Mixed enzyme liquid for lung tissue dissociation and kit and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to mixed enzyme liquid for lung tissue dissociation and a kit and application of the mixed enzyme liquid. The mixed enzyme liquid comprises neutral protease, deoxyribonuclease I and collagenase I. Under the action of the mixed enzyme liquid, the survival rate of the prepared lung single cells can reach 84.4%, the yield of the separated lung single cells reaches 2.00 * 10 < 6 >, the cell agglomeration rate is as low as 3.12%, and the method is suitable for accurate sequencing of single cell transcriptome, and has the advantages of high specificity, high specificity and high sensitivity. And the change of the single cell level in the occurrence and development process of the lung-related diseases can be known in combination with subsequent bioinformatics analysis, so that the precise medical treatment process in the lung field can be promoted.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a mixed enzyme solution for lung tissue dissociation, a kit and an application thereof. Background Art

[0002] In recent years, with the development and progress of biomedical research, single-cell sequencing technology has emerged. Among them, the most popular is the study of single-cell transcriptome sequencing (scRNA-seq), which can reveal the characteristics and connections between different cells in tissues or organs at the single-cell level, and answer the problem of heterogeneity between cells that was previously covered up by the whole transcriptome high-throughput sequencing technology. Compared with traditional high-throughput sequencing, single-cell sequencing technology has higher accuracy and a larger amount of genetic data obtained at a single time. Combined with the current bioinformatics analysis algorithms and statistical theories, single-cell omics data can be analyzed, integrated and visualized, which can restore biological integrity to the maximum extent and understand the details of biological processes (such as diseases), so that these data can serve the vast scientific research community more effectively. Nowadays, single-cell sequencing technology has been widely used in precision medicine research. For example, for major chronic diseases, we draw single-cell multi-omics information maps of diseases, discover new heterogeneous cell subpopulations, cell populations with specific functions and genes, and reveal the occurrence and development mechanisms of important diseases; discover new therapeutic targets for diseases and evaluate the effects of drug treatments; develop ultra-sensitive, high-throughput, and highly specific precision molecular diagnostic methods for multi-dimensional omics for rapid detection and diagnosis of major diseases. The preparation of single cells is a prerequisite for single-cell sequencing and is also a key part of the experiment. Summary of the invention

[0003] The purpose of the present invention is to provide a mixed enzyme solution for lung tissue dissociation, wherein lung single cells are obtained after dissociation by the enzyme solution, and can be used for lung single cell transcriptome sequencing, wherein the mixed enzyme solution includes neutral protease, deoxyribonuclease I, and collagenase I, and through the action of the mixed enzyme solution, lung single cells with high viability, high yield, and low agglomeration rate can be obtained, and thus better used for single cell related research on various lung diseases.

[0004] The technical solution of the present invention is as follows:

[0005] A mixed enzyme solution for lung tissue dissociation, comprising: 1-3 mg / mL collagenase I, 1-3 mg / mL neutral protease, and 5-30 U / mL deoxyribonuclease I.

[0006] Preferably, a solvent is further included, and preferably, the solvent is a culture medium.

[0007] Another object of the present invention is to protect a kit for lung tissue dissociation, comprising the above-mentioned mixed enzyme solution.

[0008] Furthermore, the above kit further comprises at least one of the following reagents:

[0009] Buffer;

[0010] Culture medium;

[0011] Fetal bovine serum;

[0012] Red blood cell lysis buffer;

[0013] PBS solution containing BSA.

[0014] Preferably, the buffer is PBS buffer;

[0015] And / or, the culture medium is RPMI 1640 culture medium;

[0016] And / or, the PBS solution containing BSA is a PBS solution containing 0.04% BSA.

[0017] Furthermore, it also includes tissue preservation fluid.

[0018] Another object of the present invention is to protect the use of the above kit in lung tissue dissociation.

[0019] Preferably, the application is to perform the following operations in a sterile environment:

[0020] (1) removing lung samples from mice and washing them with a buffer solution; placing the washed lung samples in a culture dish, adding a culture medium solution to the culture dish to infiltrate the lung samples, and then mincing the lung samples to obtain lung tissue;

[0021] (2) transferring the lung tissue described in step (1) to the mixed enzyme solution for lung tissue dissociation and digesting it; adding fetal bovine serum to terminate the digestion, thereby obtaining digested lung tissue;

[0022] (3) The lung tissue digested in step (2) is filtered through a cell sieve, the obtained free cells are centrifuged, the supernatant is discarded, PBS buffer is added to the obtained cell pellet, and then red blood cell lysis buffer is added, resuspended and mixed, after standing at room temperature for 5 minutes, PBS buffer is added, inverted and mixed, centrifuged, the supernatant is discarded, the obtained pellet is resuspended with a PBS solution containing BSA, centrifuged, and the supernatant is discarded to obtain single cells of lung tissue.

[0023] Preferably, the digestion conditions in step (2) are: shaking in a constant temperature water bath at 37°C, a shaking speed of 100 rpm / min, a digestion time of 30 min, and blowing once every 15 min.

[0024] Preferably, the cell sieve filtration in step (3) is performed by superimposing 70 μm and 30 μm cell sieves.

[0025] The invention provides a mixed enzyme for lung tissue dissociation. Collagenase I, neutral protease and deoxyribonuclease I are selected from a large number of enzymes to form a mixed enzyme. The mixed enzyme is used to digest and prepare a single cell suspension of lung tissue on the basis of a mechanical method. After optimizing the most suitable ratio and digestion time and other conditions, a cell suspension that is relatively in line with the single cell sequencing machine standard is obtained. By observing the cell morphology of the single cell suspension, counting the number of cells, and detecting the cell activity, key data such as the cell viability, the concentration of the single cell suspension, and the cell agglomeration rate are obtained. The optimal conditions and ratios obtained by the dissociation mixed enzyme solution and the kit thereof are suitable for efficient dissociation of lung tissue.

[0026] Collagenase I is an enzyme that specifically targets collagen, which is the main component of connective tissue in lung tissue. Collagenase I is highly efficient in degrading collagen fibers, which helps to effectively dissociate cells and disperse tissue. Collagenase I degrades collagen with relatively little impact on the integrity of cells. This helps maintain cell viability and morphology, thereby reducing cell damage and death during the dissociation process.

[0027] Since collagenase I acts primarily on collagen, it has less impact on other cellular components. This means that when collagenase I is used to dissociate tissue, cell survival rates are higher and the biological properties of cells are more likely to be preserved.

[0028] The specific degradation of collagen by collagenase I helps to obtain stable experimental results. In contrast, the use of other types of collagenases (such as collagenase II or collagenase IV) may introduce variability because they may show different effects on different types of collagen isoforms.

[0029] Collagenase II and IV may have an effect on a wider range of matrix components when dissociating tissue, including other proteins and extracellular matrix. This may lead to cell damage and disruption of the extracellular matrix, which may affect cell survival and morphology. Due to the broad range of action of collagenase II and IV, they may cause greater damage to cells during the dissociation process. This may lead to an increase in cell mortality, thereby reducing the possibility of obtaining high-quality cells.

[0030] Therefore, collagenase I has more advantages in dissociating lung tissue because of its specific collagen degradation ability, cell integrity protection, low cytotoxicity and stable experimental results. In contrast, collagenase II and collagenase IV may introduce more uncertainty and cell damage during the dissociation process, thus affecting the reliability of subsequent experiments and the stability of the results. Choosing collagenase I as the dissociation enzyme may help to obtain better cell separation effects and experimental results.

[0031] Beneficial effects of the present invention

[0032] The mixed enzyme and concentration screened and optimized by the present invention are more targeted and efficient, and are more cost-effective and time-saving than the method of using several broad-spectrum collagenases in combination. The present invention provides a good tool for single-cell related research on lung tissue samples.

[0033] The beneficial effects of using a mixed enzyme of collagenase I, neutral protease and DNase I to dissociate mouse lung tissue are mainly reflected in: obtaining lung single cells with sufficient dispersion, high yield, high activity, clean background, good morphology and low agglomeration rate; and after AOPI staining to detect the activity, the viability of the prepared lung single cells can be as high as 84.4%, and the yield of the separated lung single cells can be as high as 2.00×10 6 The cell clumping rate is as low as 3.12%, which is suitable for precise sequencing of single-cell transcriptomes. Combined with subsequent bioinformatics analysis, it can understand the changes at the single-cell level in the occurrence and development of lung-related diseases, thus promoting the process of precision medicine in the lung field. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results of the automatic cell counter detecting lung single cell suspension after AOPI staining are shown in the figure below. The left side is a bright field image showing all the cells obtained, and the right side is a fluorescence image, with green spots indicating living cells.

[0035] Figure 2 This is a statistical chart of lung cell viability at different digestion times using mixed enzyme digestion solution;

[0036] Figure 3 This is a statistical diagram of lung cell yield at different digestion times using mixed enzyme digestion solution;

[0037] Figure 4 This is a statistical chart of the lung cell agglomeration rate at different digestion times using mixed enzyme digestion solution. DETAILED DESCRIPTION

[0038] In order to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be noted that the embodiments described herein are based on the technical solution of the present invention, and provide detailed implementation methods and specific operating processes, but the protection scope of the present invention is not limited to the embodiments. On the contrary, the purpose of providing these embodiments is to make the content of the present invention more thorough and comprehensive.

[0039] Example 1 (collagenase I+neutral protease+deoxyribonuclease I, digestion time 30 min)

[0040] In the embodiments of the present invention, the collagenase is 17100-017, GIBCO; the neutral protease is 4942078001-1, Roche; the deoxyribonuclease I is D5025-15KU, Sigma;

[0041] PBS buffer (Phosphate Buffered Saline) (SRB003-500ML, SRB Biotechnology);

[0042] RPMI 1640 medium (C11875500BT, GIBCO);

[0043] Fetal bovine serum (164210-50, Procell);

[0044] ACK red blood cell lysis buffer (A1049201, ThermoFisher);

[0045] PBS solution containing 0.04% BSA (PBS, 1× (Phosphate Buffered Saline) buffer + 0.04% BSA).

[0046] (1) Enzyme concentration:

[0047] Collagenase I: the working concentration of collagenase I (17100-017, GIBCO) in Example 1 is 2 mg / mL; neutral protease: the concentration of neutral protease (4942078001-1, Roche) in Example 1 is 1 mg / mL; deoxyribonuclease I: the concentration of deoxyribonuclease I (D5025-15KU, Sigma) in Example 1 is 25 U / mL;

[0048] (2) Sampling: Obtain mouse lung samples and place them in tissue storage solution (MACS Tissue Storage Solution, 130-100-008);

[0049] (3) Rinse: Remove the tissue from the preservation solution and rinse it with PBS, 1× (Phosphate Buffered Saline) buffer (SRB003-500ML, SRB Biosciences) to remove the residual preservation solution;

[0050] (4) Weighing: Weigh 0.2 g of sample tissue into a 6 cm culture dish;

[0051] (5) Mincing tissue: Perform aseptic operation in a biological safety cabinet, add RPMI 1640 culture medium solution (C11875500BT, GIBCO) to the culture dish, soak the weighed lung tissue, and quickly mince the sample with sterilized ophthalmic scissors and forceps until there is no obvious block of tissue observed by naked eye;

[0052] (6) Tissue digestion: The minced tissue was transferred to a 15 mL centrifuge tube containing 2 mL of digestion solution (collagenase I + neutral protease + deoxyribonuclease I). The centrifuge tube was placed in a 37°C constant temperature water bath shaker with the shaker speed set at 100 rpm / min for 30 min.

[0053] (7) Termination of digestion: Add termination medium fetal bovine serum (164210-50, Procell) into the centrifuge tube containing the digestion solution to terminate the digestion;

[0054] (8) Filter sieving: After the digestion is terminated, the solution is filtered using a 70 μm and 30 μm cell sieve stacked to remove impurities and incompletely digested tissues, centrifuged at 400 g for 5 min, and the supernatant is discarded;

[0055] (9) Red blood cell lysis and washing: First add 1 ml of PBS to the cell pellet, then add 3 mL of ACK red blood cell lysis buffer (A1049201, ThermoFisher), resuspend and mix, let stand at room temperature for 5 min, add PBS 3 times the volume of ACK red blood cell lysis buffer, invert and mix, centrifuge at 400 g for 5 min, discard the supernatant, resuspend the cell pellet with PBS solution containing 0.04% BSA (PBS, 1× (Phosphate Buffered Saline) buffer + 0.04% BSA), centrifuge at 400 g for 5 min, and discard the supernatant;

[0056] (10) Cell observation and counting: The cell pellet was resuspended in 0.04% BSA PBS solution (PBS, 1× (Phosphate Buffered Saline) buffer + 0.04% BSA) to obtain a single cell suspension of lung tissue. The cell morphology was observed under an optical microscope and the cells were counted using an automatic cell fluorescence analyzer (Countstar Rigel) to obtain the viability.

[0057] Examples 2-6 and Comparative Examples 1-15

[0058] Examples 2-6 and Comparative Examples 1-11 are the same as Example 1, the only difference being the type and concentration of the enzyme. Compared with Example 1, Comparative Examples 12-15 are different in that the dissociation object is changed from mouse lung tissue to mouse fallopian tube tissue, and the concentration of the enzyme is also changed accordingly. The specific differences and corresponding results are shown in the following table:

[0059] Table 1 Comparison of conditions and results of Examples 1-6 and Comparative Examples 1-15

[0060]

[0061] Note: The data in the table are the results of at least three repeated experiments.

[0062] The comparison results in Table 1 show that the cell viability obtained by digestion with the mixed enzyme of the present invention is good, the total number of single cells obtained under the digestion conditions of 37° C. and 30 min is the largest (the number of cells far exceeds that of other embodiments and comparative examples, that is, embodiment 1 has the best effect) and the cell agglomeration rate is the lowest.

[0063] According to the above experimental results, the present invention explored the digestion effects of different types of enzymes and compared the feasibility of the composition ratio of the mixed enzymes. After comparing the data results of the single cell suspension obtained by various digestion methods, collagenase I (2mg / ml) + neutral protease (1mg / ml) + deoxyribonuclease I (25U / ML) was screened and optimized as the optimal enzyme combination, and the digestion conditions of 37°C and 30min were established to obtain the maximum number of cells, higher cell activity and lower cell agglomeration rate, which can provide a favorable single cell suspension sample for subsequent single cell machine detection experiments. And under the same conditions, the oviduct tissue of mice was dissociated and it was found that under this condition, compared with the oviduct tissue of mice, the dissociation of the lung tissue of mice was more specific, with the maximum number of cells, higher cell activity and lower cell agglomeration rate.

[0064] The above describes the specific embodiments of the invention. It should be understood that the invention is not limited to the above specific embodiments, and the devices and structures not described in detail should be understood to be implemented in a common manner in the art; those skilled in the art can make various modifications or modifications within the scope of the claims, and make some simple deductions, deformations or substitutions, which does not affect the essence of the invention.

Claims

1. A mixed enzyme solution for lung tissue dissociation, characterized in that: The mixed enzyme solution includes: 1-3 mg / mL collagenase I, 1-3 mg / mL neutral protease, and 5-30 U / mL deoxyribonuclease I.

2. The mixed enzyme solution according to claim 1, characterized in that: A solvent is also included. Preferably, the solvent is a culture medium.

3. A kit for lung tissue dissociation, characterized in that: The invention comprises the mixed enzyme solution as claimed in claim 1 or 2.

4. The kit according to claim 3, characterized in that Also includes at least one of the following reagents: Buffer; Culture medium; Fetal bovine serum; Red blood cell lysis buffer; PBS solution containing BSA.

5. The kit according to claim 4, characterized in that The buffer is PBS buffer; And / or, the culture medium is RPMI 1640 culture medium; And / or, the PBS solution containing BSA is a PBS solution containing 0.04% BSA.

6. The kit according to any one of claims 3 to 5, characterized in that Also includes tissue preservation solution.

7. Use of the kit described in claims 3-6 in lung tissue dissociation.

8. The use according to claim 7, characterized in that The application is to perform the following operations in a sterile environment: (1) removing lung samples from mice and washing them with a buffer solution; placing the washed lung samples in a culture dish, adding a culture medium solution to the culture dish to infiltrate the lung samples, and then mincing the lung samples to obtain lung tissue; (2) transferring the lung tissue described in step (1) to the mixed enzyme solution for lung tissue dissociation and digesting it; adding fetal bovine serum to terminate the digestion, thereby obtaining digested lung tissue; (3) The lung tissue digested in step (2) is filtered through a cell sieve, the obtained free cells are centrifuged, the supernatant is discarded, PBS buffer is added to the obtained cell pellet, and then red blood cell lysis buffer is added, resuspended and mixed, after standing at room temperature for 5 minutes, PBS buffer is added, inverted and mixed, centrifuged, the supernatant is discarded, the obtained pellet is resuspended with a PBS solution containing BSA, centrifuged, and the supernatant is discarded to obtain single cells of lung tissue.

9. The use according to claim 8, characterized in that: The digestion conditions in step (2) are as follows: shaking in a constant temperature water bath at 37°C, a shaking speed of 100 rpm / min, a digestion time of 30 min, and blowing once every 15 min.

10. The use according to claim 8, characterized in that: The cell sieve filtration in step (3) is performed by superimposing a 70 μm and a 30 μm cell sieve.

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