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US 2023/0053160 A1Published application
Selective and High-Resolution Printing of Single Cells
AssigneeThe Regents of the University of California
InventorsAdam R. Abate, Pengfei Zhang, Russell Cole
Life of this patent
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Abstract
Methods for on-demand printing discrete entities including, e.g., cells, media or reagents to substrates are provided. In certain aspects, the methods include manipulating qualities of the entities or biological components thereof. In some embodiments, the methods may be used to create arrays of microenvironments and/or for two and three-dimensional printing of tissues or structures and/or for in situ printing for microsurgeries. Systems and devices for practicing the subject methods are also provided.
Claims231
A method of delivering discrete entities to a substrate, the method comprising:
- flowing a plurality of discrete entities through an air flow via a microfluidic device comprising an air/liquid co-flow junction;
- directing the air flow and one or more of the plurality of discrete entities through the air/liquid co-flow junction to the substrate;
- and
- affixing the one or more of the plurality of discrete entities to the substrate via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof.
1 more element
A method of sorting microdroplets, the method comprising:
- flowing a plurality of microdroplets through a microfluidic device comprising an air/liquid co-flow junction;
- detecting via a detector a property of one or more of the plurality of microdroplets;
- and
- applying an electric field to selectively deflect one or more of the plurality of microdroplets based on the detection of the property.
1 more element
A method of treating a wound area comprising printing one or more cell layers on the wound area, wherein the method comprises:
- encapsulating cells in droplets comprising an aqueous fluid to provide cell-comprising droplets;
- flowing a plurality of droplets comprising the cell-comprising droplets through an air flow via a microfluidic device comprising an air/liquid co-flow junction;
- directing the air flow and a plurality of the cell-comprising droplets through the air/liquid co-flow junction to the wound area;
- and
- affixing the plurality of the cell-comprising droplets to the wound area via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof, to provide a first layer of cell-comprising droplets.
2 more elements
A method of printing one or more cell layers, the method comprising:
- encapsulating cells in droplets comprising an aqueous fluid to provide cell-comprising droplets;
- flowing a plurality of droplets comprising the cell-comprising droplets through an air flow via a microfluidic device comprising an air/liquid co-flow junction;
- directing the air flow and a plurality of the cell-comprising droplets through the air/liquid co-flow junction to a substrate;
- and
- affixing the plurality of the cell-comprising droplets to the substrate via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof, to provide a first layer of cell-comprising droplets.
2 more elements
A method of printing and detecting one or more cells, the method comprising:
- encapsulating cells in droplets comprising an aqueous fluid to provide cell-comprising droplets;
- flowing a plurality of droplets comprising the cell-comprising droplets through an air flow via a microfluidic device comprising an air/liquid co-flow junction;
- directing the air flow and a plurality of the cell-comprising droplets through the air/liquid co-flow junction to a substrate;
- affixing the plurality of the cell-comprising droplets to the substrate via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof, to provide a first layer of cell-comprising droplets;
- and
- detecting one or more of the cells in the affixed cell-comprising droplets, a component of one or more of the cells in the affixed cell-comprising droplets or a product of one or more of the cells in the affixed cell-comprising droplets.
3 more elements
A method of printing a three-dimensional structure, the method comprising:
- flowing discrete entities through an air flow via a microfluidic device comprising an air/liquid co-flow junction;
- directing the air flow and a first plurality of the discrete entities through the air/liquid co-flow junction to a substrate;
- affixing the first plurality of the discrete entities to the substrate via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof, to provide a first layer thereon;
- directing the air flow and a second plurality of the discrete entities through the air/liquid co-flow junction to the first layer;
- affixing the second plurality of the discrete entities to the substrate via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof, to provide a second layer thereon;
- and
- one or more additional directing steps in which a plurality of the discrete entities are directed through the air/liquid co-flow junction to an immediately preceding layer to provide a subsequent layer thereon, wherein a multilayer, three-dimensional structure is provided.
4 more elements
A method of delivering droplets from an air/liquid co-flow junction, the method comprising:
- flowing a plurality of droplets through an air flow via a microfluidic device, wherein the microfluidic device comprises an air/liquid co-flow junction and a sorter;
- detecting one or more of the plurality of droplets to provide one or more detected droplets;
- sorting via the sorter the one or more detected droplets from the plurality of droplets;
- and
- directing the air flow and the one or more detected droplets through the air/liquid co-flow junction.
2 more elements
A method of affixing a droplet to a substrate, the method comprising:
- delivering a droplet in an air flow from a microfluidic device comprising an air/liquid co-flow junction, through the air/liquid co-flow junction, to a substrate surface;
- positioning the droplet on the substrate surface;
- and
- affixing the droplet to the substrate surface via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof.
1 more element
A method of moving an affixed droplet on a substrate, the method comprising:
- delivering a droplet in an air flow from a microfluidic device comprising an air/liquid co-flow junction, through the air/liquid co-flow junction, to a substrate surface;
- affixing the droplet to the substrate surface via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof;
- and
- modulating the force so as to move the droplet from its affixed location to another location and/or applying a second force, which is sufficient, either alone or in combination with the modulated force, to move the droplet from its affixed location to another location.
1 more element
A method of adding reagents to a droplet, the method comprising:
- delivering a first droplet in an air flow from a microfluidic device comprising an air/liquid co-flow junction, through the air/liquid co-flow junction, to a substrate surface;
- affixing the droplet to the substrate surface via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof;
- delivering a second droplet comprising a reagent to the same location as the first droplet affixed to the substrate surface or a location adjacent the first droplet on the substrate surface;
- and
- coalescing the first droplet and the second droplet such that the contents of the first droplet and the second droplet are combined.
2 more elements
A method of adding reagents to a droplet, the method comprising:
- delivering a droplet in an air flow from a microfluidic device comprising an air/liquid co-flow junction, through the air/liquid co-flow junction, to a substrate surface;
- affixing the droplet to the substrate surface via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof;
- inserting an orifice fluidically connected to a reagent source into the droplet;
- and
- injecting via the orifice one or more reagents into the droplet.
2 more elements
A method of recovering all or a portion of the affixed droplet, the method comprising:
- delivering a droplet in an air flow from a microfluidic device comprising an air/liquid co-flow junction, through the air/liquid co-flow junction, to a substrate surface;
- affixing the droplet to the substrate surface via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof;
- and
- recovering all or a portion of the affixed droplet.
1 more element
A method of manipulating an affixed droplet, the method comprising:
- delivering a droplet in an air flow from a microfluidic device comprising an air/liquid co-flow junction, through the air/liquid co-flow junction, to a substrate surface;
- affixing the droplet to the substrate surface via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof;
- and
- modulating the immediate environment of the droplet, thereby modulating the contents of the droplet.
1 more element
A method of manipulating an affixed droplet, the method comprising:
- delivering a droplet in an air flow from a microfluidic device comprising an air/liquid co-flow junction, through the air/liquid co-flow junction, to a substrate surface;
- affixing the droplet to the substrate surface via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof;
- at least partially solidifying the affixed droplet;
- adding a miscible fluid to the affixed droplet;
- and modulating a chemical composition of the miscible fluid, thereby modulating the affixed droplet.
2 more elements
A method of porating a cell within an affixed droplet, the method comprising:
- delivering a droplet in an air flow from a microfluidic device comprising an air/liquid co-flow junction, through the air/liquid co-flow junction, to a substrate surface, wherein the droplet comprises a cell;
- positioning the droplet on the substrate surface;
- affixing the droplet to the substrate surface via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof;
- and
- porating the cell within the droplet.
2 more elements
A method of delivering discrete entities to a substrate, the method comprising:
- flowing a plurality of first discrete entities in an air flow through a microfluidic device comprising a first air/liquid co-flow junction;
- directing the one or more of the plurality of first discrete entities through the first air/liquid co-flow junction to the substrate;
- flowing a plurality of second discrete entities through a second microfluidic comprising a second air/liquid co-flow junction device;
- directing the one or more of the plurality of second discrete entities through the second air/liquid co-flow junction to the substrate;
- and
- affixing the one or more of the plurality of first discrete entities and the one or more of the plurality of second discrete entities to the substrate via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof.
3 more elements
A method of delivering discrete entities to a substrate, the method comprising:
- flowing a plurality of discrete entities in an air flow through a microfluidic device comprising an air/liquid co-flow junction, wherein the microfluidic device comprises a plurality of air/liquid co-flow junctions;
- directing the one or more of the plurality of discrete entities through a first air/liquid co-flow junction of the plurality of air/liquid co-flow junctions to the substrate;
- directing a second one or more of the plurality of discrete entities through a second air/liquid co-flow junction of the plurality of air/liquid co-flow junctions to the substrate;
- and
- affixing the first one or more of the plurality of first discrete entities and the second one or more of the plurality of discrete entities to the substrate via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof.
2 more elements
A method of synthesizing a polymer on a substrate, the method comprising:
- flowing a first droplet in an air flow through a microfluidic device comprising an air/liquid co-flow junction, wherein the first droplet comprises a first polymer or a first monomer;
- directing the first droplet through the air/liquid co-flow junction to the substrate;
- affixing the first droplet to the substrate via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof;
- flowing a second droplet in an air flow through the microfluidic device, wherein the second droplet comprises a second polymer or a second monomer;
- directing the second droplet through the air/liquid co-flow junction to the first droplet affixed at the predetermined location;
- and
- incubating the first and second droplets under conditions sufficient for the contents of the first and second droplets to come into contact and for the first polymer or first monomer to form a covalent bond with the second polymer or monomer, thereby generating a synthesized polymer.
4 more elements
A method of printing microarrays, the method comprising:
- delivering a plurality of droplets in an air flow from a microfluidic device comprising an air/liquid co-flow junction, through the air/liquid co-flow junction, to a substrate surface, wherein each of the plurality of droplets comprises a molecule;
- positioning the droplets on the substrate surface;
- affixing the droplets at predetermined locations to the substrate surface via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof;
- and
- incubating the substrate under conditions suitable for chemical bonding of the molecules comprised by the affixed droplets to the substrate surface, thereby providing an array of substrate-bound molecules.
2 more elements
A method of manipulating cells or embryos, the method comprising:
- flowing a plurality of droplets in an air flow through a microfluidic device comprising an air/liquid co-flow junction, wherein each droplet of the plurality of droplets comprises an aqueous fluid and a fertilized egg cell or embryo;
- directing the plurality of droplets through the air/liquid co-flow junction to a substrate;
- affixing the plurality of droplets to the substrate via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof, wherein the substrate comprises on a surface thereof a layer of fluid;
- detecting within the affixed plurality of droplets the development of one or more embryos;
- and
- selecting and recovering an embryo from the affixed droplets.
3 more elements
A method of manipulating cells or embryos, the method comprising:
- flowing a plurality of droplets in an air flow through a microfluidic device comprising an air/liquid co-flow junction, wherein each droplet of the plurality of droplets comprises an aqueous fluid and an unfertilized egg cell;
- directing the plurality of droplets through the air/liquid co-flow junction to a substrate;
- fertilizing one or more of the egg cells in the plurality of droplets;
- affixing the plurality of droplets to the substrate, via a force, wherein the force is selected from a gravitational force, an electrical force, a magnetic force, and combinations thereof, wherein the substrate comprises on a surface thereof a layer of fluid;
- detecting within the affixed droplets the development of an embryo;
- and
- selecting and recovering specific embryos from the affixed droplets.
4 more elements
A droplet printer comprising:
- a microfluidic device comprising an air/liquid co-flow junction;
- one or more droplet makers and one or more flow channels, wherein the one or more flow channels are fluidically connected to the one or more droplet makers and configured to receive one or more droplets therefrom;
- the air/liquid co-flow junction connected to one or more of the one or more flow channels and one or more air channels, wherein the one or more air channels are connected to one or more air flow controllers;
- a droplet sorter, which selectively sorts droplets;
- and
- an automated system integrated with the air/liquid co-flow junction, wherein the automated system (a) selectively positions the air/liquid co-flow junction in proximity to a substrate during operation or (b) selectively positions the substrate in proximity to the air/liquid co-flow junction during operation, such that a droplet can be ejected from the air/liquid co-flow junction and deposited on the substrate.
3 more elements
A microfluidic device comprising:
- an air/liquid co-flow junction;
- one or more flow channels;
- one or more air channels;
- one or more air flow controllers connected to the one or more air channels;
- and
- an electrode configured to selectively apply an electric field.
3 more elements
Dependencies are inferred from the claim text.