Development of mechanical isolation technique for target cell and its applications

Abstract

Cells are the basic structural and functional unit of organism and it contains many valuable information. The single cells have heterogeneity even in isogenic cultures with respect to the size, growth state, and gene expression. To obtain the correct information, separation and isolation of specific cells in heterogeneous cell mixture have been studied in biology, medicine, and biopharmaceuticals using microfluidic device. Basically, the cells are affected by the motion of fluid because cells live and exist in biological fluid. When the fluid flows in specific space, the cells are dragged by fluid shear force. Therefore, controlling of shear force can be one of the important factors to separate the target cells. In this study, the strategies to modify the flow pattern were proposed to study the cell movement and to show its applicability. First, the fabrication methods were studied to induce the specific flow pattern. Not only the general method (photo- and soft- lithography) to fabricate the PDMS-based microfluidic device, but also the CNC milling and 3D printing technique to produce the 3D structures were adopted to generate the desired flow patterns. Secondly, isolation of bacteria cell was achieved by only using the microstructures in channel. The combination of hydrogel and microfluidic system have been adopted to observe the response of cell. Typically, the polymer-based hydrogel is used to provide the specific environment. However, adoption of hydrogel leads the cellular aggregation and reduces the efficiency of bacteria separation. To resolve the bacteria aggregation and the single-cell level of separation, we adopted structures for the flow control. Micropillar was installed in microfluidic channel and it makes the flow modification. Polymer induced cell aggregates were re-dispersed by the collision with pillar, shear force and elongation force around the pillar. The fraction of single cells contained in a single droplet depends on the cell dispersion in channel. By the adoption of pillar, the cells were well dispersed and encapsulated up to 70% of single cell in the droplets. As a related investigation, shear and elongation forces enhanced device was designed and combined with droplet PCR technique. Evaluation of the design was achieved by computational fluid dynamics program. Parabolic contraction structure also showed the effective cell dispersion capability. Moreover, dispersed cell was eventually encapsulated in droplet and identified as foodborne pathogen by droplet PCR technique. Thirdly, circulating flow inducement and its applications were investigated using external vibration force. The filtration system have been used broadly in many chemical and biological industries. However, the incidence of filter fouling brought the serious issues to the system, such as dismantlement of the device or filter change. Also, the blood solution which contains red blood cell, white blood cell and plasma leads the filter clogging issue. The blood is known as the mixture of important biomarkers. Among the blood cells, white blood cell can be used as a marker for viral infection (Ebola, Influenza, and Dengue fever etc.). Furthermore, the sepsis can be diagnosed by the detection of the bacteria in the blood. The size-based mechanical filtration has been considered as the most noted method due to the size differences of red blood cell, white blood cell and bacteria. However, the use of high concentrated cell mixture inevitably lead the pore clogging with loss of its permeability. To overcome such problems, shear force was intentionally increased using circulating flow by vibration. The blood cells are differently influenced by shear force based on cell size and density, and therefore, easily separated through the filter membrane. Based on these studies, the fluidic device which has the desired fluid pattern can be fabricated using various methods including photolithography, mechanical milling and 3D printing. Modification of internal structures and external force were adopted to induce the desired flow patterns. The effect of the modified flow patterns on cell separation was analyzed and applied to single cell isolation and filtration. These flow inducement techniques would be useful to resolve the inefficiency and to improve the conventional chemical and biological processes.