Many students have faced this common issue: their cell cultures were doing well for a while, but after several generations, they started to decline and eventually stopped working. This problem often comes down to one key factor—improper digestion. Every time you digest the cells, you risk damaging them, and over time, this damage accumulates, making the situation worse.
It's important to understand that digestion isn't just a routine step—it's a critical test for the survival of your cells. If done incorrectly, it can lead to irreversible damage or even cell death. So, how can you avoid this? The answer lies in adapting your digestion method based on the condition of your cells.
Not all cells adhere to the surface equally. Some are tightly attached, while others come off more easily. Treating them all the same way isn’t fair and can cause unnecessary harm. That’s why a more personalized approach is needed. Here’s a simple yet effective method called the "four-step digestion technique," especially useful for difficult-to-digest cells.
Step-by-step Digestion Method:
- Washing without trypsin: Remove the old medium and gently wash the cells with fresh medium 1–2 times. This helps remove dead or floating cells and prepares the culture for the next steps.
- Gentle detachment: Add a small amount of fresh medium and pipette up and down to detach loosely attached cells. Collect this suspension and transfer it to a new flask. This allows you to compare the effect of trypsin with non-trypsinized cells.
- Moderate trypsin treatment: After removing the remaining liquid, add about 0.3 ml of trypsin, let it sit for a short time, then aspirate it. Add around 1 ml of trypsin and observe under the microscope. When the cells begin to detach and the gaps between them become visible, stop the digestion and rinse with fresh medium. Gently pipette to collect the cells and transfer them to a new flask.
- Fully detach remaining cells: Add the remaining trypsin back into the flask and continue digestion. Observe until the cells are fully detached. Then, collect the suspension and transfer it to a new flask. This ensures that even the most stubborn cells are properly removed without over-digesting the rest.
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