Organotypic Culture of Adult Vascularized Porcine Retina Explants In Vitro on Nanotube Scaffolds

Development of novel explant culture models for retinal diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Investigating retinal explant models cultured in static and perfused systems to test the performance of exosomes secreted from retinal organoids

This study is to give a brief introduction of immobilized enzyme reactor (IMER) in on-line LC and its application in drug screening. The literature of immobilization techniques, immobilization supports and determination of immobilized enzyme activity were reviewed; the application in the drug screening is briefly introduced. It was found that IMER increased the enzymatic stabilization, strikingly shortens reaction time and can be used to perform fast screening of enzyme inhibitor. IMER has wide fields in drug screening application.

It has been a challenge to develop skin models which exhibit the key characteristics of the human skin and facilitate high‐throughput drug screening. In this study, a method is developed to construct microspheric skin organoids based on spinning bioreactors. The organoid consists of a core‐shell structure to mimic the bilayered skin structure, where the shell is composed of cultured human keratinocytes, resembling the epidermis, and the core, which mimics the dermis, comprises human dermal fibroblasts and collagen. In fabrication of the organoid, the cores are cultured with keratinocytes in spinner flasks under defined conditions to facilitate epidermal growth and differentiation to form mature barrier. To enable efficient drug screening, the organoid is equipped with a luciferase reporter to detect canonical Wnt activation, and Minoxidil is identified to induce epidermal Wnt/beta‐catenin pathway signaling. Thus, the study has developed a novel method for efficient preparation of uniform microspheric skin organoids with potential applications for high‐throughput drug testing and screening.

Tissue-engineered heart chambers as a platform technology for drug discovery and disease modeling.

Background: For disease modeling and drug screening using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), matured iPSC-CMs are required to understand the underlying mechanism of diseases and to get the candidate drug’s deleterious features. We have shown that synthetic mRNAs encoding a fluorescent protein tagged with complementary sequences against specifically expressed microRNA (miRNA-switch) can be efficiently used for purification. Using miRNA-switch, we evaluated the selection method of matured iPSC-CMs from iPSC-CMs. Methods: We used miR-208a as a specific miRNA of CMs and miR-Matured CM (miR-MCM). We synthesized miR-208a-responsive GFP-coding mRNA (miR-208a-GFP switch), and miR-MCM-iRFP670 switch and control tagBFP mRNA and transfected them into iPSC-CMs. Results: After transfection of these mRNA, 25±5% of miR-208a positive cells were positive for miR-MCM. We also compared miR-208a positive and miR-MCM positive cells to miR-208a positive and miR-MCM negative cells by qPCR and electron microscopy (EM). MiR-208a-positive and miR-MCM-positive cells showed significantly higher expression of matured CM-related genes such as MYH7 and MYL2. In EM observation, miR-208a-positive and miR-MCM-positive cells showed M-bands, while miR-208a-positive and miR-MCM-negative cells did not display M-bands. Conclusions: We demonstrated that miR-MCM could be used for the marker of matured iPSC-CMs. The synthetic miR-switch for miR-MCM efficiently isolate matured iPSC-CMs, enabling its application in disease modeling and drug screening using differentiated cells from iPSCs.

Droplet-based 3D bioprinting for drug delivery and screening.

Graphene-loaded polymannose-chitosan scaffolds have emerged as promising candidates for drug screening applications due to their unique properties. The combination of polymannose and chitosan, supplemented with graphene, offers a versatile platform with potential applications including drug screening. The aim of this study is to fabricate and characterize the biocompatible graphene oxide loaded polymannose chitosan scaffold (PM-Chi-GO) for potential drug screening applications. The scaffold was meticulously prepared by combining oxidized polymannose with chitosan hydrochloride and graphene oxide, employing gelation techniques. Characterization involved Fourier Transform Infrared Spectroscopy (FTIR) for functional group analysis and Scanning Electron Microscopy (SEM) for morphological studies. The biocompatibility of the scaffold was assessed using Peripheral Blood Mononuclear Cells (PBMCs). FTIR analysis revealed distinctive peaks at 3296, 1624, 1528, 1389, 1060, and 808 cm-1, corresponding to specific functional groups within the scaffold. SEM displayed a porous morphological structure. Biocompatibility testing with PBMCs demonstrated favorable responses, confirming the scaffold's potential for in vitro drug screening applications. The synthesized PM-Chi-GO is characterized by its unique structural and biocompatible properties and holds significant promise for future drug screening endeavors. This study establishes a foundation for the utilization of this scaffold in drug screening applications.

As a necessary pathway to man-made organs, organ-on-chips (OOC), which simulate the activities, mechanics, and physiological responses of real organs, have attracted plenty of attention over the past decade. As the maturity of three-dimensional (3D) cell-culture models and microfluidics advances, the study of OOCs has made significant progress. This review article provides a comprehensive overview and classification of OOC microfluidics. Specifically, the review focuses on OOC systems capable of being used in preclinical drug screening and development. Additionally, the review highlights the strengths and weaknesses of each OOC system toward the goal of improved drug development and screening. The various OOC systems investigated throughout the review include, blood vessel, lung, liver, and tumor systems and the potential benefits, which each provides to the growing challenge of high-throughput drug screening. Published OOC systems have been reviewed over the past decade (2007–2018) with focus given mainly to more recent advances and improvements within each organ system. Each OOC system has been reviewed on how closely and realistically it is able to mimic its physiological counterpart, the degree of information provided by the system toward the ultimate goal of drug development and screening, how easily each system would be able to transition to large scale high-throughput drug screening, and what further improvements to each system would help to improve the functionality, realistic nature of the platform, and throughput capacity. Finally, a summary is provided of where the broad field of OOCs appears to be headed in the near future along with suggestions on where future efforts should be focused for optimized performance of OOC systems in general.

Human-induced pluripotent stem cells (hiPSCs) with their differentiation protocols, constitute a potential tool to investigate the various biological mechanisms of different human cells, such as those of the central nervous system. With the advent of such technique, we have increased the knowledge of biological mechanisms of neural diseases and, new therapies are now emerging. In particular, three-dimensional (3D) neural cell culture models including brain organoids and neurospheroids are increasingly used as in vitro platforms for studying human brain cell biology and drug screening, in genome engineering and transplantation as potential treatment for some neurodegenerative diseases. In this work, we exploited a particular differentiation protocol to generate engineered excitatory cortical neurospheroids of human origin. To assess functional network activity, we used standard Micro Electrodes Arrays (60 channels) and for the first time CMOS based devices (4096 channels). Sample cultures showed electrophysiological activity in 4 weeks and these first results suggest future possible applications for drug screening and transplantation.

Background: Current tumor models face challenges in accurately replicating the intricate and dynamic conditions of the tumor microenvironment (TME), limiting predictions of drug efficacy in drug development and personalized treatment. Furthermore, the continuous and real-time observation of drug responses variations resulting from tumor heterogeneity poses challenges in existing models, including patient-derived organoids and animal models. To address these limitations, our study introduces a novel approach through a tumor-microenvironment-on-chip (TMoC) that combines 3D tissue cultivation and a circulation system, incorporating physiological gradients of oxygen and nutrients. Serving as a tool to faithfully replicate the intricate TME, TMoC facilitates highly accurate drug screening for enhanced therapeutic precision. Method: Based on organ-on-chip and microfluidic systems, our platform reconstructs the cellular heterogeneity of the TME using tumor tissue-dissociated cells in collagen I or Matrigel within a strip-shaped 3D cultivation space, enabling simple analysis of regional responses. A circulation system, powered by a peristaltic pump, aids medication and immune cell entry for combination immunotherapy assessment. Real-time apoptosis analysis via fluorescence microscopy and sample retrieval for in-depth analysis are enabled. In drug screening, TMoC results are compared with mouse model outcomes and medication records for various cancers and treatments. Result: TMoC reconstructed physiological-like gradients, including oxygen levels, and facilitated the infiltration of cytotoxic CD8+ T cells, providing a distinctive model for assessing the synergistic impact of immune checkpoint inhibitors and chemotherapy drugs. Additionally, tissues cultured in TMoC retained their original cellular composition. TMoC demonstrated efficacy in drug screening across various cancer models, including breast, pancreatic, and colorectal cancers. In clinical studies, tumor tissue-derived cells from patients were cultivated on the TMoC, and the treating results are subsequently compared with clinical outcomes. Whether in animal models or clinical cases, the drug screening outcomes of TMoC consistently aligned with in vivo drug responses. Conclusion: The TMoC offering a platform that modeling some complex characteristics of TME. With applications in drug screening and clinical studies, it shows promising correlations between TMoC and in vivo responses. Citation Format: Chiao-Min Lin, Hsaun-Yu Mu, Li-An Chu, Ya-Hui Lin, Ji Li, Chao-Yu Liu, Hsi-Chien Huang, Sheng-Liang Cheng, Tsung-Ying Lee, Hsin Mei Lee, Hsin-Min Chen, Yun-Jen Tsai, Tzu-Hung Hsiao, Kee-Ming Man, Yunching Chen, Jen-Huang Huang. Tumor-microenvironment-on-chip: an ex vivo drug screening platform enabling real-time observation of regional tumor responses during drug development and clinical treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6771.

Hepatitis E virus (HEV) is a quasi-enveloped virus with a single-stranded positive-sense RNA genome belonging to the family Hepeviridae. Studies of the molecular aspects of HEV and drug screening have benefited from the discovery of bioluminescent reporter genes. However, the stability of large foreign genes is difficult to maintain after insertion into the viral genome. Currently, ribavirin is used to treat HEV-infected patients who require antiviral therapy. This has several major drawbacks. Thus, the development of novel anti-HEV drugs is of great importance. We developed a system consisting of recombinant infectious HEV harboring a small luciferase gene (nanoKAZ) in the hypervariable region (HVR) of the open reading frame 1 (ORF1) (HEV-nanoKAZ). It replicated efficiently in cultured cells, was genetically stable, and had morphological characteristics similar to those of the parental virus. Both membrane-associated (eHEV-nanoKAZ) and membrane-unassociated (neHEV-nanoKAZ) particles were infectious. HEV particles circulating in the bloodstream and attaching to hepatocytes in HEV-infected patients are membrane-associated; thus, eHEV-nanoKAZ was applied in drug screening. The eHEV-nanoKAZ system covers at least the inhibitor of HEV entry and inhibitor of HEV RNA replication. Four drugs with anti-HEV activity were identified. Their effectiveness in cultured cells was confirmed in naive and HEV-producing PLC/PRF/5 cells. Two hit drugs (azithromycin and ritonavir) strongly inhibited HEV production in culture supernatants, as well as intracellular expression of ORF2 protein, and may therefore be candidate novel anti-HEV drugs. The HEV-nanoKAZ system was developed and applied in drug screening and is expected to be useful for investigating the HEV life cycle. IMPORTANCE Bioluminescent reporter viruses are essential tools in molecular virological research. They have been widely used to investigate viral life cycles and in the development of antiviral drugs. For drug screening, the use of a bioluminescent reporter virus helps shorten the time required to perform the assay. A system, consisting of recombinant infectious HEV harboring the nanoKAZ gene in the HVR of ORF1 (HEV-nanoKAZ), was developed in this study and was successfully applied to drug screening in which four hit drugs with anti-HEV activity were identified. The results of this study provide evidence supporting the use of this system in more variable HEV studies. In addition, both forms of viral particles (eHEV-nanoKAZ and neHEV-nanoKAZ) are infectious, which will enable their application in HEV studies requiring both forms of viral particles, such as in the investigation of unknown HEV receptors and the elucidation of host factors important for HEV entry.

High-Throughput CMOS MEA System with Integrated Microfluidics for Cardiotoxicity Studies

Cultured skeletal myotubes are a powerful in vitro system for identifying mechanisms of skeletal muscle development and disease. However, skeletal myotubes routinely delaminate from conventional culture substrates after approximately 1 week, which significantly hampers their utility for in vitro disease modeling and drug screening. To address this problem, we fabricated micromolded gelatin hydrogels as culture substrates that are more biomimetic than conventional substrates. On micromolded gelatin hydrogels, C2C12 skeletal myoblasts align and differentiate into skeletal myotubes that are stable in culture for multiple weeks. With this protocol, we detail three key steps: (1) Fabrication of micromolded gelatin hydrogels; (2) Culture of mouse C2C12 myoblasts and differentiation into myotubes; and (3) Quantification of myotube morphology. These substrates have many applications for skeletal muscle disease modeling and drug screening over longer time scales.