Isolation of primary human gingival epithelial cell culture
การแยกเพาะเลี้ยงเซลล์เยื่อบุผิวปฐมภูมิจากชิ้นเนื้อเหงือกมนุษย์
Ureporn Kedjarune, Asst. Prof., D. of Oral Biology and Occlusion, F. of Dentistry, PSU.
Supreya Pongprerachok, D. of Oral Biology and Occlusion, F. of Dentistry, PSU.
Preamjit Arpornmaeklong, Asst. Prof., D. of Surgery, F. of Dentistry, PSU.
Kiattisak Ungkusonmongkhon, D. of Conservative Dentistry, F. of Dentistry, PSU.
Corresponding e-mail : kureporn@ratree.psu.ac.th
Grant : Prince of Songkla University
Published : Research Report
Key words : cell culture, keratinocyte, gingiva, direct exlant technique, enzymatic method
The objectives of this study were to investigate the difference in the percentage of keratinocyte isolation between the direct explant technique and the enzymatic method of human gingival epithelial cell culture and to examine the effect of age and sex of the subjects providing the tissue samples on the success in cultivation and the growth patterns of gingival keratinocytes. Gingival tissue was obtained from healthy human subjects. This tissue was used for keratinicyte isolation using the direct explant technique and the enzymatic method. Epithelial cell cultures from each of the two culture techniques were randomly selected for flow cytometry analysis for cell expression of vimentin and cytokeratin. Growth rate assays were also conducted. The success rate from the direct explant technique was 82%, which was not significantly associated with either the age or the sex of the subjects providing the
tissue. Four of the 10 tissue samples where the cells did not grow were due to contamination. The average weight of the tissue samples used was about 0.13 gram (range from 0.02-0.7). The success
rate of the enzymatic method was 57.9%, which was also not significantly associated with either the age or the sex of the subjects providing the tissue. Four of the 8 cases that failed in cultivation were
due to contamination. The average weight of the tissue samples used was about 0.16 g (range from
0.05-0.36 g). From flow cytometry, the average percentage of cells that were positive to anti-pan cytokeratin were nearly the same for each method at about 97%. It was noted that the cells from the enzymatic method gave significantly higher percentage of cells that were positive to anti-pan cyto-keratin only. In conclusion, both the direct explant technique and the enzymatic method can isolate
and culture human oral keratinocytes. The direct explant technique appeared to be a more successful means of culturing human oral keratinocytes than the enzymatic method, although there were limi-tations found with both methods. The age and sex of the subject providing the gingival samples did
not appear to be a factor influencing the success rate in culturing the keratinocytes, however conta-mination by oral flora from the gingival tissue samples remained an ever present problem.
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