Co-culture Of Cells Research Articles

Bone cells influence the degradation interface of pure Mg and WE43 materials: Insights from multimodal in vitro analysis

In this study, the interaction of pure Mg and WE43 alloy under the presence of osteoblast (OB) and osteoclast (OC) cells and their influence on the degradation of materials have been deeply analyzed. Since OB and OC interaction has an important role in bone remodeling, we examined the surface morphology and dynamic changes in the chemical composition and thickness of the corrosion layers formed on pure Mg and WE43 alloy by direct monoculture and coculture of pre-differentiated OB and OC cells in vitro. Electrochemical techniques examined the corrosion performance. The corrosion products were characterized using a combination of the focused ion beam (FIB), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). Cell viability and morphology were assessed by fluorescent microscopy and SEM. Our findings demonstrate cell spread and attachment variations, which differ depending on the Mg substrates. It was clearly shown that cell culture groups delayed degradation processes with the lowest corrosion rate observed in the presence of OBOC coculture for the WE43 substrate. Ca-P enrichment was observed in the outer-middle region of the corrosion layer but only after 7 days of OBOC coculture on WE43 and after 14 days on the pure Mg specimens. Statement of significanceMagnesium metallic materials that can degrade over time provide distinct opportunities for orthopedic application. However, there is still a lack, especially in elucidating cell-material interface characterization. This study investigated the influence of osteoblast-osteoclast coculture in direct Mg-material contact. Our findings demonstrated that pre-differentiated osteoblasts and osteoclasts cocultured on Mg substrates influenced the chemistry of the corrosion layers. The cell spread and attachment were Mg substrate-dependent. The findings of coculturing bone cells directly on Mg materials within an in vitro model provide an effective approach for studying the dynamic degradation processes of Mg alloys while also elucidating cell behavior and their potential contribution to the degradation of these alloys.

Dialog between mantle cell lymphoma cells and lymphoma-associated macrophages underlies ibrutinib resistance

IntroductionPatients with mantle cell lymphoma (MCL) frequently develop resistance to ibrutinib. Lymphoma-associated macrophages (LAMs) may play a causal role in this resistance but remain underexplored in current literature. ObjectivesTo elucidate the role of LAMs in mediating ibrutinib resistance in MCL. MethodsWe investigated macrophage polarization through multiparameter flow cytometry (MPFC) using antibodies against CD206 and CD86 in blood and tissue samples from patients with MCL, both resistant and sensitive to ibrutinib. Subsequently, we developed an in vitro co-culture model utilizing MCL cell lines to identify cytokines associated with ibrutinib resistance and macrophage M2 polarization. The mechanisms underlying resistance were examined using MPFC, RNA sequencing, and Western blot analysis. Additionally, we assessed whether SB225002, a CXCR2 inhibitor, could reverse ibrutinib resistance through CCK-8 and caspase-3 assays, as well as in a mouse xenograft model involving an ibrutinib-resistant MCL cell line. ResultsIn patients exhibiting ibrutinib resistance, the ratio of M2 to M1 LAMs was significantly higher compared to sensitive patients. In co-cultures of LAMs and MCL cells, the percentage of M2 macrophages, the IC50 value for ibrutinib, and the concentrations of IL-8 and CXCL5 were significantly elevated. Mechanistically, CXCL5 secreted by LAMs interacted with the CXCR2 on MCL cells, leading to the activation of the Akt, p38, and STAT3 signaling pathways in the presence of ibrutinib; this activity was diminished upon blockade of the CXCL5/CXCR2 axis. The combination of SB225002 and ibrutinib significantly enhanced MCL cell apoptosis, suppressed lymphoma growth in the xenograft model, and reprogrammed macrophage phenotype compared to treatment with ibrutinib alone. ConclusionOur data indicate that M2-polarized LAMs are associated with ibrutinib resistance in a model of MCL, and that a CXCR2 inhibitor can reverse this resistance. These findings suggest a potential new therapeutic strategy.

Follicular Helper T Cells in Peyer's Patches and Galactose- Deficient Iga1 Contribute to Iga Nephropathy.

Common primary glomerulonephritis with aberrant mucosal immunity is IgA nephropathy (IgAN). T follicular helper (TFH) cells are essential in regulating B cell differentiation. Peyer's patches (PPs) are the main site where IgA+ plasmablasts differentiate. Our study aimed to investigate the TFH cell's potential contribution to the etiology of IgA nephropathy. In PPs from IgAN mouse models, the ratio of the TFH cell, B220+IgA+, B220+IgM+, and B220-IgA+ lymphocytes were assessed. Then, we used Western blot to assess the expression of Bcl-6, Blimp- 1, and IL-21 proteins in PPs and used RTPCR to assess the expression of IL-21 and TGF-β1 mRNA. TFH cells coculture with spleen cells to measure the degree of IL-21 and the ratio of activation marker CD69 on the TFH cells. Naive B cells (CD27-IgD+) from children suffering from IgAN were cultured with TFH cell-related cytokines. The supernatant was detected to assess the excretion of galactose-deficient IgA1 (Gd-IgA1). IgAN mice developed noticeably increased degrees of IL-21 and CD69 on TFH cells than controls did, as well as higher percentages of B220+IgA+, B220+IgM+, B220+IgA+, TGF- β1, and IL-21 mRNA and Bcl-6, IL-21 proteins in PPs. The Gd-IgA1 level in the supernatant and IgAN- positive children's serum were noticeably higher than those of the healthy controls (P < 0.05). PPs provide the microenvironment to induce the production of IgA-secreting plasmablasts. TFH cells may be a key moderator to induce B cell differentiation into IgAsecreting plasmablasts and produce Gd-IgA1, which plays a significant part in IgAN's pathogenesis. It could be a new therapeutic target in the future.

A three-dimensional valve-on-chip microphysiological system implicates cell cycle progression, cholesterol metabolism and protein homeostasis in early calcific aortic valve disease progression

BackgroundCalcific aortic valve disease (CAVD) is one of the most common forms of valvulopathy, with a 50 % elevated risk of a fatal cardiovascular event, and greater than 15,000 annual deaths in North America alone. The treatment standard is valve replacement as early diagnostic, mitigation, and drug strategies remain underdeveloped. The development of early diagnostic and therapeutic strategies requires the fabrication of effective in vitro valve mimetic models to elucidate early CAVD mechanisms. MethodsIn this study, we developed a multilayered physiologically relevant 3D valve-on-chip (VOC) system that incorporated aortic valve mimetic extracellular matrix (ECM), porcine aortic valve interstitial cell (VIC) and endothelial cell (VEC) co-culture and dynamic mechanical stimuli. Collagen and glycosaminoglycan (GAG) based hydrogels were assembled in a bilayer to mimic healthy or diseased compositions of the native fibrosa and spongiosa. Multiphoton imaging and proteomic analysis of healthy and diseased VOCs were performed. ResultsCollagen-based bilayered hydrogel maintained the phenotype of the VICs. Proteins related to cellular processes like cell cycle progression, cholesterol biosynthesis, and protein homeostasis were found to be significantly altered and correlated with changes in cell metabolism in diseased VOCs. This study suggested that diseased VOCs may represent an early, adaptive disease initiation stage, which was corroborated by human aortic valve proteomic assessment. ConclusionsIn this study, we developed a collagen-based bilayered hydrogel to mimic healthy or diseased compositions of the native fibrosa and spongiosa layers. When the gels were assembled in a VOC with VECs and VICs, the diseased VOCs revealed key insights about the CAVD initiation process. Statement of significanceCalcific aortic valve disease (CAVD) elevates the risk of death due to cardiovascular pathophysiology by 50 %, however, prevention and mitigation strategies are lacking, clinically. Developing tools to assess early disease would significantly aid in the prevention of disease and in the development of therapeutics. Previously, studies have utilized collagen and glycosaminoglycan-based hydrogels for valve cell co-cultures, valve cell co-cultures in dynamic environments, and inorganic polymer-based multilayered hydrogels; however, these approaches have not been combined to make a physiologically relevant model for CAVD studies. We fabricated a bi-layered hydrogel that closely mimics the aortic valve and used it for valve cell co-culture in a dynamic platform to gain mechanistic insights into the CAVD initiation process using proteomic and multiphoton imaging assessment.

NNMT switches the proangiogenic phenotype of cancer-associated fibroblasts via epigenetically regulating ETS2/VEGFA axis.

Cancer-associated fibroblasts (CAFs) are known to promote angiogenesis in oral squamous cell carcinoma (OSCC). However, the epigenetic mechanisms through which CAFs facilitate angiogenesis within the tumor microenvironment are still poorly characterized. Nicotinamide N'-methyltransferase (NNMT), a member of the N-methyltransferase family, was found to be a key molecule in the activation of CAFs. This study shows that NNMT in fibroblasts contributes to angiogenesis and tumor growth through an epigenetic reprogramming-ETS2-VEGFA signaling axis in OSCC. Single-cell RNA Sequencing (scRNA-seq) analysis suggests that NNMT is mainly highly expressed in fibroblasts of head and neck squamous cell carcinoma (HNSCC). Moreover, analysis of the TCGA database and multiple immunohistochemical staining of clinical samples also identified a positive correlation between NNMT and tumor angiogenesis. This research further employed an assembled organoid model and a fibroblast-endothelial cell co-culture model to authenticate the proangiogenic ability of NNMT. At the molecular level, high expression of NNMT in CAFs was found to promote ETS2 expression by regulating H3K27 methylation level through mediating methylation deposition. Furthermore, ETS2 was verified to be an activating transcription factor of VEGFA in this study. Collectively, our findings delineate an epigenetic molecular regulatory network of angiogenesis and provide a theoretical basis for exploring new targets and clinical strategy in OSCC.

Chemopreventive effect of modified zeng-sheng-ping on oral squamous cell carcinoma by regulating tumor associated macrophages through targeting tnf alpha induced protein 6

BackgroundOral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck. Zeng-Sheng-Ping, composed of Sophora tonkinensis Gagnep., Bistorta officinalis Delarbre, Sonchus arvensis L., Prunella vulgaris L., Dioscorea bulbifera L., and Dictamnus dasycarpus Turcz., was regarded as an anti-cancer drug with significant clinical efficacy, but was discontinued due to liver toxicity. Our research group developed a modified Zeng-Sheng-Ping (ZSP-M) based on original Zeng-Sheng-Ping that exhibited high efficiency and low toxicity in preliminary investigations, although its pharmacodynamic mechanism is still unclear. Here, we aimed to elucidate the pharmacodynamic material basis of ZSP-M and investigate its chemopreventive effect on OSCC by modulating tumor associated macrophages (TAMs).MethodsComponents of ZSP-M were characterized using ultra-performance liquid chromatography-mass spectrometry. Chemopreventive effect induced by ZSP-M against experimental oral cancer was investigated using the 4-nitroquinoline N-oxide precancerous lesion mouse model. RNA sequencing analysis was used to gain a global transcriptional view of the effect of ZSP-M treatment. A cell co-culture model was used to study the targeted effect of ZSP-M on TAMs and the biological properties of OSCC cells and to detect changes in TAM phenotypes. The binding of ZSP-M active compounds to TNF alpha induced protein 6 (TNFAIP6) protein was analyzed by molecular docking and dynamic simulation.ResultsForty main components of ZSP-M were identified, the most abundant of which were flavonoids. ZSP-M inhibited the degree of epithelial dysplasia in precancerous lesions by inhibiting the expression of the TNFAIP6 and CD163 proteins in the precancerous lesions of the tongue. ZSP-M inhibited proliferation, colony formation, migration and invasion of SCC7 cells by targeting TAMs. ZSP-M reduced the expression of CD163+ cells, inhibited the expression of TNFAIP6 protein, Arg1 mRNA and Il10 mRNA in TAMs, and reduced IL-10 cytokine release in the co-culture environment. This effect was maintained after the addition of recombinant TNFAIP6 protein. Computer simulations showed that trifolirhizin and maackiain are well-connected to TNFAIP6.ConclusionsZSP-M counteracts the immunosuppressive action of TAMs by specific targeting of TNFAIP6, thereby exerting chemopreventive activity of OSCC.

The use of stem cells in treating xerostomia: a systematic review.

The complex nature of xerostomia prevents the establishment of a definite cure. Recently, research has pivoted towards stem cell transplantation for glandular reconstruction. The aim of this study is to provide an updated review of the existing research, to highlight the encountered challenges and research pathways, potentially enhancing the therapeutic applications of stem cell transplantation. This is a systematic review according to PRISMA guidelines, using the following databases: PubMed (PMC), PMC Europe, Scopus, Medline, Research Gate, Elsevier. The main question was whether stem cell therapies can contribute to the treatment of xerostomia. 19 of 226 publications met the criteria for this review, including 'in vivo', 'in vitro' studies and clinical trials. All 19 studies described thoroughly the stem cell source and the transplantation method, and documented results based on analytical and statistical methods of confirmation. Data show that the various sources of stem cells play a significant role, with bone marrow or adipose tissue-derived pluripotent blasts being the most utilized. Human transplants in mice have also been accepted and reversed hyposalivation. The effects have been beneficial especially in models undergone radiotherapy (IR) or exhibit Sjogren Syndrome-like symptoms (SS), suggesting that with appropriate treatment and enrichment techniques, stem cell transplantation seems effective regardless of the cause of the disorder. Extracts and co-cultures of gland and stem cells also seem to improve gland function. Although in its initial stages, the use of stem cells seems to be a promising therapy to alleviate xerostomia regardless of its cause.

Toxicological evaluation of primary particulate matter emitted from combustion of aviation fuel

Recently, Sustainable Aviation Fuel (SAF) blends and novel combustion technologies have been introduced to reduce aircraft engine emissions. However, there is limited knowledge about the impact of combustion technology and fuel composition on toxicity of primary Particulate Matter (PM) emissions, comparable to regulated non-volatile PM (nvPM).In this study, primary PM was collected on filters using a standardised approach, from both a Rich-Quench-Lean (RQL) combustion rig and a bespoke liquid fuelled Combustion Aerosol Standard (CAST) Generator burning 12 aviation fuels including conventional Jet-A, SAFs, and blends thereof. The fuels varied in aromatics (0–25.2%), sulphur (0–3000 ppm) and hydrogen (13.43–15.31%) contents. Toxicity of the collected primary PM was studied in vitro utilising Air-Liquid Interface (ALI) exposure of lung epithelial cells (Calu-3) in monoculture and co-culture with macrophages (differentiated THP-1 cells). Cells were exposed to PM extracted from filters and nebulised from suspensions using a cloud-based ALI exposure system. Toxicity readout parameters were analysed 24 h after exposure.Results showed presence of genotoxicity and changes in gene expression at dose levels which did not induce cytotoxicity. DNA damage was detected through Comet assay in cells exposed to CAST generated samples. Real-Time PCR performed to investigate the expression profile of genes involved in oxidative stress and DNA repair pathways showed different behaviours after exposure to the various PM samples. No differences were found in pro-inflammatory interleukin-8 secretion. This study indicates that primary PM toxicity is driven by wider factors than fuel composition, highlighting that further work is needed to substantiate the full toxicity of aircraft exhaust PM inclusive of secondary PM emanating from numerous engine technologies across the power range burning conventional Jet-A and SAF.

Para rubber seed oil and its fatty acids alleviate photoaging and maintain cell homeostasis.

Para rubber seed oil was indicated for skin dullness and hair loss in regard to its cutaneous beneficial fatty acids. Nonetheless, the oil's potency against photoaging remains unexplored. We proposed that para rubber seed oil could alleviate photoaging. Para rubber seed oil was investigated in cocultures of human HaCaT cells and dermal fibroblasts (HDF). Photoaging protectant efficiency was monitored in terms of IL-6 and IL-8 as well as MMP-1 (collagenase) and MMP-9 (gelatinase) in a comparison with its fatty acid components. Para rubber seed oil standardized in fatty acids was indicated as the promising plant oil for photoaging treatment. Its photoprotection mechanism was demonstrated in the coculture system of keratinocyte and fibroblast cells for the first time. Where the oil and its fatty acid constituents (100 μg/mL) were indicated to be safe and efficiently protect the cocultures against UV damage. The oil significantly (p < 0.001) suppressed UV-induced IL-6, IL-8, MMP-1 and MMP-9 secretions. The revealed photoprotection proficiency was abided by its fatty acids, particularly the unsaturated C18 ones. The oil was indicated on its potential to maintain skin homeostasis and would alleviate senescence ageing in regard to its photoprotection abilities exhibited. Para rubber seed oil is warranted as a new generation of photoaging protectant agent with the profiled safety and efficacy demonstrated in the epidermal coculture system. The findings encourage the development of innovative anti-ageing products containing the oil, which is categorizable as a sustainable specialty material for photoaging treatment.

CCR7/DUSP1 signaling Axis mediates iCAF to regulates head and neck squamous cell carcinoma growth

ObjectiveC-C motif chemokine receptor 7 (CCR7) significantly influences tumors onset and progression, yet its impact on the tumor microenvironment (TME) and specific mechanisms remain elusive. Inflammatory Cancer-Associated Fibroblasts (iCAF), a vital subtype of Cancer-Associated Fibroblasts (CAF), play a critical role in regulating the TME and tumor growth, though the underlying molecular mechanisms are not fully understood. This study aims to determine whether CCR7 participates in tumor regulation by iCAF and to elucidate the specific mechanisms involved. MethodsDifferential gene analysis of CAF subtypes in CCR7 knockout and wild-type groups was conducted using single-cell data. Animal models facilitated the extraction of primary iCAF cells via flow cytometry sorting. Changes in DUSP1 expression and the efficiency of lentivirus-mediated knockdown and overexpression were examined through qPCR and Western Blot. MOC1 and MOC2 cells were co-cultured with iCAF, with subsequent validation of changes in tumor cell proliferation, migration, and invasion using CCK8, EdU, and wound healing assays. ELISA was employed to detect changes in TGF-β1 concentration in the iCAF supernatant. ResultsCAF was categorized into three subtypes—myCAF, iCAF, and apCAF—based on single-cell data. Analysis revealed a significant increase in DUSP1 expression in iCAF from the CCR7 knockout group, confirmed by in vitro experiments. Co-culturing MOC1 and MOC2 cells with iCAF exhibiting lentivirus-mediated DUSP1 knockdown resulted in inhibited tumor cell proliferation, invasion, and migration. In contrast, co-culture with iCAF overexpressing DUSP1 enhanced these capabilities. Additionally, the TGF-β1 concentration in the supernatant increased in the DUSP1 knockdown iCAF group, whereas it decreased in the DUSP1 overexpression group. ConclusionThe CCR7/DUSP1 signaling axis regulates tumor growth by modulating TGF-β1 secretion in iCAF.

Mesenchymal Stem Cell and Hematopoietic Stem and Progenitor Cell Co-Culture in a Bone-Marrow-on-a-Chip Device toward the Generation and Maintenance of the Hematopoietic Niche.

Bone marrow has raised a great deal of scientific interest, since it is responsible for the vital process of hematopoiesis and is affiliated with many normal and pathological conditions of the human body. In recent years, organs-on-chips (OoCs) have emerged as the epitome of biomimetic systems, combining the advantages of microfluidic technology with cellular biology to surpass conventional 2D/3D cell culture techniques and animal testing. Bone-marrow-on-a-chip (BMoC) devices are usually focused only on the maintenance of the hematopoietic niche; otherwise, they incorporate at least three types of cells for on-chip generation. We, thereby, introduce a BMoC device that aspires to the purely in vitro generation and maintenance of the hematopoietic niche, using solely mesenchymal stem cells (MSCs) and hematopoietic stem and progenitor cells (HSPCs), and relying on the spontaneous formation of the niche without the inclusion of gels or scaffolds. The fabrication process of this poly(dimethylsiloxane) (PDMS)-based device, based on replica molding, is presented, and two membranes, a perforated, in-house-fabricated PDMS membrane and a commercial poly(ethylene terephthalate) (PET) one, were tested and their performances were compared. The device was submerged in a culture dish filled with medium for passive perfusion via diffusion in order to prevent on-chip bubble accumulation. The passively perfused BMoC device, having incorporated a commercial poly(ethylene terephthalate) (PET) membrane, allows for a sustainable MSC and HSPC co-culture and proliferation for three days, a promising indication for the future creation of a hematopoietic bone marrow organoid.

Nanoparticles in liposomes: a platform for increased antibiotic selectivity in multidrug resistant bacteria in respiratory tract infections.

Antibiotic resistance is a cause of serious illness and death, originating often from insufficient permeability into gram-negative bacteria. Nanoparticles (NP) can increase antibiotic delivery in bacterial cells, however, may as well increase internalization in mammalian cells and toxicity. In this work, NP in liposome (NP-Lip) formulations were used to enhance the selectivity of the antibiotics (3C and tobramycin) and quorum sensing inhibitor (HIPS-1635) towards Pseudomonas aeruginosa by fusing with bacterial outer membranes and reducing uptake in mammalian cells due to their larger size. Poly (lactic-co-glycolic) acid NPs were prepared using emulsion solvent evaporation and incorporated in larger liposomes. Cytotoxicity and uptake studies were conducted on two lung cell lines, Calu-3 and H460. NP-Lip showed lower toxicity and uptake in both cell lines. Then formulations were investigated for suitability for oral inhalation. The deposition of NP and NP-Lip in the lungs was assessed by next generation impactor and corresponded to 75% and 45% deposition in the terminal bronchi and the alveoli respectively. Colloidal stability and mucus-interaction studies were conducted. NP-Lip showed higher diffusion through mucus compared to NPs with the use of nanoparticle tracking analyzer. Moreover, the permeation of delivery systems across a liquid-liquid interface epithelial barrier model of Calu-3 cells indicated that NP-Lip could cause less systemic toxicity upon in-vivo like administration by aerosol deposition. Monoculture and Pseudomonas aeruginosa biofilm with Calu-3 cells co-culture experiments were conducted, NP-Lip achieved highest toxicity towards bacterial biofilms and least toxicity % of the Calu-3 cells. Therefore, the NP- liposomal platform offers a promising approach for enhancing antibiotic selectivity and treating pulmonary infections.

The 3D organ-on-a-chip model unveils a dual role of GDF-15 in vascular growth

Pathological conditions such as oxidative stress or inflammation may alter the homeostasis of adventitia triggering vascular wall remodeling and abnormal angiogenesis, what can lead to development of atherosclerosis. Growth differentiation factor-15 (GDF-15) is a stress‐responsive cytokine and metabolic regulator, but its role in angiogenesis is not yet fully defined. Here we utilized an organ-on-a-chip technology to analyze endothelial sprouting in an adventitia-resembling microenvironment. We analyzed angiogenic responses to growth factor gradient across the extracellular matrix-resembling fibrin gel and in cell co-culture in response to GDF-15-treated adventitial fibroblasts. We observed that GDF-15 enhanced the pro-angiogenic effect of vascular endothelial growth factor. On the other hand, GDF-15-treated adventitial fibroblasts decreased endothelial sprouting. GDF-15 seems to indirectly affect endothelial cells and, depending on the microenvironment, its effect can be either pro- or anti-angiogenic.

An Adaptable Protocol to Generate a Murine Enteroid-Macrophage Co-Culture System.

Impairment of the intestinal epithelial barrier is frequently seen as collateral damage in various local and systemic inflammatory conditions. The inflammatory process is characterized by reciprocal interactions between the host intestinal epithelium and mucosal innate immune cells, e.g., macrophages. This article provides step-by-step instructions on how to set up a murine enteroid-macrophage co-culture by culturing cellular elements in proximity separated by a porous membrane. Unlike previously published co-culture systems, we have combined enteroids grown from C57BL6j mice with syngeneic bone marrow-derived macrophages to preclude potential allo-reactions between immune cells and epithelium. Transformation of intestinal crypts into proliferative enteroids was achieved by cultivation in Wnt3a-Noggin-R-Spondin-conditioned medium supplemented with ROCK inhibitor Y-27632. The differentiated phenotype was promoted by the use of the Wnt3-deprived EGF-Noggin-R-Spondin medium. The resulting co-culture of primary cells can be employed as a basic model to better understand the reciprocal relationship between intestinal epithelium and macrophages. It can be used for in vitro modelling of mucosal inflammation, mimicked by stimulation of macrophages either while being in co-culture or before being introduced into co-culture, to simulate enterogenic sepsis or systemic conditions affecting the intestinal tract.

CX 3 CR1 + macrophages interact with HSCs to promote HCC through CD8 + T-cell suppression.

HSCs contribute to HCC progression by regulating multiple factors. However, the entire immunoregulatory functions of HSCs are still obscure. Here, we aim to investigate whether HSCs impose CX 3 CR1 + macrophages to protumorigenic properties in the peritumoral area. In single-cell RNA-sequencing analysis of patients with HCC, a subpopulation of macrophages specifically expressed Arg1 and Cx3cr1 in the peritumoral area and were highly enriched with retinol metabolism-related genes. Flow cytometry analysis showed significantly increased frequencies of CD14 + CD11b + HLA-DR - macrophages with CX 3 CR1 in the HCC adjacent region where α-smooth muscle actin-expressing activated hepatic stellate cells (aHSCs) showed colocalized expression of CX 3 CL1. Accordingly, in tumor-bearing mice, Cx3cl1 mRNA expression was notably increased in aHSCs within the adjacent HCC, where infiltration of CX 3 CR1 + Ly6C + macrophages was mostly observed with decreased CD8 + T cells. In adoptive transfer and in vitro coculture of myeloid cells, we demonstrated that CX 3 CR1 + Ly6C + macrophages migrated and highly expressed arginase-1 by interacting with retinoid-enriched aHSCs in the adjacent HCC. Direct treatment of retinoids or coculturing with retinol-storing mouse aHSCs or human LX-2 cells significantly increased arginase-1 expression in CX 3 CR1 + Ly6C + macrophages and human blood CD14 + cells, leading to the suppression of CD8 + T-cell proliferation. Moreover, genetic deficiency of CX 3 CR1 in myeloid cells or pharmacological inhibition of retinol metabolism remarkably attenuated HCC development. We showed that CX 3 CR1 + Ly6C + macrophages migrate and interact with aHSCs in the peritumoral region where retinoids induce arginase-1 expression in CX 3 CR1 + Ly6C + macrophages, subsequently depriving CD8 + T cells of arginine and promoting HCC.

Intestine-On-Chip: Enhancing In Vitro Intestinal Models Using Caco-2 and HT29-MTX Cocultures Under Different Flow Conditions

This study explores the development of in vitro intestinal models on chip using cocultures of Caco-2 and HT29-MTX cells over time under different flow conditions. For this purpose, cocultures in the chip were connected to a syringe pump with a characteristic shear stress of the intestinal epithelium, 0.02dyn/cm2 for 4 days and, compared to cocultures with non-specific flow by rocker for 21 days. The effect of different flow conditions in the intestinal permeability was assessed following the Lucifer Yellow assay. Coculture under specific flow conditions showed similar permeability values after 4 days to those under non-specific flow conditions after 14 days. Therefore, optimalization and further characterization of this model, may lead to a promising alternative for intestinal in vitro studies.

Engineering of gelatin scaffold by extracellular matrix of Sertoli cells for embryonic stem cell proliferation

Mimicking the microenvironment of seminiferous tubules plays an indispensable role in directing differentiation of stem cells toward germ cells in vitro. In this work, we fabricated electrospun gelatin (EG) mats (i.e., with diameter <500 nm) conditioned with Sertoli cells' extracellular matrix (ECM) to simulate both 3D structures and composition of normal testis tissue. Sertoli cells were isolated from mice testis and represented through immunocytochemistry (ICC) staining for expression of vimentin, a specific marker of Sertoli cells. The morphological characteristics of ECM-coated scaffold were investigated under scanning electron microscope (SEM). The efficient elimination of cells was confirmed by MTT assay. Furthermore, the cyto/biocompatibility of ECM-conditioned EG scaffold was determined for Sertoli cells and embryonic stem cells (ESCs), alone and as in co-culture. According to the results, the designed scaffold provided a mat for cell proliferation with negligible toxicity (almost 100% cell viability). SEM micrographs displayed cells with elongated shape and complete stretching morphology when compared with those cultured on scaffold without ECM. Moreover, an enhanced differentiation of ESCs toward sperm-generating cells was obtained through co-culturing of Sertoli cells and ESCs, where cell viability was found almost 100%. Our findings introduce the ECM-conditioned EG scaffold as a potentially influential engineered substrate for in vitro guidance of stem cells differentiation by mimicking the native microenvironment.

Dragon's blood attenuates LPS-induced intestinal epithelial barrier dysfunction via upregulation of FAK-DOCK180-Rac1-WAVE2-Arp3 and downregulation of TLR4/NF-κB signaling pathways.

Inflammation-induced intestinal epithelial barrier (IEB) dysfunction is one of the important reasons for the occurrence and development of intestinal inflammatory-related diseases, including ulcerative colitis (UC), Crohn's disease and necrotizing enterocolitis (NEC). Dragon's blood (DB) is a traditional Chinese medicine and has been clinically used to treat UC. However, the protective mechanism of DB on intestinal inflammatory-related diseases has still not been elucidated. The present study aimed to explore the protection mechanism of DB on IEB dysfunction in rat ileum and human colorectal adenocarcinoma cells (Caco-2)/human umbilical vein endothelial cells (HUVECs) coculture system induced by lipopolysaccharide (LPS). DB could ameliorate rat ileum mucosa morphological injury, reduce the accumulation of lipid-peroxidation products and increase the expression of junction proteins. DB also alleviated LPS-induced Caco-2 cells barrier integrity destruction in Caco-2/ HUVECs coculture system, leading to increased trans-endothelial electrical resistance (TEER), reduced cell permeability, and upregulation of expressions of F-actin and junction proteins. DB contributed to the assembly of actin cytoskeleton by upregulating the FAK-DOCK180-Rac1-WAVE2-Arp3 pathway and contributed to the formation of intercellular junctions by downregulating TLR4-MyD88-NF-κB pathway, thus reversing LPS-induced IEB dysfunction. These novel findings illustrated the potential protective mechanism of DB on intestinal inflammatory-related diseases and might be useful for further clinical application of DB.

Selective anticancer effects of plasma-activated saline in 3D tumor model co-culturing of normal cells and cancer cells

Compared with conventional two-dimensional (2D) cell culture model, the 3D tumor model constructed in vitro is better representative of the tumor microenvironment in vivo. Here, we proposed the utilization of 3D tumor model of co-cultured cancer cells and normal cells to evaluate the selective anticancer effects of cold atmospheric plasma-activated saline (PAS), and expected to provide more precise information about PAS-tumor interactions. By cell sorting, we clarified that A375 melanoma cells and HaCaT normal skin cells purified from the 3D multicellular tumor model differ in sensitivity and responsiveness to PAS compared to the 2D culture model. And during the optimization of PAS treatment parameters, we further found that A375 cells were almost completely killed while HaCaT cells were still present in large numbers after 5 d of certain PAS treatment. Our experiment innovatively carries out the selective study of plasma technology in 3D co-culture system and provides a theoretical basis for further clinical and practical applications of PAS.

Decoding host cell interaction– and fluconazole-induced metabolic alterations and drug resistance in Candida auris

ABSTRACT Candida auris is an emerging drug-resistant pathogen associated with high mortality rates. This study aimed to explore the metabolic alterations and associated pathogenesis and drug resistance in fluconazole-treated Candida auris–host cell interaction. Compared with controls, secreted metabolites from fluconazole-treated C. auris and fluconazole-treated C. auris–host cell co-culture demonstrated notable anti-Candida activity. Fluconazole caused significant reductions in C. auris cell numbers and aggregated phenotype. Metabolites produced by C. auris with potential fungal colonization, invasion, and host immune evasion effects were identified. Metabolites known to enhance biofilm formation produced during C. auris–host cell interaction were inhibited by fluconazole. Fluconazole enhanced the production of metabolites with biofilm inhibition activity, including behenyl alcohol and decanoic acid. Metabolites with potential Candida growth inhibition activity such as 2-palmitoyl glycerol, 1-tetradecanol, and 1-nonadecene were activated by fluconazole. Different patterns of proinflammatory cytokine expression presented due to fluconazole concentration and host cell type (fibroblasts versus macrophages). This highlights the immune response’s complexity, emphasizing the necessity for additional research to comprehend cell-type-specific responses to antifungal therapies. Both host cell interaction and fluconazole treatment increased the expression of CDR1 and ERG11 genes, both associated with drug resistance. This study provides insights into pathogenesis in C. auris due to host cell interaction and fluconazole treatment. Understanding these interactions is crucial for enhancing fluconazole sensitivity and effectively combating C. auris.