类器官的研究进展及应用前景

类器官（organoids）来源于自组织和自我更新的干细胞，是利用干细胞的自组织特性进行体外3D培养后形成的细胞团，与来源器官密切相关，再现了来源器官的三维细胞结构，并为探索来源器官的发病机制提供了新的模型。类器官系统是由自分泌、旁分泌或邻分泌信号调节下的细胞，或者外源性添加的细胞外基质（extracellular matrix，ECM）底物、小分子和生长因子等衍生而来，这些因素的相互作用创造了一个动态的环境，指导干细胞的自我更新和分化，以及细胞在类器官中的自我组装。诱导多能干细胞（induced pluripotent stem cells，iPSC）重编程方法结合3D类器官工具，使患者来源的类器官作为动物模型和人类临床试验之间的桥梁，是对细胞研究和在体试验的补充。在研究来源器官发育、生物学和病理生理学方面，类器官不仅是一种比传统细胞培养更具生理相关性的体外模型，而且还是再生医学和个性化医学领域中的新模型，有望成为研究营养素、药物、毒物及毒素等的作用机制及药物的筛选、再生医学等领域的重要模型。总之，类器官技术的发展增强了人们对器官和组织生理生化功能的认知。作者对肠、脑、肺脏、肝脏、子宫、卵巢等类器官培养和应用的研究进展进行综述，以期为类器官相关科研及应用提供参考。     

Probiotic Lactobacillus acidophilus bacteria or synthetic TLR2 agonist boost the growth of chicken embryo intestinal organoids in cultures comprising epithelial cells and myofibroblasts

The intestinal epithelial cells reside in close proximity to myofibroblasts and microbiota, which are supposed to have an impact on intestinal stem cells fate and to influence processes of tissue maturation and regeneration. Mechanism underlying these phenomena and their diversity among vertebrates can be studied in 3D organoid cultures. We investigated the growth of chicken embryo intestinal epithelial organoids in Matrigel with and without Toll-like receptors (TLRs) stimulation. The organoid cultures contained also some myofibroblasts with potential to promote intestinal stem cell survival. Organoid cells, expressing TLR4, TLR2 type 1 and TLR2 type 2 were incubated with their agonists (lipopolysaccharide – LPS and Pam3CSK4) or co-cultured with Lactobacillus acidophilus bacteria (LA-5). Pam3CSK4 and LA-5 promoted organoid growth, which was demonstrated by comparing the morphological parameters (mean number and area of organoids). The profile of prostaglandins (PG), known to promote intestinal regeneration, in supernatants from organoid and fibroblast cultures were evaluated. Both PGE₂ and PGD₂ were detected. As compared to unstimulated controls, supernatants from the Pam3CSK4-stimulated organoids contained twice as much of PGE₂ and PGD₂. The changes in production of prostaglandins and the support of epithelial cell growth by myofibroblasts are factors potentially responsible for stimulatory effect of TLR2 activation.     

Gut Organoid as a New Platform to Study Alginate and Chitosan Mediated PLGA Nanoparticles for Drug Delivery

Intestinal organoids can be used as an ex vivo epithelial model to study different drug delivery effects on epithelial cells’ luminal surface. In this study, the impact of surface charge on the delivery of 5-ASA loaded PLGA nanoparticles into the lumen of organoids was investigated. Alginate and chitosan were used to coat the nanoparticles and provide negative and positive charges on the particles, respectively. The organoid growth and viability were not affected by the presence of either alginate- or chitosan-coated nanoparticles. It was shown that nanoparticles could be transported from the serosal side of the organoids to the lumen as the dye gradually accumulated in the lumen by day 2–3 after adding the nanoparticles to the Matrigel. By day 5, the dye was eliminated from the lumen of the organoids. It was concluded that the positively charged nanoparticles were more readily transported across the epithelium into the lumen. It may be attributed to the affinity of epithelial cells to the positive charge. Thus, the organoid can be utilized as an appropriate model to mimic the functions of the intestinal epithelium and can be used as a model to evaluate the benefits of nanoparticle-based drug delivery.     

The potential of organoids in toxicologic pathology: Histopathological and immunohistochemical evaluation of a mouse normal tissue-derived organoid-based carcinogenesis model

Recently, we introduced an organoid-based chemical carcinogenesis model using mouse normal tissue-derived organoids. In the present review article, the histopathological and immunohistochemical characteristics of mouse normal tissue-derived organoids and tumors derived from these organoids after their in vitro treatment with genotoxic carcinogens and injection into nude mouse are reviewed. In organoids treated in vitro with genotoxic carcinogens, we confirmed macroscopic tumorigenicity and histopathological findings, including neoplastic characteristics, such as multilayered epithelia and/or invasion of epithelia into the surrounding interstitium. In contrast glandular/cystic structures with monolayered epithelia were clearly demarcated from the surrounding Matrigel/interstitium in the untreated control groups. In addition to macroscopic tumorigenicity, these microscopic epithelial changes, which are characteristic of the early stages of carcinogenesis, are included in the requirements for carcinogenicity-positive judgement of the organoid-based carcinogenesis model. Immunohistochemistry of cytokeratins (CKs), used to determine the origin of epithelia and distribution of extraductal invasive lesions, or oncogenic kinases, which reflect molecular activation in epithelia following chemical treatment, is helpful for accurate diagnosis and molecular evaluation in the early stages of carcinogenesis. This information improves our biological understanding of organoid-based chemical carcinogenesis models.     

The potential of organoids in toxicologic pathology: role of toxicologic pathologists in in vitro chemical hepatotoxicity assessment

The development of in vitro toxicity assessment methods using cultured cells has gained popularity for promoting animal welfare in animal experiments. Herein, we briefly discuss the current status of hepatoxicity assessment using human- and rat-derived hepatocytes; we focus on the liver organoid method, which has been extensively studied in recent years, and discuss how toxicologic pathologists can use their knowledge and experience to contribute to the development of in vitro chemical hepatotoxicity assessment methods for drugs, pesticides, and chemicals. We also propose how toxicological pathologists should assess toxicity regarding the putative distribution of undifferentiated and differentiated cells in the organoid when liver organoids are observed in hematoxylin and eosin–stained specimens. This was done while considering the usefulness and limitations of in vitro studies for toxicologic pathology assessment.