{"title":"Advancing marine invertebrate cell line research: four key knowledge gaps.","authors":"Baruch Rinkevich, Shirley A Pomponi","doi":"10.1007/s11626-025-01029-y","DOIUrl":"https://doi.org/10.1007/s11626-025-01029-y","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":1.5,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143735794","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"5-Azacytidine inhibits endoplasmic reticulum stress and apoptosis of nucleus pulposus cells by preserving PPARγ via promoter demethylation.","authors":"Peng Cheng, Huan Li, Hai-Wei Chen, Zhi-Qiang Wang, Pei-Wu Li, Hai-Hong Zhang","doi":"10.1007/s11626-025-01021-6","DOIUrl":"https://doi.org/10.1007/s11626-025-01021-6","url":null,"abstract":"<p><p>Low back pain (LBP) is a common symptom of intervertebral disc degeneration (IDD). However, the pathogenesis of IDD is not well understood. Several studies have shown that patients with IDD experience aberrant changes in DNA methylation. 5-Azacytidine (5Aza) is a nucleoside-based DNA methyltransferase inhibitor that inhibits DNA methylation. Therefore, this study investigated whether 5Aza can improve the apoptosis of nucleus pulposus (NP) cells and ER stress (ERS) induced by il-1β by inhibiting PPARγ methylation and its potential pathogenesis. NP cell viability was detected using Cell Counting Kit-8 (CCK-8). Methylation-specific PCR (MSP) was used to evaluate the DNA methylation level. TUNEL was used to evaluate the apoptosis of NP cells. Western blot determined the expression levels of DNMT1, DNMT3a, PPARγ proteins, and ERS-related indexes (C/EBP homology protein (CHOP), GRP78, ATF-6) and apoptosis-related indexes (Bcl-2, Bax, Caspase-3) protein expression levels. 5Aza can inhibit the expression of DNMT1 and DNMT3a and promote PPARγ by modifying the methylation of PPARγ promoter. Western blot (Bcl-2, Bax, Caspase-3, CHOP, GRP78, ATF-6), TUNEL, and CHOP immunofluorescence results showed that 5Aza attenuated IL-1β-induced apoptosis and ERS of NP cells. When pretreated with PPARγ inhibitor (T0070907), the protective effect of 5Aza on IL-1β-induced apoptosis and ERS in NP cells is weakened, suggesting that 5Aza inhibits IL-1β-induced NP cell apoptosis and ERS by promoting the expression of PPARγ. 5Aza preserves PPARγ by inhibiting the expression of DNMT1/DNMT3a, which can significantly reduce IL-1β damage in NP cells. Our findings suggest that preserving PPARγ through DNA demethylation may be an attractive strategy for preventing or treating IDD.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":1.5,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657114","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Franziska Annabelle Hecker, Bruno Leggio, Tim Koenig, Karsten Niehaus, Sven Geibel
{"title":"Cell Painting of insect gut cells for exploration of molecular responses of insect epithelia to insecticides.","authors":"Franziska Annabelle Hecker, Bruno Leggio, Tim Koenig, Karsten Niehaus, Sven Geibel","doi":"10.1007/s11626-025-01028-z","DOIUrl":"https://doi.org/10.1007/s11626-025-01028-z","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":1.5,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143648372","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Resolvin D1 suppresses inflammation in human fibroblast-like synoviocytes via the p-38, NF-κB, and AKT signaling pathways.","authors":"Makoto Yanoshita, Naoto Hirose, Sayuri Nishiyama, Eri Tsuboi, Naoki Kubo, Daiki Kita, Kotaro Tanimoto","doi":"10.1007/s11626-024-01008-9","DOIUrl":"https://doi.org/10.1007/s11626-024-01008-9","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":1.5,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143596978","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bayan Jamal Khaleel, Hayder Ridha-Salman, Haitham Mahmood Kadhim, Omeed M Hassan, Ammar Kubba, Hayder B Sahib
{"title":"Inhibitory effects of carbohydrazide indole derivative on micro-blood vessel growth using ex vivo, in vivo, and in vitro assays.","authors":"Bayan Jamal Khaleel, Hayder Ridha-Salman, Haitham Mahmood Kadhim, Omeed M Hassan, Ammar Kubba, Hayder B Sahib","doi":"10.1007/s11626-025-01019-0","DOIUrl":"https://doi.org/10.1007/s11626-025-01019-0","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":1.5,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143566494","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"The combination of neurotropic B vitamins (B1, B6, and B12) is superior to individual B vitamins in promoting neurite growth in vitro.","authors":"Melissa L D Rayner, Arnaud J Ruiz, Christian Viel","doi":"10.1007/s11626-025-01024-3","DOIUrl":"https://doi.org/10.1007/s11626-025-01024-3","url":null,"abstract":"","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":1.5,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143556752","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"The effect of IGF-1 on cartilage injury in bone marrow mesenchymal stem cells through the BMP2-Smad1/5 signaling pathway.","authors":"HuiYue Ye, Liang Shao","doi":"10.1007/s11626-025-01015-4","DOIUrl":"https://doi.org/10.1007/s11626-025-01015-4","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":1.5,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143566678","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jing Yang, Rongrong Zhou, Mengjiao Zhou, Xinghuan Li
{"title":"Atorvastatin inhibits ischemia‒reperfusion-associated renal tubular cell ferroptosis by blocking the PGE2/EP4 signaling pathway.","authors":"Jing Yang, Rongrong Zhou, Mengjiao Zhou, Xinghuan Li","doi":"10.1007/s11626-025-01020-7","DOIUrl":"https://doi.org/10.1007/s11626-025-01020-7","url":null,"abstract":"<p><p>Renal ischemia‒reperfusion (I/R) injury is the main cause of acute kidney injury, and its pathological features are manifested primarily by renal tubular epithelial cell injury. The underlying mechanism involves ferroptosis of renal tubular epithelial cells. Atorvastatin (ATO) regulates ferroptosis, and this study explored its role in I/R-induced ferroptosis of renal tubular epithelial cells. We constructed a renal I/R rat model with bilateral renal pedicles using noninvasive arterial clips and placed HK-2 cells in hypoxia/reoxygenation (H/R) incubators to construct the cell model. The damage to rat kidney tissues and HK-2 cells was assessed using enzyme-linked immunosorbent assay (ELISA), hematoxylin and eosin (H&E) staining, and flow cytometry, and the presence of associated proteins was identified through western blotting. Administering ATO markedly lessened the acute kidney damage caused by I/R, decreased the levels of blood urea nitrogen (BUN) and creatinine (CRE), and prevented apoptosis in renal tubular epithelial cells. Treatment with ATO additionally suppressed the production of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and markers linked to ferroptosis (Fe<sup>2+</sup>, ROS, MDA, ACSL4, and COX2), thereby reducing acute kidney damage associated with I/R. The expression of PGE2 in renal I/R injury is related to the degree of renal injury, and it mainly regulates ferroptosis by binding to EP4. ATO effectively inhibited the expression of PGE2 and EP4. Overall, this study revealed that ATO inhibited ferroptosis of renal tubular epithelial cells by blocking the PGE2/EP4 signaling pathway, thereby alleviating I/R-induced kidney injury.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":1.5,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370821","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ashaq Sultan Dar, Fayaz Ahmad, Feroz Ahmad Shah, Syed Shariq Nazir Qadiri, Keezia Khurshid
{"title":"Development and characterization of a cell line from the caudal fin of Schizothorax niger (Heckel, 1838) for in vitro toxicity testing.","authors":"Ashaq Sultan Dar, Fayaz Ahmad, Feroz Ahmad Shah, Syed Shariq Nazir Qadiri, Keezia Khurshid","doi":"10.1007/s11626-025-01018-1","DOIUrl":"https://doi.org/10.1007/s11626-025-01018-1","url":null,"abstract":"<p><p>Here, we successfully grew the SNCF (Schizothorax niger caudal fin) cell line from the caudal fin explants of S. niger, an important cold-water fish of the Himalayas. The cells were successfully grown up to 22 passages by planting explant tissues in DMEM medium supplemented with FBS. We observed optimal cell growth at a concentration of 18% FBS. We observed the steady generation of cells from explants from days 2 to 5 of seeding, and obtained a complete monolayer at days 7-10. We tested various temperatures, including 10 °C, 13 °C, 16 °C, 19 °C, 22 °C, 25 °C, and 28 °C, and found that 22 °C was the optimal temperature for cell growth. We examined the response to various doses of epidermal growth factor (EGF) and fibroblast growth factor (FGF) (0, 2, 4, 6, 8, and 10 ng/mL) on cell colony growth at an optimal temperature of 22 °C. We characterized the cell line using karyotyping at the 14th and 20th passages. The cell line showed epithelial cell-like growth by morphology, which was confirmed by immunotyping. We further used the cell line to study the impact of three pesticides (chlorpyrifos, dimethoate, and endosulfan), and a fungicide (mancozeb) and bacterial extracellular product (ECP). The DAPI stain assay and MTT assay confirmed the pesticides toxic effects on the cells, revealing disintegration of the cell nuclei by the formation of micronuclei and LC<sub>50</sub> concentrations. ECP treatment showed disruption of the monolayer within 0-36 hrs.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":1.5,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143187760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}