Human organoids: New strategies and methods for analyzing human development and disease.

For decades, insight into fundamental principles of human biology and disease has been obtained primarily by experiments in animal models. While this has allowed researchers to understand many human biological processes in great detail, some developmental and disease mechanisms have proven difficult to study due to inherent species differences. The advent of organoid technology more than 10 years ago has established laboratory-grown organ tissues as an additional model system to recapitulate human-specific aspects of biology. The use of human 3D organoids, as well as other advances in single-cell technologies, has revealed unprecedented insights into human biology and disease mechanisms, especially those that distinguish humans from other species. This review highlights novel advances in organoid biology with a focus on how organoid technology has generated a better understanding of human-specific processes in development and disease.

Controlled X-chromosome dynamics defines meiotic potential of female mouse in vitro germ cells.

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X-chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X-inactivation and reactivation dynamics using a tailor-made in vitro system of primordial germ cell-like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X-inactivation in PGCLCs in vitro and in germ cell-competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X-inactivation is followed by step-wise X-reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X-inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine-tuned X-chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.

Application of the C3 inhibitor compstatin in a human whole blood model designed for complement research - 20 years of experience and future perspectives.

The complex molecular and cellular biological systems that maintain host homeostasis undergo continuous crosstalk. Complement, a component of innate immunity, is one such system. Initially regarded as a system to protect the host from infection, complement has more recently been shown to have numerous other functions, including involvement in embryonic development, tissue modeling, and repair. Furthermore, the complement system plays a major role in the pathophysiology of many diseases. Through interactions with other plasma cascades, including hemostasis, complement activation leads to the broad host-protective response known as thromboinflammation. Most complement research has been limited to reductionistic models of purified components and cells and their interactions in vitro. However, to study the pathophysiology of complement-driven diseases, including the interaction between the complement system and other inflammatory systems, holistic models demonstrating only minimal interference with complement activity are needed. Here we describe two such models; whole blood anticoagulated with either the thrombin inhibitor lepirudin or the fibrin polymerization peptide blocker GPRP, both of which retain complement activity and preserve the ability of complement to be mutually reactive with other inflammatory systems. For instance, to examine the relative roles of C3 and C5 in complement activation, it is possible to compare the effects of the C3 inhibitor compstatin effects to those of inhibitors of C5 and C5aR1. We also discuss how complement is activated by both pathogen-associated molecular patterns, inducing infectious inflammation caused by organisms such as Gram-negative and Gram-positive bacteria, and by sterile damage-associated molecular patterns, including cholesterol crystals and artificial materials used in clinical medicine. When C3 is inhibited, it is important to determine the mechanism by which inflammation is attenuated, i.e., whether the attenuation derives directly from C3 activation products or via downstream activation of C5, since the mechanism involved may determine the appropriate choice of inhibitor under various conditions. With some exceptions, most inflammatory responses are dependent on C5 and C5aR1; one exception is venous air embolism, in which air bubbles enter the blood circulation and trigger a mainly C3-dependent thromboembolism, with the formation of an active C3 convertase, without a corresponding C5 activation. Under such conditions, an inhibitor of C3 is needed to attenuate the inflammation. Our holistic blood models will be useful for further studies of the inhibition of any complement target, not just C3 or C5. The focus here will be on targeting the critical complement component, activation product, or receptor that is important for the pathophysiology in a variety of disease conditions.

A novel in vitro model for investigating oligodendroglial maturation and myelin deposition under demyelinating and remyelinating conditions: Impact of microglial depletion and repopulation.

Experimental models of multiple sclerosis (MS) have significantly contributed to our understanding of pathophysiology and the development of therapeutic interventions. Various in vivo animal models have successfully replicated key features of MS and associated pathophysiological processes, shedding light on the sequence of events leading to disease initiation, progression, and resolution. Nevertheless, these models often entail substantial costs and prolonged treatment periods. In contrast, in vitro models offer distinct advantages, including cost-effectiveness and precise control over experimental conditions, thereby facilitating more reproducible results. We have developed a novel in vitro model tailored to the study of oligodendroglial maturation and myelin deposition under demyelinating and remyelinating conditions, which encompasses all the cell types present in the central nervous system (CNS). Of note, our model enables the evaluation of microglial cell commitment through a protocol involving their depletion and subsequent repopulation. Given that the development and survival of microglia are critically reliant on colony-stimulating factor-1 receptor (CSF-1R) signaling, we have employed CSF-1R inhibition to effectively deplete microglia. This versatile model holds promise for the assessment of potential therapies aimed at promoting oligodendroglial differentiation to safeguard and repair myelin, hence mitigate neurodegenerative processes.

Establishment and Application of a Novel In Vitro Model of Microglial Activation in Traumatic Brain Injury.

Mechanical impact-induced primary injury after traumatic brain injury (TBI) leads to acute microglial pro-inflammatory activation and consequently mediates neurodegeneration, which is a major secondary brain injury mechanism. However, the detailed pathologic cascades have not been fully elucidated, partially because of the pathologic complexity in animal TBI models. Although there are several in vitro TBI models, none of them closely mimic post-TBI microglial activation. In the present study, we aimed to establish an in vitro TBI model, specifically reconstituting the pro-inflammatory activation and associated neurodegeneration following TBI. We proposed three sets of experiments. First, we established a needle scratch injured neuron-induced microglial activation and neurodegeneration in vitro model of TBI. Second, we compared microglial pro-inflammatory cytokines profiles between the in vitro TBI model and TBI in male mice. Additionally, we validated the role of injured neurons-derived damage-associated molecular patterns in amplifying microglial pro-inflammatory pathways using the in vitro TBI model. Third, we applied the in vitro model for the first time to characterize the cellular metabolic profile of needle scratch injured-neuron-activated microglia, and define the role of metabolic reprogramming in mediating pro-inflammatory microglial activation and mediated neurodegeneration. Our results showed that we successfully established a novel in vitro TBI model, which closely mimics primary neuronal injury-triggered microglial pro-inflammatory activation and mediated neurodegeneration after TBI. This in vitro model provides an advanced and highly translational platform for dissecting interactions in the pathologic processes of neuronal injury-microglial activation-neuronal degeneration cascade, and elucidating the detailed underlying cellular and molecular insights after TBI.SIGNIFICANCE STATEMENT Microglial activation is a key component of acute neuroinflammation that leads to neurodegeneration and long-term neurologic outcome deficits after TBI. However, it is not feasible to truly dissect primary neuronal injury-induced microglia activation, and consequently mediated neurodegeneration in vivo Furthermore, there is currently lacking of in vitro TBI models closely mimicking the TBI primary injury-mediated microglial activation. In this study, we successfully established and validated a novel in vitro TBI model of microglial activation, and for the first time, characterized the cellular metabolic profile of microglia in this model. This novel microglial activation in vitro TBI model will help in elucidating microglial inflammatory activation and consequently associated neurodegeneration after TBI.

Human lung organoid: Models for respiratory biology and diseases.

The human respiratory system, consisting of the airway and alveoli, is one of the most complex organs directly interfaced with the external environment. The diverse epithelial cells lining the surface are usually the first cell barrier that comes into contact with pathogens that could lead to deadly pulmonary disease. There is an urgent need to understand the mechanisms of self-renewal and protection of these epithelial cells against harmful pathogens, such as SARS-CoV-2. Traditional models, including cell lines and mouse models, have extremely limited native phenotypic features. Therefore, in recent years, to mimic the complexity of the lung, airway and alveoli organoid technology has been developed and widely applied. TGF-ß/BMP/SMAD, FGF and Wnt/ß-catenin signaling have been proven to play a key role in lung organoid expansion and differentiation. Thus, we summarize the current novel lung organoid culture strategies and discuss their application for understanding the lung biological features and pathophysiology of pulmonary diseases, especially COVID-19. Lung organoids provide an excellent in vitro model and research platform.

Omic Studies on In Vitro Cystinosis Model: siRNA-Mediated CTNS Gene Silencing in HK-2 Cells.

Cystinosis is an autosomal recessive disease caused by mutations in the CTNS gene encoding a protein called cystinosine, which is a lysosomal cystine transporter. Disease-causing mutations lead to accumulation of cystine crystals in the lysosomes, thereby causing dysfunction of vital organs. Determination of the increased leukocyte cystine level is one of the most used methods for diagnosis. However, this method is expensive, difficult to perform, and may yield different results in different laboratories. In this study, a disease model was created with CTNS gene-silenced HK2 cells, which can mimic cystinosis in cell culture, and multiomics methods (ie, proteomics, metabolomics, and fluxomics) were implemented at this cell culture to investigate new biomarkers for the diagnosis. CTNS-silenced cell line exhibited distinct metabolic profiles compared with the control cell line. Pathway analysis highlighted significant alterations in various metabolic pathways, including alanine, aspartate, and glutamate metabolism; glutathione metabolism; aminoacyl-tRNA biosynthesis; arginine and proline metabolism; beta-alanine metabolism; ascorbate and aldarate metabolism; and histidine metabolism upon CTNS silencing. Fluxomics analysis revealed increased cycle rates of Krebs cycle intermediates such as fumarate, malate, and citrate, accompanied by enhanced activation of inorganic phosphate and ATP production. Furthermore, proteomic analysis unveiled differential expression levels of key proteins involved in crucial cellular processes. Notably, peptidyl-prolyl cis-trans isomerase A, translation elongation factor 1-beta (EF-1beta), and 60S acidic ribosomal protein decreased in CTNS-silenced cells. Additionally, levels of P0 and tubulin α-1A chain were reduced, whereas levels of 40S ribosomal protein S8 and Midasin increased. Overall, our study, through the utilization of an in vitro cystinosis model and comprehensive multiomics approach, led to the way toward the identification of potential new biomarkers while offering valuable insights into the pathogenesis of cystinosis.

Identifying and mathematically modeling the time-course of extracellular metabolic markers associated with resistance to ceftolozane/tazobactam in Pseudomonas aeruginosa.

Extracellular bacterial metabolites have potential as markers of bacterial growth and resistance emergence but have not been evaluated in dynamic in vitro studies. We investigated the dynamic metabolomic footprint of a multidrug-resistant hypermutable Pseudomonas aeruginosa isolate exposed to ceftolozane/tazobactam as continuous infusion (4.5 g/day, 9 g/day) in a hollow-fiber infection model over 7-9 days in biological replicates (n = 5). Bacterial samples were collected at 0, 7, 23, 47, 71, 95, 143, 167, 191, and 215 h, the supernatant quenched, and extracellular metabolites extracted. Metabolites were analyzed via untargeted metabolomics, including hierarchical clustering and correlation with quantified total and resistant bacterial populations. The time-courses of five (of 1,921 detected) metabolites from enriched pathways were mathematically modeled. Absorbed L-arginine and secreted L-ornithine were highly correlated with the total bacterial population (r -0.79 and 0.82, respectively, P<0.0001). Ribose-5-phosphate, sedoheptulose-7-phosphate, and trehalose-6-phosphate correlated with the resistant subpopulation (0.64, 0.64, and 0.67, respectively, P<0.0001) and were likely secreted due to resistant growth overcoming oxidative and osmotic stress induced by ceftolozane/tazobactam. Using pharmacokinetic/pharmacodynamic-based transduction models, these metabolites were successfully modeled based on the total or resistant bacterial populations. The models well described the abundance of each metabolite across the differing time-course profiles of biological replicates, based on bacterial killing and, importantly, resistant regrowth. These proof-of-concept studies suggest that further exploration is warranted to determine the generalizability of these findings. The metabolites modeled here are not exclusive to bacteria. Future studies may use this approach to identify bacteria-specific metabolites correlating with resistance, which would ultimately be extremely useful for clinical translation.

An in vitro model for osteoarthritis using long-cultured inflammatory human macrophages repeatedly stimulated with TLR agonists.

Osteoarthritis (OA) is characterized by an abundance of inflammatory M1-like macrophages damaging local tissues. The search for new potential drugs for OA suffers from the lack of appropriate methods of long-lasting inflammation. Here we developed and characterized an in vitro protocol of long-lasting culture of primary human monocyte-derived macrophages differentiated with a combination of M-CSF+GM-CSF that optimally supported long-cultured macrophages (LC-Mϕs) for up to 15 days, unlike their single use. Macrophages repeatedly stimulated for 15 days with the TLR2 ligand Pam3CSK4 (LCS-Mϕs), showed sustained levels over time of IL-6, CCL2, and CXCL8, inflammatory mediators that were also detected in the synovial fluids of OA patients. Furthermore, macrophages isolated from the synovia of two OA patients showed an expression profile of inflammation-related genes similar to that of LCS-Mϕs, validating our protocol as a model of chronically activated inflammatory macrophages. Next, to confirm that these LCS-Mϕs could be modulated by anti-inflammatory compounds, we employed dexamethasone and/or celecoxib, two drugs widely used in OA treatment, that significantly inhibited the production of inflammatory mediators. This easy-to-use in vitro protocol of long-lasting inflammation with primary human macrophages could be useful for the screening of new compounds to improve the therapy of inflammatory disorders.

Utility of human induced pluripotent stem cell-derived small intestinal epithelial cells for pharmacokinetic, toxicological, and immunological studies.

The small intestine, which plays a crucial role in the absorption and metabolism of drugs and foods, serves as a target organ for drug-induced toxicity and immune interactions with functional foods and intestinal bacteria. Current alternative models of the human small intestine, such as Caco-2 cells and experimental animals, have limitations due to variations in the expression levels of metabolic enzymes, transporters, and receptors. This study presents investigations into the utility of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) for pharmacokinetic, toxicological, and immunological studies, respectively. While hiSIECs displayed small intestinal epithelial cell characteristics and barrier function, they demonstrated pharmacokinetic properties such as cytochrome P450 3A4/5 activity equivalent to human primary enterocytes and stable P-glycoprotein activity. These cells also demonstrated potential for assessing two forms of intestinal toxicity caused by anticancer drugs and gamma-secretase inhibitors, displaying immune responses mediated by toll-like and fatty acid receptors while serving as an inflammatory gut model through the addition of tumor necrosis factor alpha and interferon gamma. Overall, hiSIECs hold promise as an in vitro model for assessing pharmacokinetics, toxicity, and effects on the intestinal immunity of pharmaceuticals, functional foods, supplements, and intestinal bacteria.

Progresses in the establishment, evaluation, and application of in vitro blood-brain barrier models.

The blood-brain barrier (BBB) is a barrier between the circulatory system and the central nervous system (CNS), contributing to CNS protection and maintaining the brain homeostasis. Establishment of in vitro BBB models that are closer to the microenvironment of the human brain is helpful for evaluating the potential and efficiency of a drug penetrating BBB and thus the clinical application value of the drug. The in vitro BBB models not only provide great convenience for screening new drugs that can access to CNS but also help people to have a deeper study on the mechanism of substances entering and leaving the brain, which makes people have greater opportunities in the treatment of CNS diseases. Up to now, although much effort has been paid to the researches on the in vitro BBB models and many progresses have been achieved, no unified method has been described for establishing a BBB model and there is much work to do and many challenges to be faced with in the future. This review summarizes the research progresses in the establishment, evaluation, and application of in vitro BBB models.

Induction of in vivo-like ciliation in confluent monolayers of re-differentiated equine oviduct epithelial cells.

We recently developed re-differentiated equine oviduct epithelial cell (REOEC) monolayers demonstrating various in vivo morphological characteristics, but lacking secondary ciliation. In this study, we evaluated the effects of fetal bovine serum, reproductive steroid hormones, Wnt- and Notch ligands and inhibitors, and different EOEC seeding densities, in both conventional wells and on microporous membranes, on EOEC morphology and, in particular, secondary ciliation. REOEC monolayers were assessed by confocal microscopy after combined staining of nuclei, cilia and the cytoskeleton. Only Wnt ligands, Notch inhibitors and oviduct explant cell concentration affected EOEC morphology. Undesirable epithelial-mesenchymal transition was observed in REOEC monolayers exposed to Wnt3a containing medium and Wnt ligand CHIR 99021. With respect to secondary ciliation, only the combined effect of oviduct explant cell concentration and Notch inhibition steered REOEC monolayers to in vivo-like ciliation patterns. De-differentiated EOECs, formed 10 days after oviduct explant cell seeding, were reseeded on inserts; only at initial oviduct explant cell concentrations of 1 and 5 x106 cells per well was the formation of REOEC monolayers with a high rate of diffuse ciliation supported. Within 1 month after air-liquid interface introduction, >40% and > 20% of the REOECs showed secondary cilia, respectively. At higher oviduct explant cell seeding densities secondary ciliation was not supported after re-differentiation. Additionally, Notch inhibition helped boost secondary ciliation rates to >60% in REOEC monolayers with diffuse ciliation only. These monolayers demonstrated higher clathrin expression under follicular phase conditions. Overall, the ciliated REOEC monolayers better resemble in vivo oviduct epithelial cells than previous models.

Arrangement into layers and mechanobiology of multi-cell co-culture models of the uterine wall.

STUDY QUESTION: Can a co-culture of three cell types mimic the in vivo layers of the uterine wall? SUMMARY ANSWER: Three protocols tested for co-culture of endometrial epithelial cells (EEC), endometrial stromal cells (ESC), and myometrial smooth muscle cells (MSMC) led to formation of the distinct layers that are characteristic of the structure of the uterine wall in vivo. WHAT IS KNOWN ALREADY: We previously showed that a layer-by-layer co-culture of EEC and MSMC responded to peristaltic wall shear stresses (WSS) by increasing the polymerization of F-actin in both layers. Other studies showed that WSS induced significant cellular alterations in epithelial and endothelial cells. STUDY DESIGN, SIZE, DURATION: Human EEC and ESC cell lines and primary MSMC were co-cultured on a collagen-coated synthetic membrane in custom-designed wells. The co-culture model, created by seeding a mixture of all cells at once, was exposed to steady WSS of 0.5 dyne/cm2 for 10 and 30 min. PARTICIPANTS/MATERIALS, SETTING, METHODS: The co-culture of the three different cells was seeded either layer-by-layer or as a mixture of all cells at once. Validation of the models was by specific immunofluorescence staining and confocal microscopy. Alterations of the cytoskeletal F-actin in response to WSS were analyzed from the 2-dimensional confocal images through the Z-stacks following a previously published algorithm. MAIN RESULTS AND THE ROLE OF CHANCE: We generated three multi-cell in vitro models of the uterine wall with distinct layers of EEC, ESC, and MSMC that mimic the in vivo morphology. Exposure of the mixed seeding model to WSS induced increased polymerization of F-actin in all the three layers relative to the unexposed controls. Moreover, the increased polymerization of F-actin was higher (P-value < 0.05) when the length of exposure was increased from 10 to 30 min. Furthermore, the inner layers of ESC and MSMC, which are not in direct contact with the applied shearing fluid, also increased their F-actin polymerization. LARGE SCALE DATA: N/A. LIMITATIONS, RESONS FOR CAUTION: The mixed seeding co-culture model was exposed to steady WSS of one magnitude, whereas the uterus is a dynamic organ with intra-uterine peristaltic fluid motions that vary in vivo with different time-dependent magnitude. Further in vitro studies may explore the response to peristaltic WSS or other physical and/or hormonal perturbations that may mimic the spectrum of pathophysiological aspects. WIDER IMPLICATIONS OF THE FINDINGS: Numerous in vitro models were developed in order to mimic the human endometrium and endometrium-myometrium interface (EMI) region. The present co-culture models seem to be the first constructed from EEC, ESC, and MSMC on a collagen-coated synthetic membrane. These multi-cell in vitro models better represent the complex in vivo anatomy of the EMI region. The mixed seeding multi-cell in vitro model may easily be implemented in controlled studies of uterine function in reproduction and the pathogenesis of diseases. STUDY FINDING/COMPETING INTEREST(S): This study was supported in part by Tel Aviv University funds. All authors declare no conflict of interest.

Chronic arsenic exposure induces malignant transformation of human HaCaT cells through both deterministic and stochastic changes in transcriptome expression.

Biological processes are inherently stochastic, i.e., are partially driven by hard to predict random probabilistic processes. Carcinogenesis is driven both by stochastic and deterministic (predictable non-random) changes. However, very few studies systematically examine the contribution of stochastic events leading to cancer development. In differential gene expression studies, the established data analysis paradigms incentivize expression changes that are uniformly different across the experimental versus control groups, introducing preferential inclusion of deterministic changes at the expense of stochastic processes that might also play a crucial role in the process of carcinogenesis. In this study, we applied simple computational techniques to quantify: (i) The impact of chronic arsenic (iAs) exposure as well as passaging time on stochastic gene expression and (ii) Which genes were expressed deterministically and which were expressed stochastically at each of the three stages of cancer development. Using biological coefficient of variation as an empirical measure of stochasticity we demonstrate that chronic iAs exposure consistently suppressed passaging related stochastic gene expression at multiple time points tested, selecting for a homogenous cell population that undergo transformation. Employing multiple balanced removal of outlier data, we show that chronic iAs exposure induced deterministic and stochastic changes in the expression of unique set of genes, that populate largely unique biological pathways. Together, our data unequivocally demonstrate that both deterministic and stochastic changes in transcriptome-wide expression are critical in driving biological processes, pathways and networks towards clonal selection, carcinogenesis, and tumor heterogeneity.

A 3D in vitro model for assessing the influence of intraocular lens: Posterior lens capsule interactions on lens epithelial cell responses.

Posterior Capsule Opacification (PCO), the most frequent complication of cataract surgery, is caused by the infiltration and proliferation of lens epithelial cells (LECs) at the interface between the intraocular lens (IOL) and posterior lens capsule (PLC). According to the "no space, no cells, no PCO" theory, high affinity (or adhesion force) between the IOL and PLC would decrease the IOL: PLC interface space, hinder LEC migration, and thus reduce PCO formation. To test this hypothesis, an in vitro hemisphere-shaped simulated PLC (sPLC) was made to mimic the human IOL: PLC physical interactions and to assess their influence on LEC responses. Three commercially available IOLs with different affinities/adhesion forces toward the sPLC, including Acrylic foldable IOL, Silicone IOL, and PMMA IOL, were used in this investigation. Using the system, the physical interactions between IOLs and sPLC were quantified by measuring the adhesion force and interface space using an adhesion force apparatus and Optical Coherence Tomography, respectively. Our data shows that high adhesion force and tight binding between IOL and sPLC contribute to a small interface space (or "no space"). By introducing LECs into the in vitro system, we found that, with small interface space, among all IOLs, acrylic foldable IOLs permitted the least extent of LEC infiltration, proliferation, and differentiation (or "no cells"). Further statistical analyses using clinical data revealed that weak LEC responses are associated with low clinical PCO incidence rates (or "no PCO"). The findings support that the in vitro system could simulate IOL: PLC interplays and predict IOLs' PCO potential in support of the "no space, no cells, no PCO" hypothesis.

Evaluation of renovated double J stents using ureter models with and without stenosis.

PURPOSE: Commercial double J stents (DJS) have a uniform shape regardless of the specific nature of various ureteral diseases. We tested renovated DJS and compared them with conventional DJS using ureter models. METHODS: One straight ureter model included stenosis at the distal ureter near the ureterovesical junction and the other did not. We used conventional DJS and renovated 5- and 6-Fr soft DJS for ureter stones and 6-, 7-, and 8.5-Fr hard DJS for tumors. The DJS comprised holes in the upper, middle, or lower one-third of the shaft (length, 24 cm; 2-cm-diameter coils at both ends). More holes were created along the shaft based on the ureteral disease location. Conventional DJS had holes spaced 1 cm apart along the shaft. Renovated DJS had holes spaced 1 cm apart along the shaft with 0.5-cm intervals on the upper, middle, or lower one-third of the shaft. Urine flow was evaluated. RESULTS: As the DJS diameter increased, the flow rate decreased. The flow rates of DJS with holes in the lower shaft were relatively lower than those of conventional DJS and DJS with holes in the upper and middle shafts. In the ureter model without stenosis, 6-, 7-, and 8.5-Fr renovated stents exhibited significantly higher flow rates than conventional stents. In the ureter model with stenosis, 5-, 6-, 7-, and 8.5-Fr renovated stents did not exhibit significantly higher flow rates than conventional stents. CONCLUSION: Renovated stents and conventional stents did not exhibit significant differences in urine flow with stenosis.

Isolation and propagation of bovine blood-derived macrophages using a mixed culture with bovine endothelial B46 cells.

Macrophages are innate immune cells with multiple functions such as phagocytosis, cytokine production, and antigen presentation. Since macrophages play critical roles in some bacterial infectious diseases in cattle, including tuberculosis, paratuberculosis, and brucellosis, the in vitro culturing of bovine macrophages is useful for evaluating host-pathogen interactions at the cellular and molecular levels. We have previously reported the establishment of two immortalized bovine liver sinusoidal cell lines, endothelial B46 cells and myofibroblast-like A26 cells (Cell Biology International, 40, 1372-1379, 2016). In this study, we investigated the use of these cell lines as feeder cells that support the proliferation of bovine blood-derived macrophages (BBMs). Notably, the B46 cell line efficiently acts as feeder cells for the propagation of BBMs. Compared with primary cultured vascular endothelial cells, the infinite proliferation ability of B46 cells is more beneficial for preparing confluent feeder layers. In conclusion, this study provides a simple and efficient protocol for the isolation and propagation of BBMs using a primary mixed culture of bovine whole blood with B46 feeder cells. Isolated BBMs are expected to be useful for developing in vitro models for studying the interactions between bovine pathogens and host immune cells.

Functional hydrogels for hepatocellular carcinoma: therapy, imaging, and in vitro model.

Hepatocellular carcinoma (HCC) is among the most common malignancies worldwide and is characterized by high rates of morbidity and mortality, posing a serious threat to human health. Interventional embolization therapy is the main treatment against middle- and late-stage liver cancer, but its efficacy is limited by the performance of embolism, hence the new embolic materials have provided hope to the inoperable patients. Especially, hydrogel materials with high embolization strength, appropriate viscosity, reliable security and multifunctionality are widely used as embolic materials, and can improve the efficacy of interventional therapy. In this review, we have described the status of research on hydrogels and challenges in the field of HCC therapy. First, various preparation methods of hydrogels through different cross-linking methods are introduced, then the functions of hydrogels related to HCC are summarized, including different HCC therapies, various imaging techniques, in vitro 3D models, and the shortcomings and prospects of the proposed applications are discussed in relation to HCC. We hope that this review is informative for readers interested in multifunctional hydrogels and will help researchers develop more novel embolic materials for interventional therapy of HCC.

Characterization of the Secretome, Transcriptome, and Proteome of Human ß Cell Line EndoC-ßH1.

Early diabetes research is hampered by limited availability, variable quality, and instability of human pancreatic islets in culture. Little is known about the human ß cell secretome, and recent studies question translatability of rodent ß cell secretory profiles. Here, we verify representativeness of EndoC-ßH1, one of the most widely used human ß cell lines, as a translational human ß cell model based on omics and characterize the EndoC-ßH1 secretome. We profiled EndoC-ßH1 cells using RNA-seq, data-independent acquisition, and tandem mass tag proteomics of cell lysate. Omics profiles of EndoC-ßH1 cells were compared to human ß cells and insulinomas. Secretome composition was assessed by data-independent acquisition proteomics. Agreement between EndoC-ßH1 cells and primary adult human ß cells was ∼90% for global omics profiles as well as for ß cell markers, transcription factors, and enzymes. Discrepancies in expression were due to elevated proliferation rate of EndoC-ßH1 cells compared to adult ß cells. Consistently, similarity was slightly higher with benign nonmetastatic insulinomas. EndoC-ßH1 secreted 783 proteins in untreated baseline state and 3135 proteins when stressed with nontargeting control siRNA, including known ß cell hormones INS, IAPP, and IGF2. Further, EndoC-ßH1 secreted proteins known to generate bioactive peptides such as granins and enzymes required for production of bioactive peptides. EndoC-ßH1 secretome contained an unexpectedly high proportion of predicted extracellular vesicle proteins. We believe that secretion of extracellular vesicles and bioactive peptides warrant further investigation with specialized proteomics workflows in future studies.

Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

BACKGROUND: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. METHODS: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. RESULTS: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. CONCLUSION: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.