I have been attempting to recreate an organotypic skin culture using primary fibroblasts and primary keratinocytes. I start by embedding fibroblasts in a collagen gel at 200,000-300,000 cells/mL. Then, I add a glass ring to the top of the gel. After 1-2 days, I add primary keratincotyes on top of the culture (within the glass ring) at a density of 100,000-200,000/cm^2. After 1-2 days, a dense monolayer is formed. I then switch to a high Ca media and airlift. However, the monolayer disappears! After 5 days post airlift, a glob of keratinocytes is formed on only 1 small part of the gel surface. It appears yellow-ish. I'm not sure where the monolayer went.
Hi dear Mehra, ADM has nothing to do with the question.
Nicholas, what do you mean by airlift?
All my cultures are conducted in an insert. When the culture is airlifted, media is only fed from the bottom---the top of the gel is exposed to air. Prior to this, media is fed from the top and bottom.
Interesting, so there are no active serum supply to the top cell layer?
Yes, the cells are exposed to air. From the bottom, a paracrine feedback loop between the keratinocytes and fibroblasts takes place (in theory).
So is this a wound healing model?
I would imagine that keratinocytes are not happy being exposed to air. Are they at least kept damp with diluted serum or plasma?
Maybe the glob you are seeing is crosslinked keratin made by the cells that couldn't survive the air exposure.
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