Stasis technology for storage and transport of natural and engineered tissues - This proposal is focused on developing innovative new cryopreservation techniques employing ice-free
preservation methods that will lead to the ability to bank complex vascularized tissue and organ grafts. Banking
of large or complex tissues, such as limbs, and organs using current tissue banking practices employing
conventional cryopreservation by freezing is not possible due to the well known damage caused by intra and
extra-cellular ice formation. The company team has developed vitrification, cryopreserved storage in a “glassy”
rather than crystalline ice phase, for banking of small tissue samples including blood vessels, heart valve
tissues and cartilage. However, scale up to larger tissues, more complex tissues and organs needs better ice
control during rewarming with less risk of cytotoxicity. Our collaborators, Professor Duman, University of Notre
Dame, and Professor Ben, University of Ottawa, have developed natural and synthetic antifreeze compounds
respectively that demonstrate benefits in frozen cell systems. Duman has developed recombinant insect-
derived antifreeze proteins and isolation methods for antifreeze glycolipids from several insects and a plant
that are very effective at ice control. Ben has developed a chemical library that was originally based upon an
antifreeze glycoprotein derived from a deep sea teleost. We will evaluate the best of these compounds during
ice-free cryopreservation using a vascular tissue model to determine if they improve ice control during
rewarming and reduce the risk of cryoprotectant toxicity by allowing lower cryoprotectant concentrations to be
used. There are three specific aims in this Phase I feasibility study to identify potentially marketable methods to
increase efficacy and minimize tissue damage during cryopreservation of complex natural and engineered
tissues: In Specific Aim 1 we will prepare of test compounds, antifreeze glycolipids from natural sources and
synthesize large quantities of synthetic antifreeze compounds. A stock of recombinant insect-derived
antifreeze proteins is already available. In Specific Aim 2 we will evaluate the impact of antifreeze compounds
individually upon ice-free cryopreserved blood vessels and in Specific Aim 3 the best antifreeze supplements
will be combined. Evaluations will include ice control during ice-free vitrification and tissue viability and function
assays. The technology developed in this proposal will be equally effective for stasis, storage, of engineered
tissue constructs as natural tissues. This work will lead to the development of banking, by us and other
cryobiology groups, for internal organs, such as livers and hearts, vascularized composite allografts, such as
larynx, trachea, abdominal wall, knee, skeletal muscles, facial tissues and legs, as well as improved methods
for critical natural and engineered tissues such as skin, heart valves and blood vessels.
