085 Freeze-dried cells and tissues

Abstract

Cryopreservation and freeze drying are widely accepted as the preferred techniques for long-term storage of biological materials in medical applications. Whereas cryopreserved samples are typically stored in liquid nitrogen, dried samples can be stored at room temperature, which has clear advantages for banking and transport. Freeze-drying is widely used to preserve biomolecules and macromolecular assemblies, and has been implicated as a method to preserve cells and tissues in the dry state. Freeze-drying involves a freezing and a drying step both imposing stress on biomaterials. Freezing may cause irreversible structural changes in biomolecules due to ice crystal formation, osmotic dehydration, and mechanical forces. Drying causes removal of bound water surrounding biomolecules. Non-reducing disaccharides have been shown to stabilize biomolecules during drying by forming direct hydrogen bonding interactions and by forming a glassy state. Attempts to stabilize cells in the dry state have focused on disaccharides as main stabilizing compound. The major challenge in using disaccharides for drying of cells is to overcome the impermeability of cell membranes to disaccharides, to provide intracellular protection. Freeze-drying of tissues has not been widely explored. Decellularized tissues, however, can be freeze-dried and used as matrix for tissue regeneration. In this presentation, feasibility of freeze-drying for stabilization of cells and decellularized tissues in the dry state will be discussed. Various methods to incorporate disaccharides into mammalian cells will be highlighted including a novel method making use of freezing-induced membrane phase transitions. This is illustrated for platelets, red blood cells, and sperm. Furthermore, it is shown how disaccharides can be used for freeze-drying of decellularized heart valve scaffolds. Diffusion of protective agents in the scaffolds has been studied by Fourier transform infrared spectroscopy, and the effects of freeze-drying on biomechanical and ultrastructural properties are shown. Source of funding: This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy). Conflict of interest: None declared. wolkers@imp.uni-hannover.de