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Journal articles on the topic '3D organoid'
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1
Du, Yuhong, Xingnan Li, Qiankun Niu, Xiulei Mo, Min Qui, Tingxuan Ma, Calvin J. Kuo, and Haian Fu. "Development of a miniaturized 3D organoid culture platform for ultra-high-throughput screening." Journal of Molecular Cell Biology 12, no. 8 (July 17, 2020): 630–43. http://dx.doi.org/10.1093/jmcb/mjaa036.
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Abstract The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery. However, organoid culturing with extracellular matrix to support 3D architecture has been challenging for high-throughput screening (HTS)-based drug discovery due to technical difficulties. Using genetically engineered human colon organoids as a model system, here we report our effort to miniaturize such 3D organoid culture with extracellular matrix support in high-density plates to enable HTS. We first established organoid culturing in a 384-well plate format and validated its application in a cell viability HTS assay by screening a 2036-compound library. We further miniaturized the 3D organoid culturing in a 1536-well ultra-HTS format and demonstrated its robust performance for large-scale primary compound screening. Our miniaturized organoid culturing method may be adapted to other types of organoids. By leveraging the power of 3D organoid culture in a high-density plate format, we provide a physiologically relevant screening platform to model tumors to accelerate organoid-based research and drug discovery.
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Matsumoto, Miki, Yuya Morimoto, Toshiro Sato, and Shoji Takeuchi. "Microfluidic Device to Manipulate 3D Human Epithelial Cell-Derived Intestinal Organoids." Micromachines 13, no. 12 (November 26, 2022): 2082. http://dx.doi.org/10.3390/mi13122082.
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In this study, we propose a microfluidic organoid-trapping device used to immobilize human intestinal organoids and apply fluidic stimuli to them. The proposed device has a microchannel with a trapping region with wall gaps between the channel walls and the bottom surface, and a constriction to clog the organoids in the channel. Since the introduced culture medium escapes from the gap, organoids can be cultured without excessive deformation by hydrostatic pressure. Owing to the characteristics of the organoid-trapping device, we succeeded in trapping human intestinal organoids in the channel. Furthermore, to demonstrate the applicability of the device for culturing intestinal organoids, we induced organoid fusion to form large organoids by aligning the organoids in the channel and applying fluidic shear stress to the organoids to regulate their surface structures. Therefore, we believe that organoid-trapping devices will be useful for investigating organoids aligned or loaded with fluidic stimulation.
3
Van Hemelryk, Annelies, Lisanne Mout, Sigrun Erkens-Schulze, Pim J. French, Wytske M. van Weerden, and Martin E. van Royen. "Modeling Prostate Cancer Treatment Responses in the Organoid Era: 3D Environment Impacts Drug Testing." Biomolecules 11, no. 11 (October 22, 2021): 1572. http://dx.doi.org/10.3390/biom11111572.
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Organoid-based studies have revolutionized in vitro preclinical research and hold great promise for the cancer research field, including prostate cancer (PCa). However, experimental variability in organoid drug testing complicates reproducibility. For example, we observed PCa organoids to be less affected by cabazitaxel, abiraterone and enzalutamide as compared to corresponding single cells prior to organoid assembly. We hypothesized that three-dimensional (3D) organoid organization and the use of various 3D scaffolds impact treatment efficacy. Live-cell imaging of androgen-induced androgen receptor (AR) nuclear translocation and taxane-induced tubulin stabilization was used to investigate the impact of 3D scaffolds, spatial organoid distribution and organoid size on treatment effect. Scaffolds delayed AR translocation and tubulin stabilization, with Matrigel causing a more pronounced delay than synthetic hydrogel as well as incomplete tubulin stabilization. Drug effect was further attenuated the more centrally organoids were located in the scaffold dome. Moreover, cells in the organoid core revealed a delayed treatment effect compared to cells in the organoid periphery, underscoring the impact of organoid size. These findings indicate that analysis of organoid drug responses needs careful interpretation and requires dedicated read-outs with consideration of underlying technical aspects.
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Jee, Joo Hyun, Dong Hyeon Lee, Jisu Ko, Soojung Hahn, Sang Yun Jeong, Han Kyung Kim, Enoch Park, et al. "Development of Collagen-Based 3D Matrix for Gastrointestinal Tract-Derived Organoid Culture." Stem Cells International 2019 (June 13, 2019): 1–15. http://dx.doi.org/10.1155/2019/8472712.
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Organoid is a cell organization grown in a three-dimensional (3D) culture system which represents all characteristics of its origin. However, this organ-like structure requires supporting matrix to maintain its characteristics and functions. Matrigel, derived from mouse sarcoma, has often been used as the supporting matrix for organoids, but the result may not be desirable for clinical applications because of the unidentified components from the mouse sarcoma. On the other hand, natural characteristics of collagen emphasize toxic-free friendly niche to both organoid and normal tissue. Hence, this study attempts to develop a new, collagen-based matrix that may substitute Matrigel in organoid culture. Collagen-based matrix was made, using type 1 collagen, Ham’s F12 nutrient mixture, and bicarbonate. Then, characteristics of mouse colon organoids were analyzed by morphology and quantitative messenger RNA (mRNA) expression, revealing that the mouse colon organoids grown in the collagen-based matrix and in Matrigel had quite similar morphology, specific markers, and proliferative rates. Mouse small intestine–derived organoids, stomach-derived organoids, and human colon–derived organoids were also cultured, all of which were successfully grown in the collagen-based matrix and had similar properties compared to those cultured in Matrigel. Furthermore, possibility of organoid transplantation was observed. When mouse colon organoids were transplanted with collagen matrix into the EDTA-colitis mouse model, colon organoids were successfully engrafted in damaged tissue. For that reason, the use of collagen-based matrix in organoid culture will render organoid cultivation less expensive and clinically applicable.
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Sibuea, Christine Verawaty, Jeanne Adiwinata Pawitan, and Radiana Antarianto. "Pengaruh Penggantian Medium terhadap Viabilitas Hepatosit Kultur 3D Organoid Hati." Nommensen Journal of Medicine 7, no. 2 (February 28, 2022): 39–42. http://dx.doi.org/10.36655/njm.v7i2.625.
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Background : Liver organoids can be used as materials for Bioartificial Liver, to study the mechanism of liver disease and as drug test toxicity. Reconstruction of liver organoids requires optimal culture methods, culture medium and cellular components to construct liver organoids that resemble liver microstructure in vivo with optimal function. 3D culture method using hepatocytes and stem cells with PRP supplemented William's E can reconstruct liver organoids with liver function. Medium exchange is an usual method to maintain the required nutrients and to eliminate waste products, but it requires a sufficient supply of medium and supplementation. Method and the use of effective and efficient medium with optimal hepatocyte viability are urgently needed in the reconstruction of liver organoids.
Objective : This study was aimed to compare the viability of primary hepatocytes in culture medium exchange liver organoids and monoculture and without culture medium exchange.
Methods : Primary hepatocytes isolated from Sprague Dawley-Rats mice (250gr, n=3) were co-cultured with umbilical cord mesenchymal stem cells, cord blood CD34+ stem cells and LX2 in PRP-supplemented William's E for 14 days. The culture medium was exchanged at 48 hours, day 7 and day 14 and no culture medium exchanged in the control group. Hepatocyte viability was analyzed using the Trypan Blue Exclusion Test at 48 hours, day 7 and day 14.
Results : Hepatocyte viability in culture medium exchange liver organoids was higher than without culture medium exchange, especially in monoculture, but there was no significant difference (p value> 0.05).
Conclusion: Hepatocyte viability in culture medium exchange liver organoids was not significantly different from no culture medium exchange liver organoids. Culture medium exchange in monoculture supported hepatocyte viability up to day 14.
Keywords: hepatocytes, liver organoids, viability, culture medium
ABSTRAK
Latar belakang : Organoid hati dapat digunakan sebagai bahan Bioartificial Liver, mempelajari mekanisme penyakit hati dan uji toksisitas obat. Rekonstruksi organoid hati membutuhkan metode kultur, medium kultur dan komponen seluler yang optimal untuk menghasilkan organoid hati yang menyerupai mikrostruktur hati in vivo dengan fungsi yang optimal. Metode kultur 3D menggunakan hepatosit dan sel punca mesenkimal dengan William’s E yang disuplementasi PRP dapat merekonstruksi organoid hati dengan fungsi hati. Pergantian medium merupakan metode yang sering dilakukan untuk mempertahankan nutrisi yang dibutuhkan dan untuk membuang sisa metabolit sel, tetapi membutuhkan persediaan medium dan suplementasi yang cukup banyak. Metode dan penggunaan medium yang efektif dan efisien dengan viabilitas hepatosit yang optimal sangat dibutuhkan dalam rekonstruksi organoid hati.
Tujuan : Penelitian ini bertujuan untuk mengetahui perbandingan viabilitas hepatosit primer pada organoid hati dengan pergantian medium kultur dan tanpa pergantian medium kultur.
Metode : Hepatosit primer yang diisolasi dari tikus Sprague Dawley-Rats (250gr, n=3) diko-kultur dengan sel punca mesenkimal asal tali pusat, sel punca CD34+ asal darah tali pusat dan LX2 dalam William’s E yang disuplementasi PRP selama 14 hari. Medium kultur diganti pada 48 jam, hari ke-7 dan hari ke-14 dan tidak dilakukan pergantian medium pada kelompok kontrol. Viabilitas hepatosit dianalisa dengan menggunakan Trypan Blue Exclusion Test pada 48 jam, hari ke-7 dan hari ke-14.
Hasil : Viabilitas hepatosit pada organoid hati dengan pergantian medium kultur tampak lebih banyak dibandingkan tanpa pergantian medium kultur khususnya pada monokultur, tetapi tidak terdapat perbedaan yang signifikan (nilai p>0,05).
Kesimpulan : Viabilitas hepatosit pada organoid hati dengan pergantian medium kultur tidak berbeda secara signifikan dengan organoid hati tanpa pergantian medium kultur. Pergantian medium kultur pada monokultur mendukung viabilitas hepatosit hingga hari ke-14.
Kata Kunci : Hepatosit, organoid hati, viabilitas, medium kultur
6
Li, Junzhi, Jing Chu, Vincent Chi Hang Lui, Shangsi Chen, Yan Chen, and Paul Kwong Hang Tam. "Bioengineering Liver Organoids for Diseases Modelling and Transplantation." Bioengineering 9, no. 12 (December 13, 2022): 796. http://dx.doi.org/10.3390/bioengineering9120796.
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Organoids as three-dimension (3D) cellular organizations partially mimic the physiological functions and micro-architecture of native tissues and organs, holding great potential for clinical applications. Advances in the identification of essential factors including physical cues and biochemical signals for controlling organoid development have contributed to the success of growing liver organoids from liver tissue and stem/progenitor cells. However, to recapitulate the physiological properties and the architecture of a native liver, one has to generate liver organoids that contain all the major liver cell types in correct proportions and relative 3D locations as found in a native liver. Recent advances in stem-cell-, biomaterial- and engineering-based approaches have been incorporated into conventional organoid culture methods to facilitate the development of a more sophisticated liver organoid culture resembling a near to native mini-liver in a dish. However, a comprehensive review on the recent advancement in the bioengineering liver organoid is still lacking. Here, we review the current liver organoid systems, focusing on the construction of the liver organoid system with various cell sources, the roles of growth factors for engineering liver organoids, as well as the recent advances in the bioengineering liver organoid disease models and their biomedical applications.
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Suhito, Intan Rosalina, and Tae-Hyung Kim. "Recent advances and challenges in organoid-on-a-chip technology." Organoid 2 (April 15, 2022): e4. http://dx.doi.org/10.51335/organoid.2022.2.e4.
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Conventional 2-dimensional cell culture poorly mimics human-relevant models, which is considered a major challenge in biological research. Organoids are a recent breakthrough in 3-dimensional (3D) in vitro tissue engineering that better reflect the physiological, morphological, and functional properties of in vivo organs (e.g., brain, heart, kidney, lung, and liver). Consequently, organoids are extensively used in various impactful biomedical applications including organ development, disease modeling, and clinical drug testing. However, organoid technology still has several limitations, including low reproducibility, vascularization, limited nutrient uptake and distribution (affecting the level of organoid maturation), lack of standardization, and intra-clonal variability. Efforts have been made to overcome these shortcomings of organoid culture. Microfluidic technology has successfully facilitated the establishment of organoid-on-a-chip systems, which effectively improve the structural and physiological features of organoids in a controlled manner. This review discusses the recent advances and developments in organoid-on-a-chip technology. We hope that this study will motivate researchers to explore the possible engagement between microfluidic devices and self-assembled 3D cell cultures to leverage the enhanced quality of organoids, which will have favorable impacts on future tissue regeneration and regenerative therapies.
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Chia, Shirley Pei Shan, Sharleen Li Ying Kong, Jeremy Kah Sheng Pang, and Boon-Seng Soh. "3D Human Organoids: The Next “Viral” Model for the Molecular Basis of Infectious Diseases." Biomedicines 10, no. 7 (June 28, 2022): 1541. http://dx.doi.org/10.3390/biomedicines10071541.
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The COVID-19 pandemic has driven the scientific community to adopt an efficient and reliable model that could keep up with the infectious disease arms race. Coinciding with the pandemic, three dimensional (3D) human organoids technology has also gained traction in the field of infectious disease. An in vitro construct that can closely resemble the in vivo organ, organoid technology could bridge the gap between the traditional two-dimensional (2D) cell culture and animal models. By harnessing the multi-lineage characteristic of the organoid that allows for the recapitulation of the organotypic structure and functions, 3D human organoids have emerged as an essential tool in the field of infectious disease research. In this review, we will be providing a comparison between conventional systems and organoid models. We will also be highlighting how organoids played a role in modelling common infectious diseases and molecular mechanisms behind the pathogenesis of causative agents. Additionally, we present the limitations associated with the current organoid models and innovative strategies that could resolve these shortcomings.
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Ren, Ya, Xue Yang, Zhengjiang Ma, Xin Sun, Yuxin Zhang, Wentao Li, Han Yang, et al. "Developments and Opportunities for 3D Bioprinted Organoids." International Journal of Bioprinting 7, no. 3 (June 28, 2021): 364. http://dx.doi.org/10.18063/ijb.v7i3.364.
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Organoids developed from pluripotent stem cells or adult stem cells are three-dimensional cell cultures possessing certain key characteristics of their organ counterparts, and they can mimic certain biological developmental processes of organs in vitro. Therefore, they have promising applications in drug screening, disease modeling, and regenerative repair of tissues and organs. However, the construction of organoids currently faces numerous challenges, such as breakthroughs in scale size, vascularization, better reproducibility, and precise architecture in time and space. Recently, the application of bioprinting has accelerated the process of organoid construction. In this review, we present current bioprinting techniques and the application of bioinks and summarize examples of successful organoid bioprinting. In the future, a multidisciplinary combination of developmental biology, disease pathology, cell biology, and materials science will aid in overcoming the obstacles pertaining to the bioprinting of organoids. The combination of bioprinting and organoids with a focus on structure and function can facilitate further development of real organs.
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Kiaee, Kiavash, Yasamin A. Jodat, Nicole J. Bassous, Navneet Matharu, and Su Ryon Shin. "Transcriptomic Mapping of Neural Diversity, Differentiation and Functional Trajectory in iPSC-Derived 3D Brain Organoid Models." Cells 10, no. 12 (December 5, 2021): 3422. http://dx.doi.org/10.3390/cells10123422.
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Experimental models of the central nervous system (CNS) are imperative for developmental and pathophysiological studies of neurological diseases. Among these models, three-dimensional (3D) induced pluripotent stem cell (iPSC)-derived brain organoid models have been successful in mitigating some of the drawbacks of 2D models; however, they are plagued by high organoid-to-organoid variability, making it difficult to compare specific gene regulatory pathways across 3D organoids with those of the native brain. Single-cell RNA sequencing (scRNA-seq) transcriptome datasets have recently emerged as powerful tools to perform integrative analyses and compare variability across organoids. However, transcriptome studies focusing on late-stage neural functionality development have been underexplored. Here, we combine and analyze 8 brain organoid transcriptome databases to study the correlation between differentiation protocols and their resulting cellular functionality across various 3D organoid and exogenous brain models. We utilize dimensionality reduction methods including principal component analysis (PCA) and uniform manifold approximation projection (UMAP) to identify and visualize cellular diversity among 3D models and subsequently use gene set enrichment analysis (GSEA) and developmental trajectory inference to quantify neuronal behaviors such as axon guidance, synapse transmission and action potential. We showed high similarity in cellular composition, cellular differentiation pathways and expression of functional genes in human brain organoids during induction and differentiation phases, i.e., up to 3 months in culture. However, during the maturation phase, i.e., 6-month timepoint, we observed significant developmental deficits and depletion of neuronal and astrocytes functional genes as indicated by our GSEA results. Our results caution against use of organoids to model pathophysiology and drug response at this advanced time point and provide insights to tune in vitro iPSC differentiation protocols to achieve desired neuronal functionality and improve current protocols.
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Amatngalim, Gimano D., Lisa W. Rodenburg, Bente L. Aalbers, Henriette HM Raeven, Ellen M. Aarts, Dounia Sarhane, Sacha Spelier, et al. "Measuring cystic fibrosis drug responses in organoids derived from 2D differentiated nasal epithelia." Life Science Alliance 5, no. 12 (August 3, 2022): e202101320. http://dx.doi.org/10.26508/lsa.202101320.
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Cystic fibrosis is caused by genetic defects that impair the CFTR channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing–derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described CFTR function assays in 3D airway organoids were not fully optimal, because of inefficient organoid differentiation and limited scalability. In this report, we therefore describe an alternative method of culturing nasal-brushing–derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air–liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1β and interleukin-1β, which enabled consistent detection of CFTR modulator responses in nasal-airway organoid cultures from subjects with cystic fibrosis.
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Maier, Christopher Fabian, Lei Zhu, Lahiri Kanth Nanduri, Daniel Kühn, Susan Kochall, May-Linn Thepkaysone, Doreen William, et al. "Patient-Derived Organoids of Cholangiocarcinoma." International Journal of Molecular Sciences 22, no. 16 (August 12, 2021): 8675. http://dx.doi.org/10.3390/ijms22168675.
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Cholangiocarcinoma (CC) is an aggressive malignancy with an inferior prognosis due to limited systemic treatment options. As preclinical models such as CC cell lines are extremely rare, this manuscript reports a protocol of cholangiocarcinoma patient-derived organoid culture as well as a protocol for the transition of 3D organoid lines to 2D cell lines. Tissue samples of non-cancer bile duct and cholangiocarcinoma were obtained during surgical resection. Organoid lines were generated following a standardized protocol. 2D cell lines were generated from established organoid lines following a novel protocol. Subcutaneous and orthotopic patient-derived xenografts were generated from CC organoid lines, histologically examined, and treated using standard CC protocols. Therapeutic responses of organoids and 2D cell lines were examined using standard CC agents. Next-generation exome and RNA sequencing was performed on primary tumors and CC organoid lines. Patient-derived organoids closely recapitulated the original features of the primary tumors on multiple levels. Treatment experiments demonstrated that patient-derived organoids of cholangiocarcinoma and organoid-derived xenografts can be used for the evaluation of novel treatments and may therefore be used in personalized oncology approaches. In summary, this study establishes cholangiocarcinoma organoids and organoid-derived cell lines, thus expanding translational research resources of cholangiocarcinoma.
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Jackson, Tim R., Miniver Oliver, Daniel Appledorn, Tim Dale, and Kalpana Barnes. "Abstract 3084: Label-free, real-time live cell assays for 3D organoids embedded in Matrigel®." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3084. http://dx.doi.org/10.1158/1538-7445.am2022-3084.
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Abstract Organoid technologies are increasingly being used as in-vitro models of human development and disease as they exhibit structural, morphogenetic, and functional properties that recapitulate in vivo pathophysiology. To successfully use these models across a variety of research disciplines and applications, approaches that reduce variability and technology pipelines to image & quantify these complex cell models are required. Here, we demonstrate simple, robust workflows for monitoring and automatically quantifying features, such as morphology, growth and death of organoids using real time live cell analysis. To quantitatively optimize and characterize organoid cultures in-vitro, mouse hepatic, intestinal and pancreatic organoids were embedded in Matrigel® domes (50% or 100%) in 24-well plates and imaged over time in an Incucyte® Live Cell System. Organoid growth, differentiation, and maturation was measured using Incucyte’ s automated Organoid Software Analysis Module, which tracks changes in size and morphology. Integrated metrics enabled objective determination of cell-type specific growth conditions and passaging regimes. To illustrate the utility of the Incucyte® Organoid Analysis Software Module to track organoid growth and death in 96-well plates, intestinal and hepatic organoid fragments were embedded in Matrigel® (50%) for 3 days prior to treatment with protein kinase inhibitor staurosporine (1 µM, STP). Vehicle treated organoids increased in size (10-fold; intestinal or 3-fold; hepatic) over time while marked reduction was observed in the presence of STP. Using label-free size and morphology metrics we could distinguish between cytotoxic and cytostatic mechanisms of action (MoA) of known chemotherapeutic compounds. STP, cisplatin (CIS, DNA synthesis inhibitor) or fluorouracil (5-FU, thymidylate synthetase inhibitor) exhibited concentration dependent inhibition of hepatic organoid growth, yielding IC50 values of 3 nM for STP, 9.7 µM for CIS and 0.78 µM for 5-FU. Whilst attenuation of size was observed across all compounds, increases in eccentricity and darkness indictive of 3D structure disruption and cell death respectively were only observed in CIS and STP-treated organoids (cytotoxic MoA). Differences between the size and morphology readouts illustrated the cytostatic mechanism of 5-FU. Use of this approach was extended to visualize and quantify CFTR function. Following forskolin stimulation, a concentration-dependent increase in intestinal organoid size was observed. In the presence of CFTR inhibitor CFTRinh-172 the maximal response was reduced by >50% (~150% at 10 µM) illustrating that swelling was CFTR-dependent. These data demonstrate the capability to kinetically visualize and quantify distinct organoid morphologies, assess drug-induced cellular changes label-free and illustrates the amenability of this approach across a range of disease areas. Citation Format: Tim R. Jackson, Miniver Oliver, Daniel Appledorn, Tim Dale, Kalpana Barnes. Label-free, real-time live cell assays for 3D organoids embedded in Matrigel® [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3084.
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Sundar, Swetha, Sajina Shakya, Lisa Wallace, Austin Barnett, Andrew Sloan, Violette Recinos, and Christopher Hubert. "TAMI-16. THREE-DIMENSIONAL ORGANOID CULTURE UNVEILS RESISTANCE TO CLINICAL THERAPIES IN ADULT AND PEDIATRIC GLIOBLASTOMA." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi201. http://dx.doi.org/10.1093/neuonc/noab196.800.
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Abstract BACKGROUND Glioblastoma (GBM) is the most common primary brain tumor with a dismal prognosis. The inherent cellular diversity and interactions within tumor microenvironments represent a significant challenge to effective treatment. Traditional culture methods may mask the complexity of such interactions while three-dimensional (3D) organoid culture systems derived from patient cancer stem cells (CSCs) can preserve cellular complexity and microenvironments. Our objective was to determine whether organoid cultures show increased patterns of resistance to potential clinical therapies compared to traditional sphere cultures. METHODS Adult and pediatric surgical specimens were collected and established as 3D organoids. We created organoid microarrays and visualized bulk and spatially defined differences in cell proliferation using immunohistochemistry (IHC) staining, as well as cell cycle analysis by flow cytometry with 3D regional labeling. We tested the response of CSCs grown in each culture method to temozolomide, ibrutinib, lomustine, ruxolitinib, and radiotherapy using proliferative and viability assays. RESULTS Compared to sphere cultures from the same patient, organoids showed diverse proliferative cell populations and broad resistance to all therapies tested, albeit with both intraspecimen and interspecimen variability in the extent of resistance. Organoid specimens demonstrated a blunt response to current GBM standard of care therapy (combination temozolomide and radiotherapy) and maintained both cellular proliferation in their outer rim and overall structure and viability compared to the matched sphere specimens. CONCLUSIONS Our results suggest that growth of tumor specimens as organoid cultures may better reflect the cellular diversity and clinical reality of GBM therapeutic response. Patient-derived GBM organoids offer a valuable complement to traditional culture methods and may have powerful predictive capability of personalized drug sensitivities and therapeutic resistance.
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Ding, Beichen, Guoliang Sun, Shiliang Liu, Ejun Peng, Meimei Wan, Liang Chen, John Jackson, and Anthony Atala. "Three-Dimensional Renal Organoids from Whole Kidney Cells: Generation, Optimization, and Potential Application in Nephrotoxicology In Vitro." Cell Transplantation 29 (January 1, 2020): 096368971989706. http://dx.doi.org/10.1177/0963689719897066.
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The kidney function of patients with chronic kidney disease (CKD) is impaired irreversibly. Organ transplantation is the only treatment to restore kidney function in CKD patients. The assessment of new potential therapeutic procedures relies heavily on experimental animal models, but it is limited by its human predictive capacity. In addition, the frequently used two-dimensional in vitro human renal cell models cannot replicate all the features of the in vivo situation. In this study, we developed a three-dimensional (3D) in vitro human renal organoid model from whole kidney cells as a promising drug screening tool. At present, the renal tissue generated from human pluripotent stem cells (hPSCs) exhibits intrinsic tumorigenicity properties. Here we first developed a 3D renal organoid culture system that originated from adult differentiated cells without gene modification. Renal organoids composed of multiple cell types were created under optimal experimental conditions and evaluated for morphology, viability and erythropoietin production. As a novel screening tool for renal toxicity, 3D organoids were exposed to three widely used drugs: aspirin, penicillin G and cisplatin. The study results showed this 3D renal organoid model can be used as a drug screening tool, a new in vitro 3D human kidney model, and provide hope for potential regenerative therapies for CKD.
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Salmon, Idris, Sergei Grebenyuk, Abdel Rahman Abdel Fattah, Gregorius Rustandi, Thomas Pilkington, Catherine Verfaillie, and Adrian Ranga. "Engineering neurovascular organoids with 3D printed microfluidic chips." Lab on a Chip 22, no. 8 (2022): 1615–29. http://dx.doi.org/10.1039/d1lc00535a.
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Organoid vascularization using an open-well microfluidic chip allows for interaction of organoids with vasculature in a temporally and spatially controllable manner. Chip are printed using a consumer-grade 3D printer, making the approach inexpensive and widely accessible.
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Takano, Atsushi, Isabel Koh, and Masaya Hagiwara. "3D Culture Platform for Enabling Large-Scale Imaging and Control of Cell Distribution into Complex Shapes by Combining 3D Printing with a Cube Device." Micromachines 13, no. 2 (January 21, 2022): 156. http://dx.doi.org/10.3390/mi13020156.
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While organoid differentiation protocols have been widely developed, local control of initial cell seeding position and imaging of large-scale organoid samples with high resolution remain challenging. 3D bioprinting is an effective method to achieve control of cell positioning, but existing methods mainly rely on the use of synthetic hydrogels that could compromise the native morphogenesis of organoids. To address this problem, we developed a 3D culture platform that combines 3D printing with a cube device to enable an unrestricted range of designs to be formed in biological hydrogels. We demonstrated the formation of channels in collagen hydrogel in the cube device via a molding process using a 3D-printed water-soluble mold. The mold is first placed in uncured hydrogel solution, then easily removed by immersion in water after the gel around it has cured, thus creating a mold-shaped gap in the hydrogel. At the same time, the difficulty in obtaining high-resolution imaging on a large scale can also be solved as the cube device allows us to scan the tissue sample from multiple directions, so that the imaging quality can be enhanced without having to rely on higher-end microscopes. Using this developed technology, we demonstrated (1) mimicking vascular structure by seeding HUVEC on the inner walls of helix-shaped channels in collagen gels, and (2) multi-directional imaging of the vascular structure in the cube device. Thus, this paper describes a concerted method that simultaneously allows for the precise control of cell positioning in hydrogels for organoid morphogenesis, and the imaging of large-sized organoid samples. It is expected that the platform developed here can lead to advancements in organoid technology to generate organoids with more sophisticated structures.
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Pan, Fong Cheng, Todd Evans, and Shuibing Chen. "Modeling endodermal organ development and diseases using human pluripotent stem cell-derived organoids." Journal of Molecular Cell Biology 12, no. 8 (July 11, 2020): 580–92. http://dx.doi.org/10.1093/jmcb/mjaa031.
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Abstract Recent advances in development of protocols for directed differentiation from human pluripotent stem cells (hPSCs) to defined lineages, in combination with 3D organoid technology, have facilitated the generation of various endoderm-derived organoids for in vitro modeling of human gastrointestinal development and associated diseases. In this review, we discuss current state-of-the-art strategies for generating hPSC-derived endodermal organoids including stomach, liver, pancreatic, small intestine, and colonic organoids. We also review the advantages of using this system to model various human diseases and evaluate the shortcomings of this technology. Finally, we emphasize how other technologies, such as genome editing and bioengineering, can be incorporated into the 3D hPSC-organoid models to generate even more robust and powerful platforms for understanding human organ development and disease modeling.
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Kulkeaw, Kasem, Alisa Tubsuwan, Nongnat Tongkrajang, and Narisara Whangviboonkij. "Generation of human liver organoids from pluripotent stem cell-derived hepatic endoderms." PeerJ 8 (October 19, 2020): e9968. http://dx.doi.org/10.7717/peerj.9968.
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Background The use of a personalized liver organoid derived from human-induced pluripotent stem cells (HuiPSCs) is advancing the use of in vitro disease models for the design of specific, effective therapies for individuals. Collecting patient peripheral blood cells for HuiPSC generation is preferable because it is less invasive; however, the capability of blood cell-derived HuiPSCs for hepatic differentiation and liver organoid formation remains uncertain. Moreover, the currently available methods for liver organoid formation require a multistep process of cell differentiation or a combination of hepatic endodermal, endothelial and mesenchymal cells, which is a major hurdle for the application of personalized liver organoids in high-throughput testing of drug toxicity and safety. To demonstrate the capability of blood cell-derived HuiPSCs for liver organoid formation without support from endothelial and mesenchymal cells. Methods The peripheral blood-derived HuiPSCs first differentiated into hepatic endoderm (HE) in two-dimensional (2D) culture on Matrigel-coated plates under hypoxia for 10 days. The HE was then collected and cultured in 3D culture using 50% Matrigel under ambient oxygen. The maturation of hepatocytes was further induced by adding hepatocyte growth medium containing HGF and oncostatin M on top of the 3D culture and incubating the culture for an additional 12–17 days. The function of the liver organoids was assessed using expression analysis of hepatocyte-specific gene and proteins. Albumin (ALB) synthesis, glycogen and lipid storage, and metabolism of indocyanine were evaluated. The spatial distribution of albumin was examined using immunofluorescence and confocal microscopy. Results CD34+ hematopoietic cell-derived HuiPSCs were capable of differentiating into definitive endoderm expressing SOX17 and FOXA2, hepatic endoderm expressing FOXA2, hepatoblasts expressing AFP and hepatocytes expressing ALB. On day 25 of the 2D culture, cells expressed SOX17, FOXA2, AFP and ALB, indicating the presence of cellular heterogeneity. In contrast, the hepatic endoderm spontaneously formed a spherical, hollow structure in a 3D culture of 50% Matrigel, whereas hepatoblasts and hepatocytes could not form. Microscopic observation showed a single layer of polygonal-shaped cells arranged in a 3D structure. The hepatic endoderm-derived organoid synthesis ALB at a higher level than the 2D culture but did not express definitive endoderm-specific SOX17, indicating the greater maturity of the hepatocytes in the liver organoids. Confocal microscopic images and quantitative ELISA confirmed albumin synthesis in the cytoplasm of the liver organoid and its secretion. Overall, 3D culture of the hepatic endoderm is a relatively fast, simple, and less laborious way to generate liver organoids from HuiPSCs that is more physiologically relevant than 2D culture.
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Gu, Shutong, Li Chen, and Shichang Zhang. "Current Advances in Intestinal Organoids: Methods and Scaffolds." Journal of Biomaterials and Tissue Engineering 9, no. 11 (November 1, 2019): 1477–84. http://dx.doi.org/10.1166/jbt.2019.2179.
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Intestinal organoids are three-dimensional (3D) cultures with a long history of being studied in vivo. They constitute a model that has been widely applied to many aspects of bioengineering and regenerative medicine. Recently, increasing demand has created an urgent need for more efficient approaches to organoid culture. Here, we present an overview of some vital factors that may affect organoid formation and maturation and explain some currently available advanced methods using pluripotent stem cells (PSCs) and scaffolding to produce intestinal organoids that best resemble the organ of origin. These methods are compared with traditional two-dimensional (2D) culturing methods. In addition, we note some remaining challenges and explore other potential factors that may help improve 3D culture technology in the future.
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Gassl, Vincent, Merel R. Aberle, Bas Boonen, Rianne D. W. Vaes, Steven W. M. Olde Damink, and Sander S. Rensen. "Chemosensitivity of 3D Pancreatic Cancer Organoids Is Not Affected by Transformation to 2D Culture or Switch to Physiological Culture Medium." Cancers 14, no. 22 (November 16, 2022): 5617. http://dx.doi.org/10.3390/cancers14225617.
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Organoids are increasingly used to investigate patient-specific drug responsiveness, but organoid culture is complex and expensive, and carried out in rich, non-physiological media. We investigated reproducibility of drug-responsiveness of primary cell cultures in 2D versus 3D and in conventional versus physiological cell culture medium. 3D pancreatic ductal adenocarcinoma organoid cultures PANCO09b and PANCO11b were converted to primary cell cultures growing in 2D. Transformed 2D cultures were grown in physiological Plasmax medium or Advanced-DMEM/F12. Sensitivity towards gemcitabine, paclitaxel, SN-38, 5-fluorouacil, and oxaliplatin was investigated by cell viability assays. Growth rates of corresponding 2D and 3D cultures were comparable. PANCO09b had a shorter doubling time in physiological media. Chemosensitivity of PANCO09b and PANCO11b grown in 2D or 3D was similar, except for SN-38, to which PANCO11b cultured in 3D was more sensitive (2D: 8.2 ×10−3 ± 2.3 ×10−3 vs. 3D: 1.1 ×10−3 ± 0.6 ×10−3, p = 0.027). PANCO09b and PANCO11b showed no major differences in chemosensitivity when cultured in physiological compared to conventional media, although PANCO11b was more sensitive to SN-38 in physiological media (9.8 × 10−3 ± 0.7 × 10−3 vs. 5.2 × 10−3 ± 1.8 × 10−3, p = 0.015). Collectively, these data indicate that the chemosensitivity of organoids is not affected by culture medium composition or culture dimensions. This implies that organoid-based drug screens can be simplified to become more cost-effective.
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Depla, Josse A., Lance A. Mulder, Renata Vieira de Sá, Morgane Wartel, Adithya Sridhar, Melvin M. Evers, Katja C. Wolthers, and Dasja Pajkrt. "Human Brain Organoids as Models for Central Nervous System Viral Infection." Viruses 14, no. 3 (March 18, 2022): 634. http://dx.doi.org/10.3390/v14030634.
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Pathogenesis of viral infections of the central nervous system (CNS) is poorly understood, and this is partly due to the limitations of currently used preclinical models. Brain organoid models can overcome some of these limitations, as they are generated from human derived stem cells, differentiated in three dimensions (3D), and can mimic human neurodevelopmental characteristics. Therefore, brain organoids have been increasingly used as brain models in research on various viruses, such as Zika virus, severe acute respiratory syndrome coronavirus 2, human cytomegalovirus, and herpes simplex virus. Brain organoids allow for the study of viral tropism, the effect of infection on organoid function, size, and cytoarchitecture, as well as innate immune response; therefore, they provide valuable insight into the pathogenesis of neurotropic viral infections and testing of antivirals in a physiological model. In this review, we summarize the results of studies on viral CNS infection in brain organoids, and we demonstrate the broad application and benefits of using a human 3D model in virology research. At the same time, we describe the limitations of the studies in brain organoids, such as the heterogeneity in organoid generation protocols and age at infection, which result in differences in results between studies, as well as the lack of microglia and a blood brain barrier.
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Martins, Jorge Miguel Faustino, Lan Vi Ngoc Nguyen, and Mina Gouti. "Humane neuromuskuläre Organoide — Anwendung und Perspektive." BIOspektrum 27, no. 2 (March 2021): 135–38. http://dx.doi.org/10.1007/s12268-021-1553-0.
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AbstractOrganoids are miniature, organ-like structures derived from stem cells. While techniques for developing organoids for different tissues have advanced in the past decade, it has remained a challenge to simultaneously grow two different tissues into a single functional organoid. We have recently developed a 3D neuromuscular organoid that allows the simultaneous generation of spinal neurons and skeletal muscles. Here we discuss their future applications in modeling neuromuscular disorders.
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Sanders, Karin, Femke C. A. Ringnalda, Marc L. van de Wetering, Hans S. Kooistra, Björn P. Meij, Hans Clevers, and Sara Galac. "Canine Pituitary Organoids as 3D In Vitro Model for Cushing Disease." Journal of the Endocrine Society 5, Supplement_1 (May 1, 2021): A533. http://dx.doi.org/10.1210/jendso/bvab048.1085.
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Abstract Cushing disease (CD) is a serious endocrine disorder that is most often caused by an ACTH-secreting pituitary adenoma. Patients can be treated medically when surgery is not an option or was unsuccessful. However, currently used pituitary-targeting drugs are effective in only 40% of patients. To efficiently identify new pituitary-targeting treatment options, we need an in vitro system that closely mimics in vivo conditions. We therefore aimed to establish organoid cultures of normal anterior pituitary and corticotroph adenomas. Organoids or tumoroids are miniature three-dimensional (3D) structures grown from stem cells, that closely resemble the organ or tumor they originate from. Because CD is a thousand times more prevalent in dogs than in humans, and hypophysectomy is the treatment of choice, we used canine tissues. Normal anterior pituitary glands were collected from three healthy dogs that were euthanized for reasons unrelated to the current study. Corticotroph adenomas were collected from six dogs that underwent transsphenoidal hypophysectomy at our University Clinic. The dogs were diagnosed with CD based on clinical signs, endocrine testing, and CT scan imaging. Normal anterior pituitary and corticotroph adenoma cells were cultured in a 3D matrix (basement membrane extract) with anterior pituitary organoid medium containing specific growth factors and ligands, which was refreshed twice a week. The organoids and tumoroids were characterized with histopathology and RT-qPCR. Structures resembling organoids or tumoroids grew from all nine samples (3 normal, 6 adenoma) that were put in culture. Both cystic and dense structures were observed. The organoids and tumoroids expanded rapidly, and could be passaged once every week. The organoids and tumoroids were successfully cultured up until passage number 10, and were then frozen down. Histopathology showed that the organoid or tumoroid cells morphologically resembled healthy anterior pituitary or corticotroph adenoma cells. All organoids cultures expressed mRNA of pituitary stem cell markers SOX2 and SOX9. This study shows that corticotroph adenomas can be cultured as tumoroids in vitro, something not previously published in any species. Based on the many opportunities in organoid culture (e.g., high-throughput drug screenings, gene editing, studying developmental processes), we expect that this in vitro model will pave the way to efficiently and reliably identify new treatment options for CD. Not only for humans, but also for our best friends: dogs.
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Chambers, Brooke E., Nicole E. Weaver, and Rebecca A. Wingert. "The “3Ds” of Growing Kidney Organoids: Advances in Nephron Development, Disease Modeling, and Drug Screening." Cells 12, no. 4 (February 8, 2023): 549. http://dx.doi.org/10.3390/cells12040549.
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A kidney organoid is a three-dimensional (3D) cellular aggregate grown from stem cells in vitro that undergoes self-organization, recapitulating aspects of normal renal development to produce nephron structures that resemble the native kidney organ. These miniature kidney-like structures can also be derived from primary patient cells and thus provide simplified context to observe how mutations in kidney-disease-associated genes affect organogenesis and physiological function. In the past several years, advances in kidney organoid technologies have achieved the formation of renal organoids with enhanced numbers of specialized cell types, less heterogeneity, and more architectural complexity. Microfluidic bioreactor culture devices, single-cell transcriptomics, and bioinformatic analyses have accelerated the development of more sophisticated renal organoids and tailored them to become increasingly amenable to high-throughput experimentation. However, many significant challenges remain in realizing the use of kidney organoids for renal replacement therapies. This review presents an overview of the renal organoid field and selected highlights of recent cutting-edge kidney organoid research with a focus on embryonic development, modeling renal disease, and personalized drug screening.
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Qu, Molong, Liang Xiong, Yulin Lyu, Xiannian Zhang, Jie Shen, Jingyang Guan, Peiyuan Chai, et al. "Establishment of intestinal organoid cultures modeling injury-associated epithelial regeneration." Cell Research 31, no. 3 (January 8, 2021): 259–71. http://dx.doi.org/10.1038/s41422-020-00453-x.
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AbstractThe capacity of 3D organoids to mimic physiological tissue organization and functionality has provided an invaluable tool to model development and disease in vitro. However, conventional organoid cultures primarily represent the homeostasis of self-organizing stem cells and their derivatives. Here, we established a novel intestinal organoid culture system composed of 8 components, mainly including VPA, EPZ6438, LDN193189, and R-Spondin 1 conditioned medium, which mimics the gut epithelium regeneration that produces hyperplastic crypts following injury; therefore, these organoids were designated hyperplastic intestinal organoids (Hyper-organoids). Single-cell RNA sequencing identified different regenerative stem cell populations in our Hyper-organoids that shared molecular features with in vivo injury-responsive Lgr5+ stem cells or Clu+ revival stem cells. Further analysis revealed that VPA and EPZ6438 were indispensable for epigenome reprogramming and regeneration in Hyper-organoids, which functioned through epigenetically regulating YAP signaling. Furthermore, VPA and EPZ6438 synergistically promoted regenerative response in gut upon damage in vivo. In summary, our results demonstrated a new in vitro organoid model to study epithelial regeneration, highlighting the importance of epigenetic reprogramming that pioneers tissue repair.
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Tejchman, Anna, Agnieszka Znój, Paula Chlebanowska, Aneta Frączek-Szczypta, and Marcin Majka. "Carbon Fibers as a New Type of Scaffold for Midbrain Organoid Development." International Journal of Molecular Sciences 21, no. 17 (August 19, 2020): 5959. http://dx.doi.org/10.3390/ijms21175959.
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The combination of induced pluripotent stem cell (iPSC) technology and 3D cell culture creates a unique possibility for the generation of organoids that mimic human organs in in vitro cultures. The use of iPS cells in organoid cultures enables the differentiation of cells into dopaminergic neurons, also found in the human midbrain. However, long-lasting organoid cultures often cause necrosis within organoids. In this work, we present carbon fibers (CFs) for medical use as a new type of scaffold for organoid culture, comparing them to a previously tested copolymer poly-(lactic-co-glycolic acid) (PLGA) scaffold. We verified the physicochemical properties of CF scaffolds compared to PLGA in improving the efficiency of iPSC differentiation within organoids. The physicochemical properties of carbon scaffolds such as porosity, microstructure, or stability in the cellular environment make them a convenient material for creating in vitro organoid models. Through screening several genes expressed during the differentiation of organoids at crucial brain stages of development, we found that there is a correlation between PITX3, one of the key regulators of terminal differentiation, and the survival of midbrain dopaminergic (mDA) neurons and tyrosine hydroxylase (TH) gene expression. This makes organoids formed on carbon scaffolds an improved model containing mDA neurons convenient for studying midbrain-associated neurodegenerative diseases such as Parkinson’s disease.
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Richiardone, Elena, Valentin Van den Bossche, and Cyril Corbet. "Metabolic Studies in Organoids: Current Applications, Opportunities and Challenges." Organoids 1, no. 1 (June 13, 2022): 85–105. http://dx.doi.org/10.3390/organoids1010008.
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Organoid technologies represent a major breakthrough in biomedical research since they offer increasingly sophisticated models for studying biological mechanisms supporting human development and disease. Organoids are three-dimensional (3D) physiological in vitro systems that recapitulate the genetic, histological and functional features of the in vivo tissues of origin more accurately than classical cell culture methods. In the last decade, organoids have been derived from various healthy and diseased tissues and used for a wide range of applications in basic and translational research, including (cancer) tissue biology, development, regeneration, disease modeling, precision medicine, gene editing, biobanking and drug screening. Here, we report the current applications of organoid models to study (stem) cell metabolism in several pathophysiological contexts such as cancer and metabolic diseases. More precisely, we discuss the relevance and limitations of these 3D cultures to model and study metabolic (dys)functions associated with hepatic, renal or pancreatic disorders, as well as tumor development and progression. We also describe the use of organoids to understand the dynamic interaction between diet, microbiota and the intestinal epithelium. Finally, this review explores recent methodological improvements in organoid culture that may help to better integrate the influence of microenvironmental conditions in the study of tumor cell metabolic phenotypes.
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Mäkinen, Lotta, Markus Vähä-Koskela, Matilda Juusola, Harri Mustonen, Krister Wennerberg, Jaana Hagström, Pauli Puolakkainen, and Hanna Seppänen. "Pancreatic Cancer Organoids in the Field of Precision Medicine: A Review of Literature and Experience on Drug Sensitivity Testing with Multiple Readouts and Synergy Scoring." Cancers 14, no. 3 (January 21, 2022): 525. http://dx.doi.org/10.3390/cancers14030525.
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Pancreatic ductal adenocarcinoma (PDAC) is a silent killer, often diagnosed late. However, it is also dishearteningly resistant to nearly all forms of treatment. New therapies are urgently needed, and with the advent of organoid culture for pancreatic cancer, an increasing number of innovative approaches are being tested. Organoids can be derived within a short enough time window to allow testing of several anticancer agents, which opens up the possibility for functional precision medicine for pancreatic cancer. At the same time, organoid model systems are being refined to better mimic the cancer, for example, by incorporation of components of the tumor microenvironment. We review some of the latest developments in pancreatic cancer organoid research and in novel treatment design. We also summarize our own current experiences with pancreatic cancer organoid drug sensitivity and resistance testing (DSRT) in 14 organoids from 11 PDAC patients. Our data show that it may be necessary to include a cell death read-out in ex vivo DSRT assays, as metabolic viability quantitation does not capture actual organoid killing. We also successfully adapted the organoid platform for drug combination synergy discovery. Lastly, live organoid culture 3D confocal microscopy can help identify individual surviving tumor cells escaping cell death even during harsh combination treatments. Taken together, the organoid technology allows the development of novel precision medicine approaches for PDAC, which paves the way for clinical trials and much needed new treatment options for pancreatic cancer patients.
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Kang, Suwon, Eun Kyung Bong, Hyo-Min Kim, and Tae-Young Roh. "Technical advances in pluripotent stem cell-derived and tumorigenic organoids." Organoid 2 (August 18, 2022): e18. http://dx.doi.org/10.51335/organoid.2022.2.e18.
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Cell culture systems have been widely used to address fundamental questions in biology without sacrificing animals. Three-dimensional (3D) organoids provide more information on in vivo conditions than traditional culture systems because multiple cell types in organoids interact with each other in 3D structures. Despite extensive research and advances in the organoid field, some important limitations remain and need further consideration. In this review, we summarize how organoids are generated from pluripotent stem cells and describe the recent technical progress that has made organoids more similar to in vivo tissues for the application of organoids to modeling cancer. Lastly, we briefly discuss some limitations that have been raised in this field.
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Camp, J. Gray, Farhath Badsha, Marta Florio, Sabina Kanton, Tobias Gerber, Michaela Wilsch-Bräuninger, Eric Lewitus, et al. "Human cerebral organoids recapitulate gene expression programs of fetal neocortex development." Proceedings of the National Academy of Sciences 112, no. 51 (December 7, 2015): 15672–77. http://dx.doi.org/10.1073/pnas.1520760112.
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Cerebral organoids—3D cultures of human cerebral tissue derived from pluripotent stem cells—have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.
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Chlebanowska, Paula, Anna Tejchman, Maciej Sułkowski, Klaudia Skrzypek, and Marcin Majka. "Use of 3D Organoids as a Model to Study Idiopathic Form of Parkinson’s Disease." International Journal of Molecular Sciences 21, no. 3 (January 21, 2020): 694. http://dx.doi.org/10.3390/ijms21030694.
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Organoids are becoming particularly popular in modeling diseases that are difficult to reproduce in animals, due to anatomical differences in the structure of a given organ. Thus, they are a bridge between the in vitro and in vivo models. Human midbrain is one of the structures that is currently being intensively reproduced in organoids for modeling Parkinson’s disease (PD). Thanks to three-dimensional (3D) architecture and the use of induced pluripotent stem cells (iPSCs) differentiation into organoids, it has been possible to recapitulate a complicated network of dopaminergic neurons. In this work, we present the first organoid model for an idiopathic form of PD. iPSCs were generated from peripheral blood mononuclear cells of healthy volunteers and patients with the idiopathic form of PD by transduction with Sendai viral vector. iPSCs were differentiated into a large multicellular organoid-like structure. The mature organoids displayed expression of neuronal early and late markers. Interestingly, we observed statistical differences in the expression levels of LIM homeobox transcription factor alpha (early) and tyrosine hydroxylase (late) markers between organoids from PD patient and healthy volunteer. The obtained results show immense potential for the application of 3D human organoids in studying the neurodegenerative disease and modeling cellular interactions within the human brain.
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Chu, J., O. Pieles, CG Pfeifer, V. Alt, C. Morsczeck, and D. Docheva. "Dental follicle cell differentiation towards periodontal ligament-like tissue in a self-assembly three-dimensional organoid model." European Cells and Materials 42 (July 12, 2021): 20–33. http://dx.doi.org/10.22203/ecm.v042a02.
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Periodontitis remains an unsolved oral disease, prevalent worldwide and resulting in tooth loss due to dysfunction of the periodontal ligament (PDL), a tissue connecting the tooth root with the alveolar bone. A scaffold-free three-dimensional (3D) organoid model for in vitro tenogenesis/ligamentogeneis has already been described. As PDL tissue naturally arises from the dental follicle, the aim of this study was to investigate the ligamentogenic differentiation potential of dental follicle cells (DFCs) in vitro by employing this 3D model. Human primary DFCs were compared, in both two- and three-dimensions, to a previously published PDL- hTERT cell line. The 3D organoids were evaluated by haematoxylin and eosin, 4′,6-diamidino-2-phenylindole and F-actin staining combined with detailed histomorphometric analyses of cell-row structure, angular deviation and cell density. Furthermore, the expression of 48 tendon/ligament- and multilineage-related genes was evaluated using quantitative polymerase chain reaction, followed by immunofluorescent analyses of collagen 1 and 3. The results showed that both cell types were successful in the formation of scaffold-free 3D organoids. DFC organoids were comparable to PDL-hTERT in terms of cell density; however, DFCs exhibited superior organoid morphology, cell-row organisation (p < 0.0001) and angular deviation (p < 0.0001). Interestingly, in 2 dimensions as well as in 3D, DFCs showed significantly higher levels of several ligament- related genes compared to the PDL-hTERT cell line. In conclusion, DFCs exhibited great potential to form PDL-like 3D organoids in vitro suggesting that this strategy can be further developed for functional PDL engineering.
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Kim, Seungil, Sarah Choung, Ren X. Sun, Nolan Ung, Natasha Hashemi, Emma J. Fong, Roy Lau, et al. "Comparison of Cell and Organoid-Level Analysis of Patient-Derived 3D Organoids to Evaluate Tumor Cell Growth Dynamics and Drug Response." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 7 (April 30, 2020): 744–54. http://dx.doi.org/10.1177/2472555220915827.
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3D cell culture models have been developed to better mimic the physiological environments that exist in human diseases. As such, these models are advantageous over traditional 2D cultures for screening drug compounds. However, the practicalities of transitioning from 2D to 3D drug treatment studies pose challenges with respect to analysis methods. Patient-derived tumor organoids (PDTOs) possess unique features given their heterogeneity in size, shape, and growth patterns. A detailed assessment of the length scale at which PDTOs should be evaluated (i.e., individual cell or organoid-level analysis) has not been done to our knowledge. Therefore, using dynamic confocal live cell imaging and data analysis methods we examined tumor cell growth rates and drug response behaviors in colorectal cancer (CRC) PDTOs. High-resolution imaging of H2B-GFP-labeled organoids with DRAQ7 vital dye permitted tracking of cellular changes, such as cell birth and death events, in individual organoids. From these same images, we measured morphological features of the 3D objects, including volume, sphericity, and ellipticity. Sphericity and ellipticity were used to evaluate intra- and interpatient tumor organoid heterogeneity. We found a strong correlation between organoid live cell number and volume. Linear growth rate calculations based on volume or live cell counts were used to determine differential responses to therapeutic interventions. We showed that this approach can detect different types of drug effects (cytotoxic vs cytostatic) in PDTO cultures. Overall, our imaging-based quantification workflow results in multiple parameters that can provide patient- and drug-specific information for screening applications.
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Bock, Nathalie, Farzaneh Forouz, Luke Hipwood, Julien Clegg, Penny Jeffery, Madeline Gough, Tirsa van Wyngaard, et al. "GelMA, Click-Chemistry Gelatin and Bioprinted Polyethylene Glycol-Based Hydrogels as 3D Ex Vivo Drug Testing Platforms for Patient-Derived Breast Cancer Organoids." Pharmaceutics 15, no. 1 (January 12, 2023): 261. http://dx.doi.org/10.3390/pharmaceutics15010261.
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3D organoid model technologies have led to the development of innovative tools for cancer precision medicine. Yet, the gold standard culture system (Matrigel®) lacks the ability for extensive biophysical manipulation needed to model various cancer microenvironments and has inherent batch-to-batch variability. Tunable hydrogel matrices provide enhanced capability for drug testing in breast cancer (BCa), by better mimicking key physicochemical characteristics of this disease’s extracellular matrix. Here, we encapsulated patient-derived breast cancer cells in bioprinted polyethylene glycol-derived hydrogels (PEG), functionalized with adhesion peptides (RGD, GFOGER and DYIGSR) and gelatin-derived hydrogels (gelatin methacryloyl; GelMA and thiolated-gelatin crosslinked with PEG-4MAL; GelSH). Within ranges of BCa stiffnesses (1–6 kPa), GelMA, GelSH and PEG-based hydrogels successfully supported the growth and organoid formation of HR+,−/HER2+,− primary cancer cells for at least 2–3 weeks, with superior organoid formation within the GelSH biomaterial (up to 268% growth after 15 days). BCa organoids responded to doxorubicin, EP31670 and paclitaxel treatments with increased IC50 concentrations on organoids compared to 2D cultures, and highest IC50 for organoids in GelSH. Cell viability after doxorubicin treatment (1 µM) remained >2-fold higher in the 3D gels compared to 2D and doxorubicin/paclitaxel (both 5 µM) were ~2.75–3-fold less potent in GelSH compared to PEG hydrogels. The data demonstrate the potential of hydrogel matrices as easy-to-use and effective preclinical tools for therapy assessment in patient-derived breast cancer organoids.
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Gamboa, Christian Moya, Yujue Wang, Huiting Xu, Katarzyna Kalemba, Fredric E. Wondisford, and Hatem E. Sabaawy. "Optimized 3D Culture of Hepatic Cells for Liver Organoid Metabolic Assays." Cells 10, no. 12 (November 24, 2021): 3280. http://dx.doi.org/10.3390/cells10123280.
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The liver is among the principal organs for glucose homeostasis and metabolism. Studies of liver metabolism are limited by the inability to expand primary hepatocytes in vitro while maintaining their metabolic functions. Human hepatic three-dimensional (3D) organoids have been established using defined factors, yet hepatic organoids from adult donors showed impaired expansion. We examined conditions to facilitate the expansion of adult donor-derived hepatic organoids (HepAOs) and HepG2 cells in organoid cultures (HepGOs) using combinations of growth factors and small molecules. The expansion dynamics, gluconeogenic and HNF4α expression, and albumin secretion are assessed. The conditions tested allow the generation of HepAOs and HepGOs in 3D cultures. Nevertheless, gluconeogenic gene expression varies greatly between conditions. The organoid expansion rates are limited when including the TGFβ inhibitor A8301, while are relatively higher with Forskolin (FSK) and Oncostatin M (OSM). Notably, expanded HepGOs grown in the optimized condition maintain detectable gluconeogenic expression in a spatiotemporal distribution at 8 weeks. We present optimized conditions by limiting A8301 and incorporating FSK and OSM to allow the expansion of HepAOs from adult donors and HepGOs with gluconeogenic competence. These models increase the repertoire of human hepatic cellular tools available for use in liver metabolic assays.
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Sakib, Sadman, Aya Uchida, Paula Valenzuela-Leon, Yang Yu, Hanna Valli-Pulaski, Kyle Orwig, Mark Ungrin, and Ina Dobrinski. "Formation of organotypic testicular organoids in microwell culture†." Biology of Reproduction 100, no. 6 (March 30, 2019): 1648–60. http://dx.doi.org/10.1093/biolre/ioz053.
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Abstract Three-dimensional (3D) organoids can serve as an in vitro platform to study cell–cell interactions, tissue development, and toxicology. Development of organoids with tissue architecture similar to testis in vivo has remained a challenge. Here, we present a microwell aggregation approach to establish multicellular 3D testicular organoids from pig, mouse, macaque, and human. The organoids consist of germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells forming a distinct seminiferous epithelium and interstitial compartment separated by a basement membrane. Sertoli cells in the organoids express tight junction proteins claudin 11 and occludin. Germ cells in organoids showed an attenuated response to retinoic acid compared to germ cells in 2D culture indicating that the tissue architecture of the organoid modulates response to retinoic acid similar to in vivo. Germ cells maintaining physiological cell–cell interactions in organoids also had lower levels of autophagy indicating lower levels of cellular stress. When organoids were treated with mono(2-ethylhexyl) phthalate (MEHP), levels of germ cell autophagy increased in a dose-dependent manner, indicating the utility of the organoids for toxicity screening. Ablation of primary cilia on testicular somatic cells inhibited the formation of organoids demonstrating an application to screen for factors affecting testicular morphogenesis. Organoids can be generated from cryopreserved testis cells and preserved by vitrification. Taken together, the testicular organoid system recapitulates the 3D organization of the mammalian testis and provides an in vitro platform for studying germ cell function, testicular development, and drug toxicity in a cellular context representative of the testis in vivo.
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Wang, Yue, Glauco R. Souza, and Robert J. Amato. "3D Organoids from Milligrams of Genitourinary Cancer Patients Tissue Retain Key Features of Original Tumors." Journal of Clinical Oncology 36, no. 6_suppl (February 20, 2018): 268. http://dx.doi.org/10.1200/jco.2018.36.6_suppl.268.
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268 Background: 3D organoid culture, especially that generated from patient samples, is becoming prevalent as 2D cell culture models fail to provide clinical relevance. However, a relatively large amount of starting tissue is still required for this process, and the turnover rate and overall success rate vary greatly if relying solely on spontaneous aggregation. Methods: Here, we report the successful use of magnetic 3D bioprinting to drive organoid formation derived from fresh tumor samples of patients with prostate cancer, renal cell cancer, and urothelial cancer. Results: Only milligrams to tens of milligrams of starting tissues (from fine-needle biopsies, core biopsies, or surgical excision) were used. Most organoids formed overnight, with a very high overall success rate ( > 95%, n = 25). We have demonstrated that these cultures can be maintained, passed, and cryopreserved like conventional 2D cultures. More importantly, we characterized the organoids derived from prostatic adenocarcinomas and neuroendocrine tumors by the means of immunostaining and showed that the organoids expressed the same signature proteins as the originating tumors. This clinical concordance is crucial for downstream translational and biological research. We also showed that these adenocarcinoma cells responded to enzalutamide, an androgen receptor inhibitor that is used clinically to treat prostatic adenocarcinoma. Conclusions: In summary, we have established an efficient and robust system to create patient-derived 3D organoids; molecular characterization shows clinical concordance with original patient tumors; and our system shows great potential in drug screening with patient-derived 3D cultures.
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Sart, Sébastien, Raphaël F. X. Tomasi, Antoine Barizien, Gabriel Amselem, Ana Cumano, and Charles N. Baroud. "Mapping the structure and biological functions within mesenchymal bodies using microfluidics." Science Advances 6, no. 10 (March 2020): eaaw7853. http://dx.doi.org/10.1126/sciadv.aaw7853.
Full textAbstract:
Organoids that recapitulate the functional hallmarks of anatomic structures comprise cell populations able to self-organize cohesively in 3D. However, the rules underlying organoid formation in vitro remain poorly understood because a correlative analysis of individual cell fate and spatial organization has been challenging. Here, we use a novel microfluidics platform to investigate the mechanisms determining the formation of organoids by human mesenchymal stromal cells that recapitulate the early steps of condensation initiating bone repair in vivo. We find that heterogeneous mesenchymal stromal cells self-organize in 3D in a developmentally hierarchical manner. We demonstrate a link between structural organization and local regulation of specific molecular signaling pathways such as NF-κB and actin polymerization, which modulate osteo-endocrine functions. This study emphasizes the importance of resolving spatial heterogeneities within cellular aggregates to link organization and functional properties, enabling a better understanding of the mechanisms controlling organoid formation, relevant to organogenesis and tissue repair.
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Yusro, Muhammad, and Isnaini Nurisusilawati. "Forecasting Approach to Investigate Dynamic Growth of Organoid within 3D Matrix for Distinct Perspective." Journal of Biomimetics, Biomaterials and Biomedical Engineering 59 (February 14, 2023): 107–17. http://dx.doi.org/10.4028/p-99od29.
Full textAbstract:
Organoid as a 3D structured model in vitro has difficulty in controlling its size. This issue becomes problematic when it is applied in a microfluidic source and sink-based because different dimension leads to different exposure to morphogen resulting in different cell fate. As a model used for biomedical purposes, this problem could lead to a discrepancy. This research is imposed to implement the forecasting method to study the dynamic of organoid growth profile. This approach could help a better understanding via spatiotemporal perspective complemented with a mathematical formula. The forecasting approach that clarifies the trend of this organoid growth by assessing whether the decided trend fits in every (or particular) stage (or not) has not been informed yet. Neural tube organoids have four different mechanical stiffness (0,5 kPa, 2 kPa, 4 kPa, 8kPa) which are documented in three days by time-lapse microscopy used in this experiment. These objects are mapped in a spatiotemporal fashion investigated in the profile and assessed by exponential trend. The actual phenomenon and forecasted result are evaluated by Mean Absolute Percentage Error (MAPE). Based on the result, the profile of organoid growth indicates that the organoid develops mostly following an exponential profile with the highest R2 value of 0,9868 and the lowest being 0,8734. Based on the MAPE value calculation it could be confirmed that the MAPE value on day 3 is the highest among the others indicating that the extended time of growth tends to have a different profile rather than the exponential trend after day 2. It should be noted that on the lowest stiffness (0,5 kPa) the mechanical properties do not significantly affect the organoid size during the development. Almost all (11 by 12 data or 91,6%) of the MAPE value is in excellent criteria (the value is less than 10%). Only one data does not belong to that classification which is in 8 kPa on day 3. Indicating that the higher stiffness the stronger effect on the system. From the axis development perspective, the organoid does not follow any specific pattern. This research could be a reference for a better understanding of the organoid growth profile in the 3D matrix environment which is nowadays become a hot topic in biomedical applications.
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Panwar, Amit, Prativa Das, and Lay Poh Tan. "3D Hepatic Organoid-Based Advancements in LIVER Tissue Engineering." Bioengineering 8, no. 11 (November 14, 2021): 185. http://dx.doi.org/10.3390/bioengineering8110185.
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Liver-associated diseases and tissue engineering approaches based on in vitro culture of functional Primary human hepatocytes (PHH) had been restricted by the rapid de-differentiation in 2D culture conditions which restricted their usability. It was proven that cells growing in 3D format can better mimic the in vivo microenvironment, and thus help in maintaining metabolic activity, phenotypic properties, and longevity of the in vitro cultures. Again, the culture method and type of cell population are also recognized as important parameters for functional maintenance of primary hepatocytes. Hepatic organoids formed by self-assembly of hepatic cells are microtissues, and were able to show long-term in vitro maintenance of hepato-specific characteristics. Thus, hepatic organoids were recognized as an effective tool for screening potential cures and modeling liver diseases effectively. The current review summarizes the importance of 3D hepatic organoid culture over other conventional 2D and 3D culture models and its applicability in Liver tissue engineering.
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Caipa Garcia, Angela L., Jill E. Kucab, Halh Al-Serori, Rebekah S. S. Beck, Franziska Fischer, Matthias Hufnagel, Andrea Hartwig, et al. "Metabolic Activation of Benzo[a]pyrene by Human Tissue Organoid Cultures." International Journal of Molecular Sciences 24, no. 1 (December 29, 2022): 606. http://dx.doi.org/10.3390/ijms24010606.
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Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.
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Halder, Tithi Ghosh, Shelby Rheinschmidt, Trason Thode, Samuel Sampson, Ryan Rodriguez del Villar, Serina Ng, Alexis Weston, et al. "Abstract 1878: 3D Tumor models in drug discovery for high throughput analysis of immune cell infiltration." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1878. http://dx.doi.org/10.1158/1538-7445.am2022-1878.
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Abstract The interaction between tumor cells and immune system has been studied mostly in animal models and co-cultures with cell lines. However, it is difficult to study the unique features of human immune cells in mice, and cell lines are often transformed or immortalized by genetic modification. Organoid platform has emerged as a powerful tool which maintains the architectures and distinctive functions of a specific organ. Although organoids still fail to model immune system accurately, co-cultures of organoids and lymphocytes may have promising applications. In this study, we developed a unique methodology to use organoids for understanding the role of epithelial cell-immune cell interactions in cancer. Using this immune-organoid tool we tested the infiltration and efficacy of TILs generated from patient tumor cells. We also tested if checkpoint blockade could amplify the immune cell infiltration in tumor using this organoid platform bringing hope for further application of this technology in immunotherapy. Single cell suspensions were prepared from patient tumor biopsy and seeded in ultra-low attachment (ULA) microplates in appropriate media to generate organoids. TILs were isolated from the same tumor and rapidly expanded in appropriate media. Tumor organoids and TILs were incubated with PD-L1 and CTLA-4 monoclonal antibody respectively for check point blockade. RFP-stained TILs were then seeded into inserts of a transwell receiver plate using specific tumoroid:TIL ratio. After 48 h organoids were analyzed by 3D Z-stack imaging and morphometric analysis with Cytation 5 software to quantify the lymphocyte infiltration. Using this immune organoid model, we were able to quantify the number of TILs that infiltrates inside the tumor organoids generated from a melanoma and a pancreatic cancer patient. We also found that PDL-1 checkpoint Blockade in tumor cells enhanced the TIL infiltration inside organoids. Moreover, we were able to isolate the infiltrated lymphocytes from the tumor organoids and characterized them by analyzing different markers and cytokines using FACS, RT-PCR and Immunohistochemistry. Therefore, this study has potential to be an excellent preclinical model for human tumors, translating basic cancer research to clinical practice as well as providing a new meaning to precision medicine by identifying and validating novel immune therapies for individual cancer patients. Citation Format: Tithi Ghosh Halder, Shelby Rheinschmidt, Trason Thode, Samuel Sampson, Ryan Rodriguez del Villar, Serina Ng, Alexis Weston, Mohan Kaadige, Jeffery Trent, John Altin, Justin Moser, Erkut Borazanci, Michael Gordon, Anna Larson, Raffaella Soldi, Sunil Sharma. 3D Tumor models in drug discovery for high throughput analysis of immune cell infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1878.
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Li, Haixin, Hongkun Liu, and Kexin Chen. "Living biobank-based cancer organoids: prospects and challenges in cancer research." Cancer Biology & Medicine 19, no. 7 (July 21, 2022): 965–82. http://dx.doi.org/10.20892/j.issn.2095-3941.2021.0621.
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Biobanks bridge the gap between basic and translational research. Traditional cancer biobanks typically contain normal and tumor tissues, and matched blood. However, biospecimens in traditional biobanks are usually nonrenewable. In recent years, increased interest has focused on establishing living biobanks, including organoid biobanks, for the collection and storage of viable and functional tissues for long periods of time. The organoid model is based on a 3D in vitro cell culture system, is highly similar to primary tissues and organs in vivo, and can recapitulate the phenotypic and genetic characteristics of target organs. Publications on cancer organoids have recently increased, and many types of cancer organoids have been used for modeling cancer processes, as well as for drug discovery and screening. On the basis of the current research status, more exploration of cancer organoids through technical advancements is required to improve reproducibility and scalability. Moreover, given the natural characteristics of organoids, greater attention must be paid to ethical considerations. Here, we summarize recent advances in cancer organoid biobanking research, encompassing rectal, gastric, pancreatic, breast, and glioblastoma cancers. Living cancer biobanks that contain cancerous tissues and matched organoids with different genetic backgrounds, subtypes, and individualized characteristics will eventually contribute to the understanding of cancer and ultimately facilitate the development of innovative treatments.
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Xu, Jie, and Zhexing Wen. "Brain Organoids: Studying Human Brain Development and Diseases in a Dish." Stem Cells International 2021 (September 9, 2021): 1–16. http://dx.doi.org/10.1155/2021/5902824.
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With the rapid development of stem cell technology, the advent of three-dimensional (3D) cultured brain organoids has opened a new avenue for studying human neurodevelopment and neurological disorders. Brain organoids are stem-cell-derived 3D suspension cultures that self-assemble into an organized structure with cell types and cytoarchitectures recapitulating the developing brain. In recent years, brain organoids have been utilized in various aspects, ranging from basic biology studies, to disease modeling, and high-throughput screening of pharmaceutical compounds. In this review, we overview the establishment and development of brain organoid technology, its recent progress, and translational applications, as well as existing limitations and future directions.
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García-Rodríguez, Inés, Adithya Sridhar, Dasja Pajkrt, and Katja C. Wolthers. "Put Some Guts into It: Intestinal Organoid Models to Study Viral Infection." Viruses 12, no. 11 (November 11, 2020): 1288. http://dx.doi.org/10.3390/v12111288.
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The knowledge about enteric viral infection has vastly increased over the last eight years due to the development of intestinal organoids and enteroids that suppose a step forward from conventional studies using cell lines. Intestinal organoids and enteroids are three-dimensional (3D) models that closely mimic intestinal cellular heterogeneity and organization. The barrier function within these models has been adapted to facilitate viral studies. In this review, several adaptations (such as organoid-derived two-dimensional (2D) monolayers) and original intestinal 3D models are discussed. The specific advantages and applications, as well as improvements of each model are analyzed and an insight into the possible path for the field is given.
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Jung, Kyle L., Un Yung Choi, Angela Park, Suan-Sin Foo, Stephanie Kim, Shin-Ae Lee, and Jae U. Jung. "Single-cell analysis of Kaposi’s sarcoma-associated herpesvirus infection in three-dimensional air-liquid interface culture model." PLOS Pathogens 18, no. 8 (August 17, 2022): e1010775. http://dx.doi.org/10.1371/journal.ppat.1010775.
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The oral cavity is the major site for transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV), but how KSHV establishes infection and replication in the oral epithelia remains unclear. Here, we report a KSHV spontaneous lytic replication model using fully differentiated, three-dimensional (3D) oral epithelial organoids at an air-liquid interface (ALI). This model revealed that KSHV infected the oral epithelia when the basal epithelial cells were exposed by damage. Unlike two-dimensional (2D) cell culture, 3D oral epithelial organoid ALI culture allowed high levels of spontaneous KSHV lytic replication, where lytically replicating cells were enriched at the superficial layer of epithelial organoid. Single cell RNA sequencing (scRNAseq) showed that KSHV infection induced drastic changes of host gene expression in infected as well as uninfected cells at the different epithelial layers, resulting in altered keratinocyte differentiation and cell death. Moreover, we identified a unique population of infected cells containing lytic gene expression at the KSHV K2-K5 gene locus and distinct host gene expression compared to latent or lytic infected cells. This study demonstrates an in vitro 3D epithelial organoid ALI culture model that recapitulates KSHV infection in the oral cavity, where KSHV undergoes the epithelial differentiation-dependent spontaneous lytic replication with a unique cell population carrying distinct viral gene expression.
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He, Juan, Xiaoyu Zhang, Xinyi Xia, Ming Han, Fei Li, Chunfeng Li, Yunguang Li, and Dong Gao. "Organoid technology for tissue engineering." Journal of Molecular Cell Biology 12, no. 8 (April 6, 2020): 569–79. http://dx.doi.org/10.1093/jmcb/mjaa012.
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Abstract For centuries, attempts have been continuously made to artificially reconstitute counterparts of in vivo organs from their tissues or cells. Only in the recent decade has organoid technology as a whole technological field systematically emerged and been shown to play important roles in tissue engineering. Based on their self-organizing capacities, stem cells of versatile organs, both harvested and induced, can form 3D structures that are structurally and functionally similar to their in vivo counterparts. These organoid models provide a powerful platform for elucidating the development mechanisms, modeling diseases, and screening drug candidates. In this review, we will summarize the advances of this technology for generating various organoids of tissues from the three germ layers and discuss their drawbacks and prospects for tissue engineering.
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Weeber, Fleur, Marc van de Wetering, Marlous Hoogstraat, Krijn K. Dijkstra, Oscar Krijgsman, Thomas Kuilman, Christa G. M. Gadellaa-van Hooijdonk, et al. "Preserved genetic diversity in organoids cultured from biopsies of human colorectal cancer metastases." Proceedings of the National Academy of Sciences 112, no. 43 (October 12, 2015): 13308–11. http://dx.doi.org/10.1073/pnas.1516689112.
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Tumor organoids are 3D cultures of cancer cells. They can be derived from the tumor of each individual patient, thereby providing an attractive ex vivo assay to tailor treatment. Using patient-derived tumor organoids for this purpose requires that organoids derived from biopsies maintain the genetic diversity of the in vivo tumor. In this study tumor biopsies were obtained from 14 patients with metastatic colorectal cancer (i) to test the feasibility of organoid culture from metastatic biopsy specimens and (ii) to compare the genetic diversity of patient-derived tumor organoids and the original tumor biopsy. Genetic analysis was performed using SOLiD sequencing for 1,977 cancer-relevant genes. Copy number profiles were generated from sequencing data using CopywriteR. Here we demonstrate that organoid cultures can be established from tumor biopsies of patients with metastatic colorectal cancer with a success rate of 71%. Genetic analysis showed that organoids reflect the metastasis from which they were derived. Ninety percent of somatic mutations were shared between organoids and biopsies from the same patient, and the DNA copy number profiles of organoids and the corresponding original tumor show a correlation of 0.89. Most importantly, none of the mutations that were found exclusively in either the tumor or organoid culture are in driver genes or genes amenable for drug targeting. These findings support further exploration of patient-derived organoids as an ex vivo platform to personalize anticancer treatment.
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Millard, Melissa, Natalie A. Williams, Ashley K. Elrod, and Teresa M. DesRochers. "Abstract 3086: Organoids standardized to a clinically validated drug response assay for truly predictive in vitro drug response profiling." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3086. http://dx.doi.org/10.1158/1538-7445.am2022-3086.
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Abstract Unlike cell lines, organoids maintain most of the biological properties of the parental tissue from which the starting cells were isolated including the histology and gene expression. When organoids include clinical annotation, they become a useful, renewable tool for clinical correlation studies, but to be truly predictive the drug profiling assays utilized to screen organoid response must have measurable correlation with patient response. 3D Predict™ is a highly accurate assay that is 89% and 85% predictive of response in first-line ovarian cancer and high-grade gliomas (HGG) respectively. We have developed a panel of organoids that are clinically annotated, include correlative primary tissue 3D Predict™ drug response data, and have been assessed for the recapitulation of primary tissue histology and genomics. Additionally, our organoid models incorporate matched immune cells, a key component of the tumor microenvironment, making them an ideal model for immune-oncology studies. Here we present data on 15 available organoid models across HGG, breast, colorectal and bladder cancer. We have applied these models to drug response studies, including checkpoint inhibitors and shown correlation to primary patient response. The assurance of predictive capacity is unique to KIYATEC’s organoids and is significant because it avoids the pitfalls of comparing drug responses across non-concordant assay platforms while providing assurance that the models are reflective of individual patient response and outcomes. Citation Format: Melissa Millard, Natalie A. Williams, Ashley K. Elrod, Teresa M. DesRochers. Organoids standardized to a clinically validated drug response assay for truly predictive in vitro drug response profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3086.