Cytotechnology最新文献

β-lactoglobulin (BLG) and carboxymethylcellulose (CMC) were conjugated by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The BLG-CMC conjugates with different CMC content and molecular weights were prepared. Confirmation of conjugation was carried out by SDS-PAGE. CD spectra revealed that the secondary structure of BLG had maintained in the conjugates. Fluorescence studies indicated that the conformation around Trp had not changed in the conjugates. Retinol-binding activity indicated that the retinol-binding site of BLG changed by the conjugation. Equilibrium constants (K_AS) of anti-BLG monoclonal antibodies (mAbs) to BLG after conjugating with CMC by competitive ELISA indicated that the structure around ¹⁵Val-²⁹Ile and ⁸Lys-¹⁹Trp maintained their native structure, and the structure around ¹²⁵Thr-¹³⁵Lys changed by conjugation. By conjugation with CMC, emulsifying property of BLG in the acidic pH region and in the presence of NaCl were much improved. Because acidic pH and salt are frequently used in food, the BLG-CMC conjugates are considered to be useful for food applications. Immunogenicity of BLG in BALB/c mice was reduced by this conjugation. In particular, there was a marked improvement in both emulsifying property and reduced immunogenicity in the BLG-high molecular weight (HMW) CMC conjugate. Therefore, conjugation with CMC is an effective way to improve BLG's function, and CMC with a high molecular weight is preferable.

[This corrects the article DOI: 10.1007/s10616-024-00698-z.].

Subretinal fibrosis is a main cause of visual loss in patients with neovascular age-related macular degeneration (nAMD), for whom there has been a lack of effective medication. Metformin can improve inflammation and angiogenesis in eye diseases. This study aimed to investigate the mechanism by which metformin inhibits subretinal fibrosis. A subretinal fibrosis cell model was induced by treating human retinal pigment epithelial cells (ARPE-19) with TGF-β1, a subretinal fibrosis mouse model was induced by a laser, and both cells and mice were treated with metformin. Cell proliferation, migration, and invasion were detected by CCK-8, scratch, and Transwell assays. Western blotting and immunofluorescence were used to evaluate protein expression levels, and RT‒qPCR was used to detect gene expression levels. HE and Masson staining were used to observe the morphological changes in retinal and choroidal tissues. Metformin treatment inhibited the TGF-β1-induced proliferation, migration, invasion and epithelial‒mesenchymal transition (EMT) of ARPE-19 cells and effectively ameliorated laser-induced subretinal fibrosis in mice. Mechanistically, metformin inhibits the expression of miR-126-5p, promotes Klotho synthesis, slows the progression of subretinal fibrosis, and miR-126-5p targets and negatively regulates Klotho. Metformin activates Klotho by inhibiting miR-126-5p, thereby reversing TGF-β1-induced ARPE-19 cell EMT and improving laser-induced subretinal fibrosis in mice.

Rheumatoid arthritis (RA) is a chronic, progressive, autoimmune inflammatory joint disease. The cause of synovitis in rheumatoid arthritis involves the interaction between immune cells/macrophages and fibroblast-like synoviocytes (FLSs-RA). The impact of circular RNAs on FLSs and their role in RA pathology is still unknown. This study aimed to investigate the roles and molecular mechanisms of circular RNA FNDC3B in regulating cell injury and glucose metabolism of FLSs in RA. We demonstrated that circFNDC3B was significantly upregulated and miR-125a-5p was significantly downregulated in FLSs from RA patients. When circFNDC3B was silenced or miR-125a-5p was overexpressed, it reduced FLSs-RA glucose metabolism and increased oxidative stress-induced cell injury. Through bioinformatics analysis, RNA pull-down, and luciferase assays, it was found that circFNDC3B sponged miR-125a-5p to create a ceRNA network in FLSs-RA. The glucose metabolism rate was elevated in FLSs-RA, showing a glucose-dependent characteristic compared to normal FLSs. The enzyme hexokinase 2 (HK2), which is crucial for glucose metabolism, was identified as a direct target of miR-125a-5p in FLSs. In rescue experiments, restoring miR-125a-5p in circFNDC3B-overexpressing FLSs-RA successfully counteracted the circFNDC3B-promoted glucose metabolism and resistance to cell injury. In conclusion, this study highlighted the important roles and molecular mechanisms of circFNDC3B in accelerating glucose metabolism and preventing cell apoptosis in fibroblast-like synoviocytes during rheumatoid arthritis by modulating the miR-125a-5p-HK2 axis. Targeting the circFNDC3B-mediated glucose metabolism pathway could be a promising strategy for rheumatoid arthritis therapy.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00745-3.

Diabetic nephropathy (DN) is a severe complication of diabetes, characterized by chronic inflammation, metabolic disturbances, and progressive renal damage. Natural perennial herb, such as Epimedium, has shown potential therapeutic effects on DN, but its underlying mechanisms remain unclear. This study aimed to explore the pharmacological mechanisms of Epimedium in the treatment of DN through network pharmacology, molecular docking, and experimental validation. Active components of Epimedium were identified using TCMSP and SwissTargetPrediction databases, while DN-related targets were retrieved from GeneCards, DisGeNET, OMIM, and TTD databases. Overlapping targets were analyzed via PPI network and Cytoscape's cytoHubba plugin to identify hub genes. GO and KEGG enrichment analyses were conducted to explore functional pathways. Molecular docking validated the binding affinity between key targets and active components. Finally, high-glucose-induced HK-2 cell injury models were used to verify the protective effects of Epimedium through RT-qPCR, western blotting, and mitochondrial function assays. A total of 224 overlapping targets were identified, with AKT1, TNF, HSP90AA1, and SRC serving as key hub genes. GO and KEGG analyses revealed significant enrichment in pathways such as the PI3K-Akt signaling pathway and lipid metabolism. Molecular docking demonstrated strong interactions between Epimedium components and hub targets. Experimental validation showed that Epimedium restored nephrin and WT1 protein levels, mitigated mitochondrial dysfunction, and reversed high-glucose-induced overexpression of key targets. Epimedium exerts therapeutic effects on DN through multi-target interactions, primarily via the PI3K-Akt pathway, highlighting its potential as a novel treatment for DN.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00748-0.

High-risk human papillomavirus (HPV), especially HPV16 and HPV18, are closely linked to the onset of cervical cancer (CC). Astragalin (AST), a bioactive flavonoid, has been reported to impede CC HeLa cell proliferation. Nevertheless, the mechanism by which AST exerts its tumor-suppressive role in CC remains unclear. HeLa (HPV18-positive) and CaSki (HPV16-positive) cells were exposed to various concentrations of AST. CCK-8 assay, flow cytometry analysis, wound healing, and Transwell assays were employed to examine the AST functions on CC cell aggressiveness. Protein levels were assessed by western blotting. Immunofluorescence staining was used to detect E6, E7, p53, and p-pRb expression. Animal experiments were performed to validate the anti-CC role in vivo. The results showed that AST dose-dependently impaired HeLa and CaSki cell viability and elicited G1 cell cycle arrest. AST restrained CC cell migration and invasiveness. AST inhibited the growth of HeLa-derived xenograft tumors in mice and repressed E6/E7 oncoprotein expression in CC cells and mouse tumor tissues. In conclusion, AST suppresses CC progression by downregulating E6/E7 oncoprotein expression to attenuate CC cell aggressiveness.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00742-6.

Recombinant monoclonal antibodies (mAbs) are commonly produced using Chinese hamster ovary (CHO) cells and the cell culture medium used in bioreactors influences the yield and quality attributes of the protein drug products. The COVID 19 pandemic revealed a vulnerability in the supply chain for necessary reagents (such as culture medium and raw material) for maintaining un-interrupted production of protein drugs with consistent quality. The supply interruption for the cell culture medium ActiPro™ optimized for producing VRC01, an IgG1-κ mAb, from a CHO-K1 cell line, necessitated the search for alternate media. VRC01 mAb is highly glycosylated and can broadly neutralize several strains of Human Immunodeficiency Virus (HIV). We investigated to see if an alternate medium can be used in the production without impacting quality attributes like glycosylation. In our strategy, we used 3 different commercially available media, performed two sets of experiments-with and without media supplements, Cell boost 7a and Cell boost 7b. Cell growth, volumetric production of the mAb protein and glycosylation pattern were compared to identify an alternative medium. Among the tested media based on cell growth, mAb production potential and glycosylation analysis, ActiCHO™ P was found to be a better alternate medium to ActiPro™ medium than EX-CELL® 325 PF CHO medium to produce VRC01 mAb. Overall, the approach used here to establish the impact of variation in medium on protein therapeutic attributes may be used during product development to build in supply chain resilience in drug manufacturing.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00733-7.

Platelet-rich plasma (PRP) has emerged as a promising biological therapy in regenerative medicine due to its high concentration of growth factors and cytokines, which promote tissue healing and regeneration. In recent years, its application in cartilage tissue engineering has garnered significant attention. This study explores the synergistic interaction between PRP and cartilage organoids, a novel three-dimensional in vitro culture system that closely mimics the structural and functional properties of native cartilage. Cartilage organoids serve as a physiologically relevant model for studying cartilage development, disease progression, and regeneration. By integrating PRP with cartilage organoids, this review aims to enhance chondrogenesis, extracellular matrix synthesis, and cellular proliferation within the organoids. Emerging evidence suggests that PRP supplementation significantly improves chondrocyte viability, growth, and differentiation in cartilage organoids, thereby accelerating their maturation. This combination holds great potential for advancing cartilage repair strategies, providing a robust platform for preclinical studies, and paving the way for innovative therapeutic approaches for cartilage-related injuries and degenerative diseases. These key aspects-chondrogenesis, matrix synthesis, and cellular proliferation-were specifically selected due to their fundamental roles in cartilage tissue engineering and regeneration. Chondrogenesis is crucial for chondrocyte differentiation and maintenance, matrix synthesis ensures the structural integrity and functional properties of regenerated cartilage, and cellular proliferation supports tissue viability and repair. Addressing these factors is essential, as current cartilage regeneration strategies often suffer from limited long-term efficacy and inadequate extracellular matrix production. By elucidating the synergistic effects of PRP and cartilage organoids in these areas, this study seeks to bridge existing knowledge gaps and provide valuable insights for improving regenerative approaches in clinical applications, particularly for osteoarthritis and cartilage defects.

Inflammatory bowel disease (IBD) is a chronic, progressive, immune-mediated, gastrointestinal inflammatory disease with increasing occurrences in children. Collagen triple helix repeat containing 1 (CTHRC1), a migration-promoting protein, acts as a tumor-promoting factor in malignant tumors. However, functions and mechanisms of CTHRC1 in children with IBD remain unclear. This study aimed to determine the effects and mechanisms of CTHRC1 on dextran sodium sulfate (DSS)-treated HT-29 cells. HT-29 control cells were exposed to 2% DSS to develop an in vitro IBD model. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to assess CTHRC1 expression in serum of children with IBD and HT-29 cells. Cell viability and apoptosis were assessed using MTT and flow cytometry (FCM). Expressions of cleaved-Caspase3 and Caspase3 were determined by western blotting. The cytokine production (TNF-α, IL-1β and IL-6) in HT-29 cells was measured by ELISA assay. Activation or inactivation of NF-κB signaling pathway was confirmed by western blot assay. Results showed that CTHRC1 expression was upregulated in the IBD serum and HT-29 control cells. The level of CTHRC1 was lower in CTHRC1-siRNA transfected cells than in control siRNA-treated cells. Notably, silence of CTHRC1 markedly enhanced HT-29 cells viability, decreased apoptotic cells, suppressed cleaved-Caspase3 expression, inhibited cleaved-Caspase3/Caspase3 ratio, reduced the production of inflammatory cytokines, and blocked NF-κB signaling pathway induced by DSS. However, these effects were reversed following diprovocim treatment. Thus, that knockdown of CTHRC1 alleviated DSS-induced HT-29 cell injury by inhibiting the NF-κB signaling pathway in vitro, providing a new therapeutic target for IBD in children.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00705-x.

In conventional Chinese hamster ovary (CHO) cell line development, static culture is used for early-stage screening, whereas suspension culture is generally used for the manufacturing process. Shaken flask (SF) fed-batch culture allows evaluation with culture mode, which is closer to the final process. However, due to its laborious and low-throughput characteristics, only a limited number of clones can be evaluated. To attain high-throughput fed-batch culture evaluation, a shaken 24-deep-well plate (24 DWP) culture process was developed. 24 DWP culture allows multiple plates to be run in parallel, and is therefore suitable for early-stage screening. One challenge of well plate culture is the nonuniform evaporation rate among wells, which may result in unnecessary bias on clone evaluation. The 'sandwich lid system' introduced here provides a uniform evaporation rate, and showed no significant difference in cell culture performance by well location. 192 antibody-producing CHO clones were evaluated by 24 DWP fed-batch culture, and 30 clones were selected. On comparison of clone sets selected by 24 DWP fed-batch culture and the conventional scheme, average antibody concentration in SF fed-batch culture was 3.6 g/L and 2.9 g/L, respectively. 24 DWP fed-batch culture process showed a high correlation ratio with SF fed-batch culture in antibody productivity and similar cell culture characteristics. These characteristics-high-throughput and sufficient culture volume to support cell culture performance monitoring-indicate that 24 DWP fed-batch culture can be applied in the clone selection stage in place of SF, and will shorten the time required for cell line development.