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EP-4739762-A2 - CELL SUBSTRATE FOR RECEIVING BIOLOGICAL CELLS, AND METHOD FOR USE THEREOF

EP4739762A2EP 4739762 A2EP4739762 A2EP 4739762A2EP-4739762-A2

Abstract

A cell substrate (100), configured to receive biological cells (1), comprises a substrate body (10) with a structured substrate surface (20), wherein the structured substrate surface (20) has a multiplicity of cell-receiving segments (21) arranged next to one another and each with a longitudinal extent, adjacent cell-receiving segments (21) are each separated by a wall region (22) protruding from the substrate body (10), and the wall regions (22) between the adjacent cell-receiving segments (21) each have a wall profile, delimited by segment wall surfaces, perpendicular to the longitudinal extent of the adjoining cell-receiving segments (21), wherein the wall profile of the wall regions (22) between the adjacent cell-receiving segments (21) in each case has a wall tip (23) which protrudes from the substrate body (10) and which inhibits arrangement of cells on the wall region (22). Uses of the cell substrate (100) are also described.

Inventors

  • HERING, STEFFEN

Assignees

  • Chanpharm GmbH

Dates

Publication Date
20260513
Application Date
20240626

Claims (20)

  1. 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 next to one another, each with a longitudinal extension, - adjacent cell uptake segments (21, 21A - 21G) each 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 extension 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) protruding from the substrate body (10) which suppresses a cell arrangement on the wall region (22).
  2. 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. 3. Cell substrate (100) according to claim 2, wherein - the segment wall surfaces (22E) enclose the acute angle and run obliquely relative to each other and/or relative to the extension of the substrate body (10).
  4. 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. 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) protruding from the substrate body (10), which forms the wall tip (23) in the wall profile of the wall region (22).
  6. 6. Cell substrate (100) according to claim 5, wherein - the vertical rib (25) extends over the entire height of the wall area (22).
  7. 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 face each other and form a segment slot (27) which extends along the longitudinal extent of the cell receiving segment (21, 21A - 21G).
  8. 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. 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. 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. 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. 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. 13. Cell substrate (100) according to claim 12, wherein - the electrode arrangement (30) comprises at least two electrodes (31, 32) which are arranged at a distance from one another along the longitudinal extent of the cell receiving segment.
  14. 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. 15. Cell substrate (100) according to one of claims 12 to 14, in which - 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. 16. Cell substrate (100) according to one of claims 12 to 15, wherein - the electrode arrangement (30) comprises a CM OS-ME A.
  17. 17. Cell substrate (100) according to one of claims 12 to 16, in which - the electrode arrangement (30) is coupled to a measuring and evaluation device (40) which is configured to characterize cell contacts between the cells (1) as a function of electrophysiological potentials detected by the electrodes (31, 32).
  18. 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 at its longitudinal ends with an end wall (22A), and - at least one of the cell receiving segments (21, 21A - 21G) has a varying width along its longitudinal extent.
  19. 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. 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.

Description

CELL SUBSTRATE FOR THE ABSORPTION OF BIOLOGICAL CELLS AND METHOD FOR USE THEREOF field of the invention The invention relates to a cell substrate that is configured to receive and preferably examine biological cells, particularly preferably for electrophysiological (electrical) measurements on the biological cells. The invention further 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 the present description, reference is made to the following prior art, which illustrates the technical background of the invention: [1] A. S. T. 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] V. J. Kujala 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] T.C. 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; [10] WO 2014/074067 Al; [11] F. Navaee et al. "Celis" 2023, 12, 576; [12] J.P. Kucera et al. in "Circ Arrhythm Electrophysiol." 2017; 10:e004665; [13] Bartels et al. in "Be Rep. " 2021; 11:9269; [14] M. Hippier et al. in "Be Adv. " 2020 Sep; 6(39): eabc2648. [15] K. Weißenbruch et al. in "Curr Opin Biotechnol. " 2022 Feb; 73:290-299; [16] Q. Akolawala et al. in "ACS Appl. Mater. Interfaces" 2022, 14, 18, 20778-20789; [17] SH Yagoub et al. in "Journal of Assisted Reproduction and Genetics" Vol. 39, pp. 1503-1513 (2022); and [18] DE 102008047399 Al. It is well known that the provision of differentiated cells, such as cardiac cells or neurons, by differentiation of human pluripotent stem cells (hPSCs) enables the development of tissue models, such as cardiac muscle or nerve tissue-like tissues. Tissue models can be used in drug development to study the effect of drugs on tissues ("tissue-on-a-chip" technologies). For example, electrophysiological measurements and/or optical measurements and/or measurements of cardiac cell contractions can be carried out on cardiac cells using high-throughput methods (see, for example, [1] to [3]). There is interest in studying the 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 fiber structure in the myocardium (see e.g. [1], [2]). Testing drugs on laminar, fibrous heart tissue structures on groove structures or on directed nerve fibers in grooves has brought initial progress in drug testing (see e.g. [2], [4] or [11]). For example, the recording of the heart cells on a cell substrate with a large number of surface grooves (hereinafter also referred to as cell recording segments) was realized, in which a fiber-like arrangement and alignment of the heart cells is created (see e.g. [1], [4] and [5]), with neighboring grooves separated by partitions. Surface grooves are formed, for example, using printing processes with a height in the range 1 pm to 100 pm, a width in the range 1 pm to 100 pm and a length of 1 mm or more. The partitions consist, 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 processes (see e.g. [1], [2]) or by microcontact printing or by means of 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, which 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 investigations. For example, in [6] a substrate is described in which point-shaped nanoelectrodes are arranged at the bottom of cell recording segments, which penetrate into 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 derivation of membrane potentials and not for the measurement of surface potentials. However, there is great interest in measuring surface potentials, since the temporal length of field potentials and the shape of measured T waves (such as determining the prolongation of the ECG parameter Q-T time) on fiber surfaces are highly informative with regard to drug effects and are similar to an ECG measurement. Measur