K. Brown1, Y. Ning2, M. Kang1, M. Farenholtz2, J. Grande-Allen1, S. Keswani1,2 1Rice University,Bioengineering,Houston, TX, USA 2Texas Children’s Hospital,Department Of Surgery,Houston, TX, USA
Introduction: Turbulent flow in the Left Ventricular Outflow Tract (LVOT) is considered the main cause of Discrete Subaortic Stenosis (DSS). DSS is a heart disease characterized by the formation of a fibrotic membrane encircling the LVOT. Our lab is using DSS patient echocardiographic data to mimic pathologic conditions ex-vivo in a parallel plate flow loop bioreactor. We aim to understand the mechanism of DSS by studying cellular behavior under varying flow conditions. To understand cellular behavior, we must have an appropriate system for cells to adhere to. In this study, we compare the efficiency of cell adherence to different matrices when exposed to flow as modeled on patient echo data.
Methods: To compare the efficiency of cell adherence with different matrices, we created gelatin, gelatin methacryloyl (gelMA), and fibronectin coatings on functionalized glass slides. Organic residues were removed from glass slides with sulfuric acid. The slides were then treated with 3-(Trimethoxysilyl)propyl methacrylate to functionalize the surface. Endocardial endothelial cells (EECs) were isolated from porcine left ventricle tissue. We then plated 1×10^6 of EECs on each slide and subjected them to flow rates observed in the LVOT. We analyzed cell adherence using a computational algorithm to assess the efficiency of each matrix with an image analysis pipeline to assess cell morphology and cell counting. CD31 IHC is used to validate endothelial phenotype.
Results: We created a bioreactor that could mimic flows at high, low, and static conditions. EECs were confirmed to be near 100% endothelial lineage by CD31 and DAPI staining. Cells were seeded onto gelatin, gelMA, and fibronectin matrices. Gelatin was observed to have 100% adherence at static conditions, 80% adherence at low shear, and 75% adherence at high shear. Cells under high shear flow on gelMA matrix showed a dissociation of the CD31 from the cell membrane in response to high shear as compared to low shear.
Conclusion: Cells adhered to gelatin with optimal adherence under high flow. This gel composition in the bioreactor allows us to the investigate flow disturbances in the LVOT. Interestingly, CD31 is suggested to be affected by differential shear forces and may have a role in mechanotransduction in LVOT pathology. We hope this project will open new avenues for studying DSS and other heart diseases influenced by turbulent flow.