Forschungsprojekte

Drittmittelprojekt: Hirsch foundation, 50.000 EUR

Kliniken/Institute:
Reproduktionsmedizinische Einheit der Kliniken

Projektdetails:
Decellularized heart valve tissues can be used to replace a malfunctioning heart valve. This option is particularly suitable for young patients, because decellularized tissues have regeneration potential. We have pioneered in developing methods for dry preservation of mammalian cells and tissues and demonstrated that this can be done using disaccharides such as sucrose or trehalose which can be found at high concentrations in anhydrobiotic organisms that naturally survive drying. Drying of decellularized heart valves may alter the properties of the extracellular matrix and impair their in vivo efficacy. The central aim of this project is to test if sucrose can be used for dry preservation of heart valves so that they can be safely used for transplantation. We already established methods to freeze-dry heart valves, but their storage stability needs to be demonstrated. In this project we plan to evaluate if vacuum-drying, which lacks a damaging freezing step, can be used as an alternative drying method. The second objective is to study the effects of (freeze-)drying on scaffold structure, and matrix biomolecules directly after drying as well during storage under normal (4°C or room temperate) and accelerated aging (37°C/high relative humidity) storage conditions. The general tissue architecture will be studied on hematoxylin-eosin stained tissue sections, whereas Fourier transform infrared spectroscopy (FTIR) will be used to obtain spectral fingerprints of tissues during storage. For the latter principal component analysis of the spectra will be used to evaluate tissue modifications during storage. Furthermore, accumulation of oxidative damage will be determined. We postulate that overall tissue structural appearance, and therewith functionality, are compromised by ice crystals, and that this can be minimized by reducing the tissue water content prior to freezing, or to avoid ice formation by using vacuum drying.

Drittmittelprojekt: DFG<br>WO 1735/6-2, 210.000 EUR

Kliniken/Institute:
Reproduktionsmedizinische Einheit der Kliniken

Projektdetails:
The central aim of the first phase of our project was to correlate subzero membrane phase and permeability properties of sperm with their ability to survive freezing and thawing. In addition, biomolecular stability of freeze-dried sperm and physical properties of glasses for dry preservation have been investigated. We discovered that ice formation triggers a membrane phase transition, which is dependent on the ice nucleation temperature, the cooling rate, and the type of cryoprotective agent that is used. Freezing-induced membrane phase changes were used to investigate the cell membrane permeability to water allowing prediction of optimal cooling rates for cryopreservation. Furthermore, we discovered that membranes become permeable for molecules for which they are normally impermeable during freezing, while the cells survive freezing zu beladen. We found that simply exposing cells to freezing can thus be used to load cells with membrane impermeable lyoprotective agents, such as sucrose or trehalose, which preserves chromatin in freeze-dried sperm even under accelerated aging conditions. Whereas in the first phase of this project water and solute transport processes have been predominantly studied at the cellular and membrane level, in the next phase this will be extended to the tissue level and multiple component solutions. Membrane transport parameters of oocytes for water and cryoprotective agents will be determined from cell volume responses in a microfluidic device. Membrane permeabilization during loading cells with cryoprotective agents will be investigated by studying uptake of membrane-impermeable molecules. It is planned to investigate if sodium ions also pass membranes during freezing and if freezing in reduced sodium increases cryosurvival. Diffusion of protective molecules in ovarian tissues and concomitant dehydration will be investigated to develop a mass transport model, which will allow to correlate distribution of protectants with cryosuriviva. Storage stability of cryopreserved specimens will be investigated by studying molecular mobility and membrane transport processes near the glass transition temperature with the aim to develop formulations allowing cryogenic storage at higher temperatures.