Protocol for clonal isolation of gene-edited hiPSCs using droplet and microfluidic sorting

Area of Science:

• Stem Cell Biology
• Gene Editing Technologies
• Disease Modeling

Background:

• Human induced pluripotent stem cells (hiPSCs) are crucial for disease modeling and drug discovery.
• Efficient generation of clonal hiPSC lines after gene editing remains a significant challenge in regenerative medicine.

Purpose of the Study:

• To present a streamlined protocol for generating clonal hiPSC lines post-gene editing.
• To demonstrate the utility of electrostatic droplet- and microfluidics-based sorting for single-cell isolation.

Main Methods:

• Culture of hiPSCs and CRISPR-RNP electroporation for gene editing.
• Single-cell deposition and expansion using electrostatic droplet or microfluidics sorting platforms.
• Generation and validation of gene-edited clonal hiPSC lines.

Main Results:

• Successful generation of over 100 clonal hiPSC lines across seven distinct knock-in/knock-out experiments.
• Demonstrated broad utility and reproducibility of the protocol for various gene editing strategies.
• Efficient isolation and expansion of single gene-edited cells into clonal populations.

Conclusions:

• The presented protocol offers an efficient and reproducible method for generating clonal hiPSC lines after gene editing.
• This advancement facilitates disease modeling and accelerates drug discovery pipelines.
• The protocol is adaptable to different gene editing techniques and cell sorting technologies.