1. A digestive enzyme for efficiently preparing a single cell suspension of human kidney tissue, wherein the digestive enzyme comprises ColII, ColIV, ColI, DNase I and hyaluronidase.

2. The digestive enzyme of claim 1, wherein the ratio of concentrations of ColII, ColIV, ColI, DNase I and hyaluronidase in the digestive enzyme is: 40-80:5-10:5-10:4-10:4-10.

3. The digestive enzyme of claim 2, wherein the ratio of concentrations of ColII, ColIV, ColI, DNase I and hyaluronidase in the digestive enzyme is: 40:5:10:2:2.

4. The digestive enzyme of any one of claims 1 to 2, wherein the concentrations of ColII, ColIV, ColI, DNase I and hyaluronidase in the digestive enzyme are 1 to 2mg/mL, 0.125 to 0.5mg/mL, 0.1 to 0.5mg/mL and 0.1 to 0.5mg/mL, respectively.

5. The digestive enzyme of claim 4, wherein the concentrations of ColII, ColIV, ColI, DNase I and hyaluronidase in the digestive enzyme are 2mg/mL, 0.25mg/mL, 0.5mg/mL, 0.1mg/mL and 0.1mg/mL, respectively.

6. Use of a digestive enzyme according to any of claims 1 to 5 for the preparation of a single cell suspension of human kidney tissue.

7. A method for efficiently preparing a single cell suspension of human kidney tissue, which is characterized by comprising the following steps:

1) a sample processing step: taking an in vitro kidney tissue sample, cutting the in vitro kidney tissue sample, washing with PBS (phosphate buffer solution), and centrifuging to remove a supernatant;

2) an enzyme digestion step: adding the digestive enzyme of any one of claims 1-5, incubating and mixing;

3) resuspension purification step: the supernatant was centrifuged off and purified after resuspension.

8. The method for efficiently preparing the single cell suspension of human kidney tissue according to claim 7, wherein the step 1) comprises: taking an in vitro kidney tissue sample, cutting the sample in an EP centrifuge tube, washing the sample with PBS, and centrifuging to remove a supernatant; preferably: taking an in vitro puncture tissue sample by using a puncture needle, putting the puncture tissue sample into a centrifuge tube, washing the puncture tissue sample by PBS (phosphate buffer solution), centrifuging the puncture tissue sample at 300 Xg for 5min, removing supernatant, cutting the puncture tissue sample by scissors, washing the puncture tissue sample by PBS, and centrifuging the puncture tissue sample to remove the supernatant.

9. The method for efficiently preparing human kidney tissue single cell suspension enzyme according to any one of claims 7 to 8, wherein in the step 2), after adding digestive enzyme, incubating at 37 ℃ for 10 to 20min, and uniformly mixing once every 5 to 8min, and stopping digestion by using DMEM complete culture medium; preferably, digestion is stopped in DMEM complete medium after addition of digestive enzymes and incubation at 37 deg.C for 20min, with mixing once every 5 min.

10. The method for efficiently preparing human kidney tissue single cell suspension enzyme according to any one of claims 7 to 9, wherein the step 3) is that the obtained cell suspension is filtered, centrifuged to remove supernatant, and purified after being resuspended in DMEM medium; preferably, the cell suspension obtained is filtered through a 70um cell sieve, centrifuged at 300 Xg for 5min, the supernatant is removed, and the cell suspension is purified after being resuspended in a DMEM medium.

Background

With the development and innovation of scRNA-seq research and the application of flow cytometry experiments in kidney tissues. The preparation of single cells is a key part for carrying out experiments such as single cell transcriptomics, flow cytometry, cell primary culture and the like. The kidney tissue digestion is generally performed by an enzyme digestion method mainly using collagenase, but the cell suspension obtained by the enzyme digestion method reported in the prior art contains a large amount of cell masses, and the requirement of single cell suspension preparation cannot be met.

At present, the preparation method of the single cell suspension of common tissues comprises a mechanical method and an enzyme digestion method. The mechanical method has great damage to tissues and is easy to cause cell damage and loss; the enzyme digestion method requires less tissue and is suitable for specimens containing more connective tissue components. Mechanical separation methods are simple, but are less frequently used due to incomplete tissue separation, more tissue blocks, and a small number of cells to be separated. The method for preparing the human kidney single cell suspension by selecting an appropriate method according to tissue types, research purposes and requirements is an important experimental link, and has important significance for researching the cytological mechanism of kidney diseases and discussing the prevention strategy.

The invention is provided in view of the above.

Disclosure of Invention

The invention aims to find a digestive enzyme system or a formula of a high-efficiency human kidney single cell suspension. In order to achieve the purpose, the invention designs an orthogonal combination test on the basis of a single-factor experiment, optimizes the conditions by using different combination digestive enzymes, and finally obtains a high-efficiency human kidney single cell suspension digestive enzyme system.

The invention firstly provides a digestive enzyme for efficiently preparing a human kidney tissue single cell suspension, which is characterized by comprising ColII, ColIV, ColI, DNase I and hyaluronidase.

Further, the concentration ratio of ColII, ColIV, ColI, DNase I and hyaluronidase in the digestive enzyme is as follows: 40-80:5-10:5-10:4-10:4-10.

In some preferred embodiments, the ratio of the concentrations of ColII, ColIV, ColI, DNase I and hyaluronidase in the digestive enzyme is: 40:5:10:2:2.

Furthermore, the concentrations of ColII, ColIV, ColI, DNase I and hyaluronidase in the digestive enzyme are respectively 1-4mg/mL, 0.125-0.5mg/mL, 0.125-0.5mg/mL, 0.1-0.5mg/mL and 0.1-0.5 mg/mL.

In some preferred embodiments, the concentration of ColII, ColIV, ColI, DNase I, and hyaluronidase in the digestive enzymes is 2mg/mL, 0.25mg/mL, 0.5mg/mL, 0.1mg/mL, and 0.1mg/mL, respectively.

The invention also provides an application of the digestive enzyme in preparation of the human kidney tissue single cell suspension.

Further, the application comprises the following steps:

1) a sample processing step: taking an in vitro kidney tissue sample, cutting the in vitro kidney tissue sample, washing with PBS (phosphate buffer solution), and centrifuging to remove a supernatant;

2) an enzyme digestion step: adding the digestive enzyme of any one of claims 1-5, incubating and mixing;

3) resuspension purification step: the supernatant was centrifuged off and purified after resuspension.

Further, the step 1) is as follows: taking an in vitro kidney tissue sample, cutting the sample in an EP centrifuge tube, washing the sample with PBS, and centrifuging to remove the supernatant.

Further, the step 2) is that after adding digestive enzyme, incubating for 10-20min at 37 ℃, uniformly mixing once every 5-8min, and stopping digestion by using a DMEM complete culture medium.

Further, in the step 3), the obtained cell suspension is filtered and centrifuged to remove supernatant, and the cell suspension is purified after being resuspended in a DMEM medium.

The invention also provides a method for efficiently preparing the human kidney tissue single cell suspension, which is characterized by comprising the following steps:

1) a sample processing step: taking an in vitro kidney tissue sample, cutting the in vitro kidney tissue sample, washing with PBS (phosphate buffer solution), and centrifuging to remove a supernatant;

2) an enzyme digestion step: adding the digestive enzyme, incubating and mixing uniformly;

3) resuspension purification step: the supernatant was centrifuged off and purified after resuspension.

Further, the step 1) is as follows: taking an in vitro kidney tissue sample, cutting the sample in an EP centrifuge tube, washing the sample with PBS, and centrifuging to remove a supernatant;

in some preferred modes: the step 1) is as follows: taking an in vitro puncture tissue sample by using a puncture needle, putting the puncture tissue sample into a centrifuge tube, washing the puncture tissue sample by PBS (phosphate buffer solution), centrifuging the puncture tissue sample at 300 Xg for 5min, removing supernatant, cutting the puncture tissue sample by scissors, washing the puncture tissue sample by PBS, and centrifuging the puncture tissue sample to remove the supernatant.

Further, the step 2) is that after adding digestive enzyme, incubating for 10-20min at 37 ℃, uniformly mixing once every 5-8min, and stopping digestion by using a DMEM complete culture medium;

in some preferred modes, the step 2) is to incubate at 37 ℃ for 20min after adding the digestive enzyme, mix the mixture every 5min, and stop the digestion by using DMEM complete medium.

Further, in the step 3), the obtained cell suspension is filtered and centrifuged to remove supernatant, and a DMEM culture medium is purified after being resuspended;

in some preferred modes, the step 3) is to filter the obtained cell suspension by using a 70um cell sieve, centrifuge the cell suspension at 300 Xg for 5min, remove the supernatant, and purify the cell suspension after the DMEM medium is resuspended.

Compared with the prior art, the invention has at least the following advantages:

1) by adopting the digestive juice of the invention to treat the frozen kidney puncture tissue, the number of single cells can be extracted to be 3.2 multiplied by 10⁶And the percentage of living cells in the obtained single cell suspension is over 45 percent, and the effect advantage is extremely obvious.

2) Compared with the traditional formula, the single cell obtained by digesting the kidney tissue with the digestive juice has less cell agglomeration proportion and is suitable for single cell detection application.

3) The invention provides an effective method for preparing human kidney tissue single cell suspension by improving a compound enzyme digestion method, and provides conditions for researching cell types and disease generating mechanisms in kidneys from a single cell level.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 the effect of various types of collagenases on the digestion of kidney tissue;

FIG. 2 the effect of various types of complex collagenases on kidney tissue digestion (cell number);

FIG. 3 the effect of various types of complex collagenases on kidney tissue digestion (cell survival);

FIG. 45 shows flow cytograms of human kidney single cell suspensions under the action of complex enzyme formula No. 45, complex enzyme formula No. 10 and complex enzyme formula No. 11;

FIG. 55 Complex enzyme formulation, 10 Complex enzyme formulation and 11 Complex enzyme digested unicells counted under microscope, 200X; white circles represent dead cells.

FIG. 65 shows the count analysis of the cell mass of the single cells digested by the complex enzyme formula No. 65, the complex enzyme formula No. 10 and the complex enzyme formula No. 11 under the microscope. The ordinate in the figure represents the percentage of cell mass after digestion.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The following terms or definitions are provided only to aid in understanding the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.

Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.

The terms "about" and "substantially" in the present invention denote an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The following are specific embodiments.

Materials and principal reagents of the invention

The fresh human kidney tissue puncture sample is an in vitro sample preserved in a tissue preservation solution for 7 days; DMEM medium, collagenase type i, collagenase type ii, collagenase type iv (Gibco, usa), hyaluronidase, DNase i (Sigma, usa), flow cytometric staining solution, PI dye (BD, usa).

The basic method of the invention

1) Method for decomposing tissue into single cells

In a culture dish, the in vitro tissue is subjected to outer membrane removal and fat removal, and a fresh sample is put into tissue preservation solution (AQIX) for preservation. Placing the tissue block into a 50mL centrifuge tube, taking an isolated puncture tissue sample by using a puncture needle, soaking the sample in FRS for 30 minutes, adding 1mL of CS10 frozen stock solution, standing overnight at-80 ℃, and preserving by using liquid nitrogen. The puncture sample was placed in a 5mL centrifuge tube, washed with 3mL PBS, centrifuged at 300 × g for 5min, the supernatant was removed, the puncture sample was cut into pieces with scissors, washed with PBS, and centrifuged to remove the supernatant. Adding digestive enzyme, incubating at 37 deg.C for 20min, mixing every 5min, terminating digestion with DMEM complete medium, filtering the obtained cell suspension with 70um cell sieve, centrifuging at 300 Xg for 5min, removing supernatant, and resuspending with 3mL DMEM medium.

2) Detection index and statistical analysis

The number of cells contained in the 15uL suspension and the cell viability were measured by a cell counter. Taking 1mL of cell suspension, centrifuging to remove supernatant, re-suspending the cell precipitate with 1mL of flow-type staining solution, adding 2uL of pI labeled cells, and detecting by an up-flow cytometer to obtain the cell activity of the mononuclear cells. Data were collected and plotted using Graphpad prism 6 software, analyzed using sps software, and all measurements expressed as (x ± SD).

Specific examples are as follows.

EXAMPLE 1 Effect of collagenase on the preparation of Single cell suspensions

The preparation method of the digestive enzyme comprises the following steps: adding collagenase type I, collagenase type II and collagenase type IV into a DMEM high-sugar culture medium to prepare stock solution of 8 mg/mL.

A fresh kidney puncture sample preserved for 5 days in AQIX was taken, 80ul of collagenase type I, collagenase type II and collagenase type IV at concentrations of 0.125, 0.25, 0.5, 1, 2 and 4mg/mL were added, FMCase was used as a control, and the sample was incubated at 37 ℃ for 20min and mixed once every 5 min.

Different collagenases have different effects on the digestive effect of kidney tissue. As can be seen from FIG. 1, 2mg/mL collagenase type II has higher activity on digested single cells of kidney tissue, which can reach 85%, low concentration collagenase type IV has strong effect on kidney tissue, the same concentration collagenase type II has higher activity on digested single cells of kidney tissue than collagenase type I, and when the concentration of three collagenases is more than 1mg/mL, the activity on digested single cells of kidney tissue is higher than FCMase.

EXAMPLE 2 Effect of Complex collagenase on the preparation of Single cell suspensions

According to the results of single factors, the total concentration of collagenase is 2mg/mL, the digestion capacity of tissues is strongest, the collagenase type II has the best effect, a higher concentration is preferably used, collagenase type I and collagenase type IV are auxiliary effect enzymes, a low concentration ratio is preferably used, and the total concentration of collagenase is not more than 4mg/mL because the activity of cells tends to be reduced when the collagenase exceeds 2 mg/mL.

By using L₉(3⁴) The orthogonal table is designed into 9 groups of experiments, and the types, concentrations and ratios of the added collagenase are shown in the table 2 in the concentration design of the experiments in the tables 1 to 9. The method is characterized in that a compound collagenase is used as a basic digestive enzyme, 0.1mg/mLDNase I and 0.1mg/mL hyaluronidase are added, collagenase can digest extracellular matrixes by breaking peptide bonds, type I DNase prevents cell aggregation, hyaluronidase is used for cutting the glycosidic bonds to break the extracellular matrixes, kidney tissues are mostly digested by different types of collagenase at the present stage, and DNase I and hyaluronidase are applied at the concentration of 0.1-0.5mg/mL and are used for assisting digestion. And (3) taking the frozen puncture tissue samples, repeating each group, and counting the total number of the single cells and the cell activity rate under the digestion of various complex enzymes after the single cells are prepared under the same digestion conditions.

TABLE 1 orthogonal experiments of collagenase on digestion of Kidney tissue

TABLE 2 Experimental design of the digestive action of Complex collagenase on Kidney tissue

The number and activity of the digested single cells are used as dependent variables, the influence of the combination of three collagenases, namely collagenase type I, collagenase type II and collagenase type IV on the digestion effect of kidney tissues is respectively inspected, and the statistics of the results are shown in fig. 2 and fig. 3; the data analysis was performed using SPSS 19.0 software, with the range analysis results shown in table 3, table 4.

As can be seen from Table 3, the effect of collagenase type I, collagenase type II and collagenase type IV on the number of cells digested into single cells is as follows: col I > col IV > col II, i.e., col I, is a major factor affecting the digestion of tissues into sufficient numbers of cells. Comparing the K value, determining the optimal hormone combination as 2mg/mLcol II +0.25mg/mLcol IV +0.25mg/mLcol I.

As can be seen from Table 4, the effects of collagenase type I, collagenase type II and collagenase type IV on the survival rate of single cells are as follows: col II > col I > col IV, i.e., col II is a major factor affecting tissue digestion into high-activity single cells. Comparing the K value, and determining that the optimal hormone combination is 2mg/mLcol II +0.25mg/mLcol IV +0.5mg/mLcol I; in fig. 2, the number of single cells obtained by digesting the tissue single cell with the group 5 complex enzyme formula in the 9 experiments is the largest, i.e. the average cell number of the single cell obtained by digesting the tissue single cell with 2mg/mLcol ii +0.25mg/mLcol iv +0.5mg/mLcol i can reach 3160000 + -95393.92, while the average cell number of the single cell obtained by digesting the tissue single cell with the reference enzyme in the group 10 is 3133333.33 + -241269.78, the average cell number of the single cell obtained by digesting the broad spectrum complex enzyme fcmask in the group 11 control is 1596666.66 + -146780.25, the number of the single cell obtained by digesting the enzyme in the group 5 and the group 10 is not significant, and the P value is 0.916, compared with the group 11, the difference is significant, and the P value is 0.001. The influence of each complex enzyme on the survival rate of the single cells is not obvious.

TABLE 3 very poor analysis of the number of cells obtained by the digestion of kidney tissue with complex collagenases

TABLE 4 very poor analysis of cell survival rate of kidney tissue digestion by complex collagenase

Example 3 optimal Complex enzyme System establishment

The advantageous complex enzyme system of the invention was established on the basis of the above examples 1 and 2:

1-2mg/mL of colII, 0.125-0.5mg/mL of ColIV, 0.125-0.5mg/mL of colI, 0.1-0.5mg/mL of DNase I and 0.1-0.5mg/mL of H enzyme;

wherein the optimal complex enzyme system is as follows:

2mg/mL colII +0.25mg/mL ColIV +0.5mg/mL colI +0.1mg/mL DNase I +0.1mg/mL Henzyme.

The frozen 1 fresh punctured kidney tissue (about 10mg) was treated with the optimal complex enzyme system described above. As shown in the above FIGS. 2 and 3, it is statistically demonstrated that the number of single cells extracted by the enzyme system (i.e., the No. 5 complex enzyme) is 3.2X 10⁶The percentage of viable cells in the obtained single cell suspension is over 45 percent.

Example 4 comparison of the Effect with commercial digestive enzymes

The optimal complex enzyme system (No. 5) and the commercial enzyme system (No. 10) Liberase TM0.025mg/mL +0.05mg/mLDNase I and (No. 11) FCMASe +0.1mg/mLDNase I are respectively adopted to carry out effect comparison under the same treatment condition.

As a result, it can be seen from the flow analysis in FIG. 4 that after the treatment with the optimal Complex enzyme formulation No. 5, the number of PI positive cells is small, the representative cells with large particle size clump, and the number of cell clumps is small. The ratio of dead cells obtained by the formula of the No. 5 complex enzyme in the figure 5 is less, the ratio of cell clusters digested by the No. 5 enzyme in the figure 6 is 12 percent, and the number of the cell clusters is lower than that of the cell clusters digested by the No. 10 enzyme, and the ratio of cell clusters of single cells obtained by digesting kidney tissues by the enzyme is less than that of the formula of the enzyme Liberase TM. The kidney single cell mass ratio digested by No. 11 complex enzyme is also lower, but as can be seen from FIG. 2, the total number of single cells digested by No. 11 enzyme is lower, so that in conclusion, the number of single cell cells digested by No. 5 complex enzyme is more, the death rate is lower, and the cell mass is less, so that the method is suitable for preparing the suspension of human kidney single cells, and the effect of the method is beyond the experimental expectation in a certain sense.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.