CN116396923A - Extraction method of mouse testis tissue exosome - Google Patents

Abstract

The invention discloses a method for extracting mouse testis tissue exosomes, which comprises the following steps: (1) Fresh mouse testis tissue is taken and washed 3 times by a PBS solution which is precooled at 4 ℃ and contains 1% of green-streptomycin, and the tissue is sheared on ice; (2) Placing the sheared tissues in a complete culture medium without exosomes for culture; (3) collecting the culture supernatant; (4) removing tissue fragments from the supernatant by centrifugation; (5) Centrifuging the supernatant to remove cell debris in the supernatant; (6) Taking the centrifuged supernatant, and filtering and sterilizing the supernatant by a 0.22 mu m filter; (7) centrifuging the filtrate to obtain exosome precipitate; (8) suspending the precipitate in pre-chilled PBS for washing; (9) Centrifuging the exosome suspension to obtain purified exosome; (10) The exosome pellet was resuspended in pre-chilled PBS to give an exosome suspension. The method can efficiently extract high-concentration and high-purity exosomes from the supernatant of the testis tissue culture of the mice.

Description

A method for extracting exosomes from mouse testis tissue

【Technical field】

The invention belongs to the field of biotechnology, and in particular relates to a method for extracting exosomes from testicular tissue of mice.

【Background technique】

Exosomes are tiny membrane vesicles with a lipid bilayer membrane with a diameter of about 30-150 nm secreted by most cells in the body. As a kind of membranous vesicles ubiquitous in body fluids, exosomes can participate in signal communication and material exchange between cells, mediate physiological and pathological processes, and may also play an important role in the testis. Exosomes contain many types of components, such as miRNA, protein, mRNA, lncRNA, etc. Exosomes can specifically recognize target cells and transfer these components to recipient cells, and regulate the biological activities of recipient cells through the miRNA and other components carried by them, and participate in cell differentiation, cell migration, immune response, Physiological or pathological processes such as tumor invasion.

With the increasing research on exosomes, the related research on exosomes from various cell sources is also increasing. However, at present, the extraction methods of exosomes at home and abroad mainly focus on the cells, and the extraction methods of exosomes from testicular tissue need to be further explored.

【Content of invention】

In view of the problem that there are relatively few studies on the extraction methods of testicular tissue-derived exosomes in the prior art, the present invention provides a method for extracting exosomes from mouse testicular tissue, which can extract and purify exosomes to the greatest extent.

The object of the present invention is achieved through the following technical solutions:

A method for extracting mouse testicular tissue exosomes, comprising the steps of:

1) Take a fresh mouse testis tissue sample and wash it 3 times with 4°C pre-cooled PBS solution containing 1% (v/v) penicillin-streptomycin, and cut the tissue on ice;

2) Culture the shredded tissue in 1% (v/v) penicillin-streptomycin-free exosome-free complete medium;

3) collecting the culture supernatant;

4) removing tissue fragments in the supernatant by first centrifugation;

5) The supernatant obtained by the first centrifugation is taken for the second centrifugation, and the cell debris in the supernatant is removed by the second centrifugation;

6) Take the supernatant obtained by the second centrifugation, and use a 0.22 μm filter to filter and sterilize;

7) centrifuging the filtrate obtained in the above step for the third time to obtain exosome precipitates;

8) suspending the exosome pellet obtained in the above step in pre-cooled PBS and washing to obtain an exosome suspension;

9) Centrifuging the exosome suspension obtained in the above step for the fourth time to obtain purified exosomes;

The exosome pellet was resuspended in pre-cooled PBS to obtain the exosome suspension, and the exosome WB, electron microscopy and particle size identification were performed.

In the present invention:

In step 2), culture in 1% (v/v) penicillin-streptomycin-free complete exosome-free medium, culture condition: 37°C, 5% CO ₂ , culture for 4 hours.

For the first centrifugation described in step 4), the centrifugation condition is: centrifuge at 4° C. at a speed of 1000 g for 5 min.

For the second centrifugation described in step 5), the centrifugation condition is: centrifuge at 4° C. at a speed of 10000 g for 10 min.

For the third centrifugation described in step 7), the centrifugation condition is: centrifuge at 4° C. at a speed of 100,000 g for 70 min.

Step 8) described precooling, preferably 4 ℃.

For the fourth centrifugation described in step 9), the centrifugation condition is: centrifugation at 4° C. at a speed of 100,000 g for 70 min.

Compared with the prior art, the present invention has the following advantages:

A method for extracting exosomes from mouse testicular tissue according to the present invention has strong operability and truly solves the difficulty in extracting tissue-derived exosomes and the fact that exosome extraction reagents are expensive, time-consuming, and the purity of the product is not high. advanced questions. Therefore, using the method of the present invention greatly improves the extraction quality and extraction efficiency of exosomes.

【Description of drawings】

FIG. 1 is a diagram of exosome precipitation extracted by a method for extracting exosomes from mouse testicular tissue according to an embodiment of the present invention;

Fig. 2 is a WB result diagram of a method for extracting exosomes from mouse testicular tissue according to an embodiment of the present invention;

3 is an electron micrograph of exosomes extracted by a method for extracting exosomes from testicular tissue of mice described in an embodiment of the present invention;

Fig. 4 is a diagram of the particle size of exosomes extracted by using a method for extracting exosomes from mouse testicular tissue according to an embodiment of the present invention.

【Detailed ways】

The specific implementation of the present invention will be further described below in conjunction with the examples. The following examples are intended to illustrate the present invention without further limiting the invention. Those who did not indicate the specific conditions in the experimental example can be carried out according to the conventional method.

Example 1:

A method for extracting exosomes from mouse testis tissue, the specific extraction steps are as follows:

(1) Take a fresh mouse testis tissue sample and wash it 3 times with 4°C pre-cooled PBS solution containing 1% (v/v) penicillin-streptomycin, and cut the tissue on ice;

(2) Acquisition of mouse testicular tissue supernatant: place the shredded tissue in 1% (v/v) penicillin-streptomycin-free exosome-free complete medium for culture, put 3ml of culture medium into each 1g of tissue, and place Incubate for 4 hours at 37°C in a 5% CO ₂ incubator;

(3) Collect the culture supernatant after 4 hours, perform the first centrifugation, 4°C, 1000g, centrifuge for 5 minutes, transfer the supernatant to another centrifuge tube, and remove tissue fragments;

(4) Take the above supernatant, and perform a second centrifugation at 4° C., 10,000 g, for 10 minutes, and transfer the supernatant to another centrifuge tube to remove cell debris;

(5) Take the supernatant obtained from the second centrifugation, filter and sterilize with a 0.22 μm filter, and collect the filtrate into an ultrahigh-speed centrifuge tube;

(6) Centrifuge the filtrate for the third time at 4°C and 100,000 g for 70 min, discard the supernatant, and obtain exosome precipitates;

(7) Add pre-cooled PBS into the ultra-high-speed centrifuge tube, pipette the exosomes with a 1000ul pipette tip, resuspend the exosomes in the PBS solution, and wash the exosomes;

(8) Centrifuge the above-mentioned exosome suspension for the fourth time, 4°C, 100,000g, centrifuge for 70min, and a yellowish precipitate appears at the bottom of the ultra-high-speed centrifuge tube, which is the purified exosome (Figure 1);

(9) Take 1ml of pre-cooled PBS and pipette exosomes to precipitate to obtain 1ml of exosomes resuspension, CD9 and CD63 are common markers of exosomes, WB results (Figure 2) and exosomes extracted by the extraction method described in the present invention Electron micrograph of exosomes (Figure 3), indicating that exosomes were successfully extracted;

(10) The diameter of exosomes membranous vesicles secreted by living cells is about 30-150 nm. It can be seen from Table 1 that the average particle size of exosomes is 86 nm and the average concentration is 1.64×10 ¹⁰ Particles/mL. The particle size diagram of exosomes extracted by the extraction method (Figure 4), it can be seen that the particle size of most of the exosomes is in the range of 60-100nm, indicating the purity and concentration of exosomes extracted by the extraction method of the present invention are higher.

Fig. 1 is an exosome sedimentation diagram extracted by a method for extracting exosomes from mouse testicular tissue described in this embodiment;

Fig. 2 is the WB result figure of the extraction method of a kind of mouse testis tissue exosome described in this embodiment;

FIG. 3 is an electron micrograph of exosomes extracted by a method for extracting exosomes from mouse testicular tissue described in this embodiment;

FIG. 4 is a particle size diagram of exosomes extracted by a method for extracting exosomes from mouse testicular tissue described in this example.

Table 1:

Sample name Average particle size (nm) Concentration (Particles/mL)

Mouse 86.0 1.64×10 ¹⁰

Example 2:

A method for extracting mouse testicular tissue exosomes, comprising the steps of:

1) Take a fresh mouse testis tissue sample and wash it 3 times with 4°C pre-cooled PBS solution containing 1% (v/v) penicillin-streptomycin, and cut the tissue on ice;

2) Culture the shredded tissue in 1% (v/v) penicillin-streptomycin complete medium without exosomes, the culture conditions are: 37°C, 5% CO ₂ , culture for 4 hours;

3) collecting the culture supernatant;

4) Remove the tissue fragments in the supernatant by the first centrifugation, the centrifugation conditions are: centrifugation at 4°C at a speed of 1000g for 5min;

5) The supernatant obtained by the first centrifugation was taken for the second centrifugation, the cell debris in the supernatant was removed by the second centrifugation, and centrifuged at 10000g for 10 minutes at 4°C;

6) Take the supernatant obtained by the second centrifugation, and use a 0.22 μm filter to filter and sterilize;

7) Centrifuge the filtrate obtained in the above step for the third time to obtain exosome precipitates, and centrifuge at 100000g for 70min at 4°C;

8) suspending the exosome pellet obtained in the above step in pre-cooled PBS and washing to obtain an exosome suspension;

9) Centrifuge the exosome suspension obtained in the above step for the fourth time to obtain purified exosomes. The centrifugation conditions are: centrifuge at 4°C at a speed of 100,000g for 70min;

The exosome pellet was resuspended in pre-cooled PBS to obtain the exosome suspension, and the exosome WB, electron microscopy and particle size identification were performed.

Comparative example 1:

The existing method for extracting exosomes from mouse testis tissue adopts the ultrafiltration method, including the following steps:

(1) Grind mouse testis tissue, dilute the tissue with 3-5 times the volume of trehalose solution, shake and mix for 10 minutes. 1000g, centrifuge at 25°C for 10min, and collect the supernatant;

(2) First filter the supernatant with a 0.8um filter head, and then filter with a 0.22nm filter head;

(3) Add the ultrafiltration tube and centrifuge for 10-20min, concentrate to 100ul and collect the concentrated solution as the exosome working solution.

Comparative example 2:

Existing methods for extracting exosomes, using size exclusion chromatography, include the following steps:

(1) Grinding mouse testis tissue, adding 3-5 times the volume of PBS buffer, mixing, centrifuging at 1000g, 4°C for 10min, and collecting the supernatant;

(2) Take the above supernatant and put it into secondary centrifugation, centrifuge at 10000g, 4°C for 10min, collect the supernatant, and filter the supernatant with a 0.22nm filter head;

(3) The filtrate flows through the chromatographic column, and substances with a size larger than the pore size of the gel particles cannot enter the pore size, and the exosome working solution is eluted.

Results and summary:

1. Through the comparison of Example 1 and Comparative Example 1, it can be seen that the method of Comparative Example 1 needs to be carried out with centrifugation, but the biggest problem of Comparative Example 1 is that during the filtration process, the retentate on one side of the filter membrane continues to accumulate, making the filtration The efficiency is getting lower and lower. In addition, cholesterol that is close to the size of exosomes will also be trapped, affecting the purity of the product.

2. Through the comparison of Example 1 and Comparative Example 2, it can be seen that the eluate of the method of Comparative Example 2 will dilute exosomes, resulting in a low concentration; in addition, the method of Comparative Example 2 is a widely accepted method for The technique of isolating exosomes in body fluids is not used for the extraction of tissue exosomes, and this method takes a long time and is not suitable for processing a large number of samples.

The above is a further detailed description of the present invention in conjunction with specific embodiments, but the protection of the present invention is not limited thereto. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, any changes or substitutions of the technical features of the technical solution that can be conceived shall be deemed to belong to the protection scope of the present invention.

Claims (6)

1. a method for extracting mouse testis tissue exosomes, characterized in that: comprising the steps of: 1) Take a fresh mouse testis tissue sample and wash it 3 times with 4°C pre-cooled PBS solution containing 1% v/v penicillin-streptomycin, and cut the tissue on ice; 2) Culture the shredded tissue in 1% v/v penicillin-streptomycin-free exosome-free complete medium; 3) collecting the culture supernatant; 4) removing tissue fragments in the supernatant by first centrifugation; 5) The supernatant obtained by the first centrifugation is taken for the second centrifugation, and the cell debris in the supernatant is removed by the second centrifugation; 6) Take the supernatant obtained by the second centrifugation, and use a 0.22 μm filter to filter and sterilize; 7) centrifuging the filtrate obtained in the above step for the third time to obtain exosome precipitates; 8) suspending the exosome pellet obtained in the above step in pre-cooled PBS and washing to obtain an exosome suspension; 9) Centrifuging the exosome suspension obtained in the above step for the fourth time to obtain purified exosomes; The exosome pellet was resuspended in pre-cooled PBS to obtain the exosome suspension, and the exosome WB, electron microscopy and particle size identification were performed. 2. the method for extracting exosomes of a kind of mouse testis tissue according to claim 1, is characterized in that: step 2) described is placed in 1% v/v penicillin-streptomycin and is completely cultured without exosomes basal culture, the culture conditions are: 37°C, 5% CO ₂ , culture for 4h. 3. A method for extracting exosomes from mouse testicular tissue according to claim 1, characterized in that: the first centrifugation described in step 4), the centrifugation condition is: centrifugation at 4°C at a speed of 1000g for 5min . 4. A method for extracting exosomes from mouse testis tissue according to claim 1, characterized in that: the second centrifugation described in step 5), the centrifugation condition is: centrifugation at 4°C at a speed of 10000g for 10min . 5. A method for extracting exosomes from mouse testis tissue according to claim 1, characterized in that: the third centrifugation described in step 7), the centrifugation condition is: centrifugation at 4°C at a speed of 100,000g for 70min . 6. A method for extracting exosomes from mouse testis tissue according to claim 1, characterized in that: the fourth centrifugation described in step 9), the centrifugation condition is: centrifugation at 4°C at a speed of 100,000g for 70min .

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