Co-culture Of Endothelial Cells Research Articles

3-HKA Promotes Vascular Remodeling After Stroke by Modulating the Activation of A1/A2 Reactive Astrocytes.

Ischemic stroke is the most common cerebrovascular disease and the leading cause of permanent disability worldwide. Recent studies have shown that stroke development and prognosis are closely related to abnormal tryptophan metabolism. Here, significant downregulation of 3-hydroxy-kynurenamine (3-HKA) in stroke patients and animal models is identified. Supplementation with 3-HKA improved long-term neurological recovery, reduced infarct volume, and increased ipsilateral cerebral blood flow after distal middle cerebral artery occlusion (MCAO). 3-HKA promoted angiogenesis, functional blood vessel formation, and blood-brain barrier (BBB) repair. Moreover, 3-HKA inhibited A1-like (neurotoxic) astrocyte activation but promoted A2-like (neuroprotective) astrocyte polarization. Proteomic analysis revealed that 3-HKA inhibited AIM2 inflammasome activation after stroke, and co-labeling studies indicated that AIM2 expression typically increased in astrocytes at 7 and 14 days after stroke. Consistently, in co-cultures of primary mouse brain microvascular endothelial cells and astrocytes, 3-HKA promoted angiogenesis after oxygen-glucose deprivation (OGD). AIM2 overexpression in astrocytes abrogated 3-HKA-driven vascular remodeling in vitro and in vivo, suggesting that 3-HKA may regulate astrocyte-mediated vascular remodeling by impeding AIM2 inflammasome activation. In conclusion, 3-HKA may promote post-stroke vascular remodeling by regulating A1/A2 astrocyte activation, thereby improving long-term neurological recovery, suggesting that supplementation with 3-HKA may be an efficient therapy for stroke.

In vivo vessel connection of pre-vascularised 3D-bioprinted gingival connective tissue substitutes.

Producing oral soft tissues using tissue engineering could compensate for the disadvantages of autologous grafts (limited availability and increased patient morbidity) and currently available substitutes (shrinkage). However, there is a lack of in vitro-engineered oral tissues due to the difficulty of obtaining stable pre-vessels that connect to the host and enable graft success. The main objective was to assess the connection of pre-vascularised 3D-bioprinted gingival substitutes to the host vasculature when subcutaneously implanted in immunodeficient mice. This study produced vascularised connective tissue substitutes using extrusion-based 3D-bioprinting of primary human gingival fibroblasts (hGF) and fluorescent human endothelial cells (RFP-HUVEC) cocultures. Pre-vascularised (hGF+RFP-HUVEC -CC grids) and control (hGF only -HG grids) grids were bioprinted and pre-cultivated for 14 days to enable pre-vessels formation. In vitro vessel formation follow-up was performed. Eight-week-old female NOG mice were used for in vivo experiments. One grid per mouse was subcutaneously implanted in 20 mice (10HG/10CC). The fluorescent activity of RFP-HUVEC was monitored. Samples were retrieved at 7, 14 and 21 days. Histological, immunohistochemical, and immunofluorescent staining was performed. CC-grids formed efficient and stable pre-vessel networks within 14 days of static pre-culture. HG-grids did not contain any vessel, while CC-grids successfully connected to the host vasculature by presenting erythrocytes within the vessel lumen inside the grids starting day 7. From days 7 to 21, vessel density was stable. Human pre-vessels were present at 7 days and were progressively replaced by murine endothelial cells. This study showed that primary hGF-HUVEC co-cultures can be successfully 3D-bioprinted within biomimetic hydrogels having a close composition to the gingival connective tissue, and HUVEC organise themselves into pre-vessel networks that connect to the murine vasculature when implanted in vivo. This approach represents a promising strategy to enhance current and future oral soft tissue substitutes for prospective clinical applications.

Selective Gamma-Secretase Inhibition by CHF5074 Attenuates Inflammation and Neovascularization in a Murine Model of Choroidal Neovascularization

Purpose Chronic inflammation plays an important role in the pathogenesis of choroidal neovascularization (CNV). This study aimed to investigate the effect of the CHF5074, a γ-secretase inhibitor, on angiogenesis in a laser-induced CNV model and elucidate its possible molecular mechanism. Methods Male C57/BL6J mice aged between 6 to 8 weeks were employed to set up a laser-induced model of CNV. Then, CHF5074 was injected intraperitoneally on the day after laser modeling, as well as on the second, third, and fourth days. Immunofluorescence staining was used to evaluate the retinal and choroidal complex. The markers used were CD31 for neovascularization and IBA1 for microglia staining in ocular tissue slices. Fundus fluorescein angiography on days 3d, 7d, and 14d analyzed neovascularization and leakage areas. Inflammatory indicators were examined by Western blot (WB) and enzyme-linked immunosorbent assay (ELISA). High-throughput whole-tissue sequencing of retinal choroids identified relevant cell pathways. Key regulatory factors modulated by CHF5074 were identified via WB. Co-culture of BV2 cells and human umbilical vein endothelial cells (HUVEC) were used to explore the function of CHF5074 on the inhibition of tube formation. Results CHF5074 significantly decreased CD31 expression in the choroid on 3d, 7d, and 14d post-laser modeling (p < 0.05) and decreased both neovascularization and leakage areas (p < 0.05). Additionally, CHF5074 significantly lowered TNF-α, IL-10, and IL-1β expression levels in the choroid (p < 0.05), as demonstrated by WB analysis and ELISA. High-throughput whole-tissue sequencing identified P38-MAPK, JNK, and Wnt signaling pathways associated with neovascularization. CHF5074 decreased P38 protein phosphorylation (p < 0.05) as confirmed by WB analysis. CHF5074 inhibited the tube formation of HUVECs co-cultured with LPS and ATP-treated BV2 cells. Conclusion CHF5074 significantly suppresses angiogenesis in laser-induced choroidal neovascularization models, suggesting its potential as a novel agent for preventing and treating CNV.

Development of Prevascularized Synthetic Block Graft for Maxillofacial Reconstruction.

Cranio-maxillofacial bone reconstruction, especially for large defects, remains challenging. Synthetic biomimetic materials are emerging as alternatives to autogenous grafts. Tissue engineering aims to create natural tissue-mimicking materials, with calcium phosphate-based scaffolds showing promise for bone regeneration applications. This study developed a porous calcium metaphosphate (CMP) scaffold with physicochemical properties mimicking natural bone, aiming to create a prevascularized synthetic bone graft. The scaffold, fabricated using sintered monocalcium phosphate with poly (vinyl alcohol) as a porogen, exhibited pore sizes ranging from 0 to 400 μm, with the highest frequency between 80 and 100 μm. The co-culture of endothelial cells (ECs) with human alveolar osteoblasts (aHOBs) on the scaffold led to the formation of tube-like structures and intrinsic VEGF release, reaching 10,455.6 pg/mL This level approached the optimal dose for vascular formation. Conversely, the co-culture with mesenchymal stem cells did not yield similar results. Combining ECs and aHOBs in the CMP scaffold offers a promising approach to developing prevascularized grafts for cranio-maxillofacial reconstruction. This innovative strategy can potentially enhance vascularization in large tissue-engineered constructs, addressing a critical limitation in current bone regeneration techniques. The prevascularized synthetic bone graft developed in this study could significantly improve the success rate of maxillofacial reconstructions, offering a viable alternative to autogenous grafts.

Down-regulation of platelet-derived growth factor receptor β in pericytes increases blood-brain barrier permeability and significantly enhances α-synuclein in a Parkinson's Disease 3D cell model in vitro under hyperglycemic condition.

Parkinson's Disease (PD) often presents with a compromised blood-brain barrier (BBB), which hyperglycemia may exacerbate. Pericytes, a key cell for BBB integrity, are potential therapeutic targets for neurodegenerative disorders. Few studies have developed 3D PD cell models incorporating neurovascular units (NVU) through the co-culture of human endothelial, pericytes, astrocytes, and SH-SY5Y cells to evaluate BBB impairment and the role of pericytes under hyperglycemic condition. A 3D PD like cell model was developed using 6-OHDA-affected SH-SY5Y cells, combined with endothelial cells, pericytes, and astrocytes through the Real Architecture for Tissue (RAFT) 3D co-culture system. PD incorporating reduced (30 % and 89 %) PDGFRβ NVU (RPN) with or without hyperglycemic model (HM) were also established. BBB permeability to sodium fluorescein was assessed, and BBB impairment was evaluated using BBB-associated proteins (ZO-1, CD54, CD144), cell-specific proteins (CD31, GFAP, PDGFRβ, CD13), tyrosine hydroxylase (TH), α-synuclein, oligomeric α-synuclein, and α-synuclein (ser9). PD 3D cell models incorporating RPN with or without hyperglycemia were successfully established in vitro. Graduately increased BBB impairment was observed in PD, PD with RPN, and PD with RPN combined with HM, indicated by decreased BBB-associated and cell-specific proteins, reduced TH, and increased α-synuclein, oligomeric α-synuclein, and α-synuclein (ser9) compared to the NVU model. Reduced pericyte PDGFRβ could increase BBB permeability, accelerate PD progression, and exacerbate under hyperglycemic condition.

Vascularized tumor on a microfluidic chip to study mechanisms promoting tumor neovascularization and vascular targeted therapies.

The cascade of events leading to tumor formation includes induction of a tumor supporting neovasculature, as a primary hallmark of cancer. Developing vasculature is difficult to evaluate in vivo but can be captured using microfluidic chip technology and patient derived cells. Herein, we established an on chip approach to investigate the mechanisms promoting tumor vascularization and vascular targeted therapies via co-culture of cancer spheroids and endothelial cells in a three dimensional environment. Methods: We investigated both, tumor neovascularization and therapy, via co-culture of human derived endothelial cells and adjacently localized metastatic renal cell carcinoma spheroids on a commercially available microfluidic chip system. Metastatic renal cell carcinoma spheroids adjacent to primary vessels model tumor, and induce vessels to sprout neovasculature towards the tumor. We monitored real time changes in vessel formation, probed the interactions of tumor and endothelial cells, and evaluated the role of important effectors in tumor vasculature. In addition to wild type endothelial cells, we evaluated endothelial cells that overexpress Prostate Specific Membrane Antigen (PSMA), that has emerged as a marker of tumor associated neovasculature. We characterized the process of neovascularization on the microfluidic chip stimulated by enhanced culture medium and the investigated metastatic renal cell carcinomas, and assessed endothelial cells responses to vascular targeted therapy with bevacizumab via confocal microscopy imaging. To emphasize the potential clinical relevance of metastatic renal cell carcinomas on chip, we compared therapy with bevacizumab on chip with an in vivo model of the same tumor. Results: Our model permitted real-time, high-resolution observation and assessment of tumor-induced angiogenesis, where endothelial cells sprouted towards the tumor and mimicked a vascular network. Bevacizumab, an antiangiogenic agent, disrupted interactions between vessels and tumors, destroying the vascular network. The on chip approach enabled assessment of endothelial cell biology, vessel's functionality, drug delivery, and molecular expression of PSMA. Conclusion: Observations in the vascularized tumor on chip permitted direct and conclusive quantification of vascular targeted therapies in weeks as opposed to months in a comparable animal model, and bridged the gap between in vitro and in vivo models.

IPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease.

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC's, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

EV-Elute: A universal platform for the enrichment of functional surface marker-defined extracellular vesicle subpopulations.

Intercellular communication via extracellular vesicles (EVs) has been identified as a vital component of a steadily expanding number of physiological and pathological processes. To accommodate these roles, EVs have highly heterogeneous molecular compositions. Given that surface molecules on EVs determine their interactions with their environment, EV functionality likely differs between subpopulations with varying surface compositions. However, it has been technically challenging to examine such functional heterogeneity due to a lack of non-destructive methods to separate EV subpopulations based on their surface markers. Here, we used the Design-of-Experiments (DoE) methodology to optimize a protocol, which we name 'EV-Elute', to elute intact EVs from commercially available Protein G-coated magnetic beads. We captured EVs from various cell types on these beads using antibodies against CD9, CD63, CD81 and a custom-made protein binding phosphatidylserine (PS). When applying EV-Elute, over 70% of bound EVs could be recovered from the beads in a pH- and incubation-time-dependent fashion. EV subpopulations showed intact integrity by electron microscopy and Proteinase K protection assays and showed uptake patterns similar to whole EV isolates in co-cultures of peripheral blood mononuclear cells (PBMCs) and endothelial cells. However, in Cas9/sgRNA delivery assays, CD63+ EVs showed a lower capacity to functionally deliver cargo as compared to CD9+, CD81+ and PS+ EVs. Taken together, we developed a novel, easy-to-use platform to isolate and functionally compare surface marker-defined EV subpopulations. This platform does not require specialized equipment or reagents and is universally applicable to any capturing antibody and EV source. Hence, EV-Elute can open new opportunities to study EV functionality at the subpopulation level.

Engineering Granular Hydrogels without Interparticle Cross-Linking to Support Multicellular Organization.

Advancing three-dimensional (3D) tissue constructs is central to creating in vitro models and engineered tissues that recapitulate biology. Materials that are permissive to cellular behaviors, including proliferation, morphogenesis of multicellular structures, and motility, will support the emergence of tissue structures. Granular hydrogels in which there is no interparticle cross-linking exhibit dynamic properties that may be permissive to such cellular behaviors. However, designing granular hydrogels that lack interparticle cross-linking but support cellular self-organization remains underexplored relative to granular systems stabilized by interparticle cross-linking. In this study, we developed a polyethylene glycol-based granular hydrogel system, with average particle diameters under 40 μm. This granular hydrogel exhibited bulk stress-relaxing behaviors and compatibility with custom microdevices to sustain cell cultures without degradation. The system was studied in conjunction with cocultures of endothelial cells and fibroblasts, known for their spontaneous network formation. Cross-linking, porosity, and cell-adhesive ligands (such as RGD) were manipulated to control system properties. Toward supporting cellular activity, increased porosity was found to enhance the formation of cellular networks, whereas RGD reduced network formation in the system studied. This research highlights the potential of un-cross-linked granular systems to support morphogenetic processes, like vasculogenesis and tissue maturation, offering insights into material design for 3D cell culture systems.

Inhibition of soluble epoxide hydrolase reverses bone loss in periodontitis by upregulating EMCN and inhibiting osteoclasts

BackgroundImproving the microenvironment to augment endogenous regenerative potential has emerged as a fundamental concept for stimulating and expediting periodontal tissue repair and regeneration. Previous studies have demonstrated that TPPU, a soluble epoxide hydrolase inhibitor (sEHi), mediates the suppression of inflammatory bone loss in periodontitis models. However, the underlying mechanisms remain largely elusive.MethodsIn this study, we constructed a human umbilical vein endothelial cell (HUVEC) and periodontal ligament stem cell (PDLSC) coculture system in vitro and tested the anti-inflammatory effect of TPPU under inflammatory conditions. The roles of HIF-1α and Endomucin (EMCN) in the anti-inflammatory effects of TPPU were analyzed. The effects of TPPU on osteogenesis and osteoclastogenesis in cocultured cells were examined. The in vivo periodontitis model further verified the effects of TPPU on inhibiting neutrophil adhesion and inflammation and inhibiting osteoclasts.ResultsOur in vitro experiments demonstrated that TPPU enhances the interaction between mesenchymal stem cells and vascular endothelial cells to enhance anti-inflammatory and osteogenic differentiation effects and revealed a new anti-inflammatory mechanism of TPPU involving the upregulation of EMCN in endothelial cells to prevent lymphocyte recruitment. We also confirmed that TPPU inhibits osteoclast activity. Our in vivo findings showed that TPPU inhibits osteoclast activity and neutrophil adhesion and enhances periodontal tissue repair and regeneration.ConclusionsTPPU promotes local regeneration in periodontitis by inhibiting inflammation and bone resorption. Thus, targeting soluble epoxide hydrolase represents an endogenous regenerative strategy for periodontitis treatment.

Engineering Biomimetic Microvascular Capillary Networks in Hydrogel Fibrous Scaffolds via Microfluidics-Assisted Co-Axial Wet-Spinning.

The microvascular bed plays a crucial role in establishing nutrient exchange and waste removal, as well as maintaining tissue metabolic activity in the human body. However, achieving microvascularization of engineered 3D tissue constructs is still an unsolved challenge. In this work, we developed biomimetic cell-laden hydrogel microfibers recapitulating oriented microvascular capillary-like networks by using a 3D bioprinting technique combined with microfluidics-assisted coaxial wet-spinning. Highly packed and aligned bundles embedding a coculture of human bone marrow-derived mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) were produced by simultaneously extruding two different bioinks. To this aim, core-shell fibers were wet-spun in a coagulation bath to collect the scaffolds later on a rotary drum. Initially, the versatility of the proposed system was assessed for the extrusion of multimaterial core-shell hydrogel fibers. Subsequently, the platform was validated for the in vitro biofabrication of samples promoting optimal cell alignment along the fiber axis. After 3 weeks of culture, such fiber configuration resulted in the development of an oriented capillary-like network within the fibrin-based core and in the endothelial-specific CD31 marker expression upon MSC/HUVEC maturation. Synergistically, the vertical arrangement of the coaxial nozzle coupled with the rotation of the fiber collector facilitated the rapid creation of tightly packed bundles characterized by a dense, oriented, and extensively branched capillary network. Notably, such findings suggest that the proposed biofabrication strategy can be used for the microvascularization of tissue-specific 3D constructs.

Deciphering influence of donor age on adipose-derived stem cells: in vitro paracrine function and angiogenic potential

Background: As fat grafting is commonly used as a filler, Adipose-derived stem/stromal cells (ASC) have been reported to be key player in retention rate. Paracrine and differentiation potential of those cells confer them strong pro-angiogenic capacities. However, a full characterization of the influence of aging on ASC has not been reported yet. Here we’ve investigated the effect of age on paracrine function, stemness and angiogenic potential of ASC. Methods: ASC were extracted from young and old adult donors. We assessed stromal vascular fraction cell populations repartition, ASC stemness potential, capability to differentiate into mesenchymal lineages as well as their secretome. Angiogenic potential was assessed using a sprouting assay, an indirect co-culture of ASC and dermal microvascular endothelial cells (EC). Total vascular sprout length was measured, and co-culture soluble factors were quantified. Pro-angiogenic factors alone or in combination as well as ASC-conditioned medium (CM) were added to EC to assess sprouting induction. Results: Decrease of endothelial cells yield and percentage is observed in cells extracted from adipose tissue of older patients, whereas ASC percentage increased with age. Clonogenic potential of ASC is stable with age. ASC can differentiate into adipocytes, chondrocytes and osteoblasts, and aging does not alter this potential. Among the 25 analytes quantified, high levels of pro-angiogenic factors were found, but none is significantly modulated with age. ASC induce a significantly longer vascular sprouts compared to fibroblasts, and no difference was found between young and old ASC donors on that parameter. Higher concentrations of FGF-2, G-CSF, HGF and IL-8, and lower concentrations of VEGF-C were quantified in EC/ASC co-cultures compared to EC/fibroblasts co-cultures. EC/ASC from young donors secrete higher levels of VEGF-A compared to old ones. Neither soluble factor nor CM without cells are able to induce organized sprouts, highlighting the requirement of cell communication for sprouting. CM produced by ASC supporting development of long vascular sprouts promote sprouting in co-cultures that establish shorter sprouts. Conclusion: Our results show cells from young and old donors exhibit no difference in all assessed parameters, suggesting all patients could be included in clinical applications. We emphasized the leading role of ASC in angiogenesis, without impairment with age, where secretome is a key but not sufficient actor.

Expression of soluble guanylate cyclase (sGC) and its ability to form a functional heterodimer are crucial for reviving the NO-sGC signaling in PAH

In order to determine the underpinnings of a dysfunctional NO-sGC signal pathway which occurs in pulmonary arterial hypertension (PAH), we investigated pulmonary arterial smooth muscle cells (PASMCs) derived from PAH patients. We found low expression of sGC, a poor sGCα1β1 heterodimer and this correlated with low expression of its facilitator chaperon, hsp90. Treating PASMCs overnight (16 h) with low micromolar doses of a slow release NO donor DETANONOate, reinstated the sGCα1β1 heterodimer and restored its NO-heme dependent activity. Transwell co-culture of HEK cells stably expressing eNOS with PAH PASMCs also restored the sGC heterodimer and its heme-dependent activity with sGC stimulator, BAY 41–2272. To determine whether the dysfunctionality in the NO-sGC pathway stems from a dysfunctional eNOS producing negligible NO, we did transwell co-cultures of pulmonary arterial endothelial cells (PAECs) with PASMCs. Our results indicated that PAECs from both control and PAH samples when activated for eNOS restored both sGC heterodimer and its heme-dependent sGC activity in the corresponding PASMCs, suggesting that PAECs from PAH can also generate NO. In line with these results expression of eNOS, its support chaperon hsp90, its specific kinase Akt, p-Akt or post-translational modifications (PTMs) like OGlcNAc or phospho-tyrosine were unchanged in PAH relative to controls. Additionally there was uniform expression of Hbα/β and Mb in PASMCs or PAECs in PAH or controls and these globins can effectively scavenge the eNOS generated NO, as there was evidence of strong eNOS-Hb/Mb interactions. Our studies suggest that factors such as globin NO scavenging along with vascular remodeling in PAH can cause hampered vasodilation which in the face of poor NO levels as occurs in PAH are additional impediments for effective vasodilation. However importantly our studies suggests that future therapies can use low doses of NO along with sGC stimulators as a potential drug for PAH subjects.

Using Polycaprolactone Nanofibers for the Proof-of-Concept Construction of the Alveolar-Capillary Interface.

The alveolar-capillary interface is the key functional element of gas exchange in the human lung, and disruptions to this interface can lead to significant medical complications. However, it is currently challenging to adequately model this interface invitro, as it requires not only the co-culture of human alveolar epithelial and endothelial cells but mainly the preparation of a biocompatible scaffold that mimics the basement membrane. This scaffold should support cell seeding from both sides, and maintain optimal cell adhesion, growth, and differentiation conditions. Our study investigates the use of polycaprolactone (PCL) nanofibers as a versatile substrate for such cell cultures, aiming to model the alveolar-capillary interface more accurately. We optimized nanofiber production parameters, utilized polyamide mesh UHELON as a mechanical support for scaffold handling, and created 3D-printed inserts for specialized co-cultures. Our findings confirm that PCL nanofibrous scaffolds are manageable and support the co-culture of diverse cell types, effectively enabling cell attachment, proliferation, and differentiation. Our research establishes a proof-of-concept model for the alveolar-capillary interface, offering significant potential for enhancing cell-based testing and advancing tissue-engineering applications that require specific nanofibrous matrices.

Bone Spheroid Development Under Flow Conditions with Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells in a 3D Porous Hydrogel Supplemented with Hydroxyapatite.

Understanding the niche interactions between blood and bone through the in vitro co-culture of osteo-competent cells and endothelial cells is a key factor in unraveling therapeutic potentials in bone regeneration. This can be additionally supported by employing numerical simulation techniques to assess local physical factors, such as oxygen concentration, and mechanical stimuli, such as shear stress, that can mediate cellular communication. In this study, we developed a Mesenchymal Stem Cell line (MSC) and a Human Umbilical Vein Endothelial Cell line (HUVEC), which were co-cultured under flow conditions in a three-dimensional, porous, natural pullulan/dextran scaffold that was supplemented with hydroxyapatite crystals that allowed for the spontaneous formation of spheroids. After 2 weeks, their viability was higher under the dynamic conditions (>94%) than the static conditions (<75%), with dead cells central in the spheroids. Mineralization and collagen IV production increased under the dynamic conditions, correlating with osteogenesis and vasculogenesis. The endothelial cells clustered at the spheroidal core by day 7. Proliferation doubled in the dynamic conditions, especially at the scaffold peripheries. Lattice Boltzmann simulations showed negligible wall shear stress in the hydrogel pores but highlighted highly oxygenated zones coinciding with cell proliferation. A strong oxygen gradient likely influenced endothelial migration and cell distribution. Hypoxia was minimal, explaining high viability and spheroid maturation in the dynamic conditions.

APPLICATION OF IN VITRO MODELS: INCORPORATING BLOOD BRAIN BARRIER MODELS WITH GLIOMA, AND THE USE OF MICROflUIDICS

Abstract AIMS The blood brain barrier (BBB) presents one of the main obstacles for the development of novel glioblastoma treatments. The various transporters and enzymes within the BBB reduce the disposition of novel therapies into the brain. There is a need for an in vitro BBB-glioma model with proven efficacy of predicting CNS delivery at earlier stages in the drug discovery pipeline. METHOD A BBB-glioma lab-on-a-chip model could enable rapid identification of novel therapies that can cross the BBB at earlier stages of the drug development process, before going to clinical trials. The application of microfluidic technology allows for the detection of soluble tissue-derived factors and the assessment of the levels of soluble markers released from malignant tissue. Adding ‘flow’ to the model, is more reflective of the dynamic flow present at the BBB. RESULTS Validation studies of the current BBB model and converting this model over to a lab-on-a-chip model have shown the presence of an endothelial cell barrier and the tight junction proteins Zo-1 and Claudin-1, through western blotting, immunocytochemistry, and permeability experiments using varying molecular weights of FitC-dextran. These validation studies have been completed using brain endothelial monolayers on polycarbonate transwell inserts, with the immortalised cell line HCMEC/d3, and the short-term culture cells HBMEC. Also, validation studies using a co-culture of endothelial cells, astrocytes and pericytes have begun, using the same techniques to show a barrier has formed, on both the current BBB model and the lab-on-a-chip model. CONCLUSION The aim of this study is to combine the use of an all-human BBB-glioma model with lab-on-a-chip technology to produce a high throughput model to test novel therapies at an earlier stage. The lab-on-a-chip model will be validated for the purpose of measuring permeability and drug response and to investigate the expression of novel targets verses expression in conventional models.

Zinc and gallium doped borate bioactive glasses influence in-vitro angiogenesis: New evidence in cell co-culture studies

Borate bioactive glasses doped with 1 mol% ZnO or Ga2O3 were produced by melt-quenching. The cytocompatibility of fibroblast and endothelial cells in response to glass extracts was assessed. The angiogenic potential was examined through fibroblast-derived vascular endothelial growth factor secretion and tubular formation analysis in an indirect co-culture of fibroblast and endothelial cells. The results confirm that Zn and Ga ion-releasing bioactive glasses are attractive biomaterials with angiogenic properties.

Bruton's tyrosine kinase inhibition suppresses pathological retinal angiogenesis.

Pathological retinal angiogenesis is a typical manifestation of vision-threatening ocular diseases. Many patients exhibit poor response or resistance to anti-vascular endothelial growth factor (VEGF) agents. Bruton's tyrosine kinase (BTK) controls the proliferation and function of immune cells. Therefore, we examined the anti-inflammatory and anti-angiogenic effects of BTK inhibition on retinal angiogenesis. Retinal neovascularisation and vascular leakage in oxygen-induced retinopathy in C57/BL6J mice were assessed by whole-mount retinal immunofluorescence. PLX5622 was used to deplete microglia and Rag1-knockout mice were used to test the contribution of lymphocytes to the effects of BTK inhibition. The cytokines, activation markers, inflammatory and immune-regulatory activities of retinal microglia/macrophages were detected using qRT-PCR and immunofluorescence. NLRP3 was detected by western blotting, and the effects of BTK inhibition on the co-culture of microglia and human retinal microvascular endothelial cells (HRMECs) were examined. BTK inhibition suppressed pathological angiogenesis and vascular leakage, and significantly reduced retinal inflammation, which involved microglia/macrophages but not lymphocytes. BTK inhibition increased anti-inflammatory factors and reduced pro-inflammatory cytokines that resulted from NLRP3 inflammasome activation. BTK inhibition suppressed the inflammatory activity of microglia/macrophages, and acted synergistically with anti-VEGF without retinal toxicity. Moreover, the supernatant of microglia incubated with BTK-inhibitor reduced the proliferation, tube formation and sprouting of HRMECs. BTK inhibition suppressed retinal neovascularisation and vascular leakage by modulating the inflammatory activity of microglia and macrophages. Our study suggests BTK inhibition as a novel and promising approach for alleviating pathological retinal angiogenesis.

Modelling the maternal-fetal interface: An invitro approach to investigate nutrient and drug transport across the human placenta.

The placenta plays a critical role in maternal-fetal nutrient transport and fetal protection against drugs. Creating physiological invitro models to study these processes is crucial, but technically challenging. This study introduces an efficient cell model that mimics the human placental barrier using co-cultures of primary trophoblasts and primary human umbilical vein endothelial cells (HUVEC) on a Transwell®-based system. Monolayer formation was examined over 7 days by determining transepithelial electrical resistance (TEER), permeability of Lucifer yellow (LY) and inulin, localization of transport proteins at the trophoblast membrane (immunofluorescence), and syncytialization markers (RT-qPCR/ELISA). We analysed diffusion-based (caffeine/antipyrine) and transport-based (leucine/Rhodamine-123) processes to study the transfer of physiologically relevant compounds. The latter relies on the adequate localization and function of the amino-acid transporter LAT1 and the drug transporter P-glycoprotein (P-gp) which were studied by immunofluorescence microscopy and application of respective inhibitors (2-Amino-2-norbornanecarboxylic acid (BCH) for LAT1; cyclosporine-A for P-gp). The formation of functional monolayer(s) was confirmed by increasing TEER values, low LY transfer rates, minimal inulin leakage, and appropriate expression/release of syncytialization markers. These results were supported by microscopic monitoring of monolayer formation. LAT1 was identified on the apical and basal sides of the trophoblast monolayer, while P-gp was apically localized. Transport assays confirmed the inhibition of LAT1 by BCH, reducing both intracellular leucine levels and leucine transport to the basal compartment. Inhibiting P-gp with cyclosporine-A increased intracellular Rhodamine-123 concentrations. Our invitro model mimics key aspects of the human placental barrier. It represents a powerful tool to study nutrient and drug transport mechanisms across the placenta, assisting in evaluating safer pregnancy therapies.

Empowering High-Throughput High-Content Analysis of Microphysiological Models: Open-Source Software for Automated Image Analysis of Microvessel Formation and Cell Invasion.

The primary aim of this study was to develop an open-source Python-based software for the automated analysis of dynamic cell behaviors in microphysiological models using non-confocal microscopy. This research seeks to address the existing gap in accessible tools for high-throughput analysis of endothelial tube formation and cell invasion in vitro, facilitating the rapid assessment of drug sensitivity. Our approach involved annotating over 1000 2mm Z-stacks of cancer and endothelial cell co-culture model and training machine learning models to automatically calculate cell coverage, cancer invasion depth, and microvessel dynamics. Specifically, cell coverage area was computed using focus stacking and Gaussian mixture models to generate thresholded Z-projections. Cancer invasion depth was determined using a ResNet-50 binary classification model, identifying which Z-planes contained invaded cells and measuring the total invasion depth. Lastly, microvessel dynamics were assessed through a U-Net Xception-style segmentation model for vessel prediction, the DisPerSE algorithm to extract an embedded graph, then graph analysis to quantify microvessel length and connectivity. To further validate our software, we reanalyzed an image set from a high-throughput drug screen involving a chemotherapy agent on a 3D cervical and endothelial co-culture model. Lastly, we applied this software to two naive image datasets from coculture lumen and microvascular fragment models. The software accurately measured cell coverage, cancer invasion, and microvessel length, yielding drug sensitivity IC50 values with a 95% confidence level compared to manual calculations. This approach significantly reduced the image processing time from weeks down to h. Furthermore, the software was able to calculate cell coverage, microvessel length, and invasion depth from two additional microphysiological models that were imaged with confocal microscopy, highlighting the versatility of the software. Our free and open source software offers an automated solution for quantifying 3D cell behavior in microphysiological models assessed using non-confocal microscopy, providing the broader Cellular and Molecular Bioengineering community with an alternative to standard confocal microscopy paired with proprietary software.This software can be found in our GitHub repository: https://github.com/fogg-lab/tissue-model-analysis-tools. The online version contains supplementary material available at 10.1007/s12195-024-00821-2.