High Throughput Intracellular Delivery Using a 2D Cell-Squeezing Mechanoporation Device and Its Analysis by a Deep...

Area of Science:

• Biotechnology
• Microfluidics
• Cell Biology

Background:

• Cell-squeezing mechanoporation offers intracellular delivery but faces throughput limitations with 1D constrictions.
• Developing high-throughput methods is crucial for advancing single-cell delivery applications.

Purpose of the Study:

• To design and fabricate a 2D microfluidic device for parallel, high-throughput cell-squeezing mechanoporation.
• To demonstrate efficient intracellular delivery of various molecules into diverse cell types using the novel device.

Main Methods:

• Fabrication of a 2D microfluidic device with an array of vertical through-holes (8-15 µm diameter) on a thin SU-8 membrane within a PDMS structure.
• Intracellular delivery via rapid cell shearing through microfluidic constrictions, enabling diffusion-based molecule uptake.
• Analysis of delivery efficiency using image cytometry, combining instance segmentation and rule-based image processing for automated single-cell state evaluation.

Main Results:

• Achieved high-throughput dextran (4-40 kDa) delivery into HeLa and Jurkat cells at rates up to 3 million cells/min.
• Successfully delivered small interfering ribonucleic acid (siRNA) and plasmids into primary human mesenchymal stem cells (hMSCs) and human gingival fibroblasts (hGFs).
• Automated deep learning-based analysis system enabled single-cell resolution quantification of mechanoporation-based intracellular delivery.

Conclusions:

• The 2D cell-squeezing microfluidic device significantly increases throughput for intracellular delivery compared to 1D methods.
• The platform demonstrates broad applicability for delivering molecules and genetic material into various cell types, including primary cells.
• The integrated automated analysis system provides precise, single-cell resolution quantification, validating the device's potential for therapeutic applications.