Method for organoid subculturing using microplate well units

Abstract

Disclosed are various embodiments related to automated cell subculture of spheroids, tuberoids, organoids, and / or other multicellular bodies. Organoids can be cultured in hydrogels that are placed in well units of microwell plates. The well units include a primary well section that is fluidly connected to a secondary well section through at least one channel, and the hydrogel is placed in the primary well section of the well units. The hydrogel is dissipated into hydrogel fragments, thereby separating the organoids from the hydrogel. The hydrogel fragments are removed from the well units, while the organoids remain in the well units. The organoids that remain are broken into organoid fragments and corresponding debris. A fresh culture environment is generated using the organoid fragments.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application is filed as a PCT international patent application on February 18, 2022, claiming the priority and benefit of U.S. Provisional Application No. 63 / 151,082, filed on February 19, 2021, the entire disclosure of which is incorporated by reference in its entirety.

Background Art

[0002] Culturing cells in a three - dimensional (3D) environment results in cell behavior and morphological structures that more closely match those observed in the human body. The 3D hydrogels / hydroscaffolds used for this type of culturing have unique attributes, namely, cells can be deposited within specific locations in 3D space and can remain in place over long periods. This enables a co - culture environment in which the generation of structures (e.g., embryoid bodies, fused embryoid bodies, spheroids, tumoroids, organoids, and / or other multicellular bodies), and cell - to - cell interactions and growth over time can be observed.

Summary of the Invention

Means for Solving the Problems

[0003] Aspects of the present disclosure relate to the automated sub - culturing of spheroids, tumoroids, organoids, and / or other multicellular bodies. According to various embodiments, since organoids and other multicellular bodies grow and proliferate over time within the wells of a microplate, sub - culturing is required to remove debris associated with dead cells and toxic by - products, reduce the total cell number, obtain a homogeneous population of smaller organoids, and promote continuous cell growth and propagation. The sub - culturing method of the present disclosure improves conventional methods associated with the sub - culturing of organoids or multicellular bodies by reducing time, reducing cost, and eliminating the need for additional laboratory equipment (e.g., conical tubes, centrifuges, etc.) and centrifugation.

[0004] In one embodiment, in particular, a method for subculturing organoids includes the step of culturing one or more organoids in a hydrogel placed within a well unit of a microwell plate. The well unit includes a primary well section, which is fluidly connected to a secondary well section via at least one channel, and the first hydrogel is placed within the primary well section of the well unit. The hydrogel is released into hydrogel fragments, thereby separating one or more organoids from the hydrogel. The hydrogel fragments are removed from the well unit, while one or more organoids remain within the well unit. One or more organoids are crushed into organoid fragments and corresponding fragments. A fresh culture environment containing organoid fragments is generated.

[0005] In one or more aspects, at least one channel is formed by at least one gap between the bottom surface of the well unit and the bottom portion of the shared sidewall of the primary well section and the secondary well section. In some embodiments, the height of the at least one gap may be specified between about 10 microns and up to about 100 microns. In various aspects, the step of dispersing the first hydrogel into the hydrogel fragments further includes a step of cooling the hydrogel. The step of cooling the hydrogel may further include a step of dispensing the liquid medium into the well unit. In some aspects, the liquid medium is at a temperature of about 10 degrees Celsius or below. In some aspects, the temperature is about 4 degrees Celsius or below.

[0006] In various aspects, the method further includes the steps of collecting one or more organoids from the primary well section of a well unit via a liquid handler, and applying at least one shear force to one or more organoids in the liquid handler. One or more organoids may be broken into organoid fragments and corresponding fragments as a result of at least one shear force. In various aspects, the method further includes the step of depositing the organoid fragments and corresponding fragments in the primary well section of a well unit via a liquid handler. In one or more aspects, the step of applying at least one shear force includes operating the liquid handler to move one or more organoids vertically within the liquid handler.

[0007] In various aspects, the step of removing hydrogel fragments may include inserting a liquid handler into the secondary well section of a well unit, transferring hydrogel fragments from the primary well section of the well unit into the secondary well section via at least one channel, and collecting the hydrogel fragments from the secondary well section via the liquid handler. In one or more aspects, the method may include flushing at least one channel to remove at least one of the fragmentary fragments or organoid fragments from at least one channel. In one or more aspects, the method may include moving the corresponding fragments into the secondary well section of the well unit via at least one channel.

[0008] In various aspects, the step of generating a fresh culture environment containing organoid fragments further includes the steps of collecting the organoid fragments via a liquid handler and depositing fresh hydrogel onto the bottom surface of a primary well section in another well unit or one of the well units. In some aspects, the organoid fragments are embedded in the fresh hydrogel prior to the step of depositing the fresh hydrogel onto the bottom surface of the primary well section. In other aspects, the organoid fragments are embedded in the fresh hydrogel after the fresh hydrogel has been deposited onto the bottom surface of the primary well section. In one or more aspects, the microwell plate containing the well units is placed in an incubator.

[0009] In another embodiment, the method includes, among other things, the step of separating one or more cell bodies from a hydrogel placed on the bottom surface of a first well unit of a first microplate. The first well unit includes a culture well and a supply well, which are fluidly connected to each other via at least one channel, and the first hydrogel is placed in the culture well of the first well unit. The hydrogel can be removed from the first well unit via the supply well of the well unit, while one or more cell bodies remain in the culture well of the first well unit. One or more cell bodies can be fragmented into cell body fragments and corresponding fragments. The corresponding fragments can be removed from the supply well of the first well unit, while the cell body fragments remain in the culture well of the first well unit. A fresh culture environment is generated in either the first well unit or a second well unit, and the fresh culture environment includes the cell body fragments.

[0010] In various aspects, at least one channel is sized and shaped to prevent the passage of objects larger than approximately 25 microns between the culture well and the supply well. In various aspects, the step of separating one or more cell bodies from the hydrogel further includes the step of cooling the hydrogel to a temperature that liquefies the hydrogel. In one or more aspects, the method includes the steps of removing one or more cell bodies from the culture well via a pipette and applying one or more shear forces to one or more cell bodies via a pipette. One or more cell bodies may be fragmented into cell body fragments and corresponding fragments based on the applied shear force. In various aspects, the step of generating a fresh culture environment within one of the first or second well units further includes the steps of depositing a fresh hydrogel within one of the first or second well units, wherein the cell body fragments are embedded in the fresh hydrogel, and culturing the cell body fragments embedded in the fresh hydrogel.

[0011] Other systems, methods, features, and advantages of this disclosure will be apparent to those skilled in the art, or will become apparent, upon examination of the following drawings and detailed description. All such additional systems, methods, features, and advantages are contained within this description, within the scope of this disclosure, and are intended to be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the embodiments described are available in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, and all optional and preferred features and modifications of the embodiments described, are combinable and interchangeable with each other. The present invention provides, for example, the following: (Item 1) A method for subculturing organoids, wherein the method is The method involves culturing one or more organoids in a hydrogel placed within a well unit of a microwell plate, wherein the well unit comprises a primary well section fluidly connected to a secondary well section via at least one channel, and the hydrogel is placed within the primary well section of the well unit. The hydrogel is dispersed into a plurality of hydrogel fragments, thereby separating one or more organoids from the hydrogel. The removal of the plurality of hydrogel fragments from the well unit, wherein one or more organoids remain within the well unit. The process of crushing one or more organoids into multiple organoid fragments and corresponding pieces, To generate a fresh culture environment comprising the aforementioned multiple organoid fragments. Methods that include... (Item 2) The method according to item 1, wherein the at least one channel is formed by at least one gap between the bottom surface of the well unit and the bottom portion of the shared side wall of the primary well section and the secondary well section. (Item 3) The method according to item 2, wherein the height of at least one gap is in the range of approximately 10 microns to a maximum of approximately 100 microns. (Item 4) The method according to any one of items 1-3, wherein the dispersal of the hydrogel into a plurality of hydrogel fragments further comprises cooling the hydrogel. (Item 5) The method according to item 4, further comprising cooling the hydrogel by dispensing a liquid medium into the well unit, wherein the liquid medium is at a temperature of about 10 degrees Celsius or below. (Item 6) The temperature is approximately 4 degrees Celsius or lower, as described in item 5. (Item 7) Collecting one or more organoids from the primary well section of the well unit via a liquid handler, Applying at least one shear force to one or more organoids in the liquid handler, wherein the one or more organoids are crushed into the plurality of organoid fragments and the corresponding fragments as a result of the at least one shear force, The plurality of organoid fragments and the corresponding fragments are deposited into the primary well section of the well unit via the liquid handling device. The method described in any one of items 1-6, further including the method described in any one of items 1-6. (Item 8) The method according to item 7, wherein applying the at least one shear force involves operating the liquid handler to move the one or more organoids vertically within the liquid handler. (Item 9) Removing the aforementioned plurality of hydrogel fragments further, Inserting the liquid handling device into the secondary well section of the well unit, Transferring the plurality of hydrogel fragments from the primary well section of the well unit into the secondary well section via at least one channel, The plurality of hydrogel fragments are collected from the secondary well section via the liquid handling device. The method described in any one of items 1-8, including the method described in item 1. (Item 10) The method according to any one of items 1-9, further comprising flushing the at least one channel to remove at least one of the fragmentary fragments or organoid fragments from the at least one channel. (Item 11) The method according to any one of items 1-10, further comprising moving the corresponding fragment to the secondary well section of the well unit through the at least one channel. (Item 12) To generate the fresh culture environment comprising the plurality of organoid fragments, Collecting the multiple organoid fragments via a liquid handling device, Depositing fresh hydrogel on the bottom surface of another well unit or the primary well section of one of the well units. The method described in any one of items 1-11, including the method described in item 1. (Item 13) The method according to item 12, wherein the plurality of organoid fragments are embedded in the fresh hydrogel prior to depositing the fresh hydrogel on the bottom surface of the primary well section. (Item 14) The method according to item 12, wherein the plurality of organoid fragments are embedded in the fresh hydrogel after the fresh hydrogel has been deposited on the bottom surface of the primary well section. (Item 15) The method according to any one of items 1-14, further comprising placing the microwell plate, which comprises the well units, inside an incubator. (Item 16) It is a method, The method involves separating one or more cell bodies from a hydrogel placed on the bottom surface of a first well unit of a first microwell plate, wherein the first well unit comprises culture wells and a supply well, which are fluidly connected to each other via at least one channel, and the hydrogel is placed within the culture wells of the first well unit. Removing the hydrogel from the first well unit via the supply well of the first well unit, wherein one or more cell bodies remain in the culture well of the first well unit. The process of fragmenting one or more cell bodies into multiple cell body fragments and corresponding fragments, Removing the corresponding fragments from the supply well of the first well unit, wherein the plurality of cell body fragments remain in the culture well of the first well unit, The method involves generating a fresh culture environment within either the first well unit or the second well unit, wherein the fresh culture environment comprises the plurality of cell body fragments. Methods that include... (Item 17) The method according to item 16, wherein the at least one channel is sized and shaped to prevent the passage of an object sized to more than about 25 microns between the culture well and the supply well. (Item 18) The method according to any one of items 16 or 17, wherein separating the one or more cell bodies from the hydrogel further comprises cooling the hydrogel to a temperature that liquefies it. (Item 19) Removing one or more cell bodies from the culture well via pipette, Applying one or more shear forces to one or more cell bodies via the pipette, wherein the one or more cell bodies are at least partially fragmented into the plurality of cell body fragments and the corresponding fragments based on the one or more applied shear forces. The method described in any one of items 16-18, further including the method described in any one of items 16-18. (Item 20) Creating the fresh culture environment within either the first well unit or the second well unit is further: The method involves depositing a fresh hydrogel into one of the first or second well units, wherein the plurality of cell body fragments are embedded within the fresh hydrogel. The process involves culturing the multiple cell body fragments embedded in the fresh hydrogel. The method described in any one of items 16-19, including the method described in item 16-19. [Brief explanation of the drawing]

[0012] Many aspects of this disclosure can be better understood by referring to the following drawings. The components in the drawings are not necessarily to scale, but rather the focus is on clearly illustrating the principles of this disclosure. Furthermore, in the drawings, similar reference numbers designate corresponding parts throughout several drawings.

[0013] [Figure 1] Figure 1 illustrates an example of a cross-section of a microplate well unit associated with cell culture according to various embodiments of the present disclosure.

[0014] [Figure 2A]Figures 2A-2B illustrate the workflow for removing the liquid culture medium from the well unit of Figure 1, illustrating cross-sectional views of the well unit of Figure 1 according to various embodiments of the present disclosure. [Figure 2B] Figures 2A-2B illustrate the workflow for removing the liquid culture medium from the well unit of Figure 1, illustrating cross-sectional views of the well unit of Figure 1 according to various embodiments of the present disclosure.

[0015] [Figure 3A] Figures 3A–3H illustrate examples of cross-sectional views of the well unit of Figure 1 according to various embodiments of the present disclosure, each figure representing an example of a workflow step associated with cell subculturing. [Figure 3B] Figures 3A–3H illustrate examples of cross-sectional views of the well unit of Figure 1 according to various embodiments of the present disclosure, each figure representing an example of a workflow step associated with cell subculturing. [Figure 3C] Figures 3A–3H illustrate examples of cross-sectional views of the well unit of Figure 1 according to various embodiments of the present disclosure, each figure representing an example of a workflow step associated with cell subculturing. [Figure 3D] Figures 3A–3H illustrate examples of cross-sectional views of the well unit of Figure 1 according to various embodiments of the present disclosure, each figure representing an example of a workflow step associated with cell subculturing. [Figure 3E] Figures 3A–3H illustrate examples of cross-sectional views of the well unit of Figure 1 according to various embodiments of the present disclosure, each figure representing an example of a workflow step associated with cell subculturing. [Figure 3F] Figures 3A–3H illustrate examples of cross-sectional views of the well unit of Figure 1 according to various embodiments of the present disclosure, each figure representing an example of a workflow step associated with cell subculturing. [Figure 3G] Figures 3A–3H illustrate examples of cross-sectional views of the well unit of Figure 1 according to various embodiments of the present disclosure, each figure representing an example of a workflow step associated with cell subculturing. [Figure 3H] Figures 3A–3H illustrate examples of cross-sectional views of the well unit of Figure 1 according to various embodiments of the present disclosure, each figure representing an example of a workflow step associated with cell subculturing.

[0016] [Figure 4] Figure 4 illustrates flowcharts of exemplary methods related to cell subculturing according to various embodiments of the present disclosure. [Modes for carrying out the invention]

[0017] Detailed explanation This disclosure relates to a method for automated subculturing of spheroids, tumoroids, organoids, and / or other multicellular bodies. Microplates with microwells are used in vitro for the proliferation, culture, monitoring, and analysis of embryoid bodies, fused embryoid bodies, spheroids, organoids, and / or other multicellular bodies. According to various embodiments, since spheroids, tumoroids, organoids, and other multicellular bodies proliferate and grow over time within the wells of a microplate, subculturing is required to remove debris associated with dead cells and toxic byproducts, reduce the total cell number, obtain a smaller homogeneous population of organoids, and promote continuous cell proliferation and reproduction. The subculturing method of this disclosure improves upon conventional methods associated with subculturing spheroids, tumoroids, organoids, or other multicellular bodies by reducing time, reducing costs, and eliminating the need for additional laboratory equipment (e.g., conical tubes, centrifuges, etc.) and centrifugation. In addition, according to various embodiments, the subculturing method of the present disclosure can control and regulate the size range of organoids or multicellular organisms, thereby improving conventional methods by providing a more controlled and homogeneous population.

[0018] According to various embodiments, the subculturing method of the present disclosure uses a microplate having a well unit comprising two conjugated wells fluidly connected to each other via at least one channel, and includes a microplate as described in U.S. Provisional Application No. 63 / 094,946, filed October 22, 2020, titled "Microplates for Automating Organoid Cultivation," and U.S. Provisional Application No. 63 / 131,123, filed December 28, 2020, titled "MICROPLATE WELLS FOR CELL CULTIVATION" (both of which are incorporated herein by reference as a whole). Figure 1 illustrates an example of a well unit 100 of a microplate that may be used in connection with the subculturing method of the present disclosure. In particular, Figure 1 illustrates an exemplary diagram of a microplate well unit 100 that may be used to grow, culture, monitor, and chemically analyze embryoid bodies, fused embryoid bodies, spheroids, organoids, or other multicellular organisms according to various embodiments of the present disclosure.

[0019] As shown in Figure 1, cell bodies 103 (e.g., spheroids, tumoroids, organoids, etc.) are embedded in hemispheres of hydrogel 106 (e.g., Matrigel®) which are placed in well units 100 and surrounded by liquid medium 109. According to various embodiments, the liquid medium 109 may contain appropriate growth factors and complements to generate and / or stimulate the proliferation of desired multicellular bodies. Exemplary growth factors that may be suitable include angiopoietin, bone morphogenetic protein (BMP), ciliary neurotrophic factor, colony-stimulating factor, ephrin, epidermal growth factor, erythropoietin, fibroblast growth factor, glial neurotrophic factor, hepatocyte growth factor, insulin, insulin-like growth factor, interleukin, leukemia suppressor factor, keratinocyte growth factor, neuregulin, neurotrophin, platelet-derived growth factor, transforming growth factor, tumor necrosis factor (α), vascular endothelial growth factor, and / or similar.

[0020] According to various embodiments, the well unit 100 in Figure 1 comprises a primary well section 112 and a secondary well section 115. In various embodiments, the primary well section 112 and the secondary well section 115 are fluidly connected to each other via at least one channel 118 to facilitate the gravitational flow of liquid (e.g., liquid medium 109) between the primary well section 112 and the secondary well section 115 in response to the tilting of the microplate. Exchanging the liquid medium 109 between the primary well section 112 and the secondary well section 115 removes toxic byproducts and supplies fresh nutrients to the growing cell culture. In the embodiment of Figure 1, the hydrogel 106 and cell bodies 103 are placed in the primary well section 112 of the well unit 100.

[0021] According to various embodiments, at least one channel 118 is further sized and molded to prevent objects having dimensions (diameter, height, width, etc.) of a certain size (e.g., greater than about 25 microns (μ)) from passing from one well section to the other. In some embodiments, the height of at least one channel 118 may be sized between about 10 microns and about 100 microns. In other embodiments, the height of at least one channel may be sized between about 10 microns and about 25 microns. For example, if the height of at least one channel 118 is about 25 microns, objects present in the primary well section 112 that are about 25 μm or larger (e.g., spheroids, tumoroids, organoids, organoid fragments, etc.) will be prevented from moving into the secondary well section 115. However, corresponding fragments present in the primary well section 112 that are smaller than or equal to a certain size (e.g., about 25 μm) or have dimensions (e.g., diameter, height, width, etc.) are possible to pass through at least one channel 118 into the secondary well section 115. These fragments include toxic by-products, dead cells, other corresponding fragments resulting from the step of culturing cell bodies 103, and cell body fragments removed as a result of the step of reducing the total number of cells to obtain a smaller homogeneous population of organoids. As a result, the corresponding fragments can be aspirated using a liquid handler (e.g., a pipette) or otherwise collected via the secondary well section 115 without inhibiting the cell bodies 103 in the primary well section 112.

[0022] According to various embodiments, the primary well section 112 is sized and shaped to support deposited cell bodies 103 (e.g., cell aggregates) that can be embedded in a hydrogel 106 deposited within the primary well section 112. For example, the primary well section 112 can be considered a culture well used for growing embryoid bodies, fused embryoid bodies, spheroids, organoids, and / or other multicellular organisms, as can be understood. According to various embodiments, depending on several well units 100 in the microplate, the width of the primary well section 112 can be up to about 8 millimeters (mm) (e.g., for a 96-well plate), up to 11 mm (e.g., for a 48-well plate), up to about 17 mm (e.g., for a 24-well plate), and / or other sizes as can be understood. In addition, the depths of the primary well section 112 and the secondary well section 115 are defined so that the microplate can be tilted to allow fluid exchange within the well unit 100 without causing fluid to overflow out of the individual primary well section 112 or secondary well section 115 of each well unit 100.

[0023] The secondary well section 115, located within the primary well section 112, may be used to supply nutrient delivery medium and / or other nutrients, which may be used to deliver nutrients to a growing cell aggregate. In addition, the secondary well section 115 may be used to collect supernatant from the cell aggregate, as can be understood. For example, the secondary well section 115 can be considered a supply well, containing nutrient delivery medium and / or other nutrients, which may be used by the growing cell culture in the primary well section 112. The secondary well section 115 is sized and molded to hold fluid, which may be replaced by the primary well section 112 according to various embodiments of this disclosure. According to various embodiments, depending on several well units 100 in the microplate, the width of the secondary well section 115 may be up to about 8 millimeters (mm) (for example, with respect to a 96-well plate), up to 11 mm (for example, with respect to a 48-well plate), up to about 17 mm (for example, with respect to a 24-well plate), and / or other sizes as can be understood.

[0024] According to various embodiments, the size and shape of the primary well section 112 and the secondary well section 115 may differ from each other. In some embodiments, the primary well section 112 is larger than the secondary well section 115 (in terms of dimensions, e.g., diameter or volume). In other embodiments, the secondary well section 115 is larger than the primary well section 112. In some embodiments, the primary well section 112 has a different shape from the secondary well section 115.

[0025] According to various embodiments, the well unit 100 further comprises a bottom layer sheet 121 positioned beneath the well plate body of the microplate. The bottom layer sheet 121 is attached beneath the well plate body, forming the bottom surface of the well unit 100. In various embodiments, the bottom layer sheet 121 comprises an optically transparent viewing window, as can be understood, to enable imaging of spheroids, organoids, or other cell cultures cultured within the well unit 100 of the microplate. The viewing window may be suitable for microscopic observation, whether or not it is bright-field, phase-contrast, fluorescence, confocal, two-photon, or any other microscopy imaging modality known in the art.

[0026] In various embodiments, the bottom layer sheet 121 comprises a gas-permeable sheet configured to increase oxygen supply for growing spheroids, organoids, or other cell bodies within the well units 100 of the microplate. The gas-permeable sheet can be formed from a material comprising polytetrafluoroethylene (PTFE), PEFP, polyimide, polydimethylsiloxane (PDMS), polycarbonate, and / or other materials that may be understood. According to various embodiments, the gas-permeable sheet can have a thickness of about 5 to 70 microns. According to various embodiments, the gas-permeable sheet can have multiple pores. In other embodiments, the gas-permeable sheet can allow molecules to pass through by diffusion. Alternatively, the gas-permeable sheet can have several other thicknesses, pore diameters, and pore densities.

[0027] Now, looking at Figures 2A and 2B, what is shown are examples of workflows associated with the step of removing the liquid medium 109 from the well unit 100 according to various embodiments of the present disclosure. In particular, as with Figure 1, Figure 2A illustrates a culture environment including cell bodies 103 embedded in a hydrogel 106 placed on the bottom surface (e.g., bottom layer sheet 121) of the primary well section 112 of the well unit 102. The liquid medium 109 is also illustrated in both the primary well section 112 and the secondary well section 115 of the well unit 100. As can be understood, the liquid medium 109 may contain appropriate growth factors and complements to produce the desired cell bodies 103.

[0028] At least one channel 118 of the well unit 100 provides a fluid connection between the primary well section 112 and the adjacent secondary well section 115. At least one channel 118 further provides the ability to facilitate a continuous gravity flow of liquid medium 109 via the tilting of the corresponding microplate, enabling pre-nutrient delivery to cell bodies 103 (e.g., organoids or other multicellular organisms). In various embodiments, the liquid medium 109 (e.g., nutrient delivery medium and / or other nutrients) can be introduced into the secondary well section 115 and finally into the primary well section 112 via at least one channel 118. In various embodiments, the liquid medium 109 can be added to and / or removed from one of the well sections (e.g., secondary well section 115) using a liquid handler 200 (e.g., a pipette) without disturbing the environment within the well of interest (e.g., primary well section 112).

[0029] Figure 2B illustrates an example of the well unit 100 after the liquid medium 109 has been removed using the liquid handler 200. As can be understood, the liquid medium 109 can be removed without disturbing the environment of the primary well section 112 (e.g., the cell bodies 103 embedded in the hydrogel 106).

[0030] Next, an overview of the subculture workflow of this disclosure is provided with reference to Figures 3A–3H. In particular, Figures 3A–3H illustrate cross-sectional views of the well unit 100 of a microplate. Each figure corresponds to an individual step associated with the subculture workflow according to various embodiments. According to various embodiments, since the cell bodies 103 proliferate over time within the well unit 100 of the microplate, subculture is required to remove dead cells and debris associated with toxic byproducts, reduce the total cell number, obtain a smaller organoid homogeneous population, promote continued cell proliferation, and maintain the overall health of the cell bodies 103.

[0031] As illustrated in Figure 3A, the hemispheres of the hydrogel 106 can be broken down into multiple hydrogel fragments 303 or otherwise into a liquid form. For example, the solid hydrogel 106 can be converted into a liquid, thereby separating the cell bodies 103 from the hydrogel 106. In some embodiments, the solid hydrogel 106 is broken down into multiple hydrogel fragments 303 or otherwise into a liquid form by adjusting the temperature of the hydrogel 106 based on the melting properties of the hydrogel 106. In some embodiments, the hydrogel 106 can be broken down into a liquid form at a temperature below approximately 10 degrees Celsius (°C). In this embodiment, as shown in Figure 3A, a liquid culture medium 109 having a temperature below approximately 10 degrees Celsius (°C) (or below approximately 4°C) can be introduced into the well unit 100 via the primary well section 112 or the secondary well section 115 using a liquid handler 200. In some embodiments, the microplate can be positioned on a temperature control device (e.g., a cooling plate) designed to regulate the temperature of the microplate so that the hydrogel 106 deposited in the corresponding well unit 100 begins to liquefy.

[0032] Next, looking at Figure 3B, the hydrogel fragments 303 or otherwise hydrogel 106 in liquid form can be removed from the well unit 100 along with the liquid medium 109 via the liquid handler 200. As shown in Figures 3A and 3B, multiple hydrogel fragments 303 or otherwise decomposed hydrogel 106 in liquid form can pass through channels 118 formed between the primary well section 112 and the secondary well section 115. In addition, the dimensions of at least one channel 118 prevent the passage of cell bodies 103, which are sized and molded with dimensions exceeding the dimensions of at least one channel 118. Thus, the cell bodies 103 will remain in the primary well section 112 while the liquefied hydrogel 106 and hydrogel fragments 303 pass through at least one channel 118 and into the secondary well section 115. To protect the cell bodies 103 separated from the hydrogel 106, the liquefied hydrogel 106, hydrogel fragments 303, and / or liquid culture medium 109 can be removed from the secondary well section 115 of the well unit 100.

[0033] In some embodiments, the workflow steps associated with Figures 3A and 3B can be repeated to allow the hydrogel 106 to be completely degraded and to allow for the complete removal of the hydrogel 106 from the well unit 100. In other words, the washing of the cell suspension, accompanied by the introduction and removal of the liquid medium 109, can be repeated multiple times to remove the hydrogel 106 from the well unit 100.

[0034] Once the hydrogel 106 is removed, only the cell bodies 103 that were originally embedded within the hydrogel 106 remain in the well unit 100. This is illustrated in Figure 3C. In some embodiments, a low level of liquid medium 109 may still be present in the primary well section 112 of the well unit. This liquid medium 109 can be removed as needed. For example, the microplate can be tilted to allow the excess medium 109 to flow into the secondary well section 115 of a given well unit 100 for removal. The liquid medium 109 can be removed using a liquid handler 200, as can be understood.

[0035] Now looking at Figure 3D, what is shown is an example of a workflow step associated with the step of disrupting the cell bodies 103 using coarse pipette dispensing. In various embodiments, liquid medium 109 can be introduced into well units 100 containing the cell bodies 103. A liquid handler 200 can be used to aspirate or otherwise collect the cell bodies 103 that remain in the primary well section 112 after the removal of the decomposed or otherwise liquefied hydrogel 106. Once the cell bodies 103 are aspirated into the liquid handler 200, a shear force is applied to the cell bodies 103, thereby causing the cell bodies 103 to decompose into multiple cell body fragments 306 (e.g., spheroid fragments, tumoroid fragments, organoid fragments, etc.) and corresponding fragments 309. The cell body fragments 306 comprise multicellular fragments of the cell bodies 103 (e.g., spheroids, tumoroids, organoids, etc.).

[0036] The applied shear force is the result of manual or automated operation of the liquid handler 200 and can cause the cell body 103 to move up and down along the longitudinal axis of the liquid handler 200. The shear force can cause the cell body 103 to decompose into multiple cell body fragments 306 and corresponding fragments. The shear force can be applied to the cell body 103 until the desired size of the cell body 103 is obtained. In some embodiments, enzymes and / or chemicals (e.g., trypsin, mild dissociation medium, etc.) may be added to the well unit 100 or otherwise to the cell body 103 prior to the step in which the cell body is aspirated by the liquid. In some embodiments, once the cell body 103 has decomposed into multicellular body fragments 306 (e.g., organoid fragments), the cell body fragments 306 can be placed in the well unit of a microplate and incubated in a cell dissociation reagent for a predetermined amount of time (e.g., up to about 20 minutes).

[0037] In various embodiments, the preferred size of the cell body fragment 306 is approximately 25–500 μm. However, it should be noted that fragments of other sizes may also be obtained. The corresponding fragment 309 may comprise dead cells associated with the cell body 103, and toxic byproducts that grow during the culture of the cell body 103.

[0038] Moving to Figure 3E, the multiple cell body fragments 306 and their corresponding fragments 309 can be reintroduced into the primary well section 112 of the well unit 100, i.e., into the liquid medium 109. In particular, Figure 3E illustrates the multiple cell body fragments 306 and their corresponding fragments 309, which are the result of shear forces applied to the cell bodies 103 that were originally embedded in the hydrogel 106. The corresponding fragments 309, sized to be smaller than the dimensions of at least one channel 118, would be able to pass through at least one channel 118 and flow into the secondary well section 115. In various embodiments, tilting of the microplate may be required to induce a gravitational flow of the liquid medium 109, thereby allowing the corresponding fragments 309 to enter the secondary well section 115.

[0039] Now, looking to Figure 3F, what is shown is an example of the removal of corresponding fragments 309 according to various embodiments of the present disclosure. In particular, corresponding fragments 309 having dimensions smaller than the dimensions of at least one channel 118 can pass through at least one channel 118 and enter into a secondary well section 115 where they can be removed via the liquid handler 200. In various embodiments, tilting the microplate can cause a gravity flow of liquid medium 109 to move between two wells, allowing the liquid medium 109 and corresponding fragments 309 to move through the channel 118 and into different wells. In some embodiments, the addition of liquid medium 109 and / or the removal of liquid medium 109 and fragments 309 can be repeated as necessary to remove all fragments 309 from the well unit 100.

[0040] Once the fragments are removed from the well unit 100, only the cell body fragments 306 remain in the well unit 100, along with some residual liquid medium 109 in some embodiments. In some embodiments, additional liquid medium 109 may be provided in the well unit 100, as shown in Figure 3G, and the cell body fragments 306 may be aspirated into the liquid handler 200. Removal of the cell body fragments 306 allows for the preparation of a fresh environment in the well unit 100.

[0041] In particular, once the cell body fragments 306 are removed from the well unit 100, the hydrogel 106 can be deposited in the primary well section 112 of the well unit 100. In some embodiments, the cell body fragments 306 can be embedded in the hydrogel 106 prior to the step of depositing the hydrogel 106 in the primary well section 112 of the well unit 100 of the microplate. In various embodiments, a new microplate will be used. In other embodiments, the cell body fragments 306 are returned to the well unit 100 and deposited on the hydrogel 106 after the hydrogel 106 has been deposited in the well unit 100. Figure 3H illustrates an embodiment of the step of depositing the cell body fragments 306 on top of the deposited hydrogel 106. In some embodiments, the microplate can be placed on a tray, heated to a desired temperature (e.g., about 37 degrees Celsius), and incubated for a desired amount of time (e.g., up to about 20 minutes).

[0042] Once the hydrogel 106 and cell body fragments 306 are introduced into the well unit 100, liquid medium 109 is added to the well unit 100, thereby providing a fresh environment and enabling the cell body fragments 306 to proliferate and grow as desired. The microplate is placed in an incubator to stimulate the proliferation and growth of the cell body fragments. This subculturing method can be repeated as needed.

[0043] Now, looking at Figure 4, we see a flowchart illustrating exemplary methods related to cell subculturing of a microplate well unit 100 according to various embodiments of the present disclosure.

[0044] Beginning in step 403, the cell bodies 103 are cultured in the primary well section 112 of the microplate well unit 100, which can provide proliferation and growth of spheroids, tumoroids, organoids, and / or other multicellular bodies. According to various embodiments, the cell bodies 103 can be embedded in a hydrogel 106. Furthermore, one or more growth factors and complements can be introduced into the microplate well unit 100 in the form of liquid medium 109. Exemplary growth factors that may be preferred include angiopoietin, bone morphogenetic protein (BMP), ciliary neurotrophic factor, colony-stimulating factor, ephrin, epidermal growth factor, erythropoietin, fibroblast growth factor, glial neurotrophic factor, hepatocyte growth factor, insulin, insulin-like growth factor, interleukin, leukemia suppressor factor, keratinocyte growth factor, neuregulin, neurotrophin, platelet-derived growth factor, transforming growth factor, tumor necrosis factor (α), vascular endothelial growth factor, and / or similar. As the cell bodies 103 proliferate and grow over time within the well units 100 of the microplate, subculturing may be required to remove dead cells and debris 309 associated with toxic byproducts, promote continued cell proliferation, reduce the total cell number, obtain a smaller homogeneous population of cell bodies (e.g., spheroids, tumoroids, organoids, etc.), and maintain the overall health of the cell bodies 103.

[0045] In step 406, the hemispheres of the hydrogel 106 are broken down to be converted into multiple hydrogel fragments 303 and / or into a liquid form, or otherwise dispersed. For example, the hemispheres of the hydrogel 106 are converted into a liquid, thereby allowing the cell bodies 103 to be separated from the hydrogel hemispheres. In some embodiments, based on the melting properties of the hydrogel 106, the step of adjusting the temperature of the hydrogel 106 (e.g., about 10°C or below, about 4°C or below) may cause the hydrogel 106 to be dispersed into multiple hydrogel fragments 303 or otherwise into a liquid form.

[0046] According to various embodiments, following the culture of cell bodies 103, the culture liquid medium 109 present in the well unit 100 may be removed from the well unit 100, and a dissociated liquid medium 109 (e.g., a mildly dissociated medium) may be introduced into the well unit 100. In some embodiments, the microplate may be placed in an incubator for a desired amount of time (e.g., up to about 20 minutes). In this embodiment, the dissociated liquid medium 109, having a temperature below or equal to a desired temperature, may be introduced into the well unit 100 via the primary well section 112 or secondary well section 115 using a liquid handler 200, as shown in Figure 3A. In some embodiments, the microplate may be positioned on a temperature control device (e.g., a cooling plate) designed to regulate the temperature of the microplate so that the hydrogel 106 deposited in the corresponding well unit 100 begins to liquefy. As the hydrogel 106 is reduced to a liquid form and / or hydrogel fragment 303, the cell bodies 103 are separated from the hydrogel 106.

[0047] In step 406, the hydrogel fragment 303 or otherwise the hydrogel 106 in liquid form can be removed from the secondary well section 115 of the well unit 100. In particular, as the hydrogel is reduced to fragment 303 or otherwise in liquid form, the hydrogel 106 can pass through at least one channel 118 that fluidly connects the secondary well section 115 to the primary well section 112. In some embodiments, the microplate can be tilted to facilitate fluid flow from the primary well section 112 into the secondary well section 115.

[0048] For example, a microplate can be mounted on a rocking or tilting device. Such a device may have a flat surface configured to receive a microwell plate, such as the one described herein, which is operably connected to a motor, and “tilt” the microwell plate about a certain axis, thereby raising the height of one side relative to the opposite side, thereby facilitating fluid flow from the primary well section 112 (e.g., culture well) to the secondary well section 115 (e.g., culture well) of the well unit 100, or vice versa. If the cell bodies are sized to exceed the dimensions of at least one channel 118, the cell bodies 103 remain within the primary well section 112 of the well unit 100, as can be understood.

[0049] The hydrogel 106 (e.g., hydrogel fragment 303) and the liquid medium 109 can be removed from the secondary well section 115 using the liquid handler 200, as shown in Figure 3B. The hydrogel 106 and the liquid medium 109 can also be removed from the primary well section 112, but it is preferable to remove the liquid medium 109 and hydrogel 106 from the secondary well section 115 in order to preserve the cell bodies 103 placed in the primary well section 112 and to avoid them being inhibited.

[0050] In step 412, it can be determined whether all of the hydrogel 106 has been removed from the well unit 100. If all (or an acceptable amount) of the hydrogel 106 has been removed, the process proceeds to step 415. Otherwise, the process returns to step 406. In particular, the liquid medium 109 may be reintroduced into the well unit 100 to allow the hydrogel 106 to be properly broken down for removal. These steps of the workflow can be repeated as necessary to allow the removal of the hydrogel 106 from the well unit 100. In addition, any cell bodies remaining in the primary well section 112 of the well unit 100 can be washed, as can be understood, by introducing the liquid medium 109 into the well unit 100 and then removing it. This washing process may be performed up to approximately three times.

[0051] In step 415, the cell bodies 103 that remain in the primary well section 112 of the well unit 100 are aspirated or otherwise collected in the liquid handler 200, as shown in Figure 3D.

[0052] In step 418, a liquid medium 109 (e.g., dissociation or culture medium) may be added to the liquid handler 200, and the cell body 103 will be broken down into multiple cell body fragments 306 and fragments 309. In various embodiments, the liquid medium 109 can be used to accelerate the breakdown of the cell body 103. For example, a shear force may be applied to the cell body 103 as a result of rough pipetting, causing the cell body 103 to move back and forth within the body of the liquid handler 200. In this embodiment, the liquid handler 200 may be operated several times (e.g., 20 times) to allow the cell body 103 to move within the body of the liquid handler 200, thereby allowing the cell body 103 to be broken down into multiple cell body fragments 306 and fragments 309 (e.g., dead cells, toxic byproducts, etc.). In various embodiments, the preferred size of the cell body fragments 306 is about 25-500 μm. However, it should be noted that, as can be understood, fragments of other sizes may also be available.

[0053] In step 421, the cell body fragments 306 and fragments 309 are returned to the primary well section 112 of the well unit 100, as shown in Figure 3E. In step 424, the fragments 309 are removed from the well unit 100. In various embodiments, a liquid medium 109 may be introduced, as shown in Figure 3F, to allow the fragments 309 to be washed away from the cell body fragments 306 and to proceed into the secondary well section 115 through at least one channel 118. In particular, the microplate may be tilted to induce a gravitational flow of fluid through at least one channel 118 and between the two well sections. Fragments 309, sized to be smaller than the dimensions of at least one channel 118, can, as can be understood, pass through the channel 118 and flow into the secondary well section 115. Similarly, the cell body fragments 306 are sized to exceed the dimensions of at least one channel 118, thereby preventing them from passing through at least one channel 118 and remaining within the primary well section 112. In various embodiments, the addition of liquid medium 109, the movement of the fragments 309 into the secondary well section 115, and / or the removal of the fragments 309 using the liquid handler 200 may be repeated as necessary to ensure the removal of the fragments 309.

[0054] In step 427, the cell body fragments 306 remaining in the primary well section 112 can be aspirated or otherwise collected using the liquid handler 200, as shown in Figure 3G. In various embodiments, liquid culture medium 109 is added to the well unit 100 to enable safe handling and collection of the cell body fragments 306.

[0055] In step 430, the hydrogel 106 is deposited in the primary well section 112 of the well unit 100, creating a fresh environment for culturing cells. As can be understood, the hydrogel 106 as described herein may include Matrigel® (a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, Corning® Life Sciences). In other words, the hydrogel or scaffold as described herein may include one or more extracellular matrix components, such as collagen or fibronectin, bioink, gelatin, alginate, Biomimesys®, cellulose-based hydrogel, basement membrane extract (BME), or other hydrogels, scaffolds, or solutions without a scaffold as can be understood.

[0056] In step 433, the cell body fragments 306 are deposited within the hydrogel 106. In various embodiments, the cell body fragments 306 can be deposited within the hydrogel 106 by any preferred technique, including, among other things, bioink droplet printing, microcontact printing, photolithography, dip-pen nanolithography, and / or pipette dispensing. In some embodiments, sieves or other filtration devices can be used to control the size of the cell body fragments 306 deposited within the hydrogel 106 and / or pre-mixed with it. For example, if any cell body fragments 306 are specified to be larger than desired, sieves can be used to restrict the use of larger cell body fragments 306 in a fresh and clean environment. This method discusses a cell body fragment 306 deposited within the hydrogel 106 after the hydrogel 106 has been deposited in the well unit 100. However, it should be noted that in some embodiments, the cell body fragment 306 may be deposited or otherwise embedded within the hydrogel 106 prior to the step of depositing the hydrogel 106 in the well unit 100. In various embodiments, the hydrogel 106 (with or without the cell body fragment 306) can be deposited on a preheated microplate that is heated to a desired temperature (e.g., about 37 degrees Celsius), and the microplate (including the cell body fragment) can be placed in an incubator for a desired amount of time (e.g., up to about 20 minutes).

[0057] In step 436, the liquid medium 109 can be introduced into the well unit 100. The liquid medium 109 can be introduced into the well unit 100 via the primary well section 112 or the secondary well section 115. For example, the liquid medium 109 can be delivered from the secondary well section 115 to the primary well section 112 by a fluid flow driven through at least one channel 118 of the well unit 100 of the microplate. Such a fluid flow can be further facilitated by other means, such as by manually tilting the microplate plate at intervals desired by the user for a period of time desired by the user, or by placing the microplate on an automated tilting or oscillating device.

[0058] In step 439, the microplate is placed in a tissue culture incubator, allowing for the proliferation and growth of cell body fragments 306 (e.g., spheroids, tumoroids, organoids, and / or other multicellular bodies). The subculturing process can then proceed to completion. (cell)

[0059] The cells described herein may include stem cells (e.g., pluripotent stem cells), supporting cells, adult somatic stem cells (ASCs), patient-derived materials (e.g., chumoroid), and / or equivalents.

[0060] The cells relating to this disclosure may be mammalian cells, in particular human, rat, or mouse cells. The cells may include various immortalized cell lines or primary cell lines (e.g., HUVECs) that are typically used in research known to those skilled in the art. The cells relating to this disclosure may be pluripotent or pluripotent stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, adult somatic stem cells (ASCs), patient-derived material (e.g., chumoroid), or mesenchymal stem cells, but not limited to these). In embodiments, the stem cells relating to this disclosure may be mouse or human stem cells that are commercially available to those skilled in the art through direct sales outlets such as ATCC® or other commercial companies known in the art. The stem cells relating to this disclosure may also be human, mouse, or rat (or another organism) stem cells that have been reprogrammed by a user from a primary cell source using any number of reprogramming methods and / or kits available in the art.

[0061] Supporting cells may be, for example, those known to support stem cell cultures. Without limiting themselves, such cells may include, but are not limited to, mouse embryonic fibroblasts, induced pluripotent stem cell (iPSC) lines, embryonic stem cell lines (e.g., E5, E7, etc.), adult somatic stem cells (ASCs), patient-derived materials (e.g., chumoroids), patient-derived organoids (e.g., intestinal, liver, LPSC-derived organoids, etc.), spheroids, embryoid bodies, chumoroid xenografts, various organisms (e.g., Drosophila, zebrafish, etc.), or others known in the art.

[0062] The culture of various cells / cell bodies at different stages of culture can be performed using standard media and techniques known in the art (e.g., Dulbecco's modified Eagle medium, stem cell medium, mTeSR). TM , lntestiCult TMThis can be carried out using culture media containing intestinal organoid culture medium or its variants, fetal bovine serum, and leukemia suppressor factors for mouse iPSCs. Differentiation of stem cells into somatic or tissue target cells of endodermal, ectodermal, and mesodermal lineages is also known and described in the art and can be employed according to the methods of this disclosure. (Hydrogel / Scaffold)

[0063] Hydrogels and / or scaffolds can be employed in microwell plates, systems, kits, and methods as described herein to facilitate 3D culture, proliferation, and chemical analysis of cell bodies as described herein.

[0064] In embodiments, the hydrogel or scaffold described herein may include Matrigel® (a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, Corning® Life Sciences). In other aspects, the hydrogel or scaffold described herein may include one or more extracellular matrix components, such as collagen or fibronectin, bioink, gelatin, alginate, Biomimesys®, cellulose-based hydrogel, basement membrane extract (BME), or other hydrogels, scaffolds, or solutions without a scaffold as can be understood.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having the meaning consistent with their meanings in the context of this specification and the relevant art, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0066] Many aspects of this disclosure can be better understood by referring to the following appendices, which are incorporated herein by reference as a whole. The elements in the drawings are not necessarily to scale, but rather the focus is on clearly illustrating the principles of this disclosure.

[0067] As used herein, the terms “about,” “approximately,” “in or about,” and “substantially” mean that the quantity or value is its exact value or may be a value that provides equivalent results or effects as enumerated in the claims or taught herein. That is, quantities, sizes, formulations, parameters, and other quantities and characteristics are not, and do not need to be, exact, but may be approximate and / or greater or less as desired, and are understood to reflect tolerances, conversion factors, rounding, measurement errors, and equivalents and other factors known to those skilled in the art in which equivalent results or effects are obtained. In some circumstances, a value that provides equivalent results or effects cannot be reasonably determined. In such cases, as used herein, “about” and “in or about” mean nominal values ​​that are indicated as ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, or ±5% of the specified value, for example, about 1 inch is generally understood to refer to the range of 0.8 inches to 1.2 inches, 0.8 inches to 1.15 inches, 0.9 inches to 1.1 inches, 0.91 inches to 1.09 inches, 0.92 inches to 1.08 inches, 0.93 inches to 1.07 inches, 0.94 inches to 1.06 inches, or 0.95 inches to 1.05 inches, unless otherwise indicated or inferred. When “about,” “approximately,” or “in or about” is used before a quantitative value, it is understood that the parameter also includes the specific quantitative value itself, unless otherwise specifically stated.

[0068] Any ratio, concentration, quantity, and other numerical data may be expressed herein in range form. Such range forms are used for convenience and brevity and should therefore be interpreted in a flexible form that includes not only the numbers explicitly listed as limits of the range, but also all individual numbers or sub-regions that are contained within that range, as if explicitly listed. For example, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly listed concentrations of about 0.1% by weight to about 5% by weight, but also the individual concentrations (e.g., 1%, 2%, 3%, and 4%) and sub-regions (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. Where a described range includes one or both limits, ranges that exclude either or both of the limits they contain are also included in this disclosure; for example, the phrase “x to y” includes the range of “x” to “y”, and the range above “x” and below “y”. This range may also be expressed as an upper limit, for example, "about x, y, z, or less than that," and should be interpreted to include the specific ranges of "about x," "about y," and "about z," as well as the ranges of "less than x," "less than y," and "less than z." Similarly, the phrase "more than x, y, z, or more than that" should be interpreted to include the specific ranges of "about x," "about y," and "about z," as well as the ranges of "more than x," "more than y," and "more than z." In some respects, the term "about" may include conventional rounding according to the significant figures of the numbers. In addition, the phrase "about x ~ y" includes "about x ~ about y."

[0069] The term "substantially" is meant to permit deviations from descriptive terms that do not negatively affect the intended purpose. All descriptive terms used herein are implicitly understood to be modified by the term "substantially," even if they are not explicitly modified by the term.

[0070] Any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this disclosure, but various methods and materials suitable for use in combination with the various disclosures disclosed herein are described herein. Functions or constructions well known in the art may not be described in detail for the sake of brevity and / or clarity.

[0071] Disjunctive terms such as "at least one of X, Y, or Z" are generally understood in the context of their use to indicate that an item, term, etc., could be any one of X, Y, or Z, or any combination thereof (e.g., X, Y, and / or Z), unless otherwise specifically described. Therefore, such disjunctive terms are not intended to imply, in general, that a particular embodiment requires the presence of at least one of X, at least one of Y, or at least one of Z, respectively.

[0072] It should be emphasized that the embodiments described above in this disclosure are merely examples that can be considered as possible implementations for a clear understanding of the principles of this disclosure. Many modifications and alterations may be made to the embodiments described above without substantially departing from the spirit and principles of this disclosure. All such modifications and alterations are incorporated herein by reference within the scope of this disclosure and are intended to be protected by the following claims.

Claims

1. A method for subculturing organoids, wherein the method is The method involves culturing one or more organoids in a hydrogel placed within a well unit of a microwell plate, wherein the well unit comprises a primary well section fluidly connected to a secondary well section by at least one channel, the at least one channel being formed as a gap between the bottom surface of the well unit and the bottom portion of the shared side wall between the primary well section and the secondary well section, the opening of the at least one channel having a vertical dimension of 10 to 100 microns, and the hydrogel being placed within the primary well section of the well unit. By cooling the hydrogel to a temperature that causes it to fragment, the hydrogel is broken down into a plurality of hydrogel fragments, thereby separating one or more organoids from the hydrogel. The removal of the plurality of hydrogel fragments from the well unit via the secondary well section of the well unit, wherein one or more organoids remain within the well unit. The process involves crushing one or more organoids into multiple organoid fragments, thereby generating multiple fragments containing dead cells and toxic byproducts. The process involves moving the plurality of fragments through at least one channel to the secondary well section of the well unit, and removing the plurality of fragments from the well unit through the secondary well section of the well unit, wherein the plurality of organoid fragments remain within the primary well section of the well unit. To generate a fresh culture environment containing the aforementioned multiple organoid fragments. Methods that include...

2. The method according to claim 1, wherein cooling the hydrogel further comprises dispensing a liquid culture medium into the well unit, the liquid culture medium being at a temperature of about 10 degrees Celsius or less.

3. The method according to claim 2, wherein the temperature is approximately 4 degrees Celsius or lower.

4. The method described above is: Collecting one or more organoids from the primary well section of the well unit via a liquid handling device, Applying at least one shear force to one or more organoids in the liquid handler, wherein the one or more organoids are crushed into the plurality of organoid fragments and the plurality of pieces as a result of the at least one shear force, The plurality of organoid fragments and the plurality of fragments are deposited into the primary well section of the well unit via the liquid handling device. The method according to any one of claims 1 to 3, further comprising:

5. The method according to claim 4, wherein applying the at least one shear force includes operating the liquid handler to move one or more organoids vertically within the liquid handler.

6. Removing the aforementioned plurality of hydrogel fragments is Inserting the liquid handling device into the secondary well section of the well unit, Transferring the plurality of hydrogel fragments from the primary well section of the well unit into the secondary well section via at least one channel, The plurality of hydrogel fragments are collected from the secondary well section via the liquid handling device. The method according to any one of claims 1 to 5, further comprising:

7. The method according to any one of claims 1 to 6, further comprising removing at least one of fragmentary fragments or organoid fragments from the at least one channel by rinsing the at least one channel.

8. To generate the fresh culture environment containing the plurality of organoid fragments, Collecting the multiple organoid fragments via a liquid handling device, Depositing fresh hydrogel on the bottom surface of another well unit or the primary well section of one of the well units. The method according to any one of claims 1 to 7, further comprising:

9. The method according to claim 8, wherein the plurality of organoid fragments are embedded in the fresh hydrogel prior to depositing the fresh hydrogel on the bottom surface of the primary well section.

10. The method according to claim 8, wherein the plurality of organoid fragments are embedded in the fresh hydrogel after the fresh hydrogel has been deposited on the bottom surface of the primary well section.

11. The method according to any one of claims 1 to 10, further comprising placing the microwell plate comprising the well unit inside an incubator.

12. A method for subculturing a multicellular organism, wherein the method is: The method involves separating one or more multicellular organisms from a hydrogel disposed on the bottom surface of a first well unit of a first microwell plate, wherein the first well unit comprises a culture well and a supply well fluidly connected to each other via at least one channel, the at least one channel being formed as a gap between the bottom surface of the well unit and the bottom portion of a shared side wall between the culture well and the supply well, the opening of the at least one channel having a vertical dimension of 10 to 100 microns, and the hydrogel being disposed within the culture well of the first well unit. The hydrogel is removed from the first well unit via the supply well of the first well unit, wherein the one or more multicellular organisms remain in the culture well of the first well unit. The process involves fragmenting one or more multicellular cells into multiple single-cell fragments or multiple multicellular fragments, thereby generating multiple fragments containing dead cells and toxic byproducts. The process involves moving the plurality of fragments from the culture well to the supply well through the opening of the at least one channel, and removing the plurality of fragments from the supply well of the first well unit, wherein the plurality of single-cell fragments or the plurality of multicellular fragments remain in the culture well of the first well unit. The method involves generating a fresh culture environment within either the first well unit or the second well unit, wherein the fresh culture environment includes the plurality of single-cell fragments or the plurality of multicellular fragments. Methods that include...

13. The method according to claim 12, wherein separating one or more multicellular organisms from the hydrogel further comprises cooling the hydrogel to a temperature at which it liquefies.

14. The method described above is: Removing one or more multicellular organisms from the culture well via a pipette, The method involves applying one or more shear forces to one or more multicellular organisms via the pipette, wherein the one or more multicellular organisms are fragmented into a plurality of single-cell fragments or a plurality of multicellular fragments and a plurality of pieces, at least partially based on the one or more applied shear forces. The method according to any one of claims 12 to 13, further comprising:

15. Creating the fresh culture environment within either the first well unit or the second well unit is: The method involves depositing a fresh hydrogel into one of the first or second well units, wherein the plurality of single-cell fragments or the plurality of multicellular fragments are embedded within the fresh hydrogel. The process involves culturing the plurality of single-cell fragments or the plurality of multicellular fragments embedded in the fresh hydrogel. The method according to any one of claims 12 to 14, further comprising:

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