Search results for: human cells line

Authors: Monika Verma, Renuka Sharma, B. R. Gulati, Namita Singh

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In combination therapy, two chemotherapeutic agents work together in a collaborative action. It has appeared as one of the promising approaches to improve anti-cancer treatment efficacy. In the present investigation, piperine (P-NPS), paclitaxel (PTX NPS), and a combination of both, piperine-paclitaxel nanoparticle (Pip-PTX NPS), were made by the nanoprecipitation method and later characterized by PSA, DSC, SEM, TEM, and FTIR. All nanoparticles exhibited a monodispersed size distribution with a size of below 200 nm, zeta potential ranges from (-30-40mV) and a narrow polydispersity index (>0.3) of the drugs. The average encapsulation efficiency was found to be between 80 and 90%. In vitro release of drugs for nanoparticles was done spectrophotometrically. FTIR and DSC results confirmed the presence of the drug. The Pip-PTX NPS significantly inhibit cell proliferation as compared to the native drugs nanoparticles in the breast cancer cell line MCF-7. In addition, Pip-PTX NPS suppresses cells in colony formation and soft gel agar assay. Scratch migration and Transwell chamber invasion assays revealed that combined nanoparticles reduce the migration and invasion of breast cancer cells. Morphological studies showed that Pip-PTX NPS penetrates the cells and induces apoptosis, which was further confirmed by DNA fragmentation, SEM, and western blot analysis. Taken together, Pip-PTX NPS inhibits cell proliferation, anchorage dependent and anchorage independent cell growth, reduces migration and invasion, and induces apoptosis in cells. These findings support that combination therapy using Pip-PTX NPS represents a potential approach and could be helpful in the future for breast cancer therapy.

Keywords: piperine, paclitaxel, breast cancer, apoptosis

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Authors: Lie-Sian Yap, Ming-Chien Yang

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This study demonstrated a novel injectable hydrogel scaffold composing of Pluronic F127, carboxymethyl hexanoyl chitosan (CA) and glutaraldehyde (GA) for encapsulating human fetal osteoblastic cells (hFOB) 1.19. The hydrogel was prepared by mixing F127 and GA in CA solution at 4°C. The mechanical properties and cytotoxicity of this hydrogel were determined through rheological measurements and MTT assay, respectively. After encapsulation process, the hFOB 1.19 cells morphology was examined using fluorescent and confocal imaging. The results indicated that the Tgel of this system was around 30°C, where sol-gel transformation occurred within 90s and F127/CA/GA gel was able to remain intact in the medium for more than 1 month. In vitro cell culture assay revealed that F127/CA/GA hydrogels were non-cytotoxic. Encapsulated hFOB 1.19 cells not only showed the spherical shape and formed colonies, but also reduced their size. Moreover, the hFOB 1.19 cells showed that cells remain alive after the encapsulation process. Based on these results, these F127/CA/GA hydrogels can be used to encapsulate cells for tissue engineering applications.

Keywords: carboxymethyl hexanoyl chitosan, cell encapsulation, hFOB 1.19, Pluronic F127

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Authors: H. M. Imam, H. M. Rezk, A. F. Tohamy

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Background: Human umbilical cord blood (HUCB) is considered as a unique source for stem cells. HUCB contain different types of progenitor cells which could differentiate into hepatocytes. Aims: To investigate the potential of rat's liver damage repair using human umbilical cord mesenchymal stem cells (hUCMSCs). We investigated the feasibility for hUCMSCs in recovery from liver damage. Moreover, investigating fibrotic liver repair and using the CCl4-induced model for liver damage in the rat. Methods: Rats were injected with 0.5 ml/kg CCl4 to induce liver damage and progressive liver fibrosis. hUCMSCs were injected into the rats through the tail vein; Stem cells were transplanted at a dose of 1×106 cells/rat after 72 hours of CCl4 injection without receiving any immunosuppressant. After (6 and 8 weeks) of transplantation, blood samples were collected to assess liver functions (ALT, AST, GGT and ALB) and level of Procollagen III as a liver fibrosis marker. In addition, hepatic tissue regeneration was assessed histopathologically and immunohistochemically using antihuman monoclonal antibodies against CD34, CK19 and albumin. Results: Biochemical and histopathological analysis showed significantly increased recovery from liver damage in the transplanted group. In addition, HUCB stem cells transdifferentiated into functional hepatocytes in rats with hepatic injury which results in improving liver structure and function. Conclusion: Our findings suggest that transplantation of hUCMSCs may be a novel therapeutic approach for treating liver fibrosis. Therefore, hUCMSCs are a potential option for treatment of liver cirrhosis.

Keywords: carbon tetra chloride, liver fibrosis, mesenchymal stem cells, rat

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Authors: Merry Meryam Martgrita, Marselina Irasonia Tan

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One of several aggresive cancer is cancer that overexpress c-erbB2 receptor along with the expression of estrogen receptor. Components of extracellular matrix play an important role to increase cancer cells proliferation, migration and invasion. Both components can affect cancer development by regulating the signal transduction pathways in cancer cells. In recent research, SKOV-3 ovarian cancer cell line, that overexpress c-erbB2 receptor was cultured on type IV collagen and treated with estradiol-17β, to reveal the role of both components on RNA and protein level of c-erbB2 receptor. In this research we found a modulation phenomena of increasing and decreasing of c-erbB2 RNA level and a stabilisation phenomena of c-erbB2 protein expression due to estradiol-17β and type IV collagen. It seemed that estradiol-17β has an important role to increase c-erbB2 transcription and the stability of c-erbB2 protein expression. Type IV collagen has an opposite role. It blocked c-erbB2 transcription when it bound to integrin receptor in SKOV-3 cells.

Keywords: c-erbB2, estradiol-17β, SKOV-3, type IV collagen

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Authors: Gupta Renu, T. S. Roy

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Introduction: For the prospect of successful replacement therapies in treatment of Diabetes mallitus it is necessary to know events occurring during normal human pancreas development. Literature of human pancreas development are few in number as well as mainly related to first trimester because of ethical and technical difficulties. So the study was conducted on 12 fetuses from 12 gestational weeks (GW) to 5 months of infant to know normal development of exocrine and endocrine part of human pancreas. Material and Methods: Human fetalpancreases were screened by haematoxyline and eosin staining and done electron microscopy for suitable specimens to know ultrastructural detail of fetal pancreas. Results:It was observed arborized tubules, the cells budding out from these tubules differentiated into primitive acini and islets in 12thGW. At 14 weeks scanty granules were observed in the endocrine cells which coincided with the capillary invasion of the islets. The ducts and acini were surrounded by well-organized connective tissue. The acinihad elongated cells, small amount of cytoplasm and large open face euchromatic nuclei with single nucleolus. The mature form of islets of Langerhans was observed close to the acini and duct in 20 GW fetus. Connective tissue around the duct was well organized.No significant developmental change was observed early postnatal, infant. Conclusion: The development of both component exocrine as well as endocrine part of human fetal pancreas was studied by light and electron microscopy. Observations suggested that the fetal pancreas contained mainly ducts, few acini, many centroacinar cells, and large undifferentiated tissue.

Keywords: gestational weeks (GW), acini, islets of Langerhans, ducts

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Authors: Jianli Dong, Fangling Xu, Gengming Huang

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Human endogenous retroviral elements (HERVs) comprise approximately 8% of the human genome. They are thought to be germline-integrated genetic remnants of retroviral infections. Although HERV sequences are highly defective, some, especially the K type (HERV-K), have been shown to be expressed and may have biological activities in the pathogenesis of cancer, chronic inflammation and autoimmune diseases. We found that HERV-K GAG and ENV proteins were strongly expressed in pleomorphic melanoma cells. We also detected a critical role of HERV-K ENV in mediating intercellular fusion and colony formation of melanoma cells. Interestingly, we found that levels of HERV-K GAG and ENV expression correlated with the activation of ERK and loss of p16INK4A in melanoma cells, and inhibition of MEK or CDK4, especially in combination, reduced HERV-K expression in melanoma cells. We also performed a reverse transcription-polymerase chain reaction (RT-PCR) assay using DNase I digestion to remove “contaminating” HERV-K genomic DNA and examined HERV-K RNA expression in plasma samples from HIV-1 infected individuals. We found a covariation between HERV-K RNA expression and CD4 cell counts in HIV-1 positive samples. Although a causal link between HERV-K activation and melanoma development, and between HERV-K activation, HIV-1 infection and CD4 cell count have yet to be determined, existing data support the further research efforts in HERV-K.

Keywords: CD4 cell, HERV-K, HIV-1, melanoma

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Authors: Arieh Moussaieff, Bénédicte Elena-Herrmann, Yaakov Nahmias, Daniel Aberdam

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Differentiation of pluripotent stem cells is a slow process, marked by the gradual loss of pluripotency factors over days in culture. While the first few days of differentiation show minor changes in the cellular transcriptome, intracellular signaling pathways remain largely unknown. Recently, several groups demonstrated that the metabolism of pluripotent mouse and human cells is different from that of somatic cells, showing a marked increase in glycolysis previously identified in cancer as the Warburg effect. Here, we sought to identify the earliest metabolic changes induced at the first hours of differentiation. High-resolution NMR analysis identified 35 metabolites and a distinct, gradual transition in metabolism during early differentiation. Metabolic and transcriptional analyses showed the induction of glycolysis toward acetate and acetyl-coA in pluripotent cells, and an increase in cholesterol biosynthesis during early differentiation. Importantly, this metabolic pathway regulated differentiation of human and mouse embryonic stem cells. Acetate delayed differentiation preventing differentiation-induced histone de-acetylation in a dose-dependent manner. Glycolytic inhibitors upstream of acetate caused differentiation of pluripotent cells, while those downstream delayed differentiation. Our data suggests that a rapid loss of glycolysis in early differentiation down-regulates acetate and acetyl-coA production, causing a loss of histone acetylation and concomitant loss of pluripotency. It demonstrate that pluripotent stem cells utilize a novel metabolism pathway to maintain pluripotency through acetate/acetyl-coA and highlights the important role metabolism plays in pluripotency and early differentiation of stem cells.

Keywords: pluripotency, metabolomics, epigenetics, acetyl-coA

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Authors: Samir Dekali, David Crouzier

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Due to their new physico-chemical properties engineered nanoparticles (ENPs) are increasingly employed in numerous industrial sectors (such as electronics, textile, aerospace, cosmetics, pharmaceuticals, food industry, etc). These new physico-chemical properties can also represent a threat for the human health. Consumers can notably be exposed involuntarily by different routes such as inhalation, ingestion or through the skin. Several studies recently reported a possible biodistribution of these ENPs on the blood-brain barrier (BBB). Consequently, there is a great need for developing BBB in vitro models representative of the in vivo situation and capable of rapidly and accurately assessing ENPs toxic effects and their potential translocation through this barrier. In this study, several in vitro models established with micro-endothelial brain cell lines of different origins (bEnd.3 mouse cell line or a new human cell line) co-cultivated or not with astrocytic cells (C6 rat or C8-B4 mouse cell lines) on Transwells® were compared using different endpoints: trans-endothelial resistance, permeability of the Lucifer yellow and protein junction labeling. Impact of NIST diesel exhaust particles on BBB cell viability is also discussed.

Keywords: nanoparticles, blood-brain barrier, diesel exhaust particles, toxicology

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Authors: Jer-Yuh Liu, Je-Chiuan Ye, Jin-Ming Hwang

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Protein kinase C alpha (PKCα) is a key signaling molecule in human cancer development. As a therapeutic strategy, targeting PKCα is difficult because the molecule is ubiquitously expressed in non-malignant cells. PKCα is regulated by the cooperative interaction of the transcription factors myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) in human cancer cells. By conducting tissue array analysis, herein, we determined the protein expression of MZF-1/Elk-1/PKCα in various cancers. The data show that the expression of MZF-1/Elk-1 is correlated with that of PKCα in hepatocellular carcinoma (HCC), but not in bladder and lung cancers. In addition, the PKCα down-regulation by shRNA Elk-1 was only observed in the HCC SK-Hep-1 cells. Blocking the interaction between MZF-1 and Elk-1 through the transfection of their binding domain MZF-160–72 decreased PKCα expression. This step ultimately depressed the epithelial-mesenchymal transition potential of the HCC cells. These findings could be used to develop an alternative therapeutic strategy for patients with the PKCα-derived HCC.

Keywords: protein kinase C alpha, myeloid zinc finger 1, ets-like protein-1, hepatocellular carcinoma

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Authors: Thanunthon Bowornsakulwong, Charukorn Charukarn, Franck Courtes, Panit Kitsubun, Lalintip Horcharoen

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Vero cell is a continuous cell line that is widely used for the production of viral vaccines. However, due to its adherent characteristic, scaling up strategy in large-scale production remains complicated and thus limited. Consequently, suspension-like Vero cell culture processes based on microcarriers have been introduced and employed while also providing increased surface area per volume unit. However, harvesting Vero cells from microcarriers is a huge challenge due to difficulties in cells detaching, lower recovery yield, time-consuming and dissociation agent carry-over. To overcome these problems, we developed a dissociation-association platform technology for detaching and re-attaching cells during subculturing from microcarriers to microcarriers, which will be conveniently applied to seed trains strategies in large scale bioreactors. Herein, Hillex-2 was used to culture Vero cells in serum-containing media using spinner flasks as a scale-down model. The overall confluency of cells on microcarriers was observed using inverted microscope, and the sample cells were daily detached in order to obtain the kinetics data. The metabolites consumption and by-products formation were determined by Nova Biomedical BioprofileFlex.

Keywords: dissociation-reassociation, microcarrier, scale up, Vero cell

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Authors: M. M. Rahman, A. Liu, A. Frostegard, J. Frostegard

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The human immune system plays an essential role in cardiovascular disease (CVD) and atherosclerosis. Our earlier studies showed that major immunocompetent cells including T cells are activated by phosphorylcholine epitope. Further, we have determined for the first time in a clinical cohort that antibodies against phosphorylcholine (anti-PC) are negatively and independently associated with the development of atherosclerosis and thus a low risk of cardiovascular diseases. It is still unknown whether activated T cells play a role in anti-PC production. Here we aim to clarify the role of T cells in anti-PC production. B cell alone, or with CD3 T, CD4 T or with CD8 T cells were cultured in polystyrene plates to examine anti-PC IgM production. In addition to mixed B cell with CD3 T cell culture, B cells with CD3 T cells were also cultured in transwell co-culture plates. Further, B cells alone and mixed B cell with CD3 T cell cultures with or without anti-HLA 2 antibody were cultured for 6 days. Anti-PC IgM was detected by ELISA in independent experiments. More than 8 fold higher levels of anti-PC IgM were detected by ELISA in mixed B cell with CD3 T cell cultures in comparison to B cells alone. After the co-culture of B and CD3 T cells in transwell plates, there were no increased antibody levels indicating that B and T cells need to interact to augment anti-PC IgM production. Furthermore, anti-PC IgM was abolished by anti-HLA 2 blocking antibody in mixed B and CD3 T cells culture. In addition, the lack of increased anti-PC IgM in mixed B with CD8 T cells culture and the increased levels of anti-PC in mixed B with CD4 T cells culture support the role of helper T cell for the anti-PC IgM production. Atherosclerosis is a major cause of cardiovascular diseases, but anti-PC IgM is a protection marker for atherosclerosis development. Understanding the mechanism involved in the anti-PC IgM regulation could play an important role in strategies to raise anti-PC IgM. Studies suggest that anti-PC is T-cell independent antibody, but our study shows the major role of T cell in anti-PC IgM production. Activation of helper T cells by immunization could be a possible mechanism for raising anti-PC levels.

Keywords: anti-PC, atherosclerosis, aardiovascular diseases, phosphorylcholine

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Authors: Kheireddine El-Boubbou

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Drug delivery to target human acute myeloid leukemia (AML) using a nanoparticulate chemotherapeutic formulation that can deliver drugs selectively to AML cancer is hugely needed. In this work, we report the development of a nanoformulation made of polymeric-stabilized multifunctional magnetic iron oxide nanoparticles (PMNP) loaded with the anticancer drug Doxorubicin (Dox) as a promising drug carrier to treat AML. Dox@PMNP conjugates simultaneously exhibited high drug content, maximized fluorescence, and excellent release properties. Nanoparticulate uptake and cell death following addition of Dox@PMNPs were then evaluated in different types of human AML target cells, as well as on normal human cells. While the unloaded MNPs were not toxic to any of the cells, Dox@PMNPs were found to be highly toxic to the different AML cell lines, albeit at different inhibitory concentrations (IC₅₀ values), but showed very little toxicity towards the normal cells. In comparison, free Dox showed significant potency concurrently to all the cell lines, suggesting huge potentials for the use of Dox@PMNPs as selective AML anticancer cargos. Live confocal imaging, fluorescence and electron microscopy confirmed that Dox is indeed delivered to the nucleus in relatively short periods of time, causing apoptotic cell death. Importantly, this targeted payload may potentially enhance the effectiveness of the drug in AML patients and may further allow physicians to image leukemic cells exposed to Dox@PMNPs using MRI.

Keywords: magnetic nanoparticles, drug delivery, acute myeloid leukemia, iron oxide, cancer nanotherapy

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Authors: Feng Guo

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Human brain organoids, 3D brain tissue cultures derived from human pluripotent stem cells, hold promising potential in modeling neuroinflammation for a variety of neurological diseases. However, challenges remain in generating standardized human brain organoids that can recapitulate key physiological features of a human brain. Here, this study presents a series of organoids-on-a-chip systems to generate better human brain organoids and model neuroinflammation. By employing 3D printing and microfluidic 3D cell culture technologies, the study’s systems enable the reliable, scalable, and reproducible generation of human brain organoids. Compared with conventional protocols, this study’s method increased neural progenitor proliferation and reduced heterogeneity of human brain organoids. As a proof-of-concept application, the study applied this method to model substance use disorders.

Keywords: human brain organoids, microfluidics, organ-on-a-chip, neuroinflammation

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Authors: Maha Azzam, Mahmoud Gabr, Mahmoud Zakaria, Ayman Refaie, Amani Ismail, Sherry Khater, Sylvia Ashamallah, Mohamed Ghoniem

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Evidence was provided that human bone marrow-derived mesenhymal stem cells (HBM-MSCs) could be differentiated to form insulin-producing cells (IPCs). Transplantation of these cells was able to cure chemically-induced diabetes in nude mice. The efficacy of these cells to control diabetes in large animals was carried out to evaluate the sufficient number of cells needed/Kg body weight and to determine the functional longevity in vivo. Materials/Methods: Ten male mongrel dogs weighing 15-20 Kg were used in this study. Diabetes was chemically-induced in 7 dogs by a mixture of alloxan and streptozotocin. Three non-diabetic served as normal controls. Differentiated HBM-MSCs (5 million/Kg) were encapsulated in theracyte capsules and transplanted beneath the rectus sheath. Each dog received 2 capsules. One dog died 4 days postoperative from inhalation pneumonia. The remaining 6 dogs were followed up for 6-18 months. Results: Four dogs became normoglycemic within 6-8 weeks with normal glucose tolerance curves providing evidence that the transplanted cells were glucose-sensitive and insulin-responsive. In the remaining 2 dogs, fasting blood glucose was reduced but did not reach euglycemic levels. The sera of all transplanted dogs contained human insulin and c-peptide but negligible levels of canine insulin. When the HBM-MSCs loaded capsules were removed, rapid return of diabetic state was noted. The harvested capsules were examined by immunofluorescence. IPCs were seen and co-expression of with c-peptide was confirmed. Furthermore, all the pancreatic endocrine genes were expressed by the transplanted cells. Conclusions: This study provided evidence that theracyte capsules could protect the xenogenic HBM-MSCs from the host immune response. This is an important issue when clinical stem cell therapy is considered for definitive treatment for T1DM.

Keywords: diabetes, mesenchymal stem cells, dogs, Insulin-producing cells

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Authors: Alejandra Betancur Sánchez, Carmen Elena Zapata Sánchez, Juan Bautista López Ortiz

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Adverse effects and increased air pollution has raised concerns about regulatory policies and has fostered the development of new air quality standards; this is due to the complexity of the composition and the poorly understood reactions in the atmospheric environment. Toxic compounds act as environmental agents having various effects, from irritation to death of cells and tissues. A toxic agent is defined an adverse response in a biological system. There is a particular class that produces some kind of alteration in the genetic material or associated components, so they are recognized as genotoxic agents. Within cells, they interact directly or indirectly with DNA, causing mutations or interfere with some enzymatic repair processes or in the genesis or polymerization of proteinaceous material involved in chromosome segregation. An air pollutant may cause or contribute to increased mortality or serious illness and even pose a potential danger to human health. The aim of this study was to evaluate the effect on the viability and the genotoxic potential on the cell lines CHO-K1 and Jurkat and peripheral blood of particulate matter PM T lymphocytes 2.5 obtained from filters collected three monitoring stations network air quality Aburrá Valley. Tests, reduction of MTT, trypan blue, NRU, comet assay, sister chromatid exchange (SCE) and chromosomal aberrations allowed evidence reduction in cell viability in cell lines CHO-K1 and Jurkat and damage to the DNA from cell line CHOK1, however, no significant effects were observed in the number of SCEs and chromosomal aberrations. The results suggest that PM2.5 material has genotoxic potential and can induce cancer development, as has been suggested in other studies.

Keywords: PM2.5, cell line Jurkat, cell line CHO-K1, cytotoxicity, genotoxicity

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Authors: Sina Mahdavi

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Multiple sclerosis (MS) is a progressive inflammatory autoimmune disease of the CNS that affects the myelination process in the central nervous system (CNS). Complex interactions of various "environmental or infectious" factors may act as triggers in autoimmunity and disease progression. The association between viral infections, especially human immunodeficiency virus (HIV) and MS is one potential cause that is not well understood. This study aims to summarize the available data on human HIV infection in MS disease progression. In this study, the keywords "Multiple sclerosis", "Human immunodeficiency virus ", and "Central nervous system" in the databases PubMed, and Google Scholar between 2017 and 2022 were searched and 15 articles were chosen, studied, and analyzed. Revealed histologic signs of "MS-like illness" in the setting of HIV, which comprised widespread demyelination with reactive astrocytes, foamy macrophages, and perivascular infiltration with inflammatory cells, all of which are compatible with MS lesions. Human immunodeficiency virus causes dysfunction of the immune system, especially characterized by hypergammaglobulinemia and chronic activation of B cells. Activation of B cells leads to increased synthesis of immunoglobulin and finally to an excess of free light chains. Free light chains may be involved in autoimmune responses against neurons. There is a high expression of HIV during the course of MS, which indicates the relationship between HIV and MS, that this virus can play a role in the development of MS by creating an inflammatory state. Therefore, measures to modulate the expression of HIV may be effective in reducing inflammatory processes in demyelinated areas of MS patients.

Keywords: multiple sclerosis, human immunodeficiency virus, central nervous system, autoimmunity

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Authors: Mariyah Poonawala, Selvan Ravindran, Anuradha Vaidya

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Despite much progress in understanding the regulatory factors and cytokines that support the maturation of the various cell lineages of the hematopoietic system, factors that govern the self-renewal and proliferation of hematopoietic stem cells (HSCs) is still a grey area of research. Hematopoietic stem cell transplantation (HSCT) has evolved over the years and gained tremendous importance in the treatment of both malignant and non-malignant diseases. However, factors such as graft rejection and multiple organ failure have challenged HSCT from time to time, underscoring the urgent need for development of milder processes for successful hematopoietic transplantation. An emerging concept in the field of stem cell biology states that the interactions between the bone-marrow micro-environment and the hematopoietic stem and progenitor cells is essential for regulation, maintenance, commitment and proliferation of stem cells. Understanding the role of mesenchymal stromal cells in modulating the functionality of HSCs is, therefore, an important area of research. Trans-resveratrol has been extensively studied for its various properties to combat and prevent cancer, diabetes and cardiovascular diseases etc. The aim of the present study was to understand the effect of trans-resveratrol on HSCs using single and co-culture systems. We have used KG1a cells since it is a well accepted hematopoietic stem cell model system. Our preliminary experiments showed that low concentrations of trans-resveratrol stimulated the HSCs to undergo proliferation whereas high concentrations of trans-resveratrol did not stimulate the cells to proliferate. We used a murine fibroblast cell line, M210B4, as a stromal feeder layer. On culturing the KG1a cells with M210B4 cells, we observed that the stimulatory as well as inhibitory effects of trans-resveratrol at low and high concentrations respectively, were enhanced. Our further experiments showed that low concentration of trans-resveratrol reduced the generation of reactive oxygen species (ROS) and nitric oxide (NO) whereas high concentrations increased the oxidative stress in KG1a cells. We speculated that perhaps the oxidative stress was imposing inhibitory effects at high concentration and the same was confirmed by performing an apoptotic assay. Furthermore, cell cycle analysis and growth kinetic experiments provided evidence that low concentration of trans-resveratrol reduced the doubling time of the cells. Our hypothesis is that perhaps at low concentration of trans-resveratrol the cells get pushed into the G0/G1 phase and re-enter the cell cycle resulting in their proliferation, whereas at high concentration the cells are perhaps arrested at G2/M phase or at cytokinesis and therefore undergo apoptosis. Liquid Chromatography-Quantitative-Time of Flight–Mass Spectroscopy (LC-Q-TOF MS) analyses indicated the presence of trans-resveratrol and its metabolite(s) in the supernatant of the co-cultured cells incubated with high concentration of trans-resveratrol. We conjecture that perhaps the metabolites of trans-resveratrol are responsible for the apoptosis observed at the high concentration. Our findings may shed light on the unsolved problems in the in vitro expansion of stem cells and may have implications in the ex vivo manipulation of HSCs for therapeutic purposes.

Keywords: co-culture system, hematopoietic micro-environment, KG1a cell line, M210B4 cell line, trans-resveratrol

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Authors: Nunthawan Nowwarote, Waleerat Sukarawan, Prasit Pavasant, Thanaphum Osathanon

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Interleukin 6 (IL-6) is a multifunctional cytokine, regulating various biological responses in several tissues. A Recent study shows that IL-6 plays a role in stemness maintenance in stem cells isolated from human exfoliated deciduous teeth (SHEDs). However, the role of IL-6 on cell differentiation in SHEDs remains unknown. The present study investigated the effect of IL-6 on SHEDs differentiation. Cells were isolated from dental pulp tissues of human deciduous teeth. Flow cytometry was used to determined mesenchymal stem cell marker expression, and the multipotential differentiation (osteogenic, adipogenic and neurogenic lineage ) was also determined. The mRNA was determined using real-time quantitative polymerase chain reaction, and the phenotypes were confirmed by chemical and immunofluorescence staining. Results demonstrated that SHEDs expressed CD44, CD73, CD90, CD105 but not CD45. Further, the up-regulation of osteogenic, adipogenic and neurogenic marker genes was observed upon maintaining cells in osteogenic, adipogenic and neurogenic induction medium, respectively. The addition of IL-6 induced osteogenic by up-regulated osteogenic marker gene also increased in vitro mineralization. Under neurogenic medium supplement with IL-6, up-regulated neurogenic marker. Whereas, an addition of IL-6 attenuated adipogenic differentiation by SHEDs. In conclusion, this evidence implies that IL-6 may participate in cells differentiation ability of SHEDs.

Keywords: SHEDs, IL-6, cell differentiations, dental pulp

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Authors: Francesca Lombardi, Francesca Rosaria Augello, Benedetta Cinque, Maria Grazia Cifone, Paola Palumbo

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Glioblastoma multiforme (GBM) is a high-grade primary brain tumor refractory to current forms of treatment. The survival benefits of patients with GBM remain unsatisfactory due to the intrinsic or acquired resistance to temozolomide (TMZ), an alkylating agent, used as the first-line chemotherapeutic drug to treat GBM patients. Its cytotoxic effect is visualized by the induction of O6-methylguanine (O6MeG) within DNA. Cyclooxygenase-2 (COX-2), an inflammation-associated enzyme, has been implicated in tumorigenesis and progression of GBM, its inhibition shows anticancer activities. In the present study, it was verified if the combination of a COX-2 selective inhibitor, NS398, with TMZ could counteract the TMZ resistance. In particular, the effect of NS398 mixed with TMZ was investigated in the GBM TMZ-resistant cell line, T98G. Cells were pretreated with NS398 (100µM, 24 hours) and then exposed to TMZ alone (200µM), NS398 alone, or both for 72 hours, after which cell growth rate and cycle phases, as well as apoptosis level, were evaluated. Coadministration of NS398 and TMZ caused a significant decrease in cell growth and a progressive increase of dead cells detected by trypan blue staining. Moreover, a significant level of apoptotic cell percentage and alteration of cell cycle phases were observed in T98G treated with TMZ-NS398 combination when compared to untreated cells or TMZ-treated cells. TMZ-resistant tumors, as GBM, express elevated levels of DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). The mixture drastically reduced MGMT expression in the TMZ-resistant cell line T98G, known to express high levels of MGMT basically. Moreover, while TMZ alone did not influence the COX-2 protein expression, the combination successfully reduced it. In conclusion, these results demonstrated that NS398 enhanced the efficacy of TMZ through cell number reduction, apoptosis induction, and decreased MGMT levels, suggesting the ability of drug combination to reduce the chemoresistance. This drug combination deserves attention and could be considered as a promising therapeutic strategy for GBM patients.

Keywords: COX-2, COX-2 inhibitor, glioblastoma, NS398, T98G, temozolomide

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Authors: Mehmet Savsar

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Several factors affect productivity of Food Processing Assembly Lines (FPAL). Engineers and line managers usually do not recognize some of these factors and underutilize their production/assembly lines. In this paper, a special food processing assembly line is studied in detail, and procedures are presented to illustrate how productivity and efficiency of such lines can be increased. The assembly line considered produces ten different types of freshly prepared salads on the same line, which is called mixed model assembly line. Problems causing delays and inefficiencies on the line are identified. Line balancing and related tools are used to increase line efficiency and minimize balance delays. The procedure and the approach utilized in this paper can be useful for the operation managers and industrial engineers dealing with similar assembly lines in food processing industry.

Keywords: assembly lines, line balancing, production efficiency, bottleneck

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Authors: Ahmad Shah Farhat

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Background: Prenatal asphyxia or birth asphyxia is the medical situation resulting from a newborn infant that lasts long enough during the birth process to cause physical harm, usually to the brain. Human umbilical cord blood (UCB) is a well-established source of hematopoietic stem/progenitor cells (HSPCs) for allogeneic stem cell transplantation. These can be used clinically to care for children with malignant diseases. Low O2 can cause in proliferation and differentiation of stem cells. Method: the cord blood of 11 infants with 3-5 Apgar scores or need to cardiac pulmonary Resuscitation as an asphyxia group and ten normal infants with more than 8 Apgar scores as the normal group was collected, and after isolating hematopoietic stem cells, the cells were cultured in enriched media for 14 days to compare the numbers of colonies by microscope. Results: There was a significant difference in the number of RBC precursor colonies (red colonies) in cultured media with 107 cord blood hematopoietic stem cells of infants who were exposed to hypoxemia in two wells of palate. There was not a significant difference in the number of white cell colonies in the two groups in the two wells of the plate. Conclusion: Hypoxia in the perinatal period can cause the increase of hematopoietic stem cells of cord blood, special red precursor stem cells in vitro, like an increase of red blood cells in the body when exposed to low oxygen conditions. Thus, it will be usable.

Keywords: asphyxia, neonre, stem cell, red cell

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Authors: Husain S. Yawer, Vasim Raja Panwar, Nidhi Priya

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The objective of this study is to evaluate and compare the role of nano-gelatin and Bioengineered Scaffolds on the attachment, proliferation, and osteogenic differentiation of human dental pulp stem cells (DPSCs). Tooth decay and early fall have each been one of the most prevailing dental disorders which cause physical and emotional suffering and compromise the patient's quality of life. The design of novel scaffolding materials will be based on mimicking the architecture of natural dental extracellular matrix which may provide as in vivo environments for proper cell growth. This methodology will involve the combination of nano-fibred gelatin as well as biodegradable hydrogel based tooth scaffold. We have measured and optimized the Dental Pulp Stem Cells growth profile in cultures carried out on collagen-coated plastic surface, however, for tissue regeneration study, we aim to develop an enhanced microenvironment for stem cell growth and dental tissue regeneration. We believe biomimetic cell adhesion and scaffolds might provide a near in vivo growth environment for proper growth and differentiation of human DPSCs, which further help in dentin/pulp tissue regeneration.

Keywords: nano-gelatin, stem cells, dental pulp, scaffold

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Authors: Maria Borawska, Patryk Nowakowski, Sylwia K. Naliwajko, Renata Markiewicz-Zukowska, Anna Puscion-Jakubik, Krystyna Gromkowska-Kepka, Justyna Moskwa

Abstract:

Coprinus comatus (O. F. Müll.) Pers.) should not be confused with the common Ink Cap, which contains coprine and can induce coprine poisoning. We study the possibility of applying coprinus mushroom (Coprinus comatus), available in Poland, as food product supporting the treatment of human glioblastoma cells. The U87MG and T98 glioblastoma cell lines were exposed to water (CW) or ethanol 95° (CE) Cantharellus extracts (50-500 μg/ml), with or without temozolomide (TMZ) during 24, 48 or 72 hours. The cell division was examined by the H³-thymidine incorporation. The statistical analysis was performed using Statistica v. 13.0 software. Significant differences were assumed for p < 0.05. We found that both, CW and CE, administrated alone, had inhibitory effect on cell lines growth, but the CE extract had a higher degree of growth inhibition. The anti-tumor effect of TMZ (50 μM) on U87MG was enhanced by mushroom extracts, and the effect was lower to the effect after using Coprinus comatus extracts (CW and CE) alone. A significant decrease (p < 0.05) in pro-MMP2 (82.61 ± 6.3% of control) secretion in U87MG cells was observed after treated with CE (250 μg/ml). We conclude that extracts of Coprinus comatus, edible mushroom, present cytotoxic properties on U87MG and T98 cell lines and may cooperate with TMZ synergistically enhancing its growth inhibiting activity against glioblastoma U87MG cell line.

Keywords: anticancer, glioma, mushroom, temozolomide

Procedia PDF Downloads 167

Authors: Reshu Saxena, R. K. Tripathi

Abstract:

HIV-1 transmission and spread involves significant host-virus interaction. Potential targets for prevention of HIV-1 lies at the site of mucosal barriers. Thus a better understanding of how HIV-1 infects target cells at such sites and lead their invasion is required, with prime focus on the host determinants regulating HIV-1 spread. HIV-1 Nef is important for viral infectivity and pathogenicity. It promotes HIV-1 replication, facilitating immune evasion by interacting with various host factors and altering cellular pathways via multiple protein-protein interactions. In this study nef was sequenced from HIV-1 patients, and showed specific mutations revealing sequence variability in nef. To explore the difference in Nef functionality based on sequence variability we have studied the effects of HIV-1 Nef in human SupT1 T cell line and (THP-1) monocyte-macrophage cell lines through proteomics approach. 2D-Gel Electrophoresis in control and Nef-transfected SupT1 cells demonstrated several differentially expressed proteins with significant modulation of alpha-enolase. Through further studies, effects of Nef on alpha-enolase regulation were found to be cell lineage-specific, being stimulatory in macrophages/monocytes, inhibitory in T cells and without effect in HEK-293 cells. Cell migration and invasion studies were employed to determine biological function affected by Nef mediated regulation of alpha-enolase. Cell invasion was enhanced in THP-1 cells but was inhibited in SupT1 cells by wildtype nef. In addition, the modulation of enolase and cell invasion remained unaffected by a unique nef variant. These results indicated that regulation of alpha-enolase expression and invasive property of host cells by Nef is sequence specific, suggesting involvement of a particular motif of Nef. To precisely determine this site, we designed a heptapeptide including the suggested alpha-enolase regulating sequence of nef and a nef mutant with deletion of this site. Macrophages/monocytes being the major cells affected by HIV-1 at mucosal barriers, were particularly investigated by the nef mutant and peptide. Both the nef mutant and heptapeptide led to inhibition of enhanced enolase expression and increased invasiveness in THP-1 cells. Together, these findings suggest a possible mechanism of host invasion by HIV-1 through Nef mediated regulation of alpha-enolase and identifies a potential therapeutic target for HIV-1 entry at mucosal barriers.

Keywords: HIV-1 Nef, nef variants, host-virus interaction, tissue invasion

Procedia PDF Downloads 384

Authors: Ipsita Biswas, Arnab Sarkar, Ashikur Rahaman, Gopeswar Mukherjee, Subhrangsu Chatterjee, Shamee Bhattacharjee, Deba Prasad Mandal

Abstract:

One of the major reasons for the high mortality rate of lung cancer is the substantial delays in disease detection at late metastatic stages. It is of utmost importance to understand the detailed molecular signaling and detect the molecular markers that can be used for the early diagnosis of cancer. Several studies explored the emerging roles of long noncoding RNAs (lncRNAs) in various cancers as well as lung cancer. A long non-coding RNA LINC00273 was recently discovered to promote cancer cell migration and invasion, and its positive correlation with the pathological stages of metastasis may prove it to be a potential target for inhibiting cancer cell metastasis. Comparing real-time expression of LINC00273 in various human clinical cancer tissue samples with normal tissue samples revealed significantly higher expression in cancer tissues. This long intergenic noncoding RNA was found to be highly expressed in human liver tumor-initiating cells, human gastric adenocarcinoma AGS cell line, as well as human non-small cell lung cancer A549 cell line. SiRNA and shRNA-induced knockdown of LINC00273 in both in vitro and in vivo nude mice significantly subsided AGS and A549 cancer cell migration and invasion. LINC00273 knockdown also reduced TGF-β induced SNAIL, SLUG, VIMENTIN, ZEB1 expression, and metastasis in A549 cells. Plenty of reports have suggested the role of microRNAs of the miR200 family in reversing epithelial to mesenchymal transition (EMT) by inhibiting ZEB transcription factors. In this study, hsa-miR-200a-3p was predicted via IntaRNA-Freiburg RNA tools to be a potential target of LINC00273 with a negative free binding energy of −8.793 kcal/mol, and this interaction was verified as a confirmed target of LINC00273 by RNA pulldown, real-time PCR and luciferase assay. Mechanistically, LINC00273 accelerated TGF-β induced EMT by sponging hsa-miR-200a-3p which in turn liberated ZEB1 and promoted prometastatic functions in A549 cells in vitro as verified by real-time PCR and western blotting. The similar expression patterns of these EMT regulatory pathway molecules, viz. LINC00273, hsa-miR-200a-3p, ZEB1 and TGF-β, were also detected in various clinical samples like breast cancer tissues, oral cancer tissues, lung cancer tissues, etc. Overall, this LINC00273 mediated EMT regulatory signaling can serve as a potential therapeutic target for the prevention of lung cancer metastasis.

Keywords: epithelial to mesenchymal transition, long noncoding RNA, microRNA, non-small-cell lung carcinoma

Procedia PDF Downloads 130

Authors: Venkat Garigapati

Abstract:

Cell-based therapies are limited due to underlying host immune system activity. Microencapsulation of living cells to overcome this issue has some serious drawbacks, such as limitations of nutrient and oxygen diffusion, which pose a threat to the function and longevity of cells. The conformal coating could overcome the issues which are generally involved in traditional microencapsulation. Some of the theoretical advantages of conformal coating include superior nutrient and oxygen supply to cells, prolonged lifespan, improved drug-secreting cell functionality and an opportunity to load high cell doses in small volumes. Despite several advantages to the conformal coating, there are no suitable methods available to apply to living cells. The ultra-thin conformal coating was achieved utilizing click-reactive methacryloyloxyethyl phosphorylcholine (MPC) polymers, which are capable of specifically reacting one polymer to another at neutral pH in the aqueous isotonic system at the desired temperature suitable for living cells without the need of deleterious initiators. ARPE-19 (Adult Retinal Pigment Epithelial cell line-19) cell-spheroids and rat pancreatic islets were used in the formulation studies. The in vitro studies of coated ARPE-19 cell-spheroids and rat islets indicate that the coat was intact; cells were viable and functioning. The in vitro study results revealed that the conformal coating technology seems promising and in vivo studies are being planned.

Keywords: cells, hydrogel, conformal coating, microencapsulation, insulin

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Authors: Muñiz-Lino Marcos Agustín, Vazquez Borbolla Jessica, Licéaga-Escalera Carlos

Abstract:

The ectomesenchymal tissues involved in tooth development and their remnants are the origin of different odontogenic lesions, including tumors and cysts of the jaws, with a wide range of clinical behaviors. Dentigerous cyst (DC) represents approximately 20% of all cases of odontogenic cysts, and it has been demonstrated that it can develop benign and malignant odontogenic tumors. DC is characterized by bone destruction of the area surrounding the crown of a tooth which has not erupted and it contain is liquid. The treatment of odontogenic tumors and cysts usually are partial or total removal of the jaw, causing important secondary co-morbidities. However, molecules implicated in DC pathogenesis as well in its development to odontogenic tumors remains unknown. A cellular model may be useful to study these molecules, but that model has not been established yet. Here, we reported the establishment of a cell culture derived from a dentigerous cyst. This cell line was named DeCy-1. In spite of its ectomesenchymal morphology, DeCy-1 cells express epithelial markers such as cytokeratins 5, 6, and 8. Furthermore, these cells express the ODAM protein, which is present in odontogenesis and in dental follicle, indicating that DeCy-1 cells derived from odontogenic epithelium. Analysis by electron microscopy of this cell line showed that it has a high vesicular activity, suggesting that DeCy-1 could secrete molecules that may be involved in DC pathogenesis. Thus, secreted proteins were analyzed by PAGE-SDS, where we observed approximately 11 bands. In addition, the capacity of these secretions to degrade proteins was analyzed by gelatin substrate zymography. A degradation band of about 62 kDa was found in these assays. Western blot assays suggested that the matrix metalloproteinase 2 (MMP-2) is responsible of this protease activity. Thus, our results indicate that the establishment of a cell line derived from DC is a useful in vitro model to study the biology of this odontogenic lesion and its participation in the development of odontogenic tumors.

Keywords: dentigerous cyst, MMP20, cancer, cell culture

Procedia PDF Downloads 113

Authors: B. Fazekas, I. Korolov, K. Kutasi

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Plasma medicine, which involves the use of gas discharge plasmas for medical applications is a rapidly growing research field. The use of non-thermal atmospheric pressure plasmas in dermatology to assist tissue regeneration by improving the healing of infected and/or chronic wounds is a promising application. It is believed that plasma can activate cells, which are involved in the wound closure. Non-thermal atmospheric plasmas are rich in chemically active species (such as O and N-atoms, O2(a) molecules) and radiative species such as the NO, N2+ and N2 excited molecules, which dominantly radiate in the 200-500 nm spectral range. In order to understand the effect of plasma species, both of chemically active and radiative species on wound healing process, the interaction of physical plasma with the human skin cells is necessary. In order to clarify the effect of plasma radiation on the wound healing process we treated keratinocyte cells – that are one of the main cell types in human skin epidermis – covered with a layer of phosphate-buffered saline (PBS) with a low power atmospheric pressure plasma. For the generation of such plasma we have applied a plasma needle. Here, the plasma is ignited at the tip of the needle in flowing helium gas in contact with the ambient air. To study the effect of plasma radiation we used a plasma needle configuration, where the plasma species – chemically active radicals and charged species – could not reach the treated cells, but only the radiation. For the comparison purposes, we also irradiated the cells using a UV-B light source (FS20 lamp) with a 20 and 40 mJ cm-2 dose of 312 nm. After treatment the viability and the proliferation of the cells have been examined. The proliferation of cells has been studied with a real time monitoring system called Xcelligence. The results have indicated, that the 20 mJ cm-2 dose did not affect cell viability, whereas the 40 mJ cm-2 dose resulted a decrease in cell viability. The results have shown that the plasma radiation have no quantifiable effect on the cell proliferation as compared to the non-treated cells.

Keywords: UV radiation, non-equilibrium gas discharges (non-thermal plasmas), plasma emission, keratinocyte cells

Procedia PDF Downloads 579

Authors: R. Stalin, D. Karthick, H. Haseena Banu, T. P. Sachidanandam, P. Shanthi

Abstract:

Background: Breast cancer is emerging as one of the leading cause of cancer related deaths and hence there arises the need to look out for drugs which are more targets specific with minimal side effects. In recent times, there is a shift towards alternative medicine due to low cost and less side effects. Siddha system of medicine is one the oldest system of medicine practiced against various ailments. Tridham (TD) is a herbal formulation prepared in our laboratory consisting of Terminalia chebula, Elaeocarpus ganitrus and Prosopis cineraria in a definite ratio (TD) and its anticancer potential is evaluated in terms of induction of apoptosis. Objective: The present study was designed to investigate the anti proliferative effect of TD and 1,2,3,4,6-penta-O-galloyl-b-D-glucose (PGG), a pure compound isolated from TD on human mammary carcinoma cell line (MCF-7). Materials and Methods: Cell viability was studied using MTT analysis and trypan blue staining. Mitochondrial membrane potential was studied using DAPI staining. The protein and mRNA expressions of pro-apoptotic and anti- apoptotic markers namely Bax, Bad, Bcl-2 and caspases were also assessed by Western Blotting and RT PCR. Results: Viability studies of TD and PGG treated MCF-7 cells showed an inhibition in cell growth in time and dose dependent manner. The alteration in mitochondrial membrane potential was restored through treatment with TD and PGG which was confirmed by DAPI staining. The protein and mRNA expression of pro-apoptotic markers was found to be significantly increased in TD and PGG treated cells with a concomitant decrease in anti-apoptotic markers. Conclusion: The results of the study suggest that TD and PGG exhibit their anticancer effect through its membrane stabilizing property and activation of apoptotic cascade in MCF-7 cells.

Keywords: apoptosis, mammary carcinoma, MCF-7, penta galloyl glucose, Tridham

Procedia PDF Downloads 286

Authors: Halima Albalushi, Mohadese Boroojerdi, Murtadha Alkhabori

Abstract:

Introduction: Maintenance of stem cell properties during culture necessitates the recreation of the natural cell niche. Studies reported the promising outcome of mesenchymal stem cells (MSC) properties maintenance after using extracellular matrix such as CELLstart™, which is the recommended coating material for stem cells cultured in serum-free and xeno-free conditions. Laminin-521 is known as a crucial adhesion protein, which is found in natural stem cell niche, and plays an important role in facilitating the maintenance of self-renewal, pluripotency, standard morphology, and karyotype of human pluripotent stem cells (PSCs). The aim of this study is to investigate the effects of Laminin-521 on human umbilical cord-derived mesenchymal stem cells (UC-MSC) characteristics as a step toward clinical application. Methods: Human MSC were isolated from the umbilical cord via the explant method. Umbilical cord-derived-MSC were cultured in serum-free and xeno-free conditions in the presence of Laminin-521 for six passages. Cultured cells were evaluated by morphology and expansion index for each passage. Phenotypic characterization of UC-MSCs cultured on Laminin-521 was evaluated by assessment of cell surface markers. Results: Umbilical cord derived-MSCs formed small colonies and expanded as a homogeneous monolayer when cultured on Laminin-521. Umbilical cord derived-MSCs reached confluence after 4 days in culture. No statistically significant difference was detected in all passages when comparing the expansion index of UC-MSCs cultured on LN-521 and CELLstart™. Phenotypic characterization of UC-MSCs cultured on LN-521 using flow cytometry revealed positive expression of CD73, CD90, CD105 and negative expression of CD34, CD45, CD19, CD14 and HLA-DR.Conclusion: Laminin-521 is comparable to CELLstart™ in supporting UC-MSCs expansion and maintaining their characteristics during culture in xeno-free and serum-free culture conditions.

Keywords: mesenchymal stem cells, culture, laminin-521, xeno-free serum-free

Procedia PDF Downloads 45