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Tissue Disaggregation of Human Renal Cell Carcinoma with Further Isopyknic and Isokinetic Gradient Purification¹

Departments of Surgery [G. P. H., P. G. E.], Microbiology [G. P. H.], and Pathology [T. G. P.], University of Alabama in Birmingham, Birmingham, Alabama 35294

Five different human renal cell carcinomas were disaggregated with three combinations of enzymes. Significant tumor heterogeneity in response to the enzyme disaggregation was observed. A combination of collagenase (0.5 mg/ml) and trypsin (0.25%) was then used for routine disaggregation of 11 additional tumors. The viability of cells in suspension ranged between 63 and 98% with a mean viability of 83.2 ± 10.7% (S.D.). The mean yield of total viable cells per g of tissue was 17.4 ± 14.2 x 10⁶. Tumor cells were further fractionated in isopyknic and isokinetic gradients. After isokinetic sedimentation, significant heterogeneity among tumors was seen, but lymphocytes were consistently located in Fraction 7 ± 2, whereas tumor cells were predominantly in Fraction 22 ± 1. Malignant epithelial cells were enriched to a 85.8 ± 9.4% (range, 69.5 to 92.5%) purity by isokinetic gradient centrifugation. Lymphocytes could be successfully separated from tumor cells using an isopyknic gradient. Controlled rate freezing of cells provided material for repeated experiments while short-term tissue culture prior to cell separation increased the proportion of viable cells in the suspension. Disaggregation of human renal cell carcinoma and separation of malignant cells from tumor lymphocytes provides the foundation for characterizing these tumors biochemically and for analyzing hormonal responsiveness and the immunological characteristics of these tumors in vitro.

¹ Supported by Veterans Administration Grant SSN 111340132, NIH Young Investigators Award CA 2487502, and American Cancer Society Fellowship Award JFCF 398B.

² To whom requests for reprints should be addressed, at Department of Surgery, Division of Urology, University of Alabama in Birmingham Medical Center, 606-A, MEB, University Station, Birmingham, Ala. 35294.

³ Recipient of NIH Grant CA 23922 and American Cancer Society Grant PDT 126.

Received 9/ 4/79. Accepted 12/12/79.