Incubation system and method for automated cell culture and testing - Patents.com

Abstract

Disclosed are incubation systems and methods for automated cell culture and / or testing. The incubation system may include a housing forming a chamber. A rack may define storage positions for supporting an array of sample holders inside the chamber. A detection robot may be configured to capture one or more images of cells contained by one or more wells of each sample holder while the sample holders remain in one of the storage positions of the rack. A fluid handling station may be configured to add and / or remove fluid from one or more wells of each of the sample holders inside the housing. At least one plate robot may be configured to move the sample holders between the rack and the fluid handling station. A computer may control the operation of the detection robot, the fluid handling station, and the at least one plate robot.

Description

[Background technology]

[0001] A typical incubator for cell culture is little more than a temperature-controlled box, often with the ability to adjust the level of carbon dioxide. The box forms a humidified incubation chamber and holds the culture vessel containing the cells. To gain access to the culture vessel and the cells therein for any purpose, such as viewing, feeding, or splitting, the incubation chamber must be opened to the surrounding environment. However, opening the chamber reduces the stability of its atmosphere and increases the possibility of contamination. Furthermore, the user must be physically present for the operation of the cell culture, which may necessitate trips to and from the laboratory at night and on weekends. Improved incubators for cell culture are needed. Summary of the Invention [Means for solving the problem]

[0002] The present disclosure provides an incubation system and method for automated cell culture and / or testing. One exemplary incubation system, also referred to as an incubator, may include a housing forming a chamber. A rack may define storage positions for supporting an array of sample holders (e.g., microplates) inside the chamber. A detection robot may be configured to capture one or more images of cells contained by one or more wells of each sample holder while the sample holders remain in one of the storage positions of the rack. A fluid handling station may be configured to add and / or remove fluids from one or more wells of each of the sample holders inside the housing. At least one plate robot may be configured to move the sample holders between the rack and the fluid handling station. A computer may control the operation of the detection robot, the fluid handling station, and the at least one plate robot. The present invention provides, for example, the following items. (Item 1) An automated incubation system (50, 150) for culturing and / or testing cells, comprising: a housing (52) defining a chamber (54); a rack (56) defining storage locations (124) for supporting an array of sample holders (58) inside said chamber (54); a detection robot (60) for capturing one or more images of cells (154) contained by one or more wells (152) of each sample holder (58) while the sample holder remains in one of the storage positions (124) of the rack (56); a fluid handling station (72) configured to add fluid to and / or remove fluid from the one or more wells (152) of each of the sample holders (58) inside the housing (52); at least one plate robot (88) for moving sample holders (58) between said racks (56) and said fluid handling stations (72); a computer (96) for controlling the operation of said detection robot (60), said fluid handling station (72), and said at least one plate robot (88); An incubation system comprising: (Item 2) 2. The incubation system (50, 150) according to item 1, wherein the detection robot (60) is configured to capture images by lensless imaging. (Item 3) 3. The incubation system (50, 150) of item 2, wherein the detection robot (60) is configured to lenslessly capture one or more holograms representing cells (154) located in one or more wells (152) of each sample holder (58), and the computer (96) is configured to use the captured holograms to reconstruct images of the cells (154) located in the wells (152). (Item 4) 4. The incubation system (50, 150) according to item 3, wherein the detection robot (60) is configured to illuminate the cells (154) using light propagated through a waveguide (178), the outlet of the waveguide (178) being configured to travel together with the detection robot (60) to each of the storage positions (124) of the rack (56). (Item 5) 2. The incubation system (50, 150) of item 1, wherein the fluid handling station (72) is configured to dispense fluid into and remove fluid from the wells (152) of the sample holder (58) inside the chamber (54). (Item 6) 2. The incubation system (50, 150) of claim 1, wherein the chamber (54) is an incubation chamber and the housing (52) forms an entry / exit chamber (122) sized to contain one or more sample holders (58), the incubation chamber communicating with the entry / exit chamber (122) via an inner door (118), and the entry / exit chamber (122) being accessible from outside the housing (52) via an outer door (120). (Item 7) 2. The incubation system (50, 150) of claim 1, wherein the fluid handling station (72) includes one or more storage locations for a pipette (78) and a container for holding a removable tip (82) for the pipette (78). (Item 8) 8. The incubation system (50, 150) of item 7, wherein the fluid handling station (72) includes a first stack position for a container holding a new tip for the pipette (78) and a second stack position for a container holding a tip already used by the pipette (78). (Item 9) 2. The incubation system (50, 150) of item 1, wherein the fluid handling station (72) includes a receiving area for the sample holder (58) and at least one of an assay plate (86) and / or a master plate (292). (Item 10) 2. The incubation system (50, 150) of item 1, wherein the rack (56) has a first side opposite a second side, the plate robot of the at least one plate robot (88) is configured to move sample holders (58) in and out of the storage positions (124) via the first side of the rack (56), and the detection robot (60) is configured to operatively access the sample holders (58) in the storage positions (124) via the second side of the rack (56). (Item 11) The incubation system (50, 150) of item 1, wherein the rack (56) includes a plurality of heaters (126) operatively associated with the storage locations (124) of the rack (56) and configured to heat lids (160) of sample holders (58) in the storage locations (124) to reduce condensation prior to image capture using the detection robot (60). (Item 12) 2. The incubation system (50, 150) of claim 1, wherein the computer (96) is configured to transmit one or more of the captured images to a remote user via the Internet, and wherein the computer (96) is configured to receive instructions from the remote user as to whether and / or how to treat a culture of cells (154) depicted in the transmitted image. (Item 13) 2. The incubation system (50, 150) of item 1, wherein the computer (96) is configured to determine whether and / or when to add fluid to and / or remove fluid from one or more wells (152) of each sample holder (58) at the fluid handling station (72) based on images captured by the detection robot (60). (Item 14) Item 14. An incubation system (50, 150) according to item 13, wherein the computer (96) is configured to determine characteristics of the cells (154) contained by one or more wells (152) of each sample holder (58) from images captured by the detection robot (60) and, based on said characteristics, determine whether and / or when to add fluid to and / or remove fluid from such wells (152). (Item 15) 2. The incubation system (50, 150) of item 1, further comprising a sample holder (58) including a pH sensor device and / or an oxygen sensor device (162) disposed in one or more wells (152) of the sample holder (58), and the incubation system (50, 150) including an optical sensor (64) configured to detect photoluminescence from the sensor device (162). (Item 16) 1. An automated cell culture and / or testing method comprising: capturing one or more images of cells (154) contained within one or more wells (152) of each sample holder of a plurality of sample holders (58), the plurality of sample holders (58) being stored in storage positions (124) defined by a rack (56) inside an incubator (50, 150), the sample holder (58) remaining in its storage position (124) within the rack (56) when the one or more images are captured for the sample holder (58); moving the sample holders (58) from the racks (56) to a fluid handling station (72) inside the incubator (50, 150) using a plate robot (88); removing fluid from and / or adding fluid to at least one well (152) of the sample holder (58) at the fluid handling station (72); A method comprising: (Item 17) 20. The method of claim 16, wherein capturing the one or more images is performed lenslessly. (Item 18) 17. The method of claim 16, wherein capturing one or more images comprises capturing one or more images for each well of a plurality of wells of the sample holder and processing the one or more images to obtain a value corresponding to the confluence and / or density and / or number of cells in the wells, and wherein the moving step is performed for the sample holder if a value for one or more wells of the plurality of wells satisfies one or more predefined criteria. (Item 19) The method further comprises: transmitting to a user one or more images of the cells resulting from the step of capturing the one or more images; receiving a command from the user to perform the moving step; removing and / or adding fluid at said fluid handling station (72) in response to said commands; 17. The method according to item 16, comprising: (Item 20) 17. The method of claim 16, further comprising locally and / or temporarily applying heat to each sample holder (58) within the incubator (50, 150) to reduce condensation on a lid (160) of each sample holder (58) prior to capturing one or more images of the sample holder (58). [Brief description of the drawings]

[0003] [Figure 1] FIG. 1 is a schematic top view of an exemplary smart incubator for culturing and / or testing biological cells according to aspects of the present disclosure, the smart incubator having a shared chamber for storing sample holders and handling fluids. [Diagram 2] FIG. 2 is a schematic top view of another exemplary smart incubator for culturing and / or testing biological cells according to aspects of the present disclosure, the smart incubator having separate but connected chambers for storing sample holders and handling fluids. [Diagram 3] FIG. 3 is a somewhat schematic, partial cross-sectional view of a microplate containing a cell culture and operatively arranged to a detection module of the detection robot of the smart incubator of FIG. 1 or FIG. 2, in accordance with an aspect of the present disclosure. [Figure 4] FIG. 4 is a somewhat schematic, partial cross-sectional view of a microplate containing a cell culture and operatively arranged relative to the lensless imaging module of the detection robot of FIG. 1 or FIG. 2, in accordance with an aspect of the present disclosure. [Diagram 5]FIG. 5 is an elevational view of selected sides of an exemplary embodiment of the smart incubator of FIG. 1 with a side wall of the incubator housing removed to reveal internal components, the incubator operatively loaded with microplates and other consumables in accordance with aspects of the present disclosure. [Figure 6] 6 is a cross-sectional top view of the operatively loaded incubator of FIG. 5 taken generally along line 6-6 of FIG. [Figure 7] 7 is a cross-sectional view of the operatively loaded incubator of FIG. 5 taken generally along line 7-7 of FIG. [Figure 8] 8 is a fragmentary cross-sectional view of the operatively loaded incubator of FIG. 5 taken generally along line 8-8 of FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0004] The present disclosure provides an incubation system and method for automated cell culture and / or testing. An exemplary incubation system may include a housing forming a chamber. A rack may define storage positions for supporting an array of sample holders (e.g., microplates) inside the chamber. A detection robot may be configured to capture one or more images of cells contained by one or more wells of each sample holder while the sample holders remain in one of the storage positions of the rack. A fluid handling station may be configured to add and / or remove fluids from one or more wells of each of the sample holders inside the housing. At least one plate robot may be configured to move the sample holders between the rack and the fluid handling station. A computer may control the operation of the detection robot, the fluid handling station, and the at least one plate robot.

[0005] An exemplary method of automated cell culturing and / or testing is provided. In the method, one or more images of cells contained in one or more wells of each sample holder of a plurality of sample holders may be captured. The plurality of sample holders may be stored in storage positions defined by a rack inside an incubator. The sample holder may remain in its storage position in the rack when one or more images for the sample holder are captured. The sample holder may be moved from the rack to a fluid handling station inside the incubator using a plate robot. Fluid may be removed from and / or added to at least one well of the sample holder at the fluid handling station.

[0006] The state of the art in incubators does not include any built-in intelligence and / or decision-making tools or attributes. Furthermore, incubators are not remotely controlled and cells within the incubator cannot be followed up to check for viability or the need for media changes, addition of test compounds, or any other steps that the user may need to perform.

[0007] The incubator of the present disclosure may enable automated short-term and long-term culture in a sample holder such as a microplate, and may enable monitoring of multiple parameters over the time course of the culture. If data collected by one or more sensors of the incubator meet one or more predefined criteria, the control computer may automatically take action or alert laboratory personnel. For example, if a captured image of the cell culture indicates that action is needed, the computer may feed the cells (e.g., change the growth medium), split the cells, add a test compound(s) to the cells, create an assay mixture, or the like, or notify one or more laboratory personnel (i.e., users) of the situation requiring attention. The incubator may automate cell culture, improve cell health, and provide better all-in-situ experiments with more detailed knowledge of cell health and cell growth.

[0008] The smart incubators disclosed herein can be used for any suitable purpose. They can reduce labor and optimize workflow for assay development and compound testing. Clonal populations of cells can be cultured, fed, and assayed. Organotypic cultures on chips can be cultured and tested in a non-microplate setting. Miniature bioreactors can provide for cell line development, where a mixture of cells and media is added.

[0009] Further aspects of the present disclosure are described in the following sections: (I) Definitions, (II) Overview of the Smart Incubator, (III) Methods of Sample Incubation and Processing, and (IV) Examples.

[0010] I. Definition 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 explained below.

[0011] Chemical sensor - a device and / or compound (e.g., a sensor dye) that detects or measures the concentration of a chemical analyte, such as free protons (relative to pH), oxygen, carbon dioxide, or the like. The chemical sensor may have optical properties that are sensitive to the concentration of the chemical analyte. The optical properties may include photoluminescence intensity, photoluminescence lifetime, photoluminescence polarization, photoluminescence quenching / energy transfer, chemiluminescence intensity, absorbance, or the like. The optical properties of the chemical sensor may be read using the optical sensor with or without illumination using an associated light source to measure the concentration of the chemical analyte. In some embodiments, the device may include a chemical sensor compound that is trapped within a substrate and locates the sensor compound within a larger fluid volume (e.g., the volume of culture medium in a microplate well). The substrate may be attachable to a surface, such as with an adhesive. The surface may be the interior surface of a well, and the optical properties may be detected through the walls of the well.

[0012] Computer - an electronic device for storing and processing data, typically in binary form, according to instructions that may be provided by a variable program. Exemplary computers, also referred to as computing devices, include desktop computers, laptop computers, tablets, smartphones, and the like.

[0013] Inspection Region - a region located on the optical axis of a detection system where an object, such as a sample (eg, biological cell), may be optically inspected (eg, imaged using an image sensor).

[0014] Image - a representation of the light detected by an image sensor in a two- or three-dimensional array of locations (e.g., a raw image), or a processed form thereof (e.g., a reconstructed image). A raw image can be, among other things, an optically focused image or a hologram created by lensless imaging (i.e., a lensless image).

[0015] Image Sensor - an optical sensor capable of detecting spatial variations in light (e.g., variations in intensity) in two dimensions, where the light is incident on a light-sensitive detection area of ​​the sensor. The image sensor may be a two-dimensional array sensor, such as a charge-coupled device (CCD) sensor, an active pixel sensor (e.g., a complementary metal-oxide semiconductor (CMOS) sensor), a hybrid CCD-CMOS sensor, or the like. The image sensor may create a raster image (i.e., a bitmap) as a rectangular array of pixels, and may be configured to create color images, grayscale (monochrome) images, or both.

[0016] Lensless imaging - the capture of a diffraction pattern(s) for an object(s) (e.g., a sample containing biological cells) using an image sensor. Lensless imaging is performed without a lens being interposed between the object and the image sensor. The captured diffraction pattern (hologram) can be referred to as an image of the object(s) (e.g., an image of a cell) even if the image is not an optically focused image. Because the image is not optically focused, the captured image is generally neither magnified nor demagnified relative to the object(s) (i.e., the capture involves a "unit magnification"). Thus, the photosensitive area of ​​the image sensor should be at least as large as the bottom of a well if the entire bottom is to be covered with the same lensless image. However, an exemplary well to be imaged may have a bottom area larger than the photosensitive area of ​​the image sensor. For example, the well may be provided by a 6-well microplate or a single-well (e.g., rectangular) sample holder, among others. Thus, multiple lensless images of overlapping regions of the same well can be stitched together to generate a larger image that represents more or all of the bottom of the well. Lensless imaging can include digital holographic reconstruction imaging, shadow imaging, or fluorescent imaging, among others.

[0017] Lensless imaging may be performed using any suitable light source, among others, which may epi-illuminate or transilluminate the sample. The light source may illuminate the sample with coherent light (e.g., from a laser), partially coherent light (e.g., from a light emitting diode), or incoherent light (e.g., by the use of an incoherent light emitting element and / or a diffuser in the illumination path). In some embodiments, the light source may transilluminate the sample with approximately plane wave illumination from a light source that is at least partially coherent. For example, the light source may be significantly farther from the sample along the z-axis than the image sensor, such as at least 5 or 10 times farther. In some embodiments, the light source may include a light emitting element optically coupled to a waveguide. The exit of the waveguide may be mounted on an arm, which may be rotatable to change the sample illumination angle and / or linearly movable to introduce a sub-pixel shift into the captured image.

[0018] The lensless images can be processed by pixel super-resolution techniques, which can generate sub-pixel resolution by laterally shifting the light source, image sensor, and / or sample to create "sub-pixel" images, which can then be merged to obtain the smaller effective size of the pixel.

[0019] Lensless images can be processed by phase retrieval techniques as well or alternatively. The captured raw image is an in-line hologram containing intensity data. Lensless imaging can be used to reconstruct quantitative phase contrast, which uses pixel intensities to provide a volumetric representation of an object (e.g., a cell). Furthermore, lensless imaging can provide a large depth of field, and thus may not require focusing. To retrieve the amplitude and phase of a sample, the phase retrieval algorithm(s) used may require more than one hologram (due to the bi-image problem). Thus, the calculation for phase retrieval is advantageously performed using holographic image data from multiple holograms with similar fields of view (e.g., holograms captured with different sample / image sensor distances, different illumination angles, and / or different illumination wavelengths (e.g., using tunable lasers or light sources (e.g., laser diodes or LEDs) emitting at different wavelengths).

[0020] Lensless imaging can be advantageous for the disclosed systems and methods because bulky collection / detection optics are not required. Hence, microplates can be stored closer to each other within a vertical row of microplates because less space is required for image capture under each microplate. The resulting incubation system is more compact and space efficient.

[0021] Light - optical radiation, including ultraviolet radiation, visible radiation (i.e., visible light), and / or infrared radiation.

[0022] Light source - a device that generates light, optionally as a beam of light, and optionally powered by electricity. A light source includes at least one light emitting element and may also include any associated optical element(s) for shaping, sizing, filtering, polarizing, scattering, directing, and / or otherwise interacting with the light emitted by the light emitting element(s). These optical elements may include any combination of at least one waveguide (e.g., optical fiber or liquid light guide), lenses, mirrors, filters, diffusers, masks, apertures, beam splitters, gratings, prisms, polarizers, and / or the like. Exemplary light emitting elements include semiconductor devices, lasers (e.g., excimer lasers, gas lasers, dye lasers, solid state lasers, semiconductor crystal or diode lasers, free electron lasers, etc.), arc lamps, and / or the like. Exemplary semiconductor light emitting elements include laser diodes, light emitting diodes (LEDs), and superluminescent diodes, among others.

[0023] Microplate - a sample holder containing a plurality of connected wells. The terms "microplate" and "plate" are synonymous herein. The wells may be arranged in a planar rectangular array with equal uniform spacing along orthogonal horizontal axes to form a plurality of rows and columns. The wells in a microplate may be substantially identical to one another, may be joined to one another, and / or may hold any suitable volume of fluid, such as 1 microliter to 10 milliliters. The dimensions of the microplate may conform to American National Standards Institute (ANSI) and Laboratory Automation and Screening Society (SLAS) (i.e., ANSI / SLAS) standards. For example, a microplate may have a 2 x 3 array of wells (such as 6, 12, 24, 48, 96, 384, or 1,536 wells), a width of about 85 mm, a length of about 128 mm, a height of about 14 mm, and / or an inter-well separation inversely proportional to the total number of wells. Exemplary well spacings include 3.6 cm, 1.8 cm, 9 mm, 4.5 mm, 2.25 mm, and 1.125 mm, among others. Each well can have a flat bottom to facilitate imaging. The microplate can be formed from a transparent polymer.

[0024] The microplate may include a lid for covering each well of the microplate, the lid being removable to allow dispensing and aspirating from the wells of the microplate.

[0025] Optical system - a set of optical elements of an imaging system, which may be arranged along an optical axis between a light source and an inspection area (illumination optics) and / or along an optical axis between an inspection area and an optical sensor (collection optics). An optical element may be any device or structure that interacts with light, such as collecting, directing, focusing, filtering, polarizing, scattering, collimating, and / or partially blocking light. An optical element may function by any suitable mechanism, such as reflection, refraction, scattering, diffraction, absorption, and / or filtering, among others. Exemplary optical elements include lenses, mirrors, diffusers, gratings, prisms, filters, apertures, masks, beam splitters, wave guides, polarizers, and the like.

[0026] Optical Sensor - A device that creates a signal (e.g., an electrical signal) in response to incident light. An optical sensor may be a point sensor or may have an array of light-sensitive elements and detect spatial differences in the incident light. The array may be a one-dimensional array, as in a linear sensor, a two-dimensional array, as in an image sensor, or the like.

[0027] Robot - a device capable of moving and performing a series of actions under computer control. Exemplary robots include detection robots for detecting light and / or capturing images from a plate, plate robots for transporting plates, pipette robots for transferring liquids (e.g., into and out of wells of a plate), lid robots for removing and replacing lids, and / or the like.

[0028] Sample - a specimen of any suitable nature. A sample may be organic and / or inorganic, natural and / or manufactured, and may include any suitable collection, material, substance, isolate, extract, particle, or the like. In an exemplary embodiment, the sample includes biological cells. The biological cells may be eukaryotic (e.g., mammalian cells) or prokaryotic (e.g., bacterial cells). Exemplary biological cells include established cells (cell lines), stem cells, primary cells, cells of a tissue sample, transfected cells, cells from a clinical sample (e.g., blood sample, fluid aspirate, tissue section, etc.), clones of cells, or the like. A cell culture may optionally include a collection of cells contained by a well and in contact with (e.g., immersed in) any suitable liquid medium. The liquid medium may be an aqueous medium, which may include, among other things, moisture, salts, buffers, glucose, detergents, dyes, proteins, amino acids, or any combination thereof. The liquid medium may be a growth medium for the cells.

[0029] Sample holder - a device for holding at least one sample or any array of spatially isolated samples, and optionally allowing the sample(s) to be imaged through a horizontal transparent wall of the device (e.g., the bottom wall of a well). An exemplary sample holder is a culture vessel containing one or more wells, such as a microplate, a Petri dish, a cell culture flask, etc.

[0030] II. Overview of Smart Incubators This section provides an overview of an automated incubation system ("smart incubator") for processing samples held by a sample holder (such as exemplified herein with a microplate) such as culturing and / or assaying biological cells held within the wells of the sample holder (see Figures 1-4).

[0031] 1 illustrates in schematic form an exemplary smart incubator 50. The incubator 50 includes a housing 52 (e.g., a box) that defines a main chamber 54, which may remain closed during operation of the incubator. The incubator may also have a storage structure 56 (interchangeably referred to as a rack) for holding and organizing multiple sample holders, such as microplates 58, within the main chamber 54 while cells contained by the wells of the sample holders are cultured.

[0032] Optical detection may be performed in the main chamber 54. A robot may be used to move at least one optical sensor to every well of each microplate 58 (or other sample holder) for each microplate (or sample holder) stored in the rack 56. More specifically, while the sample holder remains in its individual storage location in the rack 56, the detection robot 60 may optically sense the contents of the sample holder, in particular the contents in each of its wells. For example, the detection robot 60 may capture images of cells growing in the wells. The detection robot may include a detection module having at least one light source 62 for illumination of the wells and / or their contents, and at least one optical sensor, such as an image sensor 64, arranged to detect optical radiation from the illuminated wells and / or contents, as described in more detail below. The detection robot 60 may be controllable to drive the movement of its detection module with at least three degrees of freedom (e.g., along three orthogonal axes) for optical alignment with each well of each microplate 58 (or other sample holder) in the rack 56. More specifically, the detection robot and / or its detection module may travel horizontally, i.e., parallel to the array of storage locations defined by the rack 56 as indicated by the arrow at 66, horizontally in and out of the rack 56, i.e., orthogonal to the vertical array as indicated by the double arrow at 68, and vertically. In some embodiments, the detection robot 60 may have separate, individually controllable motors (e.g., servo motors) corresponding to each translational degree of freedom. Alternatively, or in addition, the contents of the microplate 58 or other sample holder may be optically sensed at a detection station 70 in the main chamber 54 (or elsewhere in the incubator 50), the detection station being separate from the rack 56. The detection station 70 may have a light source(s) and optical sensor(s) as described herein with respect to the detection robot 60, but may be relatively fixed within the main chamber 54 (or elsewhere in the incubator 50).

[0033] The detection robot 60 (and / or detection station 70) allows the growth or development of cells to be monitored. The detection robot may provide a compact, label-free imaging system for capturing one or more images of cells from every well of every microplate 58 (or other sample holder). The imaging system is movable along three orthogonal axes and may reach every well of each microplate 58 supported by the rack 56. This ensures that the optical axis (defined by the centers of all microplate wells to be imaged) is vertical at all times.

[0034] The detection robot 60 may include at least one light source 62 to be placed above and / or in each well of each microplate 58 (or other sample holder). The light source 62 may be a single light source with one light outlet optically coupled to a single upstream light emitting element or multiple upstream light emitting elements (e.g., of different colors). The light source may have compact collimation optics and / or may utilize a diffuser near or at the light outlet. In some embodiments, the light source 62 may be coupled or configured to be coupled to a specialized plate lid with a light guide that reaches into the entire well, deep enough to touch the liquid medium in the well, thereby avoiding the optical effects of a liquid meniscus. In some embodiments, the light source 62 may transmit light from a light outlet with a small diameter (e.g., in the range of the size of a cell (such as a point source with a diameter of less than about 100, 50, 25, or 10 μm)), which provides a light source with high brightness. For example, the light source may include a laser diode mounted at a fixed location. A flexible optical fiber may be optically coupled to the laser diode and configured to act as a light guide for the propagation of light from the laser diode through the fiber to the light outlet above the microplate well to be imaged. The light outlet may be movable relative to the optical axis in the horizontal plane to adjust the angle of illumination, which allows for higher resolution from computer-aided image reconstruction. In some embodiments, the array of compact point light sources each having a small diameter (e.g., less than about 100, 50, 25, or 10 μm), such as an organic LED display, may be included as a matrix of small light sources that can be individually controlled. This configuration allows for single point illumination or patterns of illumination by the combination of the matrix of light sources, which may provide higher resolution from computer-aided image reconstruction and allow for illumination enhancement strategies to compensate for the adverse effects of liquid meniscus.

[0035] The detection robot 60 may also include at least one image sensor 64 to be installed under each microplate (or other sample holder). Each image sensor 64 may be configured to capture an image of the entire bottom of the microplate well (and the cells thereon). Alternatively, the entire bottom (and the cells thereon) may be imaged by capturing multiple tiled images. The image sensor may be provided by a compact camera with integrated compact optics (e.g., a small CMOS camera with compact optics). In other embodiments, the image sensor may be exposed to illumination light beams from one or more point sources above the microplate directly, without any intervening optics to focus a beam of light between the bottom of the microplate 58 and the image sensor. In some embodiments, the image sensor 64 may be exposed to illumination light beams lensless from a source of diffuse light located above the microplate when only low optical resolution is required. However, imaging may be performed with any type of imaging in any of the incubation systems disclosed herein.

[0036] A fluid handling station 72 in the main chamber 54 may perform transfer of fluids in and out of the wells of the microplate 58 and / or other plates. The fluid handling station has a plate dock 74 with one or more plate receiving stations (interchangeably referred to as a docking station) to hold one or more microplates and / or other plates in place. The plate dock 74 may be fixed or movable. The plate dock may provide a location for a master plate, typically with deep wells, which may be loaded into the incubator through a dedicated door or through one of the other doors described elsewhere herein. A lid robot 76 may be configured to remove a lid from a plate located in the plate dock 74, allow for the addition and / or removal of fluids to and / or from the wells of the plate using at least one pipette 78, and replace the lid when fluid transfer is complete. Each pipette 78 may include a pump 80 for driving fluid into and / or out of the end of the pipette, a motorized positioner for precisely moving the end of the pipette in three dimensions, and / or a tip extractor for removing the pipette tip 82 from the end of the pipette 78 after use. The pipette(s) may be operatively accessible to each well of each plate located in the plate dock 74 for addition and / or removal of fluid. The pipette tips 82 may be stored in at least one of two different stack positions (e.g., for creating respective stacks of tip boxes) within the fluid handling station. At least one of the stack positions may be employed to store new pipette tips not yet used by a pipette. At least the other of the stack positions may be employed to store pipette tips already used by a pipette. Any suitable culture media and reagents 84 may be accessible to the pipettes 78 of the fluid handling station 72 for uptake and / or dispensing. The media and reagents may be contained in containers (eg, bottles), which may be stored inside or outside the chamber 54 and / or housing 52.Exemplary reagents include buffered saline, trypsin, assay solution, test compounds, and the like. At least one stack of assay plates 86 (and / or transfer plates) is stored within the fluid handling station 72 and can be individually transferred (e.g., by a plate robot) as needed to one of the plate receiving locations of the plate dock 74 when an assay mixture is to be created in the wells of the assay plates 86.

[0037] The assay plate 86 may be used to test cell culture supernatants contained within the wells of the microplate 58 for the presence / level of a given analyte and / or activity (e.g., monoclonal antibody binding activity). Each microplate 58 may be moved to the plate dock 74 and samples of the supernatants contained in one or more of the wells may be transferred to one or more wells of the assay plate 86. Each assay plate may be an ELISA plate having wells coated with a reagent (e.g., antibody, epitope, or the like). In other examples, each assay plate 86 may be an uncoated plate that is moved out of the incubator to an external liquid handler. In yet other examples, each assay plate 86 may receive supernatants and reagents from wells to support the same types of tests that may be performed inside or outside the incubator.

[0038] Test compounds and / or liquids for cell delivery may be added to and / or removed from the wells of the microplate 58 at the fluid handling station 72 or at a separate dedicated station inside the incubator, or while the microplate 58 remains in the rack 56. Thus, compound addition and / or cell delivery (e.g., changing medium by removing old medium and adding new medium) may be performed using a reagent dispenser (fluid addition only), a low volume pipettor (fluid addition and removal), or a microfluidic plate (fluid addition and removal). The microfluidic plate may be hydraulically connected to each storage position of the rack 56, to the fluid handling station 72, or to a separate dedicated station. Single cell dispensing may be performed in the incubator at or near the fluid handling station 72.

[0039] The plate robot 88 may transport microplates 58 within and, optionally, out of the main chamber 54. The plate robot may transport microplates 58 to and from individual storage locations within the racks 56. More generally, the plate robot 88 may move microplates 58 in, between, and across the racks 56, the detection stations 70, and / or the fluid handling stations 72. The plate robot may also move boxes of pipette tips 82 and / or assay plates 86 within the fluid handling stations 72 and / or the chambers 54. The plate robot 88 may be controllable to drive the movement of its plate-gripping structure 90 with three translational degrees of freedom (e.g., along three orthogonal axes). For example, the plate-gripping structure 90 may travel horizontally along the racks 56 as indicated by arrow 92, horizontally in and out of the racks 56 as indicated by double arrow 94, and vertically. In some embodiments, the plate robot can have separate and individually controllable motors (eg, servo motors) corresponding to each degree of freedom.

[0040] A computer 96, such as a local computing device, uses a processor 98 to control and automate the operation of the incubator 50. The computer may be connected in a wired or wireless manner, shown at 100, to each of the stations, robots, systems, and electrical devices of the incubator 50. These connections may enable the computer to receive and / or send signals from any suitable combination of the stations, robots, systems, and / or devices of the incubator. Thus, the computer may coordinate the operation of the incubator 50 and interface directly with a local user. The computer 96 may also or alternatively interface with a user via a communications network, such as a wide area (telecommunications / computer) network (WAN) 102 (e.g., the Internet) and a remote computing device 104 operated by the user.

[0041] Computer 96 may have any suitable hardware to facilitate communication with and / or operation of processor 98. Exemplary hardware includes memory 106 that stores instructions for processor 98 to perform and / or control any suitable procedures as described herein. An exemplary user interface that may be suitable includes input devices 108 (e.g., keyboard, keypad, mouse, touch screen, etc.) and output devices 110 (e.g., monitor, printer, touch screen, etc.). In some embodiments (e.g., involving a touch screen), the same device may handle input from a user and output from the processor.

[0042] The incubator 50 may include any suitable sensors and perform automated plate and fluid handling, as well as imaging of the cells. The sensors and automation may include, but are not limited to, one or more image sensors, pH sensors, O2 sensors, robotic arms, fluid level sensors, cell media hygiene sensors, temperature sensors, CO2 sensors, automated cell culture media replenishment, and pressure sensors. All of these sensors are remotely operable by a user, and results from any of the sensors may be monitored by a user at any location and at any time via the internet via a remote computing device 104, which may be a mobile device.

[0043] The computer 96 may be capable of acquiring all measurement data and controlling and coordinating the workflow of culturing / assaying cells. Input data may be collected by the computer 96. Exemplary input data includes, among others, any combination of the following: (1) captured images of single or multiple cells in all microplate wells, (2) cell count or cell density / confluency data per well from the captured images, (3) pH and / or oxygen data per well (e.g., for a particular cell culture cycle, if required), (4) colorimetric information from an indicator in the cell culture medium, (5) loading data for transfer of single or multiple cells from bulk solution to the microplate 58 (optional if the process begins with monoclonal cells), and / or (6) potential contamination data for the incubator.

[0044] The computer may perform various actions automatically, as necessary, based on the input data. For example, the computer may determine whether, when, and / or how to, among other things, (1) pump cells into each well when appropriate, (2) correct the pH and / or oxygen levels in the wells by adding appropriate reagents, (3) remove excess medium from the wells, (4) transfer medium / cells from the wells of the microplate 58 to the wells of the assay plate, such as when a defined confluency is reached, (5) report when the microplate 58 is ready for use at a defined level of confluency in at least one well of the microplate 58, (6) pass the cells into one or more new sample holders (e.g., using trypsin or a similar reagent) and restart the incubation process, (7) transfer the cells into a container for storing or freezing the cells outside the system, and / or (8) add a reagent or compound when a defined confluency is reached.

[0045] The computer 96 can generate output data from the input data. Exemplary output data includes any combination of: (1) the growth rate of cells in each well of each microplate, (2) the identification of wells that do not contain viable cells, (3) the identification of contaminated wells, (4) the level of each reagent available, and (5) the response of each cell culture in each well to the addition of test compound(s) and / or reagent(s), if applicable.

[0046] The main chamber 54 may have an atmosphere controlled by the atmosphere control system 112. Exemplary atmosphere parameters of the main chamber 54 that may be monitored and / or adjusted by the atmosphere control system 112 include temperature, gas levels (e.g., CO2, oxygen, etc.), humidity, particulate levels (e.g., by filtering), airborne, and / or surface microbial levels (e.g., by ultraviolet radiation), any combination thereof, or the like. The atmosphere control system may include a thermal control system 114, which may consist of one or more heaters for heating (and maintaining) the main chamber 54 above ambient temperature at a temperature set point, one or more temperature sensors, a set point controller, one or more fans for circulating gas inside the chamber, or the like. The atmosphere control system may also include, among others, a water source for humidification, one or more humidity sensors, a carbon dioxide source (such as a CO2 tank), a CO2 sensor(s), one or more air / gas filters, at least one ultraviolet light source for killing microorganisms in the main chamber before / during use of the incubator, or any combination thereof.

[0047] The main chamber 54 can be accessed via one or more doors, which are represented by the thicker lines in Figs. 1 and 2. A larger maintenance door 116 provides access to all areas inside the incubator and may allow for introduction / removal of consumables (e.g., plates, tips, reagents, etc.), cleaning, disinfection, inspection, or the like. Thus, the maintenance door 116 may be opened while the incubator is being prepared for use and then remain closed while cells are being cultured / assayed. A pair of smaller doors, the inner door 118 and the outer door 120, may allow items (e.g., plates) to be passed in and out of the main chamber 54 via an entry / exit chamber 122 (interchangeably referred to as an interface chamber) while the incubator 50 is operating, without opening the chamber 54 directly to the outside environment. The entry / exit chamber may be sized, among other things, to hold, for example, only one plate or a stack of two or more plates. The entry / exit chambers may include racks for organizing and / or vertically separating plates. The entry / exit chambers 122 communicate with the main chamber 54 via an inner door 118 and with the outside environment via an outer door 120. The doors 118, 120 may be opened and closed sequentially to allow the entry / exit chambers 122 to be loaded and then emptied. For example, the inner door 118 may be opened and closed first to allow automated loading of the entry / exit chambers 122 from the main chamber 54 (e.g., using the plate robot 88), and then the outer door 120 may be opened to allow manual or automated unloading of the entry / exit chambers 122 from the outer incubator 50. Alternatively, the outer door 120 may be opened and closed first to allow manual or automated loading of the entry / exit chamber 122 from the outer incubator 50, and then the inner door 118 may be opened to allow automated loading and unloading of items from the entry / exit chamber 122 into the main chamber 54 (e.g., using the plate robot 88), and then closed. The opening and closing of the or each door 118, 120 may be automatically controlled, and optionally driven by a motor.

[0048] The rack 56 has a plurality of storage locations 124 and supports microplates 58 or other sample holders. The storage locations 124 may be arranged in an array, generally at least a two- or three-dimensional array of such locations 124. For example, at least a subset of the storage locations may be arranged in a plurality of vertical columns and a plurality of horizontal rows, each of which has at least two, three, or more storage locations 124. In some embodiments, the rack 56 may be open on opposite sides such that each storage location 124, and / or the microplates 58 supported by the rack 56 at the storage locations, may be operatively accessed from opposite sides by the detection robot 60 and the plate robot 88, optionally simultaneously with one another. For example, in FIG. 1, the detection robot 60 and the plate robot 88 access each storage location 124 from the right and left sides of the rack 56, respectively, as further described below. Thus, the detection robot 60 and the plate robot 88 may have substantially non-overlapping ranges of motion such that both robots may perform their respective functions simultaneously without interfering with one another.

[0049] A number of local heaters 126 may be incorporated into the rack 56 to allow the lids of the microplates 58 (or other sample holders) to be heated prior to image capture to reduce condensation on the inner surface of each lid. Water droplets on the inner surface may scatter incident optical radiation received from a transillumination light source positioned above the lid. This scattering may degrade the quality of images captured from cells contained in the microplate wells. Each storage location 124 may include a dedicated heater 126, which may be located in an upper portion of the storage location above the corresponding stored microplate 58. For example, the heater may be vertically arranged above the lids of the microplates supported in the storage location, close enough to heat the lids of the microplates when the heaters are energized, but spaced far enough apart to allow the detection robot 60 operative access to each well of the microplate (see also below). The heaters 126 are optionally individually (i.e., independently) controllable for each storage location 124 using the computer 96. Each heater 126 associated with a storage location 124 may be preferably energized prior to imaging of cells located within the storage location to allow sufficient heating time for condensation to be eliminated. For example, the heater may be turned on about 10, 20, or 30 minutes, or 1 hour before imaging is performed. Exemplary heaters that may be suitable include resistive heaters (e.g., sheet heaters), thermoelectric heaters, optical heaters, or the like. Each heater may be under a layer of insulation to minimize undesired heat transfer to the microplate located above the storage location 124 to be heated.

[0050] FIG. 2 shows in schematic form another exemplary smart incubator 150. The incubator 150 may have any of the components described above with respect to the incubator 50, as indicated by the same reference numbers as in FIG. 1. However, the incubator 150 differs from the incubator 50 by separating the storage of the microplates in the racks 56 and the fluid transfer with the fluid handling station 72 into the culture chamber 54a and the fluid handling chamber 54b inside different portions of the housing 52. The culture chamber 54a may be atmospherically controlled by a dedicated atmospheric control system 112, while the fluid handling chamber 54b may or may not be atmospherically controlled by the same or a different atmospheric control system 112, as indicated by the dashed rectangle. Movement of the microplate 58 and other plates inside the chambers 54a, 54b may be performed by respective plate robots 88a, 88b, each of which is capable of gripping the microplate and other plates and moving the plates in three dimensions.

[0051] Each chamber 54a, 54b may be accessed through one or more doors. A large key maintenance door 116a or 116b may provide access to each chamber 54a, 54b as described above with respect to the maintenance door 116 of the incubator 50. The chambers 54a, 54b may be connected to each other through at least one door, which may be controlled by the computer 96. In the depicted embodiment, the chambers 54a, 54b are connected by an inner door 118 and an outer door 120, each of which opens to an entry / exit chamber 122a. The doors 118, 120 may operate as described above with respect to the incubator 50. The fluid handling chamber 54b may be accessed from the outer incubator 150 through the inner door 118 and the outer door 120, each of which opens to an entry / exit chamber 122b as described above with respect to the incubator 50. The microplate 58 may be removed from chamber 54a during operation of the incubator and the plate robot 88a used to transport the microplate to entry / exit chamber 122a and then the plate robot 88b used to transport the microplate to entry / exit chamber 122b. Alternatively, the microplate may be removed from the incubator via entry / exit chamber 122a and maintenance door 116b if atmospheric control and / or contamination within fluid handling chamber 54b is of less concern.

[0052] In some embodiments, detection station 70 may be located within fluid handling chamber 54b. Assay plates prepared in fluid handling chamber 54b may also be incubated in the chamber, optionally at elevated temperatures, and then transported to detection station 70 within chamber 54b for reading the results of the assay.

[0053] 3 shows an exemplary microplate 58 having a plurality of wells 152, each containing a culture of biological cells 154 disposed in a liquid growth medium 156. The wells 152 are formed by a transparent body 158 and covered by a transparent lid 160. The cells are located on the bottom of the wells and, optionally, can be adhered thereto and arranged in a monolayer.

[0054] The incubator 50 may also have an additional detection assembly to monitor the pH, oxygen, and / or carbon dioxide levels of the growth medium inside each well 152 of each microplate 58. The detection assembly may be provided by the detection robot 60 or by the detection station 70, among others. Each microplate well 152 may contain at least one chemical sensor 162 (e.g., at least one sensor spot) to sense the pH, oxygen, and / or carbon dioxide levels as photoluminescence (e.g., fluorescence) from the sensor(s). Each chemical sensor 162 may be mounted on the bottom of the well (shown in solid outline) if single cell detection is not required, or on a sidewall of the well (shown in phantom outline) to allow imaging of the entire bottom of the well. The chemical sensor 162 may be placed in the well 152 when the well 152 is empty and before the cells 154 are added into the liquid growth medium 156. In other embodiments, the chemical sensors 162 may be mounted on additional rods that reach from the microplate lid 160 into the medium 156. Each chemical sensor 162 may be configured to sense, among other things, pH, oxygen, or carbon dioxide when excited with suitable optical radiation. The excitation may induce a detectable photoluminescence characteristic that corresponds to the pH, oxygen concentration, or carbon dioxide concentration within the medium 156. Exemplary commercially available chemical sensors that may be suitable are the self-adhesive pH, oxygen, or CO2 sensor spots manufactured by PreSens Precision Sensing GmbH.

[0055] 3 illustrates one exemplary detection module 164 for the detection robot 60 (or detection station 70). The detection module 164 is operatively matched with a pair of wells 152 of a microplate 58 for imaging of one well and optical detection of photoluminescence from a chemical sensor 162 in an adjacent well 152. The detection module may include a housing 166 (interchangeably referred to as a frame) having an upper arm 168 and a lower arm 170, which may be rigidly attached to one another or actively movable relative to one another (e.g., along a vertical axis).

[0056] The arms 168, 170 are shown fragmentarily here with dashed lines to focus attention on the optical components supported by the arms. At least one trans-illumination light source 62a may be mounted on the upper arm 168 to illuminate each well 152 from above the microplate, as described elsewhere herein. The lower arm 170 may support an objective lens 172 and an image sensor 64. The objective lens may collect and optionally focus optical radiation from the light source 62a that propagates downward through the wells. The image sensor 64 captures images of the cells 154 by detecting the optical radiation.

[0057] The detection module 164 may also be configured to detect photoluminescence from the cells 154, and optionally from the chemical sensor 162. An epi-illumination light source 62b may be supported by the lower arm 170. The light source 62b is shown generating optical radiation for excitation of photoluminescence from the cells 154 in FIG. 3. The optical radiation may propagate along an optical axis 174 to a beam splitter 176, which reflects the optical radiation upward for propagation through the objective lens 172 to the cells 154. The photoluminescence from the cells 154 may be collected by the objective lens 172 for downward propagation through the beam splitter 176 to the image sensor 64.

[0058] The optical properties of the chemical sensor 162 may be detected using any suitable illumination source and optical sensor. Photoluminescence from the chemical sensor 162 may be induced by excitation with any suitable light source, such as the trans-illumination source 62a, the epi-illumination source 62b, or a different epi-illumination source 62c, among others. The photoluminescence may be detected using the image sensor 64 (e.g., in a captured image representative of the cell and the chemical sensor 162, or only in the chemical sensor 162). Alternatively or in addition, the optical properties of the chemical sensor 162 may be detected using a separate optical sensor 177 (e.g., a point sensor), which, along with the source 62c, may be optically coupled to the waveguide 178 via a coupler 180. This arrangement allows the excitation light and the emitted light to propagate in opposing directions along the waveguide 178. In the depicted embodiment, the waveguide 178 and the image sensor 64 are aligned with the chemical sensor 162 and the cells 154 in the adjacent well. In other embodiments, the waveguide 178 and image sensor 64 may be closer to each other so that they can be aligned in parallel with the chemical sensor 162 and the cells 154 in the same well 152. Light from the chemical sensor 162 propagating downward through the bottom wall of the well 152 may be detected as shown here. Alternatively, if the chemical sensor 162 is located on a sidewall (as shown in phantom in FIG. 3), light passing laterally from the well 152 through that sidewall may be detected. In that case, the waveguide 178 may be suitably oriented (e.g., diagonally) and positioned for efficient optical coupling with the chemical sensor according to its sidewall location.

[0059] FIG. 4 illustrates one exemplary lensless imaging configuration for the detection module 164 of the detection robot 60. The bottom of the well 152 and the cells 154 thereon may be illuminated at only one angle or at different angles in a sequence to facilitate capture of an image at each angle. The light source 62a may include an array of light generating elements that can be selectively energized to change the illumination angle or pattern, or the outlet of the light source 62a may be movable to change the illumination angle. The image sensor 64 may be supported by a lower arm 170 below and adjacent to the well 152 of the microplate 58. The cells 154 in the well 152 may be imaged by the image sensor 64 lensless, i.e., without an intervening lens to collect / focus light below the cells (e.g., without the objective lens 172 of FIG. 3). It may be necessary or desirable to remove the lid using the plate robot 88 prior to capture of the lensless image. Further aspects of lensless imaging that may be suitable are described in Section I above.

[0060] III. Sample Incubation and Processing Methods This section describes exemplary methods of sample incubation and processing performed using the smart incubator disclosed herein, as exemplified using incubator 50 (see FIGS. 1-4). The method steps described in this section may be performed in any suitable order and combination, using any of the smart incubator configurations and features of the present disclosure.

[0061] The incubator 50 may be cleaned and decontaminated prior to the start of a cell culture cycle. System liquids (e.g., liquids for the fluid handling station 72) may be filled. Materials such as pipette tips 82 and assay plates 86 in a box may be loaded into the incubator's main chamber 54 via the maintenance door 116. A microplate 58 with a lid 160 may be loaded into the incubator 50. In some cases, a microplate 58 containing cells in liquid growth medium may be prepared outside the incubator 50 and then loaded directly into the appropriate storage location 124 of the rack 56. Alternatively, an empty microplate 58 including a lid 160 may be placed into the incubator 50, such as in the appropriate storage location 124 of the rack 56. The incubator may then automatically load a single cell or multiple cells with the appropriate volume of growth medium into individual wells 152 of the microplate 58. In some embodiments, the cells may be loaded from a bulk solution or from the wells of a master plate. The microplate 58 may be equipped with chemical sensor devices 162 to measure pH and oxygen concentration in all wells 152. An appropriate cell culture protocol may be entered into controlling computer 96 and the process may then commence after an initial priming of chambers 54 with gas and humidity.

[0062] Images may be captured of cells contained within the wells 152 of a microplate 58 (or other sample holder). An image for each microplate may be captured by the image sensor 64 of the detection robot 60 while the microplate remains in its storage position 124 in the rack 56 inside the incubator 50. In other words, the detection robot 60 may move the image sensor 64 into vertical alignment with each well 152 of the microplate while the microplate is supported by the rack 56, and then one or more images of the cells within the well may be captured. Each image may cover the entire bottom of the well or only a portion thereof (e.g., less than one-half the area of ​​the bottom).

[0063] Prior to image capture for a given microplate 58, the lid 160 of the microplate (or other sample holder) may be heated using a heater 126 located above the lid in the rack 56. The heater 126 may be a dedicated heater for only one microplate storage location 124 in the rack 56 and / or may be independently controllable from the heaters 126 for the other microplate storage locations 124. Thus, heating may be performed locally within the incubator and only for short periods of time, minimizing temperature fluctuations within the chamber 54.

[0064] Microplates 58 (or other sample holders) may be moved from racks 56 to a fluid handling station 72 inside the incubator using a plate robot 88. Each microplate may be moved in anticipation of fluid transfer to / from one or more wells 152 of the microplate. Thus, a microplate may be moved if one or more captured images of cells contained by the microplate meet one or more predefined criteria indicating that fluid transfer is necessary or appropriate. The predefined criteria may relate to cell number / confluency / density, morphology, size, or other measurable parameters of cells in one or more wells of the microplate. For example, a microplate may be moved to the fluid handling station 72 for feeding (using a removed volume of culture supernatant), splitting, exposure to test compounds, and / or assaying if one or more wells of the microplate have cells of at least a threshold number / confluency / density. In some embodiments, a user can view the captured images, optionally via the Internet, and determine if / when the computer 96 should move a given microplate to the fluid handling station 72 for fluid addition / removal.

[0065] Fluids may be transferred in and / or out of at least one well of a microplate (or other sample holder) in the fluid handling station 72. The transferred fluid may include liquid growth (culture) medium. For example, an old volume of culture medium in at least one well may be replaced with a new volume of culture medium. In other cases, the transferred fluid may contain a test compound. For example, a test compound may be added to at least one well in a volume of carrier liquid without changing the culture medium. In still other cases, the transferred fluid may contain a volume of supernatant and / or cells from at least one well and transferred to a well of another plate. Supernatants may be transferred for testing in any suitable type of assay. Cells may be transferred for testing and / or subculture.

[0066] IV. Working Examples This section describes selected aspects and embodiments of the present disclosure related to smart incubators and methods implemented using smart incubators. Any suitable aspects of the incubators and methods described in this section may be combined with each other and / or with any suitable aspects of the incubators and methods disclosed anywhere in this disclosure. These examples are intended for illustrative purposes only and should not limit or define the overall scope of the present disclosure. EXAMPLES

[0067] Smart Incubator Embodiment This example illustrates selected aspects of one embodiment 250 of the smart incubator 50 of Section II (see FIGS. 5-8).

[0068] Figures 5, 6 and 7 respectively show a side view, a cross-sectional top view and a cross-sectional end view of an incubator 250. The incubator is generally divided into a storage station 252 on the left side in Figures 5 and 6 and a fluid handling station 72 on the right side.

[0069] The storage station 252 includes a rack 56 that holds a plurality of microplates 58 arranged in horizontal rows and vertical columns. The rack has a frame made of vertical wall members 254 mounted on the bottom of the enclosure 52. The horizontal wall members 256 attach adjacent vertical wall members 254 to one another above the bottom. A respective heater 126 is located on the bottom side of each horizontal wall member 256. A bracket 258 is mounted to the vertical wall members 254 to create a storage location 124 for supporting a microplate 58 below each heater 126. The relative vertical positions of the horizontal wall members 256 and brackets 258 create suitable clearance around the microplate 58 in the storage location 124 for access by the detection robot 60 (above and below the microplate) and the plate robot 88 (below and / or adjacent opposite lateral sides of the microplate).

[0070] The detection robot 60 and the plate robot 88 are also operable within the storage station 252. The robots 60, 88 are configured to access the microplates 58 from respective opposite sides of the rack 56. The detection robot 60 has a carrier 260 that can be driven horizontally, indicated at 66, along the front side of the rack 56 on a front rail 262 (see Figs. 6 and 7). Similarly, the plate robot 88 has a carrier 264 that can be driven horizontally, indicated at 92, along the back side of the rack 56 on a back rail 266. The back rail 266 is longer than the front rail 262, allowing the plate robot 88 to travel outside the storage station 252 within the chamber 54. This greater range of travel allows the plate robot 88 to move plates (such as the microplates 58) between the rack 56 and the fluid handling station 72, within the fluid handling station, and / or to / from the door.

[0071] The detection robot 60 and the plate robot 88 each have a respective tower 268, 270 supported by lateral rails 272 or 274 of the carrier 260 or 264. The tower 268 can be driven in a horizontal direction indicated at 68 along the rails 272 (see FIG. 7). The tower 270 can be driven in a horizontal direction indicated at 94 along the rails 274. The detection module 164 of the detection robot 60 can be driven in a vertical direction indicated at 276 along the tower 268. Similarly, the gripping head 278 of the plate robot 88 can be driven in a vertical direction indicated at 280 along the tower 270. The gripping head 278 has a pair of jaws 282 for gripping and / or supporting a respective microplate, such as a gripped microplate 284, during its transport.

[0072] 5 and 6 also show exemplary features of the fluid handling station 72 in further detail. The plate dock 74 may have storage locations for more than one plate (see FIG. 6). For example, in the depicted embodiment, a microplate 58 (with deep wells) and a master plate 292 are stored within the plate dock 74. The master plate 292 may be substituted for one of the assay plates 86 within the plate dock 74 when the fluid handling station 72 is tasked with setting up an assay. The box of tips 82c has been moved into the plate dock 74 from the stack of boxes containing new tips 82a. Tips 82c are positioned midway between the plates 58, 292 for utilization by the pipette 78.

[0073] The pipette 78 can be driven along three orthogonal axes via a nested series of three carriages (and associated motors) that travel along respective sets of rails (see Figures 5 and 6). The z-carrier 294 travels along rails 296, as indicated by arrow 298, to raise and lower the working end of the pipette 78 (see Figure 5). The x-carrier 300 travels along rails 302, as indicated by arrow 304, to move the pipette 78 horizontally along the x-axis. The y-carrier 306 travels along rails 308, as indicated by arrow 310, to move the pipette 78 horizontally along the y-axis.

[0074] 7 and 8 show exemplary features of the detection robot 60 in further detail. The arms 168, 170 of the detection module 164 are positioned above and below the microplate 58, respectively. The trans-illumination light source 62a and the image sensor 64 are aligned with one of the wells 152 of the microplate 58. The light source 62a illuminates the cells 154 through the lid 160 of the microplate 58 with a light beam 312. The image sensor 64 is positioned very close to the bottom of the well 152 without an intervening lens for lensless imaging of the cells 154. EXAMPLES

[0075] SELECTED EMBODIMENTS The present examples describe selected systems and methods of the present disclosure as a series of indexed paragraphs.

[0076] Paragraph A1. An incubation system for automated cell culturing and / or testing comprising: (i) a housing forming a chamber; (ii) a rack defining storage positions for supporting an array of sample holders (such as microplates) inside the chamber; (iii) a detection robot for capturing one or more images of cells contained by one or more wells (e.g., each well) of each sample holder while the sample holder remains in one of the storage positions of the rack; (iv) a fluid handling station configured to add and / or remove fluid from one or more wells (e.g., each well) of each of the sample holders inside the housing; (v) at least one plate robot for moving the sample holders between the rack and the fluid handling station; and (vi) a computer for controlling operation of the detection robot, the fluid handling station, and the at least one plate robot.

[0077] Paragraph A2. The incubation system of paragraph A1, wherein the detection robot is configured to capture images by lensless imaging.

[0078] Paragraph A3. An incubation system as described in paragraph A1 or A2, wherein the detection robot is configured to lenslessly capture holograms representing cells located in one or more wells of each sample holder, and the computer is configured to use the captured holograms to reconstruct one or more images of cells located in the one or more wells.

[0079] Paragraph A4. An incubation system as described in paragraph A3, wherein the detection robot is configured to illuminate cells located in one or more wells of each sample holder with light that is at least partially coherent.

[0080] Paragraph A5. An incubation system as described in paragraph A3 or A4, wherein the detection robot is configured to illuminate the cells using light propagated through the waveguide, and the outlet of the waveguide (e.g., an optical fiber or liquid light guide) is configured to travel together with the detection robot to each of the storage positions of the rack.

[0081] Paragraph A6. The incubation system of paragraph A5, wherein the outlet of the waveguide has a diameter of less than about 100 micrometers.

[0082] Paragraph A7. The incubation system of paragraph A5 or A6, wherein the light source is optically coupled to the waveguide such that light generated by the light source is transmitted from an outlet of the waveguide.

[0083] Paragraph A8. The incubation system of paragraph A7, wherein the light source is configured to be supported by the detection robot and travel with the detection robot to each of the storage locations.

[0084] Paragraph A9. The incubation system of paragraph A7, wherein the light source is configured to remain stationary while the detection robot advances to each of the storage locations.

[0085] Paragraph A10. An incubation system described in any of paragraphs A1 to A9, wherein the detection robot is configured to illuminate cells in one or more wells of each sample holder using light generated by a point source.

[0086] Paragraph A11. An incubation system described in any of paragraphs A1 to A10, wherein the detection robot is configured to illuminate cells in one or more wells of each sample holder using light generated by a light emitting diode, a laser diode, or a diode-pumped solid-state laser.

[0087] Paragraph A12. An incubation system according to any of paragraphs A1 to A11, wherein the detection robot includes an image sensor, and the image sensor includes a CCD sensor or a CMOS sensor.

[0088] Paragraph A13. An incubation system according to any of paragraphs A1-A12, wherein the fluid handling station is configured to dispense fluids into and remove fluids from wells of a sample holder inside the chamber.

[0089] Paragraph A14. An incubation system as described in paragraph A13, wherein the chamber is an incubation chamber, the housing forms an entry / exit chamber sized to contain one or more of the sample holders, the incubation chamber communicates with the entry / exit chamber via an inner door, and the entry / exit chamber is accessible from outside the housing via an outer door.

[0090] Paragraph A15. The incubation system of any of paragraphs A1-A14, wherein the fluid handling station includes one or more storage locations for pipettes and containers that hold removable tips for the pipettes.

[0091] Paragraph A16. The incubation system of paragraph A15, wherein the fluid handling station includes a first stack position for a container holding a new tip for the pipette and a second stack position for a container holding a tip already used by the pipette.

[0092] Paragraph A17. An incubation system according to any of paragraphs A1-A16, wherein the fluid handling station includes a sample holder and a receiving area for one of the assay plate and / or master plate.

[0093] Paragraph A18. The incubation system of any of paragraphs A1-A17, wherein the fluid handling station includes a lid robot for removing and replacing the lid of the sample holder.

[0094] Paragraph A19. An incubation system described in any of paragraphs A1 to A18, wherein the at least one plate robot includes a first plate robot and a second plate robot, the housing has a door that bounds a portion of the chamber, the first plate robot configured to transport the sample holder from the rack to or near the door, and the second plate robot configured to transport the sample holder from or near the door to a fluid handling station outside the chamber.

[0095] Paragraph A20. The incubation system described in paragraph A19, wherein the enclosure has an inner door and an outer door, and the first plate robot and the second plate robot are configured to cooperate to move each sample holder from the rack to the fluid handling station through the inner door and the outer door.

[0096] Paragraph A21. The incubation system of paragraph A20, wherein the chamber is a first chamber and the fluid handling station is located within a second chamber that communicates with the first chamber via an inner door and an outer door.

[0097] Paragraph A22. An incubation system described in any of paragraphs A1 to A21, wherein the rack has a first side opposite a second side, and a plate robot among the at least one plate robot is configured to move sample holders in and out of the storage position via the first side of the rack, and a detection robot is configured to operatively access the sample holders in the storage position via the second side of the rack.

[0098] Paragraph A23. An incubation system as described in paragraph A22, wherein the storage locations of the racks are arranged in horizontal rows and vertical columns, and the plate robot and the detection robot are collectively capable of simultaneously operatively accessing a pair of storage locations in the same horizontal row and simultaneously operatively accessing a pair of storage locations in the same vertical column.

[0099] Paragraph A24. An incubation system described in any of paragraphs A1 to A23, wherein the rack includes a plurality of heaters operatively associated with the storage positions and configured to heat lids of sample holders in the storage positions to reduce condensation prior to image capture using the detection robot.

[0100] Paragraph A25. The incubation system of paragraph A24, wherein a subset of the heaters are controllable independently of one another using a computer.

[0101] Paragraph A26. The incubation system of paragraph A24 or A25, wherein different heaters of the plurality of heaters are operatively associated with each storage location of the rack and are computer controllable independently of each other heater of the plurality of heaters.

[0102] Paragraph A27. The incubation system of any of paragraphs A1-A26, further comprising a thermal control system configured to maintain the chamber at a predefined incubation temperature above room temperature.

[0103] Paragraph A28. The incubation system of any of Paragraphs A1-A27, further comprising a carbon dioxide source connected to the chamber.

[0104] Paragraph A29. The incubation system of any of paragraphs A1-A28, further comprising a water source configured to humidify the chamber.

[0105] Paragraph A30. An incubation system described in any of paragraphs A1-A29, wherein the storage locations of the rack are arranged in horizontal rows and vertical columns, each horizontal row and each vertical column having at least two, at least three, or more of the storage locations.

[0106] Paragraph A31. An incubation system described in any of paragraphs A1 to A30, further comprising a detection station configured to detect light from one or more wells (e.g., each well) of each sample holder, and at least one plate robot configured to transport each of the sample holders between the rack and the detection station.

[0107] Paragraph A32. The incubation system of paragraph A31, wherein the detection station is configured to detect photoluminescence from one or more wells (e.g., each well) of each sample holder.

[0108] Paragraph A33. An incubation system as described in paragraph A31 or A32, wherein the detection station includes a lid robot for removing and replacing the lid of the sample holder, and the detection station includes a waveguide, such as an optical fiber, configured to be placed within each well of each sample holder.

[0109] Paragraph A34. An incubation system according to any of paragraphs A1-A33, wherein the computer is configured to transmit captured images of cells in the wells of the sample holder to a remote user via the Internet.

[0110] Paragraph A35. An incubation system as described in paragraph A34, wherein the computer is configured to receive instructions from a remote user via the Internet, the instructions telling the computer whether to treat and / or how to treat the culture of cells depicted in the transmitted image.

[0111] Paragraph A36. An incubation system as described in paragraph A34 or A35, wherein the computer is configured to receive, via the Internet, instructions from a remote user for processing the culture of cells depicted in the transmitted image.

[0112] Paragraph A37. An incubation system described in any of paragraphs A1-A36, wherein the computer is configured to determine whether and / or when to add fluid to and / or remove fluid from one or more wells of each sample holder at the fluid handling station based on the captured image.

[0113] Paragraph A38. An incubation system as described in paragraph A37, wherein the computer is configured to determine characteristics such as density / confluency / cell number of cells contained by one or more wells of each sample holder from one or more images captured by the detection robot, and based on the characteristics, determine whether and / or when to add fluid to and / or remove fluid from such wells.

[0114] Paragraph A39. An incubation system described in any of paragraphs A1 to A38, further comprising a sample holder including a pH sensor device and / or an oxygen sensor device disposed within one or more wells of the sample holder, wherein the incubation system includes an optical sensor configured to detect photoluminescence from the sensor device(s).

[0115] Paragraph A40. The incubation system of paragraph A39, wherein the detection robot includes an optical sensor and is configured to illuminate the sensor device to induce photoluminescence.

[0116] Paragraph A41. An incubation system according to any of Paragraphs A1 to A40, configured to carry out any method or method step of any of Paragraphs B1 to B25.

[0117] Paragraph B1. A method of automated cell culturing and / or testing comprising: (i) capturing one or more images of cells contained within one or more wells of each sample holder of a plurality of sample holders, the plurality of sample holders being stored in storage positions defined by a rack inside an incubator, the sample holder remaining in its storage position in the rack when an image is captured for the sample holder; (ii) moving the sample holder from the rack using a plate robot to a fluid handling station inside the incubator; and (iii) removing fluid from and / or adding fluid to at least one well of the sample holder at the fluid handling station.

[0118] Paragraph B2. The method of paragraph B1, wherein capturing the one or more images is performed lenslessly.

[0119] Paragraph B3. The method of paragraph B1 or B2, wherein capturing one or more images includes illuminating the cells with at least partially coherent light.

[0120] Paragraph B4. The method of any of paragraphs B1-B3, wherein illuminating the cell includes propagating light for illumination through a waveguide upstream of the cell.

[0121] Paragraph B5. The method of paragraph B3, wherein capturing one or more images includes capturing two or more images, the two or more images being of at least a similar field of view of the illuminated cell, but representing (i) different z-positions of an image sensor capturing the two or more images, (ii) illumination at different angles of incidence, (iii) illumination at different wavelengths, and / or (iv) a sub-pixel shift in lateral position of the image sensor and / or light source.

[0122] Paragraph B6. The method of any of paragraphs B1-B5, wherein capturing one or more images includes capturing a hologram.

[0123] Paragraph B7. The method of paragraph B6, further comprising reconstructing an image of the cell by digitally processing data from the captured hologram.

[0124] Paragraph B8. The method of paragraph B7, wherein reconstructing the image includes reading out a phase from the captured hologram.

[0125] Paragraph B9. The method of any of paragraphs B1-B8, wherein capturing one or more images includes capturing one or more images for each well of a plurality of wells of the sample holder and processing the one or more images to obtain a value corresponding to confluence / density / cell number of cells in the well, and the moving step is performed for the sample holder if the value(s) for one or more wells meet one or more predefined criteria.

[0126] Paragraph B10. A method as described in any of paragraphs B1-B9, further comprising transmitting to a user one or more images of the cells resulting from the step of capturing one or more images, receiving a command from the user to perform a moving step, and removing and / or adding fluid at the fluid handling station in response to the command.

[0127] Paragraph B11. The method described in paragraph B10, wherein the capturing and moving of one or more images is controlled by a local computer and the transmitting and receiving steps are performed by communication between a remote computing device operated by a user and communicating with the local computer via the Internet.

[0128] Paragraph B12. The method of paragraphs B10 or B11, further comprising receiving instructions from a user to remove and / or add fluid.

[0129] Paragraph B13. The method of any of paragraphs B10-B12, wherein the capturing and moving of the one or more images is controlled by a local computer, and the method further includes displaying the one or more images to a user using a monitor of the local computer.

[0130] Paragraph B14. The method of any of paragraphs B1-B13, wherein two or more of the sample holders are vertically aligned with one another within the rack.

[0131] Paragraph B15. The method of any of paragraphs B1-B14, further comprising locally and / or temporarily applying heat to each sample holder in an incubator to reduce condensation on its lid prior to capturing one or more images of the sample holder.

[0132] Paragraph B16. The method of paragraph B15, wherein the applying of heat is performed using a dedicated heater for each sample holder.

[0133] Paragraph B17. The method of paragraphs B15 or B16, wherein the applying of heat is performed using multiple heaters, each positioned above a different sample holder in the rack.

[0134] Paragraph B18. The method of any of paragraphs B15-B17, wherein the applying of heat is performed for a predefined period of time before one or more images are captured of the sample holder.

[0135] Paragraph B19. The method of any of paragraphs B1-B18, wherein removing and / or adding fluid includes transferring fluid from at least one well to a well of the assay plate.

[0136] Paragraph B20. The method of paragraph B19, further comprising performing the assay in a well of an assay plate.

[0137] Paragraph B21. The method of paragraph B20, wherein the assay comprises an ELISA assay.

[0138] Paragraph B22. The method of paragraph B20 or B21, further comprising reading the results from the wells of the assay plate outside the incubator.

[0139] Paragraph B23. The method of any of paragraphs B19-B22, wherein capturing one or more images is performed within a main chamber of an incubator, and the method further comprises using a plate robot to transport the assay plate from the main chamber to an entry / exit chamber of the incubator.

[0140] Paragraph B24. The method of any of paragraphs B1-B23, wherein removing and / or adding fluid includes removing old culture medium from at least one well and adding new culture medium to at least one well and delivering cells into at least one well.

[0141] Paragraph B25. The method of any of Paragraphs B1-B24, wherein removing and / or adding fluid includes adding a test compound contained within a fluid to at least one of the wells.

[0142] Paragraph B26. The method of any of paragraphs B1-B25, implemented using any of the systems or system features recited in paragraphs A1-A40.

[0143] The term "exemplary" as used in this disclosure means to "illustrate" or "serve as an example." Similarly, the term "exemplary" means to "illustrate by giving an example." Neither term implies desirability or superiority.

[0144] The disclosure described above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein should not be considered in a limiting sense, since numerous variations are possible. The subject matter of the invention includes all novel and non-obvious combinations and subcombinations of the various elements, features, functions, and / or properties disclosed herein. The following claims specifically point out certain combinations and subcombinations that are considered to be novel and non-obvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and / or properties may also be claimed in applications claiming priority from this or related applications. Such claims are also considered to be included within the subject matter of the invention of the present disclosure, whether directed to different or the same invention, and whether broader, narrower, equal, or different in scope relative to the original claims. Furthermore, ordinal indicators such as first, second, or third for identified elements are used to distinguish between the elements and do not indicate a particular position or order of such elements, unless specifically stated otherwise.

Claims

1. An automated incubation system (50, 150) for culturing and / or testing cells, comprising: a housing (52) forming a chamber (54); a rack (56) defining a plurality of storage positions (124) for supporting a plurality of sample holders (58) inside the chamber (54), each sample holder (58) of the plurality of sample holders (58) comprising one or more wells (152); a detection robot (60) for capturing one or more images of cells (154) contained by the one or more wells (152) of each sample holder (58) while the sample holder remains in one of the plurality of storage positions (124) of the rack (56), the detection robot (60) being movable along three orthogonal axes to reach each of the one or more wells (152) of each sample holder (58), the three orthogonal axes being an x-axis extending horizontally parallel to the plurality of storage positions (124) of the rack (56), a y-axis extending horizontally into and out of the rack (56) orthogonal to the x-axis, and a z-axis extending perpendicular to the rack (56) orthogonal to the x-axis and y-axis; a fluid handling station (72) configured to add fluid to and / or remove fluid from the one or more wells (152) of each of the sample holders (58) inside the housing (52); at least one plate robot (88) for moving sample holders (58) between said racks (56) and said fluid handling stations (72); a computer (96) for controlling the operation of said detection robot (60), said fluid handling station (72), and said at least one plate robot (88); An incubation system (50, 150).

2. The incubation system (50, 150) of claim 1, wherein the detection robot (60) is configured to capture images by lensless imaging.

3. 3. The incubation system (50, 150) of claim 2, wherein the detection robot (60) is configured to lenslessly capture one or more holograms representing cells (154) located in the one or more wells (152) of each sample holder (58), and the computer (96) is configured to use the captured holograms to reconstruct images of the cells (154) located in the wells (152).

4. 4. The incubation system (50, 150) of claim 3, wherein the detection robot (60) is configured to illuminate cells (154) using light propagated through a waveguide (178), and an outlet of the waveguide (178) is configured to travel with the detection robot (60) to each of the plurality of storage positions (124) of the rack (56).

5. 2. The incubation system (50, 150) of claim 1, wherein the fluid handling station (72) is configured to dispense fluid into and remove fluid from the one or more wells (152) of the sample holder (58) inside the chamber (54).

6. 2. The incubation system (50, 150) of claim 1, wherein the chamber (54) is an incubation chamber, the housing (52) forms an entry / exit chamber (122) sized to contain one or more sample holders (58), the incubation chamber communicates with the entry / exit chamber (122) via an inner door (118), and the entry / exit chamber (122) is accessible from outside the housing (52) via an outer door (120).

7. 2. The incubation system (50, 150) of claim 1, wherein the fluid handling station (72) includes one or more storage locations for a pipette (78) and a container for holding a removable tip (82) for the pipette (78).

8. 8. The incubation system (50, 150) of claim 7, wherein the one or more storage locations include a first stack position for a container holding a new removable tip (82) for the pipette (78) and a second stack position for a container holding a removable tip (82) already used by the pipette (78).

9. 2. The incubation system (50, 150) of claim 1, wherein the fluid handling station (72) includes a receiving location for at least one of the sample holders (58) and an assay plate (86).

10. 2. The incubation system (50, 150) of claim 1, wherein the rack (56) has a first side opposite a second side, the at least one plate robot (88) comprises a first plate robot configured to move sample holders (58) in and out of the plurality of storage positions (124) via the first side of the rack (56), and the detection robot (60) configured to operatively access sample holders (58) in the plurality of storage positions (124) via the second side of the rack (56).

11. 2. The incubation system (50, 150) of claim 1, wherein the rack (56) includes a plurality of heaters (126) operatively associated with the plurality of storage locations (124) of the rack (56) and configured to heat lids (160) of sample holders (58) in the plurality of storage locations (124) to reduce condensation prior to image capture using the detection robot (60).

12. 2. The incubation system (50, 150) of claim 1, wherein the computer (96) is configured to transmit one or more of the captured images to a remote user via the Internet, and the computer (96) is configured to receive instructions from the remote user regarding whether and / or how to treat a culture of cells (154) depicted in the transmitted images.

13. The incubation system (50, 150) of claim 1, wherein the computer (96) is configured to determine whether and / or when to add fluid to and / or remove fluid from the one or more wells (152) of each sample holder (58) at the fluid handling station (72) based on images captured by the detection robot (60).

14. The incubation system (50, 150) of claim 13, wherein the computer (96) is configured to determine characteristics of the cells (154) contained by the one or more wells (152) of each sample holder (58) from images captured by the detection robot (60) and, based on the characteristics, determine whether and / or when to add fluid to and / or remove fluid from the one or more wells (152).

15. 2. The incubation system (50, 150) of claim 1, further comprising a sample holder (58) including a pH sensor device and / or an oxygen sensor device (162) disposed in the one or more wells (152) of the sample holder (58), and the incubation system (50, 150) including an optical sensor (64) configured to detect photoluminescence from the pH sensor device and / or the oxygen sensor device (162).

Images