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The Study of Cell Capture and Analysis of Drug Effects on Microfluidic Device

Author: GaoDan
Tutor: GuoGuangSheng；LinJinMing
School: Beijing University of Chemical Technology
Course: Chemical Engineering and Technology
Keywords: microfluidic cell isolation immunomagnetic photolithography cell apoptosis mass spectrometry drug metabolism
CLC: R96
Type: PhD thesis
Year: 2011
Downloads: 134
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Abstract

Cells are the basic structural units and functional units of living organisms. Cell research is the basis for life science, and is of great significance for human development. The study of life activities including cell morphology, propagate and differentiation, decrepitude and apoptosis, migration, information transfer between cells, cellular secretion is very valuable. It is helpful to explore the laws and nature of life activities, diagnosis and treatment of disease, and so on. However, traditional cell research methods have several difficulties as follows:(1) It is difficult to manipulate the microscale cell; (2) The intercellular contents are small and complex. It contains a large number of biological information which requires high-throughput analysis and high sensitivity detection; (3) Traditional cell culture methods could not offer complex and microscale growing environment. Fortunately, the appearance of microfluidic technology can conquer these disadvantages in some extent. Microfluidic chip due to its characteristic of microscale has several advantages including small reagents consumption, fast analysis, ease of integration and miniaturization, high degree of automation. It has been widely used in several areas, such as analytical chemistry and life science. In recent years, the application of microfluidic technology in cell research has been attracted increased attention.The thesis focused on the urgent requirement of cell analysis in the field of life sciences, and the aim of solving the problems in traditional cell analysis methods. We carried out cell-based researches related to disease diagnosis and drug development on microfluidic device. The contents were summarized as follows:1. Magnetic bead based immunoassay for sorting of CD4+ T lymphocytes on microfluidic device for AIDS diagnosis. A Y-intersection microchannel containing an enlarged reaction chamber was flexible designed for separating and counting CD4+T lymphocytes by using a one-step immunomagnetic method. The cell sample was obtained from the thymus of mouse. Anti-CD4 magnetic beads were firstly immobilized in the reaction chamber under external magnetic field, and then CD4+T lymphocytes were captured and separated by antibody and cell surface antigen. CD4+ T lymphocytes count was only needed an ordinary microscope. In addition, the enlarged reaction chamber with two symmetrical cone-shaped ends was used to decrease the dead volume of the fluid, which is helpful for cell capture. In order to obtain the maximum cell capture efficiency, relative parameters including section area of the reaction chamber and injection flow rate of the cell suspension were investigated. It avoided the complex sample pre-treatment in our experiments, and the entire analysis time only needed about 15 min. This established CD4+ T lymphocytes counting platform had the potential to reduce the cost for HIV diagnosis which promoted the use in resource-limited setting areas.2. A microfluidic approach for anticancer drug analysis based on hydrogel encapsulated cells. The coupling of photolithography with microfluidic chip realizes the fabrication of three dimensional hydrogel microstructures in the microchannels with controlled position and shape by using a fluorescence microscope. By using this approach, human hepatoma HepG2 cells and human lung epithelial A549 cells were simultaneously immobilized inside three-dimensional hydrogel microstructures on a same array, and they were identified with different shapes. Microarrays of hydrogel encapsulated many kinds of living cells could also be fabricated in a microchannel, which offered the potential for high-throughput assays. In addition, the prepolymer composition and crosslinking parameters that influenced cell viability inside photocrosslinked hydrogels were investigated. By optimizing these conditions, the cell viabilities were nearly reached up to 100%. For the long-term culture of the encapsulated cells, the majority of these cells could keep viable for at least three days, which were able to carry out cell-based assays. In addition, two anticancer drugs were used to stimulate HepG2 and A549 cells encapsulated inside hydrogel microstructures. The variation of two intracellular redox parameters containing glutathione and reactive oxygen species was investigated. The results showed that these two drugs exhibited distinct effects on intercellular redox state. It is indicated that the selectivity of these drugs in inducing cell apoptosis. This established platform provides a simple, fast and high-throughput method for monitoring the effect of anticancer drugs on tumor cells, which has an important application in fundamental biomedical research.3. We developed a new technology by the combination of microfluidic device with mass spectrometry for the study of drug metabolism. The microfluidic device contains two different functional units:(1) a circular cell culture chamber; (2) a micro-solid phase extraction channel for sample cleanup and concentration prior to mass analysis. By connecting the two separated microchannels with polyethylene tubes, drug metabolism studies related to functional units, containing cell culture, metabolism generation, sample pretreatment and detection were all integrated into a microfluidic device. In order to assess the feasibility of a drug metabolism study on the microfluidic device, the metabolism of Vitamin E in A549 cells which was used as a model was studied. Two metabolites were successfully detected with ESI-Q-TOF-MS, and the entire analysis time only needed 8 min. By integrating several parallel channels, multiple samples can be simultaneously desalted and concentrated. The total sample pretreatment time only needed about 15 min, and solvent consumption was reduced to less than 100μL. All this demonstrated that the established microchip and mass spectrometry combination system has a potential application in drug development. 4. An integrated microfluidic device was developed for high-throughput cell cytotoxicity screening and drug metabolite characterization with an online electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF-MS) simultaneously. The microfluidic device was consisted of a multiple gradient generator following by array of microscale cell culture chambers, and integrated on-chip SPE columns. By using the combination system, the process of liquid diffusion and mixing, cell cultivation, cell stimulation, cell labeling, drug absorption and metabolism generation can be realized. To validate the feasibility of simultaneous study of apoptosis, drug absorption and metabolism on the microfluidic device, metabolism of methotrexate and its effect on cells were firstly investigated. With the increasing concentration of drugs, the percentage of apoptotic cells appeared in the dose-dependent fashion. Due to the contents of metabolites exceeded the lowest detection limit of ESI-Q-TOF MS, the contents of drug absorption in cells was finally detected. In addition, intracellular higher drug concentration, which resulted in increased cell cytotoxicity, was also proved on this device. In addition, two kinds of microchannel configurations (three-layer microfluidic device and integrated pneumatic microvalves on-chip) were designed, so that the procedures of two different functional units could be realized on a microfluidic system, which provided an automated plateform for drug analysis.

The Study of Cell Capture and Analysis of Drug Effects on Microfluidic Device

Abstract

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