红细胞在保护剂添加和洗涤时的非平衡渗透响应实验研究

在红细胞低温保存或冻干保存中，高浓度保护剂的添加和洗涤会使细胞体积收缩或膨胀，造成溶血损失，但其体积并非单调收缩或膨胀到最后平衡体积，而是要经历更严重的体积变化后再趋于平衡。本研究以NaCl溶液来模拟保护剂的添加与洗涤过程．将38种不同浓度的NaC;溶液，以一步直接法、4步等体积法或4步等摩尔浓度变化法添加到红细胞中，测定其溶血率。若以溶血率10％为标准，红细胞所能承受的最小渗透压在161mOsmol／k附近，而最大渗透压与溶液添加方式有关，等体积添加法效果较好（其值约为5680mOsmol／kg）。研究中还将12％、18％NaCl溶液以4步等体积法加到红细胞中，再用生理盐水以三种方式洗涤，发现等摩尔浓度变化法洗涤效果最好；洗涤后溶血率大大增加，说明很多细胞虽然在保护剂添加时未溶血，但膜脆性已改变，难以恢复到等渗时体积。     

透析法去除红细胞渗透性低温保护剂的实验研究

要实现人类红细胞深低温保存,必须在冷冻前添加、复温后去除低温保护剂.本研究提出了低温保护剂的新型的透析去除法,与常规的离心洗涤法比较而言,该方法能够快速、有效、安全地去除红细胞内的低温保护剂(甘油).使用该方法冰冻.复温.洗涤后的红细胞计数回收率为(89.17±2.46)%,血红蛋白回收率为(84.93±4.64)%,上清游离血红蛋白的含量为(0.66 ±0.13)g/L,所得冰冻-复温-洗涤后的红细胞悬液的渗透压(残余甘油的含量)为(340.33±20.56)mOsm,均达到了国家对冰冻洗涤红细胞的质量标准要求.     

冻干红细胞复水时体积响应显微观测及复水损伤机理探讨

冻干红细胞复水时造成的细胞损伤是目前红细胞冻干复水后回收率偏低的重要原因,主要表现复水时样品吸水,细胞环境溶液渗透压变化,细胞体积变化剧烈.了解复水时细胞体积响应对于了解冻干细胞复水损伤机理非常重要.本文中利用一种研究冻干细胞复水时体积响应的方法,即在环境扫描电镜下通过调整相对湿度来控制复水速度,显微跟踪观测细胞尺寸变化.结果 表明,在复水初始阶段红细胞厚度和直径先增大后减小,厚度方向变形能力和幅度比径向大.当体积偏移到一定程度时即发生溶血.因此为减小复水时溶血率,应控制复水条件、减小复水时细胞体积变化.     

血管冷冻保护剂的临床应用

背景：冷冻保存的同种异体血管在血管移植修复中蕴藏着广泛的临床应用前景。深低温冻存是实现同种异体血管长期保存的常用方法。在低温保存过程中,有效的冷冻保护剂对于防止血管组织低温损伤和保持细胞活力至关重要,但对于最佳保护剂的选择并未达成共识。目的：总结近年来各大血管组织库中使用的冷冻保护剂,比较其临床应用效果,以期为将来深低温冻存同种异体血管的应用提供参考。方法：由第一作者检索中国知网、万方、PubMed和Google Scholar数据库2015-2022年发表的相关文献,中文检索词为“同种异体血管,动脉,静脉,深低温冻存,冷冻保护剂,冷冻损伤,血管移植”,英文检索关键词为“cryopreservation,blood vessels,vascular allografts,arteries,veins,cryoinjury,cryoprotectants,cryoprotective agents”,检索国内外有关应用于临床的血管冷冻保护剂及其近远期结果的相关文献,最终选取54篇文献进行综述。结果与结论：(1)在血管的深低温冻存研究中,常使用包含渗透性和非渗透性的冷冻保护剂组合,通过程序...     

生殖细胞的细胞膜渗透特性研究进展

生殖细胞的低温保存在多个领域具有重要应用价值。为提高保存后细胞的存活率与活性,研究细胞膜对水和低温保护剂的渗透特性十分必要。本文综述了近年来动物生殖细胞的细胞膜渗透特性研究概况,列出了典型的精子细胞和卵母细胞的细胞膜对水和常见低温保护剂的渗透系数,分析了这些细胞的渗透特性对制定其低温保存方案的影响。论文还介绍了细胞膜渗透特性的最新测量方法。     

低温保护剂渗透关节软骨研究进展

玻璃化保存关节软骨具有临床治疗和科学研究的双重意义。如何在保证对软骨细胞造成的损伤最小的前提下,将足够多的低温保护剂载入软骨组织,是玻璃化保存关节软骨的关键环节之一,而低温保护剂渗透关节软骨的特性是指导设计低温保护剂加载处理过程的重要依据。从实验研究和数学建模两方面,系统评述低温保护剂渗透关节软骨的研究进展,指出现有研究存在的不足,并对今后低温保护剂渗透关节软骨的研究方向提出展望。     

微流体装置线性去除猪MII期卵母细胞冷冻保护剂的实验研究

低温保存后的卵母细胞在使用前必须要去除冷冻保护剂,目前常用的分步法去除步骤繁琐,容易丢失细胞,而且会对细胞造成致命的渗透损伤。为减小细胞渗透损伤,设计制作适合卵母细胞保护剂去除的微流体装置,研究微流控线性法去除猪MII期卵母细胞低温保护剂时在不同时间(6、8、10 min)下卵母细胞的体积变化,以及对卵母细胞存活率与发育率的影响;并与传统的去除方法(一步法和分步法)进行比较。结果表明,采用微流体装置线性去除冷冻保护剂,8 min为实验中的最优去除时间;线性法能够明显减小细胞的渗透损伤,其最大归一化渗透膨胀体积为1.12±0.07,卵母细胞的存活率、卵裂率及囊胚率分别达到83.6%、72.4%、21.5%,均显著高于一步法和分步法(P<0.05)。因此,微流控线性去除冷冻保护剂能够显著减小细胞的渗透损伤,为卵母细胞低温保存技术提供新思路。     

微流控法去除低温保护剂对卵母细胞发育的影响

为了减小低温保护剂去除过程对卵母细胞造成的渗透损伤和毒性损伤,本文利用微流控芯片对猪二次减数分裂中期(MⅡ-stage)卵母细胞低温保护剂的去除方案进行了优化研究。首先分析了微流控去除方法中去除时间、去除液成分及浓度对卵母细胞存活率及体外发育情况的影响,然后将微流控去除方法与传统的一步法、两步法进行了比较。研究结果表明,微流控法中去除总时间为8 min时,卵母细胞存活率(95.99%±4.64%)及桑椹胚率(74.17%±1.18%)与新鲜细胞(98.53%±2.94%;78.22%±1.34%)相比,差异无统计学意义;1 mol/L蔗糖去除液最有利于卵母细胞低温保护剂去除后的存活及体外发育;微流控法去除低温保护剂后,卵母细胞的存活率、体外发育情况等,均好于传统去除方法。本文研究结果提示,以微流控法去除低温保护剂可减小对卵母细胞的损伤,从而可能进一步提高卵母细胞的低温保存效果。     

纳米颗粒对猪GV 期卵母细胞低温保存效果的影响

纳米低温保存技术很可能是新一代低温保存技术的重要发展方向,但纳米颗粒应用于卵母细胞玻璃化保存的报道较少。本研究将羟基磷灰石（HA）、二氧化硅、三氧化二铝、二氧化钛等4种纳米颗粒添加到低温保护剂中,使用Cryotop法冷冻猪GV期卵母细胞,使用形态观察和荧光染色的方法, 研究其对细胞存活率和发育率的影响。结果显示在实验浓度范围内,HA较其它纳米颗粒对猪卵母细胞的毒性低,当浓度低于0.5%时,细胞发育率为100%;低温保护剂中添加0.1%HA纳米颗粒,发育率比其它组显著提高,可以达到22%, 且HA的粒径对结果影响不大;当低温保护剂中添加0.05%粒径为60 nm的HA颗粒时,卵母细胞冷冻复温后发育率会进一步从14.7%提高达到30.4%。在低温保护剂中添加适宜浓度的HA纳米颗粒,可以减少复温过程中的重结晶现象,促进细胞的冷冻存活率和发育率,保存效果与浓度相关,而与纳米颗粒的粒径关系不大。     

微流控法低温保护剂添加及去除线型优化研究

在卵母细胞低温保存过程中,低温保护剂的添加与去除是必不可少的步骤。近年来微流控芯片被用于低温保护剂的添加及去除,而不同线型添加和去除低温保护剂会影响卵母细胞存活率及体外发育情况。利用以微流控芯片为中心的微混合系统,分别设计线性加载和去除保护剂的方案,并以分段线性衍生出凸型、凹型保护剂加载和去除方案,最终用上述方案共组合出9种添加-去除联用方案,研究不同线型添加-去除保护剂对卵母细胞存活率及体外发育情况的影响。结果表明:凹型添加-凸型去除联用方案下,猪MII期卵母细胞的存活率及桑椹胚率达到97.22%、46.03%,均显著优于其他添加-去除方法(P<0.05);低温保护剂添加及去除过程中,连续性添加及去除方案间具有一定的匹配关系,这将为优化微流控法连续性添加-去除低温保护剂方案提供新思路。     

海藻糖和蛋黄在人类精子冻于保存中的保护作用

人类精子冻干保存中损伤除了来自环境溶液对精子生理活性的损伤外，还会来自于冷冻和干燥过程。目前精子冻干保存中保护剂选取基本上都从生理化学角度考虑，目的为抑制精子内酶活性，没有考虑对冷冻和干燥损伤的抑制。大量文献表明蛋黄能减小冷冻对精子的损伤，而二糖（特别是海藻糖）在细胞干燥中具有特殊保护作用。本研究在前人一般使用的精子冻干保护剂中加入海藻糖，或同时加入海藻糖和蛋黄，通过伊红Y染色、低渗膨胀和电镜显微观测实验，比较不同组成的保护剂对精子结构和膜的保护作用。结果表明，通过加入海藻糖修正的保护剂能提高对精子结构的保护效果，当同时加入海藻糖和蛋黄时效果更好。     

生物羊膜冷冻干燥工艺优化研究

为探索羊膜冷冻干燥保存的最佳工艺条件,取健康剖宫产产妇胎盘,钝性剥离羊膜。以保存羊膜组织形态变化、胶原蛋白酶降解速度、生物力学特征、细胞因子含量为考察指标,对羊膜冷冻干燥工艺中的关键因素进行优化。结果显示,改良冷冻干燥羊膜上皮细胞、纤维基质层形态结构与新鲜羊膜基本相同,但纤维基质厚度略有增加,上皮细胞表面微绒毛略有减少;Ⅳ型胶原酶在溶液中降解速度有所加快;生物力学特征与新鲜羊膜无明显差别;6种细胞因子含量明显低于新鲜羊膜。结合前期工作,与常规冷冻干燥法比较,改良冷冻干燥工艺对保存羊膜组织结构和生物学活性因子影响较小,并能使保存羊膜具有更好的生物力学特性。     

Andrology: Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol

Use of a cryoprotective agent is indispensable to prevent injuryto human spermatozoa during the cryopreservation process. However,addition of cryoprotective agents to spermatozoa before coolingand their removal after warming may create severe osmotic stressfor the cells, resulting in injury. The objective of this studywas to test the hypothesis that the degree (or magnitude) ofhuman sperm volume excursion can be used as an independent indicatorto evaluate and predict possible osmotic injury to spermatozoaduring the addition and removal of cryoprotective agents. Glycerolwas used as a model cryoprotective agent in the present study.To test this hypothesis, first the tolerance limits of spermatozoato swelling in hypoosmotic solutions (iso-osmotic medium dilutedwith water) and to shrinkage in hyperosmotic solutions (iso-osmoticmedium with sucrose) were determined. Sperm plasma membraneintegrity was measured by fluorescent staining, and sperm motilitywas assessed by computer-assisted semen analysis before, duringand after the anisosmotic exposure. The results indicate firstlythat motility was much more sensitive to anisosmotic conditionsthan membrane integrity, and secondly that motility was substantiallymore sensitive to hypotonic than to hypertonic conditions. Basedon the experimental data, osmotic injury as a function of spermvolume excursion (swelling or shrinking) was determined. Thesecond step, using these sperm volume excursion limits and previouslymeasured glycerol and water permeability coefficients of humanspermatozoa, was to predict, by computer simulation, the cellosmotic injury caused by different procedures for the additionand removal of glycerol. The predicted sperm injury was confirmedby experiment. Based on this study, an analytical methodologyhas been developed for predicting optimal protocols to reduceosmotic injury associated with the addition and removal of hypertonicconcentrations of glycerol in human spermatozoa.     

The protective effects of cell-free fluid exudate on cartilage degradation in vitro

In a short-term culture system, polymorphonuclear neutrophils (PMNs) were shown to cause significant loss of glycosaminoglycan (GAG) from cartilage. In addition, a lysate of these cells, and in particular cells stimulated with a phorbol ester, greatly enhanced this loss of GAG. This loss could be partially or completely inhibited by the addition of cell-free fluid exudate to the system. In other long-term culture experiments, IL-1a was shown to enhance fibroblast-induced GAG loss from cartilage. In this system too the breakdown was inhibited by cell-free fluid exudate. Initial characterization of the protective agent or agents shows them to be heat-stable at 56 degrees C and to be non-dialysable. It is proposed that in the chronic inflammatory joint disease, rheumatoid arthritis, the fluid phase of an inflammatory response may have a protective effect against the underlying pathological processes.     

The effects of freeze drying and freeze drying additives on the prothrombin time and the international sensitivity index

AIM: To determine whether freezing, freeze drying protective additives, or freeze drying of plasma samples from patients on coumarin treatment and from normal individuals affects prothrombin times or the international sensitivity index (ISI) calibration. METHODS: The effect of the addition of the protective additives singly and combined on the prothrombin time of coumarin samples and normal samples before and after freeze drying was observed using high and low ISI reference thromboplastins. ISI values were also determined. RESULTS: Freezing caused a prolongation of prothrombin time in the normal plasma samples with both reagents, which was significant with the low ISI human. Prolongation (non-significant) of the prothrombin time in coumarin plasma samples occurred with the human reagent only. Significant prolongation of normal prothrombin time by some of the protective additives before and after freeze drying was observed with both thromboplastins but to a greater extent with the human. Significant prolongation of prothrombin time in coumarin plasma samples was observed, but again was more marked with human thromboplastin. An approximate ISI was determined on the 20 coumarin samples. The only marked ISI change was with the WHO human thromboplastin after freeze drying of plasma, where a decrease from 0.95 to 0.90 was observed, corresponding to a marked prothrombin ratio increase. CONCLUSIONS: Freeze drying additives and the freeze drying procedure prolong normal and coumarin prothrombin times, with low ISI thromboplastin. Less marked prolongations occurred with a high ISI rabbit reagent, coumarin samples showing more significant prolongations. Marked ISI change in freeze dried plasma was only recorded with the low ISI ECAA human reagent. Frozen normal plasma samples cannot be used with confidence for ISI calibrations.     

冷冻干燥法对人精子DNA的影响

目的探讨冷冻干燥法用于人类精子保存的安全性。方法取健康志愿者合格精液40份，平均分为4组，其中3组分别加入不同的冻干保护剂（ETBS；ETBS＋海藻糖：ETBS＋海藻糖＋蛋黄）后给予冷冻干燥处理，在4℃冰箱中保存3周；1组作为新鲜精液对照组。对4组标本分别以原位缺口末端标记（TUNEL）法和彗星试验进行DNA断裂精子百分率检测。结果ETBS、ETBS＋海藻糖、ETBS＋海藻糖＋蛋黄组和新鲜精液组DNA断裂精子百分率以TUNEL法检测，分别为（6．39±1．46）％、（5．75±1．29）％、（5．20±1．38）％、（4．94±1．86）％；以彗星试验检测，分别为（6．48±1．58）％、（5．83±1．48）％、（5．28±1．42）％、（5．12±1．65）％。冷冻干燥保存后的各组与新鲜精液相比较，DNA断裂精子百分率差异均无统计学意义（P〉0．05）。结论以ETBS或ETBS加海藻糖、蛋黄为保护剂的冷冻干燥法对人精子DNA无明显损伤，可有效地保护人精子的DNA。     

Methodical problems for evidence of the hepatitis-B-surface and hepatitis-B-core antigens in tissue (author's transl)]

Liver biopsy material of 22 in the serum HBsAg positive patients was tested with the fluorescent antibody technique for the localization of HBcAg and HBsAg in the liver tissue. Comparative studies were done with the following tissue preparation techniques: Cryostat technique, freeze drying, freeze substitution, cold ethanol paraffin embedding technique (SAINTE MARIE) and isolated liver cells. The investigations revealed the following results: 1. No HB-components could be detected with the cold ethanol paraffin embedding technique and freeze substitution. 2. Using the cryostat technique HBsAg could be demonstrated in 16/22 (cytoplasmatic localization) and HBcAg in 8/22 (nuclear localization). 3. With freeze drying HBsAg and HBcAg could be found in the same cases. The excellent tissue preparation allowed a correct localization of the HB-components to the cell structure. 4. In comparison to cryostat sections in isolated liver cells HBcAg could be demonstrated in 11/16 and HBcAg in 8/8 cases.     

Water removal and solute additions determining increases in renal medullary osmolality

Osmolality and solute concentrations of the mammalian renal medulla increase and decrease with changing urine osmolality. These changes are brought about by addition or removal of solute or water to or from the renal medullary tissue. In Munich-Wistar rats and Syrian hamsters, males and females, actual amounts of and the various solutes involved in these changes were determined. Kidneys were removed from animals killed in different stages of water diuresis and antidiuresis. The renal medulla was analyzed by a new method that permits determination of water and solutes on the same piece of tissue. Removal of water and addition of urea were the two most important factors in raising inner medullary osmolality. Papillary water content was inversely related to the papillary osmolality and was 50% lower in extreme antidiuresis compared with water diuresis. Rats had higher papillary water content than hamsters. In the outer medulla, water removal was significant in the hamsters but not in the rats. Addition of urea to the papillary tissue exceeded the osmotic equivalent of NaCl by a factor of 2.8 in both rats and hamsters. Females of both species showed greater changes than males in amounts of urea in the inner medulla.     

The size-dependent efficacy and biocompatibility of hyperbranched polyglycerol in peritoneal dialysis

Glucose is a common osmotic agent for peritoneal dialysis (PD), but has many adverse side effects for patients with end-stage renal disease. Recently, hyperbranched polyglycerol (HPG) has been tested as an alternative osmotic agent for PD. This study was designed to further examine the efficacy and biocompatibility of HPG over a range of different molecular weights. HPGs of varying molecular weights (0.5 kDa, 1 kDa, 3 kDa) were evaluated in a preclinical rodent model of PD. HPG PD solutions were standardized for osmolality and compared directly to conventional glucose-based Physioneal™ PD solution (PYS). The efficacy of HPG solutions was measured by their ultrafiltration (UF) capacity, solute removal, and free water transport; biocompatibility was determined in vivo by the histological analysis of the peritoneal membrane and the cell count of detached peritoneal mesothelial cells (PMCs) and neutrophils, and in vitro cytotoxicity to cultured human PMCs. All the different sized HPGs induced higher UF and sodium removal over a sustained period of time (up to 8 h) compared to PYS. Urea removal was significantly higher for 1–3 kDa than PYS, and was similar for 0.5 kDa. Our analyses indicated that the peritoneal membrane exhibited more tolerance to the HPG solutions compared to PYS, evidenced by less submesothelial injury and neutrophil infiltration in vivo, and less cell death in cultured human peritoneal mesothelial cells. Free water transport analysis of HPG indicated that these molecules function as colloids and induce osmosis mainly through capillary small pores. We attribute the differences in the biocompatibility and osmotic activity of different sized HPGs to the differences in the polymer bound water measured by differential scanning calorimetry. These preclinical data indicate that compared to PYS, low MW HPGs (0.5–3 kDa) produces superior fluid and waste removal with better biocompatibility profile, suggesting that they are promising osmotic agents for PD.