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

This study demonstrates an automated workflow for scaling two rate-limiting steps in induced pluripotent stem cell (iPSC) CRISPR engineering: single cell cloning and functional protein knockout confirmation. Using CRISPR/Cas9 knockout of beta-2-microglobulin (β2M), a target for evading host immune rejection in allogeneic cell therapies, the Pala™ single cell dispenser and the Leo™ Simple Western™ system were combined to isolate and characterize edited clones. High rates of monoclonal colony outgrowth were observed (>30 colonies/96-well plate) using Pala for single cell cloning. Fluorescence-based sorting on Pala, aided by IFNγ-induced surface β2M expression, increased enrichment of edited cells, yielding 62% β2M knockout colonies compared with 40% in untreated cultures. Quantitative automated capillary western immunoassay analysis on Leo discriminated partial from complete knockout and detected truncated and altered protein products that could not be resolved by flow cytometry alone, despite all 28 clones appearing β2M-negative by surface staining. Importantly, all recovered clones retained expression of key pluripotency markers, confirming maintenance of stem cell identity following isolation and expansion. Together, these complementary technologies address critical bottlenecks in clone generation and validation, providing a scalable, reproducible workflow for the development of engineered 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