(A-28) Mechano-memory of Pulmonary Fibroblasts Demonstrates Reversibility of Transcriptomics and Contraction Phenotypes

Introduction:: The lung is a dynamic organ comprised of a variety of cell types and through the mechanical process of breathing, performs gas exchange to provide the body with oxygen. Fibroblasts are the main structural cells of the lung, producing extracellular matrix (ECM) components that maintain the architecture and structure of the lung. In-vitro systems typically used to study these cells and disease pathologies often rely on immortalized cell lines and simplified 2D culture systems, which fail to recapitulate the innate physiology, leading to findings that are not transferrable to patient studies. Previous work has demonstrated that fibroblasts are highly responsive to the stiffness and mechanics of their culture environment. Despite this established knowledge, it is not well understood whether there is a permanence to the transcriptomic and contraction phenotype changes induced by in-vitro mechanical environments. The goal of this study was to investigate the mechano-memory of fibroblasts and how culture condition and isolation methods contribute to changes in cell responses and function.


Materials and Methods:: Primary mouse lung fibroblasts were isolated using mechanical mincing or collagenase digestion and cultured on either 2D tissue culture plates (standard cell culture) or within 3D collagen hydrogels. Fibroblasts were then passaged within the same or opposite culture condition (Fig 1A) to assess changes in gene expression, protein production, contractile behavior, and traction forces.


Results, Conclusions, and Discussions:: Fibroblasts isolated via collagenase digestion had elevated expression of ECM proteins and matrix metalloproteinases (MMPs), as well as an increase in collagen remodeling and contraction profiles (data not shown). Compared to standard 2D culture, fibroblasts cultured within 3D hydrogels exhibited reduced activation phenotypes as demonstrated by decreased global gel contraction profiles (Fig. 1B), diminished individual cell traction forces and decreased αSMA gene expression, an indicator of fibroblast activation state. 3D cultures also increased gene expression of MMPs (MMP3, MMP7), ECM remodeling and degradation proteins, as well as TGFβ, a fibroblast activating cytokine (data not shown). Culture swapping studies showed that primary fibroblast activation state is reversible. Specifically, gene expression changes and contraction profiles mirrored the phenotypic behavior of cells cultured on their most recent condition (Fig 1C, D), regardless of the history of culture dimension. In addition, culturing cells for 24hrs in 3D hydrogel culture was sufficient to reverse fibroblast activation induced by 2D culture (Fig. 1E).
Overall, these findings demonstrate that lung fibroblasts have a mechanical memory that is altered by culture condition and isolation technique, and that this phenotype is reversible through precondition of cells within a softer 3D microenvironment. These findings may demonstrate a potential way to reverse the mechanotransduction process that leads to bias induced by the standard 2D cellular isolation methods and this has the potential to significantly improve in-vitro to in-vivo translatability of studies that use primary lung fibroblasts.


Acknowledgements (Optional): : Work supported by: NIH R01HL141217, 1F32HL164020, Mistletoe Research Fellowship.


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