With the continuous development of cell genetic engineering, there are currently a variety of biological expression systems that can produce recombinant proteins on a large scale, mainly divided into two categories: prokaryotic and eukaryotic. Eukaryotic expression systems include: yeast expression system, mammalian cell expression system, and insect baculovirus expression system. In contrast, the most prominent advantage of the mammalian cell expression system is that it can promote the correct folding of proteins and post-translational modifications such as glycosylation, thereby ensuring the natural activity of proteins. At present, the mammalian cell expression system has become the preferred host for the expression and production of some protein drugs, genetically engineered antibodies and other target proteins.

Commonly used cells for mammalian protein expression are HEK293 (human embryonic kidney cells) and CHO (Chinese hamster ovary cells). These two cells are the most widely used and are the most commonly used cells in eukaryotic protein expression systems. They have the following characteristics:

(1) They have accurate post-transcriptional modification functions, and the expressed proteins are closest to the natural state in any aspect;

(2) They have the ability to efficiently amplify and express recombinant genes, and the integration of exogenous proteins is stable;

(3) They have high tolerance to shear force and osmotic pressure, and have high expression levels;

(4) CHO belongs to fibroblasts, which are non-secreting cells that rarely secrete endogenous proteins, which is conducive to the separation and purification of target proteins;

(5) HEK293 cells have higher growth density and faster growth rate, and are easy to culture and transfect.

Next, the basic principles, experimental steps and precautions for subculture of HEK293 cells and CHO cells will be introduced.

Basic Principles of Cell Culture

(1) Appropriate cell culture medium

Appropriate cell culture medium is the most important condition for cell subculture. The culture medium not only provides the basic substances needed for cell growth and reproduction in vitro, but also maintains the entire environment for cell growth. Different cells have different growth habits, so it is necessary to choose the appropriate culture medium.

(2) High-quality serum

Currently, most synthetic cell culture media have added serum, and commonly used serums include calf serum, horse serum, etc. Serum contains growth factors necessary for cell growth. As an additive for mammalian cell culture, serum is very expensive and has many problems, so people are constantly looking for substances that can replace serum.

(3) Non-toxic and sterile growth environment

Cells grown in vitro lack the ability to resist microorganisms and toxic substances. Once contaminated, they will cause cell death. Therefore, when performing cell subculture, a non-toxic and sterile culture environment is a necessary condition.

(4) Temperature and gas environment

Cell growth requires a constant temperature, and gas (oxygen and carbon dioxide) is also a necessary substance for mammalian cell culture. The cell culture conditions of HEK293 and CHO are generally 37°C and 5% carbon dioxide environment.

Cell Passaging Experiment Process

• Cell recovery

(1) Open the water bath in advance and preheat to 37℃.

(2) Add 5-10 ml cell culture medium to a 15 ml centrifuge tube and place it in a water bath to preheat.

(3) Immediately put the frozen cells into a 37℃ water bath, shake gently, and take them out after thawing (about 1-1.5 minutes).

(4) Disinfect the outer wall of the tube with 75% ethanol and place it in a clean bench. Transfer to a 15 ml centrifuge tube containing 5-10 ml culture medium and centrifuge at 800-1000 rpm for 5 minutes.

(5) Remove the supernatant and add complete culture medium to resuspend the cells. Then transfer to a culture dish and gently shake it back and forth to evenly disperse the cells in the culture dish.

(6) Label the cell name, generation number, date, culture medium, etc. and place it in a 37℃ 5% CO2 incubator for culture.

(7) Adherent cells can usually adhere to the wall on the second day. Depending on the cell growth rate, change the culture medium every 2-3 days.

(8) Centrifugation may cause damage to some cells. These cells do not need to be centrifuged. It is only necessary to reduce the DMSO concentration to below 1%. (Generally, the DMSO concentration in the cryopreservation solution is 10%, that is, 1ml of cryopreserved cells needs to be added with 9ml of culture medium.)

• Cell subculture

(1) When the cell coverage rate in the culture dish reaches 80%-90%, cell subculture is required.

(2) Aspirate the old culture medium in the culture dish.

(3) Wash the cells 1-2 times with 5ml PBS.

(4) Use 1ml of trypsin solution at 37℃ for several minutes, observe under an inverted microscope, and when the cells are round and about to separate, aspirate the trypsin solution.

(5) Tap the culture dish or culture bottle to make the cells fall off the bottle wall, and add an appropriate amount of fresh complete culture medium. Gently blow several times to disperse the cells evenly. Then transfer to a new culture bottle according to the dilution ratio and culture normally. Discard the remaining cell suspension and put it into a collection bottle.

(6) 0.02% EDTA solution can be added to the trypsin solution to achieve better dissociation effect. However, the EDTA remaining in the culture medium after digestion will affect the re-attachment of cells.

• Cell freezing

(1) Ensure that the cells are in the exponential growth phase before freezing. Take 1 mL of the 5 mL cell suspension after subculturing and inoculate it into the culture flask to continue subculturing. Take another sterile centrifuge tube, place the remaining 4 mL cell suspension in it, and centrifuge at 1000 rpm for 5 minutes (the speed should not exceed 1500 rpm).

(2) After centrifugation, pour off the upper supernatant and collect the lower cell precipitate. Add 900 ul of complete culture medium to the centrifuge tube and resuspend the cells by repeatedly blowing with a pipette tip. Take a small amount of cell suspension to count the cell concentration and pre-freezing survival rate. Generally, the cell concentration is more suitable for 2~5X10⁶ cells/mL.

(3) Transfer the cell suspension into a 1.5 mL sterile cryotube and add 100 ul of reagent-grade DMSO. Mix well to make a cell freezing suspension (the final DMSO concentration is 10%). After sealing tightly, note the cell name, generation number, and date, and then freeze.

(4) Traditional method: Place the cold storage tube at 4℃ for 10 minutes → -20℃ for 30 minutes → -80℃ for 16~18 hours (overnight) → long-term storage in liquid nitrogen tank (-20℃ should not exceed 1 hour to prevent excessive ice crystals in the cells from causing cell death).

Notes

• Large bottles for cell culture are better than small bottles, as cells can fully absorb nutrients and are conducive to uniform distribution of cells
• DEME, a cell culture medium, is a high-sugar culture medium
• Ensure that the cell culture medium is fresh, use small amounts and multiple configurations, or store the culture medium at -20°C
• Control the trypsin digestion time. Long digestion time will result in poor cell adhesion
• Control the cell passage time. The best passage is when the cells are not completely filled and there are gaps between the cells
• The principle of cell freezing and recovery is slow freezing and fast recovery to maximize the success rate of cell recovery