Three-Dimensional Human Neural Stem Cell Culture for High-Throughput Assessment of Developmental Neurotoxicity

Only a few hundred of compounds, among tens of thousands of commercially available compounds, have been tested for developmental neurotoxicity (DNT) due to the limitations of current guidelines for DNT which are based entirely on in vivo experiments. In vivo studies are highly expensive and time-consuming, which often do not correlate to human outcomes. There is a key gap in our ability to predict in vivo outcomes accurately and robustly using in vitro assays. This is particularly the case for predicting the toxicity of chemicals on the developing human brains. Conventional in vitro assays are typically performed in two-dimensional (2D) cell culture systems and use cytotoxicity assays that do not provide the information on mechanisms of toxicity. High-content imaging (HCI) assays performed on three-dimensional (3D) cell cultures can provide better understanding of mechanisms of toxicity needed to predict DNT in humans. However, current 3D cell culture systems lack the throughput required for screening DNT against a large number of chemicals. Thus, there is a need for cost-effective, high-throughput, alternative in vitro test methods based on mechanisms of toxicity. In this study, we first developed a miniaturized, 3D human NSC culture with ReNcell VM on the micropillar chip platform and established a high-throughput promoter-reporter assay system using recombinant lentiviruses on human NSC spheroids to assess cell viability, self-renewal, and differentiation. Next, we identified major ion channels and ABC-transporters expressed in ReNcell VM via RNA-seq analysis and established high-throughput ion channel and ABC-transporter assays in 3D-cultured ReNcell VM on the 384-pillar plate. In the third step, we established high-content imaging (HCI) assays in 3D-cultured ReNcell VM with multiple assays which were tested with four model compounds. Finally, we established a high-throughput metabolism-mediated neurotoxicity testing system by combining 3D-cultured ReNcell VM on the 384PillarPlate and HepaRG spheroids in a ULA 384-well plate. Alternative in vitro systems for high-throughput neurotoxicity assessment established in this study will enable researchers to screen a library of test compounds with high confidence in terms of predictability of adverse reactions in vivo from those compounds

Three-Dimensional Human Neural Stem Cell Culture for High-Throughput Assessment of Developmental Neurotoxicity

Only a few hundred of compounds, among tens of thousands of commercially available compounds, have been tested for developmental neurotoxicity (DNT) due to the limitations of current guidelines for DNT which are based entirely on in vivo experiments. In vivo studies are highly expensive and time-consuming, which often do not correlate to human outcomes. There is a key gap in our ability to predict in vivo outcomes accurately and robustly using in vitro assays. This is particularly the case for predicting the toxicity of chemicals on the developing human brains. Conventional in vitro assays are typically performed in two-dimensional (2D) cell culture systems and use cytotoxicity assays that do not provide the information on mechanisms of toxicity. High-content imaging (HCI) assays performed on three-dimensional (3D) cell cultures can provide better understanding of mechanisms of toxicity needed to predict DNT in humans. However, current 3D cell culture systems lack the throughput required for screening DNT against a large number of chemicals. Thus, there is a need for cost-effective, high-throughput, alternative in vitro test methods based on mechanisms of toxicity. In this study, we first developed a miniaturized, 3D human NSC culture with ReNcell VM on the micropillar chip platform and established a high-throughput promoter-reporter assay system using recombinant lentiviruses on human NSC spheroids to assess cell viability, self-renewal, and differentiation. Next, we identified major ion channels and ABC-transporters expressed in ReNcell VM via RNA-seq analysis and established high-throughput ion channel and ABC-transporter assays in 3D-cultured ReNcell VM on the 384-pillar plate. In the third step, we established high-content imaging (HCI) assays in 3D-cultured ReNcell VM with multiple assays which were tested with four model compounds. Finally, we established a high-throughput metabolism-mediated neurotoxicity testing system by combining 3D-cultured ReNcell VM on the 384PillarPlate and HepaRG spheroids in a ULA 384-well plate. Alternative in vitro systems for high-throughput neurotoxicity assessment established in this study will enable researchers to screen a library of test compounds with high confidence in terms of predictability of adverse reactions in vivo from those compounds

Heat Transfer Enhancement in Rotating Rayleigh-B\'enard Convection at Ra≥1010Ra\ge 10^{10}

Rotation, which stabilizes flow, can enhance the heat transfer in Rayleigh-B\'enard convection (RBC) due to Ekman pumping. In this Letter, we study this heat transfer enhancement by conducting direct numerical simulations of rotating RBC for a wide range of parameters: Rayleigh number ($Ra$) from $2\times 10^4$ to $2\times 10^{10}$, Prandtl number ($Pr$) from $1$ to $1000$, and Taylor number ($Ta$) from $0$ to $2\times 10^{12}$. We show that for each $Ra$, there exists a critical Prandtl number ($Pr_{cr}$) below which no heat transfer enhancement occurs at any rotation rate, as well as an optimal Prandtl number ($Pr_{opt}$) at which maximum heat transfer enhancement occurs at an optimal rotation rate. We demonstrate a significant heat transfer enhancement for $Ra$ up to $2\times 10^{10}$ and predict that heat transfer enhancement due to rotation not only would occur at $Ra>10^{10}$ but would also become more pronounced

Creating miniaturized tissue constructs on a micropillar/microwell chip via 3D bioprinting technology

Liver tumor tissues in the human body consist of different layers of hepatic cells including hepatoma cells and surrounding normal cells. To mimic in vivo tumor tissues, three-dimensional (3D) microarray bioprinting was demonstrated on a microwell chip via layer-by-layer printing of Hep3B human hepatoma cell line. The 3D microarray printing coupled with high-content imaging (HCI) of cell layers on the chip might open new opportunities for predictive drug screening for patients. Our goal is to demonstrate high-throughput cell printing in hydrogel layers and establish HCI of cell layers from the microwell chip for miniaturized tumor tissue engineering. To achieve this goal, Hep3B cell suspension stained with TMRM and Hoechst 33342 was mixed with alginate as well as photocrosslinkable alginate and then printed onto the microwell chip using a microarray spotter. The images of Hep3B cells encapsulated in two alginate layers were acquired by scanning the chip with a chip scanner. As a result, we successfully demonstrated two layer cell printing with Hep3B cells encapsulated in alginate and establish high-throughput HCI with fluorescently-labeled Hep3B cells at different z-focus positions. To improve imaging of cells in different layers, further optimization of gelation conditions with photocrosslinkable alginate will be necessary.https://engagedscholarship.csuohio.edu/u_poster_2015/1047/thumbnail.jp

High-Throughput Assessment of Developmental Stages of NSCs via Promoter-Reporter Assay System Using Recombinant Lentiviruses

Many drugs and chemicals currently available have not been fully evaluated for their toxic effects on the developing brain. Expensive and low-throughput in vivo studies are still being used to evaluate developmental neurotoxicity (DNT). Thus, there is a need to develop an in vitro assay system which is economically feasible and high- throughput. Among various cellular models used for in vitro assay, human neural stem cells (NSCs) are highly desired due to their ability to self-renew and differentiate into neurons, astrocytes and oligodendrocytes. In vitro assessment of developmental stages (proliferation and differentiation) of human NSC is highly important to predict the in vivo effect of various chemicals on developing brain. However, conventional in vitro assay uses immunofluorescence staining to monitor changes in cell morphology and neural cell-specific biomarkers which can either be inaccurate or cumbersome. Therefore, we have developed an in vitro promoter-reporter assay system to monitor the proliferation and differentiation of NSCs using recombinant lentiviruses. Four NSC-specific biomarkers can be monitored by infecting NSCs with recombinant lentiviruses such as synapsin1 for neuron differentiation, glial fibrillary acidic protein (GFAP) for astrocyte differentiation, myelin basic protein (MBP) for oligodendrocyte differentiation, and SOX2 for self-renewal.https://engagedscholarship.csuohio.edu/u_poster_2016/1044/thumbnail.jp

High-Throughput Assessment of Developmental Stages of NSCs via Promoter-Reporter Assay System Using Recombinant Lentiviruses

Many drugs and chemicals currently available have not been fully evaluated for their toxic effects on the developing brain. Expensive and low-throughput in vivo studies are still being used to evaluate developmental neurotoxicity (DNT). Thus, there is a need to develop an in vitro assay system which is economically feasible and high- throughput. Among various cellular models used for in vitro assay, human neural stem cells (NSCs) are highly desired due to their ability to self-renew and differentiate into neurons, astrocytes and oligodendrocytes. In vitro assessment of developmental stages (proliferation and differentiation) of human NSC is highly important to predict the in vivo effect of various chemicals on developing brain. However, conventional in vitro assay uses immunofluorescence staining to monitor changes in cell morphology and neural cell-specific biomarkers which can either be inaccurate or cumbersome. Therefore, we have developed an in vitro promoter-reporter assay system to monitor the proliferation and differentiation of NSCs using recombinant lentiviruses. Four NSC-specific biomarkers can be monitored by infecting NSCs with recombinant lentiviruses such as synapsin1 for neuron differentiation, glial fibrillary acidic protein (GFAP) for astrocyte differentiation, myelin basic protein (MBP) for oligodendrocyte differentiation, and SOX2 for self-renewal.https://engagedscholarship.csuohio.edu/u_poster_2016/1044/thumbnail.jp

P1: Assessment of Metabolism-Induced Hepatotoxicity on a 384-Pillar Plate

Microarray bioprinting technology has been explored to create miniaturized 3D cell cultures on a 384-pillar plate, which were combined with drug metabolizing enzymes (DMEs) and test compounds in a 384-well plate for metabolism-induced toxicity assays. Our goal in this study was to demonstrate rapid assessment of metabolism- induced toxicity on the 384-pillar plate and obtain reliable and highly predictive information on compound\u27s hepatotoxicity in vivo. Briefly, human cells including Hep3B human hepatoma cell line as well as human embryonic kidney 293 (HEK 293) cell were encapsulated in alginate-Matrigel on the 384-pillar plate. Test compounds and six different DMEs including cytochromes P450 (CYP450) and UDP-glucuronosyltransferase (UGT) were dispensed in the 384-well plate. By sandwiching the 384-pillar plate onto the 384-well plate, human cells were exposed to the compounds and their metabolites generated by DMEs. The cells were stained with luminescent and fluorescent dyes and IC50 values were calculated using the luminescence and fluorescence obtained. In summary, our approach allowed us to assess mechanisms of metabolism-induced toxicity in high throughput. Thus, the 384- pillar plate could be used as a high-throughput, early stage, microscale alternative to conventional in vitro multi-well plate platforms and provide a rapid and inexpensive assessment of metabolism-induced toxicity at early phases of drug development.https://engagedscholarship.csuohio.edu/u_poster_2017/1053/thumbnail.jp

Soil Classification Support for Farming Decisions

The techniques of data mining are extremely popular in the area of agriculture. Data mining involves the systematic analysis of huge information sets, and data processing in agricultural soil datasets is exciting and fashionable analysis space. The productive capability of a soil depends on soil fertility. Today, data processing is employed in a very large area and plenty of ready-to-wear data processing system product and domain specific data processing application software?s are obtainable, however data processing in agricultural soil knowledge sets may be a comparatively a young analysis field. In this paper, we offer internet base answer for the soil testing laboratories yet as free messages for the farmer that contains data like soil testing code, chemical that is important for the crop and additionally the knowledgeable recommendation. Additionally, farmers specify their next crop whereas they furnish their sample to scantiest therefore in keeping with next crop the chemical can recommend. The results supported the classification of contains that should be gif tin soil and in keeping with result report are generated