WO2025119990A1

WO2025119990A1 - Electrode-enhanced cell culture monitoring

Info

Publication number: WO2025119990A1
Application number: PCT/EP2024/084707
Authority: WO
Inventors: Saeedeh EBRAHIMI TAKALLOO; Dries Braeken
Original assignee: Interuniversitair Microelektronica Centrum vzw IMEC
Current assignee: Interuniversitair Microelektronica Centrum vzw IMEC
Priority date: 2023-12-05
Filing date: 2024-12-04
Publication date: 2025-06-12
Anticipated expiration: 2026-06-05

Abstract

A semiconductor cell culture device is disclosed, which includes a semiconductor mesh with interconnected islands defining through-pores, and a cover creating a space for cell culture The device features at least one pair of electrodes on the mesh, positioned to intersect a through-pore when connected by a straight line. Electrical connections enable electrochemical measurements between the electrodes. The electrodes are placed within 100 µm of a through-pore, meeting a specific geometrical condition for precise interaction with cells or particles passing through the pores. This arrangement allows for advanced monitoring and analysis of cell cultures, with potential applications in biological research and medical diagnostics.

Description

Electrode-Enhanced Cell Culture Monitoring

Field of the Invention

The present invention relates to the field of bioelectrical measurement devices, and more specifically to a semiconductor cell culture device equipped with an electrode configuration for detecting the trans-passage of substances through pores.

Background of the Invention

The field of cell culture technology is a critical area of research and development in the life sciences, particularly in the study of cellular behaviors, drug discovery, and tissue engineering. A fundamental aspect of this field is the ability to accurately monitor and control the environment in which cells are grown and to study the interactions between cells and their surroundings. This includes the passage of various substances, such as nutrients, gases, cells (e.g., immune cells), and signaling molecules, through the cellular membrane or between cells in a culture system.

In the context of cell culture, the trans-passage of substances is a vital process that can influence cell growth, function, and response to external stimuli. The ability to detect and measure the movement of these substances through pores or channels is essential for understanding cellular mechanisms and for the development of advanced biomedical devices.

Traditionally, various methods have been employed to monitor the trans-passage of substances in cell culture environments. These methods include, but are not limited to, optical microscopy and fluorescence-based assays. Each of these techniques has its own set of advantages and limitations in terms of sensitivity, specificity, and the ability to provide real-time data.

One of the challenges in the field is the precise measurement of the trans-passage of substances at the microscale, particularly through small pores or channels that may be present in a cell culture substrate or membrane. The spatial resolution and sensitivity of the measurement techniques are crucial for detecting subtle changes in the passage of substances, which can be indicative of important biological processes or responses to pharmacological agents. Despite the advancements in cell culture technology and measurement techniques, there remains a need for further innovation in the field. Improved methods and devices that can offer high-resolution, sensitive, real-time, label-free, and non-invasive monitoring of the trans-passage of substances in cell culture systems are needed to advance our understanding of cellular processes and to facilitate the development of new therapeutic strategies.

Summary of the Invention

It is an object of embodiments of the present invention to enable the detection of substance trans-passage through pores in a semiconductor cell culture device. This objective is accomplished by a semiconductor cell culture device and associated methods and systems for detecting the passage of entities through the device, as well as computer programs and computer-readable media related to the operation of the device according to the invention.

In the first aspect, the present invention relates to a semiconductor cell culture device comprising: a. a semiconductor mesh having islands being interconnected by bridges and defining through-pores between the islands, wherein the mesh has a top surface and a bottom surface, b. a cover facing the top surface of the semiconductor mesh and separated therefrom, thereby defining a space for a cell culture, wherein the cover has a top surface and a bottom surface, c. at least a first mesh electrode pair comprising a first and a second electrode attached to the top surface or the bottom surface of the mesh and positioned such that at least one electrically conductive point from the first electrode and at least one conductive point from the second electrode, if connected by a straight line, intersect a single through- pore, d. first electrical connections to the at least a first mesh electrode pair configured to allow the performance of an electrochemical measurement between the first and the second electrode of said pair, and wherein the at least a first mesh electrode pair satisfy a geometrical condition imposing that one or more of the at least one electrically conductive point from the first electrode and one or more of the at least one electrically conductive point from the second electrode must be located within a distance of 100 pm from said single through-pore. 100 pm is the maximum endothelial/epithelial cell radius in culture.

In the second aspect, the present invention relates to a method for detecting the passage of entities through a through-pores of a cell culture device according to any embodiments of the first aspect, the method comprising: a. Initiating an electrochemical measurement, e.g., an impedance spectroscopy measurement between both electrodes of the at least a first mesh electrode pair, thereby generating electrical outputs, b. Determining from the electrical outputs obtained from the impedance spectroscopy measurements if both electrodes of the pair measure a variation in current, indicative of the passage of entities through the cell culture.

In the third aspect, the present invention relates to a semiconductor cell culture system comprising a device according to any embodiments of the first aspect, and further comprising a processing unit adapted to perform the method of the second aspect.

In the fourth aspect, the present invention relates to a computer program comprising instructions to cause the semiconductor cell culture system of the third aspect to execute the steps of the method of the second aspect.

In the fifth aspect, the present invention relates to a computer-readable medium having stored thereon the computer program of the fourth aspect.

It is an advantage of embodiments of the present invention that precise detection of the trans-passage of substances through a pore can be facilitated by electrodes positioned on the the mesh, which are in close proximity to the edge of the pore. It is a further advantage of embodiments of the present invention that the sensitivity of measurements such as impedance spectroscopy and various electrochemical techniques can be enhanced due to the strategic placement of electrodes across the pore. It is yet another advantage of embodiments of the present invention that the closer the electrodes are to the pore, the greater the sensitivity of the measurement becomes, allowing for more accurate monitoring of cellular interactions and substance movements.

It is a further advantage of embodiments of the present invention that the geometrical condition requiring electrodes to be within a specific distance from the pore edge, such as 0-100 pm, can significantly improve the resolution and reliability of the measurements.

It is an advantage of embodiments of the present invention that the option to include cover electrodes on a cover facing the mesh allows for additional measurement configurations, enabling the detection of faults, such as holes, irregularities, disruptions, heterogeneities, or tightness inconsistencies in the cell culture above the through-pore.

It is an advantage of embodiments of the present invention that it enables the performance of detailed analysis of the electrical properties across the pore. It is a further advantage of embodiments of the present invention that it enables the detection of the passage of entities through the pores by analyzing variations in current, indicative of cellular or molecular events.

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.

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.

Brief description of the drawings

Fig.l is a schematic representation of a top view of horizontal cross-section through the semiconductor cell culture device, showing the semiconductor mesh with islands interconnected by bridges and defining through-pores, according to the prior art.

Fig.2 is a detailed top view of a schematic representation of part of the semiconductor cell culture device, illustrating the geometrical condition of the mesh electrode in relation to the through-pore, according to embodiments of the present invention. Fig.3 is a schematic representation of the semiconductor cell culture device, showing the electrical connection between two mesh electrodes through a switch, according to embodiments of the present invention.

Fig.4 is a schematic representation of a vertical cross-section through the semiconductor cell culture device, demonstrating electrodes on the top surface of the mesh, the electrical connections to the mesh, the electrical measurement system connected to the electrical connections, as well as the processing unit for performing the method according to embodiments of the present invention.

Fig.5 (left) is a schematic representation of a vertical cross-section through the semiconductor cell culture device, demonstrating electrodes on the bottom surface of the mesh. Fig. 5 (right) depicts the path taken by electric charges during an electrical measurement in the device. Also illustrated is a leak due to a fault in the cell culture above through-hole.

Fig.6 is a schematic representation of a vertical cross-sectional view of a semiconductor cell culture device, illustrating the cover facing the top surface of the semiconductor mesh and defining a space for a cell culture, and having electrodes on the bottom surface of the cover and the top of the mesh for allowing electrical measurements according to embodiments of the present invention.

Fig.7 illustrates a leak due to a fault in the cell culture above through-hole in the embodiments of Fig.6.

Fig. 8 illustrates the path taken by electric charges during an electrical measurement in a device analogous to the one illustrated in Figs. 6 and 7.

Fig. 9 is a schematic representation of a vertical cross-sectional view of a semiconductor cell culture device, illustrating the cover facing the top surface of the semiconductor mesh and defining a space for a cell culture, and having a single large electrode on the bottom surface of the cover and multiple electrodes on the top of the mesh for allowing electrical measurements according to embodiments of the present invention.

Figs. 10 and 11 are schematic representations of a vertical cross-sectional views of semiconductor cell culture devices, illustrating the cover facing the top surface of the semiconductor mesh and defining a space for a cell culture, and having a single large electrode pierced by holes on the bottom surface of the cover and multiple electrodes on the top of the mesh for allowing electrical measurements according to embodiments of the present invention and for allowing observation of the cells (e.g., spectroscopy or microscopy).

Fig.12 is a schematic representation of the semiconductor cell culture device, demonstrating electrodes on the bottom surface of the mesh, the first and second electrical connections to the mesh and cover electrodes respectively, the electrical measurement system connected to the first and second electrical connections, as well as the processing unit for performing the method according to embodiments of the present invention.

Fig. 13 is a schematic representation of a vertical cross-sectional view of a semiconductor cell culture device, illustrating the cover facing the top surface of the semiconductor mesh and defining a space for a cell culture, and having a plurality of electrodes on the bottom surface of the cover and multiple electrodes on the bottom surface of the mesh, at different distances from a through-hole, according to embodiments of the present invention.

Figs. 14 and 15 are schematic representation of detailed top views of electrodes for attachment to the bottom surface of the cover, according to embodiments of the present invention.

Fig. 16 is a schematic representation of a detailed top view of mesh electrodes for use in embodiments of the present invention.

Fig. 17 is a schematic representation of a detailed top view of mesh electrodes (left) and cover electrodes (right) for use in embodiments of the present invention.

Fig.18 is a flowchart illustrating the method for detecting the presence of a fault in a biological barrier in the semiconductor cell culture device, according to embodiments of the present invention.

In the different figures, the same reference signs refer to the same or analogous elements.

Detailed description of Illustrative Embodiments

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.

The terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequence, either temporally, spatially, in ranking or in any other manner. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

Moreover, the terms top and over and the like in the description and the claims are used for descriptive purposes and not necessarily for describing relative positions. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other orientations than described or illustrated herein.

It is to be noticed that the term "comprising", used in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. The term "comprising" therefore covers the situation where only the stated features are present and the situation where these features and one or more other features are present. The word "comprising" according to the invention therefore also includes as one embodiment that no further components are present. Thus, the scope of the expression "a device comprising means A and B" should not be interpreted as being limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

Similarly, it is to be noticed that the term "coupled" should not be interpreted as being restricted to direct connections only. The terms "coupled" and "connected", along with their derivatives, may be used. It should be understood that these terms are not intended as synonyms for each other. Thus, the scope of the expression "a device A coupled to a device B" should not be limited to devices or systems wherein an output of device A is directly connected to an input of device B. It means that there exists a path between an output of A and an input of B which may be a path including other devices or means.

"Coupled" may mean that two or more elements are either in direct physical or electrical contact, or that two or more elements are not in direct contact with each other but yet still co-operate or interact with each other.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

Similarly it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.

Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

Furthermore, some of the embodiments are described herein as a method or combination of elements of a method that can be implemented by a processor of a computer system or by other means of carrying out the function. Thus, a processor with the necessary instructions for carrying out such a method or element of a method forms a means for carrying out the method or element of a method. Furthermore, an element described herein of an apparatus embodiment is an example of a means for carrying out the function performed by the element for the purpose of carrying out the invention.

In the description provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

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. Fig.l is a schematic representation of a top view of horizontal cross-section through the semiconductor cell culture device, showing the semiconductor mesh (112) with islands (116) interconnected by bridges (118) and defining through-pores (114), according to the prior art. A mesh electrode (120) is depicted at the center of an island (116), far from the edge of any through pore.

Fig.4 is a schematic representation of a vertical cross-sectional view of a semiconductor cell culture device according to the first aspect. As used herein, and unless otherwise specified, the term "semiconductor cell culture device" refers to a device that is used for the growth and maintenance of biological cells. This device comprise semiconductor materials. Fig. 4 illustrates the cover (200) facing the top surface of the semiconductor mesh (112) and separated therefrom, thereby defining a space (202) for a cell culture (204), wherein the cover (200) has a top surface and a bottom surface. In other words, the cover (200) is a component of the semiconductor cell culture device that faces the top surface of the semiconductor mesh and is separated from it. This cover (200) also has a top surface and a bottom surface, and it defines a space (202) for cell culture (204) when positioned over the semiconductor mesh. The mesh electrodes are used for conducting electrical measurements in the semiconductor cell culture device. The mesh electrodes are attached to the top surface of the mesh but they can also be attached to the bottom of the mesh (see Fig. 5). The at least a first mesh electrode pair satisfy a geometrical condition imposing that one or more of the at least one electrically conductive point from the first electrode and one or more of the at least one electrically conductive point from the second electrode must be located within a distance of 100 pm from said single through-pore (114). As used herein, and unless otherwise specified, the term "electrically conductive point" refers to a geometric location on an electrode that is capable of conducting electricity and is involved in the transfer of electrical signals during electrochemical measurements. These points are part of the electrode's surface.

The semiconductor mesh (112) has islands (116) being interconnected by bridges (118) (not depicted) and defining through-pores (114) between the islands (116), wherein the mesh (112) has a top surface and a bottom surface. In other words, the semiconductor mesh refers to a network or fabric-like structure made from semiconductor material. This mesh includes interconnected islands and bridges, and defines through-pores between the islands. The mesh has a top surface and a bottom surface. The islands, bridges, and through- pores are all integral parts of the mesh structure. First electrical connections (110, 111) to the at least a first mesh electrode pair are present and configured to allow the performance of an electrochemical measurement between the first and the second electrode of said pair. An electrochemical measurement is depicted between the electrodes of two pairs. The electrochemical measurement is performed to detect the presence of chemical species (500) in the through pore. Such a presence or a variation thereof can be indicative of a fault in the cell culture above the pore or can be useful in the study of chemical exchanges in and out of the cell culture. Also depicted is an electrical measurement system (117) connected to the first (110) and second (111) electrical connections, as well as a processing unit (119) for performing the method according to the second aspect of the present invention.

As exemplified above, in embodiments, the semiconductor cell culture device may further comprise an electrochemical measurement system, e.g., an impedance spectroscopy measurement system, connected to the first electrical connections (110, 111) to the at least a first mesh electrode pair, configured to perform measurements between both electrodes of the pair. This embodiment integrates a specific measurement techniques into the device.

Also, in embodiments, the electrical measurement system (117) connected to the first connections can also be connected second electrical connections to the cover electrodes, configured to perform measurements between any of the at least one mesh electrode (120) and a cover electrode (220).

Fig.2 is a detailed top view of a schematic representation of part of the semiconductor cell culture device, illustrating the geometrical condition of the at least a first mesh electrode pair in relation to the through-pore (114), according to embodiments of the present invention. As used herein, and unless otherwise specified, the term "geometrical condition" refers to a specific spatial requirement that must be met by the mesh electrodes. This condition stipulates that one or more of the at least one electrically conductive point from the first electrode and one or more of the at least one electrically conductive point from the second electrode must be located within a distance of 100 pm from said single through-pore (114).

In embodiments, the distance may be 10 pm, preferably 5 pm from said single through-pore. This embodiment provides the advantage of increased measurement precision due to closer proximity to the through-pore. Most preferably, said distance may be 1 pm, or even 0 pm (e.g., one or more of the at least one electrically conductive point from the first electrode and one or more of the at least one electrically conductive point from the second electrode must be in direct contact or immediate proximity with said single through- pore so that there is substantially no distance between them and the through-pore).

In embodiments, the geometrical condition may impose that at least two distinct electrically conductive points from each electrode of the pair are comprised in the top surface of the first electrode, are located within said distance, and are equidistant from said single through-pore. This embodiment ensures symmetrical positioning of the electrodes, which can improve the accuracy of the measurements.

In embodiments, both electrodes of said at least a first mesh electrode pair electrode may have an electrically conductive boundary of their top surface having at least two distinct electrically conductive points located within said distance and equidistant from said single through-pore. This embodiment provides the advantage of a defined measurement area for improved consistency in readings.

In embodiments, the geometrical condition may impose that both electrodes of said at least a first mesh electrode pair electrode have a line (122) being at least 20 pm long and entirely located within said distance from said single through-pore. This embodiment allows for a larger area of measurement, increasing the sensitivity of the device.

In embodiments, the line (122) may run parallel to a periphery of the through-pore, said periphery being coplanar with the surface of said at least a first mesh electrode pair attached to the mesh. This embodiment ensures that the measurement is taken in a consistent orientation relative to the through-pore, which can be critical for certain types of analysis.

In embodiments, the geometrical condition further may impose that at least 50%, preferably at least 75%, more preferably at least 90% of the top periphery of said single through-pore, said top periphery being coplanar with the surface of said at least a first mesh electrode pair attached to the mesh, must be within said distance of at least one electrically conductive point of said at least a first mesh electrode pair. This embodiment ensures comprehensive coverage around the through-pore for thorough measurement. It is, however, preferred if not more than 95% of the top periphery of said single through-pore is within said distance. More than 95% would typically mean that both electrodes are in very close proximity, which is not desirable.

In embodiments, the geometrical condition further may impose that said through- pore has a top periphery being coplanar with that surface of said at least a first mesh electrode pair which is attached to the mesh, said top periphery having an average diameter (D) such that the ratio between said average diameter (D) and the distance (d) separating said at least one electrically conductive point is at least 2, preferably at least 3, more preferably at least 5, and most preferably at least 9. The higher this ratio, the closer the electrodes are to the pores, relatively to the size of the pore. This provides a higher sensitivity.

We now refer to Fig. 3. In embodiments, said first and second electrodes of said at least a first mesh electrode pair may be electrically connected through a switch. When the switch (401) is on, the connection between both electrodes is closed and both electrodes act as a single electrode. This permits an electrical measurement of the cell barrier at that pore by measuring a signal between the connected electrodes on one hand and a cover electrode on another hand. Since the connected electrode surround more of the through pore than each electrode alone the signal obtained is larger and therefor exhibits a higher sensitivity. When the switch (401) is off, the connection between both electrodes is open and both electrodes can act separately. This permits each electrode to perform an electrical measurement of the cell barrier at two different locations around the pore by measuring a signal between the each of the separated electrodes on one hand and the cover electrode on another hand. This also permits to perform measurement laterally between both electrodes, without involving the cover electrode, thereby measuring what happen between the electrodes (e.g., the presence of chemical compounds). As used herein, and unless otherwise specified, the term "switch" refers to a device that is used to make or break an electrical connection. In the context of the semiconductor cell culture device, a switch (401) is used to electrically connect two or more mesh electrodes that have at least one electrically conductive point located within a certain distance (d) from the same through-pore.

We now refer to Figs. 6-13. In embodiments, the semiconductor cell culture device may further comprise at least one cover electrode and further connections to the at least one cover electrode to allow the performance of an electrical measurement, e.g., a transepithelial-transendothelial electrical measurement between one or more electrodes from the at least a first mesh electrode pair and a cover electrode. These embodiments provide the advantage of enabling additional types of measurements that can be critical for certain cell culture studies. In particular, it enables testing the potential presence of faults in the barrier layer present above the pores.

In embodiments, the first and further electrical connections may allow the simultaneous performance of an electrical measurement between a same cover electrode and a group of mesh electrodes (e.g. a mesh electrode pair), each mesh electrode of said group satisfying said geometrical condition with respect to a same through-pore. This embodiment allows for simultaneous measurements at various mesh electrodes around a same through pore, providing a more comprehensive understanding of the electrical properties of the cell culture directly above that through-pore (114).

In embodiments, said group may be both mesh electrodes of a pair. In embodiments, said group may be both electrodes of a pair linked by a switch, wherein the switch is closed, thereby electrically connecting both electrode.

In embodiments, each through-pore may have a top periphery, and wherein each of the at least one cover electrode is positioned directly above: a. a different through-pore such that a vertical projection of the electrode on the plane of the top surface of the mesh at least partially overlap with the through-pore, or b. a plurality of through-pores such that a vertical projection of the electrode on the plane of the top surface of the mesh entirely comprises the top periphery of the plurality of through-pores. Situation b. is depicted in Fig. 9 and the cover electrode is depicted in Fig. 15 (right).

In embodiments, each of the at least one cover electrode (see e.g., Figs. 14 and 15) may have openings and may be positioned directly above a plurality of through-pores such that a vertical projection of the electrode on the plane of the top surface of the mesh entirely comprises the top periphery of the plurality of through-pores and does not comprise at least some space between two adjacent through-pores. This situation is depicted in Fig.

10. These openings permit the observation of the cell culture away from the pores (e.g., by spectroscopy or microscopy).

In embodiments, each of the at least one cover electrode may have openings and may be positioned such that a vertical projection of the openings on the plane of the top surface of the mesh entirely comprises the top periphery of the plurality of through-pores. This situation is depicted in Fig. 11. These openings permit the observation of the cell culture above the pores. The embodiments of Fig. 10 and 11 can also be combined so that the vertical projection of some openings is not overlapping with a through hole while the vertical projection of other openings do overlap with a through hole.

In embodiments, the one or more of the at least one cover electrode may have a same shape and wherein the through-pores have said same shape. This is depicted in Fig. 17, which is a schematic representation of a detailed top view of mesh electrodes around hexagonal through-holes (left) and hexagonal cover electrodes (right) for use in embodiments of the present invention.

We now refer to Fig. 12. Fig.12 is a schematic representation of the semiconductor cell culture device, demonstrating electrodes on the bottom surface of the mesh, the first (110) and second (111) electrical connections to the mesh (120) and cover (220) electrodes respectively, the electrical measurement system (117) connected to the first (110) and second (111) electrical connections, as well as the processing unit (119) for performing the method according to the second aspect of the present invention.

Hence, in embodiments where the semiconductor cell culture device comprise an electrical measurement system (117) connected to the first electrical connections (110, 111), it may also be connected to the second electrical connection and b configured to perform measurements between any of the at least one mesh electrode (120) and a cover electrode (220).

Fig. 13 is a schematic representation of a vertical cross-sectional view of a semiconductor cell culture device, illustrating the cover (200) facing the top surface of the semiconductor mesh and defining a space for a cell culture, and having a plurality of electrodes on the bottom surface of the cover (200) and multiple electrodes on the bottom surface of the mesh, at different distances from a through-hole, according to embodiments of the present invention. This permit the performance of measurements in a four electrodes configuration (e.g., two concentric mesh electrodes surrounding a through-hole and two cover electrodes, e.g. two concentric cover electrodes) wherein the current is applied between the mesh electrode (123), which is farther from the through-hole than electrode (120) and which can be at a distance larger than d from the through hole, and the inner cover electrode (220i), and the voltage is measured between the mesh electrode (120) which is closer to the through-hole than electrode (123) and which is within said distance d, and the inner cover electrode (220i).

Figs. 14 and 15 are schematic representation of detailed top views of electrodes for attachment to the bottom surface of the cover (200), according to embodiments of the present invention. Fig. 14 has openings allowing observation (e.g., spectroscopy or microscopy). Fig. 15 left is a grid electrode, which also allows observation. Fig. 15 (right) is a plain electrode as can be used in the embodiment of Fig. 9.

Fig. 16 is a schematic representation of a detailed top view of mesh electrodes for use in embodiments of the present invention. Two different shapes are represented: circular and hexagonal. The electrode (120) on the left is shown around the periphery of a through hole (302) and can be used in a two electrode configuration. The electrodes on the right are a first electrode within said distance d and a second electrode, concentric to the first, which can be farther than said distance d. These two electrodes (120, 123) can be used in a four electrodes configuration.

We now refer to Fig. 18. In the second aspect, the present invention relates to a method for detecting the passage of entities through a through-pores of a cell culture device according to any embodiments of the first aspect, the method comprising: a. Initiating an electrochemical measurement, e.g., an impedance spectroscopy measurement between both electrodes of the at least a first mesh electrode pair, thereby generating electrical outputs, b. Determining from the electrical outputs obtained from the impedance spectroscopy measurements if both electrodes of the pair measure a variation in current, indicative of the passage of entities through the cell culture. Any feature of the second aspect can be as correspondingly described in any of the other aspects.

As used herein, and unless otherwise specified, the term "processing unit" refers to a component of the semiconductor cell culture system that is adapted to perform a method for detecting the passage of entities through a through-pore. This unit may include a computer or other electronic device that is capable of processing data and executing instructions.

Any feature of the third aspect can be as correspondingly described in any of the other aspects.

Any feature of the fourth aspect can be as correspondingly described in any of the other aspects.

Any feature of the fifth aspect can be as correspondingly described in any of the other aspects.

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

1. A semiconductor cell culture device comprising: a. a semiconductor mesh (112) having islands (116) being interconnected by bridges (118) and defining through-pores (114) between the islands (116), wherein the mesh has a top surface and a bottom surface, b. a cover (200) facing the top surface of the semiconductor mesh (112) and separated therefrom, thereby defining a space (202) for a cell culture (204), wherein the cover (200) has a top surface and a bottom surface, c. at least a first mesh electrode pair (120, 120b) comprising a first (120) and a second (120b) electrode attached to the top surface or the bottom surface of the mesh and positioned such that at least one electrically conductive point (121) from the first electrode and at least one conductive point (121) from the second electrode, if connected by a straight line, intersect a single through-pore (114), d. first electrical connections (110, 111) to the at least a first mesh electrode pair (120, 120b) configured to allow the performance of an electrochemical measurement between the first and the second electrode of said pair (120, 120b), and wherein the at least a first mesh electrode pair (120, 120b) satisfy a geometrical condition imposing that one or more of the at least one electrically conductive point (121) from the first electrode and one or more of the at least one electrically conductive point (121) from the second electrode must be located within a distance (d) of 100 pm from said single through-pore (114).

2. The semiconductor cell culture device according to claim 1, wherein said distance (d) is 10 pm, preferably 5 pm from said single through-pore (114).

3. The semiconductor cell culture device according to claim 1 or claim 2, wherein said geometrical condition imposes that at least two distinct electrically conductive points (121) from each electrode of the pair (120, 120b) are comprised in the top surface of the first electrode, are located within said distance (d), and are equidistant from said single through-pore (114).

4. The semiconductor cell culture device according to any one of claims 1 to 3, wherein said geometrical condition imposes that both electrodes of said at least a first mesh electrode pair have a line being at least 20 pm long and entirely located within said distance (d) from said single through-pore (114).

5. The semiconductor cell culture device according to claim 4, wherein said line runs parallel to a periphery of the through-pore (114), said periphery being coplanar with the surface of said at least a first mesh electrode pair (120, 120b) attached to the mesh.

6. The semiconductor cell culture device according to any one of the preceding claims, wherein said geometrical condition further imposes that at least 50%, preferably at least 75%, more preferably at least 90% of the top periphery of said single through-pore (114), said top periphery being coplanar with the surface of said at least a first mesh electrode pair (120, 120b) attached to the mesh, must be within said distance (d) of at least one electrically conductive point (121) of said at least a first mesh electrode pair (120, 120b).

7. The semiconductor cell culture device according to any one of the preceding claims, wherein the geometrical condition further imposes that said through-pore (114) has a top periphery being coplanar with that surface of said at least a first mesh electrode pair (120, 120b) which is attached to the mesh, said top periphery having an average diameter (D) such that the ratio between said average diameter (D) and said distance (d) separating said at least one electrically conductive point (121) from the though-pore is at least 2, preferably at least 3, more preferably at least 5.

8. The semiconductor cell culture device according to any one of the preceding claims, wherein said first and second electrodes of said at least a first mesh electrode pair (120, 120b) are electrically connected through a switch.

9. The semiconductor cell culture device according to any one of the preceding claims, further comprising at least one cover electrode (220) and further connections to the at least one cover electrode (220) to allow the performance of an electrical measurement between one or more electrodes from the at least a first mesh electrode pair (120, 120b) and a cover electrode (220).

10. The semiconductor cell culture device according to claim 9, wherein each of said at least one cover electrode is positioned directly above: a. a different through-pore such that a vertical projection of the electrode on the plane of the top surface of the mesh at least partially overlaps with the through-pore, or b. a plurality of through-pores such that a vertical projection of the electrode on the plane of the top surface of the mesh entirely comprises a plurality of through-pores.

11. The semiconductor cell culture device according to any one of the preceding claims, further comprising an electrochemical measurement system, e.g., an impedance spectroscopy measurement system, connected to the first electrical connections (110, 111) to the at least a first mesh electrode pair (120, 120b), configured to perform measurements between both electrodes of the pair (120, 120b).

12. A method for detecting the passage of entities through a through-pore of a cell culture device according to any one of the preceding claims, the method comprising: a. Initiating an electrochemical measurement between both electrodes of the at least a first mesh electrode pair (120, 120b), thereby generating electrical outputs, b. Determining from the electrical outputs obtained from the impedance spectroscopy measurements if both electrodes of the (120, 120b) measure a variation in current, indicative of the passage of entities through the cell culture.

13. A semiconductor cell culture system comprising a device according to any one of claims 1 to 11, and further comprising a processing unit adapted to perform the method of claim 12.

14. A computer program comprising instructions to cause the semiconductor cell culture system of claim 13 to execute the steps of the method of claim 12.

15. A computer-readable medium having stored thereon the computer program of claim 14.