衣藻细胞玻璃化超低温保存技术的研究

本研究以衣藻为材料,探讨其玻璃化超低温保存的条件和方法,结果表明,衣藻经含0.25mol/L蔗糖溶液的TAP培养基预培养一天后,在玻璃化冷冻保护剂中脱水5分钟,直接投稿液氮,48小时后快速化冻,去保护剂并用含0.5mol/L蔗糖溶液的TAP培养基境培养一天,再转到ATP培养基暗培养一天,最后置光照条件下恢复培养,其存活率可达31.45％,恢复培养后衣藻细胞的生长规律与未冻存的衣藻相一致。     

甘草悬浮细胞的玻璃化法超低温保存研究

以甘草悬浮细胞为材料,进行了超低温保存技术的研究。高产黄酮的甘草悬浮细胞是一种极具价值但又难于保存的材料,为解决甘草细胞超低温保存中存在的关键问题,即如何减少渗透压的突然改变对细胞造成损伤,通过优化预培养时间、细胞年龄、预处理时间、脱水时间、洗涤液蔗糖浓度等影响因素,建立了一套适合于甘草悬浮细胞的保存方法。采用改良的2,3,5-氯化三苯基四氮唑(TTC)法检测,此法存活率可达82.9%。将保存后的细胞进行恢复培养,其恢复生长率可达80.4%。     

拟南芥悬浮细胞系的玻璃化法超低温保存

悬浮培养细胞系是植物生理生化研究的好材料之一。为了保持细胞系的遗传稳定性,需要采用超低温保存技术。玻璃化法是一种不用程序降温仪的超低温保存技术。本文报道了从模式植物拟南芥建立悬浮细胞系并对其进行玻璃化法超低温研究。细胞经过合理的预培养处理和保护剂处理,直接投入液氮贮存。复温后的细胞能恢复生长,恢复生长的细胞保持着植株再生能力。国外,拟南芥悬浮细胞系的程序降温法保存和包埋脱水法保存已经报道,玻璃化法保存尚未见报道。     

长鞭红景天悬浮培养细胞的玻璃化法超低温保存研究

对长鞭红景天悬浮培养细胞的玻璃化法超低温保存进行了初步研究.结果表明,预培养、预处理、脱水处理及冻后处理对长鞭红景天悬浮培养细胞存活率均有重要影响,方差分析结果均显示差异显著.长鞭红景天悬浮培养细胞过程中最佳培养条件是:在含5%二甲基亚砜(DMSO)的MS培养基上预培养1 h,室温下80%PVS2预处理40 min,然后用100%PVS2于0℃处理50 min,投入液氮(LN)保存1 h后在40℃水浴中迅速化冻,再用1.2 mol/L蔗糖培养液洗涤3次,每次10 min,洗涤后的悬浮培养细胞用氯化三苯四氮唑(TTC)法检测,其存活率可达72.70%.     

小球藻的玻璃化超低温保存法

先用含0.5 m01·L-1甘油和0.4 mol·L-1蔗糖的预处理液处理20min,然后用含30%蔗糖 15%乙二醇 10%二甲基亚砜 BBMG培养液的玻璃化液处理,在0℃下预冻60min后,将小球藻投入液氮.此法存活率较高,可达到60.14%,小球藻种质保存效果较好.通过试验初步建立了小球藻玻璃化法超低温保存的技术程序.     

中国红豆杉悬浮培养细胞的超低温保存

对中国红豆杉悬浮细胞超低温保存中几个主要因素进行多方面对比研究。结果表明,取培养16d的细胞进行超低温保存效果最好,10％DMSO＋8％葡萄糖作为冰冻保护剂对冷冻细胞起到最佳的保护效果;较好的降温程序是在0℃中预处理30min后移入－20℃中停留180min,然后转入－70℃中停留30min,最后投入－196℃液氮中保存。该实验还对保存后细胞的恢复性生长进行了验证。     

江西铅山红芽芋胚性愈伤组织小滴玻璃化法超低温保存技术的研究

探索江西铅山红芽芋胚性愈伤组织的小滴玻璃化法超低温保存,为其种质资源的超低温保存提供技术基础和理论依据。植物组织培养和单因子试验的方法。江西铅山红芽芋胚性愈伤组织小滴玻璃化法超低温保存的较佳程序为:约0.2 g胚性愈伤组织在25℃下转入MS+2 mg·L-1TDZ+1 mg·L-1NAA+0.5 mmol·L-1蔗糖的培养基中,于14 h·d-1光周期下预培养3 d后用MS+2 mmol·L-1甘油+0.4 mmol·L-1蔗糖在25℃下装载20min,然后用PVS2在0℃下脱水40 min,吸取5滴PVS2到铝箔条上,将脱水后的红芽芋胚性愈伤组织块转到铝箔条上的PVS2液滴里。附有胚性愈伤组织块的铝箔条在液氮里蘸一下,然后迅速将其转入2 mL装满液氮的冷冻管中,再投入液氮保存1 d。从液氮中取出冷冻管中的铝箔条,浸入用40℃温水预热过的洗涤液(MS+2 mg·L-1TDZ+1 mg·L-1NAA+1.2 mmol·L-1蔗糖)中,使胚性愈伤组织块从铝箔条上脱落下来,然后再将胚性愈伤组织块转入新鲜的洗涤液中洗涤3次,每次10 min。洗涤后转入MS+2 mg·L-1TDZ+1 mg·L-1NAA固体培养基上,先暗培养7 d再转到14 h·d-1光周期中培养。30 d后将分化出的胚状体再次转入MS+2 mg·L-1TDZ+1 mg·L-1NAA固体培养基上可再生完整植株。红芽芋胚性愈伤组织包埋玻璃化超低温保存后的平均成活率约为80%。红芽芋胚性愈伤组织冻后再生苗没有发生形态学、生理学和细胞学的变异。红芽芋胚性愈伤组织小滴玻璃化法超低温保存可以保证其遗传资源的稳定性,为江西铅山红芽芋种质资源的离体保存提供了一条新的途径。     

贡蕉胚性悬浮细胞原生质体分离的研究

目的:研究不同方法对贡蕉胚性悬浮细胞原生质体分离的影响,筛选适合用于贡蕉胚性悬浮细胞原生质体分离的方案.方法:用不同的酶浓 度、酶组合及不同的酶解时间对贡蕉胚性悬浮细胞进行原生质体分离,并对不同继代时间的胚性悬浮细胞的原生质体产量和活力进行研究.结果:贡蕉胚性悬浮细胞 在酶组合为3.5%纤维素酶R-10、1%离析酶R-10和0.15%果胶酶Y-23的酶溶液中,酶解8h可获 得高产量的原生质体,采用继代7d的贡蕉胚性悬浮细胞进行原生质体分离时获得的原生质体产量最高,达到1.2×107个/mL PCV ECS,原生质体活力达到85%以上.结论: 合适的酶组合、酶浓度和酶解时间有利于贡蕉胚性悬浮细胞的原生质体分离,继代7d 后的贡蕉胚性悬浮细胞最适合用于原生质体分离.     

抗冻蛋白应用于水稻悬浮细胞超低温保存的研究

本文将鱼类抗冻蛋白应用于植物细胞的超低温保存。结果表明,在水稻悬浮细胞的两步法保存中,浓度为0.01mg/ml的抗冻蛋白具有特别的负作用,相对高浓度的抗冻蛋白则能减小细胞存活率的波动性。在玻璃化法保存中,浓度为0.2mg/ml的抗冻蛋白能改善保存效果,更高浓度的抗冻蛋白(>5mg/ml)反而会降低保存效果。环境冰晶量、抗冻蛋白浓度、低温保护剂浓度和细胞膜组成等是影响抗冻蛋白使用效果的几大因素。作者在机理分析中认为,一方面,抗冻蛋白能和冰晶作用,抑制重冰晶,防止去玻璃化;另一方面,抗冻蛋白也能和细胞膜作用,诱发膜表面冰晶形成。     

玻璃化法超低温保存蛇莓茎尖

本文采用玻璃化法对蛇莓离体茎尖超低温保存进行了初步探讨。研究了低温锻炼时间、预培养时间、预处理时间、玻璃化液处理时间和液氮保存时间对超低温保存后成活率的影响。经优化,蛇莓的最高成活率可达（42.00±2.74）%。     

长期CO2加富对苗期红掌(Anthurium andraeanum L.)植株生长和光合作用的影响

以开顶式塑料薄膜温室为设施,研究了红掌（Anthurium andlaealzum L．）幼苗植株生长、叶片净光合速率和光合酶活性对长期高CO2浓度的响应。结果表明：处理90d时,处理组T1（（700±too）μmol·mol^-1CO2）的株高、单叶面积、株鲜重分别比对照组（（360±30）μmol·mol^-1）增加了15．76％、14．30％、29．62％,而处理组他（（1000±100）μmol·mol^-1CO2）的株高、单叶面积、株鲜重分别比对照增加了15．00％、9．63％、36．22％;处理150d时T1的株高、单叶面积、株鲜重与对照相比分别增加了16．08％、17．30％、49．09％,而他增加了16．61％、10．10％、48．87％。在各自生长环境下处理组T1、T2的净光合速率在整个处理期间均高于对照,处理150d时,T1、T2的净光合速率分别比对照高8．25％、20．62％;但处理90d时,在对照CO2浓度下测定的净光合速率处理组开始低于对照组,可能此时处理组的红掌叶片开始出现光合适应现象;CO2浓度升高促进了叶片中可溶性糖和淀粉积累,处理90d时T1、T2处理组中淀粉含量分别比对照高52．60％、67．66％;处理150d时,T1组红掌叶片中淀粉与可溶性糖含量比对照高53．43％、6．32％,T2比对照高58．44％、8．07％,叶绿素含量在处理90d时也开始低于对照组;整个实验过程中,Rubisco活性前期增加,90d以后开始下降;乙醇酸氧化酶活性则明显下降,T1、T2处理组试验结束时与对照组相比分别下降了41．28％、45．35％。一定处理时间（90d）的高浓度CO2处理提高了红掌叶片的净光合速率和碳水化合物的积累,促进了营养生长,但随着处理时间的延长,这种促进作用逐渐降低。     

10个红掌品种的抗寒性与耐热性评价

采用低温半致死温度( LT50)评价低温条件下10个红掌( Anthurium andraeanum Lind.)品种的抗寒性;并在测定高温(39℃)处理48 h后各品种叶片生理指标的基础上,对这些指标的耐热系数和相关系数进行分析,采用主成分分析和隶属函数法综合评价了10个红掌品种的耐热性。结果表明：经过-1℃、-3℃、-5℃、-7℃和-9℃的低温处理,10个品种的叶片相对电导率( REC)均逐渐增大,其中,品种‘阿拉巴马’(‘Alabama’)的REC值呈“缓慢升高—迅速升高”的趋势,其他品种的REC值均呈“缓慢升高—迅速升高—平稳升高”的趋势;依据Logistic方程获得的各品种的LT50值,10个红掌品种的抗寒性由强至弱依次排序为‘阿拉巴马’,‘皇冠’(‘Royal champion’),‘马都拉’(‘Madural ’),‘甜冠军’(‘Sweet champion ’),‘特伦萨’(‘Turenza ’),‘阿瓦托’(‘Avento ’)、‘阿瑞博’(‘Arebo’)和‘粉冠军’(‘Pink champion’),‘白冠军’(‘White champion’),‘骄阳’(‘Sierra’)。高温处理48 h对各品种叶片的生理指标均有一定影响,其中,REC值和CAT活性明显高于对照(室温)、MDA含量高于或等于对照,它们的耐热系数均大于100%;相对含水量、叶绿素和类胡萝卜素及脯氨酸含量均低于对照,它们的耐热系数均小于100%;多数品种叶片的SOD和POD活性及可溶性蛋白质含量( SPC)低于对照,耐热系数也小于100%;仅品种‘阿拉巴马’叶片的SOD和POD活性及SPC值略高于对照,耐热系数大于100%;各指标的耐热系数间均存在一定的相关性。主成分分析结果显示：前3个主成分的累计贡献率达到91.50%,其中,第1主成分定义为酶活性和渗透调节物质含量,第2主成分定义为光合色素含量,第3主成分定义为膜透性。根据耐热性综合评价值,10个红掌品种耐热性由强至弱依次排序为‘阿拉巴马’、‘皇冠’、‘阿瓦托’、‘阿瑞博’、‘马都拉’、‘骄阳’、‘特伦萨’、‘粉冠军’、‘甜冠军’、‘白冠军’。研究结果表明：品种‘阿拉巴马’的抗寒性和耐热性均较强,可以在不同气候区推广种植。     

温度对火鹤花生长发育的影响

本文就温度对火鹤花生长发育的影响进行研究,结果表明,全年中日均温15℃的时段,火鹤花产量和品质最差,并造成寒害;日均温29.5℃的时段,有轻微高温伤害;日均温26℃时段最适于火鹤花生长,此时切花品质最好.     

花烛突变体叶色嵌合性状的分化特征与保持方法

以花烛品种‘Sonate’(Anthurium andraeanum‘Sonate’)的叶色嵌合型无菌苗为材料,对顶芽失去嵌合性状的16个单株分别进行单芽培养,观察侧芽及茎基部再生过程中叶色嵌合性状的分化特征;并据此归纳花烛突变体叶色嵌合性状的保持方法。结果表明:处于增殖和生根阶段的花烛突变单株侧芽再生植株的嵌合率分别为25.0%～75.0%和25.0%～66.7%,总的嵌合率分别为48.4%和47.8%,具有嵌合性状的侧芽再生植株均萌发于嵌合叶片的叶腋处;处于生根阶段的突变单株的茎基部也能再生出嵌合植株,嵌合率为33.3%～80.0%,总的嵌合率达到64.7%。研究结果显示:通过单芽离体培养的方法可以解决花烛突变体顶芽叶色嵌合性状消失的问题,且应用侧芽再生(增殖与生根阶段)或基部再生(生根阶段)的方法可以保持突变植株的嵌合性状。     

Effective surface-based cryopreservation of human embryonic stem cells by vitrification

Human embryonic stem cells (hESCs) are candidates for many applications in the areas of regenerative medicine, tissue engineering, basic scientific research as well as pharmacology and toxicology. However, use of hESCs is limited by their sensitivity to freezing and thawing procedures. Hence, this emerging science needs new, reliable preservation methods for the long-term storage of large quantities of functional hESCs remaining pluripotent after post-thawing and culturing.Here, we present a highly efficient, surface based vitrification method for the cryopreservation of large numbers of adherent hESC colonies, using modified cell culture substrates. This technique results in much better post-thaw survival rate compared to cryopreservation in suspension and allows a quick and precise handling and storage of the cells, indicating low differentiation rates.     

TDZ和CPPU对红掌快速繁殖的影响

以红掌带主脉叶切片、叶柄切段为外植体,研究TDZ和CPPU对其快速繁殖的影响。结果表明,在愈伤组织诱导上,与6-BA相比,TDZ和CPPU具有更强的诱导外植体脱分化活性;TDZ 1.0mg/L和6-BA0.5mg/L配比是诱导红掌外植体脱分化的最佳配方,与其它培养基比较,愈伤组织诱导率提高50.0%~166.0%。在附加TDZ 0.3mg/L+NAA 0.1~0.4mg/L或CPPU 0.3mg/L+NAA 0.1~0.4mg/L的MS培养基上,愈伤组织均能很好分化,并能长成完整植株。     

6-BA对红掌组织培养中红叶变异的影响

以红掌盆栽品种‘Avo-Gloria’为试材,以MS＋0.2mg·L^-12,4-D为基本培养基,分别在添加1～10mg·L^-16-BA的10种脱分化培养基上,诱导其叶柄外植体产生愈伤组织;再以MS＋2mg·L^-16-BA＋0.2mg·L^-1 NAA为分化培养基诱导分化不定芽;以MS＋0.2mg·L^-1 NAA为生根培养基,从不定芽获得再生植株。结果显示：（1）在MS＋0.2mg·L^-12,4-D＋8～10mg·L^-16-BA的3种脱分化培养基上可产生9%～10%的绿色、质地较硬的愈伤组织;（2）愈伤组织在MS＋2mg·L^-16-BA＋0.2mg·L^-1 NAA的分化培养基上,经6～8次继代培养,可获得3%～7%的不定芽,并可生根长成再生植株;（3）再生植株定植3个月后,有3%～7%植株出现红叶变异,此红叶可终生表现为红色。     

Surface-based cryopreservation strategies for human embryonic stem cells: A comparative study

Human embryonic stem cells (hESC) hold tremendous potential in the emerging fields of gene and cell therapy as well as in basic scientific research. One of the major challenges regarding their application is the development of efficient cryopreservation protocols for hESC since current methods present poor recovery rates and/or technical difficulties which impair the development of effective processes that can handle bulk quantities of pluripotent cells. The main focus of this work was to compare different strategies for the cryopreservation of adherent hESC colonies. Slow‐rate freezing protocols using intact hESC colonies was evaluated and compared with a surface‐based vitrification approach. Entrapment within ultra‐high viscous alginate was investigated as the main strategy to avoid the commonly observed loss of viability and colony fragmentation during slow‐rate freezing. Our results indicate that entrapment beneath a layer of ultra‐high viscous alginate does not provide further protection to hESC cryopreserved through slow‐rate freezing, irrespectively of the cryomedium used. Vitrification of adherent hESC colonies on culture dishes yielded significantly higher recovery rates when compared to the slow‐rate freezing approaches investigated. The pluripotency of hESC was not changed after a vitrification/thawing cycle and during further propagation in culture. In conclusion, from the cryopreservation methods investigated in this study, surface‐based vitrification of hESC has proven to be the most efficient for the cryopreservation of intact hESC colonies, reducing the time required to amplify frozen stocks thus supporting the widespread use of these cells in research and clinical applications. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1079–1087, 2012     

Large-scale expansion of feeder-free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation

Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain the ability to contribute to all cell lineages, making them an ideal platform material for cell-based therapies. ESCs are traditionally cultured in static flasks on a feeder layer of murine embryonic fibroblast cells. Although sufficient to generate cells for research purposes, this approach is impractical to achieve large quantities for clinical applications. In this study, we have developed protocols that address a variety of challenges that currently bottleneck clinical translation of ESCs expanded in stirred suspension bioreactors. We demonstrated that mouse ESCs (mESCs) cryopreserved in the absence of feeder cells could be thawed directly into stirred suspension bioreactors at extremely low inoculation densities (100 cells/ml). These cells sustained proliferative capacity through multiple passages and various reactor sizes and geometries, producing clinically relevant numbers (10⁹ cells) and maintaining pluripotency phenotypic and functional properties. Passages were completed in stirred suspension bioreactors of increasing scale, under defined batch conditions which greatly improved resource efficiency. Output mESCs were analyzed for pluripotency marker expression (SSEA-1, SOX-2, and Nanog) through flow cytometry, and spontaneous differentiation and teratoma analysis was used to demonstrate functional maintenance of pluripotency.     

Scalable culture and cryopreservation of human embryonic stem cells on microcarriers

As a result of their pluripotency and potential for unlimited self‐renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large‐scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor‐intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel‐coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF‐microcarriers was less than that on MEF‐plates, the doubling time of hESCs on Matrigel‐microcarriers was indistinguishable from that of hESCs expanded on Matrigel‐coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier‐based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009