REQUIMTE - Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal.
PLoS One. 2011 May 4;6(5):e19337. doi: 10.1371/journal.pone.0019337.
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.
肾脏是多种外源化学物质毒性的靶器官，也极易发生恶性肿瘤。在这两种情况下，体外研究都为细胞损伤提供了深入了解，并为研究几种肾毒性物质的毒性作用机制或肾癌的治疗方法提供了合适的模型。因此，建立高效的人正常和肿瘤肾细胞模型的方法至关重要。在本研究中，提出了一种从手术标本中分离和培养人近端肾小管上皮细胞和人肾肿瘤细胞的技术简单、快速的方案。基于组织的机械分散，然后进行酶消化和通过顺序筛网进行细胞纯化，使用相同的方法处理肿瘤和正常组织。整个过程大约需要一个小时。得到的细胞制剂具有良好的活力和产率。所有标本的原代培养均成功建立。通过形态学评估确定原代培养细胞的来源。通过免疫荧光染色和特定标志物表达的逆转录-聚合酶链反应分析来确认正常细胞的纯度。
