Advances in Human Cell Culture Techniques Towards more Physiological, Differentiated Tissues

A special issue of Cells (ISSN 2073-4409).

Deadline for manuscript submissions: closed (31 December 2020) | Viewed by 92771

Special Issue Editors


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Guest Editor
Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria
Interests: 3D cell culture and pathogen interactions (fungi, HIV-1, SARS-CoV-2); lung model; mucosa; T zell zone model; innate immunity; dendritic cells; T cell polarization
Special Issues, Collections and Topics in MDPI journals

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Co-Guest Editor
Molecular and Computaional Toxicology, Vrije Universiteit, Amsterdam, The Netherland
Interests: in vitro toxicology; renal physiology; phase I metabolism; induced pluripotent stem cells; kinetics; stress respobse pathways; Nrf2

Special Issue Information

Dear Colleagues,

Advanced human cell culture systems and techniques, including induced pluripotent stem cells (iPSC), microfluidics, 3D cultures and organoids, co-culture systems and multiorgan chips have gained increasing interest in disease modeling, drug discovery, and toxicity testing. Such systems promise more human-relevant information and improved predictive value in comparison to cancer cell cultures or experiments using animals.

In the past few years, tremendous efforts have been put into designing and developing sophisticated human cell culture models to more accurately illustrate the natural microenvironment. These complex systems often take into account important parameters found in the host, such as tissue architecture and composition, shear stress, or cell motility and communication as well as primary features of cells. The simultaneous consideration of multiple parameters within the advanced human cell cultures is facilitated by the development of novel high content screening techniques and single-cell analyses and is a rapidly emerging field of research often referred to as integrative biology.

While these techniques are proving to be extremely useful tools in understanding human biology, they also hold the potential to lead to a massive reduction in whole animal experimentation or indeed the phasing out of animal experimentation entirely for extrapolation to human scenarios.

Within this Special Issue of Cells, we will highlight the challenges and future directions of advanced human cell culture models and techniques and we would highly appreciate the submission of original articles, reviews, commentaries, short reports, or protocols in this exciting field of research. Cancerous cell lines are excluded from this Special Issue of Cells.

Assoc. Prof. Dr. Doris Wilflingseder
Prof. Dr. Paul Jennings
Guest Editors

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Keywords

  • Human cell modeling
  • Stem cells
  • Gene expression analyses in 3D
  • Phenotypic analyses in 3D
  • Drug screening
  • Toxicity testing
  • Optimization of human cell models
  • Challenges of 3D models
  • Animal replacement

Published Papers (16 papers)

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Research

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17 pages, 3218 KiB  
Article
Gel-Free 3D Tumoroids with Stem Cell Properties Modeling Drug Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer
by Chiharu Sogawa, Takanori Eguchi, Yuri Namba, Yuka Okusha, Eriko Aoyama, Kazumi Ohyama and Kuniaki Okamoto
Cells 2021, 10(2), 344; https://doi.org/10.3390/cells10020344 - 6 Feb 2021
Cited by 22 | Viewed by 5042
Abstract
Researchers have developed several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids with increased properties of cancer stem cells (CSCs), also called cancer-initiating cells (CICs). Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anti-cancer drugs have [...] Read more.
Researchers have developed several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids with increased properties of cancer stem cells (CSCs), also called cancer-initiating cells (CICs). Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anti-cancer drugs have been reported using 2D culture systems, whereas 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aimed to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyrosine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter the tumoroid growth of metastatic colorectal cancer (mCRC). Gene expression signatures of highly metastatic aggregative CRC (LuM1 cells) vs. low-metastatic, non-aggregative CRC (Colon26 and NM11 cells) were analyzed using microarray. To establish a 3D culture-based multiplexing reporter assay system, LuM1 was stably transfected with the Mmp9 promoter-driven ZsGreen fluorescence reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate®, a gel-free 3D culture device. LuM1 cells highly expressed mRNA encoding ABCG2 (a drug resistance pump, i.e., CSC/CIC marker), other CSC/CIC markers (DLL1, EpCAM, podoplanin, STAT3/5), pluripotent stem cell markers (Sox4/7, N-myc, GATA3, Nanog), and metastatic markers (MMPs, Integrins, EGFR), compared to the other two cell types. Hoechst efflux stem cell-like side population was increased in LuM1 (7.8%) compared with Colon26 (2.9%), both of which were markedly reduced by verapamil treatment, an ABCG2 inhibitor. Smaller cell aggregates of LuM1 were more sensitive to Cisplatin (at 10 μM), whereas larger tumoroids with increased ABCG2 expression were insensitive. Notably, Cisplatin (2 μM) and Imatinib (10 μM) at low concentrations significantly promoted tumoroid formation (cell aggregation) and increased Mmp9 promoter activity in mCRC LuM1/m9, while not cytotoxic to them. On the other hand, 5-FU significantly inhibited tumoroid growth, although not completely. Thus, drug resistance in cancer with increased stem cell properties was modeled using the gel-free 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance. Full article
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26 pages, 9594 KiB  
Article
Choice of Differentiation Media Significantly Impacts Cell Lineage and Response to CFTR Modulators in Fully Differentiated Primary Cultures of Cystic Fibrosis Human Airway Epithelial Cells
by Vinciane Saint-Criq, Livia Delpiano, John Casement, Jennifer C. Onuora, JinHeng Lin and Michael A. Gray
Cells 2020, 9(9), 2137; https://doi.org/10.3390/cells9092137 - 21 Sep 2020
Cited by 26 | Viewed by 5445
Abstract
In vitro cultures of primary human airway epithelial cells (hAECs) grown at air–liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number [...] Read more.
In vitro cultures of primary human airway epithelial cells (hAECs) grown at air–liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media, are now available, making comparison of data between studies difficult. Here, we investigated the impact of two common differentiation media on phenotypic, transcriptomic, and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH, Ussing chamber) specifically showed that ATP12A and CFTR function were altered, impacting pH and transepithelial ion transport in CF hAECs. Importantly, the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications. Full article
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11 pages, 2417 KiB  
Article
Polysaccharide Multilayer Films in Sensors for Detecting Prostate Tumor Cells Based on Hyaluronan-CD44 Interactions
by João Batista Maia Rocha Neto, Andrey Coatrini Soares, Rogério Aparecido Bataglioli, Olívia Carr, Carlos Alberto Rodrigues Costa, Osvaldo N. Oliveira, Jr., Marisa Masumi Beppu and Hernandes F. Carvalho
Cells 2020, 9(6), 1563; https://doi.org/10.3390/cells9061563 - 26 Jun 2020
Cited by 16 | Viewed by 3766
Abstract
The increasing need for point-of-care diagnosis has sparked the development of label-free sensing platforms, some of which are based on impedance measurements with biological cells. Here, interdigitated electrodes were functionalized with layer-by-layer (LbL) films of hyaluronan (HA) and chitosan (CHI) to detect prostatic [...] Read more.
The increasing need for point-of-care diagnosis has sparked the development of label-free sensing platforms, some of which are based on impedance measurements with biological cells. Here, interdigitated electrodes were functionalized with layer-by-layer (LbL) films of hyaluronan (HA) and chitosan (CHI) to detect prostatic tumor cells (PC3 line). The deposition of LbL films was confirmed with atomic force microscopy and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), which featured the vibrational modes of the HA top layer capable of interacting specifically with glycoprotein CD44 receptors overexpressed in tumor cells. Though the CHI/HA LbL films cannot be considered as a traditional biosensor due to their limited selectivity, it was possible to distinguish prostate tumor cells in the range from 50 to 600 cells/µL in in vitro experiments with impedance spectroscopy. This was achieved by treating the impedance data with information visualization methods, which confirmed the distinguishing ability of the films by observing the absence of false positives in a series of control experiments. The CD44–HA interactions may, therefore, be exploited in clinical analyses and point-of-care diagnostics for cancer, particularly if computational methods are used to process the data. Full article
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20 pages, 1885 KiB  
Article
Human Liver-Derived Extracellular Matrix for the Culture of Distinct Human Primary Liver Cells
by Niki Alevra Sarika, Valéry L. Payen, Maximilien Fléron, Joachim Ravau, Davide Brusa, Mustapha Najimi, Edwin De Pauw, Gauthier Eppe, Gabriel Mazzucchelli, Etienne M. Sokal, Anne des Rieux and Adil El Taghdouini
Cells 2020, 9(6), 1357; https://doi.org/10.3390/cells9061357 - 30 May 2020
Cited by 10 | Viewed by 4583
Abstract
The lack of robust methods to preserve, purify and in vitro maintain the phenotype of the human liver’s highly specialized parenchymal and non-parenchymal cell types importantly hampers their exploitation for the development of research and clinical applications. There is in this regard a [...] Read more.
The lack of robust methods to preserve, purify and in vitro maintain the phenotype of the human liver’s highly specialized parenchymal and non-parenchymal cell types importantly hampers their exploitation for the development of research and clinical applications. There is in this regard a growing interest in the use of tissue-specific extracellular matrix (ECM) to provide cells with an in vitro environment that more closely resembles that of the native tissue. In the present study, we have developed a method that allows for the isolation and downstream application of the human liver’s main cell types from cryopreserved material. We also isolated and solubilized human liver ECM (HL-ECM), analyzed its peptidomic and proteomic composition by mass spectrometry and evaluated its interest for the culture of distinct primary human liver cells. Our analysis of the HL-ECM revealed proteomic diversity, type 1 collagen abundance and partial loss of integrity following solubilization. Solubilized HL-ECM was evaluated either as a coating or as a medium supplement for the culture of human primary hepatocytes, hepatic stellate cells and liver sinusoidal endothelial cells. Whereas the solubilized HL-ECM was suitable for cell culture, its impact on the phenotype and/or functionality of the human liver cells was limited. Our study provides a first detailed characterization of solubilized HL-ECM and a first report of its influence on the culture of distinct human primary liver cells. Full article
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12 pages, 1462 KiB  
Article
Human Platelet Lysate Can Replace Fetal Calf Serum as a Protein Source to Promote Expansion and Osteogenic Differentiation of Human Bone-Marrow-Derived Mesenchymal Stromal Cells
by Maria Karadjian, Anne-Sophie Senger, Christopher Essers, Sebastian Wilkesmann, Raban Heller, Joerg Fellenberg, Rolf Simon and Fabian Westhauser
Cells 2020, 9(4), 918; https://doi.org/10.3390/cells9040918 - 9 Apr 2020
Cited by 16 | Viewed by 4396
Abstract
Fetal calf serum (FCS) is frequently used as a growth factor and protein source in bone-marrow-derived mesenchymal stromal cell (BMSC) culture media, although it is a xenogenic product presenting multiple disadvantages including but not limited to ethical concerns. A promising alternative for FCS [...] Read more.
Fetal calf serum (FCS) is frequently used as a growth factor and protein source in bone-marrow-derived mesenchymal stromal cell (BMSC) culture media, although it is a xenogenic product presenting multiple disadvantages including but not limited to ethical concerns. A promising alternative for FCS is human platelet lysate (hPL), which is produced out of human platelet concentrates and happens to be a stable and reliable protein source. In this study, we investigated the influence of hPL in an expansion medium (ESM) and an osteogenic differentiation medium (ODM) on the proliferation and osteogenic differentiation capacity of human BMSC. Therefore, we assessed population doublings during cell expansion, performed alizarin red staining to evaluate the calcium content in the extracellular matrix and determined the activity of alkaline phosphatase (ALP) as osteogenic differentiation correlates. The proliferation rate of BMSC cultured in ESM supplemented with hPL exceeded the proliferation rate of BMSC cultured in the presence of FCS. Furthermore, the calcium content and ALP activity was significantly higher in samples incubated in hPL-supplemented ODM, especially in the early phases of differentiation. Our results show that hPL can replace FCS as a protein supplier in cell culture media and does not negatively affect the osteogenic differentiation capacity of BMSC. Full article
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11 pages, 1267 KiB  
Article
Three-Dimensional Cell Culture Based on Magnetic Fields to Assemble Low-Grade Ovarian Carcinoma Cell Aggregates Containing Lymphocytes
by Caroline Natânia de Souza-Araújo, Cláudia Rodrigues Tonetti, Marcella Regina Cardoso, Liliana Aparecida Lucci de Angelo Andrade, Rodrigo Fernandes da Silva, Luís Gustavo Romani Fernandes and Fernando Guimarães
Cells 2020, 9(3), 635; https://doi.org/10.3390/cells9030635 - 6 Mar 2020
Cited by 9 | Viewed by 3262
Abstract
There is a limited number of established ovarian cancer cell lines matching the low-grade serous histotype available for research purposes. Three-dimensional (3D) culture systems provide in vitro models with better tissue-like characteristics than two-dimensional (2D) systems. The goal in the study was to [...] Read more.
There is a limited number of established ovarian cancer cell lines matching the low-grade serous histotype available for research purposes. Three-dimensional (3D) culture systems provide in vitro models with better tissue-like characteristics than two-dimensional (2D) systems. The goal in the study was to characterize the growth of a given low-grade serous ovarian carcinoma cell line in a 3D culture system conducted in a magnetic field. Moreover, the culture system was evaluated in respect to the assembly of malignant cell aggregates containing lymphocytes. CAISMOV24 cell line alone or mixed with human peripheral blood mononuclear cells (PBMC) were cultured using a commercially available 3D culture system designed for 24 well plates. Resulting cell aggregates revealed the intrinsic capacity of CAISMOV24 cells to assemble structures morphologically defined as papillary, and reflected molecular characteristics usually found in ovarian carcinomas. The contents of lymphocytes into co-cultured cell aggregates were significantly higher (p < 0.05) when NanoShuttle-conjugated PBMC were employed compared with non-conjugated PBMC. Moreover, lymphocyte subsets NK, T-CD4, T-CD8 and T-regulatory were successfully retrieved from co-cultured cell aggregates at 72h. Thus, the culture system allowed CAISMOV24 cell line to develop papillary-like cell aggregates containing lymphocytes. Full article
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15 pages, 3288 KiB  
Article
Modulation of the Lipid Profile of Reconstructed Skin Substitutes after Essential Fatty Acid Supplementation Affects Testosterone Permeability
by Mélissa Simard, Pierre Julien, Julie Fradette and Roxane Pouliot
Cells 2019, 8(10), 1142; https://doi.org/10.3390/cells8101142 - 25 Sep 2019
Cited by 22 | Viewed by 4200
Abstract
Skin models with efficient skin barrier function are required for percutaneous absorption studies. The contribution of media supplementation with n-3 and n-6 polyunsaturated fatty acids (PUFAs) to the development of the skin barrier function of in vitro skin models remains incompletely understood. To [...] Read more.
Skin models with efficient skin barrier function are required for percutaneous absorption studies. The contribution of media supplementation with n-3 and n-6 polyunsaturated fatty acids (PUFAs) to the development of the skin barrier function of in vitro skin models remains incompletely understood. To investigate whether PUFAs, alpha-linolenic acid (ALA, n-3 PUFA) and linoleic acid (LA, n-6 PUFA), could enhance the impermeability of a three-dimensional reconstructed human skin model, skin substitutes were produced according to the self-assembly method using culture media supplemented with either 10 μM ALA or 10 μM LA. The impact of PUFAs on skin permeability was studied by using a Franz cell diffusion system to assess the percutaneous absorption of testosterone and benzoic acid. Our findings showed that ALA supplementation induced a decrease in the absorption of testosterone, while LA supplementation did not significantly influence the penetration of testosterone and benzoic acid under present experimental conditions. Both ALA and LA were incorporated into phospholipids of the skin substitutes, resulting in an increase in n-3 total PUFAs or n-6 total PUFAs. Collectively, these results revealed the under-estimated impact of n-3 PUFA supplementation as well as the importance of the n-6 to n-3 ratio on the formation of the skin barrier of in vitro reconstructed human skin models. Full article
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27 pages, 5139 KiB  
Article
Fisetin, a 3,7,3′,4′-Tetrahydroxyflavone Inhibits the PI3K/Akt/mTOR and MAPK Pathways and Ameliorates Psoriasis Pathology in 2D and 3D Organotypic Human Inflammatory Skin Models
by Jean Christopher Chamcheu, Stephane Esnault, Vaqar M. Adhami, Andrea L. Noll, Sergette Banang-Mbeumi, Tithi Roy, Sitanshu S. Singh, Shile Huang, Konstantin G. Kousoulas and Hasan Mukhtar
Cells 2019, 8(9), 1089; https://doi.org/10.3390/cells8091089 - 15 Sep 2019
Cited by 54 | Viewed by 8536
Abstract
Psoriasis is a chronic immune-mediated skin disease that involves the interaction of immune and skin cells, and is characterized by cytokine-driven epidermal hyperplasia, deviant differentiation, inflammation, and angiogenesis. Because the available treatments for psoriasis have significant limitations, dietary products are potential natural sources [...] Read more.
Psoriasis is a chronic immune-mediated skin disease that involves the interaction of immune and skin cells, and is characterized by cytokine-driven epidermal hyperplasia, deviant differentiation, inflammation, and angiogenesis. Because the available treatments for psoriasis have significant limitations, dietary products are potential natural sources of therapeutic molecules, which can repair the molecular defects associated with psoriasis and could possibly be developed for its management. Fisetin (3,7,3′,4′-tetrahydroxyflavone), a phytochemical naturally found in pigmented fruits and vegetables, has demonstrated proapoptotic and antioxidant effects in several malignancies. This study utilized biochemical, cellular, pharmacological, and tissue engineering tools to characterize the effects of fisetin on normal human epidermal keratinocytes (NHEKs), peripheral blood mononuclear cells (PBMC), and CD4+ T lymphocytes in 2D and 3D psoriasis-like disease models. Fisetin treatment of NHEKs dose- and time-dependently induced differentiation and inhibited interleukin-22-induced proliferation, as well as activation of the PI3K/Akt/mTOR pathway. Fisetin treatment of TNF-α stimulated NHEKs also significantly inhibited the activation of p38 and JNK, but had enhanced effect on ERK1/2 (MAPK). In addition, fisetin treatment significantly decreased the secretion of Th1/Th-17 pro-inflammatory cytokines, particularly IFN-γ and IL-17A by 12-O-tetradecanolylphorbol 13-acetate (TPA)-stimulated NHEKs and anti-CD3/CD28-activated human PBMCs. Furthermore, we established the in vivo relevance of fisetin functions, using a 3D full-thickness human skin model of psoriasis (FTRHSP) that closely mimics in vivo human psoriatic skin lesions. Herein, fisetin significantly ameliorated psoriasis-like disease features, and decreased the production of IL-17 by CD4+ T lymphocytes co-cultured with FTRHSP. Collectively, our data identify the prodifferentiative, antiproliferative, and anti-inflammatory effects of fisetin, via modulation of the PI3K-Akt-mTOR and p38/JNK pathways and the production of cytokines in 2D and 3D human skin models of psoriasis. These results suggest that fisetin has a great potential to be developed as an effective and inexpensive agent for the treatment of psoriasis and other related inflammatory skin disorders. Full article
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17 pages, 3964 KiB  
Article
The Differences in the Proteome Profile of Cannabidiol-Treated Skin Fibroblasts following UVA or UVB Irradiation in 2D and 3D Cell Cultures
by Agnieszka Gęgotek, Sinemyiz Atalay, Pedro Domingues and Elżbieta Skrzydlewska
Cells 2019, 8(9), 995; https://doi.org/10.3390/cells8090995 - 28 Aug 2019
Cited by 45 | Viewed by 6950
Abstract
Cannabidiol (CBD), as the only phytocannabinoid that has no psychoactive effect, has both antioxidant and anti-inflammatory effects, and thus might be suggested as a cytoprotective compound against UV-induced metabolic changes in skin cells. Therefore, the aim of this study was to investigate the [...] Read more.
Cannabidiol (CBD), as the only phytocannabinoid that has no psychoactive effect, has both antioxidant and anti-inflammatory effects, and thus might be suggested as a cytoprotective compound against UV-induced metabolic changes in skin cells. Therefore, the aim of this study was to investigate the level of protective CBD activity by evaluating the proteomic profile of 2D and 3D cultured skin fibroblasts models following exposure to UVA and UVB radiation. The CBD cytoprotective effect against UV-induced damage in 2D and 3D cultured fibroblasts were different. The main alterations focus on the range of cell reaction and involved different proteins associated with various molecular functions. In the 2D cultured cells, following UV radiation, the major changes were associated with proteins involved in antioxidant response and inflammation, while, in the 3D cultured fibroblasts, CBD action against UV induced changes were mainly associated with the activation of signalling pathways. Therefore, the knowledge of the CBD action in a multilayer skin cells model allowed for the prediction of changes in cell-cell interactions and skin cell metabolism. Knowledge about the lower protective effect of CBD in 3D cultured fibroblasts should be taken into account during the design of UV light protection. Full article
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15 pages, 4260 KiB  
Article
Tissue Specific Differentiation of Human Chondrocytes Depends on Cell Microenvironment and Serum Selection
by Annemarie Ecke, Anne-Helen Lutter, Jenny Scholka, Anna Hansch, Roland Becker and Ursula Anderer
Cells 2019, 8(8), 934; https://doi.org/10.3390/cells8080934 - 19 Aug 2019
Cited by 18 | Viewed by 4926
Abstract
Therapeutic options to cure osteoarthritis (OA) are not yet available, although cell-based therapies for the treatment of traumatic defects of cartilage have already been developed using, e.g., articular chondrocytes. In order to adapt cell-based therapies to treat OA, appropriate cell culture conditions are [...] Read more.
Therapeutic options to cure osteoarthritis (OA) are not yet available, although cell-based therapies for the treatment of traumatic defects of cartilage have already been developed using, e.g., articular chondrocytes. In order to adapt cell-based therapies to treat OA, appropriate cell culture conditions are necessary. Chondrocytes require a 3-dimensional (3D) environment for redifferentiation after 2-dimensional (2D) expansion. Fetal bovine serum (FBS) is commonly used as a medium supplement, although the usage of a xenogeneic serum could mask the intrinsic behavior of human cells in vitro. The aim of this study was to compare human articular chondrocytes cultivated as monolayers (2D) and the development of microtissues (3D) in the presence of FBS with those cultivated with human serum (HS). Evaluation of the expression of various markers via immunocytochemistry on monolayer cells revealed a higher dedifferentiation degree of chondrocytes cultivated with HS. Scaffold-free microtissues were generated using the agar overlay technique, and their differentiation level was evaluated via histochemistry and immunohistochemistry. Microtissues cultivated in the medium with FBS showed a higher redifferentiation level. This was evidenced by bigger microtissues and a more cartilage-like composition of the matrix with not any/less positivity for cartilage-specific markers in HS versus moderate-to-high positivity in FBS-cultured microtissues. The present study showed that the differentiation degree of chondrocytes depends both on the microenvironment of the cells and the serum type with FBS achieving the best results. However, HS should be preferred for the engineering of cartilage-like microtissues, as it rather enables a "human-based" situation in vitro. Hence, cultivation conditions might be further optimized to gain an even more adequate and donor-independent redifferentiation of chondrocytes in microtissues, e.g., designing a suitable chemically-defined serum supplement. Full article
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14 pages, 6569 KiB  
Article
Biomimetic In Vitro Model of Cell Infiltration into Skin Scaffolds for Pre-Screening and Testing of Biomaterial-Based Therapies
by Rafael Ballesteros-Cillero, Evan Davison-Kotler, Nupur Kohli, William S. Marshall and Elena García-Gareta
Cells 2019, 8(8), 917; https://doi.org/10.3390/cells8080917 - 17 Aug 2019
Cited by 7 | Viewed by 5647
Abstract
Due to great clinical need, research where different biomaterials are tested as 3D scaffolds for skin tissue engineering has increased. In vitro studies use a cell suspension that is simply pipetted onto the material and cultured until the cells migrate and proliferate within [...] Read more.
Due to great clinical need, research where different biomaterials are tested as 3D scaffolds for skin tissue engineering has increased. In vitro studies use a cell suspension that is simply pipetted onto the material and cultured until the cells migrate and proliferate within the 3D scaffold, which does not mimic the in vivo reality. Our aim was to engineer a novel biomimetic in vitro model that mimics the natural cell infiltration process occurring in wound healing, thus offering a realistic approach when pre-screening and testing new skin substitutes. Our model consists of porous membrane cell culture inserts coated with gelatin and seeded with human dermal fibroblasts, inside which two different commercially available dermal substitutes were placed. Several features relevant to the wound healing process (matrix contraction, cell infiltration and proliferation, integration of the biomaterial with the surrounding tissue, and secretion of exogenous cytokines and growth factors) were evaluated. Our results showed that cells spontaneously infiltrate the materials and that our engineered model is able to induce and detect subtle differences between different biomaterials. The model allows for room for improvements or “adds-on” and miniaturization and can contribute to the development of functional and efficient skin substitutes for burns and chronic wounds. Full article
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17 pages, 4129 KiB  
Article
Epithelial-To-Mesenchymal Transition Markers and CD44 Isoforms Are Differently Expressed in 2D and 3D Cell Cultures of Prostate Cancer Cells
by Fabrizio Fontana, Michela Raimondi, Monica Marzagalli, Michele Sommariva, Patrizia Limonta and Nicoletta Gagliano
Cells 2019, 8(2), 143; https://doi.org/10.3390/cells8020143 - 11 Feb 2019
Cited by 42 | Viewed by 7108
Abstract
Three-dimensional (3D) cell cultures allow the mimic of functions of living tissues and provide key information encoded in tissue architecture. Considered the pivotal role of epithelial-to-mesenchymal transition (EMT) in carcinoma progression, including prostate cancer (PCa), we aimed at investigating the effect of the [...] Read more.
Three-dimensional (3D) cell cultures allow the mimic of functions of living tissues and provide key information encoded in tissue architecture. Considered the pivotal role of epithelial-to-mesenchymal transition (EMT) in carcinoma progression, including prostate cancer (PCa), we aimed at investigating the effect of the 3D arrangement on the expression of some key markers of EMT in cultured human prostate cancer (PCa) cells, to better understand PCa cell behavior. PC3 and DU145 PCa cells were cultured in RPMI cell culture medium either in 2D-monolayers or in 3D-spheroids. The main EMT markers E-cadherin, N-cadherin, α-smooth muscle actin (αSMA), vimentin, Snail, Slug, Twist and Zeb1 were evaluated by confocal microscopy, real-time PCR and Western blot. Confocal microscopy revealed that E-cadherin was similarly expressed at the cell boundaries on the plasma membrane of PCa cells grown in 2D-monolayers, as well as in 3D-spheroids, but resulted up-regulated in 3D-spheroids, compared to 2D-monolayers, at the mRNA and protein level. Moreover, markers of the mesenchymal phenotype were expressed at very low levels in 3D-spheroids, suggesting important differences in the phenotype of PCa cells grown in 3D-spheroids or in 2D-monolayers. Considered as a whole, our findings contribute to a clarification of the role of EMT in PCa and confirm that a 3D cell culture model could provide deeper insight into the understanding of the biology of PCa. Full article
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27 pages, 1818 KiB  
Review
Ex Vivo Culture Models to Indicate Therapy Response in Head and Neck Squamous Cell Carcinoma
by Imke Demers, Johan Donkers, Bernd Kremer and Ernst Jan Speel
Cells 2020, 9(11), 2527; https://doi.org/10.3390/cells9112527 - 23 Nov 2020
Cited by 20 | Viewed by 4482
Abstract
Head and neck squamous cell carcinoma (HNSCC) is characterized by a poor 5 year survival and varying response rates to both standard-of-care and new treatments. Despite advances in medicine and treatment methods, mortality rates have hardly decreased in recent decades. Reliable patient-derived tumor [...] Read more.
Head and neck squamous cell carcinoma (HNSCC) is characterized by a poor 5 year survival and varying response rates to both standard-of-care and new treatments. Despite advances in medicine and treatment methods, mortality rates have hardly decreased in recent decades. Reliable patient-derived tumor models offer the chance to predict therapy response in a personalized setting, thereby improving treatment efficacy by identifying the most appropriate treatment regimen for each patient. Furthermore, ex vivo tumor models enable testing of novel therapies before introduction in clinical practice. A literature search was performed to identify relevant literature describing three-dimensional ex vivo culture models of HNSCC to examine sensitivity to chemotherapy, radiotherapy, immunotherapy and targeted therapy. We provide a comprehensive overview of the currently used three-dimensional ex vivo culture models for HNSCC with their advantages and limitations, including culture success percentage and comparison to the original tumor. Furthermore, we evaluate the potential of these models to predict patient therapy response. Full article
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19 pages, 1798 KiB  
Review
Closer to Nature Through Dynamic Culture Systems
by Tzyy-Yue Wong, Sheng-Nan Chang, Rong-Chang Jhong, Ching-Jiunn Tseng, Gwo-Ching Sun and Pei-Wen Cheng
Cells 2019, 8(9), 942; https://doi.org/10.3390/cells8090942 - 21 Aug 2019
Cited by 8 | Viewed by 4422
Abstract
Mechanics in the human body are required for normal cell function at a molecular level. It is now clear that mechanical stimulations play significant roles in cell growth, differentiation, and migration in normal and diseased cells. Recent studies have led to the discovery [...] Read more.
Mechanics in the human body are required for normal cell function at a molecular level. It is now clear that mechanical stimulations play significant roles in cell growth, differentiation, and migration in normal and diseased cells. Recent studies have led to the discovery that normal and cancer cells have different mechanosensing properties. Here, we discuss the application and the physiological and pathological meaning of mechanical stimulations. To reveal the optimal conditions for mimicking an in vivo microenvironment, we must, therefore, discern the mechanotransduction occurring in cells. Full article
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21 pages, 6535 KiB  
Review
3D-Organotypic Cultures to Unravel Molecular and Cellular Abnormalities in Atopic Dermatitis and Ichthyosis Vulgaris
by Géraldine Leman, Verena Moosbrugger-Martinz, Stefan Blunder, Petra Pavel and Sandrine Dubrac
Cells 2019, 8(5), 489; https://doi.org/10.3390/cells8050489 - 22 May 2019
Cited by 15 | Viewed by 5799
Abstract
Atopic dermatitis (AD) is characterized by dry and itchy skin evolving into disseminated skin lesions. AD is believed to result from a primary acquired or a genetically-induced epidermal barrier defect leading to immune hyper-responsiveness. Filaggrin (FLG) is a protein found in the cornified [...] Read more.
Atopic dermatitis (AD) is characterized by dry and itchy skin evolving into disseminated skin lesions. AD is believed to result from a primary acquired or a genetically-induced epidermal barrier defect leading to immune hyper-responsiveness. Filaggrin (FLG) is a protein found in the cornified envelope of fully differentiated keratinocytes, referred to as corneocytes. Although FLG null mutations are strongly associated with AD, they are not sufficient to induce the disease. Moreover, most patients with ichthyosis vulgaris (IV), a monogenetic skin disease characterized by FLG homozygous, heterozygous, or compound heterozygous null mutations, display non-inflamed dry and scaly skin. Thus, all causes of epidermal barrier impairment in AD have not yet been identified, including those leading to the Th2-predominant inflammation observed in AD. Three dimensional organotypic cultures have emerged as valuable tools in skin research, replacing animal experimentation in many cases and precluding the need for repeated patient biopsies. Here, we review the results on IV and AD obtained with epidermal or skin equivalents and consider these findings in the context of human in vivo data. Further research utilizing complex models including immune cells and cutaneous innervation will enable finer dissection of the pathogenesis of AD and deepen our knowledge of epidermal biology. Full article
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13 pages, 3711 KiB  
Protocol
Turning the World Upside-Down in Cellulose for Improved Culturing and Imaging of Respiratory Challenges within a Human 3D Model
by Viktoria Zaderer, Martin Hermann, Cornelia Lass-Flörl, Wilfried Posch and Doris Wilflingseder
Cells 2019, 8(10), 1292; https://doi.org/10.3390/cells8101292 - 21 Oct 2019
Cited by 17 | Viewed by 10277
Abstract
Polarized growth of human-derived respiratory epithelial cells on hydrogel-coated filters offers big advantages concerning detailed experiments with respect to drug screening or host pathogen interactions. Different microscopic approaches, such as confocal analyses and high content screening, help to examine such 3D respiratory samples, [...] Read more.
Polarized growth of human-derived respiratory epithelial cells on hydrogel-coated filters offers big advantages concerning detailed experiments with respect to drug screening or host pathogen interactions. Different microscopic approaches, such as confocal analyses and high content screening, help to examine such 3D respiratory samples, resulting in high-resolution pictures and enabling quantitative analyses of high cell numbers. A major problem employing these techniques relates to single-use instead of multiple-use of Transwell filters and difficulties in the digestion of collagen if subsequent analyses are needed. Up to date, cells are seeded in collagen-based matrices to the inner field of Transwell inserts, which makes it impossible to image due to the design of the inserts and hard to perform other analyses since digestion of the collagen matrix also affects Transwell grown cells. To overcome these problems, we optimized culturing conditions for monitoring cell differentiation or repeated dose experiments over a long time period. For this, cells are seeded upside-down to the bottom side of filters within an animal-free cellulose hydrogel. These cells were then grown inverted under static conditions and were differentiated in air-liquid interphase (ALI). Full differentiation of goblet (Normal Human Bronchial Epithelial (NHBE))/Club (small airway epithelia (SAE)) cells and ciliated cells was detected after 12 days in ALI. Inverted cell cultures could then be used for ‘follow-up’ live cell imaging experiments, as well as, flow-cytometric analyses due to easy digestion of the cellulose compared to classical collagen matrices. Additionally, this culture technique also enables easy addition of immune cells, such as dendritic cells (DCs), macrophages, neutrophils, T or B cells alone or in combination, to the inner field of the Transwell to monitor immune cell behavior after repeated respiratory challenge. Our detailed protocol offers the possibility of culturing human primary polarized cells into a fully differentiated, thick epithelium without any animal components over >700 days. Furthermore, this animal-free, inverted system allows investigation of the same inserts, because the complete Transwell can be readily transferred to glass-bottom dishes for live cell imaging analyses and then returned to its original plate for further cultivation. Full article
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