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Primary Culture of keratinocytes - How to start a keratinocyte culture (Jun/01/2005 )

Hi all

I begin a project and i'll need to start a primary culture of keratinocytes.
So I need a precise protocole to isolate keratinocytes from human skin explants please, help wink.gif


hi. if my english is not very well ...sorry (im from Chile). well i have work with keratinocytes....first....the tissue explants (from healthy people) from surgery,,,where washed in medium (with antibiotics) 24 h . then you have to quit all the fatty tissue. then the skin (clean of fat) you have to cut with a keratom .....a you`ll obtain pieces of skin that contain dermis and epidermis....then you have to sumerge this pieces in trypsin (0.5%) to separate this layers....then cut with sscisors the epidermis and sumerge it in trypsin(0.25%) and a celular suspension ready to use..


hi, everybody.
i'm a newer. so i'm very intereted about cell culture, specially for keratinocytes culture. i think why we share information together.
i think if you do follow khalou, i think when you culture with keratinocytes, it will implant with fibroblasts. i think it very dangerous for you, because fibroblast will overgrow and use media, so kerationocytes will difficult to grow in culture flask.
if you want keratinocyte to grow good. i think you must excess fibroblast the more, the good
i thinh my english not good so you also mean it
hi hi hi
we will discuss about it more?


I have just started to culture murine keratinocytes and found that the protocol in the paper, Isolation and Utilization of Epidermal Keratinocytes for Oncogene Research in Methods in Enzymology, Vol 254, Yuspa et. al. is quite good and i recommend using the Book Methods in Molecular Biology, vol 289, Epidermal Cells, methods and protocols. I have had some success using these culturing keratinocytes from newborn mice using conditioned media from new born fibroblasts.


[quote name='Caruyo' date='Jun 1 2005, 07:16 AM' post='16430']
Hi all

I begin a project and i'll need to start a primary culture of keratinocytes.
v150ug/ml and transfer to the laboratory as soon possible (no more than 4 hours), to separate the epithelial layer using enzymatic protocols (). Briefly, the tissue will be transferred to a 10 cm dish and rinsed with PBS without ca++ to remove as much connective tissue as possible and placed in a 3.5 cm dish and enough trypsin with EDTA will be added for full tissue coverage. After incubating overnight at 4°C the epithelium will be carefully scraped with a pair of forceps into the trypsin solution. The resulting suspension will be triturated adding the same volume of PBS, and spun down for 5 min at 800 rpm, 4°C. The pellet will be resuspended and the cells seeded in growth medium (keratinocyte-SFM) 6 well plate coated with collagen type 1 rat and incubated at 37°.