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Tissue Engineering

Tissue Engineering - Poster Session E

Poster AA19 - Controlling Muscle Development in Hydrogels via Selective Cell Sorting

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Skeletal tissue engineering involves growing new muscle in vitro to treat severe muscular injuries. The standard approach for engineering muscle entails expanding muscle progenitor cells on tissue culture polystyrene before embedding the cells directly into a scaffold for further cell growth and tissue development. However, the clinical success of engineered muscles are hampered by the inherent heterogeneity of the cells. Advanced cell manufacturing approaches, such as selective cell sorting, can improve the therapeutic efficacy of engineered muscle constructs by limiting the heterogeneity of the manufactured cell populations incorporated into the scaffold. As a marker for cell sorting, we will focus on ꞵ1 integrin, which is a surface marker that regulates cell interactions with extracellular matrices and is incorporated into various collagen, RGD, laminin, and leukocyte-specific integrin receptors. ꞵ1 integrin has also previously been shown to play a role in myotube development and maturation. Thus, the purpose of this study was to evaluate how sorting myoblasts by different magnitudes of ꞵ1 integrin affects muscle progenitor cell (i.e., myoblast) fusion and myotube maturation in hydrogels.

Materials and

Methods:  Commercially available mouse C2C12 myoblasts (ATCCⓇ) were subcultured on tissue culture polystryene and then stained with a fluorescent antibody against ꞵ1 integrin. The stained cells were then sorted via fluorescence activated cell sorting for high or low expression of ꞵ1 integrin. The sorted populations and unsorted cells (stained or unstained with the ꞵ1 integrin antibody) were encapsulated in a collagen I-Matrigel hydrogel and cast into an in-house micropillar system. To allow myotubes to form within the engineered constructs, the C2C12s were cultured for three days in growth media followed by four days in differentiation media. Following culture, the constructs were fixed and subsequently stained for DAPI, phalloidin, and myosin heavy chain (MyHC). Myotube development and maturation was evaluated between sorted conditions and to unsorted unstained and unsorted stained controls.

Results, Conclusions, and Discussions: The C2C12s were all strongly positive for ꞵ1 integrin expression by flow cytometry analysis. The ꞵ1 integrin low and high sorted groups were both 100% positive for ꞵ1 integrin, but exhibited varying expression levels of the surface marker by the mean fluorescence intensity of the antibody. All conditions successfully formed myotubes within the collagen-Matrigel hydrogels. Furthermore, both control conditions and the ꞵ1-HI condition formed highly aligned myotubes, while the ꞵ1-LO group’s cells exhibited greater variability in cell morphology (Fig. 1A,B). Specifically, some of the ꞵ1-LO cells created interconnected branching structures that lacked myosin heavy chain expression (Fig. 1B). The ꞵ1-HI group significantly outperformed the ꞵ1-LO group in myotube maturation which was evident in the increased fusion index and average number of nuclei per myotube (Fig. 1C,D). The ꞵ1-LO group demonstrated a trend of having smaller myotubes both in width and length in comparison to the other groups. Additionally, the ꞵ1-HI group demonstrated a trend of having a higher fusion index and average number of nuclei per myotube than the unsorted stained control. This suggests that creating 3D engineered constructs that utilize only C2C12s with a high expression of ꞵ1 may be superior to utilizing an unsorted, heterogeneous cell population.

This study showed that sorting mouse C2C12 myoblasts by ꞵ1 integrin expression can identify subpopulations of myoblasts with varying capacity to mature and fuse into myotubes. Specifically, the ꞵ1-HI group created superior myotubes compared to the ꞵ1-LO group and a trend of enhanced maturation relative to the unsorted stained control. This suggests that selective cell sorting by high ꞵ1 integrin expression may result in superior engineered constructs. Future work will identify the muscle fibers types (fast versus slow twitch) that are formed and the muscle contractile force that is generated from the engineered constructs. Moreover, we will also explore sorting by additional surface markers to continue to manipulate myotube development and maturation.

Acknowledgements (Optional):