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Cells
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Organoids as an Experimental Tool
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Organoids as an Experimental Tool

A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Tissues and Organs".

Deadline for manuscript submissions: 10 July 2025 | Viewed by 13348

Special Issue Editors

Prof. Dr. Ralf Weiskirchen

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Guest Editor
Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, D-52074 Aachen, Germany
Interests: liver disease; fibrosis; biomarker; cytokines; chemokines; translational medicine
Special Issues, Collections and Topics in MDPI journals
Dr. Leo A. Van Grunsven

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Guest Editor
Liver Cell Biology Research Group, Vrije Universiteit Brussel, 1090 Brussel, Belgium
Interests: liver disease; in vitro models of liver fibrosis; PSC-derived liver cells; translational medicine; siRNA/miRNAs
Special Issues, Collections and Topics in MDPI journals
Dr. Anjali A. J. Roeth

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Guest Editor
Department of General, Visceral and Transplant Surgery, RWTH Aachen University Hospital, Pauwelsstrasse 30, 52074 Aachen, Germany
Interests: 3D-cell culture; organoids; immunohistochemistry; multiplex-imaging

Special Issue Information

Dear Colleagues,

Organoids are self-organized three-dimensional tissue cultures derived either from pluripotent stem cells; they can be sourced from embryonic stem cells, induced pluripotent stem cells, or tissue-derived stem cells from a specific organ. In addition, they may also be derived from progenitor or differentiated cells from healthy or diseased tissues. These cultures can replicate the complexity of an organ or focus on specific aspects of it. As a result, these three-dimensional “mini-organs” can provide valuable insights into the biological processes within an organ. They have become a powerful in vitro research tool that maintains the genetic and phenotypic characteristics of organs in vivo. The applications of organoids include modeling the development and diseases and investigating their potential for drug development and personalized medicine. Furthermore, they have shown great promise in regenerative medicine and offer a non-animal-based alternative for basic and translational research.

In this Special Issue of Cells, we invite you to contribute original research articles, reviews, or communications on all aspects related to the theme of “Organoids as an Experimental Tool”. Expert articles describing the mechanistic, functional, cellular, biochemical, or general aspects of organoid cultures and their application in basic and translational science are encouraged.

Prof. Dr. Ralf Weiskirchen
Dr. Leo A. Van Grunsven
Dr. Anjali A. J. Roeth
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Cells is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • organoid culture models
  • primary cell and cancer spheroid models
  • organotypic models
  • organoid research for animal replacement (3R)
  • chip-based methods
  • general 3D cell culture protocols
  • in silico approaches
  • microinjection apical-out organoids
  • organoid manipulation techniques
  • organoid intelligence
  • organoids in diagnostics
  • imaging in organoids

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Published Papers (7 papers)

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Research

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13 pages, 2472 KiB  
Article
The Formation of Human Arteriovenous Malformation Organoids and Their Characteristics
by Eun Jung Oh, Hyun Mi Kim, Suin Kwak, Chanhoe Huh and Ho Yun Chung
Cells 2024, 13(23), 1955; https://doi.org/10.3390/cells13231955 - 25 Nov 2024
Viewed by 1198
Abstract
Arteriovenous malformations (AVMs) are characterized by direct connections between arteries and veins without intervening capillaries, with the concomitant formation of abnormal vascular networks associated with angiogenesis. However, the current understanding of the diagnosis and treatment of AVMs is limited, and no in vitro [...] Read more.
Arteriovenous malformations (AVMs) are characterized by direct connections between arteries and veins without intervening capillaries, with the concomitant formation of abnormal vascular networks associated with angiogenesis. However, the current understanding of the diagnosis and treatment of AVMs is limited, and no in vitro disease models exist at present for studying this condition. In this study, we produced endothelial cells (ECs) in two-dimensional cultures and three-dimensional (3D) blood vessel organoids (BVOs), comparing gene expression profiles between normal and AVM organoids. The normal and AVM organoids were examined via immunofluorescence staining using CD31 and phalloidin. The AVM organoids showed significantly higher expression levels of CD31 and phalloidin than the normal organoids. Genes such as FSTL1, associated with angiogenesis, showed significantly higher expression in the AVM organoids than in the normal organoids. In contrast, the MARCKS gene exhibited no significant difference in expression between the two types of organoids. The capillaries and related CSPG4 genes exhibited the lowest expression in the 3D AVM organoids. Furthermore, hsa-mir-135b-5p, a small RNA related to AVMs, showed elevated expression in AVM tissues and significantly higher levels in 3D AVM organoids. In our study, we were able to successfully establish AVM organoids (hBVOs) containing ECs and mural cells through advancements in stem cell and tissue engineering. These organoids serve as valuable models for investigating disease mechanisms, drug development, and screening potential therapeutic interventions in drug discovery. These findings contribute essential insights for the development of treatment strategies targeting AVMs. Full article
(This article belongs to the Special Issue Organoids as an Experimental Tool)
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14 pages, 3110 KiB  
Communication
Regulation of Enterocyte Brush Border Membrane Primary Na-Absorptive Transporters in Human Intestinal Organoid-Derived Monolayers
by Jennifer Haynes, Balasubramanian Palaniappan, John M. Crutchley and Uma Sundaram
Cells 2024, 13(19), 1623; https://doi.org/10.3390/cells13191623 - 28 Sep 2024
Cited by 1 | Viewed by 1550
Abstract
In the small intestine, sodium (Na) absorption occurs primarily via two apical transporters, Na-hydrogen exchanger 3 (NHE3) and Na-glucose cotransporter 1 (SGLT1). The two primary Na-absorptive pathways were previously shown to compensatorily regulate each other in rabbit and rat intestinal epithelial cells. However, [...] Read more.
In the small intestine, sodium (Na) absorption occurs primarily via two apical transporters, Na-hydrogen exchanger 3 (NHE3) and Na-glucose cotransporter 1 (SGLT1). The two primary Na-absorptive pathways were previously shown to compensatorily regulate each other in rabbit and rat intestinal epithelial cells. However, whether NHE3 and SGLT1 regulate one another in normal human enterocytes is unknown, mainly due to a lack of appropriate experimental models. To investigate this, we generated 2D enterocyte monolayers from human jejunal 3D organoids and used small interfering RNAs (siRNAs) to knock down NHE3 or SGLT1. Molecular and uptake studies were performed to determine the effects on NHE3 and SGLT1 expression and activity. Knockdown of NHE3 by siRNA in enterocyte monolayers was verified by qPCR and Western blot analysis and resulted in reduced NHE3 activity. However, in NHE3 siRNA-transfected cells, SGLT1 activity was significantly increased. siRNA knockdown of SGLT1 was confirmed by qPCR and Western blot analysis and resulted in reduced SGLT1 activity. However, in SGLT1 siRNA-transfected cells, NHE3 activity was significantly increased. These results demonstrate for the first time the functionality of siRNA in patient-derived organoid monolayers. Furthermore, they show that the two primary Na absorptive pathways in human enterocytes reciprocally regulate one another. Full article
(This article belongs to the Special Issue Organoids as an Experimental Tool)
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16 pages, 6678 KiB  
Article
Enhanced In Vitro Recapitulation of In Vivo Liver Regeneration by Co-Culturing Hepatocyte Organoids with Adipose-Derived Mesenchymal Stem Cells, Alleviating Steatosis and Apoptosis in Acute Alcoholic Liver Injury
by Sun A Ock, Seo-Yeon Kim, Young-Im Kim, Won Seok Ju and Poongyeon Lee
Cells 2024, 13(15), 1303; https://doi.org/10.3390/cells13151303 - 4 Aug 2024
Cited by 1 | Viewed by 2293
Abstract
Hepatocyte organoids (HOs) have superior hepatic functions to cholangiocyte-derived organoids but suffer from shorter lifespans. To counteract this, we co-cultured pig HOs with adipose-derived mesenchymal stem cells (A-MSCs) and performed transcriptome analysis. The results revealed that A-MSCs enhanced the collagen synthesis pathways, which [...] Read more.
Hepatocyte organoids (HOs) have superior hepatic functions to cholangiocyte-derived organoids but suffer from shorter lifespans. To counteract this, we co-cultured pig HOs with adipose-derived mesenchymal stem cells (A-MSCs) and performed transcriptome analysis. The results revealed that A-MSCs enhanced the collagen synthesis pathways, which are crucial for maintaining the three-dimensional structure and extracellular matrix synthesis of the organoids. A-MSCs also increased the expression of liver progenitor cell markers (KRT7, SPP1, LGR5+, and TERT). To explore HOs as a liver disease model, we exposed them to alcohol to create an alcoholic liver injury (ALI) model. The co-culture of HOs with A-MSCs inhibited the apoptosis of hepatocytes and reduced lipid accumulation of HOs. Furthermore, varying ethanol concentrations (0–400 mM) and single-versus-daily exposure to HOs showed that daily exposure significantly increased the level of PLIN2, a lipid storage marker, while decreasing CYP2E1 and increasing CYP1A2 levels, suggesting that CYP1A2 may play a critical role in alcohol detoxification during short-term exposure. Moreover, daily alcohol exposure led to excessive lipid accumulation and nuclear fragmentation in HOs cultured alone. These findings indicate that HOs mimic in vivo liver regeneration, establishing them as a valuable model for studying liver diseases, such as ALI. Full article
(This article belongs to the Special Issue Organoids as an Experimental Tool)
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Review

Jump to: Research

50 pages, 4089 KiB  
Review
Colorectal Organoids: Models, Imaging, Omics, Therapy, Immunology, and Ethics
by Martina Taglieri, Linda Di Gregorio, Serena Matis, Chiara Rosa Maria Uras, Massimo Ardy, Sara Casati, Monica Marchese, Alessandro Poggi, Lizzia Raffaghello and Roberto Benelli
Cells 2025, 14(6), 457; https://doi.org/10.3390/cells14060457 - 19 Mar 2025
Viewed by 560
Abstract
Colorectal epithelium was the first long-term 3D organoid culture established in vitro. Identification of the key components essential for the long-term survival of the stem cell niche allowed an indefinite propagation of these cultures and the modulation of their differentiation into various lineages [...] Read more.
Colorectal epithelium was the first long-term 3D organoid culture established in vitro. Identification of the key components essential for the long-term survival of the stem cell niche allowed an indefinite propagation of these cultures and the modulation of their differentiation into various lineages of mature intestinal epithelial cells. While these methods were eventually adapted to establish organoids from different organs, colorectal organoids remain a pioneering model for the development of new applications in health and disease. Several basic and applicative aspects of organoid culture, modeling, monitoring and testing are analyzed in this review. We also tackle the ethical problems of biobanking and distribution of these precious research tools, frequently confined in the laboratory of origin or condemned to destruction at the end of the project. Full article
(This article belongs to the Special Issue Organoids as an Experimental Tool)
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27 pages, 2337 KiB  
Review
Three-Dimensional Models: Biomimetic Tools That Recapitulate Breast Tissue Architecture and Microenvironment to Study Ductal Carcinoma In Situ Transition to Invasive Ductal Breast Cancer
by Seema Shah, Kingsley O. Osuala, Ethan J. Brock, Kyungmin Ji, Bonnie F. Sloane and Raymond R. Mattingly
Cells 2025, 14(3), 220; https://doi.org/10.3390/cells14030220 - 4 Feb 2025
Viewed by 1311
Abstract
Diagnosis of ductal carcinoma in situ (DCIS) presents a challenge as we cannot yet distinguish between those lesions that remain dormant from cases that may progress to invasive ductal breast cancer (IDC) and require therapeutic intervention. Our overall interest is to develop biomimetic [...] Read more.
Diagnosis of ductal carcinoma in situ (DCIS) presents a challenge as we cannot yet distinguish between those lesions that remain dormant from cases that may progress to invasive ductal breast cancer (IDC) and require therapeutic intervention. Our overall interest is to develop biomimetic three-dimensional (3D) models that more accurately recapitulate the structure and characteristics of pre-invasive breast cancer in order to study the underlying mechanisms driving malignant progression. These models allow us to mimic the microenvironment to investigate many aspects of mammary cell biology, including the role of the extracellular matrix (ECM), the interaction between carcinoma-associated fibroblasts (CAFs) and epithelial cells, and the dynamics of cytoskeletal reorganization. In this review article, we outline the significance of 3D culture models as reliable pre-clinical tools that mimic the in vivo tumor microenvironment and facilitate the study of DCIS lesions as they progress to invasive breast cancer. We also discuss the role of CAFs and other stromal cells in DCIS transition as well as the clinical significance of emerging technologies like tumor-on-chip and co-culture models. Full article
(This article belongs to the Special Issue Organoids as an Experimental Tool)
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29 pages, 1074 KiB  
Review
A Comparative Analysis of Models for AAV-Mediated Gene Therapy for Inherited Retinal Diseases
by Almaqdad Alsalloum, Ekaterina Gornostal, Natalia Mingaleva, Roman Pavlov, Ekaterina Kuznetsova, Ekaterina Antonova, Aygun Nadzhafova, Daria Kolotova, Vitaly Kadyshev, Olga Mityaeva and Pavel Volchkov
Cells 2024, 13(20), 1706; https://doi.org/10.3390/cells13201706 - 15 Oct 2024
Cited by 1 | Viewed by 2994
Abstract
Inherited retinal diseases (IRDs) represent a diverse group of genetic disorders leading to progressive degeneration of the retina due to mutations in over 280 genes. This review focuses on the various methodologies for the preclinical characterization and evaluation of adeno-associated virus (AAV)-mediated gene [...] Read more.
Inherited retinal diseases (IRDs) represent a diverse group of genetic disorders leading to progressive degeneration of the retina due to mutations in over 280 genes. This review focuses on the various methodologies for the preclinical characterization and evaluation of adeno-associated virus (AAV)-mediated gene therapy as a potential treatment option for IRDs, particularly focusing on gene therapies targeting mutations, such as those in the RPE65 and FAM161A genes. AAV vectors, such as AAV2 and AAV5, have been utilized to deliver therapeutic genes, showing promise in preserving vision and enhancing photoreceptor function in animal models. Despite their advantages—including high production efficiency, low pathogenicity, and minimal immunogenicity—AAV-mediated therapies face limitations such as immune responses beyond the retina, vector size constraints, and challenges in large-scale manufacturing. This review systematically compares different experimental models used to investigate AAV-mediated therapies, such as mouse models, human retinal explants (HREs), and induced pluripotent stem cell (iPSC)-derived retinal organoids. Mouse models are advantageous for genetic manipulation and detailed investigations of disease mechanisms; however, anatomical differences between mice and humans may limit the translational applicability of results. HREs offer valuable insights into human retinal pathophysiology but face challenges such as tissue degradation and lack of systemic physiological effects. Retinal organoids, on the other hand, provide a robust platform that closely mimics human retinal development, thereby enabling more comprehensive studies on disease mechanisms and therapeutic strategies, including AAV-based interventions. Specific outcomes targeted in these studies include vision preservation and functional improvements of retinas damaged by genetic mutations. This review highlights the strengths and weaknesses of each experimental model and advocates for their combined use in developing targeted gene therapies for IRDs. As research advances, optimizing AAV vector design and delivery methods will be critical for enhancing therapeutic efficacy and improving clinical outcomes for patients with IRDs. Full article
(This article belongs to the Special Issue Organoids as an Experimental Tool)
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Graphical abstract

18 pages, 1455 KiB  
Review
Gastrointestinal Cancer Patient Derived Organoids at the Frontier of Personalized Medicine and Drug Screening
by Zhenjie Yang, Jun Yu and Chi Chun Wong
Cells 2024, 13(16), 1312; https://doi.org/10.3390/cells13161312 - 6 Aug 2024
Cited by 2 | Viewed by 2422
Abstract
Cancer is a leading cause of death worldwide. Around one-third of the total global cancer incidence and mortality are related to gastrointestinal (GI) cancers. Over the past few years, rapid developments have been made in patient-derived organoid (PDO) models for gastrointestinal cancers. By [...] Read more.
Cancer is a leading cause of death worldwide. Around one-third of the total global cancer incidence and mortality are related to gastrointestinal (GI) cancers. Over the past few years, rapid developments have been made in patient-derived organoid (PDO) models for gastrointestinal cancers. By closely mimicking the molecular properties of their parent tumors in vitro, PDOs have emerged as powerful tools in personalized medicine and drug discovery. Here, we review the current literature on the application of PDOs of common gastrointestinal cancers in the optimization of drug treatment strategies in the clinic and their rising importance in pre-clinical drug development. We discuss the advantages and limitations of gastrointestinal cancer PDOs and outline the microfluidics-based strategies that improve the throughput of PDO models in order to extract the maximal benefits in the personalized medicine and drug discovery process. Full article
(This article belongs to the Special Issue Organoids as an Experimental Tool)
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