEHVs. Future studies will address the different effects of neutral and negatively charged crowders and how crowders impact cellular phenotype and growth on valvular scaffolds.