A rapid and simple procedure for the establishment of human normal and cancer renal primary cell cultures from surgical specimens

Abstract

The kidney is a target organ for the toxicity of several xenobiotics and is also highly susceptible to the development of malignant tumors. In both cases, in vitro studies provide insight to cellular damage, and represent adequate models to study either the mechanisms underlying the toxic effects of several nephrotoxicants or therapeutic approaches in renal cancer. The development of efficient methods for the establishment of human normal and tumor renal cell models is hence crucial. In this study, a technically simple and rapid protocol for the isolation and culture of human proximal tubular epithelial cells and human renal tumor cells from surgical specimens is presented. Tumor and normal tissues were processed by using the same methodology, based on mechanical disaggregation of tissue followed by enzymatic digestion and cell purification by sequential sieving. The overall procedure takes roughly one hour. The resulting cell preparations have excellent viabilities and yield. Establishment of primary cultures from all specimens was achieved successfully. The origin of primary cultured cells was established through morphological evaluation. Normal cells purity was confirmed by immunofluorescent staining and reverse transcription-polymerase chain reaction analysis for expression of specific markers.

Figure 1. Schematic representation of the isolation procedure for HPTEC and HRTC.

Figure 2. Representative histological images of renal cell carcinoma.

(A) Clear cell renal cell carcinoma, and (B) chromophobe renal cell carcinoma.

Figure 3. Representative morphology of confluent monolayers.

(A) Primary HPTEC, (B) HK-2 cell line, (C) primary clear cell RCC, and (D) primary chromophobe RCC. Magnification: 400x.

Figure 4. Fluorescence microscopy images from immunocytochemical staining for cytokeratin.

Hoechst 33258, anti-cytokeratin antibody staining, and merged images with double staining of primary cultured HPTEC, clear cell and chromophobe RCC cells. HK-2 and A-498 cells were used as control of human normal and tumor epithelial kidney cells, respectively. Magnification: 200x.

Figure 5. RT-PCR analysis.

Uromodulin (distal tubule marker), aquaporin 3 (collecting duct marker), and aminopeptidase A (proximal tubule marker) in primary cultured HPTEC derived from 4 donors and HK-2 cells. RNA isolated from a mixture of renal cells was used as a positive control and water as a negative control. Expression of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as a house-keeping gene.