1. Maxim Gorshkov Visit my website for this slideshow: www.mgorshkov.com/mcgill/biol112

2.  The low temperature preservation of humans and animals  Goal: Successfully preserve human being, revive or take out of preservation at later point.  Fairly theoretical concept: long-term memory, personality, and identity stored in cell structures that do not require continuous brain activity to survive.

3. 1. Life can be stopped and restarted if the structure is properly preserved ◦ Certain organs (ex. Heart) can be cooled until stopped, restarted within the hour. 2. Structures can be preserved well by Vitrification ◦ Rather than freezing, certain technique can be performed 3. Methods for repairing structures are now foreseen. ◦ Nanotechnology and the like now imminent and relevant to Cryopreservation

4.  Basic testing involving Cyanobacteria  Cells in Cyanobacterial suffer stress osmotic stress/ice crystal damage during thawing/freezing processes ◦ Extrapolation to all life: damage with all living cells  Reduce effects with Cryoprotective compounds ◦ Specifically Glycerol or ~5% Methanol for Cyanobacteria

5.  Cyanobacteria represents most living cells within animals  Researchers began in the late 1990s to work with composition tissues such as cartilage.  Pioneered further Cryopreservation research

6.  Rats were anesthetised and two flaps were marked on the epigastric region.  Incision was made around one of the flaps  Tissue removed was stored at 4 degrees until preserved

7.  To study effects: ◦ Skin defect was formed, preserved flap was interposed and sutured  Conclusions: ◦ Potential expansion of technique but only 2/17 cryopreserved rats survived unique experiment. ◦ Key factors were investigated to include the preservation agent and the warming rate.

8.  Most popular/researched technique for cryopreservation  Based on premise that amphibians/insects tolerate varying amount of freezing  Since the viscosity of water is low, it can be vitrified by extremely rapid “flash-freezing”  The new structure in the polymer-like form is the “vitrified form” rather than the crystalline structure of regular frozen water.

9.  Theory: cryoprotectors would work as well as flash-freezing (or at least aid)  Study done in 1949 in which glycerol was introduced to protect bull sperm against freezing, further proven with red blood cells later on  DMSO also introduced as cryoprotectant, useful and easily passes through membranes  Mouse embryos preserved though combination of DMSO and glycerol

10. Freezing Without Cryoprotector

11. Freezing With Cryoprotector

12.  We’ve seen that freezing/warming rates of a sample affect the viability of the sample  Research has been done at University of Tennesse and has achieved a reasonable survival rate of mouse oocytes.  Through the vitrification cooling to -196 degrees Celsius and then warming to room temperature a sophisticated experiment was performed

13.  Cooling/warming rates varied to investigate survival rate  Procedure: ◦ Mature mice made to superovulate and the oocytees were collected ◦ Oocytes were vetrified in a solution of Ethylene Glyocol, Acetamide, and Ficoll. ◦ Oocytes were collected when warm and the viability was assessed.

14.  Based on the results, 70-80% of sample surivied regardless of cooling rate.  Slowest warming rate had loest percentage of sample survival.

17.  High sensitivity of suvival to warming rates  crystallization of intracellular structues during warming or growth responsible for lethality  Much more potential available for growth in the field based on this study and others.

18.  In the United States, cryonics can only be legally performed on humans after they have been pronounced legally dead as otherwise it would count as murder or assisted suicide.  Ideally this is performed with minutes of a cardiac arrest

19.  Given that a company in US offers Cryopreservation for $80,000/$200,000…  Selfish/irresponsible use of money which can provide a potential equivalent amount of money to efforts in developing countries  Social implications for Cryopreserved person, be that they are preserved: culture shock, alienation.  Big leap forward in science and can also open up many new directions with implications in many fields.

20. Given the choice, would you want to be Cryopreserved?

21. 1. Mazur P, Seki S. Survival of mouse oocytes after being cooled in a vitrification solution to −196 °C at 95° to 70,000 °C/min and warmed at 610° to 118,000 °C/min: A new paradigm for cryopreservation by vitrification. Cryobiology. 2011;62(1);1-7. 2. Jacobsen IA, Pegg DE, et al. Cryopreservation of organs: A review. Cryobiology. 1984;21(5):377-384. 3. Cryopreservation of Cyanobacteria [homepage on the Internet]. Austin: University of Austin; [cited 2011 Feb 28]. Available from: http://www-cyanosite.bio.purdue.edu/protocols/cryo.html. http://www-cyanosite.bio.purdue.edu/protocols/cryo.html 4. Cui X, Gao D, Vasconez H, Rinker B. Cryopreservation of composite tissues and transplantation: Preliminary studies. Cryobiology. 2007;55(3);295-304. 5. Merkle RC. The technical feasibility of cryonics. Medical Hypotheses. 1992;39(1);6-16 6. Cryonics: Alcor Life Extension Foundation [homepage on the Internet]. United States of America: Alcor Life Extension Foundation. Available from http://www.alcor.org.http://www.alcor.org 7. Vitrification in Cryonics [homepage on the Internet]. Available from http://www.benbest.com/cryonics/vitrify.html.