A reagent for removing cell clumps and fragments in a single cell suspension and its application

Abstract

The present invention discloses a reagent for removing cell clumps and fragments in a single cell suspension and its application, and relates to the technical field of single cell suspension preparation. A reagent for removing cell clumps and fragments in a single cell suspension comprises Percoll stock solution, 0.85% w / v NaCl, 0.5% w / v EDTA, 0.5-2.5 mg / mL ADH‑1 trifluoroacetate and 5.0-8.5 mg / mL trypsin inhibitor. The present invention adopts the above-mentioned reagent for removing cell clumps and fragments in a single cell suspension and its application, which can significantly remove the cell clumps and fragments generated during the dissociation of animal tissue into a single cell suspension, thereby obtaining a single cell suspension with high viability cells and minimal fragments, laying the foundation for subsequent single cell sequencing.

Description

Technical Field

[0001] The invention relates to the technical field of single cell suspension preparation, and in particular to a reagent for removing cell agglomerates and fragments in a single cell suspension and application thereof. Background Art

[0002] With the development of science and technology today, a variety of cell biology experimental techniques have emerged, such as cell culture, flow cytometry, and single-cell sequencing technology, which have greatly increased the demand of scientific researchers to prepare single-cell suspensions from tissues. Among them, single-cell sequencing technology can, to a certain extent, analyze in detail the specific differences between different types of cells in complex samples, analyze the functions of different cells under specific physiological and pathological conditions, and identify the role of different cells in development or disease progression, thereby revealing profound molecular mechanisms. Given the powerful function of detailed data brought by single-cell sequencing technology, this technology has revolutionized the research paradigms and basic methods in many fields since its inception, and has had a subversive impact on life science research. Preparing high-quality single-cell suspensions from tissues is a prerequisite for cell analysis, and it is also a key link in the success of downstream experiments such as single-cell sequencing.

[0003] The cell yield, cell viability, cell clumping, and cell fragments of a single cell suspension will have a decisive impact on subsequent cell analysis experiments. Therefore, removing the cell clumps and fragments produced during the dissociation of animal tissues into single cell suspensions is crucial for preparing high-quality single cell suspensions. At present, most methods of preparing single cell suspensions use low-speed centrifugation to remove cell fragments. However, this method cannot completely remove cell fragments, and it is not suitable for various types of tissues. Therefore, the present invention provides a defragmentation reagent that can be used for the dissociation of various animal tissues and a method of using the same, which separates normal living cells from cell fragments, thereby obtaining a purified single cell suspension and improving the success rate of subsequent experiments such as single cell sequencing. Summary of the invention

[0004] The purpose of the present invention is to provide a reagent for removing cell clumps and fragments in a single-cell suspension and its application, which can significantly remove cell clumps and fragments generated during the dissociation of animal tissue into a single-cell suspension, thereby obtaining a single-cell suspension with highly active cells and minimal fragments, laying the foundation for subsequent single-cell sequencing.

[0005] To achieve the above object, the present invention provides a reagent for removing cell clumps and fragments in a single cell suspension, comprising Percoll stock solution, 0.85% w / v NaCl, 0.5% w / v EDTA, 0.5-2.5 mg / mL ADH-1 trifluoroacetate and 5.0-8.5 mg / mL trypsin inhibitor.

[0006] Further, Percoll stock solution, 0.85% w / v NaCl, 0.5% w / v EDTA, 1.5 mg / mL ADH-1 trifluoroacetate, and 7.0 mg / mL trypsin inhibitor were included.

[0007] The present invention also provides the use of the above reagent in removing cell agglomerates and fragments during the process of dissociating animal tissue into single cell suspension.

[0008] The present invention also provides a method for removing cell agglomerates and fragments in a single cell suspension, comprising the following steps:

[0009] S1. Collect the single cell suspension from which the debris needs to be removed into a centrifuge tube, centrifuge at 300-500×g, 4°C for 3-5 min, then slowly pour off the supernatant to obtain the cell pellet;

[0010] S2. Add an appropriate amount of 1× PBS solution to the centrifuge tube and gently use a Pasteur pipette to resuspend the cell pellet so that the cell concentration is 1×10 6 ~1×10 7 Pieces / mL;

[0011] S3. Dilute the above reagents by 20% to 30% volume ratio using 1× PBS buffer containing calcium and magnesium ions, and then take the diluted reagents into a new centrifuge tube;

[0012] S4. Use a Pasteur pipette to slowly drip the single cell suspension with adjusted cell concentration onto the upper part of the reagent along the tube wall, while ensuring that the cell suspension and the reagent do not interfere with each other, forming a clear two-phase gradient centrifugation;

[0013] S5. Remove the upper fragments and add an appropriate amount of 1×PBS solution containing 10% FBS to resuspend the precipitate at the bottom of the centrifuge tube to obtain the purified single cell suspension.

[0014] Furthermore, in step S3, the reagent is diluted at a volume ratio of 25%.

[0015] Furthermore, in step S4, the cell suspension accounts for 30-50% of the total volume of the reagent and the cell suspension.

[0016] Furthermore, in step S4, the cell suspension accounts for 50% of the total volume of the reagent and the cell suspension.

[0017] Furthermore, in step S4, the gradient centrifugation conditions were set to 800×g, centrifugation at 4°C for 20 min, and the acceleration and deceleration speeds were set to 1.

[0018] The advantages and positive effects of the reagent for removing cell agglomerates and fragments in a single cell suspension and its application are as follows:

[0019] The defragmentation method disclosed in the present invention uses a defragmentation reagent with a density between that of live cells and cell fragments as a density gradient liquid, and centrifuges the cell mixture and the density gradient liquid together to separate live cells from fragments. During the centrifugation process, some dead cells will cause cell membrane rupture due to increased cell membrane permeability, and eventually become cell fragments and be removed, thereby improving cell viability. EDTA, ADH-1 trifluoroacetate and trypsin inhibitor contained in the defragmentation reagent can bind to cell surface proteins such as calcium-adhesive proteins, block cell-cell adhesion, and effectively reduce cell agglomeration. Compared with the traditional centrifugation method, the defragmentation method of the present invention not only removes fragments more thoroughly, reduces the cell agglomeration rate, but is also more suitable for a variety of animal tissue types, and obtains high-quality single-cell suspensions with high viability, low cell agglomeration rate and minimal fragments, ensuring the success rate of downstream cell sequencing experiments.

[0020] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The bright field images and fluorescence images of live and dead cells of the mouse liver tissue single cell suspension before and after the defragging operation in Example 1 of the present invention are shown, wherein A is the bright field image before the defragging operation, B is the fluorescence image of live and dead cells before the defragging operation, a is the bright field image after the defragging operation, b is the fluorescence image of live and dead cells after the defragging operation, ① is the bright field image after the defragging operation of the commercial product, ② is the fluorescence image of live and dead cells after the defragging operation of the commercial product, green fluorescence indicates live cells, and red fluorescence indicates dead cells;

[0022] Figure 2 The bright field images and fluorescence images of live and dead cells of the mouse tracheal tissue single cell suspension before and after the defragging operation in Example 2 of the present invention are shown, wherein A is the bright field image before the defragging operation, B is the fluorescence image of live and dead cells before the defragging operation, a is the bright field image after the defragging operation, b is the fluorescence image of live and dead cells after the defragging operation, ① is the bright field image after the defragging operation of the commercially available product, ② is the fluorescence image of live and dead cells after the defragging operation of the commercially available product, green fluorescence indicates live cells, and red fluorescence indicates dead cells;

[0023] Figure 3 The bright field images and fluorescence images of live and dead cells of the single cell suspension of the whole brain tissue of mice in Example 3 of the present invention before and after the defragging operation are shown, wherein A is the bright field image before the defragging operation, B is the fluorescence image of live and dead cells before the defragging operation, a is the bright field image after the defragging operation, b is the fluorescence image of live and dead cells after the defragging operation, ① is the bright field image after the defragging operation of the commercial product, ② is the fluorescence image of live and dead cells after the defragging operation of the commercial product, green fluorescence indicates live cells, and red fluorescence indicates dead cells;

[0024] Figure 4 The bright field images and fluorescence images of live and dead cells of the mouse skin tissue single cell suspension before and after the defragging operation in Example 4 of the present invention are shown, wherein A is the bright field image before the defragging operation, B is the fluorescence image of live and dead cells before the defragging operation, a is the bright field image after the defragging operation, b is the fluorescence image of live and dead cells after the defragging operation, ① is the bright field image after the defragging operation of the commercially available product, ② is the fluorescence image of live and dead cells after the defragging operation of the commercially available product, green fluorescence indicates live cells, and red fluorescence indicates dead cells;

[0025] Figure 5 The bright field images and fluorescence images of live and dead cells of the single cell suspension of mouse intestinal tissue in Example 5 of the present invention before and after the defragging operation are shown, wherein A is the bright field image before the defragging operation, B is the fluorescence image of live and dead cells before the defragging operation, a is the bright field image after the defragging operation, b is the fluorescence image of live and dead cells after the defragging operation, ① is the bright field image after the defragging operation of a commercial product, ② is the fluorescence image of live and dead cells after the defragging operation of a commercial product, green fluorescence indicates live cells, and red fluorescence indicates dead cells;

[0026] Figure 6 These are the bright field images and fluorescence images of live and dead cells of the single cell suspension of mouse muscle tissue before and after the defragging operation in Example 6 of the present invention, wherein A is the bright field image before the defragging operation, B is the fluorescence image of live and dead cells before the defragging operation, a is the bright field image after the defragging operation, b is the fluorescence image of live and dead cells after the defragging operation, ① is the bright field image of the commercially available product after the defragging operation, ② is the fluorescence image of live and dead cells after the defragging operation of the commercially available product, green fluorescence represents live cells, and red fluorescence represents dead cells. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0028] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0029] Unless otherwise defined, the instruments, equipment and reagents used in the present invention are conventionally commercially available.

[0030] The components of the defragmentation reagent in the following examples include: Percoll stock solution, 0.85% w / v NaCl, 0.5% w / v EDTA, 1.5 mg / mL ADH-1 trifluoroacetate, and 7.0 mg / mL trypsin inhibitor.

[0031] The debris removal product purchased on the market is Debris Removal Solution from Miltenyi Biotec, with the product number being 130-109-398.

[0032] Example 1 Experiment on preparation of single cell suspension of mouse liver tissue

[0033] Among the reagents used in this experiment, digestion solution I and digestion solution II were prepared independently, and the other reagents were common chemical reagents. Among them, 1× PBS solution did not contain calcium and magnesium ions unless otherwise specified.

[0034] Digestion Solution I formulation: 0.25% w / v trypsin.

[0035] Digestion Solution II formula: 0.1% w / v Collagenase II, 0.15% w / v Collagenase IV, 0.06% w / v Dispase II and 0.1 mg / ml DNase I.

[0036] 1. Dissect the mouse and take 30-100 mg of liver tissue. Rinse with 1× PBS solution and cut the liver tissue into 1 mm pieces using a pre-cooled surgical blade. 3 Small pieces of tissue on the left and right;

[0037] 2. Transfer the tissue to a 15 ml centrifuge tube containing 3 ml digestion solution I, mix by pipetting with a Pasteur pipette, and digest at room temperature on the operating table for 10 min, gently blowing the tissue with a Pasteur pipette every 3 min;

[0038] 3. Let stand for 30 seconds to settle the undigested large tissue pieces, aspirate the upper suspension and place it in a 4 ℃ centrifuge, centrifuge at 250 g for 5 min, discard the supernatant, add 100 μl 1× PBS solution to resuspend the cells and store them in a 4 ℃ refrigerator;

[0039] 4. Add 2 ml of digestion solution II to the remaining undigested tissue and mix thoroughly. Place in a 37°C horizontal shaker for 15 min. Set the shaker speed to 80 rpm / min.

[0040] 5. Remove the centrifuge tube and add 10 ml of pre-cooled 1× PBS mixing solution to terminate digestion;

[0041] 6. Filter the cell suspension into a new 50 ml centrifuge tube using a 40 μm cell strainer. Centrifuge the filtrate at 300 g for 5 min at 4 °C and slowly discard the supernatant.

[0042] 7. Mix the cell suspension temporarily stored in step 3 with the cell pellet obtained in step 6, add 5 ml of red blood cell lysis buffer, resuspend the pellet and place it on ice for 5 min;

[0043] 8. Add 5 ml of pre-cooled 1× PBS to stop lysis, centrifuge at 4°C, 310 g for 5 min, discard the supernatant to remove the cell lysis solution, add a small amount of pre-cooled 1× PBS to resuspend the cells, take 10 μl of the cell suspension and mix it with AOPI staining solution in a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 1 A and B in the figure), the cell viability and aggregation rate are shown in Table 1. Then, according to the cell count, an appropriate amount of 1×PBS solution containing calcium and magnesium ions was added to the centrifuge tube to adjust the cell concentration to 1×10 6 ~1×10 7 Between pc / mL;

[0044] 9. Dilute the defragmentation reagent by 25% volume using 1× PBS buffer containing calcium and magnesium ions, and then take the diluted defragmentation reagent with the same volume as the cell suspension into a new 15 mL centrifuge tube;

[0045] 10. Use a Pasteur pipette to slowly drip the cell suspension with adjusted cell concentration onto the top of the de-fragmentation reagent along the tube wall, while ensuring that the cell suspension and the de-fragmentation reagent do not interfere with each other to form a clear two-phase gradient centrifugation. The density gradient centrifugation conditions are set to a centrifugal force of 800 g, a temperature of 4 °C, centrifugation for 20 min, and both the speed of increase and speed of decrease are set to 1;

[0046] 11. Remove the upper debris, add 200-500 μl of 1× PBS solution containing 10% FBS to resuspend the sediment at the bottom of the centrifuge tube to obtain the purified liver single cell suspension. Similarly, take 10 μl of the cell suspension and mix it with AOPI staining solution at a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 1 (a, b) Cell viability and aggregation rate are shown in Table 1.

[0047] Use the fragment removal product purchased from the market, perform the experiment according to the above steps, use a cell counter to count and take pictures, the results are as follows Figure 1 As shown in ① and ②.

[0048] Example 2 Experiment on preparation of single cell suspension of mouse tracheal tissue

[0049] Among the reagents used in this experiment, the digestion solution was prepared independently, and the other reagents were common chemical reagents. Among them, 1× PBS solution did not contain calcium and magnesium ions unless otherwise specified.

[0050] Digestion solution formula: 0.1% w / v Collagenase II, 0.1% w / v Collagenase IV, 0.06% w / v Dispase II and 0.05 mg / ml DNase I.

[0051] 1. Dissect and obtain 50-100 mg of mouse tracheal tissue, rinse with 1× PBS solution, and cut the tracheal tissue into 1 mm pieces using a pre-cooled surgical blade. 3 Rinse the tissue with 1× PBS until the liquid is no longer turbid, and then remove the PBS and leave the tissue.

[0052] 2. Transfer the chopped tissue pieces to a 15 ml centrifuge tube, add 2 ml of digestion solution and mix well, then place in a 37 ℃ shaker for 20 min. Set the shaker speed to 80 rpm / min. During the digestion process, use a Pasteur pipette to mix the digestion solution every 5-8 minutes.

[0053] 3. After digestion, remove the centrifuge tube and add 10 ml of pre-cooled 1× PBS with a Pasteur pipette to terminate digestion;

[0054] 4. Use a 40 μm cell sieve to filter the enzyme-tissue mixture and collect the cell suspension in a new 50 ml centrifuge tube. Rinse the cell sieve with pre-cooled 1× PBS in advance.

[0055] 5. Centrifuge the filtrate at 300 g for 5 min at 4 °C. After all cells have been centrifuged to the bottom of the tube, slowly pour off the supernatant.

[0056] 6. Add 3 ml of red blood cell lysis buffer to resuspend the cell pellet, pipette gently to mix, and place on ice to lyse for 5 minutes;

[0057] 7. Add 5 ml of pre-cooled 1× PBS to terminate lysis, centrifuge the sample at 300 g for 5 min at 4 °C, and discard the supernatant;

[0058] 8. Add a small amount of pre-cooled 1× PBS to resuspend the cells, then take 10 μl of the cell suspension and mix it with AOPI staining solution in a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 2 A and B in the figure), the cell viability and aggregation rate are shown in Table 1. Then, according to the cell count, an appropriate amount of 1×PBS solution containing calcium and magnesium ions was added to the centrifuge tube to adjust the cell concentration to 1×10 6 ~1×10 7 Between pc / mL;

[0059] 9. Dilute the defragmentation reagent by 25% volume using 1× PBS buffer containing calcium and magnesium ions, and then take the diluted defragmentation reagent with the same volume as the cell suspension into a new 15 mL centrifuge tube;

[0060] 10. Use a Pasteur pipette to slowly drip the cell suspension with adjusted cell concentration onto the top of the de-fragmentation reagent along the tube wall, while ensuring that the cell suspension and the de-fragmentation reagent do not interfere with each other to form a clear two-phase gradient centrifugation. The density gradient centrifugation conditions are set to a centrifugal force of 800 g, a temperature of 4 °C, centrifugation for 20 min, and both the speed of increase and speed of decrease are set to 1;

[0061] 11. Remove the upper debris, add 200-500 μl of 1× PBS solution containing 10% FBS to resuspend the sediment at the bottom of the centrifuge tube to obtain the purified tracheal single cell suspension. Similarly, take 10 μl of the cell suspension and mix it with AOPI staining solution at a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 2 (a, b) Cell viability and aggregation rate are shown in Table 1.

[0062] Use the fragment removal product purchased from the market, perform the experiment according to the above steps, use a cell counter to count and take pictures, the results are as follows Figure 2 As shown in ① and ②.

[0063] Example 3 Experiment on preparation of single cell suspension of whole brain tissue of mice

[0064] The digestion solution used in this experiment was prepared independently, and the other reagents were common chemical reagents, among which 1×PBS solution did not contain calcium and magnesium ions unless otherwise specified. The digestion solution had the same formula as that in Example 2.

[0065] 1. Dissect the mouse and take the whole brain tissue (about 200-500 mg). After flushing with artificial cerebrospinal fluid, cut the brain tissue into 1 mm pieces using a pre-cooled surgical blade. 3 Then wash the chopped brain tissue once or twice with artificial cerebrospinal fluid to wash away damaged cell fragments;

[0066] 2. Transfer the cleaned brain tissue to a 15 ml centrifuge tube, add 5 ml of digestion solution and mix thoroughly, then incubate on a shaker at 37°C for 25 min with the shaker speed set to 100 rpm / min;

[0067] 3. After digestion, remove the centrifuge tube and add 5 ml of pre-cooled artificial cerebrospinal fluid to terminate digestion;

[0068] 4. Filter the cell suspension through 100 μm and 40 μm cell sieves into a new 50 ml centrifuge tube, centrifuge the filtrate at 300 g for 5 min at 4°C, and discard the supernatant;

[0069] 5. Add 3 ml of red blood cell lysis buffer to the centrifuge tube, resuspend the cell pellet, and place on ice for 5 minutes;

[0070] 6. Add 7 ml of pre-cooled artificial cerebrospinal fluid, mix by pipetting with a Pasteur pipette, centrifuge at 300 g for 5 min at 4°C, and discard the supernatant;

[0071] 7. Add a small amount of pre-cooled 1× PBS to resuspend the cells, then take 10 μl of the cell suspension and mix it with AOPI staining solution in a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 3 A and B in the figure), the cell viability and aggregation rate are shown in Table 1. Then, according to the cell count, an appropriate amount of 1×PBS solution containing calcium and magnesium ions was added to the centrifuge tube to adjust the cell concentration to 1×10 6 ~1×10 7 Between pc / mL;

[0072] 8. Dilute the defragmentation reagent by 25% volume using 1× PBS buffer containing calcium and magnesium ions, and then take the diluted defragmentation reagent with the same volume as the cell suspension into a new 15 mL centrifuge tube;

[0073] 9. Use a Pasteur pipette to slowly drip the cell suspension with adjusted cell concentration onto the top of the de-fragmentation reagent along the tube wall, while ensuring that the cell suspension and the de-fragmentation reagent do not interfere with each other to form a clear two-phase gradient centrifugation. The density gradient centrifugation conditions are set to a centrifugal force of 800 g, a temperature of 4 °C, centrifugation for 20 min, and both the speed of increase and decrease are set to 1;

[0074] 10. Remove the upper debris, add 200-500 μl of 1× PBS solution containing 10% FBS to resuspend the sediment at the bottom of the centrifuge tube to obtain the purified mouse whole brain tissue single cell suspension. Similarly, take 10 μl of the cell suspension and mix it with AOPI staining solution at a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 3 (a, b) Cell viability and aggregation rate are shown in Table 1.

[0075] Use the fragment removal product purchased from the market, perform the experiment according to the above steps, use a cell counter to count and take pictures, the results are as follows Figure 3 As shown in ① and ②.

[0076] Example 4 Experiment on preparation of single cell suspension of mouse skin tissue

[0077] Among the reagents used in this experiment, digestion solution I and digestion solution II were independently developed and prepared by our company, and the other reagents were common chemical reagents. Among them, 1×PBS solution did not contain calcium and magnesium ions unless otherwise specified.

[0078] The formulas of digestive juice I and digestive juice II are the same as those in Example 1.

[0079] 1. Dissect and obtain 100-500 mg of mouse skin tissue and place it in a sterile culture dish. After removing excess surface tissue, rinse it with 1× PBS solution and cut the skin tissue into 1 mm pieces using a pre-cooled surgical blade. 3 Then rinse the tissue with 1× PBS until the liquid is no longer turbid, and remove the PBS to leave the tissue;

[0080] 2. Transfer the chopped tissue pieces into a 15 ml centrifuge tube with 3 ml digestion solution I, pipette thoroughly to mix, and place in a 4 ℃ refrigerator for overnight digestion;

[0081] 3. After 16 hours of overnight digestion, take out the centrifuge tube and place it on the laboratory bench to let the tissue settle. Use a Pasteur pipette to remove the supernatant, add 2 ml of digestion solution II to the tissue, mix thoroughly, and place it in a shaker at 37 °C for 30 minutes. The shaker speed is set to 120 rpm / min.

[0082] 4. After digestion, remove the centrifuge tube and add 10 ml of pre-cooled 1× PBS with a Pasteur pipette to terminate digestion;

[0083] 5. Use a 40 μm cell sieve to filter the enzyme-tissue mixture and collect the cell suspension in a new 50 ml centrifuge tube. Rinse the cell sieve with pre-cooled 1× PBS in advance.

[0084] 6. Centrifuge the filtrate at 310 g for 5 min at 4 °C and slowly discard the supernatant;

[0085] 7. Add a small amount of pre-cooled 1× PBS to resuspend the cells, then take 10 μl of the cell suspension and mix it with AOPI staining solution in a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 4 A and B in the figure), the cell viability and aggregation rate are shown in Table 1. Then, according to the cell count, an appropriate amount of 1×PBS solution containing calcium and magnesium ions was added to the centrifuge tube to adjust the cell concentration to 1×10 6 ~1×10 7 Between pc / mL;

[0086] 8. Dilute the defragmentation reagent by 25% volume using 1× PBS buffer containing calcium and magnesium ions, and then take the diluted defragmentation reagent with the same volume as the cell suspension into a new 15 mL centrifuge tube;

[0087] 9. Use a Pasteur pipette to slowly drip the cell suspension with adjusted cell concentration onto the top of the de-fragmentation reagent along the tube wall, while ensuring that the cell suspension and the de-fragmentation reagent do not interfere with each other to form a clear two-phase gradient centrifugation. The density gradient centrifugation conditions are set to a centrifugal force of 800 g, a temperature of 4 °C, centrifugation for 15 min, and both the speed increase and speed decrease are set to 1.

[0088] 10. Remove the upper debris, add 200-500 μl of 1× PBS solution containing 10% FBS to resuspend the sediment at the bottom of the centrifuge tube to obtain the purified mouse skin tissue single cell suspension. Similarly, take 10 μl of the cell suspension and mix it with AOPI staining solution at a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 4 (a, b) Cell viability and aggregation rate are shown in Table 1.

[0089] Use the fragment removal product purchased from the market, perform the experiment according to the above steps, use a cell counter to count and take pictures, the results are as follows Figure 4 As shown in ① and ②.

[0090] Example 5 Experiment on preparation of single cell suspension of mouse intestinal tissue

[0091] The digestion solution used in this experiment was prepared independently, and the other reagents were common chemical reagents, among which 1×PBS solution did not contain calcium and magnesium ions unless otherwise specified. The formula of the digestion solution was the same as that of Example 2.

[0092] 1. Dissect and obtain 50-100 mg of mouse intestinal tissue and place it in a sterile culture dish. Use forceps to filter out the intestinal contents and rinse them with 1× PBS solution.

[0093] 2. Use a pre-cooled surgical blade to cut the intestinal tissue into 1 mm 3 The small tissue pieces on the left and right were rinsed again with 1× PBS solution, and the turbid washing solution was aspirated to leave the tissue pieces;

[0094] 3. Transfer the chopped tissue pieces to a 15 ml centrifuge tube, add 2 ml of digestion solution and mix thoroughly, then place in a 37°C shaker for 15 min. The shaker speed is set to 80 rpm / min. During this period, use a Pasteur pipette to fully disperse the digestion solution every 5 min.

[0095] 4. After digestion, remove the centrifuge tube and add an equal volume of pre-cooled 1× PBS solution to terminate digestion;

[0096] 5. Filter the cell-enzyme mixture through a 40 μm cell sieve into a new 50 ml centrifuge tube. Centrifuge the filtrate at 300 g for 5 min at 4 °C and discard the supernatant.

[0097] 6. Add a small amount of pre-cooled 1× PBS to resuspend the cells, then take 10 μl of the cell suspension and mix it with AOPI staining solution in a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 5 A and B in the figure), the cell viability and aggregation rate are shown in Table 1. Then, according to the cell count, an appropriate amount of 1×PBS solution containing calcium and magnesium ions was added to the centrifuge tube to adjust the cell concentration to 1×10 6~1×10 7 Between pc / mL;

[0098] 7. Dilute the defragmentation reagent by 25% volume using 1× PBS buffer containing calcium and magnesium ions, and then take the diluted defragmentation reagent with the same volume as the cell suspension into a new 15 mL centrifuge tube;

[0099] 8. Use a Pasteur pipette to slowly drip the cell suspension with adjusted cell concentration onto the top of the de-fragmentation reagent along the tube wall, while ensuring that the cell suspension and the de-fragmentation reagent do not interfere with each other to form a clear two-phase gradient centrifugation. The density gradient centrifugation conditions are set to a centrifugal force of 800 g, a temperature of 4 °C, centrifugation for 15 min, and both the speed increase and speed decrease are set to 1;

[0100] 9. Remove the upper debris, add 200-500 μl of 1× PBS solution containing 10% FBS to resuspend the sediment at the bottom of the centrifuge tube to obtain the purified mouse intestinal tissue single cell suspension. Similarly, take 10 μl of the cell suspension and mix it with AOPI staining solution at a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 5 (a, b) Cell viability and aggregation rate are shown in Table 1.

[0101] Use the fragment removal product purchased from the market, perform the experiment according to the above steps, use a cell counter to count and take pictures, the results are as follows Figure 5 As shown in ① and ②.

[0102] Example 6 Experiment on preparation of single cell suspension of mouse muscle tissue

[0103] The digestion solution used in this experiment was prepared independently, and the other reagents were common chemical reagents, among which 1×PBS solution did not contain calcium and magnesium ions unless otherwise specified. The formula of the digestion solution was the same as that of Example 2.

[0104] 1. Dissect the mouse and take 300-500 mg of leg muscle tissue and place it in a sterile culture dish. Scrape off the surface fat, rinse with 1× PBS solution, and mince the tissue block with a pre-cooled surgical blade;

[0105] 2. Transfer the chopped tissue pieces to a 15 ml centrifuge tube, add 2 ml of digestion solution and mix thoroughly, then place in a 37°C shaker for 15 min. The shaker speed is set to 100 rpm / min. During this period, take out the centrifuge tube every 5 min and use a Pasteur pipette to blow away the digestion solution.

[0106] 3. After digestion, take out the centrifuge tube, add an equal volume of pre-cooled 1× PBS solution, use a Pasteur pipette to mix well to terminate digestion, and then let it stand to precipitate the undissociated tissue;

[0107] 4. Pipette the turbid cell-enzyme mixture on the top and filter it through a 40 μm cell sieve into a new 50 ml centrifuge tube. Immediately centrifuge the filtrate at 300 g for 5 min at 4 °C and slowly pour off the supernatant.

[0108] 5. Add a small amount of pre-cooled 1× PBS to resuspend the cells, then take 10 μl of the cell suspension and mix it with AOPI staining solution in a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 6 A and B in the figure), the cell viability and aggregation rate are shown in Table 1. Then, according to the cell count, an appropriate amount of 1×PBS solution containing calcium and magnesium ions was added to the centrifuge tube to adjust the cell concentration to 1×10 6 ~1×10 7 Between pc / mL;

[0109] 6. Dilute the defragmentation reagent by 25% volume using 1× PBS buffer containing calcium and magnesium ions, and then take the diluted defragmentation reagent with the same volume as the cell suspension into a new 15 mL centrifuge tube;

[0110] 7. Use a Pasteur pipette to slowly drip the cell suspension with adjusted cell concentration onto the top of the de-fragmentation reagent along the tube wall, while ensuring that the cell suspension and the de-fragmentation reagent do not interfere with each other to form a clear two-phase gradient centrifugation. The density gradient centrifugation conditions are set to a centrifugal force of 800 g, a temperature of 4 °C, centrifugation for 15 min, and both the speed increase and speed decrease are set to 1;

[0111] 8. Remove the upper debris, add 200-500 μl of 1× PBS solution containing 10% FBS to resuspend the sediment at the bottom of the centrifuge tube to obtain the purified mouse muscle tissue single cell suspension. Similarly, take 10 μl of the cell suspension and mix it with AOPI staining solution at a volume ratio of 1:1, then use a cell counter to count and take pictures ( Figure 6 (a, b) Cell viability and aggregation rate are shown in Table 1.

[0112] Use the fragment removal product purchased from the market, perform the experiment according to the above steps, use a cell counter to count and take pictures, the results are as follows Figure 6 As shown in ① and ②.

[0113] Table 1 Cell viability and aggregation rate

[0114] ;

[0115] As shown in Table 1, compared with not using the defragmentation reagent of the present invention, the cell viability is significantly increased and the cell agglomeration rate is significantly reduced after using the defragmentation reagent of the present invention, and the most significant is the mouse muscle cells. After using the defragmentation reagent, the cell viability increases by 86% and the cell agglomeration rate decreases by nearly 70%.

[0116] Compared with existing defragmentation reagents, after using the defragmentation reagent provided in the present invention, especially in tissues such as mouse trachea, mouse whole brain, mouse skin and mouse muscle, the cell viability is significantly improved and the cell clumping rate is significantly reduced, indicating that the reagent for removing cell agglomerates and fragments in single-cell suspensions in the present invention has excellent effect, and can significantly remove cell agglomerates and fragments generated in the process of dissociating various animal tissues into single-cell suspensions. Compared with existing defragmentation reagents, it has a wider application range, thereby obtaining a single-cell suspension with highly active cells and minimal fragments, laying the foundation for subsequent single-cell sequencing.

[0117] Therefore, the present invention adopts the above-mentioned reagent for removing cell clumps and fragments in a single-cell suspension and its application, which can significantly remove the cell clumps and fragments generated during the dissociation of animal tissue into a single-cell suspension, thereby obtaining a single-cell suspension with highly active cells and minimal fragments, laying the foundation for subsequent single-cell sequencing.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A reagent for removing cell clumps and fragments in a single cell suspension, characterized in that: Includes Percoll stock solution, 0.85% w / v NaCl, 0.5% w / v EDTA, 0.5-2.5 mg / mL ADH-1 trifluoroacetate, and 5.0-8.5 mg / mL trypsin inhibitor.

2. The reagent according to claim 1, characterized in that: Includes Percoll stock solution, 0.85% w / v NaCl, 0.5% w / v EDTA, 1.5 mg / mL ADH-1 trifluoroacetate, and 7.0 mg / mL trypsin inhibitor.

3. Use of the reagent as claimed in claim 1 or 2 in removing cell clumps and fragments during the dissociation of animal tissue into single cell suspension.

4. A method for removing cell clumps and fragments from a single cell suspension, characterized in that: The following steps are involved: S1. Collect the single cell suspension from which the debris needs to be removed into a centrifuge tube, centrifuge at 300-500×g, 4°C for 3-5 min, then slowly pour off the supernatant to obtain the cell pellet; S2. Add an appropriate amount of 1× PBS solution to the centrifuge tube and gently use a Pasteur pipette to resuspend the cell pellet so that the cell concentration is 1×10 6 ~1×10 7 Pieces / mL; S3, diluting the reagent as claimed in claim 1 by 20% to 30% by volume using 1×PBS buffer containing calcium and magnesium ions, and then taking the diluted reagent into a new centrifuge tube; S4. Use a Pasteur pipette to slowly drip the single cell suspension with adjusted cell concentration onto the upper part of the reagent along the tube wall, while ensuring that the cell suspension and the reagent do not interfere with each other, forming a clear two-phase gradient centrifugation; S5. Remove the upper fragments and add an appropriate amount of 1×PBS solution containing 10% FBS to resuspend the precipitate at the bottom of the centrifuge tube to obtain the purified single cell suspension.

5. The method according to claim 4, characterized in that: In step S3, the reagent is diluted at a volume ratio of 25%.

6. The method according to claim 4, characterized in that: In step S4, the cell suspension accounts for 30-50% of the total volume of the reagent and the cell suspension.

7. The method according to claim 6, characterized in that: In step S4, the cell suspension accounts for 50% of the total volume of the reagent and the cell suspension.

8. The method according to claim 6, characterized in that: In step S4, the gradient centrifugation conditions were set to 800×g, centrifugation at 4°C for 20 min, and the acceleration and deceleration speeds were set to 1.

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