“Body-on-a-Chip” : Improving Drug Development

Michael L. SHULER 한국생물공학회 2016 한국생물공학회 학술대회 Vol.2016 No.4

Growth of Endothelial Cells on Microfabricated Silicon Nitride Membranes for an In Vitro Model of the Blood-brain Barrier

Michael L. Shuler,Sarina G. Harris 한국생물공학회 2003 Biotechnology and Bioprocess Engineering Vol.8 No.4

The blood-brain barrier (BBB) is composed of the brain capillaries, which are lined by endothelial cells displaying extremely tight intercellular junctions. Several attempts at creating an in vitro model of the BBB have been met with moderate success as brain capillary endothelial cells lose their barrier properties when isolated in cell culture. This may be due to a lack of recreation of the in vivo endothelial cellular environment in these models, including nearly constant contact with astrocyte foot processes. This work is motivated by the hypothesis that growing endothelial cells on one side of an ultra-thin, highly porous membrane and differentiating astrocyte or astrogliomal cells on the opposite side will lead to a higher degree of interaction between the two cell types and therefore to an improved model. Here we describe our initial efforts towards testing this hypothesis including a procedure for membrane fabrication and methods for culturing endothelial cells on these membranes. We have fabricated a 1 mm thick, 2.0 mm pore size, and ~55% porous membrane with a very narrow pore size distribution from low-stress silicon nitride (SiN) utilizing techniques from the microelectronics industry. We have developed a base, acid, autoclave routine that prepares the membranes for cell culture both by cleaning residual fabrication chemicals from the surface and by increasing the hydrophilicity of the membranes (confirmed by contact angle measurements). Gelatin, fibronectin, and a 50/50 mixture of the two proteins were evaluated as potential basement membrane protein treatments prior to membrane cell seeding. All three treatments support adequate attachment and growth on the membranes compared to the control.

Enhanced In-situ Mobilization and Biodegradation of Phenanthrene from Soil by a Solvent/Surfactant System

Kim, Eun Ki,Ahn, Ik Sung,L. W. Lion,M. L. Shuler 한국미생물 · 생명공학회 2001 Journal of microbiology and biotechnology Vol.11 No.4

The mobilization and biodegradation of phenanthrene in soil was enhanced by using paraffin oil, which was stabilized by the addition of a surfactant (Brij 30). The ratio of paraffin oil/Brij 30 was determined by measuring the change in the critical micelle concentration. When only surfactant was used, the stabilized paraffin oil emulsion could dissolve more phenanthrene in the water phase. Column experiment showed increased phenanthrene mobilization from the contaminated soil. The phenanthrene mobilized in the paraffin oil/Brij 30 emulsion was biodegraded faster than that in water phase or surfactant solution. This result indicates that a paraffin oil/surfactant system can be effectively used for the removal of PAH from contaminated soil.

Growth of Endothelial Cells on Microfabricated Silicon Nitride Membranes for an In Vitro Model of the Blood-brain Barrier

Harris, Sarina G.,Shuler, Michael L. The Korean Society for Biotechnology and Bioengine 2003 Biotechnology and Bioprocess Engineering Vol.8 No.4

The blood-brain barrier (BBB) is composed of the brain capillaries, which are lined by endothelial cells displaying extremely tight intercellular junctions. Several attempts at creating an in vitro model of the BBB have been met with moderate success as brain capillary endothelial cells lose their barrier properties when isolated in cell culture. This may be due to a lack of recreation of the in vivo endothelial cellular environment in these models, including nearly constant contact with astrocyte foot processes. This work is motivated by the hypothesis that growing endothelial cells on one side of an ultra-thin, highly porous membrane and differentiating astrocyte or astrogliomal cells on the opposite side will lead to a higher degree of interaction between the two cell types and therefore to an improved model. Here we describe our initial efforts towards testing this hypothesis including a procedure for membrane fabrication and methods for culturing endothelial cells on these membranes. We have fabricated a 1 $\mu\textrm{m}$ thick, 2.0 $\mu\textrm{m}$ pore size, and 55% porous membrane with a very narrow pore size distribution from low-stress silicon nitride (SiN) utilizing techniques from the microelectronics industry. We have developed a base, acid, autoclave routine that prepares the membranes for cell culture both by cleaning residual fabrication chemicals from the surface and by increasing the hydrophilicity of the membranes (confirmed by contact angle measurements). Gelatin, fibronectin, and a 50/50 mixture of the two proteins were evaluated as potential basement membrane protein treatments prior to membrane cell seeding. All three treatments support adequate attachment and growth on the membranes compared to the control.

Translational Inhibition Enhances mRNA Stability of the Retinoic Acid (RA) Responsive Keratin 18 Gene Expressed by Oral Squamous Cell Carcinoma Lines but Blocks RA Dependent Stabilization

Crowe, David L.,Shuler, Charles F. Korean Academy of Oral Biology and the UCLA Dental 1997 International Journal of Oral Biology Vol.22 No.3

The keratin intermediate filament K18 has been implicated in tumor cell invasion and resistance to chemotherapy. K18 is normally not expressed in adult stratified squamous epithelium, but is reactivated in carcinomas arising from this tissue (SCCs). To understand the role of K18 in the malignant phenotype of SCCs, it is necessary to understand how expression of this gene is regulated in these cells. The vitamin A metabolite retinoic acid (R4) regulates keratin expression through both transcriptional and posttranscriptional mechanisms. RA has been shown to stabilize keratin mRNAs by as yet undefined mechanisms. Studies have shown that translation plays an important role in regulating mRNA stability. To begin to elucidate the mechanisms by which R4 exerts its posttranscriptional effects, we examined how translational inhibition affects the mRNA stability of the R4 responsive keratin 18 gene (K18) in SCC25 cells. Cycloheximide (CHX)-induced translational inhibition enhanced K18 gene expression by mRNA stabilization. CHX blocked the R4 induced stabilization of K18 transcripts, indicating that a newly synthesized or labile protein was required for R4 to mediate this effect. These studies point to the existence of multiple mRNA stabilization pathways in these cells and provide further evidence for the existence of R4 dependent factors that regulate target gene expression at the posttranscriptional level.

아세트아미노펜 독성평가를 위한 μCCA-μGI 디바이스의 개발

장정윤,Chang Jung-Yun,Shuler Michael L. 한국약제학회 2006 Journal of Pharmaceutical Investigation Vol.36 No.4

Deficiencies in the early ADMET(absorption, distribution, metabolism, elimination and toxicity) information on drug candidate extract a significant economic penalty on pharmaceutical firms. Microscale cell culture analogue-microscale gastrointestinal(${\mu}CCA-{\mu}GI$) device using Caco 2, L2 and HEp G2/C3A cells, which mimic metabolic process after absorption occurring in humans was used to investigate the toxicity of the model chemical, acetaminophen(AAP). The toxicity of acetaminophen determined after induction of CYP 1A1/2 in Caco 2 cells was not significant. In a coculture system, although no significant reduction in viability of HEp G2/C3A and L2 cells was found, approximately 5 fold increase in the CYP 1A1/2 activity was observed. These results appear to be related to organ-organ interaction. The oral administration of a drug requires addition of the absorption process through small intestine to the current ${\mu}CCA$ device. Therefore, a perfusion coculture system was employed for the evaluation of the absolution across the small intestine and resulting toxicity in the liver and lung. This system give comprehensive and physiologic information on oral uptake and resulting toxicity as in the body. The current ${\mu}CCA$ device can be used to demonstrate the toxic effect due to organ to organ interaction after oral administration,

원보 : 아세트아미노펜 독성평가를 위한 μCCA-μGI 디바이스의 개발

장정윤 ( Jung Yun Chang ),( Michael L. Shuler ) 한국약제학회 2006 Journal of Pharmaceutical Investigation Vol.36 No.4

Investigation of portable in situ fluorescence optical detection for microfluidic 3D cell culture assays.

Choi, Jong-ryul,Sung, Jong Hwan,Shuler, Michael Louis,Kim, Donghyun Optical Society of America 2010 Optics letters Vol.35 No.9

<P>A portable fluorescence optical detection system was developed to demonstrate real-time in situ analysis of cells that are three-dimensionally cultured in an extracellular matrix under microfluidic environment. The system was designed to provide a large field of view in the lateral plane to average out cellular processes in an axial layer and simultaneously diffraction-limited axial resolution. In this proof-of-concept study, the detection system was applied to quantitative analyses of short-term measurements of cell staining and cell cytotoxicity and long-term monitoring of a cell-invasion assay. For assays, colon cancer cells were cultured in a Matrigel or alginate matrix. The measured data were largely consistent with predicted results and revealed quantitatively cell dynamics specific to 3D cell cultures. The detection system has a potential as a single package to investigate 3D cultures in a microfluidic system.</P>

Inhibition of Acidic Ribosomal Phosphoprotein P1 Expression Reduces Translational Efficiency and Cell Proliferation of Mouse Embryo Fibroblasts

Crowe, David L.,Cui, Xiao-Mei,Shuler, Charles F. Korean Academy of Oral Biology and the UCLA Dental 1998 International Journal of Oral Biology Vol.23 No.2

Acidic ribosomal proteins interact with multiple translation factors to regulate protein synthesis in prokaryotic and eukaryotic organisms. We have isolated and sequenced the complete cDNA of the mouse acidic ribosomal phosphoprotein P1 (mARP P1). The 495 base pair cDNA encodes a single open reading frame of 114 amino acids. Northern amalysis revealed a messenger RNA of appropriate size expressed in all tissues. Stable transfection of 3T3 cells with a vector containing an antisense mARP P1 construct reduced expression of the mRNA by 90%. Cells expressing the antisense construct did not incorporate radiolabeled methionine as efficiently as 3T3s transfected with vector alone. Cells expressing the antisense construct were also less prolifeeerative in vitro. Anlaysis of the deduced sequence and comparison to P proteins from other organisms revealed homology to the L12eILA protein in yeast. The carboxy termini of these proteins are highly conserved across species. The central alanine rich region of the mouse P1 protein likely evlived through duplications of a conserved sequence and may represent evolutionary modifications of an ancestral gene.

Photocatalytic hydrogen evolution from biomass conversion

Davis Kayla Alicia,Yoo Sunghoon,Shuler Eric W.,Sherman Benjamin D.,이승현,Leem Gyu 나노기술연구협의회 2021 Nano Convergence Vol.8 No.6

Biomass has incredible potential as an alternative to fossil fuels for energy production that is sustainable for the future of humanity. Hydrogen evolution from photocatalytic biomass conversion not only produces valuable carbon-free energy in the form of molecular hydrogen but also provides an avenue of production for industrially relevant biomass products. This photocatalytic conversion can be realized with efficient, sustainable reaction materials (biomass) and inexhaustible sunlight as the only energy inputs. Reported herein is a general strategy and mechanism for photocatalytic hydrogen evolution from biomass and biomass-derived substrates (including ethanol, glycerol, formic acid, glucose, and polysaccharides). Recent advancements in the synthesis and fundamental physical/mechanistic studies of novel photocatalysts for hydrogen evolution from biomass conversion are summarized. Also summarized are recent advancements in hydrogen evolution efficiency regarding biomass and biomass-derived substrates. Special emphasis is given to methods that utilize unprocessed biomass as a substrate or synthetic photocatalyst material, as the development of such will incur greater benefits towards a sustainable route for the evolution of hydrogen and production of chemical feedstocks.