Recent and advanced therapy for oral cancer

Oral cancer is a common and deadly kind of tissue invasion, has a high death rate, and may induce metastasis that mostly affects adults over the age of 40. Most in vitro traditional methods for studying cancer have included the use of monolayer cell cultures and several animal models. There is a worldwide effort underway to reduce the excessive use of laboratory animals since, although being physiologically adequate, animal models rarely succeed in exactly mimicking human models. 3D culture models have gained great attention in the area of biomedicine because of their capacity to replicate parent tissue. There are many benefits to using a drug delivery approach based on nanoparticles in cancer treatment. Because of this, in vitro test methodologies are crucial for evaluating the efficacy of prospective novel nanoparticle drug delivery systems. This review discusses current advances in the utility of 3D cell culture models including multicellular spheroids, patient-derived explant cultures, organoids, xenografts, 3D bioprinting, and organoid-on-a-chip models. Aspects of nanoparticle-based drug discovery that have utilized 2D and 3D cultures for a better understanding of genes implicated in oral cancers are also included in this review.     

Three-dimensional organoid culture unveils resistance to clinical therapies in adult and pediatric glioblastoma

BackgroundGlioblastoma (GBM) is the most common primary brain tumor with a dismal prognosis. The inherent cellular diversity and interactions within tumor microenvironments represent significant challenges to effective treatment. Traditional culture methods such as adherent or sphere cultures may mask such complexities whereas three-dimensional (3D) organoid culture systems derived from patient cancer stem cells (CSCs) can preserve cellular complexity and microenvironments. The objective of this study was to determine if GBM organoids may offer a platform, complimentary to traditional sphere culture methods, to recapitulate patterns of clinical drug resistance arising from 3D growth.MethodsAdult and pediatric surgical specimens were collected and established as organoids. We created organoid microarrays and visualized bulk and spatial differences in cell proliferation using immunohistochemistry (IHC) staining, and cell cycle analysis by flow cytometry paired with 3D regional labeling. We tested the response of CSCs grown in each culture method to temozolomide, ibrutinib, lomustine, ruxolitinib, and radiotherapy.ResultsGBM organoids showed diverse and spatially distinct proliferative cell niches and include heterogeneous populations of CSCs/non-CSCs (marked by SOX2) and cycling/senescent cells. Organoid cultures display a comparatively blunted response to current standard-of-care therapy (combination temozolomide and radiotherapy) that reflects what is seen in practice. Treatment of organoids with clinically relevant drugs showed general therapeutic resistance with drug- and patient-specific antiproliferative, apoptotic, and senescent effects, differing from those of matched sphere cultures.ConclusionsTherapeutic resistance in organoids appears to be driven by altered biological mechanisms rather than physical limitations of therapeutic access. GBM organoids may therefore offer a key technological approach to discover and understand resistance mechanisms of human cancer cells.     

Organoid models of the tumor microenvironment and their applications

A small percentage of data obtained from animal/2D culture models can be translated to humans. Therefore, there is a need to using native tumour microenvironment mimicking models to improve preclinical screening and reduce this attrition rate. For this purpose, currently, the utilization of organoids is expanding. Tumour organoids can recapitulate tumour microenvironment that is including cancer cells and non-neoplastic host components. Indeed, tumour organoids, both phenotypically and genetically, resemble the tumour tissue that originated from it. The unique properties of the tumour microenvironment can significantly affect drug response and cancer progression. In this review, we will discuss about various organoid culture strategies for modelling the tumour immune microenvironment, their applications and advantages in cancer research such as testing cancer immunotherapeutics, developing novel approaches for personalized medicine, testing drug toxicity, drug screening, study cancer initiation and progression, and we will also review the limitations of organoid culture systems.     

Circulating tumor cell-derived organoids: Current challenges and promises in medical research and precision medicine

Traditional 2D cell cultures do not accurately recapitulate tumor heterogeneity, and insufficient human cell lines are available. Patient-derived xenograft (PDX) models more closely mimic clinical tumor heterogeneity, but are not useful for high-throughput drug screening. Recently, patient-derived organoid cultures have emerged as a novel technique to fill this critical need. Organoids maintain tumor tissue heterogeneity and drug-resistance responses, and thus are useful for high-throughput drug screening. Among various biological tissues used to produce organoid cultures, circulating tumor cells (CTCs) are promising, due to relative ease of ascertainment. CTC-derived organoids could help to acquire relevant genetic and epigenetic information about tumors in real time, and screen and test promising drugs. This could reduce the need for tissue biopsies, which are painful and may be difficult depending on the tumor location. In this review, we have focused on advances in CTC isolation and organoid culture methods, and their potential applications in disease modeling and precision medicine.     

In-Depth Comparison of Matrigel Dissolving Methods on Proteomic Profiling of Organoids

Patient-derived organoids recently emerged as promising ex vivo 3D culture models recapitulating histological and molecular characteristics of original tissues, thus proteomic profiling of organoids could be valuable for function investigation and clinical translation. However, organoids are usually cultured in murine Matrigel (served as scaffolds and matrix), which brings an issue to separate organoids from Matrigel. Because of the complex compositions of Matrigel and thousands of identical peptides shared between Matrigel and organoids, insufficiently dissolved Matrigel could influence proteomic analysis of organoids in multiple ways. Thus, how to dissolve Matrigel matrix and recovery organoid cells efficiently is vital for sample preparation. Here, we comprehensively compared three popular Matrigel dissolving methods (cell recovery solution, dispase, and PBS–EDTA buffer) and investigated the effect of undissolved Matrigel proteins on proteomic profiles of organoids. By integrative analysis of label-free proteomes of Matrigel and stable isotope labeling by amino acids in cell culture proteomes of organoids collected by three methods, respectively, we found that dispase showed an optimal efficiency, with the highest peptide yield and the highest incorporation ratio of stable isotope labeling by amino acids in cell culture labels (97.1%), as well as with the least potential Matrigel contaminants. To help analysis of proteomic profiles of organoids collected by the other two methods, we identified 312 high-confidence Matrigel contaminants, which could be filtered out to attenuate Matrigel interference with minimal loss of biological information. Together, our study identifies bioinformatics and experimental approaches to eliminate interference of Matrigel contaminants efficiently, which will be valuable for basic and translational proteomic research using organoid models.     

Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury

The mechanism of how SARS-CoV-2 causes severe multi-organ failure is largely unknown. Acute kidney injury(AKI) is one of the frequent organ damage in severe COVID-19 patients. Previous studies have shown that human renal tubule cells could be the potential host cells targeted by SARS-CoV-2. Traditional cancer cell lines or immortalized cell lines are genetically and phenotypically different from host cells. Animal models are widely used, but often fail to reflect a physiological and pathogenic status because of species tropisms. There is an unmet need for normal human epithelial cells for disease modeling. In this study, we successfully established long term cultures of normal human kidney proximal tubule epithelial cells(KPTECs) in 2 D and 3 D culture systems using conditional reprogramming(CR) and organoids techniques.These cells had the ability to differentiate and repair DNA damage, and showed no transforming property. Importantly, the CR KPTECs maintained lineage function with expression of specific transporters(SLC34 A3 and cubilin). They also expressed angiotensin-converting enzyme 2(ACE2), a receptor for SARS-CoV and SARS-CoV-2. In contrast, cancer cell line did not express endogenous SLC34 A3, cubilin and ACE2. Very interestingly, ACE2 expression was around twofold higher in 3 D organoids culture compared to that in 2 D CR culture condition. Pseudovirion assays demonstrated that SARS-CoV spike(S) protein was able to enter CR cells with luciferase reporter. This integrated 2 D CR and 3 D organoid cultures provide a physiological ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation.     

Raman spectroscopy investigation of biochemical changes in tumor spheroids with aging and after treatment with staurosporine

There has been increasing use of in vitro cell culture models that more realistically replicate the three‐dimensional (3D) environment found in vivo. Multicellular tumor spheroids (MTS) using cell lines or patient‐derived organoids have become an important in vitro drug development tool, where cells are grown in a 3D “sphere” that exhibits many of the characteristics found in vivo. Significantly, MTS develop gradients in nutrients and oxygen, commonly found in tumors that contribute to therapy resistance. While MTS show promise as a more realistic in vitro culture model, there is a massive need to improve imaging technologies for assessing biochemical characteristics and drug response in such models to maximize their translation into useful applications such as high throughput screening (HTS). In this study, we investigate the potential for Raman spectroscopy to unveil biochemical information in MTS and have investigated how spheroid age influences drug response, shedding light on increased therapy resistance in developing tumors. The wealth of molecular level information delivered by Raman spectroscopy in a noninvasive manner, could aid translation of these 3D models into HTS applications.      

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.     

肝脏类器官的研究及应用进展

肝脏疾病易感性差异大且个体间的肝脏细胞存在明显的异质性,因此开发体外能够长期存活并具有代谢功能的人体类肝组织细胞模型,对治疗终末期肝病、开展肝脏致病机理研究及药物筛选具有重要意义。过去十年中,体外三维类器官模型发展迅猛,为疾病模拟、精准化治疗领域的研究提供了新的工具,显示出巨大潜力。肝脏类器官具有患者的基因表达与突变特征,在体外能够较长时间地保持肝脏细胞功能,已被应用于疾病模拟及药物有效性研究,并具有进行原位或异位移植发挥治疗作用的应用潜能。就干细胞、肝脏原代细胞等不同来源的肝脏类器官的发展及近年的研究进展作了综述,以期为肝脏类器官在疾病建模、药物发现和器官移植领域的研究和应用提供新的思路。     

Liver diseases in the dish: iPSC and organoids as a new approach to modeling liver diseases

Liver diseases negatively impact the quality of life and survival of patients, and often require liver transplantation in cases that progress to organ failure. Understanding the cellular and molecular mechanisms of liver development and pathogenesis has been a challenging task, in part for the lack of adequate cellular models directly relevant to the human diseases.Recent technological advances in the stem cell field have shown the potentiality of induced pluripotent stem cells (iPSC) and liver organoids as the next generation tool to model in vitro liver diseases. Hepatocyte-like cells and cholangiocyte are currently being generated from skin fibroblasts and mononuclear blood cells reprogrammed into iPSC and have been successfully used for disease modeling, drug testing and gene editing, with the hope to be able to find application also in regenerative medicine. Protocols to generate other liver cell types are still under development, but the field is advancing rapidly. On the other end, liver cells can now be isolated from liver specimens (liver explants or liver biopsies) and cultured in specific conditions to form polarized 3D organoids. The purpose of this review is to summarize all these recent technological advances and their potential applications but also to analyze the current issues to be addressed before the technology can reach its full potential.     

肿瘤类器官在药物筛选和个性化用药中的研究进展*

恶性肿瘤是影响人类生命健康的重大疾病之一,药物治疗是常见的治疗手段。近年来,“精准治疗”已经成为肿瘤治疗的趋势。要实现对恶性肿瘤有效、精准的药物治疗,药物筛选模型至关重要。肿瘤类器官是近年来新兴的一种三维细胞模型,具有经长期传代还保留亲本肿瘤的特征和异质性、培养成功率高、周期短和能够高通量筛选药物等优点,已被用于药物筛选、预测患者对治疗的反应以及为个性化用药提供指导等。重点介绍了肿瘤类器官在药物筛选及个性化用药中的研究进展和面临的挑战。     

The Emergence of Stem Cell‐Based Brain Organoids: Trends and Challenges

Recent developments in 3D cultures exploiting the self‐organization ability of pluripotent stem cells have enabled the generation of powerful in vitro systems termed brain organoids. These 3D tissues recapitulate many aspects of human brain development and disorders occurring in vivo. When combined with improved differentiation methods, these in vitro systems allow the generation of more complex “assembloids,” which are able to reveal cell diversities, microcircuits, and cell–cell interactions within their 3D organization. Here, the ways in which human brain organoids have contributed to demystifying the complexities of brain development and modeling of developmental disorders is reviewed and discussed. Furthermore, challenging questions that are yet to be addressed by emerging brain organoid research are discussed.     

肠道类器官在肠疾病机制研究中的运用

肠道类器官由来自肠道的隐窝或干细胞在培养基质的三维（3D）支撑下构建形成,含有肠道的所有成熟细胞,已经成为研究肠道疾病机制全新且高效的平台。相较于二维（2D）细胞培养,肠道类器官不仅可以更加有效地模拟肠道的生理结构与功能,还可以在不同体外环境下更好地还原肠道的真实生态,因此在不同肠道疾病的发病机制研究中应用更为广泛。本文介绍了肠道类器官培养方式的新进展,综述了近年来肠道类器官在炎症性肠道疾病、结肠直肠癌和乳糜泻发病机制研究中的运用及进展,同时讨论了肠道类器官在药物研发与筛选方面的应用。     

Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling

Three-dimensional(3 D) culture systems are becoming increasingly popular due to their ability to mimic tissue-like structures more effectively than the monolayer cultures. In cancer and stem cell research, the natural cell characteristics and architectures are closely mimicked by the 3 D cell models. Thus, the 3 D cell cultures are promising and suitable systems for various proposes, ranging from disease modeling to drug target identification as well as potential therapeutic substances that may transform our lives. This review provides a comprehensive compendium of recent advancements in culturing cells, in particular cancer and stem cells, using 3 D culture techniques. The major approaches highlighted here include cell spheroids, hydrogel embedding, bioreactors, scaffolds, and bioprinting. In addition, the progress of employing 3 D cell culture systems as a platform for cancer and stem cell research was addressed, and the prominent studies of 3 D cell culture systems were discussed.     

In vitro and in vivo translational models for rare liver diseases

A challenge in developing effective treatments is the modeling of the human disease using in vitro and in vivo systems. Animal models have played a critical role in the understanding of disease pathophysiology, target validation, and evaluation of novel therapeutic agents. However, as the success rate from entry into clinical testing to drug approval remains low, it is critical to have high quality and well-validated models reflective of the disease condition. Additional experimental models are being developed based on functional in vitro 3D tissue models such as organoids and 3D bioprinted tissues. Because these 3D tissue models mimic closer the architecture, cell composition and physiology of native tissues, they are now being used as screening platforms in drug discovery and development and for tissue transplant in regenerative medicine. Here we review the current state-of-art of in vitro and in vivo translational models for the development of therapies for rare diseases of the liver.     

Drug screening model meets cancer organoid technology

Tumor organoids inherit the genomic and molecular characteristics of the donor tumor, which not only bridge the gap between genome and phenotype but also circumvent the disadvantages such as genetic information change by using 2D cell lines and the mouse-specific tumor evolution in patient-derived xenograft (PDX). So, cancer organoid has been widely applied to preclinical drug evaluation, biomarker identification, biological research, and individualized therapy. Besides, cancer organoid can be preserved, resuscitated, passed infinitely, and mechanically cultured on a chip for drug screening; it has become one of the partial models for low/high-throughput drug screening in the preclinical trial in vitro. Therefore, this review presents the recent developments of tumor organoids for drug screening, which will introduce from four aspects, including the stability/credibility, types, application, deficiency and prospect of the tumor organoids model for drug screening.     

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research, the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems. Recently, researchers have been actively developing and evaluating three-dimensional (3D) cell culture-based platforms using microfluidic technologies, such as organ-on-a-chip and organoid-on-a-chip platforms, and they have achieved promising breakthroughs in stem cell engineering. In this review, we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery. In a subsequent section, we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research. In addition, some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.     

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures

Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.     

The science and engineering of stem cell-derived organoids-examples from hepatic,biliary, and pancreatic tissues

The field of organoid engineering promises to revolutionize medicine with wide-ranging applications of scientific, engineering, and clinical interest, including precision and personalized medicine, gene editing, drug development, disease modelling, cellular therapy, and human development. Organoids are a three-dimensional (3D) miniature representation of a target organ, are initiated with stem/progenitor cells, and are extremely promising tools with which to model organ function. The biological basis for organoids is that they foster stem cell self-renewal, differentiation, and self-organization, recapitulating 3D tissue structure or function better than two-dimensional (2D) systems. In this review, we first discuss the importance of epithelial organs and the general properties of epithelial cells to provide a context and rationale for organoids of the liver, pancreas, and gall bladder. Next, we develop a general framework to understand self-organization, tissue hierarchy, and organoid cultivation. For each of these areas, we provide a historical context, and review a wide range of both biological and mathematical perspectives that enhance understanding of organoids. Next, we review existing techniques and progress in hepatobiliary and pancreatic organoid engineering. To do this, we review organoids from primary tissues, cell lines, and stem cells, and introduce engineering studies when applicable. We discuss non-invasive assessment of organoids, which can reveal the underlying biological mechanisms and enable improved assays for growth, metabolism, and function. Applications of organoids in cell therapy are also discussed. Taken together, we establish a broad scientific foundation for organoids and provide an in-depth review of hepatic, biliary and pancreatic organoids.