Hi Rohan,

What method are you currently using?

If you are using small biopsies, have you tried an explant culture method which may improve viability of cells obtained but does take 2-3 weeks to see results.

I have attached my protocol for explant cultures - if your biopsies are very precious you may wish to try this method on some more readily available skin first

Best wishes, Catherine

Thank You Catherine

I am isolating cells using a partial thickness skin sample. Initially, I have used Dispase to digest the epidermal-dermal junction and then tried to isolate keratinocytes using 0.05% Trypsin + EDTA along with vigorous pipetting for 5 minutes at 37 C and 0.25% Trypsin + EDTA for 15 minutes at 37 C  in static condition. The reported literature suggest that the basal cell will get detached and can be used for seeding. But till now I am only able to isolate the cells using these methods.

I have also tried explant culture and have successfully isolated the cells. But as you have mentioned it takes a lot of time to get results. Moreover we don not have in-house facility to irradiate fibroblast cells.

I have certain queries regarding explant cultures

1. I have large number to cells in liquid media but very less cell attachment even after 48 hours?

2. Does the viability of keratinocytes is affected if the skin samples were processed after 48 hours? Till then they were kept in Saline solution at 37 C.

Appreciate your help. Thank You

You could try using feeder layer that has been treated with mitomicyn C instead of radiation.. We isolate keratinocytes usind the tripsin/EDTA method. We use scisors instead of dispase to remove as much as possible the dermal layer.. the treatment with trypsin occurs  with minced skin in the incubator 37C, 5% CO2,  under magnetic stir 30 min cycles ( I do up to 3). The trypsin is inactivated with tripsin inhibitor. The cells like feeder layer. Or maybe you could coate your plate with gelatin or collagen. I don´t think you should leave the cells in saline at 37C, but it has work fine for me at 4C in DMEM. 

 Dear  Rohan, 

You can visit this website that can help you to get isolation of keratinocytes.

https://www.lifetechnologies.com/th/en/home/references/protocols/cell-culture/primary-cell-protocols/keratinocyteo -protocols.html.,

Rohan,

1) Any keratinocytes that have not attached after 48hrs are probably dead and need removing from the culture flask as they produce substances that will adversely affect the health of the live cells.

2)  The cell viability will be affected after 48hrs but you can successfully isolate both keratinocytes and fibroblasts at this time providing the tissue is stored in medium.

On arrival we normally wash the sample in PBS containing antibiotics (antifungal only used in cases where there is a likely to be contamination from them i.e. foot skin or finger that includes a nail) - If the tissue is not to be extracted straight away then aspirate the PBS and add DMEM with 10% FBS plus antibiotics (transport medium) and place in the fridge at +4degC

3)  Be careful when using Mitomycin treated 3T3s - if the treated cells were over-confluent when the mitomycin C was used to growth arrest them we have found that sometimes they still have the capacity to proliferate.

Hope this helps.

Catherine

Thank You Clara, Juthaporn and Catherine.

Appreciate Your Help.