Non-destructive on-chip cell sorting system with real-time microscopic image processing
© Takahashi et al; licensee BioMed Central Ltd. 2004
Received: 13 December 2003
Accepted: 03 June 2004
Published: 03 June 2004
Studying cell functions for cellomics studies often requires the use of purified individual cells from mixtures of various kinds of cells. We have developed a new non-destructive on-chip cell sorting system for single cell based cultivation, by exploiting the advantage of microfluidics and electrostatic force. The system consists of the following two parts: a cell sorting chip made of poly-dimethylsiloxane (PDMS) on a 0.2-mm-thick glass slide, and an image analysis system with a phase-contrast/fluorescence microscope. The unique features of our system include (i) identification of a target from sample cells is achieved by comparison of the 0.2-μm-resolution phase-contrast and fluorescence images of cells in the microchannel every 1/30 s; (ii) non-destructive sorting of target cells in a laminar flow by application of electrostatic repulsion force for removing unrequited cells from the one laminar flow to the other; (iii) the use of agar gel for electrodes in order to minimize the effect on cells by electrochemical reactions of electrodes, and (iv) pre-filter, which was fabricated within the channel for removal of dust contained in a sample solution from tissue extracts. The sorting chip is capable of continuous operation and we have purified more than ten thousand cells for cultivation without damaging them. Our design has proved to be very efficient and suitable for the routine use in cell purification experiments.
Developing of a cell based screening assay often requires identification and isolation of particular cells from a mixture of various kinds of cells. Moreover, in order to obtain reproducible data on cells, reliable and non-destructive purification of cells is essential. Efficient and rapid sorting of cells has been accomplished with techniques such as fluorescence-activated cell sorting (FACS) , magnetic-activated cell separation (MACS), automated single-cell sorting using dual-beam optical trapping, differential adhesion cell sorting , and micro-fabricated fluorescence-activated cell sorting . These conventional cell sorters can be used for purification of individual cells, but are not ideal techniques and have a number of disadvantages.. For example, FACS can damage cells during destructive droplet generation, and the detection (based on the non-direct scattering) has poor cell recognition performance. Other conventional techniques also have disadvantages with regard to their cost, efficiency, response speed, separate resolution, and adaptability. Furthermore, if cells are to be cultivated after subsequent sorting, the damage to cells caused by to the sorting process should be minimized. Advantages offered by application of microfluidics include reduced sample volumes and reaction times, lowered space requirements and operational costs . In micro- and nano-technology liquid flow is mostly laminar and this further facilitates making microscopic sorting devises for biomedical applications. We have developed new on-chip microfluidic cell sorting system, which overcomes problems associated with the conventional cell sorting techniques. Our system directly monitors and recognizes cells with specific index using phase-contrast/fluorescence microscopy and image processing, and it can rapidly and safely sort them according to the index (shape, and spatial distribution of fluorescent dye of the specific antibody marker, and so on) in a laminar flow using the electrostatic force. The electric force is applied only to the cells which have to be removed to waste; the target cells simply flow down the laminar flow and do not receive any stimulation from non-contact forces. In this paper, we explain the design of the on-chip cell sorter system and give the results of an experimental implementation of our design.
Results and Discussion
Design of on-chip cell sorting system
Cell sorter chip design and process
Principle of sorting procedure
Our cell sorting system differs form other available systems in that only the cells, which have to be removed, were affected by the electrostatic force. The target cells had no force applied and therefore had minimal or no damage done to them. Moreover, the area of the electric field is limited to the cell sorting area, because the electric current flows only between the two gel electrodes. Thus cells out of the area receive no significant damage from the electric stimulation.
Installation of phase-contrast image recognition into the sorting system
Composition of agar-gel for low resistance electrode
Construction of filter for purification of cells and removal of dust in the sample
Sorting accuracy of the on-chip cell sorter system
We have developed anon-chip cell sorting system that can separate target cells mildly and reliably using our original method and bio-compatible materials, such as agarose gel that is used as the electrode for the generation of electrostatic force in the flow channel. Cell sorting is based on the image analysis processed automatically by computer. A micro-sized filter for a dust-free flow is fabricated in order to purify sample cells and avoid blocking of the channels by dust particles. Using our system we have achieved 100% accurate sorting. We plan to extend our approach to single cell analysis and regenerative medicine.
Financial support, in part by the Japan Science and Technology Organization (JST) and by Grants-in-Aids for Science Research from the Ministry of Education, Science and Culture of Japan, is gratefully acknowledged.
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