A Rapid and Simple Procedure for the Establishment of Human Normal and Cancer Renal Primary Cell Cultures from Surgical Specimens

Maria João Valente,Maria L Bastos,Márcia Carvalho,Carmen Jerónimo,Paula Guedes De Pinho,Vera L Costa,Félix Carvalho,Rui Henrique,Xin-Yuan Guan

doi:10.1371/journal.pone.0019337

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.

Highlights

Human proximal tubular epithelial cells (HPTEC) correspond to the major cell type in the human cortical tubulointerstitium and, most importantly, to the main target of a large number of xenobiotics, from drugs of abuse to antibiotics, antineoplastic agents, metals, and mycotoxins [1,2,3,4,5]
Cell culture medium: Dulbecco’s modified Eagle’s medium with nutrient mixture F-12 (DMEM/F-12) and GlutaMAXITM supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin/streptomycin (50 U/mL/50 mg/mL), fungizone (2.5 mg/mL), and human transferrin (5 mg/mL)
Histopathology The human renal tumor cells (HRTC) cultures were derived from RCC clear cell (4 cases) or chromophobe (2 cases) subtypes

Summary

Introduction

Human proximal tubular epithelial cells (HPTEC) correspond to the major cell type in the human cortical tubulointerstitium and, most importantly, to the main target of a large number of xenobiotics, from drugs of abuse to antibiotics, antineoplastic agents, metals, and mycotoxins [1,2,3,4,5]. Primary cultures of HPTEC can provide a well-characterized in vitro model, phenotypically representative of HPTEC in vivo. This in vitro system is endorsed for investigation on kidney cell function, transport processes, and cellular mechanisms of proximal tubular injury by xenobiotics, without interference of other factors that are associated to in vivo experiments. Several techniques have been described for isolation and culture of HPTEC. These methods have been based on time-consuming techniques like isopycnic centrifugation with Nycodenz or Percoll [6,7,8,9], or even complex microdissection protocols with or without enzymatic digestion [10,11]. The major weaknesses of these methodologies include low yields and labor intense procedures

Methods

Results

Conclusion