Submitted in fulfilment of the requirements for the degree of Master of Applied Sciences in Biotechnology, Durban University of Technology, Durban, South Africa, 2015. / In contrast to orthodox seeds, recalcitrant seeds are short-lived, shed at relatively high water contents (WCs), and are desiccation sensitive. Presently, the only option for long-term conservation of genetic resources of such plant species is by cryostorage in liquid nitrogen (LN; -196°C) or in the vapour phase over LN (at -150⁰C to -160⁰C). A number of cryopreservation protocols applied for recalcitrant zygotic embryos or embryonic axes of tropical/sub-tropical species have reported survival as either root or shoot development or callus formation, with no shoot or root production after cryopreservation. This is a consequence of the challenges encountered when optimising the WC for successful cryopreservation across species. Other shortcomings may also be the formation of ice or the sensitivity to desiccation resulting in lethal damage or poor re-growth. However, for successful cryopreservation, a normal plantlet with a shoot and a root needs to be obtained post-cryo. Specimens required for successful cryopreservation must be small; therefore embryonic axes excised from seeds have been often used as the explants of choice. However, in some cases, excised embryonic axes of mature recalcitrant seeds are too large to be cryopreserved, or, even if small, may be adversely affected by excision, dehydration and/or immersion in LN, thus failing to produce plantlets after cryopreservation. As a result, in such cases, there is a need to develop explants alternative to zygotic axes such as buds derived from in vitro shoots, shoot meristems, or shoot apices and somatic embryos. These alternative explants must have a high capacity for plantlet formation before and after cryopreservation. The present study aimed to successfully cryopreserve shoot apices of Trichilia emetica and T. dregeana, tropical recalcitrant-seeded tree species, and monitor the responses or effects of some of the procedural steps involved in cryopreservation on the survival and shoot production from these shoot apices. The main foci of the investigation were to produce vigorous plantlets after cryopreservation and ultimately develop a protocol for the successful cryopreservation of germplasm of these species. Furthermore, this study reports on a number of factors that may affect survival after cryopreservation, viz. WC of the explants, PVS2 treatment, production of reactive oxygen species (ROS) and levels of endogenous total aqueous antioxidants (TAA) during the various steps of cryopreservation. The effects of the various steps of cryopreservation on the ultra-structure of the shoot apices were also observed. Cathodic protection (by using highly reducing cathodic water; CW) of the explants was attempted to improve vigour and shoot production from the surviving shoot apices after cryopreservation as cathodic water has been reported to ameliorate the excessive burst of ROS, which often accompanies the stresses imposed by the procedural steps of cryopreservation. Experiments were also performed to optimise the medium for vigorous shoot formation from the shoot apices. Shoot apices of T. emetica in this study had an initial WC of ca. 2.2 g g -1 dry weight (DW) upon excision. Although the WC of the shoot apices decreased slightly after cryoprotection with PVS2, it did not result in sufficient dehydration before cooling. Upon retrieval from LN, 68% of the shoot apices survived and 40% of those produced shoots. Treatment of shoot apices with CW did not improve the survival or shoot production from the apices following cryo-retrieval. This could be a direct consequence of increase in WC of the shoot apices following CW treatment. Water content is not the only factor affecting successful cryopreservation; the production of ROS and the level of antioxidants may also have an impact on regrowth after cryogen exposure. Rapid changes in temperature when the samples are cryo-stored and then rewarmed result in an increase in ROS production, which could have affected the shoot production. More importantly the antioxidant activity showed a rapid decrease during recovery, especially in the CW treated shoot apices, which might have also led to the poor survival and shoot production from the shoot apices. Ultrastructural observations showed the injurious effects of PVS2 treatment typified by derangement of plastids, development of numerous small vesicles along the cell membrane and abnormalities in the structure of the nuclear envelope in the shoot apical cells both before and after cryogen exposure. Following cryo-retrieval, the meristem cells were extensively deteriorated – indicating non-survival, however, some shoot apices had areas of surviving cells which might have led to 40% shoot production after cryopreservation. Based on the studies on optimising medium composition for shoot formation from the apices, woody plant medium (WPM) with 1 mg L-1 BAP + 0.1 mg L-1 IBA was found to be the best medium which gave a higher shoot production of 67 – 70% before cryopreservation compared with only 18 – 20% shoot formation on media used previously. Therefore, this medium was used as the recovery medium. Encapsulation-dehydration of the shoot apices and the use of PVS3 instead of PVS2 for cryoprotection were also employed in an attempt to improve the survival and shoot production after post-cryo, but both methods did not result in any shoot production although 92% and 90% of the shoot apices survived cryogen immersion, respectively. While the shoot apices of T. emetica resulted in 40% shoot production following retrieval from LN and recovery on WPM with 1 mg L-1 BAP + 0.1 mg L-1 IBA, attempts to further improve the shoot production were not successful. The results of this study suggest that the shoot apices used were possibly not sufficiently developed, and with the commensurately high WC, proved to be unsuitable explants for germplasm conservation of T. emetica. The injurious effects of PVS2 treatment both before and after cryogen exposure as observed from the ultra-structural studies provide a clue to the repeated failure to cryopreserve embryonic axes of many tropical recalcitrant-seeded species after treatment with PVS2. Maintaining mother material in culture for longer durations before explant excision in order to allow better development of the axillary buds and render the cytosol more concentrated, and optimising the exposure duration to loading solution and concentration of sucrose in the loading solution might however, provide sufficient dehydration tolerance to PVS2 leading to successful vitrification up on cooling.

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programs for endangered species. Many studies have been devoted to the mechanisms of cryodamage. However, in spite of growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells often remains poor. It is known that cryopreservation causes extensive damage to membrane, results in decreased metabolism of cells, and disturbs the bioenergetical processes of cells by damage to mitochondria. Nonetheless, it has not yet been identified clearly if cryopreservation causes some disruption in the genetic integrity of reproductive cells and the safety of this approach still needs to be confirmed. The present study was undertaken on the spermatozoa of weather loach (Misgurnus tassilis) and blastomeres cells of zebrafish (Danio rerio). It was shown that survival was decreased for embryos derived from sperm after cryoprotectant treatment or cryopreservation. Some evidence has emerged that this decrease is more likely to reflect some genetic instability caused by cryopreservation of sperm. The present study showed for the first time that the DNA repair system of oocytes was activated after fertilisation with cryopreserved sperm. The effect of DNA repair system was also studied. It was found that incubation of fertilised eggs in caffeine could reverse the detrimental effects of cryopreservation of loach sperm on subsequent embryo development. On the other hand incubation of fertilised eggs with 3-aminobenzamide - inhibitor of the poly (ADP-ribose) polymerase (PARP)- brought further decrease in the survival of embryos derived from cryopreserved sperm. The effect of individual donors of sperm and eggs on overall embryo survival was also studied and these investigations revealed significant differences between different donors. Effect of cryopreservation on zebrafish blastomeres was studied at the DNA molecular level. Mitochondrial DNA was sequenced after cryopreservation and increased level of frequency of the mutation was observed. This finding showed that cryopreservation might potentially increase the instability of mtDNA genome. The significance of these changes on the subsequent function of the cells is to be elucidated. Meanwhile this study suggests that it is important to be cautious in making judgements on the safety of cryopreservation techniques in reproduction.

Studies investigating the effect of 5-HT receptors mediating a response in the neonatal intestine have been limited. There are evidences that the development of new neurones continues past postnatal term and this suggests that receptors expression may differ during maturation. Thus, 'in vitro' experiments were carried out to investigate the effects of ACh, atropine, 5-HT and its related drugs on intact intestinal segments taken from the ileum of adult and neonate rats. The application of ACh (3nM-1mM) and 5-HT (3nM-1mM) induced contractions in a concentration dependent manner in all tissues examined. The 5-HT induced contractions were only sensitive to antagonism by atropine (1μM) in segments taken from the neonates but not adults. The pre-treatment with methysergide (5-HT1/2/5-7 receptor antagonist), ritanserin (5-HT2 receptor antagonist), granisetron (5-HT3 receptor antagonist) and RS 23597 (5-HT4 receptor antagonist) at 1μM or a combination of ritanserin, granisetron, plus RS 23597 at 1μM significantly reduced or abolished contractile responses induced by 5-HT. SB 269970A (5-HT7 receptor antagonist) and WAY 100635 (5-HT1A receptor antagonist) at 1μM failed to influence contractile responses induced by 5-HT or the challenges to 5-HT receptor agonists, 5-CT (5-HT1A/7 receptor agonist) and 8-OH-DPAT (5-HT1A receptor agonist) at a concentration range of 10nM-0.1mM, indicating the unlikely involvement of 5-HT1A and 5-HT7 receptors in the mediation of contractile responses in the neonatal rat ileum. Results indicate differences in cholinergic receptor involvement during postnatal maturation and suggest the involvement of 5-HT2, 5-HT3 and 5-HT4 receptors in the mediation of contractile responses to 5-HT in the neonatal rat ileum. There is a growing need to decrease animal usage in pharmacological experiments. This may be achieved by the development of 'in vitro' cell culture models. Thus attempts were also made to develop a cell culture model of neonatal intestine to further investigate the action of pharmacologically active agents. The isolation of individual cell populations from segments taken from the intestine of rat neonates were achieved by ligation of both ends of the intestine prior to incubation in trypsin so that a gradual dissociation could be monitored. This was supported by histological procedures, determining the time required to extract large numbers of cells from different intestinal layers. Differential adhesion and selective cytotoxicity techniques were used for further purification of intestinal smooth muscle cells (ISMC), neuronal cells, and a coculture of ISMC and neuronal cells, and these were characterised through immunostaining with antibodies to α-smooth muscle actin, α-actinin and the 5-HT3 receptor. A protocol for cryopreservation of ISMC was designed in order to protect cells against genetic instability, enhance cell availability and reduce animal usage. Results showed that cells extracted from the intestine are viable for up to 4-months. ISMC functionality was analysed via the application of known pharmacologically active drugs on ISMC, which were plated onto glass and silicone elastomer substrate. The cultured ISMC responded to the application of drugs such as potassium chloride (KCl), carbachol, 5-HT and noradrenaline (NA). Large population of cocultures seeded onto silicone elastomers or cholesteric liquid crystal substrates (LC) were assessed for their ability to produce a collective response to KCl application. Attempts were made to detect any deformations of the substrate surface due to the exposure to KCl and NA. Cholesteric LC substrates seemed to be the most suitable material for investigating the cellular tensions. The availability of cell cultures allowed the development of an intestinal model of inflammation. This was achieved through the use of lipopolysaccharide (LPS)-induced inflammation and was confirmed by assessing the levels pro-inflammatory mediators interleukin (IL-8) and nitric oxide (NO), which were significantly elevated. Reduction of IL-8 ad NO was also examined using granisetron and L-NAME and Chaga mushroom extract. Granisetron and L-NAME reduced the NO production during short incubation times. However, an elevated level of NO was observed when longer treatment times were examined. The Chaga mushroom extract caused a significant reduction in NO production in the model of inflammation. This indicates that this model may be a valuable tool for the investigation of other pro-inflammatory mediators and may contribute for the investigation of more selective drugs in the management of intestinal inflammation in neonates.

published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

Preservation of tissue structure, morphology and biomarkers is of utmost importance for pathological examination of biopsy specimens for diagnostic and therapeutic purposes. However current methods employed to evade tissue degradation and preserve biomarkers have several shortcomings that include irreproducibility, morphological artifacts and altered biomarker antigenicity. These artifacts may affect the analysis and subsequent diagnosis of the tissue pathology. This creates need for developing improved preservation methods that reproducibly maintain tissue morphology and biomarker antigenicity and are simple, rapid and inexpensive. Experiments conducted for testing the hypothesis that cryopreservation procedures yield high quality morphology and antigenicity showed that cryopreservation maintains tissue structure, morphology and antigenicity at equivalent or better levels compared to standard freezing techniques. In order to understand the mechanisms of osmotic transport in cellular systems upon exposure to multi-component solutions that are prevalent in virtification protocols, experimental studies were undertaken using microfluidics for single cell manipulation. The experimental data yielded permeability parameters in binary and ternary solutions for MC3T3-E1 murine osteoblasts for the first time. The hydraulic conductivity (L(p)) decreased with increasing concentrations but the solute permeability either increased or decreased with increasing solution concentration. The changes in hydraulic conductivity were consistent with previously published trends and conform to a functional relationship in the form of Arrhenius type relationship between L(p) and solution concentration. Further a theoretical model was developed from principles of linear irreversible thermodynamics to simulate multi--‐‑component mass transport across membrane. The model was successfully validated by comparison with experimental data for murine osteoblasts and showed good agreement between the numerical predictions and experimental observations. The modeling approach can be used to investigate the transport mechanisms, which show that in multicomponent osmotic transport response, the dynamics is dictated by slower moving solute.

Cryopreservation is now a common practice in the fields of aquaculture, conservation and biomedicine. The success of cryopreservation is usually analysed in terms of cell survival, although there are other potential adverse effects that don’t necessarily result in cell death. These include DNA damage, which could result in altered gene expression. The aim of this study is to discover if cryopreservation has an impact on gene expression using zebrafish (Danio rerio) as the model organism. As the whole embryo cannot yet be successfully cryopreserved, isolated blastomeres from the embryos were used to investigate the impact of cryo-treatment on gene expression. This study sets out firstly to determine an optimum cryopreservation protocol for 50% epiboly blastomeres (Epiboly displaces the blastoderm margin to 50% of the distance between the animal and vegetal pole). Blastomeres had the highest survival level (70.2 ± 3.2%) when a mixture of 1.5 M dimethyl sulfoxide (DMSO) and 0.1 M sucrose were used as cryoprotectants. As quantitative analysis of gene expression using real-time PCR requires the use of a housekeeping gene as an internal control to normalize date, the second study aimed to identify and validate housekeeping genes for use in cryopreservation studies of zebrafish blastomeres. Seven potential housekeeping genes were analysed across a range of embryo stages and isolated blastomeres using the GeNorm and NormFinder software packages. Results indicated β-actin and EF1α housekeeping genes to be the most appropriate for cryopreservation studies on zebrafish embryos and blastomeres, and these housekeeping genes were used in the third study, the effect of cryopreservation on Pax gene expression. The results indicated that the trends (profile changes) in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryos which could have a detrimental effect on embryo development. This is the first study on the stability of housekeeping and transcription factor genes in chilled and cryopreserved embryonic cells of the zebrafish. This work will significantly enhance future studies investigating the impact of cryopreservation on gene expression in zebrafish embryos.

This work describes the development, validation and application of sensor systems to monitor phase transition events of cryoprotectant mixtures in samples and cryopreservation profiles and post-thaw recovery of Lactobacillus delbrueckii subsp. bulgaricus CFL1. Ice nucleation and glass transition (Tg) temperatures influence cell viability during cryopreservation. Knowledge of these phase changes for cryoprotectant mixtures is an essential step in optimising cryopreservation protocols for cell survival. Differential scanning calorimetry (DSC) is used to determine Tg, but the expensive nature of such instrumentation limits its widespread use. Cost-effective sensor systems have been designed to monitor ice-initiation and Tg events in small volume samples of cryoprotectants solutions. Tg values were measured for glycerol, sucrose and Me2SO (with and without NaCl supplement and ice-nucleators) in cryotubes and cryostraws, using temperature and screen-printed impedance sensors. The effect of changes to ice-initiation temperature on Tg was also investigated at different cooling and warming rates by using a Grant Asymptote (EF600) controlled rate freezer. The resulting Tg values obtained by single-channel transition monitoring system (TMS 1) were not significantly different from the values obtained by DSC reported in the literature. However multiple channelled transition monitoring system (TMS 2) requires further circuit modification and multiple screen-printed temperature probes to study the phase-change temperatures and to determine transition events in more than one sample at a time. The lactic acid bacterium (LAB) Lactobacillus delbrueckii was investigated as a model system to monitor the effect of different cryopreservation protocols on post-thaw cell metabolic activity. An important parameter for monitoring the post-thaw quality of LAB for starter culture preparation is the change in pH of the culture medium during incubation at 40 oC. Glass pH combination electrodes are the most common and widely used sensors. However, they are fragile, must be conditioned before use and are not disposable. An alternative to conventional glass electrodes are screen-printed carbon-metal electrodes. Different percentage mixtures of ruthenium and antimony pastes were tested and 54.5% carbon-antimony electrodes gave the best sensitivity and consistency in potentials at fixed pH with a screen-printed salt-bridged Ag/AgCl reference. LAB cultures were cryo-preserved at very rapid, moderate and very slow cooling rates and their post-thaw metabolic activity after overnight incubation in MRS broth was determined using screen-printed pH electrodes. Back to back testing with conventional glass pH sensors was performed to compare responses. Results indicated that early ice-initiation (by means of nucleators) prevents the cells from extensive dehydration (during cooling) and enables maximum post-thaw recovery after incubation (due to equilibrium ice formation and ice melting). In future, screen-printed pH sensors require development with integrated salt-bridged Ag/AgCl reference to make it robust in signalling response. The availability of low cost, disposable, non-fragile sensors and sensor systems to monitor transition events allows the determination of Tg of cryopreservation media during both cooling and warming cycles. A combined screen-printed (impedance + temperature) sensor is proposed for this purpose. A combined screen-printed (pH + reference) sensor would allow the monitoring of metabolic activity in post-thaw and fresh starter cultures of LAB. At present the salt-bridged pH reference is manually attached to the screen-printed pH working electrode but it requires further modifications to the method of attachment. The two sensor systems would enable optimisation of cryopreservation protocols for LAB and could enable such measurements to become routine at commercial scale.

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.

Regenerative medicine is an emerging technology and with hundreds of cell therapies currently in clinical trials there is a need to expand the limited knowledge related to their storage, shipment and preservation. The most widely used medium for human cell cryopreservation is 10%wt dimethyl sulfoxide (DMSO) in serum. However given its potential toxicity, DMSO usage is a key issue in cryopreservation. Methods specify the need to reduce cell exposure time to DMSO above 0°C as much as possible but the maximum amount of time cells can be exposed to DMSO to prevent a detrimental effect needs to be clarified. There are also regulatory issues and concerns with the xenotoxicity, ethics and supply of the other core component in the standard cryomedia formulation: Foetal Bovine Serum (FBS). Developing a viable alternative to FBS is crucial. In cryobiology literature thawing appears poorly understood. A stable process is as vital as freezing to prevent injury to cells. Protocols are currently too vague for cell therapy regulation and need improvement. The time dependent DMSO cytotoxicity was evaluated by overexposing cells to DMSO during and/or after cryopreservation. A broad investigation found that after 1 hour overexposure post thaw viability of human mesenchymal stem cells (hMSCs) was reduced from 96.3±0.6% to 74.1±4.0% and the co-expression of five key hMSC markers was changed from 97.9±1.3% to 68.3±2.6%. This significant change could cause indicate a change in product efficacy and affect patient health, to prevent this, DMSO exposure must be kept to below 1 hour. A range of alternative vehicle solutions were screened and human platelet lysate (hPL) investigated as an alternative. In depth experimentation with hPL as a cryopreservation vehicle solution and culture supplement (in place of FBS) found it to be a worthy, statistically similar alternative. With no xenological or ethical concerns, lower costs than other serum-free alternatives hPL could allow for a move away from xenological components. A heat transfer model was developed and determined that 720J is required to thaw a vial. Using the heat transfer model and additional factors such as pre-thaw stabilisation and on thaw dilution, a two-stage experiment found that the current standard process (warming in a 37°C waterbath) within the current paradigm of a 1.8mL cryovial is optimal but further work is required to define the process for scaled-up product.

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