HI
I need some advice regarding media for keratinocytes for organotypic culture.
I am using HaCaT to develop organotypic culture. My HacaT are cultured and grown in high glucose DMEM + 10% FBS +1% PenStrep.
I have gone through many protocols for Keratinocyte media and currently following this media: (stock conc & volumes used are mentioned.
High Glucose DMEM with L-glutamine (330ml) F12 (# 31765-035, GIBCO) (110ml) ,FBS (Hyclone) (50ml), Pen/strep (5ml), Transferrin (5mg/ml, T2036, Sigma) (0.5ml), Adenine (9.72mg/ml, A2786, Sigma) (1.25ml) Insulin (5mg/ml, I2643, Sigma) (0.5ml), Liothyronine (2X10^-6 M, T6397, Sigma)(0.5ml), Hydrocortisone (200ug/ml, H0888, Sigma) (1ml), Epidermal Growth factor (10ug/ ml , E9644, Sigma) (0.5ml).
Now I see my HacaTs look very differentiated in this media, looks very big, granular. I don't know what is happening. Can someone suggest what keratinocyte growth & differentiation media to use for HaCaT for organotypic culture?
Please share your suggestions and protocols if any.
Thanks
Apoorva
Hi Apoorva,
Generating skin organotypic cultures is tricky and I am afraid the major issue with your system is that the HaCaT cell line is not ideal for generating these skin equivalents since the cells already have a partially to fully differentiated phenotype under normal culture conditions due to the high calcium content of both standard media and fetal bovine serum. However, I attached a protocol for you from the guys who actually derived the HaCaT cell line (Stark 2004). They also use DMEM/FBS for cell culture and then, after seeding the cells onto the collagen gel, used TGF-b to induce terminal differentiation. If your HaCaTs appear already fully differentiated during normal culture, you might want to try thawing a new vial or even consider purchasing new cells from ATCC.
In general, I would recommend using primary human keratinocytes (also availbale from ATCC) in combination with serum and calcium-free growth medium (e.g. EpiLife https://www.thermofisher.com/order/catalog/product/MEPICF500). Cell differentiation at the air-liquid interface is then induced by adding calcium to the medium. I attached a detailed Nature Protocol paper for you. Good luck!
Dear Christian,
Thank you so much for your valuable suggesstions :) These will be very helpful.
I will probably going to culture HaCaT in specific media with low calcium.
One more thing..Is it possible that my existing HaCaTs which were in high glucose DMEM + FBS can be cultured directly in low calcium media..do these cells revert back from differentiated state?
Can you please send the Nature protocol paper.
Thanks again
Apoorva
Hi Apoorva,
I am glad you found my answer helpful. Based on your description of your HaCaT culture I assume most of your cells are terminally differentiated which is not reversible. However, if you still see some proliferation in your culture, you probably have a mixture of undifferentiated and differentiated cells and you might have a chance to recover the culture. It's worth trying if you are not too much pressed for time. The Nature Protocol paper is one of the attached files from my first reply, let me know if you can't open it. I attached another paper for you describing how HaCaTs can be grown in serum-free medium. You might want to try and give your cells a boost by adding insulin growth factor-1 (IGF-1) which should induce proliferation.
Fingers crossed,
Christian
Hello Apoorva,
I would suggest, to use KSFM (keratinocyte serum free media) for growing HaCat. It will be cost effective too and you will get rid of excessive supplements. It is a kit available from Invitrogen or Gibco. My colleague is culturing of HaCat in this media successfully since years.
*Disclaimer- The suggestions given above are not intended for any company promotions. They are based on user's personal experience only.
Hope it helps !
Dear Ajay,
ok sure! thanks a lot for suggesting the media.
The disclaimer is quite funny!! :)
Thanks
Apoorva
Dear Apoorva,
Sometimes company people suggest their products here without any reservation, so I wanted to clear that it's not for promotion ;-).
Cheers!
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