Tissue Engineering of Skeletal Muscle: Phase 1-Tissue Culture Conditions

Objectives: Skeletal muscle loss in the head and neck, regardless of cause, has both a profound physical and mental impact on the patient. One way to solve this problem is to tissue-engineer skeletal muscle that could be used to repair or replace the missing tissue. To address this issue, we have established a method to culture skeletal muscle cells that have a 3-D organization similar to intact skeletal muscle. Methods: Satellite cells were isolated from the back and thighs of 2-4 days old rats by digestion with 1.25mg/ml Pronase. The satellite cells were separated from fibroblasts by differential adhesion and expanded by culturing in high serum medium (DMEM-25%FBS). When the cultures reached semi-confluence, the satellite cells were trypsinized and transferred to new culture vessels coated with aligned collagen and cultured in low serum medium (DMEM-10%FBS). Results: The cells grew in a parallel-aligned pattern and when cultured in a low serum environment differentiated into skeletal myocytes. Over the next 4-10 days they formed myotubes with a parallel arrangement with the cultures increasing in density and complexity. The cultures possessed many of the characteristics of skeletal muscle in vivo. The majority of the cells were MyoD positive and immunohistochemical staining demonstrates a regular sarcomeric banding pattern in the uniformly arrayed muscle cells. Located between the skeletal muscle cells was a population of fibroblast-like cells that were a mixture of myoblasts and fibroblasts that in some cases appeared to be producing collagen. Conclusions: We have developed a culture substrate and conditions to cultivate skeletal muscle cells with an in vivo-like phenotype. We are currently growing the cells on an engineered collagen matrix suitable for transplantation and placing them in a rotating wall bioreactor, with the goal of developing a 3-D structure large enough to be grafted into animals. Supported by the US Army.