Q1: Can I just change the medium when cells are overgrown without any treatment?
A1: In animal cell culture, when cells are cultured in flasks, they exhibit characteristics of adherent growth and contact inhibition. If cells grow to cover the entire flask surface, they will no longer proliferate, and there will be no phenomenon of cells growing in two layers. At this point, you need to replace the culture flask and medium, otherwise cells will stop proliferating. Cancer cells are not restricted by this property. Cancer cells can continue to proliferate infinitely even after reaching confluence and contact inhibition.
Q2: Why does cell culture medium turn yellow and will it affect cell condition?
A2: The medium turns yellow because cells are growing vigorously with active metabolism, producing large amounts of lactic acid andCO2, which affects the pH value of the medium. Cells also absorb most of the nutrients in the medium, and secondary metabolic products gradually increase, making the environment increasingly unsuitable for continued cell growth. In this case, fresh medium should be replaced.
Q3: How to replace new medium or serum for cells?
A3: Possible reasons:
(1) Since serum is a natural product, there is a certain batch-to-batch variation! Sudden replacement of serum brand or batch number may cause cells to adapt poorly or even die. Recommendation: Before replacing serum, test a sample first. When replacing, it is best to prepare the old and new batch sera into culture medium separately, mix them in certain proportions, and complete the replacement after cells adapt. At the same time, purchase more of the batch with good culture results at once to ensure stable cell culture conditions for subsequent experiments.
(2) How to replace new medium for cells? If the laboratory does not have the optimal medium for a certain cell type, you can first cryopreserve some cells under original conditions, then gradually increase the proportion of new medium: 25%, 50%, 75% to 100%, and let cells adapt after 3-5 passages.
Q4: What is the role of glutamine in the medium?
A4: Almost all cells have high requirements for glutamine. Cells need glutamine to synthesize proteins. When glutamine is deficient, cells grow poorly and die. Therefore, various culture media contain large amounts of glutamine. Here comes the important point - how to use glutamine? Glutamine is very unstable in solution and should be stored at -20°C and added to the medium before use. When medium with glutamine is stored in a 4°C refrigerator for more than two weeks, the original amount of glutamine should be added again. If used correctly, additional supplementation is generally not required, such as: store at 4°C protected from light, aliquot, and do not repeatedly preheat.
Q5:What does the color change of culture medium represent in cell culture?
A5:The color of the medium mainly comes from phenol red, which is a very sensitive color indicator with a pH range of 6-8, changing from yellow to purple-red. For convenient culture observation, phenol red allows us to intuitively sense the condition of the culture medium. If the medium is acidic, it appears more yellow; if alkaline, it appears more purple. Generally speaking, when contaminated with bacteria or when medium is not changed for a long time, the medium turns yellow, mainly because bacteria and cells overgrow, producing acidic substances. When opened medium is stored in a 4°C refrigerator, carbon dioxide in the medium gradually escapes, pH becomes more alkaline, so the medium appears more purple.
For purple-colored medium, you can bubble in sterile-filtered carbon dioxide and continue using it after adjusting the pH value. It is not recommended to continue using alkaline medium that has been unused for a long time, as it may cause cell growth arrest or death.
In laboratory cell culture, the pH value of the medium has a significant impact on experimental results. Because the pH value of the medium can directly or indirectly affect nutrient absorption, respiratory metabolism, polyamine metabolism, DNA synthesis, and plant hormone transport in and out of cells, thereby affecting callus formation and morphogenesis. When the pH value is greater than 6.5, a color reaction occurs during high-temperature and high-pressure sterilization. When the pH value is 6.8, there is a qualitative change in color. Generally, the sterilization time and pressure do not affect the medium color.
Q6:What issues should be noted in cell culture?
(1) NaHCO3 must be added to cell culture medium, which functions to stabilize the pH of the solution.
(2) Using medium color changes as an indicator for medium replacement is not sufficient but quite practical. Because the indicator phenol red exists in the culture medium, changes in liquid color can indirectly indicate the growth status of cells. When cells grow vigorously and produce acid, the liquid appears yellow; conversely, it appears purple-red.
(3) When observing cells, large pH changes in the liquid can harm cells and are detrimental to cell growth.
Q7: Why use 5% CO2 for most cell cultures?
A7: The incubator used in cell culture is generally called a CO2 incubator, with a gas environment of 95% air and 5% CO2. CO2 is both a metabolic product of cells and a component needed by cells. It is mainly directly related to maintaining the pH of the culture medium. Most animal cells require a slightly alkaline environment with a pH of 7.2-7.4, preferably not exceeding 6.8-7.6. During cell culture, as CO2 release increases, the medium becomes acidic, so NaHCO3 is often added (forming a buffer pair with H2CO3 formed when CO2 dissolves in water) to adjust pH. NaHCO3 tends to release CO2, and adding CO2 can inhibit this reaction. The CO2 concentration in the incubator should balance with the NaHCO3 concentration in the medium. If the CO2 concentration in the incubator is too high, the pH will decrease; if too low, the pH will increase.
Q8:Do all cell culture media require the same CO2 concentration?
A8:Not really. The CO2 concentration depends on the NaHCO3 concentration. CO2 and -HCO3 in the solution form a buffer system that maintains the pH of the entire culture system at a weakly alkaline level (around 7), which is also the optimal pH for mammalian cell growth. When the medium is exposed to air for a long time, -HCO3 rapidly decomposes and pH quickly increases, visually changing from red to purple. When the medium is placed back in the cell culture incubator, it slowly changes from purple back to red under the effect of CO2.
Different cell culture media have different NaHCO3 concentrations, so different cell culture media should select the corresponding CO2 concentration:
NaHCO3 (g/L) concentration <1.5, required CO2 concentration is 4%;
NaHCO3 (g/L) concentration between 1.5-2.2, required CO2 concentration is 5%;
NaHCO3 (g/L) concentration between 2.2-3.4, required CO2 concentration is 7%;
NaHCO3 (g/L) >3.5, required CO2 concentration is 10%.
Q9:As cells continue to grow, the pH value gradually decreases (due to metabolic accumulation of lactic acid), and the medium turns yellow. What should we do when we find that the pH of the culture medium is changing abnormally during the culture process?
A9:Treatment methods
(1) Check if CO2 concentration is appropriate: Adjust the CO2 percentage in the incubator based on the sodium bicarbonate concentration in the medium. For sodium bicarbonate concentrations of 2.0 to 3.7 g/L, use 5-10% CO2 respectively. Or switch to a CO2-independent medium.
(2) Check if the culture flask cap is too tight: Loosen the cap by 1/4 turn.
(3) Check if bicarbonate buffering is insufficient: Add HEPES buffer to a final concentration of 10-25 mM.
(4) Check if the medium contains incorrect salts: Use Earle's salt medium under CO2 conditions, and Hanks' salt medium under atmospheric conditions.
(5) Check for bacterial, yeast, or fungal contamination: Discard the culture and medium, and attempt decontamination if possible.
Q10: Why is DMEM medium bright red?
A10: The color of the medium is mainly indicated by phenol red, which is a very sensitive color indicator with a pH range of 6-8, changing from yellow to purple-red. Phenol red is not essential and can even be redundant in some cases. Phenol red has effects similar to steroid hormones, so it is best not to add it if cell culture involves steroid hormone-related studies. However, for convenient culture observation, phenol red allows us to intuitively sense the condition of the culture medium. If the medium becomes acidic, it appears yellow; if alkaline, it appears purple.
Q11: Why does the medium turn purple when the culture dish is placed in an incubator without CO2?
A11: The buffer system used in general DMEM is NaHCO3: 2NaHCO3=Na2CO3+CO2+H2O. In an incubator without CO2, CO2 in the medium continuously escapes, carbonic acid decreases, the solution becomes alkaline, and turns purple. Similarly, the medium in a DMEM bottle that has been opened for a long time will also gradually turn purple for the same reason.
Q12: Can purple medium still be used?
A12:You can place the purple medium in a CO2 incubator with the cap loosened. After some time, you will find the color turns bright red, and then it can be used continuously. However, do not use medium that has been unused for too long and has changed color, as the medium components may have changed after oxidation in air. During cell culture, check the color and transparency changes of the medium. Yellow color indicates pH decrease; red or purple-red indicates pH increase, cell growth arrest, or death. Generally, cells with stable growth should have medium changed every 2-3 days, while slowly growing cells can have medium changed every 3-4 days.
Q13: How to prevent medium contamination?
A13: Solutions
(1) Before and after use, disinfect the outer surface of medium and other reagent bottles with 75% ethanol. In addition, do not leave reagent bottle openings exposed for too long;
(2) Aliquot the medium and other required reagents as much as possible. If an error occurs during operation, it is best not to use it again;
(3) When handling medium, use disposable sterile plastic or glass pipettes. Use each pipette only once to avoid cross-contamination;
(4) Observe cells under the microscope daily and pay close attention to the turbidity of the medium;
(5) Avoid sharing medium and other reagents with others;
(6) Use one bottle of medium for each cell line. This can effectively avoid cross-contamination;
(7) Handle only one cell line at a time to avoid cross-contamination between cells.