Rat Sertoli-spermatogenic cell co-cultures established on collagen-glycosaminoglycan crosslinked copolymers

Abstract

The role of permeable substrates on the proliferation and differentiation of rat spermatogenic cells in co-culture with Sertoli cells was evaluated. Co-cultures were prepared on substrate discs consisting of a thin, stable analog of extracellular matrix attached to a polyester mesh to facilitate handling. Substrate discs were used alone or mounted within a polysulfone reusable holder. Substrates included collagen type I alone or crosslinked with the glycosaminoglycans (GAG) chondroitin-6-sulfate (8%) or heparin (5%). Chemical crosslinking immobilizes collagen and GAG components, preserves the native triple-helical configuration of collagen type I, and renders the copolymer more resistant to degradation by cellular enzymes. Cell attachment and growth properties of Sertoli and spermatogenic cells on these substrates were compared with another permeable substrate (HATF, a mixture of cellulose esters) uncoated or coated with Matrigel (extracellular matrix material derived from EHS tumors) and with the more conventional nonpermeable glass or plastic substrates. We have found that Sertoli-spermatogenic cell co-cultures prepared from pubertal rats readily attach to collagen or collagen-GAG substrate discs as well as to HATF. However, the optical transparency of collagen or collagen-GAG, as compared to the opaque HATF substrate, facilitates monitoring cell attachment and growth by phase contrast microscopy. Substrate discs processed for light and electron microscopy using standard procedures demonstrate the organization of a structurally polarized epithelial layer with basally located Sertoli cells and spermatogenic cells associated by lateral and apical Sertoli cell surfaces. The permeable nature of the collagen-GAG substrate, its optical transparency, and the formation of an electrical-resistant, polarized Sertoli-spermatogenic cell epithelial layer opens new in vitro experimental possibilities for testing agents that may favor or disrupt the spermatogenic process.