I'm working with some ARPE19 cells but I'm having some issues when it comes to passaging them. They seem unwilling to detach from the culture flask when incubated with 1x Trypsin at 37 degrees C for 5-10 minutes. Does anyone know how best to detach these cells?
Hi Shaun,this is the method from ATCC catalog:
Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.
Remove and discard culture medium.
Briefly rinse the cell layer with 0.05% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 xg for 5 to 10 minutes. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.
Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:5 is recommended
Medium Renewal: Two to three times weekly
In my opinion 10 minutes with trypsin is way too much, you should add EDTA as they suggest, put in the incubator for 2-3 minutes and then tap the flask against the palm of your hand for 3-4times. The cells should be detached (do not rely on them saying that tey form clumps, they said the same thing regarding other cell lines I worked before, I think that lump formation could be due to over -trypsinization and cell damage, try also to spin only for 5', in this way you can improve a lot your cell survival
Ah ok, I'm not sure if the Trypsin I used had EDTA in it, probably not. Thanks for the feedback =]
Hi Shaun, you should rise briefly with PBS without Calcium and Magnesium before incubated with trypsin (5-10 minutes) otherwise its very hard to detach them.
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