Establishment of fibroblast and myofibroblast phenotypes for use in in vitro co-culture models

• Published in: Biochimie 2023-04, Vol.207, p.96-101
• Main Authors: Ramklowan, D.S.H., Snyman, C., van de Vyver, M., Niesler, C.U.
• Format: Article
• Language: English
• Subjects: Actins - genetics; Actins - metabolism; Cell Differentiation - physiology; Cells, Cultured; Coculture Techniques; Dedifferentiation; Fibroblast; Fibroblasts - metabolism; Migration; Myoblast; Myofibroblast; Myofibroblasts; Phenotype
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2022.10.017

Fibroblasts function to secrete and modify components of the extracellular matrix. During wound healing, fibroblasts migrate to the site of injury and differentiate into contractile myofibroblasts; this differentiation is characterised by an increased contractile capacity. Fully differentiated myofibroblasts can be distinguished from fibroblasts via the higher expression of α-smooth muscle actin as well as a denser cytoskeleton. Impaired wound healing has been characterised by a lack of myofibroblasts; as a result, tissue does not fully regain its strength and function. Under pathological conditions, this may be associated with the effect that a pro-inflammatory microenvironment has on fibroblast and skeletal muscle progenitor cell migration and differentiation. Given their distinct roles in tissue maintenance and repair, the communication between fibroblasts versus myofibroblasts with other cellular mediators of repair is likely to influence cell behaviour and the outcome of wound repair. An in vitro test model is required to investigate this intercellular influence, but the establishment of such a model is hampered by the difficulty in retaining the dedifferentiated fibroblastic phenotype under regular serum-containing cell culture conditions. We present a model that supports the establishment and retention in culture of fibroblast and myofibroblast phenotypes for use in a simple, inexpensive, yet relevant in vitro 2D assay. This model is then applied in a co-culture setting to determine whether the presence of myoblasts affects the ability of fibroblasts versus myofibroblasts to close an in vitro wound. Our results emphasize the importance of considering the impact of paracrine communication between all cells during wound healing. •Fibroblasts exist in either an undifferentiated or differentiated (myofibroblast) state, depending on a tissue's need for structural support or repair.•While establishing a myofibroblast population in vitro is straightforward, achieving and retaining the fibroblast phenotype is a challenge.•We optimised a protocol with which to establish and retain these two phenotypes in culture.•We then used this model to demonstrate the effect of co-cultured myoblasts on fibroblast versus myofibroblast mobility.