Addressing Four Common Challenges with Cell Culture Reagents

Reagents are indispensable "stuff" in cell culture experiments, and various problems are inevitably encountered during their use. Based on this, Pricella has summarized the common problems with reagents and their solutions as follows.

(1) Out of 10 vials of penicillin-streptomycin (P/S) solution (Cat. No. : PB180120) stored simultaneously at -20°C, 9 vials froze while 1 vial did not.

Reason: This scenario is probably due to supercooling, as water contains numerous dissolved salts. Under standard conditions, water's freezing point is 0°C. However, even below this temperature, the presence of dissolved salts can prevent freezing, resulting in supercooling. If the water is relatively pure and left undisturbed, it can remain in liquid form even at temperatures slightly below zero degrees.

Solution: When the P/S solution is out of the freezer, any agitation disturbs the stable state of the water, causing it to freeze rapidly. Furthermore, when the product is opened, the temperature difference leads to a quick release of heat from the water, resulting in a significant temperature drop and potential freezing. To address this issue, handle the solution gently to avoid agitation, and allow the vial to reach room temperature before opening to reduce temperature differentials.

(2)After high-pressure sterilization, precipitates appear at the bottom of the MEM medium (Cat. No.: PM150411).

Reason: The sequence of adding components during the preparation of the basal culture medium may lead to the formation of insoluble molecules. Calcium salts, in particular, are prone to precipitation; for instance, the reaction between CaCl2 and MgSO4 in solution results in the formation of CaSO4 crystals. High-pressure sterilization and unstable pH exacerbate this issue. Under high temperature and pressure conditions, the internal salt solution and some amino acids, vitamins, etc., undergo chemical reactions, leading to precipitation.

Solution:

1.Dissolve CaCl2 separately in deionized water before adding other culture medium components; alternatively, add a buffer solution to avoid changes in osmotic pressure.

2.High-pressure sterilization may destroy amino acids and other components in the medium, rendering most of the nutrients inactive. In such cases, the basal culture medium cannot be used and should be sterilized by passing through a 0.22μm filter.

Notes:

1.When culturing cells, always follow the instructions to select the optimal storage and operating methods for the culture medium.

2.Avoid repeated freezing and thawing.

Figure 1. Precipitation Formation in the Culture Medium

（3）Phenol red-containing trypsin (Cat. No.: PB180224) turns yellow after freezing. Reason: The solvent for this trypsin solution is PBS, a phosphate-buffered system, which turns light yellow after freezing. Additionally, if the product is transported using dry ice, a small amount of CO2 may enter the solution during transportation, causing a slight decrease in pH value.

Solution: The resulting light yellow trypsin solution will turn into an orange-red liquid after complete dissolution, indicating it can be used normally. If the solution remains yellow even after complete dissolution, it can still be used. Alternatively, adjust the pH with sterile 2M sodium hydroxide solution or contact the manufacturer for replacement.

Figure 2. Trypsin Turns Yellow after Freezing

Notes:

1.Different tissues or cells may have varied reactions to trypsin; therefore, determine the optimal digestion time based on actual conditions during operation.

2.The digestion time should be appropriate; excessively long or short digestion times can affect cell adhesion and growth.

3.Avoid prolonged storage at room temperature or under 4°C conditions.

4.Thaw at 2°C-8°C, shake well before use, and avoid repeated freezing and thawing. For small quantities, it's recommended to aliquot.

（4）L-glutamine solution (Cat. No.: PB180420) precipitates after dissolution.

Reason: After thawing at 2°C-8°C, the solution turns into an emulsion, which is a normal phenomenon. This occurs because the solubility of L-glutamine decreases due to temperature fluctuations, leading to precipitation.

Solution: Wipe the surface of the solution with 75% alcohol, shake well, and then place it in a 37°C incubator. After 30-40 minutes, the solution should dissolve into a clear and transparent state. Shake well before use. Avoid repeated freezing and thawing. For small quantities, it's recommended to aliquot.

Figure 3. Precipitation Formation after Dissolving L-glutamine Solution

Notes:

1.Avoid prolonged storage at room temperature or under 4°C conditions.

2.Dilute as needed before use.