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20 pages, 7594 KiB

Open AccessArticle

Investigating the Role of TGF-β Signaling Pathways in Human Corneal Endothelial Cell Primary Culture

by Inès Aouimeur, Tomy Sagnial, Louise Coulomb, Corantin Maurin, Justin Thomas, Pierre Forestier, Sandrine Ninotta, Chantal Perrache, Fabien Forest, Philippe Gain, Gilles Thuret and Zhiguo He

Cells 2023, 12(12), 1624; https://doi.org/10.3390/cells12121624 - 14 Jun 2023

Cited by 1 | Viewed by 1407

Abstract

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The [...] Read more.

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial–mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-βs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-β signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-β1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-β-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-β1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-β receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-β1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD. Full article

(This article belongs to the Special Issue Advances in Cell Culture Technology)

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16 pages, 1376 KiB

Open AccessArticle

Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors

by Seyyed Mehdy Elahi, Nazila Nazemi-Moghaddam, Claire Guilbault, Mélanie Simoneau and Rénald Gilbert

Cells 2023, 12(4), 619; https://doi.org/10.3390/cells12040619 - 14 Feb 2023

Cited by 2 | Viewed by 1420

Abstract

To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no [...] Read more.

To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene. Full article

(This article belongs to the Special Issue Advances in Cell Culture Technology)

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14 pages, 1626 KiB

Open AccessArticle

Cryopreservation of the Microalgae Scenedesmus sp.

by Martha Prieto-Guevara, Jany Alarcón-Furnieles, César Jiménez-Velásquez, Yamid Hernández-Julio, José Espinosa-Araujo and Víctor Atencio-García

Cells 2023, 12(4), 562; https://doi.org/10.3390/cells12040562 - 09 Feb 2023

Viewed by 2283

Abstract

Each phytoplankton species presents a different behavior and tolerance to the cryopreservation process. Therefore, in a species-specific protocol, it is essential to ensure both growth and post-thawing cell viability. In this study, we explored the effect of cryopreservation of Scenedesmus sp. with two [...] Read more.

Each phytoplankton species presents a different behavior and tolerance to the cryopreservation process. Therefore, in a species-specific protocol, it is essential to ensure both growth and post-thawing cell viability. In this study, we explored the effect of cryopreservation of Scenedesmus sp. with two cryoprotectants, dimethyl sulfoxide (DMSO) and methanol (MET), at 5% and 10% inclusion for each. In the control treatment, the microalgae were not exposed to cryoprotective agents (Control). Three post-thawing cell viability criteria were used: no cell damage (NCD), cell damage (CD), and marked lesions (LM), and mitochondrial and cell membrane damage was evaluated by flow cytometry. The study was a 2 × 2 factorial design, with five replications by treatments, population growth, and cell damage evaluated from the fifth day after thawing. On the fifth day, the highest percentage of NCD was observed when the microalgae were cryopreserved with DMSO 5% (50%); Regarding the control group, it showed 0% NCD. Flow cytometry analysis reveals minor damage at the membrane and mitochondria (9–10.7%) when DMSO is used at both inclusion percentages (5–10%) after thawing. In the exponential phase, the highest growth rates, doubling time, and yield was observed in cryopreserved cells with MET 5%. The results suggest that DMSO 5% is an ideal treatment for cryopreserving microalgae Scenedesmus sp. Full article

(This article belongs to the Special Issue Advances in Cell Culture Technology)

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15 pages, 2085 KiB

Open AccessArticle

Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell Spheroids

by Jaspal Garg, Alexandra Sporkova, Markus Hecker and Thomas Korff

Cells 2023, 12(1), 128; https://doi.org/10.3390/cells12010128 - 28 Dec 2022

Cited by 1 | Viewed by 4767

Abstract

Analyses of G-protein-mediated contraction and relaxation of vascular smooth muscle cells (VSMCs) are usually hampered by a rigid growth surface and culture conditions promoting cell proliferation and a less contractile phenotype. Our studies indicated that mouse aortic VSMCs cultured in three-dimensional spheroids acquire [...] Read more.

Analyses of G-protein-mediated contraction and relaxation of vascular smooth muscle cells (VSMCs) are usually hampered by a rigid growth surface and culture conditions promoting cell proliferation and a less contractile phenotype. Our studies indicated that mouse aortic VSMCs cultured in three-dimensional spheroids acquire a quiescent contractile status while decreasing the baseline G-protein-dependent inositolphosphate formation and increasing the expression of endothelin receptor type A (Ednra). Endothelin-1 (ET-1) promoted inositolphosphate formation in VSMC spheroids, but not in VSMCs cultured under standard conditions. To trace ET-1-mediated contraction of VSMC spheroids, we developed an assay by adhering them to collagen hydrogels and recording structural changes by time-lapse microscopy. Under these conditions, mouse and human VSMC spheroids contracted upon treatment with ET-1 and potassium chloride or relaxed in response to caffeine and the prostacyclin analogue Iloprost. ET-1 activated AKT-, MKK1-, and MKK3/6-dependent signaling cascades, which were inhibited by an overexpressing regulator of G-protein signaling 5 (Rgs5) to terminate the activity of Gα subunits. In summary, culture of VSMCs in three-dimensional spheroids lowers baseline G-protein activity and enables analyses of both contraction and relaxation of mouse and human VSMCs. This model serves as a simple and versatile tool for drug testing and investigating G-protein-depending signaling. Full article

(This article belongs to the Special Issue Advances in Cell Culture Technology)

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15 pages, 2293 KiB

Open AccessArticle

A Novel Method for Primary Blood Cell Culturing and Selection in Drosophila melanogaster

by Enikő Kúthy-Sutus, Bayan Kharrat, Erika Gábor, Gábor Csordás, Rita Sinka and Viktor Honti

Cells 2023, 12(1), 24; https://doi.org/10.3390/cells12010024 - 21 Dec 2022

Cited by 1 | Viewed by 1914

Abstract

The blood cells of the fruit fly Drosophila melanogaster show many similarities to their vertebrate counterparts, both in their functions and their differentiation. In the past decades, a wide palette of immunological and transgenic tools and methods have been developed to study hematopoiesis [...] Read more.

The blood cells of the fruit fly Drosophila melanogaster show many similarities to their vertebrate counterparts, both in their functions and their differentiation. In the past decades, a wide palette of immunological and transgenic tools and methods have been developed to study hematopoiesis in the Drosophila larva. However, the in vivo observation of blood cells is technically restricted by the limited transparency of the body and the difficulty in keeping the organism alive during imaging. Here we describe an improved ex vivo culturing method that allows effective visualization and selection of live blood cells in primary cultures derived from Drosophila larvae. Our results show that cultured hemocytes accurately represent morphological and functional changes following immune challenges and in case of genetic alterations. Since cell culturing has hugely contributed to the understanding of the physiological properties of vertebrate blood cells, this method provides a versatile tool for studying Drosophila hemocyte differentiation and functions ex vivo. Full article

(This article belongs to the Special Issue Advances in Cell Culture Technology)

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15 pages, 5923 KiB

Open AccessArticle

Easy and Affordable: A New Method for the Studying of Bacterial Biofilm Formation

by Dan Alexandru Toc, Alexandra Csapai, Florin Popa, Catalin Popa, Violeta Pascalau, Nicoleta Tosa, Alexandru Botan, Razvan Marian Mihaila, Carmen Anca Costache, Ioana Alina Colosi and Lia Monica Junie

Cells 2022, 11(24), 4119; https://doi.org/10.3390/cells11244119 - 19 Dec 2022

Cited by 4 | Viewed by 2158

Abstract

Background: Bacterial biofilm formation (BBF) proves itself to be in the spotlight of microbiology research due to the wide variety of infections that it can be associated with, the involvement in food spoilage, industrial biofouling and perhaps sewage treatment. However, BBF remains difficult [...] Read more.

Background: Bacterial biofilm formation (BBF) proves itself to be in the spotlight of microbiology research due to the wide variety of infections that it can be associated with, the involvement in food spoilage, industrial biofouling and perhaps sewage treatment. However, BBF remains difficult to study due to the lack of standardization of the existing methods and the expensive equipment needed. We aim to describe a new inexpensive and easy to reproduce protocol for a 3D-printed microfluidic device that can be used to study BBF in a dynamic manner. Methods: We used the SolidWorks 3D CAD Software (EducationEdition 2019–2020, Dassault Systèmes, Vélizy-Villacoublay, France) to design the device and the Creality3D Ender 5 printer (Shenzhen Creality 3D Technology Co., Ltd., Shenzhen, China) for its manufacture. We cultivated strains of Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. For the biofilm evaluation we used optical coherence tomography (OCT), scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy and crystal violet staining technique. Results: Based on the analysis, Enterococcus faecalis seems to produce more biofilm in the first hours while Pseudomonas aeruginosa started to take the lead on biofilm production after 24 h. Conclusions: With an estimated cost around €0.1285 for one microfluidic device, a relatively inexpensive and easy alternative for the study of BBF was developed. Full article

(This article belongs to the Special Issue Advances in Cell Culture Technology)

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14 pages, 3175 KiB

Open AccessArticle

Microfluidic Model to Evaluate Astrocyte Activation in Penumbral Region following Ischemic Stroke

by Kathryn M. Denecke, Catherine A. McBain, Brock G. Hermes, Sireesh Kumar Teertam, Mehtab Farooqui, María Virumbrales-Muñoz, Jennifer Panackal, David J. Beebe, Bolanle Famakin and Jose M. Ayuso

Cells 2022, 11(15), 2356; https://doi.org/10.3390/cells11152356 - 31 Jul 2022

Cited by 5 | Viewed by 2400

Abstract

Stroke is one of the main causes of death in the US and post-stroke treatment options remain limited. Ischemic stroke is caused by a blood clot that compromises blood supply to the brain, rapidly leading to tissue death at the core of the [...] Read more.

Stroke is one of the main causes of death in the US and post-stroke treatment options remain limited. Ischemic stroke is caused by a blood clot that compromises blood supply to the brain, rapidly leading to tissue death at the core of the infarcted area surrounded by a hypoxic and nutrient-starved region known as the penumbra. Recent evidence suggests that astrocytes in the penumbral region play a dual role in stroke response, promoting further neural and tissue damage or improving tissue repair depending on the microenvironment. Thus, astrocyte response in the hypoxic penumbra could promote tissue repair after stroke, salvaging neurons in the affected area and contributing to cognitive recovery. However, the complex microenvironment of ischemic stroke, characterized by gradients of hypoxia and nutrients, poses a unique challenge for traditional in vitro models, which in turn hinders the development of novel therapies. To address this challenge, we have developed a novel, polystyrene-based microfluidic device to model the necrotic and penumbral region induced by an ischemic stroke. We demonstrated that when subjected to hypoxia, and nutrient starvation, astrocytes within the penumbral region generated in the microdevice exhibited long-lasting, significantly altered signaling capacity including calcium signaling impairment. Full article

(This article belongs to the Special Issue Advances in Cell Culture Technology)

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13 pages, 5430 KiB

Open AccessArticle

Bronchoalveolar-Lavage-Derived Fibroblast Cell Line (B-LSDM7) as a New Protocol for Investigating the Mechanisms of Idiopathic Pulmonary Fibrosis

by Laura Bergantini, Miriana d’Alessandro, Sara Gangi, Dalila Cavallaro, Giuseppe Campiani, Stefania Butini, Claudia Landi, Luca Bini, Paolo Cameli and Elena Bargagli

Cells 2022, 11(9), 1441; https://doi.org/10.3390/cells11091441 - 24 Apr 2022

Cited by 3 | Viewed by 2532

Abstract

Background: The use of BAL to study ILDs has improved our understanding of IPF pathogenesis. BAL fluid is routinely collected and can be considered a clinical and research tool. The procedure is well tolerated and minimally invasive. No specific cell lines from BAL [...] Read more.

Background: The use of BAL to study ILDs has improved our understanding of IPF pathogenesis. BAL fluid is routinely collected and can be considered a clinical and research tool. The procedure is well tolerated and minimally invasive. No specific cell lines from BAL or immortalized cell lines from IPF patients are available commercially. A method to quickly isolate and characterize fibroblasts from BAL is an unmet research need. Materials and methods: Here we describe a new protocol by which we isolated a cell line from IPF. The cell line was expanded in vitro and characterized phenotypically, morphologically and functionally. Results: This culture showed highly filamentous cells with an evident central nucleus. From the phenotypic point of view, this cell line displays fibroblast/myofibroblast-like features including expression of alpha-SMA, vimentin, collagen type-1 and fibronectin. The results showed high expression of ROS in these cells. Oxidative stress invariably promotes extracellular matrix expression in lung diseases directly or through over-production of pro-fibrotic growth factors. Conclusions: Our protocol makes it possible to obtain fibroblasts BAL that is a routine non-invasive method that offers the possibility of having a large sample of patients. Standardized culture methods are important for a reliable model for testing molecules and eventual novel development therapeutic targets. Full article

(This article belongs to the Special Issue Advances in Cell Culture Technology)

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Review

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18 pages, 6771 KiB

Open AccessReview

Controlling Microenvironments with Organs-on-Chips for Osteoarthritis Modelling

by Louis Jun Ye Ong, Xiwei Fan, Antonia Rujia Sun, Lin Mei, Yi-Chin Toh and Indira Prasadam

Cells 2023, 12(4), 579; https://doi.org/10.3390/cells12040579 - 10 Feb 2023

Cited by 3 | Viewed by 2427

Abstract

Osteoarthritis (OA) remains a prevalent disease affecting more than 20% of the global population, resulting in morbidity and lower quality of life for patients. The study of OA pathophysiology remains predominantly in animal models due to the complexities of mimicking the physiological environment [...] Read more.

Osteoarthritis (OA) remains a prevalent disease affecting more than 20% of the global population, resulting in morbidity and lower quality of life for patients. The study of OA pathophysiology remains predominantly in animal models due to the complexities of mimicking the physiological environment surrounding the joint tissue. Recent development in microfluidic organ-on-chip (OoC) systems have demonstrated various techniques to mimic and modulate tissue physiological environments. Adaptations of these techniques have demonstrated success in capturing a joint tissue’s tissue physiology for studying the mechanism of OA. Adapting these techniques and strategies can help create human-specific in vitro models that recapitulate the cellular processes involved in OA. This review aims to comprehensively summarise various demonstrations of microfluidic platforms in mimicking joint microenvironments for future platform design iterations. Full article

(This article belongs to the Special Issue Advances in Cell Culture Technology)

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