Speaker
Description
"Introduction
One of the key challenges in the field of tissue engineering is the vascularization of tissue-engineered constructs. Until now, endothelial cells (ECs) derived from human umbilical cord have been the predominant EC type for the engineering of vascularized tissue. However, ECs of different origins display a great heterogeneity, reflecting in tissue- and organ-specific characteristics, which are important for interacting with surrounding cell types. Therefore, the use of skeletal muscle-specific microvascular endothelial cells (SkMVECs) may offer more potential for generating tissue-engineered muscle which mimic better the native muscle structure and physiology. Engineering a vascular network within an engineered tissue can be achieved by co-culturing ECs with myoblasts in a 3D co-culture setting, based on the capacity of ECs to self-assemble and form a vascular network. For this, it may be further beneficial to obtain both cell types from the same biological origin. In this work, we present the isolation of SkMVECs in combination with myoblasts from human skeletal muscle, followed by the investigation of an optimal culture medium for the co-culture of these two cell types. Finally, we demonstrate the application of SkMVECs for generating vascularized bio-artificial muscle.
Methodology
SkMVECs and satellite cell-derived myoblasts were isolated using an in-house developed protocol. Tissue was digested using an automated mechanical and enzymatic tissue dissociation procedure. Isolated single cells were cultured and separated using magnetic-activated cell sorting. Cell characterization was performed based on immunofluorescence staining and flow cytometry. Next, different media compositions varying in type and combination of growth medium and fusion medium were compared. The individual cell types were screened separately for behavior in each media composition. For SkMVECs, the formation of endothelial networks within a fibrin (1 mg/mL) hydrogel was evaluated. For myoblasts, the formation of multinucleated myotubes was assessed by performing a fusion assay. Finally, SkMVECs were applied for engineering co-culture bioartificial muscles as described in (1) using the explored culture media, and visualized using confocal microscopy.
Results
Isolated SkMVECs were found to express the endothelial-specific cell markers vWF and CD31. Isolated satellite cell-derived myoblasts were positive for desmin. The functional characteristics of the two cell types were tested and revealed endothelial network formation of SkMVECs on growth-factor reduced Matrigel, and the formation of multinucleated myotubes by isolated myoblasts. Next, two culture media consisting of a combination of serum-rich medium with a switch to serum-low medium after 3 days, were found to facilitate both a proper myotube and endothelial network formation. In a final step, the determined culture conditions were applied for the 3D co-culture of myoblasts and SkMVECs, and were demonstrated to facilitate the creation of a vascularized bioartificial muscle.
Conclusion
With the developed protocol, SkMVECs can successfully be isolated from human muscle biopsies. In addition, an optimal co-culture medium was identified which further allows the use of SkMVECs to tissue-engineer bioartificial muscles. This paves the way for the follow-up investigation of the vascular properties of SkMVECs and their potential for improving the physiological relevance of muscle tissue constructs.
References
1. Gholobova et al., Methods Mol. Biol., 169-183 (2019)."
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