Recovering cells from ancient sample - (Oct/10/2015 )

Hi, I'm curious as to the best protocol for obtaining live cells to culture from a frozen mummified sample. The current protocol I have involves techniques similar to the culturing of feeder cells.

1. Disinfecting tissue, place the sample in a conical tube with 75% alcohol and shake.

2. Pulverize tissue sample until it reaches the consistency of sludge.

3. Samples are trypsinized for 1 hour to detach epithelial cells.

4. Neutralize and wash with media x3 times.

5. Centrifuge then culture in media.

Media composition is similar to culturing fibroblasts (feeder cells).

The lab has used both trypsin and collagenase. All the researchers are using different methods to try to recover cells but so far have not yielded anything.

Any idea or proposal for better methods that could possibly yield us live cells?

I don't think there is any chance you will recover live cells from a both frozen and desiccated sample of mammalian tissue, you might from a frozen sample if it has been frozen properly (at least -80, where cells may be viable for up to two years, preferably LN2, frozen rapidly with cryoprotectant such as DMSO). I have never heard of recovering from a desiccated sample.

Some organisms can survive freezing and desiccation but they have specific molecular mechanisms that allow this to happen. Which organism are you working with?

I did not think it was possible either but I am interning with a cloning laboratory in Korea for training. They are working on the Mammoth project and I believe it started way back in 2013.

There is a bit of a language barrier so I may not understand them fully but I believe the project involves recovering live cells (attempting) to try the cloning process using ovum from a close species, I deduced it would be the Asian elephant (?). I know that it is possible to recover fragments of DNA of ancient samples (museums, etc) but I was wondering if the cells (in this case) would still be alive let alone be viable for culture. I think the objective was to attempt to "revive" cells for culture but the laboratory is also working on mapping the genome which they hope to reconstruct in the future clone.

The whole process seems a bit impractical to me (it's a personal opinion). Such samples are often highly contaminated and missing repetitive sequences so I am unsure as to how they plan on reconstructing the DNA. The live cell method (assuming there are any left) also doesn't seem very feasible since no cultures have survived thus far. I am curious to know if this lengthy trial and error culture they are doing will yield any results. Apart from that, the method we have employed seems a little crude for me considering this is a really old and probably damaged sample. So, are there better methods that could be used that may "increase" the chances of actually obtaining live cells or intact DNA from these samples? 

P/S: Not being a pessimist, just a little skeptical about the entire process. I still hope that whatever they/we are doing works or hopefully will work.

I seriously doubt that there will be any viable cells in frozen mammoths, but it might be possible to retrieve an intact cell, that you could then extract the chromosomes from and try cloning... which has a very low success rate anyway, so you would probably need a lot of cells.

I don't know about any better methods, as I'm not sure that this is a problem anyone has really considered before. It would probably be worth looking through the literature for methods for reviving insects and tardigrades, there might be some clues there.