A Mouse 3T6 Fibroblast Cell Culture Model for the Study of Normal and Protein-Engineered Collagen Synthesis and Deposition into the Extracellular Matrix

Mouse 3T6 i ro asts deposited an organize co agenous extrace u ar matrix during longterm culture in the presence of ascorbic acid. The matrix produced by the cells had a similar comprising distribution of collagen types as the mouse dermal matrix, comprising predominantly type I with smaller amounts of types III aand V collagens. By day more than 70% of the collagen in the 3T6 matrix was involved in covalent crosslinkages and required pepsin digestion for extraction. Incorporation of NaB³H₄ into reducible crosslinks and aldehydes directly demonstrated the involvement of the α1(I)CB6 an α2(I)CB3.5 in crosslinks. The pattern of reducible crosslinks in the in vitro 3T6 matrix was similar to that in mouse skin suggesting a comparable fibril organization. Processing of procollagen to collagen occurred efficiently throughout the culture period and the rate of collagen production was unaltered during 15 days of culture, indicating that the development of a collagenous matrix does not directly play a role in procollagen processing or biosynthetic regulation. The existence of a preformed matrix did however, increase the efficiency with which newly synthesized collagen was incorporated into the pericellular matrix. At day 0, when there was no measurable a matrix present, 29% of the collagen synthesised wad deposited, while by 15,88% of the collagen was laid down in the matrix. The development of this 3T6 culture system, where collagen is efficiently incorporated into an organized extracellular matrix, will facilitate detailed studies on matrix organization and regulation and provide a system in which protein-engineered mutant collagens can be expressed to determine their effects on the production of a functional extracellular matrix.