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Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution

World Journal of Hepatology 2012年第5期4卷 176-183页

作者： Mohammed Saliem Frida Holm Rosita Bergstrm Tengzelius Carl Jorns Lisa-Mari Nilsson Bo-Gran Ericzon Ewa Ellis Outi Hovatta Division of Obstetrics and Gynecology Department of Clinical ScienceIntervention and TechnologyKarolinska Institute141 86 StockholmSweden Division of Transplantation Surgery Department of Clinical ScienceIntervention and TechnologyKarolinska Institute141 86 StockholmSweden Division of Pediatrics Department of Clinical ScienceIntervention and TechnologyKarolinska Institute141 86 StockholmSweden

AIM:To optimize a xeno-free cryopreservation protocol for primary human ***:The demand for cryopreserved hepatocytes is increasing for both clinical and research *** several hepatocyte cryopreservation protocols being available,improvements are urgently *** first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the *** the polystyrene box freezing,we compared two xeno-free freezing solutions for freezing of primary human hepatocytes:a new medium(STEM-CELLBANKER,CB),which contains dimethylsulphoxide(DMSO) and anhydrous dextrose,both permeating and non-permeating cryoprotectants,and the frequently used DMSO-University of Wisconsin(DMSOUW) *** viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after *** function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms(CYPs):CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 and ***:The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol(P cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte ***,it may be a better alternative to the standard DMSO-UW *** CYP activities did not seem to be a relevant way to compare hepatocyte function

关键词： Human hepatocytes Viability Cytochrome P540 Dimethylsulphoxide cryoprotectant Cryopreservation

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Exploring novel cell cryoprotectants based on neutral amino acids

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Chinese Journal of Chemical Engineering 2020年第10期28卷 2640-2649页

作者： Xiaojie Sui Pengguang Chen Chiyu Wen Jing Yang Qingsi Li Lei Zhang Department of Biochemical Engineering School of Chemical Engineering and TechnologyTianjin UniversityTianjin 300350China Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering(MOE) School of Chemical Engineering and TechnologyTianjin UniversityTianjin 300350China Qingdao Institute for Marine Technology of Tianjin University Qingdao 266235China

cryoprotectants play a key role in cell cryopreservation because they can reduce cryoinjuries to cells associated with ice *** meet the clinical requirements of cryopreserved cells,cryoprotectants should be biocompatible,highly efficient and easily removable from cryopreserved ***,integration of these properties into one cryoprotectant still remains ***,three biocompatible neutral amino acids,includingβ-alanine,γ-aminobutyric acid andε-aminocaproic acid,are first reported to have the potential as such ideal *** results demonstrate that they can inhibit ice formation and reduce osmotic stress to provide extracellular and intracellular protection,thereby ensuring high cryopreservation efficiency for both anuclear and nucleated *** importantly,due to the remarkable osmotic regulation ability,the neutral amino acids can be rapidly removed from cryopreserved cells via a one-step method without causing observable damage to cells,superior to the current state-of-the-art cryoprotectants—dimethyl sulfoxide and *** work provides a new perspective to develop novel cryoprotectants,which may have dramatic impacts on solvent-free cryopreservation technology to support the cell-based applications,such as cell therapy and tissue engineering,etc.

关键词： Biomedical engineering Cell engineering Tissue engineering Cryopreservation cryoprotectant Neutral amino acid

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Update on techniques for cryopreservation of human spermatozoa

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Asian Journal of Andrology 2022年第6期24卷 563-569页

作者： Chuan Huang Yu-Lin Tang Jian-Ling Hu Wen-Jun Zhou Zeng-Hui Huang Xue-Feng Luo Zheng Li Wen-Bing Zhu Institute of Reproductive and Stem Cell Engineering Basic Medicine CollegeCentral South UniversityChangsha 410000China Reproductive and Genetic Hospital of CITIC-Xiangya Changsha 410000China Department of Andrology Center for Men's HealthShanghai General HospitalShanghai Key Lab of Reproductive MedicineShanghai 200025China

In the 1960s,sperm cryopreservation was developed as a method to preserve ***,techniques for the cryopreservation of human spermatozoa have been widely used in assisted ***,although sperm cryobiology has made notable achievements,the optimal method for the recovery of viable spermatozoa after cryopreservation remains *** sperm quality can be affected by cryoprotectants,ice formation,storage conditions,and osmotic stress during the freezing *** review discusses recent advances in different cryopreservation techniques,cryoprotectants,and freezing and thawing methods during cryopreservation and new indications for the use of cryopreserved spermatozoa.

关键词： cryodamage cryoprotectant dry preservation freezing and thawing sperm cryopreservation

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Influencing factors on the preservation of lytic bacteriophage VP3

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Biosafety and Health 2022年第5期4卷 314-320页

作者： Yue Xiao Pin Huang Zhenzhou Huang Keyi Yu Yang Song Ning Guo Hang Dai Mengnan Jiang Yi Xu Duochun Wang Qiang Wei Center for Human Pathogenic Culture Collection National Institute for Communicable Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China Institute of Biothermal Science&Technology the University of Shanghai for Science and TechnologyShanghai 200093China National Pathogen Resource Center Chinese Center for Disease Control and PreventionBeijing 102206China

Long-term and stable preservation of bacteriophages is of crucial *** many efforts have been made in the past decades to explore the influence of external factors on bacteriophage preservation,there is still little understanding,and a systematic description is *** this study,we explored the influence of different factors on the preservation of lytic bacteriophage VP3,one of the typing bacteriophages of Vibrio cholerae O1 biotype El Tor,and attempted to optimize its *** examined external factors,including temperature,solution,and cryoprotectant,in stable cooling/freezing conditions or alternate cooling/freezing and *** found that whether in Luria-Bertani(LB)medium or SM buffer,in terms of 20-week stable cooling or freezing,−20℃ was the most damaging while 4℃,−80℃,and−196℃ were *** cycles of alternate cooling/freezing and thawing caused a loss in the survival rates of *** addition of cryoprotectant,glycerol(30%,w/v)or dimethyl sulfoxide(DMSO,10%,w/v)significantly improved the survival rates of bacteriophages preserved at−20℃.However,at 4℃,−80℃,and−196℃,the cryoprotectant effect was only slightly positive or even *** summary,for bacteriophage VP3,the best preservation method is to directly preserve the bacteriophage stocks in LB medium at−80℃ or−196℃ instead of storing them in SM buffer or adding *** results provided insights into the external influencing factors on bacteriophage VP3 during preservation at low temperature and can be applied to the optimization of bacteriophage preservation in the future.

关键词： Influencing factors Preservation Lytic bacteriophage cryoprotectant

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Study on Cryopreservative Effect of Three Penetrating cryoprotectants with Three Kinds of Extenders on Rabbit Spermatozoa

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Journal of Northeast Agricultural University(English Edition) 2001年第1期8卷 30-36页

作者： ZHANGGui－xue LIXiao－juan 等 NortheastAgriculturalUniversity HarbinHeilongjiangPRC150030

Glycerol,dimethyl suffixod (DMSO) and acetamide were adopted to verify the cryoprotective ability of penetrating cryoprotectants with three kinds of extenders for rabbit sperm. Three extenders (A,B and C) and three level of the final concentrations of glycerol and DMSO (2%, 3% and 4% ),and acetamide 2%,4% and 5% were used with Extender A, 4% glycerol had better cryoprotective effect for rabbit sperm motility and 5% acetamide was better with Extender C. 4% acetamide was better in being combined with Extender A for sperm acrosome integrity ratio,5% acetamide was better with Extender B and 2% acetamide was better with Extende C. With extender A,there was not difference among three penetrating cryoprotectants for plasma membrane integrity. With Extender B, 3% glycerol was better than 4% acetamide for plasma membrane in- tegrity,and 3% glycerol with Extender C was better than 2% - 4% DMSO and 2% acetamide.

关键词： rabbit spermatozoa cryopreservation extender cryoprotectant

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An improved loopless mounting method for cryocrystallography

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Chinese Physics B 2010年第1期19卷 212-221页

作者：齐建勋江凡 Beijing National Laboratory for Condensed Matter Physics Institute of PhysicsChinese Academy of Sciences

Based on a recent loopless mounting method, a simplified loopless and bufferless crystal mounting method is developed for macromolecular crystallography. This simplified crystal mounting system is composed of the following components： a home-made glass capillary, a brass seat for holding the glass capillary, a flow regulator, and a vacuum pump for evacuation. Compared with the currently prevalent loop mounting method, this simplified method has almost the same mounting procedure and thus is compatible with the current automated crystal mounting system. The advantages of this method include higher signal-to-noise ratio, more accurate measurement, more rapid flash cooling, less x-ray absorption and thus less radiation damage to the crystal. This method can be extended to the flash-freeing of a crystal without or with soaking it in a lower concentration of cryoprotectant, thus it may be the best option for data collection in the absence of suitable cryoprotectant. Therefore, it is suggested that this mounting method should be further improved and extensively applied to eryocrystallographic experiments.

关键词： cryoprotectant crystal mounting data collection flash cooling

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Improving native human sperm freezing protection by using a modified vitrification method

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Asian Journal of Andrology 2021年第1期23卷 91-96页

作者： Dai Zhou Xing-Ming Wang Rui-Xue Li Yi-Ze Wang Yuan-Chi Chao Zhi-Zhong Liu Zeng-Hui Huang Hong-Chuan Nie Wen-Bing Zhu Yue-Qiu Tan Li-Qing Fan Hnstitute of Reproduction and Stem Cell Engineering School of Basic Medicine ScienceCentral South UniversityChangsha 410000China Reproductive and Genetic Hospital of CITIC-Xiangya Changsha 410000China Department of Urology Hunan Cancer HospitalChangsha 410000China

Slow freezing is the most commonly used technique for the cryopreservation of spermatozoa in clinical ***,it has been shown to have a negative impact on sperm function and *** as a successful alternative method has been proved to have better protective effects on human embryos,but vitrification of spermatozoa is still subject to low recovery *** this study,a modified vitrification method for native spermatozoa was developed.A total of 28 semen samples were included;each sample was divided into three equal parts and assigned to fresh,slow freezing,and vitrification *** vitality,motility,morphology,DNA integrity,and acrosome reaction were assessed for each of the *** results showed that vitrification achieves better results for several sperm protection parameters than slow freezing;vitrification achieves a higher recovery rate(P<0.05),motility(P<0.05),morphology(P<0.05),and curve line velocity(P<0.05)than slow ***,DNA fragmentation was decreased(P<0.05)and better acrosome protection(P<0.05)was exhibited in the spermatozoa after *** component analysis of all sperm parameters revealed that the vitrification cluster was closer to the fresh cluster,indicating that spermatozoa are better preserved through *** conclusion,while both slow freezing and vitrification have negative effects on sperm function and structure,the vitrification protocol described here had a relatively better recovery rate(65.8%)and showed improved preservation of several sperm quality parameters compared with slow freezing.

关键词： cryopreservation cryoprotectant slow freezing sperm vitrification

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Major physiological adjustments in freezing-tolerant grey tiger longicorn beetle(Xylotrechus rusticus) during overwintering period

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Journal of Forestry Research 2014年第3期25卷 653-659页

作者： Jue-wen Li Juan Shi Yu Xue Hong-bo Mao You-qing Luo Beijing Key Laboratory of Forest Pest Control Beijing Forestry University College of Forestry Northeast Forestry University Forestry Administration of Acheng District

1Xylotrechus rusticus （Linnaeus） is one of the most destruc-tive woodborers in northeast China;it damages poplar and is listed as a domestic forestry quarantine pest. Overwintering larvae were collected during October 2012 and March 2013 in Harbin, China, to quantify indi-cators related to the insect’s overwintering strategy and the major cryo-protectants. The supercooling points （SCPs）, which ranged from-14.7＆#176;C to -2.9＆#176;C, were higher than the lethal temperatures of LT50 （-33.64＆#176;C） and LT99 （-40.17＆#176;C） after 24 h exposure. , also the minimum mean daily temperature （-24.5＆#176;C） and mean monthly temperature （-18.0＆#176;C） at the sampling site in January during 2008-2012. Thus, X. rusticus is a typical freezing-tolerant insect. Glycerol serves as a major cryoprotectant for overwintering larvae , because it was the only polyol accumulated during the winter and it also had a significant negative correlation with the SCP （p=0.033, R=0.907）. The glycogen and lipid are major sources of ener-gy and their levels decreased substantially in the middle of overwintering, when glycogen had a significant correlation with the SCP （p= 0.006, R=0.971） whereas the lipid contents did not. Moreover, inter-conversions between glycerol and glycogen, as well as mannose and glycogen, were suggested by their negative correlations. The water content did not change obviously during the winter and was not correlated with the SCP. The free amino acids in the hemolymph and the total protein contents of the bodies of larvae changed significantly during winter, although both had no correlations with the SCP.

关键词： cold hardiness cryoprotectant freezing tolerant physiological variation Xylotrechus rusticus.

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Advances in Cryopreservation of Organs

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2016年第2期36卷 153-161页

作者：刘迪潘峰 Department of Urology Union HospitalTongji Medical UniversityHuazhong University of Science and Technology

Organ transplantation is an effective approach for the treatment of end-stage organ failures. Currently, the donor organs used for clinical transplantation are all preserved at above-zero temperatures. These preservation methods are well-established and simple but the storage time lasts for only 4–12 h. Some researchers tried to extend the organ storage time by improving protectant and HLA matching to raise the use of stored organs and prolong the long-term survival of organs. These efforts still fall short of the clinical demand for organ transplantation. Moreover, a great many organs were wasted due to limited storage time, HLA mismatch, patients＇ conditions or distance involved. Therefore, preserving organs for several weeks or even months and establishing Organ Bank are the tough challenges and have become a shared goal of global scholars. This article reviews some issues involved in the cryopreservation of organs, such as use of cryoprotecting agents, freezing and thawing methods in the cryopreservation of hearts, kidneys and other organs.

关键词： cryopreservation organ preservation organ transplantation cryoprotectant vitrification antifreeze proteins

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Growth and Freeze-Drying Optimization of Bifidobacterium crudilactis

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Food and Nutrition Sciences 2016年第7期7卷 616-626页

作者： Jean Tanimomo Véronique Delcenserie Bernard Taminiau Georges Daube Catherine Saint-Hubert Alain Durieux Meurice R & D Brussels Belgium Food Sciences Department Faculty of Veterinary Medicine Fundamental and Applied Research for Animal & Health (FARAH) University of Liège Liège Belgium Institut de Recherches Microbiologiques Jean-Marie Wiame Campus CERIA Brussels Belgium

Bifidobacterium crudilactis FR62/b/3 belongs to a new population of bifidobacteria isolated from raw milk and raw milk cheese. The objective of this work was to study the large scale culture of the strain and its stability in a dry formulation. Growth rate of Bifidobacterium crudilactis FR62/b/3 was optimal at a pH of 5.0 and a temperature of 37°C. At a temperature growth of 33°C and a pH of 5.0, the stationary phase was reached after 22 h, the viable cell number and the mean dry biomass concentration were respectively of 8.3 × 109 CFU/mL and of 2.1 g/L. Resistance of Bifidobacterium crudilactis FR62/b/3 to freeze-drying and effect of a variety of cryoprotectants to maintain the viability were also evaluated. Sorbitol was the most suitable cryoprotectant for freeze-drying as well as storage whereas sucrose and monosodium glutamate were only efficient during storage.

关键词： Probiotic Bifidobacteria Crudilactis cryoprotectant Freeze-Drying

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