Dulbecco's Modified Eagle Medium Research Articles

Transcriptome-wide analysis of the differences between MCF7 cells cultured in DMEM or αMEM.

MCF7 cells have been used as an experimental model for breast cancer for decades. Typically, a culture medium is designed to supply cells with the nutrients essential for their continuous proliferation. Each medium has a specific nutritional composition. Therefore, cells cultured in different media may exhibit differences in their metabolism. However, only a few studies have investigated the effects of media on cells. In this study, we compared the effects of Dulbecco's modified Eagle medium (DMEM) and minimum essential medium alpha modification (αMEM) on MCF7 cells. The two media differentially affected the morphology, cell cycle, and proliferation of MCF7 cells, but had no effect on cell death. Replacement of DMEM with αMEM led to a decrease in ATP production and an increase in reactive oxygen species production, but did not affect the cell viability. RNA-sequencing and bioinformatic analyses revealed 721 significantly upregulated and 1247 downregulated genes in cells cultured in αMEM for 48 h compared with that in cells cultured in DMEM. The enriched gene ontology terms were related to mitosis and cell proliferation. Kyoto encyclopedia of genes and genomes analysis revealed cell cycle and DNA replication as the top two significant pathways. MCF7 cells were hypoxic when cultured in αMEM. These results show that the culture medium considerably affects cultured cells. Thus, the stability of the culture system in a study is very important to obtain reliable results.

Cytotoxicity Analysis of the Effects of Heterobasidion Annosum Mycelia and Cisplatin on Colon Adenocarcinoma (CACO-2) Cell Line

Colorectal cancer ranks as the third most prevalent form of cancer and stands as the second leading cause of mortality. Both environmental and genetic risk factors contribute to its manifestation. Presently, 5-fluoruracil/leucovorin (5-FU/LV) remains the recommended course for adjuvant therapy in addressing this condition. Conversely, mushrooms, celebrated for their biologically active constituents, including valuable enzymes, have emerged as a captivating subject in diverse medical disciplines, particularly within the realm of cancer therapy, due to their promising therapeutic properties. This specific investigation aimed to conduct in vitro cytotoxic experiments using extracts obtained from Heterobasidion annosum micelles cultivated in a liquid malt extract medium. The pulverized extracts were dissolved in Dulbecco's Modified Eagle Medium (DMEM) at varied concentrations ranging from 25ng/mL to 200ng/mL and subsequently administered to colon adenocarcinoma (Caco-2) cells. The cytotoxic effects of both the fungus and cisplatin, a well-known anticarcinogenic agent, were examined at intervals of 24, 48, and 72 hours. The findings indicated a significant inhibition of cancer cell development within this timeframe. Moreover, a noteworthy discovery emerged, revealing that cisplatin, known for its efficacy in various cancer studies, substantially diminished the viability of cancer cells after 72 hours in comparison to the control group.

The antiapoptotic effects of conditioned medium from bone marrow-derived mesenchymal stromal stem cells on cyclophosphamide-induced testicular damage in rat: An experimental study.

Cyclophosphamide (CP) has some negative effects on the reproductive system. Stem cells and their metabolites are being utilized to enhance fertility after chemotherapy. This study aimed to investigate the impact of conditioned medium (CM) derived from bone marrow mesenchymal stromal stem cells (BM-MSCs) on the toxic effects of CP on testicles. BM-MSCs were isolated, a CM was collected and 25-fold concentrated. 24 male Wistar rats (8 wk, 200-250 gr) were randomly divided into following groups: control, CP, CP+ Dulbecco's Modified Eagle Medium (DMEM), CP+CM. CP was given at a single dose of 100 mg/kg. 2 wk after the CP administration, CM was injected into the testicular efferent duct. Sperm parameters, testicular histopathology, and the level of testosterone were analyzed 2 months after treatment. The expression of B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) genes were evaluated by real-time polymerase chain reaction. CP had a negative effect on testis histology (p 0.001) and sperm quality (p 0.001). It changed the expression of genes associated with apoptosis (p 0.001). Treatment with CM reduced the expression of Bax (p 0.001), while significantly increasing the expression of Bcl2 (p = 0.01). It improved sperm count (p = 0.03), viability (p 0.001), motility (p 0.001), spermatogonial count (p 0.001), and epithelial thickness of testicular tubules (p = 0.02). These findings suggest that CM produced from BM-MSCs may be valuable for therapeutic approaches in reproductive medicine and may lessen the side effects of CP.

Evaluation of Cytotoxicity of 4-Hydroxycinnamic Acid Using Tetrazolium Bromide Assay and Zebrafish Embryotoxicity: An In-Vitro Study.

Aim This study aimed to evaluate the cytotoxicity of a novel compound, 4-hydroxycinnamic acid (4-HCA), with the help of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and zebrafish embryotoxicity. Materials and methods In this in vitro study, MTT fibroblast assays using dental pulp stem cells, which were cultured in Modified Eagle's Medium or Dulbecco's Modified Eagle Medium, and zebrafish cytotoxicity and embryotoxicity were done to evaluate the cytotoxicity of the novel compound 4-HCA. The data was analyzed by plotting cell number versus absorbance, allowing quantitation of changes in cell proliferation. Results 4-HCA (40 μl) showed acceptable levels of cell viability according to the American Society for Testing and Materials standards. Cell viability is reduced with increased exposure time and concentrations of 4-HCA. Similarly, the cytotoxicity assessment in zebrafish (Danio rerio) showed an acceptable range of toxicity levels in embryonic stagesused to evaluate the mortality rate of zebrafish embryos. Conclusion Considering the constraints of this research, it can be deduced that hydroxycinnamic acid at a concentration of 40 μl was non-toxic. The findings from the MTT assay indicated a correlation between the concentration and the toxicity of the compound. Likewise, the zebrafish test demonstrated minimal toxicological effects.

Overcoming Breast Cancer Drug Resistance: A Novel Approach Using siRNA-Mediated P-glycoprotein Downregulation to Enhance Vinorelbine Efficacy.

Cancer, the second leading cause of mortality worldwide, represents a global health challenge, primarily due to drug resistance. Vinorelbine is a chemotherapeutic agent that disrupts cancer cell growth by targeting microtubules and inducing apoptosis. However, drug resistance remains a formidable obstacle. This resistance is caused by various factors including genetic mutations, drug efflux mechanisms, and DNA repair systems. Resolution of this challenge requires an innovative approach. This study investigated the potential of small interfering RNA (siRNA) to target and downregulate a vinorelbine-resistant MCF-7/ADR breast cancer cell line. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) 10% fetal bovine serum/penicillin/streptomycin. An siRNA targeting ABCB1 was designed and synthesized, and the cells were transfected with siRNA at final concentrations of 10, 20, and 30 nM. The3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess cell viability. ABCB1 mRNA expression levels were determined by real-time polymerase chain reaction (PCR). MCF-7 cells exhibited a higher sensitivity to vinorelbine than MCF-7/ADR cells. MCF-7/ADR cells exhibited resistance to vinorelbine at concentrations, 12.50 and 25.00 μM. Treatment with siRNA significantly reduced ABCB1 expression by 2.93-fold (P=0.0001). Similarly, co-treatment with siRNA and vinorelbine produced a substantial 2.89-fold decrease in ABCB1 gene expression in MCF-7 cells compared to that in MCF-7/ADR cells (P=0.0001). The results of the present study indicate that the concurrent use of siRNA and vinorelbine holds substantial promise as a therapeutic approach to overcome ABCB1-mediated multidrug resistance (MDR) in breast cancer. It is necessary to conduct comprehensive clinical trials to determine the true effectiveness of this combination therapy.

Sertoli cell-conditioned medium can improve blood-testis-barrier function and spermatogenesis in azoospermia mice induced by scrotal hyperthermia: An experimental study.

An increase in the temperature of the testis is associated with damage to the epithelium of seminiferous tubules and disruption of sperm production. The current study aimed to investigate the effect of the Sertoli cell-conditioned medium (SCCM) on the blood-testis-barrier associated genes and spermatogenesis process following scrotal hyperthermia. In this experimental study, 40 adult NMRI mice (8 wk, 25-30 gr) were allocated into 4 groups: I) control, II) DMEM (10 μl Dulbecco's Modified Eagle Medium), III) scrotal hyperthermia, and IV) scrotal hyperthermia+SCCM (10 μl SCCM). Hyperthermia was induced by placing the mice scrotum in water at 43 C for 20 min every other day for 10 days. Mice were treated every other day for 5 wk. Then the animals were euthanized, and the tails of epididymis were removed to analyze sperm parameters, testis were taken for stereological assessment, reactive oxygen spices and glutathione levels, and the expression of Ocln, Gja1, Cdh2, and Itgb1. The results of sperm analysis indicated that SCCM-treated mice significantly increased sperm count and motility and reduced DNA fragmentation. In addition, histological and molecular findings showed that the volume of testicular tissue, the number of germ cells, the glutathione level, and the expression of Ocln, Gja1, Cdh2, and Itgb1 genes were significantly increased in the SCCM-treated mice. Findings suggest that growth factors of SCCM stimulate the proliferation and differentiation of germ cells through paracrine effects and upregulate the blood-testis-barrier-associated genes in mice subjected to scrotal hyperthermia.

In-Vivo Effects of Canary (Serinus Canary) Semen Diluted with Different Semen Extenders on Eggs in Artificial Insemination

In this study, the effect of Dulbecco's Phosphate Buffered Saline (DPBS) and Dulbecco's Modified Eagle's Medium (DMEM) extenders on artificial insemination was determined.. For this purpose, the number of fertile eggs, the total number of eggs, the number of hatched eggs, and the number of hatchlings from fertile eggs were determined in the study. From these values, the fertility rate (%), egg hatch rate (%) and fertile egg hatch rate (%) were calculated. Twelve male and female canaries (Gloster breed; 1-2 years old) were used in each group. In the study, three groups, one control (C) and the other two treatment groups (T-1, T-2) were formed. Female canaries in group C were directly inseminated without using any extenders. Females in the T-1 group were diluted 1:1 with DPBS; females in the T-2 group were inseminated with semen diluted 1:1 with DMEM. Although there was a significant difference between the groups in terms of fertility rate (P0.05). The highest fertility rate (61.81%) was in group C; the lowest was in the T-1 (29.17%) group. (P

Bone marrow stem cells with or without superparamagnetic iron oxide nanoparticles as a magnetic targeting tool: Which is better in regeneration of neurolysed facial nerve? An experimental study

AimThis study was performed to evaluate neural regenerative capacities of bone marrow stem cells (BMSCs) with or without superparamagnetic iron oxide nanoparticles (SPIONs) as a magnetic targeting tool after neurolysis of the facial nerve (FN) in albino rats. MethodsThirty-eight male albino rats were selected. Two of them were euthanized for normal FN histology assessment. Thirty-six rats were injected with ethanol in the FN nerve for neurolysis induction and assessed one week post-operatively by eye blinking test. Animals were divided into three groups, each containing twelve rats: Group I (positive control) was injected with Dulbecco Modified Eagle's medium (DMEM-F12), group II was injected with BMSCs in DMEM-F12, and group III was injected with BMSCs in DMEM-F12 with poly l-lysine coated SPIONs (0.5 mmol/mL). Monitoring of SPIONs in the rat's body was carried out by MRI. A circular neodymium magnet N52 (0.57 T, 2 × 5 mm) was placed on each rat in group III just below the right ear at the site of surgery to attract SPIONs labeled BMSCs, left in place for 24 h, and then removed. From each group, six rats were euthanized at the end of the 4th and 8th week of treatment, respectively. The right FN trunks were extracted for routine histological examination using H&E stain. Immunohistochemical examination by anti-S100B was performed to characterize the thickness of the myelin sheath formed by the Schwann cells. Ultra-structural examination was performed to study changes in axons, myelin sheaths, and Schwann cells. ResultsRegeneration of nerve fibers, Schwan cells, and myelin sheaths was better in group II than in groups I and III histologically, immunohistochemically, and ultra-structurally. ConclusionBMSCs alone could ameliorate FN regeneration better than magnetic targeting treatment using BMSCs labeled with SPIONs.

Investigation of proapoptotic and cytotoxic effects of 2-aminobenzothiazole on human laryngeal carcinoma cells.

In the present study, we investigated the effects of 2-aminobenzothiazole application on human laryngeal carcinoma cells. Human larynx epidermoid carcinoma (HEp-2) (ATCC® CCL-23™) cells were purchased from American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. 2-aminobenzothiazole was prepared, and further dilutions ranging from 3.13 to 100 μM were prepared in fresh culture DMEM. HEp-2 cells on 96 well plates were incubated with the prepared dilutions of 2-aminobenzothiazole for 24, 48, and 72 hours. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test performed cytotoxicity evaluation and viability percentages. The annexin-V staining technique detected 2-aminobenzothiazole-triggered apoptosis of HEp-2 cells. The activated caspases 3/7 on HEp-2 cells after 2-aminobenzothiazole exposure were evaluated with flow cytometric analysis. The membrane potential changing of HEp-2 cells was measured following the Muse™ MitoPotential kit manufacturer instructions. MTT cytotoxicity test results showed that the viability of human laryngeal carcinoma cells decreased with an increase in the application of 2-aminobenzothiazole for 24 hours. The highest growth inhibition by 2-aminobenzothiazole for short-term application of 24 hours was detected at the highest concentration of 2-aminobenzothiazole (100 µM). The results underline that the cytotoxic effect of 2-aminobenzothiazole is dose-dependent. Cytotoxicity test results for an application time of 48 hours showed that the cytotoxicity of 2-aminobenzothiazole is dose-dependent on HEp-2 cells. The required dose of 2-aminobenzothiazole to decrease the cell viability to 50 percent has been 9-fold augmented. Annexin-V findings showed that after exposure to IC50 concentration of 2-aminobenzothiazole for 24 hours, HEp-2 cells underwent the early apoptotic stage (25.99%) and late apoptotic (16.69%), whereas 56.93% of the treated cells were alive. Only 0.39% of 2-aminobenzothiazole treated cells were necrotic. All study results showed that 2-aminobenzothiazole triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells 42.62 compared to untreated HEp-2 cells. Caspase 3/7 activation results showed that only 0.65% of control HEp-2 cells were with activated caspase 3/7, and 99.35% live cells. The analysis data from the Muse cell analyzer revealed that the percentage of cells with intact mitochondrial membranes was 21.30 after 2-aminobenzothiazole application, and 79.9% were cells with depolarized mitochondrial membranes. It has been understood that the depolarization of the inner mitochondrial membrane has been considered a dysfunction in mitochondria as a sign of apoptosis and drug toxicity. Based on all study findings, 2-aminobenzothiazole has cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner. That means that it decreased viability via inducing caspase-dependent apoptosis. Consequently, it was concluded that 2-aminobenzothiazole has good potential to lead to cytotoxicity and apoptosis on human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anticancer drugs with high efficiency.

Investigation the effect of Scrophularia striata hydroalcoholic Extract On Cell Death and Migration in Cervical Cancer Cell Line

Background: With the growing interest in plant-derived chemotherapeutic agents, there has been a significant rise in research exploring a broad range of plants in recent years. Scrophularia striata has gained attention due to its extensive medical applications. This study aimed to investigate the effect of S. striata extract on HeLa cervical cancer cells, specifically their migration, apoptosis, and necrosis. Methods: We first cultured HeLa cells in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. We then examined the cytotoxicity of S. striata extract at varying concentrations (0, 1, 10, 100, 500, and 1000 μg/mL) using the MTT assay after 24 hours. We evaluated the extent of wound healing using a scratch assay and analyzed the apoptosis activity of the extract using flow cytometry. Results: Our results showed that S. striata extract (IC50: 433.8 μg/mL) significantly enhances wound healing (P≤0.01) in cervical cancer and promotes apoptosis and necrosis of HeLa cells. Conclusion: Our findings suggest that S. striata may serve as an effective treatment for cervical cancer by inducing cell death and reducing migration.

Establishment of a gill cell line from yellowfin seabream (Acanthopagrus latus) for studying Amyloodinium ocellatum infection of fish.

Amyloodinium ocellatum is among the most devastating protozoan parasites, causing huge economic losses in the mariculture industry. However, the pathogenesis of amyloodiniosis remains unknown, hindering the development of targeted anti-parasitic drugs. The A. ocellatum invitro model is an indispensable tool for investigating the pathogenic mechanism of amyloodiniosis at the cellular and molecular levels. The present work developed a new cell line, ALG, from the gill of yellowfin seabream (Acanthopagrus latus). The cell line was routinely cultured at 28°C in Dulbecco's modified Eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS). ALG cells were adherent and exhibited an epithelioid morphology; the cells were stably passed over 30 generations and successfully cryopreserved. The cell line derived from A. latus was identified based on partial sequence amplification and sequencing of cytochrome B (Cyt b). The ALG was seeded onto transwell inserts and found to be a platform for invitro infection of A. ocellatum, with a 37.23 ± 5.75% infection rate. Furthermore, scanning electron microscopy (SEM) revealed that A. ocellatum parasitizes cell monolayers via rhizoids. A. ocellatum infection increased the expression of apoptosis and inflammation-related genes, including caspase 3 (Casp 3), interleukin 1 (IL-1), interleukin 10 (IL-10), tumour necrosis factor-alpha (TNF-α), invivo or invitro. These results demonstrated that the invitro gill cell monolayer successfully recapitulated invivo A. latus host responses to A. ocellatum infection. The ALG cell line holds great promise as a valuable tool for investigating parasite-host interactions invitro.

Mg ZX10 alloy for biomedical applications: Surface characterization and comparative corrosion studies under simulated biological conditions

Biodegradable magnesium (Mg)-based alloys emerge as potential intracorporeal implants for clinical application in bone tissue, the cardiovascular and the nervous systems. The surfaces of control (non-sterilized) and sterilized (180 °C 1 h) Mg ZX10 alloy were studied. The alloy evolution in SBF (simulated saline solution) and in DMEM (Dulbecco's Modified Eagle Medium) with fetal calf serum (FCS) was investigated using: Raman spectroscopy, FTIR-ATR, electrochemical assays and scanning electron microscopy (SEM). The Mg ZX10 microstructure analysis revealed an average grain size of 23.4 ± 11.02 and 31.6 ± 13.07 μm, considering the minor and major axis, respectively. In addition, the Mg ZX10 microstructure exhibited a homogeneous grain distribution. The contact angle formed between the water and the surface of control Mg ZX10 and sterilized were 32.24° ± 1.53 and 46.76° ± 0.83, respectively. Consequently, both groups of samples exhibited hydrophilic surfaces. Furthermore, the free surface energy of control Mg ZX10 was 63.23 ± 0.72 mN/m and sterilized Mg ZX10 was 52.31 mN/m ± 1.25, those energy values were in agreement with previous reports. Upon immersion in SBF, Raman spectroscopy detected the presence of magnesium oxides-hydroxides, phosphates and carbonates compounds in both groups of samples without significant differences. FTIR spectra of the sterilized alloys, after immersion in DMEM + FCS, showed the presence of amide and carbon-carbon bonds, magnesium-oxides, as well as phosphates and carbonates groups. These findings suggested the deposition of organic and inorganic compounds on the alloy surface. Electrochemical assays carried out in SBF demonstrated a slight reduction in the degradation of the sterilized alloy compared to control Mg ZX10. Moreover, a substantial decrease in the corrosion behavior of the sterilized alloy immersed in DMEM + FCS with respect to the same alloy immersed in SBF was observed. SEM images showed that the alloy, previously immersed in SBF, exhibited degradation cracks. Contrary, the sterilized alloy surface immersed in DMEM + FCS was more homogenous with deposits of structures similar to hybrid nanoflowers. Based on these results, this sterilization process does not alter the chemistry of Mg ZX10 alloy surface and, even, its application exhibits an advantage since the high corrosion rate of Mg in physiological media. Furthermore, the organic-inorganic structures adsorbed, after alloy immersion in DMEM + FCS, could decrease corrosion behavior.

Stability of Piceatannol in Dulbecco's Modified Eagle's Medium by In Situ UPLC-MS/MS Analysis

Piceatannol is a stilbenoid, which has shown bioactivities in various cell culture models. However, its stability in cell culture medium is not clear. Here, UPLC-MS/MS was applied in situ to analyze the degradation products of piceatannol in Dulbecco's Modified Eagle's Medium (DMEM) and cell culture to investigate the compound's stability in DMEM. During the incubation with cell culture medium (at 4 and 37 °C), several piceatannol derivatives, such as an oxidation product ( m/z 243.06), a reduction product ( m/z 247.09), dimers ( m/z 485.12 and 487.14) and trimers ( m/z 727.18) were detected, which demonstrated the instability of piceatannol in cell culture conditions. To confirm if the new products during the incubation were generated due to the instability of piceatannol, ascorbic acid was added. The presence of ascorbic acid could significantly slow the degradation rate of piceatannol and the generation of piceatannol derivatives, which proved that the new products were generated by the degradation of piceatannol and indicated that the instability of piceatannol might be related to its antioxidant activity.

Potential Use of L-Arginine Amino Acids towards Proliferation and Migratory Speed Rate of Human Dental Pulp Stem Cells.

L-arginine is a semi-essential amino acid produced by the body which has an important role in the process of stem cell regeneration. However, under inflammatory conditions, denaturation of pulp amino acids and proteins occurr resulting in a decrease in the ability of stem cells to self-renew. Therefore, in this study, L-arginine was added in vitro to the culture media Dulbecco's Modified Eagle Medium - (DMEM) of human dental pulp stem cells (hDPSCs) to analyse the potential of L-arginine on migration and proliferation by comparing between 3 concentrations, namely 300, 400, 500 μmol/L and control group (DMEM), to obtain the most optimal concentration for proliferation and migration. Serum-starved hDPSCs were divided into four groups: control: hDPSCs in DMEM; hDPSCs in 300 μmol/L of the L-Arginine based culture media group; hDPSCs in 400 μmol/L of the L-Arginine based culture media group; and hDPSCs in 500 μmol/L of the L-Arginine based culture media group, which were added in two separate 24-well-plates (5×104 cell/well) for proliferation and migration evaluation. The proliferation of all groups was measured by using a cell count test (haemacytometer and manual checker) after 24 h. The migratory speed rate of all groups was measured by using cell migration assay (scratch wound assay) after 24 h. Cell characteristics were evaluated under microscope that was then evaluated using image-J® interpretation. This image J represented the measurement of migratory speed rate (nm/h) data. Statistical analysis was conducted using one-way ANOVA and post hoc Bonferroni (p<0.05) for proliferation and post hoc LSD (p<0.05) for migration. There was a statistically significant difference in hDPSCs proliferation among various concentration groups of the L-Arginine based solution (300, 400 and 500 μmol/L) compared to the control group (p<0.05). There was a statistically significant difference in the migratory speed rate of hDPSCs at 500 μmol/L of the L-Arginine based solution group compared to lower concentrations and control group (p<0.05). All three concentrations of L-arginine can induce proliferation of hDPSCs. L-arginine at 500 μmol/L can induce higher hDPSCs proliferation and faster migration at 24 hours compared to lower concen-trations and control.

Effects of media composition and light exposure on the electrochemical current response during scanning electrochemical microscopy live cell imaging.

Scanning Electrochemical Microscopy (SECM) has been used as a non-invasive electrochemical technique for studying cellular processes. SECM enables the quantification of cellular metabolites in real-time providing a deeper understanding of cellular responses to external stimuli. SECM imaging of living cells requires maintaining an ideal physiological environment to ensure reliable data collection on cellular reactivity. The cellular response can be directly influenced by physicochemical parameters including cell media composition, temperature and light exposure. This research demonstrates the effect of media composition on the electrochemical current signal of adenocarcinoma cervical cancer (HeLa) cells during SECM measurements using ferrocenemethanol as a redox mediator. Investigated media that are commonly used as electrolyte, are phosphate buffered saline (PBS), and Dulbecco's modified Eagle's medium (DMEM) in the absence and presence of fetal bovine serum (FBS). In addition, this research demonstrates that fluctuating light illumination impacts the stability of the cellular electrochemical current response. Our findings reveal that media composition and illumination are important parameters that must be carefully considered and monitored during SECM live cell imaging.

Recombinant Methioninase Decreased the Effective Dose of Irinotecan by 15-fold Against Colon Cancer Cells: A Strategy for Effective Low-toxicity Treatment of Colon Cancer.

Irinotecan (IRN), a topoisomerase I inhibitor and pro-drug of SN-38, is first-line treatment of colon cancer as part of FOLFIRI and FOLFOXIRI combination chemotherapy. However, IRN causes dose-limiting adverse events such as neutropenia and diarrhea. Dose reductions are sometimes required, which reduce efficacy. Recombinant methioninase (rMETase) targets the fundamental basis of cancer, methionine addiction, known as the Hoffman effect, and enhances the efficacy of numerous chemotherapy drugs. The present study determined the efficacy of rMETase when administered in combination with IRN. Cell viability was assessed by cultivating the HCT-116 human colorectal cancer cell line in 96-well plates at 1×103 cells per well in Dulbecco's modified Eagle's medium (DMEM). Subsequently, HCT-116 cells were treated with increasing concentrations of SN-38, the active form of IRN, ranging from 0.5 nM to 32 nM, and/or rMETase ranging from 0.125 to 8 U/ml. After treatment for 72 h, the half-maximal inhibitory concentration (IC50) of SN-38 alone and rMETase alone for HCT-116 cells were determined. Using the IC50 concentration of rMETase, we determined the IC50 of SN-38 in combination with rMETase. Cell viability was determined with the cell-counting Kit-8 with the WST-8 reagent.. The IC50 of rMETase alone for the HCT-116 cells was 0.55 U/ml, and the IC50 of IRN (SN-38) alone was 3.50 nM. rMETase at 0.55 U/ml lowered the IC50 of SN-38 to 0.232 nM (p<0.0001), a 15-fold reduction. rMETase and IRN are strongly synergistic, giving rise to the possibility of lowering the effective dose of IRN for the treatment of patients with colon cancer, thereby reducing its severe toxicity. This new strategy will allow more patients with cancer to be effectively treated with IRN.

An evaluation of photobiomodulation effects on human gingival fibroblast cells under hyperglycemic condition: an in vitro study.

An in vitro study was designed to evaluate the effects of photobiomodulation (PBM) with 915-nm diode laser on human gingival fibroblast (HGF) cells under hyperglycemic condition. The HGF cells were cultured in Dulbecco's modified eagle medium (DMEM) medium containing 30mM glucose concentration for 48h to mimic the hyperglycemic condition. Subsequently, the cells received three sessions of PBM (915nm, continuous emission mode, 200 mW, energy density values of 3.2, 6, and 9.2J/cm2). Twenty-four hours post-irradiation, cell proliferation, expression of interleukin 6 (IL-6), and vascular endothelial growth factor (VEGF) were assessed with MTT and real-time polymerase chain reaction (PCR) tests, respectively. Also, reactive oxygen species (ROS) production was measured using CM-H2DCFDA fluorimetry. No changes were detected in the cell proliferation rate between the high glucose control group and laser-treated cells, while VEGF and IL-6 gene expression levels increased significantly after PBM in the high glucose-treated cells group. ROS level was significantly decreased in the irradiated cells in high-glucose medium compared with the high glucose control group. Our study revealed the inductive role of 915-nm-mediated PBM on VEGF and the inflammatory response while concurrently reducing reactive oxygen species production in HGF cells in hyperglycemic conditions.

Invadopodia formation assay: a promising experiment for targeting breast cancer invasion and metastasis

Abstract Introduction/Objective Invadopodia has been found to invade the extracellular matrix (ECM) in multiple cancers, including breast, lung, brain, and prostate, which is highly associated with cancer metastasis. Invadopodia have been shown to form in various cancer cells, such as the MDA-MB-231 breast cancer cell line. Objectives In-vitro testing to validate the anti-cancer invasion drugs using an invadopodia formation assay. Methods/Case Report MDA-MB-231 triple-negative breast cancer cell line was used in this study due to its highly invasive characteristics obtained from American Type Culture Collection. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), a high-glucose medium containing FBS 10% and 1% penicillin/streptomycin. Cells were maintained in a humidified atmosphere of 95% O2 and 5% CO2 incubator at 37 °C for normal conditions. Cells with low passage numbers were used in order to get accurate results. Then, the gelatin-coated coverslips were prepared for invadopodium assay, washed three times with ddH2O, and transferred to slides containing a drop of antifade, a mounting solution. For invadopodia quantification, invadopodia images on the F-actin, gelatin, and nucleus were taken using the same fluorescence microscope settings (Confocal Laser Leica fluorescent microscope, German). Cells were scored for the presence of underlying invadopodia. The mean percentage of cells with invadopodia formation was calculated for drug-validated tests. Results (if a Case Study enter NA) The study revealed how MDA-MB-231 cells penetrate the gelatin that mimics the ECM-like basement membrane in the human body; this is thought to be a key point during intravasation and extravasation steps in metastasis. The study showed actin-rich filament as red dots in the center of the MDA-MB-231 cells, representing invadopodia in a 3D image under normal conditions. In contrast, in a 2D image, invadopodia are represented as black dots on gelatin in a merged image. Conclusion Breast cancer is one of the leading causes of cancer-related death in the advanced stage. The study helps to promote using invadopodia assay to validate the effectiveness of the potential anti-cancer drugs and predict cancer metastasis in clinical studies. However, invadopodia formation assay can be especially useful for patients with early-stage cancers; it can help to predict which patients are more likely to benefit from aggressive treatment or further monitoring rather than immediate surgical intervention.

ABNORMAL CHONDROCYTE MORPHOLOGY, CLUSTERING, AND CELL SWELLING IN A SCRAPED HUMAN CARTILAGE MODEL OF OSTEOARTHRITIS

AbstractObjectivesOsteoarthritis (OA) is a complex joint disorder characterised by the loss of extracellular matrix (ECM) leading to cartilage degeneration. Changes to cartilage cell (chondrocyte) behaviour occur including cell swelling, the development of fine cytoplasmic processes and cell clustering leading to changes in cell phenotype and development of focal areas of mechanically-weak fibrocartilaginous matrix[1]. To study the sequence of events in more detail, we have investigated the changes to in situ chondrocytes within human cartilage which has been lightly scraped and then cultured with serum.MethodsHuman femoral heads were obtained with Ethical permission and consent from four female patients (mean age 74 yrs) undergoing hip arthroplasty following femoral neck fracture. Osteochondral explants of macroscopically-normal cartilage were cultured as a non-scraped control, or scraped gently six times with a scalpel blade and both maintained in culture for up to 2wks in Dulbecco's Modified Eagle's Medium (DMEM) with 25% human serum (HS). Explants were then labelled with CMFDA (5-chloromethylfluorescein-diacetate) and PI (propidium iodide) (10μM each) to identify the morphology of living or dead chondrocytes respectively. Explants were imaged using confocal microscopy and in situ chondrocyte morphology, volume and clustering assessed quantitatively within standardised regions of interest (ROI) using Imaris® imaging software.ResultsWithin 2wks of culture with HS, chondrocyte volume increased significantly from 412±9.3µm3 (unscraped) at day 0 to 724±16.6 µm3 (scraped) [N(n) = 4(380)] (P=0.0002). Chondrocyte clustering was a prominent feature of HS culture as the percentage of clusters in the cell population increased with scraping from 4.8±1.4% to 14.9±3.9% [N(n) = 4(999)] at week 2 (P=0.0116). In addition, the % of the chondrocyte population within clusters increased from approximately 38% to 60%, and the number of cells per cluster increased significantly from 3.2±0.08 to 4±0.22 (P=0.031). The development of abnormal ‘fibroblastic-like’ chondrocyte morphology demonstrating long (&gt;5µm) cytoplasmic processes also occurred, however the time course of this was more variable. For some samples, clustering occurred before abnormal morphology, but for others the opposite occurred. Typically, by the second week, 17±2.64% of the cell population had processes and this increased to 22±4.02% [N(n) = 4(759)] with scraping.ConclusionsScraping the cartilage will remove surface constituents including lubricants (e.g. lubricin, hyaluronic acid, phospholipids), extracellular matrix constituents (collagen, proteoglycans – potentially the ‘lamina splendens’) and cells (chondrocytes and mesenchymal stromal cells (MSCs)). Although we do not know which of these component(s) is important, the effect is to dramatically increase the permeation of serum factors into the cartilage matrix and signal the development of cytoplasmic processes, cell clustering and swelling. It is notable that these cellular changes are similar to those occurring in early OA[1]. This raises the interesting possibility that scraped cartilage cultured with human serum recapitulates some of the changes to in situ chondrocytes during early stages of cartilage degeneration and as such, could be a useful model for following the deleterious changes to matrix metabolism.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

F-ACTIN DISTRIBUTION OF THE CHONDROCYTE CYTOSKELETON AND ITS ASSOCIATION WITH EARLY OSTEOARTHRITIC CHONDROCYTE PROPERTIES

AbstractObjectivesOsteoarthritis (OA) is a painful and debilitating disorder of diarthroidal joints. Progressive degeneration of the cartilage extracellular matrix (ECM) together with abnormal chondrocyte characteristics occur leading to a switch to a fibroblast-like phenotype and production of mechanically-weak cartilage. Early changes to chondrocytes within human cartilage have been observed including chondrocyte swelling[1] together with the development of thin cytoplasmic processes which increase in number and length with degeneration[2]. Changes to chondrocyte phenotype in degenerate cartilage are associated with F-actin redistribution and stress fibres (SF) formation, leading to morphologically-dedifferentiated (fibroblast-like) chondrocytes[3,4]. It is unclear if these processes are a consequence of ‘passive’ cell swelling into a defective ECM or an ‘active’ event driven by changes in cell metabolism resulting in alterations to cell shape. To address this, we have quantified and compared the distribution and levels of F-actin, a key cytoskeletal protein involved in the formation of cytoplasmic processes, within in situ chondrocytes in non-degenerate and mildly degenerate human cartilage.MethodsHuman femoral head cartilage was obtained from 21 patients [15 females, 6 males, average age 69.6yrs, (range 47–90yrs)] following femoral neck fracture, with Ethical Approval and patient's permission. Cartilage explants were removed from areas graded non-degenerate grade 0 (G0) or mildly degenerate grade 1 (G1) and cultured for up to 3wks in Dulbecco's Modified Eagle's Medium (DMEM) +/− 25% human serum (HS). In situ chondrocytes were stained with CMFDA (5-chloromethylfluoresceindiacetate, Cell-Tracker Green®) and phalloidin (F-actin labelling) and imaged by confocal microscopy and analysed quantitatively using ImageJ and Imaris® software.ResultsThere were significant increases in the total amount (TA) of F-actin and its distribution [intense punctuate (IP) and intense areas (IA)] between the whole chondrocyte populations of G0 and G1 cartilage (P=0.0356; 0.0112; 0.016, respectively). Where the volume of chondrocytes was divided into normal (&lt;1000 µm³) and swollen (≥1000 µm³) cells, F-actin TA increased in swollen cells (P=0.036 within G0 and G1, and P=0.0009 between grades) compared to chondrocytes of normal volume in each grade. Moreover, IP and IA within and between G0 and G1 were higher compared to normal chondrocytes (with P&lt;0.0001 for IP and P&lt;0.001 for IA). In addition, tissue culture experiments demonstrated that 90% of chondrocytes with cytoplasmic processes had strong F-actin intensity (either IP or IA with P&lt;0.0001). Furthermore, 83% of this F-actin was associated with cytoplasmic processes, with &gt;65% situated at the base of the process (P&lt;0.0001).ConclusionsThe increases in chondrocyte F-actin levels (TA) and its localisation (IP, IA) appear to be associated with cell swelling and development of cytoplasmic processes, which are both characteristics of early OA cartilage[1]. This suggests the formation of chondrocyte cytoplasmic processes is an ‘active’ event potentially involving changes to matrix metabolism rather than a ‘passive’ cell swelling into a defective extracellular matrix.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.