Primary Cell Culture Tips and Techniques
News 24 4 月, 2025
Primary cells are initial cells obtained from living tissues through specific isolation methods. These cells are freshly isolated, not immortalized, and retain most of their original genetic and physiological traits. This makes them highly valuable for experiments requiring physiological relevance, such as drug testing, cell differentiation, and transformation studies.
1. Growth Requirements
Primary cells may be suspension or adherent. Suspension cells (e.g., peripheral blood-derived) float naturally, while adherent cells (e.g., solid tissues) need surfaces to grow.
They are usually cultured in flat plastic containers without coatings, though extracellular matrix proteins like collagen or laminin can enhance adhesion and provide growth signals. Media should include essential growth factors and be maintained at appropriate temperature (typically 37°C) and CO₂ levels (5%).
Antibiotics like gentamicin, penicillin, streptomycin, and amphotericin B are used during initial culture to suppress contamination. However, prolonged antibiotic use—especially amphotericin B—can be toxic and should be avoided.
Maintaining cell viability after isolation is crucial, as primary cells age and stop dividing after limited passages. Successful long-term culture requires excellent sterile technique and proper environmental conditions (pH, nutrients, temperature, etc.). Try to minimize the use of animal-derived components due to potential contamination risks.
2. Cellular Confluence
Confluence refers to the percentage of the culture surface covered by cells. For example, 100% confluence means complete surface coverage. Tracking confluence is essential to determine the right time for subculturing, as over-confluent cells may differentiate and slow down growth.
3. Maintenance and Subculture
Once cells adhere to the surface (usually within 24 hours), maintenance begins. Subculture should happen before reaching 100% confluence to maintain healthy growth.
Adherent cells grow in monolayers and must be enzymatically detached (e.g., trypsin/EDTA) for passaging. After detachment, cells are counted, diluted, and reseeded into fresh containers. Media composition may vary depending on the cell type.
4. Cell Counting
Trypan blue exclusion and a hemocytometer are typically used to count viable cells. The hemocytometer is a precision-engineered slide with a grid, allowing cell concentration to be calculated from known fluid volumes.
5. Cryopreservation and Recovery
Cryopreservation helps store viable cells long-term. For primary cells, freezing requires cryoprotectants like DMSO or glycerol and a slow cooling rate (e.g., -1°C/min). Store in liquid nitrogen or below -130°C.
To thaw, immerse vials in 37°C water for 1–2 minutes. Do not centrifuge immediately post-thaw. Instead, directly plate the cells and change the medium after 24 hours to remove toxic DMSO.
Common Mistakes in Primary Cell Culture
- Contamination: Avoid during tissue transfer and culture.
- pH Changes: Caused by salt imbalances, contamination, or incorrect CO₂ tension.
- Poor Adhesion: Due to lack of attachment factors or over-digestion with enzymes.
- Slow Growth: Caused by depleted nutrients, improper pH, or low-grade contamination.
- Cell Death: Due to temperature shifts, low CO₂, toxic by-products, or freeze/thaw damage.
- Precipitation: Occurs with cold media or leftover phosphate detergents.
- Clumping: Caused by calcium/magnesium in buffers or DNA release from over-digestion.
- Experimental Variability: Influenced by reagents, media, and handling techniques.