Early embryo 3D immunofluorescence rapid three-staining method
Through the early embryo 3D immunofluorescence rapid triple staining method and special Petri dishes, the problems of no coloring, loss and high cost during embryo staining are solved, and efficient and economical embryo staining effect is achieved.
Patent Information
- Application Number
- CN202411959831.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-06-06
AI Technical Summary
During the embryo immunofluorescence staining process, the embryo is prone to being unable to stain and is easily lost during the operation. Only one protein can be detected at a time, which is relatively expensive.
The early embryo 3D immunofluorescence rapid triple staining method was used, and a special Petri dish was used. The area inside the bottom of each chamber was smaller than the area inside the open plane, which was achieved by 3D culture and 3-fold immunofluorescence mixed staining.
It effectively avoids the problem of embryos not dyeing and loss, reduces the amount of reagents, reduces the cost, and improves the dyeing effect and success rate.
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Figure CN120098786A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology and relates to an early embryo 3D immunofluorescence rapid triple staining method and a special culture dish used for the method. Background Art
[0002] Immunofluorescence staining technology refers to combining fluorescent or colorable chemicals on antibodies, using the specific binding reaction between antigens and antibodies in immunological principles to detect the presence of target antigen proteins in cells or tissues through chemical reactions to make the labeled antibody color developer develop color, and to conduct localization and relative quantitative research on them.
[0003] Because human embryo samples are extremely precious, the following important issues need to be avoided as much as possible when performing immunofluorescence staining on embryos: First, the embryos fail to stain; second, the embryos are lost during the culture, washing, staining and other operations; third, one immunofluorescence staining can only detect one protein, and the amount of antibodies used is large, and the staining cost is high. Therefore, based on the creation of a new multi-purpose embryo ladder culture dish, immunofluorescence triple staining of human embryos will help advance the study of key proteins that regulate early human embryonic development. Summary of the invention
[0004] The purpose of the present invention is to provide a culture dish and method for rapid 3D immunofluorescence triple staining of early embryos.
[0005] The embryonic 3D immunofluorescence triple staining refers to first performing 3D cell culture and then performing triple immunofluorescence mixed staining on the 3D cultured cells. The chromosome process includes incubating three different primary antibodies and then adding three corresponding different secondary antibodies at the same time.
[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0007] On the one hand, the present invention provides a culture dish for improved early embryo 3D immunofluorescence rapid triple staining, characterized in that the bottom inner area (a) of each chamber of the culture dish is smaller than the opening surface inner area (b), wherein the bottom inner area (a) is surrounded by the bottom perimeter (1), and the opening surface inner area (b) is surrounded by the opening perimeter (2).
[0008] like Figure 1-2 The culture dish is a glass slide (such as Figure 3 ) based on the improvement.
[0009] The inner area of the bottom of each chamber of the culture dish (a) is smaller than the inner area of the opening surface (b), and the chamber (c) has a certain slope.
[0010] Modification 1 Figure 1 As shown, the bottom of each chamber is square (a), and the opening surface of each chamber (b) is also square.
[0011] Preferably, the inner area of the square bottom (a) is designed to be 1 / 2 of the inner area of the opening surface (b).
[0012] Preferably, the square bottom is designed to be within the projection area of the top view of the opening surface.
[0013] Preferably, the four sides of the bottom are designed to be parallel to the four sides of the opening surface.
[0014] Preferably, the square bottom and the opening surface are both square.
[0015] Preferably, the culture dish has 8 wells, 6 wells, 4 wells, 2 wells or a single well.
[0016] Modification 2 Figure 2 As shown, the bottom of each chamber is circular (a), and the opening surface of each chamber is also circular (b).
[0017] Preferably, the circular bottom (a) is designed to have an inner area that is 1 / 2 of the inner area of the circular opening surface (b).
[0018] Preferably, the circular bottom is designed to be located within the projection area of the circular opening surface in a top view.
[0019] Preferably, the circular bottom is designed to be located within the projection area of the circular opening surface in a top view and to be a concentric circle.
[0020] Preferably, the culture dish has 8 wells, 6 wells, 4 wells, 2 wells or a single well.
[0021] In another aspect, the present invention provides use of the aforementioned improved culture dish in embryo 3D culture.
[0022] Furthermore, the present invention provides the use of the aforementioned improved culture dish in simple and rapid embryo 3D immunofluorescence triple staining.
[0023] On the other hand, the present invention also provides a 3D embryo culture method for improved early embryo 3D immunofluorescence rapid triple staining, characterized in that it comprises the following steps:
[0024] 1) Embryo thawing;
[0025] 2) Remove the zona pellucida. For some embryos that have broken through the zona pellucida on their own, this step can be omitted.
[0026] 3) Embryo culture: the improved culture dish mentioned above is used for embryo culture.
[0027] Further, step 1) comprises:
[0028] ① Pipette appropriate amount (0.5-1.0 mL) of resuscitation solution TS1, resuscitation solution TS2, equilibrium solution ES and IVC1 culture medium into respective culture dishes (four-well plate, three solutions and culture medium are placed respectively). Equilibrate to room temperature.
[0029] ② Remove the straw from liquid nitrogen and expose it to air for 30 seconds.
[0030] ③ Place the straw in a 30℃ water bath for 45 seconds. Take out the straw and wipe it dry.
[0031] ④ Use aseptic technique to open the straw and carefully expel the embryo.
[0032] ⑤ Place the embryos in resuscitation solution TS1 for 5 minutes.
[0033] ⑥ Transfer the embryos to resuscitation solution TS2 for 5 minutes.
[0034] ⑦ Transfer the embryos to ES balance solution for 5 minutes.
[0035] ⑧ Transfer the embryos to 37℃ pre-equilibrated culture medium and rinse the embryos appropriately.
[0036] Tip: Make sure to carry a minimum amount (less than 1 ul) of ES equilibrated medium to avoid changing the nutrient concentration in the medium. This can be achieved by using a pipette or by transferring the embryos to a four-well dish containing IVC1 and then transferring them to the dish described in Example 1.
[0037] Further, step 2) comprises:
[0038] ① Visually check the embryos under a stereomicroscope to see if there is a zona pellucida (if some embryos have broken through the zona pellucida by themselves, omit this step). For embryos with a zona pellucida, use AT solution to remove the zona pellucida;
[0039] ② Prepare 3-5 independent AT droplets (30ul) and 3 independent M2 medium droplets (30ul) in a common round embryo culture dish, and cover each droplet with a layer of mineral oil;
[0040] ③ Transfer an embryo into a drop of AT solution;
[0041] ④ Use a pipette to blow the embryo up and down several times (gently, usually 3-5 times), and transfer the embryo to the fresh AT droplet in the culture dish.
[0042] ⑤ Repeat step 4 until the zona pellucida disappears (make a few more drops of AT solution for washing. Generally, 3 drops of AT solution are needed for one embryo to see the zona pellucida disappear).
[0043] ⑥ Transfer the embryos with zona pellucida removed into a drop of M2 medium to neutralize the AT solution;
[0044] ⑦ Transfer the embryos to a fresh drop of M2 medium and wash them repeatedly (use the egg transfer needle to pump the embryos up and down 3-5 times), and repeat this step once;
[0045] ⑧Transfer the embryos to IVC1 medium pre-equilibrated at 37°C.
[0046] Tip: After transferring embryos from droplet to droplet, there will be traces of mineral oil on the pipette tip; to avoid mineral oil carryover into the culture medium, always soak the pipette tip in IVC1 before the final transfer.
[0047] Further, step 3) comprises:
[0048] ① Transfer the zona pellucida-free embryos to a culture dish filled with 37°C pre-equilibrated IVC1 medium as described in Example 1. Place 1-2 embryos in each chamber.
[0049] ②Put CO in the culture dish 2 Incubate overnight in an incubator set to CO 2 :6%, 37℃;
[0050] ③ The next day, usually embryo D6, observe the embryo status and change the medium in each chamber (IVC1 pre-equilibrated at 37°C);
[0051] ④ Repeat this step on embryo D7.
[0052] ⑤ Embryos: Change the medium with IVC2 every day thereafter until embryo D14.
[0053] AT solution: Acid Tyrode's Solution (pH 2.5, sterile)
[0054] Prepare 10 ml of IVC-1 medium:
[0055]
[0056] Prepare 10 ml of IVC-2 medium:
[0057]
[0058] On the other hand, the present invention also provides a simple and rapid embryo 3D immunofluorescence triple staining method, characterized in that it comprises the following steps:
[0059] 1) Embryo thawing;
[0060] 2) Remove the zona pellucida. For some embryos that have broken through the zona pellucida on their own, this step can be omitted.
[0061] 3) Embryo culture: the improved culture dish mentioned above is used for embryo culture.
[0062] 4) Immunofluorescence triple staining of embryos.
[0063] The further steps 1) to 3) are the same as the aforementioned 3D embryo culture method.
[0064] A further step 4) comprises:
[0065] Fixation: The culture medium in the culture dish was aspirated with a pipette, and the dish was washed three times with PBS. 100ul of 4% paraformaldehyde was added to each chamber to cover the obtained embryos and incubated at room temperature for 30 minutes.
[0066] Washing: Slowly aspirate the paraformaldehyde and wash with sufficient amount (300ul) of PBS at room temperature for 3 times, 5 minutes each time.
[0067] Permeabilization: Add 0.1% Triton X-100, room temperature for 30 minutes
[0068] Washing: Slowly aspirate Triton X-100 and wash with sufficient amount (300ul) of PBS at room temperature for 3 times, 5 minutes each time.
[0069] Blocking: Add 5% BSA for blocking at room temperature for 30 minutes
[0070] Primary antibody incubation: dilute three antibodies from different species (mouse / rabbit / sheep) with 50ul antibody diluent according to the ratio recommended in the instructions to obtain a mixed primary antibody solution. Slowly aspirate BSA without washing, add the three antibodies to the mixed primary antibody, place in a wet box, and refrigerator at 4°C overnight.
[0071] Washing: Rewarm at room temperature for 30 minutes, slowly aspirate the mixed primary antibody, and wash three times with sufficient amount (300ul) of PBS at room temperature, 5 minutes each time.
[0072] Secondary antibody incubation: Mix three fluorescent secondary antibodies of three different species with 50 μL antibody diluent to obtain a mixed secondary antibody solution, add the mixed secondary antibody, and stand at room temperature in the dark for 1 hour.
[0073] The mixed primary antibody and mixed secondary antibody ratio system is as follows:
[0074]
[0075] Washing: Slowly aspirate the mixed secondary antibody and wash with sufficient amount (300ul) PBS at room temperature for 3 times, 5min each time.
[0076] Nuclear staining: Add 50ul of DAPI containing fluorescence quencher and incubate at room temperature in the dark for 10min.
[0077] Observation and photography: There is no need to move the embryos. Simply place the culture dish under an inverted fluorescence microscope for observation and photography.
[0078] Compared with the prior art, the present invention has the following advantages:
[0079] (1) The method of the present invention uses less reagent raw materials, saves culture medium, antibodies and other consumable reagents, and reduces costs;
[0080] (2) The samples obtained by the method of the present invention have good culture status, good staining effect, and no embryo loss;
[0081] (3) When the method of the present invention is used to stain and prepare embryos, the position of the embryos is clear and distinct, making it easier to observe. BRIEF DESCRIPTION OF THE DRAWINGS
[0082] The beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.
[0083] Figure 1 This is a top view of modified example 1 of the improved culture dish of the present invention, wherein 1 is the circumference of the bottom surface, 2 is the circumference of the opening, a is the bottom surface, and b is the opening surface.
[0084] Figure 2 This is a top view of modified example 2 of the improved culture dish of the present invention, wherein 1 is the bottom circumference, 2 is the opening circumference, a is the bottom surface, b is the opening surface, and c is the chamber wall.
[0085] Figure 3 It is a common chamber slide.
[0086] Figure 4 The embryo in vitro culture states using different culture dishes, wherein A is using a common culture dish (chamber slide), and B is using the improved culture dish of the present invention.
[0087] Figure 5 The present invention is an immunofluorescence triple staining image of a D6 human early embryo prepared using the improved culture dish of the present invention.
[0088] Figure 6 The present invention is a D11 human early embryo immunofluorescence triple staining image prepared using the improved culture dish of the present invention.
[0089] Figure 7 This is a single staining image of D6 cultured in a regular four-well plate. DETAILED DESCRIPTION
[0090] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0091] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as commonly understood by those of ordinary skill in the art to which this application belongs. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.
[0092] Example 1 An improved culture dish
[0093] An improved culture dish, such as Figure 1-2 The culture dish is a common chamber slide (such as Figure 3 ) based on the improvement.
[0094] The inner area (a) of the bottom of each chamber of the culture dish is smaller than the inner area (b) of the opening surface, and the chamber wall (c) has a certain slope, wherein the inner area (a) of the bottom is surrounded by the perimeter of the bottom (1), and the inner area (b) of the opening surface is surrounded by the perimeter of the opening (2).
[0095] Modification 1 Figure 1 As shown, the bottom of each chamber is square (a), and the opening surface of each chamber (b) is also square.
[0096] Preferably, the inner area of the square bottom (a) is designed to be 1 / 2 of the inner area of the opening surface (b).
[0097] Preferably, the square bottom is designed to be within the projection area of the top view of the opening surface.
[0098] Preferably, the four sides of the bottom are designed to be parallel to the four sides of the opening surface.
[0099] Preferably, the square bottom and the opening surface are both square.
[0100] Preferably, the culture dish has 8 wells, 6 wells, 4 wells, 2 wells or a single well.
[0101] Modification 2 Figure 2 As shown, the bottom of each chamber is circular (a), and the opening surface of each chamber is also circular (b).
[0102] Preferably, the circular bottom (a) is designed to have an inner area that is 1 / 2 of the inner area of the circular opening surface (b).
[0103] Preferably, the circular bottom is designed to be located within the projection area of the circular opening surface in a top view.
[0104] Preferably, the circular bottom is designed to be located within the projection area of the circular opening surface in a top view and to be a concentric circle.
[0105] Preferably, the culture dish has 8 wells, 6 wells, 4 wells, 2 wells or a single well.
[0106] Example 2 3D Embryo Culture
[0107] Source of embryos: discarded embryos from the Embryology Laboratory of Sichuan Jinxin Women and Children's Hospital. This study was approved by the Reproductive Medicine Ethics Committee of Chengdu Women and Children's Central Hospital (approval number: 2021017) and followed the principles of the Declaration of Helsinki. All participants signed informed consent and agreed to participate in this study.
[0108] The embryos obtained are usually D5 (fifth day after fertilization), with a small number of D3 and D4.
[0109] 1. Embryo thawing: Legal source of embryo materials:
[0110] For the cryo-thawing kit, it is recommended to use THAW-KIT1 (Vitrolife). The contents of the kit are: 1x 10mL-Thawing solution 1 (TS1), 1x 10mL-Thawing solution 2 (TS2), 1x 10mL-Equilibration solution (ES).
[0111] ① Pipette appropriate amount (0.5-1.0 mL) of resuscitation solution TS1, resuscitation solution TS2, equilibrium solution ES and IVC1 culture medium into respective culture dishes (four-well plate, three solutions and culture medium are placed respectively). Equilibrate to room temperature.
[0112] ② Remove the straw from liquid nitrogen and expose it to air for 30 seconds.
[0113] ③ Place the straw in a 30℃ water bath for 45 seconds. Take out the straw and wipe it dry.
[0114] ④ Use aseptic technique to open the straw and carefully expel the embryo.
[0115] ⑤ Place the embryos in resuscitation solution TS1 for 5 minutes.
[0116] ⑥ Transfer the embryos to resuscitation solution TS2 for 5 minutes.
[0117] ⑦ Transfer the embryos to ES balance solution for 5 minutes.
[0118] ⑧ Transfer the embryos to 37℃ pre-equilibrated culture medium and rinse the embryos appropriately.
[0119] Tip: Make sure to carry a minimum amount (less than 1 ul) of ES equilibrated medium to avoid changing the nutrient concentration in the medium. This can be achieved by using a pipette or by transferring the embryos to a four-well dish containing IVC1 and then transferring them to the dish described in Example 1.
[0120] 2. Remove the zona pellucida:
[0121] ① Visually check the embryos under a stereomicroscope to see if there is a zona pellucida (if some embryos have broken through the zona pellucida by themselves, omit this step). For embryos with a zona pellucida, use AT solution to remove the zona pellucida;
[0122] ② Prepare 3-5 independent AT droplets (30ul) and 3 independent M2 medium droplets (30ul) in a common round embryo culture dish, and cover each droplet with a layer of mineral oil;
[0123] ③ Transfer an embryo into a drop of AT solution;
[0124] ④ Use a pipette to blow the embryo up and down several times (gently, usually 3-5 times), and transfer the embryo to the fresh AT droplet in the culture dish.
[0125] ⑤ Repeat step 4 until the zona pellucida disappears (make a few more drops of AT solution for washing. Generally, 3 drops of AT solution are needed for one embryo to see the zona pellucida disappear).
[0126] ⑥ Transfer the embryos with zona pellucida removed into a drop of M2 medium to neutralize the AT solution;
[0127] ⑦ Transfer the embryos to a fresh drop of M2 medium and wash them repeatedly (use the egg transfer needle to pump the embryos up and down 3-5 times), and repeat this step once;
[0128] ⑧Transfer the embryos to IVC1 medium pre-equilibrated at 37°C.
[0129] Tip: After transferring embryos from droplet to droplet, there will be traces of mineral oil on the pipette tip; to avoid mineral oil carryover into the culture medium, always soak the pipette tip in IVC1 before the final transfer.
[0130] 3. Embryo culture
[0131] ① Transfer the zona pellucida-free embryos to a culture dish filled with 37°C pre-equilibrated IVC1 medium as described in Example 1. Place 1-2 embryos in each well.
[0132] ②Put the culture dish in CO 2 Incubate overnight in an incubator set to CO 2 :6%,37℃
[0133] ③ The next day, usually embryo D6, observe the embryo status and change the medium (pre-equilibrated IVC1 at 37°C) in each well. ④ Repeat the steps on embryo D7.
[0134] ⑤ Change the medium to IVC2 on embryo D8, and change the medium with IVC2 every day thereafter until embryo D14.
[0135] Prepare 10 ml of IVC-1 medium:
[0136]
[0137] Prepare 10 ml of IVC-2 medium:
[0138]
[0139] Example 3 Embryo immunofluorescence triple staining
[0140] Fixation: Aspirate the culture medium in the culture dish with a pipette, wash three times with PBS, add 100ul of 4% paraformaldehyde to each well to cover the obtained embryos, and incubate at room temperature for 30 minutes.
[0141] Washing: Slowly aspirate the paraformaldehyde and wash with sufficient amount (300ul) of PBS at room temperature for 3 times, 5 minutes each time.
[0142] Permeabilization: Add 0.1% Triton X-100, room temperature for 30 minutes
[0143] Washing: Slowly aspirate Triton X-100 and wash with sufficient amount (300ul) of PBS at room temperature for 3 times, 5 minutes each time.
[0144] Blocking: Add 5% BSA for blocking at room temperature for 30 minutes
[0145] Primary antibody incubation: dilute three antibodies from different species (mouse / rabbit / sheep) with 50ul antibody diluent according to the ratio recommended in the instructions to obtain a mixed primary antibody solution. Slowly aspirate BSA without washing, add the three antibodies to the mixed primary antibody, place in a wet box, and refrigerator at 4°C overnight.
[0146] Washing: Rewarm at room temperature for 30 minutes, slowly aspirate the mixed primary antibody, and wash three times with sufficient amount (300ul) of PBS at room temperature, 5 minutes each time.
[0147] Secondary antibody incubation: Mix three fluorescent secondary antibodies of three different species with 50 μL antibody diluent to obtain a mixed secondary antibody solution, add the mixed secondary antibody, and stand at room temperature in the dark for 1 hour.
[0148] The mixed primary antibody and mixed secondary antibody ratio system is as follows:
[0149]
[0150] Washing: Slowly aspirate the mixed secondary antibody and wash with sufficient amount (300ul) PBS at room temperature for 3 times, 5min each time.
[0151] Nuclear staining: Add 50ul of DAPI containing fluorescence quencher and incubate at room temperature in the dark for 10min.
[0152] Observation and photography: There is no need to move the embryos. Simply place the culture dish under an inverted fluorescence microscope for observation and photography.
[0153] The results are shown in Tables 1~2 and Figures 4 to 7 shown.
[0154] Table 1 Comparison between triple staining and single staining
[0155]
[0156] Table 2 Comparison between ordinary 8-well plate and culture dish of the present invention
[0157]
[0158] As shown in Table 1, the triple dyeing method saves more raw materials, consumables and time than the ordinary single dyeing method.
[0159] As shown in Table 2, the culture dish described in Example 1 is more economical in using less reagent raw materials, saving consumable reagents such as culture medium and antibodies, reducing costs, and having a higher success rate.
[0160] Figure 4 As shown, the culture dish described in Example 1 has fewer damaged cells because the bottom is less affected by the liquid shock during movement.
[0161] Figure 5-6 It shows that the 3D embryo three-staining effect after cultured in the culture dish described in Example 1 is very good. At the same time, using ordinary culture dishes as a control, the probability state of 50% deteriorates as shown in FIG. Figure 7 , relative to Figure 5 When shooting, the focusing effect of ordinary culture dishes is not good, and the PDL1-positive position of the single layer cannot be photographed using a confocal microscope, and it can only be judged whether there is positivity.
[0162] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.
Claims
1. An improved culture dish, characterized in that: The inner area (a) of the bottom of each chamber of the culture dish is smaller than the inner area (b) of the opening surface.
2. Use of the culture dish according to claim 1 in embryo 3D culture.
3. Use of the culture dish described in claim 1 in simple and rapid embryo 3D immunofluorescence triple staining.
4. A simple and rapid embryo 3D culture method, characterized in that: The steps include: 1) Embryo thawing; 2) Remove the zona pellucida. For some embryos that have broken through the zona pellucida on their own, this step can be omitted. 3) Embryo culture: The culture dish described in Example 1 is used for embryo culture.
5. The embryo 3D culture method according to claim 4, characterized in that: Step 3) includes placing 1 to 2 embryos in each chamber.
6. A simple and rapid embryo 3D immunofluorescence triple staining method, characterized in that: The steps include: 1) Embryo thawing; 2) Remove the zona pellucida. For some embryos that have broken through the zona pellucida on their own, this step can be omitted. 3) Embryo culture: The culture dish described in Example 1 is used for embryo culture. 4) Immunofluorescence triple staining of embryos.
7. The embryo 3D immunofluorescence triple staining method according to claim 6, characterized in that: Step 3) includes placing 1 to 2 embryos in each chamber.
8. The embryo 3D immunofluorescence triple staining method as described in claim 7, wherein step 4) comprises adding three antibodies to mix the primary antibody, and adding the corresponding fluorescent secondary antibody after incubation for a period of time.