CN113558042A

CN113558042A - Method for preserving exosome

Info

Publication number: CN113558042A
Application number: CN202110753662.6A
Authority: CN
Inventors: 赵绍军; 颜学术
Original assignee: Panas Cell Xiamen Biotechnology Co ltd
Current assignee: Panas Cell Xiamen Biotechnology Co ltd
Priority date: 2021-07-02
Filing date: 2021-07-02
Publication date: 2021-10-29

Abstract

The invention discloses a method for preserving exosome, which comprises the step of mixing ectoine with ectosome to obtain mixed solution for preservation. The mass fraction of the tetrahydropyrimidine in the mixed solution is 0.5-5%, preferably 3%. The ectosomes are preserved for a long time at a lower temperature by using the tetrahydropyrimidine, so that the stability of the ectosomes can be maintained.

Description

Method for preserving exosome

Technical Field

The invention relates to the technical field of biology, in particular to a method for preserving exosomes.

Background

Exosomes (Exosomes) are one kind of Extracellular Vesicles (EVs), have a size of 30-150 nm, are released outside cells in an exocrine form after being fused with cell membranes by intracellular multivesicular bodies (MVBs), and are widely present in cell culture supernatants and various body fluids including blood, lymph, saliva, urine, semen, milk and the like. Exosomes are widely involved in intercellular substance transport and information transfer, and regulate cell physiological activities. Meanwhile, the exosome has the effects of antigen presentation, immune escape, induction of normal cell transformation, promotion of tumorigenesis and metastasis and the like. In addition, exosomes may also be delivered as "natural nanoparticles".

The exosome has a vesicle structure with a double-layer lipid membrane, has good stability, and can protect internal biomolecules from being influenced by various enzymes in body fluid, so that the integrity and the biological activity of the exosome are maintained. However, the integrity and biological activity of the extracted exosomes may be affected by factors such as preservation medium, preservation temperature and time. Exosomes are typically suspended in Phosphate (PBS) buffer after extraction. At present, the most common storage method is cryopreservation, but cryopreservation may cause changes of the shape and physical properties of the exosome, and may also cause formation and aggregation of multilamellar vesicles, and repeated cryopreservation may cause changes of biological characteristics, content and marker composition of the exosome surface molecules. Although-80 ℃ is recognized as the optimum storage temperature for various biological specimens, such as semen, urine, milk, blood and bronchoalveolar lavage fluid, the freezing and thawing process is prone to vesicle destruction, resulting in exosomes containing large amounts of "contaminants", and it is sometimes difficult to maintain such low temperature conditions during handling or transport. Storage at 4 ℃ while maintaining good integrity of the vesicles, is prone to loss of proteins and nucleic acids from the exosomes. Therefore, how to effectively preserve exosomes has been a problem in the field of exosome research.

Disclosure of Invention

The invention aims to provide a method for preserving exosomes aiming at the defects in the prior art, wherein the ectosomes are preserved for a long time at a lower temperature by using tetrahydropyrimidine, so that the stability of the exosomes can be maintained.

Therefore, the invention adopts the following technical scheme:

a method for preserving exosome comprises mixing ectoine with tetrahydropyrimidine to obtain mixed solution, and preserving.

Preferably, the tetrahydropyrimidine is precooled for more than 30min before use.

Preferably, the particle concentration of the exosome in the mixed solution is 106-1012/mL.

Preferably, the mass fraction of the tetrahydropyrimidine in the mixed solution is 0.5-5%.

Preferably, the mass fraction of the tetrahydropyrimidine in the mixed solution is 3%.

Preferably, the preservation temperature of the mixed solution is-80-4 ℃.

Preferably, the preservation temperature of the mixed solution is 4 ℃, and the cycle is 2 weeks.

Preferably, the preservation temperature of the mixed solution is-20 ℃, and the period is 2 months.

The technical scheme has the advantages that: the ectosomes are preserved at low temperature by using the ectoin protective agent, so that the preservation effect can be improved, the stability of the ectosomes is maintained, and the change of the ectosomes in particle size, concentration and activity is avoided.

Drawings

FIG. 1 is a graph showing the particle size distribution of exosomes of experimental examples 1, 3, 4 and control example;

FIG. 2 is a graph showing the proliferation potency of exosomes of experimental examples 1, 3, 4 and control example;

FIG. 3 is a graph showing the particle size distribution of exosomes of Experimental example 3 after being stored at 4 ℃ for various times;

FIG. 4 is a graph showing the particle size distribution of exosomes of Experimental example 3 after being stored at-20 ℃ for various times;

FIG. 5 is a graph showing the particle size distribution of exosomes of the control example after storage at-80 ℃ for various periods of time;

FIG. 6 is a transmission electron microscopy image of exosomes of Experimental example 3 stored at 4 ℃ for 2 weeks;

FIG. 7 is a transmission electron microscopy image of exosomes of Experimental example 3 stored at-20 ℃ for 6 months.

Detailed Description

In order that the objects, features and advantages of the invention will be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings, which are illustrated in detail in order to provide a thorough understanding of the invention, but which may be carried out in other ways than those described. Accordingly, the invention is not limited by the specific implementations disclosed below.

Example one

In the prior art, the mesenchymal stem cells for preparing the exosomes of the mesenchymal stem cells comprise: umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, and the like. In this example, umbilical cord mesenchymal stem cells were selected as the experimental subject.

First, the present embodiment provides a method for culturing umbilical cord mesenchymal stem cells, including the following steps:

step a, taking out the umbilical cord mesenchymal stem cell cryopreservation tube of the generation P3 from a liquid nitrogen tank, and immediately placing the umbilical cord mesenchymal stem cell cryopreservation tube in a water bath at 37 ℃ for thawing for 2 minutes. After the contents are completely melted, wiping the wall of the cryopreservation tube by using 75% alcohol, and then placing the cryopreservation tube in a biological safety cabinet.

Step b, transferring the recovered umbilical cord mesenchymal stem cell suspension into a T75 culture bottle, and then adding 30mL of cell culture medium (the components are: DMEM/F12+ 10% FBS +2mM L-glutathione +100U/mL penicillin +100 mu g/mL streptomycin, and the basic culture medium of DMEM/F12 can be alpha-MEM or L-DMEM medium) which is incubated well into the T75 culture bottle to make the concentration of the stem cells be 4X 10⁶/mL, placing T75 flask at 37 deg.C, 5% CO₂In the incubator under the condition, the cells are cultured overnight, the adherence condition and the cell morphology of the cells are observed on the next day, and then the culture medium is replaced by the new culture medium for incubation.

And c, observing the result by using a microscope, and starting subculture after 70-85% of cells are fused, wherein the specific operation is as follows: washing adherent cells twice with PBS buffer solution, digesting with 0.25% pancreatin until the cells are detached from the wall, adding fresh culture medium to neutralize the pancreatin, subculturing the cells according to the ratio of 1:3, and fusing 20% of the cells after inoculation.

And d, changing the culture medium after about 3 days, changing the culture medium into 30mL of the cultured exosome collecting culture medium (DMEM/F12+ 10% exosome-free FBS +2mM L-glutathione +100U/mL penicillin +100 mu g/mL streptomycin +7 mu m pluripotency hormone after 70% -80% of cells are observed to be fused, and culturing for 48-72 hours by using an alpha-MEM or L-DMEM culture medium as the basic culture medium, and observing the fusion condition and the morphology of the cells. When the cell fusion rate reaches more than 90%, the cell culture solution is obtained.

The embodiment also provides a preparation method of the mesenchymal stem cell exosome, which comprises the following steps:

and a step a, placing the supernatant of the obtained cell culture solution into a centrifuge tube, centrifuging for 5min at the temperature of 4 ℃ by using a centrifugal force of 500g, and collecting the supernatant of the centrifuge tube.

And step b, placing the supernatant of the centrifuge tube collected in the step a into the centrifuge tube, centrifuging for 30min at the temperature of 4 ℃ by using a centrifugal force of 2000g, removing cell debris and protein polymers, and collecting the supernatant of the centrifuge tube again.

And c, passing the supernate of the centrifuge tube collected in the step b through a 0.22 mu m filter membrane to obtain a crude exosome extracting solution.

And d, centrifuging the crude exosome extract for 1h at the temperature of 4 ℃ by using a centrifugal force of 10000g to remove large apoptotic bodies, and collecting the supernate of a centrifugal tube.

And e, placing the supernatant of the centrifuge tube collected in the step d into the centrifuge tube, centrifuging for 2 hours at 4 ℃ by using 100000g of centrifugal force, then resuspending and precipitating by using 200 mu l of PBS buffer solution, and separating to obtain the mesenchymal stem cell exosome. And (4) freezing the separated mesenchymal stem cell exosome at the temperature of-80 ℃ for later use.

The prepared mesenchymal stem cell exosome adopts a particle tracking analysis (NTA) technology to measure the particle size and the concentration, and the test result is shown in the table 1:

TABLE 1 particle size and concentration of exosomes

Particle concentration (10)⁹/mL)	Standard deviation of	Particle size of NTA (nm)	Standard deviation of
				2.8	0.9	95	15.2

As can be seen from table 1, the concentration and particle size of the mesenchymal stem cell exosomes extracted by the ultracentrifugation method are consistent with those reported in the literature, i.e., the obtained mesenchymal stem cell exosomes can be applied to subsequent experiments.

Example two

This example compares the preservation effect of different concentrations of protectant.

The exosomes prepared in the first embodiment are respectively subpackaged and stored by using different protective agents, and the method comprises the following steps:

experimental example 1: after the ectoine protective agent is added and mixed, the mass fraction of ectoine in the mixed solution is 0.5 percent;

experimental example 2: after the ectoine protective agent is added and mixed, the mass fraction of ectoine in the mixed solution is 1 percent;

experimental example 3: after the ectoine protective agent is added and mixed, the mass fraction of ectoine in the mixed solution is 3 percent;

experimental example 4: after the ectoine protective agent is added and mixed, the mass fraction of ectoine in the mixed solution is 5 percent;

comparative example 1: no tetrahydropyrimidine protecting agent was added.

Precooling the ectoine protective agent for more than 30min before use.

Storage conditions are as follows: -20 ℃.

After one month, redissolving is carried out, and the concentration and the particle size of the exosomes of the experimental examples 1, 3, 4 and the comparative example are detected and analyzed through Dynamic Light Scattering (DLS), and a test result shows that the storage effect of the example 3 is best, the concentration of the exosomes of the example 3 is not changed too much after redissolving and compared with the exosomes before cryopreservation, meanwhile, the tested particle size distribution result is shown in figure 1, and the particle size of the exosomes of the example 3 is well stored.

EXAMPLE III

This example used the CCK-8 method to test the proliferative capacity of exosomes.

Taking the 12 th generation of human umbilical cord mesenchymal stem cells (the generation of the cells has weakened proliferation capacity,activity reduction) at 5 × 10 per well³Each cell was inoculated in a 96-well plate, and 10. mu.L each of the exosomes of experimental examples 1 to 4 and control example was added. The cells at 1d, 3d, 5d and 7d were detected, 10. mu.L of stain was added, and cultured for 2 hours. OD values were measured at 450nm with a microplate reader, and 6 replicates were taken for each group and averaged. The test results are shown in table 2 and fig. 2, the proliferation abilities of the exosomes are enhanced with the increase of days, the proliferation abilities of the exosomes of experimental examples 1-4 are stronger than those of the comparative example, and the proliferation ability of the exosome of experimental example 3 is strongest.

TABLE 2 average values of exosome proliferation potency

	1d	3d	5d	7d
						0％Ectoin	0.25	0.32	0.54	0.59
0.5％Ectoin	0.23	0.38	0.68	0.72
					1％Ectoin	0.25	0.42	0.76	0.85
3％Ectoin	0.26	0.43	0.77	0.89
					5％Ectoin	0.27	0.43	0.78	0.88

Example four

This example compares the retention times of the protectant under different temperature conditions.

Test group 1: experimental example 3 was separately stored in portions under different storage conditions, including:

temperature: 4 ℃, time: 2 weeks;

temperature: 4 ℃, time: 1 month;

temperature: 4 ℃, time: 3 months;

temperature: 4 ℃, time: 6 months.

Test group 2: experimental example 3 was separately stored in portions under different storage conditions, including:

temperature: -20 ℃, time: 2 weeks;

temperature: -20 ℃, time: 1 month;

temperature: -20 ℃, time: 3 months;

temperature: -20 ℃, time: 6 months.

Control group: the comparative examples were separately stored under different storage conditions, including:

temperature: -80 ℃, time: 2 weeks;

temperature: -80 ℃, time: 1 month;

temperature: -80 ℃, time: 3 months;

temperature: -80 ℃, time: 6 months.

After the

test groups

1 and 2 and the control group are completed, the concentration and the particle size of the exosome under different storage conditions in each group are analyzed through dynamic light scattering detection, the test result is shown in fig. 3-5, and after the exosome in the experimental example 3 is stored for 2 weeks at 4 ℃, the particle size is intact, so that the stability of the particle size distribution can be maintained.

The exosomes of the control example were stored in a frozen state at-80 ℃ and their particle size increased with the increase in storage time. The exosome of experimental example 3 still had intact particle size after being stored at-20 ℃ for 6 months.

The exosome of experimental example 3 stored at 4 ℃ for 2 weeks was subjected to transmission electron microscopy, and the detection result is shown in fig. 6, and the structural morphology of the exosome is complete.

The exosome of experimental example 3 stored at-20 ℃ for 6 months was subjected to transmission electron microscopy, and the detection result is shown in fig. 7, and the structural morphology of the exosome is complete.

Therefore, the fresh exosome is preserved by using the ectoine protective agent at a lower temperature, so that the preservation effect can be improved, the stability of the exosome is maintained, and the exosome is prevented from changing in particle size, concentration and activity.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for preserving exosomes is characterized in that tetrahydropyrimidine is mixed with exosomes to obtain mixed liquor for preservation.

2. The method for preserving exosomes according to claim 1, wherein the ectoine is precooled for more than 30min before use.

3. The method for preserving exosomes according to claim 1, wherein the particle concentration of exosomes in the mixed solution is 10⁶～10¹²/mL。

4. The method for preserving exosomes according to claim 1, wherein the mass fraction of the tetrahydropyrimidine in the mixed solution is 0.5 to 5%.

5. The method for preserving exosomes according to claim 4, wherein the mass fraction of the tetrahydropyrimidine in the mixed solution is 3%.

6. The method for preserving exosomes according to claim 5, wherein the preservation temperature of the mixed solution is-80 to 4 ℃.

7. A method for preserving exosomes according to claim 6, wherein the preservation temperature of the mixed solution is 4 ℃ for 2 weeks.

8. A method for preserving exosomes according to claim 6, wherein the preservation temperature of the mixed solution is-20 ℃ for 2 months.