Centimeter / decimeter-scale three-dimensional vascularization tissue engineering organoid culture device

Abstract

The invention belongs to the technical field of tissue engineering, and relates to a centimeter / decimeter-level three-dimensional vascularization tissue engineering organoid culture device which comprises a culture room, a semipermeable membrane microtube and a peristaltic pump, the semi-permeable membrane microtube is positioned in the inner cavity of the culture device; the inner ends of the culture solution inlet and outlet are respectively communicated with two ends of the semi-permeable membrane microtube; the inner ends of the vascular nutrient solution inlet and outlet are respectively communicated with the inner cavity of the culture chamber; the outer ends of the culture solution inlet and outlet are respectively communicated with a peristaltic pump and a waste liquid collector; the outer end of the blood vessel nutrient solution inlet and outlet is respectively communicated with another peristaltic pump and a waste liquid collector; the vascularization tissue engineering organoid culture system is constructed through the design of double independent circulation systems and adjustable shearing force, oxygen and nutrition can be effectively provided for centimeter / decimeter-level tissue engineering organoid in vitro, metabolites can be taken away, meanwhile, a vascular system can be formed in the cultured tissue engineering organoid, and the tissue engineering organoid can be cultured in vitro. And an efficient and controllable in-vitro culture platform is provided for vascularized tissue engineering organs.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue engineering and relates to a centimeter / decimeter level three-dimensional vascularized tissue engineering organoid culture device. Background Art

[0002] Conventional culture dishes are used for cell monolayer culture, which are all two-dimensional environments and cannot simulate the three-dimensional environment in the body, and lack hemodynamic simulation. When using the Transwell system for cell culture, the system is designed with an upper and lower layer, with a porous membrane in the middle, and the cells are cultured on the membrane. This system relies on passive diffusion to achieve material exchange, and the co-culture system adopts a physical isolation design (porous membrane separation), which hinders direct paracrine signal transmission between cells. The use of non-biocompatible materials such as PLA to print scaffolds only provides a static three-dimensional structure and cannot achieve dynamic nutrient circulation. The single-layer micro-controlled flow vascular chip system has a single microchannel structure, which leads to a gradient distribution of nutrients and uncontrollable local cell metabolic microenvironment. This system can only achieve cell adherent culture and does not integrate multi-cell and tissue co-culture functions.

[0003] Therefore, the existing cell culture devices have the following objective defects:

[0004] 1) Insufficient simulation of the dynamic physiological environment; traditional culture devices (such as culture dishes and Transwell systems) are unable to simulate mechanical stimuli such as blood flow shear force and pulsatile pressure in the vascular system, resulting in the inability of vascular endothelial cells to form a functional barrier (such as insufficient expression of tight junction proteins).

[0005] 2) The co-culture system has poor interactivity; existing co-culture devices mostly use physical isolation designs (such as porous membrane separation), which hinders the direct paracrine signal transmission between vascular cells and surrounding cells, resulting in a lack of intercellular synergy.

[0006] 3) Low precision in nutrient supply and metabolic control; static culture relies on passive diffusion, and microfluidic single-channel systems are prone to forming nutrient gradients, leading to local cell proliferation stagnation or necrosis.

[0007] 4) Real-time observation and quantitative analysis are difficult; existing devices mostly use non-transparent materials (such as porous membrane structures or opaque 3D-printed resins), which cannot track the dynamic process of angiogenesis (such as capillary sprouting, endothelial cell tube formation and connection) in real time, and the lack of positioning markers leads to large image registration errors.

[0008] Therefore, a cell culture device or method that can achieve dynamic nutrient circulation and controllable local cell metabolic microenvironment is needed to solve the above technical problems. Summary of the Invention

[0009] The technical solution adopted by the present invention to solve the technical problem is: a centimeter / decimeter-level three-dimensional vascularized tissue engineering organ culture device, comprising: a culture chamber, a culture fluid inlet pool, a culture fluid outlet pool, a diverter (porous partition), a semipermeable membrane microtube, a first peristaltic pump, and a second peristaltic pump; an external peristaltic pump (adjustable flow rate 0.1-10mL / min) drives the culture fluid to circulate in the vascular circuit, simulating pulsating shear force (10-30dyn / cm 2 )

[0010] The culture chamber is divided into a culture fluid inlet pool, a culture chamber and a culture fluid outlet pool by a diverter (porous partition);

[0011] The semipermeable membrane microtube is located in the culture chamber, and the semipermeable pore diameter of the semipermeable membrane microtube ranges from 0.1 μm to 12.0 μm;

[0012] The culture fluid inlet and the culture fluid outlet of the culture device are connected to the culture fluid inlet pool and the culture fluid outlet pool of the culture device respectively; the inner port of the culture fluid inlet is connected to the culture fluid inlet pool, and the inner port of the culture fluid outlet is connected to the culture fluid outlet pool; the inner ports of the vascular nutrient solution channels are connected to the culture chambers respectively;

[0013] There are two diverters (porous partitions). The front diverter is located in the culture chamber near the culture fluid inlet pool, and the rear diverter is located in the culture chamber near the culture fluid outlet pool. Each diverter is divided into two parts: the upper diverter and the lower diverter, for a total of four.

[0014] The semipermeable membrane microtube is inserted into the diversion hole of the front and rear diverters and fixed; the external port of the culture fluid inlet and the external port of the culture fluid outlet are connected to the first peristaltic pump and the culture fluid storage tank respectively; the external port of the vascular nutrient solution channel is connected to the second peristaltic pump and the nutrient solution storage tank respectively;

[0015] The external port of the culture fluid inlet is connected to a first peristaltic pump, the external port of the culture fluid outlet is connected to a waste liquid collector, and the first peristaltic pump is connected to a culture fluid storage tank; the external port of the vascular nutrient fluid inlet is connected to a second peristaltic pump, the external port of the vascular nutrient fluid outlet is connected to a waste liquid collector, and the second peristaltic pump is connected to a nutrient fluid storage tank; the cells are mixed with matrix gel and injected into the culture chamber, and the semipermeable membrane tube runs through the culture chamber and is connected to the external circulation system to achieve three-dimensional interaction between blood vessels and surrounding cells; a dual-circulation nutrient supply system is adopted, in which the vascular circuit (supplying nutrients required for blood vessel sprouting and growth) and the semipermeable membrane tube microcirculation (supplying nutrients required for cell growth) work together.

[0016] Preferably, the culture chamber is in the shape of a trough with an opening facing upward, and an upper cover is provided at the opening of the culture chamber.

[0017] More preferably, the upper cover is provided with an observation window made of a transparent material; there is a glass observation window on the upper cover, which supports continuous imaging with a 40x objective lens, and a laser confocal microscope or a fluorescence microscope can be used to observe the growth status of cell tissue in real time.

[0018] More preferably, the culture chamber and the upper cover are made of transparent materials; the culture chamber and the upper cover are made of transparent materials, and the growth status of the cell tissue can be observed in real time from all directions using a laser confocal microscope or a fluorescence microscope.

[0019] More preferably, the culture chamber is in the shape of a rectangular trough, the culture medium inlet and the culture medium outlet are respectively arranged on two opposite side walls in the length direction of the culture chamber, and the vascular nutrient solution channels are respectively arranged on the side walls in the width direction of the culture chamber.

[0020] Preferably, there are a plurality of semipermeable membrane microtubes, and the semipermeable membrane microtubes are arranged parallel to each other.

[0021] More preferably, the plurality of semipermeable membrane microtubes are divided into upper and lower layers according to their height.

[0022] More preferably, the culture chamber is provided with an upper layer diverter and a lower layer diverter, and the upper layer diverter and the lower layer diverter are arranged in pairs;

[0023] The diversion ports of the upper diverter are respectively connected to the two ends of the semipermeable membrane microtube of the upper layer, and the confluence ports of the upper diverter are respectively connected to the liquid inlet pool and the liquid outlet pool;

[0024] The diversion ports of the lower diverter are respectively connected to the two ends of the semipermeable membrane microtube of the lower layer, and the confluence ports of the lower diverter are respectively connected to the liquid inlet pool and the liquid outlet pool.

[0025] More preferably, the culture chamber is provided with a card slot, and the upper layer diverter and the lower layer diverter are detachably connected in the card slot.

[0026] Preferably, the first peristaltic pump is provided with a first flow rate sensor, and the second peristaltic pump is provided with a second flow rate sensor.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention constructs a vascularized tissue-engineered organoid culture system through a dual independent circulatory system and an adjustable shear force design. It can effectively provide oxygen and nutrients to centimeter / decimeter-level tissue-engineered organoids in vitro and remove metabolic products. It can also simultaneously form a vascular system within the cultured tissue-engineered organoids, providing an efficient and controllable in vitro culture platform for vascularized tissue-engineered organs.

[0029] 2. The semipermeable membrane tube-matrigel co-culture structure of the present invention can break through the physical isolation limitation, realize three-dimensional interactive culture, ensure the dynamic supply of nutrients, and avoid the accumulation of metabolic waste or local nutrient depletion caused by static culture.

[0030] 3. The integrated design of the transparent observation window of the present invention supports continuous imaging with a 40x objective lens and is compatible with high-end imaging equipment such as laser confocal microscopes and fluorescence microscopes. It can clearly capture dynamic processes such as endothelial cell tube formation and capillary sprouting.

[0031] 4. The sensor integrated design of the present invention can be linked with the peristaltic pump to achieve real-time flow control, ensuring that the shear force is stable within the target range (10-30dyn / cm 2 ) to avoid interference of flow rate fluctuations on cell function. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the external structure of a centimeter / decimeter-level three-dimensional vascularized tissue engineering organoid culture device of the present invention;

[0033] Figure 2 It is a schematic diagram of explosion decomposition of the present invention;

[0034] Figure 3 Schematic diagram of the diverter structure and internal partition structure of the culture device of the present invention;

[0035] Figure 4 4 is a working flow chart of the co-culture device of the present invention.

[0036] In the figure, 1. culture device; 2. culture fluid inlet; 3. culture fluid outlet; 4. vascular nutrient solution channel; 5. upper cover; 6. observation window; 7. ultra-white glass; 8. sealing ring; 9. semi-permeable membrane microtube; 10. upper layer diverter; 11. lower layer diverter; 12. culture chamber; 13. card slot; 14. through hole; 15. biocompatible microtube interface; 16. culture fluid inlet tank; 17. culture fluid outlet tank; 18. first peristaltic pump; 19. second peristaltic pump; 20. connecting hose; 21. first flow rate sensor, 22. second flow rate sensor, 23. waste liquid collector; 24. culture fluid storage tank; 25. nutrient solution storage tank. DETAILED DESCRIPTION

[0037] The following will provide a clear and complete description of the relevant technologies in the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0038] refer to Figures 1 to 4 As shown, this specific embodiment is as Figure 1 As shown:

[0039] The culture fluid circulation system in the culture device 1 includes:

[0040] The culture fluid inlet 2 is connected to the first peristaltic pump 18 through a connecting hose 20 (the connecting hose 20 is made of medical-grade silicone with an inner diameter of Φ2.5 mm). The first peristaltic pump is connected to the culture fluid storage tank 24 through the connecting hose 20. The culture fluid outlet 3 is connected to the waste liquid collection device through a connecting hose 20 of the same specification.

[0041] Function: To achieve dynamic circulation of culture medium with a flow rate range of 0.1-10 mL / min (regulated by the first peristaltic pump 18).

[0042] The vascular nutrient solution circulation system in the culture device 1 includes:

[0043] The vascular nutrient solution channel 4 is connected to the second peristaltic pump 19 and the waste liquid collector 23 through the connecting hose 20, and the second peristaltic pump is connected to the nutrient solution storage tank 25 through the connecting hose 20, forming a vascular circulation path.

[0044] Function: Provide nutrients for the growth and sprouting of blood vessels (such as VEGF, Angiopoietin-1 and other factors), with a flow rate range of 0.05-5 mL / min (controlled by the second peristaltic pump 19).

[0045] Figure 2 In the decomposition structure diagram:

[0046] Observation module: An observation window 6 made of ultra-white glass 7 (the size of the observation window 6 is 15 mm × 100 mm, the thickness is 0.15 mm, and the transmittance is ≥92%) is integrated into the upper cover 5, and an airtight seal is achieved with the culture chamber 12, the culture fluid inlet pool 16, and the culture fluid outlet pool 17 in the culture device 1 through a sealing ring 8 made of silicone.

[0047] Semipermeable membrane microtubule system in culture device 1:

[0048] Upper structure: Six semipermeable membrane microtubes 9 (the material of the semipermeable membrane microtubes 9 is regenerated cellulose with a molecular weight cutoff of 10kDa) pass through the through holes 14 on both sides of the culture chamber 12 and are fixed to the upper diverter 10 by a small interference fit. The upper diverter 10 is vertically embedded in the culture pool slot 13 to form a parallel microtube array.

[0049] Lower layer structure: eleven semipermeable membrane microtubes 9 are fixed to the lower layer diverter 11 in the same manner, and the lower layer diverter 11 is vertically embedded in the culture tank slot 13 to form a parallel microtube array.

[0050] Figure 3 Schematic diagram of the shunt and its internal structure:

[0051] Shunt Design: The vascular nutrient channel 4 is connected to the in vitro cultured small blood vessels via a biocompatible microtubule interface 15 (inner diameter Φ0.5mm). This is embedded within the vertical fluid channel between the upper shunt 10 and lower shunt 11. The contralateral inlet and outlet connections are similar.

[0052] Sealing structure: The sealing ring 8 is embedded in the contact surface between the upper cover 5 and the culture chamber 12 and the culture liquid inlet pool 16 and the culture liquid outlet pool 17 to meet the anti-pollution requirements of long-term culture.

[0053] Figure 4 Co-culture device workflow diagram:

[0054] Culture medium circulation process: First peristaltic pump 18 drives culture medium from culture medium reservoir 24 through culture medium inlet 2 into culture medium inlet reservoir 16. The medium then flows through semipermeable membrane microtubules 9, delivering nutrients to the surrounding matrix gel (containing cells) through diffusion. Wastewater is discharged through culture medium outlet 3 to wastewater collector 23. A first flow rate sensor 21 monitors the flow rate in real time (accuracy ±0.1 mL / min) and regulates the flow rate through first peristaltic pump 18.

[0055] Vascular nutrient solution circulation process: Second peristaltic pump 19 pumps vascular nutrient solution into the blood vessels through vascular nutrient solution channel 4 and into the blood vessels through biocompatible interface 15. After providing nutrition, waste liquid is discharged through biocompatible interface 15 and vascular nutrient solution channel 4 to waste liquid collector 23. Second flow rate sensor 22 monitors the flow rate in real time (accuracy ±0.1 mL / min) and regulates the flow rate through second peristaltic pump 19.

[0056] The key technical points of this embodiment are:

[0057] 1. Dual independent circulation nutrient supply system

[0058] Technical features: Dual system design including vascular circuit (connected to vascular nutrient solution channel 4) and semipermeable membrane tube microcirculation (connected to culture medium inlet 2 / culture medium outlet 3); the vascular circuit is driven by a peristaltic pump (flow rate 0.05-5mL / min) to simulate the pulsating shear force of human capillaries (10-30dyn / cm 2 ); the microcirculation of the semipermeable membrane tube is regulated by a peristaltic pump (flow rate 0.1-10mL / min), and combined with a diverter to achieve three-dimensional nutrient gradient control (error ±3%).

[0059] Technical effect: It can simultaneously meet the dynamic mechanical stimulation of vascular endothelial cells and the precise nutritional supply requirements of cultured cells, avoiding the nutritional gradient problem of traditional single-channel systems.

[0060] 2. Design of co-culture structure with parallel semipermeable membrane tubes

[0061] Technical features: Regenerated cellulose semipermeable membrane microtubes (pore size 0.1 μm, pore size ranges from 0.1-12.0 μm, and can be selected and adjusted according to actual needs) are used to penetrate the culture chamber 12, with six in the upper layer and eleven in the lower layer distributed in parallel; the semipermeable membrane microtubes 9 are fixed to the diverter by a small interference fit and are in direct contact with the matrix gel (containing cells); the through hole 14 and the slot 13 realize the embedded installation of the diverter.

[0062] Technical effect: Breaking the limitations of physical isolation, promoting blood vessel growth and sprouting, and promoting the connection between blood vessels and cultured cells, thereby improving the physiological relevance of the co-culture system.

[0063] 3. Real-time monitoring module integrating transparent observation window and high-precision sensor

[0064] Technical Features: Observation window 6, made of ultra-clear glass 7, is 0.15mm thick and has a light transmittance of ≥92%. It is integrated into the upper cover 5 and supports continuous imaging with a 40x objective lens. The first and second flow rate sensors 21 and 22 are linked to the circulation system to achieve closed-loop control of the dual-circulation flow rate. A silicone seal 8 ensures airtightness during the culture process. Supports real-time imaging and flow rate monitoring (glass observation window 6 + flow rate sensor).

[0065] Technical effect: It can track the dynamics of angiogenesis in real time, such as endothelial cell tube formation and capillary sprouting, and quantify metabolic microenvironment parameters, solving the observation difficulties of traditional devices.

[0066] 4. Multi-channel precision interface and embedded card slot connection solution for splitters

[0067] Technical features: The six semipermeable membrane microtubes 9 on the upper layer and the eleven semipermeable membrane microtubes 9 on the lower layer pass through the through holes 14 on both sides of the culture chamber 12, and are fixed to the diverter by a small interference fit. The diverter is vertically inserted into the culture tank slot 13 to form a parallel microtube array.

[0068] Technical effect: Achieve stable connection of high-density microtube arrays, reduce fluid resistance, and adapt to complex three-dimensional co-culture needs.

[0069] In summary, the present invention is expected to achieve functional culture of vascular endothelial cells through the dual independent circulation system and adjustable shear force design, and solve the problem of abnormal cell function in a static environment.

[0070] It should be emphasized that the above are only preferred embodiments of the present invention and do not constitute any form of limitation to the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A centimeter / decimeter-level three-dimensional vascularized tissue engineering organoid culture device, characterized in that: include: A culture chamber (12), a culture fluid inlet pool (16), a culture fluid outlet pool (17), a flow divider, a semipermeable membrane microtube (9), a first peristaltic pump (18), and a second peristaltic pump (19); The culture chamber of the culture device (1) is divided into a culture liquid inlet pool (16), a culture chamber (12) and a culture liquid outlet pool (17) by a diverter; The semipermeable membrane microtube (9) is located in the culture chamber (12), and the semipermeable pore diameter of the semipermeable membrane microtube (9) ranges from 0.1 μm to 12.0 μm; The culture liquid inlet (2) and the culture liquid outlet (3) of the culture device (1) are respectively connected to the culture liquid inlet pool (16) and the culture liquid outlet pool (17) of the culture device (1); There are two diverters, the front diverter is located in the culture chamber near the culture fluid inlet pool (16), and the rear diverter is located in the culture chamber near the culture fluid outlet pool (17), and each diverter is divided into an upper diverter (10) and a lower diverter (11); The semipermeable membrane microtube (9) is inserted into the diversion hole of the front and rear diverters and fixed; the external port of the culture fluid inlet (2) and the external port of the culture fluid outlet (3) are respectively connected to the first peristaltic pump (18) and the culture fluid storage tank (24); the external port of the vascular nutrient solution channel (4) is respectively connected to the second peristaltic pump (19) and the nutrient solution storage tank (25).

2. The centimeter / decimeter-level three-dimensional vascularized tissue engineering organoid culture device according to claim 1, characterized in that: The culture device (1) is in the shape of a trough with an opening facing upwards, and an upper cover (5) is provided at the opening of the culture device (1).

3. The centimeter / decimeter-level three-dimensional vascularized tissue engineering organoid culture device according to claim 2, characterized in that: The upper cover (5) is provided with an observation window (6) made of a transparent material.

4. The centimeter / decimeter-level three-dimensional vascularized tissue engineering organoid culture device according to claim 2, characterized in that: The culture device (1) and the upper cover (5) are made of transparent materials.

5. The centimeter / decimeter-level three-dimensional vascularized tissue engineering organoid culture device according to claim 2, characterized in that: The culture device (1) is in the shape of a rectangular trough, the culture fluid inlet (2) and the culture fluid outlet (3) are respectively arranged on two opposite side walls in the length direction of the culture device (1), and the vascular nutrient fluid channel (4) is respectively arranged on the side walls in the width direction of the culture device (1).

6. The centimeter / decimeter-level three-dimensional vascularized tissue engineering organoid culture device according to claim 5, characterized in that: The semipermeable membrane microtubes (9) are provided in plurality, and the semipermeable membrane microtubes (9) are arranged parallel to each other.

7. The centimeter / decimeter-level three-dimensional vascularized tissue engineering organoid culture device according to claim 6, characterized in that: The plurality of semipermeable membrane microtubes (9) are divided into upper and lower layers according to their height.

8. The centimeter / decimeter-level three-dimensional vascularized tissue engineering organoid culture device according to claim 7, characterized in that: The upper layer flow divider (10) and the lower layer flow divider (11) are arranged in pairs; The diversion ports of the upper diverter (10) are respectively connected to the two ends of the semipermeable membrane microtube (9) of the upper layer, and the confluence ports of the upper diverter (10) are respectively connected to the culture fluid inlet pool (16) and the culture fluid outlet pool (17); The diversion ports of the lower layer diverter (11) are respectively connected to the two ends of the semipermeable membrane microtube (9) of the lower layer, and the confluence ports of the lower layer diverter (11) are respectively connected to the culture fluid inlet pool (16) and the culture fluid outlet pool (17).

9. The centimeter / decimeter-level three-dimensional vascularized tissue engineering organoid culture device according to claim 8, characterized in that: The culture device (1) is provided with a card slot (13), and the upper layer flow divider (10) and the lower layer flow divider (11) are detachably connected in the card slot (13).

10. The centimeter / decimeter-level three-dimensional vascularized tissue engineering organoid culture device according to claim 1, characterized in that: The first peristaltic pump (18) is provided with a first flow rate sensor (21), and the second peristaltic pump (19) is provided with a second flow rate sensor (22).

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