成纤维细胞玻璃化保存的初步实验研究

成纤维细胞是构建组织工程化真皮的种子细胞,实现成纤维细胞的低温保存对成功保存组织工程化真皮有着重要的意义.采用不同组成及浓度的玻璃化溶液对成纤维细胞进行玻璃化低温保存实验,并考虑了离心次数对细胞存活率的影响.实验结果表明:玻璃化溶液的组成、浓度及复温后的离心次数等因素对细胞的存活率均有明显的影响.在实验条件下,采用组分配比为5.4 mol/LEG 1.4 mol/L DMSO的玻璃化溶液对成纤维细胞进行玻璃化保存,复温后以1000r/min的转速离心1次(5 min)后,再以800r/min的转速离心4次(每次4 min),可获得较高的细胞存活率,其值为87.1%.     

培养9天的组织工程化真皮低温保存的研究

组织工程化真皮的低温保存是皮肤组织工程的重要组成部分，对皮肤组织库的建立有重要意义。以培养一定时间的组织工程化真皮为试材，以四唑盐(MTT)比色法检测细胞存活率，辅以光学显微镜和扫描电镜观察分析，试验研究了低温保护剂预处理方式、降温程序及降温速率等条件对组织工程化真皮低温保存效果的影响。实验表明：将组织工程化真皮置于2．1mol/L的DMSO溶液中，在4℃下处理15min，再只将组织工程化真皮转入低温保存袋中，迅速热封口，以1℃／min速率降温又无恒温处理可获得较高的细胞存活率(72．8％～78％)，可满足临床应用的要求。     

人体细胞的低温保存与冷冻干燥

本文阐述了细胞和组织的低温保存和冷冻干燥的意义、现状、以及所研究的技术科学问题.包括低温损伤与溶液冻结相变的传热传质过程的关系;降温和复温过程的热分析和热控制;低温保存的玻璃化理论;水溶液的非晶态化特性的研究;一些新的技术手段和仪器设备;组织工程化细胞和组织的低温保存;活细胞的冷冻干燥.     

组织工程化骨低温保存研究现状及展望

基组织工程学是一门结合了工程学、生命科学、材料学和临床医学的交叉科学.骨组织工程研究是组织工程研究的热点.种子细胞的低温保存对临床有重要意义.从低温保存的角度综述了组织工程化骨低温保存技术以及国内外研究现状和面临的问题.最后提出了对骨组织工程低温保存研究的展望.     

用于1型糖尿病细胞治疗的胰岛低温保存进展

近年来,1型糖尿病细胞治疗技术发展迅速,但供体胰岛的缺乏及供体与受体在时间和空间上的不匹配限制了其在临床上的应用.低温保存技术可以解决该限制,但经传统低温保存方法保存的胰岛存活率和功能性均较差,难以达到临床应用的标准.本文对胰岛低温保存原理及低温保存过程中造成胰岛损伤的因素进行了分类归纳,从冰晶损伤抑制、保护剂损伤抑制...     

大鼠皮肤成纤维细胞在低密度培养条件下基质金属蛋白酶9和13基因表达的抑制

为探索体外促进原代皮肤成纤维细胞转分化为肌成纤维细胞的培养条件及原代皮肤成纤维细胞转分化前后两种状态下经肿瘤坏死因子(tumor necrosis factor alpha,TNF-α)刺激,其MMP基因的表达情况。利用Wistar大鼠真皮分离出的原代成纤维细胞为模型,分别以高密度与低密度将细胞培养于塑料界面,通过Western Blot和q RT-PCR检测肌成纤维细胞的标志物α-平滑肌肌动蛋白(α-SMA)的表达量;同时经TNF-α刺激后,利用q RT-PCR和明胶酶谱检测高密度与低密度两种状态下MMP-9和MMP-13的表达及两种状态下MMP-9的活性。发现大鼠真皮中分离出的原代成纤维细胞在低密度培养条件下,可以有效地转分化为肌成纤维细胞,其标志物α-SMA显著上调;经肿瘤坏死因子刺激,成纤维细胞在低密度培养条件下MMP-9和MMP-13对TNF-α刺激的响应程度较高密度培养时显著降低。初步表明在大鼠真皮(dermis)中,原代成纤维细胞体外转分化为肌成纤维细胞且MMP-9和MMP-13在肌成纤维细胞中下调的现象是细胞密度依赖型。     

复杂组织器官低温保存研究进展

近年来,器官移植术以及相关基础研究的快速发展对复杂组织器官的高质量保存提出了迫切需求.供体组织器官的缺乏以及供体和受体在时间和空间上的不匹配限制了其在临床上的应用,低温保存有望解决这一限制.目前,虽然已经成功实现了细胞、胚胎等小尺寸生物样本的低温保存,但对大体积的复杂组织器官来讲,现有低温保存技术仍然面临诸多瓶颈问题....     

成纤维细胞和血小板在多壁碳纳米管上粘附性

使用化学气相沉积(CVD)的方法在碳纸基底上制备了多壁碳纳米管(MWCNTs),并研究了L929小鼠成纤维细胞在多壁碳纳米管和碳纸对照组上的粘附及增殖等生长行为,以及各种血液蛋白吸附于这两种材料表面后对人皮肤成纤维细胞粘附产生的影响,同时对比了这两种材料的血小板粘附情况.结果表明:种植在多壁碳纳米管上的成纤维细胞生长明显比碳纸上的旺盛,细胞浓度从第1天的12.5×10~5/mL明显增加到第7天的4.1×10~5/mL,多壁碳纳米管对细胞无毒性反应.预吸附白蛋白、纤维蛋白原、免疫球蛋白对细胞粘附量有促进作用,并且多壁碳纳米管的血小板粘附率低于碳纸.这些结果证明多壁碳纳米管具有良好的组织相容性和一定的血液相容性.     

PDMS-玻璃微流控芯片上附着态HepG2细胞低温保存条件研究

微流控芯片上细胞样本的低温保存能够显著缩短芯片上细胞实验周期、降低实验成本,有助于芯片上细胞实验的广泛应用。本文对PDMS-玻璃微流控芯片上附着态HepG2细胞的低温保存条件进行了研究,包括降温速率、低温保护剂、低温存储时间及存储温度,确定了芯片上附着态HepG2细胞的低温保存方案。结果表明:采用不同的冷却环境、冷却介质,芯片微通道内能够实现0.36～35.12℃/min的降温速率;芯片上附着态HepG2细胞低温保存存在最优降温速率0.5～1.5℃/min;-20℃或液氮表面上方40 cm处冷却环境与-80℃冷却环境组合冻存能够显著提高芯片上冻存7 d的HepG2细胞存活率;体积浓度为20%的DMSO与0.1 mol/L海藻糖联合能够显著提高芯片上冻存HepG2细胞活性。经过上述冻存条件优化后,芯片上附着态HepG2细胞冻存7 d活性超过90%。     

细胞低温保存过程中冰晶成核的研究进展

细胞低温保存过程中,胞内冰损伤是其主要损伤因素之一,这可以通过控制冰晶成核来降低。本文总结了物理、化学及生物控制成核方法在低温保存领域的应用,归纳了各类控制成核方法的机理及对细胞产生的影响,对比得出生物及化学试剂控制成核方法较为优异。并提出最佳成核温度的概念,在该温度下,胞外冰产生且引导细胞适当脱水,减少胞内自由水,降低胞内冰的产生几率,为控制成核在细胞低温保存领域的应用指明了方向。     

Human fibroblast attachment on fibrous parylene-C thin-film substrates

Although the polymeric form of parylene-C is used in many medical devices, the mechanistic nature of cellular attachment to polymeric parylene-C is not clear. We examined the effects of (i) substrate morphology, (ii) surface wettability and (iii) presence of serum proteins on fibroblast attachment. A physicochemical vapor deposition technique was implemented to deposit flat parylene-C substrates as well as fibrous substrates of three different morphologies: slanted columnar, chevronic and chiral. Flat parylene-C surfaces were moderately hydrophobic while fibrous substrates were superhydrophobic. Pretreatment with oxygen plasma changed the substrate surfaces from hydrophobic to superhydrophilic. The attachment efficiency of human fibroblast cells to the flat and three fibrous thin-film parylene-C substrates was investigated. Fibroblast attachment was better on fibrous substrates than on flat substrates, and oxygen plasma pretreatment facilitated fibroblast attachment on all four morphologies. Serum proteins also facilitated cell attachment on all substrates. The combination of oxygen plasma pre-treatment and serum proteins increased fibroblast adhesion in an additive manner on flat, but not on fibrous parylene-C substrates. The morphology of cell–substrate interactions differed between fibrous and flat parylene-C substrates.     

Towards ready-to-use 3-D scaffolds for regenerative medicine: adhesion-based cryopreservation of human mesenchymal stem cells attached and spread within alginate–gelatin cryogel scaffolds

Cultivation and proliferation of stem cells in three-dimensional (3-D) scaffolds is a promising strategy for regenerative medicine. Mesenchymal stem cells with their potential to differentiate in various cell types, cryopreserved adhesion-based in fabricated scaffolds of biocompatible materials can serve as ready-to-use transplantation units for tissue repair, where pores allow a direct contact of graft cells and recipient tissue without further preparation. A successful cryopreservation of adherent cells depends on attachment and spreading processes that start directly after cell seeding. Here, we analyzed different cultivation times (0.5, 2, 24 h) prior to adhesion-based cryopreservation of human mesenchymal stem cells within alginate–gelatin cryogel scaffolds and its influence on cell viability, recovery and functionality at recovery times (0, 24, 48 h) in comparison to non-frozen control. Analysis with confocal laser scanning microscopy and scanning electron microscopy indicated that 2 h cultivation time enhanced cryopreservation success: cell number, visual cell contacts, membrane integrity, motility, as well as spreading were comparable to control. In contrast, cell number by short cultivation time (0.5 h) reduced dramatically after thawing and expanded cultivation time (24 h) decreased cell viability. Our results provide necessary information to enhance the production and to store ready-to-use transplantation units for application in bone, cartilage or skin regenerative therapy.     

细胞冻融策略优化及设备研究进展

低温冷冻保存是细胞治疗、辅助生殖等生物医学应用的使能技术之一.冷冻和复苏过程中,胞内外冰晶的形成和生长会对细胞产生损伤;其中,升降温速率又与冰晶的形成、生长有密切关系.本文在阐述程序化慢速冷冻和玻璃化快速冷冻两种细胞冻存方式及冷冻损伤两因素假说的基础上,对细胞特性、细胞外环境和升降温策略三个影响细胞冻存效果的要素进行分...     

In vitro evaluation of crosslinked electrospun fish gelatin scaffolds

Gelatin from cold water fish skin was electrospun, crosslinked and investigated as a substrate for the adhesion and proliferation of cells. Gelatin was first dissolved in either water or concentrated acetic acid and both solutions were successfully electrospun. Cross-linking was achieved via three different routes: glutaraldehyde vapor, genipin and dehydrothermal treatment. Solution's properties (surface tension, electrical conductivity and viscosity) and scaffold's properties (chemical bonds, weight loss and fiber diameters) were measured. Cellular viability was analyzed culturing 3T3 fibroblasts plated on the scaffolds and grown up to 7 days. The cells were fixed and observed with SEM or stained for DNA and F-actin and observed with confocal microscopy. In all scaffolds, the cells attached and spread with varying degrees. The evaluation of cell viability showed proliferation of cells until confluence in scaffolds crosslinked by glutaraldehyde and genipin; however the rate of growth in genipin crosslinked scaffolds was slow, recovering only by day five. The results using the dehydrothermal treatment were the less satisfactory. Our results show that glutaraldehyde treated fish gelatin is the most suitable substrate, of the three studied, for fibroblast adhesion and proliferation.     

Biocompatibilty of starch-based films from starch of Andean crops for biomedical applications

In this communication we report the use of starch films as cell substrates. To the best of our knowledge it is the first time that films prepared from native Andean starches are studied as biomaterials. For the present study 3T3 fibroblast cells were seeded in seventeen novel starch based films from different Andean crops. In order to analyze the use of these types of starch as biomedical materials, biocompatibility, viability and cell adhesion studies were performed at the third day of incubation on supplemented DMEM medium. After cultured, films made from starch of “tunta”, “muro-huayro” potato and white carrot showed the highest level of living cells and cell viability. These results indicate that native starches from Andean crops can be used for biomedical applications.     

Cryobank of plant genetic resources in Russian Academy of Sciences

Cryopreservation is the most reliable method for long-term storage of plant genetic resources. A review of cell injury by ice crystals and dehydration during a freeze–thaw cycle is given. For successful regeneration of plants and cultures after cryopreservation of their cells, the development of reliable cryopreservation procedure is required including preliminary cultivation, treatment by cryoprotectors, freezing by different methods, thawing and recultivation. Up to now 27 cell lines successfully resumed their growth after storage in liquid nitrogen and preserved their specific features and biosynthetic potential. Besides, shoot tips of 40 cultivars of potato, rose, strawberry and raspberry regenerated plants both in vitro and in vivo after cryopreservation. The longest storage duration was 25 years. Now in liquid nitrogen we continuously store 24 cell strains of rare medicinal plants, shoot tips of seven cultivars of strawberry and raspberry and seeds of 250 endangered plant species collected over all Russian territory.