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1.
Int J Mol Sci ; 22(5)2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33802539

RESUMO

The purpose of this study is to investigate the effectiveness of sphingosine-1-phosphate (S1P) and Z-VAD-FMK (Z-VAD) as anti-apoptotic agents to preserve ovarian function and prevent tissue damage during ovarian tissue cryopreservation and transplantation. This study consisted of two steps, in vitro and in vivo. In the first step, human ovarian tissues were cryopreserved using slow-freezing media alone, S1P, or Z-VAD (control, S1P, Z-VAD group); based on the outcomes in these groups, Z-VAD was selected for subsequent xenotransplantation. In the second step, human frozen/thawed ovarian tissues were grafted into fifty mice divided into three groups: slow-freezing/thawing and transplantation without an anti-apoptotic agent (Trans-control) and xenotransplantation with or without Z-VAD injection (Trans-Z-VAD-positive and Trams-Z-VAD-negative groups, respectively). In the first step, the Z-VAD group had a significantly higher primordial follicular count than the S1P (p = 0.005) and control groups (p = 0.04). Transplanted ovarian tissues were obtained 4 weeks after transplantation (second step). Angiogenesis was significantly increased in the Z-VAD-negative (p = 0.03) and -positive (p = 0.04) groups compared to the control group. This study demonstrated that slow-freezing and transplantation with Z-VAD is an effective method for preserving primordial follicle counts, decreasing double-strand DNA breaks, and increasing angiogenesis in a mouse model. Further molecular and clinical studies are needed to confirm these results.


Assuntos
Apoptose/efeitos dos fármacos , Xenoenxertos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Adolescente , Adulto , Clorometilcetonas de Aminoácidos/uso terapêutico , Animais , Criopreservação/métodos , Feminino , Congelamento , Humanos , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos SCID , Folículo Ovariano/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Transplante Heterólogo/métodos , Adulto Jovem
2.
Nat Commun ; 12(1): 2412, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893303

RESUMO

The development of a widely adopted cryopreservation method remains a major challenge in Drosophila research. Here we report a robust and easily implemented cryopreservation protocol of Drosophila melanogaster embryos. We present innovations for embryo permeabilization, cryoprotectant agent loading, and rewarming. We show that the protocol is broadly applicable, successfully implemented in 25 distinct strains from different sources. We demonstrate that for most strains, >50% embryos hatch and >25% of the resulting larvae develop into adults after cryopreservation. We determine that survival can be significantly improved by outcrossing to mitigate the effect of genetic background for strains with low survival after cryopreservation. We show that flies retain normal sex ratio, fertility, and original mutation after successive cryopreservation of 5 generations and 6-month storage in liquid nitrogen. Lastly, we find that non-specialists are able to use this protocol to obtain consistent results, demonstrating potential for wide adoption.


Assuntos
Criopreservação/métodos , Drosophila melanogaster/embriologia , Embrião não Mamífero/embriologia , Reaquecimento/métodos , Vitrificação , Animais , Crioprotetores/farmacologia , Drosophila melanogaster/genética , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/ultraestrutura , Feminino , Fertilidade/genética , Larva/genética , Larva/metabolismo , Microscopia Eletrônica , Permeabilidade/efeitos dos fármacos , Temperatura , Fatores de Tempo
3.
J Vis Exp ; (169)2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33843932

RESUMO

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.


Assuntos
Criopreservação/métodos , Preservação da Fertilidade/métodos , Infertilidade Feminina/terapia , Oócitos/transplante , Ovário/transplante , Insuficiência Ovariana Primária/terapia , Feminino , Humanos , Oócitos/citologia , Oócitos/fisiologia , Ovário/citologia , Ovário/fisiologia , Indução da Ovulação , Gravidez
4.
Int J Mol Sci ; 22(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673561

RESUMO

The demand for organs to be transplanted increases pressure on procurement centers, to the detriment of organ quality, increasing complications. New preservation protocols are urgently needed, requiring an in-depth understanding of ischemia-reperfusion mechanisms. We performed a proteomic analysis using LC-MS/MS-TOF data analyzed through R software and Cytoscape's ClueGO application, comparing the proteome of kidney endothelial cells, key cell type, subjected to 3, 6, 12, 19, and 24 h of cold ischemia and 6 h reperfusion. Critical pathways such as energy metabolism, cytoskeleton structure/transport system, and gene transcription/translation were modulated. Important time windows were revealed: a-during the first 3 h, central proteins were upregulated within these pathways; b-the majority of these upregulations were maintained until 12 h cold ischemia time (CIT); c-after that time, the overall decrease in protein expression was observed; d-at reperfusion, proteins expressed in response to cold ischemia were all downregulated. This shows that cold ischemia is not a simple slowing down of metabolism, as deep changes take place within the proteome on major pathways. Time-sensitive expression of key protein reveals possible quality biomarkers as well as potential targets for new strategies to maintain or optimize organ quality.


Assuntos
Isquemia Fria/efeitos adversos , Criopreservação/métodos , Células Endoteliais/metabolismo , Rim/metabolismo , Proteoma/metabolismo , Traumatismo por Reperfusão/metabolismo , Cromatografia Líquida , Células Endoteliais/patologia , Ensaios de Triagem em Larga Escala , Humanos , Rim/patologia , Soluções para Preservação de Órgãos , Proteoma/análise , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Espectrometria de Massas em Tandem
5.
Transplant Cell Ther ; 27(3): 270.e1-270.e6, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33781537

RESUMO

SARS-CoV-2 has spread rapidly worldwide, but the full impact of the COVID-19 pandemic on the field of hematopoietic cell transplantation (HCT) remains unknown. To understand this better, an 18-item online survey was disseminated by the Worldwide Network for Blood & Marrow Transplantation with questions exploring SARS-CoV-2 testing algorithms, mobilization, and cryopreservation strategies and COVID-19 infections in allogeneic related and autologous hematopoietic progenitor cell (HPC) donors. The aim of this survey was to assess the impact of the outbreak on policies relating to HPC mobilization, collection, and processing with respect to changes in daily routine. A total of 91 individual responses from distinct centers in 6 continents were available for analysis. In these centers, the majority (72%) of allogeneic related and autologous donors are routinely tested for SARS-CoV-2 before HPC collection, and 80% of centers implement cryopreservation of allogeneic HPC grafts before commencing conditioning regimens in patients. Five related and 14 autologous donors who tested positive for COVID-19 did not experience any unexpected adverse events or reactions during growth factor administration (eg, hyperinflammatory syndrome). These data are limited by the small number of survey respondents but nonetheless suggest that centers are following the recommendations of appropriate scientific organizations and provide some preliminary data to suggest areas of further study.


Assuntos
Transplante de Medula Óssea/estatística & dados numéricos , Transplante de Células-Tronco Hematopoéticas/estatística & dados numéricos , Pandemias , Algoritmos , Aloenxertos , Transplante de Medula Óssea/tendências , /prevenção & controle , /estatística & dados numéricos , Criopreservação/métodos , Seleção do Doador/normas , Saúde Global , Pesquisas sobre Serviços de Saúde , Mobilização de Células-Tronco Hematopoéticas/estatística & dados numéricos , Transplante de Células-Tronco Hematopoéticas/tendências , Padrões de Prática Médica/estatística & dados numéricos , Utilização de Procedimentos e Técnicas/estatística & dados numéricos , Preservação de Tecido/métodos , Transplante Autólogo , Doadores não Relacionados/estatística & dados numéricos
6.
Am J Phys Anthropol ; 174(4): 670-685, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33595836

RESUMO

OBJECTIVES: Flow cytometry is a powerful tool for investigating immune function, allowing for the quantification of leukocytes by subtype. Yet it has not been used extensively for field work due to perishable reagents and the need for immediate analysis of samples. To make flow cytometry more accessible, we devise and evaluate a field protocol for freezing capillary blood. MATERIALS AND METHODS: We collected finger prick blood samples from 110 volunteers, age 18 to 42. Blood samples were analyzed immediately for 18 cell surface markers. Aliquots of whole blood were frozen in the vapor phase of a liquid nitrogen tank with 10% dimethyl sulfoxide in medium. Samples were analyzed on a Guava EasyCyte HT flow cytometer after 2, 4, or 14 weeks. RESULTS: Major lymphocyte fractions in frozen samples were correlated with fresh values (T-cells: r = 0.82; Natural Killer [NK] cells: r = 0.64; CD4: r = 0.67; CD8: r = 0.82; Naïve CD4: r = 0.73, Naïve CD8: r = 0.71; B-cells: r = 0.73; all p < 0.001), and mean values were similar to those from fresh samples. However, correlations for smaller subsets of CD4 and B cells were generally poor. Some differences resulted from changes in non-specific binding for some antibody-conjugate pairs. Cryopreservation also resulted in a reduction in granulocytes more than lymphocytes. DISCUSSION: Our results suggest that antibody/fluorochrome combinations should be validated before use on frozen samples, and that functional changes in cells may affect some cell markers. However, this simple freezing protocol utilizing finger pricks, whole blood, and a liquid nitrogen shipping tank is viable for obtaining samples for flow cytometry under field conditions.


Assuntos
Coleta de Amostras Sanguíneas/métodos , Criopreservação/métodos , Citometria de Fluxo/métodos , Adolescente , Adulto , Sobrevivência Celular , Dedos/irrigação sanguínea , Humanos , Contagem de Leucócitos/métodos , Adulto Jovem
7.
Cryobiology ; 99: 46-54, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33524338

RESUMO

Antioxidant defense is essential for animals to cope with homeostasis disruption during hibernation. The present study aimed to investigate the antioxidant defense response of juvenile soft-shelled turtle Pelodiscus sinensis during hibernation and following arousal. Turtle brain, liver, and kidney samples were collected at pre-hibernation (17 °C mud temperature; MT), during hibernation (5.8 °C MT) and after arousal (20.1 °C MT) in the field. Transcript levels of NF-E2-related factor 2 (Nrf2) decreased significantly during hibernation and recovered after arousal in all tissues. Cerebral and nephric copper-zinc superoxide dismutase (Cu/Zn SOD), catalase (CAT), glutathione peroxidase 3 (GPx3) and nephric GPx4 mRNA showed similar changing patterns as Nrf2. Cerebral Mn SOD, GPx1 and nephric GPx1 up-regulated after arousal. Hepatic Cu/Zn SOD, GPx1 and GPx3 mRNA kept stable, except hepatic GPx4 increased during hibernation. Hepatic Mn SOD and CAT increased after arousal. In the GSH system, mRNA levels of glutathione synthetases (GSs) kept stable during hibernation and up-regulated after arousal in most tissues except nephric GS2 mRNA remained unchanged. Gene expressions of glutathione reductase (GR) exhibited a tissue specific changing pattern, while those of glutathione-S-Transferase (GST) shared a similar pattern among tissues: remained stable or down-regulated during hibernation then recovered in arousal. In contrast to these diverse responses in gene expressions, most of the antioxidant enzyme activities maintained high and stable. Overall, no preparation for oxidative stress (POS) strategy was found in enzymatic antioxidant system in P. sinensis juveniles during hibernation, the Chinese soft-shelled turtles were able to stay safe from potential oxidative stress during hibernation by maintaining high level activities/concentrations of the antioxidant enzymes/antioxidants.


Assuntos
Hibernação , Tartarugas , Animais , Antioxidantes , Nível de Alerta , Catalase/genética , Catalase/metabolismo , China , Criopreservação/métodos , Estresse Oxidativo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Tartarugas/genética
8.
Cryobiology ; 99: 20-27, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33545147

RESUMO

Cooling rate is a critical parameter affecting the success of cell cryopreservation. Fast cooling can result in intracellular ice formation (IIF), while slow cooling can bring solution effects injury, both are detrimental to the cells. Whilst most of the studies have investigated how IIF affects cells, solution effects injury has received little attention. Here, we studied the solution effects injury of human T lymphocytes by cryomicroscopy and tested the osmoprotective ability of some frequently used cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, trehalose, urea and l-proline. We further investigated the relationship between cell volume, latent heat and solution effects cell injury. We found that solution effects injury during interrupted slow cooling was caused by high concentration of the extracellular solution rather than eutectic formation and solutes precipitation. DMSO, glycerol and trehalose can protect cells from solution effects injury, while l-proline and urea cannot under the same condition. The cell volume and latent heat are not crucial for causing solution effects injury in cells. This work confirms that high osmotic pressure, rather than eutectic formation, leads to cell injury. It also suggests that cell volume and latent heat may not be a key factor for explaining solution effects injury and its prevention in the cryopreservation of human T lymphocytes.


Assuntos
Criopreservação , Gelo , Criopreservação/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Congelamento , Humanos , Linfócitos T
9.
Cryobiology ; 99: 1-10, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33556359

RESUMO

Mosquito-borne diseases are responsible for millions of human deaths every year, posing a massive burden on global public health. Mosquitoes transmit a variety of bacteria, parasites and viruses. Mosquito control efforts such as insecticide spraying can reduce mosquito populations, but they must be sustained in order to have long term impacts, can result in the evolution of insecticide resistance, are costly, and can have adverse human and environmental effects. Technological advances have allowed genetic manipulation of mosquitoes, including generation of those that are still susceptible to insecticides, which has greatly increased the number of mosquito strains and lines available to the scientific research community. This generates an associated challenge, because rearing and maintaining unique mosquito lines requires time, money and facilities, and long-term maintenance can lead to adaptation to specific laboratory conditions, resulting in mosquito lines that are distinct from their wild-type counterparts. Additionally, continuous rearing of transgenic lines can lead to loss of genetic markers, genes and/or phenotypes. Cryopreservation of valuable mosquito lines could help circumvent these limitations and allow researchers to reduce the cost of rearing multiple lines simultaneously, maintain low passage number transgenic mosquitoes, and bank lines not currently being used. Additionally, mosquito cryopreservation could allow researchers to access the same mosquito lines, limiting the impact of unique laboratory or field conditions. Successful cryopreservation of mosquitoes would expand the field of mosquito research and could ultimately lead to advances that would reduce the burden of mosquito-borne diseases, possibly through rear-and-release strategies to overcome mosquito insecticide resistance. Cryopreservation techniques have been developed for some insect groups, including but not limited to fruit flies, silkworms and other moth species, and honeybees. Recent advances within the cryopreservation field, along with success with other insects suggest that cryopreservation of mosquitoes may be a feasible method for preserving valuable scientific and public health resources. In this review, we will provide an overview of basic mosquito biology, the current state of and advances within insect cryopreservation, and a proposed approach toward cryopreservation of Anopheles stephensi mosquitoes.


Assuntos
Anopheles , Mosquitos Vetores , Animais , Abelhas , Criopreservação/métodos , Humanos , Resistência a Inseticidas/genética , Controle de Mosquitos , Mosquitos Vetores/genética
10.
Cryobiology ; 99: 28-39, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33529683

RESUMO

Ice-binding proteins (IBPs) inhibit the growth of ice through surface adsorption. In some freeze-resistant fishes and insects, circulating IBPs serve as antifreeze proteins to stop ice growth by lowering the freezing point. Plants are less able to avoid freezing and some use IBPs to minimize the damage caused in the frozen state by ice recrystallization, which is the growth of large ice grains at the expense of small ones. Here we have accurately and reproducibly measured the ice recrystallization inhibition (IRI) activity of over a dozen naturally occurring IBPs from fishes, insects, plants, and microorganisms using a modified 'splat' method on serial dilutions of IBPs whose concentrations were determined by amino acid analysis. The endpoint of IRI, which was scored as the lowest protein concentration at which no recrystallization was observed, varied for the different IBPs over two orders of magnitude from 1000 nM to 5 nM. Moreover, there was no apparent correlation between their IRI levels and reported antifreeze activities. IBPs from insects and fishes had similar IRI activity, even though the insect IBPs are typically 10x more active in freezing point depression. Plant IBPs had weak antifreeze activity but were more effective at IRI. Bacterial IBPs involved in ice adhesion showed both strong freezing point depression and IRI. Two trends did emerge, including that basal plane binding IBPs correlated with stronger IRI activity and larger IBPs had higher IRI activity.


Assuntos
Proteínas de Transporte , Gelo , Animais , Proteínas Anticongelantes/metabolismo , Criopreservação/métodos , Cristalização , Peixes , Congelamento , Insetos
11.
Cryobiology ; 99: 11-19, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33577784

RESUMO

Despite studies on the effects of medicinal plants on reproductive performance, the effect of extracts broccoli and caraway on serum and testicular oxidative biomarkers, testicular structure and function and sperm quality before and after cryopreservation has not been studied. Sixty-three male mice were divided into nine controlled and treated groups as follow: control, broccoli (200 mg/kg), broccoli (300 mg/kg), caraway (200 mg/kg), caraway (300 mg/kg), broccoli -caraway (200 mg/kg), broccoli (300 mg/kg)-caraway (200 mg/kg), broccoli (200 mg/kg)-caraway (300 mg/kg), broccoli-caraway (300 mg/kg). After 42 days of treatment, the animals were sacrificed and blood sample and testicular tissue were collected for biochemical and histological measurements. Sperm quality was also measured before and after cryopreservation. The results showed that the diameter and number of spermatogonium, primary spermatocytes, spermatids and sperm count were significantly increased by broccoli (300 mg/kg), while level of them were significantly decreased by caraway (300 mg/kg) compared to other groups (p < 0.01). Sperm viability and motility after thawing significantly improved by broccoli (300 mg/kg) compared to control. Testosterone levels significantly increased by broccoli (300 mg/kg) compared to control and caraway (300 mg/kg). The serum and testicular SOD and CAT activity significantly increased by broccoli (300 mg/kg) compared to other groups (p < 0.05). MDA and DNA fragmentation levels significantly increased by caraway (200 and 300 mg/kg) compared to others (p < 0.01). It can be concluded that broccoli extract in a dose-dependent manner than caraway extract could improve serum and testes oxidative biomarkers, testicular structure and function, and sperm quality before and after cryopreservation.


Assuntos
Brassica , Carum , Animais , Biomarcadores/metabolismo , Criopreservação/métodos , Masculino , Camundongos , Estresse Oxidativo , Extratos Vegetais/farmacologia , Contagem de Espermatozoides , Motilidade Espermática , Espermatozoides/metabolismo , Testículo
12.
Nat Protoc ; 16(3): 1581-1599, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33580232

RESUMO

The endodermal germ layer gives rise to respiratory epithelium, hepatocytes, pancreatic cells and intestinal lineages, among other cell types. These lineages can be differentiated from human pluripotent stem cells (hPSCs) via a common definitive endoderm (DE) intermediate that is characterized by the co-expression of the cell surface markers CXCR4, c-KIT and EPCAM and the transcription factors SOX17 and FOXA2. Here we provide a detailed protocol for mass production of DE from hPSCs in scalable and easy-to-handle suspension culture using a rotating Erlenmeyer flask or a sophisticated, fully controllable, 150-ml stirred tank bioreactor. This protocol uses two different media formulations that are chemically defined and xeno free and therefore good manufacturing practice ready. Our protocol allows for efficient hPSC-derived DE specification in multicellular aggregates within 3 days and generates up to 1 × 108 DE cells with >92% purity in one differentiation batch when using the bioreactor. The hPSC-derived DE cells that are generated can be cryopreserved for later downstream differentiation into various endodermal lineages. This protocol should facilitate the flexible production of mature DE derivatives for physiologically relevant disease models, high-throughput drug screening, toxicology testing and cellular therapies.


Assuntos
Técnicas de Cultura de Células/métodos , Endoderma/citologia , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Criopreservação/métodos , Meios de Cultura , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Hepatócitos/citologia , Humanos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia
13.
Methods Mol Biol ; 2218: 37-47, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606221

RESUMO

Cryopreservation as a method that enables long-term storage of biological material has long been used for the conservation of valuable zebrafish genetic resources. However, currently, only spermatozoa of zebrafish can be successfully cryopreserved, while protocols for cryopreservation of eggs and embryos have not yet been fully developed. Transplantation of germline stem cells (GSCs) has risen as a favorable method that can bypass the current problem in cryopreservation of female genetic resources and can lead to reconstitution of fish species and lines through surrogate production. Here, we describe essential steps needed for the cryopreservation of spermatogonial stem cells (SSCs) and their utilization in the conservation of zebrafish genetic resources through SSC transplantation and surrogate production.


Assuntos
Células-Tronco Germinativas Adultas/citologia , Criopreservação/métodos , Espermatogônias/citologia , Espermatozoides/citologia , Transplante/métodos , Células-Tronco Germinativas Adultas/efeitos dos fármacos , Animais , Crioprotetores/farmacologia , Masculino , Espermatogônias/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Testículo/citologia , Testículo/efeitos dos fármacos , Peixe-Zebra/fisiologia
14.
Methods Mol Biol ; 2218: 75-83, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606224

RESUMO

Primordial germ cells (PGCs) are unique cells in an embryo. These cells contain all genetic information and therefore represent the best source to store maternal and paternal genomes until embryo cryopreservation is achieved. However, the number of these cells in an embryo is very low limiting their potential application in cryopreservation and surrogate production. However, it was assumed that the induction of fish PGCs in vitro is not possible because in vivo they inherit germ plasm. In this chapter, we describe a successful differentiation protocol explaining the crucial factors and steps for in vitro PGC generation.


Assuntos
Células Germinativas/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Criopreservação/métodos , Embrião não Mamífero/citologia , Feminino , Masculino , Peixe-Zebra/fisiologia
15.
Methods Mol Biol ; 2218: 99-115, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606226

RESUMO

Cryopreservation of sperm cells is currently the most efficient tool for managing large and small collections of valuable genetic resources. Cryopreservation minimizes expenses for animal and facility maintenance such as personnel, water, power, and space. It extends the time offspring can be produced from individual organisms, reduces the need to maintain live populations, provides flexibility for planning future experiments and research projects, and can prevent catastrophic loss of irreplaceable research lines. In this chapter, we present the sperm collection, dilution, cryopreservation, thawing, and in vitro fertilization procedures used at the Zebrafish International Resource Center (ZIRC).


Assuntos
Criopreservação/métodos , Preservação do Sêmen/métodos , Espermatozoides/citologia , Animais , Crioprotetores/farmacologia , Fertilização In Vitro/métodos , Masculino , Motilidade Espermática/efeitos dos fármacos , Motilidade Espermática/fisiologia , Espermatozoides/efeitos dos fármacos , Peixe-Zebra/fisiologia
16.
Cryobiology ; 99: 40-45, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33508264

RESUMO

Addressing the establishment of biobanks for the conservation of wild hystricomorph rodents' germplasm, we verified the effects of different extenders and distinct concentrations of non-permeant cryoprotectants on the sperm parameters of Spix's yellow-toothed cavies. Nine testis-epididymis complexes were used for sperm collection by retrograde washing using Tris or a powdered coconut water extender (ACP®-116c). Spermatozoa were diluted and frozen with the same extenders supplemented with egg yolk or Aloe vera at a 10% or 20% concentration. After recovery and cryopreservation, all samples were evaluated for sperm kinetic parameters, morphology, membrane integrity, osmotic response, and sperm-binding capability using an egg yolk perivitelline membrane assay. After recovery, no differences were observed between Tris and ACP®-116c that provided 515.4 × 106 sperm/mL and 561.6 × 106 sperm/mL, presenting >65% motile sperm, respectively. After cryopreservation, most effective preservation of sperm kinetic parameters (68.1 ± 5.9% motile sperm) and membrane integrity (48.2 ± 7.4%) was provided by Tris extender supplemented with 10% egg yolk. However, both extenders supplemented with any concentration of egg yolk or Aloe vera presented similar preservation of osmotic response and sperm-binding ability after cryopreservation. In summary, we suggest the use of a Tris extender supplemented of 10% egg yolk for cryopreservation of Spix's yellow-toothed cavy epidydimal sperm.


Assuntos
Aloe , Preservação do Sêmen , Animais , Cocos , Criopreservação/métodos , Crioprotetores/farmacologia , Gema de Ovo , Epididimo , Cobaias , Masculino , Análise do Sêmen , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides , Água
17.
Cryobiology ; 99: 103-105, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33400960

RESUMO

PURPOSE: The aim is to report the first live-birth following ICSI using spermatozoa previously vitrified in a Stripper Tip. PRINCIPAL RESULTS: A 34-year-old cryptozoospermic man was enrolled in a sperm vitrification program. After failure of conventional freezing technique, spermatozoa were vitrified using two carriers: a commercially-available, Cell Sleeper, and a "home-made" one, Stripper Tip. This man and his 30-year-old wife underwent an ICSI attempt using vitrified-warmed spermatozoa from these devices. All frozen-warmed spermatozoa were quickly recovered. Among the oocytes retrieved, six were injected with sperm from the Cell Sleeper, and seven with sperm from the Stripper tip, leading to 4 embryos in each case. Two embryos, arising from sperm frozen in the Stripper tip, were transferred, resulting in a healthy live-birth. CONCLUSIONS: This is the first successful delivery following the use of spermatozoa frozen in an original device, the Stripper Tip, giving a promising prospect for managing severe male infertilities.


Assuntos
Criopreservação , Espermatozoides , Adulto , Criopreservação/métodos , Feminino , Humanos , Nascimento Vivo , Masculino , Oócitos , Gravidez , Vitrificação
18.
Cryobiology ; 98: 46-56, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33400962

RESUMO

In the last decades, liposomes acquired a striking success in the biomedical field thanks to their biocompatibility and drug delivery ability. Many liposomal drug formulations have been already approved by the Food and Drug Administration (FDA) and used for the treatment of a wide range of pathologies with or without further engineering. Their clinical application requires strict compliance with high standard quality rules, and it is crucial to employ storage methods that do not affect the integrity of the vesicles and preventing the leakage of their cargo. In this work, the design of a suitable formulation for freeze-drying had been investigated for two different liposomes, DOPC-DOTAP and the PEGylated counterpart, DOPC-DOTAP-DSPE-PEG. The role of various cryoprotectants was evaluated paying attention to their ability to preserve the structural integrity of liposomes. At first, the study was focused on freezing and two methodologies were investigated, quenching in liquid nitrogen and shelf-ramped freezing. This analysis showed that the disaccharides (cellobiose, glucose, lactose, sucrose, and trehalose) and the polyol (mannitol) protected successfully the integrity of liposomes, while during the process, in the presence of a surfactant, liposomes were strongly damaged and fragmented by the ice crystals. Furthermore, the choice of the rate of freezing depended on the different compositions of the lipid bilayer. Finally, the effects of lyophilization on liposomes with and without additives were studied; cellobiose, lactose and trehalose showed encouraging results for the maintenance of the morpho-functional parameters of liposomes during the entire freeze-drying process.


Assuntos
Criopreservação , Lipossomos , Criopreservação/métodos , Crioprotetores , Liofilização , Congelamento , Lipídeos
19.
Cryobiology ; 98: 33-38, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33412156

RESUMO

Accidental hypothermia is associated with increased risk for arrhythmias. QRS/QTc is proposed as an ECG-marker, where decreasing values predict hypothermia-induced ventricular arrhythmias. If reliable it should also predict nonappearance of arrhythmias, observed in species like rat that regularly tolerate prolonged hypothermia. A rat model designed for studying cardiovascular function during cooling, hypothermia and subsequent rewarming was chosen due to species-dependent resistance to ventricular arrhythmias. ECG was recorded throughout the protocol. No ventricular arrhythmias occurred during experiments. QRS/QTc increased throughout the cooling period and remained above normothermic baseline until rewarmed. Different from the high incidence of hypothermia-induced ventricular arrhythmias in accidental hypothermia patients, where QRS/QTc ratio is decreased in moderate hypothermia; hypothermia and rewarming of rats is not associated with increased risk for ventricular fibrillation. This resistance to lethal hypothermia-induced arrhythmias was predicted by QRS/QTc.


Assuntos
Hipotermia Induzida , Hipotermia , Animais , Arritmias Cardíacas/etiologia , Criopreservação/métodos , Humanos , Hipotermia/terapia , Ratos , Reaquecimento , Fibrilação Ventricular/etiologia
20.
Cryobiology ; 98: 39-45, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33417948

RESUMO

We investigated the association between tumor location on multiparametric magnetic resonance imaging (mpMRI) and outcomes of prostate cancer patients after primary total prostate cryoablation (PTPC). Between March 2010 and December 2012, consecutive 192 prostate cancer patients receiving PTPC were enrolled. Tumor locations were determined and classified as anterior apex (AA), anterior midgland (AM), anterior base (AB), posterior apex (PA), posterior midgland (PM) and posterior base (PB) using mpMRI. Midline location, central location, seminal vesicle invasion, extraprostatic extension, multiple tumors, and tumor volume were also identified. Prostate local recurrence and biochemical failure were considered as primary and secondary endpoints, respectively. Tumors on mpMRI were identified in 148 (77.1%) patients. Tumor locations were most frequently noted in PM (89, 46.4%), followed by AM (55, 28.6%), PB (53, 27.6%), PA (46, 24%), AA (35, 18.2%) and AB (31, 16.1%). Midline and central tumors were observed in 34 (17.7%) and 14 (7.3%) patients, respectively. During a median follow-up duration of 81 months (range, 2-114 months), 71 (37.0%) and 29 (40.8%) patients experienced biochemical failure and local recurrence, respectively. Multivariable analyses revealed only AA tumors increased the risk of local recurrences (HR = 2.98, 95% CI. 1.36-6.49). None of location-related parameters was associated with biochemical failure. Tumor location on mpMRI has a significant association with local tumor recurrence in patients receiving PTPC. Physicians should be cautious when conducting cryoablation for prostate tumors in AA location.


Assuntos
Criocirurgia , Neoplasias da Próstata , Criopreservação/métodos , Criocirurgia/efeitos adversos , Humanos , Imagem por Ressonância Magnética , Masculino , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/cirurgia , Estudos Retrospectivos
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