Biomedical subjects

Brian Wowk

Publications and source records attributed to Brian Wowk.

5 recordsLinked to original sources

PubMed2006

Cryopreservation of complex systems: the missing link in the regenerative medicine supply chain.

Transplantation can be regarded as one form of "antiaging medicine" that is widely accepted as being effective in extending human life. The current number of organ transplants in the United States is on the order of 20,000 per year, but the need may be closer to 900,000 per year. Cadaveric and living-related donor sources are unlikely to be able to provide all of the transplants required, but the gap between supply and demand can be eliminated in principle by the field of regenerative medicine, including the present field of tissue engineering through which cell, tissue, and even organ replacements are being created in the laboratory. If so, it could allow over 30% of all deaths in the United States to be substantially postponed, raising the probability of living to the age of 80 by a factor of two and the odds of living to 90 by more than a factor of 10. This promise, however, depends on the ability to physically distribute the products of regenerative medicine to patients in need and to produce these products in a way that allows for adequate inventory control and quality assurance. For this purpose, the ability to cryogenically preserve (cryopreserve) cells, tissues, and even whole laboratory-produced organs may be indispensable. Until recently, the cryopreservation of organs has seemed a remote prospect to most observers, but developments over the past few years are rapidly changing the scientific basis for preserving even the most difficult and delicate organs for unlimited periods of time. Animal intestines and ovaries have been frozen, thawed, and shown to function after transplantation, but the preservation of vital organs will most likely require vitrification. With vitrification, all ice formation is prevented and the organ is preserved in the glassy state below the glass transition temperature (T(G)). Vitrification has been successful for many tissues such as veins, arteries, cartilage, and heart valves, and success has even been claimed for whole ovaries. For vital organs, a significant recent milestone for vitrification has been the ability to routinely recover rabbit kidneys after cooling to a mean intrarenal temperature of about -45 degrees C, as verified by life support function after transplantation. This temperature is not low enough for long-term banking, but research continues on preservation below -45 degrees C, and some encouraging preliminary evidence has been obtained indicating that kidneys can support life after vitrification. Full development of tissue engineering and organ generation from stem cells, when combined with the ability to bank these laboratory-produced products, in theory could dramatically increase median life expectancy even in the absence of any improvements in mitigating aging processes on a fundamental level.

Animals↗

PubMed2005

Anomalous high activity of a subfraction of polyvinyl alcohol ice blocker.

Low molecular weight copolymers of polyvinyl alcohol (PVA) are known to be potent inhibitors of ice formation in solutions used for cryopreservation by vitrification, even at concentrations as low as one part per million. Concentrated aqueous solutions of these polymers tend to become turbid after preparation. Condensed particles causing turbidity were isolated from a commercially available PVA-based ice blocker (X-1000) and found to consist of a polymer subfraction that is especially effective at ice blocking. Fifty seven percentage (w/w) of ethylene glycol (EG) in distilled water and 0.025% of the condensate polymer showed similar stability against devitrification as 57% EG+0.1% ordinary X-1000. At higher concentrations, 56.9% EG+0.1% condensate polymer was as effective as 56% EG+1% ordinary X-1000. All solutions containing ice blocker showed much less devitrification during warming than a 57% EG control solution. The condensate polymer was found to be strongly self-associating and less water soluble than ordinary X-1000. The mean molecular weight of the condensate polymer was approximately 1400 compared to 2100 for ordinary X-1000. Proton NMR revealed no large chemical differences. Subtle differences in composition or stereochemistry, perhaps in local regions of molecules, must be responsible for the dramatic differences in physical behavior and ice blocking effectiveness of the condensate polymer.

Antifreeze Proteins↗

PubMed2004

Improved vitrification solutions based on the predictability of vitrification solution toxicity.

Long-term preservation of complex engineered tissues and organs at cryogenic temperatures in the absence of ice has been prevented to date by the difficulty of discovering combinations of cryoprotectants that are both sufficiently non-toxic and sufficiently stable to allow viability to be maintained and ice formation to be avoided during slow cooling to the glass transition temperature and subsequent slow rewarming. A new theory of the origin of non-specific cryoprotectant toxicity was shown to account, in a rabbit renal cortical slice model, for the toxicities of 20 vitrification solutions and to permit the design of new solutions that are dramatically less toxic than previously known solutions for diverse biological systems. Unfertilized mouse ova vitrified with one of the new solutions were successfully fertilized and regained 80% of the absolute control (untreated) rate of development to blastocysts, whereas ova vitrified in VSDP, the best previous solution, developed to blastocysts at a rate only 30% of that of controls. Whole rabbit kidneys perfused at -3 degrees C with another new solution at a concentration of cryoprotectant (8.4M) that was previously 100% lethal at this temperature exhibited no damage after transplantation and immediate contralateral nephrectomy. It appears that cryoprotectant solutions that are composed to be at the minimum concentrations needed for vitrification at moderate cooling rates are toxic in direct proportion to the average strength of water hydrogen bonding by the polar groups on the permeating cryoprotectants in the solution. Vitrification solutions that are based on minimal perturbation of intracellular water appear to be superior and provide new hope that the successful vitrification of natural organs as well as tissue engineered or clonally produced organ and tissue replacements can be achieved.

Animals↗

PubMed2004

Cryopreservation of organs by vitrification: perspectives and recent advances.

The cryopreservation of organs became an active area of research in the 1950s as a result of the rediscovery of the cryoprotective properties of glycerol by Polge, Smith, and Parkes in 1949. Over the ensuing four decades of research in this area, the advantages of vitrification, or ice-free cryopreservation, have become apparent. To date, experimental attempts to apply vitrification methods to vascularized whole organs have been confined almost entirely to the rabbit kidney. Using techniques available as of 1997, it was possible to vitrify blood vessels and smaller systems with reasonable success, but not whole organs. Beginning in 1998, a series of novel advances involving the control of cryoprotectant toxicity, nucleation, crystal growth, and chilling injury began to provide the tools needed to achieve success. Based on these new findings, we were first able to show that an 8.4M solution (VMP) designed to prevent chilling injury at -22 degrees C was entirely non-toxic to rabbit kidneys when perfused at -3 degrees C and permitted perfusion-cooling to -22 degrees C with only mild additional damage. We next investigated the ability of the kidney to tolerate a 9.3M solution known as M22, which does not devitrify when warmed from below -150 degrees C at 1 degrees C/min. When M22 was added and removed at -22 degrees C, it was sometimes [corrected] fatal, but when it was perfused for 25min at -22 degrees C and washed out simultaneously with warming, postoperative renal function recovered fully. When kidneys loaded with M22 at -22 degrees C were further cooled to an average intrarenal temperature of about -45 degrees C (about halfway through the putative temperature zone of increasing vulnerability to chilling injury), all kidneys supported life after transplantation and returned creatinine values to baseline, though after a higher transient creatinine peak. However, medullary, papillary, and pelvic biopsies taken from kidneys perfused with M22 for 25min at -22 degrees C were found to devitrify when vitrified and rewarmed at 20 degrees C/min in a differential scanning calorimeter. It remains to be determined whether this devitrification is seriously damaging and whether it can be suppressed by improving cryoprotectant distribution to more weakly perfused regions of the kidney or by rewarming at higher rates. In conclusion, although the goal of organ vitrification remains elusive, the prospects for success have never been more promising.

Animals↗

PubMed2002

Inhibition of bacterial ice nucleation by polyglycerol polymers.

The simple linear polymer polyglycerol (PGL) was found to apparently bind and inhibit the ice nucleating activity of proteins from the ice nucleating bacterium Pseudomonas syringae. PGL of molecular mass 750 Da was added to a solution consisting of 1 ppm freeze-dried P. syringae 31A in water. Differential ice nucleator spectra were determined by measuring the distribution of freezing temperatures in a population of 98 drops of 1 microL volume. The mean freezing temperature was lowered from -6.8 degrees C (control) to -8.0,-9.4,-12.5, and -13.4 degrees C for 0.001, 0.01, 0.1, and 1% w/w PGL concentrations, respectively (SE < 0.2 degrees C). PGL was found to be an ineffective inhibitor of seven defined organic ice nucleating agents, whereas the general ice nucleation inhibitor polyvinyl alcohol (PVA) was found to be effective against five of the seven. The activity of PGL therefore seems to be specific against bacterial ice nucleating protein. PGL alone was an ineffective inhibitor of ice nucleation in small volumes of environmental or laboratory water samples, suggesting that the numerical majority of ice nucleating contaminants in nature may be of nonbacterial origin. However, PGL was more effective than PVA at suppressing initial ice nucleation events in large volumes, suggesting a ubiquitous sparse background of bacterial ice nucleating proteins with high nucleation efficiency. The combination of PGL and PVA was particularly effective for reducing ice formation in solutions used for cryopreservation by vitrification.

Bacterial Outer Membrane Proteins↗