Trophoblast Migration with Different Oxygen Levels in a Gel-Patterned Microfluidic System

Abstract

In the placenta, substances such as nutrients, oxygen, and by-products are exchanged between the mother and the fetus, and the proper formation of the placenta determines the success of pregnancy, including the growth of the fetus. Preeclampsia is an obstetric disease in which the incomplete formation of the placenta occurs, which is known to occur when there is an abnormality in the invasion of trophoblast cells. The invasion of trophoblast cells is controlled by oxygen concentration, and HIF-1α changes according to oxygen concentration, showing a difference in cell mobility. MMP-2 and MMP-9 are observed to be high in the endometrium involved in trophoblast invasion, and the expression is regulated according to the oxygen concentration. In this experiment, cell culture was conducted using a gel-patterned system with a hypoxic chamber. Before the chip experiment, the difference in the expression of MMP-2 and MMP-9 according to the oxygen concentration was confirmed using a hypoxia chamber. After that, trophoblast cells (HTR8/SVneo) and endothelial cells (HUVECs) were separated and cultured through a physical barrier through a hydrogel on a microfluidic chip. Cells were cultured in a hypoxic chamber under controlled oxygen levels. It was confirmed that the mobility of trophoblast cells in culture on the chip was upregulated in a hypoxic environment through oxygen control. This suggests that the formation of a hypoxic environment in the endometrium where the invasion of trophoblast cells occurs plays a role in increasing cell mobility.

Keywords: gel-patterned system; hypoxia; microfluidic system; placenta; trophoblast invasion.

Figure 1

Schematic figure of the gel-patterning microfluidic chip. (A) The cell culture areas are separated by GelMA structure. Trophoblast channel (yellow, HTR8/SVneo); endothelial cell channel (green, HUVECs). (B) The gel-patterning chip consists of a double layer of PDMS with different heights. Bottom (50 μm); top (400 μm). This height difference means the GelMA solution can only pattern according to the non-over-lapping area. (C) Comparison of trophoblast (HTR8/SVneo) migration with normoxic (21% O₂) and hypoxic (3% O₂) conditions.

Figure 2

Gel patterning of GelMA solution in the microfluidic chip. GelMA solution patterning image according to time. The GelMA solution does not leak into their overlap areas. According to the boundary of only the bottom layer channel, the GelMA solution is moved by capillary force. Scale bar: 200 μm.

Figure 3

Determination of the independence of the channel during the cell seeding process. (A) Bright-field image. (B) HTR8/SVneo with cell tracker green. (C) HUVEC with cell tracker red. Scale bar (red): 200 μm.

Figure 4

Verification of diffusion of substances in GelMA structure. The diffusion of the substance in the structure using GelMA was confirmed using an FITC-dextran. (A) Fluorescence image according to time. Scale bar: 100 μm. (B) Relative fluorescence intensity was calculated based on the trophoblast channel. Data are expressed with mean ± standard deviations (n = 3).

Figure 5

Comparison of MMP-2 and MMP-9 mRNA expression with oxygen levels in trophoblast (HTR8/SVneo). (A) MMP-2 mRNA expression in different oxygen levels. (B) MMP-9 mRNA expression in different oxygen levels. These mRNA expressions are normalized with 21% O₂ condition for control using beta action for the reference gene. Data are expressed with mean ± standard deviations (n = 3).

Figure 6

Comparison of trophoblast (HTR8/SVneo) migration in different oxygen levels. (A) Co-culture with HUVEC in normoxia (21% O₂). (B) Co-culture with HUVEC in hypoxia (3% O₂). Scale bar = 200 μm. (C) Migration distance of trophoblast in different oxygen levels.

Figure 6

Comparison of trophoblast (HTR8/SVneo) migration in different oxygen levels. (A) Co-culture with HUVEC in normoxia (21% O₂). (B) Co-culture with HUVEC in hypoxia (3% O₂). Scale bar = 200 μm. (C) Migration distance of trophoblast in different oxygen levels.