Changes to the meiotic spindle and zona pellucida of mature mouse oocytes following different cryopreservation methods
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Keywords:
Vitrification
Oocyte cryopreservation
Human chorionic gonadotropin
Vitrification
Cryoprotectant
Ice formation
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Loss of hepatocyte viability and metabolic function after cryopreservation is still a major issue. Although vitrification is a promising alternative, it has generally been proven to be unsuitable for vitrification of large cell volumes which is required for clinical applications. Here, we propose a novel bulk droplet (3–5 mm diameter) vitrification method which allows high throughput volumes (4 mL/min), while using a low preincubated CPA concentration (15% v/v) to minimize toxicity and loss of cell viability and function. We used rapid (1.25 s) osmotic dehydration to concentrate a low preincubated intracellular CPA concentration ahead of vitrification, without the need of fully equilibrating toxic CPA concentrations. We compared direct postpreservation viability, long-term viability, and metabolic function of bulk droplet vitrified, cryopreserved, and fresh hepatocytes. Simulations and cooling rate measurements confirmed an adequate concentration of the intracellular CPA concentration (up to 8.53 M) after dehydration in combination with high cooling rates (960–1320 °C/min) for successful vitrification. In comparison to cryopreserved hepatocytes, bulk droplet vitrified hepatocytes had a significantly higher viability, directly after preservation and after 1 day in culture. Moreover, bulk droplet vitrified hepatocytes had evidently better morphology and showed significantly higher metabolic activity than cryopreserved hepatocytes in long-term collagen sandwich cultures. In conclusion, we developed a novel bulk droplet vitrification method of which we validated the theoretical background and demonstrated the feasibility to use this method to vitrify large cell volumes. Moreover, we showed that this method results in improved hepatocyte viability and metabolic function as compared to cryopreservation.
Vitrification
Viability assay
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Cryopreservation of plant germplasm by vitrification technique has been appreciated extensively to date. Vitrification method consists of 5 steps mainly: loading, dehydration by protective solution of vitrification, cooling, warming and washing (namely unloading). There are hundreds of species of plants being cryopreserved by this method in the world up to now, but mainly of species of higher plants. However, few successful studies on the cryopreservation of algae germplasm by vitrification technique have been reported so far. Applying vitrification technique to cryopreservation of some species of algae may have significant potentia1.
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Germ plasm
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In comparison to the cryopreservation of preimplantation embryos,cryopreservation of oo- cytes from most mamrhalian species has been more challenging due to their extreme sensitivity to subopti- mal conditions during the cryopreservation process.Recent attempts to find a successful cryopreservation technique for oocytes have been directed to the use of vitrification by rapid cooling.They allow oocytes to be placed directly or preequilibrium for several minutes into the cryoprotectant and then plunged directly into liquid nitrogen.At present,vitrification techniques have entered the mainstream of animal reproduc- tion and many studies have been conducted in this area in recent years.In this paper,recent research on vitrification of oocytes were reviewed,with emphasis on the effects of type and concentration of cryopro- tect,cryopreservation methods and the meiotic stages of oocytes,and changes in the ultrastructure of the cell membrane,cytoskeleton,mitochondria,cortical granules and spindle.In conclusion,studies have shown that vitrification at the ultrarapid cooling rate is a promising approach for oocytes cryopreservation, but many problems remain to be solved on the damages of ultrastructure during cryopreservation process.
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Cryoprotectant
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The cryopreservation of cells has been in routine use for decades. However, despite the extensive research in the field, cryopreservation of large tissues and organs is still experimental. The present review highlights the major studies of directional freezing and vitrification of large tissues and whole organs and describes the different parameters that impact the success rate of large tissue and organ cryopreservation. Key factors, such as mass and heat transfer, cryoprotectant toxicity, nucleation, crystal growth, and chilling injury, which all have a significant influence on whole-organ cryopreservation outcomes, are reviewed. In addition, an overview of the principles of directional freezing and vitrification is given and the manners in which cryopreservation impacts large tissues and organs are described in detail.
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Cryoprotectant
Cryobiology
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Cell suspensions are valuable materials for plant physiology and biochemistry studies. To keep the genetic stability of plant cell suspensions, the cryopreservation technique is required. Vitrification is a method of cryopreservation without the usage of programmable freezers. In the present paper, cell suspensions from Arabidopsis, a model plant, were established and cryopreserved by vitrification. After suitable preculture and dehydration treatment, cells were quenched into liquid nitrogen directly. Thawed cells were able to recover, and recovered cells were able to regenerate plants. This is the first report of cryopreservation of Arabidopsis cell suspension cells by vitrification.
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Plant cell
Suspension culture
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Abstract This work highlights short-term storage of recalcitrant Lepisanthes fruticosa seeds and long-term conservation attempts of its embryonic axes (EAs) through cryopreservation. Short-term storage was carried out using fresh seeds at 54 % moisture content and stored at 8 ±1 °C and 25 ±2 °C for 7 weeks. Three variations to sterilization were attempted to optimize survival while keeping contamination low for cryopreservation. Cryopreservation using two different methods were tested, namely vitrification and the encapsulation vitrification method. Vitrification technique involved the pre-culturing of EAs overnight in different sucrose pre-culture concentrations (0, 0.2, 0.4 and 0.6 M) prior to, loading, dehydration with plant vitrification solution (PVS2), rapid immersion into liquid nitrogen (-196 °C), rapid warming, unloading and recovery. While, encapsulation vitrification involved encapsulation of the EAs using 3 % sodium alginate followed by exposure to different duration (0, 10, 20, 30, 40 and 50 minutes) of PVS2 prior to cryopreservation. L. fruticosa seeds can be safely stored for short-term with no loss in germination up to 7 weeks of storage either at 8 ±1 °C or 25 ±2 °C. This study also showed that EA of L. fruticosa was amenable to cryopreservation, 13.0 – 66.67% of viability was obtained when the EAs were cryopreserved using the vitrification technique while the best result was obtained (66.67 % viability) when the EAs were pre-cultured with 0.4 M sucrose prior to exposure to PVS2 and liquid nitrogen. Cryopreservation of EAs using the encapsulation-vitrification method was unsuccessful.
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Liquid nitrogen
Sterilization
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Cryopreservation can make the use of oocytes not constrained by time and space,and the cryopreserved oocytes can be used as materials for in vitro fertilization,nuclear transfer,transgene and other embryo engineering technology.Porcine oocytes are highly sensitive to lower temperatures due to rich lipid droplets in ooplasm.As a result,researches about cryopreservation of porcine oocytes get less progress.Vitrification is one of the effective methods for cryopreservation of oocytes.This paper discusses the effects of developmental stage of oocytes,cryopreservation carriers and cryoprotectant on cryopreservation of porcine oocytes by vitrification.
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Cryoprotectant
Oocyte cryopreservation
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In this paper, we compared three vitrification-based cryopreservation techniques, viz. vitrification, encapsulation-vitrification and droplet-vitrification for cryopreserving sugarcane somatic embryos. Viability of somatic embryos was evaluated by measuring electrolyte leakage and by regrowth on recovery medium. Droplet-vitrification was the most efficient technique. Optimal conditions included loading with a solution containing 1.5 M glycerol and 0.3 M sucrose for 30 min at 25 degree C, treatment with the PVS2 solution for 20-40 min at 0 degree C followed by rapid immersion in liquid nitrogen of clumps of somatic embryos placed in microdroplets of cryoprotectant solution. Under such conditions, viability of cryopreserved somatic embryos reached 55 percent.
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Cryoprotectant
Liquid nitrogen
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Purpose of review The objective of this review is to describe the physical and biological barriers to organ cryopreservation, historic approaches for conventional cryopreservation and evolving techniques for ice-free cryopreservation by vitrification. Recent findings Vitrification is a process whereby a biologic substance is cooled to cryogenic temperatures without the destructive phase transition of liquid to solid ice. Recent advances in cryoprotective solutions, organ perfusion techniques and novel heating technologies have demonstrated the potential for vitrification and rewarming organs on a scale applicable for human transplantation. Summary Successful strategies for organ cryopreservation could enable organ banking, which would recast the entire process in which organs are recovered, allocated, stored and prepared for transplant.
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