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Advances in 3D Cell Culture
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Advances in 3D Cell Culture

A topical collection in Cells (ISSN 2073-4409). This collection belongs to the section "Cell Methods".

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Editor

Dr. Julia Schueler

E-Mail Website
Guest Editor
Charles River Research Services Germany GmbH, Freiburg im Breisgau, Germany
Interests: tumor biology; immuno-oncology; PDX models; spheroid 3D cultures; tumor microenvironment

Topical Collection Information

Dear Colleagues,

Important scientific topics that the scientific community aims to tackle with innovative 3D cell culture platforms include angiogenesis, cell differentiation, and the blood–brain barrier. The complex crosstalk of different cellular and non-cellular tissue components in a human setting should ideally be mimicked as closely as possible. The current Special Issue aims to give an overview of the different advances using microfluidics, bioprinting, and other innovative technologies to better recapitulate human diseases. It will cover different applications of 3D cell cultures in drug discovery, precision medicine, and basic research. The implications of different cell sources as well as the applicability of image analysis, transcriptomics, or proteomics as read-outs for these platforms will help us to understand the current landscape and identify possible synergies across technologies as well as therapeutic areas.

Dr. Julia Schueler
Guest Editor

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 collection 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

  • precision medicine
  • drug discovery
  • tumor microenvironment
  • bone marrow niche
  • immune modulation
  • metastatic cascade
  • blood–brain barrier

Published Papers (17 papers)

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2023

Jump to: 2022

16 pages, 5929 KiB  
Article
A High-Throughput, High-Containment Human Primary Epithelial Airway Organ-on-Chip Platform for SARS-CoV-2 Therapeutic Screening
by Christine R. Fisher, Felix Mba Medie, Rebeccah J. Luu, Robert B. Gaibler, Thomas J. Mulhern, Caitlin R. Miller, Chelsea J. Zhang, Logan D. Rubio, Elizabeth E. Marr, Vidhya Vijayakumar, Elizabeth P. Gabriel, Landys Lopez Quezada, Chun-Hui Zhang, Karen S. Anderson, William L. Jorgensen, Jehan W. Alladina, Benjamin D. Medoff, Jeffrey T. Borenstein and Ashley L. Gard
Cells 2023, 12(22), 2639; https://doi.org/10.3390/cells12222639 - 16 Nov 2023
Viewed by 1180
Abstract
COVID-19 emerged as a worldwide pandemic in early 2020, and while the rapid development of safe and efficacious vaccines stands as an extraordinary achievement, the identification of effective therapeutics has been less successful. This process has been limited in part by a lack [...] Read more.
COVID-19 emerged as a worldwide pandemic in early 2020, and while the rapid development of safe and efficacious vaccines stands as an extraordinary achievement, the identification of effective therapeutics has been less successful. This process has been limited in part by a lack of human-relevant preclinical models compatible with therapeutic screening on the native virus, which requires a high-containment environment. Here, we report SARS-CoV-2 infection and robust viral replication in PREDICT96-ALI, a high-throughput, human primary cell-based organ-on-chip platform. We evaluate unique infection kinetic profiles across lung tissue from three human donors by immunofluorescence, RT-qPCR, and plaque assays over a 6-day infection period. Enabled by the 96 devices/plate throughput of PREDICT96-ALI, we also investigate the efficacy of Remdesivir and MPro61 in a proof-of-concept antiviral study. Both compounds exhibit an antiviral effect against SARS-CoV-2 in the platform. This demonstration of SARS-CoV-2 infection and antiviral dosing in a high-throughput organ-on-chip platform presents a critical capability for disease modeling and therapeutic screening applications in a human physiology-relevant in vitro system. Full article
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22 pages, 8138 KiB  
Article
A Subset of Colon Cancer Cell Lines Displays a Cytokine Profile Linked to Angiogenesis, EMT and Invasion Which Is Modulated by the Culture Conditions In Vitro
by Jacqueline Bersano, Kanstantsin Lashuk, Anna Edinger and Julia Schueler
Cells 2023, 12(21), 2539; https://doi.org/10.3390/cells12212539 - 29 Oct 2023
Viewed by 1364
Abstract
Colorectal cancer (CRC) is one of the deadliest cancers worldwide. The dysregulation of secretory pathways is a crucial driver of CRC progression, since it modulates cell proliferation, angiogenesis and survival. This study explores the changes in the CRC cytokine profile depending on the [...] Read more.
Colorectal cancer (CRC) is one of the deadliest cancers worldwide. The dysregulation of secretory pathways is a crucial driver of CRC progression, since it modulates cell proliferation, angiogenesis and survival. This study explores the changes in the CRC cytokine profile depending on the culture conditions and the presence of fibroblasts and macrophages as cellular components of the tumor microenvironment in 2D and in 3D formed spheroids. Upon analysis of 45 different cytokines, chemokines and growth factors, 20 CRC cell lines were categorized into high and low secretors. In the high secretor group cytokines related to angiogenesis, EMT and invasion were significantly upregulated. LIF and HFG were identified as the best discriminator between both groups. Independent of this grouping, the addition of normal as well as cancer-associated fibroblasts had a similar impact on the cytokine profile by increasing the total amount of secreted cytokines in most of the investigated cell lines. In contrast, the differentiation and polarization of macrophages was modulated differently by normal vs. cancer-associated fibroblasts. In summary, we identified two groups of CRC cell lines that differ in their cytokine profile. The dependance of this profile was analyzed in detail—not only from the tumor cell line but as well from the culture condition in vitro. Key cytokines that discriminate the two groups were identified and their importance as promising biomarker candidates for CRC discussed. Full article
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13 pages, 3756 KiB  
Article
A Protocol for Organoids from the Urine of Bladder Cancer Patients
by Simon Walz, Paul Pollehne, Ruizhi Geng, Johannes Schneider, Moritz Maas, Wilhelm K. Aicher, Arnulf Stenzl, Bastian Amend and Niklas Harland
Cells 2023, 12(17), 2188; https://doi.org/10.3390/cells12172188 - 31 Aug 2023
Viewed by 1247
Abstract
This study investigates the feasibility of establishing urine-derived tumor organoids from bladder cancer (BC) patients as an alternative to tissue-derived organoids. BC is one of the most common cancers worldwide and current diagnostic methods involve invasive procedures. Here, we investigated the potential of [...] Read more.
This study investigates the feasibility of establishing urine-derived tumor organoids from bladder cancer (BC) patients as an alternative to tissue-derived organoids. BC is one of the most common cancers worldwide and current diagnostic methods involve invasive procedures. Here, we investigated the potential of using urine samples, which contain exfoliated tumor cells, to generate urine-derived BC organoids (uBCOs). Urine samples from 29 BC patients were collected and cells were isolated and cultured in a three-dimensional matrix. The establishment and primary expansion of uBCOs were successful in 83% of the specimens investigated. The culturing efficiency of uBCOs was comparable to cancer tissue-derived organoids. Immunohistochemistry and immunofluorescence to characterize the uBCOs exhibited similar expressions of BC markers compared to the parental tumor. These findings suggest that urine-derived BC organoids hold promise as a non-invasive tool for studying BC and evaluating therapeutic responses. This approach could potentially minimize the need for invasive procedures and provide a platform for personalized drug screening. Further research in this area may lead to improved diagnostic and treatment strategies for BC patients. Full article
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16 pages, 5036 KiB  
Article
Effects of Scaffolds on Urine- and Urothelial Carcinoma Tissue-Derived Organoids from Bladder Cancer Patients
by Simon Walz, Paul Pollehne, Philipp Vollmer, Wilhelm K. Aicher, Arnulf Stenzl, Niklas Harland and Bastian Amend
Cells 2023, 12(16), 2108; https://doi.org/10.3390/cells12162108 - 20 Aug 2023
Viewed by 1655
Abstract
Organoids are three-dimensional constructs generated by placing cells in scaffolds to facilitate the growth of cultures with cell–cell and cell–matrix interactions close to the in vivo situation. Organoids may contain different types of cells, including cancer cells, progenitor cells, or differentiated cells. As [...] Read more.
Organoids are three-dimensional constructs generated by placing cells in scaffolds to facilitate the growth of cultures with cell–cell and cell–matrix interactions close to the in vivo situation. Organoids may contain different types of cells, including cancer cells, progenitor cells, or differentiated cells. As distinct culture conditions have significant effects on cell metabolism, we explored the expansion of cells and expression of marker genes in bladder cancer cells expanded in two different common scaffolds. The cells were seeded in basement membrane extract (BME; s.c., Matrigel®) or in a cellulose-derived hydrogel (GrowDex®, GD) and cultured. The size of organoids and expression of marker genes were studied. We discovered that BME facilitated the growth of significantly larger organoids of cancer cell line RT112 (p < 0.05), cells from a solid tumor (p < 0.001), and a voiding urine sample (p < 0.001). Expression of proliferation marker Ki76, transcription factor TP63, cytokeratin CK20, and cell surface marker CD24 clearly differed in these different tumor cells upon expansion in BME when compared to cells in GD. We conclude that the choice of scaffold utilized for the generation of organoids has an impact not only on cell growth and organoid size but also on protein expression. The disadvantages of batch-to-batch-variations of BME must be balanced with the phenotypic bias observed with GD scaffolds when standardizing organoid cultures for clinical diagnoses. Full article
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17 pages, 6293 KiB  
Article
Targeting Conserved Pathways in 3D Spheroid Formation of Diverse Cell Types for Translational Application: Enhanced Functional and Antioxidant Capacity
by Chia-Chi Chang, Shih-Sheng Jiang, Fang-Yu Tsai, Pei-Ju Hsu, Chen-Chan Hsieh, Li-Tzu Wang, Men-Luh Yen and B. Linju Yen
Cells 2023, 12(16), 2050; https://doi.org/10.3390/cells12162050 - 11 Aug 2023
Cited by 1 | Viewed by 1113
Abstract
Three-dimensional (3D) in vitro spheroid/organoid culture increasingly appears to better mimic physiological states than standard 2D systems. The biological consequence of 3D spheroids, however, differs for different cell types: for pluripotent embryonic stem cells (ESCs), differentiation and loss of stemness occur, while the [...] Read more.
Three-dimensional (3D) in vitro spheroid/organoid culture increasingly appears to better mimic physiological states than standard 2D systems. The biological consequence of 3D spheroids, however, differs for different cell types: for pluripotent embryonic stem cells (ESCs), differentiation and loss of stemness occur, while the converse is true for somatic and cancer cells. Despite such diverse consequences, there are likely conserved mechanisms governing 3D spheroid formation across cell types that are unknown but could be efficiently targeted for translational application. To elucidate such processes, we performed transcriptome analysis with functional validation on 2D- and 3D-cultured mouse ESCs, mesenchymal stromal/stem cells (MSCs), and cancer cells. At both the transcriptomic and functional levels, 3D spheroid formation resulted in commitment towards known cell-specific functional outcomes. Surprisingly in all cell types, downregulation of the cholesterol synthesis pathway was found during 3D spheroid formation, with modulation concomitantly affecting 3D spheroid formation and cell-specific consequences; similar results were seen with human cell types. Furthermore, improved antioxidant capacity after 3D spheroid formation across cell types was further enhanced with modulation of the pathway. These findings demonstrate the profound cell-specific consequences and the translational value of understanding conserved mechanisms across diverse cell types after 3D spheroid formation. Full article
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16 pages, 23127 KiB  
Article
Viability Analysis and High-Content Live-Cell Imaging for Drug Testing in Prostate Cancer Xenograft-Derived Organoids
by Annelies Van Hemelryk, Sigrun Erkens-Schulze, Lifani Lim, Corrina M. A. de Ridder, Debra C. Stuurman, Guido W. Jenster, Martin E. van Royen and Wytske M. van Weerden
Cells 2023, 12(10), 1377; https://doi.org/10.3390/cells12101377 - 12 May 2023
Cited by 4 | Viewed by 2111
Abstract
Tumor organoids have been pushed forward as advanced model systems for in vitro oncology drug testing, with the eventual goal to direct personalized cancer treatments. However, drug testing efforts suffer from a large variation in experimental conditions for organoid culturing and organoid treatment. [...] Read more.
Tumor organoids have been pushed forward as advanced model systems for in vitro oncology drug testing, with the eventual goal to direct personalized cancer treatments. However, drug testing efforts suffer from a large variation in experimental conditions for organoid culturing and organoid treatment. Moreover, most drug tests are restricted to whole-well viability as the sole read-out, thereby losing important information about key biological aspects that might be impacted due to the use of administered drugs. These bulk read-outs also discard potential inter-organoid heterogeneity in drug responses. To tackle these issues, we developed a systematic approach for processing organoids from prostate cancer (PCa) patient-derived xenografts (PDXs) for viability-based drug testing and identified essential conditions and quality checks for consistent results. In addition, we generated an imaging-based drug testing procedure using high-content fluorescence microscopy in living PCa organoids to detect various modalities of cell death. Individual organoids and cell nuclei in organoids were segmented and quantified using a dye combination of Hoechst 33342, propidium iodide and Caspase 3/7 Green, allowing the identification of cytostatic and cytotoxic treatment effects. Our procedures provide important insights into the mechanistic actions of tested drugs. Moreover, these methods can be adapted for tumor organoids originating from other cancer types to increase organoid-based drug test validity, and ultimately, accelerate clinical implementation. Full article
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14 pages, 5004 KiB  
Article
A Novel Hydrogel-Based 3D In Vitro Tumor Panel of 30 PDX Models Incorporates Tumor, Stromal and Immune Cell Compartments of the TME for the Screening of Oncology and Immuno-Therapies
by Bin Xue, Julia Schüler, Christopher M. Harrod, Kanstantsin Lashuk, Zoji Bomya and Kolin C. Hribar
Cells 2023, 12(8), 1145; https://doi.org/10.3390/cells12081145 - 13 Apr 2023
Cited by 1 | Viewed by 3762
Abstract
Human-relevant systems that mimic the 3D tumor microenvironment (TME), particularly the complex mechanisms of immuno-modulation in the tumor stroma, in a reproducible and scalable format are of high interest for the drug discovery industry. Here, we describe a novel 3D in vitro tumor [...] Read more.
Human-relevant systems that mimic the 3D tumor microenvironment (TME), particularly the complex mechanisms of immuno-modulation in the tumor stroma, in a reproducible and scalable format are of high interest for the drug discovery industry. Here, we describe a novel 3D in vitro tumor panel comprising 30 distinct PDX models covering a range of histotypes and molecular subtypes and cocultured with fibroblasts and PBMCs in planar (flat) extracellular matrix hydrogels to reflect the three compartments of the TME—tumor, stroma, and immune cells. The panel was constructed in a 96-well plate format and assayed tumor size, tumor killing, and T-cell infiltration using high-content image analysis after 4 days of treatment. We screened the panel first against the chemotherapy drug Cisplatin to demonstrate feasibility and robustness, and subsequently assayed immuno-oncology agents Solitomab (CD3/EpCAM bispecific T-cell engager) and the immune checkpoint inhibitors (ICIs) Atezolizumab (anti-PDL1), Nivolumab (anti-PD1) and Ipilimumab (anti-CTLA4). Solitomab displayed a strong response across many PDX models in terms of tumor reduction and killing, allowing for its subsequent use as a positive control for ICIs. Interestingly, Atezolizumab and Nivolumab demonstrated a mild response compared to Ipilimumab in a subset of models from the panel. We later determined that PBMC spatial proximity in the assay setup was important for the PD1 inhibitor, hypothesizing that both duration and concentration of antigen exposure may be critical. The described 30-model panel represents a significant advancement toward screening in vitro models of the tumor microenvironment that include tumor, fibroblast, and immune cell populations in an extracellular matrix hydrogel, with robust and standardized high content image analysis in a planar hydrogel. The platform is aimed at rapidly screening various combinations and novel agents and forming a critical conduit to the clinic, thus accelerating drug discovery for the next generation of therapeutics. Full article
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14 pages, 24041 KiB  
Article
Evaluation of 3D Human Intestinal Organoids as a Platform for EV-A71 Antiviral Drug Discovery
by Fatma Masmoudi, Nanci Santos-Ferreira, Dasja Pajkrt, Katja C. Wolthers, Jeroen DeGroot, Maria L. H. Vlaming, Joana Rocha-Pereira and Ludovico Buti
Cells 2023, 12(8), 1138; https://doi.org/10.3390/cells12081138 - 12 Apr 2023
Cited by 3 | Viewed by 3131
Abstract
Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems [...] Read more.
Enteroviruses are a leading cause of upper respiratory tract, gastrointestinal, and neurological infections. Management of enterovirus-related diseases has been hindered by the lack of specific antiviral treatment. The pre-clinical and clinical development of such antivirals has been challenging, calling for novel model systems and strategies to identify suitable pre-clinical candidates. Organoids represent a new and outstanding opportunity to test antiviral agents in a more physiologically relevant system. However, dedicated studies addressing the validation and direct comparison of organoids versus commonly used cell lines are lacking. Here, we described the use of human small intestinal organoids (HIOs) as a model to study antiviral treatment against human enterovirus 71 (EV-A71) infection and compared this model to EV-A71-infected RD cells. We used reference antiviral compounds such as enviroxime, rupintrivir, and 2′-C-methylcytidine (2′CMC) to assess their effects on cell viability, virus-induced cytopathic effect, and viral RNA yield in EV-A71-infected HIOs and cell line. The results indicated a difference in the activity of the tested compounds between the two models, with HIOs being more sensitive to infection and drug treatment. In conclusion, the outcome reveals the value added by using the organoid model in virus and antiviral studies. Full article
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37 pages, 1178 KiB  
Review
Cancer Spheroids and Organoids as Novel Tools for Research and Therapy: State of the Art and Challenges to Guide Precision Medicine
by Sanae El Harane, Bochra Zidi, Nadia El Harane, Karl-Heinz Krause, Thomas Matthes and Olivier Preynat-Seauve
Cells 2023, 12(7), 1001; https://doi.org/10.3390/cells12071001 - 24 Mar 2023
Cited by 18 | Viewed by 5889
Abstract
Spheroids and organoids are important novel players in medical and life science research. They are gradually replacing two-dimensional (2D) cell cultures. Indeed, three-dimensional (3D) cultures are closer to the in vivo reality and open promising perspectives for academic research, drug screening, and personalized [...] Read more.
Spheroids and organoids are important novel players in medical and life science research. They are gradually replacing two-dimensional (2D) cell cultures. Indeed, three-dimensional (3D) cultures are closer to the in vivo reality and open promising perspectives for academic research, drug screening, and personalized medicine. A large variety of cells and tissues, including tumor cells, can be the starting material for the generation of 3D cultures, including primary tissues, stem cells, or cell lines. A panoply of methods has been developed to generate 3D structures, including spontaneous or forced cell aggregation, air–liquid interface conditions, low cell attachment supports, magnetic levitation, and scaffold-based technologies. The choice of the most appropriate method depends on (i) the origin of the tissue, (ii) the presence or absence of a disease, and (iii) the intended application. This review summarizes methods and approaches for the generation of cancer spheroids and organoids, including their advantages and limitations. We also highlight some of the challenges and unresolved issues in the field of cancer spheroids and organoids, and discuss possible therapeutic applications. Full article
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24 pages, 7673 KiB  
Article
Perfusion Air Culture of Precision-Cut Tumor Slices: An Ex Vivo System to Evaluate Individual Drug Response under Controlled Culture Conditions
by Meng Dong, Kathrin Böpple, Julia Thiel, Bernd Winkler, Chunguang Liang, Julia Schueler, Emma J. Davies, Simon T. Barry, Tauno Metsalu, Thomas E. Mürdter, Georg Sauer, German Ott, Matthias Schwab and Walter E. Aulitzky
Cells 2023, 12(5), 807; https://doi.org/10.3390/cells12050807 - 04 Mar 2023
Cited by 2 | Viewed by 2496
Abstract
Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air–liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we [...] Read more.
Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air–liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses. Full article
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16 pages, 3011 KiB  
Article
Novel Zebrafish Patient-Derived Tumor Xenograft Methodology for Evaluating Efficacy of Immune-Stimulating BCG Therapy in Urinary Bladder Cancer
by Saskia Kowald, Ylva Huge, Decky Tandiono, Zaheer Ali, Gabriela Vazquez-Rodriguez, Anna Erkstam, Anna Fahlgren, Amir Sherif, Yihai Cao and Lasse D. Jensen
Cells 2023, 12(3), 508; https://doi.org/10.3390/cells12030508 - 03 Feb 2023
Cited by 3 | Viewed by 2396
Abstract
Background: Bacillus Calmette-Guérin (BCG) immunotherapy is the standard-of-care adjuvant therapy for non-muscle-invasive bladder cancer in patients at considerable risk of disease recurrence. Although its exact mechanism of action is unknown, BCG significantly reduces this risk in responding patients but is mainly associated with [...] Read more.
Background: Bacillus Calmette-Guérin (BCG) immunotherapy is the standard-of-care adjuvant therapy for non-muscle-invasive bladder cancer in patients at considerable risk of disease recurrence. Although its exact mechanism of action is unknown, BCG significantly reduces this risk in responding patients but is mainly associated with toxic side-effects in those facing treatment resistance. Methods that allow the identification of BCG responders are, therefore, urgently needed. Methods: Fluorescently labelled UM-UC-3 cells and dissociated patient tumor samples were used to establish zebrafish tumor xenograft (ZTX) models. Changes in the relative primary tumor size and cell dissemination to the tail were evaluated via fluorescence microscopy at three days post-implantation. The data were compared to the treatment outcomes of the corresponding patients. Toxicity was evaluated based on gross morphological evaluation of the treated zebrafish larvae. Results: BCG-induced toxicity was avoided by removing the water-soluble fraction of the BCG formulation prior to use. BCG treatment via co-injection with the tumor cells resulted in significant and dose-dependent primary tumor size regression. Heat-inactivation of BCG decreased this effect, while intravenous BCG injections were ineffective. ZTX models were successfully established for six of six patients based on TUR-B biopsies. In two of these models, significant tumor regression was observed, which, in both cases, corresponded to the treatment response in the patients. Conclusions: The observed BCG-related anti-tumor effect indicates that ZTX models might predict the BCG response and thereby improve treatment planning. More experiments and clinical studies are needed, however, to elucidate the BCG mechanism and estimate the predictive value. Full article
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13 pages, 3743 KiB  
Article
Fabrication Scaffold with High Dimensional Control for Spheroids with Undifferentiated iPS Cell Properties
by Hidetaka Togo, Kento Terada, Akira Ujitsugu, Yudai Hirose, Hiroki Takeuchi and Masanobu Kusunoki
Cells 2023, 12(2), 278; https://doi.org/10.3390/cells12020278 - 11 Jan 2023
Viewed by 1871
Abstract
Spheroids are expected to aid the establishment of an in vitro-based cell culture system that can realistically reproduce cellular dynamics in vivo. We developed a fluoropolymer scaffold with an extracellular matrix (ECM) dot array and confirmed the possibility of mass-producing spheroids with uniform [...] Read more.
Spheroids are expected to aid the establishment of an in vitro-based cell culture system that can realistically reproduce cellular dynamics in vivo. We developed a fluoropolymer scaffold with an extracellular matrix (ECM) dot array and confirmed the possibility of mass-producing spheroids with uniform dimensions. Controlling the quality of ECM dots is important as it ensures spheroid uniformity, but issues such as pattern deviation and ECM drying persist in the conventional microstamping method. In this study, these problems were overcome via ECM dot printing using a resin mask with dot-patterned holes. For dot diameters of φ 300 μm, 400 μm, and 600 μm, the average spheroid diameters of human iPS cells (hiPSCs) were φ 260.8 μm, 292.4 μm, and 330.7 μm, respectively. The standard deviation when each average was normalized to 100 was 14.1%. A high throughput of 89.9% for colony formation rate to the number of dots and 89.3% for spheroid collection rate was achieved. The cells proliferated on ECM dots, and the colonies could be naturally detached from the scaffold without the use of enzymes, so there was almost no stimulation of the cells. Thus, the undifferentiated nature of hiPSCs was maintained until day 4. Therefore, this method is expected to be useful in drug discovery and regenerative medicine. Full article
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2022

Jump to: 2023

20 pages, 13118 KiB  
Article
HyClear: A Novel Tissue Clearing Solution for One-Step Clearing of Microtissues
by S. Soroush Nasseri, Erika M. J. Siren, Jayachandran N. Kizhakkedathu and Karen Cheung
Cells 2022, 11(23), 3854; https://doi.org/10.3390/cells11233854 - 30 Nov 2022
Cited by 1 | Viewed by 1739
Abstract
3-D cell cultures are being increasingly used as in vitro models are capable of better mimicry of in vivo tissues, particularly in drug screenings where mass transfer limitations can affect the cancer biology and response to drugs. Three-dimensional microscopy techniques, such as confocal [...] Read more.
3-D cell cultures are being increasingly used as in vitro models are capable of better mimicry of in vivo tissues, particularly in drug screenings where mass transfer limitations can affect the cancer biology and response to drugs. Three-dimensional microscopy techniques, such as confocal and multiphoton microscopy, have been used to elucidate data from 3-D cell cultures and whole organs, but their reach inside the 3-D tissues is restrained by the light scattering of the tissues, limiting their effective reach to 100–200 µm, which is simply not enough. Tissue clearing protocols, developed mostly for larger specimens usually involve multiple steps of viscous liquid submersion, and are not easily adaptable for much smaller spheroids and organoids. In this work, we have developed a novel tissue clearing solution tailored for small spheroids and organoids. Our tissue clearing protocol, called HyClear, uses a mixture of DMSO, HPG and urea to allow for one-step tissue clearing of spheroids and organoids, and is compatible with high-throughput screening studies due to its speed and simplicity. We have shown that our tissue clearing agent is superior to many of the commonly used tissue clearing agents and allows for elucidating better quality data from drug screening experiments. Full article
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17 pages, 5476 KiB  
Article
Long-Term Characteristics of Human-Derived Biliary Organoids under a Single Continuous Culture Condition
by Ranan G. Aktas, Michael Karski, Biju Issac, Liang Sun, Shira Rockowitz, Piotr Sliz and Khashayar Vakili
Cells 2022, 11(23), 3797; https://doi.org/10.3390/cells11233797 - 27 Nov 2022
Cited by 1 | Viewed by 1884
Abstract
Organoids have been used to investigate the three-dimensional (3D) organization and function of their respective organs. These self-organizing 3D structures offer a distinct advantage over traditional two-dimensional (2D) culture techniques by creating a more physiologically relevant milieu to study complex biological systems. The [...] Read more.
Organoids have been used to investigate the three-dimensional (3D) organization and function of their respective organs. These self-organizing 3D structures offer a distinct advantage over traditional two-dimensional (2D) culture techniques by creating a more physiologically relevant milieu to study complex biological systems. The goal of this study was to determine the feasibility of establishing organoids from various pediatric liver diseases and characterize the long-term evolution of cholangiocyte organoids (chol-orgs) under a single continuous culture condition. We established chol-orgs from 10 different liver conditions and characterized their multicellular organization into complex epithelial structures through budding, merging, and lumen formation. Immunofluorescent staining, electron microscopy, and single-nucleus RNA (snRNA-seq) sequencing confirmed the cholangiocytic nature of the chol-orgs. There were significant cell population differences in the transcript profiles of two-dimensional and organoid cultures based on snRNA-seq. Our study provides an approach for the generation and long-term maintenance of chol-orgs from various pediatric liver diseases under a single continuous culture condition. Full article
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21 pages, 2714 KiB  
Article
Human 3D Airway Tissue Models for Real-Time Microscopy: Visualizing Respiratory Virus Spreading
by Marion Möckel, Nino Baldok, Thorsten Walles, Roland Hartig, Andreas J. Müller, Udo Reichl, Yvonne Genzel, Heike Walles and Cornelia Wiese-Rischke
Cells 2022, 11(22), 3634; https://doi.org/10.3390/cells11223634 - 16 Nov 2022
Cited by 2 | Viewed by 1823
Abstract
Our knowledge about respiratory virus spreading is mostly based on monolayer cultures that hardly reflect the complex organization of the airway epithelium. Thus, there is a strong demand for biologically relevant models. One possibility to study virus spreading at the cellular level is [...] Read more.
Our knowledge about respiratory virus spreading is mostly based on monolayer cultures that hardly reflect the complex organization of the airway epithelium. Thus, there is a strong demand for biologically relevant models. One possibility to study virus spreading at the cellular level is real-time imaging. In an attempt to visualize virus spreading under somewhat more physiological conditions, Calu-3 cells and human primary fibroblasts were co-cultured submerged or as air-liquid interface (ALI). An influenza A virus (IAV) replicating well in cell culture, and carrying a red fluorescent protein (RFP) reporter gene was used for real-time imaging. Our three-dimensional (3D) models exhibited important characteristics of native airway epithelium including a basement membrane, tight junctions and, in ALI models, strong mucus production. In submerged models, first fluorescence signals appeared between 9 and 12 h post infection (hpi) with a low multiplicity of infection of 0.01. Virus spreading further proceeded in the immediate vicinity of infected cells. In ALI models, RFP was found at 22 hpi and later. Consequently, the progression of infection was delayed, in contrast to the submerged model. With these features, we believe that our 3D airway models can deliver new insights in the spreading of IAV and other respiratory viruses. Full article
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18 pages, 1209 KiB  
Review
Tumor Organoids as a Research Tool: How to Exploit Them
by Tijmen H. Booij, Chiara M. Cattaneo and Christian K. Hirt
Cells 2022, 11(21), 3440; https://doi.org/10.3390/cells11213440 - 31 Oct 2022
Cited by 4 | Viewed by 3522
Abstract
Organoid models allow for the study of key pathophysiological processes such as cancer biology in vitro. They offer insights into all aspects covering tumor development, progression and response to the treatment of tissue obtained from individual patients. Tumor organoids are therefore not only [...] Read more.
Organoid models allow for the study of key pathophysiological processes such as cancer biology in vitro. They offer insights into all aspects covering tumor development, progression and response to the treatment of tissue obtained from individual patients. Tumor organoids are therefore not only a better tumor model than classical monolayer cell cultures but can be used as personalized avatars for translational studies. In this review, we discuss recent developments in using organoid models for cancer research and what kinds of advanced models, testing procedures and readouts can be considered. Full article
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23 pages, 4653 KiB  
Article
Physiological Mineralization during In Vitro Osteogenesis in a Biomimetic Spheroid Culture Model
by Maximilian Koblenzer, Marek Weiler, Athanassios Fragoulis, Stephan Rütten, Thomas Pufe and Holger Jahr
Cells 2022, 11(17), 2702; https://doi.org/10.3390/cells11172702 - 30 Aug 2022
Cited by 9 | Viewed by 3240
Abstract
Bone health-targeting drug development strategies still largely rely on inferior 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings. MC3T3-E1 pre-osteoblasts were cultured in α-MEM with 10% FCS, at 37 °C and [...] Read more.
Bone health-targeting drug development strategies still largely rely on inferior 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings. MC3T3-E1 pre-osteoblasts were cultured in α-MEM with 10% FCS, at 37 °C and 5% CO2 for up to 28 days, in non-adherent V-shaped plates to form uniformly sized 3D spheroids. Osteogenic differentiation was induced by 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid. Mineralization stages were assessed through studying expression of marker genes, alkaline phosphatase activity, and calcium deposition by histochemistry. Mineralization quality was evaluated by Fourier transformed infrared (FTIR) and scanning electron microscopic (SEM) analyses and quantified by micro-CT analyses. Expression profiles of selected early- and late-stage osteoblast differentiation markers indicated a well-developed 3D biomineralization process with strongly upregulated Col1a1, Bglap and Alpl mRNA levels and type I collagen- and osteocalcin-positive immunohistochemistry (IHC). A dynamic biomineralization process with increasing mineral densities was observed during the second half of the culture period. SEM–Energy-Dispersive X-ray analyses (EDX) and FTIR ultimately confirmed a native bone-like hydroxyapatite mineral deposition ex vivo. We thus established a robust and versatile biomimetic, and high-throughput compatible, cost-efficient spheroid culture model with a native bone-like mineralization for improved pharmacological ex vivo screenings. Full article
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