Human ciliary epithelia in monolayer culture

The ciliary epithelia of human (one to 12 months old) ciliary processes were isolated by trypsin and EDTA, cultured in Dulbecco's Modified Eagle Medium (DMEM) with 5% fetal calf serum and examined by phase and electron microscopy. The primary cultures were maintained for three to four months. Only a few non-pigmented epithelia adhered and none of them proliferated. After the first passage the culture seemed to consist of only the pigmented epithelia. Most cells were densely pigmented at first, then became less pigmented during successive proliferations. Half of the cells remained densely pigmented after the first subculture, another half remained less pigmented. The cells started to lose their pigment granules at four to six weeks in culture. After three months of culture, the cell sheets became entirely unpigmented. In thin section, most of the pigment granules in the cells at two weeks in culture were pre-melanosomes, and half of them were at the earliest stage of pre-melanosomes. Monolayer cells possessed basement membranes. At 14 weeks in culture, most cells established an apparent polarity, contained well-developed Golgi apparatus and rough endoplasmic reticulum and intermediate filaments, but no pigment granules. A bundle of intermediate filaments was found in the perinuclear cytoplasm. Multilayer cells presented a typical apex-to-apex and base-to-base configuration , and the extracellular material was detected only in the base-to-base intercellular spaces. Our culture system provided differentiated cells derived from the pigment epithelia of human ciliary processes.