Systems and methods for encapsulation of cells and cell clusters
Patent Information
- Application Number
- PCT/US2026/021229
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-03-27
- Filing Date
- 2026-03-27
- Publication Date
- 2026-10-01
Smart Images
Figure US2026021229_01102026_PF_FP_ABST
Abstract
Description
[0001] SYSTEMS AND METHODS FOR ENCAPSULATION OF CELLS AND CELL CLUSTERS
[0002] RELATED APPLICATIONS
[0003] This application claims priority under 35 U.S.C. § 119(c) to U.S. Provisional Patent Application No. 63 / 779,026, filed March 27, 2025, and entitled “Systems and Methods for Encapsulation of Cells and Cell Clusters,” which is incorporated herein by reference in its entirety for all purposes.
[0004] TECHNICAL FIELD
[0005] The present invention generally relates to systems and methods of cell encapsulation.
[0006] BACKGROUND
[0007] Cell encapsulation is a promising strategy for protecting transplanted cells from the host immune system of a subject while enabling essential nutrient and secretome exchange. One of the major challenges in cell encapsulation is controlling the thickness of the encapsulating shell and / or membrane. A thick membrane can hinder oxygen and nutrient diffusion, leading to hypoxia and potential cell necrosis within the core of cell clusters. This issue is particularly concerning in the context of large cell clusters, such as pancreatic islets, where maintaining viability is essential for the success of therapeutic interventions. Conventional cell encapsulation systems are typically limited in the diameters of cells and / or clusters they are able to effectively encapsulate, along with additional challenges such as the need for acidic conditions during chemical reactions which are essential to performing the encapsulation and a lack of uniformity in encapsulation membrane thickness across diverse cluster sizes. Thus, achieving ultra-thin encapsulation membranes, particularly for larger cell clusters, remains a challenge. As such, improved systems and methods for cell and cell cluster encapsulation are needed.
[0008] SUMMARY
[0009] The present invention generally relates to systems and methods of cell encapsulation. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.
[0010] In one aspect, cell encapsulation systems are provided. In some embodiments, the cell encapsulation system comprises a fluid emitter, a voltage source in electrical communication with the fluid emitter, a reservoir configured to receive a fluid comprising a plurality of livingcells in fluidic communication with the fluid emitter, and a source of compressed gas in fluidic communication with the fluid emitter, wherein the cell encapsulation system is configured for voltage driven flow of the fluid comprising the plurality of living cells.
[0011] In another aspect, methods of encapsulating living cells are provided. In some embodiments, the methods comprise applying a voltage to a tip of a fluid emitter, flowing a fluid comprising a plurality of living cells proximate the tip of the fluid emitter, and flowing a compressed gas through a nozzle in fluidic communication with the fluid emitter, thereby encapsulating the plurality of living cells.
[0012] In yet another aspect, articles such as a plurality encapsulated cells are provided. In some embodiments, the plurality of encapsulated cells comprise a core disposed within a shell, the core comprising one or more living cells, the shell comprising a non-covalently crosslinked polymer layer having a thickness of greater than or equal to 5 microns and less than or equal to 200 microns, wherein the core region is surrounded by the shell region.
[0013] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.
[0014] BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale unless otherwise indicated. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:
[0016] FIG. 1 A shows a schematic diagram of a cell encapsulation system, according to certain embodiments;
[0017] FIG. IB shows a schematic diagram of an airflow chamber region of a cell encapsulation system, according to certain embodiments;
[0018] FIG. 2 shows a schematic drawing of an airflow chamber system, according to certain embodiments;FIG. 3 shows a photograph of encapsulated cells and cell clusters, according to certain embodiments;
[0019] FIG. 4A shows solutions of alginate microcapsules, according to certain embodiments; FIG. 4B is a chart showing the relationship between air pressure and diameter of alginate microcapsules, according to certain embodiments;
[0020] FIG. 5A is a chart showing the relationship between air pressure and cluster number of encapsulated cell clusters, according to certain embodiments;
[0021] FIG. 5B is a chart showing the relationship between air pressure and diameter of encapsulated cell clusters, according to certain embodiments;
[0022] FIG. 5C is a chart showing the relationship between air pressure and shell coating diameter of encapsulated cell clusters, according to certain embodiments;
[0023] FIG. 6A is a chart showing the size distribution of large cell clusters, according to certain embodiments;
[0024] FIG. 6B is a chart showing the diameter distribution of large encapsulated cell clusters, according to certain embodiments;
[0025] FIG. 6C is a chart showing the shell thickness distribution of large encapsulated cell clusters, according to certain embodiments;
[0026] FIG. 6D is a chart showing the size distribution of small cell clusters, according to certain embodiments;
[0027] FIG. 6E is a chart showing the diameter distribution of small encapsulated cell clusters, according to certain embodiments;
[0028] FIG. 6F is a chart showing the shell thickness distribution of large encapsulated cell clusters, according to certain embodiments;
[0029] FIG. 7A shows cells encapsulated using a conventional voltage-only technique and cells encapsulated using an air-assisted technique, according to certain embodiments;
[0030] FIG. 7B is a chart showing the average diameter of encapsulated cell clusters formed using a conventional voltage-only technique and an encapsulated using an air-assisted technique, according to certain embodiments;
[0031] FIG. 7C is a chart showing the average volume of encapsulated cell clusters formed using a conventional voltage-only technique and an encapsulated using an air-assisted technique, according to certain embodiments;
[0032] FIG. 7D shows the shell thickness of encapsulated cell clusters, according to certain embodiments;FIG. 8 is a chart showing the secretion of insulin from encapsulated cell clusters during a high-glucose challenge, according to certain embodiments;
[0033] FIG. 9A shows cells encapsulated using a conventional voltage-only technique and cells encapsulated using an air-assisted technique, according to certain embodiments;
[0034] FIG. 9B is a chart showing the average shell thickness of encapsulated cell clusters formed using a conventional voltage-only technique and an encapsulated using an air-assisted technique, according to certain embodiments;
[0035] FIG. 9C is a chart showing the secretion of insulin from encapsulated cell clusters during a high-glucose challenge, according to certain embodiments;
[0036] FIG. 10A is a chart showing the secretion of insulin from encapsulated cell clusters, according to certain embodiments;
[0037] FIG. 1 OB is a chart showing the glucose-stimulated insulin secretion from encapsulated cell clusters, according to certain embodiments;
[0038] FIG. 10C is a chart showing the stimulation index of encapsulated cell clusters, according to certain embodiments;
[0039] FIG. 11 shows a simulated cell encapsulation system, according to certain embodiments; FIG. 12 shows the boundary conditions of a simulated cell encapsulation system, according to certain embodiments;
[0040] FIG. 13 shows the viscosity of an alginate solution and a fitted Cross model, according to certain embodiments;
[0041] FIG. 14 shows the physical properties of an alginate solution, according to certain embodiments;
[0042] FIG. 15A shows the Weber number of a simulated alginate solution and a gas flowing at various pressures, according to certain embodiments;
[0043] FIG. 15B shows the transition between dripping, micro-dripping, and cone-jet modes relative to gas flow velocity in a simulated cell encapsulated system, according to certain embodiments;
[0044] FIGS. 16A-16C show fluid velocities in a simulated cell encapsulation system, according to certain embodiments;
[0045] FIG. 17A shows the droplet size predicted by a simulated cell encapsulated system, according to certain embodiments; and
[0046] FIG. 17B shows the volume of a liquid spray cone predicted by a simulated cell encapsulated system, according to certain embodiments.DETAILED DESCRIPTION
[0047] In some embodiments, systems and methods for encapsulating living cells and / or clusters of living cells are provided. In some embodiments, the encapsulated living cells and / or clusters of living cells produced using the systems and / or methods as described herein may be configured to be used in cell therapy applications. The living cells and / or clusters of living cells as described herein may be encapsulated in a shell (e.g., a membrane) that is relatively thin. In some embodiments, encapsulating the living cells and / or clusters of living cells in a relatively thin membrane may advantageously allow for fast oxygen and nutrient diffusion across the membrane, thereby reducing the risks associated with hypoxia and / or cell necrosis and increasing cell viability. This may be especially advantageous for applications in which large cell clusters (e.g., pancreatic islets) are encapsulated, in which maintaining viability is challenging but important to the success of therapeutic interventions.
[0048] In some embodiments, a cell encapsulation system is provided. For example, FIG. 1 A shows a cell encapsulation system 100. The cell encapsulation system may be configured to encapsulate cells using an electrospraying mechanism. Generally, electrospraying involves atomization of a liquid into a fine spray of charged droplets of sizes ranging from several microns down to a few nanometers by the application of a sufficiently strong electric field. The fine spray can produce very smooth and uniform layers, which are formed as the charged particles are attracted to and deposited on an electrically grounded surface and / or in an electrically grounded fluid reservoir.
[0049] In some embodiments, as shown in FIG. 1 A, the cell encapsulation system 100 comprises an airflow chamber region 101. A detailed view of the airflow chamber region 101 is shown in FIG. IB. In some embodiments, the airflow chamber region may comprise a fluid emitter 120 (e.g., an electrospray). In some embodiments, the fluid emitter comprises a needle (e.g., a capillary needle). The airflow chamber may comprise an orifice through which a fluid can flow to exit the emitter and, when the fluid emitter is configured to perform an electrospraying process, be sprayed as fine droplets. In some embodiments, the orifice is located at a tip of the fluid emitter.
[0050] The fluid emitter may have any of a variety of suitable inner diameters. For example, the fluid emitter may have an inner diameter of greater than or equal to 100 microns, greater than or equal to 150 microns, greater than or equal to 200 microns, greater than or equal to 250 microns, greater than or equal to 300 microns, greater than or equal to 350 microns, greater than or equal to 400 microns, greater than or equal to 450 microns, greater than or equal to 500 microns, greater than or equal to 550 microns, greater than or equal to 600 microns, greater than or equalto 650 microns, greater than or equal to 700 microns, greater than or equal to 750 microns, greater than or equal to 800 microns, greater than or equal to 850 microns, greater than or equal to 900 microns, or greater than or equal to 950 microns. In some embodiments, the fluid emitter may have a diameter of less than or equal to 1,000 microns, less than or equal to 950 microns, less than or equal to 900 microns, less than or equal to 850 microns, less than or equal to 800 microns, less than or equal to 750 microns, less than or equal to 700 microns, less than or equal to 650 microns, less than or equal to 600 microns, less than or equal to 500 microns, less than or equal to 450 microns, less than or equal to 400 microns, less than or equal to 350 microns, less than or equal to 300 microns, less than or equal to 250 microns, less than or equal to 200 microns, or less than or equal to 150 microns. Combinations of these ranges are also possible (e.g., the fluid emitter may have a diameter of greater than or equal to 100 microns and less than or equal to 1,000 microns, or greater than or equal to 150 microns and less than or equal to 850 microns). Other ranges are also possible.
[0051] In some embodiments, the fluid emitter corresponds to a needle having a gauge of greater than or equal to 18G, greater than or equal to 20G, greater than or equal to 22G, greater than or equal to 24G, greater than or equal to 26G, or greater than or equal to 28G. In some embodiments, the fluid emitter corresponds to a needle having a gauge of less than or equal to 30G, less than or equal to 28G, less than or equal to 26G, less than or equal to 24G, less than or equal to 22G, or less than or equal to 20G. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 18G and less than or equal to 30G). Other ranges are also possible.
[0052] In some embodiments, a cell encapsulation system 100 as shown in FIG. 1 A comprises a reservoir 102 in fluidic communication with the fluid emitter. In some embodiments, the reservoir is configured to receive a fluid comprising a plurality of living cells. In some embodiments, the fluid comprising living cells is an aqueous solution comprising living cells. In some embodiments, the fluid comprises one or more salts. In some embodiments, the fluid comprises sodium chloride (e.g., in some embodiments, the fluid comprises a saline solution).
[0053] In some embodiments, the fluid comprises a polymer. In some embodiments, the polymer may be chosen to have any of a number of advantageous properties for encapsulation of living cells. For example, the polymer may be biodegradable, non-toxic, non-immunogenic, water-soluble, capable of forming a stable hydrogel and / or other structurally stable polymer network, have slow and / or tunable degradation kinetics, have tunable mechanical properties, and / or be commonly approved for use in medical and / or biological applications.In some embodiments, the polymer may comprise a natural polymer. For example, the natural polymer may comprise a biodegradable polymer (e.g., dextran), a water-soluble polymer capable of forming stable hydrogels (e.g., Pullulan, alginate), a non-toxic polymer (e.g., agarose), and / or a polymer that forms a structurally stable polymer network and / or has tunable degradation kinetics (e.g., silk fibroin). Other natural polymers having any combination of these advantageous properties could also be used, such as chitosan, collagen, fibrin, gelatin, and / or hyaluronic acid.
[0054] In some embodiments, the polymer may comprise a synthetic polymer. For example, the synthetic polymer may comprise a non-immunogenic polymer and / or a polymer that has tunable mechanical properties (e.g., polyethylene glycol), a polymer that is biodegradable and / or commonly approved for use in biological applications (e.g., polylactic acid and / or poly(lactic-co-glycolic acid), a polymer that has slow degradation kinetics (e.g., polycaprolactone), a polymer that has slow degradation kinetics (e.g., gelatin methacryloyl), and / or a highly biocompatible polymer (e.g., zwitterionic alginate). Other synthetic polymers having any combination of these advantageous properties could be used.
[0055] In some embodiments, the fluid comprises a crosslinking agent. The crosslinking agent may be configured to form crosslinks among the polymer(s) in the fluid. As used herein, the term “crosslink” refers to a connection between two polymer strands, or a connection between two points one a single polymer strand. The crosslink may either be a chemical bond, a single atom, or multiple atoms. The crosslink may be formed by reaction of a pendant group in one polymer strand with the backbone of a different polymer strand, or by reaction of one pendant group with another pendant group. Crosslinks may exist between separate polymer strands and may also exist between different points of the same polymer strand. As used herein, the term “polymer strand” refers to an oligomeric or polymeric chain of one monomer unit, or an oligomeric or polymeric chain of two or more different monomer units. As used herein, the term “prepolymer” refers to oligomeric or polymeric strands which have not undergone crosslinking to form a network.
[0056] As used herein, the term “crosslink moiety” or “crosslinking moiety” refers to the bond or atom(s) making up the crosslink between two polymer strands (or between different points on the same polymer strand). In some embodiments, the crosslink moiety comprises one or more chemical bonds, such as an ionic bond, a covalent bond, a hydrogen bond, Van der Waals interactions, and the like. The covalent bond may be, for example, carbon-carbon, carbonoxygen, oxygen-silicon, sulfur-sulfur, phosphorus-nitrogen, carbon-nitrogen, metal-oxygen, or other covalent bonds. The hydrogen bond may be, for example, between hydroxyl, amine,carboxyl, thiol, and / or similar functional groups. The ionic bond may comprise, for example, a cation (e.g., a polyvalent cation). Non-limiting examples of polyvalent cations include calcium, barium, strontium, iron, aluminum. Other polyvalent cations are also possible. In an exemplary embodiment, the polyvalent cation is calcium. In a preferred embodiment, the crosslinking moiety comprises non-covalent bonds.
[0057] In some embodiments, the crosslink moiety may be formed by mixing a polymer (or polymer precursor and / or monomer) with a crosslinking agent. Non-limiting examples of suitable crosslinking agents include polyvalent cation crosslinkers, diamine crosslinkers, dicarboxyl crosslinkers, disulfhydryl crosslinkers, dicarbonyl crosslinkers, disulfide crosslinkers, carbodiimide, NHS ester, imidoester, maleimide, haloacetyls, pryidyldisulfide, thiosulfonate, hydrazide, calcium sulphate and N,N’-bis(acryloyl)cystamine. Other crosslinking agents are also possible and those of ordinary skill in the art would be capable of selecting suitable crosslinking agents based upon the teachings of this specification.
[0058] As used herein, a “fluid” is given its ordinary meaning, i.e., a liquid or a gas. A fluid cannot maintain a defined shape and will flow during an observable time frame to fill the container in which it is put. Thus, the fluid may have any suitable viscosity that permits flow. If two or more fluids are present, each fluid may be independently selected among essentially any fluids (liquids, gases, and the like) by those of ordinary skill in the art.
[0059] As used herein, the term “hydrogel” refers to a polymer network capable of absorbing a relatively high amount of water (e.g., a high weight percentage of water as compared to the weight of the polymer network e.g., greater than 70 wt% water).
[0060] The term “non-toxic” refers to a substance that is not toxic. Toxic reagents include, e.g., oxidative stressors, nitrosative stressors, proteasome inhibitors, inhibitors of mitochondrial function, ionophores, inhibitors of vacuolar ATPases, inducers of endoplasmic reticulum (ER) stress, and inhibitors of endoplasmic reticulum associated degradation (ERAD). In some embodiments a toxic reagent selectively causes damage to nervous system tissue. Toxic reagents include compounds that are directly toxic and reagents that are metabolized to or give rise to substances that are directly toxic. It will be understood that the term “toxic compounds" typically refers to reagents that are not ordinarily present in a cell’s normal environment at sufficient levels to exert detectable damaging effects. However, in some cases, the toxic reagents may be present in a cell’s normal environment but at concentrations significantly less than present in the auxiliary materials described herein. Typically, toxic reagents exert damaging effects when present at a relatively low concentration, e.g., at or below 1 mM, e.g., at or below 500 microM, e.g., at or below 100 microM. It will be understood that a toxic reagentstypically has a threshold concentration below which it does not exert detectable damaging effects. The particular threshold concentration will vary depending on the agent and, potentially, other factors such as cell type, other agents present in the environment, etc.
[0061] In some embodiments, the cell encapsulation system 100 as shown in FIG. 1A comprises an actuator 103 that is configured to control the flow rate of a fluid out of the reservoir 102. For example, in some embodiments, the reservoir 102 may be a syringe. In some such embodiments, the actuator 103 may be configured to depress the plunger of the syringe (i.e., the reservoir), thereby causing fluid to flow out of the reservoir 102 and to the airflow chamber region 101 (e.g., to the fluid emitter 120 via joining region 121 as shown in FIG. IB). For example, in some embodiments, the actuator 103 is a syringe pump. Other configurations are also possible (e.g., the actuator 103 may comprise a valve and / or a pump configured to control the flow rate of the fluid from the reservoir to the fluid emitter).
[0062] In some embodiments, the cell encapsulation system may comprise a collection reservoir 104. In some embodiments, the collection reservoir is configured to collect a plurality of encapsulated cells and / or encapsulated cell clusters 105 which are emitted by the fluid emitter. In some embodiments, the collection reservoir may be configured to receive a fluid. In some embodiments, the fluid comprises water. In some embodiments, the fluid is an aqueous solution. In some embodiments, the fluid is a saline solution. The fluid may, in some embodiments, comprise a polymer comprising any of the polymers and / or types of polymers as described above. In some embodiments, the fluid is configured to maintain the encapsulated cells and / or encapsulated cell clusters in a suspension (e.g., the fluid may be configured to prevent substantial agglomeration of the encapsulated cells and / or cell clusters).
[0063] In some embodiments, the cell encapsulation system as shown in FIG. 1 A comprises voltage source 111. In some embodiments, the voltage source may be configured to supply a voltage of greater than or equal to 1 kV, greater than or equal to 2.5 kV, greater than or equal to 5 kV, greater than or equal to 7.5 kV, greater than or equal to 10 kV, greater than or equal to 12.5 kV, greater than or equal to 15 kV, greater than or equal to 17.5 kV, or greater than or equal to 20 kV. In some embodiments, the voltage source may be configured to supply a voltage of less than or equal to 20 kV, less than or equal to 17.5 kV, less than or equal to 15 kV, less than or equal to 12.5 kV, less than or equal to 10 kV, less than or equal to 7.5 kV, less than or equal to 5 kV, or less than or equal to 2.5 kV. Combinations of these ranges are also possible (e.g., the voltage source may be configured to supply a voltage of greater than or equal to 1 kV and less than or equal to 20 kV, or greater than or equal to 5 kV and less than or equal to 15 kV). Other ranges are also possible.In some embodiments, the voltage source may be configured to supply and / or transmit a voltage to one or more parts of the cell encapsulation system. In some embodiments, the voltage source may be configured to transmit a voltage to an electrode associated with the cell encapsulation system. In some embodiments, as shown in FIG. 1 A, the voltage source 111 may be configured to transmit a voltage to the airflow chamber region 101 (e.g., to the fluid emitter 120 as shown in FIG. IB) via an electrical conduit 112. In some embodiments, the fluid emitter itself can be capable of transporting current, and can therefore itself be an electrode. In some such embodiments, the fluid emitter may thereby electrically drive the flow of a fluid upon the application of a voltage from voltage source 111. For example, in some embodiments, the fluid emitter may comprise stainless steel. In certain embodiments, the emitter can be fabricated from a material that is only slightly electronically conductive or substantially not electronically conductive. For example, in some embodiments, the fluid emitter may comprise fused silica and / or borosilicate glass. In some such embodiments, the emitter may be coated with a conductive material (e.g., a conductive polymer, a metal) and / or connected to an electrode which runs through the fluid emitter. In some embodiments, the conductive coating and / or electrode may be capable of transporting current, thereby electrically driving the flow of the fluid upon the application of a voltage from voltage source 111. In some embodiments, transport of the electrosprayed fluid to a collection reservoir can be achieved by applying an electrical voltage between the fluid and the collector reservoir.
[0064] In some embodiments, the cell encapsulation system may be configured for voltage driven flow of a fluid comprising a plurality of living cells. For example, in some embodiments, the system may be configured such that when a voltage is applied to the fluid emitter (and / or an electrode and / or conductive coating associated with the fluid emitter), a fluid comprising a plurality of living cells may, in response, flow through the fluid emitter. In some embodiments, system may be configured such that a voltage driven flow of a fluid may flow through the fluid emitter, out of the emitter through an orifice, and be sprayed as fine droplets.
[0065] In some embodiments, a cell encapsulation system as shown in FIG. 1 A may comprise a source of compressed gas 113, which may be in fluidic communication with the fluid emitter via conduit 114. In some embodiments, the source of compressed gas may be a source of compressed air, compressed nitrogen gas, and / or compressed argon. In some embodiments, the source of compressed gas may be a compressed gas cylinder, compressed gas tank, and / or a compressor in fluidic communication with a source of low-pressure gas. In some embodiments, the source of compressed gas and conduit are configured to deliver a stream of compressed gas to the airflow chamber region 101. For example, as shown in FIG. IB, in some embodiments,the airflow chamber region 101 comprises a compressed gas receiving region 122, to which compressed gas may be delivered by the conduit 114. In some such embodiments, the compressed gas receiving region 122 may further comprise a nozzle 123 associated with and / or in fluidic communication with the fluid emitter 120. The nozzle may be configured to direct the compressed gas out of the airflow chamber region (e.g. out of the compressed gas receiving region of the fluid emitter region) ands through a flow path associated with the fluid emitter, as represented by the arrows 125. In some embodiments, the fluid emitter, compressed gas receiving region, and / or the nozzle may be at least partially contained within an airflow chamber region enclosure 124.
[0066] The nozzle may have any of a variety of suitable diameters. For example, in some embodiments, the nozzle has a diameter of greater than or equal to 0.5 mm, greater than or equal to 1 mm, greater than or equal to 1.5 mm, greater than or equal to 1.8 mm, greater than or equal to 2 mm, greater than or equal to 2.5 mm, greater than or equal to 3 mm, greater than or equal to 3.5 mm, greater than or equal to 4 mm, or greater than or equal to 4.5 mm. In some embodiments, the nozzle has a diameter of less than or equal to 5 mm, less than or equal to 4.5 mm, less than or equal to 4 mm, less than or equal to 3.5 mm, less than or equal to 3 mm, less than or equal to 2.5 mm, less than or equal to 2 mm, less than or equal to 1.5 mm, or less than or equal to 1 mm. Combinations of these ranges are also possible (e.g., the nozzle may have a diameter of greater than or equal to 0.5 mm and less than or equal to 5 mm, or greater than or equal to 1 mm and less than or equal to 4 mm). Other ranges are also possible.
[0067] In some embodiments, the system may comprise a control mechanism configured to control the flow rate and / or pressure of a compressed gas supplied to the fluid emitter region (e.g., the nozzle) via the source of compressed gas. In some embodiments, the control mechanism comprises a valve (e.g., a gate valve, a globe valve, a ball valve, a needle valve, a pinch valve, a butterfly valve, or any other suitable valve type). In some embodiments, the control mechanism may comprise a regulator (e.g., a reducing valve, a pressure regulator, etc.). In some embodiments, the control mechanism may comprise an electronic controller configured to receive a signal from and / or transmit a signal to a valve and / or a regulator in the control mechanism, thereby raising or lowering the pressure and / or flow rate of the gas.
[0068] In some embodiments, the source of compressed gas, conduit in fluidic communication with the source of compressed gas and fluid emitter, and / or control mechanism may be configured to supply gas having any suitable pressure to the compressed gas receiving region (e.g., the system may be configured to supply gas having any suitable pressure to the compressed gas receiving region). For example, the system may be configured to supply acompressed gas having a pressure of greater than or equal to 0.1 psi, greater than or equal to 0.25 psi, greater than or equal to 0.5 psi, greater than or equal to 0.75 psi, greater than or equal to 1 psi, greater than or equal to 1.25 psi, greater than or equal to 1.5 psi, greater than or equal to 1.75 psi, greater than or equal to 2 psi, greater than or equal to 2.25 psi, greater than or equal to 2.5 psi, greater than or equal to 2.75 psi, greater than or equal to 3 psi, greater than or equal to 3.5 psi, greater than or equal to 4 psi, or greater than or equal to 4.5 psi. In some embodiments, the system may be configured to supply a compressed gas having a pressure of less than or equal to 5 psi, less than or equal to 4.5 psi, less than or equal to 4 psi, less than or equal to 3.5 psi, less than or equal to 3 psi, less than or equal to 2.75 psi, less than or equal to 2.5 psi, less than or equal to 2.25 psi, less than or equal to 2 psi, less than or equal to 1.75 psi, less than or equal to 1.5 psi, less than or equal to 1.25 psi, less than or equal to 1 psi, less than or equal to 0.75 psi, less than or equal to 0.5 psi, or less than or equal to 0.25 psi. Combinations of these ranges are also possible (e.g., the system may be configured to supply a compressed gas having a pressure of greater than or equal to 0.1 psi and less than or equal to 3 psi, or greater than or equal to 0.5 psi and less than or equal to 2 psi, greater than or equal to 0.1 psi and less than or equal to 5 psi). Other ranges are also possible.
[0069] In some embodiments, the system may be configured to deliver a compressed gas to the compressed gas receiving region (e.g., to the nozzle) and apply a voltage to the fluid emitter substantially simultaneously. In some embodiments, the system is configured to output encapsulated living cells and / or encapsulated clusters of living cells having a shell (e.g., shell 105a as shown in FIG. IB) and a core comprising the individual cells and / or cell clusters (e.g., the core 105b comprising a cell cluster, as shown in FIG. IB), wherein the cell clusters comprise individual living cells (e.g., living cells 130 as shown in FIG. IB).
[0070] In some embodiments, a method of encapsulating living cells is provided. In some embodiments, the method comprises applying a voltage to the tip of a fluid emitter (e.g., via a voltage source connected to the fluid emitter via an electrical conduit, as described above). In some embodiments, the method comprises applying a voltage of greater than or equal to 1 kV, greater than or equal to 2.5 kV, greater than or equal to 5 kV, greater than or equal to 7.5 kV, greater than or equal to 10 kV, greater than or equal to 12.5 kV, greater than or equal to 15 kV, greater than or equal to 17.5 kV, or greater than or equal to 20 kV. In some embodiments, the method comprises applying a voltage of less than or equal to 20 kV, less than or equal to 17.5 kV, less than or equal to 15 kV, less than or equal to 12.5 kV, less than or equal to 10 kV, less than or equal to 7.5 kV, less than or equal to 5 kV, or less than or equal to 2.5 kV. Combinations of these ranges are also possible (e.g., the method comprises applying a voltage of greater thanor equal to 1 kV and less than or equal to 20 kV, or greater than or equal to 5 kV and less than or equal to 15 kV). Other ranges are also possible
[0071] In some embodiments, a method of encapsulating living cells comprises flowing a fluid comprising a plurality of living cells proximate the tip of the fluid emitter. For example, in some embodiments, the method comprises flowing a fluid from a reservoir containing a fluid comprising living cells (e.g., an aqueous solution comprising living cells, as described above) that is in fluidic communication with the fluid emitter, through the fluid emitter (e.g., when the fluid emitter is a needle or other emitter comprising a conduit and an orifice), and out of the fluid emitter via an orifice at the tip of the fluid emitter. In some embodiments, the fluid comprises a polymer. The polymer may be any of the polymers described above as part of a fluid comprising living cells. In some embodiments, the fluid comprises a crosslinking agent. The crosslinking agent may be any of the crosslinking agents described above as part of the fluid comprising a plurality of living cells.
[0072] In some embodiments, flowing a fluid comprising a plurality of living cells proximate the tip of the fluid emitter may comprise using an actuator (e.g., a pump, a syringe pump, a valve, and / or other actuators as described above) to drive a flow of the fluid to the tip of the fluid emitter. In some embodiments, the actuator may be used to control the flow rate of the fluid from the reservoir to the fluid emitter.
[0073] In some embodiments, flowing a fluid comprising a plurality of living cells proximate the tip of the fluid emitter comprises inducing a voltage driven flow of the fluid. For example, in some embodiments, when a voltage is applied to the tip of fluid emitter, the fluid comprising a plurality of living cells may, in response, flow through the fluid emitter. In some embodiments, the fluid may flow through the fluid emitter, out of the emitter through an orifice, and be sprayed as fine droplets.
[0074] In some embodiments, a method of encapsulating living cells comprises flowing a compressed gas through a nozzle in fluidic communication with the fluid emitter. For example, in some embodiments, the method comprises flowing compressed gas from a source of compressed gas (e.g., a compressed gas cylinder, compressed gas tank, and / or compressor in fluidic communication with a source of low-pressure gas, as described above), through a conduit in fluidic communication with the source of compressed gas, and to the nozzle. The (compressed) gas may comprise one or more of air, nitrogen, argon, helium, carbon dioxide, and / or other suitable gases. In some embodiments, the nozzle is associated with a fluid emitter region of an air pressure system, wherein the airflow chamber region comprises the fluid emitter. For example, in some such embodiments, a compressed gas receiving region of the airflowchamber region of an air pressure system may comprise the nozzle (which may be in fluidic communication with the fluid emitter). In some embodiments, the method comprises directing the compressed gas out of the airflow chamber region through a flow path associated with the fluid emitter (e.g., via the nozzle).
[0075] In some embodiments, a method of encapsulating living cells may comprise controlling the flow rate and / or pressure of a compressed gas flowed through the nozzle via the source of compressed gas. In some embodiments, controlling the flow rate and / or pressure of the compressed gas may comprise using a control mechanism (e.g. a valve and / or a regulator, as described above). In some embodiments, the control mechanism may comprise an electronic controller configured to receive a signal from and / or transmit a component of the control mechanism configured to directly modify the flow rate and / or pressure of the compressed gas (e.g., the valve, the regulator), thereby raising or lowering the pressure and / or flow rate of the gas.
[0076] In some embodiments, the method of encapsulating living cells may comprise flowing a compressed gas through the nozzle at a pressure of greater than or equal to 0.1 psi, greater than or equal to 0.25 psi, greater than or equal to 0.5 psi, greater than or equal to 0.75 psi, greater than or equal to 1 psi, greater than or equal to 1.25 psi, greater than or equal to 1.5 psi, greater than or equal to 1.75 psi, greater than or equal to 2 psi, greater than or equal to 2.25 psi, greater than or equal to 2.5 psi, greater than or equal to 2.75 psi, greater than or equal to 3 psi, greater than or equal to 3.5 psi, greater than or equal to 4 psi, greater than or equal to 4.5 psi, or greater. In some embodiments, the method may comprise flowing a compressed gas through the nozzle at a pressure of less than or equal to 5 psi, less than or equal to 4.5 psi, less than or equal to 4 psi, less than or equal to 3.5 psi, less than or equal to 3 psi, less than or equal to 2.75 psi, less than or equal to 2.5 psi, less than or equal to 2.25 psi, less than or equal to 2 psi, less than or equal to 1.75 psi, less than or equal to 1.5 psi, less than or equal to 1.25 psi, less than or equal to 1 psi, less than or equal to 0.75 psi, less than or equal to 0.5 psi, or less than or equal to 0.25 psi.
[0077] Combinations of these ranges are also possible (e.g., the method may comprise flowing a compressed gas through the nozzle at a pressure of greater than or equal to 0.1 psi and less than or equal to 3 psi, greater than or equal to 0.5 psi and less than or equal to 2 psi, or greater than or equal to 0.5 psi and less than or equal to 5 psi). Other ranges are also possible.
[0078] In some embodiments, it is believed that flowing a compressed gas through the nozzle at a high pressure (e.g., a pressure of greater than or equal to 1 psi, or a pressure of greater than or equal to 2 psi) while encapsulating living cells and / or encapsulating clusters of living cells may advantageously result in the encapsulated cell and / or encapsulated cluster of living cells having asmaller diameter and / or lower shell thickness relative to encapsulated cells and / or encapsulated clusters of living cells encapsulated while flowing a compressed gas through the nozzle at a lower pressure. The pressure of the compressed gas may therefore be used to achieve a desired diameter and / or shell thickness of an encapsulated living cell and / or encapsulated cluster of living cells.
[0079] In some embodiments, the method comprises applying a voltage to the tip of the fluid emitter and flowing a compressed gas through a nozzle in fluidic communication with the fluid emitter substantially simultaneously. In some embodiments, applying the voltage to the tip of the fluid emitter may result in the formation of small droplets of a charged liquid (e.g., may produce electrosprayed droplets which are encapsulated living cells and / or encapsulated clusters of living cells). In some embodiments, flowing a compressed gas through a nozzle in fluidic communication with the fluid emitter substantially simultaneously may allow for the size of the electrosprayed droplets to be controlled. For example, in some embodiments, the increasing the pressure of the compressed gas may allow for the formation of smaller droplets than would be formed when a lower pressure is applied.
[0080] In some embodiments, the method may comprise collecting the encapsulated living cells and / or encapsulated clusters of living cells in a collection reservoir. In some embodiments, the method may comprise forming a plurality of encapsulated cells and / or encapsulated cell clusters which are emitted by the fluid emitter and directed to the collection reservoir. In some embodiments, the reservoir comprises a fluid (e.g., water, a saline solution, etc.). The fluid may, in some embodiments, comprise a polymer comprising any of the polymers and / or types of polymers as described above (e.g., a polymer dissolved in the fluid). In some embodiments, as described above, the fluid is configured to maintain the encapsulated cells and / or encapsulated cell clusters in a suspension (e.g., the fluid may be configured to prevent substantial agglomeration of the encapsulated cells and / or cell clusters).
[0081] In some embodiments, any of the systems and methods described above may be used to form encapsulated living cells and / or encapsulated clusters of living cells. In some embodiments, a plurality of encapsulated cells is provided. In some embodiments, the encapsulated cells comprise a core comprising one or more living cells. In some embodiments, the core comprising one or more living cells may have a diameter of greater than or equal to 100 microns, greater than or equal to 200 microns, greater than or equal to 300 microns, greater than or equal to 400 microns, greater than or equal to 500 microns, greater than or equal to 600 microns, greater than or equal to 700 microns, greater than or equal to 800 microns, greater than or equal to 900 microns, greater than or equal to 1,000 microns, greater than or equal to1,100 microns, greater than or equal to 1,200 microns, greater than or equal to 1,300 microns, or greater than or equal to 1,400 microns. In some embodiments, the core may have a diameter of less than or equal to 1,500 microns, less than or equal to 1,400 microns, less than or equal to 1,300 microns, less than or equal to 1,200 microns, less than or equal to 1,100 microns, less than or equal to 1,000 microns, less than or equal to 900 microns, less than or equal to 800 microns, less than or equal to 700 microns, less than or equal to 600 microns, less than or equal to 500 microns, less than or equal to 400 microns, less than or equal to 300 microns, or less than or equal to 200 microns. Combinations of these ranges are also possible (e.g., in some embodiments, the core may have a diameter of great than or equal to 100 microns and less than or equal to 1,500 microns, greater than or equal to 200 microns and less than or equal to 1,400 microns, or greater than or equal to 500 microns and less than or equal to 1,000 microns). Other ranges are also possible.
[0082] In some embodiments, the core is disposed within a shell. The shell may, for example, comprise a crosslinked polymer layer. For example, in a preferred embodiment, the shell may comprise a non-covalently crosslinked polymer layer. The crosslinked polymer layer may comprise any of the polymers described above as part of the fluid comprising living cells (e.g., the polymer may comprise a natural polymer such as alginate and / or agarose, or a synthetic polymer such as polylactic acid and / or zwitterionic alginate). The crosslinked polymer layer may comprise any of the crosslinking moieties and / or crosslinking agents described above as part of the fluid comprising living cells (e.g., the crosslinking moiety may be a divalent ion such as calcium).
[0083] In some embodiments, the shell has a thickness of greater than or equal to 5 microns and less than or equal to 200 microns. For example, in some embodiments, the shell has a thickness of greater than or equal to 5 microns, greater than or equal to 7.5 microns, greater than or equal to 10 microns, greater than or equal to 12.5 microns, greater than or equal to 12.5 microns, greater than or equal to 15 microns, greater than or equal to 17.5 microns, greater than or equal to 20 microns, greater than or equal to 22.5 microns, greater than or equal to 25 microns, greater than or equal to 30 microns, greater than or equal to 50 microns, greater than or equal to 75 microns, greater than or equal to 100 microns, greater than or equal to 150 microns, greater than or equal to 200 microns, or greater than or equal to 250 microns. In some embodiments, the shell has a thickness of less than or equal to 300 microns, less than or equal to 250 microns, less than or equal to 200 microns, less than or equal to 150 microns, less than or equal to 100 microns, less than or equal to 75 microns, less than or equal to 50 microns, less than or equal to 30 microns, less than or equal to 27.5 microns, less than or equal to 25 microns, less than orequal to 22.5 microns, less than or equal to 20 microns, less than or equal to 17.5 microns, less than or equal to 15 microns, less than or equal to 12.5 microns, less than or equal to 10 microns, or less than or equal to 7.5 microns. Combinations of these ranges are also possible (e.g., the shell may have thickness of greater than or equal to 5 microns and less than or equal to 30 microns, or greater than or equal to 7.5 microns and less than or equal to 25 microns, greater than or equal to 5 microns and less than or equal to 200 microns). Other ranges are also possible.
[0084] The encapsulated living cells and / or encapsulated clusters of living cells may comprise any of a variety of suitable types of cells. For example, in some embodiments, a cluster of living cells may comprise an organoid (e.g., a brain organoid) and / or a cell aggregate (e.g., a cardiomyocyte cell cluster) comprising a particular cell type and / or cells having a particular functionality. In some embodiments, the encapsulated living cells and / or encapsulated clusters of living cells may comprise insulin-secreting cells. In some embodiments, the encapsulated clusters of living cells are islets (e.g., pancreatic islets). In some embodiments, the encapsulated living cells and / or clusters of living cells may be configured to be transplanted into a subject (e.g., the encapsulated living cells and / or clusters of living cells may be configured to be used in cell therapy treatments). In some embodiments, the thin shell of the encapsulated living cells and / or clusters of living cells may allow for the fast diffusion of oxygen and / or nutrients through the shell and to the living cells and to the core. This may advantageously reduce the risk of cell hypoxia and necrosis in the core, which may enhance the viability of the living cells in the core, thereby increasing the safety and efficacy of the transplantation of the encapsulated living cells and / or clusters of living cells within a subject.
[0085] As used herein, a “subject” refers to any animal such as a mammal (e.g., a human). Nonlimiting examples of subjects include a human, a non-human primate, a cow, a horse, a pig, a sheep, a goat, a dog, a cat or a rodent such as a mouse, a rat, a hamster, a bird, a fish, or a guinea pig. Generally, the invention is directed toward use with humans.
[0086] EXAMPLES
[0087] The following example is intended to illustrate certain embodiments described herein, including certain aspects of the present invention, but do not exemplify the full scope of the invention.
[0088] EXAMPLE 1
[0089] This example describes the design, building, and testing of a cell encapsulation system. The cell encapsulation system, represented by the schematic shown in FIG. 2, comprised a fluidemitter (within an airflow chamber region) that was fluidically connected to a reservoir comprising living cells. In the system of this Example, the reservoir comprising living cells was a syringe containing an aqueous sodium alginate solution and living INS-1 cells. The system comprised a syringe pump actuator configured to control the flow of the solution comprising living cells to the fluid emitter. The cell encapsulation system further comprised a voltage source connected to the fluid emitter via an electrical conduit and a source of compressed air connected to a compressed air receiving region within the airflow chamber region via a compressed air conduit. The compressed air receiving region comprised a nozzle associated with the fluid emitter and configured to apply an air pressure to the electrosprayed encapsulated cells and encapsulated cell clusters. This system was used to form encapsulated living INS-1 cells and encapsulated clusters of living INS-1 cells.
[0090] In order to form the encapsulated clusters of living INS-1 cells, a voltage of 10 kV was applied to the fluid emitter and air pressures of 0.5 psi, 1.0 psi, 1.5 psi, and 2.0 psi were applied via the compressed air source and compressed air conduit during the formation of the encapsulated cells and clusters of cells. As shown in FIG. 3, the applied air pressure had a significant impact on the size of the encapsulations. For example, at 0.5 psi air pressure, the encapsulated cell clusters had an average diameter of approximately 800 microns, and each encapsulation, on average, contained multiple distinct clusters. In contrast, the encapsulated cell clusters formed under 2.0 psi air pressure had an average diameter of approximately 250 microns, and most encapsulations contained a single distinct cluster. The relationship between the diameter of the encapsulated cell clusters and the air pressure is also shown in FIG. 5B. As clearly seen in FIG. 3, the encapsulation under 1.0 psi and 1.5 psi produced encapsulated clusters of cells having intermediate diameters and numbers of distinct clusters. These tests clearly demonstrate that increasing the air pressure applied to the fluid emitter during the formation of encapsulated cell clusters reduces the size of the encapsulated clusters. This relationship was also observed during the formation of alginate microcapsules from a solution of sodium alginate, as shown in FIG. 4A. The relationship between the air pressure and the diameter of the sodium alginate particles is shown in FIG. 4B, where it was again observed that the diameter of the microcapsules decreased with increasing air pressure.
[0091] It can also be seen in FIG. 3 that the cell clusters shown therein are encapsulated by a very thin coating (i.e., a very thin shell). For example, the cell clusters formed over 2.0 psi air pressure demonstrated an average shell thickness of approximately 20 microns. Such a thin shell increases the availability of oxygen and nutrients to the encapsulated cells, thereby improvingcell viability. The relationship between the shell thickness of the coating and the air pressure at which encapsulation is performed is also shown in FIG. 5C.
[0092] In addition to capsule diameter and shell thickness, the air pressure was also observed to affect the cluster number within each capsule and the thickness of the coating. The relationship between the cluster number and the air pressure is shown in FIG. 5 A.
[0093] Encapsulation of large clusters (having an average diameter of approximately 280 micrometers, as shown in FIG. 6A) and small clusters (having an average diameter of approximately 196 micrometers, as shown in FIG. 6D) was also performed at pressures greater than 2.0 psi. Further decreases in capsule diameter (as shown in FIG. 6B for the large clusters and FIG. 6E for the small clusters) and shell thickness (as shown in FIG. 6C for the large clusters and FIG. 6F for the small clusters) were observed for both the large clusters and the small clusters with increasing air pressure during capsule formation.
[0094] EXAMPLE 2
[0095] This Example describes testing of insulin secretion from encapsulated clusters of INS-1 cells. In this Example, clusters of INS-1 cells were formed using the cell encapsulation system described in Example 1 above using an air pressure of 2.0 psi. The secretion of insulin from these cell clusters was studied relative to the secretion of insulin from cell clusters encapsulated using a conventional, voltage-only encapsulation system. The conventionally encapsulated cells and the cells encapsulated using the encapsulation system described in Example 1 are shown in FIG. 7 A. The average diameter and volume of the capsules was determined (shown in FIGS. 7B and 7C, respectively). It was observed that the capsules formed using the encapsulation system described in Example 1 had much smaller average diameters and much smaller volumes than the capsules formed using conventional methods. It was also observed, as shown in FIG. 7D, that the encapsulated cell clusters formed using the encapsulation system described in Example 1 had a significantly thinner shell than those formed using conventional methods.
[0096] The encapsulated cell clusters were then subjected to a high glucose challenge, and secretion of insulin was measured, as shown in FIG. 8. Higher insulin secretion was observed for the encapsulated cell clusters formed using the encapsulation system described in Example 1 at the beginning and the end of the high glucose challenge relative to the conventionally formed encapsulated cell clusters, indicating that the thinner shell of the encapsulated cell clusters formed using the encapsulation system described in Example 1 led to faster diffusion of insulin through the shell.Human islets were also encapsulated using a conventional, voltage-only technique and the encapsulation system described in Example 1 with an air pressure if 2.0 psi and an air pressure of 3.0 psi, as shown in FIG. 9A. The thickness of the shell formed relative to the cluster diameter using each of these techniques is shown in FIG. 9B, which shows that the encapsulated islets formed using the encapsulation system described in Example 1 had thinner shells than those formed using conventional techniques, and that increased air pressure resulted in an even thinner shell. The insulin secretion of the encapsulated islets during a high glucose challenge is shown in FIG. 9C, in which it can be seen that the highest insulin secretion was observed from the encapsulated islets formed using the encapsulation system described in Example 1 with an air pressure of 3.0 psi, followed by the encapsulated islets formed using the encapsulation system described in Example 1 with an air pressure of 2.0 psi. This again indicates that the thinner shell led to faster diffusion of the insulin through the shell.
[0097] The long-term insulin diffusion of the encapsulated islets was also observed. FIG. 10A shows the daily secretion of insulin measured at 7 days and 14 days, showing again that the encapsulated islets with thinner shells exhibited increased insulin secretion relative to those with thicker shells. Glucose-stimulated insulin secretion was also performed for the encapsulated islets (as shown in FIG. 10B), and the stimulation index was determined (as shown in FIG. 10C). It was observed that the encapsulated islets with thinner coatings exhibited higher stimulation indices, indicating that the thinner shells of the encapsulated islets improved mass transfer of insulin secreted by the islets and functional preservation of the islets themselves.
[0098] EXAMPLE 3
[0099] This example describes computational fluid dynamics modeling of a cell encapsulation system. Fluid flow through the cell encapsulation system was modeled using COMSOL to predict the droplet sizes produced by different flow rates of an alginate solution and a gas.
[0100] In this analysis, only the region to the right of the dashed line shown in in FIG. 11 was considered. The average gas velocity was determined along this line, which served as the inlet boundary condition for each corresponding calculation. FIG. 11 illustrates the setup for an inlet pressure of 0.5 psi. The axial symmetry of the system was leveraged to further simplify the calculations, advantage of its axial symmetry. FIG. 12 shows the resulting simplified model with the relevant boundary conditions labeled.
[0101] The two working fluids in this model were taken to be nitrogen gas and a 1.5% w / v sodium alginate (in 0.9 % NaCl) solution, both assumed at room temperature. Nitrogen gas properties were taken directly from the COMSOL model library, while the properties of alginatewere derived from rheology and interpolated from literature. Sodium-alginate solutions are shear-thinning non-Newtonian fluids; as such, a Cross was used to capture this behavior.
[0102] The Cross model over the more commonly used power-law for better fitting of data over the entire range and to include the Newtonian plateau at low shear rates.
[0103] The Cross model is a special case of the Carreau- Yasuda model in which a = 1 — n and A = — . The Cross model is:
[0104] Ttr
[0105] 1-n -1 ftapp + (Po / ^co) 1 + [Eq. 1]
[0106]
[0107] where Ttris the critical stress of the fluid, pois the zero-shear viscosity, pmis the infinite-shear viscosity, y is the shear rate, n is the shear-thinning index, and pappis the apparent viscosity. To ensure that this model accurately captured the behavior of the alginate solution, rheological measurements were performed on a 1.5% w / v sodium alginate (in 0.9 % NaCl) solution. A flow sweep of 0.1 s'1to 100 s'2was performed on the solution at 20 °C using a 40 mm stainless steel 2° cone using a 50 micron truncation gam and 50 micron trim gap. The results are shown in FIG. 13, which shows that the observed apparent viscosity of the alginate solution matched well with the predictions of the Cross model. Other material properties of the sodium alginate solution that were used in the simulation are shown in FIG. 14.
[0108] For the simulation, an incompressible flow was assumed. The governing equations of the simulation were:
[0109] V. U = 0
[0110] p— + p (u. V)u = 7. [-pl + p (Vu + 7u7’) + pg + Fst
[0111]
[0112] where u is velocity, t is time, pis fluid density, p is pressure, p is viscosity, g is gravity, and Fstis external force. Interface motion of the multiphase flow was simulated with the COMSOL module Two-Phase Flow, in which the Phase Field approach was used.
[0113] In each simulation, velocity ramped from 0 to the maximum velocity in 0.1 seconds. The liquid inlet flow rate was 200 microliters per minute and simulations were performed for a gas inlet pressure of 0.5 psi, 1.0 psi, 1.5 psi, and 2.0 psi (corresponding to gas inlet velocities of 56.81 m / s, 72.28 m / s, 94.04 m / s, and 117.58 m / s, respectively). The time of the simulation was 0.35 seconds with time steps of 10'4seconds, performed using a Newton-Raphson method with the PARDISO solver. Meshing was chosen such that minimum element size had a height of 25 micrometers, with a maximum element size of 75 micrometers.The transition between dripping, micro-dripping, and cone jet flow modes was characterized using the Weber number (We = pv2llo), which is a ratio of inertial and Laplace pressure that characterizes the atomizing quality of a spray and the resulting droplet size when producing emulsions. The Weber number of the liquid was determined to be Wenq= 0.009, indicating that the liquid flow was in the capillary-controlled regime in which a stable dripping regime with no inertial jet breakup would be expected without the influence of the surrounding gas flow. The Weber number of the gas was W egas= [20.43, 40.86, 61.30, 81.73] for pressures of [0.5, 1.0, 1.5, 2.0] psi; the much higher Weber numbers of the gas indicate that the breakup of the liquid jet into droplets will be gas-driven. As shown in FIG. 15 A, it was determined that the gas pressures of 0.5 psi and 1.0 psi (Weber numbers of 20.43 and 40.86) would lead to a transient micro-dripping regime, while gas pressures of 1.5 psi and 2.0 psi (Weber numbers of 61.3 and 81.73) would lead to a cone jet regime. The transition between dripping, micro-dripping, and cone-jet modes relative to gas velocity was also determined, as shown in FIG. 15B.
[0114] The results of an example simulation are shown in FIGS. 16A-16C. A custom image! macro was developed to determine the particle size of the capsules formed in this simulation. An example of an image used to determine particle size is shown in FIG. 16A (for a gas pressure of 0.5 psi). The cone area of the spray was also determined, as shown in FIG. 16B (for a gas pressure of 0.5 psi). With increasing pressure, a decrease in the aerospray cone size of the secondary liquid phase was observed, which corresponded with a decrease in the measured droplet size, as shown in FIG. 16C. It was also observed that the flow mode changed from dripping to streaming when air flow pressure exceeded 1 psi, with dripping becoming negligible at higher pressures. FIG. 17A quantitatively shows that droplet size decreased with increasing air pressure, and FIG. 17B quantitatively shows that the volume of the liquid spray cone decreased with increasing air flow pressure, leading to smaller capsules.
[0115] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.
[0116] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
[0117] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
[0118] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
[0119] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one elementselected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
[0120] As used herein, “wt%” is an abbreviation of weight percentage. As used herein, “at%” is an abbreviation of atomic percentage.
[0121] Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way.
[0122] Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and / or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.
[0123] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.
[0124] When a portion (e.g., a layer, a structure, a region) is “on”, “adjacent”, “above”, “over”, “overlying”, or “supported by” another portion, it can be directly on the portion, or an intervening portion (e.g., layer, structure, region) may also be present. Similarly, when a portion is “below” or “underneath” another portion, it can be directly below the portion, or an intervening portion (e.g., layer, structure, region) may also be present. A portion that is “directly adjacent”, “directly on”, “immediately adjacent”, “in contact with”, or “directly supported by” another portion means that no intervening portion is present. It should also be understood that when a portion is referred to as being “on”, “above”, “adjacent”, “over”, “overlying”, “in contactwith”, “below”, or “supported by” another portion, it may cover the entire portion or a part of the portion.
[0125] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
Claims
CLAIMSWhat is claimed is:
1. A cell encapsulation system, comprising:a fluid emitter;a voltage source in electrical communication with the fluid emitter;a reservoir configured to receive a fluid comprising a plurality of living cells in fluidic communication with the fluid emitter; anda source of compressed gas in fluidic communication with the fluid emitter, wherein the cell encapsulation system is configured for voltage driven flow of the fluid comprising the plurality of living cells.
2. A method of encapsulating living cells, the method comprising:applying a voltage to a tip of a fluid emitter;flowing a fluid comprising a plurality of living cells proximate the tip of the fluid emitter; andflowing a compressed gas through a nozzle in fluidic communication with the fluid emitter, thereby encapsulating the plurality of living cells.
3. A plurality of encapsulated cells, comprising:a core disposed within a shell, the core comprising one or more living cells, the shell comprising a non-covalently crosslinked polymer layer having a thickness of greater than or equal to 5 microns and less than or equal to 200 microns, wherein the core is surrounded by the shell.
4. A system or method as in any preceding claim, wherein the fluid comprises one or more components selected from alginate, hyaluronic acid, chitosan, collagen, fibrin, and gelatin.
5. A system or method as in any preceding claim, wherein at least a portion of the living cells are in clusters.
6. A system or method as in any preceding claim, wherein the compressed gas comprises air, argon, helium, and / or nitrogen.
7. A system or method as in any preceding claim, wherein the applied voltage is greater than or equal to 5 kV and less than or equal to 15 kV.
8. A plurality of encapsulated cells as in any preceding claim, wherein the non-covalently crosslinked polymer comprises one or more of alginate, hyaluronic acid, chitosan, collagen, fibrin, and gelatin.
9. A plurality of encapsulated cells as in any preceding claim, wherein at least a portion of the living cells are in clusters having average diameter of greater than or equal to 100 microns and less than or equal to 1,500 microns.
10. A system or method as in any preceding claim, wherein the nozzle has an inner diameter of greater than or equal to 0.5 mm and less than or equal to 5 mm.
11. A system or method as in any preceding claim, wherein the fluid comprises water, alginate, sodium alginate, and / or salt.
12. A system or method as in any preceding claim, wherein the fluid comprises sodium alginate dissolved in an aqueous saline solution.
13. A method as in any preceding claim, wherein the compressed gas has a pressure of greater than or equal to 0.1 psi and less than or equal to 5 psi.