微囊化重组CHO细胞制备和培养条件的优化

微囊化重组基因细胞移植治疗肿瘤是一种新兴的肿瘤基因治疗方法,然而由于目前微囊化细胞规模化制备和培养技术还不成熟,阻碍了其在临床治疗中的推广与应用。以重组CHO细胞为模型,考察了不同的微囊制备和培养条件对微囊化细胞生长和内皮抑素表达的影响。实验表明,种子细胞所处的生长阶段和细胞接种密度对微囊化细胞生长和内皮抑素表达的影响较大,对数生长期的细胞进行包囊并且细胞接种密度为1×106~2×106cells/mL微囊时微囊内细胞生长良好、内皮抑素表达量高。微囊制备时间对细胞活性和内皮抑素表达也有较大的影响,制备时间延长对细胞的损伤增大,因此制备时间应控制在5h以内。生物微胶囊在制备过程中会造成细胞损伤,而体外培养是恢复细胞活性的良好方法,在培养过程中微囊接种量为5%时对细胞生长和内皮抑素表达有利。     

Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

Cryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage.     

Production of cell-enclosing hollow-core agarose microcapsules via jetting in water-immiscible liquid paraffin and formation of embryoid body-like spherical tissues from mouse ES cells enclosed within these microcapsules

We developed agarose microcapsules with a single hollow core templated by alginate microparticles using a jet-technique. We extruded an agarose aqueous solution containing suspended alginate microparticles into a coflowing stream of liquid paraffin and controlled the diameter of the agarose microparticles by changing the flow rate of the liquid paraffin. Subsequent degradation of the inner alginate microparticles using alginate lyase resulted in the hollow-core structure. We successfully obtained agarose microcapsules with 20-50 microm of agarose gel layer thickness and hollow cores ranging in diameter from ca. 50 to 450 microm. Using alginate microparticles of ca. 150 microm in diameter and enclosing feline kidney cells, we were able to create cell-enclosing agarose microcapsules with a hollow core of ca. 150 microm in diameter. The cells in these microcapsules grew much faster than those in alginate microparticles. In addition, we enclosed mouse embryonic stem cells in agarose microcapsules. The embryonic stem cells began to self-aggregate in the core just after encapsulation, and subsequently grew and formed embryoid body-like spherical tissues in the hollow core of the microcapsules. These results show that our novel microcapsule production technique and the resultant microcapsules have potential for tissue engineering, cell therapy and biopharmaceutical applications.     

渗透压敏感和耐高渗酵母菌在海藻酸钠-壳聚糖-海藻酸钠微囊中的生长和代谢

为了研究微囊微环境中渗透压对微囊内不同渗透压敏感性细胞生长、代谢的影响, 分别以渗透压敏感型酿酒酵母Y02724与耐高渗酵母Hansel为细胞模型, 考察了有氧条件下这两种细胞在海藻酸钠-壳聚糖-海藻酸(Alginate-chitosan-alginate, ACA)微胶囊中的生长、代谢状态。主要检测了细胞比生长速率、最大产物生成量以及代谢物乙醇、甘油分泌量等的变化。实验结果分析表明, 渗透压胁迫可能是导致不同渗透压敏感性细胞在微囊微环境中生长代谢特征变化的因素之一, 即微囊微环境内可能存在渗透压胁迫。     

复乳法制备红细胞代用品

以单甲氧基聚乙二醇聚乳酸共聚物 [methoxypoly(ethyleneglycol) poly DL lactide,PELA]为膜材 ,用W /O/W的复乳 溶剂扩散法制备了包埋牛血红蛋白 (bovinehemoglobin ,BHb)的微胶囊作为人造红细胞。研究发现复乳过程搅拌分散速率、有机溶剂种类和固化方法对BHb活性有明显影响。当搅拌分散速率小于 90 0 0r/min、以乙酸乙酯为有机相时 ,采用复乳 溶剂扩散法包埋BHb的过程对BHb活性无明显影响。当搅拌分散速率高于 12 0 0 0r/min时 ,BHb活性降低。复乳 溶剂扩散法制备微胶囊过程中固化液体积与微胶囊中BHb活性密切相关 ,通过增大固化液和复乳液的体积比可较好地保持BHb的活性。最后制得了粒径 10 μm左右、包埋率 93%、P50 和Hill系数均接近于天然BHb的微胶囊。     

SA/CS-CaCl2/PMCG微胶囊的生物相容性研究

介绍了一种新型多组份生物微胶囊体系－SA／CS－CaCl2/PMCG微胶囊。考察了PMCG和SA／CS－CaCl2/PMCG微胶囊体系对大肠杆菌和酿酒酵母生长的影响,并用SA／CS－CaCl2/PMCG微胶囊进行了固定化培养大肠杆菌和酿酒酵母的研究。结果表明,与其它合成聚阳离子类似,PMCG组分对细胞生长有明显的抑制作用,但是在制胶囊过程中以及在用SA／CS－CaCl2/PMCG微胶囊对大肠杆菌和酿酒酵母培养过程中,都显示了良好的生物相容性,因此作为整个体系来说,该微胶囊可用于微生物细胞的固定化培养。     

Synthetic seeds as an application of mass production of somatic embryos

Production of encapsulatable unit, scale-up of the production system, and encapsulation technologies were developed as an integrated technology for transplant production using somatic embryos of celery and carrot. Encapsulatable units, somatic embryos improved in their quality for encapsulation, showed 80% conversion without encapsulation and any nutrient supply on a nursery medium in a greenhouse. The high conversion ability was also shown by carrot encapsulatable unit produced with a newly developed 8-liter culture jar, where 5.09×10⁵ torpedo stage embryos were produced and 4.10×10⁴ encapsulatable units were obtained from them.A novel self-breaking gel beads and sustained release microcapsule as an artificial endosperm were developed for the encapsulation of carrot embryos. Also a whole production system for synthetic seed was developed with which 80 thousand beads could be produced per day. The encapsulated carrot embryos showed 52% conversion frequency after sowing on a humid soil in greenhouse.Abbreviations BA benzyladenine - GA gibberellic acid - MC microcapsule     

微囊化新生大鼠卵巢组织体外及异体移植

为探讨海藻酸钠-聚左赖氨酸-海藻酸钠(APA)微囊化新生大鼠卵巢组织用于治疗实验性卵巢功能丧失大鼠的可行性,应用高压静电法,用海藻酸钠-聚左赖氨酸-海藻酸钠(APA)生物膜包裹新生大鼠卵巢组织,体外培养微囊,用免疫化学分析法检测雌二醇(E2)、孕酮(P)分泌情况,透射电镜观察卵巢组织形态,并将微囊移植到去势大鼠(切除双侧卵巢的雌性大鼠)腹腔中,检测大鼠血清中雌、孕激素变化情况,同时用阴道涂片观察大鼠动情周期恢复情况,并在不同时间回收观察微囊。结果显示在相同条件下制得的微囊粒径均匀、表面光滑;体外培养条件下持续分泌E2、P;卵巢组织中颗粒细胞发育成为粒性黄体细胞;大鼠腹腔移植微囊后无异常,E2、P水平上升,动情周期未恢复;回收的微囊大部分形态完整。提示用高压静电法制备的APA微囊化新生大鼠卵巢组织能持续稳定释放E2、P,明显改善大鼠卵巢功能,在大鼠体内有良好的生物相容性。     

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche^1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment⁴ such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures². These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded⁵. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions² to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes¹. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, >100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.     

昆虫性信息素微胶囊的研究进展

微胶囊是应用昆虫性信息素进行害虫治理的主要剂型之一。文章对昆虫性信息素微胶囊常用的制备方法进行总结,并介绍其在害虫控制方面的应用进展,探讨昆虫性信息素微胶囊制备及其田间应用效果的影响因子,并对今后昆虫性信息素微胶囊的发展作了展望。