Vascularized organ chip and method for large-scale culture

Abstract

The invention belongs to the technical field of biomedical engineering and microfluidics, and particularly discloses a vascularized organ chip and method for large-scale culture, the vascularized organ chip comprises an upper runner layer and a lower culture layer, and the runner layer and the culture layer are fixedly sealed; a liquid storage pipe matched with the flow channel layer is adhered to the upper surface of the flow channel layer; according to the vascularized organ chip, the flow channel layer is used for pouring a culture solution to provide fluid stimulation and nutrients for cells; the three parallel-connected large-scale culture chambers of the culture layer are used for culturing cells, so that blood circulation of a microvascular system in a human body can be simulated, and a microenvironment in tissues can be reproduced more truly; meanwhile, the requirement for culturing a large number of tissue cells through the chip can be met, and the cell yield and the experiment scale can be effectively increased.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering and microfluidics, and specifically relates to a vascularized organ chip for large-scale culture, and also relates to a preparation and use method of the vascularized organ chip for large-scale culture. Background Art

[0002] The liver is the largest internal organ in the human body, with a complex microscopic structure and function. The liver is responsible for key functions such as filtration, detoxification, exogenous metabolism, bile acid synthesis, and protein production. After entering the blood, drugs first enter the liver. Hepatotoxicity and drug-induced liver injury caused by various drugs are the main causes of late-stage drug failure. In addition, liver disease is one of the leading causes of death in the world, with new diseases appearing every year.

[0003] Animal experiments have been used in preclinical settings to understand human drug metabolism and toxicity, but there are huge differences between animal experiments and humans due to different species. Currently, some bionic microphysiological systems, such as liver models combined with organ-on-chip technology, can better recapitulate the physiological and pathological mechanisms of the human liver and how cells are affected by various internal and external factors. Weng et al. (doi:10.1002 / adma.201701545) developed a LOC liver chip composed of primary rat hepatocytes and hematopoietic stem cells HSCs, and connected to a peristaltic pump to circulate culture medium between the cell culture chamber and the culture medium reservoir, demonstrating the feasibility of recapitulating multi-scale physiological structural hierarchies, complex drug clearance, and regional physiology from the cellular to tissue level in long-term cultured liver chips.

[0004] The human liver has strong regeneration and proliferation capabilities, but in vitro hepatocyte functions (such as albumin secretion and enzyme activity) under traditional 2D and 3D culture conditions will weaken after being removed from the liver's natural environment (within one week); the key to this problem is the lack of an effective perfusion system to continuously deliver nutrients and effectively remove metabolic waste; in order to solve this problem and further extend the life of the liver's advanced metabolism in vitro, it is necessary to vascularize the hepatocytes and perfuse the microtissues, thus proposing a vascularized liver chip, which provides a nutrient supply and waste removal system similar to that in the body, can prolong the activity of hepatocytes and maintain their function; a continuous microfluidic perfusion system can help simulate blood flow, improve the cell microenvironment, and enable cells to maintain a similar functional state in vivo; while traditional liver models often lack a microcirculatory environment in terms of drug metabolism, which affects the accurate simulation of drug absorption, metabolism and elimination processes; vascularized chips introduce vascular networks to allow drugs to flow through hepatocytes in an in vitro environment, thus being closer to the actual metabolic process in the human body; this helps to improve the accuracy of drug metabolism and toxicity testing, making it closer to human reactions.

[0005] At present, the growth and proliferation of cells in conventional organ chips are limited, making it difficult to achieve large-scale culture of cells in organ chips and maintain a relatively stable physiological state; large-scale culture of cells is conducive to maintaining the physiological functions of cells, helping to more comprehensively analyze drug effects and disease mechanisms, providing the accuracy of drug screening and toxicity testing, and significantly improving the efficiency and yield in the production of biological products such as vaccines and monoclonal antibodies. Therefore, it is very necessary to study large-scale culture chips.

[0006] In summary, in order to better realize large-scale culture of cell tissues on organ chips and conduct research on organ tissue functions, a vascularized organ chip that can be cultured on a large scale is urgently needed. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a vascularized organ chip for large-scale culture that can simulate the interaction between tissue cells and endothelial cells, achieve steady proliferation of tissue cells and construction of organoids, and also provides corresponding preparation and use methods.

[0008] Based on the above purpose, the present invention is achieved through the following technical solutions: A vascularized organ chip for large-scale culture, comprising an upper flow channel layer and a lower culture layer, the flow channel layer and the culture layer are bonded, fixed and sealed; a liquid storage tube matching the flow channel layer is adhered to the upper surface of the flow channel layer, the upper flow channel layer and the lower culture layer are fixed along the alignment holes on the chip, and the organ chip is injection molded from a high molecular polymer material, preferably polydimethylsiloxane (PDMS), the material is a transparent material, has good biocompatibility, and can reduce manufacturing costs; a culture fluid channel is provided on one side of the flow channel layer close to the culture layer, for perfusing culture fluid to provide fluid stimulation and nutrients for cells, the culture fluid channel comprises a diamond flow channel, and a plurality of serpentine pipes of different lengths are provided on the upper and lower sides of the diamond flow channel, and the serpentine pipes on the upper and lower sides are connected through the diamond flow channel; the special tiny spacing of the diamond flow channel can effectively prevent the cell gel from penetrating and leaking into the upper flow channel when injected into the lower culture chamber, thereby avoiding the blockage of the culture fluid.

[0009] Preferably, a liquid storage tube matching the flow channel layer is adhered to the upper surface of the flow channel layer, and the liquid storage tube is a glass liquid storage tube; an upper liquid injection port and an upper liquid outlet with a clearance fit are symmetrically arranged at the intersection of the serpentine pipe and the diamond-shaped flow channel.

[0010] Preferably, the left and right sides of the flow channel layer are respectively provided with an upper second glue injection inlet and an upper second glue injection outlet, the upper and lower sides of the upper second glue injection inlet are respectively provided with an upper first glue injection outlet and an upper third glue injection outlet, and the left side of the upper second glue injection inlet is provided with an upper second pressure relief port; the upper and lower sides of the upper second glue injection outlet are respectively provided with an upper first glue injection inlet and an upper third glue injection inlet, and the right side of the upper second glue injection outlet is provided with an upper first pressure relief port; the spacing between the upper second glue injection outlet and the upper first glue injection inlet and the upper third glue injection inlet is smaller than the spacing between the upper second glue injection inlet and the upper first glue injection outlet and the upper third glue injection outlet; the upper first alignment holes and the upper second alignment holes are symmetrically arranged on the diagonal line of the flow channel layer to facilitate the fixing of the relative position of the flow channel layer and the culture layer.

[0011] Preferably, three large-scale parallel arrangement and clearance-matched first culture chambers, second culture chambers and third culture chambers are provided on the side of the culture layer close to the flow channel layer; a first capillary bursting valve connected to the outside is provided on the right channel of the first culture chamber and the third culture chamber, which is used to release part of the injection pressure when injecting the gel and reduce the risk of cell gel leakage into the flow channel layer; the first capillary bursting valve includes a first pressure regulating pipeline connected to the right channel of the first culture chamber and the third culture chamber, and a lower first pressure reducing port connected to an upper first pressure reducing port is provided at the center of the first pressure regulating pipeline.

[0012] Preferably, the channel ends at both ends of the first culture chamber are respectively provided with a lower first glue injection inlet connected to the upper first glue injection inlet, and a lower first glue injection outlet connected to the upper first glue injection outlet; the channel ends at both ends of the third culture chamber are respectively provided with a lower third glue injection inlet connected to the upper third glue injection inlet, and a lower third glue injection outlet connected to the upper third glue injection outlet.

[0013] Preferably, a second capillary bursting valve is provided on the left channel of the second culture chamber, and the second capillary bursting valve includes a second pressure regulating pipeline connected to the left channel of the second culture chamber, and a lower second pressure reducing port connected to the upper second pressure reducing port is provided at the center of the second pressure regulating pipeline; the channel ends at both ends of the second culture chamber are respectively provided with a lower second glue injection inlet connected to the upper second glue injection inlet, and a lower second glue injection outlet connected to the upper second glue injection outlet.

[0014] Preferably, the culture layer is provided with a lower layer first alignment port connected to the upper layer first alignment hole, and a lower layer second alignment port connected to the upper layer second alignment hole; the flow channel layer is provided with an upper layer middle pressure relief port, and the upper layer middle pressure relief port is connected to the lower layer middle pressure relief port arranged on the culture layer; the flow channel layer is provided with an upper layer side pressure relief port, and the upper layer side pressure relief port is connected to the lower layer side pressure relief port arranged on the culture layer.

[0015] Preferably, the thickness of the flow channel layer is 1-2mm; the height of the culture fluid channel is 100μm; in the flow channel layer, the diameter of all through holes is 1.5mm; the width of the serpentine flow channel is 0.2mm, the length of each group of serpentine channels is 80mm, the length of each group of diamond flow channels is 15mm, the width is 40um, and the spacing between adjacent diamonds is 70um; the width of the first culture chamber, the second culture chamber and the third culture chamber is 4mm, and the chamber thickness is 500um; the diameter of all through holes in the culture layer is 1.5mm; the radius of the liquid storage tube is 8mm and the height is 20mm.

[0016] Different organ tissue cells can be selected for research on this platform, such as brain glioma cells, lung cells, bone cells, etc.

[0017] A method for preparing and using a vascularized organ chip comprises the following steps; S1. Use AutoCAD drawing software to draw and make the mask plate corresponding to the structure of the chip. Use soft lithography and photolithography mask processes to make silicon wafers for the flow channel layer and the culture layer respectively, and use PDMS (polydimethylsiloxane) to cast to form a single chip structure; through oxygen plasma treatment, the upper flow channel layer of the chip is bonded to the lower cell culture layer to form a complete chip structure; finally, use PDMS to attach the glass tube to the surface of the flow channel layer and heat it to obtain the target chip.

[0018] S2. The fabricated vascularized organ chip is sterilized at high temperature and then set aside.

[0019] S3. Take 250 μL of the mixed cell stock solution containing three types of cells (human umbilical vein endothelial cells HUVEC, human lung fibroblasts NHLF and hepatocytes HepG2), take 80uL of it and mix it with 8μL of thrombin to form a hydrogel, and inject it into the three culture chambers located on the culture layer from the first injection inlet on the upper layer of the flow channel layer, the second injection inlet on the upper layer and the third injection inlet on the upper layer within 20 seconds.

[0020] S4. After placing the vascularized organ chip of step S3 in a CO2 incubator for 15-20 minutes, laminin is injected from the upper injection port of the flow channel layer to infiltrate the culture medium channel, and then placed in an incubator with the same conditions for culturing for 10 minutes.

[0021] S5. Add different volumes of culture fluid to the two liquid storage tubes on the surface of the chip to form different pressure values. Under the action of gravity, the culture fluid is constantly perfusing the cell channel in both horizontal and vertical dimensions. Finally, place the chip in a culture box and transfer it to an incubator for culture.

[0022] Preferably, in step S4, the content of laminin in the culture medium channel is 15 μL; In steps S4 and S5, the CO2 content in the incubator is 5% and the temperature is 37°C; In step S5, the culture solution in one liquid storage tube is 2.5 ml, and the culture solution in the other liquid storage tube is 0.2 ml.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention can culture tissue cells on a large scale in vitro, simulate the interaction between tissue cells and endothelial cells, and achieve rapid proliferation of tissue cells and construction of tissue organoids; a single chip can be injected with more than 20 million cells, and the structure can sustain cell growth and proliferation for more than 10 days.

[0024] 2. The vascularized organ chip provides fluid stimulation and nutrients to cells by perfusing culture fluid through the flow channel layer; the large-scale culture chambers formed by three parallel connections of the culture layer are used to culture cells. The above structure can not only simulate the blood circulation of the microvascular system in the human body, but also reproduce the microenvironment in the tissue more realistically; at the same time, it can meet the needs of culturing a large number of tissue cells on the chip, and can effectively increase the cell yield and experimental scale.

[0025] 3. The serpentine pipes on the flow channel layer of the present invention are designed to have different lengths, which is conducive to controlling the hydrostatic pressure applied by the fluid to form a pressure difference, so that the culture fluid can diffuse from the diamond flow channel to each culture chamber of the culture layer, bringing fluid shear force and nutrient supply to the cells in each culture chamber.

[0026] 4. The diamond-shaped flow channel design in the flow channel layer has a specific micro-spacing. This structure can effectively prevent leakage when injecting cell gel into the lower culture chamber, thereby avoiding entering the upper flow channel layer and causing blockage of the culture channel; this micro-spacing designed flow channel provides a reliable isolation environment for cell culture, ensuring the independence of fluid flow between different layers, while ensuring the smooth penetration process of the culture fluid.

[0027] 5. The culture layer is composed of three large-scale culture chambers in parallel. The parallel culture chamber structure helps to evenly distribute fluids and nutrients, avoiding the uneven flow rate and unbalanced nutrient supply problems that may be caused by a single chamber structure, thereby improving the stability and repeatability of the entire chip system, making it suitable for long-term in vitro culture experiments and more convenient for subsequent observations.

[0028] 6. The vascularized organ chip of the present invention can be used for in vitro drug testing and acquisition of drug metabolites, and can also be used to study the distribution, transport and absorption mechanisms of drugs in the circulatory system.

[0029] 7. The chip has a simple design and can realize a cell chamber with a height of 500um, providing a large space for cell growth and good biocompatibility. Its manufacturing process is low-cost, highly repeatable, and suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is an exploded view of Example 1 of the present invention; Figure 2 is a schematic diagram of embodiment 1 of the present invention; Figure 3 This is a schematic diagram of the internal structure of Embodiment 1 of the present invention; Figure 4 This is a schematic diagram of the lower surface structure of the flow channel layer of Example 1 of the present invention; Figure 5 This is a schematic diagram of the upper surface structure of the culture layer of Example 1 of the present invention; Figure 6 A schematic diagram of cells required for the experiment of Example 3 of the present invention; Figure 7 This is a schematic diagram of tissue formation after cell injection in Example 3 of the present invention; Figure 8 This is a schematic diagram of a dyeing experiment in Example 3 of the present invention; Fig. 9 Schematic diagram of the culture experiment results of Example 4 of the present invention (n>3).

[0031] In the figure, the flow channel layer 1, the first glue injection inlet 111 of the upper layer, the first glue injection outlet 112 of the upper layer, the second glue injection inlet 122 of the upper layer, the second glue injection outlet 121 of the upper layer, the third glue injection inlet 131 of the upper layer, the third glue injection outlet 132 of the upper layer, the first pressure reducing outlet 141 of the upper layer, the second pressure reducing outlet 151 of the upper layer, the first alignment hole 161 of the upper layer, the second alignment hole 162 of the upper layer, the liquid outlet 171 of the upper layer, the liquid injection port 172 of the upper layer, the serpentine pipe 18, the diamond flow channel 19, the culture layer 2 ... first alignment hole 161 of the upper layer, the second alignment hole 162 of the upper layer, the first alignment hole 161 of the upper layer, the second alignment hole 162 of the upper A culture chamber 21, a first glue injection inlet 211 at the lower layer, a first glue injection outlet 212 at the lower layer, a second culture chamber 22, a second glue injection outlet 221 at the lower layer, a second glue injection inlet 222 at the lower layer, a third culture chamber 23, a third glue injection inlet 231 at the lower layer, a third glue injection outlet 232 at the lower layer, a first pressure regulating pipeline 24, a first pressure reducing port 241 at the lower layer, a second pressure regulating pipeline 25, a second pressure reducing port 251 at the lower layer, a first alignment port 261 at the lower layer, a second alignment port 262 at the lower layer, and a liquid storage tube 3. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below through specific examples, but the scope of the present invention is not limited thereto.

[0033] Example 1 A vascularized organ-on-a-chip for large-scale culture, e.g. Figure 1-Figure 5 As shown, it includes an upper flow channel layer 1 and a lower culture layer 2, and the flow channel layer 1 is bonded and fixed to the culture layer 2 to seal; a culture fluid channel is provided on the side of the flow channel layer 1 close to the culture layer 2, and the culture fluid channel includes a diamond flow channel 19, and a plurality of serpentine pipes 18 of different lengths are provided on the upper and lower sides of the diamond flow channel 19, and the serpentine pipes 18 on the upper and lower sides are connected through the diamond flow channel 19.

[0034] A liquid storage tube 3 matching with the flow channel layer 1 is adhered to the upper surface of the flow channel layer 1; an upper layer liquid injection port 172 and an upper layer liquid outlet port 171 with clearance fit are symmetrically arranged at the intersection of the serpentine pipe 18 and the diamond flow channel 19.

[0035] An upper second glue injection inlet 122 and an upper second glue injection outlet 121 are respectively provided on the left and right sides of the flow channel layer 1, an upper first glue injection outlet 112 and an upper third glue injection outlet 132 are respectively provided on the upper and lower sides of the upper second glue injection inlet 122, and an upper second pressure reducing outlet 151 is provided on the left side of the upper second glue injection inlet 122; an upper first glue injection inlet 111 and an upper third glue injection inlet 131 are respectively provided on the upper and lower sides of the upper second glue injection outlet 121, and an upper first pressure reducing outlet 141 is provided on the right side of the upper second glue injection outlet 121; the spacing between the upper second glue injection outlet 121 and the upper first glue injection inlet 111 and the upper third glue injection inlet 131 is smaller than the spacing between the upper second glue injection inlet 122 and the upper first glue injection outlet 112 and the upper third glue injection outlet 132; an upper first alignment hole 161 and an upper second alignment hole 162 are symmetrically provided on the diagonal line of the flow channel layer 1.

[0036] On the side of the culture layer 2 close to the flow channel layer 1, there are three large-scale parallel arranged first culture chambers 21, second culture chambers 22 and third culture chambers 23 with gaps fitted; the right channels of the first culture chamber 21 and the third culture chamber 23 are provided with first capillary bursting valves connected to the outside world; the first capillary bursting valve includes a first pressure regulating pipeline 24 connected to the right channels of the first culture chamber 21 and the third culture chamber 23, and the center of the first pressure regulating pipeline 24 is provided with a lower first pressure reducing port 241 connected to the upper first pressure reducing port 141.

[0037] The channel ends at both ends of the first culture chamber 21 are respectively provided with a lower first glue injection inlet 211 connected to the upper first glue injection inlet 111, and a lower first glue injection outlet 212 connected to the upper first glue injection outlet 112; the channel ends at both ends of the third culture chamber 23 are respectively provided with a lower third glue injection inlet 231 connected to the upper third glue injection inlet 131, and a lower third glue injection outlet 232 connected to the upper third glue injection outlet 132.

[0038] A second capillary bursting valve is provided on the left channel of the second culture chamber 22, and the second capillary bursting valve includes a second pressure regulating pipeline 25 connected to the left channel of the second culture chamber 22, and a lower second pressure reducing port 251 connected to the upper second pressure reducing port 151 is provided at the center of the second pressure regulating pipeline 25; the channel ends at both ends of the second culture chamber 22 are respectively provided with a lower second glue injection inlet 222 connected to the upper second glue injection inlet 122, and a lower second glue injection outlet 221 connected to the upper second glue injection outlet 121.

[0039] The culture layer 2 is provided with a lower first alignment opening 261 communicating with the upper first alignment hole 161 and a lower second alignment opening 262 communicating with the upper second alignment hole 162 .

[0040] The thickness of the flow channel layer 1 is 1-2mm; the height of the culture fluid channel is 100μm; in the flow channel layer 1, the diameter of all through holes is 1.5mm; the width of the serpentine flow channel is 0.2mm, the length of each group of serpentine channels is 80mm, the length of each group of diamond flow channels 19 is 15mm, the width is 40um, and the spacing between adjacent diamonds is 70um; the width of the first culture chamber 21, the second culture chamber 22 and the third culture chamber 23 is 4mm, and the chamber thickness is 500um; the diameter of all through holes in the culture layer 2 is 1.5mm; the radius of the liquid storage tube 3 is 8mm and the height is 20mm.

[0041] A method for preparing and using a vascularized organ chip comprises the following steps; S1. Draw the microchannel pattern to be processed on AutoCAD, and then process the drawing into a mask corresponding to the structure of the chip. The upper channel layer 1 is made into a silicon wafer with a microchannel structure based on the soft photolithography method (the selected channel height is 100μm), and the lower culture layer 2 is made into a silicon wafer with a microchannel structure based on the photolithography mask method (the selected channel height is 500μm), and then the silicon wafer is pasted in a culture dish to make a mold; polydimethylsiloxane (PDMS) prepolymer and curing agent are selected and evenly mixed in a ratio of 10:1 and poured into the mold, and a PDMS sheet with a microchannel structure is obtained by vacuuming, blowing bubbles, drying, peeling, etc.; Figure 1 As shown in the flow channel layer 1 and the culture layer 2; the flow channel layer 1 and the culture layer 2 are punched respectively, and then the flow channel layer 1 and the culture layer 2 are plasma bonded, the upper first alignment hole 161 corresponds to the lower first alignment port 261, and the upper second alignment hole 162 corresponds to the lower second alignment port 262; then a small amount of PDMS is used to stick the glass liquid storage tube 3 to the chip flow channel layer 1 respectively and connect it with the upper liquid injection port 172 and the upper liquid outlet port 171, and then the chip is placed in a drying oven for drying for 2 hours at a temperature of 65°C, and a vascularized organ chip with a glass liquid storage tube 3 can be obtained.

[0042] S2. The fabricated vascularized organ chip is sterilized at high temperature and then set aside.

[0043] S3, prepare 250uL of cell stock solution containing three kinds of cells: human umbilical vein endothelial cells HUVEC, human lung fibroblasts NHLF and hepatocytes HepG2, take 80μL of the cell stock solution and mix with 8μl of thrombin in turn to form a biohydrogel, and inject it into the first culture cavity 21, the second culture cavity 22 and the third culture cavity 23 located in the culture layer 2 from the upper first injection inlet 111, the upper second injection inlet 122 and the upper third injection inlet 131 on the upper surface of the flow channel layer 1 of the chip within 20s, respectively. Then the cell stock solution will fill the first culture cavity 21, the second culture cavity 22 and the third culture cavity 23 along the lower first injection inlet 211, the lower second injection inlet 222 and the lower third injection inlet 231. The air remaining in 3 is discharged through the first glue injection outlet 212 of the lower layer, the second glue injection outlet 221 of the lower layer, and the third glue injection outlet 232 of the lower layer along the first glue injection outlet 112 of the upper layer, the second glue injection outlet 121 of the upper layer, and the third glue injection outlet 132 of the upper layer respectively; by setting the first capillary bursting valve and the second capillary bursting valve, the first capillary bursting valve is decompressed through the first pressure reducing port 241 of the lower layer of the first pressure regulating pipeline 24 and along the first pressure reducing port 141 of the upper layer, and the second capillary bursting valve is decompressed through the second pressure reducing port 251 of the lower layer of the second pressure regulating pipeline 25 along the second pressure reducing port 151 of the upper layer, so that part of the injection pressure can be released when injecting the gel, thereby reducing the risk of cell gel leaking into the upper flow channel layer 1; and then the chip is placed in a cell culture incubator with a temperature of 37°C and a carbon dioxide concentration of 5% for 15 minutes.

[0044] S4. After placing the vascularized organ chip of step S3 in a CO2 incubator for 15-20 minutes, laminin is injected from the upper injection port 172 of the flow channel layer 1 to infiltrate the culture medium channel. The serpentine pipe 18 and the diamond flow channel 19 form a culture medium channel, and then placed in an incubator with the same conditions for incubation for 10 minutes; the content of laminin in the culture medium channel is 15 μL; the CO2 content in the incubator is 5%, and the temperature is 37°C.

[0045] S5. Add different volumes of culture fluid to the two liquid storage tubes 3 on the surface of the chip respectively to form different pressure differences. Under the action of gravity, the culture fluid is constantly perfusing the cell channel in both horizontal and vertical dimensions. The culture fluid in one liquid storage tube 3 is 2.5 ml, and the culture fluid in the other liquid storage tube 3 is 0.2 ml. Finally, put the chip into a culture box and transfer it to an incubator for culture.

[0046] Example 2 A vascularized organ chip for large-scale culture, the preparation method of which comprises the following steps: Draw the microchannel pattern to be processed on AutoCAD, and then process the drawing into a mask corresponding to the structure of the chip. The upper channel layer 1 is made into a silicon wafer with a microchannel structure based on soft photolithography (the selected channel height is 100μm), and the lower culture layer 2 is made into a silicon wafer with a microchannel structure based on photolithography mask method (the selected channel height is 500μm), and then the silicon wafer is pasted in a culture dish to make a mold; polydimethylsiloxane (PDMS) prepolymer and curing agent are selected and evenly mixed in a ratio of 10:1 and poured into the mold, and a PDMS sheet with a microchannel structure is obtained by vacuuming, blowing bubbles, drying, peeling, etc.; Figure 1 As shown in the flow channel layer 1 and the culture layer 2; the flow channel layer 1 and the culture layer 2 are respectively punched, and then the flow channel layer 1 and the culture layer 2 are plasma bonded; then a small amount of PDMS is used to stick the glass liquid storage tube 3 to the chip flow channel layer 1 and connect it with the upper liquid injection port 172 and the upper liquid outlet 171, and then the chip is placed in a drying oven for drying for 2 hours at a temperature of 65°C, and a vascularized organ chip with a glass liquid storage tube 3 can be obtained; before the experiment, the chip needs to be sterilized at high temperature in advance and stored in a clean bench.

[0047] Example 3 A vascularized organ chip for large-scale culture, the method of using the same comprises the following steps: S01, take 80 μL of the cell stock solution containing human umbilical vein endothelial cells HUVEC, human lung fibroblasts NHLF and hepatocytes HepG2. Figure 6 As shown, it is mixed with 8 μl of thrombin (the function of which is to make the cell stock solution cross-linked and solidified) to form a biohydrogel, and is injected from the upper first injection inlet 111, the upper second injection inlet 122 and the upper third injection inlet 131 on the upper surface of the flow channel layer 1 of the chip within 20 seconds, and then the cell stock solution will fill the first culture chamber 21, the second culture chamber 22, and the third culture chamber 23; by setting the first capillary burst valve and the second capillary burst valve, part of the injection pressure can be released when injecting the gel, reducing the risk of cell gel leaking into the upper flow channel layer 1; then the chip is placed in a cell culture incubator with a temperature of 37°C and a carbon dioxide concentration of 5% for 15 minutes.

[0048] S02, injecting laminin into the culture medium channel through the upper injection port 172 on the flow channel layer 1 to support the outward growth of cells, and then placing the chip in an incubator with the same conditions and letting it stand for 10 minutes.

[0049] S03, respectively perfuse different volumes of culture fluid into the liquid storage tubes 3 on both sides to form different pressure differences, and under the action of gravity, achieve the result that the culture fluid always perfuses the cell channel in both horizontal and vertical dimensions. Perfuse 2.5mL of culture fluid into the liquid storage tube 3 on one side, and perfuse 0.2mL of culture fluid into the liquid storage tube 3 on the other side. Replace the culture fluid every 24 hours. When replacing the culture fluid, ensure that the injection volume is exactly the same as the previous time; swap the amount of culture fluid in the two liquid storage tubes 3 every other day; because bubbles may be generated at the perfusion port, the chip should be taken out 4 hours after the culture fluid is injected to observe the flow channel. If bubbles are generated, it is necessary to remove the bubbles. Figure 7 shown.

[0050] S04. During the culture process, one of the chips injected into the culture in the same experiment was stained with DAPI every day, and the proliferation of the number of cells in the chamber was statistically analyzed by counting the cell nuclei that emitted strong blue fluorescence after staining; DAPI (4',6-diamidino-2-phenylindole) binds to the phosphate-deoxyribose backbone of DNA and is an effective and widely used cell nucleus labeling technology; polychloroformaldehyde was used to fix and permeabilize the cells in the cultured organ chip, and DAPI dye was added. The samples were incubated at room temperature for a few minutes and the samples were rinsed multiple times; Figure 8 The figure shows an image of the DAPI channel of the cell nucleus of the vascularized organ chip under a 10x microscope. By observing the relative positions of the stained cell nuclei, we can see the process of cell injection to vascularization. The cells are evenly dispersed at the time of injection, and the nuclei that differentiate into tubes on the third day are arranged along the lumen of the tube in an elliptical shape. In addition, in the DAPI fluorescence image of the chamber on the fourth day of culture, it can be clearly observed that the number of cells has almost doubled and the blood vessels have been roughly formed.

[0051] Example 4 A vascularized organ chip for large-scale culture, which is different from Example 1 in that the serpentine channel 18 is replaced by a straight channel or a meandering channel or a shunt channel or other configuration channels to buffer the speed of injecting culture fluid and simulate the transport path of microvessels. The culture experiment results are as follows Fig. 9 shown.

[0052] Example 5 A vascularized organ chip for large-scale culture is different from Example 1 in that a peristaltic pump is used to replace the liquid storage tube 3, and the peristaltic pump is set at a certain flow rate for perfusion culture.

[0053] Example 6 The vascularized organ chip for large-scale culture is different from that of Example 1 in that different organ tissue cells are selected for research on this platform, such as brain glioma cells, lung cells, bone cells, etc.

[0054] The above description is only a preferred embodiment of the present invention, but is not limited to the above examples. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A vascularized organ chip for large-scale culture, characterized in that: It includes an upper flow channel layer and a lower culture layer, and the flow channel layer is fixedly sealed to the culture layer; a culture fluid channel is provided on the side of the flow channel layer close to the culture layer, and the culture fluid channel includes a diamond flow channel, and multiple serpentine pipes of different lengths are provided on the upper and lower sides of the diamond flow channel, and the serpentine pipes on the upper and lower sides are connected through the diamond flow channel.

2. The vascularized organ chip for large-scale culture according to claim 1, characterized in that: A liquid storage tube matched with the flow channel layer is adhered to the upper surface of the flow channel layer; an upper layer liquid injection port and an upper layer liquid outlet with clearance matching are symmetrically arranged at the intersection of the serpentine pipe and the diamond-shaped flow channel.

3. The vascularized organ chip for large-scale culture according to claim 2, characterized in that: The left and right sides of the flow channel layer are respectively provided with an upper second glue injection inlet and an upper second glue injection outlet, the upper and lower sides of the upper second glue injection inlet are respectively provided with an upper first glue injection outlet and an upper third glue injection outlet, and the left side of the upper second glue injection inlet is provided with an upper second pressure reducing port; the upper and lower sides of the upper second glue injection outlet are respectively provided with an upper first glue injection inlet and an upper third glue injection inlet, and the right side of the upper second glue injection outlet is provided with an upper first pressure reducing port; the spacing between the upper second glue injection outlet and the upper first glue injection inlet and the upper third glue injection inlet is smaller than the spacing between the upper second glue injection inlet and the upper first glue injection outlet and the upper third glue injection outlet; the upper first alignment hole and the upper second alignment hole are symmetrically arranged on the diagonal line of the flow channel layer.

4. The vascularized organ chip for large-scale culture according to claim 3, characterized in that: The side of the culture layer close to the flow channel layer is provided with three first culture cavities, a second culture cavity and a third culture cavity which are arranged in parallel and have clearances; the right channels of the first culture cavity and the third culture cavity are provided with a first capillary bursting valve which is connected to the outside world; the first capillary bursting valve includes a first pressure regulating pipeline which is connected to the right channels of the first culture cavity and the third culture cavity, and the center of the first pressure regulating pipeline is provided with a lower first pressure reducing port which is connected to the upper first pressure reducing port.

5. The vascularized organ chip for large-scale culture according to claim 4, characterized in that: The channel ends at both ends of the first culture chamber are respectively provided with a lower first glue injection inlet connected to the upper first glue injection inlet, and a lower first glue injection outlet connected to the upper first glue injection outlet; the channel ends at both ends of the third culture chamber are respectively provided with a lower third glue injection inlet connected to the upper third glue injection inlet, and a lower third glue injection outlet connected to the upper third glue injection outlet.

6. The vascularized organ chip for large-scale culture according to claim 5, characterized in that: A second capillary bursting valve is provided on the left channel of the second culture chamber, and the second capillary bursting valve includes a second pressure regulating pipeline connected to the left channel of the second culture chamber, and a lower second pressure reducing port connected to the upper second pressure reducing port is provided at the center of the second pressure regulating pipeline; the channel ends at both ends of the second culture chamber are respectively provided with a lower second glue injection inlet connected to the upper second glue injection inlet and a lower second glue injection outlet connected to the upper second glue injection outlet.

7. The vascularized organ chip for large-scale culture according to claim 6, characterized in that: The culture layer is provided with a lower layer first alignment opening connected to the upper layer first alignment hole, and a lower layer second alignment opening connected to the upper layer second alignment hole.

8. The vascularized organ chip for large-scale culture according to claim 7, characterized in that: The thickness of the flow channel layer is 1-2mm; the height of the culture fluid channel is 100μm; in the flow channel layer, the diameter of all through holes is 1.5mm; the width of the serpentine flow channel is 0.2mm, the length of each group of serpentine channels is 80mm, the length of each group of diamond flow channels is 15mm, the width is 40um, and the spacing between adjacent diamonds is 70um; the width of the first culture chamber, the second culture chamber and the third culture chamber is 4mm, and the chamber thickness is 500um; the diameter of all through holes in the culture layer is 1.5mm; the radius of the liquid storage tube is 8mm and the height is 20mm.

9. A method for preparing and using a vascularized organ chip according to any one of claims 1 to 8, characterized in that: The steps include: S1. Draw the mask corresponding to the structure of the chip, and make the silicon wafers of the flow channel layer and the culture layer respectively through soft lithography and photolithography mask processes, and use PDMS casting to form a single chip structure; through oxygen plasma treatment, the upper flow channel layer of the chip is bonded with the lower cell culture layer to form a complete chip structure; finally, use PDMS to attach the glass tube to the surface of the flow channel layer and heat and fix it to obtain the target chip; S2, sterilizing the fabricated vascularized organ chip at high temperature for later use; S3, take 250 μL of the mixed cell stock solution containing three types of cells, take 80 uL of it and mix it with 8 μL of thrombin in sequence to form a hydrogel, and inject it into the three culture chambers located in the culture layer from the first injection inlet of the upper layer of the flow channel layer, the second injection inlet of the upper layer, and the third injection inlet of the upper layer within 20 seconds; S4, placing the vascularized organ chip of step S3 in a CO2 incubator for 15-20 minutes, injecting laminin into the culture medium channel from the upper injection port of the flow channel layer, and then placing the chip in an incubator under the same conditions for 10 minutes; S5. Add different volumes of culture fluid to the two liquid storage tubes on the surface of the chip to form different pressure values. Under the action of gravity, the culture fluid is constantly perfusing the cell channel in both horizontal and vertical dimensions. Finally, place the chip in a culture box and transfer it to an incubator for culture.

10. The method for preparing and using a vascularized organ chip according to claim 9, characterized in that: In step S4, the content of laminin in the culture medium channel is 15 μL; In steps S4 and S5, the CO2 content in the incubator is 5% and the temperature is 37°C; In step S5, the culture solution in one liquid storage tube is 2.5 ml, and the culture solution in the other liquid storage tube is 0.2 ml.

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