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

Abstract

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 Ϫ45°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 Ϫ45°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. 279

Organ Preservation: Current Concepts and New Strategies for the Next Decade

“…Whilst this has been a long-standing dream within the field of organ transplantation, there are very significant challenges for organ function which take place when large, three-dimensional structures are cooled below freezing point and ice inevitably starts to form [131]. Even if special protective chemicals (known as cryoprotectants) are introduced and careful control of cooling and warming are imposed, injury occurs.…”

“…Another route to ameliorating the organ injury from ice formation is to completely suppress ice nucleation by a process called vitrification [131,135]. The technology has proved very successful in cryobanking small cell clusters such as embryos in reproductive medicine [136].…”

Whole sheep ovary cryopreservation: evaluation of a slow freezing protocol with dimethylsulphoxide

“…The major difficulties with the method of whole ovary cryopreservation seems to be the vascular microsurgery [17] and to effectively cryopreserve a large tissue-mass [18]. The ovary would be regarded as an organ of high cryobiological complexity.…”

Vitrification Successfully Preserves Hepatocyte Spheroids

“…Vitrification, on the other Hepatocytes were allowed to aggregate into spheroids in spheroid-forming medium. The spheroid-forming mehand, is ice-free cryopreservation and has been shown to retain the post-warm viability and functional ability dium, prepared in deionized water, was composed of the following: 1 mg/ml bovine serum albumin (Bovostar, of various cultures such as human vascular endothelial cells (56), encapsulated hepatocytes (30,55), stem cells BioClot, Australia), 2.2 mg/ml sodium bicarbonate (US Biological, MA), 2.4 mg/ml HEPES (US Biological), to (10,42), human oocytes and embryos (24,26) at levels similar to those of fresh controls, and certain tissues and the William's E powder (Sigma Aldrich, Steinheim, Germany) 10 ng/ml epithelial growth factor (EGF) (Gibco, organs (7,8,48), which is vital for transplantation. An additional advantage of the proposed approach is that it Invitrogen, CA), 0.5 µg/ml insulin (Boeringer Mannheim, Germany), 5 nM dexamethasone, and 50 ng/ml will be less time consuming because it does not require the use of specialized equipment, making the method linoleic acid.…”