A method for constructing an in vitro brain lymphatic circulation system model that reconstructs the brain parenchyma-perivascular space structure-functional interface.

Abstract

This invention relates to a method for constructing an in vitro brain-like lymphatic circulation system model that reconstructs the structure-function interface of the "brain parenchyma-perivascular space," belonging to the fields of tissue engineering and biomanufacturing technology. The method employs a "suspension-coaxial" bio-3D printing approach, simultaneously constructing a central vascular lumen precursor, a vascular wall precursor, and a PVS precursor within a retained outer layer material. After overall shaping, sequential demolding, cell colonization, interface orientation treatment, establishment of a multicellular interface, and the establishment of a heterogeneous dual perfusion system, a continuous multilayer structure containing a central vascular lumen, vascular wall, PVS channel, and a brain parenchyma-like extracellular matrix layer is obtained in vitro. Hemodynamic boundary conditions and peripheral fluid exchange boundary conditions are established on both sides of the same vascular wall-PVS-brain parenchyma interface, respectively, for use in research on cerebrospinal fluid-interstitial fluid exchange, pathological molecular transport, and brain-like lymphatic circulation mechanisms.

Description

Technical Field

[0001] This invention relates to a method for constructing an in vitro brain-like lymphatic circulation system model that reconstructs the structure-function interface of the "brain parenchyma-perivascular space", belonging to the field of tissue engineering and biomanufacturing technology. Background Technology

[0002] The cerebral lymphatic circulation system is a crucial structure for fluid exchange and metabolic waste removal within the brain. Its core characteristic lies in the entry of cerebrospinal fluid (CSF) into brain tissue via the perivascular space, where it exchanges with interstitial fluid. Fluid transport and solute clearance are accomplished through the joint participation of the blood-brain barrier, the perivascular space, and aquaporin-4 (AQP4) on the podocytes of astrocytes. Existing research indicates that neurodegenerative diseases such as Alzheimer's and Parkinson's are often accompanied by abnormal CSF-interstitial fluid exchange, blood-brain barrier damage, altered perivascular space structure, AQP4 polarization imbalance, and hemodynamic abnormalities, thereby affecting the efficiency of clearing metabolic waste products such as β-amyloid, tau protein, and α-synuclein.

[0003] Currently, experimental models used to study the mechanisms of brain lymphatic circulation and related neurodegenerative diseases mainly include two-dimensional cell culture models, animal models, organoid models, and some microfluidic chip models. Two-dimensional culture models lack realistic three-dimensional spatial structures and multi-cellular interfaces, making it difficult to reproduce the hierarchical tissue relationships between the vascular lumen, vascular wall, perivascular space, and brain parenchyma. Although animal models can present the overall physiological process, they suffer from significant species differences, long experimental cycles, complex operations, and high costs. While existing in vitro engineered models have improved the simulation ability of the brain vascular microenvironment to some extent, they still cannot simultaneously achieve the coordinated reconstruction of the perfusionable vascular lumen, real vascular wall, annular perivascular space, and brain parenchyma microenvironment in the same system, especially in further forming a physiologically relevant "brain parenchyma-perivascular space" structure-function interface.

[0004] In recent years, studies have attempted to construct in vitro models related to brain-like lymphatic circulation using microfluidic and 3D bioprinting technologies. For example, existing microfluidic chip models can be used to study AQP4 polarization, cellular responses to amyloid stimulation, and some fluid exchange processes. However, these models are usually based on parallel microchannels or chip chambers, making it difficult to further reconstruct multi-layered concentric structures with realistic vascular walls and annular perivascular spaces, and also difficult to form a structure-function interface involving brain parenchyma, astrocyte foot processes, and perivascular spaces. On the other hand, while existing 3D bioprinting research can construct geometrically adjustable, perfusion-like cerebral vascular conduits or cerebral vascular tissue with blood-brain barrier-like characteristics, it still mainly focuses on vascular body construction or blood-brain barrier-related functions. It has not yet simultaneously formed independent perfusion-like perivascular space channels, a preserved brain parenchyma-like extrastromal layer, and a bilateral boundary condition control system acting on the same vascular wall-perfusion space-brain parenchyma interface within the same system.

[0005] Therefore, the existing technology has at least the following shortcomings: First, it is difficult to continuously reconstruct a hierarchical three-dimensional structure in vitro that simultaneously contains the vascular lumen, vascular wall, perivascular space, and brain parenchyma-like extracellular matrix; second, it is difficult to simultaneously form the central vascular lumen and the peripheral perivascular space channels during the same construction process and maintain the stable positioning of the vascular wall between them; third, it is difficult to perform localized material modification and microenvironment reconstruction only at the vascular wall-perivascular space-brain parenchyma interface without compromising the overall formability; fourth, it is difficult to establish independently controllable and coupled hemodynamic boundary conditions and fluid exchange boundary conditions on both sides of the same interface. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a method for constructing an in vitro brain lymphatic circulation system model that reconstructs the structure-function interface of the "brain parenchyma-perivascular space". The method employs a "suspension-coaxial" bio-3D printing approach to simultaneously construct the central vascular lumen precursor, vascular wall precursor, and perivascular space (PVS) precursor within a retained outer layer material. After overall shaping, sequential demolding, cell colonization, interface orientation treatment, multi-cellular interface establishment, and the establishment of a heterogeneous dual perfusion system, a continuous multilayer structure containing the central vascular lumen, vascular wall, PVS channels, and a brain parenchyma-like extracellular matrix layer is obtained in vitro. Furthermore, hemodynamic boundary conditions and peripheral fluid exchange boundary conditions are established on both sides of the same vascular wall-PVS-brain parenchyma interface, respectively, for use in research on cerebrospinal fluid-interstitial fluid exchange, pathological molecular transport, and brain lymphatic circulation-related mechanisms.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows.

[0008] A method for constructing an in vitro brain lymphatic circulation system model that reconstructs the structure-functional interface of the "brain parenchyma-perivascular space" includes the following steps:

[0009] (1) Using extracellular matrix composite bio-ink containing embedded astrocytes, a brain parenchyma-like extracellular matrix layer precursor (referred to as layer 4) was suspended and printed in the central construction area of ​​the double-chamber culture frame. (2) The inner layer sacrificial ink, the extracellular matrix composite bio-ink for embedding cerebral vascular pericytes and the PVS precursor sacrificial ink were sequentially filled into the inner, middle and outer channels of the triaxial concentric nozzle. Coaxial printing was performed using asynchronous start-up, synchronous construction and asynchronous stop-up. From the inside out, the central vascular lumen precursor (referred to as layer 1), the vascular wall precursor (referred to as layer 2) and the peripheral PVS precursor (referred to as layer 3) were formed in the extracellular matrix precursor of the brain parenchyma sample to obtain the model precursor. (3) The model precursor is fixed to gel the brain parenchyma-like extrastromal layer precursor and vascular wall layer precursor to form the brain parenchyma-like extrastromal layer and vascular wall layer. (4) The precursor of the central blood vessel formed by the inner layer sacrificial ink is perfused to remove the sacrificial material to form a continuous central blood vessel. Brain microvascular endothelial cell suspension is perfused into the central blood vessel to allow brain microvascular endothelial cells to attach to the inner surface of the central blood vessel and form an endothelial layer. (5) Selectively remove the peripheral PVS precursor formed by PVS precursor sacrificial ink to form a continuous PVS channel between the outer side of the blood vessel wall and the inner side of the brain parenchyma matrix. Introduce interface modification solution into the PVS channel to allow the interface modification components to adhere to the outer side of the blood vessel wall and the inner side of the brain parenchyma matrix. Rinse to remove unbound components, culture under oscillation conditions to form a system model. (6) Maturation of the system model under oscillating conditions; (7) Fix the mature cultured system model in the perfusion chamber, establish a central blood flow circuit connected to the central cardiovascular cavity and a peripheral cerebrospinal fluid circuit connected to the PVS channel, and form an in vitro brain lymphatic circulation system model with a structure-function interface of "brain parenchyma-perivascular space".

[0010] Preferably, in step (1), the extracellular matrix composite bio-ink for embedding astrocytes is obtained by embedding astrocytes in one or more of the following systems: extracellular matrix (BdECM)-alginate composite system, BdECM-HAMA composite system, BdECM-GelMA composite system, BdECM-hyaluronic acid composite system, BdECM-collagen composite system, BdECM-fibrin composite system, and alginate-GelMA composite system, with a cell density of 1×10^6~1×10^7 cells / mL.

[0011] Preferably, in step (2), the sacrificial material in the inner layer sacrificial ink and the PVS precursor sacrificial ink is one or more of PF-127, CPF-127, gelatin and agarose.

[0012] Preferably, in step (2), the extracellular matrix composite bio-ink for embedding cerebral vascular pericytes is obtained by embedding cerebral vascular pericytes with one or more of the following systems: VdECM-alginate composite system, VdECM-collagen-alginate composite system, VdECM, VdECM-HAMA composite system, VdECM-GelMA composite system, collagen hydrogel, fibrin hydrogel, and alginate-gelatin composite system, with a cell density of 1×10^6~1×10^7 cells / mL.

[0013] Preferably, in step (2), when asynchronously starting the flow, the flow is started sequentially in the order of intermediate layer channel, inner layer channel and outer layer channel; During synchronous construction, the nozzle moving speed is 300~900 mm / min, the deposition pressure of the inner channel is 400~550 kPa, the deposition pressure of the middle channel is 30~50 kPa, the deposition pressure of the outer channel is 160~220 kPa, the inner diameter of the formed central vascular lumen precursor is 400~600 μm, the thickness of the vascular wall precursor is 100~180 μm, and the thickness of the outer PVS precursor is 150~250 μm. When asynchronously stopping the flow, the flow is stopped sequentially in the order of outer layer channel, inner layer channel, and intermediate layer channel.

[0014] Preferably, in step (3), during the shaping process, the model precursor is placed in an incubator at 35~37℃ and incubated for 15~30min.

[0015] Preferably, in step (4), when removing the sacrificial material, culture medium is perfused into the central cardiovascular progenitor at a flow rate of 0.3~0.5 mL / min and a perfusion time of 2~5 min.

[0016] Preferably, in step (4), the cell density of the brain microvascular endothelial cell suspension is 1×10^6~1×10^7 cells / mL, the perfusion flow rate is 0.3~0.8 mL / min, the perfusion time is 4~8h, and after the perfusion is completed, it is left to stand for 1~4h.

[0017] Preferably, in step (5), when removing the sacrificial material, a culture medium compatible with the surrounding cells is perfused. After perfusion, the mixture is left to stand for 1-3 minutes, and then subjected to low-amplitude oscillation or shaking. The oscillation angle is 2-8°, the oscillation frequency is 4-10 times / min, the single oscillation time is 3-10 minutes, and the process is repeated 1-2 times.

[0018] Preferably, in step (5), the interface modification solution contains one or more of laminin, type IV collagen, and agrin; the concentration of laminin is 50~150 μg / mL, the concentration of collagen IV is 50~150 μg / mL, and the concentration of agrin is 10~50 μg / mL; after introducing the interface modification solution, low-amplitude shaking is performed with a shaking angle of 2~6°, a shaking frequency of 4~8 times / min, and a shaking time of 5~15 min; after shaking, the mixture is left to stand at 35~37℃ for 10~30 min; and the stable culture time is 6~24 h.

[0019] Preferably, in step (6), the maturation culture conditions are 37°C and 5% CO2, the central blood vessel lumen is continuously perfused with endothelial cell culture medium or co-culture medium compatible with endothelial cells, the outer matrix layer of the peripheral brain parenchyma is maintained by adding astrocyte culture medium or co-culture medium compatible with peripheral cells, the swing angle is 2~8°, the swing frequency is 4~12 times / min, the maturation culture time is 7~14 days, and the culture medium replacement cycle is 12~48 h.

[0020] An in vitro brain-like lymphatic circulation system model with a "brain parenchyma-perivascular space" structure-functional interface was constructed using the above method.

[0021] Beneficial effects (1) This invention does not merely construct a single hollow blood vessel, but rather continuously forms a central blood vessel lumen, vessel wall, perivascular space (PVS), and brain parenchyma-like extracellular matrix layer within the same construct, thereby reconstructing a "blood vessel lumen-vessel wall-PVS-brain parenchyma" structural unit with a continuous spatial hierarchy. Compared to existing two-dimensional culture models, single microfluidic channel models, and three-dimensional models that only reconstruct the blood vessel body, this invention is closer to the brain lymphatic circulation-related tissue microenvironment, and can provide a more physiologically relevant in vitro platform for cerebrospinal fluid-interstitial fluid exchange, pathological molecular transport, and brain lymphatic function research.

[0022] (2) This invention adopts a cross-interface bridging double-sacrificial-layer synchronous coaxial construction method, defining the central vascular cavity precursor and the peripheral PVS precursor separately in the same construction process, and forming a bridging independent structural unit composed of two end interface regions, a central bridging main segment and a peripheral brain parenchyma extramatrix. Compared with the existing coaxial printing method that can usually only form a single hollow cavity, this invention can not only simultaneously form two independent fluid spaces, but also pre-establish the spatial correspondence between the subsequent central perfusion pathway and the peripheral fluid exchange pathway during the printing stage, thus having significant advantages in terms of structural integrity, system integration and subsequent perfusion adaptability.

[0023] (3) This invention addresses the problems of difficulty in establishing a stable mating relationship instantaneously in the initial stage of a double-sacrificial-layer three-coaxial structure, and the tendency for eccentricity, delamination, breakage, and interface mismatch when crossing the boundary of the suspension pool. It proposes a phased construction strategy involving pre-filling, asynchronous flow start-up, boundary compensation, synchronous construction, and asynchronous flow stop. Compared to the conventional single-sacrificial-layer three-coaxial synchronous start-stop printing method, this invention significantly improves the continuity and stability of the double-sacrificial-layer structure in the initial segment, the pool entry transition segment, the bridging main segment, the pool exit transition segment, and the termination segment, thereby improving the repeatability of cross-interface bridging printing.

[0024] (4) This invention achieves parametric control of the geometric relationship between the central vascular lumen diameter, vessel wall thickness, PVS width, and total outer diameter by synergistically adjusting the nozzle specifications, first layer deposition conditions, second layer deposition conditions, third layer deposition conditions, printing speed, and target structure dimensions. Furthermore, it allows for local geometric control of different segments through localized parameter switching. Compared to existing methods that primarily rely on empirical parameter adjustments to obtain single vascular structures, this invention integrates geometric dimension control, interface position control, and dual-fluid channel connectivity control into a unified parameter system for adjustment. Therefore, it is more conducive to stably obtaining reproducible brain-like lymphatic circulation structures and provides a comparable structural basis for subsequent research on the influence of hemodynamic changes and fluid exchange state changes on interface function.

[0025] (5) This invention does not use a single general-purpose material to construct the entire structure, but rather achieves the reconstruction of the local microenvironment related to the brain's lymphatic circulation through layered material division of labor and interface-oriented treatment. Specifically, the second layer uses blood vessel-derived specific bioactive materials to form a blood vessel wall-related microenvironment, and the fourth layer uses brain-derived specific bioactive materials to form a brain parenchyma-like extracellular matrix. The fourth layer material simultaneously undertakes three functions: suspension support, tissue modeling, and cell carrier. On this basis, the blood vessel wall-PVS-brain parenchyma interface is further modified with directional materials through PVS channels, so that the interface modification components are locally enriched on both sides of the PVS, rather than uniformly distributed throughout the entire construct. Thus, this invention can form a local microenvironment that is closer to the characteristics of the natural basement membrane without destroying the overall shapeability, which is conducive to the establishment of astrocyte foot process attachment and AQP4-related distribution, and has higher specificity in terms of interface biomimicry and interface stability.

[0026] (6) This invention employs a sequential demolding method, first forming the central cardiovascular lumen and completing endothelial colonization, then forming a continuous PVS channel. After demolding, interface orientation treatment and short-term stabilization culture are performed, thereby maintaining the stable positioning of the vessel wall during the establishment of the dual-fluid channel and reducing vessel wall drift, PVS collapse, and local instability that may occur during demolding. Compared with existing methods that remove all sacrificial layers at once, this invention is more conducive to maintaining the continuity and interface integrity of the dual-channel structure and improving the structural stability during subsequent cell colonization and long-term culture.

[0027] (7) The dual-perfusion system constructed in this invention is not two identical perfusion devices, but a heterogeneous driving system consisting of central blood flow pump-driven perfusion and peripheral bilateral ventricular oscillatory fluid exchange. Therefore, hemodynamic boundary conditions and peripheral fluid exchange boundary conditions can be established on both sides of the same vessel wall-PVS-brain parenchyma interface, enabling independent control, synchronous control, and coupled analysis. Compared to existing models that can only simulate a single fluid environment or provide only unilateral perfusion conditions, this invention can simultaneously examine changes in central hemodynamics, peripheral fluid exchange status, and the synergistic effect between the two. This is beneficial for studying the influence of changes in blood flow pulsation, pressure gradient, fluid exchange frequency, and fluid residence time on cerebrospinal fluid-interstitial fluid exchange, pathology-related molecular transport, and brain lymphoid clearance efficiency.

[0028] (8) This invention uses a “suspension-coaxial” additive manufacturing method combined with layered bioactive materials for model construction, which has the advantages of controllable structure, good repeatability, high system integration and large parameter adjustment space. By adjusting the structural size, material composition, interface modification method, cell configuration and perfusion conditions, in vitro brain lymphatic circulation system models with different scales, different interface states and different pathological stimulation conditions can be constructed, and can be applied to the study of brain lymphatic related mechanisms, pathological related molecular transport studies, drug screening and biological evaluation. Attached Figure Description

[0029] Figure 1 This is a flowchart illustrating the construction method described in this invention.

[0030] Figure 2 This is a schematic diagram of the preparation process of BdECM and VdECM bio-inks in Example 1 of the present invention.

[0031] Figure 3 This is a schematic diagram of the dual-chamber culture framework structure in an embodiment of the present invention.

[0032] Figure 4 This is a schematic diagram of the in vitro brain-like lymphatic circulation system model in an embodiment of the present invention.

[0033] Figure 5This is a schematic diagram of the dual-irrigation system structure and irrigation method in an embodiment of the present invention. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments.

[0035] like Figure 1 As shown, a method for constructing an in vitro brain lymphatic circulation system model that reconstructs the structure-functional interface of the "brain parenchyma-perivascular space" includes the following steps: Using extracellular matrix composite bio-ink containing embedded astrocytes, a brain parenchyma-like extracellular matrix precursor (referred to as layer 4) was suspended and printed in the central construction area of ​​a dual-chamber culture frame. The inner sacrificial ink, the extracellular matrix composite bio-ink embedding cerebral vascular pericytes, and the PVS precursor sacrificial ink were sequentially filled into the inner, middle, and outer channels of the triaxial concentric printhead. Coaxial printing was performed using an asynchronous start-up, synchronous construction, and asynchronous stop-up method. From the inside out, the central vascular lumen precursor (referred to as layer 1), the vascular wall precursor (referred to as layer 2), and the peripheral PVS precursor (referred to as layer 3) were formed in the extracellular matrix precursor of the brain parenchyma sample to obtain the model precursor. The model precursors were subjected to shaping treatment to gel the brain parenchyma-like extrastromal layer precursors and vascular wall layer precursors, forming the brain parenchyma-like extrastromal layer and vascular wall layer. The precursor of the central blood vessel lumen formed by the inner layer sacrificial ink is perfused to remove the sacrificial material and form a continuous central blood vessel lumen. Brain microvascular endothelial cell suspension is perfused into the central blood vessel lumen so that the brain microvascular endothelial cells attach to the inner surface of the central blood vessel lumen and form an endothelial layer. Selective removal of peripheral PVS precursors formed by PVS precursor sacrificial ink was performed to form a continuous PVS channel between the outer side of the blood vessel wall and the inner side of the brain parenchyma matrix. An interface modification solution was introduced into the PVS channel to allow the interface modification components to adhere to the outer side of the blood vessel wall and the inner side of the brain parenchyma matrix. Unbound components were washed away and cultured stably under swing conditions to form a system model. Maturation of the system model under oscillating conditions; The mature cultured system model was fixed in the perfusion chamber, and a central blood flow circuit connected to the central cardiovascular cavity and a peripheral cerebrospinal fluid circuit connected to the PVS channel were established to form an in vitro brain lymphatic circulation system model with a "brain parenchyma-perivascular space" structure-function interface.

[0036] Specifically: (1) Preparation of materials and building blocks ① Prepare the inner core sacrificial material for forming the first layer of the central vascular lumen precursor; the first layer material can be selectively removed after construction to form a continuous central vascular lumen. ② Prepare the bioactive ink for forming the second vascular wall layer; the second layer material is used to form the vascular wall structure located between the central vascular lumen and the PVS channel, and pre-embeds brain perivascular cells. ③ Prepare the sacrificial material for forming the third layer of the PVS precursor; the third layer material can be selectively removed after construction to form a continuous PVS channel on the outer side of the vascular wall. ④ Prepare the outer layer material for forming the fourth layer of the brain parenchyma-like extramatrix layer; the fourth outer layer material serves as a suspension support environment during construction, providing three-dimensional support for the inner deposition structure, and is retained after construction to form the brain parenchyma-like extramatrix layer, and also serves as a carrier for astrocytes. ⑤ Prepare brain microvascular endothelial cells for subsequent colonization on the inner surface of the central vascular lumen. ⑥ Prepare an interface modification solution for PVS interface treatment, used to directionally modify the outer surface of the vascular wall and the inner surface of the brain parenchyma-like extramatrix after PVS formation. ⑦ Prepare a bilateral ventricular culture frame; the culture frame includes a central construction area, a central blood flow interface area corresponding to the central vascular cavity, and a bilateral ventricular fluid exchange area corresponding to and connected to the PVS channel, for subsequent construction of bridging independent vascular structures and access to the dual perfusion system.

[0037] Furthermore, the selection of the second vascular wall layer material and the fourth brain parenchyma-like extracellular matrix layer material depends not only on their tissue-derived biological activity, but also on their mechanical support, shape retention after demolding, perfusion stability, and interface retention. To this end, the second and / or fourth layer materials adopt a mechanically reinforced extracellular matrix composite bio-ink system, which enables the materials to retain tissue-derived biological clues while possessing structural stability suitable for suspension printing, integral molding, and subsequent perfusion culture.

[0038] (2) Cross-interface bridging double sacrificial layer synchronous coaxial construction A fourth outer layer material was injected into the central construction area of ​​the bilateral ventricular culture frame and adjusted to a state suitable for suspension support printing. This allowed the material to provide three-dimensional support and morphological maintenance for the deposited structure during printing and to retain the extracellular matrix layer resembling brain parenchyma after printing. Subsequently, a first inner core sacrificial material, a second vascular wall layer of bioactive ink, and a third PVS precursor sacrificial material were loaded into different channels of a concentric nozzle. The first and third layers were sacrificial layers to be selectively removed later, while the second layer was the functional layer forming the vascular wall.

[0039] Since layers 1 and 3 are used as sacrificial layers simultaneously, the construction process employs a phased approach of pre-filling, asynchronous initiation, synchronous construction, and asynchronous shutdown to establish and maintain a stable three-layer coaxial mating relationship. Specifically, before formal bridging printing, layers 1, 2, and 3 are driven along their respective channels to a position close to the nozzle outlet, preparing the three materials for continuous discharge. Subsequently, the nozzle is moved to the interface area on one side of the dual-ear chamber culture frame, aligning the nozzle outlet with the predetermined printing start position, and the asynchronous initiation process is initiated. First, layer 2 forms continuous discharge, then layer 1 enters the interior of layer 2 to form a stable two-layer coaxial precursor. Then, layer 3 is initiated, forming a continuous outer layer on the outside of layer 2, thereby establishing a stable three-layer mating relationship where layer 1 is in the center, layer 2 continuously covers layer 1, and layer 3 continuously covers layer 2. In the initial interface area, the nozzle is kept stationary or moves at a low speed to form a short initial segment and complete the establishment of the initial mating relationship of the three layers.

[0040] After stable material output is established in layers 1, 2, and 3, the synchronous construction phase begins. During this phase, the printhead moves continuously along a pre-defined printing path from one side of the interface area towards the central construction zone. The three coaxial precursors, having established a stable mating relationship, enter the fourth outer layer material from the interface area under continuous output. Before entering the suspended support environment, a transition section is set before the boundary. Within this section, boundary compensation is performed for changes in support conditions by reducing the printhead's movement speed and / or adjusting the deposition conditions of layers 1, 2, and 3. After entering the fourth outer layer material, the printhead continues along the pre-defined path across the central construction zone to form a bridging main body section. The innermost channel continuously deposits the first inner core sacrificial material, the middle channel continuously coats and deposits the second vascular wall layer bioactive ink, and the outermost channel continuously coats and deposits the third PVS precursor sacrificial material. This simultaneously forms, from the inside out, the central vascular lumen precursor, vascular wall layer precursor, and outer PVS precursor structure within the fourth outer layer material.

[0041] Furthermore, during the synchronous construction process, the geometric relationships between the central vascular lumen diameter, vessel wall thickness, PVS width, and total outer diameter were controlled by coordinating the nozzle specifications, the first layer deposition conditions, the second layer deposition conditions, the third layer deposition conditions, and the nozzle moving speed. Specifically, the first layer deposition pressure was controlled at 400-550 kPa, the second layer deposition pressure at 30-50 kPa, the third layer deposition pressure at 160-220 kPa, and the nozzle moving speed at 300-900 mm / min. Under these conditions, the central vascular lumen diameter was controlled at 400-600 μm, the vessel wall thickness at 100-180 μm, and the PVS width at 150-250 μm.

[0042] Furthermore, the printing path employs a bridging-type independent vessel printing method, allowing the main body of the resulting vessel segment to span the central construction area and extend at both ends into the interface areas reserved in the bilateral ventricle culture frame. Specifically, the two ends of the central vascular lumen precursor extend into the corresponding positions of the central blood flow interface area, while the two ends of the peripheral PVS precursor extend into the corresponding positions of the bilateral ventricle fluid exchange area. This pre-establishes the spatial correspondence between the subsequent central perfusion pathway and the peripheral fluid exchange pathway during the printing stage. In the bridging zone within the pool during the synchronous construction stage, the deposition conditions and nozzle movement speed of the first, second, and third layers can be locally programmed and switched according to the target geometry. This allows for the creation of differences in the central vascular lumen diameter, vessel wall thickness, and PVS width in different segments without disrupting the overall bridging continuity and the correspondence between the two end interfaces.

[0043] As the nozzle approaches the boundary of the suspension pool on the other side, a transition section is set up before the boundary. The structural stability during the process of leaving the suspension support environment is compensated by reducing the nozzle's moving speed and / or adjusting the deposition conditions of the first, second, and third layers, thus maintaining the complete coverage relationship at the end of the bridging main section. After the nozzle enters the interface area on the other side, continuous material discharge continues to form the termination connection section. After completing the predetermined bridging length, the asynchronous stop-flow stage begins. In the asynchronous stop-flow stage, instead of using a synchronous instantaneous stop-flow method for all three layers, a staged stop-flow method is adopted to smoothly transition the stable synchronous construction state to the termination state. First, the discharge of the third layer of PVS precursor sacrificial material is reduced or stopped, allowing the outer layer to gradually finish forming in the termination area. Then, the short-range synchronous discharge of the first and second layers is maintained, ensuring a stable correspondence between the central vascular lumen precursor and the vascular wall layer in the termination area. Afterward, the discharge of the first layer of inner core sacrificial material is stopped, and finally, the discharge of the second layer of vascular wall material is stopped, thus completing the construction of the termination section.

[0044] (3) Overall shaping and sequential mold removal After the above-mentioned multilayer precursor structure is constructed, the overall construct undergoes a shaping process to further gel and stabilize the second vascular wall layer and the fourth outer layer, ensuring the relative stability of the vascular wall and the surrounding brain parenchyma extramatrix during subsequent demolding and cell colonization. This shaping process is performed without causing premature instability or non-selective removal of the first and third sacrificial layers. The shaping temperature is 35-37℃, and the time is 15-30 min.

[0045] After overall shaping, the first layer of inner core sacrificial material is selectively removed via the central blood flow interface to form a continuous central vascular lumen. A culture medium or basal culture medium compatible with subsequent endothelial colonization is slowly perfused through one or both central blood flow interfaces, allowing the culture medium to pass through the central pathway formed by the first layer of inner core sacrificial material and eluting the first layer of sacrificial material from the central interface region. The perfusion flow rate is 0.3-0.5 mL / min, and the perfusion time is 2-5 min. During the removal of the first layer, the third layer of PVS precursor material remains on the outer side of the vessel wall, and together with the fourth outer layer material, maintains the lateral support of the vessel wall and the stability of the interface positioning. The central vascular lumen formed after the removal of the first layer should remain continuous and communicate with the central interface regions on both sides.

[0046] After forming a continuous central cardiovascular lumen, brain microvascular endothelial cells are introduced into the lumen via a central blood flow interface, allowing them to attach to the inner surface of the central cardiovascular lumen and form an endothelial layer. The brain microvascular endothelial cell suspension has a cell density of 1×10^6-1×10^7 cells / mL, an inoculation volume of 100-300 μL, a perfusion flow rate of 0.3-0.8 mL / min, and a continuous perfusion time of 4-8 h. After perfusion, the cells are allowed to stand for 1-4 h to promote the attachment and spread of endothelial cells on the inner surface of the central cardiovascular lumen.

[0047] After the initial establishment of the endothelial layer, the third layer of PVS precursor material is selectively removed through the bilateral interventricular fluid exchange zone, thereby forming a continuous PVS channel between the outer side of the vessel wall and the inner side of the brain parenchyma extramatrix. Cell-compatible culture medium is added to the bilateral interventricular fluid exchange zone, 0.5-1.5 mL on each side. After standing for 1-3 min, the entire sample is placed under low-amplitude oscillation or shaking conditions, allowing the culture medium to contact the third layer of PVS precursor material along the bilateral interventricular fluid exchange zone and continuously elute it. The oscillation angle is 2-8°, the oscillation frequency is 4-10 times / min, and the single oscillation removal time is 3-10 min, repeated 1-2 times. The removal process of the third layer should achieve continuous opening of the PVS channel while avoiding displacement of the second layer of vessel wall, local tearing of the fourth layer of brain parenchyma extramatrix, or local collapse of the PVS channel.

[0048] The first and third layers of sacrificial material are removed sequentially, rather than simultaneously. The removal of the first layer is used to prioritize the establishment of the central vascular lumen and endothelial colonization, while the removal of the third layer is used for subsequent establishment of the PVS channel. This sequential removal method ensures that the dual-fluid channel is established in the order of "central vascular lumen first, then peripheral PVS channel," thereby maintaining the stability of the interface between the vessel wall, PVS, and the extracellular matrix of the brain parenchyma during the dual-channel formation process.

[0049] (4) PVS interface orientation processing and interface stabilization After forming a continuous PVS channel, an interface modification solution is introduced into the PVS channel through the bilateral ventricular fluid exchange zone. This allows the interface modification components to distribute along the PVS channel and selectively act on the outer surface of the blood vessel wall and the inner surface of the extracellular matrix of the brain parenchyma, thereby forming a locally enriched interface modification layer on both sides of the PVS. The interface modification solution contains one or more of laminin, type IV collagen, and agrin; the concentration of laminin, type IV collagen, and agrin in the interface modification solution is 50-150 μg / mL, the concentration of type IV collagen is 50-150 μg / mL, and the concentration of agrin is 10-50 μg / mL.

[0050] After removing the third layer of PVS precursor material and confirming the continuous opening of the PVS channels, an interface modification solution was added to the fluid exchange areas of the left and right ventricles, allowing it to enter the PVS channels via the fluid exchange areas. The volume of interface modification solution added to each ventricle was 0.5-1.0 mL. After addition, the entire sample was placed under low-amplitude swinging or oscillation conditions to allow the interface modification solution to distribute along the PVS channels and fully contact the outer surface of the vessel wall and the inner surface of the extracellular matrix of the brain parenchyma. The low-amplitude swinging or oscillation conditions included a swing angle of 2-6°, a swing frequency of 4-8 times / min, and a treatment time of 5-15 min. After treatment, the sample was placed at 35-37°C for 10-30 min to promote the adsorption and retention of the interface modification components on both sides of the PVS interface. The interface modification did not involve uniformly incorporating the active ingredients into the fourth layer of the main body material, but rather through directional introduction and local adsorption via the PVS channels to enhance the local basement membrane-like characteristics and interfacial adhesion characteristics of the vessel wall-PVS-brain parenchyma interface.

[0051] After the interface modification is completed, the PVS channel is rinsed at a low speed to remove unbound components. Culture medium is added to the left and right intercomplex liquid exchange zones, 0.5-1.5 mL on each side, and rinsing is performed 1-2 times at a low speed to remove any unbound interface modification components remaining in the PVS channel, while avoiding strong disturbance to the adsorbed interface modification layer. After the low-speed rinsing, culture medium is added to the intercomplex liquid exchange zones again, and a short-term stabilization culture is performed to ensure the stable retention of the interface modification layer; 1.0-3.0 mL of culture medium is added to each intercomplex, and stabilization culture is continued for 6-24 h under low-amplitude oscillation conditions; the low-amplitude oscillation conditions include an oscillation angle of 2-6° and an oscillation frequency of 4-8 times / min.

[0052] (5) Establishment and maturation of multicellular interfaces After endothelial colonization and PVS interface orientation treatment, the overall construct underwent multicellular interface establishment and maturation culture. This allowed the pre-embedded perivascular cells in the second vascular wall layer and the pre-embedded astrocytes in the fourth brain parenchyma-like extrastromal layer to form a continuous multicellular interface between the vascular wall, PVS, and brain parenchyma. The maturation culture was conducted at 37°C and 5% CO2. During the culture process, the relative positions of the central vascular lumen, vascular wall, PVS channels, and brain parenchyma-like extrastromal layer should be kept stable, and excessive erosion or damage to the interface modification layer should be avoided in the early stages of culture.

[0053] After the initial formation of the endothelial layer and the completion of the PVS interface orientation treatment, the culture conditions of the extracellular matrix layer of the brain parenchyma within the central cardiovascular lumen and the perivascular layer were maintained. This allowed the perivascular cells in the second vascular wall layer to distribute along the vessel wall and form a vessel wall-related interface with the endothelial layer. Simultaneously, the astrocytes in the fourth vascular parenchyma extracellular matrix layer gradually extended outside the PVS and formed a cell distribution area adjacent to the vessel wall-PVS interface. During the culture process, a combination of intraluminal perfusion culture and peripheral culture medium maintenance was used to ensure that the endothelial layer, perivascular cells, and astrocytes each received a suitable culture environment.

[0054] During the maturation culture phase, the central blood vessel lumen is continuously perfused with endothelial cell culture medium or a co-culture medium compatible with endothelial cells to maintain the continuity and functional state of the endothelial layer; the perfusion flow rate of the central blood flow circuit is controlled at 0.5-1.0 mL / min. The peripheral extracellular matrix layer of the brain parenchyma is maintained by adding astrocyte culture medium or a co-culture medium compatible with peripheral cells to the fluid exchange area of ​​each intercostal space. The volume of culture medium added to each intercostal space is 1.0-3.0 mL, and fluid exchange is maintained under low-amplitude swinging or oscillation conditions. The swinging angle is 2-8°, and the swinging frequency is 4-12 times / min. The maturation culture time is 7-14 days, and the culture medium replacement cycle is 12-48 hours.

[0055] (6) Establishment of heterogeneous dual-irrigation system The sample, after undergoing multi-layer structure construction, sequential demolding, cell colonization, and interface orientation treatment, is fixed in the perfusion chamber, establishing a central blood flow circuit connected to the central vascular cavity and a peripheral cerebrospinal fluid-like circuit connected to the PVS channel. During sample fixation, the central vascular cavity should maintain continuous openings in the central interface areas on both sides, and the PVS channel should maintain open communication with the fluid exchange areas of both ventricles; simultaneously, torsion, compression, local stretching, or interface area collapse of the sample should be avoided during assembly.

[0056] The central blood flow circuit employs a pump-driven mechanism to provide controllable flow rate, pressure, and shear force, thereby creating hemodynamic boundary conditions within the vascular lumen. The pump-driven mechanism includes peristaltic pumps, syringe pumps, pressure-driven devices, or other methods capable of achieving continuous or pulsatile perfusion. The perfusion flow rate of the central blood flow circuit is controlled at 0.5-1.0 mL / min, corresponding to a wall shear stress of 5-15 dyne / cm². The peripheral cerebrospinal fluid-like circuit exchanges fluid through the bilateral interventricular reservoirs with a shaking or oscillating platform, creating fluid exchange boundary conditions outside the PVS. The fluid volume in each bilateral interventricular reservoir is 1.0-3.0 mL, the fluid level is 1-3 mm above the PVS channel opening, the oscillation angle is 4-12°, and the oscillation frequency is 6-18 times / min. This dual-perfusion system is a heterogeneous dual-perfusion system, rather than a dual-pump isomorphic drive or dual static culture method.

[0057] After system establishment, the central blood flow circuit and the peripheral cerebrospinal fluid-like circuit operate independently, with separate assembly and control modes. The central blood flow circuit maintains a continuous perfusion environment within the vessel lumen, while the peripheral cerebrospinal fluid-like circuit maintains a fluid exchange environment outside the PVS (platelet-like system). When changing or replenishing the culture medium, operations should be performed separately on the central blood flow reservoir and the bilateral interauricular reservoirs, avoiding significant air bubbles, fluid depletion, instantaneous high-pressure impacts, or backflow within the central blood vessel lumen and PVS channels. The central hemodynamic boundary conditions and the peripheral fluid exchange boundary conditions act on both sides of the same vessel wall-PVS-brain parenchyma interface and can be independently controlled, synchronously controlled, and coupled for analysis. Specifically, the hemodynamic state within the vessel lumen can be adjusted by changing the perfusion velocity, perfusion mode, pressure level, and perfusion fluid composition of the central blood flow circuit; the fluid exchange state outside the PVS can be adjusted by changing the volume of the bilateral interauricular reservoirs, fluid level, oscillation angle, oscillation frequency, and peripheral fluid composition.

[0058] Preferably, in step (1), the fourth outer layer material is a bioactive material that combines three-dimensional suspension support, brain parenchyma-like extracellular matrix modeling, and cell carrier functions; the material can be selected from one or more of the following: brain-derived decellularized extracellular matrix (BdECM)-alginate composite hydrogel, BdECM-HAMA composite hydrogel, BdECM-GelMA composite hydrogel, BdECM-hyaluronic acid composite system, BdECM-collagen composite system, BdECM-fibrin composite system, or alginate-GelMA composite hydrogel, and directly embeds astrocytes; the second vascular wall layer material is a material with tissue-derived specificity and The mechanically reinforced vascular wall layer composite bio-ink can be selected from one or more of the following: VdECM-alginate composite system, VdECM-collagen-alginate composite system, VdECM, VdECM-HAMA composite hydrogel, VdECM-GelMA composite hydrogel, collagen hydrogel, fibrin hydrogel, or alginate-gelatin composite hydrogel, and pre-embedded with cerebral vascular pericytes; the sacrificial materials of the first and third layers can be selected from one or more of PF-127, CPF-127, gelatin, agarose, or combinations thereof, and improve the instantaneous shaping ability during the printing process by releasing cross-linking ions or undergoing rapid cross-linking with the outer layer material.

[0059] Preferably, in step (2), a triaxial concentric nozzle is used to simultaneously deposit the first layer of central vascular lumen precursor, the second layer of vascular wall precursor, and the third layer of PVS precursor, with the fourth layer of outer material providing suspension support. The phased construction method includes: first, pre-filling the three layers of material, then asynchronously starting the flow in the interface area on one side in the order of the second, first, and third layers to form a short starting section; then entering the central construction area through the inlet transition section to form the bridging main section; and after entering the interface area through the outlet transition section on the other side boundary to form a termination connection section, and ending the discharge by asynchronous flow stopping. The inlet transition section and outlet transition section are used to compensate for the boundary conditions changing from "no suspension support" to "with suspension support" and from "with suspension support" to "no suspension support", respectively, to reduce the discontinuity of PVS precursor, wall thinning, center offset, end collapse, interface mismatch, and end swelling. The printing path starts from one side interface area, passes through the starting section, the inlet transition section, the bridging main body section, and the outlet transition section, and extends to the other side interface area, thereby forming a bridged independent structural unit; and in some embodiments, the deposition conditions and nozzle movement speed of the first, second, and third layers can be locally programmed and switched along the same continuous printing path to form a locally narrowed section, a locally wide-walled section, or a locally wide PVS section.

[0060] Preferably, in step (3), the overall shaping method includes ionic crosslinking triggered by CPF-127, calcium-containing core fluid, or other crosslinked core fluid, as well as temperature-induced gelation, photocrosslinking, enzymatic crosslinking, or a combination thereof after construction is completed; the first and third layers of material adopt a sequential demolding method, wherein the first layer is removed first through the central blood flow interface, and the third layer is removed through the bilateral ventricular fluid exchange area to ensure the stable positioning of the vascular wall during the formation of the dual fluid channel. This sequential demolding is not only used to form the central vascular lumen and PVS channel, but also to maintain the stability of the interface position between the vascular wall, PVS, and extracellular matrix of the brain parenchyma during the process of "first establishing the central vascular lumen and endothelial layer, and then establishing the peripheral PVS channel".

[0061] Preferably, in step (4), after the removal of the third layer of PVS precursor material, an interface modification solution is introduced into the PVS channel via a peripheral cerebrospinal fluid-like circuit. The interface modification solution contains one or more of laminin, type IV collagen, and agrin, and forms a local interface modification layer after subsequent low-speed rinsing and short-term stabilization culture. The interface modification does not involve uniformly incorporating the active ingredients into the outer layer material, but rather introduces them directionally through the PVS channel, allowing the interface modification components to selectively accumulate on the outer surface of the blood vessel wall and the inner surface of the extramatrix of the brain parenchyma, thereby forming a local basement membrane-like microenvironment on both sides of the PVS. In a specific embodiment, the interface modification solution can be a combination system of laminin 100 μg / mL, collagen IV 100 μg / mL, and agrin 25 μg / mL.

[0062] Preferably, in step (5), the maturation culture process employs a combination of intraluminal perfusion culture of the central blood vessel and maintenance with culture medium in the extracellular matrix layer of the peripheral brain parenchyma sample. This allows the pre-embedded perivascular cells in the second layer of the vessel wall and the pre-embedded astrocytes in the fourth layer of the extracellular matrix layer of the brain parenchyma sample to form a continuous multicellular interface between the vessel wall, PVS, and brain parenchyma. The second layer of vascular-derived material, the fourth layer of brain-derived material, and the local interface modification layers on both sides of the PVS together constitute a functional interface to promote the attachment of astrocyte foot processes and the establishment of AQP4-related distribution.

[0063] Preferably, in step (6), the dual perfusion system includes a culture frame with two ventricles, a central blood flow perfusion device, a peripheral cerebrospinal fluid reservoir, and a shaking incubator. The central blood flow circuit is driven by one or more of a peristaltic pump, syringe pump, pressure-driven pump, or microfluidic pressure control system. The peripheral cerebrospinal fluid reservoir is not driven by a pump but is connected to the PVS channel through the two ventricles and forms peripheral fluid exchange through shaking incubator oscillation or a swinging platform. The central blood flow circuit and the peripheral cerebrospinal fluid reservoir are independently configured and can independently control the flow rate, pressure, perfusion mode, and perfusion fluid composition to establish hemodynamic boundary conditions and fluid exchange boundary conditions on both sides of the same vessel wall-PVS-brain parenchyma interface, enabling independent analysis, synchronous control, and coupled analysis of the two.

[0064] Example 1 In this embodiment, BdECM and VdECM are used as the main biological substrates, combined with HBMECs, HBVPs and astrocytes, and a four-layer brain lymphatic circulation system model with central vascular lumen, vascular wall, PVS channel and brain parenchyma-like extracellular matrix layer is constructed by "suspension-coaxial" bio-3D printing method.

[0065] 1. Tissue-derived decellularized matrix and cell preparation, such as Figure 2 As shown Fresh porcine cerebral cortex and cerebral vascular tissue were collected and cut into small pieces of approximately 5 mm × 5 mm × 5 mm. These pieces were placed in sterile centrifuge tubes and rinsed three times with deionized water for 10 min each time to remove residual blood. Then, 0.1% Triton X-100 solution was added, and the mixture was shaken at 4°C for 24 h. After treatment, the tissues were rinsed three times with deionized water for 10 min each time. DNase solution was then added for 6 h, followed by treatment with 0.1% peracetic acid for 2 h to complete decellularization and sterilization. The treated tissues were washed with deionized water, lyophilized for 48 h, and ground into powder. 10 mL of 0.01 M acetic acid solution containing pepsin was added to every 100 mg of lyophilized powder, and the mixture was magnetically stirred at room temperature for 48 h for digestion. After digestion, the solution was neutralized to pH 7.0–7.4 with NaOH and 10×PBS to obtain BdECM and VdECM precursor solutions, respectively.

[0066] HBMECs were expanded and cultured using a dedicated endothelial cell culture medium at 37°C and 5% CO2; HBVPs were expanded and cultured using pericyte culture medium; and astrocytes were expanded and cultured using astrocyte culture medium. After all cell types reached 80%-90% confluence, they were digested with 0.25% trypsin, centrifuged at 1000 rpm for 5 min to collect the cell pellet, and resuspended in serum-free basal medium for later use.

[0067] 2. Preparation of Ink Materials and Interface Modification Solution The fourth outer layer material used was a hybrid BdECM system, specifically formulated as 1.0 wt% BdECM and 0.5 wt% alginate. The BdECM precursor solution was pre-cooled at 15°C, then alginate was added and mixed at low speed for 30 min. After mixing, astrocytes were added at a density of 2 × 10^6 cells / mL, and gently pipetted to mix, thus obtaining the fourth outer layer material. The volume of the fourth outer layer material used for each construct was 3.0 mL.

[0068] The second layer, the bio-ink for the vascular wall, uses a composite system of 1.0 wt% VdECM and 0.5 wt% alginate. The VdECM precursor solution was pre-cooled at 15°C, then alginate was added and mixed at low speed for 30 min. After mixing, HBVPs were added at a cell density of 2 × 10^6 cells / mL, gently pipetted to mix, and then loaded into the middle layer printing cartridge for later use.

[0069] Both the first-layer inner core material and the third-layer PVS precursor material use CPF-127. The preparation method is as follows: 50 g of PF-127 and 4 g of calcium chloride are added to 50 mL of sterile water, followed by the addition of another 50 mL of sterile water to form a liquid-solid-liquid stratified system. After autoclaving, the system is placed at 4°C for 24 h to fully dissolve, yielding 50 wt% CPF-127 sacrificial material. The obtained CPF-127 is then loaded into the inner core cartridge and the outer sacrificial sheath cartridge, respectively, for the simultaneous formation of the central vascular luminal precursor and the PVS precursor.

[0070] In this embodiment, the four layers of material from the inside out are as follows: the first inner core layer uses CPF-127 and does not embed cells; the second vessel wall layer uses a 1.0 wt% VdECM + 0.5 wt% alginate composite system embedding HBVPs; the third PVS precursor layer uses CPF-127 and does not embed cells; the fourth outer layer material uses a 1.0 wt% BdECM + 0.5 wt% alginate composite system embedding astrocytes. HBMECs are not pre-embedded in the printing material, but are implanted onto the inner surface of the vessel lumen after the continuous central vascular lumen is formed.

[0071] In this embodiment, the interface modification solution uses three components simultaneously: laminin, collagen IV, and agrin. The specific preparation method is as follows: using serum-free basal culture medium as a solvent, laminin, collagen IV, and agrin are added sequentially under ice bath conditions to prepare interface modification solutions with final concentrations of 100 μg / mL, 100 μg / mL, and 25 μg / mL, respectively. The solutions are gently inverted to mix, avoiding vigorous shaking to prevent air bubbles. After preparation, the solutions are stored at 4°C for later use and should be used on the same day.

[0072] 3. Cross-interface bridging dual-sacrificial-layer synchronous coaxial printing build, such as... Figure 3 As shown The model framework was printed using polyethylene-vinyl acetate (PVC) to create a dual-chamber culture framework. The framework's external dimensions were 20 mm × 20 mm × 2 mm; the central printing area measured 10 mm × 6 mm × 2 mm; the fluid reservoirs in both chambers measured 4 mm × 5 mm × 2 mm; the central blood flow interface had an inner diameter of 1.0 mm; and the connection between the chamber and the PVS (polyvinyl acetate) port was 1.0 mm wide. The printing parameters were: printing pressure 120 kPa, nozzle diameter 600 μm, and printing speed 10 mm / s. After printing, the framework was fixed to the bottom of a transparent culture dish, and 3.0 mL of the fourth outer layer material embedding astrocytes was added to the central printing area. The dish was then kept at 15°C to maintain fluid dynamics for later use.

[0073] Subsequently, a triaxial concentric printhead was used to construct the fourth outer layer material. The printhead specifications were 13G / 17G / 22G; the innermost channel was loaded with CPF-127 to form a central vascular luminal precursor, the middle channel was loaded with VdECM composite vascular wall bio-ink embedding HBVPs, and the outermost channel was loaded with CPF-127 to form a PVS precursor. Before printing, the first, second, and third layers of material were driven to positions close to the printhead exit to complete pre-filling.

[0074] After pre-filling, move the nozzle to the starting position of the left interface area. First, activate the intermediate vessel wall layer channel, allowing it to discharge material in situ at 35 kPa for 0.5 s; then activate the inner core CPF-127 channel, allowing it to discharge material at 500 kPa for 0.5 s, forming a stable two-layer coaxial precursor inside the intermediate layer; then activate the outer PVS precursor channel, allowing it to discharge material at 200 kPa for 0.5 s, forming a continuous outer jacket layer outside the second layer. After all three layers of material have established stable discharge, continue discharging material in situ in the left interface area for 1.0 s, forming a short initial connecting section.

[0075] After the initial connecting section is formed, the nozzle moves along a preset path from the left interface area to the central printing area. Before entering the suspension support area, a 3 mm long inlet transition section is set, and the nozzle's movement speed is reduced to 400 mm / min. The channel pressures of layers 1, 2, and 3 are adjusted to 480 kPa, 40 kPa, and 180 kPa, respectively, to compensate for changes in support conditions. After the nozzle enters the fourth outer layer material, the bridging main body parameters are restored, and it continuously crosses the central printing area along the preset path to form a 10 mm long bridging main body section. In the bridging main body section, the channel pressure of the first inner core CPF-127 layer is 500 kPa, the channel pressure of the second blood vessel wall layer is 35 kPa, and the channel pressure of the third PVS precursor layer is 200 kPa. The nozzle's movement speed is 600 mm / min.

[0076] A transition section with a length of 3 mm is set before reaching the right-side suspension pool boundary. The nozzle movement speed is reduced to 400 mm / min, and the channel pressures of the first, second, and third layers are adjusted to 450 kPa, 35 kPa, and 180 kPa, respectively, to reduce end-point instability when leaving the suspension support environment. After the nozzle enters the right-side interface area, it continues to discharge material continuously for 1.0 s, forming the termination connection section. Subsequently, an asynchronous flow-stopping method is adopted: first, the pressure of the third-layer PVS precursor channel is reduced to 100 kPa and maintained for 1.5 s, allowing the outer layer to gradually finish forming; then, the first and second layers are discharged synchronously for about 1.5 mm; then, the discharge of the first-layer core material is stopped, and finally, the discharge of the second-layer vessel wall material is stopped, thus completing the construction of the termination section.

[0077] The target structural dimensions after construction were: an inner diameter of approximately 500 μm for the central vascular lumen, a vessel wall thickness of approximately 150 μm, a PVS width of approximately 200 μm, and a total outer diameter of approximately 1.2 mm. During construction, the deposited structure achieved rapid ionic cross-linking of the alginate components in the BdECM and VdECM composite system, triggered by the rheological support of the fourth outer layer material and calcium ions released by CPF-127, resulting in immediate shaping. After the entire precursor structure was constructed, the vascular lumen precursor, vessel wall precursor, and PVS precursor were observed arranged sequentially from the inside out in the outer layer material.

[0078] 4. Overall shaping, sequential demolding, and inner skin implantation. After the bridging multilayer precursor structure was constructed, the whole sample was placed in a 37°C incubator for 20 min to allow BdECM and VdECM to undergo further temperature-induced gelation and complete the overall molding.

[0079] Subsequently, the first layer of mold removal was prioritized. Endothelial cell culture medium was slowly perfused through the central blood flow interface at a flow rate of 0.3 mL / min for a total perfusion time of 3 min to remove CPF-127 from the first layer of the core, thereby forming a continuous central vascular lumen. After the first layer of mold removal was completed, the vessel wall was still supported by the third layer of PVS precursor and the fourth layer of brain parenchyma-like extramatrix, so no overall drift occurred at this stage.

[0080] Endothelial colonization was performed immediately after the formation of the central blood vessel lumen. HBMECs were prepared into a cell suspension at a cell density of 5 × 10^6 cells / mL, and the seeding volume was 200 μL. The suspension was slowly perfused into the blood vessel lumen through the central blood flow interface at a perfusion rate of 0.5 mL / min for 6 h to allow HBMECs to adhere to the inner surface of the lumen and form a continuous endothelial layer. After perfusion, the vessel was allowed to stand for 2 h to enhance cell adhesion stability.

[0081] After initial colonization of HBMECs, the third layer of mold removal was performed. 1.0 mL of astrocyte culture medium was added to each of the two ear chamber reservoirs. After standing for 2 min, the samples were placed on a oscillating shaker and run at a low amplitude oscillation of 4° and 6 times / min for 5 min to remove CPF-127 from the third layer of PVS precursor. This process was repeated once. After this sequential mold removal, a continuous annular PVS channel was formed around the vessel wall, maintaining the stable positioning of the vessel wall within the fourth outer layer material.

[0082] 5. PVS interface-directed treatment and stabilization culture After the third layer was demolded and a continuous PVS channel was formed, interface orientation treatment was immediately performed. 0.8 mL of interface modification solution was added to each of the left and right ear chambers. The samples were then placed on a oscillating shaker and oscillated at a 4° angle and a frequency of 6 times / min for 10 min to allow laminin, collagen IV, and agrin to distribute along the PVS channel and act directionally on the outer surface of the vessel wall and the inner surface of the fourth layer of brain parenchyma matrix. The samples were then incubated at 37°C for 20 min to promote adsorption. Subsequently, 1.0 mL of astrocyte culture medium was added to each ear chamber for a low-speed rinse to remove unbound components. Then, 2.0 mL of astrocyte culture medium was added to each ear chamber, and the samples were further stabilized at a 4° angle and a frequency of 6 times / min for 12 h to promote the stable retention of the local interface modification layer.

[0083] 6. Establishment and maturation of multicellular interfaces After endothelial colonization and PVS interface orientation treatment, the maturation culture stage began. Endothelial cell culture medium was continuously perfused into the central blood flow loop at a flow rate of 0.8 mL / min using a continuous perfusion mode. Astrocyte culture medium was added to the bilateral interauricular reservoirs, with 2.0 mL added to each chamber to maintain the survival of astrocytes and interface stability in the extracellular matrix of the fourth layer of brain parenchyma. The maturation culture period was set at 14 days, with the culture medium in the central blood flow reservoir and bilateral interauricular reservoirs replaced every 24 hours. Through these steps, HBVPs can be preserved in the second layer of the vessel wall, astrocytes in the fourth layer of the extracellular matrix of brain parenchyma, and local interface modification layers can be formed on both sides of the PVS, thereby establishing a multicellular functional interface between the vessel wall, PVS, and brain parenchyma. Figure 4 As shown.

[0084] 7. Design and operation of heterogeneous dual-irrigation systems, such as... Figure 5 As shown A dual perfusion system was constructed, comprising an acrylic bioreactor, a central blood flow reservoir, peripheral fluid storage areas in both auricles, a sample perfusion chamber, central blood flow connecting tubing, a peristaltic pump, and a shaker. Matured samples were fixed in the perfusion chambers, connecting the central blood flow loop to the central blood vessel cavity and the peripheral fluid storage areas in both auricles to the PVS channel. The central blood flow connecting silicone tubing was medical-grade silicone tubing with an inner diameter of 1.0 mm and a single loop fluid storage volume of 5 mL.

[0085] During the culture phase, the central blood flow circuit employed a continuous perfusion mode driven by a peristaltic pump, with a flow rate set at 0.8 mL / min and a wall shear stress set at 10 dyne / cm² to establish stable hemodynamic boundary conditions within the vascular lumen. The peripheral cerebrospinal fluid-like circuit incorporated a cell-compatible culture system through the bilateral interventricular reservoirs, with each ventricular reservoir containing 2.0 mL of fluid, the fluid level 1-2 mm above the PVS connection. The entire apparatus was then placed on a oscillating shaker within a cell culture incubator, with an oscillation angle of 8° and an oscillation frequency of 12 times / min for continuous oscillation culture. This ensured continuous exchange of peripheral fluid between the interventricular reservoirs and the PVS channels, thereby establishing fluid exchange boundary conditions outside the PVS. The entire system was operated in a 37℃, 5% CO2 incubator. The culture medium in the central blood flow reservoir and the bilateral ear chamber reservoir was changed every 24 hours. When changing the medium, 1.8 mL of the old medium was first removed, and then 2.0 mL of the new medium was added to avoid completely draining the system and causing air to enter the channels.

[0086] When subsequent fluid exchange or pathological molecular transport experiments are required, the peripheral culture medium in the bilateral interaural ventricles is switched to artificial cerebrospinal fluid (aCSF) or cerebrospinal fluid-like perfusion fluid, with 2.0 mL added to each ventricle. The shaker parameters remain unchanged, and peripheral fluid exchange continues. The central blood flow loop maintains the perfusion medium during this stage to ensure the stability of the vascular endothelial and vascular wall interfaces. Thus, hemodynamic boundary conditions and fluid exchange boundary conditions are established on both sides of the same "vascular wall-PVS-brain parenchyma" interface, which can be further used for independent regulation, synchronous regulation, and coupled analysis.

[0087] Comparative Example 1: Conventional synchronous start-stop printing is used, without pre-filling, asynchronous start-up, boundary compensation, and asynchronous stop-up. In this comparative example, except for the absence of the phased construction strategy of "pre-filling-asynchronous start-up-synchronous construction-asynchronous stop-up" during the printing stage, the material composition, cell configuration, and target structure size are the same as in Example 1. Specifically, during printing, the three layers of material are started simultaneously, enter the bridging path simultaneously, and stop discharging synchronously at the endpoint; at the same time, no inlet and outlet transition sections are set, and no boundary compensation is performed for changes in the support conditions at the pool edge.

[0088] The results showed that in the initial stage, because both the first and third layers were sacrificial layers, it was difficult for the three layers to establish a stable fit instantaneously at the nozzle outlet. This resulted in discontinuities in the initial stage of the third layer PVS precursor, local thinning of the second layer wall, and center shift of the first layer. When entering and leaving the suspension support zone, the two ends of the bridging main section were more prone to outer layer discontinuity, end retraction, end-point swelling, and interface mismatch. Although some samples could form a mid-segment vascular-like structure, the correspondence between the interfaces at both ends was poor, making it difficult to stably obtain bridging independent units that could be used for subsequent perfusion and fluid exchange.

[0089] The above results show that the pre-filling, asynchronous start-up, boundary compensation, and asynchronous stop-up methods used in this invention are not general printing details, but key methodological innovations that enable stable implementation of the double-sacrifice layer cross-interface bridging construction. If the conventional synchronous start-up and stop-up method is still used, it is difficult to guarantee the structural integrity and repeatability of the start, transition, and end segments.

[0090] Comparative Example 2: Using non-bridging embedded printing, neither end extends into the interface area. In this comparative example, except that the printing path does not use the bridging independent blood vessel printing method, the material composition, three-layer construction method, overall shaping, sequential demolding, and culture conditions are all the same as in Example 1. Specifically, during printing, only the embedded tubular structure is formed in the central construction area, and the two ends of the blood vessel segment are not allowed to extend into the corresponding positions of the central blood flow interface area and the bilateral ventricular fluid exchange area.

[0091] The results showed that although the central vascular lumen precursor, vascular wall precursor and peripheral PVS precursor could be formed in the central construction area in this comparative model, and the vascular lumen and PVS channel were formed after sequential demolding, the subsequent central perfusion and peripheral fluid exchange were difficult to directly access because the two ends did not enter the interface area in advance. Additional puncture, drilling or local incision was often required to try to establish the pathway connection, which led to increased structural disturbance, reduced interface repeatability and easy leakage or local collapse.

[0092] The above results indicate that the innovation of the bridging independent blood vessel printing method in this invention lies in the fact that it does not simply print out the multi-layer structure, but simultaneously establishes the interface space correspondence required for subsequent perfusion and fluid exchange during the printing stage. If this innovation is missing, although a multi-layer structure can be obtained, the system integration and subsequent operation adaptability will be significantly insufficient.

[0093] Comparative Example 3: Simultaneous demodulation is used instead of sequential demodulation. In this comparative example, except for the method of removing the sacrificial layer, the other material composition, construction steps, cell configuration, and culture conditions are the same as in Example 1. Specifically, after the overall molding is completed, the first layer is not removed before the third layer; instead, the sacrificial material in the first and third layers is removed simultaneously through the central blood flow interface and the fluid exchange area of ​​the two ventricular chambers, i.e., a simultaneous molding removal method is adopted.

[0094] The results showed that although the sacrificial materials in layers 1 and 3 could be removed, local displacement of the vessel wall was more likely to occur during the simultaneous opening of the dual-fluid channels, resulting in decreased uniformity of PVS width. In some samples, local PVS channel collapse or vessel wall displacement was observed. Compared with Example 1, the interface position in the middle of the bridging segment was more likely to be unstable under the simultaneous demolding method, and the structural reproducibility during subsequent endothelial colonization and long-term culture was also significantly reduced.

[0095] The above results indicate that the sequential mold removal process of "first establishing the central vascular cavity and endothelial layer, then establishing the peripheral PVS channel" in this invention is not a general process sequence, but a key innovative step to ensure the stability of the geometric relationship and interface position of the dual channels; if it is changed to simultaneous mold removal, it is difficult to take into account both the formation of the dual channels and the stable positioning of the blood vessel wall.

[0096] Comparative Example 4: No PVS interface orientation treatment was performed, or the interface modification components were uniformly mixed into the host material. In this comparative example, except for the interface treatment method, the other construction steps, cell configuration, and maturation culture conditions were the same as in Example 1. First, after the PVS channel was formed, the interface modification solution was no longer introduced through the dual-chamber liquid exchange zone, but was directly introduced into conventional culture. Second, in another comparative example, laminin, collagen IV, and agrin were pre-mixed uniformly into the fourth outer layer material, without directional interface treatment after PVS formation.

[0097] The results showed that, although a complete four-layer structure could be formed without interface-directed treatment, the interfaces on both sides of the PVS lacked locally enriched modification layers, and the biological differences at the vessel wall-PVS-brain parenchyma interface were not prominent. When the interface modification components were uniformly mixed into the host material, the active ingredients were distributed throughout the entire outer layer, making it difficult to form a local basement membrane-like microenvironment targeting the interfaces on both sides of the PVS. Compared to Example 1, the specificity and stability of the interface regions in both comparative examples were weaker, which was not conducive to highlighting the local interface effects of astrocyte foot process attachment and AQP4-related distribution.

[0098] The above results indicate that the interface modification strategy of directional introduction and local enrichment via PVS channels in this invention, which differs from the existing approaches of no treatment and homogeneous mixing, is an important innovation for achieving biomimetic reconstruction of the blood vessel wall-PVS-brain parenchyma interface.

[0099] Comparative Example 5: Using unilateral irrigation or isomorphic dual irrigation, without establishing heteromorphic bilateral boundary conditions. In this comparative example, except for the perfusion system setup, the material composition, construction steps, cell colonization, sequential demolding, interface modification, and maturation culture conditions were all the same as in Example 1. Specifically, one comparative example retained only the central blood flow loop without setting up a peripheral fluid exchange system; in the other comparative example, although two fluid pathways were set up simultaneously, the same type of pump was used to drive perfusion on both the central and peripheral sides, instead of the heterogeneous driving method of "central pump-driven perfusion + peripheral dual-ventricle oscillatory fluid exchange".

[0100] The results showed that under unilateral perfusion conditions, the model could only establish central hemodynamic boundary conditions, but could not establish stable fluid exchange boundary conditions on the outside of the PVS. Under isomorphic dual perfusion conditions, although two fluid pathways could be formed, it was difficult to reflect the differences in driving mechanisms, fluid exchange methods, and boundary condition properties between the inner side of the vessel lumen and the outer side of the PVS. Compared with Example 1, both of these comparative examples failed to effectively simulate the synergistic effect of different physical conditions on both sides of the same vessel wall-PVS-brain parenchyma interface.

[0101] The above results indicate that the innovation of the heterogeneous dual perfusion system in this invention lies not in setting up two pathways themselves, but in establishing hemodynamic boundary conditions and peripheral fluid exchange boundary conditions on both sides of the same interface, and realizing independent regulation, synchronous regulation and coupling analysis of the two; if this innovation is missing, the model's ability to simulate bilateral coupling effects related to brain lymphatic circulation will be significantly limited.

[0102] In summary, the invention includes, but is not limited to, the above embodiments. Any equivalent substitutions or partial improvements made under the spirit and principles of this invention shall be considered to be within the protection scope of this invention.

Claims

1. A method for constructing an in vitro brain-like lymphatic circulation system model that reconstructs the structural-functional interface of the "brain parenchyma-perivascular space", characterized in that: The method steps include: (1) Using extracellular matrix composite bio-ink containing embedded astrocytes, a brain parenchyma-like extracellular matrix precursor was suspended and printed in the central construction area of ​​a dual-chamber culture frame to form a brain parenchyma-like extracellular matrix layer precursor; (2) The inner layer sacrificial ink, the extracellular matrix composite bio-ink for embedding cerebral vascular pericytes and the PVS precursor sacrificial ink were sequentially filled into the inner, middle and outer channels of the triaxial concentric nozzle. Coaxial printing was performed using asynchronous start-up, synchronous construction and asynchronous stop-up. The central vascular lumen precursor, vascular wall precursor and peripheral PVS precursor were formed from the inside to the outside in the extracellular matrix precursor of the brain parenchyma sample to obtain the model precursor. (3) The model precursor is fixed to gel the brain parenchyma-like extrastromal layer precursor and vascular wall layer precursor to form the brain parenchyma-like extrastromal layer and vascular wall layer. (4) The precursor of the central blood vessel formed by the inner layer sacrificial ink is perfused to remove the sacrificial material to form a continuous central blood vessel. Brain microvascular endothelial cell suspension is perfused into the central blood vessel to allow brain microvascular endothelial cells to attach to the inner surface of the central blood vessel and form an endothelial layer. (5) Selectively remove the peripheral PVS precursor formed by PVS precursor sacrificial ink to form a continuous PVS channel between the outer side of the blood vessel wall and the inner side of the brain parenchyma matrix. Introduce interface modification solution into the PVS channel to allow the interface modification components to adhere to the outer side of the blood vessel wall and the inner side of the brain parenchyma matrix. Rinse to remove unbound components, culture under oscillation conditions to form a system model. (6) Maturation of the system model under oscillating conditions; (7) The mature cultured system model is fixed in the perfusion chamber, and a central blood flow circuit connected to the central cardiovascular cavity and a peripheral cerebrospinal fluid circuit connected to the PVS channel are established to form an in vitro brain lymphatic circulation system model with a "brain parenchyma-perivascular space" structure-functional interface.

2. The method for constructing an in vitro brain-like lymphatic circulation system model for reconstructing the "brain parenchyma-perivascular space" structure-functional interface as described in claim 1, characterized in that: In step (1), the extracellular matrix composite bio-ink for embedding astrocytes is obtained by embedding astrocytes in one or more of the following systems: BdECM-alginate composite system, BdECM-HAMA composite system, BdECM-GelMA composite system, BdECM-hyaluronic acid composite system, BdECM-collagen composite system, BdECM-fibrin composite system, and alginate-GelMA composite system, with a cell density of 1×10^6~1×10^7 cells / mL.

3. The method for constructing an in vitro brain-like lymphatic circulation system model for reconstructing the "brain parenchyma-perivascular space" structure-functional interface as described in claim 1, characterized in that: In step (2), the sacrificial material in the inner layer sacrificial ink and the PVS precursor sacrificial ink is one or more of PF-127, CPF-127, gelatin and agarose; The extracellular matrix composite bio-ink for embedding cerebral perivascular cells is obtained by embedding cerebral perivascular cells with one or more of the following systems: VdECM-alginate composite system, VdECM-collagen-alginate composite system, VdECM, VdECM-HAMA composite system, VdECM-GelMA composite system, collagen hydrogel, fibrin hydrogel, and alginate-gelatin composite system, with a cell density of 1×10^6~1×10^7 cells / mL.

4. The method for constructing an in vitro brain-like lymphatic circulation system model for reconstructing the "brain parenchyma-perivascular space" structure-functional interface as described in claim 1, characterized in that: In step (2), when asynchronously starting the flow, the flow is started sequentially in the order of intermediate layer channel, inner layer channel and outer layer channel; During synchronous construction, the nozzle moving speed is 300~900 mm / min, the deposition pressure of the inner channel is 400~550 kPa, the deposition pressure of the middle channel is 30~50 kPa, the deposition pressure of the outer channel is 160~220 kPa, the inner diameter of the formed central vascular lumen precursor is 400~600 μm, the thickness of the vascular wall precursor is 100~180 μm, and the thickness of the outer PVS precursor is 150~250 μm. When asynchronously stopping the flow, the flow is stopped sequentially in the order of outer layer channel, inner layer channel, and intermediate layer channel.

5. The method for constructing an in vitro brain-like lymphatic circulation system model for reconstructing the "brain parenchyma-perivascular space" structure-functional interface as described in claim 1, characterized in that: In step (3), during the shaping process, the model precursor is placed in an incubator at 35~37℃ and incubated for 15~30 minutes.

6. The method for constructing an in vitro brain-like lymphatic circulation system model for reconstructing the "brain parenchyma-perivascular space" structure-functional interface as described in claim 1, characterized in that: In step (4), when removing the sacrificial material, culture medium is perfused into the central cardiovascular lumen precursor at a flow rate of 0.3-0.5 mL / min for 2-5 min. The cell density of the brain microvascular endothelial cell suspension was 1×10^6~1×10^7 cells / mL, the perfusion flow rate was 0.3~0.8 mL / min, the perfusion time was 4~8h, and after perfusion, it was left to stand for 1~4h.

7. The method for constructing an in vitro brain-like lymphatic circulation system model for reconstructing the "brain parenchyma-perivascular space" structure-functional interface as described in claim 1, characterized in that: In step (5), when removing the sacrificial material, perfuse with a culture medium compatible with the surrounding cells. After perfusing, let stand for 1-3 minutes, and then perform low-amplitude swinging or oscillation. The swing angle is 2-8°, the swing frequency is 4-10 times / min, the single swing time is 3-10 minutes, and repeat 1-2 times.

8. The method for constructing an in vitro brain-like lymphatic circulation system model for reconstructing the "brain parenchyma-perivascular space" structure-functional interface as described in claim 1, characterized in that: In step (5), the interface modification solution contains one or more of laminin, type IV collagen, and agrin; the concentration of laminin is 50-150 μg / mL, the concentration of collagen IV is 50-150 μg / mL, and the concentration of agrin is 10-50 μg / mL; after introducing the interface modification solution, low-amplitude shaking is performed with a shaking angle of 2-6°, a shaking frequency of 4-8 times / min, and a shaking time of 5-15 min. After shaking, the solution is allowed to stand at 35-37°C for 10-30 min; the stable culture time is 6-24 h.

9. The method for constructing an in vitro brain-like lymphatic circulation system model for reconstructing the "brain parenchyma-perivascular space" structure-functional interface as described in claim 1, characterized in that: In step (6), the maturation culture conditions are 37℃ and 5% CO2. The central blood vessel lumen is continuously perfused with endothelial cell culture medium or co-culture medium compatible with endothelial cells. The outer matrix layer of the peripheral brain parenchyma is maintained by adding astrocyte culture medium or co-culture medium compatible with peripheral cells. The swing angle is 2~8°, the swing frequency is 4~12 times / min, the maturation culture time is 7~14 days, and the culture medium replacement cycle is 12~48 h.

10. An in vitro brain lymphatic circulation system model with a "brain parenchyma-perivascular space" structure-functional interface, characterized in that: It is constructed by the method described in any one of claims 1 to 9.

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