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A High Throughput Micro-Chamber Array Device for Single Cell Clonal Cultivation and Tumor Heterogeneity Analysis.

Shen FM, Zhu L, Ye H, Yang YJ, Pang DW, Zhang ZL - Sci Rep (2015)

Bottom Line: Recently, single cell cloning techniques have been gradually developed benefited from their important roles in monoclonal antibody screening, tumor heterogeneity research fields, etc.In this study, we developed a high throughput device containing 1400 lateral chambers to efficiently isolate single cells and carry out long-term single cell clonal cultivation as well as tumor heterogeneity studies.The device was further used to examine tumor heterogeneity such as morphology, growth rate, anti-cancer drug tolerance as well as adenosine triphosphate-binding cassette (ABC) transporter ABCG2 protein expression level.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, P. R. China.

ABSTRACT
Recently, single cell cloning techniques have been gradually developed benefited from their important roles in monoclonal antibody screening, tumor heterogeneity research fields, etc. In this study, we developed a high throughput device containing 1400 lateral chambers to efficiently isolate single cells and carry out long-term single cell clonal cultivation as well as tumor heterogeneity studies. Most of the isolated single cells could proliferate normally nearly as long as three weeks and hundreds of clones could be formed once with one device, which made it possible to study tumor heterogeneity at single cell level. The device was further used to examine tumor heterogeneity such as morphology, growth rate, anti-cancer drug tolerance as well as adenosine triphosphate-binding cassette (ABC) transporter ABCG2 protein expression level. Except for the single cell isolation and tumor heterogeneity studies, the device is expected to be used as an excellent platform for drug screening, tumor biomarker discovering and tumor metastasis assay.

No MeSH data available.


Related in: MedlinePlus

Schematic view of the device and cell suspension injection,(a) the structure of the designed chip. (b) cell suspension injection into the chip. (c) enlarged view of the chamber array. (d) dynamic course of single cell self-loading into the lateral chamber. The arrows refer to a single cell. Scale bar in Fig. 1d is 100 μm.
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f1: Schematic view of the device and cell suspension injection,(a) the structure of the designed chip. (b) cell suspension injection into the chip. (c) enlarged view of the chamber array. (d) dynamic course of single cell self-loading into the lateral chamber. The arrows refer to a single cell. Scale bar in Fig. 1d is 100 μm.

Mentions: The designed device contains 1400 lateral square culture chambers with 500 micrometers per side connected to 100 micrometers wide perfusion channels through 30 micrometers wide lateral entrances as shown in Figs 1a,c. In this work, 30 micrometers and 500 micrometers were chosen as the optimal size of lateral entrance and chamber respectively for single cell self-loading and long-term clonal cultivation. When the size of chamber is larger than 500 micrometers, to capture the whole culture chamber area with one image becomes impossible under a 10 × objective, which leads to a labor-intensive observation process. While smaller than 500 micrometers, long-term single cell clonal cultivation will be partially restricted because of limited space of the culture chambers. As shown in Figs 1b,d, when the cell suspension was injected into the device, it was possible to load a single cell into a lateral culture chamber, the corresponding dynamic course was displayed in Fig. S1 Movie, Electronic Supplementary Information. The capacious micro-channel and lateral micro-chamber were designed to protect isolated single cells from detrimental effect of hydrodynamic shear stress293031. COMSOL Multiphysics 4.3 simulation result showed that the surface velocity within the culture chambers was extremely low (as shown in Fig. S1,), which indicated that the impact of hydrodynamic shear stress was negligible, due to a positive correlation between hydrodynamic shear stress and flow rate32. Besides, dynamic perfusion system is essential for fresh medium supplement and poisonous metabolites discharging based on the effective diffusion, which is important for long-term clonal cultivation. The diffusion process between the main channels and lateral chambers was confirmed by the diffusion experiments of red ink into and out of the lateral chambers (as shown in Fig. S2a,b). The designed structure and dynamic perfusion system guaranteed both high throughput single cell isolation efficiency and long-term clonal cultivation.


A High Throughput Micro-Chamber Array Device for Single Cell Clonal Cultivation and Tumor Heterogeneity Analysis.

Shen FM, Zhu L, Ye H, Yang YJ, Pang DW, Zhang ZL - Sci Rep (2015)

Schematic view of the device and cell suspension injection,(a) the structure of the designed chip. (b) cell suspension injection into the chip. (c) enlarged view of the chamber array. (d) dynamic course of single cell self-loading into the lateral chamber. The arrows refer to a single cell. Scale bar in Fig. 1d is 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493670&req=5

f1: Schematic view of the device and cell suspension injection,(a) the structure of the designed chip. (b) cell suspension injection into the chip. (c) enlarged view of the chamber array. (d) dynamic course of single cell self-loading into the lateral chamber. The arrows refer to a single cell. Scale bar in Fig. 1d is 100 μm.
Mentions: The designed device contains 1400 lateral square culture chambers with 500 micrometers per side connected to 100 micrometers wide perfusion channels through 30 micrometers wide lateral entrances as shown in Figs 1a,c. In this work, 30 micrometers and 500 micrometers were chosen as the optimal size of lateral entrance and chamber respectively for single cell self-loading and long-term clonal cultivation. When the size of chamber is larger than 500 micrometers, to capture the whole culture chamber area with one image becomes impossible under a 10 × objective, which leads to a labor-intensive observation process. While smaller than 500 micrometers, long-term single cell clonal cultivation will be partially restricted because of limited space of the culture chambers. As shown in Figs 1b,d, when the cell suspension was injected into the device, it was possible to load a single cell into a lateral culture chamber, the corresponding dynamic course was displayed in Fig. S1 Movie, Electronic Supplementary Information. The capacious micro-channel and lateral micro-chamber were designed to protect isolated single cells from detrimental effect of hydrodynamic shear stress293031. COMSOL Multiphysics 4.3 simulation result showed that the surface velocity within the culture chambers was extremely low (as shown in Fig. S1,), which indicated that the impact of hydrodynamic shear stress was negligible, due to a positive correlation between hydrodynamic shear stress and flow rate32. Besides, dynamic perfusion system is essential for fresh medium supplement and poisonous metabolites discharging based on the effective diffusion, which is important for long-term clonal cultivation. The diffusion process between the main channels and lateral chambers was confirmed by the diffusion experiments of red ink into and out of the lateral chambers (as shown in Fig. S2a,b). The designed structure and dynamic perfusion system guaranteed both high throughput single cell isolation efficiency and long-term clonal cultivation.

Bottom Line: Recently, single cell cloning techniques have been gradually developed benefited from their important roles in monoclonal antibody screening, tumor heterogeneity research fields, etc.In this study, we developed a high throughput device containing 1400 lateral chambers to efficiently isolate single cells and carry out long-term single cell clonal cultivation as well as tumor heterogeneity studies.The device was further used to examine tumor heterogeneity such as morphology, growth rate, anti-cancer drug tolerance as well as adenosine triphosphate-binding cassette (ABC) transporter ABCG2 protein expression level.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, P. R. China.

ABSTRACT
Recently, single cell cloning techniques have been gradually developed benefited from their important roles in monoclonal antibody screening, tumor heterogeneity research fields, etc. In this study, we developed a high throughput device containing 1400 lateral chambers to efficiently isolate single cells and carry out long-term single cell clonal cultivation as well as tumor heterogeneity studies. Most of the isolated single cells could proliferate normally nearly as long as three weeks and hundreds of clones could be formed once with one device, which made it possible to study tumor heterogeneity at single cell level. The device was further used to examine tumor heterogeneity such as morphology, growth rate, anti-cancer drug tolerance as well as adenosine triphosphate-binding cassette (ABC) transporter ABCG2 protein expression level. Except for the single cell isolation and tumor heterogeneity studies, the device is expected to be used as an excellent platform for drug screening, tumor biomarker discovering and tumor metastasis assay.

No MeSH data available.


Related in: MedlinePlus