A Microfluidic Single-Cell Cloning (SCC) Device for the Generation of Monoclonal Cells.

Pei-Tzu Lai,Chia-Hsien Hsu,Chia-Yu Tang,Chuan-Feng Yeh,Ching-Hui Lin,Hao-Chen Chang

doi:10.3390/cells9061482

Pei-Tzu Lai, Chia-Hsien Hsu + Show 4 more

Open Access

https://doi.org/10.3390/cells9061482

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Abstract

Single-cell cloning (SCC) is a critical step in generating monoclonal cell lines, which are widely used as in vitro models and for producing proteins with high reproducibility for research and the production of therapeutic drugs. In monoclonal cell line generation, the development time can be shortened by validating the monoclonality of the cloned cells. However, the validation process currently requires specialized equipment that is not readily available in general biology laboratories. Here, we report a disposable SCC device, in which single cells can be isolated, validated, and expanded to form monoclonal cell colonies using conventional micropipettes and microscopes. The monoclonal cells can be selectively transferred from the SCC chip to conventional culture plates, using a tissue puncher. Using the device, we demonstrated that monoclonal colonies of actin-GFP (green fluorescent protein) plasmid-transfected A549 cells could be formed in the device within nine days and subsequently transferred to wells in plates for further expansion. This approach offers a cost-effective alternative to the use of specialized equipment for monoclonal cell generation.

Highlights

Monoclonal cells are groups of cells originating from a single parental cell
High-resolution images of the microscale device show single cells in the trap wells (Figure 1c), eliminating concerns about cell clustering that occur in the dilution methods
We found that the GFP signal in the cells was colocated with the rhodamine phalloidin staining signal of actin–GFP-transfected A549 cells could be obtained in 18 days

Summary

Introduction

Monoclonal cells are groups of cells originating from a single parental cell. They have cognate genomic DNA sequences and express similar phenotypes. Transfected cells usually have highly diverse gene complements, due to the random insertion of target genes and the process of gene amplification [7]. As a result, screening and selecting for large numbers of monoclonal cells with a desired phenotype and high reproductive capacity are necessary [1,11,12,13]. Reducing the time of the screening and selection processes could have significant financial implications for protein productions in which a right production clone (i.e., clone that can synthesize the required protein at high productivity) usually takes months to generate [14]

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