TRANSPORT CHARACTERISTICS OF GLYCEROL AND PROPYLENE GLYCOL IN AN ENGINEERED DERMAL REPLACEMENT

摘要

The ability to cryopreserve engineered tissues is important for the clinical application of therapies based on living cells. Cryopreservation facilitates the manufacture, transport and safety of cell-based therapies. The Cryopreservation of cells and tissues had typically required the use of specialized solutions containing cryoprotective agents (CPAs). The addition of a CPA to the freezing solution may result in damage if it is not done properly. Tissues and intact organs can exhibit reduced cellular viability when exposed to sufficiently large step changes in external osmolarity resulting from introduction or removal of a Cryopreservation solution (Pegg, 1972). Not only are large step changes in osmolarity potentially damaging, tot also long-term exposure to even low concentrations of CPAs at room temperature can be lethal (Fahy et al., 1990). Exposure of cells to CPAs (in particular dimethyl sulfoxide, Me_2SO) has been associated with a loss in viability with time of exposure. Subsequent studies have quantified specific cellular changes resulting from exposure to CPA, such as cytoskeletal reorganization, cross-linking of nuclear proteins, and alterations in membrane permeability (cf. ref (Fahy et al., 1990) for review) which may account for the loss in viability. Nuclear Magnetic Resonance (NMR) is a common nondestructive technique used to quantitate the concentration of molecules inside 3D structures. Several MR techniques have been developed to quantitate the concentration of CPAs in tissues during permeation. Spectroscopic techniques have been used (Fuller et al., 1989) to determine average concentrations with time. MRI has also been used to image CPA permeation in zebra fish embryos (Hagedorn et al., 1998) or mammalian organs (Isbell et al., 1997). We have developed a rapid imaging technique that can be applied to the study of the permeation of CPA hi artificial tissues (Bidault et al., 2000; Bidault et al., 2001). A combined Fast Low Angle Shot (FLASH)-Keyhole technique was used to increase the speed and sensitivity of images obtained of CPA permeation in tissue. This technique was used to quantify the water and Me_2SO content of an engineered dermal replacement as a function of temperature, time and bulk CPA concentration. In this study, we extend this analysis to include glycerol and propylene glycol, two CPAs also commonly used for tissue preservation.