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. A previously developed amnion membrane (AM) organ-on-chip (OOC) was utilized but with dynamic flow to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 h to mimic fluid motion. A static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control representing 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 cytokeratin 18 (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 dynamic flow environment is not necessary to mimic in utero physiologic cellular conditions of an amnion membrane.

Keywords: Amniotic fluid; Dynamic flow cell culture; Fetal membrane; Microphysiological system; Organ-on-chip; Preterm birth; Shear stress.

Fig. 1

Fetal membrane of the feto-maternal interface and the intraamniotic cavity it surrounds. a Illustration of the anatomical structure 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 the 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) and 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.

Fig. 2

The dynamic flow amnion membrane organ-on-chip (AM-OOC) used for modeling the impact of amniotic fluid motion on cells. a Illustration of the dynamic flow AM-OOC and image of a device ready for testing. 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 while the dynamic flow condition is applied.

Fig. 3

Characterization of amnion membrane cells cultured by different flow conditions for 48 h in the AM-OOC. a Bright field and fluorescent microscopy showing normal condition of amnion cells in the AM-OOC with high viability. Scale bar = 10 μm. b LDH cytotoxicity assay result showing the level of LDH production from AECs and AMCs in the AM-OOC after 48 h culture (AEC - static vs. OS: p=0.0004; 50 μl/h vs. OS: p=0.0004; 200 μl/h vs. OS: p=0.0269) Data are shown as mean ± SEM (N=5). *p<.05; **p<.01; ***p<.001; ****p<.0001.

Fig. 4

Evaluating the impact of amniotic fluid motion on amnion membrane cells in the 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, cytokeratin-18 [CK-18] = red) of AECs and AMCs (top row) and cell-to-cell junction protein marker (E-cadherin = green) of AECs (bottom row). The scale bar is 10 μm. b Fluorescence intensity analysis of cell-specific markers showed minimal changes in their expression under the 48 h flow condition (AEC - static vs. OS: p=0.0008; 50 μl/h vs. OS: p = 0.0013; 200 μl/h vs. OS: p = 0.0008). Data are shown as mean ± SEM (N=5). c Amnion membrane cell morphological changes and cellular transition were determined by cell shape index analysis. AECs under static culture condition had cell shape index of 0.9 correlated to its cuboidal nature, while AECs under OS condition had a significantly lower cell shape index. AMCs did not show significant morphological changes under the dynamic flow culture (AEC - static vs. OS: p = 0.0001; 50 μl/h vs. OS: p = 0.0001; 200 μl/h vs. OS: p = 0.0001, AMC - static vs. OS: p = 0.02142). Data are shown as mean ± SEM (N=5).

Fig. 5

Scanning electron microscopy (SEM) images of hFM_AECs after 48 h of culture. Microvilli formation was observed from flow culture while static culture condition did not show any changes after 48 h culture in the AM-OOC.

Fig. 6

Production of inflammatory mediators by different culture conditions in the AM-OOC. Interleukin [IL]-6 showed flow-dependent inflammation (IL-6: static vs. OS: p = 0.0001; 50 μl/h vs. OS: p = 0.0005; 200 μl/h vs. OS: p = 0.0073) and IL-8 from AM-OOC showed increased inflammatory responses compared to 2D condition (IL-8: static vs. OS: p = 0.0001; 50 μl/h vs. OS: p = 0.0001; 200 μl/h vs. OS: p = 0.0001), but not significant. TNF-α and IL-10 did not show any changes among all conditions (TNF-α: static vs. OS: p = 0.0008; 50 μl/h vs. OS: p = 0.0238; 200 μl/h vs. OS: p = 0.0052). (IL-10: static vs. OS: p = 0.0001; 50 μl/h vs. OS: p = 0.0001; 200 μl/h vs. OS: p=0.0003). 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).