A perfusion culture chip and a perfusion system

Abstract

The present invention provides a perfusion culture chip and a perfusion system, comprising an upper sealing plate, a culture filter layer and a lower sealing plate layer, wherein the lower sealing plate is provided with an injection interface and a filtration interface, a filling channel corresponding to the position of the injection interface runs through the culture filter layer and is connected to the upper side wall of a culture chamber in the culture filter layer, and a filtration hole at the bottom of the culture chamber corresponds to the position of the filtration interface. The present invention adopts a method of filling the culture solution at the top and filtering it at the bottom for perfusion culture, and can realize continuous perfusion culture of microorganisms and cells.

Description

Technical Field

[0001] The invention belongs to the field of organ-tube chips, and in particular relates to a perfusion culture chip and a perfusion system. Background Art

[0002] Traditional microbial or cell culture is mainly carried out in centrifuge tubes, culture dishes or culture bottles, etc., and sufficient culture fluid is added at one time to achieve a certain growth cycle of culture. The microorganisms or cells cultured by this method will show different growth stages as the culture fluid is consumed, and long-term culture cannot be achieved, and cannot be used for continuous culture.

[0003] In order to realize the small volume perfusion culture of microorganisms, a miniature culture device has emerged, which can add a part of the culture solution. The Chinese patent with publication number CN210065755U and title “A multi-chambered zoned culture dish for biomedical microbiological experimental observation” has the disadvantage that it cannot realize continuous perfusion culture; the Chinese patent with publication number CN110923137A and title “A microfluidic device and culture method for in vitro 3D cell and tissue culture” adds an inflow and outflow interface to the culture chamber, but there is no filtering device, so it is impossible to culture suspended cells and semi-adherent cells, and it is impossible to realize online observation; the Chinese patent with publication number CN111548939A and title “An organoid perfusion culture chip and its use method” also cannot realize the filling and extraction of culture at any time.

[0004] Currently, there is an urgent need for a microfluidic culture chip that can perform continuous perfusion culture and is easy to observe. Summary of the invention

[0005] In order to overcome the problems of the prior art, the present invention provides a perfusion culture chip that can continuously culture adherent, semi-adherent, suspended cells, etc., which can adapt to the conventional environment on the ground and the microgravity environment in space and can be used in a space environment.

[0006] The present invention provides the following technical solutions:

[0007] A perfusion culture chip comprises, from top to bottom, an upper sealing plate, a culture filter layer and a lower sealing plate layer, wherein the lower sealing plate is provided with an injection interface and a filtration interface, a filling channel corresponding to the injection interface position passes through the culture filter layer and is connected to the upper side wall of a culture chamber in the culture filter layer, and a filtration hole at the bottom of the culture chamber corresponds to the filtration interface position.

[0008] Furthermore, a through hole is provided at the bottom of the culture chamber of the culture filter layer, and a filter chamber is provided below the culture chamber. The filter chamber is provided with a filter membrane, a filter net, and a support layer in sequence from top to bottom, and the filter hole is provided in the support layer.

[0009] Furthermore, the upper sealing plate and the corresponding positions of the culture chamber are made of transparent material.

[0010] Furthermore, the lower sealing plate is a double-layer plate, the upper plate is provided with the injection interface and the filtration interface, the lower plate is provided with channels corresponding to the interfaces, and the channels are connected to the surface of the upper sealing plate.

[0011] Furthermore, the lower sealing plate is a double-layer plate, a sealing gasket is arranged between the two plates, and the upper and lower plates are respectively provided with an injection interface and a filtration interface, and the interface positions correspond.

[0012] Furthermore, an inlet sealing ring is provided at the opening of the filling channel at the bottom of the culture filtration layer, an outlet sealing ring is provided at the outer edge of the support layer, and the upper sealing plate, culture chamber layer, filtration chamber layer and lower sealing plate layer are bonded, glued or hot-pressed.

[0013] Furthermore, the upper sealing plate, the culture chamber layer, the filter chamber layer and the lower sealing plate layer are bolted or fastened together, and a sealing ring is provided on the upper sealing plate, the position of the sealing ring corresponds to the outer edge of the culture chamber and the filling channel.

[0014] Furthermore, the perfusion culture chip is used in a space microgravity environment.

[0015] Furthermore, the upper sealing plate, culture chamber layer, filter chamber layer and lower sealing plate layer are made of one or more of glass, PMMA, ABS, and COC; the filter membrane material is cellulose acetate, cellulose nitrate, mixed cellulose, nylon 66, polyethersulfone or PET, with a pore size of 0.22um-8um; the filter screen material is polyethylene, polypropylene, polyvinyl chloride, nylon, glass fiber or quartz sand, with a pore size of 10-200um.

[0016] Furthermore, the culture filtration layer is provided with a plurality of culture chambers, and each culture chamber is provided with a corresponding filtration chamber capable of independent perfusion.

[0017] A perfusion system including a perfusion culture chip, the perfusion system includes a culture fluid storage bag, a delivery pump, the perfusion culture chip, a waste liquid bag, and a controller, the controller controls the on-off of the delivery pump, the delivery pump pumps the culture medium in the culture fluid storage bag to the perfusion culture chip, and the perfusion effluent flows out through the waste liquid bag.

[0018] Furthermore, it also includes an image acquisition device, which acquires an image of a culture target in the culture chamber above the position of the upper sealing plate corresponding to the culture chamber, and transmits the image to the controller.

[0019] By adopting the above technical solution, the present invention has the following beneficial effects:

[0020] 1. The present invention adopts the method of adding culture solution at the top and filtering it at the bottom for perfusion culture, which can realize continuous perfusion culture of microorganisms and cells.

[0021] 2. The perfusion inlet and the filtration outlet of the culture medium are arranged at the same level of the filtration layer, so as to facilitate the real-time observation of the growth status of the microorganisms and cells in the chamber through the upper sealing plate.

[0022] 3. The culture chip can be made into an independent culture module by combining the lower sealing plate with the sealing gasket and then connected to the culture device for use as needed. The upper and lower sealing plates of the culture chip are fixed with screws or a compression structure, which makes it easy to remove the upper and lower sealing plates. The upper sealing plate is detachable, which allows the addition and removal of cultured microorganisms and cells, and is convenient for subsequent detection of microorganisms and cells; the lower sealing plate is detachable, which allows the replacement of the filter membrane, making it easy to select filter materials with different materials and pores.

[0023] 4. Arraying the culture chambers and designing a multi-chamber culture chip can achieve parallel culture of different scales or compare the differences in the growth status of microorganisms and cells under different culture conditions.

[0024] 5. The perfusion culture chip of the present invention can adapt to the conventional environment on the ground, while increasing the sealing performance and adapting to the microgravity environment in space. It can be used in the space environment and meets the protection requirements of the space environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the structure of each layer of a perfusion culture chip of the present invention;

[0026] Figure 2 It is a structural schematic diagram of a perfusion culture chip of the present invention;

[0027] Figure 3 It is a structural side view of a lower sealing plate of another perfusion culture chip of the present invention;

[0028] Figure 4 It is a schematic diagram of another structure of bolt fastening of a perfusion culture chip according to the present invention.

[0029] Figure 5 Schematic diagram of the structure of the perfusion system of the present invention.

[0030] The figures are marked as follows: 1-upper sealing plate, 2-culture filtration layer, 3-lower sealing plate layer, 4-injection interface, 5-filtration interface, 6-filling channel, 7-filter membrane, 8-filter net, 9-support layer, 10-inlet sealing ring, 11-outlet sealing ring, 12-culture chamber, 13-filtration chamber, 14-perfusion channel, 15-upper plate, 16-lower plate, 17-sealing pad, 18-upper sealing plate sealing ring, 19-bolt. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the structural diagrams and specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] Example 1

[0033] like Figure 1 , 2 As shown, the present invention provides a perfusion culture chip that can culture microorganisms and cells; it includes an upper sealing plate 1, a culture filter layer 2 and a lower sealing plate layer 3 from top to bottom, the lower sealing plate is provided with an injection interface 4 and a filtration interface 5, and a filling channel 6 corresponding to the position of the injection interface passes through the culture filter layer, and is connected to the upper side wall of the culture chamber 12 in the culture filter layer, and the filtration hole at the bottom of the culture chamber corresponds to the position of the filtration interface. In this way, the culture medium flows in from the upper side wall of the culture chamber and flows out from the bottom to complete the perfusion culture. The inflow of culture solution from the top can effectively avoid the risk of microorganisms or cells inside the culture chamber entering the culture solution injection channel, and keep the microorganisms or cells in the culture chamber.

[0034] A filter chamber is provided below the culture chamber, which is connected by a channel in the middle. The filter chamber is provided with a filter membrane 7, a filter screen 8, and a support layer 9 from top to bottom, and a filter hole is provided in the support layer. The filter membrane filter screen is used to intercept suspended cells, semi-adherent cells or microorganisms such as bacteria. The filter screen has a larger pore size than the filter membrane, which plays a diversion role. The upper sealing plate and the corresponding position of the culture chamber are made of transparent material. It is convenient to observe the culture chamber.

[0035] The lower sealing plate is a double-layer plate, the upper plate 15 is provided with an injection interface 4 and a filtration interface 5, and the lower plate 16 is provided with a channel 14 corresponding to the interface, and the channel is connected to the surface of the upper sealing plate. In this way, the culture medium injection pipeline is connected from the upper sealing plate surface, and the culture medium flows from the injection interface into the filling channel through the channel and finally enters the culture chamber, and the filtrate enters the channel from the filtration interface and finally enters the waste liquid bag from the pipeline connected to the waste liquid bag on the upper sealing plate surface.

[0036] An inlet sealing ring 10 is provided at the opening of the filling channel at the bottom of the culture filter layer, an outlet sealing ring 11 is provided at the outer edge of the support layer, and the upper sealing plate, the culture filter layer and the lower sealing plate layer are bonded, glued or hot-pressed. It can be used in a space microgravity environment and meets the protection requirements of the space environment.

[0037] The upper sealing plate, culture filter layer and lower sealing plate layer are made of one or more of glass, PMMA, ABS, COC materials; the filter membrane material is cellulose acetate, cellulose nitrate, mixed cellulose, nylon 66, polyethersulfone or PET, with a pore size of 0.22um-8um; the filter mesh material is polyethylene, polypropylene, polyvinyl chloride, nylon, glass fiber or quartz sand, with a pore size of 10-200um.

[0038] Preferably, the culture filtration layer is provided with a plurality of culture chambers, each of which is provided with a corresponding filtration chamber capable of independent perfusion. Arraying the culture chambers can realize parallel culture of different scales, or compare the growth state differences of microorganisms or cells under different culture conditions.

[0039] Example 2

[0040] like Figure 3 As shown, the present invention provides another perfusion culture chip that can be used as an independent module, the lower sealing plate is a double-layer plate, a sealing gasket 17 is provided between the upper plate 15 and the lower plate 16, and the upper and lower plates are respectively provided with an injection interface and a filtration interface, and the interface positions correspond. When in use, a piercing interface or a syringe can be used to pass through the sealing gasket. In this way, each chip can be an independent unit, and the culture chamber and filter chamber of each chip are independently perfused. By splicing and replacing the chips, parallel culture of different scales can be achieved, or the differences in the growth states of microorganisms and cells under different culture conditions can be compared.

[0041] Preferably, Figure 4 As shown, the upper sealing plate, the culture filter layer and the lower sealing plate layer are fastened together by bolts 19, and a sealing ring 18 is provided on the upper sealing plate, and the position of the sealing ring corresponds to the outer edge of the culture chamber and the filling channel. It is convenient to disassemble and replace. The upper sealing plate is detachable, and operations such as adding and removing cultured microorganisms and cells can be realized, which is convenient for subsequent detection of microorganisms and cells; the lower sealing plate is detachable, and the filter membrane can be replaced, which is convenient for selecting filter materials with different materials and pores.

[0042] Example 3

[0043] like Figure 5 As shown, the present invention provides a perfusion system including the perfusion culture chip of the present invention, including a culture liquid storage bag, a delivery pump, a perfusion culture chip, a waste liquid bag, and a controller. The culture liquid storage bag is connected to the injection interface, and the waste liquid bag is connected to the filtration interface. The controller controls the on-off of the delivery pump, and the delivery pump pumps the culture medium in the culture medium storage bag to the perfusion culture chip, and the perfusion effluent flows out through the waste liquid bag.

[0044] Preferably, it also includes an image acquisition device, which acquires an image of the culture target in the culture chamber above the position of the upper sealing plate corresponding to the culture chamber, and transmits the image to the controller.

[0045] The above-mentioned embodiments only express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A perfusion culture chip, comprising, from top to bottom, an upper sealing plate, a culture filter layer and a lower sealing plate layer, characterized in that: The lower sealing plate is provided with an injection interface and a filtration interface. The filling channel corresponding to the injection interface position passes through the culture filter layer and is connected to the upper side wall of the culture chamber in the culture filter layer. The filtration hole at the bottom of the culture chamber corresponds to the filtration interface position; the bottom of the culture chamber of the culture filter layer is provided with a through hole, and a filtration chamber is provided below the culture chamber. The filtration chamber is provided with a filter membrane, a filter net, and a support layer in sequence from top to bottom, and the filtration hole is opened in the support layer.

2. The perfusion culture chip according to claim 1, characterized in that: The corresponding positions of the upper sealing plate and the culture chamber are made of transparent material.

3. The perfusion culture chip according to claim 1, characterized in that: The lower sealing plate is a double-layer plate, the upper plate is provided with the injection interface and the filtration interface, the lower plate is provided with channels corresponding to the interfaces, and the channels are connected to the surface of the upper sealing plate.

4. The perfusion culture chip according to claim 1, characterized in that: The lower sealing plate is a double-layer plate, a sealing gasket is arranged between the two plates, and the upper and lower plates are respectively provided with an injection interface and a filtration interface, and the interface positions correspond.

5. The perfusion culture chip according to any one of claims 3 or 4, characterized in that: An inlet sealing ring is provided at the opening of the filling channel at the bottom of the culture filter layer, an outlet sealing ring is provided at the outer edge of the support layer, and the upper sealing plate, culture chamber layer, filter chamber layer and lower sealing plate layer are bonded, glued or hot-pressed.

6. The perfusion culture chip according to any one of claims 3 or 4, characterized in that: The upper sealing plate, the culture chamber layer, the filter chamber layer and the lower sealing plate layer are connected by bolts or fastened. A sealing ring is arranged on the upper sealing plate, and the position of the sealing ring corresponds to the outer edge of the culture chamber and the filling channel.

7. The perfusion culture chip according to claim 4, characterized in that: The perfusion culture chip is used in a space microgravity environment.

8. The perfusion culture chip according to claim 1, characterized in that: The upper sealing plate, culture chamber layer, filter chamber layer and lower sealing plate layer are made of one or more of glass, PMMA, ABS, and COC materials; the filter membrane material is cellulose acetate, cellulose nitrate, mixed cellulose, nylon 66, polyethersulfone or PET, with a pore size of 0.22um-8um; the filter screen material is polyethylene, polypropylene, polyvinyl chloride, nylon, glass fiber or quartz sand, with a pore size of 10-200um.

9. The perfusion culture chip according to claim 1, characterized in that: The culture filtration layer is provided with a plurality of culture chambers, and each culture chamber is provided with a corresponding filtration chamber capable of independent perfusion.

10. A perfusion system comprising the perfusion culture chip according to claim 1, wherein the perfusion system comprises a culture fluid storage bag, a delivery pump, the perfusion culture chip, a waste fluid bag, and a controller, characterized in that: The controller controls the on-off of the delivery pump, and the delivery pump pumps the culture medium in the culture medium storage bag to the perfusion culture chip, and the perfusion effluent flows out through the waste liquid bag.

11. The perfusion system according to claim 10, characterized in that: It also includes an image acquisition device, which acquires an image of a culture target in the culture chamber above the position of the upper sealing plate corresponding to the culture chamber, and transmits the image to the controller.

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