22 replies to this topic

[caliban]

As usual, please bear in mind that the first thing we want these researchers to do is to focus on life-extension research, so please give them some time to answer.

[brokenportal]

Here's a question: How hard do you anticipate your project will be in comparison to other research you have done?

[jpnitya]

from a technical perspective I don't think it will be very hard since we are familiar with most of the methods to be used in this project. I suspect the hardest part is going to be the interpretation of the results, in part because so little has been done along these lines of work. in that sense, sharing the results with the LongeCity community may prove beneficial because its members may find patterns and insights in our results that we would otherwise miss.

[AgeVivo]

sharing the results with the LongeCity community may prove beneficial because its members may find patterns and insights in our results that we would otherwise miss.

can't resist to say how great this sounds!

If from time to time you can post a picture of how you do the experiment, that may also help lots of members learn/understand/suggest/project themselves in such work

[jpnitya]

If from time to time you can post a picture of how you do the experiment, that may also help lots of members learn/understand/suggest/project themselves in such work

I have to say that visually it doesn't look as exciting as it sounds. most of what we do is on small petri dishes and eppendorfs which look really dull. we could try to get some pictures of cells under the microscope, though, and post them -- these may look more exciting.

[treonsverdery]

here is a daffy question, cryopreservatives need to diffuse, so does adding a little natural membrane detergent, like some membrane lipid, make them travel more thoughouly through tissues.

also, thinking of revival effectiveness, there may be categories of value. perhaps a thawed organism would most benefit from DNA, mRNA, tRNA intactness, as compared with say membrane intactness, (others might possibly prefer membrane intactness) Thus prioritizing what you want to thaw right could guide the creation of the chemistry.

Ive read that melatonin preferentially accumulates at the nucleus, thus a cryopreservative molecule like a glycol attached to melatonin might measurably preserve the nucleus more effectively which sure sounds important or better.

memories are linked to mRNA production, thus the crypreservation of the "immediate protein environment going on" with mRNA might conserve some mood or beingness among the thawed creatures

As a rapid development approach, even if you just crypreserve a tardigrades, perhaps you could teach them to swim towards a particular color light, then show the ones with mRNA cryopreservative retained their learning more effectively. That would tell the humans there is a cryopreservative better at preserving memory.

Ive heard the chemical ethidium bromide intercalates with DNA, possibly there is a less forceful similar molecule, or possibly a generic very brief siRNA that would lazily reversibly glom mRNA making it more freezeproof.

Edited by treonsverdery, 10 February 2012 - 07:51 PM.

[jpnitya]

I think those are interesting ideas but there is a lot that we don't know about so it's very hard to say for sure. I would guess that preserving cell viability is the most important thing and for this you need membrane integrity as well as DNA integrity and maybe to a lesser degree mRNA integrity.

I don't know if memories are linked to mRNA production. My understanding is that memories are linked to synapses. Do you have a reference on this?

[AgeVivo]

Any news? difficulties so far, choices you made, etc.

[jpnitya]

Any news? difficulties so far, choices you made, etc.

we have only just started the project on July 1 because we were waiting for all the staff to be in place to begin. so we don't have much news at this stage. I can tell you that we are in the process of establishing a culture of endothelial cells in our lab and will then be trying different cryopreservation protocols on them. I would hope that by August or September we know a little bit more about how these cells behave and can give you an update with some results or information of how our experiments are going.

Cheers,
Joao Pedro

[jpnitya]

To give you an update on the progress of the project, we have been doing some preliminary experiments in HeLa cells, exposing them to various CPA’s in different concentrations, namely Dimethyl Sulfoxide and Ethylene Glycol.

The situation is that we will need to make a decision on which conditions to profile gene expression (i.e., which CPA(s), concentrations, times from exposure). As such, before we start using HUVECs, which are not as easy to cultivate as HeLa, we want to test different types and concentrations of CPA, so that when we start using HUVECs we will only need some minor adjustments. at the moment we are inclined to profile gene expression on cells exposed to Ethylene Glycol, because I think we know much less about its toxicity than DMSO. We're inclined to do this at different time points using a concentration that kills ~half the cells, but suggestions are welcomed.

Cheers,
Joao Pedro

[AgeVivo]

Just to say it: it is great to have such open-sharing of where the experiment is currently!

To rephrase and be sure we understand, you are now first planning to test the impact of Ethylene Glycol on gene expression profile in HeLa cells, to start with what works in order to make not-too big steps so that you ensure the quality of your work. You may try a few concentrations and times from exposures, so that we'll have a feeling and a good basis for further analysis. Is that right?

Then you plan to move to HUVECs when/if you are able to cultivate them correctly in the lab, and do the same, at least with one/a few points that was chosen based on HeLa results. Right?

If things work that far, then you plan to do other CPA's, doses, times from exposures. Don't hesitate to correct if I misunderstood. The clearer, the more useful it can be to have such open-science. Thank you!!!

Edited by AgeVivo, 12 October 2012 - 07:25 PM.

[jpnitya]

We can only afford one gene expression profiling experiment, so what we are testing is only cell mortality in HeLa cells. My idea is to then test the conditions that appear better in HUVECs and do the gene expression profiling on them, probably just a single concentration on a couple of different time points.

Joao Pedro

[AgeVivo]

just to say that it sounds great. if (only if) at some point some gene expression data can be shared, I imagine that there will be many people here who will be willing to to look at it.

[Mind]

Just wondering if you have any pictures to share? Believe it or not, I wouldn't mind seeing some boring pictures of the lab or petri dishes. Cells under microscope would be interesting of course. Pictures can help "transport" those of us who are not involved, to get a better feeling of community behind this research.

Also, excuse my ignorance, but will you be testing M22 - the cryoprtectant used by 21st century medicine?

[Mind]

Besides within this forum thread, if you have some questions, you could post them here: An upcoming podcast with Joao Pedro de Magalhaes: http://www.longecity...o-de-magalhaes/

[Mind]

Just a couple of pictures to share from our leading edge LongeCity-sponsored cryonics research. Dr. de Magalhaes and assistant Rui Cordeiro in the lab.

Attached Thumbnails

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[jpnitya]

Just to give you a quick update on the project, we have optimized the culture and cryopreservation conditions for the experiment and are aiming to do the gene expression profiling soon. We were hoping to have it done by now but we had a problem with the RNA extraction and need to obtain RNA from more cells, which we hope to do next week. We will then do a microarray assay for gene expression profiling. Barring any further delays we hope to have the gene expression data next month. will let you know once we have the results.

Cheers,
Joao Pedro

[AgeVivo]

Looks great. Good luck for the heavy cell work these coming days then, to get the right RNA content. Our mind is with you!

[John Schloendorn]

Woah, nice collection of HiSeq's ;-) Basic question -- can you post a link to a project summary here? Sorry I'm having a hard time finding this -- haven't checked in for a while.

[Mind]

Here is a short summary: http://www.longecity...-fundraiser-r36

The researchers might have a little to add as well.

[AgeVivo]

we hope to have the gene expression data next month. will let you know once we have the results.

any... good or bad news? having a set of data that can be analysed at home may interest some future researchers

[jpnitya]

It took longer than anticipated to generate the data and get all the results statistically analysed but that's the way it goes sometimes in research. we have now set up a website about cryopreservation in general and with information about our project:
http://cryopreservation.org.uk/

On our website you can find a brief description of the project as well as some preliminary results:
http://cryopreservat...k/research.html
Specifically, we have the list of genes differentially expressed at 24 hours ( which is when most gene expression changes take place). We are now trying to interpret the results so we can write up and published this, so any ideas or suggestions are welcomed -- any genes that look particularly interesting?

Thanks,
Joao Pedro

[Mind]

Here is the final paper on the research. A lot of quantitative information on gene expression. Ethylene glycol was not very toxic at 60% concentration after 2 hours of exposure.

Attached Files

•  Cryobiology2015.pdf   1.04MB   521 downloads