Lipoplex-Mediated Single-Cell Transfection via Droplet Microfluidics

Area of Science:

• Biotechnology
• Microfluidics
• Cell Biology

Background:

• Lipoplex (cationic lipid-nucleic acid complex)-mediated transfection is common for mammalian cells.
• Suspension cells, like lymphatic and hematopoietic cells, show low transfection efficiency (≈5%) with traditional methods.

Purpose of the Study:

• To develop an efficient and consistent lipoplex-mediated transfection method for difficult-to-transfect suspension cells.
• To enhance gene delivery using a single-cell, droplet-microfluidics approach.

Main Methods:

• Utilized droplet microfluidics for single-cell co-confinement with lipoplexes.
• Generated monodisperse lipoplexes using chaotic mixing in serpentine microchannels.
• Leveraged shear stress at the droplet pinch-off junction to increase cell membrane permeability.

Main Results:

• Achieved ≈50% transfection efficiency for K562, THP-1, and Jurkat cell lines, a tenfold increase from ≈5%.
• Significantly reduced cell-to-cell variation in transfection rates compared to bulk methods.
• Demonstrated efficient CRISPR-Cas9 gene knockout of TP53BP1 in K562 cells.

Conclusions:

• Droplet microfluidics enables highly efficient lipoplex-mediated single-cell transfection in challenging suspension cells.
• This platform offers a promising solution for gene therapy and regenerative medicine applications.
• The method provides high efficiency and low variation, overcoming limitations of current transfection techniques.