Enzymatic hydrolysate, kit, method and application thereof for preparing single cell suspension of human arterial tissue

Abstract

The invention discloses an enzymatic hydrolysis solution, a kit, a method and an application thereof for preparing a single cell suspension of human arterial tissue. The enzymatic hydrolysis solution comprises a first enzymatic hydrolysis solution and a second enzymatic hydrolysis solution, wherein the first enzymatic hydrolysis solution comprises 80-120 U / ml hyaluronidase and 30-80 U / ml pronase; the second enzymatic hydrolysis solution comprises 1-3 mg / ml collagenase II, 0.3-0.8 mg / ml dispase and 0.05-0.1 mg / ml elastase; and a kit and an enzymatic hydrolysis method prepared by using the enzymatic hydrolysis solution. The collagenase contained in the enzymatic hydrolysis solution is a commonly used dissociation enzyme, which is easy to obtain. The enzymatic hydrolysis solution ratio is suitable for single cell dissociation of human arterial tissue, the reagent components have good compatibility, are free of harmful components, and are safe and environmentally friendly. The dissociation scheme provided by the invention can obtain a high-quality single cell suspension, and can greatly improve the feasibility of single cell sequencing in arterial tissue.

Description

Technical Field

[0001] The invention belongs to the field of single cell technology, and specifically relates to an enzymatic hydrolysate, a kit, a method and applications thereof for preparing a single cell suspension of human arterial tissue. Background Art

[0002] Single-cell transcriptome sequencing is a new technology for amplifying and sequencing transcriptomes at the single-cell level. The principle is to amplify the trace whole transcriptome RNA of isolated single cells and then perform high-throughput sequencing. It is a tool for studying cell types, dynamics and functional processes in complex tissues. This technology has been widely used in the study of different types of tissues in animals and plants. By sequencing the gene expression of single cells, we can better study the cell differentiation and development of different types of tissues. There have been many successful application cases in research fields such as cancer biology, immunology and clinical diagnosis and treatment. Using single-cell transcriptome sequencing technology to screen cancer cells or specific immune cells, and comparing and analyzing the expression of key genes and signal pathways in normal cells, we can have a deeper understanding of the mechanism of disease occurrence and provide a theoretical basis for subsequent corresponding treatment plans.

[0003] Blood vessels are the channels for biological blood transport. They can be divided into arteries, veins and capillaries according to the direction of transport. In addition to the cornea, hair, nails and epithelium, the human body has dense blood vessels. Arterial and venous blood vessels are composed of three layers, rich in connective tissue, smooth muscle, elastic fibers and collagen fibers. When preparing single-cell suspensions, collagenase or trypsin is needed to destroy the proteins that tightly connect cells, so as to obtain high-quality single-cell suspensions. However, the type of collagenase used in the dissociation process will affect the acquisition of high-quality cell suspensions.

[0004] Exploring cell-specific changes in blood vessels, providing support for finding potential cellular targets for treating related diseases, and significantly improving the feasibility of single-cell sequencing in arterial tissue, it is crucial to obtain high-quality cell suspensions of arterial tissue. Therefore, it is necessary to provide a single-cell dissociation protocol for human arterial tissue. Summary of the invention

[0005] In order to solve one of the above problems, the present invention provides an enzymatic hydrolysis solution, a kit, a method and applications thereof for preparing a single cell suspension of human arterial tissue. The enzymatic hydrolysis solution and the dissociation method can fully digest human arterial tissue to obtain a high-quality cell suspension.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] The first aspect of the present invention provides an enzymatic hydrolysis solution for preparing a single cell suspension of human arterial tissue, comprising a first enzymatic hydrolysis solution and a second enzymatic hydrolysis solution, wherein the first enzymatic hydrolysis solution comprises 80-120 U / ml hyaluronidase and 30-80 U / ml pronase;

[0008] The second enzymatic solution includes 1-3 mg / ml collagenase II, 0.3-0.8 mg / ml dispase and 0.05-0.1 mg / ml elastase.

[0009] Preferably, the first enzymatic solution comprises 100 U / ml hyaluronidase and 50 U / ml pronase;

[0010] The second enzymatic solution includes 2 mg / ml collagenase II, 0.5 mg / ml dispase and 0.08 mg / ml elastase.

[0011] The second aspect of the present invention provides a use of the enzymatic hydrolysis solution described in the first aspect in a single cell dissociation kit for preparing human arterial tissue.

[0012] The third aspect of the present invention provides a kit for preparing a single-cell suspension of human arterial tissue, comprising the enzymatic hydrolysis solution described in the first aspect.

[0013] In some specific embodiments of the present invention, a washing buffer, a dilution buffer and a red blood cell lysis solution are also included; wherein the washing buffer is a PBS solution; and the dilution buffer is a 1640 culture medium.

[0014] The fourth aspect of the present invention provides a method for preparing a single cell suspension of human arterial tissue, wherein the single cell suspension of human arterial tissue is dissociated using the kit described in the third aspect, and the dissociation method comprises the following steps:

[0015] S1. Take a human arterial tissue sample and place it in a vessel containing washing buffer and cut it into pieces. The cut tissue block sample is placed in sample tube a and repeatedly washed with washing buffer;

[0016] S2, add the first enzymatic solution into sample tube a, seal it with sealing film and perform the first digestion;

[0017] S3, rinse the tissue and cells with washing buffer, and draw the cell suspension in S2 through the cell sieve, and sieve, centrifuge the cell filtrate and discard the supernatant; place the cell tissue on the sieve into sample tube b for later use;

[0018] S4, add dilution buffer to resuspend the cells, then add red blood cell lysis buffer, lyse on ice, centrifuge and discard the supernatant; resuspend the cell pellet with dilution buffer to obtain the first enzymatic single cell suspension;

[0019] S5, add the second enzymatic solution to sample tube b, seal it with sealing film and perform the second digestion;

[0020] S6, rinse the tissue and cell sieve with washing buffer, and pipette the cell suspension in S5 through the cell sieve, and centrifuge the cell filtrate to discard the supernatant;

[0021] S7, adding dilution buffer to resuspend the cells, then adding red blood cell lysis buffer, lysing on ice, centrifuging and discarding the supernatant; resuspending the cell pellet with dilution buffer to obtain the second enzymatic single cell suspension;

[0022] S8. The single-cell suspension obtained by the first enzymatic hydrolysis and the single-cell suspension obtained by the second enzymatic hydrolysis are mixed to obtain a single-cell suspension of human arterial tissue.

[0023] In some embodiments of the present invention, the first digestion and the second digestion method are: digestion for 15-25 minutes in a 37°C water bath at 80-120 rpm, and gently blowing the tissue after digestion. Preferably, the first digestion and the second digestion method are: digestion for 20 minutes in a 37°C water bath at 100 rpm, and gently blowing the tissue after digestion.

[0024] In some embodiments of the present invention, the centrifugation method is: 350-450g, 4°C, centrifugation for 5-10 min. Preferably, the centrifugation method is: 400g, 4°C, centrifugation for 5 min

[0025] In some embodiments of the present invention, the cell sieve is a 70 μm-30 μm cell sieve. Preferably, the cell sieve is a combination of a 70 μm and a 30 μm cell sieve.

[0026] The fifth aspect of the present invention provides a human arterial tissue single cell suspension prepared by the method described in the fourth aspect.

[0027] The sixth aspect of the present invention provides the use of the human arterial tissue single cell suspension described in the fifth aspect in single cell detection, such as single cell sequencing, single cell sorting detection, etc.

[0028] Beneficial effects of the present invention

[0029] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides an enzymatic hydrolysis solution, a kit, a method and an application thereof for preparing a single-cell suspension of human arterial tissue; the collagenase contained in the enzymatic hydrolysis solution used in the present invention is a commonly used dissociation enzyme and is easy to obtain; the enzymatic hydrolysis solution ratio in the present invention is suitable for single-cell dissociation of human arterial tissue; the reagent components have good compatibility, are free of harmful ingredients, and are safe and environmentally friendly; the dissociation scheme provided by the present invention can obtain a high-quality single-cell suspension, which can greatly improve the feasibility of single-cell sequencing in arterial tissue; the prepared single-cell suspension of human arterial tissue can be applied in single-cell sequencing, single-cell sorting detection and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The fluorescence field image of the cell suspension obtained in Example 1 of the present invention is shown;

[0031] Figure 2 shows a bright field image of the cell suspension obtained in Example 1 of the present invention;

[0032] Figure 3 The result diagram of single-cell sequencing in Example 3 of the present invention is shown. DETAILED DESCRIPTION

[0033] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve 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 one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0035] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0036] Example 1-2

[0037] Two parallel dissociation experiments were performed according to the protocol provided by the present invention.

[0038] S1. Take 0.5g of sample and place it in a 60×15mm culture dish containing PBS solution. Use scissors to cut the tissue into 3-4mm2 Use a Pasteur pipette to suck the tissue block into a 5mL centrifuge tube and continue to cut it into 1-2mm 2 Use a Pasteur pipette to add PBS to about 4 mL, blow the upper part of the solution with a Pasteur pipette, gently blow up the tissue, let it stand for about 10 seconds, wait for the tissue to settle to the bottom of the tube, discard the supernatant, and wash it 3 times in total. Finally, aspirate and discard the supernatant;

[0039] S2. First dissociation and digestion: Add the prepared first enzymatic solution: 100 U / ml hyaluronidase and 50 U / ml pronase to the cut sample, seal with a sealing film, and place in a 37°C water bath at 100 rpm for 20 minutes. After digestion, use a Pasteur pipette to gently blow the tissue;

[0040] S3. Rinse the tissue and cell sieve with PBS solution, and draw the cell suspension through a combined cell sieve of 70 μm and 30 μm, and centrifuge at 400 g, 4°C, 5 min; after centrifugation, take out the centrifuge tube enriched with cell sediment from the centrifuge, and discard the supernatant with a Pasteur pipette; add 200 μL 1640 culture medium to resuspend the cells, add 2 mL red blood cell lysis buffer, lyse on ice for 5 min, and centrifuge at 400 g, 4°C, 5 min;

[0041] S4. Resuspend the cell pellet in 200 μL 1640 medium to obtain the first dissociated single cell suspension. Use a cell counter to detect cell quality;

[0042] S5. Second dissociation digestion: Add the second enzymatic solution: collagenase II 2mg / ml, dispase 0.5mg / ml, elastase 0.08mg / ml, to the remaining sample, seal with a sealing film, place in a 37℃ water bath at 100rpm and continue digestion for 20min. After digestion is complete, use a Pasteur pipette to gently blow the tissue;

[0043] S6. Rinse the tissue and cell sieve with PBS solution, and draw the cell suspension through a combined cell sieve of 70 μm and 30 μm, and centrifuge at 400 g, 4°C, 5 min; after centrifugation, take out the centrifuge tube enriched with cell sediment from the centrifuge, and discard the supernatant with a Pasteur pipette; add 200 μL 1640 culture medium to resuspend the cells, add 2 mL red blood cell lysis buffer, lyse on ice for 5 min, and centrifuge at 400 g, 4°C, 5 min;

[0044] S7. Resuspend the cell pellet in 200 μL 1640 medium to obtain a second dissociated single cell suspension. Use a cell counter to detect cell quality.

[0045] S8. The single cell suspension obtained by the first dissociation and the single cell suspension obtained by the second dissociation are mixed and collected to obtain a single cell suspension of human arterial tissue. The cell quality is detected again using a cell counter and the concentration is adjusted to a suitable concentration for subsequent experiments.

[0046] Comparative Examples 1-3

[0047] The experimental steps are the same as those in Example 1, except that the second dissociation and digestion time in step S5 is extended to 40 min.

[0048] This experimental process was carried out three times in parallel as Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0049] Comparative Examples 4-5

[0050] The experimental steps are the same as those in Example 1, except that the second enzymatic solution used for the second dissociation and digestion in step S5 is: collagenase II 2 mg / ml, elastase 0.08 mg / ml.

[0051] Two parallel experiments were conducted in this experimental process as Comparative Example 4 and Comparative Example 5.

[0052] Comparative Example 6

[0053] The experimental steps are the same as those in Example 1, except that the first enzymatic solution used for the first dissociation and digestion in step S2 is: 100 U / ml of hyaluronidase, 50 U / ml of pronase, and 2 mg / ml of collagenase I.

[0054] Comparative Example 7

[0055] The experimental steps are the same as those in Example 1, except that: the first enzymatic solution used for the first dissociation and digestion in step S2 is: hyaluronidase 100U / ml, pronase 50U / ml, collagenase I 2mg / ml; the second enzymatic solution used for the second dissociation and digestion in step S5 is: collagenase II 2mg / ml, elastase 0.08mg / ml.

[0056] Comparative Examples 8-9

[0057] The experimental steps are the same as those in Example 1, except that the second enzymatic solution used for the second dissociation and digestion in step S5 is: collagenase II 2 mg / ml, elastase 0.08 mg / ml.

[0058] The remaining tissue obtained by filtration after the second dissociation and digestion step is then subjected to a third dissociation and digestion using 0.25% pancreatic enzyme according to the previous steps S5-S7. The single cell suspensions obtained by the three dissociations are mixed to obtain a single cell suspension of human arterial tissue.

[0059] The human arterial tissue single cell suspension prepared in the above Examples 1-2 and Comparative Examples 1-9 was subjected to AO / PI fluorescence staining and counting, and the results of the Countstar cell counter are shown in Table 1. The cell counting diagram of Example 1 is shown in Figure 1 and Figure 2 .

[0060] Table 1 Comparison of single cell suspension counting results obtained in Examples 1-2 and Comparative Examples 1-9

[0061] Survival rate (%) Agglomeration rate (%) Total amount (W) Example 1 94 10 35 Example 2 95 12 40 Comparative Example 1 92 5 10 Comparative Example 2 90 10 13 Comparative Example 3 93 4 9 Comparative Example 4 91 30 10 Comparative Example 5 88 33 8 Comparative Example 6 88 19 10 Comparative Example 7 94 17 10 Comparative Example 8 87 17 28 Comparative Example 9 87 19 36

[0062] From the above results, it can be seen that the cell viability and total amount of the single cell suspension obtained by dissociation according to the patent steps of the present invention in Examples 1-2 are significantly higher than those of the control examples of several different treatment methods, indicating that the tissue digestion is complete, and the final single cell viability is high and the agglomeration rate is low. Figure 1 The fluorescence field images show that the cell viability is high; Figure 2 The bright field image shows a clean background with few cell debris and residual red blood cells.

[0063] Comparative Examples 1-3 prolonged the second dissociation digestion time, and the cell viability in the final single cell suspension decreased slightly, and the agglomeration rate decreased significantly, indicating that prolonging the digestion time helps to reduce the agglomeration rate, but the final total number of cells decreased significantly.

[0064] In Comparative Example 4-5, no dispase was used, and the total cell amount of the obtained single cell suspension was significantly reduced, while the agglomeration rate was significantly increased.

[0065] Comparative Examples 6-7 used collagenase I, and the total cell amount of the obtained single cell suspension was significantly lower than that of the embodiment. Although the cell viability of Comparative Example 7 increased, the cell agglomeration rate was significantly higher than that of the embodiment.

[0066] Comparative Examples 8-9 did not use dispase for dissociation, but added pancreatic enzyme to continue digesting the tissue. The results showed that the total number of cells in the single-cell suspension was significantly higher than that of other comparative examples, which was similar to the embodiment, but the cell viability was low and the agglomeration rate was higher than that of the embodiment. The final treatment effect was not as good as that of the embodiment.

[0067] Based on the experimental results of the embodiments and comparative examples, it can be seen that the dissociation scheme provided by the present invention can fully digest the tissue, obtain high-quality cell suspension, and greatly improve the feasibility of single-cell sequencing in arterial tissue.

[0068] Example 3

[0069] The 10×Genomics technology was used to perform a single cell sequencing experiment on the single cell suspension prepared in Example 1. The cell capture was performed according to the standard process officially provided by 10×Genomics, and experiments such as oil-in-water generation and subsequent library construction were completed.

[0070] The sequencing results are shown in Table 2 and Figure 3 shown.

[0071] Table 2 Single cell sequencing results

[0072]

[0073] The sequencing results showed that the cells were of good quality and had high expression abundance, meeting the needs of subsequent differentially expressed gene screening, functional enrichment analysis and other types of data mining.

[0074] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. 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 in the present application.

Claims

1. A method for preparing a single cell suspension of human arterial tissue for single cell sequencing, characterized in that: The preparation method comprises the following steps: S1. Take a human arterial tissue sample and place it in a vessel containing washing buffer and cut it into pieces. The cut tissue block sample is placed in sample tube a and repeatedly washed with washing buffer; S2, add the first enzymatic solution into sample tube a, seal it with sealing film and perform the first digestion; S3, rinse the tissue and cell sieve with washing buffer, and draw the cell suspension in S2 through the cell sieve, and discard the supernatant after centrifuging the cell filtrate; place the cell tissue on the sieve into sample tube b for later use; S4, add dilution buffer to resuspend the cells, then add red blood cell lysis buffer, lyse on ice, centrifuge and discard the supernatant; resuspend the cell pellet with dilution buffer to obtain the first enzymatic single cell suspension; S5, add the second enzymatic solution to sample tube b, seal it with sealing film and perform the second digestion; S6, rinse the tissue and cell sieve with washing buffer, and pipette the cell suspension in S5 through the cell sieve, and centrifuge the cell filtrate to discard the supernatant; S7, adding dilution buffer to resuspend the cells, then adding red blood cell lysis buffer, lysing on ice, centrifuging and discarding the supernatant; resuspending the cell pellet with dilution buffer to obtain the second enzymatic single cell suspension; S8, mixing the first enzymatically hydrolyzed single cell suspension and the second enzymatically hydrolyzed single cell suspension to obtain a single cell suspension of human arterial tissue; Wherein, the first enzymatic solution includes 100 U / ml hyaluronidase and 50 U / ml pronase; The second enzymatic solution includes 2 mg / ml collagenase II, 0.5 mg / ml dispase, and 0.08 mg / ml elastase; The washing buffer was PBS solution; the dilution buffer was 1640 culture medium; The first and second digestion methods were as follows: digestion was performed in a 37°C water bath at 100 rpm for 20 min, and the tissue was gently blown after digestion; The centrifugation method is: 400g, 4°C, centrifugation for 5 minutes; The cell sieve is a combination of 70 μm and 30 μm cell sieves.

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