Eagle's Minimum Essential Medium Research Articles

#2716 Selenoprotein-P1 (SEPP-1) in human proximal tubule cells after ischemic-reperfusion injury: an in-vitro model

Abstract Background and Aims Novel biomarkers, anticipating serum creatinine rise, are eagerly needed to have a speedy diagnosis of acute kidney injury (AKI) and thus to mitigate and to preserve kidney function with well-timed therapeutic plans. Recently, in a pilot study on patients undergoing elective major cardiac surgery with cardiopulmonary bypass (CPB), selenoprotein-p1 (SEPP-1) has emerged as a promising candidate plasmatic biomarker for an early AKI risk stratification. SEPP-1 increased particularly between 4th to 8th hour after CPB and with a strict relation with ischemia duration (Bolignano et Al. Rev. Cardiovasc. Med. 2022). SEPP1 is primarily secreted from liver and acts as a selenium transporter supplying tissues and organs with this trace mineral which elicits the activity of specific glutathione peroxidase selenoenzymes. Additionally, renal tubular cells are able to synthetize this protein but remain unknown if, during AKI, SEPP1 increases as defeat answer to renal cells damage or if it's hepatic SEPP1 that accumulates for a reduced renal glomerular filtration rate. The primary aim of this study was to investigate the release of SEPP-1 by renal tubular cells in an experimental model of AKI induced by ischemia-reperfusion and secondarily evaluate the effect of selenium supplying on renal cells injury. Method Human proximal tubular cells (PTC), HK2 cells and human embryonic kidney 293 cells (HEK293) were cultured in Keratinocyte-SFM a complete serum-free medium supplemented with human recombinant Epidermal Growth Factor (rEGF) and Bovine Pituitary Extract (BPE) (LGC Standards s.r.l., Milan, Italy) and in Eagle's Minimum Essential Medium (E-MEM) (LGC Standards s.r.l., Milan, Italy) containing 10% fetal bovine serum, respectively. The cells were pre-incubated with medium containing Sodium Selenite 100 nM for 24 h and then were treated for 24 hours with CoCl2 (Sigma Aldrich S.r.l., Milan, Italy) a chemical hypoxia inducer (500 µM for HK-2 and 250 µM for HEK-293). After that cell viability was evaluated by MTT and trypan bleu dye exclusion assay, while, protein expression of HIF (hypoxia marker) and SEPP1 was analyzed by western blot analysis. Results Ischemia-reperfusion was simulated by the exposure of HK-2 and HEK-293 to CoCl2 and confirmed by HIF expression. The expression of SEPP-1 increased significantly in HK-2 and HEK-293 after hypoxia stress (Fig. 1). Furthermore, the treatment with CoCl2 determined a decrease of cell viability (about 50% less) and Sodium selenite alone increased cell proliferation (about 15% more) in both cell lines. Interestingly cell growth after hypoxia augmented significantly if cell lines were pre-treated with sodium-selenite, highly in HK-2 cells (p < 0.001). Conclusion These preliminary data evidenced, in vitro, that AKI is able to induce renal tubular expression of SEPP-1 and probably SEPP-1 release in human after renal injury is not only a result of a reduced filtration of the hepatic released protein. Furthermore, SEPP-1 showed a possible protective role in AKI. In fact, AKI induced by ischemia-reperfusion, in our in vitro cellular model, was able to induce an increased expression of SEPP-1 in renal tubular cells suggesting a shielding mechanism against injury related to selenium transport. This hypothesis was supported by an exalted SEPP-1 expression after Sodium-Selenite adding to cells’ culture. Supplementary experiments, including functional molecular assays in vitro and in vivo studies will allow to better characterize the role of SEPP-1 in AKI.

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.

Metformin Inefficiency to Lower Lipids in Vitamin B12 Deficient HepG2 Cells Is Alleviated via Adiponectin-AMPK Axis.

Background: Prolonged metformin treatment decreases vitamin B12 (B12) levels, whereas low B12 is associated with dyslipidaemia. Some studies have reported that metformin has no effect on intrahepatic triglyceride (TG) levels. Although AMP-activated protein kinase (AMPK) activation via adiponectin lowers hepatic TG content, its role in B12 deficiency and metformin has not been explored. We investigated whether low B12 impairs the beneficial effect of metformin on hepatic lipid metabolism via the AMPK-adiponectin axis. Methods: HepG2 was cultured using custom-made B12-deficient Eagle's Minimal Essential Medium (EMEM) in different B12-medium concentrations, followed by a 24-h metformin/adiponectin treatment. Gene and protein expressions and total intracellular TG were measured, and radiochemical analysis of TG synthesis and seahorse mitochondria stress assay were undertaken. Results: With low B12, total intracellular TG and synthesized radiolabelled TG were increased. Regulators of lipogenesis, cholesterol and genes regulating fatty acids (FAs; TG; and cholesterol biosynthesis were increased. FA oxidation (FAO) and mitochondrial function were decreased, with decreased pAMPKα and pACC levels. Following metformin treatment in hepatocytes with low B12, the gene and protein expression of the above targets were not alleviated. However, in the presence of adiponectin, intrahepatic lipid levels with low B12 decreased via upregulated pAMPKα and pACC levels. Again, combined adiponectin and metformin treatment ameliorated the low B12 effect and resulted in increased pAMPKα and pACC, with a subsequent reduction in lipogenesis, increased FAO and mitochondrion function. Conclusions: Adiponectin co-administration with metformin induced a higher intrahepatic lipid-lowering effect. Overall, we emphasize the potential therapeutic implications for hepatic AMPK activation via adiponectin for a clinical condition associated with B12 deficiency and metformin treatment.

Assessment and verification of chemical inactivation of peste des petits ruminants virus by virus isolation following virus capture using Nanotrap magnetic virus particles.

This study reports development and optimization of a new method for the assessment and verification of the inactivation of peste des petits ruminants virus (PPRV) by chemical agents, including Triton X-100 and commercially available viral lysis buffers. Virus inactivation was confirmed by virus isolation (VI) on Vero cells following capture of the potential residual viruses from treated samples using Nanotrap magnetic virus particles (NMVPs). Since chemical agents are cytotoxic, treated PPRV samples could not be used directly for VI on Vero cell monolayers; instead, they were diluted in Eagle's Minimum Essential Medium (EMEM) to neutralize cytotoxicity and then subjected to virus capture using NMVPs. The NMVPs and the captured viruses were then clarified on a magnetic stand, reconstituted in EMEM, and inoculated onto Vero cells that were examined for cytopathic effect (CPE). No CPE was observed on cells inoculated with treated viruses captured by NMVPs; but CPE was observed on cells inoculated with untreated viruses, including those captured by NMVPs. For further verification, the supernatants of the VI cultures (treated or untreated) were subjected to RNA extraction and PPRV-specific real-time RT-PCR (RT-qPCR). The cycle threshold values were undetectable for the supernatants of VI cultures inoculated with NMVPs reconstituted from treated PPRV but detectable for the supernatants of VI cultures inoculated with untreated PPRV or the NMVPs reconstituted from untreated PPRV, indicating complete inactivation of PPRV. This new method of verification of virus inactivation using NMVPs can be applied to other high impact viruses of agricultural or public health importance. IMPORTANCE Research including diagnosis on highly contagious viruses at the molecular level such as PCR and next-generation sequencing requires complete inactivation of the virus to ensure biosafety and biosecurity so that any accidental release of the virus does not compromise the safety of the susceptible population and the environment. In this work, peste des petits ruminants virus (PPRV) was inactivated with chemical agents, and the virus inactivation was confirmed by virus isolation (VI) using Vero cells. Since the chemical agents are cytotoxic, inactivated virus (PPRV) was diluted 1:100 to neutralize cytotoxicity, and the residual viruses (if any) were captured using Nanotrap magnetic virus particles (NMVPs). The NMVPs and the captured viruses were subjected to VI. No CPE was observed, indicating complete inactivation, and the results were further supported by real-time RT-PCR. This new protocol to verify virus inactivation can be applicable to other viruses.

Processing of Mg–Ca/tricalcium phosphate composites and their electrochemical properties in a cell culture medium

Biodegradable magnesium-based composites have recently attracted considerable attention for use in orthopedic implants on account of not only their biocompatibility but also the possibility of tuning their mechanical and corrosion properties. In this study, the effects of the fraction of tricalcium phosphate (TCP) and its crystal structure on the electrochemical properties in Eagle's minimal essential medium as well as the mechanical properties were examined using high-strength Mg–Ca/TCP composites with a fine-grained matrix (grain size: 1.2 µm) and relatively uniform TCP particle dispersion. The composites were prepared by extruding mixtures of the component powders at 540 K and exhibited high compressive yield strengths exceeding 290 MPa. Electrochemical tests revealed that the corrosion parameters were insensitive to both the fraction of TCP particles and their crystal structure. The fine-grained matrix may have overwhelmed the ability of the TCP dispersion to serve as a corrosion inhibitor. The chemical compositions of the cracked surface layer on the matrix and the white discontinuous corrosion products of the composites formed after the electrochemical tests were similar to those of the parent alloy.

DETERMINATION OF CYTOTOXIC EFFECT OF AMYGDALIN IN DLD-1 CELL LINE AND ANTICYTOTOXIC EFFECT IN CCD-18CO CELL LINE

Objective: Amygdalin, which is part of the aromatic cyanogenic glycoside group, is found in plant seeds such as apricot, peach, plum, apple, pear, and cherry. It has been shown that amygdalin has anti-tumor properties against many cancers such as colon, breast, and lung cancer. This study aimed to determine the cytotoxic and anticytotoxic effects of amygdalin in human colon cancer cells (DLD-1) and normal colonic epithelium (CCD-18Co) using the MTT (3-(4,5-dimethylthiazol-2-YL)-2,5-diphenyltetrazolium bromide) test. 
 Materials and Methods: DLD-1 and CCD-18Co cells were grown in flasks containing Roswell Park Memorial Institute-1640 and Eagle's Minimum Essential Medium, respectively. Both cell groups were treated with amygdalin concentrations of 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 mM for 24 hours. Then, 20% MTT dye was added to the wells of the aspirated plates and incubated for 3 hours. After the reaction was stopped with pure DMSO at the end of the period, the absorbance values of the plates were read spectrophotometrically at a wavelength of 570 nm.
 Results: The percent viability values for the DLD-1 cell line were found to be between 48.3-71.6% and the IC50 value was calculated as 74.03 mM. The viability values for the CCD-18Co cell line after the amygdalin treatment ranged from 101.6 to 117.9%.
 Conclusion: While amygdalin showed a cytotoxic effect in the DLD-1 cell line, it showed an anticytotoxic effect in the CCD-18Co cell line. In our study, it was determined that amygdalin decreased the viability of DLD-1 cancer cells in a dose-dependent manner and did not show cytotoxic effects on CCD18-Co normal epithelial cells. More comprehensive controlled clinical trials are needed to demonstrate the feasibility of using amygdalin in combination with other anti-tumor drugs and to develop the artificial synthesis of the active ingredients in amygdalin in order to increase the anti-tumor activities of these drugs.

Antitumor Effects of L-citrulline on Hela Cervical Cancer Cell Lines.

Cervical cancer is the deadliest gynecological malignancy. This study aims to examine the anticancer effects of L-citrulline on HeLa cell culture. HeLa cells were cultured in complete Eagle's minimum essential medium. HeLa cells were seeded in 96-well plates and incubated with L-citrulline. After incubation, MTT dye was added and incubated. Annexin- V technique was used to test the apoptosis. The activated caspases of HeLa cells by L-citrulline exposure were measured with the Caspase 3/7 technique. One-way variance analysis was conducted for statistical analysis by using GraphPad Prism 6.0 for Windows. L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner in application times of 24 and 48 hours. The IC50 dose of L-citrulline was 0.19 and 0.16 mg/mL at 24 and 48 hours, respectively. When HeLa cells were exposed to an IC50 dose of L-citrulline for 24 hours, the percentages of the dead, early apoptotic, and late apoptotic cells were detected to be 0.75%, 23.05%, and 12.75%, respectively. The differences in the wideness of the scratch area were observed at the initial stage and after 24 hours of applying L-citrulline. L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner. Based on Annexin and Caspase findings, it can be concluded that L-citrulline exerted a pro-apoptotic effect on HeLa cells in only a short exposure time. L-citrulline also showed a migration inhibitory effect. The findings of this study indicate L-citrulline to be worthy of investigation for its anticancer activities in vitro and in vivo, and as a candidate for cancer therapy.

Osteoimmunomodulatory potential of 3D printed submicron patterns assessed in a direct co-culture model.

Modulation of the immune response following the implantation of biomaterials can have beneficial effects on bone regeneration. This involves complex interactions between the inflammatory and osteogenic cells. Therefore, the study of cell-cell interactions using direct co-culture models integrated with biomaterials is of great interest. This research aimed to study the viability, morphology, and osteogenic activity of preosteoblasts (OBs) co-cultured with pro-inflammatory macrophages (M1s) on the 3D printed (non)patterned surfaces. OBs and M1s remained alive and proliferated actively for 14days in the mixture of Dulbecco's Modified Eagle's Medium (DMEM) and alpha Minimum Essential Medium (α-MEM) (1:1), regardless of the cell ratio in the co-cultures. The spatial organization of the two types of cells changed with the time of culture from an initially uniform cell distribution to the formation of a thick layer of OBs covered by clusters of M1s. On day 7, the expression of PGE2 and TNF-α were upregulated in the co-culture relative to the mono-culture of OBs and M1s. The inflammation decreased differentiation and matrix mineralization of OBs after 28days of culture. Interestingly, the incorporation of 3D printed submicron pillars into the direct co-culture model enhanced the differentiation of preosteoblasts, as shown by relatively higher RUNX2 expression, thereby revealing the osteoimmunomodulatory potential of such surface patterns.

Characterization of the Impact of Classical Cell‐culture Media on the Response of Electrochemical Sensors

AbstractBiocompatibility testing is usually performed through staining and imaging of cell lines. We propose here to monitor cytotoxicity through real‐time measurement of metabolites specifically issued from cell stress behaviour using a multiparametric electrochemical (bio)sensing platform. However, the composition of culture media varies widely according to the requirements of the utilized cell lines. This matter may have significant effects on the sensor's sensitivity. With this mind, the sensitivity of four electrochemical (bio)sensors (pH, hydrogen peroxide, nitric oxide/nitrite (NO and its by‐product) and lactate) is investigated in different cell culture media. The main culture media studied were Minimum Essential Medium Eagle (MEM), Dulbecco's Modified Eagle Medium (DMEM), Williams’ Medium E and RPMI 1640 medium that were the recommended culture media for the cell types to be monitored. This work shows the impact of the different cell culture media on the performances of the different sensors (limit of detection, sensitivity, selectivity, response time and dynamic range). More particularly, FBS strongly impacts the response of the amperometric (bio)sensors. Then, cellular viability testing was effected within optimized medium (FBS content) for electrochemical sensor read‐outs in the case of short‐term cultures (one day) devoted to cytotoxicity testing. Real‐time electrochemical monitoring provides important additional information about cell behaviour during biocompatibility testing that might be further implemented in different settings including pharmaceutical efficacy and biomaterials applications.

A STUDY ON PHYTOCHEMICAL SCREENING ANTIOXIDENT AND IN VITRO-ANTICANCER ACTIVITY OF Aplotaxis auriculata ROOTS EXTRACT

The current examination was done to depict the bioactive compound constituents in plant concentrate of Aplotaxis auriculate using UV range - 210-290, FTIR, absorbance was recorded in the zone of some place in the scope of 4000 and 400 cmˉ¹. The zenith 3370 (Alcoholic and Phenolic social affairs), 2947 (Alkanes), 1656 (Alkene), 1453 (Alkane) and 668 cmˉ¹ (Aromatic) exhibits the individual get-togethers, and in vitro cell fortification against illness activities of Aplotaxis auriculata methanol remove. Cell support activity was done through DPPH, Hydrogen peroxide scrounging activity and diminishing power look at. Cell support and unfriendly to sickness practices were seen that A. auriculata extended with growing obsession. The human chest torment cell line (Michigan Cancer Foundation-7 (MCF 7)) was gotten from National Center for Cell Science (NCCS), Pune and made in Eagles Minimum Essential Medium (EMEM) containing 10% fetal dairy creatures like serum (FBS). The cells were kept up in Minimal Essential Media (MEM) improved with 10% Fetal dairy animals like serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) in a humidified atmosphere of 5% CO2 at 37°C. Support social requesting were passaged following a long time after week, and the way of life medium was changed on different occasions reliably. Over piece of all authentic canny meds are standard things that perceive a principal occupation in calm improvement in medication endeavors.

Overexpression of cellular stress proteins in skin fibroblast with presenilin 1 mutation.

The accumulation and aggregation of misfolded proteins in the human brain are known to be an essential feature of many neurodegenerative diseases, and presenilin-1 (PS1) mutations linked to Alzheimer's disease (AD) have been suggested to alter the misfolded protein response (UPR) due to stress on the endoplasmic reticulum. Moreover, to deal with the stress generated by protein aggregation, cells use a series of protection mechanisms. One of these biological responses to stress involves proteins called molecular chaperones that mediate protein folding, signaling, surveillance, and cell protection. These molecular changes of cellular stress have not only been seen in the neurons of brains from AD patients but also in the peripheral cells of patients with Alzheimer's disease, emphasizing the systemic nature of this pathology. Besides, due to the difficulties in studying dynamic processes in the human brain, peripheral cells are valuable for diagnosis and understanding the molecular mechanisms of AD. We cultured fibroblasts from patients affected with familial AD with a presenilin 1 mutation (M146L or A246E) and control subjects, in Minimum Essential Medium Eagle with 15% non-inactivated fetal bovine serum. These cells were characterized by immunodetection and karyotyping. Pathways related to neurodegeneration were analyzed by western blot. Finally, we carried out a proteomic study using mass spectrometry to study the fibroblast´s protein profile from familial AD and controls. We identified differences in the expression of proteins related to the autophagic-lysosomal pathway in affected individual´s fibroblasts. We also identified changes in kinases related to hyperphosphorylation of tau protein. We analyzed the protein profile of fibroblasts derived from mass spectrometry and identified groups of proteins related to cellular stress (HSPA8, HSP90AB1, HSPD1, HSPE1) present only in fibroblasts affected with AD and not in controls. From the Western blot analysis, we confirmed the overexpression of HSP90, HSP70, and HSP60. In summary, our results indicate that fibroblasts from patients with FAD-PS1 show altered pathways associated with cellular stress, autophagy, lysosomes, phosphorylation of tau protein, which shows that fibroblasts can be useful in the search and modeling of pathways related to neurodegeneration, as well as for the identification of early biomarkers related to AD.

Assessment of intraocular lens/capsular bag biomechanical interactions following cataract surgery in a human in vitro graded culture capsular bag model

Intraocular lenses (IOLs) are implanted during cataract surgery. For optimum results, stable positioning of the IOL in the capsular bag is important. Wound-healing events following cataract surgery lead to modification of the capsular bag and secondary visual loss due to posterior capsule opacification. At present, it is unclear how these biological events can affect stability of the IOL within the capsular bag. In the present study, a human in vitro graded culture capsular bag model was the experimental system. Capsulorhexis and lens extraction performed on human donor eyes generated suspended capsular bags (5 match-paired experiments). Preparations were secured by pinning the ciliary body to a silicone ring and maintained in 6 mL of medium for 84 days using a graded culture system: days 1–3, 5% human serum and 10 ng/mL transforming growth factor β (TGFβ2); days 4–7, 2% human serum and 1 ng/mL TGFβ2; days 8–14, 1% human serum and 0.1 ng/mL TGFβ2; days 15–84, serum-free Eagle's minimum essential medium (EMEM). A CT LUCIA 611PY IOL was implanted in all preparations. Quantitative measures were determined from whole bag images captured weekly. Images were registered using FIJI and analysed in ImageJ to determine capsular bag area; distortion; angle of contact; haptic stability; capsulorhexis area; and a fusion footprint associated with connection between the anterior and posterior capsules. Cell coverage and light scatter were quantified at end-point. The transdifferentiation marker, α-SMA was assessed by immunocytochemistry. Immediately following surgery, distortion of the capsular bag was evident, such that a long axis is generated between haptics relative to the non-haptic regions (short axis). The angle of contact between the haptics and the peripheral bag appeared inversely correlated to capsular bag area. Growth on the peripheral posterior capsule was observed 1 week after surgery and beneath the IOL within 1 month. As coverage of the posterior capsule progressed this was associated with matrix contraction/wrinkles of both the central posterior capsule and peripheral capsular bag. Cells on the central posterior capsule expressed αSMA. Fusion footprints formed in non-haptic regions of the peripheral bag and progressively increased over the culture period. Within and at the edge of the fusion footprint, refractive structures resembling lens fibre cells and Elschnig's pearls were observed. Cell attachment to the IOL was limited. An impression in the posterior capsule associated with the CT LUCIA 611PY optic edge was evident; cell density was much greater peripheral to this indent. Wound-healing events following surgery reduced capsular bag area. This was associated with the long/short axis ratio and angle of contact increasing with time. In summary, we have developed a human capsular bag model that exhibits features of fibrotic and regenerative PCO. The model permits biomechanical information to be obtained that enables better understanding of IOL characteristics in a clinically relevant biological system. Throughout culture the CT LUCIA 611PY appeared stable in its position and capsular bag modifications did not change this. We propose that the CT LUCIA 611PY optic edge shows an enhanced barrier function, which is likely to provide better PCO management in patients.

Cytotoxicity and Bioactivity of Calcium Silicate-based Cements in a Culture of Stem Cells from the Apical Papilla.

The present in vitro study evaluated the cytotoxicity and bioactivity of commonly-used calcium silicate-based cements in a culture of stem cells from the apical papilla (SCAPs). NeoMTA Plus (Avalon Biomed), BiodentineTM (Septodont) and MTA HP Repair (Angelus) cements were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and sulphorhodamine-B (SRB) viability assays. Cells were seeded (1*104 cells mL-1) in 96-well plates and exposed to 1:4 diluted extract in 24 h and 72 h. For the analysis of bioactivity, alkaline phosphatase (ALP) enzyme activity and Alizarin Red S (AZR) were assessed after 24 h of cell culture in 12-well plates (1*104 cells mL-1), where cells were exposed to 1:4 diluted extract on days 1 and 7. Minimum Essential Eagle's Medium alpha modification was used as control. ANOVA and Tukey's post hoc test were used to compare the different cements at each experimental time point. No significant differences were found between the cements and the control specimens on MTT at 24 h and 72 h (P>0.05); however, the calcium silicate-based cement materials showed higher cell viability compared to the control group (P<0.05). In the 24-h SRB, NeoMTA Plus showed lower cell viability than BiodentineTM and MTA HP Repair (P<0.05), with all groups similar to the control group (P>0.05). Compared to 24-h results, only NeoMTA Plus presented increased cell viability at 72 h (P<0.05). ALP activity was similar across the materials at 1 day (P>0.05). ALP activity was higher for BiodentineTM when compared to NeoMTA Plus (P<0.05), nevertheless, it was similar to MTA HP Repair and control groups (P>0.05) at 7 days. At 1- and 7-day periods of AZR assay, BiodentineTM presented higher levels of mineralized nodule formation (P<0.05). All evaluated calcium silicate-based cements demonstrated cell viability and bioactivity, suggesting that these (bio)materials may be indicated for use in regenerative dentine-pulp complex procedures.

Adjusting the Néel relaxation time of Fe3O4/Zn x Co1−x Fe2O4 core/shell nanoparticles for optimal heat generation in magnetic hyperthermia

In this work it is shown a precise way to optimize the heat generation in high viscosity magnetic colloids, by adjusting the Néel relaxation time in core/shell bimagnetic nanoparticles, for magnetic fluid hyperthermia (MFH) applications. To pursue this goal, Fe3O4/Zn x Co1−x Fe2O4 core/shell nanoparticles were synthesized with 8.5 nm mean core diameter, encapsulated in a shell of ∼1.1 nm of thickness, where the Zn atomic ratio (Zn/(Zn + Co) at%) changes from 33 to 68 at%. The magnetic measurements are consistent with a rigid interface coupling between the core and shell phases, where the effective magnetic anisotropy systematically decreases when the Zn concentration increases, without a significant change of the saturation magnetization. Experiments of MFH of 0.1 wt% of these particles dispersed in water, in Dulbecco modified Eagles minimal essential medium, and a high viscosity butter oil, result in a large specific loss power (SLP), up to 150 W g−1, when the experiments are performed at 571 kHz and 200 Oe. The SLP was optimized adjusting the shell composition, showing a maximum for intermediate Zn concentration. This study shows a way to maximize the heat generation in viscous media like cytosol, for those biomedical applications that require smaller particle sizes.

Effects of Holder Pasteurization on Immune Composition of Human Milk.

Background: Human milk (HM) is the ideal food for newborn (NB) nutrition, it provides all macro and micronutrients for human growth and development and also contains bioactive compounds, which influence the development of the neonatal digestive and immune systems. The holder pasteurization process is essential to prevent NB infection from donated milk. Therefore, the aim of this study was to check whether or not holder pasteurization could impact the concentration of immune components in HM and the capacity to induce epithelial cell growth. Materials and Methods: The study was performed on raw and holder pasteurized (62.5°C/30 minutes) paired milk samples after submission to the freezing process in both phases. For cytokine and adipokine measurements, ELISA was performed on 40 individual samples of HM from single donors. For analyzes of epithelial cell growth, HuTu-80 cells were cultivated in Minimum Essential Eagle medium with 15% of raw or pasteurized milk, eight pairs of milk were used. Results: The results showed that no alteration was observed in the concentration of cytokine after milk holder pasteurization, and leptin concentration was reduced in holder pasteurized milk. The heat treatment also did not impact the capacity of breast milk to promote intestinal epithelial cell growth. Conclusions: The results showed that donated breast milk pasteurization has a small impact on the HM bioactive concentration compounds. This technique is important to avoid NB infection.

Effects of serum-free culture media on human apical papilla cells properties

ObjectiveAiming at more effective and safer cell therapies, the objective of this study was to evaluate the biological properties of human apical papilla cells cultured in the absence of serum supplementation in comparison to cells cultured with fetal bovine serum (FBS). DesignTwo apical papilla cell populations were isolated from third molars with incomplete rhizogenesis, and cultured in four different media: minimum essential Eagle medium – alpha modification (alpha-MEM); alpha-MEM supplemented with FBS (alpha-MEM + FBS); Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12); and DMEM/F12 supplemented with FBS (DMEM/F12 + FBS). We evaluated their proliferation, clonogenicity, and in vitro osteogenic and chondrogenic differentiation potential. ResultsApical papilla cells cultured in DMEM/F12 + FBS and alpha-MEM + FBS were more proliferative than those grown in serum-free media, and also exhibited greater efficiency in colony cell formation. Despite this, all study groups showed immunostaining for the marker of mitosis anti-PHH3. Also, alpha-MEM + FBS, alpha-MEM, and DMEM/F12 + FBS exhibited higher amount of mineralized deposits in vitro than DMEM/F12, while only cells cultured with FBS were able to form spheres in chondrogenic differentiation assay. ConclusionsOur results showed that, although the cultivation of apical papilla cells in a serum-free medium has reduced the properties of cell proliferation and differentiation, these cells are still capable of maintaining their desirable characteristics.

Culture of pig embryos

Pig embryos can be cultured using a number of different strategies including complex approaches like culture in vivo in a surrogate oviduct (rabbit, sheep, mouse), culture in mouse oviducts in organ culture, and co-culture of embryos with cells in addition to simple approaches like culture in defined media or salt solutions. Addition of serum to medium has been of particular importance where blastocyst development and hatching are required. Pig conceptuses (day 10-15), embryonic discs or cell lines derived from conceptuses can be cultured in complex media like Eagle's minimal essential medium or Dulbecco's modified Eagle's medium with serum, although embryonic discs can be cultured in the absence of serum. In contrast, early stage pig embryos (one-cell to blastocyst) are best cultured in simpler media such as those used for mouse embryos. The media that have been used are all relatively similar in composition. They contain salts and one or more energy sources such as glucose, lactate, or pyruvate with BSA as a macromolecular component. Early attempts to culture pig embryos were not very successful, but some embryos did develop to the blastocyst stage. More recent reports indicate a much higher rate of development with relatively little or no change in media composition. Some workers have reported improved development in medium lacking glucose, which is consistent with findings with laboratory animals such as hamsters. Glutamine can serve as the sole exogenous energy source in medium lacking glucose, lactate and pyruvate. Addition of taurine and hypotaurine to culture medium enhances development of pig embryos in vitro. We suggest, where possible, adoption of a standard medium that could be used by different laboratories and, perhaps, with different species. Use of one medium for different species would simplify experimental protocols, enhance studies of comparative embryonic physiology and metabolism, and expedite transfer of information obtained in different species.

Early Odontogenic Differentiation of Dental Pulp Stem Cells Treated with Nanohydroxyapatite-Silica-Glass Ionomer Cement.

This study aimed to investigate the effects of nanohydroxyapatite–silica–glass ionomer cement (nanoHA–silica–GIC) on the differentiation of dental pulp stem cells (DPSCs) into odontogenic lineage. DPSCs were cultured in complete Minimum Essential Medium Eagle—Alpha Modification (α-MEM) with or without nanoHA–silica–GIC extract and conventional glass ionomer cement (cGIC) extract. Odontogenic differentiation of DPSCs was evaluated by real-time reverse transcription polymerase chain reaction (rRT–PCR) for odontogenic markers: dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), osteocalcin (OCN), osteopontin (OPN), alkaline phosphatase (ALP), collagen type I (COL1A1), and runt-related transcription factor 2 (RUNX2) on day 1, 7, 10, 14, and 21, which were normalized to the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Untreated DPSCs were used as a control throughout the study. The expressions of DSPP and DMP1 were higher on days 7 and 10, that of OCN on day 10, those of OPN and ALP on day 14, and that of RUNX2 on day 1; COL1A1 exhibited a time-dependent increase from day 7 to day 14. Despite the above time-dependent variations, the expressions were comparable at a concentration of 6.25 mg/mL between the nanoHA–silica–GIC and cGIC groups. This offers empirical support that nanoHA–silica–GIC plays a role in the odontogenic differentiation of DPSCs.

Establishment and characterization of a cell line derived from fin of the endangered Yangtze sturgeon (Acipenser dabryanus).

Yangtze sturgeon (Acipenser dabryanus) is an endangered endemic freshwater fish of China. Cell-line is a potential means used for long-term preservation of germplasm resources and an ideal in vitro model in place of living organisms for biological studies. Here, culture condition and characterization of fin-derived cell in Yangtze sturgeon were carried out. Tissue explant techniques have been efficiently used in the Yangtze sturgeon caudal fin (YSCF) culture. The YSCF cell line showed a fibroblast-like morphology and stable growth in minimum essential medium eagle's (MEME) supplemented with 10-20% fetal bovine serum at 25°C. Cells were cryopreserved with preservative DMSO in liquid nitrogen and grew normally after recovery. No bacterial, fungal, or mycoplasma contamination was detected in the YSCF cells. Karyotype analysis of the YSCF cells showed that the chromosome numbers of the YSCF ranged from 242 to 273, and the modal chromosome number was identified as 264 at passage 9. The YSCF cells were confirmed from A. dabryanus by assay of 16S rRNA and COI. Furthermore, GFP reporter gene was successfully transferred into YSCF cells and expressed. The established YSCF cell lines will contribute to the preservation of germplasm resources and provide a useful vitro tool for further biological studies in sturgeon species.

Pad-printed Prussian blue doped carbon ink for real-time peroxide sensing in cell culture

Abstract Hydrogen peroxide has important roles within cellular functions, as a prevalent form of Reactive Oxygen Species, detection within mammalian cells is of metabolic importance; typically requiring cell lysis or fluorescence-based methods to quantify. Herein, we explore the novel use of Prussian blue mediated, pad printed carbon electrodes to allow the indirect detection of cellular peroxides in bulk culture media, which facilitates non-invasive, real-time detection. Electrodes demonstrated capacity to detect H2O2 with a linear range of 1-200 μM in CMEM (R2 = 0.9988), enabling detection of peroxides found in culture media and lysate. Developed electrodes had a Limit of Detection (LOD) of 0.41 μM H2O2 in Britton-Robinson Buffer (BRB), 0.38 μM in Eagle's Minimum Essential Medium (EMEM) and 9.19 μM in Dulbecco's Modified Eagles Medium (DMEM). Electrodes were tested in a conventional 5% serum supplemented EMEM (CMEM) and demonstrated an LOD of 0.5 μM and LOQ of 0.9 μM. The results demonstrate proof of concept for monitoring H2O2 in complex culture media with potential long-term use and reusability using simple, pad printed Prussian Blue / Carbon electrodes. The lack of further modification, and cost-effectiveness of these disposable electrodes could offer great advancement to monitoring of peroxides in complex media.