Organ-on-a-chip (OOC) and Micro-physiological system (MPS) are cutting-edge technologies in the field of bioengineering. These platforms mimic the function of human organs on a micro-scale through the convergence of bioprinting, microfluidic chip, and tissue engineering technologies, providing models close to the physiological conditions of the human body for drug screening and disease research, and aiming to more accurately recapitulate the physiological and pathological properties of organs.
MPS further extends this concept by simulating a three-dimensional environment for cells and tissues, bridging the gap between traditional two-dimensional cell culture and animal models. Microfluidic systems play a key role in MPS, enabling precise control of fluids at the microscale to provide a dynamic hydrodynamic environment for cells, which is particularly important when modeling the vascular system.
Therefore, in vitro reconstruction of the vascular system becomes a central challenge for organ-on-a-chip and MPS construction, where microvascular network design is particularly critical.
Microfluidics has significant advantages in reconstructing the vascular system: first, it can precisely control the fluid flow in the channel in time and space to provide controlled stimulation for endothelial cells to study the mechanism of the vascular system; second, its channel size can be matched with the microstructure of the blood vessels, which, combined with three-dimensional culturing and tissue engineering techniques, enables reconstruction of functional vascular networks in vitro.
Method for performing in vitro remodeling of blood vessels in microfluidic chips
Microfluidic chips are widely used to reconstruct blood vessels in vitro, often called “microvascular chips”, which can accurately mimic the function of the human vascular system and can be used to construct disease models, drug screening and toxicity assessment.
1) Formation of a monolayer barrier by direct culture of vascular endothelial cells on a two-dimensional plane in a microfluidic chip
Culturing vascular endothelial cells directly on microfluidic chips or using porous membranes to form a monolayer barrier is one of the most important means of reconstructing blood vessels in vitro and mimicking human vascular function. The researchers used specific surface treatment and cell culture techniques to attach endothelial cells to the inner wall of microchannels to form a continuous cell monolayer.
Barriers with specific pore size and distribution can also be constructed using porous membranes, mimicking the underlying structure of the vascular wall. These endothelial cells exhibit in vivo-like morphology and functions, such as the formation of tight junctions, thus mimicking a real vascular barrier, and are widely used to study intercellular interactions, vascular permeability, drug delivery, and vascular function in pathological states. This method not only supports cell attachment and growth, but also controls intercellular material exchange.
Fabrication of microfluidic chips and integration of porous membranes by photolithography and micromachining technologies has become one of the mainstream methods for forming endothelial barriers in the field of organ-on-a-chip at present because of the controllable and reproducible process, which is easy to observe. However, the endothelial cell layer formed in this way is a two-dimensional monolayer and lacks three-dimensional lumen structure, which makes it difficult to fully reproduce the three-dimensional morphology of blood vessels and their related functions in vivo.
In addition, many physiological phenomena such as vascular neovascularization and outgrowth are difficult to reproduce on such models due to the lack of extracellular matrix encapsulation similar to that in vivo. Therefore, the development of techniques and methods capable of controllably constructing three-dimensional lumen morphology of blood vessels in vitro is receiving increasing attention.
2) Formation of blood vessels by wall culture of endothelial cells in hollow tubes shaped by gel micromodeling
Unlike the construction of endothelial cell monolayers on two-dimensional planar or porous membranes, microstructures of collagen or other matrices can be precisely fabricated on silicon wafers or PDMS molds using soft lithography and other microfabrication techniques.
After removing the mold and encapsulating the gel to form a channel, the vascular endothelial cells are then inoculated to form an in vitro vascular model. Through microfabrication, researchers are able to construct complex vascular networks, providing an important tool for deeper understanding of the mechanisms of the vascular system and the development of new therapeutic approaches.
This micromachined shaping-based approach provides precise control over the size, shape and pattern of the vascular network, ensures a high degree of reproducibility and is compatible with a wide range of materials, making it highly effective in simulating in vivo vascular structures, tissue engineering, disease modeling and drug testing.
However, the technique suffers from several shortcomings, such as cumbersome steps, high cost, non-circular tube geometry, and potential biocompatibility issues. In addition, the blood vessels formed are still composed of a single layer of cells, which makes it difficult to reproduce special physiological phenomena such as in vivo vascular tissue neogenesis.
3) Formation of blood vessels by wall culture of endothelial cells after lumen formation using sacrificial molds or gels
To simplify the cumbersome steps of micromachining shaping and enhance experimental efficiency, researchers have developed the method of removing templates after molding them for forming microvascular models in vitro. The method is typically a two-step process: first, microchannels or networks are created using a sacrificial mold; then endothelial cells are inoculated within the channels to form a complete endothelial layer.
Current template forming and removal techniques mainly include the needle-based template forming method and the sacrificial template collagen forming method, which are the current mainstream techniques for constructing blood vessels or other luminal structures in the field of bio-3D printing.
The sacrificial template method enables the formation of more complex vascular networks in vitro than the simple microneedle-removal template molding method. In this method, a two- or three-dimensional vascular network mold is first fabricated using an easily dissolvable gel or solid material that is encapsulated in a three-dimensional hydrogel. Once the mold is formed, the template material flows out of the cured hydrogel by dissolving or melting the degradable template, thereby forming a network of interconnected channels within the hydrogel.
4) Spontaneous formation of microvascular networks by directly cultured cells in microfluidic chip collagen
Studies based on the mechanism of microvessel formation have found that endothelial cells are able to self-organize into tubular structures resembling real blood vessels in collagenous matrices such as laminin and fibronectin under specific culture conditions and signal induction. These hydrogels, such as collagen, matrix gels, gelatin and fibronectin, contain 90% to 99% water and are highly permeable to biomolecules.
Current studies have focused on co-culturing vascular endothelial cells and fibroblasts in collagen or fibrin gels in vitro, with the latter secreting cytokines such as vascular endothelial growth factor (VEGF), which induces the endothelial cells to spontaneously form microvessels and endolumens in the collagen, so that microfluidic fluids can flow through the vasculature in vitro.
Another important application of in vitro vascular models is in disease research, especially in tumor biology. Cancer development goes through multiple stages, including initial tumor growth, immune cell infiltration, angiogenesis, tumor cell endocytosis, cancer cell vascular transit, distal extravasation and metastatic tumor growth. The ability of microvascular tumor models to reproduce these processes in vitro provides an important tool for cancer research and serves as a platform for low-cost anticancer drug screening.
Self-assembly of vascular networks in collagen based on a paracrine mechanism in endothelial and stromal cells has resulted in stable and perfusable microvascular networks in vitro.
Its main advantage is that the multicellular ecosystem can survive for a long period of time in three-dimensional culture and can be applied to microfluidic chips for tissue engineering experiments, which more closely mimic the natural formation process of human microvessels. This technology has important applications in drug screening and disease modeling, especially in tumor research.
However, there are challenges to this approach, such as the difficulty in 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 properties of the hydrogel that may impose limitations on network formation and function.
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