Cell culture incubator with integrated cell manipulation system

Abstract

Aspects of the present invention relate to automated cell culture incubators. In some embodiments, the automated cell culture incubator includes an integrated manipulator with one or more cell scrapers.

Description

[0001] Related applications

[0002] This invention claims the benefit of U.S. Provisional Application USSN 62 / 141,191, filed March 31, 2015, entitled “Cell Culture Incubators With Integrated Cell Manipulation Systems”, pursuant to 35 U.SC119(e), the entire contents of which are incorporated herein by reference. Technical Field

[0003] Several aspects of the present invention relate to cell culture apparatus and methods of using such apparatus. Background Technology

[0004] Cell culture is a useful technique in both research and clinical settings. For example, mammalian cell culture is often performed to establish clonal cell lines, tissue preparations, in vitro fertilization preparations, or to expand stem cell populations. However, maintaining cell cultures in currently available cell culture vessels is a laborious process, requiring highly trained personnel and strict aseptic conditions. For instance, to passage adherent cells with adequate cell coverage, the adherent cells must be detached from the cell culture dish in which they have multiplied and attached. Typically, this process involves removing the cell culture dish from a controlled environment (e.g., a culture vessel) to a cell culture hood and manually passaged the cells. This high level of human involvement can introduce contaminants into the culture or damage the cells, reducing culture efficiency and reproducibility. Summary of the Invention

[0005] Currently available cell culture equipment presents several obstacles to long-term cell culture in production. For example, many currently available cell culture equipment require the removal of culture plates from the culture vessel for cell manipulation. Generally, removing cell culture plates from the culture vessel increases the threat of contamination of the culture because, upon removal, the culture is exposed to non-sterile conditions and / or changes in the physical environment (e.g., changes in temperature, humidity, etc., or any combination thereof).

[0006] This article provides a cell culture system with an integrated manipulation device, such as having one or more cell scrapers, which can reduce the exposure of the culture to contaminants, external environment and / or changes in the culture environment (e.g., passage of these cells) by eliminating the need to remove the culture dish from the culture vessel to maintain the cell culture.

[0007] Therefore, in some aspects, this document provides a cell culture apparatus comprising: a culture cabinet including an internal chamber for culturing cells in one or more cell culture dishes (e.g., wherein the internal chamber is configured to hold the one or more cell culture dishes); an external section providing access from the external environment to the internal chamber; an imager (e.g., an imager and an imaging position) configured to image cells inside the internal chamber (e.g., when the one or more cell culture dishes are in the imaging position); a manipulator (e.g., a manipulator and a manipulator position) having one or more cell scrapers for manipulating cells in the one or more cell culture dishes inside the internal chamber (e.g., detaching adherent cells from the cell culture dishes, cleaning the cell culture dishes); and a cell culture dish transfer device for moving the one or more cell culture dishes between multiple positions inside the internal chamber (e.g., from the imaging position to the manipulator position or from the manipulator position to the imaging position).

[0008] In some embodiments, the manipulator includes one or more cell scrapers. In some embodiments, each cell scraper includes a handle portion comprising an elongated member extending from a proximal region to a distal region, the proximal region being attachable or connectable to a base of the manipulator, and the distal region including a scraping edge. In some embodiments, each cell scraper includes a continuous structure (e.g., a molded structure) including a scraping edge. In some embodiments, each cell scraper includes one or more interconnected portions. In some embodiments, each cell scraper includes a handle having an interface for removably attaching a scraping edge assembly to the handle. Thus, in some embodiments, a disposable scraper edge that is detachable or releasable from the scraper handle is provided. In some embodiments, each cell scraper includes a scraper edge that is contactable with a surface of a cell culture dish and configured to scrape cells adhered to the surface substantially without killing the cells. In some embodiments, the culture dish further includes a controller configured to control the manipulator to adjust the contact pressure between the scraping edge of the cell scraper and the surface of the cell culture dish. In some embodiments, the culture vessel further includes a sensor (e.g., a strain gauge sensor) connected to the cell scraper, the sensor providing a signal to a controller informing the controller of sensed pressure between the scraping edge of the cell scraper and the surface of the cell culture vessel, wherein the controller is configured to transmit a control signal to the manipulator to increase or decrease the pressure between the scraping edge of the cell scraper and the surface of the cell culture vessel in response to the sensed pressure. In some embodiments, each cell scraper is easily removable from the manipulator. In some embodiments, each cell scraper is configured to perform scraping and liquid handling functions. In some embodiments, each cell scraper includes a scraping edge configured to allow a defined range of scraping edge deflection upon contact with the cell culture vessel. In some embodiments, each cell scraper includes one or more components formed of a polymer. In some embodiments, each cell scraper further includes an opening configured for aspirating cells and / or cell culture medium, wherein the opening is positioned immediately adjacent to the scraping edge.

[0009] In some embodiments, the cell scraper is disposable. In some embodiments, the cell scraper is configured to perform scraping and liquid disposal functions. In some embodiments, the cell scraper includes a scraping edge configured to allow a defined range of scraping edge deflection upon contact with a cell culture vessel. In some embodiments, the scraping edge is formed of a polymer. In some embodiments, the edge includes a geometry configured to excise certain cells (e.g., predifferentiated cells) from a larger cell population (e.g., a healthy stem cell colony). In some embodiments, the scraping edge further includes an opening (e.g., a pore) configured for delivering (e.g., aspirating, depositing, or aspirating and depositing) cells and / or cell culture medium, wherein the opening (e.g., the pore) is positioned immediately adjacent to the scraping blade.

[0010] In some embodiments, the opening is an opening (e.g., a hole). In some embodiments, the opening is configured for delivering (e.g., aspirating, depositing, or aspirating and depositing) cells and / or cell culture medium and forms part of a channel (e.g., connected to the channel). In some embodiments, the channel is integrated into a cell scraper (e.g., extending inside or outside along the cell scraper handle and / or scraper blade). In some embodiments, the culture device further includes one or more additional manipulators, each additional manipulator having at least one cell scraper. In some embodiments, the at least one cell scraper (e.g., the one or more manipulators) is at least 2, 3, 4, 5, 10, 15, 20, 50, 100, 200, 300, 500, or up to 1000 cell scrapers.

[0011] In some embodiments, the cell culture apparatus further includes a controller for manipulating the cells. In some embodiments, the controller is configured to quantify and adjust the contact force between the scraper edge and the surface of the culture dish to which the cells are attached. In some embodiments, the controller is located outside the culture apparatus cabinet. In some embodiments, the controller is inside the culture apparatus cabinet or integrated into the culture apparatus cabinet. In some embodiments, the controller includes a computer.

[0012] In some embodiments, an imager is provided in the culture cabinet. In some embodiments, the imager is configured to allow selective scraping of cells using the manipulator while simultaneously imaging the cells to be scraped or the scraped cells. In some embodiments, the imager is a holographic microscope. In some embodiments, the imager is a bright-field microscope. In some embodiments, the imager is a fluorescence microscope. In some embodiments, the imager is a phase-contrast microscope. In some embodiments, the cell culture chamber further includes a second imager, or a second imager and a third imager. In some embodiments, the cell culture chamber has three imagers, including: a holographic microscope, a bright-field microscope, and a fluorescence microscope. In some embodiments, the cell culture chamber has three imagers, including: a phase-contrast microscope, a holographic microscope, and a fluorescence microscope. In some embodiments, the imager is used to analyze cells and automatically determine areas to be scraped. In other embodiments, the operator modifies the automatic selection of areas to be scraped. In other embodiments, the operator manually selects areas to be scraped from multiple images acquired by these imagers.

[0013] In some embodiments, the one or more cell culture vessels are narrow-necked flasks, suspension flasks, spin flasks, plates, culture dishes, and / or bags. In some embodiments, the one or more cell culture vessels include reference markers to facilitate alignment of the one or more cell culture vessels with the imager and the manipulator.

[0014] In some embodiments, the manipulator for manipulating the cells is a cell picker. In some embodiments, the cell culture apparatus further includes a controller for the manipulator for manipulating the cells. In some embodiments, the one or more cell culture dishes are substantially aligned when the one or more cell culture dishes are moved from an imaging position to a manipulation position or from a manipulation position to an imaging position.

[0015] In some aspects, this document provides a cell culture apparatus comprising: a culture cabinet including an internal chamber for culturing cells in one or more cell culture dishes, wherein the internal chamber is configured to hold the one or more cell culture dishes; a door leading to the internal chamber; a holographic imager including a first imaging position, the holographic imager being configured to image the cells inside the internal chamber when the one or more cell culture dishes are in the first imaging position; a second imager including a second imaging position, the imager being configured to image the cells inside the internal chamber when the one or more cell culture dishes are in the second imaging position; a manipulator for manipulating the cells in the one or more cell culture dishes in the second imaging position; and a cell culture dish transfer device for moving the one or more cell culture dishes from the first imaging position to the second imaging position or from the second imaging position to the first imaging position.

[0016] In some embodiments, the holographic imager is a holographic microscope. In some embodiments, the second imager is a bright-field microscope. In some embodiments, the second imager is a fluorescence microscope. In some embodiments, the cell culture apparatus further includes a third imager. In some embodiments, the cell culture apparatus includes three imagers. In some embodiments, the cell culture apparatus has three imagers, including: a holographic microscope, a bright-field microscope, and a fluorescence microscope. Attached Figure Description

[0017] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown in different figures may be represented by similar numbers. For clarity, not every component in every figure may be labeled. Different embodiments of the invention will now be described by way of example with reference to the accompanying drawings, in which:

[0018] Figure 1 This is a schematic diagram of an illustrative embodiment of a cell culture apparatus having an imager and manipulators;

[0019] Figure 2A-2B This is a schematic diagram of an illustrative embodiment of a cell culture apparatus; Figure 2A A schematic diagram of a cell culture apparatus with a second imager is shown. Figure 2B A schematic diagram of a cell culture apparatus is shown, in which the imaging position and the manipulation position are the same; and

[0020] Figure 3 This is a schematic diagram depicting another component of a cell culture apparatus.

[0021] Figures 4A-4D This is a schematic diagram of an illustrative embodiment of a cell culture apparatus; Figure 4A A schematic diagram of a cell culture apparatus with a manipulator, the manipulator including a cell scraper, is shown. Figure 4B A schematic diagram of a manipulator including multiple cell scrapers is shown; Figure 4C This is a schematic diagram of two manipulators, each of which includes a cell scraper; Figure 4D It is a schematic depiction of the range of motion of the manipulator, including the cell scraper, along the x, y, and z axes.

[0022] Figures 5A-5B This is a schematic diagram of an illustrative embodiment of a cell scraper. Figure 5A A schematic diagram of a cell scraper with a proximal end and a distal end is shown, the distal end including a scraper blade. Figure 5B A schematic diagram of a cell scraper with a scraper blade is shown, the scraper blade including an opening and a channel (e.g., for aspirating cells and / or cell culture medium).

[0023] Figures 6A-6B This is a schematic diagram illustrating an example of a cell scraper. Figure 6A A side view schematic diagram of a cell scraper with a proximal end and a distal end is shown, the distal end including the scraper blade. Figure 6B A side view schematic diagram of a cell scraper with a scraper blade is shown, the scraper blade including an opening and, for example, a channel for aspirating cells and / or cell culture medium.

[0024] Figures 7A-7B This is a schematic diagram of an illustrative embodiment of a cell scraper. Figure 7A A side view schematic diagram of a cell scraper with a proximal end and a distal end is shown, the distal end including the scraper blade. Figure 7B A side view schematic diagram of a cell scraper with a scraper blade is shown, the scraper blade including an opening and, for example, a channel for aspirating cells and / or cell culture medium.

[0025] Figures 8A-8B This is a schematic diagram illustrating an example of a cell scraper. Figure 8A A rear view (e.g., from proximal to distal) of a cell scraper with a proximal end and a distal end is shown, the distal end including the scraper blade. Figure 8B A rear view (e.g., from proximal to distal) of a cell scraper with a scraper blade is shown, the scraper blade including openings and channels (e.g., for aspirating cells and / or cell culture medium).

[0026] Figures 9A-9B This is a schematic diagram of an illustrative embodiment of a cell scraper. Figure 9A A schematic diagram of a cell scraper with a proximal end and a distal end is shown, the distal end including a scraper blade. Figure 9B A schematic diagram of a cell scraper with a scraper blade is shown, the scraper blade including an opening and a channel (e.g., for aspirating cells and / or cell culture medium).

[0027] Figures 10A-10B This is a schematic diagram of an illustrative embodiment of a cell scraper. Figure 10A A schematic diagram of a cell scraper with a proximal end and a distal end is shown, the distal end including a scraper blade. Figure 10B A schematic diagram of a cell scraper with a scraper blade is shown, the scraper blade including an opening and a channel (e.g., for aspirating cells and / or cell culture medium).

[0028] Figure 11 A schematic diagram of a cell scraper with a proximal end and a distal end is shown, the distal end including a scraper blade.

[0029] Figure 12 A schematic diagram of a cell scraper with a scraper blade is shown, the scraper blade including an opening and a channel (e.g., for aspirating cells and / or cell culture medium).

[0030] Figures 13A-13C This is a schematic diagram of an illustrative embodiment of a cell scraper having a proximal end and a distal end, the distal end including a scraper blade.

[0031] Figure 14 This is a schematic diagram of a manipulator including a cell scraper, depicting the range of motion of the manipulator including the cell scraper along the x, y, and z axes.

[0032] Figures 15A-15C This is a schematic diagram of an illustrative embodiment of a cell scraper. Figure 15B A front view schematic diagram of the scraper blade is shown. Figure 15C A side view of the scraper blade is shown. Detailed Implementation

[0033] Current cell culture apparatuses present obstacles to successful cell culture. For example, many cell culture apparatuses require the removal of cell culture dishes and subsequent manual handling for imaging and manipulation. Removing cultured cells from the protected environment provided by the apparatus increases the exposure of the culture to potential contaminants and environmental changes that could interfere with cell growth. In some cases, removing cultures from the apparatus exposes laboratory personnel to pathogenic organisms being cultured within. Furthermore, manual handling of cultures by operators introduces the possibility of contamination due to human error, such as inappropriate sterilization techniques. This article provides cell culture apparatuses with integrated manipulator devices (e.g., apparatuses including cell scrapers).

[0034] In some aspects, this document relates to a cell culture apparatus comprising: a culture cabinet including an internal chamber for culturing cells in one or more cell culture dishes, wherein the internal chamber is configured to hold the one or more cell culture dishes; an external section providing access from an external environment to the internal chamber; an imager and an imaging position, the imager being configured to image cells inside the internal chamber when the one or more cell culture dishes are in the imaging position; a manipulator and a manipulator position for manipulating the cells in the one or more cell culture dishes inside the internal chamber; and a cell culture dish transfer device for moving the one or more cell culture dishes from the imaging position to the manipulator position or from the manipulator position to the imaging position. In some embodiments, the manipulator includes one or more cell scrapers.

[0035] As used herein, a “culture vessel cabinet” is a housing that includes one or more chambers configured to hold one or more cell culture vessels. In some embodiments, the culture vessel cabinet includes a transfer chamber and an inner chamber, one or both of which are configured to hold one or more cell culture vessels. In some embodiments, the culture device may include one or more other components, such as: one or more gas sources (e.g., compressed gas cylinders or ozone generators); conduits (e.g., for conveying one or more liquids or gases, such as water, distilled water, deionized water, cell culture medium, air, carbon dioxide, ozone, and oxygen); airflow mechanisms (e.g., valves, release valves, pinholes, gas regulators, and mass flow regulators); pressure mechanisms (e.g., pumps, such as dry vortex pumps, rotary pumps, momentum transfer pumps, diffusion pumps, or diaphragm pumps; suction conduits; vacuum systems; and blowers); environmental monitors and controls (e.g., gas sensors and / or monitors for sensing and / or controlling the concentration of gases such as carbon dioxide, oxygen, and ozone; heat sources or heat sinks; temperature monitors and controls; humidity monitors; gas scrubbers; air filters; instruments for measuring particulate matter; pressure gauges; and flow meters); doors (e.g., openings or panels); windows (e.g., Optical windows made of glass, plastic, composite materials, or other substantially transparent materials for observing areas inside the culture chamber; ports (e.g., to allow the introduction or removal of one or more gases or liquids); light sources (e.g., lamps, bulbs, lasers, and diodes); optical elements (e.g., microscope objectives, mirrors, lenses, filters, apertures, wave plates, windows, polarizers, optical fibers, beam splitters, and beam combiners); imaging elements (e.g., barcode readers, cameras, etc.); electrical elements (e.g., circuits, cables, power lines, and power sources such as batteries, generators, and DC or AC power); computers; mechanical elements (e.g., motors, wheels, gears, robotic elements, and actuators such as pneumatic actuators, electromagnetic actuators, cam-driven motors, piezoelectric actuators, and motors with lead screws); and control elements (e.g., turntables, buttons, keys, triggers, switches, pointers, screws, dials, screens, and touchscreens). In some embodiments, one or more of these other elements are part of the culture unit but are located outside the culture chamber. In some embodiments, one or more of these other elements are contained within a culture cabinet.

[0036] As used herein, an "inner chamber" is a chamber arranged within a culture tank. An inner chamber may include one or more windows (e.g., optical windows made of glass, plastic, composite materials, or other substantially transparent materials) for observing areas inside the culture tank. An inner chamber may include at least one door (e.g., to allow the transfer of items into or out of the inner chamber). In some embodiments, the at least one door may be arranged between the inner chamber and a transfer chamber. In some embodiments, interlocking elements prevent the door from opening unintentionally (e.g., when a portion of the culture tank is opened to the surrounding environment, thus preventing contaminants from entering the inner chamber). An inner chamber may have any suitable size and geometry. In some embodiments, a culture tank may include more than one inner chamber. In other embodiments, an inner chamber may include one or more partitions to define different areas of the inner chamber. One or more inner chambers or their partitions may have different environmental conditions. The environment inside the inner chamber (e.g., air pressure, gas content, temperature, light, and humidity) may be measured and / or controlled by one or more meters, monitors, sensors, controls, pumps, valves, orifices, and / or light sources. In some embodiments, the interior chamber may have a gas-proof or airtight seal, for example, around one or more windows or doors. In certain embodiments, sealants (e.g., grease) and / or mechanical components (e.g., O-rings, gaskets, diaphragms, KF, LF, QF, quick couplings) or other sealing mechanisms may be used to establish one or more gas-proof seals. In some embodiments, grooves, recesses, protrusions, and / or molded plastic elements may facilitate the establishment of one or more gas-proof seals.

[0037] The interior chamber can be made of any useful material. In some embodiments, the interior chamber may include one or more plastics, polymers, metals, or glass.

[0038] As used herein, a "door" is an element that, when opened, allows communication between two or more environments or areas, and when closed, prevents communication between those environments or areas. Doors can be of any type, such as sliding doors, concealed doors, swing doors, hinged doors, revolving doors, pivoting doors, or folding doors. Doors can be manually, mechanically, or electrically operated. For example, an operator can open or close a door by manually grabbing, pulling, pushing, and / or otherwise interacting with the door or its components (e.g., handles), or by operating mechanical controls (e.g., buttons, triggers, dials, keys, switches, pointers, screws, rotary dials, screens, or touchscreens). In some embodiments, a door can be electrically or digitally controlled, such as by a computer. Doors can be automatically opened. For example, a door can include sensors, such as pressure, infrared, motion, or remote sensors, that detect whether the door is open or closed and / or control when the door opens or closes. Doors can be opened mechanically, pneumatically, electrically, or otherwise. In some embodiments, one or more doors can include one or more locking mechanisms. In a specific environment, one or more doors may include one or more interlocking elements (e.g., mechanical interlocking elements, such as pins, levers, or locks; or electrical interlocking elements, such as switches) to prevent one or more doors from opening at an undesirable time (e.g., when one or more rooms open to the outside environment).

[0039] A transfer device for moving one or more items can be used to move items between a transfer chamber and an inner chamber. In some embodiments, the transfer device includes a conveyor belt or other similar means for manipulating items. Non-limiting examples of items that can be moved by a transfer device include cell culture dishes, pipettes, containers, syringes, and other materials and instruments used in cell culture. In some embodiments, more than one transfer device may be included. In some embodiments, one or more transfer devices may be located in a transfer chamber and / or an inner chamber. In some embodiments, the transfer device may include one or more robotic elements. For example, the transfer device may include one or more robotic arms capable of grasping, lifting, pushing, gripping, sliding, rotating, translating, releasing, raising, lowering, and / or tilting one or more items (e.g., pipettes).

[0040] In some embodiments, the transfer device is a cell culture vessel transfer device. As used herein, "cell culture vessel transfer device" means a device that can transfer one or more cell culture vessels from a first position to a second position. In some embodiments, the transfer device is anchored inside an inner chamber. In some embodiments, the transfer device can transfer one or more items from or to multiple positions within a culture cabinet. For example, a cell culture vessel transfer device can be used to move cell culture vessels from a transfer chamber to an inner chamber, and / or from a storage position to an imaging position. In some embodiments, the culture cabinet includes more than one transfer device for moving one or more items (e.g., separate devices for transferring items between and within multiple chambers). A cell culture vessel transfer device may include one or more elements, such as valves (e.g., solenoid valves or pneumatic valves), gears, motors (e.g., electric motors or stepper motors), stages (e.g., xy or xyz stages), pistons, brakes, cables, ball screw assemblies, rack and pinion arrangements, grippers, arms, pivot points, connectors, translational elements, or other mechanical or electrical components. In some embodiments, the cell culture vessel transfer device may include one or more robotic elements. For example, the cell culture vessel transfer device may include a robotic arm capable of gripping, lifting, pushing, holding, sliding, rotating, translating, releasing, raising, lowering, and / or tilting one or more cell culture vessels. In some cases, the cell culture vessel transfer device selectively and releasably grips one or more cell culture vessels. In some embodiments, the cell culture vessel transfer device may include an arm coupled to a mechanical gripper. For example, the arm may include a mechanical gripper for releasably gripping the cell culture vessel at or near one end, and the arm is securely coupled to a surface or element of the culture vessel at or near the other end. In some embodiments, the robotic arm includes a pivot point along the arm (at which the mechanical gripper is coupled to the arm) and one or more pivoting and / or translating joints to allow a portion of the arm to rotate and translate flexibly. In this way, the robotic arm can access one or more cell culture vessels at different horizontal and vertical positions within a culture vessel cabinet (e.g., within a storage array in an inner chamber).

[0041] In some embodiments, the cell culture vessel transfer device is an automated transfer device. For example, the automated transfer device may be a computer-controlled robotic arm programmed to move the cell culture vessel from a storage location inside the culture vessel to an imaging location inside the culture vessel. In some embodiments, the cell culture vessel transfer device is manually operated. For example, a robotic arm located inside the culture vessel can be operated from a location outside the culture vessel's interior chamber using a user-controlled joystick to move the cell culture vessel from a storage location inside the culture vessel to an imaging location inside the culture vessel.

[0042] As used herein, a "cell culture dish" is a device comprising a housing and one or more chambers for culturing cells. In some embodiments, the housing is a frame. The frame may be coupled to a lid. The one or more chambers may include cell culture medium comprising one or more membranes. In some embodiments, the cell culture dish may include nutrients for promoting cell growth. In some embodiments, the cell culture dish may completely encapsulate one or more cells or cell populations. The housing of the cell culture dish may include one or more pores or openings to allow gas exchange between the cell culture dish and its surrounding environment. Non-limiting examples of cell culture dishes include narrow-necked flasks, suspension flasks, spin flasks, plates, culture dishes, and / or bags. In some embodiments, the cell culture dish includes a transparent or optically transparent window. For example, a lid coupled to the housing of the cell culture dish may include an optically transparent portion for observing cells, for example, with a microscope or other imager. In some embodiments, the cell culture dish includes one or more substantially non-reflective portions. In some embodiments, the cell culture dish is barcoded. Therefore, in some embodiments, the culture dish includes a barcode reader.

[0043] In some embodiments, cell culture vessels may be pre-equipped with one or more reagents desired for a specific purpose, such as for cell growth, for cell differentiation, for subjecting cells to specific assay conditions, etc. In some embodiments, the pre-equipped cell culture vessels contain reagents useful for performing a specific experiment (e.g., cell growth medium, growth factors, selectants, labeling reagents, etc.) prior to the experiment. Pre-equipped cell culture vessels can facilitate experimental protocols by providing ready-to-culture vessels without the need for reagent addition. For example, progenitor cells from a patient may be added to a cell culture vessel pre-equipped with reagents for cell differentiation to expand a differentiated cell population for autologous cell therapy. Pre-equipped cell culture vessels can be stored at any suitable temperature determined by recommended storage parameters of the reagents within the pre-equipped cell culture vessel. In some embodiments, the pre-equipped cell culture storage vessels are stored at a temperature between about -80°C and about 37°C prior to use. In some embodiments, the pre-equipped cell culture storage vessels are stored at a temperature between about -80°C and about -20°C prior to use. In some embodiments, the pre-equipped cell culture storage vessel is stored at a temperature between about -20°C and about 4°C before use. In some embodiments, the pre-equipped cell culture storage vessel is stored at a temperature between about 4°C and about 37°C before use. In some embodiments, the pre-equipped cell culture vessel is disposable. In some embodiments, the pre-equipped cell culture vessel is reusable and / or refillable.

[0044] As used herein, a “storage location” refers to a location where one or more cell culture dishes are stored (e.g., within a culture cabinet). For example, one or more cell culture dishes may be stored in a storage location and subsequently transferred to a different location (e.g., an imaging location). The storage location may be arranged within the interior chamber of a culture cabinet. The storage location may be configured to store multiple cell culture dishes. For example, a storage location may include one or more storage arrays, supports, shelves, pigeonholes, cubes, trays, slots, or other locations or mechanisms. In some embodiments, the storage location may be configured to store cell culture dishes horizontally, while in other embodiments, the storage location may be configured to store cell culture dishes vertically. For example, the storage location may include multiple slots for receiving cell culture dishes stacked vertically on top of each other. The storage location may be configured to hold 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or any other number of cell culture dishes. In some embodiments, the storage location may be configured to hold more than 100 cell culture dishes. In some embodiments, the storage location may include mechanisms for moving one or more storage arrays, supports, shelves, pigeonholes, cubes, trays, slots, or other locations or mechanisms. For example, the storage location may include one or more motors and movable stages (e.g., xy or xyz stages) for moving storage supports from one location within the interior to another, for example, to facilitate access to one or more cell culture dishes stored in different locations. In some embodiments, the culture cabinet may include one or more cell culture dish transfer devices for moving one or more cell culture dishes.

[0045] The storage location can be configured to securely hold or receive one or more cell culture vessels. For example, one or more components of the storage location may include one or more locking mechanisms having one or more adhesive, magnetic, electrical, and / or mechanical components (e.g., snaps, fasteners, locks, buckles, washers, O-rings, diaphragms, springs, and other engaging members). In some embodiments, the storage location and / or the cell culture vessel may include one or more recesses or depressions, and / or may involve molded plastic parts. For example, the cell culture vessel may include one or more protruding features (e.g., edges or buttons) molded for insertion into one or more corresponding recesses, holes, or depressions at the storage location. In some cases, the cell culture vessel may include one or more recesses, holes, or depressions molded to mate with one or more corresponding protruding features at the storage location.

[0046] As used herein, an "imager" refers to an imaging device for measuring light (e.g., transmitted or scattered light), color, morphology, or other detectable parameters (e.g., number of elements), or combinations thereof. An imager may also be referred to as an imaging device. In some embodiments, an imager includes one or more lenses, optical fibers, cameras (e.g., charge-coupled device cameras or CMOS cameras), apertures, mirrors, light sources (e.g., lasers or lamps), or other optical elements. An imager may be a microscope. In some embodiments, the imager is a bright-field microscope. In other embodiments, the imager is a holographic imager or microscope. In other embodiments, the imager is a fluorescence imager or microscope. In other embodiments, the imager is a phase-contrast microscope.

[0047] As used herein, a "fluorescence microscope" refers to an imaging device capable of detecting light emitted from fluorescent markers present on and / or on the surface of cells or other biological entities, the markers emitting light of a specific wavelength in response to the absorption of light of different wavelengths.

[0048] As used herein, a "bright-field microscope" is an imager that illuminates a sample and produces an image based on the light absorbed by the sample. Any suitable bright-field microscope can be used in conjunction with the culture cabinets provided herein.

[0049] As used herein, a "phase-contrast microscope" is an imager that converts the phase shift of light passing through a transparent specimen into a change in brightness in an image. The phase shift itself is invisible, but becomes visible when displayed as a change in brightness. Any suitable phase-contrast microscope can be used in conjunction with the culture apparatus provided herein.

[0050] As used herein, a “holographic imager” is an imager that provides information about an object (e.g., a sample) by measuring the intensity and phase information of electromagnetic radiation (e.g., a wavefront). For example, a holographic microscope measures the light transmitted after passing through a sample and the interference pattern (e.g., phase information) obtained by combining the beam transmitted through the sample with a reference beam.

[0051] A holographic imager may also be a device that, without interfering with a separate reference beam, records, via one or more radiation detectors, a pattern of electromagnetic radiation directly diffracted or scattered by the object to be imaged from a substantially coherent source, with or without any refractive or reflective optical elements between the substantially coherent source and the detector.

[0052] In some embodiments, the cell culture chamber includes a single imager. In some embodiments, the cell culture chamber includes two imagers. In some embodiments, the two imagers are of the same type (e.g., two holographic imagers, two bright-field microscopes, or two phase-contrast microscopes). In some embodiments, the first imager is a bright-field microscope, and the second imager is a holographic imager. In some embodiments, the cell culture chamber includes more than two imagers. In some embodiments, the cell culture chamber includes three imagers. In some embodiments, the cell culture chamber has three imagers, including: a holographic microscope, a bright-field microscope, and a fluorescence microscope. In some embodiments, the cell culture chamber has three imagers, including: a phase-contrast microscope, a holographic microscope, and a fluorescence microscope.

[0053] As used herein, “imaging position” is the location where an imager images one or more cells. For example, the imaging position may be above a light source and / or vertically aligned with one or more optical elements (e.g., lenses, apertures, mirrors, objectives, and condensers).

[0054] As used herein, a “reference mark” refers to a feature that facilitates the alignment of one or more components. In some embodiments, a reference mark may include one or more apertures on a fluorescent medium, or a printed or imprinted fluorescent material. In other embodiments, a reference mark may include a grid, lines, or symbols. In some embodiments, one or more cell culture vessels include one or more reference marks to facilitate the alignment of one or more cell culture vessels with an imager. In some embodiments, a reference mark may be associated with moving parts, including transfer devices and robotic devices.

[0055] In some embodiments, the cell culture dish is substantially aligned with the imager. In some embodiments, the cell culture dish is substantially aligned with the imager by using at least one reference marker. As used herein, the term “substantially aligned” implies that one or more elements are substantially overlapping, identical, and / or collinear with each other. Substantially aligned one or more cell culture dishes at one or more locations (e.g., imaging locations) can facilitate the analysis of the sample by allowing overlapping images of the cell culture dishes to be obtained. For example, the cell culture dish may be imaged by a first imager at a first imaging location and subsequently by a second imager at a second imaging location. If the imaging fields of view of the respective imagers are substantially aligned, the images recorded by the first and second imagers can be combined (“stitched together”) for analysis. One or more reference markers present on one or more cell culture dishes can facilitate substantially aligned. In some cases, one or more reference markers present at one or more imaging or other locations (e.g., manipulation or holding locations) can facilitate substantially aligned.

[0056] As used herein, a "manipulator for manipulating cells" refers to a manipulator for manipulating cells inside an internal chamber. The manipulator may include one or more needles, capillaries, pipettes, and / or micromanipulators. In some embodiments, the manipulator includes one or more cell scrapers. As used herein, a "cell scraper" refers to a device including a scraping edge adapted to scrape cells from a surface. In some embodiments, the cell scraper includes a handle portion comprising an elongated member extending from a proximal region to a distal region, the proximal region being attachable or connectable to a base of the manipulator, and the distal region including the scraping edge. In some embodiments, the cell scraper is a continuous structure (e.g., a molded structure) including the scraping edge. However, in some embodiments, the cell scraper includes one or more interconnected portions. For example, in some embodiments, the cell scraper includes a handle having an interface for interchangeably attaching or attaching a scraping edge or scraping edge assembly to the handle. In some embodiments, the scraper edge is a portion of a cell scraper that can contact the surface of a cell culture vessel or other surface, and is suitably configured to scrape material from the surface substantially without killing the cells (e.g., mechanically lysing the cells) to clean the surface and / or scrape cells adhering to the surface. In some embodiments, it is desirable that the scraper edge or scraper edge assembly be disposable to prevent cross-contamination between cell cultures. Therefore, in some embodiments, the scraper edge or scraper edge assembly is disposable.

[0057] In some embodiments, the scraper edge includes a blade, a wiping element, or in other cases, a substantially flat surface including an edge (e.g., a beveled edge) configured to remove cells from the surface of a cell culture vessel when pushed or pulled along its surface. In some embodiments, the scraper edge may be made of a polymer or polymer combination (e.g., plastic, silicone), glass, metal, or any other suitable material. However, in some embodiments, the mechanical / material properties of the scraper edge allow for a defined range of scraper edge contact deflection, which allows for close control of the contact angle with the cell culture vessel surface / adherent cells. In some embodiments, the edge of the cell scraper is formed of a polymer. Examples of polymers used to form the scraper edge include, but are not limited to, silicone, polyurethane, polyethylene, polyester, polypropylene, polybutene, polystyrene, polyvinyl chloride (PVC), and nylon. In some embodiments, the blade includes a geometry configured to excise certain cells (e.g., predifferentiated cells) from a larger cell population (e.g., a healthy stem cell colony).

[0058] In some embodiments, the cell scraper is configured to perform both scraping and fluid handling functions. For example, in some embodiments, the scraper further includes one or more openings (e.g., one or more orifices) immediately adjacent to the scraping edge (e.g., a blade), and accompanying ports leading to a pipette tip or other fluid movement device (e.g., a pump, a vacuum chamber), thus allowing simultaneous scraping and aspiration of cellular material. In some embodiments, such a configuration is useful for cell removal or colony collection (e.g., in a stem cell isolation setting).

[0059] In some embodiments, openings (e.g., orifices) configured for delivery (e.g., aspiration, deposition, or aspiration and deposition) of cells and / or cell culture media form part of a channel. As used herein, a “channel” refers to a partially enclosed conduit (e.g., cylindrical, tubular, or cubic pathway) designed to allow the transport of an object (e.g., cell culture medium, cells) from one location to another. In some embodiments, the channel is integrated into a cell scraper (e.g., extending inside or outside the cell scraper handle and / or scraper blade). For example, in some embodiments, the cell scraper includes a hollow handle configured to receive a channel, the channel including an opening located in or on the scraper blade (e.g., scraper edge) of the cell scraper. The channel located inside the hollow scraper handle (e.g., in the scraper handle cavity) can have a volume ranging from about 0.1% of the volume within the handle cavity to about 99% of the volume within the handle. For example, the channel can be a separate capillary tube or a hollow needle (e.g., a needle with a gauge between 28 and 10) directly connected to or attached to the wall of the hollow scraper handle (e.g., extending along the inner or outer sidewall). In some embodiments, the entire volume within the hollow scraper handle forms the channel. For example, in some embodiments, the cell scraper includes a hollow handle and a scraper blade having an opening that connects to the hollow interior of the handle (e.g., the channel). In some embodiments, internal structures (e.g., sieves, grids) are used to facilitate the mixing or separation of aggregated cells. (Frames, screens, or star-shaped structures) are integrated into cells Scraper handle and / or scraper blade In the passage It is not intended to be limited by any specific theory, including the ability of the cell scraper with openings and channels to simultaneously perform cell scraping and liquid handling functions (e.g., simultaneously grinding cells and removing debris, such as ground cells and cell culture medium).

[0060] In some embodiments, the manipulator includes at least one cell scraper. For example, the manipulator may include a number of cell scrapers between about 1 and about 100, about 10 and about 100, about 20 and about 1000, or about 50 and about 500. In some embodiments, the manipulator includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 24, 48, 96, 384, or 1536 cell scrapers. In some embodiments, a manipulator having a plurality of cell scrapers is referred to as having a “set” of cell scrapers, such as… Figure 4B As shown. In some embodiments, the manipulator includes a single cell scraper. In some embodiments, the culture device may include multiple manipulators, each manipulator having at least one cell scraper (e.g., as shown). Figure 4C (As depicted).

[0061] In some embodiments, a cell scraper can be used to separate adherent cells from the surface (or surfaces of) the cell culture vessel. For example, a cell scraper can be used manually or mechanically to loosen the protein binding of adherent cells to the culture vessel. In some cases, this is achieved by using enzymes (e.g., trypsin or...) This can be aided by mechanical separation via scraping. However, in some embodiments, cell scraping is a preferred cell separation method, primarily because using enzymes as the primary separation method requires exposing cells to the enzymes for a relatively long period to ensure adequate cell separation from the matrix. In some embodiments, scraping is a preferred method for passage of stem cells during expansion.

[0062] Manipulators may include cell pickers. Manipulators for manipulating cells can operate by detecting the presence of desired cells or cell populations at a first location based on predetermined criteria and transferring the desired cells or cell populations from the first location to a second location. Cell pickers can detect, pick up, and / or transfer desired or unwanted (e.g., predifferentiated cell removal) cells or cell populations based on manual or automated analysis. In some embodiments, information generated by an imager can be analyzed to detect desired or unwanted cells. The cell picker can then transfer the desired or unwanted cells to the second location. For example, an imager can image cells in or on a cell culture dish at an imaging location and use the image to identify desired or unwanted cells or cell populations. The cell picker can then transfer the desired or unwanted cells, for example by contacting each or all of the desired cells with a needle, capillary, pipette, or micromanipulator, and completing the transfer of this or these cells from their first location to a second location in or on a cell culture dish or elsewhere in an internal chamber. In some embodiments, the first location of the cells may be in or on a cell culture dish. In a particular embodiment, a cell picker transfers cells from the first location in or on a cell culture dish to a second location on the same cell culture dish. In other embodiments, a cell picker transfers cells from the first location in or on a first cell culture dish to a second location in or on a second cell culture dish. In some other embodiments, a cell picker transfers cells from the first location in or on a cell culture dish to a second location in an inner chamber, not in or on a cell culture dish.

[0063] In some embodiments, the manipulator includes at least one microelectrode. As used herein, the term "microelectrode" refers to an electrical conductor used to deliver electrical stimulation to cells. For example, a microelectrode can be used to deliver genetic material into cells via electroporation. In some embodiments, the manipulator includes at least one microinjector. Generally, the microinjector is a glass micropipette that has been stretched to form a sharp, hollow structure capable of piercing the cell membrane and serving as a conduit for introducing genetic material into the cell.

[0064] In some embodiments, the manipulator is manually operated. For example, a manipulator having a cell picker located inside the inner chamber of a culture cabinet can be electronically linked to and controlled by a user-guided joystick located outside the inner chamber of the culture cabinet. In some embodiments, the user-guided joystick is connected to a display device. In some embodiments, the display device shows images captured by an imaging device inside the inner chamber of the culture cabinet.

[0065] In some embodiments, the manipulator is automated. For example, a manipulator located inside the interior of a culture cabinet may be electronically connected to a controller located outside the culture cabinet, which is electronically connected to a computer that directs the manipulator. In some embodiments, the controller is connected to a hardware interface configured to quantify and adjust the contact force between the scraper edge and the cell-adhered surface of the cell culture dish. For example, the manipulator or cell scraper may further include a sensor (e.g., a pressure sensor) that provides a signal to the controller informing it of the sensed pressure, in response to which the controller signals (e.g., to the manipulator) to increase or decrease the pressure applied by the manipulator on the scraper edge (e.g., the blade) of the cell scraper. In some embodiments, the controller includes software and / or hardware configured to be programmed to achieve 360° rotation of the scraper tip / blade, thus allowing the scraping motion to be programmed according to linear movement along the x, y, and z axes, such that a constant angle of attack is maintained between the scraper blade and the scraped cells, independent of the geometry of the culture dish. In some embodiments, the controller is connected to one or more components or hardware interfaces configured to allow for the localization and identification of cell clusters, for example via imaging, thus allowing a cell scraper controlled by the manipulator to subsequently scrape / aspirate.

[0066] One or more components of a manipulator used to manipulate cells may be sterilized prior to manipulation, for example by using sterilizing ingredients or methods (e.g., ethanol or ozone gas, UV light, hydrogen peroxide).

[0067] As used herein, "manipulation location" refers to the location where a cell is manipulated by a manipulator (e.g., a cell picker). In some embodiments, the manipulation location may be the same as the imaging location.

[0068] According to one aspect, a cell culture apparatus includes a culture cabinet having an imaging position and a manipulating position. Cells in the cell culture dish are imaged by an imager at the imaging position and manipulated by a manipulator at the manipulating position. In some embodiments, the imaging position and the manipulating position are two distinct locations within the culture cabinet. The cell culture apparatus may include a transfer device for moving the cell culture dish between the imaging position and the storage position. In other embodiments, the imaging position and the manipulating position are the same, such that the cells in the culture dish are imaged at the manipulating position.

[0069] In some embodiments, an imager may be used in conjunction with a manipulator. For example, the imager may image cells in or on a cell culture dish at an imaging location and use the image to identify desired cells or cell populations. The cell picker (which may or may not be at the imaging location) may then transfer desired or unwanted cells, for example by contacting each or all desired cells with a needle, capillary, pipette, or micromanipulator, and completing the transfer of this or these cells from their first location to a second location elsewhere in or on the cell culture dish or in an inner chamber. In some embodiments, the cell picker transfers cells from a first location in or on the cell culture dish to a second location on the same cell culture dish. In other embodiments, the cell picker transfers cells from a first location in or on a first cell culture dish to a second location in or on a second cell culture dish. In some other embodiments, the cell picker transfers cells from a first location in or on the cell culture dish to a second location in an inner chamber, not in or on the cell culture dish.

[0070] In some embodiments, a single location within the culture chamber can serve as both an imaging location and a manipulation location. In one embodiment, cells are imaged while being manipulated by a manipulator. In some embodiments, the imaging location and the imaging position can be separate locations within the culture chamber.

[0071] In some embodiments, the manipulator includes sensors that allow it to report its position and determine when it has touched the bottom of the cell culture dish. In some embodiments, an imager may be used to guide the manipulator to achieve repeatability and accuracy. In some embodiments, compliance (e.g., elasticity) in the manipulator may be used to mitigate the need for extremely high mechanical accuracy.

[0072] Turn to the attached diagram. Figure 1An illustrative embodiment of a cell culture apparatus is depicted. The cell culture apparatus includes a culture cabinet having an internal chamber (100) for culturing cells in one or more cell culture dishes. The culture cabinet includes an outer section (101) that opens and closes to allow communication between the external environment and the culture cabinet. In some embodiments, the outer section opens and closes to allow communication between the external environment and the internal chamber. The internal chamber is configured to hold one or more cell culture dishes. The one or more cell culture dishes are stored in a storage location (102). In some embodiments, the storage location is a freestanding structure. For example, the storage location may be a tube or culture flask rack that can be removed from the internal chamber of the culture apparatus for loading and unloading culture dishes. In some embodiments, the storage location is fixed to a surface of the internal chamber. For example, the storage location may be a series of supports or shelves attached to the wall or floor of the internal chamber and therefore cannot be removed from the culture cabinet.

[0073] In some embodiments, the cell culture apparatus includes a cell culture vessel transfer device (103) for moving one or more cell culture vessels. The cell culture transfer device may be fixed to any suitable surface of the interior chamber of the culture apparatus. For example, the cell culture vessel transfer device may be fixed to the top or top plate of the interior chamber. Alternatively, the cell culture vessel transfer device may be fixed to a side wall of the interior chamber. In some embodiments, the cell culture vessel transfer device is not fixed to a wall of the interior chamber. For example, the cell culture vessel transfer device may be resting on a wheeled tripod or other movable structure that can move around the interior chamber.

[0074] In some embodiments, the transfer device moves one or more cell culture dishes from a storage location (102) to an imaging location (105) or to a manipulation location (107). The transfer device (103) may also move one or more cell culture dishes from the imaging location (105) to the manipulation location (107) or from the manipulation location (107) to the imaging location (105). When imaging or manipulation is complete, the transfer device (103) moves one or more cell culture dishes from the imaging location (105) or the manipulation location (107) back to the storage location (102).

[0075] In some embodiments, the culture cabinet includes a first imaging position (105) and a manipulating position (107). In some embodiments, one or more imaging positions are positioned on a surface of the inner chamber opposite the imager. In some embodiments, the imaging position is a platform, either freestanding or fixed to a surface of the inner chamber. In some embodiments, the platform is movable. For example, the movable platform may be fixed to two or more rods that allow the platform to move left, right, forward, backward, up, or down relative to the imager. In some embodiments, the movable platform is motorized.

[0076] In some embodiments, the culture dish includes a first imager (104) that images the cells in the cell culture dishes when the dishes are in a first imaging position (105). In some embodiments, the first imager is a bright-field microscope. In some embodiments, the first imager is a holographic microscope. In some embodiments, the first imager is a phase-contrast microscope.

[0077] In some embodiments, the manipulator (106) manipulates the cells in the cell culture vessels when the vessels are in the manipulation position (107). In some embodiments, the manipulator has an array of needles, capillaries, pipettes, and / or micromanipulators. For example, the manipulator may include a cell picker. In some embodiments, the manipulator includes one or more cell pickers. In some embodiments, the manipulator may include a cell scraper. In some embodiments, the manipulator includes one or more cell scrapers. Generally, the manipulation position and the imaging position share many features as described herein.

[0078] Figure 2A An illustrative embodiment of a cell culture apparatus is depicted. In some embodiments, the apparatus cabinet has a second imager (108). The second imaging position may be at or near the manipulation position (107). In some embodiments, the second imaging position and the manipulation position (107) may be the same position. In some embodiments, the second imager (108) images the cells in the cell culture dish while the cells are manipulated by a manipulator (106). In some embodiments, the second imager is a bright-field microscope. In some embodiments, the second imager is a holographic microscope. In some embodiments, the first imager is a phase-contrast microscope.

[0079] Figure 2B An illustrative embodiment of a cell culture apparatus is depicted. In some embodiments, the cell culture apparatus has an imaging position and a manipulation position, which are the same position (105).

[0080] Figure 3 An illustrative embodiment depicting further components of a cell culture apparatus is provided. Additional components are those not included in the description of the culture apparatus. Figure 1-2A Or any of the components listed in 2B. In some embodiments, the cell culture apparatus includes a cell culture dish (109) with a barcode. Thus, in some embodiments, the cell culture apparatus has a barcode scanner (110) positioned inside an interior chamber of the culture apparatus cabinet. In some embodiments, the barcode reader communicates with a computer (111) to forward information associated with a cell culture dish whose barcode has been scanned. In some cases, the barcode scanner may be fixed to any surface of the interior chamber. For example, the barcode scanner may be fixed to the wall of the interior chamber adjacent to the imaging location (105).

[0081] In some embodiments, the cell culture apparatus includes at least one detector and / or at least one sensor (113) for measuring environmental conditions inside the inner chamber. Examples of detectors for measuring environmental conditions include, but are not limited to, temperature detectors, pressure detectors, carbon dioxide (CO2) sensors, oxygen (O2) sensors, and relative humidity sensors. In some embodiments, the at least one detector and / or at least one sensor is located within an apparatus housing (112). The at least one detector and / or at least one sensor is connected to a controller (114). In some embodiments, the controller (114) communicates with a computer (111). Furthermore, the controller (114) may communicate with a fluid distribution system (115). For example, if the CO2 sensor indicates a low CO2 level in the inner chamber, the controller (114) may instruct the fluid distribution system (116) to inject CO2 gas into the inner chamber to increase the CO2 level in the inner chamber.

[0082] Figure 4 illustrates a schematic diagram of an illustrative embodiment of a cell culture apparatus. In some embodiments, the manipulator (106) includes one or more cell scrapers (116), such as Figure 4A As shown. In some embodiments, the manipulator (106) includes a plurality of cell scrapers (e.g., a set of cell scrapers (117)), as Figure 4B As shown. In some embodiments, the culture device includes two manipulators (1061 and 1062), wherein each manipulator includes a cell scraper (116), as shown. Figure 4C As shown. In some embodiments, the manipulator (106) is controlled by the controller ( Figure 3 The object (114) is controlled by a controller configured to program the 360° rotation of the tip / blade of the cell scraper (116), thus allowing the scraping motion to be programmed according to linear motion along, for example, x-, y- and z- axes, so that a constant angle of attack is maintained between the scraper blade and the scraped cell regardless of the geometry of the culture vessel.

[0083] Figure 5 illustrates a schematic diagram of an illustrative embodiment of a cell scraper. In some embodiments, the cell scraper includes a proximal end (118) and a distal end (119), such as Figures 5A-5B As shown. In some embodiments, the distal end of the cell scraper includes a scraping blade (e.g., a scraping edge) (120), as... Figures 5A-5B As shown. In some embodiments, the cell scraper includes an opening (e.g., an orifice) configured for delivery (e.g., aspiration of cells and / or cell culture medium) (121), as... Figure 5B As shown. In some embodiments, the cell scraper includes an opening connected to a channel (122), as... Figure 5B As shown. In some embodiments, the channel is integrated into the cell scraper (e.g., extending inside or outside along the cell scraper handle and / or scraper blade).

[0084] Figure 6 illustrates a schematic diagram of an illustrative embodiment of a cell scraper. In some embodiments, the cell scraper includes a proximal end (118) and a distal end (119), such as Figures 6A-6B As shown. In some embodiments, the distal end of the cell scraper includes a scraping blade (e.g., a scraping edge) (120), as... Figures 6A-6B As shown. In some embodiments, the cell scraper includes an opening (e.g., an orifice) configured for delivering (e.g., aspirating, depositing, or aspirating and depositing) cells and / or cell culture medium (121), as shown. Figure 6B As shown. In some embodiments, the scraper includes an opening connected to a channel (122), as... Figure 6B As shown. In some embodiments, the channel is anchored to the wall of the cell scraper.

[0085] Figure 7 illustrates a schematic diagram of an illustrative embodiment of a cell scraper. In some embodiments, the cell scraper includes a proximal end (118) and a distal end (119), such as Figures 7A-7B As shown. In some embodiments, the distal end of the cell scraper includes a scraping blade (e.g., a scraping edge) (120), as... Figures 7A-7B As shown. In some embodiments, the scraper edge is angled relative to the scraper handle. In some embodiments, the cell scraper includes an opening (e.g., an orifice) configured for delivering (e.g., aspirating, depositing, or aspirating and depositing) cells and / or cell culture medium (121), as... Figure 7B As shown. In some embodiments, the cell scraper further includes an opening connected to the channel (122), as... Figure 7B As shown.

[0086] Figure 8 illustrates a schematic diagram of an illustrative embodiment of a cell scraper. In some embodiments, the cell scraper includes a proximal end (118) and a distal end (119), such as Figures 8A-8BAs shown. In some embodiments, the distal end of the cell scraper includes a scraping blade (e.g., a scraping edge) (120), as... Figures 8A-8B As shown. In some embodiments, the scraper edge is angled relative to the scraper handle. In some embodiments, the cell scraper includes an opening (e.g., an orifice) configured for delivering (e.g., aspirating, depositing, or aspirating and depositing) cells and / or cell culture medium (121), as... Figure 8B As shown. In some embodiments, the cell scraper includes an opening connected to a channel (122), as... Figure 8B As shown. In some embodiments, the channel wall is formed by the inner wall of a cell scraper, such as... Figure 8B As shown.

[0087] Figure 9 illustrates a schematic diagram of an illustrative embodiment of a cell scraper. In some embodiments, the cell scraper includes a proximal end (118) and a distal end (119), such as Figures 9A-9B As shown. In some embodiments, the distal end of the cell scraper includes a scraping blade (e.g., a scraping edge) (120), as... Figures 9A-9B As shown. In some embodiments, the scraper edge is angled relative to the scraper handle. In some embodiments, the cell scraper includes an opening (e.g., an orifice) configured for delivering (e.g., aspirating, depositing, or aspirating and depositing) cells and / or cell culture medium (121), as... Figure 9B As shown. In some embodiments, the opening further includes a channel (122), as... Figure 9B As shown. In some embodiments, the channel wall is formed by the inner wall of a cell scraper, such as... Figure 9B As shown.

[0088] Figure 10 illustrates a schematic diagram of an illustrative embodiment of a cell scraper. In some embodiments, the cell scraper includes a proximal end (118) and a distal end (119), such as Figures 10A-10B As shown. In some embodiments, the distal end of the cell scraper includes a scraping blade (e.g., a scraping edge) (120), as... Figures 10A-10B As shown. In some embodiments, the scraper edge is angled relative to the scraper handle. In some embodiments, the cell scraper includes an opening (e.g., an orifice) configured for delivering (e.g., aspirating, depositing, or aspirating and depositing) cells and / or cell culture medium (121), as... Figure 10B As shown. In some embodiments, the cell scraper includes an opening connected to a channel (122), as... Figure 10B As shown. In some embodiments, the channel wall is formed by the inner wall of a cell scraper, such as... Figure 10B As shown.

[0089] Figure 11A schematic diagram depicting an illustrative embodiment of a cell scraper is provided. In some embodiments, the cell scraper includes a proximal end (118) and a distal end (119). In some embodiments, the distal end of the cell scraper includes a scraping blade (e.g., a scraping edge) (120). In some embodiments, the scraping edge includes a plurality of protrusions.

[0090] Figure 12 A schematic diagram depicting an illustrative embodiment of a cell scraper is provided. In some embodiments, the cell scraper includes a proximal end (118) and a distal end (119). In some embodiments, the distal end of the cell scraper includes a scraping blade (e.g., a scraping edge) (120). In some embodiments, the scraping edge includes a plurality of protrusions. In some embodiments, the cell scraper includes an opening (e.g., an orifice) configured for delivering (e.g., aspirating, depositing, or aspirating and depositing) cells and / or cell culture medium (121). In some embodiments, the cell scraper includes an opening connected to a channel (not shown). In some embodiments, the channel wall is formed by the inner wall of the cell scraper.

[0091] Figure 13 illustrates a schematic diagram of an illustrative embodiment of a cell scraper. In some embodiments, the cell scraper includes a proximal end (118) and a distal end (119), such as Figures 13A-13C As shown. In some embodiments, the distal end of the cell scraper includes a scraping blade (e.g., a scraping edge) (120), as... Figures 13A-13C As shown. In some embodiments, the scraper edge is positioned at an angle relative to the scraper handle.

[0092] Figure 14 A schematic diagram depicts a manipulator (106) including a cell scraper (116). In some embodiments, the manipulator (106) is controlled by a controller (not shown) configured to program the scraper tip / blade of the cell scraper (116) to achieve 360° rotational or linear oscillations, thus allowing the scraping motion to be programmed according to linear motion along, for example, x- and y- axes and rotational motion about the z- axis, such that a constant angle of attack is maintained between the scraper blade and the scraped cells regardless of the geometry of the culture dish.

[0093] Figure 15 illustrates a schematic diagram of an illustrative embodiment of a cell scraper (116). In some embodiments, the distal end of the cell scraper includes a scraping blade (e.g., a scraping edge) (120), such as Figure 15B-15C As shown.

[0094] Automated cell culture

[0095] This document relates to culturers and methods for culturing, manipulating, and / or monitoring cells under controlled conditions, such as aseptic and / or sterile conditions. In some aspects, the culturers and methods include automated components. In some aspects, the culturers and methods are useful for long-term cell culture (e.g., growing and maintaining cells for recombinant protein expression, or growing and / or differentiating cells for therapeutic applications, such as implantation). In some embodiments, cell cultures are grown within culture dishes in the culturers described herein.

[0096] Petri dishes

[0097] Cell culture vessels can be configured to culture different types of cells, including eukaryotic or prokaryotic cells. In some embodiments, the cells are mammalian cells (e.g., human cells, canine cells, bovine cells, sheep cells, cat cells, or rodent cells, such as rabbit cells, mouse cells, or rat cells). In some embodiments, the cells are insect cells, avian cells, microbial cells (e.g., yeast cells, such as Saccharomyces cerevisiae, Kluyveromyces lactis, or Pichia pastoris; or bacterial cells, such as Escherichia coli, Bacillus subtilis, or Corynebacterium cells), insect cells (e.g., Drosophila cells, or Sf9 or Sf21 cells), plant cells (e.g., algal cells), or any other type of cell.

[0098] In some embodiments, cells are cultured to produce natural products (e.g., paclitaxel, pigments, fatty acids, biofuels, etc.). In some embodiments, cells are cultured to express recombinant products (e.g., recombinant protein products such as antibodies, hormones, growth factors, or other therapeutic peptides or proteins). In some embodiments, the expansion and / or differentiation of cells is for therapeutic purposes, such as implantation into a subject (e.g., a human subject) to provide or supplement the subject's missing or defective cellular, tissue, or organ functions.

[0099] In some embodiments, the cells are derived from immortalized cell lines. Non-limiting examples of cell lines include human cells, such as HeLa cells, prostate cancer cells (e.g., DU145, PC3, and / or Lncap cells), breast cancer cells (e.g., MCF-7, MDA-MB-438, and / or T47D cells), acute myeloid leukemia cells (e.g., THP-1 cells), glioblastoma cells (e.g., U87 cells), neuroblastoma cells (e.g., SHSY5Y cells), bone cancer cells (e.g., Saos-2 cells), and chronic myeloid leukemia cells (e.g., KBM-7 cells). In some embodiments, the cell lines include primate cell lines, rodent cell lines (e.g., rat or mouse cell lines), canine cell lines, cat cell lines, zebrafish cell lines, Xenopus laevis cell lines, plant cell lines, or any other cell type. In some embodiments, the cells are human 293 cells (e.g., 293-T or HEK 293 cells), mouse 3T3 cells, Chinese hamster ovary (CHO) cells, CML T1 cells, or Jurkat cells.

[0100] In some embodiments, the cells are primary cells, feeder cells, or stem cells. In some embodiments, the cells are isolated from a subject (e.g., a human subject). In some embodiments, the cells are primary cells isolated from tissue or biopsy samples. In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are stem cells, such as embryonic stem cells, mesenchymal stem cells, cancer stem cells, etc. In some embodiments, the cells are isolated from a tissue or organ (e.g., human tissue or organ) (including, but not limited to, solid tissues and organs). In some embodiments, the cells may be isolated from the placenta, umbilical cord, bone marrow, liver, blood (including cord blood), or any other suitable tissue. In some embodiments, patient-specific cells are isolated from the patient for culture (e.g., for cell expansion and optional differentiation) and subsequently re-implanted into the same patient or a different patient. Thus, in some embodiments, cells grown in the culturer described herein may be used for allogeneic or autologous therapy. In some embodiments, cells grown in the culturer disclosed herein may be genetically modified, expanded, and re-introduced into a patient for the purpose of providing immunotherapy (e.g., chimeric antigen receptor therapy (CAR-T) or delivery of CRISPR / Cas modified cells).

[0101] In some embodiments, the primary cell culture includes epithelial cells (e.g., corneal epithelial cells, mammary epithelial cells, etc.), fibroblasts, myoblasts (e.g., human skeletal muscle myoblasts), keratinocytes, endothelial cells (e.g., microvascular endothelial cells), nerve cells, smooth muscle cells, hematopoietic cells, placental cells, or a combination of two or more of these.

[0102] In some embodiments, the cells are recombinant cells (e.g., hybridoma cells, or cells expressing one or more recombinant products). In some embodiments, the cells are infected with one or more viruses.

[0103] Primary cell isolation

[0104] In some embodiments, cells are isolated from tissue or biological samples for in vitro culture in a culture vessel provided herein. In some embodiments, cells (e.g., leukocytes) are isolated from blood. In some embodiments, cells are released from tissue or biological samples using physical and / or enzymatic destruction. In some embodiments, one or more enzymes, such as collagenase, trypsin, or protease, are used to digest the extracellular matrix. In some embodiments, tissue or biological samples are placed in a culture medium (e.g., with or without physical or enzymatic destruction), and cells released and grown in the culture medium can be isolated for further culture.

[0105] Cell culture

[0106] As used herein, cell culture refers to the procedure of maintaining and / or growing cells under controlled conditions (e.g., in vitro). In some embodiments, cells are cultured under conditions that promote cell growth and replication, conditions that promote the expression of recombinant products, conditions that promote differentiation (e.g., differentiation into one or more tissue-specific cell types), or combinations of two or more of these conditions.

[0107] In some embodiments, cell culture dishes are configured for culturing suspended cells. In some embodiments, cell culture dishes are configured for culturing adherent cells. In some embodiments, cell culture dishes are configured for 2D or 3D cell culture. In some embodiments, cell culture dishes include one or more surfaces or microcarriers to support cell growth. In some embodiments, these cell culture dishes are coated with extracellular matrix components (e.g., collagen, fibrous proteins, and / or laminin components) to increase adhesion properties and provide additional signals required for growth and differentiation. In some embodiments, cell culture dishes contain one or more synthetic hydrogels, such as polyacrylamide or polyethylene glycol (PEG) gels, to support cell growth. In some embodiments, cell culture dishes include a solid support with embedded nutrients (e.g., gels or agar, for example, for certain bacterial or yeast cultures). In some embodiments, cell culture dishes contain a liquid culture medium.

[0108] In some embodiments, cells are cultured in one of any suitable culture media. Different culture media with varying ranges of pH, glucose concentration, growth factors, and other supplements may be used for different cell types or for different applications. In some embodiments, custom-made cell culture media or commercially available cell culture media may be used, such as Dulbecco's Modified Eagle Medium, minimum essential medium, RPMI medium, HA or HAT medium, or other media available from Life Technologies or other commercial sources. In some embodiments, the cell culture medium contains serum (e.g., fetal bovine serum, calf serum, horse serum, porcine serum, or other serum). In some embodiments, the cell culture medium is serum-free. In some embodiments, the cell culture medium contains human platelet lysate (hPL). In some embodiments, the cell culture medium contains one or more antibiotics (e.g., actinomycin D, ampicillin, carbenicillin, cefotaxime, phosphatamicin, gentamicin, kanamycin, neomycin, penicillin, penicillin streptomycin, polymyxin B, streptomycin, tetracycline, or any other suitable antibiotic, or any combination of two or more of these). In some embodiments, the cell culture medium contains one or more salts (e.g., balanced salts, calcium chloride, sodium chloride, potassium chloride, magnesium chloride, etc.). In some embodiments, the cell culture medium contains sodium bicarbonate. In some embodiments, the cell culture medium contains one or more buffers (e.g., HEPES or other suitable buffers). In some embodiments, one or more supplements are included. Non-limiting examples of supplements include reducing agents (e.g., 2-mercaptoethanol), amino acids, cholesterol supplements, vitamins, transferrin, surfactants (e.g., nonionic surfactants), CHO supplements, primary cell supplements, yeast solutions, or any combination of two or more of these. In some embodiments, one or more growth or differentiation factors are added to the cell culture medium. Growth or differentiation factors (e.g., WNT family proteins, BMP family proteins, IGF family proteins, etc.) can be added alone or in combination, for example as a differentiation mixture containing different factors for inducing differentiation into a specific lineage. Growth or differentiation factors and other aspects of the liquid culture medium can be added using an automated liquid processor integrated within the culture vessel.

[0109] In some respects, the apparatus and methods described herein provide and maintain a suitable temperature and gas mixture for cell growth. It should be understood that different cell types require different growth conditions, and the apparatus described herein can be programmed to maintain different conditions. In some embodiments, conditions of approximately 37°C and 5% CO2 are used for mammalian cells.

[0110] In some embodiments, the apparatus and methods described herein are used to monitor or determine nutrient depletion, pH changes, temperature changes, accumulation of apoptotic or necrotic cells, and / or cell density in a culture medium. For example, the manipulator (106) may include a sensor monitoring the culture medium. In some embodiments, the apparatus and methods described herein are used to modify or change the culture medium or conditions as appropriate and / or for cell passage. In some embodiments, these apparatus and methods are automated (e.g., controlled by a controller (114) and / or a computer (111), such as... Figure 3 (As shown).

[0111] In some embodiments (e.g., for adherent cell cultures), the culture medium can be removed directly by aspiration and replaced with fresh medium. In some embodiments (e.g., for non-adherent / suspension cultures), changing the culture medium may involve centrifuging the cell culture, removing the old medium, and replacing it with fresh medium. In some embodiments, the centrifuge is located in an internal chamber of the culture vessel. In some embodiments, the culture vessel allows for continuous replacement of the culture medium. In some embodiments, the culture vessel described herein may include different aspects that can be used to process, replace, supply, and / or maintain the culture medium to support one or more components of the cells. The culture vessel may include reservoirs for containing waste culture medium and / or reservoirs for containing fresh culture medium. Such reservoirs may be located (e.g., for temporary storage) within a cooler inside the culture vessel or within a refrigerated section of the culture vessel. In some embodiments, one or more reservoirs are provided externally to the culture vessel, and piping is provided for access to and exit from the culture vessel space for supplying or removing fluids to a liquid handling unit (e.g., a liquid handling unit with aspirators) or temporary reservoirs within the culture vessel to facilitate cell feeding, culture medium replacement, and other related needs. For suspension cells, a device (e.g., one or more centrifuges to facilitate cell granulation) can be provided within the culture vessel for separating cells from waste culture medium to facilitate automatic replacement of the culture medium as part of the culture vessel provided herein. In some embodiments, this document provides a system comprising a cell culture vessel connected to a computer capable of automatically monitoring and adjusting cell culture conditions to achieve optimal growth of the cell culture.

[0112] In some embodiments, cells are passaged within the culture vessel described herein. In some embodiments, cell cultures are split and subsets of cell cultures are transferred to fresh culture dishes for further growth. In some embodiments (e.g., for adherent cell cultures), cells are detached from the surface (e.g., mechanically, such as by gentle scraping, and / or enzymatically, such as using trypsin-EDTA or one or more other enzymes) before being transferred to fresh culture dishes. In some embodiments (e.g., for suspension cell cultures), small volumes of cell cultures are transferred to fresh culture dishes.

[0113] In some embodiments, cell cultures are manipulated in other ways during culture in the culture vessels and containers described herein. For example, cell cultures may be transfected with nucleic acids (e.g., DNA or RNA) or exposed to viral infection (e.g., using recombinant viral particles to deliver DNA or RNA).

[0114] Aseptic techniques can be used to prevent or minimize contamination of cell cultures during growth and manipulation. In some embodiments, suitable techniques are used to sterilize equipment used for cell culture (e.g., pipettes, fluid handling devices, manipulation devices, other automated devices, or robotic devices, etc.). Non-limiting techniques include heat exposure (e.g., autoclaving), surface disinfection (e.g., using alcohol, bleach, or other disinfectants), irradiation, and / or exposure to disinfecting gases (e.g., ozone, hydrogen peroxide, etc.), as described herein. In some embodiments, suitable techniques are used to sterilize the culture medium. Non-limiting techniques include heat exposure (e.g., autoclaving), antimicrobial / antiviral treatment, filtration, and / or irradiation.

[0115] In some embodiments, the manipulation of cell cultures is performed under sterile conditions, such as in an environment that has been sterilized and the air has been filtered to remove potential contaminants (e.g., inside a culture chamber).

[0116] In some embodiments, cell cultures are grown and maintained under GMP-compliant conditions, including using GMP-compliant culture media or GMP-compliant liquid handling equipment and performing the methods in accordance with standard operating procedures (SOPs).

[0117] In some embodiments, cell cultures can be monitored and / or evaluated to detect contamination. In some embodiments, contamination from cells of different types of organisms can be detected. In some embodiments, any suitable technique can be used to detect contamination of mammalian cell cultures by mycoplasma, bacteria, yeast, or viruses. In some embodiments, cell culture contamination can be detected by measuring changes or rates of change in one or more culture properties (e.g., pH, turbidity, etc.) that are characteristic of the contamination (e.g., by bacteria or yeast) rather than of the cells growing in the culture (e.g., mammalian cells). In some embodiments, contamination (e.g., mycoplasma, bacteria, yeast, viruses, or other contaminants) can be detected using one or more molecular assays (e.g., PCR, ELISA, RNA labeling, or other enzymatic techniques) or cell-based assays.

[0118] In some embodiments, cell cultures can be monitored and / or evaluated to detect contamination by similar types of cells (e.g., human cell lines contaminated by different human cells or different mammalian cells). In some embodiments, DNA sequencing or DNA fingerprinting (e.g., short tandem repeat (STR) fingerprinting), isoenzyme analysis, human lymphocyte antigen (HLA) typing, chromosome analysis, karyotype analysis, cell morphology, or other techniques can be used to evaluate cell cultures and their potential contamination.

[0119] In some embodiments, cells produced using the apparatus and methods provided herein can be frozen to preserve them for later use and / or for transport. In some embodiments, cells are mixed with cryopreservation components after growth and / or differentiation and before freezing. The cryopreservation components can be added to cell culture dishes, or cells can be transferred from cell culture dishes to cryopreservation dishes together with the cryopreservation components. Non-limiting examples of cryoprotectants that may be included in the cryopreservation components include DMSO, glycerol, PEG, sucrose, trehalose, and dextrose. In some embodiments, a refrigerator may be present in or near the culture vessel to facilitate freezing of cells isolated from cell cultures.

[0120] Cell culture apparatus:

[0121] This document relates to culturers and methods for culturing, manipulating, and / or monitoring cells under controlled conditions (e.g., under aseptic and / or sterile conditions). In some embodiments, the cell culture culturer provided herein includes a culturer cabinet defining an internal chamber for culturing cells in one or more cell culture dishes, wherein the internal chamber is configured to hold the one or more cell culture dishes. In some cases, in addition to an internal door leading from a transfer chamber to the internal chamber, the culturer includes at least one external section (e.g., 1, 2, 3, 4, or more external sections) that opens directly from the external environment to the internal chamber, for example, to provide an alternative pathway to the internal chamber during periods when the culturer is not in operation, such as during culturer maintenance. In some embodiments, the culturer includes a storage location within the internal chamber for storing one or more cell culture dishes. In some embodiments, a cell culture dish transfer device is provided in the culturer for moving one or more cell culture dishes from a first imaging position to a storage position and / or from a storage position to a first imaging position.

[0122] In some embodiments, the culture unit or culture unit cabinet provided herein is rectangular or cubic in shape. In some embodiments, the culture unit or culture unit cabinet provided herein has a 1ft... 2 up to 16ft 2 The rectangular footprint within the specified area. In some embodiments, the culture unit or culture unit cabinet provided herein has a maximum area of ​​approximately 1 ft. 2 2ft 2 3ft 2 4ft 2 5ft 2 6ft 2 7ft 2 8ft 2 9ft 2 10ft 2 11ft 2 12ft 2 13ft 2 14ft 2 15ft 2 or 16ft 2 The rectangular footprint. In some embodiments, the culture unit or culture unit cabinet provided herein has a 1 ft... 3 up to 100ft 3 The total chamber volume within the range. In some embodiments, the culture unit or culture unit cabinet provided herein has a maximum volume of approximately 1 ft. 3 5ft 3 10ft 3 25ft 3 50ft 3 or 100ft 3The chamber volume. In some embodiments, the culture unit or culture unit cabinet provided herein has a volume of 0.09m³. 2 Up to 1.78m 2 The rectangular footprint within the specified area. In some embodiments, the culture unit or culture unit cabinet provided herein has a maximum area of ​​approximately 0.1m. 2 0.2m 2 0.3m 2 0.4m 2 0.5m 2 0.6m 2 0.7m 2 0.8m 2 0.9m 2 1.0m 2 1.1m 2 1.2m 2 1.3m 2 1.4m 2 1.5m 2 1.6m 2 or 1.7m 2 The rectangular footprint. In some embodiments, the culture unit or culture unit cabinet provided herein has a footprint of 0.03m². 3 up to 3m 3 The total chamber volume within the range. In some embodiments, the culture unit or culture unit cabinet provided herein has a maximum volume of approximately 0.03 m³. 3 0.1m 3 0.3m 3 1m 3 or 3m 3 The room volume.

[0123] Material

[0124] In some embodiments, the culture chamber cabinet has a single-walled structure. In some embodiments, the culture chamber has double-walled structures. In some embodiments, insulation is provided between the double walls of the culture chamber cabinet to control heat loss from the cabinet and to facilitate overall temperature control of the cabinet. In some embodiments, the outer wall of the culture chamber cabinet comprises sheet metal, such as cold-rolled steel of gauges 14-20. In some embodiments, the inner wall of the culture chamber cabinet (e.g., the chamber surface) comprises electropolished stainless steel. In some embodiments, the inner wall of the culture chamber cabinet (e.g., the chamber surface) comprises corrosion-resistant materials such as titanium, cobalt-chromium, tantalum, platinum, zirconium, niobium, stainless steel, and alloys thereof. However, in some embodiments, the chamber surface of the culture chamber cabinet comprises a polymeric material such as polytetrafluoroethylene (PTFE) or a polymeric material known by the trade name Parylene. In some embodiments, the chamber surface may have antimicrobial properties, such as copper or silver or an antimicrobial complex incorporated into a polymeric surface coating.

[0125] Monitoring equipment

[0126] In some embodiments, the environment within the culture vessel is controlled by a control system configured to control temperature, humidity, carbon dioxide, oxygen, and other gaseous components (e.g., sterilizing gases such as ozone and hydrogen peroxide) within the culture vessel (e.g., in one or more internal chambers). In some embodiments, the control system correspondingly controls the environmental conditions (e.g., temperature, humidity, carbon dioxide, oxygen, and other gaseous components) within each internal chamber. For example, to protect sensitive mechanical, electronic, and optical components, the humidity in an internal chamber may be maintained at a lower level than that in an internal chamber with a storage location. In some embodiments, the culture vessel is further equipped with a monitoring system with predefined sensors. Examples of monitoring devices include, but are not limited to, oxygen monitors, carbon dioxide monitors, ozone gas detectors, hydrogen peroxide monitors, and multi-gas monitors. For example, in some embodiments, the culture vessel advantageously includes multiple sensors responsive to various parameters related to cell growth, which may include temperature, air purity, contaminant levels, pH, humidity, N2, CO2, O2, and light. With this monitoring system, parameters within the culture vessel can be measured continuously using the sensors over the duration of the culture or process. In some embodiments, the parameters measured by the sensors are transmitted by the monitoring system via a line to a computer-controlled monitoring and control system for further processing as discussed herein.

[0127] In some embodiments, an environmental monitoring system may be used in conjunction with the culture unit described herein. In some embodiments, one or more sensors that provide measurements of system temperature, air composition (e.g., CO2 concentration, O2 concentration, etc.), and / or humidity may be associated with the culture unit (e.g., mounted inside a culture unit cabinet). In some embodiments, one or more such sensors may be incorporated as part of the culture unit (e.g., attached to, integrated into, or otherwise connected to the internal walls or doors of the culture unit). In some cases, one or more sensors may be positioned at any suitable location outside or inside the culture unit cabinet (e.g., inside the transfer chamber and / or interior chamber, for example, attached to an interior wall, and / or an upper or lower interior surface).

[0128] In some embodiments, a gas sensor is provided that can provide real-time readings of the concentration of a gas in contact with the sensor (e.g., gas in a cabinet, or ambient air) in parts per million or any other standard unit. Gas sensors used in the methods and cultures provided herein include CO2 sensors, O2 sensors, N2 sensors, ozone gas detectors, hydrogen peroxide monitors, multi-gas monitors, and CO sensors. Such sensors are available from a variety of commercial sources. In some cases, the environment of the culturer can be regulated or controlled based on information provided by the sensors described herein. For example, the CO2 level in the culturer can be increased based on an indication from a CO2 sensor that the presence of CO2 at a concentration below a desired level in the culturer.

[0129] In some embodiments, one or more heating or cooling elements may be incorporated into the culture vessel (e.g., on the inner surface of the cabinet or door, and / or integrated into one or more of its walls and / or the base of the cabinet) for the purpose of controlling the temperature within the culture vessel. In some embodiments, heating elements may be used to thaw liquids, such as cell culture media or other reagents.

[0130] In some embodiments, one or more air or oxygen sources, carbon filters, and / or one or more humidification or dehumidification systems are connected to the culture medium and configured to control the levels of oxygen, carbon dioxide, and / or humidity within the culture medium (e.g., in response to signals from one or more sensors in or attached to the culture medium). In some embodiments, one or more controllers are attached to these sensors and other systems to control the internal environment of the culture medium.

[0131] In some embodiments, the culture unit may include one or more light sources (e.g., incandescent bulbs, LEDs, UV, or other light sources). These light sources may be placed inside the culture unit to illuminate areas within the unit. In some embodiments, a camera or other photosensitive device, which may be placed inside or outside the culture unit, is used to monitor the operation of the culture system. In embodiments, the light source is a sterilizing light source. For example, a UV lamp may be positioned inside the transfer chamber and / or the inner chamber of the culture unit.

[0132] In some embodiments, the culture vessel includes a transparent object (e.g., a window) that allows visible light or other light wavelengths from inside the culture vessel to be detected by a camera or other photosensitive device placed outside the culture vessel. In some embodiments, the inner surface of the transparent object (e.g., from inside the cabinet) may be wiped to prevent or remove condensation droplets (e.g., due to humid air inside the culture vessel) that may accumulate on the inner surface and interfere with system monitoring. In some embodiments, the surface may be wiped using a wiping device that is automatically controlled by a controller.

[0133] Seals

[0134] In some embodiments, the culture cabinet includes windows, doors, or openings that are sealed when closed to maintain sterility after the culture cabinet has been sterilized. In some embodiments, each seal of the culture cabinet is airtight, reaching a threshold pressure level (e.g., up to 1 atm). In some embodiments, gaskets are provided to ensure the desired level of sealing capability. Generally, a “gasket” is understood as a mechanical seal that fills the space between two objects, typically used to prevent leakage between the two objects when under compression. Gaskets are typically cut from sheet materials such as gasket paper, rubber, silicone, metal, cork, felt, neoprene, nitrile rubber, fiberglass, or plastic polymers (such as polychlorotrifluoroethylene). It is generally desirable that gaskets are made of a material that provides a certain degree of yield, allowing them to deform and tightly fill the space they are designed for (including any slight irregularities). In some embodiments, gaskets may be used in conjunction with a sealant applied directly to the gasket surface to function properly. In some embodiments, the gasket material may include closed-cell neoprene foam that does not react with carbon dioxide or ozone.

[0135] Transfer device

[0136] The culture apparatus disclosed herein typically includes one or more transfer devices for moving one or more items, such as from a first position to a second position, within the culture apparatus. In some embodiments, the one or more items are one or more cell culture dishes. In other embodiments, the one or more items are useful for maintaining one or more cell culture dishes and include, but are not limited to, pipettes, capillaries, liquids (e.g., cell culture media), nutrients, and other materials. In some embodiments, the transfer device includes a robotic arm. In some embodiments, the robotic arm includes a platform within the culture apparatus cabinet that can move along a track or conveyor extending in different directions along an inner surface of the culture apparatus cabinet (e.g., an inner wall, a base, etc.). In some embodiments, the culture apparatus cabinet may be configured with more than one (e.g., 2, 3, 4, or 5 or more) robotic arms to increase the throughput of the apparatus and provide redundancy in the event of failure of one of the robotic arms.

[0137] In some embodiments, the transfer device further includes a gripper assembly coupled to a robotic arm. In some embodiments, the gripper assembly includes one or more grippers mounted on the end of the robotic arm, each gripper having two or more (e.g., 3, 4, 5, or more) gripping fingers. In some embodiments, each gripping finger on the robotic arm has a groove, friction plate, rubber pad, or other gripping surface. The gripping surface can allow these fingers to grip and transport various types of containers (e.g., culture dishes) within the cabinet. In some embodiments, the robotic arm may have an absolute encoder coupled to the gripper assembly or the platform, or a separate absolute encoder for each of the gripper assembly and / or the platform, to determine whether the robotic arm is in a position where it can safely return to its original position (e.g., return to the parking or storage configuration and / or the origin of the position or operating coordinate system) without colliding with obstacles.

[0138] In some embodiments, because it may be desirable in certain situations that the reach of the robotic arm does not extend into certain areas of the culture cabinet, the robotic arm may instead reach these locations by inserting or removing containers into a shuttle or conveyor belt, which is located, for example, on the bottom plate or other surface of the culture cabinet, moves along an axis (e.g., the x-axis, y-axis) and provides access to some of the locations that the robotic arm cannot reach.

[0139] In some embodiments, the culture cabinet is designed for use in conjunction with an external measurement or laboratory automation system. For example, in some embodiments, the culture cabinet may have a door with an opening large enough to allow gripping arms to pivot outside the culture cabinet, enabling these fingers to extend sufficiently to transport culture dishes or other containers or components from the laboratory automation system's conveyor lines into the culture cabinet, or to transport external measurement components into and / or out of the culture cabinet.

[0140] In some embodiments, the robotic arm is designed to carry (among other things) culture dishes, in which case the movement of the robotic arm is controlled to prevent such dishes from being jostled or accelerated, or other movements that could cause samples to splash out of the dishes. In some embodiments, the robotic arm is designed to carry (among other things) culture dishes, in which case the movement of the robotic arm is controlled to prevent such dishes from moving in a manner that would cause cells newly added to the plate to aggregate / concentrate in specific areas of the culture dish.

[0141] In some embodiments, because the robotic arm transports dishes or other containers between specific locations within a culture cabinet, the robotic arm, or other components of the culture cabinet, can be designed to precisely track the position of the dishes or other containers. In some cases, within a culture cabinet that can be used with a robotic arm, there may be areas where, for example, other components of the culture cabinet or the walls of the culture cabinet are located, and therefore, some movements of the robotic arm may be restricted. In these cases, a homing mechanism can be used for each of the different motors in the arm (e.g., the x-motor, the θ-motor, and the z-motor) to properly position the robotic arm to a known position after it is powered on, or before resuming operation if the robotic arm collides with another object.

[0142] In some embodiments, an uninterruptible power supply (“UPS”) is attached to or contained within the culture cabinet to allow for the orderly shutdown of the culture unit, including the preservation of various automation and sample information and the completion of any ongoing transport or transfer processes (e.g., transporting a container or vessel being carried by a robotic arm to its destination). The operator may be alerted to unauthorized opening of the culture unit by audible signals, visual signals, electronic signals (e.g., email or text messages), or in some other manner.

[0143] In some embodiments, sensors or other features are provided to detect when one or more doors of the culture unit are opened (e.g., when the doors of a culture unit cabinet, such as the exterior or interior doors, are opened). Such features are useful because they allow the operator to keep track of or be alerted to any unplanned or unauthorized opening of the culture unit (e.g., the culture unit cabinet) that may compromise sterility, disrupt production, impair assays or experiments, etc.

[0144] In some embodiments, radio frequency beacons or other signal sources are positioned within the culture unit (e.g., within a culture unit cabinet), which can be used to determine the location of one or more devices within the culture unit cabinet (e.g., devices with sensors that can detect signals and use those signals to determine their location). In some embodiments, these devices may have signal sources, and the one or more sensors may be located inside one or more chambers of the culture unit cabinet (e.g., on the inner surface of an inner chamber).

[0145] In some embodiments, optical signals or lasers (e.g., a grid of laser signals) can be used to determine the location of one or more devices or components within the culture tank. Such information can be transmitted, for example, wired or wirelessly, to an external computer or monitoring station. This information can be used to control the operation of transfer devices, such as robotic arms, within the culture tank to ensure that the transfer devices can properly grasp, manipulate, or control devices or items within the culture tank.

[0146] In some embodiments, before a container or dish is placed in a culture cabinet, the user can select an automation system option based on the specific container, dish, ingredient, or cell being inserted into the culture cabinet. Relevant information relating to the culturer and / or one or more culturer components, and the growing cells, can be entered into the data system. For example, one or more identifiers, such as barcodes (e.g., 1D or 2D barcodes), can be placed on the container or dish, indicating other important information, such as the type of container, its contents, and what measurement or manipulation will be performed on the sample in the container. In some embodiments, information relating to the culturer system and / or cells can be contained in one or more barcodes, on a separate data system, or in a combination thereof. The user can also enter information identifying the dimensions (e.g., height, diameter) of the dish or other container, or the system itself can be configured to determine the height or other dimensions of the dish or other container. Using this information, for example, when the analysis module is ready to perform a measurement or other manipulation on the cells growing in the dish, or when the measurement or manipulation has been performed, a robotic arm can be requested to deliver a specific container.

[0147] Computers and control equipment

[0148] The culture apparatus described herein includes several components, including sensors, an environmental control system, robots, etc., which can work together in the direction of a computer, processor, microcontroller, or other controller. These components may include, for example: transfer devices (e.g., robotic arms), liquid handling devices, delivery systems for delivering culture dishes or other components to or from the culture chamber, environmental control systems for controlling the temperature and other environmental aspects of the culture chamber, door operating systems, imaging or detection systems, and cell culture assay systems.

[0149] In some cases, operations such as control of cell culture apparatus and / or components provided herein or interfaced with thereto can be implemented using hardware, software, or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or set of processors, whether it is provided in a single component or distributed among multiple components. Such a processor can be implemented as an integrated circuit, wherein one or more processors are present in the integrated circuit component. The processor can be implemented using a circuit system in any suitable format.

[0150] Computers can be implemented in any of a variety of forms, such as rack-mounted computers, desktop computers, laptop computers, or tablet computers. Furthermore, computers can be embedded in devices that are not typically considered computers but have appropriate processing capabilities, including personal digital assistants (PDAs), smartphones, or any other suitable portable, mobile, or stationary electronic devices, including the grower itself.

[0151] In some cases, a computer may have one or more input and output devices. These devices may be used, in particular, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or displays for visually presenting output, and speakers or other sound-generating devices for audibly presenting output. Examples of input devices that can be used for a user interface include keyboards and pointing devices such as mice, touchpads, and digital writing tablets. In other instances, a computer may receive input information via speech recognition or in other audible formats, via visible gestures, via tactile input (e.g., including vibration, touch, and / or other forces), or any combination thereof.

[0152] One or more computers can be interconnected through one or more networks in any suitable form, including as a local area network (LAN) or wide area network (WAN), such as a corporate network or the Internet. Such networks can be based on any suitable technology and can operate according to any suitable protocol, and can include wireless networks, wired networks, or fiber optic networks.

[0153] The different methods or processes outlined in this article can be encoded as software executable on one or more processors employing any of a wide variety of operating systems or platforms. Such software can be written using any of a variety of suitable programming languages ​​and / or programming or scripting tools, and can be compiled into executable machine language code or intermediate code that executes on an architecture or virtual machine.

[0154] One or more algorithms for controlling the methods or processes provided herein may be implemented as a readable storage medium (or multiple readable media) (e.g., computer memory, one or more floppy disks, optical discs (CDs), digital video discs (DVDs), magnetic tapes, flash memory, field-programmable gate arrays or other semiconductor devices, or other tangible storage media), said readable storage medium being encoded with one or more programs that, when executed on one or more computers or other processors, perform methods for implementing the various methods or processes described herein.

[0155] In some embodiments, a computer-readable storage medium may retain information for a sufficient time to provide computer-executable instructions in a non-transitory form. One or more such computer-readable storage media may be transportable, such that one or more programs stored thereon may be loaded onto one or more different computers or other processors to implement different aspects of the methods or processes described herein. As used herein, the term "computer-readable storage medium" covers only computer-readable media that can be considered an article of manufacture (e.g., a product of manufacture) or a machine. Alternatively or additionally, the methods or processes described herein may be implemented as computer-readable media other than computer-readable storage media, such as propagating signals.

[0156] The terms “program” or “software” are used herein in a general sense, referring to any type of code or set of executable instructions that can be adopted to program a computer or other processor to implement different aspects of the methods or processes described herein. Furthermore, it should be understood that, according to one aspect of this embodiment, one or more programs that implement the methods or processes described herein when executed do not need to reside on a single computer or processor, but can be distributed in a modular manner across multiple different computers or processors to execute different programs or operations.

[0157] Executable instructions can take many forms, such as program modules, that are executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc., that perform specific tasks or implement specific abstract data types. Typically, the functionality of program modules can be combined or allocated as needed in different embodiments.

[0158] Furthermore, data structures can be stored in any suitable form on a computer-readable medium. Non-limiting examples of data storage include structured, unstructured, localized, distributed, short-term, and / or long-term storage. Non-limiting examples of protocols that can be used to transmit data include proprietary and / or industry-standard protocols (e.g., HTTP, HTML, XML, JSON, SQL, web services, text, spreadsheets, etc., or any combination thereof). For simplicity, a data structure can be shown as having fields related by their location within the data structure. Such relationships can also be established by specifying the storage locations of fields in a computer-readable medium, which convey the relationships between the fields. However, any suitable mechanism can be used to establish relationships between information in the fields of a data structure, including the use of pointers, labels, or other mechanisms to establish relationships between data elements.

[0159] In some embodiments, information relating to the operation of the culturer (e.g., temperature, humidity, gas composition, images, cell culture conditions, etc., or any combination thereof) can be obtained from one or more sensors associated with the culturer (e.g., located inside or outside the culturer cabinet), and this information can be stored in a computer-readable medium to provide information about conditions during cell culture. In some embodiments, the readable medium includes a database. In some embodiments, the database contains data from a single culturer. In some embodiments, the database contains data from multiple culturers. In some embodiments, the data is stored in a manner that makes it tamper-proof. In some embodiments, all data generated by the apparatus (e.g., the culturer) is stored. In some embodiments, a subset of the data is stored.

[0160] In some embodiments, the component (e.g., a computer) controls multiple different processes performed within the culture vessel. For example, the computer may directly control devices (e.g., manipulators, imagers, fluid handling systems, etc.). In some embodiments, the computer controls imaging of cell cultures, cell pickup, cell removal (e.g., cell cluster removal), monitoring of cell culture conditions, adjustment of cell culture conditions, tracking of movement of cell culture dishes within the culture vessel, and / or planning of previous processes.

[0161] Cell assay

[0162] In some embodiments, the culture cabinets provided herein are configured with microscopes or other imagers, or other devices for monitoring cell growth, cell viability, or other purposes. In some embodiments, the microscopes or imagers are used in conjunction with assays performed within the culture cabinet (e.g., image-based phenotypic screening or assays).

[0163] In some embodiments, the culture apparatus provided herein is configured to allow one or more assays to be performed within a culture apparatus cabinet or in a chamber operatively connected to the culture apparatus cabinet (e.g., a separate assay chamber as part of the culture apparatus). In some embodiments, the culture apparatus provided herein is configured to allow the performance of cell counting assays, replication marker assays, cell membrane integrity assays, ATP-based viability assays, mitochondrial reductase activity assays, caspase activity assays, annexin V staining assays, DNA content assays, DNA degradation assays, nuclear fragmentation assays, or combinations thereof. Other exemplary assays include BrdU, EdU, or H3-thymidine incorporation assays; DNA content assays using nucleic acid dyes such as Hoechst dye, DAPI, actinomycin D, 7-aminoactinomycin D, or propidium iodide; cell metabolism assays such as AlamarBlue, MTT, XTT, and CellTitre Glo; nuclear fragmentation assays; cytoplasmic histone-associated DNA fragmentation assays; PARP cleavage assays; and TUNEL staining assays.

[0164] Treatment and experimental intervention

[0165] In some embodiments, the culture apparatus provided herein is configured to allow high-throughput screening (HTS) within a culture cabinet. In some embodiments, HTS refers to testing up to, for example, 100,000 complexes per day. In some embodiments, screening assays can be performed in multi-well formats (e.g., 96-well, 384-well, or 1,536-well formats) and can be performed using automated methods. In such high-throughput assays, it is possible to screen thousands of different complexes or components in a single day. Specifically, each well of a microtiter plate can be used for individual assays of selected test complexes, or multiple wells can contain test samples of a single complex if concentration or culture time effects are to be observed. It is possible to assay many plates daily; using these assays, assay screening can be performed on up to approximately 6,000, 20,000, 50,000, or more than 100,000 different complexes. Typically, the HTS implementation of the assays described herein involves the use of automation. In some embodiments, an integrated robotic system including one or more robotic arms delivers assay microplates between multiple assay stations for the addition, mixing, cultivation, and final readout or detection of complexes, cells, and / or reagents. In some aspects, HTS assays may include the simultaneous preparation, cultivation, and analysis of many plates, thereby further accelerating the data collection process.

[0166] In some embodiments, the assay may include test cells, control cells, and one or more test complexes, such as 10, 100, 1000, 10,000, or more test complexes. The cells and test reagents may be arranged in one or more dishes in a manner suitable for evaluating the effect of the one or more test complexes on the cells. These assays may be performed in one or more culture cabinets of one or more culturers described herein. Typically, these dishes contain suitable tissue culture media, and the test complexes are present in the tissue culture media and may be delivered automatically to the culture media within the culture cabinet of the culturer provided herein. A culture medium suitable for culturing a specific cell type may be selected for use. In some embodiments, the culture medium is free of or substantially free of serum or tissue extracts, while in other embodiments, such components are present. In some embodiments, cells are cultured on a plastic or glass surface.

[0167] The above aspects and embodiments can be used in any suitable combination, as the invention is not limited in this respect.

[0168] It should be understood that aspects of the invention have been described herein with reference to certain illustrative embodiments and accompanying drawings. The illustrative embodiments described herein are not necessarily intended to illustrate all aspects of the invention, but are used to describe several illustrative embodiments. Therefore, aspects of the invention are not intended to be interpreted narrowly in relation to these illustrative embodiments. Furthermore, it should be understood that aspects of the invention can be used alone or in any suitable combination with other aspects of the invention.

[0169] Having described several aspects of at least one embodiment of the invention, it should be understood that various changes, modifications, and improvements will readily occur to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to fall within the spirit and scope of the invention. Therefore, the foregoing description and drawings are by way of example only.

[0170] While several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision a wide variety of other devices and / or structures to perform the functions described herein, and / or obtain the results and / or one or more of the advantages, and each of such changes and / or modifications is considered to fall within the scope of the invention. More broadly, those skilled in the art will readily recognize that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and actual parameters, dimensions, materials, and / or configurations will depend on the specific application of the teachings of this invention. Those skilled in the art will recognize or be able to identify many equivalents of the specific embodiments of the invention described herein using only conventional experimentation. Therefore, it should be understood that the foregoing embodiments are presented by way of example only, and the invention may be practiced differently from the specific descriptions and claims within the scope of the appended claims and their equivalents. The invention relates to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of this invention if such features, systems, articles, materials, and / or methods do not contradict each other.

[0171] As used herein, the indefinite articles “a” and “an” should be understood to mean “at least one” unless explicitly indicated to the contrary.

[0172] As used herein in the specification and in the claims, the phrase “and / or” should be understood to mean “one or both” of the elements so combined that multiple elements coexist in some cases and exist separately in others. Other elements may optionally exist in addition to those specifically indicated by the “and / or” phrase, whether related to or unrelated to those specifically indicated, unless expressly indicated to the contrary. Thus, as a non-limiting example, when used in conjunction with open-ended language such as “comprising,” reference to “A and / or B” in one embodiment may refer to A without B (optionally including elements other than B), in another embodiment to B without A (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), and so on.

[0173] As used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when dividing multiple items in a list, “or” or “and / or” should be interpreted as inclusive, e.g., including multiple elements or at least one element in a list of elements, but also including more than one element, and optionally including additional unlisted items. Only terms that clearly indicate the opposite, such as “only one” or “exact one” or, when used in the claims, “consisting of…”, refer to including multiple elements or an exact one element in a list of elements. In general, when preceded by an exclusive term such as “any one,” “one of,” “only one of…,” or “exact one of…,” the term “or” as used herein should be interpreted only as indicating an exclusive alternative (e.g., “one or the other but not both”). When used in the claims, “consisting of…” should have its ordinary meaning as used in the field of patent law.

[0174] As used herein in the specification and claims, the phrase "at least one" referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of all the elements specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those expressly specified in the list of elements as referred to by the phrase "at least one," whether related to or unrelated to those expressly specified elements. Therefore, as a non-limiting example, "at least one of A and B" (or equivalently "at least one of A or B", or equivalently "at least one of A and / or B") in one embodiment may refer to at least one (optionally including more than one) A, where B is absent (and optionally includes elements other than B); in another embodiment may refer to at least one (optionally including more than one) B, where A is absent (and optionally includes elements other than A); in yet another embodiment may refer to at least one (optionally including more than one) A, and at least one (optionally including more than one) B (and optionally includes other elements), and so on.

[0175] In the claims and the description above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “possessing,” etc., should be understood as open-ended, for example, meaning “including but not limited to.” As described in Section 2111.03 of the U.S. Patent Examination Procedure Manual, only the transitional phrases “consisting of” and “consisting substantially of” should be closed or semi-closed transitional phrases, respectively.

[0176] The use of sequential terms such as “first,” “second,” and “third” to modify a claim element does not imply any priority, precedence, or order of one claim element relative to another, or the chronological order in which the actions of a method are performed. Rather, it is merely used as a marker to distinguish one claim element with a certain name from another element with the same name (but using sequential terms).

[0177] It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are stated.

Claims

1. A cell culture incubator comprising: an incubator cabinet comprising an interior chamber for incubating cells in one or more cell culture vessels; a door leading from an external environment to the interior chamber; an imager configured to image the cells inside the interior chamber; a manipulator for manipulating the cells in the one or more cell culture vessels inside the interior chamber, wherein the manipulator comprises one or more cell scrapers, wherein each cell scraper comprises a handle portion comprising an elongate member extending from a proximal region attachable or connectable to a manipulator base to a distal region comprising a scooping edge, a channel extending from the proximal region to the distal region, and an opening at the scooping edge and in communication with the channel for delivering at least one of cells or cell media relative to the cell culture vessel; a controller configured to control the manipulator to adjust a contact pressure between the scooping edge of the cell scraper and a surface of a cell culture vessel; a sensor coupled to the cell scraper that provides a signal to the controller indicative of a sensed pressure between the scooping edge of the cell scraper and a surface of a cell culture vessel, wherein the controller is configured to transmit a control signal to the manipulator to increase or decrease the pressure between the scooping edge of the cell scraper and the surface of a cell culture vessel in response to the sensed pressure; wherein the controller is configured to coordinate rotation of the scraper with linear movement of the scraper in x-, y-, and z-axes such that a constant angle of attack is maintained between the scraper blade and the cells being scooped independent of the geometry of the cell culture vessel; and a cell culture vessel transfer device for moving the one or more cell culture vessels between locations inside the interior chamber.

2. The incubator of claim 1, wherein the imager is a holographic microscope.

3. The incubator of claim 1, wherein the imager is a brightfield microscope.

4. The incubator of claim 1, wherein the imager is a fluorescence microscope.

5. The incubator of any one of claims 1-4, wherein the one or more cell culture vessels comprise fiducial markers to facilitate alignment of the one or more cell culture vessels with the imager and the manipulator.

6. The incubator of any one of claims 1-5, further comprising an imaging location, wherein the imaging location comprises fiducial markers.

7. The incubator of any one of claims 1-6, wherein the manipulator for manipulating the cells further comprises a cell picker.

8. The incubator of any one of claims 1-7, wherein the cell scraper comprises a top portion for connection to the manipulator and a bottom portion for scooping, and wherein the bottom portion is removable from the top portion to replace the cell scraper. ​ 9. The incubator of claim 1, wherein the controller is configured to locate and determine cell aggregates via imaging to allow subsequent scraping and / or aspiration using a scraper.

10. The incubator of claim 1, wherein the controller comprises a computer.

11. A cell culture incubator comprising: an incubator cabinet comprising an interior chamber for incubating cells in one or more cell culture vessels; a door to the interior chamber; a holographic imager and a first imaging position configured to image the cells inside the interior chamber when the one or more cell culture vessels are in the first imaging position; a second imager and a second imaging position configured to image the cells inside the interior chamber when the one or more cell culture vessels are in the second imaging position; a manipulator for manipulating the cells in the one or more cell culture vessels at the first imaging position and / or the second imaging position, wherein the manipulator comprises one or more cell scrapers, wherein each cell scraper comprises a handle portion comprising an elongate member extending from a proximal region attachable or connectable to a manipulator base to a distal region comprising a scraping edge, a channel extending from the proximal region to the distal region, and an opening at the scraping edge and in communication with the channel for delivering at least one of cells or cell media relative to the cell culture vessel; a controller configured to control the manipulator to adjust a contact pressure between the scraping edge of the cell scraper and a surface of a cell culture vessel; a sensor coupled to the cell scraper that provides a signal to the controller indicative of a sensed pressure between the scraping edge of the cell scraper and a surface of a cell culture vessel, wherein the controller is configured to transmit a control signal to the manipulator to increase or decrease the pressure between the scraping edge of the cell scraper and the surface of a cell culture vessel in response to the sensed pressure; wherein the controller is configured to coordinate rotation of the scraper with linear movement of the scraper in x-, y-, and z-axes such that a constant angle of attack is maintained between the scraper blade and cells being scraped independent of the geometry of the cell culture vessel; and a cell culture vessel transfer device for moving the one or more cell culture vessels from the first imaging position to the second imaging position or from the second imaging position to the first imaging position.

12. The incubator of claim 11, wherein the holographic imager is a holographic microscope.

13. The incubator of claim 11 or 12, wherein the second imager is a brightfield microscope or a fluorescence microscope. ​ 14. The incubator of claim 11, wherein each cell scraper includes a continuous structure including a scraping edge.

15. The incubator of claim 14, wherein each cell scraper includes a molded structure including a scraping edge.

16. The incubator of any one of claims 11-15, wherein each cell scraper includes an interconnecting portion including a top portion for connection to the manipulator and a bottom portion for scraping, and wherein the bottom portion is removable from the top portion to replace a cell scraper.

17. The incubator of any one of claims 11-16, wherein each cell scraper includes a handle having an interface for replaceably connecting a scraping edge assembly to the handle.

18. The incubator of any one of claims 11-17, wherein each cell scraper includes a scraper edge that is contactable with a surface of a cell culture vessel and is configured to scrape cells adhered to the surface without substantially killing the cells.

19. The incubator of claim 11, wherein the sensor is a strain gauge sensor.

20. The incubator of any one of claims 11-19, wherein each cell scraper is readily removable from the manipulator.

21. The incubator of any one of claims 11-20, wherein each cell scraper is configured to perform a scraping function and a liquid handling function.

22. The incubator of any one of claims 11-21, wherein each cell scraper includes a scraping edge configured to allow a defined range of scraping edge deflection when in contact with a cell culture vessel.

23. The incubator of any one of claims 11-22, wherein each cell scraper includes one or more components formed from a polymer.

24. The incubator of claim 23, wherein the opening is configured to aspirate cells and / or cell media, wherein the opening is positioned proximate to a scraping edge.

25. The incubator of claim 11, wherein the controller is configured to locate and determine cell colonies via imaging to allow subsequent scraping and / or aspiration using a scraper.

26. The incubator of claim 11, wherein the controller is internal to or integrated into the incubator cabinet.

27. The incubator of claim 1 or any one of claims 11-26, wherein the incubator further includes a second manipulator having at least one cell scraper.

28. The incubator of any one of claims 1-27, wherein the incubator is configured to move the one or more cell culture vessels from an imaging position to a manipulation position or from a manipulation position to an imaging position, the one or more cell culture vessels being substantially aligned.

29. The incubator of any one of claims 1-28, wherein the cell culture vessel transfer device is configured for removing one or more cell culture vessels inside the interior chamber between an imaging position and a manipulation position and / or between a manipulation position and an imaging position.

30. The incubator of any one of claims 1-29, wherein the one or more cell culture vessels comprise fiducial markers to facilitate alignment of the one or more cell culture vessels with the imager and the manipulator.

Images