A method for rapid dissociation of zebrafish larvae gut for single cell sequencing

Abstract

The application belongs to the technical field of biology, and discloses a method for rapidly dissociating zebrafish larvae intestine for single-cell sequencing, which comprises the following steps: anesthetizing zebrafish larvae, placing the zebrafish larvae in an inclined manner, and sucking off the anesthetic; and dissecting and separating from the abdominal swelling part to obtain the zebrafish larvae intestine. The method of the application omits the step of fixing the zebrafish larvae by using an insect needle, optimizes the removal mode of the anesthetic, adopts the inclined plate and the syringe suction mode, effectively reduces the movement of the zebrafish larvae, and does not need the positioning mode of fixing the zebrafish larvae by using the insect needle, thereby simplifying the operation process. The application significantly improves the processing efficiency and is better in repeatability and generalizability. Since the positioning, liquid suction and dissection modes adopted are more standardized, the application is more conducive to the reproduction and general application among laboratories.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for rapid dissociation of the intestine of juvenile zebrafish for single-cell sequencing. Background Technology

[0002] Zebrafish (Danio rerio) have become an important model organism for studying vertebrate intestinal development, enteric nervous system formation, intestinal diseases, and drug screening due to their rapid embryonic development, transparent body surface, ease of genetic manipulation, and high reproductive capacity. Zebrafish intestinal development is a highly ordered process: endoderm formation begins approximately 10 hours after fertilization (10 hpf) and gradually develops into intestinal primordia; between 24 and 48 hpf, the intestine transforms from a solid structure into a tubular structure with a lumen; at 72 hpf (3 days post-fertilization, 3 dpf), the mouth opens and feeding begins; between 96 and 120 hpf (4-5 dpf), intestinal epithelial cells differentiate completely, the enteric nervous system begins to colonize, and the intestine essentially acquires digestive and absorptive functions.

[0003] With the rapid development of technologies such as single-cell RNA sequencing (scRNA-seq), flow cytometry sorting (FACS), and spatial omics, establishing high-quality and efficient methods for isolating zebrafish intestinal tissue and preparing single cells is of great significance for a deeper understanding of intestinal development mechanisms, intestinal disease pathogenesis, related drug screening, and the impact of pollutants on the intestinal health of juvenile fish. By 10 days post-fertilization (10 dpf), the juvenile fish's intestines are more mature, with denser tissue structure, more complex cellular composition, and a more complete enteric nervous system network. Furthermore, the yolk sac has largely disappeared, making it more suitable for intestinal disease modeling, flow cytometry sorting, and single-cell RNA sequencing research.

[0004] Currently, the main method for isolating the intestines of early-developing zebrafish larvae (5 dpf) for single-cell RNA sequencing involves fixing the larvae with insect needles, absorbing the surrounding fluid with absorbent materials, and then separating the intestines one by one under a microscope using insect needles. This dissection procedure is cumbersome, heavily reliant on precise manual manipulation, and has limited sample processing efficiency. Therefore, there is still room for improvement in the applicability and convenience for higher developmental stages such as 10 and 15 dpf larvae. Summary of the Invention

[0005] The present invention aims to solve at least the following technical problems existing in the prior art: (1) Solving the problems of cumbersome operation process, heavy manual burden and low efficiency of anesthetic removal method. The prior art usually requires the use of insect needles to fix the juvenile fish on the agarose plate, and then use insect needles to perform fine dissection of the intestine one by one. The overall operation steps are many, and the requirements for the skill level of the experimental personnel are high, which is not conducive to the rapid processing of large batches of samples; the use of absorbent paper to absorb the liquid around the juvenile fish is slow and easy to come into contact with the body of the juvenile fish, causing the juvenile fish to move or even be absorbed, further increasing the difficulty of operation and prolonging the dissection time. The present invention uses a tilted plate combined with a syringe to remove the anesthetic liquid around the juvenile fish, replacing the absorbent paper and insect needle fixation method in the prior art. (2) Solving the problem that the dissection tool is not conducive to rapid cutting and separation. In the prior art, insect needles are generally used for intestinal separation. Insect needles are thin and flexible, which are more suitable for picking, but not conducive to rapid dissection with cutting function; when it is necessary to separate the intestine completely and quickly from the body wall and surrounding tissues, the operation efficiency is low. This invention uses a syringe needle instead of an insect needle, and uses the sharp edge of the needle to separate and cut the intestine from the surrounding tissue. Compared with an insect needle, the syringe needle has both separating and cutting functions, which is more suitable for quickly obtaining the complete intestine. (3) It solves the problem of insufficient optimization of the intestinal separation path. This invention selects the ventral swollen part as the preferred dissection entry point and cuts the body wall from the ventral swollen part of the juvenile fish, which is more conducive to complete separation along the intestinal direction, reduces intestinal breakage, and improves the efficiency of complete intestinal recovery. (4) It solves the problem of limited sample processing efficiency and insufficient adaptation to the preparation of single-cell samples of 10dpf juvenile fish. This invention provides a highly efficient dissection and separation method for the intestine of zebrafish juvenile fish, which is suitable for the preparation of single-cell samples of 10dpf juvenile fish, significantly improves the efficiency of juvenile fish intestinal separation per unit time, and obtains high-quality samples suitable for subsequent flow cytometry sorting and single-cell RNA sequencing.

[0006] The first objective of this invention is to provide a method for dissecting and separating the intestines of juvenile zebrafish.

[0007] The second aspect of the present invention aims to provide the application of the method of the first aspect of the present invention in disease model construction and / or drug screening.

[0008] The object of a third aspect of the present invention is to provide the application of the method of the first aspect of the present invention in the preparation of intestinal single cells.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a method for dissecting and separating the intestines of juvenile zebrafish, comprising the following steps: Anesthetize the juvenile zebrafish, place them at an angle, and then remove the anesthetic. Dissecting and separating the swollen area on the ventral side yields the intestines of juvenile zebrafish.

[0010] In some embodiments of the present invention, the developmental period of the zebrafish juveniles is 5-15 dpf.

[0011] In some preferred embodiments of the present invention, the developmental period of the zebrafish juveniles is 5-12 dpf.

[0012] In some preferred embodiments of the present invention, the developmental period of the zebrafish juveniles is 10 dpf.

[0013] In some embodiments of the present invention, the zebrafish juveniles are placed at an angle on an agarose plate.

[0014] In some embodiments of the present invention, the method for preparing the agarose plate includes the following steps: dissolving agarose in E3 embryo culture medium to prepare an agarose solution; pouring the agarose solution into a petri dish and allowing it to solidify to obtain the agarose plate.

[0015] In some embodiments of the present invention, the concentration of the agarose solution is 1.5%-2.5% w / v.

[0016] In some preferred embodiments of the present invention, the concentration of the agarose solution is 1.6%-2% w / v.

[0017] In some more preferred embodiments of the present invention, the concentration of the agarose solution is 1.8% w / v.

[0018] In some embodiments of the present invention, the anesthetic agent includes at least one selected from tricaine, eugenol, benzocaine, metoprolol, etomidate, lidocaine, propofol, alfasalone, and isoflurane.

[0019] In some embodiments of the present invention, the anesthetic is tricaine.

[0020] In some embodiments of the present invention, the concentration of the anesthetic is 0.01%-0.02% w / v.

[0021] In some preferred embodiments of the present invention, the concentration of the anesthetic is 0.015%-0.018% w / v.

[0022] In some preferred embodiments of the present invention, the concentration of the anesthetic is 0.016% w / v.

[0023] In some embodiments of the present invention, the tool for aspirating the anesthetic includes at least one of a syringe, a pipette, a micropipette, a capillary tube, and a handheld vacuum aspiration controller.

[0024] In some embodiments of the present invention, the tool for aspirating the anesthetic is a syringe.

[0025] In some embodiments of the present invention, the dissection tool includes at least one of a syringe needle, a fine needle, a microscalpel, surgical scissors, and a micro-dissecting needle.

[0026] In some embodiments of the present invention, the tool used for dissection and separation is a syringe needle.

[0027] In some embodiments of the present invention, the method further includes an intestinal cleansing step.

[0028] In some embodiments of the present invention, the intestinal cleansing includes removing non-intestinal tissue attached to the intestine, the non-intestinal tissue including at least one of skin, fat and liver.

[0029] In some embodiments of the invention, the intestinal cleansing includes removing non-intestinal tissue using a needle tip.

[0030] In some embodiments of the invention, the method further includes transferring the intestine into a buffer solution containing fetal bovine serum.

[0031] In some embodiments of the present invention, the buffer solution includes PBS buffer.

[0032] In some embodiments of the present invention, the concentration of the fetal bovine serum is 5-15% v / v.

[0033] In some preferred embodiments of the present invention, the concentration of the fetal bovine serum is 8-12% v / v.

[0034] In some more preferred embodiments of the present invention, the concentration of the fetal bovine serum is 10% v / v.

[0035] A second aspect of the present invention provides the application of the method of the first aspect of the present invention in disease model construction and / or drug screening.

[0036] In some embodiments of the present invention, the disease model includes an intestinal disease model.

[0037] A third aspect of the present invention provides the application of the method of the first aspect of the present invention in the preparation of intestinal single cells.

[0038] The beneficial effects of this invention are: The zebrafish juvenile intestinal dissection and separation method provided by this invention is applicable to the intestinal dissection and separation of zebrafish juveniles older than 10 dpf. At this stage, the intestinal development is more mature and the function is more complete, which has more important value in intestinal development, disease modeling and drug screening research, and fills the gap in efficient intestinal dissection methods for juvenile fish older than 10 dpf.

[0039] This invention eliminates the need for securing juvenile fish with insect needles. By optimizing the anesthetic removal method and employing a tilted plate and syringe aspiration, it effectively reduces juvenile fish movement, simplifying the operation process and eliminating the need for insect needle fixation. The tilted plate combined with syringe aspiration is faster and more stable than absorbent paper, and is less likely to cause juvenile fish adhesion or displacement. The syringe needle also serves as a dissection and cutting tool, making it more suitable for rapid and complete intestinal separation than traditional insect needles, reducing dissection difficulty and improving the ease of dissection. By selecting the ventral swollen area as the preferred entry path, it is easier to remove the intestine completely, reducing sample breakage and increasing the recovery rate of intact intestine.

[0040] This invention significantly improves processing efficiency, enabling the intestinal dissection of 60 5-day-of-life (5dpf) juvenile fish within 30 minutes (an average rate of 2 fish / minute), approximately 1.46 times faster than existing technologies; and the intestinal dissection of 100 10-day-of-life (10dpf) juvenile fish within 30 minutes (a rate of 3.33 fish / minute), also 3.33 times faster than existing technologies. After subsequent dissection, the intestinal samples obtained by this invention show a cell viability of 94.6% and a total cell count of approximately 180,000, sufficient for single-cell RNA sequencing. The method of this invention offers better reproducibility and scalability. Due to the more standardized positioning, aspiration, and dissection methods employed, this invention is more conducive to inter-laboratory reproduction and widespread application. Attached Figure Description

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram illustrating the steps of the zebrafish juvenile gut dissociation method of the present invention.

[0042] Figure 2 The images show the cell viability results after dissociation of intestinal cells from 100 10 dpf zebrafish juveniles. In Figure A, cell viability is shown under a microscope (scale bar: 100 µm); Figure B shows the cell diameter distribution, FL1 green fluorescence intensity distribution, and FL2 red fluorescence intensity distribution.

[0043] Figure 3 The diagram shows the dissection speed (A) and speed enhancement factor (B) of Embodiment 1 and Comparative Example 1 of the present invention. Detailed Implementation

[0044] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0045] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0046] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0047] Example 1: Intestinal dissection and separation of juvenile zebrafish 1. Preparations before dissection (1) Preparation of agarose plates: Dissolve 0.45 g of agarose in 25 mL of E3 embryo culture medium to prepare a 1.8% agarose solution; pour the agarose solution into a petri dish and let it solidify; prepare about 10 agarose plates before the experiment and place them in a 4℃ refrigerator for pre-cooling.

[0048] (2) Preparation of anesthetic: Prepare a 0.016% tricaine (product number E10521, Sigma-Aldrich) solution with nuclease-free water.

[0049] (3) Collection tube preparation: Prepare a centrifuge tube containing PBS + 10% (v / v) fetal bovine serum (FCS) and place it on ice for later use.

[0050] (4) Preparation of instruments and tools: stereomicroscope (MZ62, Guangzhou Mingmei Optoelectronic Technology Co., Ltd.), fine forceps, 1mL syringe (for removing anesthetic and dissection).

[0051] 2. Dissection steps (see) Figure 1 ) (1) Anesthesia and placement: Five 5dpf / 10dpf juvenile fish (wild-type AB strain zebrafish) were anesthetized in 0.016% tricaine solution. Under a stereomicroscope, the anesthetized juvenile fish were arranged in rows on pre-cooled 1.8% agarose plates. Two plates were used alternately, and two experimenters worked together. One person was responsible for anesthetizing and placing the juvenile fish, and the other person was responsible for dissecting the juvenile fish under a stereomicroscope. Before the formal experiment, the cooperation was practiced in advance.

[0052] (2) Remove excess anesthetic: tilt the plate to allow the anesthetic to concentrate on one side; use a 1mL syringe to suck up all the anesthetic around the juvenile fish, being careful not to touch the juvenile fish's body.

[0053] (3) Separate the intestine: Use a 1mL syringe needle to tear open the body wall of the swollen ventral side of the juvenile fish; separate the intestine from other organs; completely remove the skin, a small amount of yolk and other non-intestinal tissues; the sharp side of the syringe needle can be used as a blade to cut the intestine off the juvenile fish body part and quickly remove the entire intestine.

[0054] (4) Clean the sample: Examine the isolated intestine; use a needle tip to remove any non-intestinal tissue attached to it (such as skin, fat, liver, etc.).

[0055] (5) Collecting the intestine: Use tweezers to pick up the separated intestine; transfer it to a pre-cooled PBS / 10%FCS collection tube, place it on ice, and collect the intestine of the juvenile fish. Try not to freeze the dissociated intestine to maximize cell viability.

[0056] In this embodiment, the intestinal dissection of approximately 60 5dpf juvenile fish can be completed within 30 minutes, with a processing rate of approximately 2,000 fish / minute; the intestinal dissection of approximately 100 10dpf juvenile fish can be completed within 30 minutes, with a processing rate of approximately 3,330 fish / minute.

[0057] Example 2: AOPI cell viability detection 1. Intestinal dissociation: 100 10dpf juvenile fish intestines collected in Example 1 were enzymatically digested, filtered, centrifuged, and resuspended using conventional methods. The specific process is as follows: (1) After all intestinal dissections were completed, the microcentrifuge tubes were immediately centrifuged at the highest speed (13,800×g) for 30 seconds. PBS / 10% FCS was removed, leaving approximately 100 μL of liquid to prevent intestinal drying. 500 μL of 2.17 mg / mL papain (HY-P1645, MedChemExpress) solution containing CaCl2 and MgCl2 in HBSS (C0219, Beyotime) was added for cell dissociation. 2.5 μL of 1M cysteine ​​(HY-Y0337, MedChemExpress) was added to activate the papain.

[0058] (2) Place the intestine in a 37°C metal bath and incubate for 10 minutes. At the 5th minute of incubation, blow and agitate the intestine several times to promote enzymatic digestion.

[0059] (3) Pre-wet a 70 μm cell filter with 0.5 mL PBS / 10% FCS, then pass the digested cells from step (2) through a 40 μm cell filter and transfer them to a FACS tube. Rinse the filter with 2 mL PBS / 10% FCS in 4 portions (0.5 mL each time). Centrifuge at 4°C and 700×g for 5 minutes. Discard the supernatant and resuspend the cell pellet in 300 μL PBS / 2% FCS.

[0060] 2. AOPI cell viability detection (1) Take 10µL of cell suspension, add 10µL of AOPI staining solution (AO / PI cell viability assay kit, C2017S, Beyotime), mix gently, and incubate at room temperature in the dark for 1 minute.

[0061] (2) Take 20µL of the above 20µL stained cell suspension and load it into the special counting plate of the cell counter (Countstar Rigel S2, Shanghai Ruiyu Biotechnology Co., Ltd.). Read and record the cell viability (the proportion of live cells to nucleated cells), nucleation rate (the proportion of nucleated cells to total cells), clumping rate (the proportion of cell clumps to total cells) and total cell number according to the instrument operation procedure.

[0062] The results are as follows Figure 2 As shown, under a fluorescence microscope, nucleated cell nuclei emitting green fluorescence and dead cells emitting red fluorescence were observed. Figure 2 In A), the cells exhibit strong green fluorescence ( Figure 2 (C in the middle), while the red fluorescence signal is weaker ( Figure 2 (D in the text). The cell counting results showed that the cell viability was 94.6%, the clumping rate was 24.76%, the nucleation rate was 80.34%, and the cell quantity was about 180,000, which meets the requirements for single-cell sequencing and can be performed.

[0063] Comparative Example 1: Intestinal dissection and separation of juvenile zebrafish 1. Preparations before dissection (1) Agarose plate: Dissolve 0.45 g of agarose in 25 mL of E3 embryo culture medium to prepare a 1.8% agarose solution, and pour it into a petri dish to solidify.

[0064] (2) Anesthetic: Prepare a 0.016% tricaine solution (Catalog number E10521, Sigma-Aldrich) using E3 medium.

[0065] (3) Collection tube: Prepare a microcentrifuge tube placed on ice, containing PBS + 10% fetal bovine serum (FCS).

[0066] (4) Tools: fine insect needles, fine tweezers, stereomicroscope, absorbent paper.

[0067] 2. Dissection steps (1) Anesthetize 5dpf / 10dpf zebrafish juveniles with 0.016% tricaine, arrange 6-10 juveniles on 1.8% agarose plates and observe them under a dissecting microscope.

[0068] (2) Fix the head with an insect needle, use a paper towel to remove all remaining E3 embryo culture medium, use another needle to separate the intestine, and remove the yolk and other non-intestinal tissues (such as skin, fat, liver).

[0069] (3) The collected intestines were placed in a centrifuge tube containing PBS + 10% fetal bovine serum (FCS) and placed on ice.

[0070] By comparing the dissection speed and speed improvement factor of Example 1 with Comparative Example 1 (prior art), the results are as follows: Figure 3 As shown. The method in Example 1 can complete the intestinal dissection of approximately 60 5dpf juvenile fish within 30 minutes, with a processing rate of approximately 2 fish / minute; and can complete the intestinal dissection of approximately 100 10dpf juvenile fish within 30 minutes, with a processing rate of approximately 3.33 fish / minute. The method in Comparative Example 1 can complete the intestinal dissection of approximately 41 5dpf juvenile fish within 30 minutes, with a processing rate of approximately 1.37 fish / minute; and can complete the intestinal dissection of approximately 30 10dpf juvenile fish within 30 minutes, with a processing rate of approximately 1 fish / minute. Compared with Comparative Example 1, the method in Example 1 improves the intestinal dissection and separation efficiency of 5dpf juvenile fish by approximately 1.46 times and the intestinal dissection and separation efficiency of 10dpf juvenile fish by approximately 3.33 times.

[0071] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for dissecting and separating the intestines of juvenile zebrafish, characterized in that, Includes the following steps: Anesthetize the juvenile zebrafish, place them at an angle, and then remove the anesthetic. Dissect the swollen area on the ventral side and separate the intestine to obtain the intestine of the zebrafish juvenile.

2. The method according to claim 1, characterized in that, The developmental period of the zebrafish juveniles is 5-15 days postpartum (dpf).

3. The method according to claim 1, characterized in that, The tools for aspirating anesthetics include at least one of a syringe, pipette, micropipette, capillary tube, and handheld vacuum aspiration controller.

4. The method according to any one of claims 1-3, characterized in that, The tools used for dissection include at least one of syringe needles, fine needles, microscalpels, surgical scissors, and micro-dissecting needles.

5. The method according to any one of claims 1-3, characterized in that, The method also includes a bowel cleansing step.

6. The method according to claim 5, characterized in that, The intestinal cleansing includes the removal of non-intestinal tissue attached to the intestine, including at least one of skin, fat, and liver.

7. The method according to claim 5, characterized in that, The method further includes transferring the intestine into a serum-containing buffer; and / or, the buffer includes PBS buffer; and / or, the serum includes fetal bovine serum.

8. The method according to claim 7, characterized in that, The concentration of the fetal bovine serum was 5-15% v / v.

9. The application of the method according to any one of claims 1-9 in disease model construction and / or drug screening.

10. The use of the method of any one of claims 1-9 in the preparation of intestinal single cells.

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