published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

Cryopreservation in suspension is commonplace for a variety of cell types. However, cryopreservation of adherent cells has achieved limited success. This research aimed to cryopreserve adherent nerve cell networks in vitro in a manner that preserved network morphology and physiology. Successful implementation would enable long term storage of adherent neuronal networks on microelectrode arrays and on-demand access for use in pharmacological and toxicological testing. Based upon morphological assessments, excellent post-thaw preservation was obtained and post-thaw cultures survived in a transitional medium for up to 3.5 hours. However, transitions to native culture medium post-thaw presented difficulties, ultimately resulting in necrosis. A discussion of methods to supplement the current research and increase post-thaw viability is included in the thesis.

M.Sc. (Zoology) / Please refer to full text to view abstract

The effects of two types of cryopreservation, conventional freezing and vitrification, on the in vitro and in vivo function of a pancreatic substitute were investigated. Conventional freezing uses low concentrations of cryoprotective agents (CPAs), slow cooling and rapid warming and allows ice formation. Vitrification requires high concentrations of CPAs coupled with rapid cooling and warming to achieve a vitreous, or ice-free, state. A previously published mathematical model describing the mass transfer of CPAs through the alginate matrix of the substitute and the cell membrane was expanded to incorporate heat transfer as well as CPA cytotoxicity. Our results indicate that temperature of exposure is the most critical parameter for the proper design of CPA addition and removal protocols. The use of a mathematical model is critical to ensure CPA equilibration and minimize CPA exposure. Properly designed CPA addition and removal protocols were used for vitrification. The effects of cryopreservation on the biomaterial and the cellular function of a pancreatic substitute consisting of murine insulinomas encapsulated in calcium alginate/poly-L-lysine/alginate beads were assessed. In vitro results indicate that both vitrification and conventionally frozen perform comparably to fresh. However, in vivo studies reveal that vitrified beads perform worse than both conventionally frozen and fresh beads. With adjustments, it may be possible to improve the performance of the vitrified beads. Nevertheless, for this pancreatic substitute, conventional freezing is the better method and allows successful cryopreservation.

Key to the success of natural and engineered tissues becoming clinically available until needed is their long-term storage at low temperatures. This can be implemented by means of freezing or vitrification. To this end, vitrification offers an attractive approach for tissue banking by forming an amorphous glass both intra- and extracellularly and thereby avoiding the harmful effects of ice formation. Generally, high concentrations of cryoprotectants (CPAs) are used in conjunction with high cooling and warming rates to achieve this. However, hurdles associated with applying this technique include the ability to adequately deliver and remove CPAs due to cellular osmotic and cytotoxic effects as well as achieving adequate cooling and warming rates throughout the tissue to avoid ice formation. The aim of this work was to account for these factors in designing cryopreservation protocols for native and engineered tissues that had intrinsically different characteristics, including tissue size and extracellular matrix properties. The tissues investigated were two types of three-dimensional, cell encapsulated systems consisting of murine insulinomas and murine embryonic stem cells, and native articular cartilage. A mathematical 3-D CPA transport model was developed to predict cell volume excursions and intracellular CPA equilibration and applied to cryopreserve an engineered tissue. This thesis established a systematic methodology to design cryopreservation protocols using experimental measurements and a mathematical model for tissues.

Long-term storage of tissue by cryopreservation is necessary for the efficient mass production of tissue engineered products, and for reducing the urgency and cost of organ transplantation procedures. The goal of this work was to investigate the physical processes thought result in damage during tissue cryopreservation towards development of tissue cryopreservation strategies. Although mathematical models of cell dehydration and intracellular ice formation (IIF) have been successfully used to optimize cryopreservation procedures for cell suspensions, it is not currently possible to use this approach with tissue because of the lack of tissue-specific permeability parameters for predicting cell dehydration during tissue freezing, and because of the increased complexity of the IIF process in tissue. We have measured the membrane permeability properties of tissue comprising a cell monolayer using a fluorescence quenching technique, and compared the results to the corresponding cell suspensions, revealing significant differences in the membrane transport kinetics between monolayers and suspensions. These data enabled the prediction cell dehydration during freezing of cell monolayers. Whereas the mechanisms of IIF are relatively well understood in cell suspensions, tissue is susceptible to new IIF mechanisms. In particular, cell-cell interactions have been shown to increase the IIF probability by enabling the propagation of ice between neighboring cells. We investigated the effect of cell-cell interactions on IIF using genetically modified cells expressing different levels of intercellular junction proteins. A new IIF mechanism was observed in these cells associated with penetration of extracellular ice into the cell-cell interface, and the incidence of this IIF mechanism was reduced in cells expressing the tight junction protein occludin. In addition, we investigated the effect of the cytoplasm supercooling and viscosity on the kinetics of IIF in tissue. We found that increasing the viscosity or decreasing the supercooling significantly decreased the kinetics of IIF, suggesting that IIF protocols for tissue can be optimized by modulating the cytoplasm supercooling and viscosity. Together, these data represent an important step towards developing cryopreservation strategies for tissue.

Thesis (M.S.)--University of Melbourne, Dept. of Surgery (St.Vincent's Hospital),Faculty of Medicine, Dentistry and Health Sciences, 2006. / Typescript. Includes bibliographical references (leaves 118-141).

Thesis (M.Sc. (Plant Science))--Mahidol University, 2006. / LICL has E-Thesis 0018 ; please contact computer services.

Thesis (PhD)--University of Mastericht, the Netherlands, 2007. / Includes bibliographical references.

D.Sc. (Zoology) / The spermatogenesis of two freshwater species from South Africa, Barbus marequensis and B. polylepis, and three fresh water species from Israel, B. canis, B. longiceps and Capoeta damascina, was studied. A histological comparison of the process of spermatogenesis was undertaken. The breeding cycle of B. marequensis, B. polylepis, B. canis, B. longiceps and C. damascina was similar to the breeding cycle of B. aeneus and four phases occured within the cycle. The four distinctive phases are post spawning phase, rest phase, pre-spawning phase and the spawning phase (Vlok, 1986) . During the post spawning phase a decline in sperm development is observed and possible lisosomal activity is responsible for the resorption of sperm cells not shed during the spawning phase. The presence of collagen structure provides a distinctive character to the tissue of the testis. The resting phase is characterised by the absence of the lobular structure and the testis is dominated by the collagen tissue. The testis is small and unobtrusive in the abdominal cavities of both species. At the onset of the pre-spawning phase, the testis is filled with spermatogonia. The lobular structure becomes more prominent and the interstitial tissue can be distinguished. Later during the phase, the synchronised development of sperm cells in the cysts of the lobules can be observed, whilst sperm cells in adjacent lobules are in different stages of development. During the spawning phase the testis of all species studied contain mature sperm . cells in the lumens of the lobules.