Cryopreservation of Complex Systems: The Missing Link in the Regenerative Medicine Supply Chain

Interestingness: 6

By Gregory M. Fahy, Brian Wowk, and Jun Wu. Rejuvenation Research. Summer 2006, 9(2): 279-291. doi:10.1089/rej.2006.9.279.

They pump the idea of using cryogenics to store pre-produced organs for use in transplants. They start with some interesting numbers regarding yearly US deaths that could be prevented if there were available organs:

• Heart  710,760
• Lung 122,009
• Kidney 37,251
• Liver 16,214

in total forming 36.2% of all yearly US deaths. They seem to be assigning all heart disease deaths as solvable by a heart transplant, etc. They also have a corresponding graph showing survival curves (ie age vs percentage alive) for status quo vs with those transplants, assuming no deaths during the transplants, with the transplant curve really diverging after 70 years of age with the transplant survival to status quo survival ratio of two at 80 and of 18 at 90. I don't know why they chose to show numbers that are so clearly not conservative.

They see cryopreservation as a necessary tool to manage the supply chain of organs manufactured through regenerative medicine to make mass organ availability viable. This is seen by them as the interim solution until in-situ recreation of the organ becomes a possibility.

They then describe a brief history of the cryopreservation of organs by straight freezing:

• 1950s - Freezing of guinea pig uteri to dry ice temperature (< -79C) and back. Contractile responses in vitro.
• 1973 - Freezing of dog intestines to liquid nitrogen temperature (< -196C). Long term survival of small parts of it.
• 2002 - Freezing of rat ovaries. Survival of some and offspring in one after transplantation back into rats. They say this is not a good test since the ovaries can regenerate even if completely screwed up.
• 2003 - Freezing of sheep ovaries. Vascular patency (unobstructed) retained in 3 of 11 grafts and follicle-stimulating hromone levels kept to normal limits. Second report claiming higher patency rates using directional freezing.

They claim straight-freezing of vital organs won't work because the vascular system is too easily damaged by freezing. This leads to methods that try to avoid freezing damage by avoiding ice formation.

In 1965, red blood cells and guinea pig uteri were lowered to -79C by lowering the freezing point of water and keeping it liquid all the way to the target temperature. Then in the 1980s, vitrification experiments started where the material is cooled into a glass without forming ice, starting with the vitrification of mouse embryos in 1985.

Experiments with vitrifications suggest that toxicity of cryoprotectant is due to reduced water availability for biomolecules. "Winner" of the cryoprotectants is M22 which seems to be used only below -22C with some other cryoprotectant (VMP) used to go from room temperature to -22C. Not much idea of what M22 consists of but it has a carrier solution that is meant to lower the toxicity combined with many anti-nucleating substances.

When ten rabbit kidneys were cooled to -22C, M22ised, deM22ised and rewarmed, then transplanted into rabbits, all rabbits survived (the non-cooled kidney is extracted in the experiments to make sure that the rabbits depend on the transplanted kidney), but there is more injury to the M22-ised kidneys than what is seen when just cooling them to -3C with VMP. Eight rabbit kidneys taken to -50C using M22 seem fine once rewarmed/deM22ised. Extra injury in M22-ised kidneys might be due to extra handling or extra time at cold temperature.

Newest experiment (2005) does the same rabbit kidney vitrification and transplantation on one rabbit with the rabbit surviving for 48 days after transplantation before it is killed. The cryoprotected kidney is cooled to below the glass transition temperature for M22 (-123C). Lots of damage to the kidney on this one, with hints that more even perfusion is needed.