Preparation: Cell Passaging, Expansion and Culture Contamination

Preparation of cell culture encompasses the fundamental techniques necessary to ensure confidence in experimental results. The quality of your cell cultures directly translates into the reproducibility of your experiments. Some fundamental preparation considerations include:

  • Contamination prevention--no matter the scale of your culture
  • Sterile technique for the preparation of media and reagents and for the sterile passaging of cells
  • Effective techniques and solutions for freezing and thawing cell lines

For biological insights you can trust, we offer cell culture tools that enable you to set the stage for trustworthy experimental data, improve your productivity, and/or ensure you are choosing the right solutions.

Preparing Media

The cell culture media you choose is one of the most important aspects of your experimental design. Your choice should depend on the type of cells to be cultured and the purpose of the culture. Different cell lines and cell types have highly specific growth requirements, and a number of media formulations are available to meet the growth requirements of the cells you’ve chosen. You can buy ready-made media or prepare it from a powered form in your lab. In latter case, water is a major component of the media, buffers, and additives you use with your cells, and the quality of water plays a vital role in any cell culture experiment.

Use high quality water that is free of contaminants to ensure your cells are healthy and behave consistently from one experiment to the next. 

Contamination from chemicals, or other biological agents, that are undetectable by the unaided eye, such as endotoxins, inorganic ions, and organic compounds, may affect the growth, morphology, and behavior of cells. Typical mains water contains 1-10 IU/mL of endotoxin contamination, but international standards<link> dictate <0.1 IU/mL of endotoxin contamination in cell culture water. This means that your water purification system needs to eliminate this endotoxin from the water you use for your cell culture. Using the right water filtration system helps you grow healthy cells for consistent and reproducible cell culture experiments.
 

Tools to Make Your Media and Buffer Preparation Reliable and Free of Contamination

Arium® Mini Ultrapure Water System

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Arium® Pro Ultrapure Water System

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Midi Plus Pipetting Controller

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Secura Micro Balance

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Passaging Cells

Whether you are working with adherent or suspension lines, cultured cells need to be regularly passaged or subcultured. Unfortunately, passaging presents a contamination challenge because the culture flask is opened, cells are handled, and new media and components are added. All of these steps allow the chance of introducing contaminants.

Following proper aseptic technique, working quickly, and using the best tools and uncontaminated reagents will help reduce the chance of introducing contaminants into your cell cultures. In particular, using proper aseptic pipetting technique, with sterile filter pipettes and pipette tips that allow you to work quickly and efficiently, is essential.

Tools That Make Aseptic Technique Easy

Tacta Mechanical Pipette

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Midi Plus Pipetting Controller

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Sterile Filter Tips

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Picus Electronic Pipette

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Centrisart Centrifuge

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Avoiding Contamination

Stop contamination before it stops your experiment. One of the major challenges in cell culture is the risk of contamination. Contamination comes in many forms: bacteria, yeast, mold, chemicals, other biological agents, cell line cross-contamination, or a combination of the above. Contamination may affect the growth, morphology, and behavior of your cells, and ultimately, compromise your experimental results and/or slow your project.

Passage cells completely outside the hood: avoid introducing contaminants and improve efficiency

You can passage cells aseptically without ever needing to open a flask or enter a hood, with our innovative cell expansion system. To prevent contamination during passages outside the biosafety cabinet, this system combines tubing for aseptic fluid transfer and a filter cartridge for gas exchange in the cap pre-assembled on plastic Erlenmeyer shake flasks.

You will observe equivalent growth rates between our MYCAP CCX system and traditional Erlenmeyer flasks. Media fill, inoculation, sampling, and transfer from flasks can all be done aseptically outside the biosafety cabinet by using tube welders or aseptic connectors.

Eliminate the need for back-up flasks and redundant passages

The features of MYCAP CCX system allow you to almost completely eliminate contamination risks during pasaging which means more consistent results and increased efficiency, regardless of the scale of your experiments. Eliminate your need for a biosafety cabinet during passaging, thus lowering your experimental costs and streamlining your operations. 

Tools That Stop Bacterial and Mycoplasma Contamination

Arium® Mini Ultrapure Water System

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Sartolab Vacuum Filtration System

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Microsart Mycoplasma Rapid Detection Kits

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Minisart Syringe Filter

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Arium® Pro Ultrapure Water System

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Bacterial contamination

Bacteria are the most common contaminants. Bacterial contamination is usually visually evident: The culture becomes cloudy or turbid and/or the medium color changes, indicating a drop in pH. Most bacteria are also visible under the microscope.

Once bacterial contamination has been observed, the culture must be replaced and  decontamination begun, an expensive and time-consuming process. 

To avoid bacterial contamination:

  • Be diligent with your technique
  • Use only sterile reagents
  • Beware general lab stocks
  • Use sterile filtration
  • Use fresh ultrapure water for your stocks

Mycoplasma contamination

Mycoplasma are small (0.15 µm-0.3 µm), with no cell wall and a flexible membrane. These characteristics make mycoplasma contamination difficult to detect with a microscope. Their small size allows them to proliferate to high concentrations without any obvious change to the culture’s appearance.

Elimination of mycoplasma contamination from a culture is almost impossible. They are resistant to most antibiotics and can survive liquid nitrogen without cryopreservation, allowing them to contaminate other cells stored in the same liquid nitrogen container. 

Using a mycoplsma-specific test on your cell cultures allows you to know immediately whether your cells are contaminated, preventing wasted time, reagents, and effort.

To prevent mycoplasma contamination of your cell lines:

  • Routinely test cultures
  • Quarantine and test any new cultures from outside sources
  • Discard mycoplasma positive cultures

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