[A new method for extracting adult mouse cardiac fibroblasts more efficiently and stably]

Cardiac fibroblasts (CFs) play a central role in myocardial remodeling and fibrosis. Efficient isolation of CFs is a prerequisite for investigating related mechanisms. However, current methods for isolating primary adult mouse CFs suffer from prolonged processing time, low yield, and poor viability. This study aims to establish a rapid, high-yield, and stable isolation protocol for adult mouse CFs by optimizing the synergistic effect of enzymatic digestion and mechanical dissociation parameters.

Using the gentleMACS^® Octo Dissociator with Heaters, we selected different types and concentrations of collagenase, trypsin, and nuclease as the enzymatic digestion system for CFs extraction. We explored the optimal extraction conditions and compared the results with the commercial Multi Tissue Dissociation Kit 2. The cell yield was quantified using a high-content imaging analysis system by counting the number of adherent cells per field after 72 h of culture. The CFs purity was assessed using immunofluorescence staining for vimentin. The trans-differentiation activity of the CFs was evaluated with transforming growth factor β1 (TGF-β1).

Omitting any component of the digestion solution (collagenase Ⅱ/Ⅳ, trypsin or DNaseⅠ), significantly prolonged extraction time and reduced cell yield. In contrast, the optimized protocol outperformed the commercial kit, reducing digestion time by 32.2 min and significantly increasing cell yield, and with comparable obtained CFs purity. After TGF-β1 stimulation, CFs exhibited enhanced proliferative capacity and upregulated expression of α-smooth muscle actin (α-SMA), collagen type Ⅰ (ColⅠ), and fibronectin (FN), confirming the differentiation potential of CFs isolated via the optimized method.

This study systematically optimized an enzymatic digestion method combining collagenase, trypsin, and nuclease in conjunction with mechanical dissociation using a tissue dissociator, leading to the efficient and stable isolation of adult mouse CFs. By fine-tuning enzyme concentrations and digestion conditions, we successfully reduced processing time, improved cell yield, and enhanced cell viability compared with conventional isolation methods. These findings validate the physiological relevance of the isolated CFs and demonstrate that the optimized protocol provides a reliable and reproducible method for studying myocardial fibrosis and remodeling. This protocol can serve as a valuable tool for researchers investigating CFs biology and its role in cardiovascular diseases.