Abstract: Devices and methods are presented for delivery of various macrostructures into cells, such as depleted cells as well as methods of generating engineered cells using said devices and methods. Cells are placed on a porous membrane. A force is applied by a configurable actuator to a deformable fluid reservoir that generates an applied pressure to macrostructures in a solution, causing the macrostructures to pass through the porous membrane and triggering uptake of at least some of the macrostructures into the cells to form transfected cells. Said devices and methods may be used in a process to replace defective endogenous mtDNA with corrected mtDNA to generate cellular-based therapeutics for administration to a patient. The customizable actuator may be configured with parameters to optimize efficiency for a given cell type and material to be transfected. Machine learning techniques also may be utilized to optimize transfection parameters.

Abstract: Methods are provided for transfecting cells with large cargo using a poly(beta-amino ester) (PBAE) molecule, and achieving high efficiency and viability. A method is provided of transfecting cells with a cargo, by forming a complex of the cargo with a (PBAE) molecule, mixing the complex with a first buffer and contacting the complex with the cells, wherein the cargo has a dimension of at least 0.1 ?m. The PBAE molecule may be formed by reacting an amine with a di(acrylate ester). In some aspects, the PBAE molecule is poly(1,4-butanediol diacrylate-co-4-amino-1-butanol). In some aspects, the PBAE molecule is capped with 1-(3-aminopropyl)-4-methylpiperazine.

Abstract: A transfection device suitable for delivery of various macrostructures (e.g., mitochondria, bacteria, liposomes) is described and uses mechanical force to thereby induce active endocytosis in a target cell. Contemplated devices are able to achieve high throughput of transfected cells that remain viable and are capable of producing colonies.

Abstract: Systems and methods for using microfluidic devices to concentrate cells, to perform buffer changes, to sort cells based on size, and/or to isolate particular types of cells in a rapid manner, are presented. Cells flow into a matrix of posts, wherein the posts are distributed along diagonal lines in the chamber. The cells are deflected in a lateral manner, towards a side of a chamber and are collected upon exiting the chamber.

Abstract: Systems and methods are provided for transfecting cells, such as mammalian cells and nonmammalian cells, using an electroporation apparatus having an electroporation chamber including an upper micromesh electrode, a lower micromesh electrode and a path defined in the electroporation chamber. The electroporation apparatus includes a first input allowing passage of cells into the electroporation chamber and a first output allowing passage of electroporated cells from the electroporation chamber. The first input and the first output are separated by an offset distance.

Abstract: Methods for preparing antigen-loaded dendritic cells (DCs) for treatment of cancer in a subject are provided, using an electroporation apparatus having an electroporation chamber including an upper electrode, a lower electrode and a path defined in the electroporation chamber. The electroporation apparatus includes a first input allowing passage of DCs into the electroporation chamber and a first output allowing passage of antigen-loaded DCs from the electroporation chamber. The first input and the first output are separated by an offset distance. Methods of treating cancer by administering antigen-loaded DCs are also provided.

Abstract: Systems and methods for transfection devices are contemplated for delivery of various complex macrostructures. Preferred systems and methods are suitable for mRNA reprogramming and genome editing and use mechanical force to induce uptake of the macrostructures in a target cell. Contemplated devices are able to achieve high throughput of transfected cells in remarkably short time that remain viable and are capable of producing colonies.

Abstract: A transfection device suitable for delivery of various macrostructures (e.g., mitochondria, bacteria, liposomes) is described and uses mechanical force to thereby induce active endocytosis in a target cell. Contemplated devices are able to achieve high throughput of transfected cells that remain viable and are capable of producing colonies.

Abstract: Methods and devices are provided for focusing and/or sorting activated T cells. The device comprises a microfluidic channel comprising a plurality of electrodes arranged to provide dielectrophoretic (DEP) forces that are perpendicular to forces from hydrodynamic flows along the channel. The device may be configured to apply voltages to a plurality of electrodes in a first upper region of the microfluidic channel to focus the cells into a single flow, and to apply different voltages to a plurality of electrodes in a second downstream region of the microfluidic channel to sort cells based on size. The output of the microfluidic channel may diverge into multiple channels, wherein cells of various sorted sizes are directed into the appropriate output channel.