1. A preparation method of a fish tissue single cell suspension is characterized by comprising the following steps:

1) preparing a reagent and a culture medium, wherein the culture medium comprises a serum-free culture medium and a complete culture medium, preparing a cell cleaning solution, and preparing a mixed enzyme dissociation stock solution;

2) pretreating tissues, namely adding a proper amount of 1-DPBS solution into a sterile RNase-free cell culture dish; cutting the tissue into 0.5-1mm²Sucking the small pieces into a 15ml test tube containing 1 × DPBS solution, and flushing 1 × DPBS solution for 1-2 times until the redundant tissues are completely removed;

3) digesting and dissociating tissues, sucking out redundant 1 × DPBS solution, and adding 3-5ml of mixed enzyme dissociation stock solution; digesting and incubating, and performing water bath at 37 ℃ for 15 min;

4) termination of digestion: adding an isovolumetric culture medium solution prepared in the early stage, slightly sucking, beating and uniformly mixing, visually observing a small section of a tissue in the mixed solution, and transferring the small section of the tissue to a 50ml test tube;

5) filtering by a micron cell filter, uniformly mixing the cell suspension obtained in the step 4), slowly sucking and blowing the cell suspension into a new test tube through the cell filter, and marking on the tube wall of the test tube;

6) obtaining a target tissue single cell suspension;

7) and (3) measuring the yield of the living cells, sucking AO/PI dye solution, fully and uniformly mixing the AO/PI dye solution with the equal volume of the target tissue single cell suspension in the step 6), sucking and pumping the mixture into a pool in a counting plate, and measuring the ratio of the living cells by using a cell counter.

2. The method for preparing a suspension of single cells of fish tissue according to claim 1, wherein the method comprises the following steps: every 100ml of the serum-free culture medium contains 99ml of basic culture medium and 1ml of streptomycin qing mixed liquor, every 100ml of the culture medium contains 89ml of basic culture medium, 10ml of FBS and 1ml of streptomycin qing mixed liquor, and the basic culture medium is M199-HEPES culture medium.

3. The method for preparing a suspension of single cells of fish tissue according to claim 1, wherein the method comprises the following steps: the cell washing solution is HBSS-5% FBS solution, and the HBSS-5% FBS solution contains 95ml of HBSS solution and 5ml of FBS solution.

4. The method for preparing a suspension of single cells of fish tissue according to claim 1, wherein the method comprises the following steps: the mixed enzyme dissociation stock solution comprises collagenase II freeze-dried powder, DispaseII freeze-dried powder and a serum-free basic culture medium, wherein 10mg of collagenase II freeze-dried powder and 5mg of DispaseII freeze-dried powder are added into every 10ml of the serum-free basic culture medium to prepare the mixed enzyme dissociation stock solution, and the serum-free basic culture medium is an M199-HEPES culture medium.

5. The method for preparing a suspension of single cells of fish tissue according to claim 1, wherein the method comprises the following steps: the cell filter in the step 5) is a 40-micron cell filter.

6. The method for preparing a suspension of single cells in fish tissue according to claim 1, wherein the method for obtaining the suspension of single cells in the target tissue in step 6) is as follows:

resuspending and counting, namely, uniformly mixing the cell suspension filtered in the step 5), centrifuging at 1100rpm for 5min, then discarding the supernatant, and adding 200-500 ul of HBSS cell cleaning solution containing 5% FBS for resuspension; sucking 10ul of reselected cell suspension and 10ul of AO/PI dye solution, mixing uniformly, pumping into a counting plate, and counting by a cell counter to obtain the concentration of the original single cell suspension.

Obtaining single cell suspension of target tissue, and diluting original single cell suspension to 8.5 x 10 concentration by HBSS cell washing solution of 5% FBS⁵～1.8*10⁶cells/ml。

7. The method for preparing a suspension of single cells of fish tissue according to claim 1, wherein the method comprises the following steps: the redundant tissues in the step 2) comprise blood and fat.

8. A suspension of single cells of fish tissue prepared by the method of any one of claims 1 to 7.

9. The use of a suspension of single cells of fish tissue according to claim 8 for single cell sequencing.

Background

In recent years, with the rapid development of technologies in the field of biological research, single cell sequencing technology has come to be developed, which is a new technology for performing high-throughput sequencing analysis on genomes, transcriptomes and epigenomes on the level of single cells. Single cell sequencing can reveal the gene structure and gene expression of single cell, and can reflect the heterogeneity of cells, and may be used in tumor, development biology, immunology, microbiology and other fields.

Different tissue cells have unique physiological characteristics, which causes the tissue cell culture to be a worldwide problem. The existing experimental technology can not obtain tissue single cell suspension with better cell viability, and restricts the research work of tissue cell culture, cell line establishment, functional gene verification and the like. Therefore, the efficient preparation of the tissue single cell suspension is beneficial to establishing and optimizing the in vitro culture technology of the adult tissue cells, provides important reference data for the establishment of a cell line, and has important theoretical and practical significance in the research fields of functional genes, genetic breeding and the like.

Disclosure of Invention

The invention provides a preparation method of a fish tissue single cell suspension.

The scheme of the invention is as follows:

a preparation method of a fish tissue single cell suspension comprises the following steps:

1) preparing a reagent and a culture medium, wherein the culture medium comprises a serum-free culture medium and a complete culture medium, preparing a cell cleaning solution, and preparing a mixed enzyme dissociation stock solution;

2) pretreating tissues, namely adding a proper amount of 1-DPBS solution into a sterile RNase-free cell culture dish; cutting the tissue into 0.5-1mm²Sucking the small pieces into a 15ml test tube containing 1 × DPBS solution, and flushing 1 × DPBS solution for 1-2 times until the redundant tissues are completely removed;

3) digesting and dissociating tissues, sucking out redundant 1 × DPBS solution, and adding 3-5ml of mixed enzyme dissociation stock solution; digesting and incubating, and performing water bath at 37 ℃ for 15 min;

4) termination of digestion: adding an isovolumetric culture medium solution prepared in the early stage, slightly sucking, beating and uniformly mixing, visually observing a small section of a tissue in the mixed solution, and transferring the small section of the tissue to a 50ml test tube;

5) filtering by a micron cell filter, uniformly mixing the cell suspension obtained in the step 4), slowly sucking and blowing the cell suspension into a new test tube through the cell filter, and marking on the tube wall of the test tube;

6) obtaining a target tissue single cell suspension;

7) and (3) measuring the yield of the living cells, sucking AO/PI dye solution, fully and uniformly mixing the AO/PI dye solution with the equal volume of the target tissue single cell suspension in the step 6), sucking and pumping the mixture into a pool in a counting plate, and measuring the ratio of the living cells by using a cell counter.

Preferably, the serum-free medium comprises 99ml of basic medium and 1ml of streptomycin qing mixed solution per 100ml of the serum-free medium, and the basic medium comprises 89ml of basic medium, 10ml of FBS and 1ml of streptomycin qing mixed solution per 100ml of the serum-free medium, wherein the basic medium is M199-HEPES medium.

In a preferred embodiment, the cell washing solution is an HBSS-5% FBS solution, and the HBSS-5% FBS solution contains 95ml of HBSS solution and 5ml of FBS solution.

As a preferable technical scheme, the mixed enzyme dissociation stock solution comprises collagenase II freeze-dried powder, DispaseII freeze-dried powder and a serum-free basic culture medium, 10mg of collagenase II freeze-dried powder and 5mg of DispaseII freeze-dried powder are added into every 10ml of the serum-free basic culture medium to prepare the mixed enzyme dissociation stock solution, and the serum-free basic culture medium is an M199-HEPES culture medium.

Preferably, the cell filter in the step 5) is a 40 μm cell filter.

As a preferred technical scheme, the method for obtaining the single cell suspension of the target tissue in the step 6) comprises the following steps:

resuspending and counting, namely, uniformly mixing the cell suspension filtered in the step 5), centrifuging at 1100rpm for 5min, then discarding the supernatant, and adding 200-500 ul of HBSS cell cleaning solution containing 5% FBS for resuspension; sucking 10ul of reselected cell suspension and 10ul of AO/PI dye solution, mixing uniformly, pumping into a counting plate, and counting by a cell counter to obtain the concentration of the original single cell suspension.

Obtaining single cell suspension of target tissue, and diluting original single cell suspension to 8.5 x 10 concentration by HBSS cell washing solution of 5% FBS⁵～1.8*10⁶cells/ml。

Preferably, the redundant tissue in step 2) includes blood and fat.

The invention also discloses a fish tissue single cell suspension.

The invention also discloses application of the fish tissue single cell suspension in single cell sequencing.

The preparation method of the fish tissue single cell suspension adopts the technical scheme that 1) a reagent and a culture medium are prepared, the culture medium comprises a serum-free culture medium and a complete culture medium, a cell cleaning solution is prepared, and a mixed enzyme dissociation stock solution is prepared; 2) pretreating tissues, namely adding a proper amount of 1-DPBS solution into a sterile RNase-free cell culture dish; cutting the tissue into 0.5-1mm²Sucking the small pieces into a 15ml test tube containing 1 × DPBS solution, and flushing 1 × DPBS solution for 1-2 times until the redundant tissues are completely removed; 3) digesting and dissociating tissues, sucking out redundant 1 × DPBS solution, and adding 3-5ml of mixed enzyme dissociation stock solution; digesting and incubating, and performing water bath at 37 ℃ for 15 min; 4) termination of digestion: adding an isovolumetric culture medium solution prepared in the early stage, slightly sucking, beating and uniformly mixing, visually observing a small section of a tissue in the mixed solution, and transferring the small section of the tissue to a 50ml test tube; 5) filtering by a micron cell filter, uniformly mixing the cell suspension obtained in the step 4), slowly sucking and blowing the cell suspension into a new test tube through the cell filter, and marking on the tube wall of the test tube; 6) obtaining a target tissue single cell suspension; 7) and (3) measuring the yield of the living cells, sucking AO/PI dye solution, fully and uniformly mixing the AO/PI dye solution with the equal volume of the target tissue single cell suspension in the step 6), sucking and pumping the mixture into a pool in a counting plate, and measuring the ratio of the living cells by using a cell counter.

The method for preparing the fish tissue single cell suspension has the advantages that the fish tissue single cell suspension can be obtained quickly and efficiently at low cost, the dissociated cells can be guaranteed to keep good survival rate, the method can be applied to dissociation, separation and purification of the fish cells and establishment of cell lines, and important reference data is provided for research on fish single cells, gene functions and genetic breeding.

Drawings

FIG. 1 is a flow chart of the preparation of the present invention;

FIG. 2 is a diagram showing the quality inspection results (AO/PI) of single cell suspensions of fish tissues according to an embodiment of the present invention.

Detailed Description

In order to make up for the above deficiencies, the present invention provides a method for preparing a single cell suspension of fish tissue to solve the above problems in the background art.

A preparation method of a fish tissue single cell suspension comprises the following steps:

1) preparing a reagent and a culture medium, wherein the culture medium comprises a serum-free culture medium and a complete culture medium, preparing a cell cleaning solution, and preparing a mixed enzyme dissociation stock solution;

2) pretreating tissues, namely adding a proper amount of 1-DPBS solution into a sterile RNase-free cell culture dish; cutting the tissue into 0.5-1mm²Sucking the small pieces into a 15ml test tube containing 1 × DPBS solution, and flushing 1 × DPBS solution for 1-2 times until the redundant tissues are completely removed;

3) digesting and dissociating tissues, sucking out redundant 1 × DPBS solution, and adding 3-5ml of mixed enzyme dissociation stock solution; digesting and incubating, and performing water bath at 37 ℃ for 15 min;

4) termination of digestion: adding an isovolumetric culture medium solution prepared in the early stage, slightly sucking, beating and uniformly mixing, visually observing a small section of a tissue in the mixed solution, and transferring the small section of the tissue to a 50ml test tube;

5) filtering by a micron cell filter, uniformly mixing the cell suspension obtained in the step 4), slowly sucking and blowing the cell suspension into a new test tube through the cell filter, and marking on the tube wall of the test tube;

6) obtaining a target tissue single cell suspension;

7) and (3) measuring the yield of the living cells, sucking AO/PI dye solution, fully and uniformly mixing the AO/PI dye solution with the equal volume of the target tissue single cell suspension in the step 6), sucking and pumping the mixture into a pool in a counting plate, and measuring the ratio of the living cells by using a cell counter.

Every 100ml of the serum-free culture medium contains 99ml of basic culture medium and 1ml of streptomycin qing mixed liquor, every 100ml of the culture medium contains 89ml of basic culture medium, 10ml of FBS and 1ml of streptomycin qing mixed liquor, and the basic culture medium is M199-HEPES culture medium.

The cell washing solution is HBSS-5% FBS solution, and the HBSS-5% FBS solution contains 95ml of HBSS solution and 5ml of FBS solution.

The mixed enzyme dissociation stock solution comprises collagenase II freeze-dried powder, DispaseII freeze-dried powder and a serum-free basic culture medium, wherein 10mg of collagenase II freeze-dried powder and 5mg of DispaseII freeze-dried powder are added into every 10ml of the serum-free basic culture medium to prepare the mixed enzyme dissociation stock solution, and the serum-free basic culture medium is an M199-HEPES culture medium.

The cell filter in the step 5) is a 40-micron cell filter.

The method for obtaining the target tissue single cell suspension in the step 6) comprises the following steps:

resuspending and counting, namely, uniformly mixing the cell suspension filtered in the step 5), centrifuging at 1100rpm for 5min, then discarding the supernatant, and adding 200-500 ul of HBSS cell cleaning solution containing 5% FBS for resuspension; sucking 10ul of reselected cell suspension and 10ul of AO/PI dye solution, mixing uniformly, pumping into a counting plate, and counting by a cell counter to obtain the concentration of the original single cell suspension.

Obtaining single cell suspension of target tissue, and diluting original single cell suspension to 8.5 x 10 concentration by HBSS cell washing solution of 5% FBS⁵～1.8*10⁶cells/ml。

The redundant tissues in the step 2) comprise blood and fat.

The invention also discloses a fish tissue single cell suspension.

The invention also discloses application of the fish tissue single cell suspension in single cell sequencing.

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

Example 1:

the first step is as follows: preparation of reagents and culture media

1. The following two media were prepared separately

(1) Serum-free medium: the subpackaged M199-HEPES culture medium contains 1% of Penicilin-Streptomycin; 99ml M199-HEPES Medium was added 1ml of Penicilin-Streptomyces

(2) Complete medium: the subpackaged M199-HEPES medium contains 10% FBS and 1% Penicillin-Streptomycin; 89ml of M199-HEPES medium was added with 10ml of FBS and 1ml of Penicilin-Streptomyces;

2. preparing a cell cleaning solution: HBSS-5% FBS solution: mixing 95ml HBSS solution and 5m FBS solution;

3. preparing a mixed enzyme dissociation stock solution: dissolving 10mg of collagenase type II lyophilized powder (Sigma, cat # C6885-1G) and 5mg of dispaseII lyophilized powder (Sigma, cat # D4693-1G) in 10ml of serum-free M199-HEPES medium (Sigma, cat # 12340030), filtering, sterilizing, and storing in a freezer at-20 deg.C to avoid repeated freeze thawing.

The second step is that: tissue pretreatment

Appropriate amount of 1 × DPBS solution was added to sterile rnase-free cell culture dishes. Cutting the tissue into 0.5-1mm²After the small pieces, the pieces were aspirated into 15mltube containing 1 × DPBS solution and washed 1-2 times with 1 × DPBS solution until the excess tissue, such as blood, fat layer, etc., was removed.

The third step: tissue digestion dissociation

Excess 1 × DPBS solution was aspirated off and 3-5ml of mixed enzyme dissociation stock was added. Digestion incubation: water bath at 37 deg.C for 15min

The fourth step: terminating digestion

The same volume of the prepared medium solution was added, gently pipetted and mixed (no tissue fragments visible to the naked eye), and transferred to a 50 mltube.

The fifth step: mu m cell filter filtration of the cell suspension to be obtained

The mixture was slowly poured through a 40 μm cell filter into a clean tube and labeled on the tube wall.

And a sixth step: obtaining single cell suspension of target tissue

And (3) performing heavy suspension counting, namely uniformly mixing the cell suspension filtered in the step 5), centrifuging at 1100rpm for 5min, then removing supernatant, and adding 200-500 ul of HBSS cell cleaning solution containing 5% FBS for heavy suspension. Sucking 10ul of reselected cell suspension and 10ul of AO/PI dye liquor, mixing uniformly, pumping into a counting plate, and counting by a cell counter to obtain the concentration of the original single cell suspension.

Obtaining single cell suspension of target tissue, and diluting original single cell suspension to 8.5 x 10 concentration by HBSS cell washing solution of 5% FBS⁵～1.8*10⁶cells/ml；

The seventh step: determination of viable cell yield

Sucking 10ul AO/PI dye liquor and single cell suspension with the same volume, fully mixing uniformly, sucking 20ul and filling into a pool in a counting plate. The viable cell ratio was determined by AO-PI quality assay from FL20314 of counterstar.

(shown in FIG. 2)

The results are shown in Table 1 below, with total cell numbers ranging from 1.49 to 1.68 x 10⁶About 1.24-1.38 × 10 viable cells⁶Between cells/ml, the viable cell rate is between 79.73 and 83.98 percent. Wherein, one specimen is respectively used for WT wild type and Mu mutant, and the 2 specimens are successfully constructed and wait for on-machine sequencing.

Table 1 is as follows:

the foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.