Cell culture method

Abstract

The invention relates to the field of cells, in particular to a cell culture method which comprises the following steps: S1, taking out a cryopreservation tube from liquid nitrogen; s2, quickly putting the mixture into a water bath, and shaking the mixture from time to time to quickly melt the mixture; s3, wiping and disinfecting a cryopreservation tube with alcohol, opening a cover, injecting the cell suspension into a centrifugal tube by using a suction tube, dropwise adding a culture solution, and carrying out low-speed centrifugation; s4, pouring out the supernate, and adding a culture solution to suspend the cells; s5, sucking the cells into a culture dish filled with a culture solution, and slightly shaking all around, so that the cells in the culture dish are uniformly distributed; s6, placing the culture dish in an incubator at 37 DEG C for culturing; and S7, during culture, replacing a culture solution in the culture dish through a solution replacement mechanism arranged on the culture box in a closed state of the culture box. According to the invention, the culture solution of the culture dish can be replaced when the culture box is closed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cells, in particular to a cell culture method. BACKGROUND

[0002] Cell culture is also called cell cloning technology, and its formal name in biology is cell culture technology. Cell culture is an essential process for the entire biological engineering technology, as well as the biological cloning technology, and cell culture itself is a large-scale cloning of cells. Cell culture technology can make a cell become a simple single cell or a few differentiated multicellular through a large number of culture, which is an essential link of cloning technology, and cell culture itself is cell cloning. A large number of cells or their metabolites are obtained through cell culture. Because biological products are obtained from cells, it can be said that cell culture technology is the most core and basic technology in biotechnology.

[0003] In cell culture, the cell culture solution needs to be replaced, but in the existing culture method, when the culture solution is replaced, the culture dish needs to be taken out of the incubator, and then replaced, so as to change the cell culture environment and increase the risk of cell infection. SUMMARY

[0004] The purpose of the present application is to provide a cell culture method which can replace the culture solution of the culture dish in the closed state of the incubator.

[0005] The purpose of the present application is achieved by the following technical solutions:

[0006] A cell culture method comprises the following steps:

[0007] S1. Taking the cryopreservation tube out of liquid nitrogen;

[0008] S2. Quickly placing it in a water bath and shaking it from time to time to make it melt rapidly;

[0009] S3. After the cryopreservation tube is disinfected with alcohol, the cover is opened, the cell suspension is injected into the centrifugal tube with a pipette, and the culture solution is added dropwise for low-speed centrifugation;

[0010] S4. Discard the supernatant and add culture solution to suspend the cells;

[0011] S5. Suck the cells into the culture dish containing the culture solution and shake it gently to make the cells in the culture dish evenly distributed;

[0012] S6. Placing the culture dish in a 37℃ incubator for culture;

[0013] S7. Replacing the culture solution of the culture dish in the closed state of the incubator through the liquid replacement mechanism provided on the incubator during culture.

[0014] Preferably, in the step S2, the water bath temperature is 36-37℃.

[0015] Preferably, in the step S2, the melting operation is completed within 30-80s.

[0016] Preferably, in the step S3, the alcohol used is 70% alcohol.

[0017] Preferably, in the step S3, the low-speed centrifugation speed is 500-1000r / min.

[0018] Preferably, in the step S3, the low-speed centrifugation time is 5min.

[0019] Preferably, in the step S6, the temperature in the incubator is 37℃.

[0020] Preferably, in the step S6, the incubator contains 5% carbon dioxide.

[0021] Preferably, in the step S7, the culture medium is replaced every 2-3 days according to the cell growth rate.

[0022] Preferably, the incubator is provided with a camera. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a flowchart of embodiment one of a cell culture method;

[0024] Figure 2 is a flowchart of embodiment two of a cell culture method;

[0025] Figure 3 is a structure diagram of the incubator closed;

[0026] Figure 4 and Figure 5 is a structure diagram of the incubator opened;

[0027] Figure 6 is a structure diagram of the incubator;

[0028] Figure 7 is a structure diagram of the supporting plate;

[0029] Figure 8 is a partial structure diagram of the medium replacement mechanism;

[0030] Figure 9 is a partial sectional structure diagram of Figure 8 ;

[0031] Figure 10 is a structure diagram of the front cover;

[0032] Figure 11 This is a structural diagram of the locking frame.

[0033] In the picture:

[0034] 101; 102; 103; 104; 105;

[0035] Pallet 201; tray 202; control screw 203;

[0036] Sliding seat 301; L-shaped thin tube 302; threaded tube 303; screw I 304; screw II 305; baffle 306; connecting groove 307;

[0037] Rotating bracket 401; front cover 402; hook plate 403; edge 404; locking bracket 405; spring 406. Detailed Implementation

[0038] like Figure 1 As shown, here is an example of a cell culture method:

[0039] A cell culture method, comprising the following steps:

[0040] S1. Remove the cryovials from the liquid nitrogen;

[0041] S2. Quickly place it in a water bath and shake it occasionally to melt it rapidly;

[0042] S3. After wiping the cryopreservation tube with alcohol for disinfection, open the cap, use a pipette to inject the cell suspension into the centrifuge tube, add culture medium, and centrifuge at low speed.

[0043] S4. Discard the supernatant and add culture medium to suspend the cells;

[0044] S5. Aspirate the cells into a culture dish containing culture medium and gently shake it back and forth and side to side to distribute the cells evenly in the culture dish.

[0045] S6. Place the petri dish in an incubator at 37°C for incubation;

[0046] S7. During cultivation, the culture medium is replaced in the culture dish using the medium exchange mechanism set on the incubator while the incubator is closed.

[0047] In step S2, the water bath temperature is 36℃-37℃.

[0048] In step S2, the melting operation is completed within 30-80 seconds.

[0049] In step S3, the alcohol used is 70% alcohol.

[0050] In step S3, the low-speed centrifugation speed is 500 r / min.

[0051] In step S3, the low-speed centrifugation time is 5 minutes.

[0052] In step S6, the temperature inside the incubator is 37°C.

[0053] In step S6, the incubator contains 5% carbon dioxide.

[0054] During the S7 culturing process, the culture medium should be changed every 2-3 days depending on the cell growth rate, generally after the cells have adhered to the culture medium.

[0055] The incubator is equipped with a camera.

[0056] like Figure 2 As shown, here is an example of a cell culture method:

[0057] A cell culture method, comprising the following steps:

[0058] S1. Remove the cryovials from the liquid nitrogen;

[0059] S2. Quickly place it in a water bath and shake it occasionally to melt it rapidly;

[0060] S3. After wiping the cryopreservation tube with alcohol for disinfection, open the cap, use a pipette to inject the cell suspension into the centrifuge tube, add culture medium, and centrifuge at low speed.

[0061] S4. Discard the supernatant and add culture medium to suspend the cells;

[0062] S5. Aspirate the cells into a culture dish containing culture medium and gently shake it back and forth and side to side to distribute the cells evenly in the culture dish.

[0063] S6. Place the petri dish in an incubator at 37°C for incubation;

[0064] S7. During cultivation, the culture medium is replaced in the culture dish using the medium exchange mechanism set on the incubator while the incubator is closed.

[0065] In step S2, the water bath temperature is 36℃-37℃.

[0066] In step S2, the melting operation is completed within 30-80 seconds.

[0067] In step S3, the alcohol used is 70% alcohol.

[0068] In step S3, the low-speed centrifugation speed is 1000 r / min.

[0069] In step S3, the low-speed centrifugation time is 5 minutes.

[0070] In step S6, the temperature inside the incubator is 37°C.

[0071] In step S6, the incubator contains 5% carbon dioxide.

[0072] During the S7 culturing process, the culture medium should be changed every 2-3 days depending on the cell growth rate, generally after the cells have adhered to the culture medium.

[0073] The incubator is equipped with a camera.

[0074] like Figures 3-11 As shown:

[0075] The incubator includes a box body 101, a camera 103, a tray 201, and a front cover 402. The box body 101 has an opening on the front side, a tray 201 at the lower end of the box body 101, a camera 103 at the middle of the upper end of the box body 101, and a front cover 402 at the opening of the box body 101.

[0076] During cultivation, the culture dish is placed on the tray 201, and then the front cover 402 is used to seal the opening on the front side of the chamber 101. The environment inside the chamber 101 is then adjusted to 37°C and 5% carbon dioxide. The cells in the culture dish are recorded by the camera 103, so that the cells can be observed on the monitor.

[0077] The incubator also includes a side seat 104, a rotating frame 401, a hook plate 403, a side edge 404, a locking frame 405, and a spring 406; the side seat 104 is provided on one side of the box body 101, the rotating frame 401 rotates on the side seat 104, the front cover 402 slides through the rotating frame 401, one end of the front cover 402 is provided with a side edge 404, the other end of the front cover 402 is fixed with a hook plate 403, the upper end of the box body 101 is slidably provided with a locking frame 405, and a spring 406 is provided between the locking frame 405 and the box body 101.

[0078] When sealing the opening on the front side of the box 101, pull the hook plate 403, so that the hook plate 403 drives the rotating frame 401 to rotate on the side seat 104 through the front cover 402 until the front cover 402 is attached to the opening of the box 101 to seal the opening. At this time, the hook edge of the hook plate 403 is locked on the locking frame 405, and the spring force of the spring 406 makes the locking frame 405 press against the hook edge of the hook plate 403 to form a lock on the front cover 402. At the same time, the edge 404 on the front cover 402 presses against the rotating frame 401 to ensure that the hook plate 403 on the front cover 402 and the locking frame 405 limit each other, thus completing the locking of the front cover 402.

[0079] When opening, the locking bracket 405 is pulled to overcome the elastic force of the spring 406 and slide, so that the locking bracket 405 is separated from the hook plate 403. Then the hook plate 403 is pulled to open the front cover 402. At the same time, when opening, the front cover 402 can slide inside the locking bracket 405, thereby changing the rotation trajectory of the front cover 402, making it easier to open the front cover 402.

[0080] The fluid exchange mechanism includes a fixed tube 102, a sliding seat 301, an L-shaped thin tube 302, a threaded tube 303, a screw I 304, a screw II 305, and a connecting groove 307. The fixed tube 102 is fixed to the side of the housing 101. The sliding seat 301 slides through the fixed tube 102. The L-shaped thin tube 302 slides through the sliding seat 301. The outer end of the sliding seat 301 is provided with a connecting groove 307, which communicates with the L-shaped thin tube 302. The threaded tube 303 is fixed on the L-shaped thin tube 302 and is slidably connected to the sliding seat 301. The screw I 304 rotates on the side of the housing 101 and is threadedly connected to the sliding seat 301. The screw II 305 rotates on the sliding seat 301 and is threadedly connected to the threaded tube 303.

[0081] A baffle 306 rotates on the screw II 305, a tray 202 rotates on the support plate 201, and two control screws 203 rotate through the housing 101, both of which are threadedly connected to the support plate 201.

[0082] When changing the culture medium, the first motor drives two control screws 203 to rotate simultaneously. This causes the threaded drive plate 201 to rise and fall within the chamber 101, thereby raising and lowering the culture dish. The lower end of the L-shaped capillary tube 302 is then inserted into the culture dish. By connecting the end of the pipette to the connecting groove 307, waste liquid in the culture dish is aspirated. New culture medium is then added to the culture dish through the L-shaped capillary tube 302. This allows for culture medium replacement without opening the incubator, ensuring a suitable environment within the incubator, and preventing cell contamination and environmental changes from affecting cell culture.

[0083] Furthermore, by rotating the screw II 305, the threaded drive tube 303 drives the L-shaped capillary tube 302 to slide within the sliding seat 301, thereby adjusting the lower end position of the L-shaped capillary tube 302 laterally, ensuring that the lower end of the L-shaped capillary tube 302 contacts the area with fewer cells, and thus aspirating the waste liquid; at the same time, it avoids the lower end of the L-shaped capillary tube 302 from interfering with the camera 103's recording.

[0084] Meanwhile, by rotating the screw I 304, the threaded transmission sliding seat 301 slides inside the fixed tube 102, which in turn drives the L-shaped thin tube 302 to move again, increasing the adjustment range of the lower end position of the L-shaped thin tube 302 and enhancing the adaptability of the incubator;

[0085] Furthermore, by activating the second motor installed on the tray 201, the tray 202 is driven to rotate, thereby allowing the lower end of the L-shaped tube 302 to reach any position on the culture dish.

Claims

1. A cell culture method, characterized in that: Includes the following steps: S1. Remove the cryovials from the liquid nitrogen; S2. Quickly place it in a water bath and shake it occasionally to melt it rapidly; S3. After wiping the cryopreservation tube with alcohol for disinfection, open the cap, use a pipette to inject the cell suspension into the centrifuge tube, add culture medium, and centrifuge at low speed. S4. Discard the supernatant and add culture medium to suspend the cells; S5. Aspirate the cells into a culture dish containing culture medium and gently shake it back and forth and side to side to distribute the cells evenly in the culture dish. S6. Place the petri dish in an incubator for incubation; S7. During cultivation, the culture medium is replaced in the culture dish using the medium exchange mechanism set on the incubator while the incubator is closed.

2. The cell culture method according to claim 1, characterized in that: In step S2, the water bath temperature is 36℃-37℃.

3. The cell culture method according to claim 1, characterized in that: In step S2, the melting operation is completed within 30-80 seconds.

4. The cell culture method according to claim 1, characterized in that: In step S3, the alcohol used is 70% alcohol.

5. The cell culture method according to claim 1, characterized in that: In step S3, the low-speed centrifugation speed is 500-1000 r / min.

6. The cell culture method according to claim 1, characterized in that: In step S3, the low-speed centrifugation time is 5 minutes.

7. The cell culture method according to claim 1, characterized in that: In step S6, the temperature inside the incubator is 37°C.

8. A cell culture method according to claim 1, characterized in that: In step S6, the incubator contains 5% carbon dioxide.

9. A cell culture method according to claim 1, characterized in that: During the S7 culturing process, the culture medium should be changed every 2-3 days depending on the cell growth rate.

10. A cell culture method according to claim 1, characterized in that: The incubator is equipped with a camera (103).

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