What is the advantage of single-cell passaging versus common colony passaging? Is there a desired confluency for single-cell passaging that leads to colony formation and vice-versa? Can you send pictures on how they should look after passaging?
Hi Adriana, thank you for your question! I apologize for the delay. Some advantages of single-cell passaging include gene-editing and cloning purposes; cells in singlets have more available surface area for transfection reagents to enter the cell membrane. Freezing is also much more straightforward in a single cell format-- survival is better, and you can have an accurate count of the cells going into your vial. As we generate our cell lines, we use single-cell passaging regularly, but clump passaging can be used successfully with our released lines.
The desired confluence for single-cell passaging is ~70-75%, as is pictured in our WTC culture protocol found here: https://www.allencell.org/uploads/8/1/9/9/81996008/aics_wtc_culture_sop_external_v1-6_2019_190115.pdf (see Figure 1b and f)
Additional details on single cell passaging are covered in one of our video tutorials: https://www.allencell.org/instructional-videos-and-tutorials-for-cell-methods.html#ACM-Single-cell-passaging
My teammate Angel, who is more familiar with clump passaging than I am, mentioned the following about confluence and clump passaging without ROCK inhibitor (to address a question of yours in a previous thread):
The criteria for passaging changes a bit with clump passaging. The maturity of the colonies is more critical and telling when you need to passage. Passaging an under-confluent culture in clumps is ok, as long as the colonies are nicely matured. The ideal confluence for a clump passage is probably around 60-70% – you want to avoid the colonies running into each other because those points of contact can lead to differentiation.
While passaging in clumps, you do NOT need ROCK inhibitor. At a freeze or a thaw step, you can use it for added survival, but not at the regular passage steps. One thing to add: if you’re using dispase or collagenase to dissociate, you should add in an extra wash with PBS or media before plating cells.
If anyone on the forum can add insight to this in the meantime, please do!
See related thread: When should I use ROCK inhibitor?