Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai.
Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai;
J Vis Exp. 2023 Jun 2(196). doi: 10.3791/64368.
Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues. This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process. The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.
心力衰竭仍然是全球范围内导致死亡的主要原因，因此迫切需要更好的人类心脏临床前模型。组织工程对于基础科学心脏研究至关重要；体外人类细胞培养消除了动物模型的种间差异，而更类似于组织的 3D 环境（例如，具有细胞外基质和异细胞偶联）比传统的二维塑料培养皿培养在更大程度上模拟体内条件。然而，每个模型系统都需要专门的设备，例如，定制的生物反应器和功能评估设备。此外，这些方案通常很复杂，劳动强度大，并且容易使小而脆弱的组织失败。本文描述了一种使用诱导多能干细胞衍生的心肌细胞生成稳健的人工程心脏组织（hECT）模型系统的过程，用于组织功能的纵向测量。六个具有线性条形几何形状的 hECT 平行培养，每个 hECT 悬挂在一对力感应聚二甲基硅氧烷（PDMS）柱上，该柱连接到 PDMS 架上。每个柱都用黑色 PDMS 稳定柱跟踪器（SPoT）覆盖，这是一个新的功能，可提高易用性、吞吐量、组织保留率和数据质量。该形状允许可靠地光学跟踪柱的挠度，从而产生具有绝对主动和被动张力的改进抽搐力迹线。该盖几何形状消除了由于 hECT 从柱上滑落而导致的组织失效，并且由于它们涉及 PDMS 架制造后的第二步，因此可以将 SPoT 添加到现有的基于 PDMS 柱的设计中，而无需对生物反应器制造工艺进行重大更改。该系统用于演示在生理温度下测量 hECT 功能的重要性，并显示在数据采集过程中稳定的组织功能。总之，我们描述了一种最先进的模型系统，该系统可再现关键生理条件，以提高用于体外应用的工程心脏组织的生物逼真度、效率和严格性。
