A device for fluorescent labeling of organoids and a method of using the same

Abstract

The invention discloses a device for fluorescent labeling of organoids and a method for using the device, which relates to the field of fluorescent labeling of organoids. The device comprises three layers, namely, an upper layer, a middle layer and a lower layer, wherein both the upper layer and the lower layer are circular annular glass, the middle layer is a nylon filter membrane, the thickness of the circular annular glass of the lower layer is greater than that of the circular annular glass of the upper layer, and one side of the circular annular glass of the lower layer is open: the organoid can be fixed in the liquid surface, while reducing the damage to the organoid, and the dye can directly contact the organoid, which is convenient for the organoid immunofluorescence labeling experiment. By combining bright field imaging and fluorescent imaging, the imaging images are 3D reconstructed and merged into one image, the nylon film and the organoid structure are clearly visible, the organoid is attached to the nylon film, the budding structure of the intestinal organoid is obvious, and good imaging of the villin protein is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid fluorescent labeling, and in particular to a device for organoid fluorescent labeling and a method for using the same. Background Art

[0002] As an in vitro three-dimensional life system, organoids can accurately replicate the internal structure and function of organs, providing an experimental platform for studying organogenesis, physiological and pathological processes, and drug screening. This cutting-edge technology has greatly promoted the depth and breadth of scientific research and injected new vitality into the biomedical field.

[0003] Organoids are 3D in vitro models that are established in hydrogels or matrix gels during culture. Conventional immunofluorescence dyes have difficulty penetrating colloids for fluorescent labeling. Based on this, several commonly used methods have been developed to solve the problem of fluorescent labeling of organoids, using labeling after slicing, natural sedimentation, or repeated centrifugation to achieve the purpose of labeling targets. However, the slicing method is a 2D layer labeling and cannot restore the 3D imaging results; the natural sedimentation method has the risk of losing a large number of suspended organoids when discarding the waste liquid, and some liquid needs to be retained during labeling, which will dilute the dye and reduce the labeling effect; repeated centrifugation will consume a lot of time and cause serious damage to the organoid structure during the centrifugation process. Therefore, it is necessary to develop products that allow organoids to be fixed in the liquid surface, while reducing organoid damage, and the dye can directly contact the organoid.

[0004] To this end, we provide a device for fluorescent labeling of organoids and a method for using the same to solve the above problems. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the present invention provides a device for fluorescent labeling of organoids and a method for using the same, wherein organoids can be fixed in the liquid surface while reducing damage to the organoids, and the dye can directly contact the organoids, thereby facilitating the conduct of organoid immunofluorescence labeling experiments.

[0006] In order to achieve the above object, the present invention adopts an organoid fluorescent labeling device, comprising:

[0007] There are three layers: upper, middle and lower. The upper and lower layers are both circular ring-shaped glass, and the middle layer is a nylon filter membrane.

[0008] The thickness of the circular ring-shaped glass in the lower layer is greater than that of the circular ring-shaped glass in the upper layer, and one side of the circular ring-shaped glass in the lower layer is open.

[0009] As a further optimization of the above solution, the inner diameter of the upper circular ring glass is 10 mm, the outer diameter of the ring is 18 mm, and the thickness of the ring is 0.2 mm.

[0010] As a further optimization of the above scheme, the diameter of the nylon filter membrane in the middle layer is 18 mm and the membrane pore size is 41 μm.

[0011] As a further optimization of the above solution, the inner diameter of the circular ring of the lower layer of ring-shaped glass is 10 mm, the outer diameter of the ring is 18 mm, the opening of the ring is 4 mm, and the thickness of the ring is 2 mm.

[0012] As a further optimization of the above scheme, the circular ring design of the upper circular ring glass and the lower circular ring glass provides a cylindrical channel in the middle, and the upper and lower circular ring glasses bond and fix the nylon filter membrane in the middle layer to the cylindrical channel in the middle.

[0013] The present invention also discloses an assembly process of an organoid fluorescent labeling device, comprising the following steps:

[0014] First, place the lower circular ring glass flat on the bottom layer, then apply UV-curing glue to the upper surface of the lower circular ring glass, then place the middle layer of nylon filter membrane and stick it to the upper surface of the lower circular ring glass coated with UV-curing glue, wait until the nylon film is fully attached to the lower circular ring glass, then apply UV-curing glue to the nylon film attached to the circular ring of the lower circular ring glass, then aim at the circular ring of the lower circular ring glass, and stick the upper circular ring glass to the nylon film coated with UV-curing glue, finally use UV light with a wavelength of 365-400nm to irradiate for 10-30 seconds to cure the glue, and complete the assembly of the organoid fluorescent labeling device.

[0015] The present invention also discloses a method for using a device for fluorescent labeling of organoids, comprising:

[0016] The organoid fluorescent labeling device was applied to mouse intestinal organoids, and the organoid fluorescent labeling device was used as a carrier to implement the organoid fluorescent labeling procedure:

[0017] Step 1: Use mouse intestinal organoids that have been passaged for more than 3 times to conduct experiments, culture the intestinal organoids until the 7th day, and obtain mature intestinal organoids with more budding structures;

[0018] Step 2: Resuspend the organoids in ice-cold PBS and centrifuge at 290xg, 4°C for 5 minutes to fully remove the matrix gel. Discard the supernatant after centrifugation.

[0019] Step 3: Gently resuspend the organoids in 100 μL of ice-cold PBS and then load the organoid suspension onto the center of the nylon mesh of the fluorescently labeled organoid device.

[0020] Step 4: Start the programmed fluorescent labeling of organoids. All liquid addition and removal procedures are performed from the side wall of the 12-well plate. First, use 4% PFA to fix the organoids for 30 minutes. After fixation, use ice-cold PBS to wash 3 times.

[0021] After washing, add permeabilization solution containing 0.1% Triton X-100 to permeabilize the organoids for 5-10 minutes. After permeabilization, wash three times with ice-cold PBS.

[0022] Add PBS containing 10% goat serum and block the organoids for 30 minutes at room temperature;

[0023] Discard the blocking solution and incubate the organoids with the primary antibody at 4°C overnight;

[0024] The next day, the primary antibody was discarded, the cells were washed three times with PBS at room temperature, and fluorescent secondary antibodies were added and incubated at room temperature for 1 hour;

[0025] After the secondary antibody incubation, the secondary antibody was discarded, the cells were washed three times with PBS at room temperature, and DAPI dye was added to label the cell nuclei and incubated at room temperature for 5 minutes;

[0026] Discard the DAPI stain and wash three times with PBS at room temperature;

[0027] Step 5: Complete the fluorescent labeling of organoids using the organoid fluorescent labeling device, place the device upside down in a 12-well plate or confocal dish containing PBS, and place it under a fluorescence microscope or laser confocal microscope for observation, imaging, and collection.

[0028] As a further optimization of the above scheme, in the second step, the fluorescently labeled organoid device is placed upright in a 12-well plate while centrifuging, and the organoids at the bottom of the tube cannot be touched after centrifugation.

[0029] The device for fluorescent labeling of organoids and the method for using the same of the present invention have the following beneficial effects:

[0030] The present invention provides an organoid fluorescent labeling device and a method of using the same. The organoid can be fixed in a liquid surface while reducing damage to the organoid, and the dye can directly contact the organoid, thereby facilitating the implementation of an organoid immunofluorescence labeling experiment.

[0031] Using a combination of bright field imaging and fluorescence imaging, the images were reconstructed in 3D and merged into one image. The nylon film and organoid device were clearly visible, the organoids were attached to the nylon film, the budding structure of the intestinal organoids was obvious, and good imaging of the villin protein was achieved.

[0032] With reference to the following description and drawings, a specific embodiment of the present invention is disclosed in detail, indicating the manner in which the principles of the present invention can be adopted. It should be understood that the scope of the embodiments of the present invention is not limited thereby, and within the spirit and scope of the appended claims, the embodiments of the present invention include many changes, modifications and equivalents. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the upper, middle and lower three-layer device of the organoid fluorescent labeling device of the present invention;

[0034] Figure 2 This is a schematic diagram of the device after the upper, middle and lower layers of the fluorescently labeled organoid device of the present invention are assembled;

[0035] Figure 3 A schematic diagram of the assembly process of the device for fluorescent labeling of organoids of the present invention;

[0036] Figure 4 It is a schematic diagram of the chimeric organoid simulation of the present invention;

[0037] Figure 5 It is a schematic diagram of the simulation of adding liquid and discarding liquid of the present invention;

[0038] Figure 6 is a flow chart of immunofluorescence labeling of the present invention;

[0039] Figure 7 It is a fluorescence positive image of the intestinal organoid marker of the present invention;

[0040] Figure 8 It is a fluorescence inversion image of the intestinal organoid marker of the present invention;

[0041] Fig. 9 This is the Villin confocal 3D reconstruction image of the present invention. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail below through the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0043] It should be noted that when an element is referred to as being "disposed on, provided with" another element, it may be directly on the other element or there may also be a central element. When an element is considered to be "connected, connected to" another element, it may be directly connected to the other element or there may be a central element at the same time. "Fixed connection" means a fixed connection. There are many ways of fixed connection, which are not within the scope of protection of this article. The terms "vertical", "horizontal", "left", "right" and similar expressions used in this article are for illustrative purposes only and do not represent the only implementation method.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0045] Several existing immunofluorescence labeling methods have certain defects:

[0046] First, the 2D slice method cannot achieve 3D labeling;

[0047] Second, the natural sedimentation method is prone to losing suspended organoids, and some liquid needs to be retained during labeling, which will dilute the dye and reduce the labeling effect;

[0048] 3. Repeated centrifugation is time-consuming and labor-intensive, and can cause serious damage to the organoid structure during the centrifugation process.

[0049] Therefore, it is necessary to develop products that allow organoids to be fixed in the liquid surface while reducing damage to the organoids and allowing the dye to directly contact the organoids to facilitate organoid immunofluorescence labeling experiments.

[0050] Please refer to the instruction manual Figures 1 to 9 The present invention provides a technical solution: a device for fluorescent labeling of organoids, which is composed of three layers: upper, middle and lower layers: Figure 1 and Figure 2 As shown in the figure, the upper layer is a circular ring of glass, the middle layer is a nylon filter membrane, and the lower layer is a thicker circular ring of glass with one side of the ring open.

[0051] It should be noted that glass can also be replaced by other materials to fix the filter membrane:

[0052] The dimensions of each layer of the device for fluorescent labeling of organoids are as follows: the inner diameter of the circular ring of the glass ring in the upper layer is 10 mm, the outer diameter of the ring is 18 mm, and the thickness of the ring is 0.2 mm;

[0053] The diameter of the nylon filter membrane in the middle layer is 18 mm, and the pore size is 41 μm;

[0054] It should be noted that the pore size of the nylon filter membrane can be changed according to the source of the organoid. The pore size is usually between 40μm and 70μm. The pore size of the filter membrane around 40μm is suitable for most budding organoids.

[0055] The inner diameter of the ring of the thicker circular ring glass at the bottom layer is 10mm, the outer diameter of the ring is 18mm, the ring opening is 4mm, and the ring thickness is 2mm.

[0056] Design details of the device for fluorescent labeling of organoids: The circular design of the upper and lower circular ring glasses provides a cylindrical channel in the middle. The bonding of the upper and lower circular ring glasses can fully fix the middle nylon filter membrane to the middle cylindrical channel; the pore size of the 41μm filter membrane is much larger than the size of mature budding organoids, the organoids will not penetrate the filter membrane, and the budding organoids can be fixed on the surface of the nylon filter membrane; the lower circular ring glass has a thickness of 2mm, and the opening design facilitates the flow of liquid from the middle of the cylinder during the fluorescent labeling procedure, ensuring that the liquid flows from top to bottom If there is no opening in the lower circular ring glass, the liquid may flow back and wash the organoid off the nylon filter membrane; the upper circular ring glass adopts a 0.2mm thin glass design, the purpose is to invert the device for organoid observation after the staining is completed. Since the inverted laser confocal microscope has a certain working distance limitation, this design provides 0.2mm space for the inverted organoid, and the thickness of the organoid after inversion makes it possible for the organoid to almost directly contact the bottom of the confocal dish, which is convenient for starting laser confocal 3D layer scanning from the bottom of the organoid.

[0057] It should be noted that an inverted laser confocal microscope can also be used as an ordinary inverted fluorescence microscope. If conditions permit, it is recommended to use a confocal microscope for imaging.

[0058] refer to Figure 3 As shown, the assembly process of the organoid fluorescent labeling device is as follows: first, place the lower circular ring glass flat on the bottom layer, then apply ultraviolet (UV) curing glue to the upper surface of the lower circular ring glass, then place the middle layer of nylon filter membrane and adhere it to the upper surface of the lower circular ring glass coated with ultraviolet (UV) curing glue, wait until the nylon film is fully adhered to the lower circular ring glass, then apply ultraviolet (UV) curing glue to the nylon film adhered to the circular ring of the lower circular ring glass, then align it with the circular ring of the lower circular ring glass, and adhere the upper circular ring glass to the nylon film coated with ultraviolet (UV) curing glue, finally use UV light with a wavelength of 365-400nm to irradiate for 10-30 seconds to cure the glue, and complete the assembly of the organoid fluorescent labeling device.

[0059] The working principle of the fluorescent labeling device for organoids is:

[0060] The design of the device for fluorescent labeling of organoids is inspired by the fact that there is an intractable problem in the existing organoid 3D fluorescent labeling method, that is, it is difficult to fix the organoid in the liquid after removing the matrix gel, and the flow of the liquid will cause the organoid to suspend randomly, which is not conducive to fluorescent labeling of the organoid. During the fluorescent labeling process, a pipette is used to add cleaning solution, remove cleaning solution, or add labeling dye, remove labeling dye, etc. Many liquid aspiration and liquid abandonment steps will cause the suspended organoid to be sucked into the pipette tip, resulting in the loss of the organoid. In addition, the direct impact of the liquid will cause serious damage to the structure of the organoid, which is not conducive to the maintenance of the original structure of the organoid. Therefore, it is necessary to design a carrier to fix the organoid and provide stable pre-staining labeling conditions. The invention of the device for fluorescent labeling of organoids provides a carrier to stabilize the organoid in the liquid surface, using a design of nylon film + upper and lower circular ring glass. The upper and lower circular ring glass play the role of fixing the nylon film. The nylon film is fixed using ultraviolet (UV) curing glue, which can fix this organoid carrier film in the liquid surface. Since intestinal organoids have budding structures, this structure can be embedded in a nylon membrane with a pore size of 40μm-70μm. Based on this, this mosaic property can be used in combination with a nylon membrane as a carrier for fluorescent staining of organoids, and then liquid is added and discarded through the side wall of the culture plate (or culture dish), completing the fluorescent labeling of organoids while greatly reducing the damage to the organoid structure.

[0061] The organoid chimeric simulation in the present invention can be seen Figure 4 As shown in the figure, the addition and disposal of liquid are simulated. Figure 5 as shown in .

[0062] Application of organoid immunofluorescence labeling on the device:

[0063] 1. Complete process display

[0064] Immunofluorescence labeling workflow reference Figure 6 As shown, mouse intestinal organoids were used as test subjects, and the organoid fluorescent labeling device was used as a carrier to implement the organoid fluorescent labeling procedure of the device:

[0065] first step:

[0066] The experiment was conducted using mouse intestinal organoids that had been passaged for more than 3 times. The intestinal organoids were cultured until the 7th day to obtain mature intestinal organoids with more budding structures.

[0067] Step 2:

[0068] Resuspend the organoids in ice-cold PBS and centrifuge at 290xg, 4°C for 5 minutes to fully remove the matrix gel (while centrifuging, place the fluorescently labeled organoid device upright in the 12-well plate). After centrifugation, discard as much supernatant as possible, while being careful not to touch the organoids at the bottom of the tube;

[0069] Step 3:

[0070] Gently resuspend the organoids in 100 μL of ice-cold PBS and then load the organoid suspension onto the center of the nylon mesh of the fluorescently labeled organoid device.

[0071] Step 4:

[0072] Start programmed fluorescent labeling of organoids. All liquid addition and removal procedures are performed from the side wall of the 12-well plate. First, use 4% PFA to fix the organoids for 30 minutes. After fixation, wash them three times with ice-cold PBS; after washing, add permeabilization solution containing 0.1% Triton X-100 to permeabilize the organoids for 5-10 minutes. After permeabilization, wash them three times with ice-cold PBS; add PBS containing 10% goat serum (replace the blocking solution according to the source of the primary antibody) to block the organoids at room temperature for 30 minutes; discard the blocking solution and incubate the organoids with the primary antibody at 4°C overnight; discard the primary antibody on the next day, wash three times with PBS at room temperature, add fluorescent secondary antibody and incubate at room temperature for 1 hour; after the secondary antibody incubation, discard the secondary antibody, wash three times with PBS at room temperature, add DAPI dye to label the cell nucleus, and incubate at room temperature for 5 minutes; discard the DAPI staining solution and wash three times with PBS at room temperature.

[0073] Step 5:

[0074] Complete the fluorescent labeling of organoids through the organoid fluorescent labeling device, place the device upside down in a 12-well plate or confocal dish containing PBS, and place it under a fluorescence microscope or laser confocal microscope for observation, imaging, and collection.

[0075] (II) Display of labeling results of the organoid fluorescent labeling device

[0076] (1) Physical example:

[0077] The device for fluorescent labeling of organoids is placed upright in a 12-well plate. After adding organoids to the center of the nylon film, PBS, permeabilization solution, blocking solution, primary antibody, secondary antibody, DAPI, etc. are added along the side walls of the wells. The waste liquid is aspirated along the bottom of the side walls of the wells to complete the organoid labeling operation. Then, the device is inverted in a 12-well plate containing PBS and the results are observed under a fluorescence microscope; or the device is inverted and transferred to a confocal dish containing PBS.

[0078] (2) Display of marking results:

[0079] ①12-well plate observation labeling results:

[0080] After loading the organoids onto the upright device and completing the fluorescent labeling of the intestinal organoid marker protein Villin, the device was upright and fluorescence observation was performed. It can be seen that the nylon film blocked the fluorescence observation of the organoids (intestinal organoid marker fluorescence upright image, see Figure 7 ). The upright device will also cause the working distance between the microscope objective and the organoid to be too large, which is not conducive to imaging. Therefore, it is necessary to invert it for observation. After the device is inverted, the device embedded in the organoid is placed under a fluorescence microscope for observation. The results show that the intestinal organoids are well labeled with Villin (inverted fluorescence image of intestinal organoid markers, see Figure 8 ), no nylon film blocking the field of view was seen.

[0081] ② Confocal dish observation labeling results:

[0082] After immunofluorescence labeling, the device embedded with the organoid was inverted and transferred to a confocal dish containing PBS, and placed under a laser confocal microscope for 3D layer scanning and 3D image reconstruction. Intestinal organoids labeled with Villin protein were used for imaging here. In order to more clearly expose the spatial position of the nylon film and organoids on the device, a combination of bright field imaging + fluorescence imaging was used to reconstruct the images in 3D and merge them into one image. The results show that the nylon film and organoid structure are clearly visible, the organoids are attached to the nylon film, the budding structure of the intestinal organoids is obvious, and good imaging of the villin protein Villin was achieved (Villin confocal 3D reconstruction, see Fig. 9 ), indicating that the device is feasible for fluorescent labeling of organoids, especially budding organoids.

[0083] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A device for fluorescent labeling of organoids, characterized in that: include: There are three layers: upper, middle and lower. The upper and lower layers are both circular ring-shaped glass, and the middle layer is a nylon filter membrane. The thickness of the circular ring-shaped glass in the lower layer is greater than that of the circular ring-shaped glass in the upper layer, and one side of the circular ring-shaped glass in the lower layer is open; The circular ring design of the upper circular ring glass and the lower circular ring glass provides a cylindrical channel in the middle, and the upper and lower circular ring glasses adhere and fix the nylon filter membrane in the middle layer to the cylindrical channel in the middle.

2. The device for fluorescent labeling of organoids according to claim 1, characterized in that: The inner diameter of the circular ring of the upper layer of glass is 10 mm, the outer diameter is 18 mm, and the thickness is 0.2 mm.

3. The device for fluorescent labeling of organoids according to claim 1, characterized in that: The diameter of the nylon filter membrane in the middle layer is 18 mm and the pore size is 41 μm.

4. The device for fluorescent labeling of organoids according to claim 1, characterized in that: The inner diameter of the circular ring of the lower layer of glass is 10 mm, the outer diameter of the ring is 18 mm, the opening of the ring is 4 mm, and the thickness of the ring is 2 mm.

5. An assembly process for an organoid fluorescent labeling device, characterized in that: The device for fluorescently labeling an organoid according to any one of claims 1 to 4, further comprising the following steps: First, place the lower circular ring glass flat on the bottom layer, then apply UV-curing glue to the upper surface of the lower circular ring glass, and then place the middle layer of nylon filter membrane to fit the upper surface of the lower circular ring glass coated with UV-curing glue. After the nylon film fully fits the lower circular ring glass, apply UV-curing glue to the nylon film attached to the circular ring of the lower circular ring glass, and then align it with the circular ring of the lower circular ring glass, and attach the upper circular ring glass to the nylon filter membrane coated with UV-curing glue. Finally, use UV light with a wavelength of 365-400nm to irradiate for 10-30 seconds to cure the glue, and complete the assembly of the organoid fluorescent labeling device.

6. A method for using a device for fluorescent labeling of organoids, characterized in that: The device for fluorescent labeling of organoids according to any one of claims 1 to 4, further comprising: The organoid fluorescent labeling device was applied to mouse intestinal organoids, and the organoid fluorescent labeling device was used as a carrier to implement the organoid fluorescent labeling procedure: Step 1: Use mouse intestinal organoids that have been passaged for more than 3 times to conduct experiments, culture the intestinal organoids until the 7th day, and obtain mature intestinal organoids with more budding structures; Step 2: Resuspend the organoids in ice-cold PBS and centrifuge at 290xg, 4°C for 5 minutes to fully remove the matrix gel. Discard the supernatant after centrifugation. Step 3: Gently resuspend the organoids in 100 μL of ice-cold PBS and then load the organoid suspension onto the center of the nylon filter of the fluorescently labeled organoid device. Step 4: Start the programmed fluorescent labeling of organoids. All liquid addition and removal procedures are performed from the side wall of the 12-well plate. First, use 4% PFA to fix the organoids for 30 minutes. After fixation, use ice-cold PBS to wash 3 times. After washing, add permeabilization solution containing 0.1% Triton X-100 and permeabilize the organoids for 5-10 minutes. After permeabilization, wash three times with ice-cold PBS. Add PBS containing 10% goat serum and block the organoids for 30 minutes at room temperature; Discard the blocking solution and incubate the organoids with the primary antibody at 4°C overnight; The next day, the primary antibody was discarded, the cells were washed three times with PBS at room temperature, and fluorescent secondary antibodies were added and incubated at room temperature for 1 hour; After the secondary antibody incubation, the secondary antibody was discarded, the cells were washed three times with PBS at room temperature, and DAPI dye was added to label the cell nuclei and incubated at room temperature for 5 minutes; Discard the DAPI stain and wash three times with PBS at room temperature; Step 5: Complete the fluorescent labeling of organoids using the organoid fluorescent labeling device, place the device upside down in a 12-well plate or confocal dish containing PBS, and place it under a fluorescence microscope or laser confocal microscope for observation, imaging, and collection.

7. The method for using the organoid fluorescent labeling device according to claim 6, characterized in that: In the second step, place the fluorescently labeled organoid device upright in the 12-well plate while centrifuging, and do not touch the organoids at the bottom of the tube after centrifugation.

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