两种培养液对小鼠胰岛细胞培养后的形态和功能的影响

目的:考察两种胰岛细胞培养液对小鼠胰岛细胞培养后的细胞形态和功能的影响。方法:分别以RPMI1640和低糖型DMEM为基础配制两种胰岛细胞培养液,与小鼠胰岛细胞共培养24 h,用FD-PI染色观察胰岛活性,透射电镜观察胰岛细胞内部形态,并用连续葡萄糖灌流刺激试验考察胰岛素释放功能。结果:两种胰岛细胞培养液24h内对培养的胰岛活性影响无差异,但透射电镜结果显示以低糖型DMEM为基础的培养液培养的胰岛细胞间隙较小,且葡萄糖灌流结果显示其培养的胰岛细胞后续胰岛素分泌能力显著增加。结论:以低糖型DMEM为基础配制的胰岛细胞培养液能较好地保持培养胰岛的结构和功能。     

内凝胶藻酸盐固定酵母细胞

本文研究了内凝胶藻酸盐固定酵母细胞的方法。其原理是在藻酸盐内部含有不溶性钙化合物的特定部位上发生凝胶,在此溶液中随着D-葡糖酸-1.5内酯水解而伴随溶液的pH值降低,这时钙离子从不溶性钙化合物中释放出来,促使藻酸盐凝胶化。用内凝胶方法能够制备任意形状的胶粒,又可组成新类型发酵反应器。使用标准的发酵程序评价了内凝胶固定化细胞的颗粒的特性。新的凝胶方法在发酵速率和凝胶强度方面均显示出人们所希望的特性。     

聚乙烯醇复合水凝胶外用膜剂的制备与性能*

摘要目的：考察PVA/ 葡聚糖/ 羧甲基纤维素钠复合水凝胶外用膜剂的制备方法,并与纯PVA 水凝胶贴膜进行对比,考察本膜 剂在物理性能和药物体系的体外释放行为上所具备的优越性。方法：利用冷冻-解冻物理交联方法制备水凝胶装载胰岛素模型药 物的外用膜剂,通过万能拉力机和差示扫描量热法考察膜剂的物理性能,利用高效液色谱法考察该膜剂的体外释放行为。结果： PVA复合水凝胶外用贴膜相较于纯PVA水凝胶贴膜的韧性减小、刚性增加,体外释放变好。结论：通过将具有材料友好性的PVA 和多糖葡聚糖、羧甲基纤维素钠合并使用制备胰岛素复合水凝胶贴膜,既能保证贴膜具有良好的物理性能,又具有较好地释放行 为,优于目前文献报道的纯PVA水凝胶贴膜性能,有望继续研究优化性能。     

聚乙烯醇复合水凝胶外用膜剂的制备与性能

目的：考察PVA／葡聚糖／羧甲基纤维素钠复合水凝胶外用膜剂的制备方法,并与纯PVA水凝胶贴膜进行对比,考察本膜剂在物理性能和药物体系的体外释放行为上所具备的优越性。方法：利用冷冻-解冻物理交联方法制备水凝胶装载胰岛素模型药物的外用膜剂,通过万能拉力机和差示扫描量热法考察膜剂的物理性能,利用高效液色谱法考察该膜剂的体外释放行为。结果：PVA复合水凝胶外用贴膜相较于纯PVA水凝胶贴膜的韧性减小、刚性增加,体外释放变好。结论：通过将具有材料友好性的PVA和多糖葡聚糖、羧甲基纤维素钠合并使用制备胰岛素复合水凝胶贴膜,既能保证贴膜具有良好的物理性能,又具有较好地释放行为,优于目前文献报道的纯PVA水凝胶贴膜性能,有望继续研究优化性能。     

活菌制剂冻干保护剂的研究进展

冷冻干燥技术广泛应用于细菌性活疫苗生产和菌种保藏等方面。细菌细胞在冷冻干燥过程中会出现损伤,甚至死亡。通过添加适合的冻干保护剂可以最大程度减小细胞损伤,保持活菌制剂的活力和性能。就冻干过程对细菌的损伤机制、冻干保护剂的作用机理以及保护剂的筛选方法等方面进行了阐述,对活菌制剂保护剂的筛选有所启示。     

乳酸菌冻干保藏相关影响因素的研究进展

乳酸菌在冻干保藏过程中受到多种因素的作用,采取适宜的保护措施,在很大程度上可避免或减轻冷冻干燥对细胞带来的损伤.主要介绍了影响乳酸菌冷冻干燥及保藏相关因素的研究进展,这些因素包括:菌种自身的性质、生长因素、亚致死处理、冻干保护介质和复水条件.     

兔肝细胞体外分离及培养方法的实验研究

目的：研究体外兔肝细胞分离及培养方法,比较不同培养基条件下兔肝细胞培养过程。方法：采用非灌注胶原酶消化法分离兔肝细胞,分别采用RPIM1640培养液（含10％新生牛血清）,DMEM培养液（含10％新生牛血清）,DMEM培养液（含10％胎牛血清）培养,计数法观察原代细胞增殖变化,MTF法观察传代细胞增殖情况。培养细膨采用PAS染色法鉴定,电镜观察细胞超微结构。结果：分离的肝细胞细胞活率大于85％;DMEM培养液（含10％胎牛血清）培养肝细胞生长状态较另两种培养液中的肝细胞强,DMEM培养液（含10％新生牛血清）中的细胞增殖能力较RPIM1640培养液（含10％新生牛血清）高,具有统计学意义。PAS染色和透射电镜观察培养细胞胞质中有大量糖原颗粒。结论：本实验采用的分离方法可获得较纯的肝细胞,而且操作简便实用。DMEM培养液较RPIM1640培养液更加适宜原代培养肝细胞生长,胎牛血清对培养肝细胞的生长促进作用明显高于新生牛血清。     

负载白藜芦醇自组装多肽水凝胶的抗菌性能研究

[目的]制备一种负载白藜芦醇的自组装多肽水凝胶并探讨其抗菌性能。[方法]通过自组装制备多肽(FmocFFGGRGD)水凝胶和载有白藜芦醇的多肽水凝胶(Pep/RES);通过扫描电子显微镜和透射电子显微镜观察水凝胶的形貌和内部结构;通过流变仪检测水凝胶的流变性质;通过高效液相色谱检测Pep/RES的释放速率;通过细胞毒性试验研究该水凝胶的生物相容性;通过抑菌圈实验和活死细菌染色研究Pep/RES对大肠杆菌和金黄色葡萄球菌的抗菌性能。[结果]多肽溶液可在30 min内自组装形成稳定的水凝胶,水凝胶内部的三维结构密度随多肽浓度的增加而增加,2.0wt%浓度的多肽水凝胶稳定效果最好。白藜芦醇从Pep/RES水凝胶中缓慢释放7 d释放量达到50%,Pep/RES浸泡液对NIH/3T3细胞表现出良好的生物相容性。Pep/RES水凝胶中负载的白藜芦醇浓度为512μg/m L时,对金黄色葡萄球菌的抑菌圈直径即可达到5.41±0.18 mm,但即使白藜芦醇浓度达到1 024μg/m L,对大肠杆菌的抑菌圈直径仅为4.27±0.22 nm。[结论]Pep/RES结构稳定,安全无毒,能缓释白藜芦醇,并对金黄色葡萄球菌具有显著抑制作用。     

海藻酸-SiO2杂化凝胶固定化洋葱伯克霍尔德茵脂肪酶的研究

利用四乙氧基硅烷（TEOS）原位水解法将SiO2掺杂于海藻酸（ALG）凝胶中,通过双交联制备出新型ALG—SiO2杂化凝胶以固定化洋葱伯克霍尔德菌脂肪酶。结果表明,固定化酶的最优条件：质量分数为2．0％的ALG、0．2mol／LCaCl2、V（ALG）／V（TEOS）为5、加酶量为1gALG加100mg酶粉、固定化60min、采用直径为0．8mm的针头滴定、真空冷冻干燥。在此条件下,酶蛋白的包埋率可达100％,酶活回收率可达91％。固定化酶的最适pH为8．0,最适作用温度为50℃,重复使用8次后,酶活性仍能保持80％以上。ALG—Si02杂化凝胶的场扫描电镜（FESEM）观察发现凝胶的整体构造仍然是海藻酸凝胶骨架;与ALG凝胶平滑的内部相比较,杂化凝胶仍具有完整的网络结构,但内部更为粗糙,结构更为致密。     

MEM培养基对带状疱疹减毒活疫苗卡那霉素残留量的影响

目的:分析比较MEM培养基对带状疱疹减毒活疫苗卡那霉素残留量的影响。方法:分别使用海克隆、日水2种MEM培养基培养2BS细胞,经传代、感染、洗涤、收获、合并、冻干等工序制备带状疱疹减毒活疫苗,分析比较MEM培养基对带状疱疹减毒疫苗卡那霉素残留量的影响。结果:日水MEM培养基制备的带状疱疹减毒活疫苗卡那霉素残留量一般是海克隆MEM培养基制备疫苗的5-10倍。结论:美国海克隆实验公司的MEM培养基卡那霉素残留量明显小于日水MEM培养基。     

明胶/海藻酸钠/沙蒿胶复合水凝胶的制备及表征

为探究明胶（G）、海藻酸钠（SA）,沙蒿胶（ASKG）对复合水凝胶的力学性能、溶胀和保湿性能的影响,采用共混-离子交联法制备海藻酸钠/明胶/沙蒿胶复合水凝胶,并对制得的水凝胶进行结构表征和溶血率测试。结果表明:当G质量分数为2.5%,SA为1.5%,ASKG为0.7%时,复合水凝胶压缩强度达到427.2 kPa,拉伸强度达到563.449 kPa,断裂伸长率为117%,溶胀率为744%,且具有较好的保湿性能。红外光谱表明,由于沙蒿胶中存在大量羟基,因此加入沙蒿胶后在3 300 cm^-1～3 600 cm^-1羟基峰形变宽。G/SA/ASKG复合水凝胶溶血率低于5%,具有较好的网络孔结构和血液相容性,为复合水凝胶在医用敷料方面的应用提供一定的参考价值。     

Immobilization of cells by electrostatic droplet generation: a model system for potential application in medicine

The process of electrostatic extrusion as a method for cell immobilization was investigated that could be used for potential applications in medicine. An attempt was made to assess the effects of cell addition and polymer concentration on the overall entrapment procedure, ie, on each stage of immobilization: polymer-cell suspension rheological characteristics, electrostatic extrusion process, and the process ofgelation. The findings should contribute to a better understanding of polymer-cell interactions, which could be crucial in possible medical treatments. Alginate-yeast was used as a model system for carrier-cells. The electrostatic extrusion was considered as a complex two-phase flow system and the effects of cell and alginate concentrations on the resulting microbead size and uniformity were assessed. Under investigated conditions, microbeads 50-600 microm in diameter were produced and the increase in both alginate and cell concentrations resulted in larger microbeads with higher standard deviations in size. We attempted to rationalize the findings by rheological characterization of the cell-alginate suspensions. Rheological characterization revealed non-Newtonian, pseudoplastic behavior of cell-alginate suspensions with higher viscosities at higher alginate concentrations. However, the presence of cells even at high concentrations (5x10(8) and 1x10(9) cells/mL) did not significantly affect the rheological properties of Na-alginate solution. Lastly, we investigated the kinetics of alginate gelation with respect to the quantity of Ca2+ ions and cell presence. The gelation kinetics were examined under conditions of limited supply with Ca2+ ions, which can be essential for immobilization of highly sensitive mammalian cells that require minimal exposure to CaCl2 solution. The molar ratio of G units to Ca2+ ions of 3.8:1 provided complete crosslinking, while the increase in alginate concentration resulted in prolonged gelation times but higher strength of the resulting gel. The cell presence decreased the rate of network formation as well as the strength of the obtained Ca-alginate hydrogel.     

Some properties of alginate gels derived from algal sodium alginate

Alginic acid in soluble sodium alginate turns to insoluble gel after contact with divalent metal ions, such as calcium ions. The sodium alginate character has an effect on the alginate gel properties. In order to prepare a suitable calcium alginate gel for use in seawater, the effects of sodium alginate viscosity and M/G ratio (the ratio of D-mannuronate to L-guluronate) on the gel strength were investigated. The wet tensile strengths of gel fibers derived from high viscosity sodium alginate were higher than those from low viscosity sodium alginate. The tensile strength increased with diminishing sodium alginate M/G ratio. Among the gel fibers tested, the gel fiber obtained from a sodium alginate I-5G (1% aqueous solution viscosity = 520 mPa·s, M/G ratio = 0.6) had the highest wet tensile strength. After 13 days treatment in seawater, the wet tensile strength of the gel fiber retained 36% of the original untreated gel strength. For sodium alginates with similar viscosities, the seawater tolerance of low M/G ratio alginate was greater than that of the high M/G ratio one. This study enables us to determine a suitable calcium alginate gel for use in seawater.     

Research on the Long Time Swelling Properties of Poly (vinyl alcohol)/Hydroxylapatite Composite Hydrogel

Poly(vinyl alcohol)/hydroxylapatite(PVA/HA)composite hydrogel was prepared by repeated freezing and thawing.Thewater loss properties of the resultant hydrogel were investigated by using optical microscope.Long time immersion tests ofPVA/HA composite hydrogel were carried out in the diluted calf serum solution to study the change laws of swelling propertieswith the freezing-thawing cycles and HA content.The micro-morphologies of PVA/HA composite hydrogel after long timeimmersion were observed by means of the high-accuracy 3D profiler.The results show that the swelling process of PVA/HAcomposite hydrogel is the converse process of its water loss.Long time swelling ratio curves of PVA/HA composite hydrogel inthe calf serum solution are manifested as four stages of quick increase,decrease,slow decrease and stable balance,and itsequilibrium swelling ratio decreases with the increase of freezing-thawing cycles and HA content.It is revealed that the networkstructure of the composite hydrogel immersed for a long period is significantly improved with the increase of HA content.Perfect network structures of PVA/HA composite hydrogel as well as full and equilibrium tissues after swelling equilibrium areobtained when the HA content is 3% and the number of freezing-thawing cycles is 7.     

Mechanical spectroscopy and relaxometry on alginate hydrogels: a comparative analysis for structural characterization and network mesh size determination

The structure of calcium-saturated alginate hydrogels has been studied by combining rheological determinations and relaxometry measurements. The mechanical spectroscopy analyses performed on alginate gel cylinders at different polysaccharide concentration allowed estimating their main structural features such as the average mesh size. The calculation was based on the introduction of a front factor in the classical rubber elasticity approach which was correlated to the average length of the Guluronic acid blocks along the polysaccharide chain. Transverse relaxation time (T(2)) determinations performed on the cylinders revealed the presence of two relaxation rates of the water entrapped within the hydrogel network. The cross-correlation of the latter data with the rheological measurements allowed estimating the mesh size distribution of the hydrogel network. The results obtained for the hydrogel cylinders were found to be consistent with the relaxometric analysis performed on the alginate microbeads where, however, only one type of water bound into the network structure was detected. A good correlation was found in the average mesh size determined by means of relaxometric measurements on alginate microbeads and by a statistical analysis performed on TEM micrographs. Finally, the addition of a solution containing calcium ions allowed investigating further the different water relaxation modes within alginate hydrogels.     

Enhancing the Viability of Lactobacillus plantarum Inoculum by Immobilizing the Cells in Calcium-Alginate Beads Incorporating Cryoprotectants

Many literature reports have cited the importance of the rehydration conditions of lyophilized cultures in determining viability. The rate of rehydration and the volume of fluid used have been identified as two important factors. One possible means of controlling these is by immobilizing the cells before lyophilization within a gel matrix in which the subsequent rehydration rate and fluid volume would be controlled by the properties of the gel. In this study Lactobacillus plantarum was immobilized and lyophilized in Ca-alginate beads in which 1 M glycerol or 0.75 M adonitol with skim milk were incorporated as a cryoprotectant. The properties of these Ca-alginate beads were examined before and after lyophilization and rehydration. The beads incorporating glycerol were smaller and stronger than those with adonitol. After lyophilization, size decreased and strength increased but to a greater extent in the beads with glycerol, indicating that the microenvironment within the two bead types was probably different. The protective effect of the bead microenvironment on immobilized L. plantarum was also examined. Lyophilization and rehydration within the alginate beads with either polyol yielded higher survival rates than that attained with free cell cultures during rehydration in optimal or suboptimal conditions. During rehydration under suboptimal conditions, the immobilized cell survival was greatest when 0.75 M adonitol was the incorporated cryoprotectant.     

Investigation of asphalt aging behaviour due to oxidation using molecular dynamics simulation

An attempt is made to connect the link between internal chemical and molecular mechanical property change and external physical, rheological and mechanical property change for asphalt before and after oxidative aging using molecular dynamics (MD) simulation. Intermolecular interactions, density, bulk modulus and zero shear viscosity changes of model asphalt systems before and after oxidative aging and mechanical property changes of the asphalt systems under different compressive and tensile stress rates are investigated at room temperature (298 K). Simulation results demonstrate that oxidised functional groups in asphalt molecules increase the strength of intermolecular bonds and the bulk modulus of asphalt, which further contribute to the hardening of the oxidised asphalt. The internal property change is consistent with the external physical and rheological property change after oxidation, which is revealed by the increase of density and viscosity. In addition, both the unoxidised and oxidised asphalts deform more and fail faster with an increase in both compressive and tensile stress rates, especially under tensile stresses. The oxidised asphalt is stiffer than the unoxidised asphalt, which shows less deformation.     

Cytotoxicity and biocompatibility evaluation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel for drug delivery application

In this paper, covalently cross-linked hydrogel composed of N,O-carboxymethyl chitosan and oxidized alginate was developed intending for drug delivery application. In vitro/vivo cytocompatibility and biocompatibility of the developed hydrogel were preliminary evaluated. In vitro cytocompatibility test showed that the developed hydrogel exhibited good cytocompatibility against NH3T3 cells after 3-day incubation. According to the results of acute toxicity test, there was no obvious cytotoxicity for major organs during the period of 21-day intraperitoneal administration. Meanwhile, the developed hydrogel did not induce any cutaneous reaction within 72 h of subcutaneous injection followed by slow degradation and adsorption with the time evolution. Moreover, the extraction of developed hydrogel had nearly 0% of hemolysis ratio, which indicated the good hemocompatibility of hydrogel. Based on the above results, it may be concluded that the developed N,O-carboxymethyl chitosan/oxidized alginate hydrogel with non-cytotoxicity and good biocompatibility might suitable for the various drug delivery applications.     

Alginate Hydrogel Protects Encapsulated Hepatic HuH-7 Cells against Hepatitis C Virus and Other Viral Infections

Cell microencapsulation in alginate hydrogel has shown interesting applications in regenerative medicine and the biomedical field through implantation of encapsulated tissue or for bioartificial organ development. Although alginate solution is known to have low antiviral activity, the same property regarding alginate gel has not yet been studied. The aim of this work is to investigate the potential protective effect of alginate encapsulation against hepatitis C virus (HCV) infection for a hepatic cell line (HuH-7) normally permissive to the virus. Our results showed that alginate hydrogel protects HuH-7 cells against HCV when the supernatant was loaded with HCV. In addition, alginate hydrogel blocked HCV particle release out of the beads when the HuH-7 cells were previously infected and encapsulated. There was evidence of interaction between the molecules of alginate hydrogel and HCV, which was dose- and incubation time-dependent. The protective efficiency of alginate hydrogel towards HCV infection was confirmed against a variety of viruses, whether or not they were enveloped. This promising interaction between an alginate matrix and viruses, whose chemical mechanisms are discussed, is of great interest for further medical therapeutic applications based on tissue engineering.     

Incorporation of DMSO and dextran-40 into a gelatin/alginate hydrogel for controlled assembled cell cryopreservation

A new cell cryopreservation strategy for cell-assembling constructs was proposed. With this strategy, different concentrations of dimethysulfoxide (DMSO) and dextran-40 were directly incorporated into the cell/gelatin/alginate systems, prototyped according to a predesigned structure, cryopreserved at −80 °C for 10 days and followed a thawing process at 17 °C. The rheological properties, bonding water contents and melting points of the gelatin/alginate hydrogel systems were changed with the addition of different amounts of DMSO. The microscopy analysis, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide (MTT) and hematoxylin and eosin (HE) staining indicated that the cell numbers were progressively in a selected DMSO concentration range. With DMSO 5% (v/v) alone, the metabolic rate in the construct attained (81.3 ± 5.7)%. A synergistic effect was achieved with the combination of the DMSO/gelatin/alginate and dextran-40/gelatin/alginate hydrogel systems. These results indicated that the inclusion of DMSO and dextran-40 in the hydrogel could effectively enhance the cell preservation effects. This cryopreservation strategy holds the ability to be widely used in organ manufacturing techniques.