CN114958721A - Extraction method of stem cell exosomes - Google Patents
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Abstract
The invention provides an extraction method of stem cell exosomes, belongs to the technical field of cell extraction, and is used for solving the technical problems of low extraction efficiency and low purity of the existing stem cell exosomes. The method comprises the steps of firstly, carrying out two times of aseptic culture on stem cells to obtain a relatively pure extracting solution with a relatively high exosome content; impurities can be removed twice, so that impurities and vesicles can be effectively removed, and the purity of exosomes is guaranteed; then, screening treatment is carried out, so that residual cells, platelets and a slightly larger extracellular capsule membrane can be removed, and the purity is further improved; then, incubating the exosome; and finally, recovering the exosome through low-speed centrifugation to obtain the high-purity stem cell exosome. Screening purification treatment is carried out to the extract after the edulcoration, has improved the purity of exosome, and the extract after the purification adopts PEG solution to incubate after carry out low-speed centrifugation sediment and can obtain stem cell exosome, convenient operation, weak point consuming time has improved the extraction efficiency and the purity of stem cell exosome, is fit for the mass and draws.
Description
Technical Field
The invention belongs to the technical field of cell extraction, and relates to an extraction method of a stem cell exosome.
Background
The stem cell exosome is a cell secretion from stem cells, and the exosome is a tiny vesicle (30-150 nm wide) secreted by the stem cells, carries bioactive chemical substances such as protein, mRNA and microRNA of the stem cells and has some functions of the stem cells. The exosome is defined by the nanoparticle, and the stem cell exosome can penetrate through a blood brain barrier, is a key substance for transferring intermediate information content between stem cells and other cells, and is beneficial to the completion of the functions of the stem cells. They resemble intercellular "communication soldiers" that can transfer DNA, RNA or proteins between cells, thereby affecting the function of recipient cells, and exosomes are being used to treat diseases ranging from heart disease to respiratory disease in the stem cell and regenerative medicine fields.
The stem cell exosome has similar biological performance with the stem cell, but the stem cell exosome is safer, more stable and more efficient, the exosome does not respond to the damaged microenvironment, has stronger and complex signal molecule transporting and regulating capacity, and has the capacity of changing extracellular matrix, changing transcriptome and proteome of receptor cells, and regulating apoptosis, growth, proliferation and differentiation pathways. Exosomes contain a variety of biological factors that have been found to induce migration and proliferation of fibroblasts and collagen synthesis; has important biological functions of reducing apoptosis, relieving inflammatory reaction, promoting angiogenesis, inhibiting fibrosis, improving tissue repair potential and the like, and has good application prospect in the aspects of tissue injury repair, chronic wound healing, wound scar formation inhibition, aging resistance and the like. Thereby obtaining the high-purity stem cell exosome and developing the premise of application and research.
At present, the separation and extraction of the stem cell exosomes are commonly used by a centrifugation method, a chromatography method, a density gradient centrifugation method and a magnetic bead immunization method; the chromatography has the problems of difficult large-scale production and low efficiency; the density gradient centrifugation method is also not suitable for large-scale production due to the complex and time-consuming operation; the method for separating the exosome based on the ultracentrifugation has the problems of low yield and purity, membrane integrity loss, long time consumption and the like; the magnetic bead immunization method is low in efficiency, the biological activity of the exosome is easily influenced by pH and salt concentration, downstream experiments are not facilitated, and the magnetic bead immunization method is difficult to widely popularize.
Based on the method, the method for extracting the stem cell exosomes can improve the purity and the extraction efficiency.
Disclosure of Invention
The invention aims to provide a method for extracting a stem cell exosome aiming at the problems in the prior art, and the technical problems to be solved by the invention are as follows: how the purity of the stem cell exosomes is high and the extraction efficiency is high.
The purpose of the invention can be realized by the following technical scheme:
an extraction method of stem cell exosomes comprises the following steps:
step one, primary sterile culture of stem cells: culturing stem cells in a culture medium containing serum in a constant-temperature sterile environment, and extracting the stem cells after culturing for a certain time;
step two, secondary sterile culture of stem cells: culturing the extracted stem cells in a serum-free culture medium in a constant-temperature sterile environment for a certain time, and extracting culture supernatant;
step three, primary impurity removal treatment: performing first centrifugation treatment on the culture supernatant obtained in the step two, and extracting the supernatant to remove impurity cells and apoptotic cell debris;
step four, secondary impurity removal treatment: performing second centrifugation treatment on the supernatant obtained in the third step, and extracting the supernatant to remove larger vesicles;
step five, screening treatment: screening the supernatant obtained in the fourth step by using a cell sorting device, and removing residual cells, platelets and a slightly larger extracellular sac membrane to obtain a purified extracting solution containing exosomes;
step six, exosome incubation: mixing the five obtained extracting solutions by adopting a PEG solution, and incubating at the aseptic constant temperature of 4 ℃;
step seven, exosome extraction: and precipitating and recovering the exosome from the incubated extracting solution through low-speed centrifugation to obtain the high-purity stem cell exosome.
Preferably, in the step one, the sterile culture time of the stem cells is (15-18) h; and in the second step, the secondary sterile culture time of the stem cells is (18-24) h.
Preferably, the first centrifugation treatment of the supernatant in the third step is 300g of centrifugal force, the centrifugation treatment (8-10) min is adopted, and the low-speed centrifugation treatment is adopted to remove the impurity cells and the apoptotic cell debris.
Preferably, the second centrifugation treatment of the supernatant in the fourth step adopts 2000g of centrifugal force for centrifugation treatment (8-10) min; by adopting a differential centrifugation mode, larger vesicles in the supernatant can be removed.
Preferably, in the screening treatment in the fifth step, two cell sorting devices connected in series are used for screening to purify the extracting solution, each cell sorting device consists of a microfluidic channel and acoustic transducers at two ends, the acoustic frequency of the first cell sorting device is low and is used for removing residual cells and platelets in the sample, the acoustic frequency of the second cell sorting device is high, and exosomes in the sample are separated from a slightly larger extracellular vesicle membrane to obtain the purified extracting solution containing exosomes.
The cell sorting device is an acoustic wave screening device developed based on a novel acoustic wave fluid technology, the cell sorting device is composed of a micro-fluid channel and acoustic transducers at two ends, and standing waves formed by the mutual contact of acoustic waves generated by the two acoustic transducers can generate a series of pressure nodes; when a cell or particle flows through the channel and encounters a node, the cell is guided slightly off-center by the pressure, and the distance of displacement of the cell depends on other properties such as the size and compressibility of the cell; in order to screen the exosomes, two cell sorting devices are connected in series, the sound wave frequency of the second device is set to be low and used for residual cells and platelets in a sample, the sound wave frequency of the second device is set to be high, the exosomes in the sample are separated from a slightly larger extracellular cyst membrane, and an extracting solution is purified, so that the high-purity exosomes can be obtained conveniently. And the device can be used for completing the screening work of 100ml samples in less than 25 minutes, so that the extraction speed and the purity of exosomes can be greatly improved.
Preferably, the concentration of the PEG solution in the sixth step is 0.25 g/ml.
The preparation method of the PEG solution in the sixth step comprises the following steps: weighing PEG8000 according to the concentration ratio of 0.25g/ml, dissolving in Milli-Q ultrapure water, and performing moist heat sterilization at 121 ℃ for 30 min; the obtained PEG solution is stored in a constant temperature sterile environment at 4 ℃.
Step six is based on a polymer precipitation technology, high molecular weight polyethylene glycol (PEG8000) is adopted as a polymer for incubation, the polyethylene glycol can be combined with hydrophobic protein and lipid molecules for coprecipitation to precipitate exosomes, and the method has the advantages of small influence on the separated exosomes, neutral pH and the like. And after the PEG solution is incubated, the exosome can be obtained through low-speed and short-time centrifugal precipitation, so that the time consumption is short, and the operation is convenient.
Screening and then incubating exosomes, purifying the extracting solution by using a cell sorting device, avoiding the problem of low purity of stem cell exosomes obtained by a polymer precipitation technology, and effectively solving the defects of high speed separation, long time consumption and low efficiency when exosomes are extracted by using differential centrifugation and ultracentrifugation technologies in the prior art by using PEG solution incubation and then carrying out low speed centrifugal precipitation separation; simple operation, high purity, high speed and high efficiency, and can be used for batch extraction.
Preferably, the centrifuge conditions for exosome extraction in step seven are: centrifuging at 500g for 8-10 min.
Compared with the prior art, the extraction method of the stem cell exosome has the following advantages:
1. the extraction method carries out two times of sterile culture on the stem cells, can obtain a relatively pure extracting solution with high exosome content, and is ready for the subsequent steps; impurities and vesicles can be effectively removed by impurity removal twice, so that the purity of exosomes is ensured; screening to remove residual cells, platelets and slightly larger extracellular capsule membrane to obtain purified extract containing exosomes, thereby further improving the purity of exosomes; the exosome is incubated, so that the exosome can be finally extracted conveniently, the extraction is convenient, the consumed time is short, and the extraction speed of the stem cell exosome can be improved.
2. According to the extraction method, the extracting solution is purified by adopting the cell sorting device after secondary impurity removal, the purity of the exosome can be improved, the operation time is short, and the operation is convenient; the purified extracting solution is incubated by a PEG solution and then is subjected to low-speed centrifugal precipitation separation to obtain the stem cell exosomes, the extraction is carried out without long-time ultrahigh-speed centrifugal separation, the purity of the extracted exosomes is high, and the extraction efficiency can be greatly improved.
3. The extraction method has the advantages of simple operation of the whole operation steps, short time consumption, high purity of the extracted stem cell exosomes and suitability for batch extraction.
Drawings
FIG. 1 is a schematic flow diagram of the extraction step of the present invention;
Detailed Description
The technical solution of the present patent will be described in further detail with reference to the following embodiments.
Referring to fig. 1, the present embodiment provides a method for extracting stem cell exosomes, which includes the following steps:
step one, primary sterile culture of stem cells: culturing stem cells in a culture medium containing serum in a constant-temperature sterile environment, and extracting the stem cells after culturing for 16 hours;
step two, secondary sterile culture of stem cells: culturing the extracted stem cells in a serum-free culture medium in a constant-temperature sterile environment for 20 hours, and extracting culture supernatant;
step three, primary impurity removal treatment: and (3) carrying out first centrifugation treatment on the culture supernatant obtained in the step two, extracting the supernatant, wherein in the step, centrifugal treatment is carried out for 10min by adopting a centrifugal force of 300g, and impurity cells and apoptotic cell debris are removed by adopting low-speed centrifugation treatment.
Step four, secondary impurity removal treatment: carrying out second centrifugation treatment on the supernatant obtained in the third step, and extracting the supernatant; in the step, centrifugal treatment is carried out for 10min by adopting a centrifugal force of 2000 g; and (5) matching with the third step, and removing larger vesicles in the supernatant by adopting a differential centrifugation mode.
Step five, screening treatment: screening the supernatant obtained in the fourth step by using a cell sorting device, and removing residual cells, platelets and a slightly larger extracellular sac membrane to obtain a purified extracting solution containing exosomes;
step six, exosome incubation: mixing the five obtained extracting solutions by adopting a PEG solution, and incubating at the aseptic constant temperature of 4 ℃;
step seven, exosome extraction: and precipitating and recovering the exosome from the incubated extracting solution through low-speed centrifugation to obtain the high-purity stem cell exosome. In this step, the centrifuge conditions for exosome extraction were: centrifuging at 500g for 10 min.
And in the fifth step, two cell sorting devices connected in series are adopted for screening, the extracting solution is purified, the sound wave frequency of the first cell sorting device is lower and is used for removing the residual cells and platelets in the sample, the sound wave frequency of the second cell sorting device is higher, and the exosomes in the sample are separated from a slightly larger extracellular cyst membrane, so that the purified extracting solution containing the exosomes is obtained.
The cell sorting device is an acoustic wave screening device developed based on a novel acoustic wave fluid technology, the cell sorting device is composed of a micro-fluid channel and acoustic transducers at two ends, and standing waves formed by the mutual contact of acoustic waves generated by the two acoustic transducers can generate a series of pressure nodes; when a cell or particle flows through a channel and encounters a node, the pressure will direct the cell slightly off-center, and the distance the cell is displaced depends on other properties such as cell size and compressibility. In order to screen the exosomes, two cell sorting devices are connected in series, the sound wave frequency of the second device is set to be low and used for removing residual cells and platelets in a sample, the sound wave frequency of the second device is set to be high, the exosomes in the sample are separated from a slightly larger extracellular cyst membrane, and an extracting solution is purified, so that the high-purity exosomes can be obtained conveniently. And the device can be used for completing the screening work of 100ml samples in less than 25 minutes, so that the extraction speed and the purity of exosomes can be greatly improved.
In the sixth step, the concentration of the PEG solution is 0.25g/ml, and the preparation method of the PEG solution comprises the following steps: weighing PEG8000 according to the concentration ratio of 0.25g/ml, dissolving in Milli-Q ultrapure water, and performing moist heat sterilization at 121 ℃ for 30 min; the obtained PEG solution was stored in a constant temperature sterile environment at 4 ℃.
In this embodiment, the centrifugation device and the cell sorting device used in the centrifugation process in the above process steps are all products in the prior art, and the using method and the operation mode thereof belong to means in the prior art, and detailed descriptions of the specific principle and the operation process are omitted.
The extraction method carries out two times of sterile culture on stem cells, can obtain a relatively pure extracting solution with high exosome content, and is ready for the subsequent steps; the impurity removal treatment is carried out twice, so that cell impurities and vesicles can be effectively removed, and the purity of exosomes is ensured; then screening treatment is carried out, so that residual cells, platelets and a slightly larger extracellular sac can be removed, a purified extracting solution containing exosomes is obtained, and the purity of the exosomes is further improved; polyethylene glycol with high molecular weight is adopted as a polymer for incubation, and the polyethylene glycol can be combined with hydrophobic protein and lipid molecules for coprecipitation to precipitate exosomes, so that the method has the advantages of small influence on the separated exosomes, neutral pH and the like. And after the PEG solution is incubated, the exosome can be obtained through low-speed and short-time centrifugal precipitation, so that the time consumption is short, and the operation is convenient. The exosome is incubated after screening treatment, the extracting solution is purified by adopting a cell sorting device, the problem of low purity of the stem cell exosome obtained by a polymer precipitation technology can be solved, and the low-speed centrifugal precipitation separation is carried out after PEG solution incubation, so that the defects of ultrahigh-speed separation, long consumed time and low efficiency in the existing process of extracting exosomes by adopting differential centrifugation and ultracentrifugation technologies are effectively overcome; thereby improving the extraction efficiency and purity of the stem cell exosomes.
Although the preferred embodiments of the present patent have been described in detail, the present patent is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present patent within the knowledge of those skilled in the art.
Claims (9)
1. A method for extracting a stem cell exosome is characterized by comprising the following steps:
step one, primary sterile culture of stem cells: culturing stem cells in a culture medium containing serum in a constant-temperature sterile environment, and extracting the stem cells after culturing for a certain time;
step two, secondary sterile culture of stem cells: culturing the extracted stem cells in a serum-free culture medium in a constant-temperature sterile environment for a certain time, and extracting culture supernatant;
step three, primary impurity removal treatment: performing first centrifugation treatment on the culture supernatant obtained in the step two, and extracting the supernatant to remove impurity cells and apoptotic cell debris;
step four, secondary impurity removal treatment: performing second centrifugation treatment on the supernatant obtained in the third step, and extracting the supernatant to remove larger vesicles;
step five, screening treatment: screening the supernatant obtained in the fourth step by using a cell sorting device, and removing residual cells, platelets and a slightly larger extracellular sac membrane to obtain a purified extracting solution containing exosomes;
step six, exosome incubation: mixing the five obtained extracting solutions by adopting a PEG solution, and incubating at the aseptic constant temperature of 4 ℃;
step seven, exosome extraction: and precipitating and recovering the exosome from the incubated extracting solution through low-speed centrifugation to obtain the high-purity stem cell exosome.
2. The method for extracting exosomes from stem cells according to claim 1, wherein the sterile culture time of the stem cells in the first step is (15-18) h.
3. The method for extracting exosomes from stem cells according to claim 2, wherein the time for the second sterile culture of the stem cells in the second step is (18-24) h.
4. The method for extracting the stem cell exosomes according to claim 1, wherein the first centrifugation treatment of the supernatant in the third step is performed by using a centrifugal force of 300g for 8-10 min.
5. The method for extracting exosomes from stem cells according to claim 5, wherein the second centrifugation treatment of the supernatant in the fourth step is performed by centrifugation at 2000g for 8-10 min.
6. The method for extracting stem cell exosomes according to claim 1, wherein in the step five screening treatment, the extract is purified by screening with two cell sorting devices connected in series, each cell sorting device comprises a microfluidic channel and acoustic transducers at two ends, the acoustic frequency of the first cell sorting device is low to remove residual cells and platelets in the sample, and the acoustic frequency of the second cell sorting device is high to separate exosomes in the sample from a slightly larger extracellular vesicle membrane, so as to obtain a purified extract containing exosomes.
7. The method for extracting exosomes from stem cells according to claim 1, wherein the concentration of the PEG solution in the sixth step is 0.25g/ml, and the preparation method of the PEG solution is as follows: weighing PEG8000 according to the concentration ratio of 0.25g/ml, dissolving in Milli-Q ultrapure water, and performing moist heat sterilization at 121 ℃ for 30 min; the obtained PEG solution was stored in a constant temperature sterile environment at 4 ℃.
8. The method for extracting exosomes from stem cells according to claim 7, wherein the time for incubating exosomes in the sixth step is (8-10) h.
9. The method for extracting exosomes from stem cells according to claim 1, wherein the centrifugation conditions for exosome extraction in the seventh step are as follows: centrifuging at 500g for 8-10 min.
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