Dedifferentiation, redifferentiation and bundle formation of smooth muscle cells in tissue culture: the influence of cell number and nerve fibres.

Smooth muscle from newborn guinea-pig vas deferens was enzymically dispersed into single cells or small clumps and grown in culture in the presence or absence of sympathetic ganglion expiants. Most single smooth muscle cells gradually lost their typical ultrastructural features and contractile properties during the first few days in culture. At 7 days of culture these dedifferentiated smooth muscle cells underwent extensive proliferation. If sufficient cells were present in the culture inoculate, a continuous monolayer formed at about 9 days of culture and redifferentiation of smooth muscle began. At 11-12 days of culture the cells reaggregated into clumps, began to contract spontaneously, and formed into well-organized muscle bundles in two layers at right angles, resembling the muscle layer organization of the in vivo vas deferens. In cultures where a continuous monolayer was not formed at 9 days, isolated cells did not redifferentiate. The process of dedifferentiation and proliferation was delayed in those smooth muscle cells which had sympathetic nerve fibres in close association. Clumps of vas deferens tissue which were not fully dispersed by the enzyme treatment did not dedifferentiate with time in culture but muscle bundles were disrupted and asynchronous contractions resulted. After 8-12 days of culture the muscle bundles reformed and foci of synchronous contractions developed. Nerve fibres appeared to accelerate bundle and nexus formation in this situation, with synchronous contractions resuming at 3-5 days. The relation of these findings to the process of wound healing in smooth muscle tissues in vivo is discussed.