组织器官三维构建及原位组织工程概念——组织工程连载之四

组织器官三维构建就是把种子细胞和支架材料结合而获得设计的组织或器官,属于组织工程的核心内容,也最能体现组织工程的技术水平,如血管、气管的构建。由于传统组织工程存在缺陷,Shimizu于1998年首先提出了原位组织工程的概念,它是运用组织工程学基本原理,通过各种方法诱导移植的外源性的种子细胞或内源性的缺损组织局部细胞发生迁移、增殖、分化形成新生组织修复缺损。原位组织工程最大的特点是不依赖体外的细胞培养装置--生物反应器。原位组织工程是传统离体组织工程的有益补充。离体组织工程仍具有广阔的发展前景。     

骨组织工程血管化的研究进展

血管化是组织工程骨成活的关键,组织工程骨的血管化过程与生理情况下的血管发生相似,但又有其独特性,并受多种因素影响。基于对组织工程骨血管化过程的认识,研究者通过联合细胞培养、促血管化生长因子、显微外科等方法重建血运。本文综述了当前骨组织工程的血管化研究进展。     

VEGF-VEGFR2 信号轴对糖尿病血管生成反应的调控研究进展

血管内皮生长因子(Vacularendothelial growth factor,VEGF)-血管内皮生长因子受体-2(VEGF receptor2,VEGFR-2)信号轴调控血管生成反应。糖尿病病理状态下,氧化应激异常激活、NO等血管活性物质功能受损、以及晚期糖基化终末产物增加,该信号轴功能失调,使得血管生成反应在一些器官组织中呈增强状态,如视网膜和肾;然而在另一些组织中却是受到抑制,如外周血管等。不正常的血管生成反应最终导致糖尿病性心血管并发症的发生。因此,阐明血管生产反应功能障碍,将为糖尿病心血管并发症靶向治疗提供依据。     

细胞三维培养：组织工程的关键技术突破口

组织工程是有望从根本上解决组织,器官缺损或失能的医学难题的一门新兴边缘学科,组织,器官发育的细胞和分子机制的进一步揭示和体外构建工程组织,器官的细胞培养技术的进步将使组织工程在新的千年成为广泛应用的新的治疗模式。细胞三维培养要成为体外构建工程组织,器官的成熟技术体系需先解决以下问题;（1）细胞;（2）生物材料;（3）培养基;（4）培养装置;（5）异型细胞的共培养;（6）细胞三维培养物血管化。     

骨类器官的构建及应用进展

骨骼疾病如骨质疏松、骨关节炎等已成为重要的人类健康问题,需要更深入地了解相关疾病的发病机制并开发更有效的治疗方法。由于2D细胞培养和动物实验等常规研究方法的局限性,近年来发展的类器官技术受到了极大关注。类器官作为干细胞衍生的自组织3D细胞簇,可以在体外更真实地模拟组织器官的复杂结构和生物功能。目前间充质干细胞、多能干细胞等衍生的骨类器官已逐步建立,不仅为疾病建模、药物筛选和生理病理基础研究提供了良好平台,还有望为骨缺损修复带来新希望。现对不同骨类器官模型的构建及主要应用进行概述,同时讨论了骨类器官培养面临的挑战,并对其未来发展进行展望,为构建结构功能更完善的骨类器官并将其应用于生物医学研究提供参考。     

血管化类器官的构建和研究进展

类器官在结构和功能上更接近真实器官,已经成为一种应用较为广泛的补充模型。但由于缺乏血液灌注,现有的类器官模型依然存在生长发育受限、内部坏死严重、结构功能不完善等问题。作为不少器官的重要组成成分,血管不仅能解决类器官的物质运输问题,还能提供血流灌注的机械力影响。此外,血管内皮能分泌多种因子,调节类器官的发育成熟。因此,血管化类器官具有更重要的应用意义。本文综述了构建血管化类器官的细胞来源,介绍了如何通过干细胞诱导共分化、多种细胞共培养自组装、以及直接使用微血管片段、利用移植后体内再血管化、运用组织工程手段等方法获得血管化类器官。     

类器官的应用研究进展

类器官是利用干细胞的自我更新和分化能力,在体外培养形成的一种微小组织器官类似物,在很大程度上具有体内相应器官的功能。迄今为止,在3D培养条件下,已经成功培养出多种类器官如肺、胃、肠、肝和肾等类器官。它们不仅可作为组织器官的替代品用于药物和临床研究,还可用于体内器官移植。本文综述了类器官在药物毒性检测、药效评价和新药筛选中的作用以及利用类器官建立疾病模型、研究组织器官发育和类器官在精准医疗、再生医学中的价值。     

红豆杉器官,组织,细胞培养和基因工程的研究进展

本文综述了红豆杉器官、组织、细胞培养和基因工程的研究文献,并对这些领域存在的问题及其发展前景进行了讨论。     

血管内皮细胞间紧密连接的研究进展

紧密连接是物质经旁细胞途径转运的结构和功能基础。近年来研究发现,在脑、视网膜、肺及肾等多个器官的微血管内皮细胞以及主动脉等大血管内皮细胞表达的紧密连接对维持血管稳态发挥了重要作用,一些细胞因子或环境刺激可通过调节紧密连接的表达、分布、结构和功能进而改变内皮通透性,从而影响血管及相应器官和组织的功能。本文重点综述了内皮细胞间紧密连接的研究进展,为防治与紧密连接相关的血管疾病提供新的思路。     

细胞联合培养在血管构建中的作用

组织工程三大要素为种子细胞、支架材料和信号分子,干细胞因其多分化潜能成为热门的种子细胞。血管化问题是制约工程化组织应用于临床的问题之一。利用干细胞构建组织工程血管的手段之一是在分离培养得到足够的种子细胞后,通过生长因子、细胞外基质、外力作用、其他细胞等的调控实现内皮向分化。只有实现了成功的血管构建,工程化组织才能正常的发挥作用。近年来不少国内外专家学者通过细胞联合培养的方法,观察细胞间的相互作用对血管构建的影响,结果表明,细胞联合培养在血管的形成、存活、稳定方面起到了重要的作用,为组织工程血管化提供了有效的途径,本文就部分细胞联合培养在血管构建中的作用作一综述。     

Human primary co-culture angiogenesis assay reveals additive stimulation and different angiogenic properties of VEGF and HGF

Therapeutic angiogenesis aims to induce blood vessel growth in acute or chronic ischemic tissues and has gained tremendous interest over the last years. To study factors and combinations thereof that potentially induce or modify angiogenesis and to evaluate their therapeutic potential, various in vitro assays have been developed. Although endothelial cells have attracted most attention in these assays, they alone cannot complete vessel maturation since extracellular matrix (ECM) components and mesenchymal cells also play an important role in vascular development. To address this complexity we focussed on a human co-culture angiogenesis assay comprising primary endothelial cells as well as primary ECM-producing fibroblasts. In this assay HGF and VEGF as single factors and combined were tested for the potential to induce an angiogenic response, which was detected by image analysis assessing the area, length and branches of the formed vascular structures. The results show that the cytokines HGF and VEGF both promote angiogenesis in this co-culture assay by inducing distinguishable patterns of vascular structures. VEGF increases the length, area and branch point number of induced vessels whereas HGF mediates exclusively vascular area growth resulting in vascular structures of enlarged diameter. Moreover, the combination of both cytokines results in an additive increase of vascular diameter.     

Three-dimensional co-culture model employing silica nonwoven fabrics to enhance cell-to-cell communication of paracrine signaling between hepatocytes and fibroblasts

The realization that soluble factors secreted by heterotypic cells play an importanta role in paracrine signaling, which facilitates intercellular communication, enabled the development of physiologically relevant co-culture models for drug screening and the engineering of tissues, such as hepatic tissues. The most crucial issues confronting the use of conventional membrane inserts in segregated co-culture models that are used to study paracrine signaling between heterotypic cells have been identified as long-term viability and retention of cell-specific functions, especially when isolated primary cells are used. Herein, we present an in vitro segregated co-culture model consisting of a well plate incubated with rat primary hepatocytes and normal human dermal fibroblasts which were segregated using a membrane insert with silica nonwoven fabric (SNF) on it. SNF, which mimics a physiological environment much more effectively than a two-dimensional (2D) one, promotes cell differentiation and resultant paracrine signaling in a manner that is not possible in a conventional 2D culture, owing to high mechanical strength generated by its inorganic materials and interconnected network structure. In segregated co-cultures, SNF clearly enhanced the functions of hepatocytes and fibroblasts, thereby showing its potential as a measure of paracrine signaling. These results may advance the understanding of the role played by paracrine signaling in cell-to-cell communication and provide novel insights into the applications of drug metabolism, tissue repair, and regeneration.     

Activation of CD4+ lymphocytes by syngeneic brain microvascular smooth muscle cells

Splenocyte proliferation as measured by [3H]thymidine incorporation was detected when brain microvessel smooth muscle cells (SM) were cocultured with syngeneic spleen cells. This report focuses on the role of different lymphocyte populations in this activation. The central role of CD4+ T cells in the proliferation response has been established by different sets of experiments. The phenotypic characterization of splenic lymphocytes before and after the co-culture showed that the only cell type present in higher number after the co-culture than before is the CD4+ T cell. When CD4+ cells were purified by flow microfluorimetry and co-cultured with SM a strong proliferative response was detected. In contrast, purified CD8+ cells in co-culture with SM cells did not proliferate. The activation of CD4+ cells by SM required direct cell-to-cell contact and could be detected on the fourth day, reaching maximal levels at the 6th and 7th days of the co-culture. The activation is more pronounced in the syngeneic system than under allogeneic conditions and is inhibited by anti-MHC II mAb, but not by anti-MHC II mAb. The finding that vascular smooth muscle cells can activate syngeneic T cells may have important implications concerning the mechanism of induction of vasculitis.     

Fibroblast growth factor-2 facilitates rapid anastomosis formation between bioengineered human vascular networks and living vasculature

Many common diseases involve the injury, loss, or death of organ tissues. For these patients, organ transplantation is often the only viable solution. Nonetheless, organ transplantation is seriously limited by the relative scarcity of living and non-living donors, a situation that is worsening with aging of the world population. Tissue Engineering (TE) is a research discipline in regenerative medicine that aims to generate tissues in the laboratory that can replace diseased and damaged tissues in patients. Crucially, engineered tissues must have a vascular network that guarantees adequate nutrient supply, gas exchange, and elimination of waste products. Therefore, the search for clinically relevant sources of vasculogenic cells and the subsequent development of methods to achieve rapid vascularization is of utmost importance. We and others have previously shown that human blood-derived endothelial colony-forming cells (ECFCs) have the required vasculogenic capacity to form functional vascular networks in vivo. These studies demonstrated that, in the presence of an appropriate source of perivascular cells, ECFCs can self-assemble into microvascular networks and connect to the host vasculature, a process that takes approximately 7days in vivo. The prospect is to incorporate these vascular networks into future engineered tissues. However, engineered tissues must have a functional vasculature immediately after implantation in order to preserve viability and function. Thus, it is critical to further develop strategies for rapid formation of perfused vascular network in vivo. Here, we describe a methodology to deliver ECFCs and bone marrow-derived mesenchymal stem cells (MSCs) subcutaneously into immunodeficient mice in the presence of fibroblast growth factor-2 (FGF-2). This approach significantly reduces the time needed to achieve functional anastomoses between bioengineered human blood vessels and the host vasculature. This methodology includes (1) isolation, characterization and culture of ECFCs, (2) isolation, characterization and culture of MSCs, and (3) implantation of ECFCs and MSCs, in the presence of FGF-2, into immunodeficient mice to generate perfused vascular networks.     

Bioengineering human microvascular networks in immunodeficient mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs); however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs (5-7). We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.     

Prospects for the use of nuclear transfer in human transplantation

The successful application of nuclear transfer techniques to a range of mammalian species has brought the possibility of human therapeutic cloning significantly closer. The objective of therapeutic cloning is to produce pluripotent stem cells that carry the nuclear genome of the patient and then induce them to differentiate into replacement cells, such as cardiomyocytes to replace damaged heart tissue or insulin-producing beta cells for patients with diabetes. Although cloning would eliminate the critical problem of immune incompatibility, there is also the task of reconstituting the cells into more complex tissues and organs in vitro. In the review, we discuss recent progress that has been made in this field as well as the inherent dangers and scientific challenges that remain before these techniques can be used to harness genetically matched cells and tissues for human transplantation.     

Practical choice for robust and efficient differentiation of human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have the distinct advantage of being able to differentiate into cells of all three germ layers. Target cells or tissues derived from hPSCs have many uses such as drug screening, disease modeling, and transplantation therapy. There are currently a wide variety of differentiation methods available. However, most of the existing differentiation methods are unreliable, with uneven differentiation efficiency and poor reproducibility. At the same time, it is difficult to choose the optimal method when faced with so many differentiation schemes, and it is time-consuming and costly to explore a new differentiation approach. Thus, it is critical to design a robust and efficient method of differentiation. In this review article, we summarize a comprehensive approach in which hPSCs are differentiated into target cells or organoids including brain, liver, blood, melanocytes, and mesenchymal cells. This was accomplished by employing an embryoid body-based three-dimensional (3D) suspension culture system with multiple cells co-cultured. The method has high stable differentiation efficiency compared to the conventional 2D culture and can meet the requirements of clinical application. Additionally, ex vivo co-culture models might be able to constitute organoids that are highly similar or mimic human organs for potential organ transplantation in the future.     

Co-culture of rat luteal cells with islet cells enhances islet viability and revascularization

Islet cell transplantation is a major treatment strategy for type I diabetes, and has proven to be effective for maintaining glucose homeostasis. However, this treatment requires an extended period of immunosuppression to prevent rejection and recurrent transplantation to maintain function. Thus, to enhance the properties of transplanted islet cells, we examined the effect of the co-culture of luteal cells, which secrete progesterone, on islet cell viability, functionality, and revascularization. It was found that islet viability and functionality were higher in the co-cultured group than in single cultures of islets at 48 and 96 h, in parallel with increased progesterone and vascular endothelial growth factor (VEGF) secretion from luteal cells. In the co-culture groups, VEGF levels at 48 and 96 h and CD31 levels at 48 h were significantly higher than those in the islet groups (p?<?0.001 and p?<?0.05, respectively), and basic fibroblast growth factor (bFGF) levels were increased at 96 h (p?<?0.001). Thus, co-culture with luteal cells may increase islet vascularity by enhancing VEGF and bFGF levels for up to 96 h, which could help to markedly increase the pre-transplantation time to allow for effective immunosuppression therapy. This method may also promote islet cell viability and functionality. Progesterone and angiogenic factors secreted from luteal cells may be responsible for these positive effects.     

Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes

To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.     

Localization of GFP in frozen sections from unfixed mouse tissues: immobilization of a highly soluble marker protein by formaldehyde vapor.

Green fluorescent protein (GFP) and its variants, such as enhanced GFP (EGFP), have been introduced into mammalian cells by transgenes, e.g., to distinguish donor from host cells after transplantation. Free GFP is extremely soluble and leaks out from liquid-covered cryostat sections so that fixation of whole organs before sectioning has been mandatory. This precludes the analysis of serial sections with respect to fixation-sensitive enzyme activities and antigens. We describe here a vapor fixation for sections from unfixed cryostat blocks of tissue that allows unrestricted enzyme and immunohistochemistry on adjacent sections, as demonstrated for cross-striated muscle and other tissues from EGFP transgenic "green mice" and for a transplantation experiment.