Muscle Cells in Fibrin Matrix Actively Collapse the Network Leading to Rapid Allignment and Formation of Muscle Bundles.

Abstract

The goal of in vitro organogenesis has been hampered by cell culture techniques that promote two dimensional cell proliferation. Since cell growth stops once confluence is achieved, three-dimensional organization does not occur. Possibly due to a lack of normal spatial orientation and cell-to-cell interactions, cells grown in such systems often assume morphologies significantly different from those assumed in normal biological tissues. We have recently developed a simple technique for growing cells in fibrin gels. This is accomplished by suspending cells in fibrinogen solutions and subsequently mixing the fibrinogen-cell suspension with buffered thrombin. The fibrinogen solution can be prepared from purified protein or from plasma diluted with growth media. The mixture rapidly forms a fibrin gel to which the cells are attached. When muscle cells are cultured in fibrin matrices, they initially form a latticework of proliferating cells. Over the next forty-eight hours, the cells begin to collapse the edge of the structure in a process similar to platelet induced clot retraction. As the fibrin network collapses, muscle cells are brought into linear alignment to form structures closely resembling muscle bundles. This process is relatively rapid. Within forty-eight to seventy-two hours, three-dimensional structures morphologically similar to muscle tissue are formed. By manipulating the gel structure through the imposition of outside forces, the alignment of muscle cells can be caused to occur even more rapidly. This system is simple to use and rapidly results in a muscle culture structure that resembles muscle tissue. This technique holds promise to significantly advance the goal of true in vitro “tissue” formation.