Cell co-culture system and method

Abstract

The present invention relates to a method and apparatus for in vitro three-dimensional cell co-culture, the method comprising the steps of seeding a first magnetic scaffold with a plurality of cells of a first cell type and seeding a second scaffold with a plurality of cells of a second cell type while maintaining the first and second magnetic scaffolds physically separated from each other, and bringing the first and second scaffolds closer to each other under the action of a magnetic field generated by a magnetic field generating unit until they contact on at least one surface.

Description

[Technical field]

[0001] The present invention relates to systems and methods for in vitro three-dimensional cell co-culture. [Background technology]

[0002] As is known, cell-cell interactions and cell-matrix interactions play fundamental roles for cell survival, growth, proliferation, migration and differentiation in living tissues. In particular, cell-cell interactions are regulated by the secretion of signaling molecules and by direct contact between cells.

[0003] Unlike single cell culture, multicellular co-culture systems allow interactions between cells of different populations in one system. Therefore, such systems can be used to reproduce complex biological structures in vitro, where two or more different cell populations interact with each other. For example, the above-mentioned systems can be used to model biological structures such as neuromuscular junctions and islets of Langerhans, or pathological conditions of organisms, such as the invasion of tumor cells in healthy tissue.

[0004] Currently available co-culture systems can be divided into "direct" and "indirect" co-culture systems, where cells can exchange biochemical signals but do not come into direct contact with each other. For example, WO2007021919 describes a multi-chamber co-culture system, where different populations of cells are separated into separate chambers and only communicate by exchanging signaling molecules through a semi-permeable membrane. It is clear that this type of system completely excludes any phenomena related to physical contact between cells and therefore only reproduces a part of the natural interactions between cells.

[0005] Conversely, direct co-culture systems allow for more complete reproduction of the interactions between different cells, since they also allow physical contact between cells belonging to different populations. However, systems currently known in the relevant field are based on co-culture of cells of different populations on the same carrier, e.g. three-dimensional porous structures known in the field of tissue engineering under the name "scaffolds", or on overlaying monolayers of cells belonging to different populations in a "sandwich" manner (e.g. as described in the scientific publication by Suhaeri et al. "Novel Platform of Cardiomyocyte Culture and Coculture via Fibroblast-Derived Matrix-Coupled Aligned Electrospun Nanofiber", ACS applied materials & interfaces 9.1 (2016): 224-235").

[0006] Systems based on seeding different cell populations on the same carrier do not allow for a choice of the time to incorporate different cell populations in the co-culture system, which, for example, does not allow for separate culturing of 3D structures for each cell type according to a strict type-related procedure, e.g., does not allow for the selection of carriers or cell culture media for each cell type, and furthermore does not allow for a 3D structure with a predefined spatial distribution of cells.

[0007] On the other hand, sandwich-type co-culture systems are unable to generate complex three-dimensional structures that can accurately reproduce the anatomy of an organ or device.

[0008] Therefore, in view of the above considerations, the technical problem underlying the present invention is to provide a cell co-culture system and method having a configuration that can overcome the limitations of the above-mentioned conventional techniques. Summary of the Invention

[0009] It is an object of the present invention to solve these and other problems by providing an in vitro cell co-culture utilizing magnetic interactions between scaffolds.

[0010] A further object of the present invention is to provide an in vitro cell co-culture system capable of carrying out the above method.

[0011] The idea of ​​the solution to the above problem is to use the magnetic interactions between magnetic scaffolds in the presence of a magnetic field to bring the three-dimensional structures of different cells growing on the scaffolds into contact with each other.

[0012] The solution proposed in this application allows stable contact of three-dimensional structures of different types of cells, thereby making it possible to easily create complex three-dimensional structures in which at least two cell types coexist.

[0013] Other advantageous features of the invention are set out in the accompanying claims.

[0014] The basic characteristics and further advantages of the invention will become more apparent upon reading the following description of preferred embodiments of the invention, which are illustrated in the accompanying drawings, which are provided only as non-limiting examples and not as a restrictive enumeration, in which: [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram of a system of the present invention. [Figure 2a] FIG. 1 illustrates a scaffold of one preferred solution. [Figure 2b] FIG. 1 shows a scanning electron microscope (SEM) image of a scaffold of a preferred solution. [Diagram 3] 1 is a schematic diagram of a part of a system according to a preferred embodiment of the present invention; [Figure 4] FIG. 2 shows a schematic diagram of the steps of the method of a preferred solution; [Diagram 5]FIG. 1 shows schematic diagrams of some possible arrangements of scaffolds in the co-culture system of the present invention. [Figure 6] FIG. 1 is a schematic diagram of a preferred embodiment of the system of the present invention. [Figure 7] FIG. 1 shows a schematic diagram of a part of the system of the present invention and a preferred embodiment of the method described above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Before proceeding with the detailed description, it should be noted that when the description refers to an "embodiment" or "implementation," this means that a certain configuration, structure, or feature is included in at least one embodiment of the present invention. Thus, although expressions such as "in one embodiment" may be used in various places in the description, this does not necessarily refer to the same embodiment. Furthermore, any specific configuration, structure, or feature may be appropriately combined in one or more embodiments. Therefore, the following description is merely for the purpose of simplifying the description and does not limit the scope of protection or the scope of various embodiments.

[0017] Referring to the description above and FIG. 1, the system 1 for in vitro cell co-culture of the present invention comprises at least one first scaffold 10 comprising a magnetizable material suitable for supporting the growth of a first cell type of cells, at least one second scaffold 20 comprising a magnetizable material suitable for supporting the growth of a second cell type of cells, a magnetic field generating unit 31 configured to generate a magnetic field that generates an attractive magnetic force between the first scaffold 10 and the second scaffold 20, and a cell co-culture chamber 300, in which both scaffolds 10, 20 are exposed to the magnetic field generated by the magnetic field generating unit 31 and approach each other until at least one contact surface is generated.

[0018] The scaffolds 10, 20 include a magnetizable material, making them susceptible to magnetization, i.e., capable of being magnetized when subjected to a magnetic field. Preferably, the scaffolds 10, 20 include at least one material having ferromagnetic properties and / or at least one material having superparamagnetic properties.

[0019] The scaffolds 10, 20 have selected structural properties to support the growth of each co-cultured cell type as described in the literature, for example, properties such as those described in the scientific publication "3D Stem Cell Niche Engineering via Two-Photon Laser Polymerization" by Nava et al. (Methods in Molecular Biology 1612 (2017): 253).

[0020] The scaffolds 10, 20 are three-dimensional porous structures created using scaffold manufacturing techniques known in the tissue engineering field (e.g., multiphoton polymerization, 3D printing, electrospinning, injection molding, etc.).

[0021] Preferably, the scaffolds 10, 20 are made from a biocompatible polymeric material (e.g., collagen) or a hybrid polymeric / ceramic material (e.g., Ormocomp® manufactured by Nanoscribe) and include a magnetic material (e.g., in the form of nanoparticles, thin film coatings, etc.) to facilitate magnetization of the scaffolds 10, 20.

[0022] Preferably, the scaffolds 10, 20 have a substantially prismatic shape, such as a prism whose base is a simple polygonal, concave or convex base (e.g., a square, rectangular, or hexagonal base, or a prism whose base has a more complex shape).

[0023] In a preferred embodiment, the first scaffold 10 and the second scaffold 20 have different shapes, allowing the cell cultures in the co-culture system 1 to be differentiated without the need for dyes or other tracers on the cells.

[0024] Preferably, the first scaffold 10 and the second scaffold 20 have complementary shapes to facilitate fixation of the two scaffolds to one another.

[0025] The magnetic field generator 31 in the co-culture chamber 300 is, for example, a permanent magnet or an electromagnet. Preferably, the magnetic field generator 31 is a magnetized ferromagnetic element (plate-shaped, leaf-shaped, cylindrical, etc.) confined to the co-culture chamber 300. More preferably, the magnetic field generator 31 is directly integrated into at least one of the scaffolds 10, 20, for example, the magnetic field generator 31 is integrated into the structure of at least one of the scaffolds 10, 20 as a polymer matrix of the scaffolds 10, 20 containing nanoparticles of magnetic material. The nanoparticles may be, for example, beads of ferromagnetic material with a diameter in the range of 50-100 nm, or beads of superparamagnetic material with a diameter in the range of 3-20 nm.

[0026] The co-culture chamber 300 can be a container or a cell culture plate, or can be defined as a partitioned space within a more complex structure (e.g., a microfluidic chip) connected to another environment by appropriate connection techniques.

[0027] Furthermore, the co-culture system 1 may also include additional culture chambers, which may be physically separate from the co-culture chamber 300 or which may be in communication with the co-culture chamber 300.

[0028] <Example 1> In a first variant of the invention, the co-culture system 1 comprises at least one first scaffold 10 and at least one second scaffold 20 having superparamagnetic properties and an electromagnetic field generator 31 having ferromagnetic properties.

[0029] As shown in FIG. 2, the scaffolds 10, 20 are hexagonal-based prismatic structures having horizontal central support structures and vertical lateral support structures.

[0030] The horizontal support structure has four equidistantly spaced concentric hexagonal rings (where the sides of the larger hexagonal ring have a length of 60 μm), which are interconnected at their centers by diagonal elements connecting the vertices of the hexagons.

[0031] The side support structure is a square grid (side length 60 μm) made by vertical and horizontal support elements arranged to form a plurality of square openings, and a rectangular material containing two square openings. As shown in Figure 2, the side support structure has eight small square openings with side lengths of 30 μm and 7.5 μm in the center, and four large square openings (two above and two below the eight small openings) with long sides of 30 μm and short sides of 15 μm, and the square material with two square openings is alternately arranged on the top or bottom of the side support structure, its side lengths are 30 μm and 15 μm, and the openings have side lengths of 30 μm and 7.5 μm.

[0032] The scaffolds 10, 20 are made of a composite material obtained by incorporating superparamagnetic nanoparticles into a negative photoresist that is sensitive to ultraviolet light. In particular, 10 mg of iron oxide superparamagnetic nanoparticles (also known as "SPIONs") are mixed evenly into 1 ml of IP-L 780 negative photoresist (manufactured by Nanoscribe) by ultrasonication at 8 W for 2 minutes. The superparamagnetic nanoparticles are beads with a diameter of 3 nm, and generally, the allowable range of nanoparticle diameters can be 3 to 20 nm.

[0033] The scaffolds 10, 20 were obtained by two-photon polymerization of a superparamagnetic resist deposited on a glass substrate coated with a sacrificial layer of polyvinyl alcohol (PVA), also known as "direct laser writing". The PVA sacrificial layer was obtained by depositing on the glass substrate 0.5 ml of PVA at a concentration of 2 mg / ml, maintained at a temperature of 80° C. for 5 minutes.

[0034] Two-photon polymerization of scaffolds 10 and 20 was achieved by using a direct-write laser lithography system (Photonic Professional System available from Nanoscribe, Inc.) with a 63x immersion lens, a numerical aperture (NA) of 1.4, a 780 nm laser beam, a writing speed of 10 mm / s, and a laser power of 70.2 mW. To ensure removal of the uncured photoresist, the scaffolds were developed in SU-8 developer (MicroChem) for 30 min and washed with isopropyl alcohol and deionized water.

[0035] The magnetic field generating unit 31 is a long and narrow plate made by two-photon polymerization of photoresist containing magnetized ferromagnetic particles (as shown in FIG. 3).

[0036] <Example 2> In a second embodiment, the co-culture system 1 comprises at least one first scaffold 10 having superparamagnetic properties and at least one second scaffold 20 having ferromagnetic properties.

[0037] At least one first scaffold 10 is made of a composite material obtained by inserting superparamagnetic nanoparticles into a negative photoresist sensitive to UV light, as described in Example 1.

[0038] At least one second scaffold 20 is made of a composite material obtained by placing magnetized ferromagnetic nanoparticles in a negative photoresist sensitive to ultraviolet light, according to the technique described in Example 1, but instead of the iron oxide superparamagnetic nanoparticles, iron oxide ferromagnetic nanoparticles with a diameter of 50 nm are used. However, the allowable range of the nanoparticle diameter can be 50-100 nm. In this example, the second scaffold 20 acts as a magnetic field generator, which can cause magnetization of the superparamagnetic nanoparticles of the first scaffold 10.

[0039] <Example 3> In another embodiment, the co-culture system 1 comprises at least one first scaffold 10 and at least one second scaffold 20 having ferromagnetic properties. Both scaffolds 10, 20 are made of a composite material obtained by placing magnetized ferromagnetic nanoparticles in a negative photoresist sensitive to UV light, according to the same technique described in embodiment 1, but instead of the iron oxide superparamagnetic nanoparticles, iron oxide ferromagnetic nanoparticles with a diameter of 50 nm are used. However, the acceptable range of the nanoparticle size can be 50-100 nm.

[0040] In this modified embodiment, both scaffolds 10, 20 act as magnetic field generators 31 due to the magnetized ferromagnetic nanoparticles.

[0041] Of course, other variations of the system of the present invention are possible, and therefore the present invention is not limited to the exemplary embodiments illustrated herein, but rather many changes, modifications or substitutions of equivalent parts and elements may be made without departing from the basic idea of ​​the invention as defined in the claims.

[0042] The in vitro three-dimensional cell co-culture method of the present invention is described below.

[0043] The method of the present invention comprises the following steps: - culturing a plurality of cells of a first cell type on at least one first scaffold 10 comprising a magnetizable material and a plurality of cells of a second cell type on at least one second scaffold 20 comprising a magnetizable material while maintaining them physically separated; - bringing the scaffolds 10, 20 closer together under the action of a magnetic field until they come into contact at least at one contact surface.

[0044] In other words, the method is based on culturing at least two different cell cultures separately on magnetic scaffolds 10, 20 and then merging the two cell cultures by using a magnetic field generated by a magnetic field generating unit 31 to bring both scaffolds 10, 20 closer to each other until they achieve complete contact on at least one surface.

[0045] This allows individual cell cultures to be grown separately and then subsequently assembled into 3D co-culture structures.

[0046] The above method can be implemented by using a system 1 similar to that described herein above.

[0047] In one example embodiment, depicted generally in Figure 4, cells of a first cell type are seeded onto at least one scaffold 10 in a first culture chamber 100, and cells of a second cell type are seeded onto at least one scaffold 20 in a second culture chamber 200 separate from the first culture chamber 100. The scaffolds are then transferred to a co-culture chamber 300 and left to interact under the action of a magnetic field inside the chamber.

[0048] By releasing the scaffolds 10, 20 into the co-culture chamber 300 in different release orders, scaffold assemblies 10, 20 with different scaffold distributions, number of layers, aspect ratios and shapes can be obtained (some examples are shown in FIG. 5). Magnetic field generating elements 31 (e.g., magnetized leaves of ferromagnetic material, etc.) constrained to the bottom of the co-culture chamber 300 can be used to define the basic shape of the scaffold assemblies 10, 20 (a schematic example is shown in FIGS. 6a and 6b).

[0049] In another embodiment shown in Figure 7, the scaffolds 10, 20 are inserted into a microfluidic chip. First, at least one first scaffold 10 and at least one second scaffold 20 are placed in separate chambers of the microfluidic chip connected to a co-culture chamber 300 by passages, and then, by adjusting the flow through the above-mentioned passages, the scaffolds are transported into the co-culture chamber 300 and interact under the action of a magnetic field.

Claims

1. A system (1) for in vitro three-dimensional cell co-culture, comprising: a first prismatic and porous scaffold (10) comprising a magnetizable material suitable for supporting the growth of a first cell type; a second prismatic and porous scaffold (20) comprising a magnetizable material suitable for supporting the growth of a second cell type, the second scaffold (20) having a shape complementary to the shape of the first prismatic and porous scaffold (10); A magnetic field generating unit (31) configured to generate a magnetic field that generates an attractive magnetic force between the first scaffold (10) and the second scaffold (20); A cell co-culture chamber (300); Equipped with The system (1), characterized in that the first scaffold (10) and the second scaffold (20) are exposed to the magnetic field generated by the magnetic field generating unit (31) in the cell co-culture chamber (300).

2. the magnetizable material included in the first scaffold (10) and / or the second scaffold (20) is a ferromagnetic material or a superparamagnetic material; The system (1) according to claim 1.

3. The magnetic field generating unit (31) includes at least one permanent magnet. A system (1) according to claim 1 or 2.

4. the at least one permanent magnet being a strip-shaped member of magnetized ferromagnetic material; The width of the member of ferromagnetic material is equal to the width of the scaffold (10, 20); The system (1) according to claim 3.

5. The magnetic field generating unit (31) is included in the structure of at least one of the first and second scaffolds (10, 20), A system (1) according to any one of claims 1 to 3.

6. The magnetic field generating unit (31) includes a plurality of magnetized ferromagnetic nanoparticles. The system (1) according to claim 5.

7. The magnetic field generating unit (31) is identical to at least one of the first and second scaffolds (10, 20); A system (1) according to any one of claims 1 to 3.

8. The system (1) comprises a first incubation chamber (100) for at least one of the first scaffolds (10) and a second incubation chamber (200) for at least one of the second scaffolds (20), wherein the first scaffold (10) and the second scaffold (20) are not influenced by mutual attractive magnetic forces; A system (1) according to any one of claims 1 to 7.

9. The first culture chamber (100) and the second culture chamber (200) communicate with the co-culture chamber (300); The system (1) according to claim 8.

10. The system (1) according to any one of claims 1 to 9, wherein the co-culture chamber (300) is provided in a microfluidic chip.

11. 1. A method for in vitro three-dimensional cell co-culture, comprising: seeding a first prismatic and porous scaffold (10) comprising a magnetizable material suitable for supporting the growth of a first cell type and a second prismatic and porous scaffold (20) comprising a magnetizable material suitable for supporting the growth of a second cell type while maintaining the first scaffold (10) physically separated from the second prismatic and porous scaffold (20) comprising a magnetizable material suitable for supporting the growth of a second cell type, wherein the second prismatic and porous scaffold (20) has a shape complementary to the shape of the first prismatic and porous scaffold (10); A step of approaching the first scaffold (10) to the second scaffold (20) under the action of a magnetic field generated by a magnetic field generating unit (31) until the first scaffold (10) and the second scaffold (20) come into contact with each other on at least one surface; The method according to claim 1, further comprising:

12. the magnetizable material included in the first scaffold (10) and / or the second scaffold (20) is a ferromagnetic material or a superparamagnetic material; The method of claim 11.

13. The magnetic field generating unit (31) includes at least one permanent magnet.

13. The method according to claim 11 or 12.

14. The magnetic field generating unit (31) includes at least one elongated member made of magnetized ferromagnetic material, The width of the member of ferromagnetic material is equal to the width of the scaffold (10, 20); 14. The method of claim 13.

15. The magnetic field generating unit (31) is included in at least one of the first and second scaffolds (10, 20), 14. The method according to any one of claims 11 to 13.

16. The magnetic field generating unit (31) is included in at least one of the first scaffold (10) and the second scaffold (20) as a plurality of magnetized ferromagnetic nanoparticles.

16. The method of claim 15.

17. The magnetic field generating unit (31) is identical to at least one of the first and second scaffolds (10, 20); The method of claim 11.

18. The step of bringing the first scaffold (10) into close proximity with the second scaffold (20) is performed in a cell co-culture chamber (300); 18. The method according to any one of claims 11 to 17.

19. The cell co-culture chamber (300) is provided in a microfluidic chip.

20. The method of claim 18.

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