Phenotypic selection in pseudo-quiescence inducing culture of primary human fibroblasts
Fibrotic disease contributes to 45% of all cause deaths. Understanding of the disease pathogenesis is minimal despite extensive research due to limitations of mechanistic information that comes from animal models and 2D cell assays. Microphysiological systems (MPS) aim to recapitulate the native tissue environment ex vivo through organ on a chip modeling. These models have been slow to develop due to a lack of understanding of model inputs, namely the cells. The goal of this study was to improve the understanding of phenotypic selection of primary human fibroblasts in culture prior to incorporation in more complex fibrosis models to ensure a physiologically relevant baseline. Prior work has focused on reduction of fetal bovine serum (FBS) in the culture medium as a method for pseudo quiescence induction of fibroblasts in culture. This work standardizes and conclusively quantifies the effects of serum reduction on primary fibroblast proliferation in culture. We quantified the proliferation of cells in culture through cell cycle marker, Ki67, imaging. Upon reduction, the cells exhibited significant reduction in their proliferation. Addressing senescence, irreversible exit from the cell cycle, we restimulated cells with serum to monitor return to the cell cycle. A trend towards an increase in proliferation following restimulation signaled quiescence over senescence. We utilized human dermal fibroblasts to compare effects on different organ phenotypes. Dermal fibroblasts displayed lower proliferation reduction and a greater return of Ki67 index following restimulation. We performed gene expression analysis to further quantify and define the phenotypic selection that occurs upon serum reduction in culture. The gene profiles for both lung fibroblasts and dermal fibroblasts displayed increased levels of fibronectin, collagen type I (Col-1), and alpha smooth muscle actin (SMA) expression. The same analysis showed minimal changes in the regulatory pathways of these genes. Taken together, our findings support the hypothesis that serum reduction in culture selects for a lineage of cells with high baseline expression of SMA and Col-1, suggestive of profibrotic myofibroblast potential. More experiments are needed to confirm the phenotype and functionality of these cells; however, cells of SMA/Col-I expressing lineage are thought to be the most relevant for drug development.