Development of a Microfluidic Device for Modeling Human Placenta

Abstract

The placenta is a multifunctional organ essential for fetal development and survival and plays an important role in hormone production and the exchange of substances such as nutrients, gases, and waste products between the mother and fetus. Research on the mechanism of human placental development is important for elucidating the cause of infertility and intrauterine growth restriction and developing new medicines for them, but little is known about the mechanism. Studying the human placenta has been limited because of large species differences, the limited resources of the human placenta, and also the lack of an in vitro cell culture system that can be a good model of the human placenta. To accelerate the study of the human placenta, we recently established a human trophoblast stem (TS) cell lime from the human placenta [1]. This TS cell line could differentiate into the major placental trophoblast subpopulations, i.e. extravillous cytotrophoblast (EVT), and syncytiotrophoblast (ST). In vivo, EVT cells can invade the endometrium and remodel the maternal blood vessels, and ST cells can produce large quantities of placental hormones and mediate the maternal-fetal gas, nutrient, and waste exchange. The purpose of this study is to develop an in vitro human placental model that resembles the human placenta in vivo, using the TS cell line and microfabrication technology. In particular, we have attempted to fabricate a microfluidic device that recapitulates the function of fetal EVT cells that invade the endometrium and remodel the maternal blood vessels. This device could be an excellent model for studying the development and the function of the human placenta (Fig.1a). A two-layer microfluidic device was designed to imitate the fetal and maternal compartments in the placenta and to recapitulate the function of in vivo EVT cells regarding the invasion and remodeling. The upper layer has a small well(Φ4mm) for culturing EVT cells. The lower layer has a microchannel for the formation of perfusable vascular networks of human umbilical vein endothelial cells (HUVEC), as a cell culture space mimicking the maternal environment. A porous membrane was set between the upper layer and the lower layer, and this co-culture system was fabricated to aim to evaluate the migration and interaction of each cell in both layers. This two-layer microfluidic device was made using Polydimethylsiloxane (PDMS). To make each layer, PDMS was poured into silicon substrate molds that were made by photolithography and then cured. The polyester (PET) porous membrane underwent the silane-coupling-treatment and was inserted between the upper and lower layers. To bond all three components thoroughly, they were treated with oxygen plasma before assembly (Fig.1b). In the upper layer, for culturing EVT cells, TS cells were seeded to the small well and were differentiated into EVT cells by culturing cells in a differentiation medium containing Matrigel. In the lower layer, to form vascular networks of HUVEC, HUVEC suspension and a mixed solution of fibrin gel and thrombin were introduced into a channel. Two channels with multi micro-posts were also made in the lower layer to perfuse the fresh medium into the vascular system. The cells in the device were subjected to immunostaining and analyzed by fluorescence microscopy. Results of fluorescence microscopy showed that most of the TS cells in the upper layer were differentiated into EVT cells, which expressed an EVT cell marker HLA-G (Fig1.c). Furthermore, in the lower layer, HUVEC formed vascular networks with branched luminal structures. The fresh medium was successfully introduced into the networks through channels with micro-posts, and the vascular networks of HUVEC were able to be maintained for a long period of the time in this novel EVT-HUVEC co-cultured system (Fig.1c). In this device, these two types of cells are separated by a porous membrane and we would like to analyze in detail how these cells interact through the membrane in the future. Through the analysis, we hope to reveal the mechanism by which fetal EVT cells remodel the uterine vessels. We believe that this device would be an excellent model to examine the role of EVT cells in the human placenta, and we hope it will be a critical step in constructing a human placenta model.[1] Okae. H, Toh. H, Sato. T, Hiura. H, Takahashi. S, Shirane. K, Kabayama. Y, Suyama. M, Sasaki. H, and Arima. T, "Derivation of Human Trophoblast Stem Cells.", Cell Stem Cell 22, 50, 2018. Figure 1