An organoid chip for time series analysis and preparation method thereof

Abstract

The present invention belongs to the technical field of organoid chips in biomedicine, and specifically relates to an organoid chip for time series analysis and a preparation method thereof. The organoid chip includes a detachable upper chip and a lower chip; the upper chip is provided with a flow channel inlet, a flow channel and a flow channel outlet; the lower chip is provided with an organoid culture hole; the flow channel inlet, the organoid culture hole, the flow channel and the flow channel outlet are connected. Compared with the existing organoid chip technology, the present invention realizes the construction of multi-channel independent chambers, and can realize the same environment culture of organoids in each chamber. Extracting the organoid of a chamber alone will not interfere with the generation of other chambers, and allows the acquisition and evaluation of organoids under different time processing. The above method is economical and fast, and will have an important role in promoting precision medicine, drug evaluation, disease models, especially the in vitro evaluation simulation of traditional Chinese medicine, and is suitable for promotion and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical organoid chips, and specifically relates to an organoid chip for time series analysis and a preparation method thereof. Background Art

[0002] Organoids and organoid chips are new in vitro bionic models that have emerged in recent years. Organoids have a variety of complex cells and tissues. In 2009, Hans Clevers' team used a single mouse intestinal stem cell to induce in vitro intestinal organoids with intestinal crypt-villus structures, and this type of organ entered the stage of biological models. In 2010, the first lung organ chip was built. Subsequently, researchers combined organoids with organ chip technology to provide a maintenance environment for organoid culture, which is a new technology to mimic the physiological, pathological and pharmacological effects in vivo. Physiological activities, disease processes and drug effects are a dynamic process. The analysis of a certain time section can no longer meet the needs of scientific research. It is necessary to comprehensively analyze the physiological changes, disease progression and drug intervention processes to discover new therapeutic targets and the best drug intervention links.

[0003] At present, in vitro time series analysis generally uses static and independently cultured organoids. Because the environment is not uniform, even if theoretically completely consistent conditions are used to take samples at different time points, there will be deviations between samples. At the same time, in vivo time series analysis generally uses animal models, and it is difficult to obtain samples at continuous time nodes from human specimens.

[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 provides an organoid chip for time series analysis and a preparation method thereof. The present invention utilizes multi-channel mixing microfluidics and detachable parallel chambers in series to achieve a consistent culture environment and sample acquisition at different time points.

[0006] The solution of the present invention is to provide an organoid chip for time series analysis, which comprises an upper chip and a lower chip that can be detachably arranged; wherein:

[0007] The upper chip is provided with a flow channel inlet, a flow channel and a flow channel outlet;

[0008] The lower chip is provided with organoid culture holes;

[0009] The flow channel inlet, the organoid culture hole, the flow channel and the flow channel outlet are connected.

[0010] Preferably, the number of the organoid culture wells is 2 to 20.

[0011] Preferably, the upper chip is further provided with an organoid culture cover, and the organoid culture cover and the organoid culture hole can be covered.

[0012] Preferably, the thickness of the upper chip is 1-15 mm, and the thickness of the lower chip is 2-20 mm.

[0013] Preferably, the width of the flow channel is 0.01-10 mm, and the depth of the flow channel is 0.01-10 mm.

[0014] Preferably, the flow channel includes a first-stage flow channel, a second-stage flow channel, ..., an N-th stage flow channel arranged in layers and connected in sequence; wherein:

[0015] The first-stage flow channel is connected to the organoid culture well;

[0016] The Nth stage flow channel is connected to the flow channel outlet.

[0017] Preferably, each level of flow channel comprises a plurality of flow channel units; wherein:

[0018] The flow channel unit is bent in an S shape.

[0019] Preferably, the number of the flow channel units decreases from the first-level flow channel to the N-th-level flow channel.

[0020] Preferably, a converging flow channel is horizontally arranged between any two adjacent flow channels.

[0021] Based on the same technical concept, another embodiment of the present invention is to provide a method for preparing an organoid chip for time series analysis, the method comprising the following steps:

[0022] (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;

[0023] (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;

[0024] (3) mixing the biocompatible material and the curing agent to obtain a biocompatible material solution;

[0025] (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.

[0026] To facilitate understanding of the present invention, the preparation method is described in more detail:

[0027] In step (1), the parameters of the pre-baking treatment are: treatment at 95° C. for 18 minutes.

[0028] In step (1), the parameters of the UV exposure are: at 15.2 mW / cm 2 The conditions were processed for 8 seconds.

[0029] In step (2), the parameters of the post-baking treatment are: treating at 65°C and 95°C for 1 minute and 5 minutes respectively.

[0030] In step (2), the parameters of the hardening treatment are: treatment at 150° C. for 30 minutes.

[0031] In step (3), the biocompatible material is PDMS or PMMA.

[0032] In step (3), the curing agent is a Sylgard curing agent.

[0033] In step (3), the mass ratio of the biocompatible material to the curing agent is 8-12:1.

[0034] In step (4), the heating and curing temperature is 85° C.; the heating and curing time is: 50 minutes for the upper chip and 20 minutes for the lower chip.

[0035] The beneficial effects of the present invention are:

[0036] Compared with the existing organoid chip technology, the present invention realizes the construction of multi-channel independent chambers, and can realize the same environment culture of organoids in each chamber. The individual extraction of organoids from a chamber will not interfere with the other chambers, allowing the acquisition and evaluation of organoids under different time treatments. The above method is economical and fast, and will have an important driving effect on the fields of intensive medicine, drug evaluation, disease models, especially the in vitro evaluation simulation of traditional Chinese medicine, and is suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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.

[0038] Figure 1 This is a schematic diagram of the structure of the organoid chip.

[0039] Figure 2 An exploded view of an organoid chip.

[0040] Figure 3 An exploded view of another organoid chip.

[0041] Figure 4 This is a bottom view of the upper chip.

[0042] Figure 5 This is another bottom view of the upper chip.

[0043] Figure 6 This is another bottom view of the upper chip.

[0044] Figure 7 These are cardiac organoids (4×) collected from different culture wells and at different time points.

[0045] Figure 8 These are liver organoids (4×) collected from different culture wells and at different time points.

[0046] The accompanying drawings in the figure are marked as follows:

[0047] 1-upper chip; 11-channel inlet; 12-channel; 121-first-level channel; 122-second-level channel; 123-Nth-level channel; 124-channel unit; 125-collecting channel; 13-channel outlet; 14-organoid culture cover; 2-lower chip; 21-organoid culture hole. DETAILED DESCRIPTION

[0048] 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.

[0049] Example 1

[0050] refer to Figure 1~Figure 4 This embodiment provides an organoid chip for time series analysis, which includes an upper chip 1 and a lower chip 2 that can be detachably arranged; wherein:

[0051] The upper chip 1 is provided with a flow channel inlet 11, a flow channel 12 and a flow channel outlet 13;

[0052] The lower chip 2 is provided with an organoid culture hole 21;

[0053] The flow channel inlet 11, the organoid culture hole 21, the flow channel 12 and the flow channel outlet 13 are connected.

[0054] When the organoid chip is actually used, 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 11 and the flow channel outlet 13 to close the entire system, and then the peristaltic pump is started, and the culture fluid will circulate in the entire system. That is, with such a setting, the culture fluids from different organoid culture holes 21 can be fully collided and mixed multiple times after converging, and then refluxed back to the organoid culture hole 21 to achieve the same environment culture of the organoids in each organoid culture hole 21.

[0055] As an optional embodiment, the number of the organoid culture holes 21 is 2 to 20, more preferably 4 to 10. Among them, the cross-section of the organoid culture hole 21 can be a variety of shapes, such as circular, rectangular, elliptical, etc., and the present embodiment is preferably circular. The height of the organoid culture hole 21 can be selected from 2 to 15 mm, more preferably 3 to 8 mm. Such a setting enables the culture fluid to achieve co-culture between the various organoids to realize the system timing analysis function.

[0056] As an optional embodiment, the upper chip 1 is further provided with an organoid culture cover 14, and the organoid culture cover 14 can be arranged to cover the organoid culture hole 21. Such an arrangement can facilitate the placement and sealing of the organoid.

[0057] As an optional implementation, the thickness of the upper chip 1 is 1-15 mm, more preferably 4-8 mm; the thickness of the lower chip 2 is 2-20 mm, more preferably 3-8 mm.

[0058] As an optional implementation, the width of the flow channel 12 is 0.01-10 mm, more preferably 0.1-1 mm; the depth of the flow channel 12 is 0.01-10 mm, more preferably 0.1-0.2 mm.

[0059] The organoid chip used for time series analysis is made of PDMS, PMMA or other biocompatible materials.

[0060] Example 2

[0061] refer to Figure 5~Figure 6 Based on Example 1, as an optional implementation, the flow channel 12 includes a first-level flow channel 121, a second-level flow channel 122, ..., and an N-th-level flow channel 123 which are arranged in layers and connected in sequence; wherein:

[0062] The first-stage flow channel 121 is connected to the organoid culture hole 21;

[0063] The Nth stage flow channel 123 is connected to the flow channel outlet 13 .

[0064] As an optional implementation, each level of flow channel includes a plurality of flow channel units 124; wherein:

[0065] The flow channel unit 124 is bent in an S shape.

[0066] As an optional implementation, the number of the flow channel units 124 decreases from the first-level flow channel to the N-th-level flow channel.

[0067] As an optional implementation, a converging flow channel 125 is horizontally arranged between any two adjacent flow channels.

[0068] Such a setting can increase the length of the flow channel 12, making it have multiple flow channels and fully mixed, and also achieve the effect of multi-hole series connection. More importantly, it can make the culture fluid converge from the flow channel inlet 11 to the flow channel outlet 13, and then repeat the cycle many times, so that the culture fluid in the organoid chip remains in a completely uniform state.

[0069] In this embodiment, N=5.

[0070] Example 3

[0071] This embodiment provides a method for preparing an organoid chip for time series analysis, the preparation method comprising the following steps:

[0072] (1) Spin-coat a layer of SU-8 photoresist on the surface of a 4-inch silicon wafer;

[0073] (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;

[0074] (3) Repeat steps 1 to 2 to obtain SU-8 molds with two different structural heights, which can be used as the upper chip and the lower chip respectively;

[0075] (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;

[0076] (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);

[0077] (6) PDMS and Sylgard curing agent are mixed in a ratio of 10:1 to obtain PDMS liquid;

[0078] (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: 50 minutes for the upper chip and 20 minutes for the lower chip);

[0079] (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;

[0080] (9) The treated PDMS part and the organoid culture cover are plasma treated. After treatment, the two are aligned and bonded to obtain a complete organ chip.

[0081] Example 4

[0082] As a specific operation, this embodiment provides a method for inducing cardiac organoids:

[0083] On day -2, iPSCs were separated into single cells using Accutase and resuspended in E8 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. The cells were centrifuged at 300g for 5 minutes at 4°C to aggregate. On day -1, the cells formed spherical structures, 50 μL of medium was removed and 200 μL of fresh E8 medium was added to make the final volume per well 250 μL; on day 0, 2 / 3 (166 μL) of the medium was removed, and 166 μL of RPMI 1640 medium supplemented with B-27 minus insulin, 6 μM CHIR99021, 1.875 ng / mL BMP4 and 1.5 ng / mL Activin A was added for induction for 24 h. On day 1, 166 μL of culture medium was removed and 166 μL of RPMI 1640 medium supplemented with B-27 minus insulin was added for 24 h. On day 2, 166 μL of culture medium was removed and 166 μL of RPMI 1640 medium supplemented with B-27 minus insulin and 3 μM Wnt-C59 was added for 48 h. On day 4, 166 μL of culture medium was removed and 166 μL of RPMI 1640 medium supplemented with B-27 minus insulin was added for 48 h. On day 6, 166 μL of culture medium was removed and 166 μL of RPMI 1640 medium supplemented with B-27 was added for 24 h. On day 7, 166 μL of culture medium was removed and 166 μL of RPMI 1640 medium supplemented with B-27 and 3 μM CHIR99021 was added. After 1 h of induction, 166 μL of culture medium was removed and 166 μL of RPMI 1640 medium supplemented with B-27 and 3 μM CHIR99021 was added. μL of RPMI 1640 medium supplemented with B-27 was added for 48 h; after day 9, 166 μL of medium was removed and 166 μL of RPMI 1640 medium supplemented with B-27 was added every 2 days until the organoids were ready for analysis or processing.

[0084] Example 5

[0085] Based on Example 4, a temporal analysis of doxorubicin exposure in cardiac organoids is provided, specifically:

[0086] Mature cardiac organoids were aspirated from the ultra-low attachment U-shaped 96-well plate and inoculated into the organoid chip. The cardiac organoid maintenance medium containing 10 μM doxorubicin was passed through the flow channel. The morphology of the cardiac organoids was photographed at 6 time points and the cardiac organoids were obtained from the chamber for analysis. The results are shown in Figure 2. Figure 7 shown.

[0087] Depend on Figure 7 It can be seen that culturing organoids on a chip can maintain the normal morphology of cells and keep the cells active. By mixing the culture medium and providing a stable flow rate, the organoids in each flow channel can obtain uniform culture conditions for subsequent time-series analysis.

[0088] In fact, the cardiac organoid is just one example, and a variety of organoids can be analyzed on a chip in a temporal manner:

[0089] Furthermore, the organoids include iPSC-derived organoids, animal-derived organoids, or clinical patient-derived organoids;

[0090] Furthermore, in addition to organoids, the chip of the present invention can also be used for time-series analysis of 2D cells and 3D cell spheroids;

[0091] Furthermore, the temporal analysis performed in the organoid chip may include analysis of various substances that affect the organoid, including drugs, environmental pollutants, nanomaterials, etc.

[0092] Example 6

[0093] According to the above method, the liver organoids were analyzed, and the results were as follows Figure 8 As shown. Figure 8 It can be seen that:

[0094] Culturing organoids on a chip can maintain the normal morphology of cells and keep the cells active. By mixing the culture medium and providing a stable flow rate, the organoids in each flow channel can obtain uniform culture conditions for subsequent time-series analysis.

[0095] Methods for inducing liver organoids:

[0096] On day -1, iPSCs were dissociated into single cells using Accutase and resuspended in E8 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 by centrifugation at 300 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 −7Long-term culture of hepatic organoids with M-dexamethasone. Culture medium was changed every 2 days until the organoids were ready for analysis or processing.

[0097] 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. An organoid chip for time series analysis, characterized in that: It includes an upper chip and a lower chip that can be detachably arranged; wherein: The upper chip is provided with a flow channel inlet, a flow channel and a flow channel outlet; The lower chip is provided with organoid culture holes; The flow channel inlet, the organoid culture hole, the flow channel and the flow channel outlet are connected; The upper chip is also provided with an organoid culture cover, and the organoid culture cover and the organoid culture hole can be covered and arranged; The flow channel includes a first-level flow channel, a second-level flow channel, ..., and an N-th-level flow channel arranged in layers and connected in sequence; wherein: The first-stage flow channel is connected to the organoid culture well; The Nth level flow channel is connected to the flow channel outlet; Each level of flow channel includes several flow channel units, among which: The flow channel unit is bent in an S shape; The number of the flow channel units decreases from the first-level flow channel to the N-th-level flow channel; A converging flow channel is horizontally arranged between any two adjacent flow channels.

2. The organoid chip for time series analysis according to claim 1, characterized in that: The number of the organoid culture wells is 2 to 20.

3. The organoid chip for time series analysis according to claim 1, characterized in that: The thickness of the upper chip is 1-15 mm, and the thickness of the lower chip is 2-20 mm.

4. The organoid chip for time series analysis according to claim 1, characterized in that: The width of the flow channel is 0.01-10 mm, and the depth of the flow channel is 0.01-10 mm.

5. The method for preparing an organoid chip for time series analysis 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 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.

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