Patent Description:
<CIT> discloses a cell culture incubator comprising: an incubator cabinet having an internal chamber for incubation of cells in one or more cell culture vessels, a holographic imager configured for imaging said cells within said internal chamber when said one or more cell culture vessels are at a first imaging location. A storage location within said internal chamber for storing said one or more cell culture vessels and a cell culture vessel transfer device for moving said one or more cell culture vessels from said first imaging location to said storage location or from said storage location to said first imaging location are provided.

The present disclosure provides an incubation system and method for automated cell culture and/or testing. The incubation system, also referred to as an incubator, comprises a housing forming a chamber. The incubation system further comprises the following: A rack defining storage positions to support an array of sample holders (e.g., microplates) inside the chamber. A detection robot configured to capture one or more images of cells contained by one or more wells of each sample holder while the sample holder remains at one of the storage positions of the rack, wherein the detection robot is movable along three orthogonal axes, to reach every well of each sample holder supported by the rack. A fluid handling station configured to add fluid to, and/or remove fluid from, one or more wells of each of the sample holders
inside the housing. At least one plate robot configured to move sample holders between the rack and the fluid handling station. A computer configured to control operation of the detection robot, the fluid handling station, and the at least one plate robot.

The present disclosure provides an incubation system and method for automated cell culture and/or testing. An exemplary incubation system may comprise a housing forming a chamber. A rack may define storage positions to support 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 holder remains at one of the storage positions of the rack. A fluid handling station may be configured to add fluid to, 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 sample holders between the rack and the fluid handling station. A computer may control operation of the detection robot, the fluid handling station, and the at least one plate robot.

An exemplary method of automated cell culture and/or testing is provided. In the method, one or more images may be captured of cells contained in one or more wells of each sample holder of a plurality of sample holders. The plurality of sample holders may be stored at storage positions defined by a rack inside an incubator. The sample holder may remain in its storage position in the rack as the one or more images are captured for the sample holder. 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.

The current state of the art in incubators does not include any built-in intelligence and/or decision-making tools or attributes. Moreover, incubators are not remote-controlled, and cells in an incubator cannot be followed to check for viability or the need for media replacement, addition of test compounds, or any other step a user may have to perform.

The incubator of the present disclosure may enable automated short-term and long-term culture in sample holders, such as microplates, to allow monitoring of multiple parameters over the time-course of cultivation. A controlling computer may automatically take action, or alert lab personnel, if data collected by one or more sensors of the incubator meet one or more predefined criteria. For example, the computer may feed cells (e.g., change the growth medium), split cells, add a test compound(s) to cells, create assay mixtures, or the like, if captured images of cell cultures indicate an action is needed, or may notify one or more lab personnel (i.e., users) of a situation needing attention. The incubator can automate cell culture, improve the health of cells, and provide better in situ experimentation all by better knowledge of cell health and cell growth.

The smart incubators disclosed herein may be used for any suitable purpose. They can reduce labor and optimize workflow for assay development and compound testing. Clonal populations of cells may be cultured, fed, and assayed. Organ-on-chip cultures may be cultivated and tested in non-microplate settings. Mini-bioreactors may provide cell line development, with mixing of cells and media added.

Further aspects of the present disclosure are described in the following sections: (I) definitions, (II) overview of smart incubators, (III) methods of sample incubation and processing, and (IV) examples.

Technical terms used in this disclosure have meanings that are commonly recognized by those skilled in the art. However, the following terms may be further defined as described below.

Chemical sensor-a device and/or a compound (e.g., a sensor dye) that detects or measures the concentration of a chemical analyte, such as free protons (for pH), oxygen, carbon dioxide, or the like. The chemical sensor may have an optical property that is sensitive to the concentration of the chemical analyte. The optical property may include photoluminescence intensity, photoluminescence lifetime, photoluminescence polarization, photoluminescence quenching/energy transfer, chemiluminescence intensity, absorbance, or the like. The optical property of the chemical sensor may be read using an 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 in a matrix, to localize the sensor compound within a larger fluid volume (e.g., a volume of culture medium in a microplate well). The matrix may be attachable to a surface, such as with an adhesive. The surface may be an inside surface of a well, and the optical property may be detected through a wall of the well.

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

Examination region - an area located on an optical axis of a detection system at which an object, such as a sample (e.g., biological cells), can be examined optically (e.g., imaged with an image sensor).

Image - a representation of light detected at a two-dimensional or three-dimensional array of positions by an image sensor (e.g., a raw image), or a processed form thereof (e.g., a reconstructed image). A raw image may be an optically focused image or a hologram (i.e., a lensless image) created by lensless imaging, among others.

Image sensor - an optical sensor capable of detecting spatial variations (e.g., variations in intensity) of light in two dimensions, where the light is incident on a photosensitive 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 (monochromatic) images, or both.

Lensless imaging - capture of a diffraction pattern(s) for an object(s) (e.g., a sample including biological cells) using an image sensor. Lensless imaging is performed without interposing a lens between the object and the image sensor,. The captured diffraction pattern (a hologram) can be called an image of the object(s) (e.g., an image of cells), even though the image is not an optically focused image. Since the image is not optically focused, the captured image generally is neither magnified nor minified relative to the object(s) (i.e., capture is with "unit magnification"). Accordingly, the photosensitive area of the image sensor should be at least as large as the floor of a well, if the entire floor is to be covered with the same lensless image. However, exemplary wells to be imaged may have a larger floor area than the photosensitive area of the image sensor. For example, the wells may be provided by six-well microplates or a single-well (e.g., rectangular) sample holder, among others. Accordingly, multiple lensless images for overlapping regions of the same well may be stitched together to generate a larger image representing more or all of the well's floor. Lensless imaging can include digital holographic reconstruction imaging, shadow imaging, or fluorescence imaging, or among others.

Lensless imaging may be performed with any suitable light source, which may epi-illuminate or trans-illuminate a sample, among others. 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., with an incoherent light-emitting element and/or use of a diffuser in the illumination path). In some embodiments, the light source may trans-illuminate the sample with substantially plane wave illumination from an at least partially coherent light source. For example, the light source may be significantly farther than the image sensor from the sample along a z-axis, such as at least five or ten times farther. In some embodiments, the light source may include a light-emitting element optically coupled to a waveguide. The outlet of the waveguide may be mounted to an arm, which may be rotatable to change the sample illumination angle, and/or may be movable linearly to introduce sub-pixel shifts in the captured image.

Lensless images may be processed by pixel super-resolution techniques. Pixel super-resolution can produce sub-pixel resolution by laterally shifting the light source, the image sensor, and/or the sample, to create "sub-pixel" images, and then these sub-pixel images can be merged to obtain a smaller effective size of pixel.

Lensless images also or alternatively may be processed by phase-retrieval techniques. The captured raw image is an inline hologram containing intensity data. Lensless imaging can be used to reconstruct quantitative phase-contrast, which yields a representation for the volume of an object (e.g., cell) by means of pixel intensity. Moreover, lensless imaging can provide a large depth of field, so there may be no need for focusing. To retrieve the amplitude and phase of the sample, the phase-retrieval algorithm(s) used may require more than one hologram (due to the twin-image problem). Accordingly, the calculations for phase retrieval are advantageously performed with holographic image data from multiple holograms with similar fields of view (e.g., holograms captured with different sample-to-image sensor distances, different illumination angles, and/or different illumination wavelengths (e.g., using a tunable laser or light sources (e.g., laser diodes or LEDs) emitting at different wavelengths)).

Lensless imaging may be advantageous for the systems and methods of the present disclosure because bulky collection/detection optics are not required. Accordingly, microplates can be stored closer to one another in a vertical column of the microplates, because less space is needed for image capture under each microplate. The resulting incubation system is more compact and space-efficient.

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

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 also may include any associated optical element(s) to shape, size, filter, polarize, scatter, direct, and/or otherwise interact with light emitted by the light-emitting element(s). These optical elements may include any combination of at least one waveguide (e.g., a fiber optic or liquid light guide), lens, mirror, filter, diffuser, mask, aperture, beam-splitter, grating, prism, polarizer, and/or the like. Exemplary light-emitting elements include a semiconductor device, laser (e.g., excimer laser, gas laser, dye laser, solid-state laser, semiconductor crystal or diode laser, free electron laser, etc.), arc lamp, and/or the like. Exemplary semiconductor light-emitting elements include laser diodes, light-emitting diodes (LEDs), and superluminescent diodes, among others.

Microplate - a sample holder including a plurality of connected wells. The terms "microplate" and "plate" are interchangeable herein. The wells may be arranged in a planar, rectangular array, with the same uniform spacing along orthogonal horizontal axes, to form a plurality of rows and columns The wells within 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 between <NUM> microliter and <NUM> milliliters. The dimensions of the microplate may be according to American National Standards Institute (ANSI) and Society for Laboratory Automation and Screening (SLAS) (i.e., ANSI/SLAS) standards. For example, the microplate may have a <NUM> by <NUM> array of wells (such as <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> wells), a width of about <NUM>, a length of about <NUM>, a height of about <NUM>, and/or a well-to-well separation that is inversely related to the total number of wells. Exemplary well spacings include <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, among others. Each well may have a flat bottom to facilitate imaging. The microplate may be formed of a transparent polymer.

The microplate may include a lid to cover each well of the microplate. The lid is removable to permit dispensing to, and aspiration from, wells of the microplate.

Optics - a set of optical elements of an imaging system, which may be arranged along an optical axis between a light source and an examination region (illumination optics) and/or along an optical axis between the examination region and an optical sensor (collection optics). An optical element may be any device or structure that interacts with light, such as to collect, direct, focus, filter, polarize, scatter, collimate, and/or partially block 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, waveguides, polarizers, and the like.

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 to detect spatial differences in 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.

Robot - a device capable of moving and carrying out a series of actions under the control of a computer. Exemplary robots include a detection robot to detect light and/or capture images from plates, a plate robot to transport plates, a pipette robot to transfer liquid (e.g., into or out of wells of plates), a lid robot to remove and replace lids, and/or the like.

Sample - a specimen having any suitable properties. The sample may be organic and/inorganic, natural and/or manufactured, and may include any suitable assembly, material, substance, isolate, extract, particles, or the like. In exemplary embodiments, 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., a blood sample, a fluid aspirate, a tissue section, etc.), clones of cells, or the like. A cell culture may include a set of cells, optionally 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 water, salt, buffer, glucose, detergent, dye, protein, amino acids, or any combination thereof, among others. The liquid medium may be a growth medium for the cells.

Sample holder - a device for holding at least one sample or any array of spatially isolated samples, and optionally permitting the sample(s) to be imaged through a horizontal, transparent wall of the device (e.g., the bottom wall of a well). Exemplary sample holders are culture vessels including one or more wells, such as microplates, Petri dishes, cell culture flasks, etc..

This section provides an overview of automated incubation systems ("smart incubators") for processing samples held by sample holders, such as culturing and/or assaying biological cells held in wells of sample holders (as exemplified herein with microplates); see <FIG>.

<FIG> shows an exemplary smart incubator <NUM> in schematic form. Incubator <NUM> includes a housing <NUM> (e.g., a box) defining a main chamber <NUM>, which may remain closed during operation of the incubator. The incubator also may have a storage structure <NUM> (interchangeably called a rack) for holding and organizing multiple sample holders, such as microplates <NUM>, within main chamber <NUM>, while cells contained by wells of the sample holders are being cultured.

Optical detection may be conducted in main chamber <NUM>. Robotics may be used to move at least one optical sensor to every well of each microplate <NUM> (or other sample holder), for every microplate (or sample holder) stored in rack <NUM>. More particularly, a detection robot <NUM> may optically sense contents of the sample holders, and particularly contents in each well thereof, while the sample holders remain in their respective storage sites within rack <NUM>. For example, a detection robot <NUM> may capture images of cells growing in the wells. The detection robot may include a detection module having at least one light source <NUM> for illumination of a well and/or contents thereof, and at least one optical sensor, such as an image sensor <NUM>, arranged to detect optical radiation from the illuminated well and/or contents, as described in more detail below. Detection robot <NUM> may be controllable to drive movement of the detection module thereof with at least three degrees of freedom (e.g., along three orthogonal axes), for optical alignment with each well of each microplate <NUM> (or other sample holder) of rack <NUM>. More particularly, the detection robot and/or the detection module thereof, may travel horizontally, parallel to an array of storage locations defined by rack <NUM>, indicated by arrows at <NUM>, horizontally into and out of rack <NUM>, orthogonal to the vertical array, indicated by a double-headed arrow at <NUM>, and vertically. In some embodiments, detection robot <NUM> may have a separate, individually controllable motor (e.g., a servomotor) corresponding to each degree of translational freedom. Alternatively, or in addition, contents of microplates <NUM> or other sample holders may be optically sensed at a detection station <NUM> in main chamber <NUM> (or elsewhere in incubator <NUM>), where the detection station is separate from rack <NUM>. Detection station <NUM> may have a light source(s) and an optical sensor(s) as described herein for detection robot <NUM> but may be relatively fixed within main chamber <NUM> (or elsewhere in in incubator <NUM>).

Detection robot <NUM> (and/or detection station <NUM>) allows cell growth or development to be monitored. The detection robot provides a, preferably compact, label-free, imaging system for capturing one or more images of cells from every well of every microplate <NUM> (or other sample holder). The imaging system is movable along three orthogonal axes, to reach every well of each microplate <NUM> supported by rack <NUM>. This ensures that the optical axis (defined by the center of every microplate well to be imaged) is always vertical.

Detection robot <NUM> may include at least one light source <NUM> to be placed above and/or into each well of each microplate <NUM> (or other sample holder). Light source <NUM> may be a single light source having one light outlet optically coupled to a single upstream light-emitting element or a plurality of upstream light-emitting elements (e.g., of different color). The light source may have compact collimation optics and/or may utilize a diffuser near or at the light outlet. In some embodiments, light source <NUM> may be coupled or configured to be coupled to a special plate lid having a light guide reaching down into every well, deep enough to touch a liquid medium in the well, thereby avoiding an optical effect of the liquid meniscus. In some embodiments, light source <NUM> may transmit light from a light outlet having a small diameter (e.g., in the range of the size of a cell (such as a point source having a diameter of less than about <NUM>, <NUM>, <NUM>, or <NUM>)), which provides a light source with high brilliance. For example, the light source may include a laser diode, mounted at a fixed place. A flexible fiber optic may be optically coupled to the laser diode and configured to act as a light guide for propagation of light from the laser diode, through the fiber, to a light outlet above a microplate well to be imaged. The light outlet may be movable in a horizontal plane with respect to the optical axis, to adjust the angle of illumination, which allows higher resolution from computer-aided image reconstruction. In some embodiments, the light source may include an array of compact point light sources each having a small diameter (e.g., less than about <NUM>, <NUM>, <NUM>, or <NUM>), for example, an organic LED display as a matrix of small light sources that can be controlled individually. This configuration permits single point illumination or a pattern of illumination by a combination of light sources of the matrix, which may provide higher resolution from computer-aided image reconstruction as well as enable illumination enhancement strategies for compensating negative effects of the liquid meniscus.

Detection robot <NUM> also may include at least one image sensor <NUM> to be placed under each microplate (or other sample holder). Each image sensor <NUM> may be configured to capture an image of the entire floor of a microplate well (and the cells thereon). Alternatively, the entire floor (and cells thereon) may be imaged by capturing a plurality of 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 directly to the illumination rays from one or multiple point sources above the microplate, without any intervening optics between the bottom of microplate <NUM> and the image sensor to focus light rays. In some embodiments, image sensor <NUM> may be lenslessly exposed to the illumination rays from a source of diffuse light located above the microplate, if only low optical resolution is needed. However, imaging may be performed with any type of imaging in any of the incubation systems disclosed herein.

A fluid handling station <NUM> in main chamber <NUM> may perform transfer of fluid into and out of wells of microplates <NUM> and/or other plates. The fluid handling station may have a plate dock <NUM> with one or more plate receiving sites (interchangeably called docking sites) to hold one or more microplates and/or other plates at predefined positions. Plate dock <NUM> may be fixed or movable. The plate dock may provide a position for a master plate, typically with deep wells, which may be loaded into the incubator via a dedicated door, or via one of the other doors described elsewhere herein. A lid robot <NUM> may be configured to remove lids from plates located in plate dock <NUM>, to permit fluid addition to, and/or removal from, wells of the plates with at least one pipette <NUM>, and to replace the lids when fluid transfer is complete. Each pipette <NUM> may include a pump <NUM> to drive fluid into and/or out of an end of the pipette, a motor-driven positioner to move the end of the pipette precisely in three dimensions, and/or a tip ejector to remove pipette tips <NUM> from the end of pipette <NUM> after use. The pipette(s) may be able to operatively access each well of each plate located in plate dock <NUM>, for fluid addition and/or removal. Pipette tips <NUM> may be stored in at least one of two different stack positions (e.g., to create respective stacks of tip boxes) in the fluid handling station. At least one of the stack positions may be employed for storing new pipette tips that have not yet been used by the pipette. At least one other of the stack positions may be employed for storing pipette tips that have already been used by the pipette. Any suitable culture media and reagents <NUM> may be accessible to pipette <NUM> of fluid handling station <NUM> for uptake and/or dispensing. The media and reagents may be contained in vessels (e.g., bottles), which may be stored inside or outside chamber <NUM> and/or housing <NUM>. Exemplary reagents include buffered saline, trypsin, assay solutions, test compounds, and the like. At least one stack of assay plates <NUM> (and/or transfer plates) may be stored in fluid handling station <NUM> and transferred individually (e.g., by a plate robot), as needed, to one of the plate receiving sites of plate dock <NUM> when assay mixtures are to be created in wells of assay plates <NUM>.

Assay plates <NUM> may be used to test the supernatant of cell cultures contained in wells of microplates <NUM>, for the presence/level of a given analyte and/or activity (e.g., a binding activity of a monoclonal antibody). Each microplate <NUM> may be moved to plate dock <NUM> and a sample of the supernatant contained in one or more of the wells may be transferred to one or more wells of an assay plate <NUM>. Each assay plate may be an ELISA plate having wells coated with a reagent (e.g., an antibody, an epitope, or the like). In other examples, each assay plate <NUM> may be an uncoated plate that will be moved out of the incubator to an external liquid handler. In yet other examples, each assay plate <NUM> may receive supernatant from a well and reagents to support a homogeneous test, which may be performed inside or outside the incubator.

Test compounds and/or liquid for feeding cells may be added to and/or removed from wells of microplates <NUM> at fluid handling station <NUM>, or in a separate, dedicated station inside the incubator, or while microplates <NUM> remain in rack <NUM>. Accordingly, compound addition and/or feeding cells (e.g., changing media by removal of old media and addition of new media) may be performed using a reagent dispenser (fluid addition only), a low-volume pipettor (fluid addition and removal), or microfluidics plates (fluid addition and removal). The microfluidics plates may be hydraulically connected at each storage position of rack <NUM>, at fluid handling station <NUM>, or in a separate dedicated station. Single cell dispensing may be performed in the incubator by or near fluid handling station <NUM>.

A plate robot <NUM> may transport microplates <NUM> within main chamber <NUM>, and optionally out of the main chamber. The plate robot may transport microplates <NUM> to and from individual storage positions within rack <NUM>. More generally, plate robot <NUM> may move microplates <NUM> within, between, or among rack <NUM>, detection station <NUM>, and/or fluid handling station <NUM>. The plate robot also may move boxes of pipette tips <NUM> and/or assay plates <NUM> within fluid handling station <NUM> and/or chamber <NUM>. Plate robot <NUM> may be controllable to drive movement of a plate-grasping structure <NUM> thereof with three degrees of translational freedom (e.g., along three orthogonal axes). For example, plate-grasping structure <NUM> may travel horizontally along rack <NUM>, indicated by arrows at <NUM>, horizontally into and out of rack <NUM>, indicated by a double-headed arrow at <NUM>, and vertically. In some embodiments, the plate robot may have a separate, individually controllable motor (e.g., a servomotor) corresponding to each degree of freedom.

A computer <NUM>, such as a local computing device, controls and automates operation of incubator <NUM> using a processor <NUM>. The computer may be connected, in a wired or wireless fashion, indicated at <NUM>, to each of the stations, robots, systems, and electrical devices of incubator <NUM>. These connections may permit the computer to receive signals from and/or send signals to any suitable combination of stations, robots, systems, and/or devices of the incubator. Accordingly, the computer coordinates operation of incubator <NUM>, and may interface with a local user directly. Computer <NUM> also or alternatively may interface with a user via a communications network, such as a wide area (telecommunications/computer) network (WAN) <NUM> (e.g., the Internet), and a remote computing device <NUM> operated by the user.

Computer <NUM> may have any suitable hardware to facilitate communication with, and/or operation of, processor <NUM>. Exemplary hardware includes memory <NUM> storing instructions for processor <NUM> to perform and/or control any suitable procedures, as described herein. Exemplary user interfaces that may be suitable include an input device <NUM> (e.g., a keyboard, keypad, mouse, touchscreen, etc.) and an output device <NUM> (e.g., a monitor, printer, touchscreen, etc.). In some embodiments (e.g., with a touchscreen), the same device may handle input from the user and output from the processor.

Incubator <NUM> may include any suitable sensors and may perform automated plate and fluid handling and imaging of cells. The sensors and automation may include, but are not limited to, one or more image sensors, pH sensors, O<NUM> sensors, robotic arms, fluid level sensors, cell media health sensors, temperature sensors, CO<NUM> 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 anywhere, anytime by a user over the Internet via a remote computing device <NUM>, which may be a mobile device.

Computer <NUM> may obtain all measurement data and be able to control and coordinate the workflow of cultivating/assaying cells. Input data may be collected by computer <NUM>. Exemplary input data includes any combination of the following: (<NUM>) captured images of single or multiple cells in every microplate well, (<NUM>) cell count or cell density/confluence data for each well from captured images, (<NUM>) pH and/or oxygen data for every well (e.g., if needed for a specific cell culture cycle), (<NUM>) colorimetric information from an indicator in the cell culture medium, (<NUM>) loading data for transfer of single cells or cells from a bulk solution to microplates <NUM> (optional if run starts with monoclonal cells), and/or (<NUM>) potential contamination data for the incubator, among others.

The computer may perform various actions automatically, as needed, based on input data. For example, the computer may decide whether, when, and/or how to perform any of the following: (<NUM>) feeding cells in each well when appropriate, (<NUM>) correcting pH and/or oxygen levels in wells by adding appropriate reagents, (<NUM>) removing excess media from wells, (<NUM>) transferring media/cells from wells of microplates <NUM> to wells of assay plates, such as when a defined confluency is reached, (<NUM>) reporting when microplates <NUM> are ready to be used at a defined level of confluency in at least one well of microplate <NUM>, (<NUM>) passaging cells (e.g., using trypsin or similar reagents) into one or more new sample holders to restart the incubation process, (<NUM>) transferring cells into containers for storing or freezing cells outside the system, and/or (<NUM>) adding reagents or compounds once a defined confluency is reached, among others.

Computer <NUM> may create output data from input data. Exemplary output data includes any combination of (<NUM>) the growth rate of cells in each well of each microplate, (<NUM>) identification of wells not containing living cells, (<NUM>) identification of contaminated wells, (<NUM>) the level of each reagent available, and (<NUM>) the response, if any, of each cell culture in each well to addition of a test compound(s) and/or reagent(s).

Main chamber <NUM> may have a climate that is controlled by a climate control system <NUM>. Exemplary climate parameters of main chamber <NUM> that may be monitored and/or regulated by climate control system <NUM> include temperature, gas levels (e.g., CO<NUM>, oxygen, etc.), humidity, particulate levels (e.g., by filtering), airborne and/or surface microorganism levels (e.g., by ultraviolet radiation), any combination thereof, or the like. The climate control system may include a thermal control system <NUM>, which may be composed of one or more heaters to heat (and maintain) main chamber <NUM> above the ambient temperature at a temperature set point, one or more temperature sensors, a set point controller, one or more fans to circulate gas inside the chamber, or the like. The climate control system also may include a source of water for humidification, one or more humidity sensors, a source of carbon dioxide (such as a CO<NUM> tank), a CO<NUM> sensor(s), one or more air/gas filters, at least one ultraviolet light source to kill microorganisms within the main chamber before/during use of the incubator, or any combination thereof, among others.

Main chamber <NUM> can be accessed via one or more doors, which are represented in <FIG> and <FIG> by heavier lines. A larger maintenance door <NUM> may provide access to all areas inside the incubator, to allow introduction/removal of consumables (e.g., plates, tips, reagents, etc.), cleaning, disinfection, servicing, or the like. Accordingly, maintenance door <NUM> may be opened while the incubator is being readied for use, and then may remain closed while cells are being cultured/assayed. A pair of smaller doors, namely, an inner door <NUM> and an outer door <NUM>, may permit items (e.g., plates) to be passed into and out of main chamber <NUM> via an entry/exit chamber <NUM> (interchangeably called an interface chamber), while incubator <NUM> is operating, and without opening chamber <NUM> directly to the external environment. The entry/exit chamber may, for example, be sized to hold only one plate or a stack of two or more plates, among others. The entry/exit chamber may include a rack to organize and/or vertically separate plates. Entry/exit chamber <NUM> communicates with main chamber <NUM> via inner door <NUM> and with the external environment via outer door <NUM>. Doors <NUM>, <NUM> may be opened and closed sequentially to allow entry/exit chamber <NUM> to be loaded and then emptied. For example, inner door <NUM> may be opened and closed first, to allow automated loading of entry/exit chamber <NUM> from main chamber <NUM> (e.g., using plate robot <NUM>), and then outer door <NUM> may be opened to allow manual or automated unloading of entry/exit chamber <NUM> from outside incubator <NUM>. Alternatively, outer door <NUM> may be opened and closed first, to allow manual or automated loading of entry/exit chamber <NUM> from outside incubator <NUM>, and then inner door <NUM> may be opened, to allow automated unloading of items (e.g., using plate robot <NUM>), from entry/exit chamber <NUM> into main chamber <NUM>, and then closed. Opening and closing of inner door <NUM> or each door <NUM>, <NUM> may be controlled automatically, and optionally driven by a motor.

Rack <NUM> has a plurality of storage positions <NUM> to support microplates <NUM> or other sample holders. Storage positions <NUM> may be arranged in an array, generally at least a two-dimensional or a three-dimensional array of such positions <NUM>. For example, at least a subset of the storage positions may be arranged in a plurality of vertical columns and a plurality of horizontal rows, with each column and row having at least two, three, or more storage positions <NUM>. In some embodiments, rack <NUM> may be open on opposite sides, such that each storage position <NUM> and/or a microplate <NUM> supported by rack <NUM> at the storage position, can be operatively accessed from the opposite sides by detection robot <NUM> and plate robot <NUM>, optionally at the same time as one another. For example, in <FIG>, detection robot <NUM> and plate robot <NUM> access each storage position <NUM> from the right and left sides, respectively, of rack <NUM>, as described further below. Accordingly, detection robot <NUM> and plate robot <NUM> may have ranges of motion that do not substantially overlap, so that both robots can perform their respective functions at the same time without interfering with one another.

A plurality of local heaters <NUM> may be incorporated into rack <NUM> to enable lids of microplates <NUM> (or other sample holders) to be heated before image capture, to reduce condensation on the inside surface of each lid. Water droplets on the inside surface can scatter incident optical radiation received from a trans-illumination light source positioned above the lid. This scattering may degrade the quality of images captured from the cells contained in microplate wells. Each storage position <NUM> may include a dedicated heater <NUM>, which may be located in the upper portion of the storage position, above the corresponding stored microplate <NUM>. For example, the heater may be arranged vertically above the lid of a microplate supported in the storage position, close enough to heat the microplate lid when the heater is energized, but spaced sufficiently to permit detection robot <NUM> to operatively access each well of the microplate (also see below). Heaters <NUM> are controllable with computer <NUM>, optionally individually (i.e., independently) for each storage position <NUM>. Each heater <NUM> associated with a storage position <NUM> may be energized suitably in advance of imaging cells located in the storage position, to allow sufficient heating time for condensation to be eliminated. For example, the heater may be turned on about <NUM>, <NUM>, or <NUM> minutes or one 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 underlie a layer of insulation, to minimize undesired heat transfer to a microplate located above the storage position <NUM> to be heated.

<FIG> shows another exemplary smart incubator <NUM> in schematic form. Incubator <NUM> may have any of the components described above for incubator <NUM>, as indicated by the same reference numbers as in <FIG>. However, incubator <NUM> differs from incubator <NUM> by separating microplate storage in rack <NUM>, and fluid transfer with fluid handling station <NUM>, to a culture chamber 54a and a fluid handling chamber 54b inside different portions of housing <NUM>. Culture chamber 54a may be climate-controlled by a dedicated climate control system <NUM>, while fluid handling chamber 54b may or may not be climate controlled by the same or a different climate control system <NUM>, as indicated by a dashed rectangle. Movement of microplates <NUM> and other plates inside chambers 54a, 54b may be performed by respective plate robots 88a, 88b, each capable of grasping microplates and other plates and moving them in three dimensions.

Each chamber 54a, 54b may be accessed through one or more doors. A large maintenance door 116a or 116b may provide access to respective chambers 54a, 54b, as described above for maintenance door <NUM> of incubator <NUM>. Chambers 54a, 54b may be connected to one another via at least one door, which may be controlled by computer <NUM>. In the depicted embodiment, chambers 54a, 54b are connected by an inner door <NUM> and an outer door <NUM>, each of which opens to an entry/exit chamber 122a. Doors <NUM>, <NUM> may operate as described above for incubator <NUM>. Fluid handling chamber 54b may be accessed from outside incubator <NUM> via an inner door <NUM> and an outer door <NUM>, each of which opens to an entry/exit chamber 122b, as described above for incubator <NUM>. A microplate <NUM> may be removed from chamber 54a during operation of the incubator using plate robot 88a to transport the microplate to entry/exit chamber 122a, and then plate robot 88b to transport the microplate to entry/exit chamber 122b. Alternatively, the microplate may be removed from incubator via entry/exit chamber 122a and maintenance door 116b if climate control and/or contamination in fluid handling chamber 54b is of less concern.

In some embodiments, detection station <NUM> may be located in fluid handling chamber 54b. Assay plates prepared in fluid handling chamber 54b also may be incubated in the chamber, optionally at an elevated temperature, and then transported within chamber 54b to detection station <NUM> for reading results of assays.

<FIG> shows an exemplary microplate <NUM> having a plurality of wells <NUM> each containing a culture of biological cells <NUM> disposed in a liquid growth medium <NUM>. Wells <NUM> are formed by a transparent body <NUM> and covered by a transparent lid <NUM>. Cells may be located on the floor of the well, optionally adhered thereto, and arranged in a monolayer.

Incubator <NUM> also may have an additional detection assembly to monitor the pH, oxygen, and/or carbon dioxide level of growth media inside each well <NUM> of each microplate <NUM>. The detection assembly may be provided by detection robot <NUM> or by detection station <NUM>, among others. Each microplate well <NUM> may contain at least one chemical sensor <NUM> (e.g., at least one sensor spot) to sense pH, oxygen, and/or carbon dioxide levels as photoluminescence (e.g., fluorescence) from the sensor(s). Each chemical sensor <NUM> may be mounted on the floor of the well (shown in solid outline), if single cell detection is not required, or on a side wall of the well (shown in phantom outline) to allow imaging of the entire well bottom. Chemical sensors <NUM> may be placed into wells <NUM> when they are empty, before cells <NUM> are added in liquid growth medium <NUM>. In other embodiments, chemical sensors <NUM> may be mounted on additional rods reaching into medium <NUM> from microplate lid <NUM>. Each chemical sensor <NUM> may be configured to sense pH, oxygen, or carbon dioxide, among others, when excited with suitable optical radiation Excitation may induce a detectable photoluminescence characteristic that corresponds to the pH, oxygen concentration, or carbon dioxide concentration in medium <NUM>. Exemplary commercially-available chemical sensors that may be suitable are self-adhesive pH, oxygen, or CO<NUM> sensor spots from PreSens Precision Sensing GmbH.

<FIG> shows an exemplary detection module <NUM> for detection robot <NUM> (or detection station <NUM>). Detection module <NUM> is operatively aligned with a pair of wells <NUM> of microplate <NUM> for imaging of one well and optical detection of photoluminescence from chemical sensor <NUM> in an adjacent well <NUM>. The detection module may include a housing <NUM> (interchangeably termed a frame) having an upper arm <NUM> and a lower arm <NUM>, which may be firmly attached to one another or actively movable relative to one another (e.g., along a vertical axis).

Arms <NUM>, <NUM> are shown as dashed and fragmentary here to focus attention on the optical components supported by the arms. At least one trans-illumination light source 62a may be mounted to upper arm <NUM> for illuminating each well <NUM> from above the microplate, as described elsewhere herein. Lower arm <NUM> may support an objective <NUM> and an image sensor <NUM>. The objective may collect and optionally focus optical radiation from light source 62a that has propagated downward through the well. Image sensor <NUM> captures images of cells <NUM> by detecting the optical radiation.

Detection module <NUM> also may be configured to detect photoluminescence from cells <NUM>, and, optionally, chemical sensor <NUM>. An epi-illumination light source 62b may be supported by lower arm <NUM>. Light source 62b is shown generating optical radiation for excitation of photoluminescence from cells <NUM> in <FIG>. The optical radiation may propagate along an optical axis <NUM> to a beam-splitter <NUM>, which reflects the optical radiation upward, for propagation through objective <NUM> to cells <NUM>. Photoluminescence from cells <NUM> can be collected by objective <NUM> for propagation downward through beam-splitter <NUM> to image sensor <NUM>.

An optical property of chemical sensor <NUM> may be detected using any suitable illumination source and optical sensor. Photoluminescence from chemical sensor <NUM> may be induced by excitation with any suitable light source, such as trans-illumination source 62a, epi-illumination source 62b, or a different epi-illumination source 62c, among others. Photoluminescence may be detected with image sensor <NUM> (e.g., in a captured image representing cells and chemical sensor <NUM>, or chemical sensor <NUM> alone). Alternatively, or in addition, an optical property of chemical sensor <NUM> may be detected using a separate optical sensor <NUM> (e.g., a point sensor), which may be optically coupled to a waveguide <NUM>, along with source 62c, via a coupler <NUM>. This arrangement allows excitation light and emitted light to propagate in opposite directions along waveguide <NUM>. In the depicted embodiment, waveguide <NUM> and image sensor <NUM> are aligned with chemical sensor <NUM> and cells <NUM> in adjacent wells. In other embodiments, waveguide <NUM> and image sensor <NUM> may be closer to one another, such that they can be concurrently aligned with chemical sensor <NUM> and cells <NUM> in the same well <NUM>. Light from chemical sensor <NUM> that has propagated downward through the bottom wall of well <NUM> may be detected, as shown here. Alternatively, light may be detected that has passed laterally from well <NUM> through a side wall thereof, if chemical sensor <NUM> is located on the side wall (as shown in phantom in <FIG>). In that case, waveguide <NUM> may be oriented and positioned suitably (e.g., obliquely) for efficient optical coupling with the chemical sensor according to its side wall location.

<FIG> shows an exemplary lensless imaging configuration for detection module <NUM> of detection robot <NUM>. The floor of well <NUM> and cells <NUM> thereon may be illuminated at only one angle, or successively at different angles to facilitate capture of an image at each angle. Light source 62a may include an array of light-generating elements that can be selectively energized to change the illumination angle or pattern, or an outlet of light source 62a may be movable to change the illumination angle. Image sensor <NUM> may be supported by lower arm <NUM> under and adjacent a well <NUM> of microplate <NUM>. Cells <NUM> in well <NUM> may be imaged lenslessly by image sensor <NUM>, that is, without an intervening lens to gather/focus light below the cells (e.g., without objective <NUM> of <FIG>). It may be necessary or desirable to remove the lid with plate robot <NUM> before capture of lensless images. Further aspects of lensless imaging that may be suitable are described above in Section I.

This section describes exemplary methods of sample incubation and processing performed with the smart incubators disclosed herein, as exemplified with incubator <NUM> (see <FIG>). 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.

Incubator <NUM> may be cleaned and decontaminated before the start of a cell culture cycle. System liquids (e.g., liquids for fluid handing station <NUM>) may be filled. Materials, such as pipette tips <NUM> in boxes and assay plates <NUM>, may be loaded into main chamber <NUM> of the incubator via maintenance door <NUM>. Microplates <NUM> having lids <NUM> may be loaded into incubator <NUM>. In some cases, microplates <NUM> containing cells in liquid growth medium may be prepared outside incubator <NUM> and then loaded directly into the appropriate storage positions <NUM> of rack <NUM>. Alternatively, empty microplates <NUM>, including lids <NUM>, may be placed into incubator <NUM>, such as in appropriate storage positions <NUM> of rack <NUM>. The incubator then may automatically load single cells or multiple cells into individual wells <NUM> of microplate <NUM> along with the appropriate volume of growth medium. In some embodiments, the cells may be loaded from bulk solution or from wells of a master plate. Microplates <NUM> may be equipped with chemical sensor devices <NUM> for measuring pH and oxygen concentration in every well <NUM>. An appropriate cell culture protocol may be inputted to control computer <NUM>, and then the run may be started after initial priming of chamber <NUM> with gas and humidity.

Images may be captured of cells contained in wells <NUM> of microplates <NUM> (or other sample holders). The images for each microplate may be captured by an image sensor <NUM> of a detection robot <NUM>, while the microplate remains in its storage position <NUM> in a rack <NUM> inside incubator <NUM>. In other words, detection robot <NUM> may move image sensor <NUM> into vertical alignment with each well <NUM> of a microplate while the microplate is supported by rack <NUM>, and then one or more images of cells in the well may be captured. Each image may cover the entire floor of the well, or only a portion thereof (e.g., less than one-half the floor's area).

Before image capture for a given microplate <NUM>, lid <NUM> of the microplate (or other sample holder) may be heated with a heater <NUM> located above the lid in rack <NUM>. Heater <NUM> may be a dedicated heater for only one microplate storage position <NUM> within rack <NUM>, and/or may be controllable independently of heaters <NUM> for other microplate storage positions <NUM>. Accordingly, heating may be performed locally within the incubator, and for only a short time, to minimize temperature fluctuations in chamber <NUM>.

Microplates <NUM> (or other sample holders) may be moved from rack <NUM> to fluid handling station <NUM> inside the incubator using a plate robot <NUM>. Each microplate may be moved in anticipation of fluid transfer to/from one or more wells <NUM> of the microplate. Accordingly, the 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 needed or appropriate. The predefined criteria may relate to cell number/confluence/density, morphology, size, or other measurable parameters of cells in one or more wells of the microplate. For example, the microplate may be moved to fluid handling station <NUM> for feeding splitting, exposure to a test compound, and/or assaying (using a removed volume of the culture supernatant), if one or more wells of the microplate have at least a threshold number/confluence/density of cells. In some embodiments, a user may view captured images, optionally via the Internet, and decide whether/when computer <NUM> should move a given microplate to fluid handling station <NUM> for fluid addition/removal.

Liquid may be transferred into and/or out of at least one well of the microplate (or other sample holder) at fluid handling station <NUM>. The fluid transferred may include liquid growth (culture) medium. For example, an old volume of culture medium in the at least one well may be replaced with a new volume of culture medium. In other cases, the fluid transferred may contain a test compound. For example, the test compound may be added to the at least one well in a volume of carrier liquid, without changing the culture medium. In yet other cases, the fluid transferred may contain a volume of supernatant and/or cells from the at least one well, and may be transferred to a well of another plate. Supernatant may be transferred for testing in any suitable type of assay. Cells may be transferred for testing and/or subculturing.

This section describes selected aspects and embodiments of the present disclosure related to smart incubators and methods performed with smart incubators. Any suitable aspects of incubators and methods described in this section may be combined with one another and/or with any suitable aspects of the incubators and methods disclosed elsewhere in the present disclosure. These examples are intended for illustration only and should not limit or define the entire scope of the present disclosure.

This example describes selected aspects of an embodiment <NUM> of smart incubator <NUM> of Section II; see <FIG>.

<FIG>, <FIG>, and <FIG> respectively show a side view, a sectional top view, and a sectional end view of incubator <NUM>. The incubator is generally divided into a storage station <NUM>, on the left in <FIG> and <FIG>, and a fluid handling station <NUM> on the right.

Storage station <NUM> includes a rack <NUM> holding a plurality of microplates <NUM> arranged in horizontal rows and vertical columns. The rack has a frame composed of vertical wall members <NUM> mounted on a floor of housing <NUM>. Horizontal wall members <NUM> attach adjacent vertical wall members <NUM> to one another above the floor. A respective heater <NUM> is located on a bottom side of each horizontal wall member <NUM>. Brackets <NUM> are mounted to vertical wall members <NUM> to create a storage position <NUM> for supporting a microplate <NUM> under each heater <NUM>. Suitable clearance around microplate <NUM> in storage position <NUM> for access by a detection robot <NUM> (above and below the microplate) and a plate robot <NUM> (below and/or adjacent opposite lateral sides of the microplate) is created by the relative vertical positions of horizontal wall members <NUM> and brackets <NUM>.

Detection robot <NUM> and plate robot <NUM> also are operative in storage station <NUM>. Robots <NUM>, <NUM> are configured to access microplates <NUM> from respective opposite sides of rack <NUM>. Detection robot <NUM> has a carriage <NUM> that can be driven horizontally, indicated at <NUM>, along the front side of rack <NUM>, on front rails <NUM> (see <FIG> and <FIG>). Similarly, plate robot <NUM> has a carriage <NUM> that can be driven horizontally, indicated at <NUM>, along the back side of rack <NUM>, on back rails <NUM>. Back rails <NUM> are longer than front rails <NUM>, to permit plate robot <NUM> to travel outside storage station <NUM> in chamber <NUM>. This larger range of travel allows plate robot <NUM> to move plates (such as microplates <NUM>) between rack <NUM> and fluid handling station <NUM>, within the fluid handling station, and/or to/from a door.

Each of detection robot <NUM> and plate robot <NUM> has a respective tower <NUM>, <NUM> supported by transverse rails <NUM> or <NUM> of carriage <NUM> or <NUM>. Tower <NUM> can be driven horizontally along rails <NUM>, indicated at <NUM> (see <FIG>). Tower <NUM> can be driven horizontally along rails <NUM>, indicated at <NUM>. A detection module <NUM> of detection robot <NUM> can be driven vertically along tower <NUM>, indicated at <NUM>. Similarly, a grasping head <NUM> of plate robot <NUM> can be driven vertically along tower <NUM>, indicated at <NUM>. Grasping head <NUM> has a pair of jaws <NUM> to grip and/or support each microplate, such as gripped microplate <NUM>, during transport thereof.

<FIG> and <FIG> also show exemplary features of fluid handling station <NUM> in more detail. Plate dock <NUM> may have seating positions for two or more plates (see <FIG>). For example, in the depicted embodiment, a microplate <NUM> and a master plate <NUM> (with deep wells) are seated in plate dock <NUM>. Master plate <NUM> may be replaced in plate dock <NUM> with one of assay plates <NUM> when fluid handling station <NUM> is tasked with setting up an assay. A box of tips 82c has been moved into plate dock <NUM> from a stack of boxes containing new tips 82a. Tips 82c are located intermediate plates <NUM>, <NUM> for utilization by pipette <NUM>.

Pipette <NUM> can be driven along three orthogonal axes via a nested series of three carriages (and associated motors) that move along respective sets of rails (see <FIG> and <FIG>). A z-carriage <NUM> travels along rails <NUM>, indicated by an arrow at <NUM>, to raise and lower the working end of pipette <NUM> (see <FIG>). An x-carriage <NUM> travels along rails <NUM>, indicated by an arrow at <NUM>, to move pipette <NUM> horizontally along an x-axis. A y-carriage <NUM> travels along rails <NUM>, indicated by an arrow at <NUM>, to move pipette <NUM> horizontally along a y-axis.

<FIG> and <FIG> show exemplary features of detection robot <NUM> in more detail. Arms <NUM>, <NUM> of detection module <NUM> are positioned respectively above and below a microplate <NUM>. A trans-illumination light source 62a and an image sensor <NUM> are aligned with one of wells <NUM> of microplate <NUM>. Light source 62a is illuminating cells <NUM> through a lid <NUM> of microplate <NUM> with a light beam <NUM>. Image sensor <NUM> is positioned very close to the bottom of well <NUM>, without an intervening lens, for lensless imaging of cells <NUM>.

Claim 1:
An incubation system (<NUM>, <NUM>) for automated cell culture and/or testing, the system comprising:
a housing (<NUM>) forming a chamber (<NUM>);
a rack (<NUM>) defining storage positions (<NUM>) to support an array of sample holders (<NUM>) inside the chamber (<NUM>);
a detection robot (<NUM>) to capture one or more images of cells (<NUM>) contained by one or more wells (<NUM>) of each sample holder (<NUM>) while the sample holder remains at one of the storage positions (<NUM>) of the rack (<NUM>), wherein the detection robot (<NUM>) is movable along three orthogonal axes, to reach every well (<NUM>) of each sample holder (<NUM>) supported by the rack (<NUM>);
a fluid handling station (<NUM>) configured to add fluid to, and/or remove fluid from, the one or more wells (<NUM>) of each of the sample holders (<NUM>) inside the housing (<NUM>);
at least one plate robot (<NUM>) to move sample holders (<NUM>) between the rack (<NUM>) and the fluid handling station (<NUM>); and
a computer (<NUM>) to control operation of the detection robot (<NUM>), the fluid handling station (<NUM>), and the at least one plate robot (<NUM>).