人胆囊类器官培养体系的建立与鉴定

目的 构建人胆囊上皮细胞类器官的培养体系,实现人胆囊类器官体外稳定快速扩增,并鉴定其具有的特性。 方法 分离人来源胆囊组织上皮细胞,运用三维培养体系,将细胞嵌入基质胶中进行3D培养。观察胆囊类器官生长过程中球囊样结构的面积、周长与形态。利用免疫细胞荧光染色技术检测胆囊类器官的干性指标和胆管上皮细胞标志物表达情况。使用BrdU摄入实验,检测小分子CHIR-99021和Blebbistatin对类器官的增殖特性的影响。 结果 胆囊类器官在体外培养过程中,类器官形成的球囊面积逐渐增大,第1天为（1.15±0.12）×10^4μm2,第7天为（18.97±1.64）×10^4μm2,（P<0.01）,第7天时其形状因子增高至（0.83±0.003）,表明类器官在体外稳定生长（P<0.01）,且形状逐渐趋于一个完美的圆。免疫荧光染色可见类器官表达胆管上皮细胞标志物。在培养基中加入小分子CHIR-99021和Blebbistatin后,BrdU检测其增殖特性明显增高（P<0.01）。 结论 在体外能够成功培养获得了具有增殖能力的人胆囊类器官,其具有胆管上皮细胞的特性,可用于胆管疾病的研究与建模。     

Development of patient derived organoids for cancer drug screening applications

Cancer is a disease characterised by abnormal cell growth that can invade or spread to other regions of the body. Organoids are three-dimensional ex vivo tissue cultures made from embryonic stem cells, induced pluripotent stem cells, progenitor cells or tissue that serve as a physiological model for cancer research. These are designed to recapitulate the in vivo properties of tumours. Importantly, effective recapitulation of the structure of tissues and function is believed to predict patient response, allowing for the creation of personalised therapy in a timely manner that may be used in the clinic. This Review discusses the pre-clinical model and different types of human organoids as models for the development of high throughput drug screening and also aims to highlight how organoids are shaping the future of cancer research.     

微流控肝、肾芯片在中药毒理研究中的应用

微流控肝、肾芯片是近年来进行新药研发、药效毒理研究和机制探索、疾病模型构建的优选模型载体。在美国食品药品监督管理局允许当动物疾病模型难以构建时,用体外模型数据代替动物模型数据进行新药申报的大背景下,微流控芯片因为具有高通量、能高度仿生生命体特征、可方便进行重复给药的正常或病理状态下的药物毒性评价且允许对培养物培养过程进行实时诱导、监测过程数据实时采集分析等优点而得到广泛的关注。在毒理学研究中,肝、肾芯片可以通过结合不同物种来源的2D单培养和共培养、3D培养、球状体/类器官细胞、精密切割肝、肾切片、永生化细胞系或夹心培养细胞系等培养物,构建适合不同物质药效毒理学检测的体外模型。该模型最大化模拟或保留肝脏和肾脏的脏器功能和体内微环境,包括特定的生理组织结构、多细胞相互作用/串扰、多器官相互协作/反馈等,以得到与体内实验数据相近或相同的结果,减少了不同种属之间的差异;同时大大减少实验动物的使用,降低了成本。微流控技术提供的微流体不仅能为内容物培养提供必要的剪切力微环境,还能解决目前由于肝、肾芯片培养过程中组织供氧不足、营养物质的缺失、代谢物堆积,导致的细胞凋亡甚至组织坏死纤维化,难以长期维持...     

Acute cytomegalovirus infection modulates the intestinal microbiota and targets intestinal epithelial cells

Primary and recurrent cytomegalovirus (CMV) infections frequently cause CMV colitis in immunocompromised as well as inflammatory bowel disease (IBD) patients. Additionally, colitis occasionally occurs upon primary CMV infection in patients who are apparently immunocompetent. In both cases, the underlying pathophysiologic mechanisms are largely elusive - in part due to the lack of adequate access to specimens. We employed the mouse cytomegalovirus (MCMV) model to assess the association between CMV and colitis. During acute primary MCMV infection of immunocompetent mice, the gut microbial composition was affected as manifested by an altered ratio of the Firmicutes to Bacteroidetes phyla. Interestingly, these microbial changes coincided with high-titer MCMV replication in the colon, crypt hyperplasia, increased colonic pro-inflammatory cytokine levels, and a transient increase in the expression of the antimicrobial protein Regenerating islet-derived protein 3 gamma (Reg3γ). Further analyses revealed that murine and human intestinal epithelial cell lines, as well as primary intestinal crypt cells and organoids represent direct targets of CMV infection causing increased cell death. Accordingly, in vivo MCMV infection disrupted the intestinal epithelial barrier and increased apoptosis of intestinal epithelial cells. In summary, our data show that CMV transiently induces colitis in immunocompetent hosts by altering the intestinal homeostasis.     

小鼠肠腺瘤类器官培养及其辐射敏感性

目的 建立小鼠肠腺瘤类器官的体外培养方法,观察其对电离辐射的反应。方法 采用氧化偶氮甲烷(azoxymethane,AOM)和葡聚糖硫酸钠(detrain sodium sulfate,DSS)诱导小鼠产生肠腺瘤。体外分离腺瘤类隐窝结构,接种于基质胶。通过培养基筛选,确定肠腺瘤类器官的体外培养条件,采用免疫组化染色检测Ki67和β-catenin表达水平。进一步采用X线照射,观察肠腺瘤类器官损伤情况,比较其与大、小肠类器官的辐射敏感性。 结果 经AOM/DSS诱导,小鼠肠腺瘤成瘤率达95%,肿瘤均位于结肠靠近直肠处,肠腺瘤类器官在改良的小肠类器官中生长良好,Ki67阳性腺瘤细胞比例高且β-catenin入核特征明显。经X线照射,各类器官存活比例随辐射剂量增加而降低。9 Gy照射7天后,腺瘤类器官存活率为11.96%±1.42%,高于同剂量大肠类器官的5.46%±1.22% (t=6.0082,P＜0.01),小肠类器官几乎未见存活。腺瘤类器官剂量存活曲线较大、小肠类器官右移,提示其辐射敏感性低于大肠和小肠。 结论 在AOM/DSS诱导产生的小鼠肠腺瘤中成功分离培养出腺瘤类器官,其辐射敏感性低于大、小肠类器官。     

视网膜类器官治疗视网膜退行性疾病的研究进展

视网膜退行性疾病是导致视力受损与失明的重要原因。目前,对于感光细胞大量丧失的疾病晚期阶段尚无有效的治疗方案。近年来,大量研究提供了感光细胞的移植替代治疗的新思路,而视网膜3D培养技术产生的视网膜类器官能够在体外产生移植所需的感光细胞及组织,为视网膜退行性疾病的移植替代治疗奠定了基础。本文通过综述视网膜3D培养技术以及感光细胞移植的发展,着重阐述视网膜类器官在视网膜退行性疾病移植替代治疗中的现有运用策略以及局限性,以期为视网膜3D培养技术在感光细胞替代治疗中的优化提供理论参考。     

Newly-found functions of metformin for the prevention and treatment of age-related macular degeneration

AIM: To explore the temporal mitochondrial characteristics of retinal pigment epithelium (RPE) cells obtained from human embryonic stem cells (hESC)-derived retinal organoids (hEROs-RPE), to verify the optimal period for using hEROs-RPE as donor cells from the aspect of mitochondria and to optimize RPE cell-based therapeutic strategies for age-related macular degeneration (AMD). METHODS: RPE cells were obtained from hEROs and from spontaneous differentiation (SD-RPE). The mitochondrial characteristics were analyzed every 20d from day 60 to 160. Mitochondrial quantity was measured by MitoTracker Green staining. Transmission electron microscopy was adopted to assess the morphological features of the mitochondria, including their distribution, length, and cristae. Mitochondrial membrane potentials (MMPs) were determined by JC-1 staining and flow cytometry. ROS levels were evaluated by flow cytometry, and ATP levels were measured by a luminometer. Differences between two groups were analyzed by the independent-samples t-test, and comparisons among multiple groups were made using one-way ANOVA or Kruskal-Wallis H test when equal variance is not assumed. RESULTS: hEROs-RPE and SD-RPE cells from day 60 to 160 were successfully differentiated from hESCs and expressed RPE-specific markers (Pax6, mitf, Bestrophin-1, RPE65, Cralbp). RPE features, including a cobblestone-like morphology with tight junctions (ZO-1), pigments and microvilli, were also observed in both hERO-RPE and SD-RPE cells. The mitochondrial quantities peaked in both hEROs-RPE and SD-RPE cells at day 80. However, the cristae of hEROs mitochondria were less mature and abundant than those of SD mitochondria at day 80, with hEROs mitochondria becoming mature at day 100. Both hEROs-RPE and SD-RPE cells showed low ROS levels from day 100 to 140 and maintained a normal MMP during this period. However, hEROs mitochondria maintained a longer time to produce high levels of ATP (from day 120 to 140) than SD-RPE cells (only day 120). CONCLUSION: Mitochondria of hEROs-RPE cells develop slower and maintain a longer time to supply high-levels of energy than SD-RPE cells. From a mitochondrial aspect, hEROs-RPE cells from day 100 to 140 are an optimal cell source for treating AMD.     

Combining Automated Organoid Workflows with Artificial Intelligence-Based Analyses: Opportunities to Build a New Generation of Interdisciplinary High-Throughput Screens for Parkinson's Disease and Beyond

Parkinson's disease (PD) is the second most common neurodegenerative disease and primarily characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. Despite decades of research and the development of various disease model systems, there is no curative treatment. This could be due to current model systems, including cell culture and animal models, not adequately recapitulating human PD etiology. More complex human disease models, including human midbrain organoids, are maturing technologies that increasingly enable the strategic incorporation of the missing components needed to model PD in vitro. The resulting organoid-based biological complexity provides new opportunities and challenges in data analysis of rich multimodal data sets. Emerging artificial intelligence (AI) capabilities can take advantage of large, broad data sets and even correlate results across disciplines. Current organoid technologies no longer lack the prerequisites for large-scale high-throughput screening (HTS) and can generate complex yet reproducible data suitable for AI-based data mining. We have recently developed a fully scalable and HTS-compatible workflow for the generation, maintenance, and analysis of three-dimensional (3D) microtissues mimicking key characteristics of the human midbrain (called “automated midbrain organoids,” AMOs). AMOs build a reproducible, scalable foundation for creating next-generation 3D models of human neural disease that can fuel mechanism-agnostic phenotypic drug discovery in human in vitro PD models and beyond. Here, we explore the opportunities and challenges resulting from the convergence of organoid HTS and AI-driven data analytics and outline potential future avenues toward the discovery of novel mechanisms and drugs in PD research. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society     

Muramyl dipeptide: Not just another brick in the wall

The robust expression of microbial pattern recognition receptors such as TLR4 and Nod2 in intestinal stem cells reflects an active communication dynamic between the host and the gut microbiota. A new study reveals that muramyl dipeptide, the bacterial cell wall peptidoglycan motif, activates Nod2 within crypt base columnar Lgr5-positive stem cells and promotes their survival. Apart from the immediate relevance to the growth of organoids for in vitro experiments, the study raises new questions about the molecular mechanisms whereby gut microbes influence intestinal physiology.