FIELD OF THE INVENTION

The present invention concerns methods and apparatus for the growth of skeletal muscle in vitro.

BACKGROUND OF THE INVENTION

Volumetric muscle loss due to a variety of causes, including congenital and acquired conditions, invasive surgical procedures, and traumatic injury, produces a physiological deficit for which there are currently no effective clinical treatments. Management involves the use of existing host tissue to construct muscular flaps or grafts. However, this approach is not always feasible, delaying the rehabilitation process and restoration of tissue function. The ability to create clinically relevant autologous tissue engineered muscle repair (TEMR) constructs in vitro for restoration of muscle mass and function in vivo would remove a major hurdle to the successful skeletal muscle reconstructive procedures required to repair complex extremity and facial injuries suffered by injured individuals. See, e.g., Yoo et al., US Patent Application Publication No. US2006/0239981 (Oct. 26, 2006).

SUMMARY OF THE INVENTION

Provided herein are methods of culturing organized skeletal muscle tissue from precursor muscle cells by cyclically stretching and relaxing said muscle cells on a support in vitro for a time sufficient to produce said organized skeletal muscle tissue, including reseeding said organized skeletal muscle tissue by contacting additional precursor muscle cells to said organized skeletal muscle tissue on said solid support, and then repeating said step of cyclically stretching and relaxing said muscle cells in said support in vitro for time sufficient to enhance the density (i.e., increased number of nuclei and number of multinucleated cells) of said organized skeletal muscle tissue on said support.

In some embodiments, the reseeding step is carried out under static conditions. In some embodiments, the reseeding step is carried out by contacting a solution carrying precursor muscle cells to said organized skeletal muscle tissue for a time of 10 minutes to two days.

In some embodiments, the reseeding step is carried out by contacting a solution carrying precursor muscle cells to said organized skeletal muscle tissue inside a mold configured to confine a cell suspension on top of one or more of the supports and/or supports seeded with cells.

In some embodiments, the cyclically stretching and relaxing said muscle cells on a support in vitro comprises: (a) providing precursor muscle cells on a support in a tissue media; then (b) cyclically stretching and relaxing said support at least twice along a first axis during a first time period; and then (c) maintaining said support in a substantially static position during a second time period; and then (d) repeating steps (b) and (c) for a number of times sufficient to enhance the functionality of the muscle tissue or produce organized skeletal muscle tissue on said solid support from said precursor muscle cells (e.g., increase the number of multinucleated cells, increase myotube width, enhance cellular alignment or orientation along an axis, etc.).

In some embodiments, the cyclically stretching and relaxing is carried out at least three times (e.g., from 3 or 4 to 10 or 20 times) during said first time period.

In some embodiments, the stretching comprises extending said support to a dimension between 5% and 15% greater in length than said static position. In some embodiments, the stretching comprises extending said support to a dimension of between 8% and 12% greater in length than said static position.

In some embodiments, the first time period is from 2 to 10 minutes in duration; and wherein said second time period is from 5 to 40 minutes in duration. In some embodiments, the repeating of steps (b) and (c) is carried out for a time of five days to three weeks. In some embodiments, the reseeding step is repeated one, two, three, four or five or more times.

Multi-layered skeletal muscle tissues produced by the processes disclosed herein are also provided. In some embodiments, the tissue comprises elongated multi-nucleated muscle fibers or cells (e.g., from 5, 0 or 15 to 100, 200, or 400 multinucleated cells per square millimeter of tissue or support surface area). In some embodiments, the tissue expresses acetylcholine (ACh) receptors (e.g., aggregated ACh receptors). In some embodiments, the tissue is suturable. In some embodiments, the construct further comprises or includes activated satellite cells or myoblasts.

Also provided are methods of treating a skeletal muscle injury in a patient in need thereof comprising grafting a skeletal muscle tissue (e.g., produced by the processes disclosed herein) into said patient in a treatment-effective configuration. Further provided is the use of a skeletal muscle tissue as described herein for treating a skeletal muscle injury.

