The Ultimate Buyer's Guide for Purchasing 24 well cell culture plates

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13. What is the general composition of cell culture medium?

Cell culture media consist of a complex mixture of salts, carbohydrates, vitamins, amino acids, metabolic precursors, growth factors, hormones, and trace elements. Formulations range from simple mixtures with the minimum requirements for many cell lines to complex serum-free mixtures for specific cell lines. Carbohydrates are primarily supplied as glucose. Galactose may sometimes be used as it metabolizes slower, reducing lactic acid buildup. Other carbon sources include amino acids (especially L-glutamine) and pyruvate.

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ATCC Media Brochure

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14. What is meant by the phrase "complete medium?"

Most cell culture media are sold as basal media, which is a complex mixture of salts, carbohydrates, vitamins, amino acids, metabolic precursors, and trace elements. To enhance cell growth, you must add growth factors, hormones, and other proteins, usually by supplementing with serum at the specified concentration on the product sheet. Additional supplements may also be needed as indicated on the cell culture's product sheet or catalog description.

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ATCC Animal Cell Culture Guide

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15. How does the sodium bicarbonate-carbon dioxide system buffer the pH of cell culture medium?

Sodium bicarbonate is used to stabilize pH in cell cultures. Cells produce CO2 but need only small amounts for growth and survival. CO2 levels affect the medium's pH; increasing CO2 lowers pH, and decreasing CO2 raises pH. As cells metabolize, they produce CO2, making the medium more acidic. Phenol red can indicate pH changes by turning yellow. CO2 dissolves in media, forming carbonic acid, thereby buffering the medium. Sodium bicarbonate dissociates into sodium and bicarbonate ions, helping to raise pH when more bicarbonate is added. The concentration of sodium bicarbonate must match the CO2 level in the incubator. For media with 1.5 to 2.2 g/L sodium bicarbonate, use 5% CO2; for 3.7 g/L, use 10% CO2. If sodium bicarbonate is too high for the CO2 level, the medium becomes more alkaline.

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ATCC Animal Cell Culture Guide

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16. Are there tissue culture media that do not require a CO2 incubator?

Some cell lines can be maintained on alternative media, such as:

• CRCM-30 medium
• L-15 medium
• CO2-independent medium (Invitrogen Life Technologies Cat. No. 18045088)

You can determine if a medium is satisfactory by testing it with the cell line for 3 to 5 passages. However, very low concentration cultures (e.g., cloning) often require CO2 in the gas phase. Alternatively, a 5% CO2 gas tank can stream filtered CO2 into the gas phase above the medium before sealing the flask.

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ATCC Animal Cell Culture Guide

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17. What is insulin and why is it used in cell culture media?

Insulin is a polypeptide hormone found in vertebrates and invertebrates. It promotes glucose and amino acid uptake. The Dictionary of Cell Biology defines insulin as:

1. Hormone found in mammalian serum.

2. Secreted by B cells of the pancreas in response to high blood sugar levels.

3. A mitogen-activator that commits the cell to G2 and mitosis.

4. Has sequence homologies with other growth factors.

5. Frequently added to cell culture media for demanding cell types.

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18. Why are antibiotics or antimycotics added to cell culture medium?

Antibiotics and antimycotics are added to prevent contamination, cure contamination, induce recombinant protein expression, or maintain selective pressure on transfected DNA. ATCC avoids using antibiotics/antimycotics routinely but acknowledges their necessity in specific situations. If you choose to use them, penicillin-streptomycin solution is recommended at final concentrations of 50-100 I.U./ml penicillin and 50-100 μg/ml streptomycin.

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ATCC Animal Cell Culture Guide

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19. What are the advantages of serum-free media over regular, serum-containing media?

Serum-free media have several advantages:

• Serum is an undefined supplement with batch-to-batch variability.
• Serum-free media maintain physiological consistency with known components.
• Availability is not dependent on animal conditions.
• No risk of contamination from viruses found in serum.
• Allows for creating selective media for specific cell types.
• Facilitates the regulation of proliferation and differentiation.

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ATCC Media Brochure

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20. What is conditioned medium? Why is it recommended for my cells?

Conditioned medium is spent media from cultured cells containing secreted metabolites, growth factors, and matrix proteins. Many cell lines are dependent on these components. For example, LADMAC conditioned medium contains CSF-1, aiding macrophage progenitor proliferation and differentiation. Mouse embryonic stem cells benefit from LIF, maintaining their undifferentiated state. Omission of conditioned medium can lead to unfavorable results like slow growth or quiescence. Conditioned media often provide more than one essential growth factor, crucial for maintaining cell health.

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ATCC Animal Cell Culture Guide

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21. What medium formulation should be used for culturing cell lines?

ATCC generally lists either the medium recommended by the originator of the cell line or a standard effective medium formulation. Variations in media formulations among suppliers can affect growth and function. Always read catalog descriptions and labels carefully. The recommended media for a cell line can be found on ATCC's product page under the "Culture Method" tab.

For Hyclone media formulations of common cell culture media, visit here.

ATCC Animal Cell Culture Guide

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22. How do I determine the amount of medium to add or the split ratio when culturing suspension cell lines?

The amount of medium to add or the decision to split your suspension culture depends on the optimal density range for logarithmic growth, the number of cells in your flask, and the amount of medium needed to reduce the cell density. Follow these steps:

• Perform a cell count and determine viability by dye exclusion on the day you will add medium or split the culture.
• Calculate viable cells/ml and determine how many milliliters of fresh medium to add to lower the cell density.
• Decide whether to add fresh medium to the same vessel or split the suspension into multiple vessels.

For example, if your suspension cell line should be maintained between 2 x 10^5 and 1 x 10^6 viable cells/ml, and your medium volume is 10 ml with a cell count of 3 x 10^5 viable cells/ml, you have several options:

• Wait another day before manipulating the culture.
• Centrifuge and resuspend the cells in the same volume.
• Add 5 ml of fresh medium to reduce the concentration to 2 x 10^5 viable cells/ml.

If you chose option #3, your vessel would now hold 15 ml of medium. Depending on the vessel size, that is probably okay. However, if your cell density is 1 x 10^6 viable cells/ml, you would need to add 40 ml to lower the density to 2 x 10^5 viable cells/ml, which would require splitting the culture into multiple vessels.

More info

ATCC Animal Cell Culture Guide

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