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: 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 provide improved or altered performance with biomaterials.

Abstract: A system and a method for dynamically optimizing an instrument system workflow based on operational monitoring and managing of a workflow for a hardware system. The system includes instrument resources and sample chambers, each resource and chamber with a dedicated sensor configured to acquire data. The system further includes a computing device communicatively connected to the instrument resources and sample chambers. The computing device includes a software application or program comprising a workflow builder, an execution engine, an analytics engine, a virtual system modeling engine, and an optional machine learning engine.

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: The present disclosure relates to an optical apparatus for imaging and/or manipulating micro-objects in a microfluidic device, such as a light-actuated microfluidic (LAMF) device, and related systems and methods. The optical apparatus can comprise a structured light modulator, a first and a second tube lens, an objective lens, a dichroic beam splitter, and an image sensor. The structured light modulator can be configured to receive unstructured light beams and transmit structured light beams for illuminating micro-objects located within an enclosure of the microfluidic device and/or selectively activating one or more of a plurality of dielectrophoresis (DEP) electrodes of the microfluidic device. The image light beams received by the image sensor can be used to form an image of at least a portion of the microfluidic device.

Abstract: Methods of sorting T lymphocytes in a microfluidic device are provided. The methods can include flowing a fluid sample comprising T lymphocytes through a region of a microfluidic device that contains an array of posts. The array of posts can be configured to have a critical size (Dc) that separates activated T lymphocytes from naïve T lymphocytes. Also provided are microfluidic devices having an array of posts configured to separate activated T lymphocytes from naïve T lymphocytes, compositions enriched for T lymphocytes, particularly activated T lymphocytes that are known to be reactive to an antigen of interest, and methods of treating subjects suffering from a pathogenic disorder or cancer by administering such compositions.

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: 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: A microfluidic device can include a base an outer surface of which forms one or more enclosures for containing a fluidic medium. The base can include an array of individually controllable transistor structures each of which can comprise both a lateral transistor and a vertical transistor. The transistor structures can be light activated, and the lateral and vertical transistors can thus be photo transistors. Each transistor structure can be activated to create a temporary electrical connection from a region of the outer surface of the base (and thus fluidic medium in the enclosure) to a common electrical conductor. The temporary electrical connection can induce a localized electrokinetic force generally at the region, which can be sufficiently strong to move a nearby micro-object in the enclosure.

Abstract: A microfluidic device can comprise at least one swept region that is fluidically connected to unswept regions. The fluidic connections between the swept region and the unswept regions can enable diffusion but substantially no flow of media between the swept region and the unswept regions. The capability of biological micro-objects to produce an analyte of interest can be assayed in such a microfluidic device. Biological micro-objects in sample material loaded into a microfluidic device can be selected for particular characteristics and disposed into unswept regions. The sample material can then be flowed out of the swept region and an assay material flowed into the swept region. Flows of medium in the swept region do not substantially affect the biological micro-objects in the unswept regions, but any analyte of interest produced by a biological micro-object can diffuse from an unswept region into the swept region, where the analyte can react with the assay material to produce a localized detectable reaction.

Abstract: Disclosed are methods, systems, and articles of manufacture for performing a process on biological samples. An analysis of biological samples in multiple regions of interest in a microfluidic device and a timeline correlated with the analysis may be identified. One or more region-of-interest types for the multiple regions of interest may be determined; and multiple characteristics may be determined for the biological samples based at least in part upon the one or more region-of-interest types. Associated data that respectively correspond to the multiple regions of interest in a user interface for at least a portion of the biological samples in the user interface based at least in part upon the multiple identifiers and the timeline. A count of the biological samples in a region of interest may be determined based at least in part upon a class or type of data using a convolutional neural network (CNN).

Abstract: Incubators including an enclosure with an internal chamber configured to support a cell culture plate comprising a plurality of wells are disclosed. The enclosure includes a plurality of openings configured to allow access to the wells. The incubators include a sealing element configured to seal the plurality of openings in the enclosure. The sealing element comprises a plurality of openings corresponding to at least a subset of the plurality of openings in the enclosure. Access to the internal chamber can be provided by aligning the plurality of openings in the sealing element with the plurality of openings in the enclosure. Methods for using the incubators are also provided.

Abstract: In situ-generated microfluidic capture structures incorporating a solidified polymer network, methods of preparation and use, compositions and kits therefor are described. Microfluidic capture structures may be advantageously used for assays performed within the microfluidic environment, providing flexibility in assaying micro-objects such as biological cells. Assay reagents and analytes may be incorporated within the microfluidic capture structures.

Abstract: Incubators are disclosed which include an enclosure with an internal chamber configured to support a cell culture plate and provide an environment suitable for maintaining and/or culturing biological cells. The enclosure can include one or more openings configured to allow access to the cell culture plate. The incubators can further include a structure having a plurality of openings configured to be aligned with a corresponding plurality of wells in the cell culture plate, and a sealing element configured to moveably seal the plurality of openings in the structure. The sealing element can comprise a plurality of openings corresponding to at least a subset of the plurality of openings of the structure. Access to the internal chamber can be provided by aligning the plurality of openings in the sealing element with the plurality of openings in the structure. Methods for using the incubators are also provided.

Abstract: Optically-actuated microfluidic devices permit the use of spatially-modulated light to manipulate micro-objects such as biological cells. Systems and methods are described for providing sequences of light patterns to move and direct a plurality of micro-objects within the environment of a microfluidic device. The sequenced light patterns provide improved efficiency in directing the transport of the plurality of micro-objects. Other embodiments are described.

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: Individual biological cells can be selected in a micro-fluidic device and moved into isolation pens in the device. The cells can then be lysed in the pens, releasing nucleic acid material, which can be captured by one or more capture objects in the pens. The capture objects with the captured nucleic acid material can then be removed from the pens. The capture objects can include unique identifiers, allowing each capture object to be correlated to the individual cell from which the nucleic acid material captured by the object originated.

Abstract: Biological activity in holding pens in a micro-fluidic device can be assayed by placing in the holding pens capture objects that bind a particular material of interest produced by the biological activity. The biological material of interest that binds to each capture object can then be assessed, either in the micro-fluidic device or after exporting the capture object from the micro-fluidic device. The assessment can be utilized to characterize the biological activity in each holding pen. The biological activity can be production of the biological material of interest. Thus, the biological activity can correspond to or arise from one or more biological cells. Biological cells within a holding pen can be clonal cell colonies. The biological activity of each clonal cell colony can be assayed while maintaining the clonal status of each colony.

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.