Search results for: in vitro cell line study

Authors: Adil Elrayah, Jie Weng, Esra Suliman

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The mineral in human bone is not pure stoichiometric calcium phosphate (Ca/P) as it is partially substituted by in organic elements. In this study, the copper ions (Cu²⁺) substituted hydroxyapatite (CuHA) powder has been synthesized by the co-precipitation method. The CuHA powder has been used to fabricate CuHA fiber scaffolds by sol-gel process and the following sinter process. The resulted CuHA fibers have slightly different microstructure (i.e. porosity) compared to HA fiber scaffold, which is denser. The mechanical properties test was used to evaluate CuHA, and the results showed decreases in both compression strength and hardness tests. Moreover, the in vitro used endothelial cells to evaluate the angiogenesis of CuHA. The result illustrated that the viability of endothelial cell on CuHA fiber scaffold surfaces tends to antigenic behavior. The results obtained with CuHA scaffold give this material benefit in biological applications such as antimicrobial, antitumor, antigens, compacts, filling cavities of the tooth and for the deposition of metal implants anti-tumor, anti-cancer, bone filler, and scaffold.

Keywords: fiber scaffold, copper ions, hydroxyapatite, in vitro, mechanical property

Procedia PDF Downloads 129

Authors: Tomás Sobrino, Lara García-Varela, Marta Aramburu-Núñez, Mónica Castro, Noemí Gómez-Lado, Mariña Rodríguez-Arrizabalaga, Antía Custodia, Juan Manuel Pías-Peleteiro, José Manuel Aldrey, Daniel Romaus-Sanjurjo, Ángeles Almeida, Pablo Aguiar, Alberto Ouro

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Introduction: Alzheimer’s disease (AD) and other tauopathies are primary causes of dementia, causing progressive cognitive deterioration that entails serious repercussions for the patients' performance of daily tasks. Currently, there is no effective approach for the early diagnosis and treatment of AD and tauopathies. This study suggests a theragnostic approach based on the importance of phosphorylated tau protein (p-Tau) in the early pathophysiological processes of AD. We have developed a novel theragnostic monoclonal antibody (mAb) to provide both diagnostic and therapeutic effects. Methods/Results: We have developed a p-Tau mAb, which was doped with deferoxamine for radiolabeling with Zirconium-89 (89Zr) for PET imaging, as well as fluorescence dies for immunofluorescence assays. The p-Tau mAb was evaluated in vitro for toxicity by MTT assay, LDH activity, propidium iodide/Annexin V assay, caspase-3, and mitochondrial membrane potential (MMP) assay in both mouse endothelial cell line (bEnd.3) and cortical primary neurons cell cultures. Importantly, non-toxic effects (up to concentrations of p-Tau mAb greater than 100 ug/mL) were detected. In vivo experiments in the tauopathy model mice (PS19) show that the 89Zr-pTau-mAb and 89Zr-Fragments-pTau-mAb are stable in circulation for up to 10 days without toxic effects. However, only less than 0.2% reached the brain, so further strategies have to be designed for crossing the Brain-Blood-Barrier (BBB). Moreover, an intraparenchymal treatment strategy was carried out. The PS19 mice were operated to implement osmotic pumps (Alzet 1004) at two different times, at 4 and 7 months, to stimulate the controlled release for one month each of the B6 antibody or the IgG1 control antibody. We demonstrated that B6-treated mice maintained their motor and memory abilities significantly compared with IgG1 treatment. In addition, we observed a significant reduction in p-Tau deposits in the brain. Conclusions /Discussion: A theragnostic pTau-mAb was developed. Moreover, we demonstrated that our p-Tau mAb recognizes very-early pathology forms of p-Tau by non-invasive techniques, such as PET. In addition, p-Tau mAb has non-toxic effects, both in vitro and in vivo. Although the p-Tau mAb is stable in circulation, only 0.2% achieve the brain. However, direct intraventricular treatment significantly reduces cognitive impairment in Alzheimer's animal models, as well as the accumulation of toxic p-Tau species.

Keywords: alzheimer's disease, theragnosis, tau, PET, immunotherapy, tauopathies

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Authors: Hyo-Min Kim, Seokjin Ham, Mi-Joung Yoo, Minseon Kim, Tae-Young Roh

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The gastrointestinal (GI) tract of most animals, including murine, is highly compartmentalized epithelia which also provide distinct different functions of its own tissue. Nevertheless, these epithelia share certain characteristics that enhance immune responses to infections and maintain the barrier function of the intestine. GI tract epithelia also undergo regeneration not only in homeostatic conditions but also in a response to the damage. A full turnover of the murine gastrointestinal epithelium occurs every 4-5 day, a process that is regulated and maintained by a minor population of Lgr5+ adult stem cell that commonly conserved in the bottom of crypts through GI tract. Maintenance of the stem cell is somehow regulated by epigenetic factors according to recent studies. Chromatin vacancy, remodelers, histone variants and histone modifiers could affect adult stem cell fate. In this study, Lgr5-EGFP reporter mouse was used to take advantage of exploring the epigenetic dynamics among Lgr5 positive mutual stem cell in GI tract. Cells were isolated by fluorescence-activated cell sorting (FACS), gene expression levels, chromatin accessibility changes and histone modifications were analyzed. Some notable chromatin structural related epigenetic variants were detected. To identify the overall cell-cell interaction inside the stem cell niche, an extensive genome-wide analysis should be also followed. According to the results, nevertheless, we expected a broader understanding of cellular niche maintaining stem cells and epigenetic barriers through conserved stem cell in GI tract. We expect that our study could provide more evidence of adult stem cell plasticity and more chances to understand each stem cell that takes parts in certain organs.

Keywords: adult stem cell, epigenetics, LGR5 stem cell, gastrointestinal tract

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Authors: Mahboubeh Davoudi Pahnekolayi

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Begonia rex cv. DS-EYWA is a rare, unique cultivar of begonia rex with curly colorful leaves. Optimization of an in vitro efficient regeneration protocol by focusing on transverse Thin Cell Layer (tTCL) petiole explants for high-scale production of such a beautiful cultivar was considered as our main purpose in this experiment. Thus, various concentrations of Plant Growth Regulators (PGRs) including 6-Benzylaminopurine (BAP), Thidiazuron (TDY), and –Naphthaleneacetic Acid (NAA), were selected in a Completely Randomized Design (CRD) to establish and optimize the direct organogenesis efficiency of this cultivar. Cultivation of 1 mm tTCL petiole explants in noted treatments showed that 1.5 mgl-1 BAP + 0.5 mgl-1 NAA can induce the highest number of direct regenerated shoots and lower concentration of BAP (0.5 mgl-1) can be suggested for shoot elongation before rooting stage. Elongated shoots were successfully rooted in MS free basal medium and acclimatized in 1:1 peat moss: perlite sterilized pot mixture.

Keywords: begonia rare cultivar, direct organogenesis, explant type, regeneration, thin cell layering (TCL)

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Authors: Mala Nath, Nagamani Kompelli, Partha Roy, Snehasish Das

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Two new metal-based anticancer chemotherapeutic agents, [(Ph2Sn)2(HGuO)2(phen)Cl2] 1 and [(Ph3Sn)(HGuO)(phen)]- Cl.CH3OH.H2O 2, were designed, prepared and characterized by analytical and spectral (IR, ESI-Mass, 1H, 13C and 119Sn NMR) techniques. The proposed geometry of Sn(IV) in 1 and 2 is distorted octahedral and distorted trigonal-bipyramidal, respectively. Both 1 and 2 exhibit potential cytotoxicity in vitro against MCF-7, HepG-2 and DU-145 cell lines. The intrinsic binding constant (Kb) values of 1 (2.33 × 105 M-1) and 2 (2.46 × 105 M-1) evaluated from UV-Visible absorption studies suggest non-classical electrostatic mode of interaction via phosphate backbone of DNA double helix. The Stern-Volmer quenching constant (Ksv) of 1 (9.74 × 105 M-1) and 2 (2.9 × 106 M-1) determined by fluorescence studies suggests the groove binding and intercalation mode for 1 and 2, respectively. Effective cleavage of pBR322 DNA is induced by 1. Their interaction with DNA of cancer cells may account for potency.

Keywords: anticancer agents, DNA binding studies, NMR spectroscopy, organotin

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Authors: R. L. Shirole, N. L. Shirole, R. B. Patil, M. N. Saraf

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The present investigation aimed to elucidate the probable mechanism of antiasthmatic action of essential oil of Pistacia integerrima J.L. Stewart ex Brandis galls (EOPI). EOPI was investigated for its potential antiasthmatic action using in vitro antiallergic assays mast cell degranulation and soyabean lipoxidase enzyme activit, and spasmolytic action using isolated guinea pig ileum preparation. In vivo studies included lipopolysaccharide-induced bronchial inflammation in rats and airway hyperresponsiveness in ovalbumin in sensitized guinea pigs using spirometry. Data was analysed by GraphPad Prism 5.01 and results were expressed as means ± SEM. P < 0.05 was considered to be significant. EOPI inhibits 5-lipoxidase enzyme activity, DPPH scavenging activity and erythropoietin- induced angiogenesis. It showed dose dependent anti-allergic activity by inhibiting compound 48/80 induced mast cell degranulation. The finding that essential oil induced inhibition of transient contraction of acetylcholine in calcium free medium, and relaxation of S-(-)-Bay 8644-precontracted isolated guinea pig ileum jointly suggest that suggesting that the L-subtype Cav channel is involved in spasmolytic action of EOPI. Treatment with EOPI dose dependently (7.5, 15 and 30 mg/kg i.p.) inhibited lipopolysaccharide- induced increased in total cell count, neutrophil count, nitrate-nitrite, total protein, albumin levels in bronchoalveolar fluid and myeloperoxidase levels in lung homogenates. Mild diffused lesions involving focal interalveolar septal, intraluminal infiltration of neutrophils were observed in EOPI (7.5 &15 mg/kg) pretreated while no abnormality was detected in EOPI (30 mg/kg) and roflumilast (1mg/kg) pretreated rats. Roflumilast was used as standard. EOPI reduced the respiratory flow due to gasping in ovalbumin sensitized guinea pigs. The study demonstrates the effectiveness of EOPI in bronchial asthma possibly related to its ability to inhibit L-subtype Cav channel, mast cell stabilization, antioxidant, angiostatic and through inhibition of 5-lipoxygenase enzyme.

Keywords: asthma, lipopolysaccharide, spirometry, Pistacia integerrima J.L. Stewart ex Brandis, essential oil

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Authors: Nima Samie, Sekaran Muniandy, Zahurin Mohamed, M. S. Kanthimathi

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Although number of indole containing compounds have been reported to have anticancer properties in vitro but only a few of them show potential as anticancer compounds in vivo. The current study was to evaluate the mechanism of cytotoxicity of selected indolic compound in vivo and in vitro. In this context, we determined the potency of the compound in the induction of apoptosis, cell cycle arrest, and cytoskeleton rearrangement. HT-29, WiDr, CCD-18Co, human monocyte/macrophage CRL-9855, and B lymphocyte CCL-156 cell lines were used to determine the IC50 of the compound using the MTT assay. Analysis of apoptosis was carried out using immunofluorescence, acridine orange/ propidium iodide double staining, Annexin-V-FITC assay, evaluation of the translocation of NF-kB, oxygen radical antioxidant capacity, quenching of reactive oxygen species content, measurement of LDH release, caspase-3/-7, -8 and -9 assays and western blotting. The cell cycle arrest was examined using flowcytometry and gene expression was assessed using qPCR array. Results displayed a potent suppressive effect on HT-29 and WiDr after 24 h of treatment with IC50 value of 2.52±0.34 µg/ml and 2.13±0.65 µg/ml respectively. This cytotoxic effect on normal, monocyte/macrophage and B-cells was insignificant. Dipping in the mitochondrial membrane potential and increased release of cytochrome c from the mitochondria indicated induction of the intrinsic apoptosis pathway by the compound. Activation of this pathway was further evidenced by significant activation of caspase-9 and 3/7. The compound was also shown to activate the extrinsic pathways of apoptosis via activation of caspase-8 which is linked to the suppression of NF-kB translocation to the nucleus. Cell cycle arrest in the G1 phase and up-regulation of glutathione reductase, based on excessive ROS production were also observed. These findings were further investigated for inhibitory efficiency of the compound on colonic aberrant crypt foci in male rats. Rats were divided in to 5 groups: vehicle, cancer control, positive control groups and the groups treated with 25 and 50 mg/kg of compounds for 10 weeks. Administration of compound suppressed total colonic ACF formation up to 73.4%. The results also showed that treatment with the compound significantly reduced the level of malondialdehyde while increasing superoxide dismutase and catalase activities. Furthermore, the down-regulation of PCNA and Bcl2 and the up-regulation of Bax was confirmed by immunohistochemical staining. The outcome of this study suggest sthat the indolic compound is a potent anti-cancer agent against colon cancer and can be further evaluated by animal trial.

Keywords: indolic compound, chemoprevention, crypt, azoxymethane, colon cancer

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Authors: Avadhesh Kumar

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Background: The cell phone has rapidly become an integral, and, for some, an essential communication tool that is being used worldwide. Their use without any knowledge of their harmful effects like cancers and other health effects is not ‘quite’ safe. Studies on cancers due to electromagnetic radiations from cell phones are available, but there is a need to research on the detrimental physical and psychological effects on users like students. This study focused on certain psychological or mental health effects of cell phone usage amongst students. Materials and methods: The present study will be carried out on all the students of Banaras Hindu University. Students of both sexes from urban and rural backgrounds were selected at random and administered a pre- tested questionnaire which included aspects related to few common adverse psychological health signs and symptoms attributed to cell phone over-usage. Results: Stress was found to be the commonest symptom (51.47%) followed by irritability/anger (43.79%). Other common mental symptoms included lack of concentration and academic performance, insomnia, anxiety etc. Suggestions: This study confirms that the younger generation, who are the most frequent cell phone users, needs to be aware of the adverse health effects of cell phone usage especially the mental aspects and take preventive measures to minimize and control the same. Less dependence on the device, a curtailing time period spent on talking, communicating more by texting, etc. are some of the practical measures suggested.

Keywords: cell phones, psychological health effects, students, mental health

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Authors: Chengcao Sun, Shujun Li, Cuili Yang, Yongyong Xi, Liang Wang, Feng Zhang, Dejia Li

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Recently, the long non-coding RNA (lncRNA) NEAT1 has been identified as an oncogenic gene in multiple cancer types and elevated expression of NEAT1 was tightly linked to tumorigenesis and cancer progression. However, the molecular basis for this observation has not been characterized in progression of non-small cell lung cancer (NSCLC). In our studies, we identified NEAT1 was highly expressed in NSCLC patients and was a novel regulator of NSCLC progression. Patients whose tumors had high NEAT1 expression had a shorter overall survival than patients whose tumors had low NEAT1 expression. Further, NEAT1 significantly accelerates NSCLC cell growth and metastasis in vitro and tumor growth in vivo. Additionally, by using bioinformatics study and RNA pull down combined with luciferase reporter assays, we demonstrated that NEAT1 functioned as a competing endogenous RNA (ceRNA) for has-miR-377-3p, antagonized its functions and led to the de-repression of its endogenous targets E2F3, which was a core oncogene in promoting NSCLC progression. Taken together, these observations imply that the NEAT1 modulated the expression of E2F3 gene by acting as a competing endogenous RNA, which may build up the missing link between the regulatory miRNA network and NSCLC progression.

Keywords: long non-coding RNA NEAT1, hsa-miRNA-377-3p, E2F3, non-small cell lung cancer, tumorigenesis

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Authors: H. Jeries, N. Volkova, C. G. Iglesias, M. Najjar, M. Rosenblat, M. Aviram, T. Hayek

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Background: Synthetic forms of glucocorticoids (e.g., prednisone, prednisolone) are anti-inflammatory drugs which are widely used in clinical practice. The role of glucocorticoids (GCs) in cardiovascular diseases including atherosclerosis is highly controversial, and their impact on macrophage foam cell formation is still unknown. Our aim was to investigate the effects of prednisone or its active metabolite, prednisolone, on macrophage oxidative stress and lipid metabolism using in-vivo, ex-vivo and in-vitro systems. Methods: The in-vivo study included C57BL/6 mice which were intraperitoneally injected with prednisone or prednisolone (5mg/kg) for 4 weeks, followed by lipid metabolism analyses in the mice aorta, and in peritoneal macrophages (MPM). In the ex-vivo study, we analyzed the effect of serum samples obtained from 9 healthy volunteers before or after treatment with oral prednisone (20mg for 5 days), on J774A.1 macrophage atherogenicity. In-vitro studies were conducted using J774A.1 macrophages, human monocyte derived macrophages (HMDM) and fibroblasts. Cells were incubated with increasing concentrations (0-200 ng/ml) of prednisone or prednisolone, followed by determination of cellular oxidative status, triglyceride and cholesterol metabolism. Results: Prednisone or prednisolone treatment resulted in a significant reduction in triglycerides and mainly in cholesterol cellular accumulation in MPM or in J774A.1 macrophages incubated with human serum. Similar resulted were noted in HMDM or in J774A.1 macrophages which were directly incubated with the GCs. These effects were associated with GCs inhibitory effect on triglycerides and cholesterol biosynthesis rates, throughout downregulation of diacylglycerol acyltransferase1 (DGAT1) expression, and of the sterol regulatory element binding protein (SREBP2) and HMGCR expression, respectively. In parallel to prednisone or prednisolone induced reduction in macrophage triglyceride content, paraoxonase 2 (PON2) expression was significantly upregulated. GCs-induced reduction of cellular triglyceride and cholesterol mass was mediated by the GCs receptors on macrophages since the GCs receptor antagonist (RU 486) abolished these effects. In fibroblasts, unlike macrophages, prednisone or prednisolone showed no anti-atherogenic effects. Conclusions: Prednisone or prednisolone are anti-atherogenic since they protected macrophages from lipid accumulation and foam cell formation.

Keywords: atherosclerosis, cholesterol, foam cell, macrophage, prednisone, prednisolone, triglycerides

Procedia PDF Downloads 118

Authors: Hatairuk Tungkasen, Somrudee Phetchrid, Suwapat Jaidee, Supinya Yoomak, Chantana Kankamol, Mayuree Pumipaiboon, Mayuva Areekijseree

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Ovaries of reproductive female pigs were obtained from local slaughterhouses in Nakorn Pathom Province, Thailand. Follicular fluid of medium follicle (5-6 diameters) and large follicles (7-8 mm and 10 mm in diameter) were aspirated and collected by sterile technique and analyzed protein pattern. The follicular fluid protein bands were found by SDS-PAGE which has no protein band in difference compared to standard protein band. So we chose protein band molecular weight 50, 62-65, 75-80, 90, 120-160, and >220 kDa were analyzed by LC/MS/MS. The result was found immunoglobulin gamma chain, keratin, transferrin, heat shock protein, and plasminogen precursor, ceruloplasmin, and hemopexin, and protease, respectively. All proteins play important roles in promotion and regulation on growth and development of reproductive cells. The result of this study found many proteins which were useful and important for in vitro oocyte maturation and embryonic development of cell technology in animals. The further study will be use porcine follicular fluid protein of medium and large follicles as feeder cells in in vitro condition to promote oocyte and embryo maturation.

Keywords: follicular fluid protein, LC/MS/MS, porcine oocyte, SDS-PAGE

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Authors: Valeria Arcucci, Musarat Ishaq, Steven A. Stacker, Greg J. Goodall, Marc G. Achen

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Metastasis is the lethal aspect of cancer for most patients. Remodelling of lymphatic vessels associated with a tumour is a key initial step in metastasis because it facilitates the entry of cancer cells into the lymphatic vasculature and their spread to lymph nodes and distant organs. Although it is clear that vascular endothelial growth factors (VEGFs), such as VEGF-C and VEGF-D, are key drivers of lymphatic remodelling, the means by which many signaling pathways in endothelial cells are coordinately regulated to drive growth and remodelling of lymphatics in cancer is not understood. We seek to understand the broader molecular mechanisms that control cancer metastasis, and are focusing on microRNAs, which coordinately regulate signaling pathways involved in complex biological responses in health and disease. Here, using small RNA sequencing, we found that a specific microRNA, miR-132, is upregulated in expression in lymphatic endothelial cells (LECs) in response to the lymphangiogenic growth factors. Interestingly, ectopic expression of miR-132 in LECs in vitro stimulated proliferation and tube formation of these cells. Moreover, miR-132 is expressed in lymphatic vessels of a subset of human breast tumours which were previously found to express high levels of VEGF-D by immunohistochemical analysis on tumour tissue microarrays. In order to dissect the complexity of regulation by miR-132 in lymphatic biology, we performed Argonaute HITS-CLIP, which led us to identify the miR-132-mRNA interactome in LECs. We found that this microRNA in LECs is involved in the control of many different pathways mainly involved in cell proliferation and regulation of the extracellular matrix and cell-cell junctions. We are now exploring the functional significance of miR-132 targets in the biology of LECs using biochemical techniques, functional in vitro cell assays and in vivo lymphangiogenesis assays. This project will ultimately define the molecular regulation of lymphatic remodelling by miR-132, and thereby identify potential therapeutic targets for drugs designed to restrict the growth and remodelling of tumour lymphatics resulting in metastatic spread.

Keywords: argonaute HITS-CLIP, cancer, lymphatic remodelling, miR-132, VEGF

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Authors: Pamita Awasthi, Kirna, Shilpa Dogra, Manu Vatsal, Ritu Barthwal

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Doxorubicin, also known as adriamycin, is an anthracycline class of drug used in cancer chemotherapy. It is used in the treatment of non-Hodgkin’s lymphoma, multiple myeloma, acute leukemias, breast cancer, lung cancer, endometrium cancer and ovary cancers. It functions via intercalating DNA and ultimately killing cancer cells. The major side effects of doxorubicin are hair loss, myelosuppression, nausea & vomiting, oesophagitis, diarrhoea, heart damage and liver dysfunction. The minor modifications in the structure of compound exhibit large variation in the biological activity, has prompted us to carry out the synthesis of sulfonamide derivatives. Sulfonamide is an important feature with broad spectrum of biological activity such as antiviral, antifungal, diuretics, anti-inflammatory, antibacterial and anticancer activities. Structure of the synthesized compound N-(1-methyl-2-oxo-2-N-methyl anilino-ethyl)benzene sulfonamide confirmed by proton nuclear magnetic resonance (1H NMR),13C NMR, Mass and FTIR spectroscopic tools to assure the position of all protons and hence stereochemistry of the molecule. Further we have reported the binding potential of synthesized sulfonamide analogues in comparison to doxorubicin drug using Auto Dock 4.2 software. Computational binding energy (B.E.) and inhibitory constant (Ki) has been evaluated for the synthesized compound in comparison of doxorubicin against Poly (dA-dT).Poly (dA-dT) and Poly (dG-dC).Poly (dG-dC) sequences. The in vitro cytotoxic study against human breast cancer cell lines confirms the better anticancer activity of the synthesized compound over currently in use anticancer drug doxorubicin. The IC50 value of the synthesized compound is 7.12 µM where as for doxorubicin is 7.2 µ.

Keywords: Doxorubicin, auto dock, in silco, in vitro

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Authors: Noor Shafifiyaz Mohd Yazid, Najihah Mohd Hashim, Hapipah Mohd Ali, Syam Mohan, Rosea Go

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Artocarpus kemando is a plant species from Moraceae family. This plant is used as household utensil by the local and the fruits are edible. The plants’ bark was used for the extraction process and yielded the chloroform crude extract which was used to screen for anticancer potential. The cytotoxic effect of the extract on CAOV-3 and WRL 68 cell lines were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT assays. Qualitative AO/PI assay was performed to confirm the apoptosis and necrosis process. Meanwhile, the measurement of cell loss, nuclear morphology, DNA content, cell membrane permeability, mitochondrial membrane potential changes and cytochrome c release from mitochondria were detected through cytotoxicity 3 assay. In MTT assay, A. kemando inhibited 50% growth of CAOV-3 cells at 27.9 ± 0:03, 20.1± 0:03, 18.21± 0:04 µg/mL after 24, 48 and 72 hour, respectively. The morphology changes can be seen on CAOV-3 with a production of cell membrane blebbing, cromatin condensation and apoptotic bodies. Evaluation of cytotoxicity 3 on CAOV-3 cells after treated with extract resulting loss of mitochondrial membrane potential and release of cytochrome c from mitochondria. The results demonstrated A. kemando has potentially anticancer agent, particularly on human ovarian cancer.

Keywords: anticancer, Artocarpus kemando, ovarian cancer, cytotoxicity

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Authors: Esra Cansever Mutlu, Muge Sennaroglu Bostan, Fatemeh Bahadori, Ebru Toksoy Oner, Mehmet S. Eroglu

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Paclitaxel is used in treatment of different cancer types mainly breast, ovarian, lung and Kaposi’s sarcoma. It is poorly soluble in water; therefore, currently used formulations tremendously show side-effects and high toxicity. Encapsulation of the drug in a nano drug carrier which causes both reducing side effects and increasing drug activity is a desired new approach for the nano-medicine to target the site of cancer. In this study, synthesis of a novel nano paclitaxel formulation made of a new amphiphilic monomer was followed by the investigation of its pharmacological properties. UV radical polymerization was carried out by using the monomer Lecithin-2-Acrylamido-2-methylpropane (L-AMPS) and the drug-spacer, to obtain sterically high stabilized, biocompatible and biodegradable phospholipid nanoparticles, in which the drug paclitaxel (Pxl) was encapsulated (NanoPxl). Particles showed high drug loading capacity (68%) and also hydrodynamic size less than 200 nm with slight negative surface charge. The drug release profile was obtained and in vitro cytotoxicity test was performed on MCF-7 cell line. Consequently, these data indicated that paclitaxel loaded Lecithin-AMPS/PCL-MAC nanoparticles can be considered as a new, safe and effective nanocarrier for the treatment of breast cancer.

Keywords: paclitaxel, nanoparticle, drug delivery, L-AMPS

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Authors: Jienny Lee, In-Soo Cho, Sang-Ho Cha

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Adult mesenchymal stem cells (MSCs) have been investigated using preclinical approaches for tissue regeneration. Porcine MSCs (pMSCs) are capable of growing and attaching to plastic with a fibroblast-like morphology and then differentiating into bone, adipose, and cartilage tissues in vitro. This study was conducted to investigate the proliferating abilities, differentiation potentials, and multipotency of miniature pig adipose tissue-derived MSCs (mpAD-MSCs) with or without long-term cryopreservation, considering that cryostorage has the potential for use in clinical applications. After confirming the characteristics of the mpAD-MSCs, we examined the effect of long-term cryopreservation (> 2 years) on expression of cell surface markers (CD34, CD90 and CD105), proliferating abilities (cumulative population doubling level, doubling time, colony-forming unit, and MTT assay) and differentiation potentials into mesodermal cell lineages. As a result, the expression of cell surface markers is similar between thawed and fresh mpAD-MSCs. However, long-term cryopreservation significantly lowered the differentiation potentials (adipogenic, chondrogenic, and osteogenic) of mpAD-MSCs. When compared with fresh mpAD-MSCs, thawed mpAD-MSCs exhibited lower expression of mesodermal cell lineage-related genes such as peroxisome proliferator-activated receptor-g2, lipoprotein lipase, collagen Type II alpha 1, osteonectin, and osteocalcin. Interestingly, long-term cryostoraged mpAD-MSCs exhibited significantly higher cell viability than the fresh mpAD-MSCs. Long-term cryopreservation induced a 30% increase in the cell viability of mpAD-MSCs when compared with the fresh mpAD-MSCs at 5 days after thawing. However, long-term cryopreservation significantly lowered expression of stemness markers such as Oct3/4, Sox2, and Nanog. Furthermore, long-term cryopreservation negatively affected expression of senescence-associated genes such as telomerase reverse transcriptase and heat shock protein 90 of mpAD-MSCs when compared with the fresh mpAD-MSCs. The results from this study might be important for the successful application of MSCs in clinical trials after long-term cryopreservation.

Keywords: mesenchymal stem cells, cryopreservation, stemness, senescence

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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: Zelikha Labbani

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Since its creation in 1964 by Guha and Maheshwari in India on Datura innoxia Mill, in vitro androgenesis has become the method of choice in the production of doubled haploid in many species. However, in durum wheat, the Doubled haploid plant breeding programs remained limited due to the low production of androgenetic embryos and converting them into fertile green plants. We describe here an efficient method for inducing embryos and regenerating green plants directly from isolated microspores of durum wheat.

Keywords: durum wheat, haploid embryos, on in vitro, pretreatment

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Authors: Van Huong Do, Simon Walton, German Amaya, Madeline Batsiokis, Sally Catt, Andrew Taylor-Robinson

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Identification of the most suitable stages of bovine in vitro-derived blastocysts (early, expanded and hatching) prior to vitrification is a straightforward process that facilitates the decision as to which blastocyst stage to use for transfer of fresh and vitrified embryos. Research on in vitro evaluation of suitable stages has shown that the more advanced developmental stage of blastocysts is recommended for fresh embryo transfer while the earlier stage is proposed for embryo transfer following vitrification. There is, however, limited information on blastocyst stages using in vivo assessment. Hence, the aim of the present study was to determine the optimal stage of a blastocyst for vitrification and embryo transfer through a two-step procedure of embryo transfer followed by pregnancy testing at 35, 60 and 90 days of pregnancy. 410 good quality oocytes aspirated by the ovum pick-up technique from 8 donor cows were subjected to in vitro embryo production, vitrification and embryo transfer. Good quality embryos were selected, subjected to vitrification and embryo transfer. Subsequently, 77 vitrified embryos at different blastocyst stages were transferred to synchronised recipient cows. The overall cleavage and blastocyst rates of oocytes were 68.8% and 41.7%, respectively. In addition, the fertility and blastocyst production of 6 bulls used for in vitro fertilization was examined and shown to be statistically different (P<0.05). Results of ongoing pregnancy trials conducted at 35 days, 60 days and 90 days will be discussed. However, preliminary data indicate that individual bulls demonstrate distinctly different fertility performance in vitro. Findings from conception rates would provide a useful tool to aid selection of bovine in vitro-derived embryos for vitrification and embryo transfer in commercial settings.

Keywords: blastocyst, embryo transfer, in vitro-derived embryos, ovum pick-up, vitrification

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Authors: Qazi Salman Khalid, Muhammad Khalid, Shahid Maqsood

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This paper presents a solution for optimizing the cycle time in an assembly line with human-robot collaboration and diverse operators. A genetic algorithm with tailored parameters is used to address the assembly line balancing problem in the automobile sector. A mathematical model is developed, depicting the problem. Currently, the firm runs on the largest candidate rule; however, it causes a lag in orders, which ultimately gets penalized. The results of the study show that the proposed GA is effective in providing efficient solutions and that the cycle time has significantly impacted productivity.

Keywords: line balancing, cycle time, genetic algorithm, productivity

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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: Yuanyuan Zhang, Yujuan Zheng, Giuseppina Barutello, Sumako Kameishi, Kungchun Chiu, Katharina Hennig, Martial Balland, Federica Cavallo, Lars Holmgren

Abstract:

Angiogenesis describes that new blood vessels migrate from pre-existing ones to form 3D lumenized structure and remodeling. During directional migration toward the gradient of pro-angiogenic factors, the endothelial cells, especially the tip cells need filopodia to sense the environment and exert the pulling force. Of particular interest are the integrin proteins, which play an essential role in focal adhesion in the connection between migrating cells and extracellular matrix (ECM). Understanding how these biomechanical complexes orchestrate intrinsic and extrinsic forces is important for our understanding of the underlying mechanisms driving angiogenesis. We have previously identified Angiomotin (Amot), a member of Amot scaffold protein family, as a promoter for endothelial cell migration in vitro and zebrafish models. Hence, we established inducible endothelial-specific Amot knock-out mice to study normal retinal angiogenesis as well as tumor angiogenesis. We found that the migration ratio of the blood vessel network to the edge was significantly decreased in Amotec- retinas at postnatal day 6 (P6). While almost all the Amot defect tip cells lost migration advantages at P7. In consistence with the dramatic morphology defect of tip cells, there was a non-autonomous defect in astrocytes, as well as the disorganized fibronectin expression pattern correspondingly in migration front. Furthermore, the growth of transplanted LLC tumor was inhibited in Amot knockout mice due to fewer vasculature involved. By using MMTV-PyMT transgenic mouse model, there was a significantly longer period before tumors arised when Amot was specifically knocked out in blood vessels. In vitro evidence showed that Amot binded to beta-actin, Integrin beta 1 (ITGB1), Fibronectin, FAK, Vinculin, major focal adhesion molecules, and ITGB1 and stress fibers were distinctly induced by Amot transfection. Via traction force microscopy, the total energy (force indicater) was found significantly decreased in Amot knockdown cells. Taken together, we propose that Amot is a novel partner of the ITGB1/Fibronectin protein complex at focal adhesion and required for exerting force transition between endothelial cell and extracellular matrix.

Keywords: angiogenesis, angiomotin, endothelial cell migration, focal adhesion, integrin beta 1

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Authors: Reine Suci Wulandari, Rosa Suryantini

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Albizia (Paraserianthes falcataria) is a woody plant species that has a high economic value and multifunctional. Albizia is important timber, medicinal plants and can also be used as a plant to rehabilitate critical lands. The demand value of Albizia is increased so that the large quantities and high quality of seeds are required. In vitro propagation techniques are seed propagation that can produce more seeds and quality in a short time. In vitro cultures require growth regulators that can be obtained from biological agents such as endophytic fungi. Endophytic fungi are micro fungi that colonize live plant tissue without producing symptoms or other negative effects on host plants and increase plant growth. The purposes of this research were to isolate and identify endophytic fungi isolated from the root of Albizia and to study the effect of endophytic fungus on the growth of Albizia in vitro. The methods were root isolation, endophytic fungal identification, and inoculation of endophytic fungi to Albizia plants in vitro. Endophytic fungus isolates were grown on PDA media before being inoculated with Albizia sprouts. Incubation is done for 4 (four) weeks. The observed growth parameters were live explant percentage, percentage of explant shoot, and percentage of explant rooted. The results of the research showed that 6 (six) endophytic fungal isolates obtained from the root of Albizia, namely Aspergillus sp., Verticillium sp, Penicillium sp., Trichoderma sp., Fusarium sp., and Acremonium sp. Statistical analysis found that Trichoderma sp. and Fusarium sp. affect in vitro growth of Albizia. Endophytic fungi from the results of this research were potential as plant growth promoting. It can be applied to increase productivity either through increased plant growth and increased endurance of Albizia seedlings to pests and diseases.

Keywords: Albizia, endophytic fungi, propagation, in vitro

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Authors: Sandeep Kumar Agrawal, Aditi Bhattacharya, Janvie Manhas, Krushna Bhatt, Yatin Kholakiya, Nupur Khera, Ajoy Roychoudhury, Sudip Sen

Abstract:

Autologous cultured explants of human oral mucosal epithelial cells (OMEC) are a potential therapeutic modality for limbal stem cell deficiency (LSCD). Injury or inflammation of the ocular surface in the form of burns, chemicals, Stevens Johnson syndrome, ocular cicatricial pemphigoid etc. can lead to destruction and deficiency of limbal stem cells. LSCD manifests in the form of severe ocular surface diseases (OSD) characterized by persistent and recurrent epithelial defects, conjuntivalisation and neovascularisation of the corneal surface, scarring and ultimately opacity and blindness. Most of the cases of OSD are associated with severe dry eye pertaining to diminished mucin and aqueous secretion. Mycophenolate mofetil (MMF) has been shown to upregulate the mucin expression in conjunctival goblet cells in vitro. The aim of this study was to evaluate the effects of MMF on mucin expression in primary cultures of oral mucosal epithelial cells. With institutional ethics committee approval and written informed consent, thirty oral mucosal epithelial tissue samples were obtained from patients undergoing oral surgery for non-malignant conditions. OMEC were grown on human amniotic membrane (HAM, obtained from expecting mothers undergoing elective caesarean section) scaffold for 2 weeks in growth media containing DMEM & Ham’s F12 (1:1) with 10% FBS and growth factors. In vitro dosage of MMF was standardised by MTT assay. Analysis of stem cell markers was done using RT-PCR while mucin mRNA expression was quantified using RT-PCR and q-PCR before and after treating cultured OMEC with graded concentrations of MMF for 24 hours. Protein expression was validated using immunocytochemistry. Morphological studies revealed a confluent sheet of proliferating, stratified oral mucosal epithelial cells growing over the surface of HAM scaffold. The presence of progenitor stem cell markers (p63, p75, β1-Integrin and ABCG2) and cell surface associated mucins (MUC1, MUC15 and MUC16) were elucidated by RT-PCR. The mucin mRNA expression was found to be upregulated in MMF treated primary cultures of OMEC, compared to untreated controls as quantified by q-PCR with β-actin as internal reference gene. Increased MUC1 protein expression was validated by immunocytochemistry on representative samples. Our findings conclude that OMEC have the ability to form a multi-layered confluent sheet on the surface of HAM similar to a cornea, which is important for the reconstruction of the damaged ocular surface. Cultured OMEC has stem cell properties as demonstrated by stem cell markers. MMF can be a novel enhancer of mucin production in OMEC. It has the potential to improve dry eye in patients undergoing OMEC transplantation for bilateral OSD. Further clinical trials are required to establish the role of MMF in patients undergoing OMEC transplantation.

Keywords: limbal stem cell deficiency, mycophenolate mofetil, mucin, ocular surface disease

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Authors: Norah Al Hokbany, Ibrahim Al Jammaz, Basem Al Otaibi, Yousif Al Malki, Subhani M. Okarvi

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Objective: The folate receptor is over-expressed in a wide variety of human tumors. Conjugates of folate have been shown to be selectively taken up by tumor cells via the folate receptor. In an attempt to develop new folate radiotracers with favorable biochemical properties for detecting folate receptor-positive cancers. Methods: we synthesized ⁶⁴Cu-NOTA- and ⁶⁴Cu-NOTAM-folate conjugates using a straightforward and simple one-step reaction. Radiochemical yields were greater than 95% (decay-corrected) with a total synthesis time of less than 20 min. Results: Radiochemical purities were always greater than 98% without high-performance liquid chromatography (HPLC) purification. These synthetic approaches hold considerable promise as a rapid and simple method for ⁶⁴Cu-folate conjugate preparation with high radiochemical yield in a short synthesis time. In vitro tests on the KB cell line showed that significant amounts of the radio conjugates were associated with cell fractions. Bio-distribution studies in nude mice bearing human KB xenografts demonstrated a significant tumor uptake and favorable bio-distribution profile for ⁶⁴Cu-NOTA- and ⁶⁴Cu-NOTAM-folate conjugate. The uptake in the tumors was blocked by the excess injection of folic acid, suggesting a receptor-mediated process. Conclusion: These results demonstrate that the ⁶⁴Cu-NOTAM-folate conjugate may be useful as a molecular probe for the detection and staging of folate receptor-positive cancers, such as ovarian cancer and their metastasis, as well as monitoring tumor response to treatment.

Keywords: folate, receptor, tumor imaging, ⁶⁴Cu-NOTA-folate, PET

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Authors: Ashleigh Williams

Abstract:

Micro- and nanoplastics’ (MNLPs) omnipresence and ecological accumulation is evident when surveying recent environmental impact studies. For example, in 2014 it was estimated that at least 52.3 trillion plastic microparticles are floating at sea, and scientists have even found plastics present remote Arctic ice and snow (5,6). Plastics have even found their way into precipitation, with more than 1000 tons of microplastic rain precipitating onto the Western United States in 2020. Even more recent studies evaluating the chemical safety of reusable plastic bottles found that hundreds of chemicals leached into the control liquid in the bottle (ddH2O, ph = 7) during a 24-hour time period. A consequence of the increased abundance in plastic waste in the air, land, and water every year is the bioaccumulation of MNLPs in ecosystems and trophic niches of the animal food chain, which could potentially cause increased direct and indirect exposure of humans to MNLPs via inhalation, ingestion, and dermal contact. Though the detrimental, toxic effects of MNLPs have been established in marine biota, much less is known about the potentially hazardous health effects of chronic MNLP ingestion in humans. Recent data indicate that long-term exposure to MNLPs could cause possible inflammatory and dysbiotic effects. However, toxicity seems to be largely dose-, as well as size-dependent. In addition, the transcytotic uptake of MNLPs through the intestinal epithelia in humans remain relatively unknown. To this point, the goal of the current study was to investigate the mechanisms of micro- and nanoplastic uptake and transcytosis of Polystyrene (PE) in human stem-cell derived, physiologically relevant in vitro intestinal model systems, and to compare the relative effect of particle size (30 nm, 100 nm, 500 nm and 1 µm), and concentration (0 µg/mL, 250 µg/mL, 500 µg/mL, 1000 µg/mL) on polystyrene MNLP uptake, transcytosis and intestinal epithelial model integrity. Observational and quantitative data obtained from confocal microscopy, immunostaining, transepithelial electrical resistance (TEER) measurements, cryosectioning, and ELISA cytokine assays of the proinflammatory cytokines Interleukin-6 and Interleukin-8 were used to evaluate the localization and transcytosis of polystyrene MNPs and its impact on epithelial integrity in human-derived intestinal in vitro model systems. The effect of Microfold (M) cell induction on polystyrene micro- and nanoparticle (MNP) uptake, transcytosis, and potential inflammation was also assessed and compared to samples grown under standard conditions. Microfold (M) cells, link the human intestinal system to the immune system and are the primary cells in the epithelium responsible for sampling and transporting foreign matter of interest from the lumen of the gut to underlying immune cells. Given the uptake capabilities of Microfold cells to interact both specifically and nonspecific to abiotic and biotic materials, it was expected that M- cell induced in vitro samples would have increased binding, localization, and potentially transcytosis of Polystyrene MNLPs across the epithelial barrier. Experimental results of this study would not only help in the evaluation of the plastic toxicity, but would allow for more detailed modeling of gut inflammation and the intestinal immune system.

Keywords: nanoplastics, enteroids, intestinal barrier, tissue engineering, microfold (M) cells

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Authors: Simon Grossemy, Peggy P. Y. Chan, Pauline M. Doran

Abstract:

Nerve tissue engineering is the main field of research aimed at finding an alternative to autografts as a treatment for nerve injuries. Scaffolds are used as a support to enhance nerve regeneration. In order to successfully design novel scaffolds and in vitro cell culture systems, a deep understanding of the factors affecting nerve regeneration processes is needed. Physical and biological parameters associated with the culture environment have been identified as potentially influential in nerve cell differentiation, including electrical stimulation, exposure to extracellular-matrix (ECM) proteins, dynamic medium conditions and co-culture with glial cells. The mechanisms involved in driving the cell to differentiation in the presence of these factors are poorly understood; the complexity of each of them raises the possibility that they may strongly influence each other. Some questions that arise in investigating nerve regeneration include: What are the best protein coatings to promote neural cell attachment? Is the scaffold design suitable for providing all the required factors combined? What is the influence of dynamic stimulation on cell viability and differentiation? In order to study these effects, scaffolds adaptable to bioreactor culture conditions were designed to allow electrical stimulation of cells exposed to ECM proteins, all within a dynamic medium environment. Gold coatings were used to make the surface of viscose rayon microfiber scaffolds (VRMS) conductive, and poly-L-lysine (PLL) and laminin (LN) surface coatings were used to mimic the ECM environment and allow the attachment of rat PC12 neural cells. The robustness of the coatings was analyzed by surface resistivity measurements, scanning electron microscope (SEM) observation and immunocytochemistry. Cell attachment to protein coatings of PLL, LN and PLL+LN was studied using DNA quantification with Hoechst. The double coating of PLL+LN was selected based on high levels of PC12 cell attachment and the reported advantages of laminin for neural differentiation. The underlying gold coatings were shown to be biocompatible using cell proliferation and live/dead staining assays. Coatings exhibiting stable properties over time under dynamic fluid conditions were developed; indeed, cell attachment and the conductive power of the scaffolds were maintained over 2 weeks of bioreactor operation. These scaffolds are promising research tools for understanding complex neural cell behavior. They have been used to investigate major factors in the physical culture environment that affect nerve cell viability and differentiation, including electrical stimulation, bioreactor hydrodynamic conditions, and combinations of these parameters. The cell and tissue differentiation response was evaluated using DNA quantification, immunocytochemistry, RT-qPCR and functional analyses.

Keywords: bioreactor, electrical stimulation, nerve differentiation, PC12 cells, scaffold

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Authors: Jie Ding, Yingying Pan, Shammy Raj, Lindy Schaffrick, Jolene Wong, Antoinette Nguyen, Sharada Manchikanti, Larry Unsworth, Peter Kwan, Edward E. Tredget

Abstract:

Background: Exosomes (EXOs) have been considered a new target that is thought to be involved in and treat wound healing. More research is needed to fully understand the EXO characteristics and mechanisms of EXO-mediated wound healing, especially wound healing after burn injury. Methods: Total EXOs were isolated from 85 serum samples of 29 burn patients and 13 healthy individuals. We characterized the EXOs for morphology and density, serum concentration, protein level, marker expression, size distribution, and cytokine content. After confirmation of EXO uptake by dermal fibroblasts, we also explored functional regulation of primary human normal skin and hypertrophic scar fibroblast cell lines by the EXOs in vitro, including cell proliferation and apoptosis. Results: EXOs dynamically changed their morphology, density, size, and cytokine level during wound healing in burn patients, which were correlated with burn severity and the stages of wound healing. EXOs from both burn patients and healthy individuals stimulated dermal fibroblast proliferation and apoptosis. Conclusion: EXO features may be important signals that influence wound healing after burn injury; however, to understand the mechanisms by which EXOs regulated the fibroblasts in healing wounds, further studies will be required in the future.

Keywords: exosome, burn, wound healing, hypertrophic scarring, cytokines

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Authors: Ivan Tolj

Abstract:

Water produced during electrochemical reaction in Proton Exchange Membrane (PEM) fuel cell can be used for internal humidification of reactant gases; hydrogen and air. On such a way it is possible to eliminate expensive external humidifiers and simplify fuel cell balance-of-plant (BoP). When fuel cell operates at constant temperature (usually between 60 °C and 80 °C) relatively cold and dry ambient air heats up quickly upon entering channels which cause further drop in relative humidity (below 20%). Low relative humidity of reactant gases dries up polymer membrane and decrease its proton conductivity which results in fuel cell performance drop. It is possible to maintain such temperature profile throughout fuel cell cathode channel which will result in close to 100 % RH. In order to achieve this, passive heat exchanger was designed using commercial CFD software (ANSYS Fluent). Such passive heat exchanger (with variable surface area) is suitable for small scale PEM fuel cells. In this study, passive heat exchanger for single PEM fuel cell segment (with 20 x 1 cm active area) was developed. Results show close to 100 % RH of air throughout cathode channel with increased fuel cell performance (mainly improved polarization curve) and improved durability.

Keywords: PEM fuel cell, passive heat exchange, relative humidity, thermal management

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Authors: Seyedeh Nasim Mirbahari, Taha Azad, Mehdi Totonchi

Abstract:

Oncolytic viruses, which only replicate in tumor cells, are being extensively studied for their use in cancer therapy. A particular virus known as the vaccinia virus, a member of the poxvirus family, has demonstrated oncolytic abilities glioma. Treating Glioma with traditional methods such as chemotherapy and radiotherapy is quite challenging. Even though oncolytic viruses have shown immense potential in cancer treatment, their effectiveness in glioblastoma treatment is still low. Therefore, there is a need to improve and optimize immunotherapies for better results. In this study, we have designed oncoVV-TT, which can more effectively target tumor cells while minimizing replication in normal cells by replacing the thymidine kinase gene with a luc-p2a-GFP gene expression cassette. Human glioblastoma cell line U251 MG, rat glioblastoma cell line C6, and non-tumor cell line HFF were plated at 105 cells in a 12-well plates in 2 mL of DMEM-F2 medium with 10% FBS added to each well. Then incubated at 37°C. After 16 hours, the cells were treated with oncoVV-TT at an MOI of 0.01, 0.1 and left in the incubator for a further 24, 48, 72 and 96 hours. Viral replication assay, fluorescence imaging and viability tests, including trypan blue and crystal violet, were conducted to evaluate the cytotoxic effect of oncoVV-TT. The finding shows that oncoVV-TT had significantly higher cytotoxic activity and proliferation rates in tumor cells in a dose and time-dependent manner, with the strongest effect observed in U251 MG. To conclude, oncoVV-TT has the potential to be a promising oncolytic virus for cancer treatment, with a more cytotoxic effect in human glioblastoma cells versus rat glioma cells. To assess the effectiveness of vaccinia virus-mediated viral therapy, we have tested U251mg and C6 tumor cell lines taken from human and rat gliomas, respectively. The study evaluated oncoVV-TT's ability to replicate and lyse cells and analyzed the survival rates of the tested cell lines when treated with different doses of oncoVV-TT. Additionally, we compared the sensitivity of human and mouse glioma cell lines to the oncolytic vaccinia virus. All experiments regarding viruses were conducted under biosafety level 2. We engineered a Vaccinia-based oncolytic virus called oncoVV-TT to replicate specifically in tumor cells. To propagate the oncoVV-TT virus, HeLa cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 10 MOI virus was added. After 48 h, cells were harvested by scraping, and viruses were collected by 3 sequential freezing and thawing cycles followed by removal of cell debris by centrifugation (1500 rpm, 5 min). The supernatant was stored at −80 ◦C for the following experiments. To measure the replication of the virus in Hela, cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 5 MOI virus or equal dilution of PBS was added. At the treatment time of 0 h, 24 h, 48 h, 72 h and 96 h, the viral titers were determined under the fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). Fluorescence intensity was quantified using the imagej software according to the manufacturer’s protocol. For the isolation of single-virus clones, HeLa cells seeded in six-well plates (5×105 cells/well). After 24 h (100% confluent), the cells were infected with a 10-fold dilution series of TianTan green fluorescent protein (GFP)virus and incubated for 4 h. To examine the cytotoxic effect of oncoVV-TT virus ofn U251mg and C6 cell, trypan blue and crystal violet assay was used.

Keywords: oncolytic virus, immune therapy, glioma, vaccinia virus

Procedia PDF Downloads 51