BIOSTASIS RESEARCH TODAY

Research now points to the fact that with the proper protocols whole human brains can repair the damage incurred while reaching, maintaining and returning from a state of supercooled biostasis.  The damage not repaired by innate cellular stress response processes will be rendered harmless by replacing glucose, key metabolites and enzymes, and certain amino acids during the reanimation process.  Here are some of the reasons we feel the age of elective biostasis surgery will be soon upon us.

Today, tens of thousands of viable human blastocysts (aged 5 to 8 days) are held in biostasis at liquid nitrogen (LN2) temperatures (-196 degrees C).  After years in suspended animation the blastocysts are reanimated and implanted into soon-to-be mothers, with most of the implanted blastocysts leading to healthy children.  There can be little question that human cells fully recover genetic and epigenetic functionality after being reanimated from biostasis under the right protocols.

What will it take to achieve these results in human cephalons and eventually, entire adult patients?  The answer, in part, is that we need to "harden" cells with certain additives one or two days before the patient enters biostasis, so that cells can more easily recover from cryoprotectant related toxicity damage, and not experience the growth of ice-crystals seeded by bacterial and endogenous proteins.  This will be achieved by repurposing existing drug molecules and using tissue specific delivery vectors carrying RNAi sequences to modulate the genomic and proteomic pathways that impact cell viability during the supercooling and reperfusion protocols.

Human blastocysts however, do not require genomic cell hardening therapies (though some protocols do include a pre-treatment stage of loading the blastocysts with dietary amino acids, to support the cells' repair machinery upon rewarming), yet still manage to recover from LN2 biostasis at -196 C.  Since our experiments are targeting a long-term biostasis temperature of -130 C (to minimize the risk of tissue fracturing from mechanical stresses built up from uneven cooling at deep temperatures), we should have even less of a need for cell hardening therapeutics than blastocysts, yet this is not the case.  The reason is that cryoprotectants in very high concentrations (the concentrations needed to vitrify tissues with cryoprotectant mixtures range from 55% to 65% w/v) can be toxic to cells in both specific and non-specific ways.  We can reduce the concentrations required with faster cooling rates and certain non-toxic carrier solutions for cryoprotectants, or by adding high efficiency ice-blocker molecules (with the trade-off being that known ice-blockers increase the viscosity of the perfusate, increasing the amount of time cells spend at relatively high perfusion-friendly temperatures, thus incurring more toxicity damage), but still more needs to be done. 

Luckily, toxicity damage can be controlled using fast-cooling protocols that work well with small tissue samples, and even with whole rodent cephalons (see below).  However, larger canine, swine and human bodies take too long to cool to the safe biostasis temperature of - 130 C, where toxicity damage and almost all molecular activity effectively stops for many decades and probably several centuries.  The Research section more fully describes the approaches (cell membrane additives to speed tissue loading, MWCNT venous catheter heat sinks, ventricular system hi-flow convective cooling, low temp perfusion (with planned testing of n-pentane, fluorocarbon mixes, silicone oils, chilled O2 or He gas venous persufflation)) we are taking to reduce the time that human bodies spend at the relatively high temperatures where toxicity can occur.   

After medical biostasis induction becomes routine, we will still face the more difficult stage of reanimation.  The increased difficulty has to do with the fact that today's cryoprotectants only stop ice formation during the most dangerous temperatures for ice crystal growth (antifreeze protectants are effective from 0 C to about -60 C).  Ice crystal growth will slowly continue down to around -80 C, but the quantity of ice formed between -60 C to -80 C is insufficient to degrade tissues.  The problem is actually not being able to prevent the (during the cooling stage) harmless formation of small nidi (water molecules as ice nuclei) that keep accumulating till 15 degrees C below the vitrification temperature (e.g. -115 C) for protectant saturated tissues.  It is these nidi that begin the dangerous ice-crystal formation during rewarming if the critical rewarming rate is not exceeded (i.e. if tissue heating occurs too slowly).  The good news is that several (multispectral) oscillating RF and microwave based illumination techniques hold incredible potential to homogeneously warm large volumes of tissue at sufficiently high rates to avoid ice-damage.  We believe that this is effectively a solved problem that requires a limited amount of engineering to achieve the isotropic field generation on the scales we require.  Additionally, several of the rapid cooling deanimation approaches we are pursuing would also allow for the rapid rewarming needed to avoid reanimation damage (e.g. chilled gas persufflation leaves the vascular system empty during biostasis, ready to be used for rapid warming during reanimation).

An area that we are relying on routine medical best practices to optimize is the critical care protocol for rapidly bringing a patient's core temperature down to 0 degrees C, using a chilled perfusate that replaces the blood supply, while ensuring microvasculature health for our perfusion needs.  This is because today the fields of trauma medicine and circulatory arrest hypothermic heart surgery are already preparing patients against ischemic damage during temperature drops to 15 C.  We see 2012 as the first year when trauma surgery techniques start partially mirroring the vitrification based biostasis perfusion protocols, bringing patients down to a temperature of 9 C, without risking tissue edema.  Dr. Alam Hasan, a Professor of Surgery at Harvard, has already shown great success using whole swine models at these temperatures.  Again, not surprising given the fact that over 40 years ago, Dr. RJ White of the Cleveland Clinic, showed that a perfused mammalian whole brain preparation can be returned from temperatures as low as 3 degrees C with its electrophysiology intact.

Our modern understanding of the obstacles encountered during the breakthrough cephalon biostasis experiments highlighted below, makes us very optimistic about our chances of bringing biostasis into the world's modern operating rooms.

MAMMALIAN NEURONAL SLICE

2005 Pichugin, Fahy, Morin

"In conclusion, our results provide the first demonstration that the viability of organized adult [rat] brain tissue neural networks can be well preserved by vitrification. Our results support the possibility of preserving hippocampal slices for pharmacological and physiological testing and provide new support for the possibility of neural system transplantation for medical applications. Now that viability has been demonstrated, it is feasible and appropriate to proceed to detailed neurophysiological examination of hippocampal slices after vitrification and rewarming to conclusively demonstrate their potential utility for the screening of psychoactive drugs, and experiments of this kind are currently in progress."

RODENT BIOSTASIS RESEARCH

2011 Pichugin (personal communication)

"We perfused the upper part of a rat with CI-VM-1 using [our protocol] (with modification of the Blood Brain Barrier [using cell membrane disrupter additives]), cooled it to -129 C, kept it at -129 C overnight, warmed it to -40 C [using conductive warming], performed post-whole brain perfusion (by reperfusion of the upper part), washed it out at 0 C, extracted the hippocampi from the washed rat brain, cut it into thin slices, incubated the slices in the Oslo chamber to reanimate them and evaluated slice viability by the K+/Na+ ratio test.  As I have informed you I was able to obtain up to 70% slice survival.  I could obtain better results [estimated at 95%] if I had better equipment."

2011 Fahy (personal communication, paraphrased)

A whole rabbit cephalon was vitrified to -130 C and then sliced thinly.  The reanimated slices successfully maintained organized neuronal firing patterns.

FURTHER READING

For a more complete description (with extensive referencing) of the cellular damage that vitrification based biostasis prevents during supercooling of organs and cells, please see this article written by one of the leading vitrification experimentalists working with large animal and human models, Mike Darwin. 

A general article on the chemistry of cryoprotectants and ice-active (high hysteresis) molecules was put together by Ben Best, President of the Cryonics Institute.

Experimental physicist Brian Wowk of 21st Century Medicine, a long time deep thinker on the phenomenon of vitrification, published a thorough review of the thermodynamic aspects of supercooling resulting in vitrification.

Besides the tardigrada which can withstand the vacuum of space and storage in LN2 (-196 C), the Alaskan beetle's larvae have been shown to use natural vitrification based biostasis to survive down to temperatures of -100 C.