Abstract
BackgroundHuman Tenon’s fibroblasts (HTFs) play a crucial role in wound healing. They cause postoperative scarring of the filtering bleb and are thus responsible for trabeculectomy failure. This study aimed to find an effective and fast protocol for HTF isolation from trabeculectomy biopsies. The protocol was compared with the commonly recommended HTF isolation procedure, which uses Dulbecco’s modified Eagle’s medium (DMEM). We used Eagle’s minimum essential medium (EMEM) enriched with fibroblast growth factor (FGF), which selectively promoted the proliferation of HTF cells. A secondary goal was to compare HTF morphology, metabolism and growth during parallel cultivation of the isolated cells in FGF-enriched EMEM and DMEM.ResultsStandard procedures for HTF isolation from tissue biopsies require a 20- to 30-day culture of the explants to obtain the first monolayer. Our protocol yielded the first monolayer after approx. 15 days. More importantly, the majority of the cells were fibroblasts with only individual epithelium-derived cells present. Using FGF-enriched EMEM allowed 1.3 × 106 vimentin-positive fibroblasts to be obtained from a single biopsy within approx. 25 days. Using DMEM resulted in isolation failure and required exchange to FGF-enriched medium to recover the fibroblast culture. HTFs maintained in FGF-enriched EMEM also showed faster proliferation and a different type I collagen production ability compared to HTFs cultured in DMEM. Thus, FGF-enriched EMEM is recommended for fast propagation of HTFs unless the aim of the study is to assess the effect of a tested agent on proliferation ability or type I collagen production.ConclusionsOur fast protocol for HTF isolation allows easy setup of cell banks by researchers under laboratory conditions and could be very useful during testing of novel ophthalmologic anti-fibrotic agents in vitro. Molecular analysis of HTFs isolated from patients with known treatment histories may provide valuable information on the effects of some medications taken before glaucoma surgery on the subsequent wound-healing process and potential for trabeculectomy failure.
Highlights
Human Tenon’s fibroblasts (HTFs) play a crucial role in wound healing
We propose a simple “outgrowth” method without collagenase digestion, using basal essential medium (EMEM) supplemented with key factors for fibroblast proliferation: fibroblast growth factor, insulin and vitamin C
Application of 5% fibroblast growth factor (FGF)-EMEM allowed for achievement of the first monolayer in a single well of a 12well plate after an average time of 15 days of culture (Table 2), which is very short time taking into account the single 2–3 mm × 1 mm biopsy used for the isolation
Summary
Human Tenon’s fibroblasts (HTFs) play a crucial role in wound healing. They cause postoperative scarring of the filtering bleb and are responsible for trabeculectomy failure. We used Eagle’s minimum essential medium (EMEM) enriched with fibroblast growth factor (FGF), which selectively promoted the proliferation of HTF cells. The procedures applied to decrease intraocular pressure include glaucoma medications (e.g., biomatoprost, betaxolol or levobunolol), laser therapy and trabeculectomy, which is known as glaucoma filtering surgery [2,3,4]. Human Tenon’s fibroblasts (HTFs) are the main cells responsible for initiation and mediation of wound healing and scarring after a trabeculectomy [5]. Agents receiving special attention have included 5-fluorouracil [7, 8], mitomycin C [7], bevacizumab [3, 9,10,11], and ranibizumab [4]
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