Establishment of human scleral fibroblasts-seeded collagen matrix and its mechanical properties

Authors: Hu Shoulong,  Li Li,  Cui Dongmei,  Zeng Junwen

DOI: 10.3760/cma.j.issn.2095-0160.2017.02.004
Published 2017-02-10
Cite as Chin J Exp Ophthalmol, 2017,35(2): 108-113.

Abstract

Background

Excessive elongation of axis and expansion of sclera is one of the hot topics in the study of the pathogenesis of high myopia.To establish a human scleral fibroblasts (HSFs)-collagen matrix culture model is helpful for understanding the reciprocal and adaptive interactions between HSFs and the collagen matrix in tissue.

Objective

The aim of this study was to establish a HSFs-seeded collagen three-dimension culture system that may mimic the sclera remolding in myopia.

Methods

HSFs were isolated and cultured from donor eyes by explant culture and purified by passages culture in vitro.The expressions of vimentin and keratin in the cells were detected by immunofluorescence technique to identify the cells.Rat tail tendon was obtained from 8-week-old SPF SD rats to prepare the collagen matrix.The mixed solution of 400 μl collagen matrix and 1 100 μl PBS, 200 μl nutrient medium, 50 μl NaOH and HSF suspension were mixed to prepare the collagen gel three-demension culture system.The growth and morphology of the cells in the culture system were observed under the inverted phase contrast microscope, and IPP-5 software was used to measure the contraction area of collagen gel, and the mechanical creep properties of the HSFs-seeded collagen matrix were measured by a biomechanics test instrument.

Results

HSFs emigrated from tissue 7 days after culture and passage could be performed 14 days after culture.The expression of keratin was absent in HSFs, while vimentin was positively expressed.The free-cell collagen gel was clear and unchanged in the experimental duration.However, the cells were obviously increased on the three-demension culture system and showed a tissue-like structure of net-like arrangement on dozens of layers.In 7-14 days after culture, the collagen gel area in a three-demension collagen matrix revealed a decrease of 90%.Duotriode-like and fusiform cells were seen 24 hours after culture.The biomechanical creep curve of HSFs-seeded collagen matrix consisted of the nonlinear section (0-100 seconds) and linear section (100-600 seconds), and the former appeared to be an elastic change of the gel under the temporal stress, and the latter was the creeping of the gel with the time.

Conclusions

Rat tail collagen appears to have a good biocompatibility to HSFs.HSFs-seeded collagen matrix can retain the mechanical creep properties, and it may be a good tool for the study on the relationship between HSFs and extracellular matrix or intercellular biological behaviour for scleral remodeling.

Key words:

Fibroblasts/cytology; Sclera; Humans; Collagen; Rats, Sprague-Dawley; Biomechanical phenomena; Cell culture technique/methods; Human scleral fibroblasts-seeded collagen matrix

Contributor Information

Hu Shoulong
Department of Ophthalmology, Beijing Children’s Hospital, Capital Medical University, Beijing 100056, China
Li Li
Department of Ophthalmology, Beijing Children’s Hospital, Capital Medical University, Beijing 100056, China
Cui Dongmei
Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou 510060, China
Zeng Junwen
Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou 510060, China
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