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Effect of Liposome Charge and Composition on the Delivery of Trehalose into Red Blood Cells 脂质体电荷和组成对海藻糖进入红细胞的影响
Pub Date : 2008-11-12 DOI: 10.1089/CPT.2008.0008
J. Holovati, M. Gyongyossy-Issa, J. Acker
Although the disaccharide trehalose has been shown to protect cells during freezing and desiccation when present intracellularly, a major obstacle to using intracellular sugars for biopreservation applications is the impermeability of plasma membranes. We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of stabilizing sugars into mammalian cells. Previous work has shown that the mechanism of red blood cell (RBC)–liposome interaction includes both liposome fusion with the RBC membranes, as well as tight adsorption of the vesicles onto the RBC surface. However, the fusion efficiency of liposomes was low, with only micromolar concentrations of trehalose delivered to the RBC cytosol. The purpose of this study is to enhance the efficacy of liposomes’ delivery of trehalose into RBCs with minimal detrimental effects on RBC membrane quality, by manipulating liposome physical properties and liposome–RBC incubation conditions. Charged and uncharged un...
虽然双糖海藻糖已被证明在细胞内存在时可以在冷冻和干燥期间保护细胞,但将细胞内糖用于生物保存应用的主要障碍是质膜的不渗透性。我们正在研究脂质体的使用,脂质体是一种合成的微囊泡,用于在细胞内将稳定糖输送到哺乳动物细胞中。先前的研究表明,红细胞-脂质体相互作用的机制包括脂质体与红细胞膜的融合,以及囊泡在红细胞表面的紧密吸附。然而,脂质体的融合效率很低,只有微摩尔浓度的海藻糖传递到RBC细胞质。本研究的目的是通过控制脂质体的物理性质和脂质体-红细胞的孵育条件,提高脂质体将海藻糖递送到红细胞的功效,同时对红细胞膜质量的不利影响最小。带电的和不带电的…
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引用次数: 17
Artificial Collapse of Blastocysts Before Vitrification: Mechanical vs. Laser Technique and Effect on Survival, Cell Number, and Cell Death in Early and Expanded Blastocysts 玻璃化前囊胚的人工塌陷:机械与激光技术及其对早期囊胚存活、细胞数量和细胞死亡的影响
Pub Date : 2008-11-12 DOI: 10.1089/CPT.2008.0007
N. Desai, J. Szeptycki, M. Scott, F. Abdelhafez, J. Goldfarb
The purpose of this study was to systematically examine blastocoelic fluid reduction prior to vitrification and its potential benefits. In addition, we compared artificial collapse (AC) by laser pulse to a mechanical method. Mouse and dicarded human blastocysts were used in this study. Blastocysts were collapsed using either a 10 ms pulse with a laser (LAC) or else mechanical puncture with a microneedle (MAC). Blastocysts were vitrified on cryoloops using a two-step ethylene glycol/dimethyl sulfoxide protocol. We examined the effects of AC on specific outcome parameters such as overall survival, reexpansion, cell proliferation, and DNA damage. Unlike others, we report overall high survival rates with expanded blastocysts even without fluid reduction. We did detect a significant increase in blastomeres showing signs of DNA damage in the control group (13%) in comparison to blastocysts AC prior to vitrification (LAC 3%, MAC 5%; p < 0.001). Control blastocysts exhibited a lower rate of reexpansion. Within 3 ...
本研究的目的是系统地检查玻璃化前囊胚腔液减少及其潜在的益处。此外,我们还比较了激光脉冲人工塌陷(AC)和机械塌陷(AC)。本研究使用小鼠和丢弃的人胚泡。用激光(LAC)脉冲10ms或用微针(MAC)机械穿刺使囊胚破裂。囊胚采用乙二醇/二甲基亚砜两步冷冻法玻璃化。我们检查了AC对特定结局参数的影响,如总生存、再扩张、细胞增殖和DNA损伤。与其他研究不同的是,我们报告了即使没有液体减少,扩大囊胚的总体存活率也很高。与玻璃化前的囊胚AC (LAC 3%, MAC 5%)相比,我们确实检测到对照组中显示DNA损伤迹象的囊胚(13%)显著增加。P < 0.001)。对照囊胚的再膨胀率较低。在3…
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引用次数: 29
Tumor Grade Predicts Tumor Proportion in Fresh Frozen Tissue Samples for Breast Cancer Research 肿瘤分级预测乳腺癌研究中新鲜冷冻组织样本中的肿瘤比例
Pub Date : 2008-11-12 DOI: 10.1089/CPT.2008.0010
R. Gandour-Edwards, Sandra L. Taylor, L. Beckett
Breast cancer research frequently requires frozen cancer tissue samples for potential biomarker detection. Ninety-one breast cancer frozen tumor tissue samples were examined for factors contributing to the proportion of the tumor cells per sample. The strongest predictor of high proportion tumor cells was tumor grade as determined by the Scarff–Bloom–Richardson scoring system, followed by tumor size. Because the low-grade tumors had fewer tumor cells per specimen, the type of surgery and experience level of the sample collector was not significant. This variable should be considered by investigators when evaluating potential biomarkers.
乳腺癌研究经常需要冷冻癌组织样本来检测潜在的生物标志物。对91个乳腺癌冷冻肿瘤组织样本进行了检查,以确定影响每个样本中肿瘤细胞比例的因素。由Scarff-Bloom-Richardson评分系统确定的肿瘤分级是高比例肿瘤细胞的最强预测因子,其次是肿瘤大小。由于低级别肿瘤的每个标本的肿瘤细胞较少,因此手术类型和标本收集者的经验水平不显著。研究者在评估潜在的生物标志物时应该考虑这个变量。
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引用次数: 1
Glycine Protects Bile Physiology and Biliary-Specific Liver Cell Metabolism from Ischemia-Reperfusion Injury: A 1H NMR Study 甘氨酸保护胆汁生理和胆道特异性肝细胞代谢免受缺血再灌注损伤:一项1H NMR研究
Pub Date : 2008-11-12 DOI: 10.1089/CPT.2008.0006
T. Hafez, H. Sheth, G. Glantzounis, H. Parkes, A. Seifalian, B. Fuller, B. Davidson
Liver ischemia-reperfusion (I/R) injury is a major complication of liver resection and transplantation. Cytokine release by activated Kupffer cells (KCs) play a central role in the inflammatory cascade of I/R injury. There is evidence that glycine may protect against liver I/R injury by inhibition of KC activity. However, its effect on bile flow, an established marker of hepatic function, and bile composition is not known. A rabbit model of hepatic lobar warm I/R was used. Under general anesthesia, the sham group (n = 6) underwent laparotomy alone for 7 h. The I/R group (n = 6) underwent 60 min of left and median lobe inflow occlusion and 6 h of reperfusion. The glycine + I/R group (n = 6) underwent a same procedure to controls after receiving glycine 5 mg/kg intravenous infusion for over 15 min. Bile flow was collected, measured, and analyzed by proton magnetic resonance spectroscopy. Glycine prevented the significant reduction in bile flow seen in I/R at 6 h reperfusion (145.0 ± 11.4 vs. 108.3 ± 28.2 μL...
肝缺血再灌注(I/R)损伤是肝切除和移植的主要并发症。激活的库普弗细胞(KCs)释放细胞因子在I/R损伤的炎症级联反应中起核心作用。有证据表明甘氨酸可能通过抑制KC活性来保护肝脏I/R损伤。然而,其对胆汁流量(一种已确立的肝功能指标)和胆汁成分的影响尚不清楚。采用兔肝大叶热性I/R模型。假手术组(n = 6)在全麻下单独开腹7 h, I/R组(n = 6)左、中叶流闭塞60 min,再灌注6 h。甘氨酸+ I/R组(n = 6)在甘氨酸5 mg/kg静脉输注超过15分钟后,与对照组进行相同的程序。通过质子磁共振波谱法收集、测量和分析胆汁流量。甘氨酸阻止了再灌注6 h时I/R胆流量的显著减少(145.0±11.4比108.3±28.2 μL)。
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引用次数: 3
Room Temperature Storage of Cultured Human Articular Chondrocytes 培养的人关节软骨细胞的室温贮藏
Pub Date : 2008-11-12 DOI: 10.1089/CPT.2008.0009
V. García, Eva García-Pérez, E. Belyakova, S. Llames, M. Pevida, F. Tévar, J. Otero, Á. Meana
Cell therapy requires proper techniques to store cells in good viability conditions. Liquid nitrogen containers may store cells indefinitely, but samples may suffer cross-contamination or bacterial contamination when they are manipulated. Cultured human chondrocytes are commonly employed in cell therapy. In this study, we show a simple system to store them at room temperature. We investigated 12 strains of cultured chondrocytes. First, we studied four strains, which were stored in different conditions. Later, we studied the other eight strains, which were maintained in the optimal viability condition obtained from the first experiment (room temperature with DMEM and HAM-F12 as maintenance medium). Using HAM-F12 as the maintenance medium, the viability after 15 days was similar to that of the control cells. After 30 days, the average viability was 87%. In both cases, cells were able to reach exponential growth when restored to 37oC. They could also synthesize specific cartilage extracellular matrices after...
细胞治疗需要适当的技术将细胞储存在良好的生存条件下。液氮容器可以无限期地储存细胞,但样品在操作时可能会受到交叉污染或细菌污染。培养的人软骨细胞通常用于细胞治疗。在这项研究中,我们展示了一种在室温下储存它们的简单系统。我们研究了12株培养的软骨细胞。首先,我们研究了在不同条件下储存的四种菌株。随后,我们研究了其他8株菌株,将其维持在第一次实验获得的最佳生存条件下(室温下,以DMEM和HAM-F12作为维持培养基)。以HAM-F12作为维持培养基,15天后的细胞活力与对照细胞相似。30 d后,平均存活率为87%。在这两种情况下,当恢复到37℃时,细胞都能达到指数增长。它们还可以合成特定的软骨细胞外基质。
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引用次数: 2
Long-Term Preservation at Room Temperature of Freeze-Dried Human Tumor Samples Dedicated to Nucleic Acids Analyses 用于核酸分析的冻干人肿瘤样品的室温长期保存
Pub Date : 2008-11-12 DOI: 10.1089/CPT.2008.0003
C. Leboeuf, P. Ratajczak, Weili Zhao, L. Plassa, Magali Court, H. Pisonero, H. Murata, J. Cayuela, J. Ameisen, J. Garin, A. Janin
Tumor banks are the only means for researchers to study in situ human lesions in a reproducible manner. Large amounts of money are spent at national and international levels for proper collection, in respect of both quality control and ethics, most importantly for shared use of the human samples. Preservation as whole tissue, and not simply as extracted DNA or RNA, allows additional scientific use of the samples. However, economic issues influence long-term development of biobanks since low-temperature cryopreservation with long-term storage of numerous samples and transfers avoiding RNA degradation are particularly costly. We optimized freeze-drying protocols for tumor samples dedicated to molecular analyses in our biobank using directly snap-frozen samples as references. We then compared different long-term storage conditions for preservation of nucleic acids in tumor samples to corresponding normal tissues and human cell lines. When stored for 1 year at room temperature with dessicant and no light, cel...
肿瘤库是研究人员以可重复的方式研究人体原位病变的唯一手段。在质量控制和道德规范方面,在国家和国际一级花费了大量资金进行适当的收集,最重要的是共享使用人类样本。作为整个组织保存,而不是简单地作为提取的DNA或RNA保存,允许对样本进行额外的科学使用。然而,经济问题影响了生物库的长期发展,因为大量样本的低温冷冻保存和避免RNA降解的转移成本特别高。我们优化了我们生物库中用于分子分析的肿瘤样本的冷冻干燥方案,使用直接快速冷冻的样本作为参考。然后,我们比较了不同的长期保存条件保存的核酸在肿瘤样品,相应的正常组织和人类细胞系。常温下加干燥剂,无光照保存1年,细胞…
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引用次数: 16
Evaluation of Freeze Thaw Cycles on stored plasma in the Biobank of the Norwegian Mother and Child Cohort Study. 冻融循环对挪威母婴队列研究生物库中储存血浆的评价。
Pub Date : 2008-09-01 DOI: 10.1089/cpt.2008.0012
Liv Paltiel, Kjersti S Rønningen, Helle M Meltzer, Susan V Baker, Jane A Hoppin

BACKGROUND: In many cohort studies, biological specimens are being stored without specific plans for analyses. In the Norwegian Mother and Child Cohort Study biological specimens (DNA, plasma, and whole blood) are stored on 96-well plates and as a result may undergo multiple freeze-thaw cycles. METHODS: To explore the impact of multiple freeze-thaw cycles on chemical constituents, we conducted a quality control study using pooled EDTA-plasma. Over a two-year period, samples stored at -80 degrees C were subjected up to 100 freeze-thaw cycles. Specimens were analyzed in triplicate for sodium, cholesterol, triglycerides, vitamin E, aspartate aminotransferase (AST), and free fatty acids. We assessed the percent change of analyte concentration from the values for the first freeze-thaw cycle, because this is the baseline for all stored specimens. RESULTS: With the exception of free fatty acids, there was little change over the first 10 freeze-thaw cycles. A majority of analytes showed no significant changes until 30 freeze-thaw cycles. After 30 freeze-thaw cycles, the largest percent change was observed for free fatty acids (+32%), AST (+21%), and triglycerides (-19%). CONCLUSIONS: Human plasma can go through several freeze-thaw cycles before analysis without influencing sample integrity for the selected analytes.

背景:在许多队列研究中,生物标本的储存没有具体的分析计划。在挪威母亲和儿童队列研究中,生物标本(DNA、血浆和全血)储存在96孔板上,因此可能经历多次冻融循环。方法:为了探讨多次冻融循环对化学成分的影响,我们使用池edta等离子体进行了质量控制研究。在两年的时间里,储存在-80摄氏度的样品经受了多达100次的冻融循环。样品一式三份,分析钠、胆固醇、甘油三酯、维生素E、天冬氨酸转氨酶(AST)和游离脂肪酸。我们评估了第一次冻融循环时分析物浓度变化的百分比,因为这是所有储存标本的基线。结果:除游离脂肪酸外,在前10次冻融循环中几乎没有变化。大多数分析物在30个冻融循环之前没有明显变化。冻融循环30次后,游离脂肪酸(+32%)、AST(+21%)和甘油三酯(-19%)变化最大。结论:人血浆在分析前可经过多次冻融循环而不影响所选分析物的样品完整性。
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引用次数: 46
Quality Assurance in Tissue Resources Supporting Biomedical Research. 支持生物医学研究的组织资源质量保证。
Pub Date : 2008-07-30 DOI: 10.1089/cpt.2008.9993.
William E Grizzle, Katherine C Sexton, Walter C Bell

Modern biomedical research requires access to high quality specimens of human tissue with or without extensive clinical annotation. Multiple types of organizations have developed to supply human tissues to support biomedical research. These organizations follow different models including the specific models of 1) prospective collection, 2) tissue banking, and 3) tissue collection associated with clinical trials as well as the model of 4) a tissue resource that incorporates features of the other models. These types of organizations devoted to supplying tissues for research have chosen different goals to meet the different tissue and informational needs of the investigators to whom they supply tissue. In order to provide high quality tissues to support research, all models should rely on a strong quality assurance program with extensive quality control of the tissues being provided to support research. In addition to facilities which collect, process, store and provide tissues, the need for a rigorous QA program applies to all resources and infrastructures used to support biomedical research. The UAB Tissue Collection and Banking Facility which provides human tissue to support biomedical research has been functioning and developing since 1979. To our knowledge, similar programs in providing tissues from animals are less developed, but could easily follow the models which UAB and other institutions providing human tissues have established, including the approaches of UAB and others to QA and QC. This manuscript reviews the current concepts of QA and QC in use in organizations supplying tissue to support biomedical research as well as new approaches in QA and QC that have been proposed.

现代生物医学研究需要获得高质量的人体组织标本,有或没有广泛的临床注释。多种类型的组织已经发展为提供人体组织来支持生物医学研究。这些组织遵循不同的模型,包括1)前瞻性收集的特定模型,2)组织库,3)与临床试验相关的组织收集,以及4)结合其他模型特征的组织资源模型。这些致力于为研究提供组织的组织选择了不同的目标,以满足他们提供组织的研究人员的不同组织和信息需求。为了提供高质量的组织来支持研究,所有模型都应该依赖于强有力的质量保证计划,对所提供的组织进行广泛的质量控制,以支持研究。除了收集、处理、储存和提供组织的设施外,严格的质量保证程序也适用于用于支持生物医学研究的所有资源和基础设施。自1979年以来,UAB组织收集和银行设施一直在运作和发展,为生物医学研究提供人体组织支持。据我们所知,提供动物组织的类似项目还不太发达,但可以很容易地遵循UAB和其他机构提供人体组织的模式,包括UAB和其他机构的QA和QC方法。本文回顾了目前在组织中使用的QA和QC概念,以支持生物医学研究,以及已经提出的QA和QC新方法。
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引用次数: 0
Investigating Interactions of Trehalose-Containing Liposomes with Human Red Blood Cells 研究含海藻糖脂质体与人红细胞的相互作用
Pub Date : 2008-07-30 DOI: 10.1089/CPT.2008.0004
J. Holovati, M. Gyongyossy-Issa, J. Acker
A major obstacle in using intracellular sugars for the cryopreservation of mammalian cells is the inability of cells to synthesize or actively accumulate these sugars. We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of stabilizing sugars into mammalian cells. This study examines the interactions of trehalose-containing liposomes with human red blood cells (RBCs). Unilamellar liposomes were synthesized using an extrusion method to contain trehalose in the aqueous core. Liposomal preparations were labeled with a lipophilic fluorophore rhodamine B chloride (R18) at quenching concentrations (4 mol%), or the 5(6)-carboxyfluorescein [5(6)-CF] marker to label the intraliposomal aqueous phase. Flow cytometry and fluorescent microscopy were used to assess the interactions between fluorescently labeled liposomes and RBCs. The delivery of liposomal contents into RBCs was assessed by spectrophotometric measurement of intracellular trehalose. The res...
使用细胞内糖进行哺乳动物细胞低温保存的一个主要障碍是细胞无法合成或主动积累这些糖。我们正在研究脂质体的使用,脂质体是一种合成的微囊泡,用于在细胞内将稳定糖输送到哺乳动物细胞中。本研究探讨了海藻糖脂质体与人红细胞的相互作用。采用挤压法制备单层脂质体,使其在水核中含有海藻糖。脂质体制剂用猝灭浓度(4 mol%)的亲脂荧光团罗丹明B氯(R18)或5(6)-羧基荧光素[5(6)-CF]标记物标记脂质体内水相。用流式细胞术和荧光显微镜评估荧光标记脂质体与红细胞之间的相互作用。通过分光光度法测量细胞内海藻糖来评估脂质体内容物进入红细胞的递送。res……
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引用次数: 11
Loading Trehalose into Red Blood Cells by Improved Hypotonic Method 改进低渗法将海藻糖装入红细胞
Pub Date : 2008-07-30 DOI: 10.1089/CPT.2008.0001
Xinli Zhou, Hui He, Baolin Liu, T. Hua
As a first step toward long-term preservation of red blood cells (RBCs) by using freeze-drying, we present an improved hypotonic method for loading RBCs with trehalose. The method is based on the o...
作为冷冻干燥长期保存红细胞的第一步,我们提出了一种改进的低渗方法,用海藻糖装载红细胞。该方法是基于…
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引用次数: 8
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Cell Preservation Technology
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