Automated cell passage

Abstract

Disclosed is a method for identifying the location of extracellular matrix structures on the surface of a culture plate using a substantially automated process. Also disclosed is an automated method for removing cells from extracellular matrix structures in which cells are growing. The method includes providing cultured cells within extracellular matrix structures on the surface of a culture plate and disrupting the extracellular matrix structures at X,Y positions on the surface of the culture plate using a pipette tip associated with a liquid handler, using a substantially automated process.

Description

Technical Field

[0001] The present disclosure relates to the culturing of cells. More specifically, the present disclosure relates to the use of procedures for subculturing and culturing cells such as organoids.

[0002] (Cross - Reference to Related Applications) This application claims the benefit and priority of PCT International Application No. PCT / US2023 / 064645, filed on March 17, 2023, and U.S. Provisional Application No. 63 / 511,930, filed on July 5, 2023, which are each hereby incorporated by reference in their entirety. This application was filed as a PCT international application on March 16, 2024.

Background Art

[0003] Culturing cells in a three - dimensional (3D) environment results in cell behavior and morphology that more closely matches that observed in the human body. The 3D hydrogels / hydro scaffolds used for this type of culturing have unique properties, and cells can be deposited at specific locations within the 3D space and remain in place over long periods of time. This enables the creation of co - culture environments in which structures (such as spheroids, tumoroids, organoids, and / or other multicellular bodies) and cell - cell interactions and development over time can be observed.

[0004] Organoids are three - dimensional miniature models of organs that are grown ex vivo (in a laboratory setting) from stem cells or tissue samples. They mimic the cellular or tissue structure and function of actual organs, providing a unique platform for studying human biology and disease. Organoids should not be considered organ replicas. Organoids are not vascularized, i.e., they lack blood vessels and other system components.

[0005] Organoids typically originate from pluripotent stem cells (such as embryonic stem cells or induced pluripotent stem cells) or adult stem cells (such as intestinal stem cells). These cells are induced to differentiate into specific cell types and self-organize into complex structures resembling organs.

[0006] Organoids consist of multiple cell types and exhibit an organized tissue architecture containing distinctly different cell layers, similar to the organs they model. Depending on the organ being studied, they may include functional units such as blood vessels, glands, or neural networks. [Overview of the Initiative] [Means for solving the problem]

[0007] In a first aspect, the present disclosure provides a substantially automated process for identifying the location of extracellular matrix (ECM) structures on the surface of a culture plate.

[0008] In a second aspect, the disclosure provides an automated method for removing cells from extracellular matrix structures on the surface of a culture plate on which cells are grown. The substantially automated process is used to disrupt the extracellular matrix structures at X,Y positions on the surface of the culture plate using a pipette tip associated with a liquid handler.

[0009] In a third aspect, the present disclosure provides a method for organoid fragmentation. The method includes the steps of: centrifuging a cell solution in a container to form a cell pellet and a supernatant; aspirating a first volume of the supernatant at a first fixed height and a first fixed velocity from the bottom of the container; and aspirating a second volume of the supernatant at a second fixed height and a second fixed velocity from the bottom of the container.

[0010] In a fourth aspect, the present disclosure provides a method for organoid fragmentation. The method comprises an organoid pellet and a first volume of supernatant in a container, and includes the steps of: a) sequentially aspirating and dispensing a second volume multiple times at a first fixed height from the bottom of the container; b) aspirating a third volume at a second fixed height from the bottom of the container and dispensing the third volume back into the container; and c) repeating steps a) and b).

[0011] In a fifth aspect, the present disclosure provides a method for seeding a cell solution. The method includes a) mixing about 60% of the volume of the cell solution multiple times via aspiration and dispensing; b) aspirating a portion of the mixed solution and providing the aspirated solution and the remaining unaspirated solution; c) dispensing at least a portion of the aspirated solution into a culture plate that has not been previously used to culture cells; and d) repeating steps a) to c) with the remaining unaspirated solution.

[0012] Further aspects and embodiments are provided in the aforementioned drawings, detailed description, and claims. [Brief explanation of the drawing]

[0013] The following drawings are provided to illustrate an embodiment described herein. The drawings are merely illustrative and are not intended to limit the scope of the claimed invention or to show all potential features or embodiments of the claimed invention. The drawings are not necessarily drawn to scale, and in some cases, elements of the drawings may be enlarged relative to other elements of the drawings for illustrative purposes.

[0014] [Figure 1] Figure 1 is a schematic representation of one embodiment of a substantially automated process for determining the x,y coordinates of various ECM structures.

[0015] [Figure 2]Figure 2 is an example of an image of a cell culture surface that is used to create an image mask.

[0016] [Figure 3] Figure 3 is an example of a set of image masks that are used to generate a series of x, y coordinates that define the location of an ECM structure.

[0017] [Figure 4] Figure 4 is a representation of the step of transferring the x, y coordinates of an ECM structure to a liquid handler.

[0018] [Figure 5] Figure 5 is a schematic representation of one embodiment of a substantially automated process for creating an image mask.

[0019] [Figure 6] Figure 6 is an example of contrast inversion for an image of a culture plate.

[0020] [Figure 7] Figure 7 is an example of image blurring for an image of a culture plate.

[0021] [Figure 8] Figure 8 is an example of mask creation for an image of a culture plate.

[0022] [Figure 9] Figure 9 is an example of a combination of masks of individual cells and / or cell clusters for generating a mask for the overall ECM structure for an image of a culture plate.

[0023] [Figure 10] Figure 10 depicts a micrograph showing a comparison of wells before and after passage using a liquid handler. Most of the organoids have been recovered. What remains in them is a monolayer of cells that is not as dense as the organoids and adheres to the bottom of the well.

[0024] [Figure 11] Figure 11 shows exemplary pipette movement patterns at a given location, calculated for hydrogel dome rupture using the aforementioned formula, involving 6 steps (upper left panel), 7 steps (upper right panel), 8 steps (left panel of the second row), and 9 steps (right panel of the second row). When using a theta value of 15.7, a spiral pattern of steps is formed for each pattern. Also depicted are exemplary pipette movement patterns at a given location, calculated for hydrogel dome rupture using the aforementioned formula, involving 10 steps (left panel of the third row), 11 steps (right panel of the third row), 12 steps (lower left panel), and 13 steps (lower right panel). When using a theta value of 15.7, a spiral pattern of steps is formed for each pattern.

[0025] [Figure 12] Figure 12 shows an exemplary pipette movement pattern calculated for hydrogel dome rupture using the aforementioned formula, where diameter (mm) = 8, number of steps = 13, alpha = diameter / 1.8, and theta = 30. When using a theta value of 30, the step pattern does not appear very spiral, and a star-like pattern begins to emerge.

[0026] [Figure 13] Figure 13 shows a depiction of 17 fracture locations.

[0027] [Figure 14] Figure 14 shows a 96-deep well plate (left) and an adapter for cooling (right).

[0028] [Figure 15] Figure 15 is a micrograph showing the growth of organoids subculturing and seeding via a liquid handler on (A) day 1, (B) day 2, (C) day 5, and (D) day 7.

[0029] [Figure 16] Figure 16 is a table showing the compositions required to prepare 50% Matrigel.

[0030] [Figure 17] Figure 17 is a table showing the compositions required to produce 80% Matrigel.

[0031] [Figure 18] Figure 18 shows an example of a mask generated from IN Carta to detect organoids from the background and measure the mask area.

[0032] [Figure 19] Figure 19 is a table detailing the area corresponding to Figure 18. [Modes for carrying out the invention]

[0033] Detailed explanation The following description enumerates various aspects and embodiments of the invention disclosed herein. No particular embodiment is intended to define the scope of the invention. Rather, the embodiments provide non-limiting examples of various compositions and methods that fall within the scope of the claimed invention. The description is to be read carefully from the perspective of those skilled in the art. Therefore, information that is well known to those skilled in the art is not necessarily included.

[0034] definition The following terms and phrases have the meanings set forth below unless otherwise provided herein. This disclosure may adopt other terms and phrases not expressly defined herein. Such other terms and phrases shall have the meanings they would have to a person skilled in the art within the context of this disclosure. In some cases, a term or phrase may be defined in singular or plural form. In such cases, understand that any term in the singular form includes its plural counterpart, and vice versa, unless expressly indicated otherwise.

[0035] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly determines otherwise. For example, a reference to “substituent” includes a single substituent as well as two or more substituents and their equivalents.

[0036] The term "and / or" refers to and encompasses all possible combinations of one or more of the listed items that are associated with each other.

[0037] The term "approximately" means that when referring to measurable values ​​such as the amount of a compound, dosage, time, temperature, and equivalents, it includes variations of 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0038] As used herein, “for example,” “for instance,” “such as,” or “including” means introducing examples that further clarify a more general subject matter. Unless otherwise expressly indicated, such examples are provided solely as an aid to understanding the embodiments illustrated in this disclosure and are not intended to be limiting in any way. Furthermore, these phrases do not indicate any preference of any kind regarding the embodiments disclosed.

[0039] The terms “comprises” and / or “comprising,” when used herein, specify the presence of the described features, integers, steps, actions, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, actions, elements, components, and / or groups thereof. Unless otherwise defined, all terms, including technical and scientific terms used in the description, have the same meaning as those generally understood by those skilled in the art in which this disclosure is made. In the event of any conflict in technical terminology, this specification shall prevail.

[0040] As used herein, "substantially automated" refers to a process, system, or task in which a machine or algorithm handles the majority of the work with little to no human intervention required for its core function. Human supervision or input may still be necessary, but this is limited to exceptional circumstances, specific decision points, or for quality control.

[0041] All patents, patent applications, and publications referenced herein are incorporated as a whole by reference.

[0042] The embodiments described in one aspect of this disclosure are not limited to the described aspect. The embodiments may also apply to other aspects of this disclosure, provided that they do not prevent those aspects of this disclosure from working for their intended purpose.

[0043] Organoids, spheroids, tumoroids, and three-dimensional (3D) cell culture models are useful in many applications, including disease modeling and regenerative medicine. 3D cell models, such as organoids and spheroids, can be useful for a deeper understanding of complex biology in physiologically relevant contexts, as cells often retain their natural shape and appropriate spatial orientation, while 2D models of cells grown in sheets or monolayers may be less successful. Gene and protein expression in 3D cell cultures can more closely mimic gene and protein expression. For example, 3D cell cultures may be useful for drug target identification, lead compound identification, compound optimization, preclinical validation, solid tumor modeling, genetic disease modeling, drug discovery, precision medicine, biofunctional chips, and bioprinting.

[0044] The term "spheroid" refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate consisting of one or more cell types that grow and proliferate, may exhibit enhanced physiological responses, but does not undergo differentiation or self-organization. Common cell sources for spheroids are primary tissues or immortalized cell lines. Spheroids can fill the gap between monolayers and complex organs.

[0045] The term "tumoroid" typically refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate consisting of one or more cell types derived from a primary tumor taken from a cancer patient, which can mimic the human tumor microenvironment. Tumoroids may be useful for research on novel anticancer drugs or for use in precision medicine in the field of oncology. Cancer cell lines may include, for example, bladder, breast, colon, hematopoietic and lymphatic systems, liver, lung, ovary, prostate, skin, and equivalents.

[0046] The term "stem cell" refers to an undifferentiated cell that has 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 adult human cells can be reprogrammed into an embryonic stem cell-like state called induced pluripotent stem cells (iPSCs). For example, pluripotent stem cells found in adults or in the umbilical cord of newborns can develop into cells that make up the organ systems from which they originate. When grown under certain cell culture conditions, pluripotent stem cells may remain undifferentiated. To produce differentiated cells, the chemical composition of the culture medium may be altered, the surface of the culture dish may be changed, or the cells may be modified by forcing the expression of certain genes.

[0047] As used herein, “organoid” means a self-organizing structure exhibiting multiple cell types that replicates the architecture and function of a specific organ or tissue. An organoid is a three-dimensional (3D) multicellular in vitro tissue culture aggregate consisting of one or more cell types, where the cells spontaneously self-organize into appropriately differentiated functional cell types and progenitor cells that resemble their in vivo counterparts in at least one aspect. Organoids mimic their corresponding in vivo organs. Organoids may 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 formulated to resemble many of the complexities of organs and are therefore useful for studying the pathogenesis and treatment of diseases. Organoid technology has recently emerged as an essential tool for both basic and biomedical research. Organoid cultures can be selected from different types of target organs, such as the lungs, small intestine, colon, stomach, pancreas, liver, kidney, skin, bone marrow, blood-brain barrier, brain, heart, and equivalents.

[0048] Development: Organoids typically originate from pluripotent stem cells (such as embryonic stem cells or induced pluripotent stem cells) or adult stem cells (such as intestinal stem cells). These cells are induced to differentiate into specific cell types and self-organize into complex structures resembling organs.

[0049] Structure and Complexity: Organoids consist of multiple cell types and exhibit an organized tissue architecture, containing distinctly different cell layers, similar to the organs they model. Depending on the organ being studied, they may include functional units such as blood vessels, glands, or neural networks.

[0050] Disease Modeling: Organoids are typically used in disease research by providing a more accurate representation of human physiology compared to conventional cell cultures or animal models. Researchers can generate organoids from patient-derived cells, enabling them to study diseases in a personalized manner and investigate the underlying mechanisms, drug responses, and potential therapeutic strategies.

[0051] Drug Discovery and Testing: Organoids offer significant predictive value for drug development. They can be used to test the safety and efficacy of potential drugs before clinical trials, reducing reliance on animal studies and potentially improving the success rate of clinical trials. Organoids also enable the study of drug responses in specific patient populations, leading to personalized drug approaches.

[0052] Regenerative Medicine: Organoids hold promise in regenerative medicine and tissue engineering. By using patient-derived stem cells, scientists aim to create functional organoids that can be implanted into individuals with organ damage or impairment and serve as personalized, functional replacement tissues or organs.

[0053] Organoids do not fully replicate the complexity of entire organs, and they lack interactions with the body's circulatory, immune, and nervous systems. Furthermore, scaling organoid production to a clinically relevant scale remains a challenge.

[0054] Organoid types: There are numerous types of organoids developed to study different organs, including brain organoids (cerebral organoids), liver organoids, kidney organoids, intestinal organoids, lung organoids, and more.

[0055] Organoid research is a rapidly developing field with enormous potential to advance our understanding of human biology, disease mechanisms, and therapeutic development. It bridges the gap between overly simplistic in vitro models and in vivo studies, contributing to advancements in personalized medicine and regenerative therapy.

[0056] Organoids are propagated through a series of steps, involving initial establishment of the organoid culture and subsequent subculturing or subculturing to maintain and expand the organoid population. The specific propagation method may vary depending on the organoid type used and the research protocol. However, a general overview of the process is as follows:

[0057] Establishment of organoid cultures: Isolation and preparation of stem cells or tissues: Organoids may be derived from pluripotent stem cells (PSCs) or adult stem cells, depending on the organ being studied. PSCs can be obtained from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), while adult stem cells are often isolated from specific tissues. Organoids can also be obtained from existing organoids, for example, by fragmentation of existing organoids.

[0058] For example, gastric organoid tissue cultures may be derived from sources such as adult mice, adults, hPSCs, and equivalents. Depending on the source, the gastric organoid tissue cultures may employ stem cell culture conditions (niche factors) that include culture medium components such as one or more of EGF, Noggin, R-spondin, Wnt-3A, FGF10, and equivalents. Differentiation culture conditions may include EGF, R-spondin, EGF, R-spondin, and equivalents, depending on the source.

[0059] In another example, the small intestinal organoid tissue culture may be derived from sources such as adult mice, adults, hPSCs, and equivalents. Depending on the source, the small intestinal organoid tissue culture may employ stem cell culture conditions (niche factors) including one or more culture medium components such as EGF, Noggin, R-spondin, Wnt-3A, TGF-beta inhibitors, p38 inhibitors, and equivalents. Differentiation culture conditions may include EGF, Noggin, TGF-beta inhibitors, and equivalents, depending on the source.

[0060] As a further example, the colon organoid tissue culture may be derived from sources such as adult mice, adults, and equivalents. Depending on the source, the colon organoid tissue culture may employ stem cell culture conditions (niche factors) that include one or more culture medium components such as EGF, Noggin, R-spondin, Wnt-3A TGF-beta inhibitor, p38 inhibitor, and equivalents. Depending on the source, the differentiation culture conditions may include EGF, Noggin, TGF-beta inhibitor, and equivalents.

[0061] In another example, pancreatic organoid tissue cultures may be derived from sources such as adult mice, adults, and equivalents. Depending on the source, the pancreatic organoid tissue cultures may employ stem cell culture conditions (niche factors) that include one or more culture medium components such as EGF, Noggin, R-spondin, Wnt-3A, FGF10, nicotinamide, and equivalents. Differentiation culture conditions may include EGF, Noggin, R-spondin, Wnt-3A, and equivalents, depending on the source.

[0062] As a further example, liver organoid tissue cultures may be derived from sources such as adult mice, adults, and equivalents. Depending on the source, the liver organoid tissue cultures may employ stem cell culture conditions (niche factors) that include one or more culture medium components such as EGF, Noggin, R-spondin, Wnt-3A, FGF10, HGF, nicotinamide, and equivalents. Differentiation culture conditions may include EGF, Noggin, R-spondin, Wnt-3A, FGF10, TGF-beta inhibitors, Notch inhibitors, BMP7, and equivalents, depending on the source.

[0063] Differentiation and encapsulation: Stem cells can be induced to differentiate into desired cell types using specific growth factors and culture conditions. Differentiated cells can then be embedded or encapsulated in a supporting extracellular matrix (ECM), such as Matrigel or other hydrogels, which mimics the specific microenvironment of the organ.

[0064] Culture medium: Organoids require specialized culture media that provide the necessary nutrients, growth factors, and signaling molecules for their growth and development. Culture media are typically supplemented with factors that promote organ-specific differentiation and expansion. Different media components may be required depending on the type of source cells used and the type of differentiation to be achieved. Growth factors such as EGF, Noggin (NOG), R-spondin (RSPO1), HGF, BMP, FGF, and their equivalents may be essential components of organoid media. Tissue culture media may contain growth factors. Growth factors may be produced by supporting cells. Growth factors may be recombinant growth factors. Recombinant growth factor proteins for organoid culture may include, for example, recombinant human EGF protein, recombinant HGF proteins such as human HGF protein, cynomolgus monkey HGF protein, human FGF10, human Noggin / NOG protein, human RSPO1 protein, human BMP-2 protein, and their equivalents. Additional recombinant growth factors for organoid culture may include, for example, EGF, FGF2, FGF7, FGF9, FGF10, HGF, NOG, RSPO1, RSPO3, activin A, BMP2, and BMP4, and their equivalents. Recombinant growth factor proteins for tissue culture media or organoid culture may be commercially available, for example, from Sino Biological, Inc. or Thermo Fisher Scientific.

[0065] 3D cell models such as organoids and spheroids can be cultured in tissue culture media containing hydrogels, such as in a hydrogel dome within the culture medium.

[0066] The term "hydrogel" or "multiple hydrogels" refers to an extracellular matrix useful for culturing organoids. Hydrogels may include, for example, mouse EHS sarcoma matrix commercially available as Matrigel (Corning), Cultex (Trevigen), Geltrex (Gibco), type I collagen, fibrin, hyaluronic acid (HA), gelatin methacrylate (GelMA), decellularized matrix, or biopolymers such as alginic acid, silk, nanocellulose, self-assembling peptides such as polyethylene glycol (PEG) and RADA16 / PuraMatrix bQ13, engineered materials such as poly(lactic acid / (co)glycol) acids, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptides, ELP (elastin-like proteins), or combinations of these polymers.

[0067] Organoid Passaging or Subculture: Organoid Dissociation: To passage organoids, they can first be enzymatically or mechanically dissociated into smaller clusters or single cells. This step aims to degrade the organoid structure and disperse the cells for further culture.

[0068] Re-implantation or re-seeding: Dissociated organoid cells may then be re-implanted or re-seeded in unused ECM and / or re-seeded in new culture vessels, depending on the specific protocol. Re-implantation involves mixing the cells with unused ECM, allowing them to self-organize into new organoids. Re-seeding may involve transferring the cells onto a new culture dish or plate.

[0069] One example of ECM is a hydrogel. The term "hydrogel" or "multiple hydrogels" refers to an extracellular matrix useful for culturing organoids. Hydrogels may include, for example, commercially available mouse EHS sarcoma matrix such as Matrigel (Corning), Cultex (Trevigen), and Geltrex (Gibco), type I collagen, fibrin, hyaluronic acid (HA), gelatin methacrylate (GelMA), decellularized matrix, or biopolymers such as alginic acid, silk, and nanocellulose, self-assembling peptides such as polyethylene glycol (PEG) and RADA16 / PuraMatrix bQ13, engineered materials such as poly(lactic acid / (co)glycol) acids, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptides, ELP (elastin-like proteins), or combinations of these polymers.

[0070] Growth and Differentiation: After passaging, organoid cells are cultured in unused medium to allow for growth and maturation. Culture conditions may be adjusted to promote specific differentiation pathways or maintain the desired cell type.

[0071] Maintenance and long-term culture:

[0072] Regular feeding and medium changes: Cells require regular feeding and medium changes to maintain optimal conditions for growth and survival. This typically involves replacing a portion of the culture medium with unused medium containing the necessary nutrients and growth factors.

[0073] Monitoring and Quality Control: Cells should be regularly monitored for their growth, structural integrity, and cell type composition. Quality control measures, such as genetic or protein analysis, may be employed to ensure that organoids maintain their desired properties and functionality.

[0074] It is important to note that propagation methods may vary depending on the specific research protocol, organoid type, and desired application. Protocol optimization and customization are often performed to achieve the best results for each specific organoid model.

[0075] Typically, cell passaging is performed manually. This is often time-consuming. In addition, manual passaging can be inconsistent and less precise due to individual tendencies and procedures. Nevertheless, general protocols for passaging exist. The main steps of the manual process for passaging cells growing in ECM (Corning) (登録商標) Matrisol (登録商標) This includes the steps of destroying the cells, dissociating / fragmenting them, and seeding the cells.

[0076] Partly due to the time-consuming nature of manually subculturing cells grown in the ECM, what is described herein is a method for subculturing cells. The main step of the process is subculturing the cells in the ECM (e.g., Corning (登録商標) Matrisol (登録商標) This includes steps of destroying structures (such as domes), dissociating / fragmenting cells, and seeding cells.

[0077] Identifying the ECM location

[0078] The methods described herein are for the removal of cells from extracellular matrix structures. These methods include the steps of: providing cultured cells within an extracellular matrix structure on the surface of a culture plate; identifying the location of the extracellular structure on the surface of the culture plate; and disrupting the extracellular matrix structure by puncture or cutting using a substantially automated process. The methods described herein may be used in any combination with other methods described herein, including, but not limited to, methods for removing cells from the ECM, methods for removing supernatant from a cell pellet, methods for fragmenting cell clusters, and methods for seeding cell solutions.

[0079] During cell culture within an ECM, the cell culture surface may retain one or more ECM structures on which the cells are growing. One embodiment of an ECM structure is a dome. The methods and systems described herein may include a step of identifying the location of an ECM structure prior to the step of disrupting the ECM structure. In embodiments, the identification of the ECM structure location may be performed primarily by a human or using a substantially automated process.

[0080] When a human is locating an ECM structure on the surface of a cell culture plate, the location may be described in x,y coordinates corresponding to the location on the surface. The x,y coordinates are recorded in a format that is machine-readable / readable. In a non-limiting embodiment, a human may identify the locations of various ECM structures on the culture plate through visual inspection (e.g., using a microscope or other device). The human may then record or have the x,y locations of the ECM structures recorded. Embodiments of human recording include writing or inputting the locations into any type of storage device. When a human has the x,y locations recorded, this may be through the pressing of a button or other signal that results in the automatic recording of the x,y locations determined by visual inspection.

[0081] If the step of locating the ECM structure is performed using a substantially automated process, the location may be described in x,y coordinates corresponding to the location on the surface. The x,y coordinates are recorded in a format that is machine-readable / readable. Embodiments of locating using a substantially automated process include, but are not limited to, detection methods using one or more sensors. For example, the x,y coordinates of various ECM structures may be detected using optical, electromagnetic, or physical / mechanical sensors. In the case of optical sensors, the x,y coordinates of various ECM structures may be located by distinguishing them from the background using standard image processing or artificial intelligence / machine learning techniques. In one embodiment, an image of the cell culture surface is captured and used to identify the x,y coordinates of various ECM structures. If physical / mechanical sensors are used, the x,y coordinates of various ECM structures may be located by deforming or hindering a probe moving across or near the surface of the culture plate. In a further embodiment, the x,y coordinates of various ECM structures may be identified by the use of electromagnetic sensors. Examples of such sensors include, but are not limited to, laser, lidar, radar, infrared, optical diffraction, and / or stimulus / emission-based sensors.

[0082] In some embodiments, the x,y coordinates of various ECM structures may be identified by the location of any cell. If cells are primarily located within and / or clustered within the ECM structure, the identification of cells and / or clusters of cells provides the location of the ECM structure. In embodiments, the identification of cell locations may be by any means, including detection using optical, electromagnetic, or physical / mechanical sensors, as described with respect to the direct detection of the ECM structure.

[0083] Figure 1 provides a schematic representation of one embodiment of a substantially automated process for determining the x,y coordinates of various ECM structures. The first step provided herein is to image a culture plate. The image may be tiled, if required, for masking or for setting up a coordinate grid. The tiled image may be used to cover cells and thus create a mask for identifying the location of ECM structures. Figure 2 provides an embodiment of an image of a cell culture surface used to create an image mask.

[0084] Once a mask is created, the x,y coordinates of the mask may be generated. Figure 3 provides an example of a set of image masks used to generate a set of x,y coordinates that define the location of an ECM structure. Once the x,y coordinates of the ECM structure are determined, the location may optionally be provided to another device, such as a liquid handler. The transfer of coordinates to the liquid handler is depicted in Figure 4.

[0085] One embodiment of the creation of an image mask for an ECM structure is schematically represented in Figure 5. In this embodiment, a culture plate is imaged, the image is tiled, and then the contrast of the image is inverted. An example of such contrast inversion for an image of a culture plate is provided in Figure 6. Once the inverted contrast image is obtained, the image may be blurred to fill in the spaces between cells and between them in the image. An example of such image blurring is provided in Figure 7. A mask may then be generated from the blurred image. An example of such mask creation from an image of cells is provided in Figure 8. Masks of individual cells and / or cell clusters may then be combined to generate a mask for an overall ECM structure. An example of combining masks of individual cells and / or cell clusters to generate a mask for an overall ECM structure is provided in Figure 9.

[0086] As will be understood, any combination of the steps described above is assumed herein. If the six steps described in Figure 5 are steps A to F in order, a particular embodiment includes steps A, B, and F. If the six steps described in Figure 5 are steps A to F in order, a particular embodiment includes steps A, C, and F. If the six steps described in Figure 5 are steps A to F in order, a particular embodiment includes steps A, D, and F. If the six steps described in Figure 5 are steps A to F in order, a particular embodiment includes steps A, E, and F. If the six steps described in Figure 5 are steps A to F in order, a particular embodiment includes steps A, B, C, and F. If the six steps described in Figure 5 are steps A to F in order, a particular embodiment includes steps A, B, D, and F. If the six steps described in Figure 5 are steps A to F in order, a particular embodiment includes steps A, B, E, and F. If the six steps described in Figure 5 are steps A through F in order, a particular embodiment includes steps A, C, D, and F. If the six steps described in Figure 5 are steps A through F in order, a particular embodiment includes steps A, C, E, and F. If the six steps described in Figure 5 are steps A through F in order, a particular embodiment includes steps A, D, E, and F. If the six steps described in Figure 5 are steps A through F in order, a particular embodiment includes steps A, B, C, D, and F. If the six steps described in Figure 5 are steps A through F in order, a particular embodiment includes steps A, B, C, E, and F. If the six steps described in Figure 5 are steps A through F in order, a particular embodiment includes steps A, B, D, E, and F. If the six steps described in Figure 5 are steps A through F in order, a particular embodiment includes steps A, C, D, E, and F. If the six steps shown in Figure 5 are steps A through F in order, then a particular embodiment includes steps A, B, C, D, E, and F.

[0087] Cell removal from ECM

[0088] Described herein is a substantially automated process for removing cells from an ECM structure. In one embodiment, a liquid handler is provided with the x,y coordinates of an ECM structure on a cell culture plate. In some aspects, the x,y coordinates of an ECM structure may be obtained via a substantially automated process as described herein, or may be provided through an unautomated process. The liquid handler uses an associated pipette tip to disrupt the ECM structure at the x,y coordinates and thus release the cells from within the ECM structure. In examples, the pipette tip may disrupt the ECM structure in any manner, including, but not limited to, puncturing the ECM structure, destroying the ECM structure, or cutting through the ECM structure with the pipette tip and / or dragging it. The methods described herein may be used in any combination with other methods described herein, including, but not limited to, methods for identifying the location of an ECM structure, removing supernatant from a cell pellet, fragmenting cell clusters, and seeding a solution of cells.

[0089] Figure 10 is a micrograph showing a comparison of wells before (left) and after (right) removal from the ECM as described herein. The majority of the cells have been recovered. What remains is a monolayer of cells that adhere to the surface of the cell culture plate, at a lower density than other cell clusters.

[0090] In the methods described herein, the x,y coordinates may be located at or near the x,y center of the ECM structure. For example, the pipette tip may be located at the center of the ECM structure, or within 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm from the center of the x,y center of the ECM structure, or within 0 to 2 mm or 0.01 to 1.5 mm.

[0091] The method involves moving the pipette tip through the dome in a number of steps, each step including predetermined X,Y positions on a cell culture plate, disrupting / disrupting the ECM structure, optionally liquefying the hydrogel dome, and releasing target cells from the ECM structure. At one or more, two or more, numerous, or each of the predetermined positions, the pipette tip may be aspirated and / or the contents of the pipette tip may be dispensed.

[0092] The predetermined positions X and Y within the well may be calculated to include the following, X (number of steps) = X well center + alpha / step × number of steps × COS(theta / step × number of steps), and Y (number of steps) = Y well center + alpha / step × number of steps × SIN(theta / step × number of steps), where alpha = ECM structure diameter (mm) / 1.8, step = the number of steps required to move the pipette tip through the ECM structure, X well center = x position of the ECM structure center on the surface of the culture plate, Y well center = y position of the ECM structure center on the surface of the culture plate, and theta = a constant value between 5 and 50. In some cases, the number of steps is an integer between 2 and 20, 3 and 18, 4 and 17, 5 and 15, 6 and 12, or 8 and 10. In other cases, theta is a constant between 5 and 50, 10 and 45, 15 and 40, 15 and 35, or any number in between. In some cases, theta is selected from a group consisting of 5, 10, 12, 13, 14, 15, 15.7, 16, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, or 50, or any number in between. The x,y center position of the ECM structure in the well may be determined based on the original seeding position in the well, the volume of the ECM structure deposited in the well, and / or imaging of the ECM structure. The pipette tip may be used to puncture the ECM structure and / or aspirate and / or dispense liquid within the ECM structure. For example, the pipette tip may be used to apply, remove, dispense, or aspirate liquid or gas (e.g., air) within the ECM structure. Inputs to the system may include the x,y coordinates of the seeding position in the well and the known volume of the ECM structure. Another input may be the number of steps for ECM structure disruption or pipette aspiration / dispensing. The diameter of the ECM structure may be calculated based on its volume. The volume of the ECM structure may be determined by any method. For example, the volume may be determined based on the volume of ECM deposited to form the ECM structure, or by determining one or more of the overall shape, height, and circumference of the ECM structure. The diameter of the ECM structure may be determined by imaging. The diameter of the ECM structure may be any suitable diameter.For example, the diameter of the ECM structure may be 1–12 mm, 2–10 mm, 3–8 mm, or 3–6 mm. The number of steps may be any appropriate number. For example, the number of steps may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or 2–20, 3–18, 4–17, 5–15, 6–12, or 8–10. For example, the input includes the diameter of the hydrogel dome, which in this case is 8 mm, the number of steps, which is 8, and the X,Y coordinates of the well center position. In some cases, the number of steps / positions may be minimized to effectively break up the hydrogel in order to minimize the process time. The output may include an automated sequence / pattern of puncture / aspiration positions within the dome using a pipette with a liquid handler. In some embodiments, the pattern of puncture / aspiration positions may be a helical pattern. In some embodiments, the pattern of puncture / aspiration positions is a star pattern. In some embodiments, the pattern of puncture / aspiration positions is a zigzag pattern. At each position, the automated pipette may aspirate within the dome. The pipette tip may be dragged below the surface of the dome between steps / positions. For example, the pipette tip position on the Z axis may be maintained below the surface of the hydrogel dome between steps / positions for efficient dome disruption. Between steps, the pipette tip position on the Z axis may be maintained at or near the bottom of the hydrogel dome. For example, the pipette tip may be maintained below the surface between steps or during aspiration, within 0.5, 1.0, 1.5, or 2 mm from the surface, at 0.5, 1.0, 1.5, or 2 mm near the center of the Z axis at the bottom, or within 0.5, 1.0, 1.5, or 2 mm, or within 0.5, 1.0, 1.5, or 2 mm from the bottom of the dome. The Z-axis position may be determined by the volume of the ECM structure or by imaging the cell culture surface. Exemplary Figure 11 shows the output sequence of helical X,Y coordinates of an automated pipette in a well, using a well center position, an 8 mm dome diameter, and 6-13 step inputs, with a theta of 15.7 (Figure 12) or 30 (Figure 12), resulting in effective disruption of the hydrogel dome.For example, a solid hydrogel can be converted into a liquid, thereby allowing cells to be separated from the hydrogel 106. The ECM structure can be converted into a liquid, for example, by vigorous pipetting or shear force of a liquid handler, with or without a decrease in temperature from the incubation temperature (approximately 37°C) to approximately 4°C to approximately 10°C, or to approximately 10°C. Dissociation reagents can be added to dissociate cell clusters such as organoids into stem cells and several additional cell types.

[0093] In certain embodiments, the fracture point includes a central fracture point at the center of the ECM structure and several other fracture points radially arranged around the central fracture point. Multiple concentric radial arrangements of fracture points may be arranged around the central fracture point, depending on the size of the ECM structure. In certain embodiments, one, two, three, or more concentric radial arrangements may be present. An example of a fracture point with two concentric radial arrangements is provided in Figure 13.

[0094] In some embodiments, the radius of the radial arrangement of fracture points is adjustable. The flexibility to change the radius of the circles ensures that ECM structures with different sizes (volumes) can be efficiently fractured, and multiple concentric radial arrangement designs ensure that ECM structures with larger sizes can be efficiently fractured.

[0095] In embodiments, the x,y positions of multiple ECM structures may be known. If multiple ECM structures are present on the surface of a culture plate, imaging or other techniques as described herein may be used to determine a pattern of predetermined x and y positions located over or on all of the ECM structures on the culture plate. Such patterns include radial arrangements, spiral patterns, star patterns, zigzag patterns, and combinations thereof. Movement of the pipette tip to each of the predetermined x,y positions may then cause over or on all of the ECM structures on the culture plate to be disrupted.

[0096] In one embodiment, the x,y locations of multiple ECM structures on the surface of a cell culture plate are determined as described herein. A substantially automated process may be used to create patterns for the disruption of one or more or all of the ECM structures. Various combinations of patterns may be used to disrupt the ECM structures. In an embodiment of such an embodiment, the pipette tip moves in a helical pattern to each of the multiple ECM structures, and while in each ECM structure, the pipette tip moves through a star pattern of predetermined x,y locations within each ECM structure to disrupt it, and then continues in a radial pattern to the next ECM structure.

[0097] The method may further include the step of disrupting the extracellular matrix structure by dispensing a liquid, such as culture medium, at a fixed height above the bottom of the culture plate and at a fixed speed to at least one location within the extracellular matrix.

[0098] In some embodiments, liquid aspiration / dispensing is performed by an automated / computer-controlled liquid handler / pipette / pipette. In embodiments, the entire method of removing cells from extracellular matrix structures is automated and / or performed without human intervention. In certain embodiments, the method is performed by / within an automated cell culture apparatus.

[0099] In some embodiments, the height at which the pipette is held above the surface of the culture plate is referred to as the fixed height. In some embodiments, the fixed height is approximately 1 mm to 3 mm above the bottom of the culture plate on which the ECM structure is supported. In some embodiments, the fixed height is approximately 2 mm above the bottom of the culture plate. In certain embodiments, the fixed height is 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 mm above the bottom of the culture plate. In some embodiments, the liquid is dispensed at a rate of approximately 400 μL / sec to 600 μL / sec. In some embodiments, the culture medium is dispensed at a rate of approximately 500 μL / sec. In certain embodiments, the culture medium is dispensed at a rate of 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, or 600 μL / second. In some embodiments, the cell dissociation reagent is added to the center of the well. In some embodiments, the cell dissociation reagent is added at a fixed height of approximately 0.05 mm to approximately 0.3 mm above the bottom of the culture plate surface. In some embodiments, the cell dissociation reagent is added at a fixed height of approximately 0.1 mm above the bottom of the culture plate surface.

[0100] In certain embodiments, the method includes the following steps: 1) Using a 1,000 μL tip, aspirate 500 μL of culture medium from the culture plate along the side of the well at a fixed height of 0 mm from the bottom. The x,y coordinate aspiration position sequence is pre-set along the side of the well to avoid aspirating the ECM structure that is concentrated in the center of the well. 2) Add 500 μL of Gentle Cell Dissociation Reagent or Corning Cell Recovery Solution to the center of the well at a fixed height of 0.1 mm from the bottom. 3) Wait for 60 seconds, then proceed to the next step. 4) The x,y predetermined position sequence is configured for subsequent use (see, for example, Figures 11-13). 500 μL of solution is aspirated from the side of the well at a fixed position 0 mm above the bottom and dispensed at a rate of 500 μL / sec (fastest rate) into one or more of the predetermined x,y positions at a fixed height 2 mm above the bottom to disrupt the ECM structure. This step is repeated until all predetermined x,y positions are covered. 6) The plate is tiled at 10°. 7) 500 μL of cell solution is aspirated at a fixed height 0.1 mm above the bottom and transferred to the wells in the 96-deep well plate at a fixed height 0.5 mm above the bottom. 8) The plate is returned to the horizontal position. 9) Steps 2, 5, 6, and 7 are repeated for a second wash and any residual cells are collected.

[0101] Removal of supernatant from cell pellet The method described herein is for removing supernatant from a cell pellet. The method includes the steps of: centrifuging a cell solution in a container to form a cell pellet and supernatant; aspirating a first volume of the supernatant at a first fixed height and a first fixed speed from the bottom of the container; and aspirating a second volume of the supernatant at a second fixed height and a second fixed speed from the bottom of the container. The method described herein may be used in any combination with other methods described herein, including, but not limited to, methods for identifying ECM structural locations, methods for removing cells from the ECM, methods for fragmenting cell clusters, and methods for seeding cell solutions.

[0102] In some embodiments, the cell pellet includes organoids. In certain embodiments, the organoids are obtained by removing cells (e.g., organoids) from an ECM structure as described herein. In some embodiments, aspiration and dispensing of the culture medium are performed by an automated / computer-controlled liquid handler / pipette / pipette. In some embodiments, the entire method of removing the supernatant from the cell pellet is automated and / or performed without human intervention. In certain embodiments, this method is performed by / in an automated cell culture apparatus.

[0103] In some embodiments, the pipette is held at a first height above the bottom of the container for aspirating a first volume of supernatant, and then moved to a lower height for aspirating a second volume of supernatant. This variable pipette operating speed scheme is designed to ensure that as much supernatant as possible is aspirated while minimizing pellet loss at the bottom of the container. In some embodiments, the first volume of supernatant may be discarded or retained in the pipette prior to aspirating the second volume of supernatant.

[0104] In some embodiments, the first fixed height is approximately 20 mm to 40 mm. In some embodiments, the first fixed height is approximately 30 mm. In certain embodiments, the first fixed height is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 mm. In some embodiments, the second fixed height is approximately 2 mm to 8 mm. In some embodiments, the second fixed height is approximately 5 mm. In certain embodiments, the second fixed height is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 mm. In some embodiments, the first volume is approximately 400 μL to approximately 600 μL. In certain embodiments, the first volume is 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, or 600 μL. In some embodiments, the first volume is about 500 μL. In some embodiments, the second volume is about 300 μL to about 500 μL. In some embodiments, the second volume is about 400 μL. In certain embodiments, the second volume is 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 μL.

[0105] Computer-controlled pipettes are capable of aspirating and dispensing liquids at various speeds. Aspirating at too high a speed, or at too close a distance to organoids, can result in the loss of those organoids; therefore, the aspiration speed was carefully determined to minimize damage. In some embodiments, the first speed is approximately 200 μL / sec to approximately 300 μL / sec. In some embodiments, the first speed is approximately 250 μL / sec. In certain embodiments, the first speed is 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 μL / sec. In some embodiments, the second speed is approximately 35 μL / sec to approximately 65 μL / sec. In some embodiments, the second rate is approximately 50 μL / sec. In certain embodiments, the second rate is 35, 40, 45, 50, 55, 60, or 65 μL / sec.

[0106] In one embodiment, the container is a well in a 6, 12, 24, or 96-well plate.

[0107] Fragmentation of cell clusters The method described herein is for the fragmentation of cell clusters. Non-limiting examples of cell clusters include organoids, spheroids, and tumoroids. The method comprises, in a container comprising a cell cluster pellet and a first volume of supernatant, a) sequentially aspirating and dispensing a second volume multiple times at a first fixed height from the bottom of the container; b) aspirating a third volume at a second fixed height from the bottom of the container and dispensing the third volume back into the container; and c) repeating steps a) and b). The method described herein may be used in any combination with other methods described herein, including, but not limited to, methods for identifying ECM structural locations, methods for removing cells from the ECM, methods for removing supernatant from a cell pellet, and methods for seeding a cell solution.

[0108] In some embodiments, cell clusters are obtained by removing cell clusters (e.g., organoids) from an ECM structure as described herein. In some embodiments, aspiration and dispensing of the culture medium are performed by an automated / computer-controlled liquid handler / pipette / pipette. In some embodiments, the entire method of cell cluster fragmentation is automated and / or performed without human intervention. In certain embodiments, the method is performed by / in an automated cell culture apparatus.

[0109] In some embodiments, the first volume is approximately 75 μL to approximately 125 μL. In some embodiments, the first volume is approximately 100 μL. In certain embodiments, the first volume is 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125 μL.

[0110] In some embodiments, the first fixed height is about 0.1 mm to about 0.3 mm. In some embodiments, the first fixed height is about 0.2 mm. In certain embodiments, the first fixed height is 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.3 mm. In some embodiments, the second volume is about 70 μL to about 90 μL. In some embodiments, the second volume is about 80 μL. In certain embodiments, the second volume is 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 μL.

[0111] In some embodiments, the second fixed height is approximately 0.1 mm to approximately 0.3 mm. In some embodiments, the second fixed height is approximately 0.2 mm. In certain embodiments, the second fixed height is 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.3 mm. In some embodiments, the third volume is approximately 75 μL to approximately 105 μL. In some embodiments, the third volume is approximately 90 μL. In certain embodiments, the third volume is 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 μL.

[0112] In some embodiments, in step a), the second volume is aspirated and dispensed about 40 to 60 times. In some embodiments, in step a), the second volume is aspirated and dispensed about 50 times. In certain embodiments, in step a), the second volume is aspirated and dispensed 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 times.

[0113] In some embodiments, steps a) and b) are repeated 2 to 10 times. In some embodiments, steps a) and b) are repeated 4 times. In a particular embodiment, steps a) and b) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0114] In one embodiment, the container is a well in a 6, 12, 24, or 96-well plate.

[0115] In a particular embodiment, the organoid fragmentation step includes: 1) providing 100 μL of organoid solution; 2) fragmenting the cell cluster pellet by pipetting 80 μL 50 times at a fixed height of 0.2 mm from the bottom using a 300 μL pipette tip, and then dispensing 90 μL to be aspirated at a fixed height of 0.2 mm from the bottom and returned to the well; 3) repeating step 15 four times, which results in a total of approximately 200 pipetting operations.

[0116] Fragmentation of bulk cell clusters helps ensure a uniform distribution of cell clusters within the passage vessel. In some embodiments, the pipette tip is a 300 μL tip. The 300 μL tip is used to assist in the fragmentation of bulk cell clusters. In other embodiments, other sizes of pipette tips are also used; for example, in some embodiments, a 1,000 μL pipette tip is used. Cell cluster fragmentation may be performed after two steps, centrifugation and supernatant removal, to improve organoid recovery. This step requires a balance between over-fragmentation and under-fragmentation of cell clusters. Compared to manual passage, which requires repeated fragment size checks using a microscope, the overall fragment size distribution of cell clusters can be standardized with respect to the total number of pipetting operations.

[0117] Seeding of cell solution The method described herein is for seeding a cell solution. The method includes a) mixing approximately 60% of the volume of the cell solution multiple times via aspiration and dispensing; b) aspirating a portion of the mixed solution and providing the aspirated solution and the remaining unaspirated solution; c) dispensing at least a portion of the aspirated solution into a new culture plate; and d) repeating steps a) to c) with the remaining unaspirated solution. The method for seeding cells described herein may be carried out in a substantially automated process. For example, one or more of the above steps a) to c) may be carried out by an automated liquid handler with minimal or no human intervention. The method described herein may be used in any combination with other methods described herein, including, but not limited to, methods for identifying ECM structural locations, methods for removing cells from the ECM, methods for fragmenting cell clusters, and methods for removing supernatant from a cell pellet.

[0118] Figure 15 is a micrograph showing the growth of organoids subculturing and seeding via a liquid handler on (A) day 1, (B) day 2, (C) day 5, and (D) day 7.

[0119] In embodiments, the cell solution may contain cell clusters or fragments thereof. In embodiments, organoids are obtained by removing cells (e.g., organoids) from an ECM structure as described herein. In further embodiments, fragments of cell clusters may be obtained by a cell cluster fragmentation method described herein. In some embodiments, aspiration and dispensing of the culture medium are performed by an automated / computer-controlled liquid handler / pipette / pipette. In embodiments, the entire method of seeding the cell solution is automated and / or performed without human intervention. In certain embodiments, the method is performed by / in an automated cell culture apparatus.

[0120] In some embodiments, the cell solution includes an extracellular matrix. In certain embodiments, the extracellular matrix is ​​a Matrigel or other hydrogel that mimics the organ's intrinsic microenvironment. The amount of Matrigel or other hydrogel may be varied to provide different ECM concentrations for seeding. For example, Figure 16 provides components for creating a 50% Matrigel ECM structure. In further embodiments, Figure 17 provides components for creating an 80% Matrigel ECM structure.

[0121] In some embodiments, mixing via aspiration and dispensing is performed at a fixed height of approximately 2 mm to approximately 4 mm from the bottom of the container in which the cell solution is located. In embodiments, mixing via aspiration and dispensing is performed at a fixed height of approximately 3 mm from the bottom of the container in which the cell solution is located. In certain embodiments, mixing via aspiration and dispensing is performed at a fixed height of 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 2.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4 mm from the bottom of the container in which the cell solution is located. In some embodiments, mixing via aspiration and dispensing is performed at least five times.

[0122] In the embodiment, when step d) is performed, the aspiration and dispensing are carried out at a fixed height from the bottom of the container in which the cell solution is located, lower than the steps preceding steps a) and b). In a particular embodiment, step d) is repeated until the seeding of the cell solution is complete, and each time step d) is performed, the aspiration and dispensing of step a) is carried out at a fixed height lower than the steps immediately preceding steps a) and b). In a more specific embodiment, each time step d) is performed, the aspiration and dispensing of step a) is carried out at a fixed height one-third lower than the steps immediately preceding steps a) and b).

[0123] In an embodiment, step c) includes dispensing at least a portion of the aspirated solution into a culture plate that has not been previously used to culture cells, by dispensing the aspirated solution at a distance of about 0.1 mm to about 0.5 mm from the surface of the culture plate. In an embodiment, step c) includes dispensing at least a portion of the aspirated solution into a culture plate that has not been previously used to culture cells, by dispensing the aspirated solution at a distance of about 0.3 mm from the surface of the culture plate. In a particular embodiment, step c) includes dispensing at least a portion of the aspirated solution into a culture plate that has not been previously used to culture cells, [ka] The process includes the step of dispensing a solution that has been aspirated at a distance of .

[0124] In some embodiments, step c) includes dispensing at least a portion of the aspirated solution into a culture plate that has not been previously used to culture cells, with the step of dispensing the aspirated solution at a rate of approximately 5 μL / sec to approximately 9 μL / sec. In some embodiments, step c) includes dispensing at least a portion of the aspirated solution into a culture plate that has not been previously used to culture cells, with the step of dispensing the aspirated solution at a rate of approximately 7 μL / sec. In certain embodiments, step c) includes dispensing at least a portion of the aspirated solution into a culture plate that has not been previously used to culture cells, with the step of dispensing the aspirated solution at a rate of 5, 5.1, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, or 9 μL / sec.

[0125] To achieve uniform seeding of cell clusters or fragments, it is often beneficial to mix the organoids in solution. Small pipette tips often assist in mixing cell clusters or fragments in solution. In certain embodiments, a 300 μL tip is used for seeding, with the advantage of a large volume for mixing and a small tip opening for small volume seeding.

[0126] In some embodiments, the ECM / cell cluster fragment solution is mixed using aspiration and dispensing of 0.6 × Vremaining, with a maximum of 500 μL of Vremaining being the remaining volume to ensure uniform seeding. In some embodiments, an excess of 40 μL is aspirationed for seeding in addition to the required volume, with the formula Vtotal = nseeding × Vseeding + 40 μL, where nseeding is the number of seedings per process and Vseeding is the volume of the dome per seeding, ensuring sufficient solution for the last seeding in one process. In some embodiments, 10 μL is pre-dispensed to be returned to the cell solution to ensure successful small-volume seeding (such as 7 μL) in the first well between seedings in one process. In some embodiments, the aspiration in step b) occurs at 3 mm from the bottom when Vremaining >= 2 × Vtotal, and at 1 mm from the bottom when Vremaining < 2 × Vtotal, allowing for uniform cell cluster seeding on the destination plate and reducing dead volume.

[0127] In certain embodiments, the method is performed as follows, i.e., as an example, by seeding twice per step into a 96-deep-well plate with a total of 500 μL of Matrigel / cell cluster solution per well and one or more 24-well destination plates: Using a 300 μL tip, mix 300 μL over 5 cycles at a fixed height of 3 mm from the bottom, then aspirate 120 μL at a fixed height of 3 mm from the bottom. Dispense 10 μL back into the 96-deep plate at a slow rate of 7 μL / sec to avoid any air bubbles. Dispense 40 μL into the wells of the destination plate at a slow rate of 7 μL / sec at a fixed height of 0.3 mm from the bottom. Loop until one step of two wells is complete. Discard the tip along with the remaining Matrigel / cell cluster solution. When looping through the remaining wells in the destination plate, the inventors mix with 60% of the volume of the remaining Matrigel / cell cluster solution in the 96 deep plate before aspiration. When the volume of the remaining Matrigel / cell cluster solution is less than 240 μL, the inventors aspiration at a lower height of 1 mm from the bottom.

[0128] In all embodiments, a 96-well deep plate may be used for liquid handling. Instead of using 15 ml or 2 ml tubes that require cap removal, a 96-well deep plate may be used for liquid handling. Here, two objectives exist: 1) The 96-well plate is more suitable for automation with respect to cap removal and transport, while the tubes require cap removal that requires special tools. In addition, an adapter for the 96-well plate allows for cooling of the plate at 4°C, as required for Matrigel (Figure 14).

[0129] Organoid Recovery Rate Analysis: IN Carta software was used as a tool to develop an analytical workflow that quantifies the area of ​​organoids, which is then used to calculate the organoid recovery rate after passage. An example is shown in Figure 18, with the corresponding area values ​​in Figure 19.

[0130] The methods described herein may be used in any combination with other methods described herein. Such methods include: A) Method for identifying the location of ECM structures B) Method for removing cells from the ECM C) Method for removing supernatant from cell pellet D) Methods for fragmenting cell clusters E) Method of seeding cell solution

[0131] The methods described herein include A and B as defined above. The methods described herein include A and C as defined above. The methods described herein include A and D as defined above. The methods described herein include A and E as defined above. The methods described herein include B and C as defined above. The methods described herein include B and D as defined above. The methods described herein include B and E as defined above. The methods described herein include C and D as defined above. The methods described herein include C and E as defined above. The methods described herein include C and E as defined above. The methods described herein include D and E as defined above. The methods described herein include A, B, and C as defined above. The methods described herein include A, B, and D as defined above. The methods described herein include A, B, and E as defined above. The methods described herein include A, C, and D as defined above. The methods described herein include A, C, and E as defined above. The methods described herein include A, D, and E as defined above. The methods described herein include B, C, and D as defined above. The methods described herein include B, C, and E as defined above. The methods described herein include B, C, and E as defined above. The methods described herein include B, D, and E as defined above. The methods described herein include C, D, and E as defined above. The methods described herein include A, B, C, and D as defined above. The methods described herein include A, B, C, and E as defined above. The methods described herein include A, C, D, and E as defined above.The methods described herein include B, C, D, and E as defined above.

[0132] As will be understood by those skilled in the art, various aspects of this technology may be embodied as systems, methods, or computer program products. Therefore, some aspects of this technology may take the form of entirely hardware embodiments, entirely software embodiments (including firmware, resident software, microcode, etc.), or combinations of hardware and software aspects, all of which may be generally referred to herein as circuits, modules, systems, and / or networks. Furthermore, various aspects of this technology may take the form of computer program products embodied in one or more computer-readable media, including computer-readable program code embodied thereon.

[0133] Any combination of one or more computer-readable media may be used. The computer-readable media may be a computer-readable signal medium or a physical computer-readable storage medium. The physical computer-readable storage medium may, for example, be an electronic, magnetic, optical, crystalline, polymer, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any preferred combination thereof. Non-limited embodiments of the physical computer-readable storage medium may include, for example, an electrical connection including one or more wires, a portable computer diskette, a hard disk, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), flash memory, optical fiber, compact disc read-only memory (CD-ROM), optical processor, magnetic processor, or any preferred combination thereof. In the context of this document, the computer-readable storage medium may be any tangible medium that contains or can store programs or data for use by, or in connection with, an instruction execution system, apparatus, and / or device.

[0134] Computer code embodied on a computer-readable medium may be transmitted using any suitable medium, including, but not limited to, wireless, wired, fiber optic cable, radio frequency (RF), or any preferred combination thereof. Computer code for performing operations for aspects of this technology may be written in any static language, such as the C programming language or other similar programming languages. The computer code may run entirely on the user's computing device, partially on the user's computing device, as a standalone software package, partially on the user's computing device and partially on a remote computing device, or entirely on a remote computing device or server. In the latter scenario, the remote computing device may be connected to the user's computing device through any type of network or communication system, including, but not limited to, a local area network (LAN) or wide area network (WAN), or a converged network, or the connection may be to an external computer (e.g., via the Internet using an Internet service provider).

[0135] Various aspects of this technology may be described above with reference to flowcharts and / or block diagrams of methods, apparatus, systems, and computer program products. It should be understood that each block in a flowchart and / or block diagram, and combinations of blocks within a flowchart and / or block diagram, can be implemented by computer program instructions. These computer program instructions may be provided to a general-purpose computer, a special-purpose computer, or a processing device (processor) of another programmable data processing device, in order to produce a machine, such that instructions, which can be executed via a processing device or other programmable data processing device, generate means for implementing the actions / behaviors defined within the blocks of the flowchart and / or block diagram.

[0136] Some computer program instructions may also be stored in computer-readable media, which may instruct a computer, other programmable data processing device, or other device to operate in a particular manner to produce a product, including instructions that implement actions / behaviors defined in a flowchart and / or block diagram. Some computer program instructions may also be loaded onto a computing device, other programmable data processing device, or other device, which may cause a series of operational steps to be performed on the computing device, other programmable device, or other device, to produce a computer implementation process, which may be executed by a computer or other programmable device, to provide one or more processes for implementing actions / behaviors defined in a flowchart and / or block diagram.

[0137] The flowcharts and / or block diagrams in the above figures may illustrate the architecture, functionality, and / or operation of possible implementations of devices, systems, methods, and / or computer program products according to various aspects of this technology. In this regard, blocks in the flowcharts or block diagrams may represent modules, partitions, or portions of code that may contain one or more executable instructions for implementing one or more defined logical functions. It should also be noted that, in some alternative aspects, some functions described within a block may occur in an order other than that described in the diagram. For example, two blocks shown consecutively may actually be executed substantially in parallel, or blocks may be executed in reverse order from time to time, depending on the operations involved. It should also be noted that blocks in block diagrams and / or flowcharts, or combinations of blocks in block diagrams and / or flowcharts, may be implemented by special-purpose hardware-based systems that can perform one or more defined operations or actions, or combinations of special-purpose hardware and computer instructions.

[0138] The first aspect includes an automated method for identifying the location of extracellular matrix structures, which includes identifying the X,Y positions of extracellular matrix structures on the surface of a culture plate using a substantially automated process.

[0139] The second aspect involves disrupting extracellular matrix structures at predetermined X,Y positions on the surface of a culture plate using a substantially automated process with a pipette tip associated with a liquid handler.

[0140] The third aspect includes an automated method for identifying the location of an extracellular matrix structure and disrupting the extracellular matrix structure, the method comprising using a substantially automated process to identify a predetermined location X,Y within the extracellular matrix structure on the surface of a culture plate, and using a substantially automated process to disrupt the extracellular matrix structure at the predetermined location X,Y within the extracellular matrix structure on the surface of the culture plate using a pipette tip associated with a liquid handler.

[0141] The fourth aspect involves identifying the location of extracellular matrix structures on the surface of the culture plate using a substantially automated process prior to disruption.

[0142] A fifth aspect includes the extracellular matrix structure of any aspect listed herein being in the form of a dome.

[0143] The sixth aspect includes the extracellular matrix structure of any aspect listed herein being a hydrogel.

[0144] The seventh aspect includes the fact that a given position X,Y of any aspect listed herein is located at or near the center of an extracellular matrix structure.

[0145] The eighth aspect includes the fact that a predetermined X,Y position of the extracellular matrix structure in any aspect enumerated herein is determined by one or more parameters selected from the group consisting of the original seeding position on the surface of the culture plate and imaging of the extracellular matrix structure on the surface of the culture plate.

[0146] The ninth aspect includes the fact that the diameter of the extracellular matrix structure for any aspect enumerated herein is determined by the volume of extracellular matrix deposited on the surface of the culture plate or by imaging of the extracellular matrix structure on the surface of the culture plate.

[0147] The tenth aspect, in any aspect listed herein, involves moving the pipette tip in a number of steps to release cells from the extracellular matrix structure, each of which moving steps involves additional movement to a predetermined X,Y position within the extracellular matrix structure.

[0148] An eleventh aspect includes the surface of the culture plate of any aspect listed herein being a cell culture well.

[0149] A twelfth aspect includes the fact that the culture plate of any aspect listed herein is a primary well of a well unit of an isolation well microplate.

[0150] The 13th aspect includes, in any aspect listed herein, calculating each of the predetermined X,Y locations on the cell culture surface and any additional predetermined X,Y locations via: X (number of steps) = center of X well + alpha / step × number of steps × cos(theta / step × number of steps), and Y (number of steps) = center of Y well + alpha / step × number of steps × sin(theta / step × number of steps), in the equation, Alpha = extracellular matrix structure diameter (mm) / 1.8, Step = The number of steps required to move the pipette tip through the extracellular matrix structure, which can be any integer between 2 and 20. The center of the X-well is the x-position of the center of the dome within the well. The center of the Y-well is the y-position of the center of the dome within the well. Theta is a constant value between 5 and 50.

[0151] The fourteenth aspect includes, in any aspect listed herein, that the number of predetermined positions is selected from 2 to 20, 3 to 18, 4 to 17, 5 to 15, 6 to 12, or 8 to 10 predetermined positions on X,Y.

[0152] A 15th aspect includes, in any aspect enumerated herein, that a predetermined position X,Y and additional X,Y positions form a pattern within and / or through the extracellular matrix structure, selected from the group consisting of helical patterns, star patterns, and zigzag patterns.

[0153] The sixteenth aspect includes, in any aspect listed herein, that a given X,Y position comprises at least nine X,Y positions, and eight of the X,Y positions are arranged in a circle of a fixed radius from the center of the extracellular matrix.

[0154] The 17th aspect includes, in any aspect enumerated herein, a given position X,Y comprises at least 17 given positions, and two groups of eight given positions X,Y are arranged in two circles of different radii.

[0155] The 18th aspect is that in any aspect listed herein, each of the numerous steps of moving the pipette tip individually is: Move the pipette tip through the extracellular matrix structure to disrupt the extracellular matrix structure, and / or This includes the option of removing the pipette tip from the extracellular matrix structure prior to transfer.

[0156] A 19th aspect is that, in any aspect listed herein, the liquid handler is In one or more, two or more, numerous, or each of the predetermined positions of X and Y, within the extracellular matrix structure, Aspirate the tip of the pipette, and / or This includes dispensing liquid culture medium from the tip of a pipette.

[0157] The 20th aspect involves repeatedly aspirating and dispensing at a given position of each X,Y, once or more than once, two or more than two times, or a number of times, in any aspect listed herein.

[0158] The 21st aspect includes, in any aspect listed herein, aspirating or dispensing at a fixed speed at a fixed height above the bottom of the culture plate.

[0159] The 22nd aspect includes, in any aspect listed herein, that the fixed height is approximately 1 mm to approximately 3 mm above the surface of the culture plate.

[0160] The 23rd aspect includes, in any aspect listed herein, that the fixed height is approximately 2 mm above the surface of the culture plate.

[0161] The 24th aspect includes, in any aspect listed herein, that the liquid medium is dispensed at a rate of about 1 μL / second to about 600 μL / second.

[0162] The 25th aspect includes, in any aspect listed herein, that the culture medium is dispensed at a rate of approximately 500 μL / second.

[0163] The 26th aspect includes, in any aspect listed herein, adding a cell dissociation reagent to an extracellular matrix structure.

[0164] The 27th aspect includes, in any aspect listed herein, adding a cell dissociation reagent to the extracellular matrix structure at a fixed height of about 0.05 mm to about 0.3 mm above the surface of the culture plate.

[0165] The 29th aspect, in any aspect listed herein, includes adding a cell dissociation reagent to the extracellular matrix structure at a fixed height of approximately 0.1 mm above the surface of the culture plate.

[0166] The 30th aspect includes, in any aspect listed herein, aspirating the culture medium from the culture plate prior to dispensing the culture medium.

[0167] The 31st aspect, either alone or in combination with any aspect listed herein, The cell solution is centrifuged in a container to form a cell pellet and a supernatant, A first volume of the supernatant is aspirated at a first fixed height and a first fixed velocity from the bottom of the container, This includes aspirating a second volume of the supernatant at a second fixed height and a second fixed velocity from the bottom of the container.

[0168] Aspect 32 includes, in any aspect enumerated herein, that the cell pellet includes organoids.

[0169] The 33rd aspect includes, in any aspect listed herein, that the first volume is about 400 μL to about 600 μL.

[0170] The 34th aspect includes, in any aspect listed herein, that the first volume is about 500 μL.

[0171] The 35th aspect includes, in any aspect listed herein, a first fixed height of approximately 20 mm to approximately 40 mm from the bottom.

[0172] The 36th aspect includes, in any aspect listed herein, a first fixed height of approximately 30 mm.

[0173] The 37th aspect includes, in any aspect enumerated herein, that the first rate is about 200 μL / sec to about 300 μL / sec.

[0174] The 38th aspect includes, in any aspect enumerated herein, that the first rate is about 250 μL / second.

[0175] The 39th aspect includes, in any aspect listed herein, that the second volume is between approximately 300 μL and approximately 500 μL.

[0176] The 40th aspect includes, in any aspect listed herein, that the second volume is about 400 μL.

[0177] The 41st aspect includes, in any aspect listed herein, a second fixed height of about 2 mm to about 8 mm.

[0178] The 42nd aspect includes, in any aspect listed herein, a second fixed height of approximately 5 mm.

[0179] The 43rd aspect includes, in any aspect enumerated herein, that the second rate is approximately 35 μL / sec to approximately 65 μL / sec.

[0180] The 44th aspect includes, in any aspect enumerated herein, that the second rate is about 50 μL / second.

[0181] The 45th aspect includes, in any aspect enumerated herein, that the container is a well in a 96-well plate.

[0182] The 46th aspect is to provide a container comprising an organoid pellet and a first volume of supernatant, either alone or in combination with any aspect listed herein. a) Aspirating and dispensing a second volume multiple times sequentially at a first fixed height from the bottom of the container, b) At a second fixed height from the bottom of the container, a third volume is drawn out and the third volume is dispensed so as to return to the container, c) including repeating steps a) and b).

[0183] Aspect 47 includes, in any aspect enumerated herein, that the organoid pellet is obtained by aspect 31.

[0184] The 48th aspect includes, in any aspect listed herein, that the first volume is about 75 μL to about 125 μL.

[0185] The 49th aspect includes, in any aspect listed herein, that the first volume is about 100 μL.

[0186] The 50th aspect includes, in any aspect listed herein, that the second volume is about 70 μL to about 90 μL.

[0187] The 51st aspect includes, in any aspect listed herein, that the second volume is about 80 μL.

[0188] The 52nd aspect includes having a first height of about 0.1 mm to about 0.3 mm in any of the aspects listed herein.

[0189] The 53rd aspect includes having a first height of about 0.2 mm in any aspect listed herein.

[0190] The 54th aspect includes, in any aspect listed herein, that the third volume is about 75 μL to about 125 μL.

[0191] The 55th aspect includes, in any aspect listed herein, that the third volume is about 90 μL.

[0192] The 56th aspect includes, in any aspect listed herein, having a second height of approximately 0.1 mm to approximately 0.3 mm.

[0193] The 57th side includes having a second height of approximately 0.2 mm in any of the sides listed herein.

[0194] The 58th aspect includes, in any aspect enumerated herein, step a) comprising aspirating and dispensing the second volume about 40 to about 60 times.

[0195] The 59th aspect includes, in any aspect enumerated herein, that the second volume is aspirated and dispensed about 50 times.

[0196] The 60th aspect includes, in any aspect listed herein, step c) being repeated 2 to 10 times.

[0197] The 61st aspect includes, in any aspect enumerated herein, step c) being repeated four times.

[0198] The 62nd aspect includes, in any aspect enumerated herein, that the container is a well in a 96-well plate.

[0199] Aspect 63, either alone or in combination with any aspect listed herein, a) Mixing approximately 60% of the cell solution volume multiple times via aspiration and dispensing, b) Aspirate a portion of the mixed solution and provide the aspirated solution and the remaining unaspirated solution, c) Dispense at least a portion of the aspirated solution into a culture plate that has not been previously used to culture cells, d) Repeat steps a) to c) using the remaining unaspirated solution.

[0200] Aspect 64 includes, in any aspect listed herein, that the cell solution comprises an organoid or a fragment of an organoid.

[0201] Aspect 65 includes, in any aspect enumerated herein, that the cell solution includes an extracellular matrix.

[0202] Aspect 66, in any aspect enumerated herein, includes the mixing via aspiration and dispensing being performed at a fixed height of about 3 mm from the bottom of the container in which the cell solution is located.

[0203] Aspect 67, in any aspect listed herein, includes the mixing via aspiration and dispensing being performed at a fixed height of about 2 mm to about 4 mm from the bottom of the container in which the cell solution is located.

[0204] Aspect 68 includes, in any aspect listed herein, mixing by aspiration and dispensing being performed at least five times.

[0205] Aspect 69, in any aspect enumerated herein, includes aspirating and dispensing any remaining unaspirated solution from the bottom of the container in which the cell solution is located, at a fixed height lower than the step prior to aspiration and dispensing.

[0206] Aspect 70, in any aspect enumerated herein, includes aspirating and dispensing any remaining unaspirated solution from the bottom of the container in which the cell solution is located, at a fixed height which is one-third of the height of the step prior to aspiration and dispensing.

[0207] The 71st aspect includes, in any aspect listed herein, dispensing at least a portion of the aspirated solution into a second culture plate, wherein the dispensing of the aspirated solution is performed at a distance of about 0.3 mm from the surface of the culture plate.

[0208] The 72nd aspect involves dispensing the aspirated solution at a distance of about 0.1 mm to 0.5 mm from the surface of the culture plate, in any aspect listed herein.

[0209] The 73rd aspect includes dispensing the aspirated solution at a rate of about 7 μL / second in any aspect listed herein.

[0210] The present invention has been described with reference to various specific and preferred embodiments and techniques. Nevertheless, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the invention.

Claims

1. An automated method for identifying the location of an extracellular matrix structure and disrupting the extracellular matrix structure, wherein the method is Using a substantially automated process, the predetermined positions X and Y within the extracellular matrix structure on the surface of the culture plate are identified, Using a substantially automated process, the extracellular matrix structure at predetermined locations X and Y on the surface of the culture plate is to be disrupted using a pipette tip associated with a liquid handler. Methods that include...

2. The method according to claim 1, further comprising identifying the location of the extracellular matrix structure on the surface of the culture plate using a substantially automated process prior to disruption.

3. The method according to any one of claims 1 to 2, wherein the extracellular matrix structure is a hydrogel.

4. The method according to any one of claims 1 to 3, wherein the predetermined positions of X and Y are located at the center of the extracellular matrix structure or near the center of the extracellular matrix structure.

5. The method according to any one of claims 1 to 4, wherein the predetermined positions X and Y of the extracellular matrix structure are determined by one or more parameters selected from the group consisting of the original seeding position on the surface of the culture plate and imaging of the extracellular matrix structure on the surface of the culture plate.

6. The method according to any one of claims 1 to 5, wherein the diameter of the extracellular matrix structure is determined by the volume of the extracellular matrix deposited on the surface of the culture plate or by imaging of the extracellular matrix structure on the surface of the culture plate.

7. The method according to any one of claims 1 to 6, further comprising moving the tip of the pipette in a number of steps to release cells from the extracellular matrix structure, each of the number of moving steps including additional movement of X, Y to predetermined locations within the extracellular matrix structure.

8. The method according to any one of claims 1 to 7, wherein the surface of the culture plate is a cell culture well.

9. Each of the predetermined positions of X and Y within the cell culture well and the additional predetermined positions of X and Y are calculated to include the following: X (number of steps) = X well center + alpha / step × number of steps × COS (theta / step × number of steps), Y (number of steps) = center of Y well + alpha / step × number of steps × sin(theta / step × number of steps), and in the formula, Alpha = extracellular matrix structure diameter (mm) / 1.8, Step = The number of steps required to move the pipette tip through the extracellular matrix structure, which is an integer between 2 and 20. The center of the X-well is the x-position of the center of the dome within the well. The center of the Y-well is the y-position of the center of the dome within the well. The method according to any one of claims 7 and 8, wherein theta is a constant value between 5 and 50.

10. The method according to any one of claims 7 to 9, wherein the predetermined positions of X and Y and the additional X and Y positions form a pattern selected from the group consisting of a helical pattern, a star pattern and a zigzag pattern within and / or through the extracellular matrix structure.

11. Each of the numerous steps of moving the pipette tip individually is, a) Moving the tip of the pipette through the extracellular matrix structure to disrupt the extracellular matrix structure, and / or b) Removing the pipette tip from the extracellular matrix structure prior to moving it. The method according to any one of claims 7 to 10, including selection from.

12. The aforementioned liquid handler is In one or more, two or more, or a large number of the predetermined positions of X and Y, or in each of them, within the extracellular matrix structure, Aspirating the tip of the pipette, and / or Dispensing liquid culture medium from the tip of the pipette. The method according to any one of claims 7 to 11, wherein the method is performed.

13. The method according to claim 12, wherein aspiration and / or dispensing are repeated one or more, two or more, or a large number of times at predetermined locations X, Y, respectively.

14. The method according to any one of claims 12 and 13, wherein the aspiration or dispensing occurs at a fixed height above the bottom of the culture plate and at a fixed speed.

15. The method according to any one of claims 14, wherein the fixed height is approximately 1 mm to approximately 3 mm above the surface of the culture plate.

16. The method according to any one of claims 12 to 15, wherein the liquid culture medium is dispensed at a rate of about 1 μL / second to about 600 μL / second.

17. The method according to claim 14, wherein the cell dissociation reagent is added to the extracellular matrix structure at a fixed height of about 0.05 mm to about 0.3 mm above the surface of the culture plate.

18. The method according to claim 12, wherein at least one aspiration of the tip of the pipette is performed to aspirate liquid from the culture plate.

19. The above method further, The cell solution is centrifuged in a container to form a cell pellet and a supernatant, A first volume of the supernatant is aspirated at a first fixed height and a first fixed velocity from the bottom of the container, The second volume of the supernatant is aspirated at a second fixed height and second fixed speed from the bottom of the container. The method according to any one of claims 1 to 18, including the method described above.

20. The method according to claim 19, wherein the cell pellet includes organoids.

21. The method according to claim 19, wherein the first volume is approximately 400 μL to approximately 600 μL.

22. The method according to claim 19, wherein the first fixed height is approximately 20 mm to approximately 40 mm from the bottom.

23. The method according to claim 19, wherein the first rate is approximately 200 μL / second to approximately 300 μL / second.

24. The method according to claim 19, wherein the second volume is approximately 300 μL to approximately 500 μL.

25. The method according to claim 19, wherein the second fixed height is approximately 2 mm to approximately 8 mm.

26. The method according to claim 19, wherein the second rate is approximately 35 μL / second to approximately 65 μL / second.

27. The above method further, To provide a container comprising an organoid pellet and a first volume of supernatant, a) Successively aspirating and dispensing a second volume multiple times at a first fixed height from the bottom of the container, b) At a second fixed height from the bottom of the container, a third volume is drawn out and the third volume is dispensed so as to return to the container, c) Repeating steps a) and b) above The method according to any one of claims 1 to 18, including the method described above.

28. The method according to claim 27, wherein the organoid pellet is obtained by the method according to claims 7 to 9.

29. The method according to claim 27, wherein the first volume is approximately 75 μL to approximately 125 μL.

30. The method according to claim 27, wherein the second volume is approximately 70 μL to approximately 90 μL.

31. The method according to claim 27, wherein the first height is approximately 0.1 mm to approximately 0.3 mm.

32. The method according to claim 27, wherein the third volume is approximately 75 μL to approximately 125 μL.

33. The method according to claim 27, wherein the second height is approximately 0.1 mm to approximately 0.3 mm.

34. The method according to claim 27, wherein step a) comprises aspirating and dispensing the second volume about 40 to about 60 times.

35. The above method further, a) Mixing approximately 60% of the cell solution volume multiple times via aspiration and dispensing, b) To aspirate a portion of the mixed solution and provide the aspirated solution and the remaining unaspirated solution, c) Dispense at least a portion of the aspirated solution into a culture plate that has not been previously used to culture the cells, d) Repeat steps a) to c) using the remaining unaspirated solution. The method according to any one of claims 1 to 17, including the method described in any one of claims 1 to 17.

36. The method according to claim 35, wherein the cell solution comprises an organoid or a fragment of an organoid.

37. The method according to claim 35, wherein the cell solution includes an extracellular matrix.

38. The method according to claim 35, wherein the mixing via aspiration and dispensing is performed at a fixed height of about 2 mm to about 4 mm from the bottom of the container in which the cell solution is located.

39. The method according to claim 35, wherein when step d) is performed, the aspiration and dispensing are performed at a fixed height from the bottom of the container in which the cell solution is located, lower than the steps prior to steps a) and b).

40. The method according to claim 35, wherein when step d) is performed, the aspiration and dispensing are performed at a fixed height from the bottom of the container in which the cell solution is located, which is about one-third of the height of the first steps a) and b).

41. The method according to claim 35, wherein dispensing at least a portion of the aspirated solution onto the second culture plate is performed at a distance of about 0.1 mm to 0.5 mm from the surface of the culture plate that has not been previously used to culture cells.

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