A Dynamic Flow Fetal Membrane Organ-on-a-Chip System for Modeling the Effects of Amniotic Fluid Motion

Abstract

Fetal membrane(amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. An amnion membrane (AM) organ-on-chip (OOC) was utilized to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 hours flow culture to mimic fluid motion. Static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control for pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to CK-18 ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a flow culture environment is not necessary to mimic any in utero physiologic cellular conditions of fetal membrane-derived cells.

Keywords: Fetal membrane; amniotic fluid; microphysiological system; organ-on-chip; preterm birth; shear stress.

Figure 1

Fetal membrane of the feto-maternal interface and the intraamniotic cavity it surrounds. a Illustration of the anatomical structures and cellular components of fetal membrane depicting three fetal cell layers and one maternal cell layer. The highlighted amnion membrane is composed of the amnion epithelial layer and amnion mesenchymal layer. Amnion mesenchymal cells secret type I and III collagen, creating an extracellular matrix layer, as well as organized collagen in the fibroblast layer, tightly packed fibrillar collagen in the spongy layer, loose collagen of the reticular layer. The basement membrane is attached to the underlying chorion trophoblast cells. b Amnion membrane lines the human intrauterine cavity that supports fetus development during gestation. During the entire pregnancy, fetus is continuously exposed to the amniotic (shear) fluid surrounded by the amniotic sac and protected from external changes.

Figure 2

The amnion membrane organ-on-chip (AM-OOC) used for modeling the impact of in vitro amniotic fluid on cells. a Illustration of the AM-OOC and a real device image that mimic the amnion membrane. It consists of two cell culture chambers and a media reservoir. The two bottom cell culture chambers are interconnected by an array of microchannel. The top reservoir block is punched to match with inlets and outlet of the bottom cell culture chamber. Scale bar = 1 cm. b The two amnion membrane cells (hFM_AECs and hFM_AMCs) cultured in the AM-OOC, observed using bright field microscopy. Scale bar = 25 μm. c The experiment flow of creating in vitro amniotic fluid flow that mimics the in vivo condition. Syringe pumps and control units are connected to the AM-OOC device, where the device is incubated for cell co-culture whereas the dynamic flow condition is applied.

Figure 3

Characterization of amnion membrane cells cultured by different conditions for 48 h in the AM-OOC. a Bright field and fluorescent microscopy showing normal condition of amnion cells in AM-OOC with high viability. The scale bar is 10 μm. b LDH cytotoxicity assay result showing the percentage of dead AECs and AMCs in the AM-OOC after 48 h culture. Data are shown as mean ± SEM (N=5).

Figure 4

Evaluating amniotic fluid motion on amnion membrane cells in AM-OOC. a Fluorescence microscopy showing the expression of in utero amnion membrane cell specific markers after 48 h culture. These measurements included cytoskeletal marker (vimentin; green and cytokeratin-18 [CK-18]; red) of AECs and AMCs on the top and cell to cell junction protein marker (E-cadherin; green) of AECs on the bottom. The scale bar is 10 μm. b Intensity analysis of immunocytochemical cell specific markers documented minimal changes in expression by flow over 48 hr with different culture conditions on-chip. Data are shown as mean ± SEM (N=5). c Amnion membrane cells morphological changes and cellular transition were determined by cell shape index analysis. AECs under static culture condition averaged cell shape index of 0.9 correlated to its cuboidal nature, while AECs under OS condition average significantly decreased index. AMCs did not show significant morphological changes under dynamic flow culture. Data are shown as mean ± SEM (N=5).

Figure 5

Scanned electron microscopy (SEM) image of hFM_AECs after 48 h. SEM technology was utilized to see the microvilli formation from shear flow. Microvilli formation was observed from flow culture while static culture condition did not show any changes after 48 h in the AM-OOC.

Figure 6

Production of inflammatory mediators by different culture conditions in the AM-OOC. Interleukin [IL]-6 showed flow-dependent inflammation and IL-8 from AM-OOC showed increased inflammatory responses compared to 2D condition, but not significant. TNF-α and IL-10 did not show any changes among all conditions. Regardless of the culture condition (static vs. flow) and flow rate (50 μl/hr vs. 200 μl/hr), amnion epithelial cells do not produce inflammatory mediators for 48 h culture compared to 2D control static culture (p>.05) and OS condition (p=0.00001). Data are shown as mean ± SEM (N=5).