Comparison of lymphocyte isolation methods for endoscopic biopsy specimens from the colonic mucosa

An ideal method of immune cell isolation should provide maximum cell yield without disturbing functional properties. Intestinal endoscopic biopsies, in contrast to surgical samples, allow the study of all disease stages but have the drawback of a minimum amount of tissue available, making protocol optimization mandatory. We compared for the first time two methods of separation of colonic epithelium and five methods of lamina propria cell isolation for colonic biopsy specimens (mechanical, enzymatic and organ culture protocols). Lymphocyte number, viability and phenotype (CD45 +, CD103 +, CD3 +, CD4 +, CD8 +, CD19 +, CD16-56 +) were analyzed by flow cytometry. Neither of the two epithelial detachment protocols achieved proper epithelial separation, though the high intensity ion chelation method was more accurate. Maximum cell yield of lamina propria lymphocytes without phenotypic modification was obtained with overnight smooth enzymatic digestion. High dose collagenase incubation caused a marked decrease in CD4 + lymphocytes of the lamina propria as compared to low enzymatic method (p = 0.004). Mechanical and biopsy culture are not advisable methods because of the low cell yield, and phenotypic alterations and high contamination rate, respectively.

► A method based on high intensity ion chelation demonstrated the best epithelial separation capacity.
► Overnight incubation method with low-dose enzyme achieved the best lymphocyte recovery without phenotypic disturbance.
► The high-dose enzymatic method caused a dramatic decrease in CD4 + cells.
► Low cell yield and phenotypic alterations were found with mechanical methods.
► A high contamination rate was found with culture methods.