In vitro growth characteristics of human Tenon capsule fibroblasts from patients with bleb scaring after antiglaucoma filtration surgery

Abstract

Background Human Tenon fibroblasts (HTFs) are the main cell component in scar tissue after antiglaucomatous filtrating surgery.To study the in vitro growth and proliferation features will offer an approach to the preventing and treating of scarring following antiglaucomatous filtrating surgery.Objective The goal of present study was to investigate the growth characteristics of HTFs in vitro from patients with bleb scarring after antiglaucomatous filtrating surgery.Methods This study process was approved by Ethic Committee of Beijing Chaoyang Hospital,and informed consent was obtained from each subject prior to the initaion of the study.The specimens of scaring tissue were obtained during the secondary trabeculectomy from 8 eyes with bleb scaring after initial antiglaucomatous filtrating surgery with the tissue size of 4 mm×5 mm,and the normal Tenon capsule specimens were acquired during the strabismus surgery from 8 eyes in the same way.HTFs were primarily cultured and passaged by tissue explants adherent method and identified using immunoinfluorescence technique with vemintin antibody.Nest PCR was used to exclude the mycoplasma infection during the culturing process.The morphology,growth curve and population doubling time (PDT) of the fifth generation of cells were examined and calculated by luminescence method cell vitality method 0,4,7,11,14 days and compared between the patients and normal groups.The stability of the cell growth was assessed by comparing the morphology,growth curve and PDT between the fifth generation and fifteenth generation of HTFs.Results Primarily cultured cells reached confluence 1-2 weeks with the similar shape to HTFs.The growth properties of the cells were same between the scarring group and normal group and showed positive response for vemintin antibody.No react band for mycoplasma was detected.The PDT was (20.54±3.51) hours and (18.86±2.91) hours in the scarring group and normal group,respectively,showing insignificant difference between them (t=0.634,P=0.561).No significant differences were found in the number of cells in 4,7,11 and 14 days after passage (t =2.663,P =0.081; t =0.194,P =0.863 ; t =3.338,P =0.053 ; t =0.627,P =0.565),with a consistent growth curve with the lapse of time between the two groups.The HTFs from scarred Tenon capsule fibrosis were cultured and passaged until the fifteenth generation.The PDT was gained to be (20.54 ±3.51) hours in the fifth generation of cells and (22.84±4.15) hours in the fifteenth generation of cells,without significant difference between them (t =0.733,P =0.505).The number of cellsin 4,7,11 and 14 days was not significantly different between the fifteenth generation and the fifth generation of cells (t=1.528,P=0.235;t=0.269,P=0.786;t=1.954,P=0.139;t =0.730,P =0.506).In addition,a good and stable growth curve also was exhibited in the fifteenth generation of cells compared with the fifth generation of cells.Conclusions Bleb scar-derived HTFs present good and stable growth in vitro.This result demonstrates HTFs were target cells in antifibrosis study after antiglaucoma filtrating surgery. Key words: Human; Fibroblast/cytology; Tenon capsule/cytology; Glaucoma; Cell culture; Cell proliferation