Subculturing, also known as passaging, is the process of transferring cells from one culture vessel to another to maintain healthy cell growth and prevent over-confluence. However, issues can arise during subculturing, and troubleshooting may be necessary. Here are some common problems and potential solutions:
- Poor cell attachment or spreading after subculture:
- Make sure the culture surface is properly coated, if required, and that the coating has not expired or degraded.
- Ensure that the trypsin-EDTA solution is inactivated by adding an appropriate volume of serum-containing medium or a trypsin inhibitor.
- Check that the culture medium is suitable for the specific cell type and is not contaminated, expired, or improperly stored.
- Allow more time for cells to attach, as some cell types may take longer to adhere to the culture surface.
- Slow cell growth or low viability after subculture:
- Ensure that the culture medium is appropriate for the specific cell type and is not contaminated, expired, or improperly stored.
- Verify that the incubator’s temperature, CO2, and humidity levels are stable and within the appropriate range for the cell type.
- Avoid over-trypsinization, which can damage cells; instead, use the minimum trypsin-EDTA exposure necessary for cell detachment.
- Reduce the cell density during subculture to provide more room for cell growth.
- Contamination in the cell culture:
- Regularly clean and decontaminate the cell culture workspace, incubator, and equipment.
- Always practice aseptic techniques, such as using sterile gloves, wiping surfaces with ethanol, and working in a laminar flow hood.
- Discard any contaminated cultures and start with a new, uncontaminated vial of cells.
- Use antibiotics in the culture medium, if necessary, to minimize the risk of bacterial contamination.
- Cell clumping after subculture:
- Gently pipette the cell suspension up and down to break up cell clumps during subculturing.
- Avoid over-trypsinization, which can damage cells and cause them to stick together.
- Use a cell strainer to remove clumps before seeding the cells in a new culture vessel.
- Inconsistent cell morphology or behavior:
- Regularly monitor cell morphology under an inverted microscope and compare it to the expected morphology for the cell type.
- Ensure that the culture medium and incubation conditions are consistent between passages.
- Avoid over-confluence or extended periods without passaging, as this can change cell behavior.
By following these troubleshooting procedures and maintaining strict aseptic techniques, you can address and prevent many common issues encountered during cell subculturing.
