Cells

2024

Jump to: 2023, 2022, 2021

20 pages, 9382 KiB

Open AccessArticle

Enhancing the Antibody Production Efficiency of Chinese Hamster Ovary Cells through Improvement of Disulfide Bond Folding Ability and Apoptosis Resistance

by Chen Zhang, Yunhui Fu, Wenyun Zheng, Feng Chang, Yue Shen, Jinping Niu, Yangmin Wang and Xingyuan Ma

Cells 2024, 13(17), 1481; https://doi.org/10.3390/cells13171481 - 4 Sep 2024

Viewed by 158

Abstract

The complex structure of monoclonal antibodies (mAbs) expressed in Chinese hamster ovary (CHO) cells may result in the accumulation of unfolded proteins, triggering endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). If the protein folding ability cannot maintain ER homeostasis, the [...] Read more.

The complex structure of monoclonal antibodies (mAbs) expressed in Chinese hamster ovary (CHO) cells may result in the accumulation of unfolded proteins, triggering endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). If the protein folding ability cannot maintain ER homeostasis, the cell will shut down protein translation and ultimately induce apoptosis. We co-overexpressed HsQSOX1b and survivin proteins in the antibody-producing cell line CHO-PAb to obtain a new cell line, CHO-PAb-QS. Compared with CHO-PAb cells, the survival time of CHO-PAb-QS cells in batch culture was extended by 2 days, and the antibody accumulation and productivity were increased by 52% and 45%, respectively. The proportion of (HC-LC)₂ was approximately doubled in the CHO-PAb-QS cells, which adapted to the accelerated disulfide bond folding capacity by upregulating the UPR’s strength and increasing the ER content. The results of the apoptosis assays indicated that the CHO-PAb-QS cell line exhibited more excellent resistance to apoptosis induced by ER stress. Finally, CHO-PAb-QS cells exhibited mild oxidative stress but did not significantly alter the redox status. This study demonstrated that strategies based on HsQSOX1b and survivin co-overexpression could facilitate protein disulfide bond folding and anti-apoptosis ability, enhancing antibody production efficiency in CHO cell lines. Full article

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2023

Jump to: 2024, 2022, 2021

20 pages, 4408 KiB

Open AccessArticle

High-Level Production of scFv-Fc Antibody Using an Artificial Promoter System with Transcriptional Positive Feedback Loop of Transactivator in CHO Cells

by Binbin Ying, Yoshinori Kawabe, Feiyang Zheng, Yuki Amamoto and Masamichi Kamihira

Cells 2023, 12(22), 2638; https://doi.org/10.3390/cells12222638 - 16 Nov 2023

Cited by 1 | Viewed by 2125

Abstract

With the increasing demand for therapeutic antibodies, CHO cells have become the de facto standard as producer host cells for biopharmaceutical production. High production yields are required for antibody production, and developing a high-titer production system is increasingly crucial. This study was established [...] Read more.

With the increasing demand for therapeutic antibodies, CHO cells have become the de facto standard as producer host cells for biopharmaceutical production. High production yields are required for antibody production, and developing a high-titer production system is increasingly crucial. This study was established to develop a high-production system using a synthetic biology approach by designing a gene expression system based on an artificial transcription factor that can strongly induce the high expression of target genes in CHO cells. To demonstrate the functionality of this artificial gene expression system and its ability to induce the high expression of target genes in CHO cells, a model antibody (scFv-Fc) was produced using this system. Excellent results were obtained with the plate scale, and when attempting continuous production in semi-continuous cultures using bioreactor tubes with high-cell-density suspension culture using a serum-free medium, high-titer antibody production at the gram-per-liter level was achieved. Shifting the culture temperature to a low temperature of 33 °C achieved scFv-Fc concentrations of up to 5.5 g/L with a specific production rate of 262 pg/(cell∙day). This artificial gene expression system should be a powerful tool for CHO cell engineering aimed at constructing high-yield production systems. Full article

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19 pages, 4276 KiB

Open AccessArticle

Impact of the Physical Cellular Microenvironment on the Structure and Function of a Model Hepatocyte Cell Line for Drug Toxicity Applications

by Benjamin Allcock, Wenbin Wei, Kirsty Goncalves, Henry Hoyle, Alisha Robert, Rebecca Quelch-Cliffe, Adam Hayward, Jim Cooper and Stefan Przyborski

Cells 2023, 12(19), 2408; https://doi.org/10.3390/cells12192408 - 5 Oct 2023

Viewed by 1278

Abstract

It is widely recognised that cells respond to their microenvironment, which has implications for cell culture practices. Growth cues provided by 2D cell culture substrates are far removed from native 3D tissue structure in vivo. Geometry is one of many factors that differs [...] Read more.

It is widely recognised that cells respond to their microenvironment, which has implications for cell culture practices. Growth cues provided by 2D cell culture substrates are far removed from native 3D tissue structure in vivo. Geometry is one of many factors that differs between in vitro culture and in vivo cellular environments. Cultured cells are far removed from their native counterparts and lose some of their predictive capability and reliability. In this study, we examine the cellular processes that occur when a cell is cultured on 2D or 3D surfaces for a short period of 8 days prior to its use in functional assays, which we term: “priming”. We follow the process of mechanotransduction from cytoskeletal alterations, to changes to nuclear structure, leading to alterations in gene expression, protein expression and improved functional capabilities. In this study, we utilise HepG2 cells as a hepatocyte model cell line, due to their robustness for drug toxicity screening. Here, we demonstrate enhanced functionality and improved drug toxicity profiles that better reflect the in vivo clinical response. However, findings more broadly reflect in vitro cell culture practises across many areas of cell biology, demonstrating the fundamental impact of mechanotransduction in bioengineering and cell biology. Full article

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18 pages, 3113 KiB

Open AccessArticle

Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies

by Christian Schulz, Natalie Herzog, Stefan Kubick, Friedrich Jung and Jan-Heiner Küpper

Cells 2023, 12(17), 2140; https://doi.org/10.3390/cells12172140 - 24 Aug 2023

Cited by 1 | Viewed by 1615

Abstract

In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 [...] Read more.

In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising “humanised” in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine. Full article

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20 pages, 2805 KiB

Open AccessArticle

Anti-Cancer Prodrug Cyclophosphamide Exerts Thrombogenic Effects on Human Venous Endothelial Cells Independent of CYP450 Activation—Relevance to Thrombosis

by Anne Krüger-Genge, Susanne Köhler, Markus Laube, Vanessa Haileka, Sandy Lemm, Karolina Majchrzak, Sarah Kammerer, Christian Schulz, Joachim Storsberg, Jens Pietzsch, Jan-Heiner Küpper and Friedrich Jung

Cells 2023, 12(15), 1965; https://doi.org/10.3390/cells12151965 - 29 Jul 2023

Cited by 5 | Viewed by 2051

Abstract

Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, [...] Read more.

Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation. Full article

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2022

Jump to: 2024, 2023, 2021

20 pages, 1540 KiB

Open AccessFeature PaperReview

Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives

by Ricardo Alva, Georgina L. Gardner, Ping Liang and Jeffrey A. Stuart

Cells 2022, 11(19), 3123; https://doi.org/10.3390/cells11193123 - 4 Oct 2022

Cited by 18 | Viewed by 4662

Abstract

Most conventional incubators used in cell culture do not regulate O₂ levels, making the headspace O₂ concentration ~18%. In contrast, most human tissues are exposed to 2–6% O₂ (physioxia) in vivo. Accumulating evidence has shown that such hyperoxic conditions in [...] Read more.

Most conventional incubators used in cell culture do not regulate O₂ levels, making the headspace O₂ concentration ~18%. In contrast, most human tissues are exposed to 2–6% O₂ (physioxia) in vivo. Accumulating evidence has shown that such hyperoxic conditions in standard cell culture practices affect a variety of biological processes. In this review, we discuss how supraphysiological O₂ levels affect reactive oxygen species (ROS) metabolism and redox homeostasis, gene expression, replicative lifespan, cellular respiration, and mitochondrial dynamics. Furthermore, we present evidence demonstrating how hyperoxic cell culture conditions fail to recapitulate the physiological and pathological behavior of tissues in vivo, including cases of how O₂ alters the cellular response to drugs, hormones, and toxicants. We conclude that maintaining physioxia in cell culture is imperative in order to better replicate in vivo-like tissue physiology and pathology, and to avoid artifacts in research involving cell culture. Full article

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19 pages, 3260 KiB

Open AccessArticle

Investigation of Radiotracer Metabolic Stability In Vitro with CYP-Overexpressing Hepatoma Cell Lines

by Sandy Lemm, Susanne Köhler, Robert Wodtke, Friedrich Jung, Jan-Heiner Küpper, Jens Pietzsch and Markus Laube

Cells 2022, 11(15), 2447; https://doi.org/10.3390/cells11152447 - 7 Aug 2022

Cited by 3 | Viewed by 2129

Abstract

The characterization of novel radiotracers toward their metabolic stability is an essential part of their development. While in vitro methods such as liver microsome assays or ex vivo blood or tissue samples provide information on overall stability, little or no information is obtained [...] Read more.

The characterization of novel radiotracers toward their metabolic stability is an essential part of their development. While in vitro methods such as liver microsome assays or ex vivo blood or tissue samples provide information on overall stability, little or no information is obtained on cytochrome P450 (CYP) enzyme and isoform-specific contribution to the metabolic fate of individual radiotracers. Herein, we investigated recently established CYP-overexpressing hepatoblastoma cell lines (HepG2) for their suitability to study the metabolic stability of radiotracers in general and to gain insight into CYP isoform specificity. Wildtype HepG2 and CYP1A2-, CYP2C19-, and CYP3A4-overexpressing HepG2 cells were incubated with radiotracers, and metabolic turnover was analyzed. The optimized protocol, covering cell seeding in 96-well plates and analysis of supernatant by radio thin-layer-chromatography for higher throughput, was transferred to the evaluation of three ¹⁸F-labeled celecoxib-derived cyclooxygenase-2 inhibitors (coxibs). These investigations revealed time-dependent degradation of the intact radiotracers, as well as CYP isoform- and substrate-specific differences in their metabolic profiles. HepG2 CYP2C19 proved to be the cell line showing the highest metabolic turnover for each radiotracer studied here. Comparison with human and murine liver microsome assays showed good agreement with the human metabolite profile obtained by the HepG2 cell lines. Therefore, CYP-overexpressing HepG2 cells provide a good complement for assessing the metabolic stability of radiotracers and allow the analysis of the CYP isoform-specific contribution to the overall radiotracer metabolism. Full article

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16 pages, 1952 KiB

Open AccessArticle

Deep Learning-Based Image Analysis for the Quantification of Tumor-Induced Angiogenesis in the 3D In Vivo Tumor Model—Establishment and Addition to Laser Speckle Contrast Imaging (LSCI)

by Paulina Mena Kuri, Eric Pion, Lina Mahl, Philipp Kainz, Siegfried Schwarz, Christoph Brochhausen, Thiha Aung and Silke Haerteis

Cells 2022, 11(15), 2321; https://doi.org/10.3390/cells11152321 - 28 Jul 2022

Cited by 13 | Viewed by 4370

Abstract

(1) Background: angiogenesis plays an important role in the growth and metastasis of tumors. We established the CAM assay application, an image analysis software of the IKOSA platform by KML Vision, for the quantification of blood vessels with the in ovo chorioallantoic membrane [...] Read more.

(1) Background: angiogenesis plays an important role in the growth and metastasis of tumors. We established the CAM assay application, an image analysis software of the IKOSA platform by KML Vision, for the quantification of blood vessels with the in ovo chorioallantoic membrane (CAM) model. We added this proprietary deep learning algorithm to the already established laser speckle contrast imaging (LSCI). (2) Methods: angiosarcoma cell line tumors were grafted onto the CAM. Angiogenesis was measured at the beginning and at the end of tumor growth with both measurement methods. The CAM assay application was trained to enable the recognition of in ovo CAM vessels. Histological stains of the tissue were performed and gluconate, an anti-angiogenic substance, was applied to the tumors. (3) Results: the angiosarcoma cells formed tumors on the CAM that appeared to stay vital and proliferated. An increase in perfusion was observed using both methods. The CAM assay application was successfully established in the in ovo CAM model and anti-angiogenic effects of gluconate were observed. (4) Conclusions: the CAM assay application appears to be a useful method for the quantification of angiogenesis in the CAM model and gluconate could be a potential treatment of angiosarcomas. Both aspects should be evaluated in further research. Full article

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22 pages, 11753 KiB

Open AccessArticle

A 3D In Vivo Model for Studying Human Renal Cystic Tissue and Mouse Kidney Slices

by Eva-Marie Bichlmayer, Lina Mahl, Leo Hesse, Eric Pion, Victoria Haller, Andreas Moehwald, Christina Hackl, Jens M. Werner, Hans J. Schlitt, Siegfried Schwarz, Philipp Kainz, Christoph Brochhausen, Christian Groeger, Felix Steger, Oliver Kölbl, Christoph Daniel, Kerstin Amann, Andre Kraus, Björn Buchholz, Thiha Aung and Silke Haerteis Show full author list Hide full author list

Cells 2022, 11(15), 2269; https://doi.org/10.3390/cells11152269 - 22 Jul 2022

Cited by 3 | Viewed by 3334

Abstract

(1) Background: Autosomal dominant polycystic kidney disease (ADPKD) is a frequent monogenic disorder that leads to progressive renal cyst growth and renal failure. Strategies to inhibit cyst growth in non-human cyst models have often failed in clinical trials. There is a significant need [...] Read more.

(1) Background: Autosomal dominant polycystic kidney disease (ADPKD) is a frequent monogenic disorder that leads to progressive renal cyst growth and renal failure. Strategies to inhibit cyst growth in non-human cyst models have often failed in clinical trials. There is a significant need for models that enable studies of human cyst growth and drug trials. (2) Methods: Renal tissue from ADPKD patients who received a nephrectomy as well as adult mouse kidney slices were cultured on a chorioallantoic membrane (CAM) for one week. The cyst volume was monitored by microscopic and CT-based applications. The weight and angiogenesis were quantified. Morphometric and histological analyses were performed after the removal of the tissues from the CAM. (3) Results: The mouse and human renal tissue mostly remained vital for about one week on the CAM. The growth of cystic tissue was evaluated using microscopic and CT-based volume measurements, which correlated with weight and an increase in angiogenesis, and was accompanied by cyst cell proliferation. (4) Conclusions: The CAM model might bridge the gap between animal studies and clinical trials of human cyst growth, and provide a drug-testing platform for the inhibition of cyst enlargement. Real-time analyses of mouse kidney tissue may provide insights into renal physiology and reduce the need for animal experiments. Full article

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15 pages, 705 KiB

Open AccessReview

Restenosis after Coronary Stent Implantation: Cellular Mechanisms and Potential of Endothelial Progenitor Cells (A Short Guide for the Interventional Cardiologist)

by Tommaso Gori

Cells 2022, 11(13), 2094; https://doi.org/10.3390/cells11132094 - 30 Jun 2022

Cited by 15 | Viewed by 3065

Abstract

Coronary stents are among the most common therapies worldwide. Despite significant improvements in the biocompatibility of these devices throughout the last decades, they are prone, in as many as 10–20% of cases, to short- or long-term failure. In-stent restenosis is a multifactorial process [...] Read more.

Coronary stents are among the most common therapies worldwide. Despite significant improvements in the biocompatibility of these devices throughout the last decades, they are prone, in as many as 10–20% of cases, to short- or long-term failure. In-stent restenosis is a multifactorial process with a complex and incompletely understood pathophysiology in which inflammatory reactions are of central importance. This review provides a short overview for the clinician on the cellular types responsible for restenosis with a focus on the role of endothelial progenitor cells. The mechanisms of restenosis are described, along with the cell-based attempts made to prevent it. While the focus of this review is principally clinical, experimental evidence provides some insight into the potential implications for prevention and therapy of coronary stent restenosis. Full article

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20 pages, 5661 KiB

Open AccessArticle

Leonurine Protects Bone Mesenchymal Stem Cells from Oxidative Stress by Activating Mitophagy through PI3K/Akt/mTOR Pathway

by Bingkun Zhao, Qian Peng, Dan Wang, Rong Zhou, Raorao Wang, Yizhun Zhu and Shengcai Qi

Cells 2022, 11(11), 1724; https://doi.org/10.3390/cells11111724 - 24 May 2022

Cited by 24 | Viewed by 3158

Abstract

Osteoporosis bears an imbalance between bone formation and resorption, which is strongly related to oxidative stress. The function of leonurine on bone marrow-derived mesenchymal stem cells (BMSCs) under oxidative stress is still unclear. Therefore, this study was aimed at identifying the protective effect [...] Read more.

Osteoporosis bears an imbalance between bone formation and resorption, which is strongly related to oxidative stress. The function of leonurine on bone marrow-derived mesenchymal stem cells (BMSCs) under oxidative stress is still unclear. Therefore, this study was aimed at identifying the protective effect of leonurine on H₂O₂ stimulated rat BMSCs. We found that leonurine can alleviate cell apoptosis and promote the differentiation ability of rat BMSCs induced by oxidative stress at an appropriate concentration at 10 μM. Meanwhile, the intracellular ROS level and the level of the COX2 and NOX4 mRNA decreased after leonurine treatment in vitro. The ATP level and mitochondrial membrane potential were upregulated after leonurine treatment. The protein level of PINK1 and Parkin showed the same trend. The mitophage in rat BMSCs blocked by 3-MA was partially rescued by leonurine. Bioinformatics analysis and leonurine-protein coupling provides a strong direct combination between leonurine and the PI3K protein at the position of Asp841, Glu880, Val882. In conclusion, leonurine protects the proliferation and differentiation of BMSCs from oxidative stress by activating mitophagy, which depends on the PI3K/Akt/mTOR pathway. The results showed that leonurine may have potential usage in osteoporosis and bone defect repair in osteoporosis patients. Full article

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26 pages, 17394 KiB

Open AccessArticle

Allogeneic Serum and Macromolecular Crowding Maintain Native Equine Tenocyte Function in Culture

by Andrea Rampin, Ioannis Skoufos, Michael Raghunath, Athina Tzora, Nikolaos Diakakis, Nikitas Prassinos and Dimitrios I. Zeugolis

Cells 2022, 11(9), 1562; https://doi.org/10.3390/cells11091562 - 5 May 2022

Cited by 4 | Viewed by 2498

Abstract

The absence of a native extracellular matrix and the use of xenogeneic sera are often associated with rapid tenocyte function losses during in vitro culture. Herein, we assessed the influence of different sera (equine serum and foetal bovine serum) on equine tenocyte morphology, [...] Read more.

The absence of a native extracellular matrix and the use of xenogeneic sera are often associated with rapid tenocyte function losses during in vitro culture. Herein, we assessed the influence of different sera (equine serum and foetal bovine serum) on equine tenocyte morphology, viability, metabolic activity, proliferation and protein synthesis as a function of tissue-specific extracellular matrix deposition (induced via macromolecular crowding), aging (passages 3, 6, 9) and time in culture (days 3, 5, 7). In comparison to cells at passage 3, at day 3, in foetal bovine serum and without macromolecular crowding (traditional equine tenocyte culture), the highest number of significantly decreased readouts were observed for cells in foetal bovine serum, at passage 3, at day 5 and day 7 and without macromolecular crowding. Again, in comparison to traditional equine tenocyte culture, the highest number of significantly increased readouts were observed for cells in equine serum, at passage 3 and passage 6, at day 7 and with macromolecular crowding. Our data advocate the use of an allogeneic serum and tissue-specific extracellular matrix for effective expansion of equine tenocytes. Full article

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15 pages, 2029 KiB

Open AccessArticle

The Role of Transglutaminase 2 in the Radioresistance of Melanoma Cells

by Julia Aepler, Johanna Wodtke, Robert Wodtke, Cathleen Haase-Kohn, Reik Löser, Jens Pietzsch and Sandra Hauser

Cells 2022, 11(8), 1342; https://doi.org/10.3390/cells11081342 - 14 Apr 2022

Cited by 5 | Viewed by 2450

Abstract

Transglutaminase 2 (TG2) is a protein expressed in many tissues that exerts numerous, sometimes contradictory, intra- and extracellular functions, under both physiological and pathophysiological conditions. In the context of tumor progression, it has been found to be involved in cell adhesion, DNA repair [...] Read more.

Transglutaminase 2 (TG2) is a protein expressed in many tissues that exerts numerous, sometimes contradictory, intra- and extracellular functions, under both physiological and pathophysiological conditions. In the context of tumor progression, it has been found to be involved in cell adhesion, DNA repair mechanisms, induction of apoptosis, and mesenchymal transdifferentiation, among others. Here, we hypothesized that TG2 also contributes to the radioresistance of two human melanoma cell lines, A375 and MeWo, which can be seen to differ in their basal TG2 biosynthesis by examining their proliferation and clonal expansion after irradiation. For this purpose, cellular TG2 biosynthesis and TG2 activity were modulated by transfection-induced overexpression or TG2 knock-out and application of TG2-selective inhibitors. Proliferation and clonal expansion of TG2-overexpressing cells was not enhanced over wildtype cells, suggesting that increased TG2 biosynthesis does not further enhance the radioresistance of melanoma cells. Conversely, TG2 knock-out in A375 cells reduced their proliferation, as well as clonal and spheroidal expansion after irradiation, which indicates a contribution of TG2 to the radioresistance of melanoma cells. Since TG1, TG3, and partly also, TG6 biosynthesis was detectable in A375 and MeWo cells, it can be assumed that these other members of the TG family may exert a partially compensatory effect. Full article

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19 pages, 2504 KiB

Open AccessArticle

Proteomic Analysis of the Role of the Adenylyl Cyclase–cAMP Pathway in Red Blood Cell Mechanical Responses

by Elif Ugurel, Evrim Goksel, Neslihan Cilek, Elif Kaga and Ozlem Yalcin

Cells 2022, 11(7), 1250; https://doi.org/10.3390/cells11071250 - 6 Apr 2022

Cited by 9 | Viewed by 2879

Abstract

Red blood cell (RBC) deformability is modulated by the phosphorylation status of the cytoskeletal proteins that regulate the interactions of integral transmembrane complexes. Proteomic studies have revealed that receptor-related signaling molecules and regulatory proteins involved in signaling cascades are present in RBCs. In [...] Read more.

Red blood cell (RBC) deformability is modulated by the phosphorylation status of the cytoskeletal proteins that regulate the interactions of integral transmembrane complexes. Proteomic studies have revealed that receptor-related signaling molecules and regulatory proteins involved in signaling cascades are present in RBCs. In this study, we investigated the roles of the cAMP signaling mechanism in modulating shear-induced RBC deformability and examined changes in the phosphorylation of the RBC proteome. We implemented the inhibitors of adenylyl cyclase (SQ22536), protein kinase A (H89), and phosphodiesterase (PDE) (pentoxifylline) to whole blood samples, applied 5 Pa shear stress (SS) for 300 s with a capillary tubing system, and evaluated RBC deformability using a LORRCA MaxSis. The inhibition of signaling molecules significantly deteriorated shear-induced RBC deformability (p < 0.05). Capillary SS slightly increased the phosphorylation of RBC cytoskeletal proteins. Tyrosine phosphorylation was significantly elevated by the modulation of the cAMP/PKA pathway (p < 0.05), while serine phosphorylation significantly decreased as a result of the inhibition of PDE (p < 0.05). AC is the core element of this signaling pathway, and PDE works as a negative feedback mechanism that could have potential roles in SS-induced RBC deformability. The cAMP/PKA pathway could regulate RBC deformability during capillary transit by triggering significant alterations in the phosphorylation state of RBCs. Full article

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14 pages, 2272 KiB

Open AccessArticle

Interactions between the Nociceptin and Toll-like Receptor Systems

by Lan Zhang, Ulrike M. Stamer, Melody Ying-Yu Huang and Frank Stüber

Cells 2022, 11(7), 1085; https://doi.org/10.3390/cells11071085 - 23 Mar 2022

Cited by 3 | Viewed by 2384

Abstract

Nociceptin and the nociceptin receptor (NOP) have been described as targets for treatment of pain and inflammation, whereas toll-like receptors (TLRs) play key roles in inflammation and impact opioid receptors and endogenous opioids expression. In this study, interactions between the nociceptin and TLR [...] Read more.

Nociceptin and the nociceptin receptor (NOP) have been described as targets for treatment of pain and inflammation, whereas toll-like receptors (TLRs) play key roles in inflammation and impact opioid receptors and endogenous opioids expression. In this study, interactions between the nociceptin and TLR systems were investigated. Human THP-1 cells were cultured with or without phorbol myristate acetate (PMA 5 ng/mL), agonists specific for TLR2 (lipoteichoic acid, LTA 10 µg/mL), TLR4 (lipopolysaccharide, LPS 100 ng/mL), TLR7 (imiquimod, IMQ 10 µg/mL), TLR9 (oligonucleotide (ODN) 2216 1 µM), PMA+TLR agonists, or nociceptin (0.01–100 nM). Prepronociceptin (ppNOC), NOP, and TLR mRNAs were quantified by RT-qPCR. Proteins were measured using flow cytometry. PMA upregulated ppNOC mRNA, intracellular nociceptin, and cell membrane NOP proteins (all p < 0.05). LTA and LPS prevented PMA’s upregulating effects on ppNOC mRNA and nociceptin protein (both p < 0.05). IMQ and ODN 2216 attenuated PMA’s effects on ppNOC mRNA. PMA, LPS, IMQ, and ODN 2216 increased NOP protein levels (all p < 0.05). PMA+TLR agonists had no effects on NOP compared to PMA controls. Nociceptin dose-dependently suppressed TLR2, TLR4, TLR7, and TLR9 proteins (all p < 0.01). Antagonistic effects observed between the nociceptin and TLR systems suggest that the nociceptin system plays an anti-inflammatory role in monocytes under inflammatory conditions. Full article

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11 pages, 3433 KiB

Open AccessArticle

Shear-Stress-Gradient and Oxygen-Gradient Ektacytometry in Sickle Cell Patients at Steady State and during Vaso-Occlusive Crises

by Camille Boisson, Elie Nader, Céline Renoux, Alexandra Gauthier, Solène Poutrel, Yves Bertrand, Emeric Stauffer, Emilie Virot, Arnaud Hot, Romain Fort, Giovanna Cannas, Philippe Joly and Philippe Connes

Cells 2022, 11(3), 585; https://doi.org/10.3390/cells11030585 - 8 Feb 2022

Cited by 4 | Viewed by 1958

Abstract

Oxygen gradient ektacytometry (oxygenscan) measures the changes in red blood cell (RBC) deformability in normoxia and during deoxygenation. We investigated the changes in RBC deformability, measured by both oxygenscan and classical shear-stress-gradient ektacytometry, in 10 patients with sickle cell disease (SCD) during vaso-occlusive [...] Read more.

Oxygen gradient ektacytometry (oxygenscan) measures the changes in red blood cell (RBC) deformability in normoxia and during deoxygenation. We investigated the changes in RBC deformability, measured by both oxygenscan and classical shear-stress-gradient ektacytometry, in 10 patients with sickle cell disease (SCD) during vaso-occlusive crisis (VOC) versus steady state. Oxygenscan and shear-stress-gradient ektacytometry parameters were also measured in 38 SCD patients at steady state on two different occasions. Shear-stress-gradient ektacytometry parameters, maximal RBC deformability at normoxia and the minimum RBC deformability during deoxygenation were lower during VOC compared to steady state. The oxygen partial pressure at which RBCs started to sickle (PoS) was not significantly affected by VOC, but the results were very heterogeneous: the PoS increased in 5 in 10 patients and decreased in 4 in 10 patients. Both oxygenscan and shear-stress-gradient ektacytometry parameters remained unchanged in patients at steady state between two sets of measurements, performed at 17 ± 8 months intervals. In conclusion, the present study showed that both oxygen gradient ektacytometry and shear-stress-gradient ektacytometry are sensitive to disease activity in SCD, and that both techniques give comparable results; however, the oxygen-dependent propensity of RBCs to sickle was highly variable during VOC. Full article

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21 pages, 17218 KiB

Open AccessReview

Current Progress in Vascular Engineering and Its Clinical Applications

by Hatem Jouda, Luis Larrea Murillo and Tao Wang

Cells 2022, 11(3), 493; https://doi.org/10.3390/cells11030493 - 31 Jan 2022

Cited by 18 | Viewed by 8949

Abstract

Coronary heart disease (CHD) is caused by narrowing or blockage of coronary arteries due to atherosclerosis. Coronary artery bypass grafting (CABG) is widely used for the treatment of severe CHD cases. Although autologous vessels are a preferred choice, healthy autologous vessels are not [...] Read more.

Coronary heart disease (CHD) is caused by narrowing or blockage of coronary arteries due to atherosclerosis. Coronary artery bypass grafting (CABG) is widely used for the treatment of severe CHD cases. Although autologous vessels are a preferred choice, healthy autologous vessels are not always available; hence there is a demand for tissue engineered vascular grafts (TEVGs) to be used as alternatives. However, producing clinical grade implantable TEVGs that could healthily survive in the host with long-term patency is still a great challenge. There are additional difficulties in producing small diameter (<6 mm) vascular conduits. As a result, there have not been TEVGs that are commercially available. Properties of vascular scaffolds such as tensile strength, thrombogenicity and immunogenicity are key factors that determine the biocompatibility of TEVGs. The source of vascular cells employed to produce TEVGs is a limiting factor for large-scale productions. Advanced technologies including the combined use of natural and biodegradable synthetic materials for scaffolds in conjunction with the use of mesenchyme stem cells or induced pluripotent stem cells (iPSCs) provide promising solutions for vascular tissue engineering. The aim of this review is to provide an update on various aspects in this field and the current status of TEVG clinical applications. Full article

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16 pages, 12353 KiB

Open AccessArticle

Acrylonitrile and Pullulan Based Nanofiber Mats as Easily Accessible Scaffolds for 3D Skin Cell Models Containing Primary Cells

by Markus Rimann, Astrid Jüngel, Sara Mousavi, Nicole Moeschlin, Maurizio Calcagni, Karin Wuertz-Kozak, Florian Brunner, Stefan Dudli, Oliver Distler and Christian Adlhart

Cells 2022, 11(3), 445; https://doi.org/10.3390/cells11030445 - 27 Jan 2022

Cited by 4 | Viewed by 3946

Abstract

(1) Background: Three-dimensional (3D) collagen I-based skin models are commonly used in drug development and substance testing but have major drawbacks such as batch-to-batch variations and ethical concerns. Recently, synthetic nanofibrous scaffolds created by electrospinning have received increasing interest as potential alternatives due [...] Read more.

(1) Background: Three-dimensional (3D) collagen I-based skin models are commonly used in drug development and substance testing but have major drawbacks such as batch-to-batch variations and ethical concerns. Recently, synthetic nanofibrous scaffolds created by electrospinning have received increasing interest as potential alternatives due to their morphological similarities to native collagen fibrils in size and orientation. The overall objective of this proof-of-concept study was to demonstrate the suitability of two synthetic polymers in creating electrospun scaffolds for 3D skin cell models. (2) Methods: Electrospun nanofiber mats were produced with (i) poly(acrylonitrile-co-methyl acrylate) (P(AN-MA)) and (ii) a blend of pullulan (Pul), poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) (Pul/PVA/PAA) and characterized by scanning electron microscopy (SEM) and diffuse reflectance infrared Fourier transform (DRIFT) spectra. Primary skin fibroblasts and keratinocytes were seeded onto the nanofiber mats and analyzed for phenotypic characteristics (phalloidin staining), viability (Presto Blue HS assay), proliferation (Ki-67 staining), distribution (H/E staining), responsiveness to biological stimuli (qPCR), and formation of skin-like structures (H/E staining). (3) Results: P(AN-MA) mats were more loosely packed than the Pul/PVA/PAA mats, concomitant with larger fiber diameter (340 nm ± 120 nm vs. 250 nm ± 120 nm, p < 0.0001). After sterilization and exposure to cell culture media for 28 days, P(AN-MA) mats showed significant adsorption of fetal calf serum (FCS) from the media into the fibers (DRIFT spectra) and increased fiber diameter (590 nm ± 290 nm, p < 0.0001). Skin fibroblasts were viable over time on both nanofiber mats, but suitable cell infiltration only occurred in the P(AN-MA) nanofiber mats. On P(AN-MA) mats, fibroblasts showed their characteristic spindle-like shape, produced a dermis-like structure, and responded well to TGF

β

stimulation, with a significant increase in the mRNA expression of PAI1, COL1A1, and αSMA (all p < 0.05). Primary keratinocytes seeded on top of the dermis equivalent proliferated and formed a stratified epidermis-like structure. (4) Conclusion: P(AN-MA) and Pul/PVA/PAA are both biocompatible materials suitable for nanofiber mat production. P(AN-MA) mats hold greater potential as future 3D skin models due to enhanced cell compatibility (i.e., adsorption of FCS proteins), cell infiltration (i.e., increased pore size due to swelling behavior), and cell phenotype preservation. Thus, our proof-of-concept study shows an easy and robust process of producing electrospun scaffolds for 3D skin cell models made of P(AN-MA) nanofibers without the need for bioactive molecule attachments. Full article

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Graphical abstract

14 pages, 2206 KiB

Open AccessArticle

Innovative Platform for the Advanced Online Monitoring of Three-Dimensional Cells and Tissue Cultures

by Sebastian Kreß, Roland Schaller-Ammann, Jürgen Feiel, Joachim Wegener, Joachim Priedl, Wolf Dietrich, Cornelia Kasper and Dominik Egger

Cells 2022, 11(3), 412; https://doi.org/10.3390/cells11030412 - 25 Jan 2022

Cited by 3 | Viewed by 3570

Abstract

The use of 3D cell cultures has gained increasing importance in medical and pharmaceutical research. However, the analysis of the culture medium is hardly representative for the culture conditions within a 3D model which hinders the standardization of 3D cultures and translation of [...] Read more.

The use of 3D cell cultures has gained increasing importance in medical and pharmaceutical research. However, the analysis of the culture medium is hardly representative for the culture conditions within a 3D model which hinders the standardization of 3D cultures and translation of results. Therefore, we developed a modular monitoring platform combining a perfusion bioreactor with an integrated minimally invasive sampling system and implemented sensors that enables the online monitoring of culture parameters and medium compounds within 3D cultures. As a proof-of-concept, primary cells as well as cell lines were cultured on a collagen or gelatin methacryloyl (GelMA) hydrogel matrix, while monitoring relevant culture parameters and analytes. Comparing the interstitial fluid of the 3D models versus the corresponding culture medium, we found considerable differences in the concentrations of several analytes. These results clearly demonstrate that analyses of the culture medium only are not relevant for the development of standardized 3D culture processes. The presented bioreactor with an integrated sampling and sensor platform opens new horizons for the development, optimization, and standardization of 3D cultures. Furthermore, this technology holds the potential to reduce animal studies and improve the transferability of pharmaceutical in vitro studies by gaining more relevant results, bridging the gap towards clinical translation. Full article

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16 pages, 4719 KiB

Open AccessArticle

Freeze-Dried Curdlan/Whey Protein Isolate-Based Biomaterial as Promising Scaffold for Matrix-Associated Autologous Chondrocyte Transplantation—A Pilot In-Vitro Study

by Katarzyna Klimek, Marta Tarczynska, Wieslaw Truszkiewicz, Krzysztof Gaweda, Timothy E. L. Douglas and Grazyna Ginalska

Cells 2022, 11(2), 282; https://doi.org/10.3390/cells11020282 - 14 Jan 2022

Cited by 11 | Viewed by 2720

Abstract

The purpose of this pilot study was to establish whether a novel freeze-dried curdlan/whey protein isolate-based biomaterial may be taken into consideration as a potential scaffold for matrix-associated autologous chondrocyte transplantation. For this reason, this biomaterial was initially characterized by the visualization of [...] Read more.

The purpose of this pilot study was to establish whether a novel freeze-dried curdlan/whey protein isolate-based biomaterial may be taken into consideration as a potential scaffold for matrix-associated autologous chondrocyte transplantation. For this reason, this biomaterial was initially characterized by the visualization of its micro- and macrostructures as well as evaluation of its mechanical stability, and its ability to undergo enzymatic degradation in vitro. Subsequently, the cytocompatibility of the biomaterial towards human chondrocytes (isolated from an orthopaedic patient) was assessed. It was demonstrated that the novel freeze-dried curdlan/whey protein isolate-based biomaterial possessed a porous structure and a Young’s modulus close to those of the superficial and middle zones of cartilage. It also exhibited controllable degradability in collagenase II solution over nine weeks. Most importantly, this biomaterial supported the viability and proliferation of human chondrocytes, which maintained their characteristic phenotype. Moreover, quantitative reverse transcription PCR analysis and confocal microscope observations revealed that the biomaterial may protect chondrocytes from dedifferentiation towards fibroblast-like cells during 12-day culture. Thus, in conclusion, this pilot study demonstrated that novel freeze-dried curdlan/whey protein isolate-based biomaterial may be considered as a potential scaffold for matrix-associated autologous chondrocyte transplantation. Full article

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14 pages, 2707 KiB

Open AccessArticle

Characterization of Oxygen Levels in an Uninfected and Infected Human Blood-Cerebrospinal-Fluid-Barrier Model

by Alexander Martens, Nicole de Buhr, Hiroshi Ishikawa, Horst Schroten and Maren von Köckritz-Blickwede

Cells 2022, 11(1), 151; https://doi.org/10.3390/cells11010151 - 4 Jan 2022

Cited by 1 | Viewed by 2121

Abstract

The host–pathogen interaction during meningitis can be investigated with blood-cerebrospinal-fluid-barrier (BCSFB) cell culture models. They are commonly handled under atmospheric oxygen conditions (19–21% O₂), although the physiological oxygen conditions are significantly lower in cerebrospinal fluid (CSF) (7–8% O₂). We [...] Read more.

The host–pathogen interaction during meningitis can be investigated with blood-cerebrospinal-fluid-barrier (BCSFB) cell culture models. They are commonly handled under atmospheric oxygen conditions (19–21% O₂), although the physiological oxygen conditions are significantly lower in cerebrospinal fluid (CSF) (7–8% O₂). We aimed to characterize oxygen levels in a Streptococcus (S.) suis-infected BCSFB model with transmigrating neutrophils. A BCSFB model with human choroid plexus epithelial cells growing on transwell-filters was used. The upper “blood”-compartment was infected and blood-derived neutrophils were added. S. suis and neutrophils transmigrated through the BCSFB into the “CSF”-compartment. Here, oxygen and pH values were determined with the non-invasive SensorDish^® reader. Slight orbital shaking improved the luminescence-based measurement technique for detecting free oxygen. In the non-infected BCSFB model, an oxygen value of 7% O₂ was determined. However, with S. suis and transmigrating neutrophils, the oxygen value significantly decreased to 2% O₂. The pH level decreased slightly in all groups. In conclusion, we characterized oxygen levels in the BCSFB model and demonstrated the oxygen consumption by cells and bacteria. Oxygen values in the non-infected BCSFB model are comparable to in vivo values determined in pigs in the CSF. Infection and transmigrating neutrophils decrease the oxygen value to lower values. Full article

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2021

Jump to: 2024, 2023, 2022

14 pages, 698 KiB

Open AccessReview

Sulfated Hydrogels in Intervertebral Disc and Cartilage Research

by Emily Lazarus, Paola Bermudez-Lekerika, Daniel Farchione, Taylor Schofield, Sloan Howard, Iskender Mambetkadyrov, Mikkael Lamoca, Iris V. Rivero, Benjamin Gantenbein, Christopher L. Lewis and Karin Wuertz-Kozak

Cells 2021, 10(12), 3568; https://doi.org/10.3390/cells10123568 - 17 Dec 2021

Cited by 5 | Viewed by 4675

Abstract

Hydrogels are commonly used for the 3D culture of musculoskeletal cells. Sulfated hydrogels, which have seen a growing interest over the past years, provide a microenvironment that help maintain the phenotype of chondrocytes and chondrocyte-like cells and can be used for sustained delivery [...] Read more.

Hydrogels are commonly used for the 3D culture of musculoskeletal cells. Sulfated hydrogels, which have seen a growing interest over the past years, provide a microenvironment that help maintain the phenotype of chondrocytes and chondrocyte-like cells and can be used for sustained delivery of growth factors and other drugs. Sulfated hydrogels are hence valuable tools to improve cartilage and intervertebral disc tissue engineering. To further advance the utilization of these hydrogels, we identify and summarize the current knowledge about different sulfated hydrogels, highlight their beneficial effects in cartilage and disc research, and review the biofabrication processes most suitable to secure best quality assurance through deposition fidelity, repeatability, and attainment of biocompatible morphologies. Full article

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19 pages, 13827 KiB

Open AccessArticle

Restoring Osteochondral Defects through the Differentiation Potential of Cartilage Stem/Progenitor Cells Cultivated on Porous Scaffolds

by Hsueh-Chun Wang, Tzu-Hsiang Lin, Che-Chia Hsu and Ming-Long Yeh

Cells 2021, 10(12), 3536; https://doi.org/10.3390/cells10123536 - 14 Dec 2021

Cited by 15 | Viewed by 3131

Abstract

Cartilage stem/progenitor cells (CSPCs) are cartilage-specific, multipotent progenitor cells residing in articular cartilage. In this study, we investigated the characteristics and potential of human CSPCs combined with poly(lactic-co-glycolic acid) (PLGA) scaffolds to induce osteochondral regeneration in rabbit knees. We isolated CSPCs from human [...] Read more.

Cartilage stem/progenitor cells (CSPCs) are cartilage-specific, multipotent progenitor cells residing in articular cartilage. In this study, we investigated the characteristics and potential of human CSPCs combined with poly(lactic-co-glycolic acid) (PLGA) scaffolds to induce osteochondral regeneration in rabbit knees. We isolated CSPCs from human adult articular cartilage undergoing total knee replacement (TKR) surgery. We characterized CSPCs and compared them with infrapatellar fat pad-derived stem cells (IFPs) in a colony formation assay and by multilineage differentiation analysis in vitro. We further evaluated the osteochondral regeneration of the CSPC-loaded PLGA scaffold during osteochondral defect repair in rabbits. The characteristics of CSPCs were similar to those of mesenchymal stem cells (MSCs) and exhibited chondrogenic and osteogenic phenotypes without chemical induction. For in vivo analysis, CSPC-loaded PLGA scaffolds produced a hyaline-like cartilaginous tissue, which showed good integration with the host tissue and subchondral bone. Furthermore, CSPCs migrated in response to injury to promote subchondral bone regeneration. Overall, we demonstrated that CSPCs can promote osteochondral regeneration. A monophasic approach of using diseased CSPCs combined with a PLGA scaffold may be beneficial for repairing complex tissues, such as osteochondral tissue. Full article

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21 pages, 4251 KiB

Open AccessFeature PaperArticle

Optimized 3D Culture of Hepatic Cells for Liver Organoid Metabolic Assays

by Christian Moya Gamboa, Yujue Wang, Huiting Xu, Katarzyna Kalemba, Fredric E. Wondisford and Hatem E. Sabaawy

Cells 2021, 10(12), 3280; https://doi.org/10.3390/cells10123280 - 24 Nov 2021

Cited by 18 | Viewed by 5153

Abstract

The liver is among the principal organs for glucose homeostasis and metabolism. Studies of liver metabolism are limited by the inability to expand primary hepatocytes in vitro while maintaining their metabolic functions. Human hepatic three-dimensional (3D) organoids have been established using defined factors, [...] Read more.

The liver is among the principal organs for glucose homeostasis and metabolism. Studies of liver metabolism are limited by the inability to expand primary hepatocytes in vitro while maintaining their metabolic functions. Human hepatic three-dimensional (3D) organoids have been established using defined factors, yet hepatic organoids from adult donors showed impaired expansion. We examined conditions to facilitate the expansion of adult donor-derived hepatic organoids (HepAOs) and HepG2 cells in organoid cultures (HepGOs) using combinations of growth factors and small molecules. The expansion dynamics, gluconeogenic and HNF4α expression, and albumin secretion are assessed. The conditions tested allow the generation of HepAOs and HepGOs in 3D cultures. Nevertheless, gluconeogenic gene expression varies greatly between conditions. The organoid expansion rates are limited when including the TGFβ inhibitor A8301, while are relatively higher with Forskolin (FSK) and Oncostatin M (OSM). Notably, expanded HepGOs grown in the optimized condition maintain detectable gluconeogenic expression in a spatiotemporal distribution at 8 weeks. We present optimized conditions by limiting A8301 and incorporating FSK and OSM to allow the expansion of HepAOs from adult donors and HepGOs with gluconeogenic competence. These models increase the repertoire of human hepatic cellular tools available for use in liver metabolic assays. Full article

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23 pages, 4771 KiB

Open AccessArticle

Controlled Growth Factor Delivery and Cyclic Stretch Induces a Smooth Muscle Cell-like Phenotype in Adipose-Derived Stem Cells

by Brandan Walters, Paul A. Turner, Bernd Rolauffs, Melanie L. Hart and Jan P. Stegemann

Cells 2021, 10(11), 3123; https://doi.org/10.3390/cells10113123 - 11 Nov 2021

Cited by 11 | Viewed by 3466

Abstract

Adipose-derived stem cells (ASCs) are an abundant and easily accessible multipotent stem cell source with potential application in smooth muscle regeneration strategies. In 3D collagen hydrogels, we investigated whether sustained release of growth factors (GF) PDGF-AB and TGF-β1 from GF-loaded microspheres could induce [...] Read more.

Adipose-derived stem cells (ASCs) are an abundant and easily accessible multipotent stem cell source with potential application in smooth muscle regeneration strategies. In 3D collagen hydrogels, we investigated whether sustained release of growth factors (GF) PDGF-AB and TGF-β1 from GF-loaded microspheres could induce a smooth muscle cell (SMC) phenotype in ASCs, and if the addition of uniaxial cyclic stretch could enhance the differentiation level. This study demonstrated that the combination of cyclic stretch and GF release over time from loaded microspheres potentiated the differentiation of ASCs, as quantified by protein expression of early to late SMC differentiation markers (SMA, TGLN and smooth muscle MHC). The delivery of GFs via microspheres produced large ASCs with a spindle-shaped, elongated SMC-like morphology. Cyclic strain produced the largest, longest, and most spindle-shaped cells regardless of the presence or absence of growth factors or the growth factor delivery method. Protein expression and cell morphology data confirmed that the sustained release of GFs from GF-loaded microspheres can be used to promote the differentiation of ASCs into SMCs and that the addition of uniaxial cyclic stretch significantly enhances the differentiation level, as quantified by intermediate and late SMC markers and a SMC-like elongated cell morphology. Full article

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12 pages, 2309 KiB

Open AccessArticle

Dermal Pericytes Exhibit Declined Ability to Promote Human Skin Regeneration with Ageing in 3D Organotypic Culture Models

by Lizhe Zhuang, Rahul M. Visalakshan and Pritinder Kaur

Cells 2021, 10(11), 3051; https://doi.org/10.3390/cells10113051 - 6 Nov 2021

Cited by 5 | Viewed by 2327

Abstract

The well documented decline in the regenerative ability of ageing human skin has been attributed to many factors including genomic instability, telomere shortening, poor nutrient sensing, cellular senescence, and stem cell exhaustion. However, a role for the dermal cellular and molecular microenvironment in [...] Read more.

The well documented decline in the regenerative ability of ageing human skin has been attributed to many factors including genomic instability, telomere shortening, poor nutrient sensing, cellular senescence, and stem cell exhaustion. However, a role for the dermal cellular and molecular microenvironment in skin ageing is just emerging. We previously showed that dermal pericytes co-operate with fibroblasts to improve human skin regeneration in an organotypic skin culture model, and even do so in the absence of fibroblasts. Here, we report that the number of dermal cells, particularly pericytes, declines significantly in human skin of donors aged > 50 years. Notably, aged pericytes promoted epidermal regeneration of neonatal keratinocytes in organotypic cultures and the resulting epithelium exhibited a Ki67⁺/ΔNp63⁺ basal layer and terminal differentiation. However, the epithelium lacked several features of homeostasis displaying lower levels of ΔNp63 expression, decreased LAMA5 deposition at the dermo-epidermal junction, and the absence of basement membrane and hemi-desmosome assembly. We conclude that a decline in pericyte incidence and function contribute to an impaired epidermal microenvironment and poor skin regeneration with ageing in the human skin. Full article

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32 pages, 10495 KiB

Open AccessArticle

Hypoxic Incubation Conditions for Optimized Manufacture of Tenocyte-Based Active Pharmaceutical Ingredients of Homologous Standardized Transplant Products in Tendon Regenerative Medicine

by Annick Jeannerat, Cédric Peneveyre, Florence Armand, Diego Chiappe, Romain Hamelin, Corinne Scaletta, Nathalie Hirt-Burri, Anthony de Buys Roessingh, Wassim Raffoul, Lee Ann Applegate and Alexis Laurent

Cells 2021, 10(11), 2872; https://doi.org/10.3390/cells10112872 - 25 Oct 2021

Cited by 10 | Viewed by 2447

Abstract

Human fetal progenitor tenocytes (hFPT) produced in defined cell bank systems have recently been characterized and qualified as potential therapeutic cell sources in tendon regenerative medicine. In view of further developing the manufacture processes of such cell-based active pharmaceutical ingredients (API), the effects [...] Read more.

Human fetal progenitor tenocytes (hFPT) produced in defined cell bank systems have recently been characterized and qualified as potential therapeutic cell sources in tendon regenerative medicine. In view of further developing the manufacture processes of such cell-based active pharmaceutical ingredients (API), the effects of hypoxic in vitro culture expansion on key cellular characteristics or process parameters were evaluated. To this end, multiple aspects were comparatively assessed in normoxic incubation (i.e., 5% CO₂ and 21% O₂, standard conditions) or in hypoxic incubation (i.e., 5% CO₂ and 2% O₂, optimized conditions). Experimentally investigated parameters and endpoints included cellular proliferation, cellular morphology and size distribution, cell surface marker panels, cell susceptibility toward adipogenic and osteogenic induction, while relative protein expression levels were analyzed by quantitative mass spectrometry. The results outlined conserved critical cellular characteristics (i.e., cell surface marker panels, cellular phenotype under chemical induction) and modified key cellular parameters (i.e., cell size distribution, endpoint cell yields, matrix protein contents) potentially procuring tangible benefits for next-generation cell manufacturing workflows. Specific proteomic analyses further shed some light on the cellular effects of hypoxia, potentially orienting further hFPT processing for cell-based, cell-free API manufacture. Overall, this study indicated that hypoxic incubation impacts specific hFPT key properties while preserving critical quality attributes (i.e., as compared to normoxic incubation), enabling efficient manufacture of tenocyte-based APIs for homologous standardized transplant products. Full article

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13 pages, 2162 KiB

Open AccessArticle

Modulation of Inherent Niches in 3D Multicellular MSC Spheroids Reconfigures Metabolism and Enhances Therapeutic Potential

by Li-Chi Chen, Hsin-Wen Wang and Chieh-Cheng Huang

Cells 2021, 10(10), 2747; https://doi.org/10.3390/cells10102747 - 14 Oct 2021

Cited by 22 | Viewed by 3094

Abstract

Multicellular spheroids show three-dimensional (3D) organization with extensive cell–cell and cell–extracellular matrix interactions. Owing to their native tissue-mimicking characteristics, mesenchymal stem cell (MSC) spheroids are considered promising as implantable therapeutics for stem cell therapy. Herein, we aim to further enhance their therapeutic potential [...] Read more.

Multicellular spheroids show three-dimensional (3D) organization with extensive cell–cell and cell–extracellular matrix interactions. Owing to their native tissue-mimicking characteristics, mesenchymal stem cell (MSC) spheroids are considered promising as implantable therapeutics for stem cell therapy. Herein, we aim to further enhance their therapeutic potential by tuning the cultivation parameters and thus the inherent niche of 3D MSC spheroids. Significantly increased expression of multiple pro-regenerative paracrine signaling molecules and immunomodulatory factors by MSCs was observed after optimizing the conditions for spheroid culture. Moreover, these alterations in cellular behaviors may be associated with not only the hypoxic niche developed in the spheroid core but also with the metabolic reconfiguration of MSCs. The present study provides efficient methods for manipulating the therapeutic capacity of 3D MSC spheroids, thus laying solid foundations for future development and clinical application of spheroid-based MSC therapy for regenerative medicine. Full article

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25 pages, 2324 KiB

Open AccessReview

Clinical Applications of Cell-Scaffold Constructs for Bone Regeneration Therapy

by Venkata Suresh Venkataiah, Yoshio Yahata, Akira Kitagawa, Masahiko Inagaki, Yusuke Kakiuchi, Masato Nakano, Shigeto Suzuki, Keisuke Handa and Masahiro Saito

Cells 2021, 10(10), 2687; https://doi.org/10.3390/cells10102687 - 8 Oct 2021

Cited by 22 | Viewed by 4394

Abstract

Bone tissue engineering (BTE) is a process of combining live osteoblast progenitors with a biocompatible scaffold to produce a biological substitute that can integrate into host bone tissue and recover its function. Mesenchymal stem cells (MSCs) are the most researched post-natal stem cells [...] Read more.

Bone tissue engineering (BTE) is a process of combining live osteoblast progenitors with a biocompatible scaffold to produce a biological substitute that can integrate into host bone tissue and recover its function. Mesenchymal stem cells (MSCs) are the most researched post-natal stem cells because they have self-renewal properties and a multi-differentiation capacity that can give rise to various cell lineages, including osteoblasts. BTE technology utilizes a combination of MSCs and biodegradable scaffold material, which provides a suitable environment for functional bone recovery and has been developed as a therapeutic approach to bone regeneration. Although prior clinical trials of BTE approaches have shown promising results, the regeneration of large bone defects is still an unmet medical need in patients that have suffered a significant loss of bone function. In this present review, we discuss the osteogenic potential of MSCs in bone tissue engineering and propose the use of immature osteoblasts, which can differentiate into osteoblasts upon transplantation, as an alternative cell source for regeneration in large bone defects. Full article

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13 pages, 3944 KiB

Open AccessArticle

Parametric Imaging of Contrast-Enhanced Ultrasound (CEUS) for the Evaluation of Acute Gastrointestinal Graft-Versus-Host Disease

by Antonia-Maria Pausch, Sylvia Kammerer, Florian Weber, Wolfgang Herr, Christian Stroszczynski, Ernst Holler, Matthias Edinger, Daniel Wolff, Daniela Weber, Ernst-Michael Jung and Tobias Wertheimer

Cells 2021, 10(5), 1092; https://doi.org/10.3390/cells10051092 - 3 May 2021

Cited by 8 | Viewed by 2663

Abstract

In recent years contrast-enhanced ultrasound (CEUS) has been an emerging diagnostic modality for the detection of acute gastrointestinal (GI) graft-versus-host disease (GvHD) in patients after allogeneic stem cell transplantation. However, broad clinical usage has been partially limited by its high dependence on the [...] Read more.

In recent years contrast-enhanced ultrasound (CEUS) has been an emerging diagnostic modality for the detection of acute gastrointestinal (GI) graft-versus-host disease (GvHD) in patients after allogeneic stem cell transplantation. However, broad clinical usage has been partially limited by its high dependence on the expertise of an experienced examiner. Thus, the aim of this study was to facilitate detection of acute GI GvHD by implementing false color-coded parametric imaging of CEUS. As such, two inexperienced examiners with basic knowledge in abdominal and vascular ultrasound analyzed parametric images obtained from patients with clinical suspicion for acute GvHD in a blinded fashion. As diagnostic gold standard, histopathological GvHD severity score on intestinal biopsies obtained from lower GI tract endoscopy was performed. The evaluation of parametric images by the two inexperienced ultrasound examiners in patients with histological confirmation of acute GI GvHD was successful in 17 out of 19 patients (89%) as opposed to analysis of combined B-mode ultrasound, strain elastography, and CEUS by an experienced examiner, which was successful in 18 out of 19 of the patients (95%). Therefore, CEUS with parametric imaging of the intestine was technically feasible and has the potential to become a valuable diagnostic tool for rapid and widely accessible detection of acute GvHD in clinical practice. Full article

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20 pages, 3324 KiB

Open AccessArticle

Immunomodulatory Factors in Primary Endometrial Cell Cultures Isolated from Cancer and Noncancerous Human Tissue–Focus on RAGE and IDO1

by Joanna Tkaczuk-Włach, Witold Kędzierski, Ilona Jonik, Ilona Sadok, Agata Filip, Marta Kankofer, Wojciech Polkowski, Piotr Ziółkowski, Andrzej Gamian and Magdalena Staniszewska

Cells 2021, 10(5), 1013; https://doi.org/10.3390/cells10051013 - 25 Apr 2021

Cited by 3 | Viewed by 3047

Abstract

Background: Immune modulatory factors like indoleamine 2,3-dioxygenase 1 (IDO1) generating kynurenine (Kyn) and receptor for advanced glycation end-products (RAGE) contribute to endometrial and cancer microenvironment. Using adequate experimental models is needed to learn about the significance of these molecular factors in endometrial biology. [...] Read more.

Background: Immune modulatory factors like indoleamine 2,3-dioxygenase 1 (IDO1) generating kynurenine (Kyn) and receptor for advanced glycation end-products (RAGE) contribute to endometrial and cancer microenvironment. Using adequate experimental models is needed to learn about the significance of these molecular factors in endometrial biology. In this paper we study IDO1 activity and RAGE expression in the in vitro cultured primary human endometrial cells derived from cancerous and noncancerous tissue. Methods: The generated primary cell cultures from cancer and noncancerous endometrial tissues were characterized using immunofluorescence and Western Blot for expression of endometrial and cancer markers. IDO1 activity was studied by Kyn quantification with High Performance Liquid Chromatography with Diode Array Detector. Results: The primary cultures of endometrial cells were obtained with 80% success rate and no major genetic aberrations. The cells retained in vitro expression of markers (mucin MUC1 and HER2) or immunomodulatory factors (RAGE and IDO1). Increased Kyn secretion was associated with cancer endometrial cell culture in contrast to the control one. Conclusions: Primary endometrial cells express immune modulatory factors RAGE and IDO1 in vitro associated with cancer phenotype of endometrium. Full article

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20 pages, 53498 KiB

Open AccessArticle

Hyaluronic Acid as Macromolecular Crowder in Equine Adipose-Derived Stem Cell Cultures

by Sergio Garnica-Galvez, Stefanie H. Korntner, Ioannis Skoufos, Athina Tzora, Nikolaos Diakakis, Nikitas Prassinos and Dimitrios I. Zeugolis

Cells 2021, 10(4), 859; https://doi.org/10.3390/cells10040859 - 9 Apr 2021

Cited by 14 | Viewed by 4881

Abstract

The use of macromolecular crowding in the development of extracellular matrix-rich cell-assembled tissue equivalents is continuously gaining pace in regenerative engineering. Despite the significant advancements in the field, the optimal macromolecular crowder still remains elusive. Herein, the physicochemical properties of different concentrations of [...] Read more.

The use of macromolecular crowding in the development of extracellular matrix-rich cell-assembled tissue equivalents is continuously gaining pace in regenerative engineering. Despite the significant advancements in the field, the optimal macromolecular crowder still remains elusive. Herein, the physicochemical properties of different concentrations of different molecular weights hyaluronic acid (HA) and their influence on equine adipose-derived stem cell cultures were assessed. Within the different concentrations and molecular weight HAs, the 10 mg/mL 100 kDa and 500 kDa HAs exhibited the highest negative charge and hydrodynamic radius, and the 10 mg/mL 100 kDa HA exhibited the lowest polydispersity index and the highest % fraction volume occupancy. Although HA had the potential to act as a macromolecular crowding agent, it did not outperform carrageenan and Ficoll^®, the most widely used macromolecular crowding molecules, in enhanced and accelerated collagen I, collagen III and collagen IV deposition. Full article

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