Cell mass culture device and circulation system for cell mass growth

Abstract

The utility model discloses a cell mass culture device and a circulation system for cell mass growth, and relates to the technical field of cell culture devices.The scheme is that the cell mass culture device comprises a box body, the interior of the box body is of a hollow structure, first openings are formed in the two sides of the box body, a first through hole is formed in the top of the box body, and the first through hole is communicated with the interior of the box body; the growth patch is arranged at the first through hole, the growth patch is formed by arranging a plurality of microstructures in an array mode, the top surface of each microstructure is sunken towards the center, and the growth patch is used for cell cluster growth and enables air to penetrate through; the liquid storage plate is arranged at the top of the growth patch, a culture solution is stored in the liquid storage plate, the culture solution is in contact with the growth patch, and the growth patch prevents the culture solution from permeating; and the top cover is arranged at the top of the liquid storage plate and used for covering the liquid storage plate. Therefore, the device can continuously supply oxygen for the growth of the cell cluster through the box body, and is suitable for long-period culture of the high-oxygen-consumption cell cluster in a liquid culture environment.

Description

Technical Field

[0001] The utility model relates to the technical field of cell culture devices, in particular to a cell cluster culture device and a circulation system for the growth of cell clusters. Background Art

[0002] In current biomedical research, the in vitro culture of organoids has become an important technological platform. In order to ensure that organoids have more bionic structures and functions, conditions that simulate the in vivo environment must be provided. Physical factors such as the stimulation of blood fluid, oxygen environment, bionic mechanical environment similar to tissues, and spatial geometric constraints will affect the development of tissues and the function of organs. For example, the culture of organoids such as the pancreas, liver, and brain requires a higher oxygen supply, while a low oxygen environment can cause cell necrosis. Therefore, in order to achieve long-term culture and functional maturation of cell clusters, it is necessary to construct an organoid culture device to provide a bionic gas, liquid, and mechanical environment, and provide a geometric structure to promote the formation of organoids.

[0003] However, current organoid culture molds lack oxygen exchange, making the molds unsuitable for long-term culture of high-oxygen-consuming organoids. At the same time, current organoid molds are usually made of plastic materials, which have a high mechanical modulus. Although these materials can provide geometric constraints for organoids or cell clusters and promote the formation of cell clusters, they cannot be used for long-term culture of cell clusters. Utility Model Content

[0004] The utility model aims to solve at least one problem in the above-mentioned background technology, and provides a cell cluster culture device and a circulation system for cell cluster growth.

[0005] To achieve the above-mentioned purpose, the utility model provides a cell cluster culture device, comprising:

[0006] A box body, the interior of which is a hollow structure, with first openings provided on both sides of the box body, and a first through hole provided on the top of the box body, the first through hole being connected to the interior of the box body;

[0007] A growth patch, which is arranged at the first through hole, the growth patch is formed by arranging a plurality of microstructures in an array, the top surface of the microstructure is concave toward the center, and the growth patch allows air to pass through;

[0008] A liquid storage plate, which is arranged on the top of the growth patch, wherein the culture liquid is stored in the liquid storage plate, the culture liquid is in contact with the growth patch, and the growth patch prevents the culture liquid from penetrating;

[0009] A top cover is arranged on the top of the liquid storage plate, and the top cover is used to cover the liquid storage plate.

[0010] Preferably, the depth of the depression at the top of the microstructure is in the range of 250-650 μm.

[0011] Preferably, the microstructure is arranged in an inverted pyramid shape, and the distance between two adjacent microstructures is in the range of 8-12 μm.

[0012] Preferably, the growth patch is made of soft polymer material.

[0013] Preferably, the thickness of the growth patch is in the range of 1-5 mm.

[0014] Preferably, the liquid storage plate comprises a plurality of liquid storage tanks arranged at intervals, and the liquid storage tanks penetrate the liquid storage plate;

[0015] The culture solution is stored in the liquid storage tank.

[0016] Preferably, a plurality of the first through holes are provided, and the plurality of the first through holes are provided correspondingly to the plurality of the liquid storage tanks;

[0017] The growth patches are respectively arranged between the first through holes and the liquid storage tanks.

[0018] Preferably, the top cover is made of transparent material.

[0019] Preferably, the growth patch is made of polydimethylsiloxane material.

[0020] To achieve the above object, the utility model also provides a circulation system for growing cell clusters, comprising any one of the cell cluster culture devices described above, and further comprising:

[0021] Hoses, gas circulation pumps and gas cylinders;

[0022] One end of the hose is connected to the first openings on both sides of the box body, and the other end is connected to the gas circulation pump, and the hose, the cell cluster culture device and the gas circulation pump form an air circulation;

[0023] The gas circulation pump is connected to the gas cylinder, and the gas circulation pump is used to control the gas flow rate.

[0024] Based on this, the beneficial effects of the utility model are:

[0025] 1. According to the scheme of the utility model, the culture device includes a box body, the interior of the box body is hollow, and first openings are arranged on both sides thereof for air flow, a first through hole is arranged on the top of the box body, and a growth patch is arranged at the first through hole, the growth patch can be used for the growth of cell clusters, and the growth patch is air-permeable and water-impermeable, and can be effectively used for the long-term culture of high oxygen-consuming cells;

[0026] 2. Through the solution of the utility model, the growth patch is made of PMDS material. The patch is soft and bendable and not easy to break. It has high light transmittance, is non-cytotoxic and chemically inert, and allows gas to pass through while being impermeable to water, thereby replenishing oxygen for the cell clusters to maintain the liquid culture environment of the reservoir. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0028] Figure 1 A disassembled diagram schematically showing a cell cluster culture device according to one embodiment of the utility model;

[0029] Figure 2 An enlarged schematic diagram schematically showing a growth patch according to an embodiment of the utility model;

[0030] Figure 3 A schematic diagram schematically showing the structure of a circulation system according to one embodiment of the utility model;

[0031] Explanation of the reference numerals: box body 10 , first opening 101 , first through hole 102 , growth patch 20 , microstructure 201 , liquid storage plate 30 , liquid storage tank 301 , top cover 40 , hose 50 , gas circulation pump 60 , gas cylinder 70 . DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0033] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0034] It should be noted that the directional words such as "upper", "lower", "left", and "right" described in the embodiments of the present application are described at the angles shown in the drawings and should not be understood as limiting the embodiments of the present application. In addition, in the context, it should also be understood that when it is mentioned that an element is formed "on" or "under" another element, it can not only be directly formed "on" or "under" another element, but also be indirectly formed "on" or "under" another element through an intermediate element.

[0035] Figure 1 A disassembly diagram schematically showing a cell cluster culture device according to an embodiment of the present invention, Figure 2 An enlarged schematic diagram schematically shows a growth patch according to an embodiment of the present invention, as shown in FIG. Figure 1 , 2 As shown, a cell cluster culture device of the utility model comprises:

[0036] The box body 10 has a hollow structure inside. First openings 101 are provided on both sides of the box body 10. The first openings 101 are used for air circulation. A first through hole 102 is provided on the top of the box body 10. The first through hole 102 is connected to the inside of the box body 10.

[0037] A growth patch 20 is disposed at the first through hole 102. The growth patch 20 is formed by a plurality of microstructures 201 arranged in an array. The top surface of the microstructure 201 is concave toward the center. The growth patch 20 is used for the growth of cell clusters. The growth patch 20 allows air to pass through.

[0038] A liquid storage plate 30 is disposed on the top of the growth patch 20. The liquid storage plate 30 stores culture fluid inside. The culture fluid contacts the growth patch 20. The growth patch 20 prevents the culture fluid from penetrating.

[0039] The top cover 40 is disposed on the top of the liquid storage plate 30 , and is used to cover the liquid storage plate 30 .

[0040] Specifically, the growth patch 20 is located between the box body 10 and the liquid storage plate 30. The liquid storage plate 30 can provide culture fluid to achieve a liquid culture environment, and the air circulation inside the box body 10 can continuously provide oxygen. The growth patch 20 is breathable and water-impermeable, so that the device can achieve long-term and stable culture of organoids or cell clusters.

[0041] At the same time, the growth patch 20 is formed by an array of multiple microstructures 201, and the top surface of the microstructure 201 is concave toward the center. When cells are placed on the growth patch 20, the top surface concave toward the center provides geometric space restrictions for the monodispersed cells, prompting the cells to aggregate into clusters. In addition, multiple microstructures 201 are the same size, and multiple cell clusters or organoids of uniform size can be efficiently generated.

[0042] Furthermore, if Figure 2 As shown, the distribution range of the microstructures 201 in the growth patch 20 is 100-700 / The thickness ranges from 1 to 5 mm, the side length of the microstructure 201 ranges from 200 to 500 μm, the depth of the depression at the top ranges from 250 to 650 μm, and the spacing between two adjacent microstructures 201 ranges from 8 to 12 μm.

[0043] In one embodiment of the present invention, the area of ​​the growth patch 20 is 9.07 , with a thickness of 3 mm, and composed of 5800 microstructures 201. The microstructure 201 is arranged in an inverted pyramid shape, the top surface is square, the side length is 400 μm, the depression depth of the top surface is 400 μm, and the spacing between two adjacent microstructures 201 is 10 μm.

[0044] Furthermore, the growth patch 20 is made of a soft, inert polymer material such as air-permeable and water-impermeable PDMS or F127DA hydrogel, preferably a polydimethylsiloxane (PDMS) material. The growth patch 20 formed using this material is soft, bendable, not easy to break, transparent and light-transmitting, non-cytotoxic, non-chemically inert, air-permeable and water-impermeable, and can replenish sufficient oxygen for the cell clusters to maintain a liquid culture environment.

[0045] Furthermore, if Figure 1 As shown:

[0046] The liquid storage plate 30 includes a plurality of liquid storage tanks 301 arranged at intervals, and the liquid storage tanks 301 penetrate the liquid storage plate 30;

[0047] When the growth patch 20 is located at the bottom of the liquid storage plate 30 , the culture solution is stored in the liquid storage tank 301 .

[0048] Specifically, a plurality of first through holes 102 are provided at the top of the box body 10, and the plurality of first through holes 102 are provided correspondingly to the plurality of liquid storage tanks 301;

[0049] The growth patch 20 is respectively arranged between the first through hole 102 and the liquid storage tank 301. When the culture solution is placed in the liquid storage tank 301, the culture solution is not penetrated by the growth patch 20. The culture solution can provide a liquid culture environment for the cells on the growth patch 20. The box body 10 is filled with flowing air. Through the first through hole 102, oxygen can penetrate from the growth patch 20 to provide sufficient oxygen for the cell clusters in the growth patch 20 to achieve their growth.

[0050] At the same time, the first through hole 102 on the top of the box body 10 is composed of a plurality of small holes, and the overall structure is a cross hollow bracket structure, which can effectively provide support and attachment basis for the growth patch 20 thereon.

[0051] Furthermore, the top cover 40 is made of a transparent material. When the top cover 40 covers the liquid storage plate 30, closed culture of the cell cluster is achieved, and the growth of the cell cluster on the growth patch 20 can also be directly observed from the outside of the device.

[0052] Further, Figure 3 A schematic diagram schematically shows the structure of a circulation system according to an embodiment of the present invention, as shown in FIG. Figure 3 As shown, the utility model also provides a circulation system for growing cell clusters, including the cell cluster culture device as described above, and further comprising:

[0053] Hose 50, gas circulation pump 60 and gas cylinder 70;

[0054] One end of the hose 50 is connected to the first openings 101 on both sides of the box body 10, and the other end is connected to the gas circulation pump 60. The hose 50, the cell cluster culture device and the gas circulation pump 60 form an air circulation;

[0055] The gas circulation pump 60 is in communication with the gas cylinder 70 , and the gas circulation pump 60 is used to control the gas flow rate.

[0056] In this way, by operating the gas circulation pump 60, air can continuously flow inside the box body 10 to provide the oxygen required for long-term cultivation of the growth patch 20. At the same time, the gas circulation pump 60 can control the gas flow rate and provide different amounts of oxygen supply according to actual cultivation needs.

[0057] Furthermore, based on the above circulation system, the steps of forming the cell cluster are:

[0058] S01: Assemble the box body 10, the growth patch 20, the liquid storage plate 30 and the top cover 40 to form a cell cluster culture device, and seal the first openings 101 on both sides of the box body 10 with rubber plugs.

[0059] S02: Perform anti-adsorption treatment on the cell cluster culture device. Remove the top cover 40 in the clean bench or biosafety cabinet, add 2 mL of anti-adhesion flushing solution to each of the multiple liquid reservoirs 301, centrifuge the cell cluster culture device at a speed of 1300xg for 5 minutes, and then rinse each liquid reservoir 301 with warm basal culture medium.

[0060] S03: Cultivating and digesting liver cells. When the growth confluence of the liver cells cultured in the growth patch 20 corresponding to each reservoir 301 reaches 80%, a cell digestion operation is performed, specifically:

[0061] S301: removing the cell culture medium in the liquid reservoir 301 in a clean bench or a biosafety cabinet, and washing each liquid reservoir 301 with 2 mL of DPBS buffer;

[0062] S302: Add 2 mL of trypsin digestion solution and digest for 3-5 minutes, then add 1 mL of stem cell culture medium to terminate digestion, collect cells by centrifugation, and resuspend the cells in stem cell culture medium;

[0063] S303: Count the cells and adjust the cell density of the cell suspension to 1.5× / mL;

[0064] S304: Add 2 mL of cell suspension to each storage tank 301 and cover it with a top cover 40;

[0065] S305: The cell pellet culture device is centrifuged at a speed of 200 g, and then placed in a cell culture incubator for 48 hours.

[0066] S04: Cell high oxygen culture, take out the rubber plugs on the first openings 101 on both sides of the box body 10, connect the hose 50, insert the other end of the hose 50 into the gas circulation pump 60, select a gas flow rate of 30mL / hour, the gas circulation pump 60 is outside the cell culture box, and the cell cluster culture device is inside the cell culture box. Continue to culture until the time required by the experiment, and then end the culture.

[0067] In summary, the cell cluster culture device of the present invention includes a box body 10, the interior of the box body 10 is hollow, and first openings 101 are provided on both sides thereof for air flow. A first through hole 102 is provided on the top of the box body 10, and a growth patch 20 is provided at the first through hole 102. The growth patch 20 can be used for cell cluster growth, and the growth patch 20 is air permeable but not water permeable, so that the device can be effectively used for long-term culture of high oxygen-consuming cells.

[0068] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the technical scope involved in the present application is not limited to the technical solution formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the technical concept. For example, the above features are replaced with the technical features with similar functions disclosed in this application (but not limited to) by each other.

Claims

1. A cell cluster culture device, characterized in that: include: A box body, the interior of which is a hollow structure, with first openings provided on both sides of the box body, and a first through hole provided on the top of the box body, the first through hole being connected to the interior of the box body; A growth patch, which is arranged at the first through hole, the growth patch is formed by arranging a plurality of microstructures in an array, the top surface of the microstructure is concave toward the center, and the growth patch allows air to pass through; A liquid storage plate, which is arranged on the top of the growth patch, wherein the culture liquid is stored in the liquid storage plate, the culture liquid is in contact with the growth patch, and the growth patch prevents the culture liquid from penetrating; A top cover is arranged on the top of the liquid storage plate, and the top cover is used to cover the liquid storage plate.

2. A cell cluster culture device according to claim 1, characterized in that: The depth of the depression at the top of the microstructure is in the range of 250-650 μm.

3. The cell cluster culture device according to claim 1, characterized in that: The microstructure is arranged in an inverted pyramid shape, and the distance between two adjacent microstructures is in the range of 8-12 μm.

4. The cell cluster culture device according to claim 1, characterized in that: The growth patch is made of soft polymer material.

5. The cell cluster culture device according to claim 1, characterized in that: The thickness of the growth patch ranges from 1 to 5 mm.

6. The cell cluster culture device according to claim 1, characterized in that: The liquid storage plate comprises a plurality of liquid storage tanks arranged at intervals, and the liquid storage tanks penetrate the liquid storage plate; The culture solution is stored in the liquid storage tank.

7. The cell cluster culture device according to claim 6, characterized in that: A plurality of the first through holes are provided, and the plurality of the first through holes are provided correspondingly to the plurality of the liquid storage tanks; The growth patches are respectively arranged between the first through holes and the liquid storage tanks.

8. The cell cluster culture device according to claim 1, characterized in that: The top cover is made of transparent material.

9. The cell cluster culture device according to claim 4, characterized in that: The growth patch is made of polydimethylsiloxane material.

10. A circulation system for growing cell clusters, characterized in that: A cell cluster culture device comprising any one of claims 1 to 9, further comprising: Hoses, gas circulation pumps and gas cylinders; One end of the hose is connected to the first openings on both sides of the box body, and the other end is connected to the gas circulation pump, and the hose, the cell cluster culture device and the gas circulation pump form an air circulation; The gas circulation pump is connected to the gas cylinder, and the gas circulation pump is used to control the gas flow rate.

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