Cell Contamination, Containment, and Correction: Good Cell Culture Practices in 3D | Cell Cultures | Corning

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Cell contamination is a major concern in 3D cell culture labs as well as with more traditional 2D monolayer cultures. If biological contaminants are undetected, they can significantly affect your resulting data, rendering it inaccurate or useless, or make any products manufactured by the cultures unusable.

How do you devise cell culture protocols that prevent contamination? While contamination can be controlled, it cannot be totally eliminated. So, what do you do when contamination inevitably happens?

What Are the Causes of Cell Contamination?

The major causes of contamination in the cell culture system are typically either chemical or biological. In 3D cell culture, contamination can occur in various components, such as the bio-ink or the extracellular matrix.

Chemical Contamination

Chemical contamination is the presence of a nonliving substance that may cause unwanted effects on the cell culture. Most chemical contaminants are found in the cell culture media and originate from either reagents, the water used to make the reagents, or additives to supplement reagents.

Chemical contaminants may include: metal ions, impurities, or endotoxins in media, sera, and/or water; impurities in gases used in CO2 incubators; plasticizers used in tubing and bottles; free radicals in media generated by photoactivation of various components; residue from disinfectants or chemicals used to clean equipment; or deposits left on instruments, glassware, or other equipment.

Biological Contamination

Biological contaminants fall into two categories: those that are easy to detect (such as bacteria, molds, and yeast), and those that are difficult to detect (such as viruses, protozoa, mycoplasmas, other cell lines, and insects).

Bacteria, molds, and yeast are the most common contaminants in cell culture environments. They are readily detected when antibiotics are not present, but when antibiotics are used routinely in cell culture protocols, it can cause a slow-growing infection that is difficult to detect. By the time this type of contaminant is discovered, it may have already compromised the cell culture.

Contamination Prevention in the Lab

Biological contaminants often enter cell cultures by use of nonsterile supplies and media. Sterilization procedures using autoclaving or dry heat sterilization must be followed properly.

Ensure proper maintenance and operation of sterilization autoclaves and ovens. Don't overpack the autoclave, which can cause uneven heating and thus fail to sterilize the entire contents of the autoclave. Be sure to use a long enough sterilization cycle for the materials and liquids you are sterilizing — often, a viscous liquid or large volume of liquid is understerilized, which can lead to contamination. To maintain sterility, store the supplies and solutions properly in a dust- and insect-free area.

Using good aseptic technique is also important, of course. Airborne particle risk can be minimized by washing hands, and donning gloves and clean lab coats. Minimize antibiotic use. Work with one cell line at a time. Make sure to monitor routinely for contamination.

What Are Proper Post-Contaminant Procedures?

When you've discovered contamination, autoclaving is recommended to cure the contaminated culture and prevent it from spreading to other cultures. Occasionally, you may need to attempt to save a valuable culture that has become contaminated. This is usually only tried with mycoplasma infections, as fungus and yeast contaminants are unlikely to be eradicated, bacterial contaminants are often already antibiotic-resistant, and many cultures lose critical characteristics after cleanup. Mycoplasma contamination is most often treated with antibiotics, but no approach is totally successful at eradication. What's more, treatment does not typically completely eliminate infection, but pushes it to below-detectable levels. Over time, the remaining mycoplasma that survive can recover and develop into a more serious contamination problem.

Understanding cell culture contamination best practices ensures that your research quality does not have to suffer due to an unexpected occurrence, whether big or small. The most important takeaway, however? Prevention is indeed the best medicine.