Скачать или смотреть Trypsinization Process (Animal Cell Culture )

Trypsinization is a crucial step in cell culture used to detach adherent cells from the surface of culture flasks or plates for passaging or experimentation. The process involves using trypsin, a serine protease, to break down proteins that anchor cells to the culture surface. Below is a step-by-step explanation:
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Step-by-Step Trypsinization Procedure
1. Prepare the Necessary Materials
• Phosphate-buffered saline (PBS) (calcium- and magnesium-free)
• Trypsin-EDTA solution (typically 0.25% trypsin with 1 mM EDTA)
• Complete cell culture medium (with serum to neutralize trypsin)
• Sterile pipettes and centrifuge tubes
• CO₂ incubator
• Inverted microscope
• Biosafety cabinet
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2. Pre-warm the Trypsin Solution
• Warm the trypsin-EDTA solution and complete culture medium to 37°C in a water bath or incubator.
• This ensures optimal enzymatic activity and minimizes thermal shock to cells.
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3. Remove Old Culture Medium
• Under a biosafety cabinet, aspirate the old medium from the culture flask using a sterile pipette.
• Discard the used medium properly to avoid contamination.
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4. Wash the Cells with PBS
• Add a sufficient volume of calcium- and magnesium-free PBS (e.g., 5 mL for a T-25 flask).
• Gently swirl to remove residual serum, which contains trypsin inhibitors.
• Aspirate the PBS completely.
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5. Add Trypsin-EDTA Solution
• Add enough trypsin-EDTA to cover the cell monolayer (e.g., 1–2 mL for a T-25 flask).
• Gently tilt the flask to ensure even distribution of trypsin.
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6. Incubate at 37°C for Detachment
• Place the flask in a 37°C CO₂ incubator for 1–5 minutes.
• Monitor under an inverted microscope every 1–2 minutes to check cell detachment.
• Cells will round up and detach from the surface.
Tip: Avoid overexposure to trypsin, as prolonged incubation may damage cell surface proteins.
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7. Neutralize Trypsin
• Once the cells are detached, add complete medium containing serum (e.g., 3–5 mL) to stop trypsin activity.
• Serum contains trypsin inhibitors, preventing further proteolytic damage to cells.
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8. Collect and Centrifuge Cells
• Gently pipette up and down to resuspend the cells.
• Transfer the cell suspension to a 15 mL centrifuge tube.
• Centrifuge at 300–400 × g for 5 minutes to pellet the cells.
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9. Resuspend and Count the Cells
• Discard the supernatant and resuspend the pellet in fresh medium.
• Mix well by gentle pipetting.
• Count the cells using a hemocytometer or an automated cell counter.
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10. Proceed with Further Use
• Seed the required number of cells into new flasks for passaging.
• Use the cells for experiments as needed.
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Precautions During Trypsinization
✔️ Avoid excessive trypsin exposure (can damage cell surface proteins).
✔️ Monitor cell detachment closely under the microscope.
✔️ Ensure even trypsin distribution to avoid uneven detachment.
✔️ Use serum-containing medium to neutralize trypsin at the right time.