Relationship between delayed luminescence emission and mitochondrial status in Saccharomyces cerevisiae

Delayed luminescence (DL) is gradually used in various detection of biological systems as a rapid detection technique, however, its biological mechanism was still not clear. In this study, a new model of DL detection system for liquid biological samples is established to investigate the DL emission of Saccharomyces cerevisiae cells cultured in different glucose concentrations. We analyzed the relationship between the DL emission and cell growth, cell vitality, mitochondrial morphology, mitochondrial DNA (mtDNA) copy number, adenosine triphosphate (ATP), oxygen consumption rate (OCR), as well as mitochondria membrane potential (MMP) in S. cerevisiae cells cultured with 0.01, 0.05, 0.15, 3, 10 and 20 g/L glucose respectively. It was found that the DL emission had strong correlation with mitochondrial morphology, OCR, and MMP. The results suggested that DL is an indicator of mitochondria status under different glucose supply conditions, and may be an effective method to detect mitochondrial metabolism related disorders.

Transplantation of collagen sponge-based three-dimensional neural stem cells cultured in the RCCS system facilitate locomotor functional recovery in spinal cord injury animals

Numerous studies have indicated that microgravity induces various changes in the cellular functions of neural stem cells (NSCs), and the use of microgravity to culture tissue engineering seed cells for...

Phenotypic Profile of Peripheral Blood NK Cells under Culturing with Trophoblast Cells and IL-15 and IL-18 Cytokines

Natural killer cells (NK cells) are innate immunity lymphocytes. NK cell differentiation is controlled by the cellular microenvironment and locally produced cytokines, including IL-2, IL-15 and IL-18. NK cells are present in various tissues, forming pools of tissue-resident NK cells, e.g., decidual NK cell pool. Peripheral blood NK cells (pNK cells) are considered a supposed source of cells for decidual NK cell differentiation. In the uterus, NK cells contact with trophoblast cells, which can affect their phenotype. Contribution of trophoblast cells and IL-2, IL-15 and IL-18 cytokines to the pNK cell phenotype regulation is scarcely studied. In this regard, the aim of our research was to evaluate the effect of trophoblast cells on the phenotype of pNK cells when cultured in medium with IL-2, IL-15, and IL-18. We used mononuclear cells obtained from peripheral blood of healthy non-pregnant women at their reproductive age, with regular menstrual cycle (n = 21). Mononuclear cells were cultured in presence of IL-2, and either of cytokines regulating NK cell differentiation (IL-15, or IL-18). JEG-3 cells were used as trophoblast cells. We evaluated expression of CD45, CD3, CD56, CD14, KIR3DL1, KIR2DL3, KIR2DL4, KIR2DS4, NKp44, CD215, CD122, CD127, NKG2D, KIR2DL1, NKG2C receptors by pNK cells. It was found that pNK cells cultured in presence of trophoblast cells (JEG-3 cell line) were characterized by lower intensity of CD56 receptor expression, compared to pNK cells cultured without trophoblast cells. These changes were detected upon culturing both in medium supplied by IL-15, and with IL-18. A reduced number of NKG2C+ pNK cells was detected in presence of JEG-3 trophoblast cells, compared to NK cells cultured without trophoblast cells in medium with IL-15. The detected changes in the CD56 and NKG2C expression by pNK cells in presence of trophoblast cells proved to be opposite to those previously detected for NK cells derived from NK-92 cell line. Along with trophoblast cells, the monocytes isolated among mononuclear cells and being affected by cytokines, can apparently influence the phenotype of pNK cells in the model system used. Since monocytes/macrophages are present in decidua, further research is required to study the effect of cytokines and cellular microenvironment, including monocytes, on pNK cells.

Characterization of increased mucus production of HT29-MTX-E12 cells grown under Semi-Wet interface with Mechanical Stimulation

The intestinal mucus layer plays a crucial role in human health. To study intestinal mucus function and structure in vitro, the mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used. However, this cell line produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured under Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2, concomitant with a thicker mucus layer, compared to cells cultured conventionally. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased MUC2 production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell membranes for 14 days under static and SWMS conditions (after cell seeding and attachment) were subjected to transcriptome analysis to investigate underlying molecular pathways at gene expression level. Caco-2 and LS174T cell lines were included as references. We characterized how SWMS conditions affected HT29-MTX-E12 cells in terms of epithelial barrier integrity, by measuring transepithelial electrical resistance, and cell metabolism, by monitoring pH and lactate production per molecule glucose of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions at gene and protein level and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms by which MUC2 production is enhanced under SWMS and propose potential applications of this model in future studies.

Inhibition of matrix stiffness relating integrin β1 signaling pathway inhibits tumor growth in vitro and in hepatocellular cancer xenografts

Background Cancer development is strictly correlated to composition and physical properties of the extracellular matrix. Particularly, a higher matrix stiffness has been demonstrated to promote tumor sustained growth. Our purpose was to explore the role of matrix stiffness in liver cancer development. Methods The matrix stiffness of tumor tissues was determined by atomic force microscopy (AFM) analysis. In vitro, we used a tunable Polyacrylamide (PA) hydrogels culture system for liver cancer cells culture. The expression level of integrin β1, phosphorylated FAK, ERK1/2, and NF-κB in SMMC-7721 cells was measured by western blotting analysis. We performed MTT, colony formation and transwell assay to examine the tumorigenic and metastatic potential of SMMC-7721 cells cultured on the tunable PA hydrogels. SMMC-7721 cancer xenografts were established to explore the anticancer effects of integrin inhibitors. Results Our study provided evidence that liver tumor tissues from metastatic patients possessed a higher matrix stiffness, when compared to the non-metastatic group. Liver cancer cells cultured on high stiffness PA hydrogels displayed enhanced tumorigenic potential and migrative properties. Mechanistically, activation of integrin β1/FAK/ ERK1/2/NF-κB signaling pathway was observed in SMMC-7721 cells cultured on high stiffness PA hydrogels. Inhibition of ERK1/2, FAK, and NF-κB signaling suppressed the pro-tumor effects induced by matrix stiffness. Combination of chemotherapy and integrin β1 inhibitor suppressed the tumor growth and prolonged survival time in hepatocellular cancer xenografts. Conclusion A higher matrix stiffness equipped tumor cells with enhanced stemness and proliferative characteristics, which was dependent on the activation of integrin β1/FAK/ERK1/2/NF-κB signaling pathway. Blockade of integrin signals efficiently improved the outcome of chemotherapy, which described an innovative approach for liver cancer treatment.