AIM: To culture astrocytes from human donor eyes in order to understand the function of astrocytes in remodelling events in the glaucomatous optic nerve head (ONH). METHODS: Primary cultures were prepared by explantation of human ONH tissue in order to get astrocytes. Laminar criborsa (LC) cells were prepared concurrently for com- parison. Astrocyte cultures could be separated from LC cells by selecting medium. Similar procedures were used for LC. RESULTS: Primary cells grew from human optic nerve head explants 4-8 weeks after explantation. Astrocytes had different morphologies and growth characteristics from LC cells. Type 1B astrocyte cells could grow in medium without FBS. Purified cultures were obtained by second passage and could be harvested by third to fifth passage, which were prepared to use for further study, including being characterized by positive glial fibrillary acidic protein (GFAP) and neural cell adhesion molecule (NCAM) staining. CONCLUSION: Precise dissection of fragment is the most important step to get clear explants for primary culture. Economic and rapid method could be useful to select cells by different mediums, which will help us to get more purified cells for further study.