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Fig. 6 | BMC Cancer

Fig. 6

From: Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine

Fig. 6

Schematic of workflow to generate matched VHLmut and VHLwt cultures from surgically resected ccRCC samples. Tumor tissue that has been surgically resected is digested with enzymes to generate a single cell suspension, as described in the Methods. A portion of the tissue is used to extract DNA for VHL gene sequencing. The cell suspension can be viably frozen until sequencing results are obtained, if desired. An aliquot of cells is cultured in DSFM to generate a VHLwt culture. Remaining cells are stained with antibodies to CD45 and CD31 to allow exclusion of contaminating immune and endothelial cells, and the CA9+ and CA9- fractions are isolated by FACS and plated in media containing FBS. We showed that when both methods for generating a VHLwt culture were performed, the success rate increased to 90 % (9 out of 10 attempts; Table 1). After at least 2 passages, DNA is isolated from the cultured cells and sequenced to verify their identity as VHLmut cells and VHLwt cells. Cultures should be monitored every few passages to ensure identity and VHL mutation status

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