Abstract
Intracellular delivery of functional macromolecules, such as DNA and RNA, across the cell membrane and into the cytosol, is a critical process in both biology and medicine. Herein, we develop and use microfluidic chips containing post arrays to induce microfluidic vortex shedding, or μVS, for cell membrane poration that permits delivery of mRNA into primary human T lymphocytes. We demonstrate transfection with μVS by delivery of a 996-nucleotide mRNA construct encoding enhanced green fluorescent protein (EGFP) and assessed transfection efficiencies by quantifying levels of EGFP protein expression. We achieved high transfection efficiency (63.6 ± 3.44% EGFP + viable cells) with high cell viability (77.3 ± 0.58%) and recovery (88.7 ± 3.21%) in CD3 + T cells 19 hrs after μVS processing. Importantly, we show that processing cells via μVS does not negatively affect cell growth rates or alter cell states. We also demonstrate processing speeds of greater than 2.0 × 106 cells s−1 at volumes ranging from 0.1 to 1.5 milliliters. Altogether, these results highlight the use of μVS as a rapid and gentle delivery method with promising potential to engineer primary human cells for research and clinical applications.
Highlights
Biomicrofluidics are used to isolate[1], enrich[1], modify[2,3], culture[4] and qualify cells[5], lending to the development and manufacturing of gene-modified cell therapy (GMCT) where these processes are vital
Our results indicated that six rows of circular posts of 40 μm diameter with spacing between each post approximately twice the diameter of a human T cells (~9.0 μm, 8 to 12 μm) and an applied gauge pressure of 120 per square inch (PSI) was ideal for delivery of mRNA (Fig. 1c,d) to T cells from a single donor when using compressed nitrogen
It was necessary to evaluate if build-up caused by cell debris resulted in constriction-based cell poration, which may be the cause of any transfection not accounted for by μVS
Summary
Biomicrofluidics are used to isolate[1], enrich[1], modify[2,3], culture[4] and qualify cells[5], lending to the development and manufacturing of gene-modified cell therapy (GMCT) where these processes are vital. In terms of processing, low throughput transfection methods, such as single cell micro-needle injection, would require 108 seconds, or three years, to deliver a single autologous GMCT without expansion[17,28,29] To overcome these limitations, generation transfection methods need to preserve high cell viabilities and desired cell states prior to infusion, while implementing strategies to increase processing throughput (i.e. sample size, number and processing speed), diminish production times, and minimize processing steps. Generation transfection methods need to preserve high cell viabilities and desired cell states prior to infusion, while implementing strategies to increase processing throughput (i.e. sample size, number and processing speed), diminish production times, and minimize processing steps As it stands, the current state of microfluidic and mechanical intracellular delivery methods described above fail to meet the needs of GMCT development and clinical-level manufacturing
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