Hello every one,
We are working with normal human epidermal cells, growing them in dermal cell basal medium, to which we add a keratinocyte growth kit.
Although this medium is formulated to inhibit fibroblast growth, as it has a low calcium concentration that slows differentiation, We are noticing fibroblast growth.
I wanted to ask if anyone else is working with these cells and has encountered this issue?
if so, i would appreciate any help on how to overcome and eliminate fibroblast growth.
Thanks.
Hello Yasmin Yosif
When fibroblasts contamination is present in culture, differential trypsinization can be used to get rid of them. It consists of faster trypsinization of fibroblasts compared to the cells of interest.
Protocol in brief:
You first trypsinize the culture with low concentration of trypsin say 0.01% for 5 minutes at 37-degree C. Tap the flask or plate to remove the loosely adherent cells. Fibroblasts will detach first and float. Collect the floating fibroblast cells and give a wash to the rest of the adherent cells left in the plate/flask with PBS (sterile). Use a higher concentration of trypsin say (0.25%) for 5 mins at 37-degree C to detach the rest of the adhered cells. Centrifuge these cells and re-plate the cells for a few hours in the incubator. If there are fibroblasts still left in culture which you may have missed out, they will adhere to the substratum earlier than the cells of interest. Complete adhesion of fibroblasts onto the surface is dependent on the culture environment but is commonly observed in over 30 minutes.
You may collect the rest of the floating cells and let the fibroblasts grow in the plate/flask. So, you may have fibroblast cells collected in two sets, one which you have collected earlier as floating cells and now you will have another set of fibroblasts adhered to the tissue culture plate or flask.
You may want to refer to the article attached below for another method.
https://www.sciencedirect.com/science/article/abs/pii/0014482789903200
Best.
- Badges
- Science topic