Induced pluripotent stem cells (iPSCs) are cells created from adult cells, such as those found in skin or blood. These adult cells are reprogrammed back to an embryonic-like state, giving them the ability to develop into nearly any cell type in the human body, from heart muscle to nerve cells. This process enables their use in understanding diseases and developing new treatments. To cultivate these cells in a laboratory, a specific liquid environment called cell culture medium is required. This medium provides all the necessary elements for iPSCs to survive, grow, and maintain their undifferentiated state.
Core Components of iPSC Medium
The foundation of iPSC culture medium is a “basal medium,” such as Dulbecco’s Modified Eagle Medium/F-12 (DMEM/F12). This base provides fundamental ingredients like inorganic salts, glucose (a sugar for energy), and amino acids (the building blocks for proteins). These nutrients support the cells’ metabolic activities and structural integrity.
Beyond these foundational elements, specific “growth factors” are incorporated to regulate iPSC behavior. Basic fibroblast growth factor (bFGF or FGF2) is an important example, acting as a signaling molecule that binds to cell surface receptors. This instructs the iPSCs to remain in their pluripotent, undifferentiated state and to continue self-renewing. Because bFGF is unstable and has a short functional half-life, frequent medium changes are necessary to ensure a continuous supply.
Additional “supplements and small molecules” are added to enhance cell survival and stability. An example is a Rho-associated protein kinase (ROCK) inhibitor. This small molecule helps prevent a type of programmed cell death, or apoptosis, that iPSCs are prone to experience, especially when they are dissociated into single cells. The ROCK inhibitor mitigates cellular stress, improving cell survival and attachment efficiency after passaging.
Feeder-Dependent vs. Feeder-Free Systems
Historically, induced pluripotent stem cells were cultured using a “feeder-dependent” system, where iPSCs were grown on a layer of inactivated supporting cells. These feeder cells secrete growth factors and extracellular matrix components into the medium, maintaining iPSC pluripotency and robust growth. However, this method introduces variability between feeder cell batches and carries a risk of introducing animal-derived contaminants into the human iPSCs.
The development of “feeder-free” systems was an advancement in iPSC culture methodology. In this approach, culture dishes are coated with specific proteins that mimic the extracellular matrix, such as Matrigel, a basement membrane extract, or recombinant human proteins like Vitronectin, allowing iPSCs to attach directly. The medium used in feeder-free systems is formulated to be more complex, providing all the necessary growth factors and supplements.
Feeder-free systems offer greater control and reproducibility by eliminating the biological variability of feeder cells. This also reduces the labor involved in preparing and maintaining feeder layers, making large-scale iPSC culture more feasible. The transition to feeder-free culture has been a fundamental shift towards standardized and scalable methods for iPSC research and clinical applications.
The Shift to Defined and Xeno-Free Media
Early cell culture media contained “undefined” components, with Fetal Bovine Serum (FBS) being an example. FBS is a complex mixture of proteins, growth factors, and hormones whose composition varies significantly from one batch to another. This inherent variability can lead to inconsistent experimental results and make studies difficult to reproduce.
To overcome these challenges, there has been a push towards “defined” media, where every component is known and quantified, and “xeno-free” media, which contain no non-human animal-derived products. Xeno-free formulations are important for applications in regenerative medicine and clinical translation, as they minimize the risk of immune reactions when iPSC-derived cells are transplanted into patients. They also reduce the potential for transmitting pathogens from animal sources to human cells.
This evolution has led to the development of popular commercial media formulations designed for human iPSCs, such as mTeSR1 and Essential 8 (E8). These media are formulated to be both defined and xeno-free, providing a consistent and safe environment for iPSC expansion while maintaining their pluripotency. For example, E8 medium is a simplified formulation containing only eight essential components, showing that a highly effective medium can be achieved with precise, controlled ingredients.
Selecting the Appropriate iPSC Medium
Choosing the appropriate iPSC medium depends on the specific goals of the research or application. Basic research focused on understanding iPSC biology might tolerate a broader range of media options, while preclinical studies aiming for therapeutic development demand consistent and safe formulations. The ultimate application, such as drug screening, disease modeling, or cell therapy, influences the decision.
The specific iPSC line being cultured is another consideration, as some lines may exhibit different growth characteristics or sensitivities to particular medium formulations. Researchers assess how well a given iPSC line maintains its morphology and pluripotency markers in various media before choosing. This ensures the chosen medium supports the stability and functionality of the cells.
Laboratory budget also plays a role, as commercially prepared defined and xeno-free media can be more expensive than preparing media in-house. However, the time savings, convenience, and reduced variability offered by commercial options often outweigh the cost. Experimental reproducibility is paramount, guiding scientists to select media that consistently support healthy, pluripotent iPSC cultures over many passages.