WO2017087698A1 - Incubator insert for automated culture experimentation - Google Patents

Abstract

An incubator insert system includes a support platform configured to fit internally within an incubator defined by an incubator length, an incubator height, and an incubator width. The system includes a robotic pipettor attached to the support platform for inserting a fluid into one or more fluid containers, a perfusion system attached to the support platform and in fluid communication with the robotic pipettor, and a microscope module attached to the support platform and configured to capture images of samples on the support platform.

Description

INCUBATOR INSERT FOR AUTOMATED CULTURE EXPERIMENTATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application Serial No. 62/257,043, filed on November 18, 2015, and U.S. Provisional Patent Application Serial No. 62/368,804, filed on July 29, 2016, each of which is hereby incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant no. HHSF223201310079 awarded by the U.S. Department of Defense, Defense Advanced Research Projects Agency ("DARPA"). The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates generally to biological experiments, and, more particularly, to compact platform for insertion into a standard incubator.

BACKGROUND OF THE INVENTION

[0004] Although robotic fluid handlers already exist (e.g., Tecan, Beckton Coulter, etc.), their cost, complexity, and size make them suitable only for laboratories and companies with dedicated space and staff. For example, Opentrons, a recent startup company, is selling a low-cost robotic pipettor that is bulky and that is not amenable for cell culture conditions or for ex vivo studies with cultured small embryos, living tissues, or organ explants.

[0005] Furthermore, none of these systems is currently suitable for microfluidic applications without additional significant modifications. For microfluidic applications, several products enable microfluidic cell culture inside incubators: Nortis' culture chips, Fluigent's Microfluidic Flow Controller, and the ever-present syringe and peristaltic pumps from various companies. However, these systems do not provide the interfacing necessary for complex experimentation in an automated fashion, since a user must manually change reagents.

[0006] In another example, at the Wyss Institute, an "Interrogator" device was developed as part of the DARPA Organs on Chips program. The Interrogator device represents a prototype of an instrument that attempts to address pitfalls of existing technologies. In response to use of prototype instruments, additional needs were identified. According to one need, instrument stability and user-friendliness were difficult to address based on the complexity of the instruments and the reliance on pressure-driven flow in a bubble-prone system. According to another need, it was determined that the instruments take up an excessive amount of lab space and that the each of the instruments can only handle a small number of organ chips, e.g., 12 organ chips. According to yet another need, when transitioning from manual experiments to the Interrogator devices, many workflows, protocols, and even reagents required modification for proper cell viability based on the transition to a pressurized perfusion system.

[0007] Accordingly, present embodiments are directed to solving the above and other needs. For example, some exemplary embodiments are directed to a simple platform that enables users to add on automation and imaging to otherwise standard microfluidic experiments that are provided in any range of chip geometries. According to other exemplary embodiments, a system includes a platform that is configured as an incubator insert, regardless of additional space constraints, and that provides a low-cost and scalable, widely deployed product.

SUMMARY OF THE INVENTION

[0008] Medical devices for management and treatment of medical conditions, such as asthma and anaphylaxis, include needs in which many biological experiments are automated for improved accuracy, increased throughput, and reduced risk when working with pathogens or other laboratory hazards. As such, some of needs for a generalized experimental system include a system for performing fluid transfers and a method for imaging samples for analysis or monitoring. For microfluidic systems, perfusion of fluid within microfluidic devices (such as organs on chips) and associated chip-to-world interfacing are additional requirements. An exemplary system for achieving one or more of these needs includes a compact and low-cost platform that is inserted into a standard incubator and that enables automated experiments on organs on chips and other devices or culture systems.

[0009] According to one aspect of the present invention, an incubator insert system includes a support platform configured to fit internally within an incubator space defined by an incubator length, an incubator height, and an incubator width. The system includes a robotic pipettor attached to the support platform for inserting a fluid into one or more fluid containers, a perfusion system attached to the support platform and in fluid communication with the robotic pipettor, and a microscope module attached to the support platform and configured to capture images of samples on the support platform.

[0010] According to another aspect of the present invention, an incubator insert system includes a support platform that is configured to fit within an internal incubator space and has a generally rectangular support base. The support base has a base length of in the range of about 470 millimeters to about 607 millimeters, and a base width in the range of about 500 millimeters to about 757 millimeters. The support platform also has a support frame extending upwards along a back edge of the support base. The support frame has a frame height in the range of about 533 millimeters to about 681 millimeters. The system further includes one or more experimental modules mounted to the support platform. The experimental modules include a robotic pipettor module mounted to the support frame and having a pipette head movable along one or more of a plurality of axes, a peristaltic pump module mounted to the support base along a front edge of the support base, and a microscope module mounted to the support base adjacent to the peristaltic pump module.

[0011] Additional aspects of the invention will be apparent to those of ordinary skill in the art in view of the detailed description of various embodiments, which is made with reference to the drawings, a brief description of which is provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a side view illustrating an experimental insert positioned in a standard incubator, according to one embodiment.

[0013] FIG. 2 is a cross-sectional isometric view illustrating the experimental insert in the standard incubator of FIG. 1.

[0014] FIG. 3 is a front isometric view of an experimental insert for an incubator, according to another embodiment.

[0015] FIG. 4 is a back isometric view of the experimental insert of FIG. 3.

[0016] FIG. 5 is a front isometric view of the experimental insert illustrating a robotic pipettor module.

[0017] FIG. 6 is a front isometric view of the experimental insert illustrating a peristaltic pump module.

[0018] FIG. 7 is a front isometric view of the experimental insert illustrating a microscope module.

[0019] FIG. 8 is an isometric view of the microscope module of FIG. 7. [0020] FIG. 9 is an isometric view of a fluidics plate assembly, according to one embodiment.

[0021] FIG. 10 is a top view illustrating the fluidics plate assembly of FIG. 9 without well plates.

[0022] FIG. 11 A is a display screen with flow rate settings for a pump user interface.

[0023] FIG. 1 IB is a display screen with flush rate settings for the pump user interface of FIG. 11 A.

[0024] FIG. 11C is a display screen with direction settings for the pump user interface of FIG. 11 A.

[0025] FIG. 11D is a display screen with flush interval settings for the pump user interface of FIG. 11 A.

[0026] FIG. HE is a display screen with flush duration settings for the pump user interface of FIG. 11 A.

[0027] FIG. 1 IF is a display screen with manual flush settings for the pump user interface of FIG. 11 A.

[0028] FIG. 12A is an isometric view of a fluidics plate assembly, according to another embodiment.

[0029] FIG. 12B is an exploded view of the fluidics plate assembly of FIG. 12A.

[0030] FIG. 12C is a partial view of the fluidics plate assembly of FIG. 12A, showing two pairs of individual cartridges.

[0031] While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION

[0032] While this invention is susceptible of embodiment in many different forms, there is shown in the drawings and will herein be described in detail preferred embodiments of the invention with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and is not intended to limit the broad aspect of the invention to the embodiments illustrated. For purposes of the present detailed description, the singular includes the plural and vice versa (unless specifically disclaimed); the words "and" and "or" shall be both conjunctive and disjunctive; the word "all" means "any and all"; the word "any" means "any and all"; and the word "including" means "including without limitation." Where a range of values is disclosed, the respective embodiments includes each value between the upper and lower limits of the range.

[0033] Referring to FIGs. 1 and 2, an incubator insert system 100 is in the form of a miniaturized and inexpensive experiment automation platform that is designed to fit inside a standard incubator 102 for scalable deployment. For example, the incubator insert system 100 is configured to fit internally within an incubator space generally shaped as a cube and having dimensions of up to about 800 millimeters ("mm") in each of an incubator length CL, an incubator height (CH), and a an incubator width CW. The term "about" includes +/- 10% of a nominal value of a dimension. By way of further specific examples, the incubator insert system 100 is configured to fit internally within an incubator space in which the CLxCHxCW dimensions include incubators of 541x681x508 mm, 470x607x530 mm, 607x670x583 mm, 508x533x635 mm, 584x656x757 mm, 470x607x576 mm, and 500x600x500 mm. The incubator insert system 100 is used with microfluidic or non-mi croflui die systems, including systems for standard well plate cultures, and is constructed from a range of materials, including steel, plastics, aluminum, other metals, glass, ceramics, and/or wood.

[0034] Referring to FIGs. 3 and 4, the incubator insert system 100 includes a support platform 104 that is configured to fit within the internal incubator space (defined generally by the length CL, the height CH, and the width CW). The support platform 104 has a generally rectangular support base 106 with a base length BL and a base width BW that are each sized in accordance with the respective size and shape of the incubator. For example, in one example the base length BL is less than about 607 mm and the base width BW is less than about 757 mm. The support platform 104 further has a support frame 108 that extends upward along a back edge of the support base 106. The support frame 104 has a frame height FH, which according to one example is less than about 670 mm.

[0035] The incubator insert system 100 further includes one or more experimental modules that are mounted to the support platform 104. The experimental modules include a robotic pipettor module 110 that is mounted to the support frame 108 and which has a pipette head 112 that is movable along one or more of a plurality of axes. The experimental modules also include a perfusion system 114 that, in accordance with one example, is in the form of a peristaltic pump module and that is mounted to the support base 106 along a front edge of the support base 106.

[0036] The experimental modules further include a microscope module 116 that is mounted to the support base 106 adjacent to the peristaltic pump module 114, and a chip holder module 118 that is mounted to the support base 106 above the microscope module 116. In accordance with the experimental modules, and as applicable, the incubator insert system 100 includes one or more stepper motors, servo motors, pneumatics, hydraulics, electronic actuation (e.g., solenoids), and/or other actuation systems for position control of the pipettor, pump, and/or microscope in the respective robotic pipettor module 110, the peristaltic pump module 114, and/or the microscope module 116. Optionally, the experimental modules include control modules having a GRBL shield, stepper motor drivers, and/or custom drivers.

[0037] As more clearly illustrated in FIG. 4, the incubator insert system 100 includes miniaturized microscope motion control hardware, with integration and layout of electronics 120 into a back plate 122 of the incubator insert system 100. The integration of the electronics 120 into the incubator insert system 100 avoids, or reduces, long-distance current delivery issues and electromagnetic interference (EMI) generation. Furthermore, the electronics 120 are designed to handle non-condensing humidity.

[0038] The layout of the plurality of hardware components is designed to minimize space requirements, e.g., base length BL is less than the cube length CL. The incubator insert system 100 further includes a belt drive system 124 with reduced footprint having motors move along a static belt. The microscope module 116 has optical components that provide dual imaging capability in a small footprint. The peristaltic pump module 114 includes hardware components, software components, and a control box.

[0039] The incubator insert system 100 consists of at least five main modules: (1) the robotic pipettor module 110, which handles insertion of fluids, (2) the peristaltic pump module 114 for perfusion, (3) the microscope module 116, which is a miniaturized fluorescence/brightfield microscope with 3 axis positioning for imaging, (4) the chip holder module 118, which is a platform for holding organ chips and associated reservoirs (as well as well plates and pipette boxes), and (5) a software module for integrating the hardware modules in a user-friendly manner. The incubator insert system 100 has dimensions of up to an approximately 450 millimeter ("mm") cube, allowing the platform to fit inside standard incubator designs, such as the standard incubator 102 illustrated in FIGs. 1 and 2. [0040] To facilitate a networked connection, a server box is optionally positioned outside of the standard incubator 102 to connect features of the incubator insert system 100 to a webapp interface. Lower level electronics, including motorized axis controls and a camera, are mounted to the incubator insert system 100 and located within the standard incubator 102 when experiments are performed. For connection purposes, and by way of example, a standard USB cable and a power cable are the only connections necessary for operation of any of the features of the incubator insert system 100 within the standard incubator 102.

[0041] In alternative configurations, the incubator insert system 100 is in whole or in part oriented horizontally or rotated up to 90 degrees in either direction, such as for embryo culture chips. By way of example, the incubator insert system 100 has multiple modules at different positions/angles relative to each other to offer improved access, including insertion of bays with multiple culture devices for greater modularity and speed.

[0042] Referring to FIG. 5, the robotic pipettor module 110 includes one or more features, including, by way of example, a 3-axis positioning of a commercial pipette head 112 (e.g., a Incontinent pipettor or a Z series pipettor), with approximately 300 mm by 300 mm range of lateral motion along each lateral axis X and Y, and approximately 150 mm vertical range of motion along a vertical axis Z. Optionally, the robotic pipettor module 110 includes position and acceleration control through GRBL 3-axis shield, belt driven X and Y axes for speed, and/or a screw-driven Z-axis for added precision.

[0043] According to optional or alternative embodiments, the robotic pipettor module 110 is optionally custom-made in whole or in part, and/or relies on air or liquid displacement. According to other optional or alternative embodiments, the incubator insert system 100 includes multiple pipettors or pipette tip adapters for faster operation.

[0044] Referring to FIG. 6, the peristaltic pump module 114 includes one or more features, including, by way of example, a commercial pump head (2x12 channels, Langer Instruments), an integrated 27: 1 geared stepper motor for high torque and precision speed control, and/or an optional standalone control box for use outside of an automated system. Optionally, the peristaltic pump module 114 further includes perfusion controlled through experimental design software, enables perfusion of up to 12 organ chips (2 channels each), slides out to a set distance PY for ease of access, and/or offers variable flow rates and flush cycles for debris/bubble removal, which bubble development in microfluidic. Optionally, yet, flushing with variable pump flow control enables reduction/elimination of bubble development in the culture devices. Yet another feature of the peristaltic pump module 114 is that the electronics are optionally run through a microcontroller board (such as Arduino Uno) for a standalone module, and, as such, this configuration allows stacking of "shields" and includes a touchscreen shield for user control and a motor driver shield for motor control.

[0045] Optionally or alternatively, a syringe, vacuum, pressure, electrochemical, acoustofluidic, and/or other pumping system is used for perfusion instead of or in addition to the peristaltic pump module 114. Alternatively, the system does not have perfusion or includes multiple perfusion means. Alternatively, yet, perfusion is optionally identical for all culture devices or is individually controlled.

[0046] Some benefits of the peristaltic pump module 114 is that it provides a perfusion device sufficiently compact to fit in the standard incubator 102 and within the incubator insert system 100, that is capable of external control for software integration, that is capable of 12 x2 channel culture (e.g., 12 organ chips), and that allows a low flow rate (e.g., approximately 60 microliters/hour) and flush cycles to remove bubbles or debris. These benefits greatly improve the experimental results and are in stark contrast with existing syringe systems or peristaltic pumps that are plagued by many problems, including that they cannot flow many chips, have variable flow rate problems, cannot do flush cycles, are extremely bulky, are very expensive, are difficult to interface programmatically, and produce significant heat.

[0047] Referring to FIGs. 7 and 8, the microscope module 116 integrates with hardware and software components, and includes one or more features. By way of example, some of the features include a standard Nikon lOx objective (interchangeable) at 90 degrees relative to an epifluorescence optical path for compact form factor, servos (geared 200: 1, 20 mm travel) for Y axis and focusing, and/or a stepper motor belt drive for fast X axis travel. By way of further example, the microscope module 116 optionally includes compact mounting for axes to fit under microfluidic chip rack, and/or provides epifluorescence and brightfield imaging capabilities. In yet another example, the microscope module 116 includes light- emitting diodes ("LEDs") used for excitation, and/or external LEDs mounted above chips for brightfield imaging. In a further example, the microscope module is controlled and automated through a platform interface, and/or includes periodic image-based analysis.

[0048] Referring to FIGs. 9 and 10, the chip holder module 118 includes one or more features, including, by way of example, a holder for up to 12 standard microscope slides (25 x 75 mm), or a holder for 24 inlet and 24 outlet reservoirs, e.g., standard syringe bodies and custom reservoirs. In other examples, the incubator insert system 100 is configured to accommodate any number of culture compartments/containers, e.g., the range of culture compartments/containers includes a range of 1 to 12, a range from 10 to 100, or a range that exceeds 100. The range is based on the dimensions of the compartments/containers.

[0049] By way of another example, the chip holder module 118 includes a holder that is removable to allow for chip/reservoir/tubing setup in sterile conditions in a biosafety hood and held in position by 4 alignment pins. In yet other examples, the chip holder module 118 includes additional configurable holders for pipette tip box, waste bin, standard plates, and standard tubes for fluid handling needs, and/or additional well plates located above microfluidic cultures for increased capacity.

[0050] Referring to FIGs. 11A-11E, a pump user interface 126 is in the form of a touchscreen display via which various pump settings of the peristaltic pump module 114 are adjusted. For example, adjustments to the pump settings include a flow rate adjustment (FIG. 11 A), a flush rate adjustment (FIG. 11B), a direction adjustment (FIG. 11C), a flush interval adjustment (FIG. 11D), a flush duration (FIG. HE), and a manual flush adjustment (FIG. 1 IF). The pump user interface 126 is typically used in a standalone operation. In an alternative embodiment, platform software controls the peristaltic pump module 114 as part of an experiment run.

[0051] Many and various optional features and elements are included in the incubator insert system 100, in addition or instead of the features described above. According to alternative embodiments, a belt, drive rod, air bearing, electromagnet, and/or other approach is used to transduce force to the pipettor or microscope in the respective robotic pipettor module 110 or microscope module 116. According to other alternative embodiments, the incubator insert system 100 uses automated robotic samplers to transfer liquids to external instruments (e.g., Mass Spectroscopy) for off-line analysis. According to yet other alternative embodiments, the incubator insert system 100 performs any number and type of automated assays on the cultured tissues or on the supernatant, including using reagents stored in well plates or elsewhere on a platform.

[0052] In yet other alternative embodiments, all components of the incubator insert system 100 are removable for sterilization and/or decontamination. In further alternative embodiments, multiple layers are implemented for greater density of resources (e.g., well plates, pipette tip boxes, etc.). The layers are optionally static and/or automated. In further alternative embodiments, the incubator insert system 100 is used for working with hazardous materials and/or pathogens, where minimal volumes and remote handling are desired. In other examples, the incubator insert system 100 is used for organ chips, other microfluidic systems, tissue/organ explants, embryos, organoids, and/or whole organisms (e.g., C. elegans, zebrafish, Drosophila, mosquitos, etc.).

[0053] In accordance with another embodiment, the incubator insert system 100 incorporates a gripper arm for positioning of items, is integrated with PCR and/or other analytical systems, and/or is used for diagnostics applications. By way of further examples, the incubator insert system 100 is used for 3D printing, aerosol delivery, topical ointment delivery, microsurgery/dissection, precision injury models, and other methods for manipulating tissue/organs/organisms.

[0054] In accordance with another embodiment, the incubator insert system 100 is used for automated recovery of, for example, tissue or embryos, using pipettor or another attachment, which could be used for DNA/RNA analysis or transfer for external growth of organisms or cells. In another example, workflow and/or protocol changes are minimized in experiments by superimposing automation on existing microfluidic or other culture systems rather than necessitating special disposables.

[0055] Referring to FIGs. 12A-12C, a chip holder module 218 includes one or more features, including, by way of example, cartridges 220 that are individual removable units loaded in a tray 222. In this exemplary embodiment, the chip holder module 218 includes a total of 40 cartridges 220, each cartridge 220 being spring-loaded as pairs in the tray 222. One benefit of the mounting manner of the cartridges 222 is that the loading as individual units facilitates flexibility in handling and imaging of the cartridges 222 independent of each other. Another benefit of the mounting manner of the cartridges 222 is that the spring- loading facilitates locking each unit in the tray 222, for imaging and robust handling, while still permitting easy removal of the cartridges 222.

[0056] Each of these embodiments and obvious variations thereof is contemplated as falling within the spirit and scope of the claimed invention, which is set forth in the following claims. Moreover, the present concepts expressly include any and all combinations and subcombinations of the preceding elements and aspects.

Claims

CLAIMS What is claimed is:

1. An incubator insert system, comprising:

a support platform configured to fit internally within an incubator space defined by an incubator length, an incubator height, and an incubator width;

a robotic pipettor attached to the support platform for inserting a fluid into one or more fluid containers;

a perfusion system attached to the support platform and in fluid communication with the robotic pipettor; and

a microscope module attached to the support platform and configured to capture images of samples on the support platform.

2. The incubator insert system of claim 1, wherein the incubator space is shaped as a cube, each of the incubator length, the incubator height, and the incubator width being up to 800 millimeters.

3. The incubator insert system of claim 1, wherein the dimensions for the incubator length, the incubator height, and the incubator width are selected from a group consisting of 541x681x508 mm, 470x607x530 millimeters, 607x670x583 millimeters, 508x533x635 millimeters, 584x656x757 millimeters, 470x607x576 millimeters, and 500x600x500 millimeters.

4. The incubator insert system of claim 1, wherein the perfusion system includes a peristaltic pump.

5. The system of claim 1, wherein the support platform includes a holder device.

6. The system of claim 5, wherein the holder device holds up to 12 microscope slides.

7. The system of claim 5, wherein the holder device is configured to include 24 inlet reservoirs and 24 outlet reservoirs.

8. The system of claim 5, wherein the holder device is removable.

9. The system of claim 8, wherein the holder device is removably held in position with a plurality of alignment pins.

10. The system of claim 1, wherein the robotic pipettor includes a head movable along three axes.

11. The system of claim 10, wherein the head has a movable range of approximately 300 millimeters along lateral axes X and Y and approximately 150 millimeters along vertical axis Z.

12. The system of claim 1, wherein the robotic pipettor is belt-driven along lateral axes X and Y and is screw-driven along a vertical axis Z.

13. The system of claim 1, wherein the peristaltic pump provides variable flow rates and flush cycles for debris or bubble removal.

14. The system of claim 1, wherein the peristaltic pump enables perfusion of up to 12 organ chips, with each of the organ chips having two channels.

15. The system of claim 1, wherein the microscope module includes a stepper-motor belt drive for travel along a lateral axis.

16. The system of claim 1, wherein the microscope module provides epifluorescence and brightfield images.

17. The system of claim 1, wherein the microscope module includes external light- emitting diodes for brightfield imaging.

18. An incubator insert system, comprising:

a support platform configured to fit within an internal incubator space and having a generally rectangular support base with a base length in the range of about 470 millimeters to about 607 millimeters, and with a base width in the range of about 500 millimeters to about 757 millimeters, and a support frame extending upwards along a back edge of the support base, the support frame having a frame height in the range of about 533 millimeters to about 681 millimeters; and

one or more experimental modules mounted to the support platform and including a robotic pipettor module mounted to the support frame and having a pipette head movable along one or more of a plurality of axes, a peristaltic pump module mounted to the support base along a front edge of the support base, and

a microscope module mounted to the support base adjacent to the peristaltic pump module.

19. The incubator insert system of claim 18, further comprising a chip holder module mounted to the support base above the microscope module.

20. The incubator insert system of claim 19, wherein the chip holder module includes at least 12 microscope slide receptacles, each of the receptacles being connected to a respective pair of inlet reservoirs and outlet reservoirs.

21. The incubator insert system of claim 18, wherein the pipette head is movable up to about 300 millimeters along each of two lateral axes and up to about 150 millimeters along a vertical axis.

22. The incubator insert system of claim 18, wherein the robotic pipettor module is mounted above the microscope module.

23. The incubator insert system of claim 18, wherein the microscope module includes a microscope objective movable along a plurality of axes.