Cell freezing is a crucial step in preserving cell lines for long-term storage and use in various research applications. Freezing cells helps maintain their viability, prevents contamination, and allows for easier transportation. Here is a general protocol for cell freezing:
Materials:
- Cell culture flask or plate containing the cells
- Trypsin-EDTA or other suitable cell detachment reagent
- Cell culture media with serum
- Freezing medium (e.g., cell culture media with 10% dimethyl sulfoxide (DMSO) and 20% fetal bovine serum (FBS))
- Sterile centrifuge tubes
- Cryovials (sterile and pre-chilled)
- Pipettes and tips
- Cell counter (e.g., hemocytometer or automated cell counter)
- Incubator (37°C, 5% CO2)
- Sterile biosafety cabinet
- Centrifuge
- Ice or ice-water bath
- Freezer (-80°C)
- Liquid nitrogen storage system
Procedure:
- Begin by working in a sterile biosafety cabinet to prevent contamination.
- Gently aspirate the media from the cell culture flask or plate.
- Wash the cell monolayer with sterile PBS or a suitable buffer to remove any residual media.
- Add an appropriate volume of trypsin-EDTA to detach the cells from the surface. Incubate the flask or plate for 2-5 minutes at 37°C.
- Periodically check for cell detachment under a microscope. Gently tap the flask or plate to facilitate cell detachment if necessary.
- Once the cells have detached, add an equal volume of cell culture media with serum to neutralize the trypsin.
- Transfer the cell suspension to a sterile centrifuge tube.
- Centrifuge the cell suspension at 200-300 x g for 5 minutes to pellet the cells.
- Carefully aspirate the supernatant without disturbing the cell pellet.
- Resuspend the cell pellet in an appropriate volume of pre-chilled freezing medium. Mix gently but thoroughly.
- Determine the cell concentration and viability using a cell counter.
- Adjust the cell concentration to the desired level (e.g., 1 x 10^6 cells/mL) with the freezing medium.
- Aliquot the cell suspension into pre-chilled cryovials (1-2 mL per vial).
- Place the cryovials in an ice-water bath for 10-15 minutes to begin the cooling process.
- Transfer the cryovials to a controlled-rate freezing container or a -80°C freezer for 24 hours. This step allows for a slow decrease in temperature, which is essential for optimal cell viability.
- After 24 hours, transfer the cryovials to a liquid nitrogen storage system for long-term storage.
Remember to always work under sterile conditions and follow specific guidelines for the cell line you are working with. This protocol is a general guideline, and specific conditions may vary depending on the cell type and the requirements of your laboratory.