Cell Culture Approaches Research Articles

Synergistic Antiproliferative Effects of Chondroitin Sulfate and Fucoidan in Tumor-derived Spheroids: Insights From a 3D Cell Culture Approach

Abstract The aim of the study was to investigate the effects of chondroitin sulfate (CS) and fucoidan (F), as well as their combination (CS + F) on the growth and viability of spheroids derived from the non-tumor cell line NIH3T3 and the tumor cell line Hepa1c1c7 using a three-dimensional (3D) cell culture model. Spheroids were formed using the liquid overlay method and treated with different concentrations of substances. Flow cytometry was used to quantitatively assess the representation of live, apoptotic, necrotic, and dead cells in the spheroids after treatment. Spheroids from the non-tumor cell line NIH3T3 after treatment with CS and fucoidan were nontoxic to the cells. By light microscopy, we observed a significant proliferative effect in the case of CS and combination of fucoidan and CS (F + CS). On the contrary, in spheroids from the Hepa1c1c7 tumor cell line, after treatment with our compounds, the compounds, especially fucoidan and a combination of CS and fucoidan, showed antiproliferative effects, which confirms their synergistic antiproliferative effect. According to flow cytometry in non-tumor NIH3T3 spheroids, CS had the most significant effect on the proportion of viable cells, which confirmed its proliferative effect (66.43 ± 4.43%). In spheroids derived from the Hepa1c1c7 tumor cell line, flow cytometry revealed the highest number of dead cells in spheroids after treatment with a combination of CS and fucoidan (19.84 ± 5.80%). Based on our results, we can conclude that a combination of CS and fucoidan showed a synergistic antiproliferative effect on Hepa1c1c7 cell cultures. The 3D cell cultures provide a more physiologically relevant platform for drug testing, highlighting the importance of understanding cell–extracellular matrix interactions in cancer research.

Persistence of human Aichi virus infectivity from raw surface water to drinking water.

Human Aichi virus 1 (AiV-1) is a water- and food-borne infection-associated picornavirus that causes gastroenteritis in humans. Recent studies on environmental waters showed a high frequency and abundance of AiV-1, suggesting that it might be an appropriate indicator of fecal contamination. We screened 450 surface and drinking water samples from a Tunisian drinking water treatment plant (DWTP) and the Sidi Salem dam for AiV-1 by real time reverse transcriptase PCR (RT-qPCR). The persistence of infectious particles was evaluated using an integrated cell culture approach coupled with quantitative molecular detection (ICC-RT-qPCR). In all, 85 (18.9%) water samples were positive for AiV-1 with viral loads ranging from 0.47 to 11.62 log10 cp/L and a median of 4.97 log10 cp/L, including 30/100 raw, 18/50 decanted, 14/50 flocculated, 9/100 treated, 1/50 tap, and 13/100 surface water samples. Of these, 15 (17.6%) samples contained infectious AiV-1 genotype A particles, including five raw, four decanted, one surface, three flocculated, and two treated water samples. Our data suggest that the persistence of infectious AiV-1 particles in environmental waters might represent a potential threat to public health. This study also indicates that the ICC-RT-qPCR is a practical tool for monitoring human waterborne viral risk in aquatic environments.IMPORTANCEHuman Aichi virus 1 (AiV-1) is a water- and food-borne infection-associated picornavirus that causes gastroenteritis in humans. Its high frequency and abundance in environmental waters would suggest that it might be an appropriate indicator of fecal contamination. The analysis of surface and drinking water samples from a Tunisian drinking water treatment plant (DWTP) and the Sidi Salem dam using an integrated cell culture approach coupled with a quantitative molecular detection (ICC-RT-qPCR) confirmed the persistence of infectious AiV-1 particles in samples at all stages of the treatment process, except in tap water. This suggests that the persistence of AiV-1 infectivity in environmental waters might represent a potential threat to public health. This study also indicates that the ICC-RT-qPCR is a practical tool for monitoring human waterborne viral risk in aquatic environments.

Shrinking Cancer Research Barriers: Crafting Accessible Tumor‐on‐Chip Device for Gene Silencing Assays

Tumor‐on‐chip (ToC) is crucial to bridge the gap between traditional cell culture experiments and in vivo models, allowing to recreate an in vivo‐like microenvironment in cancer research. ToC use microfluidics to provide fine‐tune control over environmental factors, high‐throughput screening, and reduce requirements of samples and reagents. However, creating these microfluidic devices requires skilled researchers and dedicated manufacturing equipment, making widespread adoption cumbersome and difficult. To address some bottlenecks and improve accessibility to ToC technology, innovative materials and fabrication processes are required. Polystyrene (PS) is a promising material for microfluidics due to its biocompatibility, affordability, and optical transparency. Herein, a fabrication process based on direct laser writing on thermosensitive PS, allowing the swift and economical crafting of devices with easy pattern alterations, is presented. For the first time, a device for cell culture fabricated only by PS is presented, allowing customizing and optimization for efficient cell culture approaches. These biochips support 2D and 3D cultures with comparable viability and proliferation kinetics to traditional 96‐well plates. The data show that gene and protein silencing efficiencies remain consistent across both chip and plate‐based cultures, either 2D culture or 3D spheroid format. Although simple, this approach might facilitate the use of customized chip‐based cancer models.

Impaired 11β-hydroxysteroid dehydrogenase type 2 activity in kidney disease disrupts 11-oxygenated androgen biosynthesis.

11-oxygenated androgens are a group of adrenal-derived steroids that require peripheral activation. In vitro data highlight a putative role for 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) in 11-oxygenated androgen biosynthesis, converting 11β-hydroxyandrostenedione (11OHA4) to 11-ketoandrostenedione (11KA4), the direct precursor of the potent androgen 11-ketotestosterone (11KT). As the kidney is the major site of HSD11B2 expression, we hypothesized that patients with chronic kidney disease (CKD) would have reduced 11-oxygenated androgen biosynthesis due to impaired HSD11B2 activity. To determine the role of HSD11B2 in 11-oxygenated androgen biosynthesis using a human CKD cohort alongside complementary cell culture and computational modeling approaches. Cross-sectional observational study of patients with CKD (n=85) and healthy controls (n=46) measuring serum and urinary concentrations of glucocorticoids, classic and 11-oxygenated androgens by liquid chromatography-tandem mass spectrometry. A computational model of peripheral 11-oxygenated androgen biosynthesis was fitted to the serum data to calculate relative HSD11B2 expression levels for each participant. HSD11B2 activity declined with eGFR, evidenced by higher cortisol (F)/cortisone (E) ratios in CKD patients compared to controls (p<0.0001). Serum concentrations of E, 11KA4, 11KT and 11β-hydroxytestosterone were lower in patients with CKD compared to controls (p<0.0001 for each). A computational model based on enzyme kinetic parameters of HSD11B2, 11β-hydroxysteroid dehydrogenase type 1, 17β-hydroxysteroid dehydrogenase type 2 and aldo-keto reductase 1C3 confirmed HSD11B2 as the key enzyme responsible for reduced 11-oxygenated androgen biosynthesis in CKD. Predicted HSD11B2 expression correlated with eGFR. This is the first in vivo study to confirm a central role for renal HSD11B2 in 11-oxygenated androgen biosynthesis. Determining the clinical implications of this observation for patients with CKD requires further research.

Hyaluronic Acid Influences Amino Acid Metabolism via Differential L‐Type Amino Acid Transporter 1 Expression in the U87‐Malignant Glioma Cell Line

The glioblastoma (GBM) tumor microenvironment is heterogeneous, complex, and being increasingly understood as a significant contributor to tumor progression. In brain tumors, the extracellular matrix contains a large concentration of hyaluronic acid (HA) that makes it important to study its role in cancer progression. In particular, abnormal accumulation of HA is observed in gliomas and is often associated with poor prognosis. In addition, HA is a polymer and its molecular weight (MW) distribution may influence tumor cell activity. Herein, the influence of the MW of HA on tumor cell metabolism is evaluated. A 2D cell culture approach is used to expose the U87‐MG (medium glucose [MG]) cell line to different HA MWs (10, 60, and 500 kDa) and glucose concentrations (0, 5.5, and 25 mm). Notably, it is found that HA influences GBM amino acid metabolism via reduction in LAT1 transporter protein expression. Also an influence on mitochondrial respiration levels and a difference in the accumulation of some key products of cell metabolic activity (lactic acid, glutamic acid, and succinic acid) are reported. Overall, in these results, it is indicated that HA MW can influence GBM metabolic state, with implications for cell invasion and tumor progression.

Forming Single-Cell-Derived Colon Cancer Organoid Arrays on a Microfluidic Chip for High Throughput Tumor Heterogeneity Analysis.

Single-cell-derived tumor organoids (STOs) possess a distinct genetic background, making them valuable tools for demonstrating tumor heterogeneity. In order to fulfill the high throughput demands of STO assays, we have developed a microfluidic chip containing 30 000 microwells, which is dedicated to a single cell culture approach for selective expansion and differential induction of cancer stem cells. The microwells are coated with a hydrophilic copolymer to eliminate cell adhesion, and the cell culture is supported by poly(ethylene glycol) (PEG) to establish a nonadhesive culture environment. By utilizing an input cell density of 7 × 103·mL-1, it is possible to construct a 4000 single cell culture system through stochastic cell occupation. We demonstrate that the addition of 15% PEG10000 in the cell culture medium effectively prevents cell loss while facilitating tumor stem cell expansion. As were demonstrated by HCT116, HT29, and SW480 colon cancer cells, the microfluidic approach achieved a STO formation rate of ∼20%, resulting in over 800 STOs generated from a single culture. Comprehensive analysis through histomorphology, immunohistochemistry, drug response evaluation, assessment of cell invasion, and biomarker detection reveals the heterogeneity among individual STOs. Specifically, the smaller STOs exhibited higher invasion and drug resistance capabilities compared with the larger ones. The developed microfluidic approach effectively facilitates STO formation and offers promising prospects for investigating tumor heterogeneity, as well as conducting personalized therapy-focused drug screening.

Spheroid trilineage differentiation model of primary mesenchymal stem/stromal cells under hypoxia and serum-free culture conditions.

Due to their unique properties, human mesenchymal stem/stromal cells (MSCs) possess tremendous potential in regenerative medicine, particularly in cell-based therapies where the multipotency and immunomodulatory characteristics of MSCs can be leveraged to address a variety of disease states. Although MSC-based cell therapeutics have emerged as one of the most promising medical treatments, the clinical translation is hampered by the variability of MSC-based cellular products caused by tissue source-specific differences and the lack of physiological cell culture approaches that closely mimic the human cellular microenvironment. In this study, a model for trilineage differentiation of primary adipose-, bone marrow-, and umbilical cord-derived MSCs into adipocytes, chondrocytes and osteoblasts was established and characterized. Differentiation was performed in spheroid culture, using hypoxic conditions and serum-free and antibiotics-free medium. This platform was characterized for spheroid diameter and trilineage differentiation capacity reflecting functionality of differentiated cells, as indicated by lineage-specific extracellular matrix (ECM) accumulation and expression of distinct secreted markers. The presented model shows spheroid growth during the course of differentiation and successfully supports trilineage differentiation for MSCs from almost all tissue sources except for osteogenesis of umbilical cord-derived MSCs. These findings indicate that this platform provides a suitable and favorable environment for trilineage differentiation of MSCs from various tissue sources. Therefore, it poses a promising model to generate highly relevant biological data urgently required for clinical translation and therefore might be used in the future to generate in vitro microtissues, building blocks for tissue engineering or as disease models.

In-situ monitoring of cellular H2O2 within 3D cell clusters using conductive scaffolds

Accurately monitoring H2O2 concentrations in 3D cell clusters is challenging due to limited diffusion and rapid degradation of H2O2 in the culture medium. Despite the incorporation of three-dimensional cell culture approaches, the detection technology has largely remained as a 2D planar system. In this study, we present a versatile approach of 3D electrochemical sensing utilizing carbon nanotubes as conductive scaffolds for in-situ monitoring of H2O2 in cell clusters. These scaffolds enabled direct contact between H2O2 released from cells and the electrodes, thereby improving sensitivity and ensuring biocompatibility for cell aggregates. The scaffolds exhibited electrocatalytic behavior with a limit of detection of 6.7nMH2O2. Additionally, the electrochemical responses of cell clusters with the scaffolds exhibited significantly higher current compared to clusters without scaffolds when stimulated with model drugs. This study underscores the potential of conductive scaffolds for real-time monitoring of H2O2 released from cell clusters in 3D microenvironments.

Umbilical cord mesenchymal stem cell exosomes alleviate necrotizing enterocolitis in neonatal mice by regulating intestinal epithelial cells autophagy.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease that affects premature infants. Although mounting evidence supports the therapeutic effect of exosomes on NEC, the underlying mechanisms remain unclear. To investigate the mechanisms underlying the regulation of inflammatory response and intestinal barrier function by umbilical cord mesenchymal stem cell (UCMSCs) exosomes, as well as their potential in alleviating NEC in neonatal mice. NEC was induced in 5-d-old C57BL/6 pups through hypoxia and gavage feeding of formula containing lipopolysaccharide (LPS), after which the mice received human UCMSC exosomes (hUCMSC-exos). The control mice were allowed to breastfeed with their dams. Ileal tissues were collected from the mice and analyzed by histopathology and immunoblotting. Colon tissues were collected from NEC neonates and analyzed by immunofluorescence. Molecular biology and cell culture approaches were employed to study the related mechanisms in intestinal epithelial cells. We found that autophagy is overactivated in intestinal epithelial cells during NEC, resulting in reduced expression of tight junction proteins and an increased inflammatory response. The ability of hUCMSC-exos to ameliorate NEC in a mouse model was dependent on decreased intestinal autophagy. We also showed that hUCMSC-exos alleviate the inflammatory response and increase migration ability in intestinal epithelial cells induced by LPS. These results contribute to a better understanding of the protective mechanisms of hUCMSC-exos against NEC and provide a new theoretical and experimental foundation for NEC treatment. These findings also enhance our understanding of the role of the autophagy mechanism in NEC, offering potential avenues for identifying new therapeutic targets.

Umbilical cord mesenchymal stem cell exosomes alleviate necrotizing enterocolitis in neonatal mice by regulating intestinal epithelial cells autophagy

BACKGROUND Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease that affects premature infants. Although mounting evidence supports the therapeutic effect of exosomes on NEC, the underlying mechanisms remain unclear. AIM To investigate the mechanisms underlying the regulation of inflammatory response and intestinal barrier function by umbilical cord mesenchymal stem cell (UCMSCs) exosomes, as well as their potential in alleviating NEC in neonatal mice. METHODS NEC was induced in 5-d-old C57BL/6 pups through hypoxia and gavage feeding of formula containing lipopolysaccharide (LPS), after which the mice received human UCMSC exosomes (hUCMSC-exos). The control mice were allowed to breastfeed with their dams. Ileal tissues were collected from the mice and analyzed by histopathology and immunoblotting. Colon tissues were collected from NEC neonates and analyzed by immunofluorescence. Molecular biology and cell culture approaches were employed to study the related mechanisms in intestinal epithelial cells. RESULTS We found that autophagy is overactivated in intestinal epithelial cells during NEC, resulting in reduced expression of tight junction proteins and an increased inflammatory response. The ability of hUCMSC-exos to ameliorate NEC in a mouse model was dependent on decreased intestinal autophagy. We also showed that hUCMSC-exos alleviate the inflammatory response and increase migration ability in intestinal epithelial cells induced by LPS. CONCLUSION These results contribute to a better understanding of the protective mechanisms of hUCMSC-exos against NEC and provide a new theoretical and experimental foundation for NEC treatment. These findings also enhance our understanding of the role of the autophagy mechanism in NEC, offering potential avenues for identifying new therapeutic targets.

Patient-Derived Conditionally Reprogrammed Cells in Prostate Cancer Research.

Prostate cancer (PCa) remains a leading cause of mortality among American men, with metastatic and recurrent disease posing significant therapeutic challenges due to a limited comprehension of the underlying biological processes governing disease initiation, dormancy, and progression. The conventional use of PCa cell lines has proven inadequate in elucidating the intricate molecular mechanisms driving PCa carcinogenesis, hindering the development of effective treatments. To address this gap, patient-derived primary cell cultures have been developed and play a pivotal role in unraveling the pathophysiological intricacies unique to PCa in each individual, offering valuable insights for translational research. This review explores the applications of the conditional reprogramming (CR) cell culture approach, showcasing its capability to rapidly and effectively cultivate patient-derived normal and tumor cells. The CR strategy facilitates the acquisition of stem cell properties by primary cells, precisely recapitulating the human pathophysiology of PCa. This nuanced understanding enables the identification of novel therapeutics. Specifically, our discussion encompasses the utility of CR cells in elucidating PCa initiation and progression, unraveling the molecular pathogenesis of metastatic PCa, addressing health disparities, and advancing personalized medicine. Coupled with the tumor organoid approach and patient-derived xenografts (PDXs), CR cells present a promising avenue for comprehending cancer biology, exploring new treatment modalities, and advancing precision medicine in the context of PCa. These approaches have been used for two NCI initiatives (PDMR: patient-derived model repositories; HCMI: human cancer models initiatives).

Evaluation of the Effect of Botulinum Toxin A on the Lymphatic Endothelial Cells

IntroductionBotulinum toxin A (BoTA) is a neurotoxin formed by Clostridium botulinum, with a broad medical application spectrum. While the primary effect of BoTA is on the muscles, the effects of BoTA in other systems including the blood vasculature have already been examined, revealing unexpected actions. However, no studies exist to the best of our knowledge regarding the potential effects of BoTA on the lymphatic vascular system, possessing a critical role in health and disease. Isolated human lymphatic endothelial cells (LECs) were cultured in dedicated in vitro culture systems. The analysis including imaging and cell culture approaches as well as molecular biology techniques is performed to examine the LEC alterations occurring upon exposure to different concentrations of BoTA.Materials and MethodsHuman LECs were cultured and expanded on collagen-coated petri dishes using endothelial basal medium and the commercial product Botox from Allergan as used for all our experiments. Harvested cells were used in various in vitro functional tests to assess the morphologic and functional properties of the BoTA-treated LECs. Gene expression analysis was performed to assess the most important lymphatic system-related genes and pathways.ResultsConcentrations of 1, 5 or 10 U of BoTA did not demonstrate a significant effect regarding the proliferation and migration capacity of the LECs versus untreated controls. Interestingly, even the smallest BoTA dose was found to significantly decrease the cord-like-structure formation capacity of the seeded LECs. Gene expression analysis was used to underpin possible molecular alterations, suggesting no significant effect of BoTA in the modification of gene expression versus the starvation medium control.ConclusionLECs appear largely unaffected to BoTA treatment, with an isolated effect on the cord-like-structure formation capacity. Further work needs to assess the effect of BoTA on the smooth-muscle-cell-covered collecting lymphatic vessels and the possible aesthetic implications of such an effect, due to edema formation.Level of Evidence VThis journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

A three-dimensional cell culture approach to investigate cytotoxic effects and production of inflammatory mediators by epoxy resin-based and calcium silicate-based endodontic sealer.

The aim of the present study was to assess the cytocompatibility of epoxy resin-based AH Plus Jet (Dentsply De Trey, Konstanz, Germany), Sealer Plus (MK Life, Porto Alegre, Brazil), calcium silicate-based Bio-C Sealer (Angelus, Londrina, PR, Brazil), Sealer Plus BC (MK Life) and AH Plus BC (Dentsply) through a tridimensional (3D) culture model of human osteoblast-like cells. Spheroids of MG-63 cells were produced and exposed to fresh root canal sealers extracts by 24h, and the cytotoxicity was assessed by the Lactate Dehydrogenase assay (LDH). The distribution of dead cells within the microtissue was assessed by fluorescence microscopy, and morphological effects were investigated by histological analysis. The secreted inflammatory mediators were detected in cell supernatants through flow luminometry (XMap Luminex). Cells incubated with AH Plus Jet, AH Plus BC, Sealer Plus BC and Bio-C Sealer extracts showed high rates of cell viability, while the Sealer Plus induced a significant reduction of cell viability, causing reduction on the spheroid structure. Sealer Plus and Seaker Plus BC caused alterations on 3D microtissue morphology. The AH Plus BC extract was associated with the downregulation of secretion of pro-inflammatory cytokines IL-5, IL-7, IP-10 and RANTES. The new AH Plus BC calcium silicate-based endodontic sealer did not reduce cell viability in vitro, while led to the downregulation of pro-inflammatory cytokines. Choosing the appropriate endodontic sealer is a crucial step. AH Plus BC demonstrated high cell viability and downregulation of pro-inflammatory cytokines, appearing reliable for clinical use, while Sealer Plus presented lower cytocompatibility.

Design approaches for 3D cell culture and 3D bioprinting platforms.

The natural habitat of most cells consists of complex and disordered 3D microenvironments with spatiotemporally dynamic material properties. However, prevalent methods of in vitro culture study cells under poorly biomimetic 2D confinement or homogeneous conditions that often neglect critical topographical cues and mechanical stimuli. It has also become increasingly apparent that cells in a 3D conformation exhibit dramatically altered morphological and phenotypical states. In response, efforts toward designing biomaterial platforms for 3D cell culture have taken centerstage over the past few decades. Herein, we present a broad overview of biomaterials for 3D cell culture and 3D bioprinting, spanning both monolithic and granular systems. We first critically evaluate conventional monolithic hydrogel networks, with an emphasis on specific experimental requirements. Building on this, we document the recent emergence of microgel-based 3D growth media as a promising biomaterial platform enabling interrogation of cells within porous and granular scaffolds. We also explore how jammed microgel systems have been leveraged to spatially design and manipulate cellular structures using 3D bioprinting. The advent of these techniques heralds an unprecedented ability to experimentally model complex physiological niches, with important implications for tissue bioengineering and biomedical applications.

FEAR antiviral response pathway is independent of interferons and countered by poxvirus proteins.

The human facilitates chromatin transcription (FACT) complex is a chromatin remodeller composed of human suppressor of Ty 16 homologue (hSpt16) and structure-specific recognition protein-1 subunits that regulates cellular gene expression. Whether FACT regulates host responses to infection remained unclear. We identify a FACT-mediated, interferon-independent, antiviral pathway that restricts poxvirus replication. Cell culture and bioinformatics approaches suggest that early viral gene expression triggers nuclear accumulation of SUMOylated hSpt16 subunits required for the expression of E26 transformation-specific sequence-1 (ETS-1)-a transcription factor that activates virus restriction programs. However, biochemical studies show that poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting structure-specific recognition protein-1 binding to SUMOylated hSpt16 and by tethering SUMOylated hSpt16 to microtubules. Furthermore, A51R antagonism of FACT enhances poxvirus replication in human cells and virulence in mice. Finally, we show that FACT also restricts rhabdoviruses, flaviviruses and orthomyxoviruses, suggesting broad roles for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 antiviral response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

The Use of Human Platelet Lysate as a Coating Substance for Adipose-Derived Stem Cell Expansion.

Large-scale production of mesenchymal stromal cells is essential for sufficient therapeutic doses in regenerative medicine. However, long-term cultivation encounters limited cell growth and cellular aging. Therefore, an alternative cell culture approach that promotes proliferation and attenuates cell senescence is required. Human platelet lysate (HPL) is a potent supplement for in vitro cell expansion. Applying HPL as a coating material can potentially improve mesenchymal stromal cell cultures. To examine the capacity of HPL, it was used to pre-coat a tissue culture plate for in vitro adipose-derived mesenchymal stromal cell expansion. Alterations in biological features of adipose-derived stem cells (ADSCs) were investigated, including cell adhesion assays, cell proliferation, population doubling time, and cellular senescence. ADSCs cultured on HPL-coated plates significantly increased cell adhesion rate, shortened population doubling time, and stimulated cell growth. The senescent cells were significantly decreased in ADSCs cultured in an HPL-coated plate, and the expression levels of senescence-associated genes, including p16, p21, and p53, were downregulated. Furthermore, Western blotting analysis revealed that HPL was enriched with fibronectin and vitronectin, essential cell adhesive proteins. HPL was effectively used as a coating material for ADSC expansions. Cellular cultivation on the HPL coating is an alternative approach for producing mesenchymal stromal cells.

Establishment of a Human Immunocompetent 3D Tissue Model to Enable the Long-Term Examination of Biofilm-Tissue Interactions.

Different studies suggest an impact of biofilms on carcinogenic lesion formation in varying human tissues. However, the mechanisms of cancer formation are difficult to examine in vivo as well as in vitro. Cell culture approaches, in most cases, are unable to keep a bacterial steady state without any overgrowth. In our approach, we aimed to develop an immunocompetent 3D tissue model which can mitigate bacterial outgrowth. We established a three-dimensional (3D) co-culture of human primary fibroblasts with pre-differentiated THP-1-derived macrophages on an SIS-muc scaffold which was derived by decellularisation of a porcine intestine. After establishment, we exposed the tissue models to define the biofilms of the Pseudomonas spec. and Staphylococcus spec. cultivated on implant mesh material. After 3 days of incubation, the cell culture medium in models with M0 and M2 pre-differentiated macrophages presented a noticeable turbidity, while models with M1 macrophages presented no noticeable bacterial growth. These results were validated by optical density measurements and a streak test. Immunohistology and immunofluorescent staining of the tissue presented a positive impact of the M1 macrophages on the structural integrity of the tissue model. Furthermore, multiplex ELISA highlighted the increased release of inflammatory cytokines for all the three model types, suggesting the immunocompetence of the developed model. Overall, in this proof-of-principle study, we were able to mitigate bacterial overgrowth and prepared a first step for the development of more complex 3D tissue models to understand the impact of biofilms on carcinogenic lesion formation.

Isolation of Rift Valley Fever Virus from Field Specimens Using Cell Culture Approaches.

Several techniques have been developed to diagnose Rift Valley fever infection. Viral isolation is one of the most difficult techniques to apply but offers great opportunities for further research. It is useful, for example, for the development of an accurate diagnostic test suitable for screening for Rift Valley fever virus infection, specific treatments by testing known antiviral molecules that act on the replication cycle to assess their therapeutic or even prophylactic potential, therapeutic applications, and vaccine candidates. Understanding how the virus replicates and interacts with the host cell and organism and identifying biomarkers of infection or new targets for the development of treatments are made possible through field virus isolates. Biosafety level 3 conditions are a pre-requisite for viral isolation by a trained staff member. Here, we describe the procedure to isolate Rift Valley fever virus from field samples by cell culture.

External RF-EMF alters cell number and ROS balance possibly via the regulation of NADPH metabolism and apoptosis.

The influence of weak radio-frequency electromagnetic field (RF-EMF) on living organisms raises new concern because of the Industrial, Scientific, and Medical (ISM) frequency band at 6.78 MHz being promoted by the AirFuel Alliance for mid-range wireless power transfer (WPT) applications and product development. Human exposure to the RF-EMF radiation is unavoidable. In this study, we employed in vitro cell culture and molecular biology approach coupled with integrated transcriptomic and proteomic analyses to uncover the effects of RF-EMF on cells at molecular and cellular levels. Our study has demonstrated that weak RF-EMF is sufficient to exert non-thermal effects on human umbilical vein endothelial cells (HUVEC). Exposure of weak RF-EMF promotes cell proliferation, inhibits apoptosis and deregulates ROS balance. Alteration of several signaling pathways and key enzymes involved in NADPH metabolism, cell proliferation and ferroptosis were identified. Our current study provide solid evidence for the first time that the present safety standards that solely considered the thermal effect of RF-EMF on cell tissue are inadequate, prompt response and modification of existing Guidelines, Standards and Regulation are warranted.