Hanging-drop Method Research Articles

Effect of viruses infection symptoms on pollen viability in different horticultural crop species

A preliminary in vitro investigation was carried out to assess the impact of virus infection symptoms on in vitro pollen germination in different horticultural crop species. Pollen from healthy and virus-infected papaya (papaya ring spot virus—PRSV), gherkin (Ilar virus), okra (Ilar virus), bottle gourd (mosaic virus), chow-chow (mosaic virus), okra (yellow vein mosaic virus -YVMV), watermelon (mosaic), pumpkin (mosaic), watermelon (bud necrosis) and bitter gourd (yellow mosaic) flowers was collected, and pollen was extracted and to assessed viability in vitro via the hanging drop method. After incubation, the viability profiles of the pollen were computed. There was a significant reduction in the pollen germination profiles of pollen from infected plants compared to those from healthy plants. The impact of virus infection on pollen production, germination and viability is discussed in light of the overall reproductive ability of virus-affected plants.

Therapeutic effect of three-dimensional hanging drop cultured human umbilical cord mesenchymal stem cells on osteoarthritis in rabbits

BackgroundMesenchymal stem cells (MSCs) have shown a positive effect on Osteoarthritis (OA), but the efficacy is still not significant in clinical. Conventional two-dimensional (2D) monolayer culture method is prone to cause MSCs undergoing replication senescence, which may affect the functions of MSCs. Three-dimensional (3D) culture strategy can sustain cell proliferative capacity and multi-differentiation potential. This study aimed to investigate the therapeutic potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) cultured by 3D hanging drop method on OA.MethodshUC-MSCs were isolated from umbilical cord and cultured by 3D hanging drop method for 48 h. Scanning electron microscopy (SEM) was used to observe gross morphology 2D and 3D hUC-MSCs. Transcriptome comparison of gene expression differences between 2D and 3D hUC-MSCs. GO enrichment analysis, KEGG pathway enrichment analysis and GSEA enrichment analysis were used to analyze the impact of 3D hanging drop culture on the biological functions of hUC-MSCs. Female New Zealand rabbits (n = 12) were divided into 4 groups: Normal group, Model group, 2D hUC-MSCs treatment group and 3D hUC-MSCs treatment group. After 8 weeks, the gross and histological appearance of the cartilage was evaluated by safranin O-fast green staining and Mankin scoring system. The expression of type I collagen and type II collagen was detected by immunohistochemistry. The levels of IL-6, IL-7, TNFα, TGFβ1 and IL-10 in the knee joint fluid were tested by ELISA.Results3D hanging drop culture changed cell morphology but did not affect phenotype. The MSCs transcriptome profiles showed that 3D hanging drop culture method enhanced cell-cell contact, improved cell responsiveness to external stimuli and immunomodulatory function. The animal experiment results showed that hUC-MSCs could promote cartilage regeneration compared with Model group. 3D hUC-MSCs treatment group had a higher histological score and significantly increased type II collagen secretion. In addition, 3D hUC-MSCs treatment group increased the expression of anti-inflammatory factors TGFβ1 and IL-10.ConclusionThe above experimental results illustrated that 3D hanging drop culture method could enhance the therapeutic effect of hUC-MSCs, and showed a good clinical application prospect in the treatment of OA.

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Shaping in the Third Direction: Colloidal Photonic Crystals with Quadratic Surfaces Self-Assembled by Hanging-Drop Method.

High-quality, 3D-shaped, SiO2 colloidal photonic crystals (ellipsoids, hyperboloids, and others) were fabricated by self-assembly. They possess a quadratic surface and are wide-angle-independent, direction-dependent, diffractive reflection crystals. Their size varies between 1 and 5 mm and can be achieved as mechanical-resistant, free-standing, thick (hundreds of ordered layers) objects. High-quality, 3D-shaped, polystyrene inverse-opal photonic superstructures (highly similar to diatom frustules) were synthesized by using an inside infiltration method as wide-angle-independent, reflective diffraction objects. They possess multiple reflection bands given by their special architecture (a torus on the top of an ellipsoid) and by their different sized holes (384 nm and 264 nm). Our hanging-drop self-assembly approach uses setups which deform the shape of an ordinary spherical drop; thus, the colloidal self-assembly takes place on a non-axisymmetric liquid/air interface. The deformed drop surface is a kind of topological interface which changes its shape in time, remaining as a quality template for the self-assembly process. Three-dimensional-shaped colloidal photonic crystals might be used as devices for future spectrophotometers, aspheric or freeform diffracting mirrors, or metasurfaces for experiments regarding space-time curvature analogy.

The Effects of Endometrial Mesenchymal Stem Cells on The In Vitro Maturation of Germinal Vesicle Oocytes in Hanging Drop and Sodium Alginate Hydrogel Co-Culture Systems.

The aim of this study is to investigate the co-culture effects of human endometrial mesenchymal stem cells (EnMSCs) with mouse oocytes to enhance their maturation and development by using the hanging drop and sodium alginate hydrogel methods. In this experimental study, we prepared human EnMSCs (2.5×105 cells/mL) and co-cultured them with partially denuded mouse oocytes by the hanging drop (n=120) and sodium alginate hydrogel (n=120) methods. Control oocytes (n=230, total) were cultured in both systems in the absence of human EnMSCs for 18 hours. Both survival and maturation rates of the oocytes were analysed morphologically. After insemination with capacitated sperm, the fertilization and development of the embryos up to the blastocyst stage were assessed and compared statistically for all of the study groups via one-way ANOVA and the t tests. Oocytes cultured in the hanging drop method had a significantly higher survival rate than their control group (92.60 ± 4.36% vs. 84.20 ± 3.12%, P=0.018). There were no significant differences between the two experimental groups in terms of survival. The mean percent of oocytes that reached the metaphase II (MII) stage was 64.35 ± 3.19% and fertilised was 62.25 ± 4.43% in the hanging drop method; these rates were 63.43 ± 1.92% and 58.14 ± 4.14 in sodium alginate hydrogel method, respectively. These rates were higher than their controls (P<0.050), but there were no statistical differences between the two experimental groups (P>0.050). Among the studied groups, the highest significant blastocyst rate (32.55 ± 2.18%) was observed in the hanging drop experimental group (P=0.0017). The results of this study show that human EnMSCs improve the survival, maturation, and development rates of oocytes and they could have future clinical applications.

SpheroMold: modernizing the hanging drop method for spheroid culture

The hanging drop method is a cost-effective approach for 3D spheroid culture. However, obtaining numerous spheroids in a limited area becomes challenging due to the risk of droplet coalescence, primarly during Petri dish handling. In this study, we describe a general method to fabricate a 3D printing-based support called SpheroMold that facilitates Petri dish handling and enhances spheroid production per unit area. As a proof-of-concept, we designed a digital negative mold which comprised 37 pegs within a 13.52 cm2 area, and then printed it using stereolithography; the density of pegs can be adjusted according to user requirements. The SpheroMold was created by pouring the base and curing agent (10:1) (Sylgard® 184 silicone) into the mold, curing it at 80°C, and then attaching it to the lid of a Petri dish. Our SpheroMold effectively prevented droplet coalescence during Petri dish inversion, enabling the production of numerous 3D spheroids while simplifying manipulation. Unlike conventional techniques, our design also facilitated a larger volume of culture medium per drop compared to a standard Petri dish, potentially decreasing the necessity for frequent medium exchange to sustain cellular health and reducing labor intensity.

Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells.

Embryonic stem cells (ESCs) serve as a crucial ex vivo model, representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos. ESCs exhibit a unique combination of self-renewal potency, unlimited proliferation, and pluripotency. The latter is evident by the ability of the isolated cells to differentiate spontaneously into multiple cell lineages, representing the three primary embryonic germ layers. Multiple regulatory networks guide ESCs, directing their self-renewal and lineage-specific differentiation. Apoptosis, or programmed cell death, emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development. However, the molecular mechanisms underlying the dynamic interplay between differentiation and apoptosis remain poorly understood. To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells, using mouse ESC (mESC) models - mESC-B-cell lymphoma 2 (BCL-2), mESC-PIM-2, and mESC-metallothionein-1 (MET-1) - which overexpress the anti-apoptotic genes Bcl-2, Pim-2, and Met-1, respectively. mESC-T2 (wild-type), mESC-BCL-2, mESC-PIM-2, and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation. The hanging drop method was adopted to generate embryoid bodies (EBs) and induce terminal differentiation of mESCs. The size of the generated EBs was measured in each condition compared to the wild type. At the functional level, the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control. At the molecular level, quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers: Troponin T, GATA4, and NKX2.5. Additionally, troponin T protein expression was evaluated through immunofluorescence and western blot assays. Our findings showed that the upregulation of Bcl-2, Pim-2, and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs, in comparison with their wild-type counterpart. Additionally, a decrease in the count of beating cardiomyocytes among differentiated cells was observed. Furthermore, the mRNA expression of three cardiac markers - troponin T, GATA4, and NKX2.5 - was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line. Moreover, the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression. Our findings revealed that the upregulation of Bcl-2, Pim-2, and Met-1 genes altered cardiac differentiation, providing insight into the intricate interplay between apoptosis and ESC fate determination.

Investigations of cardiac fibrosis rheology by in vitro cardiac tissue modeling with 3D cellular spheroids

Cardiac fibrosis refers to the abnormal accumulation of extracellular matrix within the cardiac muscle, leading to increased stiffness and impaired heart function. From a rheological standpoint, knowledge about myocardial behavior is still lacking, partially due to a lack of appropriate techniques to investigate the rheology of in vitro cardiac tissue models. 3D multicellular cardiac spheroids are powerful and versatile platforms for modeling healthy and fibrotic cardiac tissue in vitro and studying how their mechanical properties are modulated. In this study, cardiac spheroids were created by co-culturing neonatal rat ventricular cardiomyocytes and fibroblasts in definite ratios using the hanging-drop method. The rheological characterization of such models was performed by Atomic Force Microscopy-based stress-relaxation measurements on the whole spheroid. After strain application, a viscoelastic bi-exponential relaxation was observed, characterized by a fast relaxation time (τ1) followed by a slower one (τ2). In particular, spheroids with higher fibroblasts density showed reduction for both relaxation times comparing to control, with a more pronounced decrement of τ1 with respect to τ2. Such response was found compatible with the increased production of extracellular matrix within these spheroids, which recapitulates the main feature of the fibrosis pathophysiology. These results demonstrate how the rheological characteristics of cardiac tissue vary as a function of cellular composition and extracellular matrix, confirming the suitability of such system as an in vitro preclinical model of cardiac fibrosis.

Abstract 4269: Effect of PAX3-FOXO1 fusion gene on cell-extracellular matrix interaction of Rhabdomyosarcoma

Abstract Background Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma, categorized by two subtypes, fusion protein-positive (FPRMS) and negative (FNRMS), based on the existence of PAX3-FOXO1 fusion gene (P3F). Different features between the two subtypes could guide the use of distinct treatment approaches. We discovered that FNRMS exhibited enrichment of gene sets related to cell-extracellular matrix (ECM) interaction than FPRMS. ECM, providing physical support as a scaffold, regulates cancer development. We hypothesize that a distinct molecular feature, P3F, may cause the difference in cell-ECM interaction between FPRMS and FNRMS. Methods To confirm the higher cell-ECM interaction of FNRMS vs. FPRMS, we used confocal reflection microscopy (CRM), enabling us to monitor the fibrous structures of ECM. We generated the spheroids with FNRMS and FPRMS cell lines by hanging drop method and embedded them into collagen type I. Then, we assessed orientation and dynamic changes in the structure of collagen fibers surrounding spheroids. Also, we analyze the transcriptome of FPRMS cells with P3F knockdown to confirm if P3F regulates cell-ECM interaction negatively via RNA seq. Additionally, we examined ECM surrounding spheroids of P3F knockdown FPRMS cells if P3F modulates fibrous structures of ECM, indicating cell-ECM interaction. Results We showed that collagen fibers are oriented more perpendicular to the FNRMS spheroid surface, indicating the strong pulling and pushing effect of cells. However, fibers surrounding FPRMS spheroids showed parallel alignment to their surfaces, reflecting passively pushed by the spheroids. Additionally, fibers near FNRMS spheroids showed greater displacement than those near FNRMS spheroids, which suggests stronger cell-ECM interaction in FNRMS than in FPRMS. Furthermore, P3F knockdown FPRMS cells exhibited significant enrichment of gene sets related to cell-ECM interaction. Their CRM results showed that collagen fiber orientation was significantly perpendicular to the surfaces of spheroids of P3F knockdown FPRMS cells than those of control cells. P3F knockdown cell spheroids also displayed enhanced displacement of ECM fibers than control cells. These results suggest that P3F suppresses cell-ECM interaction, consistent with higher cell-ECM interaction of FNRMS than FPRMS. Conclusions The PAX3-FOXO1 gene may regulate cell-ECM interaction negatively, causing distinct features between FPRMS and FNRMS, which is crucial for various cellular functions and suggest different treatment between the two RMS subtypes. Citation Format: Chandra Kaladhar Vemula, Ivan Chavez, Anthony Chronopoulos, Jinseok Park. Effect of PAX3-FOXO1 fusion gene on cell-extracellular matrix interaction of Rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4269.

Heart rhythm in vitro: measuring stem cell-derived pacemaker cells on microelectrode arrays.

Cardiac arrhythmias have markedly increased in recent decades, highlighting the urgent need for appropriate test systems to evaluate the efficacy and safety of new pharmaceuticals and the potential side effects of established drugs. The Microelectrode Array (MEA) system may be a suitable option, as it provides both real-time and non-invasive monitoring of cellular networks of spontaneously active cells. However, there is currently no commercially available cell source to apply this technology in the context of the cardiac conduction system (CCS). In response to this problem, our group has previously developed a protocol for the generation of pure functional cardiac pacemaker cells from mouse embryonic stem cells (ESCs). In addition, we compared the hanging drop method, which was previously utilized, with spherical plate-derived embryoid bodies (EBs) and the pacemaker cells that are differentiated from these. We described the application of these pacemaker cells on the MEA platform, which required a number of crucial optimization steps in terms of coating, dissociation, and cell density. As a result, we were able to generate a monolayer of pure pacemaker cells on an MEA surface that is viable and electromechanically active for weeks. Furthermore, we introduced spherical plates as a convenient and scalable method to be applied for the production of induced sinoatrial bodies. We provide a tool to transfer modeling and analysis of cardiac rhythm diseases to the cell culture dish. Our system allows answering CCS-related queries within a cellular network, both under baseline conditions and post-drug exposure in a reliable and affordable manner. Ultimately, our approach may provide valuable guidance not only for cardiac pacemaker cells but also for the generation of an MEA test platform using other sensitive non-proliferating cell types.

Evaluation of the Most Effective Plant Extracts, Bioagents and Chemical Fungicides In vitro against Powdery Mildew of Pea

The present investigation was carried during 2022-23 at Plant Pathology Section, College of Agriculture, Pune-05 with the objectives of evaluation of the most effective plant extracts, bioagents and chemical fungicides in vitro. Effect of six plant extracts, bioagents and chemical fungicides were studied in vitro by hanging drop method where the study revealed that garlic (Allium sativum), followed by neem (Azadiracta indica) showed highest inhibition of spore germination for powdery mildew. After 48 hours of incubation, the spore germination rate ranged from 3.15 % to 34.45 %, where garlic showed the highest inhibition at 95.30 %, followed by neem at 91.03 %. The efficacy of bioagents was evaluated by preparing the bacterial and fungal culture filtrates where Trichoderma harzianum and Pseudomonas fluorescens were found to be the most effective with tune of 56.86% and 50.72% of efficacy respectively against Erysiphe pisi. Among all chemical fungicides, hexaconazole (0.05 %) resulted as the most effective chemical fungicide against powdery mildew of pea. These findings can be useful in developing safer and more eco-friendly methods to control powdery mildew and protect crops.

Morphological, cytotoxic and apoptotic effects of different concentrations of nisin ZP on MG63 cells in a 3-dimensional culture system

Osteosarcoma (OS) is a primary bone cancer that commonly affects young individuals. Despite treatment, it is known to be highly resistant to therapy. Nisin, a bacteriocin released by Lactococcus lactis has been shown to exhibit an apoptotic effect on cancer cells. To better represent in vivo cancer cell constructs, 3-dimensional (3D) culture systems have been used in vitro. This study aims to compare the effects of nisin on OS cell culture in both monolayered and 3D gas-permeable VECELL® G-Plate. Osteosarcoma cells (MG63) were cultured on 3D culture (G-Plate) and normal culture plates. Morphological assessments were carried out through light and scanning electron microscopic examinations. The results showed that the use of a 3D system is more reliable in the development of 3D osteosarcoma models compared to the 2-dimensional (2D) culture system. Specifically, the 3D culture demonstrated the formation of MG63 spheroids, indicating the reliability of the hanging drop methods to produce spheroidal cell morphology. Additionally, the cell viability assay showed a higher IC50 value for 2D groups compared to the 3D groups, with values of 102.56 µg/mL and 33.96 µg/mL, respectively. Finally, a dose-dependent apoptosis was noticed for the apoptotic assay, further demonstrating the effectiveness of the 3D culture system. The effectiveness of nisin ZP as an anticancer agent was observed in both 2D and 3D culture systems. However, the 3D culture system required a higher dose of nisin ZP to demonstrate its potential, indicating that this system could be a promising approach in OS intervention studies.

Principles of Hanging Drop Method (Spheroid Formation) in Cell Culture.

A type of three-dimensional (3D) cell culture models which is simple and easy is hanging drop method. The hanging drop method emerges as a pivotal technique with diverse applications in cancer research and cell biology. This method facilitates the formation of multicellular spheroids, providing a unique environment for studying cell behavior dynamics. The hanging drop method's theoretical underpinning relies on gravity-enforced self-assembly, allowing for cost-effective, reproducible 3D cell cultures with controlled spheroid sizes. The advantages of this approach include its efficiency in producing cellular heterogeneity, particularly in non-adherent 3D cultures, and its ability to create hypoxic spheroids, making it a suitable model for studying cancer. Moreover, the hanging drop method has proven valuable in investigating various aspects such as tissue structure, signaling pathways, immune activation of cancer cells, and notably, cell proliferation. Researchers have utilized the hanging drop method to explore the dynamics of cell proliferation, studying the effects of mesenchymal stem cells (MSC) secretome on cancer cells. The method's application involves co-culturing different cell lines, assessing spheroid formations, and quantifying their sizes over time. These studies have unveiled intricate cell behavior dynamics, demonstrating how the MSC secretome influences cancer cell growth and viability within a three-dimensional co-culture paradigm.

Исследование композитов для создания стабильной красящей пищевой системы эмульсионного типа

Composites to create a food system of the W/O emulsion type with a maximum yield of coloring substances were determined. Refined deodorized sunflower oil, beet juice and its concentrates with a dry matter content of 12.23; 18.73; 30.12 and 64.61, a mixture of mono-, diglycerides and glycerin were studied. By gas chromatography, the mass fraction of fatty acid in sunflower oil was determined from the sum of saturated, mono- and polyunsaturated fatty acids, including palmitic (6.5%), oleic (20.9%), and linoleic (66.4%). The content of betanin, conformational changes in mono-, di- and polysaccharide components, and the presence of peptide structures were determined by spectrophotometric methods in beet juice and concentrates. Flow curves of composites for the emulsion were studied by rheometric methods, and interfacial tension was studied by the hanging drop method. To check the physical and sensory properties of the emulsion, the selected composites were dispersed: sufficient conditions for the process were set for its duration – 10 minutes at a rotation speed of 6000 rpm. It is shown that, to create a food emulsion W/O with stable color characteristics during storage, beet juice concentrates with a dry matter content of 64.61% and betanin 0.54% can be used as a dispersed phase. The critical concentration of micelle formation of the emulsion is achieved by adding 0.5% surfactant. The phenomenon of syneresis at a storage temperature of 4 ±0.5℃ for 30 days was not detected.

Comparison of three-dimensional cell culture techniques of dedifferentiated liposarcoma and their integration with future research.

Background: Dedifferentiated liposarcoma is a formidable sarcoma subtype due to its high local recurrence rate and resistance to medical treatment. While 2D cell cultures are still commonly used, 3D cell culture systems have emerged as a promising alternative, particularly scaffold-based techniques that enable the creation of 3D models with more accurate cell-stroma interactions. Objective: To investigate how 3D structures with or without the scaffold existence would affect liposarcoma cell lines growth morphologically and biologically. Methods: Lipo246 and Lipo863 cell lines were cultured in 3D using four different methods; Matrigel® ECM scaffold method, Collagen ECM scaffold method, ULA plate method and Hanging drop method, in addition to conventional 2D cell culture methods. All samples were processed for histopathological analysis (HE, IHC and DNAscope™), Western blot, and qPCR; moreover, 3D collagen-based models were treated with different doses of SAR405838, a well-known inhibitor of MDM2, and cell viability was assessed in comparison to 2D model drug response. Results: Regarding morphology, cell lines behaved differently comparing the scaffold-based and scaffold-free methods. Lipo863 formed spheroids in Matrigel® but not in collagen, while Lipo246 did not form spheroids in either collagen or Matrigel®. On the other hand, both cell lines formed spheroids using scaffold-free methods. All samples retained liposarcoma characteristic, such as high level of MDM2 protein expression and MDM2 DNA amplification after being cultivated in 3D. 3D collagen samples showed higher cell viability after SAR40538 treatment than 2D models, while cells sensitive to the drug died by apoptosis or necrosis. Conclusion: Our results prompt us to extend our investigation by applying our 3D models to further oncological relevant applications, which may help address unresolved questions about dedifferentiated liposarcoma biology.

3D stem cell spheroids with urchin-like hydroxyapatite microparticles enhance osteogenesis of stem cells.

Cell therapy (also known as cell transplantation) has been considered promising as a next-generation living-cell therapy strategy to surpass the effects of traditional drugs. However, their practical clinical uses and product conversion are hampered by the unsatisfied viability and efficacy of the transplanted cells. Herein, we propose a synergistic enhancement strategy to address these issues by constructing 3D stem cell spheroids integrated with urchin-like hydroxyapatite microparticles (uHA). Specifically, cell-sized uHA microparticles were synthesized via a simple hydrothermal method using glutamic acid (Glu, E) as the co-template with good biocompatibility and structural antimicrobial performance (denoted as E-uHA). Combining with a hanging drop method, stem cell spheroids integrated with E-uHA were successfully obtained by culturing bone marrow mesenchymal stem cells (BMSCs) with a low concentration of the E-uHA suspensions (10 μg mL-1). The resulting composite spheroids of BMSCs/E-uHA deliver a high cellular viability, migration activity, and a superior osteogenic property compared to the 2D cultured counterpart or other BMSC spheroids. This work provides an effective strategy for integrating a secondary bio-functional component into stem cell spheroids for designing more cell therapy options with boosted cellular viability and therapeutic effect.

Avaliação da viabilidade do pólen em algumas variedades de crisântemo em spray no período de armazenamento

Abstract In producing chrysanthemum hybrids, more seed sets per fruit are preferred. Only successful pollination and fertilization allow seeds to de-velop. High pollen viability and pollen germination rate are intimately correlated with successful fertilization. Chrysanthemum pollen and their storage duration have only been the subject of a relatively few investigations. The study aimed to determine pollen's viability and germination during the 4 days, which were kept at 24 ºC. In the present study, two different Chrysanthemum species (Chrysanthemum coronarium L. and Chrysanthemum segetum L.) and two commercial Chrysanthemum varieties (Chic and Haydar) that belong to Chrysanthemum morifolium Ramat. species as spray chrysanthemum pollens were used for experimental material. The grains pollen were stored in an incubator which was 24 ºC and 60% humidity. The pollen viability was tested with TTC (2,3,5 Triphenyl Tetrazolium Chloride) staining test and pollen germination was evaluated hanging drop method with modified ME3-m medium, daily for 4 days including day 0. The results showed that the viability and germination of all pollens used in this study decreased day by day. Depending on the species/varieties, the viability rates ranged from 12.83% to 32.04% on the first day and between 0.57-2.33% on the last day. Pollen germination rates differed between 16.76% - 3.45% on the 0th day and 0.0-0.17% on the 4th day.

Prostate Cancer Organoids for Tumor Modeling and Drug Screening.

Prostate cancer (PCa) is the second most common malignancy and the fifth leading cause of cancer death in men worldwide. Despite its prevalence, the highly heterogenic PCa has shown difficulty to establish representative cell lines that reflect the diverse phenotypes and different stages of the disease in vitro and hence hard to model in preclinical research. The patient-derived organoid (PDO) technique has emerged as a groundbreaking three-dimensional (3D) tumor modeling platform in cancer research. This versatile assay relies on the unique ability of cancer stem cells (CSCs) to self-organize and differentiate into organ-like mini structures. The PDO culture system allows for the long-term maintenance of cancer cells derived from patient tumor tissues. Moreover, it recapitulates the parental tumor features and serves as a superior preclinical model for in vitro tumor representation and personalized drug screening. Henceforth, PDOs hold great promise in precision medicine for cancer. Herein, we describe the detailed protocol to establish and propagate organoids derived from isolated cell suspensions of PCa patient tissues or cell lines using the 3D semisolid Matrigel™-based hanging-drop method. In addition, we highlight the relevance of PDOs as a tool for evaluating drug efficacy and predicting tumor response in PCa patients.

The role of c-Jun for beating cardiomycyte formation in prepared embryonic body

BackgroundMorbidity and mortality associated with cardiovascular diseases, such as myocardial infarction, stem from the inability of terminally differentiated cardiomyocytes to regenerate, and thus repair the damaged myocardial tissue structure. The molecular biological mechanisms behind the lack of regenerative capacity for those cardiomyocytes remains to be fully elucidated. Recent studies have shown that c-Jun serves as a cell cycle regulator for somatic cell fates, playing a key role in multiple molecular pathways, including the inhibition of cellular reprogramming, promoting angiogenesis, and aggravation of cardiac hypertrophy, but its role in cardiac development is largely unknown. This study aims to delineate the role of c-Jun in promoting early-stage cardiac differentiation.MethodsThe c-Jun gene in mouse embryonic stem cells (mESCs) was knocked out with CRISPR-Cas9, and the hanging drop method used to prepare the resulting embryoid bodies. Cardiac differentiation was evaluated up to 9 days after c-Jun knockout (ko) via immunofluorescence, flow cytometric, and qPCR analyses.ResultsCompared to the wild-type control group, obvious beating was observed among the c-Jun-ko mESCs after 6 days, which was also associated with significant increases in myocardial marker expression. Additionally, markers associated with mesoderm and endoderm cell layer development, essential for further differentiation of ESCs into cardiomyocytes, were also up-regulated in the c-Jun-ko cell group.ConclusionsKnocking out c-Jun directs ESCs toward a meso-endodermal cell lineage fate, in turn leading to generation of beating myocardial cells. Thus, c-Jun plays an important role in regulating early cardiac cell development.