Separation of invasive subpopulation from primary human renal cell carcinoma via in vitro invasion assay / 第二军医大学学报

ABSTRACT

Objective:To isolate the invasive and non-invasive cells from primary human renal cell carcinoma(RCC) in vitro.Methods: Fresh RCC surgical specimens from 32 primary RCC patients were primarily cultured following enzyme digestion or mechanical minimization in vitro.In vitro invasion assay using the Transwell cultures coating Matrigel was performed for separation and recovery of invasive and non-invasive cells from the primary culture of 3 RCC patients.The concentration of Matrigel,recovery time and trypsinization were subsequently optimized.Results: The successful rate of primary culture was(90.6%)(29/32).Recovery of invasive cells was performed ideally when matrigel(diluted into 1.0 mg/ml and 20 ?l) was coated onto the filter of the well;cell suspension was at a concentration of 5?10~(5)/ml and invasive cells were recovered on the 5th day of culture.The growth of non-invasive cells was scattered,while that of the invasive cells was focal.The doubling time of invasive cells was 36.1 h and that of non-invasive was 50.6 h.Conclusion: The in vitro invasion assay using the Transwell is able to separate and recover the highly invasive primary RCC cells.The primary cells represent intact subpopulation composition,but it can hardly get through the life span of human primary tumor cells.