Cell culture is a fundamental technique for studying cellular physiology, producing recombinant proteins, testing drug candidates, and manufacturing cell therapies. Success depends on aseptic technique, appropriate media, and controlled environmental conditions.

Biosafety and Aseptic Technique

All cell culture work is performed in a Class II biological safety cabinet (BSC) using sterile technique. The BSC protects both the user and the culture by filtering air through a HEPA filter and creating a downward laminar airflow.

Key aseptic practices:

• Spray all items with 70% ethanol before placing them in the BSC.
• Do not block the air grilles at the front or back of the cabinet.
• Work from clean to dirty — arrange reagents on one side and waste on the other.
• Never touch the inside of a cap or the rim of a sterile bottle.
• Change gloves after handling non-sterile items.

Growth Media

Most mammalian cells are cultured in complex media that supply nutrients, growth factors, and a pH buffer system. Common formulations:

• DMEM (Dulbecco’s Modified Eagle Medium): high glucose (4.5 g/L) or low glucose (1 g/L). Used for HeLa, HEK 293, fibroblasts.
• RPMI-1640: developed for suspension cells and lymphocytes. Commonly used for hematopoietic cells and hybridomas.
• F-12 / Ham’s F-12: rich medium for serum-free cloning and CHO cells.
• FBS (fetal bovine serum): added at 5–20% (typically 10%) to provide growth factors, hormones, and adhesion factors. Batch variability can affect reproducibility — test and reserve a consistent lot.

Environmental Control

Mammalian cells are incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. The CO₂ dissolves in the medium to form carbonic acid, which with the bicarbonate buffer system maintains pH at ~7.4. The incubator humidifier pan should be filled with sterile water and cleaned regularly to prevent mold and bacterial growth.

Cell Types

• Adherent cells: attach to the culture vessel (e.g., HeLa, HEK 293, MDCK, primary fibroblasts). Require trypsinization for passaging.
• Suspension cells: grow floating in the medium (e.g., Jurkat, HL-60, many hybridomas). Passaged by diluting with fresh medium.
• Primary cells: isolated directly from tissue. Have a finite lifespan (Hayflick limit). More physiologically relevant but harder to culture.
• Immortalized cell lines: derived from tumors or transformed in vitro. Can be passaged indefinitely but may diverge genetically from the original tissue.

Passaging (Subculturing)

Adherent cells are passaged when they reach 70–90% confluence. The standard protocol: remove medium, wash with PBS (Ca²⁺/Mg²⁺-free), add trypsin-EDTA, incubate at 37 °C for 2–5 minutes until cells detach, add fresh medium to neutralize trypsin, count, and reseed at the appropriate density.

Record the passage number each time. Cells should be used within a defined passage window (e.g., passages 3–20 for most immortalized lines) to minimize phenotypic drift.