University of California

Harvard College

The Brigham and Women's Hospital, Inc.

Nanyang Technological University

Steven J Jonas

Paul S Weiss

Xiaobin Xu

Qing Yang

Natcha Wattanatorn

Jason N Belling

Joanna Aizenberg

Hsian-Rong Tseng

Nam-Joon Cho

Alireza Khademhosseini

Xu Hou

Leonardo Scarabelli

Gail Vinnacombe

Logan A Stewart

Liv Heidenreich

Yao Gong

Naihao Chiang

Adam Z Stieg

Dan Wilkinson

Chuanzhen Zhao

Joshua A A Jackman

A microfluidic device is disclosed that is used to process cells for the intracellular delivery of molecules or other cargo. The device includes one or more microchannels disposed in a substrate or chip and is fluidically coupled to an inlet configured to receive a solution containing the cells and the molecules or other cargo to be delivered intracellularly to the cells. Each of the one or more microchannels has one or more constriction regions formed therein, wherein the inner surface(s) of the microchannels and the one or more constriction regions have a lipid bilayer disposed thereon. In some embodiments, multiple microfluidic devices operating in parallel are used to process large numbers of cells. The device and method have particularly applicability to delivering gene-editing molecules intracellularly to cells.

High-throughput system and method for the temporary permeabilization of cells using lipid bilayers

A method of transporting biomolecular cargo intracellularly into cells includes the operations of providing magnetic nanostructures (e.g., nanospears, nanostars, nanorods, and other nanometer-sized structures) carrying the biomolecular cargo thereon and applying an external magnetic field to move the magnetic nanostructures into physical contact with at least some of the cells (or the cells into the magnetic nanostructures). The magnetic nanostructures move into physical contact with a single cell, a subset of cells, or all cells. The external magnetic field may be applied by a moving permanent magnet although an electromagnetic may also be used. The biomolecular cargo may include a molecule, a plurality of molecules, or higher order biological constructs. For example, the biological construct may include DNA plasmids, small interfering RNA, proteins, or targeted nuclease gene-editing cargo such as zinc-finger nucleases, transcription activator-like effector nucleases, Cas9 protein, Cas9 mRNA, and associated guide RNA sequences.

Guided magnetic nanostructures for targeted and high-throughput intracellular delivery

Systems and methods are disclosed that utilize metal nanostructures that are synthesized in situ along the internal surfaces of a microfluidic device. The nanostructures are formed by initial deposition of metallic seeds followed by flowing growth and reducing agent solutions into the capillaries/microfluidic channels to grow the nanostars. The nanostructures may optionally be functionalized with a capture ligand. The capture ligand may be used to selectively bind to certain cells (e.g., circulating tumor cells). The cells may be removed by a beam of light (e.g., laser beam) that induces localized heating at the surface location(s) containing the nanostructures. The plasmonic nature of the nanostructures can be used to heat the nanostructure(s) locally for the selective removal of one or certain cells. The nanostructures may be used to acquire Raman spectra of molecules or other small objects that are bound thereto for identification and quantification.

Systems and methods of using anisotropic nanostructures in microfluidic devices for binding and optional release of molecules and cells

A microfluidic device for processing cells for the intracellular delivery of molecules or other cargo includes a plurality of microchannels disposed in a substrate or chip and fluidically coupled to an inlet configured to receive a solution containing the cells and the molecules or other cargo to be delivered intracellularly to the cells. Each of the plurality of microchannels has one or more constriction regions therein, wherein the constriction regions comprise an omniphobic, superhydrophilic, or superhydrophobic surface. In some embodiments, multiple microfluidic devices operating in parallel are used to process large numbers of cells. The device and method has particularly applicability to delivering gene-editing molecules intracellularly to cells.

High-throughput system and method for the temporary permeabilization of cells

A microfluidic-based device and system is disclosed for the high-throughput intracellular delivery of biomolecular cargo to cells (eukaryotic or prokaryotic) or enveloped viruses. Cargo integration occurs due to transient membrane permeabilization by exposure to bulk acoustic waves (BAWs) transduced from surface acoustic waves (SAWs) generated by a rapidly oscillating piezoelectric substrate. In this approach, temporary pores are established across the cellular membrane as cells are partially deformed and squeezed or subject to shearing forces as they travel through the vibrational modes created within the microfludic channel(s) of the device.

Device and method for intracellular delivery of biomolecular cargo via acoustic wave exposure

A robust and general fabrication/manufacturing method is described herein for the fabrication of periodic three-dimensional (3D) hierarchical nanostructures in a highly scalable and tunable manner. This nanofabrication technique exploits the selected and repeated etching of spherical particles that serve as resist material and that can be shaped in parallel for each processing step. The method enables the fabrication of periodic, vertically aligned nanotubes at the wafer scale with nanometer-scale control in three dimensions including outer/inner diameters, heights/hole-depths, and pitches. The method was utilized to construct 3D periodic hierarchical hybrid silicon and hybrid nanostructures such as multi-level solid/hollow nanotowers where the height and diameter of each level of each structure can be configured precisely as well as 3D concentric plasmonic supported metal nanodisk/nanorings with tunable optical properties on a variety of substrates.

Methods for multiple-patterning nanosphere lithography for fabrication of periodic three-dimensional hierarchical nanostructures

Growing community of inventors

Hawthorne, CA, United States of America

Steven J Jonas