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| Title | Tissue | Keywords | Material | Year | Authors | Affliations | Origin | Cell Name | Disease | In Vitro Model | DOI | Abstract | More details | hf:tax:tissues | hf:tax:disease_taxonomy | hf:tax:in_vitro_model_taxonomy |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Time-course characterization of whole-transcriptome dynamics of HepG2/C3A spheroids and its toxicological implications | Liver | 3D cell culture; TempO-Seq; cell proliferation; gene expression; metabolism. | Publication | 2024 |
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| HepG2/C3A | Cell line | 10.1016/j.toxlet.2024.10.004 | Physiologically relevant in vitro models are a priority in predictive toxicology to replace and/or reduce animal experiments. The compromised toxicant metabolism of many immortalized human liver cell lines grown as monolayers as compared to in vivo metabolism limits their physiological relevance. However, recent efforts to culture liver cells in a 3D environment, such as spheroids, to better mimic the in vivo conditions, may enhance the toxicant metabolism of human liver cell lines. In this study, we characterized the dynamic changes in the transcriptome of HepG2/C3A hepatocarcinoma cell spheroids maintained in a clinostat system (CelVivo) to gain insight into the metabolic capacity of this model as a function of spheroid size and culture time. We assessed morphological changes (size, necrotic core), cell health, and proliferation rate from initial spheroid seeding to 35 days of continuous culture in conjunction with a time-course (0, 3, 7, 10, 14, 21, 28 days) of the transcriptome (TempO-Seq, BioSpyder). The phenotypic characteristics of HepG2/C3A growing in spheroids were comparable to monolayer growth until ~Day 12 (Day 10-14) when a significant decrease in cell doubling rate was noted which was concurrent with down-regulation of cell proliferation and cell cycle pathways over this time period. Principal component analysis of the transcriptome data suggests that the Day 3, 7, and 10 spheroids are pronouncedly different from the Day 14, 21, and 28 spheroids in support of a biological transition time point during the long-term 3D spheroid cultures. The expression of genes encoding cellular components involved in toxicant metabolism and transport rapidly increased during the early time points of spheroids to peak at Day 7 or Day 10 as compared to monolayer cultures with a gradual decrease in expression with further culture, suggesting the most metabolically responsive time window for exposure studies. Overall, we provide baseline information on the cellular and molecular characterization, with a particular focus on toxicant metabolic capacity dynamics and cell growth, of HepG2/C3A 3D spheroid cultures over time. | pubmed.ncbi.nlm.nih.gov/39368564/ | liver | cell-line | |||
| Overcoming obstacles in three-dimensional cell culture model establishment: Approaches for growing A549 non-small cell lung cancer spheroids using a clinostat system | Lung | A549 cell line, Cancer, Lung | Publication | 2024 | Charity M. Mabela1, Chrisna Gouws2, Wihan Pheiffer1 |
| A549 | Cancer | Cell line | doi.org/10.1016/j.vascn.2024.107564 | Non-small cell lung cancer (NSCLC) accounts for 80–85 % of lung cancer cases globally. And the A549 cell line is widely used in pharmacological and toxicity screening. Due to its popularity as a NSCLC model, it was inevitable that three-dimensional (3D) cultures of A549 cells would be established. 3D models increase physiological relevance, and their advanced structure allows researchers to obtain more translatable and reliable results. However, establishing this cell line as a 3D model may come with challenges, like clumping. | www.sciencedirect.com/science/article/abs/pii/S1056871924000741 | lung | cancer | cell-line | |
| Optimisation of 3D iPSC organoid cultures for enhanced hepatocyte differentiation | Liver | Organoid cultures, hepatocytes | Poster | 2024 | Adelina Rogowska-Wrzesinska1, Vilde H. Bal1, Xenia S. Hermansen1, Karoline L. Mikkelsen1, Annemette Thulesen1, Tanja Christensen1 |
| IPSC | Stem cells | A human-specific model accurately representing hepatic physiology is critical for improving the predictive power of drug toxicity testing. Current models often fail to recapitulate the complexity of human liver tissue, leading to a demand for advanced methods such as iPSC-based hepatic organoids | celvivo.com/wp-content/uploads/2024/08/Poster-iPSC-organoids-ARogowska-Wrzesinska.pdf | liver | stem-cell | ||||
| Device-free isolation of photoreceptor cells from patient iPSC-derived retinal organoids | Retina | IPSC, Retina, Organoids, Patient-derived | Publication | 2024 | Nicholas E. Stone1,2, Laura R. Bohrer1,2, Nathaniel K. Mullin1,2, Jessica Cooke1,2, Allison T. Wright1,2, Edwin M. Stone1,2, Robert F. Mullins1,2, Budd A. Tucker1,2,3 |
| IPSC | Stem cells | doi.org/10.1101/2024.05.02.592255 | Autologous photoreceptor cell replacement therapy shows great promise for treating patients with multiple forms of inherited retinal degenerative blindness. Specifically, in disorders such as retinitis pigmentosa and Stargardt’s disease, selective death of photoreceptor cells results in irreversible blindness. Induced pluripotent stem cell (iPSC) derived retinal organoids, which faithfully recapitulate the structure of the neural retina, are an ideal source of photoreceptor cells required for these therapies. However, in addition to photoreceptor cells, retinal organoids also contain many other retinal cell types. Therefore, approaches for isolating fate committed photoreceptors from dissociated retinal organoids are desirable to produce photoreceptor cell replacement therapies. In this work, we present a partial dissociation strategy, which leverages the high level of organization found in retinal organoids to enable selective enrichment of photoreceptor cells without the use of specialized equipment or reagents such as antibody labels. Given that this technique can be performed with only standard plasticware and cGMP compliant reagents, it is an ideal candidate for use in the preparation of clinical cell therapies. | www.biorxiv.org/content/10.1101/2024.05.02.592255v1.full | retina | stem-cell | |||
| Advancements in 3D spheroid imaging: Optimised cryosectioning and immunostaining techniques | Spheroids, Organoid,s Antigen retrieval, Cryosectioning, Immunostaining, Fluorescent microscopy | Publication | 2023 | Claire Charlet-Faure1,2, Annemette Præstegaard Thulesen1, Adelina Rogowska-Wrzesinska1 |
| Cryosectioning | 10.1016/j.mex.2023.102415 | This article presents a modified protocol for embedding and sectioning spheroids and organoids, which are increasingly used in research due to their ability to emulate living tissue. The modifi- cations aim to reduce the distortion and damage of these fragile structures during the embedding and sectioning process. The new method involves using optimized embedding containers, a modi- fied embedding protocol, and optimized temperatures for cryosectioning. A heat-induced antigen retrieval protocol was tested and found to significantly increase immunostaining intensity with- out compromising spheroid integrity. The combined approach allowed for the creation of thinner cryosections, leading to clearer and more detailed images. The results suggest that the modified protocol could be widely adopted to enhance the imaging of spheroids and organoids. • Paraformaldehyde fixation of spheroids • Antigen retrieval treatment of spheroids • Embedding in freezing medium and cryosectioning | pubmed.ncbi.nlm.nih.gov/37860046/ | |||||||
| Omics Studies of Tumor Cells under Microgravity Conditions | microgravity; weightlessness; space; omics studies; cancer; cancer cells; genomics; transcriptomics; proteomics; metabolomics | Publication | 2024 | Jenny Graf1, Herbert Schulz1,2 , Markus Wehland1,2 , Thomas J. Corydon3,4 , Jayashree Sahana3, |
| Cancer | Stem cells | doi.org/10.3390/ijms25020926 | Cancer is defined as a group of diseases characterized by abnormal cell growth, expansion, and progression with metastasis. Various signaling pathways are involved in its development. Malignant tumors exhibit a high morbidity and mortality. Cancer research increased our knowledge about some of the underlying mechanisms, but to this day, our understanding of this disease is unclear. High throughput omics technology and bioinformatics were successful in detecting some of the unknown cancer mechanisms. However, novel groundbreaking research and ideas are necessary. A stay in orbit causes biochemical and molecular biological changes in human cancer cells which are first, and above all, due to microgravity (μg). The μg-environment provides conditions that are not reachable on Earth, which allow researchers to focus on signaling pathways controlling cell growth and metastasis. Cancer research in space already demonstrated how cancer cell-exposure to μg influenced several biological processes being involved in cancer. This novel approach has the potential to fight cancer and to develop future cancer strategies. Space research has been shown to impact biological processes in cancer cells like proliferation, apoptosis, cell survival, adhesion, migration, the cytoskeleton, the extracellular matrix, focal adhesion, and growth factors, among others. This concise review focuses on publications related to genetic, transcriptional, epigenetic, proteomic, and metabolomic studies on tumor cells exposed to real space conditions or to simulated μg using simulation devices. We discuss all omics studies investigating different tumor cell types from the brain and hematological system, sarcomas, as well as thyroid, prostate, breast, gynecologic, gastrointestinal, and lung cancers, in order to gain new and innovative ideas for understanding the basic biology of cancer. | celvivo.com/wp-content/uploads/2024/01/240115_GrafJ-GrimD_Omics-studies-of-tumorunder-microgravity_kw.pdf | cancer | stem-cell | ||||
| Planimetry measurements of spheroids in a ClinoReactor using FIJI – A step-by-step guide | Planimetry, FIJI, ClinoReactor, 3D constructs | Application note | 2024 | Annemette Præstegaard Thulesen1, Karoline Lindgaard Mikkelsen1, Helle Sedighi Frandsen PhD1 |
| Planimetry | celvivo.com/wp-content/uploads/2024/01/111_Application-note_Planimetry-measurements-of-spheroids-in-a-ClinoReactor-using-FIJI.pdf | |||||||||
| Assembling Spheroids of Rat Primary Neurons Using a Stress-Free 3D Culture System | Brain | Neurons, Nerve Tissue Engineering, Primary neuralcells, neurospheres, | Publication | 2023 | Meaghan E. Harley-Troxell and Madhu Dhar | Tissue Engineering and Regenerative Medicine, Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA | Rat | Primary cells | 10.3390/ijms241713506 | Neural injuries disrupt the normal functions of the nervous system, whose complexities limit current treatment options. Because of their enhanced therapeutic effects, neurospheres have the potential to advance the field of regenerative medicine and neural tissue engineering. Methodological steps can pose challenges for implementing neurosphere assemblies; for example, conventional static cultures hinder yield and throughput, while the presence of the necrotic core, time-consuming methodology, and high variability can slow their progression to clinical application. Here we demonstrate the optimization of primary neural cell-derived neurospheres, developed using a high-throughput, stress-free, 3D bioreactor. This process provides a necessary baseline for future studies that could develop co-cultured assemblies of stem cells combined with endothelial cells, and/or biomaterials and nanomaterials for clinical therapeutic use. Neurosphere size and neurite spreading were evaluated under various conditions using Image J software. Primary neural cells obtained from the hippocampi of three-day-old rat pups, when incubated for 24 h in a reactor coated with 2% Pluronic and seeded on Poly-D-Lysine-coated plates establish neurospheres suitable for therapeutic use within five days. Most notably, neurospheres maintained high cell viability of ≥84% and expressed the neural marker MAP2, neural marker β-Tubulin III, and glial marker GFAP at all time points when evaluated over seven days. Establishing these factors reduces the variability in developing neurospheres, while increasing the ease and output of the culture process and maintaining viable cellular constructs. | www.mdpi.com/1422-0067/24/17/13506 | brain | primary-tissue | |||
| Improved triple-negative breast cancer (TNBC) model for relevant screening of therapeutics targeting cancer stem cells | Breast | Encapsulation | Application note | 2023 | Pernille Kirkegaard Kjær1 and Helle Sedighi Frandsen2 |
| Human | MDA-MB-231 | Cancer | Cell line | Breast cancer is the most prevalent cancer affecting women and the second leading cause of cancer deaths among women [1]. Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer with the lowest survival rate [2]. This application note presents data from an experiment comparing the ability to recapitulate in vivo properties of TNBC using the MDA-MB-231 cell line cultured either in ultra-low attachment (ULA) plates or as sodium alginate droplets in the ClinoStar system., Breast cancer is the most prevalent cancer affecting women and the second leading cause of cancer deaths among women [1]. Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer with the lowest survival rate [2]. This application note presents data from an experiment comparing the ability to recapitulate in vivo properties of TNBC using the MDA-MB-231 cell line cultured either in ultra-low attachment (ULA) plates or as sodium alginate droplets in the ClinoStar system. | celvivo.com/wp-content/uploads/2023/08/110_Improved-triple-negative-breast-cancer-TNBC-model-for-relevant-screening-of-therapeutics-targetin.pdf | breast | cancer | cell-line | |
| A Novel NCI-H69AR Drug-Resistant Small-Cell Lung Cancer Mini-Tumor Model for Anti-Cancer Treatment Screening | Lung | Mini-tumor, Cancer, lung | Publication | 2023 | Alandi van Niekirk1, Krzysztof Wrzesinski1,2, Dewald Steyn1, and Chrisna Gouws1 | 1 Centre of Excellence for Pharmaceutical Sciences (Pharmacen™), North-West University, Potchefstroom 2520, South Africa | Human | NCI-H69AR | Cancer | Cell line | doi.org/10.3390/cells12151980 | Small-cell lung cancer is a fast-growing carcinoma with a poor prognosis and a high level of relapse due to multi-drug resistance (MDR). Genetic mutations that lead to the overexpression of efflux transporter proteins can contribute to MDR. In vitro cancer models play a tremendous role in chemotherapy development and the screening of possible anti-cancer molecules. Low-cost and simple in vitro models are normally used. Traditional two-dimensional (2D) models have numerous shortcomings when considering the physiological resemblance of an in vivo setting. Three-dimensional (3D) models aim to bridge the gap between conventional 2D models and the in vivo setting. Some of the advantages of functional 3D spheroids include better representation of the in vivo physiology and tumor characteristics when compared to traditional 2D cultures. During this study, an NCI-H69AR drug-resistant mini-tumor model (MRP1 hyperexpressive) was developed by making use of a rotating clinostat bioreactor system (ClinoStar®; CelVivo ApS, Odense, Denmark). Spheroid growth and viability were assessed over a 25-day period to determine the ideal experimental period with mature and metabolically stable constructs. The applicability of this model for anti-cancer research was evaluated through treatment with irinotecan, paclitaxel and cisplatin for 96 h, followed by a 96 h recovery period. Parameters measured included planar surface area measurements, estimated glucose consumption, soluble protein content, intracellular adenosine triphosphate levels, extracellular adenylate kinase levels, histology and efflux transporter gene expression. The established functional spheroid model proved viable and stable during the treatment period, with retained relative hyperexpression of the MRP1 efflux transporter gene but increased expression of the P-gp transporter gene compared to the cells cultured in 2D. As expected, treatment with the abovementioned anti-cancer drugs at clinical doses (100 mg/m2 irinotecan, 80 mg/m2 paclitaxel and 75 mg/m2 cisplatin) had minimal impact on the drug-resistant mini-tumors, and the functional spheroid models were able to recover following the removal of treatment. | www.mdpi.com/2073-4409/12/15/1980 | lung | cancer | cell-line |
| Osteocyte-Like Cells Differentiated From Primary Osteoblasts in an Artificial Human Bone Tissue Model | Bone | Organoids, Osteoblasts, Osteocytes, Spheroids, Artificial Bone Tissue | Publication | 2023 | Arooj Munir1, Janne Elin Reseland1, Hanna Tiainen,1, Håvard Jostein Haugen1, Pawel Sikorski2, Emil Fran Christiansen2, Finn Per Reinholt3, Unni Syversen4, Lene Bergendal Solberg3,5 | 1Department of Biomaterials, Institute of Clinical Dentistry, University of Oslo, Oslo, Norway 2 Department of Physics, Norwegian University of Science and Technology (NTNU), Trondheim, Norway 3 Department of Pathology, Oslo University Hospital, Oslo, Norway 4 Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway 5 Division of Orthopedic Surgery, Oslo University Hospital, Oslo, Norway | Human | Primary cells | doi.org/10.1002/jbm4.10792 | In vitro models of primary human osteocytes embedded in natural mineralized matrix without artificial scaffolds are lacking. We have established cell culture conditions that favored the natural 3D orientation of the bone cells and stimulated the cascade of signaling needed for primary human osteoblasts to differentiate into osteocytes with the characteristically phenotypical dendritic network between cells. Primary human osteoblasts cultured in a 3D rotating bioreactor and incubated with a combination of vitamins A, C, and D for up to 21 days produced osteospheres resembling native bone. Osteocyte-like cells were identified as entrapped, stellate-shaped cells interconnected through canaliculi embedded in a structured, mineralized, collagen matrix. These cells expressed late osteoblast and osteocyte markers such as osteocalcin (OCN), podoplanin (E11), dentin matrix acidic phosphoprotein 1 (DMP1), and sclerostin (SOST). Organized collagen fibrils, observed associated with the cell hydroxyapatite (HAp) crystals, were found throughout the spheroid and in between the collagen fibrils. In addition to osteocyte-like cells, the spheroids consisted of osteoblasts at various differentiation stages surrounded by a rim of cells resembling lining cells. This resemblance to native bone indicates a model system with potential for studying osteocyte-like cell differentiation, cross-talk between bone cells, and the mineralization process in a bonelike structure in vitro without artificial scaffolds. In addition, natural extracellular matrix may allow for the study of tissue-specific biochemical, biophysical, and mechanical properties. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. | asbmr.onlinelibrary.wiley.com/doi/10.1002/jbm4.10792 | bone | primary-tissue | |||
| How to optimize respiratory models for SARS-CoV-2 research | Lung | SARS-CoV-2, Lung | Publication | 2022 | Wilfried Posch1, Stefanie Dichtl1, Viktoria Zaderer1, Cornelia Lass-Flörl1, Doris Wilflingseder1 | 1Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Austria | Human | Infectious Diseases | Primary cells | doi.org/10.26124/bec:2022-0009 | Sophisticated 3D cell culture tissue modelsexperienced a boomin the last years and in particular human cell culture and 3D respiratory systems greatly supported the development of novel drugs and vaccines during the SARS-CoV-2 pandemic. These models provide multiple benefits in terms of similarities in differentiation, metabolism, receptor expression, polarity, and infectivity compared to human tissues and thus provide excellent models to study the first interactions with the host during pathogen entry. Dependingon the experimental approach, the use of 3D models isbeneficial–apical-out lung organoids forhigh content screening (HCS) of treatment optionsandair-liquid interphase (ALI) models foreasy incorporation of immune cells, screeningof epithelial integrityor mucociliary clearance. This review givesan overview on the models established in our laboratory and their applications. | www.bioenergetics-communications.org/index.php/bec/article/view/posch_2022/97 | lung | infectious-diseases | primary-tissue | |
| Characterization of an Australian outbreak Japanese Encephalitis virus genotype 4 isolate in mice and human cortical brain organoids | Brain | Japanese Encephalitis Virus, neuroscience | Publication | 2023 | Wilson Nguren1, Romal Stewart1, Bing Tang1, Kexin Yan1, Thibaut Larcher2, Cameron Bishop1, Narayan Gyawali1, Gregor Devine1, Andreas Suhrbier1,3, Daniel J. Rawle1. |
| Human | Infectious Diseases | Cell line | 10.1038/s44298-024-00025-5 | Human infections with the Japanese encephalitis virus (JEV) are a leading cause of viral encephalitis. An unprecedented outbreak of JEV genotype 4 was recently reported in Australia, with an isolate (JEVNSW/22) obtained from a stillborn piglet brain. Herein we conduct a thorough characterization of JEVNSW/22 in three different mouse strains and in human cortical brain organoids (hBOs), and determined the ability of JEVNSW/22 to be neutralized by sera from humans vaccinated with IMOJEV. JEVNSW/22 was less virulent than JEVFU (genotype 2) and JEVNakayama (genotype 3) in C57BL/6J mice and in interferon regulatory factor 7 deficient (Irf7−/−) mice, with infection of wild-type and knockout murine embryonic fibroblasts indicating JEVNSW/22 is more sensitive to type I interferon responses. Irf7−/− mice provide a new model for JEVNSW/22, showing higher viremia levels compared to C57BL/6J mice, and allowing for lethal neuroinvasive infection. All JEV strains were universally lethal in Ifnar−/− mice by day 3, with histological signs of brain hemorrhage, but no other lesions. There were no indications of brain infection in Ifnar−/− mice, with viral protein detected in blood vessels, but not neurons. All JEV isolates showed robust cytopathic infection of human cortical brain organoids, albeit lower for JEVNSW/22. IMOJEV vaccination in humans induced antibodies capable of neutralizing JEVNSW/22, although, for all JEV strains, cross-neutralization titers declined with increasing divergence from IMOJEV in the envelope amino acid sequences. Overall, our study establishes JEVNSW/22 mouse and hBO models of infection, allowing for possible lethal neuroinvasive infection in mice that was rarer than for other JEV genotypes. JEV vaccination regimens may afford protection against this newly emerged JEV genotype 4 strain, although neutralizing antibody responses are sub-optimal. | www.nature.com/articles/s44298-024-00025-5 | brain | infectious-diseases | cell-line | |
| Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue | Liver | Histone Post-Translational Modifications, LC/MS | Protocol | 2022 | Jazmine-Saskya N. Joseph-Chowdhury1, Stephanie Stransky1, Sarah Graff1, Ronald Cutler1, Dejauwne Young1, Julie S. Kim1, Carlos Madrid-Aliste1, Jennifer T. Aguilan1, Edward Nieves1, Yan Sun1, Edwin J. Yoo1, Simone Sidoli1 |
| Human | HepG2/C3A | Cell line | DOI: 10.3791/63606-v | This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state. | www.jove.com/v/63606/global-level-quantification-histone-post-translational-modifications | liver | cell-line | ||
| A review of challenges and prospects of 3D cell-based culture models used for studying drug induced liver injury during early phases of drug development | Liver | DILI, HepG2/C3A, Drug development | Publication | 2023 | John K Chipangura1, Yonela Ntamo2, Bert Mohr1 and Nireshni Chellan2, 3 |
| Human | Toxicity | Primary cells | doi.org/10.1177/09603271221147884 | Drug-induced liver injury (DILI) is the leading cause of compound attrition during drug development. Over the years, a battery of in-vitro cell culture toxicity tests is being conducted to evaluate the toxicity of compounds prior to testing in laboratory animals. Two-dimensional (2D) in-vitro cell culture models are commonly used and have provided a great deal of knowledge; however, these models often fall short in mimicking natural structures of tissues in-vivo. Testing in humans is the most logical method, but unfortunately there are ethical limitations associated with human tests. To overcome these limitations better human-relevant, predictive models are required. The past decade has witnessed significant efforts towards the development of three-dimensional (3D) in-vitro cell culture models better mimicking in-vivo physiology. 3D cell culture has advantages in being representative of the interactions of cells in-vivo and when validated can act as an interphase between 2D cell culture models and in-vivo animal models. The current review seeks to provide an overview of the challenges that make biomarkers used for detection of DILI not to be sensitive enough during drug development and explore how 3D cell culture models can be used to address the gap with the current models. | journals.sagepub.com/doi/10.1177/09603271221147884 | liver | toxicity | primary-tissue | |
| Recombinant irisin enhances the extracellular matrix formation, remodeling potential, and differentiation of human periodontal ligament cells cultured in 3D | Bone | Stem cells, hPDL, hDPC | Publication | 2023 | Yang Yang 1, Tianxiang Geng 1, Athina Samara 1, Ole Kristoffer Olstad 2, Jianying He 3, Anne Eriksson Agger 1, Bjørn Helge Skallerud 3, Maria A Landin 4, Catherine Anne Heyward 4, Helen Pullisaar 5, Janne Elin Reseland 1 |
| Human | human periodontal ligament cells | Primary cells | DOI: 10.1111/jre.13094 | Background: Irisin is expressed in human periodontal ligament (hPDL), and its administration enhances growth, migration and matrix deposition in hPDL cells cultured in monolayers in vitro. Objectives: To identify whether irisin affects the gene expression patterns directing the morphology, mechanical properties, extracellular matrix (ECM) formation, osteogenic activity and angiogenic potential in hPDL cell spheroids cultured in 3D. Materials and methods: Spheroids of primary human hPDL cells were generated in a rotational 3D culture system and treated with or without irisin. The gene expression patterns were evaluated by Affymetrix microarrays. The morphology of the spheroids was characterized using histological staining. Mechanical properties were quantified by nanoindentation. The osteogenic and angiogenic potential of spheroids were assessed through immunofluorescence staining for collagen type I, periostin fibronectin and von Willebrand factor (vWF), and mRNA expression of osteogenic markers. The secretion of multiple myokines was evaluated using Luminex immunoassays. Results: Approximately 1000 genes were differentially expressed between control and irisin-treated groups by Affymetrix. Several genes related to ECM organization were differentially expressed, and multiple deubiquitinating enzymes were upregulated in the irisin-exposed samples analyzed. These represent cellular and molecular mechanisms indicative of a role for irisin in tissue remodeling. Irisin induced a rim-like structure on the outer region of the hPDL spheroids, ECM-related protein expression and the stiffness of the spheroids were enhanced by irisin. The expression of osteogenic and angiogenetic markers was increased by irisin. Conclusions: Irisin altered the morphology in primary hPDL cell-derived spheroids, enhanced its ECM deposition, mechanical properties, differentiation and remodeling potential. Keywords: extracellular matrix; irisin; mechanical properties; osteogenesis; periodontal ligament cells; three-dimensional culture. | pubmed.ncbi.nlm.nih.gov/36625247/ | bone | primary-tissue | ||
| SARS-CoV-2 omicron BA.5 and XBB variants have increased neurotropic potential over BA.1 in K18-hACE2 mice and human brain organoids | Brain | Covid-19, neuroscience, brain, organoid, XBB, K18-hACE2 | Publication | 2023 | Romal Stewart1, Sevannah A. Ellis2, Kexin Yan1, Troy Dumenil1, Bing Tang1, Wilson Nguyen1, Cameron Bishop1, Thibaut Larcher3, Julian De Jun Sng4, Rhys Parry4, Robert K. P. Sullivan2, Mary Lor1, Alexander A. Khromykh4, Frédéric A. Meunier2, Daniel J. Rawle1, Andreas Suhrbier1 |
| Human | iPSC | Infectious Diseases | Stem cells | 10.3389/fmicb.2023.1320856 | The reduced pathogenicity of the omicron BA.1 sub-lineage compared to earlier variants is well described, although whether such attenuation is retained for later variants like BA.5 and XBB remains controversial. We show that BA.5 and XBB isolates were significantly more pathogenic in K18-hACE2 mice than a BA.1 isolate, showing increased neurotropic potential, resulting in fulminant brain infection and mortality, similar to that seen for original ancestral isolates. BA.5 also infected human cortical brain organoids to a greater extent than the BA.1 and original ancestral isolates. In the brains of mice, neurons were the main target of infection, and in human organoids neuronal progenitor cells and immature neurons were infected. The results herein suggest that evolving omicron variants may have increasing neurotropic potential. | www.ncbi.nlm.nih.gov/pmc/articles/PMC10706942/ | brain | infectious-diseases | stem-cell |
| Bovine blastocyst like structures derived from stem cell cultures | Embryo | Blastocyst, trophoblast stem cells, embryonic stem cells | Publication | 2023 | Carlos A. Pinzón-Arteaga1, Yinjuan Wang2, Yulei Wei1,3, Leijie Li4,3, Ana Elisa Ribeiro Orsi1,5, Giovanna Scatolin2, Lizhong Liu1, Masahiro Sakurai1, Jianfeng Ye6, Leqian Yu1,7,8, Bo Li6,4, Zongliang Jiang2,9, Jun Wu1,10,11 |
| Bovine | IPSC, trophoblast stem cells, embryonic stem cells | Stem cells | doi.org/10.1016/j.stem.2023.04.003 | Understanding blastocyst formation and implantation is critical for improving farm animal reproduction but is hampered by a limited supply of embryos. We developed an efficient method to generate bovine blastocyst-like structures (termed blastoids) via the assembly of trophoblast stem cells and expanded potential stem cells. Bovine blastoids resemble blastocysts in morphology, cell composition, single-cell transcriptomes, and represent an accessible in vitro model for studying bovine embryogenesis. | www.cell.com/cell-stem-cell/fulltext/S1934-5909(23)00121-2 | embryo | stem-cell | ||
| HepG2/C3A and HMEC1 co-culture spheroids with forced aggregation | Liver | Co-culture | Application note | 2021 | Helle Sedighi Frandsen1 |
| Human | HepG2, HMEC-1 | Cancer | Cell line | celvivo.com/wp-content/uploads/simple-file-list/Application-notes/109_Co-culture_HEPG2-C3A_and_HMEC1_forced_aggregation.pdf | liver | cancer | cell-line | ||
| Creating Sodium Alginate beads directly in ClinoReactor using a Triple-Negative Breast Cancer (TNBC) cell line | Breast | Encapsulation | Application note | 2022 | Helle Sedighi Frandsen1 and Louise Leth Hefting1 |
| Human | MDA-MB-231 | Cancer | Cell line | Sodium alginate embedding provides a means to create 3D cell conglomerates [1]. It is particularly useful for production of constructs of cells which are challenging in single cell suspension or pre-aggregation setup [2]. Constructs created using this protocol, are of similar size and contain similar starting cell number. These constructs provide a structural microenvironment for minimum 28 days, preserve cell viability, and allow cell growth. Constructs made using colored sodium alginate have increased visibility for easier handling. It is important to note that during all preparation steps, solutions without calcium and magnesium ions should be used, as those ions otherwise will trigger sodium alginate polymerization. | celvivo.com/wp-content/uploads/simple-file-list/Application-notes/108_Sodium-alginate-dotted-into-CR-using-Triple-Negative-Breast-Cancer-Cell-Line.pdf | breast | cancer | cell-line | |
| Spheroid Co-culture of Breast Cancer Cell Line (MCF7) and Normal Breast Epithelial Cell Line (MCF-12A) | Breast | Co-culture | Application note | 2022 | José Luis Cortés Sánchez1, Michalina Maria Czaplicka1, Jenny Graf1, Louise Leth Hefting2 |
| Human | MCF-7, MCF-12A | Cancer | Cell line | This application note describes the procedure for culturing the breast cancer cell line MCF-7 together with the MCF-12A normal breast epithelial cell line in the CelVivo ClinoStar System from a single cell suspension subculture. Co-culturing of different cell types adds to the complexity of the 3D tissue, and it is possible to study cellular communication. It is convenient to be able to track if the cell type of interest is present during live culture. The application note at hand describes a method using transient transfection of CellLight™ ER-GFP to track MCF-12A. | celvivo.com/wp-content/uploads/simple-file-list/Application-notes/107_Co-culture-of-Breast-MCF-7-MCF-12A.pdf | breast | cancer | cell-line | |
| Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line | Liver | Metabolic alterations, Drug treatment | Publication | 2012 | Stephen J. Fey1 and Krzysztof Wrzesinsk1 |
| Human | HepG2 | Toxicity | Cell line | DOI: 10.1093/toxsci/kfs122 | Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal of this study was to investigate whether this observation could be extended to the determination of LD(50) values and whether 3D data could be correlated to in vivo observations. We developed a noninvasive means to estimate the amount of protein present in a 3D spheroid from it is planar area (± 21%) so that a precise dose can be provided in a manner similar to in vivo studies. This avoided correction of the actual dose given based on a protein determination after treatment (when some cells may have lysed). Conversion of published in vitro LC(50) data (mM) for six common drugs (acetaminophen, amiodarone, diclofenac, metformin, phenformin, and valproic acid) to LD(50) data (mg compound/mg cellular protein) showed that the variation in LD(50) values was generally less than that suggested by the original LC(50) data. Toxicological analysis of these six compounds in 3D spheroid culture (either published or presented here) demonstrated similar LD(50) values. Although in vitro 2D HepG2 data showed a poor correlation, the primary hepatocyte and 3D spheroid data resulted in a much higher degree of correlation with in vivo lethal blood plasma levels. These results corroborate that 3D hepatocyte cultures are significantly different from 2D cultures and are more representative of the liver in vivo. | www.ncbi.nlm.nih.gov/pmc/articles/PMC3355318/pdf/kfs122.pdf | liver | toxicity | cell-line |
| After trypsinisation, 3D spheroids of C3A hepatocytes need 18 days to re-establish similar levels of key physiological functions to those seen in the liver | Liver | Metabolic alterations | Publication | 2013 | Krzysztof Wrzesinski1 and Stephen J. Fey1 |
| Human | HepG2 | Cell line | doi.org/10.1039/C2TX20060K | The introduction of trypsinisation in the 1950's was a paradigm shift which helped instigate cell culture. We demonstrate here that human hepatocyte cell line HepG2–C3A needs at least 18 days after trypsinisation to re-establish key ultrastructural and physiological traits. After trypsinisation, cells start to recover these traits at similar rates in both monolayer (2D) or spheroid (3D) growth environments. While this development is restarted by trypsinisation of 2D cultures (typically after 5 days), recovery continues in 3D cultures up until 15–18 days when changes in growth rate, adenylate kinase, ATP, urea and cholesterol all suggest that spheroids undergo some type of physiological transition. Several other cell lines (e.g. Caco-2, HT 29, MDCK, MCF-10A and HepG2 used to model the small and large intestine, kidney, breast acini and liver respectively) are reported in the literature to exhibit very similar changes, on a similar timescale to those reported here. These changes may thus represent a ubiquitous recovery process after trypsinisation rather than differentiation. This would partially explain the common observation that cells grown in 3D exhibit physiological capabilities that are closer to those seen in the intact tissue or organ. | pubs.rsc.org/en/content/articlelanding/2013/tx/c2tx20060k | liver | cell-line | ||
| HepG2/C3A 3D spheroids exhibit stable physiological functionality for at least 24 days after recovering from trypsinisation | Liver | Metabolic alterations | Publication | 2013 | Krzysztof Wrzesinski, Maria Chiara Magnone, Line Visby Hansen, Marianne Ehrhorn Kruse, Tobias Bergauer, Maria Bobadilla, Marcel Gubler, Jacques Mizrahi, Kelan Zhang, Christina M. Andreasen, Kira Eyð Joensen, Signe Marie Andersen, Jacob Bastholm Olesen, Ove B. Schaffalitzky de Muckadell, Stephen J. Fey | Human | HepG2 | Cell line | doi.org/10.1039/c3tx20086h | Primary human hepatocytes are widely used as an in vitro system for the assessment of drug metabolism and toxicity. Nevertheless a cell system with higher stability of physiological functions is required for the investigation of drugs' mode of action, pathway analyses and biomarkers evaluations. We recently discovered that the human hepatocellular carcinoma cell line, HepG2/C3A, cultured as spheroids in a 3D system can recover their main functions after trypsinisation within about 18 days. The objective of this study was to investigate whether the spheroids' metabolic functions remained stable after this recovery period. Therefore we evaluated physiological capabilities of the spheroids (cell survival, growth rate, glycogenesis, ATP, cholesterol and urea synthesis and drug metabolism) and the expression of key genes related to the main liver pathways in spheroids cultured for an additional 24 days after full recovery (day 18). Here we show that after the recovery period, the 3D spheroid culture can provide a metabolically competent homeostatic cell model which is in equilibrium with its culture environment for more than 3 weeks. Such a stable system could be used for the assessment of the drugs' mode of action, for biomarkers evaluation and for any systems biology studies which require medium- to long-term stability of metabolic functions. | academic.oup.com/toxres/article/2/3/163/5575408?login=false | liver | cell-line | |||
| Determination of acute lethal and chronic lethal dose thresholds of valproic acid using 3D spheroids constructed from the immortal human hepatocyte cell line HepG2/C3A | Liver | Chronic toxicity | Publication | 2012 | Fey, S.J.,1 Wrzesinski, K1. |
| Human | HepG2 | Toxicity | Cell line | ISBN: 978-162417952-5 | Valproic acid (VPA) is a broad-spectrum antiepileptic drug that is now used commonly for several other neurological and psychiatric indications. While VPA is usually well tolerated, on rare occasions, it has been associated with severe, and sometimes, fatal liver injuries. These complications may also arise due to acute VPA overdose. As a branched chain carboxylic acid, VPA is readily metabolized in the liver via glucuronic acid conjugation, mitochondrial β- and cytosolic ω-oxidation to produce multiple metabolites, and it is probably some of these metabolites that are involved in its toxicity. While the actual mechanism of VPA hepatotoxicity is still not understood, two pathways have been in focus: the first involves the formation of reactive metabolites of VPA (and their subsequent covalent binding to cellular proteins) and the second is the development of oxidative stress in the cell. We describe here a culture system based on 3D spheroid culture of immortal hepatocytes which can determine the toxicity of valproic acid (or structurally or functionally related molecules) in vitro. The spheroids were used to follow changes in ATP production, glucose uptake and adenylate kinase following treatment and can be treated repeatedly with VPA to determine both acutely-lethal and chronically-lethal doses. This system is homeostatic and has been shown to be metabolically stable over at least 24 days and can therefore be used to follow recovery after treatment. © 2013 Nova Science Publishers, Inc. All rights reserved. | www.scopus.com/record/display.uri?eid=2-s2.0-84891981522&origin=inward&txGid=3811173f6556a8dc4eb561c5cbe16f9c | liver | toxicity | cell-line |
| Microgravity spheroids as a reliable, long-term tool for predictive toxicology | Liver | Long term culture | Publication | 2014 | Stephen J.Fey1 Krzysztof Wrzesinski1 |
| Human | HepG2 | Cell line | doi.org/10.1016/j.toxlet.2013.05.318 | www.sciencedirect.com/science/article/abs/pii/S0378427413005183?via%3Dihub | liver | cell-line | |||
| Heteromer score-using internal standards to assess the quality of proteomic data | Liver | Bioinformatics | Publication | 2014 | Adelina Rogowska-Wrzesinska 1, Krzysztof Wrzesinski1, Stephen J Fey1 |
| Human | HepG2 | Cell line | DOI: 10.1002/pmic.201300457 | In the cell, the majority of proteins exist in complexes. Most of these complexes have a constant stoichiometry and thus can be used as internal standards. In this rapid communication, we show that it is possible to calculate a correlation coefficient that reflects the reproducibility of the analytical approach used. The abundance of one subunit in a heterodimer is plotted against the abundance of the other, and this is repeated for all subunits in all heteromers found in the data set. The correlation coefficient obtained (the "heteromer score") is a new bioinformatic tool that is independent of the method used to collect the data, requires no special sample preparation and can be used retrospectively on old datasets. It can be used for quality control, to indicate when a change becomes significant or identify complexes whose stoichiometry has been perturbed during the experiment. Keywords: Bioinformatics; Cell biology; Heterodimer; Heteromer score; Internal standard; Quality control. | analyticalsciencejournals.onlinelibrary.wiley.com/doi/abs/10.1002/pmic.201300457 | liver | cell-line | ||
| The Cultural Divide: Exponential Growth in Classical 2D and Metabolic Equilibrium in 3D Environments | Liver | Metabolic alterations | Publication | 2013 | Krzysztof Wrzesinski, 1 Adelina Rogowska-Wrzesinska, 2 Rattiyaporn Kanlaya, 2 ,Kamil Borkowski, 3 Veit Schwämmle, 2 Jie Dai, 2 Kira Eyd Joensen, 1 Katarzyna Wojdyla, 2 Vasco Botelho Carvalho, 1 and Stephen J. Fey 1 |
| Human | HepG2 | Cell line | DOI: 10.1371/journal.pone.0106973 | Introduction: Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different. Results: Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell--along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex. Summary: We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance. | www.ncbi.nlm.nih.gov/pmc/articles/PMC4164521/ | liver | cell-line | ||
| Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns | Liver | Proteomics | Publication | 2015 | Andrey Tvardovskiy 1, Krzysztof Wrzesinski 2, Simone Sidoli 1, Stephen J Fey 2, Adelina Rogowska-Wrzesinska 1, Ole N Jensen 3 |
| Human | HepG2 | Cell line | DOI: 10.1074/mcp.M115.048975 | Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive analysis of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Analysis of co-existing PTMs revealed negative crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3. | www.mcponline.org/article/S1535-9476(20)32828-0/fulltext | liver | cell-line | ||
| From 2D to 3D--a New Dimension for Modelling the Effect of Natural Products on Human Tissue | Liver | Metabolic alterations | Publication | 2015 | Krzysztof Wrzesinski, Stephen J Fey 1 |
| Human | HepG2 | Cell line | DOI: 10.2174/1381612821666151002114227 | Natural products, or their synthetic derivatives are a treasure trove to find potential candidates for novel drugs for human treatment. The selection of diamonds from the huge pile of worthless stone is a critical – and difficult - stage in the discovery pipeline. Of all the factors to be considered, perhaps the most important, is that the compound should have the desired effect on the tissue in vivo. Since it is not possible (or ethical) to test all compounds in vivo one must preselect using a surrogate assay system. While animal models have the advantage of being holistic and current 3D culture systems are reductionistic, they at least can be constructed from human cell types. In this review we will consider some of the evidence demonstrating that cells grown in 3D cultures have physiological performances that mimic functions seen in human tissues significantly better than cells grown using classical 2D culture systems. We will discuss advantages and disadvantages of these new culture technologies and highlight theoretical reasons for the differences. 3D cell culture technologies are more labour intensive than 2D culture systems and therefore their introduction is a trade-off between the value of obtaining data that is more relevant to the human condition against their through-put. It is already clear that future in vitro 3D systems will become more complex, using multiple cell types to more faithfully represent a particular tissue or even organ system. And one thing is sure – the diamonds are not easy to find! | www.eurekaselect.com/article/70843 | liver | cell-line | ||
| Acetaminophen-induced S-nitrosylation and S-sulfenylation signalling in 3D cultured hepatocarcinoma cell spheroids | Liver | Drug treatment | Publication | 2016 | Katarzyna Wojdyla 1, Krzysztof Wrzesinski 2, James Williamson 1, Stephen J Fey 2, Adelina Rogowska-Wrzesinska 1 |
| Human | HepG2 | Toxicity | Cell line | DOI: 10.1039/c5tx00469a | Acetaminophen (APAP) is possibly the most widely used medication globally and yet little is known of its molecular effects at therapeutic doses. Using a novel approach, we have analysed the redox proteome of the hepatocellular cell line HepG2/C3A treated with therapeutic doses of APAP and quantitated both individual protein abundance and their reversible S-nitrosylation (SNO) and S-sulfenylation (SOH) modifications by mass spectrometry. APAP treatment results in a late, transient increase in ATP production and a multiplicity of alterations in protein abundance and modifications. The majority of the differentially SNO or SOH modified proteins are found in the endoplasmic reticulum and cytosol, suggesting that the source of reactive species is there. The cellular response indicates: constraint of fatty acid metabolism; reduction in ribosome construction and protein synthesis (to conserve ATP); maintenance of glutathione levels (by increased synthetic capacity); and an increased NADPH production (via the pentose phosphate pathway). This response appears to be coordinated, directly or indirectly, by the canonical Wnt and Nrf2 signalling pathways. Combined with the known role of NAPQI, these studies suggest that the physiological and toxicological responses form a continuum: therapeutic doses of APAP produce reactive species and NAPQI in the cytoplasm but result in little permanent damage. The cell mounts a multifaceted response which minimises disruption and repairs are effected within a day or two. Higher doses of APAP lead to intensified reactive species production, which increasingly disturbs mitochondrial function and eventually leads to cell death. | www.ncbi.nlm.nih.gov/pmc/articles/PMC6072433/ | liver | toxicity | cell-line |
| Recent advances in three-dimensional cell culturing to assess liver function and dysfunction: from a drug biotransformation and toxicity perspective | Liver | DILI | Publication | 2018 | Carlemi Calitz 1, Josias H Hamman 1, Stephen J Fey 2, Krzysztof Wrzesinski 2, Chrisna Gouws 1 |
| Human | HepG2 | Toxicity | Cell line | DOI: 10.1080/15376516.2017.1422580 | The liver is a vital organ fulfilling a central role in over 500 major metabolic functions, including serving as the most essential site for drug biotransformation. Dysfunction of the drug biotransformation processes may result in the exposure of the liver (and other organs) to hepatotoxins, potentially interacting with cellular constituents and causing toxicity and various lesions. Hepatotoxicity can be investigated on a tissue, cellular and molecular level by employing various in vivo and in vitro techniques, including novel three-dimensional (3 D) cell culturing methods. This paper reflects on the liver and its myriad of functions and the influence of drug biotransformation on liver dysfunction. Current in vivo and in vitro models used to study liver function and dysfunction is outlined, emphasizing their advantages and disadvantages. The advantages of novel in vitro 3 D cell culture models are discussed and the possibility of novel models to bridge the gap between in vitro and in vivo models is explained. Progression made in the field of cell culturing methods such as 3 D cell culturing techniques over the last decade promises to reduce the use of in vivo animal models in biotransformation and toxicological studies of the liver. Keywords: 3D models; drug biotransformation; drug toxicity; in vitro models; liver dysfunction. | www.tandfonline.com/doi/abs/10.1080/15376516.2017.1422580?journalCode=itxm20 | liver | toxicity | cell-line |
| The amended cell singnalling of in vivo-mimetic 3D structures | Liver | Drug Treatment | Poster | 2020 | Krzysztof Wrzesinski1 and Stephen J. Fey1 |
| Human | HepG2 | Cell line | celvivo.com/wp-content/uploads/2022/12/200921_Conference_Poster_final_header.pdf | liver | cell-line | ||||
| Cell-free DNA in a three-dimensional spheroid cell culture model: A preliminary study | Liver | cfDNA | Publication | 2017 | Janine Aucamp 1, Carlemi Calitz 2, Abel J Bronkhorst 3, Krzysztof Wrzesinski 4, Sias Hamman 2, Chrisna Gouws 2, Piet J Pretorius 3 |
| Human | HepG2 | Cell line | DOI: 10.1016/j.biocel.2017.06.014 | Background: Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo state, may be of significant benefit for cfDNA research. Methods: CfDNA was isolated from the growth medium of C3A spheroid cultures in rotating bioreactors during both normal growth and treatment with acetaminophen. Spheroid growth was monitored via planimetry, lactate dehydrogenase activity and glucose consumption and was related to isolated cfDNA characteristics. Results: Changes in spheroid growth and stability were effectively mirrored by cfDNA characteristics. CfDNA characteristics correlated with that of previous two-dimensional (2D) cell culture and human plasma research. Conclusions: 3D spheroid cultures can serve as effective, simplified in vivo-simulating "closed-circuit" models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. Biological significance: 3D cell cultures can be used to translate "closed-circuit" in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth environment. Combining 3D culture and cfDNA research could, therefore, optimize both research fields. Keywords: 3D cell culture; Capillary electrophoresis; Cell-free DNA; Rotating bioreactors; Spheroid. | www.sciencedirect.com/science/article/abs/pii/S1357272517301528?via%3Dihub | liver | cell-line | ||
| Metabolic Reprogramming and the Recovery of Physiological Functionality in 3D Cultures in Micro-Bioreactors | Liver | Metabolic reprogramming | Publication | 2018 | Krzysztof Wrzesinski 1, 2, Stephen J Fey 3, 4 |
| Human | HepG2 | Cell line | DOI: 10.3390/bioengineering5010022 | The recovery of physiological functionality, which is commonly seen in tissue mimetic three-dimensional (3D) cellular aggregates (organoids, spheroids, acini, etc.), has been observed in cells of many origins (primary tissues, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and immortal cell lines). This plurality and plasticity suggest that probably several basic principles promote this recovery process. The aim of this study was to identify these basic principles and describe how they are regulated so that they can be taken in consideration when micro-bioreactors are designed. Here, we provide evidence that one of these basic principles is hypoxia, which is a natural consequence of multicellular structures grown in microgravity cultures. Hypoxia drives a partial metabolic reprogramming to aerobic glycolysis and an increased anabolic synthesis. A second principle is the activation of cytoplasmic glutaminolysis for lipogenesis. Glutaminolysis is activated in the presence of hypo- or normo-glycaemic conditions and in turn is geared to the hexosamine pathway. The reducing power needed is produced in the pentose phosphate pathway, a prime function of glucose metabolism. Cytoskeletal reconstruction, histone modification, and the recovery of the physiological phenotype can all be traced to adaptive changes in the underlying cellular metabolism. These changes are coordinated by mTOR/Akt, p53 and non-canonical Wnt signaling pathways, while myc and NF-kB appear to be relatively inactive. Partial metabolic reprogramming to aerobic glycolysis, originally described by Warburg, is independent of the cell's rate of proliferation, but is interwoven with the cells abilities to execute advanced functionality needed for replicating the tissues physiological performance. Keywords: 3D cell culture; Warburg; aerobic glycolysis; bioreactors; glutaminolysis; hypoxia; metabolic reprogramming; organoids; physiological performance; spheroids. | www.mdpi.com/2306-5354/5/1/22 | liver | cell-line | ||
| Toxicity and anti-prolific properties of Xysmalobium undulatum water extract during short-term exposure to two-dimensional and three-dimensional spheroid cell cultures | Liver | Drug treatment | Publication | 2022 | Carlemi Calitz 1, Josias H Hamman 1, Alvaro M Viljoen 2, Stephen J Fey 3, 4, Krzysztof Wrzesinski 3, 4, Chrisna Gouws 1 |
| Human | HepG2 | Toxicity | Cell line | DOI: 10.1080/15376516.2018.1485805 | Xysmalobium undulatum (Uzara) is one of the most widely used indigenous traditional herbal remedies in Southern Africa. Commercially available Uzara plant material was used to prepare a crude aqueous extract, of which the toxicity potential was investigated in the hepatic HepG2/C3A cell line in both traditional two-dimensional (2D) and rotating three-dimensional (3D) spheroid cell cultures. These cultures were treated over a period of 4 days at concentrations of 200, 350, 500, and 750 mg/kg plant extract to protein content. Basic physiological parameters of the cell cultures were measured during exposure, including cell proliferation, glucose uptake, intracellular adenosine triphosphate levels, and adenylate kinase release. The results indicated that all physiological parameters monitored were affected in a dose dependent manner, with the highest concentration of Uzara crude water extract (750 mg/kg) resulting in toxicity. Anti-proliferating effects of Uzara crude water extract were observed in both the 2D and 3D cell cultures, with the most pronounced effects at concentrations of 350, 500, and 750 mg/kg. Discrepancies between results obtained from the 2D and 3D cell culture models may be attributed to the type of repair system that is initiated upon exposure, depending on where cells are within the cell cycle. DNA repair systems differ in cells within the G1 phase and non-diving cells, (i.e. cells found predominantly in in vitro 3D and the in vivo situation). Keywords: C3A; Uzara; Uzarin; cell culturing; hepatotoxicity; herbal medicine; spheroid; three-dimensional cell culture, Drug-Induced Liver Injury DILI | www.tandfonline.com/doi/abs/10.1080/15376516.2018.1485805?journalCode=itxm20 | liver | toxicity | cell-line |
| Establishing dynamic spheroid cultures as mini-tumour models for treatment evaluation | Liver | Long term culture | Webinar | 2022 | Professor Chrisna Gouws1 |
| Human | HepG2, LS180, H69V, H69AR | Cancer | Cell line | In this presentation, the process to establish three-dimensional spheroid models in rotating bioreactors, and benchmarking their suitability to study cancer treatments will be discussed. These mini-tumour models aim to bridge the gap between in vitro studies and the human in vivo system, by mimicking cancer cell behaviour in vivo better. Once established, each model is characterized in terms of growth and viability characteristics, after which it is benchmarked through treatment with a standard chemotherapeutic drug. | zoom.us/webinar/register/WN_m7M88BfxSI64zWAv2pHm1Q | liver | cancer | cell-line | |
| Toxicity and anti-prolific properties of Xysmalobium undulatum water extract during short-term exposure to two-dimensional and three-dimensional spheroid cell cultures | Liver | Drug-Induced Liver Injury | Publication | 2018 | Carlemi Calitz 1, Josias H Hamman 1, Alvaro M Viljoen 2, Stephen J Fey 3,4, Krzysztof Wrzesinski 3, 4, Chrisna Gouws 1 |
| Human | HepG2 | Toxicity | Cell line | DOI: 10.1080/15376516.2018.1485805 | Xysmalobium undulatum (Uzara) is one of the most widely used indigenous traditional herbal remedies in Southern Africa. Commercially available Uzara plant material was used to prepare a crude aqueous extract, of which the toxicity potential was investigated in the hepatic HepG2/C3A cell line in both traditional two-dimensional (2D) and rotating three-dimensional (3D) spheroid cell cultures. These cultures were treated over a period of 4 days at concentrations of 200, 350, 500, and 750 mg/kg plant extract to protein content. Basic physiological parameters of the cell cultures were measured during exposure, including cell proliferation, glucose uptake, intracellular adenosine triphosphate levels, and adenylate kinase release. The results indicated that all physiological parameters monitored were affected in a dose dependent manner, with the highest concentration of Uzara crude water extract (750 mg/kg) resulting in toxicity. Anti-proliferating effects of Uzara crude water extract were observed in both the 2D and 3D cell cultures, with the most pronounced effects at concentrations of 350, 500, and 750 mg/kg. Discrepancies between results obtained from the 2D and 3D cell culture models may be attributed to the type of repair system that is initiated upon exposure, depending on where cells are within the cell cycle. DNA repair systems differ in cells within the G1 phase and non-diving cells, (i.e. cells found predominantly in in vitro 3D and the in vivo situation). Keywords: C3A; Uzara; Uzarin; cell culturing; hepatotoxicity; herbal medicine; spheroid; three-dimensional cell culture. Drug-Induced Liver Injury (DILI) | www.tandfonline.com/doi/abs/10.1080/15376516.2018.1485805?journalCode=itxm20 | liver | toxicity | cell-line |
| A sub-chronic Xysmalobium undulatum hepatotoxicity investigation in HepG2/C3A spheroid cultures compared to an in vivo model | Liver | DILI | Publication | 2019 | Carlemi Calitz 1, Josias H Hamman 1, Stephen J Fey 2, Alvaro M Viljoen 3, Chrisna Gouws 4, Krzysztof Wrzesinski 5 |
| Human | HepG2 | Toxicity | Cell line | DOI: 10.1016/j.jep.2019.111897 | Ethnopharmacology relevance: Traditional herbal medicines are utilized by 27 million South Africans. Xysmalobium undulatum (Uzara) is one of the most widely used traditional medicinal plants in Southern Africa. A false belief in the safety of herbal medicine may result in liver injury. Herb-induced liver injury (HILI) range from asymptomatic elevation of liver enzymes, to cirrhosis and in certain instances even acute liver failure. Various in vitro and in vivo models are available for the pre-clinical assessment of drug and herbal hepatotoxicity. However, more reliable and readily available in vitro models are needed, which are capable of bridging the gap between existing models and real human exposure. Three-dimensional (3D) spheroid cultures offer higher physiological relevance, overcoming many of the shortcomings of traditional two-dimensional cell cultures. Aims of this study: This study investigated the hepatotoxic and anti-prolific effects of the crude X. undulatum aqueous extract during a sub-chronic study (21 days), in both a 3D HepG2/C3A spheroid model and the Sprague Dawley rat model. Methods: HepG2/C3A spheroids were treated with a known hepatotoxin, valproic acid, and crude X. undulatum aqueous extract for 21 days with continuous evaluation of cell viability and proliferation. This was done by evaluating cell spheroid growth, intracellular adenosine triphosphate (ATP) levels and extracellular adenylate kinase (AK). Sprague Dawley rats were treated with the same compounds over 21 days, with evaluation of in vivo toxicity effects on serum chemistry. Results: The results from the in vitro study clearly indicated hepatotoxic effects and possible liver damage following treatment with valproic acid, with associated growth inhibition, loss of cell viability and increased cytotoxicity as indicated by reduced intracellular ATP levels and increased AK levels. These results were supported by the increased in vivo levels of AST, ALT and LDH following treatment of the Sprague Dawley rats with valproic acid, indicative of hepatic cellular damage that may result in hepatotoxicity. The in vitro 3D spheroid model was also able to predict the potential concentration dependant hepatotoxicity of the crude X. undulatum aqueous extract. Similarly, the results obtained from the in vivo Sprague Dawley model indicated moderate hepatotoxic potential. Conclusion: The data from both the 3D spheroid model and the Sprague Dawley model were able to indicate the potential concentration dependant hepatotoxicity of the crude X. undulatum aqueous extract. The results obtained from this study also confirmed the ability of the 3D spheroid model to effectively and reliably predict the long-term outcomes of possible hepatotoxicity. Keywords: Cell spheroids; Herb-induced liver injury (HILI); Herbal medicine; In vivo model; Microgravity; Three-dimensional cell culturing; Traditional medicine; Uzara; Valproic acid. | www.sciencedirect.com/science/article/abs/pii/S0378874118343411?via%3Dihub | liver | toxicity | cell-line |
| Response to and recovery from treatment in human liver-mimetic clinostat spheroids: a model for assessing repeated-dose drug toxicity | Liver | Drug treatment, cronic toxicity | Publication | 2020 | Stephen J Fey 1, Barbara Korzeniowska 1, Krzysztof Wrzesinski 1 |
| Human | HepG2 | Toxicity | Cell line | DOI: 10.1093/toxres/tfaa033 | Medicines are usually prescribed for repeated use over shorter or longer times. Unfortunately, repeated-dose animal toxicity studies do not correlate well with observations in man. As emphasized by the '3Rs' and the desire to phase-out animal research, in vitro models are needed. One potential approach uses clinostat-cultured 3D HepG2-C3A liver-mimetic spheroids. They take 18 days to recover in vivo physiological functionality and reach a metabolic equilibrium, which is thereafter stable for a year. Acute and chronic repeated-dose studies of six drugs (amiodarone, diclofenac, metformin, phenformin, paracetamol and valproic acid) suggest that spheroids are more predictive of human in vivo toxicity than either 2D-cultured HepG2 cells or primary human hepatocytes. Repeated non-lethal treatment results in a clear response and return to equilibrium. Mitochondrial toxic compounds can be identified using a galactose-based medium. Some drugs induced a protective (or stress) response that intensifies after the second treatment. This 3D spheroid model is inexpensive, highly reproducible and well-suited for the determination of repeated-dose toxicity of compounds (naturally or chemically synthesized). Drug treatment: 3D cell culture; HepG2–C3A; acetaminophen; amiodarone; clinostat; diclofenac; drug toxicity; metformin; phenformin; recovery from treatment; repeated-dose; valproic acid. | academic.oup.com/toxres/article/9/4/379/5856213?login=false | liver | toxicity | cell-line |
| Vitamin K2 Modulates Vitamin D-Induced Mechanical Properties of Human 3D Bone Spheroids In Vitro | Bone | Osteoporosis | Publication | 2020 | Maria Schröder 1, Elisabeth Aurstad Riksen 1, Jianying He 2, Bjørn Helge Skallerud 2, Mona Elisabeth Møller 3, Aina-Mari Lian 4, Unni Syversen 4, 5, 6, Janne Elin Reseland 1 |
| Human | Osteoblast | Osteoporosis | Primary cells | DOI: 10.1002/jbm4.10394 | Rotational culture promotes primary human osteoblasts (hOBs) to form three-dimensional (3D) multicellular spheroids with bone tissue-like structure without any scaffolding material. Cell-based bone models enable us to investigate the effect of different agents on the mechanical strength of bone. Given that low dietary intake of both vitamin D and K is negatively associated with fracture risk, we aimed to assess the effect of these vitamins in this system. Osteospheres of hOBs were generated with menaquinone-4 (MK-4; 10μM) and 25-hydroxyvitamin D3 [25(OH)D3; 0.01μM], alone and in combination, or without vitamins. The mechanical properties were tested by nanoindentation using a flat-punch compression method, and the mineralized extracellular bone matrix was characterized by microscopy. The in vitro response of hOBs to MK-4 and 25(OH)D3 was further evaluated in two-dimensional (2D) cultures and in the 3D bone constructs applying gene expression analysis and multiplex immunoassays. Mechanical testing revealed that 25(OH)D3 induced a stiffer and MK-4 a softer or more flexible osteosphere compared with control. Combined vitamin conditions induced the same flexibility as MK-4 alone. Enhanced levels of periostin (p < 0.001) and altered distribution of collagen type I (COL-1) were found in osteospheres supplemented with MK-4. In contrast, 25(OH)D3 reduced COL-1, both at the mRNA and protein levels, increased alkaline phosphatase, and stimulated mineral deposition in the osteospheres. With the two vitamins in combination, enhanced gene expression of periostin and COL-1 was seen, as well as extended osteoid formation into the central region and increased mineral deposition all over the area. Moreover, we observed enhanced levels of osteocalcin in 2D and osteopontin in 3D cultures exposed to 25(OH)D3 alone and combined with MK-4. In conclusion, the two vitamins seem to affect bone mechanical properties differently: vitamin D enhancing stiffness and K2 conveying flexibility to bone. These effects may translate to increased fracture resistance in vivo. | asbmr.onlinelibrary.wiley.com/doi/epdf/10.1002/jbm4.10394 | bone | osteoporosis | primary-tissue |
| Hepatocellular carcinoma (HepG2/C3A) cell-based 3D model for genotoxicity testing of chemicals | Liver | Cytotoxicity, Genotoxicity | Publication | 2021 | Martina Štampar 1, Helle Sedighi Frandsen 2, Adelina Rogowska-Wrzesinska 3, Krzysztof Wrzesinski 4, Metka Filipič 5, Bojana Žegura 6 |
| Human | HepG2 | Toxicity | Cell line | DOI: 10.1016/j.scitotenv.2020.143255 | The major weakness of the current in vitro genotoxicity test systems is the inability of the indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds, which consequently can lead to misleading results. Thus, there is a significant emphasis on developing hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behaviour and offer more accurate and predictive data for human exposures. In this study, we developed an approach for genotoxicity testing with 21-day old spheroids formed from human hepatocellular carcinoma cells (HepG2/C3A) using the dynamic clinostat bioreactor system (CelVivo BAM/bioreactor) under controlled conditions. The spheroids were exposed to indirect-acting genotoxic compounds, polycyclic aromatic hydrocarbon [PAH; benzo(a) pyrene B(a)P], and heterocyclic aromatic amine [PhIP]) at non-cytotoxic concentrations for 24 and 96 h. The results showed that both environmental pollutants B(a)P and PhIP significantly increased the level of DNA strand breaks assessed by the comet assay. Further, the mRNA level of selected genes encoding metabolic enzymes from phase I and II, and DNA damage responsive genes was determined (qPCR). The 21-day old spheroids showed higher basal expression of genes encoding metabolic enzymes compared to monolayer culture. In spheroids, B(a)P or PhIP induced compound-specific up-regulation of genes implicated in their metabolism, and deregulation of genes implicated in DNA damage and immediate-early response. The study demonstrated that this model utilizing HepG2/C3A spheroids grown under dynamic clinostat conditions represents a very sensitive and promising in vitro model for genotoxicity and environmental studies and can thus significantly contribute to a more reliable assessment of genotoxic activities of pure chemicals, and complex environmental samples even at very low for environmental exposure relevant concentrations. | www.sciencedirect.com/science/article/pii/S0048969720367863 | liver | toxicity | cell-line |
| Anticancer Potential of Sutherlandia frutescens and Xysmalobium undulatum in LS180 Colorectal Cancer Mini-Tumors | Colon | Colorectal Cancer, Encapsulation | Publication | 2021 | Chrisna Gouws 1, Tanya Smit 1, Clarissa Willers 1, Hanna Svitina 1, Carlemi Calitz 2, Krzysztof Wrzesinski 1, 3 |
| Human | LS180 | Cancer | Cell line | DOI: 10.3390/molecules26030605 | Colorectal cancer remains to be one of the leading causes of death worldwide, with millions of patients diagnosed each year. Although chemotherapeutic drugs are routinely used to treat cancer, these treatments have severe side effects. As a result, the use of herbal medicines has gained increasing popularity as a treatment for cancer. In this study, two South African medicinal plants widely used to treat various diseases, Sutherlandia frutescens and Xysmalobium undulatum, were evaluated for potential activity against colorectal cancer. This potential activity for the treatment of colorectal cancer was assessed relative to the known chemotherapeutic drug, paclitaxel. The cytotoxic activity was considered in an advanced three-dimensional (3D) sodium alginate encapsulated LS180 colorectal cancer functional spheroid model, cultured in clinostat-based rotating bioreactors. The LS180 cell mini-tumors were treated for 96 h with two concentrations of each of the crude aqueous extracts or paclitaxel. S. frutescens extract markedly decreased the soluble protein content, while decreasing ATP and AK per protein content to below detectable limits after only 24 h exposure. X. undulatum extract also decreased the soluble protein content, cell viability, and glucose consumption. The results suggested that the two phytomedicines have potential to become a source of new treatments against colorectal cancer. | www.mdpi.com/1420-3049/26/3/605 | colon | cancer | cell-line |
| FNDC5/irisin is expressed and regulated differently in human periodontal ligament cells, dental pulp stem cells and osteoblasts | Bone | Stem cells, hPDL, hDPC | Publication | 2022 | Yang Yang1, Helen Pullisaar2, Maria A.Landin1, Catherine Anne Heyward3, Maria Schröder1, Tianxiang Geng1 Maria Grano4 , Janne Elin Reseland1 |
| Human | human periodontal ligament cells | Primary cells | DOI: 10.1016/j.archoralbio.2021.105061 | Objective To examine the expression and regulation of fibronectin type III domain-containing protein 5/irisin (FNDC5/irisin) in primary human periodontal ligament (hPDL) cells, dental pulp stem cells (hDPCs) and osteoblasts (hOBs). Methods FNDC5/irisin was identified in sections of paraffin embedded rat maxillae, cryo-sections of 3D cultured spheroids hPDL cells, hDPCs and hOBs, 2D cultured hPDL cells, hDPCs and hOBs by immunohistochemistry. The expression of FNDC5/irisin was identified by qPCR, followed by sequencing of the qPCR product. Regulation of FNDC5/irisin expression in hPDL cells, hDPCs and hOBs were evaluated after administration of different concentrations of irisin and all-trans retinoic acid (ATRA). qPCR and ELISA were used to identify expression and secretion of FNDC5/irisin in odontoblast-like differentiation of hDPCs. Results FNDC5/irisin was confirmed to be present in rat periodontium and dental pulp regions, as well as in 2D and 3D cultured hPDL cells, hDPCs and hOBs. BLAST analyses verified the generated nucleotide alignments matched human FNDC5/irisin. FNDC5/irisin gene expression was enhanced during odontoblast-like differentiation of hDPCs whereas the secretion of the protein was decreased compared to control. The protein signals in rat periodontal and pulpal tissues were higher than that of alveolar bone, and the expression of FNDC5/irisin was differently regulated by recombinant irisin and ATRA in hPDL cells and hDPCs compared to hOBs. Conclusions FNDC5/irisin expression was verified in rodent periodontium and dental pulp, and in hPDL cells, hDPCs and hOBs. The FNDC5/irisin expression was regulated by recombinant irisin and ATRA. Finally, expression and secretion of FNDC5/irisin were affected during odontoblast-like differentiation of hDPCs. | www.sciencedirect.com/science/article/pii/S0003996921000248 | bone | primary-tissue | ||
| In vitro Characterization of Insulin-Producing β-Cell Spheroids | Pancreas | Beta-Cells, Diabetes model | Publication | 2021 | Yonela Ntamo 1 2, Ebrahim Samodien 1, Joleen Burger 1 3, Nolan Muller 4, Christo J F Muller 1 2 3, Nireshni Chellan 1 3 |
| Human | INS-1 | Diabetes | Cell line | doi: 10.3389/fcell.2020.623889 | Over the years, immortalized rodent β-cell lines such as RIN, HIT, MIN, βTC, and INS-1 have been used to investigate pancreatic β-cell physiology using conventional two-dimensional (2D) culture techniques. However, physical and physiological limitations inherent to 2D cell culture necessitates confirmatory follow up studies using sentient animals. Three-dimensional (3D) culture models are gaining popularity for their recapitulation of key features of in vivo organ physiology, and thus could pose as potential surrogates for animal experiments. In this study, we aimed to develop and characterize a rat insulinoma INS-1 3D spheroid model to compare with 2D monolayers of the same cell line. Ultrastructural verification was done by transmission electron microscopy and toluidine blue staining, which showed that both 2D monolayers and 3D spheroids contained highly granulated cells with ultrastructural features synonymous with mature pancreatic β-cells, with increased prominence of these features observed in 3D spheroids. Viability, as assessed by cellular ATP quantification, size profiling and glucose utilization, showed that our spheroids remained viable for the experimental period of 30 days, compared to the limiting 5-day passage period of INS-1 monolayers. In fact, increasing ATP content together with spheroid size was observed over time, without adverse changes in glucose utilization. Additionally, β-cell function, assessed by determining insulin and amylin secretion, showed that the 3D spheroids retained glucose sensing and insulin secretory capability, that was more acute when compared to 2D monolayer cultures. Thus, we were able to successfully demonstrate that our in vitro INS-1 β-cell 3D spheroid model exhibits in vivo tissue-like structural features with extended viability and lifespan. This offers enhanced predictive capacity of the model in the study of metabolic disease, β-cell pathophysiology and the potential treatment thereof. | www.ncbi.nlm.nih.gov/pmc/articles/PMC7876261/ | pancreas | diabetes | cell-line |
| Clinostat 3D Cell Culture: Protocols for the Preparation and Functional Analysis of Highly Reproducible, Large, Uniform Spheroids and Organoids | Liver | Clinostat | Publication | 2021 | Krzysztof Wrzesinski 1 2, Helle Sedighi Frandsen 3, Carlemi Calitz 4, Chrisna Gouws 5, Barbara Korzeniowska 6, Stephen J Fey 6 |
| Human | HepG2 | Cell line | DOI: 10.1007/978-1-0716-1246-0_2 | Growing cells as 3D structures need not be difficult. Often, it is not necessary to change cell type, additives or growth media used. All that needs to be changed is the geometry: cells (whether primary, induced pluripotent, transformed or immortal) simply have to be grown in conditions that promote cell-cell adhesion while allowing gas, nutrient, signal, and metabolite exchange. Downstream analysis can become more complicated because many assays (like phase contrast microscopy) cannot be used, but their replacements have been in use for many years. Most importantly, there is a huge gain in value in obtaining data that is more representative of the organism in vivo. It is the goal of the protocols presented here to make the transition to a new dimension as painless as possible. Grown optimally, most biopsy derived organoids will retain patient phenotypes, while cell (both stem cell, induced or otherwise or immortalized) derived organoids or spheroids will recover tissue functionality. | link.springer.com/protocol/10.1007/978-1-0716-1246-0_2 | liver | cell-line | ||
| 3D-ViaFlow: A Quantitative Viability Assay for Multicellular Spheroids | Liver | FACS, 3D-ViaFlow | Publication | 2021 | Joel Mario Vej-Nielsen 1, Adelina Rogowska-Wrzesinska 2 |
| Human | HepG2 | Cell line | DOI: 10.1007/978-1-0716-1246-0_11 | Three-dimensional cell culture became an essential method in molecular and cell biology research. Accumulating results show that cells grown in 3D, display increased functionality and are capable of recapitulating physiological functions that are not observed in classical in vitro models. Spheroid-based cell culture allows the cells to establish their own extracellular matrix and intricate intercellular connections promoting a tissue-like growth environment.In this paper we present the 3D-ViaFlow method that combines an optimised dual live-dead cell staining with flow cytometry to deliver a quantitative estimation of viability of cells in multicellular spheroids. The method is optimised for monolayer cultures and multicellular spheroids created from HepG2/C3A human hepatocytes or coculture of HepG2/C3A and endothelial cell line HMEC-1. It includes protocol for spheroids disassembling, labeling of cells with fluorescein diacetate and propidium iodide and instructions for flow cytometry gating optimized for analysis of heterogeneous cell populations form spheroids. | link.springer.com/protocol/10.1007/978-1-0716-1246-0_11#DOI | liver | cell-line | ||
| Method to Disassemble Spheroids into Core and Rim for Downstream Applications Such as Flow Cytometry, Comet Assay, Transcriptomics, Proteomics, and Lipidomics | Liver | Comet assay, Flow Cytometry | Publication | 2021 | Helle Sedighi Frandsen 1, Martina Štampar 2, Joel Mario Vej-Nielsen 1, Bojana Žegura 2, Adelina Rogowska-Wrzesinska 3 |
| Human | HepG2 | Cell line | DOI: 10.1007/978-1-0716-1246-0_12 | Cells cultured in a monolayer have been a central tool in molecular and cell biology, toxicology, biochemistry, and so on. Therefore, most methods for adherent cells in cell biology are tailored to this format of cell culturing. Limitations and disadvantages of monolayer cultures, however, have resulted in the ongoing development of advanced cell culturing techniques. One such technique is culturing cells as multicellular spheroids, that had been shown to mimic the physiological conditions found in vivo more accurately. This chapter presents a novel method for separation of the spheroid rim and core in mature spheroids (>21 days) for further analysis using advanced molecular biology techniques such as flow cytometry, viability estimations, comet assay, transcriptomics, proteomics and lipidomic. This fast and gentle disassembly of intact spheroids into rim and core fractions, and further into viable single-cell suspension provides an opportunity to bridge the gap from 3D cell culture to current state-of-the-art analysis methods. | link.springer.com/protocol/10.1007/978-1-0716-1246-0_12 | liver | cell-line | ||
| A Purpose-Built System for Culturing Cells as In Vivo Mimetic 3D Structures | Clinostat, | Publication | 2022 | Krzysztof Wrzesinski1, Søren Alnøe1, Hans H. Jochumsen1, Karoline Mikkelsen1, Torsten D. Bryld1, Julie S. Vistisen1, Peter Willems Alnøe1 and Stephen J. Fey1 |
| HepG2 | DOI: 10.5772/intechopen.96091 | Culturing cells in 3D is often considered to be significantly more difficult than culturing them in 2D. In practice, this is not the case: the situation is that equipment needed for 3D cell culture has not been optimised as much as equipment for 2D. Here we present a few key features which must be considered when designing 3D cell culture equipment. These include diffusion gradients, shear stress and time. Diffusion gradients are unavoidably introduced when cells are cultured as clusters. Perhaps the most important consequence of this is that the resulting hypoxia is a major driving force in the metabolic reprogramming. Most cells in tissues do not experience liquid shear stress and it should therefore be minimised. Time is the factor that is most often overlooked. Cells, irrespective of their origin, are damaged when cultures are initiated: they need time to recover. All of these features can be readily combined into a clinostat incubator and bioreactor. Surprisingly, growing cells in a clinostat system do not require specialised media, scaffolds, ECM substitutes or growth factors. This considerably facilitates the transition to 3D. Most importantly, cells growing this way mirror cells growing in vivo and are thus valuable for biomedical research. | www.intechopen.com/chapters/75252 | |||||||
| A novel NCI-H69V small cell lung cancer functional mini-tumor model for future treatment screening applications | Lung | Cancer, SCLC | Publication | 2022 | Liezaan van der Merwe1, Hanna Svitina1,2, Clarissa Willers1, Krzysztof Wrzesinski1,3, Chrisna Gouws1 |
| Human | H69V | Cancer | Cell line | DOI: 10.1002/btpr.3253 | Small cell lung cancer (SCLC) is aggressive and despite multiple clinical trials, its standard of care is unchanged for the past three decades. In vitro cancer models are crucial in chemotherapy development, and three-dimensional (3D) models aim to bridge the gap between two-dimensional (2D) flat cultures and in vivo testing. Functional 3D spheroids can better represent the in vivo situation and tumor characteristics than 2D models. An NCI-H69V SCLC mini-tumor model was developed in a clinostat-based rotating bioreactor system. Spheroid growth and viability were characterized for 30 days, and the ideal experimental window with mature and metabolically stable spheroids was determined. Application of the model for anticancer treatment screening was validated with the standard chemotherapeutic drug irinotecan, for an exposure period of 72 h. The following parameters were measured: soluble protein content, planar surface area measurements, intracellular adenosine triphosphate and extracellular adenylate kinase levels, and glucose consumption. Histological morphology of the spheroids was observed. The established model proved viable and stable, while treatment with irinotecan caused a decrease in cell growth, viability, and glucose consumption demonstrating reactivity of the model to chemotherapy. Therefore, this NCI-H69V SCLC functional spheroid model could be used for future anticancer compound screening. | aiche.onlinelibrary.wiley.com/doi/10.1002/btpr.3253 | lung | cancer | cell-line |
| How to culture uniform mini liver-like constructs from HepG2/c3a using the ClinoStar system | Liver | Spheroid, Hepatocytes | Application note | 2022 | Helle Sedighi Frandsen1 |
| Human | HepG2 | Cell line | 3-dimensional cell culture has gained profound interest over the last few years. Culturing cells as spheroids is significantly advantageous. Spheroids consist of complex cellular networks, allowing for decreased rates of proliferation. This enables the culture to spend its energy establishing cell-to-cell contact and communication, which permits increased in vivo like performance and function compared to standard flat culture. | celvivo.com/wp-content/uploads/simple-file-list/Application-notes/101_How-to-culture-uniform-mini-liver-like-constructs-from-HepG2c3a-using-the-ClinoStar-system.pdf | liver | cell-line | |||
| Improved toxicology prediction by utilising hepatic spheroids | Liver | Toxicology, Hepatacyte, Acetaminophen | Application note | 2022 | Helle Sedighi Frandsen1 |
| Human | HepG2 | Toxicity | Cell line | Primary human hepatocytes are the gold standard of drug toxicology testing. However, this notion is challenged by high prices, donor variation and decreased tissue function in flat culture [1]. 3-dimensional cultures enable key liver functions to be regained, unlike 2D culture. Generating spheroids from a hepatic cell line using the ClinoStar system allows creation of uniform constructs which can more reliably mimic in-vivo standards of toxicological determination. | celvivo.com/wp-content/uploads/simple-file-list/Application-notes/102_Improved-toxicology-prediction-by-utilising-hepatic-spheroids.pdf | liver | toxicity | cell-line | |
| 3D multicellular models to study cancer – from spheroids to microfluidics | Breast | Spheroids, Cysts, Organoids, Microfluidics | Webinar | 2022 | Dr. Dominika Czaplinska1 & Dr. Roxane Crouigneau1 |
| Human | MCF7 | Cell line | The development of three-dimensional (3D) cell cultures has provided new tools for basic research and pre-clinical approaches, allowing the culture of cancer cells under conditions that closely resemble tumour growth in a living organism. We will provide an overview of the main 3D techniques used in our lab, including spheroids, cysts, organoids and microfluidics technology. We will discuss the advantages and limitations of the classical 3D models as well as recent advances in 3D culture techniques, focusing on how these culture methods have been used to study cancer progression. | us06web.zoom.us/webinar/register/rec/WN_eqv_h-3LR0i9ypWQZqNi4Q?meetingId=kwPE_tggNtGn_afpf0UwSZAf8bLkHs2HVpPhGuQ45no1Z2jx8Xa9kwhZjBiHG8NH.q9WKpgewj8mYicjb&playId=&action=play&_x_zm_rtaid=FJUYMlb4QG-aiIxv7ak_Zg.1671104677266.573549425effd0c536c03923a3ec780d&_x_zm_rhtaid=476 | breast | cell-line | |||
| Nthy-ori 3-1 thyroid multicellular spheroids in the ClinoStar system. | Thyroid | Thyroid model, scaffold-free | Application note | 2022 | José Luis Cortés Sánchez1, Michalina Maria Czaplicka1, Louise Leth Hefting2 |
| Human | Nthy-ori 3-1 | Cell line | This protocol describes the procedure for culturing the follicular thyroid cell line Nthy-Ori in the CelVivo Clinostar system from a single cell suspension subculture. | celvivo.com/wp-content/uploads/2022/06/104_DG-Nthy-Ori-cells-in-the-CelVivo-Clinostat.pdf | thyroid | cell-line | |||
| FTC-133 thyroid cancer multicellular spheroids in the ClinoStar system. | Thyroid | Spheroids, Thyroid cancer | Application note | 2022 | José Luis Cortés Sánchez1, Michalina Maria Czaplicka1, Louise Leth Hefting2 |
| Human | FTC-133 | Cancer | Cell line | This protocol describes the procedure for culturing the follicular thyroid cancer cell line FTC-133 in the CelVivo Clinostar system from a single cell suspension subculture. | celvivo.com/wp-content/uploads/simple-file-list/Application-notes/103_DG-FTC-133-cells-in-the-CelVivo-Clinostat.pdf | thyroid | cancer | cell-line | |
| Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue | Liver | Chromatin modifications, 3D culture, Tandem-MS | Publication | 2022 | Jazmine-Saskya N Joseph-Chowdhury1, Stephanie Stransky1, Sarah Graff1, Ronald Cutler1, Dejauwne Young1, Julie S Kim1, Carlos Madrid-Aliste1, Jennifer T Aguilan1, Edward Nieves1, Yan Sun1, Edwin J Yoo1, Simone Sidoli1 |
| Human | HepG2, MS, Bioinformatics | Cell line | 10.3791/63606 | Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown. | www.jove.com/t/63606/global-level-quantification-histone-post-translational-modifications | liver | cell-line | ||
| NCI-H69V Small Cell Lung Cancer (SCLC) mini-tumor model in the ClinoStar system | Lung | Lung Cancer, SCLC | Application note | 2022 | Liezaan van der Merwe1, Chrisna Gouws1, Louise Leth Hefting2 and Reeham W. Motaher3 |
| Human | H69V | Cancer | Cell line | This application note details the process of developing an SCLC tumor model using the NCI-H69V cell line in the ClinoStar. The established model is reactive to treatment with the standard chemotherapy drug irinotecan, leading to a reduction in cell growth, viability, and glucose consumption. Viability of the model is demonstrated by measuring the following parameters: soluble protein content, planar surface area, intracellular adenosine triphosphate, extracellular adenylate kinase levels, and glucose consumption. | celvivo.com/wp-content/uploads/simple-file-list/Application-notes/105_NCI-H69V-Small-Cell-Lung-Cancer-SCLC.pdf | lung | cancer | cell-line | |
| How to Grow HUB Derived Upper and Lower Respiratory Lung Organoids in the ClinoStar System | Lung | Organoid, Virus infection, SARS-CoV-2 | Application note | 2022 | Dr. Stefanie Dichtl1, Prof. Doris Wilflingseder1 and Louise Leth Hefting2 |
| Human | Primary cells | This application note details a protocol using the ClinoStar and ClinoReactor system to culture upper and lower respiratory lung organoids. The ClinoStar culture system enables a dynamic culture that can be maintained in a closed, contamination-free environment for long periods of time. Our ClinoStar culture method produce 3D lung organoids without further use of matrices. | celvivo.com/wp-content/uploads/simple-file-list/Application-notes/106_HUB-Derived-Upper-and-Lower-Respiratory-Lung-Organoids.pdf | lung | primary-tissue | ||||
| Mapping Proteome and Lipidome Changes in Early-Onset Non-Alcoholic Fatty Liver Disease Using Hepatic 3D Spheroids | Liver | NAFLD, Lipidomics, Proteomics | Publication | 2022 | Helle Sedighi Frandsen1,2, Joel Mario Vej-Nielsen1,2, Lauren Elizabeth Smith1, Lang Sun3,4, Karoline Lindgaard Mikkelsen1,5, Annemette Præstegaard Thulesen1, Christina Erika Hagensen1, Fuquan Yang3,4, Adelina Rogowska-Wrzesinska1 |
| Human | HepG2 | Non-Alcoholic Fatty Liver Disease | Cell line | 10.3390/cells11203216 | Non-alcoholic fatty liver disease affects one-fourth of the world's population. Central to the disease progression is lipid accumulation in the liver, followed by inflammation, fibrosis and cirrhosis. The underlying mechanism behind the early stages of the disease is poorly understood. We have exposed human hepatic HepG2/C3A cells-based spheroids to 65 μM oleic acid and 45 μM palmitic acid and employed proteomics and lipidomics analysis to investigate their effect on hepatocytes. The treatment successfully induced in vivo hallmarks of NAFLD, as evidenced by intracellular lipid accumulation and increased ATP levels. Quantitative lipidome analysis revealed an increase in ceramides, LPC and saturated triglycerides and a decrease in the ratio of PC/PE, similar to the changes observed in patients' liver biopsies. The proteomics analysis combined with qPCR showed increased epithelial to mesenchymal transition (EMT) signalling. Activation of EMT was further validated by transcriptomics in TGF-β treated spheroids, where an increase in mesenchymal cell markers (N-cadherin and collagen expression) was found. Our study demonstrates that this model system thus closely echoes several of the clinical features of non-alcoholic fatty liver disease and can be used to investigate the underlying molecular changes occurring in the condition. | www.mdpi.com/2073-4409/11/20/3216 | liver | nafld | cell-line |
| Investigation of reversible histone acetylation and dynamics in gene expression regulation using 3D liver spheroid model | Liver | Epigenetics; Liver; Spheroids; Transcriptome | Publication | 2022 | Stephanie Stransky1, Ronald Cutler1,2, Jennifer Aguilan3, Edward Nieves1,4 & Simone Sidoli5 |
| Human | HepG2 | Cell line | 10.1186/s13072-022-00470-7 | Background Three-dimensional (3D) cell culture has emerged as an alternative approach to 2D flat culture to model more accurately the phenotype of solid tissue in laboratories. Culturing cells in 3D more precisely recapitulates physiological conditions of tissues, as these cells reduce activities related to proliferation, focusing their energy consumption toward metabolism and homeostasis. Results Here, we demonstrate that 3D liver spheroids are a suitable system to model chromatin dynamics and response to epigenetics inhibitors. To delay necrotic tissue formation despite proliferation arrest, we utilize rotating bioreactors that apply active media diffusion and low shearing forces. We demonstrate that the proteome and the metabolome of our model resemble typical liver functions. We prove that spheroids respond to sodium butyrate (NaBut) treatment, an inhibitor of histone deacetylases (HDACi), by upregulating histone acetylation and transcriptional activation. As expected, NaBut treatment impaired specific cellular functions, including the energy metabolism. More importantly, we demonstrate that spheroids reestablish their original proteome and transcriptome, including pre-treatment levels of histone acetylation, metabolism, and protein expression once the standard culture condition is restored after treatment. Given the slow replication rate (> 40 days) of cells in 3D spheroids, our model enables to monitor the recovery of approximately the same cells that underwent treatment, demonstrating that NaBut does not have long-lasting effects on histone acetylation and gene expression. These results suggest that our model system can be used to quantify molecular memory on chromatin. Conclusion Together, we established an innovative cell culture system that can be used to model anomalously decondensing chromatin in physiological cell growth and rule out epigenetics inheritance if cells recover the original phenotype after treatment. The transient epigenetics effects demonstrated here highlight the relevance of using a 3D culture model system that could be very useful in studies requiring long-term drug treatment conditions that would not be possible using a 2D cell monolayer system. | epigeneticsandchromatin.biomedcentral.com/articles/10.1186/s13072-022-00470-7 | liver | cell-line | ||
| Characterization of an Alginate Encapsulated LS180 Spheroid Model for Anti-colorectal Cancer Compound Screening | Colon | Colon Cancer, Cells, Encapsulation, Embedding | Publication | 2020 | Tanya Smit1, Carlemi Calitz1, Clarissa Willers1, Hanna Svitina1, Josias Hamman1, Stephen J Fey2, Chrisna Gouws1, Krzysztof Wrzesinski1,2 |
| Human | LS180 | Cancer | Cell line | DOI: 10.1021/acsmedchemlett.0c00076 | Colorectal cancer is one of the leading causes of cancer-related deaths. A main problem for its treatment is resistance to chemotherapy, requiring the development of new drugs. The success rate of new candidate cancer drugs in clinical trials remains dismal. Three-dimensional (3D) cell culture models have been proposed to bridge the current gap between in vitro chemotherapeutic studies and the human in vivo, due to shortcomings in the physiological relevance of the commonly used two-dimensional cell culture models. In this study, LS180 colorectal cancer cells were cultured as 3D sodium alginate encapsulated spheroids in clinostat bioreactors. Growth and viability were evaluated for 20 days to determine the ideal experimental window. The 3- (4,5- dimethylthiazol- 2- yl)-2,5-diphenyltetrazolium bromide assay was then used to establish half maximal inhibitory concentrations for the standard chemotherapeutic drug, paclitaxel. This concentration was used to further evaluate the established 3D model. During model characterization and evaluation soluble protein content, intracellular adenosine triphosphate levels, extracellular adenylate kinase, glucose consumption, and P-glycoprotein (P-gp) gene expression were measured. Use of the model for chemotherapeutic treatment screening was evaluated using two concentrations of paclitaxel, and treatment continued for 96 h. Paclitaxel caused a decrease in cell growth, viability, and glucose consumption in the model. Furthermore, relative expression of P-gp increased compared to the untreated control group. This is a typical resistance-producing change, seen in vivo and known to be a result of paclitaxel treatment. It was concluded that the LS180 sodium alginate encapsulated spheroid model could be used for testing new chemotherapeutic compounds for colorectal cancer. | pubs.acs.org/doi/10.1021/acsmedchemlett.0c00076 | colon | cancer | cell-line |
| Hepatocellular carcinoma (HepG2/C3A) cell-based 3D model for genotoxicity testing of chemicals | Liver | Cytotoxic, Genotoxic, Comet assay | Publication | 2020 | Martina Štampar1,2, Helle Sedighi Frandsen3, Adelina Rogowska-Wrzesinska3, Krzysztof Wrzesinski4, Metka Filipič1 and Bojana Žegura1 |
| Human | HepG2 | Toxicity | Cell line | DOI: 10.1007/s12272-014-0502-6 | The major weakness of the current in vitro genotoxicity test systems is the inability of the indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds, which consequently can lead to misleading results. Thus, there is a significant emphasis on developing hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behaviour and offer more accurate and predictive data for human exposures. In this study, we developed an approach for genotoxicity testing with 21-day old spheroids formed from human hepatocellular carcinoma cells (HepG2/C3A) using the dynamic clinostat bioreactor system (CelVivo BAM/bioreactor) under controlled conditions. The spheroids were exposed to indirect-acting genotoxic compounds, polycyclic aromatic hydrocarbon [PAH; benzo(a) pyrene B(a)P], and heterocyclic aromatic amine [PhIP]) at non-cytotoxic concentrations for 24 and 96 h. The results showed that both environmental pollutants B(a)P and PhIP significantly increased the level of DNA strand breaks assessed by the comet assay. Further, the mRNA level of selected genes encoding metabolic enzymes from phase I and II, and DNA damage responsive genes was determined (qPCR). The 21-day old spheroids showed higher basal expression of genes encoding metabolic enzymes compared to monolayer culture. In spheroids, B(a)P or PhIP induced compound-specific up-regulation of genes implicated in their metabolism, and deregulation of genes implicated in DNA damage and immediate-early response. The study demonstrated that this model utilizing HepG2/C3A spheroids grown under dynamic clinostat conditions represents a very sensitive and promising in vitro model for genotoxicity and environmental studies and can thus significantly contribute to a more reliable assessment of genotoxic activities of pure chemicals, and complex environmental samples even at very low for environmental exposure relevant concentrations. | www.sciencedirect.com/science/article/pii/S0048969720367863 | liver | toxicity | cell-line |