journal article·2023-09-06Open access

Fluorinated Silane-Modified Filtroporation Devices Enable Gene Knockout in Human Hematopoietic Stem and Progenitor Cells

Isaura M. Frost, Alexandra M. Mendoza, Tzu-Ting Chiou, Philseok Kim, Joanna Aizenberg, Donald B. Kohn, Satiro N. De Oliveira, Paul S. Weiss, Steven J. Jonas

ACS Applied Materials & Interfaces· Volume 15· Issue 35

Abstract

High Resolution Image Download MS PowerPoint Slide Intracellular delivery technologies that are cost-effective, non-cytotoxic, efficient, and cargo-agnostic are needed to enable the manufacturing of cell-based therapies as well as gene manipulation for research applications. Current technologies capable of delivering large cargoes, such as plasmids and CRISPR-Cas9 ribonucleoproteins (RNPs), are plagued with high costs and/or cytotoxicity and often require substantial specialized equipment and reagents, which may not be available in resource-limited settings. Here, we report an intracellular delivery technology that can be assembled from materials available in most research laboratories, thus democratizing access to intracellular delivery for researchers and clinicians in low-resource areas of the world. These filtroporation devices permeabilize cells by pulling them through the pores of a cell culture insert by the application of vacuum available in biosafety cabinets. In a format that costs less than $10 in materials per experiment, we demonstrate the delivery of fluorescently labeled dextran, expression plasmids, and RNPs for gene knockout to Jurkat cells and human CD34 + hematopoietic stem and progenitor cell populations with delivery efficiencies of up to 40% for RNP knockout and viabilities of >80%. We show that functionalizing the surfaces of the filters with fluorinated silane moieties further enhances the delivery efficiency. These devices are capable of processing 500,000 to 4 million cells per experiment, and when combined with a 3D-printed vacuum application chamber, this throughput can be straightforwardly increased 6–12-fold in parallel experiments.
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Research context

OpenAlex topics: RNA Interference and Gene Delivery · Virus-based gene therapy research · CAR-T cell therapy research

SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.

Affiliations

Department of Bioengineering, University of California, Los Angeles, Los Angeles, California 90095, United States

UCLA Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095, United States

Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095, United States

Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095, United States

California NanoSystems Institute, University of California, Los Angeles, Los Angeles, California 90095, United States

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, United States

Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, California 90095, United States

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, United States;

Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, United States;

Department of Materials Science and Engineering, University of California, Los Angeles, Los Angeles, California 90095, United States;

Children’s Discovery and Innovation Institute, University of California, Los Angeles, Los Angeles, California 90095, United States

Funding

National Science Foundation

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