I am new to collagen gel and tried to look for protocols for collagen gel film making. Briefly, the collagen solution (in acetic acid) is neutralized and then left at 37 degree for about 30-60 min for fibrillogenesis (if we tend to go for self-cross-linking). I was just wondering if we don't neutralize the solution and put the gel for fibrillogenesis, would it not make a hydrogel? I understand that the acetic acid could be harmful for the cells cultured atop collagen gel but I assume that during swelling and washing with PBS, it could be removed easily. Also can you suggest some literature on collagen gel (not the hybrid gels) swelling ratios? What are the main differences between a self-cross linked collagen gel and a collagen gel cross-linked with the help of cross-linker.
Hi Rizwan,
I am not directly working with collagen, but with hydrogels in general. Collagen is soluble only in acidic pH and get precipitated at basic pH. This has to do with protonation of amines in the protein. If you take high concentration of collagen (15 to 20mg/mL) and neutralize it you will get a viscous solution which you can call as hydrogel. You can also try to dissolve solid material in PBS directly and check the pH later, this could help as well. This however, is not as stable as chemically crosslinked one.
Hi Oommen, thanks for your input. Lets wait what others have to say. I am more interested in knowing what will happen if I dont neutralize the solution and put it for fibrillogenesis!
My experience with collagen gels is only with fibroblasts. It was long ago since I've measured fibroblasts' contractile ability following treatment but perhaps this gel recipe would work for the corneal cells as well.
A solution of bovine collagen types I (97%) and III (3 mg collagen/ml Vitrogen
100; Collagen Corp., Palo Alto, CA) was mixed with triple strength DMEM,
containing 20 mM Hepes buffer (Gibco, Grand Island, NY) to maintain neutral pH,
calf serum (Tissue Culture Biologicals, Tulare, CA), cells (detached by trypsin from
monolayer confluent cultures)
It is more commonly described as fibrillogenesis and is as described above due to self assembly of collagen monomers as the charge profile changes with differing pH
Neutralization of collagen with 10x PBS promotes fibrillogenesis of collagen fibres . Hence if you want are interested to study fibrillogenesis, I think neutralization of collagen solution is required.
See the attach files
Please check my post at the following RG discussion: https://www.researchgate.net/post/Can_anyone_suggest_me_the_buffer_conditions_or_methods_for_solubilising_collagen_powder_other_than_acidic_condition
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