In-Vitro Human Cell Behavior: Using Cell Culture to Assess Biocompatibility and Cell Proliferation on Artacent Products

Introduction:<br/>Cell culture is a useful method of determining cell interactions and assessing product safety in-vitro before completing in-vivo testing, such as animal models. In this study, cell culture is utilized to assess the proliferation and attachment of various cell types on amniotic scaffolds. The selected cell types represent cells that are known to play a part in the wound healing process and include Human Umbilical Vein Endothelial Cells (HUVEC), Neonatal Human Dermal Fibroblasts (NHDF), Normal Human Epidermal Keratinocytes (NHEK) and L929 cells (an adherent type of mouse fibroblast cell line).<br/><br/>Methods:<br/><br/>Cell Culture<br/><br/>All cell lines are maintained in appropriate culture media supplemented with necessary growth factors, antibiotics, and serum per manufacturer’s protocol. These cells were incubated at optimal conditions in a humidified cell culture incubator.<br/><br/>Cell Treatment<br/><br/>Optimal seeding density for each cell type was determined from preliminary testing. Determined seeding densities were then used to seed each cell type on circular 6mm diameter samples from each test group. Cells were seeded for 2,3,7 and 9 days in 96 well Low cluster microplates.<br/><br/>Alamer Blue Assay<br/><br/>After the treatment period, the culture medium was removed from each well or plate and Alamar blue was added to the wells. The cells were treated with the Alamar Blue reagent for 6 hours and under appropriate conditions. The absorbance of these wells was measured using a microplate reader at 570nm and 600nm.<br/><br/>Microscopy<br/><br/>To validate the Alamer Blue Assay results, the cells seeded on Artacent product were labelled with DAPI and Vimentin 647 and observed under Zeiss LSM 800 with Airyscan.<br/><br/>Results:<br/>Graphs summarize the Alamer Blue reduction (%) for the following test groups at the following time points. Chosen time points reflect when the most Alamer Blue reduction was observed for each cell type.<br/>- HDF at 7 day time point<br/>- NHEK at 7 day time point<br/>- HUVEC at 9 day time point<br/><br/>Discussion:<br/>HDF cells showed improved proliferation and attachment on Artacent AC compared to other test groups.<br/>NHEK cells showed improved proliferation and attachment on Artacent Wound compared to other test groups.<br/>NHEK cells showed improved proliferation and attachment on Artacent Wound compared to other test groups.<br/><br/>Conclusion:<br/>Different types of amniotic scaffolds are shown to be optimal for the proliferation and attachment of different cell types.<br/>- HDF cells showed improved proliferation and attachment on Artacent AC.<br/>- NHEK cells showed improved proliferation and attachment on Artacent Wound.<br/>- NHEK cells showed improved proliferation and attachment on Artacent Wound.