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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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The overall CTC isolation and purification processes: (I) fluorescent microscopic observation was performed on 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)-(IV) fluorescent microscopy operations were performed to observe the leukocytes (red dots), cancer cells (green dots), and all nucleated cells (blue dots) for cancer cell positioning purposes; (V) a hollow circular light image was used to enclose the target cancer cells, and a long rectangular light bar was used to manipulate the leukocytes; (VI)-(VII) the long rectangular light bar was moved to sweep all unenclosed leukocytes to one side of the main microchannel, leaving the enclosed cancer cells in the same positions; (VIII) the circular light image was moved to manipulate enclosed the cancer cells to the side microchannel for collection; (IX)-(X) another moving rectangular light bar was used to transport the cancer cells collected to a site near the through-hole for harvesting; (XI)-(XII) immunofluorescent microscopic observations were performed to examine the purity of cancer cells [the leukocytes (red dots), cancer cells (green dots), and all nucleated cells (blue dots)].(Three video clips are provided as the 1st, 2nd and 3rd video clips).
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f5: The overall CTC isolation and purification processes: (I) fluorescent microscopic observation was performed on 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)-(IV) fluorescent microscopy operations were performed to observe the leukocytes (red dots), cancer cells (green dots), and all nucleated cells (blue dots) for cancer cell positioning purposes; (V) a hollow circular light image was used to enclose the target cancer cells, and a long rectangular light bar was used to manipulate the leukocytes; (VI)-(VII) the long rectangular light bar was moved to sweep all unenclosed leukocytes to one side of the main microchannel, leaving the enclosed cancer cells in the same positions; (VIII) the circular light image was moved to manipulate enclosed the cancer cells to the side microchannel for collection; (IX)-(X) another moving rectangular light bar was used to transport the cancer cells collected to a site near the through-hole for harvesting; (XI)-(XII) immunofluorescent microscopic observations were performed to examine the purity of cancer cells [the leukocytes (red dots), cancer cells (green dots), and all nucleated cells (blue dots)].(Three video clips are provided as the 1st, 2nd and 3rd video clips).

Mentions: To test the performance of the presented method for CTC isolation and purification, a cell suspension sample was prepared by spiking PC-3 cells (500 cells) into a whole blood sample (8 ml), followed by a negative selection-based CTC isolation process. The cell pellets obtained were stained with immunofluorescent dyes, and then re-suspended in 30 μl of sucrose solution. The treated cell suspension sample was subsequently loaded into the microfluidic system for further CTC isolation and purification based on the working scheme described in Fig. 2. Figure 5 shows the overall CTC isolation processes, from cancer cell identification and positioning using fluorescent microscopic observations [Fig. 5(I–IV)] to cancer cell separation via ODEP force-based cell manipulation with the aid of light field microscopic imaging [Fig. 5(V–X)]. By repeating these processes, the cancer cells isolated and temporarily collected in the side microchannel were checked for cancer cell purity using fluorescent microscopic observation. Figure 5 (XI) and (XII) showed that the cells in the side microchannel were all EpCAM marker (green dots)- and Hoechst dye (blue dots)-positive cancer cells without the existence of CD45 marker (red dots)-positive leukocytes. Overall, the results demonstrated that the proposed method was capable of isolating cancer cells with cell purity as high as 100%, which is currently impossible using existing CTC isolation techniques. Moreover, the recovery rate of cancer cells, and minimum number of cancer cells per millilitre of blood that can be isolated were experimentally evaluated to be 41.5%, and 15 cells ml−1, respectively. In addition to the abovementioned cancer cell line model tested, the use of the proposed method for the isolation of real CTCs from a cancer patient’s blood was also successfully demonstrated. Result (Supplementary figure; Fig. S2) revealed that the presented method was able to harvest 100% pure real CTCs, in which no leukocyte [red dots: Fig. S2-(b)-(II)] was found in the cells harvested. In the future application, it can be reasonably believed that the recovery rate of target cells, and working throughput can be further improved through the integration of a programmable image analysis system, and automation mechanism in the proposed system.


Application of optically-induced-dielectrophoresis in microfluidic system for purification of circulating tumour cells for gene expression analysis- Cancer cell line model
The overall CTC isolation and purification processes: (I) fluorescent microscopic observation was performed on 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)-(IV) fluorescent microscopy operations were performed to observe the leukocytes (red dots), cancer cells (green dots), and all nucleated cells (blue dots) for cancer cell positioning purposes; (V) a hollow circular light image was used to enclose the target cancer cells, and a long rectangular light bar was used to manipulate the leukocytes; (VI)-(VII) the long rectangular light bar was moved to sweep all unenclosed leukocytes to one side of the main microchannel, leaving the enclosed cancer cells in the same positions; (VIII) the circular light image was moved to manipulate enclosed the cancer cells to the side microchannel for collection; (IX)-(X) another moving rectangular light bar was used to transport the cancer cells collected to a site near the through-hole for harvesting; (XI)-(XII) immunofluorescent microscopic observations were performed to examine the purity of cancer cells [the leukocytes (red dots), cancer cells (green dots), and all nucleated cells (blue dots)].(Three video clips are provided as the 1st, 2nd and 3rd video clips).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016898&req=5

f5: The overall CTC isolation and purification processes: (I) fluorescent microscopic observation was performed on 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)-(IV) fluorescent microscopy operations were performed to observe the leukocytes (red dots), cancer cells (green dots), and all nucleated cells (blue dots) for cancer cell positioning purposes; (V) a hollow circular light image was used to enclose the target cancer cells, and a long rectangular light bar was used to manipulate the leukocytes; (VI)-(VII) the long rectangular light bar was moved to sweep all unenclosed leukocytes to one side of the main microchannel, leaving the enclosed cancer cells in the same positions; (VIII) the circular light image was moved to manipulate enclosed the cancer cells to the side microchannel for collection; (IX)-(X) another moving rectangular light bar was used to transport the cancer cells collected to a site near the through-hole for harvesting; (XI)-(XII) immunofluorescent microscopic observations were performed to examine the purity of cancer cells [the leukocytes (red dots), cancer cells (green dots), and all nucleated cells (blue dots)].(Three video clips are provided as the 1st, 2nd and 3rd video clips).
Mentions: To test the performance of the presented method for CTC isolation and purification, a cell suspension sample was prepared by spiking PC-3 cells (500 cells) into a whole blood sample (8 ml), followed by a negative selection-based CTC isolation process. The cell pellets obtained were stained with immunofluorescent dyes, and then re-suspended in 30 μl of sucrose solution. The treated cell suspension sample was subsequently loaded into the microfluidic system for further CTC isolation and purification based on the working scheme described in Fig. 2. Figure 5 shows the overall CTC isolation processes, from cancer cell identification and positioning using fluorescent microscopic observations [Fig. 5(I–IV)] to cancer cell separation via ODEP force-based cell manipulation with the aid of light field microscopic imaging [Fig. 5(V–X)]. By repeating these processes, the cancer cells isolated and temporarily collected in the side microchannel were checked for cancer cell purity using fluorescent microscopic observation. Figure 5 (XI) and (XII) showed that the cells in the side microchannel were all EpCAM marker (green dots)- and Hoechst dye (blue dots)-positive cancer cells without the existence of CD45 marker (red dots)-positive leukocytes. Overall, the results demonstrated that the proposed method was capable of isolating cancer cells with cell purity as high as 100%, which is currently impossible using existing CTC isolation techniques. Moreover, the recovery rate of cancer cells, and minimum number of cancer cells per millilitre of blood that can be isolated were experimentally evaluated to be 41.5%, and 15 cells ml−1, respectively. In addition to the abovementioned cancer cell line model tested, the use of the proposed method for the isolation of real CTCs from a cancer patient’s blood was also successfully demonstrated. Result (Supplementary figure; Fig. S2) revealed that the presented method was able to harvest 100% pure real CTCs, in which no leukocyte [red dots: Fig. S2-(b)-(II)] was found in the cells harvested. In the future application, it can be reasonably believed that the recovery rate of target cells, and working throughput can be further improved through the integration of a programmable image analysis system, and automation mechanism in the proposed system.

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