Quantitation and optimization of enzymatic and mechanical procedures to produce high‐yield single cell suspensions from human gingiva

Abstract

Existing enzymatic and mechanical techniques of cell release from human gingiva were quantitated in regard to: cell yield; viability; freedom from cell clumping; and debris formation. To optimize these parameters methodologies were modified and combined with new procedures which included incubation with a variety of enzymes. Fibrous and inflamed tissues from 17 patients were examined. The optimal procedure that was selected included thorough mincing of the tissue plus the addition of DNA‐ase, elastase and hyaluronidase to a standard bacterial collagenase mixture. Consistently, up to 3.4 × 106 cells per 100 mg of tissue were released after tissue digestion, with a mean viability of 85%. The cell suspensions were relatively free of debris and cell clumps. Data from Coulter particle counting was in close agreement with hemocytometer counts. The composition of the cell suspension was not significantly different from the original tissue sample. Tissue remnants following the digestion procedure only contained an estimated 1 % of the original number of cells in the sample. The single cell suspensions produced using this method were found to be suitable for analysis by flow cytometry. The possible selective cytotoxicity of the enzymatic technique on cell populations in the DNA‐synthesis phase of the cell cycle was examined in healthy gingival tissue of a Cynomolgus monkey. The gingiva was injected with 3H‐thymidine and the percentage of labeled fibroblasts was enumerated in radioautographs either of sections prepared from undigested tissue or in smears of single cell suspensions. There was no significant difference in the percentage of labeled cells between sections or smears. The data suggest that the method reported here can be applied to flow cytometry analysis of progenitor cell populations in gingival tissues.