Subculture of Semi-Adherent Cell Lines

 

Aim
Some cultures grow as a mixed population (e.g. B95-8 - marmoset) where a proportion of cells do not attach to the tissue culture flask and remain in suspension. Therefore to maintain this heterogeneity both the attached cells and the cells in suspension must be subcultured.

Materials

• Media� pre-warmed to 37ºC (refer to the ECACC Cell Line Data Sheet for the correct medium)
• 70% ethanol in water (Prod. No. R8382 )
• PBS without Ca₂₊ /Mg₂₊ (Prod. No. D8537 )
• 0.25% trypsin/EDTA in HBSS, without Ca₂₊ /Mg₂₊ (Prod. No. T4049 )
• Trypsin (Prod. No. T4424 )
• Soybean trypsin Inhibitor(Prod. No. T6414 )

 

Equipment

• Personal protective equipment (sterile gloves, laboratory coat, safety visor)
• Waterbath set to 37ºC
• Microbiological safety cabinet at the appropriate containment level
• Centrifuge
• Inverted phase contrast microscope
• CO₂ incubator
• Haemocytometer (Bright-line, Prod. No. Z359629 , Improved Neubauer Grid, Camlab CCH.AC1)
• Pre-labeled flasks
• Tissues

 

Procedure

1. View cultures using an inverted phase contrast microscope to assess the degree of confluency and confirm the absence of bacterial and fungal contaminants. Give the flask a gentle knock first, this may dislodge the cells from the flask and remove the need for a trypsinisation step with the subsequent loss of some cells due to the washings.
2. Decant spent medium into a sterile centrifuge tube and retain.
3. Wash any remaining attached cells with PBS without Ca₂₊ /Mg₂₊ (Prod. No. D8537 ) using 1-2ml for each 25cm₂ of surface area. Retain the washings.
4. Pipette trypsin/EDTA (Prod. No. T4049 ) onto the washed cell monolayer using 1ml per 25cm₂ of surface area. Rotate flask to cover the monolayer with trypsin. Decant the excess trypsin.
5. Return flask to incubator and leave for 2-10 minutes.
6. Examine the cells using an inverted microscope to ensure that all the cells are detached and floating. The side of the flasks may be gently tapped to release any remaining attached cells.
7. Transfer the cells into the centrifuge tube containing the retained spent medium and cells.
8. Centrifuge the remaining cell suspension at 150g for 5 minutes. Also centrifuge the washings from Number 3 above if they contain significant numbers of cells.
9. Decant the supernatants and resuspend the cell pellets in a small volume (10-20mls) of fresh culture medium. Pool the cell suspensions. Count the cells.
10. Pipette the required number of cells to a new labeled flask and dilute to the required volume using fresh medium (refer to ECACC Cell Line Data Sheet for the required seeding density).
11. Repeat this process every 2-3 days as necessary.

 

Key Points

1. Although most cells will detach in the presence of trypsin alone the inclusion of EDTA is used to enhance the activity of the enzyme.
2. Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all traces of serum from the culture medium by washing the monolayer of cells with PBS without Ca₂₊ /Mg₂₊ (Prod. No. D8537 ). Repeated warming to 37ºC also inactivates trypsin.
3. Cells should only be exposed to trypsin/EDTA (Prod. No. T4049 ) long enough to detach cells. Prolonged exposure could damage surface receptors. In general a shorter time of exposure to trypsin is required for semi adherent cell lines.
4. Trypsin should be neutralized with serum prior to seeding cells into new flasks otherwise cells will not attach.
5. Trypsin may also be neutralized by the addition of Soybean trypsin Inhibitor (Prod. No. T6414 ), where an equal volume of inhibitor at a concentration of 1mg/ml is added to the trypsinised cells. The cells are then centrifuged, resuspended in fresh culture medium and counted as above.
6. If a CO₂ incubator is not available gas the flasks for 1-2 minutes with 5% CO₂ in 95% air filtered through a 0.25m filter.