Cellular neuroprotection as a modern treatment approach for optic neuropathy

Aim. To develop technology to create 3D-spheroid multipotent mesenchymal stem cells (MMSC) of limbal cadaveric human eyes, capable of safe and long-term secretion of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF).

Materials and methods. MMSC were obtained by cultivation of limbal fragments, released from cadaveric donor human eye. Cultivation was carried out in DMEM/F12 medium, supplemented with L-glutamine, penicillin, streptomycin, amphotericin B, HEPES, insulin, dexamethasone and 10 vol.% FBS under standard conditions (5% СО2, 37 °C), medium change was performed every 3 days. To determine the phenotype of the received cell culture the method of immunophenotyping by marker proteins to MMSC (CD73, CD105, CD19, CD90, CD133) was used. Stimulation of neurotrophic factor secretion was performed via a twostep procedure. 3D-cell spheroids were created with the help of agarous plates for three groups of comparison, where group I was control group, spheroids of intact 2D-culture MMSK; group II – spheroids of previously induced 2D-culture MMSC; group III – spheroids of 2D-MMSC induced on the 1st day of cultivation. Cell cultures supernatants were selected in different periods for NGF and BDNF follow-up study by ELISA procedure.

Results. Induction of 3D-spheroids of limbal MMSC promotes short-term increase of the level of BDNF and NGF, but further, the secretion of these factors significantly decreases. Induction leads to a change in the morphology of spheroids: loss of compactness and emergence of «fringed» (debris). Such changes indicate of frailty of received constructions. Spheroids from previously induced MMSC are capable of stable NTF secretion, but the level of secretion is much less as compared to the control group.

Conclusion. 3D-cell culture of intact 2Dculture of limbal MMSC can be considered as cellular medication for a safe and long-term neuroprotection in optic neuropathy treatment.

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