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

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.


Introduction
Human fetal membrane (amniochorion) lines the intra-amniotic cavity during pregnancy (Menon, Richardson et al. 2019).This membrane is composed of chorion trophoblast cells, amnion mesenchymal cells, and amnion epithelial cells, and plays a signi cant role in maintaining pregnancy and fetal development (Menon, Richardson et al. 2019, Richardson, Kim et al. 2020).The fetal membranes surround the fetus, enclose amniotic uid, and protect the fetus during gestation from mechanical and pathological alterations, such as stress, stretch, infection, and in ammation (Manabe, Himeno et al. 1991, Verbruggen, Oyen et al. 2017).The amniotic uid bathes the innermost amnion epithelial layer and is a shock absorber during fetal movement and growth (1970( , Schmidt 1992).Unlike unidirectional blood ow (Martin 1965, Yang 1998, Prefumo, Campbell et al. 2019), amniotic uid subtly rocks back and forth, and thus, amnion epithelial cells are constantly exposed to shear stress from uid undulation, but at a very low level (Gilbert andBrace 1993, Modena andFieni 2004).The ripple effect may impact cells of the amnion membrane; however, amniotic uid is enriched in growth factors and other nutrients needed for cell survival and growth; hence, any aberrations impacted by the uid are likely repaired to avoid any disruption in the membrane function (Borgida, Mills et al. 2000).Nevertheless, uid motion may impact shear stress in conditions like polyhydramnios or oligohydramnios due to abnormal uid volume impacting membrane function (Sherer 2002, Rabie, Magann et al. 2017).Prior to testing the impact of pathologic uid volume changes and its motion effect on amnion, a normal baseline understanding of changes that can be induced by uid motion is needed.
In the human body, physiological shear stress plays an important role in regulating cell differentiation, modulating phenotypes (i.e., epithelial, or mesenchymal), and cellular functionalities (i.e., tight junction expression) based on its frequency, duration, and intensity (Colgan, Ferguson et al. 2007 inducing non-uniform levels of shear ow and stress, unlike directional blood ow.However, no in vitro model systems have tested the impact of amniotic uid shear stress on amnion cells. For the past decades, microphysiological system (MPS), also known as organ-on-chip (OOC), has been developed, validated, and utilized as more physiologically relevant in vitro models than conventional 2dimensional (2D) cell culture platforms, such as well plate or trans-well platforms (Kang, Park et al. 2021, Cao, Zhang et al. 2023).MPS allows better cell-to-cell communications for understanding human physiology and pathophysiology, as well as testing drug e cacy and toxicity, as well as potential harmful effects of environmental toxicants, to name a few examples.Moreover, OOC platforms often integrate microchannels, micropillars, or pneumatic valves to provide biomechanical cues to the cell culture systems such as shear stress, stretch, or contraction that cannot be easily applied in conventional 2D platforms (Cao, Zhang et al. 2023).
In our previously developed amnion membrane (AM) OOC model, using standard culture conditions, we investigated cellular transitions under normal (physiologic) and oxidatively stressed (pathologic) conditions (Richardson, Jeong et al. 2019).The AM-OOC model contained human fetal membrane amnion epithelial cells (hFM_AECs) and amnion mesenchymal cells (hFM_AMCs) separated by type IV collagen lled microchannels modeling the amnion basement membrane, recreating the amnion membrane during human pregnancy.These microchannel arrays allow for the biochemical transportation of signaling molecules from one chamber to another while preventing initial cell mixing between chambers (i.e., although they do allow for active cell migration between chambers due to epithelial-to-mesenchymal transition [EMT]), enabling co-culture of multiple different types of cells.This structure also allows biochemicals such as drugs or environmental toxicants to propagate between cell layers, modeling physiological human pharmacokinetics of stimulants.The use of this platform led to several new ndings: 1) amnion membrane cells can migrate (non-reversible) through the microchannels, known EMT and vice versa mesenchymal -to-epithelial transition (MET) and integrate themselves into the emigrated environment; 2) oxidative stress (OS) induces EMT or MET but inhibited migration of cells and pro-in ammatory environment; 3) inhibition of OS by antioxidants and functional inhibitors of stress signaler p38 mitogen-activated protein kinase (MAPK) restore the migration property, suggesting OS and p38 MAPK downstream signaling could regulate cellular migration.This study only focused on visualizing cellular transition and migration; thus, further studies have been proposed such as conducting ow culture to ll the knowledge gap regarding the remodeling of the fetal membrane.Here, we evaluated the effect of amniotic uid motion on AECs grown within an AM-OOC to glean better insight of in utero pathology and pathophysiology induced by oxidative stress.

Human fetal membrane cell lines for OOC experiment
We used established immortalized human fetal membrane cell lines as reported in our prior publications 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 lled with 25% 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.

Creating simulated amniotic uid movement in AM-OOC
To create a simulated amniotic uid 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 uid motion as expected in amniotic cavity (Brace 1997, Modena andFieni 2004), the pump was programmed to infuse culture media initially from 0 µl/h to a target ow rate, and then maintain for 2 h.Then, the ow gradually decreased to 0 µl/h within 2 min.Once infusion ow stops, ow starts back to opposite direction, maintain the ow 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), where, is the shear stress, is the volumetric ow rate, is the dynamic uidic viscosity, and indicate the width and height of the channel, respectively.

Creating an oxidative stress (OS) model as pathological positive control
To identify how uid 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 ltering ask containing 25 mL of hFM_AECs media to obtain 100% CSE.Then, the stock CSE solution was ltered using a 0.

Cytotoxicity assessment
To assess the cytotoxic effect of ow 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 ow culture experiment.

Microscopy
After 48 h of static and ow culture in the AM-OOC devices, bright eld microscopy was performed (BZ-X800E microscope; 4x, 10x, and 20x magni cation, Keyence, Osaka, Osaka, Japan) to determine cell morphology, intermediate lament 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 modi ed (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 ve images) per culture condition for cell circularity using the Image J software.CSI was calculated using the following formula: CSI = 4π x area/perimeter 2 , 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.

Scanning electron microscope (SEM) imaging
Cells were xed by 4% PFA at room temperature for 15 min after 48 h cultivation in static or ow culture condition.Glass substrates were then detached from the PDMS chamber layer and rinsed with 1X PBS three times.A dehydration step was rst 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).

Multiplex assays for in ammatory cytokine markers analysis using Luminex
To assess the changes in in ammatory 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 ow 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 uorescence values that were obtained to the standard curve by linear regression analysis.

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.

Modeling amniotic uid movement within the AM-OOC
Fetus drifts in the amniotic uid during gestation and support fetal organ development, such as lung, digestive system, and protect from bacterial infection and environmental alterations, such as temperature change, and injury (Shamsnajafabadi and Soheili 2022).It also allows the movement of fetus, which permits the musculoskeletal development (Fitzsimmons and Bajaj 2023).Unlike other ow in vivo as summarized in Table 1, amniotic uid can be in uenced by various situations, such as reduction of the amount of amniotic uid through swallowing (Ross and Nijland 1997) and absorbance (Brace and Cheung 2011) of fetus, or maternal abdomen trauma.We established experimental set up for uid motion by utilizing programmed pressure-driven pumps and generated two distinctive ow conditions, representing a moderate ow (i.e., 50 µl/h; 5.3 dyne/cm 2 ) and a much higher ow condition (i.e., 200 µl/h; 22 dyne/cm 2 ).Speci cally, after the cells have reached 70-80% con uency in the AM-OOC, a 3 mL syringe (Luer Lock, BD, Franklin Lakes, NJ, USA) containing complete KSFM media was placed on the syringe control unit and then tubing (EW-06420, Tygon, Irvine, CA, USA) was plugged to both the syringe and the inlet of the device to start a ow within the outer compartment for continuous shear force application.The tubing, connected to the outlet of the outer culture compartment, was immersed in a complete KSFM media in 15 mL conical tube to avoid air bubbles coming into the device during the entire ow culture condition.

Low levels of shear stress do not induce AEC cytotoxicity
AEC and AMC viability was assessed by calcein acetoxymethyl (AM) (Live; green) and ethidium homodimer (EthD)-1 (Dead; red) staining after 48 h of on-chip cultivation.Both cell types showed good viability, with calcein AM expression and proper cell morphology (i.e., AEC -cuboidal; AMCbroblastoid) (Fig. 3a).AECs had low levels of cytotoxicity (< 20%) under both static and uid ow conditions, while OS induced signi cant levels of cytotoxicity (AEC -static vs. OS: p = 0.0004; 50 µl/h vs. OS: p = 0.0004; 200 µl/h vs. OS: p = 0.0269), but AMCs showed no signi cant change (Fig. 3b).These results con rmed that the applied ow and associated shear stress did not induce cell cytotoxicity, whereas the control experiment with OS induced cytotoxicity only in AECs as expected.After 48 h of static and ow culture conditions, intermediate lament expressions (vimentin for mesenchymal characteristics; CK-18 for epithelial characteristics) and localization in AECs and AMCs were evaluated by immunocytochemistry.AECs under all conditions co-expressed both vimentin and CK-18, highlighting that they were in "meta state", an in-between state of cellular transition that is an innate characteristic of AECs in utero (Martin, Richardson et al. 2018) (i.e., vimentin; green, CK-18; red, and DAPI; blue; Fig. 4a).Static and ow cultures did not affect vimentin or CK-18 expression, as seen by nonchanging vimentin/CK-18 ratio levels (Fig. 4b).However, OS treatment did induce vimentin re-localization toward the leading edge of cells (white arrow) and a signi cant increase in vimentin/CK-18 ratio compared to all other conditions (Fig. 4a  This con rmed our prior reports where we showed that CSE-induced OS in AEC can cause EMT (Richardson, Jeong et al. 2019).

Flow does not affect the epithelial nature of AECs but does induce microvilli formation
E-cadherin was evaluated by immunocytochemistry to measure barrier function and tight junction expression between AECs within the AM-OOC.AECs under static and ow culture conditions stained positive for E-cadherin (green) but not in OS condition, and ow culture induced some increase in Ecadherin expression with more group of epithelial morphologies (Fig. 4a, E-cadherin: green, DAPI: blue).
Microvilli expression on-chip was assessed by SEM.After 48 h culture, static culture did not induce microvilli formation, while uid ow induced microvilli expression on the apical surface of the AECs (Fig. 5), as seen in in utero ( In summary, these results indicate that uid ow does not affect AEC's epithelial properties (i.e., cuboidal morphology [high CSI], meta state [no change in Vim/CK-18 ratio], and tight junction expression [Ecadherin]) but produced microvilli of AECs.Microvilli are expressed in absorptive epithelial cells but not necessarily required feature from cells in 2D culture or in a static state as they supplemented nutrient directly from culture media unlike cells in 3D environment where absorb material from the uid owing through, and thus their appearance in a ow environment is an expected phenomenon.

Discussion
Human body is consisting of up to 60% water, which circulates inside and outside the cells by constituting intracellular and extracellular uid.Blood circulates human body and acts on the endothelial surface of vessels by inducing shear stress.This mechanical phenomenon impacts various biological functions and processes.Thus, various types of in vitro models have been developed with ow-based cultures to recreate organ-or tissue-speci c phenomenon and conditions that mimic the in vivo environment.However, amniotic uid behaves differently and its role and impact in microphysiological system of feto-maternal interfaces have not been previously studied, due in part lack of information on the in vivo uid conditions.Amniotic uid uctuates during gestation from 1-1.5 ml in the rst week to 1.5 L at term.The maintenance of fetal and fetal organ growth as well as amnion cell nourishing is performed by the nutrient and growth factor enriched in amniotic uid.The low (oligohydramnios) and high (polyhydramnios) amniotic uid volume is pathologic and associated with pregnancy-associated adverse pregnancy outcomes such as growth restrictions and preterm birth (Fortunato, Menon et al. 2004, Kacerovsky, Musilova et al. 2014).Under these conditions, the ow, motion and stress are expected to differ, which can contribute to cytopathologic changes to amniotic cells.
Amniotic epithelial cells undergo multitudes of changes during pregnancy, which include senescence, EMT, and autophagy.All these are natural and physiologic processes required for membrane integrity.
However, premature senescence, terminal state of EMT (i.e., lack of MET to recycle cells), and accumulation of autophagosome complexes or incomplete autophagy are pathologic process that destabilizes membranes and predispose them to adverse events.Stress, stretch, scratch, and senescence can all cause change in amnion functions inducing in ammation.The impact of uid and its motion is aiding membrane function; however, under pathologic environment, the impact of uid motion on amnion cells and fetal responses are unknown due to di culties in studying such phenomenon in vivo.
The study presented here established and in vitro experimental model to recreate amniotic uid motion and determined the effect of amniotic uid motion on fetal epithelial layer.We utilized a pressure-driven pump providing a back and forth ow operation to mimic the rocking movement, recapitulating amniotic uid motion for the rst time in an organ-on-a-chip model.The use of this model was used to provide better insight into fetal membrane homeostasis during pregnancy.We report that shear stress has minimal impact on amniotic epithelial cells.Speci cally, regardless of the ow rate, AECs did not undergo cytotoxic changes, exhibited expected morphology, metastatic status, absence of overabundance of transitioning cells with mesenchymal morphology, and did not produce in ammation.
Although not measured, lack of in ammation is also indicative of absence of senescence-associated secretory phenotype (SASP), an in ammatory signature associated with amnion epithelial cell senescence.These data are reassuring that the motion induced by amniotic uid is not expected to cause any cytopathic changes in AECs.
Using the micro uidic OOC platform enables overcoming the limitations of conventional cell culture platform, such as lack of multicellular communication, inadequate physiologic environment, applicability of various operational components (such as pump and valves) that control the cellular culture environment.Incorporating ow dynamics into OOC platform has helped better understand endothelial and epithelial cell behavior and recreating vasculature ow and relevant uidic kinetic studies as it mimics more realistic environment.Here, rocking amniotic uid motion was established through a syringe pump system by programming the infusion and withdraw function (2 h back and forth cycle for 48 h) with different shear stress level.One of the limitations of this study is that the actual level of amniotic uid motion is unknown, as in utero this motion is primarily affected by the movement of fetus.Therefore, it uctuates all the time, which is di cult to exactly mimic.In addition, in this study we have used regular AEC culture media instead of that mimicking amniotic uid.Thus, changes in amniotic uid constituents under normal and pathologic events and fetal movement under hostile intrauterine environment was not adequately replicated in this model.These factors may impact AECs functions not studied in this work, leaving such studies to be conducted in the future.
In conclusion, we established an easy and simple amniotic uid motion organ-on-a-chip model by applying moderate to high level of shear ow to the amnion epithelial layer and monitored their cellular responses.Within the AM-OOC device, AECs maintained their phenotypic expressions without cellular transition under all ow culture conditions and did not show in ammatory responses commonly seen in pathologic condition.This indicates that pregnancy maintenance and fetal development is not affected by mechanical stimulations induced by uid motion in the intrauterine environment under normal circumstances.Furthermore, this study provides evidence that an inclusion of shear ow within amnion layers is not a requirement when studying amniochorion functions using OOC.

Declarations Figures
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 broblast layer, tightly packed brillar 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) uid surrounded by the amniotic sac and protected from external changes.Syringe pumps and control units are connected to the AM-OOC device, where the device is incubated for cell co-culture whereas the dynamic ow condition is applied.

Supplementary Files
, Anderson and Van Itallie 2009, Cucullo, Hossain et al. 2011, Delon, Guo et al. 2019).During pregnancy, the innermost epithelial cell layer of the fetal membrane is continuously exposed to the amniotic uid that gently uctuates by fetal movements (Brace 1997, Brace and Cheung 2011, Brace, Anderson et al. 2014) thus Highly elastic (due to rich presence of elastin) AECs protect the fetus by forming a watertight barrier between the amniotic uid and the distal cellular layers (Shamsnajafabadi and Soheili 2022, Truong, Menon et al. 2023).This occurs through its maintains of epithelial characteristics such as intermediate lament organization, tight junction regulation, and microvilli expression (Anderson and Van Itallie 2009, Kobayashi, Kadohira et al. 2010).

Figure 2 The
Figure 2
22 µm Sterile ip lter (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 con uency in the AM-OOC, culture media in the reservoir layer was removed and re lled with CSE in the AECs compartment, then incubated for 72 h to model OS condition.
(Richardson, Jeong et al. 2019The AM-OOC devices were washed with 1X PBS and then treated with NucBlue® Fixed Ready Probes Reagent (R37606, Thermo Fisher Scienti c, 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).
Approximately 5 µL of supernatant were used to perform the cytotoxicity assay according to the manufacturer's protocol.Brie y, 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 After 48 h, cells were xed 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 speciesspeci c 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,

Table 1
Summary of in vivo Shear Flow in Human Body AMC -static vs. OS: p = 0.02142) con rmed the ndings of no intermediate lament expression changes, suggesting that uid ow does not induce cellular transitions (i.e., EMT) in AECs.In contrast, OS treatment for 48 h (positive control) does induce vimentin re-localization, increase in vimentin/CK-18 ratio, and increase in broid morphology (high CSI), therefore indicating EMT in AECs.