Abstract

Genome-engineering of human iPSCs is often hampered by low single-cell cloning efficiency and high uncertainty of culture clonality, which can render the generation of high-quality cell lines laborious, expensive, and time-consuming.
Here, we assessed a novel microfluidic cell culture approach which can be used to obtain clonal hiPSC lines with high efficiency after CRISPR/Cas9 genome editing. Exploiting interfacial tension, small-scale cell culture chambers are fabricated on polystyrene surfaces using matrix coating and an immiscible translucent fluorocarbon overlay. Each chamber utilises less than 1 µl of cell culture medium to cultivate single cells into clonal colonies. The microfluidic chambers’ unique optical properties allow the identification of single cells directly after cell plating, even if the cells are close to the edge of the chamber.
Using a refined cloning workflow as part of our automated cloning platform, we achieved up to 90% cloning efficiency across several genetically distinct hiPSC lines. Phenotyping of clonal hiPSC colonies revealed that cells maintained the expression of pluripotency markers and their genomic integrity.
We successfully applied the microfluidic cell culture approach to a CRISPR/Cas9-mediated GFP to BFP conversion assay in a hiPSC line carrying two copies of the eGFP gene in the AAVS1 locus. Three days after transfection of a sgRNA targeting the eGFP sequence and a ssODN containing two nucleotide changes sufficient to convert GFP to BFP, transfected cells were seeded into the small cell culture chambers. Chambers containing a single cell were tracked over seven days during which 86% of single cells grew into colonies. Fluorescence imaging of individual chambers revealed that all hiPSC colonies consisted of homogenous cell populations. Around 36% contained cells which only expressed BFP, suggesting that both copies of the eGFP gene had been converted. 48% expressed neither GFP nor BFP consistent with indel events. In the case of editing only one allele, cells of respective colonies expressed both GFP and BFP simultaneously, which overall occurred in less than 1%. Together, this nicely exemplifies that the novel microfluidic cell culture approach can be readily used as part of CRISPR/Cas9-mediated genome engineering workflows with high efficiency.