• Applied Microscopy
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• v.42 no.2
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• pp.111-114
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• 2012

The scanning electron microscopy is an ideal technique for examining plant surface at high resolution. Most hydrate samples, however, must be fix and dehydrate for observation in the scanning electron microscope. Because the microscopes operate under high vacuum, most specimens, especially biological samples, cannot withstand water removal by the vacuum system without morphological distortion. Cryo-techniques can observe in their original morphology and structure without various artifacts from conventional sample preparation. Rapid cooling is the method of choice for preparing plant samples for scanning electron microscopy in a defined physiological state. As one of cryo-technique, high-pressure freezing allows for fixation of native non-pretreated samples up to $200{\mu}M$ thick and 2 mm wide with minimal or no ice crystal damage for the freezing procedure. In this study, we could design to optimize structural preservation and imaging by comparing cryo-SEM and convention SEM preparation, and observe a fine, well preserved Arabidopsis stem's inner ultrastructure using HPF and cryo-SEM. These results would suggest a useful method of cryo-preparation and cryo-SEM for plant tissues, especially intratubule and vacuole rich structure.

• Restorative Dentistry and Endodontics
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• v.34 no.4
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• pp.356-363
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• 2009

The purpose of this study was to evaluate the viability of periodontal ligament cells in rat teeth using slow cryo-preservation method under pressure by means of MTT assay and WST-1 assay. Eighteen teeth of Sprague-Dawley white female rats of 4 week-old were used for each group. Both sides of the first and second maxillary molars were extracted as atraumatically as possible under Tiletamine anesthesia. The experimental groups were group 1 (Immediate control), group 2 (Cold preservation at $4^{\circ}C$for 1 week), group 3 (Slow freezing), group 4 (Slow freezing under pressure of 3 MPa). F-medium and 10% DMSO were used as preservation medium and cryo-protectant. For cryo-preservation groups, thawing was performed in $37^{\circ}C$water bath, then MTT assay and WST-1 assay were processed. One way ANOVA and Tukey method were performed at the 95% level of confidence. The values of optical density obtained by MTT assay and WST-1 were divided by the values of eosin staining for tissue volume standardization. In both MTT and WST-1 assay, group 4 showed significantly higher viability of periodontal ligament cells than group 2 and 3 (p < 0.05), but showed lower viability than immediate control group. By the results of this study, slow cryo-preservation method under pressure suggests the possibility for long term cryo-preservation of the teeth.

• Journal of Chest Surgery
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• v.28 no.8
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• pp.731-741
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• 1995

Cardiac valve allografts have been used as replacements for diseased valves and right ventricular outflow tract reconstruction, the long term follow-up of which has been reported satisfactory. For a good long-term result, it is essential that the allograft be viable at implantation. In this study, we aimed at preparing the cardiac valve allografts aseptically, preserving them at cold- and cryo-conditions, and testing the viability of the allografts after preservation by four methods. We tested the viability of the cardiac valve allografts preserved in cold refrigerated state[4$^{\circ}$C in nutrient media & in liquid nitrogen tank[cryopreservation under -149$^{\circ}$C for pre-planned time periods. The testing methods were 1 glucose utility test 2 tissue culture 3 thymidine uptake test and 4 histologic evidence by light microscopy. We observed no differences in the viability between cold- & cryo-groups and similar results among the methods for testing the viability. In conclusion, there was no difference in the viability between cold- and cryopreserved-allografts at least for 14 days of preservation. And glucose utility test and thymidine uptake test were satisfactory in the evaluation of the allograft viability, since they were easy and rapid with relatively quantitative results.

• Archives of Plastic Surgery
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• v.40 no.4
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• pp.374-379
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• 2013

Background To date, various types of acellular dermal matrix (ADM) have been developed for clinical use. AlloDerm is the most familiar type of ADM to most surgeons in breast reconstruction. It is prepared by freeze-drying. CG CryoDerm is the first form of ADM that requires no drying process. Therefore, theoretically, it has a higher degree of preservation of the dermal structures than AlloDerm. We conducted this study to compare the clinical course and postoperative outcomes of patients who underwent direct-to-implant breast reconstructions using AlloDerm and those who did using CG CryoDerm. Methods We performed a retrospective analysis of the medical records in a consecutive series of 50 patients who underwent direct-to-implant breast reconstruction using AlloDerm (n=31) or CryoDerm (n=19). We then compared the clinical course and postoperative outcomes of the two groups based on the overall incidence of complications and the duration of drainage. Results The mean follow-up period was 16 months. There were no significant differences in the overall incidence of complications (seroma, infection, skin flap necrosis, capsular contracture, and implant loss) between the two groups. Nor was there any significant difference in the duration of drainage. Conclusions CG CryoDerm has the merits of short preparation time and easy handling during surgery. Our results indicate that CG CryoDerm might be an alternative allograft material to AlloDerm in direct-to-implant breast reconstruction.

• Journal of Chest Surgery
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• v.29 no.11
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• pp.1173-1181
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• 1996

The causes of degenerative changes in allograft cardiac valves are not well known to this day. Today's preserved allografts possess highly viable endothelial cells and degeneration of allografts can be facilitated by immune reaction which may be mediated by these viable cells. To test the antigenicity of endothelial cells, pieces from aortic wall were obtained from fresh and cryo-preserved rat allograft. Timings of sampling were prior to sterilization, after sterilization, after 1, 2, 7, 14 days of fresh preservation and cryopreservation. Endothelial cells were tested by immunohistochemical methods using monoclonal antibodies to MHC class I(MRC OX-18), class II(MRC OX-6) and ICAM-1 antigens. After transplantation of each group of aortic allograft at the subcutaneous layers of rats, population of CD4$^{+}$ T cell and CD8$^{+}$ T cell were analyzed with monoclonal antibodies after 1, 2, 3, 4, 6 and 8 weeks. MHC class I expression was 23.95% before preservation and increased to 35.53~48.08% after preservation(p=0.0183). MHC Class II expression was 9.72% before preservation and 10.13~13.39% after preservation(P=0.1599). ICAM-1 expression was 15.02% before preservation and increased to 19.85~35.33% after preservation(P=0.001). The proportion of CD4$^{+}$ T-cell was 42.13% before transplantation. And this was 49.23~36.8% after transplantation in No treat group (p=0.955), decreased to 29.56~32.80% in other group(p=0.0001~0.008). In all the groups, the proportion of CD8$^{+}$ T-cell increased from 25.57% before transplantation to 42.32~58.92% after transplantation(p=0.000l~0.0002). The CD4$^{+}$/CD8$^{+}$ ratio decreased from 1.22~2.28 at first week to 0.47~0.95 at eighth week(p=0.0001). The results revealed that the expression of MHC class I and ICAM-1 in aortic allograft endothelium were increased but that of MHC class II were not changed, despite the different method of preservation. During 8 weeks after transplantation of aortic allograft, the subpopulations of CD4$^{+}$ T cell were not changed or only slightly decreased but those of CD8$^{+}$ T cell were progressively increased.ely increased.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.225-227
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• 1996

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.256-258
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• 1996

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.63-69
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• 2012

The objective of this study was to investigate the effectiveness of cryopreservation methods for the effect of various vitrification containers, such as EM-grid, OPS, or cryo-loop on the survival and developmental rate of vitrified mouse pronuclear embryos, and mouse cleavage embryo, at 21, 24, 27 and 30 hr after hCG injection. Post-thaw cleavage was similar among treatments, while the developmental rates of mouse blastocyst and hatched blastocyst were higher ($p$ <0.05) in 27 hr and 30 hr than 21 hr. The developmental rate of hatched blastocyst at vitrified cleavage mouse embryos in cryo-loop was significantly higher than vitrified pronuclear embryos of control group as well as EM-grid and OPS ($p$ <0.05). The developmental rate using cryo-loop was higher than EM-grid, but in case of OPS at vitrified cleavage and mouse pronuclear embryos, no significant difference was noticed. These results of our study show that the developmental rates of mouse embryos were unaffected by various vitrification containers, but in case of mouse embryos and hatched blastocysts at late vitrified pronuclear embryos the developmental rates were higher than early vitrified pronuclear embryos. Moreover, the developmental rate of hatched blastocyst at vitrified cleavage mouse embryos was significantly higher than vitrified pronuclear embryos. For better execution of this study, it will be mandatory to include improvement of vitrification containers, cryopreservation methods and conditions, higher survival rate, safe preservation, contamination and embryo loss.

• Reproductive and Developmental Biology
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• v.41 no.2
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• pp.41-50
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• 2017

Until today, success in germline cells and tissue cryopreservation is limited mainly due to the poor understanding of the complex physiological processes can lead to cell damage during cryopreservation. Germline cells, from both male and female, have unique ability to differentiate into one or more cell lines and thus it becomes a crucial point to store them in subzero temperature with the minimal damage of their functional properties and maximum recovery of unchanged and viable cells when thawed. In the past three decades, a vast research has been performed using various different animal models which in fact have led to development of new methodologies and optimization of older one. However, successful use of animal model has provided the opportunity in research with human germline cells and tissues preservation, but not in all the cases. Therefore, the use of new cryo-protective chemicals and modified protocols have been often found in different groups of researchers based on the types, physical structures, utility and animal species of the specimens to be cryopreserved. This review discusses about the basics of different types of cryopreservation methodologies and commonly used optimized protocols and cryoprotectants for germline cells and tissues preservation.

• Restorative Dentistry and Endodontics
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• v.35 no.4
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• pp.285-294
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• 2010

The purpose of this study was to evaluate the viability of periodontal ligament cells of rat teeth after low-temperature preservation under high pressure by means of MTT assay, WST-1 assay. 12 teeth of Sprague-Dawley white female rats of 4 week-old were used for each group. Both side of the first and second maxillary molars were extracted as atraumatically as possible under tiletamine anesthesia. The experimental groups were group 1 (Immediate extraction), group 2 (Slow freezing under pressure of 3 MPa), group 3 (Slow freezing under pressure of 2 MPa), group 4 (Slow freezing under no additional pressure), group 5 (Rapid freezing in liquid nitrogen under pressure of 2 MPa), group 6 (Rapid freezing in liquid nitrogen under no additional pressure), group 7 (low-temperature preservation at $0^{\circ}C$ under pressure of 2 MPa), group 8 (low-temperature preservation at $0^{\circ}C$ under no additional pressure), group 9 (low-temperature preservation at $-5^{\circ}C$ under pressure of 90 MPa). F-medium and 10% DMSO were used as preservation medium and cryo-protectant. For cryo-preservation groups, thawing was performed in $37^{\circ}C$ water bath, then MTT assay, WST-1 assay were processed. One way ANOVA and Tukey HSD method were performed at the 95% level of confidence. The values of optical density obtained by MTT assay and WST-1 were divided by the values of eosin staining for tissue volume standardization. In both MTT and WST-1 assay, group 7 ($0^{\circ}C$/2 MPa) showed higher viability of periodontal ligament cells than other group (2-6, 8) and this was statistically significant (p < 0.05), but showed lower viability than group 1, immediate extraction group (no statistical significance). By the results of this study, low-temperature preservation at $0^{\circ}C$ under pressure of 2 MPa suggest the possibility for long term preservation of teeth.