(D-147) Characterization of Effects of inkjet Bioprinting of Human Fibroblasts into Stem Cell Environment

Introduction:: Thermal inkjet bioprinting (TIB) has emerged as a powerful tool with many potential applications, such as organ regeneration, drug testing, and cell differentiation, among others. Despite the forces and stress that cells are subjected to during the printing process, there is little research that investigates in detail the effects of the TIB process on cells. Furthermore, a cell’s biological environment greatly influences its behavior. Therefore, understanding the effects of bioprinting on cells in a particular environment at a genetic level can provide clues regarding changes in cell characteristics. We hypothesize that using a thermal inkjet bioprinter to bioprint human fibroblasts into induced pluripotent stem cell (iPSC) media will stress the cells, induce disruption of their membranes, and encourage them to upregulate genes in response to their environment, including stem cell related genes.

Materials and Methods:: In this study, we bioprinted fibroblasts and manually seeded controls into iPSC qualified media onto a iPSC qualified ECM gel coating and conducted RNA extraction and sequencing at different time points post printing. We analyzed RNA sequencing data for differential gene expression using Trimmomatic, RSEM with Bowtie-2, and DESeq2 in R. We also bioprinted fibroblasts and cultured them using the hang drop method, and then performed immunocytochemistry for well-established stem cell markers on the bioprinted fibroblasts and manually seeded controls.

Results, Conclusions, and Discussions:: Our preliminary results from RNA sequencing experiments conducted on a fibroblast cell line showed differential expression of genes, between bioprinted cells and control cells, involved in pluripotency relevant pathways and upregulation of some genes associated with stemness. In addition, some of these genes showed distinct levels of expression at different time points, suggesting active changes in the cells. Immunocytochemistry experiments revealed expression of NANOG, Sox-2, and Oct-4 in bioprinted cells but not in manually seeded controls. Overall, printed cells seem to adopt some behavior attributed to iPSC, signifying that they may be beginning possible differentiation into stem cells. Notably, genes in the following gene ontology (GO) term groups were significantly upregulated: regulation of cell population proliferation, negative regulation of apoptotic processes, angiogenesis, rRNA processing, cell migration, and MAPK cascade. These results point to wide ranging changes in gene and protein expression that should be analyzed further. Additional experiments in progress seek to analyze the level of DNA methylation in printed vs non-printed cells to characterize epigenetic changes caused by the printing process. Understanding how genes are up- or downregulated when cells are subjected to the bioprinting process can provide valuable information that may allow manipulation of cells using bioprinting in order to achieve transformation into different cell types.

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