Dissertations / Theses on the topic 'Cryopreservation'
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Kazem, Rahnuma. "Oocyte cryopreservation." Thesis, University of Aberdeen, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282706.
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A questionnaire based survey was done to assess the views of fertile individuals, infertile individuals, egg donors and recipients towards gamete donation. The survey showed that fertile individuals were significantly less inclined towards the use of donated eggs in research and treatment, compared to infertile individuals. Acceptability of gamete donation was found to be very high in all groups regardless of their fertility, but the majority of individuals, whether fertile or infertile, were opposed to the use of fetal and cadaveric sources of obtaining eggs. The effect of modifications of the freeze-thaw process was investigated in the mouse model. It was seen that slight modifications of the slow freeze protocol affected survival rates and that ultrarapid freezing achieved better survival rates than slow freezing. Human oocyte cryopreservation was performed using a slow freeze-rapid thaw protocol. In total, 34.4% of oocytes survived cryopreservation and these were randomly allocated for fertilisation by conventional in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). Resulting embryos were spread for chromosomal analysis. ICSI significantly improved the rates of normal fertilisation (43.2% versus 2.7%) compared to IVF (P<0.001). A normal diploid karyotype was achieved by ICSI. These studies show that oocyte donation is acceptable to the majority of both fertile and infertile individuals. Further research is required to improve the methods of oocyte cryopreservation. Once the techniques of cryopreservation have been established, ICSI may successfully be applied to enhance subsequent fertilisation rates.
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Vogel, Martin Joseph. "Proteomic profiling following cryopreservation." Diss., Online access via UMI:, 2004. http://wwwlib.umi.com/dissertations/fullcit/1424168.
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Bradley, Leanne Teresa. "Cryopreservation of bovine spermatozoa." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0016/MQ47313.pdf.
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Beaty, Myron H. "Cryopreservation of eukaryote algae." Thesis, This resource online, 1991. http://scholar.lib.vt.edu/theses/available/etd-07282008-135156/.
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Huang, Yu-Jen. "Cryopreservation of female fertility." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=92216.
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Preservation of female fertility is an important issue today. There are a few effective clinical options for preserving female fertility. Conventional IVF followed by embryo cryopreservation is the only established procedure but is not applicable to all women. Oocyte cryopreservation avoids the ethical and moral concerns related to cryopreservation of embryos but conventional slow freezing methods are associated with low survival rate of oocytes. The main objective of this translational research thesis was to develop an efficient and safe methodology for oocyte cryopreservation that is clinically applicable for female fertility preservation. Specific research objectives were to investigate cryobiology of oocytes in terms of: 1) cryoprotectant (CPA) toxicity effect on oocyte ultra-structures and embryonic developmental potential; 2) Vitrification versus conventional slow freezing of oocytes and their effects of oocyte structures and embryonic developmental potential; 3) Vitrification of embryos using the McGill Cryoleaf and its effect of embryonic development and DNA fragmentation; 4) Clinical efficacy of oocyte vitrification in prospective clinical trials; 5) Effects of oocyte vitrification in terms of the clinical obstetrical and perinatal outcomes; 6) Clinical applications of vitrification of oocytes for preservation of female fertility. The CPA mixture of ethylene glycol (EG) and 1,2-propanediol (PROH) was found to be the most suitable combination for oocyte vitrification, resulting in high embryo development and the least DNA fragmentation. Vitrification of oocyte using the CPA mixture of EG and PROH in combination with the McGill Cryoleaf system is superior to the conventional slow-cooling method, resulting in better preservation of egg ultra-structures and functions. The reduced embryonic development potential of cryopreserved oocyte is related to increased DNA fragmentation and activation of caspase enzymes. Vitrification of human oocytes using the McGPréservation de la fécondité est un sujet important à ce jour. Il y a peu de traitements effectifs pour préserver la fécondité des femmes. La fécondation in vitro (FIV) conventionnelle suivie par la cryoconservation des embryons est la seule procédure bien établie. Cependant, celle-ci n'est pas possible pour certaines femmes. La cryopréservation des ovocytes évite les problèmes éthiques et moraux reliés à la cryoconservation des embryons. Cependant, les méthodes de congélation lente sont associées à des taux de survie des ovocytes faible. Les objectifs principaux de cette thèse de recherche translationnelle était de développer une méthode efficace et sécuritaire pour la cryopréservation des ovocytes qui est cliniquement applicable pour la préservation de la fécondité des femmes. Les objectifs spécifiques de cette recherche étaient d'étudier la cryobiologie des ovocytes à propos de : 1) l'effet toxique du cryoprotectant sur l'ultra-structures des ovocytes et le potentiel de développement des embryons; 2) la vitrification versus la congélation lente des ovocytes et leur effets sur la structure des ovocytes et le potentiel de développement embryonnaire; 3) la vitrification des embryons en utilisant le McGill Cryoleaf et son effet sur le développement embryonnaire et la fragmentation de l'ADN; 4) l'efficacité clinique de vitrification des ovocytes dans des études prospectives cliniques; 5) les effets de long terme de vitrification des ovocytes au niveau des résultats obstétriques et périnatals; 6) l'application clinique de vitrification des ovocytes pour la préservation de la fécondité des femmes. Le mélange de CPA de l'éthylène glycol (EG) et de 1-2 propanediol (PROH) a été trouvé d'être la combinaison la plus convenable pour la vitrification des ovocytes en donnant des résultats de développement embryonnaire supérieur et à un taux de fragmentation de l'ADN diminué. La vitrification des ovocytes en utilisant l
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Moawad, Adel Reda. "Cryopreservation of ovine oocytes." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/27948/.
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Oocyte cryopreservation represents one of the most recent developments in the field of reproductive technologies. However, despite of significant progress, the efficiency of oocyte cryopreservation is still very low. Cryopreservation of mature metaphase II (MIl) oocytes has been reported to induce disorganization of the meiotic spindle and chromosome damage. However, cryopreservation of immature oocyte at germinal vesicle (GV) stage may provide an alternative which avoids these problems. Slow freezing protocols have more recently been replaced by vitrification approaches. In this thesis, recovery, viability and subsequent developmental potential following in vitro fertilisation (IVF), parthenogenetic activation or somatic cell nuclear transfer (SCNT) of ovine oocytes vitrified at GV stage and matured in vitro were studied. Solid surface vitrification (SSV) and cryoloop technologies share the advantages of using a containerless system and small volumes of solution (less than t J.ll) which favours rapid cooling. Maturation, fertilisation, cleavage and blastocyst development were significantly decreased in SSV vitrified oocytes as compared to controls. Following cryoloop vitrification, frequencies of in vitro maturation (43.4 vs 63.2%), oocytes with normal spindle and chromosome configuration (50.0 vs 70.4%) and fertilisation (54.0 vs 74. t %) did not differ significantly between vitrified and control oocytes. Numbers of cleaved embryos that developed to the blastocyst stage following IVM/IVF/IVC did not differ significantly between vitrified and control groups (29.4 vs 45.1 %). In vitro matured ovine oocytes vitrified at GV stage using cryoloop were activated by two different protocols (I) a combination of calcium ionophore (A 23187), cycloheximide and cytochalasin 13 (CA+CHX/CI3), (2) strontium and CB (Sr/Cll). No blastocysts developed in vitrified oocytes activated by CA+CHX/CB; however, 3.8% were obtained following Sr/CI3 activation. Developmental competence of ovinc oocytes vitrified at GV stage and used as cytoplast recipients for SCNT was evaluated. Although the frequencies of cleaved embryos were significantly decreased in vitrified oocytes as compared to control, development to morula and blastocyst stage embryos was not significantly different. No significant differences were observed in total cell numbers, number of apoptotic nuclei as detected by Hoechst and TUNEL assay and proportions of diploid embryos in day 7 blastocysts produced following IVF or seNT of vitrified oocytes as compared to control. Pre-treatment of ovine GV-oocytes with cytochalasin 13 (7.5 J.lglml for 60 min) or demecolcine (0.1 flg/ml for 20 min) prior to vitrification improved frequencies of maturation, fertilisation and subsequent development following IVF or parthenogenetic activation. Caffeine treatment during IVM (10 mM for 6 h) increased the frequencies of blastocyst development in vitrified/thawed GV ovine oocytes. Taken together, these studies suggest that, ovine oocytes vitrified at GV stage can be matured, fertilised and develop in vitro to blastocyst stage embryos. Cryoloop vitrification resulted in higher maturation, fertilisation and subsequent development as compared to SSV. Strontium can be used effectively for parthenogenetic activation of vitrified/thawed ovine GV oocytes. Ovine oocytes vitrified at GV stage can be used effectively as cytoplast recipients for SCNT.
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Gryshkov, O., V. Mutsenko, M. Tymkovych, D. Tarusin, V. Sirotinskaya, I. Braslavsky, О. Г. Аврунін, and B. Glasmacher. "Advances in cryopreservation of alginate-encapsulated stem cells and analysis of cryopreservation outcome." Thesis, Інститут проблем кріобіології та кріомедицини НАН України, 2018. http://openarchive.nure.ua/handle/document/8338.
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Chan, Gene Yel. "Cryopreservation of porcine heart valves." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ60420.pdf.
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Marsland, T. P. "Dielectric measurements for organ cryopreservation." Thesis, University of Cambridge, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.373270.
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Pandolfi, Susan M. "Cryopreservation of microencapsulated bovine spermatozoa." Thesis, This resource online, 1996. http://scholar.lib.vt.edu/theses/available/etd-11012008-063722/.
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Wilkinson, Timothy John. "Cryopreservation of rare and endangered species." Thesis, University of Reading, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312121.
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Song, Ying Ching. "Cryopreservation of arteries with dimethyl sulphoxide." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315284.
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Terry, Claire. "Cryopreservation of rat and human hepatocytes." Thesis, King's College London (University of London), 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420048.
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Sugimoto, Miki. "Studies on Cryopreservation of Mammalian Ovaries." Kyoto University, 2001. http://hdl.handle.net/2433/150806.
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Kagawa, Keiko Sompop Prathanturarug. "Cryopreservation of dendrobium cruentum Rchb. f. /." Abstract, 2006. http://mulinet3.li.mahidol.ac.th/thesis/2549/cd394/4738650.pdf.
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Gale, Samantha. "The cryopreservation of Picea sitchensis germplasm." Thesis, Abertay University, 2005. https://rke.abertay.ac.uk/en/studentTheses/460ff7ff-fbd6-4e9b-93dd-addb29b13c77.
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Picea sitchensis is an important tree species for UK forestry and is at the forefront of prototype clonal breeding programs. These can only be implemented using in vitro, culture therefore cryopreservation technology development is imperative such that elite germplasm can be conserved, without compromise to genetic integrity, whilst phenotypic validation of selected genotypes is undertaken. Three explant types were transferred from the Northern Research station, Roslin to the University of Abertay, Dundee where cryopreservation testing was initiated. Each explant comprised of different anatomical complexities varying from the simplest dedifferentiated embryogenic suspensor masses, to matured somatic embryos and whole tissue shoot-tip apices. Before cryopreservation was initiated in vitro cultures were stabilised and characterised at UAD. Shoot cultures showed different growth responses between genotypes and between culture locations, but biochemical profiling of oxidative stress markers, ethylene and DNA methylation did not confirm stress or epigenetic change as the cause of these differences and physiological recalcitrance. A cryopreservation protocol, using a programmable freezer (Planar), was successfully developed and is reported for the first time for P. sitchensis embryogenic suspensor masses (ESM). Post-LN survival rates of up to 100% were observed in several genotypes. Encapsulation-dehydration was successfully utilised to cryopreserve P. sitchensis mature somatic embryos reported for the first time. Recovered embryos were able to re-initiate into dedifferentiated non-embryogenic masses (NEM) (up to 100%) and embryogenic suspensor masses (ESM) (up to 20% post -LN) as a source of material to mass multiply cryopreserved clonal offspring. Major steps were progressed for the most recalcitrant conifer explant in the project, shoot-tip apices, with essential pre-treatment steps established. Critical cryogenic factors were determined through thermal analysis using Differential Scanning Calorimetry. The study concluded with the initiation of technology transfer of cryopreservation methods to the Northern Research Station to establish the UK’s first conifer cryobank. These systems were preliminary validated. Further implementation will proceed out with the timescale of this project but based on recommendations generated from this thesis.
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Moreira, Vanessa. "Improving biosecurity of bovine in vitro embryo production and cryopreservation." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources, 2009. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=25708.
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Carroll, John. "Cryopreservation and development of the mammalian oocyte /." Title page, contents and abstract only, 1991. http://web4.library.adelaide.edu.au/theses/09PH/09phc319.pdf.
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Onions, Vicki Jean. "Development of whole ovary cryopreservation in sheep." Thesis, Nottingham Trent University, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444616.
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McLaughlin, Eilenn Anne. "The effect of cryopreservation on human spermatozoa." Thesis, University of Bristol, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358122.
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Eisenberg, David Phillip. "Solid Mechanics Effects in Cryopreservation Via Vitrification." Research Showcase @ CMU, 2015. http://repository.cmu.edu/dissertations/633.
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Cryopreservation via vitrification is the most promising means for long-term preservation of bulky tissues and organs. While the road to successful cryopreservation is paved with obstacles, of paramount importance is thermo-mechanical stress, which can lead to fracture. In order to model the mechanical stress within a vitrifying domain during cryopreservation, the relevant thermal and mechanical properties must be explored. Toward that end, mechanical properties, such as coefficient of thermal expansion and Young’s modulus were measured in the cryogenic temperature range for a new class of cryoprotective cocktail involving DP6 (a less concentrated variant of VS55 without formamide) combined with synthetic ice modulators. A synthesis of material models and properties, some measured in the current research work and others available from the literature, were incorporated into finite element models in order to model thermo-mechanical stress. An array of representative cases has been selected to investigate the effects of a spectrum of thermal histories on the resultant mechanical stress. In particular, a temperature hold near the glass transition temperature (i.e. annealing) has shown to significantly decrease the amount of mechanical stress. Other representative cases have been used to investigate the effects of volumetric warming on mechanical stress. It was shown that internal heating using a volumetric rewarming method reduces the stress to tolerable levels, even in cases involving high cooling and rewarming rates. Additionally, photoelastic experiments were performed using an in-house proprietary device known as the cryomacroscope. These photoelastic experiments allowed for the in-situ visualization of the stress field in real time. Photoelasticity experiments were used to validate a key finding from finite element analysis presented in this thesis. Finally, a new phenomenon associated with the stress history was discovered, where stresses are seen to increase during rewarming around the glass transition temperature and an explanation was proposed based on the Narayanaswamy model of glasses.
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Uçar, Ömer. "Acrosome reaction and cryopreservation of dog spermatozoa." Thesis, University of Bristol, 2000. http://hdl.handle.net/1983/60c06232-353d-4d6c-9ca7-3073a2712d2d.
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The use of AI in dogs has been limited by the lack of effective and reliable means of cryopreservation of semen and by the poor correlation between traditional methods of post-thaw assessment of semen quality and fertility. In order to address these problems, the present study focuses upon methods of cold storage and cryopreservation of dog spermatozoa by undertaking comparative evaluations of post-thaw motility and in vitro induction of acrosome reaction. Split-ejaculate protocols were used to compare the effect of storage at +4°C and cryopreservation upon (i) the maintenance of spermatozoa motility and (ii) spontaneous or A23187-induced acrosome reactions during incubation at 39°C (in 5% CO2 in the humidified air) for 60 or 120 min. The assessments of samples were made by Bright-Field, Phase Contrast (PC), Differential Interference Contrast (DIC), Scanning (SEM) and Transmission (TEM) Electron Microscopy. The interaction between the process of glycerolisation and the presence of seminal plasma is one of the key limitors for success in cryopreservation of dog spermatozoa. Interactions between the effects of removal of seminal plasma (by centrifugation), dilution rate, the temperature at which glycerolisation took place and the concentration of glycerol upon survival of spermatozoa at +4°C were studied in a series of split-ejaculate experiments. Spermatozoa were suspended in Tris-fructose-citric acid extender containing 20% (v/v) egg yolk and 8% (v/v) glycerol at +4°C for 48 h. Survival was assessed as the percentage of spermatozoa displaying progressive motility. Survival of spermatozoa was higher (P < 0.05) after glycerolisation at +4°C than at the room temperature. At dilution rates of 1:1 and 1:2 (semen: extender), the survival was higher (P < 0.05) in samples that were centrifuged and glycerolised at +4°C than the samples that were neat and glycerolised at the room temperature. While at the dilution rate of 1:16 it was higher (P < 0.05) in samples that were neat plus glycerolised at +4°C than all samples that were glycerolised at the room temperature. Concentrations of glycerol that were > 2% (v/v) resulted in lower (P < 0.05) survival than at lower concentrations. Following the initial stage of the investigations, the optimisation and validation of a method for in vitro induction of acrosome reactions were required. Suspensions of spermatozoa in TALP medium were incubated in the presence of a logarithmic. series of concentrations of the calcium ionophore, A23187. Induction of acrosome reactions was assessedb y bright-field (using naphthol yellow S/aniline blue stain, NA) and phase contrast (PC) microscopy. Using these methods, it was determined that incubation in the presence of 1 μM/1 A23187 for a period of 30-45 min was optimal for inducing acrosomer eactions in fresh semen. It was also noted that the assessments of acrosome reactions by using NA staining, were highly correlated with PC microscopy. In consequence, the simple procedure of NA staining might be an acceptable alternative to PC microscopy for use in the field. Subsequently, the effect of chilling and glycerolisation upon in vitro induction of acrosome reactions by A23187 was assessed. Acrosome reactions were studied as these have been described in the literature as providing accurate bioassay of spermatozoal functionality in vivo. Acrosome reactions were assessed by using DIC microscopy. The acrosomal integrity was impaired after chilling, which accelerated the A23187-induced acrosome reaction such that a lower concentration (0.1 μM/1) of A23187 was also effective to induce the reaction within 60 min of incubation. However, the presence of 2% glycerol (v/v, final) in standard Tris extender, containing 20% egg yolk, did not significantly affect the sequence of acrosome reaction. The optimal freezing regimen (from +4°C to -120°C) was determined by using a programmable biological freezer in a series of experiments, in which various cooling rates were combined in a Latin square design. Semen was diluted in standard Tris extender containing 20% egg yolk and 2% glycerol (v/v, final) and packed in 0.25 ml French paiettes (straws). The optimal cooling regimen was -0.5°C/min from +4°C to -9°C, -40°C/min to -20°C, -100°C/min to -120°C, followed by direct immersion of the straws in liquid nitrogen. Changes in temperatures within an individual straw were continuously measured and these data were found to be highly correlated with the eventual post-thaw motility of frozen-thawed spermatozoa. Although freezing and thawing resulted in major acrosomal deterioration, there were no significant differences between freezing regimens on the basis of in vitro induction of acrosome reactions, as assessed by DIC microscopy. Finally, ultrastructural studies, using SEM and TEM, upon chilled (as 'ready to freeze') and frozen-thawed spermatozoa subjected to A23187-induced acrosome reaction demonstrated that freeze-thawing provoked the acrosome reaction such that, with TEM (i) the plasma membrane was usually damaged or missing, (ii) the acrosomal changes (including the loss of acrosomal content, as seen by decondensation and swelling) except vesiculation of the acrosomal membranes, exceeded to the equatorial segment and (iii) a further damage occurred to the post-acrosomal region. In summary, these results show that semen should be; (i) centrifuged for dilutions of < 1:8, (ii) diluted at 1:8 in Trisfructose-citric acid extender containing 20% egg yolk, (iii) glycerolised at +4°C at a final concentration of 2% glycerol (v/v), (iv) cooled at -0.5°C/min from +4°C to -9°C, at -40°C/min to -20°C and at -100°C/min to -120°C, followed by direct immersion of the straws in liquid nitrogen for cryopreservation and (i) introduced to 1 μM/1 A23187 in TALP, (ii) incubated for at least 30-45 min for induction of acrosome reaction in vitro and, thereby, demonstrated that optimisation of cryopreservation and in vitro induction of acrosome reaction of dog spermatozoa are possible.
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Morris, Timothy J. "Exploring the improvement of human cell cryopreservation." Thesis, Loughborough University, 2015. https://dspace.lboro.ac.uk/2134/19279.
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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.
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Tan, Sze-Ping. "Cryopreservation for the conservation of Grevillea species." Thesis, Tan, Sze-Ping (1998) Cryopreservation for the conservation of Grevillea species. Honours thesis, Murdoch University, 1998. https://researchrepository.murdoch.edu.au/id/eprint/42489/.
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Western Australia has one of the most diverse and vulnerable flora in the world, with over 300 species on the rare and endangered list and over 1000 more prioritised for further action - about 40% of described species. Due to a lack of resources or protection in situ, most species must be conserved ex situ. Traditional ex situ methods are limited in their ability to store an adequately diverse pool of genetic material, while maintaining its genetic integrity. The advent of cryostorage as a means of genetic conservation potentially provides a highly secure means of storing genetic material in the long term. This study in particular investigated the suitability of cryopreservation as a method of long term conservation of somatic tissue of Western Australian species, with the aim of application to large groups of taxa. The broad aims of this study were to research the applicability of specific cryostorage methods to larger groups of taxa, in this case the genus Grevillea, a woody shrub genus.
The first section of this study investigated the applicability of the cryostorage protocol elucidated for G. scapigera on itself and four other species without modification: G. cirsiifolia, G. dryandroides, G. flexuosa and the hybrid G. bipinnitifida X biternata (BxB). Shoot apices were subjected to a modified vitrification protocol. All five species utilised survived the treatment with significant variation in success. Survival values ranged from 3.6% for G. BxB to 92.3% for G. cirsiifolia. Observations seemed to indicate it was not the freeze-thaw process itself that was responsible for low survival rates seen, and that it was more likely the contribution of the cyototoxicity of cryoprotectectant compounds used.
The age of the explants after subculture was investigated as a factor in improvement of the established protocol for broader applicability. Shoot apices were excised at 7, 14, 21 and 28 days after subculture for the abovementioned five species. All five species tested showed a significant increase in survival post-thaw when the shoot apices were excised at 21 days after subculture, as in the unmodified G. scapigera protocol. Histological studies through GMA sectioning showed no obvious differences in cell characteristics between the ages tested, and no gross disruption in cellular integrity was seen post-thaw. It was postulated that damage caused was at a biochemical level.
The preculture of cells in cryopreservation is a step performed to optimise tissue condition for survival of the freeze-thaw process. It was found that the survival of three species of Grevillea - G. cirsiifolia, G. dryandroides and G. BxB – could be improved by variation from the stipulated protocol in type or concentration of the compound used. G. dryandroides showed a significant increase in survival from 18% to 75 % when incubated on a preculture medium containing 1.2 M glycerol. G . cirsiifolia showed survival of 24 % when incubated with 0.4 M sorbitol. G. BxB however showed a survival at the stipulated 0.6 M sorbitol of 15.6 %. These results were believed to be due to differing requirements between the species tested.
The results of this study showed that a specific established protocol could be applied with minor modifications to a wider range of species. Although only intrageneric similarities in cryopreservation requirements were tested, there is no reason to doubt that protocols could be applicable to wider groups of species. More research is recommended, particularly in the fields of preculture and recovery post-thaw, to improve existing protocols for the purposes of conservation.
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Ahangari, Yousef Jarari. "Cryopreservation of ram semen for artificial insemination." Thesis, Bangor University, 1992. https://research.bangor.ac.uk/portal/en/theses/cryopreservation-of-ram-semen-for-artificial-insemination(25836205-fd80-43ad-a725-ac602fb33b87).html.
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A theoretical study showed that Al can greatly affect the efficiency of sheep breeding schemes provided fertility is maintained at the highest levels. Factors that affect the survival of ram spermatozoa during preservation were studied. A pH range between 6 and 7 was well tolerated. The addition of 4% (v/v) glycerol to the diluted ram semen in Tris buffer lowered the motility and survival of spermatozoa during 5 hours of storage at 30'C. Following insemination of chilled ram semen, with and without glycerol in the diluent, lambing percentages of 59% and 73% respectively were obtained. Ram semen was frozen in 0.25 ml straws using various cooling combinations. The optimal procedure was found to be to cool rapidly from 5'C to -120'C at -20'C/min. When semen so treated was compared in a fertility trial with semen frozen by the pellet method of Evans and Maxwell (1987), lambing percentages of 14% and 18% respectively were obtained. Attempts were made to formulate a vitrifying diluent for ram semen. A method was developed for the assessment of semen in highly concentrated cryoprotective solutions. Semen tolerated 10% concentrations of each of glycerol, acetamide and propylene glycol applied together, but when concentrations were raised above this level sperm mortality was very high. A simple spectrophotometric procedure for the objective assessment of vigour of ram semen was developed and tested. Raffinose 66 mM in the freezing diluent improved the post-thawing revival rate of spermatozoa from 46% to 71%, and increased the post-thawing recovery of the swimmingup vigour (P< 0.01). Raffinose treatment reduced the ATP content of semen but did not reduce the rate of glucose oxidation by diluted spermatozoa at either the pre-freezing or post-thawing stages. Frozen storage of ram spermatozoa as pellets was best achieved using two volumes of Tris buffer diluent containing 18% (v/v) egg-yolk, 6% (v/v) glycerol and 66 mM raffinose to one volume of semen. The diluted semen was chilled to 5'C and frozen as 0.10 ml pellets on dry ice. For frozen storage of ram semen in 0.25 ml straws, best results were obtained when the Tris buffer diluent contained 18% (v/v) egg-yolk, 9% (v/v) glycerol and 66 mM raffmose, and cooling was at a rate of -30'C/min from 5'C to -120'C. Non-return rates were 21%, 20% and 31% for ewes inseminated with semen samples frozen as standard 0.20 ml pellets, as raffinose containing 0.10 ml pellets, and as raffinose containing 0.25 ml straws respectively. Of the in vitro tests, only the swim-up test was correlated with non-return rates (r=0.904, P< 0.1). Post-thawing survival of the spermatozoa was improved by the addition of raffinose which had no deleterious effect on fertility.
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Varesi, S. "CANINE EPIDIDYMAL SPERMATOZOA: CHARACTERISTICS, COLLECTION AND CRYOPRESERVATION." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/219126.
Full textAbstract:
Epididymal spermatozoa represent the only source of genetic material when the
male dies unexpectedly or undergoes orchiectomy for medical reasons. Yet, in
individuals of high genetic or emotional value that cannot mate or ejaculate semen, the
collection of spermatozoa from in situ epididymides might be an option to obtain
progeny.
The aims of the studies presented here were to describe the characteristics of
spermatozoa collected from the different regions of the epididymis (caput, corpus, and
cauda) and to investigate the feasibility of percutaneous epididymal sperm aspiration
(PESA) in dogs. Furthermore, the preservation of DNA integrity in canine epididymal
frozen spermatozoa and the potential protective effect of the antioxidant melatonin on
post-thaw sperm quality were evaluated.
Overall results showed that canine epididymal spermatozoa undergo several
modifications in the epididymis, leading to mature cells which are stored in the
epididymal cauda. From this site, spermatozoa can be retrieved by PESA and the quality
of the sperm population is similar to that of spermatozoa collected in vitro, although a
wide variation amongst animals was observed.
The cryopreservation of epididymal spermatozoa showed that the DNA integrity
is well preserved, but a protective effect of melatonin on post-thaw sperm quality has
not been demonstrated.
The DNA stability after thawing is particularly relevant for epididymal
spermatozoa which potential use in assisted reproductive techniques is mainly after
storage. However, other sperm characteristics as motility and acrosomal integrity are
compromised by freezing and further investigations should be focused on their
preservation.
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Heutelbeck, Anna [Verfasser]. "Cryopreservation of stallion sperm: correlating hypo-osmotic resistance and cryosurvival, and use of density centrifugation for delayed cryopreservation / Anna Heutelbeck." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2013. http://d-nb.info/1037830989/34.
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Carnevale, Kevin A. "Finite-Difference Model of Cell Dehydration During Cryopreservation." Thesis, Georgia Institute of Technology, 2004. http://hdl.handle.net/1853/7258.
Full textAbstract:
A numerical model for describing the kinetics of intracellular water transport during cryopreservation was developed. As ice is formed outside the cell, depleting the extracellular liquid of water, the cell will experience an osmotic pressure difference across its membrane, which causes cell dehydration and concomitant shrinkage. Although Mazur (1963) has previously modeled this phenomenon as a two-compartment system with membrane limited transport, the assumption of well-mixed compartments breaks down at large Biot numbers. Therefore, we have developed a numerical solution to this moving-boundary problem, including diffusive transport in the intracellular liquid, in addition to the osmotically driven membrane flux. Our model uses a modified Crank-Nicolson scheme with a non-uniform Eulerian-Lagrangian grid, and is able to reproduce predictions from Mazurs model at low Biot numbers, while generating novel predictions at high Biot numbers. Given that cell damage may result from excessive water loss, our model can be used to predict freezing methods that minimize the probability of cell injury during the cryopreservation process.
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Starr, Perry Louise. "The effects of cryopreservation on human ovarian follicles." Thesis, University of Warwick, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441573.
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Bedaiwy, Mohamed Ali. "Ovarian tissue cryopreservation and transplantation : approaches and techniques /." Cleveland, Ohio : Cleveland Clinic, 2007. http://www.loc.gov/catdir/toc/ecip082/2007042633.html.
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Gao, Renli. "Cryopreservation of pancreatic fragments for transplantation in rats." Thesis, McGill University, 1987. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=63948.
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Sipen, Philip. "Tissue Culture and Cryopreservation of Banana {Musa spp.)." Thesis, University of Nottingham, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.523611.
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Goswami, Mohar. "Embryo cryopreservation : the clinical outcome and couples' perspectives." Thesis, University of Newcastle upon Tyne, 2013. http://hdl.handle.net/10443/2481.
Full textAbstract:
Aim: Embryo freezing is a standard practice in most fertility units. According to the latest Human Fertilisation Embryology Authority data, 2,032 babies were born in 2010 from 10,548 cycles using frozen-thawed embryos in the UK. However, the practical benefit of embryo freezing in the National Health Service (NHS) context, and the psychological impact of this practice are unknown, and need to be reviewed in the light of increasing demand for NHS support for assisted conception. Therefore, this thesis investigates the answer to the question, “Should we be freezing embryos?” from two aspects: the influence on in vitro fertilization (IVF) success rates from embryo freezing and the decision-making process by which couples decide whether or not to freeze any surplus embryos. Methods: Analysis of the cumulative pregnancy rate (CPR) following three cycles of IVF treatment including embryo freezing was performed using life table analysis. A qualitative interview study involving IVF couples was performed aiming to explore the personal and social factors that couples consider when deciding about embryo freezing. Results: It was found that embryo freezing imparts a modest benefit of about 4% increase in the overall CPR. The qualitative study showed that regardless of the practical benefits of freezing embryos and the ethical and other reservations that couples have about it, the vast majority of IVF couples wish to avail themselves of the opportunity to freeze any surplus embryos, and use every additional opportunity to maximize their chances to have a baby. The decision-making process was complex and nuanced, and was fully appreciated only on reflection. Conclusion: Findings from this study will inform couples who face the difficult decisions about embryo freezing. Although this study indicates that more detailed information may not have influenced their decision, it provides the basis for further study comparing the influence of more targeted information on freezing decisions.
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Jahangir, Jahanbeen. "Development of sensor systems for application in cryopreservation." Thesis, University of Bedfordshire, 2014. http://hdl.handle.net/10547/556475.
Full textAbstract:
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.
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Gryshkov, O., M. Tymkovych, О. Г. Аврунін, and B. Glasmacher. "Cryopreservation of stem cells within intact alginate microspheres." Thesis, IUPESM, Prague, Czech Republic, 2018. http://openarchive.nure.ua/handle/document/8335.
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Nordskov, Harder FIE BARBARA. "Effect of Dextrans on Cryopreservation of Human Spermatozoa." Thesis, Malmö universitet, Fakulteten för hälsa och samhälle (HS), 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-24295.
Full textAbstract:
Cryopreservation is a process where a sample, e.g. cells or tissue, is preserved by cooling to sub-zero temperatures, usually -196°C. Upon freezing these materials, ice crystals form, which eventually results in cell death. In order to reduce the formation of ice crystals, cryoprotectants are added to the storage solution. Current cryoprotective agents have several weaknesses, making the development of cryoprotective agents with advanced properties of great interest. In this study, two possible non-penetrating cryoprotectants termed Dextran Sulphate Sodium Salt 80 (DSSS 80) and Dextran Sulphate Sodium Salt (DSSS 140), were developed. The cryopreservative effect of DSSS 80 and DSSS 140 was studied on human spermatozoa regarding cryorecovery, using a simple cryopreservation procedure. Moreover, the new compound’s cryoprotective properties were compared to conventional types of dextrans, which in previous studies have proven effective. The percentage of sperm motility was assayed before freezing by the optical microscopic method using a haemocytometer as counter chamber. After thawing, the cells were stained with trypan blue and post-thaw motility was determined. It was found that all sperm cells lost their motility, regardless cryoprotectant, concentration or in the absence of dextrans, which indicated that the applied cryopreservation was unfitting. Thus, since the method used in study was not optimal for cryopreservation of sperm cells, it was not possible to determine whether DSSS 80 or DSSS 140 display cryoprotective properties or not, nor if they are superior to conventional types of cryoprotectants.
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AlMulhem, Norah. "Cryopreservation and Hypothermal Storage of Hematopoietic Stem Cells." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1439307244.
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Chen, Shi. "Cryopreservation of human embryonic stem cells and hepatocytes." Thesis, University of Oxford, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711665.
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Plachynta, Maksym. "Studies on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling." Thesis, University of Bedfordshire, 2007. http://hdl.handle.net/10547/622016.
Full textAbstract:
Cryopreservation of fish germ cells has important applications in aquaculture, conservation of endangered species and human genomic studies. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. The objective of the present study was to develop successful cryopreservation protocol for zebrafish oocytes at temperature of liquid nitrogen (-196°C), or if unachieved, to investigate the limiting factors associated with fish oocytes cryopreservation. In this study, the effects of cryoprotectants exposure and enzymatic treatments on oocytes survival were studied, and new viability tests for zebrafish oocytes were developed. The effects of controlled slow cooling with different cryoprotective agents, in different freezing media and at different cooling rates on cryosurvival of zebrafish (D. rerio) oocytes were investigated. Cryomicroscopic observations on zebrafish oocytes were also carried out. Three reliable vital tests -trypan blue (TB) staining, ATP assay, and in vitro maturation followed by germinal vesicle breakdown observation (GVBD) were found suitable for assessment of oocytes viability. Vitellogenesis (stage III) was found to be the optimal developmental stage for cryopreservation. Methanol was found to be the best CPA for zebrafish oocytes. Combination of 4M methanol and 0.2M glucose in potassium chloride (KCI) buffer was found to be the optimal cryoprotective solution. Controlled slow cooling at 0.3°C/min rate, combined with seeding at -12.5°C and plunge to liquid nitrogen (LN) at-40°C were found to be the optimal conditions for cryopreservation of stage III oocytes. However, even with the optimal protocol, TB-assessed viability, Le. the ratio of oocytes with intact plasma membrane after cooling to -196°C was 19.6±8%. Furthermore, GVBD experiments showed that none of the cryopreserved oocytes can be matured in vitro, and their ATP levels were decreased dramatically, indicating that successful cryopreservation of fish oocytes at liquid nitrogen temperature still remains elusive. Cryomicroscopic observations demonstrated, that the damages of oocytes are associated with intracellular ice formation (lIF). IIF occurred simultaneously with extracellular ice formation (ElF) in nearly 100% of the cases, and formation of lethal hexagonal type of ice was observed. This study was the first systematic attempt to cryopreserve fish oocytes at liquid nitrogen temperature. The results provided will undoubtedly assist successful protocol design for cryopreservation of fish oocytes in the future.
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Kaity, Adam. "Genetic, Epigenetic and Morphological Evaluation of the Effects of Cryopreservation on Papaya." Thesis, Griffith University, 2010. http://hdl.handle.net/10072/365796.
Full textAbstract:
A vitrification-based cryopreservation technique for storage of in vitro shoot tips of papaya has been tested to ensure applicability across a range of genotypes and to assess the stability of both genotype and phenotype of clonal material following cryopreservation. Shoot tips of twelve female genotypes were cryopreserved, regeneration percentages were determined and resultant plants were screened for genetic and epigenetic changes. Genomic DNA structure was studied using polymerase chain reaction (PCR) based randomly amplified DNA fingerprinting (RAF), and methylation patterns were monitored using the amplified DNA methylation polymorphism (AMP) PCR technique. Plantlets were regenerated following cryopreservation in all but one genotype and regeneration percentages of 61-73% were obtained from 6 genotypes. The regenerated plantlets showed varying levels of genomic DNA modifications (0-10.07%), and methylation modifications (0.52-6.62%) for detected markers. These findings have not been reported previously for papaya, and indicate some genotype dependent variability in DNA modifications occurred following cryopreservation which could result in somaclonal variation.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
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Keating, Jean. "Physiological and morphological studies on cryostored and untreated human spermatozoa." Thesis, University of Hull, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363275.
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Penfold, Justin David John. "Controlled electromagnetic rewarming of cryopreserved biological materials." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.319611.
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Brock, M. Kelly. "Cryopreservation of semen of the American kestrel Falco sparverius." Thesis, McGill University, 1986. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=65449.
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Medrano, Hernandez Jose Alfredo. "The importance of individual variation in Boar semen cryopreservation." Thesis, Royal Veterinary College (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299995.
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Feig, Justin S. G. "Advancements in Cryomacroscopy with Applications to Cryopreservation by Vitrification." Research Showcase @ CMU, 2014. http://repository.cmu.edu/dissertations/439.
Full textAbstract:
The role of cryopreservation for tissue banking and transplant medicine is undisputable, being the only practical method for long-term storage of high quality biomaterials. Ideally, cells, tissues, and organs could be preserved indefinitely at cryogenic temperatures, where metabolic activity and the natural degradation mechanisms of the biomaterial are arrested. Techniques for successful cryopreservation have been developed over the past five decades for several tissue types. However, successful techniques are generally related to small specimens, in the scale-range of cell clusters to small-organized tissues (μm to mm range), with stem cells and corneas as examples. Cryopreservation of large specimens (cm and above) has been accomplished only in cases where the mechanical functionality has a higher priority than the recovery of biological functionality, with heart valves as an example. Yet, the science and technology of cryopreservation have dramatically advanced in recent years, and cryopreservation of large tissues and organs is closer to becoming a practical reality than ever before. In classical cryopreservation, the low-concentration cryoprotective agents (CPAs) are used to control ice formation- the cornerstone of cryoinjury. However, classical cryopreservation has proven ineffective in preventing damage to complex mammalian tissues, where vitrification is investigated as an alternative cryopreservation application (vitreous means glassy in Latin). Vitrification is achieved by rapidly cooling high-concentration CPAs, resulting in a sample trapped in glassy domain. The high concentrations and rapid cooling rates applied have created additional obstacles, such as toxicity and thermal stresses, with potentially detrimental outcome. In this context cryopreservation research is focused on finding a balance between these three competing effects. The subject matter of this study is the development of new prototype devices to provide real-time visualization and documentation of cryopreservation of large samples in situ; these devices belong to a unique classification termed cryomacroscopy. In the absence of cryomacroscopy techniques, common practices for macro-scale visualization are based on end-state observations, at room and storage temperatures. Due to the path dependent nature of vitrification, this practice may miss many physical phenomena associated with cryopreservation that could be captured with cryomacroscopy such as: (i) heterogeneous ice nucleation frequently associated with low-concentration CPAs and slow cooling rates; (ii) homogeneous ice nucleation frequently associated with high-concentration CPAs and rapid cooling; (iii) deformation of CPAs due to thermal contraction; (iv) undesired solute precipitation effects; and (v) fracture formation due to thermo-mechanical stress. The implementation of polarized light is introduced in this study, serving as a contrast enhancement technique for the improved visualization of contaminants and ice crystals in the CPA domain. Furthermore, polarized light is used for the first time to create photoelasticity effects in the study of thermo-mechanical stresses during vitrification. Photoelasticity techniques are presented in this study to investigate the conditions leading to fracturing, as well as the formation of stress concentrations. Photoelasticity is also demonstrated in the study of annealing-where stress relaxation is facilitated by an intermediate temperature-hold above glass transition. A thermal model is presented in this study to correlate observed phenomena with the thermal history within the specimen. Related investigations include: (i) crystallization within the CPA; (ii) removal of dissolved gases by pre-freezing in an effort to eliminate potential ice nucleation sites; (iii) incorporation of the enthalpy method to track the development of a freezing front; (iv) identifying conditions of solute precipitation from the vehicle solutions used to mitigate osmotic effects during cell permeation; (v) cooling rate effects on the developing stresses during vitrification; (vi) identifying the optimal temperatures to enable stress relaxation; and (vii) evaluation of thermal gradients in the domain during annealing. Lastly, cryomacroscopy observations are demonstrated on a special class of chemicals compound, recently termed synthetic ice modulators (SIMs), which represent the cutting edge in the field of cryobiology. SIMs are may be added to the CPA cocktail to inhibit ice growth. SIMs may permit vitrification in the presence of lower concentration CPAs, which may reduce toxicity effects. In conclusion, the cryomacroscope prototypes presented in this study may be developed as tools: (1) to facilitate the development of new materials and protocols; (2) to aid in the quality control of mass production of cryopreservation products; and (3) to facilitate advanced cryopreservation research.
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Wiswedel, Klaus. "Sperm cryopreservation and artificial insemination at Groote Schuur Hospital." Master's thesis, University of Cape Town, 1987. http://hdl.handle.net/11427/25895.
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The diagnosis and therapy managed by the specialities of infertile couples is traditionally of Gynaecology and Andrology. The latter is a subspeciality, which should combine the knowledge of urologists and gynaecologists in the treatment of sub or infertile men.
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Zhang, Tiantian. "Investigations into the cryopreservation of zebrafish (Brachydanio rerio) embryos." Thesis, University of Bedfordshire, 1994. http://hdl.handle.net/10547/622045.
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Cryopreservation of fish embryos was studied using zebrafish embryo as a model system. Cryoprotectant toxicity, chilling sensitivity and embryo membrane permeability were investigated with a view to developing optimum protocols for cryopreservation of the embryos using controlled slow cooling, non-freezing storage and vitrification. Methanol was found to be the most effective cryoprotectant for controlled slow cooling and non-freezing storage of zebrafish embryos with 11 %heart beat stage embryos surviving after controlled slow cooling to -25°C. Zebrafish embryos were found to be very chilling sensitive with early developmental stages being the most sensitive to chilling injury. Embryo developmental stages after closure of the blastopore (>12-h), especially post heart beat stages were much more resistant to cryoprotectant toxicity and chilling injury. Heart beat stage (27-h) embryos proved to be the best embryo developmental stage for controlled slow cooling and non-freezing storage. Dechorionated embryos are more sensitive to cryoprotectant toxicity and chilling injury. The sensitivity to chilling injury of zebrafish embryos limited the use of controlled slow cooling and non-freezing storage for long term cryopreservation. The attempts at cryopreservation of zebrafish embryos using vitrification produced no embryo survival, although up to 32 % embryos remained morphologically intact immediately after vitrification. Poor cryoprotectant permeation, dehydration and consequently ice formation within the egg are probably the main factors on effecting embryo survival. The results of zebrafish embryo permeability studies demonstrated that the chorion of the embryos was permeable to water and cryoprotectants, whilst the vitelline (plasma) membrane was an effective permeability barrier. The inability to achieve sufficient penetration of the vitelline membrane by cryoprotectants poses severe problems for long term cryopreservation, which need to be overcome, possibly by permeabilisation of the vitelline membrane, before successful cryopreservation can be achieved.
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Cagol, Nicola. "Cell-laden hydrogels for biofabrication: matrices processing and cryopreservation." Doctoral thesis, Università degli studi di Trento, 2018. https://hdl.handle.net/11572/367623.
Full textAbstract:
In this dissertation, a report of my PhD research activity is provided. The activity was carried out in Biotech Research Center, part of the Industrial Engineering Department, of the University of Trento (Italy), under the supervision of Prof. Claudio Migliaresi and Dr. Devid Maniglio. Biofabrication, an approach to the bottom-up paradigm of tissue engineering, represents the research topic. This technology is defined as the production of complex biological constructs using cells, components of the ECM, biomolecules, and biomaterials that are assembled with different techniques in an engineered tissue fragment. The general aim of the work was to address some of the problems that currently limited the development and applicability of biofabrication. In particular, two issues were considered in the experimental part: the cryopreservation of cell-laden hydrogel constructs and the development of novel building blocks containing cells using alginate-based hydrogels. Alginate was the material of choice for investigation, as an accepted support for different tissue engineering applications that can sustain several modification and fabrication methods.
In the first chapter, the concepts of bottom-up tissue engineering and biofabrication are introduced. The role and state of the art of hydrogels to manufacture cell-laden building blocks, the techniques for cell encapsulation and the commonly used fabrication strategies for biofabrication and bioprinting are reviewed together with their applications. Moreover, the limitations that currently restrict the applicability of hydrogel-based tissue engineering are discussed.
In chapter two, the role of alginate hydrogels in tissue engineering and biofabrication is described. In particular, its chemical content, crosslinking behavior, manufacturing capacity, and applications are reviewed with emphasis on the possible modification of alginate hydrogels in order to enhance biocompatibility and functionality of encapsulated cells.
The experimental part is described in the following chapters. Chapter three introduces the concept of cryopreservation and in particular the issues concerning the preservation of cell-laden building blocks. Subsequently, the impact of cryopreservation on the viability and functionality of cells encapsulated in alginate matrices is evaluated comparing different cryoprotective agents. The experimental methods for manufacturing and preserving cell-laden alginate fibers and for performing the biological and structural tests are reported. The results are presented, discussed and compared with the state of the art.
In chapter four, a novel method for encapsulating cells within alginate-based hydrogel films with micrometer thickness is described. The procedure for immobilizing cells within hydrogel films with different composition is described, together with the performed biological assays aimed at selecting the best matrix composition. The results are reported and discussed, emphasizing the potential applications and future developments of the proposed method.
49
Cagol, Nicola. "Cell-laden hydrogels for biofabrication: matrices processing and cryopreservation." Doctoral thesis, University of Trento, 2018. http://eprints-phd.biblio.unitn.it/3044/1/complete_05-28_revision_.pdf.
Full textAbstract:
In this dissertation, a report of my PhD research activity is provided. The activity was carried out in Biotech Research Center, part of the Industrial Engineering Department, of the University of Trento (Italy), under the supervision of Prof. Claudio Migliaresi and Dr. Devid Maniglio. Biofabrication, an approach to the bottom-up paradigm of tissue engineering, represents the research topic. This technology is defined as the production of complex biological constructs using cells, components of the ECM, biomolecules, and biomaterials that are assembled with different techniques in an engineered tissue fragment. The general aim of the work was to address some of the problems that currently limited the development and applicability of biofabrication. In particular, two issues were considered in the experimental part: the cryopreservation of cell-laden hydrogel constructs and the development of novel building blocks containing cells using alginate-based hydrogels. Alginate was the material of choice for investigation, as an accepted support for different tissue engineering applications that can sustain several modification and fabrication methods.
In the first chapter, the concepts of bottom-up tissue engineering and biofabrication are introduced. The role and state of the art of hydrogels to manufacture cell-laden building blocks, the techniques for cell encapsulation and the commonly used fabrication strategies for biofabrication and bioprinting are reviewed together with their applications. Moreover, the limitations that currently restrict the applicability of hydrogel-based tissue engineering are discussed.
In chapter two, the role of alginate hydrogels in tissue engineering and biofabrication is described. In particular, its chemical content, crosslinking behavior, manufacturing capacity, and applications are reviewed with emphasis on the possible modification of alginate hydrogels in order to enhance biocompatibility and functionality of encapsulated cells.
The experimental part is described in the following chapters. Chapter three introduces the concept of cryopreservation and in particular the issues concerning the preservation of cell-laden building blocks. Subsequently, the impact of cryopreservation on the viability and functionality of cells encapsulated in alginate matrices is evaluated comparing different cryoprotective agents. The experimental methods for manufacturing and preserving cell-laden alginate fibers and for performing the biological and structural tests are reported. The results are presented, discussed and compared with the state of the art.
In chapter four, a novel method for encapsulating cells within alginate-based hydrogel films with micrometer thickness is described. The procedure for immobilizing cells within hydrogel films with different composition is described, together with the performed biological assays aimed at selecting the best matrix composition. The results are reported and discussed, emphasizing the potential applications and future developments of the proposed method.
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Maffei, S. "OVARIAN TISSUE CRYOPRESERVATION FOR THE SAFEGUARD OF FEMALE FERTILITY." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/230860.
Full textAbstract:
Ovarian tissue cryobanking is considered an available method for preserving female fertility and represents the best option in young pre-pubertal cancer patients. Cryopreservation can be performed on ovarian cortical fragments or on the entire organ. To move forward, the cryopreservation of both avascular ovarian fragments and of whole ovaries must be improved. To this purpose, we performed a detailed comparison between conventional slow freezing and directional freezing on both cortical fragments and whole ovary.
We provide that directional freezing improves the viability of cryopreserved ovarian tissue both in whole ovaries and cortical fragments, but we observed a better preservation of follicles when the samples were frozen as entire organ.
We also developed a perfusion system for ex vivo culture of whole ovary demonstrating that it is possible to maintain alive sheep ovary outside the body for up 4 days. This perfusion culture system could provide a future alternative for women at risk of ovarian involvement due to the threat of reintroducing malignant cells.