Method for Automatic Embedding of Embryoid Bodies into Hydrogels Using a Separated Well Microplate

Abstract

Methods are provided for the automated embedding of embryoid bodies into hydrogels, medium exchange, culturing of organoids, and monitoring the development of organoids such as brain organoids. Methods for the automated testing of compounds and toxic effects are also provided. The methods include embedding the embryoid bodies into a liquid hydrogel to encapsulate the embryoid bodies, enabling careful, efficient, and automatable embedding of the embryoid bodies into the hydrogel by use of a separate well microplate, solidifying the hydrogel encapsulating the embryoid bodies, and suspending the solidified hydrogel encapsulating the embryoid bodies in a medium within the separate well microplate.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application was filed on July 20, 2023 as a PCT international patent application and claims the benefit and priority of U.S. Provisional Application No. 63 / 393,567, filed on July 29, 2022, the entire content of which is incorporated herein by reference.

Background Art

[0002] Background of the Invention The method of embedding embryoid bodies into hydrogels for culturing brain organoids is difficult to automate. It is necessary to transport the organoids together with the culture medium, but an excessive amount of the culture medium may liquefy the hydrogel (e.g., Matrigel) used to encapsulate the embryoid bodies and organoids.

[0003] Lancaster & Knoblich (Nat Protoc. 2014 October; 9(10):2329 - 2340) describes a method for generating cerebral organoids from human pluripotent stem cells (hPSCs). A labor - intensive process for transferring appropriately differentiated embryoid bodies (EBs) into Matrigel droplets is described, and for the generation of Matrigel droplets, a dented parafilm substrate needs to be manually prepared. Careful but rapid handling of the EBs is required to avoid damage to the aggregates or premature polymerization of Matrigel. An improved automated method for embedding embryoid bodies into hydrogels for culturing brain organoids is desired.

Prior Art Documents

Non - Patent Documents

[0004]

Non - Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0005] Gist of the Invention An automated method for culturing brain organoids by embedding embryoid bodies (EBs) in a hydrogel, and an automated brain organoid screening method are provided.

[0006] A method for culturing brain organoids is provided, which includes embedding embryoid bodies in a liquid hydrogel to encapsulate the embryoid bodies. The use of a separated well microplate enables careful, efficient, and automatable embedding of embryoid bodies into the hydrogel. This method may include solidifying the hydrogel encapsulating the embryoid bodies and suspending the solidified hydrogel encapsulating the embryoid bodies in a culture medium within the separated well microplate. A method is provided for shaping the liquid hydrogel encapsulating the embryoid bodies to approximate a spherical shape, solidifying the hydrogel encapsulating the EBs, and suspending the hydrogel embedded with the EBs in a culture medium. Methods are provided for transporting the hydrogel embedded with the EBs to a bioreactor, feeding nutrients, differentiating the EBs into brain organoids, and testing the brain organoids.

[0007] A method for culturing organoids, which includes adding embryoid bodies to a first culture medium in a main well of a separated well microplate including a plurality of well units, each well unit including a main well, a secondary well, and one or more microchannels connecting the main well and the secondary well; removing the first culture medium from the well unit through the secondary well; adding a liquid hydrogel to encapsulate the embryoid bodies; incubating the plate for a first period to solidify the hydrogel encapsulating the embryoid bodies; and adding a second culture medium to the secondary well to detach the solidified hydrogel encapsulating the embryoid bodies from the plate and suspend it in the second culture medium.

[0008] In some examples, adding embryoid bodies to the first medium in the main well includes performing an automated pipetting operation.

[0009] In some examples, a method of culturing an organoid includes tilting a separation well microplate to move embryoid bodies to a corner of the main well before removing the first medium, and optionally, this tilting further includes automatically tilting the separation well microplate.

[0010] Tilting this separation well microplate can include moving embryoid bodies towards the microchannel, and optionally, this tilting includes tilting the plate at an angle of 1 to 60 degrees, 5 to 45 degrees, 5 to 35 degrees, 5 to 25 degrees, 5 to 15 degrees, 7 to 12 degrees, or about 10 degrees from the horizontal position.

[0011] Tilting this separation well microplate can include moving embryoid bodies away from the microchannel, and optionally, tilting includes tilting the plate at an angle of 1 to 60 degrees, 5 to 15 degrees, 7 to 12 degrees, or about 10 degrees from the horizontal position.

[0012] In some examples, a method of culturing an organoid further includes returning the microplate to a horizontal position before removing the second medium.

[0013] In some examples, adding a liquid hydrogel to encapsulate embryoid bodies includes an image-guided pipetting operation of the liquid hydrogel, and optionally, this image-guided pipetting operation is an automated image-guided pipetting operation that includes delivering the liquid hydrogel onto at least a portion of the embryoid bodies.

[0014] In some examples, a method of culturing an organoid further includes cooling the microplate before adding the liquid hydrogel, and optionally, this cooling is an automatic cooling to a temperature below room temperature or within a temperature range of 4 to 8 °C.

[0015] In some examples, the method of culturing organoids further includes heating the microplate to the incubation temperature before adding the liquid hydrogel, and optionally, this heating is automatic heating.

[0016] In some examples, the method of culturing organoids includes, after adding the second medium, further tilting the microplate to separate the solidified hydrogel encapsulating the embryoid bodies from the microchannels, and optionally, the further tilting includes automatically further tilting.

[0017] In some examples, incubating to solidify the hydrogel occurs over a first period selected from the group consisting of about 5 minutes to about 90 minutes, about 10 minutes to about 60 minutes, about 20 minutes to about 40 minutes, and about 30 minutes, and optionally, the microplate is held at room temperature for a second period prior to the incubation step.

[0018] In some examples, the method of culturing organoids further includes periodically replacing the second medium in the well unit through the secondary well to nutrient supplement the embryoid bodies.

[0019] In some examples, the method of culturing organoids further includes introducing a biocompatible oil into the main well after adding the liquid hydrogel to encapsulate the embryoid bodies and before incubating, and optionally, this introducing includes an automatic pipetting operation of the biocompatible oil into the main well.

[0020] In some examples, this introducing includes placing a pipette tip within the hydrogel and aspirating the hydrogel encapsulating the embryoid bodies and the oil into the pipette tip, and optionally, this placing is image-guided placement. This aspirating can be performed gently over a period of at least 0.3 seconds, at least 0.5 seconds, at least 0.7 seconds, or at least 1 second.

[0021] In some examples, the method of culturing an organoid further includes pushing back an oil and a hydrogel encapsulating the embryoid body from a pipette tip into a main well. This pushing back can be gently performed over a period of at least 0.3 seconds, at least 0.5 seconds, at least 0.7 seconds, or at least 1 second.

[0022] In some examples, introduction of a biocompatible oil into the main well causes the hydrogel encapsulating the embryoid body to approach a spherical shape.

[0023] In some examples, the method of culturing an organoid includes removing a biocompatible oil from the main well through a secondary well after solidifying the hydrogel encapsulating the embryoid body; and

[0024] washing the well unit with a wash medium before adding a second medium, and optionally, this wash medium is at room temperature, further including washing.

[0025] In some examples, the method of culturing an organoid further includes transferring the encapsulated embryoid body embedded in the solidified hydrogel to a bioreactor for maturation.

[0026] In some examples, the method of culturing an organoid further includes transferring a hydrogel encapsulating the embryoid body and a biocompatible oil to an oil pool warmed to an incubation temperature to solidify the hydrogel encapsulating the embryoid body.

[0027] In some examples, this oil pool includes a stream for transferring the hydrogel encapsulating the embryoid body to a bioreactor, optionally including a filter.

[0028] In some examples, the method of culturing organoids comprises discharging oil from the bioreactor, wherein the filter retains the solidified hydrogel encapsulating the embryoid bodies; washing the bioreactor and the retained solidified hydrogel encapsulating the embryoid bodies; and further comprising adding a third medium to the bioreactor to nourish the washed hydrogel encapsulating the embryoid bodies.

[0029] In some examples, the method of culturing organoids comprises a stream of an oil pool transferring a hydrogel encapsulating embryoid bodies into the bioreactor, wherein the stream contacts a third medium, and the method further comprises separating the mixture of the oil and the third medium into layers; removing the oil layer through a filter system to retain the solidified hydrogel encapsulating the embryoid bodies in the third medium; and guiding the solidified hydrogel encapsulating the embryoid bodies in the third medium into the bioreactor.

[0030] In some examples, the method of culturing organoids is an automated method, and adding embryoid bodies to the first medium, removing the first medium from the well unit, adding a liquid hydrogel, and adding the second medium each comprise automated pipetting operations.

[0031] The present organoids can be brain organoids. The present organoids can be liver organoids.

[0032] In some examples, the present embryoid bodies are differentiated embryoid bodies showing germ layer differentiation.

[0033] In some examples, the present organoids are brain organoids. The brain organoids can be cerebral organoids.

[0034] In some examples, the hydrogel is an extracellular matrix. For example, the hydrogel can be selected from the group consisting of mouse Engelbreth-Holm-Swarm (EHS) sarcoma matrix, type I collagen, fibrin, hyaluronic acid (HA), gelatin methacrylate (GelMA), decellularized matrix, alginate, silk, nanocellulose, polyethylene glycol (PEG), self-assembling peptides, poly(lactic / (co)glycolic) acid, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptide, and ELP (elastin-like protein).

[0035] In some examples, the liquid hydrogel is a cold liquid hydrogel, optionally a cold liquid hydrogel at a temperature of less than about 10 °C or in the temperature range of 4 - 8 °C. In some examples, the biocompatible oil has a density different from that of water. This biocompatible oil may have a density higher than that of water. The biocompatible oil having a density higher than that of water can be a halogenated hydrocarbon, optionally a perfluorocarbon. In some examples, the biocompatible oil has a density lower than that of water. The biocompatible oil having a density lower than that of water can be a silicone oil, a mineral oil, or a vegetable oil.

[0036] An automated method of culturing brain organoids, comprising adding differentiated embryoid bodies to a first medium in a main well of a separated well microplate comprising a plurality of well units, each well unit comprising a main well, a secondary well, and one or more microchannels connecting the main well and the secondary well; tilting the separated well microplate to move the embryoid bodies to a corner of the main well; removing the first medium from the well unit via the secondary well; cooling the microplate and adding a liquid hydrogel to encapsulate the embryoid bodies at a corner of the main well of the tilted microplate; incubating the plate for a first period to solidify the hydrogel encapsulating the embryoid bodies; and adding a second medium to the secondary well to detach the solidified hydrogel encapsulating the embryoid bodies from the plate and suspend them in the second medium.

Brief Description of the Drawings

[0037]

Figure 1-1

[0038] Figure 1B shows a second view of an exemplary single well unit 100 in a separated well microplate useful for horizontal co-culture for cell / organoid nourishment. The main well 112 is separated from the secondary well 115 by at least one microchannel 118. While the separated well microplate is placed on a rocker, medium exchange is performed by gravity flow through the microchannel 118.

[0039]

Figure 1-2

[0040]

Figure 2

[0041]

Figure 3

[0042]

Figure 4

[0043]

Figure 5

[0044]

Figure 6

[0045] Figure 6B shows an embryoid body embedded in a spherical GrowDex® cellulose hydrogel with Fluorinert® FC-40 biocompatible oil at the top inside the pipette tip.

[0046] Figure 6C shows an embryoid body embedded in a spherical collagen I (rat) hydrogel with Fluorinert® FC-40 biocompatible oil at the top inside the pipette tip. By using this biocompatible oil, a more spherical hydrogel is obtained in each hydrogel tested.

DETAILED DESCRIPTION OF THE INVENTION

[0047] Detailed Description of the Invention An automated method for embedding embryoid bodies into hydrogels, an organoid culture method, and an organoid screening method are provided.

[0048] An automated method for preparing a 3D cell model representing brain tissue has been developed.

[0049] The present disclosure provides an automated method of integrated cell culture using a high-content imaging system that enables automated monitoring, maintenance, characterization of organoids, and testing of the effects of various compounds. This integrated system includes a confocal imaging system, an automated incubator, an automated liquid handler, and a collaborative robot. This equipment is controlled by integrated software that enables process setting. As described herein, an example of a process for monitoring cells during processing is shown in FIG. 5. The development of EBs and brain organoids can be monitored by imaging. For example, plates are moved from the incubator to the imaging system (e.g., ImageXpress Confocal HT.ai, Molecular Devices LLC) for brightfield imaging and then returned to the incubator. This process can be incorporated into a schedule and the plates that need to be imaged can be listed to facilitate batch processing. Complex routines can also be implemented, including a liquid handler for medium exchange (nutrient replenishment).

[0050] Methods for automatically embedding EBs in hydrogels, methods for medium exchange, and methods for monitoring the development of brain organoids are provided. In addition, these methods enable automated testing of compounds and toxic effects.

[0051] Definitions

[0052] The singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0053] The term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0054] The term "about", when referring to measurable values such as amounts of compounds, times, temperatures, and the like, is meant to encompass variations of 10%, 5%, 1%, 0.5%, and even 0.1% of the specified amount.

[0055] Unless otherwise specified, the term "percent" or "%" refers to weight percent.

[0056] Unless otherwise specified, the term "density" (d) refers to the mass of a substance per unit volume at 25°C.

[0057] As used herein, the term "biocompatible oil" refers to an oil that, when used according to the methods of the present disclosure, does not adversely affect the development, differentiation, or survival of embryoid bodies or organoids. A biocompatible oil can be a non-toxic oil when used according to the methods of the present disclosure. A biocompatible oil can have a density different from that of water.

[0058] As used herein, the term "composition" can refer to one or more of a compound, mixture, blend, alloy, polymer, and / or copolymer.

[0059] The term "room temperature" refers to a temperature in the range of 20 - 25°C (average 23°C).

[0060] The term "cold" in "cold liquid hydrogel" etc. refers to a temperature below room temperature, or a temperature within the range of 0 - 10°C, 1 - 8°C, or about 4°C.

[0061] The terms "incubate", "incubating", or "incubation temperature" refer to about 37°C.

[0062] As defined herein, ranges are intended to include at least the numbers defining the boundaries of the range.

[0063] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entirety.

[0064] The term "feeder cell" refers to a cell that provides extracellular secretions to assist in the proliferation, growth, differentiation, and / or maintenance of identity of another cell. Feeder cells can support the growth of target cells in culture by providing a complex mixture of extracellular matrix (ECM) components and growth factors. In some cases, feeder cells may be non-dividable, i.e., cell growth may have stopped. The growth of feeder cells can be arrested by, for example, any suitable method known in the art. The growth of feeder cells can be arrested by chemical fixation (e.g., chemical fixation with mitomycin-C or glutaraldehyde). The growth of feeder cells can be arrested by physical methods (e.g., gamma irradiation, x-ray irradiation, or electrical pulses). For example, feeder cells used in the co-culture of target cells such as embryonic stem cells (ESCs) can be fibroblasts with inactivated mitosis, thereby maintaining survival. In some cases, target cells can grow in the presence of dividable feeder cells. Some living feeder cells (e.g., human fibroblasts) can also become target cells such as in the case of induced pluripotent stem cells (iPSCs) by reprogramming. Feeder cells can be, for example, non-dividable arrested feeder cells. The selection of feeder cells can depend on the target cells. Feeder cells can be, for example, non-arrested fibroblasts. Feeder cells can be arrested fibroblasts, epithelial cells, mesenchymal cells, muscle cells, stromal cells, spleen cells, or amniotic cells. Fibroblasts can be, for example, human dermal fibroblasts, 3T3 fibroblasts, human fetal fibroblasts, mouse fetal fibroblasts, and the like. Epithelial cells can be, for example, human mature fallopian tube epithelial cells, human amniotic epithelial cells, HeLa cells (human cervical cancer epithelial cells), and the like. Mesenchymal cells can be adipose-derived mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells, human bone marrow-derived mesenchymal cells, human amniotic mesenchymal stem cells, and the like. Stromal cells can be, for example, human bone marrow stromal cells or mouse bone marrow stromal cells. Amniotic cells can be, for example, human amniotic cells or mouse amniotic cells.

[0065] The term "target cell" refers to the cells for the automated cell culture applications of the present disclosure, and for example, refers to embryoid bodies, organoids, tumor organoids, spheroids, stem cells, or production cell lines. In some embodiments, the target cells are embryoid bodies, organoids, and / or other multicellular bodies. The organoid can be a brain organoid. The brain organoid can be a cerebral organoid. The organoid can be a liver organoid. In some embodiments, the target cells can be stem cells. The stem cells can be human induced pluripotent stem cells (hiPSCs). In some embodiments, the target cells can be production cell lines. The target cells can be derived from a target tissue. The target tissue can be a mammalian primary tissue, or an organoid, or a tumor organoid. The mammalian tissue can be derived from a patient biopsy sample. The target tissue can be derived from target organs such as, for example, the lungs, the intestines such as the small intestine, the colon, the stomach, the pancreas, the liver, the kidneys, the skin, the bone marrow, the blood-brain barrier, the brain, the heart, and the like.

[0066] Organoids, spheroids, tumor organoids, and three-dimensional (3D) cell culture models are useful in many applications such as disease modeling and regenerative medicine. 3D cell models such as organoids and spheroids can be useful for understanding complex biology in physiologically relevant contexts because cells often retain their natural shape and proper spatial orientation in aggregates or spheroids, etc., while 2D models of cells grown in sheets or monolayers may not be as successful. Gene and protein expression in 3D cell culture can more closely mimic gene and protein expression. For example, 3D cell culture can be useful for drug target identification, lead compound identification, compound optimization, preclinical proof-of-concept, solid tumor modeling, genetic disease modeling, drug discovery, precision medicine, organ-on-a-chip, and bioprinting.

[0067] The term "spheroid" refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate composed of one or more cell types that can grow and proliferate and exhibit enhanced physiological responses, but do not undergo differentiation or self-organization. Common cell sources for spheroids are primary tissues or immortalized cell lines. Spheroids can bridge the gap between monolayers and complex organs.

[0068] The term "organoid" refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate composed of one or more cell types, in which the cells spontaneously self-organize into appropriately differentiated functional cell types and progenitor cells similar to their in vivo counterparts. Organoids mimic their corresponding in vivo organs. Organoids can be derived from pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), neonatal tissue stem cells, embryonic stem cells (ESCs), adult stem cells, or primary tissues. Organoid cultures can be made to resemble much of the complexity of organs and are thus useful for studying the etiology and treatment of diseases. Organoid technology has emerged in recent years as an essential tool for both basic and biomedical research. Organoid cultures can be selected from various types of target organs. Organoids can be, for example, brain organoids, liver organoids, lung organoids, intestinal organoids (e.g., small intestine organoids), colon organoids, stomach organoids, pancreatic organoids, kidney organoids, skin organoids, heart organoids, bone marrow, the blood-brain barrier, and the like. In the present disclosure, the organoid can be a brain organoid. In some examples, the organoid can be a liver organoid.

[0069] The term "brain organoid" refers to a 3D tissue model that represents one or more regions of the brain. Brain organoids can be, for example, cerebral organoids, cortical organoids, forebrain organoids, ventral organoids, ventral forebrain organoids, thalamic organoids, hypothalamic organoids, midbrain organoids, hindbrain organoids, cerebellar organoids, medial ganglionic eminence (MGE) organoids, neuromuscular organoids, and the like. Forebrain organoids can include cortical organoids, ventral forebrain organoids, and thalamic organoids. Hindbrain organoids can include cerebellar organoids.

[0070] Brain organoids can be produced using induction protocols and non-induction protocols, as described, for example, in Kim et al., 2021, iScience 24, 102063, February 19, 2021 (incorporated herein by reference in its entirety). Non-induction protocols rely on endogenous signaling and do not use specific chemicals or inhibitors. For example, cerebral organoids can self-organize and naturally differentiate from hPSCs without the use of exogenous factors and in the presence of an extracellular matrix (ECM) hydrogel such as Matrigel. The ECM hydrogel can serve a function of supporting 3D structure and expansion of neuroepithelium. In the induction method, exogenous signaling factors or inhibitors such as signaling activators are used to generate region-specific brain organoids. For example, exogenous signaling factors can include various combinations of SMADi, SHH, insulin, BMP7, MAPK / ERKi, Wnt, FGF2, FGF8, FGF19, SDF1, Noggin, rhDkk1, SB431542, LDN193189, XAV939, WNT3A, purmorphamine, and the like.

[0071] The structure, transcriptome, and function of brain organoids can be assayed in multiple ways. For example, the structure can be assayed by immunostaining, tissue clearing, or an imaging system. Transcriptome evaluation can include qPCR, bulk RNAseq, or scRNAseq methods. Function evaluation can include Ca2+ imaging, microelectrode array (MEA), or patch clamp methods.

[0072] When cultured, human induced pluripotent stem cells (hiPSCs) mature over time and differentiate into various nerve cells that resemble the structures of various brain regions. 3D cerebral organoids are a developing technology that can be used to understand human brain development and nervous system diseases and can be used to test the effects of compounds and genetic mutations. Brain organoids can be cerebral organoids. Cerebral organoids can be grown based on the Lancaster and Knoblich protocol (2014). Lancaster and Knoblich, Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014 October; 9(10):2329-2340 (incorporated herein by reference in its entirety).

[0073] The term "stem cell" refers to undifferentiated cells that have the potential to develop into many different cell types that perform various functions. Pluripotent stem cells, such as those found in embryos, can give rise to all types of cells, such as cells of the brain, bone, heart, and skin. Some human mature cells can be reprogrammed into a state similar to embryonic stem cells called induced pluripotent stem cells (iPSCs). iPSCs can be human iPSCs. For example, pluripotent stem cells found in adult or neonatal umbilical cords are cells that can develop into cells that make up the organ system from which this cell originated. When grown under certain cell culture conditions, pluripotent stem cells can remain undifferentiated. To generate differentiated cells, the chemical composition of the culture medium can be changed, the surface of the culture dish can be altered, or the cells can be modified by forcing the expression of certain genes.

[0074] The term "embryoid body" (EB) refers to a three-dimensional aggregate of pluripotent stem cells. EBs can be formed in suspension by pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). EB differentiation can be used as a common platform for generating specific cell lineages from PSCs. Many EB formation protocols utilize components such as fetal bovine serum (FBS) or knockout serum replacement (KOSR), or albumin products. Many human PSCs cannot survive in suspension unless they are in aggregates or under ROCK inhibitor treatment (e.g., Rock Inhibitor, Tocris).

[0075] A method for culturing three-dimensional brain organoids from human iPSCs using an automated system, the method including the automated embedding of embryoid bodies into a hydrogel, is provided. In some examples, iPSCs can be grown and separated into single cells, and on days 0 to 4, they can be re-aggregated in a medium such as hES medium to form embryoid bodies (EBs). The cells can be monitored, for example, by imaging with respect to EB formation and germ layer differentiation. On days 4 to 8, the EBs are transferred to a 96-well U-bottom plate, and the medium is exchanged with a medium such as a neural induction medium. Nutrition supplementation and monitoring are carried out. After induction of the neuroectoderm at about days 8 to 9, the EBs can be transferred to a separate well microplate and embedded in a hydrogel such as Matrigel. A medium such as a differentiation medium can be added. The differentiation medium may initially not contain vitamin A (retinoic acid). After the progression of the expansion of neuroepithelial buds, the medium exchange can include a differentiation medium containing vitamin A (retinoic acid). On days 10 to 50, or around that time, the organoids can be maintained and, if necessary, compounds can be added. The separate well microplate can be transferred to a nutrition supplementation incubator equipped with a tilting function and a monitoring ability. This nutrition supplementation incubator can be, for example, a large nutrition supplementation incubator having a capacity of 10 to 1000 plates, 20 to 800 plates, or larger. On days 50 to 51, the brain organoids can be assayed using a high-throughput cell screening instrument such as the FLIPR Penta High-Throughput Cellular Screening System (Molecular Devices LLC). This cell screening instrument can enable high-throughput screening of brain organoids for toxicology and the identification of lead compounds. This cell screening instrument can include an internal plate handler, a camera, a pipetter head, a heated stage, lenses for excitation and emission of fluorescent labels, and a computer controller.

[0076] Using an automated liquid handling system, automated seeding of EB and addition of hydrogel droplets for embedding EB are enabled, followed by automatic addition and replacement of the culture medium. Imaging in transmitted light was used to monitor the development of organoids. Machine learning-based image analysis then enabled the detection of organoids and the characterization of their size and density. For endpoint measurements, the organoids could be stained with fluorescently labeled antibodies or viability dyes and imaged using an automated confocal imaging system. Advanced image analysis is enabled by the characterization of the size and complexity of the organoids, the morphology and viability of the cells, and the 3D reconstruction and complex phenotypic evaluation of the organoid structure, such as the presence of differentiation markers.

[0077] The method of the present disclosure utilizes a separated well microplate having a well shape including a main well and a secondary well connected by at least one channel. For example, the culturing method of the present disclosure may utilize a separated well microplate having a plurality of well units each including a main well and a secondary well connected by at least one channel. For example, each well unit may include the well shape shown in FIG. 1A, including a main well fluidly connected to the secondary well via at least one channel. The separated well microplate is described in U.S. Patent Application No. 17 / 580,193, titled "MICROPLATES FOR AUTOMATING ORGANOID CULTIVATION," filed on October 22, 2021, and PCT International Application No. PCT / IB2021 / 062356, titled "MICROPLATE WELLS FOR CELL CULTIVATION," filed on December 27, 2021, each of which is hereby incorporated by reference in its entirety.

[0078] The size and morphology of developing brain microtissues can be monitored using an automated transmission light microscope. The size, shape, and density of the tissues can be defined using an AI-based image analysis (IN Carta software) approach. For example, the microtissues can be analyzed at various stages of development using confocal imaging by the expression of Sox2 marker, TuJ1 marker, and GFAP marker, or other markers using, for example, antibody fluorescent dye conjugates. The organoids can be fixed and stained with Hoechst (a blue fluorescent dye used to stain DNA) and Sox2 (radial glia). The analysis of brightfield images can be performed using deep learning-based segmentation (SINAP). The maturation of the organoids can be monitored using brightfield imaging and analyzed using IN Carta SINAP. For example, a custom deep learning model can be trained and then applied to a dataset. The SINAP model enables the segmentation of organoids with various shapes and sizes.

[0079] Figure 1A shows an example of a well unit 100 of a separated well microplate that can be used in connection with the methods of the present disclosure. Figure 1A shows an exemplary view of a well unit 100 of a microplate that can be used for the growth, culture, monitoring, and assay of embryoid bodies, fused embryoid bodies, spheroids, organoids, or other multicellular bodies, according to various embodiments of the present disclosure. For example, a single well unit 100 of a separated well microplate is useful for horizontal co-culture for cell / organoid nourishment. The separated well microplate used in the methods according to the present disclosure may include a plurality of well units, and each well unit includes a main well 112 separated from a secondary well 115 by a common sidewall, and the main well and the secondary well may be fluidly connected by at least one microchannel 118. In some embodiments, at least one microchannel 118 may include a removable barrier for separating the main well from the secondary well. In some embodiments, at least one microchannel 118 may be opened to enable media exchange between the main well 112 and the secondary well 115. The well unit 100 of the separated well microplate includes a main well 112 that may be fluidly connected to the secondary well 115 by at least one microchannel 118. In some examples, the main well 112 may be used as a culture well, and the secondary well 115 may be used as a nourishment well. The microchannel 118 may include a removable barrier. At least one microchannel 118 may be closed or opened by a removable barrier. One or more microchannels 118 between the main well 112 and the secondary well 115 may be closed by a removable barrier, for example, closed by an air gap, a hydrogel seal, or a silicone seal. One or more microchannels 118 between the main well 112 and the secondary well 115 may be closed, for example, by an air gap. This air gap may be formed by, for example, air bubbles.

[0080] Figure 1B shows a well unit 100 of another example of a separation well microplate that can be used in connection with the method of the present disclosure. In Figure 1B, the well unit 100 includes a main well 112 and a secondary well 115 that can be fluidly connected by at least one open microchannel 118. When open, due to the shape of at least one channel 118, exchange of medium, expressed proteins such as hormones, nutrients, growth factors, waste products, and debris can occur between the main well and the secondary well, while at the same time, target cells such as organoids are retained in the main well 112 and feeder cells are retained in the secondary well 115. The dimensions of at least one microchannel 118 can prevent multicellular target cells (e.g., organoids, tumor organoids, etc.) from passing through the microchannel 118. In some examples, the shape of at least one microchannel 118 can include a slit structure having a channel width of 150 - 500 microns or greater, or 200 - 400 microns, or about 300 microns × 10 - 100 microns, or a height of 10 - 75 microns. During the method of the present disclosure, at least one microchannel 118 between the main well and the secondary well may be closed by a removable barrier, or may be opened to allow exchange of medium, secreted factors, hormones, growth factors, nutrients, waste products, and debris between the main well 112 and the secondary well 115, for example, by gravity feed while rocking the separation well microplate. In some examples, at least one microchannel can be opened by removing a removable barrier such as removing an air gap using a pipette as a pump (e.g., pressing the pipette tip against the secondary well to force liquid flow between the two wells).

[0081] Figure 1C shows a well unit 100 of a further example of a separation well microplate that can be used in connection with the method of the present disclosure. This well unit 100 includes a main well section 112, a secondary well section 115, at least one microchannel 118, and an embryoid body 103 embedded in a suspended hydrogel 111 disposed within the main well section 112 of the well unit 100. The main well 112 and the secondary well 115 contain a culture medium 109. According to various embodiments, the well unit 100 may further include a bottom layer sheet 121 disposed on the lower side of the well plate body of the microplate. The bottom layer sheet 121 is attached to the lower side of the well plate body that forms the bottom surface of the well unit 100. In various examples, the bottom layer sheet 121 includes an optically transparent observation window to enable imaging of embryoid bodies or organoids, or other cell cultures being cultured in the well unit 100 of the microplate, as can be understood. This observation window can be a window suitable for microscopic observation, whether in bright field, phase contrast, fluorescence, confocal, two-photon, or any other microscopy imaging modality known in the art. In some examples, the bottom layer sheet 121 may include a gas permeable sheet configured to increase the oxygen supply to embryoid bodies and organoids, or other cell bodies growing within the well unit 100 of the microplate. This gas permeable sheet can be formed from a material including polytetrafluoroethylene (PTFE), PEFP, polyimide, polydimethylsiloxane (PDMS), polycarbonate, and / or other materials, as can be recognized. According to various examples, this gas permeable sheet can have a thickness of about 5 to 70 microns. According to various examples, this gas permeable sheet may include a plurality of pores. In other examples, this gas permeable sheet can allow the passage of molecules by diffusion. Alternatively, this gas permeable sheet can include some other thickness, pore diameter, and pore density.

[0082] The secondary well section 115 can be used to grow feeder cells and supply nutrient medium and / or other nutrients, which can be used to supply nutrients to the growing cell aggregates placed within the primary well section 112. The secondary well section 115 can be regarded as a supply well containing nutrient medium and / or other nutrients that can be used by the cell culture growing in the primary well section 112. The secondary well section 115 can be sized and shaped to hold a fluid that can be exchanged with the primary well section 112 according to various embodiments of the present disclosure.

[0083] According to various embodiments, the sizes and shapes of the primary well section 112 and the secondary well section 115 can be different from each other. In some examples, the primary well section 112 is larger (in terms of dimensions such as diameter, cross-sectional area, or volume) than the secondary well section 115. In other examples, the secondary well section 115 is larger than the primary well section 112. In some examples, the primary well section 112 includes a shape different from the shape of the secondary well section 115.

[0084] According to various examples, the well unit of the separated well microplate includes at least one microchannel 118 sized and shaped to prevent an object having a certain size (e.g., larger than about 25 microns (μm)) (such as diameter, height, width, etc.) from passing from one well section to another well section.

[0085] In some examples, the height of at least one microchannel 118 can be sized from about 10 microns to about 100 microns, or from about 10 microns to about 75 microns, or from about 10 microns to about 25 microns. The shape of at least one microchannel 118 can include a channel width of 150 - 500 microns or greater, or 200 - 400 microns, or about 300 microns × 10 - 100 microns, or a slit structure having a height of 10 - 75 microns. For example, the height of at least one microchannel 118 can be such that an object (e.g., embryoid body, organoid, organoid fragment, etc.) sized about 25 μ or greater present within the main well section 112 is prevented from moving into the secondary well section 115. The microchannel 118 can be closed, for example, by an air gap, a hydrogel seal, or a silicone seal. The microchannel 118 can be opened, for example, by removing the air gap or removing the hydrogel seal or silicone seal.

[0086] In some embodiments of the present disclosure, the main well 112 can be utilized as a culture chamber. First, at least one channel 118 can be closed, and for example, EBs can be cultured within the main well 112 of a separation well microplate without mixing, for example. Optionally, feeder cells can be cultured and attached to the bottom in the secondary well 115 of the separation well microplate without mixing, for example. After the feeder cells have attached to the secondary well, at least one channel 118 that is optionally closed can be opened. The separation well microplate can be placed on a rocker to allow for the exchange of medium, hormones, growth factors, nutrients, waste products, and debris by gravity feed. In some examples, feeder cells can be cultured in the secondary chamber 115, thereby enabling the EBs embedded in the hydrogel cultured in the main chamber 112 to be nutrient-supplemented through at least one open channel.

[0087] Figure 2 shows a workflow method of embryoid body embedding that shows a single well unit of a separation well microplate over time, as shown in panels 200 - 210. First, in a single well unit 200, the differentiated embryoid body 203 is pipetted into the main well 212 of a separation well microplate containing the first medium 209. Second, in the same single well unit 202, the plate is tilted at an angle (e.g., about 10 degrees) to move the embryoid body 203 to the corner of the main well near the channel 218. Third, in the same single well unit 204, the plate is repositioned to the horizontal position, and the first medium 209 is gently removed from the secondary well 215 (nutrient supply well) by the pipette 220. Fourth, in the same single well unit 206, the plate is tilted (about 10 degrees), and a liquid hydrogel such as cold liquid Matrigel 211 is gently added to the main well 212 at the corner near the channel 218 by the pipette 220 to encapsulate the embryoid body 203. The plate is held for about 5 minutes to return to room temperature and then transferred to an incubator for about 30 minutes to solidify the Matrigel 209. Fifth, in the same single well unit 208, after the Matrigel 209 is solidified, the second medium 213 is added from the secondary well 215 (nutrient supply well) to suspend the embryoid body 203 embedded in the Matrigel 211. Sixth, in the same single well unit 210, the plate containing the embryoid body 203 embedded in the Matrigel 211 is placed in an incubator, and the second medium 213 is periodically replaced using the secondary well 215 (nutrient supply well) to avoid material loss. The first medium and the second medium may be different media. The first medium and the second medium may be the same medium. The first medium and the second medium may differ in one or more components, or two or more components. The medium may be a liquid medium at any working temperature range (e.g., cold temperature, room temperature, incubation temperature).

[0088] Regular automated medium exchanges can be performed, for example, every 24 - 72 hours, every 30 - 60 hours, every 36 - 54 hours, or about every 36 hours, about every 48 hours, or about every 54 hours. In some examples, the medium exchange can be performed about every 48 hours.

[0089] As shown in FIG. 2, target cells in the form of multicellular bodies 203 (e.g., embryoid bodies) can be embedded in a hydrogel 211 (e.g., Matrigel®) by adding a liquid hydrogel (e.g., cold liquid hydrogel) onto the embryoid bodies in the main well 212 to encapsulate the embryoid bodies 203. At this stage, the hydrogel (e.g., Matrigel®) is typically crescent - shaped. According to various embodiments, a second liquid medium 213 containing appropriate growth factors, additives, and nutritional supplements for generating the desired multicellular bodies and / or for stimulating this growth is added. This liquid medium 213 can be added via the open channel 218 through the secondary well 215.

[0090] In the formation of EBs, a common approach for differentiating iPSCs into various cell types can be utilized.

[0091] Prior to automatic embedding into the hydrogel, embryoid bodies can be formed by any suitable method. Heterogeneous methods can include stirred flask culture, liquid suspension culture, rotating cell culture system (RCCSS). Homogeneous methods can include low attachment U-bottom multiwell plates, hanging drop culture, solid micro surfaces with irregularities (Aggrewell (trademark)), or other methods. Generation of embryoid bodies from iPSCs can include an in vitro approach for pluripotency assessment. EB is a three-dimensional aggregate of cells that can differentiate to show the three germ layers. To evaluate the pluripotency of hiPSCs for forming EB, downstream evaluations can be performed to demonstrate the ability to form representative germ layers. The evaluation can include, for example, histological analysis of EB, which can include assessment of tissue differentiation by investigation of tissue organization, cell morphology, and / or local protein expression, or assessment of tissue differentiation by expression of germ layer-specific genes, for example, to distinguish the presence of germ layer-specific gene markers and the simultaneous loss of pluripotency gene markers.

[0092] The methods of the present disclosure can utilize one or more, two or more, three or more, or four or more different media to generate embryoid bodies and brain organoids. The media can be selected from any suitable media known in the art. For example, the media can be as described in Lancaster and Knoblich, Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014 October; 9(10):2329-2340 (incorporated herein by reference in its entirety).

[0093] The medium can be any suitable medium known in the art. In some examples, the medium can be selected from an hES medium, a neural induction medium, a cerebral organoid differentiation medium without vitamin A (retinoic acid), and a cerebral organoid differentiation medium containing vitamin A (retinoic acid). This medium can be based on, for example, minimal essential media such as DMEM medium, Essential Basal Media, Essential 6 Media, commercially available from Invitrogen or Life technologies.

[0094] The medium can be an hES medium. For example, the hES medium can be used for generating embryoid bodies up to the stage of germ layer differentiation from human PSCs. The hES medium can contain DMEM-F12 (Dulbecco’s Modified Eagle Media / F12, (Thermo Fisher Scientific), KOSR, FBS, Glutamax (e.g., Gibco(®) GlutaMAX(®) nutritional supplement, an L-alanyl-L-glutamine dipeptide substitute for glutamine, Thermo Fisher Scientific), MEM-NEAA (Minimum Essential Medium - Non-Essential Amino Acids, Thermo Fisher Scientific), and can have 2-mercaptoethanol, bFGF (fibroblast growth factor - basic, e.g., FGF-basic(143 - 288), ACROBiosystems) added immediately before use. The hES medium can be prepared as described, for example, in Lancaster and Knoblich 2014.

[0095] The medium can be a neural induction medium. For example, the neural induction medium can be used for inducing neuroectoderm in EBs. The neural induction medium can contain DMEM-F12 containing N-2 nutritional supplement (e.g., 1 vol / vol%) (serum-free nutritional supplement based on Bottenstein’s N-1 formulation, Thermo Fisher Scientific), Glutamax (e.g., 1 v / v%), MEM-NEAA (e.g., 1 v / v%), and heparin (e.g., 1 μg / mL).

[0096] The medium can be, for example, a cerebral organoid differentiation medium containing DMEM-F12 medium, Neurobasal™ medium (Thermo Fisher Scientific), N-2 supplement, insulin, MEM-NEAA, penicillin-streptomycin, Gibco™ B27 supplement (optimized serum-free neuronal cell culture supplement, Thermo Fisher Scientific), and / or 2-mercaptoethanol. The differentiation medium can be used without vitamin A (retinoic acid) or together with vitamin A (retinoic acid).

[0097] The term "hydrogel" refers to an extracellular matrix useful for culturing organoids. Hydrogels include, for example, mouse EHS sarcoma matrices commercially available as Matrigel (Corning), Cultex (Trevigen), Geltrex (Gibco), type I collagen, fibrin, hyaluronic acid (HA), gelatin methacrylate (GelMA), decellularized matrices, or biopolymers such as alginate, silk, nanocellulose; artificial materials such as polyethylene glycol (PEG), self-assembling peptides such as RADA16 / PuraMatrix bQ13, poly(lactic / (co)glycolic) acid, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptide, ELP (elastin-like protein), or combinations of these polymers. The hydrogel can be Matrigel. Corning Matrigel® matrix is a soluble basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins including laminin (major component), collagen IV, heparan sulfate proteoglycan, entactin / nidogen, and several growth factors. The hydrogel can be Corning® Matrigel® Growth Factor Reduced (GFR) basement membrane mix. The hydrogel can be a collagen hydrogel. The hydrogel can be a type I collagen hydrogel. For example, the hydrogel can be a rat tail type I collagen scaffold (ibibi GmbH, Germany). The collagen scaffold can be 1 - 5 mg / mL, or 2 - 2.5 mg / mL.

[0098] The hydrogel can be an animal-free hydrogel. The animal-free hydrogel can include a polysaccharide matrix. The hydrogel can include a cellulose matrix. The hydrogel can include cellulose fibers. The cellulose can be nanofibrillated cellulose. The hydrogel can have cellulose fibers that are neutral. The hydrogel can have anionic cellulose fiber charges. The hydrogel can be GrowDex® animal-free hydrogel (UPM Biomedicals). The hydrogel can be a transparent hydrogel. The hydrogel can include a working concentration range of 0.1 to 1.5 wt% or 0.2 to 1.0 wt%. The hydrogel can be a liquid hydrogel. The liquid hydrogel can be a cold liquid hydrogel.

[0099] The animal-free hydrogel can include a self-assembling peptide (SAP). The SAP can be PuraMatrix® synthetic peptide hydrogel. The SAP can be, for example, RADA16 peptide or RLDL-16 peptide. RADA16 is H-(ArgAlaAspAla)4-OH. Spontaneous and reversible self-assembly of RADA16 molecules occurs in an acidic solution (pH 2 to 4, pH less than about 3.7), generating nanofibers. The chemical structure of RADA16 has 16 amino acids with a sequence of 4 amino acids repeated continuously. It includes R residues (positively charged arginine), A residues (hydrophobic alanine), and D residues (negatively charged aspartic acid). The SAP can be RLDL-16 which is H-(ArgLeuAspLeu)4-OH. Extracellular matrix-like fibers form a viscous and transparent aqueous solution at a low concentration, for example, about 0.1 to about 2.5 wt%.

[0100] Hydrogels can be liquid hydrogels under certain conditions. For example, hydrogels such as Matrigel maintain a liquid state at low temperatures below room temperature, or at 0 - 10 °C, 1 - 8 °C, or approximately 4 °C. When warmed to room temperature or incubation temperature, Matrigel solidifies. Regarding self - assembling peptides, under neutral conditions, for example, RADA16 is mainly fibrous, and these fibers consist of stacked beta - pleated sheets. Under acidic conditions, RADA16 fibers decompose into a single layer.

[0101] In some methods of the present disclosure, biocompatible oils can be utilized. Biocompatible oils can be useful for making the suspended hydrogel encapsulating the embryoid bodies more spherical. Biocompatible oils can be useful for the transport of the suspended hydrogel encapsulating the embryoid bodies.

[0102] Biocompatible oils typically have a density different from that of water. Biocompatible oils can have a density different from that of water. Biocompatible oils can have a density higher or lower than that of water.

[0103] Biocompatible oils can have a density higher than that of water. In some examples, this biocompatible oil can be a halogenated hydrocarbon oil. This halogenated hydrocarbon oil can be a fluorinated hydrocarbon. In some examples, the biocompatible oil can be 1 - methoxyheptafluoropropane (e.g., HFE7000 or Novec™ 7000, 3M), or 3 - ethoxy - 1,1,1,2,3,4,4,5,5,6,6,6 - dodecafluoro - 2 - trifluoromethyl - hexane (e.g., Novec™ 7500, 3M).

[0104] Fluorinated hydrocarbons can be perfluorocarbons. The term "perfluorocarbon" refers to an organic fluorine compound containing only carbon - fluorine and C - C bonds, as well as potential functional groups, heteroatoms, and associated hydrogen atoms. Common functional groups can include - OH, - CO2H, - Cl, O, and SO3H.

[0105] The perfluorinated hydrocarbon may have a density (d) at 25 °C greater than 1, 1.2, 1.3, or 1.5, or may be in the range of 1.1 to 2.0, 1.2 to 1.95, or 1.5 to 1.95. The perfluorinated hydrocarbon may be a 3M (trademark) Fluorinert (trademark) FC perfluorinated compound (3M Company). The Fluorinert FC compound may be Fluorinert FC-40, a perfluorinated compound, C5-C18 (CAS RN 86508-42-1), density (d) 1.85 g / cm 3 , Fluorinert FC-70 (CAS RN 338-84-1), tris(undecafluoropentyl)amine, d 1.94 g / cm 3 , Fluorinert FC-770, d 1.79 g / cm 3 , Fluorinert FC-43 (CAS RN: 311-89-7) perfluorotributylamine, d 1.86, and the like. In some embodiments, the perfluorinated hydrocarbon has a kinematic viscosity at 25 °C greater than 0.7 cSt, 1.1 cSt, or 2.0 cSt.

[0106] In some examples, the biocompatible oil has a boiling point greater than 37 °C or greater than 40 °C. The biocompatible oil may have a density lower than that of water. The biocompatible oil having a density lower than that of water may be, for example, silicone oil or mineral oil. Silicone oil is a term used to describe a group of hydrophobic polymers and monomeric compounds containing silicon-oxygen bonds and is also referred to as organosiloxane. Silicone oil may be any liquid polymeric siloxane having an organic side chain. For example, silicone oil may be polydimethylsiloxane (d 0.96 g / cm 3) can be. The silicone oil can be a cyclic siloxane such as decamethylcyclopentasiloxane (d 0.96 at 20 °C) or cyclomethicone D6 (d 0.98). The side chains can include not only methyl but also phenyl groups, vinyl groups, and / or trifluoropropyl groups. The differences in silicone oils can include molecular weight (MW), linear chain length, side chain type, chain ends, and chain size distribution. Silicone oils with high viscosities may have a low emulsification tendency. Barca et al., 2014, Silicone oil: different physical properties and clinical applications, BioMed Res Int, article id 502143. The silicone oil can have a viscosity in the range of about 5 to 10,000 cSt, 100 cSt to 5,000 cSt, or about 1,000 cSt to about 5,000 cSt at 25 °C. The biocompatible oil can be an oil for tissue culture, for example, liquid paraffin oil, white mineral oil (e.g., ORIGIO liquid paraffin for tissue culture, CooperSurgical®). The density of white mineral oil is 0.8 - 0.87 g / cm 3 is. In some examples, the biocompatible oil can be a vegetable oil. The vegetable oil can be, for example, olive oil, corn oil, argan oil, camelina oil, or a mixture thereof. Vegetable oils and mixtures are described, for example, in Said et al., 2013, Can J Physiol Pharmacol 91: 812 - 817. Vegetable oils can be commercially available, for example, from Sigma - Aldrich, St. Louis, Missouri, USA.

[0107] The embodiments described in one aspect of the present disclosure are not limited to this described aspect. This embodiment can also be applied to different aspects of the present disclosure as long as these aspects of the present disclosure do not prevent it from operating for its intended purpose.

Examples

[0108] (Example 1) Embedding of embryoid bodies using a separated well microplate In this example, automatic embedding of embryoid bodies into a hydrogel was performed. The embryoid bodies were prepared according to the Lancaster and Knoblich protocol (2014) and then subjected to the automated method of the present disclosure outlined in FIG. 5.

[0109] As outlined in FIG. 5, the generation of EBs from human iPSCs was initiated on day 0 using hES medium in an automated stem cell culture plate, the medium was exchanged, and the development of the EBs was monitored by imaging for germ layer differentiation from day 0 to day 4.

[0110] Briefly, from day 0 to day 4, automated stem cell culture was started from induced pluripotent stem cells (iPSC-derived cells) grown in 2D culture to approximately 70-80% confluence using an exemplary 6-well culture plate in hES medium. The colonies were disrupted by short exposure to enzymes and EDTA (e.g., dispase, wash, then trypsin / EDTA, wash, and resuspend) to obtain a cell suspension. The EBs were fed and exposed to a first medium (e.g., hES medium containing a ROCK inhibitor (1:100) and low bFGF 4 / ng / mL). The medium was exchanged regularly. The development of the cells was monitored by imaging. In some examples, when the diameter of the EBs was about 350-600 μm, the medium was exchanged to hES without a ROCK inhibitor. When the diameter of the EBs was about 500-600 μm, the appearance of the EBs became bright and the edges became smooth, indicating germ layer differentiation.

[0111] On about day 4, single cells were transferred to a low-adhesion U-bottom 96-well plate. From day 4 to day 8, the medium was exchanged to a neural induction medium, and this neural induction medium was exchanged regularly. The cells were monitored by imaging for the induction of neuroectoderm. On about day 8-9, the outside of the EBs became bright, indicating neuroectoderm differentiation. The resulting differentiated EBs were transferred by automatic pipetting to the main wells of a separated well microplate for embedding in cold liquid hydrogel (Matrigel).

[0112] The automatic embedding of EBs was performed as outlined in FIG. 2. On approximately the 8th to 9th day, the differentiated embryoid bodies (EBs) 203 were transferred by a pipette arm to the main wells 212 of a separation well microplate 200 containing the first medium 209, and during differentiation, the EBs were nourished by periodic automated medium exchanges through the secondary wells 215 via the microchannels 218. When the preparation for the embedding of the embryoid bodies 203 was complete, the plate 202 was tilted for several seconds to position the organoids near the channel 218 (the central wall in the figure). Then, the plate 204 was repositioned to its normal flat position, and the first medium 209 was removed from the nourishing well. When the medium was depleted from the well, the plate 206 was tilted again, and cold Matrigel 211 was added by a pipette arm at the corner near the EB 203 and the channel 218. After 5 minutes at room temperature, the plate was transferred to an incubator at 37° C. for approximately 30 minutes. When the Matrigel solidified, the second medium 213 (a cerebral organoid differentiation medium without vitamin A) was added from the secondary (nourishing) well 215 via the channel 218 to resuspend the solidified hydrogel encapsulating the embryoid bodies in the second medium 213 of the separation well microplate 210. The separation well microplate was transferred to a large nourishing incubator equipped with a tilting function and an automated medium exchange (a cerebral organoid differentiation medium containing vitamin A) and monitored by imaging over 10 - 50 days. The resulting cerebral organoids were ready for compound testing, including the addition and assay of compounds on the 50th - 51st day, using image analysis by high-throughput cell screening equipment such as, for example, an ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices LLC) or a FLIPR Penta High-Throughput Cellular Screening System (Molecular Devices LLC).

[0113] Typically, the following steps were utilized for the automatic embedding of EBs into the hydrogel. 1. Pipette the embryoid bodies into the main well of the separation plate and tilt the plate to move the embryoid bodies into the separation channel. 2. Remove the medium through the nutrient replenishment well in a flat position. 3. Tilt the plate and add liquid Matrigel to the main well to encapsulate the embryoid bodies. 4. Warm the plate to 37 °C over 30 minutes to solidify the Matrigel. 5. Add medium through the nutrient replenishment channel to detach the embedded embryoid bodies from the bottom of the plate. 6. (Optional step) Tilt the plate in the other direction to release the embryoid bodies from the separation channel. 7. (Optional step) Add additional medium to nourish the embryoid bodies. 8. (Optional step) Transfer the embryoid bodies to a bioreactor for maturation.

[0114] Representative images of embryoid bodies appropriately differentiated into brain organoids are shown in Figure 3. From day 10, the organoids were embedded in Matrigel to support the expansion of the neuroepithelium. In this case, the aforementioned workflow was used (Figure 2). Note that on the third day after embedding, numerous epithelial buds were formed, indicating good differentiation.

[0115] (Example 2) Embedding of embryoid bodies using a separation well microplate and oil In this example, the automatic embedding of embryoid bodies into a hydrogel was performed using a biocompatible oil (e.g., Matrigel) with a different density from this hydrogel.

[0116] This procedure is similar to that shown in Figure 2, except that after transferring Matrigel onto the embryoid body by pipette and embedding the EB in Matrigel, a biocompatible oil with a density different from that of Matrigel was added to the main well by pipette to reform the Matrigel from a semi - lunar shape to a more spherical shape. Two biocompatible oils were tested. Fluorinert™ FC40 (a C5 - C18 perfluorinated compound, density 1.845 - 1.895 g / mL at 25 °C, b.p. 165 °C) with a density and specific gravity higher than that of water, and silicone oil with a density / specific gravity lower than that of water. The same pipette tip was placed inside the Matrigel, and this pipette tip was used to gently aspirate the Matrigel and the oil. As shown in Figure 4, the Matrigel was present as a separate layer on top of the pipette tip and on top of the more dense biocompatible oil. Gently, the oil containing the organoids and the Matrigel were pushed back into the well. Then, the plate was warmed to an incubation temperature of 37 °C to solidify the Matrigel. The oil was removed via the secondary well (feeder well), and the well was washed with medium at room temperature. As shown in the lower - right panel of Figure 4, additional medium was added to nourish the embryoid body. The separated - well microplate was transferred to a large - scale nourishment incubator with an inclination function for nourishing and maintaining the organoids and monitored over 10 - 50 days.

[0117] Typically, the following steps were utilized. 1. The embryoid body was placed in the main well of the separated - well plate. 2. The plate was tilted to move the embryoid body far from the separation channel (to the opposite side) so that the organoids were not lost during medium removal. 3. In a flat position, the medium was removed via the nourishment well. 4. The plate was tilted and cold liquid Matrigel was added to the main well at the corner to encapsulate the embryoid body. 5. Biocompatible oil was added to the main well to reform the liquid hydrogel from a semi - lunar shape to a more spherical shape. 6. Place the same pipette tip into Matrigel and gently aspirate the Matrigel and oil. 7. Then, the Matrigel is located on top of the tip, and the oil, due to its density, is located more inferiorly (Figure 4, lower middle panel). 8. Gently push back the oil and Matrigel containing the organoids into the main well. 9. Warm the plate to 37 °C for 30 minutes to solidify the Matrigel. 10. Remove the oil through the nutrient supply channel and then wash once with medium at room temperature. 11. Finally, add additional medium to nourish the embryoid bodies. 12. (Optional step) Transfer the embryoid bodies to a bioreactor for maturation.

[0118] (Example 3) Use of various hydrogels for embedding of embryoid bodies In addition to Matrigel® (Corning), in the methods of the present disclosure, GrowDex® , a plant-derived hydrogel, and collagen I hydrogel (rat-derived) were each utilized. Matrigel, the plant-derived hydrogel, and the collagen I hydrogel each exhibited similar behavior. Each of these hydrogels was less dense than the biocompatible oil and thus formed into spherical hydrogels when exposed to the biocompatible oil. Images of embryoid bodies within the hydrogels on top of the biocompatible oil in a pipette tip are shown in FIGS. 6A-6C. FIG. 6A shows a cerebral embryoid body embedded in a spherical Matrigel® hydrogel in the upper Fluorinert™ FC-40 biocompatible oil in a pipette tip. FIG. 6B shows a cerebral embryoid body embedded in a spherical GrowDex® cellulose hydrogel in the upper Fluorinert™ FC-40 biocompatible oil in a pipette tip. FIG. 6C shows a cerebral embryoid body embedded in a spherical collagen I (rat) hydrogel in the upper Fluorinert™ FC-40 biocompatible oil in a pipette tip. The use of the biocompatible oil resulted in the EB being embedded in the spherical hydrogel in each of the hydrogels tested.

[0119] (Example 4) Embedding of embryoid bodies using standard 96-well plates and advanced pipetting protocols

[0120] In this example, automated embryoid body embedding with oil is performed with immediate transfer to the bioreactor. The following automated steps are performed. 1. Add embryoid bodies to the medium in the main wells of a separation well microplate. 2. Tilt this plate to move the embryoid bodies far from the separation channel (to the opposite side) so that the organoids are not lost during medium removal. 3. After repositioning this plate to a flat horizontal position, remove the medium through the nutrient replenishment wells. 4. Tilt this plate and add cold liquid Matrigel to the main well by pipetting at the corner to encapsulate the embryoid bodies. 5. Add a biocompatible oil to the main well to reform the hydrogel from a semi - lunar shape to a more spherical shape. 6. Place the same pipette tip into the Matrigel and use this pipette tip to gently aspirate the Matrigel and the oil. 7. Then, the Matrigel is located at the top of the tip, and the oil, due to its density, is located lower (Figure 4, lower - middle panel). 8. Transfer the EBs embedded in Matrigel and the oil to an oil pool warmed to 37 °C, which had a stream that carried the embedded organoids to the bioreactor. 9. When the Matrigel with the embryoid bodies (= future organoids) solidifies, empty the bioreactor containing the filter, wash it once to remove any remaining oil, and add the final medium. 10. (Alternative to Step 9) The organoids move into the fluid system and the organoids embedded in the oil stream come into contact with the normal medium. Due to the different densities, the oil is removed by the filter system. When the embedded organoids are located in the complete medium, they are redirected to the bioreactor.

[0121] Clause Article 1 A method for culturing organoids, comprising: adding embryoid bodies to a first medium in the main well of a separated - well microplate containing a plurality of well units, each well unit including a main well, a secondary well, and one or more microchannels connecting the main well and the secondary well; removing the first medium from the well unit through the secondary well; adding a liquid hydrogel to encapsulate the embryoid bodies; incubating the plate for a first period to solidify the hydrogel encapsulating the embryoid bodies; and Adding the second medium to the secondary well, detaching the solidified hydrogel encapsulating the embryoid bodies from the plate, and suspending it in the second medium A method comprising the steps of:

[0122] The method according to claim 1, wherein adding the embryoid bodies to the first medium in the main well comprises performing an automated pipetting operation.

[0123] The method according to claim 1 or 2, further comprising tilting the separation well microplate to move the embryoid bodies to the corner of the main well before removing the first medium, wherein tilting, optionally, comprises automatically tilting the separation well microplate.

[0124] The method according to claim 3, wherein tilting comprises moving the embryoid bodies towards the microchannel, and wherein tilting, optionally, comprises tilting the plate at an angle of 1 to 60 degrees, 5 to 45 degrees, 5 to 35 degrees, 5 to 25 degrees, 5 to 15 degrees, 7 to 12 degrees, or about 10 degrees from the horizontal position.

[0125] The method according to claim 3, wherein tilting comprises moving the embryoid bodies away from the microchannel, and wherein tilting, optionally, comprises tilting the plate at an angle of 1 to 60 degrees, 5 to 15 degrees, 7 to 12 degrees, or about 10 degrees from the horizontal position.

[0126] The method according to any one of claims 3 to 5, further comprising returning the microplate to the horizontal position before removing the second medium.

[0127] The method according to any one of claims 1 to 6, wherein adding the liquid hydrogel to encapsulate the embryoid bodies comprises performing an image-guided pipetting operation of the liquid hydrogel, and wherein the image-guided pipetting operation, optionally, is an automated image-guided pipetting operation comprising delivering the liquid hydrogel onto at least a portion of the embryoid bodies.

[0128] The method according to any one of claims 1 to 7, further comprising cooling the microplate before adding the liquid hydrogel, and optionally, the cooling is automatic cooling to a temperature below room temperature or within a temperature range of 4 to 8 °C.

[0129] The method according to any one of claims 1 to 7, further comprising heating the microplate to the incubation temperature before adding the liquid hydrogel, and optionally, the heating is automatic heating.

[0130] The method according to any one of claims 1 to 9, further comprising, after adding the second medium, further tilting the microplate to release the solidified hydrogel encapsulating the embryoid bodies from the microchannels, and optionally, the further tilting includes automatically further tilting.

[0131] The method according to any one of claims 1 to 10, wherein incubating to solidify the hydrogel occurs over a first period selected from the group consisting of about 5 minutes to about 90 minutes, about 10 minutes to about 60 minutes, about 20 minutes to about 40 minutes, and about 30 minutes, and optionally, the microplate is held at room temperature for a second period prior to the incubation step.

[0132] The method according to any one of claims 1 to 11, further comprising periodically replacing the second medium in the well unit through the secondary well to supply nutrients to the embryoid bodies.

[0133] The method according to any one of claims 1 to 12, further comprising introducing a biocompatible oil into the main well after adding the liquid hydrogel to encapsulate the embryoid bodies and before incubating, and optionally, the introducing includes automatic pipetting of the biocompatible oil into the main well.

[0134] The method according to claim 13, wherein introducing comprises placing a pipette tip within the hydrogel and aspirating into the pipette tip a hydrogel encapsulating the embryoid body and oil, and optionally, placing is image-guided placement.

[0135] The method according to claim 14, wherein aspirating is performed gently over a period of at least 0.3 seconds, at least 0.5 seconds, at least 0.7 seconds, or at least 1 second.

[0136] The method according to claim 14 or 15, further comprising pushing back from the pipette tip into the main well the oil and the hydrogel encapsulating the embryoid body.

[0137] The method according to claim 16, wherein pushing back is performed gently over a period of at least 0.3 seconds, at least 0.5 seconds, at least 0.7 seconds, or at least 1 second.

[0138] The method according to any one of claims 13 to 17, further comprising: removing biocompatible oil from the main well via a secondary well after solidifying the hydrogel encapsulating the embryoid body; and washing the well unit with a wash medium, optionally at room temperature, before adding the second medium. The method according to any one of claims 13 to 17, further comprising washing.

[0139] The method according to any one of claims 13 to 18, wherein introduction of biocompatible oil into the main well causes the hydrogel encapsulating the embryoid body to approach a spherical shape.

[0140] The method according to any one of claims 1 to 19, further comprising transferring the encapsulated embryoid body embedded in the solidified hydrogel to a bioreactor for maturation.

[0141] The method according to any one of claims 13 to 20, further comprising transferring a hydrogel encapsulating an embryoid body and a biocompatible oil to an oil pool warmed to an incubation temperature to solidify the hydrogel encapsulating the embryoid body.

[0142] The method according to claim 21, wherein the oil pool comprises a stream for transferring the hydrogel encapsulating the embryoid body to a bioreactor optionally containing a filter.

[0143] The method according to claim 22, further comprising discharging oil from the bioreactor, wherein the filter retains the solidified hydrogel encapsulating the embryoid body; washing the bioreactor and the retained solidified hydrogel encapsulating the embryoid body; and adding a third medium to the bioreactor to nourish the washed hydrogel encapsulating the embryoid body. The method according to claim 22, further comprising.

[0144] The method according to claim 22, wherein the stream of the oil pool transferring the hydrogel encapsulating the embryoid body into the bioreactor contacts the third medium, and the method comprises separating the mixture of the oil and the third medium into layers; and removing the oil layer through a filter system to retain the solidified hydrogel encapsulating the embryoid body in the third medium; and directing the solidified hydrogel encapsulating the embryoid body in the third medium to the bioreactor. The method according to claim 22, further comprising.

[0145] The method according to any one of claims 1 to 24, wherein the method is an automated method, and adding the embryoid body to the first medium, removing the first medium from the well unit, adding the liquid hydrogel, and adding the second medium each comprise an automated pipetting operation.

[0146] Item 26. The method according to any one of Items 1 to 25, wherein the embryoid body is a differentiated embryoid body showing germ layer differentiation.

[0147] Item 27. The method according to any one of Items 1 to 26, wherein the organoid is a brain organoid.

[0148] Item 28. The method according to Item 27, wherein the brain organoid is a cerebral organoid.

[0149] Item 29. The method according to any one of Items 1 to 28, wherein the hydrogel is selected from the group consisting of mouse Engelbreth-Holm-Swarm (EHS) sarcoma matrix, type I collagen, fibrin, hyaluronic acid (HA), gelatin methacrylate (GelMA), decellularized matrix, alginate, silk, nanocellulose, polyethylene glycol (PEG), self-assembling peptide, poly(lactic / (co)glycolic) acid, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptide, and ELP (elastin-like protein).

[0150] Item 30. The method according to any one of Items 1 to 29, wherein the liquid hydrogel is a cold liquid hydrogel, and optionally, a cold liquid hydrogel at a temperature of less than about 10°C.

[0151] Item 31. The method according to any one of Items 13 to 30, wherein the biocompatible oil has a density different from that of water.

[0152] Item 32. The method according to Item 31, wherein the biocompatible oil has a density higher than that of water.

[0153] Item 33. The method according to Item 32, wherein the biocompatible oil is a halogenated hydrocarbon, and optionally, a perfluorocarbon.

[0154] Item 34. The method according to Item 31, wherein the biocompatible oil has a density lower than that of water.

[0155] The method according to claim 34, wherein the biocompatible oil is silicone oil, mineral oil, or vegetable oil.

Claims

1. A method for culturing organoids, Adding embryoid bodies to a first culture medium in the main well of an isolation well microplate containing a plurality of well units, wherein each well unit includes a main well, a secondary well, and one or more microchannels connecting the main well and the secondary well; Removing the first culture medium from the well unit via the secondary well; Encapsulating the embryoid body by adding a liquid hydrogel; Incubating the plate for a first period of time to solidify the hydrogel that encapsulates the embryoid body; and The second culture medium is added to the secondary well, and the solidified hydrogel encapsulating the embryoid body is peeled off the plate and suspended in the second culture medium. Includes, and as needed, Adding the embryoid body to the first culture medium in the main well includes performing an automated pipetting operation. method.

2. Before removing the first culture medium, the isolation well microplate is tilted to move the embryoid body to the corner of the main well, wherein the tilting, if necessary, includes automatically tilting the isolation well microplate, and, if necessary, the tilting includes moving the embryoid body toward the microchannel. If necessary, the tilting includes tilting the plate to an angle of 1 to 60 degrees, 5 to 45 degrees, 5 to 35 degrees, 5 to 25 degrees, 5 to 15 degrees, 7 to 12 degrees, or approximately 10 degrees from the horizontal position. The method according to claim 1.

3. The method according to claim 2, wherein the tilting includes moving the embryoid body away from the microchannel, and optionally the tilting includes tilting the plate at an angle of 1 to 60 degrees, 5 to 15 degrees, 7 to 12 degrees, or about 10 degrees from the horizontal position.

4. The method of claim 2, further comprising returning the microplate to a horizontal position before removing the second culture medium.

5. The method according to claim 1, wherein adding the liquid hydrogel to encapsulate the embryoid body includes an image-guided pipetting operation of the liquid hydrogel, and optionally the image-guided pipetting operation is an automated image-guided pipetting operation that includes the delivery of the liquid hydrogel onto at least a portion of the embryoid body.

6. The method according to claim 1, further comprising cooling the microplate before adding the liquid hydrogel, wherein, if necessary, the cooling is auto-cooled to a temperature below room temperature or within a temperature range of 4 to 8°C.

7. The method according to claim 1, further comprising heating the microplate to an incubation temperature before adding the liquid hydrogel, wherein the heating is, if necessary, automated heating.

8. The method according to claim 1, comprising, after adding the second culture medium, further tilting the microplate to separate the solidified hydrogel encapsulating the embryoid body from the microchannel, wherein, if necessary, the further tilting includes automatic further tilting.

9. The method according to claim 1, wherein the incubation to solidify the hydrogel occurs over a first period selected from the group consisting of about 5 to about 90 minutes, about 10 to about 60 minutes, about 20 to about 40 minutes, and about 30 minutes, and optionally the microplate is kept at room temperature for a second period prior to the incubation step.

10. The method according to claim 1, further comprising periodically replacing the second culture medium in the well unit via the secondary wells to nourish the embryoid body.

11. The method according to claim 1, further comprising adding the liquid hydrogel to encapsulate the embryoid body and before incubation, wherein the introduction optionally includes an automated pipetting operation of the biocompatible oil into the main well, and optionally the introduction of the biocompatible oil into the main well causes the hydrogel encapsulating the embryoid body to become more spherical.

12. The method according to claim 11, wherein the introduction includes placing a pipette tip in the hydrogel and aspirating the hydrogel and oil encapsulating the embryoid body into the pipette tip, wherein the placement is, if necessary, an image-guided placement, and if necessary, the aspiration is performed slowly over a period of at least 0.3 seconds, at least 0.5 seconds, at least 0.7 seconds, or at least 1 second.

13. The method according to claim 12, further comprising pushing the oil and the hydrogel encapsulating the embryoid body back from the pipette tip into the main well, wherein, if necessary, the pushing is carried out slowly over a period of at least 0.3 seconds, at least 0.5 seconds, at least 0.7 seconds, or at least 1 second.

14. After the hydrogel encapsulating the embryoid body has solidified, the biocompatible oil is extracted from the main well via the secondary well; and The well unit is washed with a washing medium before adding the second culture medium, and if necessary, the washing medium is at room temperature. The method according to claim 11 or 12, further comprising:

15. The method according to any one of claims 1 to 13, further comprising transferring the encapsulated embryoid body embedded in the solidified hydrogel to a bioreactor for maturation.

16. The method according to any one of claims 11 to 13, further comprising transferring the hydrogel encapsulating the embryoid bodies and the biocompatible oil to an oil pool heated to an incubation temperature to solidify the hydrogel encapsulating the embryoid bodies, wherein the oil pool optionally includes a stream for transferring the hydrogel encapsulating the embryoid bodies to a bioreactor optionally including a filter.

17. Discharging the oil from the bioreactor, wherein the filter holds the solidified hydrogel that encapsulates the embryoid body; Washing the bioreactor and the solidified hydrogel that holds and encapsulates the embryoid body; and Adding a third culture medium to the bioreactor provides nutrients to the washed hydrogel that encapsulates the embryoid body. The method according to claim 16, further comprising:

18. The stream of the oil pool into which the hydrogel encapsulating the embryoid body is transferred comes into contact with the third culture medium, and the method Separating the mixture of oil and the third medium into layers; and Removing the oil layer by passing it through a filter system and retaining the solidified hydrogel encapsulating the embryoid body in the third culture medium; and The solidified hydrogel in the third culture medium, which encapsulates the embryoid body, is introduced into the bioreactor. The method according to claim 16, further comprising:

19. The method according to any one of claims 1 to 13, wherein the method is an automated method, and the addition of the embryoid body to the first medium, the removal of the first medium from the well unit, the addition of the liquid hydrogel, and the addition of the second medium each include an automated pipetting operation.

20. The method according to any one of claims 1 to 13, wherein the embryoid body is a differentiated embryoid body showing germ layer differentiation, and optionally the organoid is a brain organoid, and optionally the brain organoid is a cerebral organoid.

21. The method according to any one of claims 1 to 13, wherein the hydrogel is selected from the group consisting of mouse Angelbreth-Holm-Swarm (EHS) sarcoma matrix, type I collagen, fibrin, hyaluronic acid (HA), methacrylate gelatin (GelMA), decellularized matrix, alginate, silk, nanocellulose, polyethylene glycol (PEG), self-assembling peptide, poly(milk / (co)glycol) acid, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptide, and ELP (elastin-like protein), and optionally the liquid hydrogel is a cold liquid hydrogel, and optionally a cold liquid hydrogel at a temperature of less than about 10°C.

22. The method according to any one of claims 11 to 13, wherein the biocompatible oil has a density different from that of water.

23. The method according to claim 22, wherein the biocompatible oil has a higher density than water, and optionally the biocompatible oil is a halogenated hydrocarbon, and optionally a perfluoro hydrocarbon.

24. The method according to claim 22, wherein the biocompatible oil has a density lower than that of water, and optionally the biocompatible oil is a silicone oil, mineral oil, or vegetable oil.

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