人工膜与胰岛移植

人工膜是目前应用于组织移植和细胞移植领域中一种免疫隔离物质.包括渗透性小腔、中空纤维管、海藻酸钠—多聚赖氨酸—海藻酸钠微囊三种.它们均在一定程度上对免疫细胞、淋巴因子及自身抗体起到免疫隔离作用,其中以海藻酸钠微囊应用比较广泛.微囊化胰岛在同种及异种胰岛移植中,可有效地控制糖尿病模型的血糖浓度,延长移植物存活时间.     

载β-TC3细胞新型葡萄糖敏感微囊的生物学性能CSCD

背景:构建一种能够感应外界葡萄糖浓度变化而控制胰岛素释放的系统对有效地控制糖尿病的发生与发展具有重要意义。目的:研究负载β-TC3细胞的葡萄糖敏感海藻酸钠/改性壳聚糖/海藻酸钠微囊的性能。方法:通过层层自组装方法制备葡萄糖敏感海藻酸钠/改性壳聚糖/海藻酸钠微囊,观察并评价其性能;进一步应用制备的葡萄糖敏感微囊包裹β-TC3细胞,观察微囊内细胞的增殖情况。结果与结论:实验制备的葡萄糖敏感海藻酸钠/改性壳聚糖/海藻酸钠微囊温水浴振荡48h后完整微囊仍达到95%,且微囊硬度变小、弹性增大;通透性测试结果显示实验制备的微囊可将大分子物质牛血清白蛋白、免疫球蛋白G截留在微囊外;体外释放实验显示,随着周围环境葡萄糖缓冲液浓度的增加,微囊内胰岛素释药量增大。说明实验制备的葡萄糖敏感海藻酸钠/改性壳聚糖/海藻酸钠微囊具有良好的机械强度、葡萄糖敏感特性与免疫隔离功能。进一步将β-TC3细胞微囊化,发现β-TC3细胞在微囊中生长良好,增殖高峰滞后于未微囊化的细胞。可见该生物微囊具有良好的细胞相容性。     

微囊化细胞移植的研究进展

微囊技术为组织 /细胞移植开辟了新途径 ,它有效地避免了移植后的免疫排斥反应 ,并解决了移植物来源稀少的问题。微囊的包裹材料有多种 ,以海藻酸钠—多聚赖氨酸—海藻酸钠 (APA)应用最为广泛 ,可通过提高其生物相容性 ,从而减弱免疫排斥反应。微囊具有良好的免疫隔离作用 ,体现在对免疫活性细胞及部分细胞因子的阻挡作用 ,使移植物能存活下来并能发挥其功能。目前对微囊化人工细胞的研究取得了很大的进展 ,特别是基因工程细胞日益成为研究的焦点。微囊技术是一新兴的、尚需进一步改进的技术 ,它在异体组织 /细胞移植等方面必将有广阔的应用前景     

微囊化细胞移植的研究进展

微囊技术为组织／细胞移植开辟了新途径,它有产地避免了移植后的免疫排斥反应,并解决了移植物来源稀少的问题。微囊的包裹材料有多种,以海藻酸钠－多聚赖氨酸－海藻酸钠（APA）应用最为广泛,可通过提高其生物相容性,从而减弱免疫排斥反应。微囊具有良好的免疫隔离作用。体现在对免疫活性细胞及部分细胞因子的阻挡作用,使移植物能存活下来并能发挥其功能,目前对微囊化人工细胞的研究取得了很大的进展,特别是基因工程细胞日益成为研究的焦点。微囊技术是一新兴的,尚需进一步改进的技术,它在异体组织／细胞移植等方面必将有广阔的应用前景。     

微囊化技术在医学领域中的应用与进展

背景:微囊的免疫隔离保护和可控缓释的特性得到了医学领域的重视,并已广泛应用于糖尿病、帕金森症、肝衰竭、镇痛、肿瘤等疾病治疗过程中。目的:综述微囊化技术的特性以及在医学研究领域中的应用。方法:由第一作者检索1964/2009PubMed数据库(网址http://www.ncbi.nlm.nih.gov/PubMed),2000/2009万方数据库(网址http://www.wanfangdata.com.cn)有关微囊化移植,微囊化可控缓释,微囊化技术在医学应用方面的应用方面的文献,英文关键词为"microencapsulated,transplantation,controlled release"。中文关键词为"微囊化"。结果与结论:微囊作为一种载体,为细胞提供了一个三维培养基质,改善细胞间的作用,提高细胞的功能;为异体细胞提供了免疫屏障,阻挡免疫细胞的识别,但又不妨碍营养物质、氧气及代谢产物运输;为机体提供可控的长期稳定缓释有效成分治疗疾病。微囊化技术在糖尿病方面研究比较多,在帕金森症、肝衰竭、脊髓损伤、甲状旁腺功能低下、骨科领域、肿瘤癌症等方面也有一定的探索,但微囊化技术从个别动物试验成功到真正临床广泛应用还有很漫长的历程,在拓宽微囊化技术在医学领域运用的同时,需进一步加强理想刚性结构、良好生物相容性和可控半透膜性的探索。     

微囊化细胞的低温保存

背景：微胶囊是一种有效的免疫隔离工具,低温冷冻方便了微囊的保存与运输,有利于微囊化技术在临床的推广应用。 目的：综述微囊低温保存技术中微囊低温保存特点、不同保存方法优缺点、研究方法及微囊低温保存实验现状。 方法：检索1980/2010 PubMed数据库,1991/2010万方数据库、维普数据库有关微囊低温保存研究,低温保存工艺理论及专用仪器,微囊低温保存技术医学应用的文献。 结果与结论：微囊化细胞在低温保存过程中的损伤特点与细胞、组织有很大不同。微囊相对较大的尺寸与复杂的囊壁半透膜结构,使得微囊结构与囊内细胞更容易受到溶质损伤与冰晶损伤,因此不能简单套用单细胞悬液的低温保存方案。慢速冷却法与玻璃化法是两种常见的微囊化细胞低温保存方法,各有利弊。微囊化细胞低温保存技术尚未成熟,在开发新型微囊低温保存设备,优化设计微囊低温保存方案的同时,需进一步加强对微囊冻存机制更深层次的探索。     

细胞微囊技术及其在基因治疗领域的研究进展

利用重组工程细胞进行基因治疗无疑为多种难治性疾病的有效治疗提供了新的思路。但宿主对异源细胞产生的移植排斥反应却是其在临床上应用的主要障碍。现代细胞微囊技术因其具有良好的免疫隔离屏障作用，使克服这一障碍成为可能。近年来，细胞微囊技术在基因治疗方面的发展迅速，已在血友病，帕金森病、肿瘤等多种难治性疾病的治疗应用中显示出广阔的应用前景。本文就细胞微囊技术的微囊膜材料、制作工艺、微囊内重组细胞的构建以及其在基因治疗方面的研究作一综述。     

微囊化细胞冻存的研究进展

背景:细胞微囊化为细胞大规模、高活性体外培养及长期存储提供了新的途径,低温保存是目前保存细胞的重要方法,技术日新月异,复苏细胞在临床和基础研究中的应用越来越多。目的:分析近年来微囊化细胞冻存的相关研究,对微囊化细胞冻存技术的发展作一总结。方法:以"微囊,冻存"为检索词,检索中国期刊网(1979/2010)及维普数据库(1989/2010),限定文章语言种类为中文;以"Cryopreservation,Microencapsule"为检索词,检索PubMed数据库(1979/2010),限定文章语言种类为English。纳入含有微囊化细胞冻存的研究,排除其他形式细胞冻存的研究,结果以各种细胞微囊化后进行冻存处理后产生作用为指标,共检索到43篇文献。结果与结论:随着生物人工肝与以及其他细胞移植研究的深入,对微囊化细胞的需要量将显著增加,微囊化细胞的低温保存技术已成为保正其顺利应用的关键技术之一。目前,微囊化细胞的低温保存技术已经开展了不少研究,有多项研究结果表明复苏后微囊化细胞的存活率和生物学功能都能保持较好的水平,但相关机制仍未完全阐清,各种冻存方法还需要优化。     

微囊化人肾上腺嗜铬细胞瘤细胞在人工脑脊液中的生长和分泌特征

背景：微囊化细胞是目前常用的免疫隔离工具,可以解决细胞蛛网膜下腔移植存在的免疫排斥问题,然而微囊化对人嗜铬细胞瘤细胞分泌功能的影响尚不明确。 目的：观察微囊化的人嗜铬细胞瘤细胞在人工脑脊液中的生长及分泌功能。 方法：取手术切除的人嗜铬细胞瘤组织,采用连续分次胶原酶消化法分离人嗜铬细胞瘤细胞,并用人工脑脊液进行原代细胞培养。用海藻酸盐-聚赖氨酸-海藻酸盐微囊包裹原代培养的人嗜铬细胞瘤细胞,用倒置相差显微镜观察人工脑脊液中的微囊化和非微囊化的人嗜铬细胞瘤细胞的形态;用CCK-8试剂盒测定人嗜铬细胞瘤细胞的增殖情况;用Elisa试剂盒检测微囊化和非微囊化的人嗜铬细胞瘤细胞分泌的去甲肾上腺素和甲硫氨酸脑啡肽浓度。 结果与结论：微囊化的人嗜铬细胞瘤细胞镜下呈悬浮、聚集生长状态,细胞突起观察不明显。与非微囊化组相比,微囊化人嗜铬细胞瘤细胞增殖较快,甲硫氨酸脑啡肽和去甲肾上腺素的分泌量较多。但不同病例来源的细胞,甲硫氨酸脑啡肽和去甲肾上腺素的分泌量差异较大。提示微囊化的人嗜铬细胞瘤细胞在人工脑脊液中具有良好的生长和分泌功能,并且不同病例来源的人嗜铬细胞瘤细胞可以相对稳定地分泌甲硫氨酸脑啡肽和去甲肾上腺素。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

APA微囊细胞移植的免疫隔离作用

随着器官移植研究的深入,诸如移植物排斥反应、供体缺乏等一系列问题出现在人们面前。1980年,Lim等[1]首次采用微囊免疫隔离技术进行胰岛微囊化移植研究,为人类解决这些问题提供了新思路。微囊化免疫隔离技术近些年取得了众多成就,目前研究表明由海藻酸钠—多聚赖氨酸—海藻酸钠(APA)构成的微胶囊具有制作简便、体积小、生物相容性好等优点,是应用前景较好的免疫隔离膜[2]。本文将就其免疫隔离效果的研究进展作如下综述。1发展史60年代Chang[3]首次提出人工细胞的概念,即用具有生物相容性的半透膜包裹组织细胞,该膜允许小分子营养物、代谢…     

The effect of covalent cross-links between the membrane components of microcapsules on the dissemination of encapsulated malignant cells

Stem cells and immortalized cells have considerable therapeutic potential but present risks of malignant transformation. Cell microencapsulation allows transplantation without immunosuppression. We have developed a method for microencapsulating living cells within covalently cross-linked membranes that are chemically and mechanically extremely resistant. We provide herein direct evidence that these microcapsules can prevent malignant cell dissemination. When 20,000 or more nonencapsulated EL-4 thymoma cells were implanted intraperitoneally in mice, all recipients died with widespread metastasis within 26.3+/-1.0 days. All recipients of 250,000 EL-4 cells microencapsulated in covalently cross-linked membranes were living and disease-free, 150 days post-implantation. Encapsulation in standard microcapsules only slightly delayed the recipient death. Pancreatic islets transplanted using either type of microcapsule presented similar survival. We conclude that microencapsulation in covalently cross-linked membranes prevents malignant cell dissemination.     

A quantitative luminescence assay for measuring cell uptake of aqueous-based microcapsules in vitro

We recently developed a system of microencapsulation consisting of aqueous-based polymers (e.g. alginate) and aqueous amines (e.g. spermine). We found that microencapsulation enhanced virus-specific protective immune responses. In addition, we found that microencapsulation may enhance virus-specific immune responses by selecting for antigen-presenting cells (APC) that are more efficient at processing and presenting viral antigens than those involved after natural infection. To determine the intracellular trafficking patterns and fate of microcapsules within APC, we developed a luminescence assay that permits the determination of specific quantities of proteins introduced into cells by microcapsules. We found that the time-dependent uptake of horseradish peroxidase (HRP)-labeled microcapsules was accurately detected in lysates of peritoneal exudate cells using luminol. The amplitude of HRP-catalyzed chemiluminescence in cell lysates correlated with the capture efficiency and retention kinetics of HRP in three different microcapsule preparations. HRP was most efficiently captured and retained by linking biotinylated HRP to microcapsulses chemically modified at the amine moiety with egg avidin. This preparation yielded more accurate and sensitive quantitation of HRP contained within cells than preparations capturing HRP or HRP-conjugated goat antibody into the microcapsular matrix by ionic interactions.     

Photo-crosslinkable microcapsules formed by polyelectrolyte copolymer and modified collagen for rat hepatocyte encapsulation

New anionic polyelectrolyte tetra-copolymers with photo-crosslinkable 4-(4-methoxycinnamoyl)phenyl methacrylate monomer in addition to a HEMA-MMA-MAA ter-copolymer system were synthesized. The tetra-copolymers were used to form photo-crosslinkable microcapsules with modified collagen by complex coacervation for rat hepatocytes encapsulation. The hepatocytes were encapsulated within a two-layered membrane comprising of modified collagen as the inner core and an outer photo-crosslinkable copolymer shell. Upon photo-crosslinking of the microcapsules with UV-Vis light irradiation, the mechanical strength and chemical stability of the microcapsules, and the cellular functions of the encapsulated hepatocytes were enhanced. Particularly, the mechanical stability of the microcapsules was dramatically strengthened. The new photo-crosslinkable tetra-copolymer formulation described in this article has opened a way to the development of hepatocyte microencapsulation technology for bioartifical liver assist device.     

Induction dopamine releasing cells from mouse embryonic stem cells and their long-term culture

Cell transplantation therapy using dopaminergic neurons derived from embryonic stem (ES) cells for the treatment of Parkinson's disease has been proposed as one of the major applications for stem cell-based therapy. However, the low collection efficiency of neurons from a culture dish and the rejection of cells after transplantation are expected to limit their future clinical applications. To overcome these problems, we examined the induction of neurogenesis of ES cells under free-floating conditions and microencapsulation of the obtained cell aggregates into an agarose hydrogel. Cell aggregates from ES cells were cultured in various media under the free-floating condition. Immunohistochemical staining for tyrosine hydroxylase (TH) and RT-PCR analyses for TH and Nurr1 showed that dopaminergic neurons were induced in ES cell aggregates cultured in a 1:2 mixture of conditioned medium of PA6 stromal cells and Glasgow minimum essential medium (GMEM) after 16 days in culture. The cell aggregates could be collected and were encased within agarose microcapsules without loss of dopaminergic neurons. The cell aggregates with/without microencapsulation were maintained in CM/GMEM for an additional period. KCl stimulation assays were done at day 23, 30, 37, 44, 51, and 58 to examine dopamine release. Dopamine release abilities were well maintained during 58 days of observation. Amounts of dopamine release from encapsulated cell aggregates were slightly higher than those of unencapsulated cell aggregates from day 16 to 58. Although efficacy for immunoisolation of the agarose microcapsules still remains for future in vivo studies, microencapsulation did not adversely affect viability and functions of the dopamine releasing ES cell progeny.     

Microfabrication of homogenous, asymmetric cell-laden hydrogel capsules

Cell encapsulation has been broadly investigated as a technology to provide immunoprotection for transplanted endocrine cells. Here we develop a new fabrication method that allows for rapid, homogenous microencapsulation of insulin-secreting cells with varying microscale geometries and asymmetrically modified surfaces. Micromolding systems were developed using polypropylene mesh, and the material/surface properties associated with efficient encapsulation were identified. Cells encapsulated using these methods maintain desirable viability and preserve their ability to proliferate and secrete insulin in a glucose-responsive manner. This new cell encapsulation approach enables a practical route to an inexpensive and convenient process for the generation of cell-laden microcapsules without requiring any specialized equipment or microfabrication process.     

Microencapsulation of living cells in semi-permeable membranes with covalently cross-linked layers

Microencapsulation in semi-permeable membranes protects transplanted cells against immune destruction. Microcapsule strength is critical. We describe a method to microencapsulate living cells in alginate-poly-L-lysine (PLL)-alginate membranes with covalent links between adjacent layers of microcapsule membranes, while preserving the desired membrane molecular weight cut-off (MWCO) and microencapsulated cell viability. A heterobifunctional photoactivatable cross-linker, N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) was used. The N-hydroxysuccinimide ester group of ANB-NOS was covalently linked to PLL. Islets of Langerhans were immobilized in alginate beads, incubated in PLL-ANB-NOS and again in alginate. Upon illumination with UVA, covalent links were created between the phenyl azide residue of ANB-NOS and alginate from both the core bead and the outer coating. Covalently linked microcapsules remained intact after 3 years in a strong alkaline buffer (pH 12), whereas standard microcapsules disappeared within 45 s in the same solution. A standardized mechanical stress broke 22-fold more standard than covalently linked microcapsules. The MWCO and microencapsulated cell viability were similar with standard and covalently linked microcapsules. These microcapsules, extremely resistant to chemical and mechanical stresses, will be useful in numerous applications.     

Alginate-based microcapsules generated with the coaxial electrospray method for clinical application

Alginate-based microencapsulation of cells has made a significant impact on the fields of regenerative medicine and tissue engineering mainly because of its ability to provide immunoisolation for the encapsulated material. This characteristic has allowed for the successful transplantation of non-autologous cells in several clinical trials for life threatening conditions, such as diabetes, myocardial infarction, and neurodegenerative disorders. Methods for alginate hydrogel microencapsulation have been well developed for various types of cells and can generate microcapsules of different diameters, degradation time, and composition. It appears the most prominent and successful method in clinical applications is the coaxial electrospray method, which can be used to generate both homogenous and non-homogeneous microcapsules with uniform size on the order of 100 μm. The present review aims to discuss why alginate hydrogel is an ideal biomaterial for the encapsulation of cells, how alginate-based microcapsules are generated, and methods of modifying the microcapsules for specific clinical treatments. This review will also discuss clinical applications that have utilized alginate-based microencapsulation in the treatment of diabetes, ischemic heart disease, and neurodegenerative diseases.     

Biocompatibility of alginate-poly-L-lysine microcapsules for cell therapy

Cell microencapsulation holds promise for the treatment of many diseases by the continuous delivery of therapeutic products. The biocompatibility of the microcapsules and their biomaterials components is a critical issue for the long-term efficacy of this technology. The objective of this paper is to provide detailed information about the principal factors affecting the biocompatibility of alginates and alginate-poly-l-lysine microcapsules, which are the most frequently employed biomaterials and encapsulation devices for cell immobilization, respectively. Some of these factors include the alginate composition and purification, the selection of the polycation, the interactions between the alginates and the polycation, the microcapsule fabrication process, the uniformity of the devices and the implantation procedure. Improved knowledge will lead to the production of standardized transplantation-grade biomaterials and biocompatible microcapsules.     

Immune plasma-dependent cytotoxicity of immune and non-immune peripheral lymphoid cells for target cells coated with bacterial outer unit membrane.

The development of a model system for use in the study of lymphoid cell cytotoxicity to bacterial membrane antigens was attempted. In this system 51 Cr-labelled chicken red blood target cells were coated with pieces of the outer unit membrane of leptospirae rather than with soluble antigens. Using the model system to study dog peripheral immune lymphoid cell cytotoxicity to coated target cells we found that both immune and non-immune lymphocytes are antibody dependent for the expression of their cytotoxicity. It was also found that the unit membrane preparation from leptospirae can serve as a good antigenic stimulant to immune dog lymphoid cells as measured by increased [3H]thymidine uptake.