Aims: In vitro organ culture systems with living nerve cells are very attractive for investigating the mechanisms of neurotrauma and neurodegeneration. Astrocytes are a particular focus of research. These cells are usually isolated from animal tissue. We have developed a relatively fast and simple protocol for the isolation of astrocytes from brain biopsies with a high yield and low risk of contamination.

Method: Human astrocytes can be obtained after cranial surgery, especially in neurotrauma patients after brain nectomy. Fragments of viable tissue removed during surgery were collected under sterile conditions. The tissue was cut, minced and seeded through a mesh system. After sequential centrifugation and separation, the sediment was harvested and the cells were seeded in suspension supplemented with special media (DMEM Advanced) with high nutrient content (FBS) and antibiotics (streptomycin, penicillin). Characterization was performed and the cells were subisolated.

Results: The isolated cells remained viable and proliferated rapidly. Attachment was observed after 8 to 12 hours and proliferation after 7 days. The time to confluence was 22 days. Cell proliferation, apoptosis and cell senescence were examined after 21 days in culture. The cells were stable. Cell proliferation and cluster formation were observed under standard culture conditions. The viability of the cells was 90%. DAPI and GFAP immunohistology were performed for characterization. The cells were strongly positive and confirmed the astrocyte markers.

Conclusions: The demonstrated isolation procedure is simple, fast and inexpensive and enables viable, longterm primary cell culture. The availability of such a system allows in vitro studies of cell properties, biochemical aspects and the potential of therapeutic candidates for traumatic and neurodegenerative diseases in a well-controlled environment on a human astrocyte cell culture.

Funding: No funding received.