cell-squeezing-as-robust-microfluidic-intracellular-delivery

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Massachusetts Institute of Technology

We report a microfluidic based approach for single cell microinjection in which fluid streams direct a cell onto a fixed microneedle in contrast to moving a microneedle towards an immobilized cell, as done in conventional methods. The approach simplifies microinjection and offers the potential for flow through automated microinjection of cells.

Microfluidic based single cell microinjection.

Mechanical properties of cells have been shown to have a significant role in disease, as in many instances cell stiffness changes when a cell is no longer healthy. We present a high-throughput microfluidics-based approach that exploits the connection between travel time of a cell through a narrow passage and cell stiffness. The system resolves both cell travel time and relative cell diameter while retaining information on the cell level. We show that stiffer cells have longer transit times than less stiff ones and that cell size significantly influences travel times. Experiments with untreated HeLa cells and cells made compliant with latrunculin A and cytochalasin B further demonstrate that travel time is influenced by cell stiffness, with the compliant cells having faster transit time.

Microfluidics-based assessment of cell deformability.

The ability to straightforwardly deliver engineered nanoparticles into the cell cytosol with high viability will vastly expand the range of biological applications. Nanoparticles could potentially be used as delivery vehicles or as fluorescent sensors to probe the cell. In particular, quantum dots (QDs) may be used to illuminate cytosolic proteins for long-term microscopy studies. Whereas recent advances have been successful in specifically labeling proteins with QDs on the cell membrane, cytosolic delivery of QDs into live cells has remained challenging. In this report, we demonstrate high throughput delivery of QDs into live cell cytoplasm using an uncomplicated microfluidic device while maintaining cell viabilities of 80-90%. We verify that the nanoparticle surface interacts with the cytosolic environment and that the QDs remain nonaggregated so that single QDs can be observed.

Nonendocytic delivery of functional engineered nanoparticles into the cytoplasm of live cells using a novel, high-throughput microfluidic device.

We present a microfluidic electroporation device with a comb electrode layout fabricated in polydimethylsiloxane (PMDS) and glass. Characterization experiments with HeLa cells and fluorescent dextran show efficient delivery (∼95%) with low toxicity (cell viability ∼85%) as well as rapid pore closure after electroporation. The activity of delivered molecules is also verified by silencing RNA (siRNA) studies that demonstrate gene knockdown in GFP expressing cells. This simple, scalable approach to microfluidic, flow-through electroporation could facilitate the integration of electroporation modules within cell analysis devices that perform multiple operations.

Flow-through comb electroporation device for delivery of macromolecules.

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.

Article	Year
Microfluidic based single cell microinjection. Lab on a chip\| PubMed ID: 18651065	2008
Microfluidics-based assessment of cell deformability. Analytical chemistry\| PubMed ID: 22746217	2012
Nonendocytic delivery of functional engineered nanoparticles into the cytoplasm of live cells using a novel, high-throughput microfluidic device. Nano letters\| PubMed ID: 23145796	2012
Flow-through comb electroporation device for delivery of macromolecules. Analytical chemistry\| PubMed ID: 23259401	2013
A vector-free microfluidic platform for intracellular delivery. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23341631	2013

Andrea Adamo

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