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ACS Biomaterials Science & Engineering May 2024Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or...
Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from a necrotic core due to limited nutrient and oxygen diffusion and waste removal and have a limited -like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids were loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow velocity was maintained within perfusion wells and the pillar plate was separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging . The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in a dynamic 3D cell culture.
Topics: Cell Culture Techniques, Three Dimensional; Humans; Cell Proliferation; Reproducibility of Results; Perfusion; Hydrogels; Spheroids, Cellular; Cell Culture Techniques
PubMed: 38695610
DOI: 10.1021/acsbiomaterials.4c00179 -
MSphere May 2024Biofilm formation is an important virulence factor for methicillin-resistant (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of...
Biofilm formation is an important virulence factor for methicillin-resistant (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA that hold the biofilm together. MRSA cells secrete micrococcal nuclease (Nuc1), which degrades double-stranded DNA. In this study, we used standard methodologies to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. We quantified biofilm formation and extracellular DNA (eDNA) levels in broth and agar cultures. In some experiments, cultures were supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods and enumerating detached colony-forming units (CFUs), and biofilm sloughing was investigated by perfusing biofilms cultured in glass tubes with fresh broth and measuring the sizes of the detached cell aggregates. We found that an MRSA mutant strain produced significantly more biofilm and more eDNA than a wild-type strain, both in the absence and presence of sub-MIC amoxicillin. mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or complementing the mutant. In the sloughing assay, mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.IMPORTANCEInfections caused by antibiotic-resistant bacteria known as methicillin-resistant (MRSA) are a significant problem in hospitals. MRSA forms adherent biofilms on implanted medical devices such as catheters and breathing tubes. Bacteria can detach from biofilms on these devices and spread to other parts of the body such as the blood or lungs, where they can cause life-threatening infections. In this article, researchers show that MRSA secretes an enzyme known as thermonuclease that causes bacteria to detach from the biofilm. This is important because understanding the mechanism by which MRSA detaches from biofilms could lead to the development of procedures to mitigate the problem.
Topics: Biofilms; Methicillin-Resistant Staphylococcus aureus; Micrococcal Nuclease; Anti-Bacterial Agents; Bacterial Proteins; DNA, Bacterial; Virulence Factors; Microbial Sensitivity Tests; Amoxicillin
PubMed: 38695568
DOI: 10.1128/msphere.00126-24 -
Frontiers in Cell and Developmental... 2024Immunotherapy has changed the landscape of treatment options for patients with hepatocellular cancer. Checkpoint inhibitors are now standard of care for patients with...
Immunotherapy has changed the landscape of treatment options for patients with hepatocellular cancer. Checkpoint inhibitors are now standard of care for patients with advanced tumours, yet the majority remain resistant to this therapy and urgent approaches are needed to boost the efficacy of these agents. Targeting the liver endothelial cells, as the orchestrators of immune cell recruitment, within the tumour microenvironment of this highly vascular cancer could potentially boost immune cell infiltration. We demonstrate the successful culture of primary human liver endothelial cells in organ-on-a-chip technology followed by perfusion of peripheral blood mononuclear cells. We confirm, with confocal and multiphoton imaging, the capture and adhesion of immune cells in response to pro-inflammatory cytokines in this model. This multicellular platform sets the foundation for testing the efficacy of new therapies in promoting leukocyte infiltration across liver endothelium as well as a model for testing cell therapy, such as chimeric antigen receptor (CAR)-T cell, capture and migration across human liver endothelium.
PubMed: 38694823
DOI: 10.3389/fcell.2024.1359451 -
The Journal of Physiology May 2024Passive whole-body hyperthermia increases limb blood flow and cardiac output ( ), but the interplay between peripheral and central thermo-haemodynamic mechanisms...
Passive whole-body hyperthermia increases limb blood flow and cardiac output ( ), but the interplay between peripheral and central thermo-haemodynamic mechanisms remains unclear. Here we tested the hypothesis that local hyperthermia-induced alterations in peripheral blood flow and blood kinetic energy modulate flow to the heart and . Body temperatures, regional (leg, arm, head) and systemic haemodynamics, and left ventricular (LV) volumes and functions were assessed in eight healthy males during: (1) 3 h control (normothermic condition); (2) 3 h of single-leg heating; (3) 3 h of two-leg heating; and (4) 2.5 h of whole-body heating. Leg, forearm, and extracranial blood flow increased in close association with local rises in temperature while brain perfusion remained unchanged. Increases in blood velocity with small to no changes in the conduit artery diameter underpinned the augmented limb and extracranial perfusion. In all heating conditions, increased in association with proportional elevations in systemic vascular conductance, related to enhanced blood flow, blood velocity, vascular conductance and kinetic energy in the limbs and head (all R ≥ 0.803; P < 0.001), but not in the brain. LV systolic (end-systolic elastance and twist) and diastolic functional profiles (untwisting rate), pulmonary ventilation and systemic aerobic metabolism were only altered in whole-body heating. These findings substantiate the idea that local hyperthermia-induced selective alterations in peripheral blood flow modulate the magnitude of flow to the heart and through changes in blood velocity and kinetic energy. Localised heat-activated events in the peripheral circulation therefore affect the human heart's output. KEY POINTS: Local and whole-body hyperthermia increases limb and systemic perfusion, but the underlying peripheral and central heat-sensitive mechanisms are not fully established. Here we investigated the regional (leg, arm and head) and systemic haemodynamics (cardiac output: ) during passive single-leg, two-leg and whole-body hyperthermia to determine the contribution of peripheral and central thermosensitive factors in the control of human circulation. Single-leg, two-leg, and whole-body hyperthermia induced graded increases in leg blood flow and . Brain blood flow, however, remained unchanged in all conditions. Ventilation, extracranial blood flow and cardiac systolic and diastolic functions only increased during whole-body hyperthermia. The augmented with hyperthermia was tightly related to increased limb and head blood velocity, flow and kinetic energy. The findings indicate that local thermosensitive mechanisms modulate regional blood velocity, flow and kinetic energy, thereby controlling the magnitude of flow to the heart and thus the coupling of peripheral and central circulation during hyperthermia.
Topics: Humans; Male; Adult; Hyperthermia; Cardiac Output; Blood Flow Velocity; Regional Blood Flow; Fever; Young Adult; Hot Temperature; Hemodynamics
PubMed: 38690610
DOI: 10.1113/JP285760 -
HardwareX Jun 2024Studies of the effects of external stimuli on bone tissue, disease transmission mechanisms, and potential medication discoveries benefit from long-term tissue viability...
Studies of the effects of external stimuli on bone tissue, disease transmission mechanisms, and potential medication discoveries benefit from long-term tissue viability ex vivo. By simulating the in-vivo environment, bioreactors are essential for studying bone cellular activity throughout biological processes. We present the development of an automated 3D-printed bioreactor EnduroBone designed to sustain the ex-vivo viability of 10 mm diameter cancellous bone cores for an extended period. The device is supplied with two critical parameters for maintaining bone tissue viability: closed-loop continuous flow perfusion of 1 mL/min for nutrient diffusion and waste removal and direct mechanical stimulation with cyclic compression at 13.2 RPM (revolutions per minute) to promote cell viability which can lead to improved tissue stability during ex vivo culturing. The bioreactor addresses several limitations of existing systems and provides a versatile open-source platform for bone cancer research, orthopedic device testing, and other related applications. To validate the bioreactor, fresh swine samples were cultured ex-vivo, and their cell viability was determined to be maintained for up to 28 days. Periodic cell viability assessment through live/dead cell staining and confocal imaging at the start (0 days) and at several time points throughout the culture period (7, 14, 21, and 28 days) was used to demonstrate effectiveness in sustaining bone cell health for the extended period tested.
PubMed: 38690152
DOI: 10.1016/j.ohx.2024.e00535 -
Cellular and Molecular Life Sciences :... Apr 2024Cellular senescence of hepatocytes involves permanent cell cycle arrest, disrupted cellular bioenergetics, resistance to cell death, and the release of pro-inflammatory...
BACKGROUND AND AIM
Cellular senescence of hepatocytes involves permanent cell cycle arrest, disrupted cellular bioenergetics, resistance to cell death, and the release of pro-inflammatory cytokines. This 'zombie-like' state perpetuates harmful effects on tissues and holds potential implications for liver disease progression. Remarkably, senescence exhibits heterogeneity, stemming from two crucial factors: the inducing stressor and the cell type. As such, our present study endeavors to characterize stressor-specific changes in senescence phenotype, its related molecular patterns, and cellular bioenergetics in primary mouse hepatocytes (PMH) and hepatocyte-derived liver organoids (HepOrgs).
METHODS
PMH, isolated by collagenase-perfused mouse liver (C57B6/J; 18-23 weeks), were cultured overnight in William's E-medium supplemented with 2% FBS, L-glutamine, and hepatocyte growth supplements. HepOrgs were developed by culturing cells in a 3D matrix for two weeks. The senescence was induced by DNA damage (doxorubicin, cisplatin, and etoposide), oxidative stress (HO, and ethanol), and telomere inhibition (BIBR-1532), p53 activation (nutlin-3a), DNA methyl transferase inhibition (5-azacitidine), and metabolism inhibitors (galactosamine and hydroxyurea). SA-β galactosidase activity, immunofluorescence, immunoblotting, and senescence-associated secretory phenotype (SASP), and cellular bioenergetics were used to assess the senescence phenotype.
RESULTS
Each senescence inducer triggers a unique combination of senescence markers in hepatocytes. All senescence inducers, except hydroxyurea and ethanol, increased SA-β galactosidase activity, the most commonly used marker for cellular senescence. Among the SASP factors, CCL2 and IL-10 were consistently upregulated, while Plasminogen activator inhibitor-1 exhibited global downregulation across all modes of senescence. Notably, DNA damage response was activated by DNA damage inducers. Cell cycle markers were most significantly reduced by doxorubicin, cisplatin, and galactosamine. Additionally, DNA damage-induced senescence shifted cellular bioenergetics capacity from glycolysis to oxidative phosphorylation. In HepOrgs exposed to senescence inducers, there was a notable increase in γH2A.X, p53, and p21 levels. Interestingly, while showing a similar trend, SASP gene expression in HepOrgs was significantly higher compared to PMH, demonstrating a several-fold increase.
CONCLUSION
In our study, we demonstrated that each senescence inducer activates a unique combination of senescence markers in PMH. Doxorubicin demonstrated the highest efficacy in inducing senescence, followed by cisplatin and HO, with no impact on apoptosis. Each inducer prompted DNA damage response and mitochondrial dysfunction, independent of MAPK/AKT.
Topics: Animals; Cellular Senescence; Hepatocytes; Mice; DNA Damage; Oxidative Stress; Mice, Inbred C57BL; Cells, Cultured; Senescence-Associated Secretory Phenotype; Tumor Suppressor Protein p53; Doxorubicin; Energy Metabolism; Liver; Male
PubMed: 38684535
DOI: 10.1007/s00018-024-05230-2 -
Pharmaceutics Apr 2024To better understand ischaemia-related molecular alterations, temporal changes in angiogenic Aminopeptidase N (APN/CD13) expression and glucose metabolism were assessed...
BACKGROUND
To better understand ischaemia-related molecular alterations, temporal changes in angiogenic Aminopeptidase N (APN/CD13) expression and glucose metabolism were assessed with PET using a rat model of peripheral arterial disease (PAD).
METHODS
The mechanical occlusion of the base of the left hindlimb triggered using a tourniquet was applied to establish the ischaemia/reperfusion injury model in Fischer-344 rats. 2-[F]FDG and [Ga]Ga-NOTA-c(NGR) PET imaging performed 1, 3, 5, 7, and 10 days post-ischaemia induction was followed by Western blotting and immunohistochemical staining for APN/CD13 in ischaemic and control muscle tissue extracts.
RESULTS
Due to a cellular adaptation to hypoxia, a gradual increase in [Ga]Ga-NOTA-c(NGR) and 2-[F]FDG uptake was observed from post-intervention day 1 to 7 in the ischaemic hindlimbs, which was followed by a drop on day 10. Conforming pronounced angiogenic recovery, the NGR accretion of the ischaemic extremities differed significantly from the controls 5, 7, and 10 days after ischaemia induction ( ≤ 0.05), which correlated with the Western blot and immunohistochemical results. No remarkable radioactivity was depicted between the normally perfused hindlimbs of either the ischaemic or the control groups.
CONCLUSIONS
The PET-based longitudinal assessment of angiogenesis-associated APN/CD13 expression and glucose metabolism during ischaemia may continue to broaden our knowledge on the pathophysiology of PAD.
PubMed: 38675203
DOI: 10.3390/pharmaceutics16040542 -
Bioengineering (Basel, Switzerland) Mar 2024Reconstructive techniques to repair severe tissue defects include the use of autologous fasciocutaneous flaps, which may be limited due to donor site availability or...
Reconstructive techniques to repair severe tissue defects include the use of autologous fasciocutaneous flaps, which may be limited due to donor site availability or lead to complications such as donor site morbidity. A number of synthetic or natural dermal substitutes are in use clinically, but none have the architectural complexity needed to reconstruct deep tissue defects. The perfusion decellularization of fasciocutaneous flaps is an emerging technique that yields a scaffold with the necessary composition and vascular microarchitecture and serves as an alternative to autologous flaps. In this study, we show the perfusion decellularization of porcine fasciocutaneous flaps using sodium dodecyl sulfate (SDS) at three different concentrations, and identify that 0.2% SDS results in a decellularized flap that is efficiently cleared of its cellular material at 86%, has maintained its collagen and glycosaminoglycan content, and preserved its microvasculature architecture. We further demonstrate that the decellularized graft has the porous structure and growth factors that would facilitate repopulation with cells. Finally, we show the biocompatibility of the decellularized flap using human dermal fibroblasts, with cells migrating as deep as 150 µm into the tissue over a 7-day culture period. Overall, our results demonstrate the promise of decellularized porcine flaps as an interesting alternative for reconstructing complex soft tissue defects, circumventing the limitations of autologous skin flaps.
PubMed: 38671744
DOI: 10.3390/bioengineering11040321 -
Bioresource Technology Jun 2024Currently, the predominant method for the industrial production of 1,3-dihydroxyacetone (DHA) from glycerol involves fed-batch fermentation. However, previous research...
Currently, the predominant method for the industrial production of 1,3-dihydroxyacetone (DHA) from glycerol involves fed-batch fermentation. However, previous research has revealed that in the biocatalytic synthesis of DHA from glycerol, when the DHA concentration exceeded 50 g·L, it significantly inhibited microbial growth and metabolism, posing a challenge in maintaining prolonged and efficient catalytic production of DHA. In this study, a new integrated continuous production and synchronous separation (ICSS) system was constructed using hollow fiber columns and perfusion culture technology. Additionally, a cell reactivation technique was implemented to extend the biocatalytic ability of cells. Compared with fed-batch fermentation, the ICSS system operated for 360 h, yielding a total DHA of 1237.8 ± 15.8 g. The glycerol conversion rate reached 97.7 %, with a productivity of 3.44 g·L·h, representing 485.0 % increase in DHA production. ICSS system exhibited strong operational characteristics and excellent performance, indicating significant potential for applications in industrial bioprocesses.
Topics: Dihydroxyacetone; Cells, Immobilized; Glycerol; Bioreactors; Fermentation; Batch Cell Culture Techniques; Perfusion; Catalysis; Biocatalysis
PubMed: 38670288
DOI: 10.1016/j.biortech.2024.130734 -
Redox Biology Jun 2024Protein disulfide isomerases (PDIs) are involved in many intracellular and extracellular processes, including cell adhesion and cytoskeletal reorganisation, but their...
Protein disulfide isomerases (PDIs) are involved in many intracellular and extracellular processes, including cell adhesion and cytoskeletal reorganisation, but their contribution to the regulation of fenestrations in liver sinusoidal endothelial cells (LSECs) remains unknown. Given that fenestrations are supported on a cytoskeleton scaffold, this study aimed to investigate whether endothelial PDIs regulate fenestration dynamics in primary mouse LSECs. PDIA3 and PDIA1 were found to be the most abundant among PDI isoforms in LSECs. Taking advantage of atomic force microscopy, the effects of PDIA1 or PDIA3 inhibition on the fenestrations in LSECs were investigated using a classic PDIA1 inhibitor (bepristat) and novel aromatic N-sulfonamides of aziridine-2-carboxylic acid derivatives as PDIA1 (C-3389) or PDIA3 (C-3399) inhibitors. The effect of PDIA1 inhibition on liver perfusion was studied in vivo using dynamic contrast-enhanced magnetic resonance imaging. Additionally, PDIA1 inhibitors were examined in vitro in LSECs for effects on adhesion, cytoskeleton organisation, bioenergetics, and viability. Inhibition of PDIA1 with bepristat or C-3389 significantly reduced the number of fenestrations in LSECs, while inhibition of PDIA3 with C-3399 had no effect. Moreover, the blocking of free thiols by the cell-penetrating N-ethylmaleimide, but not by the non-cell-penetrating 4-chloromercuribenzenesulfonate, resulted in LSEC defenestration. Inhibition of PDIA1 did not affect LSEC adhesion, viability, and bioenergetics, nor did it induce a clear-cut rearrangement of the cytoskeleton. However, PDIA1-dependent defenestration was reversed by cytochalasin B, a known fenestration stimulator, pointing to the preserved ability of LSECs to form new pores. Importantly, systemic inhibition of PDIA1 in vivo affected intra-parenchymal uptake of contrast agent in mice consistent with LSEC defenestration. These results revealed the role of intracellular PDIA1 in the regulation of fenestration dynamics in LSECs, and in maintaining hepatic sinusoid homeostasis.
Topics: Animals; Male; Mice; Cell Adhesion; Cells, Cultured; Cytoskeleton; Endothelial Cells; Enzyme Inhibitors; Liver; Protein Disulfide-Isomerases
PubMed: 38669864
DOI: 10.1016/j.redox.2024.103162