A Triple Co-Culture Method to Investigate the Effect of Macrophages and Fibroblasts on Myoblast Proliferation and Migration

Abstract

The communication between nonmyogenic cells, such as macrophages and fibroblasts, and myoblasts is crucial for successful skeletal muscle repair. In vitro co-culture methods can be used to increase our understanding of these cellular interactions; however, current protocols are restricted to two, often physically separate, cell populations. Here, we demonstrate a novel, inexpensive in vitro triple co-culture method that facilitates the co-culture of at least three cell populations with some degree of cell–cell contact. Using this method, we determined the effect of macrophages and fibroblasts on myoblast proliferation and migration. A significant increase in myoblast proliferation and migration was observed following co-culture with either macrophages or fibroblasts. However, triple co-culture of macrophages, fibroblasts, and myoblasts revealed that the presence of macrophages prevented fibroblasts from maintaining this positive effect on myoblast migration. Macrophages, on the other hand, continued to promote myoblast proliferation whether in the presence of fibroblasts or not. Our triple co-culture system highlights the significance of multicellular communication in regulating myoblast proliferation and migration and emphasizes the importance of more complex co-culture systems when investigating myogenesis in vitro.

Co‐culture of pro‐inflammatory macrophages and myofibroblasts: Evaluating morphological phenotypes and screening the effects of signaling pathway inhibitors

“…Furthermore, myoblasts cultured in the presence of M1 macrophages (Mϕ + LPS + ) were present with a relative cell number of 297 ± 68% which was also not significantly different to myoblasts cultured with LPS alone. M0 macrophages (Mϕ + LPS − ) themselves (Figure 3d) continued to significantly increase ( p < 0.05) relative myoblasts numbers from 141 ± 4% to 220 ± 15% in SFM as previously shown (Venter & Niesler, 2018b). Myoblasts maintained in the presence of LPS (Mϕ − LPS + ) were present with relative cell numbers of 145 ± 6% which was not significantly different compared to the control.…”

Impact of Culture Medium on Cellular Interactions in in vitro Co-culture Systems

“…Most co-culture studies involve two cell types, owing to an increased complexity in establishing a stable system when more cell types are involved (Goers et al, 2014). There are also studies reporting the use of three (Venter and Niesler, 2018;Churm et al, 2019;Lin et al, 2019) or even four cell types (Zhang et al, 2009;DesRochers et al, 2015). Different strategies to co-culture cells in 3D exist, each allowing for a different degree of contact between the cell types.…”

Effects of External Stimulators on Engineered Skeletal Muscle Tissue Maturation

“…[ 20b ] These cells have a significant impact on myogenic migration and proliferation in vitro if cultured together, as reported by Venter et al. [ 138 ] Their experiments investigated the proliferative and migratory effects on myoblasts, compared between three experimental groups: myoblast/macrophage cocultures, myoblast/fibroblast cocultures, and a triple coculture with all three cell types. In individual cocultures, fibroblast cells would promote the migration of myoblasts whereas macrophages could only promote their proliferation.…”

“…Specifically, the promoted migratory effects seen in the myoblast/fibroblast coculture were not reproducible in the triple coculture, indicating that particular response was inhibited in some way by the presence of the macrophages. [ 138 ] The exact mechanisms for these responses are as of yet unknown.…”