LightKick: Novel bioengineering system for activity-dependent acceleration of functional maturation of human stem cell-derived cardiomyocytes

摘要 人IPSC来源的心肌细胞为心血管药物的发现和再生提供了无限的可能性 医学，但前提是它们能反映成人心脏的生理状态。目前的方案生产心肌细胞与 胚胎特征，而不是成人特征，严重限制了它们对成年人口的实用性。最近的研究表明 旨在在培养皿中重新创造环境线索的生物工程方法可以显著改善 心肌细胞的成熟度。需要新的技术工具才能将这些发现转化为实践。 使学术界和生物技术/制药行业的科学家能够产生更成熟的hiPSC来源 基于对心肌细胞的需求，我们建议设计、设计、制造和验证一种新的生物工程硬件系统 这将在很长一段时间内光学控制HiPSC来源的心肌细胞的起搏，以驱动其 以一种依赖活动的方式向成体表型成熟。拟议的成熟促进系统将 利用我们专有的石墨烯介导的光学刺激技术，实现快速、可逆和生理上的 细胞的相应激活。我们的技术有几个关键的优势，因为(1)它不需要基因修改 (vs.光遗传学)或电线/电极(vs.电场刺激)，(2)它为可调光提供了前所未有的灵活性- 受控细胞刺激协议，以及(3)符合高通量和可扩展性要求。 拟议系统(LightKick)的组件将包括石墨烯涂层微型板(G板)、多LED 用于照亮在细胞孵化器内的G板中培养的细胞的单元和外部多参数可编程控制器 定义照明参数。G型平板已经被开发和验证用于光学刺激和全光学 化验。我们将在基板硬度和微图案化方面进一步优化G板。两个方面的发展 其他组件一起组成光学刺激模块，将代表 建议的项目。为了使该模块适用于长期的光学刺激，我们将考虑到最近开发的 光发射体、G板的光电特性、心肌细胞的生物物理特性和长期细胞培养 条件。我们的光刺激模块的广泛技术能力将提供(1)可扩展性(通过提供 按需在几口井或几块板上发出刺激信号)；(2)模块无线监控和远程控制 光学调步；以及(3)在广泛的生理相关光照明参数范围内的灵活可调整性 使用用户友好的交互式图形用户界面软件。 重要的是，由LightKick实现的对HiPSC来源的心肌细胞的长期光学刺激将完全 与许多可能在成熟过程中发挥作用的环境线索相适应。LightKick系统将允许STEM 细胞专家(1)开发高效和个性化的细胞刺激模式，以产生成人特有的患者 HiPSC衍生细胞，以及(2)容易地将动态电环境线索与其他环境线索相结合，进一步 改进多参数成熟促进方案。我们预计LightKick系统将戏剧性地 简化，从而加快寻找成熟问题的根本和实际答案的进程。

ABSTRACT Human iPSC-derived cardiomyocytes offer infinite possibilities for cardiovascular drug discovery and regenerative medicine, but only if they reflect the physiology of an adult human heart. Current protocols produce cardiomyocytes with embryonic rather than adult profiles, severely limiting their utility for the adult population. Recent studies suggest that bioengineering approaches aimed at re-creating environmental cues in a dish can lead to marked improvements in cardiomyocytes’ maturation profiles. New technological tools are required to translate these findings into the practice. To enable scientists in academia and biotech/pharmaceutical industry to generate more mature hiPSC-derived cardiomyocytes on demand, we propose to design, engineer, fabricate, and validate a novel bioengineering hardware system that will optically control the pacing of hiPSC-derived cardiomyocytes over extended periods of time to drive their maturation toward the adult phenotype in an activity-dependent manner. The proposed maturation-promoting system will utilize our proprietary graphene-mediated optical stimulation technology that allows fast, reversible, and physiologically relevant activation of cells. Our technology has several critical advantages, as (1) it requires neither genetic modifications (vs. optogenetics) nor wires/electrodes (vs. electric field stimulation), (2) it offers an uncharted flexibility for tunable light- controlled cell stimulation protocols, and (3) it is compliant with high-throughput and scalability requirements. The components of the proposed system (LightKick) will include graphene-coated microplates (G-plates), a multi-LED unit to illuminate cells cultured in G-plates inside a cell incubator, and an external multiparametric programmable controller to define illumination parameters. G-plates have already been developed and validated for optical stimulation and all-optical assays. We will further optimize G-plates in terms of the substrate rigidity and micropatterning. The development of two other components, together comprising an optical stimulation module, will represent the main engineering efforts of the proposed project. To tailor this module for long-term optical stimulation, we will take into consideration recently developed light emitters, optoelectronic properties of G-plates, biophysical properties of cardiomyocytes, and long-term cell culturing conditions. Extensive technical capabilities of our optical stimulation module will offer (1) the scalability (by delivering stimulation signals in few wells or few plates on demand); (2) wireless monitoring of the module and remote control of optical pacing; and (3) the flexible tunability across the wide range of physiologically-relevant light illumination parameters using a user-friendly interactive GUI software. Importantly, long-term optical stimulation of hiPSC-derived cardiomyocytes enabled by the LightKick would be fully compatible with many environmental cues that might play in the maturation process. The LightKick system will allow stem cell experts (1) to develop efficient and individualized cell stimulation patterns for producing adult-like patient-specific hiPSC-derived cells, and (2) to easily combine dynamic electric environmental cues with other environmental cues, further improving multiparametric maturation-promoting protocols. We anticipate that the LightKick system will dramatically simplify and, thus, accelerate the process of finding fundamental and practical answers to the maturation problem.