Culturing and Maintaining Stem Cells

Stem cells—from healthy donors as well as patient-derived—are frequently used in basic research, drug discovery, and therapeutic applications, yet their culture can be challenging. This article will provide tips on how to culture stem cells in the lab to ensure reproducible and reliable results.

Under the right conditions, stem cells will either retain their stemness—self-renew—or differentiate down a specific pathway. These cells come from sources ranging from embryos to connective tissue, or can be created by de-differentiating already committed lineages.

For practical purposes, most stem cells can be divided into one of two types: adult stem cells and pluripotent stem cells (PSCs), notes James Chen, CEO of ScienCell. The latter category is made up of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). “Those cells basically use the same culture conditions,” he says.

Feed(er) me

“Culturing stem cells is an art, and we are still in its infancy,” notes Lindy O’Clair, Head of BioAnalytics Reagents and Consumables, Product Management BioAnalytics Workflows, Sartorius. “With that being said, considerations in quality source material are of utmost importance. Understanding the reagents and their impact on cell culture viability and reproducibility, as well as culturing within equipment that can be relied upon, will allow for confidence as well as reproducibility in data.”

Historically PSCs were cultured on a layer of feeder cells—typically mitotically inactivated fibroblasts—a technique still in some use today. These feeders provided not only a physical support matrix, but would also condition the media with (sometimes unidentified) growth factors needed for the stem cells to thrive and remain undifferentiated. In addition to questions of reproducibility, some drawbacks of a feeder cell system include that it is labor-intensive and requires the feeder surface to be prepared ahead of time. It can yield mixed cultures that may be challenging to separate. And in the case of cross-species feeders (mouse fibroblasts with human PSCs, for example) there is a risk of transmitting pathogens.

Feeder-free cultures are easier and less time-consuming to set up and maintain, more reproducible, more easily scalable, and do not suffer from the possible pitfalls of co-cultures.

Yet absent a feeder cell layer, PSCs will not adhere to common tissue culture surfaces. A large spectrum of matrices—typically derived from or mimicking extracellular matrix (ECM) proteins, singly or in combination—are used as a physical support, either for a (relatively) planar culture or even a more three-dimensional culture. Researchers can coat cultureware themselves using their choice of matrix at their choice of dilution, depending on the protocol they are following, or purchase cultureware pre-coated.

And absent feeder cells, conditioned medium has been replaced by a defined stem cell culture medium, based on the pioneering work of James Thomson at the University of Wisconsin. Several companies now offer similar commercial media for culturing PSCs, says Chen: “Basically this is DMEM/F12 and supplements,” with the principal supplement being fibroblast growth factor (FGF).

Culturing PSCs

While there are some select vendors from which iPSCs can be obtained, most people isolate their own—there are plenty of good protocols readily available from colleagues and online, comments Chen.

The resulting cell lines can vary one from another based on a number of factors, according to the ATCC Stem Cell Culture Guide: Tips and Techniques for Culturing Stem Cells. Among these may include genetics, genomic foot-prints, and culture conditions, as well as the method used to de-differentiate (re-program) the cells back to a pluripotent state.

But there are some nearly universal ways in which PSCs are cared for.

A few tips

Cultures should be examined daily. Things to look for include monitoring for morphological changes, which may mean those cells may be differentiating—less than 10% of the cells should be differentiating. Undifferentiated PSCs grow as compact colonies, with high nucleus to cytoplasm ratios and prominent nucleoli. Small colonies may initially seem loose but will tighten up as they grow. Because colonies tend to pile up and layer as they grow, their centers tend to appear bright.

Cells that are showing signs of differentiation, and those that are not attached, should be removed—a good way to do this is to mark the area and then suction them off using a 200 microliter pipette tip on the end of an aspirating pipette.

Media should be completely changed every day or two (depending on who you ask).

Monitor for confluence. Cells should be passaged when they reach about 80% confluence or after five days or so. Handle the cultures gingerly, using an enzyme-free method (utilizing a chelating agent like EDTA or EGTA) or a gentle enzyme solution like Accutase to lift cells off the plate. Keep the cells in clumps (they’re less likely to differentiate), and minimize pipetting cells up and down when passaging. ROCK inhibitor should be used when cells are recovering from being passaged or thawed (but is not necessary during simple media changes).

When passaging, cells should be split at a 1:4 ratio, and frozen down—in clumps—using cold stem cell freezing medium at a rate of 1 degree per minute.

Know what you’ve got

Morphology can differ based on the culture conditions as well as cell state. Many researchers rely on panels of pluripotency markers to verify their cells’ stemness, points out O’Clair—a time-consuming and costly process that requires the cells’ sacrifice. (Even more so are functional assays in which stem cells are made to differentiate to more committed phenotypes.) She “eagerly awaits the advent of technology that can identify and accurately assess stem cell culture status.”

“Other” stem cells

Adult stem cells—self-renewing multipotent but somewhat committed cells, isolated from somatic tissue—are an attractive alternative to PSCs, points out John Ludlow, Vice President, Regenerative Medicine at Zen-Bio. The raw material may be essentially unlimited, and they are often far less finicky to culture—often on regular tissue cultureware, using standard commercially available media and growth factors contained within the fetal bovine serum. Depending on the source, they can be made to differentiate—partially or fully—down selected pathways to become, for example, adipocytes or neurites, monocytes, or hepatocytes.

Adult stem cells and their progeny can be used to generate material such as enzymes, for example, Ludlow explains. They can be used for functional assays, and pharmacologically agonized or -antagonized to modulate that function. They can be made to migrate across membranes, or used in animal studies to examine their toxicity profile.

Stem cells in whatever their variety—pluripotent, adult, even cancer stem cells or those yet to be discovered—will inevitably be exploited for a host of applications ranging from gaining a better understanding of basic biology, health, and disease, to mitigating or curing disease. Growing and maintaining them, while an art, needn’t be rocket science.