To describe the maintenance of cells in culture.

Safety

Equipment

1. Laminar flow hood
2. CO₂ Incubator
3. Mechanical Pipettor
4. Inverted microscope
5. Vacuum pump and flask

Materials

1. Complete cell culture medium, appropriate for the cell line
2. Tissue culture flasks of appropriate sizes
3. Tissue culture plates - 96 well or 24 well.
4. Sterile pipets, assorted sizes
5. Multichannel pipet and sterile tips.
6. Pasteur pipets, 9¡±, sterile, unplugged.
7. 70% alcohol
8. Sterile Petri dishes.

Procedure

1. General considerations
1. Turn on the hood and allow to run for at least 10 minutes before starting.
2. Pre-warm all media in 37^oC water bath.
3. Wipe all surfaces with 70% alcohol before starting.
2. For cells in flasks.
1. Cells are split or refed every 3-4 days.
2. Remove the flasks from the incubator.  Examine flasks with inverted microscope.
3. Place flasks and medium under hood.
4. Remove an aliquot of the cell suspension for counting with a hemocytometer (SP 05-009) and viability determination (SP 09-005).
5. Label the appropriate number of new flasks with the cell line name, the passage, the slit ratio or seeding density and the date.
6. Aseptically transfer the required number of cells to the new flask.  Add fresh medium to the flask.
• For T-25 flask - maximum of 10 ml of medium.
• For T-75 flask - maximum of 50 ml of medium.
• For T-150 flask - maximum of 100 ml of medium.
• For T-225 flask - maximum of 200 ml of medium.
7. Recap the flask(s), gently shake to evenly disperse the cells.
8. Return the flasks to the incubator.  Loosen the caps 1/2 turn, if necessary.
3. For 96 well plates.
1. Plates are re-fed every 3-4 days.
2. Remove the plates from the incubator, and examine microscopically or with the mirror.  Mark wells that are positive for cell growth.
3. Place plates and medium under the hood.
4. If supes are being collected for an assay, pre-label the plates for the supernatants.
• Transfer 150 ml of supernatant to the clean, labeled plate. 
• Store all supernatant plates at 2-8⁰C until ready to assay.
5. Remove spent medium with a sterile Pasteur pipet attached to a vacuum.  Hold the pipet at a 45 degree angle about 1/2 way down the well.
6. Start at the top left corner of the plate and work back and forth across the wells to the lower right corner.
7. Repeat with all plates.
8. Aseptically transfer medium into sterile Petri dish.  Add 150-175 ml of fresh medium into each well using a multichannel pipettor.  Work from the end of the plate furthest from your hand, to minimize the number of times your hand passes over the plate.
9. Repeat with all plates.  Add additional medium to a new, clean Petri dish if more is needed.  Change pipet tips for each plate.
10. Return the plates to the incubator.
4. For 24 well plates
1. Remove plates from the incubator and examine microscopically.
2. Place plates and medium under the hood.
3. If supes are being collected for an assay, pre-labels the plates for the supernatants.
4. Transfer 1-2 ml of supernatant to the clean, labeled plate.
5. Store all supernatant plates at 2-8^oC until ready to assay.
6. Remove spent medium with a sterile Pasteur pipet attached to a vacuum.  Hold the pipet at a 45 degree angle about 1/2 way down the well.
7. Start at the top left corner of the plate and work back and forth across the wells to the lower right corner.
8. Repeat with all plates.
9. If cells need to be split, indicated by orange colored medium and the cells covering more than half the surface of the well, add 2 ml of fresh medium to the well.
• Resuspend the cells by gently pipetting.
• Transfer 0.5 ml of cell suspension to each new well.
10. Add 2 ml of fresh medium to each well.
11. Return the plates to the incubator.
5. Transferring cells from a 96 to 24 well plate.
1. Identify the clones to be expanded.
2. Using a sterile pipet tip and pipettor, resuspend the cells by pipetting.
3. Transfer 150 ml of the cell suspension one well of a pre-labeled 24 well plate.
4. NOTE:  Use only the top row of each 24 well plate for new clones to allow room to expand the cells down the plate as they grow.
5. Add 2 ml of fresh medium to each well of the 24 well plate.  Add 150 ml of fresh medium to each 96 well.