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Application of optically-induced-dielectrophoresis in microfluidic system for purification of circulating tumour cells for gene expression analysis- Cancer cell line model

View Article: PubMed Central - PubMed

ABSTRACT

Circulating tumour cells (CTCs) in a blood circulation system are associated with cancer metastasis. The analysis of the drug-resistance gene expression of cancer patients’ CTCs holds promise for selecting a more effective therapeutic regimen for an individual patient. However, the current CTC isolation schemes might not be able to harvest CTCs with sufficiently high purity for such applications. To address this issue, this study proposed to integrate the techniques of optically induced dielectrophoretic (ODEP) force-based cell manipulation and fluorescent microscopic imaging in a microfluidic system to further purify CTCs after the conventional CTC isolation methods. In this study, the microfluidic system was developed, and its optimal operating conditions and performance for CTC isolation were evaluated. The results revealed that the presented system was able to isolate CTCs with cell purity as high as 100%, beyond what is possible using the previously existing techniques. In the analysis of CTC gene expression, therefore, this method could exclude the interference of leukocytes in a cell sample and accordingly contribute to higher analytical sensitivity, as demonstrated in this study. Overall, this study has presented an ODEP-based microfluidic system capable of simply and effectively isolating a specific cell species from a cell mixture.

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Related in: MedlinePlus

Schematic illustration of the overall cancer cell isolation processes (I) Fluorescent microscopic observation was performed in the CTC isolation zone to detect the cancer cell images (green dots) in a dynamic cell suspension flow; (II) the cell suspension flow was temporarily suspended when the cancer cells (green dots) were observed in the CTC isolation zone; (III) fluorescent microscopy operations were performed to observe the leukocytes (red dots), cancer cells (green dots) and (IV) all nucleated cells (blue dots); (V) the target cancer cells [i.e., EpCAM marker- and Hoechst dye-positive (green and blue dots) and CD45 marker-negative images (non-red dots)] were precisely positioned under light field microscopic imaging through the abovementioned contradistinction; (VI) ODEP force-based cell manipulation was performed to separate the cancer cells targeted from the leukocytes: hollow circular light images (ID: 40 μm; light bandwidth: 40 μm) were used to enclose the target cancer cells, and a long rectangular light bar (bar length: 1000 μm; bar width: 150 μm) was used to manipulate the leukocytes; (VII) the long rectangular light bar was moved (moving velocity: 100 μm s−1) to sweep all unenclosed leukocytes to the one side of the main microchannel leaving the enclosed cancer cells in the same positions; (VIII) the hollow circular light images were moved (moving velocity: 50 μm s−1) to manipulate the enclosed cancer cells to the side microchannel for collection; (IX)-(X) another moving rectangular light bar (bar length: 1000 μm; bar width: 150 μm; moving velocity: 100 μm s−1) was used to transport the cancer cells collected to a site near the through-hole for harvesting.
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f2: Schematic illustration of the overall cancer cell isolation processes (I) Fluorescent microscopic observation was performed in the CTC isolation zone to detect the cancer cell images (green dots) in a dynamic cell suspension flow; (II) the cell suspension flow was temporarily suspended when the cancer cells (green dots) were observed in the CTC isolation zone; (III) fluorescent microscopy operations were performed to observe the leukocytes (red dots), cancer cells (green dots) and (IV) all nucleated cells (blue dots); (V) the target cancer cells [i.e., EpCAM marker- and Hoechst dye-positive (green and blue dots) and CD45 marker-negative images (non-red dots)] were precisely positioned under light field microscopic imaging through the abovementioned contradistinction; (VI) ODEP force-based cell manipulation was performed to separate the cancer cells targeted from the leukocytes: hollow circular light images (ID: 40 μm; light bandwidth: 40 μm) were used to enclose the target cancer cells, and a long rectangular light bar (bar length: 1000 μm; bar width: 150 μm) was used to manipulate the leukocytes; (VII) the long rectangular light bar was moved (moving velocity: 100 μm s−1) to sweep all unenclosed leukocytes to the one side of the main microchannel leaving the enclosed cancer cells in the same positions; (VIII) the hollow circular light images were moved (moving velocity: 50 μm s−1) to manipulate the enclosed cancer cells to the side microchannel for collection; (IX)-(X) another moving rectangular light bar (bar length: 1000 μm; bar width: 150 μm; moving velocity: 100 μm s−1) was used to transport the cancer cells collected to a site near the through-hole for harvesting.

Mentions: In this study, the combination of ODEP force-based cell manipulation and laminar flow in a microfluidic system was established (Fig. 1) for CTC isolation based on the working scheme described in Fig. 2. With the recent progress in Bio-MEMS (Bio-Micro-Electro-Mechanical System) technology, a wide variety of novel approaches such as acoustophoresis32, magnetophoresis33, thermophoresis34, dielectrophoresis (DEP)29, and optically induced-dielectrophoresis (ODEP)35 have been proposed for the manipulation of biological substances (e.g., cells36, bacteria37, or DNA38). Among them, DEP force-based cell manipulation has been widely adopted for various applications2939. However, it normally requires a costly, time-consuming, and technically demanding microfabrication process to create a unique metal electrode layout that is specific to the application. The key technical merit of the ODEP force-based technique is that it can easily and quickly create or modify an electrode layout through the control of optical patterns, acting as a virtual electrode40. In operation, one can simply use a commercial digital projector to display optical images on the ODEP system to manipulate cells in a simple, flexible and user-friendly manner through a computer-interfaced control36. In this study, moreover, the inherent nature of laminar flow in a microfluidic system was used to transport a cell suspension in the main microchannel (Fig. 2). This allowed the biological cells to be delivered unidirectionally without cross-contamination of the sample caused by fluidic turbulent flow. Furthermore, immunofluorescent microscopic observation was conducted in the CTC isolation zone to specifically identify and position the target cancer cells. This step was followed by implementing ODEP force to precisely separate the cells of interest from the cell mixture, as described in Fig. 2. Through the specific biochemical differentiation of cell identity, CTC isolation with higher cell purity can be achieved.


Application of optically-induced-dielectrophoresis in microfluidic system for purification of circulating tumour cells for gene expression analysis- Cancer cell line model
Schematic illustration of the overall cancer cell isolation processes (I) Fluorescent microscopic observation was performed in the CTC isolation zone to detect the cancer cell images (green dots) in a dynamic cell suspension flow; (II) the cell suspension flow was temporarily suspended when the cancer cells (green dots) were observed in the CTC isolation zone; (III) fluorescent microscopy operations were performed to observe the leukocytes (red dots), cancer cells (green dots) and (IV) all nucleated cells (blue dots); (V) the target cancer cells [i.e., EpCAM marker- and Hoechst dye-positive (green and blue dots) and CD45 marker-negative images (non-red dots)] were precisely positioned under light field microscopic imaging through the abovementioned contradistinction; (VI) ODEP force-based cell manipulation was performed to separate the cancer cells targeted from the leukocytes: hollow circular light images (ID: 40 μm; light bandwidth: 40 μm) were used to enclose the target cancer cells, and a long rectangular light bar (bar length: 1000 μm; bar width: 150 μm) was used to manipulate the leukocytes; (VII) the long rectangular light bar was moved (moving velocity: 100 μm s−1) to sweep all unenclosed leukocytes to the one side of the main microchannel leaving the enclosed cancer cells in the same positions; (VIII) the hollow circular light images were moved (moving velocity: 50 μm s−1) to manipulate the enclosed cancer cells to the side microchannel for collection; (IX)-(X) another moving rectangular light bar (bar length: 1000 μm; bar width: 150 μm; moving velocity: 100 μm s−1) was used to transport the cancer cells collected to a site near the through-hole for harvesting.
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f2: Schematic illustration of the overall cancer cell isolation processes (I) Fluorescent microscopic observation was performed in the CTC isolation zone to detect the cancer cell images (green dots) in a dynamic cell suspension flow; (II) the cell suspension flow was temporarily suspended when the cancer cells (green dots) were observed in the CTC isolation zone; (III) fluorescent microscopy operations were performed to observe the leukocytes (red dots), cancer cells (green dots) and (IV) all nucleated cells (blue dots); (V) the target cancer cells [i.e., EpCAM marker- and Hoechst dye-positive (green and blue dots) and CD45 marker-negative images (non-red dots)] were precisely positioned under light field microscopic imaging through the abovementioned contradistinction; (VI) ODEP force-based cell manipulation was performed to separate the cancer cells targeted from the leukocytes: hollow circular light images (ID: 40 μm; light bandwidth: 40 μm) were used to enclose the target cancer cells, and a long rectangular light bar (bar length: 1000 μm; bar width: 150 μm) was used to manipulate the leukocytes; (VII) the long rectangular light bar was moved (moving velocity: 100 μm s−1) to sweep all unenclosed leukocytes to the one side of the main microchannel leaving the enclosed cancer cells in the same positions; (VIII) the hollow circular light images were moved (moving velocity: 50 μm s−1) to manipulate the enclosed cancer cells to the side microchannel for collection; (IX)-(X) another moving rectangular light bar (bar length: 1000 μm; bar width: 150 μm; moving velocity: 100 μm s−1) was used to transport the cancer cells collected to a site near the through-hole for harvesting.
Mentions: In this study, the combination of ODEP force-based cell manipulation and laminar flow in a microfluidic system was established (Fig. 1) for CTC isolation based on the working scheme described in Fig. 2. With the recent progress in Bio-MEMS (Bio-Micro-Electro-Mechanical System) technology, a wide variety of novel approaches such as acoustophoresis32, magnetophoresis33, thermophoresis34, dielectrophoresis (DEP)29, and optically induced-dielectrophoresis (ODEP)35 have been proposed for the manipulation of biological substances (e.g., cells36, bacteria37, or DNA38). Among them, DEP force-based cell manipulation has been widely adopted for various applications2939. However, it normally requires a costly, time-consuming, and technically demanding microfabrication process to create a unique metal electrode layout that is specific to the application. The key technical merit of the ODEP force-based technique is that it can easily and quickly create or modify an electrode layout through the control of optical patterns, acting as a virtual electrode40. In operation, one can simply use a commercial digital projector to display optical images on the ODEP system to manipulate cells in a simple, flexible and user-friendly manner through a computer-interfaced control36. In this study, moreover, the inherent nature of laminar flow in a microfluidic system was used to transport a cell suspension in the main microchannel (Fig. 2). This allowed the biological cells to be delivered unidirectionally without cross-contamination of the sample caused by fluidic turbulent flow. Furthermore, immunofluorescent microscopic observation was conducted in the CTC isolation zone to specifically identify and position the target cancer cells. This step was followed by implementing ODEP force to precisely separate the cells of interest from the cell mixture, as described in Fig. 2. Through the specific biochemical differentiation of cell identity, CTC isolation with higher cell purity can be achieved.

View Article: PubMed Central - PubMed

ABSTRACT

Circulating tumour cells (CTCs) in a blood circulation system are associated with cancer metastasis. The analysis of the drug-resistance gene expression of cancer patients’ CTCs holds promise for selecting a more effective therapeutic regimen for an individual patient. However, the current CTC isolation schemes might not be able to harvest CTCs with sufficiently high purity for such applications. To address this issue, this study proposed to integrate the techniques of optically induced dielectrophoretic (ODEP) force-based cell manipulation and fluorescent microscopic imaging in a microfluidic system to further purify CTCs after the conventional CTC isolation methods. In this study, the microfluidic system was developed, and its optimal operating conditions and performance for CTC isolation were evaluated. The results revealed that the presented system was able to isolate CTCs with cell purity as high as 100%, beyond what is possible using the previously existing techniques. In the analysis of CTC gene expression, therefore, this method could exclude the interference of leukocytes in a cell sample and accordingly contribute to higher analytical sensitivity, as demonstrated in this study. Overall, this study has presented an ODEP-based microfluidic system capable of simply and effectively isolating a specific cell species from a cell mixture.

No MeSH data available.


Related in: MedlinePlus