The kidney is a target organ for the toxicity of several

The kidney is a target organ for the toxicity of several xenobiotics and is also highly susceptible to the development of malignant tumors. proximal tubular epithelial cells (HPTEC) correspond to the major cell type in the 1256094-72-0 human cortical tubulointerstitium and, most importantly, to the main target of a large number of xenobiotics, from drugs of abuse to antibiotics, antineoplastic brokers, metals, and mycotoxins [1]C[5]. Primary cultures of HPTEC can provide a well-characterized model, phenotypically representative of HPTEC system is usually endorsed for investigation on kidney cell function, transport processes, and cellular mechanisms of proximal 1256094-72-0 tubular injury by xenobiotics, without interference of other factors that are associated to experiments. For that purpose, it is essential to achieve highly enriched HPTEC preparations from kidney tissue. 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]C[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. In addition to xenobiotic-induced toxicity, the kidney is also susceptible to the development of benign (e.g., oncocytoma) or malignant (e.g., renal cell carcinoma, RCC) 1256094-72-0 neoplasms. RCC comprises 85% of renal cancers in adults, and more than 3% of adult malignancies. With over 30,000 new cases diagnosed annually, it is the sixth leading cause of cancer-related death in the USA, being responsible for approximately 12,000 deaths per year [12], [13]. According to its histological appearance, RCC can be divided into subtypes: conventional or clear cell, papillary, chromophobe, and unclassifiable RCC [14], [15]. Clear cell RCC is the most common form of renal cancer. It is originated from the proximal tubular epithelium, and accounts for 80 to 85% of renal cell tumor [12], [15]. Papillary RCC is the second most usual subtype of kidney cancer, with a prevalence of roughly 10% of renal malignant tumors, and is characterized by tumor cells arranged in a papillary configuration [16], [17]. Chromophobe is an uncommon subtype of RCC, with a prevalence of approximately 5% of renal malignant tumors. As clear cell RCC, it develops in the renal cortex [18], [19]. RCC etiology is usually yet unidentified, developing either as a sporadic form or as a hereditary disease, and whatever the subtype is usually, it is usually described as highly resistant to conventional radio-, chemo- and immunotherapy regimens [12], [15], [20]. Therefore, the discovery of new strategies for therapeutic intervention remains a priority. In this regard, cell culture of human renal tumor cells (HRTC) has proven to be an adequate in vitro model for the study of therapeutic approaches in RCC [15], [21]C[23]. Moreover, alongside studies in tumor cell cultures, it is necessary TMEM8 to test the toxicity of potential therapeutic agents in the normal counterpart cells. Therefore, it is the main goal of this study to present a simple and rapid method for the establishment of human kidney primary cultures, both normal (HPTEC) and tumoral (HRTC), obtained from the same organ. The procedure presented herein has been adapted from previously established methods [6], [9], [24]C[26] and used to process normal and tumor tissues. It is usually based on mechanical disaggregation of the tissue followed by enzymatic digestion and cell purification by sequential sieving. This technique allows the separation of a cellular fraction that is highly enriched in HPTEC or HRTC from respectively normal 1256094-72-0 renal cortex and tumoral kidney tissue, with far higher yield and cell viability than other established isolation procedures. The overall procedure is usually technically simple, enabling its easy implementation in cell culture laboratories. Materials and Methods Materials The following materials were obtained from GIBCO? Invitrogen (Barcelona, Spain) unless stated otherwise. Cell culture medium: Dulbecco’s altered Eagle’s medium with nutrient mixture F-12 (DMEM/F-12) and GlutaMAX-I? supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin/streptomycin (50 U/mL/50 g/mL), fungizone (2.5 g/mL), and human transferrin (5 g/mL). Hank’s buffered salt answer (HBSS) without CaCl2 and MgCl2. Collagenase answer: dissolve 50 mg of collagenase type 2, 315 U/mg (Worthington, Lakewood, NJ) in.