In Vitro Cellular & Developmental Biology. Animal最新文献

Aquaculture is essential to satisfying the world's increasing demand for seafood. Likewise, overfishing is becoming more common across the world, inflicting tremendous damage to the marine environment. There is a critical need for protecting sustainable fishing resources to fulfil the increasing demand for seafood. The current work focuses on the cells derived from Asian seabass muscle (SBM) and Asian seabass fin (SBF) for producing cell-based fish meat. SBM and SBF cells were seeded separately in the TubeSpin bioreactor and placed on a 3D orbital rocker. Cell sheets formed on the TubeSpin were detached and formed spheroid-like structures. These structures aggregated and formed visible tissue-like structures on 45 d of culture. Immunotyping results revealed that the presence of myosin in the cells of muscle and fin tissue, and indicating that these cells might have originated from myoblasts. The origin of cultured tissue from SBM and SBF cell lines was confirmed by amplification and sequencing of the L. calcarifer specific mitochondrial larger subunit rRNA gene. Additionally, these cells could be cultivated in multilayered forms that were appropriate for large-scale production. This approach provides a new method for the production of cell-based, laboratory-grown meat from the Asian seabass muscle and fin cell lines.

Insect cell lines are valuable for basic and applied biological research. In this study, we established 27 cell lines from various insect species, including Hemiptera: Nilaparvata lugens, Coleoptera: Sitophilus oryzae, Hymenoptera: Allantus luctifer and Trichogramma ssp., Diptera: Culicoides oxystoma, Lepidoptera: Spodoptera litura, Mythimna separata, Bombyx mori, Agrius convolvuli, Plodia interpunctella, and Cryptophlebia horii. This is the first report of cell lines derived from A. luctifer, C. oxystoma, A. convolvuli, and C. horii. Additionally, cell lines from S. litura and M. separata were established from different tissues including the hemocytes, fat bodies, embryos, and Malpighian tubules. Eighteen cell lines were successfully adapted to commercial culture media, with the population doubling time ranging from 1 to 8 d. The identities of the cell lines were confirmed using DNA barcoding. These established cell lines could be valuable for various research applications.

Although cell cultures from marine invertebrates have great potential as valuable tools in various scientific fields, nearly all attempts to culture these cells in vitro have consistently failed, and the reasons for this remain unclear. The ongoing failure to develop stable, long-term cell cultures from marine invertebrates, despite varied species and methods employed, highlights significant knowledge gaps in understanding their in vitro requirements. These gaps impede progress, underscoring the complexity of marine invertebrate cells and the need for innovative approaches to overcome challenges in the field. When reviewing recent literature on the key data deficiencies and challenges behind the failure to develop marine invertebrate cell cultures, we identified and discussed four major knowledge gaps: (1) optimizing culture media, (2) strategies to extend stemness of isolated cells, (3) using "omics" to enhance cell culture, and (4) selecting suitable cell types for in vitro cultures. Bridging these gaps is crucial for advancing marine invertebrate cell culture systems. Yet, given the current state-of-the-art, addressing these gaps and advancing the discipline necessitate comprehensive, integrated, and species- or cell-specific strategies, along with close collaboration among laboratories working on diverse species.

Cell Painting is a sophisticated high-content imaging technique that has been predominantly applied to mammalian cells. Recent advancements have extended its applicability to the first insect cell line, the ovarian cell line Sf9, revealing significant insights into similarities and differences in cellular responses between different taxonomic groups. This study explores the utility of Cell Painting in Helicoverpa zea gut-derived cells, specifically the RP-HzGUT-AW1 cell line, to assess the specifics of insect epithelial cells in response to chemical treatments. Upon adaptation of the analysis pipeline to accommodate their unique morphology and characteristics, our investigations revealed distinct responses of RP-HzGUT-AW1 cells compared to the ovarian insect cell line Sf9. Variations were obtained not only in the dose-response behavior to treatments but also in the overall detectability of specific modes of action. Specifically, processes that relate to osmoregulation and the formation of epithelial structures showed the most significant and distinct responses. This suggests that the specific morphological and physiological attributes of these gut-derived insect cells contribute to unique phenotypic profiles, which enables in-depth interpretation of drug efficacy and safety in these models.

Defective angiogenesis is a characteristic of many diseases, notably cancer and immune-mediated conditions. Numerous shortcomings in anti-angiogenic therapies, including undesirable effects, drug resistance, and cancer recurrence, encouraged the development of innovative medicines with improved anti-angiogenic efficacy. Indole analogues are thought to interact with the mitotic spindle, preventing malignant human cells from multiplying and invading. N'-(1-Benzyl-2-oxoindolin-3-ylidene)-5-bromo-1H-indole-2-carbohydrazide (N-5-BIC) represents one of these chemicals exhibiting remarkable anti-angiogenesis and anti-proliferation features. The study aimed to investigate the antiangiogenic, antioxidant, and antiproliferative activities of a carbohydrazide indole derivative, N-5-BIC. The ex vivo rat aorta ring (RAR), DPPH, and chick chorioallantois membrane (CAM) assays were employed to assess the N-5-BIC antiangiogenic and antioxidant activities. The MTT assay investigated the anti-proliferative activity in the human umbilical vascular endothelial cells (HUVEC) cell line. The VEGF gene expression level in the colon cancer (HCT116) cell line was evaluated using quantitative real-time polymerase chain reaction (RT-PCR). N-5-BIC demonstrated a substantial and dose-dependent inhibition of blood vessel growth, resulting in an 87.37% reduction at a concentration of 100 μg/ml compared to the negative control (DMSO 1%) in the RAR assay. Additionally, N-5-BIC exhibited a significant decrease in DPPH free radicals in a concentration-dependent manner, with an IC50 value of 129.6 µg/ml. The in vivo CAM assay confirmed a significant regression in blood vessels compared to the negative control. Furthermore, N-5-BIC demonstrated low to non-toxic effects on the HUVEC cell line, with an IC50 value of 1681 μg/ml. The RT-PCR study revealed a significant reduction in VEGF gene expression at doses of 200 and 400 µg/ml as compared to control cells. N-5-BIC has resilient anti-angiogenic properties, which may be attributed to its extensive anti-proliferative and free radical neutralizing properties.

The objective of this study is to explore how adipose-derived stem cells (ASCs) regulate mitochondrial structure and function and the impact of this regulation on slowing cellular senescence. HFF-1 cells were induced by H₂O₂ to establish a cellular senescence model, and ASCs or Mdivi-1 (mitochondrial fission inhibitor) was added. MTT examined the cell proliferation; flow cytometry detected mitochondrial membrane potential as well as apoptosis and cell cycle; kit measured ATP production; ELISA analyzed the levels of interleukin-6 (IL-6), interleukin 1 beta (IL-1β), tumor necrosis factor alpha-like (TNF-α), glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD); Western blotting and qRT-PCR detected the expression of protein and mRNA levels; and β-galactosidase staining observed the degree of cellular senescence. Compared to normal HFF-1 cells, senescent HFF-1 cells exhibited weaker proliferative capacity, marked apoptosis, and G0-G1 cell cycle arrest. These cells also showed lower mitochondrial membrane potential and ATP production, higher expression of inflammatory factors, oxidative damage, and increased levels of senescence. Treatment with Mdivi-1 or ASCs enhanced HFF-1 cell proliferation, reduced apoptosis and cell cycle arrest, increased mitochondrial membrane potential and ATP production, decreased the expression of inflammatory factors, and mitigated oxidative stress, thereby reducing the degree of cellular senescence. Concurrent intervention with Mdivi-1 and ASCs further diminishes the impacts of cellular senescence. In conclusion, ASCs regulate mitochondrial dynamics (promoting mitochondrial fusion and inhibiting mitochondrial fission), enhance ATP production, and upregulate mitochondrial membrane potential, thereby alleviating cell cycle arrest, apoptosis, inflammatory responses, and oxidative stress induced by senescence in HFF-1 cells.

The objective of this study is to analyze the effect of insulin-like growth factor-1 (IGF-1) in bone marrow mesenchymal stem cells (BMSCs) on cartilage injury and explore the regulatory mechanism of IGF-1 on the bone morphogenetic protein 2 (BMP2)-Smad1/5 signaling pathway. We cultivated rat BMSCs in vitro and observed their cell morphology using an inverted microscope. Flow cytometry was used to identify the surface antigen expression of BMSCs. IL-1β is used to induce rat chondrocyte ATDC5 to construct a cartilage injury model. We integrated IGF-1 overexpressed BMSCs, empty vector transfected BMSCs, and BMSCs with IL-1, respectively. IL-1β-induced ATDC5 cells were co-cultured for 24 h. We recorded them as BMSCs + IGF-1 group, BMSCs + empty vector group, BMSCs group, and normal cultured ATDC5 cells as the control group. qRT-PCR and Western blot were used to detect IGF-1 mRNA and protein levels in each group. CCK-8 experiment and flow cytometry were used to detect cell proliferation and apoptosis in each group. ELISA is used to detect the levels of TNF-α, IL-8, and IL-6. Western blot was used to detect protein levels of Bax, Bcl-2, Cleaved Caspase-3, Aggrescan, Col II, MMP-1, MMP-13, BMP2, and p-Smad1/5 in each group. Fifty rats were randomly divided into a control group, a model group, a BMSCs group, a BMSCs + empty body group, and a BMSCs + IGF-1 group using a random number table method, with 10 rats in each group. We evaluated cartilage repair using the O'Driscoll scoring system and Mankin's scoring system. HE staining was used to observe pathological changes in cartilage tissue. qRT-PCR and Western blot were used to detect the expression levels of cartilage repair-related genes OC, GSK-3β, and Runx2 in various cartilage tissues. Overexpression of IGF-1 in BMSCs could enhance IL-1β-induced ATDC5 cell survival rate and the protein level of Bcl-2; reduce apoptosis rate and the protein levels of Bax and Cleaved Caspase-3; decrease the levels of IL-6, TNF-α, and IL-8; increase the protein levels of BMP2, p-Smad1/5, Aggrescan, and Col II; and reduce the protein levels of MMP-1 and MMP-13 (P < 0.05). Compared with the model group, the O'Driscoll score in the BMSCs group, the BMSCs + empty body group, and the BMSCs + IGF-1 group was increased; Mankin's score was decreased; and the expression levels of OC, GSK-3β, and Runx2 were decreased (P < 0.05). Compared with the BMSCs group and BMSCs + empty body group, the O'Driscoll score in the BMSCs + IGF-1 group was increased, Mankin's score was decreased, and the expression levels of OC, GSK-3β, and Runx2 were decreased (P < 0.05). Overexpression of IGF-1 in BMSCs could inhibit IL-1β-induced chondrocyte apoptosis, promote cell proliferation, reduce the secretion of inflammatory factors, alleviate chondrocyte damage, and promote cartilage tissue repair. Its mechanism may be related to the activation of the BMP2-Smad1/5 signaling pathway.

Synovitis represents the initial pathological change in osteoarthritis and contributes to its progression. Resolvin D1 (RV-D1) is a novel and endogenous docosahexaenoic acid-derived lipid mediator, which regulates the duration and magnitude of inflammation by downregulating pro-inflammatory genes and mediators. However, the effects of RV-D1 on synovitis remain unknown. The aim of the present study was to investigate the anti-inflammatory effects of RV-D1 in human fibroblast-like synoviocytes (HFLSs) and the underlying mechanisms. The expression of the HFLS formyl peptide receptor 2 (ALX/FPR) was examined via immunocytochemical analysis. HFLSs were treated with 1 ng/mL recombinant human interleukin-1β (IL-1β) and RV-D1. The gene expression of interleukin-1β (IL1B), matrix metalloproteinase 3 (MMP3), and MMP13 was examined using real-time reverse transcription-polymerase chain reaction after treatment with IL-1β and RV-D1. The effect of RV-D1 on apoptosis was examined based on fluorescence intensity. Phosphorylation of p-38, extracellular signal-regulated kinase, c-Jun N-terminal kinase, nuclear factor kappa B (NF-κB), and AKT was analyzed via western blotting. ALX/FPR staining was observed on the cell surface. RV-D1 significantly suppressed the IL-1β-induced increase in gene and protein expression of IL-1β, MMP-3, and MMP-13. Pretreatment with 100 nM RV-D1 significantly increased the fluorescence intensity compared to that in the non-treatment group. Furthermore, pretreatment with RV-D1 significantly suppressed the phosphorylation of p-38, NF-κB, and AKT. Whereas WRW4, an antagonist of ALX/ FPR2, treatment weakened the effect of RV-D1, resulting in p-38, NF-κB, and AKT phosphorylation and the protein expression of MMP-13 at levels comparable to those in the IL-1β without RV-D1. In conclusion, RV-D1 suppressed IL-1β and MMP expression by inhibiting the phosphorylation of p-38, NF-κB, and AKT in inflammation in HFLSs. RV-D1 can be used to develop treatments for osteoarthritis and other inflammatory disorders.