Organ-like microballoon stent and method for culturing organ-like microballoons by using same

Abstract

The invention relates to a device-tube-like microsphere bracket and a method for culturing organoid microspheres by using the bracket, and solves the problems that device-tube-like microspheres are difficult to accurately place in the center of a U-shaped hole of a culture plate during dropwise adding in the culture process of the conventional U-shaped hole culture plate, so that the subsequent microscopic observation and high-throughput screening are influenced; and the sphericity of the microspheres is difficult to guarantee. Comprising a base, a device-like tube microsphere positioning cylinder and a supporting and positioning cylinder, a device-like tube microsphere positioning groove is formed in the top end of the device-like tube microsphere positioning cylinder, a supporting and positioning part is arranged at the top end of the supporting and positioning cylinder, and during use, cell matrix gel mixed liquid is dropwise added into the device-like tube microsphere positioning groove of the stent to form device-like tube microspheres; placing the U-shaped hole culture plate on a bracket, and waiting for solidification of the matrigel; the bracket is overturned and placed on the corresponding culture plate, and the vessel-like microspheres fall into the centers of the U-shaped holes of the culture plate through a centrifugal method, so that the sphericity of the vessel-like microspheres is ensured, the vessel-like microspheres cannot be lost, and microscopic observation is facilitated.

Description

Technical Field

[0001] This invention relates to the fields of cell culture and organoid culture technology, specifically to an organoid microsphere scaffold and a method for culturing organoid microspheres using the scaffold. Background Technology

[0002] Existing organoid culture methods mostly involve culturing single or small numbers of organoid microspheres, relying on multiple culture dishes and complex auxiliary equipment. This is cumbersome and inefficient, especially during medium changes and recovery, which can easily lead to microsphere loss or contamination. Currently, organoid microsphere culture typically involves adding microspheres dropwise into the U-wells of a U-shaped culture plate. Commonly used U-shaped culture plates are 96-well and 384-well plates. However, it is difficult to precisely center the microspheres in the wells during addition, affecting subsequent microscopic observation and high-throughput screening. It also makes it difficult to ensure the sphericity of the microspheres, impacting the reliability of experimental results. Furthermore, microspheres are often lost due to adhesion to the plate walls during addition. Summary of the Invention

[0003] The purpose of this invention is to solve the problem that existing U-shaped well culture plates are prone to causing difficulties in accurately placing organoid microspheres into the center of the U-shaped well during the culture process, which affects subsequent microscopic observation and high-throughput screening, makes it difficult to ensure the sphericity of the microspheres, and affects the reliability of experimental results.

[0004] The technical solution adopted to solve the technical problem proposed in this invention is as follows: The organoid microsphere support of this invention includes a base, on which one or more organoid microsphere positioning cylinders and one or more supporting positioning cylinders are provided. The organoid microsphere positioning cylinders and supporting positioning cylinders are both configured to cooperate with the U-shaped holes of the U-shaped culture plate. The top end of the organoid microsphere positioning cylinder is provided with a concave organoid microsphere positioning groove with an arc-shaped cross section. The surface of the organoid microsphere positioning groove is covered with a hydrophobic material. The top end of the supporting positioning cylinder is provided with an outwardly convex arc-shaped supporting positioning part, which is higher than the organoid microsphere positioning groove.

[0005] A method for culturing organoid microspheres using the organoid microsphere scaffold of the present invention, the method comprising the following steps: Step A: Preparation stage: Sterilize the scaffold and culture plate, pre-cool and prepare the matrix gel, prepare the cell suspension and culture medium; Step B: Mix the cells and matrix gel evenly as required for the experiment; Step C: The cell matrix gel mixture is dropped into the organoid tube microsphere positioning groove of the scaffold to form organoid tube microspheres; Step D: Place the U-shaped culture plate, which matches the positioning cylinder of the organoid microsphere and the supporting positioning cylinder of the support, on the support and place it in an incubator at a certain temperature until the matrix gel solidifies. Step E: Invert the scaffold and place it on the corresponding culture plate. Centrifuge the microspheres to fall into the center of the U-shaped hole in the culture plate and remove the scaffold. Step F: After adding an appropriate amount of culture medium, place the culture plate in an incubator for culture, and change the culture medium regularly to maintain the nutrient supply to the organoid microspheres; Step G: Observe the organoid microspheres under a microscope during the cultivation process.

[0006] The technical solutions that further define the present invention include: The hydrophobic material is fluorosilicone modified silica nanoparticles.

[0007] The hydrophobic material coating has a thickness of 10 to 100 nm and a contact angle ≥110°.

[0008] The two or more microsphere positioning cylinders are evenly arranged, and the two or more supporting positioning cylinders are evenly arranged.

[0009] The outer circumference of the microsphere positioning cylinder is provided with a ring of supporting positioning cylinder.

[0010] In step C, the cell matrix gel mixture is dropped into the organelle tube microsphere positioning groove of the scaffold using a multichannel or manual pipette, with each groove having a volume of 10 to 50 µL.

[0011] In step B, the concentration of the matrix adhesive is 70%-85%.

[0012] In step D, the U-shaped well culture plate is a U-shaped 96-well culture plate or a 384-well culture plate. When the organoid microsphere positioning cylinder and the supporting positioning cylinder are inserted into the U-shaped 96-well culture plate or the 384-well culture plate, the space between the organoid microsphere positioning groove and the U-shaped groove of the U-shaped 96-well culture plate or the 384-well culture plate forms the accommodating space for the organoid microsphere.

[0013] In step F, when changing the culture medium, insert the organoid microsphere positioning cylinder and the support positioning cylinder of the scaffold into the U-shaped well of the U-shaped 96-well culture plate or 384-well culture plate, then turn it over to pour out the culture medium, then turn it over again to place the organoid microsphere in the center of the U-shaped well of the culture plate, and then add new culture medium.

[0014] The beneficial effects of the present invention through the above technical solution are as follows: The organelle microsphere scaffold base of the present invention is provided with one or more organelle microsphere positioning cylinders and one or more supporting positioning cylinders. The organelle microsphere positioning cylinders and supporting positioning cylinders are all configured to cooperate with the U-shaped holes of the U-shaped well culture plate. In use, the cell matrix gel mixture is dropped into the organelle microsphere positioning groove of the scaffold to form organelle microspheres; the U-shaped well culture plate that cooperates with the organelle microsphere positioning cylinders and supporting positioning cylinders of the scaffold is placed on the scaffold and placed in an incubator at a certain temperature until the matrix gel solidifies; the scaffold is flipped and placed on the corresponding culture plate, and the organelle microspheres are made to fall into the center of the U-shaped hole of the culture plate by centrifugation, ensuring the sphericity of the organelle microspheres. At the same time, it can accurately place the organelle microspheres in the center of the U-shaped hole of the culture plate, so that the organelle microspheres will not be lost due to wall adhesion, nor will wall adhesion affect microscopic observation and high-throughput screening, ensuring the reliability of experimental results. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the structure of a type of microsphere scaffold of the present invention.

[0016] Figure 2 This is a cross-sectional structural diagram of a type of microsphere scaffold according to the present invention.

[0017] Figure 3 This is a schematic diagram of the structure of a type of microsphere scaffold and a U-shaped culture plate according to the present invention.

[0018] Among them, the base 1, the arc-shaped structure 11, the organoid microsphere positioning cylinder 2, the organoid microsphere positioning groove 21, the supporting positioning cylinder 3, the supporting positioning part 31, the U-shaped well culture plate 4, and the U-shaped well 41. Detailed Implementation

[0019] The structure of the present invention will be further described below with reference to the accompanying drawings.

[0020] Reference Figures 1 to 3The organoid microsphere scaffold of the present invention includes a base 1, on which one or more organoid microsphere positioning cylinders 2 and one or more supporting positioning cylinders 3 are provided. Both the organoid microsphere positioning cylinders 2 and the supporting positioning cylinders 3 are configured to mate with the U-shaped holes 41 of the U-shaped culture plate 4. The top end of the organoid microsphere positioning cylinder 2 is provided with a concave, arc-shaped organoid microsphere positioning groove 21. The surface of the organoid microsphere positioning groove 21 is coated with a hydrophobic material. In this embodiment, the hydrophobic material is fluorosilicone-modified silica nanoparticles. The hydrophobic material coating thickness is 10 to 100 nm, and the contact angle is ≥110°. This treatment can reduce surface tension, promote the formation of standard spheres in the matrix adhesive, and improve the uniformity of the microspheres. The top end of the supporting positioning cylinder 3 is provided with a convex, arc-shaped supporting positioning part 31, which is higher than the organoid microsphere positioning groove 21. The support and positioning part is used for positioning the support and culture plate during insertion. In use, when the organoid microsphere positioning cylinder 2 and the support and positioning cylinder 3 are inserted into the U-shaped hole of the U-shaped culture plate, the support and positioning cylinder 3 is higher than the organoid microsphere positioning groove 21, thus creating a space for the organoid microspheres between the organoid microsphere positioning groove 21 and the U-shaped hole of the U-shaped culture plate. In this embodiment, a ring of support and positioning cylinders 3 is provided around the outer periphery of the organoid microsphere positioning cylinder 2, thereby ensuring stable support and positioning.

[0021] In this embodiment, two or more organoid microsphere positioning cylinders 2 are evenly arranged, and two or more supporting positioning cylinders 3 are evenly arranged. The organoid microsphere positioning cylinders and supporting positioning cylinders can be arranged as needed. When used with a U-shaped 96-well culture plate or a 384-well culture plate, organoid microsphere positioning cylinders and supporting positioning cylinders of appropriate size and height can be set to fit and insert with the U-shaped 96-well culture plate or the 384-well culture plate.

[0022] In this embodiment, the base 1 has a hollow annular cross-section, and both the microsphere positioning cylinder 2 and the supporting positioning cylinder 3 are hollow structures. The support can be printed using a 3D printer. The hollow structure ensures sufficient strength support for the base and facilitates the setting of uniform wall thickness, resulting in a smoother and flatter printing surface. In this embodiment, the base 1 has an arc-shaped structure 11 around its perimeter, which enhances the base's strength. In practical implementation, the base can also be made without a hollow structure; the microsphere positioning cylinder and the supporting positioning cylinder can be made hollow, with holes opened at the bottom of the support, and can be manufactured using an injection mold.

[0023] A method for culturing organoid microspheres using the organoid microsphere scaffold described in this invention includes the following steps: Step A: Preparation stage: Sterilize the scaffold and culture plate, pre-cool and prepare the matrix gel, prepare the cell suspension and culture medium; the scaffold and culture plate can be thoroughly sterilized by soaking in 70% ethanol or by UV disinfection for 30 minutes.

[0024] Step B: Mix the cells and matrix gel thoroughly as needed for the experiment; mix gently to avoid air bubbles. In this example, the matrix gel concentration is 70%-85%. Other suitable matrix gel concentrations can be used as needed in specific implementations.

[0025] Step C: The cell matrix gel mixture is dropwise added into the organelle-like microsphere positioning groove 21 of the scaffold to form organelle-like microspheres. In this embodiment, a multi-channel or manual pipette is used to dropwise add the cell matrix gel mixture into the organelle-like microsphere positioning groove 21 of the scaffold, with each groove having a volume of 10 to 50 µL, ensuring that the matrix gel forms spherical shapes. In specific implementations, other suitable volumes can be used as needed.

[0026] Step D: Place the U-shaped culture plate, which mates with the microsphere positioning cylinder 2 and the supporting positioning cylinder 3 of the support, on the support and place it in an incubator at 37°C for about 15-30 minutes until the matrix gel solidifies. Step E: Invert the support and place it on the corresponding culture plate. Centrifuge to allow the organoid microspheres to fall into the center of the U-shaped well of the culture plate, then remove the support. In this embodiment, the U-shaped well culture plate is a U-shaped 96-well or 384-well culture plate. When the organoid microsphere positioning cylinder 2 and the supporting positioning cylinder 3 are inserted into the U-shaped 96-well or 384-well culture plate, the space between the organoid microsphere positioning groove 21 and the U-shaped groove of the U-shaped 96-well or 384-well culture plate forms the receiving space for the organoid microspheres. Centrifugation and other methods are used to ensure that the microspheres fall accurately into the center of the well of the culture plate, guaranteeing the sphericity of the organoid microspheres. At the same time, it can accurately place the organoid microspheres in the center of the U-shaped well of the culture plate, so as not to lose the organoid microspheres due to wall adhesion, nor to affect microscopic observation and high-throughput screening due to wall adhesion, thus ensuring the reliability of experimental results.

[0027] Step F: After adding an appropriate amount of culture medium, place the culture plate in an incubator for incubation. Change the culture medium regularly to maintain the nutrient supply to the organoid microspheres. When changing the culture medium, insert the organoid microsphere positioning cylinder and support positioning cylinder of the scaffold into the U-shaped wells of the U-shaped 96-well or 384-well culture plate. Then, invert the plate to pour out the culture medium, and then invert it again to place the organoid microspheres in the center of the U-shaped wells before adding new culture medium. This process prevents damage to the organoid microspheres during replacement.

[0028] Step G: Observe the organoid microspheres under a microscope during the cultivation process. This invention ensures the sphericity of the prepared organoid microspheres and prevents them from sticking to the sidewalls of the U-shaped wells in the U-shaped well culture plate, thus facilitating microscopic observation and high-throughput screening, and ensuring the reliability of experimental results.

[0029] Although specific embodiments of the present invention have been described in detail with reference to the accompanying drawings, this should not be construed as limiting the scope of protection of the present invention. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims still fall within the scope of protection of the present invention.

Claims

1. A tubular microsphere stent, characterized by: The support comprises a base, and one or more organ tubule microsphere positioning cylinders and one or more support positioning cylinders are arranged on the base, the organ tubule microsphere positioning cylinders and the support positioning cylinders are arranged in cooperation with the U-shaped holes of the U-shaped hole culture plate, the top end of the organ tubule microsphere positioning cylinder is provided with an organ tubule microsphere positioning groove with a concave cross section in the shape of an arc, the surface of the organ tubule microsphere positioning groove is coated with a hydrophobic material, and the top end of the support positioning cylinder is provided with a support positioning part in the shape of an arc and protruding outward, and the support positioning part is higher than the organ tubule microsphere positioning groove.

2. The stent of claim 1, wherein: the stent is a tubular stent. The hydrophobic material is fluorosilicon modified silicon dioxide nanoparticles.

3. A stent as claimed in claim 2, wherein: the stent is a tubular microstent. The hydrophobic material has a coating thickness of 10-100 nm and a contact angle of ≥110°.

4. The microtubular stent according to claim 1, wherein: The two or more organ tubule microsphere positioning cylinders are arranged uniformly, and the two or more support positioning cylinders are arranged uniformly.

5. A stent as claimed in claim 4, wherein: the stent is a tubular microstent. The outer periphery of the organ tubule microsphere positioning cylinder is provided with a ring of support positioning cylinders.

6. A method for culturing organotube microspheres using the organotube microsphere scaffold according to any one of claims 1 to 5, characterized by: The culture method comprises the following steps: Step A: preparation stage: sterilize the support and the culture plate, prepare the matrigel, prepare the cell suspension and prepare the culture medium; Step B: mix the cells and the matrigel uniformly according to the experimental requirements; Step C: drop the cell-matrigel mixture into the organ tubule microsphere positioning groove of the support to form organ tubule microspheres; Step D: place the U-shaped hole culture plate matched with the organ tubule microsphere positioning cylinder and the support positioning cylinder of the support on the support, and place it in a culture box at a certain temperature, and wait for the matrigel to solidify; Step E: turn over the support and place it on the corresponding culture plate, and make the organ tubule microspheres fall into the center of the U-shaped hole of the culture plate by centrifugation, and then take out the support; Step F: after adding an appropriate amount of culture medium, place the culture plate in a culture box for culture, replace the culture medium regularly, and maintain the nutrient supply of the organ tubule microspheres; Step G: observe the organ tubule microspheres by microscope during the culture process.

7. The method for culturing organoid microspheres as described in claim 6, characterized in that: In step C, a multi-channel or manual pipette is used to drop the cell-matrigel mixture into the organ tubule microsphere positioning groove of the support, and the volume of each groove is 10-50 µL.

8. The method for cultivating organoid microspheres as described in claim 6, characterized in that: In step B, the concentration of the matrigel is 70%-85%.

9. The method for culturing organoid microspheres as described in claim 6, characterized in that: In step D, the U-shaped hole culture plate is a U-shaped 96-well culture plate or a 384-well culture plate, and when the organ tubule microsphere positioning cylinder and the support positioning cylinder are inserted into the U-shaped 96-well culture plate or the 384-well culture plate, the space between the organ tubule microsphere positioning groove and the U-shaped groove of the U-shaped 96-well culture plate or the 384-well culture plate forms a containing space for the organ tubule microspheres.

10. The method of claim 6, wherein the microspheres are organotypic. 5 In step F, when the culture medium is replaced, the organ tubule microsphere positioning cylinder and the support positioning cylinder of the support are inserted into the U-shaped hole of the U-shaped 96-well culture plate or the 384-well culture plate, then the culture medium is poured out by turning over, then the organ tubule microspheres are placed in the center of the U-shaped hole of the culture plate by turning over again, and then new culture medium is added.

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