10th Anniversary of Biomedicines—3D In Vitro Models as an Alternative to Animal Experimentation: Advantages and Pitfalls

A special issue of Biomedicines (ISSN 2227-9059). This special issue belongs to the section "Gene and Cell Therapy".

Deadline for manuscript submissions: closed (31 October 2023) | Viewed by 10425

Special Issue Editor

Tissue Biology Research Unit, Department of Surgery, University Children’s Hospital Zurich, 8032 Zurich, Switzerland
Interests: human skin; keratinocytes; endothelial cells; skin substitutes; stem cells; melanocytes; adipose-dervied stem cells; fat tissue; skin inflammation; immune cells; skin adipocyte progenitors
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Special Issue Information

Dear Colleagues,

Animal experimentation has long been used in science to study complex biological phenomena that cannot be studied using two-dimensional in vitro models. With time, it emerged that animal models do not fully resemble the real (patho-) physiological tissue environment, in particular when translated to human patients. Recently, 3D cell culture systems have been improved with new techniques and formulations that allow the culture of cell lines as well as organoids from primary tissues. In these conditions, tissue-engineered micro-tissues can orient themselves spatially, creating niches enriched in stem or differentiated cells specific to the tissue of origin. These tools allow precise and direct monitoring of physiological and pathological mechanisms and are often much more informative and versatile than in vivo tests. Thus, three-dimensional cell culture represents an excellent alternative to the animal model by mimicking the in vivo microenvironment. These innovative in vitro models can recapitulate the complexity of the tissue of origin, with different cellular components (epithelium, stroma, leukocytes) colonizing a matrix that reproduces the spatial conformation of the tissue vitro by mimicking the in vivo microenvironment without the use of animal models that are usually quite expensive and, in some instances, do not fully resemble the real pathophysiological environment. These tools can be essential for analyzing the physiological behavior of healthy cells and patient cells derived from several diseases such as cancers, metabolic diseases, neurodegenerative disorders, autoimmune diseases, and inherited pathologies allowing for a more accurate personalized medicine approach.

Dr. Agnes Klar
Guest Editor

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Keywords

  • application of organoids
  • self-assembly method
  • dynamic perfusion bioreactor approach for tissue engineering
  • magnetic levitation
  • microfluidic 3D cell culture
  • body-on-a-chip
  • adipocyte in vitro platform to model metabolic diseases
  • skin irritation models
  • vascular blood-brain barrier in vitro
  • iPSC-derived models of healthy and diseased tissues
  • personalized medicine
  • integrated 3D cell culture models for preclinical drug development

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Published Papers (5 papers)

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Research

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18 pages, 2588 KiB  
Article
Characterization of Distinct Chondrogenic Cell Populations of Patients Suffering from Microtia Using Single-Cell Micro-Raman Spectroscopy
by Dominika Zielinska, Hesham K. Yosef, Tilo Zollitsch, Johann Kern, Yvonne Jakob, David Gvaramia, Nicole Rotter, Luca Pontiggia, Ueli Moehrlen, Thomas Biedermann and Agnes S. Klar
Biomedicines 2023, 11(9), 2588; https://doi.org/10.3390/biomedicines11092588 - 21 Sep 2023
Viewed by 805
Abstract
Microtia is a congenital condition of abnormal development of the outer ear. Tissue engineering of the ear is an alternative treatment option for microtia patients. However, for this approach, the identification of high regenerative cartilage progenitor cells is of vital importance. Raman analysis [...] Read more.
Microtia is a congenital condition of abnormal development of the outer ear. Tissue engineering of the ear is an alternative treatment option for microtia patients. However, for this approach, the identification of high regenerative cartilage progenitor cells is of vital importance. Raman analysis provides a novel, non-invasive, label-free diagnostic tool to detect distinctive biochemical features of single cells or tissues. Using micro-Raman spectroscopy, we were able to distinguish and characterize the particular molecular fingerprints of differentiated chondrocytes and perichondrocytes and their respective progenitors isolated from healthy individuals and microtia patients. We found that microtia chondrocytes exhibited lower lipid concentrations in comparison to healthy cells, thus indicating the importance of fat storage. Moreover, we suggest that collagen is a useful biomarker for distinguishing between populations obtained from the cartilage and perichondrium because of the higher spectral contributions of collagen in the chondrocytes compared to perichondrocytes from healthy individuals and microtia patients. Our results represent a contribution to the identification of cell markers that may allow the selection of specific cell populations for cartilage tissue engineering. Moreover, the observed differences between microtia and healthy cells are essential for gaining better knowledge of the cause of microtia. It can be useful for designing novel treatment options based on further investigations of the discovered biochemical substrate alterations. Full article
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20 pages, 5312 KiB  
Article
3D Spheroid Cultivation Alters the Extent and Progression of Osteogenic Differentiation of Mesenchymal Stem/Stromal Cells Compared to 2D Cultivation
by Anne Wolff, Marcus Frank, Susanne Staehlke, Armin Springer, Olga Hahn, Juliane Meyer and Kirsten Peters
Biomedicines 2023, 11(4), 1049; https://doi.org/10.3390/biomedicines11041049 - 29 Mar 2023
Cited by 3 | Viewed by 1842
Abstract
Mesenchymal stem/stromal cells (MSC) are capable of progenitor cell fraction renewal or tissue-specific differentiation. These properties are maintained during in vitro cultivation, making them an interesting model system for testing biological and pharmacological compounds. Cell cultivation in 2D is commonly used to study [...] Read more.
Mesenchymal stem/stromal cells (MSC) are capable of progenitor cell fraction renewal or tissue-specific differentiation. These properties are maintained during in vitro cultivation, making them an interesting model system for testing biological and pharmacological compounds. Cell cultivation in 2D is commonly used to study cellular responses, but the 2D environment does not reflect the structural situation of most cell types. Therefore, 3D culture systems have been developed to provide a more accurate physiological environment in terms of cell–cell interactions. Since knowledge about the effects of 3D culture on specific differentiation processes is limited, we studied the effects on osteogenic differentiation and the release of factors affecting bone metabolism for up to 35 days and compared them with the effects in 2D culture. We demonstrated that the selected 3D model allowed the rapid and reliable formation of spheroids that were stable over several weeks and both accelerated and enhanced osteogenic differentiation compared with the 2D culture. Thus, our experiments provide new insights into the effects of cell arrangement of MSC in 2D and 3D. However, due to the different culture dimensions, various detection methods had to be chosen, which in principle limits the explanatory power of the comparison between 2D and 3D cultures. Full article
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15 pages, 5450 KiB  
Article
Treatment of 3D In Vitro Tumoroids of Ovarian Cancer Using Photochemical Internalisation as a Drug Delivery Method
by Layla Mohammad Hadi, Katerina Stamati, Elnaz Yaghini, Alexander J. MacRobert and Marilena Loizidou
Biomedicines 2023, 11(2), 572; https://doi.org/10.3390/biomedicines11020572 - 15 Feb 2023
Cited by 2 | Viewed by 1534
Abstract
Photochemical internalisation (PCI) is a means of achieving spatio-temporal control of cytosolic drug delivery using sub-lethal photodynamic therapy (PDT), with a photosensitiser that can be activated by non-ionising visible light. Various 3D models including those developed at our laboratory, where spheroids are grown [...] Read more.
Photochemical internalisation (PCI) is a means of achieving spatio-temporal control of cytosolic drug delivery using sub-lethal photodynamic therapy (PDT), with a photosensitiser that can be activated by non-ionising visible light. Various 3D models including those developed at our laboratory, where spheroids are grown in a compressed collagen matrix, have been used for studying anti-cancer drug effects. However, the use of a more biomimetic tumouroid model which consists of a relatively hypoxic central cancer mass surrounded by its microenvironment (stroma) has not yet been explored in either toxicity or phototoxicity studies involving PCI. Here, we examined the efficacy of PCI using a porphyrin photosensitiser and a cytotoxin (Saporin) on ovarian cancer tumouroids, with HEY ovarian cancer cells in the central cancer compartment, and HDF fibroblast cells and HUVEC endothelial cells in the surrounding stromal compartment. The efficacy was compared to tumouroids treated with either Saporin or PDT alone, or no treatment. PCI treatment was shown to be effective in the tumouroids (determined through viability assays and imaging) and caused a considerable decrease in the viability of cancer cells both within the central cancer mass and those which had migrated into the stroma, as well as a reduction in the cell density of surrounding HUVEC and HDFs. Post-treatment, the mean distance of stromal invasion by cancer cells from the original cancer mass following treatment with Saporin alone was 730 μm vs. 125 μm for PCI. PDT was also effective at reducing viability in the central cancer mass and stroma but required a higher photosensitiser dose and light dose than PCI. Tumouroids, as tissue mimics, are suitable models for interrogating multicellular events following pharmacological assault. Full article
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22 pages, 3264 KiB  
Article
A Human Stem Cell-Derived Neurosensory–Epithelial Circuitry on a Chip to Model Herpes Simplex Virus Reactivation
by Pietro Giuseppe Mazzara, Elena Criscuolo, Marco Rasponi, Luca Massimino, Sharon Muggeo, Cecilia Palma, Matteo Castelli, Massimo Clementi, Roberto Burioni, Nicasio Mancini, Vania Broccoli and Nicola Clementi
Biomedicines 2022, 10(9), 2068; https://doi.org/10.3390/biomedicines10092068 - 24 Aug 2022
Cited by 3 | Viewed by 1863
Abstract
Both emerging viruses and well-known viral pathogens endowed with neurotropism can either directly impair neuronal functions or induce physio-pathological changes by diffusing from the periphery through neurosensory–epithelial connections. However, developing a reliable and reproducible in vitro system modeling the connectivity between the different [...] Read more.
Both emerging viruses and well-known viral pathogens endowed with neurotropism can either directly impair neuronal functions or induce physio-pathological changes by diffusing from the periphery through neurosensory–epithelial connections. However, developing a reliable and reproducible in vitro system modeling the connectivity between the different human sensory neurons and peripheral tissues is still a challenge and precludes the deepest comprehension of viral latency and reactivation at the cellular and molecular levels. This study shows a stable topographic neurosensory–epithelial connection on a chip using human stem cell-derived dorsal root ganglia (DRG) organoids. Bulk and single-cell transcriptomics showed that different combinations of key receptors for herpes simplex virus 1 (HSV-1) are expressed by each sensory neuronal cell type. This neuronal–epithelial circuitry enabled a detailed analysis of HSV infectivity, faithfully modeling its dynamics and cell type specificity. The reconstitution of an organized connectivity between human sensory neurons and keratinocytes into microfluidic chips provides a powerful in vitro platform for modeling viral latency and reactivation of human viral pathogens. Full article
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Review

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36 pages, 3176 KiB  
Review
Replacement, Reduction, and Refinement of Animal Experiments in Anticancer Drug Development: The Contribution of 3D In Vitro Cancer Models in the Drug Efficacy Assessment
by Elena M. Tosca, Davide Ronchi, Daniele Facciolo and Paolo Magni
Biomedicines 2023, 11(4), 1058; https://doi.org/10.3390/biomedicines11041058 - 30 Mar 2023
Cited by 15 | Viewed by 3421
Abstract
In the last decades three-dimensional (3D) in vitro cancer models have been proposed as a bridge between bidimensional (2D) cell cultures and in vivo animal models, the gold standards in the preclinical assessment of anticancer drug efficacy. 3D in vitro cancer models can [...] Read more.
In the last decades three-dimensional (3D) in vitro cancer models have been proposed as a bridge between bidimensional (2D) cell cultures and in vivo animal models, the gold standards in the preclinical assessment of anticancer drug efficacy. 3D in vitro cancer models can be generated through a multitude of techniques, from both immortalized cancer cell lines and primary patient-derived tumor tissue. Among them, spheroids and organoids represent the most versatile and promising models, as they faithfully recapitulate the complexity and heterogeneity of human cancers. Although their recent applications include drug screening programs and personalized medicine, 3D in vitro cancer models have not yet been established as preclinical tools for studying anticancer drug efficacy and supporting preclinical-to-clinical translation, which remains mainly based on animal experimentation. In this review, we describe the state-of-the-art of 3D in vitro cancer models for the efficacy evaluation of anticancer agents, focusing on their potential contribution to replace, reduce and refine animal experimentations, highlighting their strength and weakness, and discussing possible perspectives to overcome current challenges. Full article
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