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Separable bilayer microfiltration device for viable label-free enrichment of circulating tumour cells.

Zhou MD, Hao S, Williams AJ, Harouaka RA, Schrand B, Rawal S, Ao Z, Brenneman R, Gilboa E, Lu B, Wang S, Zhu J, Datar R, Cote R, Tai YC, Zheng SY - Sci Rep (2014)

Bottom Line: Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture.Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably.In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis.

View Article: PubMed Central - PubMed

Affiliation: Micro &Nano Integrated Biosystem (MINIBio) Laboratory, Department of Biomedical Engineering and Materials Research Institute, Pennsylvania State University, University Park, PA 16802, U.S.A.

ABSTRACT
The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 10(3)), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

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Related in: MedlinePlus

On-chip cell proliferation demonstrated with various cell lines (DU145, Hela, LnCaP and PC3).The images in each column are bright field microscopic images of cell growth on Petri dish as positive control and day 0, 4, and 8 on SB microfilters after enrichment.
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f4: On-chip cell proliferation demonstrated with various cell lines (DU145, Hela, LnCaP and PC3).The images in each column are bright field microscopic images of cell growth on Petri dish as positive control and day 0, 4, and 8 on SB microfilters after enrichment.

Mentions: In the second approach to demonstrate cell viability after filtration, wild adenovirus type 5 was modified with green fluorescence expression gene eGFP under the control of CMV promoter. It has been shown that modified adenovirus can preferably infect and express in tumour cells and thus can be used as a tool for viable CTC detection39. In this experiment, MCF-7 cells were modified with a red fluorescence expressing mCherry gene. As shown in Figure 4H–4K, cells with both red and green fluorescence (mCherry+GFP+) represented virus-infected viable MCF-7 cells, while cells with only red fluorescence (mCherry+GFP−) are presumably non-viable cells. The virus infection assay probed the cell's ability to actively express proteins (GFP in this case). Therefore, it is a different viability indicator compared with the measurement of membrane permeability (EthD-1) and enzyme activity (Calcein-AM). It should be noted that, assigning all the mCherry+GFP− as non-viable tumour cells made an assumption that the efficiency of virus infection and GFP expression in viable tumour cells was 100%. Based on the initial test, the virus infection efficiency was measured to be 90% ~ 95% depending on the tumour cell lines (data not shown).


Separable bilayer microfiltration device for viable label-free enrichment of circulating tumour cells.

Zhou MD, Hao S, Williams AJ, Harouaka RA, Schrand B, Rawal S, Ao Z, Brenneman R, Gilboa E, Lu B, Wang S, Zhu J, Datar R, Cote R, Tai YC, Zheng SY - Sci Rep (2014)

On-chip cell proliferation demonstrated with various cell lines (DU145, Hela, LnCaP and PC3).The images in each column are bright field microscopic images of cell growth on Petri dish as positive control and day 0, 4, and 8 on SB microfilters after enrichment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260227&req=5

f4: On-chip cell proliferation demonstrated with various cell lines (DU145, Hela, LnCaP and PC3).The images in each column are bright field microscopic images of cell growth on Petri dish as positive control and day 0, 4, and 8 on SB microfilters after enrichment.
Mentions: In the second approach to demonstrate cell viability after filtration, wild adenovirus type 5 was modified with green fluorescence expression gene eGFP under the control of CMV promoter. It has been shown that modified adenovirus can preferably infect and express in tumour cells and thus can be used as a tool for viable CTC detection39. In this experiment, MCF-7 cells were modified with a red fluorescence expressing mCherry gene. As shown in Figure 4H–4K, cells with both red and green fluorescence (mCherry+GFP+) represented virus-infected viable MCF-7 cells, while cells with only red fluorescence (mCherry+GFP−) are presumably non-viable cells. The virus infection assay probed the cell's ability to actively express proteins (GFP in this case). Therefore, it is a different viability indicator compared with the measurement of membrane permeability (EthD-1) and enzyme activity (Calcein-AM). It should be noted that, assigning all the mCherry+GFP− as non-viable tumour cells made an assumption that the efficiency of virus infection and GFP expression in viable tumour cells was 100%. Based on the initial test, the virus infection efficiency was measured to be 90% ~ 95% depending on the tumour cell lines (data not shown).

Bottom Line: Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture.Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably.In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis.

View Article: PubMed Central - PubMed

Affiliation: Micro &Nano Integrated Biosystem (MINIBio) Laboratory, Department of Biomedical Engineering and Materials Research Institute, Pennsylvania State University, University Park, PA 16802, U.S.A.

ABSTRACT
The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 10(3)), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

Show MeSH
Related in: MedlinePlus