Cell Growth Conditions Research Articles

Effect of Rat C6 Glioma Cells in Varying Concentrations of Cultured in Isorhamnetin

Objective: To investigate the effects of different concentrations of isorhamnetin on C6 rat glioma cells in vitro from January 2015 to June 2015. Methods: The blank control group, blank solvent control group and four concentration groups were used to observe the cell growth status under a microscope. MTT colorimetric assay was used to detect the effect of isorhamnetin on C6 glioma cells in vitro and the cell inhibition rate And survival rate were measured. The apoptotic and apoptotic rates were measured by flow cytometry in the treatment group and the control group. The relationship between the different concentrations of isorhamnetin and C6 glioma cell apoptosis was analyzed the total protein was extracted and the total AKT protein and Ser473 AKT protein content were detected by Western blotting. The rat model of glioma was constructed by SD rats.Five days of isorhamnetin was continuously fed and the plasma was detected by high-performance liquid chromatography,liver, brain tissue isorhamnetin content.Â

Suspension culture process for H9N2 avian influenza virus (strain Re-2)

H9N2 avian influenza virus has caused huge economic loss for the Chinese poultry industry since it was first identified. Vaccination is frequently used as a control method for the disease. Meanwhile suspension culture has become an important tool for the development of influenza vaccines. To optimize the suspension culture conditions for the avian influenza H9N2 virus (Re-2 strain) in Madin-Darby Canine Kidney (MDCK) cells, we studied the culture conditions for cell growth and proliferation parameters for H9N2 virus replication. MDCK cells were successfully cultured in suspension, from a small scale to industrial levels of production, with passage time and initial cell density being optimized. The influence of pH on the culture process in the reactor has been discussed and the process parameters for industrial production were explored via amplification of the 650L reactor. Subsequently, we cultivated cells at high cell density and harvested high amounts of virus, reaching 10log2 (1:1024). Furthermore an animal experiment was conducted to detect antibody. Compared to the chicken embryo virus vaccine, virus cultured from MDCK suspension cells can produce a higher amount of antibodies. The suspension culture process is simple and cost efficient, thus providing a solid foundation for the realization of large-scale avian influenza vaccine production.

Intermolecular interactions involving an acidic patch on immunoglobulin variable domain and the γ2 constant region mediate crystalline inclusion body formation in the endoplasmic reticulum.

Full-length immunoglobulins (Igs) are widely considered difficult to crystallize because of their large size, N-linked glycosylation, and flexible hinge region. However, numerous cases of intracellular Ig crystallization are reported in plasma cell dyscrasias. What makes some Ig clones more prone to crystallize during biosynthesis as well as the biochemical and cell biological requirements for this cryptic event are poorly understood. To investigate the underlying process of intracellular Ig crystallization we searched for model IgGs that can induce crystalline inclusions during recombinant overexpression. By testing various subunit combinations through mixing and matching of individual subunit chains derived from a panel of human IgG clones, we identified one secretion competent IgG2λ that induced needle-like crystalline inclusions in transfected HEK293 cells. Ig crystallization rarely occurred at steady-state cell growth conditions but was easily induced when ER-to-Golgi transport was pharmacologically blocked. Homology modeling revealed the presence of a prominent negatively-charged patch on the variable domain surface. The patch was composed of eight aspartic acids, of which five were in the heavy chain variable region and three were in the light chain. Crystallization occurred only when the two subunits were co-transfected and the intracellular crystals co-localized with ER resident proteins. Furthermore, subtype switching from IgG2 to IgG1 and stepwise neutralization of the acidic patch independently abrogated Ig crystallization events. The evidence supported that the formation of needle-like crystalline inclusions in the ER was underscored by multivalent intermolecular interactions between the acidic patch and undefined determinants present on the γ2 subunit constant region.

Abstract 5897: Single cell-derived analysis of desmoid tumors for studying tumor-stroma interactions

Abstract Cancer associated fibroblasts play an important role in the maintenance and remodeling of the tumor microenvironment, providing the appropriate conditions for neoplastic cell growth and invasion. Desmoid tumors (DT), also called aggressive fibromatosis, are rare, locally invasive soft tissue tumors that consist of fibroblastic cells embedded in extracellular matrix. Identification of the stromal cells to study tumor-stroma interactions is difficult due to both populations displaying a fibroblastic phenotype, and no cell marker exists that reliably differentiates between the two populations. Majority of DT arise sporadically due to somatic activating mutations in beta-catenin (CTNNB1), a major effector molecule of canonical Wnt signaling. We established single cell derived colonies from multiple DT samples and characterized the beta-catenin mutation status of each clone by Sanger sequencing. Indeed, we were able to establish both mutant and non-mutant colonies from DT samples. Quantitative PCR for beta-catenin targets AXIN2 and LEF1 confirmed differential activity between the mutant and non-mutant colonies. The specific CTNNB1 codon mutation had no difference on beta-catenin transcriptional activity. We next performed a high throughput surface antigen screen to identify cell markers that can distinguish between the two subpopulations. Our screen found CD142 to be uniquely expressed by the mutant colonies, while the non-mutant colonies uniquely expressed Podoplanin. Quantitative PCR confirmed the differential expression of these markers. Furthermore, the CD142-positive population in heterogeneous DT samples correlated with their mutation frequency. Importantly, CD142-based cell sorting allowed the isolation of the mutant subpopulation even in samples that appeared as wild-type by Sanger sequencing. We also studied the expression of secreted factors in our mutant and non-mutant populations. We observed that CTHRC1, a ligand related to the Wnt/PCP pathway, is highly elevated exclusively in the mutant subpopulations. Recombinant CTHRC1 increased the proliferation rate of DT primary cultures, as measured by BrdU incorporation, while neutralizing antibodies against CTHRC1 decreased cell proliferation. The importance of tumor-stroma interactions cannot be studied without first identifying and characterizing the two populations. This has been especially difficult in soft tissue sarcomas where both the neoplastic and stromal cells exhibit a mesenchymal phenotype. Our study offers a novel method for identifying the mutant and non-mutant subpopulations within desmoid tumors to study how they may interact. Rapidly quantifying tumor composition will also support efforts to understand the natural progression of disease and how it responds to therapy. Citation Format: Mushriq Al-Jazrawe, Steven Xu, Qingxia Wei, Raymond Poon, Benjamin Alman. Single cell-derived analysis of desmoid tumors for studying tumor-stroma interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5897. doi:10.1158/1538-7445.AM2017-5897

Study of composite vascular scaffold combining with differentiated VSMC- and VEC-like cells invitro and invivo.

Although research into the tissue engineering of vessels has proceeded at a tremendous pace, many deficiencies still need to be resolved. A well-adopted constructed vessel requires both functional and structural properties to stimulate the native vessel and resist stress and tension invivo. In the present study, we developed a novel three-layer composite vascular scaffold consisting of differentiated vascular smooth muscle cell-, vascular endothelial cell-like cells, and a rabbit acellular vascular matrix (ACVM)-0.25% HLC-I scaffold. HE staining, immunohistochemical assays, immunofluorescence assays (IFAs), and scanning electron microscopy were performed to monitor the growth status of cells on the scaffold material invitro. After the vascular endothelial cell -vascular smooth muscle cell-scaffold was implanted into nude mice for three, six, and nine weeks, samples were harvested from the implanted mice and observed visually or by HE staining and IFAs for cell viability and morphology. Additionally, burst pressure resistance experiments were used to assess the maximal pressure that the engineered vessel could resist. We found that the engineered vascular endothelial cell-vascular smooth muscle cell-scaffold vessel possessed favorable biocompatibility and considerable strength, matching native vessels invivo and invitro, and may be significant in the future clinical implantation of tissue-engineered vasculature.

Production and characterization of polyhydroxyalkanoic acid from Bacillus thuringiensis using different carbon substrates

Polyhydroxyalkanoic acids (PHA) are biodegradable microbially synthesised polymers which can act as alternatives to conventional petrochemical based plastics. Bacterial isolates from soil and sewage samples were screened for the ability to accumulate PHA using Sudan Black B dye, and growth conditions of the best PHA accumulating bacterium was optimized. The PHA produced was characterized by FTIR spectroscopy. Eight PHA-producing bacteria were isolated and isolate SBC4 which had the highest percentage accumulation of 30% was genotypically identified as Bacillus thuringiensis and selected for further studies. Glucose and ammonium sulfate supplementation afforded the optimum cultural conditions for cell growth and PHA accumulation yielding 0.33g/l and 0.40g/l respectively. B. thuringiensis SBC4 utilized corn bran, corn cob and wheat bran for PHA production, with corn cob yielding the highest PHA content of 21.05%. The optimum pH, temperature and incubation time for PHA productions were 7.0, 37°C and 48h. The use of all the optimum conditions in a newly modified medium increased the PHA content, with an overall increase in PHA accumulation from 30% to 44.96% equivalent to an increase of 50% in PHA yield. The presence of major peaks such as CO stretching, CH vibrations, COC stretching confirmed PHA production.

Degradation of phytosterols in tobacco waste extract by a novel Paenibacillus sp.

Phytosterols have been demonstrated to be precursors of polycyclic aromatic hydrocarbons (PAHs) formed during biomass pyrolysis. Here, a novel Paenibacillus sp. was evaluated for its ability to degrade phytosterols in tobacco waste extract (TWE). The optimal conditions for cell growth and stigmasterol (a representative of phytosterols) degradation were 37 °C, pH 7.0, 1.0g/L yeast extract, and 6.0g/L glucose. Paenibacillus sp. could degrade stigmasterol under high concentrations of glucose (up to 130g/L) and tolerate wide pH (5.0-9.0) and temperature (25-42 °C) ranges. The new strain could degrade stigmasterol completely into CO2 and H2 O, and no intermediate steroids were detected during the degradation process. Phytosterol degradation in TWE was demonstrated by high-performance liquid chromatography-tandem mass spectrometry. Under optimal conditions (37 °C, pH 7.0, with the exponential-phase cells), the total degradation ratio of phytosterols reached 38.5% in TWE, including 45.2% of stigmasterol, 37.4% of β-sitosterol, 27.3% of campesterol, and 28.7% of cholesterol. These results showed that Paenibacillus sp. is a candidate for phytosterol degradation in TWE and other biomass and is potentially useful in reducing the PAHs generated from biomass pyrolysis.

MP24-18 DETRUSOR BIOENGINEERING USING COMPRESSED COLLAGEN, ADIPOSE-DERIVED STEM CELLS AND SMOOTH MUSCLE CELLS

You have accessJournal of UrologyTrauma/Reconstruction/Diversion: Ureter (including Pyeloplasty) and Bladder Reconstruction (including fistula), Augmentation, Substitution, Diversion I1 Apr 2017MP24-18 DETRUSOR BIOENGINEERING USING COMPRESSED COLLAGEN, ADIPOSE-DERIVED STEM CELLS AND SMOOTH MUSCLE CELLS Jakub Smolar, Maya Horst, and Daniel Eberli Jakub SmolarJakub Smolar More articles by this author , Maya HorstMaya Horst More articles by this author , and Daniel EberliDaniel Eberli More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3312AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Conditions impairing bladder function in children and adults often need urinary diversion or augmentation cystoplasty as when untreated they may cause severe bladder dysfunction and kidney failure. Currently, the gold standard therapy of end-stage bladder disease refractory to conservative management is enterocystoplasty, which despite providing functional improvement is associated with significant long-term complications. Therefore, there is a strong clinical need for alternative therapies for these reconstructive procedures. The aim of this study is to develop functional smooth muscle tissue for the detrusor muscle repair combining various cell types in hydrogel scaffolds. METHODS Rat bladder smooth muscle (SMC) and adipose-derived stem cells (ADSC) were isolated, expanded and characterized using flow cytometry. ADSC were pre-differentiated into SMC-like cells (pADSC). Cells were combined in ratios 1:1, 1:2 and 1:3 (SMC:pADSC) and embedded in compressed collagen (CC). After 1, 2 and 3 weeks, cells in CC scaffolds, direct and indirect 2D co-cultures were analyzed for viability, proliferation, morphology, SMC-marker expression and functionality. RESULTS Cell growth conditions have been optimized and cells have shown high viability and good proliferation in the CC scaffolds. Interconnected microtissues and cell layers have developed all over the CC already after 1 week of co-culture. At 1 and 2 week timepoints cells in CC showed strong expression of the SMC markers calponin, MyH11 and smoothelin. Direct cell co-culture resulted in significantly increased cellular proliferation. Microtissues consisted of a SMC-core surrounded by pADSC. Indirect co-culture resulted in an increased pADSC survival and ratio-dependent increase in SMC-proliferation. pADSC proliferation rate also improved, but remained unaffected by the cell ratio, with 1:1 showing the most consistent results. SMC-marker expression normalized between the different ratios after 2 weeks of co-culture and reached almost the SMC monoculture expression levels. The 1:1 co-culture contracted significantly better than the other ratios after 24h. CONCLUSIONS We have shown that a SMC–pADSC co-culture results in an improved cell survival, proliferation, microtissue and cell layer formation without any significant changes in phenotype and functionality. The combination of SMC and pADSC with CC may help to engineer functional detrusor muscle tissue by solving the major issues of tissue engineering, namely poor cell survival, proliferation, phenotype instability and functionality. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e308-e309 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Jakub Smolar More articles by this author Maya Horst More articles by this author Daniel Eberli More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cellular responses and gene expression profile changes due to bleomycin-induced DNA damage in human fibroblasts in space.

Living organisms in space are constantly exposed to radiation, toxic chemicals or reactive oxygen species generated due to increased levels of environmental and psychological stresses. Understanding the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage is essential for assessing the radiation risk for astronauts and the mutation rate in microorganisms. In a study conducted on the International Space Station, confluent human fibroblasts in culture were treated with bleomycin for three hours in the true microgravity environment. The degree of DNA damage was quantified by immunofluorescence staining for γ-H2AX, which is manifested in three types of staining patterns. Although similar percentages of these types of patterns were found between flight and ground cells, there was a slight shift in the distribution of foci counts in the flown cells with countable numbers of γ-H2AX foci. Comparison of the cells in confluent and in exponential growth conditions indicated that the proliferation rate between flight and the ground may be responsible for such a shift. We also performed a microarray analysis of gene expressions in response to bleomycin treatment. A qualitative comparison of the responsive pathways between the flown and ground cells showed similar responses with the p53 network being the top upstream regulator. The microarray data was confirmed with a PCR array analysis containing a set of genes involved in DNA damage signaling; with BBC3, CDKN1A, PCNA and PPM1D being significantly upregulated in both flight and ground cells after bleomycin treatment. Our results suggest that whether microgravity affects DNA damage response in space can be dependent on the cell type and cell growth condition.

Investigation of the Best Saccharomyces cerevisiae Growth Condition.

IntroductionSaccharomyces cerevisiae is known as one of the useful yeasts which are utilized in baking and other industries. It can be easily cultured at an economic price. Today the introduction of safe and efficient carriers is being considered. Due to its generally round shape, and the volume that is enclosed by its membrane and cell wall, it is used to encapsulate active materials to protect them from degradation or to introduce a sustained release drug delivery system. Providing the best conditions in order to achieve the best morphological properties of Saccharomyces cerevisiae as a carrier.MethodsIn this research, the most suitable growth condition of yeast cells which provides the best size for use as drug carriers was found by a bioreactor in a synthetic culture medium. Yeast cell reproduction and growth curves were obtained, based on pour plate colony counting data and UV/Visible sample absorption at 600 nm. Yeast cell growth patterns and growth rates were determined by Matlab mathematical software.ResultsResults showed that pH=4 and dissolving oxygen (DO) 5% was the best condition for yeast cells to grow and reproduce. This condition also provided the largest size (2 × 3 μ) yeast cells.ConclusionOwing to the yeast cells’ low-cost production and their structural characteristics, they could be used as potent drug carriers.FundingThis work was supported by a grant from the Vice Chancellor of Research of Mashhad University of Medical Sciences.

A rapid and high-quality method for total RNA isolation from Haematococcus pluvialis.

Haematococcus pluvialis, as the most potential natural source of astaxanthin, which is a powerful antioxidant with high economic value, has attracted more and more scientific attention in recent years. An in-depth understanding of the mechanism for how H. pluvialis produces astaxanthin requires the intensive investigations on its genetic information. In particular, many reported studies were based on a variety of RNA analyses. However, it is difficult to extract RNA with high quality and quantity from H. pluvialis, because of the blockage from its thick cell wall and contamination by a large quantity of pigments, polysaccharides, and lipids. Therefore, we proposed an optimized Trizol-based RNA extraction method for H. pluvialis by investigating the effect of cell wall broken ways, algal strains, and cell growth status on total RNA isolation. Using this rapid, convenient, and cost-saving method, isolated H. pluvialis RNA had high quantity and quality (with an RNA integrity number of 7.0 and a concentration of 1604.1 ng/μL) equivalent to that isolated by commercial kit, enabling its applications into downstream RNA analyses.

Mixing theory for culture and harvest in bioreactors of human mesenchymal stem cells on microcarriers

The use of human mesenchymal stem cells (hMSCs) in regenerative medicine is a potential major advance for the treatment of many medical conditions, especially with the use of allogeneic therapies where the cells from a single donor can be used to treat ailments in many patients. Such cells must be grown attached to surfaces and for large scale production, it is shown that stirred bioreactors containing ~200 μm particles (microcarriers) can provide such a surface. It is also shown that the just suspended condition, agitator speed NJS, provides a satisfactory condition for cell growth by minimizing the specific energy dissipation rate, eT, in the bioreactor whilst still meeting the oxygen demand of the cells. For the cells to be used for therapeutic purposes, they must be detached from the microcarriers before being cryopreserved. A strategy based on a short period (~7 min) of very high eT, based on theories of secondary nucleation, is effective at removing >99% cells. Once removed, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. This approach is shown to be successful for culture and detachment in 4 types of stirred bioreactors from 15 mL to 5 L.

Gene and miRNA expression signature of Lewis lung carcinoma LLC1 cells in extracellular matrix enriched microenvironment.

BackgroundThe extracellular matrix (ECM), one of the key components of tumor microenvironment, has a tremendous impact on cancer development and highly influences tumor cell features. ECM affects vital cellular functions such as cell differentiation, migration, survival and proliferation. Gene and protein expression levels are regulated in cell-ECM interaction dependent manner as well. The rate of unsuccessful clinical trials, based on cell culture research models lacking the ECM microenvironment, indicates the need for alternative models and determines the shift to three-dimensional (3D) laminin rich ECM models, better simulating tissue organization. Recognized advantages of 3D models suggest the development of new anticancer treatment strategies. This is among the most promising directions of 3D cell cultures application. However, detailed analysis at the molecular level of 2D/3D cell cultures and tumors in vivo is still needed to elucidate cellular pathways most promising for the development of targeted therapies. In order to elucidate which biological pathways are altered during microenvironmental shift we have analyzed whole genome mRNA and miRNA expression differences in LLC1 cells cultured in 2D or 3D culture conditions.MethodsIn our study we used DNA microarrays for whole genome analysis of mRNA and miRNA expression differences in LLC1 cells cultivated in 2D or 3D culture conditions. Next, we indicated the most common enriched functional categories using KEGG pathway enrichment analysis. Finally, we validated the microarray data by quantitative PCR in LLC1 cells cultured under 2D or 3D conditions or LLC1 tumors implanted in experimental animals.ResultsMicroarray gene expression analysis revealed that 1884 genes and 77 miRNAs were significantly altered in LLC1 cells after 48 h cell growth under 2D and ECM based 3D cell growth conditions. Pathway enrichment results indicated metabolic pathway, MAP kinase, cell adhesion and immune response as the most significantly altered functional categories in LLC1 cells due to the microenvironmental shift from 2D to 3D. Comparison of the expression levels of selected genes and miRNA between LLC1 cells grown in 3D cell culture and LLC1 tumors implanted in the mouse model indicated correspondence between both model systems.ConclusionsGlobal gene and miRNA expression analysis in LLC1 cells under ECM microenvironment indicated altered immune response, adhesion and MAP kinase pathways. All these processes are related to tumor development, progression and treatment response, suggesting the most promising directions for the development of targeted therapies using the 3D cell culture models.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2825-9) contains supplementary material, which is available to authorized users.

Repair of human periodontal bone defects by autologous grafting stem cells derived from inflammatory dental pulp tissues.

BackgroundRecently, stem cells derived from inflammatory dental pulp tissues (DPSCs-IPs) have demonstrated regenerative potential, but the real effect remains to be examined. This pilot study attempted to isolate DPSCs-IPs from two patients and to evaluate the feasibility and the effect of reconstructing periodontal intrabone defects in each patient.MethodsDPSCs-IPs were harvested from two patients with periodontal intrabone defects with their approval. After discussing the biological characteristics of DPSCs-IPs in each patient, DPSCs-IPs were loaded onto the scaffold material β-tricalcium phosphate and engrafted into the periodontal defect area in the root furcation. After 1, 3, and 9 months, the outcome was evaluated by clinical assessment and radiological study. Furthermore, new samples were collected and the biological characteristics of DPSCs-IPs were further studied compared with normal dental pulp stem cells. The primary cell culture success rate, cell viability, cell cycle analysis, and proliferation index were used to describe the growth state of DPSCs-IPs. In-vitro differentiation ability detection was used to further discuss the stem cell characteristics of DPSCs-IPs.ResultsAs expected, DPSCs-IPs were able to engraft and had an effect of regeneration of new bones to repair periodontal defects 9 months after surgical reconstruction. Although the success rate of primary cell culture and growth status was slightly inhibited, DPSCs-IPs expressed comparable levels of stem cell markers as well as retaining their multidifferentiation ability.ConclusionsWe developed a standard procedure that is potentially safe and technological for clinical periodontal treatment using human autologous DPSCs-IPs.Trial registrationAccording to the editorial policies, the present study is a purely observational study, so trial registration is not required.

Dysregulated JAK2 expression by TrkC promotes metastasis potential, and EMT program of metastatic breast cancer.

Metastatic breast cancers are aggressive tumors associated with high levels of epithelial-mesenchymal transition (EMT) markers, activation of IL6/JAK2/STAT3 and PI3K/AKT pathways for cell growth, mobility, invasion, metastasis, and CSC status. We identified a new molecular and functional network present in metastasis that regulates and coordinates with TrkC. Inhibition of SOCS3-mediated JAK2 degradation by TrkC increases total JAK2/STAT3 expression, and then leads to upregulation of Twist-1 through activation of JAK2/STAT3 cascade. Also, TrkC increases secretion and expression of IL-6, suggesting that this autocrine loop generated by TrkC maintains the mesenchymal state by continued activation of the JAK2/STAT3 cascade and upregulation of Twist expression. Moreover, TrkC interacts with the c-Src/Jak2 complex, which increases Twist-1 and Twist-2 levels via regulation of JAK2/STAT3 activation and JAK2/STAT3 expression. Furthermore, TrkC enhances metastatic potential of breast cancer via induction of EMT by upregulating Twist-1 and Twist-2. Additionally, TrkC significantly enhances the ability of breast cancer cells to form pulmonary metastases and primary tumor formation. Unexpectedly, we found that TrkC expression and clinical breast tumor pathological phenotypes show significant correlation. These findings suggest that TrkC plays a central role in tumorigenicity, metastasis, and self-renewal traits of metastatic breast cancer.

High-efficient and high-content cytotoxic recording via dynamic and continuous cell-based impedance biosensor technology.

Cell-based bioassays were effective method to assess the compound toxicity by cell viability, and the traditional label-based methods missed much information of cell growth due to endpoint detection, while the higher throughputs were demanded to obtain dynamic information. Cell-based biosensor methods can dynamically and continuously monitor with cell viability, however, the dynamic information was often ignored or seldom utilized in the toxin and drug assessment. Here, we reported a high-efficient and high-content cytotoxic recording method via dynamic and continuous cell-based impedance biosensor technology. The dynamic cell viability, inhibition ratio and growth rate were derived from the dynamic response curves from the cell-based impedance biosensor. The results showed that the biosensors has the dose-dependent manners to diarrhetic shellfish toxin, okadiac acid based on the analysis of the dynamic cell viability and cell growth status. Moreover, the throughputs of dynamic cytotoxicity were compared between cell-based biosensor methods and label-based endpoint methods. This cell-based impedance biosensor can provide a flexible, cost and label-efficient platform of cell viability assessment in the shellfish toxin screening fields.

Discovery of a Family of Genomic Sequences Which Interact Specifically with the c-MYC Promoter to Regulate c-MYC Expression.

G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III1 of the c-MYC promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII1 located upstream of the P1 promoter regulates up-to 80% of c-MYC transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII1 sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII1 resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the c-MYC and SOX2 promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common regulatory mechanism for genes whose promoters contain these sequences.

Reversible Immortalization Enables Seamless Transdifferentiation of Primary Fibroblasts into Other Lineage Cells.

Fibroblasts can be transdifferentiated directly into other somatic cells such as cardiomyocytes, hematopoietic cells, and neurons. An advantage of somatic cell differentiation without first generating induced pluripotent stem cells (iPSCs) is that it avoids contamination of the differentiated cells with residual iPSCs, which may cause teratoma. However, since primary fibroblasts from biopsy undergo senescence during repeated culture, it may be difficult to grow transdifferentiated cells in sufficient numbers for future therapeutic purposes. To circumvent this problem, we reversibly immortalized primary fibroblasts by using the piggyBac transposon to deliver the human telomerase reverse transcriptase (hTERT) gene hTERT plus SV40 Large T. Both approaches enabled fibroblasts to grow continuously without senescence, and neither caused teratoma formation in immunodeficient mice. However, fibroblasts immortalized with hTERT plus SV40 large T antigen accumulated chromosomal rearrangements, whereas fibroblasts immortalized with hTERT retained the normal karyotype. To transdifferentiate hTERT-immortalized fibroblasts into other somatic lineage cells, we transiently transfected them with episomal OCT4 and cultured them under neural cell growth condition with transposase to remove the transposon. Tripotent neural progenitor cells were seamlessly and efficiently generated. Thus, reversible immortalization of primary fibroblasts with hTERT will allow potential autologous cell-based therapeutics that bypass and simulate iPSC generation.

Abstract 18: Galactose impacts cell growth and redox status of glioblastoma multiforme U87 cell lines

Abstract Glioblastoma multiforme (GBM) is the most common and malignant form of brain cancer in adults, with a median survival time of 12-15 months. Characterizing the metabolic alterations of GBM is a crucial step towards identifying potential targets for treatment, with mitochondria, the electron transport chain, and associated reactive oxygen species presenting lucrative targets for therapy. However, the Warburg effect, in which cells generate ATP mainly via glycolysis despite adequate oxygen and functional mitochondria, can render cancer cells resistant to antagonists of the electron transport chain. This prompts investigations into other nodes of oncogenic metabolism such as the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase pathway which can regulate glucose metabolism. The present work defined the sensitivity of GBM U87MG, U87OE (over-expressed EGFR), and U87vIII (hyper-activated vIII mutated EGFR) cell lines to media supplemented with galactose versus conventional glucose concentrations. Cells were transfected with a mitochondrial-targeted Glrx-roGFP2 redox biosensor, a mutated GFP protein whose fluorescence reflects the local redox potential, and incubated in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with either 25 mM glucose (DMEM-Glu) or 10 mM galactose with and without 1 mM pyruvate (DMEM-Gal±Pyr). Glucose deprivation combined with galactose supplementation allows metabolic isolation of mitochondrial contributions to ATP generation because the glycolytic metabolism of galactose yields no net ATP, forcing cells to rely on increased mitochondrial respiration. Cells were exposed to differential media conditions and assessed via flow cytometry to monitor mitochondrial redox homeostasis after 24 h of media exposure. Wild type U87 cells exhibited a slight reduction upon glycolytic blockade, while mitochondria in the mutant EGFR U87OE and U87vIII cells exhibited extreme oxidation. The sulforhodamine B assay, which determines cell density based on a colorimetric measurement of cellular protein content, was performed to assess cell concentration in different media after 72 h of media exposure. Mutant EGFR U87OE and U87vIII cells experienced much greater growth inhibition and cell death compared to wild type U87MG cells. Thus, EGFR-activating mutations increase cellular dependence on glycolytic ATP generation for cell growth and redox homeostasis. Future work will build on these findings to characterize how the metabolic phenotypes and preferences of these cells are linked to their migratory behavior in an engineered 3D hydrogel environment. Citation Format: Jan C. Lumibao, Vladimir Kolossov, Matthew T. Leslie, H R. Gaskins. Galactose impacts cell growth and redox status of glioblastoma multiforme U87 cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 18.

Feasibility of bacterial cellulose membrane as biological scaffold for construction of tissue engineering corneal epithelium

Background Corneal transplantation is a primary method for the treatment of serious corneal diseases, but its application is limited because of the shortage of corneal donor. The study on tissue engineering corneal epithelium provides a new approach to corneal transplantation, and the biological scaffold materials for tissue engineering corneal epithelium is an issue of increasing concern. Bacterial cellulose membrane has been used in medical field, but its application in tissue engineering corneal epithelium deserves more researching. Objective This study was to evaluate the biocompatibility of bacterial cellulose membrane as a biological scaffold of tissue engineering corneal epithelium. Methods Corneal epithelium was isolated from 1 month-old New Zealand White rabbit. Corneal epithelial cells were cultured using explant method and identified by detecting the CK-3 expression using immunofluorescence technique. The second generation cells were inoculated on bacterial cellulose membrane and culture plate, respectively, and the growth status of the cells were examined and compared under the optical microscope. The cell activity/toxicity test was performed by LIVE/DEAD cell staining kit at the third day after inoculation to evaluate the survival rate. The ultrastructure of the cell surface was examined under the scanning electron microscope. The study was performed in accordance with the ARVO Statement. Results Rabbit corneal epithelial cells grew well 1 week after primarily cultured with a cobblestone-like appearance and positive response for CK3 antibody. The cells on the bacterial cellulose membrane presented a round shape and regular arrangement and showed the green fluorescence for LIVE/DEAD test, with the survival rate 100%. Abundant leafy protrusion, microvilli and intercellular junction were seen under the scanning electron microscope. In addition, mitosis phase of cells and many filopodia between the cells and bacterial cellulose membrane were also exhibited. Conclusions Rabbit corneal epithelial cells can grow well in bacterial cellulose membrane. Bacterial cellulose membrane has good biocompatibility, indicating that bacterial cellulose membrane can be used as new biological material for tissue engineering corneal epithelium. Key words: [Key words] Epithelium, corneal/cytology; Tissue engineering; Tissue scaffolds/microbiology; Biocompatible materials; Cells, cultured; Rabbits; Bacterial cellulose