Cultivation and identification of keratoconus associated-fibroblasts

Objective To culture and identify corneal fibroblasts from human keratoconus patients (HKCs). Methods HKCs and corneal fibroblasts from human healthy controls (HCFs) were cultured by tissue block adherence method.Cellular morphology and ultrastructure were observed by inverted phase contrast microscope and electron microscopy respectively.Cell viability was detected by cell counting kit-8 (CCK-8) assay.α-Smooth muscle actin (α-SMA), collagen type 1 alpha 1 (COL1A1) and collagen type 3 alpha 1 (COL3A1) protein expression levels were detected by Western blot.This study protocol was approved by Ethic Committee of Xi'an No.1 Hospital (No.1504). Results Compared with HCFs, HKCs showed several distinguishing properties.First of all, its growth speed was faster, with collagen fibers decreased and attenuated.At the same time, mitochondrion swelled and mitochondrial cristae disappeared.Additionally, Golgi apparatus presented significant expansion and endoplasmic reticulum displayed severe swelling.There were statistically significant differences in A values between the two kinds of corneal fibroblasts at different time points after culture (Fgroup=5 023.13, P<0.01; Ftime=38 518.16, P<0.01), the A value of HKCs was significantly higher than that of HCFs at the same time point, and the difference was statistically significant (all at P<0.01). The relative expression of α-SMA, COL3A1 and COL1A1 was 120.00±5.77, 158.33±4.41 and 88.33±1.67, respectively in HKCs, the relative expression of α-SMA, COL3A1 and COL1A1 was 100.00±0.00, 100.00±0.00 and 100.00±0.00, respectively in HCFs, the relative expressions of α-SMA and COL3A1 were significantly increased in HKCs than those in HCFs, the relative expression of COL1A1 was significantly decreased in HKCs than that in HCFs, with significant differences between them (t=-3.46, P<0.05; t=-13.23, P<0.01; t=7.00, P<0.05). Conclusions HKCs are cultured and identied, which is suitable for establishing in vitro cell model of keratoconus. Key words: Keratoconus; Fibroblasts; Cell culture; Identification