In vitro placenta barrier model using primary human trophoblasts, underlying connective tissue and vascular endothelium

Accepted Manuscript In vitro placenta barrier model using primary human trophoblasts, underlying connective tissue and vascular endothelium Akihiro Nishiguchi, Catherine Gilmore, Aman Sood, Michiya Matsusaki, Gavin Collett, Dionne Tannetta, Ian L. Sargent, Jennifer McGarvey, Nagaraj D. Halemani, Jon Hanley, Fiona Day, Simon Grant, Catherine Murdoch-Davis, Helena Kemp, Paul Verkade, John D. Aplin, Mitsuru Akashi, C Patrick Case PII:

S0142-9612(18)30579-9

DOI:

10.1016/j.biomaterials.2018.08.025

Reference:

JBMT 18831

To appear in:

Biomaterials

Received Date: 12 June 2018 Revised Date:

7 August 2018

Accepted Date: 8 August 2018

Please cite this article as: Nishiguchi A, Gilmore C, Sood A, Matsusaki M, Collett G, Tannetta D, Sargent IL, McGarvey J, Halemani ND, Hanley J, Day F, Grant S, Murdoch-Davis C, Kemp H, Verkade P, Aplin JD, Akashi M, Case CP, In vitro placenta barrier model using primary human trophoblasts, underlying connective tissue and vascular endothelium, Biomaterials (2018), doi: 10.1016/ j.biomaterials.2018.08.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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In vitro placenta barrier model using primary human

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endothelium

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trophoblasts, underlying connective tissue and vascular

Akihiro Nishiguchi1,10, Catherine Gilmore2,10, Aman Sood2, Michiya Matsusaki1,3, Gavin Collett4, Dionne Tannetta4, Ian L Sargent4, Jennifer McGarvey5, Nagaraj D

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Halemani5, Jon Hanley5, Fiona Day6, Simon Grant6, Catherine Murdoch-Davis7, Helena Kemp7, Paul Verkade8, John D Aplin9, Mitsuru Akashi1*, C Patrick Case2 1

Department of Applied Chemistry, Graduate School of Engineering, Osaka University 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan. 2 Musculoskeletal Research Unit,

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School of Clinical Sciences, University of Bristol, Southmead Hospital, Bristol BS10 5NB, UK. 3 PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan 4 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, UK. 5 School of Biochemistry, University

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of Bristol, Bristol, UK. 6 Department of Obstetrics and Gynaecology, Southmead Hospital, Bristol, UK. 7 Department of Clinical Pathology, Southmead Hospital, Bristol, UK. 8 Department of Biochemistry, University of Bristol, Bristol, UK. 9 Maternal and Fetal Health Research Group, University of Manchester, 5th Floor Research, St Mary’s

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Hospital, Manchester, UK. 10 These authors are equally contributed. Correspondence and requests for materials should be addressed to M.A. (email: [email protected]).

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Abstract Fetal development may be compromised by adverse events at the placental interface

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between mother and fetus. However, it is still unclear how the communication between mother and fetus occurs through the placenta. In vitro - models of the human placental

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barrier, which could help our understanding and which recreate three-dimensional (3D)

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structures with biological functionalities and vasculatures, have not been reported yet. Here we present a 3D-vascularized human primary placental barrier model which can be constructed in 1 day. We illustrate the similarity of our model to first trimester human placenta, both in its structure and in its ability to respond to altered oxygen and to

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secrete factors that cause damage cells across the barrier including embryonic cortical neurons. We use this model to highlight the possibility that both the trophoblast and the

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endothelium within the placenta might play a role in the fetomaternal dialogue.

Keywords: Placenta, Signalling, 3D-tissue model, Vascularization, Layer-by-layer

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The placenta at the maternal fetal interface plays a key role in regulating fetal growth and development including neurodevelopmental outcome [1]. Adverse events in

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the placenta including hypoxia, reoxygenation, and infection, may damage the development of the fetus [2-4] and cause intrauterine growth restriction [5,6]. They

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increase the risk of diseases such as cerebral palsy [7] and even schizophrenia [8] in

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later life. In animal models, nanoparticle exposure will also cause intrauterine growth restriction [9,10]. The placenta both secretes and allows the exchange of substances between mother and fetus [6]. Maternal infection at later gestational ages modifies placental inflammatory and endocrine mediators that may adversely affect the growth of

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developing neurons in affected offspring [1]. We have shown that secretions from human placental explants or choriocarcinoma trophoblast barrier models after hypoxia

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or reoxygenation or toxin exposure in vitro may cause DNA damage in fibroblasts [11]

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and alter the morphology of neurons in tissue culture [12]. Several experimental models, including animal models and in vitro cell culture

models have been used to study the maternofetal interface [13-15]. These have potential drawbacks as the placental anatomy is significantly different between species. In vitro models of the trophoblast barrier require the use of cancer cell lines, rather than primary cells, to make them confluent. These cell line models provide an incomplete model of

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the placental barrier, since they lack connective tissue and vasculature. Placental explants possess the many cell types of the placental barrier as well as the structural

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organization observed in vivo, however this makes it difficult to decipher which cell type significantly contributes to placental signaling to the fetus.

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Here, we describe the rapid construction of an in vitro primary placental barrier

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model which contains blood capillary networks which can be used to examine the

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mechanism of fetomaternal dialogue during pregnancy (Fig. 1).

Fig. 1. Reconstitution of vascularized placental barriers for damage-signalling assay to neurons. (a) Schematic illustration of a human placental villus and an engineered vascularized placental barrier model. The TB bilayer that covers the placental villus responds to oxidative stress, from the maternal side, by secreting damaging signaling molecules to the fetus. Thus, oxidative stress can indirectly damage the fetus. The engineered barriers, constructed by a bottom-up approach using ECM nanofilms, were 4

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able to model these signaling processes in vitro. (b) Schematic illustration of the blood-capillary model construction. Primary fibroblasts (normal human dermal

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fibroblast cell, NHDF) and endothelial cells (human umbilical vein endothelial cell, HUVEC) were coated in FN and G nanofilms, and, for placental barrier model, pCTBs were coated in Col and LN nanofilms.

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1. Materials and methods

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1.1 Isolation of primary CTB and explants

Human placenta from 1st and 3rd trimester was obtained with ethical approval

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and patient consent from patients with normal pregnancies at voluntary termination of pregnancy and at elective caesarean section. Primary villous CTBs were isolated from

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3rd trimester placenta using the protocol reported previously [16]. To obtain explants from placental villi, 1st trimester placenta (9 weeks 4 days gestation) were cut into 2.5 x 2.5 mm pieces. These isolated primary CTBs and explants were used without

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preliminary culture.

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1.2. Placental barrier fabrication

Human pCTB or BeWo cells from human placental carcinoma were suspended

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in a 0.04 mg ml-1 of ColIV (Mw = 5.4 × 105, Sigma-Aldrich, USA) and LN (Mw = 8.5 × 105, Sigma-Aldrich, USA) / Tris-HCl solution (50 mM, pH = 7.4) and alternately incubated for 1 min with washing step. The centrifugation was performed at 400 xg for 1 min at each step. After 9 steps of coating, about 10 nm of ColIV-LN nanofilms were coated onto each cell surfaces. To construct bilayer structures of pCTB, 5 x 105 cells suspended 0.3 ml of in Dulbecco’s modified eagle’s medium (DMEM) nutrient mixture 6

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F-12 Ham with 10% FBS, 5% gentamicin, 0.6% streptomycin, and 0.6% L-glutamine were seeded into 24 well trans-well inserts (area: 0.33 cm2, pore size: 0.4 µm, pore

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density: 4 x 106 pores/cm2, 3470, Corning, NY, USA). The 1.4 ml of media were added to the basal chambers. After 1 day of incubation in 5% CO2 at 37 oC, human pCTB

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barriers with bilayers were constructed. In the case of BeWo cells, we seeded 2 x 105

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BeWo cells to construct bilayered structures and cultured under the same conditions as pCTB. With regard to oxygen concentration, the samples exposed to normoxia (21% to 21%) were cultured under 21% oxygen conditions and the samples exposed to hypoxia

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or reoxygenation were cultured under 2% conditions for 1 day to construct the tissues.

1.3. Morphological analysis

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For hisological observation, placental barriers were fixed with 10% formaline

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and processed into 4 µm thick paraffin-embbeded sections. The immunostaining for hCG and CD45 (Dako, Glostrup, Denmark) were performed using DAB substrate kit (Vector, PE, UK). For fluorescent obsercvation, prepared barriers were fixed with 4% PFA and then treated with 5% BSA, 5% goat serum, and 0.25% Triton-X for 30 min. The barriers were incubated overnight with the primary antibodies (1:100 for E-cadherin antibody (Abcam, CB, UK), 1:200 for Connexin43 antibody (Life

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Technologies, Carlsbad, OR)). After washing steps, the secondary antibodies (1:200 for E-cadherin, 1:2500 for Connexin43) were added to the tissues. The images were

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captured by confocal laser scanning microscopy (Leica, WZ, Germany). For transmission electron microscopy (TEM) observation, briefly, prepared barriers were

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fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 30 min and then replaced

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with ultrapure water. The membranes were cut from plastic supports and were subsequently post-fixed with osmium tetraoxide (2%, 0.1 M PIPES buffer pH = 7.2) and dehydrated with ethanol before embedding in resin. 50 – 70 nm thick cross-sections

1.4. Dye transfer

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were cut and stained for TEM observation with uranyl acetate and lead citrate.

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Fluorescein isothiocyanate (FITC)-labelled albumin (Sigma, St Louis, MO) with an average molecular weight of 66 kDa were used to exam the integrity of pCTB

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barriers and BeWo barriers. The 100 µl of 3 µM FITC-albumin and 600 µl of 3 µM albumin were added to the apical and basal chambers of 24 well trans-well inserts respectively. Plates were incubated for 3 hours at 37 oC and 5% CO2. 50 µl of media were collected from each basal chamber and add into a black 96-well plate and read at excitation 485 nm and emission 520 nm on a FluoSTAR OPTIMA fluorescence

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microplate reader. All values were blank-corrected and media concentrations were

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determined using a dilution standard curve.

1.5. Damage signalling assay to neurons

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The prepared barriers and explants were placed at 37 oC in a Ruskin hypoxic chamber to expose to 4 different oxygen conditions, 21% or 2% for 48 hours, or transfer

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from 2% for 24 hours to either 21% or 8% for 24 hours. After 24 hours of incubation, media were changed to fresh media and the apical and basal media were collected after further 24 hours of incubation at hypoxia or reoxygenation conditions. The obtained

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conditioned media were used for neurons growth assay. Neurons from P0 and E18 rat cerebral cortices were seeded onto coverslips covered with poly-L-lysine and cultured

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for 12 days in neurons growth media. To test damage signalling, neurons were exposed

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to the 4 types of conditioned media (21%, 2%, 2 to 8%, and 2 to 21%) and incubated for 6 days. When neurons were exposed to the conditioned media, we exchanged half of the existing culture media with conditioned media (0.5 mL each) so as to not to remove all of important factors that keeps neurons in a functional state. After exposure, neurons were fixed with cold methanol at -20 oC for 10 min. After blocking treatment, neurons were incubated overnight with a 1:2000 dillution of guinea pig anti-MAP2 antibody

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(Millipore, Billerica, MA). After washing steps, samples were treated with a 1:500 dillution of Alexa Fluor 488-conjugated goat anti-guinea pig IgG antibody (Life

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Technologies) for 1 hour. Cell nuclei were stained with DAPI (Vector) and images were

measured using ImgaeJ.

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1.6. Vascularized placental barrier fabrication

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captured by fluorescent microscopy (Leica, Germany). The dendrite length was

To construct NHDF multilayered tissues with blood-capillary networks, sandwich culture was employed. Briefly, 4 x 105 of NHDF coated with FN (Sigma, St

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Louis, MO, Mw = 4.6 × 105) and G (Wako, Osaka, Japan, Mw = 1.0 × 105) nanofilms (about 10 nm thickness after 9 steps) were seeded into 24 well trans-well inserts to

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obtain 4L-NHDF tissues after 1 day of incubation. HUVEC layers were formed on

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NHDF tissues in the same manner. After 1 day, FN-G coated NHDFs were accumulated on them again and a sandwich culture was performed. After 3 days of incubation, 5 x 105 cells ColIV-LN coated pCTB were seeded onto blood-capillary models to fabricate vascularized placental barrier models after 1 day. The NHDFs (passages: 4–10, Lonza, NJ, USA) were cultured in DMEM with 10% FBS. The HUVECs (passages: 3–8, Lonza) were cultured in endothelial basal medium (EGM-2MV, Lonza). To observe the

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structures of vascularized barriers, fluorescent staining with an anti-CD31 antibody for

by confocal laser scanning microscopy (Leica, WZ, Germany).

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1.7. DNA damage assay

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HUVECs and cell tracker orange for pCTB were performed. The images were captured

DNA damage was measured in fibroblast cells using the alkaline comet assay

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according to previous protocols [11]. CoCr nanoparticles (80 nm in diameter) were washed in 100% ethanol, heat-sterilized at 180 oC for 4 h and sonicated. Barriers, which were constructed in a 24 well transwell insert, were exposed to CoCr nanoparticles

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(0.36 mg/cm2) for 1 day. 1.4 mL of conditioned media below the barrier were collected. 5 x 104 NHDFs (fetal model) which had been cultured in 12 well plates for 1 day, were

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then exposed to the conditioned media for 1 day. The NHDFs were collected with the

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trypsinization and embedded in agarose hydrogels for the alkaline comet assay. A total of 100 cells for each treatment (nine repetitions) were scored at ×400 magnification using a fluorescence microscope (Olympus BX-50) with an excitation filter of 515–560 nm and barrier filter of 590 nm and image analysis software (COMET III, Perceptive Instruments). DNA damage was evaluated by the tail moment (product of comet length and tail intensity).

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2. Results 2.1. Structural analysis of human primary placental barrier models

placenta,

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The placenta is the key interface between the mother and foetus [17, 18]. In the form

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multinucleate

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syncytiotrophoblasts (STB) on the villus surface. Early in pregnancy, both cell types

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cover the placental villi to form a bi-layered trophoblast barrier, interconnected by connexin 43 gap junction channels. As pregnancy progresses, the placenta enlarges and the cytotrophoblasts become relatively sparser. As a result, the cytotrophoblast layer becomes thinner and predominantly mono-layered at term, but it maintains its structural

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integrity [19]. Here, we focused on constructing a bi-layered barrier that modelled the placental barrier in early pregnancy, when the fetus is most vulnerable to external

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stimuli. Since damage signalling to neurons depends on the thickness of placental

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barrier [11], we fabricated bi-layered structures which is similar to placental barrier at the early pregnancy. The bi-layered pCTB barrier was recreated by the cell-accumulation technique using nanometer-sized extracellular matrix (ECM) films formed onto cell surfaces [20-22]. In view of the relationship of the placental villi with basement membrane, we have used collagen type IV (Col) and laminin (LN) as basement membrane components for adhesive ECM-scaffolds [23], instead of

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fibronectin (FN) and gelatin (G) which have been used for fibroblast and endothelial cells (Fig. 1b). The isolated pCTB were coated with approximately 10 nm of ColIV-LN

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nanofilms using a process of ‘layer-by-layer assembly’ [24,25]. The 5 x 105 pCTB coated with ColIV-LN nanofilms were seeded into 24 well transwell inserts to form

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bi-layered barrier structures after 1 day of incubation. The same technique was also used

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to produce a bi-layered barrier model using the choriocarcinoma trophoblast cell line, BeWo. The similarity of the engineered pCTB barrier to human placental villi was examined using light microscopy, immunohistochemistry and TEM observation. Like the human placenta, the pCTB barriers were bi-layered (Fig. 2a). As in the human

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placenta, the barrier cells were interconnected with adherens and gap junctions, visualized by anti-E-cadherin and anti-Connexin-43 immunostaining, respectively (Fig.

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2b,c). TEM revealed the presence of junctional complexes between cells in the barriers,

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with tight junctions and well-developed microvilli along the barrier surface, with an apical-basal polarity (Fig. 2d). The pCTB barrier models suppressed the transfer of FITC-albumin (66 kDa) dye through the barrier under various oxygen concentration and displayed a similar barrier integrity as compared with those of BeWo barriers (Fig. 2e). Resistance of the pCTB barriers increased during culture (Fig. S1). Importantly, E-cadherin expression of pCTB barriers decreased during the incubation time and

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especially it disappeared after 6 days, indicating that syncytialization proceeded during the culture and this model showed similar morphology to physiological tissues (Fig. 2f).

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To evaluate the basic function of placental barrier, we confirmed hCG expression (Fig. 2g). The hCG secretion from pCTB barrier increased during the cultivation partly

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because of spontaneous syncytialization (Fig. 2h).

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Fig. 2. Histological observation and basic functions of pCTB barriers. (a) HE staining images of engineered placental barrier models. (b) E-cadherin and (c) connexin 43 immunofluoresecnt staining images of engineered placenta barriers. (d) TEM observation of whole image of bi-layered structure (upper), microvilli at apical surface (left), tight junctions (right). (e) Barrier integrity tests of pCTB barrier and cell line (BeWo) barrier models exposed to reoxygenation using FITC-albumin transfer (n = 3). 15

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(f) E-cadherin immunofluoresecnt staining images of pCTB barriers during 6 days of culture. (g,h) hCG staining image and hCG secretion from pCTB barrier models. ***P < 0.001 (n = 3).

2.2. Secretions from placental explants, primary trophoblast barriers, and cell line

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barrier models

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In previous work, we have shown that the placental barrier secretes factors in response to different oxygen conditions. Tissue culture media conditioned by placental explants or BeWo barriers exposed to hypoxia can cause retraction of the dendritic

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arborisation of dissociated embryonic neurons from the cerebral cortex [12] in vitro and cause DNA damage in fibroblasts [11] in vitro. Nanoparticles caused signalling within the bi-layered trophoblast barriers through connexin and pannexin channels, with

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purinergic transmission and involving Ca2+ wave activation and a subsequent release of

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DNA damaging molecules from the undersurface of the BeWo cells to the fibroblasts below the barrier. We therefore compared the functional effects of tissue culture media that were conditioned by our models under different oxygen levels to examine whether they imitated the placenta in these respects (Fig. 3a). To mimic the indirect signaling process in pregnancy, explants, primary pCTB, and cell line barriers barrier were used as a placenta model and the cultured embryonic cortical neurons were used as a fetal 16

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brain model. Each model was cultured under 21% oxygen for 48 hours, 2% oxygen for 48 hours (hypoxic culture), or transferred from 2% for 24 hours to either 21% or 8% for

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24 hours (reoxygenation). The embryonic cortical neurons were cultured on coverslips and were exposed to conditioned media for 6 days. Their morphology was evaluated by

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immunofluorescence. There was a marked decrease in the dendrite lengths of the

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cultured embryonic cortical neurons when they were exposed to the conditioned media from placental explants and pCTB barriers after hypoxia or reoxygenation (Fig. 3b,c and Fig. S2). The number of cell bodies were approximately the same. On the other hand, there were no significant differences in dendrite length after exposure of neurons

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to media conditioned by BeWo barriers also constructed using the cell accumulation technique. The media below the pCTB barriers (i.e. basal, non-microvillous surface)

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was more effective in causing dendrite retraction than the media above the barriers (i.e.

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apical, microvillous surface) when the barriers were exposed to hypoxia or reoxygenation (Fig. 3c). These medias both contained an increase in glutamate, which was shown in our previous work to be an active factor in causing dendrite shortening [12] (Fig. 3d). Since the basal surface of the cytotrophoblast layer in a human first trimester placenta is the one facing the fetus, it highlights the potential relevance of the cytotrophoblast barrier in the maternofetal dialogue. There were no significant

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differences in cell viability and cell number within the placental barriers in response to hypoxia or reoxygenation (Fig. S3a-c). Interestingly a 2 to 8% hypoxia reoxygenation

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increased the immunostaining of connexin 43 in the barrier (and hence potentially its ability to signal) (Fig. S3d,e), potentially explaining how hypoxia may suppress the

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differentiation of CTB and sequential fusion [26,27]. Moreover, signalling from pCTB

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barrier caused morphological changes in separately cultured endothelial cells (human umbilical vein endothelial cell, HUVEC) (Fig. S4). Conditioned media secreted from pCTB barriers under hypoxia or reoxygenation conditions activated endothelial cells and increased tubular length, tubular number, and branching point number, indicating

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the interaction of placental tissues with not only neurons but also endothelial cells.

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Fig. 3. Comparison of damage-signalling to neurons under hypoxia or reoxygenation conditions between explant, primary engineered barrier, and BeWo cell line barrier models. (a) Experimental process of damage-signaling assay to neurons. The dendrite length of neurons exposed to the conditioned media for 6 days were measured from fluorescent images. (b) Fluorescent images of the embryonic cortical neurons exposed

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to the conditioned media below each placental model cultured under normoxia (21%) and reoxygenation (2 to 21%) conditions. (c) Dendrite length of neurons exposed to the conditioned media collected from each placental model cultured under normoxia (21%), hypoxia (2%), and reoxygenation (2 to 8% and 2 to 21%) conditions. Media above and below barriers were separately collected from primary and cell line barrier models. *P < 0.05, **P < 0.01 when compared with 21% and #P < 0.05, ##P < 0.01 when compared with 2% (n = 3). (d) Release of glutamate in the media above and below pCTB barrier models. *P < 0.05, **P < 0.01 when compared with 21% (n = 3).

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2.3. Reconstruction of primary placental barriers with blood-capillary networks

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To improve the primary trophoblast cell model and make it more like a placental villous, we explored the possibility of creating a trophoblast barrier with an underlying fibroblast stroma containing a network of endothelial tubes (albeit not blood

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carrying). We first created vascularized bi-layered BeWo barriers which lay beneath 1

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layer (1L), 5 layers (5L) or 10 layers (10L) of normal human dermal fibroblasts (NHDF) containing HUVEC tubular networks, by seeding Col-LN coated BeWo cells onto fibroblast/HUVEC tissues. Briefly, 8 x 105 of NHDF and 1 x 105 HUVECs were

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coated with FN-G nanofilms and were seeded into 24 well transwell inserts, with the HUVEC layer sandwiched between the fibroblasts. After 3 days of incubation, ColIV-LN coated BeWo cells were seeded onto the blood-capillary tissue models to

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fabricate vascularized placental barrier models after 1 day (Fig. 4a). In this vascularized

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placental barrier model, BeWo cells possessed epithelial morphology (Fig. 4a(i)) and the HUVECs formed capillary-like structures with lumens (Fig. 4a(ii), b). This process was repeated using primary human trophoblasts with or without the combination of layers of human fibroblasts and HUVECs. The isolated pCTBs were coated with ColIV-LN nanofilms and seeded onto the blood-capillary models of NHDFs and HUVECs, and then cultured for 1 day to form vascularized pCTB barriers. The 20

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vascularized barriers displayed dense tubular networks within the fibroblast layers (Fig.

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4c-e).

Moreover, to investigate whether fibroblast layers cause damage signalling when exposed to metal nanoparticles, we exposed multilayered fibroblasts with

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underlying BeWo barrier to CoCr nanoparticles and observed the signalling using the

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alkaline comet assay. We confirmed the construction of BeWo bilayers lying underneath 5L- and 10L-fibroblast layers, while the BeWo cells in 1L-NHDF sample intermingled with each other (Fig. S5a). In fact, no significant DNA damage was

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observed in fibroblasts below CoCr nanoparticle exposed barriers of 5 layers and 10 layers of fibroblasts on BeWo barriers, whilst there was when BeWo cell barriers alone or BeWo barriers with 1 layer of intermingled fibroblasts were used, in keeping with

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our previous results (Fig. S5b). We also showed that cell barriers comprised only of 1, 5

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or 10 layers of fibroblasts and 1L-BeWo barriers did not induce DNA damage in underlying fibroblasts, and the exposure time enhanced damage signalling (Fig. S5c-e). These results revealed that DNA damage signalling only occurred when barriers of trophoblasts were exposed to nanoparticles and not when barriers of fibroblasts were exposed.

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Fig. 4. Creation of cell line and pCTB barrier models with blood-capillary networks. (a) 3D-reconstructed CLSM images of BeWo-10L-NHDF barriers with HUVEC tubular networks. CLSM images in (i) and (ii) displayed the morphologies of BeWo cells and HUVECs at the xy plane, respectively. BeWo cells and HUVECs were immunostained with an anti-EpCAM antibody (green) and an anti-CD31 antibody (red). Nuclei were stained with DAPI (blue). (b) CD31 staining image of BeWo-NHDF tissue with HUVECs show lumen structures in the NHDF tissue. (c) CLSM images of pCTB barrier models with HUVEC tubular networks. The pCTBs and nuclei were stained with cell tracker orange (red) and DAPI (blue). HUVECs were immunostained with an anti-CD31 antibody (green). (d,e) 3D-reconstructed CLSM images of pCTB barrier with HUVECs networks when seen from top and bottom.

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2.4. Vascularized pCTB barrier induce damage signalling to neurons To explore the role of HUVEC tubular networks on signalling to neurons, we

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addressed signalling experiments using pCTB barriers. When barriers were created of layers of fibroblasts and networks of endothelial cells and without trophoblast layers

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and then were exposed to hypoxia or reoxygenation conditions, the tubular networks of

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endothelial cells were maintained and significant changes were not seen in them (Fig. 5a). However, when neurons were exposed to the media that were conditioned by these models barrier by hypoxia reoxygenation, we found that there was a decrease of total dendrite length (Fig. S6a) and an increase of beaded dendrites (Fig. 5b) without any

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change in the number of cell bodies (Fig. S6b) as compared to equivalent barriers of fibroblasts but without the endothelial cell network. This suggests that the endothelium

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might release a specific factor that cause dendrite beading rather than the retraction. We

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then created cell barriers with primary trophoblasts and underlying fibroblasts containing endothelial tubular networks. Media conditioned by the vascularized pCTB barriers, which contained HUVECs and which were subjected to hypoxia reoxygenation, caused a reduction in dendrite length as compared to media conditioned by these barriers under 21% oxygen (Fig. 5d, shown in Direct-HUVEC). In contrast, media conditioned by non-vascularized pCTB barriers, which contained fibroblasts but no

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HUVEC network, caused only minimal non-significant reductions of dendrite length (Fig. 5c, shown in Direct-without). These results suggest that the endothelial tubular

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networks contributed to this change in response to reoxygenation. In order to explore the interaction between the primary trophoblast layers and the fibroblast and endothelial

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cell layers in this signalling process, the barrier was created in two separate halves, a

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barrier of primary trophoblast cells which was placed above an underlying barrier of fibroblasts and endothelial cells (Indirect). These two barriers were close to each other but not in direct contact. Both the trophoblast cell barrier and the underlying barrier of fibroblasts and HUVECs were exposed to hypoxia reoxygenation. The media sampled

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beneath the trophoblast barrier caused a reduction of dendrite lengths as before. However, the media below the second layer of fibroblasts and endothelial cells (i.e a

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barrier exposed to the secretions from the trophoblast barriers) caused even more

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reductions in dendrite lengths suggesting that there is a synergy in cell signalling between the underlying vascular connective tissue and the overlying trophoblast.

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Fig. 5. Vascularized placental barrier models composed of human primary cells allow for damage assay to developing neurons in vitro. (a) Fluorescent observation of HUVEC tubules stained with an anti-CD 31 antibody. (b) Beading dendrite length of neurons exposed to the conditioned media collected from the placental barriers with HUVEC networks cultured under normaxia (21%), hypoxia (2%), and reoxygenation (2 to 21%) conditions. *P < 0.05 when compared with fibroblast tissues (n = 3). (c) Dendrite length of neurons below the vascularized barriers directly adhered with pCTB

3. Discussion

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and separately cultured under normaxia and reoxygenation conditions. *P < 0.05, **P < 0.01 when compared with control (n = 3).

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There is much interest in discovering the underlying mechanisms of disorders

of normal human reproduction at the tissue, cellular, and molecular level, so that they could be diagnosed and treated or even prevented successfully. Significant and frequent disorders include implantation failure, occult pregnancy loss, recurrent miscarriage, pre-eclampsia, abruption placenta, fetal growth restriction, and premature parturition [29]. In addition, the early life environment is recognised as a key factor which 25

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contributes to disease susceptibility in later life [30], including cardiometabolic and psychiatric diseases [31]. The placenta is thought to play an important role in this

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process of early life or developmental ‘programming’ as it is the interface between mother and fetus. Because tissue cannot be typically obtained from pregnant women in

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a manner that allows a study of problems as they develop, it is important to study these

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disorders with models [29].

One of the major challenges of human gynecologic and toxicological research is to find an appropriate model for the human placenta. The human placenta contains fetal villi which are anchored into the basal plate of maternal endometrium and are

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surrounded by the maternal blood space [32]. The villous mesenchyme contains a complex, branching blood vessel system consisting of stem, intermediate, and terminal

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villous capillaries [33]. This specific placental type does not exist in most experimental

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animals. Those with hemochorial placentation do not have fetal villi, but a labyrinthine interdigitation pattern [34]. Several models of the human placenta in vivo have been used. These include placenta-derived cell lines, mainly trophoblastic from choriocarcinoma cells including BeWo (as used here), isolated primary placenta cells, animal models, human placenta tissue explants, and single (maternal) or dual side placenta perfusion [35]. Each of these models have advantages and limitations.

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Choriocarcinoma trophoblast cells continue to proliferate in tissue culture unlike primary trophoblast cells [36]. As a result, they provide relatively simple models of

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mono-layered or multilayered trophoblast barriers which have been used successfully to study nanoparticle transport [37] or trophoblast signalling [11, 28]. Although these

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trophoblastic cells lines share numerous characteristics of primary trophoblast cells,

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they differ in several aspects due to their malignant transformation [38]. This problem is overcome by using primary trophoblasts isolated from human placenta. Unfortunately, the lack of maintained proliferation of these cells in culture has not allowed them to be successfully and predictably grown as barriers in the past. Hemmings did describe a

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method using EGF to stimulate growth but it required three repeated isolations and seeding of trophoblast from placenta over 12 days and did not result in a predictable

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number of layers [39]. More recently, Huang et al produced a mono-layered primary

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trophoblast barrier model, by seeding the trophoblasts onto transwell inserts coated in matrigel. However, these models did not incorporate the added complexity of a vascularized network.

In the in vivo environment, trophoblasts exist in close proximity to multiple cell types [40], however, primary trophoblasts in culture have mostly been studied in isolation, without considering the potential influence of these other cell types. The

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proper development of the placenta depends upon autocrine and paracrine signalling between trophoblasts and vascular endothelial cells for example [41]. Several studies

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suggest that crosstalk between the cells may be necessary for appropriate growth, development, and remodeling. This paracrine signalling between trophoblast and

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endothelium has previously been imitated using conditioned media, however it would

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be ideal to co-culture the two cell types in vitro, while maintaining some of the topographical arrangement observed in placental villi in vivo [42]. The advancement in our understanding of the cellular environment of the placental chorionic villous has been delayed by the lack of an in vitro model that facilitates these cellular interactions

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[43,44]. There are distinct anatomical and functional differences between human and other eutherian animal placentas. There are up to 30-fold differences in the placenta

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transport of hydrophilic drugs [45]. Systemic reactions to toxic chemicals differ widely

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[46]. Some authors have gone so far to say that the differences between placentation in humans and rodents are sufficient to render human pregnancy unique and to justify ignoring data generated using mice [29]. The anatomical relationships within the villi are maintained [36] in human placental explants, which can be prepared from both 1st trimester and term placenta. However, the limitation here is that exposures of these tissues to toxins are not polarised

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and it is not possible to study transport of chemicals across the placenta. This difficulty is overcome by the use of placental perfusion models, in which the in vivo placental

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metabolism is intact [47]. Here it is possible to perfuse the maternal side of the circulation ex vivo and study transport to the fetal side. However, this can only be done

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with late third trimester placenta. As such this model is considered a ‘worst case

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scenario’ [35] because the permeability of drugs increases throughout pregnancy as the trophoblast barrier changes from a thick bilayer to a thin and incomplete monolayer [11, 47]. Moreover, this technique is laborious, dependent on fresh placenta [48] and has a high failure rate due to physical damage to the placenta during delivery, presence of

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clots and inaccessible vasculature [45]. The implementation of the REACH legislation results in the increased use of animals for toxicity testing, especially for reproduction

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and developmental toxicity testing [48]. However, there has been a paradigm shift in

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toxicology research from the use of animal studies towards alternative models with high relevance to human beings [49]. The basis for a new system has emerged over the past two decades. Early cell culture-based experiments have evolved with the use of three dimensional (organotypic) cultures that resemble organs in structure and function. As one of the last big challenges, the establishment of cell source of primary human cells

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can be overcome by the generation of human stem cells and appropriate differentiation induction [46], although this in turn presents its own challenge.

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We present a model of the human placenta for the first time which contains a confluent barrier of primary human trophoblast cells with a predictable number of layers and with

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an underlying fibroblast stroma and vasculature which can be created within 1 day

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whilst the primary cells are most viable. We have coated the primary trophoblasts with collagen and laminin nanofilms to imitate the basement membrane. This as we show supports cellular adhesion and function without disrupting cell to cell communication, including the formation of gap junctions. Our model which can be conveniently divided

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into segments using several transwells allows a study of cross talk between different cell types. Indeed, preliminary evidence was provided that crosstalk exists between the

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trophoblast and endothelium in our model. We have systematically compared our model

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to human placental explants in order to compare appropriate vectoral arrangements under the same tissue culture conditions with or without alterations of oxygen or nanoparticle exposure. We demonstrate similarities in phenotype and signalling in response to these insults. This model can be used to clarify the mechanism of cellular signalling across the barriers which affects various organs including brain and cardiovascular functions [50-52].

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This model, being polarised and using primary human cells, avoids many of the disadvantages of other models. It can therefore be used to study disorders of

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reproduction and early life events in the hope of better treatment and diagnosis. It has been suggested that increased knowledge of the placenta and placenta-based

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interventions to improve the course of pregnancy might someday improve the health of

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multiple generations [53]. If this model could be expanded on a much bigger scale it might be possible sometime in the future to create an artificial placenta. This idea was first proposed over 50 years ago with the hope of recreating the intrauterine environment with extracorporeal oxygenation instead of mechanical ventilation in order

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related lung failure.

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to avoid the complications of premature labour on the infant, for example prematurity

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Acknowledgments

We thank Ms. Chihiro Kamimura in Osaka University for partial fabrication of vascularized tissues. This work was supported by the Bilateral Programs (JSPS), the NEXT Program (LR026), a Grant-in-Aid for Scientific Research (S:A232250040, B:17H02099), JST, PRESTO (15655131), the SENTAN-JST Program (13A1204), and Grand-in-Aid for JSPS Fellows (24 622). We also thank Dr. C. Abbott in University of Bristol for histological experiments. Author contributions

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A.N. designed and carried out the studies and wrote the paper. C.G. performed the barrier construction. A.S. carried out and organized the studies. M.M. designed the

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concept and organized the experiments. G.C., D.T, and I.S. isolated the human primary pCTB. J.M., N.H., and J.H. isolated the rat embryonic cortical neurons. F.D. and S.G. provided the whole placenta for explant models. C.M. and H.K. performed the amino acid analysis. P.V. performed the TEM observations. J.A. and M.A. supervised the project. P.C. supervised the project and edited the paper. All authors reviewed the manuscript.

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Present Addresses

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Akihiro Nishiguchi; Polymeric Biomaterials Group, Biomaterials Field, Research Center for Functional Materials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan Mitsuru Akashi; Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, 565-0871, Osaka, Japan. Competing financial interests: The authors declare no competing financial interests.

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Appendix A. Supplementary data

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Supplementary data related to this article can be found at

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