Cell culture application method using separation pore microwell plate

Abstract

Automated methods for horizontal co-culture of cells using a single separation well microwell plate are provided. The present disclosure also provides improved methods of quantifying secreted factors from cultured cells, as well as methods of isolating target cells from organoids in a single isolation well microplate.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application was filed as a PCT international application on March 17, 2023, and claims the benefit and priority of U.S. Provisional Application No. 63 / 321,373 filed on March 18, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Culturing cells in a three-dimensional (3D) environment results in cell behaviors and morphologies that are closer to those observed in the human body. The 3D hydrogels / hydroscaffolds used for such cultures have a unique property: cells can be deposited at specific locations in 3D space and remain in place for long periods of time. This enables the creation of structures (e.g., spheroids, tumoroids, organoids, and / or other multicellular bodies) and co-culture environments in which cell interactions and development over time are observed.

[0004] Until now, vertical co-cultures usually require transwells. However, transwell handling can be relatively complex, difficult to automate, and difficult to image the cells. The supernatant can be transferred to different assay plates for analysis using ELISA or different methods. However, kinetic measurements and direct comparisons of different hormones secreted by organoids have been impractical so far. Until now, adult stem cells could be captured if an additional capture plate was used for washing.

[0005] For in vitro toxicity studies, the presence of several cell types is often required: for example, cardiac cells and hepatocytes that metabolize chemicals, neurons and astrocytes, cardiac cells and endothelial cells, enterocytes and hepatocytes, etc. Although co-culture of different cells or transfer of supernatants are current solutions, it is difficult to obtain error-free data due to the need to distinguish between different cells or to evaluate the effects of a small number of secreted factors.

[0006] The ideal approach would be to use a single separation well microplate that would allow not only physical separation but also fluid communication between the different cell types and ease of detection and evaluation of effects over time. The ideal approach would be to use a single separation well microplate that would allow evaluation of in vivo type situations in a single assay, simultaneously evaluating immune cell migration, infiltration, and target tumor cell killing. Summary of the invention

[0007] The present disclosure provides methods for co-culturing different cell types in separate compartments that allow for efficient cross-talk and interaction 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 engraftment, inflammation, and secretion of multiple growth factors and metabolites.

[0008] The present disclosure provides methods for using split-well microplates that support horizontal co-culture suitable for automated means. Split-well microplates support culturing different cell types in adjacent wells that can be hydraulically connected using, for example, a pipette as a pump, or connected by laminar flow with appropriate microchannel geometry.

[0009] The use of split-well microplates allows cell culture, cell washing and (adult) stem cell capture to be performed in a single split-well microplate without the need for additional plates or special washing equipment. The use of split-well microplates also allows multiple readouts to be performed simultaneously in a kinetic manner.

[0010] The present disclosure provides a method for co-culturing target cells, the method comprising introducing one or more target cells into the main well of a well unit of a separation well microplate, and performing an initial incubation in a liquid culture medium without mixing to allow the target cells to adhere to the bottom of the main well, the main well being separated from the secondary well of the well unit of the separation well microplate by at least one closed microchannel, the closed microchannel comprising a removable barrier; introducing one or more feeder cells into the secondary well, and performing an initial incubation without mixing in a liquid culture medium 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; shaking the separation well microplate to allow mixing and medium exchange between the main well and the secondary well by gravity flow through the opened microchannel; detaching the target cells from the main well; and allowing the detached target cells to settle in the main well away from the opened microchannel. The method for co-culturing target cells can be a horizontal method for co-culturing target cells. The method of co-culturing target cells may further include mixing the detached target cells and allowing the detached target cells to further settle in the main well away from the opened microchannel; washing the settled target cells; and collecting the washed target cells by a liquid handler.

[0011] The method of co-culturing target cells may include wherein introducing one or more target cells into the main well and introducing one or more feeder cells into the secondary well are performed simultaneously or substantially simultaneously. Simultaneously can be within about 5, about 10, or about 15 minutes. Substantially simultaneously can be within about 30, about 60, or about 90 minutes.

[0012] The method of co-culturing target cells may include wherein at least one enclosed microchannel is enclosed by a removable barrier within or adjacent to the microchannel, the removable barrier comprising an air gap, a hydrogel seal, or a silicone seal. The method of co-culturing target cells may include wherein at least one enclosed microchannel is enclosed during at least a portion of an initial incubation period.

[0013] The method of co-culturing target cells can include wherein the removable barrier includes an air gap, and removing the removable barrier includes eliminating the air gap after at least a portion of an initial incubation period, optionally including using a pipette as a pump to force liquid medium exchange between the primary well and the secondary well.

[0014] The method of co-culturing target cells may include wherein the detachment includes exposing the target cells to a dissociation reagent to detach the target cells without detaching the feeder cells. The dissociation reagent may include a detachment enzyme, optionally a physiological buffer solution of the detachment enzyme. In some embodiments, the detachment 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.

[0015] In some embodiments, the target cell may be a stem cell or an organoid. In some embodiments, the feeder cell is a fibroblast.

[0016] The method of co-culturing target cells may include wherein the target cells are embedded in a hydrogel dome disposed in a liquid culture medium in a main well of a well unit. The method of co-culturing target cells may include wherein at least one enclosed microchannel is located between a bottom surface of the well unit and a bottom of a shared sidewall of the main well and the secondary well.

[0017] The method of co-culturing target cells may include using a separation well microplate comprising a plurality of well units, each well unit comprising at least one microchannel capable of fluidically connecting a main well and a secondary well in the well unit, wherein the height of at least one microchannel is in the range of about 10 microns to about 100 microns or about 10 microns to about 75 microns; optionally, wherein the width of the microchannel is in the range of about 150 to about 500 microns, about 200 to about 400 microns, or about 300 microns. The separation well microplate may include a plurality of well units, such as 384 well units, 96 well units, 48 ​​well units, 24 well units, 12 well units, 8 well units, or 6 well units.

[0018] The present disclosure provides a method for quantifying secreted factors from cultured target cells, comprising performing kinetic measurements over time in a single separation well microplate, the method comprising: culturing target cells in a liquid culture medium in a main well of a well unit of the separation well microplate during an initial incubation period, the main well being separated from a secondary well of a well unit of the separation well microplate by at least one closed microchannel, the microchannel comprising a removable barrier, and the secondary well comprising an immobilized capture reagent; after the initial incubation period, removing the removable barrier to open at least one closed microchannel, thereby allowing the secreted factors to diffuse between the main well and the secondary well through the opened microchannel; allowing the secreted factors to be captured by the immobilized capture reagent; adding a labeled secondary antibody specific to the captured secreted factors 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.

[0019] The method for quantifying secreted factors from cultured target cells may include wherein the immobilized capture reagent is selected from the group consisting of a primary antibody specific for the captured secreted factor, avidin, and biotin.

[0020] The method for quantifying secreted factors from cultured target cells may also include adding a first antibody conjugate to the secondary well with the avidin-immobilized capture reagent before removing the removable barrier, wherein the first antibody conjugate includes a secreted factor-specific primary antibody conjugated to biotin.

[0021] The method for quantifying secreted factors from cultured target cells may further include adding a secondary antibody conjugate to the secondary well with the biotin-immobilized capture reagent prior to removing the removable barrier, wherein the secondary antibody conjugate includes a secreted factor-specific primary antibody conjugated to avidin.

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

[0023] The method for quantifying secreted factors from cultured target cells may also include adding a labeled secondary antibody specific to the captured secreted factor, immobilized capture reagent or primary antibody at a third time point to form a third labeled complex; and detecting the third labeled complex with a microplate reader.

[0024] The method for quantifying secreted factors from cultured target cells may include wherein quantification includes multiplexed detection using imaging and uniform detection using a microplate reader. The method for quantifying secreted factors from cultured target cells may include wherein diffusion of the secreted factors includes shaking the separation well microplate to allow the secreted factors to diffuse between the main well and the secondary well by gravity flow through the opened microchannel.

[0025] The method for quantifying secreted factors from cultured target cells may include wherein the target cells are multicellular target cells. In some embodiments, the multicellular target cells are selected from the group consisting of organoids and tumoroids. In some embodiments, the multicellular target cells are derived from target tissues, biopsy samples of patients, stem cells, tumor-like fragments, or organoid fragments.

[0026] A method for quantifying secreted factors from cultured target cells may include wherein the cultured organoids are embedded within a hydrogel dome in liquid culture medium within a main well of a well cell.

[0027] The method for quantifying secreted factors from cultured target cells may include wherein the removable barrier comprises an air gap, a hydrogel seal, or a silicone seal; optionally, wherein the removable barrier is located within or adjacent to the microchannel.

[0028] The method for quantifying secreted factors from cultured target cells may include wherein removing the removable barrier includes eliminating the air gap; optionally including using a pipette as a pump to allow liquid medium exchange to occur between the primary well and the secondary well.

[0029] The method for quantifying secreted factors from cultured target cells may include wherein an immobilized capture reagent is fixed to the bottom of the secondary well. In some embodiments, the immobilized capture reagent includes a plurality of primary antibodies specific for the plurality of secreted factors, wherein the primary antibodies are arranged in the form of a detection array at the bottom of the secondary well. The method for quantifying secreted factors from cultured target cells may include wherein the secreted factors are selected from the group consisting of hormones, antibodies, cytokines, chemokines, and growth factors.

[0030] The present disclosure provides a method for separating a single cell type from a culture of a multicellular target cell culture in a single separation well microplate, the method comprising: culturing a multicellular target cell in a liquid culture medium in a main well of a well unit of a separation well microplate during an initial incubation period, the main well being separated from a secondary well of a well unit of the separation well microplate by at least one enclosed microchannel, the enclosed microchannel comprising a removable barrier, the secondary well comprising a surface functionalized with an immobilized capture reagent; dissociating the multicellular target cell in the main well to form a mixture of single cells containing a single cell type and a non-target cell; removing the removable barrier to open the microchannel, thereby allowing the mixture of single cells to travel through the open microchannel to the secondary well, and allowing the single cell type to bind to the immobilized capture reagent in the secondary well to form a fixed complex; washing the main well and the secondary well to remove unbound non-target cells; and separating the single cell type, which comprises dissociating the fixed complex. Allowing a single cell to travel through the open microchannel may include using a pipette as a pump, diffusion, or laminar flow with an appropriate microchannel geometry.

[0031] The method of isolating a single cell type from a cultured multicellular target cell culture may also include re-seeding the isolated single cell type in a main 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.

[0032] In some embodiments, the multicellular target cell is an organoid.

[0033] The method of isolating a single cell type from a cultured multicellular target cell culture may include wherein an immobilized capture reagent is disposed on the bottom of a secondary well or on a support within the secondary well.

[0034] In some embodiments, the immobilized capture reagent can be selected from a primary antibody specific for a surface marker of a single cell type, avidin, and biotin.

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

[0036] The method of isolating a single cell type from a culture of a multicellular target cell may further comprise: before removing the removable barrier, adding a secondary antibody conjugate to the secondary well having the biotin-immobilized capture reagent, wherein the secondary antibody conjugate comprises a primary antibody specific for a surface marker of the single cell type conjugated to avidin.

[0037] The method of isolating a single cell type from a culture of a multicellular target cell may include wherein the multicellular target cell is cultured in a hydrogel dome in a liquid culture medium within a main well.

[0038] The present disclosure provides a method for isolating stem cells from a multicellular target cell culture in a single separation well microplate, the method comprising: culturing the multicellular target cells in a first hydrogel dome in a liquid culture medium in a main well of a well unit of the separation well microplate during an initial incubation period, the main well being separated from a secondary well of a well unit of the separation well microplate by at least one closed microchannel, the closed microchannel comprising a removable barrier; dissociating the multicellular target cells in the main well to form a mixture of single cells comprising stem cells and non-target cells; placing an immobilized cell containing a specificity for the stem cells in the first hydrogel dome; Magnetic beads of capture reagent are added to the main well and further incubated to allow the magnetic beads to bind to the stem cells; a magnet is applied below the main well to retain the magnetic bead-stem cell complex; the removable barrier is removed to open the microchannel, and the retained magnetic bead-stem cell complex is washed by aspirating liquid culture medium through the secondary well to remove unbound non-target cells; a second hydrogel dome is added to the magnetic bead-stem cell complex retained after washing in the main well; the magnet is removed from below the main well; the stem cells are dissociated from the magnetic beads; and a magnet is applied above the main well to float the magnetic beads and separate the stem cells in the second hydrogel dome.

[0039] In some embodiments, the method of isolating stem cells from a multicellular target cell culture further comprises: removing the magnet from under the main well; transferring the magnetic bead-stem cell complex retained after washing to a new main well in a new well unit of a new separation well microplate; and reapplying the magnet under the new main 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 the stem cell, avidin, and biotin.

[0040] The present disclosure provides a method for selecting a therapy for cancer treatment for a subject in need thereof, the method comprising: in an initial incubation period, culturing multicellular target cells derived from a subject in a liquid culture medium in a main well of a well unit of a separation well microplate, the main well being separated from a secondary well of a well unit of the separation well microplate by at least one closed microchannel, the closed microchannel comprising a removable barrier, and the secondary well comprising a candidate cancer therapeutic immune cell, a monoclonal antibody, an immune system modulator or an anti-tumor agent; in a second incubation period, removing the removable barrier to open at least one closed microchannel to allow diffusion between the main well and the secondary well through the opened microchannel; after the second incubation period, imaging the secondary well and optionally the main well to quantify cell migration or multicellular target cell growth; and selecting a candidate therapy based on the following factors: increased immune cell migration within the secondary well or between the secondary well and the main well compared to a control well unit without multicellular target cells; or decreased multicellular target cell growth in the main well compared to a control well unit without the candidate therapy.

[0041] The method for selecting a therapy for cancer treatment may include wherein the multicellular target cells are embedded in a hydrogel dome disposed in a liquid culture medium in a main well of a well unit. The method for selecting a therapy for cancer treatment may include wherein the multicellular target cells are derived from a biopsy sample or tumor-like fragment from a subject. In some embodiments, the immune cell may be a CAR-T cell.

[0042] The present disclosure provides an automated method for disrupting a hydrogel dome, the method comprising: culturing a first cell in a hydrogel dome in a liquid culture medium in a well of a microplate during an initial incubation period to provide a multicellular target cell; after the initial incubation period, piercing the hydrogel dome at or near the x, y center of the dome in the well with a pipette tip associated with a liquid handler; moving the pipette tip through the dome in multiple steps, each step including an x, y predetermined position in the well to disrupt the hydrogel dome, and optionally liquefying the hydrogel dome to release the target cell from the hydrogel dome in the well. The pipette can aspirate at one or more, two or more, multiple or each predetermined position.

[0043] The present disclosure provides an automated method for disrupting a hydrogel dome and isolating a target cell, the method comprising: culturing a first cell in a hydrogel dome in a liquid culture medium in a well of a microplate during an initial incubation period to provide a multicellular target cell; after the initial incubation period, piercing the hydrogel dome with a pipette tip associated with a liquid handler at or near the x, y center of the dome in the well; moving the pipette tip through the dome in multiple steps to release the target cell from the hydrogel dome, each step including an x, y predetermined position in the well to disrupt the hydrogel dome, and optionally liquefying the hydrogel dome; and isolating one or more target cells from the well. The pipette can aspirate at one or more, two or more, or each predetermined position.

[0044] Each of the X,Y predetermined positions within the hole can be calculated, including: X(number of steps) = X hole center + α / step * number of steps * cos(θ / step * number of steps); and

[0045] Y(number of steps) = Y well center + α / step * number of steps * SIN(θ / step * number of steps), where α = dome diameter (mm) / 1.8; steps = number of steps to move the pipette tip 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 θ = a constant value between 5 and 50. In some cases, the number of steps is an integer between 2-20, 3-18, 4-17, 5-15, 6-12, or 8-10. In some cases, θ is a constant between 5-50, 10-45, 15-40, 15-35, or any number in between. In some cases, θ 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.

[0046] In some cases, the liquid handler draws the pipette tip within the hydrogel dome and / or the pipette tip pierces the hydrogel dome at one or more, two or more, a plurality of, or each predetermined location.

[0047] In some cases, the well is a main well of a well unit of a separation well microplate.

[0048] The x,y center position of the hydrogel dome may be determined by one or more parameters selected from the group consisting of the original seeding position in the well, the volume of hydrogel deposited in the well, and imaging of the hydrogel dome in the well.

[0049] The diameter of the hydrogel dome can be determined by the volume of hydrogel deposited in the well and / or imaging of the hydrogel dome within the well.

[0050] The X,Y predetermined locations may form a pattern within and / or through the hydrogel dome in the hole selected from the group consisting of a spiral pattern, a star pattern, and a sawtooth pattern.

[0051] Between each position, the pipette tip can be dragged below the surface of the hydrogel dome. Between each position, the pipette tip can be lifted above the surface of the hydrogel dome.

[0052] The target cell may be a multicellular target cell, optionally selected from the group consisting of a spheroid, a tumoroid, and an organoid.

[0053] In some cases, the method further comprises dissociating the multicellular target cells from each other to provide single target cells prior to separating. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1A A first view of an exemplary single well unit 100 in a split well microplate that can be used for horizontal co-culture of cells / organoids is shown. The main well 112 is separated from the secondary well 115 by at least one microchannel 118. In some embodiments, at least one microchannel 118 can be open to allow medium exchange between the main well 112 and the secondary well 115. Alternatively, at least one microchannel 118 can include a removable barrier to isolate the main well and the secondary well from each other.

[0055] Figure 1B A second view of an exemplary single well unit 100 in a split well microplate for horizontal co-culture that can be used for cell / organoid feeding is shown. The primary well 112 is separated from the secondary well 115 by at least one microchannel 118. When the split well microplate is on a rocker, medium exchange is performed by gravity flow through the microchannel 118.

[0056] Figure 1C An example of a cross section of a single well unit 100 of a separation well microplate associated with a cell culture method according to various embodiments of the present disclosure is shown. The main well 112 is separated from the secondary well 115 by a shared sidewall, which includes at least one microchannel 118 located between the main well 112 and the secondary well 115. The main well 112 may contain target cells (e.g., organoids) embedded in a hydrogel dome 106. The main well 112 and the secondary well 115 may be at least partially filled with a liquid culture medium 109. The bottom of the well unit 100 includes a bottom sheet 121, which includes an optically transparent observation window to allow imaging.

[0057] Figure 2A representative schematic diagram of an exemplary method for detecting multiple secreted factors from target cells (e.g., organoids) is shown, which allows cell culture and kinetic measurement of secreted factors within a single separation well microplate. Target cells (e.g., organoids) are cultured in primary wells 112. Target cells produce secreted factors (e.g., hormones) that are allowed to diffuse through microchannels 118. The secreted factors are captured in secondary wells 115, which include an array of coated / surface-functionalized immobilized antibodies specific for the secreted factors.

[0058] Figure 3 An exemplary method is shown for single cell isolation (e.g., stem cells) from a multicellular target cell culture (e.g., organoids) in a single separation well microplate comprising a surface functionalized with an immobilized capture reagent (e.g., a stem cell-specific antibody, avidin, or biotin). The multicellular target cells (e.g., organoids) are cultured in a hydrogel dome 106 in a liquid culture medium 109 in a main well 112, the secondary well 115 is coated / surface functionalized with an immobilized antibody specific for the target cell (e.g., stem cell), and the microchannel 118 is closed by a removable barrier (310). After a period of time, the multicellular target cells (e.g., organoids) are dissociated to form a mixture of single cells including the target cells (320). The barrier is removed from the microchannel 118, and the mixture of single cells is able to travel through the open microchannel 118, for example, by hydraulic pressure using a pipette as a pump, or by laminar flow with appropriate microchannel geometry. The target cells are captured by the immobilized specific antibodies in the secondary wells 115, and non-target cells are washed away from the microwells (330). The target cells (e.g., stem cells) are dissociated from the antibody / cell complex (340). The target cells (e.g., stem cells) can be used to re-inoculate new wells (350).

[0059] Figure 4An exemplary method is shown for stem cell isolation from organoid cultures in a single separation well microplate including an antibody-coated scaffold. Multicellular target cells (e.g., organoids) are cultured in a hydrogel dome 106 in a liquid culture medium 109 in a main well 112, a secondary well 115 includes a scaffold containing a coated / surface-functionalized immobilized antibody specific to the target cell (e.g., stem cell), and a microchannel 118 is closed by a removable barrier (410). After a period of time, the multicellular target cells (e.g., organoids) are dissociated to form a mixture of single cells including target cells (420). The barrier is removed from the microchannel 118, and the mixture of single cells can pass through the open microchannel 118, for example, by using a pipette as a pump, or by laminar flow with an appropriate microchannel geometry. The target cells are captured by the scaffold containing immobilized specific antibodies in the secondary well 115, and non-target cells are washed out of the microwell (430). The target cells (e.g., stem cells) are dissociated from the antibody / cell complex in the scaffold (440). Cells of interest (eg, stem cells) may be used to re-seed new wells (450).

[0060] Figure 5 An exemplary passaging method is shown, which breaks multicellular target cells (e.g., organoids) to the single cell level in a single separation cell microplate, and separates the desired single cells (e.g., stem cells) using magnetic beads coated with a capture reagent (e.g., cell-specific antibodies or biotin). Multicellular target cells (e.g., organoids) are cultured in a well unit of a separation well microplate, which includes a main well 112, which contains an organoid 103 (e.g., intestinal organoid) in a hydrogel dome 106 in a liquid culture medium 109 disposed on the bottom surface of the main well 112, and a closed microchannel 118 located between the main well and the secondary well includes a removable barrier. After a period of time, the multicellular target cells (e.g., organoids) are dissociated to form a mixture of single cells including the target cells (520). Magnetic beads surface-functionalized with an immobilized capture reagent (e.g., an antibody or biotin specific for a stem cell marker) are added to the mixture of single cells in the main well unit (530). The magnetic beads are allowed to incubate with the mixture of single cells to allow binding of the desired single cells (e.g., stem cells) (540). A magnet is applied to the bottom of the main well to retain the magnetic bead-capture reagent-stem cell complex, the microchannel 118 is opened by removing the barrier, and the unbound cells and debris are washed by (e.g.) aspirating liquid culture medium through the secondary well 115 with a liquid handler. A new hydrogel dome can be formed in the main well so that the stem cells and magnetic beads are embedded in the hydrogel (560), and the magnet is removed from under the main well (570). A magnet can be placed on top of the main well to float the stem cells in the hydrogel (580).

[0061] Fig. 6A An exemplary method is shown for evaluating cell (e.g., lymphocyte) function and toxicology studies in a single separation well microplate. Multicellular target cells (e.g., tumor-like, organoid) are cultured in a hydrogel dome 106 in a liquid culture medium 109 in the main well 112 of the well unit 100 of the separation well microplate. The secondary well 115 contains immune cells (e.g., CAR-T cells, CAR-NK cells, other lymphocytes). Chemical attractants can be discharged by multicellular target cells and diffuse through the open microchannel 118, which can cause certain immune cells in the secondary well 115 to migrate, even to the main well 112, depending on the configuration of the microchannel 118. The microchannel 118 can have a channel dimension of 5 microns, while the lymphocytes have a dimension of 7-10 microns. In some embodiments, the multicellular target cells can discharge chemical attractants, which can cause immune cells to migrate to the main well 112 according to the configuration of the microchannel 118.

[0062] Figure 6B An exemplary method is shown for evaluating cell (e.g., lymphocyte) function and toxicology studies in a single separation well microplate, in which an inclined position can be used to impair immune cell migration. Multicellular target cells (e.g., tumoroids, organoids) are cultured in a hydrogel dome 106 in a liquid culture medium 109 in the main well 112 of the well unit 100 of the separation well microplate. The secondary well 115 contains immune cells (e.g., CAR-T cells, CAR-NK cells, other lymphocytes). Chemical attractants can be expelled by the multicellular target cells and diffuse through the open microchannel 118, which can cause certain immune cells in the secondary well 115 to migrate, and even migrate into the main well 112, depending on the configuration of the microchannel 118. The separation well microplate can be maintained in an inclined position to impair the migration of immune cells to the main well 112.

[0063] Fig. 7A An exemplary spreadsheet calculation of the hydrogel dome rupture pattern for an X, Y predetermined position of an automated pipette motion calculated using the following formula: X(number of steps) = X-hole center + α / step*number of steps*COS(θ / step*number of steps); and

[0064] Y(number of steps) = Y hole center + α / step * number of steps * SIN(θ / step * number of steps), where dome diameter (mm) = 8, number of steps = 9, α = diameter / 1.8, and θ = 15.7. Figure 7B As shown, a spiral pattern of steps is formed (lower right).

[0065] Figure 7BExemplary pipette motion patterns for predetermined locations to rupture the hydrogel dome calculated using the previous formula are shown, including 6 steps (upper left), 7 steps (upper right), 8 steps (lower left), and 9 steps (lower right). When a θ value of 15.7 is used, each pattern forms a spiral pattern of steps.

[0066] Figure 7C Exemplary pipette motion patterns for predetermined locations to rupture the hydrogel dome calculated using the previous formula are shown, including 10 steps (upper left), 11 steps (upper right), 12 steps (lower left), and 13 steps (lower right). When a θ value of 15.7 is used, each pattern forms a spiral pattern of steps.

[0067] Fig.7D An exemplary pipette motion pattern for rupturing a hydrogel dome is shown, calculated using the previous formula, where Diameter (mm) = 8, Number of Steps = 13, α = Diameter / 1.8, θ = 30. When a θ value of 30 is used, the pattern of steps does not look like a spiral, and a star-shaped pattern begins to appear. DETAILED DESCRIPTION

[0068] Studying co-cultures and cell interactions involves multiple barriers to culture and data analysis. Isolating different cell types in separate compartments can allow for efficient cross-talk and interaction 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 engraftment, inflammation, and secretion of multiple growth factors and metabolites.

[0069] Methods for cell / organoid co-culture assays are provided, comprising the use of split-well microplates. Split-well microplates support horizontal co-culture suitable for automated formats.

[0070] The present disclosure provides methods for co-culturing different cell types in adjacent wells that can be connected hydraulically using a pipette as a pump or via laminar flow through microchannels between the primary and secondary wells.

[0071] In support of the present disclosure, methods for culturing organoids and cells using separate well microplates were studied. For example, organoids were dissociated and passaged to new wells. Long-term organoid culture showed good condition of cells cultured in passage plates.

[0072] Fibroblast adhesion was assessed on plasma-treated split-well microplates. Fibroblast adhesion and normal behavior were observed. Migration of fibroblasts through the channels was tested using chemoattractants.

[0073] The method of the present disclosure uses a separation well microplate having a well geometry, wherein the well geometry includes a main well and a secondary chamber connected by at least one channel. For example, the culture method of the present disclosure can utilize a separation well microplate having a plurality of well units, each well unit including a main well and a secondary chamber connected by at least one channel. For example, each well unit may include a well geometry as shown in Figure 1, including a main well connected to a secondary well fluid via at least one channel. The separation well microplate is described in U.S. Provisional Application No. 63 / 151,082, entitled “Methods and Apparatus for Cell Passaging Using Microplate Well Units,” filed on February 19, 2021, U.S. Patent Application No. 17 / 580,193, entitled “Microplate for Automated Organoid Culture,” filed on October 22, 2021, U.S. Provisional Application No. 63 / 300,294, entitled “3D Printing of Organoid Passaging Plates,” filed on January 18, 2022, and PCT International Application No. PCT / IB2021 / 062356, entitled “Microplate Wells for Cell Culture,” filed on December 27, 2021, each of which is incorporated herein by reference in its entirety.

[0074] Figure 1A An example of a well unit 100 of a separation well microplate that can be used in conjunction with the method of the present disclosure is shown. Specifically, Figure 1AAn example diagram of a well unit 100 of a microplate according to various embodiments of the present disclosure is shown, which can be used to grow, culture, monitor and analyze embryoids, fused embryoids, cell spheres, organoids or other multicellular bodies. For example, a single well unit 100 of a separation well microplate can be used for horizontal co-culture of cell / organoid feeding. The separation well microplate used in the method according to the present disclosure may include a plurality of well units, each well unit including a main well 112 separated from a secondary well 115 by a shared side wall, and the main well and the secondary well may be connected by at least one microchannel 118 fluid. In some embodiments, at least one microchannel 118 may include a removable barrier to isolate the main well and the secondary well from each other. In some embodiments, at least one microchannel 118 may be open to allow the exchange of culture medium between the main well 112 and the secondary well 115. The well unit 100 of the separation well microplate includes a main well 112, which may be connected to the secondary well 115 fluid by at least one microchannel 118. In some examples, the main hole 112 can be used as a culture hole, and the secondary hole 115 can be used as a feed hole. The microchannel 118 can include a removable barrier. At least one microchannel 118 can be closed by a removable barrier or can be open. One or more microchannels 118 between the main hole 112 and the secondary hole 115 can be closed by a removable barrier, such as by an air gap, a hydrogel seal or a silicone seal. One or more microchannels 118 between the main hole 112 and the secondary hole 115 can be closed, for example, by an air gap. The air gap can be formed by, for example, bubbles.

[0075] Figure 1B Another example of a well unit 100 of a separation well microplate that can be used in conjunction with the methods of the present disclosure is shown. Figure 1B, the well unit 100 includes a main well 112 and a secondary well 115, which can be fluidically connected by at least one open microchannel 118. When open, the geometry of the at least one channel 118 can allow for the exchange of culture medium, expressed proteins (such as hormones), nutrients, growth factors, waste and debris between the main well and the secondary well, while retaining, for example, target cells (such as organoids) in the main well 112 and feeder cells in the secondary well 115. The dimensions of the at least one microchannel 118 can prevent multicellular target cells (such as organoids, tumoroids, etc.) from passing through the microchannel 118. In some examples, the geometry of the at least one microchannel 118 can include a slit configuration with a channel width of 150-500 microns or more, or 200-400 microns, or about 300 microns x 10-100 microns, or 10-75 microns in height. During the methods of the present disclosure, at least one microchannel 118 between the primary well and the secondary well can be closed by a removable barrier, or can be opened to allow exchange of culture medium, secreted factors, hormones, growth factors, nutrients, waste and debris between the primary well 112 and the secondary well 115, for example, by gravity feeding while shaking the separation well microplate. In some examples, at least one microchannel can be opened by, for example, removing a removable barrier, such as by using a pipette as a pump to eliminate the air gap, such as sealing the secondary well with a pipette tip to allow liquid flow between the two wells.

[0076] Figure 1CAnother example of a well unit 100 of a split well microplate that can be used in conjunction with the methods of the present disclosure is shown. The well unit 100 includes a main well section 112, a secondary well section 115, at least one microchannel 118, and a first hydrogel dome 106 that can be disposed in the main well section 112 of the well unit 100. According to various embodiments, the well unit 100 also includes a bottom sheet 121 disposed on the underside of the well body of the microplate. The bottom sheet 121 is attached to the underside of the well body to form the bottom surface of the well unit 100. In various examples, the bottom sheet 121 includes an optically transparent viewing window to allow imaging of cell spheres, organoids, or other cell cultures cultured in the well unit 100 of the microplate, as can be understood. The viewing window can be a window suitable for microscopic observation, whether bright field, phase contrast, fluorescence, confocal, two-photon, or other microscopic imaging methods known in the art. In some examples, the bottom sheet 121 may include a breathable sheet configured to increase the oxygen supply to the cell spheres, organoids or other cell bodies grown in the well unit 100 of the microplate. The breathable sheet can be formed by materials including polytetrafluoroethylene (PTFE), PEFP, polyimide, polydimethylsiloxane (PDMS), polycarbonate and / or other materials that can be understood. According to different examples, the breathable sheet can have a thickness of about 5-70 microns. According to different examples, the breathable sheet can include multiple holes. In other examples, the breathable sheet can allow molecules to pass through by diffusion. Alternatively, the breathable sheet can include some other thicknesses, pore sizes and pore densities.

[0077] like Figure 1C As shown, target cells in the form of multicellular bodies 103 (e.g., spheroids, tumoroids, organoids, etc.) can be embedded in a hydrogel 106 (e.g., ) is disposed in a dome of a well unit 100, which is disposed in a main well 112 in the well unit 100 and is surrounded by a liquid culture medium 109. According to various embodiments, the liquid culture medium 109 may contain appropriate growth factors and supplements to produce and / or stimulate the growth of the desired multicellular bodies. Applicable exemplary growth factors include angiogenin, bone morphogenetic protein (BMP), ciliary neurotrophic factor, colony stimulating factor, ephrin, epidermal growth factor, erythropoietin, fibroblast growth factor, glial-derived neurotrophic factor, hepatocyte growth factor, insulin, insulin-like growth factor, interleukin, leukemia inhibitory factor, keratinocyte growth factor, neuromodulin, neurotrophic factor, platelet-derived growth factor, transforming growth factor, tumor necrosis factor (α), vascular endothelial growth factor, and the like.

[0078] According to different implementation schemes, Figure 1CThe well unit 100 includes a main well segment 112 and a secondary well segment 115. In various examples, the main well segment 112 and the secondary well segment 115 are isolated from each other by closed microchannels. In some embodiments, the microchannel 118 may be closed by, for example, an air gap, a hydrogel, or a silicone. In some embodiments, the microchannel 118 may be open so that the main well segment 112 and the secondary well segment 115 are fluidly connected to each other through at least one microchannel 118, for example, to facilitate gravity flow of a liquid (e.g., a liquid culture medium 109) between the main well segment 112 and the secondary well segment 115 in response to tilting of the microplate. Exchanging the liquid culture medium 109 between the main well segment 112 and the secondary well segment 115 may allow for the removal of toxic byproducts and may provide fresh nutrients for growing cell cultures. Figure 1C In the example shown in FIG. 1 , the hydrogel 106 and the cell body 103 are disposed in the main pore section 112 of the pore unit 100 .

[0079] According to different embodiments, the method according to the present disclosure uses a separation hole microplate, which includes a plurality of hole units 100, such as 384 hole units, 96 hole units, 48 ​​hole units, 24 hole units, 12 hole units, 8 hole units or 6 hole units. Depending on the number of hole units 100 in the microplate, the width of the main hole segment 112 can be up to about 8 millimeters (mm) (for example, for a 96-hole plate), up to 11mm (for example, for a 48-hole plate), up to about 17mm (for example, for a 24-hole plate) and / or other sizes that can be understood. In addition, the depth of the main hole segment 112 and the secondary hole segment 115 is specified so that the microplate can be tilted to allow fluid exchange within the hole unit 100 without causing the fluid to overflow from the main hole segment 112 or the secondary hole segment 115 of each hole unit 100.

[0080] According to various embodiments, the well unit 100 of the separation well microplate includes a main well section 112, which is sized and shaped to support deposited cell bodies 103 (e.g., cell aggregates), which can be embedded in the hydrogel 106 deposited in the main well section 122. For example, the main well section 112 can be considered a culture well for growing target cells (e.g., embryoid bodies, fused embryoid bodies, cell spheres, organoids, and / or other multicellular bodies).

[0081] The secondary pore segment 115 can be used to grow feeder cells, supply feeder culture medium and / or other nutrients that can be used to feed cell aggregates grown in the main pore segment 112. In some embodiments, the secondary pores can be coated with primary antibodies specific to secreted factors (e.g., secreted factors from target cells). In some embodiments, the secondary pore segment 115 can be used to harvest supernatant from cell aggregates. For example, the secondary pore segment 115 can be viewed as a supply pore that contains feeder culture medium and / or other nutrients that can be used by cell cultures grown in the main pore segment 112. According to various embodiments of the present disclosure, the size and shape of the secondary pore segment 115 can accommodate fluids that can be exchanged with the main pore segment 112.

[0082] According to different embodiments, and depending on the number of well units 100 in the microplate, the width of the secondary well segment 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 dimensions as may be appreciated.

[0083] According to different embodiments, the size and shape of the main bore section 112 and the secondary bore section 115 can be different from each other. In some examples, the main bore section 112 is larger (in dimension, such as diameter, cross-section, or volume) than the secondary bore section 115. In other examples, the secondary bore section 115 is larger than the main bore section 112. In some examples, the shape of the main bore section 112 is different from the shape of the secondary bore section 115.

[0084] According to various examples, the well unit of the separation well microplate includes at least one microchannel 118 whose size and shape are designed to prevent objects having dimensions (e.g., diameter, height, width, etc.) of a specific size (e.g., greater than about twenty-five (25) microns (μm)) from passing from one well segment to another.

[0085] In some examples, the height dimension of at least one microchannel 118 can be between about ten (10) microns and about one hundred (100) microns, or about 10 microns to about 75 microns, or about ten (10) microns to about twenty-five (25) microns. The geometry of at least one microchannel 118 can include a slit configuration with a channel width of 150-500 microns or greater, or 200-400 microns, or about 300 microns x 10-100 microns, or 10-75 microns high. For example, the height of at least one microchannel 118 can prevent objects (e.g., cell spheres, tumoroids, organoids, organoid fragments, etc.) with a size of about 25 μm or greater present in the main pore section 112 from migrating to the secondary pore section 115. The microchannel 118 can be closed by, for example, an air gap, a hydrogel seal, or a silicone seal. The microchannel 118 can be opened by, for example, eliminating the air gap or removing the hydrogel seal or silicone seal.

[0086] In some examples of the present disclosure, the main hole 112 can be used as a culture chamber. For example, initially, at least one channel 118 can be closed, for example, target cells can be cultured without mixing and allowed to adhere to the bottom of the main hole 112 of the separation hole microplate. For example, feeder cells can be cultured without mixing and allowed to adhere to the bottom of the secondary hole 115 of the separation hole microplate. After the target cells and / or feeder cells adhere to the hole, the closed at least one channel 118 can be opened. The separation hole microplate can be placed on a rocker to allow culture medium, hormones, growth factors, nutrients, waste and debris to be exchanged by gravity supply. In some examples, feeder cells can be cultured in the secondary chamber 115, allowing the target cells cultured in the main chamber 112 to be fed through at least one channel that is opened.

[0087] definition

[0088] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0089] The singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0090] The term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items.

[0091] When referring to a measurable value of an amount of a compound, dosage, time, temperature, etc., the term "about" is meant to encompass variations of 10%, 5%, 1%, 0.5% and even 0.1% of the specified amount.

[0092] The term "antibody conjugate" refers to a specific antibody conjugated to a chromophore or fluorophore molecule, avidin (eg, streptavidin) or biotin, wherein the antibody is specific for a target antigen (eg, a secreted factor).

[0093] The terms "comprises" and / or "comprising", when used in this specification, specify the presence of the features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof. Unless otherwise defined, all terms, including technical and scientific terms used in the specification, have the same meaning as commonly understood by ordinary technicians in the field to which the present disclosure belongs. In the event of a conflict in terms, the present specification shall prevail.

[0094] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0095] The embodiments described in one aspect of the present disclosure are not limited to the described aspects. As long as the embodiments do not prevent these aspects of the present disclosure from operating for their intended purposes, these embodiments can also be applied to different aspects of the present disclosure.

[0096] "Treating" or "treatment" of a disease state or condition includes: (i) preventing the disease state or condition, i.e., preventing the clinical symptoms of the disease state or condition in a subject who may be exposed to or susceptible to the disease state or condition but does not yet experience or show 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 its clinical symptoms, or (iii) palliating the disease state or condition, i.e., causing temporary or permanent regression of the disease state or condition or its clinical symptoms.

[0097] The term "feeder cell" refers to a cell that provides extracellular secretions to help another cell proliferate, grow, differentiate and / or maintain characteristics. Feeder cells can support the growth of target cells in culture by providing a complex mixture of extracellular matrix (ECM) components and growth factors. In some cases, feeder cells may not be able to divide, that is, cell growth is inhibited. The growth of feeder cells can be inhibited by any appropriate method known in the art, for example. The growth of feeder cells can be inhibited by chemical fixation (for example, chemical fixation by mitomycin C or glutaraldehyde). The growth of feeder cells can be inhibited by physical methods (for example, gamma ray irradiation, x-ray irradiation or electric pulses). For example, feeder cells for co-culture of target cells (such as embryonic stem cells (ESC)) may be fibroblasts, which may not have mitotic activity, but still survive. In some cases, target cells can grow in the presence of feeder cells that can divide. Some live feeder cells (such as human fibroblasts) may also become target cells, just like the case of induced pluripotent stem cells (iPSC) after reprogramming. Feeder cells may be suppressed feeder cells, for example, feeder cells may not be able to divide. The selection of feeder cells may depend on the target cells. For example, feeder cells can be uninhibited fibroblasts. Feeder cells can be inhibited fibroblasts, epithelial cells, mesenchymal cells, muscle cells, stromal cells, spleen cells or amniotic cells. Fibroblasts can be (for example) human skin fibroblasts, 3T3 fibroblasts, human fetal fibroblasts, mouse embryonic fibroblasts, etc. Epithelial cells can be (for example) human adult fallopian tube epithelial cells, human amniotic epithelial cells, HeLa cells (human cervical cancer tumor epithelial cells), etc. Mesenchymal cells can be adipose-derived mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells, human bone marrow-derived mesenchymal cells, human amniotic mesenchymal stem cells, etc. Stromal cells can be (for example) human bone marrow stromal cells or mouse bone marrow stromal cells. Amniotic cells can be (for example) human amniotic cells or mouse amniotic cells.

[0098] The term "target cell" refers to a cell used for automated cell culture applications of the present disclosure, such as an organoid, a tumoroid, a spheroid, a stem cell, or a production cell line. In some embodiments, the target cell is a spheroid, a tumoroid, an organoid, and / or other multicellular bodies. In some embodiments, the target cell can be a stem cell. In some embodiments, the target cell can be a production 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 tumoroid. Mammalian tissue can be derived from a patient biopsy sample. The target tissue can be derived from a target organ, such as a lung, intestine (such as the small intestine), colon, stomach, pancreas, liver, kidney, skin, bone marrow, blood-brain barrier, brain, heart, etc.

[0099] Organoids, spheroids, tumoroids, and three-dimensional (3D) cell culture models are useful in many applications such as disease modeling and regenerative medicine. 3D cell models such as organoids and spheroids may help to better understand complex biology in a physiologically relevant context because cells often maintain their natural shape and appropriate spatial orientation, such as in aggregates or spheroids, while 2D models of cells grown in sheets or monolayers may not be as successful. Gene and protein expression in 3D cell cultures can more closely mimic gene and protein expression. For example, 3D cell cultures can be used for drug target identification, lead compound identification, compound optimization, preclinical qualification, solid tumor modeling, genetic disease modeling, drug discovery, precision medicine, organs-on-chips, and bioprinting.

[0100] The term "spheroid" refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate composed of one or more cell types that grow and proliferate, may exhibit enhanced physiological responses, but do not undergo differentiation or self-organization. Common cell sources for spheroids are primary tissues or immortalized cell lines. Spheroids can bridge the gap between monolayers and complex organs.

[0101] The term "organoid" refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate composed of one or more cell types, in which cells spontaneously self-organize into appropriately differentiated functional cell types and progenitor cells similar to their in vivo counterparts in at least one aspect. Organoids mimic their corresponding in vivo organs. Organoids can be derived from pluripotent stem cells (PSC), induced pluripotent stem cells (iPSC), neonatal tissue stem cells, embryonic stem cells (ESC), adult stem cells or primary tissues. Organoid cultures can be elaborated to be similar to the complexity of organs, and are therefore useful for the study of disease etiology and treatment. Organoid technology has recently become an important tool for basic research and biomedical research. Organoid cultures can be selected from different types of target organs, such as lungs, intestines (such as small intestine), colon, stomach, pancreas, liver, kidney, skin, bone marrow, blood-brain barrier, brain, heart, etc.

[0102] For example, gastric organoid tissue cultures can be derived from, for example, adult mice, adult humans, hPSCs, etc. Gastric organoid tissue cultures can use stem cell culture conditions (niche factors), including culture medium components such as one or more of EGF, Noggin, R-spondin, Wnt-3A, FGF10, etc., depending on the source. Differentiation culture conditions can include EGF, R-spondin-EGF, R-spondin, etc., depending on the source.

[0103] As another example, intestinal organoid tissue cultures can be derived from, for example, adult mice, adult humans, hPSCs, etc. Intestinal organoid tissue cultures can use stem cell culture conditions (niche factors), including culture medium components such as one or more of EGF, Noggin, R-spondin, Wnt-3A TGF-β inhibitors, p38 inhibitors, etc., depending on the source. Differentiation culture conditions can include EGF, Noggin, TGF-β inhibitors, etc., depending on the source.

[0104] As another example, colon organoid tissue cultures can be derived from, for example, adult mice, adult humans, etc. Colon organoid tissue cultures can use stem cell culture conditions (niche factors), including culture medium components such as one or more of EGF, Noggin, R-spondin, Wnt-3A TGF-β inhibitors, p38 inhibitors, etc., depending on the source. Differentiation culture conditions can include EGF, Noggin, TGF-β inhibitors, etc., depending on the source.

[0105] As another example, pancreatic organoid tissue cultures can be derived from, for example, adult mice, adult humans, etc. Pancreatic organoid tissue cultures can use stem cell culture conditions (niche factors), including culture medium components such as one or more of EGF, Noggin, R-spondin, Wnt-3A, FGF10, nicotinamide, etc., depending on the source. Differentiation culture conditions can include EGF, Noggin, R-spondin, Wnt-3A, etc., depending on the source.

[0106] As another example, liver organoid tissue cultures can be derived from, for example, adult mice, adult humans, etc. Liver organoid tissue cultures can employ stem cell culture conditions (niche factors), including culture medium components such as one or more of EGF, Noggin, R-spondin, Wnt-3A, FGF10, HGF, nicotinamide, etc., depending on the source. Differentiation culture conditions can include EGF, Noggin, R-spondin, Wnt-3A, FGF10, TGF-β inhibitors, Notch inhibition, BMP7, etc., depending on the source.

[0107] The term "tumoroid" refers to a three-dimensional (3D) multicellular in vitro tissue culture aggregate composed of one or more cell types that can mimic the human tumor microenvironment, and these cell types are usually derived from primary tumors collected from tumor patients. Tumoroids can be used to study new cancer drugs or for precision medicine in the field of oncology. Cancer cell lines can be (for example) bladder, breast, colon, hematopoietic and lymphoid, liver, lung, ovary, prostate, skin, etc.

[0108] The term "stem cell" refers to undifferentiated cells that have the potential to develop into many different cell types and perform different functions. Pluripotent stem cells (such as those found in embryos) can give rise to any type of cell, such as those in the brain, bone, heart, and skin. Some human adult cells can be reprogrammed to an embryonic stem cell-like state, called induced pluripotent stem cells (iPSCs). For example, pluripotent stem cells found in the umbilical cord of an adult or infant may develop into cells that make up the organ system from which they originated. Pluripotent stem cells can remain in an undifferentiated state when grown under certain cell culture conditions. To produce differentiated cells, the chemical composition of the culture medium can be changed, the surface of the culture dish can be altered, or the cells can be modified by forcing the expression of certain genes.

[0109] The term "production cell line" refers to a cell line that can be used to produce enzymes, vaccines, monoclonal antibodies, cytokines, peptides, therapeutic toxins, coagulation 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 can include Chinese hamster ovary (CHO) cell lines, mouse myeloma cell lines (e.g., NS0, Sp2 / 0), mouse C127, human embryonic kidney (HEK) cell lines (e.g., HEK293 cell lines), human fibrosarcoma cell lines (e.g., HT-1080 cell lines), human embryonic retinal cell lines (e.g., PER.C6 cell lines), baby hamster kidney (BHK) cell lines (e.g., BHK21 cell lines), yeast cell lines (e.g., Saccharomyces cerevisiae, Pichia pastoris), insect cell lines infected with viral vector baculovirus (baculovirus-insect cell expression system) (e.g., Sf9 cell lines), and plant cells.

[0110] The term "secreted factor" or "secreted factors" refers to a factor that can be secreted by a target cell during the automated cell culture application process 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 coagulation factor, or an Fc fusion protein.

[0111] "Transwell" culture, also known as direct co-culture, can include vertically arranged wells with a lower compartment and an upper compartment, such as a transwell insert, separated by, for example, a 0.4 μM microporous membrane to minimize direct contact between producer cells and target cells. In some embodiments, the tissue culture methods of the present disclosure do not employ transwell culture.

[0112] Each type of source cell and the type of differentiation to be achieved may require different culture medium components. Growth factors such as EGF, Noggin (NOG), R-spondin (RSPO1), HGF, BMP, FGF, etc. may be essential components of organoid culture medium. Tissue culture medium may include growth factors. Growth factors may be produced by feeder cells. Growth factors may be recombinant growth factors. Recombinant growth factor proteins used for organoid culture may include (for example) recombinant human EGF protein, recombinant HGF protein (for example, human HGF protein, cynomolgus monkey HGF protein), human FGF10, human Noggin / NOG protein, human RSPO1 protein, human BMP-2 protein, etc. Other recombinant growth factors used 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 can be purchased from, for example, Sino Biological Co., Ltd. or Thermo Fisher Scientific.

[0113] 3D cell models, such as organoids and spheroids, can be cultured in tissue culture media containing hydrogels, for example, in hydrogel domes within culture media.

[0114] The term "hydrogel" or "hydrogels" refers to an extracellular matrix that can be used to culture organoids. Hydrogels can include mouse EHS sarcoma matrix, for example, commercially available Matrigel (Corning), Cultex (Trevigen), Geltrex (Gibco), type I collagen, fibrin, hyaluronic acid (HA), gelatin methacrylate (GelMA), decellularized matrix or biopolymers such as alginate, silk, nanocellulose; engineered materials such as polyethylene glycol (PEG), self-assembling peptides such as RADA16 / PuraMatrix bQ13, polylactic acid / (co)glycolic acid, polycaprolactone, polyacrylamide, oligo(ethylene glycol)-substituted polyisocyanopeptides, ELP (elastin-like protein), or combinations of these polymers.

[0115] Horizontal co-culture of cells / organoids

[0116] A horizontal co-culture method for cell / organoid feeding is provided. Figure 1AThe method comprises: allowing one or more target cells to adhere to the bottom of the main well 112 of the well unit 100 of the separation well microplate, including incubation without mixing, the main 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 incubation without mixing; opening the at least one closed microchannel; shaking the separation well microplate to allow mixing and medium exchange between the main well 112 and the secondary well 115 by gravity flow through the at least one opened microchannel 118, as shown in FIG. Figure 1B As shown; detaching the target cells from the main well 112; and allowing the detached target cells to settle in the main well 112 away from the opened microchannel. The method may also include: mixing the detached target cells and allowing them to further settle in the main well away from the opened microchannel; washing the settled target cells; and collecting the washed target cells by a liquid processor. Washing may include suspending the settled target cells, including introducing and removing liquid culture medium, and washing may optionally be repeated multiple times. In some embodiments, during the initial incubation period, allowing one or more target cells to adhere to the bottom of the main 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.

[0117] Initially, at least one microchannel can be closed, for example, by an air gap in or adjacent to the microchannel. After at least a portion of the initial incubation period, the closed at least one microchannel 118 can be opened by eliminating the air gap. For example, the at least one microchannel can be opened by using a pipette as a pump to exchange liquid culture medium between the main hole and the auxiliary hole. In order to use the pipette as a pump, the outer surface of the pipette engages with the edge of the auxiliary hole to form a seal. Then, the liquid in the pipette is allowed to enter and pass through the microchannel 118, thereby removing the air gap.

[0118] Dissociating the target cells from the main well 112 may include exposing the target cells to a dissociation agent to dissociate the target cells without dissociating the feeder cells, optionally wherein the dissociation agent includes EDTA. The dissociation agent may include a physiological buffer solution of a dissociation enzyme. For example, the dissociation enzyme may be selected from the group consisting of trypsin, collagenase, elastase, catalase, superoxide dismutase, and dispase.

[0119] In some examples, the target cells may be stem cells or organoids. In some examples, the feeder cells may be fibroblasts. In some examples, the target cells are embedded in a hydrogel disposed in a main well of the well unit.

[0120] Detection of secreted factors from cells allows kinetic measurements within a single microplate well

[0121] Target cells can produce secreted factors. For example, Figure 2 As shown, intestinal organoids can secrete hormones 230, or production cell lines (such as CHO cells) can secrete antibodies. A method is provided for detecting multiple secreted factors from cells to allow kinetic measurements over time in a single microplate. For example, multiple secreted factors can be detected at different time points after organoid formation from stem cells or organoid fragments (kinetic measurements) 270, 280, 290.

[0122] Figure 2 A method for detecting secreted factors from target cells is shown, including kinetic measurements over time in a single separation well microplate. In some embodiments, the secreted factors can be selected from hormones, antibodies, cytokines, chemokines, or growth factors. Patient biopsy samples 210 can be obtained, which can be a source of target cells, such as multicellular target cells, such as organoids or tumors; other organoid sources, such as organoid fragments or stem cells, can be used. The culture of target cells such as organoids can be carried out in the main wells of the well unit of the separation well microplate 220. The organoids can secrete hormones into the liquid culture medium 230. During the initial incubation period, the main well is separated from the secondary well by at least one closed microchannel, and the bottom of the secondary well is coated / spotted with a primary antibody (Ab) 240 specific for the secreted factor. After the initial incubation period, the barrier in the microchannel is removed to allow the secreted factor to diffuse between the main well and the secondary well through the opened microchannel 250. The secreted factor is captured 260 by the primary antibody in the secondary well. A labeled secondary antibody (2'Ab) specific for the primary antibody or captured secreted factor is added to the secondary well at a first time point, and bound 2'Ab 270 is detected in the secondary well using a microplate reader. Diffusion of the secreted factor can include shaking the separation well microplate to allow the secreted factor to diffuse between the primary well and the secondary well by gravity flow through the open microchannels. Detection can include multiplexing detection using imaging and uniform detection using a microplate reader.

[0123] like Figure 2 As shown, after further incubation of the target cells in the microplate 275, a further labeled secondary antibody (2'Ab) specific to the primary antibody or the captured secreted factor is added at a second time point and detected with a microplate reader 280. Optionally, after further incubation of the target cells in the microplate 285, a further labeled secondary antibody (2'Ab) specific to the primary antibody or the captured secreted factor is added at a third time point and detected with a microplate reader 290. Further continuous incubation and detection operations can be performed at the fourth, fifth, sixth, seventh and other time points. This method allows for kinetic measurement of secreted factors over time in the same separate well microplate used to culture target cells.

[0124] The labeled antibody can be labeled with any suitable marker, such as a fluorescent dye. Different antibodies can be differentially labeled with different fluorophores. The primary antibody (Ab) and the labeled secondary antibody (2'Ab) can be purchased commercially, for example, from (for example) Beckman Coulter Co., Ltd., Abcam or Novus Biologicals. The labeled secondary antibody (2'Ab) can be prepared using antibody labeling methods known in the art, for example, by N-hydroxysuccinimide esters (NHS esters), heterobifunctional reagents or carbodiimides.

[0125] In some embodiments, the target cell is an organoid, which can be derived from a target tissue selected from the group consisting of a patient biopsy sample, a stem cell, or an organoid fragment. The organoid can be cultured in a hydrogel dome in the main well of the well unit. For example, an organoid can be grown from a stem cell or an organoid fragment in a hydrogel dome (such as Matrigel) in the main (culture) well.

[0126] Initially, the main hole and the secondary hole are separated by, for example, air trapped in the microchannel (or by other means), thereby avoiding any non-specific binding on the detection array. Optionally, during at least a portion of the initial incubation period, at least one closed microchannel may be closed by a barrier comprising an air gap, a hydrogel seal or a silicone seal, which is located in or adjacent to the microchannel. When the microchannel is closed by a barrier comprising an air gap, the opening of the closed microchannel may include eliminating the air gap after at least a portion of the initial incubation period; optionally including using a pipette as a pump to exchange liquid culture medium between the main hole and the secondary hole. For example, after the organoid is fully developed, liquid flow can be generated between the main hole and the secondary hole by using a pipette as a pump (sealing the pipette tip to the feed hole), thereby eliminating the air gap. For example, the air gap can be eliminated by pumping the liquid through the microchannel between the main hole and the secondary hole. In another example, gravity flow can be used to exchange culture medium between the holes to transport the excretion products of the organoid to the secondary hole containing the dotted / coated antibody.

[0127] The secondary wells can be dotted / coated with a plurality of different primary antibodies specific for a plurality of different secreted factors, and the primary antibodies can be arranged at the bottom of the secondary wells in the form of a detection array. The type of secreted factor detected can be located in a specific area of ​​the detection array. The amount of hormone can be measured by the intensity of the fluorescent signal after washing. In some embodiments, different time points can be recorded with the aid of different fluorophores, for example, each time point is a different fluorescent color. For example, by using different fluorophores, the concentration of the capture antibody (Ab) can be normalized.

[0128] Measurements can be done with a microscope or a microplate reader. Microplate readers have the advantage of allowing a high dynamic range, but may require about 1 mm2 per array spot for detection. Imagers can detect a greater number of array spots, are able to observe smaller array spots but may have a more limited dynamic range.

[0129] Functionalized surfaces with antibodies for stem cell isolation

[0130] Some organoid types require that the organoids be disaggregated to the single-cell level for passaging. In this case, it is important to capture the desired (adult) stem cells to be able to reconstitute new organoids.

[0131] Another embodiment of the present disclosure provides a method for passaging organoids using single cell passaging and should have secondary wells whose surfaces are functionalized with antibodies to specifically capture stem cells during the wash process at the end of each culture cycle. Figure 3 An exemplary passaging method is shown for isolating desired stem cells by disassembling organoids to the single-cell level. Operation 310 includes culturing organoids in a well unit of a separation well microplate having a secondary well 115 coated or surface-functionalized with an immobilized primary antibody (Ab) specific for stem cells (e.g., Lgr5+), a main well 112 containing an organoid 103 (e.g., intestinal organoid) within a hydrogel dome 106 disposed on the bottom surface of the main well 112, and an initially closed microchannel 118. Liquid culture medium 109 is also shown in the main well section 112 and the secondary well section 115 of the well unit 100. The liquid culture medium 109 may contain appropriate growth factors and supplements to support the growth of the organoid 103. Operation 320 includes disassembling the dome 106 of the hydrogel into a plurality of hydrogel fragments 320 or other liquid forms to release the organoid 103 into the liquid culture medium 109.

[0132] The present disclosure provides an automated process for disrupting a hydrogel dome 320. In the prior art, disrupting a hydrogel dome is typically performed manually using a pipette, with random repeated injections / punctures of the dome (for example) about 20 times to help disrupt it.

[0133] An automated method 320 for disrupting a hydrogel dome is provided, the method comprising: culturing a first cell in a hydrogel dome in a liquid culture medium in a well of a microplate during an initial incubation period to provide a multicellular target cell; after the initial incubation period, piercing the hydrogel dome with a pipette tip associated with a liquid handler at or near the x, y center of the dome in the well; moving the pipette tip through the dome in multiple steps, each step including an x, y predetermined position in the well to disrupt 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. After culturing the target cell, the automated pipette tip can be positioned at or near the center of the hydrogel dome. In some cases, the first cell can be a target cell or a stem cell. The liquid handler can aspirate the pipette at one or more, two or more, or each of the multiple steps. For example, the pipette tip can 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 of the x, y center of the hydrogel dome, or within 0-2 mm, or 0.01-1.5 mm of the center. Each of the X, Y predetermined positions within the well can be calculated, including: X(number of steps) = X well center + α / step * number of steps * cos(θ / step * number of steps); and

[0134] Y(number of steps) = Y well center + α / step * number of steps * SIN(θ / step * number of steps), where α = dome diameter (mm) / 1.8; steps = number of steps to move the pipette tip 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 θ = a constant value between 5 and 50. In some cases, the number of steps is an integer between 2-20, 3-18, 4-17, 5-15, 6-12, or 8-10. In some cases, θ is a constant between 5-50, 10-45, 15-40, 15-35, or any number in between. In some cases, θ 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 in the hole can be determined based on the original inoculation position in the hole, the volume of the hydrogel deposited in the hole, and / or the imaging of the hydrogel dome. The pipette tip can be used to pierce the dome and / or aspirate within the dome. For example, the pipette can be used to apply, remove or aspirate liquid or air within the dome. The input of the system can include the inoculation position x, y coordinates in the hole and the known volume of the dome. Another input can be the number of steps of the dome pipette piercing or pipette aspiration. 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 2-20, 3-18, 4-17, 5-15, 6-12, or 8-10. Fig. 7AAs shown, the input includes the diameter of the hydrogel dome, which in this case is 8 mm, the number of steps is 8, and the X, Y coordinates of the center position of the hole. In some cases, the number of steps / positions can be minimized to effectively destroy the hydrogel, thereby minimizing the processing time. The output can include an automated sequence / pattern of puncture / aspiration positions within the dome by the liquid handler using a pipette. In some embodiments, the pattern of puncture / aspiration positions can 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 can aspirate within the dome. Between steps / positions, the pipette tip can be dragged under the surface of the dome. For example, between steps / positions, the position of the pipette tip in the Z axis can be maintained below the surface of the hydrogel dome to effectively destroy the dome. Between steps, the position of the pipette tip in the Z axis can be maintained at or near the bottom of the hydrogel dome. For example, between steps or during aspiration, the pipette tip can be held below the surface, within 0.5, 1.0, 1.5, or 2 mm of the surface, at or near the center of the Z axis, at the bottom, 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 the volume of the hydrogel dome or by imaging the well. Exemplary Figures 7B-7D The input using the hole center position, 8mm dome diameter and 6-13 steps is shown, using a θ of 15.7 ( Figure 7B -C) or 30( Fig.7D ), the output series of spiral X, Y coordinates of the automatic pipette in the hole causes the effective rupture of the hydrogel dome. For example, the solid hydrogel 106 can be converted into a liquid, thereby separating the organoid 103 from the hydrogel 106. The hydrogel can be converted into a liquid by, for example, vigorous pipetting or shear force of a liquid handler, and the temperature can be reduced or not reduced, for example, from the culture temperature (about 37 degrees Celsius) to about 4 degrees Celsius to about 10 degrees Celsius, or about 10 degrees Celsius. A dissociation reagent can be added to the main hole 112 to dissociate the organoid into stem cells and multiple other cell types. After the dissociated organoid is formed, the microchannel 118 is opened, for example by removing the air gap. After the organoid is disassembled, liquid aspiration is performed through the secondary hole. The stem cells are allowed to be captured by the primary antibody (Ab) in the secondary hole 115. Operation 330 includes washing the well unit 100 to remove unwanted cells and debris. Operation 340 includes dissociating the separated stem cells from the antibody / cell complex in the secondary hole. Operation 350 includes reseeding the isolated stem cells into a new hydrogel dome 106 within a new main well 112 .

[0135] Alternatively, at operation 310, the secondary wells 115 can be coated or surface functionalized with an immobilized capture reagent (e.g., a biotin capture reagent or an avidin capture reagent). With respect to the biotin immobilized capture reagent, after treating the organoids with the dissociation reagent at 320, the dissociated organoids can be treated in a solution with an antibody specific for the target stem cell, which is coupled to a reagent having a strong affinity for the capture reagent, such as streptavidin (e.g., an antibody-streptavidin conjugate), to form a stem cell-antibody-streptavidin complex. The microchannel 118 is opened by removing the removable barrier and allowing the stem cell antibody-streptavidin complex to bind to the immobilized biotin in the secondary wells 115. After washing away unbound cells and debris in operation 330, the stem cells can be dissociated from the complex. The remaining operations 340 and 350 can be performed as described herein. Alternatively, an antibody conjugate comprising a stem cell-specific primary antibody conjugated to streptavidin can be allowed to bind to an immobilized biotin capture reagent prior to removing the removable barrier.

[0136] Functionalized scaffolds with antibodies for stem cell isolation

[0137] Some organoid types require disaggregation of the organoids to the single-cell level for passaging. Additional methods are provided to capture the desired (adult) stem cells to be able to reconstitute new organoids.

[0138] Another embodiment of the present disclosure provides a method for organoid passaging using single cell passaging and should have a secondary well including a scaffold coated with immobilized antibodies or surface functionalized to capture specific stem cells during the wash process at the end of each culture cycle.

[0139] Figure 4 An exemplary passaging method is shown, which isolates desired stem cells by disaggregating organoids to the single-cell level. Operation 410 includes culturing organoids in a well unit of a separation well microplate, the separation well microplate comprising a main well 112 and a secondary well 115, the main well 112 containing an organoid 103 (e.g., intestinal organoid) disposed within a hydrogel dome 106 on the bottom surface of the main well 112, a closed microchannel 118 between the main well and the secondary well, and the secondary well 115 having a porous scaffold coated or surface-functionalized with a primary antibody (Ab) specific for stem cells (e.g., Lgr5+). Liquid culture medium 109 is also shown in the main well section 112 and the secondary well section 115 of the well unit 100. The liquid culture medium 109 may contain appropriate growth factors and supplements to support the growth of the organoid 103.

[0140] Operation 420 includes breaking down the dome of the hydrogel 106 into a plurality of hydrogel fragments 320 or other liquid forms to release the organoid 103 into the liquid culture medium 109. For example, the solid hydrogel 106 can be converted into a liquid, thereby separating the organoid 103 from the hydrogel 106. The hydrogel can be converted into a liquid by, for example, vigorous pipetting or shear force of a liquid handler, and the temperature can be reduced or not reduced, for example, from the culture temperature (about 37 degrees Celsius) to about 4 degrees Celsius to about 10 degrees Celsius, or about 10 degrees Celsius. A dissociation reagent is added to the main well 112 to dissociate the organoid into stem cells and a plurality of other cell types. After the dissociated organoid is formed, the microchannel 118 is opened, for example by removing the air gap. After the organoid is broken down, liquid aspiration is performed through the secondary well. The stem cells are allowed to be captured by the first antibody (Ab) in the secondary well 115.

[0141] 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 into a new hydrogel dome 106 in a new primary well 112.

[0142] Magnetic beads for stem cell isolation

[0143] Some organoid types require disaggregation of the organoids to the single-cell level for passaging. After disaggregation of the organoids, magnetic beads coated with antibodies specific for stem cells can be added to the master wells to capture the desired (adult) stem cells, enabling the reconstruction of new organoids.

[0144] During the washing operation, a magnet is placed below the main well to retain the (adult) stem cells within the main well. Antibody binding can be disrupted in several ways, for example enzymatically (eg by trypsin), or by adding an enzymatically digestible linker.

[0145] Figure 5 An exemplary passaging method is shown for isolating desired stem cells by disaggregating organoids to the single cell level. Operation 510 includes culturing organoids in a well unit of a separation well microplate, the separation well microplate comprising a main well 112 containing an organoid 103 (e.g., an intestinal organoid) within a hydrogel dome 106 disposed on the bottom surface of the main well 112, and having a closed microchannel 118 between the main well and the secondary well. Liquid culture medium 109 is also shown in the main well section 112 and the secondary well section 115 of the well unit 100. The liquid culture medium 109 may contain appropriate growth factors and supplements to support the growth of the organoid 103.

[0146] Operation 520 includes breaking up the hydrogel dome 106 into a plurality of hydrogel fragments 320 or other liquid forms to release the organoid 103 into the liquid medium 109. For example, the hydrogel 106 can be converted to a liquid to separate the organoid 103 from the hydrogel 106. The hydrogel can be converted to a liquid by, for example, shear forces or vigorous pipetting of a liquid handler, and the temperature can be reduced or not, such as from the incubation temperature (about 37 degrees Celsius) to about 4 degrees Celsius to about 10 degrees Celsius, or about 10 degrees Celsius. A dissociation reagent is added to the main well 112 to dissociate the organoid into stem cells and a plurality of other cell types.

[0147] Operation 530 includes adding magnetic beads labeled with antibodies specific for stem cell markers. Operation 540 includes allowing the magnetic beads coated with antibodies to incubate with the dissociated organoids to allow the binding of the desired stem cells. Operation 550 includes applying a magnet to the bottom of the main well, opening the microchannel 118, and washing unbound cells and debris by (for example) aspirating liquid culture medium through the secondary well with a liquid handler. Optionally, operation 550 can also include transferring the captured cells after washing to a new well unit of a new separation well microplate, including removing the magnet to allow the captured cells to be transferred to a new well unit of a new separation well microplate. The magnet should be reapplied under the new well unit before continuing with operation 560. Operation 560 includes adding a new hydrogel to the main well, embedding the stem cells and magnetic beads in the hydrogel, and removing the magnet from under the main well. Operation 580 includes placing a magnet on top of the main well to float the stem cells in the hydrogel. Immunotherapy of Tumoroids and Organoids for Toxicology Studies

[0148] CAR T cells and other cell therapies may be beneficial in the treatment of cancer. Tissue migration, invasion, and tumor-specific cell killing can be evaluated. The assay includes several readouts that may be useful in evaluating immunotherapies.

[0149] Another embodiment of the present disclosure is a method for organoid / tumoroid culture and observing how fibroblasts, macrophages or other cells migrate through microchannels to the main organoid dome. The role of these cells can be evaluated by observing the phenotypic changes of the organoid / tumoroid.

[0150] Fig. 6A and 6B Methods for evaluating cell (eg, lymphocyte) function and toxicology studies in single split-well microplates are shown. Fig. 6AA view of a well unit 100 of a separation well microplate is shown, which contains multicellular target cells (e.g., organoids) in a hydrogel dome 106 in a liquid culture medium 109 in a main well 112 of a microplate microplate. The secondary well 115 contains chimeric antigen receptor T cells (CAR-T cells), CAR natural killer cells (CAR-NK cells), or other lymphocytes. The microchannel 118 can have a channel size of 5 microns, while the lymphocytes have a size of 7-10 microns. The organoids can release chemical attractants, which may cause immune cells to migrate into the main chamber, depending on the size of the microchannel. Figure 6B The effect of tilting a separation well microplate is shown, where the tilted position can be used to impair the migration of immune cells to the main well 112 .

[0151] Other assays

[0152] In addition to the assays described above, the split-well microplate architecture provides at least two major advantages for cell-based assay development and increasing the sophistication of cell-based assays.

[0153] The first major advantage is the ability to allow cells to move or migrate between the main and secondary wells of a well cell. The distance of movement or extension is short, and the main and secondary wells can be effectively separated. Separate well microplates do not have to include a matrix or force liquid flow.

[0154] Many applications illustrate this advantage. To study angiogenesis, tumor microtissue or growth factors would be present in one well of the well cell, while an endothelial cell layer would be present in the other well of the well cell. The endothelial cells would have the ability to migrate into the first chamber and potentially form angiogenesis sprouts.

[0155] To study neurogenesis - nerve regeneration, the effects of growth factors or neurotoxins on neurite outgrowth can be developed. Neurons will be placed in one chamber and extend into a second chamber in the presence of growth factors in the second chamber. The process will depend on the therapeutic agent. The advantage is the ability to study growth in a given direction.

[0156] The movement of normal or tumor cells into different chambers can be studied to study tumor cell escape / invasion, migration, wound healing. Compared with traditional cell migration assays, the migrated cells can be counted more easily and accurately in different cells of the cell unit.

[0157] To study T cell invasion or monocyte migration, immune cells can be placed in one chamber and chemoattractants from the microtissue can be placed in a second chamber. When chemoattracted, the immune cells will move to the other chamber.

[0158] The second major advantage of the separate well microplate structure is the ability to co-culture and study the crosstalk of different cell types and microtissues. In this model, cells representing different tissues will be separated in different wells of the well unit, and the microplate will allow medium exchange between the main and secondary wells of the well unit, so the effects of added compounds and secreted factors can be studied. For example: hepatocytes may secrete factors that affect cardiac cells. Endothelial cells may secrete cytokines in response to inflammation, thereby attracting immune cells, or may cause hepatocyte or neuronal cell damage (cirrhosis, stroke model). Some other applications may include: multi-tissue toxicity evaluation of organ tissue pairs such as liver-brain, liver-heart, kidney-liver, and intestine-liver. The differentiation and self-renewal of stem cells in response to compounds, toxins, growth factors, inflammation, and metabolic conversion can be studied. In some embodiments, the evaluation of secreted factors or cell movement only can be performed by using a horizontal slit configuration to allow cell migration, or using a vertical wider slit configuration for feeding and medium-growth factor exchange.

Claims

1. A method for co-culturing target cells, the method comprising: Introducing one or more target cells into a main well of a well unit of a separation well microplate and performing an initial incubation in a liquid culture medium without mixing to allow the target cells to adhere to the bottom of the main well, the main well being separated from the secondary wells of the well unit of the separation well microplate by at least one enclosed microchannel, the enclosed microchannel comprising a removable barrier; introducing one or more feeder cells into the secondary well and incubating initially without mixing in liquid culture medium to allow the feeder cells to adhere to the bottom of the secondary well; removing the removable barrier to open the at least one closed microchannel; shaking the separation well microplate to allow gravity flow through the opened microchannels to mix and exchange media between the primary well and the secondary well; allowing the target cells to detach from the main well; and The detached target cells are allowed to settle in the main well away from the opened microchannel.

2. The method according to claim 1, further comprising mixing the detached target cells and allowing the detached target cells to further settle in the main well away from the opened microchannel; washing the settled target cells; and The washed target cells are collected by a liquid handler.

3. The method according to any one of claims 1 or 2, wherein introducing one or more target cells into the main well and introducing one or more feeder cells into the secondary well are performed simultaneously or substantially simultaneously.

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

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

6. The method of claim 1, wherein the detaching comprises exposing the target cells to a dissociation agent to detach the target cells without detaching the feeder cells, optionally wherein the dissociation agent comprises EDTA.

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

8. The method according to 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. The method according to any one of claims 1 to 9, wherein the target cells are embedded in hydrogel domes disposed in a liquid culture medium in the main well of the well unit, optionally wherein the method further comprises disrupting the hydrogel domes prior to detaching the target cells.

11. The method according to any one of claims 1 to 10, wherein the at least one closed microchannel is located between a bottom surface of the well unit and a bottom of a shared side wall of the primary well and the secondary well.

12. The method of any one of claims 1 to 11, wherein the height of the at least one microchannel is in the range of about 10 microns to about 100 microns or about 10 microns to about 75 microns; optionally, wherein the width of the microchannel is in the range of about 150 to about 500 microns, about 200 to about 400 microns, or about 300 microns.

13. A method for quantifying secreted factors from cultured target cells, comprising performing kinetic measurements over time in a single well microplate, the method comprising: During an initial incubation period, culturing the target cells in a liquid culture medium in a main well of a well unit of a separation well microplate, the main well being separated from a secondary well of a well unit of the separation well microplate by at least one enclosed microchannel, the microchannel comprising a removable barrier, the secondary well comprising an immobilized capture reagent; After an initial incubation period, removing the removable barrier to open the at least one closed microchannel, thereby allowing the secreted factor to diffuse between the primary pore and the secondary pore through the opened microchannel; allowing the secreted factor to be captured by an 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 The first labeled complex is quantified using an ELISA reader.

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

15. The method according to claim 14, further comprising: Prior to removing the removable barrier, a primary antibody conjugate is added to the secondary well with avidin-immobilized capture reagent, wherein the primary antibody conjugate comprises a secreted factor-specific primary antibody conjugated to biotin.

16. The method according to claim 14, further comprising: Prior to removing the removable barrier, a secondary antibody conjugate is added to the secondary well with the biotin-immobilized capture reagent, wherein the secondary antibody conjugate comprises a secreted factor-specific primary antibody conjugated to avidin.

17. The method according to any one of claims 13 to 16, further comprising: adding a labeled secondary antibody specific for the captured secreted factor, the immobilized capture reagent, or the primary antibody at a second time point to form a second labeled complex; and quantifying the second labeled complex using an ELISA reader; Optionally also include: adding a labeled secondary antibody specific to the captured secreted factor, the immobilized capture reagent, or the primary antibody at a third time point to form a third labeled complex; and The third labeled complex is detected using an ELISA reader.

18. The method according to any one of claims 13 to 15, wherein the quantification comprises multiplexed detection by imaging and uniform detection using the microplate reader.

19. The method according to any one of claims 13 to 19, wherein the diffusion of the secreted factor comprises shaking the separation well microplate to allow the secreted factor to flow by gravity through the opened microchannel to diffuse between the main well and the sub-well.

20. The method according to 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 tumoroid.

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

22. The method of claim 20 or 21, wherein the cultured organoids are embedded in hydrogel domes in liquid culture medium within the main 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 located within or adjacent to the microchannel.

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

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

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

27. The method according to 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. A method for isolating a single cell type from a multicellular target cell culture in a single separation well microplate, the method comprising: culturing the multicellular target cells in a liquid culture medium in a main well of a well unit of the separation well microplate during an initial incubation period, the main well being separated from a secondary well of a well unit of the separation well microplate by at least one enclosed microchannel, the enclosed microchannel comprising a removable barrier, the secondary well comprising a surface functionalized with an immobilized capture reagent; dissociating the multicellular target cells in the master well to form a mixture of single cells comprising a single cell type and non-target cells; removing the removable barrier to open the microchannel, thereby allowing a mixture of single cells to travel through the opened microchannel to the secondary well and allowing the single cell type to bind to the immobilized capture reagent in the secondary well to form an immobilized complex; Washing the main well and the secondary well to remove unbound non-target cells; and The single cell types are isolated, which includes dissociating the immobilized complexes.

29. The method according to claim 28, further comprising: The isolated single cell types were reseeded in the main wells of the separate well microplate.

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

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

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

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. The method of claim 33, further comprising: Prior to removing the removable barrier, a primary antibody conjugate is added to the secondary well with the avidin-immobilized capture reagent, wherein the primary antibody conjugate comprises a primary antibody specific for a single cell type surface marker conjugated to biotin.

35. The method of claim 33, further comprising: Prior to removing the removable barrier, a secondary antibody conjugate is added to the secondary well with the biotin-immobilized capture reagent, wherein the secondary antibody conjugate comprises a primary antibody specific for a single cell type surface marker conjugated to avidin.

36. The method of any one of claims 28 to 35, wherein the multicellular target cells are cultured in hydrogel domes in the liquid culture medium within the main well, optionally wherein the method further comprises disrupting the hydrogel domes prior to dissociating the multicellular target cells.

37. A method for isolating stem cells from a multicellular target cell culture in a single separation well microplate, the method comprising: culturing multicellular target cells in a first hydrogel dome in a liquid culture medium in a main well of a well unit of the separation well microplate during an initial incubation period, the main well being separated from a secondary well of a well unit of the separation well microplate by at least one enclosed microchannel, the enclosed microchannel comprising a removable barrier; destroying the first hydrogel dome; dissociating the multicellular target cells in the main well to form a mixture of single cells comprising the stem cells and non-target cells; adding magnetic beads containing an immobilized capture agent specific for the stem cells to the main wells, and further incubating to allow the magnetic beads to bind to the stem cells; applying a magnet below the main 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 culture medium through the secondary wells to remove unbound non-target cells; adding a second hydrogel dome to the magnetic bead-stem cell complex retained in the main well after washing; removing the magnet from beneath the main aperture; dissociating the stem cells from the magnetic beads; and A magnet is applied over the main well to float the magnetic beads and isolate the stem cells in the second hydrogel dome.

38. The method of claim 37, wherein Removing the removable barrier to open the microchannel and washing the retained magnetic bead-stem cell complexes by aspirating liquid culture medium through the secondary aperture to remove unbound non-target cells further comprises: removing the magnet from beneath the main aperture; Transfer the magnetic bead-stem cell complexes retained after washing to a new master well in a new well unit of a new separation well microplate; and Reapply the magnet under the new primary well in a new well cell of a new separation well microplate.

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

40. A method for selecting a therapy for cancer treatment for a subject in need thereof, the method comprising: During an initial incubation period, culturing multicellular target cells derived from a subject in a liquid culture medium in a main well of a well unit of a separation well microplate, the main well being separated from a secondary well of a well unit of the separation well microplate by at least one enclosed microchannel, the enclosed microchannel comprising a removable barrier, the secondary well comprising a candidate cancer therapeutic immune cell, monoclonal antibody, immune system modulator, or anti-tumor agent; During a second incubation period, removing the removable barrier to open the at least one closed microchannel to allow diffusion between the primary pore and the secondary pore through the opened microchannel; After the second incubation period, imaging the secondary wells and optionally the primary wells to quantify cell migration or multicellular target cell growth; and Candidate therapies are selected based on the following factors: The migration of immune cells within the secondary well or between the secondary well and the main well is increased compared to a control well unit without multicellular target cells; or The multicellular target cell growth in the master wells is reduced compared to control wells not treated with the candidate therapy.

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

42. The method of claim 40 or 41, wherein the multicellular target cells are derived from a biopsy sample or tumor-like fragments of a subject.

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

44. An automated method for disrupting a hydrogel dome, the method comprising: culturing first cells in the hydrogel domes in liquid culture medium in the wells of the microplate for an initial incubation period to provide multicellular target cells; After the initial incubation period, piercing the hydrogel dome with a pipette tip associated with a liquid handler at or near the x, y center of the dome in the well; moving a pipette tip through the dome in a plurality of steps to release the target cells from the hydrogel dome, each step comprising disrupting the hydrogel dome at an X, Y predetermined location within the well, and optionally liquefying the hydrogel dome; and Optionally, one or more of the target cells are isolated from the well.

45. The method of claim 44, wherein the liquid handler aspirates the pipette tip at one or more, two or more, a plurality of, or each predetermined location within the hydrogel dome.

46. ​​The method of claim 44 or 45, wherein calculating each of the X, Y predetermined positions within the aperture comprises: X(number of steps) = X hole center + α / step * number of steps * cos(θ / step * number of steps); and Y(number of steps) = Y hole center + α / step * number of steps * SIN(θ / step * number of steps), where α = dome diameter (mm) / 1.8; Steps = the number of steps to move the pipette tip through the hydrogel dome, optionally an integer from 2 to 20; X hole center = the x position of the center of the dome in the hole; Y hole center = the y position of the center of the dome inside the hole; and θ = a constant value between 5 and 50.

47. The method according to any one of claims 44 to 46, wherein the well is a main well of a well unit of a separation well microplate.

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

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

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

51. The method of any one of claims 44 to 49, wherein the predetermined 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 sawtooth pattern.

52. The method of any one of claims 44 to 51, wherein between each of the positions the pipette tip is dragged beneath the surface of the hydrogel dome.

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

54. The method of any one of claims 44 to 53, wherein the multicellular target cells are selected from the group consisting of spheroids, tumoroids, and organoids.

55. The method according to any one of claims 44 to 54, further comprising: Prior to separation, the multicellular target cells are dissociated to provide single target cells.

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