Shcherbakova Anastasia, Utkina Marina, Valyaeva Anna, Pachuashvili Nano, Bondarenko Ekaterina, Urusova Liliya, Popov Sergey, Mokrysheva Natalya
Department of General, Molecular and Population Genetics, Endocrinology Research Centre, Dm. Ulyanova St., 11, 117292 Moscow, Russia.
A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskye Gory, House 1, Building 40, 119992 Moscow, Russia.
Biology (Basel). 2024 Aug 27;13(9):665. doi: 10.3390/biology13090665.
The enzymatic dissociation of human solid tissues is a critical process for disaggregating extracellular matrix and the isolation of individual cells for various applications, including the immortalizing primary cells, creating novel cell lines, and performing flow cytometry and its specialized type, FACS, as well as conducting scRNA-seq studies. Tissue dissociation procedures should yield intact, highly viable single cells that preserve morphology and cell surface markers. However, endocrine tissues, such as adrenal gland tumors, thyroid carcinomas, and pituitary neuroendocrine tumors, present unique challenges due to their complex tissue organization and morphological features. Our study conducted a morphological examination of these tissues, highlighting the intricate structures and secondary degenerative changes that complicate the dissociation process. We investigated the effects of various dissociation parameters, including the types of enzymes, incubation duration, and post-dissociation purification procedures, such as debris removal and nontarget blood cell lysis, on the viability of cells derived from different tumor types. The findings emphasize the importance of optimizing tissue digestion protocols to preserve cell viability and integrity, ensuring reliable outcomes for downstream analyses.
人实体组织的酶解是分解细胞外基质以及分离单个细胞的关键过程，可用于多种应用，包括永生化原代细胞、创建新型细胞系、进行流式细胞术及其特殊类型荧光激活细胞分选（FACS）以及开展单细胞RNA测序（scRNA-seq）研究。组织解离程序应产生完整、高活力的单细胞，这些细胞要保持形态和细胞表面标志物。然而，内分泌组织，如肾上腺肿瘤、甲状腺癌和垂体神经内分泌肿瘤，由于其复杂的组织结构和形态特征，带来了独特的挑战。我们的研究对这些组织进行了形态学检查，突出了使解离过程复杂化的复杂结构和继发性退行性变化。我们研究了各种解离参数的影响，包括酶的类型、孵育时间以及解离后纯化程序，如去除碎片和非靶血细胞裂解，对不同肿瘤类型来源细胞活力的影响。研究结果强调了优化组织消化方案以保持细胞活力和完整性的重要性，确保下游分析有可靠的结果。
