cell-culture-basics

Overview

cell-culture-basics covers the essential practices of mammalian cell culture: aseptic technique, media and supplements, passaging, counting, cryopreservation, and contamination detection and response. Cell culture is a craft — most failures come from technique drift and unvalidated assumptions, not from bad luck.

When to use

• Setting up culture: hood technique, media selection, serum considerations.
• Routine maintenance: feeding schedules, confluency, passaging, splitting ratios.
• Cryopreservation and thawing: DMSO protocols, viability, cell banking.
• Contamination: detection (bacterial, fungal, mycoplasma), response, prevention.
• Cell line authentication: STR profiling, when and how often.
• Counting and viability: hemocytometer, automated counters, trypan blue discipline.
• Scaling: from dishes to flasks to bioreactors.

Core concepts

• Aseptic technique. Work in the biosafety cabinet with organized workflow (clean → dirty), 70% ethanol on everything entering, minimal talking, no clutter blocking airflow. Technique is the contamination control — antibiotics are not (they mask low-level contamination and select resistance; use antibiotic-free culture when possible).
• Media. Basal media (DMEM, RPMI, MEM) matched to cell type; serum (FBS 5-10%) provides growth factors but introduces variability — batch-test serum lots, consider serum-free defined media for sensitive work. L-glutamine degrades (use GlutaMAX or fresh aliquots); check pH (phenol red) and osmolarity when troubleshooting growth issues.
• Passaging. Split at 70-80% confluency (not 100% — contact inhibition and differentiation change phenotypes); use appropriate detachment (trypsin-EDTA with timed exposure, non-enzymatic for sensitive cells); consistent split ratios for reproducible growth curves. Record passage number — high-passage cells drift genetically and phenotypically.
• Counting. Hemocytometer with trypan blue (count ≥100 cells per count for statistics); automated counters need validation against manual counts. Viability <90% at thaw or <95% in routine culture warrants investigation.
• Cryopreservation. Freeze slowly (1°C/min, isopropanol containers or controlled-rate freezers) in 5-10% DMSO + serum/media; thaw rapidly (37°C water bath, <2 min) and dilute out DMSO promptly. Bank early-passage master stocks + working stocks — never let the lab's only stock reach passage 50.
• Mycoplasma. The invisible contaminant: no turbidity, just slow growth and altered responses. Test regularly (PCR or luminescence kits, monthly for active lines); positive = discard and thaw clean stock (treatment rarely fully clears and selects weirdness). Quarantine new lines until tested.
• Authentication. STR profiling for human lines (HeLa contamination is the classic catastrophe — an estimated significant fraction of lines are misidentified). Authenticate on receipt, after banking, and periodically. A paper built on the wrong cell line is worthless.
• Documentation. Passage number, split ratios, media lots, freeze/thaw dates, test results — per flask, in the ELN. "The cells looked fine" is not a record.

Practical workflow

1. Setup. Validated hood technique; appropriate media/serum lot; antibiotic-free unless justified.
2. Thaw. Rapid thaw, prompt DMSO removal, plate at recommended density; expect 24-48h lag.
3. Maintain. Feed per schedule; passage at 70-80% confluency; consistent ratios; log everything.
4. Bank. Master stock at low passage (≥10 vials), working stocks from it; test banked vials for viability and mycoplasma.
5. Monitor. Monthly mycoplasma testing; STR on receipt and periodically; growth-rate tracking (sudden changes = investigate).
6. Respond. Contamination: identify, discard affected cultures, decontaminate incubators/ hoods, thaw clean stock. Never "rescue" with antibiotics and carry on silently.

Common pitfalls

• Passaging at 100% confluency (phenotype drift).
• Antibiotic-masked contamination carried for months.
• No mycoplasma testing (the most common undetected problem in cell culture).
• Misidentified cell lines (no STR authentication).
• Single stock at high passage (no clean backup).
• Serum lot changes without testing (growth characteristics shift).
• Poor records making troubleshooting impossible.
• CO₂ levels unchecked (pH drift when CO₂ doesn't match the medium's buffer system).
• Cells left in trypsin too long (surface-protein damage, viability loss).
• Incubator water baths and humidity neglected (evaporation concentrating media).