Scaling cell therapy engineering with microfluidic single cell dispensing and automated capillary-based western immunoassays

Generating stable, edited induced pluripotent stem cell (iPSC) lines depends on two rate-limiting steps: single cell clone isolation and confirmation of functional protein knockout. Using CRISPR/Cas9 knockout of beta-2-microglobulin (β2M), a target for evading immune rejection in allogeneic cell therapies, automated single cell dispensing was combined with quantitative capillary western immunoassay. Although all 28 clones were classified as β2M-negative by surface staining, whole cell protein analysis distinguished complete from partial knockouts and resolved truncated and altered protein products undetected by flow cytometry. Fluorescence-based sorting, aided by IFNγ-induced surface expression, increased knockout recovery from 40% to 62% while preserving pluripotency-marker expression. These findings indicate that surface-expression screening can overestimate functional knockout frequency, and that combining single cell isolation with protein-level validation offers a scalable, reproducible approach for engineering iPSC-derived cell therapies and disease models.

What you will learn

01

How combining fluorescence-based single cell dispensing (Pala) with automated capillary western immunoassay (Leo) addresses two major bottlenecks in iPSC CRISPR engineering: single cell cloning and functional knockout confirmation

02

Why IFNγ-induced surface β2M expression improves fluorescence-based enrichment of edited clones, increasing knockout recovery to 62% versus 40% in untreated cultures

03

How Simple Western analysis discriminates partial from complete β2M knockout and detects truncated or altered protein products missed by flow cytometry, even when all clones appear negative by surface staining

04

Why whole cell protein analysis is a valuable orthogonal method to surface-expression screening, which can overestimate the true frequency of functional knockout

05

How Pala's gentle sorting supports high monoclonal colony yields (>30 colonies/96-well plate) while preserving pluripotency, with all clones exceeding 70% co-expression of NANOG, OCT4, and SSEA4

06

How this combined workflow provides a scalable, reproducible strategy for generating and validating engineered iPSC-derived cell therapies and disease models

Key interests

iPSC CRISPR engineering β2M knockout Single cell cloning Simple Western Capillary immunoassay Allogeneic cell therapy Clone characterization Pluripotency markers Flow cytometry