Search Results (72)

Primary hepatocytes are widely used in preclinical drug development, with their transporter expression being well-characterized. However, less is known about non-parenchymal liver cells (NPCs), which constitute 40% of the liver’s cell population and include sinusoidal endothelial cells and Kupffer cells. This study aimed to characterize transporter expression in murine NPCs compared to hepatocytes. Cell fractions were isolated using collagenase perfusion, density gradient centrifugation, and magnetic-activated cell sorting (MACS) with F4/80 and CD146 antibodies. Transporter expression and separation quality were analyzed via RT-qPCR. Results showed NPC-specific genes were significantly lower in hepatocytes and vice versa. Importantly, NPCs exhibited higher expression of several transporters: Abcc1/Mrp1 (87-fold), Abcc4/Mrp4 (4-fold), Abcc5/Mrp5 (40-fold), as well as Slc15a2/PepT2 (16-fold), Slc28a2/Cnt2 (20-fold), Slco3a1/Oatp3a1 (15-fold), and Slco4a1/Oatp4a1 (13-fold), compared to hepatocytes. Hepatocytes showed dominant expression of Abcc2/Mrp2, Abcg2/Bcrp, Slc22a1/Oct1, and others. Minimal differences in transporter expression were found between Kupffer and endothelial cells. In conclusion, the efflux transporters Abcc1/Mrp1 and Abcc5/Mrp5 are predominantly expressed in NPCs. This suggests that NPCs are potentially relevant for the transport of certain drugs and should be included in in vitro preclinical testing. Full article

Objective: Dendritic cells (DCs) are the most potent antigen-presenting cells, serving as a bridge between innate and adaptive immunity. Activation of the stimulator of interferon genes (STING) pathway by pathogen-derived DNA induces type I interferon responses and promotes CD8⁺ cytotoxic T cell activity. This study aimed to establish a protocol for generating immature DCs from murine bone marrow, optimize their maturation in vitro with a STING agonist, and evaluate their ability to prime naïve T cells for potential use in cancer immunotherapy. Methods: Bone marrow cells from C57BL/6 mice were differentiated into immature DCs under growth factor–supplemented conditions. Maturation was induced using a STING agonist and B16 tumor-derived DNA. Naïve CD4⁺ and CD8⁺ T cells were isolated via magnetic-activated cell sorting (MACS) and co-cultured with the stimulated DCs. Culture conditions were optimized to enhance DC maturation efficiency, and T cell proliferation was assessed following co-culture. Results: Optimization of the culture system markedly increased the yield of mature DCs. Importantly, co-culture of STING agonist-stimulated DCs with naïve T cells resulted in strong CD8⁺ T cell proliferation, indicating effective priming. Conclusions: These findings demonstrate the feasibility of generating functional DCs in vitro and highlight their capacity to prime T cells through STING pathway activation. This proof-of-concept supports the development of DC-based platforms as a promising strategy for novel cancer immunotherapies. Full article

Background: Type 2 diabetes mellitus (DM) is a major cardiovascular risk factor that induces monocyte dysfunction and contributes to their accumulation in atherosclerotic lesions. Monocyte recruitment and accumulation in the tissues contribute to chronic inflammation and are essential to the pathobiology of diabetes-induced atherosclerosis. However, the mechanisms that drive the accumulation of monocytes in the diabetic environment are not clearly understood. Methods: Primary monocytes from type 2 (T2) DM and non-T2DM individuals were isolated using magnet-assisted cell sorting. To examine the influence of a diabetic milieu on monocyte function, monocytes from T2DM patients, db/db mice, or human monocytes subjected to hyperglycaemia were analysed for their responses to pro-atherogenic cytokines using Boyden chamber assays. Furthermore, the interactions of non-diabetic and diabetic monocytes with TNFα-inflamed endothelium were studied using live-cell imaging under physiological flow conditions. RT-qPCR and FACS were used to study the expression of relevant molecules involved in monocyte-endothelium interaction. Results: CD14⁺CD16⁻ monocytes isolated from T2DM patients or monocytes exposed to hyperglycaemic conditions showed reduced chemotactic responses towards atherosclerosis-promoting cytokines, CCL2 and CX3CL1, indicating monocyte dysfunction. Under flow conditions, the transendothelial migration (TEM) capacity of T2DM monocytes was significantly reduced. Even though these monocytes adhered to the endothelial monolayer, only a few transmigrated. Interestingly, the T2DM monocytes and monocytes exposed to hyperglycaemic conditions accumulated in the ablumen following transendothelial migration. The time period in the ablumen of T2DM cells was prolonged, as there was a significant impairment of the reverse transendothelial migration (rTEM). Mechanistically, the T2DM milieu specifically induced the activation of monocyte integrins, Macrophage-1 antigen (Mac-1; integrin αMβ2 consisting of CD11b and CD18), and Lymphocyte function-associated antigen 1 (LFA-1; αLβ2 consisting of CD11a and CD18). Furthermore, elevated levels of CD18 transcripts were detected in T2DM monocytes. Junctional Adhesion Molecule 3 (JAM-3)–MAC-1 interactions are known to impede rTEM and T2DM milieu-potentiated JAM-3 expression in human coronary artery endothelial cells (HCAEC). Finally, the overexpression of JAM-3 on HCAEC was sufficient to completely recapitulate the impaired rTEM phenotype. Conclusions: Our results revealed for the first time that the enhanced T2DM monocyte accumulation in the ablumen is not secondary to the elevated transmigration through the endothelium. Instead, the accumulation of monocytes is due to the direct consequence of a dysfunctional rTEM, potentially due to enhanced JAM3-MAC1 engagement. Our results highlight the importance of restoring the rTEM capacity of monocytes to reduce monocyte accumulation-dependent inflammation induction and atherogenesis in the T2DM environment. Full article

Sorting and isolating specific cells from heterogeneous populations are crucial for many biomedical applications, including drug discovery and medical diagnostics. Conventional methods such as Fluorescent Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS) face limitations in throughput, cost, and the ability to separate subtly different cells. Cell partitioning in Aqueous Two-Phase Systems (ATPSs) offers a biocompatible and cost-effective alternative, particularly when combined with continuous-flow microfluidics. However, it remains challenging to rationally design microfluidic ATPS devices and operation to separate cells with similar origin but different phenotypes. In this paper, using a model ATPS, polyethylene glycol (PEG)—Dextran (Dex) system, and model cells, human chondrocytes (hChs), and carboxylated polystyrene (PS) microparticles, we systematically characterized the material properties affecting cell partitioning in ATPSs, such as surface energies of the solutions and cells and solution viscosities. We developed an energy balance approach between interfacial energy and viscous dissipation to estimate the interface translocation dynamic of cells partitioning into the preferred phase. Combining the experimental measurement and the energy balance model, our calculation reveals that the time required for complete cell partitioning at the ATPS interface can be exploited in microfluidic ATPS devices to separate hChs with different phenotypes (healthy and diseased). We expect our dynamic energy approach to provide a basis and a design strategy for optimizing microfluidic ATPS devices to achieve the efficient separation of phenotypically similar cell populations and further expand the potential of microfluidic cell separation. Full article

Background: Oxidative stress is a critical factor contributing to male infertility, impairing spermatogonial stem cells (SSCs) and disrupting normal spermatogenesis. This study aimed to isolate and characterize human SSCs and to investigate oxidative stress-related gene expression, protein interaction networks, and developmental trajectories involved in SSC function. Methods: SSCs were enriched from human orchiectomy samples using CD49f-based magnetic-activated cell sorting (MACS) and laminin-binding matrix selection. Enriched cultures were assessed through morphological criteria and immunocytochemistry using VASA and SSEA4. Transcriptomic profiling was performed using microarray and single-cell RNA sequencing (scRNA-seq) to identify oxidative stress-related genes. Bioinformatic analyses included STRING-based protein–protein interaction (PPI) networks, FunRich enrichment, weighted gene co-expression network analysis (WGCNA), and predictive modeling using machine learning algorithms. Results: The enriched SSC populations displayed characteristic morphology, positive germline marker expression, and minimal fibroblast contamination. Microarray analysis revealed six significantly upregulated oxidative stress-related genes in SSCs—including CYB5R3 and NDUFA10—and three downregulated genes, such as TXN and SQLE, compared to fibroblasts. PPI and functional enrichment analyses highlighted tightly clustered gene networks involved in mitochondrial function, redox balance, and spermatogenesis. scRNA-seq data further confirmed stage-specific expression of antioxidant genes during spermatogenic differentiation, particularly in late germ cell stages. Among the machine learning models tested, logistic regression demonstrated the highest predictive accuracy for antioxidant gene expression, with an area under the curve (AUC) of 0.741. Protein oxidation was implicated as a major mechanism of oxidative damage, affecting sperm motility, metabolism, and acrosome integrity. Conclusion: This study identifies key oxidative stress-related genes and pathways in human SSCs that may regulate spermatogenesis and impact sperm function. These findings offer potential targets for future functional validation and therapeutic interventions, including antioxidant-based strategies to improve male fertility outcomes. Full article

Exosomes, small extracellular vesicles secreted by various cell types, have gained significant attention in cancer investigations. Isolation and characterization of exosomes derived from DOK (dysplastic oral keratinocyte), SCC (squamous cell carcinoma) and HaCaT (normal skin keratinocyte) cell lines and microRNA profiling were conducted. Magnetic sorting was applied to obtain pure exosomes. Morphology and size were characterized by transmission electron microscopy and nanoparticle tracking analysis. Validation of membrane exosomal markers (CD9, CD63) was performed via Western blotting. MiR-21, miR-31, and miR-133 levels were analyzed in exosomes and parent cells by qPCR. Biological effects of the exosomes were tested by adding them to fibroblast cultures and determining the expression of relevant carcinogenesis markers by qPCR. Exosomes appeared as cup-shaped nano-sized particles, and there was no difference regarding particle diameter and concentration between the three types of exosomes. The oncogenic miR-21 was significantly upregulated both in SCC and SCC-derived exosomes compared to DOK and HaCaT cells and their respective exosomes. However, miR-31 unexpectedly showed the highest expression in normal cells and the lowest in HaCaT exosomes. MiR-133, the tumor suppressor miRNA, was downregulated in both SCC and DOK cells compared to normal (HaCaT) cells, while the opposite situation was observed in exosomes, with HaCaT cells showing the lowest levels of miR-133. The differences in exosome content were reflected in signaling pathway activation in exosome-treated fibroblasts, with SCC exosomes exerting the most potent effect on several cancer-related pathways, notably PIK3/AKT, PTEN, and NOTCH signaling cascades. Full article

Background: Human cytomegalovirus (CMV) is a major pathogen that poses significant risks to immunocompromised individuals and neonates. The tegument protein pp71, encoded by the UL82 gene, plays a pivotal role in initiating viral lytic replication and evading host immune responses. Despite its clinical relevance, standardized monoclonal antibodies (mAbs) for pp71 remain limited, prompting the need to expand the available repertoire of antibodies targeting this critical protein. Methods: In this study, we constructed a diverse human single-chain variable fragment (scFv) library using RNA derived from the B cells of four healthy donors. The library was expressed in Saccharomyces cerevisiae, and iterative rounds of magnetic-activated cell sorting (MACS) were performed against recombinant pp71. Clonal enrichment was monitored using flow cytometry. Results: Among the isolated clones, one designated ID2 exhibited high sensitivity and specificity for pp71, as demonstrated by flow cytometry, immunofluorescence, an enzyme-linked immunosorbent assay (ELISA), and biolayer interferometry (BLI). Conclusions: Collectively, these findings establish a novel pp71-specific mAb and underscore the utility of yeast surface display combined with MACS for expanding the antibody toolkit available for CMV research and diagnostics. Full article

Tracking T cells in vivo using MRI is a major challenge due to the difficulty of labeling these non-phagocytic cells with a sufficient contrast agent to generate a detectable signal change. In this study, we explored CD4-Superparamagnetic iron oxide nanoparticles (SPION), which is commonly used in magnetic cell sorting, as a potential receptor-mediated, specific CD4⁺ T cell MRI labeling agent. We optimized the labeling protocol for maximal CD4⁺ cell labeling and viability. Cell health was confirmed with trypan blue assay, and labeling efficacy was confirmed with Prussian blue staining, transmission electron microscopy, and MRI of labeled cell pellets. Key cell functionality was assessed by flow cytometry. Next, CD4-SPION-labeled T cells or unlabeled T cells were delivered via intravenous injection in naïve mice. Liver MRIs pre-, 24 h, and 72 h post-T cell injection were performed to determine in vivo tracking ability. Our results show that CD4-SPION induces significant attenuation of T2 signals in a concentration-dependent manner, confirming their potential as an effective MRI contrast agent. In vitro, analyses showed that CD4⁺ T cells were able to uptake CD4-SPION without affecting cellular activity and key functions, as evidenced by Prussian blue staining and flow cytometric analysis of IL-2 receptor and the IL-7 receptor α-chains, CD69 upregulation, and IFN-γ secretion. In vivo, systemically distributed CD4-SPION-labeled T cells could be tracked in the liver at 24 and 72 h after injection, contrary to controls. Histological staining of tissue sections validated the findings. Our results showed that SPION CD4⁺ T cell sorting coupled with longitudinal MR imaging is a valid method to track CD4⁺ T cells in vivo. This safe, specific, and sensitive approach will facilitate the use of SPION as an MRI contrast agent in clinical practice, allowing for non-invasive tracking of adoptive cell therapies in multiple disease conditions. Full article

It has been demonstrated that S100B actively participates in neuroinflammatory processes of different diseases of the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE), a recognized animal model for multiple sclerosis (MS). The inhibition of S100B activity using pentamidine and of S100B synthesis using arundic acid are able to determine an amelioration of the clinical and pathologic parameters of MS with milder and delayed symptoms. This study further goes in detail on the role of S100B, and in particular of astrocytic S100B, in these neuroinflammatory processes. To this aim, we used a model of S100B knockout (KO) mice. As expected, S100B protein levels were significantly reduced in the S100B KO mouse strain resulting in an amelioration of clinical and pathological parameters (clinical and morphological analyses). To dissect the potential mechanisms that could explain the role of S100B in the development of EAE, we sorted, cultured, and compared glial subpopulations (astrocytes, oligodendrocytes, and microglia) derived from S100B KO and wild type mice, through flow cytometric panels and ELISA. Glial cells were analyzed for proinflammatory molecules showing a significant reduction of TNFα protein in mice where S100B was silenced. To dissect the role of S100B in MS, we cultured astrocytes and microglial cells magnetically sorted and enriched from the brains of EAE-affected animals, both from KO and wild type animals. Both genetic silencing of S100B and pharmacological inhibition with S100B-targeting compounds demonstrated a direct impact on specific subpopulations of astrocytes (mainly), oligodendrocytes, and microglia. The present results further individuate astrocytic S100B as a key factor and as a potential therapeutic target for EAE neuroinflammatory processes. Full article

Oligodendrocytes form myelin in the central nervous system, and their dysfunction can cause severe neurological symptoms, as large-scale analyses have highlighted numerous gene expression alterations in pathological conditions. Although in vivo functional gene analyses are preferable, they have several limitations, especially in large-scale studies. Therefore, standardized in vitro systems are needed to facilitate efficient and reliable functional analyses of genes identified in such studies. Here, we describe a practical and efficient method for oligodendrocyte precursor cell (OPC) isolation from mouse brains on postnatal day 6–8 and a gene delivery method for the isolated OPCs. By modifying the magnetic-activated cell sorting (MACS) procedure with reduced processing volumes, we simplified OPC isolation, allowing simultaneous handling of multiple samples and improving workflow efficiency. We also optimized electroporation parameters to achieve robust transfection efficiency with minimal cell death. Transfected OPCs are suitable for both monoculture-based differentiation assays and co-culture with dorsal root ganglion (DRG) explants, in which they reliably differentiate into mature oligodendrocytes and myelinate along the axons. This system enables stable and reproducible in vitro analysis of oligodendrocyte function, supports investigations into both intrinsic differentiation and neuron–glia interactions, and provides a powerful platform for oligodendrocyte research with efficient and timely gene manipulation. Full article

Droplet-based microfluidics (DBM) has emerged as a powerful tool for a wide range of biochemical applications, from single-cell analysis and drug screening to diagnostics and tissue engineering. This review provides a comprehensive overview of the latest advancements in droplet generation and trapping techniques, highlighting both passive and active approaches. Passive methods—such as co-flow, cross-flow, and flow-focusing geometries—rely on hydrodynamic instabilities and capillary effects, offering simplicity and integration with compact devices, though often at the cost of tunability. In contrast, active methods exploit external fields—electric, magnetic, thermal, or mechanical—to enable on-demand droplet control, allowing for higher precision and throughput. Furthermore, we explore innovative trapping mechanisms such as hydrodynamic resistance networks, microfabricated U-shaped wells, and anchor-based systems that enable precise spatial immobilization of droplets. In the final section, we also examine active droplet sorting strategies, including electric, magnetic, acoustic, and thermal methods, as essential tools for downstream analysis and high-throughput workflows. These manipulation strategies facilitate in situ chemical and biological analyses, enhance experimental reproducibility, and are increasingly adaptable to industrial-scale applications. Emphasis is placed on the design flexibility, scalability, and biological compatibility of each method, offering critical insights for selecting appropriate techniques based on experimental needs and operational constraints. Full article

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease. The pathomechanism of RA depends on both B and T cells. Regulatory B cells (Breg) have been shown to suppress T-cell immune responses and play a key role in modulating autoimmune processes. We aimed to investigate the possibility of utilizing PD-L1⁺ Breg cells in downregulating the Th cells’ immune response in healthy individuals and RA patients. We hypothesized that the PD-1/PD-1L interaction plays a key role in this process, which may be defective in autoimmune diseases. We separated T and B cells from the peripheral blood of healthy volunteers and RA patients by magnetic cell sorting, and Th cells and Treg cells were isolated by fluorescence-activated cell sorting. The cytokine production by CD4⁺ Th cells was detected by intracellular flow cytometry. CpG and CD40L stimulations were applied to induce PD-L1^hi expressing Breg cells. We found that the frequency of PD-L1^hi cells is significantly lower in all B-cell subsets in RA compared to healthy controls. Functional analysis of induced PD-L1⁺ Breg cells in coculture with activated autologous Th cells has shown that healthy control samples containing higher levels of PD-L1^hi Breg cells significantly inhibit IFN-ү and IL-21 production by Th cells. In contrast, RA patients’ samples with lower levels of PD-L1^hi Breg cells failed to do so. Since the expression of PD-L1 on B cells can be modulated in vitro to induce Breg cell suppressive capacity, these data may provide new perspectives for future therapy for RA. Full article

Background/Objectives: Treatment of knee osteoarthritis (OA) with autologous stem cells from microfragmented adipose tissue (MFAT) has shown promising but varying results. Multiple stem cell types, including CD34⁺, CD146⁺, and CD271⁺ stem cells, have been identified within MFAT. Patient-specific heterogeneity in stem cell populations and the content of highly potent cells may be determining factors for a successful treatment outcome. The current study aimed to identify the most promising stem cell type in MFAT to treat OA, focusing on their chondrogenic and osteogenic differentiation performance. Methods: CD34⁺, CD146⁺, and CD271⁺ stem cells from the MFAT of eight patients with knee OA were separated using magnetic-activated cell sorting (MACS) and analyzed as subtypes. Unsorted cells were used as a control. Chondrogenic and osteogenic in vitro differentiation were assessed through Safranin-O and H&E staining, pellet size, and qPCR for chondrogenesis, as well as Alizarin Red S staining and qPCR for osteogenesis. Results: CD34⁺, CD146⁺, and CD271⁺ stem cells were doubled using MACS. All subtypes were able to undergo osteogenic differentiation with Alizarin Red S staining, revealing a significant increase in calcium deposits of induced cells compared to non-induced controls. CD146⁺ stem cells showed higher calcium deposition compared to CD34⁺, CD271⁺, and unsorted stem cells. All cell types could form chondrogenic pellets. CD271⁺ stem cells produced more proteoglycans, as shown by Safranin-O staining, than CD34⁺ and CD146⁺ stem cells, but not more than the unsorted stem cells. After differentiation induction, all cell types showed an upregulation of most chondrogenic and osteogenic biomarkers. Conclusions: CD146⁺ stem cells showed the highest osteogenic differentiation performance for calcium deposition, while CD271⁺ stem cells showed the greatest chondrogenic differentiation performance for proteoglycan formation. The prevalence of these stem cell types may play a critical role in the clinical effectiveness when treating OA. Full article

Background: Male infertility, accounting for nearly half of infertility cases worldwide, has spurred significant research into its causes, diagnosis, and treatment strategies. Genetic abnormalities, social causes, environmental exposures, lifestyle, and further health conditions are key contributors. Methods: Essential to improving the outcomes of ART is, among other things, the selection of high-quality sperm, which requires methods that assess sperm motility, morphology, DNA integrity, and oxidative stress levels. Results: Traditional techniques such as semen analysis, swim-up, and density gradient centrifugation (DGC) are still widely used, but there is ongoing discussion regarding the limitations in detecting DNA damage and oxidative stress. Advanced methods like magnetic-activated cell sorting (MACS) and microfluidic sorting have emerged as more precise tools for selecting sperm with better genetic integrity, although they face challenges in terms of their standardization, cost, and clinical adoption. Emerging technologies such as artificial intelligence (AI) and Raman spectroscopy offer the potential for more automated, accurate sperm selection, minimizing human error and variability. However, the integration of these methods into clinical practice requires further validation through large-scale studies, including assessments of their long-term safety and cost-effectiveness. Conclusions: Future research should focus on refining sperm selection techniques, tailoring them to personalized infertility approaches, and addressing the gaps in the evidence to improve ART outcomes and patient care. Full article

Oral squamous cell carcinoma (OSCC) is a highly aggressive malignancy with poor prognosis, mainly due to the presence of cancer stem cells (CSCs), a small subpopulation of cells that contribute to therapy resistance and tumor progression. The principal objective of this study was to investigate the role of miRNA-21 in the maintenance of cancer cell stemness and the possibility of altering it. The CD44 antigen was used as a marker for CSC isolation from oral cancer cell cultures. CD44+ and CD44− populations were sorted via magnetic separation. miRNA-21 inhibition was performed in CD44+ cells via transfection. CD44+ cells possessed a significantly higher migration and invasion potential compared to CD44− cells, higher levels of miRNA-21 (p = 0.004) and β-catenin (p = 0.005), and lower levels of BAX (p = 0.015). miRNA-21 inhibition in CD44+ cells reduced migration, invasion, and colony formation while increasing apoptosis. Stemness markers were significantly downregulated following miRNA-21 inhibition: OCT4 (p = 0.013), SOX2 (p = 0.008), and NANOG (p = 0.0001), as well as β-catenin gene (CTNNB1) (p < 0.05), an important member of WNT signaling pathway. Apoptotic activity was enhanced, with a significant downregulation of the antiapoptotic Bcl-2 (p = 0.008) gene. In conclusion, miRNA-21 plays a critical role in the regulation of oral cancer CD44+ cells properties. Targeting and inhibiting miRNA-21 in CD44+ cells could represent a promising novel strategy in OSCC treatment. Full article