An organoid molding culture device

Abstract

The application discloses an organoid forming culture device. The device comprises a hydrogel container and a nutrient maintenance system. The hydrogel container is provided with an inlet and an outlet. The hydrogel container contains a polymer hydrogel capable of carrying living cells. The polymer hydrogel is provided with perfusion channels, forming a horizontal microchannel structure. The polymer hydrogel is also provided with vertical culture holes. The vertical culture holes are located around the perfusion channels. The inlet and outlet of the perfusion channels are respectively connected with the inlet and outlet of the hydrogel container. The nutrient maintenance system is connected with the inlet of the hydrogel container. The nutrient solution is transported to the hydrogel container through the inlet of the hydrogel container. The organoid forming culture device can assist the formation of organoids in vitro and can long-term culture organoids in vitro.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of organoids and cell culture, and particularly relates to an organoid forming culture device. BACKGROUND

[0002] Since the 21st century, organoid technology has been developed and rapidly developed due to the development of three-dimensional cell culture technology. In short, an organoid is a three-dimensional assembly of cells with some characteristics of a part of an organ in a living body. At present, various organoids such as hair follicle, intestine, liver, kidney, brain and lung have been successfully cultivated in vitro. The research progress of various organoids is different, and some organoid research can only reproduce part of the morphological structure and function of the organ in vivo. Compared with them, the development of hair follicle organoids is more mature. The hair follicle organoids constructed by Toyoshima et al. can continue to develop after being transplanted to the body surface of a nude mouse and eventually obtain a mature hair follicle with hair fiber secretion, hair follicle cycle, accessory sebaceous gland and erector muscle.

[0003] However, the process of organoid construction is easily disturbed by various factors, resulting in a great randomness of the final organoids, which limits the hair follicle organoid technology to basic research and drug testing, and makes it difficult to be clinically transformed and applied. In order to reduce randomness, Abaci limits the construction process of hair follicle organoids within a designed physical boundary. However, compared with boundaryless culture, the existence of physical boundary also limits the energy metabolism and material metabolism of cells, making the organoids prone to necrosis.

[0004] It is of great significance to develop a novel organoid forming culture device to provide a new platform for organoid pathology, developmental biology and drug development. SUMMARY

[0005] The technical problem to be solved by the present application is to provide an organoid forming culture device which can assist in the formation of organoids in vitro and can long-term culture organoids in vitro, in view of the deficiencies of the prior art.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is:

[0007] The present application provides an organoid forming culture device, which comprises a hydrogel container and a nutrient maintenance system. The hydrogel container is provided with an inlet and an outlet. The hydrogel container contains a polymer hydrogel capable of carrying living cells. The polymer hydrogel is provided with perfusion channels, forming a horizontal microchannel structure. The polymer hydrogel is also provided with vertical culture holes around the perfusion channels. The inlet and outlet of the perfusion channels are respectively connected with the inlet and outlet of the hydrogel container. The nutrient maintenance system is connected with the inlet of the hydrogel container, and the nutrient solution is transported to the hydrogel container through the inlet of the hydrogel container.

[0008] According to the above scheme, the nutrient maintenance system includes a circulation pump for circulating the nutrient solution containing nutrients, and a nutrient solution storage device for storing the nutrient solution. The nutrient solution storage device has an inlet and an outlet. The inlet of the circulation pump is connected to the outlet of the nutrient solution storage device through the upper section of an inlet circulation pipe, and the outlet of the circulation pump is connected to the inlet of the hydrogel container through the lower section of an inlet circulation pipe. The outlet of the hydrogel container is connected to the inlet of the nutrient solution storage bottle, forming a closed loop that continuously delivers the nutrient solution to the hydrogel body, providing the nutrients required for organoid growth and development. The nutrient solution in the closed loop flows unidirectionally under the drive of the circulation pump.

[0009] According to the above scheme, the nutrient solution storage device is also equipped with a gas exchange pipeline to connect the internal and external gases, balance the gas pressure in the entire system with the atmospheric pressure, and prevent injection failure caused by the imbalance of internal and external pressure.

[0010] According to the above scheme, the gas exchange pipeline connects the internal and external gases through a gas filtration and sterilization device.

[0011] According to the above scheme, the shape of the infusion channel can be designed as a grid-shaped, straight, serpentine, or other cylindrical or rectangular channel, preferably a serpentine cylindrical channel that surrounds the vertical culture well and is not connected to the vertical culture well.

[0012] Furthermore, the aforementioned polymeric hydrogel can be formed by crosslinking one or more polymeric materials or their modified products. These polymeric materials include natural and synthetic polymers, possessing good biocompatibility and biostability. Specifically, these materials include gelatin, hyaluronic acid, collagen, chitosan, silk fibroin, and their modified forms. The aforementioned polymeric materials and their modified products can be crosslinked using methods such as photocrosslinking, chemical crosslinking, and ionic crosslinking to form the desired hydrogel.

[0013] Furthermore, the hydrogel thickness is 4-6 mm.

[0014] Furthermore, the diameter of the injection channel is 400-600 μm.

[0015] Furthermore, the diameter of the culture wells is between 250 and 600 μm.

[0016] According to the above scheme, the height of the vertical culture well is preferably 2-3 mm, and preferably accounts for 60%-80% of the thickness of the hydrogel.

[0017] Furthermore, preferably, the culture well is a frustum-shaped structure with a small upper opening diameter and a large bottom diameter, similar to the shape of a hair follicle, wherein the diameter of the outlet pore is 250-350 μm and the bottom diameter is 400-600 μm.

[0018] In this invention, the injection channels and culture wells can be formed in the hydrogel using molding methods such as the die-casting method and the sacrificial die-casting method. However, it is preferred that the injection channels and culture wells be formed using the sacrificial die-casting method.

[0019] The living cells can be related cells from the hair follicle organs and vascular tissues of any mammal, including but not limited to dermal fibroblasts, keratinocytes, dermal papilla cells, hair follicle stem cells, and vascular endothelial cells. These cells can be distributed arbitrarily within the hydrogel, on the surface of the hydrogel, or in the porous structures of the hydrogel.

[0020] During operation, the nutrient solution flows from the perfusion channels through the organoid culture device, contacting the hydrogel on the pore walls. In this process, nutrients gradually permeate into the hydrogel and eventually reach the vicinity of the living cells contained within it. Waste products from the growth and metabolism of the living cells also permeate into the liquid through the hydrogel. The nutrient solution can be DMEM or F12 culture medium containing a certain proportion of fetal bovine serum, or a synthetically produced liquid containing clearly defined nutrients.

[0021] The hydrogel holding device is a container with a volume slightly larger than the hydrogel it holds, and nutrient liquid inlets and outlets are set according to the position of the infusion orifice on the surface of the polymer hydrogel held in it.

[0022] The beneficial effects of this invention are:

[0023] This invention combines tissue engineering and organoid culture technology to design an organoid culture and shaping system with vertical culture wells and a horizontal microchannel network. The vertical culture wells provide a culture environment for the organoids and allow for more controllable cell assembly by adding cells sequentially. The horizontal microchannel network is connected to an external nutrient maintenance system, which can deliver the nutrient solution required for cell growth to the hydrogel container for organoid shaping and culture. Furthermore, through the design of an external nutrient solution circulation system, culture medium can be continuously supplied to the hydrogel culture device to maintain the long-term energy and material metabolism of hair follicle cells, enabling long-term in vitro culture of organoids.

[0024] The device designed in this invention is suitable for organoid formation and culture, providing a new platform for research on the pathology and developmental biology of organoids such as hair follicles, as well as drug development. This invention can assist in the in vitro formation of hair follicle organoids and enable long-term in vitro culture of hair follicle organoids, providing a research and application platform for model building, drug screening, and drug toxicity testing in hair follicle-related fields. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the organoid formation and culture device of the present invention;

[0026] Figure 2 This is a schematic diagram of the structure of the hydrogel used in this invention;

[0027] In the diagram: 1. Gas exchange pipe; 2. Hydrogel container; 3. Upper section of liquid inlet circulation pipe; 4. Liquid outlet circulation pipe; 5. Storage bottle; 6. Circulation pump; 7. Lower section of liquid inlet circulation pipe; 8. Liquid inlet; 9. Hydrogel; 10. Infusion channel; 11. Infusion channel inlet; 12. Infusion channel outlet; 13. Vertical culture well; 14.

[0028] The invention will now be described with reference to the accompanying drawings, by way of exemplary and non-limiting embodiments. Detailed Implementation

[0029] like Figure 1 As shown in the figure, an organoid culture apparatus according to an embodiment of the present invention includes a hydrogel container and a nutrient maintenance system. The hydrogel container is provided with an inlet 8 and an outlet 9. The hydrogel container contains a polymeric hydrogel capable of carrying living cells. The polymeric hydrogel is provided with perfusion channels 11, forming a horizontal microchannel structure. The polymeric hydrogel is also provided with vertical culture wells 14, which are located around the perfusion channels. The inlet 12 and outlet 13 of the perfusion channels are respectively connected to the inlet and outlet of the hydrogel container. The nutrient maintenance system... The system includes a circulation pump 6 for circulating a nutrient solution containing nutrients, and a nutrient solution storage bottle 5 for storing the nutrient solution. The nutrient solution storage bottle has an inlet and an outlet. The inlet of the circulation pump is connected to the outlet of the nutrient solution storage bottle via an upper section 3 of an inlet circulation pipe, and the outlet of the circulation pump is connected to the inlet of the hydrogel container via a lower section 7 of an inlet circulation pipe. The outlet 9 of the hydrogel container is connected to the inlet of the nutrient solution storage bottle, forming a closed-loop circuit that continuously delivers the nutrient solution to the hydrogel body, providing various nutrients required for organoid growth and development. The nutrient solution in the closed-loop circuit flows unidirectionally under the drive of the circulation pump. The nutrient solution storage bottle is also equipped with a gas exchange pipe 1, which connects internal and external gases through a gas filtration and sterilization device to balance the gas pressure within the entire system with atmospheric pressure, preventing infusion failure caused by pressure imbalance.

[0030] The circulation pump 6 is responsible for providing power for the flow of nutrient solution in the entire pipeline. The nutrient solution flows from the storage bottle 5 through the upper section 3 of the inlet circulation pipeline to the lower section 7 of the inlet circulation pipeline, and finally enters the hydrogel 10 in the hydrogel holding device 2 through the inlet 8. It is connected to the internal pipeline of the hydrogel and continuously supplies nutrient solution to the hydrogel.

[0031] like Figure 2As shown, the hydrogel 10 has a multiple channel structure, including a serpentine infusion channel 11 and four rows of culture wells 14 arranged around the infusion channel, with five culture wells in each row.

[0032] This invention combines tissue engineering and organoid culture technology to design an organoid culture and shaping system with vertical culture wells and a horizontal microchannel network. The vertical culture wells provide a culture environment for the organoids and allow for more controlled cell assembly by adding cells sequentially. The horizontal microchannel network is connected to an external nutrient maintenance system that delivers the nutrient solution required for cell growth to the hydrogel container for organoid shaping and culture. Furthermore, through the design of an external nutrient solution circulation system, culture medium can be continuously supplied to the hydrogel culture device to maintain the long-term energy and material metabolism of hair follicle cells, enabling long-term in vitro culture of organoids.

[0033] In a specific example of hair follicle organoid culture, the hydrogel uses methacrylated gelatin (GelMA) as the base material, and the colloidal portion of the hydrogel is loaded with 100,000 to 200,000 living dermal fibroblasts per milliliter.

[0034] Add a drop of culture medium containing 3,000-8,000 dermal papilla cells to the opening of each culture well. Let it stand for a period of time to allow the cells to naturally settle to the bottom of the culture well. Use cell-free culture medium to rinse the dermal papilla cells that have not entered the well.

[0035] A culture medium containing one to two million keratinocytes was dropped onto the surface of the hydrogel, and the mixture was left to stand until the cells naturally deposited onto the surface of the hydrogel and into the culture wells.

[0036] After completing the above steps, transfer the hydrogel to a hydrogel container and connect it to a nutrient maintenance system. Then, transfer the entire apparatus to a cell culture incubator at 37°C and 5% CO2 concentration for continuous perfusion culture for 1-3 weeks. On the first day of perfusion, maintain the culture medium flow rate at 100 μL / min, subsequently increasing the flow rate to 200 μL / min. Change the culture medium in the storage bottle weekly. Use the above method to culture hair follicle organoids.

[0037] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention, as long as they do not depart from the spirit and scope of the technical solutions of the present invention, should be covered within the scope of the claims of the present invention.

Claims

1. An organoid culture apparatus, characterized in that: The device includes a hydrogel container and a nutrient maintenance system. The hydrogel container has an inlet and an outlet. The hydrogel container contains a polymeric hydrogel capable of carrying live cells. The polymeric hydrogel has perfusion channels, forming a horizontal microchannel structure. The polymeric hydrogel also has vertical culture wells located around the perfusion channels. The inlet and outlet of the perfusion channels are connected to the inlet and outlet of the hydrogel container, respectively. The nutrient maintenance system is connected to the inlet of the hydrogel container and delivers nutrient solution to the hydrogel container through the inlet.

2. The organoid formation and culture device according to claim 1, characterized in that: The nutrient maintenance system includes a circulation pump for circulating a nutrient solution containing nutrients, and a nutrient solution storage device for storing the nutrient solution. The nutrient solution storage device has an inlet and an outlet. The inlet of the circulation pump is connected to the outlet of the nutrient solution storage device through the upper section of the inlet circulation pipe, and the outlet of the circulation pump is connected to the inlet of the hydrogel container through the lower section of the inlet circulation pipe. The outlet of the hydrogel container is connected to the inlet of the nutrient solution storage device, forming a closed loop that continuously delivers the nutrient solution to the hydrogel body, providing the nutrients required for the growth and development of organoids.

3. The organoid formation and culture device according to claim 2, characterized in that: The nutrient solution storage device is also equipped with a gas exchange pipeline that connects to internal and external gases.

4. The organoid formation and culture apparatus according to claim 3, characterized in that: The gas exchange pipeline connects internal and external gases through a gas filtration and sterilization device.

5. The organoid formation and culture apparatus according to claim 1, characterized in that: The infusion channels can be in the shape of a grid, a straight line, or a serpentine shape, and are evenly arranged around the vertical culture wells without being connected to them.

6. The organoid formation and culture apparatus according to claim 1, characterized in that: The injection channel is a serpentine cylindrical channel.

7. The organoid formation and culture apparatus according to claim 1, characterized in that: The diameter of the injection channel is 400-600 μm.

8. The organoid formation and culture apparatus according to claim 1, characterized in that: The diameter of the vertical culture wells is 250-600 μm.

9. The organoid formation and culture apparatus according to claim 1, characterized in that: The culture wells are shaped like a frustum with a small opening diameter and a large bottom diameter, similar to the shape of a hair follicle. The diameter of the outlet pore is 250-350 μm, and the bottom diameter is 400-600 μm.

10. The organoid formation and culture apparatus according to claim 1, characterized in that: The height of the vertical culture wells is 2-3 mm, accounting for 60%-80% of the hydrogel thickness.

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