Multi-channel device for organoid co-culture and drug sensitivity detection and application method of multi-channel device

Abstract

The multi-channel device comprises an outer placement assembly, a plurality of culture storage assemblies and a rotary valve, a culture channel is formed in the middle of the culture assembly, and the culture assembly is provided with a side wall inner channel communicated with the culture channel. A culture medium inlet and a culture medium outlet are correspondingly formed in the side wall inner channels on the two sides respectively; an annular exchange membrane and a culture main body are arranged in the culture channel, and the inner side and the outer side of the annular exchange membrane are attached to the inner wall of the culture channel and the outer wall of the culture main body respectively; two ends of the culture main body are respectively provided with an outer extending end and an inner extending end, the outer extending end is provided with a culture channel inlet, and the inner extending end is provided with a culture channel outlet; a middle mixing cavity and a plurality of side connecting holes connected with the middle mixing cavity are formed in the middle of the rotary valve; the rotary valve is rotated to enable the side connecting hole to be communicated with or disconnected from the culture channel of the culture assembly, so that culture liquid in the middle mixing cavity interacts with tissues in the culture channel.

Description

Technical Field

[0001] The present invention relates to organoid co-culture and drug sensitivity detection technology, and in particular to a multi-channel device for organoid co-culture and drug sensitivity detection and an application method thereof. Background Art

[0002] At present, organoids have been widely used as important preclinical models for studying organ transplantation, tumor mechanisms, drug development, and precision medicine. The U.S. FDA explicitly requires that drug development must be verified by organoid models before entering clinical trials. There are currently several major "bottleneck" problems in organoid research: First, it is difficult for organoids to simulate microvessels similar to those in the body, making it difficult for organoids to grow continuously compared to tumors and other tissues in the body; second, the construction activity rate of certain specific organoids, such as liver cancer organoids and immune organoids, is not high; third, there is a systematization problem, that is, organoids are often cultured in a single or single species, which cannot reflect the actual situation of the interdependence of multiple organs in the body.

[0003] The patent document with application number "CN202311656684.6" discloses the field of culture or maintenance of undifferentiated human, animal or plant cells, such as cell lines, and discloses a method for co-culture of different types of organoids, which uses an organoid co-culture device, including the steps of placing different organoids or cells in two second culture plates for culture, respectively, and the two second culture plates are connected by splicing male parts and splicing female parts, and the micro-environment is connected by the microporous arrays on the splicing male parts and the splicing female parts, which provides the possibility for simulating organoid co-culture. The organoid co-culture method of the present invention has a modular design for the co-culture device, which can realize the assembly of different components and the co-culture of different organoids. In this way, a disease model of multiple organ interactions can be established, providing a model basis for subsequent research.

[0004] The patent document with application number "CN202111058849.0" discloses a three-dimensional organoid co-culture device, culture system and culture method, which relates to the field of cell technology. The present invention utilizes a constricting ring to improve the fixation method of organoid hydrogel filaments, making the inoculation of organoid hydrogel filaments more convenient. The provided co-culture device can meet the experiments of different cell combinations and has strong scalability. The co-culture device can realize the prediction of drug-induced autoimmune diseases, help reduce research and development costs, and also help to improve the success rate of new drug research and development. In addition, the co-culture device can simulate the normal substance exchange and interaction between cells, tissues, organs, and environment in vivo in vitro.

[0005] The above patent document, combined with the prior art, reveals that the existing multi-channel device for organoid co-culture and drug sensitivity detection has the following defects:

[0006] 1. Due to limited space and material exchange in commercial chips, the culture cycle of organoid spheres is relatively short, or the activity rate of organoids is low.

[0007] 2. Commercialized microfluidic chips are often single-channel. To achieve high-throughput drug testing, multiple chips are often required to be used in combination, which is costly and imposes a heavy burden on medical insurance. It is not conducive to the clinical application and promotion of organoids for precise drug screening after surgery for cancer patients.

[0008] 3. The future application trend of organoids is systematic culture. As far as the field of oncology is concerned, the occurrence, development, and metastasis of human tumors are not isolated, but are under the action of multiple cells and systems, such as liver metastasis of colorectal cancer, brain metastasis of lung cancer, and co-culture of tumors and engineered immune cells. These in vitro simulation processes often require the co-culture of tumor organoids with organoids or cell tissues of other systems. At present, the co-culture in vitro models commonly used in basic research often use traditional Transwells chambers. However, the disadvantages of this system are also obvious, and it often only allows two different cells to be co-cultured.

[0009] In summary, existing technologies have problems such as difficulty in simulating vascularization, inability to simulate the internal environment, often co-culture and co-delivery with other tissue cells, less physical isolation and exchange, poor activity, narrow space, and not conducive to long-term culture. Summary of the invention

[0010] In order to overcome the deficiencies of the prior art, the present invention provides a multi-channel device for organoid co-culture and drug sensitivity detection, which solves the problem of multi-channel devices for organoid co-culture and drug sensitivity detection.

[0011] The first aspect of the present invention is to provide a multi-channel device for organoid co-culture and drug sensitivity detection, comprising an external placement component, a plurality of culture storage components, and a rotary valve, wherein the plurality of culture storage components are arranged around the outside of the rotary valve, the external placement component is provided with a placement inner cavity, and the culture storage component and the rotary valve are both located in the placement inner cavity; the culture component is provided with a culture channel along the middle part, and the culture component is provided with side wall inner channels connected to the culture channel along both sides of the culture channel, and the side wall inner channels on both sides are respectively provided with a culture medium inlet and a culture medium outlet; the culture channel inner cavity An annular exchange membrane and a culture body are provided, and the inner and outer sides of the annular exchange membrane are respectively in contact with the inner wall of the culture channel and the outer wall of the culture body; the two ends of the culture body are respectively provided with an outer protruding end and an inner protruding end, the outer protruding end is provided with a culture channel inlet, and the inner protruding end is provided with a culture channel outlet; the rotary valve is provided with a middle mixing chamber and a plurality of side connecting holes connected to the middle mixing chamber along the middle part; the rotary valve is rotated to connect or disconnect the side connecting holes with the culture channel of the culture component, thereby allowing the culture liquid in the middle mixing chamber to interact with the tissue of the culture channel.

[0012] In the first aspect of the present invention, as a preferred embodiment, the culture component is provided with a through hole along the lower portion of the culture channel, and the through hole is communicated with the culture channel.

[0013] In the first aspect of the present invention, as a preferred embodiment, the culture component is cylindrical or rectangular.

[0014] In the first aspect of the present invention, as a preferred embodiment, the outer protruding end has the same structure as the inner protruding end, and the outer protruding end is cylindrical.

[0015] In the first aspect of the present invention, as a preferred embodiment, the end of the outer protruding end is provided with an arc-shaped end surface.

[0016] In the first aspect of the present invention, as a preferred embodiment, the rotary valve and the middle mixing chamber are both cylindrical.

[0017] In the first aspect of the present invention, as a preferred embodiment, the number of the culture components is the same as the number of the side connecting holes, and the number of the culture components is 4-32.

[0018] In the first aspect of the present invention, as a preferred embodiment, a connection end is provided at the lower portion of the culture component, and the connection end is provided with a connection channel for connecting with a side connection hole.

[0019] In the first aspect of the present invention, as a preferred embodiment, the number of the culture components is 8 / 16 / 24 / 32.

[0020] A second aspect of the present invention provides an application method of a multi-channel device for organoid co-culture and drug sensitivity detection, comprising the following steps:

[0021] Step S10: placing the culture body into the culture channel of each culture component;

[0022] Step S20: placing the drug to be tested into the middle mixing chamber;

[0023] Step S30: Through the chip system, the side connection holes of the rotary valve are controlled to be connected or disconnected with each culture component, so that the drug to be tested comes into contact with the organoid.

[0024] The external placement component is provided with a placement inner cavity, and the culture storage component and the rotary valve are both located in the placement inner cavity; the culture component is provided with a culture channel along the middle part, and the culture component is provided with side wall inner channels connected to the culture channel along both sides of the culture channel, and the side wall inner channels on both sides are respectively provided with a culture medium inlet and a culture medium outlet; an annular exchange membrane and a culture body are provided in the culture channel, and the inner and outer sides of the annular exchange membrane are respectively fitted with the inner wall of the culture channel and the outer wall of the culture body; the two ends of the culture body are respectively provided with an outer protruding end and an inner protruding end, the outer protruding end is provided with a culture channel inlet, and the inner protruding end is provided with a culture channel outlet; the rotary valve is provided with a middle mixing chamber and a plurality of side connecting holes connected to the middle mixing chamber along the middle part; the rotary valve is rotated to connect or interrupt the side connecting holes with the culture channel of the culture component, thereby allowing the culture liquid in the middle mixing chamber to interact with the tissue of the culture channel. By using the device of the present application, various interactions can be achieved by controlling the switch of the rotary valve. When it is in the closed state, the sensitivity of multiple drugs to organoids from the same patient can be tested, and the different killing intensities of a certain drug at different concentrations on organoids from the same patient can be explored. When it is in the open state, a multi-system co-culture system of organoids can be carried out.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] By using the device of the present application, various interactions can be achieved by controlling the switch of the rotary valve 300. When it is in the closed state, the sensitivity test of multiple drugs on organoids from the same patient can be carried out, and the different killing strengths of a certain drug at different concentrations on organoids from the same patient can also be explored. When it is in the open state, a multi-system co-culture system of organoids can be carried out. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A perspective view of the present invention;

[0028] Figure 2A top view of the present invention;

[0029] Figure 3 It is a partial cross-sectional view of the present invention;

[0030] Figure 4 is a cross-sectional view of the present invention;

[0031] Figure 5 is another cross-sectional view of the present invention;

[0032] Figure 6 It is another cross-sectional view of the present invention;

[0033] Figure 7 It is a sentence stereogram of the present invention;

[0034] Figure 8 is a flow chart of the present invention;

[0035] Fig. 9 This is a modular mosaic application diagram.

[0036] In the figure: 200, culture storage component; 10, external placement component; 11, placement inner cavity; 20, culture component; 201, culture channel; 21, side wall inner channel; 211, culture medium inlet; 212, culture medium outlet; 22, connecting end; 221, connecting channel; 30, annular exchange membrane; 40, culture body; 41, external extension end; 42, internal extension end; 300, rotary valve; 301, middle mixing chamber; 302, side connecting hole. DETAILED DESCRIPTION

[0037] Below, in conjunction with the accompanying drawings and specific embodiments, the invention is further described. It should be noted that, under the premise of no conflict, the various embodiments described below or the various technical features can be arbitrarily combined to form a new embodiment. Except as otherwise specified, the materials and equipment used in this embodiment can be purchased from the market. Examples of the embodiments are shown in the accompanying drawings, in which the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0038] In the description of the present application, it should be understood that the terms "upper", "lower", "front", "back", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific position, be constructed and operated in a specific position, and therefore cannot be understood as a limitation on the present application. In the description of the present application, "plurality" means two or more, unless otherwise precisely and specifically specified.

[0039] In the description of this application, it should be noted that, unless otherwise clearly specified and limited, the terms "connected", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, or it can be connected through an intermediary medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0040] The terms "first", "second", etc. in the specification and claims of this application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0041] like Figure 1-7As shown, the first aspect of the present invention is to provide a multi-channel device for organoid joint culture and drug sensitivity detection, including an external placement component 10, a plurality of culture storage components 200, and a rotary valve 300. The plurality of culture storage components 200 are arranged around the outside of the rotary valve 300, and the external placement component 10 is provided with a placement inner cavity 11, and the culture storage components 200 and the rotary valve 300 are both located in the placement inner cavity 11; the culture component 20 is provided with a culture channel 201 along the middle part, and the culture component 20 is provided with side wall inner channels 21 connected to the culture channel 201 along both sides of the culture channel 201, and the side wall inner channels 21 on both sides are respectively provided with a culture medium inlet 211 and a culture medium outlet 212; the culture channel 20 1 is provided with an annular exchange membrane 30 and a culture body 40, and the inner and outer sides of the annular exchange membrane 30 are respectively attached to the inner wall of the culture channel 201 and the outer wall of the culture body 40; the two ends of the culture body 40 are respectively provided with an outer protruding end 41 and an inner protruding end 42, the outer protruding end 41 is provided with a culture channel inlet, and the inner protruding end 42 is provided with a culture channel outlet; the rotary valve 300 is provided with a middle mixing chamber 301 and a plurality of side connecting holes 302 connected with the middle mixing chamber 301 along the middle part; the rotary valve 300 is rotated to connect or disconnect the side connecting holes 302 with the culture channel 201 of the culture component 20, so that the culture liquid in the middle mixing chamber 301 interacts with the tissue of the culture channel 201. By using the device of the present application, various interactions can be achieved by controlling the switch of the rotary valve 300. When it is in the closed state, the sensitivity test of multiple drugs on organoids from the same patient can be carried out, and the different killing strengths of a certain drug at different concentrations on organoids from the same patient can also be explored. When it is in the open state, a multi-system co-culture system of organoids can be carried out.

[0042] Specifically, by opening and closing the integrated microfluidic rotary valve, the chip is connected to a modular structure and remotely controlled. In addition, the annular exchange membrane 30 uses a photocurable exchange membrane to replace the physical partition of the traditional microfluidic chip, thereby increasing the material exchange area and increasing the activity rate of the organoid.

[0043] Specifically, the construction process of the light-cured exchange membrane is as follows:

[0044] 1-1 First, a heat press is used to integrate the pre-designed and engraved mold.

[0045] 1-2 Mix the light curing agent with phosphate buffer solution (PBS) at a ratio of 4%. Hydroxypropyl methylcellulose acrylate HAMA colloid in this ratio exhibits the best light transmittance and structural stability. Subsequently, the mixed colloid is injected into the prepared mold.

[0046] 1-3 Inject 4% HAMA glue light-curing film into the mold, and use the new name "optical path precision control platform" to accurately design and control the illumination area and shape of the HAMA glue surface. What does "optical path precision control platform" specifically refer to? Please explain and elaborate

[0047] 1-4 Through the preset illumination trajectory, ultraviolet rays are irradiated for 15 minutes to form a fixed form of HAMA glue with high light transmittance and structural stability, thereby preparing a light-cured exchange membrane that meets the design requirements. The membrane is used to isolate the organoid culture channel from the culture medium channel and serve as a medium for material exchange.

[0048] 1-5 Finally, the mold is rinsed to remove the uncured liquid HAMA glue and retain the cured exchange membrane.

[0049] The specific application scenarios of this application are as follows:

[0050] Application scenario 1: When the rotary control valve is closed, multiple drugs are tested for sensitivity to organoids from the same patient:

[0051] In this application scenario, the chip system is used to test the drug sensitivity of multiple drugs to organoids from the same patient. By closing the rotary valve, each drug is introduced into a separate channel and contacted with the organoid. The organoids in each channel are treated with different drugs to evaluate their response to different drugs. This configuration allows researchers to screen multiple drugs simultaneously under the same experimental conditions to quickly determine the most effective treatment plan.

[0052] Application scenario 2: When the rotary valve is closed, the killing intensity of a certain drug on organoids from the same patient at different concentration gradients:

[0053] In this application scenario, the rotary valve is set to the closed state, so that the organoids from the same patient are exposed to different concentration gradients of the drug in multiple independent channels. In this way, the dose-effect relationship of the drug can be accurately evaluated under the same experimental conditions, thereby determining the most effective drug concentration and evaluating the drug's lethality and potential dose-dependent toxicity.

[0054] Application scenario three: When the rotary control valve is open, the multi-system joint culture system of organoids:

[0055] In the future trend of organoid research, systematic culture, especially in the co-culture of organoids of multiple systems, will play an increasingly important role. The occurrence, development and metastasis of tumors do not occur alone, but are closely related to multiple systems and cell types. For example, common liver metastases of colorectal cancer, brain metastases of lung cancer, and the interaction between tumors and immune cells all show the complex relationship between tumors and other organs and systems.

[0056] In the first aspect of the present invention, as a preferred embodiment, the culture component 20 is provided with a through hole along the lower portion of the culture channel 201 , and the through hole is communicated with the culture channel 201 .

[0057] In the first aspect of the present invention, as a preferred embodiment, the culture component 20 is cylindrical or rectangular. The outer extension end 41 has the same structure as the inner extension end 42, and the outer extension end 41 is cylindrical. The end of the outer extension end 41 is provided with an arc-shaped end surface. The outer extension end 41 and the inner extension end 42 are both provided with an inlet and an outlet for the culture channel of the organoid.

[0058] In the first aspect of the present invention, as a preferred embodiment, the rotary valve 300 and the middle mixing chamber 301 are both cylindrical. The number of the culture components 20 is the same as the number of the side connection holes 302, and the number of the culture components 20 is 4-32. Specifically, the lower part of the culture component 20 is provided with a connection end 22, and the connection end 22 is provided with a connection channel 221 for connecting with the side connection hole 302.

[0059] In the first aspect of the present invention, as a preferred embodiment, the number of the culture components 20 is 8 / 16 / 24 / 32.

[0060] Please refer to Figure 8-9 The second aspect of the present invention provides an application method of a multi-channel device for organoid co-culture and drug sensitivity detection, comprising the following steps:

[0061] Step S10: placing the culture body into the culture channel of each culture component;

[0062] Step S20: placing the drug to be tested into the middle mixing chamber;

[0063] Step S30: Through the chip system, control the side connection holes of the rotary valve to connect or disconnect with each culture component, so that the drug to be tested comes into contact with the organoid. The multi-channel integrated photocuring microfluidic rotary control chip of the present invention can realize the co-culture of organoids from multiple different systems on the same platform by opening the rotary control valve. The chip can connect organoids from different sources, such as liver, lung, heart, kidney and other systems, to each other, simulating the interaction of multiple systems in the body. This configuration not only allows researchers to observe the biological behavior of multiple systems on a single experimental platform, but also allows in-depth research on the metastasis mechanism of tumors in different organ systems, as well as the mutual influence between organoids.

[0064] Compared with traditional Transwells chambers, this chip greatly expands the possibility of co-culture and provides a more complex and realistic in vitro model. This multi-system co-culture method has important application prospects for exploring the impact of multi-organ interactions on tumor progression, especially in drug screening and personalized treatment.

[0065] In summary, this application has the following advantages:

[0066] 1. Photocurable exchange membrane, with larger exchange area and better permeability;

[0067] 2. The activity rate of organoid culture is higher and the survival time is prolonged;

[0068] 3. Meet the requirements of multi-channel culture of organoids and high-throughput drug testing and screening;

[0069] 4. Realize a multi-system organoid culture co-culture system;

[0070] 5. Modular design facilitates subsequent chip upgrades and can connect to the perfusion system and remote visualization system, ultimately realizing the "Lab on line" modular mosaic application.

[0071] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A multi-channel device for organoid co-culture and drug sensitivity detection, characterized in that: It comprises an external placement component, a plurality of culture storage components, and a rotary valve, wherein the plurality of culture storage components are arranged around the outer side of the rotary valve, the external placement component is provided with a placement inner cavity, and the culture storage components and the rotary valve are both located in the placement inner cavity; The culture component is provided with a culture channel along the middle part, and the culture component is provided with side wall inner channels connected with the culture channel along both sides of the culture channel, and the side wall inner channels on both sides are respectively provided with a culture medium inlet and a culture medium outlet; The culture channel is provided with an annular exchange membrane and a culture body, and the inner and outer sides of the annular exchange membrane are respectively in contact with the inner wall of the culture channel and the outer wall of the culture body; the two ends of the culture body are respectively provided with an outer extension end and an inner extension end, the outer extension end is provided with a culture channel inlet, and the inner extension end is provided with a culture channel outlet; The rotary valve is provided with a central mixing chamber and a plurality of side connection holes connected with the central mixing chamber along the middle; the rotary valve is rotated to connect or disconnect the side connection holes with the culture channel of the culture component, thereby allowing the culture liquid in the central mixing chamber to interact with the tissue of the culture channel.

2. The multi-channel device for organoid co-culture and drug sensitivity detection according to claim 1, characterized in that: The culture component is provided with a through hole along the lower part of the culture channel, and the through hole is communicated with the culture channel.

3. The multi-channel device for co-culture of organoids and drug sensitivity detection according to claim 2, characterized in that: The culture component is cylindrical or rectangular.

4. The multi-channel device for co-culture of organoids and drug sensitivity detection according to claim 1, characterized in that: The outer protruding end has the same structure as the inner protruding end, and the outer protruding end is cylindrical.

5. The multi-channel device for co-culture of organoids and drug sensitivity detection according to claim 4, characterized in that: The end of the outer protruding end is provided with an arc-shaped end surface.

6. The multi-channel device for co-culture of organoids and drug sensitivity detection according to claim 1, characterized in that: The rotary valve and the middle mixing chamber are both cylindrical.

7. The multi-channel device for co-culture of organoids and drug sensitivity detection according to claim 1, characterized in that: The number of the culture components is the same as the number of the side connection holes, and the number of the culture components is 4-32.

8. The multi-channel device for co-culture of organoids and drug sensitivity detection according to claim 7, characterized in that: A connection end is provided at the lower part of the culture component, and the connection end is provided with a connection channel for connecting with the side connection hole.

9. The multi-channel device for co-culture of organoids and drug sensitivity detection according to claim 7, characterized in that: The number of the culture components is 8 / 16 / 24 / 32.

10. A method for using the multi-channel device for co-culture of organoids and drug sensitivity detection according to any one of claims 1 to 9, characterized in that: The following steps are involved: Step S10: placing the culture body into the culture channel of each culture component; Step S20: placing the drug to be tested into the middle mixing chamber; Step S30: Through the chip system, the side connection holes of the rotary valve are controlled to be connected or disconnected with each culture component, so that the drug to be tested comes into contact with the organoid.

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