Primary cell cultures of human lamina cribrosa astrocytes were obtained from the eye bank of the Ludwig-Maximilians-University, Munich, Germany. Monolayer cultures were established from eyes of five human donors between 56 and 68 years of age. These eyes, without any history of eye diseases, were obtained 4 to 12 hours postmortem. Methods of securing human tissue were humane, included proper consent and approval, complied with the Declaration of Helsinki, and were approved by the local ethics committee. Astrocytes of the ONH were prepared, grown, and classified as described previously.
6 53 54 In brief, eyes were cut equatorially behind the ora serrata and the ONH was isolated from the neighboring tissues. The ONH was sagittally dissected under a microscope and the lamina cribrosa was identified. Discs of lamina cribrosa were prepared by dissection from the pre- and postlaminar regions, subsequently cut into three to four explants and placed in Petri dishes with 2 mL Dulbecco’s modified Eagle’s medium (DMEM)/F-12 supplemented with 10% fetal bovine serum (FBS; Gibco-Life Science Technology, Karlsruhe, Germany), 5 ng/mL human basic pituitary fibroblast growth factor (bFGF; Sigma, Deisenhofen, Germany), 5 ng/mL human platelet-derived growth factor-A chain (PDGF AA; Sigma), 50 U/mL penicillin and 50 μg/mL streptomycin (Gibco-Life Science Technology) at 37°C in a 5% CO
2 incubator. To isolate ONH astrocytes, the primary cell cultures were first plated in serum-free astrocytes growth medium (AGM; Cambrex Bio Science, Verviers, Belgium) for 24 hours and then changed to AGM containing 5% FBS.
55 Other cell populations, such as lamina cribrosa cells, failed to attach in serum-free medium and were removed with medium change. Subsequently, cultured ONH astrocytes were maintained in DMEM/F-12 with 10% FBS. ONH astrocytes were distinguished from adjacent cells by their morphology and immunohistochemical staining (data not shown).
6 54 Primary human ONH astrocytes were characterized by positive immunostaining for glial fibrillary acidic protein (GFAP; Sigma), neural cell adhesion molecule (NCAM; Serotec, Düsseldorf, Germany), vimentin (Sigma), desmin (Abcam, Cambridge, UK), S100 (Invitrogen, Karlsruhe, Germany), and Pax2 (Abcam), and negative immunostaining for A2B5 (Chemicon International, Hampshire, UK) and smooth muscle actin (smA; Dako, Glostrup, Denmark).
6 53 56 57 58 Only cell cultures, which were at least 95% positive for GFAP, NCAM, vimentin, desmin, S100, and Pax2, and negative for A2B5 and smA, were used in this study.
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