High throughput intracellular delivery by viscoelastic mechanoporation

Brief pulses of electric field (electroporation) and/or tensile stress (mechanoporation) have been used to reversibly permeabilize the plasma membrane of mammalian cells and deliver materials to the cytosol. However, electroporation can be harmful to cells, while efficient mechanoporation strategies have not been scalable due to the use of narrow constrictions or needles which are susceptible to clogging. Here we report a high throughput approach to mechanoporation in which the plasma membrane is stretched and reversibly permeabilized by viscoelastic fluid forces within a microfluidic chip without surface contact. Biomolecules are delivered directly to the cytosol within seconds at a throughput exceeding 250 million cells per minute. Viscoelastic mechanoporation is compatible with a variety of biomolecules including proteins, RNA, and CRISPR-Cas9 ribonucleoprotein complexes, as well as a range of cell types including HEK293T cells and primary T cells. Altogether, viscoelastic mechanoporation appears feasible for contact-free permeabilization and delivery of biomolecules to mammalian cells ex vivo.


Statistics
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Data analysis
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All manuscripts must include a data availability statement This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or or web links for publicly available datasets -A description of of any restrictions on on data availability -For clinical datasets or or third party data, please ensure that the statement adheres to to our policy Derin Sevenler & Mehmet Toner Nov 15, 2023 Rheotool (https://github.com/fppimenta/rheoTool)and OpenFOAM v9 v9 (https://openfoam.org/) were used for computational fluid dynamics simulations.
Plotting and statistical tests were performed with MATLAB v2019a.Flow cytometry analysis was performed with Imagestream IDEAS version 6.2.
Source data are provided with this paper.Raw videos, raw flow cytometry data, and raw numerical simulation solutions are available from the corresponding author upon reasonable request.
For intracellular delivery studies, n=1 or n=2 replicates were initially used (as indicated) for preliminary screening across the potentially vast design parameter space (flow rate, geometry height, width, length, hyaluronic acid concentration, biomolecule concentration, buffer composition).For characterization experiments, n=3 replicates per condition were used to identify potential outliers (ultimately no outliers were excluded).No statistical tests were used to determine the required sample size.
No data were excluded from analysis.
All experiments were performed independently.The number of replicates of each experiment are included in the manuscript.The authors have reproduced the experimental findings with consistent results.
For each test, cells from a single mixed population were randomly allocated to the experimental conditions.
Blinding was not always possible since one scientist designed, performed, and analyzed all experiments.
FITC anti-human TCR !/" Antibody, Clone IP26 https://www.biolegend.com/en-us/products/fitc-anti-human-tcr-alpha-beta-antibody-772 Describe the methods by which all novel plant genotypes were produced.This includes those generated by transgenic approaches, gene editing, chemical/radiation-based mutagenesis and hybridization.For transgenic lines, describe the transformation method, the number of independent lines analyzed and the generation upon which experiments were performed.For gene-edited lines, describe the editor used, the endogenous sequence targeted for editing, the targeting guide RNA sequence (if applicable) and how the editor was applied.was applied.was applied.
Report on the source of all seed stocks or other plant material used.If applicable, state the seed stock centre and catalogue number.If plant specimens were collected from the field, describe the collection location, date and sampling procedures.Cells were washed by by centrifugation, resuspended in in 50 50 uL uL of of phosphate buffered saline with 2 ug/mL of of propidium iodide, and incubated at at room temperature for 10-30 minutes before measurement.

Amnis ImageStream Mark II
Imagestream IDEAS version 6.2 All input cells were analyzed (the cells of of interest represented 100% of of input sample).
The default ImageStream gating approach was used, specifically: 1. 1. Gating of of in-focus images by by image gradient RMS (~99% of of images); 2. 2. Gating of of single cells yb yb feature size and aspect ratio (~90% or or more of of images); 3. 3. Gating of of viable cells to to determine population viability.For FITC-dextran delivery experiments, another sequential gate was used on on the FITC-channel Describe any authentication procedures for each seed stock used or novel genotype generated.Describe any experiments used to Describe any authentication procedures for each seed stock used or novel genotype generated.Describe any experiments used to Describe any authentication procedures for each seed stock used or novel genotype generated.Describe any experiments used to assess the effect of a mutation and, where applicable, how potential secondary effects (e.g.second site T-DNA insertions, mosiacism, off-target gene editing) were examined.