Cells including stem cells are grown in liquid solutions called media and stem cell culture medium composition is crucial since the ingredients have a powerful impact on the cells. The media affects the cells’ identities and functions. It also impacts the risks of using specific regenerative products.
My first job in science was isolating and growing human umbilical cord vein endothelial cells or HUVECs. We call this cell culture or tissue culture.
Ever since one of my favorite things in research has been to grow cells and take pictures of them on the microscope. Immunofluorescently-stained cells are like bio-art under the microscope.
Most often with stem cell culture, we are trying to keep their “stemness” intact. Then if want them to turn into specific cells, we switch the media composition to induce a certain type of differentiation. Inducing differentiation into a specific type of neuron, muscle, or blood cell, for example, in each case requires an exact media formula. Overall, cell culture media have major implications for both basic research and clinical science.
In today’s post, I’m going through all the key info about stem cell media and how they are unique.
Cell culture media generally
In general, cell culture media contains a few core components.
There is the base medium, which is the liquid that is the starting place for making any media recipe. These base media go by funny names, sometimes reflecting who devised them. There’s DMEM, MEM, RPMI, and so forth. For example, DMEM is an acronym that stands for “Dulbecco’s Modified Eagle Medium.” It was named after Renato Dulbecco an Italian-American scientist who won the Nobel Prize. He didn’t win for devising DMEM, but rather for his work on cancer-causing viruses. Physician scientist Harry Eagle is also part of that media name and deserves major credit here for pioneering media studies.
No matter which media type, think of the base medium as the starting broth for our stem cell soup. These media bases usually contain buffers to avoid wild swings in pH, color indicators that readily show if the pH is changing, sugar, salts, and other core components.
To this base, we add additional ingredients that particular cells need. These additives can include growth factors, glutamine, antibiotics, and other specific chemicals that particular cells need.
Often we add a very general ingredient called fetal bovine serum or FBS. For example, adding FBS to make it represent 10% of the final bottom of cell culture medium is very common. The reason for adding FBS is that it has high concentrations of growth factors that stimulate cells to proliferate. FBS has millions of molecules, many at functionally meaningful concentrations, so adding it can complicate things too.
When I first grew cells as a lab tech at UCSD, the HUVECs we worked with required human serum rather than FBS. Getting the human serum was an unusual experience. But that’s a story for another time.
Stem cell culture medium composition
What’s different about stem cell culture medium composition?
It typically has some of the same ingredients as regular cell culture medium. However, much depends on the type of stem cells. Certain kinds of stem cells cannot tolerate the presence of FBS, for example. They lose their stemness as serum contains pro-differentiation factors. In such cases, we often add purified growth factors into the media instead. Some vendors also sell defined media that needs no addition of FBS as they already have growth factors. You can add a few specific growth factors to media too yourself. Defined media is very helpful for growing cells for potential clinical applications.
Pluripotent stem cells like iPS cells or embryonic stem cells are highly sensitive so they can require specific combinations of growth factors. Recently we were comparing two media types for our human iPS cells as you can see in the photo above. We’ll see if one is better than the other.
We even sometimes grow iPS cells literally on top of other cells called “feeder cells.”
These feeder cells, as their name suggests, feed the stem cells by releasing lots of growth factors. The feeders can also provide other good stuff like extracellular matrix that stem cells love. Feeder cells are typically mouse embryonic fibroblasts or MEFs that have been irradiated. Nuking them first at a non-lethal dose makes the MEFs unable to grow. However, they are still alive and secrete the good stuff. Taking away their ability to grow keeps their contribution to the overall mixed cell culture to a minimum. However, we sometimes take extra steps to try to remove them prior to experiments.
While feeders aren’t technically part of the stem cell culture medium composition, in a way they are functionally part of it because their secretions supplement the media.
Clinical use and stem cell culture medium composition
Stem cells intended for clinical use are typically grown in growth factor-rich media rather than on feeders. Ideally, this would also be without FBS as you typically do not want bovine proteins in the mix for a cell therapy for people.
Some stem cells are also grown without antibiotics as they are sensitive to those chemicals as well. This makes life tricky though because we use antibiotics to keep our cultures from getting contaminated with bacteria. Even under careful cell culture conditions, there can be some bacteria around that can get into the media. Other potential pathogens are also of concern for stem cells intended for clinical use too including fungi. It’s such a bad feeling to check on your cells in the morning and find they are contaminated.
As one differentiates stem cells into a specific product that will be transplanted into patients, it’s crucial to make sure there are no remaining stem cells present. You also need to know that your cell therapy product is ideally just one desired cell type like retinal cells or a certain kind of neuron. Unless your cell therapy is intended to be a mixture of cells.
One of the concerns about unproven stem cell clinics, especially those that grow cells before giving them to patients, is that the personnel may not be using good manufacturing practices (GMP, cGMP, etc.) We’ve seen in FDA inspection reports and warning letters that some clinics or suppliers often fail on many levels with cell culture or even just cell isolation. The results can be disastrous such as with dozens of people getting septic from a Liveyon product.
Overall, stem cell culture media are a fascinating element of the regenerative medicine field. Media research probably needs even more attention.