Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 179
Filtrar
1.
Cryobiology ; 99: 106-113, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33382993

RESUMO

Laboratory rearing of mosquitoes is commonly practiced by researchers studying mosquito-borne infectious diseases and vector control methods. In the absence of cryopreservation methods to stabilize unique or genetically modified strains, mosquito lines must be continuously maintained, a laborious process that risks selection effects, contamination, and genetic drift. Towards the development of a cryopreservation protocol, several commonly used cryoprotectants were systematically characterized here both individually and as cocktails. Among first instar, feeding-stage An. gambiae and An. stephensi larvae, cryoprotectant toxicity followed the order of dimethyl sulfoxide > ethylene glycol > methanol. The resulting LD50 values were used as the basis for the development of cryoprotectant cocktail solutions, where formulation optimization was streamlined using Taguchi methods of experimental design. Sensitivity to hypothermia was further evaluated to determine the feasibility of cryoprotectant loading at reduced temperatures and slow cooling approaches to cryopreservation. The information described here contributes to the knowledge base necessary to inform the development of a cryopreservation protocol for Anopheles larvae.


Assuntos
Anopheles , Hipotermia , Animais , Criopreservação/métodos , Crioprotetores/toxicidade , Larva , Mosquitos Vetores
2.
Biochim Biophys Acta Gen Subj ; 1865(1): 129749, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32980500

RESUMO

BACKGROUND: Cryopreservation is a key method of preservation of biological material for both medical treatments and conservation of endangered species. In order to avoid cellular damage, cryopreservation relies on the addition of a suitable cryoprotective agent (CPA). However, the toxicity of CPAs is a serious concern and often requires rapid removal on thawing which is time consuming and expensive. SCOPE OF REVIEW: The principles of Cryopreservation are reviewed and recent advances in cryopreservation methods and new CPAs are described. The importance of understanding key biophysical properties to assess the cryoprotective potential of new non-toxic compounds is discussed. MAJOR CONCLUSIONS: Knowing the biophysical properties of a particular cell type is crucial for developing new cryopreservation protocols. Similarly, understanding how potential CPAs interact with cells is key for optimising protocols. For example, cells with a large osmotically inactive volume may require slower addition of CPAs. Similarly, a cell with low permeability may require a longer incubation time with the CPA to allow adequate penetration. Measuring these properties allows efficient optimisation of cryopreservation protocols. GENERAL SIGNIFICANCE: Understanding the interplay between cells and biophysical properties is important not just for developing new, and better optimised, cryopreservation protocols, but also for broader research into topics such as dehydration and desiccation tolerance, chilling and heat stress, as well as membrane structure and function.


Assuntos
Permeabilidade da Membrana Celular , Criopreservação/métodos , Crioprotetores/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/química , Crioprotetores/toxicidade , Humanos , Estabilidade Proteica/efeitos dos fármacos
3.
Cryobiology ; 88: 98-105, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30826335

RESUMO

Cryoprotective agents (CPAs) are used in cryopreservation protocols to achieve vitrification. However, the high CPA concentrations required to vitrify a tissue such as articular cartilage are a major drawback due to their cellular toxicity. Oxidation is one factor related to CPA toxicity to cells and tissues. Addition of antioxidants has proven to be beneficial to cell survival and cellular functions after cryopreservation. Investigation of additives for mitigating cellular CPA toxicity will aid in developing successful cryopreservation protocols. The current work shows that antioxidant additives can reduce the toxic effect of CPAs on porcine chondrocytes. Our findings showed that chondroitin sulphate, glucosamine, 2,3,5,6-tetramethylpyrazine and ascorbic acid improved chondrocyte cell survival after exposure to high concentrations of CPAs according to a live-dead cell viability assay. In addition, similar results were seen when additives were added during CPA removal and articular cartilage sample incubation post CPA exposure. Furthermore, we found that incubation of articular cartilage in the presence of additives for 2 days improved chondrocyte recovery compared with those incubated for 4 days. The current results indicated that the inclusion of antioxidant additives during exposure to high concentrations of CPAs is beneficial to chondrocyte survival and recovery in porcine articular cartilage and provided knowledge to improve vitrification protocols for tissue banking of articular cartilage.


Assuntos
Cartilagem Articular/citologia , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Crioprotetores/toxicidade , Animais , Ácido Ascórbico/farmacologia , Cartilagem Articular/efeitos dos fármacos , Fenômenos Fisiológicos Celulares , Sulfatos de Condroitina/farmacologia , Criopreservação/métodos , Crioprotetores/farmacologia , Glucosamina/farmacologia , Pirazinas/farmacologia , Suínos , Bancos de Tecidos , Vitrificação
4.
Mol Cell Biochem ; 450(1-2): 43-52, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29790115

RESUMO

The protective activity of N-(2-hydroxyphenyl)acetamide (NA-2) and NA-2-coated gold nanoparticles (NA-2-AuNPs) in glycerol-treated model of acute kidney injury (AKI) in mice was investigated. NA-2 (50 mg/kg) and NA-2-AuNPs (30 mg/kg) were given to the animals for four days followed by 24-h water deprivation and injection of 50% glycerol (10 ml/kg im). The animals were sacrificed on the next day. Blood and kidneys were collected for biochemical investigations (urea and creatinine), histological studies (hematoxylin and eosin; and periodic acid-Schiff staining), immunohistochemistry (actin and cyclooxygenase-2, Cox-2), and real-time RT-PCR (inducible nitric oxide synthase, iNOS; nuclear factor-κB p50, NFκB; hemeoxygenase-1, HO-1; and kidney injury molecule-1, Kim-1). NA-2 protected renal tubular necrosis and inflammation, though the result of NA-2-AuNPs was better than compound alone and it also exhibited the activity at far less dose. The test compound and its gold nano-formulation decreased the levels of serum urea and creatinine level in the treated animals. Both NA-2 and NA-2-AuNPs also conserved actin cytoskeleton, and lowered COX-2 protein expression. Moreover, the mRNA expressions of iNOS and NFkB p50 were down-regulated, and HO-1 and Kim-1 genes were up-regulated. We conclude that NA-2 and NA-2-AuNPs ameliorates kidney inflammation and injury in glycerol-induced AKI animal model via anti-oxidant and anti-inflammatory mechanisms which make it a suitable candidate for further studies. We believe that these findings will contribute in the understanding of the mechanism of action of paracetamol-like drugs and can be considered for clinical research for the prevention of AKI.


Assuntos
Acetanilidas/farmacologia , Lesão Renal Aguda/prevenção & controle , Glicerol/toxicidade , Ouro/química , Inflamação/prevenção & controle , Nanopartículas Metálicas/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Rabdomiólise/prevenção & controle , Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/metabolismo , Animais , Apoptose , Crioprotetores/toxicidade , Modelos Animais de Doenças , Inflamação/metabolismo , Masculino , Nanopartículas Metálicas/química , Camundongos , Camundongos Endogâmicos BALB C , Rabdomiólise/induzido quimicamente , Rabdomiólise/metabolismo
5.
Cryobiology ; 86: 71-76, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30527584

RESUMO

We have looked at the effects of the cryoprotectant M22 upon viability in the model organism C. elegans. M22 is a well-known vitrification solution which has been successfully used in the laboratory to preserve organs destined for transplantation. M22 reduces survival of C. elegans in a concentration-dependent manner. M22 at concentrations of 10% (v/v) or higher inhibits progeny production and development. A few mutants in the ILS (insulin-like signaling) pathway of C. elegans are more resistant to the toxic effect of M22 compared to wild-type worms. Afatinib, an anti-cancer drug, protects against M22 toxicity. Afatinib by itself does not increase longevity.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Vitrificação/efeitos dos fármacos , Afatinib/química , Animais , Proteínas de Caenorhabditis elegans , Crioprotetores/toxicidade , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Formamidas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
6.
Cryobiology ; 86: 84-88, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30476465

RESUMO

The natural population of the edible red sea urchin, Loxechinus albus, is decreasing due to overfishing. The embryos and larvae of the species are highly useful for monitoring marine pollution, which makes it necessary to conserve gametes, embryos and larvae to facilitate their use in diverse areas of aquaculture and environmental quality monitoring. This need can be met by cryopreserving individuals representing the different developmental stages to provide an ongoing supply of genetic material of the species. The present study establishes a reproducible protocol for cryopreserving red sea urchin blastula and larvae. Toxicity tests were conducted in the first stage of this study using two permeable cryoprotectors, dimethyl sulfoxide (Me2SO) and propylene glycol (PG), at three concentrations (5%, 10% and 15%). The tests were repeated in the second stage, but mixing the cryoprotectors with 0.04 M of trehalose (TRE), a non-permeable cryoprotector. Cryopreservation tests were conducted in the third stage employing different freezing rates: 2 °C/min, 3 °C/min, 3.5 °C/min, 4 °C/min and 4.5 °C/min, using the cryoprotectors that yielded the highest post-incubation survival rates. The highest post-freezing survival rates for blastula (76 ± 7%) and larvae (79 ± 7%) were obtained with Me2SO at 10% + 0.04 M of trehalose, with freezing rates of 3 °C/min and 4.5 °C/min, respectively.


Assuntos
Blástula/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Larva/efeitos dos fármacos , Ouriços-do-Mar/embriologia , Animais , Conservação dos Recursos Naturais , Crioprotetores/toxicidade , Dimetil Sulfóxido/farmacologia , Pesqueiros , Congelamento , Propilenoglicol/farmacologia , Trealose/farmacologia
7.
Cryobiology ; 86: 95-102, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30458175

RESUMO

We report here a new, unbiased forward genetic method that uses transposon-mediated mutagenesis to enable the identification of mutations that confer cryoprotectant toxicity resistance (CTR). Our method is to select for resistance to the toxic effects of M22, a much-studied whole-organ vitrification solution. We report finding and characterizing six mutants that are resistant to M22. These mutants fall into six independent biochemical pathways not previously linked to cryoprotectant toxicity (CT). The genes associated with the mutations were Gm14005, Myh9, Nrg2, Pura, Fgd2, Pim1, Opa1, Hes1, Hsbp1, and Ywhag. The mechanisms of action of the mutations remain unknown, but two of the mutants involve MYC signaling, which was previously implicated in CT. Several of the mutants may up-regulate cellular stress defense pathways. Several of the M22-resistant mutants were also resistant to dimethyl sulfoxide (Me2SO), and many of the mutants showed significantly improved survival after freezing and thawing in 10% (v/v) Me2SO. This new approach to overcoming CT has many advantages over alternative methods such as transcriptomic profiling. Our method directly identifies specific genetic loci that unequivocally affect CT. More generally, our results provide the first direct evidence that CT can be reduced in mammalian cells by specific molecular interventions. Thus, this approach introduces remarkable new opportunities for pharmacological blockade of CT.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Crioprotetores/toxicidade , Células-Tronco Embrionárias/citologia , Estresse Fisiológico/genética , Supressão Genética/genética , Animais , Linhagem Celular , Elementos de DNA Transponíveis/genética , Dimetil Sulfóxido/farmacologia , Dimetil Sulfóxido/toxicidade , Etilenoglicol/farmacologia , Etilenoglicol/toxicidade , Formamidas/farmacologia , Formamidas/toxicidade , Congelamento , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese/genética , Estresse Fisiológico/efeitos dos fármacos , Vitrificação/efeitos dos fármacos
8.
Andrology ; 6(6): 836-845, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30105872

RESUMO

INTRODUCTION: Human sperm freezing is very widely used for male fertility preservation. This procedure consists in adding cryoprotectants to the spermatozoa followed by cooling and storing the spermatozoa at a subzero temperature. Many standardized cryopreservation media are available on the market. However, these media differ in their chemical composition and there are no sufficient data to optimize their classification. Therefore, the aim of this study was to compare five commercially available sperm cryopreservation media, which have not been compared together, in terms of motility, morphology and DNA integrity. MATERIALS AND METHODS: One-hundred semen samples were obtained from 10 fertile participants and 90 infertile men. Each sample was evaluated before freezing for motility, morphology and DNA fragmentation index (DFI). Then, it was equally divided into five aliquots. Each aliquot was cryopreserved using one of the five media (A, B, C, D, and E). The same parameters were re-evaluated after the addition of the cryopreservation media in the fertile group, and after sperm thawing in fertile and infertile groups. RESULTS: The results showed that the five selected cryopreservation media had negative effects on sperm motility and morphology per se. In the infertile group, the cryosurvival factor was significantly lower in cryomedium A when compared to the four other media (p < 0.001). In addition, a significantly higher percentage of sperm with coiled tail was detected in cryomedium E compared to cryomedium A (p < 0.05) and to cryomedium B (p < 0.001) after thawing, in the infertile group. Furthermore, the sperm DFI was significantly higher in cryomedia A (p < 0.001), B (p < 0.001), C (p < 0.01), D (p < 0.01) and E (p < 0.05) compared to that of the fresh semen derived from infertile participants. CONCLUSION: This study indicates that the recovery rate of competent spermatozoa, after cryopreservation, is still critical in infertile men. Therefore, frozen semen sample should be used only when necessary.


Assuntos
Forma Celular/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Dano ao DNA/efeitos dos fármacos , Infertilidade Masculina/patologia , Preservação do Sêmen/métodos , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Estudos de Casos e Controles , Crioprotetores/toxicidade , Congelamento , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Estudos Prospectivos , Medição de Risco , Preservação do Sêmen/efeitos adversos , Espermatozoides/metabolismo , Espermatozoides/patologia
9.
Sci Rep ; 8(1): 4279, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29523856

RESUMO

Diatoms constitute the most diverse group of microalgae and have long been recognised for their large biotechnological potential. In the wake of growing research interest in new model species and development of commercial applications, there is a pressing need for long-term preservation of diatom strains. While cryopreservation using dimethylsulfoxide (DMSO) as a cryoprotective agent is the preferred method for long-term strain preservation, many diatom species cannot be successfully cryopreserved using DMSO. Therefore, in this study, we studied cryopreservation success in six different diatom species, representing the major morphological and ecological diatom groups, using a range of DMSO concentrations and Plant Vitrification Solution 2 (PVS2) as an alternative cryoprotectant to DMSO. In addition, we tested whether suppressing bacterial growth by antibiotics accelerates the post-thaw recovery process. Our results show that the effects of cryoprotectant choice, its concentration and the addition of antibiotics are highly species specific. In addition, we showed that PVS2 and antibiotics are useful agents to optimize cryopreservation of algae that cannot survive the traditional cryopreservation protocol using DMSO. We conclude that a species-specific approach will remain necessary to develop protocols for diatom cryopreservation and to increase their representation in public culture collections.


Assuntos
Criopreservação/métodos , Diatomáceas/efeitos dos fármacos , Crioprotetores/toxicidade , Dimetil Sulfóxido/toxicidade
10.
Anim Reprod Sci ; 192: 185-192, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29576468

RESUMO

This study focused on the quality of sperm obtained from spermatophores of cadaveric shrimp after long-term storage. Spermatophores were collected using the stripping method, which has resulted in maximum sperm viability when this approach was previously used. Cryoprotectants toxicity assessment of samples was conducted using dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG), glycerol (Gly), dimethyl acetamide (DMA) and propylene glycol (PG) at different concentrations (5%, 10% and 30% v/v), prepared in Ca-F saline. Based on the results from the cryoprotectant toxicity assay, DMSO and MeOH were used individually as well as in combination for the subsequent study. Samples along with cryoprotectants were subjected to slow and fast freezing protocols (i.e. -0.5, and -10 °C/min to a final temperature of -80 °C) and were subsequently stored in LN2 (196 °C). Similarly, vitrification was performed by plunging the samples directly in to LN2. Samples of control and cryopreserved spermatophores that were stored for 45 days had sperm viabilities of 91.4 ±â€¯3.6% and 53.9 ±â€¯4.7%, respectively. Further observations with HOST and DNA integrity analyses of the cryopreserved sperm, resulted in percentages of 45.6 ±â€¯4.2%; 58.1 ±â€¯1.7% compared to control values of 82.3 ±â€¯4.8%; 94.3 ±â€¯1.9%, respectively. Use of the one-step slow freezing protocol at the rate of -0.5 °C/min between 4 °C and -80 °C in LN2 with DMSO (5%) + MeOH (5%) was a desirable preservation strategy of spermatophores, compared to other freezing protocols. Unlike sperm viability, the HOST results affirm the fertility potential of the sperm that have the capacity to participate in the fertilization process. Thus, the results of this study demonstrate that long term storage of sperm in spermatophores of Fenneropenaeus indicus collected from cadaveric specimens can result in viable sperm after cryopreservation if extender (Ca-F saline) containing DMSO and MeOH are used.


Assuntos
Criopreservação , Penaeidae/fisiologia , Espermatogônias/fisiologia , Espermatozoides/fisiologia , Animais , Crioprotetores/farmacologia , Crioprotetores/toxicidade , Masculino
11.
Theranostics ; 7(19): 4735-4752, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29187900

RESUMO

Rationale: Dimethyl sulfoxide (DMSO) is commonly used as a solvent for water-insoluble substances, a vehicle for drug therapy, and a cryoprotectant for cultured cells. DMSO induced embryonic defects and its mechanism of action remains unclear. The rationale is based on the assumption that DMSO supplementation should induce long-term negative effects on both pre- and post-implantation embryo development. Methods: DMSO induced oxidative stress, ER stress, autophagy, mitophagy, signaling responsible genes and proteins were determined by RT-qPCR, Western blotting, immunofluorescence, and confocal microscopy. DMSO induced mitochondrial dysfunction was measured by transmission electron microcopy and JC-1 assay. Apoptosis was estimated using TUNEL and comet assay. Post-implantation embryo developmental capability was estimated by implantation site and fetus numbers. Results: Exposure to DMSO induced an early oxidative stress response within 0.5 to 2 h in 1-cell zygotes by disrupting the balance of pro- and anti-oxidants. Notably, DMSO-treated 2-cell embryos showed increased expression of unfolded protein response genes such as Hspa5, Hsp90b1, Ddit3, Atf4, and Xbp1. As a result, the development of many embryos is arrested at the 2-cell, 4-cell, or morula stages in a dose-dependent manner. Further, DMSO-induced endoplasmic reticulum stress increased mitochondrial Ca2+ levels, induced mitochondrial depolarization/dysfunction, and induced apoptotic cell death via the JNK/ATF2-dependent pathway. Consequently, treatment with DMSO increased the expression of autophagy initiation-, phagophore elongation-, and autophagosome formation-related genes, as well as localization of PINK1/Parkin, which are the main mediators of mitophagy, in mitochondria. Interestingly, DMSO causes cytotoxic effects in preimplantation embryos by inducing extensive mitophagy and autophagy. Especially, DMSO treatment decreased the inner cell mass and trophectoderm cell numbers as well as mRNA expression of B3gnt5 and Wnt3a in developed blastocysts, which decreased the implantation and developmental rates of full-term offspring after being transferred into pseudopregnant mice. Conclusion: These results provide a significant contribution to finding effective protective agents to combat DMSO mediated reproductive toxicity for application in human embryos in the near future.


Assuntos
Blastocisto/efeitos dos fármacos , Crioprotetores/toxicidade , Dimetil Sulfóxido/toxicidade , Desenvolvimento Embrionário/efeitos dos fármacos , Animais , Apoptose , Autofagia , Blastocisto/metabolismo , Cálcio/metabolismo , Células Cultivadas , Feminino , Camundongos , Dinâmica Mitocondrial , Estresse Oxidativo , Resposta a Proteínas não Dobradas
12.
Cryobiology ; 78: 1-7, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28803845

RESUMO

Low survival of cryopreserved sperm impedes the application of cryopreservation technique in spermcasting oyster species. This study developed a simple method of liquid nitrogen vapor freezing to improve post-thaw sperm survival in the spermcasting oyster Ostrea angasi. The results indicate that the permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were non-toxic to sperm up to 20% concentration and 90 min exposure whereas methanol at 10% or higher was toxic to sperm for any exposure over 30 min. Among the treatments with permeable cryoprotectants, 15% EG produced the highest post-thaw sperm motility. Sperm motility was further improved by the addition of non-permeable cryoprotectants (trehalose and glucose), with 15% EG + 0.2 M trehalose resulting in the highest post-thaw sperm motility among all the combinations evaluated. The durations of 20, 30 and 60 min equilibrations produced a higher post-thaw sperm motility and plasma membrane integrity (PMI) than 10 min. Higher post-thaw motility and PMI were achieved by freezing sperm at the 8 cm height from the liquid nitrogen surface than at the 2, 4, 6, 10 or 12 cm height. Holding sperm for 10 min in liquid nitrogen vapor produced higher post-thaw motility and PMI than for 2, 5 or 20 min. The cryopreservation protocol developed in this study improved both post-thaw motility and PMI of O. angasi sperm at least 15% higher than those cryopreserved using programmable freezing method. Liquid nitrogen vapor freezing might have greater applicability in improving post-thaw sperm quality of spermcasting oyster species.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/toxicidade , Preservação do Sêmen/métodos , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Dimetil Sulfóxido/toxicidade , Etilenoglicol/farmacologia , Etilenoglicol/toxicidade , Glucose/farmacologia , Glucose/toxicidade , Masculino , Metanol/farmacologia , Metanol/toxicidade , Ostrea , Propilenoglicol/farmacologia , Propilenoglicol/toxicidade , Trealose/farmacologia , Trealose/toxicidade
13.
Reprod Biomed Online ; 35(3): 311-313, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28645837

RESUMO

There have been 60 births after transplantation of cryopreserved ovarian tissue: 58 using the slow freezing method, and two using the vitrification method. DMSO and EG are widely used as cryoprotectants. However DMSO is a known epimutagen, and EG has been reported to be toxic in high concentrations. In this study, we measured residual DMSO and EG in ovarian tissue after vitrification and slow freezing. Cryoprotectants remained at a high concentration in the vitrified/warmed ovarian tissue just before transplantation (DMSO: 9.8 mg/g, EG: 9.8 mg/g). We must consider the impact of the cryoprotectants on the mother and the baby.


Assuntos
Criopreservação , Dimetil Sulfóxido/farmacologia , Resíduos de Drogas/toxicidade , Etilenoglicol/farmacologia , Oócitos/efeitos dos fármacos , Ovário , Células Cultivadas , Criopreservação/métodos , Crioprotetores/farmacologia , Crioprotetores/toxicidade , Dimetil Sulfóxido/toxicidade , Transferência Embrionária/efeitos adversos , Transferência Embrionária/métodos , Etilenoglicol/toxicidade , Feminino , Congelamento , Humanos , Recém-Nascido , Exposição Ocupacional/efeitos adversos , Oócitos/química , Oócitos/citologia , Folículo Ovariano/química , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Ovário/química , Ovário/efeitos dos fármacos , Gravidez , Vitrificação
14.
Cryobiology ; 75: 68-74, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28192075

RESUMO

High concentrations of cryoprotective agents are required for cryopreservation techniques such as vitrification. Glycerol is a common cryoprotective agent used in cryopreservation protocols but this agent is toxic at high concentrations. This work is an attempt to mitigate the toxic effects of high concentrations of glycerol on intact chondrocytes in human knee articular cartilage from total knee arthroplasty patients by simultaneous exposure to glycerol and a variety of additive compounds. The resulting cell viability in the cartilage samples as measured by membrane integrity staining showed that, in at least one concentration or in combination, all of the tested additive compounds (tetramethylpyrazine, ascorbic acid, chondroitin sulphate, glucosamine sulphate) were able to reduce the deleterious effects of glycerol exposure when examination of membrane integrity took place on a delayed time frame. The use of additive compounds to reduce cryoprotectant toxicity in articular cartilage may help improve cell recovery after cryopreservation.


Assuntos
Condrócitos , Criopreservação/métodos , Crioprotetores/farmacologia , Crioprotetores/toxicidade , Glicerol/toxicidade , Idoso , Idoso de 80 Anos ou mais , Ácido Ascórbico/farmacologia , Cartilagem Articular/citologia , Sobrevivência Celular/efeitos dos fármacos , Sulfatos de Condroitina/farmacologia , Glucosamina/farmacologia , Humanos , Pessoa de Meia-Idade , Pirazinas/farmacologia , Vitrificação
15.
J Pediatr Hematol Oncol ; 39(5): e297-e299, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28121745

RESUMO

Dimethyl sulfoxide (DMSO) is a cryoprotective agent used in storage of frozen stem cells in stem cell transplantation. Central nervous system side effects of DMSO such as epileptic seizures, stroke, transient global amnesia, and temporary leucoencephalopathy are rarely seen. Here, we report a pediatric patient who developed seizures after DMSO-cryopreserved stem cell infusion and whose magnetic resonance imaging of the brain demonstrated parietal and occipital focal cortical T2-signal intensity increase. DMSO toxicity should be kept in mind in patients who received cryopreserved stem cell infusion and magnetic resonance imaging may be helpful in differential diagnosis of central nervous system involvement.


Assuntos
Crioprotetores/toxicidade , Dimetil Sulfóxido/toxicidade , Síndromes Neurotóxicas/etiologia , Transplante de Células-Tronco/efeitos adversos , Adolescente , Aloenxertos , Criopreservação/métodos , Diagnóstico Diferencial , Dimetil Sulfóxido/uso terapêutico , Feminino , Humanos , Síndromes Neurotóxicas/diagnóstico por imagem
16.
J Immunol Methods ; 439: 74-78, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27594593

RESUMO

Although cryopreserved cell specimens are used throughout biomedical research, the process for thawing samples is labor-intensive and prone to error. Here we describe a small laboratory device that couples an uncapped vial of frozen cells to a conical tube containing warm cell culture media. The entire complex is loaded directly into a centrifuge; within 5min, cells are thawed and diluted out of toxic cryopreservation medium. The recovery and viability of cells are slightly reduced compared to the common (traditional) method. However, antigen-specific T-cell function is not affected. Since no technician time is required (beyond uncapping of vials), our device allows the parallel processing of as many samples as a centrifuge can hold (up to 96, in some models). Moreover, since the samples are not thawed manually in a water bath, the problems associated with technician-to-technician differences in sample handling are minimized, as is the potential for contamination. Importantly, the elimination of substantial labor involving subjective decisions standardizes this process and can reduce variability in results from cryopreserved specimens.


Assuntos
Centrifugação/instrumentação , Criopreservação , Linfócitos T/fisiologia , Automação Laboratorial , Sobrevivência Celular , Centrifugação/normas , Crioprotetores/toxicidade , Meios de Cultura/química , Desenho de Equipamento , Citometria de Fluxo , Humanos , Linfócitos T/efeitos dos fármacos , Fatores de Tempo
17.
PLoS One ; 11(6): e0158074, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27348312

RESUMO

Meiotic maturation of mammalian oocytes is a precisely orchestrated and complex process. Dimethyl sulfoxide (DMSO), a widely used solvent, drug, and cryoprotectant, is capable of disturbing asymmetric cytokinesis of oocyte meiosis in mice. However, in pigs, DMSO's effect on oocyte meiosis still remains unknown. We aimed to evaluate if DMSO treatment will affect porcine oocyte meiosis and the underlying molecular changes as well. Interestingly, we did not observe the formation of the large first polar body and symmetric division for porcine oocytes treated with DMSO, contrary to findings reported in mice. 3% DMSO treatment could inhibit cumulus expansion, increase nuclear abnormality, disturb spindle organization, decrease reactive oxygen species level, and elevate mitochondrial membrane potential of porcine oocytes. There was no effect on germinal vesicle breakdown rate regardless of DMSO concentration. 3% DMSO treatment did not affect expression of genes involved in spindle organization (Bub1 and Mad2) and apoptosis (NF-κB, Pten, Bcl2, Caspase3 and Caspase9), however, it significantly decreased expression levels of pluripotency genes (Oct4, Sox2 and Lin28) in mature oocytes. Therefore, we demonstrated that disturbed cumulus expansion, chromosome alignment, spindle organization and pluripotency gene expression could be responsible for DMSO-induced porcine oocyte meiotic arrest and the lower capacity of subsequent embryo development. Our results provide new insights on DMSO's effect on porcine oocyte meiosis and raise safety concerns over DMSO's usage on female reproduction in both farm animals and humans.


Assuntos
Ciclo Celular , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Meiose , Oócitos/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Animais , Apoptose , Núcleo Celular/efeitos dos fármacos , Crioprotetores/toxicidade , Dimetil Sulfóxido/toxicidade , Feminino , Mitocôndrias/efeitos dos fármacos , Oócitos/citologia , Suínos
18.
Reprod Fertil Dev ; 28(5): 599-607, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25209652

RESUMO

Embryos produced from vitrified feline oocytes have resulted in pregnancies, but the efficiency of oocyte vitrification in cats is still low. Our objectives were to evaluate the effects of exposing feline oocytes to ethylene glycol (EG), propanediol (PrOH) and dimethyl sulfoxide (DMSO) on changes in intracellular free-calcium concentrations ([Ca(2+)]i), the time needed for enzymatic digestion of the zona pellucida (ZP), the incidence of parthenogenetic activation and degeneration and embryonic development following in vitro fertilisation (IVF). All of the chemicals tested altered [Ca(2+)]i, but changes in [Ca(2+)]i, resistance of the ZP to enzymatic digestion and the incidence of parthenogenetic activation (<5% for all treatments) were not affected (P>0.05) by extracellular Ca(2+). Exposure to EG (>44.1%) and DMSO (19.7%) increased (P<0.05) oocyte degeneration compared with control oocytes and oocytes exposed to PrOH (≤2.5%). Following exposure to a combination of PrOH and DMSO (10% v/v each), blastocyst development (per cleaved embryo; 52.1%) was similar (P>0.05) to control oocytes (64.4%). When oocytes were vitrified with PrOH and DMSO, 28.3% of surviving (intact plasma membrane) oocytes cleaved following IVF, but no blastocyst developed. When a non-permeating cryoprotectant (galactose, 0.25M) was added to the vitrification medium, 47.7% of surviving oocytes cleaved and 14.3% developed to the blastocyst stage.


Assuntos
Cálcio/metabolismo , Permeabilidade da Membrana Celular , Criopreservação , Crioprotetores/farmacologia , Fertilização In Vitro , Técnicas de Maturação in Vitro de Oócitos , Oócitos/efeitos dos fármacos , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Blastocisto/patologia , Gatos , Células Cultivadas , Fase de Clivagem do Zigoto/efeitos dos fármacos , Crioprotetores/toxicidade , Dimetil Sulfóxido/farmacologia , Técnicas de Cultura Embrionária , Etilenoglicol/farmacologia , Feminino , Oócitos/metabolismo , Oócitos/patologia , Partenogênese/efeitos dos fármacos , Propilenoglicóis/farmacologia , Vitrificação
19.
PLoS One ; 10(11): e0142828, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26605546

RESUMO

Ice-free cryopreservation, known as vitrification, is an appealing approach for banking of adherent cells and tissues because it prevents dissociation and morphological damage that may result from ice crystal formation. However, current vitrification methods are often limited by the cytotoxicity of the concentrated cryoprotective agent (CPA) solutions that are required to suppress ice formation. Recently, we described a mathematical strategy for identifying minimally toxic CPA equilibration procedures based on the minimization of a toxicity cost function. Here we provide direct experimental support for the feasibility of these methods when applied to adherent endothelial cells. We first developed a concentration- and temperature-dependent toxicity cost function by exposing the cells to a range of glycerol concentrations at 21°C and 37°C, and fitting the resulting viability data to a first order cell death model. This cost function was then numerically minimized in our state constrained optimization routine to determine addition and removal procedures for 17 molal (mol/kg water) glycerol solutions. Using these predicted optimal procedures, we obtained 81% recovery after exposure to vitrification solutions, as well as successful vitrification with the relatively slow cooling and warming rates of 50°C/min and 130°C/min. In comparison, conventional multistep CPA equilibration procedures resulted in much lower cell yields of about 10%. Our results demonstrate the potential for rational design of minimally toxic vitrification procedures and pave the way for extension of our optimization approach to other adherent cell types as well as more complex systems such as tissues and organs.


Assuntos
Criopreservação/métodos , Crioprotetores , Células Endoteliais , Animais , Bovinos , Técnicas de Cultura de Células , Sobrevivência Celular , Crioprotetores/toxicidade , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Glicerol , Modelos Teóricos , Pressão Osmótica , Temperatura , Vitrificação
20.
Cryo Letters ; 36(3): 158-64, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26510333

RESUMO

BACKGROUND: Signaling of Insulin-like growth factor-I (IGF-I) is involved in development, growth, reproduction and aging of organisms. OBJECTIVE: The work investigated the toxicity of glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol (EG) to hen clam (Mactra chinensis) embryos, as well as the possible role of the insulin-like growth factor-I (IGF-I) during the development and growth of embryos after freeze. MATERIALS AND METHODS: Effects of glycerol, DMSO and EG at different concentrations were tested. The relationship between larval viability and signaling of IGF-I receptor after cryoprotective treatment and/or freezing was examined using immuno-blot analysis. RESULTS: Glycerol had the highest toxicity, followed by DMSO or EG. No embryo survived freeze and thaw without CPAs. After freeze, the activation of the IGF-I signaling pathway, including the IGF-I receptor (IGF-IR) ß-subunit, could be detected in freeze-thawed embryos. The level of IGF-IR expression was very weak in freeze-thawed embryos. CONCLUSION: The survival and developmental rate of embryos was closely related to CPA concentration. IGF-IR was activated and regulated the downstream IGF-I signaling in embryos. The reduced activation of IGF-IR could be related to the death of hen clam embryos.


Assuntos
Bivalves/embriologia , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Glicerol/farmacologia , Receptor IGF Tipo 1/metabolismo , Animais , Bivalves/efeitos dos fármacos , Criopreservação , Crioprotetores/toxicidade , Dimetil Sulfóxido/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/fisiologia , Etilenoglicol/toxicidade , Glicerol/toxicidade , Transdução de Sinais/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...