Lodrini Alessandra M, Groeneveld Esmee J, Palmen Meindert, Hjortnaes Jesper, Smits Anke M, Goumans Marie-José
Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands.
Department of Cardiothoracic Surgery, Leiden University Medical Center, Leiden, Netherlands.
Curr Protoc. 2024 Dec;4(12):e70068. doi: 10.1002/cpz1.70068.
Living myocardial slices (LMS) are ultrathin sections of adult myocardium that can be maintained in culture. These slices provide a unique platform for studying interactions between cardiomyocytes (CMs), other cardiac cell types, and the extracellular matrix while maintaining the cytoarchitecture and electrical phenotype of CMs over extended periods. Despite their advantages over other cardiac models, LMS have limitations, particularly their reliance on slice quality. The primary factor influencing the quality of the slices is the method used to process the cardiac tissue block. Current methods typically require immediate slice preparation following the excision of the tissue block, which restricts the timing of experiments. To address this limitation, we developed a simple procedure for cryopreserving human adult myocardium, allowing the preparation of LMS at a later stage. The protocol provides a list of required equipment and reagents, as well as a detailed description of the methodology for processing the myocardium and slice preparation. We present typical results demonstrating that cryopreserved human cardiac tissue retains biomass and structural integrity comparable to freshly obtained myocardium. Furthermore, we assessed the LMS derived from both fresh and cryopreserved samples. Histological analysis confirmed the preservation of viability, normal cytomorphology, and gap junctions between CMs in all LMS after 24 h and up to 5 days of culture in the absence of electrical stimulation. Cryopreservation extends the interval between tissue collection and LMS preparation, facilitating longer-term and more complex experiments. Further research into the impact of cryopreservation on various cardiac cell types will promote better donor organ management and efficient banking of cardiac samples from a multitude of donors and disease states. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation and preservation of human adult myocardium Basic Protocol 2: Preparation of adult living myocardial slices from cryopreserved blocks.
活性心肌切片（LMS）是成年心肌的超薄切片，可在培养中维持。这些切片为研究心肌细胞（CMs）、其他心脏细胞类型和细胞外基质之间的相互作用提供了一个独特的平台，同时能在较长时间内维持CMs的细胞结构和电表型。尽管LMS比其他心脏模型具有优势，但也存在局限性，尤其是对切片质量的依赖。影响切片质量的主要因素是处理心脏组织块所采用的方法。目前的方法通常要求在切除组织块后立即制备切片，这限制了实验的时间安排。为解决这一局限性，我们开发了一种简单的程序来冷冻保存人类成年心肌，以便在后期制备LMS。该方案提供了所需设备和试剂的清单，以及处理心肌和制备切片方法的详细描述。我们展示了典型结果，表明冷冻保存的人类心脏组织保留的生物量和结构完整性与新鲜获得的心肌相当。此外，我们评估了来自新鲜和冷冻保存样本的LMS。组织学分析证实，在无电刺激的情况下培养24小时及长达5天的所有LMS中，CMs的活力、正常细胞形态和间隙连接均得以保留。冷冻保存延长了组织采集和LMS制备之间的间隔时间，便于进行更长期、更复杂的实验。对冷冻保存对各种心脏细胞类型影响的进一步研究将促进更好地管理供体器官，并有效地储存来自众多供体和疾病状态的心脏样本。© 2024作者。由Wiley Periodicals LLC出版的《Current Protocols》。基本方案1：人类成年心肌的制备和保存 基本方案2：从冷冻保存的组织块制备成年活性心肌切片
