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Biofabrication enables efficient interrogation and optimization of sequential culture of endothelial cells fibroblasts and cardiomyocytes for formation of vascular cords in cardiac tissue engineering

机译：生物制造能够用于在心脏组织中形成工程血管帘线的有效询问和内皮细胞成纤维细胞和心肌细胞的序贯培养的优化

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We previously reported that preculture of fibroblasts (FBs) and endothelial cells (ECs) prior to cardiomyocytes (CMs) improved the structural and functional properties of engineered cardiac tissue compared to culture of CMs alone or co-culture of all three cell types. However, these approaches did not result in formation of capillary-like cords, which are precursors to vascularization in vivo. Here we hypothesized that seeding the ECs first on Matrigel and then FBs 24 h later to stabilize the endothelial network (sequential preculture) would enhance cord formation in engineered cardiac organoids. Three sequential preculture groups were tested by seeding ECs (D4T line) at 8%, 15% and 31% of the total cell number on Matrigel-coated microchannels and incubating for 24 h. Cardiac FBs were then seeded (32%, 25% and 9% of the total cell number, respectively) and incubated an additional 24 h. Finally, neonatal rat CMs (60% of the total cell number) were added and the organoids were cultivated for seven days. Within 24 h, the 8% EC group formed elongated cords which eventually developed into beating cylindrical organoids, while the 15% and 31% EC groups proliferated into flat EC monolayers with poor viability. Excitation threshold (ET) in the 8% EC group (3.4 ± 1.2 V cm−1) was comparable to that of the CM group (3.3 ± 1.4 V cm−1). The ET worsened with increasing EC seeding density (15% EC: 4.4 ± 1.5 V cm−1; 31% EC: 4.9 ± 1.5 V cm−1). Thus, sequential preculture promoted vascular cord formation and enhanced architecture and function of engineered heart tissues.
机译：我们之前曾报道过，与单独培养CM或共培养所有三种细胞类型相比，在心肌细胞（CM）之前进行成纤维细胞（FBs）和内皮细胞（EC）的预培养可以改善工程心脏组织的结构和功能特性。但是，这些方法并未导致形成毛细血管样的帘线，而毛细血管样的帘线是体内血管形成的先兆。在这里，我们假设先将ECs接种在Matrigel上，然后在24小时后将FBs播种以稳定内皮网络（顺序预培养），将增强工程心脏类器官中的脐带形成。通过在包有Matrigel的微通道上分别以总细胞数的8％，15％和31％播种EC（D4T系）来测试三个连续的预培养组，并孵育24小时。然后接种心脏FB（分别占总细胞数的32％，25％和9％），再孵育24小时。最后，添加新生大鼠CM（占细胞总数的60％）并将类器官培养7天。在24小时内，8％的EC组形成了细长的帘线，最终发展成跳动的圆柱状类器官，而15％和31％的EC组则增殖为具有较弱存活力的扁平EC单层。 8％EC组（3.4±1.2 V cm -1 ）的励磁阈值（ET）与CM组（3.3±1.4 V cm -1 ）的励磁阈值相当）。 ET随着EC播种密度的增加而恶化（15％EC：4.4±1.5 V cm -1 ； 31％EC：4.9±1.5 V cm -1 ）。因此，顺序的预培养促进了血管索的形成并增强了工程心脏组织的结构和功能。

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