Abstract: Objective To compare the effects of several methods to isolate mouse primary thymus epithelial cells (TECs), so as to screen out a simple, efficient, and practical method. MethodsMouse thymus tissues were digested using the following enzymes: collagenase Ⅰ (ColIE, 1 mg/ml), dispase Ⅱ (DisⅡ, 3 mg/ml), ColIE+low-concentration DisⅡ (ColIE/DisⅡ-L, 0.75 mg/ml), Col IE+medium concentration DisⅡ (ColIE/DisⅡ-M, 1.5 mg/ml), Col IE+high-concentration DisⅡ (Col IE/DisⅡ-H, 3 mg/ml), with or without Percoll density gradient centrifugation. Then, the number of cells were counted using a blood cell counter plate, and the cell viability was detected by trypan blue staining. The proportions of thymus stromal cells (TSCs) and TECs were detected by flow cytometry. ResultsCompared with enzyme digestion with Col IE or DisⅡ alone, a combination of Col IE and different concentrations of DisⅡ could rapidly digest thymus tissue (P<0.01), resulting in an increased number of thymus total cells (P<0.05 or P<0.01), especially in Group Col IE/DisⅡ-M. Compared with enzyme digestion with Col IE or DisⅡ alone, the combination of Col IE and different concentrations of DisⅡ could present an increased proportion of TSC and TEC in Groups Col IE/DisⅡ-M and Col IE/DisⅡ-H (P<0.01). Furthermore, the proportions of TSC and TEC were improved after Percoll density gradient centrifugation (P<0.05). In addition, all the methods of isolation mentioned above could result in higher cell viability. ConclusionsBased on the combined use of Col IE and DisⅡ followed by Percoll density gradient centrifugation, mouse primary TECs can be obtained with higher cell viability.