Hello everyone,
My lab regularly extracts collagen from rat tails of normal rats, and we would like to include steps for sterilization. I have seen some protocols but I am unsure what step or steps actually perform the sterilization? Does chloroform sterilize the collagen? I have seen others include antibiotics and antimycotics during the dissolution step. I understand that some people UV sterilize, but I worry what that will do to the protein. I have heard of sterilization of lyophilized collagen by ethylene oxide gas but I am unsure of its effects. What will I notice during cell culture if we fail to adequately sterilize/purify the collagen?
I have used filter sterilization through a 0.2 micron filter from millipore. The filters are low protein binding and you can use the ones that attach to a syringe, and just push the solution through with the syringe plunger. The luer lock of the syringe attaches to the filter disk for a good seal. I would filter the solution into a container that is also sterile, and you can do this under a hood too.
Peracetic acid has also been used to sterilize biomaterials. http://www.ncbi.nlm.nih.gov/pubmed/15225206
Antibiotics and antimycotics will not sterilize. I haven't filtered collagen, but filter sterilizing fibrinogen incurs a massive loss of material along with a change in molecular weight distribution. If all you want to do is coat some plates though, this is perhaps the expedient way to go. UV & ethylene oxide can work well, but the normally the ECM needs to be dry (after coating, perhaps). Gaurav's & Ali's suggestions are good for soluble collagen, and may work quite well. Try them, and then toss a little bit of the sterilized material into L broth (rich e. coli medium) and see if anything grows. Gamma irradiation & electron beam are alternatives, but require access to appropriate equipment.
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