Low-loss cell sphere passage device, microfluidic device and cell passage method

Abstract

The invention belongs to the field of cell culture, and discloses a low-loss cell ball passage device, a micro-fluidic device and a cell passage method. The passage device comprises an open cavity and a top cover, and the open cavity is connected with the top cover to form a sealed environment; a cell ball forming bottom plate is arranged at the bottom of the open cavity and consists of a plurality of cell chambers which are arranged in an array, and the open cavity is made of a visible material; the microfluidic device comprises a plurality of cell ball passage devices which are arranged in a layered manner according to an exponential growth mode, and one cell ball passage device is connected with two inverted cell ball passage devices at the downstream through a valve controller; when the cell balls are digested based on the device, the passage of the cell balls is completed by overturning and shaking the corresponding device. Therefore, the passage method basically based on the device is easy to automation and more efficient, and the cell loss is lower.

Description

Technical Field

[0001] The present invention belongs to the technical field related to cell culture, and more specifically, relates to a low-loss cell ball passage device, a microfluidic device and a cell passage method. Background Art

[0002] Cell culture technology plays an important role in biomedical research, drug development, regenerative medicine, and tissue engineering. The culture of cell spheroids (or cell aggregates) is a cell culture method that has emerged in recent years. It has attracted widespread attention due to its advantages in simulating the in vivo microenvironment, improving cell-to-cell interactions, and enhancing cell activity. However, the current cell spheroid passaging process often faces problems such as cell damage, low recovery rate, and complex operation. These problems directly affect the growth state of cells and the repeatability of subsequent experiments, limiting the further development of cell culture technology.

[0003] Traditional cell spheroid passaging methods usually rely on manual operations, during which the cell spheroid culture position needs to be repeatedly blown to remove it from the microwell and other environments. This method not only increases the risk of cell damage, but may also lead to reduced cell activity, affecting the reliability of experimental results.

[0004] In order to overcome the above challenges, it is urgent to develop a low-loss cell spheroid passaging method and device. The device should have an efficient cell spheroid recovery mechanism that can minimize cell damage during passaging, while simplifying the operation process and improving reproducibility and consistency. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a low-loss cell ball passage device, a microfluidic device and a cell passage method. The present invention improves the efficiency and safety of cell ball transmission by optimizing the passage structure and operation. The passage device involved establishes a closed integrated passage environment through an open mouth and a top cover, and a culture chamber is provided at the bottom of the container; after the cell balls that can be passed are formed, the method correspondingly turns and shakes the device so that the cell balls in the culture chamber are transferred to the digestive fluid to complete the cell passage operation, thereby solving the technical problems of low efficiency and high loss of cell ball passage in the prior art through the device and method of the present invention.

[0006] To achieve the above-mentioned purpose, according to the first aspect of the present invention, a low-loss cell ball passaging device is provided, comprising an open cavity and a top cover, wherein the open cavity and the top cover are connected to form a sealed environment; a cell ball forming base plate is provided at the bottom of the open cavity, which is composed of a plurality of cell chambers arranged in an array, and the material of the open cavity is a visible material.

[0007] As a preferred embodiment of the present invention, a flow channel extending from the top of the open cavity to the bottom is provided on the inner wall of the open cavity.

[0008] As a preferred embodiment of the present invention, a rubber ring is provided inside the top cover to reversibly combine with the open cavity.

[0009] As a preferred embodiment of the present invention, the cell chamber is a patterned microwell array or a low-absorption microwell plate.

[0010] To achieve the above object, according to the second aspect of the present invention, there is provided a microfluidic device for cell spheroid passage, comprising a plurality of cell spheroid passage devices according to any one of the first aspect of the present invention and a plurality of valve controls; the plurality of cell spheroid passage devices are arranged in n layers, and the number of cell spheroid passage devices arranged in the i-th layer is A. i =2 i-1 , and the j-th cell ball passage device in the i-th layer is connected to the (2j-1)-th and 2j-th cell ball passage devices in the i+1-th layer through the valve control; wherein n is the number of layers in which the cell ball passage devices are arranged, Ai is the number of cell ball passage devices corresponding to the i-th layer, 1≤i≤n, 1≤j≤2 i-1 .

[0011] As a preferred embodiment of the present invention, the microfluidic device further comprises a flipping mechanism connected to each of the cell spheroid passaging devices and used for controlling the cell spheroid passaging devices to flip.

[0012] As a preferred embodiment of the present invention, each cell ball passage device in the microfluidic device is provided with a liquid inlet and a liquid outlet, and are respectively connected to a liquid pump.

[0013] To achieve the above object, according to the third aspect of the present invention, based on the cell culture device of the first aspect of the present invention, a cell sphere cell culture method is provided, comprising the following steps:

[0014] (1) adding a single cell suspension into a cell chamber in a cell spheroid passaging device and culturing the cell spheroids to be passable;

[0015] (2) After the culture medium is aspirated, a digestion solution is continuously added to the cell spheroid passaging device, and then the top cover is closed, and the cell spheroid passaging device is turned over and shaken until the cell spheroids fall out of the cell chamber and mix with the digestion solution to digest the cell spheroids;

[0016] (3) Adding stop solution to the cell spheroid passaging device to complete the passaging of the cell spheroid.

[0017] To achieve the above object, according to a fourth aspect of the present invention, based on the cell spheroid passaging microfluidic device of the second aspect of the present invention, a cell spheroid passaging method is provided, comprising: adding a single cell suspension into a cell chamber in a cell spheroid passaging device of a first layer for passaging operation, and then, in a progressive order from the first layer to the nth layer, opening a valve control on the cell spheroid passaging device of the i-th layer, distributing the single cell suspension after the i-th passaging operation in the i-th layer to the cell spheroid passaging device of the i+1th layer, and performing the i+1th passaging according to the passaging operation until the nth passaging is completed;

[0018] The passaging operation is specifically as follows: adding a single cell suspension into a cell chamber in a cell ball passaging device, and culturing until a cell ball can be passed; after aspirating the culture medium, continuing to add a digestion solution into the cell ball passaging device, then covering the top cover, flipping and shaking the cell ball passaging device until the cell ball falls off from the cell chamber and mixes with the digestion solution to digest the cell ball; adding a stop solution into the cell ball passaging device to complete the passaging of the cell ball and form a single cell suspension after passaging.

[0019] As a preferred embodiment of the third aspect or the fourth aspect of the present invention, flipping and shaking the cell spheroid passaging device specifically comprises:

[0020] Flipping the cell spheroid passaging device in a vertical direction at the bottom thereof, and shaking the cell spheroid in the vertical direction thereof, and adjusting the number of flipping and shaking according to the digestion state of the cell spheroid;

[0021] Alternatively, the cell spheroid passaging device is controlled to flip in the vertical direction of the bottom by a flipping mechanism connected to the cell spheroid passaging device, and to shake along the vertical direction of the flipping, and the number of flipping and shaking is adjusted according to the digestion status of the cell spheroid.

[0022] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0023] (1) The cell culture device of the present invention establishes a sealed environment during cell spheroid cell culture by means of an open cavity and a top cover, and by optimizing the structure of the device, can reduce cell damage caused by human operation during cell spheroid cell culture, and improve cell recovery rate and activity, thereby optimizing cell culture efficiency.

[0024] (2) The microfluidic device of the present invention comprises a plurality of cell passaging devices arranged in layers in an exponential growth manner, and a cell sphere passaging device is connected to two cell sphere passaging devices in the next layer through a valve control device, thereby completing semi-automatic or fully automatic cell passaging, further improving the cell recovery rate and activity during the cell sphere passaging process, and improving the cell culture efficiency.

[0025] (3) The microfluidic device of the present invention preferably includes a flipping mechanism, and each passage device in the microfluidic device is provided with a liquid inlet and a liquid outlet, and the communication of adjacent passage environments is controlled by valve control, so that the cell balls can be transferred in a sealed environment to continuously and safely complete the culture and passage process, while ensuring safety and improving efficiency. Specifically, semi-automatic means manually controlling the flipping and shaking of the cell balls during digestion, manually controlling the addition and aspiration of the culture solution and digestion solution, and manually controlling the valve control to complete the continuous and automatic transfer process; fully automatic means embedding the automation system, and fully automatically completing the flipping and shaking of the cell balls during digestion, pumping in or out the culture solution, automatically adding the digestion solution, and controlling the valve control switch, etc.

[0026] (4) The device of the present invention has a simple design and a clear operation process, which can significantly reduce the requirements for operator skills and reduce the possibility of human operating errors.

[0027] (5) In the process of using the cell passaging device or microfluidic device of the present invention, the cell spheres can be more efficiently mixed with the digestion solution by flipping and gently shaking, thereby accelerating the cell digestion speed, making the overall cell passaging process faster, and saving experimental time. Specifically, the device assembly flips and shakes the cell sphere passaging device during the cell passaging and digestion process, so that the cell spheres can be separated from the cell chamber and mixed with the digestion solution to be digested into a single cell suspension. Preferably, the device is flipped in the vertical direction of the bottom, and shaken along the vertical direction of the flip, and the order and number of flipping and shaking are adjusted according to the digestion state of the cell spheres.

[0028] (6) The present invention can be applied to various types of cell spheroids based on the use of a transfer device or a microfluidic device, has broad application prospects, and can meet the research needs of different fields, including but not limited to drug screening, regenerative medicine, and tissue engineering.

[0029] In summary, the device of the present invention not only improves the overall efficiency and reliability of cell culture, but also provides more effective technical support for biomedical research, and has broad application prospects and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure is a schematic diagram of the structure of a cell spheroid passaging device according to an example of the present invention.

[0031] Figure 2 A flow chart of a low-loss cell spheroid passaging method exemplified by the present invention.

[0032] Figure 3 is a schematic structural diagram of a microfluidic device of the present invention, wherein Figure 3 (a) is an exploded view of the microfluidic device, (b) is a plan view of the microfluidic device, and (c) is a cross-sectional view of the microfluidic device.

[0033] Throughout the drawings, the same reference numerals are used to refer to the same structures, wherein:

[0034] 1. Cell ball forming bottom plate; 2. In the chamber of the cell transfer device; 3. Flow channel; 4. Top cover; 5. Rubber ring; 6. Sample inlet or liquid inlet; 7. Simplified module of the cell ball transfer device; 8. Valve control; 9. Mixing channel; 10. Sample outlet; 11. Fluid channel; 12. Secondary liquid outlet. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions 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 specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0036] In a first aspect of the present invention, Figure 1 As shown, a low-loss cell spheroid passage device is provided, comprising an open cavity and a top cover that forms a sealed environment when combined with the open cavity. A cell spheroid formation base plate is provided at the bottom of the open cavity, which is composed of a plurality of cell chambers arranged in an array; and the material of the open cavity is a visible material.

[0037] The following is an example of the steps for making a low-loss cell spheroid passaging device:

[0038] (1) First, prepare a 1mm thick polymethyl methacrylate sheet, peel off the film on both sides, and use a laser scanning relief machine to scan the Figure 1 The microwell array structure shown;

[0039] (2) Prepare another piece of 2mm polymethyl methacrylate sheet, peel off the film on one side, stick it with special double-sided tape, and use a laser engraving machine to engrave it as follows Figure 1 The structure shown in , serves as a culture medium storage chamber;

[0040] (3) aligning the engraved culture medium storage chamber with the micropore array structure and bonding them through the double-sided adhesive layer at the bottom of the culture medium storage chamber;

[0041] (4) Put the bonded whole structure into a hot press together, with the upper and lower pressing surfaces at 60°C for 5 minutes;

[0042] (5) After hot pressing, wait for the temperature to return to room temperature, place it in pure water for 24 hours, and blow it dry with a nitrogen gun.

[0043] Based on the low-loss cell spheroid passaging device provided above, the digestion and passaging of cells are completed by flipping and shaking. Figure 2 As shown, the following steps are included:

[0044] A cell spheroid passaging device is prepared according to the preparation method described above, and the cell spheroids are cultured until they can be passed on; after the culture medium is aspirated, an appropriate amount of digestion solution is continued to be added to the culture module of the passaging device, and then the top cover is closed; the entire device is turned over and shaken until the cell spheroids are completely detached from the cell chamber and mixed with the digestion solution to digest the cell spheroids; a stop solution is added to the device, and the formed single cell suspension is added to other passaging devices, thereby completing the passaging of the cell spheroids.

[0045] In the cell ball passage device of the present invention, the culture medium storage chamber has a certain height, and one or more flow channels are provided on the inner wall from the top of the cavity to the bottom. The flow channel is open or channel-type, and is used to introduce the culture medium, digestion solution or stop solution from the top of the cavity to the bottom of the cavity through the flow channel to avoid damaging the cells in the cavity.

[0046] In the cell spheroid passage device of the present invention, a rubber ring is arranged inside the top cover, which can be tightly combined with the culture module in a reversible manner, thereby preventing liquid leakage during operation.

[0047] In the cell sphere passaging device of the present invention, the cell chamber is selected from a patterned microwell array or a low-absorption microwell plate.

[0048] According to the low-loss cell sphere passaging device provided above, a manual cell sphere passaging method is provided. The use of the device is specifically described below in conjunction with breast tumor sphere passaging. The manufacturing steps and parameters of the cell sphere passaging device are described in detail above and will not be repeated here. The method of using the device specifically includes the following:

[0049] (1) Prepare 500 μL of breast tumor sphere cell suspension with 50 cells per microwell in the microwell array and add it to the cell sphere passaging device and culture it for 3 days until it is ready for passaging.

[0050] (2) The culture medium was aspirated from the flow channel on the inner wall of the chamber, 500 μL of digestion solution was added, and then the top cover was closed.

[0051] (3) The entire device is turned over and gently shaken, and the cell spheroids in the microwell array are effectively transferred to the digestion solution. The mixing of the digestion solution and the cell spheroids promotes cell digestion.

[0052] (4) After 2 minutes, add stop solution to terminate the digestion process.

[0053] (5) The single-cell suspension is added to several other cell culture devices. After settling for 10 min, the culture medium is replaced from the flow channel to complete the cell culture.

[0054] In a second aspect of the present invention, a microfluidic device for cell spheroid passage is provided, comprising a plurality of cell spheroid passage devices as described in the first aspect and a plurality of valve controls. The plurality of cell spheroid passage devices are arranged in n layers, and the number of cell spheroid passage devices arranged in the i-th layer is A. i =2 i-1 , and the j-th cell ball passage device in the i-th layer is connected to the (2j-1)-th and 2j-th cell ball passage devices in the i+1-th layer through valve control; wherein n is the number of layers in which the cell ball passage devices are arranged, Ai is the number of cell ball passage devices corresponding to the i-th layer, 1≤i≤n, 1≤j≤2 i-1 .

[0055] Based on the cell spheroid passaging microfluidic device provided above, the cell spheroid passaging device can be deformed as a single module, and connected in parallel or in series using microfluidic technology or fluid channels to form a passaging chip such as Figure 3 As shown in (a) and (b), semi-automatic operation is achieved by manual flipping, wherein the last serial unit sealing layer of the chip is removed, and the chip can be directly subjected to, including but not limited to, drug sensitivity testing, toxicity testing, and biological mechanism research.

[0056] Specifically, the digestion and passaging of cells are completed by flipping and shaking. Specifically, the following steps are included: prepare a cell ball passaging device according to the preparation method described above, and assemble it into a cell ball passaging microfluidic device according to the above arrangement through valve control; add the single cell suspension into the cell chamber of the cell ball passaging device of the first layer for passaging, and then, in a progressive order from the first layer to the nth layer, open the valve control on the cell ball passaging device of the i-th layer, distribute the single cell suspension after the i-th passaging of the i-th layer to the cell ball passaging device of the i+1th layer, and perform the i+1th passaging according to the above passaging steps until the nth passaging is completed;

[0057] Among them, the specific passaging is as follows: adding a single cell suspension into the cell chamber in the cell ball passaging device, and culturing until a cell ball can be passaged; after aspirating the culture medium, continue to add a digestion solution into the cell ball passaging device, then cover the top cover, flip and shake the cell ball passaging device until the cell ball falls off from the cell chamber and mixes with the digestion solution to digest the cell ball; adding a stop solution into the cell ball passaging device to complete the passaging of the cell ball and form a single cell suspension after passaging.

[0058] In the above method, except for the first passaging process corresponding to the first layer, only one cell ball passaging device in the first layer can be flipped and shaken to digest the cell ball. i The cell spheroid passage devices can be operated by selecting one or more of them. The digestion and passage operations of the cell spheroids in the multiple cell spheroid passage devices do not need to be performed synchronously.

[0059] In the operation of the cell spheroid passaging microfluidic device, the cell spheroid passaging device is flipped and shaken, specifically:

[0060] The cell spheroid passaging device was flipped in the vertical direction at the bottom, and shaken in the vertical direction of flipping, and the flipping and shaking times were adjusted according to the digestion status of the cell spheroids.

[0061] In the cell spheroid passaging microfluidic device of the present invention, the microfluidic device also includes a flipping mechanism connected to each cell spheroid passaging device for controlling the cell spheroid passaging device to flip and shake, thereby achieving semi-automatic digestion and passaging operations.

[0062] In the cell spheroid passage microfluidic device of the present invention, each cell spheroid passage device is provided with a liquid inlet and a liquid outlet, which are respectively connected to a liquid pump, and the liquid is pumped into the liquid inlet and discharged from the liquid outlet to complete the addition and discharge of the culture liquid, the digestion liquid and the stop liquid. Figure 3 In the structural design, all the liquid outlets can be collected into one outflow pipe through a hose and discharged from one liquid outlet.

[0063] The cell spheroid passage microfluidic device of the present invention can be integrated into an automated passage system to achieve fully automated passage operation. For example, the automated system is electrically connected to the flip mechanism and valve control, and can be used for a corresponding fully automated passage process; the automated passage system integrates a temperature control module, a carbon dioxide valve, and a UV sterilization system, which further optimizes the environment for cell spheroid passage.

[0064] Specifically, the following examples illustrate two passaging methods, namely, a method in which a spheroid passaging device is integrated into a microfluidic device to realize semi-automatic passaging as a passaging chip, and a fully automated method.

[0065] A low-loss cell spheroid passaging device is integrated into a cell spheroid passaging microfluidic device to achieve a semi-automatic passaging method, wherein the method for making the cell passaging device is not described in detail. Figure 3 As shown in the figure, this method is suitable for semi-automated expansion of cell spheroids. The specific steps are as follows:

[0066] (1) Prepare 500 μL of breast tumor sphere cell suspension with 50 cells per microwell in the microwell array, add it to the liquid inlet / sample inlet of the cell sphere passaging microfluidic device, and pass it into the first layer of the cell sphere passaging device for 3 days until it is ready for passaging.

[0067] (2) Aspirate or drain the culture medium in the first layer of the cell spheroid passaging device, and continue to add 500 μL of digestion solution to the first layer of the cell spheroid passaging device.

[0068] (3) The entire cell spheroid passaging device is turned over and gently shaken. The cell spheroids in the microwell array are effectively transferred to the channel, and the mixing of the digestion solution and the cell spheroids promotes cell digestion.

[0069] (4) After 2 minutes, add the stop solution, open the valve control on the first layer of the cell spheroid passaging device, mix the cell suspension and the newly introduced stop solution in the mixing channel 4, and then enter the two parallel cell spheroid passaging devices. After settling for 10 minutes, adjust the valve control channel, discharge the digestion solution and stop solution from the outlet, and introduce culture medium for cultivation.

[0070] (5) The subsequent transfer operations are the same as (2)(3)(4), and the valve controls are opened in sequence.

[0071] A low-loss cell spheroid passaging device is integrated into a cell spheroid passaging microfluidic device to achieve a fully automatic passaging method, wherein the manufacturing method of the cell passaging device is not described in detail. The method of fully automated cell expansion culture is as follows:

[0072] (1) Place the cell spheroid passage microfluidic device into an automated device, and connect the centrifuge tube containing the cell suspension, the culture medium, the digestion solution, and the stop solution to the fluid channel of the device.

[0073] (2) After entering the chamber, the pipeline is automatically connected to the liquid inlet and outlet of the cell ball passage microfluidic device, the valve control device is connected to the valve of the microfluidic device, and the hatch is connected to the flip system.

[0074] (3) All operations of the semi-automatic cell culture method are completed by the device through the microfluidic device to obtain a large number of highly uniform cell spheroids, which can be led out through the liquid outlet at the end, and subsequent operations can also be performed directly on the microfluidic device.

[0075] The device and method of the present invention are applicable to the subculture in synthetic biology including but not limited to organoid culture, stem cell culture, cell therapy, and cell production, and the subculture includes but is not limited to one-to-one, one-to-two, and one-to-many.

[0076] The cell sphere passaging operation is applicable to cell spheres and organoids including but not limited to those formed by immortalized cell lines, the cell spheres include but are not limited to breast, gastrointestinal tract, liver, kidney, lung and other tissue sources, species include but are not limited to mice, rats, monkeys, humans, and sources include but are not limited to normal or pathological.

[0077] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the present invention and its equivalent technologies, the present invention is also intended to include these changes and variations. The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and their protection scope is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are all within the protection scope of the present invention.

Claims

1. A low-loss cell spheroid passaging device, characterized in that: It includes an open cavity and a top cover, and the open cavity and the top cover are connected to form a sealed environment; a cell ball forming bottom plate is provided at the bottom of the open cavity, which is composed of a plurality of cell chambers arranged in an array, and the material of the open cavity is a visible material.

2. The cell spheroid passaging device according to claim 1, characterized in that: A flow channel extending from the top of the open cavity to the bottom is arranged on the inner wall of the open cavity.

3. The cell spheroid passaging device according to claim 1, characterized in that: A rubber ring is arranged inside the top cover so as to be reversibly combined with the open cavity.

4. The cell spheroid passaging device according to claim 1, characterized in that: The cell chamber is a patterned microwell array or a low-absorption microwell plate.

5. A microfluidic device for cell spheroid passage, characterized in that: comprising a plurality of cell ball passage devices according to any one of claims 1 to 4 and a plurality of valve controls; the plurality of cell ball passage devices are arranged in n layers, and the number of cell ball passage devices arranged in the i-th layer is A i =2 i-1 , and the j-th cell ball passage device in the i-th layer is connected to the (2j-1)-th and 2j-th cell ball passage devices in the i+1-th layer through the valve control; wherein n is the number of layers in which the cell ball passage devices are arranged, Ai is the number of cell ball passage devices corresponding to the i-th layer, 1≤i≤n, 1≤j≤2 i-1 .

6. The cell spheroid passage microfluidic device according to claim 5, characterized in that: The microfluidic device further comprises a flipping mechanism connected to each of the cell ball passage devices and used for controlling the cell ball passage devices to flip.

7. The cell spheroid passage microfluidic device according to claim 5, characterized in that: Each cell ball passage device in the microfluidic device is provided with a liquid inlet and a liquid outlet, which are respectively connected to a liquid pump.

8. A cell sphere passaging method, characterized in that: The cell spheroid passaging device according to any one of claims 1 to 4 comprises the following steps: (1) adding a single cell suspension into a cell chamber in a cell spheroid passaging device and culturing the cell spheroids to be passable; (2) After the culture medium is aspirated, a digestion solution is continuously added to the cell spheroid passaging device, and then the top cover is closed, and the cell spheroid passaging device is turned over and shaken until the cell spheroids fall out of the cell chamber and mix with the digestion solution to digest the cell spheroids; (3) Adding stop solution to the cell spheroid passaging device to complete the passaging of the cell spheroid.

9. A cell sphere passaging method, characterized in that: The cell spheroid passaging microfluidic device according to any one of claims 5 to 7 comprises: after adding a single cell suspension into a cell chamber in a cell spheroid passaging device of a first layer for passaging operation, in a progressive order from the first layer to the nth layer, by opening the valve control on the cell spheroid passaging device of the i-th layer, the single cell suspension after the i-th passaging operation in the i-th layer is distributed to the cell spheroid passaging device of the i+1th layer, and the i+1th passaging is performed according to the passaging operation until the nth passaging is completed; The passaging operation is specifically as follows: adding a single cell suspension into a cell chamber in a cell ball passaging device, and culturing until a cell ball can be passed; after aspirating the culture medium, continuing to add a digestion solution into the cell ball passaging device, then covering the top cover, flipping and shaking the cell ball passaging device until the cell ball falls off from the cell chamber and mixes with the digestion solution to digest the cell ball; adding a stop solution into the cell ball passaging device to complete the passaging of the cell ball and form a single cell suspension after passaging.

10. The cell sphere passaging method according to claim 8 or 9, characterized in that: Invert and shake the spheroid passaging apparatus by: Flipping the cell spheroid passaging device in a vertical direction at the bottom thereof, and shaking the cell spheroid in the vertical direction thereof, and adjusting the number of flipping and shaking according to the digestion state of the cell spheroid; Alternatively, the cell spheroid passaging device is controlled to flip in the vertical direction of the bottom by a flipping mechanism connected to the cell spheroid passaging device, and to shake along the vertical direction of the flipping, and the number of flipping and shaking is adjusted according to the digestion status of the cell spheroid.

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