Cell Culture Media Change Protocol | Corning

Cell culture media contains the amino acids, carbohydrates, inorganic salts, pH buffering, and growth factors that cells need to thrive. Changing the media during application is a common and essential task in any cell culture lab.

Here's what you need to know about cell culture media change protocol, plus some pointers and best practices for maintaining healthy cell cultures.

How Often Should You Change Cell Culture Media?

Hilary Sherman, Senior Scientist at Corning Life Sciences, explained that, for most cell lines grown in standard basal media, it's okay to change media on a schedule or as needed. The choice depends on the researcher's preferences and their knowledge of the cells they are growing.

However, she noted that "some cell types are extremely sensitive or are grown in media that includes components that degrade very quickly at 37°C. Those cells will typically require media changes daily or every other day." Stem cells have, historically, been an example of this, but new media formulations with more stable growth factors allow for slightly less frequent media changes in stem cell cultures.

Keep an eye on the color of your culture media. Cell culture media includes a buffering system, but cells release acidic waste products over time, and the pH of the media will eventually drop as waste accumulates. Many cell culture media include phenol red, a pH indicator that appears red when the media pH is within a healthy range but will take on orange or yellow hues as the pH drops. You should change the media if you see this color change. More metabolically active cells will accumulate waste more quickly and will likely require earlier media changes.

Cell Culture Solutions, Steps, and Techniques Guide

What Happens If You Forget to Change Media?

If culture media is not changed when needed, nutrients in the media will be depleted and waste products will build up. This will eventually impact the health of cultured cells and stop them from growing.

"In the case of stem cells," Sherman said, "they're very sensitive, and there are components in that media that communicate to the cells to maintain their pluripotency. If that media is not getting changed, those cells might not get those signaling molecules and, thus, could start to differentiate into an undesired cell type, or it could impact the health of the cells."

Cell Culture Media Change Steps

For most cell culture media changes, there are standard steps to be followed. First, prepare by warming the new media to 37°C, or at least to room temperature, before adding it to the cells. This is important because cold media can "shock" cells. Either warm the new media in a water bath or simply leave it at room temperature for several hours.

One disadvantage of a water bath is that it can be a contamination risk. "Water baths can be a potential source of contamination because all the things that our cells love — warm liquid, nutrients — are also loved by bacteria, yeast, and fungi. It's really important to make sure you're cleaning that water bath and maintaining it," Sherman said.

When the media has come to temperature, remove the cell cultures from the incubator.

Inspecting Cells

Sherman explained that it's important to visually inspect cultures before bringing them into the tissue culture hood. This can be done either with the naked eye or under a microscope. Check for signs of potential contamination, such as cloudy media or visible fungal growth.

"The last thing I want to do if I have a contaminated culture is then bring that into my laminar flow hood, use my reagents, and risk potentially spreading that contamination to other cultures," she said.

Also, check to make sure cells have normal morphology and the overall culture has a healthy appearance.

While looking under a microscope before a media change is optional, it can provide helpful information, including a better understanding of the health of cells and their growth rate. This can help researchers plan the next steps in their experiments.

Work Within the Laminar Flow Hood or Biosafety Cabinet

Next, transfer cells to a laminar flow hood or biosafety cabinet. Use aseptic technique for all steps performed in the hood. Doing so will help prevent bacterial or fungal contamination as well as cross-contamination with other cell lines.

Then, remove the old media. The procedure for doing this differs slightly between adherent and suspension cell cultures. With adherent cells, simply aspirate away the spent media using a pipet or aspirator. Avoid touching the growth surface to prevent any damage to cells.

With non-adherent cells, either wait until cells have settled to the bottom of the vessel by gravity or use a centrifuge to pellet the cells. Then, carefully remove the old media while avoiding disrupting the cells.

Next, add new media. As a starting point for most vessels, the volume of media should typically be between 0.2 and 0.3 mL of media per square centimeter of cell culture surface. Using too little media can lead to rapid waste buildup and pH drop, potentially harming cells. Using too much media can make it difficult for gases to diffuse down to the cells. Media can be expensive, so regularly using too much can also have a financial impact.

Return cells to the incubator. Finally, clean up the area, properly dispose of spent media and any other waste, and refrigerate unused media.

What About Switching Cells from One Media Type to Another?

A media change in cell culture is possible, but Sherman suggests transitioning the cultures gradually so as not to "shock" the cells when switching cells from one media formulation to another.

"For example, if you're trying to reduce the serum concentration in your culture, instead of going from 10% FBS to immediately 2% FBS," Sherman explained, "you might do it in a gradual exposure where each day or every couple of days you change the media to slightly less FBS."

Encourage Cell Culture Success with Corning

Although these steps represent a good general procedure for most cell culture media change protocol, some aspects of media change procedures may differ based on the specific needs of an experiment or cell type.

To learn more about cell culture and find protocols, resource guides, and product information, visit Corning's Cell Culture Products webpage. Or, explore the Corning product catalog to find media options and an extensive selection of cell culture supplies.