More Than 25 Tips For Gaining Well-cultured Cells

Cell culture is not an easy thing which needs more care. Paying more attention to following tips, you will do it better.

Tip 1: How to thaw cooling tubes?
Cooling tubes should be put into the 37 ℃ water tank immediately for rapid thaw after taking out, shaking to melt within 1 minute. They key point is t keep the water level lower than cooling tubes’ covers in order to avoid pollution. One more thing, after taking out cooling tubes from liquid nitrogen bucket, extra concern should be needed to prevent the burst for your safety.

Tip 2: Should DMSO be removed immediately while thawing?
Except for a small number of cells that have been particularly specified to be sensitive to DMSO, most cell lines (including suspension cells), after thawing, should be placed directly into the cell culture flask with 10 ~ 15 ml fresh medium, and we should replace the medium the next day in order to remove DMSO, and then most of the thawed cells’ growing or sticking problems can be avoided.

Tips 3: Can I use mediums with different culture conditions from original ones ?
Absolutely not! Each cell line has its specific and adapted cell culture medium, mediums with different culture conditions, to which most cells can not adapt immediately, will result in cells death.

Tip 4: Can I use serum with different types from original one?
Of course not! Serum is a very important source of nutrition in cell culture, its type and quality will have a great impact on cell growth. Different species have different substances or molecular amount in serum. Inappropriate serum will cause cell survival problem.

Tip 5: What is FBS, FCS, CS, HS?
FBS (fetal bovine serum) and FCS (fetal calf serum) share the same meaning—fetal bovine serum. FCS, an incorrect expression, should not be used. CS (calf serum) stands for calf serum and HS (horse serum) for horse serum.

Tip 6: Should I use 5% CO2 or 10% CO2 when culturing cells? Or have no effect at all?
Generally speaking, HCO3- / CO32- / H + is used as the pH buffer system in most of the culture medium, and the content of NaHCO3 in the culture medium will determine the CO2 concentration while culturing. When NaHCO3 content is 3.7 g/liter in the medium, 10% CO2 is needed; when its content is 1.5 g/liter, 5% CO2 is applied.

Tip 7: When should I change the medium? Is it necessary to add antibiotics in the medium?
This depends on cell growth density, or changing medium on time according to the replacement time on the basic data of the cell line. Under normal culture state, no antibiotics should be added to the culture medium, except in a special screening system.

Tip 8: What is the trypsin-EDTA concentration when the adherent cells are subcultured? How to handle it?
Generally-used trypsin-EDTA concentration is 0.05% trypsin-0.53 mM EDTA.4 Na. After opening the bottle for the first time, trypsin-EDTA should be stored respectively in sterile test tubes in a small amount at -20 ℃, to do so, we can avoid trypsin activity decreased caused by repeated freezing and thawing and reduce the risk of pollution.

Tip 9: What are the subculture processes for suspended cells?
Generally, what we only to do is to adding fresh culture medium to the original culture bottle continuously to dilute the cell concentration. If the culture medium is too much, we can slightly raise the mouth of the culture bottle until it can not be accommodated. Transferring a part of the cell-containing culture medium to another new culture flask when you are separating the culture medium, and adding fresh medium to dilute to the appropriate concentration, repeating the previous steps.

Tip 10: How much rotating speed should be to centrifuge general animal cells?
The centrifugal rate is generally 1,000 rpm and 5 to 10 minutes to recover animal cells. Excessive speed will cause cell death.

Tip 11: What is the inoculated density of cells?
Inculated density can be in accordance with the density of cell lines’ basis data or the proportion of dilution. The small number of cells or diluting too much is also one of the important negative causes of cell growth.

Tip 12: What is the composition of cell freezing medium?
The most commonly-used fresh frozen medium for cryopreserving animal cells contains 5% ~10% DMSO (dimethyl sulfoxide) and 90%~95% medium for the original cell growth mixed evenly.

Tip 13: What is the level of DMSO and the way of sterile filtration?
DMSO grade for frozen storage must be tissue culture grade, and it is sterile itself. After opening the bottle for the first time, you should store DMSO respectively in sterile test tubes in a small amount at 4 ℃, and then harmful substances released by DMSO cleavage through repeated freezing and thawing can be avoided, and also the risk of pollution will be reduced. And you can use DMSO-resistant Nylon filter to filter DMSO.

Tip 14: How to cryopreserve cells? And what is the cell concentration in cooling tubes during cryopreservation?
Cryopreservation method A: placing cooling tubes at 4 ℃ for 30~60 minutes → (-20 ℃ for 30 minutes) →-80 ℃ for 16 ~ 18 hours (or overnight) →liquid nitrogen tank vapor phase for long-term storage

Cryopreservation method B: placing cooling tubes in the automatic cooling machine, whose program has been set to drop 1 ~ 3 ℃ per minute, until to -80 ℃ or below. And then you should place it into liquid nitrogen tank vapor phase for long-term storage. Storing at -20 ℃ can not be more than 1 hour in order to prevent too large ice crystals and a large number of dead cells. This step can also be ignored, and placing tubes into -80 ℃ refrigerator and lowering survival rate slightly.

The number of cells in the cooling tube is generally 1×106 cells/ml vial, and that of fusion tumor cells is preferably 5×106 cells/ml vial.

Tip 15: How to avoid cell contamination? How to deal with microbial contamination?
Cell contamination can be classified into bacteria, yeasts, molds, viruses and mold bacteria. The main causes of contamination are improper operation of aseptic technique, poor environment of operating room, contaminated serum, contaminated cells and so on. The best ways to reduce cell contamination are mainly depending on strict aseptic technique, clean environment, well-qualified cell sources and medium preparation.

When microbial contamination occurs, we should add appropriate antibiotics, sterilize and discard cells directly.

Tip 16: Can I observe the abnormal state of mycoplasma contaminated cells with naked eyes and its effect on the cell culture?
We can not observe the abnormal state of mycoplasma contaminated cells with naked eyes. Except very experienced experts, general researchers can not distinguish most mycoplasma contaminated cell lines according to their appearance.

Mycoplasma contamination can affect almost all cell growth parameters, metabolize and any other study data. Therefore, before the experiment performed, we must confirm that the cells are mycoplasma-free, and then the experimental result data can be meaningful.

Mycoplasma contaminated cells lines should be directly sterilized and discarded to avoid contaminating other cell lines.

Tip 17: Why the medium color will be dark red and the pH will be more and more alkaline when stored in the refrigerator at 4 ℃?
When storing in the refrigerator at 4℃, CO2 in the culture medium will gradually overflow, and this will cause the medium get more and more alkaline, the color of the acid-base indicator (usually phenol red) in the culture medium will also be darker with the alkaline and then become dark red. Alkaline culture medium will result in cell growth stagnation or death. Filtering CO2 aseptically to adjust the pH value should be a proper method when culture medium gets alkaline.

Tip 18: Are the dish and flask same for all kinds of cell culture?
Dishes or flasks from different brands coat different polymer and have different manufacturing processes. Although there is no great impact on most cells, only a few cells may have growth differences due to the use of dishes or flasks from different brands.

Tip 19: Possible causes for cell death or poor survival rate? After the cell freezing tube thawed, why are there a small number of cells?
Common causes for cells’ poor survival rate: improper use of medium or poor quality of medium; improper use of serum or poor-quality serum; thawing process error; washing and centrifuging frozen cells after thawing; misreading suspension cells as dead cells; improper culturing temperature; long-time storage at -80 ℃.

The small number of cells in the frozen cell culture is mostly due to error operation of the centrifugal, this results in cells’ physical damage and cell loss. Do not immediately centrifuge the cells after thawing, and should replace medium overnight after cell growth.

Tip 20: Reasons for rupture frozen tube bottle, cracked cap or fall-off cap?
Rupture frozen tube bottle and cracked cap may be due to the improper force when operator held the frozen tube. The hemostatic clamp is recommended to use and held carefully. As for fall-off caps, it is because of the physical phenomenon—heat makes it expand and cold makes it contract, and cooling tubes may be contaminated. So whether it is put into or taken out of the liquid nitrogen bucket, cooling tubes should be immediately tightened.

Tip 21: How to choose a special cell line culture medium?
There is no fixed culture condition for a certain cell type. Cells cultured in MEM are likely to grow well in DMEM or M199 as well. In a word, it will be a good start to choose MEM for adherent cell culture, RPMI-1640 for suspension cell culture. The best serum-free medium for various purposes prefers AIM V (12005) culture medium (SFM).

Tip 22: Is L-glutamine important in cell culture? Is it unstable in solution?
L-glutamine plays a very crucial role in cell culture. After removal of the amino group, L-glutamine can be used as a source of energy for cultured cells, involved in protein synthesis and nucleic acid metabolism. L-glutamine will be degraded in solution in a period of time, but the exact degradation rate has not been finalized. The degradation of L-glutamine leads to the formation of ammonia, and the ammonia is toxic to some cells.

Tip 23: What is GlutaMAX-I? How to use GlutaMAX-I in cell culture? How about its stability?
The GlutaMAX-I dipeptide is a derivative of L-glutamine, and its unstable alpha-amino group is protected by L-alanine. A certain peptidase gradually cleaves the dipeptide to release GlutaMAX-I for use. GlutaMAX-I dipeptide is very stable, it degrades a little even sterilized at 121 pounds for 20 minutes. Under the same condition, L-glutamine is almost completely degraded.

Tip 24: What medium can work well without phenol red? What is the role of sodium pyruvate in the culture medium?
We always use Phenol red as a indicator of pH in the medium: it will be red when neutral, yellow when acidic and purple when alkaline. Studies have shown that phenol red can mimic the role of steroid hormones (especially estrogen). In order to avoid the steroid reaction, we should culture cells especially mammalian cells in no phenol red medium. And since the phenol red interfers detection, some researchers use mediums without phenol red to do flow cytometry.

Sodium pyruvate can be used as an alternative carbon source in cell culture. Although cells tend to use glucose as a carbon source, cells can also metabolize sodium pyruvate if there is no glucose.

Tip 25: Why does not Hank’s balanced salt solution (HBS) used in the air need for CO2 incubators? What are the essential functional differences between HBS and Earle’s balanced salt solution (EBS)?
The main difference between HBS and EBS is the level of sodium bicarbonate, and it is much higher in Eagles (2.2 g/L) than in Hanks (0.35 g/L). Sodium bicarbonate requires a high level of CO2 to maintain the pH of the solution. Eagles liquid will become alkali in the air level of CO2, hanks solution in the CO2 incubator will become acidic. We can choose eagles solution for storing tissue in a CO2 incubator, and hanks solution for cleaning tissue stored in the cell culture medium.

Tip 26: Does divalent ions inhibit trypsin activity? What is the purpose of adding EDTA when using trypsin?
Divalent ions can indeed inhibit trypsin activity. EDTA is used to chelate free magnesium ions and calcium ions in order to maintain inhibition of trypsin activity. So it is recommended that cells be washed with EDTA to remove all divalent ions from the medium before dealing the cells with trypsin.

Tip 27: Other precautions
1.We should apply at most 50% concentration of antibiotics used in serum medium to serum-free medium. Serum proteins will combine and inactivate some antibiotics. Under the serum-free culture conditions, if antibiotics are not inactivated and they may be toxic to cells.
2.Once you add serum and antibiotics to fresh medium, you should used it within two to three weeks. Because some basic components of the antibiotics and serum will begin to degrade after thawing.
3.Most of the additives and reagents can be repeatedly frozen up to 3 times at most, many times of operation will cause a certain level of degradation and precipitation in protein solution, and this will affect its performance.
4.Within a week after dissolution, we should use the liquid trypsin solution stored in a refrigerator at 4 ℃. Trypsin may begin to degrade at 4 ℃ and become unstable at room temperature for more than 30 minutes.
5.In order to keep the water tray in the CO2 incubator clean, you must replace (at least once every two weeks) with it sterile distilled water or sterile deionized water.

All above are some tips for cell culture. If you got another tips, you can discuss with us.

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