Stem cells are unique cells within the body possessing two fundamental properties: the ability to indefinitely self-renew and the capacity to develop into many different specialized cell types. Researchers grow these cells outside of the living organism, a process called cell culture, to study their behavior and harness their potential. This in vitro environment allows for the controlled expansion and manipulation of stem cells to create models of human development and disease. The success of this technique hinges on recreating the chemical and physical conditions that allow these cells to thrive and maintain their unspecialized state.
Fundamental Components for Cell Growth
Sustaining stem cells relies on a specialized nutrient broth called culture media, which provides sustenance for survival and proliferation. The media consists of a basal solution containing essential components like amino acids, vitamins, salts, and a carbohydrate source, typically glucose, to fuel cellular metabolism. The liquid environment requires a buffering system, often using bicarbonate and a 5% carbon dioxide atmosphere, to maintain a specific $\text{pH}$ range, generally between 7.2 and 7.4.
To prevent spontaneous differentiation and promote self-renewal, the media must be supplemented with specific signaling molecules known as growth factors. For example, Fibroblast Growth Factor 2 (FGF-2) and Activin A are commonly added to human embryonic and induced pluripotent stem cell cultures to maintain their unspecialized status. Mouse embryonic stem cells often require Leukemia Inhibitory Factor (LIF) for the same purpose.
Beyond the liquid medium, stem cells require a physical surface to anchor themselves, which mimics the extracellular matrix found in the body. Historically, researchers used a layer of mitotically inactivated cells, such as mouse embryonic fibroblasts, known as a feeder layer, for attachment and necessary conditioning factors. Modern techniques use defined, feeder-free systems where the culture dish is coated with synthetic or purified biological matrices, like fibronectin or basement membrane gel, eliminating the variability associated with animal-derived components.
Maintaining a Sterile Environment and Cell Health
The delicate nature of stem cells necessitates a strictly controlled physical environment and rigorous laboratory practices to prevent contamination and maintain viability. All cell handling procedures must use aseptic technique within a biosafety cabinet, or laminar flow hood, which uses filtered air to create a sterile workspace. Contaminants such as bacteria, fungi, or the hard-to-detect mycoplasma can rapidly destroy a culture or skew experimental results.
Cells are housed in specialized incubators that regulate temperature and atmospheric composition to replicate the body’s internal conditions. These chambers maintain a constant temperature of 37°C and control humidity and the concentration of carbon dioxide to ensure the media’s $\text{pH}$ remains stable. Monitoring the physical environment is important because even minor fluctuations can stress the cells and induce unwanted differentiation.
As cells multiply, they must be regularly “passaged,” or split, into new culture dishes to prevent overcrowding, which triggers differentiation and cell death. This process involves using enzymatic solutions, like trypsin or Accutase, or non-enzymatic dissociation reagents to gently detach the cells from their substrate. Pluripotent stem cells are particularly sensitive to dissociation, so they are often scraped and replated in small clumps, or aggregates, rather than as single cells, which significantly enhances their survival rate.
Different Stem Cell Sources Used in Culture
Stem cell research utilizes cells harvested from three distinct sources, each requiring specific culture protocols dictated by their unique origin and potential for specialization.
Adult Stem Cells (ASCs)
ASCs, such as Mesenchymal Stem Cells (MSCs) found in bone marrow or adipose tissue, are multipotent, meaning they can only differentiate into a limited number of cell types, such as bone, cartilage, or fat cells. These cells are generally the least demanding to culture, often growing readily on standard tissue culture plastic using less complex, commercially available media formulations.
Embryonic Stem Cells (ESCs)
ESCs are derived from the inner cell mass of the blastocyst and are pluripotent, possessing the capacity to form every cell type in the adult body. Their culture is technically challenging because they have a strong tendency to spontaneously differentiate without precise conditions. Maintaining pluripotency requires specialized matrices and daily media changes supplemented with specific concentrations of growth factors like FGF-2.
Induced Pluripotent Stem Cells (iPSCs)
iPSCs are laboratory-created cells generated by genetically reprogramming specialized adult cells, such as skin fibroblasts, back to an ESC-like pluripotent state. This reprogramming is achieved by introducing a cocktail of transcription factor genes, including Oct3/4, Sox2, and Klf4, effectively turning back the cell’s developmental clock. iPSCs are highly valued because they share the pluripotency and self-renewal of ESCs while circumventing ethical concerns and allowing for the creation of patient-specific cell lines.
Primary Applications of Cultured Cells
The ability to culture and expand stem cells in the laboratory provides powerful tools for advancing biomedical research, primarily through disease modeling and drug testing.
Disease Modeling
Cultured cells, particularly patient-derived iPSCs, can be directed to differentiate into specific cell types relevant to a disease, such as neurons for neurodegenerative conditions like Alzheimer’s or dopaminergic cells for Parkinson’s disease. Researchers use these cells to study the cellular mechanisms and progression of the disease outside the complex environment of the human body. This high fidelity to human biology makes cultured stem cells an important platform for understanding a wide range of inherited and complex disorders.
Drug Screening and Toxicity Testing
The cultured cells are also routinely used for high-throughput drug screening and toxicity testing, significantly streamlining the drug development process. By exposing these cell models—or multicellular structures called organoids—to thousands of potential new drug compounds, researchers can quickly assess a compound’s effectiveness and safety profile. This is especially important for pre-screening compounds for cardiotoxicity, as iPSC-derived heart cells can accurately predict drug-induced arrhythmia risk in humans, potentially reducing the high failure rate of drugs in clinical trials.