Key result
A cell culture protocol using 8-kPa PDMS-coated coverslips maintains cardiac fibroblast quiescence in vitro, enabling the assessment of activation status in response to fibrogenic stimuli.
Why the study?
Evaluating cardiac fibroblast signaling and activation in vitro has been technically challenging because traditional cell culture substrates exhibit super-physiologic stiffness that triggers high basal fibroblast activation.
This protocol enables the in vitro study of cardiac fibroblast activation under physiologic stiffness, overcoming the high basal activation seen with traditional culture substrates.
In vitro cardiac fibroblast models support mechanistic exploration; leaves open clinical translation to fibrosis therapies.
Cardiac fibroblasts (CF) are an essential cell type in cardiac physiology, playing diverse roles in maintaining structural integrity, extracellular matrix (ECM) synthesis, and tissue repair. Under normal conditions, these cells reside in the interstitium in a quiescent state poised to sense and respond to injury by synthesizing and secreting collagen, vimentin, hyaluronan, and other ECM components. In response to mechanical and chemical stimuli, these "resident" fibroblasts can undergo a transformation through a continuum of activation states into what is commonly known as a "myofibroblast," in a process critical for injury response. Despite progress in understanding the contribution of fibroblasts to cardiac health and disease, much remains unknown about the signaling mediating this activation, in part owing to technical challenges in evaluating CF function and activation status in vitro. Given their role in monitoring the ECM, CFs are acutely sensitive to stiffness and pressure. High basal activation of isolated CFs is common due to the super-physiologic stiffness of traditional cell culture substrates, making assays dependent on quiescent cells challenging. To overcome this problem, cell culture parameters must be tightly controlled, and the use of dishes coated with biocompatible reduced-stiffness substrates, such as 8-kPa polydimethylsiloxane (PDMS), has shown promise in reducing basal activation of fibroblasts. Here, we describe cell culture protocol for maintaining CF quiescence in vitro to enable a dynamic range for the assessment of activation status in response to fibrogenic stimuli using PDMS-coated coverslips. Our protocol provides a cost-effective tool to study fibroblast signaling and activity, allowing researchers to better understand the underlying mechanisms involved in cardiac fibrosis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 8-kPa polydimethylsiloxane (PDMS)/gelatin-coated coverslips for cardiac fibroblast cell culture Basic Protocol 2: Isolation of adult cardiac fibroblasts and plating onto PDMS coverslips Basic Protocol 3: Assessment of cardiac fibroblast activation by α smooth muscle actin (αSMA) immunocytochemistry.
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Goldsmith et al. (2024) studied Cardiac fibrosis. 8-kPa polydimethylsiloxane (PDMS)/gelatin-coated coverslips vs. Traditional cell culture substrates was evaluated on Cardiac fibroblast activation status. A cell culture protocol using 8-kPa PDMS-coated coverslips maintains cardiac fibroblast quiescence in vitro, enabling the assessment of activation status in response to fibrogenic stimuli.
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