AbstractCryopreservation of biological tissues with storage in liquid or vapour phase nitrogen (LN) has frequently been adopted as the in vitro preservation method of choice. It offers a cost effective technique for the long-term maintenance of cultures and effectively guarantees genetic stability. To improve post-thaw viability levels and to preserve freeze-recalcitrant organisms, components of the cryogenic process which predispose and/or subject cells to stress and injury require to be determined. In this study markers of stress and cryoinjury were identified using flow cytometry and cryomicroscopy, they include: intracellular ice formation, cryoprotectant toxicity, osmotic shock (due to excessive cryodehydration), freeze-fracture events and morphological changes. Further studies investigated: inhibition of photosynthetic oxygen evolving capacity, products of lipid peroxidation, hydroxyl radical production, changes in antioxidant status [superoxide dismutase (SOD), catalase, peroxidase, glutathione reductase] and levels of protein and non-protein bound sulfhydryl groups (SH) in treated and untreated cells.
The morphological and physiological diversity of the algae influenced their responses to stresses and injury induced during cryopreservation. Compartmentalisation of ice nucleation contributed to the ability of Enteromorpha intestinalis (L.) Link CCAP 320/1 to survive freezing. In contrast, propagation of intracellular ice throughout the coenocytic alga Vaucheria sessilis (Vaucher) De Candolle ex Collins CCAP 745/1C enhanced its freeze-recalcitrance. Lipid deposition and inhibition of photosynthetic capacity on chilling were also detected in this alga.
An effective protocol, which avoided lethal cryoinjury, was developed for the previously freeze-recalcitrant alga Euglena gracilis Klebs CCAP 1224/5Z. This alga experienced non-lethal responses to the cryopreservation protocol including inhibition of photosynthetic oxygen evolution. Oxidative stress was also identified and the production of highly toxic hydroxyl radicals (OH) was assessed using a novel nondestructive gas chromatographic technique. Cells were demonstrated to experience both lethal and non-lethal stresses. A mechanism contributing to the relative freeze-recalcitrance of E. gracilis is proposed involving increased SOD activity, which increases the availability of H2O2 , thus contributing to the generation of the highly cytotoxic hydroxyl radical through Fenton chemistry. Elevated antioxidant activities were also detected for those enzymes which remove H2O2, these changes in antioxidant status, when poorly regulated, may in themselves contribute to oxidative stress and imbalances in their complex interactions may result in cryoinjury. Protection by the preferential oxidation of non-protein bound SH groups and a possible acclimation response enhancing protein SH levels were also detected in cryopreserved algae. In Haematococcus pluvialis Flotow CCAP 34/8 a regulated antioxidant response and high carotenoid composition were considered to contribute to freeze-tolerance.
Novel techniques (encapsulation/two-step cooling) and the use of exogenous antioxidants were investigated and these offer considerable scope for the preservation of presently freeze- recalcitrant protists. The benefits of using investigative rather than speculative approaches to develop cryoprotocols are discussed.
|Date of Award||May 1998|
|Sponsors||Esmee Fairbaim Charitable Trust|
|Supervisor||David Bremner (Supervisor)|