Mechanomemory of pulmonary fibroblasts demonstrates reversibility of transcriptomics and contraction phenotypes

Fibroblasts are cells responsible for producing extracellular matrix (ECM) components, which provides physical support for organs. Although these mesenchymal cells are responsive to mechanical cues in their environment, the permanence of these mechano-phenotypes is not well defined. We investigated the mechano-memory of lung fibroblasts and determined how switching culture conditions modulate cell responses and function. Primary murine lung fibroblasts were isolated and cultured on 2D tissue culture plates or within 3D collagen hydrogels and were then passaged within the same or opposite culture condition to assess changes in gene expression, protein production, fibroblast subpopulation, contractile behavior, and traction forces. Compared to fibroblasts isolated on 2D tissue culture plates, fibroblasts within 3D hydrogels exhibited a decreased activation phenotype including reduced contraction profiles, diminished cell traction forces and decreased aSMA gene expression. Cells initially isolated via 2D culture and then cultured in 3D hydrogels exhibited a reversal in activation phenotype as measured by gene expression and contraction profiles. Bulk RNAseq identified groups of genes that exhibit reversible and non-reversable expression patterns. Overall, these findings indicate that lung fibroblasts have a mechanical memory that is altered by culture condition and can be reversible through precondition of cells within a softer 3D microenvironment. Overall design: To investigate the impact of culture dimension on primary murine fibroblast profiles we isolated primary murine fibroblasts from lung tissue using collagenase digestion. These cells were then separated into the following conditions: 1) sorted for fibroblast populations using Paramagnetic particles to eliminate CD45+, EpCam+, and CD31+ cells. The flow through was then collected for RNAseq and labeled with Sort_Fib 1-4. 2) collagenase digests were placed onto 2D tissue culture plates for 4 days, and 3) collagenase digests were encapsulated into 3D collagen hydrogels for 4 days. After 4 days of culture, the cells were then passaged into the same or alternative culture conditions for 2 days prior to collection for RNAseq, giving rise to the following 4 sample conditions: 1) 2D-2D, 2) 2D-3D, 3) 3D-2D, and 4) 3D-3D.