Method and system for culturing cells in medium exchange wells

Abstract

Methods, systems, and apparatus for culturing cells in medium exchange wells. In an exemplary method of cell culture, a device (50) may be selected that includes a row of wells (52) and a first reservoir (56) and a second reservoir (58) located at opposite ends of the row of wells (52). Each well (52) may have a lower portion and an upper portion. The lower portion of each well (52) of at least two of the wells (52) may contain a cell culture in contact with a liquid. Liquid may be transferred between the first reservoir (56) and the second reservoir (58) at least partially along a flow path (62) defined by the device and extending through the upper portion of each well (52) of the row of wells (52) so that medium molecules are exchanged between or within the at least two wells (52).

Description

[Background technology]

[0001] (Related Applications) This application claims priority to U.S. Provisional Application No. 62 / 882,363, filed Aug. 2, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] There is a pressing need to increase the biological relevance of cell-based assays. To meet this need, researchers have turned to organoids for increased interest. Organoids are three-dimensional populations of different types of cells that are produced in vitro and have certain similarities to organs, such as exhibiting realistic histology of organ-specific tissues. A population of cells can be generated by seeding a small number of stem cells onto a matrix, such as a hydrogel. The stem cells then proliferate, differentiate, and self-organize within the matrix, using the matrix as a scaffold.

[0003] Organoids resembling tissues from the brain, heart, intestine, kidney, liver, and stomach, among others, have been generated in "organ-on-a-chip" systems. In some cases, multiple different types of organoids, such as organoids representing the brain, heart, and liver, are cultured in the same device under conditions of medium exchange. This creates a "body-on-a-chip" system in which a set of organoids representing an organ system can interact with each other at a distance via signaling molecules. Both organ-chip and body-chip systems have produced promising results, thereby driving a fundamental shift from animal experiments to three-dimensional (3D) cell-based models for studying biological processes, modeling diseases, and testing drugs. These 3D cell-based models are attractive to researchers because they can reduce hands-on time and costs for experiments. However, currently available techniques for organoid culture are typically highly complex, not very reproducible, often not compatible with automation, and poorly characterized.

[0004] New methods, systems, and devices are needed to cultivate separate cell cultures under conditions of medium exchange and to allow interaction between the cultures. Summary of the Invention [Means for solving the problem]

[0005] The present disclosure provides a method, system, and apparatus for culturing cells in a medium exchange well. In an exemplary method of cell culture, a device may be selected that includes a row of wells and a first and a second reservoir located at opposite ends of the row of wells. Each well may have a lower portion and an upper portion. The lower portion of each well of at least two of the wells may contain a cell culture in contact with a medium. Liquid may be transferred between the first and second reservoirs at least partially along a flow path defined by the device and extending through the upper portion of each well of the row of wells such that molecules of the medium are exchanged between or within the at least two wells. The present invention provides, for example, the following items. (Item 1) 1. A method of cell culture, the method comprising: (a) selecting a device (50) including a row of wells (52) and a first reservoir (56) and a second reservoir (58) located at opposite ends of the row of wells (52), each well (52) having a lower portion (70) and an upper portion (68), the lower portion of each well (52) of at least two of the wells (52) including a cell culture in contact with a liquid (104); (b) transferring the liquid (104) between the first reservoir (56) and the second reservoir (58) at least partially along a flow path (62) defined by the device (50) and extending through the upper portion of each well (52) of the row of wells (52), whereby the liquid (104) is exchanged between or among the at least two wells (52); A method comprising: (Item 2) 2. The method of claim 1, wherein the flow path (62) extends through a plurality of passages (64), each passage (64) creating fluid communication between upper portions of a pair of adjacent wells (52) in the row of wells (52) or between an upper portion of one of the reservoirs and the upper portion of an adjacent well (52) in the row of wells (52). (Item 3) 2. The method of claim 1, wherein transferring the liquid (104) includes generating turbulence within the lower and upper portions of each well (52) in the row of wells (52) to promote in-well mixing of the liquid. (Item 4) 2. The method of claim 1, wherein transporting the liquid (104) comprises using gravity to drive the liquid flow. (Item 5) 5. The method of claim 4, wherein driving liquid flow comprises tilting the device (50) and raising the first reservoir (56) relative to the second reservoir (58) such that the liquid (104) flows along the flow path (62) from the first reservoir (56) to the row of wells (52) and from the row of wells (52) to the second reservoir (58). (Item 6) 6. The method of claim 5, wherein transferring the liquid (104) comprises agitating the device (50) such that gravity drives flow of the liquid (104) alternately in opposite directions along the flow path (62) between the first reservoir (56) and the second reservoir (58). (Item 7) 2. The method of claim 1, further comprising introducing at least one three-dimensional population of cells into at least one of said at least two wells (52). (Item 8) A device (50) for cell culture, said device (50) comprising: (a) an array of wells (52), each having a lower portion and an upper portion, the lower portion being configured to contain a cell culture; (b) a first reservoir (56) and a second reservoir (58) located at opposite ends of the row of wells (52); Equipped with The device (50) defines a flow path (62) for liquid, the flow path (62) extending from the first reservoir (56) to the second reservoir (58) through the row of wells (52) and passing through the upper portion of each well (52) in the row of wells (52). (Item 9) 9. The device (50) of claim 8, wherein the lower portion of each well (52) is configured to receive liquid only through the upper portion of the well (52). (Item 10) 9. The device (50) of claim 8, wherein one or more of the wells (52) in the row of wells (52) have a flat bottom on the inside of the one or more of the wells (52). (Item 11) 9. The device (50) of claim 8, wherein the lower portion of at least one well (52) in the row of wells (52) includes a transparent lateral optical window (90) having outer and inner surfaces that are planar, each of the outer and inner surfaces being parallel to the longitudinal axis (60) of the device (50). (Item 12) 9. The device (50) of claim 8, wherein at least one well (52) in the row of wells (52) has a concave inner surface area at the bottom of the well (52). (Item 13) Item 13. The device (50) of item 12, wherein the concave inner surface region is concave in a vertical plane that includes a longitudinal axis (60) defined by the row of wells (52). (Item 14) Item 13. The device (50) of item 12, wherein the concave inner surface region is concave in a vertical plane perpendicular to a longitudinal axis (60) defined by the row of wells (52). (Item 15) 9. The device (50) of item 8, further comprising a lid (100) configured to cover the row of wells (52), the first reservoir (56), and the second reservoir (58). (Item 16) 9. The device (50) of claim 8, wherein each well (52) of the one or more wells (52) has a bottom outer surface region that is flat or rounded in a pair of mutually perpendicular vertical planes. (Item 17) 10. An apparatus comprising a frame (42) for removably holding a device (50) according to item 8 in alignment with another device (50). (Item 18) 18. The device of item 17, wherein the center-to-center spacing of wells (52) within each row (52) of wells is the same as the center-to-center spacing of wells (52) between adjacent rows (52) of wells. (Item 19) 20. The apparatus of claim 17, further comprising a lid (100) configured to fit over at least one of the devices (50) and cover the at least one well (52) of the device (50). (Item 20) 20. The apparatus of claim 19, wherein the lid (100) is configured to cover all of the wells (52) and each of the reservoirs of the device (50). [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 is an isometric view of an exemplary cell culture device including multiple replicas of culture vessels held in alignment with one another by a standard microplate-format frame, each replica of the culture vessel defining a flow path for liquid through the upper region of a row of communicating wells, allowing exchange of medium, and in particular molecular and / or cellular products therein, between two or more of the wells containing respective cell cultures. [Diagram 2] FIG. 2 is an isometric view of one of the replicated culture vessels of FIG. 1 taken in isolation. [Diagram 3] FIG. 3 is a top view of the culture vessel of FIG. [Figure 4] FIG. 4 is a side view of the culture vessel of FIG. [Diagram 5] 5 is a cross-sectional view of the culture vessel of FIG. 2 taken generally along line 5-5 of FIG. [Figure 6] 6 is another cross-sectional view of the culture vessel of FIG. 2 taken generally along line 6-6 of FIG. [Figure 7] 7 is yet another cross-sectional view of the culture vessel of FIG. 2 taken generally along line 7-7 of FIG. [Figure 8] FIG. 8 is a diagram of a selected side of an exemplary light sheet microscopy setup for capturing images of cells contained within one of the wells of the culture vessel of FIG. [Figure 9] FIG. 9 is a cross-sectional view of the culture vessel of FIG. 2 as obtained in FIG. 5 where the culture vessel is oriented horizontally, each well and first reservoir of the device holds a separate volume of medium, and each well contains a separate 3D cell culture. [Figure 10] FIG. 10 is another cross-sectional view of the culture vessel of FIG. 2 as taken in FIG. 9, except that the culture vessel is supported at an angle by the rocking platform after net flow of liquid through the row of wells from the first reservoir to the second reservoir of the culture vessel. [Figure 11] FIG. 11 is yet another cross-sectional view of the culture vessel of FIG. 2 generally as taken in FIG. 10, except that the culture vessel is supported at a fixed incline to drive the flow of liquid from the first reservoir to the second reservoir using gravity, and a pump is utilized to return fluid from the second reservoir to the first reservoir at least partially via an external flow path. [Figure 12] FIG. 12 is a flow chart of exemplary steps that may be performed in a method of cell culture using any of the culture vessels of the present disclosure. [Figure 13]FIG. 13 is a diagram of another exemplary culture vessel defining a flow path for liquid, such as medium, through an upper region of a row of wells. [Figure 14] FIG. 14 is a side view of the culture vessel of FIG. [Figure 15] 15 is a cross-sectional view of the culture vessel of FIG. 13 taken generally along line 15-15 of FIG. [Figure 16] FIG. 16 is a diagram of an exemplary culture vessel defining a serpentine flow path for liquid, such as medium, through the upper region of a row of wells. [Figure 17] FIG. 17 is a side view of the culture vessel of FIG. [Figure 18] 18 is a cross-sectional view of the culture vessel of FIG. 16 taken generally along line 18-18 of FIG. [Figure 19] FIG. 19 is a top view of the culture vessel of FIG. 16 showing the serpentine flow path. [Figure 20] 20 is a cross-sectional view of the culture vessel of FIG. 16 taken generally along line 20-20 of FIG. [Figure 21] FIG. 21 is a fragmentary longitudinal cross-sectional view of a row of wells from another exemplary culture vessel defining a flow path for liquid, such as medium, through an upper region of the row of wells, the cross-section of which corresponds to that of FIG. 5. [Figure 22] 22 is a cross-sectional view of one of the wells of the culture vessel of FIG. 21 taken generally along line 22-22 of FIG. [Figure 23] FIG. 23 is a fragmentary, partial longitudinal cross-sectional view of a row of wells from another exemplary culture vessel defining a flow path for liquid, such as medium, through an upper region of the row of wells, the cross-section of which corresponds to that of FIG. 21. [Figure 24] 24 is a transverse partial cross-sectional view of one of the wells of the culture vessel of FIG. 23 taken generally along line 24-24 of FIG. [Diagram 25] 25 is a fragmentary longitudinal cross-sectional view of a row of wells from another exemplary culture vessel defining a flow path for liquid, such as medium, through an upper region of the row of wells, the cross-section of which corresponds to that of FIG. 21. [Figure 26]26 is a cross-sectional view of one of the wells of the culture vessel of FIG. 25 taken generally along line 26-26 of FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] The present disclosure provides a method, system, and device for culturing cells in a medium exchange well. In an exemplary method of cell culture, a device may be selected that includes a row of wells and a first and a second reservoir located at opposite ends of the row of wells. Each well may have a lower portion and an upper portion. The lower portion of each well of at least two of the wells may contain a cell culture in contact with a medium. A liquid may be transferred between the first and second reservoirs at least partially along a flow path defined by the device and extending through the upper portion of each well of the row of wells, thereby causing molecules of the medium to be exchanged between or within the at least two wells.

[0008] The methods, systems, and devices disclosed herein have at least some advantages over known culture methods and systems. The culture vessels of the present disclosure define a flow path through the upper portion of each well instead of the lower portion. The flow path is spaced from, and thus avoids, the cell culture located at the bottom of the well. Thus, there is less chance of unwanted migration of cells to different wells that would contaminate the cell culture. Furthermore, the flow path does not require elongated microfluidic channels that can become clogged with cells or debris. Furthermore, the use of the flow path can generate sufficient turbulence to exchange medium and / or molecules and / or cell products therein within the lower portion of the well without disturbing the cell culture therein.

[0009] Replica culture vessels can be held by a frame to produce plates having a microplate format of wells, and microplate handling robotics and fluidics can be utilized to provide automated operation and substantially higher throughput than with known culture vessels with medium exchange wells.

[0010] Further advantages are provided by the design of the wells and / or reservoirs. For example, the wells may have one or more planar side windows optimized for light sheet fluorescence microscopy, facilitating imaging of sub-millimeter 3D cell cultures such as spheroids or organoids with diameters of about 100-600 micrometers in one or more of the wells. The shape of the wells may differ from traditional U-shaped or V-shaped wells. Each well may have quasi-vertical planar sidewalls in the lower portion of the well. The sidewalls may be transparent to allow light sheet illumination. Alternatively or additionally, each of the reservoirs may be configured to substantially flow into the row of wells on its own at a relatively small incline of the culture vessel.

[0011] This disclosure provides assay designs and consumables for Organ-on-a-Chip and / or Body-on-a-Chip models, either as hardware alone, with detailed protocols, or as plates prepared with live cells and ready for compound testing. These methods and systems can provide valuable information related to the biological model of choice, are compatible with imaging and automation, and are easy to set up.

[0012] The present disclosure also provides a culture vessel suitable for organ-on-chip and body-on-chip assays that are compatible with imaging systems, plate readers, and other instruments. The culture vessel may have wells with shapes (e.g., U-shaped or V-shaped) for forming spheroids / organoids. The culture vessel may contain spheroids or organoids up to 300-600 μm in size (or larger) that may be formed by introducing appropriate primary or induced pluripotent stem cell (iPSC)-derived cells into the wells with or without a preformed substrate already present in the wells. The introduced cells may form organoids in, for example, 2-4 days while the wells are liquid-isolated from each other (i.e., no medium exchange occurs between or within the wells). At any desired time, the culture vessel allows interaction of different cell types with each other by medium exchange (i.e., exchange of medium components such as molecules and / or cell products between / in the wells). Liquid flow for medium exchange between or within the wells may be driven by gravity flow (e.g., using a rocking platform) and / or with a pump. (Cells can be grown in culture vessels while supported by a rocking platform inside an incubator.) Examples of interacting cells include liver-heart models, liver-heart-brain models, tumor-liver models, gut-liver-kidney models, or other multi-organ models in any desired format and combination.

[0013] The culture vessel can be a strip of wells connected to each other by channels. When tilted, medium flows from a reservoir to a first well, a second well, etc., until all excess medium is collected in another reservoir at the opposite end of the strip.

[0014] Culturing cells with medium exchange as disclosed herein allows for the investigation of interactions between different (normal / transformed) cell types or tissues cultured in 2D or 3D formats, respectively. These interactions can be mediated by the exchange of cells and / or cell products transported within the liquid, including, but not limited to, cytokines, chemokines, growth factors, lipids, circulating DNA / RNA, eukaryotic cells, bacteria, and / or viruses.

[0015] Exemplary studies that can be performed using the culture vessels and culture methods disclosed herein are listed below, without limitation.

[0016] (1) Liver and cardiac spheroids can be cultured with medium exchange in different wells of the same culture vessel. Compounds that do not produce a direct effect on the cardiac spheroids can be tested for cardiac effects resulting from the metabolism of the compound by the liver spheroids into potentially active derivatives.

[0017] (2) Liver and kidney cells or tissues can be cultured with medium exchange in the same culture vessel to study drug metabolism.

[0018] (3) Heart, liver, and neuronal cells can be cultured with medium exchange in the same culture vessel to study the toxic effects of chemicals.

[0019] (4) Tumor cells and normal liver, nerve, and cardiac cells can be cultured with medium exchange in the same culture vessel to study the effects of anticancer drugs and associated toxicities.

[0020] (5) Vascular cells and cardiac cells (or other tissues) can be cultured with medium exchange in the same culture vessel to study inflammation and its effects in different tissues.

[0021] (6) The effects of bacteria or viruses on different tissues can be investigated.

[0022] (7) The effects of T cells or other immune cells on normal cells can be investigated.

[0023] (8) The effects of T cells or other immune cells on malignant tissues can be studied.

[0024] (9) More than four tissue types representing fundamental tissues can be cultured with medium exchange according to the “body-on-a-chip” concept.

[0025] Further aspects of the present disclosure are described in the following sections: (I) Definitions, (II) Overview of Systems, Devices, and Methods, (III) Examples, and (IV) Selected Aspects.

[0026] (I. Definition) The technical terms used in this disclosure have meanings commonly recognized by those of ordinary skill in the art. However, the following terms may be further defined as follows:

[0027] (Cell) - the basic structural, functional, and biological unit of an organism. Cells can be eukaryotic or prokaryotic. Exemplary cells include stem cells, established cells (e.g., cell lines), primary cells, cells of a tissue sample, transfected cells, cells from a clinical sample (e.g., blood samples, fluid aspirates, tissue sections, etc.), and the like.

[0028] (Cell Culture) - A set of cells cultured together in the same compartment, such as in the same well. The set of cells may be in contact with and surrounded by one and / or multiple media.

[0029] A 2D (two-dimensional) cell culture is a cell culture with its cells arranged at least primarily along the surface of a compartment such as a well, and optionally attached to the surface. A 2D cell culture typically has an average thickness measured perpendicularly (e.g., vertically) to the surface that is only a small fraction of the length and width of the cell culture measured along the surface (e.g., horizontally). The average thickness can be about one or two cells or less. The surface of the compartment can be planar or concave, among others. A 2D cell culture can be a monoculture (i.e., containing substantially only one type of cell) or a coculture (i.e., containing two or more different types of cells).

[0030] 3D (three-dimensional) cell culture is a cell culture that forms a three-dimensional cluster of cells. The average thickness of the 3D cell culture that is measured perpendicularly (e.g., perpendicularly) to the adjacent support surface can be at least 5 or 10 cells, among others. The cluster can be described as an aggregate. Exemplary 3D cell cultures include spheroids and organoids. Thus, 3D cell cultures can be a monoculture of a single cell type or a coculture of different cell types.

[0031] (Cell Product) - any molecule, molecular complex, macromolecular assembly, cellular organelle, or particle produced by at least one cell. Cell products may be released into the liquid surrounding the cell culture by any suitable process, such as secretion (e.g., exocytosis), budding, cell lysis, etc. Cell products and / or cells may be exchanged between / within the wells.

[0032] (cell culture) - Promoting the survival, health, growth, and / or proliferation of living cells within an artificial environment.

[0033] (Culture vessel) - a device for the cultivation of at least one cell culture or any series of cell cultures. The devices disclosed herein can be single-use devices (consumables) or can be reused.

[0034] (Light) - Optical radiation, including ultraviolet radiation, visible radiation (i.e., visible light), and / or infrared radiation.

[0035] (Culture medium) - an aqueous composition for culturing cells. The composition may be liquid or semi-solid. The composition may include, among others, a carbon source (e.g., glucose), inorganic salts, vitamins, and growth regulators. The term "culture medium" as used herein means at least one culture medium and may refer, for example, to separate volumes of culture medium, a first medium and a second medium of different composition, medium of substantially the same composition in contact with different / separate cell cultures, or a combination / mixture of previously isolated volumes of the same medium.

[0036] (Organoid) - a three-dimensional aggregate of different types of cells produced in vitro that has certain similarities to organs, such as exhibiting realistic histology of organ-specific tissues. A population of cells can be generated by seeding a small number of stem cells onto a matrix, such as a hydrogel. The stem cells then proliferate, differentiate, and self-organize within the matrix, using the matrix as a scaffold.

[0037] (Spheroid) - A three-dimensional collection of a single type of cell.

[0038] II. Overview of Systems, Apparatus, and Methods This section provides an overview of systems, devices, and methods for culturing cells in wells that are structurally in communication with one another and allow for fluid sharing between the wells in a controllable manner via gravity-driven and / or pump-driven flow (see Figures 1-12). Further aspects of systems, devices, and methods that may be suitable are described elsewhere herein, such as before Section I and in Sections I, III, and IV.

[0039] FIG. 1 shows an exemplary cell culture device 40 (also called a plate) including a frame 42 and replicas of a culture vessel 50 having wells 52 in communication with one another. FIGS. 2-7 show a single, isolated culture vessel 50. The frame 42 can be configured to removably hold the replicas of the culture vessel 50 in alignment with one another to form a rectangular array of wells 52. The rectangular array can have standard ANSI / SBS microplate well spacing (e.g., 18, 9, or 4.5 mm center-to-center well spacing). Each culture vessel 50 can have a row 54 of at least two, three, four, or five wells 52 for containing at least two different cell cultures, among others. In the depicted embodiment, the row 54 has eight wells 52. The wells 52 may (or may not) be uniformly spaced from one another within the row 54 and may (or may not) be substantially uniform in size and / or shape.

[0040] First and second reservoirs 56, 58 may be located at opposite ends of the row 54 for fluid communication with adjacent wells 52. The reservoirs 56, 58 may be structured as wells, as shown here, and thus may be fluid-loadable from above using a pipette. Thus, in some embodiments, the wells 52 may be described as cell wells and the reservoirs 56, 58 as reservoir wells. The size and / or shape of the reservoirs 56, 58 may differ from the wells 52. For example, the reservoirs 56, 58 may have a larger fluid holding capacity than the wells 52 and / or may be configured to drain more efficiently and / or completely, as described below. In the plate of FIG. 1, each reservoir 56, 58 replaces two wells of a standard microplate format (e.g., a 96-well microplate, forming an 8×12 array of wells). In other examples, each reservoir may replace only one or at least three wells of a standard microplate format.

[0041] The culture vessel 50 may be elongated so as to define a longitudinal axis 60, which may be linear (see Figures 1 and 2). The wells 52 and reservoirs 56, 58 may be arranged along the longitudinal axis 60. Thus, the culture vessel may be described as a strip.

[0042] A flow path 62 for fluid progression between wells 52 is defined by culture vessel 50. The flow path may be formed by a channel. In the depicted embodiment, flow path 62 extends through row 54 of wells 52 from first reservoir 56 to second reservoir 58, or vice versa. Flow path 62 may at least generally follow longitudinal axis 60. In the depicted embodiment, flow path 62 lies on longitudinal axis 60. In other embodiments, flow path 62 may be parallel to but offset from longitudinal axis 60, linear but not parallel to longitudinal axis 60, or non-linear (serpentine, etc. (see Example 2)), among others.

[0043] The flow paths 62 may extend through passages 64 (synonymously referred to as openings) that allow the wells 52 to communicate with each other and with the reservoirs 56, 58 (see Figs. 2, 3, and 5-7). Each passage 64 may provide fluid communication between an adjacent pair of wells 52, or between a well 52 at one end of a row 54 and a reservoir 56 or 58. The passages 64 may be located in an upper region 66 of the culture vessel 50 in an upper portion 68 of the well 62, and the cells may be contained within a lower portion 70 of the well 62. This arrangement allows for control of the flow of liquid between the wells 52 and / or between the row 54 and the reservoirs 56, 58. For example, liquid progression can be controlled by adjusting the level of liquid in one or more of the wells and / or reservoirs and / or by adjusting the orientation of the vertical axis 60 (e.g., from horizontal to inclined, or vice versa). The lower portion of each well may be referred to as a cell-receiving portion, and the upper portion of each well may be referred to as an exchange portion.

[0044] The passages 64 may have any suitable location, size, and shape. Each passage 64 may be formed in the wall 72 between adjacent wells 52 or between a well 52 and one of the reservoirs 56, 58. The passages may extend to the upper edge 74 of the wall 72 and / or to the upper edge 76 of the well 62 and / or reservoir 56, 58 to form a recess 78, such as a slot, in the upper surface 80 of the culture vessel 50. Alternatively, each passage 64 may be positioned below and spaced apart from the upper edge 74, upper edge 76, and upper surface 80. The passages 64 may have a passage width that is substantially less than the well width of an adjacent well 52, each measured parallel to a horizontal transverse axis 82 (see FIG. 3). For example, the passage width may be less than about ½, ⅓, or ¼ of the well width. Narrow passages 64 may advantageously promote more turbulent flow of the liquid, facilitating more mixing of the liquid within each well 52 and thus more efficient exchange of, among other things, cellular products, additives (e.g., reagents and / or test compounds), and / or suspended cells between wells 52. Each passage 64 may or may not be elongated at least approximately vertically (i.e., within 45 degrees of exactly vertical). Passages 64 may be disposed along a straight line (e.g., longitudinal axis 60 in the depicted embodiment) or at least a portion of the passage may be offset laterally from a straight line to promote more turbulence (see Example 2).

[0045] Each well 52 may have any suitable shape. The well may narrow stepwise and / or taper in a vertical longitudinal plane (including the longitudinal axis 60) and / or in a vertical transverse plane (parallel to the transverse axis 82) as it extends downward between the top and bottom of the well. This narrowing may be configured to encourage the population of introduced cells at the bottom of the well to form a 3D cell culture. In some embodiments, the well may taper with the same constant slope in the vertical longitudinal and transverse planes to produce a lower portion 70 having a conical (e.g., frustoconical) shape (see, e.g., Example 3). In some embodiments, the well may taper with a decreasing slope on one or both of these surfaces to form a lower portion 70 having a concave surface 84 that is concave in the vertical longitudinal plane (see FIG. 5) and / or transverse plane. Thus, the bottom interior surface region 86 (i.e., the floor surface) of the well can be flat (see, e.g., Example 3) or non-planar, such as rounded (on one or both sides) (see, e.g., Figures 5 and 7, and Examples 1, 3, and 5). Similarly, the outer bottom surface region 88 opposite the interior surface region 86 can be flat (see, e.g., Examples 1 and 3) or rounded (e.g., convex on one or both sides (see, e.g., Figures 5 and 7, and Examples 1, 2, 3, and 5)). The area of ​​the interior surface region 86 can be much smaller than the interior horizontal top area of ​​the well 52, such as less than about 20%, 10%, or 5% of the top area.

[0046] The geometry of each well 52 may be chosen according to its intended use for cell culture (e.g., for 2D vs. 3D cell culture) and imaging (e.g., conventional imaging vs. light sheet microscopy). The wells 52 of the culture vessel 50 allow for lateral illumination and / or lateral collection (e.g., for light sheet microscopy) at least approximately parallel to the horizontal axis 82. Each well 52 may have at least one or a pair of transparent optical windows 90 in the lower portion 70 formed by the opposing sides 92. Each optical window 90 may be approximately planar and at least approximately vertical (i.e., within exactly 45 degrees of vertical, such as within 30, 20, or 10 degrees of vertical) (see also Example 1). When a pair of transparent optical windows 90 is formed by the wells 52, they may be disposed opposite each other and spaced apart from each other parallel to the horizontal axis 82.

[0047] 8 illustrates a light sheet imaging configuration for capturing images of cells contained within one or more wells 52 of a culture vessel 50. One of the wells 52 may be illuminated with at least one light sheet 94 that enters and is transmitted into the well through one or both optical windows 90. In the depicted embodiment, one well 52 is illuminated from both sides by a pair of horizontal light sheets 94 formed by cylindrical lenses. Light, such as fluorescence induced by the light sheets 94, may be collected with an objective lens 96 located below the well.

[0048] The culture vessel 50 can be configured to be stable and support itself in a horizontal orientation on a horizontal surface (see FIGS. 2, 4, and 6). For example, the culture vessel 50 can have a base portion for contacting the horizontal surface and, optionally, elevating the row 54 of wells 52 above the horizontal surface. In the depicted embodiment, the base portion is formed by a pair of spaced apart feet 98a, 98b (optionally adjacent and / or at least partially formed by the reservoirs 56, 58) located at opposite ends of the culture vessel.

[0049] The culture vessel 50 may be covered with a removable lid 100 (see FIG. 4) that functions like a standard lid for a tissue culture dish. The lid may be placed on top of the culture vessel 50 to cover the row 54 of wells 52 and, optionally, the reservoirs 56, 58, and then removed when fluids are added to or removed from the culture vessel. The lid 100 may be sized to cover only one copy or multiple copies of the culture vessel 50. Thus, all wells 52 of the cell culture device 40 may be covered with a single lid 100 and / or a separate lid may be used to cover each copy of the culture vessel 50 (see also FIG. 1).

[0050] 9 and 10 illustrate an exemplary configuration of a culture vessel 50 containing multiple cell cultures 102 and liquid 104 (e.g., medium) in wells 52. Each cell culture 102 may consist of any suitable number of cells 106 and may contain one, two, or more different cell types, each may be 2D or 3D. Each cell culture 102 may be of a different type, or some of the cell cultures may be replicates (e.g., a well 52 may contain four different types of cell cultures 102 each present twice, two different types each present four times, etc.). Although eight cell cultures 102 are shown here, in some cases, as few as two cell cultures may be used. Thus, before liquid transfer is initiated between the wells, one, two, or more wells 52 may contain liquid 104 but may not contain any cells, or may contain neither liquid nor cells. The liquid 104 may be of essentially the same composition (except for different cell products) prior to liquid transfer within the wells 52, or may be a medium of different composition.

[0051] 9 shows the culture vessel 50 prior to the transfer of liquid 104 in the wells 52. The culture vessel 50 is horizontal and the level of liquid 104 is below each passage 64. Thus, the contents of each well 52 (liquid, cell culture, cell products, etc.) are isolated from the contents of each other well 52 and reservoir 56, 58.

[0052] FIG. 10 shows the culture vessel 50 after the net transfer of liquid 104 from the first reservoir 56 to the second reservoir 58 (compared to FIG. 9). The culture vessel 50 is mounted on a rocking platform 108 that alternately rocks the culture vessel in opposite rotational directions indicated by the double-headed rotational arrows at 110. When the culture vessel 50 is rotated from the horizontal orientation of FIG. 9 to the inclined orientation of FIG. 10, the first reservoir 56 becomes elevated relative to the well 52 and the second reservoir 58, and the liquid 104 enters the passage 64 and flows along the flow path 62 indicated by the representative flow arrows at 112, 114, and 116 (see also FIGS. 2 and 3). More specifically, the liquid 104 flows from the first reservoir 56 to the adjacent well 52, indicated at 112, between the adjacent wells 52, and from the adjacent wells 52 into the second reservoir 58, indicated at 114. The rocking platform 108 may then rotate in the opposite direction, raising the second reservoir 58 above the first reservoir 56, which drives the flow of liquid 104 along the flow path 62 in the opposite direction, thereby returning a volume of liquid 104 to the first reservoir 56. In this process, the suspended / soluble contents of each well 52 are distributed throughout all of the wells, allowing the cellular products of the cell cultures 102 to affect each other (e.g., enhance / inhibit growth, proliferation, differentiation, migration, organization, apoptosis, etc.).

[0053] Each reservoir 56, 58 may have a slope 118 that slopes downward from an adjacent passage 64 at one end of the row 54 (see FIG. 9). The angle of inclination for the slope 118 relative to the horizontal may be, for example, less than about 50, 45, 40, 35, or 30 degrees, among others. The slope 118 may allow each reservoir 56, 58 to be substantially drained of liquid 104 by tilting the culture vessel 50 at least to the slope of the slope of the slope 118 (e.g., as shown in FIG. 10). As a result, liquid flow within the culture vessel 50 can be efficiently driven by the rocking platform 108 using a smaller range of angular motion.

[0054] FIG. 11 (compared to FIG. 10) illustrates a pump-based strategy for returning liquid from the second reservoir 58 to the first reservoir 56. The culture vessel 50 may be supported at a fixed tilt on a tilted platform 120 such that the first reservoir 56 is elevated relative to the second reservoir 58 as depicted. This tilted orientation of the culture vessel 50 encourages gravity-driven flow of liquid 104 along the flow path 62 (see also FIGS. 2 and 2) shown at 112, 114, and 116 from the first reservoir 56 to the second reservoir 58. A pump 122 may be utilized to actively convey liquid 104 from the second reservoir 58 back to the first reservoir 56 via an at least partially external flow path 124 defined by the pump 122 and associated upstream and downstream conduits 126, 128. The liquid flow rate along the exterior flow path 124 driven by pump 122 can substantially match that along the vessel-defining flow path driven by gravity. The tilt of the tilted platform 120 can be adjusted to increase or decrease the flow rate of liquid 104 along the interior flow path 62, if necessary. In some embodiments, the culture vessel 50 can be supported on a horizontal surface instead of a tilted platform when pump 122 is utilized.

[0055] 12 is a flow chart of exemplary steps that may be performed in a method 130 of cell culture using culture vessel 50 or any of the other culture vessels of the present disclosure. The steps may be performed in any suitable order and combination, and may be modified by or combined with any other suitable steps and features disclosed elsewhere herein.

[0056] A device such as culture vessel 50 is shown and may be selected at 132. The device may have any suitable combination of features and characteristics described herein.

[0057] Cells and liquid (i.e., medium) can be introduced into the wells of the device, shown at 134. At least one cell can be introduced into at least two, three, four, five, or more wells of the device. Optionally, a separate volume of the same or different medium containing at least one cell to be introduced can be dispensed into each well. The volume of medium introduced into the wells can be small enough to keep the level of the medium in each well below the flow path defined by the device. In some embodiments, a preformed three-dimensional population of cells, such as preformed spheroids or organoids, can be introduced into one or more wells of the device. In some embodiments, a substrate for organoid formation can also be optionally placed (e.g., formed in) in at least one well in which organoids are to be formed before cells are introduced into the at least one well. The substrate can be located on the inside bottom of the well.

[0058] A liquid may be introduced into one or more reservoirs of the device, shown at 136. The liquid may be culture medium.

[0059] The device and its contents may be incubated, shown at 138. Incubation may be at a suitable temperature for culturing the cells, optionally in a controlled atmosphere. During incubation, the wells and reservoirs of the device may be liquid-isolated from one another. In other words, no liquid is transferred within the wells and reservoirs. Incubation may be performed for any suitable length of time, such as at least 0.5, 1, 2, 3, 4, or more days, to allow the cells to settle, settle, aggregate, grow, divide, etc. Incubation may be performed before or after step 136 above, and may be performed before liquid (e.g., medium) is transferred within the cells and reservoirs.

[0060] Fluid (e.g., medium), shown at 140, can be transferred between (two) wells or within (three or more) wells of the device. Fluid transfer generally drives the flow of liquid in one direction or in alternating directions along a flow path defined by the device. Flow can be driven by gravity (e.g., on a rocking platform), pumps (e.g., device is horizontal or inclined), or both, among others. In some cases, test compounds or other test substances can be added to the wells / reservoirs of the device, and fluid transfer can allow the test compounds / substances to interact with the cells in each of the wells.

[0061] The cells within the device may be imaged and / or assays may be performed on the cells / liquid (e.g., medium) within the device, as shown at 142. For example, conventional imaging, confocal imaging, or light sheet imaging, among others, may be performed to obtain at least one optical or recorded image of at least a portion of the cell culture contained within at least one of the wells. Exemplary instruments for imaging include conventional microscopes, confocal microscopes, light sheet fluorescence microscopes, ImageXpress® Micro or ImageXpress® Pico imaging systems, and the like.

[0062] III. EXAMPLES This section describes further examples of methods, systems, and devices for culturing cells in wells configured to exchange liquid (e.g., medium) in a controllable manner. These examples are intended for illustrative purposes only and should not limit the overall scope of the invention. Aspects and features of these examples may be combined with each other and with those described elsewhere herein, such as in Sections I, II, and IV, in any suitable combination.

[0063] Example 1. Culture vessel for light sheet microscopy This example describes another exemplary culture vessel 150 configured for medium exchange in wells 152 and imaging by light sheet microscopy. See Figures 13-15.

[0064] Culture vessel 150 is related to culture vessel 50 (see, e.g., FIGS. 1-7). For example, culture vessel 150 has a row of U-shaped wells 152 and a first reservoir 156 and a second reservoir 158 at opposite ends of row of wells 152. Passages 164 provide communication between wells 152 and between row of wells 152 and the ends of reservoirs 156, 158.

[0065] Each well 152 may have a flat (planar, horizontal) outer surface area 188 at the bottom of the well (see FIG. 15). At least one or a pair of transparent optical windows 190 may be formed on both sides of the well (see FIGS. 13-15). The optical windows 190 may serve a similar purpose for light sheet illumination as the optical windows 90 of the culture vessel 50 (see, for example, FIG. 8). However, the orientation of the outer window surface 191 may be offset from vertical than the corresponding window surface of the culture vessel 50 and less parallel to the inner window surface 193 (see FIGS. 7 and 15). The orientation of these inner and outer window surfaces allows a light sheet 195 to enter the well from the side and be refracted by the window surfaces such that illumination of the inner bottom of the U-shaped well is possible (see FIG. 15).

[0066] 3D cell populations, such as spheroids or organoids, are most likely to be found near or at the inner bottom of the well. The populations can be very small (100-300 μm). The bottom of the U-shaped well is already elevated about 160 μm from the center to the side wall. The U-shape is advantageous as it can help the cell populations form a sphere.

[0067] Example 2. Culture vessel with serpentine channels This example describes an exemplary culture vessel 250 configured for controllable medium exchange within wells 252 and defining a serpentine flow path 262 between reservoirs 256, 258. See Figures 16-20.

[0068] The culture vessel 250 has passages 264 formed as lateral at least approximately vertical slots. Each passage is formed at least partially in one of the lateral walls 272 in an upper region of the culture vessel 250. The passage is located between a pair of adjacent wells 252 or between the end well 252 and one of the reservoirs 256, 258. Each passage 264 may be an at least approximately vertically elongated slot. The passage may extend to the upper edge of the lateral wall 272 or its upper extent may be spaced below this upper edge. Each passage 264 may be defined cooperatively by the lateral wall 272 and one of the opposing vertical side walls 273 of the culture vessel 250 or completely by the lateral wall 272, as depicted, among others.

[0069] The flow path 262 is non-linear (see FIG. 19, compare with the flow path 62 of the culture vessel 50 of FIG. 2). More specifically, at least a portion of the passage 264 may be offset from a central vertical plane that includes the longitudinal axis defined by the culture vessel 250. In the depicted embodiment, each passage 264 is offset laterally (none are visible in the central cross-sectional view of FIG. 18). The culture vessel 250 may define a serpentine flow path as shown. For example, the passages 264 may be continuously offset in alternating horizontal directions from the central vertical plane. The serpentine flow of medium along the flow path 262 may generate more turbulence than flow along the linear flow path 62 (see FIG. 2), which may enhance medium exchange when driving flow along the flow path between the reservoirs 256, 258 (e.g., by rocking the culture vessel 250).

[0070] Each well 252 of the culture vessel 250 has a rounded shape in the bottom region of the lower portion 270 in the longitudinal vertical plane. In the depicted embodiment, in this bottom region, the inner and outer well surfaces oriented transversely to the longitudinal vertical plane are cylindrical. This rounded shape at the inner bottom of the well may limit the movement of three-dimensional populations, such as spheroids, within the well when the culture vessel is cultured to drive fluid flow.

[0071] Each well 252 has a pair of lateral transparent optical windows 290 opposite those in the lower portion 270, allowing for light sheet illumination (see Figs. 16, 17, and 20). Each optical window 290 has an outer plane 291 and an inner plane 293, each parallel to the longitudinal axis of the culture vessel 250, but inclined relative to one another (see Fig. 20). The pair of inner planes 293 may be inclined relative to one another such that the inner width of the lower portion 270 tapers downward (see Fig. 20). For example, an angle of about 10-20 degrees may be formed between the inner planes 293. This taper in the inner well width in the horizontal vertical plane, together with the rounded inner shape in the vertical vertical plane, may encourage the settling of cells in the medium into a narrow slot formed in the central area of ​​the inner bottom region 286 of each well. This geometry may promote the formation of a three-dimensional cell population.

[0072] The outer plane 291 may be approximately vertical (e.g., less than about 2 degrees from vertical) to minimize refraction of the illumination light at the air-polymer interface. A slight offset from vertical may facilitate removal of the culture vessel from the mold during manufacturing. A larger offset of the inner plane 293 from vertical may be less critical when performing light sheet microscopy due to smaller refractive index changes at the polymer-medium interface inside the well.

[0073] As long as the illumination light sheet hits a nearly vertical surface, all may be well. However, if the light sheet approaches the very bottom inside the well and scans a 3D cell population (e.g., 200 μm spheroids), the light sheet may hit the edge due to the rounded shape of the well, which may generate stray light and adversely affect image quality. Thus, there may be a trade-off for the well geometry between a smaller radius, which creates better conditions for spheroid formation and less movement during rocking, and a larger radius or flat bottom for light sheet microscopy.

[0074] Example 3. Culture vessel with flat bottom wells This example describes an exemplary culture vessel 350 configured for controllable medium exchange in wells 352, where each well has a bottom that is flat on the inside and outside of the well. See Figures 21 and 22.

[0075] The culture vessel 350 may have any of the elements and features described above with respect to other culture vessels. For example, each well 352 may have structural communication with adjacent wells 352 in its row 354 via passages 364. The passages 364 may allow for controllable liquid flow between upper portions 368 of adjacent wells 352.

[0076] When the culture vessel 350 is horizontal, the cells may be contained within a lower portion 370 of each of the wells 352 that is below the passages 364 (at a lower elevation than the passages 364). Each lower portion 370 may be conical as shown (see FIGS. 21 and 22) with a flat bottom wall 371. More specifically, the bottom wall 371 may include a horizontal planar inner bottom region 386 and a horizontal planar outer bottom region 388. The well may taper sharply downward, producing an area of ​​the interior surface region 386 that is much smaller than the horizontal interior area at the top of the well 35, such as only 10%, 5%, or 3% of this top area. For example, the inside diameter at the top of the well may be about 6 mm, and the inside diameter may be less than about 1, 0.5, or 0.25 mm at the bottom of the well. This geometry may concentrate cells on the inner bottom region 386 as they settle, thus encouraging the formation of 3D cell cultures. In other cases, 2D cell cultures may be cultured on the inner bottom region 386. The flat bottom wall 371 allows for conventional imaging configurations such as epifluorescence microscopy, transillumination microscopy, and confocal microscopy, among others.

[0077] Example 4. Culture vessel with plate bonded to body This example describes an exemplary culture vessel 450 configured for controllable medium exchange in wells 452, each well having a bottom 471 that is flat on the inside and outside of the well and is formed by a plate 497 joined to an overlying body 499. See Figures 23 and 24.

[0078] Culture vessel 450 may have any of the elements and features described above with respect to other culture vessels, such as culture vessel 350 (see Example 3). For example, each well 452 may have structural communication with adjacent wells 452 in its row 454 via passages 464. Passages 464 may allow for controllable liquid flow between upper portions 468 of adjacent wells 452.

[0079] When the culture vessel 450 is horizontal, the cells may be contained within a lower portion 470 of each of the wells 452 that is below (at a lower elevation than) the passages 464. Each lower portion 470 may be conical as shown (see FIGS. 23 and 24 ), with a flat bottom wall 471. More specifically, the bottom wall 471 may include a horizontal, planar inner bottom region 486 and a horizontal, planar outer bottom region 488.

[0080] Plate 497 and body 499 may be formed separately from one another and then bonded together. Plate 497 may be flat with planar top and bottom surfaces. The plate may be formed from any transparent optical grade material, generally polymer or glass. Body 499 may define a plurality of compartments 501, precursors to wells 452, prior to attachment to plate 497. Each compartment 501 may be an opening defining a vertical through axis. Compartments 501 may have an open top end and an open bottom before plate 497 is bonded to body 499. The open bottom end of each compartment 501 may be closed by plate 497, which may form a fluid-tight seal with body 499 at the compartment's bottom end to create wells 452.

[0081] An integrally formed culture vessel may have wells with various bottom thicknesses due to undesirable dimensional variations in the corresponding molds used to form the vessels. This lack of consistent bottom thickness may affect the ability to accurately focus on the cells within the wells when imaging. One method to reduce the variability in well bottom thickness is to utilize the same plate 497 (glass or polymer) to form the bottom of each well. The plate 497 may be manufacturable with less thickness variability than the bottom thickness of the wells 452.

[0082] Example 5. Culture vessel with rounded bottom wells This example describes an exemplary culture vessel 550 configured for controllable medium exchange in wells 552, each well having a rounded bottom 571. See Figures 25 and 26.

[0083] The culture vessel 550 may have any of the elements and features described above with respect to other culture vessels. For example, each well 552 may have structural communication with an adjacent well 552 in the row 554 via a passage 564. The passage 564 may allow for controllable liquid flow between upper portions 568 of adjacent wells 552. The lower portions 568 of the wells 552 may be rounded in the bottom region of the well in a pair of mutually orthogonal vertical planes. For example, the bottom inner surface region 586 and the bottom outer surface region 588 of each well 552 may be spherical in shape (i.e., corresponding to a portion of a perfect sphere). In other cases, the bottom inner surface region 586 may be rounded in the orthogonal vertical plane and the bottom outer surface region 588 may be flat and horizontal.

[0084] (IV. SELECTED ASPECTS) This section describes selected aspects of this disclosure as a series of indexed paragraphs.

[0085] A1. A method of cell culture comprising: (a) selecting a device including a row of wells and a first and a second reservoir located at opposite ends of the row of wells, each well having a lower portion and an upper portion, the lower portion of each well of at least two of the wells containing a cell culture in contact with a medium; and (b) transferring a liquid (such as a medium) between the first and second reservoirs at least partially along a flow path defined by the device and extending through the upper portion of each well of the row of wells, such that molecules of the medium are exchanged between or within the at least two wells.

[0086] A2. The method of paragraph A1, wherein the flow path extends through a plurality of passages, each passage creating fluid communication between upper portions of a pair of adjacent wells in the row of wells, or between an upper portion of one of the reservoirs and an upper portion of an adjacent well in the row of wells.

[0087] A3. The method of paragraph A2, wherein at least one passageway (or each passageway) of the plurality of passageways intersects an upper edge of an upper portion of each well of an adjacent pair of wells.

[0088] A4. The method of paragraphs A2 or A3, wherein a wall is shared between upper portions of adjacent pairs of wells in the row of wells, and a passageway in the plurality of passageways extends through the wall.

[0089] A5. The method of any of paragraphs A2-A4, wherein at least one passageway (or each passageway) of the plurality of passageways is formed on a top surface of the device.

[0090] A6. The method of any of paragraphs A2-A5, wherein at least one passageway (or each passageway) of the plurality of passageways is a slot.

[0091] A7. The method of paragraph A6, wherein the slot is vertically elongated.

[0092] A8. The method of any of paragraphs A1-A7, wherein the flow path is non-linear, optionally a tortuous flow path.

[0093] A9. The method of any of paragraphs A1-A8, wherein transferring the liquid includes generating turbulence within lower and upper portions of each well of the row of wells to promote in-well mixing of the liquid.

[0094] A10. The method of any of paragraphs A1-A9, wherein transporting the liquid includes using gravity to drive the flow of the liquid.

[0095] A11. The method of paragraph A10, wherein driving the flow of liquid includes tilting the device and raising the first reservoir relative to the second reservoir so that liquid flows along a flow path from the first reservoir to the row of wells and from the row of wells to the second reservoir.

[0096] A12. The method of paragraph A11, wherein transferring the liquid includes rocking the device such that gravity drives flow of the liquid alternately in opposite directions along the flow path between the first and second reservoirs.

[0097] A13. The method of any of paragraphs A1-A10, wherein the flow path is a first flow path, and transporting the liquid includes moving the liquid from a first reservoir to a second reservoir along the first flow path, and transporting the liquid includes using a pump to move the liquid from the second reservoir to the first reservoir along a second flow path located at least partially outside the device.

[0098] A14. The method of any of paragraphs A1-A13, wherein transferring liquid includes moving one or more replicas of at least one cell product produced by a cell culture in one of the at least two wells to another of the at least two wells.

[0099] A15. The method of paragraph A14, wherein the at least one cellular product comprises a secreted factor (e.g., a cytokine, chemokine, or growth factor), lipid, DNA, RNA, protein, virus, and / or bacteria.

[0100] A16. The method of any of paragraphs A1-A15, wherein at least one of the at least two wells contains at least one three-dimensional population of cells.

[0101] A17. The method of paragraph A16, wherein at least one three-dimensional population of cells comprises an organoid.

[0102] A18. The method of any of paragraphs A1-A17, wherein at least two wells contain two, three, four, or more different types of organoids within each well.

[0103] A19. The method of paragraph A18, wherein the different types of organoids include organoids corresponding to any combination of brain, heart, intestine, kidney, stomach, and liver tissue.

[0104] A20. The method of any of paragraphs A16-A19, wherein at least one three-dimensional population of cells comprises a spheroid.

[0105] A21. The method of paragraph A20, wherein at least two wells contain two, three, or more different types of spheroids.

[0106] A22. The method of any of paragraphs A1-A21, wherein at least one of the at least two wells comprises a two-dimensional cell culture.

[0107] A23. The method of any of paragraphs A1-A22, wherein the liquid comprises a compound being tested for an effect on one or more of the cell cultures.

[0108] A24. The method of any of paragraphs A1-A23, further comprising placing one or more progenitor cells for each cell culture into at least two wells prior to selecting.

[0109] A25. The method of any of paragraphs A1-A24, further comprising imaging at least a portion of the cell culture contained within one of the at least two wells.

[0110] A26. The method of paragraph A25, wherein imaging includes imaging at least a portion of a three-dimensional population of cells.

[0111] A27. The method of paragraph A26, wherein the three-dimensional population of cells comprises a spheroid or an organoid.

[0112] A28. The method of any of paragraphs A25-A27, wherein imaging includes illuminating at least a portion of the single well from above or below the single well.

[0113] A29. The method of any of paragraphs A25-A28, wherein imaging includes detecting light propagated through the top or bottom of one of the wells.

[0114] A30. The method of any of paragraphs A25-A29, wherein the imaging includes fluorescence imaging, phase contrast imaging, confocal imaging, and / or transmitted light imaging.

[0115] A31. The method of any of paragraphs A25-A30, wherein imaging includes illuminating at least a portion of one of the wells with light propagated through a side optical window of the one of the wells.

[0116] A32. The method of any of paragraphs A25-A31, wherein imaging includes detecting light propagated through a side optical window of one of the wells.

[0117] A33. The method of paragraph A31 or A32, wherein the imaging is performed by light sheet microscopy.

[0118] A34. The method of any of paragraphs A1-A33, wherein each reservoir has a slope extending from a bottom region of the reservoir to a passageway located on the flow path at the end of the row of wells.

[0119] A35. The method of paragraph A34, wherein the beveled surface is inclined at an angle of less than 45 degrees from the horizontal when the device has a horizontal orientation.

[0120] A36. The method of paragraphs A34 or A35, wherein transferring the liquid includes moving an inclined surface of at least one of the reservoirs in a generally horizontal orientation.

[0121] A37. The method of any of paragraphs A1-A36, wherein each reservoir is configured to hold more liquid than each well in the row of wells.

[0122] A38. The method of any of paragraphs A1-A37, further comprising placing a lid onto the device to cover the row of wells.

[0123] A39. The method of paragraph A38, wherein transferring the liquid is performed while a lid is covering the row of wells, and optionally both reservoirs.

[0124] A40. The method of any of paragraphs A1-A39, wherein the device is according to any of paragraphs B1-B41.

[0125] B1. A device for cell culture comprising: (a) a row of wells, each having a lower portion and an upper portion, the lower portion configured to contain a cell culture; and (b) a first reservoir and a second reservoir located at opposite ends of the row of wells, the device defining a flow path for a liquid, the flow path extending from the first reservoir to the second reservoir through the row of wells and passing through the upper portion of each well in the row of wells.

[0126] B2. The device of paragraph B1, wherein a lower portion of each well is configured to receive liquid only through an upper portion of the well.

[0127] B3. The device of paragraphs B1 or B2, wherein one or more wells (e.g., each well) in the row of wells has a flat bottom on the outside of the well.

[0128] B4. The device of any of paragraphs B1-B3, wherein one or more wells (e.g., each well) in the row of wells has a flat bottom on the inside of the well.

[0129] B5. The device of any of paragraphs B1-B4, wherein a lower portion of one or more wells (e.g., each well) in the row of wells is internally conical.

[0130] B6. The device of any of paragraphs B1-B4, wherein a lower portion of at least one well (e.g., each well) in the row of wells includes a transparent side optical window having outer and inner surfaces that are planar, each of the outer and inner surfaces being parallel to a longitudinal axis of the device.

[0131] B7. The device of paragraph B6, wherein the exterior and interior surfaces are not parallel to one another.

[0132] B8. The device of paragraphs B6 or B7, wherein a lower portion of at least one well has a pair of transparent side optical windows spaced apart from one another across the row of wells.

[0133] B9. The device of any of paragraphs B1-B8, wherein the row of wells defines a longitudinal axis and a lower portion of one or more wells (e.g., each well) in the row of wells is elongated parallel to the longitudinal axis in a horizontal plane.

[0134] B10. The device of any of paragraphs B1-B9, wherein at least one well (eg, each well) in the row of wells has a concave inner surface area at the bottom of the well.

[0135] B11. The device of paragraph B10, wherein the concave inner surface region is concave in a vertical plane that includes a longitudinal axis defined by the row of wells.

[0136] B12. The device of paragraph B10, wherein the concave inner surface region is concave in a vertical plane perpendicular to a longitudinal axis defined by the row of wells.

[0137] B13. The device of any of paragraphs B1-B12, wherein the flow path follows a straight line through the row of wells.

[0138] B14. The device of any of paragraphs B1-B12, wherein the flow path is a non-linear flow path, such as a serpentine flow path through a row of wells.

[0139] B15. A device of any of paragraphs B1-B14, wherein the flow path extends through a plurality of passages, each passage creating fluid communication between an upper portion of a pair of adjacent wells in the row of wells, or between an upper portion of one of the reservoirs and an upper portion of an adjacent well in the row of wells.

[0140] B16. The device of paragraph B15, wherein at least one passageway (or each passageway) of the plurality of passageways intersects an upper edge of an upper portion of each well of an adjacent pair of wells.

[0141] B17. The device of paragraphs B15 or B16, wherein a wall is shared between upper portions of adjacent pairs of wells in the row of wells, and a passageway in the plurality of passageways extends through the wall.

[0142] B18. The device of any of paragraphs B15-B17, wherein at least one passageway (or each passageway) of the plurality of passageways is formed in a top surface of the device.

[0143] B19. The device of any of paragraphs B15-B17, wherein at least one passageway (or each passageway) of the plurality of passageways is formed below and spaced from a top surface of the device.

[0144] B20. The device of any of paragraphs B15-B19, wherein at least one passageway (or each passageway) of the plurality of passageways is a slot.

[0145] B21. The device of paragraph B20, wherein the slot is elongated at least generally vertically.

[0146] B22. The device of any of paragraphs B1-B21, wherein at least one of the first and second reservoirs has a different size and / or shape than each well of the row of wells.

[0147] B23. The device of paragraph B22, wherein at least one reservoir has a capacity for holding liquid greater than each well in the row of wells.

[0148] B24. The device of paragraph B23, wherein the larger volume is at least 2, 3, 4, or 5 times the liquid holding volume of each well in the row of wells.

[0149] B25. The device of any of paragraphs B1-B24, wherein the device is a single piece (with no movable / removable parts).

[0150] B26. The device of paragraph B25, wherein the row of wells, the first reservoir, and the second reservoir are integrally formed with one another.

[0151] B27. The device of any of paragraphs B1-B26, wherein the device is made of a polymer and is optionally injection molded.

[0152] B28. The device of any of paragraphs B1-B27, wherein a top surface of the row of wells is in the same plane as an edge of each reservoir.

[0153] B29. The device of any of paragraphs B1-B28, wherein the device is elongated parallel to the row of wells.

[0154] B30. The device of any of paragraphs B1-B29, wherein the device has a single row of wells.

[0155] B31. The device of any of paragraphs B1-B30, further comprising a lid configured to cover the row of wells and each reservoir.

[0156] B32. The device of any of paragraphs B1-B31, wherein the device is packaged in substantially sterile conditions.

[0157] B33. The device of any of paragraphs B1-B32, wherein one or more of the wells (such as each of the wells) have a bottom interior surface region that is rounded in a pair of mutually perpendicular vertical planes, optionally spherical.

[0158] B34. A device according to any of paragraphs B1-B33, wherein each well of the one or more wells (such as each well of the device) has a bottom outer surface region that is flat or rounded (optionally spherical) in a pair of mutually perpendicular vertical planes.

[0159] B35. A device according to any of paragraphs B1-B25 and B27-B34, wherein the device includes a body and at least one plate joined to a bottom of the body, the at least one plate forming a bottom region of one or more of the wells (such as each of the wells of the device).

[0160] B36. The device of paragraph B35, wherein at least one plate forms a floor of each well of the one or more wells and forms a fluid-tight seal with the body at a bottom region of the well.

[0161] B37. A device according to paragraph B35 or B36, wherein, in the absence of a plate, the body optionally includes a plurality of compartments, each defining a vertical through axis and each having an open top and an open bottom, and at least one plate converts each compartment into a well having a closed bottom.

[0162] B38. The device of any of paragraphs B35-B37, wherein at least one plate forms a fluid-tight seal with the body at a bottom region of each well of the one or more wells.

[0163] B39. The device of any of paragraphs B35-B38, wherein the body is made of a polymer and is optionally injection molded.

[0164] B40. The device of any of paragraphs B35-B39, wherein the body is formed from a preformed body having a plurality of wells by cutting the preformed body and removing a bottom end of one or more of the wells of the preformed body.

[0165] B41. The device of any of paragraphs B35-B40, wherein the body is a polymer body and the plate is a polymer plate or a glass plate.

[0166] C1. An apparatus comprising: (a) a plurality of devices according to any of paragraphs B1-B41; and (b) a frame for removably holding the plurality of devices in alignment with one another.

[0167] C2. The apparatus of paragraph C1, wherein the apparatus includes a rectangular array of wells formed by a row of wells of a plurality of devices.

[0168] C3. The apparatus of paragraphs C1 or C2, wherein the center-to-center spacing of wells within each row of wells is the same as the center-to-center spacing of wells between adjacent rows of wells.

[0169] C4. The apparatus of paragraph C3, wherein both the center-to-center spacing of wells within each row of wells and the center-to-center spacing of wells between adjacent rows of wells is 18, 9, or 4.5 mm.

[0170] C5. The apparatus of any of paragraphs C1-C4, further comprising a lid configured to fit over at least one of the devices and cover the well of at least one of the devices.

[0171] C6. The apparatus of paragraph C5, wherein the lid is configured to cover all of the wells and each of the reservoirs of the device.

[0172] C7. The device of any of paragraphs C1-C6, wherein the device is packaged in a sterile condition.

[0173] The term "exemplary" as used in this disclosure means "illustrative" or "serving as an example." Similarly, the term "exemplary" means "to illustrate by giving an example." Neither term implies desirability or superiority.

[0174] Although the present invention has been described through the above examples and features, it will be understood by those skilled in the art that a wide variety of modifications, combinations, and alterations of the examples and features may be made without departing from the inventive concept disclosed herein. Moreover, the present invention should not be considered limited to any specific object or embodiment described herein, but rather should be considered applicable to achieve a wide variety of objects other than those described herein. This disclosure has described some examples of the technology with reference to the accompanying drawings, in which only some of the possible examples are shown. However, other aspects may be embodied in many different forms, and should not be construed as being limited to the examples described herein, even if not expressly illustrated in combination. Rather, these examples are provided so that this disclosure will be thorough and complete, and will fully convey the scope of possible embodiments to those skilled in the art.

Claims

1. 1. A method of cell culture, the method comprising: (a) selecting a device (50) including a row of wells (52) and a first reservoir (56) and a second reservoir (58), each of the row of wells (52) having a first end and a second end, the first end opposite the second end, the first reservoir (56) located at the first end and the second reservoir (58) located at the second end, each well (52) having a lower portion (70) and an upper portion (68), the lower portion of each well (52) of at least two of the wells (52) containing a cell culture in contact with a liquid (104); (b) transferring the liquid (104) between the first reservoir (56) and the second reservoir (58) at least partially along a flow path (62) defined by the device (50) and extending through the upper portion of each well (52) of the row of wells (52), whereby the liquid (104) is exchanged between or among the at least two wells (52); wherein the lower portion of each well (52) in the row of wells (52) includes a transparent lateral optical window (90) having outer and inner surfaces that are planar, each of the outer and inner surfaces being parallel to a longitudinal axis (60) of the device (50).

2. 2. The method of claim 1, wherein the flow path (62) extends through a plurality of passages (64), each passage (64) creating fluid communication between upper portions of a pair of adjacent wells (52) in the row of wells (52) or between an upper portion of one of the reservoirs and the upper portion of an adjacent well (52) in the row of wells (52).

3. 2. The method of claim 1, wherein transferring the liquid includes generating turbulence within the lower and upper portions of each well in the row of wells to promote in-well mixing of the liquid.

4. The method of claim 1 , wherein transporting the liquid (104) comprises using gravity to drive liquid flow.

5. 5. The method of claim 4, wherein driving liquid flow includes tilting the device (50) and raising the first reservoir (56) relative to the second reservoir (58) such that the liquid (104) flows along the flow path (62) from the first reservoir (56) to the row of wells (52) and from the row of wells (52) to the second reservoir (58).

6. 6. The method of claim 5, wherein transferring the liquid (104) includes rocking the device (50) such that gravity drives flow of the liquid (104) alternately in opposite directions along the flow path (62) between the first reservoir (56) and the second reservoir (58).

7. The method of claim 1, further comprising introducing at least one three-dimensional population of cells into at least one of said at least two wells (52).

8. A device (50) for cell culture, said device (50) comprising: (a) an array of wells (52), each having a lower portion and an upper portion, the lower portion being configured to contain a cell culture; (b) a first reservoir (56) and a second reservoir (58), each of the rows of wells (52) having a first end and a second end, the first end being opposite the second end, the first reservoir (56) being located at the first end and the second reservoir (58) being located at the second end; Equipped with the device (50) defines a flow path (62) for liquid, the flow path (62) extending from the first reservoir (56) to the second reservoir (58) through the row of wells (52) and passing through the upper portion of each well (52) in the row of wells (52); A device (50), wherein the lower portion of at least one well (52) in the row of wells (52) includes a transparent lateral optical window (90) having outer and inner surfaces that are planar, each of the outer and inner surfaces being parallel to a longitudinal axis (60) of the device (50).

9. The device (50) of claim 8, wherein the lower portion of each well (52) is configured to receive liquid only through the upper portion of the well (52).

10. The device (50) of claim 8, wherein one or more of the wells (52) in the row of wells (52) have a flat bottom on the inside of the one or more of the wells (52).

11. The device (50) of claim 8, wherein at least one well (52) in the row of wells (52) has a concave inner surface area at a bottom of the well (52).

12. The device (50) of claim 11, wherein the concave inner surface region is concave in a vertical plane that includes a longitudinal axis (60) defined by a row of the wells (52).

13. The device (50) of claim 11, wherein the concave inner surface region is concave in a vertical plane perpendicular to a longitudinal axis (60) defined by the row of wells (52).

14. 9. The device (50) of claim 8, further comprising a lid (100) configured to cover the array of wells (52), the first reservoir (56), and the second reservoir (58).

15. 9. The device (50) of claim 8, wherein each well (52) of the one or more wells (52) has a bottom outer surface region that is flat or rounded in a pair of mutually perpendicular vertical planes.

16. An apparatus comprising a frame (42) for removably holding a device (50) according to claim 8 in alignment with another device (50).

17. 17. The apparatus of claim 16, wherein the center-to-center spacing of wells (52) within each row (52) of wells is the same as the center-to-center spacing of wells (52) between adjacent rows (52) of wells (52).

18. 17. The apparatus of claim 16, further comprising a lid (100) configured to fit over at least one of the devices (50) and cover the at least one well (52) of the device (50).

19. 20. The apparatus of claim 18, wherein the lid (100) is configured to cover all of the wells (52) and each of the reservoirs of the device (50).

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