Culturing primary human gingival epithelial cells: comparison of two isolation techniques.
BACKGROUND
Cultured epithelial cells offer many potential clinical applications. There have generally been two techniques that have been used to cultivate oral keratinocytes, which include the direct explant technique and the enzymatic method. Little work has been done comparing these two techniques and their capacity to isolate and cultivate oral keratinocytes.


OBJECTIVE
The objectives of this study were to (1) 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 (2) to examine the effect of age and sex of the subjects providing the tissue samples on (a) the success in cultivation and (b) the growth patterns of gingival keratinocytes.


METHODS
Gingival tissue was obtained from healthy human subjects and was used for keratinocyte isolation using the direct explant technique or the enzymatic method. Epithelial cell cultures from each of the two culture techniques were selected randomly for flow cytometry analysis for cell expression of vimentin and cytokeratin. Growth rate assays were also conducted.


RESULTS
The success rate for cultivation from the direct explant technique was higher (82%) than in the enzymatic method (57.9%). The success rate of both methods was not significantly associated with either age or sex of the subjects providing the tissue. From flow cytometry, the average percentage of cells that was positive to anti-pan cytokeratin was nearly the same for both methods at about 97%. It was noted that the cells from the enzymatic method gave significantly higher percentages of cells that were positive to anti-pan cytokeratin only.


CONCLUSIONS
Both the direct explant technique and the enzymatic method can be used for isolating and culturing human oral keratinocytes. The direct explant technique appeared to be more successful in culturing human oral keratinocytes than the enzymatic method, although there were limitations found with both methods. The age and sex of the subjects providing the gingival samples did not appear to be a factor influencing the success rate in culturing the keratinocytes. However, contamination by oral microbiological flora from the gingival tissue samples remained an ever present problem. Further studies are needed in the investigation of clinical applications of these two epithelial cell isolation methods.
Kedjarune
U
U
Department of Oral Biology and Occlusion Faculty of Dentistry, Prince of Songkla University, Hat Yai, Thailand. kureporn@ratree.psu.ac.th
Pongprerachok
S
S
Arpornmaeklong
P
P
Ungkusonmongkhon
K
K
D000293:Adolescent; D000328:Adult; D000367:Age Factors; D000368:Aged; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D016009:Chi-Square Distribution; D002648:Child; D005260:Female; D005434:Flow Cytometry; D005881:Gingiva / Q000166:cytology*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D007633:Keratins / Q000096:biosynthesis; D008297:Male; D008875:Middle Aged; D012737:Sex Factors; D018709:Statistics, Nonparametric; D014746:Vimentin / Q000096:biosynthesis
D014746:Vimentin; D007633:Keratins