Microfluidic devices for investigating epithelial and / or endothelial barrier function, such as placental-like or duct-like 3D microvascular tissue.

Abstract

The present invention relates to a microfluidic device configured to study epithelial and / or endothelial barrier function in a human 3D cell microtissue model, while simultaneously allowing for the application of mechanical stimuli such as luminal flow and / or lateral flow. Embodiments include three cultures representing the maternal-fetal interface (placental barrier): human umbilical vein endothelial cells (HUVEC), human placental fibroblasts (HPF), and human placental pericytes (HPP), as well as a ductal interface composed of human primary mammary gland-derived endothelial cells, fibroblasts, and epithelial layer (MCF10). The model is expandable to include tumors (cell line-derived or patient-derived). The present invention further relates to a method for fabricating a human 3D vascular microtissue model using a microfluidic device according to the present invention, and to the use of a human 3D vascular microtissue model for, for example, studying the effects of cell barrier function (solute permeability), interstitial flow, luminal flow and / or lateral flow, and epithelial and / or endothelial barrier function (solute permeability), and extravascular matrix properties (diffusivity, stiffness, transport of molecules, antibodies and / or cells, and matrix proteins). The present invention further relates to a method for presenting a pre-eclampsia (disease) model of the placental barrier, and means for testing drug therapies.

Description

[Technical Field]

[0001] The present invention relates to a microfluidic device configured to study epithelial barrier function and / or endothelial barrier function in a human 3D cell microtissue model, while simultaneously allowing for the application of mechanical stimuli such as luminal flow and / or lateral flow. Embodiments include a tri-culture of human umbilical vein endothelial cells (HUVEC), human placental fibroblasts (HPF), and human placental pericytes (HPP), representing the maternal-fetal interface (placental barrier), as well as a mammary duct interface composed of human primary mammary-derived endothelial cells, fibroblasts, and an inclusion of the epithelial layer (MCF10). The model is expandable to include tumors (cell line-derived or patient-derived). The present invention further relates to a method for fabricating a human 3D vascular microtissue model using a microfluidic device according to the present invention, and to the use of a human 3D vascular microtissue model for studying, for example, the effects of cell barrier function (solute permeability), interstitial flow, luminal flow and / or lateral flow, and epithelial and / or endothelial barrier function (solute permeability), and extravascular matrix properties (diffusivity, stiffness, transport of molecules, antibodies and / or cells, and matrix proteins). The present invention further relates to a method for representing a pre-eclampsia (disease) model of the placental barrier, and means for testing drug therapies. [Background technology]

[0002] The human placenta is a highly vascular organ, and its blood vessels are constantly growing and remodeling to ensure efficient exchange between the maternal and fetal blood circulation systems (Non-Patent Literature 1). The majority of essential solute exchange (oxygen, nutrients, hormones, antibodies) takes place through a thin layer of epithelial cells in the terminal villous tree, which includes the fetal-placental capillary network. This dense capillary network arises from the elongation and branching of existing blood vessels formed by angiogenesis (formation of new blood vessels) by approximately 6 weeks of gestation. From 25 weeks post-conception until full term, villous vessel growth switches from branched angiogenesis to unbranched angiogenesis, and coiled capillary structures are generated within the terminals of the fetal-placental vascular tree (Non-Patent Literature 2).

[0003] The formation of a proper vascular tree in the early placenta is crucial for ensuring optimal blood volume loading and preventing placental dysfunction and adverse fetal outcomes. Structural chorionic microangiological abnormalities are a common feature of pregnancy complications associated with perinatal morbidity and mortality, such as pre-eclampsia and fetal growth restriction (Non-patent Literature 3, 4). Understanding the underlying mechanisms of fetal-placental angiogenesis and regulation is essential to identifying the causes of these changes.

[0004] Studying fetal-placental vascular development is challenging, primarily due to ethical considerations and the difficulty of accessing tissue, especially in early pregnancy. Current knowledge regarding the development and function of the chorionic vascular network comes from observations of human placental tissue samples obtained at various stages of pregnancy (Non-Patent Documents 5, 6), ultrasound examinations (Non-Patent Document 7), and computer (in silico) modeling (Non-Patent Documents 8, 9). However, these methods cannot directly measure the physical and chemical signals that occur during placental angiogenesis and neovascularization, such as mechanical shear stress and associated mechanical transmissions. In fact, developing blood vessels in vivo are subjected to constant mechanical stimuli, including fluid shear stress caused by interstitial flow (IF) and intraluminal blood flow (Non-Patent Document 10).

[0005] Several in vivo and in vitro studies have documented the effects of flow-induced shear stress on vascular morphogenesis, demonstrating flow-induced control of angiogenesis and lumen formation through endothelial cell migration, alignment, and apical deformation (Non-Patent Literature 11-14). Hemodynamic forces induce vascular remodeling through cell rearrangement and regulate vascular permeability through the regulation of endothelial adhesion and tight junctions (Non-Patent Literature 15-18). In vivo experiments have mainly focused on umbilical circulation (Non-Patent Literature 19), and the role of human placental hemodynamics during pregnancy is not well understood due to a lack of proper characterization of placental microhemodynamics occurring at the fetal-placental interface. While full-term placentas are a useful tool for ex vivo perfusion studies (Non-Patent Literature 20), there are limitations to understanding the vascular circulation of the early placenta, necessitating the use of biomimetic models.

[0006] Three-dimensional human tissue-specific microvascular networks have recently contributed to the inventors' understanding of flow-induced vascular growth and remodeling by overcoming the limitations of simpler monolayer systems (Non-Patent Literature 14, 16, 21, 22). For example, in another tissue that is difficult to study, interstitial flow has been demonstrated to enhance angiogenesis, function, and lifespan in brain-specific microvessels composed of endothelial cells, pericytes, and astrocytes (Non-Patent Literature 23).

[0007] Non-patent document 24 (Deng P, et al. (in: Fluidic Flow Enhances the Differentiation of Placental Trophoblast-Like 3D Tissue from hiPSCs in a Perfused Macrofluidic Device. Front Bioeng Biotechnol. 2022 Jun 30;10:907104. doi: 10.3389 / fbioe.2022.907104. PMID: 35845423; PMCID: PMC9280037)) describes an in vitro placental trophoblast-like cell model created through the self-organization of human induced pluripotent stem cells (hiPSCs) in a perfused 3D culture macrofluidic device. This device enables cell seeding, in situ differentiation of trophoblast lineages, and the formation of trophoblast-like tissue from hiPSCs in a biomimetic microenvironment. This integrated extracellular matrix (ECM) and fluid flow into a single device. After trophoblast cell line differentiation, Deng et al. were able to generate 3D clusters containing major human placental cell types, including trophoblast progenitor cells, cytotrophoblasts (CTBs), differentiated subtypes, syncytiotrophoblasts (STBs), and extravillous trophoblasts (EVTs), through long-term 3D culture (approximately 23 days). Furthermore, the formed tissue showed enhanced expression of CTB, STB, and EVT-related markers at the gene and protein levels under dynamic culture conditions compared to static conditions. RNA-seq analysis revealed elevated expression of trophoblast-specific genes in the 3D tissue, indicating that fluid flow plays a crucial role in promoting trophoblast cell differentiation of hiPSCs. The disclosed device does not allow for the application of fluids such as lateral flow and cannot be used to efficiently study vascular and / or barrier function in placental tissue.

[0008] Non-patent document 25 (Nishiguchi A, et al. (in: In vitro placenta barrier model using primary human trophoblasts, underlying connective tissue and vascular endothelium. Biomaterials. 2019 Feb;192:140-148. doi: 10.1016 / j.biomaterials.2018.08.025. Epub 2018 Aug 25. PMID: 30448698)) discloses a 3D angiogenic human primary placenta barrier model (Transwell model) that includes a layer of laminin and collagen-coated trophoblast cells (BeWo or primary trophoblast cell layer) on top of a thick layer of self-organizing capillary network formed from primary fibroblasts (normal human dermal fibroblasts (NHDF)) and human umbilical vein endothelial cells (HUVEC) in a fibrin hydrogel. Vascular perfusion is not possible in this model.

[0009] Non-patent document 26 (Pu, et al. (in: A 3-dimensional microfluidic platform for modeling human extravillous trophoblast invasion and toxicological screening. Lab Chip. 2021 Feb 9;21(3):546-557. doi: 10.1039 / d0lc01013h. PMID: 33166377; PMCID: PMC8212566)) describes a chip-on placental platform that can evaluate the invasion of trophoblast cells by intraluminal flow into the center of non-luminal blood vessels within a designed PDMS 3D microfluidic chip, and further requires a geometrically defined outer layer supported by studs.

[0010] Patent Document 1 discloses a microfluidic device capable of simulating capillary blood flow on the fetal side of the device and pooled blood on the maternal side (i.e., the intervillous space) of the device, separated by a permeable membrane. The microfluidic device can reconstruct the maternal-fetal interface, extend the functionality of cell culture models, and provide an alternative to current maternal-fetal transplantation models. Similarly, Patent Document 2 generally relates to organ-mimicking devices with microchannels, as well as methods for using and manufacturing the same.

[0011] Despite the development of new in vitro placental models (outlined in Non-Patent Literature 27), there are currently no models that allow for the investigation of the effects of blood flow-related placental vascular development while simultaneously enabling the testing of future and existing pharmaceuticals and other biologically relevant compounds for use in human pregnancy. Human placental tissue is difficult to maintain and perfuse, and usually only the late-stage pregnancy state can be replicated. In recent years, several humanized in vitro models that mimic the function of the placental barrier have been developed (Non-Patent Literature 28), but they lack the combination of two important adjacent barriers that regulate maternal-fetal exchange: the trophoblastic epithelial-like barrier and the fetal vascular system beneath it.

[0012] Furthermore, several in vitro placental barrier models have been developed to measure the transfer of substances between mother and fetus (Non-Patent Literature 29). The various technologies currently in existence can be summarized as follows:

[0013] Transwell technology Next, a monolayer of trophoblast cells is grown on a plate or semipermeable membrane to investigate placental barrier properties such as hormone secretion, intercellular glucose transport, susceptibility to environmental toxins, and parasitic infections (Non-patent documents 30-32).

[0014] Micromesh cell culture technology Therefore, to study drug permeability, a multilayered placental barrier model has been created using endothelial cells, fibroblasts, and iPSC-derived trophoblast cells (Non-Patent Literature 33).

[0015] Microfluidic Technology Then, a double 2D layer of endothelial cells and trophoblast cells is seeded on both sides of a semipermeable membrane, replicating the transport barrier from maternal circulation to fetal circulation (see, for example, Non-Patent Literature 34: Rabussier G, et al. Healthy and diseased placental barrier on-a-chip models suitable for standardized studies. Acta Biomater. 2023 Jul 1;164:363-376. doi: 10.1016 / j.actbio.2023.04.033. Epub 2023 Apr 26. PMID: 37116636). Fluid flow through separate microfluidic channels on both sides of this cellular barrier allows for both cell-specific exposure to the culture medium and quantification of cross-layer transport. This enables the exchange of continuously flowing nanoparticles between trophoblast cell lines (Non-Patent Documents 35, 36) and / or quantification of cross-layer transport (Non-Patent Documents 37-39).

[0016] lastly, 3D and more complex microfluidic models This includes culturing cells in three dimensions to better replicate the complex microenvironment and intercellular interactions involved in placental development. These have been found as 3D trophoblast cell organoid models capable of self-renewal, proliferation, and differentiation (Non-Patent Literature 40, 41), rotating wall bioreactor systems for developing 3D-based cell co-culture (trophoblast cells and microvascular endothelial cells) models that simulate fluid flow and replicate the floating microgravity environment found in vivo tissues (Non-Patent Literature 42), fluid culture systems for placental villous tissue fragments to study the effects of shear stress on the structural integrity of tissues (Non-Patent Literature 43), and finally, “placenta on a chip” integrating vascular networks (Non-Patent Literature 44, Non-Patent Literature 28), or complex shapes of villous structures using gelatin-based hydrogel materials (Non-Patent Literature 45).

[0017] Previously, the present inventors established a 3D model of terminal villous microvessels using three - cell culture of stromal cells and endothelial cells (Non - Patent Document 46, which is incorporated herein by reference in its entirety for the purpose of the present invention). In this model, it was demonstrated that placental pericytes contribute to growth restriction and that it mainly depends on VEGF and angiopoietin / Tie2 signaling. On the other hand, fibroblasts contributed to increased angiogenesis, but there was a limitation in using non - specific fibroblasts in the former model.

[0018] However, in all in vitro models designed so far to mimic the aspects of the placental barrier, the combination of two important adjacent barriers involved in maternal - fetal exchange is still lacking, and not all necessary components such as endothelium, stromal tissue, trophoblast cells, etc. are included.

Prior Art Documents

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[25] [Overview of the project] [Problems that the invention aims to solve]

[0021] Therefore, the object of the present invention is to provide an improved human placental 3D vascular model that reflects in vivo conditions and can be used to appropriately investigate the effects of flow-related vascular and epithelial barrier function and cellular barrier function, such as morphology. Other objects and advantages will become apparent to those skilled in the art through further study of this disclosure. [Means for solving the problem]

[0022] In a first aspect of the present invention, the present invention provides a microfluidic device comprising the following to solve the above problems: a) Elongated central culture channel for holding mammalian cells within a suitable gel, b) at least one first media channel positioned parallel to a first side of an elongated central culture channel, and at least one second media channel extending parallel to a second side opposite to the elongated central culture channel, configured to enable a lateral flow connection from at least one first media channel to at least one second media channel via the central culture channel, and c) At least one third culture medium channel, at least in part, extending parallel to the third side of the elongated central culture channel, Here, optionally, at least one third medium channel is filled with a soluble gel barrier suitable for preventing cells from entering at least one third medium channel while the device is being prepared. This allows the cell-gel mixture to be contained within the central channel and, upon lysis, to form a monolayer of adjacent cells.

[0023] Preferably, the microfluidic device according to the present invention is advantageously a postless system, which simplifies manufacturing and allows for direct contact between the culture medium channel and the gel, i.e., no physical barrier is provided.

[0024] In the microfluidic device according to the present invention, the mammalian cells in the culture chamber are preferably selected from human cells and preferably include cells and / or cell lines derived from epithelial tissue having an epithelial layer (e.g., cancer cells and / or immortalized cell lines), vascular cells, respiratory cells, digestive cells, bladder cells, mammary gland cells, ductal cells and their cancer cells. The culture also preferably comprises three types of cells: human umbilical vein endothelial cells (HUVECs), human placental fibroblasts (HPFs), and human placental pericytes (HPPs), and a layer of human trophoblast cells located on the side of the cell culture in contact with at least one third culture medium channel, and more preferably a combination of mammary gland cells and epithelial cells.

[0025] In a second aspect of the present invention, the present invention provides a microfluidic device comprising the following to solve the above problems: a) Elongated central culture channel for holding human cells within a suitable gel, b) At least one first and second culture channel, wherein at least a portion of the first culture channel extends parallel to a first side of the elongated central culture channel, and at least a portion of the second culture channel extends parallel to a second side opposite to the elongated central culture channel, and is configured to allow a lateral flow connection from the at least one first culture channel to the at least one second culture channel via the central culture channel, and the mammalian cells in the gel include cultures of three species: human umbilical vein endothelial cells (HUVECs), human placental fibroblasts (HPFs), and human placental pericytes (HPPs).

[0026] Preferably, the microfluidic device according to the present invention is advantageously a postless system, which simplifies manufacturing and allows direct contact between the culture medium channel and the gel, i.e., no physical barrier is provided.

[0027] The microfluidic device according to the present invention preferably further comprises a barrier layer of cells, preferably trophoblast cells, located on either the first or second side of the elongated central culture channel.

[0028] The advantages of the novel design described above will allow for the investigation of transport across the entire barrier, including the trophoblast, fetal vascular system, and its interstitial mass. Furthermore, this device will enable assays to investigate transport across the maternal-fetal interface. The model should preferably be designed to replicate the physical barrier between mother and fetus and preferably include human cell-derived trophoblast cells and fetal vascular system. This system will mimic the role of the placenta in regulating the movement of solutes, antibodies, and other fetal xenobiotics.

[0029] Safety testing of pharmaceuticals and other biomolecules intended for use in human pregnancy has been severely limited due to ethical reasons and the lack of appropriate relevant models. For example, animal models do not exhibit human species-specific placental formation, and human placental tissue fragments are difficult to maintain and perfuse, usually only reflecting the late stages of pregnancy. In recent years, as mentioned above, several humanized in vitro models have been designed to mimic the in vivo aspects of the placental barrier, but these lack the combination of two key barriers that control maternal-fetal exchange (in this case, adequate amounts of trophoblast cells and the placenta-fetal vascular system). The advantage of this new design is that it allows for the investigation of transport across the entire barrier, including trophoblast cells, the fetal vascular system, and its interstitial tissue.

[0030] Following the aforementioned development of fetal microvessels, the inventors further developed novel designs (Figures 9, 10, and 11) that, in a preferred embodiment, integrate trophoblast cells into the system to generate a crucial epithelial layer that forms a barrier between maternal blood and fetal capillaries. These models may function as a barrier for the terminal villus of the placenta. A general method for fabricating the novel designs involves (1) introducing a temperature-dependent solubility gel into the “maternal” channel of the device, (2) seeding fetal microvessels on the “fetal” side, including the culture chamber, and (3) dissolving the gel and, after about 48 hours, introducing an appropriate amount of barrier cells, e.g., 1.0 x 10⁶. 6 Cells / mL or more, for example, about 1.5 x 10⁻¹⁴ 6 This involves seeding trophoblast cells at a cell / mL rate into the “maternal” channel (immortalized trophoblast cell lines BeWo and HTR-8 / SVneo, and primary isolated trophoblast cells (see Figure 13) can be used as preferred examples), or, as in (3a)(3), lysing the gel in the third or fourth medium channel and establishing barrier cells on the third and / or fourth sides of the culture chamber as described herein.

[0031] The design of this invention is unique and enables the development of a combined trophoblast layer and a perfusing fetal vascular system without the need for a separation mesh. Furthermore, it allows for perfusion of both the maternal and fetal tissues. This design allows for the generation of a pressure gradient across the entire maternal-fetal interface, enabling the examination of the properties of both the trophoblast barrier and the fetal vascular barrier.

[0032] Furthermore, in a preferred embodiment, the design is employed to be used in conjunction with other tissues / organs having the ability to integrate various cells (e.g., primary tissue from a patient) and / or having an epithelial layer as disclosed herein.

[0033] In the microfluidic device according to the present invention, HPF and HPP are cultured in an endothelial cell:stromal cell (HPF / HPP) ratio of approximately 10:1, preferably with a ratio of HPF to HPP of 1:1. More preferably, the microfluidic device according to the present invention is configured such that the device is placed on a solid support such as a biochip or coverslip, and / or several or more devices are grouped on the solid support.

[0034] In a third aspect of the present invention, the present invention provides a method for manufacturing a placenta-derived human 3D vascular microtissue model, comprising the following steps, in order to solve the above problems: a) A step of providing a microfluidic device according to the present invention (here, a "gel-barrier version" in which at least one of the culture medium channels contains a gel), b) The step of culturing the cells in the culture chamber in an appropriate culture medium, and c) Dissolving at least one of the gel barriers (gel barrier(s)) described herein and seeding appropriate mammalian barrier cells as described herein.

[0035] Preferably, the present invention relates to a method for producing a human 3D vascular microtissue model derived from the placenta, Here, in b), the tri-culture is cultured for approximately 48 hours in a suitable culture medium under essentially static conditions. b1) By applying a hydrostatic pressure gradient of preferably about 20 Pa to about 70 Pa between at least one first culture channel and at least one second culture channel, and / or by applying a mean lateral flow velocity of preferably about 0.20 μm / sec to 1.25 μm / sec across the central culture channel, a lateral flow (e.g., intermittent interstitial flow (IF)) is established across the central culture channel from at least one first culture channel to at least one second culture channel, and This lateral flow is established between approximately day 2 and day 7 of the three-culture process.

[0036] In a fourth aspect of the present invention, the present invention provides a method for manufacturing a placenta-derived human 3D vascular microtissue model, comprising the following steps, in order to solve the above problems: a) A step of providing a microfluidic device according to the present invention (hereinafter referred to as the "vascular version"), b) A step of culturing the three cultures in a suitable culture medium for approximately 48 hours under essentially static conditions. c) Establishing lateral flow (e.g., intermittent interstitial flow (IF)) across the central culture channel from at least one first culture channel to at least one second culture channel by applying a hydrostatic pressure gradient of preferably about 20 Pa to about 70 Pa between at least one first culture channel and at least one second culture channel, and / or by applying an average lateral flow velocity of preferably about 0.20 μm / sec to 1.25 μm / sec across the central culture channel, The lateral flow is established between approximately day 2 and day 7 of the three cultures, and d) Optionally, a step to test the perfusion rate following step c).

[0037] The method according to the present invention preferably further includes a step of introducing a barrier layer of cells (preferably trophoblast cells) located on either the first or second side of the elongated central culture channel ("vascular barrier version").

[0038] In the method according to the present invention, the hydrostatic pressure gradient (ΔP) established in step c) is preferably less than about 10 mmH2O, and more preferably about 2 to 7 mmH2O. More preferably, in the method according to the present invention, the hydrostatic pressure gradient is 3 mH2O or about 30 Pa, or the mean lateral flow velocity on day 2 is 0.30 ± 0.12 μm / sec.

[0039] In a fifth aspect of the present invention, the present invention provides a placental-derived human 3D vascular microtissue model manufactured according to the method of the present invention to solve the above problems. Preferably, the placental-derived human 3D vascular microtissue model according to the present invention exhibits laminar flow and / or barrier function throughout the model described herein.

[0040] In a sixth aspect of the present invention, the present invention provides the use of a microfluidic device or a placenta-derived human 3D vascular microtissue model according to the present invention to solve the above problems, the use of which is for studying placental barrier function, for drug screening, for example for evaluating novel drug targets for dysfunction in pre-eclampsia, for studying trophoblast cell infiltration, or for toxicity screening of substances and stresses that interfere with trophoblast cell infiltration, for studying the effects of placental branching angiogenesis, nutrients, hormones, and environmental factors on placental development, pregnancy disorders of fetal blood vessels, and / or vascular-related pregnancy disorders, and / or for studying the effects of epithelial and / or endothelial barrier function (solute permeability), transport of solutes, contaminants, antibodies, immune cells, and drugs through epithelial cell barriers and / or vascular cell barriers, and extravascular matrix properties (diffusivity, stiffness, and matrix proteins), in particular the effects of luminal flow, interstitial flow, and / or lateral flow on endothelial barrier function (solute permeability) and extravascular matrix properties (diffusivity, stiffness, and matrix proteins). [Brief explanation of the drawing]

[0041] [Figure 1]Figure 1 shows that interstitial flow promotes perfusion of the placental microvascular network. A) Diagram showing the culture timeline and protocol. B) Immunofluorescence images of three cultured placental vessels in orthogonal view showing open vascular lumen. C) Interstitial flow condition (shown in figure) promotes perfusion of the complete vascular bed. Perfusion is indicated by FITC-labeled dextran introduced into RFP-labeled vessels on day 7. Scale bar is 1000 μm. D) The percentage of perfused vessels is quantified across static and interstitial flow-condition vessels on day 7. A box plot with the standard error at the outer edge and a bar showing the standard deviation are shown. Significance is measured by one-way ANOVA and is indicated as ***<0.001 by Tukey mean comparison test. E) Pressure drop measured over different days of the culture period. Mean and standard deviation of three separate experiments using six or more devices are shown. [Figure 2] Figure 2 shows that interstitial flow promotes vascular connectivity and barrier function. A) Confocal images of microvessels (HUVECs including cytoplasmic RFP) on days 3 and 7 of culture under static and flowing conditions. Morphology is measured by image segmentation of RFP-labeled cytoplasmic HUVECs. For B-D, morphological parameters were measured over days 3 and 7. B) Connectivity is quantified as the ratio of vascular junctions to endpoints. C) Branch density is the number of branches per unit area. D) Effective diameter is measured as the coverage of vascular area relative to length. E) Permeability of 70 kDa dextran under static and flowing conditions on day 7. The dotted line under static conditions indicates that very few vessels were perfused and measured. Significance was measured by one-way ANOVA, and Tukey's mean comparison test is shown as *<0.05, **<0.01, ***<0.001, and # represents day-by-day significance. [Figure 3]Figure 3 shows that interstitial flow influences inflammatory and angiogenic signaling in the early stages of angiogenesis. The concentrations of inflammatory molecules A) IL8 and B) MCP1 were measured by ELISA from 3D microvessels at various time points. Angiogenic signaling molecules C) Ang2 and D) VEGF were significantly different between statically cultured vessels and early and late time points, respectively. Significance was measured by one-way ANOVA, and Tukey's mean comparison test indicates *<0.05, **<0.01, and ***<0.001. # represents significance over day 5 and day 3, and $ represents significance over day 7 and day 3. [Figure 4] Figure 4 shows that flow promotes the long-term stability of placental microvessels. A) Confocal images show the difference in vascular density between static and flow-conditioned vessels at 14 days of culture. B) Area coverage and C) Effective diameter of vessels are significantly increased by flow-conditioning. D) Branch density and E) Length were not significantly affected by flow. F) Vascular permeability is measured for fully perfused flow-conditioned vessels and for a small number of perfusing vessels remaining under static conditions. Even after 14 days post-culture, flow maintains vascular perfusion capacity (G), and area coverage (H) increases over time compared to static conditions. Significance was measured by one-way ANOVA, and Tukey's mean comparison test shows *<0.05 and **<0.01. [Figure 5] Figure 5 shows CFD simulation predictions of fetal placental microvascular tissue. A) A pipeline for calculating and predicting velocity and shear stress within microvessels embedded in a porous gel. Four independent predictions are shown for the distribution of B) velocity and C) shear stress within the microvessels and gel region, under a 7 mmH2O condition. [Figure 6]Figure 6 shows that interstitial flow alters extravascular remodeling and tissue-level stiffness. A) FRAP measurements performed in the extravascular region. ROI is the bleach of the region of interest, EVS and IVS are the extravascular and intravascular spaces, respectively. B) Diffusivity measurements calculated from FRAP experiments on statically and fluidly conditioned microvessels on day 7. C) Schematic diagram of nanoindentation on microtissue. D) Stiffness measurements performed across statically and fluidly conditioned samples. E) Populations of stromal cells from microvessels corresponding to different culture times. Significance was measured by one-way ANOVA, and Tukey's mean comparison test indicates *<0.05, **<0.01, and ***<0.001. [Figure 7-1] Figure 7 shows that flow induces EVM protein deposition and compositional changes. A) Confocal images of static and flow-conditioned microvessels (red labels shown in corner image insets) immunolabeled with collagen I, laminin, and fibronectin (green labels) at day 7. B) Fluorescence intensity of collagen I, fibronectin, and laminin was measured and normalized to vessel area. Significance was measured by one-way ANOVA and indicated by *<0.05 in Tukey mean comparison test. C) EVM protein composition is ranked in descending order of abundance based on total peptide count after 7 days of culture (top 50 most abundant proteins). Proteins are classified according to five ontological terms selected based on their association with angiogenesis and matrix remodeling. D) PCA was performed against mass spectrometry-based proteomics, and data obtained from 7-day flow-conditioned samples were normalized. E) K-mean clustering and heatmaps of 13 hit and candidate proteins. The difference in expression between static and flowing conditions is shown (expressed as the Log.2 ratio of protein amounts in each 3mmH2O and 7mm H2O sample to the average protein amount in the 0mmH2O sample). F) Volcano plot of p-values ​​and log2 protein amount differences under flowing and static conditions. Red dots are hits: FDR < 0.05, FC > 30%. Blue dots are candidates: FDR < 0.05, no FC threshold. p-values ​​are calculated from adjusted t-tests (Limma). [Figure 7-2] Same as above [Figure 7-3] Same as above [Figure 7-4] Same as above [Figure 8] Figure 8 shows an example of human fetal-like blood vessels on a microfluidic chip (according to the present invention) that includes a "vascular barrier" function. When human umbilical vein endothelial cells (HUVECs) were co-cultured with stromal cells (fibroblasts and pericytes), perfusion-capable, interconnected microvessels were formed in 5–7 days. Subsequently, trophoblast cells were incorporated into the system, generating an important epithelial layer that forms a barrier between maternal blood and fetal capillaries. In this embodiment, in a chip design established to generate a fetal vascular system using two immortalized cell lines, BeWo and HTR-8 / SVneo, a trophoblast cell monolayer (Figure 13) was first seeded adjacent to the fetal blood vessels. [Figure 9-1] Figure 9 shows that the permeability of trophoblast cells is limited in another embodiment of the device according to the present invention, which includes a temperature-soluble gel barrier (bottom culture channel) and trophoblast cells. A) Schematic diagram of a model showing fetal and maternal components in this configuration. B) Cellular structure in the gel barrier (left) and DAPI staining (right). C) Selective permeability with dextran particles of different sizes in the model shown in Figure B. Molecules larger than 1 kDa do not easily pass through the barrier (as expected in vivo). [Figure 9-2] Same as above [Figure 10] Figure 10 shows that perfusing the model in Figure 9 with sFLT-1 results in a pre-eclampsia (PE) phenotype of trophoblast barrier dysfunction. Dysfunction of the trophoblast barrier associated with increased sFLT-1 concentration allows for the transport of dextran from maternal components to fetal blood vessels. [Figure 11] Figure 11 shows that perfusing the model in Figure 9 with sFLT-1 results in a microvascular pre-eclampsia (PE) phenotype. A) Microvessels regress with increasing sFLT-1 concentration, and their normal morphology is significantly lost. B) At concentrations related to plasma levels in PE patients. [Figure 12]Figure 12 shows a CAD drawing illustrating the main features of a 3-channel post-less chip design according to the present invention. [Figure 13-1] Figure 13 outlines the method for fabricating a complete two-barrier model of the maternal-fetal interface according to the present invention. A) Photograph of the fabricated three-channel microfluidic device. Figure comparing the size of the device to a 1 euro coin. B) An experimental cell-based approach that generates images showing the maternal-fetal interface-on-chip and the interface between microvessels and trophoblast cells. C) Photograph showing that the placental barrier on-chip is permeable to a glucose fluorescent analog (2-NDBG). D) Showing decreased permeability of the placental barrier on-chip to dextran greater than 0.4 kDa. E) The placental barrier on-chip shows low permeability to immunoglobulins (G and M) and insulin even 24 hours after perfusion. Dashed lines indicate corresponding maternal concentrations. F) A population of nearly pure primary trophoblast cells (cytokeratin 7 positive) was obtained by excising chorionic villi from a tissue piece of term placenta. G) Isolated primary cytotrophoblasts spontaneously fuse and differentiate into syncytiotrophoblasts after 72 hours of culture. H) Image of the maternal-fetal interface generated by the fusion of isolated primary cytotrophoblasts (G). [Figure 13-2] Same as above [Figure 13-3] Same as above [Figure 14-1]Figure 14 shows the trophoblast barrier function assay according to the present invention. A) Schematic diagram showing the timeline and assay (below) for measuring the permeability of the trophoblast barrier. Dextran was added to the parent component of the chip. The permeability of the trophoblast barrier to 0.4 kDa dextran was monitored by time-lapse imaging. Scale bar 200 microns. B) In the permeability measurement to 0.4 kDa dextran, a significant increase (barrier dysfunction) was shown when cultured under hypoxic conditions. [Figure 14-2] Same as above [Figure 15-1] Figure 15 shows the morphology and barrier assay of fetal-placental microvessels. Because fetal-placental microvessels can serve as an indicator of developmental health, the present invention has established a method for measuring changes in vascular morphology and barrier function. A) Schematic diagram of the experimental timeline for analyzing microvascular morphology and barrier function. The figure below shows where molecules (sFLT1 and sEng) were perfused into the maternal compartment and dextran was perfused into the fetal compartment for measurement of microvascular barrier function. To mimic pre-eclampsia on the chip, molecules sEng and sFlt1 were supplemented with normal culture medium, or the entire chip was cultured under hypoxic conditions. B) Images of fetal microvessels on day 7. Shows untreated, treated with PE-related factors sFLT1, sEng, or treated under 1% O2 conditions. All scale bars are 200 microns. C) Morphological evaluation was performed by imaging vessels in bright field, segmenting the vessels using a machine learning (ML) pipeline, and outputting morphological mappings of various features (similar to the case of 98). D) One parameter, namely the mean microvessel diameter, is shown under the conditions measured in C). Under all conditions mimicking PE, vessel diameter was significantly reduced. E) Microvessel barrier function was measured as in A) and B) and was shown to be altered by sEng with 0.4 kDa dextran. Vessels became non-perfusionable under hypoxic conditioning. [Figure 15-2] Same as above [Figure 16-1]Figure 16 outlines a secondary use case of the 3-channel chip design according to the present invention as a duct-on-chip model. A) Image of the 3-channel device with reservoir and an experimental cell-based approach for generating ducts on the chip. B) Image of the interface between microvessels and epithelial monolayer. C) Image showing fluorescence images of microvessels and E-cadherin (epithelial marker). D) Schematic diagram of an experimental timeline for including hormones, tumors, and drugs. E) Tumor contained within the ductal compartment. F) The epithelial barrier of the duct on the chip is impermeable to dextran with molecular weight greater than 1 kDa. G) Permeability to various dextran sizes, measured as response to untreated (NT), added hormones (Fol = estrogen and progesterone at follicular levels), and hormones + tamoxifen (Fol + TAM). All scale bars are 250 microns unless otherwise noted. [Figure 16-2] Same as above [Modes for carrying out the invention]

[0042] As described above, the present invention provides a microfluidic device comprising: a) an elongated central culture channel for holding mammalian cells within a suitable gel; b) at least one first culture channel positioned parallel to a first side of the elongated central culture channel, and at least one second culture channel extending parallel to a second side opposite to the elongated central culture channel, configured to allow lateral flow connections from at least one first culture channel to at least one second culture channel via the central culture channel; and c) at least one third culture channel extending parallel to a third side of the elongated central culture channel, optionally having at least one third culture channel filled with a soluble gel barrier suitable for preventing cells from entering at least one third culture channel. A preferred suitable gel is a fibrinogen / thrombin gel (see the examples below). Preferred soluble gel barriers include temperature-dependent soluble gels such as gelatin.

[0043] The microfluidic device according to the present invention preferably comprises a fourth culture channel extending parallel to a fourth side of at least partially elongated central culture channel, where optionally, at least one third culture channel is filled with a soluble gel barrier suitable for preventing cells from entering at least one third culture channel.

[0044] More preferably, in the microfluidic device according to the present invention, the mammalian cells in the culture chamber are selected from human cells and preferably include cells and / or cell lines (e.g., cancer cells and / or immortalized cell lines) derived from epithelial tissue having an epithelial layer, vascular cells, respiratory cells, digestive cells, bladder cells, mammary gland cells, ductal cells and their cancer cells. And preferably, it includes cultures of three types of cells: human umbilical vein endothelial cells (HUVECs), human placental fibroblasts (HPFs), and human placental pericytes (HPPs). and includes a layer of human trophoblast cells located on the side of the cell culture that is in contact with at least one third or fourth culture medium channel.

[0045] In a second aspect of the present invention, a) a microfluidic device is provided comprising an elongated central culture channel or chamber for holding human cells within a suitable gel. In this placental model, three types of cultures—human umbilical vein endothelial cells (HUVECs), human placental fibroblasts (HPFs), and human placental pericytes (HPPs)—are carried out within this chamber. Preferred gels are hydrogels such as fibrinogen / thrombin gel (see examples below).

[0046] The device according to the present invention then comprises at least one first and second culture channel, wherein at least one first culture channel extends parallel to the first side of the elongated central culture channel, and at least one second culture channel extends parallel to the second side opposite the elongated central culture channel, with at least one portion of the channel extending parallel to the second side opposite the elongated central culture channel. These channels (at least two are present in the device) allow for controlled flow of the culture medium through the device, flowing parallel to the elongated central culture channel or chamber (see Figures 9, 12, or 13 for preferred embodiments). Any medium suitable for culturing the cells involved can be used as the culture medium, such as endothelial medium (e.g., VascuLife medium) and / or fibroblast medium and / or pericyte growth medium (see Examples below).

[0047] Importantly, the device according to the present invention is configured to allow lateral flow, such as interstitial flow, luminal flow, or lateral flow, from at least one first culture medium channel to at least one second culture medium channel via a central culture channel. In the context of the present invention, the term “lateral flow” also includes meaning a pressure gradient established over a large portion of the gel region of the culture chamber. This makes it possible to study the effects of interstitial flow (a preferred embodiment of lateral flow) or luminal flow on vascular and extravascular remodeling, as described herein.

[0048] The device of the present invention may also contain other mammalian cells within the gel of the culture chamber. These cells include any suitable cells and / or cell lines, such as cancer cell lines and / or immortalized cell lines derived from epithelial tissue having an epithelial layer, vascular cells, respiratory cells, digestive cells, bladder cells, mammary gland cells, ductal cells and their cancer cells, as well as mixtures thereof. As described above, cultures of three types of cells are preferred: human umbilical vein endothelial cells (HUVECs), human placental fibroblasts (HPFs), and human placental pericytes (HPPs).

[0049] In a preferred microfluidic device according to the present invention, the device further comprises a cell barrier layer, preferably of epithelial cells, more preferably of trophoblast cells, located on either the first or second side of the elongated central culture channel.

[0050] In contrast to cutting-edge technologies, placental fibroblasts are now fully integrated into human 3D vascular models derived from the placenta and are used to study the effects of interstitial or luminal flow in vascular and extravascular remodeling. The use of placental fibroblasts advantageously provides a model that is as close as possible to in vivo conditions. Furthermore, the system can include epithelial barrier cells and / or layers (see above). Thus, as an alternative approach to animal experiments, the present invention includes novel devices and assays that can measure the movement of substances across the maternal-fetal interface. As previously described, a human fetal-like vascular system is grown on a microfluidic chip by co-culturing human umbilical vein endothelial cells (HUVECs) in the presence of stromal cells (fibroblasts and pericytes) to form interconnected microvessels that can be perfused in 5–7 days.

[0051] Subsequently, the trophoblast cells are integrated into the system, generating a desirable and crucial epithelial layer that forms a barrier between maternal blood and fetal capillaries (see Figure 9). In this example, two immortalized cell lines, BeWo and HTR-8 / SVneo, and primary isolated trophoblast cells (see Figure 13) were used to seed a monolayer of trophoblast cells adjacent to the fetal blood vessels in a chip design after the development of the fetal vascular system.

[0052] The inventors' results obtained in connection with the present invention demonstrate that the growth and remodeling of fetal microvessels, as well as the study of epithelial and / or endothelial barrier function (permeability to solutes) and extravascular matrix properties (diffusivity, stiffness, and matrix proteins), are influenced by the presence of flow. Furthermore, fetal microvessels under flowing conditions show significant changes in angiogenesis and the expression of inflammatory cytokines. Computational fluid dynamics revealed that intraluminal velocity, pore velocity, and shear stress tend to increase with increasing flow rate (Figure 5). Interstitial flow (extravascular region) promotes early connection of networks that maintain viability and perfusionability over long (several weeks) culture periods.

[0053] Cells and microfluidic devices can be cultured under standard conditions of 37°C and 5% CO2 in a standard incubator, or in a hypoxic incubator.

[0054] The model of the present invention, which incorporates placental fibroblasts rather than co-culturing them with lung fibroblasts as in conventional methods (reference 25), required adjustment. Preferably, HPF and HPP are cultured in HPF / HPP with an endothelial-to-stromal cell ratio of approximately 10:1, and preferably with an HPF-to-HPP ratio of 1:1. Furthermore, the device preferably contains a gel containing a hydrogel such as fibrinogen / thrombin gel (fibrin gel) at a concentration of approximately 3 to approximately 3.5 mg / ml. However, other suitable gel systems such as collagen or alginate can also be used. Interstitial flow was also introduced to realize perfusing vessels. In this three-cell culture, placental fibroblasts and pericytes are bound to microvessels. This indicates that stromal cells play a clear role in the growth and remodeling of fetal-placental vessels.

[0055] In a preferred embodiment, the microfluidic device according to the present invention further comprises a culture medium reservoir, preferably a 3D-printed customized culture medium reservoir, connected to at least one of the culture medium channels. The reservoir can be connected to at least one culture medium channel via an inlet and is used to generate lateral flow based on a hydrostatic pressure gradient (ΔP). See the following examples for a preferred example. The preferred gradient established in device c) is less than about 10 mmH2O, preferably about 2 to 7 mmH2O.

[0056] More preferably, the present invention relates to a microfluidic device, wherein the device is placed on a solid support such as a biochip or coverslip, and / or several or more devices are grouped on the solid support. In this embodiment, the microfluidic device according to the present invention is used, for example, as an array to investigate (or test) the properties of a human 3D vascular microtissue model derived from placenta present on the device. The test can then be automated, for example, using a robot or automated optical analysis.

[0057] While this is a preferred embodiment of the present invention, there is still room for further improvement in the single-layer seeding on the side of the gel in the culture chamber.

[0058] Gel contraction and cell aggregation prevented the formation of a connected monolayer, at least to some extent, as observed with BeWo cell lines (see Figures 9-11). Furthermore, similar to the case with HTR-8 / SVneo cells, the distribution of culture medium across the entire gel was inhibited, resulting in poor angiogenesis and lack of perfusion.

[0059] Therefore, to overcome these drawbacks, the inventors have developed an alternative design that includes an additional fluid chamber for the epithelial interface (Figures 9A, 12, and 13). Our preliminary data show that in this system, blood vessels can form a properly connected vascular network, and the epithelial barrier mimics physiological permeability properties (i.e., 70 kDa molecules do not permeate; see Figure 11).

[0060] The method for manufacturing a microfluidic device according to the present invention is described herein and in the references (reference 25) (which is incorporated in its entirety by reference), and is disclosed in the references cited herein, and is known to those skilled in the art.

[0061] Any suitable biocompatible polymer can be used as the material for the device according to the present invention, but preferably a silicone elastomer such as polydimethylsiloxane (PDMS) elastomer (see below). This example is known to those skilled in the art and has been described in the literature (e.g., Okoshi, M., Yoshida, T. Fabrication of Silicone Rubber-Based Biochip for Disinfection Under Deep-UV Light by ArF Excimer Laser-Induced Photodissociation. Electron. Mater. Lett. 17, 68-73 (2021)).

[0062] Another important aspect of the present invention relates to a method for producing a human 3D vascular microtissue model derived from the placenta, the method comprising the step of a) providing the microfluidic device according to the present invention as described above.

[0063] The next step in the method for producing the placenta-derived human 3D vascular microtissue model of the present invention is b) incubating the cells in a culture chamber in a suitable culture medium. These include any suitable cells and / or cell lines, such as cancer cells and / or immortalized cell lines derived from epithelial tissue having an epithelial layer, vascular cells, respiratory cells, digestive cells, bladder cells, mammary gland cells, ductal cells and their cancer cells, and mixtures thereof. As mentioned above, three cultures of human umbilical vein endothelial cells (HUVECs), human placental fibroblasts (HPFs), and human placental pericytes (HPPs) are preferred, cultured for about 48 hours in a suitable culture medium under essentially static conditions. In the context of the present invention, “essentially static conditions” means that there is no substantial (forced or induced) flow of the medium within the device, or at least no (forced or induced) flow (e.g., lateral flow) of the medium within or through the central culture channel. This is done to seed the cells into the central culture channel.

[0064] In the next step of the method for producing a placenta-derived human 3D vascular microtissue model of the present invention, c) a lateral flow, such as intermittent interstitial flow (IF) or luminal flow, is established from at least one first medium channel across the central culture channel to at least one second medium channel. This is achieved by applying a suitable pressure gradient, for example, about 20 Pa to about 70 Pa, between at least one first medium channel and at least one second medium channel, and / or by applying an average lateral flow velocity of about 0.20 μm / s to 1.25 μm / s across the central culture channel, where this lateral flow is established between about 2 to about 7 days of the three cultures. Preferably, in the method according to the present invention, the flow rate is re-established daily by replenishing reservoir media volumes for 5 days after step b). However, other intervals may be used as needed, for example, twice a day or every other day.

[0065] In the next step, in the barrier-versions of the device, in the first embodiment, a barrier layer of cells, preferably trophoblast cells, is established on either the first or second side of the elongated central culture channel.

[0066] In a second embodiment of the barrier version, a barrier layer of cells, preferably trophoblast cells, is established by dissolving the soluble gel barrier of the device and seeding cells as described herein. Other methods for achieving this are known to those skilled in the art or described in the art.

[0067] Preferably, in the method according to the present invention, the hydrostatic pressure gradient (ΔP) established in step c) is less than about 10 mmH2O, preferably about 2 to 7 mmH2O. More preferably, in the method according to the present invention, the hydrostatic pressure gradient is 3 mmH2O or about 30 Pa, or the average lateral flow velocity on day 2 is 0.30 ± 0.12 μm / sec.

[0068] Another aspect of the method according to the present invention for tracking vascular development in a 3D model and / or detecting phenotypic changes in cells contained in the model further includes a step of detecting changes in angiogenesis and inflammatory cytokine and chemokine expression during or after steps c) and / or d). Each detection is described herein and in the literature.

[0069] As the next step d), the perfusion rate can be tested as needed. Methods for such testing are described herein (see Examples), cited reference 2, and other references. This test can also be performed during three cultures to track the development of blood vessels in the 3D model.

[0070] As the next step d), the permeability of materials in the barrier version of the present invention can optionally be tested. Such tests and other test methods are described herein (see Examples) and in the literature. This test can also be performed during the development of the barrier cell layer to track the development of the vascular system in a 3D model.

[0071] Another important aspect of the present invention relates to a method for producing a human 3D vascular microtissue model derived from breast tissue, the method comprising the steps of: a) providing a microfluidic device according to the present invention as described above; b) seeding human mammary vascular endothelial cells (HMVECs) or iPSC-derived endothelial cells and human mammary fibroblasts (HMFs) into an elongated central culture channel in a suitable culture medium for about 8 hours under essentially static conditions; c) introducing an epithelial cell (MCF10) monolayer into a third medium channel and optionally inserting tumor spheroids or patient-derived organoids on day 1 post-seeding; d) establishing lateral flow, e.g., intermittent interstitial flow (IF) or luminal flow, across the central culture channel from at least one first medium channel to at least one second medium channel by applying a hydrostatic pressure gradient of 3 mmH2O 24 hours post-seeding; and e) optionally testing the perfusion rate after step d). Aside from the cells, this model is generally established like other models and is tested and used as described herein.

[0072] A further important aspect of the present invention relates to a placenta-derived human 3D vascular microtissue model or a breast-derived human 3D vascular microtissue model manufactured according to the method of the present invention.

[0073] As mentioned above, these models consist of and are based on favorable designs for studying the barrier function of cells, particularly epithelial and endothelial cells.

[0074] In another embodiment, this model includes three cultures of human umbilical vein endothelial cells (HUVECs), human placental fibroblasts (HPFs), and human placental pericytes (HPPs). In contrast to state-of-the-art techniques, here placental fibroblasts are fully integrated into a human 3D vascular model derived from the placenta, which can be used to study the effects of interstitial or luminal flow on vascular and extravascular remodeling. The use of placental fibroblasts advantageously provides a model that is as close as possible to in vivo conditions.

[0075] The placenta-derived human 3D vascular microtissue model or breast-derived human 3D vascular microtissue model according to the present invention preferably exhibits laminar flow (see also above), and particularly vascular flow, throughout the entire model.

[0076] In some embodiments, the model further comprises a cell barrier layer, preferably of trophoblast cells, located on either the first or second side of the elongated central culture channel.

[0077] Advantageously, the microfluidic device and / or the placenta-derived human 3D vascular microtissue model and / or breast-derived human 3D vascular microtissue model according to the present invention enable the direct measurement of mechanical stimuli such as shear stress and physical and chemical stimuli such as corresponding cytokines and chemokines, as illustrated below herein. The model further comprises a barrier layer of cells, preferably trophoblast cells, located on either the first or second side of an elongated central culture channel.

[0078] A further important aspect of the present invention relates to studying barrier functions, such as placental barrier function, disclosed herein, for drug screening, for evaluation of novel drug targets for functional disorders such as pre-eclampsia, or for studying ductal function using breast-derived human 3D vascular microtissue models according to the present invention.

[0079] Importantly, compared to static culture, endothelial barrier function was significantly improved in flow-conditioned vessels (Figure 2E). Our results indicate that flow- and size-dependent conditions are necessary to ensure the development of perfusing fetal-placental-like vessels with improved barrier function. Vascular abnormalities have been found in placentas affected by fetal growth restriction (reference 45), which are characterized by high vascular resistance and decreased fetal-placental blood flow (hyperfusion).

[0080] A further important aspect of the present invention relates to the use of a microfluidic device or a placenta-derived human 3D vascular microtissue model according to the present invention to study trophoblast invasion, for example, through toxicity screening of substances and stresses that inhibit trophoblast cell invasion. Recently, Park et al. (in A microphysiological model of human trophoblast invasion during implantation. Nat Commun 13, 1252 (2022). https: / / doi.org / 10.1038 / s41467-022-28663-4) described a microengineered system that models a complex sequence of organized multicellular events that play a crucial role in early pregnancy. By implanting it onto their chip, they were able to remodel the three-dimensional structural tissue of the maternal-fetal interface and model the invasion of specific fetal extrachorionic trophoblast cells into the maternal uterus. Using primary human cells isolated from clinical specimens, they demonstrated the directional migration of extravillous trophoblast cells toward microengineered maternal blood vessels, similar to that in vivo, and their interaction with endothelium necessary for vascular remodeling. Through parameter changes in the cellular microenvironment and proteomic analysis of microengineered tissues, they showed the crucial role of decidual stromal cells as regulators of extravillous trophoblast migration.

[0081] A further important aspect of the present invention relates to studying branching angiogenesis of the placenta or breast, breast cancer and associated angiogenesis, the effects of nutrients, hormones, and environmental factors on placental development, pregnancy disorders of fetal blood vessels, and / or vascular-related pregnancy disorders using a microfluidic device according to the present invention, or a human 3D vascular microtissue model derived from the placenta according to the present invention, or a human 3D vascular microtissue model derived from the breast according to the present invention. Each test and assay is described herein and is known in the art.

[0082] A further important aspect of the present invention relates to studying the effects of interstitial flow, lateral flow, and luminal flow on studies of epithelial and / or endothelial barrier function (solute permeability), drug screening and testing of novel drug targets, transport of solutes, contaminants, antibodies, immune cells, and drugs across epithelial cell barriers and / or vascular cell barriers, and extravascular matrix properties (diffusivity, stiffness, and matrix proteins), as well as, in particular, the effects of interstitial flow, lateral flow, and luminal flow on studies of epithelial and / or endothelial barrier function (solute permeability) and extravascular matrix properties (diffusivity, stiffness, and matrix proteins), using the microfluidic devices according to the present invention, or the placenta-derived human 3D vascular microtissue model according to the present invention, or the breast-derived human 3D vascular microtissue model according to the present invention.

[0083] Tun et al. (in "Differences in placental capillary shear stress in fetal growth restriction may affect endothelial cell function and vascular network formation." Sci Rep 9,9876 (2019). https: / / doi.org / 10.1038 / s41598-019-46151-6) explained that vascular branching is often poor in placentas with fetal growth restriction (FGR). They hypothesized that malformations of vascular structure at the organ level alter the shear stress of microvessels, thereby inhibiting angiogenesis. Endothelial cells cultured under predicted FGR shear stress migrated significantly slower and more persistently than those cultured under predicted shear stress in a normal placenta. The behavior of these cells suggests that vascular extension is more dominant than branching. These results, taken together, suggest that (1) poor blood vessel development leads to increased shear stress in blood vessels, (2) increased shear stress induces cellular behavior that inhibits branching angiogenesis of capillaries, and (3) impaired branching angiogenesis leads to a continued increase in shear stress, further endangering angiogenesis.

[0084] In the context of this invention, the term "approximately" means a deviation of ±10% from a given value, unless otherwise specified.

[0085] Limited access to the human placenta, particularly in early pregnancy, hinders the elucidation of mechanisms related to the development of fetal placental blood vessels. To overcome this challenge, one embodiment of the present invention employs 3D placenta-specific microvascular tissue on a chip. Based on a conventional model (referenced 25), the inventors generated the entire placental vascular microtissue and incorporated a fluid reservoir, thereby characterizing the angiogenesis and extravascular remodeling processes in response to fluid conditions. In the present invention's model, which incorporates placental fibroblasts rather than the conventional co-culture with lung fibroblasts (referenced 25), adjustments such as the ratio of endothelial cells to stromal cells (reduced from 5:1 to 10:1), fibrin gel concentration (reduced from 3 to 3.5 mg / mL), and introduction of interstitial flow were necessary to realize perfusing vessels. In this three-cell culture, placental fibroblasts are associated with microvessels, as well as pericytes, as shown in the conventional model (referenced 25). This indicates that stromal cells play a clear role in the growth and remodeling of fetal placental blood vessels.

[0086] Aside from the interstitial space, little is known about the hemodynamics of the placenta in early pregnancy, particularly the villous vessels. Ultrasonographic imaging has shown that the blood flow velocity waveforms of the umbilical artery and its branches change with the progression of pregnancy and correlate with the development of the placental villous tree and capillary network (reference 39). Despite the presence of a complete vascular network within the villi, villous circulation is not thought to be fully established until the end of the first trimester of pregnancy. Flow is gradually established in the third month of pregnancy with the perfusion of maternal blood into the intervillous space (reference 40). The precise mechanisms behind the formation and remodeling of the villous vessel network remain unclear. Nevertheless, we hypothesized that hemodynamic force and related signaling may play a role in directing angiogenesis. Previous studies have shown that interstitial flow promotes early vascular connections in the 3D vascular system on the chip (reference 16). Here, static, low, and high interstitial flow velocities ranging from approximately 0.1 to 1.2 μm / s (measured on day 2) were applied via the reservoir. To the best of our knowledge, there are no reports on the velocity of human fetal placental interstitial fluid, but our measurements fall within the physiological range (0.1 to 4.0 μm / s) found in most soft tissues (references 41, 42). In the case of these placental-like vessels, it is necessary to apply interstitial flow at an early stage to establish a fully perfusing vascular network that transitions to luminal flow by day 5 after seeding (Figure 1E). The flow conditions clearly affect the morphology of the vessels, resulting in earlier connections, decreased branching density, and increased vessel diameter (Figure 2). Importantly, compared to static culture, endothelial barrier function was significantly improved in flow-conditioned vessels (Figure 2E). Other studies conducted in perfusing placenta models have also shown shear stress (20 dyn cm) -2It has been demonstrated that 2 Pa promotes vasodilation (release of nitric oxide) and reduces vascular resistance (references 43, 44). Our results indicate that fluidity and size-dependent conditions are necessary to ensure the development of perfusing fetal-placental-like vessels with improved barrier function. In line with this, vascular abnormalities have been found in placentas affected by fetal growth restriction (reference 45), which are known to be characterized by high vascular resistance and reduced fetal-placental blood flow. Our findings may suggest that blood flow is necessary at an early stage of fetal vascular development to prevent pregnancy complications.

[0087] Mechanical forces mediated by blood flow induce changes in vascular function and remodeling by promoting inflammatory and angiogenic responses (references 46, 47); however, our understanding of flow-induced signaling in the placenta remains limited. Pro-inflammatory chemokines such as IL-8 and MCP-1 are associated with angiogenesis, as demonstrated by various studies (references 48, 49). These chemokines are continuously produced by the placenta and stimulate an immune response to potential infections during pregnancy (references 50, 51); however, their role in placental vascular development remains unclear. Our findings demonstrate that the expression of IL-8 and MCP-1 during angiogenesis in our system is strongly influenced by flow, supporting previous reports on their regulation by shear stress (references 52-54). The human placenta is also known to be an important source of locally produced angiogenic factors, including members of the VEGF family, FGF family, and angiopoietin (reference 55). We analyzed Ang-2 levels in our fetal-placental blood vessels, which are highly expressed in early pregnancy and promote vascular remodeling in the presence of VEGF (references 56, 57). Our findings indicate that flow in the early stages increases Ang-2 expression (Figure 3D), in contrast to previous studies showing a decrease in shear stress-dependent Ang-2 expression in experiments using HUVEC monolayers exposed to flow (references 58, 59). It is noteworthy that previous studies investigating Ang-2 have mainly focused on laminar flow conditions (WSS > 6 dyn cm) -2 While other studies focused on Ang-2 expression at lower shear stress levels (1 dyn cm), other studies focused on Ang-2 expression at lower shear stress levels (0.6 Pa). -2It has been demonstrated that shear stress is upregulated (reference 60). In particular, it has been previously shown that Ang-2 expression in fibroblasts induces vascular growth while maintaining a balance with the quiescent effect of angiopoietin-1 (Ang-1) produced by placental pericytes (reference 25). These observations support our findings and highlight the need for further research to fully understand the complex relationship between shear stress and Ang-2 expression. In particular, VEGF and its receptor are expressed in trophoblast cells and choriofetal blood vessels in the early stages of development, suggesting their involvement in the initiation and progression of angiogenesis (reference 61). Here, we observed a significant increase in VEGF levels in the later stages (day 7) of static culture, which has been proposed to be a compensatory mechanism to counteract the lack of fluidity. In line with this hypothesis, VEGF expression has been found to be elevated in placentas with fetal growth retardation, suggesting that reduced fetal-maternal blood circulation during placental formation enhances the expression of angiogenic factors (reference 62).

[0088] Vascular stabilization is driven by hemodynamic forces due to branch regression and shearing exposed to low blood flow, along with maintaining vascular connections that experience a flow threshold level (reference 63). After two weeks of culture under intermittent flow conditions, placental-like microvessels were perfusing and functional (maintaining relatively low permeability) (Figure 4), consistent with observations in the 3D microvessel system of the brain (reference 23). Although there was no difference in vascular branching, flow significantly maintained vascular diameter and prevented vascular stenosis. This was in contrast to vascular stenosis observed in static culture (Figure 4H), which decreased from 74.5% on day 7 to approximately 59% of the area coverage on day 14. Previous observations in co-cultures of HUVECs and lungs have also shown that continuous flow (low WSS < 1 Pa) maintains stable vascular diameter of formed vessels (reference 16). At this later stage of culture, only a small number of statically cultured vessels were perfusing, and interestingly, these few vessels maintained high barrier function (Figure 4F). However, vessels under flowing conditions showed a slight decrease in endothelial barrier capacity compared to day 7 (Figure 4F, orange stratified data plot), which we believe is due to the limited stimulation the vessels receive (every other day) when hydrostatic pressure is re-established after day 7. Furthermore, flow conditioning is essential for maintaining microvascular connectivity over the four weeks of culture, highlighting the importance of implementing flow in long-term in vitro microvascular studies.

[0089] Intravascular lumen flow was measured by particle tracking, and it was shown that the mean velocity of flow-conditioned vessels significantly increased by day 7. To obtain more accurate predictions, the flow of the vascular network was modeled using COMSOL (Figure 5). A heterogeneous flow distribution dependent on the formation of open lumens within the culture medium channels was evident within the vascular network. The shear stress of the flow-conditioned vessels was 0.32 Pa (3.2 dyne / cm²). 2 ) was predicted, but the shear stress in the EVM was 0.001 Pa (0.01 dyne / cm²). 2The CFD results differed from the experimental bead measurements by approximately one order of magnitude. It should be noted that the predictions of velocity and shear stress were obtained using 2D segment projection and did not take into account the precise spatial complexity of 3D vessels, wall effects, or bead size-dependent effects. It has been previously reported that the presence of common additives in the culture medium (reference 64), the size of the tracer beads, and properties such as deformability (reference 65) contribute to elastic no-slip boundaries at the fluid interface and affect the velocity.

[0090] The inventors hypothesized that the differences in vascular morphogenesis and long-term stability observed in the presence of flow may be due to extravascular rearrangement and ECM changes, as previously reported (reference 66). In line with this hypothesis, recent studies have shown that flow conditioning reduces cathepsin proteolytic activity and prolongs the stability of in vitro microvessels (reference 67). Flow-conditioned vessels, in contrast to static vessels, showed a significantly reduced extravascular diffusion rate and increased overall matrix / tissue stiffness (Figure 6). Despite evidence suggesting that the presence of stromal cells may contribute to tissue hardening (reference 28), the flow-dependent increase in stiffness is not attributable to changes in stromal cell number, as stromal cells maintain a stable state over time (approximately 30% of total cells) (Figure 6E). Immunostaining of proteins within the EVM revealed that flow promotes the deposition of collagen I, fibronectin, and laminin (Figures 7A-B). Several studies have reported the effects of shear stress on the control of matrix deposition and remodeling (references 68-70), but none have reported on this for placental tissue. The placental stroma and basement membrane are rich in collagen, fibronectin, and laminin, and their expression increases as pregnancy progresses, supporting the tissue structure and function during development (reference 71). The low expression of these ECM proteins observed in the absence of fluidity may explain their increasing destabilization over time. Supporting this, a recent study reported that endothelial cells seeded on a matrix derived from villous stromal fibroblasts, in which clinically observed fetal growth retardation (decreased expression of collagen I and fibronectin compared to controls), showed impaired proliferation and migration, suggesting that matrix composition is crucial for the development of fetal placental blood vessels (reference 72). Further examination of the gel / tissue composition using mass spectrometry revealed an abundance of EVM-related proteins deposited by cells during the culture period (Figure 7C).As expected, tissue protein enrichment was affected by the presence of flow, revealing changes in protein expression related to EVM tissue and vascular homeostasis. Consistent with immunofluorescence results, MS analysis revealed laminin enrichment in response to flow. This indicates flow-induced adaptations and changes in cell matrix adhesion, ultimately leading to matrix remodeling. Furthermore, multimelin 2 (cited 73, 74), an EVM molecule that controls permeability by enhancing vascular stability and stabilizing endothelial junctions, was found to be abundant in flow-conditioned tissue, suggesting a protective function that maintains vascular function and stability under mechanical shear stress. A substantial increase in prothrombin (cited 75), which functions as a precursor to thrombin and promotes fibrin formation, provides further evidence of flow-induced enhancement in EVM stiffness and subsequent matrix stabilization. Flow conditions also depleted nicotinamide phosphoribosyltransferase (NAMPT), an essential coenzyme that plays a crucial role in energy production, DNA repair, and signaling pathways. NAMPT overexpression is associated with inflammatory processes and the development of various human diseases, including acute lung injury, atherosclerosis, and cancer (reference 76). While direct evidence for the specific effects of fluidity on NAMPT expression is lacking, our findings suggest that this protein is mechanosensitive and exhibits a potential protective role against the mechanical forces of fluidity. Furthermore, fluidity affects actin dynamics, as indicated by decreased levels of profilin and fascin in fluid-conditioned tissues. Based on previous reports (references 77-79), we propose that depletion of these actin-bundling proteins may alter the structure of the cytoskeleton and inhibit cell migration. Consequently, once perfusing angiogenic tissue is formed, the lack or reduction of cell motility plays a crucial role in ensuring the functionality and stability of the fetal-placental vascular barrier.

[0091] The inventors investigated the flow in the formation of fetal placental microvessels, made possible by their 3D model. While their three-cell culture system is more physiological than conventional methods, the intermittent flow applied only partially replicates the hemodynamic forces experienced by the in vivo villous capillary network (references 19, 80). Furthermore, their model lacks the branched villous structure and crucial epithelial layer (trophoblast) that form a barrier between maternal blood and fetal capillaries. Trophoblast cells are mechanosensitive to shear stress and release angiogenic factors involved in regulating placental angiogenesis and morphogenesis (reference 81). While the role of trophoblast cells in maternal vascular remodeling processes has been extensively studied, their influence on fetal villous development remains largely unknown. Integrating trophoblast cells into the inventors' system resulted in the formation of a maternal-fetal interface.

[0092] Placental neovascularization disorders are closely related to pregnancy pathology, but our understanding of these disorders is limited due to the lack of suitable models. Both animal models and human transplant tissue pieces have limitations, and recent in vitro models lack functional vascular systems. To address this problem, we present a 3D chip-based model of human placental terminal villi, including fetal mesenchymal tissue and vascular endothelium. Fully perfusible fetal microvessels are generated by co-culturing HUVECs, placental fibroblasts, and pericytes in a microfluidic chip fitted with a fluid reservoir. By controlling the pressure gradient throughout the microtissue, fluidity has been shown to play a crucial role in the growth and remodeling of fetal placental microvessels. Fluidity conditioning leads to the early formation of an interconnected placental vascular network, which maintains its viability for extended periods (more than two weeks) during culture. Increased fluidity leads to increased vessel diameter and simultaneously increases the shear stress acting within the vessels (approximately 1.5 Pa), enhancing barrier function. Furthermore, as predicted by computational fluid dynamics simulations, the shear force measured in the interstitial space (approximately 0.3 Pa) leads to increased stiffness, deposition of associated proteins, and decreased diffusivity, all of which contribute to significant remodeling of placental blood vessels. Overall, the model of the present invention is expected to provide a means of inferring complex in vivo parameters, such as shear stress, on developing neovascular placental tissue, and to provide insights into vascular-related pregnancy disorders. Moreover, it suggests that fluid dynamics plays a crucial role in controlling fetal vascular development and remodeling, and that poor or restricted fluid conditions can adversely affect fetal-placental angiogenesis. Our findings will form the basis for further research into the mechanisms of placental vascular defects in pregnancy-related disorders associated with insufficient fluid flow.

[0093] Our in vitro placental vascular model is crucial for understanding the mechanisms underlying fetal placental angiogenesis and regulation. It allows for the direct measurement of physical and chemical signals, such as shear stress and corresponding cytokines and chemokines, in a controlled in vitro environment, mimicking parts of complex in vivo tissues. This model holds promise for studying pregnancy-related disorders and can provide insights into fetal vascular pregnancy disorders and vascular-related pregnancy disorders. A more general approach, including a barrier function version with a soluble gel, allows for the testing of cellular barrier functions related to other conditions or diseases, such as cancer, using the model.

[0094] This invention provides a unique device that offers a maternal-fetal interface model and a mammary duct model on a chip in both healthy and diseased states, and provides a method for drug screening / targeted therapy and / or toxicity / safety testing.

[0095] This invention relates to the following items:

[0096] Item 1. a) Elongated central culture channel for holding mammalian cells within a suitable gel, b) at least one first culture medium channel, at least a portion of which is positioned parallel to a first side of an elongated central culture channel, and at least one second culture medium channel, at least a portion of which is positioned parallel to a second side opposite to the elongated central culture channel, configured to allow lateral flow connections from at least one first culture medium channel to at least one second culture medium channel via the central culture channel, and c) At least one third culture channel, which extends parallel to the third side of the elongated central culture channel, Here, optionally, at least one third medium channel is filled with a soluble gel barrier suitable for preventing cells from entering at least one third medium channel. A microfluidic device equipped with the following features.

[0097] The presence of a soluble gel barrier allows the cell-gel mixture to be contained within a central channel, and as the gel dissolves, it can form a monolayer of adjacent cells.

[0098] Preferably, the microfluidic device according to the present invention is advantageously a postless system, which simplifies manufacturing and allows direct contact between the culture medium channel and the gel, i.e., no physical barrier is provided.

[0099] Item 2. The microfluidic device according to Item 1, comprising a fourth channel that at least partly extends parallel to a fourth side of an elongated central culture channel, and optionally the fourth channel is filled with a soluble gel barrier suitable for preventing cell entry into at least one third culture channel.

[0100] Item 3. A microfluidic device according to item 1 or 2, wherein at least one gel barrier comprises a temperature-dependent solubility gel.

[0101] Item 4. Mammalian cells in the culture chamber are selected from human cells and preferably include cells and / or cell lines derived from epithelial tissue having an epithelial layer, such as cancer cells and / or immortalized cell lines, vascular cells, respiratory cells, digestive cells, bladder cells, mammary gland cells, ductal cells, and their cancer cells. And preferably, a culture of three types of cells: human umbilical vein endothelial cells (HUVECs), human placental fibroblasts (HPFs), and human placental pericytes (HPPs), and a layer of human trophoblast cells located on the side of the cell culture that is in contact with at least one third or fourth culture medium channel. A microfluidic device as described in any one of items 1 to 3.

[0102] Item 5. a) Elongated central culture channel for holding human cells within a suitable gel, b) At least one first and second culture channel, wherein at least a portion of the first culture channel extends parallel to a first side of the elongated central culture channel, and at least a portion of the second culture channel extends parallel to a second side opposite to the elongated central culture channel, and is configured to allow lateral flow connections from the at least one first culture channel to the at least one second culture channel via the central culture channel, and the mammalian cells in the gel include three cultures of human umbilical vein endothelial cells (HUVECs), human placental fibroblasts (HPFs), and human placental pericytes (HPPs), A microfluidic device equipped with the following features.

[0103] Item 6. HPF and HPP are cultured with an endothelial cell:stromal cell (HPF / HPP) ratio of approximately 10:1, preferably with an HPF to HPP ratio of 1:1. A microfluidic device as described in item 4 or 5.

[0104] Item 7. The culture channel gel includes hydrogels such as fibrinogen / thrombin (fibrin) gel. A microfluidic device as described in any one of items 1 through 6.

[0105] Item 8. A microfluidic device according to any one of items 1 to 7, further comprising an additional medium reservoir, preferably a 3D-printed customized medium reservoir, connected to at least one of the medium channels.

[0106] Item 9. A microfluidic device according to any one of items 1 to 8, further comprising a barrier layer of cells, preferably trophoblast cells, located on either the first or second side of an elongated central culture channel.

[0107] Item 10. A microfluidic device according to any one of items 1 to 9, wherein the device is placed on a solid support such as a biochip or coverslip, and / or several or more devices are grouped on the solid support.

[0108] Item 11. A method for manufacturing a human 3D vascular microtissue model derived from the placenta, a) A step of providing a microfluidic device as described in items 1-4 and 8-10, b) The step of culturing the cells in the culture chamber in an appropriate culture medium, and c) Dissolving at least one of the gel barriers described herein and seeding appropriate mammalian barrier cells, Methods that include...

[0109] Item 12. A method for producing a human 3D vascular microtissue model derived from the placenta as described in Item 11, In (b), the three cultures were cultured for approximately 48 hours in a suitable culture medium under essentially static conditions. b1) By applying a hydrostatic pressure gradient of preferably about 20 Pa to about 70 Pa between at least one first culture channel and at least one second culture channel, and / or by applying an average lateral flow velocity of preferably about 0.20 μm / sec to 1.25 μm / sec across the central culture channel, a lateral flow (e.g., intermittent interstitial flow (IF) or luminal flow) is established across the central culture channel from at least one first culture channel to at least one second culture channel, and This lateral flow is established between approximately day 2 and day 7 of the three-type culture. method.

[0110] Item 13. A method for manufacturing a human 3D vascular microtissue model derived from the placenta, a) A process for providing a microfluidic device as described in items 5 to 10, b) A step of culturing the three cultures in a suitable culture medium for approximately 48 hours under essentially static conditions. c) A step of establishing a lateral flow (e.g., intermittent interstitial flow (IF) or luminal flow) across the central culture channel from at least one first culture channel to at least one second culture channel by applying a hydrostatic pressure gradient of preferably about 20 Pa to about 70 Pa between at least one first culture channel and at least one second culture channel, and / or by applying an average lateral flow velocity of preferably about 0.20 μm / sec to 1.25 μm / sec across the central culture channel, The lateral flow is established between approximately day 2 and day 7 of the three cultures, and d) Optionally, a step to test the perfusion rate following step c), Methods that include...

[0111] Item 14. The method according to Item 12 or 13, wherein the hydrostatic pressure gradient (ΔP) established in step c) is less than approximately 10 mmH2O, preferably about 2 to 7 mmH2O.

[0112] Item 15. The method according to any one of items 12-14, wherein the hydrostatic gradient is 3 mmH2O or about 30 Pa, or the mean lateral flow velocity on day 2 is 0.30 ± 0.12 μm / sec.

[0113] Item 16. The method according to any one of items 12-15, wherein the flow rate is re-established daily by replenishing the reservoir medium volume for 5 days following step b).

[0114] Item 17. The method according to any one of items 12 to 16, further comprising the step of introducing a barrier layer of cells, preferably trophoblast cells, located on either the first or second side of an elongated central culture channel.

[0115] Item 18. The method according to any one of items 12 to 17, further comprising the step of detecting changes in the expression and / or quantity of a cell analyte, such as angiogenic cytokines and inflammatory cytokines, during step c) and / or step d).

[0116] Item 19. A placental-derived human vascular microtissue model manufactured by the method described in any one of items 11-18.

[0117] Item 20. A placental-derived human vascular microtissue model described in Item 19, showing laminar flow throughout the entire model.

[0118] Item 21. A placental-derived human vascular microtissue model, as described in Item 19 or 20, exhibiting epithelial barrier function.

[0119] Item 22. Use of a microfluidic device described in any of Items 1-5 and 8-10 or a placental-derived human 3D vascular microtissue model described in Item 21 for studying placental barrier function.

[0120] Item 23. Use of a microfluidic device described in any of Items 1-10 or a placenta-derived human 3D vascular microtissue model described in any of Items 19-21 to study trophoblast cell invasion, such as toxicological screening for substances and / or stress that inhibit trophoblast cell invasion.

[0121] Item 24. Use of a microfluidic device described in any of Items 1-10 or a placental-derived human 3D vascular microtissue model described in any of Items 19-21 to study placental branching angiogenesis, the effects of nutrients, hormones, and environmental factors on placental development, fetal vascular pregnancy disorders, and / or vascular-related pregnancy disorders.

[0122] Item 25. Use of microfluidic devices described in any of Items 1-10 or placental-derived human 3D vascular microtissue models described in any of Items 19-21 for studying epithelial barrier function and / or endothelial barrier function (solute permeability), e.g., drug screening, evaluation of novel drug targets for functional disorders such as pre-eclampsia, transport of solutes, contaminants, antibodies, immune cells and drugs across epithelial cell barriers and / or vascular cell barriers, and extravascular matrix properties (diffusivity, stiffness, and matrix proteins), as well as for studying the effects of interstitial flow and / or lateral flow and / or luminal flow on studies of epithelial barrier function and / or endothelial barrier function (solute permeability) and extravascular matrix properties (diffusivity, stiffness, and matrix proteins).

[0123] The present invention will be described in more detail in the following embodiments with reference to the accompanying drawings, but will not be limited thereto. For the purposes of the present invention, all references cited are incorporated herein by reference in their entirety.

[0124] Table 1 shows a quantitative analysis of the simulation results, referring to Figure 5. Surface averaging is applied to estimate the velocity and shear stress (mean ± SD) within the regions of blood vessel + gel (tissue), blood vessels only, and gel only. [Table 1] [Examples]

[0125] Materials and methods

[0126] cell culture

[0127] Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and cultured in endothelial medium (VascuLife, Lifeline cell systems) on T75 or T150 flasks coated with 50 μg / ml rat tail collagen I (Roche). HUVECs were transduced to stably express cytoplasmic RFP (LentiBrite RFP Control Lentiviral Biosensor, Millipore Sigma Aldrich) and used from passage 6 to 9. Primary human placental fibroblasts (HPFs) were purchased from ATCC and cultured in fibroblast medium on uncoated T75 flasks (FibroLife, Lifeline cell systems) and used from passage 4 to 8. Unlabeled and GFP-labeled primary human placental microvascular pericytes (HPPs) were obtained from Angioproteomie and cultured in pericyte growth medium according to the manufacturer's protocol and used from passage 6 to 9. All cells were cultured in a standard incubator under normal conditions of 37°C and 5% CO2, dissociation was performed using TrypLE Express (Gibco), and the culture medium was completely refreshed every other day.

[0128] Device manufacturing

[0129] The devices were manufactured as previously described (reference 25). Briefly, PDMS (SYLGARD™ 184 Silicone Elastomer Kit, Dow) was mixed with elastomer in a 10:1 ratio according to the manufacturer's protocol, degassed, and poured into molds. After further degassing, the PDMS was left in a 60°C oven overnight. Individual devices were cut out, holes were punched with 1 mm and 2 mm diameter biopsy punches for the gel port and culture medium port, respectively, and the glass slides were cleaned using air-plasma bonding (Harrick systems). The assembled devices were incubated overnight at 60°C to return to their original hydrophobic state. All devices were UV sterilized for at least 30 minutes before cell seeding.

[0130] Device seeding and microangiogenesis under interstitial flow

[0131] Bovine plasma-derived fibrinogen (Sigma) was reconstituted in phosphate-buffered saline (PBS) before use to a working concentration of 7 mg / ml. Thrombin (Sigma) stock solution (100 U / ml in 0.1% w / v bovine serum albumin solution) was diluted to a 4 U / ml working solution in cold VascuLife medium. Endothelial and stromal cells were cultured to near confluence before separation and resuspended separately in thrombin to concentrations of 24 million endothelial cells / ml and 2.4 million stromal cells / ml (combination of fibroblasts and pericytes). The cell suspensions were mixed in a 1:1 volume ratio and then mixed with fibrinogen solution in a 10:1 ratio within fibrin (3.5 mg / ml) to produce final concentrations of 6 million endothelial cells / ml and 600,000 stromal cells / ml. The cell-gel mixture was injected into the central channel of the device and polymerized in a humidified chamber at 37°C for 15-20 minutes. VascuLife medium was added to each medium channel (150 ul total), and the devices were initially cultured under static conditions. By adding a 3D-printed, customized medium reservoir (Form3 printer, Formlabs), intermittent interstitial flow (IF) across the hydrogel channels was generated 48 hours after seeding. Equal volumes of medium were added differentially to the reservoir compartment to generate three hydrostatic pressure gradients (ΔP) of 0, 3, and 7 mmH2O. For 5 days after seeding, the flow was re-established daily while replenishing the medium volume in the reservoir. From day 8 onward, the vessels became fully perfusing, and the hydrostatic pressure gradient was restored every other day.

[0132] Imaging and quantification of vascular morphology

[0133] All images were acquired using a Stellaris 8 confocal microscope with LAS X software (Leica). Confocal z-stacks were acquired at all time points and used to quantify the morphology of microvascular networks cultured under different ΔP conditions, as previously described (reference 25). Briefly, a custom macro (ImageJ, NIH) was generated, and the images were processed as follows: projection of the maximum intensity of the RFP channel in the z direction, smoothing with a Gaussian filter, removal of outliers, and conversion to a binary image. Finally, using particle analysis and 2D skeletonization with ImageJ built-in plugins, branch density, network connectivity, and effective diameter (vascular area / total length) were determined. A label-free alternative method is also available, as described in reference 98.

[0134] Permeability measurement

[0135] Microvessels cultured under different flow conditions were perfused with 70 kDa FITC-labeled dextran (Merck) on day 7 or day 14 by applying hydrostatic pressure reduction across the central gel channel. Briefly, the culture medium was removed and replaced with 40 μl of fluorescent perfusion solution (FITC 0.1 mg / ml) added to one channel. After perfusion of the microvessels, convection was stopped by injecting an equal volume of dextran solution into the opposite channel. After stabilization (approximately 2-3 minutes), time-lapse confocal images were acquired (3x5 minute intervals), and permeability was measured based on these images as previously reported (reference 25).

[0136] Cytokine release

[0137] Supernatants were collected on days 3, 5, and 7 from n=3 device reservoirs for each ΔP condition of two or three independent experiments and frozen until use. VEGF, Ang-2, and MCP-1 concentrations were measured from individual samples according to the manufacturer's protocols for each Quantikine ELISA Kit (R&D Systems, DVE00, DANG20, and DCP00). Cytokine expression profiles in 2D cultures were evaluated using a human angiogenesis antibody array (Abcam). Supernatants of lung fibroblasts, placental fibroblasts, and pericytes were collected 48 hours after seeding and processed according to the manufacturer's instructions. Cytokine profiles on the array membrane were detected by chemiluminescence using Fusion FX Spectra (Vilber, France). Relative (semi-quantitative) intensity expression was standardized relative to the positive control.

[0138] Fluid dynamics property evaluation using bead tracking

[0139] The fluid velocity of statically and fluidly conditioned vessels was evaluated on day 7 by perfusing the microvascular network with 2.0 μm fluorescent beads (Fluorescent blue latex beads, Sigma Aldrich) using a reservoir with a 5 mmH2O pressure gradient. Time-lapse images (1024 x 1024 pixels, resolution 0.64 μm / pixel, frame rate 0.06 sec) over 25 seconds were acquired using a fluorescence microscope (Thunder Imager - DMi8 microscope, Leica). Tracking of individual beads (distance over time) was performed using the particle tracking plugin TrackMate (NIH ImageJ) (reference 25). First, individual beads were detected based on size using a "LoG detector" scheme with an "estimated blob diameter" of 5 μm and an intensity threshold of 2 AU. Next, a "Simple LAP Tracker" scheme is used with a "Maximum Link Distance" of 15 μm, a "Maximum Gap Close Distance" of 15 μm, and a "Maximum Frame Gap Close" of 2 frames to identify the same object over time. After applying a "Track displacement" filter to easily remove objects that are not moving, the "Analyze" function is used to obtain the average bead velocity.

[0140] Numerical fluid dynamics simulation

[0141] To estimate the range of fluid velocity and shear stress generated by the application of pressure-induced flow within microvessels and surrounding gel domains, binary masks were obtained from confocal microscope images and converted to .dxf format in Adobe Illustrator 2021. These .dxf files were imported into COMSOL multiphysics software (version 6.0). Using the laminar flow physics module with porous domain-compatible physics settings, spatial profiles of lumen and interstitial fluid velocity and shear stress were calculated for an applied pressure of 70 Pa (pressure drop ΔP, 7 mmH2O) between the inlet and outlet of four microfluidic chips, including placental microvessels. A computationally efficient, ultra-fine mesh with physical control was applied to the model, resulting in an overall computation time of approximately 2 hours.

[0142] Diffusion coefficient measurement and initial fluid velocity tracking

[0143] Diffusivity was evaluated in the extravascular space (adjacent to but outside of blood vessels observable in the image) under each flow condition using FRAP measurements with a Stellaris 8 confocal microscope (Leica). The device was perfused with 70 kDa FITC-labeled dextran and incubated at 37°C for 12 hours to allow complete diffusion throughout the gel matrix (extravascular space). A small area (30 μmφ) within the matrix was photobleached, and time-lapse images were recorded every 0.4 seconds to capture fluorescence recovery immediately after photobleaching. Diffusivity analysis was performed using the Matlab frap_analysis plugin (reference 27), as previously described (reference 28). Similarly, the inventors measured the initial IF rate (day 2 after seeding) using FRAP, as previously described (reference 16). Briefly, a pressure gradient was generated throughout the device containing the gel and cells by adding FITC dextran to the corresponding reservoir medium volume. After dextran saturation, a 30 μmφ region was bleached, and sequential time-lapse images were collected to observe the recovery of fluorescence along the direction of fluid flow. IF velocity was estimated using the Matlab frap_analysis plugin, which tracks the movement of the centroid of the bleached region.

[0144] Evaluation of microtissue hardness

[0145] The mechanical resistance of fetal placental angiogenesis microtissues cultured under different flow conditions was measured on day 7 using a Chiaro Nanoindenter (Optics 11, Amsterdam, Netherlands) as previously described (cited reference 28). Briefly, the gel was excised from the device and extracted, placed in a Petri dish, and completely immersed in VascuLife medium. Nanoindentation measurements were performed using a spherical probe tip with a radius of 25 μm and a stiffness of 0.027 N / M. After properly adjusting the probe, an indent of 12 μm depth was applied to the gel. The value of the effective Young's modulus was derived from the load-indentation curve by fitting to the standard Hertz model using the manufacturer's data analysis plugin, assuming a Poisson's ratio of 0.5. In two independent experiments, an average of ≈80 measurements were performed for each condition.

[0146] Flow cytometry

[0147] Characterization of the stromal cell population was performed by flow cytometry on days 3 and 7 after seeding. To extract cells from the device, the gel was excised and digested with a solution of Accutase (Gibco) and 50 FU / ml nattokinase (Japan Bioscience Ltd) at 37 °C for 15 - 20 minutes. An average of 6 gels were pooled at each time point. Then, single cells were stained with the pericyte marker CD140b PerCP-Cy™ 5.5 (BD Bioscience) at 4 °C for 2 hours and washed with PBS. Analysis was performed on a BD LSR II and then processed with FlowJo v.10.8.1 software. The stromal cell population was gated by excluding cell clusters (expressed only in HUVECs) using RFP + and CD140b + .

[0148] Immunostaining

[0149] On day 7, the devices were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes. The samples were then permeabilized and nonspecific binding was blocked overnight at 4°C using an orbital shaker with 0.1% v / v Triton X-100, 5% w / v BSA, and 1% v / v serum (the same as the secondary antibody) in PBS. The primary antibody was diluted with 0.5% w / v BSA PBS and added to the samples overnight at 4°C using an orbital shaker. The primary antibodies used in this study were S100A4(FSP-1) (1:100, Abcam), Collagen I (1:200, Abcam), Fibronectin (1:200, Abcam), and Laminin (1:200, Abcam). The following day, the samples were washed with 0.1% v / v Triton X-100 PBS and incubated overnight at 4°C in an orbital shaker with a suitable secondary antibody (1:200, Alexa Fluor 488 or 647, Invitrogen) and DAPI counterstain diluted in PBS. The samples were washed with PBS and stored at 4°C before imaging. To effectively wash away stain and unbound reagents, all washing and incubation steps were performed under a pressure gradient across the gel.

[0150] Characterization of matrix-derived proteins by mass spectrometry

[0151] Trypsin digestion of hydrogel / tissue samples for mass spectrometry (MS) analysis was performed on day 7 post-seeding, as described by LASawicki et al. (reference 29). Two experiments were performed, each with at least two replicates per device under static or flowing conditions. Samples were first decellularized to remove cellular structure and protein contributions. Briefly, the extracted gel matrix was washed with PBS and washing buffer (100 mM Na2HPO4, 2 mM MgCl2, 2 mM EGTA), followed by incubation in lysis buffer (8 mM Na2HPO4, 1% NP-40) at 37°C for 90 minutes. After additional washing (300 mM KCl, 10 mM Na2HPO4), the decellularized gel was degraded with collagenase (Gibco, 50 U / mL in HBSS) at 37°C for 1 hour to release all remaining proteins. The gel was then vigorously pipettered and dissolved, and stored at -80°C until lyophilized (Labconco FreeZone 2.5). The lyophilized sample was reconstituted with 25 mM NH4HCO3, reduced with DTT, and alkylated for cysteine ​​residues by addition of iodoacetamide (reference 29). The protein was digested overnight with trypsin (Promega), and the sample was acidified with formic acid according to (reference 29). The digested sample was applied to a 10 kDa cutoff spin column concentrator (Corning) to remove trypsin and collagenase. The mixed peptide was subjected to a reverse-phase cleanup step (OASIS HLB 96-well μElution plate, Waters). The peptide was dried and reconstituted with 10 μl of 400 mM Hepes / NaOH, pH 8.5, and reacted with 80 μg of TMT18plex (Thermo Scientific) dissolved in 4 μl of acetonitrile at room temperature for 1 hour. The peptides were subjected to a reversed-phase cleanup step before analysis by LC-MS / MS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific).

[0152] For this purpose, peptides were separated using an Ultimate 3000 nano RSLC system (Dionex) equipped with a trapping cartridge (Precolumn C18 PepMap100, 5 mm, inner diameter 300 μm, 5 μm, 100 Å) and an analytical column (Acclaim PepMap 100, 75 x 50 cm C18, 3 mm, 100 Å) connected to a nanospray-Flex ion source. Peptides were loaded into the trap column at 30 μl / min using solvent A (0.1% formic acid) and eluted at 0.3 μl / min over 2 hours using a gradient of 2–80% solvent B (0.1% formic acid in acetonitrile) (all solvents were LC-MS grade). The Orbitrap Fusion Lumos was operated in positive ion mode with a spray voltage of 2.2 kV and a capillary temperature of 275°C. Full scan MS spectra in the mass range of 375–1,500 m / z were acquired in profile mode with a maximum injection time of 50 ms, AGC operated in standard mode, RF lens setting of 30%, and resolution of 120,000. Fragmentation was triggered with a 3-second cycle time for peptide-like features with charge states 2–7 during the MS scan (data-dependent acquisition). Precursors were separated using a quadrupole with a 0.7 m / z window and fragmented at a normalized collision energy of 34%. Fragment mass spectra were acquired in profile mode with a resolution of 30,000. The maximum injection time was 94 ms, and the AGC target was custom-set. Dynamic exclusion was set to 60 seconds.

[0153] The acquired data were analyzed using FragPipe (reference 30) and the Uniprot Homo sapiens database (UP000005640, ID9606, entry 20594, October 26, 2022), and common contamination was also included. The following modifications were considered: carbamide methyl (C, fixed), TMT18plex (K, fixed), acetyl (N-terminus, variable), oxidation (M, variable), and TMT18plex (N-terminus, variable). The mass error tolerance for full-scan MS spectra was set to 10 ppm and for MS / MS spectra to 0.02 Da. A maximum of two cleavage errors were tolerated. At the peptide and protein levels, at least two unique peptides with a false positive rate of less than 0.01 and a length of at least 7 amino acids were required (reference 31).

[0154] MS Data Processing

[0155] The raw output file from FragPipe (protein.tsv file, (cited 30)) was processed using the R programming language (ISBN 3-900051-07-0). Contaminants were removed, and only proteins quantified with at least two intrinsic peptides were included in the analysis. 333 proteins passed through the quality control filter. Log2-converted raw TMT reporter ion intensities were first normalized using the "removeBatchEffects" function of the limma package (cited 32) to remove batch effects, and then normalized using the vsn package (variance-stabilized normalization, (cited 33)). Differential expression of proteins was examined using the limma package. Repetition information was added as a factor in the design matrix given as an argument to the "lmFit" function of limma. Proteins were annotated as hits if the false discovery rate (fdr) was less than 5% and the fold change was at least 30%, and as candidates if there was no fold change threshold and the fdr was less than 5%. Hit proteins and candidate proteins were clustered into two clusters (method kmeans) based on the Euclidean distance between normalized TMT intensities divided by the 0 mmH2O data point.

[0156] statistics

[0157] Statistical significance was analyzed using one-way ANOVA with OriginPro v.9.85, followed by post-hoc Tukey tests for multiple comparisons. Differences were judged to be statistically significant at p<0.05. Unless otherwise specified, the data presented here are from at least two independent experiments using n≧3 devices, with at least three measurements performed per device.

[0158] As an alternative approach to animal experiments, the inventors propose a novel device and assay capable of measuring the movement of substances across the maternal-fetal interface. A method for culturing human fetal-like vessels on a microfluidic chip has been previously developed (reference 25), and currently, human umbilical vein endothelial cells (HUVECs) are co-cultured in the presence of stromal cells (fibroblasts and pericytes) to form perfusionable, interconnected microvessels in 5–7 days. Next, trophoblast cells are incorporated into the system to generate a crucial epithelial layer that forms a barrier between maternal blood and fetal capillaries (Figures 9, 12, and 13). In this embodiment, a monolayer of trophoblast cells was seeded adjacent to fetal vessels in a chip design established for the generation of the fetal vascular system (reference 25). This was done using two immortalized cell lines, BeWo and HTR-8 / SVneo, or primary isolated trophoblast cells (see Figure 13).

[0159] A general method for manufacturing a new preferred design according to the present invention is as follows: (1) Introducing a temperature-dependent solubility gel into the "matrix" channel of the device, (2) Seeding fetal microvessels into the "fetal" channel (general protocol), and (3) After about 48 hours, about 1.5 x 10 6 Seed trophoblast cells at a rate of cells / mL into the “maternal” channel (see Figure 9), or, in embodiments with three channels, into the third culture medium channel (see Figure 10).

[0160] To date, immortalized trophoblast cell lines BeWo and HTR-8 / SVneo, as well as primary isolated trophoblast cells (see Figure 13), have been tested.

[0161] The system of the present invention is initially set up as a maternal-fetal interface, but can be used for other tissues as well. For example, any tissue with an epithelial layer and a vascular system beneath it can be generated on the chip. This allows it to be used as a model for organs including the respiratory and digestive tracts, the bladder, or for specific (epithelial) cancer types such as breast cancer, particularly ductal carcinoma (DCIS).

[0162] Using HTR-8 / svNeo trophoblast cells, measurements over 20 minutes demonstrated impermeability to larger molecules (molecular weights of 70 and 4 kDa) and low permeability to 0.4 kDa dextran (Figure 9B, Figure 14A). Immediate permeability to glucose homolog (GFP-labeled 2-DG) was shown as expected (Figure 13C). To approximate pre-eclampsia (PE), oxidative stress (incubation at hypoxic concentration -1% O2) or incubation with a known PE-related circulating factor (sFLT-1) was used (Figures 10, 11, 14, 15). These treatments resulted in expected negative changes in both vascular density and trophoblast barrier function.

[0163] Manufacturing protocol for generating a 3-channel postless chip design (see Figures 12 and 13A)

[0164] As outlined by Cherubini and Haase et al. (reference 99), inverse molds were created according to the outlined design (Figure 12) by either 3D printing or laser cutting using biocompatible resin. For laser cutting, individual devices were cut from thin 0.5 mm acrylic sheets and bonded to larger, thicker (>1 mm) acrylic pieces with acetone. Once the mold was formed, polydimethylsiloxane (PDMS) with an elastomer-to-crosslinking agent ratio of 10:1 was prepared and cured within the mold to produce the devices. After 24 hours at 65°C, the PDMS was removed from the mold and an inlet hole was drilled, similar to Cherubini et al. (reference 99). The PDMS devices were bonded to clean glass coverslips with air plasma and left to return to a hydrophobic state before seeding cells (Figure 13A).

[0165] Methods for generating and characterizing maternal-fetal interface models

[0166] As previously established by Cherubini et al. (reference 100), human umbilical vein endothelial cells (HUVECs), human placental fibroblasts, and human placental pericytes were seeded into a central gel channel to establish a placental-specific microvascular system. First, the lower third channel was filled with a soluble gelatin solution (10% gelatin in PBS, solid below 37°C) to ensure that a hydrogel formed only in the central channel. Forty-eight hours after dissolving the gelatin at 37°C, a monolayer of *trophotrophoblast cells (HTR-8 / SVneo) was introduced into the lower channel (Figures 9 and 13B). A concentration of 800,000 cells / mL was used for the HTR-8 / SVneo cells. The device was tilted 90 degrees and cultured for 2 hours to promote the formation of a confluent monolayer. Interstitial flow across the central channel was established in a 3D resin-printed reservoir constructed 48 hours after seeding using a 7 mmH2O pressure gradient. The culture medium was changed daily, and the pressure gradient was re-established. Placental barrier function could be evaluated after 7 days of culture by perfusing the tissue with fluorescent dextran of various molecular weights, immunoglobulins such as IgG and IgM, insulin, and glucose (using the fluorescent analog 2-NBDG) (Figure 13). The permeability of fluorescent dextran and 2-NBDG was measured by perfusing these molecules into the maternal compartment and monitoring the fluorescence signal at the interface for approximately 12 minutes using a confocal microscope. Permeability values ​​were calculated as in the same manner as Shin et al. (reference 101) (Figure 13D). The permeability of immunoglobulins and insulin was measured by perfusing these molecules into the maternal compartment and evaluating the concentrations in the supernatant collected from the fetal compartment using an ELISA assay after 24 hours. * Primary trophoblast cells are isolated from full-term placental tissue fragments and form the epithelial barrier (Figure 13F-H). These cells are obtained after dissecting the chorionic villi and performing a series of digestive steps as described by Li and Schust et al. (reference 102).

[0167] A method for generating and characterizing a model of pre-eclampsia (PE) using the maternal-fetal interface:

[0168] The PE phenotype at the maternal-fetal interface on the chip was generated using various approaches. This included culturing the device under hypoxic conditions (1% O2) for up to 7 days, or perfusing the maternal compartment with PE-related factors such as soluble endoglin (sEng) and soluble fms-like tyrosine kinase 1 (sFlt1) at concentrations comparable to those found in the plasma of pre-eclamptic patients. For hypoxic conditions, all cells (HUVECs, stromal cells, trophoblast cells) were pre-treated to 1% O2 (or the target hypoxic concentration) before integration into the 3-channel microfluidic system. PE-related factors, e.g., sEng (30 ng / mL) and sFlt1 (50 ng / mL to match plasma levels), were perfused into the maternal compartment for 48 hours starting from day 5 of culture (Figures 14 and 15).

[0169] Measurement method on day 7 (or later)

[0170] Microvascular permeability was measured according to conventional methods (reference 100). Morphological parameters of microvessels (diameter, branch density, branch length, etc.) were measured by imaging on day 7 (measurements at other time points are also possible). Bright-field images were subjected to a machine learning pipeline for vascular segmentation and spatial mapping of morphometric parameters (reference 98) (Figure 15C). Trophoblast barrier permeability was measured by perfusing tissue with fluorescent dextran of various molecular weights and taking time-lapse images at the interface between the maternal and fetal compartments. Trophoblast permeability values ​​were calculated similarly to Shin et al. (reference 101). Alternatively, whether analytes (antibodies or small molecules) perfusing the maternal compartment pass through the trophoblast barrier can be measured by collecting them in the culture channel of the fetal compartment using an ELISA assay or LC-MS.

[0171] Outline of the method for generating a milk duct model on a chip

[0172] To create a mammary duct model (Figure 16), human mammary vascular endothelial cells (HMVECs) and human mammary fibroblasts (HMFs) were seeded into the central gel channel to establish mammary-specific microvessels, as previously established by Moccia et al. (reference 103). Alternatively, HMVECs can be replaced with iPSC-derived endothelial cells. After 8 hours, a monolayer of epithelial cells (MCF10) was introduced into the lower channel. MCF10 cells were used at a concentration of 50,000 cells / mL. Patient-derived epithelial cells can also be used with further optimization. The device was tilted 90 degrees for 1 hour to promote the formation of a confluent monolayer. Interstitial flow was established using a 3 mmH2O pressure gradient in the constructed 3D resin-printed reservoir 24 hours after seeding. *Optionally, tumor spheroids or patient-derived organoids can be inserted into the lower channel when filling the reservoir on day 1 after seeding. The culture medium was changed daily, and the pressure gradient was re-established. The volumes of culture medium in the reservoir chambers were 600 μL and 240 μL, respectively. Epithelial barrier function was measured by perfusing with dextran of various molecular weights after several days of culture (or after microvessels had been established).

[0173] Interstitial flow enables the formation and function of the placental microvascular network.

[0174] The inventors previously published a 3D in vitro model of terminal placental villous microvessels capable of reproducing several aspects of placental vascular disease (cited reference 25). Now, to more accurately approximate the physiological cellular composition of placental fetal tissue, the inventors have procured primary placental fibroblasts (HPF) integrated with endothelial cells (HUVECs) and placental pericytes (HPPs) to generate a microvascular network on a chip. The cells were cultured in a single gel channel PDMS device in a 3.5 mg / ml fibrin gel with an endothelial-to-stromal cell ratio of 10:1 (Figure 1A, in contrast to the previous 5:1 and 3 mg / ml (cited reference 25)). Differences in these cultures are partly due to differences in the types of secretions from stromal cells isolated from placental tissue and lung tissue, resulting in differences in angiogenesis profiles. In particular, inflammatory molecules such as PDGF-BB and MCP-1 are increased in placental fibroblasts compared to lung fibroblasts. The formation of a connected microvascular network occurs over approximately one week (Figure 1B) (in this new tri-culture). However, the vascular network becomes fully perfusing only when a 3D-printed culture medium reservoir is added to apply interstitial flow (IF) to the entire hydrogel (shown in the upper right of Figure 1A). Placental microvessels cultured under static conditions self-organize by day 4 but are not perfusing, and by day 7 they lose connectivity and the vessels are pruned.

[0175] Pressure gradients (ΔP) representing static (0 mmH2O), low-pressure gradient (3 mmH2O or approximately 30 Pa), and high-pressure gradient (7 mmH2O or approximately 70 Pa) culture conditions were applied to microvessels (Figure 1C), and the mean interstitial fluid flow velocities were measured on day 2 as 0.13 ± 0.06 μm / s, 0.30 ± 0.12 μm / s, and 1.23 ± 0.32 μm / s, respectively. On day 7, complete vascular connectivity was demonstrated in devices cultured under flow conditions with perfusion with FITC dextran, while partial perfusion was observed in the absence of FITC dextran (Figure 1C), which was quantified as perfused vascular area (Figure 1D).

[0176] During culturing, blood vessels form over several days, creating an open, perfusing lumen, and the flow transitions from interstitial to tubular over time. This transition was measured by a decrease in body fluid volume (approximately every 4 hours). The pressure gradient initially decreases exponentially, but by day 5, it decreases linearly over 24 hours. This suggests that at this point in the culture, an open lumen is present, and the flow has shifted from interstitial to primarily tubular (Figure 1E).

[0177] Interstitial flow promotes early angiogenesis in the placenta.

[0178] Placental microvessels were quantitatively evaluated for their corresponding morphological and functional barrier properties, as daily exposure to interstitial flow increased their perfusionability. Vascular development was monitored at specific time points using confocal microscopy under static and flowing conditions (Figure 2A). In the early stages of culture (day 3), IF-conditioned vessels showed significantly increased vascular network connectivity (Figure 2B), decreased branch density (Figure 2C), and increased vessel diameter (Figure 2D). By day 7, connectivity between flowing and static conditions remained unchanged. At subsequent time points, branch density decreased in all cases compared to day 3 under the same conditions (Figure 2C). Under both conditions, diameter continued to increase over time (from day 3 to day 7), but flow-conditioned microvessels were significantly larger in diameter compared to statically cultured vessels (Figure 2D).

[0179] Next, on day 7, we perfused with 70 kDa FITC dextran to investigate the effects of static or flow conditioning on endothelial barrier function. In vessels cultured under high flow conditions, permeability values ​​were significantly reduced (increased barrier function) (Figure 2E). This indicates that intermittently applied pressure gradients are sufficient to influence the endothelial phenotype over time.

[0180] Interstitial flow promotes inflammatory signaling in placental vascular development.

[0181] While vascular formation and remodeling are closely related to inflammation and growth factor signaling, little is known about signaling in human fetal placental angiogenesis. Therefore, we investigated the release of various cytokines from statically and fluidly conditioned placental microvessels. Inflammatory cytokines such as interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1), as well as the pro-angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2), were quantified by ELISA (Figure 3). Generally, fluid conditioning increased inflammatory signals, particularly IL-8, at all time points, while MCP1 levels decreased over time (this was observed in both conditions). Ang-2 was also upregulated in fluidly conditioned vessels, and VEGF levels increased significantly over time in statically cultured vessels (Figure 3D).

[0182] Fluid flow maintains the stability of placental microvessels.

[0183] The inventors investigated whether the structure and functionality of placental microvessels could be maintained during long-term culture (>7 days) under fluid conditioning. Confocal images acquired 14 days after seeding showed a clear difference in vascular coverage between static and fluid conditions (Figure 4A). In statically cultured microvessels, the area coverage rate decreased significantly (Figure 4B), and the effective diameter also narrowed accordingly (Figure 4C). Branch density and branch length did not change between static and fluid conditions (Figures 4D, E).

[0184] Perfusion with 70 kDa FITC dextran on day 14 allowed for the characterization of barrier function in the later stages of culture. Leakage was observed to decrease at higher pressure gradients (7 mmH2O) compared to 3 mmH2O. While the permeability of statically cultured vessels was generally low, in this case only a small portion of the vessels were perfused (see Figure 4G). Over time, vessel area and barrier function decreased compared to earlier time points (see orange dots on day 7 in Figures 4H and 4F). Overall, flow conditioning resulted in increased vessel coverage and enabled the culture of connected vessels even after 3–4 weeks.

[0185] Characterization of velocity and shear stress distributions in placental microvessels

[0186] First, as an alternative to experimental measurement of flow velocity within placental microvessels, fluorescent beads were introduced into the microvessels on day 7 and tracked using a time-lapse microscope. In the flow-conditioned vessels, the average flow velocity increased significantly, corresponding to the increase in effective diameter (Figure 2C).

[0187] Next, to accurately map the velocity and shear stress distribution within microvessels, large sections of a 70kDa FITC dextran perfusion device were imaged and converted into binary masks for import as vectors into computational fluid dynamics (CFD) software. Using COMSOL and the Brinkman equations physics module for flow through porous media, both vascular and extravascular regions were modeled as separate regions. Vessels were treated as open pores with permeability of 1, and simplified fluid parameters were used for water (density = 1000 kg / m³). 3 , viscosity=1e -4 Pa*s) and the extravascular matrix (EVM) has a porosity of 0.3 and a water permeability of k=1e -15 m 2 The gel (density = 985 kg / m³) 3 , viscosity=1e -2It is treated as Pa*s), and the fibrin parameters are obtained from the following references [references 33, 34]. Note: The water permeability of cell-free fibrin gel is approximately k=1e -13 m 2 However, in our simulation, we considered cellular components (placental fibroblasts and pericytes) and estimated k=1e -15 m 2 The following was used. A pressure gradient (50 Pa or 5 mmH2O) was simulated across the gel region and set as an intermediate value of 3–7 mmH2O used in the experimental setup. From these simulations, both the velocity and shear stress distributions are predicted (Figure 5). The preferred flow pattern is shown to be heterogeneous and dependent on lumen formation at the interface between the gel and the culture medium channels.

[0188] Interstitial flow alters the biophysical properties of placental microvascular tissue.

[0189] The extracellular matrix (ECM) contains hollow blood vessels and provides the basic structure for remodeling. The ECM provides mechanical and chemical signals that contribute to the formation of the vascular network and barrier integrity (reference 35). The inventors previously demonstrated that significant changes occur in tissue stiffness and diffusion properties in response to co-culture with specific fibroblasts (reference 28). In this study, we evaluated whether these tissue-level changes can be promoted by flow conditioning alone. First, the inventors examined the diffusion rate in the extravascular region of blood vessels cultured under static conditions or flow conditions on day 7. FRAP measurements (Figure 6A) were performed to calculate the diffusion coefficient. In blood vessels cultured under flow conditions, the diffusion rate in the extravascular region was significantly reduced (Figure 6B).

[0190] Next, the inventors evaluated the effect of fluid conditioning on the stiffness of microvascular tissue. As shown in the schematic diagram in Figure 6C, the tissue was exposed on day 7, and the apparent Young's modulus was evaluated by nanoindentation. Microvascular tissue cultured under fluid conditions was significantly stiffer than that under static conditions (Figure 6D), and the stiffness depended on the magnitude of the pressure gradient (7 mmH2O vs. 3 mmH2O).

[0191] Because ECM is dynamic and constantly undergoes remodeling, activating cell proliferation and tissue morphogenesis (reference 36), the inventors evaluated whether changes in tissue-level properties were associated with changes in stromal cell populations over time. For this purpose, cells were extracted from hydrogel matrices of statically and flow-conditioned devices seven days after seeding, and the cell composition over time was compared. Cells were analyzed by flow cytometry daily, but no differences were observed in the ratio of endothelial cells to stromal cells (Figure 6E).

[0192] Flow promotes the deposition and remodeling of ECM proteins.

[0193] The increase in tissue stiffness was consistent with a decrease in the diffusion rate in the extravascular region of the placental vessels under flow conditioning. Since many ECM proteins have been identified in the developing placenta, the inventors then investigated the effect of blood flow on protein production. After 7 days of culture, microvessels were fixed and stained for type I collagen, laminin, and fibronectin (Figure 7A). Normalized mean intensities measured compared to static conditions indicated that all ECM proteins were more pronounced under flow conditions (Figure 7B). To verify these findings, the inventors performed mass spectrometry on the containers after 7 days of culture to measure changes in matrix composition under static and flow conditions. Specifically, individual samples from each condition were labeled with tandem mass tags (TMT), then multiplexed, and finally measured in mass spectrometry-based experiments. As a result, the inventors identified a total of 804 proteins, of which 333 were quantified. Based on gene ontology classification, many of these proteins showed associations with angiogenesis, extracellular matrix (ECM) composition, and organization (Figure 8A). For each protein, the multiplicative change in expression between conditions was calculated, and the ratio of protein expression was obtained by dividing the multiplicative change value of the flow-conditioned sample by the corresponding multiplicative change value of the static control. Furthermore, PCA results showed that the sample replicates under static and flow conditions were spaced and clustered, consistent with the experimental conditions. In this way, the inventors identified 26 proteins as hit proteins and candidate proteins, and clustered them into six different groups based on similarity in expression and behavior under different conditions (Figure 8B). Several hits and candidates from the protein list were found to be related to ECM composition and remodeling. For example, the matrix component laminin (LAMC1) appeared to increase significantly under flow conditions (Figure 8C), indicating major protein deposition due to fluid-mechanical stress.On the other hand, proteins known to regulate ECM stability, such as interalphatrypsin inhibitor heavy chain H2 (ITIH2) and pentraxin 3 (PTX3) (references 37, 38), showed decreased expression (statistically significant for ITIH2), suggesting matrix destabilization and subsequent remodeling caused by fluid f.

[0194] Generate a maternal-fetal interface model on a chip.

[0195] Based on the development and characterization of perfusing fetal placental microvessels (reference 100), the inventors further developed a model including a trophoblast layer representative of the maternal-fetal interface. This required the design of a novel device with a central gel channel and three independent culture medium channels. Each channel is separated only by a reduced wall that acts as a phase guide (Figures 12 and 13). By including a soluble gel (gelatin) in the third channel (without a mirroring channel), cells and fibrin hydrogel are seeded within the frame of the central channel (Figure 13). Similar to (reference 100), human umbilical vein endothelial cells (HUVECs), human placental fibroblasts, and human placental pericytes were seeded into the central gel channel to construct placenta-specific microvessels. Subsequently, after the gelatin had dissolved, trophoblast cells were added to the third channel 24–48 hours later. HTR-8 / SVneo is shown being introduced into the lower channel and forming a dense monolayer. 48 hours after sowing, interstitial flow was established in a 3D resin-printed reservoir using a 7 mmH2O pressure gradient.

[0196] This design allows for separate perfusion of the fetal and maternal compartments, enabling the simultaneous growth of two key barriers representing placental transport: the trophoblast layer and the fetal-placental microvessels beneath it.

[0197] Characteristics of the healthy mother-fetus interface

[0198] To assess whether the maternal-fetal interface functions as expected in vivo, several assays were performed, including evaluation of the trophoblast barrier's ability to restrict or allow the transport of known solutes. Normal trophoblast barrier function was assessed by perfusing maternal channels with a glucose analog (fluorescent 2-NBDG) after 7 days of culture. As expected (reference 104), 2-NBDG readily passed through the trophoblast barrier (Figure 13C). Next, the permeability of the trophoblast barrier was assessed by perfusing maternal channels with fluorescent dextrans of various molecular weights. The trophoblast barrier was able to restrict the transport of molecules larger than 1 kDa (Figure 9B), exhibiting a size-dependent effect on transport, with reduced transport of larger molecules (Figure 13D).

[0199] To further support the placental physiological barrier function, immunoglobulins including IgG and IgM, along with insulin, were perfused into the maternal compartment, and transport after 24 hours was evaluated by ELISA analysis of the collected supernatant (Figure 13E). As expected, transport was limited, and this trend was particularly pronounced for insulin, which does not cross the maternal-fetal interface.

[0200] A similar trophoblast barrier can also be formed using primary trophoblast cells (isolated trophoblast cells that can fuse with syncytial trophoblast cells in vitro). This model demonstrates that primary trophoblast cells form the barrier (Figure 13F-H).

[0201] Mimicry of pre-eclampsia in a maternal-fetal interface model.

[0202] The inventors demonstrated, according to the timeline in Figure 14, that perfusion of soluble molecules associated with pre-eclampsia (sFLT-1 and sEng) or culture under hypoxic conditions (1% O2) disrupts normal trophoblast barrier function. Permeability (measured as fluorescent dextran flux) is measured under different treatment conditions (Figure 14B). Under hypoxic culture conditions, significant dysfunction of the trophoblast barrier is induced.

[0203] In addition to measuring trophoblast barrier function, microvessels were also evaluated under these conditions (Figure 15). Microvascular network imaging was performed 2 days after treatment or 7 days after 1% O2 culture. A machine learning pipeline was used to segment the microvascular network and spatially quantify morphological features (Figure 15C). Average changes in specific vascular features, such as mean vessel diameter, can also be reported (Figure 15D). Using this approach, morphometric data demonstrate the significant effects of all treatments. Hypoxia, sFLT-1, and sEng negatively affect the development of fetal placental microvessels. Furthermore, microvascular permeability can be measured, as previously (as shown in Figure 15). Hypoxia leads to complete microvascular regression and lack of perfusion, while sEng increases leakage from microvessels.

[0204] Use as a milk duct on a 3-channel model chip

[0205] In addition to generating a maternal-fetal interface using a postless three-channel design, the inventors also demonstrated its use as a ductal model on a chip to help measure transport through the ducts (Figure 16). Microvessels derived from mammary gland tissue (see cited reference 103) are encapsulated in the central channel, followed by the encapsulation of an epithelial layer. Epithelial barrier function can be measured regardless of the presence of tumors within the ductal channel and also shows responsiveness to hormones (composite levels of estrogen and progesterone during the follicular phase, see cited reference 103) and tamoxifen (Figure 16G).

[0206] Cited literature

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[0208] Terms in the drawings Fibroblasts Fibroblasts Pericytes Pericytes Placenta stromal cells Placental stromal cells stromal Stromal Day Day Seeding Seeding Reservoirs Reservoirs Formed Formed Perfusable microvessels Day 7 Placental vessels Dextran Perfused vessels Time hours Connectivity Ratio Branch density Diameter Permeability Area Branch length Vessel area Mask Pressure Velocity Shear stress Gel region Vessels region Bleach Post-bleach After bleaching Tissue gel dissection incision nanoindentation submerged in liquid Young's Modulus Stromal cells Stromal cells Collagen I Laminin Fibronectin Fluorescent intensity relative to 0mmH2O Peptide count Gene ID Function Angiogenesis Cell-matrix adhesion ECM costituent ECM components ECM organization ECM organization Response to mechanical stimulus variability Log2 ratio Log2 ratio -log10(pvalue) -log10(p-value) log2(fold change) log2(multiple change) Trophoblasts (trophoblast cells) Seeding of trophoblasts Microvessels Fibrin + cells EC coalescence EC coalescence Fetal side Capillary Vasculature Maternal side Trophoblast monolayer (a single layer of cells with a trophoblast membrane) Fetal vessels Fetal microvessels vessels blood vessels maternal fetal fetus +5 min +5 minutes +1h30min +1h30min Untreated SECTION SCALE scale DETAIL Details hydrogel (fibrin) 2-Barriers (bright-field image) Perfusion with glucose analogue Crosses trophoblast barrier Trophoblast permeability Fetal concentration Insulin 90% pure cytotrophoblasts Cytokeratin Cytotrophoblast cells Syncytiotrophoblast syncytiotrophoblast e-cadherin Primary-derived trophoblasts + / -treatments + / -processing Seeding Barrier assays Various MWs + / - small molecules + / - small molecules Trophoblast barrier function Permeability Hypoxia Pre-eclampsia (PE)-like treatments + / -treatments + / -processing Not perfusable Microvascular morphometrics pipeline pipeline Output morphologic features map morphologic features map Tortuosity Microvascular morphology Microvascular barrier function Mammary ECs Mammary ECs Mammary FBs Mammary FBs Epithelial cells hormones / drugs tumor epithelium Perfusion Image at barrier

Claims

1. A microfluidic device comprising the following: a) Elongated central culture channel for holding mammalian cells within a suitable gel, b) at least one first culture medium channel, at least a portion of which is positioned parallel to the first side of the elongated central culture channel, and at least one second culture medium channel, at least a portion of which is positioned parallel to the second side opposite the elongated central culture channel, configured to allow lateral flow connections from at least one first culture medium channel to at least one second culture medium channel via the central culture channel, and c) At least one third culture channel, which extends parallel to the third side of the elongated central culture channel, Here, optionally, at least one third medium channel is filled with a soluble gel barrier suitable for preventing cells from entering at least one third medium channel. Here, preferably, the device comprises a fourth culture medium channel, at least a portion of which extends parallel to the fourth side of the elongated central culture channel. Here, optionally, the fourth culture medium channel is filled with a soluble gel barrier suitable for preventing cell entry into at least one fourth culture medium channel, preferably at least one soluble gel barrier comprising a temperature-dependent soluble gel. Microfluidic devices.

2. The mammalian cells in the culture chamber are selected from human cells and preferably include cells and / or cell lines derived from epithelial tissue having an epithelial layer, such as cancer cells and / or immortalized cell lines, vascular cells, respiratory cells, digestive cells, bladder cells, mammary gland cells, ductal cells, and their cancer cells. And preferably, the culture includes three types of cells: human umbilical vein endothelial cells (HUVECs), human placental fibroblasts (HPFs), and human placental pericytes (HPPs), and a layer of human trophoblast cells located on the side of the cell culture that is in contact with at least one third or fourth culture medium channel. The microfluidic device according to claim 1.

3. A microfluidic device comprising the following: a) An elongated central culture channel for holding human cells within a suitable gel, b) at least one first medium channel, at least a portion thereof extending parallel to the first side of the elongated central culture channel, and at least one second medium channel, at least a portion thereof extending parallel to the second side opposite to the elongated central culture channel, configured to be laterally flow-connected from the at least one first medium channel through the central culture channel to the at least one second medium channel, Here, the mammalian cells in the gel include cultures of three types: human umbilical vein endothelial cells (HUVECs), human placental fibroblasts (HPFs), and human placental pericytes (HPPs). Microfluidic devices.

4. HPF and HPP are cultured with an endothelial cell:stromal cell (HPF / HPP) ratio of approximately 10:1, preferably with an HPF-to-HPP ratio of 1:

1. The microfluidic device according to claim 2 or 3.

5. The culture channel gel contains hydrogels such as fibrinogen / thrombin gel. A microfluidic device according to any one of claims 1 to 4.

6. The microfluidic device according to any one of claims 1 to 5, further comprising an additional culture medium reservoir, preferably a 3D-printed customized culture medium reservoir, connected to at least one of the culture medium channels.

7. The microfluidic device according to any one of claims 1 to 6, further comprising a barrier layer of cells, preferably trophoblast cells, arranged on either the first or second side of an elongated central culture channel.

8. The device is placed on a solid support such as a biochip, and / or several or many devices are grouped on the solid support. A microfluidic device according to any one of claims 1 to 7.

9. A method for manufacturing a human 3D vascular microtissue model derived from the placenta, a) A step of providing a microfluidic device according to any one of claims 1 to 8, b) A step of culturing cells in a culture chamber in an appropriate culture medium, and c) A step of dissolving at least one of the gel barriers described herein, and d) The step of seeding appropriate epithelial cells and / or epithelial mammalian barrier cells, A method including, Here, preferably in b), the three cultures are cultured for about 48 hours in a suitable culture medium under essentially static conditions. b1) By applying a hydrostatic pressure gradient of preferably about 20 Pa to about 70 Pa between at least one first culture channel and at least one second culture channel, and / or by applying an average lateral flow velocity of preferably about 0.20 μm / sec to 1.25 μm / sec across the central culture channel, a lateral flow (e.g., intermittent interstitial flow (IF) or luminal flow) is established across the central culture channel from at least one first culture channel to at least one second culture channel, and This lateral flow is established between approximately day 2 and day 7 of the three-type culture. method.

10. A method for manufacturing a human 3D vascular microtissue model derived from the placenta, a) A step of providing a microfluidic device according to any one of claims 3 to 8, b) A step of culturing the three cultures in a suitable culture medium for approximately 48 hours under essentially static conditions. c) A step of establishing a lateral flow (e.g., intermittent interstitial flow (IF) or luminal flow) across the central culture channel from at least one first culture channel to at least one second culture channel by applying a hydrostatic pressure gradient of preferably about 20 Pa to about 70 Pa between at least one first culture channel and at least one second culture channel, and / or by applying an average lateral flow velocity of preferably about 0.20 μm / sec to 1.25 μm / sec across the central culture channel, The lateral flow is established between approximately day 2 and day 7 of the three cultures, and d) Optionally, a step to test the perfusion rate following step c), Methods that include...

11. The hydrostatic pressure gradient (ΔP) established in step c) is approximately 10 mmH ２ It is less than 0 mmH, preferably about 2 to 7 mmH ２ O is, and / or Here, the lateral flow is 3 mmH ２ The pressure is 0 or approximately 30 Pa, or the mean lateral flow velocity on day 2 is 0.30 ± 0.12 μm / sec, and / or Here, the lateral flow is re-established daily by replenishing the reservoir medium volume for 5 days after step b). The method according to claim 9 or 10.

12. The method according to any one of claims 9 to 11, further comprising the steps of introducing a barrier layer of cells, preferably trophoblast cells, located on either a first or second side of an elongated central culture channel, and / or detecting changes in the expression and / or quantity of a cell analyte, such as angiogenic cytokines and inflammatory cytokines, during steps c) and / or d).

13. A method for manufacturing a human 3D vascular microtissue model derived from breast tissue, a) A step of providing a microfluidic device according to any one of claims 1, 2, or 8, b) A step of seeding human mammary vascular endothelial cells (HMVECs) or iPSC-derived endothelial cells and human mammary fibroblasts (HMFs) into elongated central culture channels in a suitable culture medium for approximately 8 hours under essentially static conditions. c) A step of introducing a monolayer of epithelial cells (MCF10 or other cell lines) into a third culture medium channel, optionally including the insertion of tumor spheroids or patient-derived organoids on day 1 after seeding. d) 3 mmH 24 hours after sowing ２ The process involves applying a hydrostatic pressure gradient of O to establish a lateral flow across the central culture channel from at least one first medium channel to at least one second medium channel, such as an intermittent interstitial flow (IF) or luminal flow, and e) Optionally, a step to test the perfusion rate after step d), Methods that include...

14. A placental-derived human 3D vascular microtissue model manufactured by the method of any one of claims 9 to 12, or a breast-derived human 3D vascular microtissue model manufactured by the method of claim 13, wherein the model preferably exhibits laminar or luminal flow throughout and / or epithelial barrier function.

15. For studying placental barrier function, use of a microfluidic device according to any one of claims 1 to 8 or a placental-derived human 3D vascular microtissue model according to claim 14, or A human 3D vascular microtissue model derived from breast tissue, as described in claim 14, is used to study ductal function.

16. For example, use of a microfluidic device according to any one of claims 1 to 8, or a placenta-derived human 3D vascular microtissue model according to claim 13, for studying trophoblast barrier function, such as toxicological screening of substances and / or stress that inhibit trophoblast cell infiltration.

17. Use of a microfluidic device according to any one of claims 1 to 8, or a placental-derived human 3D vascular microtissue model according to claim 13, or a breast-derived human 3D vascular microtissue model according to claim 14, for studying branching angiogenesis of the placenta or breast, breast cancer and associated angiogenesis, the effects of nutrients, hormones, and environmental factors on placental development or breast development, fetal vascular pregnancy disorders, and / or vascular-related pregnancy disorders.

18. Use of a microfluidic device according to any one of claims 1 to 8, or a placenta-derived human 3D vascular microtissue model according to claim 13, or a breast-derived human 3D vascular microtissue model according to claim 14, for studying epithelial barrier function and / or endothelial barrier function (permeability to solutes), for drug screening, for evaluating novel drug targets for functional disorders such as pre-eclampsia, for studying the transport of solutes, contaminants, antibodies, immune cells, and drugs across epithelial cell barriers and / or vascular cell barriers, and for studying extravascular matrix properties (diffusivity, stiffness, and matrix proteins), and in particular for studying the effects of luminal flow, interstitial flow, and / or lateral flow on epithelial barrier function and / or endothelial barrier function (permeability to solutes) and extravascular matrix properties (diffusivity, stiffness, and matrix proteins).

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