In Vitro Cellular & Developmental Biology. Animal最新文献

Insect cell lines are effective tools used in industry and academia. For example, they are used in screening potential insecticides, in making certain proteins for biomedical applications, and in basic research into insect biology. So far, there are no cell lines derived from the black soldier fly, Hermetia illucens (BSF). This may become an issue because BSFs are employed in a range of industrial and household processes. BSFs are used in producing biodiesel, in developing cosmetics and skin creams, and in the production of some medicines and animal feeds. BSF larvae process waste streams from a variety of sources into food for some animals and are also used in household composting. Our BSF cell line, designated BCIRL-HiE0122021-SGS, was developed from eggs using the medium CLG#2 (50% L-15 + 50% EX-CELL 420, with 9% FBS and antibiotics), with many other media being tested. This cell line consists of attached cells with a variety of morphologies and its identity was authenticated using CO1 barcoding. A growth curve was generated and the resulting doubling time was 118 h. We quantified the fatty acid methyl esters (FAMES) and recorded the expected range of saturated, monounsaturated, and polyunsaturated FAMEs, with only trace levels of lauric acid being noted. The BSF cell line is available free of charge by request.

Osteoblast-derived semaphorin3A (Sema3A) has been reported to be involved in bone protection, and Sema3A knockout mice have been reported to exhibit chondrodysplasia. From these reports, Sema3A is considered to be involved in chondrogenic differentiation and skeletal formation, but there are many unclear points about its function and mechanism in chondrogenic differentiation. This study investigated the pharmacological effects of Sema3A in chondrogenic differentiation. The amount of Sema3A secreted into the culture supernatant was measured using an enzyme-linked immunosorbent assay. The expression of chondrogenic differentiation-related factors, such as Type II collagen (COL2A1), Aggrecan (ACAN), hyaluronan synthase 2 (HAS2), SRY-box transcription factor 9 (Sox9), Runt-related transcription factor 2 (Runx2), and Type X collagen (COL10A1) in ATDC5 cells treated with Sema3A (1,10 and 100 ng/mL) was examined using real-time reverse transcription polymerase chain reaction. Further, to assess the deposition of total glycosaminoglycans during chondrogenic differentiation, ATDC5 cells were stained with Alcian Blue. Moreover, the amount of hyaluronan in the culture supernatant was measured by enzyme-linked immunosorbent assay. The addition of Sema3A to cultured ATDC5 cells increased the expression of Sox9, Runx2, COL2A1, ACAN, HAS2, and COL10A1 during chondrogenic differentiation. Moreover, it enhanced total proteoglycan and hyaluronan synthesis. Further, Sema3A was upregulated in the early stages of chondrogenic differentiation, and its secretion decreased later. Sema3A increases extracellular matrix production and promotes chondrogenic differentiation. To the best of our knowledge, this is the first study to demonstrate the role of Sema3A on chondrogenic differentiation.

Bee drone brood is a beehive by-product with high hormonal activity used in natural medicine to treat male infertility. The aim of the study was to assess the effect of drone brood on stallion spermatozoa during a short-term incubation for its potential use in the equine semen extenders. Three different forms of fixed drone brood (frozen (FR), freeze-dried (FD), and dried extract (DE)) were used. Solutions of drone brood were compared in terms of testosterone, protein, total phenolic content, and antioxidant activity. The stallion semen was diluted with prepared drone brood solutions. The computer-assisted semen analysis (CASA) method was employed to evaluate the movement characteristics of the diluted ejaculate. To determine spermatozoa viability, the mitochondrial toxicity test (MTT) and Alamar Blue test were performed. In terms of testosterone content and antioxidant activity, a close likeness between FR and FD was found whereas DE's composition differed notably. FR had a positive effect mainly on progressive motility, but also on sperm distance and speed parameters after 2 and 3 h of incubation. On the contrary, FD and DE acted negatively, depending on increasing dose and time. For the first time, a positive dose-dependent effect of fixed drone brood on spermatozoa survival in vitro was demonstrated.

Differentiation of Leydig cells plays a key role in male reproductive function. Bone marrow mesenchymal stem cells (BMSCs) have emerged as a potential cell source for generating Leydig-like cells due to their multipotent differentiation capacity and accessibility. This study aimed to investigate the morphological and genetic expression changes of BMSCs during differentiation into Leydig-like cells. Testicular extract liquid, which simulates the microenvironment in vivo, induced the third passage BMSCs differentiated into Leydig-like cells. Changes in cell morphology were observed by microscopy, the formation of lipid droplets of androgen precursor was identified by Oil Red Staining, and the expression of testicular specific genes 3β-HSD and SF-1 in testicular stromal cells was detected by RT-qPCR. BMSCs isolated from the bone marrow of Sprague-Dawley (SD) rats were cultured for 3 generations and identified as qualified BMSCs in terms of morphology and cell surface markers. After 14 days of induction with testicular tissue lysate, lipid droplets appeared in the cytoplasm of P3 BMSCs by Oil Red O staining. RT-qPCR detection was performed on BMSCs on the 3^rd, 7^th, 14^th, and 21^st day after induction. Relative expression levels of 3β-HSD mRNA significantly increased after 14 days of induction, while the relative expression of SF-1 mRNA increased after 14 days of induction but was not significant. BMSCs can differentiate into testicular interstitial cells with reserve androgen precursor lipid droplets after induction by testicular tissue lysate. The differentiation ability of BMSCs provides the potential to reconstruct the testicular microenvironment and is expected to fundamentally improve testicular function and provide new treatment options for abnormal spermatogenesis diseases.

The integration of precision medicine principles into bone tissue engineering has ignited a wave of research focused on customizing intricate scaffolds through advanced 3D printing techniques. Bioceramics, known for their exceptional biocompatibility and osteoconductivity, have emerged as a promising material in this field. This article aims to evaluate the regenerative capabilities of a composite scaffold composed of 3D-printed gelatin combined with hydroxyapatite/tricalcium phosphate bioceramics (G/HA/TCP), incorporating human dental pulp-derived stem cells (hDPSCs). Using 3D powder printing, we created cross-shaped biphasic calcium phosphate scaffolds with a gelatin layer. The bone-regenerating potential of these scaffolds, along with hDPSCs, was assessed through in vitro analyses and in vivo studies with 60 rats and critical-sized calvarial defects. The assessment included analyzing cellular proliferation, differentiation, and alkaline phosphatase activity (ALP), and concluded with a detailed histological evaluation of bone regeneration. Our study revealed a highly favorable scenario, displaying not only desirable cellular attachment and proliferation on the scaffolds but also a notable enhancement in the ALP activity of hDPSCs, underscoring their pivotal role in bone regeneration. However, the histological examination of calvarial defects at the 12-wk mark yielded a rather modest level of bone regeneration across all experimental groups. The test and cell group exhibited significant bone formation compared to all other groups except the control and cell group. This underscores the complexity of the regenerative process and paves the way for further in-depth investigations aimed at improving the potential of the composite scaffolds.

BP001 is a promising small molecule compound that has been specifically designed to target and degrade Bruton's tyrosine kinases (BTK), which is known to play a crucial role in lymphoma development. Macrophages are important immune cells in inflammation regulation and immune response. In this study, we aimed to investigate the effect of BP001 on RAW264.7 macrophage activation stimulated by a high glucose environment. Our findings revealed that treatment with BP001 significantly inhibited the production of nitric oxide (NO), reactive oxygen species (ROS), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in RAW264.7 macrophages exposed to high glucose conditions. Furthermore, we observed that BP001 treatment also down-regulated the expression of BTK in these activated macrophages. To elucidate the underlying mechanism behind these observations, we investigated the phosphorylation level of NF-κB. Our results demonstrated that BP001 treatment led to decreased phosphorylation levels of NF-κB, thereby inhibiting the level of inflammation. In addition, we also found that BP001 could restore RAW264.7 macrophages from the pro-inflammatory state to the normal phenotype and reduce the occurrence of inflammation. The regulatory function of BP001 in autoimmunity is mediated through the degradation of BTK protein, thereby attenuating macrophage activation. Additionally, BTK plays a pivotal role in transcriptional regulation by inducing NF-κB activity. Consequently, it is not difficult to understand that BP001 effectively inhibits inflammation. In conclusion, the present study provides evidence that BP001, a BTK degrader, can serve as a novel immunomodulator of inflammation induced by high glucose, making it an attractive candidate for further investigation.

Heparin-binding protein 17 (HBp17), first purified in 1991 from the conditioned medium of the human A431 squamous cell carcinoma (SCC) cell line, was later renamed fibroblast growth factor-binding protein 1 (FGFBP-1). HBp17/FGFBP-1 is specifically expressed and secreted by epithelial cells, and it reversibly binds to fibroblast growth factor (FGF)-1 and FGF-2, as well as FGFs-7, -10, and -22, indicating a crucial involvement in the transportation and function of these FGFs. Our laboratory has investigated and reported several studies to elucidate the function of HBp17/FGFBP-1 in SCC cells and its potential as a molecular therapeutic target. HBp17/FGFBP-1 transgene exoression in A431-4 cells, a clonal subline of A431 that lacks tumorigenicity and does not express HBp17/FGFBP-1, demonstrated a significantly enhanced proliferation in vitro compared with A431-4 cells, and it acquired tumorigenicity in the subcutis of nude mice. Knockout (KO) of the HBp17/FGFBP-1 by genome editing significantly suppressed tumor growth, cell motility, and tumorigenicity compared with control cells. A comprehensive analysis of expressed molecules in both cell types revealed that molecules that promote epithelial cell differentiation were highly expressed in HBp17/FGFBP-1 KO cells. Additionally, we reported that 1α,25(OH)₂D₃ or eldecalcitol (ED-71), which is an analog of 1α,25(OH)₂D₃, suppresses HBp17/FGFBP-1 expression and tumor growth in vitro and in vivo by inhibiting the nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway. Here, we discuss the prospects of molecular targeted therapy targeting HBp17/FGFBP-1 with 1α,25(OH)₂D₃ or ED71 in SCC and oral SCC.

Personal lubricants intended for local or systemic delivery via the vaginal route can induce vaginal irritation, damage the vaginal epithelial barrier which can enhance microbial entry, induce inflammation, and alter the microbiome of the vaginal ecosystem. Therefore, manufacturers of personal lubricants and medical devices are required to show biocompatibility and safety assessment data to support regulatory decision-making within a specified context of use. Furthermore, due to ethical concerns and the introduction of the 7th amendment of the European Council Directive which bans animal testing for cosmetic ingredients and products coupled with the Food and Drug Administration modernization Act 2.0 guidelines, there is a wave of drive to develop alternative test methods to predict human responses to chemical or formulation exposure. In this framework, there is a potential to use three-dimensional organotypic human vaginal-ectocervical tissue models as a screening tool to predict the vaginal irritation potential of personal lubricants and medicaments. To be physiologically relevant, the in vitro tissue models need to be reconstructed using primary epithelial cells of the specific organ or tissue and produce organ-like structure and functionality that recapitulate the in vivo-like responses. Through the years, progress has been made and vaginal tissue models are manufactured under controlled conditions with a specified performance criterion, which leads to a high level of reproducibility and reliability. The utility of vaginal tissue models has been accelerated in the last 20 years with an expanded portfolio of applications ranging from toxicity, inflammation, infection to drug safety, and efficacy studies. This article provides an overview of the state of the art of diversified applications of reconstructed vaginal tissue models and highlights their utility as a tool to predict vaginal irritation potential of feminine care products.

The development and characterization of two novel humpback grouper (Cromileptes altivelis) fin cell lines are described in this study. The CA1F3Ex and CA1F4Tr cell lines were developed by explant and trypsinization methods, respectively, in Leibovitz's L15 (L-15) medium supplemented with 20% FBS (fetal bovine serum) and subcultured over 150 times. Cell lines exhibited high stability, as evidenced by the high revival rate (85-95%) and good attachment while seeding after one year of cryostorage. They displayed good seeding (91%) and plating efficiencies (15-25%). The optimum temperature for growth was recorded at 28˚C. Serum requirement decreased with increased passage and lowered to 2% FBS beyond 30-35 passages. However, higher serum concentration (2-20%) caused a concurrent increase in cell growth. Both the cell lines were fibroblast-type, and immunotyping results showed strong reactivity towards the fibroblast marker. Chromosome analysis of these cell lines revealed aneuploidy, and the authenticity was confirmed by mitochondrial Cytochrome C Oxidase Subunit I (COI) genotyping analysis. Cell cycle studies were performed utilizing the flow cytometric technique. CA1F3Ex and CA1F4Tr cell lines showed high transfection efficiency with pEGFP-N1 plasmid using Lipofectamine and cytotoxicity towards heavy metals (Hg and Cd) was also studied. Hence, these continuous cell lines could be employed as in vitro models for aquatic toxicological and genetic manipulation studies.

Wnt, a family of secreted signaling proteins, serves diverse functions in embryogenesis, organogenesis, cancer, and stem cell functions. In the context of development, Wnt has been considered a representative morphogen, forming concentration gradients to give positional information to cells or tissues. However, although gradients are often illustrated in schemata, the reality of concentration gradients, or in other words, actual spatial distribution of Wnt ligands, and their behaviors in the extracellular space still remain poorly known. To understand extracellular behavior of Wnt ligands, quantitative analyses such as fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are highly informative because Wnt dispersal involves physical and biochemical processes, such as diffusion and binding to or dissociation from cell surface molecules, including heparan sulfate proteoglycans (HSPGs). Here, I briefly discuss representative methods to quantify morphogen dynamics. In addition, I discuss molecular manipulations of morphogens, mainly focusing on use of protein binders, and synthetic biology of morphogens as indicators of current and future directions in this field.