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Cryopreservation Fundraiser 2012

fundraiser longecity cryonics donate donations

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#1 caliban

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Posted 08 January 2012 - 07:03 PM


http://www.longecity.org/forum/page/index.html/_/science/projects/cryopreservation-fundraiser-r36



LongeCity continues its proud tradition to support small-scale, high-impact life extension research. This round, in honor of the pioneer Robert Ettinger recently placed in cryostasis, we were looking to support a project dealing with cryopreservation, cryobiology, biostasis or a related topic.

The selection criteria:
A project should
- be basic or applied research but basic research should have potential for applied development
- present short updates for Members with interim data, photos from the facility etc at agreed intervals
- be led or overseen by a person with a postgraduate qualification in the relevant field or by a person with demonstrable equivalent experience
- have clearly defined interim milestones
- have a flexible project structure that can be adjusted according to the amount of money raised
- be small in scale - one or two key workers
- be short in duration - 6 months maximum
- not be confidential. LongeCity will expect open and public presentation and discussion of research results. However, confidentiality will be accepted where a manuscript is prepared for publication or where a patent is filed.


Out of several good submissions, the Board, in consultation with knowledgeable peer reviewers drew up a shortlist of two finalists, and the final selection was left to ImmInst Members in a referendum.

The winning project has now been selected:


Uncovering the mechanisms of cryoprotectant toxicity

Joao Pedro de Magalhaes (aging@liv.ac.uk), Soren Stirling
Integrative Genomics of Ageing Group, University of Liverpool, UK

Background
Vitrification led to great hopes in cryobiology, including in human cryonics. However, a large percentage of the intercellular water must be replaced by a vitrifying cryoprotective agent (CPA) to avoid the damaging effects of ice formation. Current CPAs have toxic effects on human tissues, through mechanisms still poorly understood. As such, CPA toxicity remains one of the biggest barriers to clinical application of vitrification in human tissues, including in cryonics (1). In this project we will use cell biology and genomic methods to study the mechanisms of CPA toxicity.
High-throughput gene expression profiling has proven valuable to study the molecular mechanisms of a number of processes, including ageing and cellular responses to stress (2). Surprisingly very little work has been done to identify gene expression signatures of cryopreservation or CPA stress in human tissues. Our project will employ functional genomics to study CPA toxicity and cryopreservation in order to identify promising mechanisms for pharmacological targeting.
Human vascular endothelial cells are to be interrogated in this work. These are readily grown in culture and are critical for the integrity of all vascularised tissues. They can be vitrified but suffer from moderate CPA toxicity (3). Strikingly, CPA toxicity neutralization has not been studied in this cell type, and knowing if they exhibit this characteristic is essential. These cells are also important in protecting against reperfusion injury which is critical to cryonics. Therefore, studying CPA toxicity in vascular endothelial cells will not only advance the field but findings in these cells could have important practical applications in cryonics since the integrity of endothelial cells is crucial for whole organ outcome.

The specific aims of this project are:
1) Determine if vascular endothelial cells exhibit CPA toxicity neutralization;
2) Employ large-scale gene expression profiling to identify genes and mechanisms of CPA toxicity in vascular endothelial cells.

Programme of Work
We will first optimize temperature/permeability coefficients for our cell line to avoid cell insult through osmotic effects. In accordance with the current cryopreservation protocols (4), we will cool cells through to the glass transition temperature at rapid cooling rates (1-3 degrees per minute) to achieve vitrification. CPA will be introduced and washed-out at 0 degrees.
DMSO and ethylene glycol, which are common CPAs used in cryobiology, will be studied. We will use rapid warming. The CPA in LM5 carrier solution will be introduced to cells at 0 C in exponentially increasing concentration increments to reduce osmotic stress. The cells will be held in peak concentration of CPA for 30 minutes at 0 C and then the CPA removed from the cells by exposing them to exponentially decreasing concentration increments including mannitol osmotic buffer. A mortality curve will be constructed to establish LD50, the CPA concentration at which 50% of cells are dead.
Once the above preparatory steps are performed, we will be able to carry out the required analyses to achieve the goals of the project:
Aim 1): Toxicity neutralizers previously reported in other systems, such as amides and sugars (1), will be assayed for effects on LD50.
Aim 2): For the gene expression profiling we will focus on cells before and after treatment, with and without CPA. Digital gene expression profiles will be obtained using RNA-seq (5), as already done in our lab. We will identify genes and pathways associated with 1) response to CPAs; 2) response to vitrification/rewarming. In this way, we will build up a body of data setting out critical factors in cryopreservation stress and responses to CPAs.
Complementary histological analyses will be carried out to characterize any gross changes in cells and results will be interpreted together with gene expression results.
Overall, in addition to determining if endothelial cells exhibit CPA toxicity neutralization, this promises the first detailed molecular view of the current cryopreservation limitations in endothelial cells. Discovery of drug/protein interventions to overcome them can follow, for example by chemoinformatics using the gene expression profiles obtained to identify drugs that modulate genes and pathways involved in CPA toxicity and thus translate our findings into the clinic.

Milestones
#1 Optimize experimental conditions for our cell line.
#2 Test CPA toxicity neutralization.
#3 Perform gene expression profiling.

The project is flexible to allow adjustments (e.g., eliminating one of the aims) if sufficient funds are not raised.

Budget
Graduate student (six months): $6,000
Part-time bioinformatician (two months): $2,000
Consumables: $1,000
RNA-seq: $3,000
Total: $12,000

References
1. G. M. Fahy, Cryobiology 60, S45 (2010).
2. J. P. de Magalhaes et al., Exp Gerontol 39, 1379 (2004).
3. M. C. Wusteman et al., Cryobiology 44, 24 (2002).
4. T. Takahashi et al., Cryobiology 23, 103 (1986).
5. J. P. de Magalhaes et al., Ageing Res Rev 9, 315 (2010).


We are raising funds in support of this project UNTIL MARCH 1st 2012.

In the portfolio of life extension research efforts, cryopreservation is a field that is very poorly funded.
This is precisely where even a small community like LongeCity can make a real impact.
Please consider making a donation, however small, and please spread the word as widely as possible to other potential donors.
Every cent will donated will be matched by LongeCity up to an amount of $6000.

As a SPECIAL BONUS: if, together, we manage to support the project at least to a level where it can commence (~$10.000 = 5K from public donations) then LongeCity will also support the runner-up project proposed by Ben Best, Aschwin & Chana deWolfe to study the effect of blood washout and vitrification perfusion in a small animal mode, with a seed funding contribution of $1500l!!



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#2 AgeVivo

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Posted 10 January 2012 - 10:04 PM

since the frontpage only shows the first sentences of the article, the start of the article could be more direct. Like:

LongeCity Research Project Selected! Contribute to the cause: DONATE NOW!
Two well-recognized anti-aging researchers have just been awarded a LongeCity grant for their project on....

You can also say that there is another research project to be funded, provided we donate enough. This is motivating!
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#3 brokenportal

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Posted 20 January 2012 - 01:05 AM

I agree. When you post the link in Facebook the caption it gives is

LongeCity continues its proud tradition to support small-scale, high-impact life extension research. This round, in honor of the pioneer Robert Ettinger recently placed in cryostasis, we were looking to support a project dealing with cryopreservation, cryobiology, biostasis or a related topic.


That's alright but I think that other central wording might have greater impact. I have been cutting that wording out and pasting in the following lines from the page:

CPA toxicity neutralization has not been studied in this cell type, and knowing if they exhibit this characteristic is essential. These cells are also important in protecting against reperfusion injury which is critical to cryonics.


I might go with leading sentiments for the page along the lines of, "Help us get this research up and running. Understanding cryoprotectant toxicity is an important part of increasing the chances for successful cryopreservation. Two well-recognized anti-aging researchers have just been awarded a LongeCity grant for their project on...."

#4 jpnitya

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Posted 02 February 2012 - 01:03 AM

I kept to the sidelines during the funding voting and discussion because I did not find it adequate to participate. But now that this process is finished I obviously wish to thank LongeCity and its members for supporting our project and for the fundraiser. I also checked the funding discussion page in the members area and found some good comments (inc. criticisms) regarding our project that will be useful for us to implement and develop this ambitious project.

Lastly, anyone wishing to work in cryopreservation in Liverpool or collaborate with us please feel free to contact me:
http://pcwww.liv.ac....ng/contact.html

Cheers,
Joao Pedro





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