Mammalian cell culture FAQ

1. What is the difference between primary cells and cell lines?

Primary cells are cells derived directly from human or animal tissues. Primary cells usually have a limited lifespan. Compared to cell lines, primary cells retain many of the characteristics and markers of the donor and can be used to build better disease models.

The cells of the cell line have undergone passage lasting a long time and have generally undergone genetic transformation. A cell line can usually pass through 30-80 generations, and the cell line has lost the true characteristics of the original tissue from which they were isolated compared to the primary cell. But because they are easy to work with and are supported by widely published literature, people still prefer to use these cell lines for convenience.

2. How to choose the correct type of medium？

It is generally considered that MEM is the first choice for adherent cell culture, and RPMI-1640 is the suspension cell culture, but for primary culture, the selection of medium is a very critical issue. In this case, attention should theoretically be paid to the cell type, nutritional requirements, the need for that type of cell to grow in the body, and whether there are readily available products for these optimal combinations. In fact, not only should different kinds of media be tried in primary cultures, but in some cases, it may also be necessary to try a specific type of a medium, such as DMEM+ non-essential amino acids + glutamine or DMEM w/o L-glutamine, but containing non-essential amino acids. It all depends on the type of nutrients required for a particular type of cell and the microenvironment isolated from it. Sometimes HAM's F-12 and other nutrients are also necessary. Different cells need different nutrients, it can be HGF of liver cells, RA (retinoic acid) of nerve cells, etc., so in the case of specific cell types, we need to change to a different medium.

In addition, the downstream applications of the cells need to be considered. For example, if fluorescence analysis is performed using cell lines, phenol red needs to be excluded from the medium because it is highly fluorescent and can cause background readings to be too high.

3. What is the role of serum?

Serum is usually added as an additive, at a ratio of 2-20%, to the medium to provide the cells with comprehensive nutrients, hormones, growth and attachment factors, etc. Serum also acts as a buffer for cell culture systems, preventing various factors (including pH changes, proteolytic activity or heavy metals, endotoxins, etc.) from interfering with cell growth or producing toxic effects.

Although the use of serum will bring some problems, such as batch differences, unknown ingredients, interference with downstream purification, and so on, there are indeed many use of serum-free culture scenarios, but serum has always had a place in cell culture.

4. Why should bovine serum be divided into fetal cows, newborn cows and calves? How to choose？

Bovine serum is mostly a by-product of animal husbandry, and the difference between fetal bovine serum (FBS), newborn bovine serum, or calf serum is due to different blood collection times, or cow age.

Fetal bovine serum is obtained by heart puncture from unborn fetal cows (3-8 months of age), neonatal bovine serum is obtained by venous blood collection from newborn cows 12-24 hours after birth, and calf serum is collected by arterial blood collection from calves 16-22 weeks of age. Although the age division of newborn cattle or calves may vary, it is clear that the younger the cow, the higher the proportion of serum components such as pro-growth factor, pro-adhesion factor, hormones and other active substances, and the lower the level of antibodies.

The advantage of FBS in biochemical composition makes it considered to be the gold standard of serum, and therefore the highest price. Serum selection is usually based on a combination of cell type and culture cost. For delicate and difficult cells, only fetal bovine serum can be selected, while some easily cultured tumor cell lines and conventional cell lines can consider newborn bovine or calf serum.

5. Is the appearance of black spots in cell culture pollution? How to prevent and deal with it？

First, the medium is visually observed for turbidity. If it is cloudy, it can basically be confirmed as pollution. If not, look under the microscope to see if the black spots are regular in size and shape, if they are moving, if they are doing Brownian motion, or if they are moving fast in a straight line. If the black spot is irregular in size and performs Brownian motion, it may be a cell fragment (poor cell condition or overdigestion). Protein precipitation and cell metabolites after repeated freezing and thawing of serum may also form black spots. If the black spots are the same size and moving quickly, it could be bacterial contamination.

Preventive measures:

1) Grasp the best time for cell passage, not when cells are aging.

2) Master the digestion time to prevent excessive digestion of cell fragments.

3) Reduce the repeated freezing and thawing of serum and other reagents. Adjust the pH of the medium to the optimum.

4) Strictly control the cleanliness of water and appliances.

Treatment method: If the black spot is determined to be a pollutant, treat the cell in time and discard it. If the cells are observed under the mirror and the growth state is good, there is no change compared with before the appearance of black spots, it can be treated without treatment, or you can refer to the following methods for treatment：

1) Suspension cells: The cell supernatant was collected by slow centrifugation (500-600 rpm/min, 5-6 min) and replaced with a new culture bottle.

2) Adherent cells: Wash the cells with PBS 2-3 times. When cleaning, gently tap the culture bottle to remove unfirmly attached debris and particles. The PBS is then discarded and a low concentration of trypsin (e.g. 0.05%, 1 minute) is added for digestion, so that the particles and debris in the cellular space are removed. Low concentrations of trypsin are removed and the cells are then digested normally. The collected cell suspension was centrifuged at a low speed (500-600rpm/min, 5-6min) and the culture vial was replaced with a new one. Appropriate increase of serum concentration for culture.

6. What are the symptoms of bacterial contamination? How to deal with？

Bacterial contamination is the most common contamination in cell culture and is mainly introduced by careless handling or the use of incompletely sterilized consumables and reagents. Bacterial contamination in culture is generally detected by visual observation of turbidity in the medium. Aerobic bacteria proliferate and divide rapidly, and can make the medium cloudy within 24 hours of infection. Anaerobic bacteria proliferate slowly under conventional cell culture conditions, and it takes a longer time to observe turbidity.

The most common are subtilis, Escherichia coli, pseudomonas and Staphylococcus albus. Under the microscope, the morphology is long rod, short rod and granular. When bacterial contamination occurs, if the amount of bacteria is small, the medium can be kept clear, and the bacteria can generally be seen to swim rapidly along the axial direction. In the case of granular contamination, the medium will generally be cloudy or have a white sandy precipitate.

Contamination treatment: Generally, if the culture is not very precious, it is recommended to discard it directly and revive the cell culture. In the case that the cell vitality is still maintained, the cells can be cleaned with PBS first, and then try to save with antibiotics. Due to the widespread use of antibiotics in domestic cell culture, the addition of double antibodies (cyanine and streptomycin) after contamination generally has no effect. Bacillus can be treated with 100μg/ml gentamicin for a week, granular bacteria can be treated with 25μg/ml ciprofloxacin for a week, and the effect is relatively good.

7. How to deal with fungal contamination？

The most common polluting fungi in cell culture are: Aspergillus fumigatus, Aspergillus Niger, mucor, candida albicans, yeast and so on. Mycelium growth can be seen with the naked eye, and the bud formation can be seen under the microscope with yeast pollution.

Contamination treatment: If it is not a very precious culture, it is recommended to discard it as soon as possible, and strengthen disinfection of the incubator and related links in time. If the cell culture plate is polluted by a single hole or several holes, it can be carefully removed and sealed with high concentration of sodium hydroxide to avoid the expansion of pollution and the involvement of other holes or cells in the culture bottle. Treatment with amphotericin B can be used if salvage is attempted. Note that amphotericin is highly toxic and needs to be treated before the cells have more than 50% and the state is not greatly affected.

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