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Cells
Special Issues
State-of-the-Art of Cell Cultures in Drug Validation and Toxicity Assessment
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State-of-the-Art of Cell Cultures in Drug Validation and Toxicity Assessment

A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Cell Methods".

Deadline for manuscript submissions: 31 July 2025 | Viewed by 18623

Special Issue Editors

Dr. Anna Bassi

E-Mail Website
Guest Editor
Department of Experimental Medicine (DIMES), University of Genoa, 16132 Genoa, Italy
Interests: in vitro models; hiPSC; 3D culture; millifluidic culture systems; molecular pathways
Special Issues, Collections and Topics in MDPI journals
Dr. Stefania Vernazza

E-Mail
Guest Editor
Department of Experimental Medicine (DIMES), University of Genoa, 16132 Genoa, Italy
Interests: in vitro models; hiPSC; 3D culture; millifluidic culture systems
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Chemical risk assessment has relied on animal testing for decades. The system's overall performance, sustainability, relevance and ethics are being questioned by society and the scientific community, revisiting the 'gold standard' rodent tests, for instance, in repeated-dose toxicity, carcinogenicity studies and potential adverse effects of chemicals which can be combined to form mixtures. These issues necessitate a paradigm shift towards a new toxicity testing system that evaluates biologically significant perturbations in key toxicity pathways. To achieve these goals, new prediction models should consider novel intelligent and more efficient safety assessments based on in vitro testing in combination with computational modelling. Indeed, several up-to-date scientific tools used for risk assessment are continuously enhancing through the development of new methodological approaches (NAMs) based on complex in vitro models, omic sciences, biological analysis of high-performance screening and quantitative structure–activity relationships (QSAR). The challenge of using NAMs to assess hazards and risks is aligned with the One Health approach.

 A good in vitro model should be as close as possible to in vivo conditions to better reproduce the response of the organism. Therefore, translation into 3D-reconstructed model tissues from primary human cells enables microphysiological disease modelling, drug discovery and toxicity testing, as they are increasingly integrating advanced human cell culture systems and techniques, which include induced pluripotent stem cells (iPSC), 3D cultures and organoids (from healthy or cancer cells), co-culture systems, microfluidics and multiorgan chips.

Moreover, these innovative models can mimic the key events that underlie the AOP (adverse outcoming pathway) concept, a significant step forward towards the goal of full animal replacement in toxicology.  

This Special Issue is dedicated to collating information on state-of-the-art cell culture models and techniques. We will highlight the current knowledge and future potential of cell cultures, spotlighting a unique tool to test the effect of new target drugs and study in vitro toxicity assessment, which will provide novel insights into the field of cellular biology. Both original research articles and reviews about experimental cytology are welcome. We look forward to receiving your contributions.

Dr. Anna Bassi
Dr. Stefania Vernazza
Guest Editors

Manuscript Submission Information

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

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

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

Keywords

  • human in vitro models
  • toxicity testing
  • drug development
  • mandatory requirements
  • regulatory approval
  • standardization
  • validation

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Published Papers (7 papers)

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Research

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12 pages, 3930 KiB  
Article
Standardized Protocol for Resazurin-Based Viability Assays on A549 Cell Line for Improving Cytotoxicity Data Reliability
by Jessica Petiti, Sabrina Caria, Laura Revel, Mattia Pegoraro and Carla Divieto
Cells 2024, 13(23), 1959; https://doi.org/10.3390/cells13231959 - 26 Nov 2024
Cited by 1 | Viewed by 1966
Abstract
The A549 cell line has become a cornerstone in biomedical research, particularly in cancer studies and serves as a critical tool in cytotoxicity studies and drug screening where it is used to evaluate the impact of pharmaceutical compounds on cellular viability. One of [...] Read more.
The A549 cell line has become a cornerstone in biomedical research, particularly in cancer studies and serves as a critical tool in cytotoxicity studies and drug screening where it is used to evaluate the impact of pharmaceutical compounds on cellular viability. One of the most widely adopted methods for viability assessment, which is also used in evaluating drug cytotoxicity, is the resazurin-based assay. This assay exploits the ability of living cells to convert resazurin into fluorescent resorufin, providing a reliable indicator of metabolic activity. By measuring this conversion, cell viability can be estimated. Resazurin assay is extensively used for evaluating cytotoxic effects on various cell lines, including A549 cells, thereby bridging the gap between in vitro experimentation and drug development. However, frequent data inconsistencies in pre-clinical drug screening highlight the critical need for standardization to ensure reliability and reproducibility. This manuscript addresses these challenges by describing the optimization of resazurin-based viability assays for A549 cells in both 2D cultures and 3D fibrin gel models. By optimizing this test, the study aims to enhance the reliability of cytotoxicity results and introduces a new standard operating procedure, thus providing consistent results with minimal measurement uncertainty. This standardization is crucial for advancing drug screening and ensuring robust research findings. Full article
(This article belongs to the Special Issue State-of-the-Art of Cell Cultures in Drug Validation and Toxicity Assessment)
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22 pages, 3326 KiB  
Article
Bioengineering the Human Intestinal Mucosa and the Importance of Stromal Support for Pharmacological Evaluation In Vitro
by Matthew Freer, Jim Cooper, Kirsty Goncalves and Stefan Przyborski
Cells 2024, 13(22), 1859; https://doi.org/10.3390/cells13221859 - 8 Nov 2024
Viewed by 1322
Abstract
Drug discovery is associated with high levels of compound elimination in all stages of development. The current practices for the pharmacokinetic testing of intestinal absorption combine Transwell® inserts with the Caco-2 cell line and are associated with a wide range of limitations. [...] Read more.
Drug discovery is associated with high levels of compound elimination in all stages of development. The current practices for the pharmacokinetic testing of intestinal absorption combine Transwell® inserts with the Caco-2 cell line and are associated with a wide range of limitations. The improvement of pharmacokinetic research relies on the development of more advanced in vitro intestinal constructs that better represent human native tissue and its response to drugs, providing greater predictive accuracy. Here, we present a humanized, bioengineered intestinal construct that recapitulates aspects of intestinal microanatomy. We present improved histotypic characteristics reminiscent of the human intestine, such as a reduction in transepithelial electrical resistance (TEER) and the formation of a robust basement membrane, which are contributed to in-part by a strong stromal foundation. We explore the link between stromal–epithelial crosstalk, paracrine communication, and the role of the keratinocyte growth factor (KGF) as a soluble mediator, underpinning the tissue-specific role of fibroblast subpopulations. Permeability studies adapted to a 96-well format allow for high throughput screening and demonstrate the role of the stromal compartment and tissue architecture on permeability and functionality, which is thought to be one of many factors responsible for unexpected drug outcomes using current approaches for pharmacokinetic testing. Full article
(This article belongs to the Special Issue State-of-the-Art of Cell Cultures in Drug Validation and Toxicity Assessment)
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22 pages, 8070 KiB  
Article
Preparation of Viable Human Neurites for Neurobiological and Neurodegeneration Studies
by Markus Brüll, Nils Geese, Ivana Celardo, Michael Laumann and Marcel Leist
Cells 2024, 13(3), 242; https://doi.org/10.3390/cells13030242 - 27 Jan 2024
Cited by 3 | Viewed by 2500
Abstract
Few models allow the study of neurite damage in the human central nervous system. We used here dopaminergic LUHMES neurons to establish a culture system that allows for (i) the observation of highly enriched neurites, (ii) the preparation of the neurite fraction for [...] Read more.
Few models allow the study of neurite damage in the human central nervous system. We used here dopaminergic LUHMES neurons to establish a culture system that allows for (i) the observation of highly enriched neurites, (ii) the preparation of the neurite fraction for biochemical studies, and (iii) the measurement of neurite markers and metabolites after axotomy. LUHMES-based spheroids, plated in culture dishes, extended neurites of several thousand µm length, while all somata remained aggregated. These cultures allowed an easy microscopic observation of live or fixed neurites. Neurite-only cultures (NOC) were produced by cutting out the still-aggregated somata. The potential application of such cultures was exemplified by determinations of their protein and RNA contents. For instance, the mitochondrial TOM20 protein was highly abundant, while nuclear histone H3 was absent. Similarly, mitochondrial-encoded RNAs were found at relatively high levels, while the mRNA for a histone or the neuronal nuclear marker NeuN (RBFOX3) were relatively depleted in NOC. Another potential use of NOC is the study of neurite degeneration. For this purpose, an algorithm to quantify neurite integrity was developed. Using this tool, we found that the addition of nicotinamide drastically reduced neurite degeneration. Also, the chelation of Ca2+ in NOC delayed the degeneration, while inhibitors of calpains had no effect. Thus, NOC proved to be suitable for biochemical analysis and for studying degeneration processes after a defined cut injury. Full article
(This article belongs to the Special Issue State-of-the-Art of Cell Cultures in Drug Validation and Toxicity Assessment)
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Review

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24 pages, 2160 KiB  
Review
In Vitro 3D Models of Haematological Malignancies: Current Trends and the Road Ahead?
by Carlotta Mattioda, Claudia Voena, Gianluca Ciardelli and Clara Mattu
Cells 2025, 14(1), 38; https://doi.org/10.3390/cells14010038 - 2 Jan 2025
Viewed by 2214
Abstract
Haematological malignancies comprise a diverse group of life-threatening systemic diseases, including leukaemia, lymphoma, and multiple myeloma. Currently available therapies, including chemotherapy, immunotherapy, and CAR-T cells, are often associated with important side effects and with the development of drug resistance and, consequently, disease relapse. [...] Read more.
Haematological malignancies comprise a diverse group of life-threatening systemic diseases, including leukaemia, lymphoma, and multiple myeloma. Currently available therapies, including chemotherapy, immunotherapy, and CAR-T cells, are often associated with important side effects and with the development of drug resistance and, consequently, disease relapse. In the last decades, it was largely demonstrated that the tumor microenvironment significantly affects cancer cell proliferation and tumor response to treatment. The development of biomimetic, in vitro models may promote the investigation of the interactions between cancer cells and the tumor microenvironment and may help to better understand the mechanisms leading to drug resistance. Although advanced in vitro models have been largely explored in the field of solid tumors, due to the complex nature of the blood cancer tumor microenvironment, the mimicking of haematological malignancies mostly relies on simpler systems, often limited to two-dimensional cell culture, which intrinsically excludes the microenvironmental niche, or to ethically debated animal models. This review aims at reporting an updated overview of state-of-the-art hematological malignancies 3D in vitro models, emphasizing the key features and limitations of existing systems to inspire further research in this underexplored field. Full article
(This article belongs to the Special Issue State-of-the-Art of Cell Cultures in Drug Validation and Toxicity Assessment)
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37 pages, 4688 KiB  
Review
Cell-Based Glioma Models for Anticancer Drug Screening: From Conventional Adherent Cell Cultures to Tumor-Specific Three-Dimensional Constructs
by Daria Lanskikh, Olga Kuziakova, Ivan Baklanov, Alina Penkova, Veronica Doroshenko, Ivan Buriak, Valeriia Zhmenia and Vadim Kumeiko
Cells 2024, 13(24), 2085; https://doi.org/10.3390/cells13242085 - 17 Dec 2024
Viewed by 1642
Abstract
Gliomas are a group of primary brain tumors characterized by their aggressive nature and resistance to treatment. Infiltration of surrounding normal tissues limits surgical approaches, wide inter- and intratumor heterogeneity hinders the development of universal therapeutics, and the presence of the blood–brain barrier [...] Read more.
Gliomas are a group of primary brain tumors characterized by their aggressive nature and resistance to treatment. Infiltration of surrounding normal tissues limits surgical approaches, wide inter- and intratumor heterogeneity hinders the development of universal therapeutics, and the presence of the blood–brain barrier reduces the efficiency of their delivery. As a result, patients diagnosed with gliomas often face a poor prognosis and low survival rates. The spectrum of anti-glioma drugs used in clinical practice is quite narrow. Alkylating agents are often used as first-line therapy, but their effectiveness varies depending on the molecular subtypes of gliomas. This highlights the need for new, more effective therapeutic approaches. Standard drug-screening methods involve the use of two-dimensional cell cultures. However, these models cannot fully replicate the conditions present in real tumors, making it difficult to extrapolate the results to humans. We describe the advantages and disadvantages of existing glioma cell-based models designed to improve the situation and build future prospects to make drug discovery comprehensive and more effective for each patient according to personalized therapy paradigms. Full article
(This article belongs to the Special Issue State-of-the-Art of Cell Cultures in Drug Validation and Toxicity Assessment)
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26 pages, 1516 KiB  
Review
In Vitro Models of Diabetes: Focus on Diabetic Retinopathy
by Giulia Galgani, Giorgia Bray, Alma Martelli, Vincenzo Calderone and Valentina Citi
Cells 2024, 13(22), 1864; https://doi.org/10.3390/cells13221864 - 11 Nov 2024
Cited by 1 | Viewed by 3164
Abstract
Diabetic retinopathy is a major eye complication in patients with diabetes mellitus, and it is the leading cause of blindness and visual impairment in the world. Chronic hyperglycemia induces endothelial damage with consequent vascular lesions, resulting in global vasculitis, which affects the small [...] Read more.
Diabetic retinopathy is a major eye complication in patients with diabetes mellitus, and it is the leading cause of blindness and visual impairment in the world. Chronic hyperglycemia induces endothelial damage with consequent vascular lesions, resulting in global vasculitis, which affects the small vessels of the retina. These vascular lesions cause ischemic conditions in certain areas of the retina, with a consequent increase in the release of pro-angiogenic mediators. In addition to pharmacological interventions for controlling the blood glycaemic level, the main strategies for treating diabetic retinopathy are the intravitreal injections of drugs, surgical treatments, and vitrectomies. The complexity of diabetic retinopathy is due to its close interactions with different cell types (endothelial cells, astrocytes, and Müller cells). The evaluation of the efficacy of novel pharmacological strategies is mainly performed through in vivo models. However, the use of different animal species leads to heterogenic results and ethical concerns. For these reasons, the development of new and reliable in vitro models, such as cell co-cultures and eye organoids, represents an urgent need in this area of research. This review features an overview of the in vitro models used to date and highlights the advances in technology used to study this pathology. Full article
(This article belongs to the Special Issue State-of-the-Art of Cell Cultures in Drug Validation and Toxicity Assessment)
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26 pages, 3056 KiB  
Review
Growing Role of 3D In Vitro Cell Cultures in the Study of Cellular and Molecular Mechanisms: Short Focus on Breast Cancer, Endometriosis, Liver and Infectious Diseases
by Nora Bloise, Marialaura Giannaccari, Giuseppe Guagliano, Emanuela Peluso, Elisa Restivo, Silvia Strada, Cristina Volpini, Paola Petrini and Livia Visai
Cells 2024, 13(12), 1054; https://doi.org/10.3390/cells13121054 - 18 Jun 2024
Cited by 7 | Viewed by 4666
Abstract
Over the past decade, the development of three-dimensional (3D) models has increased exponentially, facilitating the unravelling of fundamental and essential cellular mechanisms by which cells communicate with each other, assemble into tissues and organs and respond to biochemical and biophysical stimuli under both [...] Read more.
Over the past decade, the development of three-dimensional (3D) models has increased exponentially, facilitating the unravelling of fundamental and essential cellular mechanisms by which cells communicate with each other, assemble into tissues and organs and respond to biochemical and biophysical stimuli under both physiological and pathological conditions. This section presents a concise overview of the most recent updates on the significant contribution of different types of 3D cell cultures including spheroids, organoids and organ-on-chip and bio-printed tissues in advancing our understanding of cellular and molecular mechanisms. The case studies presented include the 3D cultures of breast cancer (BC), endometriosis, the liver microenvironment and infections. In BC, the establishment of 3D culture models has permitted the visualization of the role of cancer-associated fibroblasts in the delivery of exosomes, as well as the significance of the physical properties of the extracellular matrix in promoting cell proliferation and invasion. This approach has also become a valuable tool in gaining insight into general and specific mechanisms of drug resistance. Given the considerable heterogeneity of endometriosis, 3D models offer a more accurate representation of the in vivo microenvironment, thereby facilitating the identification and translation of novel targeted therapeutic strategies. The advantages provided by 3D models of the hepatic environment, in conjunction with the high throughput characterizing various platforms, have enabled the elucidation of complex molecular mechanisms underlying various threatening hepatic diseases. A limited number of 3D models for gut and skin infections have been developed. However, a more profound comprehension of the spatial and temporal interactions between microbes, the host and their environment may facilitate the advancement of in vitro, ex vivo and in vivo disease models. Additionally, it may pave the way for the development of novel therapeutic approaches in diverse research fields. The interested reader will also find concluding remarks on the challenges and prospects of using 3D cell cultures for discovering cellular and molecular mechanisms in the research areas covered in this review. Full article
(This article belongs to the Special Issue State-of-the-Art of Cell Cultures in Drug Validation and Toxicity Assessment)
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