Abstract
Fibroblasts are often used as a feeder layer for progenitor or stem cells in co-culture systems. In the heart fibroblasts are important for cardiac development, homeostasis, and remodelling. They provide cardiomyocytes and progenitor cells not only with nutrition but also secrete extracellular matrix that forms the microenvironment that ensures cell survival and function. Although different kinds of mouse fibroblasts have been used in co-cultures (embryonic, skin and cardiac fibroblasts) adult mouse cardiac fibroblasts (AMCFs) create the closest microenvironment to the adult murine heart for culturing adult mouse cardiac progenitor cells. This protocol describes the isolation of cardiac fibroblasts from adult mouse hearts as well as their maintenance in culture.
Materials and Reagents
Equipment
Procedure
Representative data
For representative photos and results please refer to Zafiriou et al. (2014).
Notes
Note that primary isolated cells as fibroblasts are differentiated when cultured for a long time period (more than 20 days) cease to divide (Bayreuther et al.,1988). Moreover, using cells from the same passage leads to better reproducibility. Therefore, we recommend using passage 4 fibroblasts for all co-culture experiments.
Recipes
Acknowledgments
This protocol was adapted by methods described by Lafontant et al. (2006) and Ohkubo et al. (1997). The HBSS recipe was retrieved from the internet site labrat.com (link: http://www.thelabrat.com/protocols/Hanks.shtml). This work was supported by the Deutsche Forschungsgemeinschaft (ZE900/1-1; ZI708/7-1, 8-1, 10-1; SFB1002; exFLIII) and the German Center for Cardiovascular Research (DZHK).
References
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