Yücel Doğacan, Solinsky Jacob, van Berlo Jop H
Department of Integrative Biology and Physiology, University of Minnesota; Lillehei Heart Institute, Department of Medicine, University of Minnesota.
Lillehei Heart Institute, Department of Medicine, University of Minnesota.
J Vis Exp. 2020 Oct 7(164). doi: 10.3791/60938.
The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and α-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.
成年哺乳动物的心脏由多种细胞类型组成，包括心肌细胞、内皮细胞和成纤维细胞。由于在组织学切片上难以可靠地识别心肌细胞核，许多研究小组依赖于在固定前分离活的心肌细胞来进行免疫染色。然而，这些活心肌细胞分离技术需要优化，以最大限度地提高样品的产量、活力和质量，尽管进行了最大程度的优化，但样品之间仍存在固有的波动。在这里，我们报告了一种可重复的方案，即在心脏酶消化之前进行固定，这可以在保持单个心肌细胞体内形态的同时实现最大产量。我们进一步开发了一个自动化分析平台，以确定单个心肌细胞的细胞核数量和每个细胞核的DNA含量。暴露胸腔后，通过在PBS中灌注60 mM KCl使心脏停搏于舒张期。接下来，将心脏固定在4%多聚甲醛（PFA）溶液中，然后用60 mg/mL胶原酶溶液消化。消化后，通过研磨使细胞分散，通过差速离心富集心肌细胞部分。对分离的心肌细胞进行肌钙蛋白T和α-肌动蛋白染色，以评估所得细胞群体的纯度。此外，我们开发了一个图像分析平台，以确定DAPI染色后心肌细胞的成核和倍性状态。基于图像的倍性评估产生了一致且可重复的结果。因此，通过该方案，可以保留单个心肌细胞的天然形态，以便进行免疫细胞化学和DNA含量分析，同时实现最大产量。
