Method for automatically embedding embryoid bodies in hydrogel by using separation pore microplate

Abstract

Methods for automatically embedding embryoid bodies in hydrogels, replacing media, culturing organoids, and monitoring the development of organoids, such as brain organoids, are provided. The invention also provides a method for automatically testing the compounds and toxic effects.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application was filed as a PCT international patent application on July 20, 2023, and claims the benefit and priority of U.S. Provisional Application No. 63 / 393,567 filed on July 29, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] The method of encapsulating embryoid bodies (EBs) in hydrogels to grow brain organoids is difficult to automate. Organoids must be shipped with culture medium, but too much culture medium may liquefy the hydrogel (such as Matrigel) used to encapsulate EBs and organoids.

[0004] Lancaster and Knoblich (Nat Protoc. 2014 October; 9 (10): 2329-2340) described a method for generating brain organoids from human pluripotent stem cells (hPSCs). A labor-intensive process for transferring properly differentiated embryoid bodies (EBs) to Matrigel droplets was described, which required manual preparation of a pitted paraffin film matrix to produce Matrigel droplets. EBs need to be handled delicately and quickly to avoid damaging aggregates or avoiding premature polymerization of Matrigel.

[0005] Improved automated methods for embedding embryoid bodies in hydrogels to culture brain organoids are needed. Summary of the invention

[0006] Provided are automated methods for embedding embryoid bodies (EBs) in hydrogels, culturing brain organoids, and automatically screening brain organoids.

[0007] A method for culturing brain organoids is provided, comprising embedding embryoid bodies in a liquid hydrogel to encapsulate the embryoid bodies. Using a separation hole microplate, the embryoid bodies can be embedded in the hydrogel in a fine, efficient and automatic manner. The method may include solidifying the hydrogel encapsulating the embryoid bodies, and suspending the solidified hydrogel encapsulating the embryoid bodies in a culture medium in the separation hole microplate. A method is provided for shaping the liquid hydrogel encapsulating the embryoid bodies into a more spherical shape, solidifying the hydrogel encapsulating the EBs, and suspending the hydrogel-embedded EBs in the culture medium. A method is provided for transporting the hydrogel-embedded EBs to a bioreactor, raising the EBs and differentiating them into brain organoids, and testing the brain organoids.

[0008] A method for culturing organoids is provided, the method comprising adding embryoid bodies to a first culture medium, the first culture medium being located in a main well of a separation well microplate, the separation 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 to 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 time period to solidify the hydrogel encapsulating the embryoid bodies; and adding a second culture medium to the secondary wells to separate the solidified hydrogel encapsulating the embryoid bodies from the plate and suspend them in the second culture medium.

[0009] In some examples, adding the embryoid bodies to the first culture medium in the main well comprises automated pipetting.

[0010] In some examples, the method for culturing organoids further comprises tilting the separation well microplate prior to removing the first culture medium to move the embryoid bodies to a corner of the main well, optionally wherein the tilting comprises automatically tilting the separation well microplate.

[0011] Tilting of the separation well microplate can comprise moving the embryoid bodies toward the microchannels, optionally wherein the tilting comprises 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 about 10 degrees from horizontal.

[0012] Tilting of the separation well microplate can include moving the embryoid bodies away from the microchannels, optionally wherein the tilting includes tilting the plate to an angle of 1 to 60 degrees, 5 to 15 degrees, 7 to 12 degrees, or about 10 degrees from horizontal.

[0013] In some examples, the method for culturing organoids further comprises returning the microplate to a horizontal position before removing the second culture medium.

[0014] In some examples, adding the liquid hydrogel to encapsulate the embryoid bodies comprises image-guided pipetting of the liquid hydrogel, optionally wherein the image-guided pipetting is automated image-guided pipetting, comprising delivering the liquid hydrogel to the top of at least a portion of the embryoid bodies.

[0015] In some examples, the method for culturing organoids further comprises cooling the microplate prior to adding the liquid hydrogel, optionally wherein the cooling is automated cooling to a temperature below room temperature, or to a temperature within a range of 4 to 8 degrees Celsius.

[0016] In some examples, the method for culturing organoids further comprises heating the microplate to an incubation temperature prior to adding the liquid hydrogel, optionally wherein the heating is automated.

[0017] In some examples, the method for culturing organoids comprises further tilting the microplate after adding the second culture medium to move the solidified hydrogel encapsulating the embryoid bodies away from the microchannels, optionally wherein the further tilting comprises automated further tilting.

[0018] In some examples, incubation is performed for a first time period to solidify the hydrogel, the time period being 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, optionally wherein the microplate is maintained at room temperature for a second time period prior to the incubation step.

[0019] In some examples, the method for culturing organoids further comprises periodically replacing a second culture medium in the well unit through the secondary well to feed the embryoid bodies.

[0020] In some examples, the method for culturing organoids further comprises introducing a biocompatible oil into the main well after adding the liquid hydrogel to encapsulate the embryoid bodies and prior to incubating, optionally wherein the introducing comprises automatically pipetting the biocompatible oil into the main well.

[0021] In some examples, the introducing comprises positioning a pipette tip within the hydrogel and aspirating the hydrogel and oil encapsulating the embryoid bodies into the pipette tip, optionally wherein the positioning is image-guided positioning. The aspiration may be 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.

[0022] In some examples, the method for culturing organoids further comprises pushing the oil and the hydrogel encapsulating the embryoid bodies from the pipette tip back into the main well. The pushing can be 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.

[0023] In some cases, the introduction of biocompatible oil into the main pores caused the hydrogel encapsulating the embryoid bodies to become more spherical.

[0024] In some examples, the method for culturing organoids further comprises, after solidifying the hydrogel encapsulating the embryoid bodies, withdrawing the biocompatible oil from the main well through the secondary well; and

[0025] Prior to adding the second culture medium, the well unit is washed with a wash medium, optionally wherein the wash medium is at room temperature.

[0026] In some examples, the method of culturing organoids further comprises transferring the encapsulated embryoid bodies embedded in the solidified hydrogel to a bioreactor for maturation.

[0027] In some examples, the method for culturing organoids further comprises transferring the hydrogel encapsulating the embryoid bodies and the biocompatible oil into an oil pool, which is heated to an incubation temperature to solidify the hydrogel encapsulating the embryoid bodies.

[0028] In some examples, the oil pool includes a split flow that transfers the hydrogel encapsulating the embryoid bodies to a bioreactor, which optionally includes a filter.

[0029] In some examples, the method for culturing organoids further comprises draining the oil from the bioreactor, wherein the filter retains the solidified hydrogel encapsulating the embryoid bodies; washing the bioreactor and retaining the solidified hydrogel encapsulating the embryoid bodies; and adding a third culture medium to the bioreactor to feed the washed hydrogel encapsulated embryoid bodies.

[0030] In some examples, the method for culturing organoids also includes, wherein the hydrogel encapsulating the embryoid bodies is transferred to an oil pool shunt of the bioreactor to meet a third culture medium; and the method also includes stratifying the mixture of the oil and the third culture medium; and removing the oil layer by a filtration system to keep the solidified hydrogel encapsulating the embryoid bodies in the third culture medium; and directing the solidified hydrogel encapsulating the embryoid bodies in the third culture medium to the bioreactor.

[0031] In some examples, the method for culturing organoids is an automated method, wherein adding embryoid bodies to a first culture medium, removing the first culture medium from the well cells, adding the liquid hydrogel, and adding the second culture medium each comprise automated pipetting.

[0032] The organoid may be a brain organoid. The organoid may be a liver organoid.

[0033] In some examples, the embryoid bodies are differentiated embryoid bodies that exhibit germ layer differentiation.

[0034] In some examples, the organoid is a brain organoid. The brain organoid can be a cerebral organoid.

[0035] 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, polylactic acid / (co)glycolic acid, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptides, and ELP (elastin-like protein).

[0036] In some examples, the liquid hydrogel is a cold liquid hydrogel, optionally at a temperature below about 10°C or in a temperature range of 4 to 8°C. In some examples, the density of the biocompatible oil is different from water. The density of the biocompatible oil can be higher than water. The biocompatible oil with a density higher than water can be a halogenated hydrocarbon, optionally a perfluorinated hydrocarbon. In some examples, the density of the biocompatible oil is less than water. The biocompatible oil with a density less than water can be a silicone oil, a mineral oil, or a vegetable oil.

[0037] An automated method for culturing brain organoids is provided, comprising adding differentiated embryoid bodies to a first culture medium located in a main well of a separation well microplate, the separation 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 to the secondary well; tilting the separation well microplate to move the embryoid bodies to a corner of the main well; removing the first culture medium from the well unit through the secondary well; cooling the microplate and adding a liquid hydrogel to encapsulate the embryoid bodies in the corner of the main well of the tilted microplate; incubating the plate within a first time period to solidify the hydrogel encapsulating the embryoid bodies; and adding a second culture medium to the secondary wells to separate the solidified hydrogel encapsulating the embryoid bodies from the plate and suspend them in the second culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1A A first view of an exemplary single well unit 100 in a split well microplate for horizontal co-culture that can be used for cell / organoid feeding is shown. The main well 112 is separated from the secondary well 115 by at least one microchannel 118. In some embodiments, at least one microchannel 118 can be opened to allow medium exchange between the main well 112 and the secondary well 115. Alternatively, at least one microchannel 118 can include a removable barrier to isolate the main well and the secondary well from each other.

[0039] Figure 1B A second view of an exemplary single well unit 100 in a split well microplate for horizontal co-culture that can be used for cell / organoid feeding is shown. The primary well 112 is separated from the secondary well 115 by at least one microchannel 118. When the split well microplate is on a rocker, medium exchange is performed by gravity flow through the microchannel 118.

[0040] Figure 1CAn example of a cross-section of a single well unit 100 of a separate well microplate associated with a cell culture method according to various embodiments of the present disclosure is shown. The main well 112 is separated from the secondary well 115 by a shared sidewall that includes at least one microchannel 118 located between the main well 112 and the secondary well 115. The main well 112 may contain target cells 103 (e.g., brain organoids) embedded in a hydrogel dome 111. The main well 112 and the secondary well 115 may be at least partially filled with a liquid culture medium 109. The bottom of the well unit 100 includes a bottom sheet 121 that includes an optically transparent observation window to allow imaging.

[0041] Figure 2 A representative schematic diagram of an exemplary automated workflow method for embedding embryoid bodies in hydrogel using a separation well microplate is shown. A cross-section of a single well unit of a separation well microplate includes a main well 212 and a secondary well 215 separated by a shared sidewall, the shared sidewall including at least one microchannel 218 located between the main well 212 and the secondary well 215. At 200, target cells 203 (e.g., embryoid bodies) are added to the main well 212 containing a first culture medium 209. At 202, the separation well microplate is tilted to move the embryoid bodies 203 toward the at least one microchannel 218. At 204, the separation well microplate is restored to a horizontal state, and the first culture medium 209 is removed via the secondary well 215 by a pipette 220. At 206, a liquid hydrogel 211 is added to the top of the embryoid bodies 203 in the main well 212 by a pipette 220, and incubated to solidify the hydrogel 211. At step 208, a second culture medium 213 is added to the secondary wells 215 to suspend the embryoid bodies 203 embedded in the hydrogel 209. The hydrogel 211 is in the shape of a half moon or teardrop. At step 210, the plate is incubated and the second culture medium 213 is replaced periodically through the secondary wells 215.

[0042] Figure 3 Representative images of brain organoids developed from properly differentiated embryoid bodies at day 0 (left), day 2 (middle), and day 3 (right) after automated embedding in hydrogels are shown. At day 10 from the start of automated stem cell culture, organoids were embedded in a hydrogel (e.g., Matrigel) to support the expansion of neuroepithelial cells. In this case, the previously described workflow ( Figure 2 ) EBs were embedded in hydrogels. Note that many epithelial buds were formed on day 3, indicating good differentiation after embedding.

[0043] Figure 4A set of pictures are shown, depicting microphotographic images of an automated method for embedding embryoid bodies in hydrogels using a 96-well separation well microplate and an advanced pipetting protocol. The upper left image (1) shows an embryoid body in culture medium in the main well, and the culture medium feed well is also shown. The upper middle image (2) shows an image of an embryoid body located at the corner of the culture medium main well after tilting the separation well microplate. The upper right image (3) shows an embryoid body located at the corner of the main well after the culture medium is removed from the main well through the feed port. The lower left image (4) shows Matrigel added to the top of the embryoid body in the main well. The lower middle image (5) shows an image of a pipette tip after slowly removing Matrigel and oil, where the embryoid body will remain in the Matrigel, the denser oil is located at the bottom of the pipette tip, and the Matrigel is now on top of the oil and becomes more spherical. The lower right image (6) shows an embryoid body successfully embedded in Matrigel.

[0044] Figure 5 An exemplary workflow of automated brain organoid culture and screening methods according to the present disclosure is shown. In short, at day 0-4, automated stem cell culture starts with induced pluripotent stem cells (iPSC-derived cells) in 2D culture, which can be grown to about 70-80% fusion using an exemplary 6-well culture plate in a culture medium such as hES culture medium. The morphology of stem cell colonies can be monitored by imaging. For example, colonies can be destroyed by brief exposure to enzymes (such as dispase, trypsin) and chelating agents (such as EDTA), washing, and suspension to obtain a single cell suspension. EBs are raised and culture medium is replaced. At day 4-8, single cells are transferred to a 96-well plate with a low-adsorption U-bottom, raised, and culture medium is regularly replaced. Low adsorption plates can help avoid EB adsorption. At day 8-9, differentiated embryoid bodies are transferred to the main wells of a passage plate (separation well microplate) to embed the embryoid bodies in a hydrogel using a separation well microplate. These plates are incubated in a large feeding incubator with a tilting function and monitored within 10-50 days to form 3D brain organoids. Through automated imaging technology, the development and growth of organoids can be monitored in Matrigel to assess the size, volume, and complexity of organoids. The addition and effect measurement of test compounds can be performed after day 50-51, for example, using imaging and 3D analysis for quantitative characterization. In this exemplary workflow, approximately 42k compounds (210k organoids) can be screened in approximately 6 months. The tilting incubator can accommodate approximately 800 plates for feeding and monitoring organoids.

[0045] Fig. 6A Fluorinert in pipette tips TM FC-40 biocompatible oil is embedded on the top of the spherical Embryoid bodies in hydrogel.

[0046] Figure 6B Fluorinert in pipette tips TM FC-40 biocompatible oil is embedded on the top of the spherical Embryoid bodies in cellulose hydrogels.

[0047] Figure 6C Fluorinert in pipette tips TM Embryoid bodies embedded on top of FC-40 biocompatible oil in spherical type I collagen (rat) hydrogels. The use of biocompatible oil resulted in more spherical hydrogels in each of the tested hydrogels. DETAILED DESCRIPTION

[0048] Automated methods for embedding embryoid bodies in hydrogels, organoid culture, and organoid screening are provided.

[0049] An automated method for preparing 3D cell models of representative brain tissue has been developed.

[0050] The present disclosure provides an automated method for integrated cell culture using an advanced imaging system that allows automated monitoring, maintenance, characterization of organoids, and testing of the effects of various compounds. The integrated system includes a confocal imaging system, an automated incubator, an automated liquid handler, and a collaborative robot. These instruments are controlled by an integrated software that can set up the process. As described herein, Figure 5 An example of a process for monitoring cells in the process is shown in . The development of EBs and brain organoids can be monitored by imaging. For example, the plate can be moved from the incubator to an imaging system (e.g., ImageXpress ConfocalHT.ai, Molecular Devices LLC) for imaging in bright field instead of returning to the incubator. The process can be pre-arranged and the plates that need to be imaged can be entered in a list to make batch processing easier. Complex routines including liquid handlers for culture medium replacement (feeding) can also be implemented.

[0051] Methods are provided for automated embedding of EBs in hydrogels and for performing media changes, as well as monitoring brain organoid development. In addition, these methods allow for automated testing of compounds and toxic effects.

[0052] definition

[0053] The singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0054] The term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items.

[0055] When referring to a measurable value, such as an amount of a compound, a time, a temperature, and the like, the term "about" is meant to encompass variations of 10%, 5%, 1%, 0.5%, and even 0.1% of the specified amount.

[0056] Unless otherwise indicated, the terms "percent" or "%" refer to percent by weight.

[0057] Unless otherwise indicated, the term "density (d)" refers to the mass of a material per unit volume at 25°C.

[0058] As used herein, the term "biocompatible oil" refers to an oil that does not adversely affect the development, differentiation or survival of embryoid bodies or organoids when used according to the methods of the present disclosure. When used according to the methods of the present disclosure, the biocompatible oil can be a non-toxic oil. The biocompatible oil can have a different density from water.

[0059] As used herein, the term "composition" may refer to one or more of a compound, a mixture, a blend, an alloy, a polymer, and / or a copolymer.

[0060] The term "room temperature" refers to a temperature in the range of 20 to 25°C, with an average of 23°C.

[0061] The "cold" in the term "cold liquid hydrogel" refers to a temperature below room temperature, or in the range of 0 to 10°C, in the range of 1 to 8°C, or about 4°C.

[0062] The terms "incubate", "incubating" or "incubation temperature" refer to about 37°C.

[0063] As used herein, ranges are intended to include at least the numbers defining the limits of the range.

[0064] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0065] The term "feeder cell" refers to a cell that provides extracellular secretions to help another cell proliferate, grow, differentiate and / or maintain characteristics. 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 not be able to divide, i.e., cell growth stagnation. The growth of feeder cells can be arrested by any appropriate method known in the art, for example. Feeder cell growth can be arrested by chemical fixation (e.g., chemical fixation by mitomycin C or glutaraldehyde). Feeder cell growth can be arrested by physical methods (e.g., gamma ray irradiation, x-ray irradiation or electric pulses). For example, feeder cells for co-culture of target cells (e.g., embryonic stem cells (ESC)) can be fibroblasts, which may not have mitotic activity, but still survive. In some cases, target cells can grow in the presence of feeder cells that can divide. Some live feeder cells (e.g., human fibroblasts) may also become target cells, just like the case of induced pluripotent stem cells (iPSC) after reprogramming. For example, feeder cells can be stagnant feeder cells that cannot divide. The selection of feeder cells can depend on the target cells. Feeder cells can be, for example, unstagnant fibroblasts. Feeder cells can be stagnant fibroblasts, epithelial cells, mesenchymal cells, muscle cells, stromal cells, spleen cells or amniotic cells. Fibroblasts can be, for example, human skin fibroblasts, 3T3 fibroblasts, human fetal fibroblasts and mouse embryonic fibroblasts, etc. Epithelial cells can be, for example, human adult fallopian tube epithelial cells, human amniotic epithelial cells, HeLa cells (human cervical cancer tumor epithelial cells), etc. 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, etc. 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.

[0066] The term "target cell" refers to a cell used for automated cell culture applications of the present disclosure, such as an embryoid body, an organoid, a tumoroid, a cell sphere, a stem cell, or a production cell line. In some embodiments, the target cell is an embryoid body, an organoid, and / or other multicellular bodies. The organoid may be a brain organoid. The brain organoid may be a cerebral organoid. The organoid may be a liver organoid. In some embodiments, the target cell may be a stem cell. The stem cell may be a human induced pluripotent stem cell (hiPSC). In some embodiments, the target cell may be a production cell line. The target cell may be derived from a target tissue. The target tissue may be a mammalian primary tissue, an organoid, or a tumoroid. Mammalian tissue may be derived from a patient biopsy sample. The target tissue may be derived from a target organ, such as a lung, intestine (such as the small intestine), colon, stomach, pancreas, liver, kidney, skin, bone marrow, blood-brain barrier, brain, heart, etc.

[0067] Organoids, spheroids, tumors, 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 may help to better understand complex biology in a physiologically relevant context because cells often maintain their natural shape and proper spatial orientation, such as in aggregates or spheroids, while 2D models of cells grown in sheets or monolayers may not be as successful. Gene and protein expression in 3D cell cultures can more closely mimic gene and protein expression. For example, 3D cell cultures can be used for drug target identification, lead compound identification, compound optimization, preclinical qualification, solid tumor modeling, genetic disease modeling, drug discovery, precision medicine, organs-on-chips, and bioprinting.

[0068] The term "spheroid" refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate composed of one or more cell types that grow and proliferate, may 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.

[0069] The term "organoid" refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate composed of one or more cell types, in which 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 (PSC), induced pluripotent stem cells (iPSC), neonatal tissue stem cells, embryonic stem cells (ESC), adult stem cells or primary tissues. Organoid cultures can be elaborated to be similar to the complexity of organs, and are therefore useful for the study of disease etiology and treatment. Organoid technology has recently become an important tool for basic research and biomedical research. Organoid cultures can be selected from different types of target organs. Organoids can be, for example, brain organoids, liver organoids, lung organoids, intestinal organoids, such as small intestine organoids, colon organoids, stomach organoids, pancreatic organoids, kidney organoids, skin organoids, heart organoids, bone marrow, blood-brain barrier, etc. In the present disclosure, organoids can be brain organoids. In some examples, organoids can be liver organoids.

[0070] The term "brain organoid" refers to a 3D tissue model representing one or more regions of the brain. Brain organoids can be, for example, cerebral organoids, cortical organoids, forebrain organoids, ventral brain organoids, ventral forebrain organoids, thalamic organoids, hypothalamic organoids, midbrain organoids, hindbrain organoids, cerebellar organoids, medial ganglionic eminence (MGE) organoids, neuromuscular organoids, etc. Forebrain organoids can include cortical organoids, ventral forebrain tissues, thalamic tissues. Hindbrain organoids can include cerebellar organoids.

[0071] Brain organoids can be formed using guided or non-guided schemes, for example, as described in Kim et al., 2021, iScience 24, 102063, February 19, 2021, the entire contents of which are incorporated herein by reference. Non-guided schemes rely on intrinsic signaling and do not use specific chemicals or inhibitors. For example, brain organoids can be self-organized and can spontaneously differentiate from hPSCs in the presence of extracellular matrix (ECM) hydrogels (such as Matrigel) in the absence of extrinsic factors. ECM hydrogels can be used to support 3D structures and neuroepithelial expansion. Guided methods use exogenous signaling factors such as signal activators or inhibitors to produce brain organoids in specific areas. 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, 2,6,9-trisubstituted purine (Purmorphamine), etc.

[0072] The structure, transcriptome, and function of brain organoids can be determined by a variety of methods. For example, architecture can be analyzed by immunostaining, tissue clearing, or imaging systems. Transcriptome assessments can include qPCR, bulk RNAseq, or scRNAseq approaches. Functional assessments can include Ca 2+ Imaging, microelectrode array (MEA) or patch clamp.

[0073] When cultured, human induced pluripotent stem cells (hiPSC) differentiate into various neural cells that mature over time and resemble the structures of various brain regions. Brain 3D organoids are a developing technology that can be used to understand human brain development, neuronal 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 according to the Lancaster and Knoblich protocol (2014). See Lancaster and Knoblich, Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014 October; 9 (10): 2329-2340, the entire contents of which are incorporated herein by reference.

[0074] The term "stem cell" refers to undifferentiated cells that have the potential to develop into many different cell types that perform different functions. Pluripotent stem cells, such as those found in embryos, can give rise to any type of cell, such as those in the brain, bone, heart, and skin. Some human adult cells can be reprogrammed to an embryonic stem cell-like state, called induced pluripotent stem cells (iPSCs). iPSCs can be human iPSCs. For example, pluripotent stem cells found in an adult or in the umbilical cord of a baby may develop into cells that make up the organ system from which they originated. Pluripotent stem cells can remain in an undifferentiated state when grown under certain cell culture conditions. To produce 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.

[0075] The term "embryoid body" (EB) refers to a three-dimensional aggregate of pluripotent stem cells. EB can be formed in suspension by pluripotent stem cells (PSC), including embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). EB differentiation can be used as a common platform for generating specific cell lineages from PSC. Many EB formation schemes use ingredients such as fetal bovine serum (FBS) or knockout serum substitutes (KOSR) or albumin products. Many human PSCs cannot survive in suspension unless in aggregates or under ROCK inhibitor treatment (such as ROCK inhibitors, Tocris).

[0076] Using an automated system, a method for culturing three-dimensional brain organoids by human iPSC is provided, including automatically embedding an embryoid body in a hydrogel. In some examples, iPSC can grow and dissociate into single cells, and on days 0-4, re-aggregate to form an embryoid body (EB) in a culture medium such as hES culture medium. Cells can be monitored by imaging, for example, EB formation and germ layer differentiation are imaged. On days 4-8, EB is transferred to a 96-well U-shaped bottom plate, and the culture medium is replaced with a culture medium such as a neural induction culture medium. Feeding and monitoring. After inducing neural ectoderm on about days 8-9, EB can be transferred to a separation well microplate and embedded in a hydrogel such as Matrigel. Culture medium such as differentiation culture medium can be added. Differentiation culture medium may not initially include vitamin A (retinoic acid). After the expansion and development of neural epithelial buds, the culture medium can be replaced with a differentiation culture medium including vitamin A (retinoic acid). On days 10-50 or before and after it, organoid maintenance and optional compound addition can be performed. The separation hole microplate can be moved to a feeding incubator with a tilting function and monitoring capability. The feeding incubator can be a large-scale feeding incubator with a capacity of, for example, 10-1000 plates, 20-800 plates or more. At the 50th-51st day, a high-throughput cell screening instrument (such as FLIPR Penta high-throughput cellular screening system (Molecular Devices LLC)) can be used to analyze brain organoids. Cell screening instruments can achieve high-throughput screening of brain organoids for toxicology and lead compound identification. Cell screening instruments can include internal plate processors, cameras, pipette heads, heating tables, optical devices and computer controllers for exciting and emitting fluorescent markers.

[0077] Using an automated liquid handling system, EBs can be automatically seeded and hydrogel droplets added to embed the EBs, followed by automated addition of culture medium and culture medium changes. Organoid development is monitored using transmitted light imaging. Machine learning-based image analysis is then able to detect organoids and characterize their size and density. For endpoint measurements, organoids can be stained with fluorescently labeled antibodies or active dyes and imaged using an automated confocal imaging system. Advanced image analysis is possible through 3D reconstruction and complex phenotypic assessment of organoid structure, including characterization of organoid size and complexity, cell morphology and viability, and the presence of differentiation markers.

[0078] The methods of the present disclosure utilize a separation well microplate having a well geometry that includes a primary well and secondary wells connected by at least one channel. For example, the culture methods of the present disclosure may utilize a separation well microplate having a plurality of well units, each well unit including a primary well and secondary wells connected by at least one channel. For example, each well unit may include Figure 1A The well geometry shown includes a main well connected to a secondary well fluid via at least one channel. The separation well microplate is described in U.S. Patent Application 17 / 580,193, entitled "Microplate for Automated Organoid Culture," filed on October 22, 2021, and PCT International Application PCT / IB2021 / 062356, entitled "Microplate Well for Cell Culture," filed on December 27, 2021, each of which is incorporated herein by reference in its entirety.

[0079] The size and morphology of developing brain microtissues can be monitored using automated transmitted light microscopy. AI-based image analysis (IN Carta software) methods can be used to define the size, shape, and density of tissues. For example, at different developmental stages, microtissues can be analyzed using confocal imaging by the expression of Sox2, TuJ1, and GFAP markers or other markers (e.g., using antibody-fluorescent dye conjugates). Organoids can be fixed and stained with Hoechst (a blue fluorescent dye for DNA staining) and Sox2 (radial glial cells). Analysis of brightfield images can be performed using deep learning-based differentiation (SINAP). The maturation of 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 allows for the differentiation of organoids with different shapes and sizes.

[0080] Figure 1A An example of a well unit 100 of a separation well microplate that can be used in conjunction with the methods of the present disclosure is shown. Figure 1AAn example diagram of a well unit 100 of a microplate according to various embodiments of the present disclosure is shown, which can be used to grow, culture, monitor and analyze embryoid bodies, fused embryoid bodies, cell spheres, organoids or other multicellular bodies. For example, a single well unit 100 of a separation well microplate can be used for horizontal co-culture of cell / organoid feeding. The separation well microplate used in the method according to the present disclosure may include a plurality of well units, each well unit including a main well 112, which is separated from a secondary well 115 by a shared sidewall, and the main well and the secondary well can be connected by at least one microchannel 118 fluid. In some embodiments, at least one microchannel 118 may include a removable barrier to isolate the main well and the secondary well from each other. In some embodiments, at least one microchannel 118 may be open to allow the exchange of culture medium between the main well 112 and the secondary well 115. The well unit 100 of the separation well microplate includes a main well 112, which can be connected to the secondary well 115 fluid by at least one microchannel 118. In some examples, the main hole 112 can be used as a culture hole, and the secondary hole 115 can be used as a feed hole. The microchannel 118 can include a removable barrier. At least one microchannel 118 can be closed by a removable barrier or can be open. One or more microchannels 118 between the main hole 112 and the secondary hole 115 can be closed by a removable barrier, such as by an air gap, a hydrogel seal or a silicone seal. One or more microchannels 118 between the main hole 112 and the secondary hole 115 can be closed, for example, by an air gap. The air gap can be formed by, for example, bubbles.

[0081] Figure 1B Another example of a well unit 100 of a separation well microplate that can be used in conjunction with the methods of the present disclosure is shown. Figure 1BIn the embodiment of the present invention, the well unit 100 includes a main well 112 and a secondary well 115, which can be fluidically connected by at least one open microchannel 118. When open, the geometry of the at least one channel 118 can allow the exchange of culture medium, expressed proteins (such as hormones), nutrients, growth factors, waste and debris between the main well and the secondary well, while retaining, for example, target cells (such as organoids) in the main well 112 and feeder cells in the secondary well 115. The dimensions of the at least one microchannel 118 can prevent multicellular target cells (such as organoids, tumoroids, etc.) from passing through the microchannel 118. In some examples, the geometry of the at least one microchannel 118 can include a slit configuration with a channel width of 150-500 microns or more, or 200-400 microns, or about 300 microns x 10-100 microns, or 10-75 microns in height. During the methods of the present disclosure, at least one microchannel 118 between the primary well and the secondary well can be closed by a removable barrier, or can be opened to allow exchange of culture medium, secreted factors, hormones, growth factors, nutrients, waste and debris between the primary well 112 and the secondary well 115, for example, by gravity feeding while shaking the separation well microplate. In some examples, at least one microchannel can be opened by, for example, removing a removable barrier, for example, by using a pipette as a pump to eliminate the air gap, for example, sealing the secondary well with a pipette tip to allow liquid flow between the two wells.

[0082] Figure 1CAnother example of a well unit 100 of a split well microplate that can be used in conjunction with the methods of the present disclosure is shown. The well unit 100 includes a main well section 112, a secondary well section 115, at least one microchannel 118, and an embryoid body 113 embedded in a suspension hydrogel 111, which is disposed in the main well section 112 of the well unit 100. The main well section 112 and the secondary well section 115 contain a culture medium. According to various embodiments, the well unit 100 may further include a bottom sheet 121 disposed on the lower side of the well plate body of the microplate. The bottom sheet 121 is attached to the lower side of the well plate body to form the bottom surface of the well unit 100. In various examples, the bottom sheet 121 includes an optically transparent viewing window to allow imaging of embryoid bodies or organoids, or other cell cultures, cultured in the well unit 100 of the microplate, as can be understood. The observation window can be a window suitable for microscopic observation, whether it is bright field, phase contrast, fluorescence, confocal, two-photon or other microscopic imaging methods known in the art. In some examples, the bottom sheet 121 can include a breathable sheet that is configured to increase the oxygen supply of the embryoid bodies and organoids or other cell bodies grown in the well unit 100 of the microwell plate. The breathable sheet can be formed by materials including polytetrafluoroethylene (PTFE), PEFP, polyimide, polydimethylsiloxane (PDMS), polycarbonate and / or other materials that can be understood. According to different examples, the breathable sheet can have a thickness of about 5-70 microns. According to different examples, the breathable sheet can include multiple holes. In other examples, the breathable sheet can allow molecules to pass through by diffusion. Alternatively, the breathable sheet can include some other thicknesses, pore sizes and pore densities.

[0083] The secondary pore segment 115 can be used to grow feeder cells, supply feeder medium and / or other nutrients that can be used to feed cell aggregates grown in the primary pore segment 112. The secondary pore segment 115 can be considered as a supply pore that contains feeder medium and / or other nutrients that can be used by cell cultures grown in the primary pore segment 112. According to various embodiments of the present disclosure, the size and shape of the secondary pore segment 115 can be designed to accommodate fluids that can be exchanged with the primary pore segment 112.

[0084] According to different embodiments, the size and shape of the main hole section 112 and the secondary hole section 115 can be different from each other. In some examples, the main hole section 112 is larger than the secondary hole section 115 (e.g., in diameter, cross-sectional or volume dimensions). In other examples, the secondary hole section 115 is larger than the main hole section 112. In some examples, the shape of the main hole section 112 is different from the shape of the secondary hole section 115.

[0085] According to various examples, the well unit of the separation well microplate includes at least one microchannel 118 whose size and shape are designed to prevent objects having a specific size (e.g., greater than about twenty-five (25) microns (μm)) in a dimension such as diameter, height, width, etc. from passing from one well segment to another.

[0086] In some examples, the height dimension of at least one microchannel 118 can be between about ten (10) microns and about one hundred (100) microns, or between about 10 microns and about 75 microns, or between about ten (10) microns and about twenty-five (25) microns. The geometry of at least one microchannel 118 can include a slit configuration with a channel width of 150-500 microns or greater, or 200-400 microns, or about 300 microns x 10-100 microns, or 10-75 microns high. For example, the height of at least one microchannel 118 can prevent objects (e.g., embryoid bodies, tumoroids, organoid fragments, etc.) with a size of about 25 μm or greater present in the main pore section 112 from migrating to the secondary pore section 115. The microchannel 118 can be closed by, for example, an air gap, a hydrogel sealant, or a silicone sealant. The microchannel 118 can be opened by, for example, eliminating the air gap or removing the hydrogel sealant or silicone sealant.

[0087] In some embodiments of the present disclosure, the main hole 112 can be used as a culture chamber. For example, initially, at least one channel 118 can be closed, for example, EBs can be cultured in the main hole 112 of the separation hole microplate without mixing. Optionally, feeder cells can be cultured without mixing and allowed to adhere to the bottom of the secondary hole 115 of the separation hole microplate. After the feeder cells adhere to the secondary hole, at least one closed channel 118 can be optionally opened. The separation hole microplate can be placed on a rocker to allow culture medium, hormones, growth factors, nutrients, waste and debris to be exchanged by gravity supply. In some examples, feeder cells can be cultured in the secondary chamber 115, allowing EBs embedded in the hydrogel to be cultured in the main chamber 112 to be fed through at least one opened channel.

[0088] Figure 2The workflow method of embryoid body embedding is shown, as shown in the group of figures 200 to 210, which show the changes of a single well unit of a separated well microplate over time. In the first step, in the single well unit 200, the differentiated embryoid body 203 is pipetted into the main well 212 of the separated well microplate containing the first culture medium 209. In the second step, in the same single well unit 202, the plate is tilted at a certain angle (e.g., ~10 degrees) to move the embryoid body 203 to the corner of the main well near the channel 218. In the third step, in the same single well unit 204, the plate is repositioned to a horizontal position, and the first culture medium 209 is gently removed from the secondary well 215 (feed well) by a pipette 220. Fourth step, in the same single well unit 206, the plate is tilted (~10 degrees) and liquid hydrogel (such as cold liquid Matrigel 211) is slowly added to the corner near the channel 218 in the main well 212 by a pipette 220 to encapsulate the embryoid body 203. The plate is kept for about 5 minutes and allowed to reach room temperature, and then the plate is moved to an incubator for about 30 minutes to solidify the Matrigel 209. Fifth step, in the same single well unit 208, after the Matrigel 209 solidifies, the second culture medium 213 is added from the secondary well 215 (feeding well) to suspend the embedded embryoid body 203 embedded in the Matrigel 211. Sixth step, in the same single well unit 210, the plate containing the embedded embryoid body 203 embedded in the Matrigel 211 is placed in an incubator, and the second culture medium 213 is regularly replaced using the secondary well 215 (feeding well) to avoid material loss. The first culture medium and the second culture medium can be different culture mediums. The first culture medium and the second culture medium can be the same culture medium. The first culture medium and the second culture medium can be different in one or more or two or more components. Culture medium can be a liquid culture medium in all operating temperature ranges (for example, at low temperature, room temperature, culture temperature).

[0089] For example, regular automatic culture medium replacement can be performed every 24 to 72 hours, 30 to 60 hours, 36 to 54 hours, or approximately every 36 hours, approximately every 48 hours, or approximately every 54 hours. In some examples, culture medium replacement can be performed approximately every 48 hours.

[0090] like Figure 2 As shown, target cells in the form of multicellular bodies 203 (e.g., embryoid bodies) can be encapsulated in hydrogel 211 (e.g., ). At this stage, the hydrogel (e.g. ) is generally half-moon shaped. According to various embodiments, a second liquid culture medium 213 is added, which contains appropriate growth factors, additives and supplements to produce and / or stimulate the growth of the desired multicellular body. The liquid culture medium 213 can be added through the open channel 218 via the secondary hole 215.

[0091] EB formation can be performed using a common method for differentiating iPSCs into different cell types.

[0092] Prior to being automatically embedded in the hydrogel, embryoid bodies may be formed by any suitable method. Heterogeneous methods may include stirred flask culture, liquid suspension culture, rotating cell culture system (RCCSS). Homogenous methods may include low-adsorption U-bottom multiwell plates, hanging drop culture, indented solid microsurface (Aggrewell TM ) or other methods. The generation of embryoid bodies by iPSC may include an in vitro method for pluripotency assessment. EB is a three-dimensional aggregate of cells that can be differentiated into three developmental germ layers. In order to assess the pluripotency of hiPSC to form EBs, downstream assessments may be performed to demonstrate the ability to form representative germ layers. Assessments may include, for example, histological analysis of EBs, which may include, by studying tissue structure, cell morphology and / or local protein expression, or by the expression of germ layer-specific genes to assess tissue differentiation, for example, to identify the presence of germ layer-specific gene markers and the concomitant loss of pluripotent gene markers.

[0093] The method disclosed herein can use one or more, two or more, three or more, or four or more different culture media to produce embryoid bodies and brain organoids. The culture media can be selected from any suitable culture media known in the art. For example, the culture media can be described in the document Lancaster and Knoblich, Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. 2014 October; 9 (10): 2329-2340, the entire contents of which are incorporated herein by reference.

[0094] The culture medium can be any suitable culture medium known in the art. In some examples, the culture medium can be selected from hES culture medium, neural induction culture medium, brain organoid differentiation culture medium without vitamin A (retinoic acid), and brain organoid differentiation culture medium containing vitamin A (retinoic acid). The culture medium can be based on a minimum essential culture medium, such as DMEM culture medium, essential basal culture medium, Essential 6 culture medium, commercially available forms, such as Invitrogen or Lifetechnologies.

[0095] The culture medium may be hES culture medium. For example, hES culture medium can be used to generate embryoid bodies from human PSCs until germ layer differentiation. hES culture medium may include DMEM-F12 (Dulbecco's modified Eagle's medium F12 (Thermo Fisher Scientific), KOSR, FBS, Glutamax (e.g., Gibco TM GlutaMAX TM Supplements, L-alanyl-L-glutamine dipeptide substitute for glutamine, Thermo Fisher Scientific), MEM-NEAA (minimum essential medium non-essential amino acids, Thermo Fisher Scientific) and mercaptoethanol, bFGF (basic fibroblast growth factor, such as FGF-basic (143-288), ACROBiosystems) can be added immediately before use. hES medium can be prepared according to the method described in, for example, Lancaster and Knoblich 2014.

[0096] The culture medium can be a neural induction medium. For example, the neural induction medium can be used to induce the neural ectoderm in EB. The neural induction medium can include DMEM-F12, which has N-2 supplement (e.g., 1% vol / vol) (serum-free supplement based on Bottenstein's N-1 preparation, Thermo Fisher Scientific), Glutamax (e.g., 1% v / v), MEM-NEAA (e.g., 1% v / v) and heparin (e.g., 1 μg / mL).

[0097] The culture medium can be a brain organoid differentiation medium, which includes, for example, DMEM-F12 medium, Neurobasal TM Culture medium (Thermo Fisher Scientific), N-2 supplement, insulin, MEM-NEAA, penicillin-streptomycin, Gibco TM B27 supplement (optimized serum-free neuronal cell culture supplement, Thermo Fisher Scientific) and / or 2-mercaptoethanol. Differentiation medium can be used without or with vitamin A (retinoic acid).

[0098] The term "hydrogel or hydrogels" refers to an extracellular matrix that can be used to culture organoids. The hydrogel can include a mouse EHS sarcoma matrix, for example, commercially available Matrigel (Corning), Cultex (Trevigen), Geltrex (Gibco), type I collagen, fibrin, hyaluronic acid (HA), gelatin methacrylate (GelMA), acellular matrix or biopolymers such as alginate, silk, nanocellulose; engineered materials such as polyethylene glycol (PEG), self-assembling peptides such as RADA16 / PuraMatrix bQ13, polylactic acid / (co)glycolic acid, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptides, ELP (elastin-like protein), or a combination of these polymers. The hydrogel can be Matrigel. Corning The matrix is ​​a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins including laminin (the major component), type IV collagen, heparin sulfate proteoglycans, entactin / nidogenin, and many growth factors. The hydrogel can be Growth factor reduced (GFR) basement membrane mixture. 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 to 5 mg / mL or 2 to 2.5 mg / mL.

[0099] The hydrogel may be an animal-free hydrogel. The animal-free hydrogel may include a polysaccharide matrix. The hydrogel may include a cellulose matrix. The hydrogel may include cellulose fibers. The cellulose may be nanofiber cellulose. The hydrogel may have neutral cellulose fibers. The hydrogel may have anionic cellulose fiber charges. The hydrogel may be Animal-free hydrogel (UPM Biomedicals). The hydrogel may be a transparent hydrogel. The hydrogel may include a working concentration range of 0.1-1.5 wt % or 0.2-1.0 wt %. The hydrogel may be a liquid hydrogel. The liquid hydrogel may be a cold liquid hydrogel.

[0100] The animal-free hydrogel may include a self-assembling peptide (SAP). The SAP may be Synthetic peptide hydrogel. SAP can be (for example) RADA16 peptide or RLDL-16 peptide. RADA16 is H-(ArgAlaAspAla) 4-OH. Spontaneous and reversible self-assembly of RADA 16 molecules occurs in acidic solutions (pH 2-4, pH ~ <3.7) to produce nanofibers. The RADA16 chemical structure has 16 amino acids as a 4-amino acid sequence repeated in sequence. Contains R (positively charged arginine), A (hydrophobic alanine) and D (negatively charged aspartic acid) residues. SAP can be RLDL-16, H-(ArgLeuAspLeu) 4 -OH. The extracellular matrix-like fibers form a viscous, transparent aqueous solution at low concentrations, such as about 0.1 to about 2.5 wt %.

[0101] Under certain conditions, the hydrogel can be a liquid hydrogel. For example, hydrogels such as Matrigel remain liquid at low temperatures below room temperature, or at 0 to 10°C, 1 to 8°C, or about 4°C. After heating to room temperature or incubation temperature, Matrigel solidifies. Regarding self-assembling peptides, under neutral conditions, for example, RADA16 is mainly in the form of fibrils, which are composed of stacked β-pleated sheets; under acidic conditions, RADA16 fibrils are decomposed into monolayers.

[0102] In some methods of the present disclosure, a biocompatible oil may be used. The biocompatible oil may be used to make the suspended hydrogel encapsulating the embryoid bodies have a shape that is closer to a spherical shape. The biocompatible oil may be used to transport the suspended hydrogel encapsulating the embryoid bodies.

[0103] The density of the biocompatible oil is typically different from that of water. The density of the biocompatible oil may be different from that of water. The density of the biocompatible oil may be higher or lower than that of water.

[0104] The density of the biocompatible oil can be higher than that of water. In some examples, the biocompatible oil can be a halogenated hydrocarbon oil. The halogenated hydrocarbon oil can be a fluorinated hydrocarbon. In some examples, the biocompatible oil can be 1-methoxyheptafluoropropane (such as HFE7000 or Novec TM 7000, 3M) or 3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-trifluoromethyl-hexane (such as Novec TM 7500, 3M).

[0105] The fluorinated hydrocarbon may be a perfluorinated hydrocarbon. The term "perfluorinated hydrocarbon" refers to an organic fluorine compound that includes only carbon-fluorine and CC bonds and potential functional groups, heteroatoms and associated hydrogen atoms. Common functional groups may include -OH, -CO 2 H, -Cl, O and SO 3 H.

[0106] The density (d) of the perfluorinated hydrocarbon at 25°C may be greater than 1, 1.2, 1.3, or 1.5, or in the range of 1.1-2.0, 1.2-1.95, or 1.5-1.95. The perfluorinated hydrocarbon may be 3M TM Fluorinert TM FC perfluorochemical (3M Company). Fluorinert FC compounds may include Fluorinert FC-40 (perfluorochemical, C5-C18 (CAS RN 86508-42-1), with a density (d) of 1.85 g / cm at 25°C. 3 ), Fluorinert FC-70 (CAS RN 338-84-1, tris(undecylfluoropentyl)amine, d 1.94 g / cm 3 ), Fluorinert FC-770(d 1.79g / cm 3 ), Fluorinert FC-43 (CAS RN: 311-89-7, perfluorotributylamine, d 1.86), etc. In some embodiments, the kinematic viscosity of the perfluorinated hydrocarbon at 25° C. is greater than 0.7 cSt, >1.1 cSt, or >2.0 cSt.

[0107] In some examples, the boiling point of the biocompatible oil is above 37°C, or above 40°C. The density of the biocompatible oil can be lower than that of water. A biocompatible oil having a density lower than that of water can be, for example, silicone oil or mineral oil. Silicone oil is a term used to describe a group of hydrophobic polymers and monomer compounds including silicon oxygen bonds, also known as organosiloxanes. Silicone oil can be any liquid polymerized siloxane with organic side chains. For example, silicone oil can be polydimethylsiloxane (d0.96 g / cm 3). The silicone oil can be a cyclosiloxane, such as decamethylcyclopentasiloxane (d of 0.96 at 20°C), or cyclomethicone D6 (d 0.98). The side chains can include not only methyl groups, but also phenyl, vinyl and / or trifluoropropyl groups. The differences in silicone oils can include molecular weight (MW), linear chain length, side chain type, chain termination, and chain size distribution. Silicone oils with higher viscosity can have a lower emulsification tendency, see Barca et al., 2014, Silicone oil: different physical properties and clinical applications, BioMed Res Int, article id 502143. The viscosity of the silicone oil at 25 degrees Celsius can be in the range of about 5-10,000 cSt, 100 cSt-5,000 cSt, or about 1,000 cSt to about 5,000 cSt. The biocompatible oil can be an oil for tissue culture, such as liquid paraffin oil, white mineral oil (e.g., ORIGIO liquid paraffin for tissue culture, ). The density of white mineral oil is 0.8-0.87g / cm 3 In some examples, the biocompatible oil can be a vegetable oil. The vegetable oil can be, for example, olive oil, corn oil, argan oil, linseed oil, or a mixture thereof. Vegetable oils and mixtures are described, for example, in S Said et al., 2013, Can J Physiol Pharmacol 91:812-817. Vegetable oils can be commercially available, for example, from Sigma-Aldrich in St. Louis, Missouri, USA.

[0108] The embodiment described in one aspect of the present disclosure is not limited to the described aspect. The embodiment can also be applied to different aspects of the present disclosure as long as the embodiment does not hinder the operation of these aspects of the present disclosure for its intended purpose.

[0109] Example

[0110] Example 1. Embryoid body embedding using a separate well microplate

[0111] In this example, an automated process of embedding embryoid bodies in hydrogel was performed. Embryoid bodies were prepared according to the Lancaster and Knoblich protocol (2014) and then processed as follows: Figure 5 The automated method of the present disclosure is shown.

[0112] like Figure 5 As shown, EB generation from human iPSCs was initiated on day 0 using hES medium in an automated stem cell culture plate, the medium was changed, and EB development was monitored by germ layer differentiation imaging on days 0-4.

[0113] In brief, automated stem cell culture was started on days 0-4 using an exemplary 6-well culture plate in hES culture medium using induced pluripotent stem cells (iPSC-derived cells) grown in 2D culture until about 70-80% fusion. The colony was disrupted, including brief exposure to enzymes and EDTA (e.g., dispase, washing, then trypsin / EDTA, washing and suspension) to obtain a cell suspension. EBs were raised and exposed to a first culture medium (e.g., hES culture medium containing ROCK inhibitor (1:100) and low bFGF 4 / ng / mL). The culture medium was replaced regularly. The development of cells was monitored by imaging. In some examples, when the EB diameter was ~350-600 μm, the culture medium was replaced with hES without ROCK inhibitor. When the EB diameter was about 500-600 μm, the appearance of the EB became brighter with smooth edges, indicating germ layer differentiation.

[0114] At about day 4, single cells were transferred to a 96-well plate with a low-adsorption U-bottom. On days 4-8, the medium was replaced with a neural induction medium and changed regularly. Cells were monitored by imaging of the induced neural ectoderm. At about day 8-9, the EBs became brighter on the outside, indicating neural ectoderm differentiation. The resulting differentiated EBs were transferred to the main wells of a separate well microplate by automated pipetting to be embedded in a cold liquid hydrogel (Matrigel).

[0115] like Figure 2As shown, automatic embedding of EBs is performed. At about day 8-9, the differentiated embryoid body (EB) 203 is transferred to the main well 212 of the separate well microplate 200 containing the first culture medium 209 by a pipette arm. During the differentiation process, the EBs are fed by the automatic culture medium that is regularly replaced from the secondary well 215 through the microchannel 218. When the embryoid body 203 is ready to be embedded, the plate 202 is tilted for a few seconds until the organoid is located near the channel 218 (the middle wall in the figure). After that, the plate 204 is repositioned in a normal horizontal position and the first culture medium 209 is removed from the feed well. When the culture medium in the well is emptied, the plate 206 is tilted again, and cold Matrigel 211 is added to the corners near the EB 203 and the channel 218 by a pipette arm. After standing at room temperature for 5 minutes, the plate is transferred to an incubator at 37°C for ~30 minutes. Once the Matrigel solidifies, a second culture medium 213 (cerebral organoid differentiation medium without vitamin A) is added from the secondary (feed) well 215 through channel 218, and the solidified hydrogel encapsulating the embryoid bodies is resuspended in the second culture medium 213 in the separation well microplate 210. The separation well microplate is moved to a large feeding incubator with a tilt function, and the culture medium (cerebral organoid differentiation medium with vitamin A) is automatically replaced and monitored by imaging on days 10-50. On days 50-51, the resulting brain organoids are ready for compound testing, including compound addition and measurement, for example, using image analysis by the ImageXpress Micro Confocal High-Content Imaging system (Molecular Devices LLC) or a high-throughput cell screening instrument such as the FLIPR Penta High-Throughput Cell Screening System (Molecular Devices LLC).

[0116] The following steps are generally used to automatically embed EBs in hydrogels.

[0117] 1. Use a pipette to move the embryoid bodies into the main wells of the separation plate, and tilt the separation plate to move the embryoid bodies to the separation channel.

[0118] 2. Remove the culture medium through the feed port in a horizontal position.

[0119] 3. Tilt the plate and add liquid Matrigel to the main wells to encapsulate the embryoid bodies.

[0120] 4. Heat the plate to 37°C and keep for 30 minutes to allow the Matrigel to solidify.

[0121] 5. Add culture medium through the feed channel to allow the embedded embryoid bodies to detach from the bottom of the plate.

[0122] 6. (Optional step) Tilt the plate in the other direction to move the embryoid bodies away from the separation channel.

[0123] 7. (Optional step) Add more medium to feed the embryoid bodies.

[0124] 8. (Optional step) Transferring the embryoid bodies to a bioreactor for maturation.

[0125] Figure 3 Representative images of correctly differentiated embryoid bodies into brain organoids are shown in Figure . Starting from day 10, organoids were embedded in Matrigel to support the expansion of neuroepithelial cells. In this case, the previously described workflow was used ( Figure 2 ). Note that at day 3 after embedding, the formation of many epithelial buds indicates good differentiation.

[0126] Example 2. Use of Split Well Microplates and Oil Embedding of Embryoid Bodies

[0127] In this example, embryoid bodies are automatically embedded in a hydrogel (eg, Matrigel) using a biocompatible oil with a different density than the hydrogel.

[0128] The process is similar to Figure 2 The procedure shown, except that after pipetting Matrigel onto embryoid bodies to embed the EBs in Matrigel, a biocompatible oil with a different density than the Matrigel was added to the main wells via pipette, thereby reshaping the Matrigel from a half-moon shape to a more spherical shape. Two types of biocompatible oils were tested. Fluorinert TM The density and specific gravity of FC40 (C5-C18 perfluorinated compound, density 1.845-1.895 g / mL at 25°C, bp 165°C) are higher than water, and the density / specific gravity of silicone oil is lower than water. The same pipette tip is placed inside the Matrigel and used to slowly aspirate the Matrigel and oil. Figure 4 As shown, Matrigel is located on top of the pipette tip in a separate layer above the high-density biocompatible oil. The oil and Matrigel containing the organoids are slowly pushed back into the wells. The plate is then heated to an incubation temperature of 37°C to solidify the Matrigel. The oil is removed through the secondary wells (feed wells) and the wells are washed with culture medium at room temperature. Figure 4 As shown in the lower right image, more medium is added to feed the embryoid bodies. The split-well microplate is moved to a large feeding incubator with a tilt function for feeding and maintaining organoids and monitoring them from day 10 to 50.

[0129] The following steps are usually taken.

[0130] 1. Place embryoid bodies in the main wells of a separate well plate.

[0131] 2. Tilt the plate so that the EBs move away from the separation channel (towards the opposite side) so that the organoids do not come loose when the medium is removed.

[0132] 3. Remove the culture medium through the feed port in a horizontal position.

[0133] 4. Tilt the plate and add cold liquid Matrigel to the corners of the main wells to encapsulate the embryoid bodies.

[0134] 5. Adding biocompatible oil to the main pores reshapes the liquid hydrogel from a half-moon shape to a more spherical shape.

[0135] 6. Place the same pipette tip inside the Matrigel and slowly aspirate the Matrigel and oil.

[0136] 7. The Matrigel will be at the top of the tip and the oil will be at the bottom due to density ( Figure 4 (lower middle picture).

[0137] 8. Slowly push the oil and Matrigel containing the organoids back into the main well.

[0138] 9. Heat the plate to 37°C for 30 minutes to solidify the Matrigel.

[0139] 10. Remove oil through the feed channel and then wash once with culture medium at room temperature.

[0140] 11. Finally, add more medium to feed the embryoid bodies.

[0141] 12. (Optional step) Transferring the embryoid bodies to a bioreactor for maturation.

[0142] Example 3. Use of various hydrogels in embryoid body embedding

[0143] Apart from (Corning), the method of the present disclosure also uses plant-based hydrogels, and type I collagen hydrogels (from rats). Matrigel, plant-based hydrogels, and type I collagen hydrogels all performed similarly. Each hydrogel had a lower density than the biocompatible oil and formed spherical hydrogels when exposed to the biocompatible oil. Figures 6A-6C Shown is an image of embryoid bodies in a hydrogel above biocompatible oil in a pipette tip. Fig. 6A Fluorinert in pipette tips TM FC-40 biocompatible oil is embedded on the top of the spherical Brain embryoid bodies in hydrogel. Figure 6B Fluorinert in pipette tips TM FC40 biocompatible oil is embedded on the top of the spherical Brain embryoid bodies in cellulose hydrogel. Figure 6C Fluorinert in pipette tips TM Brain embryoid bodies embedded on top of FC40 biocompatible oil in spherical type I collagen (rat) hydrogels. The use of biocompatible oil allowed the EBs to be embedded in the spherical hydrogels in each hydrogel tested.

[0144] Example 4. Embryoid body embedding using a standard 96-well plate and an advanced pipetting protocol

[0145] In this example, automated embryoid body embedding was performed with oil and immediate transfer to a bioreactor. The following automated steps were performed.

[0146] 1. Add embryoid bodies to culture medium in the main wells of a separate well microplate.

[0147] 2. Tilt the plate to move the EBs away from the separation channel (towards the opposite side) so that the organoids do not come loose during the removal of the medium.

[0148] 3. After repositioning the plate to a horizontal position, remove the culture medium through the feed port.

[0149] 4. Tilt the plate and add cold Matrigel via pipette to the corners of the main wells to encapsulate the embryoid bodies.

[0150] 5. Add biocompatible oil to the main wells to reshape the hydrogel from a half-moon shape to a more spherical shape.

[0151] 6. Place the same pipette tip inside the Matrigel and use to slowly aspirate the Matrigel and oil.

[0152] 7. The Matrigel will be at the top of the tip and the oil will be at the bottom due to density ( Figure 4 , lower middle figure).

[0153] 8. Transfer the EBs embedded in Matrigel and oil to an oil bath heated to 37 °C with flow that carries the embedded organoids into the bioreactor.

[0154] 9. Once the Matrigel with embryoid bodies (= future organoids) has solidified, empty the bioreactor with the filter, wash once to remove possible residual oil and add the final culture medium.

[0155] 10. (Optional Step 9) The organoids will enter the fluid system where the embedded organoids in the oil flow will meet normal culture medium. Due to the density difference, the oil will be drained out through the filtration system. When the embedded organoid is in complete culture medium, it will be redirected to the bioreactor.

[0156] Terms

[0157] Item 1. A method for culturing an organoid, the method comprising

[0158] Adding embryoid bodies to a first culture medium in a main well of a separation well microplate, wherein the separation well microplate comprises a plurality of well units, each well unit comprising a main well, a secondary well, and one or more microchannels connecting the main well to the secondary well;

[0159] removing the first culture medium from the well unit through the secondary well;

[0160] adding a liquid hydrogel to encapsulate the embryoid bodies;

[0161] incubating the plate for a first period of time to solidify the hydrogel encapsulating the embryoid bodies; and

[0162] A second culture medium is added to the secondary wells so that the solidified hydrogel encapsulating the embryoid bodies is separated from the plate and suspended in the second culture medium.

[0163] Clause 2. The method according to Clause 1, wherein adding the embryoid bodies to the first culture medium in the main well comprises automated pipetting.

[0164] Clause 3. The method according to clause 1 or 2, further comprising

[0165] Prior to removing the first culture medium, tilting the separation well microplate to move the embryoid bodies to the corners of the main wells, optionally wherein tilting comprises automatically tilting the separation well microplate.

[0166] Item 4. A method according to Item 3, wherein tilting comprises moving the embryoid bodies toward the microchannel, optionally wherein tilting comprises 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 about 10 degrees from horizontal.

[0167] Clause 5. The method of clause 3, wherein tilting comprises moving the embryoid bodies away from the microchannel, optionally, tilting comprises tilting the plate to an angle of 1 to 60 degrees, 5 to 15 degrees, 7 to 12 degrees, or about 10 degrees from horizontal.

[0168] Clause 6. The method according to any one of clauses 3 to 5, further comprising

[0169] Before removing the second medium, return the microplate to a horizontal position.

[0170] Item 7. A method according to any one of items 1 to 6, wherein adding the liquid hydrogel to encapsulate the embryoid body comprises image-guided pipetting of the liquid hydrogel, optionally wherein the image-guided pipetting is automated image-guided pipetting comprising delivering the liquid hydrogel to the top of at least a portion of the embryoid body.

[0171] Clause 8. The method according to any one of clauses 1 to 7, further comprising cooling the microwell plate prior to adding the liquid hydrogel, optionally wherein the cooling is auto-cooling to a temperature below room temperature, or to a temperature in the range of 4 to 8 degrees Celsius.

[0172] Clause 9. The method according to any one of clauses 1 to 7, further comprising heating the microplate to an incubation temperature prior to adding the liquid hydrogel, optionally wherein the heating is automated.

[0173] Clause 10. A method according to any one of clauses 1 to 9, comprising:

[0174] After adding the second culture medium, the microplate is further tilted to allow the solidified hydrogel encapsulating the embryoid bodies to move away from the microchannels, optionally wherein the further tilting comprises automatic further tilting.

[0175] Item 11. A method according to any one of items 1 to 10, wherein incubation is performed for a first time period to solidify the hydrogel, the time period being 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, wherein the microwell plate is maintained at room temperature for a second time period prior to the incubation step.

[0176] Clause 12. The method according to any one of clauses 1 to 11, further comprising

[0177] The second culture medium in the well unit was replaced regularly through the secondary wells to feed the embryoid bodies.

[0178] Clause 13. The method according to any one of clauses 1 to 12, further comprising

[0179] After adding the liquid hydrogel to encapsulate the embryoid bodies and prior to incubation, a biocompatible oil is introduced into the main wells, optionally wherein introducing comprises automatically pipetting the biocompatible oil into the main wells.

[0180] Clause 14. The method according to clause 13, wherein the method comprises

[0181] A pipette tip is positioned within the hydrogel, and the hydrogel and oil encapsulating the embryoid bodies are aspirated into the pipette tip, optionally wherein the positioning is image-guided.

[0182] Clause 15. The method of clause 14, wherein the pumping 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.

[0183] Clause 16. The method according to clause 14 or 15, further comprising

[0184] The oil and the hydrogel encapsulating the EBs were pushed from the pipette tip back into the main well.

[0185] Clause 17. The method of clause 16, wherein the pushing 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.

[0186] Clause 18. The method according to any one of clauses 13 to 17, further comprising

[0187] After the hydrogel encapsulating the embryoid bodies is solidified, extracting the biocompatible oil from the main wells through the secondary wells; and

[0188] The well unit is washed with a wash medium prior to adding the second medium, optionally wherein the wash medium is at room temperature.

[0189] Item 19. The method according to any one of items 13 to 18, wherein the introduction of the biocompatible oil into the primary pores causes the hydrogel encapsulating the embryoid bodies to become more spherical.

[0190] Clause 20. The method according to any one of clauses 1 to 19, further comprising

[0191] The encapsulated embryoid bodies embedded in the solidified hydrogel were transferred to a bioreactor for maturation.

[0192] Clause 21. The method according to any one of clauses 13 to 20, further comprising

[0193] The hydrogel encapsulating the embryoid bodies and the biocompatible oil are transferred to an oil bath, which is heated to an incubation temperature to solidify the hydrogel encapsulating the embryoid bodies.

[0194] Clause 22. The method of Clause 21, wherein the oil pool comprises a stream that transfers the hydrogel encapsulating the embryoid bodies to a bioreactor, the bioreactor optionally comprising a filter.

[0195] Clause 23. The method according to clause 22, further comprising

[0196] draining the oil from the bioreactor, wherein the filter retains the solidified hydrogel encapsulating the embryoid bodies;

[0197] washing the bioreactor and retaining the solidified hydrogel encapsulating the embryoid bodies; and

[0198] A third culture medium was added to the bioreactor to feed the washed hydrogel-encapsulated embryoid bodies.

[0199] Clause 24. The method according to clause 22, wherein the hydrogel encapsulating the embryoid bodies is transferred to the oil pool flow in the bioreactor and meets the third culture medium; and the method further comprises

[0200] allowing the mixture of the oil and the third medium to separate; and

[0201] removing the oil layer through a filtration system to maintain the solidified hydrogel encapsulating the embryoid bodies in the third culture medium; and

[0202] The solidified hydrogel encapsulating the embryoid bodies in the third culture medium is introduced into the bioreactor.

[0203] Clause 25. The method according to any one of clauses 1 to 24, wherein the method is an automated method, wherein adding embryoid bodies to the first culture medium, removing the first culture medium from the well units, adding the liquid hydrogel, and adding the second culture medium each comprise automated pipetting.

[0204] Clause 26. The method according to any one of Clauses 1 to 25, wherein the embryoid bodies are differentiated embryoid bodies exhibiting germ layer differentiation.

[0205] Clause 27. The method according to any one of Clauses 1 to 26, wherein the organoid is a brain organoid.

[0206] Clause 28. The method according to Clause 27, wherein the brain organoid is a cerebral organoid.

[0207] Item 29. A method according to any one of items 1 to 28, wherein the hydrogel is selected from rat 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, polylactic acid / (co)glycolic acid, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptides and ELP (elastin-like protein).

[0208] Clause 30. The method according to any one of Clauses 1 to 29, wherein the liquid hydrogel is a cold liquid hydrogel, optionally at a temperature below about 10°C.

[0209] Clause 31. The method according to any one of Clauses 13 to 30, wherein the biocompatible oil has a density different from that of water.

[0210] Clause 32. The method of Clause 31, wherein the biocompatible oil has a higher density than water.

[0211] Clause 33. The method according to Clause 32, wherein the biocompatible oil is a halogenated hydrocarbon, optionally a perfluorinated hydrocarbon.

[0212] Clause 34. The method of Clause 31, wherein the biocompatible oil has a density less than water.

[0213] Clause 35. The method according to Clause 34, wherein the biocompatible oil is silicone oil, mineral oil or vegetable oil.

Claims

1. A method for culturing an organoid, the method comprising: Adding embryoid bodies to a first culture medium in a main well of a separation well microplate, wherein the separation well microplate comprises a plurality of well units, each well unit comprising a main well, a subsidiary well, and one or more microchannels connecting the main well to the subsidiary well; removing a 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 of time to solidify the hydrogel encapsulating the embryoid bodies; and Adding a second culture medium to the secondary wells so that the solidified hydrogel encapsulating the embryoid bodies is separated from the plate and suspended in the second culture medium, optionally, Wherein adding the embryoid bodies to the first culture medium in the main well comprises automatic pipetting.

2. The method according to claim 1, further comprising: Prior to removing the first culture medium, tilting the separation well microplate to move the embryoid bodies to the corner of the main well, optionally, wherein the tilting comprises automatically tilting the separation well microplate, optionally, wherein the tilting comprises moving the embryoid bodies toward the microchannel, optionally, Wherein the tilting comprises 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 about 10 degrees from the horizontal position.

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

4. The method according to claim 2 or 3, further comprising: Before removing the second culture medium, the microplate is returned to a horizontal position.

5. The method of any one of claims 1 to 4, wherein adding the liquid hydrogel to encapsulate the embryoid body comprises image-guided pipetting of the liquid hydrogel, optionally wherein the image-guided pipetting is automated image-guided pipetting comprising delivering the liquid hydrogel to the top of at least a portion of the embryoid body.

6. The method according to any one of claims 1 to 5, further comprising cooling the microplate prior to adding the liquid hydrogel, optionally wherein the cooling is auto-cooling to a temperature below room temperature, or to a temperature in the range of 4 to 8 degrees Celsius.

7. The method according to any one of claims 1 to 5, further comprising heating the microplate to an incubation temperature prior to adding the liquid hydrogel, optionally wherein the heating is automated.

8. The method according to any one of claims 1 to 7, comprising: After adding the second culture medium, the microplate is further tilted to move the solidified hydrogel encapsulating the embryoid bodies away from the microchannels, optionally wherein the further tilting comprises automatic further tilting.

9. The method of any one of claims 1 to 8, wherein incubating to solidify the hydrogel is performed for a first time 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, optionally wherein prior to the incubation step, the microplate is maintained at room temperature for a second time period.

10. The method according to any one of claims 1 to 9, further comprising: The second culture medium in the well unit is regularly replaced through the secondary well to feed the embryoid bodies.

11. The method according to any one of claims 1 to 10, further comprising: After adding the liquid hydrogel to encapsulate the embryoid bodies and before the incubation, a biocompatible oil is introduced into the main well, optionally, wherein the introducing comprises automatically pipetting the biocompatible oil into the main well, optionally, wherein the introduction of the biocompatible oil into the main well causes the hydrogel encapsulating the embryoid bodies to become closer to a spherical shape.

12. The method of claim 11, wherein the introducing comprises: Positioning a pipette tip within the hydrogel and aspirating the hydrogel encapsulating embryoid bodies and the oil into the pipette tip, optionally wherein the positioning is image-guided positioning, optionally wherein 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: The oil and the hydrogel encapsulating embryoid bodies are pushed from the pipette tip back into the main well, optionally wherein the pushing 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.

14. The method according to claim 11 or 12, further comprising: After the hydrogel encapsulating the embryoid bodies is solidified, extracting the biocompatible oil from the main hole through the secondary hole; and The well unit is washed with a wash medium prior to adding the second medium, optionally wherein the wash medium is at room temperature.

15. The method according to any one of claims 1 to 14, further comprising: The encapsulated embryoid bodies embedded in the solidified hydrogel were transferred to a bioreactor for maturation.

16. The method according to any one of claims 11 to 15, further comprising: The hydrogel encapsulating embryoid bodies and the biocompatible oil are transferred to an oil pool, the oil pool is heated to an incubation temperature to solidify the hydrogel encapsulating embryoid bodies, optionally wherein the oil pool comprises a flow to transfer the hydrogel encapsulating embryoid bodies to a bioreactor, the bioreactor optionally comprising a filter.

17. The method according to claim 16, further comprising: draining the oil from the bioreactor, wherein the filter retains the solidified hydrogel encapsulating the embryoid bodies; washing the bioreactor and retaining the solidified hydrogel encapsulating the embryoid bodies; and A third culture medium was added to the bioreactor to feed the washed hydrogel-encapsulated embryoid bodies.

18. The method according to claim 16, wherein the oil pool flow that transfers the hydrogel encapsulating the embryoid bodies into the bioreactor meets a third culture medium; and the method further comprises: allowing the mixture of oil and the third culture medium to separate; and removing the oil layer through a filtration system to keep the solidified hydrogel encapsulating the embryoid bodies in the third culture medium; and The solidified hydrogel encapsulating the embryoid bodies in the third culture medium is introduced into the bioreactor.

19. The method according to any one of claims 1 to 18, wherein the method is an automated method, wherein adding the embryoid bodies to the first culture medium, removing the first culture medium from the well units, adding the liquid hydrogel, and adding the second culture medium each comprise automated pipetting.

20. The method of any one of claims 1 to 19, wherein the embryoid bodies are differentiated embryoid bodies exhibiting germ layer differentiation, optionally wherein the organoids are brain organoids, optionally wherein the brain organoids are cerebral organoids.

21. The method of any one of claims 1 to 20, wherein the hydrogel is selected from the group consisting of murine 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, polylactic acid / (co)glycolic acid, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptides, and ELP (elastin-like protein), optionally wherein the liquid hydrogel is a cold liquid hydrogel, optionally at a temperature below about 10°C.

22. The method of any one of claims 11 to 21, wherein the biocompatible oil has a density different from that of water.

23. The method of claim 22, wherein the biocompatible oil has a density higher than water, optionally wherein the biocompatible oil is a halogenated hydrocarbon, optionally a perfluorinated hydrocarbon.

24. The method of claim 22, wherein the biocompatible oil has a density less than water, optionally wherein the biocompatible oil is silicone oil, mineral oil or vegetable oil.

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