Engineering vascularized organotypic tissues via module assembly

Adequate vascularization is a critical determinant for the successful construction and clinical implementation of complex organotypic tissue models. Currently, low cell and vessel density and insufficient vascular maturation make vascularized organotypic tissue construction difficult, greatly limiting its use in tissue engineering and regenerative medicine. To address these limitations, recent studies have adopted pre-vascularized microtissue assembly for the rapid generation of functional tissue analogs with dense vascular networks and high cell density. In this article, we summarize the development of module assembly-based vascularized organotypic tissue construction and its application in tissue repair and regeneration, organ-scale tissue biomanufacturing, as well as advanced tissue modeling.


Introduction
Engineering organotypic tissues with adequate vascularization play a central role in basic and translational research on tissue development, disease progression, drug testing, and pathogenic infection models [1][2][3][4].The vascular system is known to adjust based on the physiological or pathological Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence.Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.state of the human body [5,6].As the body's major transport system, the vasculature is deeply rooted in the tissue and is essential for the transfer of oxygen, nutrients, metabolites, cytokines, and drugs within complex organisms [5,7].In addition to this transporter role, the vasculature is also responsible for extra-tissue or extra-organ communication, as observed during the immune evasion and metastasis of tumor cells [8,9].Furthermore, the morphology and function of vasculature are constantly reprogrammed by the surrounding tissue milieu [10,11].During development, the vascular system enables prolonged tissue survival, thus providing an anatomical basis for the generation of hierarchical microarchitectures and complex functions [12][13][14][15].In the course of disease progression, the vascular system, in synergy with other components of the microenvironment, develops morphological and functional abnormalities [16][17][18].During transvascular delivery after drug administration, the milieu at the vascular-tissue niche is often a 'culprit' for ineffective treatment and therapy resistance [19][20][21][22].Therefore, there is an urgent need to develop an approach for the construction of vascularized organotypic tissues that recapitulate in vivo-like cell density and organ-level functions.
To date, several strategies have been introduced for the development of three-dimensional (3D) vascularized tissues.The most direct approach is to co-culture parenchymal and non-parenchymal cells, including endothelial cells, thereby generating material-free microtissues with capillaries (such as vascularized spheroids or organoids) [23,24].However, this microtissue could grow to a limited size (micron scale).As the size of microtissue increases, the existing internal microvessels become insufficient to support the mass transfer to cells at the tissue core in a timely manner.Another method is to establish bulk material-based vascularized tissues, wherein parenchymal and endothelial cells are co-cultured and they randomly self-assemble in the bulk hydrogel material (e.g.Matrigel) [25,26].Alternatively, vascularized tissues can be developed by co-culturing parenchymal cells in hydrogels with flanking vascular channels in a microfluidic chip [27].Recently, through the use of sacrificial 3D bioprinting, it is possible to construct blood vessels of different shapes, which are embedded in cell-laden bulk hydrogels [28,29].This strategy enables the integration of extensive, branching, and perfusable vascular networks into tissues.However, in constructing these (hydrogel) material-based vascularized tissue models, while cells show a uniform distribution when initially embedded, they tend to randomly self-organize.For example, multiple cells grow along the edges of the 3D matrix blocks after a period of incubation [23,26].This usually leads to differences between tissue models prepared using the same protocol, even if they are prepared from the same batch.Compared to the in vivo tissue situation, the composition and functional differences resulting from this assembly method are distinct with regard to the following aspects.First, the presence of a matrix interface between cells leads to a relatively low cell density (10 6 -10 7 cells ml −1 ) and limited intercellular communication through direct contact [27][28][29].Second, the disordered or semi-ordered arrangement of different cells does not allow the observation of certain complex biological activities that require multicellular involvement and are spatiotemporally regulated [30,31].Third, as the structural basis for tissue growth, development, and the formation of organ-specific functions, the vascular system is mostly absent in current tissue models, lagging in formation and having a single, rigid composition (for example, straight or dendritic large vessels and reticulated capillaries) [32,33].This impedes prolonged tissue survival and the process of organotypic function formation.Furthermore, pathophysiological events and therapeutic interventions that involve the vascular system are not sufficiently recapitulated in conventional tissue models.Finally, the differences among tissue models give rise to poorly reproducible research results on disease mechanisms, drug testing, and pathogenic infection mechanisms.All of the above-mentioned methods of modeling share a common feature, i.e. the construction of 3D models is accomplished with or without the incorporation of materials, using individual cells as the base active module.We categorize these routes as 'cell-based assembly'.Recently, novel module assembly (MoA) methods (i.e.'microtissue-based assembly') have been proposed for constructing 3D vascularized organotypic tissues [34][35][36][37][38][39].Microtissues containing multiple cells (with or without matrix), rather than individual cells, are used as active basic module units for assembly into complex and advanced 3D constructs.In this approach, parenchymal and non-parenchymal cells, including endothelial cells, are first pre-assembled into homotypic or heterotypic tissue modules.These modules are then further assembled into 3D structures according to a specific spatial arrangement.The MoA platform is versatile, flexible, and scalable, bridging the gaps between techniques currently employed for building vascularized organotypic tissues to be used for tissue engineering and regenerative medicine [40][41][42][43][44][45][46].Nowadays, tissue modules of different profiles with regard to type, size, cell composition, and morphology can be prepared (figure 1).Furthermore, using different fabrication techniques, the modules could be assembled into 3D vascularized structures with distinct characteristics, e.g.multi-organ systems, organ-scale tissues, and advanced tissues.Compared to conventional cell-assembly methods for vascularized organotypic tissue construction, this approach has the following advantages [40][41][42][43][44][45][46].First, the cell density is close to that in vivo; second, this approach accelerates the formation of a vascular network and markedly improves vascular maturation; third, the vascular network interacts with other components within the microenvironment and fosters adaptive remodeling; fourth, it significantly improves tissue survival and development.Therefore, vascularized organotypic tissues constructed based on this method are more bionic, show high fidelity in drug evaluation, and can be used in a diverse basic and translational research.Hence, an overview of the advances and prospects of vascularized organotypic tissues constructed via the MoA approach would be of great value for promoting their use and applications.
In this review, the preparation and assembly techniques for different types of modules are introduced.Moreover, the applications of pre-vascularized MoA techniques, including tissue repair and regeneration, organ-scale tissue construction, as well as advanced tissue modeling, are described.

Different types of tissue modules
Modules for engineering organotypic constructs include scaffold-free and scaffold-based microtissues (figure 2).Scaffold-free microtissues might be presented in the form of cellular aggregates (also called spheroids or organoids).These are usually prepared using special culture devices to promote cell aggregation in 3D.Typical culture methods include microplate, rotatory bioreactor, hanging drop, and (guided or unguided) matrix embedding [47][48][49][50][51][52].For instance, dot-or  filament-shaped kidney organoids were generated through extrusion bioprinting [52].The scaffold-based microtissues might be cell-laden porous microspheres or solid microgels.Microspheres accommodate cells in the 3D porous structures, which are prepared via freeze drying or emulsification of the microdroplets [42,53,54].Microgels encapsulate cells in the 3D hydrogel microparticles, which is achieved using droplet microfluidics or bioprinting [55].
When used for organotypic tissue construction, different types of tissue modules show remarkable advantages over conventional cell assembly under in vitro and in vivo settings.Cellular aggregates allow for in vivo-like cell density and enhance cell-cell interactions [56,57].Microspheres facilitate oxygen and nutrient diffusion deep into tissues owing to their porous topological structures.When adjusting the pore size of hydrogel-crosslinking networks, microgels could block host immune cells outside the network and establish immune privilege [41].Furthermore, due to the incorporation of matrix scaffolds, microspheres and microgels can protect cells from shear damage when squeezed via needles (or nozzles) [58][59][60].Notably, microspheres and microgels are particularly advantageous in terms of spatial and temporal control when loading multiple cells or compounds for cell behavior regulation and therapeutic delivery [53,54].Furthermore, unlike traditional tissue engineering methods that mostly rely on biomaterials (i.e.cell inoculation in material scaffolds), the main components in engineered constructs based on MoA are pure cells.Two of the main reasons for incorporating materials during module preparation or assembly are initial structural support and rapid fusion of cell clusters.As intercellular connections are established, the materials gradually degrade.Therefore, the selection criteria for materials generally include the following characteristics: biocompatibility, degradability, and solgel transformability [53,54,[58][59][60].

Technologies for MoA
Methods for tissue MoA can be divided into device-assisted, microfluidic-assisted, and bioprinting-assisted (figure 3 and table 1).Typical devices include a microplate, microchamber, microtube, rotating or shaking bioreactor, and a hanging drop [61][62][63][64][65].In device-assisted culture, tissue modules are forced to randomly contact and fuse due to external forces (e.g.gravity and centrifugal force) or minute cultivation space.For example, homotypic or heterotypic organoids were pipetted into a microtube bottom or microchamber to promote their contact [61].Using a rotating centrifuge tube, cell-laden microspheres were aggregated into largesized tissue clumps [62].In a manner distinct from random fusion, Cui et al created an adjustable droplet-fusion approach to enable the programmed assembly of many cell spheroids into intricate multicellular architectures [63].With this method, a variety of topologies (including dual-, multi-, and hetero-spheroids) can be built in a highly compact, miniature form.Microfluidic systems provide researchers with a flexible and scalable approach for manipulating organoid fusion [66,67].For example, brain region-specific organoids were introduced into a microfluidic chip consisting of a micropillar array and a complementary microhole array [66].Various organoids could settle into the vertical microholes and fuse into cortical-hippocampal-thalamic assembloids with the desired patterning.Assembloids are biological systems that self-organize through the fusion of one kind of organoid with another or with various specialized cell types, culminating in integration [68][69][70][71].In contrast to device-or microfluidicassisted methods, bioprinting makes it possible to manipulate modules in various dimensions, including size and direction.The common strategies for module bioprinting include extrusion, aspiration, acoustic, and magnetic control [72][73][74][75][76].For example, Sun et al proposed an all-in-one method for efficiently preparing spheroid-based bioink with favorable printability and spheroid fusion ability [73].The solidified hydrogel microwell arrays were used to produce cell spheroids.Afterward, the microwell-spheroid composites were directly transferred into the bioprinter and converted to spheroid-laden soluble bioink.After extrusion printing, the cell spheroids were densely aligned to retain fusion capacity.Alternatively, by regulating the fusion of organoids in space, Ao et al enhanced the standardization of organoid assembly models using acoustofluidics [76].This procedure is a label-free and contact-free technique for efficient organoid assembly, which makes organoid assembly-based disease models and engineered tissues more uniform.

Formats of MoA for various applications
The engineering of tissues through MoA is versatile, flexible, and scalable.Based on the number of modules, the assembly can be divided into binary, ternary, and multiple fusion modes (figure 4 and table 1).Depending on the module types, the assembly can also be divided into homotypic or heterotypic fusion modes.Nowadays, through various assembly formats, different types of organotypic tissues can be developed, including multi-organ systems, organ-scale tissues, and complex tissue models.
To construct multi-organ systems, tissue modules derived from different organs or tissues can be fused to create relative physical isolation between distinct identities while also achieving direct cell-cell communication between them.Multi-organ systems consisting of healthy and disease modules offer a tool for investigating tumor invasion mechanisms.For example, Goranci-Buzhala et al reported a technique that allows the rapid and effective assessment of glioma stem cell (GSC) invasion through the assembly of GSC spheres with brain organoids [77].The assays could be employed to describe different characteristics of tumor microtubes formed between modules and to test pharmacological agents.Moreover, multi-organ systems consisting of brain tissue-specific modules provide a platform for investigating functional circuits and tissue development within the brain.As an example, by combining two distinct regionspecific organoids, Xiang et al produced reciprocal projections between the thalamus and cortex in 3D [78].This framework helped model circuit organizations as well as associated brain diseases and human thalamic development.Similarly, corticostriatal assembloids were created by Miura et al who combined striatal organoids with cerebral cortical organoids in 3D [79].When the assembloids were derived from patients, this method enabled analysis of the functional organization of corticostriatal connectivity.Additionally, Bagley et al constructed other domain-specific assembloids, such as ventral-dorsal or medial ganglionic eminence-cortical organoid fusions, to identify multimodal interneuron migration and neurotransmitter signaling between different brain regions [80][81][82].These assembloid cultures offered the possibility of analyzing neurodevelopmental defects and testing potential therapeutic compounds.Alternatively, Kim et al established ventraldorsal assembloids to promote oligodendroglia maturation, which could not be captured in ventral organoid, dorsal organoid, and organoid-oligodendrocyte co-culture models [83].This model served to reveal disease mechanisms associated with cortical myelin defects.Finally, multi-organ systems consisting of different organ-derived modules enable the investigation of functional control of one organ over another.For instance, to facilitate their mutual isolation, Fernando et al positioned retinal and brain organoids adjacent to one another [84].The development of nerve-like structures bridging retinal and brain organoids may advance research into neurological conditions that impact the eye and brain.In addition to the binary assembly, Fligor et al organized retinal organoids with cortical and thalamic organoids to create threepart assembloids [85].In response to environmental signals, retinal ganglion cells (RGCs) replicated the projections of the visual system by extending axons deep into assembloids.Furthermore, long-term assembloids improved RGC survival, addressing the drawbacks of retinal organoids in which RGCs were lost.This method would make it easier to study growth, disease, or damage of the human visual system.Using a similar method, Andersen et al created functional circuits in threepart assembloids by fusing 3D spheroids that resembled the cerebral cortex, spinal cord, and skeletal muscle [86].The assembloids were easily modified to simulate the long-term cortical regulation of muscle contraction in vitro.
Tissue modules with different sizes, shapes, and morphological profiles can be assembled for engineering complex, hierarchical, and macro-scale constructs.For example, Liu et al established macro-scale lumenized airway tubes utilizing multi-organoid patterning and fusion [87].Multi-organoid aggregates retained predetermined morphologies without an external scaffold interface, possessed a continuous lumen and experienced faster fusion in a matrix-free, floating environment.These aggregates displayed a distinct three-stage procedure of luminal material clearance, lumina connection, and inter-organoid surface integration.This platform enabled the up-scaling of organoid engineering towards massive organ tubes of tunable shape.Another example is the organoid bioprinting concept that Brassard et al presented to direct tissue development over more physiologically relevant scales [88].This approach used stem cell-derived organoids as spontaneously self-organizing building blocks with relatively random shapes.Following the geometry and limitations imposed by 3D printing, these tiny organoids might be spatially united and restructured to create interconnected and dynamic biological structures.They created a centimeter-scale intestinal tube with self-organized elements like branched vasculature, lumens, and a tubular intestine with crypts and villus domains Note: 2D, two-dimension; 3D, three-dimension; MoA, module assembly.that resemble those found in living things.They also produced a macro-scale continuous epithelial tube that included the mouse small intestine and proximal colon, a macro-scale vascular tube made of the continuous lumen with complex geometries and branched capillaries, as well as a macro-scale continuous multi-organ tube made of the intestinal part, transition zone, and stomach part.Thus, by employing this adaptable technique, macro-scale cellular structures containing important cell types can be printed to mimic the interactions between tissues, thus providing new opportunities for drug development, diagnostics, and regenerative medicine.Tissue modules representing local tissue-specific milieus, which are composed of multiple stromal cells and extracellular matrix (ECM), can be assembled for engineering well-organized, complex, and heterogeneous constructs.One strategy is to create parenchymal milieus in relatively isolated local niches first, and then fill stromal cells into the interniche voids, thereby enabling the formation of dense, multilayer, large-scale constructs.For example, Fang et al produced a large number of hepatocyte-laden microgels via droplet microfluidics [89].Afterwards, bi-phasic bioinks composed of microgels and another endothelial cell-laden hydrogel phase, which filled into the inter-microgel voids, were bioprinted into 3D heterogeneous tumor constructs.Another strategy is to create heterogeneous sub-milieus in local niches first and then splice or stack these sub-milieus.For example, Liu et al used 3D porous gelatin microspheres as building blocks for engineering scalable muscle tissues [90].Microsphere culture in spinner flasks separately facilitated the expansion of skeletal muscle satellite cells and myoblasts, in addition to triggering their spontaneous myogenesis.Using a 3D-printed mold, they assembled these hybrid microspheres into centimeterscale tissues, which exhibited similar mechanical properties and enhanced function compared to conventional 2D muscle tissues.

Pre-vascularization strategies for tissue modules
MoA provides a new approach to address the technical limitations of conventional cell-assembly methods in terms of cell density, spatial arrangement of multiple cell types, and coexistence of multiple heterogeneous niches.This technological breakthrough provides a realistic structural and compositional basis for the use of engineered tissues in research, including complex or advanced organ-level function studies and studies on improving in vivo transplantation efficacy.However, for this desired goal to be achieved, a key issue must be addressed, namely, vascularization.
As the structural basis for organotypic tissues, the vascular system has to be included and be able to form in time to participate in and promote tissue growth, development, and the formation of organ-specific functions.Endothelial cells, which are the key cellular constituent of vessels, should be incorporated into vascularized organotypic constructs.Appropriate endothelial cell sources include: (1) stem cellderived endothelial cells/progenitors; (2) endothelial cells with E-twenty-six variant 2 overexpression; (3) healthy/disease tissue-derived endothelial cells; and (4) stem cells supplemented with pro-angiogenic factors [91][92][93][94].Additionally, how endothelial cells/progenitors are incorporated is critical for the structure and maturation of the vascular system.The common strategy is to co-culture parenchymal cells with vascular endothelial cells/progenitors for pre-vascularized module preparation.In this case, the formed (micro)vascular network can uniformly distribute in or envelop the parenchymal layer.For instance, utilizing spheroids as the fundamental building block, Kim et al created 3D complex vascularized macrotissues [24].A micro-patterned device was used to create pre-vascularized spheroids composed of stem and endothelial cells.Spheroids with the core-shell structure quickly generated a vessel-like network in vitro when fused for the creation of macro-tissue.Another strategy is to co-culture the pre-established parenchymal modules with endothelial modules.Carvalho et al proposed a technique enabling spheroids to self-assemble into modular vascular beds in a directed manner [95].The spheroid-based patches distinguished themselves as artificial vascular beds because of their scalability, high surface area, and propensity for endothelial sprouting.Using a microfluidic platform, Rodoplu et al fused embryoid bodies and tumor spheroids to probe tumor angiogenesis and test an anti-angiogenic drug [96].

Pre-vascularized MoA for advanced tissue engineering and regenerative medicine
In the past decade, the engineering of vascularized tissues based on MoA has been proposed for applications in tissue engineering and regenerative medicine [97][98][99][100] (table 2).In tissue engineering, these models can be used for research into tissue development, disease, and drug testing.In regenerative medicine, these models can be employed as tissue substitutes for the repair and functional reconstruction of defective tissues.

Pre-vascularized MoA for tissue repair and regeneration
Cell therapy through the transplantation of functional cells or a tissue patch has been proposed as a potential strategy for patients with non-regenerative disease-associated defects, such as those affecting cardiac tissue, skeletal muscle tissue, and bone.However, conventional cell-assembly-based strategies face several challenges.To begin with, the biomanufactured tissue analogs lack an efficient, timely vascular supply, which hinders the successful clinical translation of cell therapy.One strategy for cell therapy is through autologous or allogeneic harvesting and transplantation.However, these procedures are associated with many issues, such as limited resources, secondary injury and complications, as well as immune rejection.Stem cells, such as human induced pluripotent stem cells (hiPSCs) and mesenchymal stem cells (MSCs), are widely used for tissue repair in preclinical studies.Current stem cell delivery methods involve direct or intravenous injection of cells into the damaged site.However, this method is limited by low cell retention, which necessitates repeated injections to ensure effective repair.Additionally, mechanical damage to the cells during injection may induce apoptosis, thereby compromising therapeutic efficacy.Recent studies are exploring feasible strategies for improving the success of cell therapy, such as through the acceleration of angiogenesis and anastomosis, thus improving graft success and reducing secondary injury [101][102][103].
The use of pre-vascularized tissue modules for transplantation might improve the efficacy of cell therapy in the treatment of myocardial defects (figure 5).For example, Liu et al used early vascular cell (EVC) spheroids as the basic modules of a myocardial patch to facilitate the formation of cardiac tissues with a well-organized microvascular system [104].EVCs were obtained through the 3D differentiation of human embryonic stem cells.Thereafter, EVC/cardiomyocyte spheroids were tightly arranged in hydrogels via bioprinting.The tissue size was scalable, and tiny or large-sized myocardial tissues could be generated.Furthermore, the density, maturation level, and formation period of microvasculature in the spheroid assembly group were significantly better than those in the cellassembly group in vitro.When transplanted into the ischemic area of mice with myocardial infarction, EVC spheroids efficiently improved cardiac function and reduced myocardial fibrosis, in contrast to EVC suspension.Therefore, using prevascularized modules might contribute to the development of a well-organized vasculature and the improved efficacy of cell therapy.Finally, vascularized tissues could provide reliable results when simulating cardiac conditions in vitro and may serve as alternative models for drug screening in cardiovascular diseases.
Pre-vascularized tissue modules might address challenges posed by skeletal muscle tissue defects.Wang et al coinjected myoblast-laden microspheres and endothelial cellladen microtubes to achieve the in situ repair of defective skeletal muscle tissue [105].Briefly, the authors employed microfluidics to prepare polylactic acid-glycolic acid (PLGA) porous microspheres and short rod-shaped hollow microfibers.The myoblast (C2C12)-laden microspheres were dynamically cultured to form 3D muscle modules, which further aggregated into large tissue clumps.Moreover, endothelial cells (human umbilical vein endothelial cell (HUVECs)) distributed along the hollow microtubes and self-assembled into a vascular network.When co-culturing the hybrid modules in vitro, the established muscle tissue recapitulated the in vivo microenvironment, accounting for cellcell and cell-environment interactions.When co-injecting the hybrid modules at sites of massive muscle defects in [140] Note: hBMSC, human bone marrow stromal cell; HUVEC, human umbilical vein endothelial cell; MSC, mesenchymal stem cell; vMoA, pre-vascularized module assembly.mice, the skeletal muscle tissue was completely repaired, and its function was restored after a 28 day injection.In contrast to tissue patches, the plastic deformation property of tissue modules enabled injection into the injury site, which provided a new method for in vivo delivery and avoided surgical injury.In addition, this MoA method accelerates tissue maturation and increases the success rate of transplantation.
The addition of therapeutic cells into pre-vascularized tissue modules can achieve effective delivery and optimize the desired bone tissue repair effect.Yuan et al developed an injectable gelatin methacrylate (GelMA) colloidal microsphere to mediate bone regeneration [106].The microsphere was a suitable delivery vehicle for therapeutic cells, having an adjustable porous structure, a shape memory function, and ideal cytocompatibility.Furthermore, the microsphere could enhance cell differentiation potential and allowed the efficient loading of bone marrow mesenchymal stem cells (hBMSCs) as well as endothelial cells (HUVECs).These GelMA microspheres promoted the sustained proliferation of hBMSCs and HUVECs, while protecting the cells during injection.Osteogenesis and angiogenesis were achieved two months after co-injecting the hybrid microspheres into mice.Similarly, Patrick et al presented a technique for creating vascularized structures utilizing injectable nanoporous microgels that enable bone regeneration in critical-sized defects [107].Microgels supported endothelial sprouting and network development while encouraging hBMSC osteogenesis.The osteogenic microgel enabled strong MSC adhesion, proliferation, and differentiation by mimicking the properties of bone matrix.However, when implanted in 3D matrices, vasculogenic microgels enhanced the endothelial phenotype and promoted the creation of vascular networks.The two different types of microgels were combined to create a hybrid construct that preserved and even improved their functionalities.The minimally invasive administration of these multifunctional microgels can replace substantial bone deficiencies in a conformal manner.This work presents concepts for the design of versatile scaffolds with specific biophysical and biochemical features for the reconstruction of vascularized interfacial tissues.Furthermore, the endothelialized scaffold-based MoA facilitates the targeted delivery and long-term retention of therapeutic cells at the injection site.
In addition to applications in heart, muscle, and bone regeneration, modular assembly strategies are also adopted for treating non-regenerative diseases, such as diabetic complications and peripheral artery disease [108][109][110].Using 'self-condensation' culture, Takahashi et al created a sophisticated method for designing vascularized islets that allow heterotypic cellular lineages, such as endothelial cells, to self-assemble with tissue fragments or organoids in a spatiotemporal manner [111].Using this technique, they were able to create complex tissues from different tissue fragments, including islets.Endothelial cells were used in the self-condensation culture of islets to boost functionalization and significantly enhance post-transplant engraftment.Vascularized islets dramatically increased the longevity of diabetic mice after transplantation, showing that blood glucose levels returned to normal more quickly than with standard islet transplantation.This strategy presents a viable way to improve the effectiveness of therapeutic tissue transplantation.In another study, Nalbach et al described a method for creating pre-vascularized islet organoids by fusing functioning native microvessels into pancreatic islet cells [112].When compared to non-pre-vascularized islet organoids, these insulinsecreting organoids exhibited considerably greater angiogenic activity in vitro, which was attributed to paracrine signaling between β-cells and microvessels.Due to the connectivity between the pre-vascularized islet organoid's autochthonous microvasculature and surrounding blood arteries, they received blood quickly after transplantation.As a result, fewer islet grafts were needed to cure diabetic mice.Pre-vascularized islet organoids may therefore be employed to increase the likelihood of successful clinical islet transplantation.Rossen et al, on the other hand, developed a sacrificial scaffold to mass-produce highly reproducible multicellular organoids [113].Pre-vascularized organoids made of endothelial cells and MSCs quickly developed perfusing vasculature when administered to healthy mice.Additionally, a mouse model of peripheral artery dysfunction was used to test the therapeutic potential of pre-vascularized organoids, and the results showed that vascular perfusion was quickly restored within 7 d.Therefore, the utilization of pre-vascularized aggregates as assembly units accelerates the formation of vascular networks and tissue maturation.

Pre-vascularized MoA for biomanufacturing organ-scale tissues
The construction of artificial organs through tissue engineering has broad application prospects.However, it is extremely difficult to create and maintain dense cellular structures (>10 8 cells ml −1 ) for therapeutic purposes.To achieve therapeutic effects, millions of cells need to be rapidly assembled into functional microstructural units during the manufacturing process.Furthermore, a vascular system needs to be embedded to deliver oxygen and nutrients to tissues.Currently, the thickness of engineered human tissues is limited to a few hundred microns owing to the lack of a vascular network, which results in the absence of large-sized structural features [114][115][116].Thus, it is difficult to achieve complex, multilevel functional organ responses in vitro.The fabrication of 3D vascularized tissues (∼1 cm in thickness) through multimaterial 3D bioprinting and stereolithography was recently reported [117][118][119][120][121][122].However, these lacked the cellular density and complex microstructure necessary to mediate relevant physiological functions.The cell density of the 3D bioprinting ink is usually 1-2 orders of magnitude lower than that of human tissue [123].
Tissues that possess the required cellular density, microstructure, and function can be achieved using the prevascularized MoA approach (figure 6) [124,125].For instance, Skylar-Scott et al suggested using the 'sacrificial writing into functional tissue (SWIFT)' method to create huge, organotypic tissues with embedded vascular channels and high cellular density [126].A large quantity of hiPSC-derived organ-building modules was produced and assembled into dense living matrices (about 2 × 10 8 cells ml −1 ).Additionally, perfusable vascular channels were introduced into the matrices through sacrificial 3D bioprinting.The blood vessels could be created in any direction to form a perfusable, branched, and hierarchical vascular network (vessel diameter range: 400 µm ∼ 1 mm).Using this method, the authors created centimeter-scale cardiac tissue that beat synchronously over a 7 day period and produced electrophysiological behaviors in response to calcium ion and electrical stimulation.Therefore, biomanufacturing through MoA allows one to quickly organize perfusable tissues at the scales required for therapeutic application.While sacrificial bioprinting has been used to vascularize large tissues in vitro, it has not been able to create dense-enough microvessel networks to perfuse huge de novo tissues up to this point.Grebenyuk et al created networks of artificial 3D capillary-scale capillaries to perfuse millimeterscale tissue structures [127].A printable hydrogel formulation that enables precision microvessel printing at scales smaller than the limit of diffusion (>200 µm) in living tissues made 3D soft microfluidics possible.During prolonged in vitro incubation, the large-scale synthetic tissues displayed complex morphogenesis.In perfused neural constructs, neuronal differentiation was dramatically accelerated.This entirely synthetic vascularization platform thus paves the way for the creation of tissue models of unprecedented complexity and scale.In another instance, Ho et al provided a roadmap for organ-scale tissue engineering by integrating the bioprinting of completely cellular bioinks with the development of billions of human cells [128].Using a stirred tank bioreactor technique, the cultivation of hiPSC-derived aggregates was made scalable.The cardiac aggregates as well as vascular organoids were differentiated from the pluripotent aggregates into derivatives of the three germ layers.To create a fully cellular bioink for 3D bioprinting, the aggregates were finally compressed.The printed aggregates were then transformed into a variety of interesting cell types, including neuronal and vascular cells.Overall, a billion-cell rapid organ engineering strategy is made possible through the aggregate bioprinting pipeline.

Pre-vascularized MoA for advanced tissue modeling
As a widespread non-parenchymal component of tissues, vasculature, in concert with surrounding components, participates in the maintenance of normal physiology, disease progression, and treatment responses as well as post-intervention tissue remodeling [129][130][131][132][133]. To date, various 3D models, such as organoids, organ chips, and bioprinted models, have been developed to mimic tissue development, study pathogenesis, and screen drugs.The lack of stroma, tissue-resident immune cells, and, most critically, vasculature, which collectively establish micro-environmental niches during development and disease, renders such models insufficient.At present, efficient recapitulation of the highly complex in vivo milieu and vasculature-mediated cell-cell and cell-environment interactions remains a major challenge.
To circumvent these drawbacks, researchers increasingly employ MoA to produce complicated vascularized organotypic tissues.For example, Wörsdörfer et al suggested the guided insertion of mesodermal progenitor cell organoids into parenchymal organoids to form vascularized tumor or brain tissue models (figure 7) [134].The generated blood arteries showed a characteristic ultrastructure and a hierarchical architecture.Additionally, during organoid development, the endothelial network displayed great plasticity with responses to both pro-and anti-angiogenic substances.After transplantation, vessels within tumor organoids were linked to host vessels.Surprisingly, the assembly process produced mesenchymal-epithelial interfaces, a crucial developmental component during organogenesis.In another study, Kim et al developed normal and cancerous bladder assembloids by using parenchymal organoids as tissue modules and incorporating other cellular components of the microenvironment, such as vascular endothelial cells, fibroblasts, and immune cells [135].In the assembloid models, tumor or healthy organoids were densely arranged and surrounded by dense stroma layers.The genomic variation and phenotype of parental tumors were well preserved in assembloids.Additionally, endothelial cells developed into a vascular network, which prolonged in vitro culture and promoted tumor growth.The tumor assembloids could also be used to study the molecular mechanism of stroma plasticity as well as that underlying the response to various anticancer drugs.Finally, vascularized normal and disease models provide a reliable tool for studying vascular-tumor interaction, exploring anti-angiogenic drugs, and modeling tissue development.Apart from assembling homotypic modules, the formed vascularized assembloids could reproduce invasion at the tumor border in hybrid MoA.Developments at the border regions are of major relevance to disease progression and treatment, with vasculature niches mediating cancer cell invasion and tumor spread.Chen et al fabricated tumor border assembloids by fusing heterotypic organoids to reproduce the inter-niche invasion interface [136].This assembloid was composed of colorectal endothelialized cancer organoids and healthy organoids, which were accurately placed in close contact through acoustic bioprinting so as to recapitulate the affected colorectum of a patient.Histological, genomic, and phenotypical characterization demonstrated that the assembloid preserved the primary cell types (cancer, fibroblast, and endothelial cells) and the border microenvironment.The organoid's invasive capacity was positively correlated with tumor spread in patients.Additionally, the significant vascular penetration in the border area was similar between the in vitro model and in patients.Overall, this approach mimics the microenvironment at the tumor border and provides an auxiliary diagnostic tool for guiding personalized medicine.More importantly, the method can be extended to the construction and study of complex physiological systems composed of heterogeneous microtissues.
Beyond recapitulating the tumor microenvironment, MoAbased 3D tumor models also unambiguously reflect the pharmacokinetics of anti-tumor therapeutics.Wang et al constructed a 3D vascularized hepatoma model based on multicellular microsphere assembly [62].In this model, human hepatoma cells (HepG2) and human endothelial cells (HUVECs) were seeded in PLGA porous microspheres.Due to intercellular interactions, 'cell bridges' were formed in inter-microspheres and tissue clumps.Compared to HUVEC or HepG2 microsphere groups, the HepG2/HUVEC model could secrete more albumin and exhibited higher multidrug resistance protein expression.This model was utilized for the evaluation of various anticancer chemotherapeutics, such as doxorubicin and cisplatin.The half-maximal inhibitory concentration of drugs against the cells under 3D culture was substantially higher than that against cells under 2D culture conditions.In another example, Agarwal et al reported a method for engineering macro-scale 3D vascularized tumors [137].The tumor cells were initially enclosed in microgels to create avascular microtumors.Endothelial cells and other stromal cells were then assembled with the microtumor modules.In comparison to avascular microtumor assemblies, the resulting 3D vascularized tumors were more resistant to doxorubicin.Furthermore, this resistance could be overcome through nanoparticle-mediated drug delivery.The 3D tumor model may therefore be useful for studying how vascularized microenvironments affect tumor progression as well as for creating potent anticancer medications.
Modeling viral infection-associated neurotrophic pathology is another research focus that would benefit from MoA.The main elements of the blood-brain barrier, i.e. cerebral pericytes and endothelial cells, express various viral entry receptors and respond to systemic inflammation [138,139].However, due to insufficient immune cells and a lack of vasculature, studies of virus-induced neurotrophic disease using conventional brain models, such as organoids, fail to mimic these symptoms.In recent investigations, vascularized assembloid models have been constructed using the MoA method for studying neurotrophic disease.Kong et al created cortical-blood vessel assembloids to supply brain organoids with vasculature and noted enhanced abundance of astrocytes and microglia [140].They also observed pathologies that were similar to those of Alzheimer's disease, influenced by the inflammatory response triggered by SARS-CoV-2 infection.These findings provide evidence for the use of MoA as an advanced platform for investigating neurotrophic diseases, including COVID-19.Similarly, Sun et al fused vessel and brain organoids to produce vascularized brain organoids [1].The engrafted vascular network-like structures and increased number of neural progenitors in the fused brain organoids are consistent with the notion that vasculature may control neural development.As a result, interactions between neuronal and non-neuronal components, notably those between the vasculature and microglia, could be modeled in vitro using fusion organoids.
MoA allows for the extra-spheroid assembly of intricately vascularized tissues.Feijo et al reported a technique for the production of vascularized dermal macro-tissue from fibroblast/endothelial progenitor spheroids [141].Spheroids embedded in fibrin provided a 3D milieu for extra-spheroid development.While endothelial progenitors self-organized into capillaries with a lumen and basal lamina, fibroblasts secreted ECM, generating a thick tissue.A complex vascular plexus was quickly established in vitro owing to the extensive connections between sprouts from nearby spheroids.Pre-vascularized spheroids with fibrin entrapment transformed into a macro-scale tissue with evident host vessel invasion after being injected into chick embryos.Quick and effective inoculation was observed between host and donor arteries.The modular construction method for producing dense vascularized tissue is thus clinically applicable and holds promise in a variety of applications.

Conclusions and future perspectives
Compared with traditional methods for biomanufacturing vascularized tissue, the utilization of microtissues as basic modules has emerged as a new strategy for the rapid generation of up-scaled functional tissue substitutes with a high cell density, well-organized microarchitecture, and complete vasculature.These features greatly promote the application of MoA in tissue engineering and regenerative medicine by enabling the construction of multi-organ systems, organ-scale tissues, as well as advanced tissue models.Moreover, the flexibility in their assembly and scalability allows for the construction of homogeneous or heterogeneous tissues with regard to shape, size, and cell types, thus expanding the application range.While the MoA approach facilitates an unprecedented step forward in the study of organ-level biological activities, several issues remain to be addressed, as summarized below.
Standardization of pre-vascularization routes.Technical routes for achieving module pre-vascularization should be standardized.Current strategies mainly include: (1) endothelial and parenchymal cell co-culture, with endothelial cells uniformly distributed in the module; (2) endothelial and parenchymal cell co-culture, with endothelial cells wrapped around the outer layer of the module; (3) endothelial and parenchymal cells separately pre-organized into modules, followed by the co-culture of these heterotypic modules; and (4) parenchymal cells pre-assembled into modules, followed by inoculation of endothelial cells into the parenchymal modules and further co-culture.These strategies may lead to differences in the morphology and maturation of vascular networks, as well as in tissue function.Overall, the advantages and shortcomings of the four above-described routes as well as their specific applications remain to be further characterized in subsequent studies.
Improving vasculature quality.In addition to the amount of vasculature, various physiological and mechanical factors are crucial for its function and maturation.Blood vessel formation undergoes two stages, namely vasculogenesis and angiogenesis, which are complex processes involving multiple cells and cytokines [142].Vasculogenesis is the process by which endothelial progenitor cells differentiate in situ into mature endothelial cells and form primitive blood vessels.Angiogenesis is the proliferation, migration, and remodeling of vascular endothelial cells to form new mature vessels on the basis of pre-existing vascular beds, with smooth muscle cells and pericytes playing an important role in the maturation and stability of blood vessels.Vasculogenesis and angiogenesis are achieved under the precise regulation of multiple proangiogenic factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor.Other essential determinants of vascularization include oxygen availability, mechanical properties, and fluid forces [143,144].MoA can employ strategies that take into account physiological and mechanical factors during module preparation and fusion in order to improve model bionics [103,145].For example, Onoe et al used a coaxial printing platform to prepare meter-long scale tubular tissue modules with different inner diameters, including cardiomyocyte, HUVEC, and cortical tissue [103].The MoA was performed by laminating these modules to form a vascularized tissue with a threelayer tubular structure.This tissue could recapitulate in vivo physiology and pathology, including cellular composition and patterning, oxygen supply, electrophysiological signaling, and fluid shear force imposed by perfusion.
Standardization of culture protocols.The protocols on culture conditions, culture devices, and assembly techniques should be subject to stringent control.Each stage of the MoA process is closely related to the functionality and reproducibility of the pre-built model, including the culture conditions before and after fusion, fusion timing, as well as assembly modes.Currently, fusion timing is generally determined based on the selection of optimal growth or development time windows for the module, which depend on the specific cell type and culture conditions.Still, further studies are needed to optimize the culture parameters at each stage for the construction of normal physiology or disease models.In addition, preserving the 3D structure of the assembled model is a great challenge.When the modules are assembled through different techniques, maintenance of the 3D structure requires the consideration of the following factors: (1) use of external forces to promote the initial passive contact and avoid structural dispersion, and (2) endogenous intercellular forces during culture to maintain the 3D structure while avoiding structural collapse caused by gravity and cell adhesion properties.Almost all existing culture devices for 3D structure are directly panned from 2D culture.More customized cross-scale culture platforms are expected to facilitate the integration and versatility of 'preparation-culture-observation'.Finally, more comprehensive research is needed to clarify which assembly technique should be chosen for a given research objective, while ensuring consistent culture conditions.
Matching assay methods and platforms.As the tissue size increases, corresponding inspection methods should be developed.Recently, real-time monitoring platforms matching these models have been developed.For example, Daly et al used muscle motion software to detect the contraction amplitude and electrophysiological conditions of the heart at the centimeter scale [146].Park et al produced a '3D multifunctional mesoscale framework' that could detect the tension of intercellular interactions within different modules [147,148].To monitor nerve conduction during tissue development throughout the process, the model or its living sections can be placed in a multielectrode array system to continuously observe dynamic changes in real time [101,149].Additional tools or platforms still need to be developed to provide multidimensional organ-level physiological or pathological information.In conclusion, MoA-based vascularized organotypic tissue fabrication represents the convergence of new manufacturing technologies, applied materials, and biomedical research.

Figure 1 .
Figure 1.Characteristics of diverse modeling routes, including cell-based and microtissue-based assembly.(a) Pre-vascularized module assembly (MoA) enables the rapid generation of up-scaled functional tissue substitutes with high cell density, well-organized microarchitecture, and complete vasculature in contrast to cell-based or avascular microtissue-based assembly methods.(b) Feasible paths and time period for constructing vascularized organotypic tissue.

Figure 2 .
Figure 2. Different types of pre-vascularized MoA and their applications.(a) Preparation of scaffold-free or scaffold-based tissue modules.(b) Tissue modules for assembly include cell aggregates, cell-laden microspheres, and cell-laden microgels.(c) Applications of pre-vascularized MoA include tissue repair and regeneration, biomanufacturing organ-scale tissues, and advanced tissue modeling.

Figure 3 .
Figure 3. Technologies for MoA.(a) Microplate-assisted methods for random module fusion.Reproduced from [79], with permission from Springer Nature.(b) Droplet-assisted methods for semi-random module fusion.[63] John Wiley & Sons.© 2020 The Authors.Advanced Materials published by Wiley-VCH GmbH.(c) Microfluidic-assisted methods for unordered or semi-ordered MoA and scalable tissue fabrication.Reproduced from [67].CC BY 4.0.(d) Extrusion bioprinting-assisted methods for random or semi-random MoA and large-scale tissue fabrication.(e) Aspiration bioprinting-assisted methods for directional MoA and up-scaled tissue fabrication.

Figure 4 .
Figure 4. Formats of MoA for various applications.(a) Schematic of the generation of forebrain circuits by integrating ventral and dorsal organoids.Reproduced from [80], with permission from Springer Nature.(b) Strategy to reconstruct visual pathway via generating retinal-brain assembloids.(c) Diagram showing how to assemble 'multi-organoid aggregate' modules to recreate the hierarchical design of branched tubular organs.Reproduced from [87].CC BY 4.0.(d) Strategy to recapitulate macro-scale tissue self-assembly through organoid bioprinting.Reproduced from [88], with permission from Springer Nature.RGCs, retinal ganglion cells.

Figure 6 .
Figure 6.Pre-vascularized MoA for biomanufacturing organ-scale tissues.(a) Sacrificial writing into functional tissue (SWIFT) printing of an endothelialized bifurcating channel and a perfusable tissue module-based living matrix.(b) Human pluripotent stem cell (hPSC) spheroids are fused into solid tissue when cultured on a 3D-printed chip with soft microfluidic capillary grids.Reproduced from [127].CC BY 4.0.(c) Principle for bioprinting and subsequent differentiation of aggregate-based bioinks.Reproduced from [128].CC BY 4.0.ECM, extracellular matrix.

Figure 7 .
Figure 7. Pre-vascularized MoA for advanced tissue modeling.(a) Generation of tissue assembloids recapitulating the pathophysiology of human urothelial carcinomas.Reproduced from [135], with permission from Springer Nature.(b) Principle for constructing an endothelialized aggregate-based tumor model and its applications for up-scaled tissue fabrication and drug screening.Reproduced from [62].CC BY 4.0.(c) Microtumors and stromal cells, including endothelial cells, are assembled using a microfluidic device to create a massive 3D vascularized tumor model.Reprinted with permission from [137].Copyright (2017) American Chemical Society.(d) Principle for constructing vessel-cortical organoids, which allows us to observe the vascular penetration and neurite sprouting.Reproduced from [140].CC BY 4.0.

Table 1 .
The characteristics of MoA technologies and formats.

Table 2 .
The characteristics of representative vMoA.