Isolation and identification of cancer stem cells in human gastric cancer primary cells

Objective: To isolate and identify the cancer stem cells in human fresh gastric cancer primary cells by combined spherical cultivation in serum-free culture media and immunomagneticbeads isolation technique. Methods: The gastric cancer primary cells in fresh gastric cancer tissues were cultured successfully, which were identified by hematoxylin- eosin (HE) stainning and carcino-embryonic antigen (CEA) immunohistochemisty. The gastric cancer cell spheres were obtained by spherical cultivation in serum-free culture media and the expressions of CD44 on the gastric cancer cell spheres were examined by immunofluorescence. The CD44-positive cells in the gastric cancer cell spheres which were separated by immunomagneticbeads isolation technique were transplanted into SCID mice. The morphology of tumor cells was observed by HE stainning, and the expressions of CD44 and CEA in the transplanted tumors were detected by immuohistochemistry. Results: Of 17 patients with gastric cancer, the gastric cancer primary cells obtained from only one case of fresh gastric cancer tissue belonged to well, moderately and poorly differentiated gastric cancer tissues, respectively. The gastric cancer primary cells from the gastric cancer cell spheres cultivated in serum-free culture media had higher expression of CD44. The CD44-positive gastric cancer cells separated from the gastric cancer cell spheres formed transplanted tumor in SCID mice more rapidly. The characteristics of cell differentiation and self-renewal of the cancer stem cells were found in the xenografts. Conclusion: The gastric cancer stem cells in fresh gastric cancer tissues can be recruitmented efficiently by combined spherical cultivation in serum-free culture media and immunomagneticbeads isolation technique through the marker of CD44. DOI:10.3781/j.issn.1000-7431.2014.08.007