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Cryopreservation Protocol (cryopreservation + protocol)

Distribution by Scientific Domains

Medical Sciences	37%
Life Sciences	25%

Selected Abstracts

Cryotherapy of shoot tips: a technique for pathogen eradication to produce healthy planting materials and prepare healthy plant genetic resources for cryopreservation

ANNALS OF APPLIED BIOLOGY, Issue 3 2009
Q.C. Wang
Abstract Cryotherapy of shoot tips is a new method for pathogen eradication based on cryopreservation techniques. Cryopreservation refers to the storage of biological samples at ultra-low temperature, usually that of liquid nitrogen (,196°C), and is considered as an ideal means for long-term storage of plant germplasm. In cryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria are eradicated from shoot tips by exposing them briefly to liquid nitrogen. Uneven distribution of viruses and obligate vasculature-limited microbes in shoot tips allows elimination of the infected cells by injuring them with the cryo-treatment and regeneration of healthy shoots from the surviving pathogen-free meristematic cells. Thermotherapy followed by cryotherapy of shoot tips can be used to enhance virus eradication. Cryotherapy of shoot tips is easy to implement. It allows treatment of large numbers of samples and results in a high frequency of pathogen-free regenerants. Difficulties related to excision and regeneration of small meristems are largely circumvented. To date, severe pathogens in banana (Musa spp.), Citrus spp., grapevine (Vitis vinifera), Prunus spp., raspberry (Rubus idaeus), potato (Solanum tuberosum) and sweet potato (Ipomoea batatas) have been eradicated using cryotherapy. These pathogens include nine viruses (banana streak virus, cucumber mosaic virus, grapevine virus A, plum pox virus, potato leaf roll virus, potato virus Y, raspberry bushy dwarf virus, sweet potato feathery mottle virus and sweet potato chlorotic stunt virus), sweet potato little leaf phytoplasma and Huanglongbing bacterium causing ,citrus greening'. Cryopreservation protocols have been developed for a wide variety of plant species, including agricultural and horticultural crops and ornamental plants, and can be used as such or adjusted for the purpose of cryotherapy. [source]

The effect of cell size distribution during the cooling stage of cryopreservation without CPA

AICHE JOURNAL, Issue 8 2010
S. Fadda
Abstract A novel model capable of quantitatively describing and predicting intracellular ice formation (IIF) as a function of temperature in a cell population characterized by a size distribution is proposed. The model overcomes the classical approach which takes into account a population of identically sized cells. The size distribution dynamics of a cell population in response to water osmosis and IIF occurrence during the cooling stage of a standard cryopreservation protocol without using cryoprotective agent (CPA) is simulated by means of a suitable population balance approach. Specifically, the model couples the classical water transport equation developed by Mazur1 to the quantitative description of nucleation and diffusion-limited growth of ice crystals in the framework of a 1-D population balance equation (PBE). It is found that IIF temperature depends on the cell size, i.e., it is higher for larger cells. Correspondingly, the probability of IIF (PIIF) results to be dependent on the initial size distribution of the cell population. Model's parameters related to the osmotic behavior of the cell population and to IIF kinetics are obtained by comparison between theoretical results and suitable experimental data of isolated rat hepatocytes available in the literature. Model reliability is successfully verified by predicting the experimental data of PIIF at different, constant cooling rates with better accuracy as compared to the theoretical approaches available in the literature. © 2009 American Institute of Chemical Engineers AIChE J, 2010 [source]

Morphological changes in mouse embryos cryopreserved by different techniques

MICROSCOPY RESEARCH AND TECHNIQUE, Issue 4 2007
A.R.S. Coutinho
Abstract Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnologies. Subjective evaluation to determine embryo viability is often used. The determination of the best cryopreservation protocol depends on morphological and molecular analysis of cellular injuries. The main objective of this study was to compare two methods of cryopreservation by assessing morphological alterations of frozen embryos using light, fluorescence, and transmission electron microscope. Fresh (control), slow frozen, and vitrified mouse embryos were composed. To evaluate the viability of the embryos, the cell membrane integrity was assessed using Hoechst33342 and propidium iodide (H/PI) staining. Morphological analyses using hematoxylin and eosin (HE) staining were performed to test different techniques (in situ, paraffin, and historesin) by both light and fluorescence microscopy. Transmission electron microscope was used to detect ultrastructural alterations in Spurr- and Araldite-embedded samples. H/PI staining detected more membrane permeability in the vitrification (69.8%) than in the slow freezing (48.4%) or control (13.8%) groups (P < 0.001). Historesin-embedded samples showed to be more suitable for morphological analyses because cellular structures were better identified. Nuclear evaluation in historesin sections showed the induction of pycnosis in slow freezing and vitrification groups. Cytoplasm evaluation revealed a condensation and an increase in eosinophilic intensity (indicating apoptosis) in the slow freezing group, and weakly eosinophilic structures and degenerated cells (indicating oncosis) in the vitrification group (P < 0.05). Ultrastructural analyses confirmed HE morphological findings. It was concluded that both cryopreservation techniques resulted in oncosis and apoptosis injuries. However, vitrification caused more severe cellular alterations and reduced embryonic viability compared to slow freezing. Microsc. Res. Tech., 2007. © 2006 Wiley-Liss, Inc. [source]

Assessment of sperm quality, DNA integrity and cryopreservation protocols in men diagnosed with testicular and systemic malignancies

ANDROLOGIA, Issue 6 2009
T. M. Said
Summary Men diagnosed with malignancy are often referred for semen banking to preserve their fertility prior to cancer treatment. The chances of cancer patients for achieving future fecundity will be determined by the sperm quality including the integrity of the genomic material in the frozen samples. The objectives of this study were to compare the sperm quality and DNA integrity in men diagnosed with testicular and systemic malignancies before receiving treatment and to identify the optimum cryopreservation protocol for their samples including a remote semen collection option. In comparison with fertile donors, patients with testicular malignancies had significantly lower sperm concentration, while both testicular and systemic malignancy patients had significantly lower sperm motility and cryosurvival rates. In addition, the SCSA defined DNA fragmentation index was significantly higher in patients with testicular and systemic malignancies compared with fertile donors. It was noted that the extent of deterioration in sperm quality and DNA integrity seen in cancer patients did not reach the previously defined statistical threshold for impaired fertility. Freezing spermatozoa with the seminal plasma offers the highest protection against cryo-injury. Nevertheless, remote semen collection can still be used as it yields adequate results. [source]

Cryopreservation of isolated blastomeres and embryonic stem-like cells of Leopard danio, Brachydanio frankei

AQUACULTURE RESEARCH, Issue 4 2010
Padmanav Routray
Abstract This study aimed at developing a suitable cryopreservation protocol for embryonic stem (ES)-like cells of a tiny freshwater fish Leopard danio (Brachydanio frankei). Embryonic stem (ES)-like cells derived from blastomeres of the early blastulae stage of the developing embryo were cultured in vitro in a medium containing Leibowitz-15 supplemented with 10% foetal bovine serum, leopard danio embryo extract, sodium bicarbonate, sodium selenite, basic fibroblast growth factor, epidermal growth factor and leukaemia inhibitory factor. The ES-like cells showed properties similar to ES cells in other species. They were morphologically small, round to polygonal and present in patches and extensively expressed alkaline phosphatase and stage-specific embryonic antigen. The toxicity and chilling sensitivity of these cells were determined using ethylene glycol (EG), propylene glycol (PG) and glycerol as cryoprotective agents at molar concentrations of 0.6, 1.0, 1.4, 1.8 and 2.0. Among them, 1.8 M EG showed 70% significant viable ES-like cells (P<0.05). The post-thawed cells retained similar properties of non-cryopreserved ES-like cells with a viability rate of 65%. Similarly, blastomeres cryopreserved following the slow cooling rate with EG and PG yielded a viability of more than 70%. [source]

Spermatophore cryopreservation and artificial insemination of black tiger shrimp, Penaeus monodon (Fabricius)

AQUACULTURE RESEARCH, Issue 5 2006
Amrit N Bart
Abstract To develop an appropriate cryopreservation protocol for spermatophores of black tiger shrimp, Penaeus monodon, three cryoprotectants (dimethyl sulphoxide (DMSO), methanol (MeOH) and ethylene glycol (EG)) at two concentrations (5% and 10%) were examined. Artificial implantation of spermatophores was also carried out to assess the fertilizing ability of fresh and post-thaw spermatophores. Spermatophores were collected during consecutive regenerations (15-day intervals) and assessed for qualitative and quantitative changes and also for fertilizing ability by implantation. The mean fertilization rate for artificial insemination using post-thaw spermatophore was 79.9±3.7%, lower than the fertilization rates observed for artificial implantation using fresh spermatophore and natural mating. Mean hatch rates for fresh spermatophore, frozen-thawed spermatophore and natural mating were 88.8±0.6%, 87.8±0.4% and 88.3±0.5%, respectively; and there was no difference among the three groups. The mean fertilization rate of spermatophores collected during the first stripping was higher (90.6±0.6) than during the second stripping (85.7±2.6), but the mean hatch rate was not different between the two strippings. The highest mean sperm viability (79.7±0.4%) was obtained from DMSO (5%), with no survival observed in the 10% MeOH treatment. Spermatophore weight, total sperm count and percentage of abnormal sperm were not different between spermatophores collected at the first and second stripping. This is the first study to report high fertilization and hatch rates from cryopreserved spermatophore using artificial implantation of spermatophore before spawning. [source]

The roles of apoptotic pathways in the low recovery rate after cryopreservation of dissociated human embryonic stem cells

BIOTECHNOLOGY PROGRESS, Issue 3 2010
Xia Xu
Abstract Human embryonic stem (hES) cells have enormous potential for clinical applications. However, one major challenge is to achieve high cell recovery rate after cryopreservation. Understanding how the conventional cryopreservation protocol fails to protect the cells is a prerequisite for developing efficient and successful cryopreservation methods for hES cell lines and banks. We investigated how the stimuli from cryopreservation result in apoptosis, which causes the low cell recovery rate after cryopreservation. The level of reactive oxygen species (ROS) is significantly increased, F-actin content and distribution is altered, and caspase-8 and caspase-9 are activated after cryopreservation. p53 is also activated and translocated into nucleus. During cryopreservation apoptosis is induced by activation of both caspase-8 through the extrinsic pathway and caspase-9 through the intrinsic pathway. However, exactly how the extrinsic pathway is activated is still unclear and deserves further investigation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]