Generating multilayered engineered heart tissue patches to mimic physiological thickness and function using open microfluidics

项目摘要/摘要 心脏病是美国的主要死亡原因，通常是由不可逆转的心脏组织损伤引起的 导致心力衰竭。心脏组织不能自然再生，因此是 组织工程学。具有某些功能的工程化心脏组织(EHT)“贴片”的研究进展 结构，如预制图案化的血管和排列，在植入时显示出有希望的结果 变成了小动物模型。此外，注射的干细胞和植入的心脏“薄片”已经显示出一些 当移植到大型动物或人类的梗塞心脏时，成功地恢复了一些心脏功能。 尽管取得了这些成功，但扩展EHT结合了重要的功能特征，如血管形成 而对齐生理厚度(厘米级)仍然是一个主要的工程挑战。 这项提议的首要目标是在体外产生具有生理厚度的心脏组织补片，可以 最终被植入患者体内，以取代受损组织。重述的两个关键考虑因素 天然组织是(1)心脏组织高度血管化，(2)细胞和细胞外基质排列 在每一生理层内都是起作用的关键。我们利用全新的开放式解决方案来应对这些挑战 微流控图案化的方法。我们开放的微流控技术进步为 例如，它几乎与任何水凝胶兼容，包括标准的细胞外基质材料 如胶原蛋白和纤维蛋白，广泛用于EHT。它也与专门的刺激反应相兼容 工程化水凝胶，为复杂的工程组织开辟了空间和时间的可能性 控制力。此外，前体液体的流动是由被动表面张力驱动的；因此，敏感的干细胞- 衍生细胞不暴露于通过针挤压或光化学交联所产生的剪应力， 这是对诸如3D生物打印的其他组织制造技术的要求。最后，大面积(厘米- 比例尺)可以用单个移液步骤来图案化，使得这种制造方法非常适合于产生 (厘米级)组织。在这项提议中，我将开放微流控模式的这些独特属性应用于EHT。 具体地说，通过标准细胞培养背景形成可酶降解凝胶的能力 细胞外基质材料，如胶原或纤维蛋白，使复杂的血管系统能够在三维上形成图案。我 还将利用先前演示的开放式微流控设备的模块化堆叠 悬浮微流体以产生对齐的EHT贴片。在这些贴片中，组织被固定在 最后，诱导ECM重塑和排列。每一层都是独立生成和对齐的。然后，他们 与前一层成一定角度堆叠在一起，形成一个模仿心脏的多层组织 螺旋组织纤维排列。因此，我将以两个独立的目标--血管化和组织--为题 使用开放式微流控技术对准生理厚度的EHT。

PROJECT SUMMARY/ABSTRACT Heart disease is the leading cause of death in the U.S., often driven by irreversible cardiac tissue damage leading to heart failure. Cardiac tissue does not naturally regenerate, and thus is an important area of focus for tissue engineering. Previous development into engineered heart tissue (EHT) "patches" with some functional architecture such as pre-patterned vasculature and alignment has shown promising results when implanted into small animal models. Moreover, injected stem cells and implanted cardiac "sheets" have shown some success in restoring some cardiac function when implanted into infarcted hearts of large animals or humans. Despite these successes, scaling EHT that incorporates important functional features such as vascularization and alignment to a physiological thickness (cm-scale) remains a major engineering challenge. The overarching goal of this proposal is to generate in vitro physiologically thick heart tissue patches that can ultimately be implanted into patients to replace damaged tissue. Two key considerations for recapitulating native tissue are (1) cardiac tissue is highly vascularized, and (2) alignment of cells and extracellular matrix within each physiological layer is critical to function. We address these challenges utilizing novel open microfluidic patterning approaches. Our open microfluidic technological advancement offers unique benefits to EHT; for example, it is compatible with virtually any hydrogel, including standard extracellular matrix material such as collagen and fibrin, used extensively for EHT. It is also compatible with specialized stimuli-responsive engineered hydrogels, opening up possibilities for complex engineered tissues with spatial and temporal control. Further, the flow of precursor fluid is driven by passive surface tension forces; thus, sensitive stem-cell- derived cells are not exposed to shear stress from extrusion through a needle or photochemical crosslinking, which are requirements for other tissue fabrication techniques such as 3D bioprinting. Finally, a large area (cm- scale) can be patterned with a single pipetting step, making this fabrication approach ideal for generating large (cm-scale) tissues. In this proposal, I apply these unique attributes of open microfluidic pattering to EHT. Specifically, the ability to pattern enzymatically degradable gels through a background of standard cell culture ECM materials such as collagen or fibrin enables the patterning of complex vasculature in three dimensions. I will also take advantage of previously demonstrated modular stacking of open microfluidic devices and suspended microfluidics to generate aligned EHT patches. In these patches, the tissue is anchored on either end, inducing ECM remodeling and alignment. Each layer is generated and aligned independently. Then, they are stacked together at an angle from the previous layer, creating a multilayered tissue mimicking the heart's helical tissue fiber alignment. As such, I will address in two independent aims, vascularization and tissue alignment of physiologically thick EHT using open microfluidics.