Hi,
I have been having problems culturing keratinocytes from 4mm human skin punch biopsies. When I recieve the biopsy I first was the samples in sterile PBS, then I remove the subcutaneous fat. I then use the miltenyi Epidermis skin digestion kit to remove the epidermis and to isolate the keratinocytes. This involves firstly treating the skin sample (dermis and epidermis) with dispase. This is left overnight at 4 degrees. The following day I peel the epidermis off and treat with collagenase and trypsin for one hour. The epidermis is then mechanically digested using a gentleMacs Dissociator. I'm getting approximately 7 x 10^4 keratinocytes per biopsy.
The issue I am having is culturing the keratinocytes long term. I plate all of my cells into a 6 well plate with supplemented EpiLife medium, however after 3 days the cells failed to adhere to the well surface and the cells began to die after 24 hours. I then tried coating the well with Sigma's coating matrix kit, which contains collagen and is optimised for primary keratinocyte culture. However, although some cells stuck to the well of the plate, the cells did not proliferate in the EpiLife medium and ultimately the cells died. I tried using different cell concentrations in different sized wells but nothing seems to work.
If anybody has any suggestions or can tell me where I am going wrong it would be greatly appreciated!
It sounded to me like the cells are not proliferating and it is usually due to the supplements. If this is the case, try adding HKGS (Gibco S0015) to your Epilife medium if you have not done so. Otherwise, I have heard multiple times that KGM-2 (from Lonza) is better in setting the culture obtained immediately from tissue source. Epilife is better in the subsequent passage and subculture. Remember to supplement the KGM-2 as well (insulin, hEGF and BPE, they should be all available from the same vendor selling the medium). You can also try changing the medium every other day during the culture to keep the desired supplement level, although I do not think this alone will change things drastically for you. And finally you can try adding FBS/FCS in the first few days. Check this paper out (Prof. Poumay's literatures are my major reference in making Keratinocyte culture)
Article Reconstruction of Normal and Pathological Human Epidermis on...
Dear Conor Smith,
I agreed with Tsemay Tse even though it could help you to accelerate the keratinocyte culture but it depends on sample sources you got, sometimes it could affect your culture anyway. In our experience, you could use another technic by doing co-culture technique die to its eminent growth rate. However, you need to perform differential trypsinisation to ensure the removal of fibroblasts from keratinocytes. Best of luck!
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