Apparatus with integrated electrodes for electrical monitoring of cell cultures

Abstract

The invention is a device, shown in Fig 1., comprising at least one well with integrated electrodes, layers forming plates and at least two integrated electrodes positioned in at least two different planes inside the well, for electrical measurements of in vitro culture of biological constructs such as cells layer, 3D tissue constructs or organoids. The apparatus comprises at least one well with at least two integrated electrodes with at least one suspended electrode of the at least two electrodes integrated into the well at different planes, or with the plurality of suspended electrodes realized on multiple suspended layers in different combinations, Fig 4., The invention is a measurement instrument, capable for electrical measurements of in vitro culture of biological construct.

Description

[0001] APPARATUS WITH INTEGRATED ELECTRODES FOR ELECTRICAL MONITORING OF CELL CULTURES

[0002] Technical Field

[0003] The invention, widely speaking, refers to devices, plate components, and kits for conducting chemical, biochemical, biomedical and / or biological assays.

[0004] More specifically the invention is an apparatus comprising at least one well with integrated electrodes, layers forming plates and at least two integrated electrodes positioned in at least two different planes inside the well for electrical measurements of in vitro culture of biological constructs such as cells layer, 3D tissue constructs or organoids.

[0005] The invention is a measurement instrument, capable of improved and more efficient electrical measurements of in vitro culture of biological constructs as disclosed, with wide industrial applicability, including but not limited to pharmaceutical and biotech industries as the invention can be used for a variety of high throughput screening applications such as drug testing, toxicity testing of different chemicals or compounds on cells, disease modelling, phenotypic screening, cell culture growing and maintaining cells in vitro, stem cell culture, alternative protein, cultivated meat, tissue engineering, immunocytochemistry, and basic cell biology.

[0006] Background arts

[0007] Scientific methods in modern science are based on requirements of accurate and precise determination of facts. The development of biology, chemistry, biochemistry, molecular biology and biomedicine goes more and more into exploring to understand the structure and functioning of cells and tissue.

[0008] The invention is widely covering the field of impedance spectroscopy and electrochemical measurements with a special extent on the specific realization of the culture of a biological construct such as cell layers, three-dimensional (3D) tissue constructs or organoids / organ-on-chip.

[0009] The invention is a measurement instrument, more specifically an apparatus comprising at least one well (chamber or reservoir) with integrated electrodes, layers forming plates and at least two electrodes placed in at least two different planes integrated inside the well for electrical measurements of in vitro culture of biological constructs such as cell layers, 3D tissue constructs or organoids, providing the accurate data referring to the three-dimensional (hereinafter: 3D) structure of biological constructs.

[0010] The measurements comprise but are not limited to impedance spectroscopy, electrochemical measurements, or TEER (transepithelial / transendothelial electrical resistance) measurements to monitor the changes of conductivity of a cell culture layer, 3D tissue constructs or organoid / organ-on-chip.

[0011] The closest prior art known to the applicants and inventors of this invention discloses a method for monitoring the electrophysiology of cell culture that relies on a multielectrode array (MEA) which involves planar electrodes situated at the bottom of a well (chamber) on which adherent cells or tissue are cultured (US9279801B2, US6376233B1, US9290756B2). Each MEA records electrical activity from the contents of the wells (and / or electrical stimulation to the contents of the wells). Despite the benefits of cost-effectiveness and time efficiency of the mentioned method, the platforms used for testing multiple cell cultures or tissue constructs simultaneously do not provide data referring to the 3D structure of biological constructs. Planar electrodes situated at the bottom of a well (chamber) on which adherent cells or tissue are cultured prevent the planar electrical measurement, which is highly influenced by such electrodes, from accurately measuring electrical parameters on 3D biological constructs and monitoring the tissue barrier. The present invention overcomes this technical deficiency by introducing the new and inventive construction of the apparatus comprising a cell culture plate that facilitates electrical measurements of 3D or 2D biological constructs grown between the electrodes integrated in the cell culture well. The present invention responds to the need of the biological sciences focused on studying biological constructs or tissue layer studies, but not excluding other aspects such as to utilize systems that would provide enhanced electrical measurements of 3D biological constructs. This can be accomplished by providing a novel and inventive apparatus that enables improved and accurate electrical measurements of 3D biological constructs to help better understanding of such constructs or tissue layer as well as allow quantification of intracellular junctions.

[0012] Patent application US7459303B2, discloses a well that consists of an upper chamber receiving a biological sample, a lower chamber having at least two electrodes, and a biocompatible porous membrane. The invention disclosed in the patent US7459303B2 is used for monitoring the migration or invasion of biological particles and does not allow carrying out electrical measurements through a biological construct since all electrodes are incorporated below the biological construct. The article titled “Cardiac microphysiological devices with flexible thin-film sensors for higher-throughput drug screenin ', Lind JU, Yadid M, Perkins I, O'Connor BB, Eweje F, Chantre CO, Hemphill MA, Yuan H, Campbell PH, Vlassak JJ, Parker KK, Oct 2017, mentions a multi- well plate with a suspended electrode onto which cardiac muscle is grown. The disclosed device uses the suspended electrode as an electric circuit, wherein such electrode is capable of measuring piezoresistive strain but not measurement of electrical parameters through a biological construct as it does not provide integrated electrodes.

[0013] Patent US10670576B2 discloses a multi-well plate for measuring impedance in organotypic tissue. The device includes bottom electrodes and top electrodes separated by a porous membrane onto which the organotypic tissue is cultured. Compared to the invention disclosed in the patent (US10670576B2), the present invention provides non-obvious advancement and differs from the prior art. Namely, the invention comprises at least two integrated electrodes (105) at fixed positions placed at two different planes, wherein at least one electrode of the at least two electrodes located in the well is suspended into the well (100). The electrodes are integrated in a cell culture well (101) (Fig.l) and a complete electrical measurement of a biological construct grown in a well requires only one cell culture well. While the invention disclosed in the patent (US10670576B2) comprises movable electrodes attached above the element with the function of a cell culture well and requires at least two cell culture chambers (top and bottom chambers inside one well) separated by a porous membrane. Compared to the closest prior art known to the inventors and the applicant, the invention discloses including but not limited to the following non-obvious, inventive advancements:

[0014] - The tolerance of the proposed solution in the US10670576B2 has a larger effect on manufacturing due to the complexity of the device that comprises two chambers with movable electrodes, bottom electrodes, and a membrane on each well. While the present invention comprises suspended electrodes inside the well at fixed positions. The invention disclosed in US 10670576B2 has a more complex structure than the present invention which can increase the tolerance range in the manufacturing process and affect the precision and reproducibility of the measurements between different fabrication batches.

[0015] - The present invention does not create any disturbance in a cell culture condition / s during measurements, such as it is with adaptable electrodes denoting that the electrodes of the current invention can be placed at any position perpendicular to the walls of the cell culture wells.

[0016] - Compared to the invention disclosed in patent US 10670576B2 which comprises a top-positioned movable electrode, the present invention enables parallel measurements between each pair of integrated electrodes, which is not technically possible with the invention in the referred patent (US10670576B2). The current invention enables simultaneous electrical measurements through cell layers or a tissue construct utilizing the different electrodes that can be placed at the different planes and the impedance measurement in the plane with electrodes located at the same plane to monitor cell growth on the surface which is not possible with the invention disclosed in US10670576B2 as the biological construct is grown on the membrane with does not integrate electrodes.

[0017] -The present invention contributes to improved electrical measurements and excitation of 3D biological constructs, with enables an effective and more precise method for capturing biological phenotypes of 3D biological constructs and provides better electrical stimulation.

[0018] The proposed apparatus with integrated electrodes as defined in the claims will overcome at least some of the problems of the prior art.

[0019] Disclosure of Invention

[0020] The invention is a measurement instrument, more specifically an apparatus comprising at least one well with integrated electrodes, layers forming plates with at least two electrodes integrated perpendicular to the wall inside the well positioned at at least two different planes for electrical measurements of in vitro culture of biological constructs such as cells layer, 3D tissue constructs or organoids, providing the accurate data referring to the three-dimensional (hereinafter: 3D) structure of biological constructs.

[0021] The invention is a measurement instrument, capable of improved and more efficient electrical measurements of in vitro culture of biological constructs as disclosed, with wide industrial applicability, including but not limited to pharmaceutical and biotech industries as the invention can be used for a variety of high throughput screening applications such as drug testing, toxicity testing of different chemicals or compounds on cells, disease modelling, phenotypic screening.

[0022] In addition, the invention can be used as a tool for monitoring any type of in vitro biological process such as during cell culture growth and maintenance, stem cell culturing, alternative protein, tissue engineering, cellular agriculture / meat cultivation and immunocytochemistry.

[0023] The invention comprises multiple embodiments, wherein one preferred embodiment is disclosed in detail. Other embodiments have been disclosed as well. All embodiments disclosed form a single general inventive concept. The first preferred embodiment (Fig. 1) discloses an apparatus comprising: at least one well (100) with integrated electrodes, a frame (101), a suspended layer

[0024] (102), a perforated layer (103), a supporting layer (104), and four integrated electrodes (105), characterized in that the frame (101) forms the upper side of the apparatus and is connected to the suspended layer (102) with two suspended electrodes (105).

[0025] The well (100), is constructed as an open cavity preferably as an open-top cylinder, wherein the open-top part of the well (100) forms an opening, that enables the insertion of biological material into the well (100). The well (100) extends from its open-top part all the way to the upper surface of the supporting layer (104), intersecting the suspended layer (102) and the perforated layer (103), forming an opening in the parts where it intersects the mentioned layers (102) and

[0026] (103). The upper surface of the supporting layer (104) closes the well (100) forming its bottom where the biological material is retained when inserted in the well (100).

[0027] The suspended layer (102) is connected to the lower surface of the frame (101) and is being connected to the perforated layer (103). Two suspended electrodes (105) are realized on the lower surface of suspended layer (102), while another two electrodes (105) are realized on the upper side of the supporting layer (104). Each electrode (105) further comprises a routing pad (105a) and a connecting pad (105b), Fig. 3.

[0028] The perforated layer (103) is connected to the suspended layer (102) with its upper surface and to the supporting layer (104) with its lower surface, wherein the perforated layer (103) is configured to act as a physical barrier between the suspended layer (102) and the supporting layer (104), making a physical distance between electrodes (105) placed on the upper surface of the supporting layer ( 104) and the suspended electrodes (105) placed on the lower surface of the suspended layer (102).

[0029] The supporting layer (104) forms the bottom side of the apparatus, and it is connected to the lower surface of the perforated layer (103), wherein the upper surface of ( 104) is configured to support a biological construct inserted inside the well (100).

[0030] Disclosed elements of the apparatus can be of different materials, sizes and shapes, as is further elaborated in the detailed description.

[0031] In other embodiments, the apparatus comprises at least one well (100) with at least two integrated electrodes, and can be realized with at least one suspended electrode of the at least two electrodes integrated into the well (100) at different planes, and with the plurality of suspended electrodes realized on multiple suspended layers (102) in different combinations. The plurality of electrodes (105) can be realized at different sides of the suspended layer (upper or lower surface or lateral side) or on the upper surface of supporting layer (104), as being further elaborated in the detailed description.

[0032] Brief description of the Drawings

[0033] Fig. 1. illustrates the preferred embodiment of the invention, the lateral perspective. The preferred embodiment comprising the well (100) with integrated electrodes, a frame (101), a suspended layer (102), a perforated layer (103), a supporting layer (104), and four integrated electrodes (105).

[0034] Fig. 2. illustrates the cross section of the preferred embodiment of the invention with the elements in 3D. The preferred embodiment comprising at least one well (100) with integrated electrodes, a frame (101), a suspended layer (102), a perforated layer (103), a supporting layer (104), and four integrated electrodes (105). Two suspended electrodes (105) are realized on the lower surface of suspended layer (102), while another two electrodes (105) are realized on the upper side of the supporting layer (104).

[0035] Fig. 3. illustrates the integrated electrodes (105). Each electrode (105) comprises a routing pad (105a) and a connecting pad (105b).

[0036] Fig. 4. illustrates an unfolded 3D view of the preferred embodiment of the invention and depicts the the assembly of a multi well plate with a number of wells (100) with integrated electrodes.

[0037] Fig. 5. illustrates another embodiment of the invention wherein: (a) one suspended electrode (105) is realized on the lower surfaces of the suspended layer

[0038] (102), wherein another integrated electrode (105) is realized on the support layer (104); (b) one suspended electrode (105) is realized on the upper surface of the suspended layer (102), and another suspended electrode (105) is realized on the lower surface of the suspended layer (102); (c) two suspended electrodes (105) are realized on the upper surface of the suspended layer (102), two suspended electrodes (105) are realized on the lower surface of suspended layer (102), and one electrode (105) is realized on the upper surface of supporting layer (104).

[0039] Fig. 6. illustrates another embodiment of the invention wherein: (a) the apparatus can be realized with plurality of the suspended layers (102) and perforated layers

[0040] (103) stuck one on the top of each other, where plurality of the integrated electrodes (105) can be realized even on the upper surface of supporting layer 104 and / or lower and / or upper surfaces of each suspended layer (102); (b) the suspended electrode (105) realized on the suspended layer (102) can be realized on the lower surface of the suspended layer (102) and / or upper surface of the suspended layer (102) and / or lateral surface of suspended layer (102) that enters the well (100). In the case where the suspended electrode contains the lateral surface of supporting layer (102), the suspended layer (102) can be realized to have the same length as perforated layer (103) (length (106) is equal to 0).

[0041] Fig. 7. illustrates example 1 of different the 2, 3 and 4-terminal measurement setup with invention: (a) 2-port measurement using one suspended electrode (105) positioned at suspended layer (102) and another electrode positioned at supporting layer (104); (b) 2-port measurement using two electrodes (105) positioned on supporting layer (104); (c) 3 -port measurement using three suspended electrodes (105) positioned on suspended layer (102); (d) 4-port measurement using four electrodes (105); (e) multiport measurement that can combine different 2, 3 and 4-terminal measurements with invention with plurality of integrated electrodes (105).

[0042] Fig. 8. illustrates the 4-terminal impedance measurement set-up.The measurement included the use of. the embodiment including: a collagen layer (201), an endothelial layer (202), a pair of stimulating electrodes (203) (one electrode realized on the supporting layer (104) and one suspended electrode realized on the suspended layer (102)), and pair of measuring electrodes (204) (another electrode realized on the supporting layer (104) and another suspended electrode realized on the suspended layer (102)).

[0043] Fig. 9. illustrates impedance measurement carried-out for 4 selected cell culture wells before and after seeding HUVEC cells on top of a previously deposited collagen matrix using different electrode configurations.

[0044] Best Mode for Carrying Out of the Invention

[0045] In the following description, a preferred embodiment of the invention is disclosed, including other functional alternatives of the invention considered as “other embodiments” of the invention.

[0046] The preferred embodiment of the invention is also referred to as "the embodiment" or “the apparatus”.

[0047] A reference to "other embodiment" or "other embodiments" does not imply that the referenced embodiment is mutually exclusive with another embodiment (in terms of its technical functionalities, shape, construction materials, etc.) described unless expressly specified otherwise. The first preferred embodiment, discloses an apparatus comprising: at least one well (100) with integrated electrodes, a frame (101), a suspended layer (102), a perforated layer (103), a supporting layer (104), and four integrated electrodes (105) wherein:

[0048] - The well (100), is constructed as an open cavity preferably an open-top cylinder, wherein the open-top part of the well (100) forms a circular opening, that enables the insertion of biological material into the well (100), the well (100) extends from its open-top part all the way to the upper surface of the supporting layer (104), intersecting the perforated layer (103) and forming circular openings in the intersected parts.

[0049] The upper surface of the supporting layer (104) closes the well (100) forming its bottom where the biological material is retained when inserted in the well (100).

[0050] The number of the wells (100) is limited by the size of the well, a plate and the apparatus as a whole.

[0051] The apparatus can be realized in a standard format with standard dimensions ofwells comprising: 6, 24, 48, 96, 192, 384, 1536, 3456 or 9600 wells (100) with integrated electrodes or in any non-standard format and dimensions.

[0052] - the frame (101), is constructed to have an upper and a lower surface, forming an upper side of the apparatus, further being connected to the suspended layer (102).

[0053] The frame (101) can be of different sizes and shapes, including but not limited to a square, rectangular or circular shape.

[0054] The frame (101) can be created by means of CNC milling laser cutting, hot embossing, injection moulding or 3D printing.

[0055] - the suspended layer (102), is constructed to have an upper and a lower surface. The suspended layer (102) is positioned between the lower surface of the frame (101) and the upper surface of the perforated layer (103).

[0056] The suspended layer (102) is being constructed as a plate with apertures with size smaller than size of the openings of the wells (100) and a suspended part (106) that enters inside the well (100).

[0057] The suspended layer ( 102) is physically longer on that side that enters the well (100) than the perforated layer (103) with its suspended part (106), to allow insertion of the suspended electrodes (105) realized on the suspended layer (102).

[0058] The suspended part (106) can have any kind of shape and geometry such as, but not limited to, cantilever, bridge, ring, circle, square, rectangle, triangle arc or a combination of the above. The length of the suspended part (106) should be more than twice smaller than the diameter of the well (100) to allow passing of biological material to the bottom of the well (100) and provide enough aperture to allow microscopic analysis.

[0059] The suspended layer (102) is configured to act as a physical carrier for the suspended electrodes (105) realized on its surface.

[0060] The suspended layer (102) can be made of various non-conductive biocompatible materials such as: glass or polymer.

[0061] The suspended layer (102) can be manufactured by CNC milling, laser cutting or 3D printing, injection moulding or hot embossing or any other compatible technology.

[0062] - the perforated layer (103), is constructed to have an upper and a lower surface, the perforated layer (103) further being intersected by the well (100) forming a cylindrical opening in the perforated layer (103). The upper surface of the perforated layer (103) is connected to the suspended layer (102). The lower surface of the perforated layer (103) is connected to the supporting layer (104). Wherein the perforated layer (103) is configured to act as a physical barrier between the suspended layer (102) and the supporting layer (104).

[0063] The perforated layer (103) can be made of any non-conductive biocompatible material such as glass, polymer, PCB, silicon.

[0064] The perforated layer (103) can be of different thicknesses to allow the incorporation of biological constructs.

[0065] The perforated layer (103) is constructed as a plate.

[0066] - the supporting layer (104), is constructed to have an upper and a lower surface. The supporting layer (104) is configured to retain a biological construct inserted through the opening of the well (100) and into the well (100), forming a bottom side of the apparatus.

[0067] Two electrodes (105) are realized on the upper surface of supporting layer (104).

[0068] The upper surface of supporting layer (104) is connected to the perforated layer (103).

[0069] The supporting layer (104) is preferably made of biocompatible, transparent, and non-conductive material. the integrated electrodes (105), are electrical conductors, constructed to detect electrical activity (such as resistance, voltage, current, conductance, impedance, capacitance) from a biological construct. Each electrode (105) comprises a routing pad (105a) and a connecting pad (105b) that enables connection of the integrated electrode (105) to a measurement setup, Fig. 3.

[0070] In the preferred embodiment two electrodes (105) are realized on the upper surface of the supporting layer (104) while another two suspended electrodes (105) are realized on the lower surface of suspended layer (102). The integrated electrodes (105) placed at different planes can be parallel to each other and can be realized with different sizes and geometry.

[0071] The integrated electrodes (105) can have different shapes including but not limited to round, interdigitated, squared, ring-like, or fractal, and can also have different sizes.

[0072] The surface and dimension of the integrated electrodes (105) are limited only by the size of the well (100) and the necessary aperture required for handling liquid or cell culture media with a pipette or other methods. In addition, the integrated electrodes (105) can provide enough aperture to allow microscopic analysis.

[0073] The process of realizing each electrode (105) relies on the semiconductor technology processes such as: thermal evaporation, sputtering, or 3D printing technologies such as inkjet or screen printing or any other methods. The integrated electrodes (105) can be made of any transparent or nontransparent material that is conductive. Preferable material is biocompatible material such as: as gold, platinum or conductive polymers and conductive inks.

[0074] In case that integrated electrodes (105) are made from a non-transparent material, an optical pathway for allowing microscopy can be assured.

[0075] Other embodiments:

[0076] In other embodiments, the well (100) can be constructed in other shapes including but not limited to a cylinder, square prism, ellipse prism, oval prism, triangle prism, or cube.

[0077] In other embodiment presented in Fig. 5a, at least one suspended electrode (105) is realized on the upper or lower surfaces of the suspended layer (102), wherein at least one electrode is realized on the support layer (104).

[0078] In other embodiment presented in Fig. 5b, at least one suspended electrode (105) is realized on the upper surface of the suspended layer (102), and at least one suspended electrode (105) is realized on the lower surface of the suspended layer (102).

[0079] In other embodiment presented in Fig. 5c, at least one suspended electrode (105) is realized on the upper surface of the suspended layer (102), at least one suspended electrode (105) is realized on the lower surface of suspended layer (102), and at least one electrode (105) is realized on the upper surface of supporting layer (104).

[0080] In other embodiment presented in Fig. 6a, the apparatus can be realized with plurality of suspended layers (102) and perforated layers (103) stuck one on the top of each other, where plurality of the integrated electrodes (105) can be realized even on the upper surface of supporting layer (104) and / or lower and / or upper surfaces of each suspended layer (102).

[0081] In other embodiment presented in Fig. 6b, the suspended electrodes (105) deposited on the suspended layer (102) can be realized on lower surface of the suspended layer (102), or / and upper surface of the suspended layer (102) or / and lateral surface of suspended layer (102) that entering into the well (100). If the suspended electrode (105) is realized on suspended layer (102) is realized in that manner to cover upper or lower surface of the suspended layer (102) and lateral side of suspended layer (102), the suspended layer can be realized to have the same length as perforated layer (103) (length (106) is equal to 0).

[0082] In other embodiment, one or all electrodes (105) comprise an additional layer, the layer being an insulating layer or / and a biological coating that can promote cell attachment such as a polymer film, biomolecules, peptides or proteins, or extracellular matrix components (or derivatives thereof).

[0083] In other embodiment, one or all suspended electrodes (105) can be coated with a material that can act as a bioreceptor to capture molecules to which target analytes can specifically bind to. The material includes but is not limited to specific proteins such as antibodies, aptamer, nucleic acid molecules, flagella, protein molecules, molecularly imprinted polymer (MIP) or combination of the above.

[0084] In other embodiment, one or all suspended electrodes (105) can be coated by or made of different nanomaterials. The material includes but is not limited to graphene, MXenes, gold or magnetic nanoparticles, nanotubes or their combination or their combination with bioreactors.

[0085] In other embodiment, an adhesive electrically conductive layer can be placed / coated between one or all suspended electrodes (105) and the suspended layer (102) or between one or all electrodes (105) and the supporting layer (104). The adhesive layer can be titanium, chromium, or any other conductive material. In other embodiment, a non-conductive double-sided adhesive layer can be applied between the suspended layer (102) and the frame (101), between the suspended layer (102) and the perforated layer (103) and between the perforated layer (103) and the supporting layer (104). The double-sided layer provides the better sealing between the referred elements, prevent any liquid leakage, while the chemical properties of the double-sided layer prevent the adverse effect on cell growth or on the response of cells during the cell culture process / experimentation while ensuring proper sealing between the different layers and parts.

[0086] In other embodiments, the frame (101), the suspended layer (102), the perforated layer (103), the supporting layer (104), and the integrated electrodes (105) are a single body manufactured using advanced techniques such as 3D printing.

[0087] Example 1:

[0088] Example 1 discloses some of the measurement modes that can be used for carrying out different types of measurements with the proposed invention, Fig. 7. This includes different types of measurements with two, three, four or number of electrodes (105), including 2, 3 and 4 measurements and multiple port measurement with plurality of the integrated electrodes. Measurements setup can be configured for impedance measurements, electrochemical measurements with cyclic voltammetry (CV), amperometry, and voltammetry, normal pulse voltammetry (NPV), differential pulse voltammetry (DPV), chronoamperometry (CA), chronopotentiometry (CP), square wave voltammetry (SWV), electrochemical impedance spectroscopy (EIS), 4 port TEER measurements as well as the multiport measurements that can combine all other methods for application in in vitro electrical measurements of 3D biological constructs and cell culture media using the invention disclosed.

[0089] Example 2:

[0090] Example 2 discloses the best mode for carrying out in vitro electrical measurements of 3D biological constructs using the invention disclosed in the preferred embodiment (Fig. 8). This mode neither limits other possible modes of performing measurements utilizing the invention nor surpasses them.

[0091] The test measurement was set up; the measurement included the use of the embodiment including: a collagen layer (201), an endothelial layer (202), a pair of stimulating electrodes (203) (one electrode (105) realized on the supporting layer (104) and one suspended electrode (105) realized on the suspended layer (102)), and pair of measuring electrodes (204) (another electrode (105) realized on the supporting layer (104) and another suspended electrode (105) realized on the suspended layer (102)). In one aspect, the device includes connection means for establishing electrical communication between the electrically conductive routes and an impedance analyser. In a preferred embodiment, the connection means include a connecting pin adapted to form the electrical contact with the contact pads (105b).

[0092] The 4-terminal impedance analysis consists of at least one pair of stimulating electrodes (203) which are diagonally opposite and at least one pair of measuring electrodes (204) which are also diagonally opposite.

[0093] In one preferred aspect, a sinusoidal signal is applied between the simulating electrodes (203), and the measuring electrodes (204) measure the impedance of the biological construct.

[0094] In one other aspect, the biological construct / tissue layer is grown between the layer (104) and all electrodes (105).

[0095] In one other aspect, the apparatus comprises a collagen coating (extracellular matrix mimicking layer) applied on the layer (104) covering the integrated electrodes (105) realized on supporting layer (104) on which cells adhere. Such adherence / growth of cells over the electrodes (105) realized on supporting layer (104) causes a change in the impedance between those electrodes which in turn produces a signal indicative of the changes in the adherence and growth of the cells on the extracellular matrix mimicking layer.

[0096] Fig.9 Illustrates impedance values obtained as a function of frequency carried-out for 4 selected cell culture wells before and after seeding HUVEC cells on top of a previously deposited collagen matrix.

[0097] Industrial Applicability

[0098] The invention is a measurement instrument, capable of improved and more efficient electrical measurements of in vitro culture of biological constructs as disclosed, with wide industrial applicability, including but not limited to pharmaceutical and biotech industries as the invention can be used for a variety of high throughput screening applications such as drug testing, toxicity testing of different chemicals or compounds on cells, disease modelling, phenotypic screening, cell culture growing and maintaining cells in vitro, stem cell culture, alternative protein, cultivated meat, tissue engineering, immunocytochemistry, and basic cell biology.

Claims

CLAIMS1. An apparatus with integrated electrodes for electrical monitoring of cell cultures characterized in that it comprises: a minimum one well (100) with integrated electrodes, a frame (101), a suspended layer (102), a perforated layer (103), a supporting layer (104) and a minimum two integrated electrodes (105) positioned in minimum two different planes, wherein at least one electrode of the at least two electrodes located in the well is suspended into the well from the wall of the well (100). wherein planes on which electrodes can be placed are the upper and lower surface of the suspended layer (102) and the top surface of the supporting layer (104) wherein the well (100) is a cavity constructed in the form of an open-top cylinder with a circular opening on its top part, the well (100) extends from its open-top part all the way to the upper surface of the supporting layer (104), intersecting the perforated layer (103), forming openings in the intersected parts, wherein the frame (101) is constructed to have an upper and lower surface, and forms an upper side of the apparatus, wherein the suspended layer (102) is, constructed to have an upper and lower surface, the suspended layer (102) is being positioned between the lower surface of the frame (101) and the upper surface of the perforated layer (103) and is connected with them, wherein the suspended layer (102) enters into the well (100) with its suspended part (106), wherein the suspended layer (102) is physical carrier for the integrated electrodes (105), wherein the perforated layer (103) is constructed to have an upper and a lower surface, the lower surface of the perforated layer (103) is connected to the upper surface of the supporting layer (104), wherein the perforated layer (103) is a physical barrier between the suspended layer (102) and the supporting layer ( 104), wherein the supporting layer (104), is constructed to have an upper and a lower surface, wherein the supporting layer (104) forms the bottom side of the apparatus, wherein the upper side of the supporting layer (104) is a physical carrier for the electrodes (105). wherein the integrated electrodes (105) comprise a routing pad (105a) and a connecting pad (105b),2. The apparatus of claim 1, wherein the suspended electrode (105) is situated on the lower surface, or upper surface, or the lateral surface of the suspended layer ( 102), or a combination thereof.

3. The apparatus of claim 1, wherein it further comprises a plurality of the suspended layers (102) and a plurality of perforated layers (103), each of the layers being stuck one on top of each other, wherein it further comprises a plurality of the suspended electrodes situated at least 2 different planes of the well (105).

4. The apparatus of claims 1 to 3, wherein at least one electrode of at least two electrodes (105) integrated into the well is constructed on the top surface of the supporting layer (104).

5. The apparatus of claims 1 to 4, wherein the position of the electrodes is selected from the group of positions comprising: the upper surface of the supporting layer (104), or upper surface of the suspended layer (102), or the lower surface of the suspended layer (102) or the combination of thereof.

6. The apparatus of claims 1 to 5, wherein at least one electrode (105) further comprises an additional layer, the additional layer being an insulating layer or biological coating or nanomaterial, or combination of thereof.

7. The apparatus of claim 6, wherein the biological coating is selected from the group consisting of: a polymer film, biomolecules, peptides or proteins extracellular matrix components.

8. The apparatus of claim 6, wherein the nanomaterial is selected from the group of nanomaterials including: graphene, MXenes, gold nanoparticles, magnetic nanoparticles, nanotubes or their combination or their combination with bioreceptor materials.

9. The apparatus of claims 1 to 6, wherein at least one electrode (105) is coated by a material selected from the group of biological bioreceptor materials consisting of: antibodies, aptamer, nucleic acid molecules, flagella, protein molecules and molecularly imprinted polymer (MIP).

10. The apparatus of any of the claims, wherein it further comprises a doublesided adhesive layer set: between the suspended layer (102) and the frame (101), and / or between the suspended layer (102) and the perforated layer (103), and / or between the perforated layer (103) and the supporting layer (104).

11. The apparatus of any of the claims, wherein the number of the wells (101) can be realized in the standard dimensions and format comprising a standard dimensions and formats for cell culture multi well dishes and cell culture plates, or any non-standard format and dimensions.

12. The apparatus of any of the claims, wherein the well ( 100), the frame (101). suspended layer (102), the perforated layer (103) and the supporting layer (104) are made of a non-conductive material.

13. The apparatus of any of the claims, wherein a non-conductive material is glass or biocompatible-polymer or PCB or silicon.

14. The apparatus of any of the claims, wherein the suspended layer is made of a conductive, biocompatible material to form the suspended electrode.

15. The apparatus of any of the claims, wherein the conductive, biocompatible material is selected for the electrodes from the group consisting of gold, platinum, silver, aluminium, conductive polymers, graphene, MXenes, carbon nanotubes.

16. The apparatus of any of the claims, wherein the frame (101), the suspended layer (102), the perforated layer (103), the supporting layer (104), and the electrodes (105) are a single body manufactured using 3D printing techniques.

17. The apparatus of any of the claims, wherein the electrodes (105) can be constructed in different shapes including circle, square, ellipse prism, oval, triangle, interdigitated or fractal, or combination of thereof.

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