Cell culture techniques let researchers study cells outside their natural environment by growing, manipulating, and analyzing them under controlled conditions. These methods are foundational for understanding cellular processes, modeling disease, and testing therapeutics.
This topic covers how to maintain cells in culture, introduce new genetic material through transfection, and sort cells based on specific characteristics.
Cell Culture Techniques
Principles of mammalian cell culture
Aseptic technique is the foundation of all cell culture work. Contamination by bacteria, fungi, or mycoplasma can ruin experiments and entire cell stocks, so every step must prioritize sterility.
- Work inside a laminar flow hood or biosafety cabinet
- Use only sterile equipment and reagents
- Wash hands thoroughly and wear PPE (gloves, lab coat)
- Regularly decontaminate work surfaces with 70% ethanol
Media preparation provides cells with the nutrients they need to survive and proliferate. Different cell types require different formulations (for example, DMEM for many adherent lines, RPMI for suspension cells like lymphocytes).
- Supplement with fetal bovine serum (FBS) or defined growth factors to supply proteins, hormones, and attachment factors
- Adjust pH (typically ~7.4) and osmolarity to match physiological conditions
- Filter-sterilize media through a 0.22 µm filter before use
Cell maintenance is a routine cycle of monitoring, feeding, and passaging:
- Incubate cells at 37°C with 5% (the buffers the bicarbonate in the medium to maintain pH).
- Monitor growth and morphology daily under a microscope. Healthy cells should have consistent shape and refractility.
- When cells approach confluency (the point where they cover most of the culture surface), subculture them: detach adherent cells with trypsin or another dissociation reagent, centrifuge to pellet, resuspend in fresh media, and seed at an appropriate density into new vessels.
- For cryopreservation, resuspend cells in media containing a cryoprotectant like dimethyl sulfoxide (DMSO), which prevents lethal ice crystal formation during freezing. Freeze cells slowly (about –1°C per minute) and store long-term in liquid nitrogen (–196°C).
Cell Types and Applications
Types of cell cultures
Primary cell cultures are isolated directly from living tissue (e.g., hepatocytes from liver, neurons from brain). They closely resemble the cells' behavior in vivo, which makes them valuable for studying tissue-specific functions and for drug screening. The tradeoff is that they have a limited lifespan and can only be passaged a finite number of times before they senesce or change character.
Immortalized cell lines have been genetically altered to bypass senescence, giving them essentially unlimited proliferative capacity. HeLa cells (derived from cervical cancer tissue) and HEK293 cells (derived from embryonic kidney) are two of the most widely used examples. They're convenient for long-term studies and high-throughput screening, but their altered genetics mean they may not perfectly reflect normal cell behavior.
Stem cell-derived cultures start from embryonic stem cells or induced pluripotent stem cells (iPSCs) and can be differentiated into specific cell types like cardiomyocytes or neural progenitors. These cultures are particularly powerful for disease modeling (especially patient-specific iPSC lines), studying developmental processes, and exploring regenerative medicine approaches.
Methods for cell transfection
Transfection is the process of introducing exogenous nucleic acids (DNA, RNA) into cells. The method you choose depends on cell type, efficiency requirements, and whether you need transient or stable expression.
Chemical methods:
- Lipid-based reagents (e.g., Lipofectamine) form liposomes that encapsulate nucleic acids. These liposomes fuse with the cell membrane and deliver their cargo into the cytoplasm. This is one of the most common approaches for adherent cell lines.
- Calcium phosphate precipitation works by mixing DNA with calcium chloride and a phosphate buffer. The DNA co-precipitates with calcium phosphate into fine particles that cells take up by endocytosis. It's inexpensive but less consistent than lipid-based methods.
Physical methods:
- Electroporation applies a brief electric field to create temporary pores in the cell membrane, allowing nucleic acids to enter. It works well for hard-to-transfect cells (like primary cells and suspension cells) but can cause significant cell death if not optimized.
- Microinjection uses a micropipette to inject nucleic acids directly into individual cells. It's precise but extremely low-throughput, so it's typically reserved for specialized applications like injecting oocytes.
Viral-based methods (transduction): Engineered viruses (lentiviruses, adenoviruses, retroviruses) carry the gene of interest and infect target cells. Lentiviruses and retroviruses integrate into the host genome, enabling stable long-term expression. Adenoviruses do not integrate, so expression is transient. Viral methods tend to have high efficiency, even in difficult cell types, but require additional biosafety precautions.
What transfection enables:
- Overexpression studies: Introduce an expression vector carrying a gene to study the protein's function or localization.
- Gene knockdown studies: Deliver small interfering RNAs (siRNAs) for transient knockdown or short hairpin RNAs (shRNAs) for stable knockdown via RNA interference (RNAi). This suppresses a target gene's expression so you can investigate what happens when that gene is lost.
Cell sorting and flow cytometry
Flow cytometry measures physical and fluorescent properties of individual cells as they pass single-file through a laser beam in a fluid stream. Cells are typically labeled with fluorescent antibodies or dyes before analysis.
The instrument's detectors measure three main parameters:
- Forward scatter (FSC): correlates with cell size
- Side scatter (SSC): correlates with internal complexity/granularity
- Fluorescence intensity: indicates the amount of a specific marker present on or in the cell
By combining these measurements, you can identify and quantify distinct cell populations within a mixed sample.
Cell sorting techniques go a step further by physically separating cells based on these measurements:
- Fluorescence-activated cell sorting (FACS) builds on flow cytometry. After laser interrogation, the fluid stream is broken into droplets, each containing a single cell. Droplets are given an electrical charge based on the cell's fluorescence profile, then deflected by an electric field into separate collection tubes. FACS can sort multiple populations simultaneously at thousands of cells per second.
- Magnetic-activated cell sorting (MACS) uses antibodies conjugated to magnetic beads. Cells expressing the target surface marker bind the beads, and when the mixture is passed through a magnetic column, labeled cells are retained while unlabeled cells flow through. MACS is simpler and faster than FACS but only sorts based on one or two markers at a time.
Common applications:
- Isolating specific populations for downstream culture or analysis (e.g., stem cells, circulating tumor cells)
- Purifying rare cell types from heterogeneous mixtures
- Analyzing cell cycle distribution (using DNA-binding dyes like propidium iodide)
- Detecting intracellular proteins, cytokines, or phosphorylated signaling molecules to assess cell state and activation