Fluidic cell culture device

Abstract

A cell culture device designed for automated in vitro experiments, integrates fluidic technology and electrophysiological monitoring. The device features a fluidic culture chamber with a lid incorporating a polished glass rod for high-quality imaging, alignment grooves for precise fitting, and ventilating air ducts for gas exchange. The device also includes inlet and outlet fluid paths for media exchange, managed by programmable fluidic pumps. A catch tray prevents overflow, protecting connected equipment. The device supports live-cell imaging and high-density microelectrode arrays (HD-MEA) for continuous monitoring of neural activity. This system enables touch-free, automated cell culture, enhancing consistency and reducing contamination risk, suitable for various biological research applications.

Description

FLUIDIC CELL CULTURE DEVICECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Patent Application Serial No. 63 / 565,893 filed on March 15, 2024, and U.S. Provisional Patent Application Serial No. 63 / 707,298 filed on October 15, 2024. The entire disclosures of the foregoing applications are incorporated by reference herein.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant 1RM1HG011543 awarded by the National Human Genome Research Institute and grant 2134955 awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND

[0003] Recently, advances in biological research have been greatly influenced by the development of organoids, a specialized form of 3D cell culture. Created from pluripotent stem cells, organoids are effective in vitro models in replicating the structure and progression of organ development, providing an exceptional tool for studying the complexities of biology. Among these, cerebral cortex organoids (hereafter “organoid”) have become particularly instrumental in providing valuable insights into brain formation, function, and pathology. Despite their potential, organoid experiments present significant challenges. Brain organoids require a rigorous, months- long developmental process, demanding substantial resources and meticulous care to yield valuable data on aspects of biology such as neural unit electrophysiology, cytoarchitecture, and transcriptional regulation.

[0004] The primary methods for generating and measuring organoids depend on media manipulations, imaging, and electrophysiological measurements, which are all labor and skillintensive, limiting the power and throughput of experiments. Cell culture feeding and data collection occur at intervals realistic for researchers. Furthermore, during manual feeding and data collection, the cell cultures are removed from the incubator, which provides a controlled gas, temperature, and humidity environment. Feeding may be aligned with the cells' metabolic cycles, and data may be collected at intervals on par with the biological phenomenon. The disturbance incurred by leaving the incubator environment is shown to increase metabolic stress and batch-to-batch variability, potentially impacting the quality of the experiment, as well as increasing contamination risk. These limitations hinder the depth of insights gained from these organoid models, particularly in studies focused on dynamic neural processes and disease modeling.

[0005] Laboratory robotics, such as liquid handling devices, offer increased precision and throughput but are primarily designed for pharmaceutical screens, limiting their adoption in research labs due to high costs, large footprints, and inflexible workflows. Moreover, many of these systems lack the ability to seamlessly integrate new technologies as they emerge. Conversely, academic research labs are benefiting from advancements in commercial and custom-made technologies, facilitated by in-house fabrication methods like 3D printing, which are enhancing their capacity to manipulate and measure biological systems. However, without an easy-to-integrate, device-agnostic robotic platform, researchers are constrained to manual operations, restricting the power and scope of their experiments. By outfitting devices to carry out automated jobs and relay data through communication networks, they acquire around-the- clock functionality and increased fidelity. The flexibility in size (number of devices perintegrated system) allows researchers to optimize for the experimental design and budget. Implementing programmable feedback loops derives precision and self-optimization by dynamically adjusting to real-time data, offering a practical alternative to complex mathematical modeling for experiment control. This approach would enable more integrated, flexible automation in research settings, broadening the scope and efficiency of experiments.

[0006] Automating multiple devices to report data presents a challenge for device management and communication, necessitating flexible and efficient infrastructure. Addressing this need for an interconnected ecosystem of devices, services, and technologies is possible through designing networks using standards defined by the Internet of Things (loT). This approach has already impacted wearables, agriculture, city infrastructure, security, and healthcare. It was recently proposed to expand this approach to biology research. Previously, researchers built a custom device and code from scratch with unique assumptions for communication and behavior. Each device operated in solitude, lacking integration and feedback with other devices. There is a need for a platform that addresses these challenges, combining electrophysiology, microscopy, fluidics, and feedback control, automated and integrated through loT technology for touch-free, in-incubator tissue research.SUMMARY

[0007] The analysis of tissue cultures, particularly brain organoids, takes a high degree of coordination, measurement, and monitoring. The present disclosure provides an automated research platform enabling independent devices to achieve collaborative objectives for feedback- driven cell culture studies. Unified by an Internet of Things (loT) architecture, our approach enables continuous, communicative interactions among various sensing and actuation devices,achieving precisely timed control of in vitro biological experiments. The framework integrates fluidics, electrophysiology, and imaging devices to maintain cerebral cortex organoids and monitor their neuronal activity.

[0008] The organoids may be cultured in custom cell culture devices, which may be 3D-printed, and are attached to microelectrode arrays (MEA) for electrophysiology monitoring. Periodic feeding is achieved using programmable fluidic pumps. The present disclosure also provides for computer vision fluid volume estimations of aspirated media, achieving high accuracy, and uses feedback to rectify deviations in fluidic perfusion during media feeding / aspiration cycles. The automated system may provide for periodic, e.g., hourly, electrophysiology recordings that may reveal temporal changes in neuron firing rates not observed in once-a-day recordings.

[0009] The fluidic cell culture device provides a microenvironment for tissue culture samples. It is a component that interfaces two systems: fluidic pumps for reagent delivery to a multielectrode array for analyzing electrical potentials. The fluidic cell culture device features inlets and outlets (e.g., threaded) for connection to auxiliary fluidic pumps, which feed into the device's fluidic channels to supply the tissue culture with nutritional media and / or reagents. Additionally, a lid with a viewing window (e.g., a glass rod) enables live-cell imaging of the tissue situated on the multielectrode array.

[0010] Prior art cell culture devices require manual handling of the precious and fragile (e.g., brain) tissue sample to perform experiments. This is laborious for researchers, incurs contamination risk, and adds human errors and inconsistencies, which degrade the study. The disclosed device solves this problem by relaying all the fluidic manipulations of the sample to a robotic pump without contacting the sample or removing it from incubation. Feeding tissue samples may occur on a schedule reasonable for researchers (e.g., every day or two); however,this is not physiologically optimal, and it has been shown that the tissue culture would have improved health if fed more often.

[0011] The disclosed system and the fluidic cell culture device provide automated fluidic feeding that can occur on a programmatic user-defined schedule with the system for measuring electric potentials (i.e., firing neurons). Adhering tissue samples to the multielectrode array is a precise and delicate process. This is prone to delamination when media is introduced to the well either by pipette or fluidics. The disclosed fluidic cell culture device has fluidic geometries that perform the media exchange in the headspace away from the tissue sample so that it is not subjected to these flow streams. There is also a physical minimum volume guard to prevent media levels from becoming too low. There is a need to image the tissue culture on the multi el ectrode array to study the character of neurons. The industry standard is a film membrane that is drawn over the lid of the multi el ectrode array well. Once inside the incubator, condensation clouds the membrane, and clear imaging is impossible leading to the need to remove the sample from incubation. The fluidic cell culture device of the present disclosure contains a polished glass rod submerged in the cell culture media that maintains optical clarity indefinitely, allowing live-cell imaging for the duration of the experiment. Light can be projected through polished glass rod to the samples, opening the applications in imaging and perturbation such as optogenetics.

[0012] The disclosed fluidic cell culture device is highly modular. The removable and reattachable feature of the lid reduces manufacturing complexity and enables use of other lids with applications beyond imaging, such as an optogenetic light source or an ion pump. Alignment grooves in the lid prevent rotation and incorrect fitting. The lid performs gas exchange with the outside atmosphere, e.g., incubator conditions, through ventilating air ducts.

[0013] The fluidic cell culture device also provides chemical compound testing. The well can be flushed multiple times in serial with a reagent of choice to introduce new compounds (e.g., a new differentiation medium) or dilute away media components (e.g., clearing a chemical of interest). Additionally, the fluidic cell culture device provides for universal fit and can slot into an existing commercially available MEA well (i.e., any circular well). Liquid-impermeable sealing of the fluidic cell culture device is made with the walls by O-ring gaskets, ensuring the retention of the culture’s media. Fresh media enters the device, filling the internal cavity and conditioned media is drawn out of the device. Custom software protocols may be used to control the fluidic pumps to deliver media and / or reagents to the tissue cultures for feeding, experimenting, and conducting assays. The change of height occurs between a distance that keeps the rod immersed in media and the biological sample (e.g., cerebral cortex organoid) guarded against high flow rates and sheer forces.

[0014] The cell culture device may be fabricated by 3D printing three pieces (e.g., a catch tray, a fluidic module, and a lid) from a biocompatible material. O-ring gaskets may then be slotted into the printed grooves. The polished glass rod is then inserted into the lid. All parts may be sterilized by autoclave to prepare them for tissue culture. The catch tray is slotted around the multi el ectrode array well in the case of overflow or leakage. The cell culture device is inserted into the multi el ectrode array well, making a liquid-tight, hermetic seal. Flexible fluidic tubing from an auxiliary fluidic pump is mated to the fluidic module with threaded fluidic couplers. A digital microscope or camera may also be used and is affixed above the cell culture device for imaging.

[0015] The present disclosure can be broadly applied to various fluidic cell culture devices. The disclosed devices are adaptable to operate across different fluidic scales, from macrofluidicdevices handling larger volumes of media to microfluidic and nanofluidic systems designed for smaller-scale applications. The principles of automated media exchange, real-time monitoring, and fluid handling can be implemented in any fluidic system, allowing the technology to be scalable for different experimental needs, whether in large tissue culture setups or precise applications involving small cellular systems, without size constraints.

[0016] According to one embodiment of the present disclosure, a cell culture device is disclosed. The cell culture device includes a fluidic culture module having a cavity configured to house a biological sample and an inlet and outlet for supplying a cell culture media into the fluidic culture module. The device also includes a lid for sealing the fluidic culture module. The lid includes an access port defined through the lid, a viewing window disposed through the access port and configured for imaging of the biological sample, and an air duct defined in the lid and in fluid communication with the cavity to allow gas exchange between the cavity and an outside atmosphere.

[0017] Implementations of the above embodiment may include one or more of the following features. According to one aspect of the above embodiment, the fluidic culture module may further include an inlet fluid path fluidly coupled to the inlet and an outlet fluid path fluidly coupled to the outlet, and the inlet and outlet fluid paths may terminate in the cavity. Each of the inlet fluid path and the outlet fluid path may have a sinuous shape. The fluidic culture module may further include an inner well and the inlet and outlet fluid paths may terminate above the inner well to direct flow of the media above the biological sample. The inner well may have a beveled or chamfered upper surface to provide for circulation of the media around the biological sample. The inlet fluid path, the outlet fluid path, and the inner well may be configured to minimize disturbance of the biological sample due to the circulation of the media. The fluidicculture module may also be configured to be inserted into a well of a substrate including a bottom viewing window for imaging of the biological sample. The fluidic culture module may also be configured to be inserted into a well of an electrophysiological device, which may include a sensor. A bottom portion of the viewing window may be submerged in the cell culture media. The cell culture device may include a tray surrounding the fluidic culture module that is configured to receive overflow of the cell culture media through the air duct.

[0018] According to another embodiment of the present disclosure, a cell culture system is disclosed. The cell culture system includes an electrophysiological device having a sensor and a well surrounding the sensor. The system also includes a cell culture device having a fluidic culture module inserted into the well. The fluidic culture module includes a cavity configured to house a biological sample and an inlet and an outlet for supplying a cell culture media into the fluidic culture module. The fluidic culture module also includes a lid for sealing the fluidic culture module, the lid having an access port defined through the lid. A viewing window may be disposed placed through the access port and configured for imaging of the biological sample. The lid may also include an air duct defined in the lid and in fluid communication with the cavity to allow gas exchange between the cavity and an outside atmosphere.

[0019] Implementations of the above embodiment may include one or more of the following features. According to one aspect of the above embodiment, the fluidic culture module may further include an inlet fluid path fluidly coupled to the inlet and an outlet fluid path fluidly coupled to the outlet, and the inlet and outlet fluid paths may terminate in the cavity. Each of the inlet fluid path and the outlet fluid path may have a sinuous shape. The fluidic culture module may further include an inner well rising above the biological sample, and the inlet and outlet fluid paths may terminate above the inner well to direct flow of the media above the biologicalsample. The inner well may have a beveled or chamfered upper surface to provide for circulation of the media around the biological sample. The inlet fluid path, the outlet fluid path, and the inner well may be configured to minimize disturbance of the biological sample due to the circulation of the media. The system may also include an imaging device positioned over the viewing window. The imaging device may be configured to image the biological sample through the viewing window. A bottom portion of the viewing window may be submerged in the cell culture media. The system may also include a tray surrounding the well of the electrophysiological device and configured to receive overflow of the cell culture media through the air duct. The system may further include a programmable fluidic pump connected to the inlet and the outlet for automated media exchange. The system may additionally include a collection reservoir configured to receive the media removed from the cell culture device and a camera module configured to measure an amount of the media inside the collection reservoir. The system may also include a computing device configured to receive measurement data from the camera module and to control the fluidic pump to adjust flow rate of the media based on the measurement data.BRIEF DESCRIPTION OF DRAWINGS

[0020] Various embodiments of the present disclosure are described herein below with reference to the figures wherein:

[0021] FIG. 1A is a schematic diagram of a fluidic system for electrophysiological monitoring and imaging of cell cultures inside a fluidic cell culture device according to an embodiment of the present disclosure;

[0022] FIG. IB is a side view with parts separated of the fluidic cell culture device disposed over an electrophysiological device and a recording unit according to an embodiment of the present disclosure;

[0023] FIG. 1C is a top view of the fluidic cell culture device and an enlarged view thereof according to an embodiment of the present disclosure;

[0024] FIG. 2 is a photograph of the fluidic cell culture devices according to an embodiment of the present disclosure setup inside an incubator with MEA wells covered by conventional membrane lids;

[0025] FIG. 3 is a photograph of the fluidic cell culture device with a lid removed according to an embodiment of the present disclosure;

[0026] FIG. 4 is a side cross-sectional view taken across a sectional line 4-4 of the fluidic cell culture device of FIG. 3;

[0027] FIG. 5 is another side cross-sectional view taken across a sectional line 5-5 of the fluidic cell culture device of FIG. 3;

[0028] FIG. 6 is a side cross-sectional view of the fluidic cell culture device of FIG. 3 disposed over a substrate having a viewing window according to an embodiment of the present disclosure;

[0029] FIGS. 7-10 are top views of the fluidic module of the cell culture device of FIG. 3 according to embodiments of the present disclosure;

[0030] FIG. 11 is a side cross-sectional view taken across a sectional line 11-11 of the fluidic module of FIG. 10;

[0031] FIG. 12 is a side cross-sectional view taken across a sectional line 12-12 of the fluidic module of FIG. 10; and

[0032] FIG. 13 is a side cross-sectional view taken across a sectional line 13-13 of the fluidic module of FIG. 10.DETAILED DESCRIPTION

[0033] The present disclosure provides for an integrated platform that automates organoid culture and data collection in individual microenvironments. The system includes a fluidic cell culture device storing a cell culture (e.g., organoids) and fluidics to control the media environment. Digital microscopy captures the morphogenic features. The neural activity is recorded by local field potential measurements using complementary-metal-oxide semiconductor (CMOS) high-density microelectrode arrays (HD-MEA). The loT cloud network brokers the communication between all devices and facilitates data storage, processing, and presentation services including an interactive webpage. Through touch-free automation, samples remain undisrupted in the incubator, increasing the consistency of images and allowing for higher frequencies of feeding and recording.

[0034] At user-defined intervals, conditioned media is aspirated by a syringe pump through a system of distribution valves, stored in a collection reservoir (without passing through the syringe pump vial), and replaced by an equivalent volume of fresh media. Both types of media are perfused through tubing, which may be formed from flexible fluorinated ethylene propylene (FEP) tubing. The flow rate may be from about 50 mm / s to about 200 mm / s and in embodiments may be from about 80 mm / s to about 150 mm / s, which leads to low shear forces.

[0035] Imaging of the cell culture inside the fluidic cell culture device may be accomplished using a digital microscope disposed over the chamber. The microscope may be secured using 3D-printed parts movably attached to one or more posts. The chamber may also be 3D printedand integrates the fluidics and HD-MEAs. A liquid-impermeable O-ring gasket ensures media retention inside the chamber. The well lid includes a polished glass rod submerged in the media, improving image quality and removing the effects of condensation. Alignment grooves in the glass rod lid prevent rotation and incorrect fitting. The lid exchanges gas with the incubator conditions through ventilating air ducts, similar to a cell culture well plate. The removable and re-attachable lid reduces manufacturing complexity and enables future use of other lids with applications beyond imaging.

[0036] The term “cell culture” or “culture” refers to the maintenance of cells in an artificial, in vitro environment. It is to be understood, however, that the term “cell culture” is a generic term and may be used to encompass the cultivation not only of individual cells, but also of tissues, organs, organoids, and non-human organisms.

[0037] The term “organoid” as used herein refers to a 3-dimensional growth of mammalian cells in culture that retains characteristics of a tissue in vivo, e.g., prolonged tissue expansion with proliferation, multilineage differentiation, recapitulation of cellular and tissue ultrastructure, etc. A primary organoid is an organoid that is cultured from an explant, i.e., a cultured explant. A secondary organoid is an organoid that is cultured from a subset of cells of a primary organoid, i.e., the primary organoid is fragmented, e.g., by mechanical or chemical means, and the fragments are replated and cultured. A tertiary organoid is an organoid that is cultured from a secondary organoid, etc.

[0038] The term “explant” is used herein to mean a piece of tissue and the cells thereof originating from mammalian tissue that is cultured in vitro, for example in a cell culture well, as described herein. The mammalian tissue from which the explant is derived may be obtained froman individual, i.e., a primary explant, or it may be obtained in vitro, e.g., by differentiation of induced pluripotent stem cells.

[0039] The phrase “mammalian cell” refers to any cell originating from mammalian tissue. The cell can be a primary cell obtained directly from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a subject. For example, the cell may be a stem cell, progenitor cell, or adult cell. Immortalized cells are also included within this definition. In some embodiments, the cell has been genetically engineered to express a recombinant protein and / or nucleic acid. The term “mammalian” includes, without limitation, human, equine, bovine, porcine, canine, feline, rodent (e.g., mice, rats, hamster), and primate.

[0040] “Biocompatible,” as used herein, refers to a property of a material that allows for prolonged contact with a tissue in a subject without causing toxicity or significant damage.

[0041] Additional information on types of cell cultures and studies that may be performed using the cell culture device according to the present disclosure may be found in an International Application Publication WO2023081229A1 “Microfluidic Well Plates and Related Methods” published on May 11, 2023, the entire disclosure of which is incorporated by reference herein.

[0042] FIG. 1 shows a system 10 for electrophysiological and imaging of cell cultures inside one or more fluidic cell culture devices 100. The culture device 100 disclosed herein may be configured to operate as a macrofluidic, microfluidic, or nanofluidic device, depending on the specific application and scale of use. As a macrofluidic device, it can handle larger volumes of media and reagents for experiments involving substantial tissue samples or larger organoids. In a microfluidic configuration, the device can manage smaller volumes with high precision, suitable for standard organoid culture and fine control of fluid dynamics. Additionally, as a nanofluidic device, it can be adapted for ultra-small-scale applications, such as experiments with single cellsor subcellular structures, where minute volumes of fluids and highly precise control are required. This flexibility in scale enables the culture device 100 to be used in a wide variety of biological research applications, from large-scale tissue cultures to intricate cell-level studies.

[0043] Macrofluidic refers to fluidic systems designed to handle and manipulate relatively large volumes of fluids, typically in the range of milliliters or more. Macrofluidic systems are used in experiments where larger samples, such as full tissue cultures or large organoids, are cultured and managed. These systems often involve larger channels, pumps, and reservoirs to accommodate the greater fluid volume.

[0044] Microfluidic refers to fluidic systems that manipulate small volumes of fluids, typically ranging from microliters to nanoliters. Microfluidic systems are characterized by microscale channels and are commonly used in cell culture, diagnostics, and other biological applications that require precise control of fluid flow at a smaller scale, such as in organoid culture or singlecell analysis.

[0045] Nanofluidic refers to systems that handle fluids at the nanoscale, typically in volumes from picoliters to nanoliters. Nanofluidic systems involve extremely fine control of fluids in nanometer-scale channels and are used in highly sensitive applications, such as single-cell or subcellular analysis, molecular sorting, or DNA sequencing, where ultra-precise manipulation of tiny fluid volumes is desired.

[0046] The system 10 includes a media source 11 (e.g., container) of fresh cell culture media, which provides the cells with the nutrients the cells need to grow and divide. The culture device 100 includes an inlet 102 and an outlet 104, each of which is coupled to a distribution valve 12 and 14, respectively. The valves 12 and 14 are coupled to a pump 16, which may be a syringe pump. The pump 16 controls flow of the fresh media into the culture device 100 through thedistribution valve 12 and the inlet 102 as well as remove conditioned media from the culture device 100 through the outlet 104 and the distribution valve 14, e.g., via aspiration. The aspirated conditioned media drawn from the culture device 100 is collected in a collection reservoir 17, which is imaged by a camera module 18 configured to measure the amount (e.g., volume) of the media inside the collection reservoir 17. The camera module 18 captures images of the aspirated conditioned media drawn from each culture and relays them to a computing device 28, which processes the volume data from the camera module 18 for volume estimation feedback to the pump 16, i.e., the computing device 28 controls operation of the pump 16.

[0047] The computing device 28 may be any suitable computing device, such as a desktop computer, a laptop, etc. Computing device 28 may include a communication interface (e.g., ethernet, WiFi, etc.) allowing for communication with a network. Computing device 28 also includes a processor, a memory, a storage device, an input device, and a display screen. The processor is connected to each of the hardware components constituting the computing device 28. The computing device 28 may also be a virtualized computer, a containerized application (e.g., Docker), a cloud server or service, or any other computing platform.

[0048] The media source 11, the collection reservoir 17, and optionally the camera module 18, may be placed inside a refrigerator 19 or any other suitable temperature-controlled apparatus. In embodiments, the refrigerator 19 may be capable of maintaining preset humidity and temperature, e.g., humidity from about 35 % to about 50 % and temperature from about -5 °C to about 10 °C, which in embodiments may be about 4 °C.

[0049] The culture device 100 may be placed inside an incubator 20 to maintain optimal cell growth conditions inside the culture device 100. The incubator 20 may be any suitable cell culture incubator capable of maintaining preset humidity and temperature, e.g., humidity fromabout 75 % to about 90 % and temperature from about 35 °C to about 40 °C. An imaging device 23, which may be a digital microscope or camera, and a light source 21 (e.g., one or more LEDs), are positioned over the culture device 100 inside the incubator 20 as shown in FIG. 2, allowing for longitudinal imaging of the cells or organoids inside the culture device 100. The computing device 28 receives, records, and processes images of the cells inside the culture device 100. Imaging properties of the imaging device 23 may also be adjusted using the computing device 28, including imaging frequency, zoom, focus, etc.

[0050] With reference to FIGS. IB and 1C, the culture device 100 is placed over an observation or measurement device, such as an electrophysiological device 24 (FIG. IB), which houses a sensor 25 (FIG. 1C) for recording electrophysiology signals, such as HD-MEA sensor, such as MaxOne Single-Well MEA available from MaxWell Biosystems. As described above, the electrophysiological device 24 may be a suitable CMOS MEA having a plurality of electrodes, e.g., 26,400 (9.3x5.45 sq-pm, 17.5 pm pitch) and may have about 1,204 readout channels with 32 simultaneous channels.

[0051] The electrophysiological device 24 may be coupled to a recording unit 26 (FIGS. 1A and IB), which is configured to output a stimulation signal for the electrophysiological device, e.g., sampling rate amplitude, etc. The recording unit 26 may also measure neurological signals of the neural tissue disposed in the electrophysiological device 24 in response to electrical stimulating signals. The recording unit 26 is also coupled to the computing device 28. The computing device 28 also receives, records, and processes the neurological signals from the recording unit 26. Some or all of the components (e.g., the camera module 18, the imaging device 23, the electrophysiological device 24, etc.) may communicate with the computing device 28 using any suitable communication protocol, such as Message Queuing Telemetry Transport (MQTT)protocol and automatically upload data to the cloud, where it is stored, processed, and presented on a web page. This setup allows for automatic recording, media exchange, collecting morphogenic and functional dynamics data of the biological sample inside the culture device 100.

[0052] With reference to FIGS. IB, 1C, and 3-5, the culture device 100 is configured to securely couple to the electrophysiological device 24 thereby forming a liquid-tight, hermetic seal therebetween. The electrophysiological device 24 includes a substrate 40 supporting the MEA sensor 25. The sensor 25 (FIG. 1C) is surrounded by a well 42 (FIG. IB) and the culture device 100 is configured to fit therein. The shape and dimensions of the circular device 100 match the shape of the electrophysiological device 24 and in particular the well 42.

[0053] The culture device 100 includes three main components: a catch tray 106, a fluidic module 110, and a lid 150. The cell culture device components may be formed from any biocompatible (i.e., not harmful to living tissue) material, such as glass, metal (e.g., titanium, cytocompatible steels including but not limited to 316 and 254 SMO grades, nickel-chromium- based alloys such as Inconel, etc.) and / or a polymer, including but not limited to one or more of polyarylethersulfones, polyaryletherketones polycarbonates, polystyrenes, polyethylene terephthalates, and the like. In embodiments, the material may be a clear biocompatible photopolymer resin, such as Biomed Clear Vlmaterial (RS-F2-BMCL-01, Formlabs) used with a 3D resin printer (e.g., Form 3B+, Formlabs). The cell culture device components may be constructed using manufacturing techniques such as subtractive manufacturing (e.g., computer numerical control (CNC) milling, machining, laser cutting), additive manufacturing (e.g., 3D printing using stereolithography or any other printer), injection molding, thermal or ultrasonic welding of walls, and the like.

[0054] With reference to FIGS. 3-5, the catch tray 106 is configured to receive any excess media that might overflow from the culture device 100 during its operation. The catch tray 106 handles media overflow, ensuring that any excess media is collected and does not spill over, which could potentially damage the electrophysiological device 24 and / or the recording unit 26. The catch tray 106 may be designed to collect any suitable amount of fluid, which may be up to 1.5 ml of overflow or approximately 200% of the capacity volume of the cavity 113, thereby maintaining the operational integrity of the electrophysiological device 24 and / or the recording unit 26.

[0055] To prevent fluid leakage, the catch tray 106, along with other components such as the fluidic module 110 and the lid 150, includes O-rings to provide secure seals. The O-rings be made from any elastomeric material, e.g., silicone, rubber, etc. The catch tray 106 includes an inner wall 107 having substantially the same shape (e.g., circular) as the well 42 of the electrophysiological device 24. The inner wall 107 defines an opening for insertion of the well 42 therethrough. An O-ring 109 is disposed on the inner wall 107, which is configured to frictionally engage the outer surface of the well 42 thereby providing a waterproof seal between the outside surface of the well 42 and the inner wall 107 of the catch tray 106.

[0056] With continued reference to FIGS. 3-5, the fluidic module 110 is inserted into the well 42 to provide for a fluid enclosure of the biological sample (e.g., organoid) disposed on the sensor 25 inside the well 42. As shown in FIG. 4, the fluidic module 110 includes a body 111 having an outer surface 112 and a bottom surface 114, both of which contact the inner surface of the well 42 when the fluidic module 110 is inserted into the well 42. The body 11 1 has substantially the same shape (e.g., circular) as the well 42 of the electrophysiological device 24 and is configured to be inserted inside thereof. The body 111 defines a cavity 113 having a top opening 115 and a bottom opening 117. Once inserted, the bottom opening 117 provides access to the sensor 25.

[0057] The fluidic module 110 also includes the inlet 102 and outlet 104, which are configured to receive microtube fluidic fittings 105 for coupling tubing (not shown) interconnecting the valves 12, 14, and the pump 16. The inlet 102 and the outlet 104 are disposed above the well 42 and are fluidly coupled to an inlet fluid path 116 and an outlet fluid path 118, respectively, which include a first portion that extends downward from the inlet 102 and the outlet 104 towards the bottom surface 114. As used herein, “fluidly coupled” refers to a connection between two or more components that allows for the transfer or flow of a fluid, such as a gas or liquid, between them. The inlet and outlet fluid paths 116 and 118 also include a second portion extending from the first portions thereof in an opposing direction and are fluidly coupled to the cavity 113. The first and second portions of the inlet and outlet fluid paths 116 and 118 are separated by partitions 119 and 121, respectively. The first and second portions of the inlet and outlet fluid paths 116 and 118 may have chamfered transitions at the turns where the first and second portions switch directions. Fresh media enters the cavity 113 through the inlet fluid path 116 thereby filling the cavity 113. Conditioned media is drawn out of the cavity 113 through the outlet fluid path 118.

[0058] The cavity 113 also includes an inner well 120 surrounding the sensor 25 and the biological sample that is placed on top thereof. The inner well 120 may have any suitable shape, e.g., rectangular, circular, oval, polygonal, etc. and extends upward from the bottom surface 114 thereby directing the fluid flow from the inlet and outlet fluid paths 116 and 118 upwards into the cavity 113, thus, forming high fluid ports 184 and 186, respectively, inside the cavity 1 13 (see FIG. 11). As used herein, the term “high fluid port” denotes a port circulating the fluid above the biological sample disposed inside the fluidic module 110. The inner well 120 may alsohave a beveled or chamfered upper surface to aid in the fluid circulation around the biological sample.

[0059] The sinuous, e.g., having multiple turns, inlet and outlet fluid paths 116 and 118 and geometry of the cavity 113 ensure minimum disturbance to the biological sample. Fresh media is delivered on top of the volume present in the cavity 113, similar to partial media changes found in manual feeding protocols. The operating range may be between 350 to 700 pL. In the case of over-aspiration, media may drop to a minimum of 170 pL before aspirating air from the headspace of the cavity 113.

[0060] The fluidic module 110 also includes an O-ring 122 configured to provide a secure, waterproof seal between the fluidic module 110 and the well 42 of the electrophysiological device 24. The O-ring 122 may be formed from any suitable elastomeric material as described above. The O-ring 122 is disposed on the outer surface 112 of the body 111. The O-ring 122 is configured to frictionally engage the inner surface of the well 42 thereby providing a waterproof seal between the fluidic module 110 and the well 42.

[0061] With continued reference to FIGS. 3-5, the lid 150 is configured to be inserted into the top opening 115 of the fluidic module 110. The lid 150 enhances performance and maintain sample integrity during in vitro experiments. The lid 150 includes an insertion portion 152 and a cover portion 154. The insertion portion 152 is insertable into the top opening 115 while the cover portion 154 rests on top of the body 111 of the fluidic module 110. The lid 150 also includes a central access port 156 (FIG. 5) having a top opening and a bottom opening. The central access port 156 is used for securing a viewing window 158, which may be a glass rod or a quartz drawn rod having a diameter of 5 mm + 0.20 mm and a length of 15 mm ± 0.20 mm.The viewing window 158 improves image quality by eliminating condensation effects andprovides for unobstructed observation of biological samples. In embodiments, the access port 156 may be used to hold any desired observation or measurement device, such as an optogenetic light source or an ion pump.

[0062] The access port 156 includes one or more (or two or more) O-rings 160, that may be formed from any suitable elastomeric material as described above and are used to frictionally engage the viewing window 158 thereby securing the viewing window 158 laterally while allowing for longitudinal adjustment of the viewing window 158 into and out the cavity 113 of the fluidic module 110. In particular, a bottom end of the viewing window 158 may be inserted into the media inside the cavity 113 to eliminate visual artifacts, e.g., due to condensation. The media level may be maintained in the cavity 113 between an upper level indicated by an arrow “high” and a lower level indicated by an arrow “low”. The upper volume corresponding to the high arrow may be about 560 pL and the lower volume corresponding to the lower arrow may be about 350 pL, which is still above the bottom surface of the viewing window 158. Thus, the operating media volume range keeps the viewing window 158 immersed in media. For microfluidic setup, the volume of the cavity 113 may be from about 100 pL to about 1,000 pL.

[0063] The insertion portion 152 also includes an O-ring 162, which may be formed from any suitable elastomeric material as described above. The O-ring 162 is disposed on the outer surface of insertion portion 152 and is configured to frictionally engage the inner surface of the cavity 113 thereby providing a waterproof seal between the insertion portion 152 and the inside surface of the cavity 1 13.

[0064] The lid 150 also includes one or more air ducts 164, which pass through the insertion portion 152 and the cover portion 154. The air ducts 164 exchange gas with the incubator conditions by fluidly connecting to the cavity 113. A top surface of the fluidic module 110includes one or more grooves 170 such that when the lid 150 is seated on top of the fluidic module 110 the air ducts 164 are fluidly coupled to the grooves 170 allowing for air flow into and out of the cavity 113. With reference to FIG. 5, the cover portion 154 may include one or more partitions 165 with openings, which divide the air ducts 164 to allow for air flow between the groove 170 and the cavity 113. In addition, the air ducts 164 also act as overflow ducts for excess media which flows into the catch tray 106. In addition, the lid 150 also includes one or more grooves 172 that are used to align the lid 150 with the fluidic module 110 during assembly by mating corresponding protrusions (not shown) on the fluidic module 110 with the grooves 172.

[0065] With reference to FIG. 6, the culture device 100 may be used with a substrate 24a rather than the electrophysiological device 24. The substrate 24a includes a viewing window 25a surrounded by a well 42a. The substrate 24a is configured to engage the culture device 100 in the same manner as the electrophysiological device 24, as described above with respect to FIGS. 3-5, such as providing for insertion of the fluidic module 110 into the well 42a, placement of the catch tray 106 around the well 42a, contacting the O-rings 109 and 122, etc. The fluidic module 110 is inserted into the well 42a to provide for a fluid enclosure of the biological sample (e.g., organoid) disposed on the viewing window 25a inside the well 42a. The viewing window 25a allows for use of the imaging device 23 from one or both of the viewing windows 158 and 25a. In embodiments, the access port 156 may be used to hold any desired observation or measurement device, such as an optogenetic light source or an ion pump while imaging may be provided through the viewing window 25a of the substrate 24a.

[0066] FIGS. 7-13 illustrate additional embodiments of the fluidic module 110 of the culture device 100. The modifications described below may be incorporated in any suitable manner intothe above-disclosed embodiment of the fluidic module 1 10. In particular, the fluidic module 1 10 may have one or more of the following: one or more high fluid ports (FIGS. 3-8 and 11), one or more low fluid ports (FIGS. 9, 10, 12, and 13), or one or more injection ports (FIGS. 7-11 and 13). As noted above, the term “high fluid port” denotes a port circulating the fluid above the biological sample disposed inside the fluidic module 110. The term “low fluid port” denotes a port circulating the fluid at or below the biological sample disposed inside the fluidic module 110. In addition, the fluidic module 110 may have any number, i.e., two or more, of inlets and outlets, for example, two inlets and two outlets as shown in FIGS. 7-10 each of which terminates in a high fluid port or a low fluid port.

[0067] FIGS. 7-9 illustrate the fluidic module 110 including a side injection port 180, which passes through a top surface of the fluidic module 110 at a diagonal line relative to a plane defined by the top surface fluidic module 110. FIG. 13 shows a cross-sectional view illustrating the structure and direction of the side injection port 180. The side injection port 180 provides direct access to the cavity 113 of the fluidic module 110 and may be threaded to allow for coupling an adapter 182 (FIGS. 7, 8, and 10) for guiding a needle (see FIG. 8) or another fluid injection instrument. The injection port 180 may be used to inject compounds (e.g., drugs) into the cavity 113. The fluidic module 110 also includes a pair of inlets 102a and 102b and a pair of outlets 104a and 104b, which may terminate inside the cavity 113 as high fluid ports 184a, 184b and / or low fluid ports 186a, 186b (FIGS. 11 and 12). As noted above, the fluidic module 110 may have any desired volume, which in microfluidic uses may be from about 100 pL to about 1,000 pL. For example, the fluidic module 110 of FIG. 7 may have a base volume of about 400 pL, the fluidic module 110 of FIG. 8, may have a base volume of about 300 pL, the fluidicmodule 1 10 of FIG. 9 may have a base volume of about 150 pL, and the fluidic module 110 of FIG. 10 may have a base volume of about 133 pL.

[0068] FIG. 11-13 illustrate cross-sectional views of the fluidic module 110 providing a detailed view of the structure of fluid paths connected to the high and low fluid ports 184a, 184b, 186a, 186b. The inlets 102a, 102b and the outlets 104a, 104b are configured to receive microtube fluidic fittings 105 for coupling tubing (not shown) interconnecting the valves 12, 14, and the pump 16. The inlets 102a, 102b and the outlets 104a, 104b are disposed above the well 42 or 42a. With reference to FIG. 11, the inlet 102a and the outlet 104a are fluidly coupled to a high inlet fluid path 116a and a high outlet fluid path 118a, respectively, which include a first portion that extends downward from the inlet 102a and the outlet 104a towards the bottom surface 114 of the fluidic module 110. The inlet and outlet fluid paths 116a and 118a also include a second portion extending from the first portions thereof in an opposing direction that are fluidly coupled to the cavity 113. The first and second portions of the inlet and outlet fluid paths 116a and 118a are separated by partitions 119 and 121, respectively. The first and second portions of the inlet and outlet fluid paths 116a and 118a may have chamfered transitions at the turns where the first and second portions switch directions. Fresh media enters the cavity 113 through the inlet fluid path 116a thereby filling the cavity 113. Conditioned media is drawn out of the cavity 113 through the outlet fluid path 118a. The fluid flow from the inlet and outlet fluid paths 116a and 118a is directed upwards into the cavity 113, thus, forming high fluid ports 184a and 186a, respectively, inside the cavity 113.

[0069] With reference to FIG. 12, the inlet 102b and the outlet 104b are fluidly coupled to a low inlet fluid path 116b and a low outlet fluid path 118b, respectively, which include a first portion that extends downward from the inlet 102b and the outlet 104b towards the bottom surface 114of the fluidic module 110. The inlet and outlet fluid paths 116b and 1 18b also include a second portion extending laterally from the first portions thereof (e.g., perpendicularly relative to the first portions) in an opposing direction and are fluidly coupled to the cavity 113. The second portions of the inlet and outlet fluid paths 116b and 118b terminate in openings defined in the bottom surface 114.

[0070] The first and second portions of the inlet and outlet fluid paths 116b and 118b may have chamfered transitions at the turns where the first and second portions switch directions. Fresh media enters the cavity 113 through the inlet fluid path 116b thereby filling the cavity 113. Conditioned media is drawn out of the cavity 113 through the outlet fluid path 118b. The fluid flow from the inlet and outlet fluid paths 116b and 118b is directed through the bottom surface 114, thus, forming low fluid ports 184b and 186b, respectively, through the bottom surface 114.

[0071] The views shown in FIGS. 11 and 12 are rotated 90° relative to each other and show phantom views of the fluid paths, namely, FIG. 11 shows the phantom view of the low inlet and outlet fluid paths 116b and 118b and FIG. 12 shows the phantom view of the high inlet and outlet fluid paths 116a and 118a.

[0072] In embodiments, the inlets 102a, 102b and outlets 104a, 104b may have either low or high fluid ports in any suitable combination, e.g., both inlets are coupled to low fluid ports and outlets are coupled to high fluid ports, or vice versa, where the outlets are coupled to the low fluid ports and the inlets are coupled to high fluid ports, or all of inlets and outlets are coupled to the same type of fluid port, or where one of the inlets or outlets is coupled to one type of the fluid port (e.g., low) where the remaining inlets or outlets are coupled to another type of the fluid port (e.g., high).

[0073] Alternate embodiments may be devised without departing from the spirit or the scope of the present technology. Additionally, well-known elements of embodiments of the systems, apparatuses, and methods have not been described in detail or have been omitted so as not to obscure the relevant details of the systems, apparatuses, and methods.

[0074] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The terms “comprises,” “comprising,” or any other variation thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by “comprises ... a” does not, without more constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element. The terms “including” and / or “having,” as used herein, are defined as comprising (i.e., open language). The terms “a” or “an”, as used herein, are defined as one or more than one. The term “plurality,” as used herein, is defined as two or more than two. The term “another,” as used herein, is defined as at least a second or more. The description may use the terms “embodiment” or “embodiments,” which may each refer to one or more of the same or different embodiments.

[0075] When the terms “coupled” and “connected,” along with their derivatives, are used, these terms are not intended as synonyms for each other. For example, “connected” may be used to indicate that two or more elements are in direct physical or electrical contact with each other. “Coupled” may mean that two or more elements are in direct physical or electrical contact (e.g., directly coupled) or that two or more elements are not in direct contact with each other but yet still cooperate or interact with each other (e.g., indirectly coupled).

[0076] For the purposes of the description, a phrase in the form “A / B” or in the form “A and / or B” or in the form “at least one of A and B” means (A), (B), or (A and B), where A and B are variables indicating a particular object or attribute. When used, this phrase is intended to and is hereby defined as a choice of A or B or both A and B, which is similar to the phrase “and / or”. Where more than two variables are present in such a phrase, this phrase is hereby defined as including only one of the variables, any one of the variables, any combination of any of the variables, and all of the variables, for example, a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C).

[0077] Relational terms such as first and second, top and bottom, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. The description may use perspective-based descriptions such as up / down, back / front, top / bottom, and proximal / distal. Such descriptions are merely used to facilitate the discussion and are not intended to restrict the application of disclosed embodiments. Various operations may be described as multiple discrete operations in turn, in a manner that may be helpful in understanding embodiments; however, the order of description should not be construed to imply that these operations are order dependent.

[0078] As used herein, the term “about” or “approximately” applies to all numeric values, whether or not explicitly indicated. These terms generally refer to a range of numbers that one of skill in the art would consider equivalent to the recited values (i.e., having the same function or result). In many instances these terms may include numbers that are rounded to the nearest significant figure. As used herein, the terms “substantial” and “substantially” means, when comparing various parts to one another, that the parts being compared are equal to or are so closeenough in dimension that one skill in the art would consider the same. Substantial and substantially, as used herein, are not limited to a single dimension and specifically include a range of values for those parts being compared. The range of values, both above and below (e.g., or greater / lesser or larger / smaller), includes a variance that one skilled in the art would know to be a reasonable tolerance for the parts mentioned.

[0079] Various embodiments of the systems, apparatuses, and methods have been described, and in many of the different embodiments many features are similar. To avoid redundancy, repetitive description of these similar features may not be made in some circumstances. It shall be understood, however, that description of a first-appearing feature applies to the later described similar feature and each respective description, therefore, is to be incorporated therein without such repetition.

[0080] From the foregoing, it will be appreciated that specific embodiments of the disclosure have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the disclosure. Accordingly, the disclosure is not limited except as by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A cell culture device comprising: a fluidic culture module including: a cavity configured to house a biological sample; and an inlet and an outlet for supplying a cell culture media into the fluidic culture module; and a lid for sealing the fluidic culture module, the lid including: an access port defined through the lid; a top viewing window placed through the access port and configured for imaging of the biological sample; and an air duct defined in the lid and in fluid communication with the cavity to allow gas exchange between the cavity and an outside atmosphere.

2. The cell culture device according to claim 1, wherein the fluidic culture module further includes an inlet fluid path fluidly coupled to the inlet and an outlet fluid path fluidly coupled to the outlet and wherein the inlet and outlet fluid paths terminate in the cavity.

3. The cell culture device according to claim 2, wherein each of the inlet fluid path and the outlet fluid path have a sinuous shape.

4. The cell culture device according to claim 3, wherein the fluidic culture module further includes an inner well and the inlet and outlet fluid paths terminate above the inner well to direct flow of the media above the biological sample.

5. The cell culture device according to claim 4, wherein the inner well has a beveled or chamfered upper surface to provide for circulation of the media around the biological sample.

6. The cell culture device according to claim 5, wherein the inlet fluid path, the outlet fluid path, and the inner well are configured to minimize disturbance of the biological sample due to the circulation of the media.

7. The cell culture device according to claim 1, wherein the fluidic culture module is configured to be inserted into a well of a substrate including a bottom viewing window for imaging of the biological sample.

8. The cell culture device according to claim 1, wherein the fluidic culture module is configured to be inserted into a well of an electrophysiological device including a sensor.

9. The cell culture device according to claim 1, wherein a bottom portion of the viewing window is submerged in the cell culture media.

10. The cell culture device according to claim 1, further comprising a tray surrounding the fluidic culture module and configured to receive overflow of the cell culture media through the air duct.

11. A cell culture system comprising: an electrophysiological device including a sensor and a well surrounding the sensor; and a cell culture device including: a fluidic culture module inserted into the well, the fluidic culture module having: a cavity configured to house a biological sample; and an inlet and an outlet for supplying a cell culture media into the fluidic culture module; and a lid for sealing the fluidic culture module, the lid having: a access port defined through the lid; a viewing window disposed through the access port and configured for imaging of the biological sample; and an air duct defined in the lid and in fluid communication with the cavity to allow gas exchange between the cavity and an outside atmosphere.

12. The system according to claim 11, wherein the fluidic culture module further includes an inlet fluid path fluidly coupled to the inlet and an outlet fluid path fluidly coupled to the outlet and wherein the inlet and outlet fluid paths terminate in the cavity.

13. The system according to claim 12, wherein each of the inlet fluid path and the outlet fluid path have a sinuous shape.

14. The system according to claim 13, wherein the fluidic culture module further includes an inner well rising above the biological sample and the inlet and outlet fluid paths terminate above the inner well to direct flow of the media above the biological sample.

15. The system according to claim 14, wherein the inner well has a beveled or chamfered upper surface to provide for circulation of the media around the biological sample.

16. The system according to claim 15, wherein the inlet fluid path, the outlet fluid path, and the inner well are configured to minimize disturbance of the biological sample due to the circulation of the media.

17. The system according to claim 11, wherein a bottom portion of the viewing window is submerged in the cell culture media.

18. The system according to claim 11, further comprising a tray surrounding the well of the electrophysiological device and configured to receive overflow of the cell culture media through the air duct.

19. The system according to claim 11, further comprising: a programmable fluidic pump connected to the inlet and the outlet for automated media exchange.

20. The system according to claim 19, further comprising: a collection reservoir configured to receive media removed from the cell culture device; and a camera module configured to measure an amount of media inside the collection reservoir.

21. The system according to claim 20, further comprising: a computing device configured to receive measurement data from the camera module and to control the fluidic pump to adjust flow rate of the media based on the measurement data.

22. The system according to claim 11, further comprising: an imaging device positioned over viewing window, the imaging device configured to image the biological sample through the viewing window.

Images