Optimisation of a technique for isolating lymphocyte subsets from human endometrium

Human endometrium is a rich source of lymphocytes which may have unique imm unoregulatory functions. The aim of this study was to compare current proce dures for endometrial tissue disaggregation, and optimise a method for isol ation of endometrial lymphocytes. Tissue was obtained from 41 women undergo ing elective hysterectomy or dilation and curettage (D&C) for reasons of be nign non-endometrial pathology. Specimens were exposed to reduction / chela tion, mechanical or enzymatic disruption. Optimal single cell suspensions o f high yields (mean 8.8 x10(6) range 3.5-18 x10(6) lymphs) and good viabili ty (60%) were obtained, using a combination of collagenase IV (200 U/ml) an d DNase I (35 U/ml). Suspensions were further purified by density gradient centrifugation. Multi-colour flow cytometry was used for analysis of endome trial lymphocyte subsets. Cell suspensions were stained with mAbs specific for CD3, CD4, CD8, CD56, CD45 and CD14, and it was clearly shown that the d eveloped method had no effect on surface glycoprotein expression. Phenotypi c analysis revealed consistent populations of endometrial large granular ly mphocytes (CD56+CD3-) 54.16%, and T-cells (CD3+) 37.73%. This technique was applicable to the characterisation of T-cell populations, including CD8+ ( 56.6%), CD4+ (44.0%), and particularly smaller populations of CD4+CD8+(3.56 %), CD4-CD8-(3.34%) and CD56+(6.3%) due to it's sensitivity. In conclusion, optimised enzymatic digestion, in combination with flow cytometry provides an effective method for phenotypic examination of small endometrial lympho cyte subpopulations.