Abstract: Microfluidic devices having an electrowetting configuration and an optimized droplet actuation surface are provided. The devices include a conductive substrate having a dielectric layer, a hydrophobic layer covalently bonded to the dielectric layer, and a first electrode electrically coupled to the dielectric layer and configured to be connected to a voltage source. The microfluidic devices also include a second electrode, optionally included in a cover, configured to be connected to the voltage source. The hydrophobic layer features self-associating molecules covalently bonded to a surface of the dielectric layer in a manner that produces a densely-packed monolayer that resists intercalation and or penetration by polar molecules or species.

Abstract: This application describes systems and methods for assaying micro-objects in a microfluidic device. These methods include contacting a reagent with a micro-object by introducing a reagent in a first fluidic medium to a flow region of a microfluidic device, wherein the microfluidic device comprises the flow region and a chamber comprising a proximal opening fluidically connecting the chamber to the flow region, diffusing the reagent from the flow region into the chamber; introducing a micro-object into the flow region of the microfluidic device, and diffusing the reagent from the chamber to the flow region to contact the reagent with the micro-object within the flow region. Other embodiments are described.

Abstract: Methods are described herein for screening an antibody producing cell within a microfluidic environment. The antibody producing cell may be a B cell lymphocyte, which may be a memory B cell or a plasma cell. An antigen of interest may be brought into proximity with the antibody producing cell and binding of the antigen by an antibody produced by the antibody producing cell may be monitored. Methods of obtaining a sequencing library from an antibody producing cell are also described.

Abstract: In biosciences and related fields, it can be useful to study cells in isolation so that cells having unique and desirable properties can be identified within a heterogenous mixture of cells. Processes and methods disclosed herein provide for encapsulating cells within a microfluidic device and assaying the encapsulated cells. Encapsulation can, among other benefits, facilitate analyses of cells that generate secretions of interest which would otherwise rapidly diffuse away or mix with the secretions of other cells.

Abstract: Systems, methods and kits are described for culturing one or more biological cells in a microfluidic device, including provision of nutrients and gaseous components configured to enhance cell growth, viability, portability, or any combination thereof. In some embodiments, culturing a single cell may produce a clonal population in the microfluidic device.

Abstract: Reagents for stabilizing the nucleic acids of a biological cell, compositions, kits and methods of use thereof are described. The stabilization reagents may prepare the nucleic acids within the biological cell for storage and preserve the representative population of the nucleic acids for later isolation and analysis.

Abstract: A method of preparing an antibody therapeutic is provided comprising: (a) providing a dissociated cell sample from at least one solid tumor sample obtained from a patient; (b) loading the dissociated cell sample into a microfluidic device having a flow region and at least one isolation region fluidically connected to the flow region; (c) moving at least one B cell from the dissociated cell sample into at least one isolation region in the microfluidic device, thereby obtaining at least one isolated B cell; and (d) using the microfluidic device to identify at least one B cell that produces antibodies capable of binding to cancer cells. The cancer cells can be the patient's own cancer cells. Also provided are methods of treating patients, methods of labeling or detecting cancer, engineered T or NK cells comprising antibodies or fragments thereof, and engineered antibody constructs.

Abstract: Microfluidic devices having an electrowetting configuration and an optimized droplet actuation surface are provided. The devices include a conductive substrate having a dielectric layer, a hydrophobic layer covalently bonded to the dielectric layer, and a first electrode electrically coupled to the dielectric layer and configured to be connected to a voltage source. The microfluidic devices also include a second electrode configured to be connected to the voltage source. The hydrophobic layer features self-associating molecules covalently bonded to a surface of the dielectric layer in a manner that produces a densely-packed monolayer that resists intercalation and or penetration by polar molecules or species.

Abstract: Disclosed herein are methods for performing assays, including general functional assays, on a biological cell. Also disclosed herein are methods of barcoding the 5? ends of RNA from a biological cell and methods of preparation of expression constructs from the barcoded RNA. The barcoded RNA can encode proteins of interest, such as B cell receptor (BCR) heavy and light chain sequences. The expression constructs can be generated individually or in a paired/multiplexed manner, allowing rapid re-expression of individual proteins or protein complexes.

Abstract: In biosciences and related fields, it can be useful to study cells in isolation so that cells having unique and desirable properties can be identified within a heterogenous mixture of cells. Processes and methods disclosed herein provide for encapsulating cells within a microfluidic device and assaying the encapsulated cells. Encapsulation can, among other benefits, facilitate analyses of cells that generate secretions of interest which would otherwise rapidly diffuse away or mix with the secretions of other cells.

Abstract: In biosciences and related fields, it can be useful to study cells in isolation so that cells having unique and desirable properties can be identified within a heterogenous mixture of cells. Processes and methods disclosed herein provide for encapsulating cells within a microfluidic device and assaying the encapsulated cells. Encapsulation can, among other benefits, facilitate analyses of cells that generate secretions of interest which would otherwise rapidly diffuse away or mix with the secretions of other cells.

Abstract: The disclosure provides methods for amplification of paired transcript sequences from a single cell, such as, but not limited to alpha and beta T cell receptor (TCR) sequences from a single T cell. The method can include: placing a single T cell into a cell lysis solution to provide a T cell lysate comprising RNA; generating first strand cDNA from the RNA; amplifying the first strand cDNA to provide amplified cDNA; and amplifying the alpha and beta TCR sequences from the amplified cDNA in a single reaction, wherein the alpha and beta TCR sequences are amplified using three sets of TCR amplification primers. In some instances, the methods can be performed using no more than six sets of primers.

Abstract: Systems, methods and kits are described for culturing one or more biological cells in a microfluidic device, including provision of nutrients and gaseous components configured to enhance cell growth, viability, portability, or any combination thereof. In some embodiments, culturing a single cell may produce a clonal population in the microfluidic device.

Abstract: Systems, methods and kits are described for culturing one or more biological cells in a microfluidic device, including provision of nutrients and gaseous components configured to enhance cell growth, viability, portability, or any combination thereof. In some embodiments, culturing a single cell may produce a clonal population in the microfluidic device.

Abstract: In biosciences and related fields, it can be useful to study cells in isolation so that cells having unique and desirable properties can be identified within a heterogenous mixture of cells. Processes and methods disclosed herein provide for encapsulating cells within a microfluidic device and assaying the encapsulated cells. Encapsulation can, among other benefits, facilitate analyses of cells that generate secretions of interest which would otherwise rapidly diffuse away or mix with the secretions of other cells.

Abstract: Methods are provided for the assay of secreted biomolecules using automated detection and characterization of micro-objects in a microfluidic device. The biomolecules can be secreted by cells, particularly immunological cells, such as T cells. The biomolecules being assayed can include cytokines, growth factors, and the like. Methods are also provided for assaying the cytotoxicity of a cell with respect to another, target cell. Also provided are kits and non-transitory computer-readable media in which programs are stored for causing a system comprising a computer to perform automated methods for detecting secreted biomolecules and/or cytotoxicity in a microfluidic device.

Abstract: Systems, methods, and kits therefor, enabling rapid protein evolution are described herein. A system useful in the methods described herein include a DNA synthesis component; a microfluidic system including a microfluidic device having a microfluidic channel and sequestration pens; and a computing component, which is configured to analyze assay results and, based upon the analysis, design improved DNA sequences for iterative protein evolution. The microfluidic system is configured to permit correlation of DNA sequence on a bead to its location within the microfluidic device, permit cell free protein expression of a DNA sequence captured to a bead, and to permit assay of the protein so produced.

Abstract: Methods are described herein for isolating clonal populations of cells having a defined genetic modification. The methods are performed, at least in part, in a microfluidic device comprising one or more sequestration pens. The methods include the steps of: maintaining individual cells (or precursors thereof) that have undergone a genomic editing process in corresponding sequestration pens of a microfluidic device; expanding the individual cells into respective clonal populations of cells; and detecting, in one or more cells of each clonal population, the presence of a first nucleic acid sequence that is indicative of the presence of an on-target genome edit in the clonal population of cells. Also described are methods of performing genome editing within a microfluidic device, and compositions comprising one or more clonal populations of cells generated according to the methods disclosed herein.

Abstract: Processes and kits are provided for producing sequence specific fragments of nucleic acid molecules, whether from a genome or transcriptome, where one end of the molecule is highly diverse and/or the full-length molecule, whether a gene or a mRNA, is too long for it to be sequenced using currently available sequencing methods. Methods of preparing a sequencing library configured for 5? or 3? anchored sequencing, wherein the opposing termini of the library molecules are differentially truncated, and methods of parallel sequencing such libraries are described.

Abstract: Microfluidic devices having an electrowetting configuration and an optimized droplet actuation surface are provided. The devices include a conductive substrate having a dielectric layer, a hydrophobic layer covalently bonded to the dielectric layer, and a first electrode electrically coupled to the dielectric layer and configured to be connected to a voltage source. The microfluidic devices also include a second electrode configured to be connected to the voltage source. The hydrophobic layer features self-associating molecules covalently bonded to a surface of the dielectric layer in a manner that produces a densely-packed monolayer that resists intercalation and or penetration by polar molecules or species.