Tumor adherent cell culture method

Abstract

The tumor adherent cell culture method comprises the following steps: 1) preparing a complete culture medium; (2) carrying out cell resuscitation; (3) culturing cells; (4) carrying out cell passage; 5) preparing a cell freezing medium; and 6) performing cell cryopreservation. According to the invention, growth conditions with sufficient nutrition are provided for tumor cells by increasing the proportion of non-essential amino acids and fetal calf serum in the complete culture medium, so that the cell adherence time can be effectively shortened, the good form of the cells is maintained, and the proliferation of the cells is promoted.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to a method for culturing tumor adherent cells. Background Art

[0002] Tumor cell in vitro culture experiments play an extremely important role in tumor biology research. Tumor cells cultured in vitro can retain the biological characteristics of primary tumor tissues, making them an indispensable tool for studying tumor invasion, metastasis and drug sensitivity.

[0003] Therefore, it is necessary to provide a method for culturing tumor adherent cells. Summary of the invention

[0004] The purpose of the present invention is to provide a method for culturing tumor adherent cells.

[0005] The present invention achieves the above-mentioned purpose through the following technical scheme: A method for culturing tumor adherent cells, 1). Preparation of complete culture medium: 39mL high-glucose basal culture medium + 10mL fetal bovine serum + 500μL 100x penicillin-streptomycin (double-antibody) additive + 500μL 100x non-essential amino acid solution, increase or decrease the four components in proportion to the cell culture demand to prepare the complete culture medium, filter with a 0.22μm sterile filter membrane to ensure the complete culture medium is sterile, and refrigerate for later use;

[0006] 2) Cell recovery: Take out the cell cryovial from liquid nitrogen, quickly place it in a 37°C water bath and shake to thaw until there is no crystal in the cryovial, then wipe the outer wall of the cryovial with 75% alcohol; pipette the cells in the cryovial into a 15mL centrifuge tube containing 5mL complete culture medium, centrifuge at 1000rpm for 5min; discard the supernatant, resuspend the precipitate with 5mL complete culture medium, inoculate it into a T25 cell culture flask, tilt the culture flask 30° and shake it crosswise, place it in a 37°C, 5% CO2 cell culture incubator with the flask mouth facing inward to prevent cell contamination;

[0007] 3) Cell culture: Observe the cell growth under an inverted microscope on the second day of recovery. If there are many free suspended dead cells, wash once with 2mL sterile 1×PBS and once with 1mL 1×PBS, shake horizontally and pour out the liquid, and continue to culture with fresh complete medium. The red color becomes lighter, indicating nutrient consumption, which is a normal cell growth phenomenon. Cells can be passaged only when they have attached to the wall and expanded to grow by at least 80%;

[0008] 4). Cell passaging;

[0009] 5). Preparation of cell freezing solution;

[0010] 6). Cell cryopreservation.

[0011] Furthermore, the refrigeration temperature in step 1) is 4°C.

[0012] Furthermore, the cell freezing solution is 90% fetal bovine serum + 10% DMSO.

[0013] Further, in step 6), when the cells grow to cover 90% of the area of ​​the culture flask, the culture medium in the T25 culture flask is discarded, and the cells are washed once with 2 mL of PBS and once with 1 mL of PBS; about 1 mL of 0.25% trypsin digestion solution is added to the culture flask (T75 plus 3 mL of trypsin), placed in an incubator for digestion for 2 minutes, and the cells are observed to shrink and become round under a microscope, and then 2 mL of complete culture medium is added to terminate the digestion, and the cells are gently blown to make them fall off, and then the suspension is transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes; the supernatant is discarded, and the precipitated cells are resuspended by blowing and suction with 2 mL of cell freezing solution, and the cells are counted with a hemocytometer, and the cell suspension is dispensed into cryopreservation tubes according to the cell amount, and the cell amount in each tube should be 1×106 cells / mL. The dispensed cell cryopreservation tubes are transferred to a program cooling box and directly placed in a -80°C refrigerator for at least 24 hours, and then transferred to a liquid nitrogen tank for long-term storage.

[0014] Compared with the prior art, the tumor adherent cell culture method of the present invention has the beneficial effect of providing tumor cells with nutritionally sufficient growth conditions by increasing the ratio of non-essential amino acids and fetal bovine serum in the complete culture medium, which can effectively shorten the cell adhesion time, maintain the good morphology of the cells and promote cell proliferation. DETAILED DESCRIPTION

[0015] A method for culturing tumor adherent cells comprises the following steps:

[0016] 1). Preparation of complete culture medium: 39mL high-glucose basal culture medium + 10mL fetal bovine serum + 500μL 100x penicillin-streptomycin (double-antibody) additive + 500μL 100x non-essential amino acid solution (20% fetal bovine serum is suitable for most adherent tumor cell culture). Add or reduce the four components in proportion to the cell culture requirements to prepare the complete culture medium, filter with a 0.22μm sterile filter membrane to ensure the sterility of the complete culture medium, and refrigerate at 4℃ for later use.

[0017] 2). Cell recovery: Take out the cell cryovial from liquid nitrogen, quickly place it in a 37℃ water bath and shake to thaw until there is no crystal in the cryovial, then wipe the outer wall of the cryovial with 75% alcohol. Transfer the cells in the cryovial to a 15mL centrifuge tube containing 5mL complete culture medium with a pipette, and centrifuge at 1000rpm for 5min. Discard the supernatant, resuspend the precipitate with 5mL complete culture medium, inoculate it in a T25 cell culture flask, tilt the culture flask 30° and shake it crosswise, and place it in a 37℃, 5% CO2 cell culture incubator with the flask mouth facing inward to prevent cell contamination.

[0018] 3). Cell culture: Observe the cell growth under an inverted microscope on the second day of recovery. If there are many free suspended dead cells, wash them once with 2mL sterile 1×PBS and once with 1mL 1×PBS. Shake horizontally and pour out the liquid. Replace with fresh complete medium for continued culture. The lightening of the red color indicates nutrient consumption, which is a normal cell growth phenomenon. Cells can be passaged only when they adhere to the wall and grow by at least 80%.

[0019] 4). Cell passaging: When the cells grow to cover more than 80% of the area at the bottom of the culture flask, discard the culture medium in the T25 culture flask, wash the cells once with 2mL 1×PBS, and once with 1mL 1×PBS. Add 1mL of 0.25% trypsin digestion solution to the culture flask (if the T75 flask is cultured, add 3mL of trypsin), place it in the incubator for digestion for 2 minutes, observe the cells shrink and become round under a microscope (if the cells have not fallen off, the digestion time can be appropriately extended), then add 2mL of complete culture medium to terminate the digestion (the volume of complete culture medium for terminating digestion should be more than 2 times the volume of trypsin), then gently blow the cells to make them fall off, avoid bubbles during the blowing process, and then transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 1000rpm for 5min. Discard the supernatant, resuspend the precipitated cells with 3mL of complete culture medium by suction, and then pass them in bottles at a ratio of 1:3 (3 T25), add new complete culture medium to 5mL / bottle, and finally culture in a 37℃, 5% CO2 incubator.

[0020] 5). Preparation of cell freezing solution: 90% fetal bovine serum + 10% DMSO.

[0021] 6) Cryopreservation of cells: When the cells grow to cover 90% of the area of ​​the culture flask, discard the culture medium in the T25 culture flask, wash the cells once with 2mL PBS, and once with 1mL PBS. Add about 1mL of 0.25% trypsin digestion solution to the culture flask (T75 plus 3mL of trypsin), place it in the incubator for digestion for 2min, observe the cells shrink and become round under a microscope, then add 2mL of complete culture medium to terminate digestion, then gently blow the cells to make them fall off, and then transfer the suspension to a 15mL centrifuge tube and centrifuge at 1000rpm for 5min. Discard the supernatant, resuspend the precipitated cells with 2mL of cell freezing solution, count the cells with a hemocytometer, and dispense the cell suspension into cryopreservation tubes according to the cell amount. The cell amount in each tube should be 1×106cells / mL. After the dispensed cell cryopreservation tubes are transferred to the program cooling box, they are directly placed in a -80℃ refrigerator for at least 24h, and then transferred to a liquid nitrogen tank for long-term storage.

[0022] The present invention provides a nutrient-sufficient growth condition for tumor cells by increasing the ratio of non-essential amino acids and fetal bovine serum in a complete culture medium, thereby effectively shortening the cell attachment time, maintaining a good cell morphology and promoting cell proliferation.

[0023] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0024] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for culturing tumor adherent cells, characterized in that: The following steps are involved: 1). Preparation of complete culture medium: 39mL high-glucose basal culture medium + 10mL fetal bovine serum + 500μL 100x penicillin-streptomycin (double-antibody) additive + 500μL 100x non-essential amino acid solution. Add or reduce the four components in proportion to the cell culture requirements to prepare the complete culture medium, filter with a 0.22μm sterile filter membrane to ensure the complete culture medium is sterile, and refrigerate for later use; 2) Cell recovery: Take out the cell cryovial from liquid nitrogen, quickly place it in a 37°C water bath and shake to thaw until there is no crystal in the cryovial, then wipe the outer wall of the cryovial with 75% alcohol; pipette the cells in the cryovial into a 15mL centrifuge tube containing 5mL complete culture medium, centrifuge at 1000rpm for 5min; discard the supernatant, resuspend the precipitate with 5mL complete culture medium, inoculate it into a T25 cell culture flask, tilt the culture flask 30° and shake it crosswise, place it in a 37°C, 5% CO2 cell culture incubator with the flask mouth facing inward to prevent cell contamination; 3) Cell culture: Observe the cell growth under an inverted microscope on the second day of recovery. If there are many free suspended dead cells, wash once with 2mL sterile 1×PBS and once with 1mL 1×PBS, shake horizontally and pour out the liquid, and continue to culture with fresh complete medium. The red color becomes lighter, indicating nutrient consumption, which is a normal cell growth phenomenon. Cells can be passaged only when they have attached to the wall and expanded to grow by at least 80%; 4). Cell passaging; 5). Preparation of cell freezing solution; 6). Cell cryopreservation.

2. The method for culturing tumor adherent cells according to claim 1, characterized in that: Step 1) The refrigeration temperature is 4°C.

3. The method for culturing tumor adherent cells according to claim 1, characterized in that: The cell freezing solution is 90% fetal bovine serum + 10% DMSO.

4. The method for culturing tumor adherent cells according to claim 1, characterized in that: Step 6) When the cells grow to cover 90% of the area of ​​the culture flask, discard the culture medium in the T25 culture flask, wash the cells once with 2mL PBS and once with 1mL PBS; add about 1mL of 0.25% trypsin digestion solution to the culture flask (T75 plus 3mL of trypsin), place it in an incubator for digestion for 2min, observe the cells shrink and become round under a microscope, then add 2mL of complete culture medium to terminate the digestion, then gently blow the cells to make them fall off, and then transfer the suspension to a 15mL centrifuge tube and centrifuge at 1000rpm for 5min; discard the supernatant, resuspend the precipitated cells by blowing and aspiration with 2mL of cell freezing solution, count the cells with a hemocytometer, and dispense the cell suspension into cryopreservation tubes according to the cell amount. The cell amount in each tube should be 1×106cells / mL. The dispensed cell cryopreservation tubes are transferred to a program cooling box and directly placed in a -80℃ refrigerator for at least 24h, and then transferred to a liquid nitrogen tank for long-term storage.