Human Gingival Lymphocytes. I. Methodology for the Isolation of Human Gingival Lymphocytes

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.

Section: Differential Ceil Countssupporting

confidence: 90%

Section: Discussion Yieldmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

McCulloch

Knowles

Overall

1987

“…Phenotypic and functional characterization of lymphocytes from periodontally diseased tissues has received attention (Seytnour, Powell & Aitken 1983, Seymour et al 1983). Mechanical and enzymatic procedures have been described for isolation of viable human gingival lymphocytes (Sinden & Walker 1979, Mackler et al 1979, Okada, Kida & Yamagami 1982. Recently, cells from chronically infiamed human gingiva were obtained by a combination of 90min incubation in a collagenase-containing medium, followed by mechanical disruption (Daly, Clancy & Cripps 1983).…”

Section: Introductionmentioning

confidence: 99%

Preparation and characterization of human gingival cells

Stoufi

Taubman²,

Ebersole³

et al. 1987

A procedure was developed for the preparation of single cell suspensions from gingival and periodontal tissues using collagenase digestion followed by gentle mechanical disruption. The procedure was evaluated on rat gingival tissues. Onehour incubation was found to produce the greatest number of cells with the highest viability, and the largest recovery of mononuclear cells. Collagenase did not appear to effect recovery, viability or B cell surface markers on human peripheral blood cells. Human gingival and periodontal tissues were obtained from 35 patients: 20 with adult periodontitis; 8 with juvenile periodontitis; 7 with normal gingivae or chronic gingivitis. Scaling, followed by probing measurements and then surgery of the affected sites was routinely performed. In single cell suspensions from tissue obtained from surgery, significantly fewer mononuclear cells were recovered from the tissues of normal/chronic gingivitis patients than cells/mg of tissue recovered from adult periodontitis or juvenile periodontitis groups. The maximum contribution of blood mononuclear cells to the gingival cells averaged 0.5 + 0.2%. This method is useful for recovering gingival (periodontal) cells for phenotypic and functional studies.

“…Nevertheless, interpretation is still diflicult in as much as the responsiveness of peripheral blood lymphocytes may not reflect that of the cells in the often localized lesions. Recognition of this fact has recently led to a number of studies designed to extract cells from periodontally diseased tissue (Mackler et al 1979, Sinden & Walker 1979, Ivanyi 1980, Daly, Clancy & Cripps 1983a, Daly, Cripps & Clancy 1983b. However, only one study has investigated the blastogenic response of these extracted cells (Ivanyi 1980).…”

Section: Introductionmentioning

confidence: 99%

Analysis of lymphocyte populations extracted from chronically inflamed human periodontal tissues

Seymour

Cole

Powell

1985

The current study was undertaken to characterize the lymphoid cell populations extracted from chronically inflamed periodontal tissues. Tissue was removed from 20 patients undergoing periodontal surgery. All patients had received repeated oral hygiene instruction and root planing prior to the surgery. The excised tissue was cut finely, digested in collagenase for 90 min, and then forced through a stainless steel grid to obtain a single cell suspension. T‐cells were identified by labelling with the monoclonal antibody UCHTI in an immunofluorescence assay. B‐cells were also identified in an immunofluorescence assay by the presence of surface membrane immunoglobulin as well as by labelling with the monoclonal antibodies FMCl, FMC4, and FMC7. Eight patients had 3 mm or less loss of attachment while 12 patients had 5 mm or more loss of attachment. UCHTI+ve cells accounted for almost 50% of the lymphocytes (a mean of 49.3 ± 1.5%), while B‐cells identified by all three of the pan B‐cell markers (Smlg, FMC1, and FMC4) accounted for over 30% of the lymphocytes. There was no statistically significant difference between each group with any of the markers used. The vast majority of B‐cells (81.4%) appeared to carry the maturation antigen identified by the FMC7 antibody. Histologically some fragments of tissues were found to contain large numbers of plasma cells. Other fragments were predominated by lymphocytes, clusters of which had the enzyme profile of T‐cells while in other foci the lymphocytes had the enzyme profile of B‐cells. This finding may reflect the presence of different disease states within the surgical field. Caution should therefore be taken before extrapolating to individual disease sites.