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1.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 43(2): 193-198, 2021 Apr 28.
Artigo em Chinês | MEDLINE | ID: mdl-33966697

RESUMO

Objective To investigate the effects of self-made carriers on the cryopreservation of ovarian tissue of sheep. Methods Thirty-two ovaries were randomly assigned to fresh group,programmed freezing group,self-made carrier I vitrification group,and self-made carrier Ⅱ vitrification group.The morphology,proliferation,apoptosis,and estrogen level of the ovarian tissue in each group were observed. Results After cryopreservation,the morphology normal rate of the primordial follicles in programmed freezing group,self-made carrier I vitrification group,and self-made carrier Ⅱ vitrification group were 74.2%,72.8%,and 72.3%,respectively,lower than that(83.7%)in the fresh group(χ2=13.079,P=0.004).The percentage of normal primary follicles in programmed freezing group was lower than that in the fresh group(χ2=12.486,P=0.000).The percentage of normal primary follicles showed no significant difference between vitrification groups and fresh group(P=1.000,P=0.972).There was no significant difference in estrogen level or the positive expression rate of PCNA among the 4 groups(F=0.363,P=0.780;χ2=0.359,P=0.949).The number of apoptotic cells in cryopreservation groups was significantly higher than that in the fresh group(F=37.584,P=0.000),and it was significantly higher in the programmed freezing group than in the two vitrification groups(F=18.992,P=0.000). Conclusion Compared with slow programmed freezing,the vitrification with self-made carriers could well preserve the activity of cells in large sheep ovarian tissue blocks.


Assuntos
Criopreservação , Vitrificação , Animais , Feminino , Congelamento , Folículo Ovariano , Ovário , Ovinos
2.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800956

RESUMO

Crosslinking of hydroxypropyl methyl cellulose (HPMC) and acrylic acid (AAc) was carried out at various compositions to develop a high-solid matrix with variable glass transition properties. The matrix was synthesized by the copolymerisation of two monomers, AAc and N,N'-methylenebisacrylamide (MBA) and their grafting onto HMPC. Potassium persulfate (K2S2O8) was used to initiate the free radical polymerization reaction and tetramethylethylenediamine (TEMED) to accelerate radical polymerisation. Structural properties of the network were investigated with Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), modulated differential scanning calorimetry (MDSC), small-deformation dynamic oscillation in-shear, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The results show the formation of a cohesive macromolecular entity that is highly amorphous. There is a considerable manipulation of the rheological and calorimetric glass transition temperatures as a function of the amount of added acrylic acid, which is followed upon heating by an extensive rubbery plateau. Complementary TGA work demonstrates that the initial composition of all the HPMC-AAc networks is maintained up to 200 °C, an outcome that bodes well for applications of targeted bioactive compound delivery.


Assuntos
Acrilamidas/química , Derivados da Hipromelose/química , Acrilatos/química , Varredura Diferencial de Calorimetria , Vidro , Teste de Materiais , Microscopia Eletrônica de Varredura , Polimerização , Espectroscopia de Infravermelho com Transformada de Fourier , Termogravimetria , Vitrificação , Difração de Raios X
3.
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
5.
Molecules ; 26(3)2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525346

RESUMO

The mode coupling theory of supercooled liquids is combined with advanced closures to the integral equation theory of liquids in order to estimate the glass transition line of Yukawa one-component plasmas from the unscreened Coulomb limit up to the strong screening regime. The present predictions constitute a major improvement over the current literature predictions. The calculations confirm the validity of an existing analytical parameterization of the glass transition line. It is verified that the glass transition line is an approximate isomorphic curve and the value of the corresponding reduced excess entropy is estimated. Capitalizing on the isomorphic nature of the glass transition line, two structural vitrification indicators are identified that allow a rough estimate of the glass transition point only through simple curve metrics of the static properties of supercooled liquids. The vitrification indicators are demonstrated to be quasi-universal by an investigation of hard sphere and inverse power law supercooled liquids. The straightforward extension of the present results to bi-Yukawa systems is also discussed.


Assuntos
Vidro/química , Plasma/química , Entropia , Transição de Fase , Vitrificação
6.
An Acad Bras Cienc ; 93(1): e20190555, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33624712

RESUMO

Cryopreservation of pineapple shoot tips has been established from various protocols, including droplet vitrification. Thus, this work aimed to evaluate the morphoanatomical conditions of the starting material over different times (30, 45 and 60 days) of culture before freezing and its correlation with the survival percentage of the cryopreserved shoot tips. Four accessions, Ananas comosus var. comosus (BGA-009); var. bracteatus (BGA-119); var. parguazensis (BGA-376), var. erectifolius (BGA-750) from the Pineapple Active Germplasm Bank (BGA Pineapple) and two hybrids from the Embrapa Genetic Breeding Program, FIB-ROX1 (var. bracteatus X var. erectifolius) and FIB-ROX2 ( var. erectifolius X var. bracteatus), recently introduced in the field from in vitro storage, were used. Histological sections before freezing and the percentages of survival after freezing were obtained taking into account the different times of cultivation of the donor plants. The results showed a significative interaction between genotypes (accessions and hybrids) and the culture period. The accessions BGA-009 and BGA-119 showed the highest survival rates, with 95% and 90% respectively for the 30-day culture time. Different results were obtained for each genotype, showing the need for improvements in the standardization of starting material, which would allow better repeatability of the protocol.


Assuntos
Ananas , Vitrificação , Criopreservação , Melhoramento Vegetal , Brotos de Planta
7.
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
8.
Cryobiology ; 99: 95-102, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33422478

RESUMO

The protocol of aseptic cryoprotectant-free vitrification on human spermatozoa is well documented. However, data about the effect of permeable cryoprotectants at this procedure is limited. Presented study aimed to test the aseptic capillary vitrification technologies using permeable cryoprotectant-included or cryoprotectant-free media. Thirty-two normal samples were included and analyzed after vitrification in three different media and thawing. Three treatment groups were formed: Group 1, basic medium; Group 2, basic medium with 0.25 M sucrose; Group 3, basic medium with glycerol. Before plunging into liquid nitrogen, capillaries were filled by 10 µl of spermatozoa suspension and isolated from liquid nitrogen by location in hermetically closed 0.25 ml straws. Progressive motility, plasma membrane integrity, total motility/viability after 24, 48 and 72 h in vitro culture, apoptosis and mitochondrial membrane potential (ΔΨm) were determined after thawing at 42 °C. Progressive motility of spermatozoa in groups 1, 2, 3 was 24.9 ± 1.7%, 34.5 ± 2.8% and 34.0 ± 1.4%, respectively (P1-2,3<0.05). The plasma membrane integrity of spermatozoa in groups 2 and 3 (48.4 ± 2.9% and 45.5 ± 3.9%, respectively) was higher than in Group 1 (33.3 ± 2.1%, P < 0.05). After 24 h, 48 h and 72 h in vitro culture, the total motility and viability of spermatozoa in Group 1 was significantly lower than Group 2 and Group 3. The apoptosis rate in Group 3 (44.5 ± 3.0%) and Group 2 (47.7 ± 4.1%) were lower than in Group 1 (52.5 ± 4.4%; P < 0.05). ΔΨm rates in Group 3 and Group 2 were higher than in Group 1 (P < 0.05) with no statistical differences between this parameter in Group 2 and Group 3 (P > 0.1). In conclusion, supplementation of medium for aseptic capillary technology for cryoprotectant-free vitrification of human spermatozoa by permeable cryoprotectant does not improve the quality of spermatozoa after warming.


Assuntos
Preservação do Sêmen , Vitrificação , Capilares , Criopreservação/métodos , Crioprotetores/farmacologia , Humanos , Masculino , Motilidade Espermática , Espermatozoides , Tecnologia
9.
Int J Mol Sci ; 22(3)2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33513717

RESUMO

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGß, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.


Assuntos
Blastocisto/metabolismo , Criopreservação/métodos , Embrião de Mamíferos/embriologia , Suínos/embriologia , Suínos/metabolismo , Transcriptoma/genética , Vitrificação , Animais , Ciclo Celular/genética , Senescência Celular/genética , Transferência Embrionária , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Ontologia Genética , Redes Reguladoras de Genes , Sistema de Sinalização das MAP Quinases/genética , Análise em Microsséries , Análise de Sequência com Séries de Oligonucleotídeos , Suínos/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
10.
Cryobiology ; 98: 80-86, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33386123

RESUMO

Coral reefs worldwide are receding because of detrimental human activities, and cryopreservation of coral larvae would ensure that their genetic biodiversity is not irremediably lost. In recent years, the vitrification and laser warming of coral propagules has demonstrated promising results. During cryopreservation, cellular membranes undergo substantial reconfigurations that may affect survival. Fat enrichment may alter the physical proprieties of cell membranes and improve resistance to low temperatures. Therefore, the aim of this study was to determine whether supplementation of exogenous lipids using liposomes would improve cryosurvival and further development of the vitrified and laser-warmed coral larvae of Seriatopora caliendrum and Pocillopora verrucosa. A vitrification solution (VS) composed of 2 M ethylene glycol (EG), 1 M propylene glycol (PG), 40% (w/v) Ficoll, and 10% gold nanoparticles (at a final concentration of 1.2 × 1018 particles/m3 and an optimised emission wavelength of 535 nm) was chosen. Coral larvae were subjected to vitrification with VS incorporating one of four lipid classes: phosphatidylcholine (PC), phosphatidylethanolamine (PE), erucic acid (EA), and linoleic acid (LA). Warming was achieved using a single laser pulse (300 V, 10 ms pulse width, 2 mm laser beam diameter). A significantly higher vitality rate was observed in S. caliendrum larvae subjected to vitrification and laser warming with EA-incorporated VS, and P. verrucosa larvae vitrified and laser warmed using PE-incorporated VS achieved a significantly higher settlement rate. Our study demonstrated that supplementation of exogenous lipids with liposomes enhances coral larvae cryotolerance and improves cryopreservation outcomes. Lipid enrichment may play a key role in cryobanking coral propagules, and in propagule development after thawing.


Assuntos
Antozoários , Nanopartículas Metálicas , Animais , Criopreservação/métodos , Suplementos Nutricionais , Ouro , Larva , Lasers , Lipídeos , Lipossomos , Vitrificação
11.
Methods Mol Biol ; 2180: 593-605, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797437

RESUMO

Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples (1-3 mL total volume) where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, permits preservation of large samples (80-100 mL total volume) with several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at -80 °C; better matrix preservation than freezing with retention of material properties; very low cell viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is not used; and reduced manufacturing costs. More recently, we have developed Protocol 4 in which VS55 is supplemented with sugars resulting in reduced concerns regarding nucleation during cooling and warming. This method can be used for large samples resulting in retention of cell viability and permits short-term exposure to -80 °C with long-term storage preferred at or below -135 °C.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Valvas Cardíacas/citologia , Vitrificação , Animais , Sobrevivência Celular , Valvas Cardíacas/química , Valvas Cardíacas/efeitos dos fármacos , Humanos , Transição de Fase
12.
Methods Mol Biol ; 2180: 639-645, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797440

RESUMO

Plant cell cultures consist of single cells or cell clusters growing as callus or suspension. Such cell cultures may be able to produce secondary metabolites and/or possess embryogenic potential. Therefore, they can be used for very different purposes in research, biotechnological applications, as well as for plant propagation. Cryopreservation is the only practical method to preserve such cultures until they are needed. Different cryopreservation approaches that have been developed for differentiated plant tissues including slow freezing, vitrification, and encapsulation/dehydration have also been applied to plant cell cultures. The controlled rate or slow-freezing approach, however, remains to be the gold standard for cell cultures. In this chapter, a standard slow-freezing cryopreservation procedure in combination with alginate immobilization is presented for long-term preservation of plant cell cultures.


Assuntos
Alginatos/química , Técnicas de Cultura de Células/métodos , Criopreservação/métodos , Crioprotetores/farmacologia , Células Vegetais/metabolismo , Plantas/química , Proliferação de Células , Células Cultivadas , Congelamento , Plantas/efeitos dos fármacos , Vitrificação
13.
Methods Mol Biol ; 2180: 3-25, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797407

RESUMO

Cryopreservation and freeze-drying can be used to preserve cells or tissues for prolonged periods. Vitrification, or ice-free cryopreservation, is an alternative to cryopreservation that enables cooling cells to cryogenic temperatures in the absence of ice. The processing pathways involved in (ice-free) cryopreservation and freeze-drying of cells and tissues, however, can be very damaging. In this chapter, we describe the principles underlying preservation of cells for which freezing and drying are normally lethal processes as well as for cells that are able to survive in a reversible state of suspended animation. Freezing results in solution effects injury and/or intracellular ice formation, whereas drying results in removal of (non-freezable) water normally bound to biomolecules, which is generally more damaging. Cryopreservation and freeze-drying require different types of protective agents. Different mechanistic modes of action of cryoprotective and lyoprotective agents are described including minimizing ice formation, preferential exclusion, water replacement, and vitrification. Furthermore, it is discussed how protective agents can be introduced into cells avoiding damage due to too large cell volume excursions, and how knowledge of cell-specific membrane permeability properties in various temperature regimes can be used to rationally design (ice-free) cryopreservation and freeze-drying protocols.


Assuntos
Permeabilidade da Membrana Celular , Criopreservação/métodos , Crioprotetores/farmacologia , Liofilização/métodos , Tecidos Suporte/química , Vitrificação , Animais , Sobrevivência Celular , Humanos , Transição de Fase
14.
Methods Mol Biol ; 2180: 285-302, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797416

RESUMO

The development of freezing and freeze-drying processes for biological samples requires knowledge of the thermophysical properties of the biomaterial and protectant solutions involved. This chapter provides an introduction on the use of differential scanning calorimetry (DSC) to study thermophysical properties of biomaterials in protective solutions. It covers specific methods to study thermal events related to freezing and drying processes including crystallization, eutectic formation, glass transition, devitrification, recrystallization, melting, molecular relaxation, and phase separation.


Assuntos
Materiais Biocompatíveis/química , Varredura Diferencial de Calorimetria/métodos , Criopreservação/métodos , Crioprotetores/farmacologia , Composição de Medicamentos , Liofilização/métodos , Vitrificação , Animais , Bioensaio , Humanos , Transição de Fase
15.
Methods Mol Biol ; 2180: 303-315, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797417

RESUMO

Quantification of the amount of cryoprotective agent (CPA) in a tissue is an essential step in the design of successful cryopreservation protocols. This chapter details two inexpensive methods to measure cryoprotective agent permeation into tissues as functions of time. One of the methods to measure the CPA permeation is to permeate a series of tissue samples from a surrounding solution at a specified concentration of CPA, each sample for a different amount of time, and then to quantitate the amount of CPA that was taken up in the tissue during that time period. The quantification is performed by equilibrating the permeated tissue with a surrounding solution and then measuring the osmolality of the solution to determine the amounts of CPAs that have come out of each tissue sample corresponding to each permeation time. An alternative method to measuring the CPA permeation as a function of time, which requires fewer tissue samples, is to measure the CPA efflux as a function of time. In the efflux method, a CPA-permeated tissue sample is placed in a surrounding solution, and solution samples are taken at different time points throughout the efflux to quantitate how much CPA has left the tissue by each time point.


Assuntos
Cartilagem Articular/citologia , Criopreservação/veterinária , Crioprotetores/farmacologia , Vitrificação , Animais , Transporte Biológico , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Criopreservação/métodos , Concentração Osmolar , Suínos
16.
Methods Mol Biol ; 2180: 27-97, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797408

RESUMO

Vitrification is an alternative to cryopreservation by freezing that enables hydrated living cells to be cooled to cryogenic temperatures in the absence of ice. Vitrification simplifies and frequently improves cryopreservation because it eliminates mechanical injury from ice, eliminates the need to find optimal cooling and warming rates, eliminates the importance of differing optimal cooling and warming rates for cells in mixed cell type populations, eliminates the need to find a frequently imperfect compromise between solution effects injury and intracellular ice formation, and can enable chilling injury to be "outrun" by using rapid cooling without a risk of intracellular ice formation. On the other hand, vitrification requires much higher concentrations of cryoprotectants than cryopreservation by freezing, which introduces greater risks of both osmotic damage and cryoprotectant toxicity. Fortunately, a large number of remedies for the latter problem have been discovered over the past 35 years, and osmotic damage can in most cases be eliminated or adequately controlled by paying careful attention to cryoprotectant introduction and washout techniques. Vitrification therefore has the potential to enable the superior and convenient cryopreservation of a wide range of biological systems (including molecules, cells, tissues, organs, and even some whole organisms), and it is also increasingly recognized as a successful strategy for surviving harsh environmental conditions in nature. But the potential of vitrification is sometimes limited by an insufficient understanding of the complex physical and biological principles involved, and therefore a better understanding may not only help to improve present outcomes but may also point the way to new strategies that may be yet more successful in the future. This chapter accordingly describes the basic principles of vitrification and indicates the broad potential biological relevance of this alternative method of cryopreservation.


Assuntos
Permeabilidade da Membrana Celular , Criopreservação/métodos , Crioprotetores/farmacologia , Liofilização/métodos , Vitrificação , Animais , Sobrevivência Celular , Humanos
17.
Methods Mol Biol ; 2180: 427-436, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797425

RESUMO

Spermatozoa cryopreservation is used for the management of infertility and some other medical conditions. Routinely applied cryopreservation techniques depend on permeating cryoprotectants and relatively slow freezing rates. Cryoprotectant-free vitrification is an alternative and cost-effective method that is based on rapid cooling of spermatozoa by direct plunging into a cooling agent to prevent lethal intracellular ice crystallization and the detrimental effects of high salt concentrations. One of the problems with this technique is that full sterilization of commercially produced liquid nitrogen, which could be contaminated with different pathogens, is not possible. Here we use a benchtop device for the production of sterile liquid air with the same temperature as liquid nitrogen (-195.7 °C). This has been used to develop aseptic technology for cryoprotectant-free vitrification of human spermatozoa.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Congelamento , Preservação do Sêmen/métodos , Espermatozoides/citologia , Vitrificação , Humanos , Masculino , Transição de Fase , Espermatozoides/efeitos dos fármacos
18.
Methods Mol Biol ; 2180: 455-468, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797427

RESUMO

Oocyte cryopreservation is a potent approach to keep female germplasm safe from epidemic diseases. In the last decade, we developed simple, cheap, and robust vitrification protocols which enable quick cryopreservation of immature porcine oocytes and zygotes in large numbers. In this chapter, we describe vitrification procedures for porcine oocytes and zygotes where they are vitrified in 1-2 µL aliquots of a defined (protein-free) vitrification medium and dropped either on a metal surface pre-cooled from the bottom with liquid nitrogen (solid surface vitrification) or directly into liquid nitrogen. Vitrified microdrops can be stored in cryo-vials in liquid nitrogen. Low concentrations of permeating cryoprotectants during equilibration and proper temperatures during equilibration and warming are crucial for achieving high survival rates. The device used for cooling does not seem to affect system efficacy as vitrification of oocytes or zygotes either on Cryotop® sheets or in microdrops were equally effective.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Congelamento , Oócitos/citologia , Vitrificação , Zigoto/citologia , Animais , Temperatura Baixa , Criopreservação/métodos , Feminino , Nitrogênio , Oócitos/efeitos dos fármacos , Transição de Fase , Suínos , Zigoto/efeitos dos fármacos
19.
Methods Mol Biol ; 2180: 437-454, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797426

RESUMO

Two basic methods for the laboratory-focused cryopreservation of mammalian oocytes are described, based on work with murine oocytes. One method uses a relatively low concentration of the cryoprotectant propanediol plus sucrose and requires controlled rate cooling equipment to achieve a slow cooling rate. This method has also produced live births from cryopreserved human oocytes. The second method, which is described here, employs a high concentration of the cryoprotectant dimethyl sulfoxide plus a low concentration of polyethylene glycol. This is a vitrification method, which involves ultra-rapid cooling by plunging standard straws into liquid nitrogen vapor, hence avoiding the need for specialized equipment, but requires technical ability to manipulate the oocytes quickly in the highly concentrated cryoprotectant solutions. Murine oocytes that have been vitrified using this technique have resulted in live births. Vitrification using other cryoprotectant mixtures is now a popular clinically accepted method for cryobanking of human oocytes.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Congelamento , Oócitos/citologia , Vitrificação , Animais , Temperatura Baixa , Feminino , Camundongos , Oócitos/efeitos dos fármacos , Transição de Fase
20.
Methods Mol Biol ; 2180: 501-515, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797430

RESUMO

Cryopreservation is one of the keystones in clinical infertility treatment. Especially vitrification has become a well-established and widely used routine procedure that allows important expansion of therapeutic strategies when IVF is used to treat infertility. Vitrification of human blastocysts allows us to maximize the potential for conception from any one in vitro fertilization cycle and prevents wastage of embryos. This goes even further toward to best utilize a patient's supernumerary oocytes after retrieval, maximizing the use of embryos from a single stimulation cycle. The technology can even be used to eliminate fresh embryo transfers for reasons of convenience, uterine receptivity, fertility preservation, preimplantation genetic diagnosis, or emergency management. In this chapter, the application of vitrification technology for cryopreserving human blastocyst will be revealed through step-by-step protocols. The results that are presented using the described protocols underscore the robustness of the vitrification technology for embryo cryopreservation.


Assuntos
Blastocisto/citologia , Criopreservação/métodos , Crioprotetores/farmacologia , Embrião de Mamíferos/citologia , Preservação da Fertilidade/métodos , Vitrificação , Blastocisto/efeitos dos fármacos , Transferência Embrionária , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Fertilização In Vitro , Humanos , Gravidez
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