A single cell suspension dissociation kit for mouse colorectal tissue and its application

Abstract

The present invention discloses a dissociation kit for mouse colorectal single cell suspension, which is composed of the following components: a first enzymatic digestion solution, a second enzymatic digestion solution, a third enzymatic digestion solution, a PBS buffer solution, an RPMI-1640 culture medium, and an erythrocyte lysate; the first enzymatic digestion solution is a 20 mM EDTA solution; the second enzymatic digestion solution is a 0.25% trypsin-EDTA solution; the concentration of neutral protease in the third enzymatic digestion solution is 100 mg / mL, the concentration of collagenase I is 100 mg / mL, and the concentration of collagenase II is 100 mg / mL; a method for extracting mouse colorectal single cell suspension using the dissociation kit is also disclosed. The reagent materials used in the dissociation solution in the kit are easy to obtain, contain no harmful components, are safe and environmentally friendly, the method is simple to operate, and the final result after the cooperation of each step has high repeatability and success rate, and can stably obtain a mouse colorectal single cell suspension with high viability and low impurities.

Description

Technical Field

[0001] The present invention belongs to the technical field of single cell technology, and particularly relates to a single cell suspension dissociation kit for mouse colorectal tissue and its application. Background Art

[0002] The colorectum is an important part of the large intestine. The colon is subdivided into the ascending colon, transverse colon, descending colon, and sigmoid colon, which mainly help the human body absorb water and inorganic salts, provide a temporary storage place for food residues after digestion and absorption, and convert food into feces. In addition, glandular cells in the colon secrete mucus to form an intestinal mucus layer to protect the inner wall of the colon from the stimulation of digestive enzymes and other harmful substances. The rectum is a relatively short section at the end of the colon, and its main function is to store feces. The colorectum is mainly composed of a mucosal layer, a cortical layer, a muscle layer, a lamina propria, and a serosa layer, which together help promote the absorption of water and inorganic salts and generate and store feces.

[0003] In single cell research, the preparation of single cell suspension is crucial for the smooth progress of subsequent experiments and the quality of downstream data. In the existing mouse colorectal single cell suspension preparation protocols, there are often problems such as low cell viability, high aggregation rate, a large number of cell debris, and a large number of background impurities, which affect subsequent experiments and ultimately affect the data quality. Therefore, based on this, the present invention scheme is proposed. Summary of the Invention

[0004] In view of the current technical problems, the present invention provides a single cell suspension dissociation kit for mouse colorectal tissue and its application. Using the kit in the technical solution of the present invention to isolate mouse colorectum, a single cell suspension with high viability and low background impurities can be obtained.

[0005] To achieve the above object, the present invention adopts the following technical means:

[0006] The first aspect of the present invention provides a dissociation kit for mouse colorectal single cell suspension, which is composed of the following components: a first enzymatic digestion solution, a second enzymatic digestion solution, a third enzymatic digestion solution, a PBS buffer solution, an RPMI-1640 medium, and an erythrocyte lysate;

[0007] The first enzymatic digestion solution is an EDTA-PBS solution with a concentration of 20 mM;

[0008] The second enzymatic digestion solution is a 0.25% trypsin-EDTA solution;

[0009] The third enzymatic digestion solution is prepared by dissolving neutral protease, collagenase I, and collagenase II in a medium, wherein the concentration of neutral protease is 100 mg / mL, the concentration of collagenase I is 100 mg / mL, and the concentration of collagenase II is 100 mg / mL.

[0010] In some embodiments of the present invention, the storage temperature of the first enzymatic hydrolysate, the second enzymatic hydrolysate, and the third enzymatic hydrolysate is -20°C.

[0011] In some embodiments of the present invention, when the first enzymatic hydrolysate is used, it is diluted to the working concentration with PBS, and when the second enzymatic hydrolysate and the third enzymatic hydrolysate are used, they are diluted to the working concentration with RPMI-1640 medium solution.

[0012] The second aspect of the present invention provides a method for preparing a single-cell suspension of mouse colorectal tissue using the dissociation kit described in the first aspect, comprising the following steps:

[0013] S1. Reagent preparation: Place the refrigerated first enzymatic hydrolysate, second enzymatic hydrolysate, and third enzymatic hydrolysate on ice for thawing. Dilute the first enzymatic hydrolysate to the working concentration with PBS; dilute the second enzymatic hydrolysate and the third enzymatic hydrolysate to the working concentration with RPMI-1640 medium.

[0014] S2. Sample preparation: Take mouse colorectal tissue without blood stains and impurities and place it in sample tube a. Process it into pieces on ice and then wash it repeatedly with PBS solution until clean.

[0015] S3. First digestion: Add 2-4 ml of the first dissociation solution to sample tube a, digest it by constant temperature water bath shaking. After taking it out, aspirate all the solution in sample tube a through a cell sieve. The remaining tissue on the sieve is put into sample tube b for standby, and the filtrate after sieving is put into sample tube c and centrifuged to discard the supernatant. Resuspend the cell pellet with PBS, centrifuge again to discard the supernatant, and then resuspend the cell pellet with RPMI-1640 medium to obtain cell suspension A.

[0016] S4. Second digestion: Add 2-4 mL of the second dissociation solution to sample tube c in S3, digest it by constant temperature water bath shaking. After taking it out, pass all the solution in sample tube c through a cell sieve. The filtrate after sieving is centrifuged to discard the supernatant, and the cell pellet is resuspended with RPMI-1640 medium to obtain cell suspension B.

[0017] S5. Third digestion: Add 2-4 mL of the third dissociation solution to sample tube b in S3, digest it by constant temperature water bath shaking. After taking it out, aspirate all the solution in sample tube b through a cell sieve. The filtrate after sieving is centrifuged to discard the supernatant, and the cell pellet is resuspended with RPMI-1640 medium to obtain cell suspension C.

[0018] S6. Red blood cell lysis: Combine cell suspension B and cell suspension C into sample tube d, add 5-10 ml of red blood cell lysis solution, lyse it at room temperature and then centrifuge to discard the supernatant.

[0019] S7. Single-cell suspension: Add PBS solution to sample tube d, pipette and mix well, centrifuge to discard the supernatant, and resuspend the cell pellet with RPMI-1640 medium to obtain the single-cell suspension.

[0020] In some embodiments of the present invention, in the first digestion step, the working concentration of the first dissociation solution is 10-20 mM.

[0021] The digestion method is as follows: place it in a water bath thermostatic shaker, shake and digest at 37°C and 120 rpm for 8-12 min; in some preferred embodiments, the working concentration of the first dissociation solution is 20 mM and the digestion is carried out for 10 min.

[0022] In some embodiments of the present invention, in the second digestion step, the working concentration of the second dissociation solution is 0.1%-0.25%.

[0023] The digestion method is as follows: place it in a water bath thermostatic shaker, shake and digest at 37°C and 120 rpm for 1-3 min; in some preferred embodiments, the working concentration of the second dissociation solution is 0.25% and the digestion is carried out for 2 min.

[0024] In some embodiments of the present invention, in the third digestion step, the working concentration of the third dissociation solution is 0.5-2 mg / mL of collagenase I, 0.5-2 mg / mL of collagenase II, and 0.5-2 mg / mL of neutral protease, and it is diluted with RPMI-1640 solution.

[0025] The digestion method is as follows: place it in a water bath thermostatic shaker, shake and digest at 37°C and 120 rpm for 8-12 min; in some preferred embodiments, the working concentration of the third dissociation solution is 1 mg / mL of collagenase I, 1 mg / mL of collagenase II, and 1 mg / mL of neutral protease; the digestion is carried out for 10 min.

[0026] In some embodiments of the present invention, in steps S3, S4, S5, and S7, the centrifugation condition is centrifugation at 400 g and 4°C; in step S6, the centrifugation condition is centrifugation at 300 g and 4°C; in steps S3, S4, and S5, the cell sieve is a 30 μm cell sieve.

[0027] The third aspect of the present invention provides a mouse colorectal single-cell suspension prepared by the method described in the second aspect.

[0028] The fourth aspect of the present invention provides the application of the mouse colorectal single-cell suspension described in the third aspect in single-cell detection. Such as applications in single-cell sequencing, single-cell sorting and detection, etc.

[0029] Advantages of the present invention

[0030] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a mouse colorectal single-cell suspension dissociation kit. The reagent materials used in the dissociation solution in this kit are easy to obtain, contain no harmful components, and are safe and environmentally friendly. The method for preparing a mouse colorectal single-cell suspension using this kit is simple to operate. It adopts a multi-step digestion method, and each operation step affects each other. The final result after coordination has a very high success rate. The obtained cell concentration is above 1×10 6 cells / mL, and the cell viability reaches more than 91%. Using this method, a mouse colorectal single-cell suspension with high viability and low impurities can be stably obtained, which can be effectively applied in single-cell sequencing and other single-cell detection aspects, and can meet the standard that the viability of the cell suspension for loading on the 10x Genomics platform reaches more than 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Shows the fluorescence field picture of the cell suspension in Example 1 of the present invention;

[0032] Figure 2 Shows the bright field picture of the cell suspension in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The following examples are used herein to demonstrate the preferred embodiments of the present invention. Those skilled in the art will understand that the technologies disclosed in the following examples represent the technologies that the inventor has found can be used to implement the present invention, and thus can be regarded as the preferred solutions for implementing the present invention. However, those skilled in the art should understand from this specification that the specific embodiments disclosed here can be modified in many ways and still obtain the same or similar results without departing from the spirit or scope of the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The materials cited herein and their citations will be incorporated by reference. Those skilled in the art will realize or can learn through routine experiments many equivalent technologies of many specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0035] The technical solutions of this patent will be further described in detail below in conjunction with the specific embodiments.

[0036] Example 1

[0037] (1) Reagent preparation: Thaw the refrigerated first digestion solution, second digestion solution, and third digestion solution on ice. The working concentration of the first digestion solution is 20 nM, the working concentration of the second digestion solution is 0.25%, and the third digestion solution is diluted to the working concentration with RPMI-1640 medium: 3 mL of the third dissociation solution contains 1 mg / mL of neutral protease, 1 mg / mL of collagenase I, and 1 mg / mL of collagenase II;

[0038] (2) Sample preparation: Take the mouse colorectal tissue into a culture dish, add PBS solution to the culture dish, pick up the tissue with forceps, and wash away blood stains and impurities; Weigh 200 mg of the washed mouse colorectal tissue into a culture dish, add PBS, and use an ophthalmic scissors to cut the tissue into pieces about 0.5 - 3 mm 2 in size on an ice plate; Then transfer the tissue to a 5 mL centrifuge tube and cut it into pieces about 1 mm 2 in size; Add PBS solution to the 5 mL centrifuge tube to about 4 mL, pipette several times, discard the supernatant, which is regarded as one wash, and wash 3 times in total. Finally, aspirate and discard the supernatant completely;

[0039] (3) First digestion: Add 3 mL of the first digestion solution: 20 mM EDTA-PBS solution to the centrifuge tube containing the tissue, place it in a 37 °C, 120 rpm water bath constant temperature shaker for digestion for 10 min; Take out the centrifuge tube from the water bath, aspirate all the tissue onto a 30 μm cell sieve, rinse the tissue on the sieve with 3 mL of PBS 2 times, and transfer the tissue on the sieve to a 5 mL centrifuge tube for standby; Centrifuge the sieving solution at 400 g, 4 °C for 5 min; After centrifugation, discard the supernatant, resuspend the cell pellet with 10 mL of PBS, centrifuge at 400 g, 4 °C for 5 min; After centrifugation, discard the supernatant, and resuspend the cell pellet with 100 μL of RPMI-1640 medium to obtain cell suspension A;

[0040] (4) Second digestion: Add 3 mL of the second digestion solution: 0.25% trypsin-EDTA solution to cell suspension 1, place it in a 37 °C, 120 rpm water bath constant temperature shaker for digestion for 2 min; Take out the sample tube, aspirate all the solution through a 30 μm cell sieve, centrifuge the sieving filtrate at 400 g, 4 °C for 5 min, then discard the supernatant, and resuspend the cell pellet with 100 μL of RPMI-1640 medium to obtain cell suspension B;

[0041] (5) Third digestion: Add 3 mL of the pre-prepared third enzyme digestion solution to the remaining tissue from the first digestion: 1 mg / mL neutral protease, 1 mg / mL collagenase I, 1 mg / mL collagenase II. Place it in a water bath thermostatic shaker at 37°C and 120 rpm for digestion for 10 min. Take out the 5 mL centrifuge tube from the water bath, aspirate all the tissue onto a 30 μm cell sieve, and rinse the tissue on the sieve with 3 mL of PBS twice. Centrifuge the filtered solution at 400 g and 4°C for 5 min. After centrifugation, discard the supernatant, and resuspend the cell pellet with 100 μL of RPMI-1640 medium to obtain cell suspension C;

[0042] (6) Red blood cell lysis: Combine cell suspension B and cell suspension C, add 5 mL of red blood cell lysis solution, lyse at room temperature for 5 min, centrifuge at 300 g and 4°C for 5 min, and discard the supernatant after centrifugation;

[0043] (7) Single cell suspension: Add 5 mL of PBS solution and pipette to mix evenly, centrifuge at 400 g and 4°C for 5 min. After centrifugation, discard the supernatant, and resuspend the cell pellet with an appropriate amount of RPMI-1640 medium to obtain the final cell suspension.

[0044] Example 2

[0045] All other operation procedures are exactly the same as those in Example 1, and a parallel experiment is conducted.

[0046] Example 3

[0047] All other operation procedures are exactly the same as those in Example 1, and a parallel experiment is conducted.

[0048] Comparative Example 1

[0049] All other operation procedures are the same as those in Example 1, except that:

[0050] The 3 mL of the third dissociation solution contains 0.2 mg / mL of neutral protease, 1 mg / mL of collagenase I, and 1 mg / mL of collagenase II.

[0051] Comparative Example 2

[0052] All other operation procedures are the same as those in Example 1, except that:

[0053] The 3 mL of the third dissociation solution contains 0.1 mg / mL of elastase, 1 mg / mL of collagenase I, and 1 mg / mL of collagenase II.

[0054] Comparative Example 3

[0055] All other operation procedures are the same as those in Example 1, except that:

[0056] The 3 mL third dissociation solution contains 50 u / mL hyaluronidase, 1 mg / mL collagenase I, and 1 mg / mL collagenase II.

[0057] Comparative Example 4

[0058] The other operation procedures are the same as those in Example 1, except that:

[0059] The 3 mL third dissociation solution contains 1 mg / mL neutral protease, 0.1 mg / mL elastase, and 1 mg / mL collagenase II.

[0060] Comparative Example 5

[0061] The other operation procedures are the same as those in Example 1, except that:

[0062] The 3 mL third dissociation solution contains 1 mg / mL neutral protease, 50 u / mL hyaluronidase, and 1 mg / mL collagenase II.

[0063] Comparative Example 6

[0064] The other operation procedures are the same as those in Example 1, except that:

[0065] The 3 mL third dissociation solution contains 1 mg / mL neutral protease, 1 mg / mL collagenase I, and 0.1 mg / mL elastase.

[0066] Comparative Example 7

[0067] The other operation procedures are the same as those in Example 1, except that:

[0068] The 3 mL third dissociation solution contains 1 mg / mL neutral protease, 1 mg / mL collagenase I, and 50 u / mL hyaluronidase.

[0069] Comparative Example 8

[0070] The other operation procedures are the same as those in Example 1, except that: the 3 mL third dissociation solution contains 1 mg / mL collagenase I and 50 u / mL hyaluronidase.

[0071] Comparative Example 9

[0072] The other operation procedures are the same as those in Example 1, except that: in the third digestion step, the digestion time is 30 min.

[0073] Comparative Example 10

[0074] The other operation procedures are the same as those in Example 1, except that: in the first digestion step, the digestion temperature is 4°C.

[0075] Comparative Example 11

[0076] The other operation procedures are the same as those in Example 1, except that in the first digestion step, the first enzymatic hydrolysate is replaced with 1 mM DTPA.

[0077] Comparative Example 12

[0078] The other operation procedures are the same as those in Example 1, except that in the first digestion step, the first enzymatic hydrolysate is an EDTA-PBS solution with a concentration of 40 nM.

[0079] Comparative Example 13

[0080] The other operation procedures are the same as those in Example 1, except that in the second digestion step, the second enzymatic hydrolysate is replaced with 0.25% papain.

[0081] Comparative Example 14

[0082] The other operation procedures are the same as those in Example 1, except that in the second digestion step, the second enzymatic hydrolysate is a trypsin-EDTA solution with a concentration of 0.5%.

[0083] Comparative Example 15

[0084] The other operation procedures are the same as those in Example 1, except that the steps of digesting with the first enzymatic hydrolysate and the second enzymatic hydrolysate are omitted.

[0085] Single cell suspensions were prepared respectively by using the preparation methods of the above Examples 1-3 and Comparative Examples 1-15. The tissue single cell suspensions prepared were subjected to AO / PI fluorescence staining and counting. The results of the Countstar cell counter are shown in Table 1. Among them, the cell counting diagram of the single cell suspension prepared in Example 1 is shown in Figure 1 and Figure 2 .

[0086] Table 1 Counting results of tissue single cell suspensions prepared by different experimental methods

[0087]

[0088]

[0089] The results show that: As can be seen from the comparison of the results in Table 1 above, the cell viability of Examples 1 to 3 is higher than that of the comparative examples, and the aggregation rate is lower than that of the comparative examples. When cells are on the verge of death, they will release DNA, which will make the surrounding of the cells more viscous and form aggregates. Therefore, the cell viability is high and the aggregation rate is low. The results of Examples 1 to 3 show that the single-cell suspensions obtained from three parallel experiments can all meet the requirements of single-cell sequencing technology for a cell viability exceeding 85%, and at the same time also meet the standard of a cell suspension viability of more than 90% for the 10x Genomics platform. However, the cell viability of Comparative Examples 1 to 15 is low and the aggregation rate is relatively high, which cannot meet the requirements of the 10x Genomics platform for loading.

[0090] It can be seen from Comparative Examples 1 to 8 that changes in the composition of neutral protease, collagenase type I and collagenase type II in the digested enzyme solution or changes in the concentration of neutral protease will not only result in fewer digested cells, but also a decrease in cell viability and an increase in the aggregation rate; it can be seen from Comparative Example 9 that the digestion time for the third digestion is too long. Although the number of digested cells increases, the final cell viability is low; it can be seen from Comparative Example 10 that the temperature for the first digestion is too low, resulting in fewer digested cells.

[0091] It can be seen from Comparative Examples 11 - 15 that the enzymes and enzyme dosages for the first digestion and the second digestion have a huge impact on the subsequent third digestion. Arbitrary changes will cause a decrease in the final cell viability and an increase in the aggregation rate.

[0092] As can be seen from the above comparative examples, in the technical solution of the present invention, the functions of each step influence each other. As a whole, they interact with each other, and finally result in a high cell viability and a low aggregation rate of the mouse colorectal single-cell suspension obtained. In the preparation method of the present invention, the composition of the enzymes in the dissociation solution, the digestion time, digestion temperature and digestion times in the digestion step are very important for the preparation of the single-cell suspension, and will significantly affect the cell concentration and cell viability of the finally obtained suspension.

[0093] The single-cell suspension prepared by using the method of the present invention, Figure 1 shows a high cell viability from the fluorescence field pictures; Figure 2 it can be seen from the bright field pictures that the background is clean and the number of cell debris and residual red blood cells is small.

[0094] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the present application.

Claims

1. A method for preparing a single-cell suspension of mouse colorectal tissue, characterized in that, It includes the following steps: S1. Reagent preparation: Thaw the refrigerated first digestion solution, second digestion solution, and third digestion solution on ice. Dilute the first digestion solution to the working concentration with PBS; dilute the second digestion solution and the third digestion solution to the working concentration with RPMI-1640 medium; S2. Sample preparation: Take mouse colorectal tissue without blood stains and impurities into sample tube a, process it into pieces on ice, and repeatedly wash it with PBS solution until clean; S3. First digestion: Add 2 - 4 ml of the first dissociation solution to sample tube a, perform digestion by shaking in a constant temperature water bath, after taking it out, aspirate all the solution in sample tube a through a cell sieve, put the remaining tissue on the sieve into sample tube b for standby, centrifuge the filtrate passed through the sieve in sample tube c and discard the supernatant, resuspend the cell pellet with PBS, centrifuge again and discard the supernatant, then resuspend the cell pellet with RPMI-1640 medium to obtain cell suspension A; S4. Second digestion: Add 2 - 4 mL of the second dissociation solution to cell suspension A in sample tube c of S3, perform digestion by shaking in a constant temperature water bath; after taking it out, pass all the solution in sample tube c through a cell sieve, centrifuge the filtrate passed through the sieve and discard the supernatant, resuspend the cell pellet with RPMI-1640 medium to obtain cell suspension B; S5. Third digestion: Add 2 - 4 mL of the third dissociation solution to the remaining tissue in sample tube b of S3, perform digestion by shaking in a constant temperature water bath; after taking it out, aspirate all the solution in sample tube b through a cell sieve, centrifuge the filtrate passed through the sieve and discard the supernatant, resuspend the cell pellet with RPMI-1640 medium to obtain cell suspension C; S6. Red blood cell lysis: Combine cell suspension B and cell suspension C into sample tube d, add 5 - 10 ml of red blood cell lysis solution, perform lysis at room temperature, then centrifuge and discard the supernatant; S7. Single cell suspension: Add PBS solution to sample tube d, pipette and mix well, centrifuge and discard the supernatant, resuspend the cell pellet with RPMI-1640 medium to obtain a single cell suspension; In the first digestion step, the working concentration of the first dissociation solution is an EDTA solution with a concentration of 10 - 20 mM, and the digestion method is: Place it in a water bath constant temperature shaker, and shake and digest at 37 °C and 120 rpm for 8 - 12 min; In the second digestion step, the working concentration of the second dissociation solution is a 0.1 - 0.25% trypsin-EDTA solution, and the digestion method is: Place it in a water bath constant temperature shaker, and shake and digest at 37 °C and 120 rpm for 1 - 3 min; In the third digestion step, the working concentration of the third dissociation solution is collagenase I 0.5 - 2 mg / mL, collagenase II 0.5 - 2 mg / mL, and neutral protease 0.5 - 2 mg / mL, and the digestion method is: Place it in a water bath constant temperature shaker, and shake and digest at 37 °C and 120 rpm for 8 - 12 min.

2. The method for preparing a single-cell suspension of mouse colorectal tissue according to claim 1, characterized in that, In steps S3, S4, S5, and S7, the centrifugation condition is centrifugation at 400 g and 4 °C; in step S6, the centrifugation condition is centrifugation at 300 g and 4 °C; in steps S3, S4, and S5, the cell sieve is a 30 μm cell sieve.

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