Cell culture insert with porous substrate and sensors

Abstract

A cell culture insert comprising a body sized for insertion into a reservoir of a cell culture device, the body having a first open end, a second end, and one or more sidewalls extending from the first open end to the second end; a substrate at the second end, the substrate comprising a plurality of separated through-pores and a plurality of sensors; and electrical connections for individually reading from each sensor.

Description

[0001] CELL CULTURE INSERT WITH POROUS SUBSTRATE AND SENSORS

[0002] Field of the Invention

[0003] The present invention relates to the field of cell culture devices, and more specifically to cell culture inserts and assemblies comprising a substrate with through-pores and a plurality of sensors.

[0004] Background of the Invention

[0005] Cell culture devices have been widely used in biological research and pharmaceutical development for studying cellular behavior, drug interactions, and tissue engineering. These devices typically consist of a reservoir or well plate in which cells can be grown and observed. Transwells, a specific type of cell culture device, have gained popularity due to their ability to separate cell cultures into apical and basolateral compartments, mimicking physiological barriers found in the body.

[0006] Traditional transwells utilize porous membranes to support cell growth and allow for the exchange of molecules between the two compartments. These membranes are typically made of materials such as polycarbonate or polyester and feature randomly distributed pores. While useful for many applications, these conventional transwells have limitations in terms of their measurement capabilities and the level of detail they can provide about cellular behavior.

[0007] There is, therefore, a need in the art for cell culture inserts overcoming at least partially the above limitations.

[0008] Summary of the Invention

[0009] It is an object of embodiments of the present invention to provide a cell culture insert with integrated sensors for measuring properties of cells, tissues or organoids cultured therein. This objective is accomplished by a cell culture insert comprising a body sized for insertion into a reservoir of a cell culture device, the body having a first open end and a second end, and one or more sidewalls extending from the first open end to the second end, a substrate at the second end, the substrate comprising a plurality of separated through-pores and a plurality of sensors, and electrical connections for individually reading from each sensor according to the invention.

[0010] In the first aspect, the present invention relates to a cell culture insert comprising: a. A body sized for insertion into a reservoir of a cell culture device, said body having a first open end and a second end, and one or more sidewalls extending from the first open end to the second end; b. A substrate at the second end, the substrate comprising: i. a plurality of separated through-pores; and ii. a plurality of sensors; and c. Electrical connections for individually reading from each sensor.

[0011] In embodiments, the second end may be defined by the entirety of the top surface of the bottom of the insert. This is typically the surface comprising the top surface of the substrate.

[0012] In embodiments, the first open end may have a larger length (e.g., inner diameter in the case of circular first and second ends) than the second end and the one or more sidewalls may be sloped. This facilitates insertion into cell culture reservoirs.

[0013] In alternative embodiments, the first open end and the second end may have substantially equal diameters, with the one or more sidewalls being substantially parallel.

[0014] The slope of the sidewalls is preferably linear and continuous, but it can also be variable, curved, stepped, textured, or a combination thereof.

[0015] In embodiments, said second end may have a length in a range from 100 pm to 1000 pm, preferably from 200 pm to 750 pm. This provides an optimal size for cell culture applications.

[0016] In embodiments, the first open end and the second end may be circular. This matches standard cell culture reservoir geometries.

[0017] In embodiments, the first open end may have a length which is from 5 to 50% larger than the second end.

[0018] In embodiments, the first open end may be surrounded by a flange for resting on the edges of the reservoir. This enables secure positioning of the insert.

[0019] In embodiments, the substrate may comprise silicon. This leverages well-established microfabrication techniques.

[0020] In embodiments, the substrate may be fabricated using standard lithography and etching techniques.

[0021] In embodiments, the thickness of the substrate may range from 1 pm to 100 pm, preferably from 5 to 50 pm, more preferably from 10 pm to 50 pm. This thickness range is specific to silicon substrates and cannot easily be achieved with polymer-based materials while keeping similar mechanical properties.

[0022] In embodiments, the insert may be embedded into a transwell device using liquid crystal polymer technology, gluing, or clamping methods.

[0023] In embodiments, the sensors may be facing towards or away from the first open end. This provides flexibility in sensor orientation.

[0024] In embodiments, the sensors may have an electrical sensing modality enabling one or more of impedance measurements, voltage measurements, current measurements, and capacitance measurements. This allows for comprehensive electrical characterization. In embodiments, the sensors may comprise electrodes, e.g., electrodes of a multielectrode array. In embodiments, the sensors may comprise a plurality of electrodes positioned adjacent to each through-hole for measuring electrical signal changes, such as impedance changes, associated with cell migration at that through-hole. In other words, the sensors may comprise a plurality of electrodes for measuring electrical signal changes associated with cell migration, said electrodes being positioned so that each through-hole has at least one electrode adjacent thereto. This embodiment enables the local, non-invasive, and real-time detection of cell migration events. It is an advantage of embodiments of the present invention that direct read-out of migration events is possible without the need for imaging-based quantifications, which can be labor-intensive and time-consuming.

[0025] In embodiments, the sensors may include impedance electrodes arranged in a planar configuration across the substrate.

[0026] In embodiments, the cell culture insert may comprise a plurality of substrates as defined herein arranged on top of one another. For instance, the insert may comprise multiple porous substrates stacked vertically, each containing an array of sensors.

[0027] In embodiments, each layer of the insert may contain its own set of electrodes for independent monitoring of cell layer formation and integrity on each level.

[0028] In embodiments, the sensors may be configured to perform cross-layer electrical measurements between electrodes on different layers of a multi-layer system.

[0029] In embodiments, the sensors may be configured to perform Electrical Impedance Spectroscopy (EIS) measurements at multiple frequencies ranging from 10 Hz to 100 kHz. For instance, this can occur between sensors present on different substrate of a plurality of substrates as defined herein arranged on top of one another.

[0030] In embodiments, the sensors may be configured to perform spatially resolved impedance measurements for mapping local variations in electrical properties across the cell culture area.

[0031] In embodiments, the electrodes may have a width of from 1 to 50 pm. This embodiment ensures that the electrodes are appropriately sized for detecting the electrical signals associated with the cells of interest.

[0032] In embodiments, the electrodes may be made of a biocompatible conductive material selected from the group consisting of gold, platinum, titanium nitride, and indium tin oxide. This embodiment ensures biocompatibility and reliable electrical signal detection.

[0033] In embodiments, the sensors may be further configured to apply a voltage and / or current. This allows for active stimulation of the cells.

[0034] In embodiments, the sensors may be arranged in a two-dimensional array. This enables high- resolution spatial mapping. In embodiments, the electrical connections may be configured to provide bus communication for receiving a plurality of electrical readings from the sensors. This allows for efficient data collection from multiple sensors.

[0035] In embodiments, the sensors may be connected to integrated circuits embedded in the substrate. This enables on-chip signal processing and data reduction.

[0036] In embodiments, the integrated circuits may comprise one or more elements selected from the list consisting of multiplexers, amplifiers, electrical signal stimulation circuitry, electrical signal recording circuitry, ADCs, and DACs. This allows for sophisticated on-chip functionality.

[0037] In embodiments, the electrical connections may include magnetic connectors. This enables easy and secure electrical interfacing.

[0038] In embodiments, the electrical connections may include a water-resistant flexible cable. This allows for reliable connections in liquid environments.

[0039] In embodiments, the electrical connections can be wireless, wired, or a combination thereof.

[0040] In the second aspect, the present invention relates to a cell culture assembly, comprising: a. A reservoir defining an interior and having an inner bottom; and b. A first cell culture insert as defined in any embodiments of the first aspect, configured to be positioned in the interior of the reservoir to retain the second end of the body above the bottom of the reservoir.

[0041] In embodiments, the reservoir may comprise a plurality of wells, each well defining an interior and having a bottom, and wherein the assembly comprises a plurality of cell culture inserts, each insert being configured to be positioned in the interior of a well to retain the second end of the body above the bottom of the well. This allows for high-throughput parallel experiments.

[0042] In embodiments, the assembly may further comprise a lid and an electrode attached to the lid. This enables additional measurement capabilities from above the cell culture.

[0043] In embodiments, the interior of the reservoir may comprise an electrode on its inner bottom and the electrode attached to the lid and the electrode on the bottom of the reservoir may be configured to perform together one or more of the following: voltage application, current application, voltage recording, and current recording. This allows for measurements across the full thickness of the cell culture.

[0044] In embodiments, the electrode attached to the lid and the electrode on the inner bottom of the reservoir may be configured to perform Electrical Impedance Spectroscopy (EIS) measurements at multiple frequencies ranging from 10 Hz to 100 kHz.

[0045] In the third aspect, the present invention relates to a measurement system for cell culture, comprising: a. A cell culture assembly as claimed in any embodiments of the second aspect; and b. An external acquisition device configured to receive data from the plurality of sensors in the cell culture insert.

[0046] In the fourth aspect, the present invention relates to a kit of parts for cell culture, comprising: a. A reservoir defining an interior and having a bottom; and b. A first cell culture insert as defined in any embodiments of the first aspect, configured to be positioned in the interior of the reservoir to retain the second end of the body above the bottom of the reservoir.

[0047] It is an advantage of embodiments of the present invention that a cell culture insert with a substrate having both through-pores and multiple integrated sensors can be achieved. It is a further advantage of embodiments of the present invention that spatial information about cell cultures can be obtained at the pore level, i.e., at proximity of the pores. It is an advantage of embodiments of the present invention that complex multi-cellular barrier structures can be studied by combining measurements between one or more top on the lid and / or bottom electrodes in the reservoir with on-chip measurements in the cell culture insert. It is a further advantage of embodiments of the present invention that defects or heterogeneities in cell layers can be precisely localized. It is an advantage of embodiments of the present invention that high-throughput measurements can be performed on cells, tissues, or organoids. It is an advantage of embodiments of the present invention that the cell culture insert can be compatible with standard multi-well plates for ease of use. It is a further advantage of embodiments of the present invention that on-chip signal processing and digitization can be achieved. It is an advantage of embodiments of the present invention that flexible and liquid-resistant electrical connections can be incorporated. It is a further advantage of embodiments of the present invention that multiple cell culture inserts can be combined in a single assembly for parallel experiments. It is a further advantage of embodiments of the present invention that the cell culture insert can be effectively used for static barrier applications, such as the bloodbrain barrier (BBB), to study the integrity and characteristics of barrier cell layer(s).

[0048] Moreover, in embodiments, the system may provide comprehensive, real-time data about cellular functions and interactions, overcoming the limitations of end-point measurements or separate analytical techniques prevalent in prior art. This capability allows for the capture of dynamic cellular processes in real-time, providing a more accurate representation of cellular behavior over time.

[0049] The high spatial resolution in measurements achieved in embodiments of the present invention enables the detection of localized changes or heterogeneity within the cell culture, a feature that was lacking in traditional systems. This advancement, coupled with the ability to obtain detailed data from each culture while maintaining high-throughput capabilities, represents a significant leap forward in cell culture technology.

[0050] Another advantage of embodiments of the present invention is its capacity to better mimic the complexity of in vivo environments, including the ability to create and monitor multi-layered cell cultures or organoids. The sophisticated measurement capabilities enable the monitoring of complex biological phenomena in advanced cell culture models, opening new avenues for research and discovery. Furthermore, the ability to study static barrier applications, particularly the blood-brain barrier, provides valuable insights into the integrity and characteristics of these critical biological interfaces, enhancing our understanding of drug penetration, cellular transport, and barrier function in vitro.

[0051] Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and with features of other dependent claims as appropriate and not merely as explicitly set out in the claims.

[0052] The above and other characteristics, features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention. This description is given for the sake of example only, without limiting the scope of the invention. The reference figures quoted below refer to the attached drawings.

[0053] Brief description of the drawings

[0054] Fig.l is a close-up view of the substrate used in the insert according to embodiments of the present invention.

[0055] Fig.2 is another close-up view of the substrate according to embodiments of the present invention.

[0056] Fig. 3 is a cross-sectional view of the cell culture insert according to embodiments of the present invention.

[0057] Fig. 4 is a cross-sectional view of the cell culture assembly according to embodiments of the present invention.

[0058] Fig. 5 is a cross-sectional view of the cell culture assembly with a lid according to embodiments of the present invention.

[0059] Fig. 6 is a schematic view of the measurement system according to embodiments of the present invention.

[0060] In the different figures, the same reference signs refer to the same or analogous elements. Detailed description of Illustrative Embodiments

[0061] The present invention will be described with respect to particular embodiments and with reference to certain drawings, but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. The dimensions and the relative dimensions do not correspond to actual reductions to practice of the invention.

[0062] The following terms are provided solely to aid in the understanding of the invention.

[0063] As used herein, and unless otherwise specified, the term "cell culture insert" refers to a device or apparatus that can be inserted into a cell culture reservoir or well to provide a surface or substrate for culturing cells. The cell culture insert typically includes a body with a first open end, a second end, and one or more sidewalls extending between the ends. Examples of cell culture inserts include Transwell inserts, Millicell inserts, and Falcon cell culture inserts.

[0064] As used herein, and unless otherwise specified, the term "substrate" refers to a material or surface on which cells can be cultured. The substrate is typically located at the second end of the cell culture insert and may include features such as through-pores and sensors. Examples of substrates include membranes, filters, and microelectrode arrays.

[0065] As used herein, and unless otherwise specified, the term "through-pores" refers to openings or holes that extend completely through the substrate, allowing for communication between the two sides of the substrate. The through-pores are typically separated from each other and may have various sizes and shapes. Typically, the through-pore are not interconnected. Examples of through- pores include circular pores having a circular cross-section, an oval cross-section, or a rectangular cross-section. The cross-section of the through-pore is preferably constant through the thickness of the substrate. In embodiments, each through-pore has a cross-section not varying by more than 10%, preferably not varying by more than 5%, around an average cross-section across the thickness of the substrate. In embodiments, the set of through-pores may be organized so that the through-pores are aligned in parallel rows and lines. In embodiments, any through-pore may be misaligned with respect to its neighboring through-pores in a line or a column by no more than 500 nm. This level of precision is achievable through lithographic manufacturing techniques. In embodiments, the width or hydraulic diameter of the through-pores may be from 100 nm to 500 pm, preferably from 200 nm to 200 pm, more preferably from 300 nm to 100 pm, and most preferably from 1 pm to 30 pm. This embodiment allows for the study of cell migration through a wide range of through-pore width, relevant to various biological contexts. Cells should typically not be able to migrate through a through-pore of 100 nm. The smallest bacteria have a diameter of 200 nm, and eukaryotic cells are at least 800 nm large. Bacteria have limited ability to squeeze through pores smaller than themselves. Having pores as small as 100 nm has the advantage to test the translocation of the smallest bacteria when they are very attracted toward the other side of the substrate. In most case, however, a lower limit at 100 nm will not be useful and a lower limit at 200 nm, or even at 300 nm will be sufficient to study the translocation of bacteria. To study the transmigration of cells of the human or animal body, a lower limit at 1 pm is advantageous to test the transmigration of the smallest body cells when they are very attracted toward the other side of the substrate. Since the smallest body cells are about 6 pm in diameter, a through-pore having a width or hydraulic diameter of 1 pm would require the body cell to squeeze through the pore. The largest through-pore do not need to be larger than 500 pm since most cells of interest are not as large. Amongst cells of interest, some cancer cells can reach 500 pm.

[0066] As used herein, and unless otherwise specified, the term "sensors" refers to devices or elements that can detect or measure properties. The sensors are typically located on or integrated into the substrate of the cell culture insert. Examples of sensors include electrodes.

[0067] As used herein, and unless otherwise specified, the term "electrical connections" refers to conductive elements or pathways that allow for the transmission of electrical signals to and from the sensors. The electrical connections may include wires, cables, conductive traces, or other conductive structures. Examples of electrical connections include flexible printed circuit cables, wire bonding, and conductive adhesives.

[0068] As used herein, and unless otherwise specified, the term "reservoir" refers to a container or vessel that can hold a volume of cell culture medium and accommodate one or more cell culture inserts. The reservoir typically has an interior space and a bottom surface. Examples of reservoirs include cell culture plates, Petri dishes, well plates, and microfluidic devices.

[0069] As used herein, and unless otherwise specified, the term "lid" refers to a cover or top portion of the cell culture assembly that can be placed over the reservoir to enclose the interior space. The lid may include additional features such as electrodes or ports for introducing or removing fluids. Examples of lids include plastic covers, glass covers, and microfluidic device tops.

[0070] As used herein, and unless otherwise specified, the term "external acquisition device" refers to a device or system that is separate from the cell culture assembly and is configured to receive and process data from the sensors in the cell culture insert. The external acquisition device may include hardware and software components for data acquisition, analysis, and storage. Examples of external acquisition devices include computers, microcontrollers, and specialized data acquisition systems.

[0071] The invention will now be described by a detailed description of several embodiments of the invention. It is clear that other embodiments of the invention can be configured according to the knowledge of persons skilled in the art without departing from the technical teaching of the invention, the invention being limited only by the terms of the appended claims. We now refer to Fig. 1, which shows a vertical cross-section through a substrate (30) used in a cell culture insert (1) according to embodiments of the present invention. The substrate (30) comprises a plurality of separated through-pores (31) and a plurality of sensors (32), e.g., arranged in a two-dimensional array. The sensors (32) in this embodiment are facing towards the first open end (11) of the body (10) of the cell culture insert (1). The sensors (32) can, for instance, be electrodes. In embodiments, the sensors (32) may have an electrical sensing modality and may enable impedance measurements, voltage measurements, current measurements, and capacitance measurements. In embodiments, the sensors (32) may also be configured to apply a voltage and / or current.

[0072] We now refer to Fig. 2, which shows a vertical cross-section through a substrate (30) used in a cell culture insert (1) according to embodiments of the present invention. The sensors (32) in this embodiment are facing away from the first open end (11) of the body (10) of the cell culture insert (1).

[0073] The electrical connections (40) for individually reading from each sensor (32) can be seen on the top side of the substrate (30) in both figures. In embodiments, these electrical connections (40) may be configured to provide bus communication for receiving a plurality of electrical readings from the sensors (32). In embodiments, the electrical connections (40) may include magnetic connectors (41) and / or a water-resistant flexible cable (42, see Fig. 3) for connecting the sensors (32) to an external acquisition device (80).

[0074] We now refer to Fig. 3, which shows a cross-sectional view of the cell culture insert (1) according to embodiments of the present invention. The cell culture insert (1) comprises a body (10) sized for insertion into a reservoir (20) of a cell culture device. The body (10) has a first open end (11), a second end (12), and one or more sidewalls (13) extending from the first open end (11) to the second end (12). The substrate (30) is located at the second end (12) of the body (10). The first open end (11) may have a larger length than the second end (12), and the one or more sidewalls (13) may be sloped. In embodiments, the second end (12) may have a length in a range from 100 pm to 1000 pm, preferably from 200 pm to 750 pm. In embodiments, the first open end (11) and the second end (12) may be circular in shape. In embodiments, the first open end (11) may also be surrounded by a flange for resting on the edges of the reservoir (20).

[0075] We now refer to Fig. 4, which shows a cross-sectional view of the cell culture assembly (70) according to embodiments of the present invention. The cell culture assembly (70) comprises a reservoir (20) defining an interior (21) and having an inner bottom (22). The first cell culture insert (1) is configured to be positioned in the interior (21) of the reservoir (20) to retain the second end (12) of the body (10) above the bottom (22) of the reservoir (20). In embodiments, the reservoir (20) may comprise a plurality of wells (23), each well (23) defining an interior (21) and having a bottom (22). In this case, the assembly (70) would comprise a plurality of cell culture inserts (1), each insert (1) being configured to be positioned in the interior (21) of a well (23) to retain the second end (12) of the body (10) above the bottom (22) of the well (23).

[0076] We now refer to Fig. 5, which shows a cross-sectional view of the cell culture assembly (70) with a lid (60) according to embodiments of the present invention. The cell culture assembly (70) further comprises a lid (60) and an electrode (61) attached to the lid (60). The interior (21) of the reservoir (20) may also comprise an electrode (62) on its inner bottom (22). In embodiments, the electrode (61) attached to the lid (60) and the electrode (62) on the bottom (22) of the reservoir (20) may be configured to perform together one or more of the following: voltage application, current application, voltage recording, and current recording.

[0077] We now refer to Fig. 6, which shows a schematic view of the measurement system (3) according to embodiments of the present invention. The measurement system (3) for cell culture comprises a cell culture assembly (70) as described in the previous figures and an external acquisition device (80) configured to receive data from the plurality of sensors (32) in the cell culture insert (1). In embodiments, the external acquisition device (80) may be connected to the electrical connections (40) of the cell culture insert (1) for individually reading from each sensor (32). In embodiments, the substrate can comprise of a set of sensors (32) connected directly to an external acquisition device / potentiostat / impedance analyser (80), or can have integrated circuits such as amplifiers, stimulation circuitry, ADCs and DACs embedded to perform measurements on the substrate and have only digital data being transferred out. In embodiments, the substrate embedding can be done by liquid crystal polymer technology, gluing or clamping so the substrate is part of the transwell-cup which can be placed into a standard well plate. In embodiments, the cell culture insert (1) may comprise a plurality of substrates (30) as defined herein arranged on top of one another.

[0078] Example 1: Fabrication and Testing of a Silicon Chip Embedded Transwell for Cell Measurements

[0079] A silicon chip is fabricated and embedded into a transwell device to enable high-throughput measurements on cells, tissues, and organoids. The chip is designed with a substrate pierced with through-pores and contains a two-dimensional array of sensors, including voltage sensors and impedance sensors.

[0080] The fabrication process involves the following steps. First, a silicon substrate is patterned with a multitude of through-pores using standard lithography and etching techniques. The pore sizes are varied to accommodate different biological applications. Next, an array of sensors is integrated onto the surface of the porous silicon substrate. These sensors include voltage sensors and impedance sensors. The sensors are arranged in a two-dimensional grid to enable spatial mapping of cellular activity and electrical properties.

[0081] To embed the silicon chip into the transwell device, liquid crystal polymer technology is employed. The chip is placed into a mold, and liquid crystal polymer is injected and cured around it, forming a seamless integration. Alternatively, the chip is also successfully embedded using gluing and clamping methods. Electrical connections are established from the sensors on the chip to an external data acquisition system. This is achieved through the integration of amplifiers, stimulation circuitry, analog-to-digital converters (ADCs), and digital-to-analog converters (DACs) directly on the chip. The on-chip electronics enable local signal processing and digitization, reducing the number of connections required to interface with external instrumentation.

[0082] The performance of the silicon chip embedded transwell is evaluated in a series of cell culture experiments. Human epithelial cells are seeded onto the porous membrane of the transwell and cultured for 7 days to form a confluent monolayer. The transepithelial electrical resistance (TEER) is measured using the integrated voltage sensors, providing a quantitative assessment of the barrier function.

[0083] The silicon chip embedded transwell developed in this study offers several advantages over existing solutions. The integration of a multitude of sensors on a porous silicon substrate enables comprehensive monitoring of cellular behavior and electrical properties with high spatial resolution. The ability to perform measurements from both the apical and basolateral sides of the transwell membrane provides a more complete understanding of cellular processes. Furthermore, the high- throughput format allows for efficient screening of multiple conditions and cell types, accelerating research in fields such as drug discovery and toxicology.

[0084] Example 2: Transwell with Integrated Electrical Sensors for Real-Time Monitoring of Cell Layer Formation and Barrier Function

[0085] A silicon chip embedded transwell is fabricated with an array of electrical sensors integrated onto the porous substrate. The electrical sensors include impedance electrodes arranged in a planar configuration across the substrate. The transwell is used to culture human epithelial cells, and the electrical sensors are employed to monitor cell layer formation and barrier function in real-time.

[0086] The impedance electrodes are used to perform Electrical Impedance Spectroscopy (EIS) measurements. As cells adhere and proliferate on the substrate, they act as insulators, increasing the impedance between the electrodes. This allows for real-time, label-free monitoring of cell coverage and confluency. The EIS measurements are taken at multiple frequencies, typically ranging from 10 Hz to 100 kHz, providing information about both the resistance and capacitance of the cell layer. Changes in the low-frequency impedance are primarily related to the formation of tight junctions between cells, indicating the development of a barrier function. High-frequency measurements are more sensitive to changes in cell membrane capacitance, which can indicate changes in cell morphology or coverage.

[0087] The transwell also incorporates electrodes on both sides of the membrane (one at the bottom of the well and one in the lid), allowing for measurements of Trans-Epithelial Electrical Resistance (TEER). TEER measurements provide quantitative information about the integrity and permeability of the cell monolayer.

[0088] The data from the electrical sensors is collected and processed using on-chip electronics and transmitted wirelessly to an external data acquisition system. This enables continuous, non-invasive monitoring of cell layer formation and barrier function without the need for manual sampling.

[0089] This system allows researchers to track the kinetics of cell layer formation, assess the quality of the epithelial barrier, and monitor its response to various stimuli or compounds in real-time. It offers valuable insights into the collective behavior of the cell population and the functional properties of the epithelial layer.

[0090] This approach is particularly useful for high-throughput screening applications in drug discovery, toxicology studies, and for modeling epithelial and endothelial barriers in vitro.

[0091] Example 3: Multi-Layered Transwell with Integrated Electrical Sensors for Studying Cell-Cell Interactions

[0092] A silicon chip embedded transwell is fabricated with multiple porous substrates stacked vertically, each containing an array of electrical sensors. The sensors include pairs of electrodes for measuring Trans-Epithelial / Endothelial Electrical Resistance (TEER) and for performing Electrical Impedance Spectroscopy (EIS). The multi-layered transwell is used to co-culture different cell types in a physiologically relevant arrangement.

[0093] For example, endothelial cells are seeded on the top layer to mimic the blood vessel lining, while astrocytes and neurons are cultured on the lower layers to represent the brain parenchyma, creating an in vitro model of the blood-brain barrier (BBB). Each layer of the transwell contains its own set of electrodes, allowing for independent monitoring of cell layer formation and integrity on each level.

[0094] The integrated electrical sensors are used to monitor the interactions between the different cell types:

[0095] TEER measurements: Electrodes on either side of each cell layer allow for continuous monitoring of barrier integrity. Changes in TEER can indicate alterations in tight junction formation or disruption, providing insights into the barrier function of the endothelial layer and how it's influenced by the presence of astrocytes and neurons.

[0096] EIS measurements: By applying alternating current at various frequencies, EIS provides information about cell coverage, adhesion, and morphology. This can be used to monitor the formation and maintenance of each cell layer independently.

[0097] Spatially resolved impedance measurements: The array of electrodes on each layer allows for mapping of local variations in electrical properties across the cell culture area. This can reveal heterogeneities in cell distribution or barrier function.

[0098] Cross-layer electrical measurements: By applying electrical signals between electrodes on different layers, it's possible to detect changes in the overall electrical properties of the multi-layer system, which may reflect complex interactions between the different cell types.

[0099] The ability to perform these electrical measurements in real-time and in a spatially-resolved manner provides new insights into the complex interplay between different cell types in a tissue-like environment. For instance, researchers can observe how the presence of astrocytes affects the development and maintenance of the endothelial barrier, or how neurons respond to changes in the endothelial layer.

[0100] This system allows for non-invasive, label-free, and continuous monitoring of cellular interactions in a physiologically relevant 3D structure. While it doesn't directly measure specific molecular factors, the electrical measurements can serve as sensitive indicators of cellular responses and tissue-level changes, making this platform valuable for studying complex biological barriers and for applications in drug screening and disease modeling.

[0101] It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for devices according to the present invention, various changes or modifications in form and detail may be made without departing from the scope of this invention. For example, any formulas given above are merely representative of procedures that may be used. Functionality may be added or deleted from the block diagrams and operations may be interchanged among functional blocks. Steps may be added or deleted to methods described within the scope of the present invention.

Claims

Claims1. A cell culture insert (1) comprising: a. A body (10) sized for insertion into a reservoir (20) of a cell culture device, said body (10) having a first open end (11) and a second end (12), and one or more sidewalls (13) extending from the first open end (11) to the second end (12); b. A substrate (30) at the second end (12), the substrate (30) comprising: i. a plurality of separated through-pores (31); and ii. a plurality of sensors (32); and c. Electrical connections (40) for individually reading from each sensor (32).

2. The cell culture insert (1) according to any one of the preceding claims, wherein the sensors (32) is for electrical sensing.

3. The cell culture insert (1) according to claim 1 or claim 2, wherein the sensors (32) are further configured to apply a voltage and / or are further configured to apply a current.

4. The cell culture insert (1) according to any one of claims 1 to 3, wherein the electrical connections (40) are configured to provide bus communication for receiving a plurality of electrical readings from the sensors (32).

5. The cell culture insert (1) according to any one of claims 1 to 4, comprising a plurality of substrates (30) as defined in claim 1 arranged on top of one another.

6. The cell culture insert (1) according to any one of claims 1 to 5, wherein the sensors (32) are connected to integrated circuits embedded in the substrate (30).

7. The cell culture insert (1) according to any one of claims 1 to 6, wherein the electrical connections (40) include a water-resistant flexible cable (42).

8. The cell culture insert (1) according to any one of claims 1 to 7, wherein the substrate (30) comprises silicon.

9. The cell culture insert (1) according to any one of claims 1 to 8, wherein the sensors (32) are arranged in a two-dimensional array.

10. A cell culture assembly (70), comprising: a. A reservoir (20) defining an interior (21) and having an inner bottom (22); and b. A first cell culture insert (1) as defined in any one of claims 1 to 9, configured to be positioned in the interior (21) of the reservoir (20) to retain the second end (12) of the body (10) above the bottom (22) of the reservoir (20).

11. The cell culture assembly (70) according to claim 10, wherein the reservoir (20) comprises a plurality of wells (23), each well (23) defining an interior (21) and having a bottom (22), and wherein the assembly comprises a plurality of cell culture inserts (1), each insert (1) being configured to be positioned in the interior (21) of a well (23) to retain the second end (12) of the body (10) above the bottom (22) of the well (23).

12. The cell culture assembly (70) according to claim 10 or claim 11, further comprising a lid (60) and an electrode (61) attached to the lid (60).

13. The cell culture assembly (70) according to claim 12, wherein the interior (21) of the reservoir (20) comprises an electrode (62) on its inner bottom (22) and wherein the electrode (61) attached to the lid (60) and the electrode (62) on the bottom (22) of the reservoir (20) are configured to perform together one or more of the following: voltage application, current application, voltage recording, and current recording.

14. A measurement system (3) for cell culture, comprising: a. A cell culture assembly (70) as claimed in any one of claims 10 to 13; and b. An external acquisition device (80) configured to receive data from the plurality of sensors (32) in the cell culture insert (1).

15. A kit of parts for cell culture, comprising: a. A reservoir (20) defining an interior (21) and having a bottom (22); and b. A first cell culture insert (1) as defined in any one of claims 1 to 9, configured to be positioned in the interior (21) of the reservoir (20) to retain the second end (12) of the body (10) above the bottom (22) of the reservoir (20).

Images