Three-dimensional culture device and method for non-destructively manipulating and monitoring internal signals of cell spheroids

Abstract

The present invention discloses a three-dimensional culture device and method capable of non-destructively manipulating and monitoring internal signals of cell spheres. The device includes: an ultra-low adhesion cell sphere culture chamber; the chamber is in a vertical tubular shape, and the lower section inside the tube is an agarose solid culture medium; at least one metal wire conductor; the outer surface of the conductor is wrapped with an insulating layer except at both ends, and a certain length in the middle is left with a completely exposed surface; the conductor penetrates through the chamber such that the exposed section in the middle of the conductor is exactly located on the upper surface of the agarose solid culture medium; a fixing bracket; the fixing bracket is used to fix the chamber and the conductor. The device and method of the present invention can not only achieve the culture of cell spheres, but also extremely conveniently achieve the manipulation of cell spheres and the monitoring of internal and external signals of cell spheres, and the entire process will not cause damage to the cell spheres. The device is simple to manufacture, has strong expandability, and can be mass-produced.

Description

Technical Field

[0001] The present invention relates to the technical field of three-dimensional cell culture, and relates to a cell sphere culture device and a method for manipulating and monitoring signals inside the cell sphere, and particularly relates to a three-dimensional culture device and method for non-destructively manipulating and monitoring signals inside the cell sphere. Background Art

[0002] In neuroscience and brain science research, neural networks are a very popular research direction. The traditional method is to culture nerve cells on a microelectrode array, record the electrical signals of the cells, and study and analyze the connections between nerve cells. However, the brain is three-dimensional, and nerve cells in the body are all in a three-dimensional environment. Therefore, in order to better simulate the in-vivo environment, more and more research has begun to focus on three-dimensional cell sphere culture, three-dimensional brain organoids, etc.

[0003] However, in traditional three-dimensional cell sphere culture methods, such as culturing cells on a hydrogel, it is very difficult to transfer the cell sphere. During the process of aspiration and release with a pipette, it is easy to damage the cell sphere or even cause it to rupture. At the same time, the signals inside the cell sphere cannot be recorded. If a probe is forcibly inserted into the cell sphere, it will cause irreversible damage to the cells. In traditional culture methods, hypoxia in the center of the cell sphere is inevitable. As the diameter of the cell sphere increases, insufficient oxygen supply to the internal cells will cause a large number of deaths, affecting the internal connections of the nerve cell sphere. Summary of the Invention

[0004] The purpose of the present invention is to provide a three-dimensional culture device and method for non-destructively manipulating and monitoring signals inside the cell sphere in view of the deficiencies of the prior art. The device and method can not only achieve the culture of the cell sphere, but also extremely conveniently achieve the manipulation of the cell sphere and the monitoring of internal and external signals of the cell sphere, and the whole process will not cause damage to the cell sphere.

[0005] The technical solution adopted by the present invention is as follows:

[0006] In a first aspect, a three-dimensional culture device for non-destructively manipulating and monitoring signals inside the cell sphere according to an embodiment of the present invention includes:

[0007] A cell sphere culture chamber with ultra-low adsorption; the chamber is in a vertical tubular shape, and the lower section inside the tube is an agarose solid medium;

[0008] At least one metal wire; the outer surface of the wire is wrapped with an insulating layer except at both ends, and a certain length in the middle is left with a completely exposed surface; the wire penetrates through the chamber and makes the exposed section in the middle of the wire exactly located on the upper surface of the agarose solid medium;

[0009] A fixing bracket; the fixing bracket is used to fix the chamber and the wire.

[0010] In the above technical solution, further, the ultra-low adsorption cell sphere culture chamber includes an ultra-low adsorption tube with openings at both ends and agarose. The ultra-low adsorption tube is thicker at the top and thinner at the bottom, and the agarose is located in the lower section of the ultra-low adsorption tube.

[0011] Further, the metal wire conductor is a platinum wire, a gold wire, a tungsten wire, a silver wire or a copper wire.

[0012] Further, the inner diameter of the tube of the ultra-low adsorption cell sphere culture chamber is 2 - 12 mm, which can be specifically determined according to the size of the cell sphere to be cultured. Generally, the most suitable inner diameter is about 5 times the diameter of the target cell sphere.

[0013] Further, the diameter of the metal wire conductor is 25 - 200 μm, which can be specifically determined according to the size of the cell sphere to be cultured. Generally, the most suitable diameter is about 1 / 10 of the diameter of the target cell sphere.

[0014] Further, the length of the exposed section of the conductor is 5 - 100 μm, which can be specifically determined according to the size of the cell sphere to be cultured. Generally, the most suitable length is about 1 / 50 of the diameter of the target cell sphere.

[0015] In a second aspect, an embodiment of the present invention provides a method for preparing any of the above three-dimensional culture devices, including:

[0016] Fabricating the ultra-low adsorption cell sphere culture chamber: First, heat the agarose until it becomes a clear and transparent liquid, suck the liquid into the middle section of the tube using the ultra-low adsorption tube, and place it at room temperature to allow the agarose to solidify.

[0017] Insulating the metal wire conductor: Fix both ends of the metal wire on the substrate with tape, evenly apply photoresist, and after masking, irradiating with light and treating with a developer using photolithography technology, form an exposed section in the middle part of the metal wire, and the remaining part is wrapped with photoresist.

[0018] Assembling the culture device: Vertically fix the ultra-low adsorption tube on the bracket, pass the wire through the ultra-low adsorption tube, clamp both ends of the wire with a fixing clip and straighten it, so that the wire is exactly in the center of the ultra-low adsorption tube, and the exposed section is exactly located on the upper surface of the agarose.

[0019] In a third aspect, an embodiment of the present invention provides a method for culturing, manipulating cell spheres and monitoring internal signals, using any of the above three-dimensional culture devices. The method includes:

[0020] Add the cell suspension along the wire into the ultra-low adsorption cell sphere culture chamber, and then place the entire device in an incubator.

[0021] Replace half of the culture medium every day, and cell spheres will be formed in 5 - 10 days and the wire will be wrapped.

[0022] Immerse the ultra-low adsorption cell sphere culture chamber into the culture medium, and slowly draw out the wire, then the cell sphere can be taken out along with it.

[0023] Place the wire carrying the cell sphere on the planar electrode, and the signals outside the cell sphere can be recorded.

[0024] Connect both ends of the wire to the signal collector, and the signals inside the cell sphere can be recorded.

[0025] In the above technical solution, further, multiple wires are provided in the three-dimensional culture device for recording signals at different positions inside the cell sphere.

[0026] The beneficial effects of the present invention are as follows:

[0027] Compared with the traditional three-dimensional cell culture method, the three-dimensional culture device of the present invention has at least the following advantages: ① The wire can be implanted into the cell sphere without damage, which is convenient for manipulating the position of the cell sphere.

[0028] ② The signals inside the cell sphere can be directly recorded conveniently by using the wire, and the cell sphere will not be damaged.

[0029] ③ It is simple to manufacture, has strong expansibility, and can be mass-produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a specific structural schematic diagram of the three-dimensional culture device in the present invention;

[0031] Figure 2 It is a photo of the cell sphere cultured by using the three-dimensional culture device of the present invention under a camera and a microscope;

[0032] Figure 3 It is the internal and external signals of the cell sphere cultured by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The technical solution of the present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0034] The present invention provides a three-dimensional culture device and method for non-destructively manipulating and monitoring internal signals of cell spheres. On the one hand, it can culture three-dimensional cell spheres and non-destructively manipulate them to reach the target position, such as a well plate, an electrode, or place two cell spheres in a very close position to observe the signal communication between them. On the other hand, it can record the external signals of the cell spheres, and can also record the electrical signals inside the cell spheres without damaging the morphology of the cell spheres, study the internal connections of the cell spheres, and compare them with the signals recorded externally.

[0035] The present invention utilizes an ultra-low adsorption surface and an agarose substrate to prevent cells from adhering to the wall, thereby forming cell spheres; and pre-sets wires to induce the cell spheres to wrap around the wires, so as to achieve the non-destructive transfer of the cell spheres and the recording of signals inside the spheres.

[0036] The three-dimensional culture device includes:

[0037] An ultra-low adsorption cell sphere culture chamber; the chamber is in a vertical tubular shape, and the lower section inside the tube is an agarose solid culture medium;

[0038] At least one metal wire; the wire is wrapped with an insulating layer on the outer surface except at both ends and a certain length in the middle is left with the surface completely exposed; the wire penetrates through the chamber and the exposed section in the middle of the wire is exactly located on the upper surface of the agarose solid culture medium;

[0039] A fixing bracket; the fixing bracket is used to fix the chamber and the wire.

[0040] Among them, the ultra-low adsorption tube can be directly purchased or can be made by oneself. Preferably, the ultra-low adsorption tube is thicker at the top and thinner at the bottom, and the agarose solid culture medium is located in the lower section of the ultra-low adsorption tube; the wire can be a platinum wire, a gold wire, a tungsten wire, a silver wire or a copper wire; the inner diameter of the tube of the ultra-low adsorption cell sphere culture chamber can be determined according to the size of the cell spheres to be cultured. Generally, the most suitable inner diameter is about 5 times the diameter of the target cell spheres; the diameter of the metal wire can be determined according to the size of the cell spheres to be cultured. Generally, the most suitable diameter is about 1 / 10 of the diameter of the target cell spheres; the length of the exposed section of the wire can be determined according to the size of the cell spheres to be cultured. Generally, the most suitable length is about 1 / 50 of the diameter of the target cell spheres.

[0041] According to a specific example of the present invention, as Figure 1 shown, the three-dimensional culture device of the present invention includes: a wire fixing device, a wire with an exposed section in the middle, a hollow glass tube with both ends open, agarose, and a glass tube fixing bracket.

[0042] The glass tube and the agarose form an ultra-low adsorption cell sphere culture chamber, the wire passes through the glass tube, and the wire fixing device includes a fixing bracket and two alligator clips, which clamp the two ends of the wire up and down and straighten it;

[0043] The wire with the exposed section in the middle will be wrapped by the cell spheres. By pulling the wire to control the movement of the cell spheres, the signals inside the cell spheres can be recorded at the same time.

[0044] The pre-processing of each component in this example:

[0045] To inhibit cell adhesion, the glass tube needs to be an ultra-low adsorption glass tube (hereinafter referred to as the glass tube): Immerse the glass tube in "anti-adhesion solution" (i.e., anti-adhesion solution) for more than 12 hours, take it out and dry it with an air gun. At this time, the surface of the glass tube becomes ultra-low adsorption, and cells cannot adhere to it.

[0046] To record the internal signal of the cell sphere, a wire with a bare middle part (hereinafter referred to as the wire) is needed: Take an extremely thin metal wire (a platinum wire with a diameter of 100 um), fix both ends of it on the silicon wafer with tape, evenly apply photoresist SU8 2010, and control its thickness to about 10 microns with a spin coater. Use lithography technology to leave a gap of about 10 um (i.e., the bare section) in the middle part of the metal wire, and remove it with a developer. By this method, as long as the bare part is placed inside the cell sphere, the internal signal of the cell sphere can be recorded. Other parts will not record signals due to the block of the photoresist, and the noise is greatly reduced.

[0047] Device construction and experimental operation in this embodiment:

[0048] Prepare 2% by mass of agarose and heat it to 100 °C until the solution becomes clear and transparent.

[0049] Use the glass tube to suck the agarose to the 1 / 3 position, place it at room temperature for one minute until it solidifies slightly, and then fix it on the rack. The specific method of "using the glass tube to suck the agarose" can be achieved by cooperating with a rubber tube and a syringe: Connect one end of the rubber tube to the glass tube and the other end to the syringe, draw the syringe to suck the agarose to the required position (such as the 1 / 3 position of the tube), keep it for about one minute until the agarose solidifies slightly, and then remove the rubber tube.

[0050] Pass the wire through the glass tube, clamp both ends of the wire up and down with a fixing clip and straighten it so that the wire is in the center of the glass tube.

[0051] Digest, centrifuge the nerve cells and resuspend them to a cell suspension of 10 7 cells / mL.

[0052] Take 100 uL of the cell suspension and slowly add it into the glass tube along the wire, and then place the whole device in the incubator.

[0053] Change half of the liquid every day, and cell spheres can be formed and the wire can be wrapped in about one week. The principle of cell sphere formation is as follows: On the agarose substrate, cells cannot adhere and grow, and the surrounding is the ultra-low adsorption glass surface, so cells will tend to aggregate to form cell spheres; the principle of the cell sphere wrapping the wire is as follows: When the agarose solidifies in the glass tube, due to the surface tension, a slightly concave structure will be formed. The wire is set in the center of the glass tube, that is, in the center of the depression, and the cell sphere will wrap it due to the gravity.

[0054] Immerse the glass tube into the culture medium and slowly withdraw the wire, and the cell sphere will also be taken out accordingly, as Figure 2 shown.

[0055] Place the cell sphere on the planar electrode to record the signals outside the cell sphere. At the same time, connect the wire to the signal collector to record the signals inside the cell sphere, as Figure 3 shown.

[0056] The parameters of the glass tube used in this example are: inner diameter 3 mm, outer diameter 5 mm, length 30 mm. In the present invention, the glass tube can be replaced by any other ultra-low adsorption tube, and its parameters are usually inner diameter 2 - 12 mm, outer diameter 4 - 14 mm, length 30 mm, and it is preferably thicker at the top and thinner at the bottom;

[0057] The parameters of the wire used in this example are: diameter 100 μm, length 4 cm, and it is a platinum wire. In the present invention, the wire can be replaced by a gold wire, a platinum wire, a copper wire, a tungsten wire, etc., and its parameters are usually diameter 25 - 200 μm, length 4 cm.

[0058] The device provided by the present invention has a simple structure, simple processing steps, good assembly performance. All components can be replaced and adjusted according to specific needs. Moreover, the diameter of the tube body used can be selected according to the size of the cell sphere to be cultured as required. Several of the above-mentioned wires can also be arranged in parallel in the tube body to record the signals at different positions of the cell sphere; the cell types cultured by using this device are not limited to nerve cells, and can be any cells to be studied, such as cancer cell spheres, etc. The cell sphere can be cultured, manipulated and monitored for signals to conduct various researches. The device of the present invention has excellent universality and is conducive to mass production.

[0059] The above are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A three-dimensional culture device capable of non-destructively manipulating and monitoring internal signals of cell spheres, characterized in that, Comprising: An ultra-low adsorption cell sphere culture chamber; the chamber is in a vertical tubular shape, and the lower section inside the tube is an agarose solid medium. When the agarose solidifies inside the tube, due to the surface tension effect, a slightly concave structure is formed; At least one metal wire; the metal wire is a platinum wire, a gold wire, a tungsten wire, a silver wire or a copper wire. The outer surface of the wire is wrapped with an insulating layer except at both ends, and a certain length in the middle is left with a completely bare surface; the wire penetrates through the chamber and makes the bare section in the middle of the wire exactly located at the center of the concave upper surface of the agarose solid medium, and the cell sphere wraps the bare section in the middle of the wire due to the gravity effect; A fixing bracket; the fixing bracket is used to fix the chamber and the wire.

2. The three-dimensional culture device capable of non-destructively manipulating and monitoring internal signals of cell spheres according to claim 1, characterized in that, The ultra-low adsorption cell sphere culture chamber includes an ultra-low adsorption tube with openings at both ends and agarose. The ultra-low adsorption tube is thicker at the upper part and thinner at the lower part, and the agarose is located in the lower section of the ultra-low adsorption tube.

3. The three-dimensional culture device capable of nondestructively manipulating and monitoring internal signals of cell spheres according to claim 1, characterized in that, The inner diameter of the tube of the ultra-low adsorption cell sphere culture chamber is 2 - 12 mm.

4. The three-dimensional culture device capable of non-destructively manipulating and monitoring internal signals of cell spheres according to claim 1, wherein The diameter of the metal wire is 25 - 200 μm.

5. The three-dimensional culture device capable of non-destructively manipulating and monitoring internal signals of cell spheres according to claim 1, characterized in that, The length of the bare section of the wire is 5 - 100 μm.

6. The preparation method of the three-dimensional culture device according to any one of claims 1-5, characterized in that, Comprising: Fabricating an ultra-low adsorption cell sphere culture chamber: First, heat the agarose until it becomes a clear and transparent liquid, suck the liquid into the middle section of the tube using the ultra-low adsorption tube, and place it at room temperature to solidify the agarose; Insulating the metal wire: Fix both ends of the metal wire on the substrate with tape, evenly apply photoresist, use photolithography technology for masking, light exposure, and treatment with a developer solution, and then form a bare section in the middle part of the metal wire, and the rest is wrapped with photoresist; Assembling the culture device: Vertically fix the ultra-low adsorption tube on the bracket, pass the wire through the ultra-low adsorption tube, clamp both ends of the wire up and down with a fixing clip and straighten it, so that the wire is exactly in the center of the ultra-low adsorption tube, and the bare section is exactly located on the upper surface of the agarose.

7. A method for culturing, manipulating cell spheres, and monitoring internal signals, characterized in that, Using the three-dimensional culture device according to any one of claims 1 - 5, the method includes: Adding the cell suspension along the wire into the ultra-low adsorption cell sphere culture chamber, and then putting the whole device into an incubator; Changing half of the liquid every day, and cell spheres are formed and wrap the wire in 5 - 10 days; Immersing the ultra-low adsorption cell sphere culture chamber into the culture solution, slowly pulling out the wire, and the cell spheres can be taken out accordingly; Putting the wire carrying the cell spheres on a planar electrode, and the signals outside the cell spheres can be recorded; Connecting both ends of the wire to a signal collector, and the signals inside the cell spheres can be recorded.

8. The method for culturing, manipulating cell spheres and monitoring internal signals according to claim 7, wherein, Multiple wires are provided in the three-dimensional culture device for recording signals at different positions inside the cell spheres.

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