Primary Cell Culture From Human Oral Tissue: Gingival Keratinocytes, Gingival Fibroblasts And Periodontal Ligament Fibroblasts

Songklanakarin J. Sci. Technol. 32 (4), 327-331, Jul. - Aug. 2010

Original Article

Primary cell culture from human oral tissue: gingival keratinocytes, gingival fibroblasts and periodontal ligament fibroblasts
Supreya Wanichpakorn* and Ureporn Kedjarune-Laggat
Department of Oral Biology and Occlusion, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Songkhla, 90112 Thailand.Received 2 February 2010; Accepted 2 August 2010

Abstract Primary cell culture of human oral tissue has many applications for oral biology research. There are two techniques in primary culture, which includes the enzymatic and direct explant technique. The objectives of this study were (1) to isolate and investigate the difference in percentage the success in culturing three cell types fromhuman oral tissue: gingival keratinocytes, gingival fibroblasts and periodontal ligament fibroblasts by using the direct explant technique; (2) to compare the effect of sex and age on the success of tissue culturing. Twenty seven tissue samples were obtained from healthy human gingival tissue, 19 female and 8 male patients aged 14-67 years (37.7±17.5). The tissue was cut into 1x1 mm pieces and placedon plastic culture plates containing Dulbecco,s Modified Eagle,s Medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 1% amphotericin B. For the keratinocytes culture, after the epithelial cells started to multiply around the gingival origin and the diameter was 2-5 mm., the fibroblasts were liminated by mechanical removal under inverted microscope toprevent fibroblast overgrowth and the medium was changed to keratinocyte-SFM (Gibco, BRL) supplemented with 5 µg/ml gentamycin. The results revealed that gingival fibroblast gave the highest success rate in culture (96.3%), followed by gingival keratinocytes (88.9%) and periodontal ligament fibroblasts (81.5%). There was no significant difference in the success rate of cultivation between younger andolder individuals, as between sex of the subjects (p>0.05). The risk of failure in culture techniques is mainly caused by microbiological contamination from the tissue samples. Keywords: primary cell culture, direct explant technique, gingival keratinocytes, gingival fibroblasts, periodontal ligament fibroblasts

1. Introduction The techniques of cell culturing are used to study the geneexpression and cytotoxicity testing. Currently, primary cell culturing of human oral tissue has many applications for oral biology research, including the study of differentiation processes, effects of drugs, and chromosomal analysis (Reid et al., 1997). Two techniques that have been used to cultivate oral tissue are the enzymatic and direct explant techniques (Bernice, 1994; Kedjarune et al., 2001;Klingbeil et al., 2009).

* Corresponding author. Email address: supreya.w@psu.ac.th

The enzymatic technique was described by Daniels et al. (1996), where they surveyed the success rate of human keratinocyte isolation with various concentrations including trypsin and dispase, the enzymatic condition, as well as the calcium concentration in the culture medium. For the keratinocytes yield, thedirect explant technique revealed higher proliferation than the enzymatic technique. The operating procedure used in the direct explant technique process involves fewer steps compared with the enzymatic technique (Klingbeil et al., 2009). The direct explant technique has also been used for 30 years in the culturing of human gingival (Lauer et al., 1991) and buccal tissues (Flaxman et al., 1967) andappeared to be more successful than the enzymatic technique in culturing human oral keratinocytes (Kedjarune

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S. Wanichpakorn & U. Kedjarune-Laggat / Songklanakarin J. Sci. Technol. 32 (4), 327-331, 2010 cells. When the primary cell culture reached confluence at 70-80% (the cell number was 1x106 cells/ml), the PGK and the PGF were detached with 0.025% trypsin-EDTA (Life Technology, USA),...