System and method for forming microparticles for culturing organoids

Abstract

The present disclosure generally relates to a system (100) and method (200) for forming microparticles (140) for culturing organoids. The system (100) comprises: a droplet generator (110) comprising a nozzle (112) for producing droplets (114); a supply apparatus (120) for communicating an aqueous solution to the droplet generator (110), the aqueous solution comprising a hydrogel, organoid cells, and a crosslinking agent; a collection apparatus (130) for collecting the droplets (114) produced from the nozzle (112), each droplet (114) comprising the aqueous solution. The collection apparatus (130) comprises a thin film of oil surfactant (132) that is immiscible with the droplets (114), such that the droplets (114) are separately collected on the thin film of oil surfactant (132) and thereby forming discrete microparticles (140). The hydrogel in the microparticles (140) is solidifiable by the crosslinking agent to form an extracellular matrix for culturing organoids from the organoid cells.

Description

[0001] SYSTEM AND METHOD FOR FORMING MICROPARTICLES FOR CULTURING ORGANOIDS

[0002] Cross Reference to Related Application(s)

[0003] The present disclosure claims the benefit of Greece Patent Application No. 20230100753 filed on 20 September 2023, which is incorporated in its entirety by reference herein.

[0004] Technical Field

[0005] The present disclosure generally relates to a system and method for forming microparticles for culturing organoids. More particularly, the present disclosure describes various embodiments of a system and a method for forming microparticles, such as microcapsules, which encapsulate organoids for their growth and development.

[0006] Background

[0007] Organoids are miniaturized, in the order of microns to millimetres, versions of an organ that are produced in 3D cellular assemblages to replicate the functional, structural, and biological characteristics of that organ. The cells employed to form organoids can be obtained by programming the differentiation of pluripotent cells, such as induced pluripotent stem cells, embryonic stem cells, and adult stem cells, or by extracting tissue-derived multipotent or differentiated cells. The process can be initiated by either single cells or cell aggregates. Organoids have considerable use in biomedical research because they are more accurate than 2D culture models, offer greater availability than animal organs and human explants, and are easy to handle.

[0008] Organoid research typically involves high throughput screening and multivariable and combinatorial assessments using organoids. Organoid research will benefit greatly from high throughput production of organoids, and increased automation of producing organoids will further make organoid research more economic and accessible. For example, microencapsulation is one way for large scale production of organoids. Microencapsulation is used in various industries including medicine, biomedical research, food, agriculture, construction, and chemical. In particular, there is profound interest in using microcapsules in biomedical research, where the microcapsules perform important roles such as immobilisation of enzymes, protective isolation of cells, controllable release of agents and carriers of drugs.

[0009] There has been increasing interest in use of microencapsulation in organoid research. Organoids usually develop either in non-adherent conditions or in an instructive matrix, which presents extracellular matrix cues for organoid development. These cues include both cellular adhesion moieties and biomechanical properties such as stiffness or modulus of elasticity. The process of microencapsulation has two stages. The first stage is dispersion stage wherein a bulk starting liquid such as a hydrogel is dispersed into multiple microcapsules. The second stage is the collection stage wherein the microcapsules solidify to maintain structural integrity. Accordingly, the hydrogel in the microcapsules hardens to confer stiffness and maintain structural integrity for culturing the organoids. Organoid culture requires hydrogel material that is in the liquid phase for cell incorporation and mixing in the first stage and undergo gelation in second stage. In order to increase production of organoids for organoid research, there is a need to provide a system and method for forming microparticles for culturing organoids.

[0010] Summary

[0011] According to a first aspect of the present disclosure, there is a system for forming microparticles for culturing organoids. The system comprises: a droplet generator comprising a nozzle for producing droplets; a supply apparatus for communicating an aqueous solution to the droplet generator, the aqueous solution comprising a hydrogel, organoid cells, and a crosslinking agent; a collection apparatus for collecting the droplets produced from the nozzle, each droplet comprising the aqueous solution. The collection apparatus comprises a thin film of oil surfactant that is immiscible with the droplets, such that the droplets are separately collected on the thin film of oil surfactant and thereby forming discrete microparticles. The hydrogel in the microparticles microcapsules is solidifiable by the crosslinking agent to form an extracellular matrix for culturing organoids from the organoid cells.

[0012] According to a second aspect of the present disclosure, there is a method for forming microparticles for culturing organoids. The method comprises: communicating an aqueous solution from a supply apparatus to a droplet generator, the aqueous solution comprising a hydrogel, organoid cells, and a crosslinking agent; producing a plurality of droplets from a nozzle of the droplet generator, each droplet comprising the aqueous solution; arranging a collection apparatus for collecting the droplets produced from the nozzle, the collection apparatus comprising a thin film of oil surfactant that is immiscible with the aqueous solution; collecting the droplets separately on the thin film of oil surfactant and thereby forming discrete microparticles; and solidifying the hydrogel in the microparticles by the crosslinking agent to form an extracellular matrix for culturing organoids from the organoid cells.

[0013] A system and method for forming microparticles for culturing organoids according to the present disclosure are thus disclosed herein. Various features and advantages of the present disclosure will become more apparent from the following detailed description of the embodiments of the present disclosure, by way of non-limiting examples only, along with the accompanying drawings.

[0014] Brief Description of the Drawings

[0015] FIG. 1A and FIG. 1 B are illustrations of a system for forming microparticles for culturing organoids, according to embodiments of the present disclosure.

[0016] FIG. 2A and FIG. 2B are illustrations of a collection apparatus of the system.

[0017] FIG. 3 is a flowchart illustration of a method for forming microparticles for culturing organoids, according to embodiments of the present disclosure.

[0018] FIG. 4 is an image of the microparticles for culturing organoids. FIG. 5 is an image from experiments on microparticles for culturing mouse intestinal and human lung organoids.

[0019] FIG. 6A to FIG. 6C are additional images of the microparticles culturing the mouse intestinal and human lung organoids.

[0020] FIG. 7A to FIG. 7D are illustrations from experiments on tracking of icroparticles containing organoids in screening.

[0021] FIG. 8A and FIG. 8B are illustrations from experiments on microparticles containing organoids with high fluorescent intensities.

[0022] FIG. 9A and FIG. 9B are illustrations from experiments on degradation rates of hydrogels.

[0023] FIG. 10A and FIG. 10B are illustrations from experiments on storage moduli of hydrogels.

[0024] FIG. 11A and FIG. 11 B are illustrations of the size distribution of monodisperse microparticles.

[0025] FIG. 12A to FIG. 12D are illustrations from experiments on microparticles containing mouse intestinal organoids.

[0026] FIG. 13A to FIG. 13D are images of the microparticles with and without thin shells.

[0027] Detailed Description

[0028] For purposes of brevity and clarity, descriptions of embodiments of the present disclosure are directed to a system and method for forming microparticles for culturing organoids, in accordance with the drawings. While parts of the present disclosure will be described in conjunction with the embodiments provided herein, it will be understood that they are not intended to limit the present disclosure to these embodiments. On the contrary, the present disclosure is intended to cover alternatives, modifications and equivalents to the embodiments described herein, which are included within the scope of the present disclosure as defined by the appended claims. Furthermore, in the following detailed description, specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be recognized by an individual having ordinary skill in the art, i.e. a skilled person, that the present disclosure may be practiced without specific details, and / or with multiple details arising from combinations of features of particular embodiments. In a number of instances, well-known systems, methods, procedures, and components have not been described in detail so as to not unnecessarily obscure features of the embodiments of the present disclosure.

[0029] In embodiments of the present disclosure, depiction of a given element or consideration or use of a particular element number in a particular figure or a reference thereto in corresponding descriptive material can encompass the same, an equivalent, or an analogous element or element number identified in another figure or descriptive material associated therewith.

[0030] References to “an embodiment / example”, “another embodiment I example”, “some embodiments I examples”, “some other embodiments / examples”, and so on, indicate that the embodiment(s) / example(s) so described may include a particular feature, structure, characteristic, property, element, or limitation, but that not every embodiment / example necessarily includes that particular feature, structure, characteristic, property, element or limitation. Furthermore, repeated use of the phrase “in an embodiment I example” or “in another embodiment / example” does not necessarily refer to the same embodiment / example.

[0031] The terms “comprising”, “including”, “having”, and the like do not exclude the presence of other features / elements I steps than those listed in an embodiment. Recitation of certain features / elements / steps in mutually different embodiments does not indicate that a combination of these features I elements I steps cannot be used in an embodiment. As used herein, the terms “a” and “an” are defined as one or more than one. The use in a figure or associated text is understood to mean “and / or” unless otherwise indicated. The term “set” is defined as a non-empty finite organization of elements that mathematically exhibits a cardinality of at least one (e.g. a set as defined herein can correspond to a unit, singlet, or single-element set, or a multiple-element set), in accordance with known mathematical definitions. The recitation of a particular numerical value or value range herein is understood to include or be a recitation of an approximate numerical value or value range.

[0032] Representative or exemplary embodiments of the present disclosure describe a system 100 for forming microparticles for culturing organoids, as shown in FIG. 1A. Microparticles are solid particles, such as microcapsules, microbeads, or microspheres, with dimensions in the order of microns to millimetres, such as ranging from 1 pm to 1 mm. The system 100 includes a droplet generator 110, a supply apparatus 120, and a collection apparatus 130.

[0033] The supply apparatus 120 is configured for communicating an aqueous solution to the droplet generator 110. The aqueous solution includes a hydrogel, organoid cells, and a crosslinking agent. The hydrogel forms an extracellular matrix for culturing organoids from the organoid cells. For example, the hydrogel may include gelatin, collagen, Matrigel®, or hyaluronic acid. The organoid cells are cells suitable for organoid culture, i.e. for growing and developing into organoids. For example, the organoid cells may include stem cells, pluripotent cells, or differentiated cells. The crosslinking agent is mixed with the hydrogel to solidify the hydrogel to form the extracellular matrix. For example, the crosslinking agent includes genipin, transglutaminase, or a photoinitiated crosslinking agent.

[0034] In some embodiments as shown in FIG. 1A, the supply apparatus 120 includes a set of pumps 121 , such as pneumatic pressure pumps or syringe pumps, for pumping the aqueous solution to the droplet generator 110. For example, the pumps 121 include a syringe pump for delivering a solution containing the hydrogel and organoid cells to the droplet generator 110. The supply apparatus 120 may include a mixer device 122 for receiving the hydrogel and organoid cells from the pumps 121. For example, the pumps 121 deliver the hydrogel and organoid cells through a 4-way connector 123 and a set of female I male luer locks to the mixer device 122. For example, the connector 123 attaches to a female luer having a 0.5 mm barb, wherein the barb is forced into the barrel 124 of a 1 ml syringe. The mixer device 122 is disposed in the syringe barrel 124 and includes an 8 turn, 0.5 mm helical polypropylene mixer. Further, at the 0.15 ml marking of the syringe barrel 124, 0.7 mm bore holes 125 at approximately 45° angle to the syringe barrel 124 are introduced. 0.7mm dispensing syringe needles are inserted into the bore holes 125. These syringe needles allow the less concentrated and low volume factors to be introduced directly to the mixer device 122. For example, a syringe needle introduces the crosslinking agent to the mixer device 122 to mix with the hydrogel. Alternatively or additionally, the crosslinking agent is delivered from the pumps 121 to the mixer device 122. The syringe luer slip 126 is inserted directly into the droplet generator 110, thus delivering the aqueous solution, comprising the mixture of the hydrogel, organoid cells, and crosslinking agent, from the mixer device 122 in the syringe barrel 124 to the droplet generator 110.

[0035] As shown in FIG. 1 B, the droplet generator 110 includes a nozzle 112 for producing droplets 114. For example, the droplet generator 110, such as the Buchi Encapsulator B-390, produces droplets 114 of sizes ranging from 150 microns to 4 mm, and at flow rates ranging from 1 ml to 40 ml per minute. The nozzle 112 preferably includes a perturbation mechanism that generates a force to alter the production of droplets 114. For example, the perturbation mechanism allows the nozzle 112 to produce droplets 114 that are smaller than gravity dripping alone.

[0036] In some embodiments, the nozzle 112 includes a coaxial airflow nozzle, wherein the perturbation mechanism is a coaxial airflow. This coaxial airflow generates a force at the nozzle 112, causing the droplets 114 to be sprayed at lower volumes, such as ranging from 0.2 mm to 2 mm compared to more than 2 mm in size by gravity dripping. Preferably, the droplet generator 110 having the coaxial airflow nozzle can produce droplets 114 of sizes ranging from 200 microns to 1 mm, and at a rate of approximately 4000 to 10,000 droplets 114 per minute. More preferably, the coaxial airflow nozzle sprays droplets of about 0.3 pl at a flow rate of 2 ml per minute, resulting in approximately 111 droplets 114 per second. Other than coaxial airflow, the perturbation mechanism at the nozzle 112 can be generated by other means such as electro dripping, resonance jet breakage or cutting jet breakage.

[0037] The collection apparatus 130 is configured for collecting the droplets 114 produced from the nozzle 112, each droplet comprising the aqueous solution. As shown in FIG. 2A and FIG. 2B, the collection apparatus 130 includes a thin film of oil surfactant 132 that is immiscible with the droplets 114, such that the droplets 114 are separately collected on the thin film 132 and thereby forming discrete microparticles 140. A thin film can be defined as a layer of material ranging from fractions of a nanometre to several nanometres in thickness. The thin film of oil surfactant 132 comprises a chemical composition of an oil and a surfactant, wherein the surfactant is mixed in the oil. For example, the oil is fluorinated oil. For example, the surfactant is fluorosurfactant. For example, the oil surfactant 132 comprises dSURF or FluoSurf™ fluorosurfactant mixed in fluorinated oil.

[0038] After collecting the droplets 114 by the collection apparatus 130 and forming the discrete microparticles 140 from the collected droplets 114, the hydrogel in the microparticles 140 is solidifiable by the crosslinking agent to form the extracellular matrix for culturing organoids from the organoid cells. Organoids may grow and develop from single cells or cell aggregates in the solidified hydrogel. Specifically, each microparticle 140 contains at least one organoid cell embedded or encapsulated within the hydrogel extracellular matrix, wherein an organoid can be grown and developed from the at least one organoid cell.

[0039] The nozzle 112 of the droplet generator 110 is preferably disposed at a low height, such as 5 cm, above the collection apparatus 130, so that the droplets 114 can land on the thin film of oil surfactant 132 without coalescence, aggregation, or flocculation to maintain the structural integrity of the discrete droplets 114. The thin film of oil surfactant 132 is immiscible with the aqueous solution in the droplets 114 and adsorbs onto the surfaces of the droplets 114. The adsorption of the oil surfactant 132 reduces the surface tension and confers stearic repulsion between droplets 114, allowing for discrete microparticles 140 to be formed. In particular, the thin film of oil surfactant 132 has a thickness that is smaller than the size of the droplets 114, so that the droplets 114 merely sit on the surface of the oil surfactant mixture and do not fully submerge into the oil surfactant mixture. For example, the thickness of the thin film of oil surfactant 132 is in the nanometre range and the size of the droplets 114 is at least 500 microns. By using a thin film of oil surfactant 132 instead of a collection bath of oil or other immiscible liquid, droplets can avoid collision among themselves which would occur if they were descending through the collection bath of oil. The latter often requires a long drop height so that the droplets have sufficient speed to descend through the oil bath. The oil bath also requires sufficient depth for the droplets to stabilize as they descend through the oil. A stirrer is sometimes used to prevent the landing droplets from colliding and coalescing, but the long drop height and mechanical mixing can damage delicate organoid cells in the droplets. Hydrogel also has low modulus, such as less than 1 kPa, for culturing organoids from the organoid cells, and such high drop heights and mechanical mixing would cause the low-stiffness hydrogel to break apart easily. Moreover, the oil bath usually uses a hydrocarbon oil, which is cheaper so it is not expensive to fill a collection bath.

[0040] In embodiments of the present disclosure, the collection apparatus 130 uses a thin film of oil surfactant, as opposed to a collection bath of oil. The low drop height and absence of mechanical mixing of the droplets 114 mitigate risk of damaging the droplets 114, such as disintegrating or rupturing the droplets 114 which can lead to loss of the delicate organoid cells. More delicate droplets 114 can be produced as the hydrogel in the droplets 114 can have lower modulus and stiffness, such as comprising low concentration of hydrogel polymer, to facilitate culturing of organoids from the organoid cells.

[0041] Preferably, the oil constituent of the thin film of oil surfactant 132 includes fluorinated oil. Fluorinated oil is more effective than hydrocarbon oil because fluorinated oil confers better emulsion stability due to rapid adsorption, low surface tension, low critical micelle concentrations, high surface activity, and stronger repulsive effects between droplets 114 due to the presence of fluorine instead of hydrogen. Although fluorinated oil is more expensive than hydrocarbon oil, it is still economically feasible to use fluorinated oil since only a thin film of oil surfactant 132 comprising the fluorinated oil is required for the collection apparatus 130. However, it will be appreciated that the collection apparatus 130 may alternatively use a thin film of oil surfactant 132 wherein the oil constituent includes hydrocarbon oil.

[0042] In some embodiments, the collection apparatus 130 includes a platform 134 for collecting the droplets 114 from the nozzle 112, wherein the platform 134 has the thin film of oil surfactant 132. In some embodiments as shown in FIG. 2A and FIG. 2B, the collection apparatus 130 includes a plurality of collection vessels 136 for collecting separate batches of droplets 114 from the nozzle 112. Each collection vessel 136 includes the thin film of oil surfactant 132 for collecting the droplets 114. More specifically, the base surface of each collection vessel 136 is coated with the thin film of oil surfactant 132, though it will be appreciated that side surfaces of the collection vessel 136 may also be coated with the thin film of oil surfactant 132.

[0043] Further, the platform 134 may be a moving platform 134 whereon the collection vessels 136 are disposed. The moving platform 134 is configured for moving the collection vessels 136 to the nozzle 112 for collecting each batch of droplets 114 in each collection vessel 136 sequentially. The droplet generator 110 may optionally include an aerosol shield 116 covering the nozzle 112 to prevent the droplets 114 from splattering outside the collection vessels 136.

[0044] For example, the moving platform 134 has a diameter of about 40 cm, and 32 collection vessels or beakers 136 are arranged around the circumference of the moving platform 134. Each collection vessels 136 has a diameter of about 3.5 cm and a height of about 3 cm. The moving platform 134 is configured to move the collection vessels 136 to the nozzle 112 and sequentially place each beaker 136 under the nozzle 112. The collection vessels 136 spatially separate the batches of droplets 114, thus reducing collision between the droplets 114 like in a collection bath. Particularly, in a collection bath where all the droplets are collected, there is a higher risk of the droplets merging, coalescing, aggregating, and / or flocculating. In some embodiments, the moving platform 134 is configured to move the collection vessels 136 to the nozzle 112 at predefined intervals. Specifically, the moving platform 134 is configured to rotate at a predefined rotational speed so that each collection vessel 136 is sequentially placed under the nozzle 112 at the predefined intervals. The rotational speed is set to minimize collisions between droplets 114 in the collection vessels 136, and may be adjusted depending on the viscosity of the aqueous solution and the flow rate from the nozzle 112.

[0045] In some embodiments, the moving platform 134 is configured to move the collection vessels 136 to the nozzle 112 in response to a set of predetermined trigger conditions. For example, the predetermined trigger conditions include emergence of droplets 114 from the nozzle 112. Specifically, after a batch of droplets 114 is released into a collection vessel 136, the moving platform 134 is triggered to turn to place the next collection vessel 136 under the nozzle 112 in response to emergence of the next batch of droplets 114from the nozzle 112. For example, the predetermined trigger conditions include stopping of droplets 114 being produced from the nozzle 112. Specifically, after the nozzle 112 has completed releasing a batch of droplets 114 into a collection vessel 136, the moving platform 134 is triggered to turn to place the next collection vessel 136 under the nozzle 112.

[0046] As described above, the hydrogel is solidifiable by the crosslinking agent into the extracellular matrix. Preferably, the crosslinking agent is biocompatible so as not to damage the organoid cells, and can solidify the hydrogel quickly to further reduce the risk of the droplets 114 coalescing and / or aggregating together. For example, the crosslinking agent includes a chemical such as genipin, or an enzyme such as microbial transglutaminase. For example, transglutaminase enzyme is used to covalently crosslink gelatin in the hydrogel.

[0047] Moreover, the crosslinking agent should be useable in a time window without clogging the nozzle 112. For example, the droplets 114 are released from the nozzle 112 within a short time after the crosslinking with the hydrogel is induced. For example, the crosslinking with the hydrogel is induced after the droplets 114 are collected on the thin film of oil surfactant 132. This inducing of the crosslinking may be by light or temperature.

[0048] In some embodiments, the crosslinking agent includes a photoinitiated crosslinking agent, such as a methacrylate polymer. The system 100 further includes a light emitting device configured for illuminating the photoinitiated crosslinking agent to solidify the hydrogel in the microparticles 140. Notably, crosslinking does not occur until the photoinitiated crosslinking agent is exposed to light of the correct wavelength range from the light emitting device. The delayed crosslinking of the hydrogel prevents the nozzle 112 from clogging since no solidification of hydrogel occurs at the nozzle 112.

[0049] In some embodiments, the crosslinking agent includes a temperature-induced crosslinking agent. The system 100 further includes an incubator for heating the microparticles 140 and induce crosslinking of the hydrogel. For example, the hydrogel is initially chilled before being supplied from the supply apparatus 120 to the droplet generator 110, so that crosslinking of the hydrogel does not occur when the droplets 114 are collected in the collection vessels 136. The moving platform 134 may move each collection vessel 136 to the incubator to heat the collection vessel 136, such as to a temperature of 37 °C, and induce crosslinking and thermogelation of the hydrogel.

[0050] In some embodiments, the system 100 further includes an actuation device for removing a collection vessel 136 comprising the microparticles 140 and replacing the removed collection vessel 136 with a new collection vessel 136. This would allow the moving platform 134 to continually rotate for multiple revolutions as collection vessels 136 with collected droplets 114 are removed and replaced with new ones periodically, thereby improving efficiency and increasing throughput.

[0051] In some embodiments, the system 100 includes a computer control module for controlling the supply apparatus 120, droplet generator 110, and collection apparatus 130, as well as optionally the actuation device, to operate cooperatively with each other. For example, the computer control module controls the pumps 122 to supply the respective materials to the droplet generator 110 at predefined or variable rates. For example, the computer control module times the droplet generator 110 and collection apparatus 130 so that the nozzle 112 only begins to release the droplets 114 after a collection vessel 136 is positioned below the nozzle 112. Each collection vessel 136 with the collected droplets 114 may represent an elution time point for downstream processes, such as for retrieving the collection vessel 136 to culture the organoids in the microparticles 140.

[0052] Embodiments of the present disclosure also describe a method 200 for forming microparticles 140 for culturing organoids, with reference to FIG. 3. The method 200 includes a step 202 of communicating an aqueous solution from a supply apparatus 120 to a droplet generator 110, the aqueous solution comprising a hydrogel, organoid cells, and a crosslinking agent. The method 200 includes a step 204 of producing a plurality of droplets 114 from a nozzle 112 of the droplet generator 110, each droplet comprising the aqueous solution. The method 200 includes a step 206 of arranging a collection apparatus 130 for collecting the droplets 114 produced from the nozzle 112, the collection apparatus 130 comprising a thin film of oil surfactant 132 that is immiscible with the aqueous solution. The method 200 includes a step 208 of collecting the droplets 114 separately on the thin film of oil surfactant 132 and thereby forming discrete microparticles 140. The method 200 includes a step 210 of solidifying the hydrogel in the microparticles 140 by the crosslinking agent to form an extracellular matrix for culturing organoids from the organoid cells.

[0053] Accordingly, the system 100 and method 200 allows for soft landing of droplets 114 on the thin film of oil surfactant 132 without collisions, thereby enabling controlled formation of microparticles 140 for culturing of organoids. Further, by collecting multiple batches of droplets 114 in multiple collection vessels 136, the system 100 and method 200 can achieve high-throughput microencapsulation of the organoids in the microparticles 140. Production of organoids can thus be increased for various applications including organoid research.

[0054] Specifically, the organoids grow and develop from single cells or cell aggregates in the hydrogel extracellular matrix that has low modulus and is soft to support organoid development in culture. Organoid culturing is a prolonged process and the low modulus microparticles 140 must stabilize and retain their integrity for long culture periods to allow the organoids to mature, such as for more than 2 weeks.

[0055] The microparticles 140 provide a suitable 3D matrix environment for culturing the organoids. For example, the hydrogel extracellular matrix may include Matrigel®, which is the trade name for the solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. For example, the hydrogel extracellular matrix may include PEG-4MAL hydrogel. For example, the hydrogel extracellular matrix may include gelatin crosslinked by transglutaminase. For example, the hydrogel extracellular matrix may include gelatin methacryloyl (GelMA) hydrogel that has methacryloyl groups crosslinked by 405 nm light exposure.

[0056] For example, FIG. 4 shows a bright field image of microparticles 140 comprising HEK293 cells in a hydrogel extracellular matrix comprising transglutaminase crosslinked 2.5% gelatin.

[0057] Various experiments were done to demonstrate culturing of mouse gastrointestinal organoids and proximal lung organoids derived from human induced pluripotent stem cells (iPSCs).

[0058] FIG. 5 shows the experimental results of bulk culturing mouse intestinal crypt cell aggregates in microparticles 140 comprising Matrigel® as the hydrogel extracellular matrix. The microparticles 140 were stabilized through thermogelation and cultured for 14 days for the intestinal organoids to form and mature.

[0059] In these experiments, microparticles 140 containing viable differentiated mouse small intestinal and lung organoids were cultured in Matrigel®. FIG. 6A shows the mouse small intestinal organoids after 7 days. FIG. 6B and FIG. 6C show the lung organoids after 5 days and 12 days, respectively. Particularly, the lung organoids show expressions of TP63+ and NKX2-1 after 12 days. These results demonstrate superb compatibility with high-throughput organoid microencapsulation process from the system 100 and method 200. Further experiments were done to show that organoids can be successfully icroencapsulated in the microparticles 140 using the system 100 and method 200 of the present disclosure. According to these experiments, approximately 6,000 to 8,000 microparticles 140 can be generated every minute, which would meet the throughput scale necessary for combinatorial screening. Combinatorial screening allows for massive parallel screening of more than 40 niche factors in up to 160,000 combinations across multiple time points. The high-throughput combinatorial screening can address existing problems of current culture protocol for mature lung organoid differentiation, which include prolonged durations, reporter dependent, and complex.

[0060] In another experiment, microparticles 140 containing organoids were tagged with specific trackers after exposure to one type of media containing specific niche factor(s). These trackers allow for traceability of each microparticle 140. As shown in FIG. 7A, four types of trackers were attached to the microparticles 140 layer by layer at different time points. FIG. 7B and FIG. 7C show the mouse small intestinal organoids and iPSC induced lung organoids (indicated by the solid-line arrows), respectively, with different layers of trackers (indicated by the broken-line arrows).

[0061] After screening, the microparticles 140 containing organoids with high fluorescent intensities were sorted using a large particle sorter. The experimental results showed that microparticles 140 of up to 2 mm in size can be efficiently screened at about 100 to 150 per minute. FIG. 8A and FIG. 8B show two exemplary microparticles 140 - ID- 34 and ID-38 - which were respectively assigned as the Lgr5+ highly green fluorescent protein and tdTomato red fluorescent protein.

[0062] The microparticles 140 were sorted into individual wells of a 96-well plate for imaging. Selected top-ranked microparticles 140 were selected to undergo a deconvolution process for the tracking of niche factors involved in the screening. The experimental results in FIG. 7D showed that there was an adequate number of trackers on the microparticles 140 after 15 days of organoid culture. Matrigel® is an exemplary hydrogel extracellular matrix suitable for organoid cultures. To facilitate applications of organoids in disease modelling and high-throughput applications such as drug or toxicity screening, other hydrogel extracellular matrices were tested using mouse small intestine organoids.

[0063] GelMA hydrogels were tested due to its high stability and low degradation rate properties. FIG. 9A and FIG. 9B show the degradation rates of DS95 GelMA and DS50 GelMA hydrogels, respectively, over 30 days with incubation at 37 °C. FIG. 10A shows the storage moduli of DS95 GelMA and DS50 GelMA hydrogels of different w / v% concentrations compared to Matrigel®. FIG. 10B shows the optimal elastic modulus range required for culturing different types of organoids. These results show that GelMA has highly tunable storage moduli ranges and is suitable for many different organoid cultures.

[0064] In addition, GelMA is crosslinked using 405 nm light exposure after droplet collection, which minimise the risk of nozzle blockage during spraying. The system 100 and method 200 are highly controllable and results in uniform size distribution of monodisperse GelMA microparticles 140, as shown in FIG. HA and FIG. 11 B.

[0065] In addition to stiffness of the extracellular matrix, the composition of the extracellular matrix is also an important criterion in organoid maturation and differentiation. Using mouse small intestinal organoids, various w / v% concentrations and compositions of GelMA were screened in comparison with Matrigel®.

[0066] FIG. 12A and FIG. 12B show the size and morphology, respectively, of the mouse small intestinal organoids cultured in 2 w / v% DS95 GelMA, supplemented with 2 mg / ml of lamininl -entactin complex, in comparison with undiluted Matrigel® over 7 days of culture. FIG. 12C shows the adult intestinal cell expression quantification by qPCR analysis. Lgr5 - stem cells; Lyz1 - Paneth cells; Muc2 - goblet cells; ChgA - enteroendocrine cells; Alpi - epithelial cells. All cell expressions were normalized to GAPDH housekeeping gene and Matrigel® control. It can be seen from the results in FIG. 12A to FIG. 12C that the supplemented GelMA hydrogel was able to achieve the size, morphology, and cell types of the mature organoids comparable to those cultured in Matrigel® hydrogel, which is the current gold standard extracellular matrix hydrogel for organoid cultures.

[0067] FIG. 12D shows an image of the mouse small intestinal organoids cultured in GelMA microparticles 140 after ? days of culture. It can be seen that the mouse small intestinal organoids are fully matured, showing that the GelMA hydrogel is compatible with the high-throughput microencapsulation process performed using the system 100 and method 200.

[0068] In some embodiments, the stability of the microparticles 140 may be improved or enhanced by adding one or more thin shells 142 on external surfaces of the microparticles 140. The thin shells 142 are formed of a shell material that is biocompatible with the organoid culture and that does not cause merging of the microparticles 140. For example, the shell material includes sodium alginate crosslinked with a low concentration of calcium chloride, such as ranging from 50 to 100 pg / ml. The thin shells 142 may be formed using the method 200 after the step 210 of solidifying the hydrogel in the microparticles 140. For example, sodium alginate solution is added to the solidified microparticles 140 and followed by addition of a low concentration of calcium chloride for crosslinking. The sodium alginate crosslinked with calcium chloride then solidifies to form the thin shells 142 around the microparticles 140. The thin shells 142 enhance the stability of the microparticles 140 containing the organoids, especially for organoids with long culture periods of 2 weeks or more. The enhanced stability also reduces risk of the microparticles 140 breaking up from the dispensing pressure during the sorting process by the large particle sorter described above.

[0069] FIG. 13A shows microparticles 140 containing lung organoids after 12 days of culture, wherein the microparticles 140 do not have the thin shells 142. FIG. 13B shows the microparticles 140 with one thin shell 142 (formed from sodium alginate crosslinked with calcium chloride), as indicated by the white arrow. FIG. 13C shows the microparticles 140 with two thin shells 142 (formed from sodium alginate crosslinked with calcium chloride), as indicated by the white arrow. Notably, the lung organoids inside the microparticles 140 with or without the thin shells 142 are comparably the same size, thus confirming the biocompatibility of sodium alginate with organoid culture and the additional thin shells 142 do not prevent nutrients from penetrating into the microparticles 1 0. The presence of the thin shells 142 was checked by mixing the sodium alginate with blue fluorescence tags. FIG. 13D also shows the microparticles 140 with two thin shells 142 indicated by the white arrow, wherein the thin shells 142 are marked by fluorescence tags indicated by the grey arrow.

[0070] The various experiments showed that the system 100 and method 200 can provide high-throughput combinatorial screening in a time-efficient and cost-efficient manner. For example, the combinatorial screening can explore various lung niche factors, to derive more efficient lung organoid differentiation. The combinatorial screening can be expanded to various types of organoids to improve organoid disease modelling and drug screening. Further, the combinatorial screening is reproducible and can be scaled up to increase throughput and screening efficiency.

[0071] In the foregoing detailed description, embodiments of the present disclosure in relation to a system and method for forming microparticles for culturing organoids are described with reference to the provided figures. The description of the various embodiments herein is not intended to call out or be limited only to specific or particular representations of the present disclosure, but merely to illustrate non-limiting examples of the present disclosure. The present disclosure serves to address at least one of the mentioned problems and issues associated with the prior art. Although only some embodiments of the present disclosure are disclosed herein, it will be apparent to a person having ordinary skill in the art in view of this disclosure that a variety of changes and / or modifications can be made to the disclosed embodiments without departing from the scope of the present disclosure. Therefore, the scope of the disclosure as well as the scope of the following claims is not limited to embodiments described herein.

Claims

Claims1. A system for forming microparticles for culturing organoids, the system comprising: a droplet generator comprising a nozzle for producing droplets; a supply apparatus for communicating an aqueous solution to the droplet generator, the aqueous solution comprising a hydrogel, organoid cells, and a crosslinking agent; a collection apparatus for collecting the droplets produced from the nozzle, each droplet comprising the aqueous solution; and the collection apparatus comprising a thin film of oil surfactant that is immiscible with the droplets, such that the droplets are separately collected on the thin film of oil surfactant and thereby forming discrete microparticles, wherein the hydrogel in the microparticles is solidifiable by the crosslinking agent to form an extracellular matrix for culturing organoids from the organoid cells.

2. The system according to claim 1 , wherein the nozzle comprises a coaxial airflow nozzle.

3. The system according to claim 1 or 2, wherein the collection apparatus comprises a plurality of collection vessels for collecting separate batches of droplets from the nozzle, each collection vessel comprising the thin film of oil surfactant for collecting the droplets.

4. The system according to claim 3, wherein the collection apparatus comprises a moving platform whereon the collection vessels are disposed, the moving platform configured for moving the collection vessels to the nozzle for collecting each batch of droplets in each collection vessel sequentially.

5. The system according to claim 4, wherein the moving platform is configured to move the collection vessels to the nozzle at predefined intervals or in response to a set of predetermined trigger conditions.

6. The system according to any one of claims 3 to 5, further comprising an actuation device for removing a collection vessel comprising the microparticles and replacing the removed collection vessel with a new collection vessel.

7. The system according to any one of claims 1 to 6, further comprising a computer control module for controlling the supply apparatus, droplet generator, and collection apparatus to operate cooperatively with each other.

8. The system according to any one of claims 1 to 7, wherein the thin film of oil surfactant comprises fluorinated oil.

9. The system according to any one of claims 1 to 8, wherein the hydrogel comprises gelatin, collagen, or hyaluronic acid.

10. The system according to any one of claims 1 to 9, wherein the crosslinking agent comprises genipin, transglutaminase, or a photoinitiated crosslinking agent.

11. The system according to claim 10, wherein the crosslinking agent comprises the photoinitiated crosslinking agent, and wherein the system further comprises a light emitting device configured for illuminating the photoinitiated crosslinking agent to solidify the hydrogel in the microparticles.

12. The system according to any one of claims 1 to 11 , further comprising an incubator configured for heating the microparticles for thermogelation of the hydrogel.

13. A method for forming microparticles for culturing organoids, the method comprising: communicating an aqueous solution from a supply apparatus to a droplet generator, the aqueous solution comprising a hydrogel, organoid cells, and a crosslinking agent; producing a plurality of droplets from a nozzle of the droplet generator, each droplet comprising the aqueous solution;arranging a collection apparatus for collecting the droplets produced from the nozzle, the collection apparatus comprising a thin film of oil surfactant that is immiscible with the aqueous solution; collecting the droplets separately on the thin film of oil surfactant and thereby forming discrete microparticles; and solidifying the hydrogel in the microparticles by the crosslinking agent to form an extracellular matrix for culturing organoids from the organoid cells.

14. The method according to claim 13, further comprising collecting separate batches of droplets from the nozzle in a plurality of collection vessels of the collection apparatus, each collection vessel comprising the thin film of oil surfactant for collecting the droplets.

15. The method according to claim 14, further comprising: arranging the collection vessels on a moving platform of the collection apparatus; moving the collection vessels to the nozzle; and collecting each batch of droplets from the nozzle in each collection vessel sequentially.

16. The method according to claim 15, further comprising moving the collection vessels to the nozzle at predefined intervals or in response to a set of predetermined trigger conditions.

17. The method according to any one of claims 14 to 16, further comprising, using an actuation device, removing a collection vessel comprising the microparticles and replacing the removed collection vessel with a new collection vessel.

18. The method according to any one of claims 13 to 17, further comprising controlling, using a computer control module, the supply apparatus, droplet generator, and collection apparatus to operate cooperatively with each other.

19. The method according to any one of claims 13 to 18, wherein the crosslinking agent comprises a photoinitiated crosslinking agent, the method further comprising illuminating the photoinitiated crosslinking agent to solidify the hydrogel in the microparticles.

20. The method according to any one of claims 13 to 19, further comprising heating the microparticles in an incubator for thermogelation of the hydrogel.21 . The method according to any one of claims 13 to 20, further comprising forming one or more thin shells on external surfaces of the microparticles for enhancing stability of the microparticles.

22. The method according to claim 21 , wherein the thin shells comprise sodium alginate crosslinked with calcium chloride.

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