Characterization of a variety of standard collagen substrates: ultrastructure, uniformity, and capacity to bind and promote growth of neurons.

Collagen substrates were characterized after preparation by the four methods most commonly used for tissue culture (saline precipitation, exposure to ammonium hydroxide vapor, exposure to ultraviolet light, and air drying). Although roughly equivalent percentages of collagen were precipitated by each technique (87 to 97%), marked differences were found in surface uniformity and ultrastructure. Substrates were quite uniform if precipitated by exposure to ammonium hydroxide or ultraviolet light, of intermediate uniformity if saline precipitated, and not at all uniform if air dried. Scanning electron microscopy revealed that (a) ammonium hydroxide and saline precipitation primarily resulted in formation of collagen fibrils, (b) air drying produced a small number of fibrils plus a large amount of amorphous material, and (c) exposure to ultraviolet light only resulted in the formation of globular, nonfibrillar collagen aggregates. The capacity of collagen substrates to bind and grow neurons differed markedly with the method of preparation and the amount of collagen plated per unit area. Quantifications of binding and growth of both cerebral and sympathetic neurons revealed that these are separate measures of the biocompatibility of a surface and that growth was uniformly inferior on globular collagen that had been precipitated by ultraviolet light. Long-term (greater than or equal to 2 wk) growth of sympathetic neurons was optimal on thick beds of saline-precipitated collagen, whereas short-term growth was best on thin layers of either saline or ammonium hydroxide-precipitated collagen. Cerebral neurons bound and grew optimally on thick collagen beds after both short- and long-term culture. In addition, cerebral neurons were found to be more dependent on the method of precipitation of the thin collagen substrates than were sympathetic neurons.