WO2023158430A1

WO2023158430A1 - Methods of making hydrogel microparticles

Info

Publication number: WO2023158430A1
Application number: PCT/US2022/016897
Authority: WO
Inventors: Ratima SUNTORNNOND; Wai Yee YEONG; Wei Long NG
Original assignee: Hewlett-Packard Development Company, L.P.; Nanyang Technological University
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2023-08-24

Abstract

The present disclosure is drawn to methods of making hydrogel microparticles, hydrogel microparticle printer components, and multi-component hydrogel microparticle printing systems. In one example, a method of making hydrogel microparticles can include jetting a plurality of droplets of a hydrogel precursor fluid into a mixture of an oil and a surfactant. The oil can be immiscible with the hydrogel precursor fluid. The droplets can be allowed to coalesce into a microparticle. The hydrogel precursor fluid can be crosslinked to form a hydrogel microparticle.

Description

METHODS OF MAKING HYDROGEL MICROPARTICLES BACKGROUND [0001] Hydrogels are hydrophilic three-dimensional polymer networks that can have a large amount of water absorbed in the hydrogel relative to the dry weight of the hydrogel. Hydrogel materials can also maintain their structure even when swollen with water because the polymer chains in the hydrogel can be crosslinked either chemically or physically. Depending on the chemical composition of a hydrogel, the hydrogel may be responsive to various stimuli such as heat, pH, light, and chemical stimulus. A wide variety of applications exist for hydrogels. Because some hydrogel materials have high biocompatibility, these materials have many uses in the biomedical field. For example, hydrogels can be used in tissue engineering, as a scaffold for tissue repair, as a drug delivery medium, and in other applications. BRIEF DESCRIPTION OF THE DRAWING [0002] FIG 1. is a flowchart illustrating an example method of making hydrogel microparticles in accordance with the present disclosure; [0003] FIGs.2A-2C show schematic views of an example process of making a hydrogel microparticle in accordance with the present disclosure; [0004] FIG.3 is a schematic cross-sectional view of an example hydrogel microparticle printer component in accordance with the present disclosure; [0005] FIG.4 is a schematic cross-sectional view of an example multi- component hydrogel microparticle printing system in accordance with the present disclosure; [0006] FIG.5 is a graph of microparticle diameter vs. total jetted volume of hydrogel precursor fluid; and [0007] FIG.6 is a graph of microparticle diameter vs. volume of oil/surfactant DETAILED DESCRIPTION [0008] The present disclosure describes methods of making hydrogel microparticles that can be used for biological applications, such as hydrogel microparticles that carry cells, drugs, proteins, or other biological materials. In one example, a method of making hydrogel microparticles includes jetting a plurality of droplets of a hydrogel precursor fluid into a mixture of an oil and a surfactant, where the oil is immiscible with the hydrogel precursor fluid. The droplets of hydrogel precursor fluid coalesce into a microparticle. The hydrogel precursor fluid is then crosslinked to form a hydrogel microparticle. The hydrogel precursor fluid can include a cell, a protein, a drug, or a combination thereof. The droplets can have a volume from 1 picoliter to 1 nanoliter. The droplets can be jetted using a fluid ejector that includes an ejection nozzle and an ejection actuator positioned to eject a droplet of the hydrogel precursor fluid from the ejection nozzle. In some examples, the ejection actuator can include a thermal resistor or a piezoelectric actuator. The oil can include mineral oil, paraffin oil, silicone oil, perfluorodecalin, or a combination thereof. The surfactant can be present in an amount from about 0.1 wt% to about 5 wt%. In some examples, the oil can be in a well of a well plate. The oil can have a depth from 2 mm to 8 mm. The total volume of the hydrogel precursor fluid jetted to form the hydrogel microparticle can be from 200 nanoliters to 1000 nanoliters. In certain examples, the hydrogel precursor fluid can be a first hydrogel precursor fluid, and the method can also include jetting a plurality of droplets of a second hydrogel precursor fluid into the mixture of oil and surfactant. The second hydrogel precursor fluid can have different ingredients than the first hydrogel precursor fluid. The droplets of the first hydrogel precursor fluid can coalesce with the droplets of the second hydrogel precursor fluid to form a multi-component microparticle that includes the ingredients of both the first hydrogel precursor fluid and the second hydrogel precursor fluid. [0009] The present disclosure also describes hydrogel microparticle printer components. In one example, a hydrogel microparticle printer component includes a reservoir of a hydrogel precursor fluid; a well plate including wells containing a mixture of an oil and a surfactant, where the oil is immiscible with the hydrogel fluid. The fluid ejector includes an ejection nozzle and an ejection actuator positioned to eject a droplet of the hydrogel precursor fluid from the ejection nozzle. The fluid ejector is positioned or positionable to eject droplets of the hydrogel precursor fluid into the mixture of oil and surfactant. In some examples, the hydrogel precursor fluid can include a cell, a protein, a drug, or a combination thereof. In further examples, the oil can include mineral oil, paraffin oil, silicone oil, perfluorodecalin, or a combination thereof. The surfactant can be present in an amount from about 0.1 wt% to about 5 wt%. [0010] The present disclosure also describes multi-component hydrogel microparticle printing systems. In one example, a multi-component hydrogel microparticle printing system includes a first reservoir of a first hydrogel precursor fluid and a second reservoir of a second hydrogel precursor fluid. The second hydrogel precursor fluid has different ingredients than the first hydrogel precursor fluid. The system also includes a container containing a mixture of an oil and a surfactant. The oil is immiscible with the first and second hydrogel precursor fluids. A first fluid ejector is connected to the first reservoir and a second fluid ejector is connected to the second reservoir. The first and second fluid ejectors include an ejection nozzle and an ejection actuator positioned to eject a droplet of the hydrogel precursor fluid from the ejection nozzle. The fluid ejectors are positioned or positionable to eject droplets of the hydrogel precursor fluid into the mixture of oil and surfactant. The system can also include a crosslinking radiation source positioned to expose hydrogel precursor fluid in the mixture of oil and surfactant to crosslinking radiation. In certain examples, the first hydrogel precursor fluid can include a first type of cells and the second hydrogel precursor fluid can include a second type of cells. [0011] It is noted that when discussing methods of making hydrogel microparticles, hydrogel microparticle printer components, hydrogel microparticle printers, and systems, these discussions can be considered applicable to other examples whether or not they are explicitly discussed in the context of that example unless expressly indicated otherwise. Thus, for example, when discussing a hydrogel precursor composition, such disclosure is also relevant to and directly supported in context of hydrogel microparticle printer components, systems, methods of making hydrogel microparticles, and vice versa. Furthermore, for mainly to certain examples. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. It will be readily apparent however, that the present disclosure can be practiced without limitation to some of these specific details. In other instances, certain methods, compounds, compositions, and structures have not been described in detail so as not to obscure the present disclosure. Methods of Making Hydrogel Microparticles [0012] The present disclosure describes methods of making hydrogel microparticles. Hydrogel microparticles can have a wide variety of uses, including in biomedical applications. For example, the hydrogel microparticles can be used as a three-dimensional scaffold for tissue growth, or for cell delivery, or drug delivery, or in drug toxicity testing, among other applications. The methods described herein are particularly useful because they allow hydrogel microparticles to be formed with a spherical shape and a controllable particle size. The methods can also allow hydrogel microparticles to be formed containing multiple different types of cells or other materials, with control over the amounts of the individual types of cells or other materials included. [0013] Some previously used methods of making hydrogel microparticles include batch emulsion processes, microfluidic emulsion processes, lithography, electrohydrodynamic spraying, and mechanical fragmentation. Batch emulsion involves mixing a hydrogel precursor fluid and an oil in which the hydrogel precursor fluid is immiscible, with sufficient mixing so that the hydrogel precursor fluid forms many small aqueous phase globules in the oil. The hydrogel precursor can then be crosslinked to form hydrogel microparticles. This type of process can be useful for preparing a large number of spherical particles quickly, but the process does not promote as much control over the size of the particles or the composition of the particles. Hydrogel microparticles produced by a batch emulsion process often have a distribution of random particle sizes. It may be possible to make hydrogel microparticles containing multiple different types of cells by including different sized microparticles. [0014] Microfludic emulsion devices can be microfluidic devices designed to form hydrogel microparticles with more consistent size and consistent amounts of microparticles and amounts of ingredients are usually not controllable by an individual microfluidic device. Therefore, if hydrogel microparticles of a different size or composition are desired, then a new microfluidic device is designed and fabricated. However, the amounts and ratios of the different types of cells in individual microparticles will typically be random. [0015] Lithography is another process that can be used to form hydrogel microparticles with fine control over the shape and size of the microparticles. However, this is a slow process that involves the use of a photomask to make the hydrogel microparticles. Changing the size or shape of the microparticles can be done by fabricating a new photomask. Furthermore, this process does not provide a way to control the composition or relative amounts of two or more types of cells in the microparticles. [0016] Electrohydrodynamic spraying involves spraying a hydrogel precursor fluid from an electrically charged needle, which forms very small electrically charged droplets that repel one another because of their similar electric charges. This process can form spherical hydrogel microparticles, but the process does not allow much control over the size of the microparticles or the amounts of multiple different types of cells or other materials in the microparticles. [0017] Mechanical fragmentation is a process of forming a larger body of hydrogel and mechanically breaking the hydrogel into smaller fragments. This process can produce a large number of hydrogel particles quickly, but the particles are not spherical. Instead, the particles have irregular shapes due to the fracturing of the larger body of hydrogel. This process also does not allow for fine control over the size of the particles or the amounts of multiple different types of cells in the particles. [0018] Unlike these other methods of making hydrogel microparticles, the methods described herein can allow the size of the microparticles to be easily tuned without fabricating any new photomasks, molds, or other equipment. Multiple different types of cells, proteins, drugs, or other materials can also be easily combined in individual hydrogel microparticles using the methods described herein. The relative amounts of the different materials can also be tuned with a fine level of control. [0019] These methods can include jetting a plurality of droplets of a hydrogel precursor fluid into a mixture of an oil and a surfactant. The oil can be immiscible coalesce to form a microparticle of the hydrogel precursor fluid surrounded by the oil. This fluid microparticle can have an approximately spherical shape. A suitable crosslinking process can then be applied to the hydrogel precursor fluid to crosslink the hydrogel precursor and convert the fluid microparticle to a gel microparticle that can retain its shape. [0020] The plurality of droplets of hydrogel precursor fluid can be jetted from a fluid ejector designed to jet very small droplets of fluid. In some examples, the fluid ejector can operate similarly to an ejector of an inkjet printer. Inkjet printers can utilize an ejection actuator such as a thermal resistor or a piezoelectric actuator to eject very small droplets of ink from a nozzle. In some cases, the individual droplets can have a volume on the order of picoliters to nanoliters. The size of the hydrogel microparticles produced using the methods described herein can be significantly larger than the individual droplets. For example, hundreds of droplets can be jetted and coalesced to form a single hydrogel microparticle. Since the exact number of droplets jetted can be controlled electronically, the size of the final hydrogel microparticle can be tuned easily by changing the number of droplets. [0021] Hydrogel microparticles can also be made with two or more different compositions combined in the individual microparticles. For example, two different hydrogel precursor fluids can be prepared with two different types of live cells in the fluids. A hydrogel microparticle can be made by jetting a desired number of droplets of the first hydrogel precursor fluid and a desired number of droplets of the second hydrogel precursor fluid. All of the droplets can then coalesce to form a single hydrogel microparticle containing both types of cells. The relative amounts of the different cells can be easily tuned by changing the number of droplets of the two different hydrogel precursor fluids. [0022] As used herein, “microparticles” refers to particles that have a particle size from 1 μm to 1 mm. For spherical particles, the particle size refers to the diameter of the spherical particles. For non-spherical particles, the particle size can refer to the longest dimension of the particle. For the microparticles described herein, the diameter or longest dimension of the microparticles can be measured using an optical microscope. The hydrogel particles formed using the methods described herein can be spherical or nearly spherical in many cases. In certain examples, the hydrogel particles can have an aspect ratio (defined as the longest dimension of the 1.2 or from 1 to 1.5. Additionally, the terms “particle” and “microparticle” can refer to a solid or liquid particle as used herein. A hydrogel that has been crosslinked and retains its own shape can be considered a solid particle. However, as mentioned above, a plurality of droplets of hydrogel precursor fluid can coalesce into a particle of hydrogel precursor fluid, which is still in the liquid form until it is crosslinked. The coalesced body of hydrogel precursor fluid is also referred to as a “particle” or “microparticle” herein. [0023] As used herein, “hydrogel” refers to a gel that includes a polymer network swelled by water. In a hydrogel, the polymer network has been crosslinked such that the hydrogel can maintain its shape under some amount of stress. A term “hydrogel precursor” refers to a polymer and/or monomer that can form a hydrogel through a crosslinking process and swelling with water. The hydrogel precursor fluids described herein can include a crosslinkable polymer and/or monomer and water in some examples. Examples of hydrogel precursors that can be used in the methods described herein include polyethylene glycol diacrylate, heat-treated saponified gelatin methacrylate, collagen methacrylamide, methacrylated hyaluronic acid, and other photocrosslinkable hydrogel precursors. [0024] As described above, the hydrogel precursor fluid can be jetted in the form of small droplets into an oil. As used herein, “oil” refers to a liquid that is nonpolar and hydrophobic, including vegetable oils, animal oils, petrochemical oils, silicone oils, and halogenated oils. The oil can be defined to be “immiscible” with water, meaning that at some ratios of the amount of water to the amount of oil in a mixture, the mixture will separate into a water phase and an oil phase. In some examples, the oil can be immiscible with water across all ratios of the amount of water to the amount of oil. In further examples, the oil can be immiscible with water in mixtures that include 1 wt% or more of water or 1 wt% or more of oil. Some specific examples of oils that can be used in the methods described herein include mineral oil, paraffin oil, silicone oil, perfluorodecalin, and others. [0025] FIG.1 is a flowchart illustrating an example method of making hydrogel microparticles 100. The method includes: jetting a plurality of droplets of a hydrogel precursor fluid into a mixture of an oil and a surfactant, wherein the oil is immiscible with the hydrogel precursor fluid 110; allowing the droplets to coalesce into a microparticle 120; and crosslinking the hydrogel precursor fluid to form a hydrogel microparticle 130. [0026] FIGs.2A-2C show schematic views of one example method being performed. FIG.2A shows a fluid ejector 210 ejecting a plurality of droplets 220 of a hydrogel precursor fluid 222. The droplets are jetted into a mixture 230 of oil and surfactant which is held in a well 240 of a well plate. In this example, the fluid ejector includes an ejection nozzle 212 and an ejection actuator 214 positioned to eject the droplets from the ejection nozzle. The ejection actuator can be a thermal resistor or piezoelectric element in various examples. [0027] FIG.2B shows the well 240 of the well plate containing the mixture 230 of oil and surfactant after the plurality of droplets of hydrogel precursor fluid have coalesced to form one larger microparticle 224 of hydrogel precursor fluid. At this point, the microparticle is still a liquid, but the microparticle forms with an approximately spherical shape due to surface tension forces at the interface between the hydrogel precursor fluid and the oil. FIG.2C shows the microparticle being exposed to ultraviolet radiation 250 from an ultraviolet radiation source 252. In this example, the hydrogel precursor is crosslinkable by ultraviolet radiation. Therefore, the exposure to ultraviolet radiation converts the liquid microparticle of hydrogel precursor fluid to a solid microparticle of hydrogel 260.The hydrogel microparticle can then be removed from the well and used as desired. [0028] As mentioned above, the hydrogel precursor fluid can include a crosslinkable polymer, monomer, or combination thereof that can be crosslinked to form a hydrogel. The hydrogel precursor can be crosslinked to form a biocompatible hydrogel. Some examples of hydrogel precursors that can be used in the methods described herein include polyethylene glycol diacrylate, heat-treated saponified gelatin methacrylate, collagen methacrylamide, methacrylated hyaluronic acid, and other photocrosslinkable hydrogel precursors. These materials can be crosslinked by exposing the hydrogel precursor fluid to ultraviolet radiation. In further examples, other polymers can also be used to form hydrogels. Additional examples include crosslinkable polyhydroxylated polymers, such as polyvinyl alcohol, cellulose, gelatin, alginate, chitosan, poly(2-hydroxyethyl acrylate), poly(2-hydroxyethyl methacrylate), poly(acrylic acid), poly(methacrylic acid), poly(N,N- dimethylacrylamide), poly(N,N-diethylacrylamide), poly(N-isopropylacrylamide), and ultraviolet radiation, as explained above, or by the addition of a suitable crosslinking agent. In certain examples, the hydrogel precursor fluid can include a polymer having a weight average molecular weight, before crosslinking, from about 1,000 Mw to about 500,000 Mw. In other examples, the molecular weight can be from about 10,000 Mw to about 300,000 Mw or from about 20,000 Mw to about 200,000 Mw. The polymer and/or monomer that forms the hydrogel can be included in the hydrogel precursor fluid in an amount from 0.5 wt% to 20 wt%, or from 0.5 wt% to 10 wt%, or from 0.5 wt% to 5 wt%, or from 1 wt% to 5 wt%, or from 2 wt% to 6 wt%, or from 3 wt% to 10 wt%, in various examples. [0029] In addition to the crosslinkable polymer and/or monomer, the hydrogel precursor fluid can include water. The water of the hydrogel precursor fluid can swell the crosslinked polymer network after the polymer has been crosslinked, thus forming the swelled hydrogel. In some examples, the amount of water in the hydrogel precursor fluid can be from 50 wt% to 99.9 wt%, or from 60 wt% to 99.5 wt%, or from 75 wt% to 99.5 wt%, or from 90 wt% to 99.5 wt%. [0030] The hydrogel precursor fluid can also include a buffer that is suitable for live cells. A variety of buffer formulations can be used. Some example buffer solutions include phosphate buffered saline (PBS), citric acid buffer, Tris buffer, HEPES buffer, CABS buffer, CAPS buffer, AMP buffer, CAPSO buffer, CHES buffer, AMPSO buffer, TABS buffer, AMPD buffer, TAPS buffer, HEPBS buffer, Bicine buffer, Gly-Gly buffer, Tricine buffer, EPPS buffer, TEA buffer, POPSO buffer, HEPPSO buffer, TAPSO buffer, MOBS buffer, DIPSO buffer, HEPES buffer, TES buffer, MOPS buffer, BES buffer, Bis-Tris Propane buffer, MOPSO buffer, PIPES buffer, ACES buffer, ADA buffer, Bis-Tris buffer, MES buffer, and combinations thereof. The concentration of the buffer solution can be an appropriate concentration for live cells. However, the hydrogel precursor fluid can also be free of ingredients that would inhibit printing using an ejector with a thermal resistor or piezoelectric actuator. In some cases, cell growth medium can include ingredients that can inhibit printing with one of these types of ejectors. Such ingredients can include amino acids, vitamins, glucose, serum (such as fetal bovine serum) which can include a variety of biological molecules such as proteins, antibodies, and others. If the hydrogel precursor fluid includes a buffer for live cells, then in some examples the buffer solution can be any type of buffer solution that does not inhibit printing with an ejector having a thermal resistor or piezoelectric actuator. [0031] Other ingredients can also be included in the hydrogel precursor fluid to increase jettability of the fluid. In the methods described herein, the hydrogel precursor fluid can be jetted from a fluid ejector that utilizes a thermal resistor or piezoelectric actuator to eject small droplets from an ejection nozzle. Certain properties of the fluid can contribute to jettability or, in other words, the ability to eject droplets of the fluid from these types of fluid ejectors consistently and accurately. In some cases, the viscosity of the hydrogel precursor fluid can affect the jettability. In some examples, the hydrogel precursor fluid can have a viscosity from about 0.1 centipoise to about 15 centipoise, or from about 0.5 centipoise to about 10 centipoise, or from about 1 centipoise to about 8 centipoise, or from about 2 centipoise to about 8 centipoise. The surface tension of the hydrogel precursor fluid can be from about 20 dyn/cm to about 60 dyn/cm, or from about 30 dyn/cm to about 50 dyn/cm, or from about 35 dyn/cm to about 50 dyn/cm, in some examples. [0032] The hydrogel precursor fluid can also include a biological or biologically active component. These can include, for example, cells, proteins, drugs, or other biologically active components. For many applications, it can be useful to include live cells in the hydrogel microparticles. In some examples, the live cells can be primary cells derived directly from humans or animals, stem cells that can differentiate into different types of cells, or cells from various cell lines. Some types of live cells can have a limited lifespan, and some types can be propagated indefinitely. In some examples, the concentration of live cells in the hydrogel precursor fluid can be from about 1 million cells/mL to about 4 million cells/mL. In other examples, the concentration of live cells can be from about 1 million cells/mL to about 2 million cells/mL, or from about 2 million cells/mL to about 4 million cells/mL, or from about 3 million cells/mL to about 4 million cells/mL. [0033] The size of the droplets of hydrogel precursor that are jetted can depend on the design of the fluid ejector used to jet the droplets. Some fluid ejectors that utilize a thermal resistor or a piezoelectric actuator can jet relatively small droplets on the order of picoliters (pL) in volume. The volume of an individual jetted droplet can be from 1 pL to 1000 pL (1 nL), or from 1 pL to 500 pL, or from 1 pL to 300 pL, or from 1 pL to 200 pL, or from 1 pL to 100 pL, or from 1 pL to 50 pL, or from various examples. A plurality of these droplets can be jetted into oil where the droplets coalesce to form a microparticle. The total volume of hydrogel precursor fluid that is jetted to form a single hydrogel microparticle can be from 200 nL to 1,000 nL, or from 200 nL to 800 nL, or from 200 nL to 600 nL, or from 300 nL to 800 nL, or from 300 nL to 600 nL, or from 400 nL to 800 nL, or from 400 nL to 600 nL, in various examples. [0034] In some cases, it can be useful to prepare hydrogel microparticles with multiple different types of cells, proteins, drugs, or other materials in the individual microparticles. It can also be useful to tune the relative amounts of the different materials in the individual microparticles. In one example, a hydrogel microparticle can be formed from two different hydrogel precursor fluids. The hydrogel precursor fluids can include different ingredients, such as different types of cells, proteins, or drugs. A plurality of droplets of the first hydrogel precursor fluid and a plurality of droplets of the second hydrogel precursor fluid can be jetted into an oil and surfactant mixture, and the droplets of both hydrogel precursor fluids can coalesce together to form a single microparticle. The relative amounts of the two hydrogel precursor fluids in the microparticle can be easily tuned by changing the number of droplets of the individual hydrogel precursor fluids that are jetted into the oil/surfactant mixture. In some examples, the individual hydrogel precursor fluids can be jetted in an amount from 100 nL to 500 nL, or from 200 nL to 500 nL, or from 200 nL to 400 nL, or from 200 nL to 300 nL. Although the example described above includes two different hydrogel precursor fluids, in further examples the microparticle can be formed from three different hydrogel precursor fluids, or four different hydrogel precursor fluids, and so on. [0035] In a particular example, a first hydrogel precursor fluid can include cells, proteins, drugs, or a combination thereof. A second hydrogel precursor fluid can be prepared without the cells, proteins, or drugs. For example, the second hydrogel precursor fluid can include water and a crosslinkable polymer, without any additional ingredients. These two hydrogel precursor fluids can be combined in any desired ratio to form a hydrogel microparticle. The concentration of the cells, protein, or drug in the microparticle can be tuned by changing the ratio of these two hydrogel precursor fluids. [0036] In some examples, multiple different hydrogel precursor fluids can be hydrogel precursor fluid can be jetted into the oil/surfactant mixture first, and then a plurality of droplets of a second hydrogel precursor fluid can be jetted afterward. The methods can also include a time delay between jetting the first and second fluids, in some examples. The time delay can help facilitate coalescing of the droplets, for example, by allowing the droplets of the first fluid time to partially or fully coalesce before jetting the second fluid. In some examples, the time delay can be from 5 seconds to 1 minute, or from 10 seconds to 45 seconds, or from 20 seconds to 40 seconds, or from 10 seconds to 30 seconds. [0037] The mixture of oil and surfactant can be held in a suitable container with an open top to allow droplets of hydrogel precursor fluid to be jetted into the mixture. In certain examples, the oil and surfactant can be held in a well of a well plate. The depth of the oil and surfactant mixture can be from 2 mm to 8 mm, or from 2 mm to 6 mm, or from 2 mm to 4 mm, or from 4 mm to 8 mm, or from 4 mm to 6 mm, or from 6 mm to 8 mm. The volume of the oil and surfactant mixture in the container (such as a well of a well plate, for example) can be from 50 μL to 500 μL, or from 100 μL to 500 μL, or from 100 μL to 250 μL, or from 100 μL to 200 μL, or from 200 μL to 500 μL, in various examples. [0038] The oil can be any type of liquid oil that is immiscible with the hydrogel precursor fluid, such that the hydrogel precursor fluid does not dissolve in the oil when jetted. Some examples of oil that can be used include mineral oil, paraffin oil, silicone oil, perfluorodecalin, or a combination thereof. The surfactant can be added to the oil to facilitate coalescing the droplets of hydrogel precursor fluid and forming a microparticle. In fact, in some cases it has been found that the hydrogel precursor fluid droplets do not coalesce to form a microparticle if oil is used without any surfactant. In some examples, the surfactant can be a non-ionic surfactant, such as SPAN® 80 from Sigma-Aldrichm (USA). The amount of surfactant in the oil/surfactant mixture can be from about 0.1 wt% to about 5 wt% with respect to the total weight of the mixture, or from about 1 wt% to about 5 wt%, or from about 2 wt% to about 5 wt%, or from about 2 wt% to about 4 wt%. Hydrogel Microparticle Printer Components [0039] The present disclosure also describes hydrogel microparticle printer components. These can be a portion of a printer that is used to make the hydrogel component can include a fluid ejector for jetting droplets of hydrogel precursor fluid as described above. The fluid ejector can be connected to a reservoir of the hydrogel precursor fluid. In some examples, the fluid ejector and the reservoir can be integrated in a print cartridge. The printer component can also include a well plate that has wells filled with a mixture of oil and surfactant. The fluid ejector can jet droplets of hydrogel precursor fluid into the wells to form hydrogel microparticles. [0040] FIG.3 is a schematic illustration of an example hydrogel microparticle printer component 300. This example includes a reservoir 320 of hydrogel precursor fluid 222 connected to a fluid ejector 210. As in the previous examples, the fluid ejector includes an ejection nozzle 212 and an ejection actuator 214 positioned to eject droplets of hydrogel precursor fluid from the ejection nozzle. The printer component also includes a well plate 340 that includes wells 240 containing a mixture 230 of oil and surfactant. The fluid ejector is positioned to eject droplets of the hydrogel precursor fluid into the mixture of oil and surfactant. The fluid ejector can also move to eject droplets into the other wells of the well plate. [0041] In some examples, the fluid ejector can have a specific clearance distance above the well plate. In particular, the ejection nozzle can have a clearance above the well plate. In some examples, the well plate can be from about 0.5 mm to about 5 mm below the ejection nozzle, or from about 0.5 mm to about 3 mm below, or from about 0.5 mm to about 2 mm below, or from about 1 mm to about 2 mm below the ejection nozzle. The oil and surfactant mixture can partially fill the wells in some examples. Therefore, the top surface of the oil/surfactant mixture can be an additional distance below the top of the well plate. In some examples, the total distance from the ejection nozzle to the top surface of the oil/surfactant mixture can be from about 3 mm to about 15 mm, or from about 4 mm to about 14 mm, or from about 6 mm to about 14 mm, or from about 8 mm to about 14 mm, or from about 10 mm to about 14 mm, or from about 12 mm to about 14 mm, or from about 8 mm to about 12 mm. In some examples, the ejection nozzle can be above the well plate and oriented to eject the hydrogel precursor fluid downward. However, in other examples, a different orientation can be used. [0042] In some examples, the ejection actuator can be a thermal resistor. The thermal resistor can heat the hydrogel precursor fluid to form a vapor bubble that forces fluid out of the ejection nozzle. Thus, the mechanical energy used to eject a thermal resistor. The thermal resistor can be made of metal, metal oxide, or another conductive material, in some examples. In certain examples, the thermal resistor can be formed by a thin-film deposition process. A stack of thin film layers can be formed that can include a thin layer of metal, metal oxide, or other conductive material to act as a thermal resistor, and additional layers can also be included such as an oxide layer, a passivation layer, electrically conductive traces, or combinations thereof. The thermal resistor can be activated by passing an electric current through the thermal resistor to generate heat. [0043] In other examples, the ejection actuator can be a piezoelectric actuator. The piezoelectric actuator can include a layer of piezoelectric material that can change shape and/or volume in response to an electric field. In certain examples, the piezoelectric material can be attached to a moveable membrane that can exert pressure on the hydrogel precursor fluid when the membrane flexes. The membrane can exert sufficient pressure on the fluid to displace a droplet of fluid out of the ejection nozzle. This type of actuator can also be formed using a deposition process similar to the thermal resistors described above. The layers deposited can include the piezoelectric material, membrane, and electrical traces for providing an electric field in the piezoelectric material. [0044] The size of elements of the fluid ejector are not particularly limited. However, in some examples the ejection nozzle can have a diameter from about 20 ^m to about 80 ^m. In further examples, the ejection nozzle can have a diameter from about 20 ^m to about 50 ^m or from about 50 ^m to about 80 ^m. The ejection actuator (such as a thermal resistor or piezoelectric actuator) can have a width or diameter from about 20 ^m to about 200 ^m in some examples, or from about 50 ^m to about 200 ^m or from about 100 ^m to about 200 ^m, or from about 50 ^m to about 100 ^m, in other examples. The fluid ejector can include an ejection chamber with an internal width, length, or height (i.e., any internal dimension) from about 20 ^m to about 400 ^m in some examples, or from about 20 ^m to about 300 ^m, or from about 50 ^m to about 300 ^m, or from about 100 ^m to about 300 ^m, or from about 200 ^m to about 400 ^m, in other examples. The ejection chamber can be connected directly to the ejection nozzle and adjacent to the ejection actuator so that the ejection actuator can exert pressure on the fluid in the ejection chamber to eject a droplet out through the ejection nozzle. [0045] Hydrogel microparticle printers can also include a variety of other components not illustrated in the figures herein. Additional components may include a print head assembly, a moveable print head carriage, a well plate transport assembly, an electronic controller to control the activation of fluid ejectors and the motion of print heads and/or well plates, a power supply, communication or networking modules, and others. In some examples, an electronic controller can include a processor, memory components including volatile and/or non-volatile memory components, electronics for communicating with and controlling fluid ejectors, print head assemblies, carriages, well plate transport assemblies, and so on. In certain examples, the electronic controller can receive data from a host system, such as a computer, and temporarily store data in memory. The data can include a representation of locations at which hydrogel precursor fluid is to be printed. The electronic controller can control the fluid ejectors to print droplets of hydrogel precursor fluid in the wells of the well plate according to this data. [0046] The well plate can be any suitable well plate, which may also be referred to as a microwell plate, microplate, and by other names. The well plate can include a plurality of wells that can hold a small volume of fluid. Any size and shape of well plate can be used. In various examples, the well plate can include an array of wells, such as a well plate having 6, 24, 96, 384, 1536, 3456, or 9600 wells. The individual wells can have a depth from about 1 mm to about 20 mm, or from about 2 m to about 16 mm, or from about 4 mm to about 16 mm, or from about 6 mm to about 16 mm, or from about 10 mm to about 16 mm. The wells can have an internal width or diameter from about 1 mm to about 20 mm, or from about 2 mm to about 16 mm, or from about 4 mm to about 12 mm, or from about 8 mm to about 12 mm. Multi-component Hydrogel Microparticle Printing Systems [0047] The present disclosure also describes multi-component hydrogel microparticle printing systems. These systems can be specifically designed to form microparticles from two or more different hydrogel precursor fluids. As explained above, the two or more different hydrogel precursor fluids can include different ingredients such as types of cells, proteins, or drugs. In some examples, a first hydrogel precursor fluid can be devoid of the live cells, proteins, or drugs. Thus, the second hydrogel precursor fluid can be added in a desired ratio with the first hydrogel precursor fluid to allow the concentration of the cells, proteins, or drugs to be tuned in the final microparticle. These systems can also allow multiple different types of cells, proteins, drugs, or other materials to be included in individual microparticles, with control over the ratios of the materials in the individual microparticles. [0048] FIG.4 shows an example multi-component hydrogel microparticle printing system 400. This example includes a first reservoir 320 of a first hydrogel precursor fluid 222 and a second reservoir 322 of a second hydrogel precursor fluid 224. The second hydrogel precursor fluid has different ingredients than the first hydrogel precursor fluid. The system also includes a container containing a mixture 230 of oil and surfactant. In this example, the container is a well 240 of a well plate 340, although other types of containers can be used. A first fluid ejector 210 is connected to the first reservoir, and a second fluid ejector 216 is connected to the second reservoir. Both fluid ejectors include an ejection nozzle 212 and an ejection actuator 214 positioned to eject droplets of hydrogel precursor fluid from the ejection nozzle. The fluid ejectors can move to be positioned above the container so that the fluid ejectors can jet droplets of both hydrogel precursor fluids into the mixture of oil and surfactant. This system also includes a crosslinking radiation source 252 that can be used to expose the microparticles of hydrogel precursor fluid to radiation such as ultraviolet radiation. [0049] In certain examples, the multi-component hydrogel microparticle printing system can include a cassette that includes the first and second hydrogel precursor fluid reservoirs and the first and second fluid ejectors. The cassette may be packaged with a first and second hydrogel precursor fluid in the reservoirs. Alternatively, the cassette can be provided empty and a user can introduce a first and second hydrogel precursor fluid into the reservoirs. The cassette can then be loaded in a dispenser that can include other components such as a power supply, electronic controller, a carriage, a substrate transport assembly, and so on. The cassette can be a disposable cassette, designed for one-time use, so that subsequent hydrogel precursor fluids are not contaminated by cells from previously used fluids. In further examples, the cassette can include four reservoirs for different hydrogel precursor fluids and four fluid ejectors. In still further examples, the cassette can include eight reservoirs and eight fluid ejectors. [0050] It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. [0051] As used herein, the term “about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “a little above” or “a little below” the endpoint. The degree of flexibility of this term can be dictated by the particular variable and can be determined based on experience and the associated description herein. [0052] As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though the individual members of the list are individually identified as separate and unique members. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. [0053] Concentrations, dimensions, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include the numerical values explicitly recited as the limits of the range, and also to include all the individual numerical values or sub-ranges encompassed within that range as if the numerical values and sub-ranges are explicitly recited. For example, a weight ratio range of 1 wt% to 20 wt% should be interpreted to include the explicitly recited limits of 1 wt% and 20 wt%, and also to include individual weights such as 2 wt%, 11 wt%, 14 wt%, and sub-ranges such as 10 wt% to 20 wt%, 5 wt% to 15 wt%, etc. EXAMPLES [0054] The following examples illustrate the technology of the present disclosure. However, it is to be understood that the following is merely illustrative of the application of the principles of the presented formulations and methods. from the scope of the present disclosure. The appended claims are intended to cover such modifications and arrangements. Thus, while the technology has been described above with particularity, the following examples provide further detail in connection with what are presently deemed to be certain acceptable examples. Example 1 – Preparation of Hydrogel Microparticles [0055] A series of hydrogel precursor fluids were prepared. The fluids included a 4 wt% aqueous solution of heat treated saponified gelatin methacrylate. To this solution was added cells (L-929 fibroblast cells) in varying concentrations from 0 to 2 million cells/mL. The hydrogel precursor fluids were printed from an HP® D300e Digital Dispenser from HP Inc. (USA), which includes a thermal inkjet-type fluid ejector configured to jet droplets with a droplet volume of about 3 pL. The oil/surfactant mixture into which the droplets were jetted was mineral oil with a surfactant (SPAN® 80 from Sigma-Aldrich (USA)) in amounts ranging from 0 wt% to 5 wt%. The droplets were allowed to coalesce into microparticles and then the microparticles were crosslinked by applying ultraviolet light using a UVA flood curing system at a wavelength of 315-400 nm with a peak intensity of 175 mW/cm² from TechnoDigm (Singapore). The ultraviolet light was applied at 60% intensity for 130 seconds. Several experiments were performed to test the effects of various factors on the formation of the hydrogel microparticles. These experiments are described in the following examples. Example 2 – Total Jetted Volume of Hydrogel Precursor Fluid [0056] A series of hydrogel microparticles were formed by jetting different volumes of hydrogel precursor fluid to determine the influence of the total jetted volume on the formation of the microparticles. The volumes used were 1 nL, 5 nL, 10 nL, 50 nL, 100 nL, 500 nL, 1000 nL, and 5000 nL. The hydrogel precursor fluid had a cell concentration of 2 million cells/mL. These volumes were jetted into ten wells to provide ten trials per volume. At a volume of 1 nL, 5 nL, and 10 nL, the individual droplets did not agglomerate to form a microparticle and instead remained as small individual droplets. At a volume of 50 mL, the droplets began to agglomerate but most of the droplets still remained separate. At 100 nL, the droplets mostly agglomerated to form a larger microparticle, with some droplets remaining separate. defined spherical microparticles. At 1000 nL, the droplets mostly agglomerated to form a larger microparticle but some droplets remained separate. At 5000 nL, multiple microparticles were formed, and the size of the multiple microparticles was not consistent. Thus, it was found that the microparticle formed best and most consistently between about 500 nL and 1000 nL. FIG.5 is a graph showing the diameter of the microparticles formed in μm for the trials. Example 3 – Depth of Oil and Surfactant Mixture [0057] The same hydrogel precursor fluid was jetted into wells holding different amounts of the oil and surfactant mixture to test the influence of the depth of the oil and surfactant mixture. The volumes of oil/surfactant mixture were: 50 μL (depth of 1.46 mm in the well); 100 μL (2.92 mm); 150 μL (4.38 mm); 200 μL (5.84 mm); 250 μL (7.31 mm); 300 μL (8.77 mm); and 350 μL (10.22 mm). Ten trials were performed at these volumes of oil/surfactant mixture. FIG.6 shows a graph of the diameter of microparticles formed from these trials. There was some variation in the diameter of the microparticles at all of the volume levels, but the variation was smallest when the volume of oil/surfactant mixture was 250 μL, corresponding to a depth of 7.31 mm. Example 4 – Concentration of Surfactant [0058] To test the influence of the concentration of the surfactant, the hydrogel precursor was printed in oil that was mixed with different concentrations of the surfactant (SPAN® 80 from Sigma-Aldrich (USA)). The concentrations of surfactant were 0 wt%, 2 wt%, 3 wt%, and 5 wt%. When no surfactant was used, the hydrogel precursor fluid did not form a spherical microparticle. Instead, the hydrogel precursor fluid sank to the bottom of the well and puddled at the bottom under the oil. The best results were observed with a surfactant concentration of 2 wt% or 3 wt%, with one larger microparticle forming and a few smaller droplets remaining around the microparticle. When the surfactant concentration was 5 wt%, a microparticle formed but the microparticle was smaller and more of the hydrogel precursor fluid remained as separate droplets around the microparticle. Example 5 – Multiple Cell Types [0059] Several hydrogel microparticles were prepared with multiple cell types in the individual microparticles. The cells were L-929 fibroblast cells that were split into two groups. One group was stained with orange cytoplasmic membrane dye (Ex/Em 549/565 nm, CELLBRITE® from Biotium (USA)) and the other group was stained with green cytoplasmic membrane dye (Ex/Em 484/501 nm, CELLBRITE® from Biotium (USA)). The two groups of cells were mixed into two hydrogel precursor fluids. The hydrogel precursors were jetted in orange/green ratios of 1:3, 1:2, 1:1, 2:1, and 3:1. The total volume of hydrogel precursor fluid jetted in the individual trials was 600 nL. All of these trials successfully formed microparticles that included mixtures of the orange and green cells at the specific ratios. [0060] While the present technology has been described with reference to certain examples, various modifications, changes, omissions, and substitutions can be made without departing from the disclosure. It is intended, therefore, that the disclosure be limited by the scope of the following claims.

Claims

CLAIMS What Is Claimed Is: 1. A method of making hydrogel microparticles, comprising: jetting a plurality of droplets of a hydrogel precursor fluid into a mixture of an oil and a surfactant, wherein the oil is immiscible with the hydrogel precursor fluid; allowing the droplets to coalesce into a microparticle; and crosslinking the hydrogel precursor fluid to form a hydrogel microparticle.

2. The method of claim 1, wherein the hydrogel precursor fluid comprises a cell, a protein, a drug, or a combination thereof.

3. The method of claim 1, wherein the droplets have a droplet volume from 1 picoliter to 1 nanoliter.

4. The method of claim 1, wherein the droplets are jetted using a fluid ejector that comprises an ejection nozzle and an ejection actuator positioned to eject a droplet of the hydrogel precursor fluid from the ejection nozzle, wherein the ejection actuator includes a thermal resistor or a piezoelectric actuator.

5. The method of claim 1, wherein the oil comprises mineral oil, paraffin oil, silicone oil, perfluorodecalin, or a combination thereof and wherein the surfactant is present in an amount from about 0.1 wt% to about 5 wt%.

6. The method of claim 1, wherein the oil is in a well of a well plate.

7. The method of claim 1, wherein the oil has a depth from 2 mm to 8 mm.

8. The method of claim 1, wherein a total volume of the hydrogel precursor fluid jetted to form the hydrogel microparticle is from 200 nanoliters to 1000 nanoliters.

9. The method of claim 1, wherein the hydrogel precursor fluid is a first hydrogel precursor fluid, and wherein the method further comprises jetting a plurality of droplets of a second hydrogel precursor fluid into the mixture of oil and surfactant, wherein the second hydrogel precursor fluid has different ingredients than the first hydrogel precursor fluid, wherein the droplets of the first hydrogel precursor fluid coalesce with the droplets of the second hydrogel precursor fluid to form a multi- component microparticle that includes the ingredients of both the first hydrogel precursor fluid and the second hydrogel precursor fluid.

10. A hydrogel microparticle printer component, comprising: a reservoir of a hydrogel precursor fluid; a well plate comprising wells containing a mixture of an oil and a surfactant, wherein the oil is immiscible with the hydrogel precursor fluid; a fluid ejector connected to the reservoir of hydrogel precursor fluid, wherein the fluid ejector comprises an ejection nozzle and an ejection actuator positioned to eject a droplet of the hydrogel precursor fluid from the ejection nozzle, wherein the fluid ejector is positioned or positionable to eject droplets of the hydrogel precursor fluid into the mixture of oil and surfactant.

11. The hydrogel microparticle printer component of claim 10, wherein the hydrogel precursor fluid comprises a cell, a protein, a drug, or a combination thereof.

12. The hydrogel microparticle printer component of claim 10, wherein the oil comprises mineral oil, paraffin oil, silicone oil, perfluorodecalin, or a combination thereof and wherein the surfactant is present in an amount from about 0.1 wt% to about 5 wt%.

13. A multi-component hydrogel microparticle printing system, comprising: a first reservoir of a first hydrogel precursor fluid; a second reservoir of a second hydrogel precursor fluid, wherein the second hydrogel precursor fluid has different ingredients than the first hydrogel precursor fluid; a container containing a mixture of an oil and a surfactant, wherein the oil is a first fluid ejector connected to the first reservoir; and a second fluid ejector connected to the second reservoir, wherein the first and second fluid ejectors comprise an ejection nozzle and an ejection actuator positioned to eject a droplet of the hydrogel precursor fluid from the ejection nozzle, wherein the first and second fluid ejectors are positioned or positionable to eject droplets of the hydrogel precursor fluid into the mixture of oil and surfactant.

14. The system of claim 13, further comprising a crosslinking radiation source positioned to expose hydrogel precursor fluid in the mixture of oil and surfactant to crosslinking radiation.

15. The system of claim 13, wherein the first hydrogel precursor fluid comprises a first type of cells and the second hydrogel precursor fluid comprises a second type of cells.