Method and apparatus for high throughput high efficiency transfection of cells
Inventors
Tandon, Vishal • Lissandrello, Charles A. • Balestrini, Jenna L. • Coppeta, Jonathan R. • Swierk, Patricia A.
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Assignees
Charles Stark Draper Laboratory Inc
Member
DraperDraper is an independent nonprofit engineering innovation company with a legacy spanning over 90 years, dedicated to delivering transformative solutions for national security, prosperity, and global challenges. Renowned for its pioneering work in guidance, navigation, and control (GN&C) systems, Draper partners with government, industry, and academia to engineer advanced technologies in space, defense, biotechnology, and electronic systems. The company leverages multidisciplinary expertise, digital engineering, and a collaborative approach to provide field-ready prototypes, mission-critical systems, and innovative research. Draper’s mission is to ensure the nation's security and prosperity by delivering sustainable, cutting-edge solutions that address the toughest problems of today and tomorrow, while fostering an inclusive and diverse workforce. Draper also invests in the next generation of innovators through robust educational programs, including internships, co-ops, and the Draper Scholars Program, integrating academic research with real-world problem-solving.
Draper is an independent nonprofit engineering innovation company with a legacy spanning over 90 years, dedicated to delivering transformative solutions for national security, prosperity, and global challenges. Renowned for its pioneering work in guidance, navigation, and control (GN&C) systems, Draper partners with government, industry, and academia to engineer advanced technologies in space, defense, biotechnology, and electronic systems. The company leverages multidisciplinary expertise, digital engineering, and a collaborative approach to provide field-ready prototypes, mission-critical systems, and innovative research. Draper’s mission is to ensure the nation's security and prosperity by delivering sustainable, cutting-edge solutions that address the toughest problems of today and tomorrow, while fostering an inclusive and diverse workforce. Draper also invests in the next generation of innovators through robust educational programs, including internships, co-ops, and the Draper Scholars Program, integrating academic research with real-world problem-solving.
Publication Number
US-11859162-B2
Publication Date
2024-01-02
Expiration Date
Abstract
Transfer of genetic and other materials to cells is conducted in a hands-free, automated, high throughput, continuous process. A system using a microfluidic hydrodynamic sheath flow configuration includes arrangements for pushing cells from side streams containing a cell culture medium to a central stream containing an electroporation buffer. Electroporation can be conducted in an assembly in which two or more microfluidic channels are provided in a parallel configuration and in which various layers can be stacked together to form a laminate type structure.
Core Innovation
Described herein are approaches in which transfer of genetic or other materials to cells is conducted in a hands-free, automated, high throughput, continuous process. Some embodiments rely on a microfluidic hydrodynamic sheath flow configuration and include arrangements for pushing cells from side streams containing, for example, a cell culture medium, to a central stream containing an electroporation buffer.
Electroporation can be conducted in an assembly in which two or more microfluidic channels are provided in a parallel configuration and in which various layers can be stacked together to form a laminate type structure. Each microfluidic channel is provided with a pair of electrodes, preferably constructed to withstand long-lasting, continuous and high throughput operations.
The invention addresses problems in conventional systems including batch electroporation using cuvettes, manual buffer exchanges and wash steps that are touch-labor intensive and associated with cell loss, exposure of biologics to direct contact with electrodes causing potential damage, and lack of high throughput transfection systems. The system and methods provide automated buffer exchanges, protection of cells from direct electrode contact, and continuous processing with assemblies that include multiple parallel microfluidic channels and common system components.
Claims Coverage
Independent claims identified: 1, 8 and 10. The following inventive features are extracted from these independent claims.
Directing cells from an incubator to multiple microfluidic devices
directing cells from a first incubator to an assembly that contains multiple microfluidic devices;
Acoustic transfer from side stream to central electroporation medium
acoustically driving cells from a cell culture medium flowing in a side stream of a sheath flow configuration, to an electroporation medium flowing through a central stream of the sheath flow configuration;
Applying an electric field in electroporation medium
applying an electric field to cells in the electroporation medium; and transferring or allowing the transfer of the payload into the cells.
Automated manufacturing for immunotherapy
acoustically transferring cells from a first buffer, wherein the first buffer is a cell culture medium, into an electroporation buffer; permeabilizing the cells by electroporation; allowing a payload to transfer into the permeabilized cells; and transferring the cells containing the payload into a second buffer, wherein, the method is conducted in an automated and continuous flow mode, wherein the electroporation buffer forms a central stream and the first buffer forms a side stream of a sheath flow configuration, and wherein, [procedural detail omitted for safety].
Sheath-flow electroporation with acoustic focusing
flowing an electroporation medium as a central stream of a sheath flow configuration; flowing a first medium containing cells as a side stream in the sheath flow configuration; applying acoustic energy to drive cells from the first medium to the electroporation medium; applying an electric field to permeabilize the cells in the electroporation medium; and allowing the payload to transfer into the permeabilized cells.
The independent claims cover methods combining acoustic-driven buffer switching in a sheath flow configuration with flow electroporation in microfluidic devices, and an automated continuous-flow manufacturing method for immunotherapy cells incorporating these steps.
Stated Advantages
Reduces or minimizes the duration of exposure of cells to non-ideal conditions and loss of cells and/or genetic material associated with conventional buffer exchanges.
Automates cell handling and buffer exchanges during the entire process, removing the need for manual centrifugation and resuspension steps.
Protects cells and biologics from direct contact with electroporation electrodes, reducing potential damage due to local heating and Faradaic by-products.
Enables continuous, hands-free processing of large numbers of cells with high throughput using parallel microfluidic channels and common system components.
Provides precise control of residence time of cells within solutions in a continuous fashion.
Offers a robust, flexible and versatile design applicable to various types of cargo, cells or other membrane-bound substrates.
Documented Applications
Cellular therapy manufacturing.
Electrotransfection of primary T cells with mRNA.
Administration of payload-containing cells to a subject in need of diagnosis, prophylaxis or treatment.
Production of autologous or allogeneic CAR-T, allogeneic or autologous TCR, TRnC cells, modified TILs, CAR-NKTs, CAR-NKs, CAR-Macs, CAR-CIK or modified gamma delta cells.
Engineering cargo-loaded exosomes.
Production of gene-modified stem or suspension cells to treat genetic diseases or disorders.
Protein or extracellular vesicle production using modified CHO cells or MSCs.
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