Cytotechnology最新文献

Fetal bovine serum (FBS), which is widely used in cell culture media, has the potential to cause medical and ethical problems. Here, an experimental study using milk or whey proteins containing essential nutrients and growth factors is presented to limit the use of FBS in cell culture media produced for cell and tissue regeneration. Study groups were formed by culturing human placenta mesenchymal stem cells, known to have high proliferation and differentiation capacity, with milk or whey solution at increasing concentrations, alone or in combination with FBS. Osteogenic and adipogenic differentiation capacities of proliferating cells were observed in FBS, milk or whey groups. Milk, whey or FBS groups obtained in P3 and after differentiation were separately analyzed for protein mRNA expression by reverse transcriptase-polymerase chain reaction (RT-qPCR). Fibroblast Growth Factor 2 (FGF2), Octamer-binding Transcription Factor 4 (OCT4), Bone Morphogenetic Protein 6 (BMP6), and adipogenic differentiation marker Peroxisome Proliferator-Activated Receptor Gamma (PPARG) were analysed by RT-qPCR. Proliferation was more pronounced in FBS alone and in its combinations with milk-whey compared to the groups in which only milk and whey were used. OCT4 mRNA and FGF2 mRNA expression decreased in differentiated cells. BMP6 mRNA expression increased with osteogenic and adipogenic stimuli. As expected, PPRG expression also increased with adipogenic stimulation. With this experimental study, evidence has been obtained that milk or whey can provide nutritional support to the culture media of repair cells and preserve the functional capacity of the cells, with a slightly more limited capacity than FBS.

Graphical abstract:

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-023-00585-z.

Gastric cancer (GC) is a heterogeneous disease and is the fifth most common cancer worldwide. Lobetyolin, as a bioactive ingredient extracted from Codonopsis pilosula (Franch.) Nannf., has been reported to exert anti-tumor effects in several cancer types. This study was aimed to investigate the role of lobetyolin in GC and the associated mechanism. MKN-45 and MKN-28 cells were incubated with concentrations of lobetyolin for 24 h. The viability and survival of GC cells were evaluated by performing MTT assay. Glutamine uptake, Adenosine Triphosphate, reactive oxygen species (ROS), and glutathione levels were measured by corresponding kits. Apoptosis and mitochondrial membrane potential of GC cells were determined by flow cytometry. Alanine, serine, cysteine-preferring transporter 2 (ASCT2) and the AKT/GSK3β/c-Myc pathway protein levels were examined by western blotting. Xenograft model and immunohistochemical staining were used to evaluate the pharmacological effects of lobetyolin in mice in vivo. We found that lobetyolin treatment suppressed the proliferative capacity of both MKN-45 and MKN-28 cells in a concentration-dependent manner. Lobetyolin reduced the uptake of glutamine and downregulated the expression levels of ASCT2 in GC cells and xenograft tumors. Lobetyolin effectively restrained the growth of tumors in vivo. In addition, lobetyolin induced the accumulation of ROS to attenuate mitochondria-mediated apoptosis via downregulation of ASCT2 expression. Lobetyolin promoted the phosphorylation of c-Myc and suppressed the phosphorylation of GSK3β and AKT in both MKN-45 and MKN-28 cells. The level of total Nrf2 protein was reduced after lobetyolin treatment. Overall, lobetyolin exerts anti-cancer effects by repressing cell proliferation and inducing cell apoptosis via downregulation of ASCT2 in GC.

miRNA expressions are altered during development of breast cancer (BC). The aim of this study is to identify novel cancer-related miRNAs and pathways to understand the mechanisms of BC subtypes. GSE59247 dataset was downloaded from gene expression omnibus (GEO) database and analyzed with GEO2R software. The differential miRNA expressions in BC cells were evaluated by miRNome PCR array. Venn diagram was used to reveal co-differentially expressed miRNAs between GSE59247 dataset and miRNome array. Clinical prognostic significance of selected miRNAs was evaluated via Kaplan Meier curve. KEGG pathway enrichment analysis was performed to find miRNA targets and results were validated by TNM plot analysis and q-RT-PCR. TargetScan database was used to predict the association of miRNAs and 3'-untranslated regions of target genes and their expressions were visualized by human protein atlas database. Venn diagram analysis showed overlap of 11 miRNAs from in silico and in vitro analysis. KEGG analysis revealed 'Lysine Degradation Pathway' as the most significantly enriched targeted pathway. q-RT-PCR results confirmed that Lysine degradation pathway related genes SETD7, SETDB2, EHHADH, SETMAR, KMT2A and SUV39H2 were differentially expressed in BC cells. Target prediction analysis identified binding sites between miR-1323-5p and 3'-UTR of SETD7, miR-129-5p and 3'-UTR of EHHADH and miR-628-5p and 3'-UTR of SETDB2 mRNA. Notably, miR-1323-5p, miR-129-5p, and miR-628-5p are differentially expressed in BC and they bind to 3'UTR of critical genes of Lysine degradation pathway, namely SETD7, SETDB2 and EHHADH. These miRNAs might serve as potential diagnostic and prognostic biomarkers for progression.

Interchain disulfide bonds in monoclonal antibodies may be reduced during large-scale mAb production using Chinese hamster ovary (CHO) cells. This reaction lowers the mAb product yield and purity; however, it may be prevented by screening cell lines that are unsusceptible to reduction and using them in mAb production. Antibody reduction susceptibility may be cell line-dependent. To the best of our knowledge, however, an efficient method of screening reduction-unsusceptible CHO cell lines has not been previously reported. Here, we report a novel screening method that can simultaneously detect and identify mAb reduction susceptibility in lysates containing ≤ 48 CHO cell lines. This evaluation system was equally effective and generated similar results at all culture scales, including 250 mL, 3 L, and 1000 L. Furthermore, we discovered that reduction-susceptible cell lines contained higher total intracellular nicotinamide adenine dinucleotide phosphate (NADPH) and NADP⁺ concentrations than reduction-unsusceptible cell lines, regardless of whether they expressed immunoglobulin (Ig)G4 or IgG1. NADPH or NADP⁺ supplementation in the lysate of reduction-unsusceptible cells resulted in mAb reduction. Application of the innovative CHO cell line screening approach could mitigate or prevent reductions in large-scale mAb generation from CHO cells.

Growing evidence demonstrates that cancer-associated fibroblasts (CAF) are responsible for tumor genesis, growth, metastasis, and treatment response. Therefore, targeting these cells may contribute to tumor control. It has been proposed that targeting key molecules and pathways of proliferative functions can be more effective than killing CAFs. In this regard, multicellular aggregates, like spheroids, can be used as human tumor models. Spheroids closely resemble human tumors and mimic many of their features. Microfluidic systems are ideal for cultivation and study of spheroids. These systems can be designed with different biological and synthetic matrices in order to have a more realistic simulation of the tumor microenvironment (TME). In this study, we investigated the effect of all-trans retinoic acid (ATRA) on 3D spheroid invasion of MDA-MB cells exposed to hydrogel matrix derived from CAFs. The number of invasive cells significantly decreased in CAF-ECM hydrogel treated with ATRA (p < 0.05), which indicates that ATRA could be effective for CAFs normalization. This experiment was done using an agarose-alginate microfluidic chip. As compared with common methods, such hydrogel casting is an easier method for chip fabrication and can even reduce costs.

Graphical abstract:

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-023-00578-y.

The stimulation of interleukin-1β (IL-1β) is the risk factor for temporomandibular joint osteoarthritis (TMJOA). We aim to investigate IL-1β stimulation-related gene and signal pathways in synovial fluid-derived mesenchymal stem cells (SF-MSCs) inflammatory activation to predict the occurrence of TMJOA. The microarray dataset GSE150057 was downloaded from the gene expression omnibus (GEO) database, and principal component analysis (PCA) was performed on the involved genes to obtain differential genes (DEGs). Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway were performed based on the DAVID database. The protein-protein interaction (PPI) network was constructed by the STRING database to identify hub genes. Based on the correlation between differential expression levels of lncRNAs and mRNAs, the co-expression network of lncRNA-mRNA was established. A total of 200 DEGs were obtained. Among 168 differential mRNAs, 126 were up-regulated and 42 were down-regulated; among 32 differential lncRNAs, 23 were up-regulated and 9 were down-regulated. Then, GO analysis showed that DEGs were mainly involved in signal transduction, inflammation, and apoptosis processes. KEGG pathway mainly involved the TNF signaling pathway, NF-κB signaling pathway, NOD-like receptor signaling pathway, and cytokine-cytokine-receptor interaction. Ten hub genes were recognized by PPI analysis, including CXCL8, CCL2, CXCL2, NFKBIA, CSF2, IL1A, IRF1, VCAM1, NFKB1, and TNFAIP3. In conclusion, our study has indicated the role of IL-1β stimulation in the progression of SF-MSCs inflammation and predicted DEGs and downstream pathways.

The plasticizer di (2-ethylhexyl) phthalate (DEHP) inhibits differentiation, impairs glucose metabolism, and decreases mitochondrial function in murine muscle satellite cells; however, if these effects are translated to human cells is unknown. The goal of this study was to evaluate changes in morphology and proliferation of primary human skeletal muscle cells exposed to DEHP. Rectus abdominis muscle samples were obtained from healthy women undergoing programed cesarean surgery. Skeletal muscle cells were isolated and grown under standard primary culture conditions, generating two independent sample groups of 25 subcultures each. Cells from the first group were exposed to 1 mM DEHP for 13 days and monitored for changes in cell morphology, satellite cell frequency and total cell abundance, while the second group remained untreated (control). Differences between treated and untreated groups were compared using generalized linear mixed models (GLMM). Cell membrane and nuclear envelope boundary alterations, loss of cell volume and presence of stress bodies were observed in DEHP-treated cultures. DEHP-treated cultures also showed a significant reduction in satellite cell frequency compared to controls. Exposure to DEHP reduced human skeletal muscle cell abundance. Statistical differences were found between the GLMM slopes, suggesting that exposure to DEHP reduced growth rate. These results suggest that exposure to DEHP inhibits human skeletal muscle cell proliferation, as evidenced by reduced cell abundance, potentially compromising long-term culture viability. Therefore, DEHP induces human skeletal muscle cell deterioration potentially inducing an inhibitory effect of myogenesis by depleting satellite cells.

Graphical abstract:

The M2 macrophages are major components in the tumor microenvironment and are closely linked to immune suppression and tumor metastasis. This work focuses on how M2 macrophage-derived extracellular vesicles (EVs) affect colorectal cancer (CRC) progression. THP-1 monocytes were induced to differentiate to M0 or M2 macrophages, and the macrophage-derived EVs (M0-EVs and M2-EVs, respectively) were collected and identified. The M2-EVs stimulation augmented proliferation, mobility, and the in vivo tumorigenic activity of CRC cells. Circular RNA_CCDC66 (circ_CCDC66) was highly enriched in M2-EVs and could be delivered into CRC cells. The RNA pull-down and luciferase assays showed that circ_CCDC66 could competitively bind to microRNA (miR)-342-3p, therefore restoring the expression of metadherin (MTDH) mRNA, a target transcript of miR-342-3p. Suppression of circ_CCDC66 in the M2-EVs or specific knockdown of MTDH in CRC significantly blocked the growth and mobility of CRC cells. However, miR-342-3p inhibition restored the malignant phenotype of cancer cells. Moreover, the MTDH knockdown was found to increase the cytotoxicity of CD8⁺ T and reduce the protein level of the immune checkpoint PDL1 in CRC cells. In summary, this study reveals that the M2-EVs augment immune evasion and development of CRC by delivering circ_CCDC66 and restoring the MTDH level.

This review aims to describe the most recent achievements and provide an insight into cartilage engineering and strategies to restore the cartilage defects. Here, we discuss cell types, biomaterials, and biochemical factors applied to form cartilage tissue equivalents and update the status of fabrication techniques, which are used at all stages of engineering the cartilage. The actualized concept to improve the cartilage tissue restoration is based on applying personalized products fabricated using a full cycle platform: a bioprinter, a bioink consisted of ECM-embedded autologous cell aggregates, and a bioreactor. Moreover, in situ platforms can help to skip some steps and enable adjusting the newly formed tissue in the place during the operation. Only some achievements described have passed first stages of clinical translation; nevertheless, the number of their preclinical and clinical trials is expected to grow in the nearest future.