2.1 Human fetal membrane cell lines for OOC experiment
We used established immortalized human fetal membrane cell lines as reported in our prior publications (Richardson, Jeong et al. 2019, Richardson, Kim et al. 2020, Richardson, Kim et al. 2020, Radnaa, Richardson et al. 2021, Tantengco, Richardson et al. 2021, Menon and Richardson 2022, Richardson, A et al. 2022, Richardson, Kammala et al. 2023) in our microfluidic in vitro model. AECs were cultured in Keratinocyte serum-free medium (KSFM) (17005042, ThermoFisher Scientific, Waltham, MA, USA) supplemented with bovine pituitary extract (30 µg/mL), epidermal growth factor (0.1 ng/mL), CaCl2 (0.4 mM), and primocin (0.5 mg/mL). AMCs were cultured in Dulbecco’s modified eagle medium/nutrient mixture F-12 (DMEM/F12) (MT10092CV, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 5% FBS, 10% penicillin/streptomycin, and 10% amphotericin B. All cells were grown at 37 ºC and 5% CO2 until they reached 80%-90% confluency. Immortalized cells have been validated against primary cells previously (Radnaa, Urrabaz-Garza et al. 2022). Only cells under passage 25 were used for experiments.
2.2 Microfluidic OOC model design, fabrication, and experimental setup
OOC design and fabrication: To model the amnion membrane (Fig. 1), the AM-OOC device previously developed, tested, and validated to mimic the in utero tissue (Richardson, Jeong et al. 2019, Richardson, Emezienna et al. 2022) was modified. Briefly, the device is composed of two cell culture compartments (250 µm height; outer compartment for AECs and inner compartment for AMCs) interconnected by 24 microchannels (5 µm in height, 30 µm in width, 600 µm in length) (Fig. 2a) not only to allow cell-to-cell communication during cultivation and localized biochemical treatment to each compartment, but also to prevent the movement of cells between compartments during the initial cell loading process. The device also contains an on-chip reservoir block where each reservoir compartment is aligned on top of the inlets and outlets of each chamber. The designed platform was fabricated in polydimethylsiloxane (PDMS; Sylgard 184, 1:10 mixture, DowDuPont, Midland, MI, USA) using a two-step photolithography master mold fabrication process, followed by soft lithography process of replica molding (Park, Jang et al. 2009). To improve bonding of the PDMS layer onto the glass substrate, the PDMS layers were treated with oxygen plasma (Harrick Plasma, Ithaca, NY, USA) for 90 s. This process was repeated for the PDMS reservoir bonding onto the PDMS cell culture layer already bonded to a glass substrate.
OOC device basement membrane coating
Before cells were loaded into the chip, the microchannels were coated with collagen to mimic the basement membrane of the amnion. Before using the AM-OOC, the devices were washed with 70% ethanol for 15 min for sterilization, washed three times with warm DMEM/F12 media to transit the culture compartment to appropriate culture condition, and then the microchannels were filled with 25% type IV collagen (Corning® Matrigel® Basement Membrane Matrix, Corning, Corning, NY, USA), dissolved in complete DMEM/F12, through an active vacuum suctioning process. The devices were then incubated for 4–12 h at 37 ºC and 5% CO2 environment.
Collection of primary amnion collagen
Methods for obtaining and preparing cell-free collagen from amniotic membrane extracellular matrix (ECM) follow our previously reported procedure (Richardson, Kim et al. 2020, Radnaa, Richardson et al. 2021, Kim, Richardson et al. 2022, Richardson, A et al. 2022).
Cell seeding in the AM-OOC
After Matrigel coating of the microchannels, the devices were washed two times with complete DMEM/F12 media before cell seeding. Immortalized AECs and AMCs (Fig. 2b) were trypsinized and loaded into the AM-OOC device (250,000 AECs for outer chamber, 45,000 AMCs cells + 20% primary collagen (Menon, Radnaa et al. 2020) + 25% Matrigel for inner chamber), mimicking the concentration ratios seen in utero.
2.3 Creating simulated amniotic fluid movement in AM-OOC
To create a simulated amniotic fluid movement environment in the outer compartment of the AM-OOC where AECs are being cultured, a pressure-driven pump controller with 4 independent syringe control units were utilized (HAPC, Harvard Apparatus, Holliston, MA, USA). To mimic the dynamic fluid motion as expected in amniotic cavity (Brace 1997, Modena and Fieni 2004), the pump was programmed to infuse culture media initially from 0 µl/h to a target flow rate, and then maintain for 2 h. Then, the flow gradually decreased to 0 µl/h within 2 min. Once infusion flow stops, flow starts back to opposite direction, maintain the flow for another 2 h, then continue the cycle throughout the 48 h incubation period (Fig. 2c). Flow rate in the outer compartment was converted to shear stress level using the Navier-Stokes Eq. (1),
$$\tau =\frac{6Q\mu }{w{h}^{2}}$$
1
where, \(\tau\) is the shear stress, \(Q\) is the volumetric flow rate, \(\mu\) is the dynamic fluidic viscosity, \(w\) and \(h\) indicate the width and height of the channel, respectively.
2.4 Creating an oxidative stress (OS) model as pathological positive control
To identify how fluid movement may affect PTB-associated signaling pathways, cigarette smoke extract (CSE) dissolved media was used as positive control to create a pathological condition. A single cigarette (Camel; R. J. Reynolds Tobacco, Winston Salem, NC, USA) was vacuumed into a filtering flask containing 25 mL of hFM_AECs media to obtain 100% CSE. Then, the stock CSE solution was filtered using a 0.22 µm Sterile flip filter (SCP00525, Millipore Sigma, Burlington, MA, USA) and diluted at a 1:25 ratio in the culture media before use. CSE was previously validated as a reproducible OS inducer in our OOC models (Menon, Boldogh et al. 2013, Bredeson, Papaconstantinou et al. 2014, Menon 2014, Menon, Boldogh et al. 2014, Polettini, Richardson et al. 2018, Richardson, Kammala et al. 2023) as well as is a major known risk factor of PTB. After cells have reached to 70–80% of confluency in the AM-OOC, culture media in the reservoir layer was removed and refilled with CSE in the AECs compartment, then incubated for 72 h to model OS condition.
2.5 Evaluating the effect of flow culture
The overall effect of flow culture in the AEC and AMC layers was examined by following cellular changes at each culture condition using: (1) lactate dehydrogenase (LDH) cytotoxicity assay, (2) immunocytochemical staining of vimentin, cytokeratin 18 (CK-18), and junction protein E-cadherin, (3) vimentin/CK-18 intensity ratio, (4) cell shape index, (5) cytokine production, and (6) scanning electron microscope (SEM) imaging.
2.6 Cytotoxicity assessment
To assess the cytotoxic effect of flow culture on the amnion epithelial cells cultured in the AM-OOC, a LDH cytotoxicity detection kit (ab197004, Abcam, Cambridge, UK) was used. Cell culture media from the cell culture chamber and reservoir in the AM-OOC was collected after the 48 h flow culture experiment. Approximately 5 µL of supernatant were used to perform the cytotoxicity assay according to the manufacturer’s protocol. Briefly, 2 µL of Developer Mix I/LDH Substrate Mix, 4 µL of Pico Probe III/Pico Probe, and 89 µL of LDH Assay buffer were mixed and then added to 5 µL of the collected supernatant in a 96-well plate. The assay plate was incubated at room temperature in the dark for 10 min. The control culture media was used as a negative control, expecting low level of cytotoxicity. Supernatant from cell culture in the AM-OOC was treated with Lysis buffer II/Cell Lysis Solution and incubated for 30 min to ensure lysis of cell membranes as Lysate control. LDH positive control buffer was mixed with 95 µL of prepared LDH Reaction Mix solution to provide positive cytotoxicity. Fluorescence was measured at 535 nm for excitation and 587 nm for emission.
2.7 Immunocytochemistry analysis
Immunocytochemical staining for vimentin (1:300; ab92547, Abcam, Cambridge, UK), CK-18 (1:800; ab668, Abcam, Cambridge, UK), and Epithelial (E)-cadherin (1:300; 24E10, Cell Signaling, Danvers, MA, USA) were used to monitor epithelial to mesenchymal transition (EMT) and tight junction expression. Antibodies were titrated to determine the appropriate dilutions to ensure specific and uniform staining. After 48 h, cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton-X, and blocked with 3% bovine serum albumin in 1X phosphate buffer silane (PBS), before incubation with primary antibodies overnight. Cells were washed three times in 1X PBS and then incubated with species-specific secondary antibodies (1:1000; Goat Anti-Rabbit IgG H&L Alexa Fluor 488, ab150073; Goat Anti-Mouse IgG H&L Alexa Fluor 594, ab150116; Donkey Anti-Rabbit IgG H&L Alexa Fluor 488, ab150073, Abcam, Cambridge, UK) for 1 h. The AM-OOC devices were washed with 1X PBS and then treated with NucBlue® Fixed Ready Probes Reagent (R37606, Thermo Fisher Scientific, Waltham, MA, USA) to stain the nucleus. The concentrations of primary and secondary antibodies were validated based on our previous AM-OOC-based study (Richardson, Jeong et al. 2019).
2.8 Microscopy
After 48 h of static and flow culture in the AM-OOC devices, bright field microscopy was performed (BZ-X800E microscope; 4x, 10x, and 20x magnification, Keyence, Osaka, Osaka, Japan) to determine cell morphology, intermediate filament expression, and tight junction.
Fluorescence image analysis
More than three random regions of interest per device (N = 5) and per chamber were used to determine overall vimentin, CK-18, and E-cadherin expression. Laser settings, brightness, contrasts, exposure times, and collection settings were uniform for all images collected. Images were not modified (brightness, contrast, and smoothing) for intensity analysis. Image J software was used to measure staining intensity.
Cell shape index (CSI) analysis: CSI was determined for AECs and AMCs by evaluating one frame from each experiment (total of five images) per culture condition for cell circularity using the Image J software. CSI was calculated using the following formula: CSI = 4π x area/perimeter2, which is an established method that was originally reported to determine vascular cell shape (Schutte and Taylor 2012). A circle would have a CSI of 1; a straight line would have a CSI of 0.
2.9 Scanning electron microscope (SEM) imaging
Cells were fixed by 4% PFA at room temperature for 15 min after 48 h cultivation in static or flow culture condition. Glass substrates were then detached from the PDMS chamber layer and rinsed with 1X PBS three times. A dehydration step was first performed using graded ethanol concentrations: 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95%, and 100%. Cells were immersed in each concentration once for 5 min and twice for 100% ethanol. Samples were gradually transferred to ethanol/hexamethyldisilane (HMDS) mixture in ratios of 3:1, 1:1, 1:3 with 30 min immersion for each step. Samples were then replenished with pure HMDS three times and dried in a chemical fume hood for at least 1 day. A thin gold layer was deposited on samples using a sputter coater (108 Manual Sputter Coater, Cressington, Watford, UK) and imaged with SEM (MIRA 3 Scanning Electron Microscope, TESCAN, Brno, Czechia).
Table 1
Summary of in vivo Shear Flow in Human Body
Organ/tissue
|
Fluid shear stress (dyne/cm2)
|
Ref.
|
Brain arterial vessels
|
10–70
|
(Wang, Xu et al. 2020)
|
Brain capillary network
|
5–23
|
(Wang, Xu et al. 2020)
|
Renal
|
0.3–1.2
2–4
|
(Ross, Gordon et al. 2021)
(Wang, Gust et al. 2022)
|
Glomerular tubule
|
30–50
5–20
|
(Wang, Gust et al. 2022)
(Ballermann, Dardik et al. 1998)
|
Podocyte slit diaphragm
|
80
|
(Wang, Gust et al. 2022)
|
Alveolar
|
0.5-3
|
(Denise C. Fernandes 2018)
|
Intestinal tract
|
0.002–0.08
|
(Denise C. Fernandes 2018)
|
Large veins
|
< 1
|
(Ballermann, Dardik et al. 1998)
|
Small arterioles
|
60–80
|
(Ballermann, Dardik et al. 1998)
|
Placenta
|
0.04–0.2
|
(Lee, Moulvi et al. 2023)
|
2.10 Multiplex assays for inflammatory cytokine markers analysis using Luminex
To assess the changes in inflammatory mediators, interleukin (IL)-6, IL-8, IL-10, and tumor necrosis (TNF)-alpha (⍺) were analyzed from the cell supernatants in the AM-OOC after 48 h of static or flow cell cultivation. Supernatants were collected from the reservoir of the device using a pipette. Standard curves were developed with duplicate samples of known quantities of recombinant proteins that were provided by the manufacturer. Sample concentrations were determined by relating the fluorescence values that were obtained to the standard curve by linear regression analysis.
2.11 Statistical analysis
All data were analyzed using Prism 8 software (GraphPad Software, La Jolla, CA, USA). Ordinary one-way ANOVA analysis of variance followed by Tukey’s multiple comparison test was used to compare normally distributed data with at least three means. Data are shown as mean ± standard error of mean. Asterisks denote p values as follows: *p < .05; **p < .01; ***p < .001; ****p < .0001.