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Biotechnology and Bioengineering May 2024The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of...
The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes.
PubMed: 38715197
DOI: 10.1002/bit.28730 -
Placenta Apr 2024The study of very early human placentation is largely limited due to ethical restrictions on the use of embryonic tissue and the fact that the placental anatomy of...
The study of very early human placentation is largely limited due to ethical restrictions on the use of embryonic tissue and the fact that the placental anatomy of common laboratory animal models varies considerably from that of humans. In recent years several promising models, including trophoblast stem cell-derived organoids, have been developed that have also proven useful for the study of important trophoblast differentiation processes. However, the consideration of maternal blood flow in trophoblast invasion models currently appears to be limited to animal models. An almost forgotten model to study the invasive behavior of trophoblasts is to culture them in vitro on the chicken chorioallantoic membrane (CAM), showing an extraembryonic vascular network in its mesenchymal stroma that is continuously perfused by the chicken embryonic blood circulation. Here, we present an extension of the previously described ex ovo CAM assay and describe the use of cavity-bearing trophoblast spheroids obtained from the first trimester cell line ACH-3P. We demonstrate how spheroids penetrated the CAM and that erosion of CAM vessels by trophoblasts led to filling of the spheroid cavities with chicken blood, mimicking initial steps of intervillous space blood perfusion. Moreover, we prove that this model is useful for state-of-the-art techniques including immunofluorescence and in situ padlock probe hybridization, making it a versatile tool to study aspects of trophoblast invasion in presence of blood flow.
PubMed: 38705802
DOI: 10.1016/j.placenta.2024.04.013 -
Biotechnology and Bioengineering May 2024The growing demand for biological therapeutics has increased interest in large-volume perfusion bioreactors, but the operation and scalability of perfusion membranes...
The growing demand for biological therapeutics has increased interest in large-volume perfusion bioreactors, but the operation and scalability of perfusion membranes remain a challenge. This study evaluates perfusion cell culture performance and monoclonal antibody (mAb) productivity at various membrane fluxes (1.5-5 LMH), utilizing polyvinylidene difluoride (PVDF), polyethersulfone (PES), or polysulfone (PS) membranes in tangential flow filtration mode. At low flux, culture with PVDF membrane maintained higher cell culture growth, permeate titer (1.06-1.34 g/L) and sieving coefficients (≥83%) but showed lower permeate volumetric throughput and higher transmembrane pressure (TMP) (>1.50 psi) in the later part of the run compared to cultures with PES and PS membrane. However, as permeate flux increased, the total mass of product decreased by around 30% for cultures with PVDF membrane, while it remained consistent with PES and PS membrane, and at the highest flux studied, PES membrane generated 12% more product than PVDF membrane. This highlights that membrane selection for large-volume perfusion bioreactors depends on the productivity and permeate flux required. Since operating large-volume perfusion bioreactors at low flux would require several cell retention devices and a complex setup, PVDF membranes are suitable for low-volume operations at low fluxes whereas PES membranes can be a desirable alternative for large-volume higher demand products at higher fluxes.
PubMed: 38702962
DOI: 10.1002/bit.28722 -
Journal of Experimental & Clinical... May 2024Peritoneal metastases from colorectal cancer (CRCPM) are related to poor prognosis. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC)...
Colorectal carcinoma peritoneal metastases-derived organoids: results and perspective of a model for tailoring hyperthermic intraperitoneal chemotherapy from bench-to-bedside.
BACKGROUND
Peritoneal metastases from colorectal cancer (CRCPM) are related to poor prognosis. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) have been reported to improve survival, but peritoneal recurrence rates are still high and there is no consensus on the drug of choice for HIPEC. The aim of this study was to use patient derived organoids (PDO) to build a relevant CRCPM model to improve HIPEC efficacy in a comprehensive bench-to-bedside strategy.
METHODS
Oxaliplatin (L-OHP), cisplatin (CDDP), mitomycin-c (MMC) and doxorubicin (DOX) were used to mimic HIPEC on twelve PDO lines derived from twelve CRCPM patients, using clinically relevant concentrations. After chemotherapeutic interventions, cell viability was assessed with a luminescent assay, and the obtained dose-response curves were used to determine the half-maximal inhibitory concentrations. Also, induction of apoptosis by different HIPEC interventions on PDOs was studied by evaluating CASPASE3 cleavage.
RESULTS
Response to drug treatments varied considerably among PDOs. The two schemes with better response at clinically relevant concentrations included MMC alone or combined with CDDP. L-OHP showed relative efficacy only when administered at low concentrations over a long perfusion period. PDOs showed that the short course/high dose L-OHP scheme did not appear to be an effective choice for HIPEC in CRCPM. HIPEC administered under hyperthermia conditions enhanced the effect of chemotherapy drugs against cancer cells, affecting PDO viability and apoptosis. Finally, PDO co-cultured with cancer-associated fibroblast impacted HIPEC treatments by increasing PDO viability and reducing CASPASES activity.
CONCLUSIONS
Our study suggests that PDOs could be a reliable in vitro model to evaluate HIPEC schemes at individual-patient level and to develop more effective treatment strategies for CRCPM.
Topics: Humans; Colorectal Neoplasms; Peritoneal Neoplasms; Hyperthermic Intraperitoneal Chemotherapy; Organoids
PubMed: 38698446
DOI: 10.1186/s13046-024-03052-5 -
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