Three-dimensional cell culture device and method based on wall-free fluid chip

Abstract

The invention discloses a three-dimensional cell culture device and method based on a device-wall-free fluid chip, the device comprises a super-hydrophobic prismatic table array substrate and a plurality of liquid marbles placed on the super-hydrophobic prismatic table array substrate, and the plurality of liquid marbles can be fused and converged to form the device-wall-free fluid chip with a pipeline structure. The method comprises the following steps: placing liquid marbles containing a cell culture fluid on a super-hydrophobic prismoid array substrate, wherein the adjacent liquid marbles are in contact with each other or are mediated by a liquid bridge to be fused and form a device-wall-free fluid chip; then performing cell culture in a sterile environment, wherein the cells are gathered on the central axis of the bottom of the fluid chip and near the central axis in the culture process; and extracting supernatant of the culture solution in the fluid chip, injecting the temperature-sensitive hydrogel solution, and transferring and dissolving the temperature-sensitive hydrogel solution after the temperature-sensitive hydrogel solution is solidified to obtain the three-dimensional multicellular aggregate. According to the device and the method, three-dimensional culture of various cells can be realized, and the centimeter-scale multicellular aggregate of which the shape is matched with the macroscopic shape of the device-wall-free fluid chip is obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell culture, and relates to a three-dimensional cell culture device, in particular to a three-dimensional cell culture device and method based on a wall-free fluidic chip. BACKGROUND

[0002] In the field of cell biology and tissue engineering research, two-dimensional cell culture system is usually constructed on a culture dish, a glass slide or other planar substrate surface. Such monolayer culture environment can effectively control the gradient distribution of nutrients, the diffusion and removal of metabolic products and the contact between cells due to its simple physical structure. The traditional two-dimensional culture has the following limitations: lack of three-dimensional spatial structure, unable to simulate the in vivo environment; difficult to maintain cell polarity, which is crucial for cell function; cell morphology is flattened and distorted, leading to significant differences between key characteristics such as cytoskeleton, signal transduction and gene expression and the actual state in vivo.

[0003] Three-dimensional cell culture technology can more truly simulate the in vivo environment and promote cell-cell interactions, providing models for disease research, organ regeneration and drug testing. Among them, the hanging drop culture method is simple and convenient, which uses gravity and surface tension to form three-dimensional aggregates inside the inverted hanging cell suspension, without the need for complex reagents or equipment to construct cell spheroids. However, the hanging drop method also has significant limitations: the spherical profile of the droplet formed under the constraint of surface tension restricts the morphology of the cell aggregates (mostly spherical), and a single morphology of cell aggregates cannot meet the construction needs of complex biological models. SUMMARY

[0004] In view of the limitations of existing three-dimensional cell culture technology, the present application aims to provide a three-dimensional cell culture device and method based on a wall-free fluidic chip suspended in the air, to solve the bottleneck problem of the hanging drop method in obtaining complex morphology cell aggregates.

[0005] To solve the above technical problems, the present application adopts the following technical solutions:

[0006] A three-dimensional cell culture device based on a wall-free fluidic chip suspended in the air, comprising a culture dish, an ultrahydrophobic prism array substrate is placed in the culture dish, a plurality of liquid marbles are placed in the V-shaped grooves of the ultrahydrophobic prism array substrate, and the plurality of liquid marbles can fuse and converge to form a wall-free fluidic chip with a pipeline structure.

[0007] The present application also includes the following technical features:

[0008] Specifically, the super-hydrophobic prism array substrate comprises a bottom plate, a plurality of convex platforms are integrally arranged on the bottom plate, and the outer surface of each convex platform is coated with a hydrophobic coating; the plurality of convex platforms are arranged in a matrix structure, the gap between two adjacent convex platforms is a V-shaped groove, a plurality of liquid marbles arranged in the V-shaped groove along the transverse direction are in communication with each other and constitute a wall-less fluid chip of pipeline structure, a plurality of liquid marbles arranged in the V-shaped groove along the longitudinal direction are in communication with each other and constitute a wall-less fluid chip of pipeline structure; there is a gap between the bottom end of the liquid marble or the wall-less fluid chip and the bottom end of the V-shaped groove, that is, the liquid marble or the wall-less fluid chip is arranged in a suspended manner.

[0009] Specifically, the material of the hydrophobic coating is silicon dioxide.

[0010] Specifically, the convex platform is a trapezoidal structure with a small upper part and a large lower part, and the axis cross-sectional shape of the V-shaped groove is an inverted triangle, and the top angle of the inverted triangle is 15° to 60°.

[0011] Specifically, the liquid marble comprises a porous hydrophobic shell layer composed of a plurality of micron or nanometer hydrophobic particles; the hydrophobic shell layer encapsulates a liquid core.

[0012] Specifically, the hydrophobic particles constituting the hydrophobic shell layer are made of polytetrafluoroethylene, silicon dioxide, polyvinylidene fluoride, lycopodium powder or glass sand.

[0013] Specifically, the particle size of the hydrophobic particles is 20 nm to 100 μm.

[0014] Specifically, the liquid core is a cell-containing culture solution or a temperature-sensitive hydrogel solution.

[0015] Specifically, the temperature-sensitive hydrogel is methacrylated gelatin.

[0016] The application also protects a three-dimensional cell culture method based on a suspended wall-less fluid chip, which is implemented by using the device described above; the method comprises the following steps: placing a liquid marble containing cell culture solution on the super-hydrophobic prism array substrate, and the adjacent liquid marbles contact each other or fuse and form a wall-less fluid chip through droplet mediation; then, cell culture is carried out in a sterile environment, and during the culture process, cells gather on the central axis at the bottom of the wall-less fluid chip and its vicinity, and grow into a three-dimensional multicellular aggregate matching the geometric shape of the wall-less fluid chip; the supernatant of the culture solution in the wall-less fluid chip is extracted, and a temperature-sensitive hydrogel solution is injected again, and after the temperature-sensitive hydrogel solution is solidified, the three-dimensional cell aggregate is fixed, and then the hydrogel is transferred to a conventional container (such as a culture dish) and heated and melted at 37°C, so that subsequent manipulation of the three-dimensional cell aggregate can be carried out in the container.

[0017] Specifically, the method comprises the following steps:

[0018] Step one, preparation of the suspended wall-free fluidic chip:

[0019] The hydrophobic shell is prepared by using hydrophobic particles, and the liquid core wrapped by the hydrophobic shell is a culture solution containing cells; the prepared liquid beads are suspended on the V-shaped groove, and the adjacent liquid beads can be fused and form a suspended wall-free fluidic chip after contacting each other or being mediated by a liquid bridge;

[0020] Step two, three-dimensional cell culture:

[0021] The super-hydrophobic prism array substrate with the wall-free fluidic chip prepared in step one is placed in a culture dish, and then sterile liquid is added to the culture dish to increase the environmental humidity, and the liquid surface is lower than the lowest point of the V-shaped groove. The culture dish is covered with a dish cover and placed in a sterile incubator for cell culture.

[0022] Step three, obtaining three-dimensional multicellular aggregates:

[0023] After the culture is completed, the covered culture dish is taken out, and it is observed that the cells have gathered at the bottom of the wall-free fluidic chip to form three-dimensional multicellular aggregates matching the shape of the wall-free fluidic chip. The supernatant of the culture solution in the wall-free fluidic chip is extracted with a pipette gun, and the same volume of a temperature-sensitive hydrogel solution is injected. The super-hydrophobic prism array substrate and the wall-free fluidic chip with the multicellular aggregates and the hydrogel solution as the liquid core are stored at 5℃ for 20min. Under low temperature conditions, the temperature-sensitive hydrogel solution solidifies. The super-hydrophobic prism array substrate is inverted to make the solidified wall-free fluidic chip fall off from the V-shaped groove in the form of a solid. The solidified material is heated to 37℃, and the temperature-sensitive hydrogel is liquefied, so that the three-dimensional multicellular aggregates can be subsequently manipulated.

[0024] Compared with the prior art, the present application has the following beneficial technical effects:

[0025] (I) The present application uses micro / nano particles to wrap liquid, and obtains a wall-free fluidic chip after processing and shaping. Because there is no solid wall constraint, the interaction of substances inside and outside the chip is very convenient, which is of great significance for biological system manipulation. Culturing cells in a suspended wall-free fluidic chip can realize three-dimensional growth of cells and obtain centimeter-scale multicellular aggregates matching the macroscopic morphological characteristics of the wall-free fluidic chip.

[0026] (II) The present application adopts the bottom-suspended wall-less fluidic chip to carry out three-dimensional cell culture. The bottom of the wall-less fluidic chip presents a curved profile under the synergistic effect of surface tension and gravity, which can induce cells to be oriented and aggregated at the central axis of the bottom of the wall-less fluidic chip and its vicinity. Through the above principle, three-dimensional culture of human osteosarcoma (143B) cells, breast cancer (HeLa) cells and other cells can be realized, and centimeter-scale multicellular aggregates with a morphology matching the macroscopic morphology of the suspended wall-less fluidic chip are obtained.

[0027] (III) The present application breaks through the bottleneck problem that it is difficult to obtain large-size and complex-morphology cell aggregates in traditional three-dimensional culture methods, and can provide a biological model with complex morphology for disease research, which is of great significance for the development of organ regeneration, tumor drug screening and drug toxicity analysis.

[0028] (IV) The device of the present application has no connection and fixation between the elements, and is convenient to disassemble and assemble, and the cell culture operation is simple. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a top view schematic diagram of the preparation process of the suspended wall-less fluidic chip.

[0030] Figure 2 It is a schematic diagram of the overall structure of the three-dimensional cell culture device based on the suspended wall-less fluidic chip.

[0031] Figure 3 It is a three-dimensional view (a) and a two-dimensional side view (b) of the "cross-shaped" suspended wall-less fluidic chip.

[0032] Figure 4 It is a schematic diagram of three-dimensional cell culture using the suspended wall-less fluidic chip.

[0033] Figure 5 The morphology of the three-dimensional cell aggregate is shown. Figure 5 M: (a) is a strip-shaped centimeter-scale multicellular aggregate obtained when the present application adopts a long strip-shaped suspended wall-less fluidic chip to carry out three-dimensional cell culture; (b) is a strip-shaped centimeter-scale multicellular aggregate picked up in the air with tweezers; (c) is a micrometer-scale multicellular aggregate in the form of a sphere obtained when three-dimensional cell culture is carried out by the hanging drop method.

[0034] The meanings of the reference numerals in the figure are as follows: 1 - culture dish, 2 - superhydrophobic prism array substrate, 3 - liquid bead, 4 - wall-less fluidic chip, 5 - sterile liquid, 6 - sterile incubator.

[0035] 201 - bottom plate, 202 - hydrophobic coating, 203 - V-shaped groove, 204 - boss.

[0036] 301 - liquid core, 302 - hydrophobic particles.

[0037] The technical solutions of the present application are further described below in combination with the embodiments. DETAILED DESCRIPTION

[0038] It should be noted that the materials or culture media used in the present application are conventional materials or culture media known in the prior art, unless otherwise specified.

[0039] In accordance with the above technical solutions, the specific embodiments of the present application are given below. It should be noted that the present application is not limited to the following specific embodiments, and any equivalent variations made on the basis of the technical solutions of the present application fall within the scope of protection of the present application.

[0040] Embodiment 1

[0041] This embodiment gives a three-dimensional cell culture device based on a suspended wall-less fluidic chip. As shown in the figure, the device comprises a culture dish 1, an ultrahydrophobic prism array substrate 2 is placed in the culture dish 1, a plurality of suspended liquid marbles 3 are placed in the V-shaped grooves 203 of the ultrahydrophobic prism array substrate 2, and the plurality of liquid marbles 3 can fuse and converge to form a pipe structure wall-less fluidic chip 4. Figure 1

[0042] As a specific scheme of this embodiment, the culture dish 1 contains sterile liquid 5; the sterile liquid 5 is sterile water, which is used to maintain the humidity of the culture environment and inhibit the evaporation of the liquid inside the wall-less fluidic chip 4 during the culture process.

[0043] As a specific scheme of this embodiment, the ultrahydrophobic prism array substrate 2 comprises a bottom plate 201 of a rectangular flat plate structure, a plurality of bosses 204 are integrally placed on the bottom plate 201, and a hydrophobic coating 202 is coated on the outer surface of each boss 204; the plurality of bosses 204 are arranged in a matrix structure, the gap between the adjacent two bosses 204 is a V-shaped groove 203, the plurality of liquid marbles 3 arranged along the transverse direction of the V-shaped groove 203 are in communication with each other and constitute a pipe structure wall-less fluidic chip 4, and there is a gap between the bottom end of the liquid marble 3 or the wall-less fluidic chip 4 and the bottom end of the V-shaped groove 203 (i.e. suspended). In this embodiment, the liquid marbles 3 contact each other or are mediated by liquid bridges, and then fuse and converge along the V-shaped groove 203 to form a pipe structure wall-less fluidic chip 4, and the suspended setting makes the bottom profile of the pipe structure wall-less fluidic chip 4 curved, thereby promoting the cells to gather in the middle line area of the bottom of the wall-less fluidic chip 4.

[0044] As a specific scheme of this embodiment, the boss 204 is a trapezoidal body structure with a small upper part and a large lower part, and the axis cross-sectional shape of the V-shaped groove 203 is a reverse triangle, and the top angle of the reverse triangle is 15° to 60°.

[0045] ​As a specific solution of the embodiment, the material of the hydrophobic coating 202 is silicon dioxide.

[0046] As a specific solution of the embodiment, the liquid bead 3 comprises a porous hydrophobic shell layer composed of a plurality of micron or nanometer hydrophobic particles 302; the hydrophobic shell layer wraps a liquid core 301 to form a spherical liquid bead.

[0047] As a specific solution of the embodiment, the hydrophobic particles 302 constituting the hydrophobic shell layer are made of polytetrafluoroethylene, silicon dioxide, polyvinylidene fluoride, lycopodium powder, or glass sand; the hydrophobic particles 302 realize the formation of the wall-free fluidic chip 4 with a pipeline structure by the retraction of the liquid core 301 under the surface tension.

[0048] As a specific solution of the embodiment, the liquid core 301 is a cell-containing culture solution or a temperature-sensitive hydrogel solution.

[0049] As a specific solution of the embodiment, the temperature-sensitive hydrogel is methacrylated gelatin.

[0050] As a specific solution of the embodiment, the particle size of the hydrophobic particles 302 is 20 nm to 100 μm.

[0051] Embodiment 2:

[0052] The present embodiment provides a three-dimensional cell culture method based on a suspended wall-free fluidic chip, which is realized by using the device of embodiment 1. The method specifically comprises the following steps:

[0053] Step 1: Preparation of a suspended wall-free fluidic chip:

[0054] The hydrophobic shell layer is prepared by using single-layer hydrophobic particles 302 (about 20 nm in diameter) made of silicon dioxide, and the liquid core 301 is a culture solution containing 143B cells (about 10 6 cells per 1 mL). The cell culture medium is specifically a basic culture medium (such as DMEM, MEM, etc.) containing 10 vt% fetal bovine serum and 100 U / mL penicillin. The prepared liquid bead 3 is suspended on the V-shaped groove 203, and the adjacent liquid beads 3 can be fused and form a wall-free fluidic chip 4 after contacting each other.

[0055] Step 2: Three-dimensional cell culture:

[0056] Put the super-hydrophobic prism array substrate 2 with the wall-less fluidic chip 4 prepared in step one into a culture dish 1, then add sterile liquid 5 into the culture dish 1 to increase the environmental humidity, and the liquid level is lower than the lowest point of the V-shaped groove 203. Cover the culture dish 1 with a dish cover and place it in a sterile incubator 6 with a temperature of 37°C, a carbon dioxide concentration of 5%, and a humidity of 90% for cell culture.

[0057] Step three, obtaining the three-dimensional multicellular aggregates:

[0058] After 24 hours of culture, take out the covered culture dish 1, and it is observed that the cells have aggregated at the bottom of the wall-less fluidic chip 4. Remove the culture solution inside the wall-less fluidic chip 4 with a pipette gun, and then inject the same volume of a warm-sensitive hydrogel solution. Place the super-hydrophobic prism array substrate 2 and the wall-less fluidic chip 4 with the liquid core being the above-mentioned hydrogel solution and the multicellular aggregates in a 5°C environment for 20 minutes. The warm-sensitive hydrogel solution solidifies under low-temperature conditions. Invert the super-hydrophobic prism array substrate 2 to make the solidified wall-less fluidic chip 4 fall off from the V-shaped groove in the form of a solid. Heat the fallen solid to 37°C to liquefy the warm-sensitive hydrogel, and obtain the three-dimensional multicellular aggregates matching the shape of the wall-less fluidic chip 4.

[0059] Example 3:

[0060] This example gives a three-dimensional cell culture method based on a suspended wall-less fluidic chip, which is implemented by using the device of Example 1. This method is completely the same as steps two and three of Example 2, and the difference lies in step one.

[0061] In this example, step one is as follows: hydrophobic particles 302 (about 35 μm in diameter) made of polytetrafluoroethylene are used to prepare a hydrophobic shell, and the liquid inner core 301 is a culture solution containing 143B cells (about 10 6 Each 1 mL). The prepared liquid beads 3 are suspended on the V-shaped groove 203. Adjacent liquid beads 3 are in contact with each other and are difficult to directly fuse, so it is necessary to mediate fusion through a water-carrying needle. The water-carrying amount of the needle is controlled in the range of 0.1-0.5 μL, and the two liquid beads 3 can be fused by contacting the needle and forming a suspended wall-less fluidic chip 4.

[0062] Example 4:

[0063] This example gives a three-dimensional cell culture method based on a suspended wall-less fluidic chip, which is implemented by using the device of Example 1. This method is completely the same as steps two and three of Example 2, and the difference lies in step one.

[0064] In this embodiment, step one is as follows: hydrophobic particles 302 (diameter about 50 μm) made of polytetrafluoroethylene are used to prepare the hydrophobic shell, and the liquid core 301 is a culture solution containing HeLa cells (about 10 6 6 In this embodiment, step one is as follows: hydrophobic particles 302 (diameter about 50 μm) made of polytetrafluoroethylene are used to prepare the hydrophobic shell, and the liquid core 301 is a culture solution containing HeLa cells (about 10

[0065] Example 5

[0066] This embodiment gives a three-dimensional cell culture method based on the suspended wall-free fluidic chip. The method is realized by using the device of Example 1. The method is completely the same as steps two and three of Example 2, and the difference is in step one.

[0067] In this embodiment, step one is as follows: hydrophobic particles 302 (diameter about 50 μm) made of polytetrafluoroethylene are used to prepare the hydrophobic shell, and the liquid core 301 is a culture solution containing HeLa cells (about 10 6 In this embodiment, step one is as follows: hydrophobic particles 302 (diameter about 50 μm) made of polytetrafluoroethylene are used to prepare the hydrophobic shell, and the liquid core 301 is a culture solution containing HeLa cells (about 10

[0068] Example 6

[0069] This embodiment gives a three-dimensional cell culture method based on the suspended wall-free fluidic chip. The method is realized by using the device of Example 1. The method is completely the same as steps two and three of Example 2, and the difference is in step one.

[0070] In this embodiment, step one is as follows: hydrophobic particles 302 (diameter about 50 μm) made of polytetrafluoroethylene are used to prepare the hydrophobic shell, and the liquid core 301 is a culture solution containing HeLa cells (about 10 6The liquid beads 3 are placed in the V-shaped groove 203, and the adjacent liquid beads 3 are difficult to directly fuse with each other, thus the fusion is mediated by the water-carrying needle, the water-carrying amount of the needle is controlled in the range of 0.1-0.5 μL, and the two liquid beads 3 contact the needle to fuse and form the suspended wall-free fluid chip 4.

[0071] Example 7

[0072] This embodiment gives a three-dimensional cell culture method based on the suspended wall-free fluid chip, which is realized by the device of Example 1. The method is completely same as steps two and three of Example 2, and the difference is that step one is different.

[0073] In this embodiment, step one is specifically as follows: the hydrophobic shell is prepared by using the hydrophobic particles 302 (diameter is about 100 μm) of glass sand material, and the liquid inner core 301 is the culture solution containing 143B cells (about 10 6 The liquid beads 3 are placed in the V-shaped groove 203, and the adjacent liquid beads 3 are difficult to directly fuse with each other, thus the fusion is mediated by the water-carrying needle, the water-carrying amount of the needle is controlled in the range of 0.1-0.5 μL, and the two liquid beads 3 contact the needle to fuse and form the suspended wall-free fluid chip 4.

Claims

1. A three-dimensional cell culture device based on a suspended-wall-less fluidic chip, comprising a culture dish (1), characterized in that: A super-hydrophobic prism array substrate (2) is placed in a culture dish (1), and a plurality of liquid marbles (3) are placed on the super-hydrophobic prism array substrate (2), the plurality of liquid marbles (3) can fuse and converge to form a wall-less fluidic chip (4) with a pipeline structure.

2. The three-dimensional cell culture device based on the suspended- wall-less fluidic chip according to claim 1, wherein, The super-hydrophobic prism array substrate (2) comprises a bottom plate (201), and a plurality of bosses (204) are integrally placed on the bottom plate (201), and the outer surface of each boss (204) is coated with a hydrophobic coating (202); The plurality of bosses (204) are arranged in a matrix structure, and the gap between two adjacent bosses (204) is a V-shaped groove (203), and the plurality of liquid marbles arranged in the transverse direction of the V-shaped groove (203) are connected to each other and constitute a wall-less fluidic chip (4) with a pipeline structure, and the plurality of liquid marbles arranged in the longitudinal direction of the V-shaped groove (203) are connected to each other and constitute a wall-less fluidic chip (4) with a pipeline structure. There is a gap between the bottom end of the liquid marble (3) or the wall-less fluidic chip (4) and the bottom end of the V-shaped groove (203).

3. The three-dimensional cell culture device based on the suspended- wall-less fluidic chip according to claim 2, wherein, The material of the hydrophobic coating (202) is silicon dioxide.

4. The three-dimensional cell culture device based on the suspended- wall-less fluidic chip according to claim 2, wherein, The boss (204) is a trapezoidal structure with a small upper part and a large lower part, and the axis cross-sectional shape of the V-shaped groove (203) is an inverted triangle, and the top angle of the inverted triangle is 15° to 60°.

5. The three-dimensional cell culture device based on the suspended- wall-less fluidic chip according to claim 1, wherein, The liquid marble (3) comprises a porous hydrophobic shell layer composed of a plurality of micron or nanometer hydrophobic particles (302), and the hydrophobic shell layer encapsulates a liquid core (301).

6. The three-dimensional cell culture device based on the suspended- wall-less fluidic chip according to claim 5, wherein, The hydrophobic particles (302) constituting the hydrophobic shell layer are made of polytetrafluoroethylene, silicon dioxide, polyvinylidene fluoride, lycopodium powder or glass sand.

7. The three-dimensional cell culture device based on the suspended- wall-less fluidic chip according to claim 5, wherein, The particle size of the hydrophobic particles (302) is 20 nm to 100 μm.

8. The three-dimensional cell culture device based on the suspended- wall-less fluidic chip according to claim 5, wherein, The liquid core (301) is a cell-containing culture solution or a temperature-sensitive hydrogel solution.

9. The three-dimensional cell culture device based on the suspended- wall-less fluidic chip according to claim 8, wherein, The temperature-sensitive hydrogel is methacrylated gelatin.

10. A three-dimensional cell culture method based on a suspended-wall-less fluidic chip, which is implemented by using the device according to any one of claims 1 to 9; characterized in that, The method comprises the following steps: placing the liquid marble (3) containing the cell culture solution on the super-hydrophobic prism array substrate (2), and the adjacent liquid marbles (3) can fuse and form a wall-less fluidic chip (4) through liquid bridge mediation after contacting each other; then, cell culture is carried out in a sterile environment, and the cells are aggregated on the central axis and its vicinity at the bottom of the wall-less fluidic chip (4) during the culture process; the supernatant of the culture solution in the wall-less fluidic chip (4) is extracted, and a temperature-sensitive hydrogel solution is injected, and after the temperature-sensitive hydrogel solution is solidified, a three-dimensional cell aggregate is fixed, and then the hydrogel is transferred to a conventional container and heated and melted at 37℃, so that subsequent manipulation of the three-dimensional cell aggregate can be carried out in the container.

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