Abstract: Proto-antigen-presenting surfaces and related kits, methods, and uses are provided. The proto-antigen-presenting surface can comprise a plurality of primary activating molecular ligands comprising a major histocompatibility complex (MHC) molecule configured to bind to a T cell receptor (TCR) of a T cell and a plurality of co-activating molecular ligands each including a TCR co-activating molecule or an adjunct TCR activating molecule, wherein an exchange factor is bound to the MHC molecules. Exchange factors include, e.g., dipeptides such as GL, GF, GR, etc. Proto-antigen-presenting surfaces can be used to rapidly prepare antigen-presenting surfaces comprising one or more peptide antigens of interest by contacting the proto-antigen-presenting surface with one or more peptide antigens so as to displace the exchange factor. As such, the disclosure facilitates rapid evaluation of the immunogenicity of peptide antigens for activating T lymphocytes.

Abstract: Methods are described herein for isolating clonal populations of T 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 T cells (or precursors thereof) that have undergone a genomic editing process in corresponding sequestration pens of a microfluidic device; expanding the T cells into respective clonal populations of T cells; detecting, in one or more T cells of each clonal population, the absence of a cell surface marker that was present in the individual T cells (or precursors thereof); and detecting, in one or more T 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 T cells. Also described are compositions comprising one or more clonal populations of T cells isolated according to the methods disclosed herein.

Abstract: The present disclosure provides methods of preparing tumor infiltrating cells engineered to express a pro-inflammatory polypeptide. The pro-inflammatory polypeptide is expressed from the tumor infiltrating cell to counter a generally immunosuppressive state in and around tumors resulting from an imbalance between the number and activation state of immune effector cells versus those of suppressor cells. Delivering the proinflammatory polypeptide via expression from the TICs, as distinct from systemic administration, reduces side effects from increased inflammation at sides remote from a tumor to be treated.

Abstract: Methods for laser-assisted repositioning of a micro-object and for culturing an attachment-dependent biological cell within a microfluidic device are described herein. Laser illumination is used to controllably create a bubble which repositions the micro-object. Further, methods of culturing an attachment-dependent biological cell are described, where the methods may include laser-assisted repositioning.

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: 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: Disclosed herein are methods for performing assays, including general functional assays, on a biological cell. The methods can include contacting a biological cell with a test agent for a period of time; lysing the biological cell while the biological cell is disposed within a sequestration pen located within an enclosure of a microfluidic device; and allowing RNA molecules released from the lysed biological cell to be captured by capture oligonucleotides linked to a capture object disposed within the sequestration pen of the microfluidic device. Each capture oligonucleotide can include a priming sequence that binds a primer, and a capture sequence. Each cDNA transcribed from a captured RNA can have an oligonucleotide sequence complementary to the captured RNA molecule, with the complementary oligonucleotide sequence being covalently linked to one of the capture oligonucleotides of the capture object.

Abstract: In biosciences and related fields, it can be useful to modify surfaces of apparatuses, devices, and materials that contact biomaterials such as biomolecules and biological micro-objects. Described herein are surface modifying and surface functionalizing reagents, preparation thereof, and methods for modifying surfaces to activate T Lymphocytes.

Abstract: Proto-antigen-presenting surfaces and related kits, methods, and uses are provided. The proto-antigen-presenting surface can comprise a plurality of primary activating molecular ligands comprising a major histocompatibility complex (MHC) molecule configured to bind to a T cell receptor (TCR) of a T cell and a plurality of of co-activating molecular ligands each including a TCR co-activating molecule or an adjunct TCR activating molecule, wherein an exchange factor is bound to the MHC molecules. Exchange factors include, e.g., dipeptides such as GL, GF, GR, etc. Proto-antigen-presenting surfaces can be used to rapidly prepare antigen-presenting surfaces comprising one or more peptide antigens of interest by contacting the proto-antigen-presenting surface with one or more peptide antigens so as to displace the exchange factor. As such, the disclosure facilitates rapid evaluation of the immunogenicity of peptide antigens for activating T lymphocytes.

Abstract: Methods are described herein for isolating clonal populations of T 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 T cells (or precursors thereof) that have undergone a genomic editing process in corresponding sequestration pens of a microfluidic device; expanding the T cells into respective clonal populations of T cells; detecting, in one or more T cells of each clonal population, the absence of a cell surface marker that was present in the individual T cells (or precursors thereof); and detecting, in one or more T 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 T cells. Also described are compositions comprising one or more clonal populations of T cells isolated according to the methods disclosed herein.

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: 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: 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: The present disclosure provides methods of preparing tumor infiltrating cells engineered to express a pro-inflammatory polypeptide. The pro-inflammatory polypeptide is expressed from the tumor infiltrating cell to counter a generally immunosuppressive state in and around tumors resulting from an imbalance between the number and activation state of immune effector cells versus those of suppressor cells. Delivering the proinflammatory polypeptide via expression from the TICs, as distinct from systemic administration, reduces side effects from increased inflammation at sides remote from a tumor to be treated.