Isolation and Expansion of Mesenchymal Stem Cells from Human Conjunctival Tissue

Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells (MSCs) from conjunctiva stromal tissue, with applications to stem cell biology and regenerative medicine. This protocol is based on an explant culture protocol for the adhesion and migration of MSCs from tissue biopsy in primary culture and expansion of the MSC population by passaging cells on the surface of plastic culture dishes. Conjunctiva mesenchymal stem cells (CJMSCs) are generally isolated from small biopsies of human conjunctival tissue. Epithelial sheets are carefully loosened and removed by enzyme treatment after 18 hr and placed in fresh medium supplemented with growth factors. When the cells migrating from tissue segments in primary cultures become nearly confluent, the culture is treated with trypsin. A purified population of CJMSCs that retain proliferation and differentiation potential for many passages can be obtained 2 weeks after the initiation of culture.