Hi

You should try this medium:

KBM-Gold ™ Keratinocyte Basal Medium - Gold without Ca - Lonza

We normally use with human keratinocytes.

A friend of mine and I did a lot of primary mouse keratinocyte cultures a few years agao. We used newborn skin as our source. Adult skin had been a different story. We liked the media from CellnTec (cellntec.com/products/skin/keratinocyte-cells/). I think, not much has changed since then. I think we did some comparisons and the cellntec keratinocyte progenitor medium did best for growing the cells without feeders.

I cannot recall how long the cells lasted. I am always impressed when people report to grow mouse keratinocytes for long periods of time. Perhaps I misread the papers and mixed up cell doublings and splittings...

Mouse cells seem to be more prone to senescence due to an increased sensitivity to oxygene. Forgot who published this and where. Reducing O2 in the incubator may help, if you know someone working on hypoxia in vitro you may find such an incubator.

I second the recommendation for the Lonza product. We used KGM-2 back when I worked with keratinocytes. It is expensive, but comes frozen with a "bullet-kit" of growth factors and other stuff that can be added just before use. It works very well and cells maintain their proper morphology/responses. This stuff: http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/keratinocytes-and-media/kgm-2-keratinocyte-growth-medium-2.aspx

Thank you Fernanda, Thomas and Emily. Am trying EMEM media(Lonza)with additives(T3, EGF, insulin,cholera toxin, 2% serum) with high percentage of CO2 as suggested by Thomas and the cells look happy. But they are growing in patches and not confluent throughout the plate.  Guess I will try KGM 2 or cellntec media if they are not covering the entire plate. 

I have used a very similar protocol to Thomas using Cellntec C57 media, with the Cellntec supplements added. We also found that mouse keratinocytes did not passage very well and struggled to get them beyond 2 passages. We also found that it was crucial to use skin from mice less than 48hrs old as after this time the yield was much lower. Older mice will have hair present and think this was affecting the yield. With older mice we always waxed/shaved first, but the the hair canal harboured bacteria, which was not entirely removed by washing and sometimes caused infection in some of our cultures.

A high % of CO2 is not the same as a low % of O2. To reduce O2 from atmospheric concentrations (~21%) you will need a hypoxia chamber as suggested.

One important question is what are you growing the cells on? Are you using using coated plates, and if so what is it? We did all our work without a cell feeder layer but coated our plates with collagen. The surface they are being grown on will have a big impact on how long they can survive -  there are probably already some papers on this, though you could test a few different types of matrix (e.g. collagen, fibronectin, etc)to see if this helps.

I coated the plate with fibronectin and have passaged the cells  to P4. But the problem now is patchy growth.

Will try  using a hypoxia chamber and will see whether the problem could be resolved.

Have successfully passaged the cells to P5. cell morphology is seemingly fine.But still the growth is slightly patchy. Think I will try plating them on smaller plates. 

Dear Ajai, the cells also dislike being "alone". Try not to split them too high otherwise many of the cells may not grow well anymore due to this "loneliness".

Dear Ajai,

I have used the medium detailed in this publication: Hager B, Bickenbach JR, Fleckman P. Long-term culture of murine epidermal

keratinocytes. J Investig Dermatol 1999;112:971–6.

and have grown the cells to about passage 9. But colleagues of mine have also used the KGM medium suggested above, with apparently with better growth characteristics. I always use Costar plates, coated with collagen IV, and plate the cells at about 80% density, less is detrimental. We use adult mice, ear and tail KC pooled-it is not necessary to use newborn mice. They are fed every 2nd day, by exchanging 50% of the medium. They grow in patches, so I split them 1:2 or 1:3 at the very most when the patches get too big (0.25% trypsin).  I wait till most of the cells come off (7-15 minutes), then inactivate the trypsin, remove the cells, and re-trypsinize if necessary. You can also continue culturing the cells that remain behind- I suspect they are more like stem cells than the ones that come off easily. The cells go thru a crisis after a while, with a senescent phenotype, but if you keep feeing them some will grow thru, and they may be the sponateously transfomed ones.

Dear Rossiter,

The cells are now happy and growing fine in low Ca EMEM media supplimented with low Ca serum(4%), T3, insulin, Cholera toxin, hydrocortisone and glutamax. The cells were going through the crisis after the trypsinisation and were patchy on the fibronectin coated BD plates till P5. From thereon cells grow vigorously with no crisis or no 'patchy'ness even on uncoated plates. They still have the morphology of undifferentiated keratinocytes(am at P7 right now), but the growth dynamics is very different from the cells before P5.

Dear Ajai,

we use CnT-07 medium from cellNTec. Passage two to three is very critical, you should not trypsinize the cells, or if you do, replate everything on the same dish size. This critical phase will last around 4-6 weeks, during this time they start to collect mutations that will eventually result in immortalization. You will see this because some cells will start to proliferate and will form colonies. Ones this phase is over, they will grow forever.

Stefanie,  Is it OK to rely on mutated keratinocytes?  How to ensure reproducibility of such mutated immortalised cell lines?