Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules.

We describe a method for fragmenting, in-situ, surface-adsorbed and immobilized DNAs on polymethylmethacrylate(PMMA)-coated silicon substrates using microfluidic delivery of the cutting enzyme DNase I. Soft lithography is used to produce silicone elastomer (Sylgard 184) gratings which form microfluidic channels for delivery of the enzyme. Bovine serum albumin (BSA) is used to reduce DNase I adsorption to the walls of the microchannels and enable diffusion of the cutting enzyme to a distance of 10mm. Due to the DNAs being immobilized, the fragment order is maintained on the surface. Possible methods of preserving the order for application to sequencing are discussed.

A Size-Selective Intracellular Delivery Platform.

Identifying and separating a subpopulation of cells from a heterogeneous mixture are essential elements of biological research. Current approaches require detailed knowledge of unique cell surface properties of the target cell population. A method is described that exploits size differences of cells to facilitate selective intracellular delivery using a high throughput microfluidic device. Cells traversing a constriction within this device undergo a transient disruption of the cell membrane that allows for cytoplasmic delivery of cargo. Unique constriction widths allow for optimization of delivery to cells of different sizes. For example, a 4 µm wide constriction is effective for delivery of cargo to primary human T-cells that have an average diameter of 6.7 µm. In contrast, a 6 or 7 µm wide constriction is best for large pancreatic cancer cell lines BxPc3 (10.8 µm) and PANC-1 (12.3 µm). These small differences in cell diameter are sufficient to allow for selective delivery of cargo to pancreatic cancer cells within a heterogeneous mixture containing T-cells. The application of this approach is demonstrated by selectively delivering dextran-conjugated fluorophores to circulating tumor cells in patient blood allowing for their subsequent isolation and genomic characterization.

Plasma membrane recovery kinetics of a microfluidic intracellular delivery platform.

Intracellular delivery of materials is a challenge in research and therapeutic applications. Physical methods of plasma membrane disruption have recently emerged as an approach to facilitate the delivery of a variety of macromolecules to a range of cell types. We use the microfluidic CellSqueeze delivery platform to examine the kinetics of plasma membrane recovery after disruption and its dependence on the calcium content of the surrounding buffer (recovery time ∼ 5 min without calcium vs. ∼ 30 s with calcium). Moreover, we illustrate that manipulation of the membrane repair kinetics can yield up to 5× improvement in delivery efficiency without significantly impacting cell viability. Membrane repair characteristics initially observed in HeLa cells are shown to translate to primary naïve murine T cells. Subsequent manipulation of membrane repair kinetics also enables the delivery of larger materials, such as antibodies, to these difficult to manipulate cells. This work provides insight into the membrane repair process in response to mechanical delivery and could potentially enable the development of improved delivery methods.

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 technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

A vector-free microfluidic platform for intracellular delivery.

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.