Thermische Prozeßführung beim Einfrieren und Wiedererwärmen humaner Erythrozyten mit Hydroxyethylstärke

The objective of this thesis was the optimisation of the thermal processing in cryopreservation of human red blood cells using the cryoprotectant hydroxyethyl starch such that cell damage induced by freezing and thawing can be reduced. The motivation for this work was the clinical application of the cryopreservation protocol, where a scale-up to bulk samples is required. In the experimental part, technical measures where tested and evaluated aiming to extend the range of cooling rates achieved during freezing with a finned freezing container. By comparing measurements of steady state and transient heat transfer, the temperature difference between the refrigerant liquid nitrogen and the finned profile turned out to be the main condition to achieve improved heat transfer. A new thawing device for rapid rewarming of cryopreserved samples was developed. The device is equipped with a double-sided field of jets impinging warmed water directly on the sample. In combination with this improved convective heat absorption, an orbital movement of the sample resulted in a clear improvement of the rewarming characteristics, as shown by temperature courses measured in a sample bag containing a test solution. A similar effect was achieved by using low molecular weight hydroxyethyl starch, resulting from the decreased viscosity of the solution. A numerical study demonstrated that the heat transfer coefficient between frozen and liquid part of a sample during thawing is the key parameter for rapid rewarming. Further improvement can be achieved by increasing the bore diameter of the jets as well as increasing the frequency of the orbital movement. The results of cryopreservation studies indicate that the new thawing procedure is a suitable method to reduce the cellular damage, if the device is used in combination with low molecular weight hydroxyethyl starch as cryoprotective agent. Therefore, the main goal of process optimisation by rapid rewarming was reached. The application of the aforementioned methods of increased cooling rates did not yield less cellular damage, if combined with rapid rewarming. Nevertheless, adjustment of the saline concentration of the cryoprotective solution turned out to be a potential measure to further optimise the cryopreservation protocol. By additional experimental studies, the determination of the oxygen transport capacity by rheooxymetry and a multiple step wash-out procedure were identified as an alternative to established vitality tests. The application of multiple freeze-thaw cycles resulted in an exponential decay of cell survival with the number of cycles indicating a stochastic process