A substrate for cell and / or tissue culture and for micro-patterning cells and / or tissues, its manufacturing method and its use

Abstract

A substrate for cell and / or tissue culture and for micro-patterning cells and / or tissues, wherein the substrate comprises a base layer (1), and a polymer compound layer (2), wherein each of the base layer (1) and the polymer compound layer (2) has an upper surface and a lower surface, each of the upper surface and the lower surface extending in an xy-plane, wherein each of the base layer (1) and the polymer compound layer (2) has a thickness in the z-direction being perpendicular to the xy-plane, wherein the polymer compound layer (2) is stacked on the upper surface of the base layer, wherein the polymer compound layer (2) comprises a first portion (2a), a second portion (2b) and a slit portion (2c), wherein the slit portion (2c) extends along the z-direction from the upper surface of the polymer compound layer (2) through the polymer compound layer (2), thereby at least partly separating the first portion (2a) from the second portion (2b), so that the second portion (2b) is removeable from the upper surface of the base layer (1) independently from the first portion (2a).

Description

DESCRIPTIONTITLEA substrate for cell and / or tissue culture and for micro-patterning cells and / or tissues, its manufacturing method and its useTECHNICAL FIELD

[0001] The present disclosure relates to a substrate for cell and / or tissue culture and for micro-patterning cells and / or tissues, its manufacturing method and its use.BACKGROUND

[0002] In current practice, the so-called “cell micro-patterning” technology is expected to be applied to various biotechnology fields such as artificial organs and / or biosensors. The term “micro-patterning” generally means to pattern (and / or incubate) biological samples and / or materials, such as biological cells and / or tissues and / or nucleotides such as DNA and RNA and / or proteins and / or lipids, in a micrometer-order shape (i.e. a shape having a structure in a micrometer range). One of the conventional methods of cell micro-patterning is to pattern a substrate surface with a material to which cells easily adhere. Document JP 6348853 B2 discloses a substrate onto which a gel containing cell culture medium is patterned, so that cells would be adhered to the gel.SUMMARY OF INVENTION

[0003] The technical problem to be solved may be formulated as to more accurately pattern cultured cells and / or tissues and / or biomolecules.

[0004] According to the first aspect of the present disclosure, a substrate for cell and / or tissue culture and for micro-patterning cells and / or tissues comprises a base layer (1), and a polymer compound layer (2). Each of the base layer (1) and the polymer compound layer (2) has an upper surface and a lower surface, each of the upper surface and the lower surface extending in an xy-plane. Each of the base layer (1) and the polymer compound layer (2) has a thickness in the z-direction being perpendicular to the xy-plane.

[0005] The x-, y-, z-directions are of the three-dimensional rectangular coordinate system, which are orthogonal to each other. The xy-plane extends in the x-direction and in the y-direction. The polymer compound layer (2) is stacked on the upper surface of the base layer (1). The base layer (1) and the polymer compound layer (2) are therefore stacked in the z-direction, and the lower surface of the base layer (1), the upper surface of the base layer (1), the lower surface of the polymer compound layer (2) and the upper surface of the polymer compound layer (2) are arranged in this order in the z-direction.

[0006] The polymer compound layer (2) comprises a first portion (2a), a second portion (2b) and a slit portion (2c). The slit portion (2c) extends along the z-direction from the upper surface of the polymer compound layer (2) through the polymer compound layer (2), thereby at least partly separating the first portion (2a) from the second portion (2b), so that the second portion (2b) is (physically and / or manually) removeable from the upper surface of the base layer (1) independently from the first portion (2a). The second portion (2b) may be able to be peeled off from the upper surface independently from the first portion (2a), for example by manually. The second portion (2b) can be solely removed and peeled off from the base layer (1). The first portion (2a) can remain attached to the base layer (1) (preferably via a sub-layer such as an adhesive layer) after having removed and / or peeled off the second portion (2b) from the base layer (1). The adhesion force between the base layer (1) and the polymer compound layer (2) is preferably within a range where the polymer compound layer (2) can be manually peeled off from the base layer (1) and / or the adhesive layer. The first potion (1) and the second portion (2) may be partly connected and / or partly continuous.

[0007] The base layer (1) may comprise a non-conductive material(s) (such as glass) and / or a conductive material(s) (such as a metal) and / or a semiconductive material(s). The base layer (1) maybe a transparent material, which maybe further advantageous for optical observation and / or controlling of cells and / or tissues and / or biomolecules on the substrate. When a conductive material is used for the base layer (1), it may be possible to further conducting electrical observation and / or controlling of cells.

[0008] The polymer compound layer (2) may comprise parylene and / or polyimide and / or photo-curable polymer and / or plastic and / or elastomer. The polymer compound layer may further comprise metals and / or fibers (such as cellulose).

[0009] The based layer (1) has a thickness preferably in a range of 0.1 pm to 3 mm, more preferably 1 mm to 3 mm. The polymer compound layer (2) has a thickness preferably 0.1 pm to 3 mm, more preferably 1 pm to 1 mm. This range maybe advantageous in that the second portion (2b) can be more efficiently and manually peeled off from the base layer (1). The polymer compound layer (2) is preferably mechanically flexible and / or formed by polymers being mechanically flexible, so that the second portion (2b) can be more efficiently removed and / or peeled off from the base layer (1).

[0010] The slit portion (2c) have a width (i.e. the shortest distance and / or the average distance between the first portion (2a) and the second portion (2b) in the x- and / or y- direction and / or in the xy-plane) preferably in a range of 0.1 pm to 1 mm, more preferably 0.1 pm to 10 pm. This range may be more preferable, as cells would not grow into the slit portion (2c). The width of the slit portion (2c) may be smaller than a dimension of biological samples such as cells and / or tissues and / or proteins to be applied to the polymer compound layer (2). The slit portion (2c) may be a cut portion and / or a notch portion.[oon] The substrate according to the present disclosure may provide one or more of the following technical advantages: Biological samples such as cells and / or tissues and / or biomolecules can be at first applied to and / or seeded on the entire upper surface of the polymer compound layer (2), and it is therefore not necessary to seed biological samples into a targeted shape prior to the incubation step. The patterning of biological samples can be achieved by removing the second portion (2b) of the polymer compound layer (2) together with the biological samples on the upper surface of the second portion (2b) from the base layer (1). The patterned cells would be thus obtained on the first portion (2a) of the polymer compound layer (2). The substrate according to the present disclosure enables to pattern cells and / or tissues and / or biomolecules more accurately in the micrometer range, especially to form into a pattern having a microstructure, while it is possible to suitably control cell densities in the incubation process.

[0012] Preferably, the first portion (2a) is completely separated from the second portion (2b). This structure may be further advantageous to more accurately remove the second portion (2b) from the base layer (1) and from the first portion (2a). Preferably, the first portion (2a) is completely surrounded by the second portion (2b). This structure may be further advantageous to more accurately micro-pattern cells and / or tissues and / or biomolecules.

[0013] Preferably, the first portion (2a) is removeable from the upper surface of the base layer (1) independently from the second portion (2b). This structure may be further advantageous in that a stack of the first portion (2a) and the cell layer (i.e. being micropatterned into the shape of the first portion (2a)) can be further removed from the base layer and integrated into an external device, such as a biosensor.

[0014] Preferably, the substrate further comprises an adhesive layer (3) between the upper surface of the base layer (1) and the lower surface of the polymer compound layer (2). The adhesive layer (3) may comprise calcium alginate and / or polyvinyl alcohol (PVA) and / or agarose amd / or Polyhydroxyethylmethacrylate (polyHEMA) and / or 2- Methacryloyl oxyethyl phosphoryl choline (MPC) polymer and / or Arginylglycylaspartic acid (RGD peptides). The adhesive layer (3) may be advantageous to stabilize the adhesivity between the base layer (1) and the polymer compound layer (2) and may further prevent from that cells would grow outside of the first portion (2a) after having removed the second portion (2b) from the base layer (1). The adhesive layer (3) may at least remain attached to the base layer (1) after having removed (peeled off) the second portion (2b) from the base layer (1). Preferably the slit portion (2c) further extends through the adhesive layer (3).

[0015] Preferably, the substrate further comprises a coating layer being stacked on the upper surface of the polymer compound layer (2) or directly on the upper surface of the polymer. The coating layer includes biomolecules such as proteins and / or peptides, forexample, fibronectin and / or laminin and / or collagen and / or proteoglycan family (such as agrin, aggrecan, syndecan, neurocan, versican, phosphacan, brevican) and / or collagen and / or tenascin C and / or tenascin R and / or thrombospondin and / or hyaluronic acid and / or fibronectin and / or laminin and / or lealin and / or nucleotides such as DNA. The coating layer may further improve adhesion of the cell layer on the polymer compound layer (2).

[0016] Preferably, the upper surface of the first portion (2a) has a polygonal shape. The upper surface of the first portion (2a) extends in the xy-plane, and the contour of the first portion (2a) viewed from the z-direction may form a polygonal shape. The polygonal shaped first portion has n vertices, wherein n is preferably an odd number.

[0017] Preferably, the first portion (2a) comprises a first sub-portion (2a-i), a junction portion (23-3) and a second sub-portion (2a-2), being arranged in this order in the y- direction. The first sub-portion (2a-i) and the second sub-portion (2a-2) are connected by the junction portion (23-3). The first sub-portion (2a-i) has a first length (h) in the x- direction, the second sub-portion (23-2) has a second length (12) in the x-direction, the junction portion (23-3) has a third length (13) in the x-direction. The third length (13) is shorter than each of the first length (h) and the second length (12).

[0018] Preferably, the area of the upper surface of the first sub-portion (2a-i) is equal to or smaller than the area of the upper surface of the second sub-portion (23-2). The term “area of the upper surface of the first / second sub-portion” means the surface area of the first / second sub-portion (23-2), which extends in the xy-plane.

[0019] Preferably, the first sub-portion (2a- 1) has a first width (wj in the y-direction, the second sub-portion (23-2) has a second width (w2) in the y-direction, the junction portion (23-3) has a third width (w3) in the y-direction. The width of the first portion (2a) being the sum of the first width (wj, the second width (w2) and the third width (w3) is longer than the first length (L). Preferably, the first width (wj is shorter than the second width (w2).

[0020] Preferably, the upper surface of the first sub-portion (2a-i) has a circular shape or an oval shape, and preferably the upper surface of the second sub-portion (23-2) has a circular shape or an oval shape. The junction portion (23-3) has a third length (13) in the x- direction. The third length (13) is shorter than each of the maximum length (h) of the first sub-portion (2a-i) in the x-direction and the maximum length (12) of the second sub-portion (23-2) in the x-direction. The maximum length of each of the first sub-portion (2a-i) and the second sub-portion (23-2) may be a diameter of the circular shape and / or the oval shape.

[0021] Preferably, each of the first length (h), the second length (12) and the third length (13) is in a range of 1 pm to 1 mm, more preferably 10 pm to 500 pm. Preferably, each of the first width (wj, the second width (w2) and the third width (w3) is in a range of 1 pm to 1 mm, more preferably in a range of 1 pm to 1 cm, more preferably 50 pm to 50 mm, more preferably 50 pm to 2 mm.

[0022] Preferably, the upper surface of the first sub-portion (2a-i) has a polygonal shape, wherein each of corners of the polygonal shape is rounded, wherein preferably the upper surface of the first sub-portion (2a-i) has a rectangular shape with rounded corners. The term “a corner being rounded” and “a rounded corner” may be for example a corner composed of a part of a circle or an arc. The contour of the first sub-portion (2a-i) (viewed from the z-direction) may include or be formed by straight lines and a part of a circle and / or a part of an arc, and each of the corners is the part of the circle and / or the arc. For example, the first and second sub-portions are rectangles with rounded corners. Namely, each of the first and second sub-portions has four corners, each of which being formed by a part of a circle or a part of an arc (connected between two straight lines).

[0023] Preferably, the upper surface of the second sub-portion (2a-2) has a polygonal shape, wherein each of corners of the polygonal shape is rounded, wherein preferably the upper surface of the second sub-portion (2a-2) has a rectangular shape with rounded corners.

[0024] According to the second aspect of the present disclosure, a method for manufacturing a substrate according to the first aspect of the present disclosure comprises a step of forming the polymer compound layer (2) on the upper layer of the base layer (1) and a step of forming the slit portion (2c) thereby separating the first portion (2a) from the second portion (2b).

[0025] Preferably, the step of forming the slit portion (2c) is performed by a photolithography process and / or electron-beam lithography process, or the step of forming the slit portion (2c) is performed by irradiating a laser beam onto the polymer compound layer (2).

[0026] Preferably, the method further comprises a step of forming the adhesive layer (3) on the upper surface of the base layer (1), prior to the step of forming the polymer compound layer (2), so that the adhesive layer (3) would be disposed between the base layer (1) and the polymer compound layer (2).

[0027] Preferably, the method further comprises a step of forming the coating layer on or directly on the upper surface of the polymer compound layer (2).

[0028] According to the third aspect of the present disclosure, a use of a substrate according to the first aspect of the present disclosure is for cell incubation and micropatterning of cells, and / or immobilizing nucleotides, and / or biosensors, and / or regenerative medicine and / or organoid formation and / or spheroid formation.

[0029] According to the fourth aspect of the present disclosure, a method of micropatterning cells or tissues by using a substrate according to the first aspect of the present disclosure comprises a step of seeding and incubating cells and / or tissues on the polymer compound layer (2) of the substrate thereby forming a cell layer (C), and a step of removinga stack of the second portion (2b) and a first part of the cell layer (C) from the upper surface of the base layer (1), the first part of the cell layer being adhered to and / or attached to and / or incubated on and / or immobilized on the second portion (2a).

[0030] Preferably, the method according to the fourth aspect further comprises a step of forming a coating layer on the upper surface of the polymer compound layer (2) prior to the step of seeding and incubating cells and / or tissues.

[0031] Preferably, the method according to the fourth aspect further comprises a step of removing a stack of the first portion (2a) and a second part of the cell layer (C) from the upper surface of the base layer (1), the second part of the cell layer (C) being adhered to the first portion (2a).

[0032] According to the fifth aspect of the present aspect, a micro-patterned cell layer obtained by the method according to the fourth aspect of the present disclosure.BRIEF DESCRIPTION OF THE DRAWING

[0033] Figs. 1A, 1B, 1C schematically illustrate a perspective view, a top view, a cross section of a substrate according to the first embodiment, respectively.Figs. 2A to 2E schematically illustrate steps of a manufacturing method for the substrate according to the first embodiment.Figs. 3A to 3E schematically illustrate steps of a use of the substrate according to the first embodiment.Figs. 4Ato 4C schematically illustrate a perspective view, a top view, a cross section of a substrate according to the second embodiment, respectively.Fig. 4D schematically illustrates a top view of a substrate according to a variation of the second embodiment.Fig. 5A shows a photo of a top view of a sample substrate (Sample 1).Fig. 5B shows a photo of a top view of another sample substrate (Sample 2) onto which cells have been incubated.Fig. 5C shows a photo of a top view of the Sample 2 from which the second portion has been removed.Fig. 5D shows a fluorescence imaging of a top view of the Sample 2 from which the second portion has been removed.Figs. 6A to 6C show fluorescence imaging of calcium signal propagations at t=o, 0.4 sec and 0.9 sec, which were observed with cells micro-patterned with the Sample 2.DETAILED DESCRIPTION OF EMBODIMENTSFirst embodiment

[0034] Figs. 1A and 1B schematically illustrate a perspective view and a top view of a substrate according to the first embodiment. Fig. 1C schematically illustrates a cross section (X1-X1 in Fig. 1A) of the substrate according to the first embodiment.

[0035] According to the first embodiment, a base layer (1) has a square or a rectangle shape and has a thickness in a range of 0.05 mm to 5 mm. The base layer (1) may be formed in any shape such as a circle or oval shape. An adhesive layer (3) and a polymer compound layer (2) are stacked in this order in the z-direction on the base layer (1). The adhesive layer (3) is optional and may be omitted. The first portion (2a) is formed at a center part of the polymer compound layer (2) and formed into a triangular shape being completely surrounded by the slit portion (2c) and the second portion (2b). A length of one side of the triangular shape of the first portion (2a) may be 1 pm to 50 mm.

[0036] In this embodiment, the substrate includes only one first portion (2a). However, the substrate may include multiple first portions (2a) being separated from each other.

[0037] The substrate according to the first embodiment may have one or more of the following technical effects: According to the substrate of the first embodiment, cell culture (including cell seeding and incubation) and / or immobilization of biomolecules can be at first performed on the entire upper surface of the polymer compound layer (2), and it is not necessary to seed and / or immobilize cells and / or tissues and / or biomolecules into a targeted shape. The patterning of cells and / or tissues and / or biomolecules can be achieved by removing the second portion (2b) of the polymer compound layer (2) together with cells being incubated on the upper surface of the second portion (2b) from the upper surface of the base layer (1). The patterned cells would be thus obtained on the first portion (2a) of the polymer compound layer. The substrate enables to pattern cells more accurately in the micrometer range, especially to form such a pattern having a microstructure with e.g. sharp edges. In particular, when the structure of the first portion (2a) (i.e. the slit portion (2c)) is formed by a photolithography process or an electron-beam lithography process, it would be further possible to provide a resolution less than 1 pm. The first portion (2a) can be therefore shaped into more accurate (geometrical) structures, and accordingly biomolecules and / or cells can be more accurately micro-patterned. By patterning cells into a polygon, cells located near the vertices are expected to become signal propagation points, which would induce signal transduction between cells. Signal propagations maybe more pronounced when cells are patterned into a polygonal shape with an odd number of vertices. Such patterned cells,for example cardiomyocytes and / or neurons, would be especially advantageous for applications, as artificial organs and / or biosensors.

[0038] < Manufacturing methods of the substrate >Figs. 2A to 2E schematically illustrates steps of manufacturing the substrate according to the first embodiment: The polymer compound layer (2) is formed on the upper surface of the base layer (1), for example by spin coating and / or chemical vapor deposition and / or spray coating and / or physical vapor deposition (such as sputtering or thermal deposition or ion plating). Optionally, the adhesive layer (3) can be formed directly on the base layer (1), prior to the step of forming the polymer compound layer (2) (Fig. 2A).

[0039] A positive photoresist is then deposited on the polymer compound layer (2) (cf. Fig. 2B) and a photolithography process is performed on the photoresist layer (4). The photoresist layer (4) is exposed under light through a mask having a pattern of the slit portion (Fig. 2C). The photoresist layer (4) is then developed to remove a part of the photoresist layer, the part having been exposed under the light. The part of the polymer compound layer (2) and the adhesive layer (3), which is not covered by the photoresist, are then etched by oxygen plasma to form the slit part (Fig. 2D).

[0040] Finally, the photoresist layer (4) was removed, for example by organic solvent, so that the polymer compound layer (2), in which the first portion (2a) is surrounded by the slit portion (2c) and separated from the second portion (2b), is exposed.

[0041] Alternative to the photolithography process, an electron-beam lithography may be used. Further alternatively, the slit portion (2) may be formed by irradiating directly the upper surface of the polymer compound layer (2) with a laser beam and cut through the polymer compound layer (2) in the z-direction and / or the adhesive layer (3) in the z- direction. Further alternatively, the slit portion (2) may be formed by direct milling, drilling (boring) processes. Forming the slit portion (2) by the laser cutter and / or direct milling / drilling may be advantageous in that the slit portion (2) can be directly formed by the laser beam, and therefore it is not necessary to form the photoresist layer and to perform multiple steps of the photolithography process. The manufacturing can be more quickly carried out. On the other hand, the photolithography process and / or the electron-beam lithography would be more advantageous to form a precise structure in the micrometer range and even in a range under 1 pm.

[0042] <Use of the substrate>Figs. 3A to 3E schematically illustrate a use of the substrate according to the substrate and illustrate steps of micropatterning of cells by using the substrate according to the first embodiment. Preferably, prior to seeding and incubating cells and / or tissues, the upper surface of the polymer compound layer (2) may be coated by a coating layer, which mayinclude proteins and / or peptides, to improve adhesivity of the cells on the polymer compound layer (2). The cells may be seeded directly on the upper surface of the polymer compound layer (2) or may be seeded on the upper surface of the coating layer. The cells are incubated and thereby a cell layer (C) is formed on the first portion (2a) and on the second portion (2c) (Fig. 3B).

[0043] Subsequently, a stack of the second part (2b) of the polymer compound layer (2) and a first part of the cell layer (C), i.e. the cell layer being adhered to the second portion (2b) (via the coating layer), is removed from the base layer (1) by e.g. manually peeling off the second portion (2b) along the slit portion (2c) from the upper surface of the base layer (Figs. 3C, 3D). In this embodiment, the slit portion (2c) completely surrounds the first portion (2a), and the second portion (2b) can be accordingly removed from the base layer (1) only by lifting the second portion (2b) in the upper direction (i.e. z-direction). Accordingly, a stack of the first part (2a) and a second part of the cell layer (C), i.e. the cell layer being adhered to the first portion (2a) (via the coating layer), remains attached to the upper surface of the base layer (1). The second part of the cell layer (C) is accordingly patterned into the shape of the first portion (2a) (Fig. 3D). The adhesive layer (3) may at least partly remain on the base layer (1) and / or at least partly removed from the base layer(1) together with the second portion (2b)(Fig. 3E). The adhesive layer (3) may contain one or more substances that would prevent cells from growing on the adhesive layer, so that the cells remain only inside of the first portion (2a). For example, the adhesive layer may include calcium alginate.

[0044] The stack of the cell layer (C) and the first part (2a) of the polymer compound layer(2) may be further removed from the base layer (1) and further integrated in a biological system such as an artificial organ and / or biosensors.

[0045] The use of the substrate is not limited to cell culture and the substrate may be also used to pattern various other biological materials and / or molecules, such as proteins and / or peptides and / or nucleotides such as DNA and / or lipids, and such micro-patterned biological materials and / or molecules may be for example further applied to biosensing technology. The substrate may be further used for regenerative medicine (for example stem cell differentiation such as differentiation of human mesenchymal stem cells) and / or applications for understanding of the impact of the microenvironment on cells and the interaction between cells and protein and / or single cell assay and / or organoid formation and / or spheroid formation.Second embodiment

[0046] Figs. 4A and 4B schematically illustrate a perspective view and a top view of a substrate according to the second embodiment, respectively. Fig. 4C schematicallyillustrates a cross section (X2-X2 in Fig. 4A) of the substrate according to the second embodiment.

[0047] The substrate according to the second embodiment is different from the first embodiment in the shape of the first portion (2a). The first portion (2a) according to the second embodiment (i.e. the upper surface of the first portion (2a) viewed from the z- direction) has a first sub-portion (2a-i), a junction portion (23-3) and a second sub-portion (2a-2), being arranged in this order in the y-direction, and the junction portion (23-3) connects the first and second sub-portions (2a-i, 2a-2). The junction portion (23-3) accordingly forms a neck of the first portion (2a), and the third length (13) of the junction portion (23-3) is shorter than each of the first length (h) of the first sub-portion (2a-i) and the second length (12) of the second sub-portion (23-2) (each of the lengths, h, 12, 13, are defined in the x-direction).

[0048] According to the second embodiment, the first length (h) and the second length (12) are the same. However, the second length (12) maybe longer than the first length (L), or vice versa. The area of the upper surface of the first sub-portion (2a-i) is smaller than the area of the upper surface of the second sub-portion (23-2). Each of the first sub-portion (2a-i) and the second sub-portion (23-2) has a rectangular shape in which each of the corners is rounded (i.e. a so-called rounded rectangle and / or curved rectangle). Alternatively, each of the first sub-portion (2a-i) and the second sub-portion (23-2) may have a square shape with rounded corners.

[0049] Fig. 4D schematically shows a top view of a variation of the second embodiment. In this variation, the first sub-portion (2a-i) has a circle shape or an oval shape, and the second sub-portion (23-2) has a circle shape or an oval shape.

[0050] Both in the second embodiment and in the variation of the second embodiment, the junction portion (23-3) is connected to the first sub-portion (2a-i), such that the center of the third length (13) of the junction portion (23-3) coincides with each of the center of the first length (h) of the first sub-portion (2a-i) and the center of the second length (12) of the second sub-portion (23-2). However, this is only an example and the junction portion may be shifted away from the center of the first sub-portion in the x-direction, as long as the junction portion has a length (13) in the x-direction being shorter than the x-directional lengths (li, 12) of the first and second sub-portions, thereby forming a neck by the junction portion at the first portion (2a) and connecting and / or separating the first and second subportions (2a-i, 2a-2).

[0051] The manufacturing method of the substrate according to the second embodiment are the same as those of the first embodiment, in which only the slit portion (2c) would be patterned into the shape having the first sub-portion, the junction portion and the secondsub-portion. The substrate according to the second embodiment can be also used in the same way as described in the first embodiment.

[0052] The substrate according to the second embodiment may have one or more of the following technical effects:

[0053] As discussed also for the first embodiment, cell culture (including cell seeding and incubation) can be at first performed on the entire upper surface of the polymer compound layer (2), the accurately patterned cells would be thus more efficiently obtained on the first portion (2a) of the polymer compound layer (2).

[0054] The substrate according to the second embodiment and the variation of the second embodiment may be especially advantageous in that it is possible to pattern cells into the particular shape having the junction portion in the micrometer range. The patterned cells having a structure in which the first sub-portion and the second sub-portion are connected by the junction portion is effective in controlling intercellular signal transduction. In particular, when the first and second sub-portions have substantially the same surface area or the same surface area, it is expected that signals between cells propagate alternately in (i) the direction from the first sub-portion through the junction portion to the second subportion and in (ii) the direction from the second sub-portion to through the junction portion the first sub-portion. When the surface area of the first sub-portion is smaller than the surface area of the second sub-portion, the cells in the first sub-portion would become a signal propagation point, i.e. the cells in the first sub-portion would initiate signals. The signals would then propagate in the direction from the first sub-portion through the junction portion and to the second sub-portion.

[0055] As discussed also in the first embodiment, if the cells are patterned in a polygonal shape, the cells near the vertex can become the signal propagation point. Therefore, the rounded corners of the first and second sub-portions according to the second embodiment may effectively prevent from that cells at the corners (and / or in vicinity of the corners) would behave as signal initiators, and may further ensure that the cells located in the first subportion would induce signals and the signals propagate in the direction from the first subportion to the second sub-portion.

[0056] When the first sub-portion (2a-i) and the second sub-portion (2a-2) have the same length in the x-direction, it may be further advantageous in that the intercellular signal propagation can be more accurately controlled and the signal may propagate homogeneously in the y-direction.

[0057] The stack of the cell layer (C) (i.e. a part of the cell layer formed on the first part) and the first part (2a) of the polymer compound layer (2) may be further removed from the base layer (1) and further integrated in a biological system such as an artificial organ and / or biosensors.

[0058] The use of the substrate is not limited to cell culture and the substrate may be also used to pattern various other biological materials and / or molecules, such as proteins and / or peptides and / or nucleotides such as DNA and / or lipids, and such micro-patterned biological materials and / or molecules may be for example further applied to biosensing technology. The substrate may be further used for regenerative medicine (for example stem cell differentiation such as differentiation of human mesenchymal stem cells) and / or applications for understanding of the impact of the microenvironment on cells and the interaction between cells and protein and / or single cell assay and / or organoid formation and / or spheroid formation.Examples

[0059] Certain aspects and embodiments of the present disclosure will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only.

[0060] < Sample preparation>A glass slide (120 pm thickness, 20 x 20 mm2square), which is an example of the base layer in the embodiments, was exposed to oxygen plasma for 10 mins. This process is for making glass hydrophilic and removes organic / inorganic contaminants. Thereafter, Na-alginate was spin coated (1500 rpm speed) into a thickness of 0.4 pm. The Na+ions were then exchanged with Ca2+ions by immersing the Na-alginate layer in Calcium solution, thereby forming a Ca-alginate layer, which is an example of an adhesive layer in the embodiments. A parylene- C was coated via chemical vapor deposition on the Ca-alginate layer into a thickness of 1.09 pm. The parylene-C layer is an example of the polymer compound layer in the embodiments.

[0061] < Lithography process >A lithography process was then applied on the parylene-C layer: A positive photoresist (S1813G) was spin coated (3000 rpm speed) on the parylene-C layer, and then baked at 90 °C for 2 min. The positive photoresist layer was exposed in light (365 nm wavelength, i2o mJ / cm2dose) under a mask and was developed with 6:1 diluted NaOH solution (Microposit™ 351) for 1 min.

[0062] < Oxygen plasma etching>The sample was then exposed under oxygen plasma (oxygen flow rate 20 ml / min with 50W power) to etch a part of the parylene-C layer and the Ca-alginate layer, at which the positive photoresist was removed by the lithography process, to form a slit part.

[0063] The photoresist layer was then removed in acetone, so that the patterned parylene-C layer with the slit portion was remained on the glass slide.

[0064] Fig. 5A shows a top view of a substrate sample (Sample 1), which is an example of the first embodiment. The top view of the first portion (2a) is formed into a triangular shape, which is separated from the second portion (2b) by the slit portion (2c). Each of the sides of the triangular shape is 233 pm.

[0065] Fig. 5B shows a part of a top view of another substrate sample (Sample 2), which is an example of the second embodiment. Cell culture was performed on the Sample 2 having the first portion (2a) with the first square sub-portion with 200 pm on each side (2a-i), the junction portion with 50 pm width and too pm length (23-3), the second rectangular subportion with 200 pm x 2000 pm on sides (2a-2): The sample substrate was sterilized under UV for 5 min and additionally washed with ethanol. The parylene-C layer was then immersed in fibronectin-collagen mixture solution for overnight to form a fibronectin-collagen-coated layer, which is an example of a coating layer in the embodiment. Cell-lines of cardiomyocytes (HL-i) or Human derived iPSCs cells (0.22 million cells) were seeded on the fibronectincollagen layer and incubated for 1 week. (Fig. 5B).

[0066] The second portion (2b) of the Sample 2 surrounding the first portion (2a) was then manually peeled off from the glass slide, thereby the first portion (2a) remained attached on the glass slide. At least a part of the Ca-alginate layer was remained attached on the glass slide. The cells on the first portion (2a) were stained with a fluorescent dye (Cal- 520® AM) to observe calcium signals (Fig. 5D). The fluorescence image was then observed by a fluorescence microscope (excitation wavelengths at 493 m, emission wavelength at 515 nm).

[0067] The Ca-imaging showed that the cells were successfully incubated on the polymer compound layer and further micro-patterned into the shape of the first portion (2a). We further observed calcium signal propagations through the micro-patterned cells (Figs. 6Ato 6C). The cells adhered to the first sub-portion (2a-i) initiates calcium signals (at t=o, Fig. 6A) and then propagates into the direction from the first sub-portion (2a-i) to the second sub-portion (2a-2) via the junction portion (23-3), as marked with triangular marks in Figs. 6B and 6C (Figs. 6B and 6C are fluorescence images at t= 0.4 sec and t=o.9 sec, respectively).

[0068] The calcium signal propagation can be also observed on the cell layer patterned by the Sample 1 into the triangular shape, by using the same method as demonstrated for the cell layer micropatterned by the Sample 2.

[0069] List of reference signs1: base layer, 2: polymer compound layer, 3: adhesive layer, 2a: first portion, 2b: second portion, 2c: slit portion, 4: photo resist layer, C: cell layer, 2a-i: first sub-portion, 2a-2: second subportion, 23-3: junction portion

Claims

Claims1. A substrate for cell and / or tissue culture and for micro-patterning cells and / or tissues, wherein the substrate comprises a base layer (1), and a polymer compound layer (2), wherein each of the base layer (1) and the polymer compound layer (2) has an upper surface and a lower surface, each of the upper surface and the lower surface extending in an xy-plane, wherein each of the base layer (1) and the polymer compound layer (2) has a thickness in the z-direction being perpendicular to the xy-plane, wherein the polymer compound layer (2) is stacked on the upper surface of the base layer, wherein the polymer compound layer (2) comprises a first portion (2a), a second portion (2b) and a slit portion (2c), wherein the slit portion (2c) extends along the z-direction from the upper surface of the polymer compound layer (2) through the polymer compound layer (2), thereby at least partly separating the first portion (2a) from the second portion (2b), so that the second portion (2b) is removeable from the upper surface of the base layer (1) independently from the first portion (2a).

2. The substrate according to claim 1, wherein the first portion (2 a) is completely separated from the second portion (2b), and / or wherein the first portion (2 a) is completely surrounded by the second portion (2b).

3. The substrate according to any one of claims 1 or 2, wherein the first portion (2a) is removeable from the upper surface of the base layer (1) independently from the second portion (2b).

4. The substrate according to any one of the preceding claims further comprisingan adhesive layer (3) between the upper surface of the base layer (1) and the lower surface of the polymer compound layer (2), wherein preferably the slit portion (2c) further extends through the adhesive layer (3).

5. The substrate according to any one of the preceding claims further comprising a coating layer being stacked on the upper surface of the polymer compound layer (2), wherein the coating layer includes proteins and / or peptides.

6. The substrate according to any one of the preceding claims, wherein the upper surface of the first portion (2a) has a polygonal shape, wherein the polygonal shaped first portion has n vertices, wherein n is preferably an odd number.

7. The substrate according to any one of the preceding claims, wherein the first portion (2 a) comprises a first sub-portion (2a-i), a junction portion (2a-3) and a second sub-portion (2a-2), being arranged in this order in the y-direction, wherein the first sub-portion (2a-i) and the second sub-portion (2a-2) are connected by the junction portion (2a-3), wherein the first sub-portion (2a-i) has a first length (L) in the x-direction, wherein the second sub-portion (2a-2) has a second length (12) in the x-direction, wherein the junction portion (2a-3) has a third length (13) in the x-direction, wherein the third length (13) is shorter than each of the first length (L) and the second length (12).

8. The substrate according claim 7, wherein the area of the upper surface of the first sub-portion (2a-i) is equal to or smaller than the area of the upper surface of the second sub-portion (2a-2).

9. The substrate according to claims 7 or 8, wherein the first sub-portion (2a-i) has a first width (wi) in the y-direction, wherein the second sub-portion (2a-2) has a second width (w2) in the y-direction, wherein the junction portion (2a-3) has a third width (w3) in the y-direction, wherein the width of the first portion (2a) being the sum of the first width (wi), the second width (w2) and the third width (w3) is longer than the first length (li) , wherein preferably the first width (wi) is shorter than the second width (w2).

10. The substrate according to any one of claims 7 to 9, wherein the upper surface of the first sub-portion (2a-i) has a circular shape or an oval shape, and the upper surface of the second sub-portion (2a-2) has a circular shape or an oval shape, wherein the junction portion (2a-3) has a third length (13) in the x-direction, wherein the third length (13) is shorter than each of the maximum length (li) of the first sub-portion (2a-i) in the x-direction and the maximum length (12) of the second subportion (2a-2) in the x-direction.

11. The substrate according to any one of claims 7 to 9, wherein the upper surface of the first sub-portion (2a-i) has a polygonal shape, wherein each of corners of the polygonal shape is rounded, wherein preferably the upper surface of the first sub-portion (2a-i) has a rectangular shape with rounded corners, and / or wherein the upper surface of the second sub-portion (2a-2) has a polygonal shape, wherein each of corners of the polygonal shape is rounded, wherein preferably the upper surface of the second sub-portion (2a-2) has a rectangular shape with rounded corners.

12. The substrate according to any one of the preceding claims,wherein the polymer compound layer (2) comprises paiylene and / or polyimide and / or photopolymer.

13. A method for manufacturing a substrate according to any one of claims 1 to 12 comprising a step of forming the polymer compound layer (2) on the upper layer of the base layer (1), a step of forming the slit portion (2c) thereby separating the first portion (2a) from the second portion (2b).

14. The method according to claim 13, wherein the step of forming the slit portion (2c) is performed by a photolithography process, or wherein the step of forming the slit portion (2c) is performed by irradiating a laser beam onto the polymer compound layer (2).

15. The method according to claims 13 or 14 further comprising a step of forming the adhesive layer (3) on the upper surface of the base layer, prior to the step of forming the polymer compound layer (2).

16. The method according to any one of claims 13 to 15 further comprises a step of forming the coating layer on the upper surface of the polymer compound layer (2).

17. A use of a substrate according to any one of claims 1 to 12 for cell incubation and micropatterning of cells, and / or immobilizing nucleotides, and / or biosensors, and / or medical devices, and / or regenerative medicine, and / or organoid formation, and / or spheroid formation.

18. A method of micro-patterning cells or tissues by using a substrate according to any one of 1 to 12 comprising a step of seeding and incubating cells and / or tissues on the polymer compound layer (2) of the substrate thereby forming a cell layer (C), a step of removing a stack of the second portion (2b) and a first part of the cell layer (C) from the upper surface of the base layer (1), the first part of the cell layer being adhered to the second portion (2a).

19. The method according to claim 18 further comprising a step of forming a coating layer on the upper surface of the polymer compound layer (2) prior to the step of seeding and incubating cells and / or tissues.

20. The method according to 18 or 19 further comprising a step of removing a stack of the first portion (2a) and a second part of the cell layer (C) from the upper surface of the base layer (1), the second part of the cell layer (C) being adhered to the first portion (2 a).

21. A micro-patterned cell layer obtained by the method of claims 18 or 19.

22. A system comprising a plurality of a substrate according to any one of claims 1 to 11.

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