Dissertations / Theses on the topic 'Tissues Cryopreservation'
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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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Vogel, Martin Joseph. "Proteomic profiling following cryopreservation." Diss., Online access via UMI:, 2004. http://wwwlib.umi.com/dissertations/fullcit/1424168.
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Mukherjee, Indra Neil. "A rational design approach for the cryopreservation of natural and engineered tissues." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/22579.
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Thesis (Ph. D.)--Chemical and Biomolecular Engineering, Georgia Institute of Technology, 2008.Committee Chair: Sambanis, Athanassios; Committee Member: Long, Jr., Robert C.; Committee Member: Ludovice, Peter J.; Committee Member: Prausnitz, Mark R.; Committee Member: Song, Ying C.
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Xu, Xia. "A study of mass transfer in cryopreservation of living tissues." Thesis, University of Oxford, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.275199.
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Els, Cecilia Lydia. "Early human follicle ultrastructure comparison after slow cryopreservation in two different cryoprotectants." Thesis, Link to the online version, 2008. http://hdl.handle.net/10019/831.
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Higgins, Adam Zachary. "Intracellular ice formation in tissue constructs and the effects of mass transport across the cell membrane." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/28166.
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Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2008.Committee Chair: Karlsson, Jens; Committee Co-Chair: Nerem, Robert; Committee Member: Meda, Paolo; Committee Member: Prausnitz, Mark; Committee Member: Sands, Jeff; Committee Member: Zhu, Cheng.
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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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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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Fuku, Eiji. "Studies on the cryopreservation of immature and in vitro matured bovine - oocytes." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41590.
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The developmental potential of mammalian oocytes cryopreserved with procedures similar to those used for embryos has been limited, inasmuch as oocytes differ from embryos in advanced stages of development, both physiologically and morphologically. The objective of this work was to elucidate the precise nature of freeze-thaw damage with the expectation that identification of specific targets will enable devising optimal procedures for cryopreservation of bovine oocytes to prevent specific damage and minimize the loss of developmental capacity.In the first series of experiments, bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Survival was assessed morphologically and also by in vitro fertilization (IVF) and culture (IVC). Morphological integrity and developmental capacity were greater in S-oocytes than in V-oocytes (P $<$ 0.05). Transfer of four embryos (2 morulae and 2 blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in the birth of twin calves.
In the second series of experiments, oocytes (GV and IVM) were exposed to a cryoprotectant solution (DAP213: 2M DMSO, 1M acetamide, 3M propanediol) for 1.5 or 5 min and viability assessed by IVM-IVF-IVC. Oocytes were also examined by transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound premature cortical granule (CG) release and vesiculation. These changes were less pronounced in oocytes exposed to DAP213 only after IVM. The results suggest that: (1) the extrusion of CG is one of the important cytological events affected by the treatment of oocytes with DAP213; (2) GV oocytes are more sensitive to the cryoprotectant than IVM oocytes.
In the third series of experiments, GV and IVM oocytes were vitrified with DAP213. On rewarming, DAP213 was removed by a one- or three-step dilution procedure and survival assessed by development after IVM-IVF-IVC. Morphology was assessed by TEM study immediately following DAP213 removal. Both assessments indicated that: (1) IVM oocytes are more tolerant to vitrification than are GV oocytes; (2) the three-step dilution is less damaging than the one-step procedure; (3) changes in the zona pellucida (loss of plasticity) of IVM oocytes following vitrification may result from the premature release of cortical granules.
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Lawson, Alison N. "Cryopreservation effects on the in vitro and in vivo function of a model pancreatic substitute." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/39540.
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The effects of two types of cryopreservation, conventional freezing and vitrification, on the in vitro and in vivo function of a pancreatic substitute were investigated. Conventional freezing uses low concentrations of cryoprotective agents (CPAs), slow cooling and rapid warming and allows ice formation. Vitrification requires high concentrations of CPAs coupled with rapid cooling and warming to achieve a vitreous, or ice-free, state. A previously published mathematical model describing the mass transfer of CPAs through the alginate matrix of the substitute and the cell membrane was expanded to incorporate heat transfer as well as CPA cytotoxicity. Our results indicate that temperature of exposure is the most critical parameter for the proper design of CPA addition and removal protocols. The use of a mathematical model is critical to ensure CPA equilibration and minimize CPA exposure. Properly designed CPA addition and removal protocols were used for vitrification. The effects of cryopreservation on the biomaterial and the cellular function of a pancreatic substitute consisting of murine insulinomas encapsulated in calcium alginate/poly-L-lysine/alginate beads were assessed. In vitro results indicate that both vitrification and conventionally frozen perform comparably to fresh. However, in vivo studies reveal that vitrified beads perform worse than both conventionally frozen and fresh beads. With adjustments, it may be possible to improve the performance of the vitrified beads. Nevertheless, for this pancreatic substitute, conventional freezing is the better method and allows successful cryopreservation.
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Stott, Shannon Leigh. "Kinetic Study of Intracellular Ice Formation in Micropatterned Endothelial Cell Cultures Using High Speed Video Cryomicroscopy." Diss., Georgia Institute of Technology, 2006. http://hdl.handle.net/1853/16256.
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Intracellular ice formation (IIF), a major cause of cryoinjury in biological cells, is significantly more pronounced during freezing of tissue than during freezing of suspended cells. While extensive studies of IIF have been conducted for single cells in suspension, few have investigated IIF in tissue. Due to the increased complexity that arises from both cell-substrate and cell-cell interactions in tissue, knowledge of cryobiology of isolated cells cannot simply be extrapolated to tissue. Different theories have been hypothesized for the mechanisms of IIF in tissue, but none have been conclusively proven. Towards the goal of developing mathematical models to accurately predict the probability of IIF in tissues of one or more cell types, we have developed a novel high-speed video cryomicroscopy system capable of image acquisition at sampling rates up to 32,000 Hz. Specifically, the effects of cell adhesion to the substrate and cell-cell interactions were investigated with experimental (micropatterned endothelial cell constructs) and mathematical models (Monte Carlo simulations). We have reported the first direct observations of the IIF process recorded at unprecedented sub-millisecond and sub-micron resolution. For the majority of our experiments, IIF nucleation was determined to occur preferentially at the cell perimeter. This observation was not consistent with the commonly accepted hypotheses of ice nucleation in suspended cells and suggests that an alternative mechanism of IIF initiation is dominant in adherent cells. In addition, the kinetics of ice nucleation were shown to be influenced by time in culture, attached cell perimeter, fibronectin coating density, and degree of cell-cell contact. Moreover, an additional phenomenon, paracellular ice penetration was identified, and the frequency of formation was correlated with focal adhesion formation. The data and mathematical models presented in this thesis bring closer the goal of elucidating the primary mechanisms contributing to IIF in tissue; providing important contributions to both the fields of cryopreservation (minimizing IIF) and cryosurgery (maximizing IIF).
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Brossollet, Louis-Joseph. "Effects of cryopreservation on the biaxial mechanical properties of canine saphenous veins." Diss., Georgia Institute of Technology, 1992. http://hdl.handle.net/1853/19241.
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Webb, Veronica Fine Gross Guenter W. "Investigation of cryopreservation methods for adherent nerve cell networks in vitro." [Denton, Tex.] : University of North Texas, 2009. http://digital.library.unt.edu/ark:/67531/metadc12213.
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Sanchez, Daniel Andres. "The effects of cryopreservation on the viscoelastic properties of the canine anterior cruciate ligament." Thesis, Georgia Institute of Technology, 1988. http://hdl.handle.net/1853/19321.
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Salinas-Flores, Liliana, and n/a. "Understanding and improving the cryopreservation of pacific oyster (Crassostrea gigas) oocytes via the use of two approaches : modification of an existing cryopreservation protocol and manipulation of the lipis fraction of the oocytes." University of Otago. Department of Food Science, 2008. http://adt.otago.ac.nz./public/adt-NZDU20080305.143446.
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Cryopreservation of gametes is a valuable tool for the fast-growing aquaculture industry in New Zealand. In the present study, research was aimed to improve the cryopreservation of Pacific oyster (Crassostrea gigas) oocytes. For this, two main approaches were used: the modification of an existing published (standard) cryopreservation protocol for oyster oocytes and the modification of the oocytes themselves prior to cryopreservation. The objectives in the chapters of this thesis were: (a) determination of the cryobiological characteristics of oyster oocytes; (b) assessment and reduction of intracellular ice formation (IIF) in oocytes; and (c) modification of the lipid fraction (cholesterol and fatty acids) of oocytes prior to cryopreservation.
Knowledge of the membrane permeability parameters in response to concentrations of water and ethylene glycol (EG), the influence of temperature upon these parameters, and the osmotic tolerance limits of oyster oocytes were used to develop computer models that simulated the cellular volume changes that oocytes underwent during EG addition and removal. The models predicted that when one part of EG was added in one step to one part of oocyte suspension and equilibrated for 20 min at 20 �C, similar volume changes in oocytes would be obtained, compared to a more complicated multi-step addition method. This method of addition resulted in similar post-thaw fertilization rates to those obtained by using the multi-step addition method, thus reducing oocyte handling.
Cryomicroscopy was used to assess the effect of cooling rates and EG concentration on the temperature at which oocytes underwent IIF. It was found that IIF occurred at higher subzero temperatures when fast cooling rates were used (30 and 5 �C min⁻�) and at EG concentrations ranged between 0 and 15%. At a relatively slower cooling rate of 0.3 �C min⁻� and with 10% EG, which are the conditions employed in the standard cryopreservation protocol, no IIF occurred.
The steps of the standard protocol that were more likely to cause oocyte damage were identified by evaluating the fertilization rate of oocytes at each step. Results showed that oocytes were most damaged by cooling them to -35 �C and followed by plunging them in liquid nitrogen. Contrary to what had been observed under the cryomicroscope, transmission electron microscopy (TEM) analysis revealed that all oocytes cryopreserved by the standard protocol contained cytoplasmic ice. In addition, it was also observed that oocytes were at two developmental stages when frozen (prophase and metaphase I). These observations prompted the development of alternative cooling programmes aimed to reduce intracellular ice. The effect of cooling rate, plunge temperature and time held at the plunge temperature were thus evaluated, based on post-thaw fertilization rate of oocytes. Overall, neither the cooling rate nor the holding time had an effect on oocyte fertility. However, the plunge temperature had an effect, where oocytes plunged at -60 �C had lower post-thaw fertilization rates than oocytes plunged at -35 �C. Through the slowing of the cooling rate, lengthening of the holding time and lowering of the plunge temperature, it was possible to reduce the amount of ice in the cytoplasm. However, the reduction of intracellular ice did not improve the post-thaw fertilization rate of the oocytes; on the contrary, post-thaw fertilization decreased notoriously. From these results, it can be suggested that oyster oocytes are more likely to be damaged by exposure to high intra and extracellular solute concentration than IIF during cryopreservation.
In an effort to modify the lipid content of oyster oocytes prior to cryopreservation and thus, making them more resistant during cryopreservation, oocytes were incubated in solutions that would add or remove cholesterol or in solutions rich in long chain fatty acids (EPA or DHA). Oocytes incubated in cholesterol-rich solutions showed a positive uptake of fluorescently labelled cholesterol and this effect was dose dependent. Nevertheless, this uptake did not improve the post-thaw fertilization rate nor did it increase the total cholesterol content of the oocytes. When oocytes were incubated in non-conjugated or conjugated EPA or DHA, no increase in the proportion of these fatty acids was identified in the fatty acid profiles of whole oocytes and no improvement of the post-thaw fertilization rate was recorded.
Given that there was no uptake of fatty acids from the incubation media by the oocytes, a different approach was taken. This involved the supplementation of lipid-rich diets to the oyster broodstock during gametogenesis (cold-conditioning) and vitellogenesis (warm-conditioning). Despite results showing that lipid content and, indeed, fatty acid profile was altered through the diet, the results also showed that fresh oocytes from broodstock fed during cold-conditioning did not show any improvement in their fertilization rates, nor did they benefit from a lipid-rich diet during warm-conditioning. On the other hand, cryopreserved oocytes did have higher post-thaw fertilisation rates when broodstock were fed during cold-conditioning and, although no effect was found from feeding broodstock with either of the lipid-rich diets during warm-conditioning, trends indicated that a diet consisting of fresh microalgae or the commercial supplement Algamac would yield the highest post-thaw fertilization rates.
This thesis has furthered the understanding of some of the factors that determine cryosurvival in oyster oocytes and has demonstrated that both physical and biological issues must be taken into consideration for cryopreservation. Specifically, the results in this thesis helped to modify an empirically developed cryopreservation protocol for Pacific oyster oocytes. In addition, the results also showed strong evidence of the survival of oyster oocytes to intracellular ice and highlighted the importance of supplying the broodstock with lipid-rich food during the periods of gamete formation and maturation in order to obtain oocytes that are more amenable to cryopreservation. These benefits could be of significant practical importance and may be extended for the development or refinement of cryopreservation protocols for other shellfish species of commercial importance to the aquaculture industry of New Zealand.
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Bathgate, Roslyn. "Studies on the cryopreservation of boar spermatozoa and its integration into assisted reproductive technologies." Connect to full text, 2004. http://hdl.handle.net/2123/1279.
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Thesis (Ph. D.)--Faculty of Veterinary Science, University of Sydney, 2005.Title from title screen (viewed 13 January 2009). Degree awarded 2005; thesis submitted 2004. Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Veterinary Science. Includes bibliographical references. Also available in print form.
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Webb, Veronica Fine. "Investigation of cryopreservation methods for adherent nerve cell networks in vitro." Thesis, University of North Texas, 2009. https://digital.library.unt.edu/ark:/67531/metadc12213/.
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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.
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Ahmad, Hajira Fatima. "Cryopreservation effects on a pancreatic substitute comprised of beta cells or recombinant myoblasts encapsulated in non-adhesive and adhesive alginate hydrogels." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/48968.
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For clinical translation of a pancreatic substitute, long-term storage is essential, and cryopreservation is a promising means to achieve this goal. The two main cryopreservation methods are conventional freezing and vitrification, or ice-free cryopreservation. However, as both methods have their potential drawbacks for cryopreservation of a pancreatic substitute, they must be systematically evaluated in order to determine the appropriate method of cryopreservation. Furthermore, previous studies have indicated benefits to encapsulation in 3-D adhesive environments for pancreatic substitutes and that adhesion affects cell response to cryopreservation. Thus, the overall goal of this thesis was to investigate cryopreservation effects on model pancreatic substitutes consisting of cells encapsulated in non-adhesive and adhesive 3-D alginate hydrogels. Murine insulinoma betaTC-tet cells encapsulated in unmodified alginate hydrogels were chosen as the model pancreatic substitute in a non-adhesive 3-D environment. Murine myoblast C2C12 cells, stably transfected to secrete insulin, encapsulated in partially oxidized, RGD-modified alginate hydrogels were chosen as the model pancreatic substitute in a 3-D adhesive environment. With respect to cryopreservation effects on intermediary metabolism of betaTC-tet cells encapsulated in unmodified alginate, results indicate that relative carbon flow through the tricarboxylic acid cycle pathways examined is unaffected by cryopreservation. Additionally, insulin secretory function is maintained in Frozen constructs. However, vitrification by a cryopreservation cocktail referred to as DPS causes impairment in insulin secretion from encapsulated betaTC-tet cells, possibly due to a defect in late-stage insulin secretion. Results from Stable C2C12 cells encapsulated in RGD vs. RGE-alginate indicate that up to one day post-warming, cell-matrix interactions do not affect cellular response to cryopreservation after vitrification or freezing. Although there are differences in metabolic activity and insulin secretion immediately post-warming for DPS-vitrified RGD-encapsulated Stable C2C12 cells relative to Fresh controls, metabolic activity and insulin secretion are maintained at all time points assayed for Frozen constructs. Overall, due to results comparable to Fresh controls and simplicity of procedure, conventional freezing is appropriate for cryopreservation of betaTC-tet cells encapsulated in unmodified alginate or Stable C2C12 cells encapsulated in partially oxidized, RGD-modified alginate.
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Letsoalo, Phutiane Thomas. "Characterisation and cryopreservation of semen from indigenous Namaqua Afrikaner sheep breed, in comparison with Dorper and Dohne Merino breeds." Thesis, University of Fort Hare, 2017. http://hdl.handle.net/10353/4759.
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The aim of this study was to characterise and cryopreserve semen of the indigenous Namaqua Afrikaner breed, and to compare it to that of Dorper and Dohne Merino sheep, whose semen is commercially frozen on a large scale. The study was conducted between January and August 2015. September 2013-born Namaqua Afrikaner (12), Dohne Merino (12) and Dorper (9) rams were used in the study. The rams were kept under kraal conditions with adequate shade, and they received a high protein, high energy diet. Originally it was envisaged to collect semen samples using the artificial vagina (AV) method, which proved to be problematic with the Namaqua Afrikaner rams. Semen samples were subsequently collected twice a week by either AV (Dohne Merino and Dorper) or electro-ejaculation (EE; all three breeds). Macroscopic sperm traits were assessed and sperm concentration determined immediately after collection. Each semen sample was diluted with Triladyl® (1:3) and subsequently frozen in liquid nitrogen vapour in straws. Frozen straws were thawed and evaluated at 7, 30 and 90 days after cryopreservation. A droplet (0.5 ml) from each thawed sample was assessed microscopically for post-thaw motility and percentage live sperm..
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Blais, Louis. "The evaluation of spermatozoal damage done at each step of the cryopreservation procedure from a line of chicken selected for high fertility, of frozen-thawed semen and a random, bred control line /." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61847.
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Chan, Chun-wai. "In-vitro study of the cryopreserved intervertebral disc." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41290380.
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Maceda, Tintaya Edwin Eddy. "Effect of three cryoconservation diluents on sperm motility and vitality in the ejaculate of bulbourethal-ectomized llamas (Lama glama), department of La Paz." BYU ScholarsArchive, 2008. https://scholarsarchive.byu.edu/etd/5391.
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The purpose of this study was to evaluate the effects of three different diluents used during the cryopreservation process on the motility and vitality of sperm cells. The three diluters used in this study were: A) trice-serum-egg yolk-glycerin, B) serum-egg yolk-glycerin, and C) Dulbecco’s serum-egg-yolk- glycerin. Diluters were tested in proportions of 64-15-15-6% (N1), 54-20-20-6% (N2), and 44-35-15-6% (N3). Llama semen was collected at the Mejoramiento Genético y Diagnóstico Clínico Del Servicio Agropecuario (SEDAG) in the Los Andes Province of the Department of La Paz. The procedure took place at the Unidad Académica Campesina de Tiahuanaco by a direct optimized bulbourethral collection method with an artificial vagina.
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Chan, Chun-wai, and 陳春慧. "In-vitro study of the cryopreserved intervertebral disc." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41290380.
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Pustowka, Cory V. "Effects of source of dietary lipid on the fatty acid composition and cholesterol content of tissues, semen cryopreservation and embryo survival in rainbow trout (Oncorhynchus mykiss)." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ35463.pdf.
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Yap, Cheng-Hon. "Factors influencing cryopreserved allograft heart valve degeneration." Connect to thesis, 2006. http://repository.unimelb.edu.au/10187/2120.
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Heart valve replacement is becoming more commonplace in developed nations. Despite this the ideal valve prosthesis has not been found. The allograft valve has been used for over 40 years and remains an important prosthesis with many advantages. However, like other biological valve prosthesis, they have a finite durability. The causes of allograft valve degeneration are still unknown. The study aims to identify factors associated with cryopreserved allograft valve degeneration. Knowledge of such factors will improve our understanding of the potential causes and mechanisms of allograft heart valve degeneration. (For complete abstract open document)
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Sumpter, Megan Louise. "Johnson-Mehl-Avrami Kinetics of Intracellular Ice Formation in Confluent Tissue Constructs." Thesis, Georgia Institute of Technology, 2004. http://hdl.handle.net/1853/7263.
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In an effort to minimize the harmful effects of intracellular ice formation (IIF) during cryopreservation of confluent tissues, computer simulations based on Monte Carlo methods were performed to predict the probability of IIF in confluent monolayers during various freezing procedures. To overcome the prohibitive computational costs of such simulations for large tissues, the well-known Johnson-Mehl-Avrami (JMA) model of crystallization kinetics was implemented as a continuum approximation of IIF in tissues. This model, which describes nucleation, growth, and impingement of crystals in a supercooled melt, is analogous to the process of intracellular ice formation and propagation in biological tissues. Based on the work of Weinberg and Kapral (1989), the JMA model was modified to account for finite-size effects, and was shown to predict accurately the results of freezing simulations in 1-D tissue constructs, for various propagation rates and tissue sizes. An initial analysis of IIF kinetics in 2-D tissues is also presented. The probability of IIF in 2-D liver tissue was measured experimentally during freezing of HepG2 cells cultured in monolayers, and compared to Monte Carlo simulations and predictions of the continuum model. The Avrami coefficient and exponent for IIF in HepG2 tissue were estimated to be k = 0.19 and n = 0.45.
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Fasano, Giovanna. "Contribution of vitrification to human assisted reproduction." Doctoral thesis, Universite Libre de Bruxelles, 2013. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209484.
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La cryopréservation, dans le domaine de la reproduction médicalement assistée, constitue depuis de nombreuses années une branche suscitant beaucoup d’intérêts et d’espoirs. En effet, de nombreuses équipes de recherche se sont attelées à mettre au point et à améliorer des protocoles permettant de conserver les gamètes, les embryons et les tissus reproducteurs.Malgré le fait que la cryopréservation soit une technique très attractive, elle peut avoir des effets délétères sur les cellules. Les protocoles expérimentaux visent donc à minimiser ces effets afin d’augmenter la survie et la compétence cellulaire après décongélation.
Les deux méthodes les plus utilisées, la congélation lente et la vitrification, présentent chacune des avantages et des inconvénients. En effet, la première ne permet pas d’éliminer la cristallisation intracellulaire. Quant à la seconde, elle empêche la formation de cristaux de glace mais pourrait provoquer une toxicité due à la forte concentration des cryoprotecteurs.
Cette thèse de doctorat propose plusieurs objectifs :
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Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished
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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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Turner, Shane. "Cryopreservation of somatic germplasm of selected Australian monocotyledonous taxa (Haemodoraceae)." Thesis, Curtin University, 2001. http://hdl.handle.net/20.500.11937/1409.
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The South West Botanical Province of Western Australia is one of the most floristically rich areas of the world with over, 8,000 species present, the majority of which (70%), are endemic to this region. Coupled with this high level of endemism, many taxa are threatened which makes them vulnerable to habitat alterations, modifications and destruction. Significant habitat alteration in many areas has resulted in 27 species becoming extinct in the South West Botanical Province, while an additional 327 species are classified as rare and endangered. In the context of stemming this loss of biodiversity, research in cryopreservation was undertaken to provide offsite protection and conservation of somatic germplasm.Cryostorage techniques were evaluated in this study to determine the key factors which may affect the ability of somatic tissues of Haemodoraceae species to survive, recover and grow following liquid nitrogen (LN) immersion and storage. Using Anigozanthos viridis as a comparator in most experiments, the base vitrification protocol was established, which involved: (1) preculturing shoot apices on 0.4 M sorbitol for 48 h; (2) incubation in a vitrification cryoprotective solution (PVS2) for 25 min at 0 degrees celsius; (3) LN immersion; (4) recovery to active growth through warming (immersion in a 40 degrees celsius water bath). Using this procedure the highest post-LN survival of shoot apices for A. viridis was 41.4 plus or minus 6.1% Four additional taxa were successfully cryopreserved with this base protocol (Anigozanthos manglesii, A. rufus, Conostylis wonganensis and A. rufus x A. pulcherrimus); a fifth taxon, Macropidia fuliginosa, however, proved unresponsive.To improve on post-LN survival, further research established that four of the six study taxa responded to the following amendments to the basic protocol: (1) longer preculture duration; (2) preculture on 0.8 M glycerol rather than sorbitol, (3) utilisation of PVS2 solutions with reduced DMSO content; and (4) incorporation of an additional loading phase (2 M glycerol plus 0.4 M sucrose for 20 mins at 0 degrees celsius).Macropidia fuliginosa, a species with poor recovery after LN exposure, was successfully cryostored using somatic embryos. Treatments which resulted in the highest survival (67.3% 5.7 plus or minus %) included preculture with 0.4 M sorbitol, and incubation in PVS2 for 30 min. Further experimentation indicated that preculture for two days on 0.8 M glycerol (replacing 0.4 M sorbitol) was more beneficial for achieving high post-LN survival.Post-LN survival was significantly correlated to the use of polyalcohols when the total number of hydroxyl (-OH) groups (regardless of molarity) present was the same as that found in 0.4 M sorbitol. It was hypothesised that hydroxyl number is more important than molarity in membrane stabilisation, during dehydration and cooling. Post-LN survival was also found to be significantly influenced by stereochemical arrangement of the -OH groups of polyalcohol molecules used in the preculture media. Finally, post-LN survival was also found to be significantly influenced by the size of the molecule, with smaller polyalcohols with more -OH groups on one flank of the carbon chain being superior as cryoprotective agents.The influence of plant growth regulators on post-LN survival and recovery growth was also investigated. The survival of shoot apices was not correlated to cytokinin or auxin treatments administered in culture media prior to cryostorage. However, in the recovery medium, a combination of cytokinin and 0.5 mu M GA(subscript)3 in the medium was found to be the most efficacious for obtaining healthy plantlets.Genetic fidelity was then examined using Amplified Fragment Length Polymorphism (AFLP). Plantlets of one done kept or maintained under the following conditions: (1) standard tissue culture conditions; (2) cold storage and (3) cryostorage, over a 12 month duration, showed no detectable genetic changes.Further, shoot apex viability evaluated at regular intervals (after 0, 3, 6 and 12 months of LN storage) suggested that medium term storage of samples cryopreservation did not reduce shoot apex viability over this time span.This study has provided a better understanding of the factors influencing post-LN survival and recovery and, as a result, the cryopreservation protocols have been refined. Consequently, the prospects for conserving threatened Haemodoraceae species from Western Australia through cryostorage is now significantly improved.
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Unhale, Sanket Anil. "Cryobiology of Cell and Tissue Cryopreservation: Experimental and Theoretical Analysis." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/202974.
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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.
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Turner, Shane. "Cryopreservation of somatic germplasm of selected Australian monocotyledonous taxa (Haemodoraceae)." Curtin University of Technology, Department of Environmental Biology, 2001. http://espace.library.curtin.edu.au:80/R/?func=dbin-jump-full&object_id=12678.
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The South West Botanical Province of Western Australia is one of the most floristically rich areas of the world with over, 8,000 species present, the majority of which (70%), are endemic to this region. Coupled with this high level of endemism, many taxa are threatened which makes them vulnerable to habitat alterations, modifications and destruction. Significant habitat alteration in many areas has resulted in 27 species becoming extinct in the South West Botanical Province, while an additional 327 species are classified as rare and endangered. In the context of stemming this loss of biodiversity, research in cryopreservation was undertaken to provide offsite protection and conservation of somatic germplasm.Cryostorage techniques were evaluated in this study to determine the key factors which may affect the ability of somatic tissues of Haemodoraceae species to survive, recover and grow following liquid nitrogen (LN) immersion and storage. Using Anigozanthos viridis as a comparator in most experiments, the base vitrification protocol was established, which involved: (1) preculturing shoot apices on 0.4 M sorbitol for 48 h; (2) incubation in a vitrification cryoprotective solution (PVS2) for 25 min at 0 degrees celsius; (3) LN immersion; (4) recovery to active growth through warming (immersion in a 40 degrees celsius water bath). Using this procedure the highest post-LN survival of shoot apices for A. viridis was 41.4 plus or minus 6.1% Four additional taxa were successfully cryopreserved with this base protocol (Anigozanthos manglesii, A. rufus, Conostylis wonganensis and A. rufus x A. pulcherrimus); a fifth taxon, Macropidia fuliginosa, however, proved unresponsive.To improve on post-LN survival, further research established that four of the six study taxa responded to the following amendments to the basic protocol: (1) longer preculture duration; (2) preculture on ++0.8 M glycerol rather than sorbitol, (3) utilisation of PVS2 solutions with reduced DMSO content; and (4) incorporation of an additional loading phase (2 M glycerol plus 0.4 M sucrose for 20 mins at 0 degrees celsius).Macropidia fuliginosa, a species with poor recovery after LN exposure, was successfully cryostored using somatic embryos. Treatments which resulted in the highest survival (67.3% 5.7 plus or minus %) included preculture with 0.4 M sorbitol, and incubation in PVS2 for 30 min. Further experimentation indicated that preculture for two days on 0.8 M glycerol (replacing 0.4 M sorbitol) was more beneficial for achieving high post-LN survival.Post-LN survival was significantly correlated to the use of polyalcohols when the total number of hydroxyl (-OH) groups (regardless of molarity) present was the same as that found in 0.4 M sorbitol. It was hypothesised that hydroxyl number is more important than molarity in membrane stabilisation, during dehydration and cooling. Post-LN survival was also found to be significantly influenced by stereochemical arrangement of the -OH groups of polyalcohol molecules used in the preculture media. Finally, post-LN survival was also found to be significantly influenced by the size of the molecule, with smaller polyalcohols with more -OH groups on one flank of the carbon chain being superior as cryoprotective agents.The influence of plant growth regulators on post-LN survival and recovery growth was also investigated. The survival of shoot apices was not correlated to cytokinin or auxin treatments administered in culture media prior to cryostorage. However, in the recovery medium, a combination of cytokinin and 0.5 mu M GA(subscript)3 in the medium was found to be the most efficacious for obtaining healthy plantlets.Genetic fidelity was then examined using Amplified Fragment Length Polymorphism (AFLP). Plantlets of one done kept ++
or maintained under the following conditions: (1) standard tissue culture conditions; (2) cold storage and (3) cryostorage, over a 12 month duration, showed no detectable genetic changes.Further, shoot apex viability evaluated at regular intervals (after 0, 3, 6 and 12 months of LN storage) suggested that medium term storage of samples cryopreservation did not reduce shoot apex viability over this time span.This study has provided a better understanding of the factors influencing post-LN survival and recovery and, as a result, the cryopreservation protocols have been refined. Consequently, the prospects for conserving threatened Haemodoraceae species from Western Australia through cryostorage is now significantly improved.
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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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Liu, Jianan. "Cryopreservation and transplantation of ovarian tissue in Japanese quail (Coturnix japonica)." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/17424.
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Cryopreservation of avian genetic material is limited in terms of practicality, efficiency, and success. The complex structure of female gametes in birds makes the application of cryopreservation extremely difficult, and the fertilization obtained from frozen/thawed poultry semen is low and unpredictable. Embryonic germ cells can be frozen and used to generate germline chimeras, but the low efficiency and complex procedures limit these techniques for genetic conservation.
The techniques of transplantation of ovaries and testes in newly hatched chicks have recently been developed and donor-derived offspring could be efficiently produced from cryopreserved testicular transplants and from fresh ovarian tissue transplants in chicken. This success in chicken gonadal transplantation provides an effective method for recuperating live birds from cryopreserved gonadal tissue. Research efforts are needed to develop cryopreservation technique for ovarian tissues and further apply cryopreservation and transplantation to the other avian species.
The Japanese quail is a much smaller bird than the chicken and has a slightly different reproductive biology. Preliminary research has demonstrated that live offspring could be obtained from fresh ovarian grafts in one-week old quail. Using the Japanese quail as a model, the objective of this thesis is to develop a feasible and reliable cryopreservation technique for ovarian tissue and to recover it by subsequent transplantation, with the hope that this protocol of cryopreservation and transplantation can attribute to female germplasm conservation for other avian species in the future.
Both slow-freezing and vitrification were used as cryopreservation approaches in this study. The efficiency was evaluated at three levels: cell viability, tissue histology and the recovery of the donor fertilities after surgical transplantation. Donor-derived offspring were obtained from the ovarian tissues that had been cryopreserved by either slow-freezing or vitrification. The vitrification protocol used in this study showed better outcomes at each level of evaluation. Thus the current study verified that the function of ovarian tissue in an avian species can be successfully preserved at subzero temperatures and recovered by transplantation. The vitrification protocol is recommended for future use concerning its low cost, high efficiency and overall simplicity in optimization to benefit more avian species.
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Al-Forkan, Mohammad. "Agrobacterium-mediated transformation of Indica rice (Oryza sativa L.)." Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342480.
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Zhang, Pu. "Human ovarian follicles and oocytes : collection, cryopreservation, culture and gene expression /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-281-0/.
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Charles, Lara Nicole. "Culture of cells from mammalian tissue cryopreserved without cryoprotection." [College Station, Tex. : Texas A&M University, 2008. http://hdl.handle.net/1969.1/ETD-TAMU-2819.
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Liu, Jianan. "Cryopreservation and transplantation of gonadal tissue for genetic conservation and biological research in avian species." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/45101.
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Avian researchers and the poultry industry have experienced a massive loss of genetic resources due to the high cost of maintaining live flocks. Cryobanking of germplasm is economical and ensures long-term availability of genetic resources. Cryopreservation and transplantation of avian gonadal tissue allow effective preservation and recovery of female germplasm and provide an alternative to semen cryobanking. A vitrification protocol including dimethyl sulphoxide, ethylene glycol and sucrose as cryoprotective agents and the use of acupuncture needles to facilitate tissue handling has been successful in preserving Japanese quail ovarian tissue. Its effectiveness in preserving testicular tissue is unknown and an efficient storage system is needed.
The vitrification protocol was first adapted to a 0.5-ml straw system and used to cryopreserve quail ovarian tissue, which showed no significant impairment of viability or vascularization compared to fresh tissue after in ovo culture. This system was then replaced by a simpler 2-ml straw system and applied to quail testicular and ovarian tissue. Normal morphology of testicular tissue was observed after in ovo culture and live offspring were produced by intramagnal insemination of the extrusion retrieved from allotransplants of cryopreserved testicular tissue. Donor-derived offspring were also efficiently produced from cryopreserved and allotransplanted ovarian tissue.
Gonadal transplantation is critical to functional recovery of cryopreserved tissue but can be limited by tissue rejection. Donor thymic tissue was implanted into recipient embryos and gonadal tissue from the same donor was transplanted ectopically to the recipient after hatching. Transplant viability and histology was examined. Thymic implantation may improve survival of the allogeneic gonadal transplants in chickens but not the xenotransplants from quail to chickens. Investigations of avian ovarian transplantation have led to intriguing additional observations. Donor-derived offspring were produced from transplanted adult quail ovarian tissue, although delayed age at first egg and reduced reproductive longevity were observed with the transplants. As well, offspring with chimeric plumage coloration were produced from cryopreserved and transplanted chicken ovarian tissue, indicating chimeric folliculogenesis.
This dissertation provides a model of cryobanking avian gonadal tissue using a simple vitrification method, and suggests future directions in improving transplantation tolerance and using gonadal transplantation in avian research.
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Anil, Siji. "Development of in-vitro culture and cryopreservation protocol for zebrafish (Danio rerio) ovarian tissue fragments." Thesis, University of Bedfordshire, 2013. http://hdl.handle.net/10547/308923.
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Cryopreservation of fish ovarian tissue fragments can be a viable alternative to cryopreservation of oocytes and embryos. The ability to cryopreserve both maternal and paternal gametes would provide a reliable source of fish genetic material for scientific and aquaculture purposes. The main aim of the present study was to develop an in-vitro culture protocol and cryopreservation protocol for zebrafish ovarian tissue fragments. In-vitro culture protocol for the tissue fragments containing stage I and stage II follicles were developed and the growth assessment of follicles were evaluated using biomarkers. To develop the cryopreservation protocol using control slow cooling method, the effect on freezing medium, cryoprotectants and cooling rate on the tissue fragments were investigated. The in-vitro culture experiments showed that L-15 medium (pH 9) containing 100mIU/ml FSH along with 20% FBS was effective for tissue fragments containing stage I and II follicles to grow in-vitro. The growth of the ovarian follicle stages was confirmed by the level of expression of p450aromA and vtg1 gene. The optimal cryopreservation protocol for the ovarian tissue fragments was found as 2M methanol+ 20%FBS in 90% L-15 medium with the cooling rate of 4°C/min. Although the highest survival rate obtained for stage II follicles within the fragments was 68.2±1.9% and stage I follicles within the fragments was 55.4±2.3% using TB staining, it showed a significant decrease in their ATP levels. This is the first study carried out on the zebrafish ovarian tissue fragments. Study on cryopreservation of the ovarian tissue fragments and development of the in-vitro culture protocol and use of biomarkers for the ovarian tissue fragments were reported here for the first time. The outcomes of this study have provided useful information for future cryopreservation protocol development.
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Huang, Haishui. "Microfluidic Generation and Manipulation of Hydrogel Microcapsules for Biomimetic 3D Tissue Culture and Cell Cryopreservation." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1461095928.
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Kaczmarczyk, Anja. "Physiological, biochemical, histological and ultrastructural aspects of cryopreservation in meristematic tissue of potato shoot tips." Berlin Köster, 2008. http://d-nb.info/991906160/04.
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Carvalho, Allan Charles Marques de. "Criopreservação de sêmen de tambaqui Colossoma macropomum em criotubo." Pós-Graduação em Zootecnia, 2013. https://ri.ufs.br/handle/riufs/6391.
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The semen cryopreservation in cryotubes reduces the time needed for filling, freezing and thawing of the samples, while optimizing the procedures for artificial fertilization. However, no study has yet been performed with tambaqui semen in this container. The aim of this study was to evaluate the influence of cryotubes (1.6 and 4.5 mL) and thawing time (60ºC/70s and 60ºC/90 s) on the quality and fertility of tambaqui cryopreserved semen. For that, semen samples were diluted in freezing solution (1:9) composed with 75% glucose 290 mOsm , 10% methylglycol and 5% egg yolk, and frozen in liquid nitrogen vapor (in a dry shipper), and stored in liquid nitrogen (-196 °C). The semen samples was thawed at 60ºC in bath water during 70s or 90s and the semen quality was evaluated (Total motility - MT; Progressive motility - MP; Curvilinear velocity - VCL; Straight-line velocity - VSL and Average path velocity - VAP). In this study was also determined the time viability of spermatozoa thawed, maintained under refrigeration at 5°C, and assessed over 24 hours. Besides the kinetic parameters were evaluated sperm morphology and membrane integrity of spermatozoa. The fertilizing capacity of semen was evaluated from the samples thawed in the best time. All parameters of sperm kinetic showed higher values when the semen samples were thawed in 90 s compared to 70 s, independently of cryotube type. No significant differences were observed in sperm kinetic parameters after thawing between samples frozen in 1.6 ml and 4.5 mL cryotubes, except for total motility that was higher in 1.6 mL (47 ± 14% ) compared with 4.5 mL cryotubes (40 ± 11%), independently of thawing time. After activation, the spermatozoa significantly reduced the values of kinetic parameters within 37 seconds, except the MT which remained constant during this period. Relying on the sperm parameters evaluated (VCL, VSL, VAP and membrane integrity for both cryotubes and MT, MP and morphology only for 1.6 mL cryotube) the frozen semen maintained the quality during 3h after thawing. The fertilization rate obtained with fresh semen (74±6%) was higher than cryopreserved semen (1.6 mL - 45±9% and 4.5 mL - 41±12%). The two cryotubes did not differ in this parameter. A high correlation (p <0.05) was observed between fertility and sperm kinetics parameters (MT - 89%, MP - 86%; VCL - 79%; VSL - APV and 69% - 78%). It is concluded that 1.6 and 4.5 mL cryotubes can be used in the cryopreservation of tambaqui semen being recommended to be thawed at 60°C for 90s and their use in fertilization procedures within 3 hours after thawing since kept at 5°C.A criopreservação de sêmen em criotubos reduz o tempo necessário para o envase, congelamento e descongelamento das amostras, além de otimizar os procedimentos de fertilização artificial. No entanto, nenhum estudo ainda foi realizado com o sêmen de tambaqui neste recipiente. Assim, o objetivo do presente trabalho foi avaliar a influência do tipo de criotubo (1,6 e 4,5 mL) e do tempo de descongelamento (60ºC/70s e 60ºC/90s) sobre a qualidade e fertilidade do sêmen de tambaqui criopreservado. Para isso, amostras de sêmen foram diluídas em solução de congelamento (1:9 v/v) composta por 75% de glicose 290 mOsm, 10% de metilglicol e 5% de gema de ovo, sendo envasadas em criotubos de 1,6 e 4,5 mL, congeladas em vapor de nitrogênio líquido no botijão dry-shipper (-175ºC) e armazenadas em botijão criogênico a -196°C. Para avaliação do tempo de descongelamento do sêmen, os criotubos foram imersas em água a 60°C durante 70 s ou 90 s e a qualidade espermática imediatamente avaliada (Motilidade total - MT; Motilidade progressiva - MP; Velocidade curvilinear - VCL; Velocidade em linha reta - VSL e Velocidade média da trajetória - VAP). Neste estudo foi determinado também o tempo de viabilidade dos espermatozoides descongelados, mantidos sob refrigeração a 5°C e avaliados durante 24 horas. Além dos parâmetros de cinética espermática foram avaliadas a morfologia e a integridade da membrana plasmática dos espermatozoides. A capacidade de fertilização do sêmen foi avaliada a partir das amostras descongeladas no melhor tempo. Todos os parâmetros de cinética espermática apresentaram valores superiores quando as amostras de sêmen foram descongeladas por 90s em relação ao tempo de 70s, independentemente do tipo de criotubo. Não foram observadas diferenças significativas nos parâmetros de cinética espermática pós-descongelamento entre as amostras congeladas nos criotubos de 1,6 e 4,5 mL, com exceção da MT que foi superior nos criotubos de 1,6 mL (47±14%) em comparação com os criotubos de 4,5 mL (40±11%), independentemente do tempo de descongelamento. Após ativação, os espermatozoides reduziram significativamente os valores dos parâmetros de cinética dentro de 37 segundos, exceto a MT que se manteve constante neste período. Baseando-se na maior parte dos parâmetros espermáticos avaliados (VCL, VSL, VAP e Integridade da membrana plásmatica para ambos os criotubos e MT, MP e Morfologia espermática somente para o criotubo de 1,6 mL) o sêmen congelado manteve sua qualidade durante 3h após o descongelamento. A taxa de fertilização obtida com o sêmen in natura (74±6%) foi superior ao sêmen criopreservado (1,6 mL - 45±9% e 4,5 mL - 41±12%). Os dois criotubos não diferiram entre si neste parâmetro. Uma alta correlação significativa (p<0,05) foi observada entre a fertilização e a cinética espermática (MT - 89%; MP - 86%; VCL - 79%; VSL - 69% e VAP - 78%). Conclui-se que os criotubos de 1,6 e 4,5 mL podem ser utilizados na criopreservação do sêmen de tambaqui, sendo recomendado seu descongelamento a 60°C por 90s e seu uso em procedimentos de fertilização dentro de 3 horas após o descongelamento desde que mantido a 5°C.
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Faria, Surian Guerios. "Estabelecimento e caracterização de um banco de células de trabalho para produção da enzima Taq DNA polimerase." Universidade Tecnológica Federal do Paraná, 2017. http://repositorio.utfpr.edu.br/jspui/handle/1/2729.
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O Instituto de Biologia Molecular do Paraná (IBMP) produz kits de diagnóstico para o Sistema Único de Saúde (SUS), que consistem em testes moleculares, realizados por reação em cadeia da polimerase (polymerase chain reaction - PCR). Essa reação ocorre pela atividade da enzima Taq DNA Polimerase (Taq). O IBMP fabrica a Taq a partir do cultivo de células bacterianas, em conformidade às Boas Práticas de Fabricação (BPF) e pretende produzi-la a partir de um banco de células provindas de um mesmo clone visando o aumento da homogeneidade e reprodutibilidade do processo produtivo. O objetivo do presente trabalho é estabelecer um banco de células de trabalho (BCT) e avaliar sua estabilidade para a produção da enzima Taq, partindo de um banco de células mestre (BCM) estabelecido em Bio-Manguinhos. O estabelecimento do BCT consiste em cultivar colônias do BCM, caracterizá-las, avaliar desempenho, e eleger células adequadas para compor o BCT. O estudo da estabilidade contempla testes para comprovar que as células retêm a capacidade de produzir a Taq ao longo do tempo (i.e., viabilidade celular, estabilidade plasmídica, cinética de crescimento, indução da expressão, extração e dosagem do DNA plasmidial, análise de restrição e avaliação plasmidial). O critério decisivo para a seleção de um dos clones para compor o BCT foi o resultado da cinética de crescimento. A estabilidade foi estudada em 10 avaliações realizadas mês a mês. Nelas, a viabilidade celular foi superior a 1,0 x 106 UFC/mL, mostrando que as células permanecem com capacidade de realizar seu metabolismo e reprodução. O crescimento até a fase exponencial se deu entre 6 e 7 horas de cultivo, com taxa específica de crescimento superior a 0,4 min-1 . Os cultivos acrescidos de 1 mM de IPTG expressaram a enzima Taq DNA Polimerase, revelando a qualificação das células para o fim proposto. A estabilidade plasmídica foi superior a 90%, indicando que o plasmídeo pBioMTaq se mantém estável e com boa capacidade de replicação. A concentração plasmidial média foi de 338 ng/μL, e em todos os meses foi maior que 100 ng/μL. Na análise de restrição os plasmídeos foram corretamente clivados e na avaliação plasmidial houve adequada amplificação do fragmento de 500 pb, demonstrando que o plasmídeo se mantém íntegro e estável, com sequências compatíveis com sua construção. O BCT foi testado como substrato para um processo fabril típico do IBMP de produção da Taq DNA polimerase, se apresentando satisfatório em todas as etapas em que foi avaliado. Esses resultados indicam que o banco estabelecido se manteve estável ao longo de 10 meses e está apto a ser utilizado como protótipo para um BCT em conformidade às BPF.The Molecular Biology Institute of Paraná (Instituto de Biologia Molecular do Paraná - IBMP) produces diagnostic kits for the Brazilian Unified National Health System (Sistema Único de Saúde - SUS), that consist in molecular tests, carried out by polymerase chain reaction (PCR). This reaction is performed by the enzyme Taq DNA Polymerase (Taq). The IBMP manufactures Taq from bacterial cell culture in accordance with Good Manufacturing Practices (GMP) and intends to produce it from a cell bank using cells from the same clone in order to increase the homogeneity and reproducibility of the production process. The objective of the present work is to establish a working cell bank (WCB) and to evaluate its stability for the production of the Taq enzyme, starting from a master cell bank (MCB) established in BioManguinhos. The WCB establishment consists of cultivating MCB colonies, characterizing them, evaluating performance, and electing proper cells to compose the WCT. The stability study contemplates tests to prove that cells retain the ability to produce Taq over time (i.e., cell viability, plasmid stability, growth kinetics, expression induction, plasmid DNA extraction and dosage, restriction analysis and plasmid evaluation). The decisive discretion for the selection of one of the clones to compose BCT was the result of growth kinetics. Stability was studied in 10 month-to-month evaluations. In them, cell viability was higher than 1,0 x 106 CFU/mL, showing that the cells remain capable of performing their metabolism and reproduction. Growth to the exponential phase occurred between 6 and 7 hours of culture, with a specific growth rate greater than 0.4 min-1 . The cultures with 1 mM IPTG expressed Taq DNA polymerase enzyme, revealing the qualification of the cells to the intended purpose. The plasmid stability was greater than 90%, indicating that the plasmid pBioMTaq remains stable and has good replication ability. The mean plasmid concentration was 338 ng/μL, and in all months it was greater than 100 ng/μL. In the restriction analysis the plasmids were correctly cleaved and in the plasmid evaluation there was adequate amplification of the 500 bp fragment, demonstrating that the plasmid remains intact and stable, with compatible sequences with its construction. The WCB was tested as substrate for a typical IBMP production process of Taq DNA polymerase, presenting satisfactory in all the stages in which it was evaluated. These results indicate that the established bank remained stable over 10 months and is apt to be used as a prototype for a WCB in compliance with GMP.
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Philpott, Megan. "The Genetic Consequences of ex situ Conservation of Exceptional Plant Species." University of Cincinnati / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1535467395352645.
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Giovani, Arlete Mazzini Miranda. "Estudo comparativo entre o tecido ósseo criopreservado e o conservado em glicerol a 98%." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/5/5140/tde-20082007-130551/.
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O Banco de Tecidos do Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo está desenvolvendo uma linha de estudos experimentais com o intuito de apresentar novos métodos de armazenamento de aloenxertos. Este estudo tem como objetivo comparar o método da criopreservação (- 80° C) com o da conservação em glicerol a 98% em temperatura ambiente. Foram analisadas tanto a capacidade de inibição de crescimento bacteriano e fúngico quanto às eventuais alterações histológicas. Para isso, foram estudadas 30 amostras de tecido ósseo trabecular coletadas de 10 pacientes submetidos à artroplastia total do quadril. Estas amostras foram divididas em três grupos de 10 espécimes, a saber: grupo controle, grupo criopreservado e grupo conservado em glicerol a 98% à temperatura ambiente. O período de armazenamento das amostras foi de um ano. Os resultados foram analisados estatisticamente pelo método de McNemar, com índice de significância de 0,10. No que diz respeito à preservação da matriz óssea, não houve variações significativas nos dois grupos estudados em relação ao grupo controle. Não ocorreu crescimento bacteriano ou de fungos nas amostras armazenadas durante um ano em glicerol a 98%. Por ser extremamente reduzido em relação à criopreservação, o custo do método da conservação em glicerol a 98% o indica para uso em Bancos de Tecidos de pequena monta. Contudo, são ainda necessários posteriores estudos sobre as propriedades biomecânicas, remoção do glicerol do tecido ósseo e o processo de integração biológica dos mesmos com o receptor.The Tissue Bank of Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da Universidade de São Paulo is developing a line of experimental studies with the intention to present new methods of allografts storage. This study has the objective to compare the method of cryopreservation (-80 ºC) with glycerol 98% preservation at room temperature. It even analyses, in such way, the inhibition capacity of bacterial and fungal growth and any eventual histological changes. For this, 30 samples of trabecular bone tissue have been collected from 10 patients submitted to total hip arthroplasty. These samples were separated in three groups of 10 specimens, as following: a control group, a cryopreserved group and a glycerol 98% preserved group at room temperature. They were stored during a period of one year. The results were analysed by the McNemar statistic method, with a significance of 0,10. Concerning to bone matrix, no significant changes occurred to the two studied groups compared to the control group. Bacterial and fungal growth do not occurred in the stored samples for one year in glycerol 98%. For being extremely reduced when compared with cryopreservation method, the preservation cost in glycerol 98% indicates its use on small sum tissue banks. However, posterior studies about biomechanical properties, glycerol removal of bone tissue and their biological process of integration with the receiver are necessary.
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CARVALHO, Maria da Conceição. "Criopreservação de tecido testicular de cães : avaliação histológica e ultraestrutural." Universidade Federal Rural de Pernambuco, 2016. http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/4805.
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Canids are part of the large number of endangered species. The survival of these species depends on the conservation of existing biodiversity. The use of gamete preservation techniques associated with reproductive technologies such as artificial insemination (AI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were developed to help propagate and preserve the genetic potential of canídeos.A testicular tissue cryopreservation might be a method used when ejaculate freezing techniques is not possible. This work set in two experimentos congelamento and vitrificaçãoin vitro, comparing different cryoprotectants (glycerol, dimetisufóxido (DMSO) and trehalose) in testicular tissue of domestic dogs (Canis familiaris). Fragments of 9 adult dogs testes were subjected to a cooling with 4 cryopreservation protocols: slow freezing with glycerol, dimethylsulfoxide (DMSO) or solid surface vitrification using glycerol or DMSO. Fragments of 3mm testicular parenchyma were separated into groups: control, subjected to slow freezing and vitrification. Histological evaluations of the fragments were performed before, after freezing and thawing by light and electron microscopy. Based on morphology and ultrastructure, the testicular tissue of slow freezing, there was no difference between the cryoprotectants. Among the vitrified groups, exposure to DMSO produced a greater structural integrity and architecture when compared to the glycerol group. Comparison of slow freezing and vitrification have shown that vitrification samples revealed more area consisting of tubular compartment, tubular lumen, seminiferous epithelium and conserved membrane. Furthermore, the intertubular compartment Leydig cells showed normal morphology and had characteristics typical of steroidogenic cells. From the results, it was concluded that vitrification DMSO is the most effective method for criopresevar testicular tissue of adult dogs, and can be used as a routine procedure.
Os canídeos fazem parte do grande número de espécies ameaçadas de extinção. A sobrevivência destas espécies depende da conservação da biodiversidade existente. O uso de técnicas de preservação de gametas associadas às tecnologias reprodutivas tais como, inseminação artificial (IA), fertilização in vitro (FIV) e injeção intracitoplasmática de espermatozoides (ICSI) foram desenvolvidas para ajudar a propagar e preservar o potencial genético dos canídeos.A criopreservação de tecido testicular pode vir a ser um método utilizado quando técnicas de congelação do ejaculado não é possível. A presente dissertação configurou-se em dois experimentoscongelamento e vitrificaçãoin vitro, comparando diferentes crioprotetores (glicerol, dimetisufóxido (DMSO) e trealose) em tecido testicular de cães domésticos (Canis familiaris). Fragmentos de testículos de 9 cães adultos foram submetidos a um arrefecimento com 4 protocolos de criopreservação: congelamento lento com glicerol, dimetilsulfóxido (DMSO), ou vitrificação superfície sólida, utilizando glicerol ou DMSO. Os Fragmentos de 3mm do parênquima testicular foram separados em grupos: controle, submetido ao congelamento lento e vitrificação. As avaliações histológicas dos fragmentos foram realizadas antes, após congelação e descongelação por microscopia óptica e eletrônica. Com base na morfologia e ultraestrutura, o tecido testicular de congelação lenta, não houve diferença entre os crioprotetores. Entre os grupos vitrificados, a exposição ao DMSO produziu maior integridade da estrutura e arquitetura quando comparado ao grupo de glicerol. A comparação do congelamento lento e vitrificação demonstraram que a vitrificação revelou amostras com mais área composta por compartimento tubular, luz tubular, epitélio seminífero e membrana conservada. Além disso, o compartimento intertubular mostrou células de Leydig tinham morfologia normal e características típicas de células esteroidogênicas. Diante dos resultados concluiu-se que a vitrificação com DMSO é o método mais eficaz para criopresevar tecido testicular de cães adultos, podendo ser utilizado como procedimento de rotina.
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Terraciano, Paula Barros. "Vitrificação versus congelamento lento não automatizado em tecido ovariano de camundongos CF1." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2016. http://hdl.handle.net/10183/151510.
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Introdução: a alta prevalência do câncer e o aumento significativo da sobrevivência em longo prazo geraram interesse quanto à preservação da fertilidade em mulheres jovens expostas a quimioterapia e radioterapia. Neste sentido estudos de congelamento de tecido ovariano para posterior transplante, abriram uma nova perspectiva de aplicação no tratamento e prevenção da infertilidade feminina. Objetivos: comparar dois protocolos de congelamento de tecido ovariano, um lento não automatizado e um por vitrificação, com o intuito de avaliar a viabilidade dos tecidos para posterior transplante autólogo. Método: Foram utilizadas 30 camundongos fêmea CF1 com aproximadamente 8 semanas e pesando 29,29g±2,9. •Os ovários extraídos foram vitrificados ou congelados, mantidos em nitrogênio líquido por 30 dias e descongelados. Após o descongelamento, o ovário esquerdo foi destinado às análises histológicas e caracterização por imuno histoquímica para o marcador mouse vasa homologue (MVH) e o ovário direito foi utilizado para os testes de viabilidade celular com exclusão por azul de trypan. Resultados: Nas análises de Hematoxilina e Eosina (HE) foram contados folículos primordiais, primários, pré-antrais e antrais. Não houve diferença significativa na proporção de folículos primordiais, primários e pré-antrais após descongelamento entre os grupos testados. A contagem de folículos antrais foi significativamente maior no grupo de vitrificação (p = 0,004). No ensaio de imunohistoquímica para o marcador MVH, folículos MVH + e MVH- foram contados e comparados com o número total de folículos. O grupo congelamento lento apresentou maior número de células não marcadas (p = 0,012). Conclusão: Embora ambos os protocolos tenham apresentado resultados semelhantes na análise histológica das contagens foliculares, o protocolo de vitrificação foi significativamente melhor para preservar a população de células tronco ovarianas.Introduction: The high prevalence of cancer and the significant increase in long-term survival have generated interest as the preservation of fertility in young women exposed to chemotherapy and radiotherapy. Experimental techniques have been tried in an attempt to reverse the ovarian failure induced by these treatments. In this regard studies of ovarian tissue freezing for subsequent transplantation disclose a new application perspective in the treatment and prevention of female infertility. Objective: two ovarian tissue freezing protocols were tested, a non-automated slow-freezing and by vitrification, in order to assess the viability of the tissues for subsequent autologous transplantation. Methods: as ovaries donors, were used 30 female CF1 mice approximately 8 weeks and weighing 29,29g±2,9. • The ovaries were vitrified or frozen, stored in liquid nitrogen for 30 days and thawed. After thawing, the left ovary was intended for histological and immunohistochemical characterization by histochemical marker for MVH and right ovary was used for the tests with cell viability by trypan blue exclusion. Results: In HE slides was counting primordial, primary, pre antral and antral follicles. No significant difference was found in the proportion of high-quality primordial, primary and pre antral follicles after thawing/warming in the slow-freezing and vitrification group, respectively. The antral follicle counting was significant higher in vitrification group (p=0,004). In immunohistochemistry assay for MVH Antibody , MVH+ and MVH- follicles were counted and compared with the total number of follicles and slow freeze group had a higher number of not marked cells (p=0,012). Conclusion: Although both protocols showed similar results in the histological analysis for follicular counts, the vitrification protocol was significantly better for preserve the ovarian stem cell population.
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Feuillassier, Lionel. "La cryoconservation : un outil performant pour la sauvegarde des coraux en danger : son application à Pocillopora damicornis." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS177/document.
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Les nombreuses pressions naturelles et anthropiques qui pèsent sur les écosystèmes coralliens font craindre leur disparition pour les années futures. Parmi les mesures de conservation, la cryoconservation permet de maintenir en sécurité les échantillons sur le long terme et à coût réduit. Les premiers travaux sur la cryoconservation des Anthozoaires incitent à développer davantage la méthode de vitrification plutôt que la congélation lente. Dans ce contexte, cette thèse propose d'expérimenter la technique de vitrification sur plusieurs formes pluricellulaires dont les apex, les planulae, les polypes primaires, les polypes isolés et les balles tissulaires (TB), toutes issues du Scléractiniaire Pocillopora damicornis. Les meilleurs résultats ont été produits avec les TB obtenues après exposition à une solution de KSW puis traitées selon la méthode V Cryo-plate. L'éthylène glycol (EG) s'est avéré le cryoprotecteur (CPA) le mieux toléré jusqu'à 4.0 M pendant 20 min à température ambiante (RT). Les mélanges binaires et ternaires de CPA ont cependant permis d'obtenir de meilleures tolérances des TB qu'avec les solutions individuelles. L'utilisation de solutions successives a permis d'obtenir des survies jusqu'à 4.5 M selon le protocole : 1.5 M EG + 0.5 M Glycérol (Gly) (5 min, RT) puis 1.5 M DMSO + 1.5 M EG + 1.5 M Gly (10 min, 0°C) et enfin 1.5 M EG + 0.5 M Gly (5 min, RT). L'intégrité des cellules épithéliales de l'ectoderme apparaît essentielle au maintien des TB durant et après les traitements. Si le protocole de vitrification n'a pu être mis au point, en revanche, l'utilisation des TB à des fins de cryoconservation apparaît très intéressante pour de futures investigationsNumerous environmental and anthropic pressures threaten reef ecosystems, rising concerns on species loss in coming years. Among conservation measures, cryopreservation ensures the safe and cost-effective long-term conservation of biological material. The first publications focusing on Anthozoa cryopreservation reported that the vitrification approach was preferable to the slow-cooling approach. In this context, this thesis aimed at investigating a vitrification technique with several pluricellular forms of the Scleractinian Pocillopora damicornis including apexes, planulae, primary polyps, isolated polyps and tissue balls (TB). The best results were obtained using TBs produced by exposing coral branches to a KSW solution. TBs were cryopreserved using the V Cryo-plate method. The highest TB tolerance was obtained after exposure to solution containing ethylene glycol (EG) concentrated to 4.0 M for 20 min at room temperature (RT). Binary and ternary cryoprotectant (CPA) solutions were better tolerated by TBs compared with individual cryoprotectant solutions. Exposure of TBs to a series of cryoprotectant solutions with progressively increased concentration allowed obtaining TB tolerance to cryoprotectant with a concentration of 4.5 M with: 1.5 M EG + 0.5 M Glycerol (Gly) (5 min, RT), 1.5 M DMSO + 1.5 M EG + 1.5 M Gly (10 min, 0°C) and then 1.5 M EG + 0.5 M Gly (5 min, RT). Epithelial cells from the ectoderm were essential to maintain TB integrity during and following CPA treatments. Successful cryopreservation was not achieved in this work; however, it demonstrated that the use of TBs constitutes a promising way for further cryopreservation research
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Commin, Loris. "Cryoconservation du tissu ovarien et production d’embryons chez la chienne." Thesis, Lyon 1, 2012. http://www.theses.fr/2012LYO10116.
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De nos jours, la cryoconservation est une technique très largement utilisée dans les protocoles d’assistance à la reproduction ou comme outil pour la sauvegarde des ressources génétiques. Toutefois, la chienne est un modèle animal complexe pour l’application des biotechnologies de la reproduction du fait de ses nombreuses singularités anatomiques et physiologiques. L’objectif de notre travail était d’étudier et de développer une méthode de cryoconservation des ressources génétiques chez la chienne par le biais de deux types de ressources : les embryons et le tissu ovarien. Après avoir mis au point une méthode de collecte d’embryons, nous nous sommes appliqués à la constitution d’un stock d’embryons cryoconservés en prévision d’un transfert embryonnaire. L’étude et le développement d’un protocole de cryoconservation du tissu ovarien ont été abordés après avoir adapté et validé nos méthodes d’analyses in vitro. L’utilisation de plans d’expériences factoriels fractionnaires a permis de mettre en évidence les facteurs les plus influents sur la qualité de la réserve folliculaire (nature du cryoprotecteur pénétrant, cinétique de congélation, étapes d’équilibration) et de proposer un protocole de cryoconservation. La combinaison du DMSO incorporé en un seul bain d’équilibration avec une vitesse de congélation de 0,3°C/min est apparue comme la combinaison la plus appropriée à la cryoconservation de tissu ovarien chez la chienne et a permis d’observer, après xénogreffe de tissu ovarien cryoconservé, une reprise de la croissance folliculaire et de l’activité hormonale du tissu grefféNowadays, cryopreservation is widely used in animal assisted reproduction or safeguarding of genetic resources. Nevertheless, the bitch is a complex animal model concerning the use of this biotechnology, due to numerous anatomical and physiological peculiarities. The aim of our research work was to investigate and develop a method of cryopreservation of genetic resources in the bitch by exploring two kinds of resources: embryos and ovarian tissue. After the setting up of a method for embryo collection, we have built up a stock of cryopreserved embryo for subsequent embryo transfer. After a preliminary validation of our in vitro assessment methods, the investigation and development of a cryopreservation protocol has been conducted. The use of fractional experimental design allowed us to highlight the main factors affecting the follicular pool quality (CPA nature, freezing rate and equilibration steps). The combination of DMSO incorporated in a unique equilibration bath with a freezing rate of 0.3°C/min appeared to be suitable for the cryopreservation of bitch ovarian tissue. Finally, Follicular growth and hormonal activity resumption have been observed after xenotransplantation of cryopreserved bitch ovarian tissue
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Mutsenko, Vitalii [Verfasser], Birgit [Akademischer Betreuer] Glasmacher, Oleksandr [Akademischer Betreuer] Gryshkov, Thomas [Akademischer Betreuer] Illig, and Stephan [Akademischer Betreuer] Kabelac. "Cryopreservation of mesenchymal stromal cells within tissue engineering approaches / Vitalii Mutsenko ; Akademische Betreuer: Birgit Glasmacher, Oleksandr Gryshkov, Thomas Illig, Stephan Kabelac ; Leibniz Universität Hannover, Institut für Mehrphasenprozesse." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2019. http://d-nb.info/1197911510/34.
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Farooque, Tanya Mahbuba. "Biochemical and mechanical stimuli for improved material properties and preservation of tissue-engineered cartilage." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/26710.
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Thesis (Ph.D)--Chemical Engineering, Georgia Institute of Technology, 2009.Committee Chair: Boyan, Barbara; Committee Chair: Wick, Timothy; Committee Member: Brockbank, Kelvin; Committee Member: Nenes, Athanasios; Committee Member: Sambanis, Athanassios. Part of the SMARTech Electronic Thesis and Dissertation Collection.