Effects of DMEM and RPMI 1640 on the biological behavior of dog periosteum-derived cells

Periosteum-derived cells (PDCs) are being extensively studied as potential tissue engineering seed cells and have demonstrated tremendous promise to date. There is convincing evidence that culture medium could modulate the biological behavior of cultured cells. In this study, we investigate the effects of DMEM (low glucose) and RPMI 1640 on cell growth and cell differentiation of PDCs in vitro. PDCs isolated from Beagle dogs were maintained in DMEM and RPMI 1640, respectively. Then, the cell migration rate of periosteum tissues was analyzed. PDCs of the third passage were harvested for the study of proliferation and osteogenic activity. Proliferation was detected by MTT assay. Alkaline phosphatase activity and mineralized nodules were measured to investigate osteogenic differentiation. Our data demonstrated that DMEM induced alkaline phosphatase activity and strongly stimulated matrix mineralization in cell culture, while similar cell migration rates and proliferation behaviors were observed in the two culture conditions. Interestingly, the osteogenic differentiation of PDCs could be enhanced in DMEM compared with that in RPMI 1640. Thus, it can be ascertained that DMEM may serve as a suitable culture condition allowing osteogenic differentiation of dog PDCs.     

Activation of Hydrogen Peroxide to Peroxytetradecanoic Acid Is Responsible for Potent Inhibition of Protein Tyrosine Phosphatase CD45

Hydrogen peroxide induces oxidation and consequently inactivation of many protein tyrosine phosphatases. It was found that hydrogen peroxide, in the presence of carboxylic acids, was efficiently activated to form even more potent oxidant - peroxy acid. We have found that peroxytetradecanoic acid decreases the enzymatic activity of CD45 phosphatase significantly more than hydrogen peroxide. Our molecular docking computational analysis suggests that peroxytetradecanoic acid has a higher binding affinity to the catalytic center of CD45 than hydrogen peroxide.     

Inhibitory effect of extracellular histidine on cobalt-induced HIF-1alpha expression

Cobalt chloride (CoCl(2)) can mimic hypoxia in inducing hypoxia-inducible factor 1 (HIF-1). Several cultured cells were examined for susceptibility to CoCl(2) in DMEM, MEM and RPMI 1640 medium. Here we report that HIF-1α expression of mammalian cells by CoCl(2) was largely dependent on the culture medium. HIF-1α protein and hypoxia response element (HRE)-dependent reporter activity were strongly induced in RPMI 1640 but not in DMEM in several cultured cells including MCF-7, a human breast cancer cell line. Analysis of causal nutrients has revealed that histidine, which is contained richer in DMEM, acts as the inhibitory nutrient for cobalt-induced HIF-1α expression of MCF-7 cells in DMEM. D-Histidine also inhibited the HIF-1α activity at the same level as L-histidine, suggesting that sequestration of free cobaltous ion by chelation with histidine was the cause of the inhibition. These results demonstrate that selection of the culture medium must be considered with caution in cell culture experiments using CoCl(2) as a hypoxia-mimetic reagent.     

Generation of dendritic cells from human peripheral blood monocytes--comparison of different culture media

Culture medium or medium supplement is one of the factors responsible for dendritic cell (DC) generation, but little is known about the influence of various media on DC culture. In our study we generated DC from adherent monocytes of human peripheral blood in the presence of GM-CSF, IL-4 and TNF-alpha. The following culture media were used: RPMI 1640 supplemented with 2% human serum albumin; RPMI 1640 supplemented with 2% TCH serum replacement; X-VIVO 15 and Panserin 501. Flow cytometry analysis revealed that in all media cells were CD83+ and lost CD14. Interestingly, the use of Panserin and RPMI with albumin preferentially gave rise to CD1a+ DC, whereas in X-VIVO and RPMI with TCH we observed both CD1a+ and CD1a-. Our results showed that RPMI with TCH yielded the highest percentage of cells expressing both CD80 and CD86 molecules and, in contrast to other media, the higher percentage of CD86+ cells in comparison to CD80+ cells.     

Reactive oxygen species induce reversible PECAM-1 tyrosine phosphorylation and SHP-2 binding

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) functions to control the activation and survival of the cells on which it is expressed. Many of the regulatory functions of PECAM-1 are dependent on its tyrosine phosphorylation and subsequent recruitment of the Src homology (SH2) domain containing protein tyrosine phosphatase SHP-2. The recent demonstration that PECAM-1 tyrosine phosphorylation occurs in cells exposed to the reactive oxygen species hydrogen peroxide (H2O2) suggested that this form of oxidative stress may also support PECAM-1/SHP-2 complex formation. In the present study, we show that PECAM-1 tyrosine phosphorylation in response to exposure of cells to H2O2 is reversible, involves a shift in the balance between kinase and phosphatase activities, and supports binding of SHP-2 and recruitment of this phosphatase to cell-cell borders. We speculate, however, that the unique ability of H2O2 to reversibly oxidize the reactive site cysteine residues of protein tyrosine phosphatases may result in transient inactivation of the SHP-2 that is bound to PECAM-1 under these conditions. Finally, we provide evidence that PECAM-1 tyrosine phosphorylation and SHP-2 binding in endothelial cells requires exposure to an "oxidative burst" of H2O2, but that exposure of these cells to sufficiently high concentrations of H2O2 for a sufficiently long period of time abrogates binding of SHP-2 to tyrosine-phosphorylated PECAM-1. These findings support a role for PECAM-1 as a sensor of oxidative stress, perhaps most importantly during the process of inflammation.     

Oxidative stress-induced expression and modulation of Phosphatase of Regenerating Liver-1 (PRL-1) in mammalian retina

The phosphatase of regenerating liver-1, PRL-1, gene was detected in a screen for foveal cone photoreceptor-associated genes. It encodes a small protein tyrosine phosphatase that was previously immunolocalized to the photoreceptors in primate retina. Here we report that in cones and cone-derived cultured cells both PRL-1 activity and PRL-1 gene expression are modulated under oxidative stress. Oxidation reversibly inhibited the phosphatase activity of PRL-1 due to the formation of an intramolecular disulfide bridge between Cys104 within the active site and another conserved Cys, Cys49. This modulation was observed in vitro, in cell culture and in isolated retinas exposed to hydrogen peroxide. The same treatment caused a rapid increase in PRL-1 expression levels in cultured cells which could be blocked by the protein translation inhibitor, cycloheximide. Increased PRL-1 expression was also observed in living rats subjected to constant light exposure inducing photooxidative stress. We further demonstrated that both oxidation and overexpression of PRL-1 upon oxidative stress are greatly enhanced by inhibition of the glutathione system responsible for cellular redox regulation. These findings suggest that PRL-1 is a molecular component of the photoreceptor's response to oxidative stress acting upstream of the glutathione system.     

Population doubling time, phosphatase activity, and hydrogen peroxide generation in Jurkat cells.

Differences in growth characteristics, phosphatase activity, and hydrogen peroxide generation in two clones of a T-cell leukemic line are described in this communication. Wurzburg cells had significantly shorter population doubling times compared with the parental Jurkat cells (16.6 +/- 2.0 h and 20.7 +/- 2.2 h, respectively; mean +/- SD, p < .0001, n = 20). In addition, total phosphatase activity was significantly decreased (p < .006) and hydrogen peroxide production was significantly increased (p < .002) in Wurzburg cells compared to Jurkat cells. That the cell line with the faster growth rate should have these latter two properties is entirely consistent with the positive effects of increased kinase activity and hydrogen peroxide on proliferative cellular responses in T cells. As originally described, Wurzburg cells were distinguished from Jurkat cells by their lack of CD45, a membrane protein tyrosine phosphatase, and their positive response to hydrogen peroxide-stimulation of NF-kappaB activation. We propose that these two clones, with their distinguishing characteristics, can be used to advantage in experiments designed to study the effects of antioxidants on signaling pathways that control cell life and death.     

Effect of media composition on the induction of chorionic gonadotropin by sodium butyrate in HeLa cells

Summary Production of the glycoprotein hormone α-subunit by HeLa cells and its induction by sodium butyrate are dependent on the choice of culture medium. Under identical growth conditions it was found that subunit synthesis in the presence of butyrate was highest in RPMI 1640, lowest in Medium 199 (M199), and intermediate in minimum essential medium (MEM) and Waymouth's MB 752/1. Cell growth was similar in all media examined and was retarded in the presence of butyrate. Alkaline phosphatase activity was also lower in M199 than in RPMI 1640, although, in general, the magnitude of this difference was less than that for the hormone subunit. Incorporation of [1-¹⁴C]butyrate by HeLa cells was simimar in both M199 and RPMI 1640, indicating that uptake and metabolism of the fatty acid were not significantly different under these conditions. In the presence of 3 mM butyrate, mixtures of RPMI 1640 and M199 gave intermediate levels of α-subunit and alkaline phosphatase compared to each medium alone. Intracellular levels of α-subunit as well as that of the culture medium were reduced in M199 compared to RPMI 1640 indicating that synthesis rather than secretion was altered. This work was supported by Grant CA 21534 from the National Institutes of Health, Bethesda, MD.     

Oxidation and generation of hydrogen peroxide by thiol compounds in commonly used cell culture media

Many studies have examined the effects of thiol compounds upon cells in culture (e.g., upon signal transduction and regulation of gene expression), but few have considered how thiols can interact with cell culture media. A wide range of thiols (cysteine, GSH, N-acetylcysteine, gamma-glutamylcysteine, cysteinylglycine, cysteamine, homocysteine) were found to interact with three commonly used cell culture media (RPMI, MEM, DMEM) to generate hydrogen peroxide with complex concentration-dependencies. Thiols added to these media rapidly disappeared, although less H(2)O(2) was generated on a molar basis than the amount of thiol lost. Studies on cellular effects of thiols, especially those on redox regulation of gene expression or protein function, need to take into account that thiols are rapidly lost, and that their oxidation generates H(2)O(2), which can have multiple concentration-dependent effects on cell metabolism.     

Analysis of the cytotoxic effects of light-exposed hepes-containing culture medium

Summary The addition ofN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) to RPMI 1640 medium markedly increases the production of cytotoxic products during exposure of the medium to visible light. The cytotoxicity has been analyzed by measuring uptake of [³H]thymidine by murine thymocytes cultured in preirradiated medium containing 25 mM HEPES. Complete inhibition of thymidine uptake was produced by exposing 50% of the culture medium to light for 3 h before addition of cells. The HEPES-mediated effect requires only that HEPES and riboflavin be exposed to light; other medium constituents are not necessary. Hydrogen peroxide is a principal cytotoxic agent produced in this system. It is demonstrated that most, but not all, of the inhibition of thymidine uptake can be attributed to hydrogen peroxide.     

Instability of, and generation of hydrogen peroxide by, phenolic compounds in cell culture media

Many papers in the literature have described complex effects of flavonoids and other polyphenols on cells in culture. In this paper we show that hydroxytyrosol, delphinidin chloride and rosmarinic acid are unstable in three commonly-used cell culture media (Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 (RPMI) and Minimal Essential Medium Eagle (MEM)) and undergo rapid oxidation to generate H₂O₂. This may have confounded some previous studies on the cellular effects of these compounds. By contrast, apigenin, curcumin, hesperetin, naringenin, resveratrol and tyrosol did not generate significant H₂O₂ levels in these media. Nevertheless, curcumin and, to a lesser extent, resveratrol (but not tyrosol) were also unstable in DMEM, so the absence of detectable H₂O₂ production by a compound in cell culture media should not be equated to stability of that compound. Compound instability and generation of H₂O₂ must be taken into account in interpreting effects of phenolic compounds on cells in culture.     

Light-dependent generation of reactive oxygen species in cell culture media

Cell culture media (RPMI 1640, Dulbecco’s Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5′-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS). The production of ROS was light dependent. The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin. These observations point to exposure of cells to ROS under in vitro culture conditions.     

Evaluation of culture media for effects on cell cycle kinetics and incidence of chromosomal aberrations in human blood cells

Peripheral blood samples from 17 apparently healthy male volunteers were set up in duplicate cultures using three commercially available media: Eagle's MEM, RPMI 1640, and TC 199. BUdR (5-bromo,2-deoxyuridine) (10 micrograms/mL) was added to one of the cultures from each person in each medium after 24 h of culture initiation. All cultures were harvested at 72 h of incubation in the presence of colcemid. RPMI 1640 stimulated the highest mitotic activity in both BUdR-treated and untreated cultures. Higher numbers of first division metaphases corresponded with the higher frequency of chromosome-type aberrations in cultures with Eagle's MEM as compared with RPMI 1640 media. On the other hand, higher numbers of chromatid-type aberrations were present in cultures with TC 199 as compared with those with Eagle's MEM. When the chromosome- and chromatid-type aberration data were pooled to score total cytogenetic abnormalities, an influence of the medium was demonstrable. While cultures with Eagle's MEM and TC 199 had the greater number of first division cells, third of subsequent division cells were most prevalent in RPMI 1640 cultures. It is inferred that the length of the cell cycle, the mitotic index, and to some degree the incidence of spontaneous cytogenetic abnormalities are variable attributes of culture media.     

Influence of hyperosmolar basal media on hybridoma cell growth and antibody production

To investigate the influence of hyperosmolar basal media on hybridoma response, S3H5/γ2bA2 and DB9G8 hybridomas were cultivated in a batch mode using hyperosmolar basal media resulting from additional sodium chloride supplementation. The basal media used in this study were IMDM, DMEM, and RPMI 1640, all of which are widely used for hybridoma cell culture. In IMDM, two hybridomas showed different responses to hyperosmotic stress regarding specific MAb productivity (q _MAb), though they showed similar depression of cell growth in hyperosmolar media. Unlike S3H5/γ2bA2 hybridoma, the q _MAb of DB9G8 hybridoma was not enhanced significantly around 390 mOsm kg^?1. The variation of basal media influenced DB9G8 hybridoma response to hyperosmotic stress regarding q _MAb. In IMDM, the q _MAb of DB9G8 hybridoma was increased by more than 200% when the osmolality increased from 281 to 440 mOsm/kg. However, in RPMI 1640 and DMEM, similar amplitude of osmolality increase resulted in less than 100% increase in q _MAb. The variation of basal media also influenced the cell growth in hyperosmolar medium. Both hybridomas were more tolerant against hyperosmotic stress in DMEM than in IMDM, which was found to be due to the high osmolality of standard DMEM. The osmolalities of standard IMDM and DMEM used for inocula preparation were 281 and 316 mOsm kg^?1, respectively. Thus, when the cells were cultivated at 440 mOsm kg^?1, the cells in IMDM experienced higher osmotic shock than in DMEM. By using the inoculum prepared at 317 mOsm kg^?1 in IMDM, S3H5/γ2bA2 cell growth at 440 mOsm kg^?1 in IMDM was comparable to that in DMEM. Taken together, the results obtained from this study show that the selection of basal media is an important factor for MAb production by employing hyperosmotic stress.     

GM1 Ganglioside Activates ERK1/2 and Akt Downstream of Trk Tyrosine Kinase and Protects PC12 Cells Against Hydrogen Peroxide Toxicity

Ganglioside GM1 at micro- and nanomolar concentrations was shown to increase the viability of pheochromocytoma PC12 cells exposed to hydrogen peroxide and diminish the accumulation of reactive oxygen species and oxidative inactivation of Na⁺,K⁺-ATPase, the effects of micromolar GM1 being more pronounced than those of nanomolar GM1. These effects of GM1 were abolished by Trk receptor tyrosine kinase inhibitor and diminished by MEK1/2, phosphoinositide 3-kinase and protein kinase C inhibitors. Hydrogen peroxide activates Trk tyrosine kinase; Akt and ERK1/2 are activated downstream of this protein kinase. GM1 was found to activate Trk receptor tyrosine kinase in PC12 cells. GM1 (100 nM and 10 µM) increased the basal activity of Akt, but did not change Akt activity in cells exposed to hydrogen peroxide. Basal ERK1/2 activity in PC12 cells was increased by GM1 at a concentration of 10 µM, but not at nanomolar concentrations. Activation of ERK1/2 by hydrogen peroxide was enhanced by GM1 at a concentration of 10 µM and to a lesser extent at a concentration of 100 nM. Thus, the protective and metabolic effects of GM1 ganglioside on PC12 cells exposed to hydrogen peroxide appear to depend on the activation of Trk receptor tyrosine kinase and downstream activation of Akt and ERK1/2.     

青海湖裸鲤体外肝胰细胞原代与传代培养

采用组织块移植培养技术,分别用DMEM和RPM11640培养基对青海湖裸鲤肝胰组织细胞进行原代培养。培养48h组织块周围有细胞迁出,并形成生长晕。培养一周可形成单层细胞。对原代培养的单层细胞用胰蛋白酶-EDTA消化后,传代培养至第四代。确立青海湖裸鲤肝胰细胞培养条件为：培养基为DMEM,培养温度为27℃,pH值为7．0—7．5,原代培养血清浓度为20％,传代培养的血清浓度为10％,无需通入CO2和添加细胞生长因子。     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum     

TCTP is a critical survival factor that protects cancer cells from oxidative stress-induced cell-death

The translationally controlled tumor protein (TCTP) displays growth-promoting and antiapoptotic properties. To gain information on the role of TCTP in cancer disease, we studied the modulation of TCTP and cell survival under stress conditions on tumor cell lines of different origins. When cancer cells were exposed to a mild oxidative stress, such low doses of Arsenic trioxide (ATO) or hydrogen peroxide (H₂O₂), up-regulation of TCTP was observed in cells survived to the treatment. Differently, a strong oxidative hit provided by ATO combined with glutathione (GSH) depletion or condition of glucose deprivation caused a down-modulation of TCTP followed by cell death.Clones with a forced expression of TCTP or with silenced TCTP were obtained from the breast cancer cell line MDA-MB-231. The sensitivity to oxidative stress was strongly enhanced in down-modulated TCTP cells while decreasing in cells with high levels of TCTP.Together these results indicate that TCTP is a survival factor that protects cancer cells from oxidative stress-induced cell-death. We propose TCTP as a “stress hallmark” that may be exploited as a therapeutic target to decrease the resistance of cancer cells to anticancer therapy.     

The effects of oxaloacetate on hydrogen peroxide generation from ascorbate and epigallocatechin gallate in cell culture media: potential for altering cell metabolism

Several phenolic compounds as well as ascorbate can oxidise in certain cell culture media (especially Dulbecco's modified Eagle's medium (DMEM)) to generate hydrogen peroxide. Addition of oxaloacetate decreased the levels of H(2)O(2) detected and the oxaloacetate was depleted. Oxaloacetate was approximately as effective as pyruvate in decreasing H(2)O(2) levels and more effective than α-ketoglutarate. Our data raise important issues to consider when interpreting the behaviour and metabolism of cells in culture (which are both altered by the oxidative stress of cell culture) and their apparent response to addition of autooxidisable compounds such as ascorbate and epigallocatechin gallate.