Hi Rizal,

Are you isolating the cells yourself? There are many factors that influence the isolation:

- donor age

- skin condition (its better to isolate as early as possible)

- source of the skin sample (my experience: samples from the foot don't work too well for example)

- isolation technique (which enzymes do you use? trypsin? dispase? incubation times and conditions?)

- was the skin sample treated with something? (unfortunately some doctors use chemicals to remove the hair before they take a biopsy)

- ...

And then there are many factors that influence the cultivation:

- use of feeder cells or use of collagen/laminin/... - coatings

- media composition

- seeding density

- Ca-level

- pH

- ...

If you can't get rid of the contamination, please check the Question-section for questions about methods to properly clean your bench and incubator. It can be very tough to get rid of that.

I hope this was a bit helpful.

Cheers!

Miriam

Hi Miriam, thanks for your suggestions.

I have isolated using mechanical methods in which only using physical isolation without any chemical digestion.

I don't think the skin was treated with any chemical as the skin that I obtained was from healthy donor and the donor age was less than 60 years old.

First the skin was sanitized using alcohol 70%, then the skin was cleaned by removing fat layer and the skin debris. After that, using a foot planner the skin will be scrapped but in my case it's difficult to scrap and alternatively, I was using scalpel to cut the skin. Then, the skin was incubated with trypsin+dpbs for 30 min and later on the skin was filtered and the epidermal cells were released by using tweezer in rpmi+2%fbs. Then, it was centrifuged at 450xg for 10 min, and later cultured according to the cell density.

Hi Rizal,

below is my protocol to isolate keratinocytes from foreskin:

- Keep the skin in Hank’s balanced salt solution or DMEM with penicillin/streptomycin (50 IU/ml) at 4°C either for one hour or overnight.

- Wash foreskin once with 70% ethanol, then twice with PBS- to remove ethanol completely.

- Cut the skin in small pieces (0,5x0,5 cm) with surgical scissors or blade

- Incubate pieces in dispase II (5 mg/ml, Boehringer Mannheim, Germany) at 4°C (15 ml/one foreskin) overnight.

- Separate epidermis from dermis using forceps

- Incubate the epidermis in 0,25% trypsin/0,02% EDTA for 15 min at 37°C (15 ml/one foreskin) and mechanically disaggregate for 5 min.

Regarding the isolation of keratinocytes from other parts of the body, first, I remove the fat layer and then keep the skin in penicillin/streptomycin solution.

Good luck!

Alessandra