A modified roller tube technique for organotypic cocultures of embryonic rat spinal cord, sensory ganglia and skeletal muscle.

The roller tube technique as initially described in the literature in 1981, was modified in several aspects for the coexplantation of embryonic rat spinal cord with attached dorsal root ganglia and skeletal muscle from newborn rats. The high metabolic activity of this coculture system required a particular culturing protocol to stabilize pH and osmotic pressure. The appropriate adjustment of the partial pressure of carbon dioxide gas in the incubator proved to be essential for the control of the pH within narrow limits (7.3 +/- 0.1). The adjustment of the osmotic pressure of the medium (290-300 mOsm) improved the growth of the cultures considerably. Roller drum speed was set to 120 revolutions per hour for enhanced flattening of the culture. A simple rating system was used to evaluate neuronal and non-neuronal outgrowth under different modifications of the culture system. Furthermore, morphological and electrophysiological criteria were defined for evaluating individual neurons. The technique described insures the growth of long-term organotypic cocultures of spinal cord, sensory ganglia and skeletal muscle.