Cell culture applications using separate well microplates

Abstract

A method for automating horizontal co-culture of cells using a single separated well microplate is presented. The present disclosure also presents an improved method for quantifying secreted factors from cultured cells, and a method for isolating target cells from organoids in a single separated well microplate. The use of a separated well microplate allows cell culture, cell washing, and (adult) stem cell capture in a single separated well microplate without the need for additional plates or special washing equipment. The use of a separated well microplate also allows multiple readings to be read simultaneously in a kinetic manner.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is filed as a PCT international application on March 17, 2023, and claims the benefit of and priority to U.S. Provisional Application No. 63 / 321,373, filed on March 18, 2022, the entire contents of which are incorporated by reference herein. [Background technology]

[0002] background Culturing cells in a three-dimensional (3D) environment results in cellular behaviors and morphologies that are more consistent with those observed in the human body. The 3D hydrogels / hydroscaffolds used in this type of culture have unique properties: cells can be deposited in specific locations in 3D space and remain there for extended periods of time. This allows the creation of structures (e.g., spheroids, tumor analogs, organoids, and / or other multicellular bodies) and co-culture environments in which cellular interactions and development over time can be observed.

[0003] So far, transwells are generally required for vertical co-culture. However, the handling of transwells is relatively complicated, can be difficult to automate, and cells are difficult to image. The supernatants can be transferred to different detection plates using ELISA or different analytical methods. However, kinetic measurements and direct comparisons of several different hormones secreted by organoids have not been practical so far. So far, adult stem cell capture is possible when an additional capture plate is used for washing.

[0004] In vitro toxicity testing often requires the presence of several cell types: for example, cardiac and hepatic cells (metabolizing chemicals), neurons and astrocytes, cardiac and endothelial cells, intestinal and hepatic cells, etc. Co-culturing different cells or transferring supernatants is the current solution, but it can be difficult to obtain clean data for different cells, since it is necessary to distinguish them or evaluate effects resulting from low amounts of secreted factors.

[0005] It would be desirable to use a single isolated well microplate to provide physical separation while allowing fluid communication between different cell types, as well as to allow easy detection and evaluation of effects over time. It would be desirable to use a single isolated well microplate to allow evaluation of an in vivo format while combining immune cell migration, infiltration, and targeted tumor cell killing in one assay. Summary of the Invention [Means for solving the problem]

[0006] Abstract Methods are presented for co-culturing different cell types within separate compartments that allow for effective mutual responses and interactions of cell types and tissues, including, but not limited to, cell migration, neurite outgrowth, angiogenesis, immune cell migration and death, metastasis, cell invasion, monocyte adhesion, apoptosis, cell differentiation, stem cell engraftment, inflammation, and secretion of multiple growth factors and metabolites.

[0007] The present disclosure presents methods for using separate-well microplates to support horizontal co-culture in an automation-friendly manner. Separate-well microplates support the culture of different cell types in adjacent wells that may be connected, for example, hydraulically using a pipette as a pump or by laminar flow with suitable microchannel geometries.

[0008] The use of separated well microplates allows cell culture, cell washing, and capture of (adult) stem cells in a single separated well microplate without the need for additional plates or special washing equipment. The use of separated well microplates also allows multiple reads to be taken simultaneously in a kinetic fashion.

[0009] The present disclosure presents a method for co-culturing target cells, comprising the steps of: introducing one or more target cells into a primary well of a well unit of a separation well microplate, and first incubating in a liquid medium without mixing to allow the target cells to adhere to the bottom of the primary well, the primary well being separated from the secondary well of the well unit of the separation well microplate by at least one closed microchannel including a removable barrier; introducing one or more feeder cells into the secondary well, and first incubating in a liquid medium without mixing to allow the feeder cells to adhere to the bottom of the secondary well; removing the removable barrier to open at least one closed microchannel; rocking the separation well microplate to allow mixing and medium exchange between the primary and secondary wells by gravity flow through the open microchannel; detaching the target cells from the primary well; and allowing the detached target cells to settle into the primary well away from the open microchannel. The method for co-culturing target cells may be a horizontal method for co-culturing target cells. The method for co-culturing target cells may further include mixing the detached target cells and further sedimenting the detached target cells into the primary well away from the open microchannel, washing the sedimented target cells, and collecting the washed target cells via a liquid handler.

[0010] The method for co-culturing target cells may include where the steps of introducing one or more target cells into a primary well and introducing one or more feeder cells into a secondary well are performed simultaneously or substantially simultaneously. Simultaneous may be within about 5, about 10, or about 15 minutes. Substantially simultaneous may be within about 30, about 60, or about 90 minutes.

[0011] The method for co-culturing the target cells may include where at least one closed microchannel is closed by a removable barrier comprising an air gap, a hydrogel seal, or a silicone seal within or adjacent to the microchannel. The method for co-culturing the target cells may include where at least one closed microchannel is closed during at least a portion of the initial incubation period.

[0012] The method for co-culturing target cells may include where the removable barrier comprises an air gap, and removing the removable barrier comprises removing the air gap after at least a portion of the initial incubation period has elapsed, and optionally includes using a pipette as a pump to exchange liquid medium between the primary well and the secondary well.

[0013] The method for co-culturing target cells may include where the detaching step is to expose the target cells to a dissociation reagent that detaches the target cells without detaching the feeder cells. The dissociation reagent may include a detaching enzyme, optionally in a physiological buffer. In some embodiments, the detaching enzyme may be selected from the group consisting of trypsin, collagenase, elastase, catalase, superoxide dismutase, and dispase. In some embodiments, the dissociation reagent includes EDTA.

[0014] In some embodiments, the target cells can be stem cells or organoids. In some embodiments, the feeder cells are fibroblasts.

[0015] The method for co-culturing target cells may include where the target cells are embedded in a hydrogel dome disposed in a primary well of a well unit in liquid medium. The method for co-culturing target cells may include where at least one closed microchannel is located between a bottom surface of the well unit and a bottom portion of a common sidewall of the primary well and the secondary well.

[0016] The method for co-culturing target cells may include the use of a separate well microplate containing a plurality of well units, each well unit containing at least one microchannel capable of fluidly connecting a primary well and a secondary well within the well unit, the height of the at least one microchannel ranging from about 10 microns to about 100 microns, or from about 10 microns to about 75 microns, and optionally, the width of the microchannel ranging from about 150 to about 500 microns, about 200 to about 400 microns, or about 300 microns. The separate well microplate may contain a plurality of well units, for example, 384 well units, 96 well units, 48 ​​well units, 24 well units, 12 well units, 8 well units, or 6 well units.

[0017] The present disclosure provides a method for quantifying secreted factors from cultured target cells, including time-course kinetic measurements in a single separated well microplate, comprising the steps of culturing target cells in a primary well of a well unit of the separated well microplate in liquid medium for an initial incubation period, the primary well being separated from a secondary well of the well unit of the separated well microplate by at least one closed microchannel comprising a removable barrier, the secondary well comprising an immobilized capture reagent; after the initial incubation period, removing the removable barrier to open at least one closed microchannel and allowing the secreted factor to diffuse between the primary and secondary wells through the open microchannel; capturing the secreted factor with the immobilized capture reagent; adding a labeled secondary antibody specific for the captured secreted factor or the immobilized capture reagent at a first time point to form a first labeled complex; and quantifying the first labeled complex using a microplate reader.

[0018] The method for quantifying secreted factors from cultured target cells can include where the immobilized capture reagent is selected from a primary antibody specific for the captured secreted factor, avidin, and biotin.

[0019] The method for quantifying secreted factors from cultured target cells may further comprise, prior to removing the removable barrier, adding a first antibody conjugate to a secondary well having an avidin-immobilized capture reagent, where the first antibody conjugate comprises a secreted factor-specific primary antibody conjugated to biotin.

[0020] The method for quantifying secreted factors from cultured target cells may further comprise, prior to removing the removable barrier, adding a second antibody conjugate to the secondary well with the biotin-immobilized capture reagent, where the second antibody conjugate comprises a secreted factor-specific primary antibody conjugated to avidin.

[0021] The method for quantifying secreted factors from cultured target cells may further include, at a second time point, adding a labeled secondary antibody specific for the captured secreted factor, the immobilized capture reagent, or the primary antibody to form a second labeled complex, and quantifying the second labeled complex using a microplate reader.

[0022] The method for quantifying secreted factors from cultured target cells may further include, at a third time point, adding a labeled secondary antibody specific for the captured secreted factor, the immobilized capture reagent, or the primary antibody to form a third labeled complex, and detecting the third labeled complex using a microplate reader.

[0023] The method for quantifying secreted factors from cultured target cells may include where the quantifying step includes multiplexed detection using imaging equivalent to a plate reader. The method for quantifying secreted factors from cultured target cells may include where the diffusion of the secreted factors includes rocking a separated well microplate to diffuse the secreted factors between the primary and secondary wells by gravity flow through the open microchannels.

[0024] The method for quantifying secretory factors from cultured target cells can include the case where target cells are multicellular target cells.In some embodiments, the multicellular target cells are selected from the group consisting of organoids and tumor analogs.In some embodiments, the multicellular target cells are derived from target tissue, biopsy samples obtained from patients, stem cells, tumor analog fragments, or organoid fragments.

[0025] A method for quantifying secreted factors from cultured target cells may include where cultured organoids are embedded within a hydrogel dome contained in liquid medium in a primary well of a well unit.

[0026] The method for quantifying secreted factors from cultured target cells may include where the removable barrier comprises an air gap, a hydrogel seal or a silicone seal, optionally where the removable barrier is disposed within or adjacent to a microchannel.

[0027] The method for quantifying secreted factors from cultured target cells may include where the step of removing the removable barrier includes removing the air gap and optionally using a pipette as a pump to exchange liquid medium between the primary well and the secondary well.

[0028] The method for quantifying secreted factors from cultured target cells may include where an immobilized capture reagent is immobilized on the bottom of the secondary well. In some embodiments, the immobilized capture reagent includes multiple primary antibodies specific for multiple secreted factors, where the primary antibodies are arrayed on the bottom of the secondary well in a detection array. The method for quantifying secreted factors from cultured target cells may include where the secreted factors are selected from the group consisting of hormones, antibodies, cytokines, chemokines, and growth factors.

[0029] The present disclosure provides a method for isolating a single cell type from a cultured multicellular target cell culture in a single separated well microplate, comprising culturing multicellular target cells in a primary well of a well unit of the separated well microplate in liquid medium for an initial incubation period, the primary well being separated from a secondary well of the well unit of the separated well microplate by at least one closed microchannel comprising a removable barrier, the secondary well comprising a surface functionalized with an immobilized capture reagent; dissociating the multicellular target cells in the primary well to form a mixture of individual cells and unrelated cells comprising a single cell type; removing the removable barrier to open the microchannel to allow the mixture of individual cells to migrate through the open microchannel to the secondary well, where the single cell type associates with the immobilized capture reagent in the secondary well to form an immobilized complex; washing the primary and secondary wells to remove unbound unrelated cells; and isolating the single cell type, comprising dissociating the immobilized complex. Enabling individual cells to migrate through open microchannels may involve the use of a pipette as a pump, diffusion, or laminar flow with suitable microchannel geometries.

[0030] The method of isolating a single cell type from a cultured multicellular target cell culture may further comprise reseeding the isolated single cell type into a primary well of a separate well microplate. In some embodiments, the single cell type is a stem cell. In some embodiments, the single cell type is a target cell.

[0031] In some embodiments, the multicellular target cell is an organoid.

[0032] A method for isolating a single cell type from a cultured multicellular target cell culture can include where an immobilized capture reagent is disposed on the bottom of a secondary well or on a scaffold within the secondary well.

[0033] In some embodiments, the immobilized capture reagent may be selected from a primary antibody specific for a surface marker of a single cell type, avidin, and biotin.

[0034] The method of isolating a single cell type from a cultured multicellular target cell culture may further comprise, prior to removing the removable barrier, adding a first antibody conjugate to the secondary well having the avidin-immobilized capture reagent, where the first antibody conjugate comprises a single cell type surface marker-specific primary antibody conjugated to biotin.

[0035] The method of isolating a single cell type from a cultured multicellular target cell culture may further comprise, prior to removing the removable barrier, adding a second antibody conjugate to the secondary well with the biotin-immobilized capture reagent, where the second antibody conjugate comprises a single cell type surface marker-specific primary antibody conjugated to avidin.

[0036] A method for isolating a single cell type from a cultured multicellular target cell culture can include where the multicellular target cells are cultured within a hydrogel dome contained in liquid medium in a primary well.

[0037] The present disclosure provides a method for isolating stem cells from a multicellular target cell culture in a single separated well microplate, comprising the steps of culturing the multicellular target cells in a first hydrogel dome contained in a liquid medium in a primary well of a well unit of the separated well microplate for a first incubation period, the primary well being separated from a secondary well of the well unit of the separated well microplate by at least one closed microchannel comprising a removable barrier; dissociating the multicellular target cells in the primary well to form a mixture of individual and unrelated cells, including stem cells; and adding magnetic beads containing immobilized capture reagent specific for stem cells to the primary well, wherein the magnetic beads capture the stem cells. a method is presented that includes further incubating to allow the magnetic beads to bind to the cells; applying a magnet under the primary well to retain the magnetic bead-stem cell complexes; removing the removable barrier to open the microchannel and washing the retained magnetic bead-stem cell complexes by aspirating liquid medium through the secondary well to remove unbound unrelated cells; adding a second hydrogel dome to the washed, retained magnetic bead-stem cell complexes in the primary well; removing the magnet from under the primary well; dissociating the stem cells from the magnetic beads; and applying a magnet above the primary well to levitate the magnetic beads and isolate the stem cells in the second hydrogel dome.

[0038] In some embodiments, the method of isolating stem cells from a multicellular target cell culture further comprises removing the magnet from beneath the primary well, transferring the washed, retained magnetic bead-stem cell complexes to a new primary well in a new well unit of a new separation well microplate, and reapplying the magnet beneath the new primary well in the new well unit of the new separation well microplate. In some embodiments, the immobilized capture reagent is selected from a primary antibody specific for a surface marker of stem cells, avidin, and biotin.

[0039] The present disclosure provides a method for selecting a therapy for cancer treatment in a subject in need thereof, comprising the steps of culturing multicellular target cells from the subject in a primary well of a well unit of a separated well microplate in a liquid medium for an initial incubation period, the primary well being separated from a secondary well of the well unit of the separated well microplate by at least one closed microchannel comprising a removable barrier, the secondary well containing a candidate cancer therapeutic immune cell, a monoclonal antibody, an immune system modulator, or an anti-tumor agent; and removing the removable barrier to separate the at least one closed microchannel from the secondary well of the well unit of the separated well microplate. A method is presented that includes opening the channel to allow diffusion between the primary and secondary wells through the open microchannels for a second incubation period, imaging the secondary wells and optionally the primary wells after the second incubation period to quantify cell migration or multicellular target cell proliferation, and selecting a therapeutic candidate based on increased immune cell migration in the secondary wells or between the secondary and primary wells compared to a control well unit without the multicellular target cells, or decreased multicellular target cell proliferation in the primary wells compared to a control well unit without the therapeutic candidate.

[0040] The method for selecting a therapy for cancer treatment may include where the multicellular target cells are embedded in a hydrogel dome disposed in a primary well of a well unit in a liquid medium. The method for selecting a therapy for cancer treatment may include where the multicellular target cells are derived from a biopsy sample or tumor analog fragment obtained from a subject. In some embodiments, the immune cells may be CAR T cells.

[0041] The present disclosure presents an automated method of disrupting hydrogel domes, comprising culturing a first cell within a hydrogel dome in liquid medium in a well of a microplate for an initial incubation period to provide a multicellular target cell, piercing the hydrogel dome with a pipette tip associated with a liquid handler after the initial incubation period at or near the x,y central location of the dome within the well, and moving the pipette tip through the hydrogel dome in multiple steps, each step including a pre-determined x,y location within the well for disrupting the hydrogel dome, and optionally liquefying the hydrogel dome to release the target cell from the hydrogel dome within the well. The pipette can be aspirated at one or more, two or more, multiple, or individual pre-determined locations.

[0042] The present disclosure presents an automated method of disrupting hydrogel domes and isolating target cells, comprising culturing a first cell within a hydrogel dome contained in a liquid medium in a well of a microplate for an initial incubation period to provide multicellular target cells, piercing the hydrogel dome with a pipette tip associated with a liquid handler after the initial incubation period at or near the x,y central location of the dome within the well, moving the pipette tip through the dome in multiple steps to release the target cell from the hydrogel dome, each step including a pre-determined x,y location within the well for disrupting the hydrogel dome and, optionally, liquefying the hydrogel dome, and isolating one or more of the target cells from the well. The pipette may be aspirated at one or more, two or more, or individual pre-determined locations.

[0043] Each of the pre-determined X,Y positions within the well is 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 = dome diameter (mm) / 1.8; steps = number of steps the pipette tip moves through the hydrogel dome; X well center = x position of the dome center within the well; Y well center = y position of the dome center within the well; 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 some cases, theta is a constant derived from 5 and 50, 10 and 45, 15 and 40, 15 and 35, or any number in between. In some cases, theta is selected from the 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.

[0044] In some cases, the liquid handler aspirates the pipette tip within the hydrogel dome and / or the pipette tip punctures the hydrogel dome at one or more, two or more, multiple, or individual pre-determined locations.

[0045] In some cases, the well is a primary well of a well unit of a split-well microplate.

[0046] The x,y central position of the hydrogel dome can be determined by one or more parameters selected from the group consisting of the initial seeding position within the well, the volume of hydrogel deposited in the well, and imaging of the hydrogel dome within the well.

[0047] The diameter of the hydrogel dome can be determined by the volume of hydrogel deposited in the well and / or by imaging the hydrogel dome within the well.

[0048] The pre-determined X,Y locations may form a pattern within the well within and / or through the hydrogel dome selected from the group consisting of a spiral pattern, a star pattern, and a zigzag pattern.

[0049] The pipette tip can be lowered below the surface of the hydrogel dome between each position.The pipette tip can be raised above the surface of the hydrogel dome between each position.

[0050] The target cell may optionally be a multicellular target cell selected from the group of a spheroid, a tumor analog, and an organoid.

[0051] In some cases, the method further comprises dissociating the multicellular target cells from one another prior to isolation to provide individual target cells. [Brief description of the drawings]

[0052] [Figure 1A] FIG. 1A shows a first view of an exemplary single well unit 100 located in a separate well microplate useful for horizontal co-culture for cell / organoid feeding. The primary well 112 is separated from the secondary well 115 by at least one microchannel 118. In some embodiments, the at least one microchannel 118 may be open to allow medium exchange between the primary well 112 and the secondary well 115. Alternatively, the at least one microchannel 118 may include a removable barrier to isolate the primary and secondary wells from each other.

[0053] [Figure 1B]1B shows a second view of an exemplary single well unit 100 located in a separate well microplate useful for horizontal co-culture for cell / organoid feeding. The primary well 112 is separated from the secondary well 115 by at least one microchannel 118. Medium exchange occurs by gravity flow through the microchannel 118 while the separate well microplate is placed on a rocking device.

[0054] [Figure 1C] FIG. 1C shows an example of a cross section of a single well unit 100 of a separated well microplate associated with the cell culture method described in various embodiments of the present disclosure. The primary well 112 is separated from the secondary well 115 by a common sidewall that includes at least one microchannel 118 between the primary well 112 and the secondary well 115. The primary well 112 may contain target cells (e.g., organoids) embedded within a hydrogel dome 106. The primary well 112 and the secondary well 115 may be at least partially filled with liquid medium 109. The bottom of the well unit 100 includes a bottom layered sheet 121 that includes an optically transparent observation window to allow imaging.

[0055] [Diagram 2] Figure 2 shows a representative schematic diagram of an exemplary method for detecting multiple secreted factors from target cells (e.g., organoids), which allows cell culture and kinetic measurement of secreted factors in a single separate well microplate. Target cells (e.g., organoids) are cultured in a primary well 112. The target cells produce secreted factors (e.g., hormones) that can diffuse through a microchannel 118. The secreted factors are captured in a secondary well 115 that contains an array of coated / surface-functionalized immobilized antibodies specific for the secreted factors.

[0056] [Diagram 3]FIG. 3 illustrates an exemplary method for performing single cell (e.g., stem cell) isolation from a multicellular target cell culture (e.g., organoids) in a single separation well microplate containing a surface functionalized with immobilized capture reagents (e.g., stem cell specific antibodies, avidin, or biotin). Multicellular target cells (e.g., organoids) are cultured in a hydrogel dome 106 contained in liquid medium 109 in a primary well 112, a secondary well 115 is coated / surface functionalized with immobilized antibodies specific for the cells of interest (e.g., stem cells), and the microchannel 118 is closed with a removable barrier (310). After a period of time, the multicellular target cells (e.g., organoids) dissociate to form a mixture of individual cells, including the cells of interest (320). The barrier is removed from the microchannel 118, and the mixture of individual cells can be moved, for example, hydrodynamically, through the open microchannel 118 by using a pipette as a pump or by laminar flow with a suitable microchannel geometry. The cells of interest are captured by the immobilized specific antibody in the secondary well 115, and the unrelated cells are washed out of the microwell (330). The cells of interest (e.g., stem cells) dissociate from the antibody / cell complex (340). The cells of interest (e.g., stem cells) can be used to seed new wells (350).

[0057] [Figure 4]FIG. 4 shows an exemplary method for performing stem cell isolation from organoid cultures in a single separate well microplate containing antibody-coated scaffolds. Multicellular target cells (e.g., organoids) are cultured in a hydrogel dome 106 contained in liquid medium 109 in a primary well 112, a secondary well 115 contains a scaffold coated / surface-functionalized with immobilized antibodies specific for the cells of interest (e.g., stem cells), and the microchannel 118 is closed by a removable barrier (410). After a period of time, the multicellular target cells (e.g., organoids) dissociate to form a mixture of individual cells, including the cells of interest (420). The barrier is removed from the microchannel 118, and the mixture of individual cells can be traversed, for example, hydraulically, through the open microchannel 118 by using a pipette as a pump or by laminar flow with a suitable microchannel geometry. The cells of interest are captured by the scaffold containing the immobilized specific antibody in the secondary well 115, and the unrelated cells are washed out of the microwell (430). The cells of interest (e.g., stem cells) dissociate from the antibody / cell complex in the scaffold (440). The cells of interest (e.g., stem cells) can be used to reseed a new well (450).

[0058] [Diagram 5]FIG. 5 illustrates an exemplary method for passaging multicellular target cells (e.g., organoids) in a manner that resolves them to the single cell level while isolating desired single cells (e.g., stem cells) in a single separated cell microplate using magnetic beads coated with a capture reagent (e.g., cell-specific antibody or biotin). Multicellular target cells (e.g., organoids) are cultured in a well unit of a separated well microplate that includes a primary well 112 containing organoids 103 (e.g., intestinal organoids) in a hydrogel dome 106 contained in a liquid medium 109 disposed on the bottom surface of the primary well 112, and has a closed microchannel 118 that includes a removable barrier between the primary and secondary wells. After a period of time, the multicellular target cells (e.g., organoids) dissociate to form a mixture of individual cells, including cells of interest (520). Magnetic beads that are surface-functionalized with immobilized capture reagents (e.g., antibodies specific for stem cell markers or biotin) are added to the mixture of individual cells in the primary well unit (530). The magnetic beads can be incubated with the mixture of individual cells to allow binding of the desired single cells (e.g., stem cells) (540). A magnet is applied to the bottom of the primary well to retain the magnetic bead-capture reagent-stem cell complex, the microchannel 118 is opened by removing the barrier, and unbound cells and debris are washed through the secondary well 115, for example, by aspirating the liquid medium with a liquid handler. A new hydrogel dome can be formed in the primary well, such that the stem cells and magnetic beads are embedded in the hydrogel (560), and the magnet is removed from beneath the primary well (570). A magnet can be placed above the primary well to levitate the stem cells within the hydrogel (580).

[0059] [Figure 6A]FIG. 6A illustrates an exemplary method for evaluating functionality of cells (e.g., lymphocytes) and performing toxicology testing in a single separated well microplate. Multicellular target cells (e.g., tumor analogs, organoids) are cultured in a hydrogel dome 106 contained in liquid medium 109 in a primary well 112 of a well unit 100 of a separated well microplate. A secondary well 115 contains immune cells (e.g., CAR-T cells, CAR-NK cells, other lymphocytes). Chemoattractants may be released by the multicellular target cells and diffuse through the open microchannels 118 (which may cause certain immune cells to migrate into the secondary well 115 or into the primary well 112, depending on the microchannel 118 configuration). The microchannels 118 may have a channel size of 5 microns, while lymphocytes have a size of 7-10 microns. In some embodiments, the multicellular target cells may release chemoattractants (which may cause immune cells to migrate into the primary well 112, depending on the microchannel 118 configuration).

[0060] [Figure 6B] FIG. 6B shows an exemplary method for evaluating the functionality of cells (e.g., lymphocytes) and performing toxicology testing in a single separation well microplate, where a tilted configuration can be adopted to prevent immune cell migration. Multicellular target cells (e.g., tumor analogs, organoids) are cultured in hydrogel domes 106 contained in liquid medium 109 in the primary wells 112 of the well units 100 of the separation well microplate. The secondary wells 115 contain immune cells (e.g., CAR-T cells, CAR-NK cells, other lymphocytes). Chemoattractants can be released by the multicellular target cells and diffuse through the open microchannels 118 (which can cause certain immune cells to migrate in the secondary wells 115 or to the primary wells 112 depending on the microchannel 118 configuration). The separation well microplate can be maintained in a tilted configuration to prevent immune cell migration to the primary wells 112.

[0061] [Figure 7A]Figure 7A shows an exemplary spreadsheet calculation of the hydrogel dome rupture pattern for a pre-determined X,Y position for an automated pipetter movement calculated using the formulas: X(steps) = X well center + alpha / step * step number * COS(theta / step * step number); and Y(steps) = Y well center + alpha / step * step number * SIN(theta / step * step number), where dome diameter (mm) = 8, step number = 9, alpha = diameter / 1.8, and theta = 15.7. As shown in Figure 7B (lower right panel), a spiral pattern of steps is formed.

[0062] [Figure 7B] 7B shows exemplary pipettor movement patterns of pre-determined positions calculated using the above formula for 6 (top left panel), 7 (top right panel), 8 (bottom left panel), and 9 (bottom right panel) steps of the hydrogel dome rupture. A helical pattern of steps is formed for each pattern using a theta value of 15.7.

[0063] [Figure 7C] 7C shows exemplary pipettor movement patterns of pre-determined positions calculated using the above formula for 10 steps (top left panel), 11 steps (top right panel), 12 steps (bottom left panel), and 13 steps (bottom right panel) for the rupture of the hydrogel dome. A helical pattern of steps is formed for each pattern when a theta value of 15.7 is used.

[0064] [Figure 7D] Figure 7D shows an exemplary pipettor movement pattern calculated using the formula above for the rupture of a hydrogel dome with diameter (mm) = 8, number of steps = 13, alpha = diameter / 1.8, and theta = 30. With a theta value of 30, the pattern of steps is less spiral and begins to look like a star. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Detailed Description When studying co-culture and cell interactions, there are several obstacles for culture and data analysis. Separating different cell types in separate compartments allows for effective mutual responses and interactions of cell types and tissues, including but not limited to cell migration, neurite outgrowth, angiogenesis, immune cell migration and killing, metastasis, cell invasion, monocyte adhesion, apoptosis, cell differentiation, stem cell transplantation, inflammation, and secretion of multiple growth factors and metabolites.

[0066] A method is presented for cell / organoid co-culture assays that involves the use of separate-well microplates, which support horizontal co-culture in an automation-friendly manner.

[0067] A method is presented for co-culturing different cell types in adjacent wells that can be connected hydraulically using a pipette as a pump or by laminar flow through a microchannel between the primary and secondary wells.

[0068] In support of this disclosure, the method of culturing organoids and cells using separate well microplates has been investigated.For example, organoids are dissociated and transferred to new wells.Long-term culture of organoids shows that the cultured cells in the subculture plate are in good condition.

[0069] Fibroblast adhesion was assessed on plasma-treated split-well microplates. Fibroblast adhesion and normal behavior were observed. Fibroblast migration through the channel was tested using chemoattractants.

[0070] The disclosed method employs a separate well microplate having a well geometry including a primary well and a secondary chamber connected by at least one channel. For example, the disclosed culture method may utilize a separate well microplate having multiple well units (each including a primary well and a secondary chamber connected by at least one channel). For example, each well unit may include a well geometry including a primary well that fluidly connects with a secondary well via at least one channel, as shown in FIG. 1. Separation well microplates are described in U.S. Provisional Patent Application No. 63 / 151,082, filed February 19, 2021, entitled "METHODS AND APPARATUSES FOR CELL PASSAGING USING MICROPLATE WELL UNITS," U.S. Provisional Patent Application No. 17 / 580,193, filed October 22, 2021, entitled "MICROPLATES FOR AUTOMATING ORGANOID CULTIVATION," U.S. Provisional Patent Application No. 63 / 300,294, filed January 18, 2022, entitled "3D PRINTING OF ORGANOID PASSAGING PLATE," and PCT International Application No. PCT / IB2021 / 062356, filed December 27, 2021, entitled "MICROPLATE WELLS FOR CELL CULTIVATION," each of which is incorporated by reference in its entirety herein.

[0071] FIG. 1A illustrates one example of a well unit 100 of a separated well microplate that can be used in conjunction with the method of the present disclosure. In particular, FIG. 1A illustrates an exemplary diagram of a well unit 100 of a microplate that can be used to grow, culture, monitor, and assay embryoid bodies, fused embryoid bodies, spheroids, organoids, or other multicellular bodies according to various embodiments of the present disclosure. For example, a single well unit 100 of a separated well microplate is useful for horizontal co-culture for cell / organoid feeding. A separated well microplate for use in a method according to the present disclosure can include multiple well units, each well unit including a primary well 112 separated from a secondary well 115 by a common sidewall, and the primary and secondary wells can be fluidically connected by at least one microchannel 118. In some embodiments, the at least one microchannel 118 can include a removable barrier to isolate the primary and secondary wells from each other. In some embodiments, at least one microchannel 118 may be open to allow medium exchange between the primary well 112 and the secondary well 115. The well unit 100 of the separated well microplate includes a primary well 112 that may be fluidly connected to a secondary well 115 by at least one microchannel 118. In some cases, the primary well 112 may be used as a culture well, and the secondary well 115 may be used as a feeding well. The microchannel 118 may include a removable barrier. The at least one microchannel 118 may be closed by a removable barrier or may be open. The one or more microchannels 118 between the primary well 112 and the secondary well 115 may be closed by a removable barrier, for example, an air gap, a hydrogel seal, or a silicone seal. The one or more microchannels 118 between the primary well 112 and the secondary well 115 may be closed by, for example, an air gap. The air gap may be formed, for example, by an air bubble.

[0072] FIG. 1B illustrates another example of a well unit 100 of a separated well microplate that can be used in conjunction with the method of the present disclosure. In FIG. 1B, the well unit 100 includes a primary well 112 and a secondary well 115 that can be fluidly connected by at least one open microchannel 118. When open, the at least one channel 118 geometry can allow for the exchange of medium, expressed proteins, such as hormones, nutrients, growth factors, waste products, and debris between the primary and secondary wells while retaining, for example, target cells, such as organoids, in the primary well 112 and feeder cells in the secondary well 115. The dimensions of the at least one microchannel 118 can prevent multicellular target cells (e.g., organoids, tumor analogs, etc.) from passing through the microchannel 118. In some cases, the at least one microchannel 118 geometry may include a slit configuration having a channel width of 150-500 microns or more, or 200-400 microns, or about 300 microns by a height of 10-100 microns or 10-75 microns. During the practice of the methods of the present disclosure, the at least one microchannel 118 between the primary and secondary wells may be closed by a removable barrier or may be opened to allow exchange of media, secreted factors, hormones, growth factors, nutrients, waste products, and debris between the primary well 112 and the secondary well 115, e.g., by gravity feeding while rocking the separated-well microplate. In some cases, the at least one microchannel may be opened, e.g., by removing the removable barrier, by eliminating an air gap, e.g., by using a pipette as a pump, by flowing liquid between the two wells while sealing the secondary well with a pipette tip, etc.

[0073] FIG. 1C illustrates a further example of a well unit 100 of a separated well microplate that may be used in connection with the methods of the present disclosure. The well unit 100 includes a primary well section 112, a secondary well section 115, at least one microchannel 118, and a first hydrogel dome 106 that may be disposed within the primary well section 112 of the well unit 100. According to various embodiments, the well unit 100 further includes a bottom layered sheet 121 disposed on the underside of the well plate body of the microplate. The bottom layered sheet 121 forms a bottom surface of the well unit 100 that is attached to the underside of the well plate body. In various instances, as will be appreciated, the bottom layered sheet 121 includes an optically transparent observation window that allows imaging of spheroids, organoids, or other cell cultures cultured within the well unit 100 of the microplate. The observation window may be a window suitable for microscopic observation, whether bright field, phase contrast, fluorescence, confocal, two-photon, or other microscopic imaging modalities known in the art. In some cases, the bottom layered sheet 121 may include a gas permeable sheet configured to increase oxygen supply for growing spheroids, organoids, or other cell bodies in the well unit 100 of the microplate. As will be appreciated, the gas permeable sheet may be formed from materials including polytetrafluoroethylene (PTFE), PEFP, polyimide, polydimethylsiloxane (PDMS), polycarbonate, and / or other materials. According to various cases, the gas permeable sheet may have a thickness of about 5-70 microns. According to various cases, the gas permeable sheet may include a plurality of pores. In other examples, the gas permeable sheet may allow molecules to pass through by diffusion. Alternatively, the gas permeable sheet may include some other thickness, pore diameter, and pore density.

[0074] As shown in FIG. 1C, target cells in the form of multicellular bodies 103 (e.g., spheroids, tumor analogs, organoids, etc.) may be placed in well units 100 in primary wells 112 and embedded within a dome of hydrogel 106 (e.g., Matrigel®) surrounded by liquid medium 109. According to various embodiments, liquid medium 109 may contain appropriate growth factors and nutritional supplements to create and / or stimulate the growth of the desired multicellular bodies. Exemplary growth factors that may be suitable include angiopoietin, bone morphogenetic proteins (BMPs), ciliary neurotrophic factor, colony stimulating factors, ephrins, epidermal growth factors, erythropoietin, fibroblast growth factors, glial derived neurotrophic factors, hepatocyte growth factors, insulin, insulin-like growth factors, interleukins, leukemia inhibitory factors, keratinocyte growth factors, neuregulins, neurotrophins, platelet-derived growth factors, transforming growth factors, tumor necrosis factor (alpha), vascular endothelial growth factors, and the like.

[0075] According to various embodiments, the well unit 100 of FIG. 1C includes a primary well section 112 and a secondary well section 115. In various cases, the primary well section 112 and the secondary well section 115 are isolated from each other by a closed microchannel. In some embodiments, the microchannel 118 may be closed, for example, by an air gap, a hydrogel, or a silicone. In some embodiments, the microchannel 118 may be open such that the primary well section 112 and the secondary well section 115 are fluidly connected to each other via at least one microchannel 118 to facilitate gravity flow of liquid (e.g., liquid medium 109) between the primary well section 112 and the secondary well section 115, for example, in response to tilting of the microplate. The exchange of liquid medium 109 between the primary well section 112 and the secondary well section 115 may allow for the removal of toxic by-products and may supply the proliferative cell culture with fresh nutrients. In the example of FIG. 1C, the hydrogel 106 and cell bodies 103 are disposed within the primary well section 112 of the well unit 100.

[0076] According to various embodiments, methods according to the present disclosure employ a separate well microplate including a plurality of well units 100, such as 384 well units, 96 well units, 48 ​​well units, 24 well units, 12 well units, 8 well units, or 6 well units. Depending on the number of well units 100 in the microplate, the width of the primary well section 112 may 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 that may be recognized. Additionally, the depth of the primary well section 112 and the secondary well section 115 are defined to allow for liquid exchange within the well units 100 by tilting the microplate without spilling liquid from each primary well section 112 or secondary well section 115 of each well unit 100.

[0077] According to various embodiments, the well units 100 of the separated well microplate include a primary well section 112, which may be sized and shaped to support deposited cell bodies 103 (e.g., cell aggregates) that may be deposited within the primary well section 112 and embedded within the hydrogel 106. For example, the primary well section 112 may be considered a culture well used to grow target cells, such as embryoid bodies, fused embryoid bodies, spheroids, organoids, and / or other multicellular bodies.

[0078] The secondary well section 115 can be used to grow feeder cells and provide feeding medium and / or other nutrients that can be used to nourish the proliferating cell aggregates disposed within the primary well section 112. In some embodiments, the secondary wells can be coated with a primary antibody specific for a secreted factor from the target cells, for example. In some embodiments, the secondary well section 115 can be used to harvest supernatant from the cell aggregates. For example, the secondary well section 115 can be considered a supply well that contains feeding medium and / or other nutrients that can be used by the proliferating cell culture within the primary well section 112. The secondary well section 115 can be sized and shaped to hold a liquid that can be exchanged with the primary well section 112 as described in various embodiments of the present disclosure.

[0079] According to various embodiments, and depending on the number of well units 100 in the microplate, the width of the secondary well section 115 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 that may be discerned.

[0080] According to various embodiments, the sizes and shapes of the primary well section 112 and the secondary well section 115 can differ from one another. In some cases, the primary well section 112 is larger (in dimensions, e.g., diameter, cross-sectional area, or volume) than the secondary well section 115. In other examples, the secondary well section 115 is larger than the primary well section 112. In some cases, the primary well section 112 includes a shape that differs from the shape of the secondary well section 115.

[0081] In various cases, the well units of a separated well microplate include at least one microchannel 118 having a size and shape that prevents objects having a dimension (e.g., diameter, height, width, etc.) of a certain size (e.g., greater than about 25 microns (μ)) from passing from one well section to another well section.

[0082] In some cases, the height of the at least one microchannel 118 may be about 10 microns to up to about 100 microns, or about 10 microns to about 75 microns, or about 10 microns to about 25 microns in size. The at least one microchannel 118 geometry may include a slit configuration having a channel width of 150-500 microns or more, or 200-400 microns, or about 300 microns by a height of 10-100 microns or 10-75 microns. For example, the height of the at least one microchannel 118 may be such that objects present in the primary well section 112 that are about 25μ or more in size (e.g., spheroids, tumor analogs, organoids, organoid fragments, etc.) are prevented from migrating to the secondary well section 115. The microchannel 118 may be closed, for example, by an air gap, a hydrogel seal, or a silicone seal. The microchannel 118 may be opened, for example, by removing the air gap or removing the hydrogel seal or silicone seal.

[0083] In some embodiments of the present disclosure, the primary well 112 may be utilized as a culture chamber. Initially, at least one channel 118 may be closed, e.g., target cells may be cultured and attached to the bottom of the primary well 112 of the separation well microplate, e.g., without mixing. Feeder cells may be cultured and attached to the bottom of the secondary well 115 of the separation well microplate, e.g., without mixing. After the target cells and / or feeder cells are attached to the well, the closed at least one channel 118 may be opened. The separation well microplate may be placed on a rocking device that allows for the exchange of medium, hormones, growth factors, nutrients, waste products, and debris by gravity feed. In some cases, feeder cells may be cultured in the secondary chamber 115, allowing the target cells to be cultured in the primary chamber 112 and fed through the open at least one channel.

[0084] definition

[0085] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0086] The singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0087] The term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items.

[0088] The term "about" when referring to a measurable quantity, such as an amount of a compound, a dose, a time, a temperature, etc., is intended to encompass variations of as much as 10%, 5%, 1%, 0.5%, or 0.1% of the stated amount.

[0089] The term "antibody conjugate" refers to a specific antibody conjugated to a chromophore or fluorophore molecule, avidin, such as streptavidin, or biotin, where the antibody is specific for a target antigen, such as a secreted factor.

[0090] The terms "comprises" and / or "comprising", as used herein, specify the presence of the stated properties, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other properties, integers, steps, operations, 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 commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict in terminology, the present specification shall control.

[0091] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

[0092] An embodiment described in one aspect of the present disclosure is not limited to the described aspect, and applies to a different aspect as well, unless the embodiment prevents the other aspect of the present disclosure from achieving its intended purpose.

[0093] "Treating" or "treatment of" a disease state or condition includes (i) preventing the disease state or condition, i.e., preventing the development of clinical symptoms of the disease state or condition in a subject that may be exposed to or susceptible to the disease state or condition, but that does not yet experience or display symptoms of the disease state or condition; (ii) inhibiting the disease state or condition, i.e., arresting the development of the disease state or condition, or clinical symptoms thereof; or (iii) relieving the disease state or condition, i.e., causing temporary or permanent regression of the disease state or condition, or clinical symptoms thereof.

[0094] The term "feeder cells" refers to cells that provide extracellular secretions that help another cell reproduce, grow, differentiate, and / or maintain identity. Feeder cells can support the growth of target cells in culture by contributing a complex mixture of extracellular matrix (ECM) components and growth factors. In some cases, feeder cells can be in a state of non-dividing, i.e., cell growth is arrested. Growth of feeder cells can be arrested by any suitable method, for example, known in the art. Growth of feeder cells can be arrested by chemical fixation, for example, by mitomycin-C or glutaraldehyde chemical fixation. Feeder cell growth can be arrested by physical methods, for example, by gamma irradiation, X-ray irradiation, or electrical pulses. For example, feeder cells used in co-culture with target cells, such as embryonic stem cells (ESCs), can be fibroblasts that can be mitotically inactivated to remain viable. In some cases, target cells can grow in the presence of feeder cells that have the ability to divide. A portion of viable feeder cells (e.g., human fibroblasts, etc.) can be target cells, as in the case of induced pluripotent stem cells (iPSCs) during reprogramming. The feeder cells can be, for example, growth-arrested feeder cells that cannot divide. The choice of feeder cells can depend on the target cells. The feeder cells can be, for example, non-growth-arrested fibroblasts. The feeder cells can be growth-arrested fibroblasts, epithelial cells, mesenchymal cells, muscle cells, stromal cells, spleen cells, or amniotic cells. The fibroblasts can be, for example, human skin fibroblasts, 3T3 fibroblasts, human fetal fibroblasts, mouse embryonic fibroblasts, etc. The epithelial cells can be, for example, human adult fallopian tube epithelial cells, human amniotic fluid epithelial cells, HeLa cells (human cervical cancer epithelial cells), etc. The mesenchymal cells can be adipose-derived mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells, human bone marrow-derived mesenchymal cells, human amniotic fluid mesenchymal stem cells, etc. The stromal cells can be, for example, human bone marrow stromal cells or mouse bone marrow stromal cells. The amniotic cells can be, for example, human amniotic cells or mouse amniotic cells.

[0095] The term "target cell" refers to a cell used in the automated cell culture application of the present disclosure, such as an organoid, a tumor analog, a spheroid, a stem cell, or a manufacturing cell line. In some embodiments, the target cell is a spheroid, a tumor analog, an organoid, and / or other multicellular body. In some embodiments, the target cell can be a stem cell. In some embodiments, the target cell can be a manufacturing cell line. The target cell can be derived from a target tissue. The target tissue can be a mammalian primary tissue, or an organoid or a tumor analog. The mammalian tissue can be derived from a patient biopsy sample. The target tissue can be derived from a target organ, such as the lung, intestine (small intestine, colon, etc.), stomach, pancreas, liver, kidney, skin, bone marrow, blood-brain barrier, brain, heart, etc.

[0096] Organoids, spheroids, tumor analogs, and three-dimensional (3D) cell culture models are useful in many applications, such as disease modeling and regenerative medicine. 3D cell models such as organoids and spheroids can be useful to better understand complex biology in a physiologically relevant context, since cells often retain their native shape and unique spatial orientation, for example, within aggregates or spheroids, whereas 2D models consisting of cells grown in sheets or monolayers may be less achievable. Gene and protein expression from 3D cell cultures may more closely mimic gene and protein expression. For example, 3D cell cultures can be useful for drug target identification, lead compound identification, compound optimization, preclinical evaluation, solid tumor modeling, genetic disease modeling, drug discovery, personalized medicine, organs-on-chips, and bioprinting.

[0097] "Spheroid" refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate composed of one or more cell types that can grow and reproduce and exhibit enhanced physiological responses, but do not undergo differentiation or self-organization. Common cell sources for spheroids are primary tissues or immortalized cell lines. Spheroids can bridge the gap between monolayers and complex organs.

[0098] The term "organoid" refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate composed of one or more cell types in which the cells spontaneously self-organize into appropriately differentiated functional cell types and precursors that resemble their in vivo counterparts in at least one aspect. Organoids mimic their in vivo counterparts. Organoids can be derived from pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), neonatal tissue stem cells, embryonic stem cells (ESCs), adult stem cells, or primary tissues. Organoid cultures can be created to resemble many of the organs in their complexity, and are therefore useful for studying disease pathogenesis and treatment. 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 lung, intestine (small intestine, colon, etc.), stomach, pancreas, liver, kidney, skin, bone marrow, blood-brain barrier, brain, heart, etc.

[0099] For example, gastric organoid tissue culture can be derived from sources such as adult mouse, adult human, hPSC, etc. Gastric organoid tissue culture can adopt stem cell culture conditions (niche factors) according to source, such as medium components such as EGF, Noggin, R-spondin, Wnt-3A, FGF10, etc. Differentiation culture conditions can include EGF, R-spondinEGF, R-spondin, etc. according to source.

[0100] In another example, small intestinal organoid tissue culture can be derived from sources such as adult mouse, adult human, hPSC, etc. Small intestinal organoid tissue culture can adopt stem cell culture conditions (niche factors) according to source, such as medium components such as EGF, Noggin, R-spondin, Wnt-3A TGF-beta inhibitor, p38 inhibitor, etc. Differentiation culture conditions can include EGF, Noggin, TGF-beta inhibitor, etc. according to source.

[0101] As another example, colon organoid tissue culture can be derived from source such as adult mouse, adult human, etc. Colon organoid tissue culture can adopt stem cell culture condition (niche factor) according to source, such as medium components such as EGF, Noggin, R-spondin, Wnt-3A TGF-beta inhibitor, p38 inhibitor, etc. Differentiation culture condition can include EGF, Noggin, TGF-beta inhibitor, etc. according to source.

[0102] In another example, pancreatic organoid tissue culture can be derived from source such as adult mouse, adult human, etc. Pancreatic organoid tissue culture can adopt stem cell culture condition (niche factor) according to source, such as medium components such as EGF, Noggin, R-spondin, Wnt-3A, FGF10, nicotinamide, etc. Differentiation culture condition can include EGF, Noggin, R-spondin, Wnt-3A, etc. according to source.

[0103] As another example, liver organoid tissue culture can be derived from source such as adult mouse, adult human, etc. Depending on source, liver organoid tissue culture can adopt stem cell culture condition (niche factor), such as medium components such as EGF, Noggin, R-spondin, Wnt-3A, FGF10, HGF, nicotinamide, etc. Differentiation culture condition can include EGF, Noggin, R-spondin, Wnt-3A, FGF10, TGF-beta inhibitor, notch inhibitor, BMP7, etc.

[0104] The term "tumor analog" refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate composed of one or more cell types typically derived from a primary tumor taken from an oncology patient and capable of mimicking the human tumor microenvironment. Tumor analogs can be useful for testing new cancer drugs or for use in personalized medicine in the field of oncology. Cancer cell lines can be, for example, bladder, breast, colon, hematopoietic and lymphatic, liver, lung, ovarian, prostate, skin, etc.

[0105] The term "stem cell" refers to an undifferentiated cell that has the ability 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 found in the brain, bone, heart, and skin. Some human adult cells can be reprogrammed to an embryonic stem cell-like state called induced pluripotent stem cells (iPSCs). For example, pluripotent stem cells found in adult or infant umbilical cords can develop into cells that make up the organ systems from which they originated. When grown under certain cell culture conditions, pluripotent stem cells can remain undifferentiated. To generate differentiated cells, the chemical composition of the culture medium can be altered, the surface of the culture dish can be modified, or the cells can be modified by expressing certain genes.

[0106] The term "production cell line" refers to a cell line that can be used in the production of enzymes, vaccines, monoclonal antibodies, cytokines, peptides, therapeutic toxins, clotting factors, Fc fusion proteins, or hormones, etc. The production cell line can be a prokaryotic or eukaryotic production cell line. The production cell line can be any production cell line suitable for protein or virus production. The prokaryotic production cell line can include any suitable prokaryotic cell line. The prokaryotic production cell line can include any suitable bacterial cell line (e.g., Escherichia coli). Eukaryotic production cell lines may include Chinese hamster ovary (CHO) cell lines, mouse myeloma cell lines (e.g., NS0, Sp2 / 0), mouse C127, human embryonic kidney (HEK) cell lines such as the HEK293 cell line, human fibrosarcoma cell lines such as the HT-1080 cell line, human embryonic retina cell lines such as the PER.C6 cell line, baby hamster kidney (BHK) cell lines such as the BHK21 cell line, yeast cell lines (e.g., Saccharomyces cerevisiae, Pichia pastoris), insect cell lines infected with the viral vector baculovirus (baculovirus-insect cell expression system) such as the Sf9 cell line, plant cells.

[0107] The term "secreted factor" or "secreted factors" refers to a factor that can be secreted from a target cell in the automated cell culture application of the present disclosure. The secreted factor can be any secreted factor including a hormone, a metabolite, an enzyme, a vaccine, a monoclonal antibody, a cytokine, a peptide, a therapeutic toxin, a clotting factor, or an Fc fusion protein.

[0108] "Transwell" culture, also known as indirect co-culture, may comprise vertically aligned wells having a lower and upper compartment, such as a transwell insert separated by a 0.4 uM microporous membrane to minimize direct contact between producer and target cells. In some embodiments, the tissue culture methods of the present disclosure do not employ transwell culture methods.

[0109] Different medium components may be required for each type of source cell used and type of differentiation to be achieved. Growth factors, such as EGF, Noggin (NOG), R-spondin (RSPO1), HGF, BMP, FGF, etc., may be essential components of organoid medium. Tissue culture medium may contain growth factors. Growth factors may be produced by feeder 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 protein, such as human HGF protein, cynomolgus monkey HGF protein, human FGF10, human Noggin / NOG protein, human RSPO1 protein, human BMP-2 protein, etc. 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, etc. Tissue culture medium or recombinant growth factor proteins for organoid culture may be commercially available, for example, from Sino Biological, Inc., or Thermo Fisher Scientific.

[0110] 3D cell models, such as organoids and spheroids, can be cultured in tissue culture medium containing hydrogels, for example, within hydrogel domes contained in the medium.

[0111] The term "hydrogel" or "hydrogels" refers to the extracellular matrix useful for culturing organoids.Hydrogels can include, for example, mouse EHS sarcoma matrix commercially available as Matrigel (Corning), Cultex (Trevigen), Geltrex (Gibco), collagen type I, fibrin, hyaluronic acid (HA), gelatin methacrylate (GelMA), decellularized matrix, or biopolymers such as alginate, silk, nanocellulose, etc.; engineered materials such as polyethylene glycol (PEG), self-assembling peptides such as RADA16 / PuraMatrix bQ13, poly(lactic / (co)glycolic) acid, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptides, ELP (elastin-like protein), or combinations of these polymers.

[0112] Horizontal co-culture for cell / organoid supply

[0113] A method for horizontal co-culture for cell / organoid feeding is presented. A well unit 100 as shown in FIG. 1A can be employed. The method includes the steps of: allowing one or more target cells to adhere to the bottom of the primary well 112 of the well unit 100 of the separation well microplate, including incubating without mixing, the primary well being separated from the secondary well 115 of the well unit of the separation well microplate by at least one closed microchannel; allowing one or more feeder cells to adhere to the bottom of the secondary well, including incubating without mixing; opening at least one closed microchannel; rocking the separation well microplate to allow mixing and medium exchange between the primary well 112 and the secondary well 115 by gravity flow through at least one open microchannel 118 as shown in FIG. 1B; detaching the target cells from the primary well 112; and allowing the detached target cells to settle into the primary well 112 away from the open microchannel. The method includes mixing the detached target cells and allowing them to further settle into the primary well away from the open microchannel, washing the settled target cells, and collecting the washed target cells via a liquid handler. The washing step may include suspending the settled target cells, including introducing and removing liquid medium, and the washing step can be repeated multiple times as necessary. In some embodiments, the steps of allowing one or more target cells to adhere to the bottom of the primary well 112 and allowing one or more feeder cells to adhere to the bottom of the secondary well 115 are performed simultaneously or substantially simultaneously during an initial incubation period.

[0114] Initially, at least one microchannel may be closed, for example, by an air gap in or adjacent to the microchannel. The closed at least one microchannel 118 may be opened by removing the air gap after at least a portion of the initial incubation period has elapsed. For example, at least one microchannel may be opened by using a pipette as a pump to allow liquid medium to be exchanged between the primary well and the secondary well. To use the pipette as a pump, the exterior surface of the pipette fits around the secondary well to ensure a tight seal. Liquid in the pipette is forced through the microchannel 118 and into it, eliminating the air gap.

[0115] The step of detaching the target cells from the primary well 112 may include exposing the target cells to a dissociation reagent, optionally including EDTA, to detach the target cells without detaching the feeder cells. The dissociation reagent may include a detaching enzyme contained in a physiological buffer. For example, the detaching enzyme may be selected from the group consisting of trypsin, collagenase, elastase, catalase, superoxide dismutase, dispase, and the like.

[0116] In some cases, the target cells can be stem cells or organoids. In some cases, the feeder cells can be fibroblasts. In some cases, the target cells are embedded in a hydrogel that is disposed in a primary well of the well unit.

[0117] Detection of secreted factors from cells allows for kinetic measurements within a single microplate.

[0118] Target cells can produce secretory factors. For example, as shown in Figure 2, intestinal organoids can secrete hormones 230, or manufacturing cell lines, such as CHO cells, can secrete antibodies. A method is presented for detecting multiple secretory factors from cells, which allows for time-dependent kinetic measurements in a single microplate. For example, multiple secretory factors can be detected at different time points (kinetic measurements) 270, 280, 290 after organoids are formed from stem cells or organoid fragments.

[0119] FIG. 2 illustrates a method for detecting secreted factors from target cells, including time-course kinetic measurements in a single separate well microplate. In some embodiments, the secreted factors can be selected from hormones, antibodies, cytokines, chemokines, or growth factors. A patient biopsy sample 210 can be obtained, which can be a source of target cells (e.g., multicellular target cells, such as organoids or tumor analogs); other sources of organoids, such as organoid fragments or stem cells, can also be utilized. Cultivation of target cells, such as organoids, can be performed in a primary well of a well unit of a separate well microplate 220. The organoids can secrete hormones 230 into liquid medium. During an initial incubation period, the primary wells are separated from the secondary wells by at least one closed microchannel, and the secondary well bottoms are coated / spotted with a primary antibody (Ab) specific for the secreted factor 240. After an initial incubation period, the barrier in the microchannel 250 is removed, allowing the secreted factors to diffuse through the open microchannel between the primary and secondary wells. The secreted factors are captured 260 by the primary antibody in the secondary wells. A labeled secondary antibody (2'Ab) specific for the primary antibody or the captured secreted factors is added to the secondary wells at a first time point, and bound 2'Ab is detected in the secondary wells using a microplate reader 270. Diffusion of the secreted factors may include rocking the separate well microplate to allow diffusion of the secreted factors between the primary and secondary wells by gravity flow through the open microchannels. Detecting may include multiplexed detection by imaging equivalent to a plate reader.

[0120] As shown in FIG. 2, after further incubation of the target cells in the microplate 275, a labeled secondary antibody (2′Ab) specific for the primary antibody or the captured secreted factor is added at a second time point and detected using a microplate reader 280. Optionally, after further incubation of the target cells in the microplate 285, a labeled secondary antibody (2′Ab) specific for the primary antibody or the captured secreted factor is added at a third time point and detected using a microplate reader 290. Successive further incubation and detection procedures can be performed at fourth, fifth, sixth, seventh, and further time points. This method allows for time-dependent kinetic measurements of secreted factors in separate well microplates identical to those used for culturing the target cells.

[0121] The labeled antibody can be labeled with any suitable label, such as a fluorescent dye. Different antibodies can be labeled differently with different fluorophores. Primary antibodies (Ab) and labeled secondary (2'Ab) antibodies can be commercially purchased, for example, from Beckman Coulter Inc., Abcam, or Novus Biologicals. Labeled secondary antibodies (2'Ab) can be prepared using antibody labeling methods known in the art, for example, via N-hydroxysuccinimide esters (NHS esters), heterobifunctional reagents, or carbodiimides.

[0122] In some embodiments, the target cell is an organoid that can be derived from target tissue selected from the group consisting of a biopsy sample from a patient, stem cell, or organoid fragment.The organoid can be cultured in a hydrogel dome in the primary well of a well unit.For example, the organoid can be grown in a primary (culture) well from stem cells or organoid fragments contained in a hydrogel dome (e.g., Matrigel, etc.).

[0123] Initially, the primary and secondary wells are separated, for example, by trapped air in the microchannel (or by other means) to avoid any non-specific binding on the detection array. At least one of the closed microchannels can be optionally closed during at least a portion of the first incubation period by a barrier comprising an air gap, a hydrogel seal, or a silicone seal in or adjacent to the microchannel. When the microchannel is closed by a barrier comprising an air gap, the step of opening the closed microchannel can include removing the air gap after at least a portion of the first incubation period has elapsed; optionally includes using a pipette as a pump to exchange liquid medium between the primary well and the secondary well. For example, after the organoids are fully developed, the air gap can be removed by using a pipette as a pump (sealing the feeding well with a pipette tip) to flow liquid between the primary and secondary wells. For example, the air gap can be removed by aspirating liquid through the microchannel between the primary and secondary wells. In another example, gravity flow can be used to perform medium exchange between wells and transfer the organoid output to a secondary well (containing spotted / coated Abs).

[0124] The secondary wells may be spotted / coated with multiple different primary antibodies specific for multiple different secreted factors, and the primary antibodies may be arrayed in the bottom of the secondary wells in a detection array. The secreted factor type detected may be located in a given area of ​​the detection array. The amount of hormone may be measured by the fluorescent signal intensity after washing. In some embodiments, different time points may be recorded with the aid of different fluorophores - e.g., different fluorescent colors, per time point. The concentration of the capture antibody (Ab) may be standardized, e.g., by using different fluorophores.

[0125] Measurements can be performed using a microscope or a plate reader. Plate readers have the advantage of allowing a wide dynamic range, but with a detection array point resolution of approximately 1 mm. 2 Imagers can detect more array points and allow for the observation of smaller array points, but the dynamic range may be more limited.

[0126] Antibody-functionalized surfaces for stem cell isolation

[0127] Some organoid types require passaging as the organoids break down to the single cell level. In this case, it is important to capture the desired (adult) stem cells so that the reconstitution of new organoids is possible.

[0128] Another embodiment of the present disclosure provides a method for passaging organoids using single cell passaging method, and includes a secondary well having a surface functionalized with an antibody to specifically capture stem cells during the washing process at the end of each culture cycle. Figure 3 shows an exemplary method for passaging organoids in a manner that breaks down to single cell level and isolates desired stem cells. Operation 310 includes culturing organoids in a secondary well 115 coated or surface functionalized with immobilized primary antibody (Ab) specific for stem cells (e.g. Lgr5+), a primary well 112 containing organoids 103 (e.g. intestinal organoids) in a hydrogel dome 106 disposed on the bottom surface of the primary well 112, and a well unit of a separated well microplate having an initially closed microchannel 118. A liquid medium 109 is also illustrated in both the primary well section 112 and the secondary well section 115 of the well unit 100. The liquid medium 109 may contain suitable growth factors and nutritional supplements to support the growth of the organoids 103. The operation 320 includes disintegrating the dome of the hydrogel 106 into a plurality of hydrogel fragments 320 or otherwise in a liquid form, releasing the organoids 103 into the liquid medium 109.

[0129] This disclosure presents an automated process for breaking up hydrogel domes 320. In the prior art, breaking up a hydrogel dome is typically performed manually using a pipette to randomly inject / puncture the dome repeatedly (e.g., about 20 times) to promote its disintegration.

[0130] An automated method 320 for breaking a hydrogel dome is presented, which includes culturing a first cell in a hydrogel dome contained in a liquid medium in a well of a microplate for an initial incubation period to provide a multicellular target cell; piercing the hydrogel dome after the initial incubation period with a pipette tip associated with a liquid handler at or near an x,y center location of the dome in the well; and moving the pipette tip through the dome in multiple steps, each step including a pre-determined x,y location in the well for breaking the hydrogel dome, and optionally liquefying the hydrogel dome to release the target cell from the hydrogel dome in the well. The target cell can then be isolated. The automated pipette tip can be positioned at or near the center of the hydrogel dome after culturing the target cell. In some cases, the first cell can be a target cell or a stem cell. The liquid handler can aspirate the pipette in one or more steps, two or more steps, or each step of the multiple steps. For example, the pipette tip may be positioned at the center of the hydrogel dome 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, or within 0-2 mm or 0.01-1.5 mm of the center of the x,y center of the hydrogel dome. Each of the predetermined X,Y locations within the well is determined by the formula: 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 = dome diameter (mm) / 1.8; steps = number of steps the pipette tip moves through the hydrogel dome; X well center = x position of the dome center within the well; Y well center = y position of the dome center within the well; 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 some cases, theta is a constant between 5 and 50, 10 and 45, 15 and 40, 15 and 35, or any number therebetween. In some cases, theta is selected from the 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 therebetween. The x,y center position of the hydrogel dome within the well can be determined based on the initial seeding position within the well, the volume of hydrogel deposited within the well, and / or imaging of the hydrogel dome. A pipette tip can be used to puncture and / or aspirate within the dome. For example, a pipette can be used to apply, remove, or aspirate liquid or air within the dome. Inputs to the system can include the seeding position x,y coordinates within the well and the known volume of the dome. Another input can be the number of steps of pipette puncture or pipette aspirate of the dome. The diameter of the dome can be calculated based on the volume. The diameter of the dome can be determined by imaging. The diameter of the dome can be any suitable diameter. For example, the diameter of the dome can be 1-12 mm, 2-10 mm, 3-8 mm, or 3-6 mm. The number of steps can be any suitable number. For example, the number of steps can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or 2 to 20, 3 to 18, 4 to 17, 5 to 15, 6 to 12, or 8 to 10. As illustrated in Figure 7A, the inputs include the diameter of the hydrogel dome (8 mm in this case), the number of steps (which is 8), and the well center location X,Y coordinates.In some cases, the number of steps / positions may be minimized to minimize process time and effectively break the hydrogel. The output may include an automated sequence / pattern of puncture / aspiration positions within the dome using the pipette by the liquid handler. In some embodiments, the pattern of puncture / aspiration positions may be a spiral 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 pulled below the dome surface between steps / positions. For example, the pipette tip position in the Z-axis may be maintained below the surface of the hydrogel dome between steps / positions to effectively break the dome. Between steps, the pipette tip position in the Z-axis may be maintained at or near the bottom of the hydrogel dome. For example, the pipette tip can be maintained between steps or during aspiration below the surface, within 0.5, 1.0, 1.5, or 2 mm of the surface, near or within 0.5, 1.0, 1.5, or 2 mm of the center of the Z axis, at or within 0.5, 1.0, 1.5, or 2 mm of the bottom of the dome. The Z axis position can be determined by imaging the volume of the hydrogel dome, or the well. Exemplary Figures 7B-7D show an output series of the helical X,Y coordinates of an automated pipettor within a well using as input a well center position, 8 mm dome diameter, and 6-13 steps, a theta of 15.7 (Figures 7B-C), or 30 (Figure 7D), which causes efficient disruption of the hydrogel dome. For example, the solid hydrogel 106 can be converted to a liquid, which separates the organoids 103 from the hydrogel 106. The hydrogel can be converted to a liquid, for example, by vigorous pipetting or shear forces of a liquid handler, with or without lowering the temperature, for example, by about 4°C to about 10°C, or about 10°C, from the incubation temperature (about 37°C).A dissociation reagent can be added to the primary well 112 to dissociate the organoids into stem cells and multiple additional cell types. After dissociated organoids are formed, the microchannel 118 is opened, for example, by removing the air gap. Liquid aspiration through the secondary well is performed after the organoids are disrupted. Stem cells can be captured by the primary antibody (Ab) in the secondary well 115. Operation 330 includes washing the well unit 100 to remove unwanted cells and debris. Operation 340 includes dissociating the isolated stem cells from the antibody / cell complex in the secondary well. Operation 350 includes reseeding the isolated stem cells into a new hydrogel dome 106 in a new primary well 112.

[0131] Alternatively, in operation 310, the secondary well 115 can be coated or surface functionalized with immobilized capture reagent, such as biotin capture reagent or avidin capture reagent. For biotin immobilized capture reagent, after treating the organoid with dissociation reagent in 320, the dissociated organoid can be treated in solution with a reagent that has a strong affinity for the capture reagent, such as an antibody specific to the stem cell of interest (e.g., antibody-streptavidin conjugate) conjugated to streptavidin, to form stem cell-antibody-streptavidin complex. The microchannel 118 is opened by removing the removable barrier, and allows the stem cell-antibody-streptavidin complex to bind with the immobilized biotin in the secondary well 115. After washing unbound cells and debris in operation 330, the stem cell can be dissociated from the complex. The remaining operations 340 and 350 can be performed as presented herein. Alternatively, prior to removing the removable barrier, an antibody conjugate comprising a stem cell-specific primary antibody conjugated to streptavidin can be allowed to bind to the immobilized biotin capture reagent.

[0132] Antibody-functionalized scaffolds for stem cell isolation

[0133] Some organoid types require passaging steps that break down the organoids to the single cell level. Additional methods are presented to capture the desired (adult) stem cells so that new organoids can be reconstituted.

[0134] Another embodiment of the present disclosure provides a method for passaging organoids using single cell passaging, which has a secondary well containing a scaffold coated or surface functionalized with immobilized antibodies to capture specific stem cells during a washing process at the end of each culture cycle.

[0135] 4 shows an exemplary method for disaggregating organoids to single cell level and passaging in a manner to isolate desired stem cells. Operation 410 includes culturing organoids in a well unit of a separated well microplate, which includes a primary well 112 containing organoids 103 (e.g., intestinal organoids) in a hydrogel dome 106 disposed on the bottom surface of the primary well 112, and a secondary well 115 having a porous scaffold coated or surface-functionalized with a primary antibody (Ab) specific for stem cells (e.g., Lgr5+), and has a microchannel 118 closed between the primary and secondary wells. A liquid medium 109 is also illustrated in both the primary well section 112 and the secondary well section 115 of the well unit 100. The liquid medium 109 may include suitable growth factors and nutritional supplements that support the growth of organoids 103.

[0136] Operation 420 includes disintegrating the dome of the hydrogel 106 into a plurality of hydrogel fragments 320 or otherwise into a liquid form, releasing the organoids 103 into the liquid medium 109. For example, the solid hydrogel 106 can be converted to a liquid, which separates the organoids 103 from the hydrogel 106. The hydrogel can be converted to a liquid, for example, by vigorous pipetting or by shear forces of a liquid handler, with or without lowering the temperature, for example, from the incubation temperature (about 37° C.) by about 4° C. to about 10° C., or about 10° C. To dissociate the organoids into stem cells and a plurality of additional cell types, a dissociation reagent is added to the primary well 112. After dissociated organoids are formed, the microchannel 118 is opened, for example, by removing the air gap. Liquid aspiration through the secondary well is performed after the organoids are disrupted. The stem cells can be captured by the primary antibody (Ab) in the secondary well 115.

[0137] Operation 430 includes washing the well unit 100 to remove unwanted cells and debris. Operation 440 includes dissociating the isolated stem cells from the antibody / cell complex in the secondary well. Operation 450 includes reseeding the isolated stem cells in a new hydrogel dome 106 in a new primary well 112.

[0138] Magnetic Beads for Stem Cell Isolation

[0139] Some organoid types require a passaging step that breaks down the organoids to the single cell level. Magnetic beads coated with antibodies specific for stem cells can be added to the primary well after disrupting the organoids to capture the desired (adult) stem cells and allow for the reconstruction of new organoids.

[0140] A magnet is placed under the primary well during the washing procedure to retain the (adult) stem cells in the primary well. Antibody binding can be disrupted by several means, such as by enzymatic means, such as trypsin, or by adding a linker that can be digested by an enzyme.

[0141] 5 shows an exemplary method for disaggregating organoids to single cell level and passaging them in a manner to isolate desired stem cells. Operation 510 includes culturing organoids in a well unit of a separated well microplate, which includes a primary well 112 containing organoids 103 (e.g., intestinal organoids) in a hydrogel dome 106 disposed on the bottom surface of the primary well 112, and has a microchannel 118 closed between the primary and secondary wells. A liquid medium 109 is also illustrated in both the primary well section 112 and the secondary well section 115 of the well unit 100. The liquid medium 109 may include suitable growth factors and nutritional supplements that support the growth of the organoids 103.

[0142] Operation 520 includes disintegrating the dome of the hydrogel 106 into a plurality of hydrogel fragments 320 or otherwise into a liquid form, releasing the organoids 103 into the liquid medium 109. For example, the hydrogel 106 can be converted to a liquid, which separates the organoids 103 from the hydrogel 106. The hydrogel can be converted to a liquid, for example, by shear forces of a liquid handler or vigorous pipetting, with or without lowering the temperature, for example, from the incubation temperature (about 37° C.) by about 4° C. to about 10° C., or about 10° C. Dissociation reagents are added to the primary wells 112 to dissociate the organoids into stem cells and / or multiple additional cell types.

[0143] Operation 530 includes adding magnetic bead-labeled antibodies specific for stem cell markers. Operation 540 includes incubating the antibody-coated magnetic beads with the dissociated organoids to allow binding of the desired stem cells. Operation 550 includes applying a magnet to the bottom of the primary well, opening the microchannel 118, and washing unbound cells and debris through the secondary well, for example by aspirating liquid medium using a liquid handler. If necessary, operation 550 may further include transferring the washed captured cells to a new well unit of a new separation well microplate, including removing the magnet to allow transfer of the captured cells to the new well unit of the new separation well microplate. The magnet shall be reapplied under the new well unit before continuing with operation 560. Operation 560 includes adding a new hydrogel to the primary well so that the stem cells and magnetic beads are embedded within the hydrogel, and removing the magnet from under the primary well. Operation 580 includes placing a magnet on top of the primary well to levitate the stem cells within the hydrogel. Immunotherapy of Tumor Analogs and Organoids for Toxicology Testing.

[0144] CAR T cells and other cell therapies can be beneficial for cancer treatment. Tissue migration, invasion, and tumor-specific cell death can be evaluated. This assay method includes several readings that can be beneficial for the evaluation of immunotherapy.

[0145] Another embodiment of the present disclosure is a method for culturing organoids / tumor analogs and observing how fibroblasts, macrophages, or other cells migrate through microchannels to the main organoid dome. The effect of such cells can be evaluated by observing the phenotypic changes of the organoids / tumor analogs.

[0146] 6A and 6B illustrate a method for evaluating the functionality and toxicology testing of cells (e.g., lymphocytes) in a single separation well microplate. FIG. 6A shows a diagram of a separation well microplate well unit 100 that contains multicellular target cells (e.g., organoids) in a hydrogel dome 106 contained in liquid medium 109 in a primary well 112 of a separation well microplate microwell unit. A secondary well 115 contains chimeric antigen receptor T cells (CAR-T cells), CAR natural killer (CAR-NK cells), or other lymphocytes. The microchannel 118 may have a channel size of 5 microns, while lymphocytes have a size of 7-10 microns. Depending on the microchannel size, the organoids may release chemoattractants that may cause migration of immune cells into the primary chamber. FIG. 6B shows the effect of tilting the separation well microplate, a tilted arrangement that may be employed to prevent immune cells from migrating into the primary well 112.

[0147] Additional assays

[0148] In addition to the assays described above, the split-well microplate architecture allows for at least two major advantages in the development and increasing complexity of cellular assays.

[0149] The first major advantage is the ability to allow cell movement or migration between the primary and secondary wells of a well unit. Although the distance of movement or elongation is short, the primary and secondary wells are effectively separable. Separate well microplates do not need to contain a matrix or forced liquid flow.

[0150] Many applications illustrate this advantage. When testing for angiogenesis, tumor microtissues or growth factors are present in one well of the well unit, while a layer of endothelial cells is present in the other well of the well unit. The endothelial cells have the ability to migrate to the first chamber and form angiogenic sprouts.

[0151] When testing for neurogenesis-nerve regeneration, the effect of growth factors or neurotoxic drugs on neurite outgrowth may be demonstrated. Neurons are placed in one chamber and when growth factors are present in the second chamber, they will outgrow into that chamber. The process is treatment dependent. An advantage is the ability to test for outgrowth in a given direction.

[0152] When testing tumor cell evasion / invasion, migration, wound healing, normal or tumor cells can be tested for their migration into different chambers, and the migrated cells can be more easily and accurately measured within different cells of the cell unit compared to traditional cell migration assays.

[0153] When testing for T cell infiltration or monocyte migration, immune cells can be placed in one chamber while a microtissue chemoattractant is placed in a second chamber. When the immune cells become chemoattractant, they migrate to the other chamber.

[0154] The second major advantage of the separated well microplate configuration is the ability to perform co-cultures and test the mutual response of different cell types and micro-tissues. In this model, cells representing different tissues are separated in different wells of the well unit, while the microplate allows for medium exchange between the primary and secondary wells of the well unit, and thus the effect of added compounds and secreted factors can be tested. For example, hepatocytes may secrete factors that affect cardiac cells. Endothelial cells may respond to inflammation and secrete cytokines that may attract immune cells or cause damage to hepatocytes or neuronal cells (liver cirrhosis, stroke models). Some other applications include multi-tissue toxicity evaluation in organ tissue pairs, such as liver-brain, liver-heart, kidney-liver, gut-liver, etc. Stem cell differentiation and self-renewal in response to compounds, toxins, growth factors, inflammation, metabolic transformation can be tested. In some embodiments, evaluation of secreted factors only or evaluation of cell movement can be applied by using either a horizontal slit configuration that allows cell movement, or a wide vertical slit configuration for nutrient supply and medium-growth factor exchange.

Claims

1. 1. A method for co-culturing target cells, comprising: introducing one or more target cells into a primary well of a well unit of a separation well microplate and initially incubating in liquid medium without mixing to allow the target cells to adhere to the bottom of said primary well, said primary well being separated from a secondary well of said well unit of said separation well microplate by at least one closed microchannel comprising a removable barrier; introducing one or more feeder cells into said secondary wells and initially incubating in liquid medium without mixing to allow said feeder cells to adhere to the bottom of said secondary wells; removing the removable barrier to open the at least one blocked microchannel; rocking the separated well microplate to allow mixing and medium exchange between the primary and secondary wells by gravity flow through open microchannels; Detaching target cells from the primary wells; allowing detached target cells to settle away from the open microchannel and into the primary well; The method includes:

2. mixing the detached target cells and allowing the detached target cells to further settle away from the open microchannel and into the primary well; washing the sedimented target cells; collecting the washed target cells via a liquid handler; The method of claim 1 further comprising:

3. 3. The method of claim 1, wherein the steps of introducing one or more target cells into the primary well and introducing one or more feeder cells into the secondary well are performed simultaneously or substantially simultaneously.

4. 4. The method of claim 3, wherein the at least one closed microchannel is optionally closed during at least a portion of the initial incubation period by a removable barrier within or adjacent to the microchannel, comprising an air gap, a hydrogel seal, or a silicone seal.

5. 5. The method of claim 4, wherein the removable barrier comprises an air gap, and removing the removable barrier comprises removing the air gap after at least a portion of an initial incubation period has elapsed, and optionally comprising using a pipette as a pump to exchange liquid medium between the primary well and the secondary well.

6. 2. The method of claim 1, wherein the detaching step comprises exposing the target cells to a dissociation reagent that detaches the target cells without detaching the feeder cells, optionally wherein the dissociation reagent comprises EDTA.

7. 7. The method of claim 6, wherein the dissociation reagent comprises a lyase enzyme in a physiological buffer, and optionally the lyase enzyme is selected from the group consisting of trypsin, collagenase, elastase, catalase, superoxide dismutase, and dispase.

8. The method of any one of claims 1 to 7, wherein the target cell is a stem cell or an organoid.

9. The method according to any one of claims 1 to 7, wherein the feeder cells are fibroblasts.

10. 10. The method of claim 1, wherein the target cells are embedded in a hydrogel dome disposed in the liquid medium within the primary well of the well unit, and optionally, the method further comprises the step of disrupting the hydrogel dome before detaching the target cells.

11. 11. The method of claim 1, wherein the at least one closed microchannel is located between a bottom surface of the well unit and a bottom portion of a common side wall of the primary well and the secondary well.

12. 12. The method of any one of claims 1 to 11, wherein the height of at least one microchannel ranges from about 10 microns to about 100 microns, or from about 10 microns to about 75 microns, and optionally the width of said microchannel ranges from about 150 to about 500 microns, from about 200 to about 400 microns, or about 300 microns.

13. 1. A method for quantifying secreted factors from cultured target cells comprising time-course kinetic measurements in a single separate well microplate, comprising: culturing target cells in a primary well of a well unit of the separation well microplate in liquid medium for an initial incubation period, the primary well being separated from a secondary well of the well unit of the separation well microplate by at least one closed microchannel comprising a removable barrier, the secondary well containing immobilized capture reagent; removing the removable barrier after the first incubation period to open the at least one closed microchannel and allow the secreted factors to diffuse between the primary well and the secondary well through the open microchannel; capturing the secreted factor with the immobilized capture reagent; adding a labeled second antibody specific for the captured secreted factor or the immobilized capture reagent at a first time point to form a first labeled complex; quantitating the first labeled complex using a microplate reader; The method includes:

14. The method of claim 13, wherein the immobilized capture reagent is selected from a primary antibody specific for the captured secreted factor, avidin, and biotin.

15. 15. The method of claim 14, further comprising the step of adding a first antibody conjugate to the secondary well having an avidin-immobilized capture reagent prior to removing the removable barrier, wherein the first antibody conjugate comprises a secreted factor-specific primary antibody conjugated to biotin.

16. 15. The method of claim 14, further comprising the step of adding a second antibody conjugate to the secondary well having a biotin-immobilized capture reagent prior to removing the removable barrier, the second antibody conjugate comprising a secreted factor-specific primary antibody conjugated to avidin.

17. At a second time point, adding a labeled secondary antibody specific for the captured secreted factor, the immobilized capture reagent, or the primary antibody to form a second labeled complex; quantitating the second labeled complex using a microplate reader; and, if necessary, at a third time point, adding a labeled secondary antibody specific for the captured secreted factor, the immobilized capture reagent, or the primary antibody to form a third labeled complex; detecting the third labeled complex using a microplate reader; 17. The method of any one of claims 13 to 16, further comprising:

18. 16. The method of any one of claims 13 to 15, wherein the quantification step comprises multiplexed detection using imaging equivalent to a plate reader.

19. 19. The method of any one of claims 13 to 18, wherein spreading the secreted factors comprises rocking the separated well microplate to spread the secreted factors between the primary wells and the secondary wells by gravity flow through the open microchannels.

20. 20. The method of any one of claims 13 to 19, wherein the target cell is a multicellular target cell, optionally selected from the group consisting of an organoid and a tumor analog.

21. 21. The method of claim 20, wherein the multicellular target cells are derived from a target tissue, a biopsy sample obtained from a patient, stem cells, a tumor analog fragment, or an organoid fragment.

22. 22. The method of claim 20 or 21, wherein the cultured organoids are embedded in a hydrogel dome contained in liquid medium in the primary well of the well unit.

23. The method of claim 13 , wherein the removable barrier comprises an air gap, a hydrogel seal, or a silicone seal, optionally wherein the removable barrier is disposed within or adjacent to the microchannel.

24. 24. The method of claim 23, wherein removing the removable barrier comprises removing the air gap and, optionally, using a pipette as a pump to exchange liquid medium between the primary well and the secondary well.

25. 25. The method of any one of claims 13 to 24, wherein the immobilized capture reagent is immobilized on the bottom of the secondary well.

26. 26. The method of claim 25, wherein the immobilized capture reagent comprises multiple primary antibodies specific for multiple secreted factors, the primary antibodies being arrayed at the bottom of the secondary wells in a detection array.

27. 27. The method of any one of claims 13 to 26, wherein the secreted factor is selected from the group consisting of hormones, antibodies, cytokines, chemokines, and growth factors.

28. 1. A method for isolating a single cell type from a multicellular target cell culture cultured in a single separate well microplate, comprising: culturing multicellular target cells in a primary well of a well unit of the separation well microplate in liquid medium for an initial incubation period, the primary well being separated from a secondary well of the well unit of the separation well microplate by at least one closed microchannel comprising a removable barrier, the secondary well comprising a surface functionalized with immobilized capture reagent; dissociating the multicellular target cells in the primary well to form a mixture of individual cells and unrelated cells comprising a single cell type; removing the removable barrier to open the microchannel and move the mixture of individual cells through the open microchannel to the secondary well, allowing the single cell type to associate with the immobilized capture reagent in the secondary well to form an immobilized complex; washing the primary wells and the secondary wells to remove unbound, non-relevant cells; isolating said single cell type, comprising dissociating said immobilized complexes; The method includes:

29. 30. The method of claim 28, further comprising the step of reseeding an isolated single cell type into a primary well of said separated-well microplate.

30. 30. The method of claim 28 or 29, wherein the single cell type is a stem cell.

31. 31. The method of any one of claims 28 to 30, wherein the multicellular target cell is an organoid.

32. 32. The method of any one of claims 28-31, wherein the immobilized capture reagent is disposed on the bottom of the secondary well or on a scaffold within the secondary well.

33. 33. The method of any one of claims 28 to 32, wherein the immobilized capture reagent is selected from a primary antibody specific for a surface marker of the single cell type, avidin, and biotin.

34. 34. The method of claim 33, further comprising the step of adding a first antibody conjugate to the secondary well having an avidin-immobilized capture reagent prior to removing the removable barrier, wherein the first antibody conjugate comprises a single cell type surface marker-specific primary antibody conjugated to biotin.

35. 34. The method of claim 33, further comprising the step of adding a second antibody conjugate to the secondary well with a biotin-immobilized capture reagent prior to removing the removable barrier, wherein the second antibody conjugate comprises a single cell type surface marker-specific primary antibody conjugated to avidin.

36. 36. The method of any one of claims 28 to 35, wherein the multicellular target cells are cultured within a hydrogel dome contained in liquid medium in the primary well, and optionally, the method further comprises the step of disrupting the hydrogel dome prior to dissociating the multicellular target cells.

37. 1. A method for isolating stem cells from a multicellular target cell culture in a single separate well microplate, comprising: culturing multicellular target cells in a first hydrogel dome contained in liquid medium in a primary well of a well unit of the separated well microplate for a first incubation period, the primary well being separated from a secondary well of the well unit of the separated well microplate by at least one closed microchannel comprising a removable barrier; destroying the first hydrogel dome; dissociating the multicellular target cells in the primary well to form a mixture of individual cells and unrelated cells, including the stem cells; adding magnetic beads containing immobilized capture reagent specific for the stem cells to the primary wells and further incubating to allow the magnetic beads to bind to the stem cells; applying a magnet beneath the primary well to retain the magnetic bead-stem cell complex; removing the removable barrier to open the microchannel and washing the retained magnetic bead-stem cell complexes by aspirating liquid medium through the secondary well to remove unbound, unrelated cells; adding a second hydrogel dome to the washed, retained magnetic bead-stem cell complexes in the primary well; removing the magnet from beneath the primary well; dissociating the stem cells from the magnetic beads; applying a magnet over the primary well to levitate the magnetic beads and isolate the stem cells within the second hydrogel dome; The method includes:

38. removing the removable barrier to open the microchannel and washing the retained magnetic bead-stem cell complexes by aspirating liquid medium through the secondary well to remove the unbound unrelated cells; removing the magnet from beneath the primary well; transferring the washed, retained magnetic bead-stem cell complexes to new primary wells in new well units of a new separate well microplate; reapplying a magnet beneath the new primary wells in the new well units of the new separated well microplate; 38. The method of claim 37, further comprising:

39. 39. The method of claim 37 or 38, wherein the immobilized capture reagent is selected from a primary antibody specific for a surface marker of the stem cell, avidin, and biotin.

40. 1. A method for selecting a therapy for the treatment of cancer in a subject in need thereof, comprising: culturing multicellular target cells from the subject in a primary well of a well unit of a separated well microplate in liquid medium for an initial incubation period, the primary well being separated from a secondary well of the well unit of the separated well microplate by at least one closed microchannel comprising a removable barrier, the secondary well containing a candidate cancer therapeutic immune cell, a monoclonal antibody, an immune system modulator, or an anti-tumor drug; removing the removable barrier to open the at least one closed microchannel and allowing diffusion between the primary well and the secondary well through the open microchannel for a second incubation period; imaging the secondary wells, and optionally the primary wells, after the second incubation period to quantify cell migration or multicellular target cell proliferation; an increase in immune cell migration within the secondary wells or between the secondary wells and the primary wells as compared to a control well unit that does not have multicellular target cells; or A reduction in multicellular target cell proliferation in said primary wells as compared to control well units not containing the therapeutic candidate. selecting a candidate therapeutic agent based on the The method includes:

41. 41. The method of claim 40, wherein in the liquid medium, the multicellular target cells are embedded within a hydrogel dome disposed within the primary well of the well unit, and optionally, the method further comprises the step of disrupting the hydrogel dome prior to removing the removable barrier.

42. 42. The method of claim 40 or 41, wherein the multicellular target cells are derived from a biopsy sample or tumor analog fragment obtained from the subject.

43. 43. The method of any one of claims 40 to 42, wherein the immune cell is a CAR T cell.

44. 1. An automated method for disrupting a hydrogel dome, comprising: culturing a first cell within a hydrogel dome contained in liquid medium in a well of a microplate for an initial incubation period to provide a multicellular target cell; After the initial incubation period, puncturing the hydrogel dome with a pipette tip associated with a liquid handler at or near the x,y center of the dome within the well; moving a pipette tip through the dome to release the target cells from the hydrogel dome in multiple steps, each step including a predetermined X,Y location within the well to break the hydrogel dome and, if necessary, liquefy the hydrogel dome; Optionally, isolating one or more of said target cells from said well. The method includes:

45. 45. The method of claim 44, wherein the liquid handler aspirates the pipette tip within the hydrogel dome at one or more, two or more, multiple, or individual pre-determined positions.

46. Each of the predetermined X,Y locations within the well is X(steps) = X well center + alpha / step * step * cos(theta / step * step), and Y (number of steps) = Y Well Center + Alpha / Step * Number of Steps * SIN (Theta / Step * Number of Steps) The calculation is based on the formula: Alpha = Dome diameter (mm) / 1.8, Steps = the number of steps the pipette tip moves through the hydrogel dome, an integer between 2 and 20 as appropriate; X well center = x position of the dome center within the well, Y well center = the y position of the dome center within the well, and 46. ​​The method of claim 44 or 45, wherein theta=a constant value between 5 and 50.

47. 47. The method of any one of claims 44 to 46, wherein the well is a primary well of a well unit of a separated well microplate.

48. 48. The method of any one of claims 44 to 47, wherein the x,y central position of the hydrogel dome is determined by one or more parameters selected from the group consisting of an initial seeding position within the well and imaging of the hydrogel dome within the well.

49. 49. The method of any one of claims 45 to 48, wherein the diameter of the hydrogel dome is determined by the volume of hydrogel deposited in the well or by imaging the hydrogel dome within the well.

50. 50. The method of any one of claims 44 to 49, wherein the number of steps is selected from 2 to 20, 3 to 18, 4 to 17, 5 to 15, 6 to 12, or 8 to 10 steps.

51. 50. The method of any one of claims 44 to 49, wherein the pre-determined locations form a pattern within and / or through the hydrogel dome selected from the group consisting of a spiral pattern, a star pattern, and a zigzag pattern.

52. 52. The method of any one of claims 44 to 51, wherein the pipette tip is lowered below the surface of the hydrogel dome between each position.

53. 52. The method of any one of claims 44 to 51, wherein the pipette tip is elevated above the surface of the hydrogel dome between each position.

54. 54. The method of any one of claims 44 to 53, wherein the multicellular target cell is selected from the group of a spheroid, a tumor analog, and an organoid.

55. 55. The method of any one of claims 44 to 54, further comprising dissociating the multicellular target cells to provide individual target cells prior to said isolating step.

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