Establishment of epithelial and fibroblast cell cultures and cell lines from primary renal cancer nephrectomies.

Ning Yi Yap,Teng Aik Ong,Glenda C Gobe,Jayalakshmi Pailoor,Retnagowri Rajandram,Christudas Morais

doi:10.1002/cbin.11150

Abstract

Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were grown from explants. This protocol has been modified from previous published reports with additional antibiotics and washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial origin expressed higher expressions of epithelial markers such as pan-cytokeratin, cytokeratin 8 and E-cadherin whereas fibroblast cells expressed high α-smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.

Highlights

Renal cell carcinoma (RCC) comprises of 2-3% of all human malignancies and the incidence is increasing worldwide [1]
Ethical approval was obtained from the University of Malaya Medical Centre (UMMC) Ethics Committee (Ref : 848.17) and written informed consent was obtained for each patient
Collagenase type II 1mg/ml in tissue collection media Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose 4.5g/L and sodium pyruvate supplemented with 10% fetal bovine serum (FBS), 1X antibioticantimycotic, Primocin 100μg/ml (Invivogen, USA) and Mycozap prophylactic 1X (Lonza, USA) DMEM with high glucose 4.5g/L and sodium pyruvate supplemented with 10% FBS and 1X antibiotic-antimycotic 0.25% Trypsin-EDTA solution DMEM plus 10% FBS and 10% dimethyl sulfoxide

Summary

Introduction

Renal cell carcinoma (RCC) comprises of 2-3% of all human malignancies and the incidence is increasing worldwide [1]. RCC with sarcomatoid or rhabdoid transformation is not a recognised subtype of RCC as sarcomatoid or rhabodoid features can be found in all histologic subtypes of RCC [3]. Both of these histological transformations in RCC are associated with aggressive tumours and poor prognosis. Immune checkpoint inhibitors are the most recent approved for treatment of metastatic RCC. Since these drugs are relatively new, the long term clinical outcome is unknown at the moment. The genetic and molecular changes leading to the pathogenesis of RCC and development of therapy resistance are still not well understood

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