Jong Hwan Sung

A microfluidic device for a pharmacokinetic–pharmacodynamic (PK–PD) model on a chip

Drug discovery is often impeded by the poor predictability of in vitro assays for drug toxicity. One primary reason for this observation is the inability to reproduce the pharmacokinetics (PK) of drugs in vitro. Mathematical models to predict the pharmacokinetics-pharmacodynamics (PK-PD) of drugs are available, but have several limitations, preventing broader application. A microscale cell culture analog (microCCA) is a microfluidic device based on a PK-PD model, where multiple cell culture chambers are connected with fluidic channels to mimic multi-organ interactions and test drug toxicity in a pharmacokinetic-based manner. One critical issue with microfluidics, including the microCCA, is that specialized techniques are required for assembly and operation, limiting its usability to non-experts. Here, we describe a novel design, with enhanced usability while allowing hydrogel-cell cultures of multiple types. Gravity-induced flow enables pumpless operation and prevents bubble formation. Three cell lines representing the liver, tumor and marrow were cultured in the three-chamber microCCA to test the toxicity of an anticancer drug, 5-fluorouracil. The result was analyzed with a PK-PD model of the device, and compared with the result in static conditions. Each cell type exhibited differential responses to 5-FU, and the responses in the microfluidic environment were different from those in static environment. Combination of a mathematical modeling approach (PK-PD modeling) and an in vitro experimental approach (microCCA) provides a novel platform with improved predictability for testing drug toxicity and can help researchers gain a better insight into the drugs mechanism of action.

Purification and Characterization of α-l-Arabinopyranosidase and α-l-Arabinofuranosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium Metabolizing Ginsenoside Rb2 and Rc

ABSTRACT Two arabinosidases, α-l-arabinopyranosidase (no EC number) and α-l-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. α-l-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 μmol/min/mg.α -l-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 μmol/min/mg. The molecular mass ofα -l-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that ofα -l-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. α-l-Arabinopyranosidase and α-l-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40°C and pH 4.5 and 45°C, respectively. Both purified enzymes were potently inhibited by Cu2+ and p-chlormercuryphenylsulfonic acid.α -l-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinopyranoside, followed by ginsenoside Rb2. α-l-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-β-galactopyranoside or p-nitrophenyl-β-d-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified α-l-arabinosidases. This is the first reported purification ofα -l-arabinopyranosidase from an anaerobic Bifidobacterium sp.

Using physiologically-based pharmacokinetic-guided ''body-on-a-chip'' systems to predict mammalian response to drug and chemical exposure

The continued development of in vitro systems that accurately emulate human response to drugs or chemical agents will impact drug development, our understanding of chemical toxicity, and enhance our ability to respond to threats from chemical or biological agents. A promising technology is to build microscale replicas of humans that capture essential elements of physiology, pharmacology, and/or toxicology (microphysiological systems). Here, we review progress on systems for microscale models of mammalian systems that include two or more integrated cellular components. These systems are described as a “body-on-a-chip”, and utilize the concept of physiologically-based pharmacokinetic (PBPK) modeling in the design. These microscale systems can also be used as model systems to predict whole-body responses to drugs as well as study the mechanism of action of drugs using PBPK analysis. In this review, we provide examples of various approaches to construct such systems with a focus on their physiological usefulness and various approaches to measure responses (e.g. chemical, electrical, or mechanical force and cellular viability and morphology). While the goal is to predict human response, other mammalian cell types can be utilized with the same principle to predict animal response. These systems will be evaluated on their potential to be physiologically accurate, to provide effective and efficient platform for analytics with accessibility to a wide range of users, for ease of incorporation of analytics, functional for weeks to months, and the ability to replicate previously observed human responses.

In vitro microscale systems for systematic drug toxicity study

After administration, drugs go through a complex, dynamic process of absorption, distribution, metabolism and excretion. The resulting time-dependent concentration, termed pharmacokinetics (PK), is critical to the pharmacological response from patients. An in vitro system that can test the dynamics of drug effects in a more systematic way would save time and costs in drug development. Integration of microfabrication and cell culture techniques has resulted in ‘cells-on-a-chip’ technology, which is showing promise for high-throughput drug screening in physiologically relevant manner. In this review, we summarize current research efforts which ultimately lead to in vitro systems for testing drug’s effect in PK-based manner. In particular, we highlight the contribution of microscale systems towards this goal. We envision that the ‘cells-on-a-chip’ technology will serve as a valuable link between in vitro and in vivo studies, reducing the demand for animal studies, and making clinical trials more effective.

Fluorescence optical detection in situ for real-time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices

We describe an in situ fluorescence optical detection system to demonstrate real‐time and non‐invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real‐time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof‐of‐concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in MatrigelTM construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in MatrigelTM with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3‐Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system. Biotechnol. Bioeng. 2009; 104: 516–525

Microtechnology for Mimicking In Vivo Tissue Environment

Microtechnology provides a new approach for reproducing the in vivo environment in vitro. Mimicking the microenvironment of the natural tissues allows cultured cells to behave in a more authentic manner, and gives researchers more realistic platforms to study biological systems. In this review article, we discuss the physiochemical aspects of in vivo cellular microenvironment, and relevant technologies that can be used to mimic those aspects. Secondly we identify the core methods used in microtechnology for biomedical applications. Finally we examine the recent application areas of microtechnology, with a focus on reproducing the functions of specific organs, or whole-body response such as homeostasis or metabolism-dependent toxicity of drugs. These new technologies enable researchers to ask and answer questions in a manner that has not been possible with conventional, macroscale technologies.

Integration of in silico and in vitro platforms for pharmacokinetic–pharmacodynamic modeling

Importance of the field: Pharmacokinetic–pharmacodynamic (PK-PD) modeling enables quantitative prediction of the dose–response relationship. Recent advances in microscale technology enabled researchers to create in vitro systems that mimic biological systems more closely. Combination of mathematical modeling and microscale technology offers the possibility of faster, cheaper and more accurate prediction of the drugs effect with a reduced need for animal or human subjects. Areas covered in this review: This article discusses combining in vitro microscale systems and PK-PD models for improved prediction of drugs efficacy and toxicity. First, we describe the concept of PK-PD modeling and its applications. Different classes of PK-PD models are described. Microscale technology offers an opportunity for building physical systems that mimic PK-PD models. Recent progress in this approach during the last decade is summarized. What the reader will gain: This article is intended to review how microscale technology combined with cell cultures, also known as ‘cells-on-a-chip’, can confer a novel aspect to current PK-PD modeling. Readers will gain a comprehensive knowledge of PK-PD modeling and ‘cells-on-a-chip’ technology, with the prospect of how they may be combined for synergistic effect. Take home message: The combination of microscale technology and PK-PD modeling should contribute to the development of a novel in vitro/in silico platform for more physiologically-realistic drug screening.

Nanomaterial-Based Biosensor as an Emerging Tool for Biomedical Applications

The combination of nanomaterials and biological sensing elements to selectively recognize chemical or biological molecules has resulted in the development of novel nanobiosensors. Nanobiosensors offer several important advantages over conventional biological procedures, and could have a significant impact on humankind. Hence, the momentum toward building miniaturized, reliable, sensitive, and selective sensing instruments has focused on combining nanomaterials with biomolecules for detection of a wide range of analytes. In this article, we present an overview of the various nanomaterial-based biosensors that utilize different biological recognition elements for biomedical applications. In this review, several types of nanomaterial-based biosensors along with their applications are discussed, including the latest developments in the field of nanobiosensors for biomedical applications.

Real-time fluorescence detection of multiple microscale cell culture analog devices in situ

We investigated multiple microscale cell culture analog (μCCA) assays in situ with a high‐throughput imaging system that provides quantitative, nondestructive, and real‐time data on cell viability. Since samples do not move between measurements, captured images allow accurate time‐course measurements of cell population response and tracking the fate of each cell type on a quantitative basis. The optical system was evaluated by measuring the short‐term response to ethanol exposure and long‐term growth of drug‐resistant tumor cell lines with simultaneous samples.

Microfluidic assay-based optical measurement techniques for cell analysis: A review of recent progress.

Since the early 2000s, microfluidic cell culture systems have attracted significant attention as a promising alternative to conventional cell culture methods and the importance of designing an efficient detection system to analyze cell behavior on a chip in real time is raised. For this reason, various measurement techniques for microfluidic devices have been developed with the development of microfluidic assays for high-throughput screening and mimicking of in vivo conditions. In this review, we discuss optical measurement techniques for microfluidic assays. First of all, the recent development of fluorescence- and absorbance-based optical measurement systems is described. Next, advanced optical detection systems are introduced with respect to three emphases: 1) optimization for long-term, real-time, and in situ measurements; 2) performance improvements; and 3) multimodal analysis conjugations. Moreover, we explore presents future prospects for the establishment of optical detection systems following the development of complex, multi-dimensional microfluidic cell culture assays to mimic in vivo tissue, organ, and human systems.