Digestion method, passage method and cryopreservation method of mouse mononuclear macrophages

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a digestion method, a passage method and a cryopreservation method of mouse mononuclear macrophages, and the method comprises the following steps: adding a phosphate buffer solution with the temperature of 1-4 DEG C into a culture for culturing the mouse mononuclear macrophages for digestion; digesting at the temperature of 1 to 4 DEG C for 5 to 10 minutes; the concentration of the phosphate buffer solution is 0.01 M to 0.015 M; the pH value of the phosphate buffer solution is 7.2-7.4. According to the method disclosed by the invention, the success rate of RAW cell passage and cryopreservation is obviously improved, the possibility of cell differentiation, denaturation, rupture and the like caused by pancreatin digestion, cell scraping and the like is reduced, and the efficiency of RAW cell experiments is effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a digestion method, a passage method and a cryopreservation method for mouse mononuclear macrophages. Background Art

[0002] Mouse mononuclear macrophages, referred to as RAW cells, are slow-heating cells and are difficult to digest. The existing digestion methods are as follows: Trypsin digestion: 1. Taking T25 culture flasks as an example, add 1mL to 2mL of trypsin and EDTA digestion solution, digest at 37℃ for about 2min to 5min, and stop digestion when the cells are basically detached under the microscope, and collect the digested cells; because the bottom cells generally have pseudopodia, they are tightly attached to the flask wall and are more difficult to digest. During digestion, the upper cells can be collected first, and then the bottom cells can be digested. The cells digested each time cannot be placed in the same flask. 2. Add PBS buffer to rinse twice, add 1mL to 2mL of trypsin and EDTA digestion solution, digest at 37℃ for about 2min to 5min, continue to digest the bottom cells, and stop digestion when the cells are basically detached under the microscope, and collect the digested cells separately. There are multiple digestions, cell chemical damage and differentiation occur, and the cells are slow to attach to the wall again, and rapid growth is delayed. Cell scraper: Wash with PBS buffer, and scrape the cells with a cell knife in 5mL PBS buffer. This method causes physical damage, which may cause cell rupture. If it is too light, the scraping may fail and cells may be lost; if it is too severe, the cells may be damaged.

[0003] If the digestion time is not enough, the cells cannot be digested. If the digestion time is too long, the cells will grow a corner after passage, that is, the cells will differentiate. In addition, the existing digestion method uses strong blowing and cell knife scraping, which will not only grow a corner after passage, but also cause many small black spots to float, that is, cell fragmentation. In addition, the inoculation density of such cells should be high during passage, otherwise it is easy to cause macrophage differentiation, cell deformation, and irreversible failure. This is a problem encountered during the culture process. In summary, the existing digestion method will cause cell differentiation and cell fragmentation after passage. Therefore, a gentle digestion method is needed to quickly digest cells and avoid cell differentiation and damage. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for digesting, passage and freezing mouse mononuclear macrophages.

[0005] A method for digesting mouse mononuclear macrophages, the method comprising adding a phosphate buffer at 1°C to 4°C to a culture of mouse mononuclear macrophages for digestion;

[0006] Digest at 1℃~4℃ for 5min~10min;

[0007] The concentration of the phosphate buffer is 0.01M to 0.015M;

[0008] The pH of the phosphate buffer is 7.2-7.4.

[0009] The present invention utilizes the flushing properties of PBS buffer and the low-temperature shrinkage properties of RAW cells to cause the cells to fall off from the culture dish / bottle, thereby reducing the possibility of cell differentiation, degeneration, rupture, etc. caused by trypsin digestion, cell knife scraping, etc.

[0010] Preferably, the amount of the phosphate buffer used is no more than the amount of the culture medium for culturing the mouse mononuclear macrophages.

[0011] Preferably, digestion is performed when the growth density of mouse mononuclear macrophages in the culture reaches 80% to 90%.

[0012] Preferably, during digestion, when 70% to 80% of mouse mononuclear macrophages shrink and become round and the intercellular spaces become larger, the cells are beaten to fall off.

[0013] Preferably, the mouse mononuclear macrophages are RAW264.7.

[0014] A method for subculturing mouse mononuclear macrophages comprises digesting mouse mononuclear macrophages using the method for digesting mouse mononuclear macrophages, collecting cell suspensions, performing centrifugation, retaining cell sediments, adding culture fluid to resuspend cells to obtain new cell suspensions, and inoculating the new cell suspensions into new cultures for culturing.

[0015] Preferably, centrifugation is performed for 3 to 5 minutes.

[0016] A method for freezing mouse mononuclear macrophages comprises the following steps: using the method for digesting mouse mononuclear macrophages to digest mouse mononuclear macrophages, collecting cell suspension, performing centrifugation, retaining cell sediment, adding freezing solution to resuspend, packaging into sterile freezing vessels, and storing in a freezing environment.

[0017] Preferably, centrifugation is performed for 3 to 5 minutes.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The method of the present invention significantly improves the success rate of RAW cell passage and cryopreservation, reduces the possibility of cell differentiation, degeneration, rupture, etc. caused by trypsin digestion, cell scraping, etc., effectively improves the efficiency of RAW cell experiments, and reduces a large amount of manpower and financial resources wasted due to cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The figure is a flow chart of the digestion method of the present invention.

[0021] Figure 2 These are the cell growth curves of four different digestion methods.

[0022] Figure 3 The cells grow over time with different digestion methods and the appearance under the microscope.

[0023] Figure 4 The proliferation of cck-8 cells in four different digestion methods. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0025] PBS buffer is usually used for rinsing in cell culture to remove some growth factors secreted by cells, to remove accumulated metabolites, and even to wash off some cells that are in poor growth state or aging. The present invention utilizes the flushing properties of PBS buffer and the low temperature shrinkage properties of RAW cells to make the cells fall off from the culture dish / bottle, such as Figure 1 shown.

[0026] The phosphate buffer used in the present invention is the phosphate buffer BL601A (Phosphate Buffered Saline, PBS buffer for short) of biosharp Company, which is a commonly used buffer solution for biological research. It is a water-based salt solution containing sodium chloride and phosphate, and its main components are 137mM sodium chloride, 10mM disodium hydrogen phosphate, 1.76mM potassium dihydrogen phosphate, and 2.7mM potassium chloride. The PBS buffer helps to maintain a constant pH value, and its osmotic pressure and ion concentration are usually close to the pH of the human body (isotonic). The pH of this product is 7.2-7.4, and the concentration after dissolution is 0.01M.

[0027] The main inventive concept of the present invention is to provide a method for digesting mouse mononuclear macrophages, comprising the following steps:

[0028] (1) Mouse mononuclear macrophages were cultured in a P75 bottle. When the cell growth density reached 80%-90%, the culture medium in the P75 bottle was discarded, and PBS buffer was added to wash 1-2 times. The bottle was gently shaken and then discarded.

[0029] (2) Add 4°C PBS buffer to the P75 bottle, which is usually slightly less than the culture medium. Place the P75 bottle in a 4°C refrigerator for digestion. After 5 to 10 minutes, take it out and observe it under an inverted microscope. When 70% to 80% of the cells are found to shrink and become round and the intercellular spaces are enlarged, tap the culture bottle to make the remaining cells fall off.

[0030] The above method can reduce the cell differentiation, degeneration and rupture caused by trypsin digestion, cell scraping and the like, and specifically includes the following embodiments:

[0031] Example 1

[0032] A method for digesting mouse mononuclear macrophages comprises the following steps:

[0033] (1) RAW 264.7 cells were cultured in a P75 bottle. When the cell growth density reached 90%, the culture medium in the P75 bottle was discarded, and PBS buffer was added to wash twice and then discarded.

[0034] (2) Add 10 mL of 4°C phosphate buffer to the P75 bottle and place the P75 bottle in a 4°C refrigerator for digestion. After 7 min, take it out and observe it under an inverted microscope. When 80% of the cells are found to have shrunk and become rounded and the intercellular spaces have become larger, tap the culture bottle to make the cells fall off.

[0035] This method avoids cell differentiation and damage and enters the growth phase earlier.

[0036] Example 2

[0037] A method for digesting mouse mononuclear macrophages comprises the following steps:

[0038] (1) RAW 264.7 cells were cultured in a P75 bottle. When the cell growth density reached 90%, the culture medium in the P75 bottle was discarded, PBS buffer was added for washing once, and then discarded.

[0039] (2) Add 10 mL of 4°C phosphate buffer to the P75 bottle and place the P75 bottle in a 4°C refrigerator for digestion. After 5 minutes, take it out and observe it under an inverted microscope. When 80% of the cells are found to have shrunk and become rounded and the intercellular spaces are enlarged, tap the culture bottle to make the cells fall off.

[0040] This method avoids cell differentiation and damage and enters the growth phase earlier.

[0041] Example 3

[0042] A method for digesting mouse mononuclear macrophages comprises the following steps:

[0043] (1) RAW 264.7 cells were cultured in a P75 bottle. When the cell growth density reached 90%, the culture medium in the P75 bottle was discarded, and PBS buffer was added to wash twice and then discarded.

[0044] (2) Add 10 mL of 4°C phosphate buffer to the P75 bottle, place the P75 bottle in a 4°C refrigerator for digestion, and take it out after 10 minutes for observation under an inverted microscope. When 80% of the cells are found to have shrunk and become rounded and the intercellular spaces are enlarged, tap the culture bottle to make the cells fall off.

[0045] This method avoids cell differentiation and damage and enters the growth phase earlier.

[0046] Based on the digestion method of mouse mononuclear macrophages, the present invention also provides a method for subculturing mouse mononuclear macrophages. During subculturing, when the cell growth density reaches 80% to 90%, subculturing can be performed, which specifically includes the following embodiments:

[0047] Example 4

[0048] The RAW264.7 adherent cell passaging method includes the following steps:

[0049] (1) RAW 264.7 cells were cultured in P75 flasks. Cell morphology and growth density were observed under an inverted microscope before subculturing. Subculturing was performed when the cell growth density reached 80%.

[0050] (2) Discard the culture medium in the P75 bottle, add PBS buffer to wash twice and then discard.

[0051] (3) Add 10 mL of 4°C PBS buffer to the P75 bottle and place the P75 bottle in a 4°C refrigerator for digestion. After 7 min, take it out and observe it under an inverted microscope. When 80% of the cells are found to have shrunk and become rounded and the intercellular spaces have become larger, tap the culture bottle to make the remaining cells fall off.

[0052] (4) Use a pipette tip to gently blow off the detached cells, aspirate all the cell suspension into a centrifuge tube, and centrifuge at 1000 rpm / min for 3 min.

[0053] (5) Discard the supernatant, add culture medium to resuspend the cells, and mix by pipetting to evenly disperse the cells.

[0054] (6) Use a pipette to absorb the cell suspension, inoculate it into a new culture bottle, replenish the culture medium, shake well, and place it in a 37°C, 5% CO2 incubator for culture.

[0055] Example 5

[0056] The RAW264.7 adherent cell passaging method includes the following steps:

[0057] (1) RAW 264.7 cells were cultured in P75 flasks. Cell morphology and growth density were observed under an inverted microscope before subculturing. Subculturing was performed when the cell growth density reached 90%.

[0058] (2) Discard the culture medium in the P75 bottle, add PBS buffer to wash twice and then discard.

[0059] (3) Add 10 mL of 4°C PBS buffer to the P75 bottle and place the P75 bottle in a 4°C refrigerator for digestion. Take it out after 5 minutes and observe it under an inverted microscope. When 80% of the cells are found to have shrunk and become rounded and the intercellular spaces have become larger, tap the culture bottle to make the remaining cells fall off.

[0060] (4) Use a pipette tip to gently blow off the detached cells, aspirate all the cell suspension into a centrifuge tube, and centrifuge at 1000 rpm / min for 5 min.

[0061] (5) Discard the supernatant, add culture medium to resuspend the cells, and mix by pipetting to evenly disperse the cells.

[0062] (6) Use a pipette to absorb the cell suspension, inoculate it into a new culture bottle, replenish the culture medium, shake well, and place it in a 37°C, 5% CO2 incubator for culture.

[0063] Example 6

[0064] The RAW264.7 adherent cell passaging method includes the following steps:

[0065] (1) RAW 264.7 cells were cultured in P75 flasks. Cell morphology and growth density were observed under an inverted microscope before subculturing. Subculturing was performed when the cell growth density reached 80%.

[0066] (2) Discard the culture medium in the P75 bottle, add PBS buffer to wash twice and then discard.

[0067] (3) Add 10 mL of 4°C PBS buffer to the P75 bottle and place the P75 bottle in a 4°C refrigerator for digestion. After 10 min, take it out and observe it under an inverted microscope. When 80% of the cells are found to have shrunk and become rounded and the intercellular spaces have become larger, tap the culture bottle to make the remaining cells fall off.

[0068] (4) Use a pipette tip to gently blow off the detached cells, aspirate all the cell suspension into a centrifuge tube, and centrifuge at 1000 rpm / min for 4 min.

[0069] (5) Discard the supernatant, add culture medium to resuspend the cells, and mix by pipetting to evenly disperse the cells.

[0070] (6) Use a pipette to absorb the cell suspension, inoculate it into a new culture bottle, replenish the culture medium, shake well, and place it in a 37°C, 5% CO2 incubator for culture.

[0071] The cryopreservation of RAW264.7 also requires a digestion process. Based on the digestion method of mouse mononuclear macrophages, the present invention also provides a cryopreservation method of mouse mononuclear macrophages, which specifically includes the following embodiments:

[0072] Example 7

[0073] The steps for freezing RAW264.7 are as follows:

[0074] (1) Cultivate RAW 264.7 in a P75 bottle, select cells in the logarithmic proliferation phase and observe the cell morphology and growth density under an inverted microscope. When the cell growth density reaches 90%, aspirate or pour out the culture medium in the P75 bottle. Add PBS buffer to wash twice and then discard.

[0075] (2) Add 10 mL of 4°C PBS buffer to the P75 bottle and place the P75 bottle in a 4°C refrigerator for digestion. After 7 min, take it out and observe it under an inverted microscope. When 70% of the cells are found to have shrunk and become rounded and the intercellular spaces have become larger, tap the culture bottle to remove the remaining cells and gently blow it with a pipette tip.

[0076] (3) Aspirate all the cell suspension into a centrifuge tube and centrifuge at 1000 rpm / min for 3 min.

[0077] (4) Discard the supernatant, add culture medium to resuspend the cells, and mix by pipetting to evenly disperse the cells.

[0078] (5) After centrifugation, discard the supernatant, add serum-free freezing solution to resuspend and mix well.

[0079] (6) Aliquot into sterile cryovials, 1.5 mL per tube.

[0080] (7) Seal with sealing film, mark, and store in a liquid nitrogen tank.

[0081] Example 8

[0082] The steps for freezing RAW264.7 are as follows:

[0083] (1) Cultivate RAW 264.7 in a P75 bottle, select cells in the logarithmic proliferation phase and observe the cell morphology and growth density under an inverted microscope. When the cell growth density reaches 90%, aspirate or pour out the culture medium in the P75 bottle. Add PBS buffer to wash twice and then discard.

[0084] (2) Add 10 mL of 4°C PBS buffer to the P75 bottle and place the P75 bottle in a 4°C refrigerator for digestion. Take it out after 5 minutes and observe it under an inverted microscope. When 70% of the cells are found to shrink and become round and the intercellular spaces are enlarged, tap the culture bottle to make the remaining cells fall off and gently blow with a pipette tip.

[0085] (3) Aspirate all the cell suspension into a centrifuge tube and centrifuge at 1000 rpm / min for 5 min.

[0086] (4) Discard the supernatant, add culture medium to resuspend the cells, and mix by pipetting to evenly disperse the cells.

[0087] (5) After centrifugation, discard the supernatant, add serum-free freezing solution to resuspend and mix well.

[0088] (6) Aliquot into sterile cryovials, 1.5 mL per tube.

[0089] (7) Seal the tube with sealing film, label it, and store it in a -80℃ refrigerator.

[0090] Example 9

[0091] The steps for freezing RAW264.7 are as follows:

[0092] (1) Cultivate RAW 264.7 in a P75 bottle, select cells in the logarithmic proliferation phase and observe the cell morphology and growth density under an inverted microscope. When the cell growth density reaches 90%, aspirate or pour out the culture medium in the P75 bottle. Add PBS buffer to wash twice and then discard.

[0093] (2) Add 10 mL of 4°C PBS buffer to the P75 bottle, place the P75 bottle in a 4°C refrigerator for digestion, and take it out after 10 minutes for observation under an inverted microscope. When 70% of the cells are found to have shrunk and become rounded and the intercellular spaces have become larger, tap the culture bottle to remove the remaining cells and gently blow with a pipette tip.

[0094] (3) Aspirate all the cell suspension into a centrifuge tube and centrifuge at 1000 rpm / min for 4 min.

[0095] (4) Discard the supernatant, add culture medium to resuspend the cells, and mix by pipetting to evenly disperse the cells.

[0096] (5) After centrifugation, discard the supernatant, add serum-free freezing solution to resuspend and mix well.

[0097] (6) Aliquot into sterile cryovials, 1.5 mL per tube.

[0098] (7) Seal with sealing film, mark, and store in a liquid nitrogen tank.

[0099] Verification test:

[0100] 1. Cell counting method

[0101] 1. Experimental steps

[0102] On the first day, a bottle of RAW cells from P175 was donated by Dr. Chen Wenjie from the Department of Hematology of Air Force Medical University. On the third day, when P175 grew to a density of about 70-80, it was mixed and passaged in three P275 bottles. On the fifth day, when the RAW cells from three P275 bottles grew to a density of about 70-80, they were mixed and passaged in 12 P325 bottles, and ranked 1-12 according to the order of inoculation. On the seventh day, according to the random number table method, starting from the first number in the sixth row, 1 group of 2 numbers were read in sequence, and the P325 numbers 1-12 were assigned values ​​of 93, 22, 53, 64, 39, 07, 10, 63, 76, 35, 87, 03. Ice PBS buffer group, trypsin group, cell scraping group, and direct blowing group were set up, and ice PBS buffer, trypsin, cell scraping, and direct blowing were used to free the cells for experiments.

[0103] Ice PBS buffer group (experimental group): The P325 bottles represented by the largest three numbers are group 1, i.e., 76, 87, and 93. The P325 bottles with serial numbers 9, 11, and 1 were washed twice with conventional room temperature PBS buffer, 3 mL / bottle of 4°C PBS buffer, sealed, and placed in a 4°C refrigerator. After 10 minutes, they were taken out and blown with a 1 mL pipette. After 5 minutes, they were blown clean and no blur was seen at the bottom of the bottle. Under the microscope, a small number of cells were observed to be scattered in a round shape and attached to the wall. Centrifuged at 1000 rpm / min for 5 minutes, 1 mL of complete culture medium (DMEM high glucose culture medium + 10% fetal bovine serum + 1% blue / chain double antibody solution) was added, resuspended, and diluted 10 times. The cell counts were 1.35×10 6 / mL, 1.42×10 6 / mL, 1.37×10 6 / mL, average 1.38×10 6 / mL, after mixing, draw 2.22mL of the stock solution, mix it in 57.78mL, and prepare 5×10 4 / mL cell suspension 60mL. Inoculate in 24-well plate, 2mL / well, for survival curve. Inoculate in 96-well plate, 5 rows × 6 lines, 0.1mL / well, for CCK-8 experiment.

[0104] Pancreatin group (control group 1): The P25 bottles represented by the smallest three numbers were divided into 2 groups, namely 03, 07, and 10, i.e., bottles 12, 6, and 7 from top to bottom. They were washed twice with conventional room temperature PBS buffer, and 1.5 mL of pancreatin was added respectively. Incubated in a 37°C incubator for 3 min, taken out, and blown with a 1 mL pipette. After 4 minutes and 20 seconds, the bottles were blown clean and no blur was seen at the bottom of the bottles. Under the microscope, a small number of cells were observed to be scattered in a circular shape and attached to the wall. Centrifuged at 1000 rpm / min for 5 min, 1 mL of complete culture medium (DMEM high glucose medium + 10% fetal bovine serum + 1% blue / chain double antibody solution) was added, resuspended, and diluted 10 times. After 10 times dilution, the cell counts were 1.71×10 6 / mL, 1.70×10 6 / mL, 1.74×10 6 / mL, average 1.72×10 6 / mL, after mixing, aspirate 1.74mL of the stock solution, mix it in 58.26mL, prepare the cell suspension, and seed it in 24- and 96-well plates.

[0105] Cell scraping group (control group 2): The P25 bottles represented by the three smallest numbers remaining were divided into 3 groups, namely 22, 35, and 39, that is, the P325 bottles with serial numbers 2, 10, and 5 were washed twice with conventional room temperature PBS buffer, and 3 mL of room temperature PBS buffer was added to scrape the cells. After 3 minutes and 20 seconds, no small blurry shadows were seen on the bottom of the bottle. Under the microscope, scattered cells or sheet-like cells were observed, which were round and attached to the wall. After centrifugation, resuspending, and counting, the number was 1.42×10 6 / mL, 1.37×10 6 / mL, 1.41×10 6 / mL, average 1.72×10 6 / mL, after mixing, take 2.14mL of the original solution, mix 57.86mL, prepare the cell suspension, and seed it in 24- and 96-well plates.

[0106] Direct blowing group (control group 3): The remaining three numbers represent P25 bottles, which are 4 groups, namely 53, 63, and 64, i.e., bottles 3, 8, and 4 from top to bottom. Wash twice with conventional room temperature PBS buffer, add 3 mL of room temperature PBS buffer, and blow with a 1 mL pipette. After 12 minutes, blow clean and see no blur at the bottom of the bottle. Under the microscope, a small number of cells are scattered and attached to the wall in a round shape. Centrifuge, resuspend, and count, the number is 1.16×10 6 / mL, 1.06×10 6 / mL, 1.11×10 6 / mL, average 1.11×10 6 / mL, mix, take 2.70mL of the original solution, mix 57.30mL, prepare the cell suspension, and seed in 24- and 96-well plates.

[0107] 2. Results

[0108] like Figure 2 and Figure 3 As shown, the ice PBS buffer group passed the impact of digestion earlier than the other three groups, entered the growth phase earlier, and was higher than the other three groups at 24h, p < 0.05; the trypsin group had faster and more cell differentiation, which affected the maintenance of cell characteristics; the cell scraping group had a large number of cell ruptures and deaths, which affected the early cell growth; the direct blowing group, the cells remained in the original culture bottle after digestion, resulting in cell loss, and the blown cells were clumps and unevenly distributed. After 72h, the cell growth of the four groups was basically the same.

[0109] 2. CCK8 cell proliferation assay

[0110] 1. Experimental steps

[0111] The cells digested in different ways were inoculated in 96-well plates, 5 rows × 6 lines, 10 4 / well, do CCK-8 experiment.

[0112] At 2 h, 24 h, 48 h, and 72 h, respectively, the solution was replaced first, and then 10 μL / well of CCK-8 was added. After 2 h, the absorbance was measured at a wavelength of 450 nm.

[0113] 2. Results

[0114] like Figure 4 As shown, at 2h, there was no difference in cell growth among the four groups, p>0.05, that is, the base number was the same. The ice PBS buffer group grew faster than the trypsin group at 24h and 48h, p<0.05.

[0115] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0116] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0117] 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 claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for digesting mouse mononuclear macrophages, characterized in that: The method comprises adding a phosphate buffer at 1°C to 4°C to culture the mouse mononuclear macrophages for digestion; Digest at 1℃~4℃ for 5min~10min; The concentration of the phosphate buffer is 0.01M to 0.015M; The pH of the phosphate buffer is 7.2-7.

4.

2. The method according to claim 1, characterized in that: The amount of the phosphate buffer used is no more than the amount of the culture medium for culturing the mouse mononuclear macrophages.

3. The method according to claim 1, characterized in that: In the culture, when the growth density of mouse mononuclear macrophages reached 80% to 90%, digestion was performed.

4. The method according to claim 1, characterized in that: During digestion, 70% to 80% of mouse mononuclear macrophages shrink and become round, and the intercellular spaces become larger, and the cells fall off when they are beaten.

5. The method according to claim 1, characterized in that The mouse mononuclear macrophages are RAW 264.

7.

6. A method for subculturing mouse mononuclear macrophages, characterized in that: After digesting mouse mononuclear macrophages using the method for digesting mouse mononuclear macrophages according to claim 1, the cell suspension is collected and centrifuged, the cell pellet is retained, culture medium is added to resuspend the cells to obtain a new cell suspension, and the new cell suspension is inoculated into a new culture medium for cultivation.

7. The method for subculturing mouse mononuclear macrophages according to claim 6, characterized in that: Centrifuge for 3 to 5 minutes.

8. A method for freezing mouse mononuclear macrophages, characterized in that: After digesting mouse mononuclear macrophages using the method for digesting mouse mononuclear macrophages according to claim 1, the cell suspension is collected and centrifuged, the cell pellet is retained, the freezing solution is added for re-suspending, and the pellet is dispensed into sterile freezing vessels and stored in a frozen environment.

9. The method for freezing mouse mononuclear macrophages according to claim 8, characterized in that: Centrifuge for 3 to 5 minutes.

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