Lung cancer remains one of the most prevalent and lethal malignancies worldwide. The advancement of its precise diagnosis and therapeutic development urgently requires in vitro models that can highly recapitulate the pathophysiological characteristics of human tissues. Organ-on-a-chip has emerged as a novel technological platform that integrates microfluidic engineering, biomaterials, and other engineering strategies with organoid culture. This platform enables precise control over the cellular microenvironment, thereby closely mimicking the three-dimensional structure and physiological functions of human organs in vitro. Organ-on-a-chip systems demonstrate significant advantages in cancer research, developmental biology, and disease modeling, as they not only preserve the heterogeneity and pathological features of patient samples but also support co-culture of various cell types to reconstruct the tumor microenvironment (TME). However, standardized construction methods and integrated analytical strategies for this technology in lung cancer research remain to be further refined. This review systematically elaborates on the key technical principles of organ-on-a-chip and its recent advances in lung cancer modeling, drug screening, and immunotherapy research. It aims to provide a theoretical foundation and technical perspective for promoting the deeper application of organ-on-a-chip in precision medicine and translational research for lung cancer.
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Humans ; Lung Neoplasms/drug therapy* ; Organoids/drug effects* ; Lab-On-A-Chip Devices ; Animals ; Tumor Microenvironment

Mutations in ion channel genes have long been implicated in a spectrum of epilepsy syndromes. However, therapeutic decision-making is relatively complex for epilepsies associated with channelopathy. Therefore, in the present study, we used a patient-derived organoid model with a novel SCN2A mutation (p.E512K) to investigate the potential of utilizing such a model as a platform for preclinical testing of anti-seizure compounds. The electrophysiological properties of the variant Nav1.2 exhibited gain-of-function effects with increased current amplitude and premature activation. Immunofluorescence staining of patient-derived cortical organoids (COs) displayed normal neurodevelopment. Multielectrode array (MEA) recordings of patient-derived COs showed hyperexcitability with increased spiking and remarkable network bursts. Moreover, the application of patient-derived COs for preclinical drug testing using the MEA showed that they exhibit differential responses to various anti-seizure drugs and respond well to carbamazepine. Our results demonstrate that the individualized organoids have the potential to serve as a platform for preclinical pharmacological assessment.
Organoids/physiology* ; NAV1.2 Voltage-Gated Sodium Channel/genetics* ; Humans ; Anticonvulsants/pharmacology* ; Epilepsy/drug therapy* ; Mutation ; Cerebral Cortex/drug effects* ; Action Potentials/drug effects* ; Carbamazepine/pharmacology*

OBJECTIVE: To elucidate the molecular mechanisms underlying sepsis-induced injury in mouse intestinal organoids and investigate the possible mechanisms or potential drug targets of myeloid differentiation factor 88 inhibitor [TJ-M2010-5 (TJ5)] on this condition. METHODS: Small intestinal organoids from C57BL/6 mice aged 6-8 weeks were established and characterized using immunofluorescence for cell growth and proliferation marker nuclear antigen Ki-67, goblet cell marker mucin-2 (MUC-2), epithelial cell marker E-cadherin, and Paneth cell marker lysozyme (Lyz). Small intestinal organoids after 3 days of passaging were divided into different groups: a normal control group treated with culture medium containing 0.2% dimethyl sulfoxide (DMSO) for 10 hours, a lipopolysaccharide (LPS) group treated with culture medium containing 200 mg/L LPS and 0.2% DMSO for 10 hours, and a TJ5 group pre-treated with 10 mmol/L TJ5 for 2 hours followed by treatment with culture medium containing 200 mg/L LPS for 10 hours. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to measure the expression levels of interleukin-6 (IL-6) and zonula occludens-1 (ZO-1) in the small intestinal organoids. RNA transcriptome sequencing was performed on the small intestinal organoids from each group to analyze differentially expressed genes between groups, and significant enrichment was analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). RESULTS: By the 7th day of primary culture, mature organoids had formed, and their growth rate increased after passaging. Immunofluorescence identification showed expressions of Ki-67, MUC-2, E-cadherin, and Lyz, indicating that the mouse small intestinal organoids maintained their cellular composition and functional characteristics under in vitro culture conditions. RT-qPCR results showed that compared with the normal control group, the mRNA expression of IL-6 in the small intestinal organoids of the LPS group was significantly increased (2^-ΔΔCT: 1.83±0.16 vs. 1.02±0.28, P < 0.05), while the mRNA expression of ZO-1 was significantly decreased (2^-ΔΔCT: 0.53±0.11 vs. 1.01±0.18, P < 0.05). In contrast, the mRNA expression trends of both IL-6 and ZO-1 were reversed in the TJ5 group, showing statistically significant differences as compared with the LPS group (2^-ΔΔCT: IL-6 mRNA was 1.24±0.01 vs. 1.83±0.16, ZO-1 mRNA was 1.97±0.29 vs. 0.53±0.11, both P < 0.05). RNA transcriptome sequencing showed 49 differentially expressed genes in the LPS group compared to the normal control group, with 42 upregulated and 7 downregulated. Compared to the LPS group, the TJ5 group showed 84 differentially expressed genes, with 47 upregulated and 37 downregulated. GO enrichment analysis of these differentially expressed genes showed that the significantly enriched biological processes of the differentially expressed genes between the normal control group and the LPS group included responses to LPS, responses to molecule of bacterial origin and responses to bacterium. The significantly enriched biological processes of the differentially expressed genes between the LPS group and the TJ5 group included glutathione metabolic processes, responses to stress cellular and responses to chemical stimulus. In molecular function groups, glutathione binding and oligopeptide binding were significantly enriched by the differentially expressed genes. In cellular component classifications, the enrichment of the differentially expressed genes was mainly observed in the cytoplasm, endoplasmic reticulum, and microsomes. KEGG pathway enrichment analysis indicated that the differentially expressed genes between the normal control group and LPS group were enriched in IL-17 signaling pathways, tumor necrosis factor (TNF) signaling pathways, viral protein interactions with cytokines and cytokine receptors signaling pathways, and cytokine-cytokine receptor interaction signaling pathways. In contrast, the differentially expressed genes between the LPS and TJ5 groups were mainly enriched in atherosclerosis signaling pathways, ferroptosis signaling pathways, glutathione metabolism signaling pathways, and cytochrome P450-mediated drug metabolism signaling pathways. CONCLUSIONS Mouse small intestinal organoids were successfully extracted and cultured. TJ5 may exert its protective effects by regulating gene expression and related signaling pathways (fluid shear stress and atherosclerosis, ferroptosis, glutathione metabolism, cytochrome P450 drug metabolism, etc.) in sepsis-injured mouse small intestinal organoids. These genes and signaling pathways may be key targets for treating sepsis-induced intestinal injury.
Animals ; Mice ; Sepsis/genetics* ; Organoids/drug effects* ; Mice, Inbred C57BL ; Intestine, Small/metabolism* ; Gene Expression Profiling ; Transcriptome ; Lipopolysaccharides

Peptide-based hydrogel, the polymer materials with a special network structure, are widely used in various fields of biomedicine due to their stable properties and biocompatibility. Environment-responsive self-assembled peptide aqueous solutions can respond to environment changes by the self-assembly of peptides into nanofiber networks. Peptide-based hydrogels well simulate the extracellular matrix and cell growth microenvironment, being suitable for 3D cell culture and organoid culture. To establish a tumor organoid culture system with peptide-based hydrogels, we cultured Panc-1, U87, and H358 cells in a 3D spherical manner using CulX Ⅱ peptide-based hydrogels in 24-well plates for 15 days. The organoids showed a 3D spherical shape, and their sizes increased with the extension of the culture time, with a final diameter ranging from 150 to 300 μm. The organoids had a large number, varying sizes, good cell viability, clear edges, and a good shape, which indicated successful organoid construction. The tumor organoid culture system established in this study with CulX Ⅱ peptide-based hydrogels provides a model for studying tumor pathogenesis, drug development, and tumor suppression.
Hydrogels/chemistry* ; Organoids ; Humans ; Peptides/pharmacology* ; Cell Line, Tumor ; Cell Culture Techniques, Three Dimensional ; Cell Culture Techniques ; Cell Survival/drug effects* ; Nanofibers/chemistry*

In order to establish a stable in vitro culture platform for chicken small intestine three-dimensional (3D) organoids, in this study, crypt cells were collected from the small intestine of 18-day-old embryos of AA broilers. On the basis of the L-WRN conditioned medium, we optimized the culture conditions of chicken small intestinal organoids by adjusting the proportions of nicotinamide, N-acetylcysteine, LY2157299, CHIR99021, Jagged-1, FGF, and other cytokines to select the medium suitable for the long-term stable growth of the organoids. The optimization results showed that the addition of 1.5 µmol/L CHIR99021 significantly improved the organoid formation efficiency and organoid diameter. When 0.5 µmol/L Jagged-1 was added, a small amount of bud-like tissue appeared in organoids. After the addition of 50 ng/mL FGF-2, the rate of organoid germination was significantly increased. The 1.5 µmol/L CHIR99021, 0.5 µmol/L Jagged-1, and 50 ng/mL FGF-2 added in the medium can cooperate with each other to improve the formation and speed up the proliferation and differentiation of organoids, while improving the stemness maintenance of cells. The morphology, cell types, and culture characteristics of chicken small intestinal organoids were studied by HE staining, transmission electron microscopy, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), indirect immunofluorescence, and immunohistochemistry. The results showed that the 3D organoids of the chicken small intestine cultured in vitro were morphologically consistent with the chicken intestinal tissue and contained differentiated epithelial cells. In summary, we successfully established an in vitro culture system for chicken small intestinal organoids, providing a new method for the subsequent research on chicken intestinal physiology, pathology, and host-pathogen interaction mechanism and the development of relevant drugs.
Animals ; Organoids/metabolism* ; Intestine, Small/drug effects* ; Chickens ; Cell Culture Techniques/methods* ; Culture Media ; Chick Embryo ; Tissue Culture Techniques/methods*

BACKGROUNDColorectal cancer (CRC) is a heterogeneous disease; current research relies on cancer cell lines and animal cancer models, which may not precisely imitate inner human tumors and guide clinical medicine. The purpose of our study was to explore and further improve the process of producing three-dimensional (3D) organoid model and impel the development of personalized therapy.

METHODSWe subcutaneously injected surgically resected CRC tissues from a patient into BALB/c-nu mice to build patient-derived xenografts (PDXs). Isolated cells from PDXs at appropriate tumor size were mingled with Matrigel, and then seeded in ultra-low attachment 96-well plates at four cell densities (500, 1000, 2000, and 4000 single cells/well). Cells were cultured with advanced Dulbecco's Modified Eagle Medium/F12 medium additional with various factors added to maintain tumor's biological traits and growth activity. The growth curves of the four cell densities were measured after 24 h of culture until 25 days. We evaluated the effects of four chemotherapeutic agents on organoid model by the CellTiter-Glo ® Luminescent Cell Viability Assay. Hematoxylin and eosin (H and E) staining of 3D organoids was performed and compared with patient and CRC PDX tissues. Furthermore, immunohistochemistry was performed, in which the organoids were stained with the proliferation marker, Ki-67. During the experimental process, a phase-contrast microscope was used.

RESULTSPhenotype experimental results showed that 3D organoids were tightly packed together and grew robustly over time. All four densities of cells formed organoids while that composed of 2000 cells/well provided an adequate cultivation system and grew approximately 8-fold at the 25 th day. The chemosensitivity of the four conventional drugs was [s]-10-hydroxycamptothecin > mitomycin C > adriamycin > paclitaxel, which can guide clinical treatment. Histological features of CRC patient's tumor tissues and mice tumor xenograft tissues were highly similar, with high-column-like epithelium and extracellular matrix. H and E-stained sections showed heterogeneous cell populations harbored in cancer organoids and were histologically similar to tumor tissues. The proliferation index was only 8.33% within spheroids, which exhibited confined proliferative cells that might be cancer stem cells.

CONCLUSIONSWe successfully constructed a CRC organoid model that grew robustly over 25 days and demonstrated that 2000 cells/well in 96-well plate was a prime seeding density for cells to form organoids. The results confirmed that organoid model can be used for agent screening and personalized medicine.

Adult ; Animals ; Antineoplastic Agents ; therapeutic use ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Colorectal Neoplasms ; drug therapy ; pathology ; Doxorubicin ; therapeutic use ; Humans ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred BALB C ; Mitomycin ; therapeutic use ; Organoids ; drug effects ; pathology ; Paclitaxel ; therapeutic use ; Rectal Neoplasms ; drug therapy ; pathology ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays