If you’ve just subcultured your cells and noticed lower-than-expected viability, don’t panic! This is a common issue in cell culture, and there are several factors that could be at play. Let’s troubleshoot the problem together—Bill Nye style! 🚀
1. Are Your Cells Stressed? 😰
Cells don’t love change. If they’re exposed to sudden shifts in temperature, pH, osmolarity, or media composition, they can experience stress, leading to poor viability.
Common Stress Factors:
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Temperature Shock – Were your cells left out too long? 🌡️
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pH Shifts – Was your media properly buffered? 💨
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Osmotic Stress – Did you switch to a different media formulation? ⚖️
🔬 Pro Tip: Always pre-warm your media and work quickly to minimize environmental fluctuations!
2. Did You Handle Your Cells Too Harshly? 🏋️♂️
Cells are delicate! Rough pipetting, excessive centrifugation, or over-trypsinization can damage cell membranes and lower viability.
Things to Watch Out For:
✔ Pipetting Too Hard – Use gentle up-and-down motions to resuspend. ✔ Over-Trypsinizing – Excessive exposure to trypsin can strip essential surface proteins. ✔ High-Speed Centrifugation – Too much force can cause cell damage; use ~300 g for most mammalian cells.
🚀 Pro Tip: If your cells are extra sensitive, try using Accutase instead of trypsin for detachment!
3. Is Your Media Fresh & Balanced? 🍽️
Nutrient depletion, expired supplements, or improper media preparation can negatively affect cell survival.
Media-Related Issues:
❌ Expired Serum – Growth factors degrade over time. ❌ Improperly Stored Supplements – Insulin, glutamine, and vitamins lose potency. ❌ Incomplete Media – Missing essential components (e.g., antibiotics, buffering agents).
🔬 Pro Tip: Always filter sterilize, use freshly prepared media, and store supplements correctly!
4. Did You Seed the Right Density? 📏
Seeding too many or too few cells can lead to viability issues.
Cell Density Matters:
📉 Too Low? – Cells may struggle to attach or grow. 📈 Too High? – Nutrient competition and waste accumulation can cause death.
🚀 Pro Tip: Refer to your cell line’s recommended seeding density to maintain optimal conditions!
5. Is Contamination Sneaking In? 🦠
Bacterial, fungal, or mycoplasma contamination can kill cells rapidly.
Contamination Clues:
⚠ Cloudy Media – Bacterial or fungal overgrowth. ⚠ pH Changes – Rapid shifts (acidic or basic) suggest microbial presence. ⚠ Odd Morphology – Cells looking strange? Check under the microscope!
🔬 Pro Tip: Use sterile techniques, change media regularly, and screen for mycoplasma contamination!
Wrapping It Up: Keep Those Cells Happy! 😊
If your cells aren’t thriving after subculture, take a systematic approach to identify the problem and make necessary adjustments. Gentle handling, proper media preparation, and optimal seeding conditions will help keep your cultures healthy and growing!
Stay curious, stay sterile, and keep those cells alive! 🚀🔬
