Fractals in tissue engineering

Abstract

The present disclosure relates to a method for producing three-dimensional cell clusters on an inorganic cell culture platform comprising a three-dimensional structure, preferably a fractal structure. Such three-dimensional structures are preferably useful for culturing cells and tissues in three dimensions. Such three-dimensional structures are preferably useful for inducing differentiation of non-embryonic stem cells. In particular, such three-dimensional (3D) structures are useful for culturing primary tissue cells.

Description

[Technical Field]

[0001] The present disclosure relates to a method for producing three-dimensional cell clusters on an inorganic cell culture platform comprising a three-dimensional structure, preferably a fractal structure. Such three-dimensional structures are preferably useful for culturing cells and tissues in three dimensions. Such three-dimensional structures are preferably useful for inducing differentiation of non-embryonic stem cells. In particular, such three-dimensional (3D) structures are useful for culturing primary tissue cells. [Background technology]

[0002] Biology, drug discovery, disease, and physiology research is often carried out in cell culture by studying cells or cell lines. In vitro cell culture is one of the breakthrough tools we have used to understand biology in health and disease. In vitro cell culture provides an accessible, controlled environment in which to study cells and perform experiments.

[0003] Over the past few decades, various cell culture techniques and cell culture templates have been developed. The majority of experiments in biology and medicine are performed in 2D cell culture. However, the growth of 3D cell cultures (spheroids) and organoids is believed to better mimic the interactions and behavior of cells in the body. This has led to the belief that in vitro 3D experiments may partially replace in vivo experiments. Another important area of ​​3D cell culture is tissue engineering, which aims to "create functional 3D tissues using scaffolds or devices that promote cell growth, organization, and differentiation."

[0004] Growth of cells in 3D as multicellular organoid complexes, preferentially as cocultures of different cell types, is still in its infancy because it usually requires specific surface modifications or culture conditions. To convert two-dimensional (2D) cell cultures to 3D, prevention of adhesion in liquid cell cultures (floating spheroids) or introduction of cells into a gel matrix is ​​required. Floating spheroids are achieved by increasing the hydrophobicity of the culture dish surface or preventing general adhesion (e.g., hanging drop culture, continuous stirring of cell suspensions, or deposition of low-adhesion polymers). Nanostructuring is used in some molds as a "coating" to introduce patterns that prevent cell attachment. 3D spheroids are formed by seeding cells into dense materials in hydrogels (e.g., Matrigel, alginate, collagen).

[0005] US 2002 / 182241 describes the preparation of a three-dimensional mold or scaffold that mimics blood vessels and serves as a template for cell adhesion and growth. Example 1 of US 2002 / 182241 describes the preparation of a scaffold from a silicon or Pyrex wafer, whereby a layer of silicon dioxide is deposited on the silicon wafer, followed by anisotropic etching of the silicon wafer to form channels. After etching, the silicon dioxide is removed, and cells are seeded and grown directly on the etched silicon or Pyrex. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US 2002 / 182241 Summary of the Invention [Problem to be solved by the invention]

[0007] These complex coating and culturing techniques, in addition to other drawbacks (long growth times, limited availability, or low numbers of spheroids), somehow limit the standardized use of 3D cell culture despite its usefulness, especially in terms of predictability for medical applications. Thus, there is a need for cell culture templates that allow cells to grow in three dimensions and can be used without prior surface treatment to better mimic the cells' natural conditions in vivo. [Means for solving the problem]

[0008] The present disclosure provides the following preferred embodiments.

[0009] The present disclosure provides a method for producing a cell culture mold having at least one three-dimensional structure having a surface for supporting a cell culture, the at least one three-dimensional structure being preferably a fractal structure, preferably produced by micro- and nano-fabrication, comprising the steps of: Step 1: Providing a single crystal substrate, preferably a single crystal silicon substrate; Step 2: removing at least one geometric feature from the single crystalline substrate to create a geometric cavity, preferably an octahedral cavity or a portion of an octahedral cavity, in the single crystalline substrate that will be the starting point for a three-dimensional structure, preferably forming one or more vertices; Step 3: growing and / or depositing a base three-dimensional structure material, preferably silicon oxide, preferably amorphous silicon dioxide, on the surfaces of the geometric features in the substrate to form the three-dimensional structure; Step 4: bonding at least one three-dimensional structure to the surface of a support base, preferably borosilicate glass, in particular whereby the support base is bonded to the at least one three-dimensional structure at the surface on which the base three-dimensional structure material was grown or deposited; Step 5: Removing a mass of the single-crystalline substrate around at least one three-dimensional structure; Including, After removing the bulk of the monocrystalline substrate, a method is provided in which cells are placed on the surface of at least one three-dimensional structure under conditions that allow growth of cells to create a cell culture template, particularly whereby the cells are placed on the at least one three-dimensional structure at a surface that comprises the underlying three-dimensional material.

[0010] Preferably, the method comprises the following steps: Step 6: Treating the single crystal substrate to form a protective layer compatible with the next step; Step 7: creating one or more holes in the protective layer, preferably at each of one or more vertices, to accommodate subsequent steps; Step 8: removing at least one geometric feature, preferably an octahedron or a portion of an octahedron, in the single-crystalline substrate through one or more holes, and then stripping off the protective layer; Further comprising: Steps 6 to 8 are performed between steps 2 and 3 of the method of claim 1, and optionally steps 6 to 8 are repeated one or more times to create at least one three-dimensional structure having a higher level of complexity; Preferably, steps 6 to 8 of the method are repeated 2 to 10 times, preferably 2 to 5 times, to create three-dimensional structures with higher complexity.

[0011] The protective layer is preferably a basic three-dimensional structural material as described herein, preferably silicon oxide or silicon nitride, more preferably silicon dioxide.

[0012] The method for producing a cell culture template comprising at least one three-dimensional structure as described herein, wherein preferably, the cavities formed in the single crystalline substrate in step 2 are accessible from the outside of the substrate through openings provided in the substrate by the directional step before removal, and preferably, the openings in the substrate have a width that is relatively large compared to the average width of the cavities, and more preferably, the openings form the widest portions of the cavities formed in the substrate.

[0013] A method for fabricating a cell culture mold comprising at least one three-dimensional structure described herein, preferably, the removing step is performed by anisotropic etching.

[0014] A method for fabricating a cell culture mold comprising at least one three-dimensional structure described herein, preferably, the prepared single-crystalline substrate is silicon, whereby a layer of silicon oxide, preferably amorphous silicon dioxide, is formed by thermal oxidation, whereby, in step 3, a layer of silicon dioxide is deposited, whereby, in step 5, the silicon mass around the formed three-dimensional structure is removed.

[0015] A method for fabricating a cell culture mold comprising at least one three-dimensional structure described herein, preferably, step 7 is omitted at the final stage of the preparation, and a three-dimensional structure with a closed vertex is fabricated.

[0016] A method for fabricating a cell culture mold comprising at least one three-dimensional structure described herein, preferably, the three-dimensional structure comprises a surface defining a regular pattern of protrusions, the protrusions are constructed from an octahedral structure, and the octahedral structure narrows towards the outside of the three-dimensional structure.

[0017] Preferably, the three-dimensional structure has the following topography: - Cone (G0), - Cone having an octahedron (G1) at the vertex, - Cone having an octahedron at the vertex and a second-layer octahedral structure (G2) at each vertex of the octahedron, - Cone having an octahedron at the vertex, a second-layer octahedral structure at each vertex of the octahedron, and a third-layer octahedral structure (G3) at each vertex of the second layer, or - Cone having an octahedron at the vertex, a second-layer octahedral structure at each vertex of the octahedron, a third-layer octahedral structure at each vertex of the second layer, and a fourth-layer octahedral structure (G4) at each vertex of the third layer. - A cone having an octahedron at a vertex, a second-level octahedral structure at each vertex of the octahedron, a third-level octahedral structure at each vertex of the second level, a fourth-level octahedral structure (G4) at each vertex of the third level, and an n-th level octahedral structure (Gn) [n is 5 to 10] at each vertex of the n-1th level. A method for producing a cell culture template comprising at least one three-dimensional structure as described herein, the method comprising:

[0018] Preferably, the method for making a cell culture template comprising at least one three-dimensional structure as described herein, wherein the three-dimensional structure is sterilized before growing cells, preferably the three-dimensional structure is sterilized by any one of UV, chemical means and high temperature treatment.

[0019] Preferably, the method for making a cell culture template comprising at least one three-dimensional structure as described herein, wherein the at least one three-dimensional structure comprises a plurality of three-dimensional structures, and the plurality of three-dimensional structures are disposed on the surface of the support base in a lattice configuration, preferably a square or hexagonal lattice configuration.

[0020] Preferably, the method for making a cell culture template described herein, wherein a block of monocrystalline substrate is partially etched away, leaving the remaining substrate at least partially covering at least one of the plurality of three-dimensional structures.

[0021] Preferably, the method for making the cell culture mold described herein, wherein a block of monocrystalline substrate is partially etched away to create multiple compartments exposing one or more three-dimensional structures.

[0022] A method for producing a cell culture template comprising at least one three-dimensional structure as described herein, preferably wherein the cells are in the form of a tissue or organoid.

[0023] Preferably, the method for making a cell culture template comprising at least one three-dimensional structure as described herein, wherein the cell culture template further comprises at least one insulator, preferably wherein the insulator is an amorphous silicon dioxide three-dimensional structure.

[0024] Preferably, the method for making a cell culture template comprising at least one three-dimensional structure as described herein, wherein the cell culture template further comprises at least one metal portion, preferably wherein the metal portion is embedded or patterned within the three-dimensional structure.

[0025] Preferably, the three-dimensional structure is used for external stimulation of the culture. A method for producing a cell culture template comprising at least one three-dimensional structure described herein.

[0026] Preferably, a method for producing a cell culture template comprising at least one three-dimensional structure as described herein, wherein an electrode is used for cell stimulation, and preferably at least a part of the three-dimensional structure functions as an electrode.

[0027] A method for making a cell culture template comprising at least one three-dimensional structure as described herein, preferably wherein the vertices are open and solutions are supplied into the cell culture through these vertices.

[0028] The present disclosure provides a cell culture template for growing and maintaining cell cultures, particularly cell cultures comprising primary cells, the cell culture template comprising cells seeded on a cell growth surface, e.g., an amorphous silicon dioxide surface, defined by at least one three-dimensional fractal structure supported on a layer of a support base, e.g., borosilicate glass.

[0029] Preferably, a cell culture template as described herein, the surface of which is defined by a multitude of, preferably at least nearly identical, three-dimensional fractal structures evenly distributed on the support layer.

[0030] Preferably, a portion of the numerous three-dimensional fractal structures on the support layer are covered with a monocrystalline substrate, and other three-dimensional fractal structures of the numerous three-dimensional fractal structures are exposed, i.e., do not have monocrystalline properties, to form a cell growth surface, in the cell culture template described herein.

[0031] Preferably, the cell culture template described herein, wherein the monocrystalline substrate is arranged to define one or more cell growth compartments having one or more exposed fractals.

[0032] Preferably, the cell culture mold described herein is provided with a lid on the side of the cell layer opposite the cell growth surface that is on top of the monocrystalline substrate and supported by the monocrystalline substrate.

[0033] The present disclosure provides a method for culturing cells, comprising the steps of providing a cell culture template obtainable by the method of the present invention and culturing cells.

[0034] Preferably, the method for culturing cells or tissues described herein, wherein the cells are primary cells, preferably primary tumor cells.

[0035] Preferably, the methods for culturing cells or tissues described herein, wherein the cells are primary cells, preferably primary tissue cells.

[0036] Preferably, the method for culturing cells or tissues described herein, wherein the cells are cancer-associated fibroblasts (CAFs).

[0037] Preferably, the methods for culturing cells or tissues described herein, wherein the cells are cancer-associated fibroblasts (CAFs) activated by the material, shape, and / or pattern of the three-dimensional structure.

[0038] Preferably, the method for culturing cells or tissues as described herein, wherein the cells are stem cells, preferably mesenchymal stem cells, adult stem cells, adipose adult stem cells and / or induced pluripotent stem cells.

[0039] Preferably, the methods for culturing cells or tissues described herein, wherein the cells form multicellular organoids or tissues.

[0040] Preferably, the method for culturing cells or tissues described herein, wherein the cells undergo stem cell differentiation initiated by the cone-shaped shape and distance of the three-dimensional structure.

[0041] Preferably, the methods for culturing cells or tissues described herein, wherein the cells are grown and stored under non-optimal growth conditions.

[0042] The present disclosure further provides a cell culture template comprising at least one three-dimensional structure obtainable by the method described herein, the three-dimensional structure being composed of amorphous silicon dioxide and cells attached to the structure. Preferably, the three-dimensional structure of amorphous silicon dioxide is composed of SiO2.

[0043] The present disclosure provides a method for fabricating three-dimensional structures for cell culture, preferably fractal structures, fabricated by micro- and nano-fabrication, comprising: Step 1: Providing a single crystal substrate, preferably a single crystal silicon substrate; Step 2: removing at least one geometric feature from the single crystalline substrate to form a geometric cavity, preferably an octahedral cavity or a portion of an octahedral cavity, in the single crystalline substrate, which is the starting point for a three-dimensional structure, preferably forming one or more vertices; Step 3: growing and / or depositing a base three-dimensional structure material, preferably silicon oxide, preferably amorphous silicon dioxide, on the surfaces of the geometric features in the substrate to form the three-dimensional structure; Step 4: Attaching at least one three-dimensional structure to the surface of a support substrate, preferably borosilicate glass; Step 5: Removing a mass of the single-crystalline substrate around at least one three-dimensional structure; Including, After removing the bulk of the single crystalline substrate, placing cells on the surface of at least one three-dimensional structure under conditions that allow growth of cells to form a cell culture template; Optionally, the following steps: Step 6: Treating the single crystal substrate to form a protective layer compatible with the next step; Step 7: creating one or more holes in the protective layer, preferably at each of one or more vertices, to accommodate subsequent steps; Step 8: removing at least one geometric feature, preferably an octahedron or a portion of an octahedron, in the single-crystalline substrate through one or more holes, and then stripping off the protective layer; Further comprising: Further provided is a method in which steps 6-8 are performed between steps 2 and 3, and optionally steps 6-8 are repeated one or more times to create at least one three-dimensional structure having a higher hierarchy of complexity. [Brief explanation of the drawings]

[0044] [Figure 1] Starting structure: A single-crystalline substrate is etched by anisotropic etching to remove at least one geometric feature or a portion of one geometric feature, thereby generating a geometric cavity. The geometric cavity shown is an octahedral cavity or a portion of an octahedral cavity. This cavity is the start of a three-dimensional structure, thereby preferably forming one or more vertices. In the middle illustration, the octahedral cavity in the single-crystalline substrate is widely accessible to the exterior of the substrate. In the right illustration, the octahedral cavity in the single-crystalline substrate has the widest point of the octahedral shape as the opening, and is accessible to the exterior of the substrate. G1: A schematic representation of the second stage of anisotropic etching, which creates an octahedral cavity at each vertex of an existing cavity in the single-crystalline substrate. [Figure 2] Scanning electron microscope images of amorphous silicon dioxide fractals. A) Square array with 20 μm pitch, B) Hexagonal array with 12 μm pitch, C) G0, D) G1, E) G2, F) G3, G) G4 structures. The size bars in A) and B) represent 20 μm, while the size bars in C)-G) are 2 μm. [Figure 3]CAFs 13 days after seeding on a hexagonally arranged inorganic fractal surface. A) Control, B) G0, C) G1, D) G2, E) G3, F) G4. The blue fluorescent signal is due to DAPI staining of the nuclei, and the red fluorescence is associated with TRITC-phalloidin, which labels actin filaments of the cytoskeleton. The underlying fractals were visualized by transmitted light. The arrows indicate elongated nuclei. The size bar represents 100 μm. [Figure 4] CAFs 8 days after seeding on a square array inorganic fractal surface. Nuclei are stained with DAPI (blue), and actin filaments are stained with TRITC-phalloidin (red). The size bar in the fluorescence microscope image represents 100 μm, and in the EM image represents 20 μm. [Figure 5] A close-up of CAFs grown for 8 days on a G3 square configuration. Nuclei are stained with DAPI (blue) and actin filaments with TRITC-conjugated phalloidin (red). Lamellipodia are brighter red due to actin accumulation. Nuclei are elongated but located between fractals. [Figure 6] Light microscopy of (A) CAFs at day 1 and (B) tumor spheroids on CAF cells at day 6 of culture on G0Sqr. [Figure 7] Optical microscopy of hADSCs grown on a square configuration after 24 hours (middle panel) and 48 hours (bottom panel). The top panel shows the corresponding fractal structure. [Figure 8] Human adipose-derived stem cells (hADSCs) after one day of culture on G2Hex. The green signal indicates nestin, a biomarker for neurospheres, and the red signal indicates the presence of NeuN, a nuclear marker for mature neurons. The blue signal is due to nuclear staining. [Figure 9] (Top panel) Optical microscopy images of COLO205 on various fractal-structured surfaces 48 hours after seeding. Cells only form 2D cell sheets. (Bottom panel) Cells also grow in sheets on low-cell-adhesive PEG6000 (Carlo Erba) coatings. [Figure 10]Selective opening of thermally grown amorphous silicon dioxide at the apex of a pyramidal depression after HF etching. Note that the stress-induced oxidation retardation is more evident at the inside corners where more than two planes intersect. [Figure 11A] Top and middle rows: 3D and top-view schematic representations of two, three, and four intersecting (111)-Si planes. Bottom row: Top-view SEM images of the intersections of two, three, and four (111)-Si planes when etched in HF: time-dependent opening of the vertices is visible. [Figure 11B] Residual oxide thickness at the apex and ribbon as a function of etching time in 1% HF (starting oxide thickness 160 nm (left) or 88 nm (right)): within the time window Δt, only the apex is open. Processing can be continued to achieve a starting oxide thickness of 25 nm. [Figure 12] The three-dimensional structure is bonded to a glass surface. The single-crystalline substrate may then be thinned before etching the single-crystalline substrate. The single-crystalline substrate can be partially etched away, making portions of the three-dimensional structure available for, for example, cell culture purposes. [Figure 13] Analysis of epithelial, stem cell, and mesenchymal markers in CAF-enriched cell populations isolated from HCC primary tumors of three patients (P1, P2, P3). The percentage of positive cells and / or mean fluorescence intensity (MFI, expressed as arithmetic (A-Mean) and geometric (G-Mean) means) of the antibody-stained cell population are reported. Fluorescence values ​​were normalized to control / isotype-related signals. [Figure 14A] Isolation of CAFs from a primary liver cancer, first passage in 2D cell culture, stained with antibodies against vimentin (red) and α-SMA (green), a marker of activated fibroblasts. Nuclei were stained with DAPI (blue). [Figure 14B] Cell clusters and spheroids formed by enriched CAFs isolated from liver cancers of three patients and cultured for 6 days on G0Hex. 100 μm. [Figure 15A]Spheroids grown on a G0Hex template. Z-stack of confocal micrographs of two spheroids on a layer of α-SMA (red)-positive 2D CAFs. Nuclei were stained with DAPI (blue). [Figure 15B] Spheroids grown on G0Hex templates. Confocal image of spheroids. Tumor cells are positive for AFP (green) and are surrounded by CAFs (arrows) that are positive for α-SMA (red). The 2D cell layer consists of CAFs, connecting the tumor with the cell layer. DAPI stains the nuclei (blue). [Figure 16] Cells on the fractal template were stained for α-SMA (red), AFP (green), and nuclei (DAPI, blue). (A) Peritumoral tissue on G0Hex. Absence of AFP signal due to absence of tumor cells. (B) Tumor tissue on G0Sqr and (C) tumor tissue on G1Hex. Absence of AFP signal due to exclusive growth of CAFs. Scale bar indicates 100 μm. [Figure 17] (A) Epifluorescence image of spheroids grown from the HLF cell line on G0Hex at day 4. The inset shows only the DAPI signal, and the arrows indicate the size of the spheroids considered for size distribution as an example. (B) Illustration of the spheroid size distribution on a fractal, determined by image analysis with ImageJ. (C) Light microscopy of HLF cell spheroids embedded in Matrigel at day 13. (D) Illustration of the size distribution of spheroids in Matrigel, determined by image analysis with ImageJ. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present disclosure provides a method for producing three-dimensional cell clusters on inorganic cell culture template, which comprises three-dimensional structure, preferably fractal structure.The cell culture template described herein can contribute to the cell culture and / or tissue engineering of primary cells.Cell culture template can be used for various cell culture purposes, such as 3D cell culture, stem cell differentiation induction and multicellular organoid culture.

[0046] The present disclosure provides a method for producing a cell culture mold having at least one three-dimensional structure having a surface for supporting a cell culture, the at least one three-dimensional structure being preferably a fractal structure, preferably produced by micro- and nano-fabrication, comprising the steps of: Step 1: Providing a single crystal substrate, preferably a single crystal silicon substrate; Step 2: removing at least one geometric feature from the single crystalline substrate to create a geometric cavity, preferably an octahedral cavity or a portion of an octahedral cavity, in the single crystalline substrate that will be the starting point for a three-dimensional structure, preferably forming one or more vertices; Step 3: growing and / or depositing a base three-dimensional structure material, preferably silicon oxide, preferably amorphous silicon dioxide, on the surfaces of the geometric features in the substrate to form the three-dimensional structure; Step 4: Attaching at least one three-dimensional structure to the surface of a support substrate, preferably borosilicate glass; Step 5: Removing a mass of the single-crystalline substrate around at least one three-dimensional structure; Including, A method is provided for creating a cell culture mold by removing the bulk of the monocrystalline substrate and then placing cells on the surface of at least one three-dimensional structure under conditions that allow cells to grow.

[0047] Preferably, the method comprises the following steps: Step 6: Treating the single crystal substrate to form a protective layer compatible with the next step; Step 7: creating one or more holes in the protective layer, preferably at each of one or more vertices, to accommodate subsequent steps; Step 8: removing at least one geometric feature, preferably an octahedron or a portion of an octahedron, in the single-crystalline substrate through one or more holes, and then stripping off the protective layer; Further comprising: Steps 6 to 8 are performed between steps 2 and 3 of the method of claim 1, and optionally steps 6 to 8 are repeated one or more times to create at least one three-dimensional structure having a higher level of complexity; Preferably, steps 6 to 8 of the method are repeated 2 to 10 times, preferably 2 to 5 times, to create three-dimensional structures with higher complexity.

[0048] A cell culture template is a product that can be used to culture and grow cells. In particular, the term "cell culture template" refers to a three-dimensional structure, particularly a scaffold, prepared by the method of the present invention, on which cells can be cultured and grown. A cell culture template comprises at least one template that can be used to grow cells in a cell culture medium. The template comprises a surface to which cells can adhere.

[0049] The cell culture template of the present disclosure includes at least one three-dimensional structure. Such a three-dimensional structure can be placed on the surface of the template. The structure can rise above the surface to increase the surface area. Preferably, the structure has a maximum height above the surface between 0.1 and 50 μm. In a preferred embodiment, the structure is oriented perpendicular to the base and has dimensions ranging from 1 nm to 100 μm, preferably 50 nm to 50 μm. In a preferred embodiment, the volume and area of ​​the three-dimensional structure are defined by the size of the first geometric cavity, and preferably the surface dimensions of the first geometric shape, also referred to as the footprint, are between 1 and 2500 μm. 2 It is between.

[0050] The cells in the cell culture template may attach to the three-dimensional structure, preferably a 3D nanostructure having nanopart structures.

[0051] In a preferred embodiment, the three-dimensional structure in the cell culture template is a fractal structure. A fractal structure exhibits similar patterns at different scales, which is called self-similar. The term "fractal," as used herein, means and includes a pattern (i.e., shape or geometric arrangement) that can be repeatedly divided into similar parts or repeatedly amplified into more meaningful parts that are identical or similar to the original pattern (i.e., shape or geometric arrangement).

[0052] The three-dimensional structure(s) of the cell culture template are fabricated by micro- and nano-fabrication. In microtechnology, the term "micro" means that the relevant dimensions are in the micrometer range, preferably, but not limited to, less than 100 μm. In nanotechnology, the term "nano" means that the relevant dimensions are less than 100 nm. In this application, the term "nano" also encompasses structures with relevant dimensions up to several hundred microns (μm), preferably between 100 and 10 microns (μm). The lower limit is about 1 nm, preferably about 5 or 100 nm.

[0053] The three-dimensional structures produced have a size between 10 nm and 100 μm. In a preferred embodiment, the three-dimensional structures have a size between 1 and 50 μm, more preferably between 1 and 25 μm.

[0054] The three-dimensional structure of the cell culture mold is created using a monocrystalline substrate. A single crystal, or monocrystalline solid, is a material in which the crystal lattice throughout the sample is continuous and unbroken toward the edge of the sample, with no grain boundaries. A monocrystalline substrate is composed of a single crystal throughout, while a polycrystalline substrate is composed of agglomerates of very small crystals with random orientation. Examples of monocrystalline materials are monocrystalline silicon, sapphire, quartz, Ge (germanium), or GaN (gallium nitride).

[0055] In a preferred embodiment, the single-crystalline substrate is single-crystalline silicon. Single-crystalline silicon is also called monocrystalline silicon, abbreviated as mono-c-Si or mono-Si. It is composed of silicon whose crystal lattice is continuous and unbroken to its edge throughout the solid, and does not contain any grain boundaries.

[0056] In the low-pressure SiO2 polymorphs quartz, tridymite, and cristobalite, and in their high-pressure polymorph coesite, silicon is tetrahedrally coordinated by oxygen. In the high-pressure SiO2 polymorph stishovite, silicon is coordinated by six oxygen atoms.

[0057] To create the three-dimensional structure, at least one or more geometric features are removed from the single crystalline substrate. The geometric features can have various shapes, such as pyramids, octahedrons, tetrahedrons, cubes, rectangular prisms, or cones. Preferably, the geometric features have one or more vertices. In a preferred embodiment, the geometric features have an octahedral shape.

[0058] In some embodiments, the geometric features can be partially removed from the substrate. For example, three-quarters, one-half, or one-quarter of the shape can be removed from the single crystalline substrate. After the geometric features are removed, a geometric cavity exists in the single crystalline substrate. This cavity is also referred to as the starting cavity. Figure 1 shows a schematic representation of a partially or fully removed octahedral structure in a single crystalline substrate.

[0059] In a preferred embodiment, the geometric cavity is an octahedral cavity in the single crystalline substrate that serves as the starting point for a three-dimensional structure, thereby preferably forming one or more vertices as depicted in FIG. 1 (starting structure).

[0060] Geometric features can be removed from a single-crystalline substrate by various methods for removing material. For example, the geometric features can be removed by a removal process performed by etching or drilling. Preferably, the removal of material from a single-crystalline substrate is performed using etching. For example, anisotropic etching is used to etch geometric cavities into the substrate. Anisotropic etching is a subtractive microfabrication technique that aims to remove material in a specific direction to obtain a geometric shape. Preferably, an etching technique can be used as anisotropic etching. Wet techniques utilize the crystalline properties of the structure to etch in a direction governed by the crystal orientation. In some embodiments, potassium hydroxide (KOH) is used for anisotropic etching of a single-crystalline substrate.

[0061] After the geometric features are removed from the single crystalline substrate, the resulting geometric cavities in the single crystalline substrate are treated to form a protective layer. In some embodiments, the basic three-dimensional structure material described herein is preferably silicon oxide or silicon nitride, more preferably silicon dioxide.

[0062] In some embodiments, the surfaces defining the cavity are formed by a layer of thermally grown oxide and a layer of silicon nitride. The silicon nitride layer can be applied by low-pressure chemical vapor deposition (LPCVD), followed by corner lithography and localized oxidation of the silicon. This is followed by selective stripping of the remaining nitride and the underlying thin oxide, followed by an anisotropic silicon etching step.

[0063] In another embodiment, the process for forming the protective layer is thermal oxidation. This amorphous silicon dioxide layer is grown to conform to the inside corners.

[0064] In some embodiments, the process for forming the protective layer is thermal oxidation. The formed geometric cavity is subjected to thermal oxidation at a temperature between 950 and 1500 degrees Celsius. At this temperature, the surface of the removed structure is oxidized. The resulting silicon oxide forms the protective layer. The thickness of the layer depends on the temperature and duration of the thermal oxidation step. In preferred embodiments, the oxide layer is at least 25 nm thick, with a preferred thickness being 160 nm. In some embodiments, the oxide layer is between 25 and 160 nm thick, and in more preferred embodiments, the oxide layer is between 88 and 160 nm thick.

[0065] In a preferred embodiment, the single-crystalline substrate is single-crystalline silicon. Thermal oxidation of the single-crystalline silicon results in a protective layer of silicon oxide. In a preferred embodiment, thermal oxidation of the silicon is performed at 1100 degrees Celsius. Oxidation of the silicon produces a conformal layer of silicon dioxide, preferably amorphous throughout the silicon crystal. This process results in a conformal layer around corners. At the intersections of multiple, e.g., three or four, planes, the oxide becomes sharp. This aspect leads to a powerful approach in which timed isotropic etching removes silicon oxide only from the vertices, while the oxide layer remains on the ribbons and planes. In some embodiments, processes such as plasma oxidation of silicon, anodization of silicon, or nitridation (by thermal conversion of silicon to nitride) can be applied to create the protective layer.

[0066] In the next step, holes are created at each and every vertex of the protective layer. These holes allow for the removal of additional layers of cavities to create multi-level three-dimensional structures. Various techniques can be used to create the holes, for example, corner lithography or timed isotropic etching.

[0067] In some embodiments, the holes are created by timed isotropic etching. In this technique, the holes are created by removing the protective layer only from the apex. This can be done by timed wet etching using hydrogen fluoride, for example, 1% hydrogen fluoride. Alternatively, other methods for hole processing, such as low-temperature oxidation and selective etching, can be applied.

[0068] Another step is performed to remove at least one geometric feature or a portion of one geometric feature in the single-crystalline substrate using one or more holes. In a preferred embodiment, the geometric shape is an octahedron. The removal is performed through one or more holes formed at one or more vertices. Figure 1 (G1) shows a schematic diagram of the second step of the removal, which creates octahedral cavities at each vertex of the existing cavities.

[0069] For example, the next stage of geometric cavities can be created by anisotropically etching in TMAH (tetramethylammonium hydroxide) to selectively etch the underlying silicon at each vertex, resulting in the formation of cavities at all vertices simultaneously.

[0070] A repeated sequence of anisotropic etching of the single-crystalline substrate, thermal oxidation, and isotropic etching of the protective layer to create pores results in a multi-level three-dimensional structure. In some embodiments, this sequence of fabrication method steps is repeated to create a three-dimensional structure with a higher level of complexity. Each subsequent layer of the structure will contain smaller geometric cavities.

[0071] After the growth and / or deposition of a protective layer on the removed geometric cavities, holes are created at each vertex of the geometrically shaped outer layer. The holes are used to perform another step of removing at least one geometric feature or a portion of one geometric feature having a geometric shape in the single-crystalline substrate. In a preferred embodiment, the geometric feature is an octahedron. The removal is performed through one or more holes formed at one or more vertices. After a new layer of the geometric cavities is formed, the protective material is stripped from the geometric cavities.

[0072] As an example, Figures 2a) and 2b) show top-view scanning electron microscope (SEM) images of two different layouts of starting structures composed of square or hexagonal lattices. Figure 2c) shows a perspective view of a single starting structural feature depicted in the rightmost image of Figure 1. Exemplary structures are shown for octahedral geometries. Figure 2C shows a simple three-dimensional structure that can be created in one removal step. Figure 2D shows a three-dimensional structure that can be created in two removal steps. Figure 2E shows a three-dimensional structure that can be created in three removal steps. Figure 2F shows a three-dimensional structure that can be created in four removal steps. And Figure 2G shows a three-dimensional structure that can be created in five removal steps.

[0073] Once the desired hierarchical complexity is reached, a new layer is grown and / or deposited throughout the geometric cavities. This layer can be made from a variety of materials. For example, the layer can be grown by oxidation or nitridation. Alternatively, the layer can be created by nitride or oxide deposition. Because cells are placed on the surface containing this layer to form at least one three-dimensional structure, the material must be compatible with cell growth. After removing the bulk of the monocrystalline structure, this created layer will form the three-dimensional structure. Therefore, this layer must be thick enough to create individual free-standing structures. Without wishing to be bound by theory, the material, the thickness of the material, and the morphology of the structure all contribute to the strength of the structure. The structure must be robust enough to support the cells that could potentially grow on it. In a preferred embodiment, the formed layer is at least 25 nm thick, more preferably at least 50 nm thick.

[0074] In some embodiments, the silicon undergoes thermal oxidation to form a layer. The formed geometric cavity is exposed to thermal oxidation at a temperature between 950 and 1500 degrees Celsius. At this temperature, the surface of the removed structure oxidizes, resulting in a silicon oxide layer. The thickness of the layer depends on the temperature and duration of the thermal oxidation step. In preferred embodiments, the oxide layer is at least 25 nm thick, with a preferred thickness being 160 nm. In some embodiments, the oxide layer is between 25 and 160 nm thick, and in more preferred embodiments, the oxide layer is between 88 and 160 nm thick.

[0075] After fabricating a three-dimensional structure, the outer side of the last grown or deposited layer forms the functional layer of the structure and becomes the outer surface. Cells use this outer surface to attach and / or grow on it. For example, when growing a layer by thermal oxidation, the layer grows from the surface of the cavity and grows outward. Thus, the outer layer that becomes the surface of the three-dimensional structure is finally formed.

[0076] The fabricated three-dimensional structure(s) are then attached to a surface, i.e., a support base, in particular the three-dimensional structure(s) are attached to a support base on the surface on which the base three-dimensional structure material was grown or deposited. Preferably, the surface is suitable for cell culture purposes. Suitable surfaces include ceramic, glass, or plastic surfaces, e.g. Ceramics: silicon nitride, alumina, zirconia, Glass: borosilicate glass and soda-lime glass, Polymers: polystyrene, permanox, polydimethylsiloxane In a preferred embodiment, one or more three-dimensional structures are attached to the surface of the borosilicate glass.

[0077] The fabricated three-dimensional structure or structures can be bonded to a surface by various techniques. In some embodiments, the structure is bonded by electrostatic bonding. In a preferred embodiment, the structure is bonded by anodic bonding, for example, anodic bonding with a Mempax glass wafer at 400°C.

[0078] The monocrystalline substrate mass is then removed from around the formed three-dimensional structure. The monocrystalline mass can be removed by a wet etching process. For example, the monocrystalline substrate mass, preferably silicon, is removed by prolonged exposure to tetramethylammonium hydroxide. At this stage, for example, the outside of the three-dimensional structure is accessible for cell attachment. After removing the monocrystalline substrate mass, cells are seeded and / or placed on the surface of the three-dimensional structure under conditions that allow growth to form a cell culture template. In particular, cells are placed on at least one three-dimensional structure on a surface comprising a base three-dimensional material, particularly silicon oxide or silicon nitride, more particularly silicon dioxide or silicon nitride.

[0079] In vitro culture of cells and tissues requires a medium and a supply of nutrients. The culture environment must be stable in terms of pH, oxygen supply, and temperature. Cell culture media often contain balanced salt solutions, amino acids, vitamins, fatty acids, and lipids to support cell and / or tissue growth. Precise medium formulations are often achieved by optimizing the concentrations of each and every component. Different cell types require different medium compositions and / or cell culture conditions.

[0080] The three-dimensional cell culture templates described herein can be used to culture various cell types, either alone or in co-culture, and can be used with various types of cell culture media. In some embodiments, the cultured cells are eukaryotic cells, preferably mammalian cells. In preferred embodiments, the cultured cells are human primary or immortalized cells. The cells can be grown in adherent culture or in suspension. In some embodiments, the cells are attached to the three-dimensional structure of the cell culture template.

[0081] Some cell types require surface modification to properly attach to the material of the cell culture template. The surface may be coated before cells are seeded. Commonly used coatings are collagen, fibronectin, and laminin. In some embodiments, the cell culture template of the present invention can be used for many cell types without surface pretreatment or coating. The three-dimensional structure allows for proper cell attachment even without a coating. However, if a coating is desired, the cell culture template having the three-dimensional structure may be coated.

[0082] In some embodiments of the methods for fabricating cell culture templates described herein, the cavities initially etched into the single-crystalline substrate are in contact with the outside of the substrate defined by the pre-etching directional process. In a preferred embodiment, the octahedral cavities in silicon are in contact with the outside of the substrate defined by the pre-etching directional process. In a more preferred embodiment, the octahedral cavities in silicon have the widest point of the octahedral shape as an opening, and are in contact with the outside of the substrate defined by the pre-etching directional process. When the etched cavities are in contact with the outside of the substrate, the fabrication of multi-layered three-dimensional structures is more optimal. Figure 1 shows a schematic side view of the etching of an octahedron in a single-crystalline substrate. The top diagram shows how the etched octahedrons can be in contact with the outside of the substrate.

[0083] In some embodiments, at least one three-dimensional structure of the cell culture template described herein is fabricated using silicon as a single-crystalline substrate. Thermal oxidation of silicon produces a silicon oxide layer. Then, in step 3 of the described method, a silicon dioxide layer is grown and / or deposited. In the final step, the silicon mass around the formed three-dimensional structure is removed. When the protective layer is created by thermal oxidation of silicon, this results in silicon oxide. Alternatively, when the protective layer is created by thermal nitridation of silicon, this results in silicon nitride.

[0084] Silicon is a chemical element.Single crystalline silicon can be used to make the three-dimensional structure described herein.Single crystalline silicon is also called single crystal silicon, and is abbreviated as mono-c-Si or mono-Si.It is composed of silicon whose crystal lattice is continuous and unbroken towards its edge throughout the solid, and does not contain any grain boundaries.

[0085] In some embodiments, the methods for producing cell culture templates described herein are used to produce three-dimensional structures with closed or open vertices. After the final preparation step of creating holes at all vertices, a three-dimensional structure with open vertices can be produced. In some embodiments, open vertices can be used to supply solutions to cell culture. After the final preparation step of forming a protective layer that also covers the vertices, a three-dimensional structure with closed vertices can be produced.

[0086] In some embodiments, the methods for producing cell culture templates described herein produce three-dimensional structures of greater complexity. To produce three-dimensional structures of greater complexity, steps 6-8 of the method are repeated 2-10 or more times, preferably 2-5 times. Each repetition of these steps results in an additional layer of octahedral structures, as illustrated in the series of Figures 2C-2G. Each subsequent layer will contain smaller geometric cavities. Preferably, each subsequent layer will contain smaller octahedra at each vertex of the existing layer.

[0087] In some embodiments, some set of steps of the fabrication method are repeated to generate three-dimensional structures with higher levels of complexity (eg, Figures 2C-2G).

[0088] After depositing a protective layer over the geometric cavities created by etching, holes are created at each vertex of the outer layer of the geometric shape. Using the holes, another step is performed to create at least one geometric feature or a portion of one geometric feature having a geometric shape in the single-crystalline base by anisotropic etching. In a preferred embodiment, the geometric shape is an octahedron. The anisotropic etching is performed through one or more holes created at one or more vertices. The new layer of geometric cavities is then protected with a protective layer.

[0089] Exemplary structures for octahedral geometries are shown in Figure 2. Figure 2C shows a simple three-dimensional structure that can be created with one step of anisotropic etching. Figure 2D shows a three-dimensional structure that can be created with two steps of anisotropic etching. Figure 2E shows a three-dimensional structure that can be created with three steps of anisotropic etching. Figure 2F shows a three-dimensional structure that can be created with four steps of anisotropic etching. And Figure 2G shows a three-dimensional structure that can be created with five steps of anisotropic etching.

[0090] In some embodiments, the methods for producing cell culture templates described herein produce three-dimensional structures with surfaces bearing a regular pattern of protrusions. These protrusions are constructed from octahedral structures that narrow toward the outside of the three-dimensional structure. The outer narrowing between structures is defined as the pitch. Among other factors, the pitch is determined by the three-dimensional hierarchy of complexity achieved by fractal generation.

[0091] The distance between fractals can vary. The distance between the centers of any two adjacent three-dimensional structures can also be referred to as the pitch. Preferably, the pitch between the three-dimensional structures is 5 to 100 μm, preferably 10 to 50 μm, more preferably 10 to 25 μm, and most preferably 12 to 20 μm. The pitch between the three-dimensional structures depends on the placement, arrangement, and size of the three-dimensional structures. For example, in a preferred embodiment, the pitch between three-dimensional structures placed in a hexagonal array is 12 μm, and the pitch between three-dimensional structures placed in a square array is 20 μm.

[0092] In some embodiments, the methods for making the cell culture template described herein comprise the following topography: - cone (G0, Fig. 2C), - a cone with an octahedron at its vertex (G1, Fig. 2D), - a pyramid with an octahedron at its vertex and a second octahedron structure at each vertex of the octahedron (G2, Fig. 2E); - a pyramid with an octahedron at its vertex, a second level of octahedral structures at each vertex of the octahedron, and a third level of octahedral structures at each vertex of the second level (G3, Fig. 2F), or - A cone with an octahedron at its vertex, a second-level octahedral structure at each vertex of the octahedron, a third-level octahedral structure at each vertex of the second level, and a fourth-level octahedral structure at each vertex of the third level (G4, Figure 2G). The present invention includes at least one three-dimensional structure having any one of the following:

[0093] Different levels of complexity affect the surface patterns of cell culture templates. These patterns become more precise when the 3D structures have higher levels of complexity. As the level of complexity increases, the spacing between the 3D structures may decrease.

[0094] In some embodiments, at least one three-dimensional structure or the entire cell culture template containing the three-dimensional structure is sterilized before growing cells. For example, the structure can be sterilized by chemical means, high temperature treatment, irradiation, such as autoclaving and UV light. In a preferred embodiment, the three-dimensional structure or the entire cell culture template is sterilized by using UV, chemical means, and / or high temperature treatment.

[0095] In some embodiments, in the methods for fabricating a cell culture template described herein, the at least one three-dimensional structure comprises a plurality of three-dimensional structures, and the plurality of three-dimensional structures are arranged in a lattice configuration. In preferred embodiments, the structures are arranged in a square or hexagonal lattice configuration, more preferably in a hexagonal array.

[0096] In some embodiments, the method for producing a cell culture mold described herein includes a step of partially removing a block of monocrystalline substrate. For this embodiment, the block of monocrystalline substrate is partially etched away around a plurality of formed three-dimensional structures. In a preferred embodiment, the block of monocrystalline substrate is partially etched away to create a plurality of compartments containing one or more three-dimensional structures. By leaving rings of the block of monocrystalline substrate unetched, these compartments can be in the form of wells. Rings of silicon separate the wells, allowing the wells to contain fluids. These wells are suitable for culturing cells. Furthermore, the structure of the remaining block of monocrystalline substrate can protect the fractal structure. The partial etching step is illustrated in FIG. 11.

[0097] The distance between fractals can vary. The distance between the centers of any two adjacent three-dimensional structures can also be referred to as the pitch. Preferably, the pitch between the three-dimensional structures is 5 to 100 μm, preferably 10 to 50 μm, more preferably 10 to 25 μm, and most preferably 12 to 20 μm. The pitch between the three-dimensional structures depends on the placement, arrangement, and size of the three-dimensional structures. In a preferred embodiment, the pitch between the three-dimensional structures placed in a hexagonal array is 12 μm, and the pitch between the three-dimensional structures placed in a square array is 20 μm.

[0098] In some embodiments, the cell culture template described herein further comprises at least one insulator. The insulator is made of a material that does not allow electrons to flow freely. As a result, little current flows through the insulator under the influence of an electric field. Amorphous silicon dioxide is a suitable material for an insulator. Therefore, the three-dimensional fractal structure described herein can function as an insulator in the cell culture template. In a preferred embodiment, the insulator is a three-dimensional structure of amorphous silicon dioxide.

[0099] In some embodiments, the method of the present invention further comprises step 9: providing an inorganic layer on the at least one three-dimensional structure, whereby the inorganic layer contacts the base three-dimensional material, i.e., the inorganic layer is provided on the surface of the at least one three-dimensional structure comprising the base three-dimensional material. Step 9 is performed after step 5 and before providing cells on the at least one three-dimensional structure under growth-enabling conditions to prepare a cell culture template. The inorganic layer is preferably provided by conformal deposition or directional deposition. More preferably, the inorganic layer is deposited on the base three-dimensional material using atomic layer deposition (ALD; in the case of conformal deposition), physical vapor deposition (PVD), or sputtering (all in the case of directional deposition). These three techniques are well known in the art.

[0100] The layer is provided on at least a portion of the three-dimensional structure, particularly on a portion of the structure on which cells will be placed, so that cells can be cultured on the layer. Thus, in a preferred embodiment of the method of the present invention, the method further comprises step 9: providing an inorganic layer on at least one three-dimensional structure, whereby step 9 is performed after step 5 and before placing cells on at least one three-dimensional structure under growth-enabling conditions to prepare a cell culture template, whereby the cells are placed on at least a portion of the structure on which the inorganic layer is provided. That is, the cells are placed on the surface of at least one three-dimensional structure comprising an inorganic layer, particularly the cells are placed on the inorganic layer. Preferably, the cells are then cultured on the layer.

[0101] The portion of the three-dimensional structure provided with the inorganic layer preferably covers at least 25% of the surface area of ​​the three-dimensional structure, and preferably, cells are then placed on at least the portion of the structure provided with the inorganic layer, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90% of the surface area of ​​the three-dimensional structure.

[0102] In one embodiment, essentially the entire surface of the three-dimensional structure is provided with an inorganic layer, and preferably thereafter cells are placed on at least the portion of the structure provided with said inorganic layer.

[0103] The inorganic layer is compatible with cell culture.

[0104] In some embodiments, the inorganic layer preferably comprises platinum, gold, silver, or a combination thereof.

[0105] In a preferred embodiment, the inorganic layer allows for surface-enhanced Raman spectroscopy measurements, for example for high-resolution molecular determination, for example for electrical simulation and recording of neuronal cells.

[0106] In some embodiments, the cell culture templates described herein further comprise at least one metal moiety, which can impart other properties to the cell culture template that can affect cell culture.

[0107] In a preferred embodiment, the metal portion is part of the three-dimensional structure of the cell culture template described herein. The metal portion can be embedded or patterned in the three-dimensional structure. The metal portion can impart other properties to the three-dimensional structure described herein. The metal portion in the three-dimensional portion can facilitate electrical current. The electrical current can affect cells in culture. For example, the electrical current can affect cell morphology and / or cell spreading in cell culture. The metal portion can also increase the flexibility of the three-dimensional structure.

[0108] In some embodiments, the metal portion of the cell culture mold described herein is used for external stimulation of cells or tissues in culture. This external stimulation can be performed by a three-dimensional structure. For example, external stimulation of cells and / or tissues in cell culture can be used to induce synthetic rhythms of waves.

[0109] The cell culture template containing a three-dimensional structure and the ability to implement external stimuli can be a great advantage for culturing muscle cells, particularly cardiomyocytes. Therefore, the cell culture template described herein can improve muscle cell and / or cardiomyocyte culture techniques. Furthermore, neurons and neuronal synapses can be stimulated by electric or fluctuating magnetic fields. Therefore, the cell culture template described herein can be used to culture neurons and / or nervous tissue and simulate these cells during cell culture.

[0110] In some embodiments, the cell culture template described herein uses electrodes for cell stimulation. In a preferred embodiment, the three-dimensional structure can function as an electrode for cell stimulation. Cells in culture can be attached to the three-dimensional structure of the cell culture template. Therefore, stimulation via these structures can directly reach the cells. Direct contact contributes to good signal transmission.

[0111] The present disclosure further provides a cell culture template for growing and maintaining cell cultures, particularly cell cultures containing primary cells. The cell culture template includes cells seeded on a cell growth surface, e.g., an amorphous silicon dioxide surface. The surface is defined by at least one three-dimensional fractal structure supported on a layer of a support substrate, e.g., borosilicate glass.

[0112] The surface of the cell culture template may be defined by a large number of, preferably at least nearly identical, three-dimensional fractal structures evenly distributed on the support layer. In some embodiments, some of the large number of three-dimensional fractal structures on the support layer are covered with a single-crystalline substrate, and other three-dimensional fractal structures among the large number of three-dimensional fractal structures are exposed, i.e., do not have single crystallinity, to form a cell growth surface. The single-crystalline substrate is arranged to define one or more cell growth compartments with one or more exposed fractals. In some embodiments, the cell culture template is provided with a lid on the side of the cell layer opposite the cell growth surface that is on top of the single-crystalline substrate and supported by the single-crystalline substrate.

[0113] The present disclosure further provides a method for culturing cells or tissues, comprising using a cell culture template produced by the methods disclosed herein, seeding cells, tissues and / or organoid structures, and culturing the seeded cells, tissues, or organoids.

[0114] Cells can be grown in adherent culture or in suspension. In some embodiments, cells are attached to the three-dimensional structure of a cell culture template. The three-dimensional structure can enhance adhesion between the cells and the cell culture template. Without wishing to be bound by theory, cell adhesion may provide signals required for growth and differentiation. Most primary cells require a surface to grow properly in vitro.

[0115] As demonstrated in the Examples herein, cell culture templates produced by the methods of the present invention allow for the purification of primary fibroblasts or other motile cells in a single step. Purification occurs by selective migration of motile cells, e.g., fibroblasts, into the vacant space of the template. This is particularly true for templates of generation G1 and above, where motile tumor cells are excluded.

[0116] Different cell types require different cell culture conditions. Some cell types require surface modification to properly attach to the material of the cell culture template. The surface may be coated before cells are seeded. Commonly used coatings are collagen, fibronectin, and laminin. The cell culture template of the present invention can be used for many cell types without surface pretreatment or coating. The three-dimensional structure allows for proper cell attachment even without a coating. However, if a coating is desired, the cell culture template having the three-dimensional structure may be coated.

[0117] The three-dimensional cell culture templates described herein can be used with various types of cell culture media.

[0118] In some embodiments, cells are dissociated before seeding and culturing in the cell culture template. Cells can be dissociated by known techniques, such as mechanical dissociation by pipetting or enzymatic dissociation by adding collagenase. Dissociated cells can be seeded as single cells in the cell culture template.

[0119] In some embodiments, the cells are seeded into the cell culture mold as multicellular tissue fragments without further processing.

[0120] In some embodiments, for the methods of culturing cells described herein, additional steps may be used to isolate specific cell types before seeding the cells into cell culture molds.

[0121] In some embodiments, the cells seeded in the cell culture template have also been cultured in another cell culture template before being seeded in the cell culture template described herein. For example, the cells may be cultured in a suspension or 2D cell culture template.

[0122] In preferred embodiments, the cultured cells or tissue are primary cells, preferably the cells are primary tissue cells. In some embodiments, the primary cells are primary tumor cells. In some embodiments, the cells are cancer-associated fibroblasts.

[0123] Primary cells are cells isolated directly from tissue. For example, these primary cells can be epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic cells, and mesenchymal stem cells. The culture can be heterogeneous. The cell culture can also be used to co-culture different cell types. In some embodiments, the primary cells cultured in the 3D cell culture mold are epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic cells, and / or mesenchymal stem cells. In some embodiments, the culture is heterogeneous and includes various cell types.

[0124] Furthermore, primary cells may be derived from healthy or diseased tissue, e.g., tumors. Primary cells derived from tumors are called primary tumor cells. These cells may be tumor cells, but they may also be cells present in the tumor microenvironment that support tumor cells, e.g., cancer-associated fibroblasts. In some embodiments, the cultured cells are cancer-associated fibroblasts.

[0125] It is known that primary cells are very sensitive to their environment. In known culture formats, these cells require additional supplies of nutrients and / or other factors, such as growth factors. These additional factors must be customized for each cell type. For example, endothelial cells have very different requirements than epithelial cells or neurons.

[0126] Although primary cells are more difficult to handle, experiments using primary cells are thought to be more relevant and reflective of the in vivo environment. Primary cells retain the morphological and functional properties of the tissue from which they originate. Therefore, these cells may accurately represent the in vivo situation in humans. For example, primary tumors preserve most tumor markers and known microRNAs.

[0127] Cell culture templates comprising at least one three-dimensional structure described herein can support the growth and survival of these primary cells. Without wishing to be bound by theory, the material, shape, and / or pattern of the three-dimensional culture template may support primary tissue cells. The cells adopt their own shape within the spatial constraints of the three-dimensional structure. As shown in the experimental section, this can potentially activate primary cells, such as cancer-associated fibroblasts.

[0128] It is known that primary cells have limited potential for self-renewal and differentiation. When these cells are cultured for a longer period of time, they exhibit morphological and functional changes. The three-dimensional culture template described herein can support primary cells. Therefore, these cells will maintain their tissue-specific properties for a longer period of time, which allows for more detailed studies on these cells.

[0129] Cancer-associated fibroblasts are non-tumor cells present in the tumor microenvironment. The tumor microenvironment is a multicellular tumor support system, containing cells of mesenchymal, endothelial, and hematopoietic origin. These cells interact closely with tumor cells and contribute to tumorigenesis. The tumor microenvironment is also a target for the development of anticancer drugs. Therefore, culturing cells from the tumor microenvironment, such as tumor-associated fibroblasts, is valuable for the study of tumor-targeted drugs.

[0130] In a preferred embodiment of the method for culturing cells or tissues described herein, the cells are stem cells, preferably mesenchymal stem cells, adult stem cells, adipose adult stem cells and / or induced pluripotent stem cells. In some embodiments, the cells are progenitor cells. In a preferred embodiment, the stem cells are not derived from an embryo or embryonic tissue. Preferably, the stem cells are not embryonic stem cells.

[0131] Stem cells can self-renew and differentiate into tissue-specific cells.Therefore, these cells have many uses, and the culture of stem cells and progenitor cells has attracted great interest.The cell culture template comprising at least one three-dimensional structure described herein can optimize the culture conditions for stem cells.Without wishing to be bound by theory, the material, shape and / or pattern of the three-dimensional culture template may support stem cells and enable them to differentiate into specific cell types.

[0132] In some embodiments, the cell culture template described herein can be used to grow or create functional 3D structure.In some embodiments, the cell in the method for culturing described herein forms complex cell aggregate, preferably multicellular organoid.

[0133] Organoid is a miniaturized and simplified version of organs that are produced in vitro in three dimensions.These organoids are multicellular and show realistic microscopic anatomical forms.They are derived from one or a few cells obtained from tissue, stem cells, or induced pluripotent stem cells.The cells in these organoids can be organized and polarized to have apical and basal sides.The three-dimensional structure of the described cell culture template can contribute to the formation of organoid structures and support the growth of these structures.

[0134] In preferred embodiments, the shape, material and / or pattern of the three-dimensional structure of culture template can support the differentiation of cell into tissue-specific cells, thus stimulating the formation of organoid.For example, the microtumor derived from patient with bystander cells can be used as the in vitro test for personalized chemotherapy.Neurosphere is the precursor of neuron, which can be used to create the transplant for spinal cord injury and other nerve injury or neurological disorder.

[0135] In some embodiments, cultured stem cells undergo differentiation when cultured in a tissue culture mold containing a three-dimensional structure. In a preferred embodiment, the cells undergo stem cell differentiation. Differentiation can be initiated by the shape, material, and / or pattern of the three-dimensional structure. In a preferred embodiment, differentiation is initiated by the cone-shaped shape and pattern of the structure. With respect to the pattern, the distance between the three-dimensional structures is important.

[0136] In vitro culture of cells and tissues requires a medium and a supply of nutrients. The culture environment must be stable in terms of pH, oxygen supply, and temperature. Cell culture media often contain balanced salt solutions, amino acids, vitamins, fatty acids, and lipids to support cell and / or tissue growth. Precise medium formulations are often achieved by optimizing the concentrations of each and every component. Different cell types require different medium compositions.

[0137] Furthermore, cell culture often requires the addition of serum. Serum is a complex mixture of proteins, peptides, growth factors, and growth inhibitors. The most commonly used serum is fetal bovine serum, which is used for a wide range of cell types. In addition, the culture medium may be supplemented with growth factors and cytokines.

[0138] During culture, cells use nutrients provided by the medium and excrete their waste products into the medium. Therefore, it is important to regularly provide fresh medium to cultured cells or tissues. The frequency of medium renewal depends on the cell type and the growth rate of the cells.

[0139] During the establishment of primary cultures, it is often necessary to include antibiotics in the growth medium to prevent contamination from host tissues.

[0140] After isolation, primary cells often undergo a senescence process, stop dividing after a certain number of cell divisions, or sense cell-cell contact. Maintaining the viability of primary cells is difficult. Appropriate culture conditions are essential for long-term cell viability. Growth factors are often supplied by adding serum to the culture medium.

[0141] In some embodiments of the methods for culturing cells or tissues described herein, the cultured cells are grown and / or stored under non-optimal growth conditions. At least one three-dimensional structure in the cell culture mold supports the cultured cells. The three-dimensional structure provides a suitable location for attachment. These conditions allow for compatibility with other culture conditions and still maintain the cell culture. Non-optimal growth conditions may include removing certain factors, such as growth factors, from the culture medium. Non-optimal growth conditions may also include maintaining the cell culture at room temperature instead of 37°C, a lower CO2 (air) percentage instead of 5%, long-term cell growth, and / or less frequent medium changes. Because cells survive under non-optimal growth conditions, the cell culture platforms described herein are suitable for transporting live cells and cell cultures. When transported outside the incubator, the cells remain healthy during transport.

[0142] In some embodiments, the cell culture mold comprising three-dimensional structure is prepared as described herein and is composed of amorphous silicon dioxide and cells attached to the structure.Amorphous silicon dioxide is the non-crystalline form of silicon dioxide.It can be deposited as a thin film, but it can also be a structure itself.Amorphous silicon does not consist of small particles, also known as crystallites.In amorphous structure, atomic positions are only limited to short-range order.In a preferred embodiment, the three-dimensional structure of amorphous silica is composed of SiO2.

[0143] At least one three-dimensional structure of the cell culture template described herein is suitable for microscopic observation purposes, and thus cells can be analyzed while attached to the three-dimensional structure.

[0144] Definition: As used herein, the term "comprise" and its conjugations are used in their open-ended sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Additionally, the verb "consisting of" may be substituted with "consisting essentially of," which means that the compound or compounds defined herein may contain additional components other than those specifically identified that do not alter the unique properties of the invention.

[0145] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the object that the article modifies. By way of example, "an element" means one element or more than one element.

[0146] The term "approximately" or "about" when used in connection with a numerical value (approximately 10, about 10), preferably means that the value is the given value of 10 or may be 1% greater or less than 10.

[0147] For clarity and conciseness of description herein, features may be described as part of the same or separate aspects or embodiments of the invention. Those skilled in the art will understand that the scope of the present invention may include embodiments having all or partial combinations of the features described herein as part of the same or separate embodiments.

[0148] All patents and references cited herein are hereby incorporated by reference in their entirety.

[0149] The present invention is further described in the following examples, which do not limit the scope of the invention but merely serve to clarify it. [Example]

[0150] Example 1 Cell culture is a "workhorse" as a testing platform for the toxicity and efficacy of new drugs towards a better understanding of biology in health and disease. While the majority of results in biology and medicine are based on 2D cell culture, it is well known that 3D cell spheroids or multicellular organoid complexes are more realistic models. There are two main methods on how to create cell spheroids: i) suspending the cell spheroids in liquid, or ii) embedding the cells in hydrogel. To create floating spheroids, it is necessary to prevent cell attachment to the culture dish surface. This is achieved by increasing the surface hydrophobicity. 1 or polymer deposition 2~4 , e.g., hanging drop culture 5 general adhesion prevention or nano- or microstructuring of the surface 6 (e.g., titanium surface texturization on implants) 7 or deposition of polymer nanomaterials 3,8 However, surface structuring may also induce differentiation in stem cells. 6 Another form of inducing floating spheroids of stem cells is pelleting, so culturing cells in Eppendorf cups was introduced by Konig and his group. 9 Cells can be seeded into hydrogels (e.g., Matrigel or other gels) or onto 3D scaffolds to form 3D spheroids, allowing for the formation of attached or better embedded spheroids. 10 The main applications of low cell adhesion surfaces in medicine are on implants or in dentistry. 3,7,8or preventing bacterial adhesion in the laboratory to study drug efficacy and toxicity under more realistic conditions. Both techniques have advantages and disadvantages. Floating spheroids are accessible to anyone for drug exposure, and released factors or extracellular vesicles can be easily harvested. However, the liquid cannot mimic the properties of the surrounding tissue. Gel-embedded spheroids respond to stimuli similar to tissue, but harvesting released factors and exposing them to defined concentrations of drugs is difficult because the surrounding gel also interacts with drug molecules, creating a concentration gradient.

[0151] We present a novel growth platform using periodically organized inorganic fractals of increasing complexity (G0-G4). We study cell growth of cancer-associated fibroblasts (CAFs) and adipose stem cells isolated from patients with liver cancer on these fractal surfaces. Our results show that certain surface structures enable cells to grow in 3D spheroids of attached but free-standing CAFs and stem cells. Other structures induce extended cell growth in 2D, with filopodia surrounding the structure.

[0152] Materials and Methods Fractal Preparation Fractal preparation was performed as described by Berenschot et al. 8 The protocol described by

[10] was followed. The surface was structured with hexagonal and square arrays of structures, which also had different interfractal distances: 12 and 20 μm pitch, respectively. Scanning electron microscope (SEM) images of the fractals and the surface covered with them are shown in Figure 2.

[0153] As the size of the fractal increases, the free distance in pitch decreases. Table 1 shows the fractal size and free distance.

[0154] [Table 1]

[0155] cell culture All procedures involving the use of patient samples were performed in accordance with relevant guidelines and regulations. The experiments were approved by the ethical committee. Patients signed informed consent.

[0156] Isolation of liver cancer tissue and cancer-associated fibroblasts (CAFs) Immediately after surgical resection, HCC tumor and peritumoral specimens were cut into 0.5-1 cm pieces and placed in MACS tissue preservation solution (130-100-008, Miltenyi). These tissue fragments were then cut into smaller pieces (1-2 mm), washed three times in Hanks' balanced salt solution (HBSS), and then incubated for 4 h at 37°C with gentle rotation in the presence of type IV collagenase (17104-019, Life Technologies) and 3 mM CaCl2 in HBSS. At the end of this step, dissociation was mechanically facilitated by pipetting the digested tissue up and down using a large-bore 50 ml pipette. Floating cells were collected, washed three times in HBSS, and kept in this solution on ice (first digestion stage). Decanted partially digested tissue specimens were subjected to the second stage of digestion (as described above). The resulting dissociated cells (second digestion stage) were washed twice with HBSS and then combined with the cells from the first digestion stage and centrifuged at 80 rcf for 5 minutes to separate epithelial and fibroblastic cells. The supernatant was centrifuged at 100 x g for 10 minutes, and the pelleted fibroblasts were purified by positive selection using anti-fibroblast MicroBeads and MS columns (Miltenyi Biotech) according to the manufacturer's instructions. CAFs were then cultured in IMDM + 20% FBS. To assess the purity of the CAF preparations, immunofluorescence or flow cytometry analysis was performed to assess the expression of mesenchymal markers, such as vitamin D and smooth muscle actin alpha (αSMA). The presence of minimal contaminating non-fibroblastic cells (mostly cancerous hepatocytes, cholangiocytes, and macrophages) was assessed using antibodies against EpCAM, CD45, and CD11b.

[0157] Trypsinize CAFs and culture 4 x 105 The cells were resuspended in complete DMEM medium at a concentration of 2 × 10 cells / ml. 50 μl of the cell suspension (2 × 10 4 Fractal-coated molds (containing cells) were seeded in triplicate onto fractal surface-coated molds (1 × 1 cm; controls (flat silicon, G0-4, square and hexagonal arrays)) placed in 6-well plates (three per well). First, the cells were incubated for 4 hours at 37°C and 5% CO2 without additional medium to allow cells to exclusively adhere to the fractal-coated surface to achieve a defined number of cells. Then, the molds were covered with 3 ml of complete medium and placed in an incubator. The medium was changed every 3 days.

[0158] On days 8 and 13, one template for each sample was fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH = 7.4 for 10 min. Fixed cells were stored at +4°C for further use.

[0159] Human adipose stem cells (hADSCs) Cell culture for hADSCs was performed as described by Legzdina et al. 12 Briefly, cells were grown in DMEM / F12 medium (Euroclone, Italy) containing 10% fetal bovine serum (FBS) (Euroclone, Italy), 20 ng / ml basic fibroblast growth factor (bFGF) (Lonza Sales, Switzerland), 2 mM L-glutamine 100 μg / ml: 100 μg / ml penicillin-streptomycin, and incubated for 37 min. o C, Cultured in a humidified atmosphere at 5% CO. Medium was changed every 3 days.

[0160] COLO 205 cells Human colon adenocarcinoma COLO 205 cell line (ATCC® CCL-222™, LGC Standards Srl, Italy), derived from metastatic ascites, was cultured in RPMI-1640 medium (Euroclone, Italy) supplemented with a final concentration of 10% fetal bovine serum (FBS South America, Euroclone, Italy), 2 mM glutamine (Euroclone, Italy), and 1% penicillin / streptomycin (Euroclone, Italy). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

[0161] HLF cells HLF (JCRB Cell Bank, JCRB0405, Osaka, Japan) is a non-differentiated hepatocellular carcinoma cell line. Cells were cultured in DMEM medium (Gibco) supplemented with 10% FBS, 1 mM pyruvate, 25 mM HEPES, and 100 U / ml penicillin-streptomycin and maintained at 37°C in an atmosphere containing 5% CO2.

[0162] Culture on fractal substrate. Three fractal-coated molds (1 cm × 1 cm) were placed in a 6-well plate if the experiment was in triplicate, or in a 24-well plate if only one mold was used, and sterilized by UV light in a laminar flow hood for 1 h. 2D cultured cells were trypsinized and plated at 4 × 10 5 The cells were resuspended in complete DMEM medium at a concentration of 2 × 10 cells / mL. 50 μL of the cell suspension (2 × 10 4(containing cells) were seeded onto sterile substrates. Each experiment was performed in triplicate. First, single cells were incubated for 4 hours at 37°C and 5% CO2 without additional medium to ensure exclusive attachment of single cells to the defined number of cells on the fractal-coated surface. The substrate was then covered with 3 mL of complete medium and placed in an incubator, with the medium changed every 3 days. Isolates from primary CAF preparations were grown for 8 and 13 days and then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.4 for 10 minutes and then processed for immunohistochemistry. HLF cells were cultured on the fractal surface for 4 days before being fixed and stained.

[0163] Each CAF cell was grown on treated 24-well plates (Corning Cellbind Surface) as a control, except for HLF, where cell growth was compared to cells grown in Matrigel.

[0164] Culture in Matrigel 30 μl of Matrigel (Corning Inc., USA) was layered on the bottom of a 96-well plate and allowed to gel for 20 minutes in a cell culture incubator (37°C, 5% CO2). 1000 hepatocellular carcinoma HLF cells were mixed with 30 μl of Matrigel, layered on top of the first Matrigel gel layer, and placed in the incubator for another 20 minutes. Finally, 90 μl of complete DMEM medium was added to the Matrigel-embedded cells, and the cells were grown for 13 days to form spheroidal multicellular structures. The medium was changed every two days.

[0165] Growth and adhesion to fractal surfaces Proliferation was assessed by cell counting in a Burker chamber. hADSCs were counted at 1.4 × 10 4 COLO 205 cells at a density of 1 x 10 cells / well 4Cells were seeded at a density of 1000 cells / well. Each cell line was grown on six different fractal templates in 24-well plates in complete medium at 37°C and 5% CO2. The control condition was cells seeded directly onto the wells of the 24-well plate. After 24 hours, cells were washed thoroughly in phosphate-buffered saline (PBS), detached with trypsin / EDTA, and counted. Values ​​were expressed as absolute cell numbers or percent variation relative to the baseline count, ± standard deviation. After 2, 24, 48, and 96 hours, cells were observed and photographed to document any differences in proliferation and adhesion abilities. Each experimental point was repeated three times.

[0166] Flow cytometry. Analysis of markers detecting HCC cancer cells and CAFs was performed using the following anti-human antibodies: Alexa Fluor 488-conjugated IgG2a against alpha fetal protein (AFP, BD Biosciences, USA), FITC-conjugated IgG1 against CD13 (Merck, Germany), FITC-conjugated IgG2b against CD44 (BD Biosciences, USA), FITC-conjugated IgG1 against CD90 (BD Biosciences, USA), FITC-conjugated IgG1 against CD133 (Miltenyi Biotec, Germany), unconjugated IgG1 against CD151 (abcam, UK), FITC-conjugated IgG2b against EpCAM (BioLegend, USA), unconjugated IgG1 against OV-6 (R&D Systems, USA), FITC-conjugated IgG1, IgG2a, and IgG2b isotype control antibodies (Miltenyi Biotec, Germany), Alexa Fluor 488-conjugated IgG isotype control antibody (abcam, UK), and Alexa Fluor 488-conjugated anti-mouse antibody.

[0167] Briefly, cells were detached using StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific, USA) and incubated with fluorophore-conjugated antibodies for surface staining of CD13, CD44, CD90, CD133, CD151, EpCAM, and OV-6 for 1 h at 4°C in the dark. For AFP staining, cells were fixed and permeabilized using Foxp3 / Transcription Factor Fixation / Permeabilization Concentrate and Diluent (eBioscience, Thermo Fisher Scientific, USA) prior to antibody incubation. A second incubation step (1 h at 4°C in the dark) with a secondary Alexa Fluor 488-conjugated antibody was performed to detect CD151 and OV-6. Fluorophore-conjugated isotype antibodies were used as controls for the detection of AFP, CD13, CD44, CD90, CD133, and EpCAM. Alexa Fluor 488-conjugated anti-mouse antibody was used as a relevant control for the detection of CD151 and OV-6. Cells were analyzed using a Navios flow cytometer, and data were processed using Kaluza software (Beckman Coulter).

[0168] Fluorescence microscopy For fluorescence imaging, fixed cells were permeabilized with 0.1% Triton X-100 in PBS (supplemented with 2% fetal bovine serum albumin) for 15 min and then incubated for 1–2 h in the presence of phalloidin-tetramethylrhodamine B isothiocyanate (TRITC, Sigma-Aldrich) to visualize the actin cytoskeleton.

[0169] To distinguish CAFs from tumor cells, cells were stained with an AFP antibody covalently conjugated to Alexa Fluor 488 (tumor) and for α-smooth muscle actin (α-SMA, CAF). Detection of α-SMA and α-fetoprotein expression by immunofluorescence imaging was performed on 4% paraformaldehyde-fixed cells. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes. Cells were washed three times with PBS and then incubated with 1% BSA in PBS (PBS + 0.1% Tween 20) for 30 minutes to block nonspecific antibody binding. Subsequently, cells were incubated overnight at 4°C with antibodies diluted in 1% BSA in PBS (α-SMA: Cell Signaling Technology, 1:100; AFP: BD Pharmingen, 1:100). Cells were washed three times in PBS, and for α-SMA, cells were incubated with secondary antibody Alexa Fluor® 488 conjugate (Invitrogen) diluted (1:50) in 1% BSA in PBS for 1 hour at room temperature in the dark.

[0170] After washing three times with PBS, the surface of the template with the adherent cells was covered with 4',6-diamidino-2-phenylindole (DAPI)-supplemented antifade mounting medium (VECTASHIELD, Vectorlabs). The cells were then stained with an anti-focal adhesion kinase 1 (FAK) antibody covalently linked to quantum dots emitting at 585 nm (SiteClick™ Qdot™ 585 Antibody Labeling Kit; ThermoFisher; order number S10451). The FAK antibody was labeled according to a modified protocol provided by the vendor.

[0171] Optical microscope observation COLO 205 and hADSC cells were visualized with an OLYMPUS CKX41 microscope equipped with a 4X / 0.25 PHP objective.

[0172] Results and Discussion Fractal preparation was performed as described by Berenschot et al. 11The protocol described by

[1999] was followed. Inorganic fractal structures were periodically deposited on a glass surface and sterilized by simple exposure to UV light for 1 hour in a laminar flow cabinet. Primary CAF cells were seeded onto the various molds without any further treatment. Primary cancer-associated fibroblasts (CAFs) isolated from liver cancer patients were seeded at 2 × 10 4 Cells were seeded onto fractal substrates of different generations and lattice configurations at different cell densities. The mold size was 1 cm x 1 cm for all generations (G0-G4) and consisted of flat-etched SiO2 (flat SiO2) grown on silicon and bonded / etched back. The molds were placed in 24-well plates without further functionalization (e.g., addition of extracellular matrix molecules). Immediately before use, they were sterilized by 1 hour of UV exposure. Plastic and flat SiO2 were used as controls. To achieve a defined number of cells on the mold, cells were allowed to attach for 4 hours before filling the wells with medium. Their growth and morphology were monitored daily by microscopy. On days 8 and 13, cells were fixed and fluorescently stained with DAPI to visualize nuclei and TRITC-phalloidin for cytoskeletal actin filaments. Representative images of CAFs on hexagonal array molds at day 13 are shown in Figure 3. CAFs in a square configuration 8 days after seeding can be seen in Figure 4.

[0173] Below we describe some interesting features observed for various cells grown on surfaces covered with periodically repeating fractals (Figs. 3 and 4).

[0174] Overall, it can be observed that the surface area covered by a single cell is greater in the square configuration than in the hexagonal configuration. There is little difference in cell morphology at days 8 and 15. CAFs on the square configuration appear round, while cells on the hexagonal configuration are elongated, with elongated nuclei (arrows in Figures 3C and 3F) and well-connected lamellipodia. While the nuclei are typically located between the fractals, it is clear that the lamellipodia actively interact with the fractals, as indicated by the high concentration of actin (red signal in Figure 5).

[0175] Detailed cellular studies on the influence of fractal microstructures on cell morphology, proliferation, viability, differentiation, and activation of each cell type (CAFs, stem cells, COLO205) are ongoing and are the scope for further publication.

[0176] Spheroid cell growth The most intriguing result of using fractal-coated surfaces as cell growth platforms was the presence of spheroid-like cell clusters by CAFs isolated from patient liver cancer tissue (Figures 3 and 4). CAFs grown on flat silicon surfaces occasionally, and always in both G0 configurations, exhibited a 2D layer of fibroblast-like cells directly attached to the fractal, and in some areas, 3D spheroid-like cell clusters with diameters greater than 100 μm attached to this 2D cell layer (Figures 3B, 7, and 5). Typically, 16–20 spheroids were observed per 1 × 1 cm template in both G0 configurations and the control consisting of a flat amorphous SiO2 surface. We observed that spheroid precursors formed as early as 1 day after seeding with single cells (Figure 6A), which subsequently grew into dense, large spheroids within 8 days (Figure 6B). Larger cell clusters exhibited only diffuse blue fluorescence within, indicating the absence of distinct nuclei. We believe this to be a necrotic core surrounded by a layer of intact cells.

[0177] Interestingly, the same results were observed for hADSCs, as can be seen in Figure 7 for the square configuration.

[0178] First, a larger number of cells were detected on the fractal-coated template than on the plastic surface of a cell culture dish. However, counting cells was not straightforward as cells became more difficult to detach by trypsinization at larger generations (G3 and G4). After 24 h, clusters of cells were forming on the G0 and G1 square configurations, while a cell layer could be observed in G2. As can be seen in the bottom panel of Figure 7, after 48 h, the clusters formed dense spheroids. In the hexagonal configuration, we observed hADSC spheroids even on the G2 template. Unlike CAF spheroids, intact cells (fluorescence image: nuclei stained with DAPI (blue); CD90, a stem cell and neuron biomarker, stained with FITC-conjugated anti-CD90 antibody (green)) could be found inside the spheroids. Further investigation confirmed that the fractal surface induced differentiation into nestin-positive neurospheres (Figure 8).

[0179] In contrast, no spheroid growth was observed for the colon adenocarcinoma cell line COLO205. COLO205 grew in 2D on all tested surfaces for up to 96 hours (Figure 9, top panel; G0–G3, both configurations). This is consistent with our observation that COLO205 generally does not form spheroids in other spheroid-producing systems after coating with low-cell-adhesive PEG6000 (Figure 9, bottom panel). 4 .

[0180] CAFs in the hexagonal lattice configuration appear astrocyte-like, with elongated nuclei and well-developed lamellipodia connecting to the fractal structures. While the cell nuclei are primarily located between the fractals, the lamellipodia interact with the fractals, as indicated by the high concentration of actin (red signal in Figure 5). Further studies on the fractal-induced triggers for cell morphology, proliferation, viability, proteomics, and genomics of primary cells are ongoing and will be published in the future.

[0181] As summarized in Table 2, we observed different responses for each fractal surface.

[0182] [Table 2]

[0183] To understand the origin of spheroid-forming cells in the case of CAF isolates, we analyzed CAF isolates from 2D cell cultures for biomarkers of various cell types by FACS (Figure 13).

[0184] CAF cell isolates contain different amounts of cells positive for markers of cancer stem cells (tumor stem cells; CD13 [14, 15], 44, 90

[15] , 133

[16] , OV6

[15] ), epithelial cells (EpCAM

[15] ), or general tumor cells (AFP

[13] ). This can also be seen in Figure 14A, where only approximately 20% of cells are positive for α-SMA (CAFs) when cultured in 2D. Interestingly, when cells isolated from three patients and characterized by FACS (Figure 13) were cultured in G0Hex for 6 days, cells from patients 2 and 3 formed spheroids (Figure 14B).

[0185] Confocal microscopy of the spheroids by Z-stack imaging confirmed the 2D layer of α-SMA-positive CAF cells and the spheroidal morphology of the microtumors (Figure 15A). Interestingly, the 2D layer appears to be located at the center height of the spheroid (50 μm from the top and bottom). This is surprising, since the height of the fractal is only 15 μm. To understand whether the spheroids digest the amorphous silica layer, the organic material (cells) was etched with piranha solution, and the underlying layer was visualized by optical and electron microscopy. No changes to the inorganic surface were observed (data not shown). Because the fractal interacts with light and induces changes in the refractive index, small portions of the microtumors embedded within the fractal appear distorted and enlarged.

[0186] The microtumors were then co-stained with AFP (green) and α-SMA (red) antibodies. The image in Figure 15B shows that a fibroblast capsule surrounded the AFP-positive microcells. No AFP signal was observed in the 2D layer, confirming that this layer consisted solely of CAFs.

[0187] While G0 templates promote the growth of complex 2D-tumor spheroid clusters, tumor cells appear to be excluded from the template at G1 and higher generations (Figures 3C-F). To explore whether these templates could be used to isolate CAFs in a single step, small pieces of peritumoral and tumor tissue were placed on the fractal templates. After 2-3 days, the first star-shaped CAF-like cells began to migrate into the fractal templates. After 20 days, the tissue was removed, and the layer of cells that had invaded the fractal surface was stained for AFP and α-SMA (Figure 16).

[0188] Neither the peritumoral tissue (free of tumor cells) on G0 (Figure 16A) nor the tumor tissue on G1 (Figure 16C) exhibits any green fluorescence for AFP. In both cases, only a 2D cell layer positive for α-SMA (red) is observed. In contrast, when the tumor fragment was in contact with G0, a strong yellow signal corresponding to the green AFP antibody co-localized with the red α-SMA antibody was detected where the tumor was in contact with the G0Sqr fractal template and later mechanically removed. Furthermore, a gradual decrease in the AFP signal was observed in more distal cells. The spot-like appearance of the AFP signal may indicate that it is due to invadosomes, which are known to be concentrated in actin and invade the microenvironment.

[0189] Finally, in the absence of fibroblasts, the G0 fractal template induces the rapid formation of spheroids in cancer cell lines, as can be seen in Figure 17A for the liver cancer cell line HLF. Spheroid formation was compared to the growth of HLF cells in Matrigel (Figure 17B).

[0190] It is noteworthy that spheroids of comparable size grew on the molds in 4 days, whereas those in Matrigel required 13 days. The average size of spheroids on the fractal substrate after 4 days was 74 ± 20 μm (N = 12), whereas those grown in Matrigel were 108 ± 57 μm (N = 52) at 13 days. Because spheroid growth in Matrigel begins with embedded single cells and is exponential (12), direct comparisons at day 4 are not possible. Therefore, it is likely that only small clusters with very few cells formed by day 4. Although HLF cells on fractals were also seeded as single cells, they began to form clusters early during attachment, as seen for CAFs in Figure 6.

[0191] conclusion We present a novel cell growth platform. This platform is particularly suitable for difficult-to-grow cells, such as stem cells and primary cells (CAFs and tumor cells). These templates, coated with periodic fractal structures, are made of inorganic materials and therefore easily sterilized. Without further processing or functionalization, such as the deposition of extracellular matrix molecules, they enhanced the growth of complex 2D spheroids of cancer-associated fibroblasts obtained from patient samples. For some structures, we observed selective growth of isolated CAFs and suppression of the growth of contaminating tumor cells, which are unavoidable during CAF isolation. However, when coculture of CAFs with tumor cells is required, for example, to test different therapeutic options for microtumors to optimize patient treatment, other fractal structures were found to support the growth or survival of 3D microtumors. These microtumors were observed after 8 days of culture, providing a more realistic model of patient tumors than 2D isolated tumor cells. For the G1-4 fractal surfaces, we observed selective growth of CAFs, enabling one-step CAF isolation from tumors. In tumor cell lines, we observed enhanced spheroidal cell growth compared to standard 3D matrix growth systems.

[0192] (References)

[0193] (Example 2 (Berenschot et al. 2016)) Exemplary preparation of three-dimensional fractal structures To be able to fabricate 3D fractals with oxide-only corner lithography, the grown amorphous silicon dioxide layer must be equal in thickness on the (100) and (111) crystallographic planes of silicon and must be compliant around external corners. If these requirements are not met, the SiO layer cannot be properly patterned by timed isotropic etching (i.e., thickness variations result in removal of SiO in locations where it should remain), or it will not function as a proper mask during selective anisotropic etching of silicon. Therefore, this simplified process uses (dry) thermal oxidation at 1100°C. Oxidation of silicon at this temperature results in fundamental differences in the grown oxide in terms of layer thickness on the (100) and (111) silicon crystallographic planes and layer conformality around external corners, compared to thermal oxidation at relatively low temperatures (≤950°C).

[0194] At low thermal oxidation temperatures (≤950°C), the oxide thickness at outside and inside corners is smaller than that at flat (100)-Si surfaces due to compressive stresses in the corner structures. [5], [6] At temperatures of 1000°C, the oxide layer formed at outside corners is not thinned relative to the thickness of the layer on the (100)-Si surface, but at this temperature, there is a difference in oxide growth rate along the main crystallographic orientation of silicon. [7] Dry thermal oxidation of silicon at 1100°C avoids the aforementioned aspects of non-conformity at outside corners and the difference in oxide thickness on the (100) and (111)-Si surfaces. [7] At inside corners, the severe compressive stresses [8] that arise are not relieved, and the associated slower oxidation rate results in locally thinner layers.

[0195] The degree of thermal oxide sharpening at inside corners depends on the amount of intersecting (111) planes. That is, the more intersecting planes, the thinner the grown oxide layer. Thus, at ribbons, i.e., two intersecting (111) planes, less oxide sharpening occurs compared to intersections of three or four (111) planes (i.e., vertices) (Figure 10). These aspects lead to a promising approach: timed isotropic etching in 1% HF removes SiO2 only from the vertices, while leaving the oxide on the ribbons and planes. This is illustrated in Figure 10.

[0196] The self-assembly procedure for 3D fractals is then very simple: after thermal curing and timed HF etching, the underlying Si is selectively etched (anisotropic etching in TMAH) at each vertex, resulting in the simultaneous formation of the next-level octahedral structure at all vertices. Repeating this simple sequence of anisotropic Si etching / thermal oxidation at 1100°C / isotropic SiO2 etching yields multilevel 3D fractal structures.

[0197] Experimental results and discussion To demonstrate the selective opening of the apex, we used KOH (25 wt %, 70 °C) to etch inverted cones in (100)-Si with slightly rectangular footprints (Figure 11, left) and square footprints (Figure 11, right). These structures were then oxidized (dry, 1100 °C for 95 min) to obtain SiO2 thicknesses of 160 nm and 155 nm on the (111) and (100) oriented faces, respectively. Figure 11 shows SEM images (top view) after 19 min + 30 s of etching in 1% HF (etch rate 4.4 ± 0.1 nm / s) and 5 min of TMAH etching (25 wt %, 70 °C) to create the appropriate openings for better SEM visibility. The residual oxide thickness on the (111) face is 74 nm.

[0198] The first indications of the available time window (Δt) for apex-only and ribbon-and-apex openings are shown in Figure 11B for starting oxide thicknesses of 88 nm and 160 nm (on the (111) plane), respectively. For each measurement point in the graph, a sample was removed from the 1% HF solution, etched in TMAH, and then examined by SEM. This sequence was repeated, and the detected apex or ribbon openings are shown in the graph. Note that there is a significant margin of error in the time windows shown due to the limited number of measurement points.

[0199] A 5 μm square footprint represents the starting point for realizing 3D fractal structures in inverted pyramids etched into (100)-Si with KOH. After growing a thermal oxide layer of known thickness (approximately 160 nm, 1 hour 35 minutes at 1100 °C), there is a time window during which only the vertices are oxide-free. To design 3D fractal structures based solely on oxide corner lithography, an etching time of 20 minutes 30 seconds in 1% HF is applied. After this HF step, etching the silicon through the vertices in TMAH (25 wt %, 70 °C) allows new octahedra to be formed by slow etching of the (111)-Si planes. For each fabrication level of the fractal structure, the oxidation and isotropic etching times remain constant, but the length of the TMAH etching step is halved for each new level (starting with an etching time of 145 minutes at level zero). This sequence of TMAH etching, 1100°C oxidation, and SiO2 etching is repeated three times, followed by a final thermal oxidation and anodic bonding to a Mempax glass wafer at 400°C to remove the bulk of the Si, resulting in a free-standing third-generation silicon oxide fractal sheet. Note that the vertices can be left closed or open depending on the final step.

[0200] (References) TIFF0007802784000003.tif101156

Claims

1. 1. A method for making a cell culture mold having at least one three-dimensional structure having a surface for supporting a cell culture, the method comprising the steps of: Step 1: providing a single crystal substrate; Step 2: Removing at least one geometric feature from the single crystalline substrate to create a geometric cavity in the single crystalline substrate that will be the starting point for a three-dimensional structure; Step 3: growing and / or depositing a base three-dimensional structure material onto the surfaces of the geometric cavities in the substrate to form a three-dimensional structure; Step 4: attaching at least one three-dimensional structure to a surface of a support base; Step 5: Removing a mass of the single-crystalline substrate around at least one three-dimensional structure; Including, A method for producing a cell culture template by removing a block of the single-crystalline substrate and then placing cells on the outer surface of at least one of the three-dimensional structures comprising a basic three-dimensional structural material under conditions that allow cells to grow on the surface of the three-dimensional structure.

2. 2. The method for producing a cell culture template comprising at least one three-dimensional structure according to claim 1, wherein the basic three-dimensional structure material is silicon nitride or silicon oxide.

3. 3. The method for producing a cell culture template comprising at least one three-dimensional structure according to claim 1 or 2, wherein the at least one three-dimensional structure is a fractal structure.

4. 4. A method for producing a cell culture template comprising at least one three-dimensional structure according to any one of claims 1 to 3, wherein the monocrystalline substrate is a monocrystalline silicon substrate.

5. 5. A method for producing a cell culture template comprising at least one three-dimensional structure according to claim 1, wherein in step 2, one or more vertices are formed.

6. 6. A method for producing a cell culture template comprising at least one three-dimensional structure according to any one of claims 1 to 5, wherein the geometric cavity is an octahedral cavity or a part of an octahedral cavity.

7. 7. A method for producing a cell culture mold comprising at least one three-dimensional structure according to any one of claims 1 to 6, wherein the support base is borosilicate glass.

8. The following steps: Step 6: Treating the single crystal substrate to form a protective layer compatible with the next step; Step 7: creating one or more holes in the protective layer to accommodate subsequent steps; Step 8: removing at least one geometric feature in the single crystalline substrate through one or more holes, and then stripping off the protective layer; Further comprising:

8. A method for producing a cell culture template comprising at least one three-dimensional structure according to any one of claims 1 to 7, wherein steps 6 to 8 are carried out between steps 2 and 3 of the method according to claim 1.

9. The method of claim 8, wherein steps 6 to 8 are repeated one or more times to create at least one three-dimensional structure having a higher level of complexity.

10. 10. The method for producing a cell culture template comprising at least one three-dimensional structure according to claim 8 or 9, wherein the protective layer is silicon oxide or silicon nitride.

11. 11. The method for preparing a cell culture template comprising at least one three-dimensional structure according to any one of claims 1 to 10, further comprising step 9: providing an inorganic layer on the at least one three-dimensional structure, whereby the inorganic layer is in contact with the base three-dimensional material, whereby step 9 is performed after step 5 and before providing the at least one three-dimensional structure with cells under growth-enabling conditions to prepare the cell culture template, whereby the cells are placed on the surface of the at least one three-dimensional structure comprising the inorganic layer.

12. 12. A method for producing a cell culture template comprising at least one three-dimensional structure according to claim 1, wherein the cavity formed in the single-crystalline substrate in step 2 is accessible from the outside of the substrate through an opening provided in the substrate.

13. 13. The method for producing a cell culture template comprising at least one three-dimensional structure according to any one of claims 1 to 12, wherein the provided monocrystalline substrate is silicon, whereby a layer of silicon oxide is generated by thermal oxidation, whereby in step 3 a layer of silicon dioxide is deposited, whereby in step 5 silicon chunks around the formed three-dimensional structure are removed.

14. 11. The method for producing a cell culture template comprising at least one three-dimensional structure according to any one of claims 8 to 10, wherein step 7 is omitted at the final stage of preparation to produce a three-dimensional structure with closed vertices.

15. The three-dimensional structure has the following topography: - Awl (G0), - a cone with an octahedron (G1) at its vertex, - a pyramid with an octahedron at its vertex and a second octahedron structure (G2) at each vertex of the octahedron; - a pyramid having an octahedron at its vertex, a second layer of octahedral structures at each vertex of the octahedron, and a third layer of octahedral structures (G3) at each vertex of the second layer, or - A pyramid having an octahedron at its vertex, a second-level octahedron structure at each vertex of the octahedron, a third-level octahedron structure at each vertex of the second level, and a fourth-level octahedron structure (G4) at each vertex of the third level; - A cone having an octahedron at a vertex, a second-level octahedral structure at each vertex of the octahedron, a third-level octahedral structure at each vertex of the second level, a fourth-level octahedral structure (G4) at each vertex of the third level, and an n-th level octahedral structure (Gn) [n is 5 to 10] at each vertex of the n-1th level.

15. A method for producing a cell culture template comprising at least one three-dimensional structure according to any one of claims 1 to 14, wherein the three-dimensional structure comprises:

16. 16. A method for fabricating a cell culture template comprising at least one three-dimensional structure according to any one of claims 1 to 15, wherein the at least one three-dimensional structure comprises a plurality of three-dimensional structures, the plurality of three-dimensional structures being disposed on a surface of a support base in a lattice configuration.

17. 17. The method for making a cell culture template of claim 16, wherein the bulk of the single crystalline substrate is partially etched away, leaving the remaining substrate at least partially covering at least one of the plurality of three-dimensional structures and / or creating a plurality of compartments exposing one or more of the three-dimensional structures.

18. 18. A method for producing a cell culture template comprising at least one three-dimensional structure according to any one of claims 1 to 17, wherein the cell culture template further comprises at least one insulator and / or the cell culture template further comprises at least one metal part.

19. The method of claim 18, wherein the insulator is a three-dimensional structure of amorphous silicon dioxide.

20. The method of claim 18, wherein the metal portion is embedded or patterned within the three-dimensional structure.

21. 21. A method for producing a cell culture template comprising at least one three-dimensional structure according to any one of claims 18 to 20, wherein the three-dimensional structure is used for external stimulation of the culture.

22. 22. A method for producing a cell culture template comprising at least one three-dimensional structure according to any one of claims 18 to 21, wherein an electrode is used for cell stimulation, and at least a part of the three-dimensional structure functions as an electrode.

23. 23. A method for producing a cell culture template comprising at least one three-dimensional structure according to any one of claims 1 to 22, wherein the vertices are open and solutions are supplied into the cell culture through these vertices.

24. 1. A cell culture template for growing and maintaining cell cultures, the cell culture template comprising cells seeded on an exterior of a cell growth surface comprising a base three-dimensional structural material, the surface being defined by at least one three-dimensional fractal structure supported on a support base.

25. 25. The cell culture template of claim 24, wherein the surface is defined by a multitude of three-dimensional fractal structures evenly distributed on the support layer.

26. 26. The cell culture template of claim 25, wherein some of the three-dimensional fractal structures among the plurality of three-dimensional fractal structures on the support layer are covered with a single-crystalline substrate, and other of the plurality of three-dimensional fractal structures are exposed, i.e., do not have single crystallinity, to form a cell growth surface.

27. 27. The cell culture template of claim 26, wherein the monocrystalline substrate is arranged to define one or more cell growth compartments having one or more exposed fractals.

28. 24. A method for culturing cells, comprising the steps of providing a cell culture template obtainable by the method of any one of claims 1 to 23, and culturing cells.

29. 29. The method for culturing cells or tissues according to claim 28, wherein the cells are primary cells.

30. A method for culturing cells or tissues according to claim 28 or 29, wherein the primary cells are primary tissues or primary tumor cells.

31. 31. The method for culturing cells or tissues according to any one of claims 28 to 30, wherein the cells are cancer-associated fibroblasts (CAFs), stem cells, mesenchymal stem cells, adult stem cells, adipose adult stem cells and / or induced pluripotent stem cells.

32. 1. A method for fabricating three-dimensional fractal structures for cell culture fabricated by micro- and nano-fabrication, comprising the steps of: Step 1: providing a single crystalline silicon substrate; Step 2: removing at least one geometric feature from the single crystalline silicon substrate to form one or more vertices, thereby forming a geometric cavity in the single crystalline silicon substrate that will be the start of a three-dimensional structure; Step 3: growing and / or depositing a silicon oxide based three-dimensional structure material on the surface of the geometric cavities in the substrate to form a three-dimensional structure; Step 4: attaching at least one three-dimensional structure to a surface of a support base; Step 5: Removing a mass of the single-crystalline silicon substrate around at least one three-dimensional structure; Including, After removing the bulk of the monocrystalline silicon substrate, at least one surface of the three-dimensional structure comprising the silicon oxide-based three-dimensional material is prepared with cells under conditions that allow cell growth, thereby forming a cell culture template; The following steps: Step 6: Treating the monocrystalline silicon substrate to form a protective layer compatible with subsequent steps; Step 7: creating one or more holes in the protective layer to accommodate subsequent steps; Step 8: removing at least one geometric feature in the monocrystalline silicon substrate through one or more holes, and then stripping off the protective layer; Further comprising: A method in which steps 6-8 are performed one or more times between steps 2 and 3 to create at least one three-dimensional structure having a higher hierarchy of complexity.

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