Preparation and Preservation of Hypoxia UW Solution

In order to explore the method to prepare hypoxia UW solution and the stability and preservation of hypoxia UW solution, UW solution was purged by argon or air for 15 min or 60 at a flow rate of 0.8 or 2 L/min, and the oxygen partial pressure of UW solution was detected. The hypoxia UW solution was exposed to the air or sealed up to preserve by using different methods, and the changes of oxygen partial pressure was tested. The results showed that oxygen partial pressure of 50 mL UW solution, purged by argon for 15 min at a flow rate of 2 L/min, was declined from 242±6 mmHg to 83±10 mmHg. After exposure to the air, oxygen partial pressure of hypoxia UW solution was gradually increased to 160±7 mmHg at 48 h. After sealed up by the centrifuge tube and plastic bad filled with argon, oxygen partial pressure of hypoxia UW solution was stable, about 88±13 mmHg at 72 h. It was concluded that oxygen of UW solution could be purged by argon efficiently. Sealed up by the centrifuge tube and plastic bag filled with argon, oxygen partial pressure of UW solution could be stabilized.     

A modified CZ-1 preserving solution for organ transplantation: comparative study with UW preserving solution

Background The University of Wisconsin colloid based preserving solution （UW solution） is the most efficient preserving solution for multiorgan transplantation. Unfortunately, unavailability of delayed organ preserving solutions hindered further progression of cardinal organ transplantation in China. In this study, we validated an organ preserving Changzheng Organ Preserving Solution （CZ-1 solution） and compared it with UW solution.
Methods A series of studies were conducted on how and how long CZ-1 solution could preserve the kidneys, livers, hearts, lungs and pancreas of New Zealand rabbits and SD rats. Morphology of transplanted organs was studied by visible microscopy and electron microscopy; biochemical and physiological functions and the survival rate of the organs during prolonged cold storage were studied.
Results There was no significant difference between CZ-1 and UW solutions in preserving the kidneys, livers, hearts or lungs of rabbits; kidneys, livers, intestinal mucosa or pancreases of SD rats or five deceased donors＇ testicles. In some aspects, such as preserving rabbits＇ hearts, rats＇ intestinal mucosa and pancreases, the effect of CZ-1 solution was superior to UW solution. CZ-1 could safely preserve kidneys for 72 hours, livers for 24 hours, hearts for 18 hours and lungs for 8 hours for SD rats. Twelve kidneys preserved in cold CZ-1 solution for 22-31 hours were transplanted successfully and the mean renal function recovery time was （3.83±1.68） days.
Conclusions CZ-1 solution is as effective as UW solution for organ preservation. The development of CZ-1 solution not only reduces costs and improves preservation of organs, but also promotes future development of organ transplantation in China.     

玻璃化法保存异体肌腱移植的实验研究

目的评介玻璃化法保存的异体肌腱移植的可行性与有效性。方法家兔60只,其中24只切取双侧跟腱48条,分为2组,分别采用玻璃化法和程序冷冻法保存2周以上。其余36只平均分为3组:A组行新鲜肌腱自体移植,B组行玻璃化法保存肌腱异体移植,C组行程序冷冻法保存肌腱异体移植。术后3、8、12周后取材,行形态学、组织学观察及生物力学测试,并进行组间比较。结果玻璃化法保存肌腱完整率高于程序冷冻法,差异有显著性。A、B组移植后较C组粘连轻,光泽好。新生血管、腱细胞成熟早,愈合进程快。术前A、B、C组生物力学性能差异无显著性,术后12周差异有显著性。结论玻璃化法保存异体肌腱操作简单,能较好保存肌腱的组织结构,移植效果优于传统的程序冷冻法保存的异体肌腱。     

降温速率对程序化降温低温保存动脉的影响

目的探讨降温速率对深低温保存兔颈总动脉力学性能、组织结构和生物活性的影响。方法取家兔双侧颈总动脉100根,均分为5组(n=20)。A组:新鲜家兔动脉作为对照组;B、C、D、E组分别以-0.5、-1、-2、-5℃/m in的降温速率进行低温保存。对各组动脉力学性能?组织结构?生物学活性进行比较。结果低温保存动脉与对照组比较,其黏弹性均有损失;随降温速率的增加,其黏弹性损失趋于增加。B、D、E组与C组的生物活性差异有显著性(P<0.05)。结论-0.5℃/m in降温速率低温冷冻保存的血管参数最接近新鲜血管,是血管低温保存的最佳降温速率。     

富含血小板血浆冰冻保存及保存效果的研究

目的：研究不同因素对冰冻保存富含血小板血浆（Cryo-PRP)在血库批量制备的整个过程中的质量和特性的影响,优化建立Cryo-PRP的批量制备技术。方法：对冰冻PRP制备的整个过程中的产品包括献血者外周血,ACD抗凝全血,新鲜富含血小板血浆（FPRP）,5％,MDSO富含血小板血浆（MPV）（DMSO－PRP）及－85℃冰冻保存2d后37℃水浴解冻后的Cryo-PRP检测其血小板计数（PLT）,血小板平均体积（MPV）,血小板分布宽度（PDW）,pH,乳酸脱氢酶（LDH）和乳酸的变化,并进行细菌污染监测。结果：（1）制备PRP可以获得全血中70％的血小板;（2）与ACD全血相比FPRP血小板群体发生了改变;（3）冰冻／复温过程中部分血小板膜的完整性受到损害不可避免。（4）在新鲜冰冻血小板制作,保存到临床应用整个过程中血小板代谢不活跃,基本处于休眠状态,有利于其长期保存。（5）冰冻血小板内未见细菌污染和繁殖。结论：冰冻保存PRP能够长期,大量,有效,安全地保存大量宝贵血小板资源,解决目前血小板供不应求的局面。     

自制长征-1号多器官保存液的动物实验和部分临床应用研究

目的：研制我国自制的多器官保存液,以满足日益发展的国内器官移植手术的需要。方法;在全面分析国外最先进的多器官保存液ＵＷ液配方的基础上,研制成长征－１号多器官保存液。改进有：（１）用低分子右旋糖酐－４０替代羟乙基淀粉’（２）用蔗糖替代木棉粮;（３）去掉ＵＷ液中添加成分;（４）添加钙离子拮抗剂维拉帕米;（５）使ＰＨ值进一步偏碱性。     

含血低钾右旋糖酐液肺保存效果的实验研究

目的:比较含与不含20%自体静脉血的低钾右旋糖酐(LPD)液的肺保存效果。方法:将12只犬分为2组,分别用含与不含20%自体静脉血的LPD液灌注,10℃保存12h后,分别在离体状态下,连续通气和温静脉血再灌注10min。比较供肺流入、流出血的氧分压、二氧化碳分压、呼吸道峰压及再灌注后离体肺的湿/干重量比,并行病理学检查。结果:两组间氧分压犤(21.55±3.94)kPa与(20.90±1.16)kPa犦、二氧化碳分压犤(4.25±0.15)kPa与(4.32±0.11)kPa犦、呼吸道峰压犤(19.00±1.41)kPa与(18.50±1.87)kPa犦及再灌注后离体肺的湿/干重量比(6.47±0.41与6.09±0.28)之间差异无统计学意义(P>0.05),病理切片均未见肺泡内出血。结论:含与不含20%自体静脉血的LPD液的肺保存效果差异无统计学意义,肺保存效果并未因灌注液中加血而明显改善。     

SMO保存液对犬肾低温保存期间皮质细胞凋亡的影响

目的:探讨自制上海多器官保存液(Shanghai multi-organ preservation solution,SMO液)对低温保存期间犬肾皮质细胞凋亡的影响,以探讨SMO液的作用机制.方法:犬肾分别以0～4℃的SMO液、高渗枸橼酸盐腺嘌呤液(HC-A)或UW液(University of Wisconsin solution)灌洗保存,在低温保存各时间点取肾皮质标本作形态学观察;采用TUNEL法检测皮质细胞凋亡发生情况;以硫代巴比妥酸法测定各保存液组肾皮质丙二醛(MDA)含量的改变,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性变化.结果:低温保存第1天光镜观察细胞水肿、胞质空泡化、肾小管坏死等改变,三组相似,但保存2 d后SMO组和UW组形态学改变明显轻于HC-A组.随低温保存时间延长,各保存液组肾皮质细胞凋亡的发生增加,而SMO组凋亡指数显著低于同时点HC-A液组 (P＜0.05).随着低温保存时间延长,各保存液组肾皮质MDA含量增加,保存1～3 d SMO液组MDA含量显著低于HC-A液组 (P＜0.05);低温保存中SMO液组、HC-A液组、UW液组肾皮质SOD酶活性无明显差异.结论:SMO液可以提高肾皮质保存效果,可能与其减少低温保存期间肾皮质细胞凋亡和抑制脂质过氧化损伤有关.     

HTK液和UW液对大鼠原代心肌细胞冷保存的保护作用

目的 研究HTK液和UW液对体外培养的大鼠原代心肌细胞冷保存中的保护作用。方法 体外培养大鼠原代心肌细胞,分别用生理盐水,UW液和HTK(液在0℃-4℃保存心肌细胞6h、8h、10h、12h。检测保存液的AST和LDH-L,台盼蓝染色法计算心肌细胞的生存率。结果 UW液组和HTK液组的AST和LDH-L均比生理盐水组极显著低下,心肌细胞的生存率则极显著的高于生理盐水组。而在12h时,HTK液组的AST和LDH-L显著低于UW液组,心肌细胞的生存率在8h和10h也明显高于UW液组。结论 HTK液和UW液均对体外培养的大鼠原代心肌细胞的冷保存有明显的保护作用。而HTK液相对于UW液更能延长对心肌细胞的冷保存时间。     

SMO液对低温下肾皮质线粒体功能的保护作用

目的:探讨自制上海多器官保存液(SMO液)对肾皮质线粒体低温缺氧损伤的保护作用.方法:以HC-A液和UW液作对照,在犬肾低温保存各时间点,取皮质标本;通过电镜观察保存期间线粒体形态改变;差速离心法分离皮质线粒体,Clark氧电极法测定其活性,描记四态呼吸曲线,计算Ⅲ态、Ⅳ态耗氧速率、线粒体呼吸控制率(RCR)及磷氧比(P/O).结果:低温保存期间, HC-A组肾皮质线粒体水肿、空泡样变明显重于SMO组和UW组,而后两者改变相似;随着低温保存时间延长,实验组和对照组肾皮质线粒体Ⅲ态呼吸耗氧速率、RCR和P/O均明显降低;保存1～3 d HC-A组Ⅲ态呼吸耗氧速率显著低于SMO组(P<0.05);保存各时间点SMO组线粒体RCR和P/O显著高于HC-A组(P<0.05),与UW组无明显差异.结论:SMO液对肾皮质线粒体低温缺氧损伤的保护作用比HC-A液强,与UW液相似.     

EDS-40玻璃化冻存小鼠卵母细胞的研究

目的设计研究玻璃化液EDS一40对冻贮小鼠卵母细胞的效果。方法①按随机分组原则，分为实验组和对照组，实验组（a）采用自行设计的EDS一40玻璃化液进行玻璃化冷冻（其配方为：40％乙二醇＋18％葡聚糖），对照组（b）采用国际上已证实效果较好的EFS-40J~璃化溶液进行玻璃化冷冻，对照组（c）采用以PROH为基础的程序冷冻液进行程序冷冻。首先对EDS一40和EFS-40玻璃化溶液，行玻璃化测试；再随机选用小鼠卵母细胞，在室温25℃下分别加入EDS-40和EFS．40玻璃化溶液，玻璃化后装管并迅速投入液氮中。对照组（c）置于程序冷冻仪中进行程序化冷冻。各组均冻存2mo-3mo后，在25℃的水浴中复温后，用培养液反复洗涤后移入培养孔板培养，观察卵母细胞的成活率。将成活的卵母细胞行体外受精，观察其受精率。结果①两种玻璃化溶液能达到玻璃化效果；②EDS一40、EFS一40及对照组冷冻复温后的存活率依次为：79．34％、67．94％、56．34％；⑤EDS一40、EFS-40及对照组冻卵的受精率分别：79．17％、66．29％、51．25％。结论①EDS一40可以用来玻璃化冷冻小鼠卵母细胞；②EDS一40较EFS一40玻璃化溶液冻存小鼠卵母细胞效果更好；③玻璃化技术比传统冷冻法效果更好。     

兔眼结膜上皮细胞和成纤维细胞的培养及冻存

目的 探讨体外培养兔眼结膜上皮细胞和成纤维细胞的最佳方法,为进一步研究结膜的生物学特性和结膜重建提供基础。 方法 分别用组织块法培养结膜上皮细胞和混合消化法培养结膜基质细胞,用倒置显微镜观察其生长方式和形态特征;收集第2代细胞,-80℃冻存。结果 兔眼结膜上皮细胞和上皮下成纤维细胞可以在体外成功培养,结膜上皮细胞呈圆形或多角形,成纤维细胞呈长梭形;冻存细胞1个月后复苏成功率达80%。结论 组织块法和混合消化法培养可成功获得结膜上皮细胞和上皮下成纤维细胞。     

Studies on the development of the multi-organ preservation solution

At PreSent, UW solution is the ~ efficient multiogn PreSent solution for ~. lhause ourcountly is laCk of Prolonged multi~Ogu PreSerVation soluhon laal UW ~on, the baber developnd of caldhaloaf haplantation is ~ctedll]. Our h~ be toexplore the multi~ogu PreSerVation solution in lop. After5 yealS effort, we succ~ developed a ~ed [J'WSOlution: NO. I Ch~ Mulh-Ogu ~ sation (CZ-l ~on) and has accoedished ~ acments and some clinical aPPlication re~.~ ~ ~ P~ ~ (l) caf ~:Inade in our hafta…     

低温灌洗UW液和Euro-Collins液保存离体鼠肺效果的组织形态学比较研究

目的　比较UW液和Euro Collins液的肺保存效果。方法　选用 2 1只Wistar大鼠 ,应用 10℃的UW液和Euro Collins液同时分别进行左、右肺动脉内灌洗 ,灌洗前主肺动脉内快速推注PGE1。灌洗结束后于肺充气状态下切取左、右肺 ,并迅速分别置于 10℃的UW液及Euro Collins液中保存。分别于 0 ,2 ,4,6h取材 ,在光镜及电镜下观察肺组织形态学变化 ,并互相比较 ,以判定组织活力 ,确定肺保存效果。结果　低温灌洗 10℃的UW液肺保存效果优于低温灌洗Euro Collins液。 6h后 ,组织损伤表现为不可逆性。结论　在肺保存不超过 6h时 ,UW液比Euro Collins液更有效     

HX-1号液保存大鼠胰腺24及48小时的胰腺移植研究

本实验将大鼠胰腺分别用HX - 1号液和CollinsⅡ液保存 2 4小时和 4 8小时 ,然后移植给药物性糖尿病大鼠 ,通过检查受鼠的血糖、糖耐量试验及胰腺病理表现。探讨HX - 1号液保存大鼠胰腺的效果。结果发现HX - 1号液和CollinsⅡ液保存大鼠胰腺 2 4小时后移植的两组受鼠的血糖 ,糖耐量试验结果无统计学差异 (p >0 0 5 ) ,与正常大鼠对照组也无统计学差异。在保存 4 8小时的两组中 ,HX - 1号液的保存效果明显优于CollinsⅡ液 ;其血糖值较保存 2 4小时的两组稍高 ,但无统计学差异 ,而糖耐量试验结果受到损害。CollinsⅡ液保存 4 8小时后移植 ,经上述各项指标检测 ,移植的胰腺已基本无功能     

Comparative study of automated cryopreservation of red blood cells

Background

Strategic blood reserves are an important component in meeting blood needs and this can be accomplished through the establishment of a frozen blood program.

Methods

One hundred units of packed RBC were glycerolized using the Haemonetics ACP 215 automated cell processor and placed in a −86 °C deep freezer for freezing and storage. Product weight, hematocrit, RBC count, WBC count and hemoglobin were recorded prior to freezing. Twenty five bags were thawed and deglycerolized after every three months starting at one year from the date of first glycerolization In addition to the earlier parameters the bags were assessed for supernatant osmolality, pH, supernatant hemoglobin, ATP levels and supernatant potassium and from these red cell recovery, percentage hemolysis, supernatant glycerol and red cell viability were estimated. All tests were repeated at the end of 7 and 14 days.

Results

The mean red cell recovery was found to be 86.12% on Day 0 and 84% on Day 14. All the bags showed residual glycerol and pH within the acceptable limits upto Day 14. Percentage hemolysis, Mean ATP levels and mean supernatant potassium levels were within acceptable limits upto Day 14. All the units were sterile upto Day 14.

Conclusion

The data in this study showed that the red cells which were glycerolized using the automated platform ACP 215, frozen at −80 °C for more than a year and deglycerolized again using the ACP 215 had excellent viability while being stored at 4 °C during the 14 days of post-thaw storage.     

Comparative study of automated cryopreservation of red blood cells

Background

Strategic blood reserves are an important component in meeting blood needs and this can be accomplished through the establishment of a frozen blood program.

Methods

One hundred units of packed RBC were glycerolized using the Haemonetics ACP 215 automated cell processor and placed in a −86 °C deep freezer for freezing and storage. Product weight, hematocrit, RBC count, WBC count and hemoglobin were recorded prior to freezing. Twenty five bags were thawed and deglycerolized after every three months starting at one year from the date of first glycerolization In addition to the earlier parameters the bags were assessed for supernatant osmolality, pH, supernatant hemoglobin, ATP levels and supernatant potassium and from these red cell recovery, percentage hemolysis, supernatant glycerol and red cell viability were estimated. All tests were repeated at the end of 7 and 14 days.

Results

The mean red cell recovery was found to be 86.12% on Day 0 and 84% on Day 14. All the bags showed residual glycerol and pH within the acceptable limits upto Day 14. Percentage hemolysis, Mean ATP levels and mean supernatant potassium levels were within acceptable limits upto Day 14. All the units were sterile upto Day 14.

Conclusion

The data in this study showed that the red cells which were glycerolized using the automated platform ACP 215, frozen at −80 °C for more than a year and deglycerolized again using the ACP 215 had excellent viability while being stored at 4 °C during the 14 days of post-thaw storage.     

脐血造血细胞分离和冻存的研究

用６％羟乙基淀粉学淀，比重１．０７７，１．０８０的Ｆｉｃｏｌｌ－泛影葡胺及比重１．０８０的Ｐｅｒｃｏｌｌ分层分离脐血有核细胞，发现以比重１．０８０的Ｐｅｒｃｏｌｌ分层后粒－单核细胞集落形成单位（ＣＦＵ－ＧＭ０回收率最高，可达１０４％，随全血加入培养体系中的少量血浆会明显影响ＣＦＵ－ＧＭ回收率的计算，６例脐血用不同保护剂及降温方法冻存证明－７０℃冰箱降温效果接近甚至优于程控降温，结果表明，用Ｐｅｒｃ     

成人脂肪间充质干细胞冻存的实验研究

目的 探寻成人脂肪间充质干细胞冻存和复苏的条件,观察冻存前后脂肪间充质干细胞的形态学特征以及向心肌细胞诱导分化的潜能.方法 自成人脂肪组织分离培养脂肪间充质干细胞,流式细胞仪检测细胞表面分子.将脂肪间充质干细胞于液氮中低温冻存,经3个月后复苏,倒置相差显微镜及透射电子显微镜下观察细胞形态.5-氮杂胞苷进行诱导,免疫荧光技术检测心肌特异性肌钙蛋白-Ⅰ.比较冻存前后脂肪间充质干细胞的形态学特征及诱导转化率.结果 脂肪间充质干细胞较幼稚,冻存前后光镜下的形态及电镜下的超微结构无明显差别,脂肪间充质干细胞呈CD29阳性表达,HLA-DR阴性表达.冻存前后脂肪间充质干细胞经5-氮杂胞苷诱导后第28天均可表达心肌特异性肌钙蛋白-Ⅰ,诱导转化率无差异.结论 脂肪间充质干细胞可耐受低温冻存,复苏后细胞的形态学特征以及诱导分化潜能无明显变化.     

上海多器官保存液保存离体大鼠肝脏的实验研究

目的:观察上海多器官保存液(Shanghai-mutil-organ solution,SMO液)对离体大鼠肝脏的保存效果,探讨应用SMO液保存离体供肝的可行性.方法:SD大鼠随机分为SMO液、UW液和HTK液保存组,建立离体肝脏单纯低温保存模型,保存液保存8、16、24、36 h分析肝脏组织能量代谢情况,观察肝组织形态学改变和肝脏细胞凋亡情况.结果:保存16、24、36 h,SMO液组肝组织三磷酸腺苷(ATP)、磷酸腺苷总量(TAN)及Atkinson能荷(AEC)均明显高于同时点HTK液组 (P＜0.05),与同时点UW液组无显著差异;形态学检查见SMO液组组织损伤较同时点HTK液组轻,除细胞肿胀较同时间点UW液组明显外,其余表现基本一致.保存24、36 h,SMO液组凋亡指数明显低于HTK液组(P＜0.05),而与UW液组无明显差异.结论:SMO液对大鼠离体肝脏的保存效果总体上与UW液相当,优于HTK液,仅在防止细胞水肿方面较UW液稍差.