• Journal of Marine Life Science
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• v.1 no.1
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• pp.70-78
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• 2016

Microalgae are of significant importance for future biotechnological applications. Many microalgae banks or laboratories attempt to maintain various microalgae for further research purposes. Cryopreservation has been preferred to reduce a labor-intensive and costly routine sub-culturing. Cryopreservation can also diminish the genetic drift risk. However, cryopreservation as a long term storage of microalgae method are still in developing progress because it cannot be generalized for all microalgae. Microalgae types, cryoprotectant agents (CPAs) types, freezing and thawing methods are the most important factors that should be considered for cryopreservation. In this short review the basic principles and the current advanced of microalgae cryopreservation methods are discussed with a suggested starting parameters for microalgae cryopreservation.

• ALGAE
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• v.21 no.1
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• pp.125-131
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• 2006

Long-term preservation of bloom-forming cyanobacteria was evaluated using cryopreservation and freeze-drying of nine strains belonging to four genera and seven species. All test strains, except Aphanizomenon flos-aquae NIER- 10028, showed partial or complete survival following cryopreservation and freeze-drying. Frozen and freeze-dried strains were preserved for more than two years and were revived monthly. Most strains showed higher post-thaw viability after cryopreservation, especially without cryoprotectant compared to freeze-drying. Microcystis aeruginosa NIER-10010, M. viridis NIER-10020, M. ichthyoblabe NIER-10023, M. novacekii NIER-10029 and Oscillatoria sancta NIER-10027 were revived after 2.5 years of cryopreservation. These results suggest that cryopreservation may be an easy and timesaving long-term preservation method for bloom-forming cyanobacteria.

• Korean Journal of Plant Resources
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• v.34 no.6
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• pp.600-607
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• 2021

Cryopreservation method using a droplet vitrification was applied to the thirty-one strawberry accessions of in vitro grown shoot tips. A protocol with 0.3 - 0.5 M preculture followed by C4 loading and B1 dehydration solutions efficiently implemented cryopreservation of twenty-six strawberry accessions. The highest regrowth rate was 85.8% for PHS0007 and others were ranged between 85.8% and 21.0%. A slightly modified protocol was applied to five accessions. With these two protocols, twenty-eight accessions obtained more than 40% regrowth rate. This study showed that the droplet vitrification method was able to practically implement cryopreservation of in vitro grown shoot tips of broad range of strawberry germplasm (105).

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.127-135
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• 1999

Cryopreservation is able to store the surplus pre-embryos for freezing and furthermore thawing and transfer in a subsequent cycle. Cryopreserving cells which are maintaining their viability are the very complex process. This study has been carried out in order to find the effects of cryopreservation steps, freezing media and embryonic stages on the rates of viability and development of cryopreserved mouse embryos. Female ICR mice ($6{\sim}8$ weeks old) were induced to superovulate by sequential intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h apart. Mouse embryos were collected according to its developmental stage after the injection of hCG. Embryos were cryopreserved not only by cryoprotectant step (1 step${\sim}$4 step) but also in a variety of media (HTF, IVF medium, D-PBS) and cell stage. The results were as follows: There is no clear advantage in these freezing media of rapid method, but 4 cell and 8 cell of slow method (2, 3, 4 step) have advantage in D-PBS. The development of embryos according to cell stage become greater in 8 cell stage. In the treatment steps of cryopreservation, the development of embryo to blastocyst was similar among rapid method, but the development of 4 cell and 8 cell embryos to blastocyst according to slow method was better than rapid method.

• Journal of Forest and Environmental Science
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• v.36 no.4
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• pp.274-280
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• 2020

As the importance of biological resources increases, the conservation technology is becoming important for rarities. This study was conducted to establish an efficient cryopreservation conditions for the Deutzia paniculata, endangered plant species, by using both cryopreservation methods of vitrification and encapsulation. As a result, the sucrose pretreatment seed viability showed up to 30.7% in the treatments. The cryoprotectant treatment improved the viability of the seeds, and was found to be excellent in the vitrification method using PVS3. The vitrification method had over 10% higher germination rate than the seeds preserved by encapsulation. In addition, the germination rate showed a significant difference according to the cryopreservation treatment time, and the germination rate of seeds decreased very much as the long time became longer. Plants germinated from preserved seed in liquid nitrogen showed poor growth compared to untreated, and good growth in PVS3 120 minutes. In addition, the growth of germinated plants by liquid nitrogen treatment time was better in the vitrification method. These results are expected to be useful for long-term preservation of D. paniculata, endangered plants.

• The Journal of Korean Obstetrics and Gynecology
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• v.32 no.2
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• pp.119-128
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• 2019

Objectives: The aim of this case is to report the effects of herbal medicine on a single woman patient with a low level of AMH (anti-$M{\ddot{u}}llerian$ Hormone) in progress of Oocyte Cryopreservation. Methods: A patient with a low level of AMH had symptom of secondary amenorrhea. For preparing oocyte cryopreservation after a long time of secondary amenorrhea, she was treated by twice a day herb medication for 10 months. And we observed the effects of treatments by improvement of symptoms and following up endometrium ultrasonography. After oocyte cryopreservation, for maintaining her menstruation, she was also treated by twice a day herb medication for two and a half months. Results: After treatments, symptom of amenorrhea was improved and the thickness of endometrium was increased as well as AMH in progress of oocyte cryopreservation. So 20 oocytes could be cryopreserved. Conclusions: This case shows that herbal medicine can be a concurrent method for a single woman patient with secondary amenorrhea in progress of oocyte cryopreservation.

• Journal of Embryo Transfer
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• v.4 no.1
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• pp.1-13
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• 1989

This paper reviews the most important steps that have generated consistent progress in principles and developmental progress of embryo cryopreservation, and also study on freezing procedure and its application by conventional method and current improved method for freezing procedure and its appilcation of embryo cryopreservation in farm animals. Four were of particular interest: 1.The transport of water across the ccli membrane (zona pellucida) during freezing and thawing accordinglyplays a role in determing whether the celi survives. This movement of water is controlied mainly by extracellular phase changes and by the nature and concentration of any cryoprotective agent present. Therates of cooling, freezing and warming, and the intervals over which they are applied are further decisi've factors in determining whether a cryopreservation procedure allows survival after thawing. 2.The first successful deep freezing experiments with sheep morula and blastocysts during the seventies were based on the early procedures used for mouse embryos.Current research during the eighties is developed with the aim of simplifying and improving current procedures such as one-step dilution and rapid or ultra-rapid cooling by using the model of laboratory animals. 3.The conventional method for the embryo cryopreservation is described. An alternative to this method which may result in high survival and also in reducing of the freezing and thawing time is done by combing a permeable cryoprotectant such as glycerol, DMSO or propanediol and a non-permeable compound such as sucrose, trehalose, raffinose or lactose. 4.Finally a different approach to the preservation of embryos, named vitrification, is introduced. This procedure depends upon the ability of concentrated solutions of cryoprotective agents such as glycerol and propanediol to supercool to very low temperature (-196$^{\circ}C$) during rapid cooling before solidifying without formation of ice. However, more complete data are necessary for successful vitrification of blastocysts.

• Horticultural Science & Technology
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• v.30 no.1
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• pp.79-84
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• 2012

An efficient protocol for cryopreservation of madder hairy root cultures has been developed using droplet-vitrification. In previous study, combining loading solution C4 (35% PVS3) and vitrification solution B5 (80% PVS3) was the most effective method. In this study, we tried three types of vitrification solution, B5, A3 (90% PVS2, on ice), and A5 (70% PVS2, on ice). Combining loading solution C4 and vitrification solution A5 (on ice) showed the best regeneration rate in this study. Histological changes of the cells within the hairy root of madder were also observed in different steps. The cells from the hairy roots of the control treatment were full and intact with different size of vacuoles and obvious cell nucleus having a dark nucleolus. After the stage of preparing for cryopreservation (after preculturing, loading, followed by dehydration solution A5 or B5), intercellular spaces had become distinct, and within cells, the cytoplasms had become denser and week plasmolyses had appeared. The cell plasmolyses were much more apparent and we measured the degree of plasmolysis by calculating, the area of cell/the area of cytoplasm. The value of plasmolysis degree was the highest in the combination of preculture, loading solution C4, and dehydration solution A5, 1.97. Because the highest regeneration rates appeared in the treatment of A5 for 20 min, we could assume that the optimal degree of plasmolysis for cryopreservation might be around 1.97. The changes in cell structure during cryopreservation might be a useful basis for the development of a proper long-term preservation method for madder germplasms.

• ALGAE
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• v.24 no.4
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• pp.257-265
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• 2009

Marine microalgae are a key diet component in finfish and shellfish aquaculture. Cryopreservation of the microalgae is suggested by many other studies as the best method for long-term storage. To test cryopreservation efficacy, 19 taxas of marine microalgal species were examined. In the first experiment we compared dimethylsulfoxide ($Me_2SO$) and glycerol, which are most widely used as cryoprotectant agents (CPAs). The cryopreservation comprised two freezing procedures. Firstly, the samples containing the CPAs were kept at $4^{\circ}C$ for 10 min before being plunged into liquid nitrogen ($-196^{\circ}C$). Secondly, samples containing CPAs were pre-cooled ($-1^{\circ}C$ $min^{-1}$ to $-80^{\circ}C$ before being plunged into liquid nitrogen. Most of the species were successfully cryopreserved using $Me_2SO$, whereas the Prasinophyceae (T. striata and T. suecica) were successfully cryopreserved using glycerol. In general, the cooling method had no influence on the survival of the microalgae except in the case of the Tetraselmis species. In the second experiment, the cultured solution was divided before cryopreservation into concentrated and non-concentrated groups to identify the effect of cell density during cryopreservation. After 12 months of storage, the samples were again divided into centrifugation and non-centrifugation groups to learn the effect of $Me_2SO$ on the culture. Viability and growth of the microalgae were not influenced by cell density or the centrifugal removal of the $Me_2SO$ after thawing.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.207-213
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• 2020

Cryopreservation is used for blastocyst preservation of most mammalian embryos and is an important technique for breeding. We aimed to compare the efficiency of the cryopreservation method using the standard Cryotop device and the ReproCarrier device, a domestic product manufactured in Korea. The efficacy of the two devices was analyzed based on the survival rate, intracellular levels of reactive oxygen species (ROS), and apoptosis of the vitrified bovine blastocysts. The survival rates of the vitrified-warmed blastocysts were similar between the ReproCarrier group (58.4 ± 17.7%) and Cryotop group (59.9 ± 14.1%). Intracellular ROS levels and apoptotic index were determined by DCFDA staining and TUNEL assay. Changes in intracellular ROS levels, number of total nuclei, and cellular apoptosis of vitrified blastocysts after cryopreservation were not significantly different between the two groups. These results indicate that the ReproCarrier device method is as effective as the standard Cryotop method for vitrification of bovine blastocysts in vitro.