Vascularized multi-organ chip and use method thereof

Abstract

The invention belongs to the technical field of biological tissue engineering, and discloses a vascularized multi-organ chip and a use method thereof.The vascularized multi-organ chip comprises a blood vessel layer, an organ-like culture layer and a porous membrane, the blood vessel layer comprises a flow channel, a liquid inlet end and a liquid outlet end, and the liquid inlet end and the liquid outlet end are communicated with the two ends of the flow channel; the first blood vessel culture unit and the second blood vessel culture unit are arranged on the flow channel and are communicated with the flow channel; the organoid culture layer comprises a liquid inlet, a liquid outlet, a first culture chamber and a second culture chamber which are separated from one another, the organoid culture layer and the blood vessel layer are stacked, the liquid inlet is communicated with the liquid inlet end, and the liquid outlet is communicated with the liquid outlet end; porous membranes are arranged between the first blood vessel culture unit and the first culture chamber as well as between the second blood vessel culture unit and the second culture chamber, and are communicated through through holes of the porous membranes, so that interaction among different organs is realized; perfusion culture simulates stimulation of human body blood flow to organoids, higher bionic performance is achieved, and the accuracy of research such as drug screening is improved.

Description

Technical Field

[0001] The present invention relates to the field of biological tissue engineering technology, and in particular to a vascularized multi-organ chip and a method for using the same. Background Art

[0002] For many years, in vitro studies of cell biology have relied on traditional 2D cell culture. However, this non-physiological cell culture method often fails to preserve the original organ function and morphology, and cannot reflect the complex structure, intercellular interactions, and blood perfusion. Organoids are tissue analogs with 3D structures that are formed by the in vitro growth and differentiation of stem cells or tumor tissue-derived cells. Compared with cells cultured using 2D technology, organoids are more similar to human tissue in structure and function. Organ-on-a-Chip (OCI), a cutting-edge technology that integrates biology, fluid mechanics, materials science, and engineering, can recreate the smallest functional units of human tissue or organs in vitro by constructing highly biomimetic micro-functional units, opening up a new technical path for pathophysiological research. This technology relies on micro-nanofabrication processes to construct a multi-chamber three-dimensional culture system and microchannel network on a chip substrate, and combines microfluidics technology for precise fluid manipulation to achieve dynamic culture.

[0003] When applied to the in vitro culture of organoids, organ-on-a-chip technology can induce organoids to self-organize and differentiate into organ-specific structures through a biomimetic microenvironment, reproducing the biological functions of natural organs in vitro. This technological advantage makes it a key tool in basic research fields such as disease modeling and drug screening, with particular application value in simulating organ physiological microenvironments, analyzing pathological mechanisms, and evaluating drug efficacy.

[0004] The human body is a complex biological system composed of tissues and organs with multiple physiological functions. Multiple organs can communicate with each other through various molecular signals mediated by blood and lymphatic circulation to maintain overall vitality and homeostasis. Similarly, the occurrence and development of many diseases are the result of the joint action of multiple organs. The occurrence and development of many diseases also involve the coordinated action of multiple organs. Current research based on single organs has the defect of insufficient physiological relevance, making it difficult to accurately evaluate the pathophysiological processes of the human body. Constructing an in vitro model of vascularized multi-organ interactions can better simulate the complexity of the human system, restore physiological structural characteristics, and provide a more accurate research model for disease mechanism analysis, drug development and personalized medicine. Summary of the Invention

[0005] The present invention aims to provide a vascularized multi-organoid chip and its use method. The vascular network structure in the chip has dual functions: first, flow perfusion culture can timely update the nutrients required for organoid growth and remove metabolic waste, thereby improving the organoid's lifespan and maturity; second, it can enable the mutual transmission of substances such as cytokines secreted by different organoids, effectively simulating the interaction between human organs or tissues.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] A vascularized multi-organ chip, comprising:

[0008] The blood vessel layer comprises a flow channel, a liquid inlet and a liquid outlet connected to both ends of the flow channel, and a first blood vessel culture unit and a second blood vessel culture unit provided on the flow channel and connected to the flow channel;

[0009] An organoid culture layer comprising a liquid inlet, a liquid outlet, a first culture chamber, and a second culture chamber separated from each other, wherein the organoid culture layer and the vascular layer are stacked, the liquid inlet is connected to the liquid inlet end, and the liquid outlet is connected to the liquid outlet end;

[0010] The porous membrane includes multiple micron-sized through holes. The porous membrane is provided between the first vascular culture unit and the first culture chamber, and between the second vascular culture unit and the second culture chamber, and they are connected through the through holes. The opening size of the through hole is smaller than the outer size of the endothelial cells and larger than the outer size of the signal molecules secreted by the organoids.

[0011] In some possible embodiments, the flow channel includes a main channel and two first branch flow channels, the vascular layer includes two liquid outlets, one end of the main channel is connected to the liquid inlet, and the other end is connected to the two first branch flow channels, the other ends of the two first branch flow channels are correspondingly connected to the two liquid outlets, and the first vascular culture unit and the second vascular culture unit are provided on each of the two first branch flow channels;

[0012] The organoid culture layer includes two first culture chambers, two second culture chambers and two liquid outlets. The two first culture chambers and the two first blood vessel culture units are correspondingly arranged, the two second culture chambers and the two second blood vessel culture units are correspondingly arranged, and the two liquid outlets and the two liquid outlet ends are correspondingly arranged.

[0013] In some possible embodiments, the flow channel includes a main channel and a plurality of second branch flow channels, the vascular layer includes a plurality of liquid outlets, one end of the main channel is connected to the liquid outlet, and the other end is connected to a plurality of second branch flow channels, and the other ends of the plurality of second branch flow channels are correspondingly connected to the plurality of liquid outlets, the main channel is provided with the first blood vessel culture unit, and the plurality of second branch flow channels are correspondingly provided with the plurality of second blood vessel culture units; the flow channel and the plurality of liquid outlets form a flow channel combination;

[0014] The organoid culture layer includes a culture chamber combination, which includes one first culture chamber, multiple second culture chambers and multiple liquid outlets. The first culture chamber and the first blood vessel culture unit are correspondingly arranged, the multiple second culture chambers and the multiple second blood vessel culture units are correspondingly arranged, and the multiple liquid outlets and the multiple liquid outlet ends are correspondingly arranged.

[0015] In some possible implementations, the flow channel further includes a plurality of intermediate branch flow channels, and the main flow channel is connected to the second branch flow channels through the intermediate branch flow channels.

[0016] In some possible implementations, the vascular layer includes a liquid inlet end and a plurality of flow channel combinations, and the flow channels of the plurality of flow channel combinations are all connected to the liquid inlet end;

[0017] The organoid culture layer includes a liquid inlet and a plurality of culture chamber assemblies, wherein the liquid inlet is connected to the liquid inlet end; the plurality of culture chamber assemblies and the plurality of flow channel assemblies are arranged in a one-to-one correspondence.

[0018] In some possible implementations, a plurality of flow channel assembly rings are arranged around the liquid inlet; and a plurality of culture chamber assembly rings are arranged around the liquid inlet.

[0019] In some possible embodiments, the first vascular culture unit and the second vascular culture unit each include a plurality of branch ports and a plurality of confluence ports, and the plurality of branch ports and the plurality of confluence ports are connected via channels.

[0020] In some possible implementations, both the first culture chamber and the second culture chamber are provided with an opening, and a detachable sealing cover is connected to the opening, and the opening is sealed by the sealing cover.

[0021] A method for using the vascularized multi-organoid chip as described above comprises:

[0022] S100, injecting the endothelial cell suspension from the liquid inlet into the flow channel of the vascular layer, so that the endothelial cell suspension enters the first vascular culture unit and the second vascular culture unit and fills the flow channel;

[0023] S200, flipping the vascularized multi-organoid chip upside down and placing it in a cell culture incubator under preset conditions, culturing it statically until the endothelial cells adhere to the porous membrane; wherein the vascular layer is located above the organoid culture layer;

[0024] S300, flipping the vascularized multi-organoid chip so that the organoid culture layer faces upward, adding Matrigel to the bottom of the first culture chamber and the second culture chamber to cover the surface of the porous membrane, and placing them in a cell culture incubator under preset conditions to incubate and solidify the Matrigel;

[0025] S400, inoculating a mixture of the first organoid and Matrigel into a first culture chamber, inoculating a mixture of the second organoid and Matrigel into a second culture chamber, and placing the cells in a cell culture incubator under preset conditions for incubation;

[0026] S500: After a preset time, the culture medium of the first organoid is added to the first culture chamber, and the culture medium of the second organoid is added to the second culture chamber. Simultaneously, the culture fluid is continuously injected from the liquid inlet into the flow channel of the vascular layer. After passing through the first vascular culture unit and the second vascular culture unit, the culture fluid flows out through the liquid outlet.

[0027] In some possible embodiments, the flow channel includes a main channel and two first branch flow channels, the vascular layer includes two liquid outlet ends, one end of the main channel is connected to the liquid inlet end, and the other end is connected to the two first branch flow channels, the other ends of the two first branch flow channels are correspondingly connected to the two liquid outlet ends, and the first vascular culture unit and the second vascular culture unit are provided on the two first branch flow channels; the organoid culture layer includes two first culture chambers, two second culture chambers and two liquid outlets, the two first culture chambers and the two first vascular culture units are correspondingly arranged, the two second culture chambers and the two second vascular culture units are correspondingly arranged, and the two liquid outlets and the two liquid outlets are correspondingly arranged;

[0028] Step S400 includes: inoculating a mixture of a first organoid and matrigel into a first first culture chamber and a second second culture chamber, and inoculating a mixture of a second organoid and matrigel into a first second culture chamber and a second first culture chamber;

[0029] Step S500 includes: adding a culture medium of the first organoid into the first first culture chamber and the second second culture chamber, and adding a culture medium of the second organoid into the first second culture chamber and the second first culture chamber respectively.

[0030] Beneficial effects of the present invention:

[0031] The present invention provides a vascularized multi-organoid chip and a method for using the same. The first and second vascular culture units are vascularized by injecting an endothelial cell suspension into the vascular layer. The first and second culture chambers are used to culture different organoids. Since the opening size of the micron-scale through-holes is larger than the signal molecules secreted by the organoids, the signal molecules secreted by the organoids cultured in the first culture chamber can pass through the through-holes of the porous membrane and enter the first vascular culture unit of the vascular layer; when the culture fluid is continuously perfused through the liquid inlet, the signal molecules in the first vascular culture unit can flow with the culture fluid, and when the signal molecules flow to the second vascular culture unit, they can enter the second culture chamber through the through-holes of the porous membrane, and the signal molecules act on the organoids in the second culture chamber; the organoids in the second culture chamber secrete signal molecules that enter the second vascular culture unit through the through-holes of the porous membrane, thereby achieving interaction between different organoids. The signal molecules produced by the organoids in the first culture chamber and the signal molecules produced by the organoids in the second culture chamber can both flow out of the liquid outlet with the culture fluid for subsequent biochemical testing. Due to the vascularized construction of the first vascular culture unit and the second vascular culture unit, the in vitro organoid model is closer to the human physiological structure. When the culture fluid is continuously perfused in the vascular layer, it can simulate the nutrient supply and fluid stimulation of the human blood flow, and can simulate the mechanical stress of the blood fluid stimulation on the organoids in the first culture chamber and the second culture chamber. Compared with traditional 2D models such as well plates, it has higher bionics, thereby improving the accuracy of research such as drug screening.

[0032] The vascular network structure formed by the vascular layer has dual functions: first, flow perfusion culture can timely update the nutrients required for organoid growth and excrete metabolic waste, thereby improving the survival cycle and maturity of organoids; second, it can realize the mutual transmission of substances such as cytokines secreted by different organoids, effectively simulating the interaction between human organs or tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a schematic diagram of a vascularized multi-organ chip provided in Example 1 of the present invention;

[0034] Figure 2 This is an exploded view of the vascularized multi-organ chip provided in Example 1 of the present invention;

[0035] Figure 3 is a top view of the vascular layer provided by the first embodiment of the present invention;

[0036] Figure 4 yes Figure 3 A magnified view of point A;

[0037] Figure 5 This is a flow chart of a method for using the vascularized multi-organ chip provided in Example 1 of the present invention;

[0038] Figure 6 Schematic diagram of a vascularized multi-organ chip provided in Example 2 of the present invention;

[0039] Figure 7 is a top view of a blood vessel layer provided by embodiment 2 of the present invention;

[0040] Figure 8 is a schematic diagram of an organoid culture layer provided in Example 2 of the present invention;

[0041] Figure 9 This is a schematic diagram of the vascularized multi-organ chip provided in Example 3 of the present invention.

[0042] In the picture:

[0043] 1. Vascular layer; 11. Flow channel; 111. Main flow channel; 112. First branch flow channel; 113. Second branch flow channel; 114. Intermediate branch flow channel; 1141. Third branch flow channel; 1142. Fourth branch flow channel; 12. Liquid inlet; 13. Liquid outlet; 14. First vascular culture unit; 141. First diversion port; 142. Second diversion port; 143. Third diversion port; 144. Fourth diversion port; 145. First confluence port; 146. Second confluence port; 147. Third confluence port; 148. Fourth confluence port; 149. Channel; 15. Second vascular culture unit; 16. Flow channel assembly;

[0044] 2. Organoid culture layer; 21. Liquid inlet; 22. Liquid outlet; 23. First culture chamber; 24. Second culture chamber; 25. Culture chamber assembly;

[0045] 3. Porous membrane;

[0046] 4. Sealing cover. DETAILED DESCRIPTION

[0047] To make the technical problems solved by the present invention, the technical solutions adopted, and the technical effects achieved more clearly, the technical solutions of the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0048] In the description of the present invention, unless otherwise expressly specified or limited, the terms "connected," "connected," and "fixed" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integration; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal communication between two components or interaction between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention in specific circumstances.

[0049] In the present invention, unless otherwise expressly specified or limited, a first feature being "above" or "below" a second feature may include the first and second features being in direct contact, or may include the first and second features being in contact not directly but through another feature between them. Furthermore, a first feature being "above," "above," and "above" a second feature may include the first feature being directly above or obliquely above the second feature, or may simply mean that the first feature is higher in level than the second feature. A first feature being "below," "below," and "below" a second feature may include the first feature being directly below or obliquely below the second feature, or may simply mean that the first feature is lower in level than the second feature.

[0050] Example 1

[0051] like Figures 1-4 As shown, this embodiment provides a vascularized multi-organoid chip, including a vascular layer 1, an organoid culture layer 2, and a porous membrane 3. The vascular layer 1 includes a flow channel 11, a liquid inlet 12 and a liquid outlet 13 connected to both ends of the flow channel 11, and a first vascular culture unit 14 and a second vascular culture unit 15 provided on the flow channel 11 and connected to the flow channel 11. The organoid culture layer 2 includes a liquid inlet 21, a liquid outlet 22, a first culture chamber 23 and a second culture chamber 24 separated from each other. The organoid culture layer 2 and the vascular layer 1 are stacked, with the liquid inlet 21 connected to the liquid inlet 12, and the liquid outlet 22 connected to the liquid outlet 13. The liquid inlet 21, the liquid inlet 12, the first vascular culture unit 14, the second vascular culture unit 15, the liquid outlet 13, and the liquid outlet 22 are connected in sequence. After being injected through the liquid inlet 21 , the endothelial cell culture fluid can sequentially pass through the liquid inlet 12 , the first blood vessel culture unit 14 , the second blood vessel culture unit 15 and the liquid outlet 13 , and flow out from the liquid outlet 22 .

[0052] The first vascular culture unit 14 is provided in correspondence with the first culture chamber 23, and the second vascular culture unit 15 is provided in correspondence with the second culture chamber 24. A porous membrane 3 is provided between the first vascular culture unit 14 and the first culture chamber 23, and between the second vascular culture unit 15 and the second culture chamber 24. For example, two porous membranes 3 are provided, which are respectively provided at the bottom of the first culture chamber 23 and the second culture chamber 24. Alternatively, there is one porous membrane 3, which can cover the bottom of the first culture chamber 23 and the second culture chamber 24 at the same time. The porous membrane 3 includes a plurality of through holes, and the first vascular culture unit 14 and the first culture chamber 23, and the second vascular culture unit 15 and the second culture chamber 24 are connected through the through holes, wherein the opening size of the through holes is smaller than the outer size of the endothelial cells and larger than the outer size of the signal molecules secreted and produced by the organoids.

[0053] The endothelial cell suspension is injected through the liquid inlet 21 and enters the flow channel 11, the first vascular culture unit 14 and the second vascular culture unit 15. The first vascular culture unit 14 and the second vascular culture unit 15 are used for cell culture, thereby achieving vascularization. The first culture chamber 23 and the second culture chamber 24 are used for co-culture of multiple organoids. The porous membrane 3 is provided with multiple micron-level through-holes, which allow material exchange between the first culture chamber 23 and the first vascular culture unit 14, the second culture chamber 24 and the second vascular culture unit 15. Because the opening size of the through-holes is smaller than the outer size of the endothelial cells, the endothelial cells in the vascular layer 1 are prevented from entering the first culture chamber 23 and the second culture chamber 24. The endothelial cells are only located in the vascular layer 1, thereby achieving vascularization. Optionally, the porous membrane 3 is a PET elastic porous membrane with a pore size of 8 μm. The diameter of the vascular endothelial cells in the flat spreading state is approximately between 20 μm and 50 μm. The pore size of the through-holes is much smaller than the diameter of the endothelial cells, thereby preventing the endothelial cells from passing through the porous membrane 3 into the organoid culture layer 2.

[0054] Because the opening size of the through-hole is larger than the signal molecules secreted by the organoids, the signal molecules secreted by the organoids cultured in the first culture chamber 23 can pass through the through-holes of the porous membrane 3 and enter the first vascular culture unit 14 of the vascular layer 1; when the culture fluid is continuously perfused through the liquid inlet 21, the signal molecules in the first vascular culture unit 14 can flow with the culture fluid, and when the signal molecules flow to the second vascular culture unit 15, they can enter the second culture chamber 24 through the through-holes of the porous membrane 3, and the signal molecules act on the organoids in the second culture chamber 24; the signal molecules secreted by the organoids in the second culture chamber 24 enter the second vascular culture unit 15 through the through-holes of the porous membrane 3, thereby realizing the interaction between different organoids. The signal molecules produced by the organoids in the first culture chamber 23 and the signal molecules produced by the organoids in the second culture chamber 24 can both flow out of the liquid outlet 22 with the culture fluid for subsequent biochemical detection. Due to the continuous perfusion of culture fluid in the vascular layer 1, the nutrient supply and fluid stimulation of the human blood flow are simulated, and the mechanical stress of the organoids in the first culture chamber 23 and the second culture chamber 24 caused by the blood fluid stimulation can be simulated, which has bionic properties and improves the research effect of the in vitro research platform.

[0055] Both the vascular layer 1 and the organoid culture layer 2 are made of PDMS. The film is poured after obtaining the master through soft lithography technology. The vascular layer 1, the organoid culture layer 2 and the porous membrane 3 located in the middle are irreversibly bonded after oxygen plasma surface treatment. The above are all conventional technical means for organ chip processing. Please refer to the existing technology and do not repeat them here.

[0056] Both the first and second culture chambers 23 and 24 have openings connected to removable sealing covers 4. These seals ensure a tight seal between the first and second culture chambers 23 and 24, ensuring fluid perfusion and culture. Furthermore, the removable sealing covers 4 facilitate organoid inoculation and sampling.

[0057] The first vascular culture unit 14 and the second vascular culture unit 15 both include multiple branch ports and multiple confluence ports, which are connected by channels 149 to simulate the intricate vascular network in human organs, further improving bionics.

[0058] In one embodiment, the first blood vessel culture unit 14 and the second blood vessel culture unit 15 each include a first diversion port 141, two second diversion ports 142, four third diversion ports 143, eight fourth diversion ports 144, eight first confluence ports 145, four second confluence ports 146, two third confluence ports 147 and one fourth confluence port 148, which are sequentially arranged along the flow channel 11 from the liquid inlet end 12 to the liquid outlet end 13; one first diversion port 141 corresponds to two second diversion ports 142; one second diversion port 142 corresponds to two third diversion ports 143, so that the two second diversion ports 142 correspond to Four third branch ports 143; one third branch port 143 corresponds to two fourth branch ports 144, so that the four third branch ports 143 correspond to eight fourth branch ports 144; the eight fourth branch ports 144 correspond to eight first confluence ports 145; the two first confluence ports 145 correspond to one second confluence port 146, so that the eight first confluence ports 145 correspond to four second confluence ports 146; the two second confluence ports 146 correspond to one third confluence port 147, so that the four second confluence ports 146 correspond to two third confluence ports 147; the two third confluence ports 147 correspond to one fourth confluence port 148. The first diversion port 141 and the second diversion port 142, the second diversion port 142 and the third diversion port 143, the third diversion port 143 and the fourth diversion port 144, the fourth diversion port 144 and the first confluence port 145, the first confluence port 145 and the second confluence port 146, the second confluence port 146 and the third confluence port 147, and the third confluence port 147 and the fourth confluence port 148 are all connected through the channel 149, and the first diversion port 141 and the fourth confluence port 148 are both connected to the flow channel 11.

[0059] The blood supply to the organs of the human body undergoes multiple diversions through large blood vessels, medium blood vessels, small blood vessels, and capillaries, and finally substances are exchanged through the capillary network.

[0060] The channel 149 between the first shunt port 141 and the second shunt port 142, and the channel 149 between the third confluence port 147 and the fourth confluence port 148 are all first channels, and the first channels simulate large blood vessels. The channel 149 between the second shunt port 142 and the third shunt port 143, and the channel 149 between the second confluence port 146 and the third confluence port 147 are all second channels, and the second channels simulate medium-sized blood vessels. The channel 149 between the third shunt port 143 and the fourth shunt port 144, and the channel 149 between the first confluence port 145 and the second confluence port 146 are all third channels, and the third channels simulate small blood vessels. The channel 149 between the fourth shunt port 144 and the first confluence port 145 is the fourth channel, and simulates capillaries. The first blood vessel culture unit 14 and the second blood vessel culture unit 15 include multiple shunt ports and multiple confluence ports, and the multi-level shunt structure of blood in human organs is simulated through the first channel, the second channel, the third channel, and the fourth channel.

[0061] Optionally, the opening size of the first channel, the opening size of the second channel, the opening size of the third channel and the opening size of the fourth channel decrease in sequence, and can be specifically designed with reference to the size of human blood vessels.

[0062] Optionally, the projection shapes of the first vascular culture unit 14 and the second vascular culture unit 15 on the vascular layer 1 are centrally symmetrical figures. In other embodiments, the projection shapes of the first vascular culture unit 14 and the second vascular culture unit 15 on the vascular layer 1 may also be irregular shapes.

[0063] In one embodiment, the flow channel 11 includes a main channel 111 and two first branch flow channels 112. The vascular layer 1 includes two liquid outlets 13. One end of the main channel 111 is connected to the liquid inlet 12, and the other end is connected to the two first branch flow channels 112. The other ends of the two first branch flow channels 112 are correspondingly connected to the two liquid outlets 13. The two first branch flow channels 112 are each provided with a first vascular culture unit 14 and a second vascular culture unit 15. The organoid culture layer 2 includes two first culture chambers 23, two second culture chambers 24, and two liquid outlets 22. The two liquid outlets 22 are correspondingly provided with the two liquid outlets 13. The liquid inlet 21, the main channel 111, a first branch flow channel 112, the first vascular culture unit 14 and the second vascular culture unit 15 on the first branch flow channel 112, and the liquid outlet 13 are connected to the liquid outlet 22. The liquid inlet 21, the main channel 111, another first branch channel 112, the first vascular culture unit 14 and the second vascular culture unit 15 on the first branch channel 112, and the liquid outlet 13 are connected to the liquid outlet 22. The endothelial cell suspension injected through the liquid inlet 21 can enter the two first branch channels 112 at the same time, and is used for cell culture on the two first vascular culture units 14 and the two second vascular culture units 15 to achieve vascularization, thereby realizing the parallel organoid co-culture channel 11 design. The two first culture chambers 23 and the two first vascular culture units 14 are correspondingly arranged, and the two second culture chambers 24 and the two second vascular culture units 15 are correspondingly arranged. The two first culture chambers 23 and the two second culture chambers 24 are all used for organoid culture, so that two groups of organoids can be cultured at the same time.

[0064] like Figure 5 As shown, this embodiment also provides a method for using the vascularized multi-organ chip as described above, comprising the following steps:

[0065] S100: Slowly inject the endothelial cell suspension into the flow channel 11 of the vascular layer 1 from the liquid inlet 21, so that the endothelial cell suspension enters the first vascular culture unit 14 and the second vascular culture unit 15 and fills the flow channel 11. The endothelial cell suspension is set to an appropriate cell density, which can be determined experimentally.

[0066] S200: The vascularized multi-organoid chip is flipped upside down and placed in a cell culture incubator at preset conditions, such as 37°C and 5% CO2. The chip is incubated for approximately 2 to 4 hours until the endothelial cells attach to the porous membrane 3. After the endothelial cells attach to the porous membrane 3, the first vascular culture unit 14 and the second vascular culture unit 15 are vascularized. The vascularized multi-organoid chip is flipped upside down so that the vascular layer 1 is located above the organoid culture layer 2.

[0067] S300: Flip the vascularized multi-organoid chip so that the organoid culture layer 2 faces upward. Add Matrigel to the bottom of the first and second culture chambers 23 and 24 to cover the surface of the porous membrane 3. Place the chip in a cell culture incubator at preset conditions, such as 37°C and 5% CO2, for incubation. This allows the Matrigel to solidify. Solidification is the process of converting the liquid Matrigel into a stable gel. The solidified Matrigel provides a certain bottom support for subsequent organoid seeding. The Matrigel solidifies for 20 minutes.

[0068] S400: 50 μL of a mixture of the first organoid and Matrigel is inoculated into the first culture chamber 23, and 50 μL of a mixture of the second organoid and Matrigel is inoculated into the second culture chamber 24. The cells are then placed in a cell culture incubator set to preset conditions, such as 37°C and 5% CO2. This allows the first organoid to be cultured in the first culture chamber 23 and the second organoid to be cultured in the second culture chamber 24. The Matrigel is Matrigel. Specifically, the sealing cover 4 on the first culture chamber 23 is opened, and the mixture of the first organoid and Matrigel is inoculated into the first culture chamber 23 through the opening. The sealing cover 4 is then sealed. The sealing cover 4 on the second culture chamber 24 is opened, and the mixture of the second organoid and Matrigel is inoculated into the second culture chamber 24 through the opening. The sealing cover 4 is then sealed.

[0069] The signal molecules secreted by the first organoid in the first culture chamber 23 pass through the porous membrane 3 into the vascular layer 1 , and the signal molecules secreted by the second organoid in the second culture chamber 24 pass through the porous membrane 3 into the vascular layer 1 .

[0070] S500, after a preset time, such as 20 minutes, 500 μL of the first organoid culture medium is added to the first culture chamber 23, and 500 μL of the second organoid culture medium is added to the second culture chamber 24; at the same time, a precision syringe pump is used at a rate of 10.0 μL·min -1The culture medium is continuously injected into the flow channel 11 of the vascular layer 1 from the liquid inlet 21 at a speed of , and the culture medium flows out through the liquid outlet 22 after passing through the first vascular culture unit 14 and the second vascular culture unit 15. Specifically, the culture medium is preheated before being added to the corresponding culture chamber. The culture medium is usually stored at 4°C, and the organoids inoculated in the culture chamber are taken out from the 37°C incubator. The culture medium is preheated and added to the chip to reduce the negative stimulation of low temperature on the growth of organoids, so as to obtain a more stable and reliable experimental model. The use of a syringe pump for stable fluid drive can better simulate the blood flow in the human body and provide shear force stimulation under physiological conditions for the development and maturation of various organs.

[0071] When the culture fluid is continuously perfused through the liquid inlet 21, the signal molecules in the first vascular culture unit 14 can flow with the culture fluid, and when the signal molecules flow to the second vascular culture unit 15, they can enter the second culture chamber 24 through the through-holes of the porous membrane 3, and the signal molecules act on the second organoid in the second culture chamber 24; the signal molecules secreted by the second organoid enter the second vascular culture unit 15 through the through-holes of the porous membrane 3, thereby realizing the interaction between different organoids. The signal molecules produced by the organoids in the first culture chamber 23 and the signal molecules produced by the organoids in the second culture chamber 24 can both flow out of the liquid outlet 22 with the culture fluid for subsequent biochemical testing. Due to the continuous perfusion of the culture fluid in the vascular layer 1, the nutrient supply and fluid stimulation of the human blood flow are simulated, and the shear force of the blood fluid stimulation on the organoids in the first culture chamber 23 and the second culture chamber 24 can be simulated.

[0072] Specifically, before step S100, the vascularized multi-organoid chip is sterilized by ultraviolet irradiation for 1 hour. The sealing cover 4 is sealed at the open end to form a sealed space between the first culture chamber 23 and the second culture chamber 24, thereby reducing external environmental interference and ensuring research results.

[0073] Furthermore, the flow channel 11 includes a main channel 111 and two first branch flow channels 112, the vascular layer 1 includes two liquid outlet ends 13, one end of the main channel 111 is connected to the liquid inlet end 12, and the other end is connected to the two first branch flow channels 112, the other ends of the two first branch flow channels 112 are correspondingly connected to the two liquid outlet ends 13, and the two first branch flow channels 112 are each provided with a first vascular culture unit 14 and a second vascular culture unit 15; the organoid culture layer 2 includes two first culture chambers 23, two second culture chambers 24 and two liquid outlets 22, the two first culture chambers 23 and the two first vascular culture units 14 are correspondingly arranged, the two second culture chambers 24 and the two second vascular culture units 15 are correspondingly arranged, and the two liquid outlets 22 and the two liquid outlet ends 13 are correspondingly arranged.

[0074] Step S400 includes: inoculating a mixture of a first organoid and Matrigel into the first first culture chamber 23 and the second second culture chamber 24, and inoculating a mixture of a second organoid and Matrigel into the first second culture chamber 24 and the second first culture chamber 23;

[0075] Step S500 includes: adding a culture medium of the first organoid into the first first culture chamber 23 and the second second culture chamber 24 , and adding a culture medium of the second organoid into the first second culture chamber 24 and the second first culture chamber 23 , respectively.

[0076] Through the above arrangement, the signal molecules secreted by the first organoid in the first first culture chamber 23 enter the vascular layer 1 and flow with the culture fluid into the first second culture chamber 24, acting on the second organoid. The signal molecules secreted by the second organoid in the second first culture chamber 23 enter the vascular layer 1 and flow with the culture fluid into the second second culture chamber 24, acting on the first organoid. This enables the signal molecules of the first organoid to act on the second organoid, and vice versa. By cross-seeding and culturing the first and second organoids and designing the parallel organoid co-culture flow channel 11, interaction between different organoids is achieved, providing a good platform and method for studying the crosstalk relationship between the first and second organoids. The above structure and method can simultaneously achieve interaction between the two organoids, saving time and improving efficiency.

[0077] Example 2

[0078] like Figure 6-Figure 8 As shown, this embodiment provides a vascularized multi-organoid chip, which has a structure substantially the same as that of the first embodiment, except that the flow channel 11 includes a main channel 111 and multiple second branch channels 113. The vascular layer 1 includes multiple liquid outlets 13. One end of the main channel 111 is connected to the liquid outlet 13, and the other end is connected to multiple second branch channels 113. The other ends of the multiple second branch channels 113 are correspondingly connected to the multiple liquid outlets 13. The main channel 111 is provided with a first vascular culture unit 14, and the multiple second branch channels 113 are correspondingly provided with multiple second vascular culture units 15. The flow channel 11 and the multiple liquid outlets 13 form a flow channel assembly 16. The organoid culture layer 2 includes a culture chamber assembly 25, which includes a first culture chamber 23, multiple second culture chambers 24, and multiple liquid outlets 22. The multiple liquid outlets 22 are arranged correspondingly to the multiple liquid outlets 13. The first culture chamber 23 is arranged correspondingly to the first vascular culture unit 14, and the multiple second culture chambers 24 are arranged correspondingly to the multiple second vascular culture units 15.

[0079] For example, the second branch flow channels 113 may be provided with two, three, four, etc., without limitation. Accordingly, the liquid outlet 13, the second blood vessel culture unit 15, the second culture chamber 24, and the liquid outlet 22 are all provided with the same number as the second branch flow channels 113. This embodiment is described by taking the example of three second branch flow channels 113, three liquid outlets 13, three second blood vessel culture units 15, three second culture chambers 24, and three liquid outlets 22.

[0080] The liquid inlet 21, the liquid inlet end 12, the main channel 111, the first blood vessel culture unit 14, the first second branch channel 113, the first second blood vessel culture unit 15, the liquid outlet end 13, and the liquid outlet 22 are connected. The liquid inlet 21, the liquid inlet end 12, the main channel 111, the first blood vessel culture unit 14, the second second branch channel 113, the second second blood vessel culture unit 15, the liquid outlet end 13, and the liquid outlet 22 are connected. The liquid inlet 21, the liquid inlet end 12, the main channel 111, the first blood vessel culture unit 14, the third second branch channel 113, the third second blood vessel culture unit 15, the liquid outlet end 13, and the liquid outlet 22 are connected.

[0081] The first culture chamber 23 and the first blood vessel culture unit 14 are set correspondingly, and the first culture chamber 23 and the first blood vessel culture unit 14 can exchange substances through the porous membrane 3. The three second culture chambers 24 and the three second blood vessel culture units 15 are set correspondingly, and the first second culture chamber 24 and the first second blood vessel culture unit 15 can exchange substances, the second second culture chamber 24 and the second second blood vessel culture unit 15 can exchange substances, and the third second culture chamber 24 and the third second blood vessel culture unit 15 can exchange substances.

[0082] The first organoid is cultured in the first culture chamber 23, the second organoid is cultured in the first second culture chamber 24, the third organoid is cultured in the second second culture chamber 24, and the fourth organoid is cultured in the third second culture chamber 24. Signal molecules secreted by the first organoid enter the vascular layer 1, flow with the culture fluid into the three second vascular culture units 15, and then enter the three second culture chambers 24, thereby acting on the second, third, and fourth organoids respectively.

[0083] During cancer progression, cross-organ communication may lead to the development of tumor metastasis, which is the main cause of cancer mortality. Tumor metastasis occurs when tumor cells break through the tissue matrix and intravasate into the vasculature, causing cancer cells to spread to multiple other organs.

[0084] Using this chip, tumor organoids (i.e., the first organoid) are seeded in the first culture chamber 23, while three tumor-targeting organoids (i.e., the second, third, and fourth organoids) are seeded in the other three second culture chambers 24. This allows for the construction of a highly realistic tumor metastasis model, providing a reliable in vitro experimental platform for studying tumor invasion of different organs and detecting metastasis rates. Compared to Example 1, the chip of this example, by enabling the co-culture of multiple organoids, is not only well suited for multi-organ disease modeling, but also has great potential for predicting cancer metastasis and testing anti-metastatic therapies, thus expanding the functionality of multi-organoid chips.

[0085] Optionally, the flow channel 11 also includes multiple intermediate branch flow channels 114. The main flow channel 111 is connected to the second branch flow channel 113 through the intermediate branch flow channels 114. The intermediate branch flow channels 114 buffer the main flow channel 111 and the second branch flow channel 113, so that the liquid in the main flow channel 111 can flow more evenly into the second branch flow channel 113.

[0086] Exemplarily, the multiple intermediate branch channels 114 include two third branch channels 1141 and four fourth branch channels 1142. The main channel 111 connects the two third branch channels 1141, and the third branch channels 1141 are correspondingly connected to the two fourth branch channels 1142, so that the two third branch channels 1141 are correspondingly connected to the four fourth branch channels 1142; the two fourth branch channels 1142 are correspondingly connected to the two second branch channels 113, and the other two fourth branch channels 1142 are each connected to another second branch channel 113.

[0087] Example 3

[0088] like Figure 9 As shown, this embodiment provides a vascularized multi-organoid chip, which has a structure basically the same as that of Example 2, except that the vascular layer 1 includes a liquid inlet 12 and multiple flow channel assemblies 16, and the flow channels 11 of the multiple flow channel assemblies 16 are all connected to the liquid inlet 12. The organoid culture layer 2 includes a liquid inlet 21 and multiple culture chamber assemblies 25, the liquid inlet 21 is connected to the liquid inlet 12; the multiple culture chamber assemblies 25 and the multiple flow channel assemblies 16 are arranged in a one-to-one correspondence. The liquid inlet 21 is connected to the liquid inlet 12 and is respectively connected to the multiple flow channel assemblies 16. Compared with Example 2, the integrated design of multiple co-culture basic units can not only meet the needs of repeated experiments of multi-organoid co-culture, but also provide the possibility of studying multiple organoid co-culture models simultaneously, with the advantage of high throughput. Specifically, it can provide more repeating unit structures for multi-organ disease and tumor metastasis research, which is conducive to parallel experimental research of the same disease model, and also provide the possibility of constructing different disease models on the same chip, greatly increasing experimental throughput.

[0089] Optionally, multiple flow channel assemblies 16 are arranged around the liquid inlet end 12; multiple culture chamber assemblies 25 are arranged around the liquid inlet 21, and the distances between the multiple flow channel assemblies 16 and the liquid inlet end 12 are the same, and the consistency is good.

[0090] Vascularized multi-organ chips have the advantages of high degree of vascularization, large throughput, wide range of applications and high degree of biomimetic. They can be used for the construction of multi-organ disease models, high-throughput drug screening and tumor metastasis research.

[0091] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A vascularized multi-organ chip, characterized in that: include: The blood vessel layer (1) comprises a flow channel (11), a liquid inlet (12) and a liquid outlet (13) connected to both ends of the flow channel (11), and a first blood vessel culture unit (14) and a second blood vessel culture unit (15) provided on the flow channel (11) and connected to the flow channel (11); An organoid culture layer (2) comprises a liquid inlet (21), a liquid outlet (22), a first culture chamber (23), and a second culture chamber (24) separated from each other; the organoid culture layer (2) and the vascular layer (1) are stacked, the liquid inlet (21) is in communication with the liquid inlet end (12), and the liquid outlet (22) is in communication with the liquid outlet end (13); The porous membrane (3) comprises a plurality of micrometer-scale through-holes. The porous membrane (3) is provided between the first vascular culture unit (14) and the first culture chamber (23), and between the second vascular culture unit (15) and the second culture chamber (24), and the two are connected through the through-holes. The opening size of the through-holes is smaller than the outer dimensions of the endothelial cells and larger than the outer dimensions of the signal molecules secreted by the organoids.

2. The vascularized multi-organ chip according to claim 1, characterized in that: The flow channel (11) includes a main flow channel (111) and two first branch flow channels (112); the blood vessel layer (1) includes two liquid outlets (13); one end of the main flow channel (111) is connected to the liquid inlet (12); the other end is connected to the two first branch flow channels (112); the other ends of the two first branch flow channels (112) are correspondingly connected to the two liquid outlets (13); and the first blood vessel culture unit (14) and the second blood vessel culture unit (15) are both provided on the two first branch flow channels (112); The organoid culture layer (2) comprises two first culture chambers (23), two second culture chambers (24) and two liquid outlets (22); the two first culture chambers (23) and the two first blood vessel culture units (14) are correspondingly arranged; the two second culture chambers (24) and the two second blood vessel culture units (15) are correspondingly arranged; and the two liquid outlets (22) and the two liquid outlet ends (13) are correspondingly arranged.

3. The vascularized multi-organ chip according to claim 1, characterized in that: The flow channel (11) includes a main flow channel (111) and a plurality of second branch flow channels (113); the blood vessel layer (1) includes a plurality of liquid outlet ends (13); one end of the main flow channel (111) is connected to the liquid outlet end (13); the other end of the plurality of second branch flow channels (113) is connected to the plurality of liquid outlet ends (13); the other ends of the plurality of second branch flow channels (113) are correspondingly connected to the plurality of liquid outlet ends (13); the main flow channel (111) is provided with the first blood vessel culture unit (14); the plurality of second branch flow channels (113) are correspondingly provided with the plurality of second blood vessel culture units (15); the flow channel (11) and the plurality of liquid outlet ends (13) form a flow channel combination (16); The organoid culture layer (2) includes a culture chamber combination (25), the culture chamber combination (25) includes one first culture chamber (23), multiple second culture chambers (24) and multiple liquid outlets (22), the first culture chamber (23) and the first blood vessel culture unit (14) are correspondingly arranged, the multiple second culture chambers (24) and the multiple second blood vessel culture units (15) are correspondingly arranged, and the multiple liquid outlets (22) and the multiple liquid outlet ends (13) are correspondingly arranged.

4. The vascularized multi-organ chip according to claim 3, characterized in that: The flow channel (11) further comprises a plurality of intermediate branch flow channels (114), and the main flow channel (111) is connected to the second branch flow channels (113) via the intermediate branch flow channels (114).

5. The vascularized multi-organ chip according to claim 3, characterized in that: The blood vessel layer (1) comprises a liquid inlet end (12) and a plurality of flow channel assemblies (16), and the flow channels (11) of the plurality of flow channel assemblies (16) are all in communication with the liquid inlet end (12); The organoid culture layer (2) comprises a liquid inlet (21) and a plurality of culture chamber assemblies (25), wherein the liquid inlet (21) is connected to the liquid inlet end (12); the plurality of culture chamber assemblies (25) and the plurality of flow channel assemblies (16) are arranged in a one-to-one correspondence.

6. The vascularized multi-organ chip according to claim 5, characterized in that: A plurality of flow channel assemblies (16) are arranged around the liquid inlet end (12); and a plurality of culture chamber assemblies (25) are arranged around the liquid inlet (21).

7. The vascularized multi-organ chip according to any one of claims 1 to 6, characterized in that: The first blood vessel culture unit (14) and the second blood vessel culture unit (15) both include a plurality of branch ports and a plurality of confluence ports, and the plurality of branch ports and the plurality of confluence ports are connected via channels (149).

8. The vascularized multi-organ chip according to any one of claims 1 to 6, characterized in that: The first culture chamber (23) and the second culture chamber (24) are both provided with an opening, and a detachable sealing cover (4) is connected to the opening, and the opening is sealed by the sealing cover (4).

9. A method for using the vascularized multi-organ chip according to any one of claims 1 to 8, characterized in that: include: S100, injecting the endothelial cell suspension into the flow channel (11) of the vascular layer (1) from the liquid inlet (21), so that the endothelial cell suspension enters the first vascular culture unit (14) and the second vascular culture unit (15) and fills the flow channel (11); S200, flipping the vascularized multi-organoid chip upside down and placing it in a cell culture incubator under preset conditions, culturing it statically until the endothelial cells adhere to the porous membrane (3); wherein the vascular layer (1) is located above the organoid culture layer (2); S300, flipping the vascularized multi-organoid chip so that the organoid culture layer (2) faces upward, adding matrigel to the bottom of the first culture chamber (23) and the second culture chamber (24) to cover the surface of the porous membrane (3), and placing the chip in a cell culture incubator under preset conditions for incubation to solidify the matrigel; S400, inoculating a mixture of the first organoid and matrigel into a first culture chamber (23), inoculating a mixture of the second organoid and matrigel into a second culture chamber (24), and placing the cells in a cell culture incubator under preset conditions for incubation; S500: After a preset time, the culture medium of the first organoid is added to the first culture chamber (23), and the culture medium of the second organoid is added to the second culture chamber (24); at the same time, the culture fluid is continuously injected into the flow channel (11) of the vascular layer (1) from the liquid inlet (21), and the culture fluid flows out from the liquid outlet (22) after passing through the first vascular culture unit (14) and the second vascular culture unit (15).

10. The method for using the vascularized multi-organ chip according to claim 9, characterized in that: The flow channel (11) includes a main flow channel (111) and two first branch flow channels (112); the blood vessel layer (1) includes two liquid outlet ends (13); one end of the main flow channel (111) is connected to the liquid inlet end (12); the other end of the two first branch flow channels (112) is connected to the two liquid outlet ends (13); the first blood vessel culture unit (14) and the second blood vessel culture unit (15) are provided on the two first branch flow channels (112); the organoid culture layer (2) includes two first culture chambers (23), two second culture chambers (24) and two liquid outlets (22); the two first culture chambers (23) and the two first blood vessel culture units (14) are correspondingly arranged; the two second culture chambers (24) and the two second blood vessel culture units (15) are correspondingly arranged; the two liquid outlets (22) and the two liquid outlet ends (13) are correspondingly arranged; Step S400 includes: inoculating a mixture of a first organoid and matrigel into a first first culture chamber (23) and a second second culture chamber (24), and inoculating a mixture of a second organoid and matrigel into a first second culture chamber (24) and a second first culture chamber (23); Step S500 includes: adding a culture medium of the first organoid into the first first culture chamber (23) and the second second culture chamber (24), and adding a culture medium of the second organoid into the first second culture chamber (24) and the second first culture chamber (23), respectively.

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