Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia; St Vincent's Clinical School, University of NSW, Kensington, NSW 2052, Australia.
Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia.
J Mol Cell Cardiol. 2022 Sep;170:47-59. doi: 10.1016/j.yjmcc.2022.05.012. Epub 2022 May 26.
Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different age-dependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2 × 10 cells/heart) and viability (70-100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell type-specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR < 0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rod-shaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.
原代心肌细胞对于理解出生后心脏发育至关重要。然而，目前缺乏一种通用的方法，可以在不使用不同年龄相关分离程序和细胞培养的情况下获得新鲜纯化的心肌细胞。在这里，我们报告了一种标准化方法的发展，该方法允许从单个新生到成年 C57BL/6J 鼠心脏快速分离和纯化高质量的心肌细胞。Langendorff 逆行灌注目前仅限于成年心脏，通过开发一种更容易的原位主动脉插管技术，被改编为适用于新生和婴儿心脏。组织消化条件进行了优化，以在可比的时间内（<14 分钟）有效地消化所有年龄段的心脏。这导致了高产量（1.56-2.2×10^6 个细胞/心脏）和分离后心肌细胞的高活力（70-100%）。然后应用免疫磁珠细胞分离步骤，以获得高纯度的心肌细胞（95%），这通过免疫细胞化学、流式细胞术和 qRT-PCR 得到证实。对于细胞类型特异性研究，可以从单个心脏中提取足够数量的心肌细胞 DNA、RNA 和蛋白质，以进行分子实验。我们首次为单个心脏的新生心肌细胞生成了转录组数据集，其中揭示了九个性染色体上编码的性别特异性基因（FDR<0.05）。最后，我们还开发了一种原位固定方案，该方案可以保留心肌细胞的固有细胞结构（分离后约 94%呈杆状），并用于评估心肌细胞成熟过程中的细胞形态，以及捕获正在进行胞质分裂的新生儿呈纺锤形的细胞。总之，这些程序允许从任何出生后年龄的单个心脏中分离和分析高质量的心肌细胞进行分子和形态分析。
