1. BACKGROUND
2. INITIAL EXPERIMENTS
3. SUMMARY OF THE DISCOVERIES FROM THE INITIAL EXPERIMENTS
4. FINAL RESULTS

I. BACKGROUND

The Hippocampal Slice Cryopreservation Project (HSCP) was a research project conducted by cryobiologists Dr. Gregory Fahy and Dr. Yuri Pichugin at the University of California in Los Angeles (UCLA) with funds provided by The Institute for Neural Cryobiology (INC) and UCLA. The objective of the project was to cryopreserve hippocampal slices through vitrification at −130ºC, with complete viability upon rewarming.

The hippocampus is the area of the brain thought to be most critical in learning new information through a process known as LTP (Long-Term Potentiation). More neurophysiological experiments are performed on hippocampal slices than on any other brain tissue.The hippocampus is also the brain tissue most easily damaged by oxygen deprivation (ischemia) and is one of the first areas of the brain affected by Alzheimer's Disease. In addition to the medical and neurophysiological significance of the hippocampus is the fact that hippocampal slices are not only easy to study & manipulate, but are so widely studied by others that considerable literature & equipment exists to assist the study.

The HSCP used hippocampal slices which Dr. Pichugin prepared from rat brains and evaluated for viability in an Oslo Chamber. An Oslo Chamber is a plastic container that looks like a casserole dish with aerated partitions where hippocampal slices can be maintained in an environment controlled for temperature, oxygenation and humidity. Hippocampal slices from an adult rat can live in Oslo Chambers for 12 hours or longer. Injuries to slices occurring during surgical removal from a rat brain may even heal in the Oslo Chamber.

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II. INITIAL EXPERIMENTS

The cryoprotectants used in this initial experiment were ethylene glycol (used in automobile antifreeze), DMSO (DiMethylSulfOxide), glycerol and V_EG (pronounced "Vee Ee Gee", not "veg"). V_EG is a modification of the cryoprotectant cocktail VS41A (VS41A is a Vitrification Solution which is a mixture of DMSO, formamide and propylene glycol) in which the propylene glycol is replaced with an equal weight of ethylene glycol (the "EG" of V_EG). [V_EG is patent 6,395,467 -- 21st Century Medicine (21CM) -- composed of 16.84% w/v ethylene glycol, 13.96% w/v formamide and 24.2% w/v DMSO, totalling 55% w/v cryoprotective solution.] DMSO showed the worst viability, ethylene glycol the next worst, and glycerol the third worst at two of the 3 temperatures studied, with V_EG being the best overall. The superiority of glycerol to DMSO was reassuring in view of unpublished experimental results of Isamu Suda showing the same superiority for whole brains (see next paragraph).

Decades ago the Japanese experimentalist I. Suda had exposed whole cat brains to 15% glycerol, stored them for 5 days at −20ºC and then demonstrated that the cat brains had EEG patterns similar to those of cat brains that had not been subject to cooling [NATURE 212:268-270 (1966) and BRAIN RESEARCH 70:527-531 (1974)]. In the spirit of Suda, hippocampal slices were subjected to 30% glycerol and then either stored for 12 hours at −20ºC, −40ºC and −76ºC or cooled to these temperatures and rewarmed without storage. In these and all subsequent experiments, the viability assay was based on the potassium/sodium ratio (K⁺/Na⁺ ratio). For neural tissue, especially, the ability of cells to maintain membrane potential with the sodium pump is an easily-measured & reliable indicator of viability. Based on this assay, although the controls exposed to 30% glycerol showed 70% viability, those cooled and stored at −20ºC were essentially dead. Those stored at −40ºC and −76ºC had slightly better viability, but not more than 15% viability, and the effects of cooling to −76ºC were not improved by warming immediately from that temperature (no storage). Using 30% V_EG rather than glycerol produced even worse results -- possibly because the temperature of introduction of V_EG was too high. V_EG needs to be added at a lower temperature than glycerol.

In addition to the ice crystal damage of freezing and the toxicity of cryoprotectant, attempts to cryopreserve organs & tissues are also hampered by a phenomenon known as chilling injury. To investigate this phenomenon hippocampal slices were cooled to 0ºC for one hour (without cryoprotectant) and then rewarmed & assayed. The slices showed 30% of the viability of controls which had been maintained at 37ºC. Hippocampal slices held at 0ºC in artificial CerebroSpinal Fluid (aCSF) for 50 minutes also showed 30% viability. This result was not changed by using modified aCSF containing mannitol (maCSF) instead of the standard aCSF, at 50 min of exposure to 0ºC. But adding 10% glycerol seemed to reduce the chilling injury somewhat -- and adding 10% V_EG resulted in a 40% viability. 25% V_EG (with & without mannitol) resulted in 50% viability. These results indicate a protective effect of V_EG against the 0ºC chilling injury.

The experiment was repeated with controls at 37ºC and 10ºC as well as with 25% V_EG slices at 10ºC and 0ºC. There was little significant difference between the 37ºC controls, the 10ºC controls or the 10ºC 25% V_EG slices, but the 0ºC 25% V_EG slices lost 30% of viability. This indicates that chilling injury occurs between 10ºC and 0ºC. Worse results were obtained with V_EG addition at 15ºC -- indicating that 10ºC is the optimum temperature for introduction of V_EG. Moreover, at 10ºC introduction of V_EG at 5-minute steps of increasing concentration produced better viability than introduction of V_EG at 10-minute steps -- indicating that toxicity damage is more important than osmotic damage at this temperature and for these times & concentrations. Experiments also indicated that 300mM mannitol is the optimum concentration for buffering osmotic damage during removal of V_EG.

Carrier solutions can enhance cryoprotection. RPS−2 (Renal Preservation Solution number 2) was developed by Dr. Fahy in 1981 as a result of studies on kidney slices. RPS−2 actually resulted in hippocampal slice viability at 10ºC which was 50% greater than that of control slices at 37ºC after one hour in the Oslo Chamber (although the difference disappeared after 2 hours). A combination of 25% V_EG with RPS−2 at 10ºC resulted in viability fully the same as that of 37ºC controls -- although 25% V_EG is insufficient concentration for vitrification. Pushing the V_EG concentration to 50% in RPS−2 at 10ºC brought the slice viability back down to 50% that of the 37ºC controls.

The next experiment was based on the idea that both chilling injury and toxicity should be reduced at sub-zero temperatures. V_EG was added at 10ºC in steps up to 25% concentration, and a final 25% V_EG (bringing the total to 50%) was added (and washed-out) at −10ºC. These 50% V_EG slices were only slightly less viable (and not statistically less viable) than the 37ºC controls. The concentration of V_EG required to vitrify is estimated to be 53%. Adding (and later washing-out) 30% rather than 25% V_EG at −10ºC (bringing the total to 55%) resulted in viability 77% of that of controls.

In the next experiment one group of slices with 53% V_EG was held at −10ºC while the other group with 53% V_EG was cooled to vitrification temperature (−130ºC) and then rewarmed. The non-vitrified 53% V_EG slices had 60% viability and the vitrified 53% V_EG slices had 56% viability as compared to untreated controls. The difference between the V_EG-treated slices and the slices that were exposed to V_EG and then vitrified was not statistically significant. But when the experiment was repeated, the vitrified group was significantly less viable.

The following experiment added V_EG at 5-minute intervals at 10ºC, but the final addition (and washout) at −10ºC was given 10 minutes for diffusion. Viability of the non-vitrified slices exposed to 53% V_EG was 65%, but when slices treated in this way were vitrified, the K/Na ratio was only 42% of control K/Na, perhaps due to inadequate equilibration. In conclusion, using 10-minute steps throughout all phases of the addition and washout procedure was able to protect completely against vitrification/devitrification injury, but was associated with increased toxicity. Nonetheless, the viability after vitrification was up to 56% of untreated control viability.

Subsequent experiments made use of ice blockers and cryoprotectant cocktails less toxic than V_EG as well as longer equilibration times prior to vitrification. The worst of these newer experiments gave results equal to the best results obtained with V_EG, and the best results of the newer experiments indicated no difference between vitrified slices and untreated controls. However, the variability of the results were very high -- up to plus or minus 25%, with an average of 75% viability of hippocampal slices which have been vitrified at −130ºC. Efforts were devoted to reducing this variability and moving the average viability closer to 100%.

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III. SUMMARY OF THE DISCOVERIES FROM THE INITIAL EXPERIMENTS

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IV. FINAL RESULTS

The final results of the HSCP were published in the April 2006 issue of the peer-reviewed journal CRYOBIOLOGY -- [CRYOBIOLOGY 52(2):228-40 (2006)]. (Also available as a PDF file: Cryopreservation of rat hippocampal slices by vitrification )

When hippocampal slices were treated with 50% w/v V_EG in aCSF carrier solution at 0ºC there was considerable damage, which was mostly eliminated by the use of RPS−2 carrier solution instead of aCSF. But 53% w/v V_EG was the minimum concentration which would vitrify at the cooling rates used in the study. An increase of V_EG concentration from 50% w/v to 53% w/v caused a considerable reduction of viability above −10ºC (but no further injury from −10ºC to −130ºC).

The viability problems were eliminated by using VM3 rather than V_EG as the cryoprotectant solution. VM3 is non-toxic (>90% K/Na ratio recovery upon exposure of tissues to VM3). Slices treated with VM3 cooled to −130ºC (vitrified) and rewarmed were as viable as slices treated with VM3 without any cooling. VM3 is composed of 16.84% w/v ethylene glycol, 12.86% w/v formamide, 22.3% w/v DMSO, 7% w/v polyvinylpyrrolidine K12, 1% w/v "Supercool X−100" ice blocker and 1% w/v "Supercool Z−100" ice blocker in a carrier solution of LM5 rather than RPS−2. LM5 maximizes the effectiveness of ice blockers better than RPS−2.

Electron Micrographs (EMs) of the CA1 and CA3 regions of the hippocampus in VM3 after cooling to −130ºC and rewarming showed complete preservation of ultrastructure, in agreement with the >90% viability assays obtained from the same treatment with VM3. Glass transition temperature (T_g) of both V_EG and VM3 are 3−4ºC above −130ºC, so the samples had vitrified (solidified).

These results demonstrate the ability to cool hippocampal slices to −130ºC and rewarm them without loss of viability.

For further discussion of cryoprotective agents, see A Summary of the First 21CM Seminars .

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