Cell Culture Parameters Research Articles

Callus induction, subculture, and browning inhibition of the anticancer plant Taxus media

In order to obtain more effective cell culture parameters of Taxus anticancer plants, we optimized the callus induction and subculture conditions of the explants (stem segments with buds) of the anticancer medicinal plant Taxus media by using the plant tissue culture technology and orthogonal test. Furthermore, we studied the method to inhibit browning in the culture. The results indicated that the optimal conditions for inducing callus was culture in the medium composed of B5+0.25 mg/L 2, 4-D+1.5 mg/L NAA+0.5 mg/L KT in the dark, which showed short induction time and a high induction rate (86.7%). The formula of the optimal medium for subculture was B5+0.5 mg/L 2, 4-D+2.0 mg/L NAA+1.5 mg/L KT.The proliferation multiple of callus cultured by subculture on the 10th day of callus growth was the highest. Activated carbon inhibited the browning in callus subculture, with the optimal inhibitory concentration of 0.8 g/L. The results of this study lay a foundation for the production of taxol by suspension culture of T. media cells.

Beyond viability: Advancing CHO cell culture process strategies to modulate host cell protein levels

Chinese Hamster Ovary (CHO) cells are widely utilized in bioprocessing industry for monoclonal antibody (mAb) production. In many instances, challenges persist in achieving sufficient clearance of Host Cell Proteins (HCPs) in the final drug substance. While purification strategies usually offer substantial HCP clearance, certain "problematic" HCPs, particularly lipases, continue to pose significant challenges. This study investigates the accumulation of various "problematic" HCPs in CHO cell culture using transcriptomics, revealing correations between cell culture parameters and HCP level. Contrary to conventional assumptions, viability alone does not reliably predict HCP levels, with factors such as clone selection, host cell line choice, media and feed compositions significantly influencing HCP accumulation. Leveraging transcriptomics-based approaches, we demonstrate the potential of upstream process control strategies to mitigate HCP presence and improve biologic product quality. Our findings underscore the importance of considering diverse cell culture parameters in bioprocess optimization to ensure product stability and quality. While promising, further research is needed to elucidate the mechanisms underlying HCP release and propagation through downstream processing stages, emphasizing the necessity of a comprehensive integrated approach to HCP control in biologics production.

Reinventing shake flask fermentation: The breathable flask.

Cultivating cells in shake flasks is a routine operation that is largely unchanged since its inception. A glass or plastic Erlenmeyer vessel with the primary gas exchange taking place across various porous plugs is used with media volumes typically ranging from 100 mL to 2 L. Oxygen limitation and carbon dioxide accumulation in the vessel is a major concern for studies involving shake flask cultures. In this study, we enhance mass transfer in a conventional shake flask by replacing the body wall with a permeable membrane. Naturally occurring concentration gradient across the permeable membrane walls facilitates the movement of oxygen and carbon dioxide between the flask and the external environment. The modified flask called the breathable flask, has shown a 40% improvement in mass transfer coefficient (kLa) determined using the static diffusion method. The prokaryotic cell culture studies performed with Escherichiacolishowed an improvement of 28%-66% in biomass and 41%-56% in recombinant product yield. The eukaryotic cell culture study performed with Pichia pastoris expressing proinsulin exhibited a 40% improvement in biomass and 115% improvement in protein yield. The study demonstrates a novel approach to addressing the mass transfer limitations in conventional shake flask cultures. The proposed flask amplifies its value by providing a membrane-diffusion-based sensing platform for the integration of low-cost, noninvasive sensing capabilities for real-time monitoring of critical cell culture parameters like dissolved oxygenand dissolved carbon dioxide.

Comprehensive assessment of goat adiposetissue-derived mesenchymal stem cells cultured in different media.

Mesenchymal stem cells (MSCs) have demonstrated potential in treating livestock diseases that are unresponsive to conventional therapies. MSCs derived from goats, a valuable model for studying orthopaedic disorders in humans, offer insights into bone formation and regeneration. Adipose tissue-derived MSCs (ADSCs) are easily accessible and have a high capacity for expansion. Although the choice of culture media significantly influences the biological properties of MSCs, the optimal media for goat ADSCs (gADSCs) remains unclear. This study aimed to assess the effects of four commonly used culture media on gADSCs' culture characteristics, stem cell-specific immunophenotype, and differentiation. Results showed that MEM, DMEM/F12, and DMEM-LG were superior in maintaining cell morphology and culture parameters of gADSCs, such as cell adherence, metabolic activity, colony-forming potential, and population doubling. Conversely, DMEM-HG exhibited poor performance across all evaluated parameters. The gADSCs cultured in DMEM/F12 showed enhanced early proliferation and lower apoptosis. The cell surface marker distribution exhibited superior characteristics in gADSCs cultured in MEM and DMEM/F12. In contrast, the distribution was inferior in gADSCs cultured in DMEM-LG. DMEM/F12 and DMEM-LG culture media demonstrated a significantly higher potential for chondrogenic differentiation and DMEM-LG for osteogenic differentiation. In conclusion, DMEM/F12 is a suitable culture medium for propagating gADSCs as it effectively maintains cell morphology, growth parameters, proliferation and lower apoptosis while exhibiting desirable expression patterns of MSC-specific markers. These findings contribute to optimising culture conditions for gADSCs, enhancing their potential applications in disease treatment and regenerative medicine.

In Vitro Assessment of Cardiac Fibroblast Activation at Physiologic Stiffness.

Cardiac fibroblasts (CF) are an essential cell type in cardiac physiology, playing diverse roles in maintaining structural integrity, extracellular matrix (ECM) synthesis, and tissue repair. Under normal conditions, these cells reside in the interstitium in a quiescent state poised to sense and respond to injury by synthesizing and secreting collagen, vimentin, hyaluronan, and other ECM components. In response to mechanical and chemical stimuli, these "resident" fibroblasts can undergo a transformation through a continuum of activation states into what is commonly known as a "myofibroblast," in a process critical for injury response. Despite progress in understanding the contribution of fibroblasts to cardiac health and disease, much remains unknown about the signaling mediating this activation, in part owing to technical challenges in evaluating CF function and activation status in vitro. Given their role in monitoring the ECM, CFs are acutely sensitive to stiffness and pressure. High basal activation of isolated CFs is common due to the super-physiologic stiffness of traditional cell culture substrates, making assays dependent on quiescent cells challenging. To overcome this problem, cell culture parameters must be tightly controlled, and the use of dishes coated with biocompatible reduced-stiffness substrates, such as 8-kPa polydimethylsiloxane (PDMS), has shown promise in reducing basal activation of fibroblasts. Here, we describe cell culture protocol for maintaining CF quiescence in vitro to enable a dynamic range for the assessment of activation status in response to fibrogenic stimuli using PDMS-coated coverslips. Our protocol provides a cost-effective tool to study fibroblast signaling and activity, allowing researchers to better understand the underlying mechanisms involved in cardiac fibrosis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 8-kPa polydimethylsiloxane (PDMS)/gelatin-coated coverslips for cardiac fibroblast cell culture Basic Protocol 2: Isolation of adult cardiac fibroblasts and plating onto PDMS coverslips Basic Protocol 3: Assessment of cardiac fibroblast activation by α smooth muscle actin (αSMA) immunocytochemistry.

MOTS-c regulates pancreatic alpha and beta cell functions in vitro

The aim of this study is to determine the influence of the mitochondrial open-reading-frame of the twelve S rRNA-c (MOTS-c) peptide on pancreatic cell physiology. Moreover, in this study, we examined the changes in MOTS-c secretion and expression under different conditions. Our experiments were conducted using laboratory cell line cultures, specifically the INS-1E and αTC-1 cell lines, which represent β and α pancreatic cells, respectively. As the pancreas is an endocrine organ, we also tested its hormone regulation capabilities. Furthermore, we assessed the secretion of MOTS-c after incubating the cells with glucose and free fatty acids. Additionally, we examined key cell culture parameters such as cell viability, proliferation, and apoptosis. The results obtained from this study show that MOTS-c has a significant impact on the physiology of pancreatic cells. Specifically, it lowers insulin secretion and expression in INS-1E cells and enhances glucagon secretion and expression in αTC-1 cells. Furthermore, MOTS-c affects cell viability and apoptosis. Interestingly, insulin and glucagon affect the MOTS-c secretion as well as glucose and free fatty acids. These experiments clearly show that MOTS-c is an important regulator of pancreatic metabolism, and there are numerous properties of MOTS-c yet to be discovered.

Оценка показателей секреции цитокинов и пролиферативной активности в культурах мононуклеарных клеток крови больных клещевыми инфекциями

Associations between the immune response and the severity of tick-borne infections are poorly understood. Objective. Assessment of the parameters of cytokines secretion, proliferative activity and cytogenetic instability in the mononuclear blood cell cultures of patients with tick-borne encephalitis febrile form and ixodid tick-borne borreliosis nonerythemal form at the onset of the disease. Patients and methods. We assessed 24 parameters in the phytohemagglutinin-stimulated mononuclear blood cell cultures from 25 patients with tick-borne encephalitis, 24 patients with ixodid tick-borne borreliosis, and from 14 healthy donors. Statistical analysis included the Mann–Whitney test, Pearson’s goodness-of-fit test, Spearman’s rank correlation, and ROC analysis. Results. The patients compared with the controls showed a suppression of the stimulated blast transformation, increases in the levels of the micronucleated T-lymphocytes and the secretion of not only pro-inflammatory cytokines – tumor necrosis factor (TNF)-α and interleukin (IL)-6, but IL-10 and IL-4 as well. Pyrexia levels were negatively correlated with the blast transformation and positively correlated with the micronucleated T-cells levels and pro-inflammatory cytokines secretion. The predictors for the diagnosis of tick-borne encephalitis compared with borreliosis were decreases in the blank and/or stimulated levels of blast transformation and the production of TNF-α, IL-6, interferon-γ as well as increases in the micronucleated T-cell levels and the secretion of IL-8 and IL-10. Conclusion. The results represent the features of the immune response reactions and its association with the severity of the clinical picture. Key words: blast transformation, ixodid tick-borne borreliosis, tick-borne encephalitis, micronucleus assay, mononuclear blood cells, phytohemagglutinin, cytokines

Design of experiments for the automated development of a multicellular cardiac model for high-throughput screening

Cardiovascular toxicity remains a major cause of drug attrition in early drug development, clinical trials, and post-market surveillance. In vitro assessment of cardiovascular liabilities often relies on single cell type-based model systems coupled with functional assays, like calcium flux and multielectrode arrays. Although these models offer high-throughput capabilities and demonstrate good predictivity for functional cardiotoxicities, they fail to consider the vital contribution of non-myocyte cells, thus limiting the potential for integrated risk assessment. Complex 3D hPSC-derived multicellular cardiac model systems have been growing in popularity; however, many of these models are limited to low-throughput with lengthy development timelines and high costs, which hampers their suitability to drug discovery.To optimize the development of an in vitro multicellular model system containing human-induced pluripotent stem-cell derived cardiomyocytes, endothelial cells and cardiac fibroblasts, we employed the Synthace platform, which enables scientists to express complex experimental intent in a simple format (e.g. Design of Experiments) and to translate this to automation protocols using no-code. Utilizing this approach, we systematically screened the impact of multiple cell culture parameters, including the co-culture of three cell types, on cardiac contractility, with minimal hands-on time. Our platform accelerates the assay development process, providing users with an efficient means to explore and optimize the experimental space for the development of multicellular models. This is particularly valuable in scenarios involving variable biological responses and limited understanding of underling mechanisms. Moreover, users can make better use of resources, streamline their workflows, and drive data-driven decision-making throughout the assay development journey.

Optical glucose sensor for microfluidic cell culture systems

Glucose is the primary energy source of human cells. Therefore, monitoring glucose inside microphysiological systems (MPS) provides valuable information on the viability and metabolic state of the cultured cells. However, continuous glucose monitoring inside MPS is challenging due to a lack of suitable miniaturized sensors. Here we present an enzymatic, optical glucose sensor element for measurement inside microfluidic systems. The miniaturized glucose sensor (Ø 1 mm) is fabricated together with a reference oxygen sensor onto biocompatible, pressure-sensitive adhesive tape for easy integration inside microfluidic systems. Furthermore, the proposed microfluidic system can be used as plug and play sensor system with existing MPS. It was characterized under cell culture conditions (37 °C and pH 7.4) for five days, exhibiting minor drift (3% day−1). The influence of further cell culture parameters like oxygen concentration, pH, flow rate, and sterilization methods was investigated. The plug-and-play system was used for at-line measurements of glucose levels in (static) cell culture and achieved good agreement with a commercially available glucose sensor. In conclusion, we developed an optical glucose sensor element that can be easily integrated in microfluidic systems and is able to perform stable glucose measurements under cell culture conditions.

Comprehensive analysis of culture conditions governing differentiation of MSCs into articular chondrocytes.

Treatment of osteoarthritic patients requires the development of morphologically and mechanically complex hyaline cartilage at the injury site. A tissue engineering approach toward differentiating mesenchymal stem cells into articular chondrocytes has been developed to overcome the drawbacks of conventional therapeutic and surgical procedures. To imitate the native micro and macro environment of articular chondrocytes, cell culture parameters such as oxygen concentration, mechanical stress, scaffold design, and growth factor signalling cascade regulation must be addressed. This review aims to illuminate the path toward developing tissue engineering approaches, accommodating these various parameters and the role these parameters play in regulating chondrogenesis for better articular cartilage development to treat osteoarthritis effectively.

Role of interleukin-6 and interleukin-10 in morphological and functional changes of the blood–brain barrier in hypertriglyceridemia

BackgroundHypertriglyceridemia is closely linked to atherosclerosis related inflammatory processes and blood–brain barrier (BBB) dysfunction. Using apolipoprotein B-100 (APOB-100) transgenic mice, an animal model of chronic hypertriglyceridemia, we analyzed BBB function and morphology in vitro and ex vivo. Our objective was to determine which BBB characteristics are produced mainly by interleukin (IL)-6, an atherosclerosis promoting cytokine, and whether these actions can be antagonized by IL-10, an anti-inflammatory cytokine.MethodsBrain endothelial and glial cell cultures and brain microvessels were isolated from wild type (WT) and APOB-100 transgenic mice and were treated with IL-6, IL-10 and their combination. First, IL-6 and IL-10 production was measured in WT and APOB-100 microvessels using qPCR. Then functional parameters of endothelial cell cultures were analyzed and immunocytochemistry for key BBB proteins was performed.ResultsIL-6 mRNA levels were higher in brain microvessels than in brain parenchyma of APOB-100 transgenic mice. Transendothelial electric resistance and P-glycoprotein activity were lower, and paracellular permeability was higher in cultured APOB-100 brain endothelial cells. These features were sensitive to both IL-6 and IL-10 treatments. A decreased P-glycoprotein immunostaining was measured in transgenic endothelial cells under control conditions and in WT cells after treating them with IL-6. This effect was antagonized by IL-10. Changes in immunostaining for tight junction proteins were observed after IL-6 exposure, which were in part antagonized by IL-10. In glial cell cultures an increase in aquaporin-4 immunolabeling in the transgenic group and an increase in microglia cell density in WT glia cultures was detected after IL-6 treatment, which was antagonized by IL-10. In isolated brain microvessels a decrease in P-glycoprotein immunolabeled area fraction was measured in APOB-100 microvessels under control conditions and in WT microvessels after every cytokine treatment. ZO-1 immunolabeling showed characteristics similar to that of P-glycoprotein. No change was seen in claudin-5 and occludin immunoreactive area fractions in microvessels. A decrease in aquaporin-4 immunoreactivity was measured in WT microvessels treated by IL-6, which was antagonized by IL-10.ConclusionIL-6 produced in microvessels contributes to BBB impairment observed in the APOB-100 mice. We showed that IL-10 partly antagonizes the effects of IL-6 at the BBB.

Preparation of Human Muscle Precursor Cells for the MyoGravity Project’s Study of Cell Cultures in Experiment Units for Space Flight Purposes

Long-time exposure to the microgravity conditions experienced during space flights induces alterations in the homeostasis of organs and tissues, including skeletal muscles, which undergo atrophy with the loss of mass and strength due to decreased size and altered composition of myofibers. Microgravity conditions can also affect the functionality of satellite cells, i.e., the adult stem cells providing the muscle precursors that are responsible for the growth and maintenance of muscle mass in adult life, as well as for muscle regeneration following a damage. The MyoGravity project, funded by Agenzia Spaziale Italiana (ASI), aimed to send human muscle precursor cells (huMPCs) on board the International Space Station (ISS) in order to study the effects of real microgravity on the differentiation capacity of this cell type. To this end, it was necessary to use a methodology to cultivate huMPCs inside dedicated space bioreactor devices (Experiment Units, EUs) specifically designed to cultivate cell cultures and perform scientific protocols in the space environment of the ISS. Here, we report the setting of several cell culture parameters to convert the EUs into suitable devices for biomedical experiments using huMPCs for space flight purposes.

The Effect of Age and Cell Culture Parameters on the Quantity and Function of Bone Marrow-derived Dendritic Cells.

Dendritic cells (DCs) are a group of bone marrow-derived cells that play a crucial role in innate and acquired immune responses. Bone marrow-derived dendritic cells (BMDC) are used in many studies, so the efficiency and purity of the differentiated cells are essential. This study aimed to investigate the effect of several parameters, including the age of mice, cell culture medium, and swirling of the culture plate, to increase the efficiency of the induced cells, considering the standard protocols. Bone marrow-derived dendritic cells were induced from both juvenile and adult mice bone marrow cells. Then, the purity of CD11c+ cells was compared between juvenile mice BMDCs and adult mice BMDCs. Cells were cultured in an enriched and non-enriched medium, and some wells were swirled when changing the medium on the 3rd day. Then the effect of enriched medium and swirling before medium replacement were evaluated based on the expression of the CD11c marker. The efficiency of DCs differentiation (CD11c+ cells) was higher when juvenile mouse bone marrow precursors were used compared to adult mice; using the enriched media with supplements and swirling the well before media replacement significantly affected the purity of immature CD11c+ cells. Due to our results, using juvenile mice, an enriched culture medium, and physical removal of granulocyte cells could significantly improve the purity and efficiency of CD11c+ cells. Therefore, considering these three items in the production protocol of these cells can probably reduce the use of lymphocyte-removing antibodies and purification methods.

Meta-analysis of in-vitro cytotoxicity evaluation studies of zinc oxide nanoparticles: Paving way for safer innovations

Nano-based products have shown their daunting presence in several sectors. Among them, Zinc Oxide (ZnO) nanoparticles wangled the reputation of providing “next-generation solutions” and are being utilized in plethora of products. Their widespread application has led to increased exposure of these particles, raising concerns regarding toxicological repercussions to the human health and environment. The diversity, complexity, and heterogeneity in the available literature, along with correlation of befitting attributes, makes it challenging to develop one systematic framework to predict this toxicity. The present study aims at developing predictive modelling framework to tap the prospective features responsible for causing cytotoxicity in-vitro on exposure to ZnO nanoparticles. Rigorous approach was used to mine the information from complete body of evidence published to date. The attributes, features and experimental conditions were systematically extracted to unmask the effect of varied features. 1240 data points from 76 publications were obtained, containing 14 qualitative and quantitative attributes, including physiochemical properties of nanoparticles, cell culture and experimental parameters to perform meta-analysis. For the first time, the efforts were made to investigate the degree of significance of attributes accountable for causing cytotoxicity on exposure to ZnO nanoparticles. We show that in-vitro cytotoxicity is closely related with dose concentration of nanoparticles, followed by exposure time, disease state of the cell line and size of these nanoparticles among other attributes.

INCREASING THE COST-EFFECTIVENESS OF IN VITRO RESEARCH THROUGH THE USE OF TITANIUM IN THE DEVICE FOR MEASURING THE ELECTRICAL PARAMETERS OF CELLS

Currently, various methods are used to assess the biocompatibility of materials. After an in-depth and detailed review of the literature, the method used in the research was selected. As part of the experiments, a method based on the analysis of the values ​​of electrical parameters of cell cultures measured in the presence of electrodes was used. The electrode is a structure made of a thin layer of metallization. It measures the change in resistance, impedance and capacity of a mixture of cells and the substance in which they are grown. The plate containing the electrode assembly is called the measurement matrix. Currently, commercially used test matrices are made of gold or platinum. However, their high price means that large-scale research is significantly limited. In order to increase the access to the widespread use of this method, it was decided that it was necessary to use cheaper materials, reducing the necessary costs of conducting experiments. Considering this, an attempt was made to use a different conductive material to build matrices compatible with the ECIS® Z-Theta measurement system. Their use would enable in vitro research on living cells. In the presented work, titanium was used as a material that may turn out to be an alternative to the materials currently used. Its application to the production of matrices will allow to study the influence of this metal on the behavior of cells.

Tissue dermal equivalent – а cellular product based on human dermal keratinocytes and fibroblasts: the properties of equivalent components and perspectives of practical application

The nature and ways of isolation and cultivation in vitro of keratinocytes and fibroblasts, the main cellular components of skin to prepare a new biomedical product, tissue dermal equivalent were considered. The main attention was payed to optimization of upbuilding dermal cell biomass including selection of medium compositions and conditions of cultivation. The information was given on main parameters of cell cultures as proliferation activity, viability and phenotype of the cells. Genotoxicity of fibroblasts and biocompatibility of the cells with organic matrixes to find the optimal carrier for cellular elements of tissue dermal equivalent were studied. The composition, the process of preparation of tissue dermal equivalent and perspectives of its practical application were discussed.

In Vitro Low-Fluence Photodynamic Therapy Parameter Screening Using 3D Tumor Spheroids Shows that Fractionated Light Treatments Enhance Phototoxicity.

The evaluation of novel photosensitizers (PSs) for photodynamic therapy (PDT) is difficult due to the limitations of two-dimensional cell culture and multiple parameters (dose, light intensity, uptake time), which complicate progression to in vivo experiments and clinical translation. Three-dimensional (3D) cell culture models like multicellular cancer tumor spheroids (MCTS) show great similarities to in vivo avascular tumor conditions, improving the speed and accuracy of screening novel compounds with various treatment combinations. In this study, we utilize C8161 human melanoma spheroids to screen PDT treatment combinations using protoporphyrin IX (PpIX) and drug-loaded carbon dot (CD) conjugates PpIX-CD and PpIX@CD at ultralow fluence values (<10 J/cm2). Conjugates show equivalent light-induced damage to PpIX from 1 μg/mL with significantly less dark cytotoxicity up to 72 h after exposure, shown by LDH release and dsDNA content. Fractionated treatments, carried out by dividing light exposure with 24 h intervals, demonstrate an enhanced PDT effect compared to single exposure at equal concentrations. Light sheet fluorescence microscopy combined with live/dead staining demonstrates that spheroids sustain extensive damage after PDT, with PpIX and PpIX-CD showing improved uptake compared to PpIX@CD. We show that PDT parameter screening can be carried out using a low-cost and convenient combination of assays to improve the efficiency of evaluating novel compounds.

Influence of Stem-cell Size and Culture Media Flowing Modality on Cell’s Fate within a Microchannel; a Numerical Analysis

The dynamic cell culture process has been widely used in tissue engineering. The success of cell culture is influenced by many factors, one of which is how the cells are transferred from the bioreactor to the scaffolds through microchannels. The risk that can reduce the success of the cell culture process is that the cells do not reach the final destination correctly. In this study, the movement of stem cells through a microchannel was theoretically analysed using discrete phase computational fluid dynamics. Three factors of cell size, fluid flow rate and fluid viscosity were investigated on their sedimentation rate before reaching the microchannel outlet. Considering four sizes of 10, 15, 20 and 30 µm for cells, four flow rates of 20, 50, 90 and 180 µl/min in addition, four viscosities of 0.001, 0.005, 0.01 and 0.025 Pa.s were selected for culture media left us a total number of 64 models. The results of the analysis showed that cells with smaller size have a better chance of reaching the microchannel outlet and larger cells are more likely to sediment. Also, higher flow velocities as well as higher fluid viscosity delivering more cells to the destination. The results of this study shed more light on the regulation and control of dynamic cell culture parameters.

SARS-CoV-2 Structural Proteins Exposure Alter Thrombotic and Inflammatory Responses in Human Endothelial Cells.

IntroductionWe have experienced a pandemic induced by the interaction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) structural proteins with innate structures. These interactions are especially prevalent for patients with underlying pathologies, such as cardiovascular diseases. However, there has been limited work to uncover the range of responses induced by SARS-CoV-2 structural proteins. Thus, our objective was to investigate how endothelial cell pro-thrombotic and pro-inflammatory responses are altered after exposure to SARS-CoV-2 spike, nucleocapsid, and membrane-envelope proteins. We hypothesized that after a short duration exposure, endothelial cells would have a heightened thrombotic and inflammatory potential. With longer exposures, this may lead to altered disease progression and the observed increased mortality and morbidity rates in patients with underlying vascular pathologies.MethodsTo test this hypothesis, human endothelial cells were exposed to SARS-CoV-2 structural proteins. After the exposure, the expression of thrombomodulin, PECAM-1, connexin-43, and gC1qR were assessed. In parallel, standard cell culture readouts were assessed to determine if these incubations altered cell growth and metabolism.Results and ConclusionsWe observed significant increases in thrombotic and inflammatory marker expression, with no change to the cell culture parameters (with the exception of a reduction in cell density in response to one SARS-CoV-2 structural protein). Importantly, these observations were dependent on the viral structural protein the cells were exposed to, suggesting that the interactions of SARS-CoV-2 with innate cells is complex and must be uncovered. Combined, this suggests that SARS-CoV-2 structural proteins can regulate inflammatory and thrombotic responses that underlie common pathologies observed during COVID-19.

3D two-photon polymerization of smart cell gelatin – collagen matrixes with incorporated ruthenium complexes for the monitoring of local oxygen tensions

The extra cellular matrix plays a major role in the biomechanical properties of tissues that impact cell behavior and fate. It is therefore crucial to mimic these complex cell-matrix interactions in 3D cell cultures. Here, two-photon polymerization is applied to produce gelatin methacryloyl (GelMA) – collagen matrixes that further enable local pO2matrix measurement, when ruthenium complexes are used as photo-activators. The fluorescence intensity of these complexes has a direct and inverse relationship with the local pO2matrix. The 3D structures reached their maximum size in cell culture conditions after 3H with a swelling factor of ~1.5. Their shape and the ruthenium fluorescence intensity of the alveoli walls stayed constant for at least 2 weeks in the absence of cells. They were used in time series to monitor the local pO2matrix adjacent to cancer cells during their division, migration and the formation of a tumor tissue mass. At the presence of these cell activities that consume O2, a significant ~3-fold increase of the ruthenium fluorescence intensity in the alveoli walls was observed. This study demonstrates that online monitoring of the local pO2matrix is possible. The ruthenium complexes provide the bio-optical sensors that are useful for further analysis of cancer and healthy cell energy metabolism in a 3D matrix that better mimics in vivo conditions and migration paths. Unraveling the cancer cell metabolic adaptations in a changing micro-environment will help the development of new therapeutic opportunities. Statement of significanceIn 3D cell cultures, monitoring pericellular pO2 is as critical as controlling pH. This facility is currently missing. Here, we take advantage of the direct and inverse relationship between pO2 and the fluorescence intensity of ruthenium complexes to generate stable gelatin-collagen matrixes able to continuously monitoring the pO2 at the pericellular level. The ruthenium complexes, which are photo-activators in the two-photon polymerization of these matrixes, became covalently bind to the collagen fibers. Indeed, local O2 consumption by cancer cells during migration, mitosis and tumor mass formation caused a 3-fold increase of the ruthenium fluorescence. In the future, incorporating ruthenium complexes with other bio-optical sensors will create new drug screening platforms that monitor cell culture parameters at the pericellular level.