人肝癌细胞Hep-G2的低温保存研究

在细胞的低温保存中,低温保护剂的种类与浓度对复温后的存活率有着重要影响。本文以人肝癌细胞Hep-G2为研究对象,利用慢速冷冻法,筛选最佳的冻存液配方。通过配比不同浓度的甘油、Me2SO以及在Me2SO中添加一定浓度的蔗糖、海藻糖,一周后复温细胞,对台盼蓝染色存活率、MTT存活率以及24 h贴壁率三种检测结果进行比较分析,结果表明:以Me2SO作为低温保护剂时,冻存液浓度为10%(v/v)的Me2SO复温后细胞的三种检测指标最优;以甘油作为低温保护剂时,冻存液浓度为20%(v/v)的甘油复温后细胞的三种检测指标最优;再将以上分别得到的最佳浓度(即体积浓度20%甘油、10%Me2SO)与5%Me2SO(v/v)+0.3 mol/L蔗糖、5%Me2SO(v/v)+0.3 mol/L海藻糖这四种低温保护剂进行冻存与比较,5%Me2SO(v/v)+0.3mol/L海藻糖检测指标高于其他实验组,并且差异显著(P0.05)。最终得到5%Me2SO(v/v)+0.3 mol/L海藻糖为慢速冷冻保存Hep-G2细胞的最优保护剂配方。     

微滴喷射玻璃化的过程损伤分析及保护剂优化

微滴喷射玻璃化保存系统产生的微滴尺寸较小,在较低浓度的低温保护剂条件下即可实现玻璃化。本文采用微滴喷射玻璃化保存系统对Hep G2细胞进行玻璃化保存,研究保护剂加载过程、喷射过程、接收过程及玻璃化/复温过程对细胞造成的损伤程度,并通过降低保护剂中Me₂SO浓度、添加适量海藻糖来优化保护剂配方。结果表明,微滴喷射玻璃化保存各过程对细胞均有损伤,保护剂加载过程、喷射过程、玻璃化及复温过程对细胞造成的损伤较大,薄片接收过程对细胞造成的损伤小。另外,随着保护剂中Me₂SO浓度的降低,低温保存后的细胞活性明显降低;保护剂浓度相同时,玻璃化保存效果较慢速冷冻效果好;适量的海藻糖能够起到增强低温保存效果的作用,过量则起到降低作用;以5%Me₂SO+0. 3 mol/L海藻糖作为低温保护剂玻璃化保存细胞时,细胞存活率达(92. 42±0. 95)%,24 h贴壁率达到(95. 64±1. 03)%,微滴喷射玻璃化效果最好。     

胞内海藻糖对红细胞冷冻干燥保存效果的影响

冷冻干燥是长期保存人体红细胞最理想的方法之一。利用孵育的方法将非渗透性保护剂海藻糖载人人体红细胞中,以含海藻糖的保护剂对红细胞进行冻干保存实验,研究胞内海藻糖浓度对红细胞冻干保存的影响。在37℃下,以不同浓度海藻糖溶液（400mM,600mM,800mM,1000mM）对红细胞孵育7h。结果表明,随胞外海藻糖浓度的增加,胞内海藻糖的浓度相应升高,试验中海藻糖浓度最高达到35mmol／L。对新鲜与孵育后红细胞在相同条件下进行冻干实验,结果表明,孵育后的红细胞冻干回收率随胞内海藻糖浓度增加而增加,表明胞内海藻糖在冻干过程中对红细胞有保护作用。     

糖醇类物质对人肝细胞低温保存的影响

将传统肝细胞冻存保护剂配方中的DMSO的浓度减为5%,并添加不同浓度的D-山梨醇、木糖醇或麦芽糖醇,置于-80℃的冰箱冻存两周,两周后将细胞快速复温,进行细胞存活率、24h贴壁率以及细胞形态学的检测,并与10%浓度DMSO组对照比较。结果表明,D-山梨醇、木糖醇、麦芽糖醇均对人肝细胞的低温保存有一定的保护作用,其中5%DMSO+0.4M D-山梨醇组复温后细胞的存活率、贴壁率均优于其他冻存液组,D-山梨醇与DMSO联合使用降低了冻存保护剂DMSO的浓度,表明两者有协同作用。     

海藻糖和胎牛血清对人乳腺细胞HBL-100低温保存的影响

建立高质量的生物样本库至关重要,低温保存过程中保护剂的种类和浓度对样本的冷冻效果有很大影响。本文以人乳腺细胞HBL-100为研究对象,在Me_2SO含量10%的DMEM溶液中分别添加不同浓度海藻糖(0~0.3 mol/L)和不同体积分数FBS(20%~60%)。采用逐步降温法,先在-80℃冰箱中慢速降温4 h,再快速投入到液氮中(-196℃),冻存7 d后,在37℃水浴锅中快速摇晃复温。分别用台盼蓝法、CCK法和24 h贴壁率法检测细胞的成活率,结果表明:与对照组相比,海藻糖和FBS对细胞的冷冻效果都有显著影响。当海藻糖浓度一定时,FBS体积分数越高,细胞的冷冻效果越好,但不添加海藻糖时,FBS的作用不明显;当FBS体积分数一定时,三种检测指标在海藻糖浓度为0.2 mol/L时最优,且高浓度海藻糖可能会抑制FBS作用。考虑成本等因素,最适宜冻存人乳腺细胞HBL-100的冻存液为10%Me_2SO+40%~60%FBS+0.2 mol/L海藻糖。     

基于蚕丝蛋白的低温保护剂用于梅山猪耳成纤维细胞的低温保存研究

细胞低温保存为临床治疗和科学研究提供优质的细胞。体积分数为10%二甲基亚砜(DMSO)和体积分数为20%胎牛血清(FBS)是目前冻存细胞常用的保护剂。但DMSO对细胞具有毒性损伤,FBS存在携带病毒、感染疾病的风险。本文以梅山猪耳成纤维细胞作为模型细胞进行冻存实验,将蚕丝蛋白用于低温保护剂中,用不同质量浓度的丝胶蛋白和不同体积分数的丝素蛋白来分别替代FBS和DMSO,验证其在细胞低温保存中的有效性。解冻复苏后用台盼蓝染色法、MTT法、24 h贴壁率等检测细胞的存活率和生长活力,筛选出最佳的基于天然蚕丝蛋白的低温保护剂配方。结果表明:含质量浓度为1%丝胶蛋白和含体积分数为20%FBS的低温保护剂对猪耳细胞的冻存效果无显著差异,说明丝胶蛋白能有效替代FBS。将体积分数为10%DMSO浓度降至5%,添加体积分数为10%丝素蛋白后,细胞的存活率和贴壁率与对照组相比无明显差异,说明丝素蛋白能降低DMSO的浓度。将质量浓度为1%丝胶蛋白与体积分数为10%丝素蛋白联用后,能达到较好的冻存效果。     

人体骨髓基质干细胞冷冻干燥的探索性实验

人体骨髓基质干细胞已成为组织工程、细胞移植和基因治疗等领域的重要研究对象,目前对它已有低温保存的研究,而通过冷冻干燥的方法对其进行保存国内外未见报导.实验尝试用冻干的方法来保存骨髓基质干细胞.文中选取海藻糖、PVP、HES等作保护剂,应用差示扫描量热仪(DSC)测量其结晶温度和玻璃化转变温度;随后对加入保护剂的细胞溶液进行冻干实验,并应用流式细胞仪对冻干样品复水后的细胞活性进行了测定,其中30%PVP 20%海藻糖对细胞的保护效果较好,细胞成活率达到16.40%.     

冷冻干燥保存人脐血实验研究

尝试用冷冻干燥法来长期保存脐血和有核细胞,并对冻干效果进行比较分析.选用聚乙烯吡咯烷酮(PVP)、蔗糖和甘露醇等作为冻干保护剂,先将样品冻结到-38℃,随后在-30℃搁板温度下进行升华干燥,最后在15℃搁板温度下进行解吸干燥.冻干后的样品用磷酸盐缓冲液复水,然后用血细胞计数板、流式细胞仪和扫描电镜进行检测.实验结果表明:在400 g/L PVP 200 g/L蔗糖 100 g/L甘露醇作为保护剂时细胞数目恢复率最高,为(75.0±4.1)%(P＜0.01),PI染色鉴定测得单个核细胞拒染率在400 g/L PVP 200 g/L蔗糖 10%胎牛血清作为保护剂时最高,为(98.57±0.68)%(P＜0.01),CD34 细胞回收率最高为35.59%.最终确定有核细胞的冻干效果比全血的要好,初步判定冷冻干燥法保存脐血是可行的.     

人血小板的冷冻干燥保存:细胞内外保护剂的作用

通过液相内吞途径将13mmol／L海藻糖载入人血小板内,采用牛血清白蛋白和海藻糖作细胞外保护剂,研究细胞内海藻糖和细胞外保护剂在血小板冻干保存中分别所起的作用。实验结果表明冻干血小板的活性主要依靠细胞外保护剂提供的保护作用而得以保存,13mmol／L的细胞内海藻糖只起到有益的补充作用。在13mmol／L细胞内海藻糖和1％牛血清白蛋白＋1％海藻糖细胞外保护剂同时存在的情况下,冻干保存40天的血小板复水后的数值恢复率约85％,显微镜观察细胞形态完整,对1U／ml凝血酶的聚集百分比为97．3％。     

甘油预处理对红细胞冷冻干燥保存作用的实验研究

探索使用甘油作保护剂提高人体红细胞冷冻干燥保存效果的可能性。甘油预处理红细胞的目的是增加细胞质浓度,减小细胞的冷冻损伤。实验分为3组:新鲜对照组、20%甘油组与40%甘油组,冻干保存前后分别检查红细胞相关指标。结果表明,20%与对照组无显著差别;40%甘油组的冻干细胞回收率与血红蛋白回收率分别维持在冷冻干燥前的55.37±4.26%和53.49±3.85%,非常显著高于对照组(P<0.01);渗透脆性与超氧化物歧化酶(SOD)活性均显著高于对照组(P<0.05),但与新鲜红细胞无显著差异。研究结果为使用甘油作红细胞的冻干保护剂提供了理论基础。     

Evaluation of alginate-chitosan semi IPNs as cartilage scaffolds

In this study, alginate and alginate:chitosan semi interpenetrating polymer network (IPN) scaffolds were prepared by freeze-drying process. Alginate scaffolds were crosslinked with different concentrations of CaCl₂, i.e. 0.5, 1 or 3% (w/v), in 96% (v/v) ethanol solutions for two different periods, i.e. 4 and 24 h, after freeze-drying. Scanning electron microscope (SEM)/ Energy Dispersive Analysis by X-ray (EDAX) analysis and swelling studies indicated that crosslinking of scaffolds with 3% (w/v) CaCl₂ for 24 h was effectively created suitable alginate scaffolds in terms of optimum porosity and mechanical stability. This is why, alginate:chitosan semi IPN scaffolds were prepared at the crosslinking condition mentioned above in 70:30, 60:40 and 50:50% (v/v) alginate:chitosan ratios. Besides the attachment and proliferation abilities of ATDC5 murine chondrogenic cells on alginate, 70:30% (v/v) alginate:chitosan and 50:50% (v/v) alginate:chitosan scaffolds, their cellular responses were assessed for chondrogenic potential. These structural and cellular outcomes demonstrate potential utility of chitosan semi IPNs in alginate scaffolds. Comparative results found in relation to alginate scaffolds, support the necessity for alginate:chitosan scaffolds for improved cartilage tissue engineering.     

甘油对人的红细胞冷冻干燥保存效果的影响

就甘油对人红细胞冷冻干燥保存效果的影响进行了实验研究。实验中先用不同浓度的甘油对红细胞进行处理，用PVP、柠檬酸钠、蔗糖和胎牛血清作为冻干保护剂对红细胞进行冻干保存，冻干后的样品用质量浓度为30g／L的NaCl复水，用血球分析仪和光镜进行检测。实验结果表明；用甘油进行处理后，红细胞的回收率明显高于没有用甘油处理的红细胞组。其中，采用质量浓度为400g／L的甘油处理后，红细胞冻干回收率达到51．4％。     

连用DCS与显微方法的冷冻干燥系统及实验研究

研制了一种真空冷冻干燥装置,并在其中引入了DSC热分析和显微观察的功能,利用此连用DSC与显微方法的冷冻干燥系统,对低温保护剂海藻糖(trehalose)和聚乙烯醇(PVA)的水溶液进行了冷冻干燥实验,主要考察冷冻速率和溶液用量等不同冷冻条件对溶液冻结特性和升华过程的影响,并通过冻干溶液多孔结构的显微照片估计溶液冻结后形成冰晶的情况.实验结果表明,加快冷冻速率会导致不完全冻结并形成大量的细小冰晶,增加样品用量同样会引起过冷度增大,导致冰晶尺寸减小,使升华阻力增大,升华速率减慢.     

In vitro characterization of chitosan scaffolds: influence of composition and deacetylation degree

In this study, the influence of degree of deacetylation (DD) and composition on some structural and biological properties of chitosan scaffolds were examined in vitro. 3D chitosan scaffolds of 2% (w/v) and 3% (w/v) composition in different DDs i.e. 75–85% and >85% were prepared by freeze-drying method at −80 °C. We noticed that >85% deacetylated chitosan scaffolds of 2% (w/v) composition has a highly interconnected morphological structure having ∼100 μm pore size with 0.0917 N/mm² compression modulus. L929 fibroblastic cells were cultured on chitosan scaffolds in order to evaluate their biocompatibilities. Cell culture studies demonstrated that fibroblastic cell attachment and proliferation is affected by DD. The higher deacetylated chitosan scaffolds strongly supported the attachment and proliferation when compared with the lower deacetylated scaffolds. MTT assay indicated that >85% deacetylated chitosan scaffolds of 2% (w/v) composition, having the highest specific growth rate 0.017 h⁻¹ of all, was found to be the most suitable for cell culture studies and a potential candidate for tissue engineering with enhanced biostability and good biocompatibility.     

High-resolution TOF-SIMS imaging of eukaryotic cells preserved in a trehalose matrix

A novel, trehalose-glycerol matrix was utilized to generate high-resolution, TOF-SIMS images of macrophages and glial cells. Viable cells incubated in 50 mM trehalose, then lyophilized in a 50 mM trehalose, 10-15% (w/w) glycerol rinse, are preserved and chemically profiled. These experiments demonstrate the utility of the disaccharide matrix as an efficient, cost-effective alternative to cryogenics for SIMS and other ultrahigh-vacuum (UHV) analyses of biological species. Cellular processes on oligodendrocytes and astrocytes, 1-3 mum in width, were well resolved for cells in the trehalose-glycerol matrix. The viscous cell matrixes were fractured and analyzed at room temperature and maintained their three-dimensional integrity under UHV. Images have been generated with a Au primary ion source near the static limit of 10(12) ions/cm2. Though these nucleated cells do not remain viable after desiccation, TOF-SIMS imaging and subsequent rehydration reveals structural and morphological preservation. Eliminating the inherent obstacles associated with cryogenic analysis opens the door to greater utility of SIMS as a bioanalytical tool, such as lipid mapping of single cells in the nervous system.     

Increasing the aqueous solubility of acetaminophen in the presence of polyvinylpyrrolidone and investigation of the mechanisms involved

It was shown that the aqueous solubility of acetaminophen in the presence of polyvinylpyrrolidone (PVP) increased. The solubility at 25°C increased from 14.3 mg mL^-1 in the absence of PVP, to 19.7 mg mL^-1 in the presence of 4% w/v PVP, and to 26.7 mg mL^-1 in the presence of 8% w/v PVP. Dialysis studies indicated that there is a potential of binding between PVP and acetaminophen in their aqueous solutions. Dialysis studies also revealed that the nature of interaction between PVP and acetaminophen is physical and reversible, and there was no strong binding between PVP and acetaminophen in their solutions. Infrared spectroscopy of acetaminophen/PVP solid dispersion indicated that the mechanism of interaction between PVP and acetaminophen is via hydrogen bonding. Therefore, the increase in solubility of acetaminophen in the presence of PVP is probably attributed to its ability to form a water-soluble complex with PVP.     

Radiation synthesis of PVP/alginate hydrogel containing nanosilver as wound dressing

Hydrogels with polyvinyl pyrrolidone (PVP) and alginate were synthesized and silver nanoparticles were incorporated in hydrogel network using gamma radiation. PVP (10?and 15?%) in combination with 0.5?and 1?% alginate was gamma irradiated at different doses of 25?and 40?kGy. Maximum gel percent was obtained with 15?% PVP in combination with 0.5?% alginate. The fluid absorption capacity for the PVP/alginate hydrogels was about 1881–2361?% at 24?h. Moisture vapour transmission rate (MVTR) of hydrogels containing nanosilver at 24?h was 278.44?g/(m²h). The absorption capacity and moisture permeability of the PVP/alginate–nanosilver composite hydrogel dressings show the ability of the hydrogels to prevent fluid accumulation in exudating wound. The hydrogels containing nanosilver demonstrated strong antimicrobial effect and complete inhibition of microbial growth was observed with 70?ppm nanosilver dressings. PVP/alginate hydrogels containing nanosilver with efficient fluid handling capacity and antimicrobial activity was found suitable for use as wound dressing.