Multi-organ chip and preparation method thereof

Abstract

The invention belongs to the technical field of organoid chips of biological medicine, and particularly relates to a multi-organoid chip and a preparation method thereof. The multi-class organ chip comprises an upper-layer chip and a lower-layer chip which are detachably arranged; 2-10 organ-like chambers are embedded in the upper-layer chip, and 2-5 porous membranes are arranged at the bottoms of the organ-like chambers; the lower-layer chip is provided with a flow channel, and the flow channel is communicated with the organ-like chamber. Compared with the existing multi-class organ chip technology, the multi-barrier construction in the independent cavity is realized, and meanwhile, the multi-class organ chip is combined with a transwell component for use, so that the personalized combination of the multi-class organ chips is realized. The method is economical and fast, can play an important role in promoting the fields of precise medicine, medicine evaluation, disease models, especially in-vitro evaluation and simulation of traditional Chinese medicine and the like, and is suitable for popularization and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical organoid chips, and specifically relates to a multi-organoid chip and a preparation method thereof. Background Art

[0002] The traditional in vitro drug evaluation model is cells, which later developed into complex organoid models, but these models still lack an extracellular environment and cannot evaluate drugs and mimic the overall effects of physiology and pathology. The combination of microfluidics and a variety of cells and organoids forms an organ chip, which can simulate the physiological environment of the target organ in vitro. In 2004, microfluidics was first used for organ and system function modeling in human physiology or disease research. In 2010, the first lung organ chip was constructed. After more than ten years of development, organ chips are moving towards multi-organ integration, integrating organoids with microfluidics and organ chip technologies to achieve more complex in vitro models. In 2022, a multi-organ chip model with independent barriers was born, which brought revolutionary technology to complex physiology, pathology and in vitro efficacy evaluation, especially the in vitro evaluation of complex compounds such as traditional Chinese medicine.

[0003] At present, multi-organoid chips have achieved long-term culture of multiple organoids connected by flow paths and isolated by independent barriers. However, a single barrier can only simulate one of the vascular endothelial barrier, blood-brain barrier or respiratory endothelial cell barrier, while the actual internal environment of the human body often has multiple barriers. For example, food enters the body fluid through the intestinal barrier and then enters the blood circulation from the vascular barrier. A single intestinal barrier also includes mechanical barriers, endothelial barriers, immune barriers, etc. Although the porous membrane barrier can isolate the culture medium environment of the organoids from the flow path, it is difficult to simulate the real environment of the human body, especially for simulating the absorption and metabolic process of oral drugs. It can only be used for in vitro efficacy evaluation of injectable drugs, while oral drugs and traditional Chinese medicines require intestinal absorption-liver metabolism processes that are difficult to achieve.

[0004] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention constructs a modular multi-barrier multi-organoid chip. Specifically, it realizes the independent culture environment of organoids, multi-barrier function simulation, multi-organoid fluid circuit series connection and matching with commercial transwell modules to achieve random combination of multiple organoids. At the same time, the chambers between the barriers can also realize the simulation of tumor cell migration and interaction between organs.

[0006] Specifically, the present invention provides a multi-organ chip, comprising an upper chip and a lower chip that can be detachably arranged; wherein: The upper chip is embedded with an organoid chamber, and a porous membrane is disposed at the bottom of the organoid chamber; The lower chip is provided with a flow channel, and the flow channel is connected to the organoid chamber; The number of the organoid chambers is 2 to 10; The porous membrane is arranged in layers; wherein: The number of layers of the porous membrane is 2 to 5, and a barrier chamber is formed between two adjacent layers of the porous membrane, and the barrier chamber is used to fill a liquid medium or an organoid.

[0007] Preferably, the upper chip is further provided with a flow channel inlet and a flow channel outlet; wherein: The flow channel inlet, the flow channel, and the flow channel outlet are connected.

[0008] Preferably, the pore size of the porous membrane is 0.1-100 μm.

[0009] Preferably, the multi-organ chip further comprises a chip cover, wherein: The chip cover is arranged to cover the upper chip.

[0010] Based on the same technical concept, the present invention further provides a method for preparing a multi-organ chip, the preparation method comprising the following steps: (1) Spin-coating photoresist on the surface of the silicon wafer, then performing pre-baking, and setting the UV exposure time for photolithography to obtain a pre-mold; (2) post-baking the pre-mold, then washing it in a developer, and finally performing a hard film treatment to obtain a hard-baked mold; (3) mixing the biocompatible material and the curing agent to obtain a biocompatible material solution; (4) placing the hard-baked mold in a container, then pouring the biocompatible material solution into it, and heating and curing it to obtain an organoid chip.

[0011] Preferably, in step (1), the parameters of the pre-baking treatment are: treating at 95°C for 18 minutes; And / or, in step (1), the parameters of the UV exposure are: at 15.2 mW / cm 2 Processing conditions for 8 seconds.

[0012] Preferably, in step (2), the post-baking treatment parameters are: treating at 65°C and 95°C for 1 minute and 5 minutes respectively; And / or, in step (2), the parameters of the hardening treatment are: treating at 150° C. for 30 minutes.

[0013] Preferably, in step (3), the biocompatible material is PDMS or PMMA; And / or, in step (3), the curing agent is a Sylgard curing agent; And / or, in step (3), the mass ratio of the biocompatible material to the curing agent is 8-12:1; And / or, in step (4), the temperature of the heating and curing is 85°C; the time of the heating and curing is: 30 minutes for the chip cover, 50 minutes for the upper chip, and 20 minutes for the lower chip.

[0014] The beneficial effects of the present invention are: Compared with the existing multi-organ chip technology, the present invention realizes the construction of multiple barriers in independent chambers, and is used in combination with transwell components to realize personalized combination of multi-organ chips. The present invention is economical and fast, and will play an important role in promoting the fields of intensive medicine, drug evaluation, disease models, especially in vitro evaluation simulation of traditional Chinese medicine, and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is an axial schematic diagram of the multi-organ chip (without the chip cover).

[0017] Figure 2 This is an exploded view of multiple organ chips.

[0018] Figure 3 This is a cross-sectional view of multiple organoid chips.

[0019] Figure 4 This is a partial cross-sectional view of the multi-organ chip.

[0020] Figure 5 A partial cross-sectional view of another multi-organoid chip.

[0021] Figure 6 This is an axial schematic diagram of the multi-organ chip (including the chip cover).

[0022] Figure 7 This is a picture of the quality control results of liver organoids in a multi-organoid chip used in the evaluation of traditional Chinese medicine.

[0023] Figure 8 This is a picture of the quality control results of cardiac organoids in multiple organ chips used in the evaluation of traditional Chinese medicine.

[0024] The accompanying drawings in the figure are marked as follows: 1: upper chip; 11: organoid chamber; 12: porous membrane; 121: first porous membrane; 122: second porous membrane; 13: flow channel inlet; 14: flow channel outlet; 15: barrier chamber; 2: lower chip; 21: flow channel; 3: chip cover; 4: first transwell chamber; 5: second transwell chamber; 6: third transwell chamber. DETAILED DESCRIPTION

[0025] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0026] Example 1 refer to Figure 1~Figure 4 This embodiment provides a multi-organ chip, which includes an upper chip 1 and a lower chip 2 that can be detachably arranged; wherein: The upper chip 1 is embedded with an organoid chamber 11, and a porous membrane 12 is disposed at the bottom of the organoid chamber 11; The lower chip 2 is provided with a flow channel 21 , and the flow channel 21 is connected to the organoid chamber 11 .

[0027] As an optional implementation, the upper chip 1 is further provided with a flow channel inlet 13 and a flow channel outlet 14; wherein: The flow channel inlet 13, the flow channel 21, and the flow channel outlet 14 are connected.

[0028] In actual use, culture fluid needs to be added, and an external device is also required for cooperation. The external device includes an external pipe and an external power device (such as a peristaltic pump). When in use, the external pipe is connected to the flow channel inlet 13 and the flow channel outlet 14 to close the entire system, and then the peristaltic pump is started, and the culture fluid will circulate in the entire system.

[0029] Alternatively, for organoid chambers11, mature commercial transwell components (chambers) can be selected.

[0030] As an optional embodiment, the number of the organoid chambers 11 is 2 to 10.

[0031] When the organoids are cultured and mature outside the chip, they can be placed in the organoid chamber 11 (i.e., the transwell component), thereby achieving any personalized functional combination of multiple organoids, and the optimal number is 2 to 10.

[0032] As an optional embodiment, the porous membrane 12 is arranged in layers; wherein: The number of layers of the porous membrane 12 is 2 to 5, and a barrier chamber 15 is formed between two adjacent layers of the porous membrane 12. The barrier chamber is used to fill a liquid medium or an organoid, and the pore size of the porous membrane is 0.1 to 100 μm.

[0033] In practical applications, multiple layers of porous membranes 12 can be used to construct multiple barriers. Barrier chambers 15 are formed between the multiple barriers, and culture medium, hydrogel and other media can be placed according to needs. The technical best number of porous membranes 12 (i.e., barriers) is 2 to 5 layers.

[0034] Furthermore, such a configuration can realize serial co-culture of each organoid chamber 11 through the flow channel 21, wherein each culture module (including the organoid chamber 11, the porous membrane 12 and the barrier chamber 15) can also maintain an independent culture environment, having multiple functions such as simulating the physiological and pathological functions of organ systems, evaluation of injection and oral drugs, and in vitro efficacy evaluation of Chinese herbal compound.

[0035] As can be seen from the picture, the number of layers of the porous membrane in this embodiment is 2, namely the first porous membrane 121 and the second porous membrane 122; the number of the organoid chambers 11 in this embodiment is 3.

[0036] Example 2 Based on Example 1, as a further extension, refer to Figure 5 The organoid chamber 11 can be set as a nested multi-chamber: for example, the first transwell chamber 4, the second transwell chamber 5, and the third transwell chamber 6 of gradually increasing sizes are nested in sequence to form a multi-barrier system, so that the organoids can be combined according to actual needs.

[0037] Example 3 refer to Figure 6 Based on Example 2, as an optional implementation, the multi-organ chip further includes a chip cover 3, wherein: The chip cover 3 is arranged to cover the upper chip 1 .

[0038] Such an arrangement can make the culture process more sealed and thus not be affected by negative external influences.

[0039] Example 4 This embodiment provides a method for preparing a multi-organ chip, the preparation method comprising the following steps: (1) Spin-coat a layer of SU-8 photoresist on the surface of a 4-inch silicon wafer; (2) Pre-bake the silicon wafer coated with SU-8 photoresist (treatment at 95°C for 18 minutes) and set the appropriate UV exposure time (at 15.2 mW / cm 2 Photolithography was performed under the conditions of 8 seconds; (3) Repeat steps 1 and 2 to obtain SU-8 molds with three different structural heights, which can be used as chip cover, upper chip and lower chip respectively; (4) Post-baking the SU-8 mold (at 65°C for 1 minute and 95°C for 5 minutes respectively), and then placing the post-baked SU-8 mold in a developer for cleaning; (5) Wash out the SU-8 mold of the microstructure and check whether the microstructure size meets the design requirements. The SU-8 mold whose microstructure size meets the requirements is hardened (treated at 150°C for 30 minutes); (6) PDMS and Sylgard curing agent are mixed in a ratio of 10:1 to obtain PDMS liquid; (7) Place the hard-baked SU-8 mold in a clean culture dish, pour the prepared PDMS liquid into the culture dish, and heat it to cure (the curing temperature is 85°C; the curing time is: 30 minutes for the chip cover, 50 minutes for the upper chip, and 20 minutes for the lower chip); (8) Peel off the cured PDMS, cut it into the required size, and use a circular mold knife to cut off the excess part to facilitate the placement of organ tissues; (9) The treated PDMS parts are plasma treated and then aligned and bonded to obtain a complete multi-organ chip.

[0040] Example 5: Cultivation and quality control of intestinal barrier Caco2 cells were seeded in 24-well transwell plates and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The medium was changed every day, and the barrier integrity was tested after 14 days.

[0041] Example 6: Culture and quality control of vascular barrier HUVEC cells were seeded in the porous membrane of the chip. The upper layer was cultured with RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The medium was changed every day. The lower layer used a microfluidic system to control the flow rate of the medium in the flow channel to provide shear stress and improve the barrier function.

[0042] Example 7 Liver organoids are generated from iPSCs. Specifically: On day -1, iPSCs were dissociated into single cells using Accutase and resuspended in mTeSR1 medium containing 10 μM Y-27632 and seeded into ultra-low attachment U-shaped 96-well plates at a density of 10,000 cells per well. Cells were aggregated using a centrifuge at 200 g for 5 minutes at 4°C. On day 0, cells formed spherical structures and RPMI 1640 medium supplemented with 1% KSR, 1% glutamine, 1% B27 supplement, and 1% penicillin-streptomycin was used as the basal medium. On days 0-5, the medium was replaced with basal medium supplemented with 100 ng / mL Activin A. On days 5-10, the medium was replaced with basal medium supplemented with 10 ng / mL FGF2 and 20 ng / mL HGF. On days 10-15, the medium was replaced with HCM medium supplemented with 10 ng / mL oncostatin M and 10 −7 M dexamethasone. From day 15, the culture medium was replaced with HCM medium supplemented with 10 −7 Long-term culture of liver organoids with M-dexamethasone. The culture medium was changed every 2 days until the organoids were used for experiments.

[0043] The results are as follows Figure 7 As shown in the figure, we can see that the IPSc-induced liver organoids were maintained in the organoid chip for 48 hours. The organoids were observed to be intact under the microscope. The fluorescent quantitative PCR method was used to detect that the liver organoid markers were clearly present, indicating that the differentiation was complete and in good condition.

[0044] Example 8 Cardiac organoids are generated from iPSCs. Specifically: On day -1, iPSCs were dissociated into single cells using Accutase and resuspended in mTeSR1 medium containing 10 μM Y-27632, and seeded into ultra-low attachment U-shaped 96-well plates at a density of 5000 cells per well. The cells were centrifuged at 200g for 5 minutes at 4°C to aggregate. On day 0, the cells formed spherical structures and were subsequently induced for 2 days using chemically defined medium (CDM) (10 mL insulin-transferrin-selenium, 1 mL GlutaMAX, 1 mL concentrated lipids, 1 mL penicillin-streptomycin, 4 μL monothioglycerol, 6 mL 150 mg / mL BSA solution, and 90 mL 1:1 mixture of F12 and IMDM per 100 mL) supplemented with FGF2 (50 ng / mL), BMP4 (10 ng / mL), Activin A (50 ng / mL), LY294002 (5 μM), CHIR99021 (8 μM), and insulin (1 μg / m). On day 2, the culture medium was changed to CDM medium supplemented with FGF2 (10 ng / mL), BMP4 (15 ng / mL), IWP2 (5 μM), VEGF-A (200 ng / mL), RA (0.5 μM), and insulin (10 μg / mL) for 4 days. On day 6, the culture medium was changed to CDM medium supplemented with FGF2 (10 ng / mL), BMP4 (20 ng / mL), VEGF-A (100 ng / mL), and insulin (10 μg / mL) for 2 days. After day 8, the culture was maintained with CDM medium containing VEGF-A (100 ng / mL). The culture medium was changed every 2 days until the organoids were used for experiments.

[0045] The results are as follows Figure 8 As shown in the figure, the heart organoids induced by IPSc were maintained in the organoid chip for 48 hours. The organoids were observed to be intact and had regular heartbeats under the microscope. The fluorescent quantitative PCR method was used to detect that the heart organoid markers were clearly present, indicating that the differentiation was complete and in good condition.

[0046] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A multi-organ chip, characterized in that: It includes an upper chip and a lower chip that can be detachably arranged; wherein: The upper chip is embedded with an organoid chamber, and a porous membrane is disposed at the bottom of the organoid chamber; The lower chip is provided with a flow channel, and the flow channel is connected to the organoid chamber; The number of the organoid chambers is 2 to 10; The porous membrane is arranged in layers; wherein: The number of layers of the porous membrane is 2 to 5, and a barrier chamber is formed between two adjacent layers of the porous membrane, and the barrier chamber is used to fill a liquid medium or an organoid.

2. The multi-organ chip according to claim 1, characterized in that: The upper chip is also provided with a flow channel inlet and a flow channel outlet; wherein: The flow channel inlet, the flow channel, and the flow channel outlet are connected.

3. The multi-organ chip according to claim 1, characterized in that: The pore size of the porous membrane is 0.1-100 μm.

4. The multi-organ chip according to claim 1, characterized in that: Also included is a chip cover, wherein: The chip cover is arranged to cover the upper chip.

5. The method for preparing the multi-organ chip according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) Spin-coating photoresist on the surface of the silicon wafer, then performing pre-baking, and setting the ultraviolet exposure time for photolithography to obtain a pre-mold; (2) post-baking the pre-mold, then washing it in a developer, and finally performing a hard film treatment to obtain a hard-baked mold; (3) mixing the biocompatible material and the curing agent to obtain a biocompatible material solution; (4) placing the hard-baked mold in a container, then pouring the biocompatible material solution into it, and heating and curing it to obtain an organoid chip.

6. The method for preparing a multi-organ chip according to claim 5, characterized in that: In step (1), the parameters of the pre-baking treatment are: treatment at 95°C for 18 minutes; And / or, in step (1), the parameters of the UV exposure are: at 15.2 mW / cm 2 Processing conditions for 8 seconds.

7. The method for preparing a multi-organ chip according to claim 5, characterized in that: In step (2), the post-baking treatment parameters are: 1 minute at 65°C and 5 minutes at 95°C; And / or, in step (2), the parameters of the hardening treatment are: treating at 150° C. for 30 minutes.

8. The method for preparing a multi-organ chip according to claim 5, characterized in that: In step (3), the biocompatible material is PDMS or PMMA; And / or, in step (3), the curing agent is a Sylgard curing agent; And / or, in step (3), the mass ratio of the biocompatible material to the curing agent is 8-12:1; And / or, in step (4), the temperature of the heating and curing is 85°C; the time of the heating and curing is: 30 minutes for the chip cover, 50 minutes for the upper chip, and 20 minutes for the lower chip.

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