5 replies to this topic

[Mixter]

Modern vitrification already works at rather well although not perfect, at macroscopic levels such as tissue levels of mm scales.

Before:
After:
(Note: different tissue, not fully comparable to the above)

Here is my idea and suggestion on how to improve vitrification on a subcellular scale, to better protect
the vitrified DNA and generally make it better:

Days to hours prior to vitrification, temporarily express very high levels of cold-shock proteins in all of the patient's tissues that should be vitrified.

How: mammalian liposome-mediated transfection with mRNA transcripts of appropriate cold-shock domain containing genes

This idea may sound ambitious, but ordering mRNA transcripts and doing liposome transfection should,
to my knowledge, not be a great cost issue, and most tests could be performed on mice, and to take
tissue size in account, some larger animals, for example. I'd be happy about any feedback regarding
feasibility and ideas in that direction which may have already been pursued at places like 21CM.


The reference paper on the transfection procedure: Cationic Liposome-Mediated RNA Transfection http://www.pnas.org/...ract/86/16/6077
Recommended reference on this mRNA transfection: http://www.springerl...38w1462u5w145r/


Which genes may be used: transcripts of a combination of harmless, cytosolic and DNA-binding genes with cold-shock domains
- DbpA, and DNA-binding protein A and B variants, which contain several cold-shock domains
- CSDA, CRA_a, Y-box binding protein
- unr (PMID: 10049359) especially for cytosolic cold-shock


Advanced ideas
- Use injections into the cerebrospinal fluid to facilitate transfection to brain tissue
- Experiment with the life cycle of the proteins; perhaps mRNA can/should be given no sooner than during perfusion
- Explore further cold-shock proteins, such as on CSDbase: http://www.chemie.un...rg.de/~csdbase/
- Add/insert specific import sequences to the chosen genes for ER, golgi, mitochondrial and nuclear availability
- The inner of the mitochondria probably need extra protection
- Improve the process using nanoapatite particles: http://lib.bioinfo.pl/pmid:16125127

Edited by mixter, 08 March 2008 - 05:20 PM.

[Mind]

Thanks for sharing your idea mixter! Cryonics (whole body and organ preservation) is a small field of research and new ideas/interest are great.

One idea I had been thinking about lately is somewhat similar. Besides using cold shock proteins, I was wondering if there were any other "biological" molecules that are liquid at room temperature, naturally occurring within all cells, and don't "clump" together (like water) into larger crystalline forms when frozen down to liquid nitrogen temps. Basically a natural non-toxic vitrification liquid (isn't that what we are all looking for). I was just thinking that there are naturally occurring components/molecules of the cell that survive freezing and if we can isolate those and perfuse them in larger quantities within cells to replace much of the water, or at least keep the water from doing damage, then it would be an ideal vitrification liquid.

This is just off the top of my head, no doubt some people have already looked into such things.

[bgwowk]

Exposure to cold per se is not much a problem with new vitrification methods that mitigate so-called "chilling injury". The most serious remaining problem is cryoprotectant toxicity, the mechanism of which is not currently understood. Chaperone proteins generally could have a beneficial role, but this has never been explored.

Mind, your suggestion is interesting because increasing the concentration of solutes naturally present in cells is exactly what happens in cryopreservation protocols that involve partial dehydration. This can be beneficial, but only to a certain extent. If the concentration of natural salts and proteins is allowed to become too high, then more damage usually happens than if non-natural solutes like ethylene glycol and DMSO are allowed to replace water inside cells instead. Natural is not always best.

Edited by bgwowk, 08 March 2008 - 06:47 PM.

[maestro949]

Exposure to cold per se is not much a problem with new vitrification methods that mitigate so-called "chilling injury". The most serious remaining problem is cryoprotectant toxicity, the mechanism of which is not currently understood. Chaperone proteins generally could have a beneficial role, but this has never been explored.

Mind, your suggestion is interesting because increasing the concentration of solutes naturally present in cells is exactly what happens in cryopreservation protocols that involve partial dehydration. This can be beneficial, but only to a certain extent. If the concentration of natural salts and proteins is allowed to become too high, then more damage usually happens than if non-natural solutes like ethylene glycol and DMSO are allowed to replace water inside cells instead. Natural is not always best.

Ironically, I just posted an article about a guy capable of meditating to survive extreme cold for abnormal periods of time. Perhaps studying these individuals' gene expression might provide more clues as to whether there is any insight that can be gleened from such individuals for cryopreservation. If there is physiological change that permits them to survive colder temperatures, perhaps less cryoprotectant would be necessary for someone treated with agents that mimic the physiological affect being produced by the meditation.

Another thought regarding the toxicity you mention bgwowk, Some species (the famous frozen frogs) are already capable of generating their own cryoprotectant. Has any research determined whether these are toxic in humans? Or perhaps even that the pathways are conserved such that we still carry the DNA for such processes?

[bgwowk]

Ironically, I just posted an article about a guy capable of meditating to survive extreme cold for abnormal periods of time. Perhaps studying these individuals' gene expression might provide more clues as to whether there is any insight that can be gleened from such individuals for cryopreservation. If there is physiological change that permits them to survive colder temperatures, perhaps less cryoprotectant would be necessary for someone treated with agents that mimic the physiological affect being produced by the meditation.

People able to survive cold through meditation techniques don't get especially cold. The key to their environmental cold tolerance is that they have learned to increase internal body heat generation through their own volition. A woman with this ability was able to swim a significant distance in Antarctic waters at 0 degC. Her measured body temperature just before she jumped in was +102 degF. This temperature was purely the result of the mental anticipation of the swim. Like other "mind over matter" biofeedback techniques, this may be a skill that can be learned.

Another thought regarding the toxicity you mention bgwowk, Some species (the famous frozen frogs) are already capable of generating their own cryoprotectant. Has any research determined whether these are toxic in humans? Or perhaps even that the pathways are conserved such that we still carry the DNA for such processes?

Frozen frogs are not frozen the way most people think. They form ice in certain compartments of their body to drive up the concentration cryoprotectant so that actual cells and tissues remain unfrozen at sub-zero temperatures. The cryoprotectant is ordinary glucose, which is not a very good cryoprotectant, except for the advantage that it can be easily made naturally. They will usually not survive temperatures colder than about -5 degC.

Edited by bgwowk, 09 March 2008 - 01:53 AM.

[maestro949]

Thanks Brian. So is there anything that could be done to better "survive" the vitrification process such that protein denaturation is minimized while still alive? Perhaps apply the process of mithridatism with the cryoprotectant?