Gas-liquid level microsphere culture device

Abstract

The utility model discloses a gas-liquid level microsphere culture device which comprises a microsphere culture unit, the microsphere culture unit comprises a plurality of sunken structures, each sunken structure is provided with an opening part and a bottom part which are opposite to each other, the bottom part is provided with a gas inlet, and the sunken structures are used for accommodating a culture medium and culturing skin microspheres; the gas pipeline unit comprises a plurality of ventilation pipelines, and the ventilation pipelines are communicated with the bottom through gas inlets; and the gas supply unit is connected with the gas pipeline unit and is used for supplying gas to the sunken structure through the ventilation pipeline within a set time interval. Ventilation is conducted on the bottom of the concave structure at regular time through the gas pipeline unit and the gas supply unit, the skin microspheres placed in the concave structure to be cultured rotate along with the concave structure, and all the positions can be exposed out of the liquid level of the culture medium and exposed in the air; and maintaining a gas-liquid level to induce epidermal cells to differentiate into cuticles, simulating a gas-liquid level culture step of epidermal differentiation of planar artificial skin, and in addition, ensuring nutrition supply of skin microspheres.

Description

Technical Field

[0001] The present application relates to the field of cell culture, and in particular to an air-liquid surface microsphere culture device. Background Art

[0002] There are currently two common artificial skins whose construction technologies have become increasingly mature, one is planar and the other is microsphere-type. The construction of planar artificial skin is often based on the Transwell air-liquid surface culture method, that is, the epidermis of the artificial skin cultured in liquid for a period of time is placed in the air, and the dermis is soaked in the culture medium, thereby inducing epidermal cell differentiation. This method is a classic standard method for inducing the stratum corneum of artificial skin. When primary epidermal cells or immortalized epidermal cell lines with good differentiation ability are used, the epidermis of the planar artificial skin has a complete stratum corneum. However, the microsphere-type artificial skin currently only has a culture method that is completely immersed in the culture concentrate, which makes it difficult to evenly differentiate the outer epidermis. Utility Model Content

[0003] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes an air-liquid surface microsphere culture device, which can make the epidermis of microsphere-type artificial skin evenly differentiate to form a stratum corneum.

[0004] In a first aspect of the present application, there is provided an air-liquid surface microsphere culture device, the air-liquid surface microsphere culture device comprising:

[0005] A microsphere culture unit, the microsphere culture unit comprises a plurality of recessed structures, the recessed structures have an opening and a bottom relative to each other, the bottom is provided with an air inlet, and the recessed structures are used to contain a culture medium and culture skin microspheres;

[0006] A gas pipeline unit, the gas pipeline unit comprises a plurality of ventilation pipelines, and the ventilation pipelines are connected with the bottom through the air inlet;

[0007] The gas supply unit is connected to the gas pipeline unit and is used to supply gas to the recessed structure through the ventilation pipeline within a set time interval.

[0008] The gas-liquid surface microsphere culture device according to the present application has the following beneficial effects:

[0009] The gas-liquid surface microsphere culture device is based on the microsphere culture unit. The gas pipeline unit and the gas supply unit are used to regularly ventilate the bottom of the recessed structure. The gas impact method allows the skin microspheres placed in the recessed structure for culture to rotate, so that each position on the surface of the skin microspheres can be exposed to the culture medium liquid surface and exposed to the air. In addition, this intermittent rotation mode can maintain the gas-liquid surface at each position for a certain period of time to induce epidermal cells to differentiate into stratum corneum, simulate the gas-liquid surface culture steps of planar artificial skin epidermal differentiation, and also ensure the nutrient supply of the skin microspheres.

[0010] In some embodiments of the present application, a limiting hole is provided at the bottom, and the limiting hole is used to fix the skin microspheres.

[0011] In some embodiments of the present application, the diameter of the limiting hole is 5 to 500 μm.

[0012] In some embodiments of the present application, the limiting hole is located in the center of the bottom.

[0013] In some embodiments of the present application, the bottom includes an arc-shaped bottom wall with the limiting hole as the lowest point.

[0014] In some embodiments of the present application, the air inlet is located inside the limiting hole.

[0015] In some embodiments of the present application, the air inlet is located at the outer edge of the limiting hole.

[0016] In some embodiments of the present application, the air inlet is provided with a one-way air inlet component.

[0017] In some embodiments of the present application, the one-way air inlet component includes a valve-type sealing ring.

[0018] In some embodiments of the present application, the gas supply unit includes:

[0019] Gas cylinders, which store gas;

[0020] Electric valve, which is located on the gas path between the gas cylinder and the ventilation pipe. The electric valve is used to open or close the gas cylinder;

[0021] Timer: The timer is used to adjust the opening and closing of the electric valve within the set time interval.

[0022] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the structure of the gas-liquid surface microsphere culture device in one embodiment of the present application.

[0024] Figure 2 It is a partial schematic diagram of the concave structure of the gas-liquid surface microsphere culture device in one embodiment of the present application. Among them, A and B are different settings of the air inlet.

[0025] Figure 3 It is a schematic structural diagram of the limiting holes and the bottom wall of the gas-liquid surface microsphere culture device in one embodiment of the present application.

[0026] Figure 4 It is a schematic diagram of the process of culturing cell microspheres using an air-liquid surface microsphere culture device in one embodiment of the present application.

[0027] Figure 5 This is the fluorescent staining result of cell microspheres cultured using the air-liquid surface microsphere culture device in one embodiment of the present application.

[0028] Figure numerals: recessed structure 100 , opening 110 , bottom 120 , air inlet 121 , side wall 130 , limiting hole 140 , ventilation pipe 200 , gas cylinder 300 , electric valve 310 , skin microspheres 400 , culture medium 500 . DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the concept of the present application and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of them. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present application.

[0030] The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0031] In the description of this application, "several" means more than one, "many" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, "above", "below", "within", etc. are understood to include the number itself, and "about" means within the range of ±20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. of the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0032] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0033] Skin is the largest organ of vertebrates, especially mammals (such as humans). The skin includes the epidermis, the dermis, and in some cases, subcutaneous tissue and skin appendages, and has functions such as physical barrier, sensing the outside world, maintaining body temperature and moisture. The skin microspheres involved in the embodiments of the present application are an in vitro model that at least partially simulates its structure and function. Therefore, the skin microspheres include a core-shell structure composed of a nuclear layer and a shell layer outside the nuclear layer. The nuclear layer includes dermal cells, that is, the dermis; the shell layer includes epidermal cells, that is, the epidermis.

[0034] In some embodiments, the dermal cells include dermal cells separated from the dermis of the skin, which may be primary dermal cells, or dermal cells after culture and expansion, or immortalized dermal cells; in addition, in other embodiments, the dermal cells include dermal cells differentiated from stem cells (such as induced pluripotent stem cells, dermal stem cells). In some embodiments, the type of dermal cells may be, for example, fibroblasts.

[0035] In some of the embodiments, the epidermal cells include epidermal cells derived from the skin or mucous membrane, which may be primary epidermal cells, or epidermal cells after culture amplification, or immortalized epidermal cells. In addition, in other embodiments, the epidermal cells include epidermal cells differentiated from stem cells (such as induced pluripotent stem cells, epidermal stem cells). In some of the embodiments, the shell layer includes one or more layers of epidermal cell layers. In some of the embodiments, the epidermal cells in one or more layers of epidermal cell layers differentiate and mature to form a stratum corneum with a complete structure. In some of the embodiments, the type of epidermal cells may be, for example, keratinocytes.

[0036] In some of the embodiments, the epidermis also includes other cells or other extracellular substances added according to different application requirements, for example, a certain proportion of melanocytes is included to study the effect of drugs on melanocytes in the epidermis of the skin, or a certain proportion of immune cells (such as Langerhans cells) is included to study the effect of drugs on the skin's defense function.

[0037] In view of the above-mentioned cultivation of skin microspheres, a gas-liquid surface microsphere cultivation device according to an embodiment of the present application is proposed. The gas-liquid surface microsphere cultivation device comprises a microsphere cultivation unit, a gas pipeline unit and a gas supply unit.

[0038] refer to Figure 1 The microsphere culture unit comprises a plurality of recessed structures 100, wherein the recessed structures 100 have an opening 110 and a bottom 120 opposite to each other, and the bottom 120 is provided with an air inlet 121. The gas pipeline unit comprises a plurality of ventilation pipes 200, and the ventilation pipes 200 are connected to the bottom 120 of the recessed structure 100 through the air inlet 121. The gas supply unit is connected to the gas pipeline unit, and is used to supply gas to the recessed structure 100 through the ventilation pipes 200 within a set time interval.

[0039] In some embodiments, the bottom 120 includes a bottom wall. In some specific embodiments, the bottom wall includes a plane perpendicular to the vertical direction. In other embodiments, the bottom wall includes an arc surface, for example, it can be hemispherical, hemispherical, nearly hemispherical, nearly hemispherical, etc. In some embodiments, the recessed structure 100 also includes a side wall 130 located between the opening 110 and the bottom 120. Therefore, in some embodiments, the recessed structure 100 is cylindrical or inverted convex cone as a whole. In other embodiments, the recessed structure 100 can also be prismatic or inverted convex cone as a whole.

[0040] In some embodiments, reference Figure 2The bottom 120 is provided with a limiting hole 140, which is a hole formed by the bottom 120 in a direction away from the opening 110. Before the gas-liquid surface culture begins, the skin microsphere 400 can be placed in the limiting hole 140, so that at least a part of the skin microsphere 400 enters the limiting hole 140 to fix the skin microsphere, for example, the limiting hole 140 contains 1 / 100, 1 / 80, 1 / 50, 1 / 40, 1 / 30, 1 / 20, 1 / 15, 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6, 1 / 4, 1 / 3, 1 / 2, 2 / 3, 3 / 4, 4 / 5, 5 / 6, 6 / 7, 7 / 8, 8 / 9, 9 / 10, or all of the volume of the skin microsphere 400. In some embodiments, the limiting hole 140 is located in the center of the bottom 120. Among them, fixation means that when the limiting hole 140 contains at least a part of the skin microsphere 400, the skin microsphere 400 does not undergo a large displacement under the action of the limiting hole 140 and the air inlet 121, but does not limit the skin microsphere 400 to not undergo any displacement or rotation at all. Through this fixing method of the limiting hole, the deviation of the skin microsphere can be avoided to a large extent, which facilitates the placement, observation and removal of the skin microsphere before and after gas-liquid surface culture, and is conducive to the implementation of automated work. In some other embodiments, the limiting hole 140 can be located in the surrounding area of ​​the center of the bottom 120.

[0041] When there is a limiting hole 140, in some embodiments, reference Figure 2 A, the air inlet 121 is located inside the limiting hole 140, so the airflow of the air inlet 121 directly blows the skin microspheres 400 vertically from directly below the skin microspheres 400 to rotate.

[0042] In some other embodiments, reference Figure 2 B, the air inlet 121 is located at the outer edge of the limiting hole 140, so the airflow of the air inlet 121 blows the skin microsphere 400 vertically from the lower side of the skin microsphere 400 to rotate. It is understandable that the air inlet 121 can also be located at other positions, such as on the side wall, so as to blow the skin microsphere 400 horizontally to rotate. In some embodiments, the bottom 120 of the recessed structure 100 includes a plurality of air inlets 121, and the plurality of air inlets 121 can be all located inside the limiting hole 140, or all located at the outer edge of the limiting hole 140, or all located on the side wall, or located at least two of the inside, outer edge, and side wall of the limiting hole 140, so that air is supplied to the bottom through these different air inlets 121, providing different rotation modes for the skin microsphere 400, so as to more accurately adjust the surface of the skin microsphere 400 exposed to the culture medium 500, so that the epidermal cells can differentiate more evenly during the gas-liquid surface culture process.

[0043] In some embodiments, the projection of the limiting hole 140 on a plane perpendicular to the vertical direction is circular, for example, it can be a hemispherical limiting hole, a semi-ellipsoidal limiting hole, a cylindrical limiting hole, or a nearly hemispherical, nearly hemispherical, or nearly cylindrical limiting hole. In some embodiments, the diameter of the limiting hole 140, that is, the diameter d of the circular projection of the limiting hole 140 on a plane perpendicular to the vertical direction is 5 to 500 μm, for example, it can be 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm. At this time, the diameter of the limiting hole 140 is substantially smaller than the diameter of the skin microsphere, so when the skin microsphere is placed in the concave structure, it can close the limiting hole 140, and then close the air inlet 121 located inside the limiting hole 140. Therefore, the culture medium can be added or removed from the concave structure and the culture medium is prevented from flowing into the ventilation channel.

[0044] In some embodiments, reference Figure 3 , the bottom 120 includes an arc-shaped bottom wall with the limiting hole 140 as the lowest point. Through the setting of this arc-shaped bottom wall, when the skin microspheres are placed in the recess, they will automatically move to the position of the limiting hole under the action of gravity and will eventually be fixed in the corresponding position by the limiting hole 140. In this embodiment, the limiting hole 140 can be a hemispherical limiting hole, a semi-ellipsoidal limiting hole, a cylindrical limiting hole, or a nearly hemispherical, nearly hemispherical, or nearly cylindrical limiting hole. When the limiting hole 140 is hemispherical or nearly hemispherical, in some specific embodiments, the curvature of the limiting hole 140 is greater than the curvature of the arc-shaped bottom wall, so that the skin microspheres 400 placed in the recessed structure 100 are more convenient to be fixed by the limiting hole 140.

[0045] In some embodiments, the air inlet 121 is provided with a one-way air inlet component. Since the culture medium 500 is contained in the recessed structure 100 during gas-liquid surface culture, when gas is fed from the air inlet 121 to the bottom 120 and before and after the gas is fed, the culture medium 500 may flow back from the air inlet 121 into the ventilation duct 200. For this reason, a one-way air inlet component is provided at the air inlet 121 to ensure that gas can only be fed from the ventilation duct 200 to the bottom 120, and to prevent the culture medium 500 from flowing from the bottom 120 into the ventilation duct 200.

[0046] In some specific embodiments, the one-way air inlet component includes a valve-type sealing ring (not shown in the figure), and the one-way inflow of gas from the ventilation duct to the inward depression is more effectively controlled by this valve structure. In some specific embodiments, the raw material of the valve-type sealing ring includes a polymer material, for example, at least one of polytetrafluoroethylene, polyethylene terephthalate, polyurethane, etc. In some other specific embodiments, the one-way air inlet component includes a hydrophobic and breathable microporous membrane, such as a hydrophobic and breathable microporous polymer film (such as a polytetrafluoroethylene microporous membrane). In some embodiments, micro-protrusions are provided on the bottom 120 and / or the limiting hole 140 to prevent the skin microspheres 400 from being too closely attached to the bottom 120 and / or the limiting hole 140, which is not conducive to the gas supply unit to blow the skin microspheres through the air inlet 121 for ventilation and rotation.

[0047] In some embodiments, the microsphere culture unit comprises a plurality of array-arranged recessed structures, for example, 4, 6, 8, 12, 24, 48, 96, 384, 1536 array-arranged recessed structures. In some specific embodiments, the microsphere culture unit is a polymer material, for example, at least one of polystyrene, polytetrafluoroethylene, etc. Similarly, in some specific embodiments, the gas pipeline unit is a polymer material, for example, at least one of polystyrene, polytetrafluoroethylene, etc.

[0048] In some embodiments, reference Figure 1 The gas supply unit includes a gas cylinder 300, an electric valve 310 and a timer (not shown in the figure). Gas is stored in the gas cylinder 300, and the electric valve 310 is located on the gas path between the gas cylinder 300 and the ventilation pipe 200. The electric valve 310 can open or close the gas cylinder 300, and the timer adjusts the switch of the electric valve 310 within a set time interval, so that the gas cylinder 300 can feed gas into the ventilation pipe 200 or stop feeding gas within a set time interval. Among them, the gas stored in the gas cylinder 300 is a gas that does not affect the processes such as cell growth, proliferation and differentiation in the skin microspheres, for example, it can be at least one of carbon dioxide, nitrogen, etc. In some specific embodiments, the ventilation duration of the gas supply unit supplying gas to the skin microspheres is 5 to 30 seconds, and the ventilation interval is 10 minutes to 4 hours. For example, each ventilation duration is 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 12 seconds, 14 seconds, 15 seconds, 16 seconds, 18 seconds, 20 seconds, 22 seconds, 24 seconds, 26 seconds, 28 seconds, and 30 seconds, and each ventilation interval is 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, and 4 hours.

[0049] The present application is described below in conjunction with specific embodiments.

[0050] Example 1

[0051] refer to Figure 1 and Figure 4 This embodiment provides a gas-liquid surface microsphere culture device, and its preparation process is as follows:

[0052] The corresponding cylindrical limiting holes and the air inlets in the limiting holes are formed at the bottom of the 8×12 cylindrical microporous recessed structure of the 96-well microplate by laser drilling.

[0053] A polytetrafluoroethylene sheet of a gas pipeline unit is formed by 3D printing, and ventilation ducts corresponding to 8×12 air inlets are arranged on the sheet.

[0054] The polytetrafluoroethylene sheet and the 96-well microplate are compounded together by adhesive, and a micro gas cylinder is connected to a port on one side of the ventilation pipe, and an electric gas valve switch and a timer are connected to the micro gas cylinder.

[0055] Therefore, the gas-liquid surface microsphere culture device provided in this embodiment includes a microsphere culture unit, a gas pipeline unit and a gas supply unit; the microsphere culture unit includes 8×12 recessed structures, the recessed structures have relative openings and bottoms, and the bottom is provided with an air inlet; the gas pipeline unit includes a ventilation pipeline connected to the 8×12 air inlets, and the gas supply unit includes a gas cylinder storing CO2, and an electric valve capable of opening or closing the gas cylinder is provided on the gas path between the gas cylinder and the ventilation pipeline, and the electric valve adjusts the opening and closing of the valve within a set time interval under the action of a timer, thereby controlling the ventilation time of the recessed structure. In addition, the 96-well microplate is also equipped with a lid for aseptic cell culture.

[0056] Example 2

[0057] This embodiment provides a method for culturing skin microspheres, referring to Figure 4 , including the following steps:

[0058] The human dermal cells cultured in vitro were mixed with a collagen hydrogel at a concentration of 2 mg / mL to obtain a mixed solution. The dilution medium of the system was an epidermal growth medium (K-SFM medium with 40 μg / mL bovine pituitary extract BPE and 50 ng / mL epidermal growth factor EGF added). The mixed solution was added to a Teflon-96 well plate sterilized by high temperature and high pressure steam containing 200 μL / well sterile 3M F3100 oil phase at 4 μL / well. The well plate was covered with a Teflon plate cover and moved into a 37°C incubator for incubation for 15 minutes to form solidified dermal microspheres.

[0059] The dermal microspheres were collected using DMEM containing FBS, transferred into a 10 cm non-adherent culture dish, and cultured statically for 1 day.

[0060] Human epidermal stem cells were digested and collected, uniformly mixed with the dermal microspheres after static culture for 1 day, replaced with epidermal growth medium, and transferred to the sterilized air-liquid surface microsphere culture device of Example 1. During the transfer, the microspheres were first transferred to be fixed to the limiting holes so that the bottom of the microspheres blocked the airway opening below the limiting holes, and then 100 μL of culture solution was added to cover the top of the microspheres.

[0061] After 4-5 days of culture, discard all the culture medium and add about 30 μL of epidermal differentiation medium to each well, so that half of the skin microspheres are exposed to air. Start the timer and set it to rest for 2 hours and ventilate for 10 seconds. Continue to culture for 5 days, changing the medium every two days. After 5 days, skin microspheres with stratum corneum are obtained.

[0062] The skin microspheres cultured in the air-liquid surface microsphere culture device for 5 days were fixed, embedded, and sliced, and the largest surface was taken for immunofluorescence staining. Figure 5 As shown in the figure, it can be seen that the cell nucleus is dark blue, the skin microsphere structure is the inner dermal cell, the outer layer wraps the epidermal cell, and the outermost cell nucleus is spindle-shaped, showing the characteristics of keratinocytes; K5 is widely expressed in the inner epidermis, indicating that this layer of cells is epidermal stem cells, similar to the basal layer in human skin; K10 is highly expressed and evenly expressed in the outer epidermis, indicating that the outer epidermal cells are obviously differentiated and the stratum corneum is evenly distributed. This result shows that the epidermal layer of the skin microspheres cultured in the air-liquid surface microsphere culture device can be evenly differentiated to form a stratum corneum.

[0063] Example 3

[0064] This embodiment provides a gas-liquid surface microsphere culture device, which is different from Embodiment 1 in that the air inlet is located at the outer edge of the limiting hole and is provided with a micro valve-type sealing ring.

[0065] Example 4

[0066] This embodiment provides an air-liquid surface microsphere culture device, which is different from Embodiment 1 in that the air inlet is located on the side wall of the recessed structure, and a water-proof and breathable PTFE film is provided at the air inlet.

[0067] The present application is described in detail above in conjunction with the embodiments, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A gas-liquid surface microsphere culture device, characterized in that: include: A microsphere culture unit, wherein the microsphere culture unit comprises a plurality of recessed structures, wherein the recessed structures have an opening portion and a bottom portion opposite to each other, wherein the bottom portion is provided with an air inlet, and the recessed structures are used to contain a culture medium and culture skin microspheres; A gas pipeline unit, the gas pipeline unit comprising a plurality of ventilation pipes, the ventilation pipes being connected to the bottom through the air inlet; A gas supply unit is connected to the gas pipeline unit and is used to supply gas to the recessed structure through the ventilation pipeline within a set time interval.

2. The gas-liquid surface microsphere culture device according to claim 1, characterized in that: The bottom is provided with a limiting hole, and the limiting hole is used to fix the skin microsphere.

3. The gas-liquid surface microsphere culture device according to claim 2, characterized in that: The diameter of the limiting hole is 5-500 μm.

4. The gas-liquid surface microsphere culture device according to claim 2, characterized in that: The limiting hole is located at the center of the bottom.

5. The gas-liquid surface microsphere culture device according to claim 2 or 4, characterized in that: The bottom comprises an arc-shaped bottom wall with the limiting hole as the lowest point.

6. The gas-liquid surface microsphere culture device according to claim 2, characterized in that: The air inlet is located inside the limiting hole.

7. The gas-liquid surface microsphere culture device according to claim 2, characterized in that: The air inlet is located at the outer edge of the limiting hole.

8. The gas-liquid surface microsphere culture device according to claim 1, characterized in that: The air inlet is provided with a one-way air inlet component.

9. The gas-liquid surface microsphere culture device according to claim 8, characterized in that: The one-way air inlet component includes a valve-type sealing ring.

10. The gas-liquid surface microsphere culture device according to claim 1, characterized in that: The gas supply unit comprises: A gas cylinder storing gas; An electric valve, the electric valve is located on the gas path between the gas cylinder and the ventilation pipe, and the electric valve is used to open or close the gas cylinder; A timer is used to adjust the switch of the electric valve within a set time interval.

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