Troubleshoot cell growth | PHCbi

Your Cells Don't Grow: Now What? Evolving Science For The Future | Articles

13 Apr 2021

Cell culture has evolved from its humble beginnings as "just another laboratory method" to the driving force behind basic research, diagnostics, cell-based assays, cellular therapies, and biomanufacturing.[1] Practically every industry involved in food, medicine, health, consumer goods, and even industrial materials relies on cell culture or cell-based assays.

As cell culture applications grew, the supplier industry responded with cultureware, media, nutritional supplements, reagents, antibiotics, and instrumentation for isolating, manipulating, expanding, and characterizing cells—not to mention cells designed specifically for research or manufacturing.

We tend to think of cell culture as formulaic—find a recipe, follow it, and voila! The reality is often very different.

Cells are living, respiring entities with their own genetic destinies. Even experienced cell biologists encounter problems keeping cells growing and functioning. Cell culture crashes are not a matter of if, but of when.

Why Cell Cultures Fail

Cell cultures fail for one of two reasons—culture conditions or the cells themselves.

The first step in solving growth problems is to check to confirm that the protocol was followed to the letter, with special attention to the timing and duration of each step. Next, make sure that reagents, labware, and anything else that comes into contact with cells has been prepared according to specifications.

Next check media, supplements, and feeds to see if any of these critical ingredients changed from previous runs. Just because you use the same medium from the same vendor doesn't mean it's the same. Batch-to-batch consistency is a significant problem in the production of culture media,[2] particularly with undefined media based on bovine serum albumin or other animal-derived products. Many manufacturers of growth media have developed serum-free or even chemically defined media to replace traditional media.[3] Because these products are now widely used in biomanufacturing they are catching on with research and development groups as well.

Variations in a medium's metal or mineral content is another source of medium inconsistency that may affect cell growth. Metals can catalyze or inhibit enzymatic processes, so too much or too little can slow down cell growth, or even cause cells to die.[4] Medium suppliers often provide a certificate of analysis that includes mineral content, but some only list concentrations of metals that are supposed to be in the formulation, not trace metals. If the current medium is from the same batch and lot as last time, metal contamination probably isn't the problem.

Microbiological Contamination

Microbial contamination is a major cause of cell culture failure, and one of the most difficult issues to diagnose.[5] Contamination takes many forms, from chemical adulteration of ingredients (e.g. trace metals), to microbiological contamination, to contaminated cell lines.

Microbial contamination has so many potential sources that even after confirming its presence, identifying the source may be next to impossible. Almost anything that comes into contact with cells may be a source, including cultureware, spatulas, contamination from work surfaces, improperly sterilized media or nutrient additives, and of course human beings.

What makes glove boxes, biosafety cabinets, incubators, and ductless hoods indispensable for cell culture also makes them suitable for the growth of unwanted microorganisms. Manufacturers of these enclosures have designed them for cleanability by eliminating nooks and crannies where molds and bacteria could take up residence.[6] Work surfaces are typically made of easily cleaned materials like stainless steel or bacteria-resistant resins. Many enclosures have built-in cleaning protocols, for example high temperature or disinfectant cycles that use ultraviolet light or gaseous hydrogen peroxide. All such enclosures can schedule the cleaning cycle to occur over nights or weekends. Protocols for disinfecting biological workspaces are readily available online.[7]

When working with such enclosures, minimize the number of times the "door" is opened to introduce or remove items to the workspace. If you suspect that contamination originates in your incubator pay special attention to the cleanliness of doors and passageways.

Beware of Mycoplasma

Of the many microbiological contaminants that destroy cell cultures, mycoplasmas are the most feared. Mycoplasma contamination not only ruins experiments, it can also shut down an entire enterprise.[8]

Mycoplasmas, which lack cell walls, are the smallest organisms and the simplest bacteria known. Their size, about 100 nm across, makes them impossible to detect with optical microscopy, so they almost always go undetected when visually inspecting cells.

Mycoplasmas enter cultures similarly to other contaminants, but the major source is the cell line itself. Up to 85% of certain cell lines may be infected.[9] Cell biologists can eliminate most of the risk of mycoplasma contamination by regularly checking cultures for infection and adhering to strict aseptic technique. Checking culture media and feeds, and all their ingredients—particularly for animal-derived components—is also a good idea.

A lab will typically enter a decontamination protocol after a mycoplasma infestation. In some cases, when cells are rare, investigators may attempt to salvage the cells through antibiotic treatment.[10]

Non-Authenticated Cells

Last but certainly not least on the list for why cells don't grow is the cells themselves. We've already touched on adherence to culture protocols, which includes proper storage, thawing, and freezing of cells. Cells expand at varying rates but whether the critical growth stage is mostly linear or mostly exponential, the fewer viable cells present, the shallower the growth curve will be.

Cell-line authenticity is a very serious problem in cell culture, and a major contributor to the reproducibility crisis in the life sciences.[11] It does not take long for even small numbers of contaminating cells to completely take over a culture. Thanks to the rapidly falling cost of genetic analysis most, if not all, commercial cell-line suppliers conduct formal cell-line authentication and supply documentation to that effect. Researchers should request similar data from non-commercial sources of cell lines as well, for example cells obtained from other research groups.


Many industries rely on cell culture for research, development, and manufacturing. Along the narrow, winding path to successful cell-line expansion even minor excursions can mean failure. Following protocols strictly improves your chances for success and can even help in identifying causes of failure when it occurs.

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