Culturing vascular Endothelial cells directly on microfluidic chips or with the help of porous membranes to form a monolayer barrier is one of the important methods to reconstruct blood vessels in vitro and simulate human vascular function.endothelial cells were attached to the inner surface of the microchannel and formed a continuous cell monolayer by specific surface treatment and cell culture techniques.in addition,by using porous membranes,researchers can create barriers with specific pore sizes and distributions that mimic the underlying structure of the vessel wall.These cells exhibit morphologies and functions similar to those in vivo,including the formation of tight junctions,which mimic the real vascular barrier,and can be used to study cell-cell interactions,vascular permeability,drug delivery,and vascular function under pathological conditions.This method allows cells to attach and grow,while also controlling the exchange of substances between cells。

In 2010,Ingber's team at Harvard University used a porous membrane made of PDMS combined with a microfluidic chip to simulate the anatomical structure of alveoli,and constructed a lung tissue chip with diastolic function^[27]。 the device consists of three PDMS layers arranged And irreversibly bonded to form two sets of three parallel microchannels,with the upper and lower channels separated by a sheet of self-fabricated 10µm thick PDMS elastic porous membrane.After permanent bonding,the PDMS etchant flows through the two side channels to selectively etch the film in the channels,forming two large side chambers.Vascular endothelial cells and alveolar epithelial cells were seeded on both sides of the porous membrane,and the structure was highly similar to the actual alveolar structure in anatomy,forming the alveolar-capillary barrier.Furthermore,physiological respiratory motion was successfully mimicked by applying a periodic vacuum to the side chamber,causing mechanical stretching of the PDMS membrane(Fig.2A).Cyclic mechanical strain was found to enhance nanoparticle uptake by epithelial and endothelial cells and stimulate their trafficking to potential microvascular channels.the research team used the lung chip model to further study,that is,to simulate a variety of normal lung functions(infection-induced immune cell recruitment,respiratory-induced nanoparticle absorption and related inflammation,interleukin-2(IL-2)-induced pulmonary edema,inflammation-induced pulmonary thrombosis,etc.)and the observed response to drug treatment^[28]。 In addition,Zhang et al.Constructed a human disease model on a lung chip based on this structure^[29]。 The human alveolar chip model co-cultures human alveolar epithelium,microvascular endothelium,and circulating immune cells under fluid flow to reproduce the main features of the alveolar-capillary barrier in normal disease.From this,the model can reproduce the process of SARS-CoV-2-induced lung injury and immune response At the organ level.at the same time,they also found that COVID-19 infection of lung tissue may induce pulmonary microvascular endothelial injury by activating human immune cells to release a large number of inflammatory factors.Using this model,they also conducted preliminary testing and evaluation of the efficacy of antiviral compounds(Figure 2B).the Vunjak-Novakovic group used a vascular monolayer on a porous membrane in combination with multiple organs to simulate multiple organ physiology^[30]。 in this system,mature human heart,liver,bone,and skin tissues and multiple iPSC-derived organs are connected by flow to form a cycle that mimics interdependent organ functions.each tissue was cultured In its own optimized environment,and a porous membrane-constructed endothelial barrier was integrated on Each organ unit.during 4 weeks of culture,these interconnected tissues maintained their molecular,structural,and functional phenotypes,reproducing the pharmacokinetic and pharmacodynamic profile of doxorubicin During normal culture(Figure 2C),confirming that vascular junctions and phenotypically stable mature human tissues may contribute to the clinical applicability of tissue chips。

in addition,microfluidic systems can precisely control the flow of fluid and simulate the shear force of blood flow on endothelial cells,which is essential for the study of hydrodynamic effects in vascular biology.Huang's group used an integrated multilayer microfluidic system to analyze umbilical vein endothelial cells cultured in the chip from phenotype to whole transcriptome sequencing under single flow shear stress and double flow shear stress^[31]。 Because the culture medium fluid can be controlled by the integrated micropump and microvalve In the chip,the direction and speed of the fluid can be accurately manipulated.Studies have shown that the expression of BMP4,BMPER,TEK,ADAM15,IL8,EDN1,ANGPT2 and CYR61 is sensitive to different fluid directions.in addition,the sequencing results showed that the RNA post-processing(alternative splicing)of NRG1 and IFI44 genes was different under different fluid conditions,which revealed that besides gene expression,RNA post-processing was also one of the strategies for endothelial cells to cope with mechanical environment。

Fabricating microfluidic chips by means of lithography and microfabrication and integrating porous membranes into them is one of the mainstream ways to form endothelial barriers in the field of organ chips because of its controllable integration process,good repeatability and convenient observation.However,the endothelial cell layer formed in this way is a two-dimensional monolayer,which does not have a three-dimensional lumen structure,and to some extent,it is difficult to reproduce the three-dimensional structure of blood vessels in vivo and the functions related to the three-dimensional structure.Moreover,due to the lack of extracellular matrix similar to the vascular structure in vivo,many physiological phenomena such as angiogenesis and sprouting are difficult to reconstruct and simulate on this model.Therefore,more and more attention has been paid to the techniques and methods that can controllably construct the three-dimensional lumen morphology of blood vessels in vitro。

Different from constructing a vascular endothelial cell monolayer on a two-dimensional plane or a porous membrane,a precise collagen or other matrix microstructure can be precisely and controllably manufactured on a silicon wafer or a PDMS mold by using a soft lithography technology and other micro-processing technologies,Removing the mold and encapsulating the gel to form the channel,vascular endothelial cells can be further seeded to form blood vessels and construct an in vitro vascular model.Through microfabrication techniques,researchers are able to create complex vascular network models,providing an important tool for in-depth understanding of the mechanisms of the vascular system and the development of new therapeutic approaches。

Based on this method,Zheng et al.Used a PDMS mold to prepare type I collagen gel with a groove structure^[32]。 the two collagen layers were sealed together to form a closed fluid structure by applying pressure inside the Plexiglas apparatus.microvascular networks were formed by culturing microchannels seeded with human umbilical vein endothelial cells into collagen.in which native type I collagen has the appropriate stiffness to allow high reproducibility of vascular microarchitecture and can also be remodeled by degradation and deposition of extracellular matrix.the morphology,mass transfer process,and long-term stability of endothelial cells are described based on the vascular network formed in a native collagen matrix;The angiogenic activity of the endothelium and the differential interaction with perivascular cells in collagen were elucidated;The nature of the nonthrombotic vascular endothelium and its transition to a prethrombotic state in response to inflammation was also demonstrated(Figure 3A).The platform can successfully reproduce these Microvascular phenomena in vitro,indicating that in vitro vascular network models based on this method have broad potential in cardiovascular biology and pathophysiology.He research group can conveniently construct various complex channel structures in hydrogel,such as branched chain,helix,serpentine and so on,by using the inherent crosslinking characteristics of hydrogel materials^[33]。 the authors attached human umbilical vein endothelial cells to the inner wall of the channel,forming a complete ring of cell layers along the entire length,while maintaining high survival and good propagation morphology(Figure 3 B).in addition,based on this cross-linking strategy,the research group established a complete blood circulation system model including large blood vessels and capillaries in the hydrogel.Compared with the single scale of other in vitro vascular models,a new concept based on multi-scale vascular model is proposed^[34]。 in clinical translation,large solid tissues are difficult and cumbersome To form metabolites In vitro due to the lack of vascular network transport.to overcome this limitation,Zhang et al.Constructed a stable but biodegradable vascular chip scaffold using layer-by-layer stacking^[35]。 this technique takes advantage of the UV polymerizability of poly(octylmethylene maleic anhydride citrate)(POMaC)to construct vascular chip scaffolds under mild conditions.A POMaC sheet with a blood vessel network pattern is superimpose on each other layer by lay under ultraviolet light irradiation,so that a complex suspended microstructure and an internal cavity can be formed.Finally,using This method,a myocardial patch with a vascular network was assembled and successfully implanted in mice。

vascular network formation based on microfabrication modeling enables precise control of the size,shape,and pattern of the vascular network,ensures a high degree of repeatability,and is compatible with a wide range of materials,making it very effective in simulating vascular structures in the human body,tissue engineering,disease models,and drug testing.However,this technique also suffers from cumbersome steps,high cost,non-circular tube geometry,and possible biocompatibility issues.in addition,the manufactured blood vessels are still composed of a single layer of cells,and it is still difficult to reconstruct and achieve special physiological phenomena such as vascular tissue regeneration in vivo^[24]。

in order to simplify the tedious steps of microfabrication molding and improve the experimental efficiency,the researchers designed a method to remove the template after template molding to form a microvascular model In vitro^[36]。 this method usually requires two steps:first,a sacrificial mold is used to create a microchannel or network;Second,endothelial cells were seeded in the channel to form the endothelial layer.based on the current preparation methods,microvascular template formation and removal can be divided into needle-Based template formation and sacrificial template collagen formation.it is worth noting that this strategy is also the mainstream technical solution for the formation of blood vessels or other cavity structures in the field of bio-3D printing.Since this paper mainly discusses the use of microfluidic chip devices to form blood vessel models in vitro,It does not involve too much discussion on the scope of biological 3D printing。

Chen's group used a template method with removable microneedles to form a vascular endothelial cavity in collagen that could be perfused for a long time^[37]。 this work demonstrates in vivo-like vascular sprouting and perfusable neovascularization in 3D collagen matrices.Microfluidic devices use acupuncture needles to form cylindrical channels of controlled size in the matrix.the two channels are respectively used as an endothelial channel and an angiogenic factor channel to display directional angiogenesis.Through the effects on angiogenesis under the action of different inducing factors,it is found that the angiogenesis process induced by different factors may have different mechanisms and targets on endothelial cells(Fig.4A).the method based on the presetting and removal of microneedles in collagen can not only form an endothelial lumen with a circular cross-section,but also allow physiological phenomena such as angiogenesis and sprouting of blood vessels in the three-dimensional structure of collagen.in order to better understand the interaction between tumor and vascular endothelium in pancreatic ductal adenocarcinoma(PDAC),Chen's team used microneedles to construct two parallel channels in collagen,one of which contained PDAC cells and was adjacent to the original blood vessel composed of endothelialized and perfused chambers.Using this model,it was observed that PDAC tumor cells could invade and remove the vascular endothelium during the process of endothelial cell ablation.the authors further used this model to confirm the critical role of the activin-ALK7 signaling pathway in mediating endothelial cell ablation in PDAC^[38]。 Kim et al.Constructed a vessel-tumor model in a microfluidic chip by a combination of microfabrication and template-based removal of microneedles^[39]。 by studying the interaction between the two,the effect of the position of lung cancer spheroids on the speed and growth direction of tumor angiogenesis was explored By controlling the direction of interstitial flow and the ratio and position of fibroblasts(Figure 4 B).Solid tumors create an immunosuppressive environment that places a tremendous burden on the immune system.Beebe's team used removable PDMS rod-like structures to create a 340-µm-diameter tube in collagen and seeded endothelial cells on the inner wall of the tube to form a vascular structure,which was perfused with culture medium to nourish the cells and mimic the vascular structure present in tumors^[40]。 the authors used the chip to assess the impact of tumor environmental stress on NK cell function,exploring the plasticity of NK cells and their ability to reverse immune exhaustion。

Compared with the single microvascular channel formed by the simple microneedle removal templating method,the sacrificial templating method can form a more complex vascular network structure in vitro.in this approach,a two-dimensional or three-dimensional vascular network mold is first formed using an easily soluble gel or solid material.the preformed template was encapsulated in a three-dimensional hydrogel.Finally,the degradable template is dissolved or melted and flows out of the solidified gel,leaving interconnected channels in the hydrogel structure.Huang's group used biocompatible sodium alginate as a sacrificial template to form an interconnected three-dimensional microfluidic vascular network in the hydrogel^[41]。 The sodium alginate solution was crosslinked by Ca²⁺and then solidified to form a sacrificial template,which was encapsulated in the PDMS prepolymer.After the prepolymer is completely solidified,the sodium alginate mold encapsulated by PDMS is dissolved with EDTA solution to obtain a vascular network of interconnected channels(Fig.4C )。

based on the mechanism of angiogenesis,it has been found that endothelial cells in collagen matrices such as laminin and fibronectin will organize themselves into tubular structures under specific culture conditions and signal induction,which are similar to real blood vessels in morphology and function.Hydrogels,such as collagen,Matrigel,gelatin and fibrin,contain 90%to 99%water and have high permeability to biomolecules.At present,the study of vascular network self-organization is mainly Based on the co-culture of vascular endothelial cells and fibroblasts in collagen or fibrin gel in vitro.endothelial cells spontaneously form microvessels and lumens in collagen under the action of cytokines such as vascular endothelial growth factor(VEGF)secreted by fibroblasts,and allow fine fluids to flow through vascular compartments in vitro^{[42⇓⇓~45]}。

Jeon et al.Designed a microfluidic device with five parallel microchannels to construct a perfusable microvascular network^[46]。 the method involves mixing umbilical vein endothelial cells and fibroblasts separately In fibrinogen solution and introducing them into different channels to promote angiogenesis(Figure 5A).this design helps keep the endothelial and stromal cell suspensions separated within the central channel,encouraging endothelial cells to self-assemble to form a microvascular network.By adjusting parameters such as fibroblast ratio,they successfully established a perfusable physiological microvascular network in the microfluidic device.This method is of great significance in the construction of microvascular networks.Using This method,Jeon's team has constructed a sustainable perfusion microvascular network with physiological morphology on a three-dimensional scale and carried out many applications.in addition,Lee and Hughes established a physiological transport model from artery to vascularized tissue and then to vein^[47]。 In this chip,the different stages of vascular development,including angiogenesis,endothelial cell lining,germinal angiogenesis and vascular anastomosis,are simulated by the spontaneous drive of endothelial cells and fibroblasts。

Another important application of in vitro vascular models is in vitro disease modeling,particularly in the field of tumor biology.the development of cancer can be divided into the following steps:initial growth of the primary tumor,infiltration of immune cells,angiogenesis,intravasation of the primary tumor cells,transvascular transport of cancer cells,distal extravasation of cancer cells,and growth of metastatic tumors^[48]。 To realistically reproduce tumor progression in vitro,microvascular tumor models provide important tools for cancer research and serve as a low-cost anticancer drug screening platform.Kamm's research group studied the extravasation of tumor cells in blood vessels by using microvessels formed by self-organization in microfluidic chips,which provided a new research method for the study and analysis of the interaction between tumor cells and blood vessels in vitro^[49]。 They demonstrated the process of tumor intravasation mediated by macrophages in a microfluidic system.the three-dimensional tumor microenvironment of this system,which mimics the interface of the tumor and endothelial layer,revealed that tumor necrosis factorα(TNF-α)secreted by macrophages directly impairs the barrier function of the endothelial layer and enhances the intravasation of breast tumors.in addition,the research team used a similar device To simulate the extravasation process of breast cancer cells in microvascularized osteoblastic tissue,and used confocal microscopic imaging to study the dynamic process of breast cancer cells entering,adhering,and metastasizing through the microvascular network.to demonstrate whether the secreted factor affects tumor extravasation,breast cancer cells were seeded on one side of the microfluidic channel and their migration through the vessel was monitored in real time.It was found that the extravasation rate of metastatic breast cancer cells into the bone microenvironment was four times higher than that of the skeletal muscle microenvironment in the control group,which is consistent with the phenomenon observed in vivo^[50]。 in the tumor microenvironment,blood vessels play an indispensable role,especially In the delivery of anticancer drugs.Jeon's team proposed that individual parts of the device could be used to form a liquid guide without contact with the bottom surface,which could confine the colloidal solution to a designated area and eventually form a collagen shape with self-organized blood vessels inside^[51]。 Using this method combined with 3D printing and micro-injection molding,Jeon's team studied the angiogenesis model induced by tumor spheroids and the migration and invasion detection of tumor spheroids,and used it for drug screening^[52,53]。 Using a similar method,Wang's team at Shanghai Jiaotong University used a simpler laser cutting technology to process PMMA resin chip devices and used the device to form patterned fibrin collagen.vascular endothelial cells were cultured by self-organization to form a vascular network,which provides a new way to prepare microfluidic devices for vascular network research without microfabrication equipment^[54]。

in order to more accurately simulate the tumor microenvironment,researchers have tried to use different types of cells,but it is still a challenge to correctly form tumor tissue for blood flow.Yokokawa's team at Kyoto University has proposed a platform for vascularization of tumor spheroids using chips,which can simulate the tumor microenvironment In vitro^[55]。 Fibroblasts within the tumor spheroid induce endothelial cells in fibrin collagen to form blood vessels and sprout structures,creating a perfusable vascular network.drug administration under perfusion conditions did not show a dose-dependent effect of anticancer drugs on tumor activity,in contrast to the results under static conditions,confirming the importance of flow in the vascular network for assessing tumor activity on a Drug screening platform(Figure 5B).in addition,Ibrahim et al.Established an in vitro model of vascularization of human peritoneal omentum and ovarian tumor microenvironment^[56]。 Based on this model,the effect of stromal cells on tumor cell attachment and growth,and the effect of tumor cells on vascular and mesothelial cell permeability in early and late metastasis were studied(fig.5C).Jeon's team constructed tumor tissue for blood flow by implanting multicellular tumor spheroids into microfluidic devices^[57]。 This work monitored blood perfusion,sphere growth,and vessel dynamics,and analyzed the contribution of internal and external vascular cells to sphere perfusion according to the cellular composition of the sphere。

Approaches to self-assemble vascular networks in collagen have achieved stable perfusable microvascular networks in vitro based on paracrine biological mechanisms intrinsic to endothelial and stromal cells^[58]。 the main advantage of this vascular network construction method is that the relevant multicellular ecosystem can survive for a long time in the three-dimensional culture system,and tissue engineering experiments can be carried out on the microfluidic chip,thus more closely simulating the natural formation process of human microvasculature.It has important application value for drug screening and disease model research,especially in the field of cancer.However,this technology also faces certain challenges,such as the difficulty of precisely controlling the geometry and distribution of the vascular network,the limited structural complexity,the lack of long-term stability and physiological function,and the physical and chemical characteristics of hydrogel materials that may limit network formation and function。