Development of a Method for Separating Primary Cell Isolation from Human Gingival Tissue Free of Cell Cross-Contamination
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Abstract
This study aimed to develop an optimized protocol for isolating and culturing human gingival fibroblasts and keratinocytes with high purity and minimal cross-contamination. Gingival tissue samples were collected from five healthy volunteers undergoing periodontal surgery at the Faculty of Dentistry, Khon Kaen University. Cells were cultured using the direct explant method, and cell types were separated using differential trypsinization with 0.25% and 0.05% Trypsin-EDTA, followed by cultivation in cell type-specific media. The efficiency of the method was evaluated by comparing the morphological characteristics of isolated cells with reference standards. All five tissue samples (100%) successfully yielded primary cell cultures by the first passage (P1). Four samples (80%) showed no cross-contamination between cell types, with isolated cells exhibiting morphological characteristics consistent with their respective cell types and achieving greater than 95% purity. Cross-contamination was observed in only one sample (20%).
This optimized method demonstrates improved efficiency for primary cell isolation from human gingival tissue, yielding highly purified cell populations with minimal cross-contamination. The protocol offers a cost-effective approach suitable for routine laboratory application, reducing potential losses associated with contaminated cell cultures.
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