Cell substrate for receiving biological cells, and method for use thereof
Patent Information
- Authority / Receiving Office
- EP · EP
- Patent Type
- Applications
- Current Assignee / Owner
- CHANPHARM GMBH
- Filing Date
- 2024-06-26
- Publication Date
- 2026-05-13
Smart Images
Figure EP2024067978_09012025_PF_FP_ABST
Abstract
Description
Cell substrate for containing biological cells and method for using it Field of the invention The invention relates to a cell substrate configured for receiving and preferably examining biological cells, particularly preferably for electrophysiological (electrical) measurements on the biological cells. Furthermore, the invention relates to methods for using the cell substrate, e.g., in the arrangement, cultivation, and / or examination of biological cells, in particular heart muscle cells (cardiomyocytes) or nerve cells (neurons). State of the art In this description, reference is made to the following prior art, which illustrates the technical background of the invention: [1] AST Smith et al. in "Nano Lett." 2020, 20, 3, pp. 1561-1570; [2] M. Kitsara et al. in "Microelectronic Engineering" 2019, 203-204, pp.44-62; [3] H. Savoji et al. in "Biomaterials" 2019, 198, pp. 3–26; [4] VJ Closing et al. in "J. Mater. Chem." B, 2016, 4, p. 3534; [5] A. Atmanli et al. in "J. Vis. Exp." (73), e50288, doi:10.3791 / 50288 (2013); [6] WO 2021 / 083885 Al; [7] TC McDevitt et al. in "J. Biomed. Mater. Res.", 2002, 60, 472-479; [8] G. Khademhosseini et al. in "Biomed. Microdevices", 2007, 9, 149-157; [9] Ma et al. in “Lab Chip” 2012, 12, 566;
[0010] WO 2014 / 074067 Al;
[0011] F. Navaee et al. Celis 2023, 12, 576;
[0012] JP Kucera et al. in "Circ Arrhythm Electrophysiol." 2017; 10:e004665;
[0013] Bartels et al. in "Six Rep. " 2021; 11:9269;
[0014] M. Hippier et al. in "You Adv. " 2020 Sep; 6(39): eabc2648.
[0015] K. Weißenbruch et al. in "Curr Opin Biotechnol. " 2022 Feb; 73:290–299;
[0016] Q. Akolawala et al. in "ACS Appl. Mater. Interfaces" 2022, 14, 18, 20778-20789;
[0017] SH Yagoub et al. in "Journal of Assisted Reproduction and Genetics" Vol. 39, pp. 1503-1513 (2022); and
[0018] DE 102008047399 Al. It is well known that the provision of differentiated cells, such as cardiac cells or neurons, through differentiation of human pluripotent stem cells (hPSCs) enables the development of tissue models, such as cardiac muscle- or nerve-like tissues. Tissue models can be applied in drug development to study the effects of drugs on tissue ("tissue-on-a-chip" technologies). For example, high-throughput methods can be used to perform electrophysiological measurements and / or optical measurements and / or measurements of cardiac cell contractions on cardiac cells (see, for example, [1] to [3]). There is interest in studying differentiated cells in a state that is as similar as possible to the cell conditions in a natural tissue environment. For example, the development of "heart-on-a-chip" technologies may involve growing heart cells on anisotropic substrate structures to mimic a fibrous structure in the myocardium (see, e.g., [1], [2]). Drug testing on laminar, fibrous heart tissue structures on groove structures or on directed nerve fibers in grooves has provided initial progress in drug testing (see, e.g., [2], [4], or
[0011] ). For example, cardiac cell imaging has been achieved on a cell substrate with a plurality of surface grooves (hereinafter also referred to as cell imaging segments), in which a fibrous arrangement and alignment of the cardiac cells is created (see, e.g., [1], [4], and [5]), with adjacent grooves separated by partitions. Surface grooves are formed, for example, using printing processes with a height in the range of 1 pm to 100 pm, a width in the range of 1 pm to 100 pm, and a length of 1 mm or more. The partitions are made, for example, of a polymer such as PDMS or a hydrogel. Alternatively, grooves are created by etching depressions into a substrate surface, microinjection molding, or lithographic techniques (see, e.g., [1], [2]), or by microcontact printing or using microfluidic structures. For the alignment of cardiac cells to form cardiac muscle fibers in vitro, cell adhesion proteins (e.g., fibronectin, laminin, or collagen) can also be used. These proteins are applied, for example, to polystyrene cell culture dishes to dictate the orientation of the cardiomyocytes ([7], [8]). It is also known to use substrates with integrated electrodes for electrophysiological studies. For example, [6] describes a substrate in which point-shaped nanoelectrodes are arranged at the bottom of cell-receiving segments, which penetrate biological cells on the substrate when they are recorded. The nanoelectrodes can be disadvantageous if they require an additional actuator on the substrate for penetration of the cell membrane. Another disadvantage can be that the nanoelectrodes are designed for the intracellular recording of membrane potentials and not for the measurement of surface potentials. However, there is considerable interest in measuring surface potentials, as the temporal length of field potentials and the shape of measured T waves (such as the determination of the prolongation of the ECG parameter QT interval) on fiber surfaces are highly informative regarding drug effects and are similar to an ECG measurement. Surface potential measurements are possible using flat electrodes arranged at the bottom of the surface grooves of a cell substrate. Cell substrates with flat electrodes can be manufactured by forming an anisotropic substrate structure, for example with surface grooves, on a so-called micro-electrode array (MEA) substrate (or MEA chip) (see, e.g., [9]). The MEAs are exposed at the bottom of the surface grooves. By positioning multiple electrodes at the bottom of a groove (see, e.g., [9]), the propagation of excitation along a cell fiber, e.g., a myocardial or nerve fiber, can be determined. This advantageously makes the use of fluorescent probe molecules for measuring action potentials obsolete. Since the use of fluorescent probes can produce cytotoxic degradation products and these fluorescent probes can also influence the effects of drugs, the direct determination of excitation propagation is particularly useful for drug research. However, a disadvantage of known MEAs is that heart muscle cells growing as a monolayer (cell layer), which are connected to each other via cell-cell contacts (nexus contacts), detach from the substrate after a relatively short time. Cell layers are initially connected to the substrate via distributed attachment points. The cells expand during the rhythmically occurring The resulting contractions move the cell layer in various spatial directions. However, forces acting in different spatial directions quickly lead to the detachment of substrate. This leads to an undesirable shortening of the time available for investigations. A further disadvantage of known cell substrates with a groove structure on an MEA substrate is the inadequate mutual separation (separation, isolation) of individual fibers in adjacent grooves. Individual fibers in adjacent grooves can inadvertently touch each other at different (undefined) positions and spontaneously form cell-cell contacts when cells adhere to the upper side of the partition between the grooves and grow into the adjacent groove. Cells are located on the upper side of the partition, for example, as a result of the deposition of cells 1' on the cell substrate 100' and / or due to cell growth or cell migration across the partition 22' (see Figure 11, prior art).Partition walls and associated partition wall tops of conventional cell substrates with grooves 21' have wall thicknesses in the range of 10 pm to 30 pm in known cell substrates to achieve sufficient stability, so that adhesion of cells with a diameter of 10 pm is favored. However, unintentional, irregular contact between individual fibers from neighboring grooves can distort electrophysiological measurements. Cells on wall structures, for example, 5 pm to 100 pm high, can establish electrical contacts between neighboring fibers. Excitations from individual fibers (e.g., spontaneous or through electrical stimulation) are then spontaneously and undefinedly conducted to neighboring fibers, thus precluding or at least distorting a measurement of excitation conduction along a defined section of a single fiber with the surface electrodes belonging to a groove. Furthermore, the irregular overgrowth of wall structures over several grooves creates tissue segments which, due to their size, increase the undesirable force effects during contraction movements in different spatial directions, so that the unintentional detachment from the substrate is further accelerated. Finally, the spontaneous and uncontrolled adhesion of cells to the wall surfaces and the transmission of excitation processes ("crosstalk") to neighboring fibers also prevent the reliable subdivision of cell culture surfaces into separate, well-defined sections. Practical studies have shown that cell-repelling nanostructures on the partition walls or a coating with strong hydrophobicity can reduce cell growth on the partition wall surface, but cannot prevent spontaneous overgrowth of the walls. For example, the structures described in [6] (see, for example, Figure 12, cited from [6]) still offer sufficient surface area for the adherent attachment of cells and the overgrowth of the partition walls 22'. Overgrowth of the partition wall surface can be promoted, among other things, by the cells deposited on the flat upper surface synthesizing extracellular matrix proteins, which then enable cell adhesion even on cell-repellent surfaces. The above-mentioned disadvantages caused by adherent cell attachment and overgrowth of the septa not only affect electrophysiological studies. Other applications of the cell substrate, from pure cell cultivation to other, such as optical or chemical, investigations, can also be compromised by cells, for example, on the surface of cell walls. In the practical application of conventional cell substrates with surface grooves, a further disadvantage was found that filling the grooves can be difficult if cells in a suspension do not reach the bottom of the grooves and the associated electrodes, but remain stuck on the intermediate walls of the grooves. Object of the invention The object of the invention is to provide an improved cell substrate for holding biological cells that is suitable for avoiding disadvantages of conventional techniques. In particular, the object of the invention is to provide a cell substrate that separates cells in different cell holding segments with increased reliability and / or reproducibility, better suppresses or excludes uncontrolled mutual interaction between cells in different cell holding segments, extends the duration and / or adhesion of the biological cells to the cell substrate, improves the reliability and meaningfulness of studies on the cells on the cell substrate, and / or enables new applications of MEA substrates. The object of the invention is also to provide improved applications of the cell substrate for holding biological cells. Summary of the invention These objects are achieved by a cell substrate for receiving biological cells having the features of claim 1. Preferred embodiments and applications of the invention emerge from the dependent claims. According to a first general aspect of the invention, the above object is achieved by a cell substrate configured to receive biological cells and comprising a substrate body with a structured substrate surface. The structured substrate surface has a plurality of cell-receiving segments arranged side by side, each with a longitudinal extension. Adjacent cell-receiving segments are each separated by a wall region protruding from the substrate body and delimited by segment wall surfaces. The wall regions between the adjacent cell-receiving segments each have a wall profile (cross-sectional shape) perpendicular to the longitudinal extension of the adjacent cell-receiving segments. According to the invention, the wall profile of the wall regions between the adjacent cell-receiving segments each has a wall tip protruding from the substrate body, which suppresses cell arrangement on the wall regions. Along the length of the wall regions, the respective wall profile with the wall tip can preferably form one, particularly preferably a single, wall apex line (or: wall edge), which is shaped as a barrier against cell deposition on the wall region and / or against cell growth or cell migration between adjacent cell-receiving segments. The wall edge can suppress cell arrangement on the wall region. Wall profiles of the wall regions can be shaped such that the wall regions adjacent to a cell-receiving segment form a funnel-shaped inlet into the cell-receiving segment (funnel slot).Advantageously, this can support the collection and / or concentration of cell material at the bottom of cell uptake segments, particularly during centrifugation of a cell suspension on an MEA substrate with cell uptake segments. According to a second general aspect of the invention, the above object is achieved by a method for using the cell substrate according to the first general aspect of the invention or one of its embodiments, wherein at least one of the following steps is provided: cultivation of biological cells in the cell receiving segments of the cell substrate, examination of the biological cells, in particular measurement of electrophysiological Potentials at biological cells in the cell-receiving segments of the cell substrate, determining an excitation propagation velocity at cells, e.g., fibers or cell processes (in particular axons and / or dendrites) along at least one of the cell-receiving segments of the cell substrate by detecting a time-delayed arrival of an electrical excitation at electrodes at the bottom of the cell-receiving segment, in particular without a fluorescence measurement, and determining an excitation propagation velocity through cells along at least one of the cell-receiving segments of the cell substrate, in particular with simultaneous fluorescence measurement for the transmission of an action potential with a potential-sensitive fluorescence probe and the intracellular calcium release with a calcium-sensitive fluorescence probe, cell contraction with video microscopy, optical measurement of a contraction of cardiac muscle cells by analyzing the shortening of cell segments,Measurements of intracellular calcium concentrations, electrical stimulation of biological cells in the cell-receiving segments of the cell substrate, biochemical stimulation of biological cells in the cell-receiving segments of the cell substrate, and operation of the cell substrate as a generator device configured to generate electrical current by membrane potential derivation at the biological cells in the cell-receiving segments of the cell substrate (see
[0018] ). The electrophysiological determination of the excitation propagation velocity can advantageously be used to determine the nexus resistance of cell-cell contacts, particularly without a fluorescence measurement. The cell substrate is generally formed by the substrate body with the structured substrate surface. The substrate body can be plate-shaped, preferably flat and / or solid (rigid). The substrate body generally comprises a bottom side, preferably formed by a substrate plate, such as an MEA substrate and / or a substrate with integrated microfluidics for supplying cells on the cell substrate, and a top side on which the structured substrate surface is exposed for receiving the cells. The substrate body and the structured substrate surface can be formed in one piece or assembled (in particular multilayered) and / or made of plastic, a glass, a semiconductor, and / or a ceramic.The substrate body and / or the structured substrate surface may be formed from a transparent material, which advantageously simplifies microscopic observation and / or examination of cells on the cell substrate. The cell receiving segments are formed by the shape of the structured substrate surface. Each cell receiving segment is generally a recess, in particular a depression in the substrate surface and / or a receptacle formed by protruding wall regions. which has the longitudinal extension. The longitudinal extension of a cell-accommodating segment refers to a geometric shape with which the cell-accommodating segment extends primarily in one direction (longitudinal direction of the cell-accommodating segment, length), while the extension of the cell-accommodating segment along the substrate surface perpendicular to the longitudinal direction (lateral extension of the cell-accommodating segment, width) is smaller than the length. For example, the width of the cell-accommodating segments can be equal to or less than 1 / 5, in particular 1 / 10, of the length of the cell-accommodating segments. Preferably, the cell-receiving segments can have a straight shape entirely or in sections, i.e., at least one of the cell-receiving segments can partially or particularly preferably form a straight, linear receptacle (also referred to as a groove, channel, or groove) along its entire length. Alternatively, at least one of the cell-receiving segments can be curved (curved) at least in sections. In the case of the continuously straight cell-receiving segment, the longitudinal extent can be formed by a straight groove shape, and in the case of a partially or continuously curved cell-receiving segment, the longitudinal extent can be formed by a locally curved groove shape. The regions of the structured substrate surface that separate immediately adjacent cell-receiving segments from one another are referred to as wall regions. In the case of a curved cell-receiving segment, wall regions are alternatively or additionally provided between different, preferably straight, segment sections of the cell-receiving segment. A wall region is generally a projection extending from the substrate body, having a longitudinal extension, and comprising adjacent cell-receiving segments (or various adjacent segment sections of curved cell-receiving segments) on both sides and bounded by the segment wall surfaces. A wall region may have a thickness (extension perpendicular to the longitudinal direction of the bounding cell-receiving segments) that may be less than, equal to, or greater than the width of the adjacent wall regions and / or less than, equal to, or greater than the depth of the bounding wall regions. The wall regions may be configured for a continuous, continuous separation of the adjacent cell-receiving segments along their longitudinal extension.The term "separation" of the adjacent cell receiving segments optionally also includes a demarcation formed in sections along the longitudinal extent of the cell receiving segments, which may have through openings (see below). In general, each wall region between adjacent cell-receiving segments (or segment sections of curved cell-receiving segments) forms a wall (segment wall) between the cell-receiving segments (or their segment sections). The cross-sectional shape of a wall region transverse to the longitudinal direction of the delimiting cell-receiving segments forms the wall profile of the wall region. Advantageously, at least one of the wall regions, preferably all wall regions, can have a constant wall profile along the respective longitudinal extent of the wall region over its entire length. Alternatively, the wall profile of a wall region and a surface structure of the wall can be variable along its longitudinal extent, for example, forming a narrower or wider receptacle at the longitudinal ends of the delimiting cell-receiving segments than in a central part of the cell-receiving segments.Alternatively or additionally, all wall profiles of all wall areas can have the same shape or different shapes. The wall profile can be formed by sloping, tapered or parallel side surfaces (segment wall surfaces) on the sides of the wall region. The wall profile can preferably have a triangular shape with the wall tip along the entire length of the respective wall region. The triangular shape, containing the protruding wall tip, refers to a shape of the wall profile that can be described by three points. The three points comprise a first point at an end of the wall region facing away from the cell substrate, where the wall tip is formed, and two (base) points on the lateral surfaces of the wall region in the depth of the delimiting cell-receiving segments. The wall profile can have a straight outline or a curved outline between the said points, i.e. the lateral surfaces of the wall regions (segment walls) can form a straight or curved, in particular concave, line.Alternatively or additionally, the wall profile can be formed by a vertical rib (also referred to as a partition or lamella) protruding from the substrate body. The inventor has found that the wall tips and / or vertical ribs can be manufactured with high stability, so that they can have a significantly thinner thickness than conventional partition walls, e.g., in the range of 0.5 μm to 20 μm. At the wall tip (if necessary at the free end of a vertical rib, see below), an extremely small radius of curvature equal to or less than 10 pm, in particular equal to or less than 5 pm, e.g. 1 pm or less, up to 100 nm, can be set, thereby preventing cell overgrowth between adjacent cell uptake segments. This effect can alternatively also with a larger radius of curvature, e.g. in the range from 10 pm to 15 pm or from 10 pm to 20 pm or above. Along the length of a respective wall area, the wall tip of the wall profile forms a wall apex line that is at least partially continuous and which is preferably exposed and / or, as explained below, can be formed by a vertical rib. The inventors have found that by designing the wall regions with the wall profile that forms the wall apex line, a cell barrier is advantageously created between delimiting cell-accommodating segments. The surface of the wall region has a shape that negligibly impedes or completely prevents cell deposition upon cell deposition on the cell substrate and / or negligibly impedes or completely eliminates cell growth or migration from one side of the wall region across the top of the wall region to the other side of the wall region.Cell deposition, cell growth and / or cell migration are inhibited to a negligible extent if remaining cells or cell components on the upper side of the wall regions do not affect the application of the cell substrate, in particular cultivation of the cells and / or examination of the cells in the cell uptake segments. A particular advantage of the invention is that the inventive shape of the wall regions creates a compartmentalization of the structured substrate surface of the cell substrate, i.e. each cell receiving segment forms an elongated vessel (compartment) for the received cells, separate from other cell receiving segments. Advantageously, the invention creates a subdivision of the structured substrate surface into individual elongated cell receptacles, which can be open at the top and are separated from one another without the individual cell receptacles having to be separated from one another by lids. A further advantage of the compartmentalization is that the yield when using cells for cell deposition on the substrate is improved, for example because all cells received on the cell substrate contribute to fiber formation. The above-described disadvantages of conventional cell substrates with surface grooves for the absorption and cultivation of biological cells, in particular heart and / or nerve cells, are eliminated by the wall areas provided according to the invention. When applying or depositing (in particular seeding) a cell suspension, cell adhesion to the upper surface of the wall areas can be prevented. At the same time, inclined surfaces adapted to a cell absorption The side walls adjacent to the segment form an elongated funnel slot, which facilitates the filling of the cell receiving segments, since the cells in the suspension inevitably collect at the bottom of the cell receiving segments, e.g., under the influence of gravity and / or centrifugal force, and are guided to the bottom by the segment wall surfaces of the wall regions. The inventive shape of the structured substrate surface thus not only provides separation of the biological cells in the cell receiving segments, but also aids in filling the cell substrate. Biological cells can be arranged more uniformly and with increased reliability in the cell receiving segments. Advantageously, the invention provides microcompartmentalization of the substrate surface, which limits the amplitude of the contractions to one direction (the longitudinal direction of the cell-receiving segments). This can substantially reduce the risk of premature cell detachment due to contractions by reducing lateral contractions and / or stretching. Longitudinal adhesion in the cell-receiving segments extends the duration of cell adhesion to the surface, with lateral contacts on the walls of the cell-receiving segments providing additional stabilizing effects. According to a preferred embodiment of the invention, the segment wall surfaces of at least one of the wall regions, preferably all of the wall regions, can enclose an acute angle distally at the wall tip between adjacent cell-receiving segments or can run parallel. The acute angle is an angle of less than 90°, preferably less than 80°. The smaller the angle at the wall tip, e.g., up to 0°, the more effective the separation of the adjacent cell-receiving segments advantageously is. Both segment wall surfaces of a wall region can be inclined relative to the substrate surface. In particular, when they enclose the acute angle, the segment wall surfaces can run obliquely relative to one another and / or relative to the extent of the substrate body (oblique segment wall surfaces).Alternatively, one of the segment wall surfaces of a wall region may be inclined relative to the substrate surface, while the other segment wall surface of the wall region is perpendicular relative to the substrate surface. According to a further alternative, both segment wall surfaces of a wall region may be perpendicular relative to the substrate surface, i.e., the wall region may form the vertical rib (see below). According to a further preferred embodiment of the invention, the segment wall surfaces can have a low roughness such that structures (protrusions and / or depressions) on the segment wall surfaces have dimensions < 100 nm, e.g. < 50 nm, in particular < 10 nm. Irregular, rugged surfaces on the pm or nm scale, and thus increased cell adhesion, can thus be advantageously suppressed or avoided. Advantageously, the segment wall surfaces and / or the wall tip along the length of the wall regions may be free of local structures, such as local tips, which could in particular form a rough topology. According to a further preferred embodiment of the invention, at least one, particularly preferably each, of the wall regions can have a vertical rib (lamella) protruding from the substrate body, which forms the wall tip in the wall profile of the wall region. The shape of the wall profile can be selected such that the wall tip is formed by the vertical rib (in particular a protruding lamella). Preferably, the vertical rib can extend over the entire height of the wall region, i.e. form an ultra-thin wall with the parallel segment wall surfaces. Alternatively, the separation of adjacent cell-receiving segments (or segment sections) can be formed by a vertical rib arranged on a base region of a wall region that is triangular in profile. Preferably, the thickness of the vertical rib can be selected in the range of 1 μm to 10 μm, for example, 1 μm to 5 μm. Alternatively, the thickness of the vertical rib can be less than 1 μm. The provision of the vertical rib has the advantage of improving the separation of the adjacent cell-receiving segments or sections of a curved cell-receiving segment. In particular, the vertical rib represents an insurmountable barrier to cell growth or cell migration from the cell-receiving segments. Additional advantages of the cell substrate may result if, according to a further embodiment of the invention, the segment wall surfaces adjacent to at least one of the cell receiving segments have lateral projections facing each other and forming a segment slot extending along the longitudinal extent of the cell receiving segment. The lateral projections can advantageously be formed such that the wall profile of the wall areas has an arrow shape, the distal end of which forms the wall tip, optionally with the vertical rib. The cross-sectional shape of a wall area can in this case be characterized by a Assemble a triangle with the top of the wall and a rectangle in the lower part of the wall area. Unlike in [6] (see Figure 11), the lateral projections are not formed by T-profiles, but rather by inclined segment wall surfaces of the wall regions that taper upwards. Cell adhesion is also prevented by a tapered top surface of the wall regions between the cell-receiving segments. Advantageously, the segment slit can fulfill several functions. First, it further improves the separation between adjacent cell-receiving segments. By forming the segment slit with the lateral protrusions on the segment wall surfaces, the compartmentalization of the structured substrate surface can be improved. Cells are retained in the cell-receiving segments more reliably and for longer periods of time. Second, the segment slit allows optical observation of cells in the cell-receiving segment from the top of the cell substrate (or illumination for microscopic procedures) while simultaneously retaining the cells in the cell-receiving segment. The segment slot can preferably have a smaller width than the width of the cell receiving segment (groove) at its base, i.e. on the substrate body. The segment slot can particularly preferably have a width equal to or less than 20 pm, in particular equal to or less than 5 pm. With these dimensions, an inclusion (trapping) of the cells, in particular cell fibers, in the cell receiving segments is advantageously achieved. The segment slot can in particular be dimensioned such that cells can be forced through the segment slot during their deposition on the cell substrate, for example under the effect of gravity or centrifugal forces, whereas in the adherent state in the cell receiving segment they cannot pass through the segment slot again without the effect of the forces occurring during deposition. This promotes fixation of the cells in the cell receiving segment.At the same time, culture medium can be supplied to the cells in the cell receiving segment through the segment slot. The lateral projections on the segment wall surfaces can preferably be dimensioned such that the segment slot has a width that is smaller than the diameter of the cells to be accommodated by the cell substrate. For example, the segment slot for accommodating cells with a diameter of 10 μm can have a width <10 μm, for example, 8 μm or less. According to a further preferred embodiment of the invention, at least one, particularly preferably each, of the wall regions can have at least one through-opening between adjacent cell-receiving segments. The at least one through-opening forms an opening at a predetermined position along the longitudinal extent of a cell-receiving segment. The at least one through-opening is preferably oriented transversely to the longitudinal extent of the adjacent cell-receiving segments. Adjacent to the at least one through-opening, the associated cell-receiving segments are separated from one another along their longitudinal extent. The at least one through-opening can extend over the entire height of the wall region perpendicular to the substrate body or form an opening exclusively in the base region of the wall region.Advantageously, H-shaped connections can be created between cell-receiving segments, providing lateral cross-linking of cell fibers (formation of tissue structures from individual fibers with lateral contacts). The at least one through-opening is preferably dimensioned such that connections of biological cells can form between the cell-receiving segments connected via the through-opening. The at least one through-opening can, for example, be at least as wide and as high as the cross-sectional shape of the cell-receiving segments. The wall regions can have through-openings at several predetermined positions, i.e., they can be interrupted, allowing targeted transmission of action potentials to neighboring fiber structures. This can be advantageous for simulating excitation processes across flat tissue structures consisting of individual fibers contacting each other at these positions. A further advantage of the defined through-openings is the possibility of investigating complex excitation processes in these tissue structures using the coordinates of the transmission of action potentials to neighboring fibers. The through-openings in the wall regions, which also delimit individual fiber segments from one another, also allow axons and dendrites of nerve cells to grow through them. According to a further advantageous embodiment of the invention, at least one, particularly preferably each, of the wall regions can have a corrugated wall surface on at least one side. A lateral structure of the side surfaces of the wall regions can improve cell adhesion in the cell-receiving segments and extend the residence time of the cells. Advantageously, the cell substrate offers the possibility of examining the cells recorded in the cell recording segments, whereby a variety of different examination methods can be implemented individually or in combination. Preferably, an optical examination, for example using a microscope, in particular a fluorescence measurement, an examination to detect contraction or other movements of the cells, a chemical examination, and / or an electrophysiological examination (detection of electrophysiological characteristics, in particular electrophysiological potentials, of the biological cells) can be provided. If the cell substrate is configured to detect electrophysiological characteristics of the biological cells, advantageously at least one of the cell receiving segments can have an electrode arrangement with at least one electrode. Preferably, at least two electrodes can be provided, which are arranged spaced apart from one another along the longitudinal extent of the cell receiving segment. Advantageously, the at least one electrode can be arranged on the side of the cell receiving segments facing the substrate body, in particular on their underside (bottom). Particularly preferably, more than two electrodes, for example three, four, five or more, can be arranged in each cell receiving segment, distributed along its longitudinal extent.Depending on the dimensions, at least 10, in particular at least 100, or even more than 1000 electrodes can be arranged in each cell receiving segment, distributed along its longitudinal extent. Alternatively, a single electrode can be arranged in each cell receiving segment. In this case, the cells are stimulated by applying a potential to an electrode in the cell receiving segment, with the potential of a reference electrode (see, for example, Figure 2) serving as the reference potential. In addition to the (measuring) electrodes mentioned, at least one reference electrode is provided on the cell substrate, which provides a reference potential, e.g., ground potential, for potential measurements on the cells. According to one embodiment of the invention, a cell receiving segment of a cell substrate may in particular comprise a first electrode at a first end of the cell receiving segment, a second electrode between the first end and a second end of the cell receiving segment, and a third electrode at the second end of the cell receiving segment. With the first Electrical stimulation of biological cells in the cell recording segment can be performed using the first electrode, after which the speed of excitation conduction through the fiber is recorded by recording field potentials with the second and third electrodes. Advantageously, the electrode arrangement can be implemented with different electrode types. The electrodes can, in particular, comprise tip-shaped electrodes configured to penetrate cell membranes, which can also be referred to as protruding electrode tips, tip electrodes, nano- or microelectrodes, and / or flat surface electrodes configured to derive field potentials at cell surfaces. To provide the electrode array of surface electrodes, an MEA substrate with an array (preferably a regular array, e.g., a matrix array with straight rows and columns) of surface electrodes, each with an electrode lead, can advantageously be used, wherein the structured substrate surface is formed on the surface of the MEA substrate. The structured substrate surface is shaped to match the array of surface electrodes such that the wall regions are formed between the electrode rows and the surface electrodes of the electrode rows are exposed at the bottoms of the compartments (cell receiving segments). Advantageously, the surface electrodes of MEAs can be used at the bottom of the compartments to measure the longitudinal excitation propagation. Advantageously, the electrode arrangement can comprise a CMOS MEA. A CMOS MEA comprises an MES in which the electrodes are formed as CMOS semiconductors. CMOS MEAs are manufactured, for example, by Multi Channel Systems MCS GmbH (Reutlingen, Germany). CMOS MEAs can have, for example, 4,000 or more electrodes on an area of one square millimeter. A cell substrate with a CMOS MEA can be manufactured, for example, by building up the wall regions on the surface of the CMOS MEA, e.g., by printing them. According to a particularly advantageous embodiment of the invention, the electrode arrangement can be coupled to a measuring and evaluation device configured to characterize cell contacts between the cells as a function of electrophysiological potentials detected by the electrodes. Advantageously, the measuring and evaluation device offers the possibility of deriving nexus contacts directly from measurement signals of the electrode arrangement of the cell substrate. Preferably, the measuring and evaluation device contains an amplifier for electrically amplifying the potentials derived at the electrodes. Advantageously, the assignment of one or more electrodes at the bottom of a cell recording segment enables a significantly more effective use of, for example, stem cell-derived nerve or muscle cells. For example, in a cell recording segment with a length of 1 mm and a width of 10 pm, only 5 to 100 cells with lengths of 200 pm (for spindle-shaped cells) or diameters of 10 pm (for round suspension cells) form a micromyocardial fiber, whose individual cells are connected by nexus contacts. Independent measurements can be performed on this fiber in the cell recording segment using optical methods (fluorescence probes) and / or electrophysiological measurements. With a seeding of approximately 50,000 cells on a conventional MEA, this means a savings of approximately 1000 times in cell material per measurement (per data point). According to further preferred embodiments of the invention, at least one of the cell-accommodating segments can have at least one branch and / or a varying width along its longitudinal extent. Advantageously, these variants can be used to simulate further in vivo environmental conditions of cells or, in particular, cell tissue, e.g., by branching or step-like widenings or narrowings of the compartments. At a step-like widening or narrowing, the width of the cell-accommodating segment can change, for example, by a factor in the range of 2 to 10. For example, anatomical structures of myocardial tissue can be reproduced (modeled). Fiber branching can, for example, be the starting point of arrhythmias, so that a cell substrate with a branched cell-accommodating segment is particularly suitable for testing antiarrhythmic drugs (see, for example,
[0012] ). A branch comprises a region of the structured substrate surface in which a segment portion of a cell-receiving segment divides into at least two segment portions and / or two segment portions merge into one segment portion. The branch can, for example, comprise at least one of a Y-shaped branch, a division forming right angles between the segment portions, and a division into curved segment portions. A branch can also occur via a lateral through-opening of the wall region (H-shaped). Optionally, at least one of the cell-receiving segments can be provided with an end wall at each of its longitudinal ends. This advantageously forms the cell-receiving segment as a compartment bounded on all sides along the lateral extent of the cell substrate. Each cell-receiving segment with end walls forms a defined vessel. According to a further variant of the invention, at least two adjacent segment sections of at least one of the cell-receiving segments can be connected via a curved segment. This embodiment advantageously makes it possible to form cell-receiving segments with a length greater than the extent of the cell substrate and / or with complex shapes. A cell-receiving segment can, for example, have at least two straight segment sections connected via at least one curved curved section, so that a cell-receiving segment can generally have a C-, S-, or meander shape. The cell-receiving segments are arranged next to one another in the substrate surface. The segment sections of a cell-receiving segment can preferably be arranged such that their longitudinal extent (longitudinal direction) runs parallel to one another. The cell substrate according to the invention is configured to accommodate the biological cells in the cell-accommodating segments. The biological cells can generally comprise plant or animal cells, preferably cells from mammals, in particular from humans, particularly preferably differentiated cells formed by differentiation of human pluripotent stem cells. The biological cells can have any shape and can be, for example, round or elongated. It is also possible to deposit contiguous cell groups (cell aggregates of two or more cells) on the cell substrate. When suspended in the culture medium, the biological cells can have typical cross-sectional dimensions in the range of 5 μm to 80 μm, in particular 10 μm to 30 μm.On the cell substrate, the cells can be arranged individually or preferably as aggregates, in particular fibers, and / or, in particular when using branched cell uptake segments, form a tissue. The cell substrate according to the invention is preferably also configured for the cultivation (in particular growth and / or differentiation) of the biological cells in the cell receiving segments. For this purpose, a supply of cultivation and / or differentiation media can be provided from the top side of the cell substrate and / or through microfluidic lines in the substrate body. Particularly advantageous for the supply of cultivation and / or differentiation media from the top side of the cell substrate is the provision of an enclosure of the area with the cell receiving segments, e.g., by an annular wall, in particular made of The enclosure can be made of glass or plastic. The enclosure can, for example, have an inner diameter of 10 mm to 15 mm and a height of 3 mm to 15 mm and be configured for the supply of a cell suspension, a culture medium, and / or test solutions from the top of the cell substrate. To accommodate the cells, the cell-accommodating segments can preferably have a width (distance between segment wall surfaces of adjacent wall areas facing the cell-accommodating segment or, if applicable, the width of the funnel slit) in the range of equal to or greater than 2 pm, in particular equal to or greater than 5 pm, and / or equal to or less than 100 pm, in particular equal to or less than 30 pm. These ranges are advantageous because smaller widths may not provide sufficient space for cell growth and a supply of cultivation and / or differentiation medium, and larger widths may not achieve sufficient restriction to fiber growth. Particularly narrow cell-accommodating segments are preferably used to accommodate skeletal muscle fibers. The cell receiving segments can preferably have a depth perpendicular to the extent of the cell substrate (wall height of the wall region) in the range of equal to or greater than 5 pm, in particular equal to or greater than 30 pm, and / or equal to or less than 500 pm, in particular equal to or less than 300 pm. These ranges are advantageous because, for smaller depths, there may not be sufficient space for cell growth and a supply of cultivation and / or differentiation medium, and for depths of width, observation of the cells, e.g., with a microscope, may be hindered. The length of the cell-accommodating segments can be selected in the range of equal to or greater than 300 pm, in particular equal to or greater than 500 pm, and / or equal to or less than 3000 pm, in particular equal to or less than 1000 pm. These ranges are advantageous because for shorter lengths, fiber growth may be too limited, and for longer lengths, fiber growth exceeds the lengths of cell fibers in a natural environment. Preferably, the cell receiving segments may have an aspect ratio formed by a quotient of a lateral width at the bottom of the cell receiving segments and a depth of the cell receiving segments and selected in the range 2 to 10. Features disclosed in connection with the cell substrate or its embodiments also represent preferred features of the methods according to the invention for use of the cell substrate or its embodiments. The aforementioned aspects and inventive and preferred features, in particular with regard to the structure of the cell substrate as well as the dimensions and compositions of individual components described with reference to the cell substrate, also apply to the methods for its application. The preferred embodiments, variants, and features of the invention described here can be combined with one another. Brief description of the drawings Further details and advantages of the invention are described below with reference to the accompanying drawings, which show schematically: Figure 1: schematic sectional views of cell substrates (partial representations) with features of preferred embodiments of the invention; Figures 2 and 3: schematic top views of an MEA substrate with integrated surface electrodes; Figure 4: further sectional views of cell substrates (partial representations) with features of further embodiments of the invention; Figure 5: a top view of a cell substrate with different shapes of cell receiving segments; Figure 6: a top view of a cell substrate with an illustration of the detection of cell contacts by an electrophysiological measurement; Figure 7: a schematic sectional view of a cell receiving segment with a surface electrode; Figure 8: a schematic sectional view of a cell receiving segment with a tip electrode; Figure 9: a schematic illustration of the uptake of cells in a cell uptake segment; Figure 10: another illustration of the uptake of cells in cell uptake segments; and Figures 11 and 12: Illustrations of conventional cell substrates (state of the art). Preferred embodiments of the invention Features of preferred embodiments of the invention are described below, in particular with reference to the shape of cell receiving segments (or their segment sections) and wall regions between them, and the design of an electrode arrangement. Details of cell substrates or their application, such as details of the seeding of cells on the cell substrate, the cultivation or differentiation of cells, and / or the evaluation of electrophysiological measurement signals, are not illustrated or described, insofar as they are known per se from the prior art. The invention is not limited to a specific embodiment of the cell substrate, but can be varied, e.g. with regard to the size and shape of the cell receiving segments (or their segment sections) and wall regions between them, or the number, arrangement, and / or design of the electrodes, or the number, arrangement, and / or design of through-openings.The application of the invention is not limited to the measurements described with reference to Figures 6 to 8. The invention can also be applied to other electrophysiological measurements or to other tasks without an electrophysiological measurement, such as spectroscopic examinations. For electrophysiological measurements, reference electrodes are provided, which are shown by way of example in Figures 7 and 8. The production of cell substrates according to the invention can be carried out using available structuring techniques for constructing the structured substrate surface (bottom up, e.g., with laser-based rapid prototyping) and / or for forming the structured substrate surface by material removal (top down, e.g., with lithographic etching, e.g., UV lithography). Preferably, for rapid prototyping, in particular for producing the substrate surface, 2-photon polymerization (2-photon polymerization printing) with laser excitation can be used (see
[0013] to
[0017] ), which has advantages for forming the cell receiving segments (or their segment sections) and wall regions with high accuracy and spatial resolution and, optionally, for forming undercuts, e.g., when providing the funnel slots. Surfaces of the cell substrate can be coated with substances such as Matrigel, fibronectin, laminin or collagen to promote local cell adhesion and growth. The above-described disadvantages of conventional cell substrates for the cultivation of myocardial or nerve fibers in particular are overcome by the wall sections shown in the figures, which taper upwards and / or are formed by vertical ribs. When a cell suspension is seeded, this prevents adhesion to the upper surface of the wall sections. At the same time, the inclined segment wall surfaces of the wall sections form an elongated funnel-shaped slot, which facilitates the filling of the cell receiving segments, as the cells inevitably collect at the bottom of the cell receiving segments due to gravity. This process can be assisted by centrifugation. Figure 1 schematically illustrates embodiments of cell substrates 100 (partial representations) in schematic sectional views, wherein a substrate body 10 and a structured substrate surface 20 are each shown with two or three cell-receiving segments 21 separated by wall regions 22 protruding from the substrate body 10. The sectional views show cross sections of the cell-receiving segments 21 perpendicular to their longitudinal extent. The wall regions 22 between the adjacent cell-receiving segments 21 each have a triangular wall profile (see Figure 1A), with which the cell-receiving segments 21 form a slotted funnel shape on at least one of the lateral segment wall surfaces 22E delimiting the wall regions 22, and / or the shape of a vertical rib (see Figures 1B, 1C, and 1D). The wall profile of the wall sections 22 has a protruding wall tip 23, which is freestanding (Figure 1A) or formed by the vertical rib 25 (Figures 1B, 1C, and 1D). The vertical rib 25 has the shape of a flat lamella or a more complex geometry with an arrow shape or a taper towards the free end of the vertical rib 25 (see Figure 4E). Comparing the cell substrates 100 according to Figure 1 with the conventional cell substrate 100' according to Figure 11 illustrates the effect of the cell substrate 100 according to the invention. In conventional cell substrates 100', cells 1' can adhere to flat upper surfaces of the partition walls 22', which typically have a height of up to 50 pm, and overgrow the upper surfaces, so that contacts are formed between fibers 2' in adjacent grooves 21' (see Figure 11), and an independent investigation of electrical processes on individual (adjacent) fibers becomes impossible. In contrast, in the cell substrate 100 according to the invention, cell arrangement on the wall region 22 is suppressed. According to Figure 1A, the shape of the upwardly tapered wall regions 22 in the form of pointed roofs prevents cells 1 from adhering to the wall regions 22 when cells 1 are seeded from a suspension 3 (partially shown). The cells 1 sink under the effect of gravity to the bottom 28 of the cell receiving segments 21, where they form fibers 2 as a result of further cultivation and / or differentiation. According to Figure 1B, the vertical ribs 25 form the wall tips 23 and thus advantageously the desired barrier for cells 1 from adjacent cell receiving segments 21, so that contact between cell receiving segments 21 is prevented. According to Figure 1C, the vertical ribs 25 extend as ultra-thin walls over the entire height of the wall regions 20. According to Figure 1D, the vertical ribs 25 are positioned as ultra-thin walls on wider, cuboid-shaped base sections 22A. In the variants shown in Figures 1 to 10, the substrate body 10 is formed, for example, from glass or a plastic, preferably with a thickness equal to or less than 1 mm. The base 28 of the cell-receiving segments 21 is formed by the substrate body 10, optionally with a coating to promote cell adhesion, e.g., with cell adhesion proteins, or an intermediate layer. The wall regions 22 are made, for example, from a polymer (see
[0013] -
[0017] ) or, for example, from PDMS. The dimensions of the structured substrate surface 20 of cell substrates 100 according to the invention can generally be selected, for example, in the following ranges: wall height hi of the wall regions 22 (see, for example, Figure 1A) > 5 pm, in particular 10 pm to 100 pm, height h 2of the vertical rib (see e.g. Figure 1B) 10 pm to 100 pm, distance Bi of the wall tips 23 to 100 pm, width Bz of the base 28 3 pm to 50 pm, thickness D of the vertical ribs 25 0.5 pm to 10 pm, e.g. 1 pm to 5 pm or 5 pm to 10 pm, and width of the base section (see e.g. Figure ID) 10 pm to 20 pm. Figure 2 schematically illustrates an embodiment of a cell substrate 100 in a schematic plan view, showing a substrate body 10 and a structured substrate surface 20 with three cell receiving segments 21. The substrate body 10 is an MEA substrate (MEA chip) with an electrode arrangement 30 comprising electrically separated surface electrodes 31 arranged spaced apart from one another along the longitudinal extent of the cell receiving segments 21, and a connecting line 33 for each surface electrode 31. For reasons of clarity, the MEA substrate is shown with only 9 surface electrodes 31. In practice, a considerably higher number of, for example, 256 up to > 1000 surface electrodes 31 can be provided. Furthermore, a CMOS MEA can be used as the electrode device 30 on the substrate 10, which, for example, has more than 4000 lead positions and is schematically illustrated in Figure 3. In this embodiment, the formation of the substrate surface with the wall regions and cell receiving segments can take place, e.g., by printing, directly on the electrodes 31 of the CMOS MEA. The electrodes 31 of the CMOS MEA can be coated, e.g., with a platinum layer ("black platinum"). The electrode density, as shown in Figure 3, can be much higher than in conventional MEAs (several thousand electrodes / mm 2 ). The printing of the wall regions and cell-receiving segments can preferably be carried out using 2-photon polymerization, unlike in Figure 2, directly on the electrodes 31. Each of the electrodes 31 is connected to a measuring and evaluation device 40. The structured substrate surface 20 is shaped with the wall regions 22 such that the surface electrodes 31 are exposed at the bottoms of each of the cell-accommodating segments 21. The wall regions 22 are shaped, for example, with the exposed wall tips 23, which form continuous wall apex lines along the length of the wall regions 22. At the longitudinal ends of the cell-accommodating segments 21, the substrate body 10 forms end walls 22A, with which the cell-accommodating segments 21 are closed in the longitudinal direction (see also Figures 4A and 4B). The surface electrodes 31 each comprise, for example, circular electrode layers made, for example, from metal, such as gold or titanium nitride, and having a diameter that is less than or equal to the width of the base 28 of the cell receiving segments 21. The connecting lines 33, via which the surface electrodes 31 are each connected to a separate measuring and evaluation device 40 (only one measuring and evaluation device 40 is shown, see also Figure 7), comprise conductor tracks on and / or in the substrate body 10. A preferred material for the conductor tracks is, for example, transparent ITO (indium tin oxide), and SiN (silicon nitride) serves, for example, as an insulation layer. Figure 2 illustrates, by way of example, an annular enclosure 11 of the region with the cell receiving segments 21, wall regions 22, and electrodes 31. The enclosure 11 has a height of, for example, 3 mm, forming a vessel for receiving culture media, cell suspensions, and / or test substances confined to this region. The enclosure 11 can, for example, be made of a plastic and glued to the surface of the substrate body 10. Furthermore, the enclosure 11 can be connected to a fluidic device for supplying or discharging liquids into or out of the enclosure 11. Further variants of the wall profile of the wall regions 22 according to embodiments of the invention are illustrated in Figure 4. The wall profiles, which are shown as cross sections, delimit adjacent cell-receiving segments 21. Partial features of the individual variants can be combined, for example, by providing the end walls 22A from Figures 4A and 4B and / or the corrugated wall surface 22C from Figure 3D in one of the remaining variants. According to Figure 4A, the wall regions 22 each have a wall profile comprising a rectangle at the base region 22B facing the substrate body 10 and a triangle with the roof peak 23 at the free end. Surface electrodes 31 are arranged on the bottoms 28 of the cell-receiving segments 21. The ends of the cell-receiving segments 21 are delimited by end walls 22A. According to the variant shown in Figures 4B and 4C, the wall regions 22 each have a wall profile that comprises a tapered trapezoid in the base region 22B facing the substrate body 10 and the triangle with the roof peak 23 at the free end. With this geometry, lateral projections 26 are formed on the segment wall surfaces 22E and segment slots (funnel slots) 27 are formed between them. The segment slots 27 have a width that is less than the diameter of the cells 1. Upon passing through the segment slot 27, a temporary deformation of the cells 1 occurs (see 1A in Figures 4C and 9), which then expand again below the segment slot 27 and form a fiber 2 (see 1B, 2 in Figures 4C and 9) and are thus captured in the cell receiving segment 21 between the base regions 22B. The geometry of the fibers to be formed can be determined by varying the height of the base region 22B and the width of the separation slot 27. Typical dimensions for the width of the separation slot 27 are 10 pm to 30 pm and for the height of the base region 22B, 1 pm to 50 pm, although these dimensions can vary depending on the fiber geometry specifications. Figure 4D shows, by way of example, further wall profiles of the wall regions 22, each with a triangular section, a wall tip 23, and a rectangular base region 22B, wherein at least one side wall in the base region 22B may have a corrugated wall surface 22C. With the corrugated wall surface 22B, the surface of the side wall is enlarged near the base 28, so that the fixation of the cells 1 and / or fibers in the cell receiving segments 21 is further improved. The vertical rib 25 shown in Figure 1B in the form of a flat slat can be provided on a wall region 22 with a wall profile according to Figure 4B, as shown in Figure 4E (left). The profile of the vertical rib 25 can have the shape of an arrow-shaped tip (Figure 4E (center)) or a tapered tip (Figure 4E (right)). In particular, according to Figure 4E (right), the radius of curvature of the vertical rib 25 can advantageously be reduced to a radius of curvature of 0.5 pm. Figure 4F shows another variant of the wall regions 22 with projections 26 formed by a rectangular base region 22B and a triangular tip. Since the wall region 22 has a smaller thickness at the base region 22B than at the projection 26 and an undercut is formed, a larger space for accommodating cells can be provided at the bottom of the cell-receiving segment 21. The variants with a narrowing segment slit 27 (Figures 4B, C, E, and F) are preferably used when deposition of the cells 1 in the cell receiving segments 21 by centrifugation is intended. Since greater forces occur during centrifugation than during sedimentation with the sole effect of gravity, the deformation of the cells 1 is supported as they pass through the segment slit 27. The top view of a cell substrate in Figure 5 schematically illustrates variants of cell receiving segments according to embodiments of the invention. Cell receiving segments can extend over the entire extent of the cell substrate 10 (cell receiving segment 21A), be shorter than the extent of the cell substrate (cell receiving segments 21B, 21C), form a C-shape with two straight segment sections and a curved section 29 (curved cell receiving segment 21D), have a stepped extension (cell receiving segment 21E), form at least one Y-shaped branch (cell receiving segment 21F), and / or form at least one H-shaped branch (cell receiving segments 21C, 21G). Cell receiving segments 21C, 21G with at least one H-shaped branch are formed by the wall region 22 having at least one through-opening 22D between adjacent cell receiving segments. The through-opening 22D forms a wall interruption, so that the adjacent cell receiving segments have an H-shape in plan view. The adjacent cell receiving segments can be connected by a plurality of through-openings 22D. For example, an MEA (Figures 2, 3) can be connected to each other at defined positions, which is particularly advantageous for simulating myocardial tissue structures. Lateral connections also exist in cardiac muscle tissue, where cell fibers can also be connected laterally via nexus contacts. The variants are shown in Figure 5 by way of example as individual or paired cell receiving segments. Combinations, sub-combinations or modifications of these variants can be formed. In practice, a plurality of cell receiving segments is preferably provided, wherein all cell receiving segments represent the same variant or cell receiving segments of different variants are combined. The cell receiving segments are formed on the substrate body 10 and arranged next to one another, so that adjacent cell receiving segments are each separated by a wall region 22 protruding from the substrate body 10. For reasons of clarity, the cell receiving segments are shown in Figure 5 with relatively large mutual distances and without the structure of the wall regions. In practice, they can alternatively be arranged with smaller distances. In any case, the material between the cell receiving segments is in the form of, for example,formed into the wall regions 22 shown in Figures 1 to 4. The cell receiving segments 21A to 21C show, by way of example, the row-like arrangement of a plurality of electrodes, e.g., surface electrodes 31. The cell receiving segment 21D illustrates, by way of example, that the cell receiving segments generally do not necessarily contain individual cells or cell fibers, but can alternatively be designed to receive planar cell groups (cell layers) and / or volumetric cell groups (cell aggregates). Figure 6 schematically shows a top view of another embodiment of the cell substrate 100, exemplarily with a straight cell receiving segment 21A and a curved cell receiving segment 21C, which are each delimited by wall regions, e.g., in the form of the wall regions 22 shown in Figures 1 to 4, preferably by wall regions 22 with projections 26 according to Figures 4B, 4C, 4E, or 4F. Fibers 2 from heart cells are held in the cell receiving segments 21A, 21C, preferably under the projections 26 on both sides of the adjacent wall regions, which makes it difficult for the fibers 2 to detach from the cell substrate 100. The straight cell recording segment 21A in the left part of Figure 6 contains a fiber 2 of cardiac cells, whose nexus contacts 4 are schematically illustrated. Measurements of field potentials and stimulations of the fibers can be made at various positions along the longitudinal extent of the Fibers. The length of fiber 2 is, for example, 3 mm. With the curved cell-receiving segment 21C in the right part of Figure 6, the length of fiber 2 can be extended to, for example, 7 mm. The curved cell receiving segment 21C in the right-hand part of Figure 6 has a first surface electrode 31A at a first end of the cell receiving segment 21C, a second surface electrode 31B in the curved section 29, and a third surface electrode 31C at the second end of the cell receiving segment 21C. With the first surface electrode 31A, a pulsed electrical stimulation of the biological cells in the cell receiving segment 21C can be carried out at a starting time using a pulse voltage source (not shown). By detecting field potentials with the second and third surface electrodes 31B, 31C, each of which is connected to the measuring and evaluation device 40, the propagation times of the excitation relative to the starting time and thus the speed of the excitation conduction through the fiber are detected. The ends of the cell recording segment 21C can alternatively or additionally be captured with an optical measuring device, in particular a microscope. The observation field ROI (region of interest) of the optical measuring device (not shown) contains the ends of the cell recording segment 21C. A stimulation pulse and the arrival of the excitation at the second end of the cell recording segment 21C can be captured, for example, by measuring fluorescence signals (e.g., with a "FluoVolt™" probe) in a camera segment (ROI, region of interest). Figures 7 and 8 schematically illustrate the arrangement of electrodes in a cell receiving segment 21. Figures 7 and 8 show a partial sectional view of a cell substrate 100 with two wall regions 22 shaped according to Figure 4B and the cell receiving segment 21 between the wall regions 22. According to Figure 7, a surface electrode 31 for measuring surface field potentials, as is known from conventional measurements with MEAs, is located on the bottom 28. The surface electrode 31 is connected to a measuring and evaluation device 40, e.g., a voltmeter, via a connecting line 33 embedded in the substrate body 10. Potential measurements on the cell 1 are performed with respect to a reference electrode (Ref), which is in electrically conductive connection with the culture medium in the interior of the cell receiving segment 21. Figure 8 schematically illustrates the measurement of intracellular potentials with an electrode tip 32, as described, for example, in [6]. The electrode tip 32 penetrates a cell membrane of a cell 1 in the cell receiving segment 21. The electrode tip 32 is mechanically moved into a perfusion pore 53 in the substrate body 10 with the aid of a coupled manipulator 51, e.g., a piezoelectric actuator, or a suction device 52, which leads to the penetration of the membrane of the cell 1 with the electrode tip 32. Alternatively, the membrane can be penetrated by electroporation. Potential measurements on the cell 1 are again carried out with respect to a reference electrode (Ref), which is in electrically conductive connection with the culture medium in the interior of the cell receiving segment 21. The suction device 52 and the perfusion pore 53 in the substrate body 10 can also be provided without the provision of an electrode tip 32, as schematically illustrated in Figure 9 by way of example. With the suction device 52, the cell 1B or fiber 2 can be additionally held in the cell receiving segment 21 and / or the filling of the cell receiving segment 21 can be accelerated. The embodiment of the cell substrate shown in Figure 8 can be used as a generator device ("biological battery") for generating electrical current by membrane potential derivation (potentials in the range of the cell membrane potential of, for example, 40 mV to 100 mV) at the biological cells 1 in the cell receiving segments 21 of the cell substrate 100. The generator device is configured, for example, like the embodiment according to Figures 2 or 3, with an MEA. For this application, a larger number of cells adapted to the function of the generator device are integrated on a correspondingly larger area and supplied with nutrient media. Myocardial cells are particularly suitable for use as a generator device due to their efficient metabolism. Figures 9 and 10 further illustrate the effect of the projections 26 to form a narrowing segment slit 27 during the deposition of cells 1 in the cell receiving segment 21. The cell 1, initially spherical in suspension (Figure 9), is forced through the segment slit 27 under the action of, for example, a centrifugation force, whereby it deforms without being damaged (Figure 1A). In the area of the undercut below the projections 26, the cell can then relax and assume its round shape (Figure 1B) or form a fiber (2), a cell layer, or a cell aggregate. The fiber 2, which extends in the longitudinal direction of a cell receiving segment 21, is also shown schematically in Figure 10. The features of the invention disclosed in the above description, the drawings and the claims may be important individually, in combination or sub-combination for the practice of the invention in its various embodiments.
Claims
Claims 1. Cell substrate (100) configured to receive biological cells (1), comprising - a substrate body (10) with a structured substrate surface (20), wherein - the structured substrate surface (20) has a plurality of cell receiving segments (21, 21A - 21G) arranged side by side, each with a longitudinal extension, - adjacent cell uptake segments (21, 21A - 21G) are each separated by a (10) projecting wall area (22) delimited by segment wall surfaces (22E), and - the wall regions (22) between the adjacent cell receiving segments (21, 21A - 21G) each have a wall profile perpendicular to the longitudinal extent of the adjacent cell receiving segments (21, 21A - 21G), characterized in that - the wall profile of the wall regions (22) between the adjacent cell-receiving segments (21, 21A - 21G) each has a wall tip (23) projecting from the substrate body (10), which suppresses a cell arrangement on the wall region (22).
2. Cell substrate (100) according to claim 1, wherein - the segment wall surfaces (22E) of at least one of the wall regions (22) distally at the wall tip (23) between adjacent cell receiving segments (21, 21A - 21G) enclose an acute angle or run parallel.
3. Cell substrate (100) according to claim 2, wherein - the segment wall surfaces (22E) enclose the acute angle and run obliquely relative to one another and / or relative to the extension of the substrate body (10).
4. Cell substrate (100) according to one of the preceding claims, in which - the segment wall surfaces (22E) have a low roughness such that structures on the segment wall surfaces have dimensions < 100 nm, in particular < 10 nm.
5. Cell substrate (100) according to one of the preceding claims, in which - at least one of the wall regions (22) has a vertical rib (25) projecting from the substrate body (10), which forms the wall tip (23) in the wall profile of the wall region (22).
6. Cell substrate (100) according to claim 5, wherein - the vertical rib (25) extends over the entire height of the wall area (22).
7. Cell substrate (100) according to one of the preceding claims, in which - the segment wall surfaces (22E) adjacent to at least one of the cell receiving segments (21, 21A - 21G) have lateral projections (26) which point towards one another and form a segment slot (27) which extends along the longitudinal extent of the cell receiving segment (21, 21A - 21G).
8. Cell substrate (100) according to claim 7, wherein - the segment slot (27) has a width equal to or less than 10 pm, in particular equal to or less than 5 pm.
9. Cell substrate (100) according to one of the preceding claims, in which - the segment wall surfaces (22E) adjacent to at least one of the cell receiving segments (21, 21A - 21G) form an elongated funnel slot.
10. Cell substrate (100) according to one of the preceding claims, in which - at least one of the wall regions (22) has at least one through-opening (22D) between the adjacent cell-receiving segments (21, 21A - 21G).
11. Cell substrate (100) according to one of the preceding claims, in which - at least one of the wall regions (22) has a corrugated wall surface (22C) on at least one side.
12. Cell substrate (100) according to one of the preceding claims, which is configured to detect electrophysiological characteristics of the biological cells (1), wherein - at least one of the cell receiving segments (21, 21A - 21G) has an electrode arrangement (30) with at least one electrode (31, 32).
13. Cell substrate (100) according to claim 12, wherein - the electrode arrangement (30) comprises at least two electrodes (31, 32) arranged spaced apart from one another along the longitudinal extent of the cell receiving segment.
14. Cell substrate (100) according to claim 12 or 13, wherein - the at least one electrode (31, 32) is arranged on a bottom (28) of at least one of the cell receiving segments (21, 21A - 21G).
15. Cell substrate (100) according to one of claims 12 to 14, wherein - the at least one electrode (31, 32) comprises at least one of surface electrodes (31) configured for planar contact with the cells (1) and protruding electrode tips (32) configured for penetration into the cells (1).
16. Cell substrate (100) according to one of claims 12 to 15, wherein - the electrode arrangement (30) comprises a CM OS-ME A.
17. Cell substrate (100) according to one of claims 12 to 16, wherein - the electrode arrangement (30) is coupled to a measuring and evaluation device (40) which is configured to characterise cell contacts between the cells (1) as a function of electrophysiological potentials recorded with the electrodes (31, 32).
18. Cell substrate (100) according to one of the preceding claims, having at least one of the features - at least one of the cell uptake segments (21, 21A - 21G) has at least one branch (21A), - at least one of the cell receiving segments (21, 21A - 21G) is provided with an end wall (22A) at its longitudinal ends, and - at least one of the cell receiving segments (21, 21A - 21G) has a varying width along its longitudinal extent.
19. Cell substrate (100) according to one of the preceding claims, in which - at least two adjacent segment sections of at least one of the cell receiving segments (21D) are connected via a curved section (29).
20. Cell substrate (100) according to one of the preceding claims, in which - the cell receiving segments (21, 21A - 21G) have an aspect ratio which is determined by a quotient of a lateral width at the bottom (28) of the cell receiving segments (21, 21A - 21G) and a depth of the cell receiving segments (21, 21A - 21G) and is selected in the range 2 to 10.
21. Use of the cell substrate (100) according to one of the preceding claims, with at least one of the steps - Cultivation of biological cells (1) in the cell uptake segments (21, 21A - 21G) of the cell substrate (100), - Examination of the biological cells (1), in particular measurement of electrophysiological potentials on biological cells (1) in the cell uptake segments (21, 21A - 21G) of the cell substrate (100), - Determining an excitation propagation speed through cells along at least one of the cell receiving segments (21, 21A - 21G) of the cell substrate (100) by detecting a time-delayed arrival of an electrical excitation at electrodes at the bottom of the cell receiving segment (21, 21A - 21G), in particular without a fluorescence measurement, - Determination of an excitation propagation speed through cells along at least one of the cell uptake segments (21, 21A - 21G) of the cell substrate (100), in particular with simultaneous fluorescence measurement for the transmission of an action potential with a potential-sensitive fluorescence probe and the intracellular calcium release with a calcium-sensitive fluorescence probe, the cell contraction with video microscopy, - optical measurement of a contraction of heart muscle cells by analyzing the shortening of cell segments, - Measurements of intracellular calcium concentrations, - electrical stimulation of biological cells (1) in the cell uptake segments (21, 21A - 21G) of the cell substrate (100), - biochemical stimulation of biological cells (1) in the cell uptake segments (21, 21A - 21G) of the cell substrate (100), and - Operating the cell substrate (100) as a generator device configured to generate electrical current by membrane potential derivation at the biological cells (1) in the cell receiving segments (21, 21A - 21G) of the cell substrate (100).