To grow cells, you also need to pay attention to these details!（1）

Cell regeneration is a heart-stirring thing. You have to treat it as carefully as a child. Take care of her and take care of her. If you pay attention to these issues when you take care of it, your cells will grow better. Let’s talk about the precautions for growing cells with you.

Preparation before cell culture

Before you put on your gloves and start cell culture, check whether the number of pipettes and bottles is sufficient, so as not to enter and exit the operating table again after starting the experiment, which can reduce the risk of cell contamination.

The cell culture medium should also be preheated first. Choose to preheat only part of the culture medium instead of heating the entire bottle. This not only saves the experiment time, but also avoids protein degradation caused by repeated heating of the medium.

After finishing the operation, don’t forget that the medium is sensitive to light and should be kept away from light as much as possible.

Periodic inspection of cell culture

Regularly checking the morphology of cultured cells, that is, their shape and appearance, is critical to the success of cell culture experiments.

In addition to confirming the health of the cells, the naked eye and microscopic examination of the cells each time the cells are manipulated can also detect signs of contamination early and prevent the contamination from spreading to other cells in the laboratory.

Signs of cell deterioration

The signs of cell deterioration include the appearance of granules around the nucleus, the dissociation of cells from the matrix, and the formation of cytoplasmic vacuoles.

These signs of deterioration may be caused by a variety of reasons, such as:

Contamination of the culture, senescence of the cell line, or the presence of toxic substances in the medium, or these signs only indicate that the culture needs to be replaced.

When the deterioration is severe, it will become an irreversible change.

Disinfection and layout of cell culture fume hood

Keep the cell culture fume hood clean and orderly, and place all items within direct sight.

Spray 70% ethanol on all items placed in the fume hood, wipe clean for disinfection.

Place the cell culture container in the open center of the fume hood; place the pipette on the right front for easy access; place the reagent and medium on the right back for easy suction; place the test tube rack at the middle and rear; place small containers on the left back for use Yu Sheng holds waste liquid.

Aseptic operation of culture flasks/dishes and other items

Aseptic culture bottles, reagent bottles, petri dishes and other items should not be opened until they are in use. They must not be exposed to the environment. After the operation is completed, cover the cover as soon as possible. When removing the cover, place the cover on the work surface with the opening facing down. superior.

Do not speak, sing, or whistle when performing aseptic operations. Complete the experiment as soon as possible to avoid contamination as much as possible.

Possible contaminants in cell culture

Cell culture contaminants are mainly divided into two categories:

Chemical contaminants, such as impurities in culture media, serum and water, endotoxins, plasticizers and detergents.

Biological contaminants, such as cross-contamination of bacteria, molds, yeasts, viruses, mycoplasma and other cell lines.

The frequency and severity of pollution can be reduced by fully understanding the source of pollution and adopting good aseptic techniques.

Confirmation of cross contamination

Although cross-contamination is not as common as microbial contamination, extensive cross-contamination with HELA cells and other fast-growing cell lines is a clear problem and can cause serious consequences.

Obtaining cell lines from a reputable cell bank, regularly checking cell line properties, and using good aseptic techniques can help avoid cross-contamination.

DNA fingerprinting, karyotype analysis and isotope analysis can confirm whether there is cross-contamination.

Precautions for cell exchange

When replacing the cells, gently add it along the side of the culture flask instead of directly adding it to the cells to avoid damage to the cells, especially when the cultured cell line is fragile.

When using sterile glass pipettes or disposable plastic pipettes and pipettes to handle liquids, each pipette can only be used once to avoid cross-contamination.

Time control of cell passage

When the cells proliferate exponentially, the adherent cultured cells have occupied all the available substrate, there is no expansion space, or the suspension cultured cells have exceeded the capacity of the culture medium and cannot grow further, the cell proliferation speed will be greatly reduced or even stopped completely .

In order to maintain the cell density at an optimal level so that the cells continue to grow and stimulate further proliferation, the cells must be passaged.

Precautions for using antibiotics in cell culture

Antibiotics should not be used for a long time in cell culture, because continuous use of antibiotics will promote the production of antibiotic-resistant cell lines, leading to the persistence of mild pollution.

Antibiotics can only be used as a last resort to deal with pollution for a short period of time and should be removed as soon as possible.

If antibiotics are used for a long time, cultures without antibiotics should be carried out at the same time to serve as a control for identifying hidden infections.

The necessity of cell cryopreservation

Continuously cultured cell lines are prone to genetic drift, and limited cell lines will eventually become senescent. All cultured cells are susceptible to microbial contamination. Even the best-functioning laboratory will encounter equipment failure.

Since the established cell line is a valuable resource, it is costly and time-consuming to replace the cell line. Therefore, it must be frozen and stored for a long time.

Cell cryopreservation matters

The cell culture should be cryopreserved when the cell concentration is high, and the number of cell passages should be as few as possible.

Make sure that the percentage of viable cells is at least 90% before freezing.

Please note that the optimal cryopreservation conditions depend on the cell line used.

The freezing and resuscitation process will have an adverse effect on most cells, so do not detach the cells by vortexing or tapping the culture flask (except when culturing insect cells), and do not centrifuge the cells at high speed.