Summary
Highlights
Begin by visually inspecting the cell culture. Ensure cells are attached to the vessel surface, there's no mold or cloudiness, and the medium color is normal. Then, examine the culture under a microscope, verifying approximately 80% surface coverage and absence of contamination.
All liquid waste must be held in disinfectant for at least 2 hours before disposal. Return trypsin and fresh culture medium to the fridge. Clean all inside surfaces of the cabinet with ethanol and dispose of all contaminated general waste and plastic wear in the biohazard waste bin.
Understand the function of a Class 2 microbiological safety cabinet, which uses filtered air for a clean working space. Clean the inside surfaces thoroughly with 70% ethanol, ensuring all corners are disinfected.
Put on gloves and sterilize them with ethanol. Sterilize all equipment with ethanol before placing it in the cabinet, arranging it efficiently to avoid disrupting airflow.
The purpose of cell culture is to replace old, nutrient-exhausted media. Use a sterile pipet to remove the old media and dispose of it in disinfectant, minimizing contamination risk by replacing the lid quickly.
Add a buffer solution without nutrients to wash off any residual old media, as this can affect subsequent cell detachment. Draw 10 mL of buffer, wash the cells, and dispose of the buffer in disinfectant.
Apply trypsin, a digestive enzyme, to disrupt proteins holding cells to the flask. Ensure the trypsin covers the cell surface. Incubate the flask at 37°C for 3-4 minutes; avoid over-incubation to protect cell surface proteins.
After incubation, visually inspect the flask under a microscope. Cells should appear spherical and beginning to detach. Gently tap the flask to dislodge cells, then re-examine to confirm they are freely floating.
Add fresh culture medium to inactivate the trypsin and prevent cell damage. Transfer the cell suspension to a centrifuge tube, ensuring a counterweight for balance. Centrifuge at 1800 RPM for 4 minutes to pellet the cells without damage.
Prepare a new flask with fresh culture medium. Invert the centrifuge tube to dispose of the old medium while preserving the cell pellet. Resuspend the cell pellet in fresh media, flushing it in and out with a pipet until a single-cell suspension is achieved.
Transfer approximately 30% of the cell suspension to the new culture flask and tighten the cap. Examine the new culture under a microscope to confirm sufficient cell density before placing it in the incubator at 37°C.