Limits...
Development of a new rapid isolation device for circulating tumor cells (CTCs) using 3D palladium filter and its application for genetic analysis.

Yusa A, Toneri M, Masuda T, Ito S, Yamamoto S, Okochi M, Kondo N, Iwata H, Yatabe Y, Ichinosawa Y, Kinuta S, Kondo E, Honda H, Arai F, Nakanishi H - PLoS ONE (2014)

Bottom Line: Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively.Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers.These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Aichi Science and Technology Foundation, Knowledge Hub Aichi, Priority Research Projects, Japan ; Department of Micro-Nano Systems Engineering, Graduate School of Engineering, Nagoya University, Japan ; Division of Oncological Pathology, Aichi Cancer Center Research Institute, Japan.

ABSTRACT
Circulating tumor cells (CTCs) in the blood of patients with epithelial malignancies provide a promising and minimally invasive source for early detection of metastasis, monitoring of therapeutic effects and basic research addressing the mechanism of metastasis. In this study, we developed a new filtration-based, sensitive CTC isolation device. This device consists of a 3-dimensional (3D) palladium (Pd) filter with an 8 µm-sized pore in the lower layer and a 30 µm-sized pocket in the upper layer to trap CTCs on a filter micro-fabricated by precise lithography plus electroforming process. This is a simple pump-less device driven by gravity flow and can enrich CTCs from whole blood within 20 min. After on-device staining of CTCs for 30 min, the filter cassette was removed from the device, fixed in a cassette holder and set up on the upright fluorescence microscope. Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively. Cell spike experiments demonstrated that the recovery rate of tumor cells from blood by this Pd filter device was more than 85%. Single living tumor cells were efficiently isolated from these spiked tumor cells by a micromanipulator, and KRAS mutation, HER2 gene amplification and overexpression, for example, were successfully detected from such isolated single tumor cells. Sequential analysis of blood from mice bearing metastasis revealed that CTC increased with progression of metastasis. Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers. These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

Show MeSH

Related in: MedlinePlus

Computational simulations of fluid dynamics of 2D and 3D Pd filter.Fluid dynamics of the 2D and 3D metal filter devices were analyzed using FEM analysis software as described in the Materials and Methods. A–B. Comparison of fluid field simulation between high pore density (pitch 34 µm) 2D (A) and 3D Pd filter devices (B) without cells showing velocity and streamline (yellow) around the area of capturing pores. C. Fluid field simulation of 3D Pd filter device with low pore density (pitch 40 µm). Pseudo color image of the flow rate around the pore as shown in the right corner reveals higher flow rate in the low pore density filter. D. Enlarged view of 3D Pd filter devices shown in B. Each calculated values are described in the bottom of each panel. Parameters such as φ, H and pitch shown in figures are follows; φ = pocket diameter, H = height of the pocket, Pitch = distance between pore.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3921253&req=5

pone-0088821-g003: Computational simulations of fluid dynamics of 2D and 3D Pd filter.Fluid dynamics of the 2D and 3D metal filter devices were analyzed using FEM analysis software as described in the Materials and Methods. A–B. Comparison of fluid field simulation between high pore density (pitch 34 µm) 2D (A) and 3D Pd filter devices (B) without cells showing velocity and streamline (yellow) around the area of capturing pores. C. Fluid field simulation of 3D Pd filter device with low pore density (pitch 40 µm). Pseudo color image of the flow rate around the pore as shown in the right corner reveals higher flow rate in the low pore density filter. D. Enlarged view of 3D Pd filter devices shown in B. Each calculated values are described in the bottom of each panel. Parameters such as φ, H and pitch shown in figures are follows; φ = pocket diameter, H = height of the pocket, Pitch = distance between pore.

Mentions: COMSOL analysis showed that when constant velocity (0.3 mm/s) was applied, the maximum flow velocity around the constricted part of the filter (pore) was slower in the high density 3D φ30-H10-pitch34 filter (18.9 mm/s, 100,000 pores/cm2) than in the low density 3D φ30-H10-pitch40 filter (24.4 mm/s, 40,000 pores/cm2) (Figure 3B and 3C), but there was no significant difference in velocity between the 2D φ30-H10-pitch34 (18.1 mm/s) and 3D φ30-H10-pitch34 filter (18.9 mm/s) (Figure 3A and 3B). This indicated that the pore number is critical for determining the flow rate of the filter, and that the higher the pore number, the lower the flow rate in the filter device. Furthermore, as expected, structures of filters such as 2D and 3D did not significantly affect the flow rate of the filter device. A similar inverse correlation was obtained between the pressure of cancer cells trapped in the filter pore and the pore number. Tension on a plugged-like cell for 2D Pd filter device with low (pitch 50 µm) and high (pitch 30 µm) pore density was calculated as 45 pascal and 18 pascal, respectively, in the case of constant flow rate of 2.5 ml/min (data not shown). Interestingly, the streamline around the capturing pore showed that recirculation and an eddy appeared in the margin of the pocket in 3D filters with φ30-H10-pitch34 parameter (Figure 3D), but not in 2D filters, suggesting that a local turbulent-like flow in the 3D filter device may be present.


Development of a new rapid isolation device for circulating tumor cells (CTCs) using 3D palladium filter and its application for genetic analysis.

Yusa A, Toneri M, Masuda T, Ito S, Yamamoto S, Okochi M, Kondo N, Iwata H, Yatabe Y, Ichinosawa Y, Kinuta S, Kondo E, Honda H, Arai F, Nakanishi H - PLoS ONE (2014)

Computational simulations of fluid dynamics of 2D and 3D Pd filter.Fluid dynamics of the 2D and 3D metal filter devices were analyzed using FEM analysis software as described in the Materials and Methods. A–B. Comparison of fluid field simulation between high pore density (pitch 34 µm) 2D (A) and 3D Pd filter devices (B) without cells showing velocity and streamline (yellow) around the area of capturing pores. C. Fluid field simulation of 3D Pd filter device with low pore density (pitch 40 µm). Pseudo color image of the flow rate around the pore as shown in the right corner reveals higher flow rate in the low pore density filter. D. Enlarged view of 3D Pd filter devices shown in B. Each calculated values are described in the bottom of each panel. Parameters such as φ, H and pitch shown in figures are follows; φ = pocket diameter, H = height of the pocket, Pitch = distance between pore.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921253&req=5

pone-0088821-g003: Computational simulations of fluid dynamics of 2D and 3D Pd filter.Fluid dynamics of the 2D and 3D metal filter devices were analyzed using FEM analysis software as described in the Materials and Methods. A–B. Comparison of fluid field simulation between high pore density (pitch 34 µm) 2D (A) and 3D Pd filter devices (B) without cells showing velocity and streamline (yellow) around the area of capturing pores. C. Fluid field simulation of 3D Pd filter device with low pore density (pitch 40 µm). Pseudo color image of the flow rate around the pore as shown in the right corner reveals higher flow rate in the low pore density filter. D. Enlarged view of 3D Pd filter devices shown in B. Each calculated values are described in the bottom of each panel. Parameters such as φ, H and pitch shown in figures are follows; φ = pocket diameter, H = height of the pocket, Pitch = distance between pore.
Mentions: COMSOL analysis showed that when constant velocity (0.3 mm/s) was applied, the maximum flow velocity around the constricted part of the filter (pore) was slower in the high density 3D φ30-H10-pitch34 filter (18.9 mm/s, 100,000 pores/cm2) than in the low density 3D φ30-H10-pitch40 filter (24.4 mm/s, 40,000 pores/cm2) (Figure 3B and 3C), but there was no significant difference in velocity between the 2D φ30-H10-pitch34 (18.1 mm/s) and 3D φ30-H10-pitch34 filter (18.9 mm/s) (Figure 3A and 3B). This indicated that the pore number is critical for determining the flow rate of the filter, and that the higher the pore number, the lower the flow rate in the filter device. Furthermore, as expected, structures of filters such as 2D and 3D did not significantly affect the flow rate of the filter device. A similar inverse correlation was obtained between the pressure of cancer cells trapped in the filter pore and the pore number. Tension on a plugged-like cell for 2D Pd filter device with low (pitch 50 µm) and high (pitch 30 µm) pore density was calculated as 45 pascal and 18 pascal, respectively, in the case of constant flow rate of 2.5 ml/min (data not shown). Interestingly, the streamline around the capturing pore showed that recirculation and an eddy appeared in the margin of the pocket in 3D filters with φ30-H10-pitch34 parameter (Figure 3D), but not in 2D filters, suggesting that a local turbulent-like flow in the 3D filter device may be present.

Bottom Line: Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively.Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers.These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Aichi Science and Technology Foundation, Knowledge Hub Aichi, Priority Research Projects, Japan ; Department of Micro-Nano Systems Engineering, Graduate School of Engineering, Nagoya University, Japan ; Division of Oncological Pathology, Aichi Cancer Center Research Institute, Japan.

ABSTRACT
Circulating tumor cells (CTCs) in the blood of patients with epithelial malignancies provide a promising and minimally invasive source for early detection of metastasis, monitoring of therapeutic effects and basic research addressing the mechanism of metastasis. In this study, we developed a new filtration-based, sensitive CTC isolation device. This device consists of a 3-dimensional (3D) palladium (Pd) filter with an 8 µm-sized pore in the lower layer and a 30 µm-sized pocket in the upper layer to trap CTCs on a filter micro-fabricated by precise lithography plus electroforming process. This is a simple pump-less device driven by gravity flow and can enrich CTCs from whole blood within 20 min. After on-device staining of CTCs for 30 min, the filter cassette was removed from the device, fixed in a cassette holder and set up on the upright fluorescence microscope. Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively. Cell spike experiments demonstrated that the recovery rate of tumor cells from blood by this Pd filter device was more than 85%. Single living tumor cells were efficiently isolated from these spiked tumor cells by a micromanipulator, and KRAS mutation, HER2 gene amplification and overexpression, for example, were successfully detected from such isolated single tumor cells. Sequential analysis of blood from mice bearing metastasis revealed that CTC increased with progression of metastasis. Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers. These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

Show MeSH
Related in: MedlinePlus