In Vitro Cellular & Developmental Biology. Animal最新文献

{"title":"Huwang Mingmu decoction protects retinal neurons in early diabetic retinopathy by suppressing the IL-1β-c-Jun-GS pathway: an in vivo and in vitro study.","authors":"Xuewei Qin, Limin Wang, Xianfeng Yao, Mei Chen, Kaijin Zheng, Li Zheng","doi":"10.1007/s11626-026-01246-z","DOIUrl":"https://doi.org/10.1007/s11626-026-01246-z","url":null,"abstract":"<p><p>Huwang Mingmu decoction (HWMMT) is a traditional Chinese herbal formula used clinically for diabetic retinopathy (DR), but its neuroprotective mechanisms remain unclear. This study aimed to investigate whether HWMMT protects retinal neurons in early DR by attenuating glutamate excitotoxicity via the IL-1β/c-Jun/glutamine synthetase (GS) signaling axis. Diabetes was induced in Sprague-Dawley rats by streptozotocin injection, and animals were randomized into control, diabetic model (DM), and HWMMT-treated diabetic (DM + HWMMT) groups. HWMMT was orally administered for 8 wk, with body weight and blood glucose monitored weekly. At weeks 4 and 8, retinal tissues and serum were collected for qRT-PCR, Western blotting, and ELISA. In vitro, primary rat retinal Müller glial cells (Müller) were cultured and assigned to five groups: blank control, HWMMT serum control, high-glucose (HG), HG + HWMMT, and HG + HWMMT + IL-1β (rescue group). Cell viability, LDH release, glutamate secretion, apoptosis, and expression of IL-1β, c-Jun, and GS were evaluated. In vivo, DM rats showed slower weight gain, sustained hyperglycemia, upregulated retinal IL-1β and c-Jun expression, downregulated GS, and elevated serum glutamate; HWMMT treatment significantly reversed these abnormalities. In vitro, HG exposure decreased Müller cell viability, increased apoptosis, LDH leakage and glutamate release, and induced IL-1β/c-Jun upregulation with GS downregulation; co-treatment with HWMMT-containing serum reversed all these changes. Notably, exogenous IL-1β supplementation significantly attenuated the cytoprotective and anti‑apoptotic effects of HWMMT and reversed its modulation of IL-1β, c-Jun, GS, and glutamate homeostasis. Collectively, these findings demonstrate that HWMMT exerts neuroprotection in early DR by suppressing the IL-1β/c-Jun signaling cascade and restoring GS expression and function in Müller cells, thereby reducing extracellular glutamate accumulation and mitigating excitotoxic damage. The rescue experiments further support that HWMMT acts specifically through this pathway, providing mechanistic insight and preclinical evidence for HWMMT as a potential therapeutic intervention for early diabetic retinopathy.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":2.0,"publicationDate":"2026-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149001768","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":"Punicic acid alleviates osteoarthritis progression through miR-29a-3p-dependent inhibition of the MAP2K6/p38 MAPK pathway.","authors":"Wei Wang, Tao Zhang, Chao Yu, Zhi-Xiong Xu, Long Luo, Liang Dong, Wen-Sheng Xu","doi":"10.1007/s11626-026-01239-y","DOIUrl":"https://doi.org/10.1007/s11626-026-01239-y","url":null,"abstract":"<p><p>Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive articular cartilage degradation. Punicic acid (PA), a plant-derived n-3 polyunsaturated fatty acid, has been reported to exert anti-inflammatory and anti-apoptotic effects. However, the molecular mechanisms underlying its effects in OA remain incompletely understood. In this study, bioinformatics analysis was performed to identify apoptosis-related differentially expressed genes (DEGs) and enriched signaling pathways associated with OA. An in vitro OA-like model was established using interleukin-1β (IL-1β)-stimulated SW1353 cells, which were subsequently treated with different concentrations of PA. PA suppressed IL-1β-induced expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3),matrix metalloproteinase-13 (MMP-13), and A Disintegrin And Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS-5) and attenuated apoptosis by modulating Bcl-2-associated X protein (BAX) and B-cell lymphoma 2 (BCL-2) expression. Notably, target prediction and dual-luciferase reporter assays confirmed that mitogen-activated protein kinase kinase 6 (MAP2K6) is a direct target of microRNA-29a-3p (miR-29a-3p), and PA increased miR-29a-3p expression in the in vitro OA-like model. Furthermore, PA inhibited MAP2K6-mediated activation of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, thereby attenuating downstream inflammatory and apoptotic signaling. These findings suggest that PA exerts chondroprotective effects by modulating the miR-29a-3p/MAP2K6/p38 MAPK signaling axis, highlighting its therapeutic potential and supporting the development of miRNA-based therapeutic strategies for OA.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":2.0,"publicationDate":"2026-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148982670","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":"SPI1 exacerbates neurogenic bladder dysfunction by enhancing NRSF-mediated suppression of HCN1 and inducing endoplasmic reticulum stress.","authors":"Kai Fu, FuMing Deng, XiangLiang Tang, RuiYu Liu, Jin Zhang, GuoChang Liu","doi":"10.1007/s11626-026-01238-z","DOIUrl":"https://doi.org/10.1007/s11626-026-01238-z","url":null,"abstract":"<p><p>Neurogenic bladder (NB) severely impacts patients' quality of life. This study was designed to explore the molecular mechanisms underlying the pathophysiology of NB-associated bladder dysfunction. A rat model of NB was established via spinal cord injury (SCI). Bladder function was evaluated by urodynamic testing and hematoxylin-eosin staining of tissue sections. The expression levels of SPI1, HCN1, neuronal restrictive silencing factor (NRSF), and endoplasmic reticulum (ER) stress-related proteins in bladder tissues or cells were examined using immunohistochemistry, Western blot, and reverse transcription quantitative polymerase chain reaction. Cell viability was measured with the Cell Counting Kit-8 assay. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry. Potential binding sites between SPI1 and the NRSF promoter were predicted using the JASPAR database and then validated by dual-luciferase reporter and chromatin immunoprecipitation assays. The role of ER stress in NB was investigated using the inhibitor 4-phenylbutyric acid or the inducer tunicamycin. Bladder tissues from NB rats exhibited increased apoptosis and a significant upregulation of ER stress-related proteins. Furthermore, expression of SPI1 and NRSF was elevated, whereas the expression of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) was reduced in bladder tissues from NB rats. Further analysis revealed that SPI1 binds to the NRSF promoter and upregulates its expression, thereby enhancing the repression of HCN1. Knockdown of SPI1 significantly alleviated bladder dysfunction, attenuated ER stress, and inhibited apoptosis. These effects were reversed by overexpression of NRSF. This study demonstrates that SPI1 contributes to bladder dysfunction by upregulating NRSF, which subsequently potentiates the inhibition of HCN1 and triggers ER stress. The findings identify promising molecular targets and signaling pathways for the intervention and treatment of NB.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":2.0,"publicationDate":"2026-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148976699","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":"Site-specific gene integration by recombinase-mediated cassette exchange in anhydrobiotic Pv11 cells.","authors":"Masahiko Hotta, Yuki Yoshida, Takahiro Kikawada","doi":"10.1007/s11626-026-01240-5","DOIUrl":"https://doi.org/10.1007/s11626-026-01240-5","url":null,"abstract":"<p><p>Pv11 cells, derived from Polypedilum vanderplanki, uniquely tolerate complete desiccation (anhydrobiosis). Although a CRISPR/Cas9-based precise integration method (CRIS-PITCh) has been developed for Pv11 cells, a CRISPR-free strategy that fixes both the genomic locus and transgene copy number has not yet been established. Here, we implement recombinase-mediated cassette exchange (RMCE) in Pv11 cells and generate master cell lines that retain anhydrobiosis following genetic engineering. We first evaluated the activity of multiple site-specific recombinases in Pv11 cells using a transient two-plasmid reporter assay. Flp, Cre, and Bxb1 recombinases all excised a test cassette and activated a green fluorescent protein (GFP) reporter, whereas phiC31 integrase mediated recombination at the DNA sequence level but did not induce reporter expression under our construct configuration. To enable genomic RMCE, we inserted an FRT/FRT3-landing pad (LP) into a previously identified genomic safe-harbor locus using CRIS-PITCh and isolated clonal master cell lines by single-cell sorting. Using the established master line, Flp-based RMCE achieved site-specific cassette exchange at the LP, producing HaloTag fluorescence and drug resistance upon successful exchange. In addition, the expected post-exchange sequence was confirmed by sequencing. We further established an all-in-one RMCE vector combining the Flp recombinase and donor cassette on a single plasmid. Together, these results demonstrate locus-defined, single-copy transgene integration in anhydrobiotic Pv11 cells via RMCE and provide a standardized, CRISPR-free workflow for routine genetic manipulation in this unique cell line. This workflow facilitates both fundamental research and applied biotechnological applications using desiccation-tolerant cells.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":2.0,"publicationDate":"2026-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148906033","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":"Ferulic acid liposomes improve diabetic EPC angiogenesis by upregulating the AMPK/KLF4/FAM3A pathway.","authors":"Xiao-Ling Zou, You-Yuan He, Xi Zhang, Wen-Xiao Hu, Si-Ying Li, Xiang-Nan Liu, Shao-Ye Qiao, Ying Wang, Biao Luo, Si-Jia Zhao, Qiao Liu, Wu Xiong","doi":"10.1007/s11626-026-01244-1","DOIUrl":"https://doi.org/10.1007/s11626-026-01244-1","url":null,"abstract":"<p><p>Diabetic vascular complications are a leading cause of disability in diabetic. This study aimed to investigate the functional improvement effects and molecular mechanisms of internalizing RGD (iRGD) peptide-modified ferulic acid-targeted liposomes (Lp-iRGD@FA) on high glucose-induced injured endothelial progenitor cells (EPCs). Lp-iRGD@FA was prepared using the thin-film hydration method and subsequently characterized. In vitro experiments were conducted using human peripheral blood-derived EPCs, in which a high glucose injury model was established. Cells were treated with free ferulic acid, non-targeted liposomes, and targeted liposomes, respectively. In vivo, a diabetic hindlimb ischemia mouse model was established, and Lp-iRGD@FA, non-targeted liposomes, or Lp-iRGD were intramuscularly injected into the ischemic muscles. The high glucose environment significantly inhibited EPC proliferation, promoted apoptosis, and impaired tube formation ability, accompanied by inhibited AMPK phosphorylation and downregulated expression of KLF4 and FAM3A. Intervention with the targeted liposomes effectively reversed these changes. However, the AMPK inhibitor Compound C blocked the Lp-iRGD@FA-induced upregulation of KLF4 and FAM3A and the associated functional improvements, whereas KLF4 supplementation partially rescued the decreased FAM3A expression and impaired tube formation caused by AMPK inhibition. In vivo, Lp-iRGD@FA treatment in diabetic mice enhanced angiogenesis in a hindlimb ischemia model and activated the AMPK/KLF4/FAM3A pathway in ischemic muscles. This study demonstrates that Lp-iRGD@FA efficiently delivers ferulic acid to restore FAM3A expression by activating the AMPK/KLF4/FAM3A signaling axis, subsequently upregulating VEGFA/VEGFR2 levels and improving the function of EPCs under high glucose stress. These findings provide a novel nanomedicine strategy and potential molecular targets for the treatment of diabetic vascular complications.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":2.0,"publicationDate":"2026-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148891603","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":"Regulatory role of lncRNA DANCR in osteogenic differentiation of bone marrow mesenchymal stem cells through targeting the miR-19a-3p/PTEN/AKT/mTOR pathway.","authors":"Chenglei Zhai, Yan Lv, Gang Sun","doi":"10.1007/s11626-026-01229-0","DOIUrl":"https://doi.org/10.1007/s11626-026-01229-0","url":null,"abstract":"<p><p>Given the involvement of long non-coding RNA-differentiation antagonizing non-protein coding RNA (lncRNA DANCR) in osteoporosis (OP), this study aimed to investigate its role in osteogenic differentiation (OD) of bone marrow mesenchymal stem cells (BMSCs) and underlying mechanisms. BMSCs were intervened with sh-DANCR, oe-DANCR, sh-phosphatase and tensin homolog (PTEN) lentivirus, miR-19a-3p agomir, or MK-2206 dihydrochlorode. The number of mature osteoblasts, osteogenic ability, and alkaline phosphatase (ALP) activity were assessed by ALP staining, alizarin red staining, and kit assay. Bioinformatics prediction, dual-luciferase assay, and RNA pull-down assay were conducted to assess the binding between lncRNA DANCR and miR-19a-3p and that between PTEN and miR-19a-3p. The levels of lncRNA DANCR, osteogenesis-related proteins (osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), osteopontin (OPN)), PTEN, phosphorylated-protein kinase B (p-AKT), AKT, phosphorylated-mammalian target of rapamycin (p-mTOR), and mTOR were determined by RNA quantitation and western blot techniques. Silencing of lncRNA DANCR potentiated the OD capability of BMSCs, as evidenced by increased ALP activity, calcium nodule formation, and elevated expression of OPN, RUNX2, and OCN. In contrast, lncRNA DANCR overexpression inhibited the OD function by downregulating miR-19a-3p. Mechanistically, lncRNA DANCR regulated PTEN expression by competitively binding to miR-19a-3p. Additionally, PTEN silencing partially reversed the anti-OD effect of lncRNA DANCR. LncRNA DANCR knockdown activated the AKT/mTOR pathway, whereas lncRNA DANCR overexpression or MK-2206 treatment disrupted this activation. Silencing of lncRNA DANCR facilitates BMSC OD by binding to miR-19a-3p and further regulating the PTEN/AKT/mTOR pathway.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":2.0,"publicationDate":"2026-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148880268","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":"Adipogenic effects of stevioside and rebaudioside A on proliferation and lipid accumulation in 3T3-L1 preadipocytes in vitro.","authors":"Cynthia P López Murillo, Rosa E Sierra Puente, Graciela Zambrano Galván, Abelardo Camacho Luis, Alma L Guerrero Barrera, Jorge A Burciaga Nava","doi":"10.1007/s11626-026-01245-0","DOIUrl":"https://doi.org/10.1007/s11626-026-01245-0","url":null,"abstract":"","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":""},"PeriodicalIF":2.0,"publicationDate":"2026-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148880263","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":"miR-135a-5p exerts tumor-suppressive effects in 769P renal carcinoma cells through the TFAP2A/KRT8 signaling axis.","authors":"Delong Xie, Jizhe Zhou, Yueyue Guo, Guangyang Wan, Feiyan Lu, Yuying Guo, Sangui Yi, Zongling Liu","doi":"10.1007/s11626-026-01188-6","DOIUrl":"10.1007/s11626-026-01188-6","url":null,"abstract":"<p><p>The oncogenic roles of microRNAs have been extensively documented across various cancer types. In the present study, we aimed to investigate the functional impact of miR-135a-5p on the clear cell renal cell carcinoma (ccRCC) cell line 769P. Through weighted gene co-expression network analysis (WGCNA), we identified a significant association between miR-135a-5p and ccRCC progression. Furthermore, miR-135a-5p expression was downregulated in both ccRCC tissues and cell lines. Functional assays demonstrated that miR-135a-5p inhibits the progression of 769P cells. Mechanistically, miR-135a-5p directly binds to the 3' untranslated region (3'UTR) of TFAP2A, reducing its mRNA stability. In turn, TFAP2A binds to the promoter region of KRT8 and activates its transcription, thereby mediating the inhibitory effect of miR-135a-5p on 769P cell progression. In conclusion, our findings provide evidence that miR-135a-5p suppresses ccRCC progression in 769P cells through the TFAP2A/KRT8 axis, highlighting its potential as a therapeutic target for ccRCC.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":"1168-1181"},"PeriodicalIF":2.0,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147837632","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":"Isoliquiritin alleviates sepsis-induced intestinal barrier dysfunction in mice potentially by promoting autophagy.","authors":"Qian Ye, Lingxuan Zhang, Xiuzhen Wang, Hong Sun, Hong Wang, Xin Wang, Junbo Zheng, Yeting Hou, Yun Wang, Yingrun Sun, Kai Liu","doi":"10.1007/s11626-026-01190-y","DOIUrl":"10.1007/s11626-026-01190-y","url":null,"abstract":"<p><p>Intestinal barrier function protection is essential to ameliorate sepsis. Isoliquiritin (ILQ) is a natural compound with broad biological activities. However, whether ILQ attenuates sepsis-induced intestinal damage remains unclear. An LPS-induced sepsis mouse model was established. ILQ inhibited body weight loss in sepsis mice. ILQ enhanced mouse intestinal function, as evidenced by decreased serum LDH levels and increased citrulline levels. Alcian blue staining showed that ILQ increased goblet cells in sepsis mouse ileum. ILQ increased the MUC2 RNA levels and decreased the claudin-2 protein expression in the sepsis mouse ileum. We subsequently examined the ILQ effects on intestinal autophagy activity. ILQ reduced the p62 protein expression and increased the conversion of LC3BI to LC3BII in sepsis mouse ileum. The LC3 protein expression was reduced in sepsis mouse ileum. An LPS-stimulated Caco-2 cell model was established. ILQ enhanced the viability of LPS-stimulated Caco-2 cells, reduced the LDH activity in cell supernatants, and increased the MUC2 RNA levels. ILQ enhanced the cellular barrier of LPS-stimulated Caco-2 cells, as indicated by the reduction in claudin-2 protein and the increase in TEER. ILQ also promoted autophagy activity in LPS-stimulated Caco-2 cells. Rescue experiments showed that an autophagy inhibitor disrupts the protective effect of ILQ on the Caco-2 cell barrier and inhibits ILQ-promoted autophagy activity. ILQ promoted the expression of LC3BII following the addition of chloroquine in LPS-stimulated Caco-2 cells. In conclusion, ILQ alleviated intestinal dysfunction and promoted autophagy activity in sepsis mice ileum, and might enhance the barrier function of LPS-stimulated Caco-2 cells via autophagy.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":"1093-1103"},"PeriodicalIF":2.0,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147837563","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":"3D HepG2 spheroid model exhibits drug-specific response profiles validating their application in therapeutic screening.","authors":"Wee Ling Choe, Ain Zubaidah Ayob, Muhammad Danish Ahmad Tarmizi, Nur'ain Salehen, Thamil Selvee Ramasamy","doi":"10.1007/s11626-026-01204-9","DOIUrl":"10.1007/s11626-026-01204-9","url":null,"abstract":"<p><p>This work establishes a robust and reproducible 96-well plate-based 3D HepG2 tumour spheroid model for drug screening applications. A 7-d culture protocol was optimised to generate HepG2 spheroid model using 96-well ultra-low attachment plates with 5 seeding densities (500, 1000, 2500, 5000 and 10,000 cells/well). Spheroids were evaluated for morphology, viability and proliferation. The optimized model was treated with doxorubicin and sorafenib at their respective IC₅₀ values (2.4 µM and 5.3 µM, determined in HepG2 monolayer). Drug-specific responses were assessed via spheroid growth, viability and expression of apoptotic and drug resistance markers. Spheroids seeded at 500 cells/well exhibited optimal characteristics, including sustained proliferative capacity (up to 30-fold increase relative to day 1) and progressive spheroidal growth (1.85-fold-change in diameter); hence, it was selected for further experiments. Day 4 was identified as the optimal treatment point based on morphology, viability and diameter progression. The 72-h IC₅₀ values were determined to be 2.4 µM for doxorubicin and 5.3 µM for sorafenib; these concentrations were chosen for subsequent treatments on HepG2-derived spheroids. Doxorubicin induced a delayed but potent cytotoxic response, while sorafenib triggered a slower, sustained effect, distinct from its rapid action in monolayer cultures. Furthermore, spheroid diameter did not correlate with viability or proliferation decline, highlighting its limitation as a sole readout. Molecular profiling revealed drug-specific responses in which ABCB1 was upregulated by doxorubicin, while ABCC2 was selectively induced by sorafenib. Sorafenib progressively reduced BAX/BCL2 ratio, suggesting potential resistance development. This study presents an optimized HepG2 spheroid model with validated culture and treatment parameters, capturing drug-specific cellular and molecular responses for therapeutic screening and mechanistic studies in hepatocellular carcinoma.</p>","PeriodicalId":13340,"journal":{"name":"In Vitro Cellular & Developmental Biology. Animal","volume":" ","pages":"1131-1142"},"PeriodicalIF":2.0,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148535793","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}