I'm growing MDCK cells on polyacrylimide gels. I am using sulfo-SANPAH to crosslink collagen to the PAAM.
Link included to some pictures I took... on my phone through the eyepiece.
There are many small round colonies that don't seem to be reaching confluence. I realize the cells are much denser than they would be on glass or plastic, and this could be slowing down confluence, and I could try seeding them at higher densities in addition to leaving them longer. However, previously the colonies seemed to eventually detach before becoming confluent.
I'm using sulfoSANPAH at 0.5 mM buffered with HEPES, I'm photoactivating with long-wave UV for long enough to see a color change and have done three rounds. I incubate overnight at 4 degrees with collagen at 10 ug/ml in PBS prior to seeding with cells. The culture media is DMEM supplemented with FBS. I'll need to lookup/calculate the gel concentration, have not tested the rigidity.
I'm not sure why the link didn't attach.
http://imgur.com/a/etjPK
10 and 20 x objectives, phase contrast, sorry for the low quality images.
http://imgur.com/a/etjPK
Hi Philip, the problem could be that most cultured cell lines, including MDCK, have been propagated using adhesion proteins, such as vitronectin, fibronectin, etc. that select for cells expressing integrins such as avb3 and avb5.
These av integrins are not good at mediating cell adhesion to intact collagen, because their RGD sequences are not accessible. If they cant adhere to a substrate, they tend to bundle up into balls and just float around.
What collagen are you using? Type I, Type IV? Can you use denatured collagen?
Hi Philip, We routinely use fibrillar collagen as a matrix for MDCK II (Heidelberg strain) cells and they adhere just fine mainly via a2b1-integrins. Which MDCK cells are you using? Another issue is the elastic modulus or stiffness of your gel. Have you tried more rigid recipe? From the low magnification images it is difficult to say whether your MDCK cells grow really as monolayer cell islands or maybe they form 3D clusters due to excessively soft PAA-gel? Have you looked at this in more detail?
Hi Aki, integrin a2b1 binds fibrillar collagen. Fibrillar collagen should not have exposed RGD sequences.
Cell lines maintained in TC usually express integrins that bind adhesion proteins with exposed RGD sequences (vitronectin, fibronectin, gelatin etc.)
Can you provide more details on how the Heidelberg strain of MDCK was developed and maintained?
Hi Steingrimur,
The (main) ligand site in fibrillar collagens for a2b1-integrins is the triple-helical GFOGER-(consensus)motif. Variants of this motif (in other molecules) can also be recognized by a2b1-integrins. However, a2b1-integrin also binds other as of yet poorly defined motifs. MDCK cells are generally grown directly on TC plastic as they efficiently produce their own basement membrane-like matrix containing Col IV, LN-332, LN-511, ColXVIII as well as other proteoglycans. Upon transformation or TGF-beta treatment they can also upregulate the expression of FN and Col I. This is typical for non-transformed epithelial cell lines. Cancer cell lines, however, may significantly vary in their capacity to produce ECM. MDCK cells were originally isolated from a kidney of a cockerspaniel sometime during 50s I think and initially they were used to propagate viruses. Several subclones of those original batches are grown around the world and I guess people have different ways how to maintain them. Generally they should be grown until passage 20-(max)30 and then start pver from an earlier passage in order to keep the culture stable. Here is a nice article by Dukes et al that clarifies some of the confusion that relates to multiple different MDCK cells being used in different laboratories http://www.biomedcentral.com/1471-2121/12/43
Here are a couple of our articles where we have characterized the integrin expression profile/functions in the MDCK II cells we are using...
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0019453
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0071485
One additional issue is that while fibronectin and vitronectin might be often used they also strongly bias the selection of cells (if primary cells are to be isolated) to favor fibroblasts (or cancer cells). Normal epithelial cells can bind RGD via their aV-class integrins such as aVb3 and aVb6 among others but the surface expression levels of these integrins are generally low unless a (partial) EMT process is activated (transformation/inflammation). Upon EMT epithelial cells often also upregulate a5b1-integrin which also binds RGD with high affinity.
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