Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, Raleigh, North Carolina.
Curr Protoc Toxicol. 2020 Dec;86(1):e99. doi: 10.1002/cptx.99.
Several cell lines of different origin are routinely used in research and drug development as important models to study human health and disease. Studying cells in culture represents an easy and convenient tool to approach complex biological questions, but the disadvantage is that they may not necessarily reflect what is effectively occurring in vivo. Human primary cells can help address this limitation, as they are isolated directly from human biological samples and can preserve the morphological and functional features of their tissue of origin. In addition, these can offer more relevant data and better solutions to investigators because they are not genetically manipulated. Human foreskin tissue discarded after surgery, for instance, represents a precious source for isolating such cells, including human foreskin fibroblasts (FSK), which are used in several areas of research and medicine. The overall health of cells is determined by the mitochondria. Alterations of cellular metabolism and cell death pathways depend, in part, on the number, size, distribution, and structure of mitochondria, and these can change under different cellular and pathological conditions. This highlights the need to develop accurate approaches to study mitochondria and evaluate their function. Here, we describe three easy, step-by-step protocols to study cellular viability and mitochondrial functionality in FSK. We describe how to use circumcision tissue obtained from the clinic to isolate FSK cells by mechanical and enzymatic disaggregation, how to use a cationic dye, crystal violet, which is retained by proliferating cells, to determine cell viability, and how to prepare samples to assess the metabolic status of cells by evaluating different mitochondrial parameters with transmission electron microscopy. We have successfully used the approaches outlined here to recapitulate physiological conditions in these cells in order to study the effects of increased intracellular levels of formaldehyde. © 2020 U.S. Government. Basic Protocol 1: Isolation and maintenance of human primary foreskin fibroblasts (FSK) Basic Protocol 2: Determination of cell viability by crystal violet staining Basic Protocol 3: Transmission electron microscopy to study cellular damage and mitochondrial dysfunction.
几种不同来源的细胞系通常被用于研究和药物开发，作为研究人类健康和疾病的重要模型。在培养的细胞中进行研究代表了一种简单方便的方法来解决复杂的生物学问题，但缺点是它们可能不一定反映体内实际发生的情况。人原代细胞可以帮助解决这个局限性，因为它们是直接从人体生物样本中分离出来的，可以保留其组织来源的形态和功能特征。此外，由于它们没有经过基因操作，因此可以提供更相关的数据和更好的解决方案。例如，手术后丢弃的人包皮组织是分离这些细胞的宝贵来源，包括人包皮成纤维细胞（FSK），它们被用于多个研究和医学领域。细胞的整体健康状况由线粒体决定。细胞代谢和细胞死亡途径的改变部分取决于线粒体的数量、大小、分布和结构，这些结构在不同的细胞和病理条件下会发生变化。这凸显了需要开发准确的方法来研究线粒体并评估其功能。在这里，我们描述了三种简单的分步协议，用于研究 FSK 中的细胞活力和线粒体功能。我们描述了如何使用从诊所获得的包皮组织通过机械和酶解聚来分离 FSK 细胞，如何使用阳离子染料结晶紫来确定增殖细胞的细胞活力，以及如何制备样品以通过评估不同的线粒体参数来评估细胞的代谢状态用透射电子显微镜。我们已经成功地使用了这里概述的方法来重现这些细胞中的生理条件，以便研究细胞内甲醛水平升高的影响。© 2020 美国政府。基本方案 1：人原代包皮成纤维细胞（FSK）的分离和培养基本方案 2：结晶紫染色法测定细胞活力基本方案 3：通过透射电子显微镜研究细胞损伤和线粒体功能障碍。
