BACKGROUND: Sertoli cells (SC) in the testis secrete factors that nourish and immunoprotect developing spermatozoa, which have made them the focus of studies that aim to generate localized tolerance, particularly for transplantation and perhaps autoimmunity. Several methods have been described to isolate these cells, which include a two-step enzymatic digestion with limited assessment of the culture. Here we describe a one-step method, and a series of tests for determining purity, viability, and function of the cultured cells. METHODS: We isolated SC from neonatal pigs using Liberase HI digestion. Viability and apoptosis of cultured cells were measured by flow cytometry with propidium iodide and annexin, respectively. Specific identification of the Sertoli type was made by immunodetection of Sox9, vimentin, and Mullerian inhibiting substance. Moreover, for functionality we were able to detect clusterin in the cultured cells by Western blot. RESULTS: Our isolation method had a yield and purity similar to previous reports measured with two-step methods. Viability was 95.22 +/- 0.57% and apoptotic cells were 10.5 +/- 0.32% after 48 h in culture. At 7 days, practically all cells expressed Sox9, Mullerian inhibiting substance, clusterin, and vimentin. CONCLUSIONS: We describe an alternative strategy for preparing and identifying cultured SC for further assays of metabolic activity or in transplantation models. Establishing a one-step Liberase-digestion method for isolation, evaluating viability and apoptosis by more sensitive methods, and detecting specific markers in culture can help to evaluate the quality of cultured cells. Specific cell markers for identifying SC may be critical when identifying SC outside the testis, in contrast with vimentin which is useful only for in situ cells.
BACKGROUND: Sertoli cells (SC) in the testis secrete factors that nourish and immunoprotect developing spermatozoa, which have made them the focus of studies that aim to generate localized tolerance, particularly for transplantation and perhaps autoimmunity. Several methods have been described to isolate these cells, which include a two-step enzymatic digestion with limited assessment of the culture. Here we describe a one-step method, and a series of tests for determining purity, viability, and function of the cultured cells. METHODS: We isolated SC from neonatal pigs using Liberase HI digestion. Viability and apoptosis of cultured cells were measured by flow cytometry with propidium iodide and annexin, respectively. Specific identification of the Sertoli type was made by immunodetection of Sox9, vimentin, and Mullerian inhibiting substance. Moreover, for functionality we were able to detect clusterin in the cultured cells by Western blot. RESULTS: Our isolation method had a yield and purity similar to previous reports measured with two-step methods. Viability was 95.22 +/- 0.57% and apoptotic cells were 10.5 +/- 0.32% after 48 h in culture. At 7 days, practically all cells expressed Sox9, Mullerian inhibiting substance, clusterin, and vimentin. CONCLUSIONS: We describe an alternative strategy for preparing and identifying cultured SC for further assays of metabolic activity or in transplantation models. Establishing a one-step Liberase-digestion method for isolation, evaluating viability and apoptosis by more sensitive methods, and detecting specific markers in culture can help to evaluate the quality of cultured cells. Specific cell markers for identifying SC may be critical when identifying SC outside the testis, in contrast with vimentin which is useful only for in situ cells.
Authors: R Esquivel-Pérez; A L Rodriguez-Ventura; L M Dorantes; B Ramírez-González; M G López-Santos; R Valdes-Gonzalez Journal: Clin Exp Immunol Date: 2011-04-19 Impact factor: 4.330
Authors: Rafael A Valdés-González; Luis M Dorantes; G Nayely Garibay; Eduardo Bracho-Blanchet; Roberto Dávila-Pérez; Luis Terán; Christopher E Ormsby; Jorge-Tonatiuh Ayala-Sumuano; Laura Copeman; David J G White Journal: J Clin Immunol Date: 2007-03-15 Impact factor: 8.542