I'm trying to carry out human keratinocyte (from adult skin) cultures. Since I need a great number of cells for setting up my experiments, I have to reach to 162-cm2 flasks, at least, in a very few passages. My problems are related to trypsinization and the relative number of cells able to adhere on plastic (around 50% of viable cells!). I've tried many protocols, using TryPLE without blocking, trypsin 0,05%, trypsin 0,05%+EDTA 0,1% (no blocking), and trypsin 0,05% blocked with Keratinocyte medium+1%BSA. In any case, I have seen a lot of cell aggregates and a very low percentage of adherent cells. Moreover, I can't rely upon the number of viable cell counted after trypsinization and those after seeding (for example setting up MTT analyses). Can anybody help me? What is the best way (protocol) to detach this kind of cells avoinding aggregates formation and cell adherence loss?
Thank you in advance!
I don't have specific experience with keratinocytes but know a little about certain cells that tend to aggregate. As a general rule, do not disturb cells during trypsinization (tapping etc). If the cells are easy to detach, add trypsin (e.g., 5 ml), let it cover all areas, and remove most (e.g. 4.5 ml). Turn side to side slowly to check detachment. Then add your regular medium for passage. This is the way we treat osteoblast cells, and the survival rate is high.
I have used trypsin/EDTA and it has worked fine. As Ke-We suggested, use minimal amount of trypsin and check for the detachment. Some cells are detached more easily than others, and it is good to take them out soon ( pipette up and dow several times and take them out the trypsin, block the trypsin with trypsin inhibitor) At the end of the trypsin period I carefully pipet up and down to dissolve any clumps and inactivate with specific trypsin inhibitor.
Hi Simona, I largely agree with all the answers above. Be careful with how much trypsin you use, and neutralise it with 2X volumes of media post detachment before you centrifuge the cells. However, specific to keratinocytes, I find that their viability and attachement becomes poor if you subculture them at confluence >80%. Therefore, if you could try and subculture them around 70-75% confluence and see if results improve? Good luck!
I routinely use 0.05% trypsin/EDTA and 2X volume of trypsin of soybean trypsin inhibitor (250 ug/mL) to stop trypsin. Don't let culture get to greater than 70% confluence as cells will start to differentiate and will stop growing (can see larger cells appear). I love Lifeline Cell Technology DermaLife K media (LS-1030). I've tried other media and this allows the best growth and health of the cells. Their freezing medium is also great. Can get 50-90% recovery. I also recommend Promega's Cell TiterGlo 2.0 viability assay over MTT. MTT is not very sensitive but the chemiluminescent assay is very sensitive. I've used Cell TiterGlo with primary keratinocytes, fibroblasts, and melanocytes and get great results. Promega offers a variety of other cell viablity/death assays as well (I don't work for them, just like their products).
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