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在微电极阵列上构建各向异性的心脏单层细胞用于分析电生理特性的非侵入式方法。

Engineering anisotropic cardiac monolayers on microelectrode arrays for non-invasive analyses of electrophysiological properties.

机构信息

Department of Biomedical Engineering, University of Miami, Coral Gables, FL, USA.

出版信息

Analyst. 2019 Dec 16;145(1):139-149. doi: 10.1039/c9an01339c.

Abstract

A standard culture of cardiac cells as unorganized monolayers on tissue culture plastic or glass does not recapitulate the architectural or the mechanical properties of native myocardium. We investigated the physical and protein cues from the extracellular matrix to engineer anisotropic cardiac tissues as highly aligned monolayers on top of the microelectrode array (MEA). The MEA platform allows non-invasive measurement of beating rate and conduction velocity. The effect of different extracellular proteins was tested by using the most common extracellular matrix proteins in the heart, fibronectin and gelatin, after aligning myocytes using a microcontact (μC) printing technique. Both proteins showed similar electrophysiological results before the monolayer began to delaminate after the sixth day of culture. Additionally, there were no significant differences on day 4 between the two microcontact printed proteins in terms of sarcomere alignment and gap junction expression. To test the effect of substrate stiffness, a micromolded (μM) gelatin hydrogel was fabricated in different concentrations (20% and 2%), corresponding to the elastic moduli of approximately 33 kPa and 0.7 kPa, respectively, to cover both spectra of the in vivo range of myocardium. Cardiac monolayers under micromolded conditions beat in a much more synchronized fashion, and exhibited conduction velocity that was close to the physiological value. Both concentrations of gelatin hydrogel conditions yielded similar sarcomere alignment and gap junction expression on day 4 of culture. Ultimately, the 3D micromolded gelatin hydrogel that recapitulated myocardial stiffness improved the synchronicity and conduction velocity of neonatal rat ventricular myocytes (NRVM) without any stimulation. Identifying such microenvironmental factors will lead to future efforts to design heart on a chip platforms that mimic in vivo environment and predict potential cardiotoxicity when testing new drugs.

摘要

标准的心肌细胞培养方式是在组织培养塑料或玻璃上形成无序的单层细胞,这种方式无法重现天然心肌的结构或机械特性。我们研究了细胞外基质的物理和蛋白质线索,以工程化各向异性的心肌组织,使其在微电极阵列(MEA)上形成高度对齐的单层。MEA 平台允许对跳动率和传导速度进行非侵入性测量。通过使用心脏中最常见的细胞外基质蛋白——纤连蛋白和明胶,在使用微接触(μC)印刷技术对齐心肌细胞后,我们测试了不同细胞外蛋白的效果。在培养的第 6 天单层细胞开始分层之前,两种蛋白都显示出类似的电生理结果。此外,在第 4 天,两种微接触印刷蛋白在肌节排列和缝隙连接表达方面没有显著差异。为了测试基底硬度的影响,我们制备了不同浓度(20%和 2%)的微成型(μM)明胶水凝胶,分别对应于大约 33 kPa 和 0.7 kPa 的弹性模量,涵盖了体内心肌范围的两个弹性模量谱。在微成型条件下,心肌单层以更同步的方式跳动,并表现出接近生理值的传导速度。在培养的第 4 天,两种浓度的明胶水凝胶条件下均产生了类似的肌节排列和缝隙连接表达。最终,模拟心肌硬度的 3D 微成型明胶水凝胶在没有任何刺激的情况下,提高了新生大鼠心室肌细胞(NRVM)的同步性和传导速度。确定这些微环境因素将有助于未来努力设计模拟体内环境的心脏芯片平台,并在测试新药时预测潜在的心脏毒性。

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