Stem Cell-based Human Placenta-on-a-Chip Using 3D Bioprinting

作为母体和胎儿血液供应之间的界面，胎盘将营养物质和氧气输送到胎儿，并将代谢废物和二氧化碳从胎儿中带走;它还产生建立和维持妊娠所必需的激素。为了执行这些不同的功能，胎盘由称为绒毛膜绒毛的功能单位组成，绒毛膜绒毛由基质细胞包围的胎儿毛细血管环组成，然后是细胞滋养层，整个包裹在合胞体滋养层单层中。随着胎盘成熟，基质细胞和细胞滋养层的数量显着减少，导致足月时形成主要由邻近合胞体滋养层单层的胎儿毛细血管组成的交换界面。胎盘功能异常与常见的和临床上显著的妊娠并发症有关，包括先兆子痫和胎儿生长受限。鉴于人类和实验上易于处理的动物模型之间胎盘结构和功能的显著差异，以及胎儿-母体界面的复杂微结构，迫切需要可用于实验性探测人类胎盘功能的体外模型。传统的系统，如绒毛膜癌细胞系、原代细胞滋养层、胎盘外植体培养物和胎盘组织的离体灌注，具有与使用恶性细胞来模拟非恶性细胞、未能重建不同细胞类型之间的复杂3D关系和/或短实验寿命相关的显著限制。最近，芯片胎盘方法已被应用，但现有的实现缺乏概括详细机制研究所需的原生微环境、解剖结构和长期功能的能力。 我们将通过在微流体平台中设计一种新型的人类胎盘芯片来应对这些挑战，这将重现人类胎盘的微观结构和功能。通过使用快速3D生物打印方法，我们能够更好地复制足月天然母胎胎盘界面的复杂微结构，并整合每种关键的人类胎盘细胞类型，包括胎盘微血管内皮细胞和原代细胞滋养层或人类滋养层干细胞。这项工作将通过以下两个目标来完成：（1）构建3D芯片上胎盘并确认组成细胞类型的空间位置、活力和身份，以及（2）对我们的平台作为胎盘功能的仿生模型进行详细评估，包括评估屏障形成以及变化的葡萄糖浓度和氧张力对生物分子转运的影响，胎盘激素的产生以及细胞内和细胞外RNA表达。在适当情况下，将这些结果与在相同条件下培养的胎盘外植体获得的结果进行比较。这项工作将产生一种经过验证的新型3D生物打印胎盘模型，可用于揭示正常和复杂妊娠中胎盘功能和功能障碍的机制。

As the interface between the maternal and fetal blood supplies, the placenta transports nutrients and oxygen to, and metabolic waste products and carbon dioxide away from, the fetus; it also produces hormones necessary for establishment and maintenance of pregnancy. To carry out these diverse functions, the placenta is comprised of functional units, called chorionic villi, which consist of loops of fetal capillaries surrounded by stromal cells, followed by cytotrophoblast, with the whole encased in a syncytiotrophoblast monolayer. As the placenta matures, the number of stromal cells and cytotrophoblast decrease significantly, resulting, at term, in an exchange interface composed primarily of fetal capillaries adjacent to the syncytiotrophoblast monolayer. Abnormalities in placental function are associated with common and clinically significant complications of pregnancy, including preeclampsia and fetal growth restriction. Given marked differences in placental structure and function between humans and experimentally tractable animal models, and the complex microarchitecture of the feto-maternal interface, there is a pressing need for in vitro models that can be used to experimentally probe the function of the human placenta. Traditional systems, such as choriocarcinoma cell lines, primary cytotrophoblast, placental explant cultures, and ex vivo perfusion of placental tissue, have significant limitations related to use of malignant cells to model non-malignant cells, failure to recreate the complex 3D relationships among different cell types, and/or short experimental life-span. Recently, placenta-on-a-chip approaches have been applied, but existing implementations lack the ability to recapitulate the native microenvironment, anatomical structure, and long-term function needed for detailed mechanistic studies. We will address these challenges by engineering a novel human placenta-on-a-chip in a microfluidic platform, which will recapitulate human placental microstructure and function. By using a rapid 3D bioprinting method, we are able to better replicate the intricate microarchitecture of the native maternal-fetal placental interface at term and incorporate each of the key human placental cell types, including placental microvascular endothelial cells, and primary cytotrophoblast or human trophoblast stem cells. The work will be accomplished in two aims by: (1) building the 3D placenta-on-a-chip and confirming the spatial placement, viability, and identity of the component cell types, and (2) performing detailed evaluation of our platform as a biomimetic model of placental function, including assessment of barrier formation, and the effects of varying glucose concentration and oxygen tension on biomolecular transport, production of placental hormones, and intracellular and extracellular RNA expression. Where appropriate, these results will be compared to those obtained from placental explants cultured in the same conditions. This work will produce a validated novel 3D bioprinted placental model that can be used to reveal the mechanisms of placental function and dysfunction in normal and complicated pregnancies.