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1.
ACS Appl Mater Interfaces ; 12(41): 45744-45752, 2020 Oct 14.
Article in English | MEDLINE | ID: mdl-32940030

ABSTRACT

Innovative technologies for intracellular delivery are ushering in a new era for gene editing, enabling the utilization of a patient's own cells for stem cell and immunotherapies. In particular, cell-squeezing platforms provide unconventional forms of intracellular delivery, deforming cells through microfluidic constrictions to generate transient pores and to enable effective diffusion of biomolecular cargo. While these devices are promising gene-editing platforms, they require frequent maintenance due to the accumulation of cellular debris, limiting their potential for reaching the throughputs necessary for scalable cellular therapies. As these cell-squeezing technologies are improved, there is a need to develop next-generation platforms with higher throughput and longer lifespan, importantly, avoiding the buildup of cell debris and thus channel clogging. Here, we report a versatile strategy to coat the channels of microfluidic devices with lipid bilayers based on noncovalent lipid bicelle technology, which led to substantial improvements in reducing cell adhesion and protein adsorption. The antifouling properties of the lipid bilayer coating were evaluated, including membrane uniformity, passivation against nonspecific protein adsorption, and inhibition of cell attachment against multiple cell types. This surface functionalization approach was applied to coat constricted microfluidic channels for the intracellular delivery of fluorescently labeled dextran and plasmid DNA, demonstrating significant reductions in the accumulation of cell debris. Taken together, our work demonstrates that lipid bicelles are a useful tool to fabricate antifouling lipid bilayer coatings in cell-squeezing devices, resulting in reduced nonspecific fouling and cell clogging to improve performance.


Subject(s)
Biofouling/prevention & control , Lab-On-A-Chip Devices , Lipid Bilayers/chemistry , Cell Adhesion , Cells, Cultured , Humans , Jurkat Cells , Molecular Structure , Particle Size , Surface Properties
2.
Proc Natl Acad Sci U S A ; 117(20): 10976-10982, 2020 05 19.
Article in English | MEDLINE | ID: mdl-32358194

ABSTRACT

Advances in gene editing are leading to new medical interventions where patients' own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells. As these technologies continue to mature, there is a need to achieve efficient, safe, nontoxic, fast, and economical processing of clinically relevant cell types. We demonstrate an acoustofluidic sonoporation method to deliver plasmids to immortalized and primary human cell types, based on pore formation and permeabilization of cell membranes with acoustic waves. This acoustofluidic-mediated approach achieves fast and efficient intracellular delivery of an enhanced green fluorescent protein-expressing plasmid to cells at a scalable throughput of 200,000 cells/min in a single channel. Analyses of intracellular delivery and nuclear membrane rupture revealed mechanisms underlying acoustofluidic delivery and successful gene expression. Our studies show that acoustofluidic technologies are promising platforms for gene delivery and a useful tool for investigating membrane repair.


Subject(s)
Gene Transfer Techniques , Genetic Therapy/methods , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic System , Stem Cells , Cell Survival , Cytoplasm , Gene Expression , Gene Transfer Techniques/instrumentation , Genetic Therapy/instrumentation , Green Fluorescent Proteins/genetics , Humans , Jurkat Cells , Plasmids , Sound
3.
FEBS Lett ; 590(1): 101-9, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26787464

ABSTRACT

Pulmonary arterial hypertension (PAH) is characterized by excessive pulmonary arterial smooth muscle cells (PASMCs) growth, partially in response to PDGF-BB but whether this is dependent on ß-catenin (ßC) activation is unclear. Compared to healthy cells, PAH PASMCs demonstrate higher levels of proliferation both at baseline and with PDGF-BB that correlate with GSK3ß dependent ßC activation. We show that ßC knockdown but not Wnt5a stimulation reduces PDGF-BB dependent growth and normalizes PAH PASMCs proliferation. These findings support that cross-talk between PDGF and Wnt signaling modulates PASMC proliferation and suggest that ßC targeted therapies could treat abnormal vascular remodeling in PAH.


Subject(s)
Familial Primary Pulmonary Hypertension/metabolism , Glycogen Synthase Kinase 3/metabolism , Muscle, Smooth, Vascular/metabolism , Proto-Oncogene Proteins c-sis/metabolism , Pulmonary Artery/metabolism , Wnt Signaling Pathway , beta Catenin/agonists , Active Transport, Cell Nucleus/drug effects , Anticoagulants/pharmacology , Becaplermin , Cell Proliferation/drug effects , Cells, Cultured , Familial Primary Pulmonary Hypertension/pathology , Gene Expression Regulation/drug effects , Genes, Reporter/drug effects , Glycogen Synthase Kinase 3 beta , Humans , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Phosphorylation/drug effects , Promoter Regions, Genetic/drug effects , Protein Processing, Post-Translational/drug effects , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-sis/chemistry , Proto-Oncogene Proteins c-sis/pharmacology , Pulmonary Artery/cytology , Pulmonary Artery/drug effects , Pulmonary Artery/pathology , RNA Interference , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Wnt Proteins/antagonists & inhibitors , Wnt Proteins/genetics , Wnt Proteins/metabolism , Wnt Signaling Pathway/drug effects , Wnt-5a Protein , beta Catenin/antagonists & inhibitors , beta Catenin/genetics , beta Catenin/metabolism
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