Hi,
I am currently isolating keratinocytes from human skin samples but I am having trouble with the speed of keratinocyte growth. I am using overnight treatment in 5mg/ml dispase to disociate the epidermis and dermis and then using 15 minute incubation with T/E to release the keraintocytes, which are then grown on FNC coated flasks in Epilife media. From the cells that attach few are dividing and those that are are doing so very slowly (~5 weeks to confluency). Has anyone got any suggestions?
Many thanks
An old recipe of EGF, cholera toxin, thyroid hormone, and insulin added every two day (no skipping) should accelerate keratinocyte growth. If you skip a change, cell differentiate and you are left with 1-2% stem cells still able to proliferate. But I do not know what is in the Epilife media. At any rate, the total cell cycle time of keratinocytes is not shorter than 24 hours under most conditions. Best. Ubaldo Armato
To the certain point, media formulation can enhance the speed of keratinocyte growth. After that few things can be done:
1. better isolation technique with larger number of basal cells in good condition
2. Larger skin sample from the younger donor
3. Place of biopsy ( not exposed to the sun)
In addition to all above mentioned, I can think of two factors:
Initial cell density – cells should be seeded at relatively high density, paracrine stimulation is very important for vitality. Try to seed X2, X3 or x4 cells or use smaller cell dishes/wells.
Excessive amount of some salt/mineral – see if you can play with reduction of mineral content in your medium. Calcium levels are the most important…Before work with human keratinocytes we always do quality screening of mediums and serums on keratinocytes derived from mice– there is a huge discrepancy depending on serum batches…
May be this will help also: http://www.labome.com/method/Cell-Culture-Media-A-Review.html
In addition to all above mentioned, I can think of two factors:
Initial cell density – cells should be seeded at relatively high density, paracrine stimulation is very important for vitality. Try to seed X2, X3 or x4 cells or use smaller cell dishes/wells.
Excessive amount of some salt/mineral – see if you can play with reduction of mineral content in your medium. Calcium levels are the most important…Before work with human keratinocytes we always do quality screening of mediums and serums on keratinocytes derived from mice– there is a huge discrepancy depending on serum batches…
May be this will help also: http://www.labome.com/method/Cell-Culture-Media-A-Review.html