CN118345029A - Extraction method of stem cell exosomes - Google Patents
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Abstract
The invention provides a stem cell exosome extraction method, which belongs to the technical field of stem cell exosome extraction, and the stem cell exosome extraction method comprises the steps of separating a sample into single cells by a trypsin hydrolysis and centrifugation mode, and separating the stem cells by stem cell factors; placing the stem cells in a culture medium to induce the stem cells to release exosomes after the stem cells are amplified; adding the stem cells into an exosome extraction component, and purifying by low-temperature chromatography, so that exosomes are fully extracted from a culture medium and separated from cell substances under the condition of not affecting the intracellular substances; separating the exosomes by density gradient centrifugation to obtain the final exosomes; the invention provides a stem cell exosome extraction method, which can solve the problems that the purity of exosome extracted by the existing method for rapidly extracting exosome from stem cells is insufficient, various other substances are contained, and death of cells is easy to cause in the extraction process.
Description
Technical Field
The invention belongs to the technical field of extraction of stem cell exosomes, and particularly relates to a stem cell exosomes extraction method.
Background
Stem cell Exosomes (Exosomes) are a class of microvesicles with diameters between 30 and 150 nanometers. These vesicles are mainly derived from a variety of cell types, such as stem cells, tumor cells, immune cells, etc., wherein stem cell exosomes have important significance in biological and medical research. The stem cell exosomes have a variety of biological functions, such as modulating cell signaling, promoting cell proliferation and differentiation, modulating immune responses, and the like. Stem cell exosomes are also considered as biomarkers and therapeutic targets of potential application value in the treatment of diseases. In recent years, with the intensive research on stem cell exosomes, related extraction methods are also being continuously developed and perfected. Common methods for extracting stem cell exosomes include differential centrifugation, ultrafiltration, centrifugation, and the like. These methods have advantages and disadvantages and need to be selected and optimized according to the specific situation. Differential centrifugation is one of the common extraction methods, and its principle is to separate cells from extracellular material using a high-speed centrifuge. The method has the advantages of simple operation, good separation effect and the like, but requires longer centrifugation time and can cause the damage of exosomes. The ultrafiltration method is a rapid and efficient extraction method, and the principle is that an ultrafiltration membrane is utilized to separate cells from extracellular substances. The method has the advantages of simple operation, rapidness, high efficiency and the like, but proper ultrafiltration membranes are required to be selected so as to avoid membrane pollution and membrane pore size selection. Centrifugation is a conventional extraction method in which cells and extracellular material are separated by a centrifuge. The method has the advantages of simple operation, good separation effect and the like, but requires longer centrifugation time and higher centrifugation speed, and can lead to the damage of exosomes. Centrifugal centrifugation is a highly efficient extraction method, the principle of which is to separate cells from extracellular material using a centrifugal centrifuge. The method has the advantages of good separation effect, simple operation and the like, but requires longer centrifugation time and higher centrifugation speed, and can lead to the damage of exosomes.
The existing method for rapidly extracting exosomes from stem cells is to directly use trypsin to crack and separate cell membranes of the stem cells, but the extracted exosomes are not pure enough, contain various other substances, and easily cause cell death in the extraction process.
Disclosure of Invention
In view of the above, the present invention provides a method for extracting exosomes from stem cells, which can solve the problems that the exosomes extracted by the existing method for rapidly extracting exosomes from stem cells have insufficient purity, contain various other substances, and easily cause death of cells in the extraction process.
The invention is realized in the following way:
the invention provides a stem cell exosome extraction method, which comprises the following steps:
S10: separating a sample into single cells by means of trypsin hydrolysis and centrifugation, and separating stem cells by stem cell factors;
s20: placing the stem cells in a culture medium to induce the stem cells to release exosomes after the stem cells are amplified;
s30: adding the stem cells into an exosome extraction component, and purifying by low-temperature chromatography, so that exosomes are fully extracted from a culture medium and separated from cell substances under the condition of not affecting the intracellular substances;
S40: and (3) separating further substances from the purified exosomes by density gradient centrifugation to obtain the final exosomes.
The technical effects of the extraction method of stem cell exosomes provided by the invention are as follows: the sample is separated into single cells by means of trypsin hydrolysis and centrifugation, and the stem cells are separated by stem cell factors, so that differentiation and proliferation of the stem cells and release and secretion of stem cell exosomes are promoted, and centrifugation is a common method for separating the cells from the mixture by centrifugation. In the stem cell exosome extraction method, centrifugation may be used to separate stem cells from stem cell exosomes; the stem cell factor can stimulate the differentiation and proliferation of stem cells, so that in the stem cell exosome extraction method, the stem cell factor can promote the differentiation and proliferation of stem cells, thereby increasing the yield of stem cell exosomes; placing the stem cells in a culture medium to induce the stem cells to release exosomes after the stem cells are amplified, and stimulating the stem cells to generate and release exosomes by utilizing various nutritional ingredients and signal molecules in the cell culture medium; the stem cells are added into an exosome extraction component to be purified by low-temperature chromatography, so that exosomes are fully extracted from a culture medium and separated from cell substances under the condition of not affecting the intracellular substances, the exosomes are extracted efficiently, controllably and reproducibly, the degradation rate of proteins is slowed down under the low-temperature condition, and the integrity and activity of the proteins in the exosomes are maintained; compared with normal temperature or high temperature, the low temperature chromatography is more suitable for maintaining the stability of bioactive substances in exosomes.
Based on the technical scheme, the stem cell exosome extraction method can be further improved as follows:
The specific steps of separating the sample into single cells by means of trypsin hydrolysis and centrifugation and separating the stem cells by stem cell factors include:
firstly, sampling tissues and blood, and adding trypsin into the samples for hydrolysis;
adding the hydrolyzed sample into a sample layer of a centrifuge tube;
thirdly, adding centrifugal liquid into a centrifugal liquid layer of the centrifugal tube, and fixing the centrifugal tube on the centrifugal machine for rotary separation;
A fourth step of sucking the separated single cells onto a medium by a pipette;
fifthly, adding stem cell factors to the culture medium to promote proliferation and differentiation of the stem cells;
sixthly, separating the stem cells from the culture medium by a molecular biological method;
The centrifugal liquid is physiological saline, the rotating speed of the centrifugal machine is 1000-3000rpm, and the centrifugal time is 5-30min;
The specific steps of sampling from tissues and blood and adding trypsin to the sample for hydrolysis comprise:
Firstly, preparing required tissue and blood samples, and dissolving and diluting trypsin powder according to the preparation concentration;
secondly, processing the sample;
Thirdly, adding the treated sample into digestion liquid containing trypsin with proper concentration, and gently shaking the culture dish to ensure that the sample is fully and uniformly mixed with the trypsin;
fourthly, carrying out hydrolysis treatment on the sample added with the trypsin at the temperature of 37 ℃;
fifth, after hydrolysis reaches the expected degree, placing the culture dish in a temperature below 20 ℃, and stopping trypsin activity by changing the temperature to prevent excessive hydrolysis;
the specific steps of adding the treated sample into digestion liquid containing trypsin with proper concentration, and gently shaking the culture dish to ensure that the sample is fully and uniformly mixed with the trypsin are as follows:
and designing a quantitative control experiment, and determining the optimal concentration according to an experimental result.
The beneficial effects of adopting above-mentioned improvement scheme are: the method has the advantages that the required components can be more conveniently obtained by the separation method, the loss of the sample in the sampling process can be reduced, the utilization rate of the sample is improved, substances with different densities can be effectively separated, and the purity of the sample is improved; through adding the centrifugate to the centrifugal liquid layer of centrifuging tube, fix the centrifuging tube on the centrifuge and carry out rotatory separation, because the rotatory of centrifuging tube, the different material of density in the mixture receives different centrifugal force effects, and the less material of density is got rid of the bottom of centrifuging tube, and the higher material of density then remains the upper portion of centrifuging tube. The centrifuge layer of the centrifuge tube can be repeatedly added with the centrifuge liquid so as to separate purer substances; the isolated single cells were pipetted onto the medium for further study and analysis; by adding stem cell factors to the culture medium to promote proliferation and differentiation of the stem cells, growth and differentiation of the stem cells can be effectively promoted; the stem cells are separated from the culture medium by a molecular biological method, so that the purity of the stem cells is improved, the risk of mutation is reduced, and the safety of the stem cells is improved.
Sample processing:
in the case of a tissue sample, the tissue is first cut into small pieces or broken into cell suspensions. This facilitates the trypsin to fully contact the cell surface and promote hydrolysis.
In the case of a blood sample, the blood may be added directly to the trypsin-containing digestion solution.
Further, the specific method for separating the stem cells from the culture medium by the molecular biological method comprises the following steps:
firstly, adding cells in a culture medium into a centrifuge for centrifugation, collecting supernatant, and re-suspending the supernatant with the culture medium to prepare a cell suspension;
Secondly, adding the cell suspension into a sample cell of a flow cytometer, and adding an anti-CD 3 marker for identifying and marking stem cells;
and thirdly, starting a flow cytometer, sequentially passing the cells in the sample cell through a cell separation channel, and carrying out cell separation according to the expression condition of the cell surface markers to obtain separated stem cells.
Anti-CD 3 (anti-CD 3 monoclonal antibody) is a common tool for studying T cell and immune system responses. anti-CD 3 antibodies can bind to CD3 molecules on the surface of T cells, thereby activating T cells and allowing them to participate in an immune response. In the laboratory, anti-CD 3 antibodies can be used to label stem cells in order to study their role in immune response and cell differentiation processes.
The method for labeling the anti-CD 3 antibody and the stem cells mainly comprises the following steps:
fluorescent labeling: the anti-CD 3 antibody is conjugated to fluorescein (e.g., alexa Fluor 647, alexa Fluor 700, alexa Fluor 800, etc.) to form a fluorescently labeled anti-CD 3 antibody. The fluorescent-labeled anti-CD 3 antibodies were then bound to stem cells and observed and detected by fluorescence microscopy.
Biotinylation: the anti-CD 3 antibody is conjugated to Biotin (Biotin) to form a biotinylated anti-CD 3 antibody. After the biotinylated anti-CD 3 antibody is bound to the stem cells, the stem cells can be detected by methods such as affinity chromatography.
Affinity chromatography: the anti-CD 3 antibody is conjugated to an avidin (e.g., strepavidin) to form an avidin-linked anti-CD 3 antibody. After the affinity-modified anti-CD 3 antibody is combined with the stem cells, the stem cells can be separated and purified by methods such as affinity chromatography.
Flow cytometry: the anti-CD 3 antibody is coupled with a fluorescent marker, and the expression of the anti-CD 3 receptor on the surface of the stem cells is detected by flow cytometry. The method can be used for rapidly and efficiently detecting and sorting a large number of cells.
Immunofluorescent staining: the anti-CD 3 antibody is coupled with a fluorescent marker, and the expression of the anti-CD 3 receptor of the stem cells is observed and detected through an immunofluorescence staining technology. The method can directly observe the morphology and structure of cells and tissues and can carry out quantitative analysis.
Further, the specific steps of placing the stem cells in a culture medium to induce the stem cells to release exosomes after the stem cells are expanded include:
Firstly, amplifying stem cells, separating the stem cells from a culture medium by a molecular biological method, repeating for a plurality of times, and improving the purity of the stem cells;
Secondly, placing the stem cells in a proper culture medium, and placing the stem cells under proper temperature and humidity conditions to promote the growth and proliferation of the stem cells;
Thirdly, adding cytokines, growth factors and medicines into the culture medium to induce the stem cells to release exosomes;
the proper temperature and humidity conditions are sterile and nontoxic environment with the temperature of 36-37 ℃ and the humidity of 70-90%, and the content ratio of air to carbon dioxide in the environment is 19:1.
Further, the exosome extraction component comprises a shell, a fluid box, an anti-splashing component, a liquid flow component and a temperature control component, wherein the shell is of a square structure, the fluid box is fixed at the central position inside the shell, fluid is placed inside the fluid box, and the stem cells enter the fluid to be subjected to chromatographic filtration through the movement of the fluid; the top of the fluid box is fixedly provided with the splash-proof assembly, and the splash-proof assembly is used for preventing fluid inside the fluid box from splashing; the liquid flow components are arranged on two sides of the top of the fluid box, are opposite to the two sides of the fluid box and are used for applying pressure to fluid in the fluid box so that the fluid moves in the fluid box from top to bottom; water molecule filtering membranes are fixed at the two sides of the fluid box and only can make water molecules pass through; films with different pore sizes are arranged between the water molecule filtering films at the two sides of the fluid box from top to bottom, and the purity of the exosome is improved by adsorbing the fluid after the fluid flows through the films from top to bottom; the temperature control assembly is arranged between the shell and the fluid box and is used for controlling the temperature of fluid in the fluid box so as to reduce the release of cell substances;
The top center and the bottom center of the fluid tank are provided with communicating pipes which are in through connection with the top center and the bottom center of the fluid tank; wherein, the communicating pipe on one side is connected with the deionized water barrel, and the communicating pipe on the other side is used for outputting the purified exosome solution fluid;
The two sides of the communicating pipe, which are far away from the fluid box, are provided with fluid pumps, and the two sides, which are close to the fluid box, are provided with electromagnetic valves, the electromagnetic valves are used for controlling the flow of fluid, and the fluid pumps are used for conveying the fluid;
The shell is made of transparent toughened glass.
The materials of the films with different pore sizes from top to bottom are different, and the films are respectively separated ligands or compounds with specific affinity for other substances, and are used for purifying exosomes in an enrichment mode.
Further, the liquid flow component comprises a fixed shell, an air pipe, an air suction valve, a compression cylinder, a nozzle and a filtering component, wherein the fixed shell is fixed at the top end of the inner wall of the shell and corresponds to the positions of two sides of the fluid tank; one end of the air pipe is connected with the top of the fixed shell in a sealing way, and the other end of the air pipe extends out of the shell and is used for sucking air into the shell; the compression cylinder is fixed in the fixed shell and corresponds to the air pipe in position and is used for compressing air sucked by the air pipe; one end of the nozzle is connected with the fixed shell in a penetrating way, and the other end of the nozzle stretches into the fluid box and is used for pumping air compressed by the compression cylinder into the fluid box; the air suction valve is fixed on the air pipe, the air outlet valve is fixed on the nozzle, and the air suction valve and the air outlet valve are used for controlling air inlet and outlet inside the fixed shell;
after the air suction valve and the air outlet valve are closed, the inside of the fixed shell is sealed and is used for rapidly compressing gas;
The air pipe is in a shape of two funnels combined into an integrated structure, and the cross-sectional area of two ends is larger than that of the middle part, so that pressure difference is formed; the area with small middle cross-sectional area of the air pipe is provided with the filter assembly, and the filter assembly is used for sterilizing and filtering air entering the liquid flow assembly;
The filter assembly is an air purifier and is used for filtering pollutants in the air;
and a pressure sensor is fixed on the inner wall of the fixed shell and used for monitoring the pressure change inside the fixed shell.
Further, the splash-proof assembly is a grid-shaped structure, and glass balls are fixed in each grid of the grid-shaped structure, and the diameter of each glass ball is the same as the length of each grid;
The periphery of the splash-proof assembly is provided with an inward-bending round corner structure, and the round corner structure is used for reflecting water drops splashed on the periphery of the splash-proof assembly back to the inside of the fluid tank.
Further, the pore size of the films with different pore sizes is changed from top to bottom to 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 500nm and 300nm, and the films are used for purifying exosomes layer by layer.
Further, the temperature control assembly comprises a flow pipe, liquid nitrogen circulates in the flow pipe, and the temperature in the fluid box is controlled to be between-10 ℃ and 4 ℃ by adjusting the distance between the flow pipe and the fluid box.
Further, the fluid placed in the fluid box is phosphate buffer solution.
Compared with the prior art, the stem cell exosome extraction method provided by the invention has the beneficial effects that: the sample is separated into single cells by means of trypsin hydrolysis and centrifugation, and the stem cells are separated by stem cell factors, so that differentiation and proliferation of the stem cells and release and secretion of stem cell exosomes are promoted, and centrifugation is a common method for separating the cells from the mixture by centrifugation. In the stem cell exosome extraction method, centrifugation may be used to separate stem cells from stem cell exosomes; the stem cell factor can stimulate the differentiation and proliferation of stem cells, so that in the stem cell exosome extraction method, the stem cell factor can promote the differentiation and proliferation of stem cells, thereby increasing the yield of stem cell exosomes; placing the stem cells in a culture medium to induce the stem cells to release exosomes after the stem cells are amplified, and stimulating the stem cells to generate and release exosomes by utilizing various nutritional ingredients and signal molecules in the cell culture medium; the stem cells are added into an exosome extraction component to be purified by low-temperature chromatography, so that exosomes are fully extracted from a culture medium and separated from the cell substances under the condition of not affecting the intracellular substances, the exosomes in the cells are extracted efficiently, controllably and reproducibly, and the degradation rate of the proteins is slowed down under the low-temperature condition, and the integrity and activity of the proteins in the exosomes are maintained; compared with normal temperature or high temperature, the low temperature chromatography is more suitable for maintaining the stability of bioactive substances in exosomes. This helps to maximize the functionality of the exosomes during the purification process; and separating further substances from the purified exosomes by density gradient centrifugation to obtain the final exosomes.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments of the present invention will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of the operation of a method for extracting stem cell exosomes;
FIG. 2 is a schematic structural view of an exosome extraction assembly;
FIG. 3 is a schematic structural view of an anti-splash assembly;
In the drawings, the list of components represented by the various numbers is as follows:
10. A housing; 20. a fluid tank; 30. an anti-splash assembly; 40. a liquid flow assembly; 41. a fixed case; 42. an air duct; 43. an air suction valve; 44. a compression cylinder; 45. a nozzle; 46. a filter assembly; 47. an air outlet valve; 50. and a temperature control assembly.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
As shown in fig. 1-3, the present invention provides an operation flow chart of a stem cell exosome extraction method, which includes the following steps:
S10: separating a sample into single cells by means of trypsin hydrolysis and centrifugation, and separating stem cells by stem cell factors;
s20: after the stem cells are amplified, placing the stem cells in a culture medium to induce the stem cells to release exosomes;
S30: adding stem cells into an exosome extraction component, purifying by low-temperature chromatography, and separating exosomes from the culture medium without affecting intracellular substances;
S40: and (3) separating further substances from the purified exosomes by density gradient centrifugation to obtain the final exosomes.
When in use, the sample is separated into single cells by means of trypsin hydrolysis and centrifugation, and the stem cells are separated out by stem cell factors; after the stem cells are amplified, placing the stem cells in a culture medium to induce the stem cells to release exosomes; adding stem cells into an exosome extraction component, purifying by low-temperature chromatography, and separating exosomes from the culture medium without affecting intracellular substances; and (3) separating further substances from the purified exosomes by density gradient centrifugation to obtain the final exosomes.
In the above technical scheme, the specific steps of separating the sample into single cells by means of trypsin hydrolysis and centrifugation, and separating the stem cells by stem cell factors include:
firstly, sampling from tissues and blood, and adding trypsin into the samples for hydrolysis;
Adding the hydrolyzed sample into a sample layer of the centrifuge tube;
thirdly, adding centrifugal liquid into a centrifugal liquid layer of the centrifugal tube, and fixing the centrifugal tube on the centrifugal machine for rotary separation;
A fourth step of sucking the separated single cells onto a medium by a pipette;
Fifthly, adding stem cell factors to the culture medium to promote proliferation and differentiation of stem cells;
sixthly, separating the stem cells from the culture medium by a molecular biological method;
The centrifugate is normal saline, the rotation speed of the centrifuge is 1000-3000rpm, and the centrifugation time is 5-30min;
The specific steps of sampling from tissue and blood and adding trypsin to the sample for hydrolysis include:
Firstly, preparing required tissue and blood samples, and dissolving and diluting trypsin powder according to the preparation concentration;
secondly, processing the sample;
Thirdly, adding the treated sample into digestion liquid containing trypsin with proper concentration, and gently shaking the culture dish to ensure that the sample is fully and uniformly mixed with the trypsin;
fourthly, carrying out hydrolysis treatment on the sample added with the trypsin at the temperature of 37 ℃;
Fifthly, after the hydrolysis reaches the expected degree, placing the culture dish in the temperature below 20 ℃, and stopping the activity of trypsin by changing the temperature to prevent excessive hydrolysis;
Adding the treated sample into digestion liquid containing trypsin with proper concentration, and gently shaking the culture dish to ensure that the sample is fully and uniformly mixed with the trypsin, wherein the specific steps are as follows:
and designing a quantitative control experiment, and determining the optimal concentration according to an experimental result.
Designing a quantitative control experiment, and determining the optimal concentration according to the experimental result comprises the following specific steps:
preparation:
Trypsin solutions of different concentrations were prepared, and concentrations of 0.05%, 0.1%, 0.15%, 0.2% and the like were selected.
The same type of tissue sample or blood sample is prepared.
Experiment design:
The tissue sample or blood sample is divided into a plurality of equal parts, and each part of sample is respectively added into trypsin digestion liquid with different concentrations to form different treatment groups.
A control group, i.e. samples without trypsin digestion, was designed for comparison.
And (3) hydrolysis treatment:
The samples of each treatment group were subjected to hydrolysis treatment under the same treatment conditions, such as temperature, treatment time, etc.
The reaction was stopped:
After the hydrolysis treatment has ended, the trypsin activity is stopped using a suitable method, such as changing the temperature or adding trypsin inhibitors.
Analysis of experimental results:
the same assay is performed on samples from each treatment group, such as to determine the activity of a particular protein or enzyme released.
The experimental results of trypsin treatment groups with different concentrations were compared to find out the concentration with the best effect.
Determining the optimal concentration:
the optimal trypsin concentration, i.e. the concentration that is able to hydrolyze the sample sufficiently without over-digestion, is determined based on the experimental results.
The experiment was repeated:
in order to verify the reliability of the experimental results, the above steps may be repeated for a plurality of experiments.
The method for confirming the optimal concentration of the digestion liquid containing trypsin for carrying out trypsin separation on the tissues in the extraction process of the stem cell exosomes of the adipose tissues comprises the following steps:
1. Preparation:
Four trypsin solutions of different concentrations, 0.05%, 0.1%, 0.15%, 0.2% were prepared.
Adipose tissue samples of the same origin were prepared.
2. Experimental group settings:
Four treatment groups were set, each with trypsin solutions of the four concentrations described above, labeled group a (0.05%), group B (0.1%), group C (0.15%), and group D (0.2%).
A control group, i.e. samples without trypsin digestion, was set and labeled as control group.
3. And (3) hydrolysis treatment:
the adipose tissue samples of each treatment group were subjected to hydrolysis treatment at 37℃for 2 hours.
4. The reaction was stopped:
after the hydrolysis treatment is completed, the trypsin activity is stopped by changing the temperature or adding a trypsin inhibitor.
5. Data analysis:
and measuring the extraction rate of exosomes or the content of related proteins of exosomes in each group of samples as evaluation indexes.
Table 1: extraction rate of exosomes in adipose tissue samples hydrolyzed by trypsin solutions of different concentrations:
| Treatment group | Trypsin concentration (%) | Exosome extraction rate |
| Control group | 0 | 1 |
| Group A | 0.05 | 0.8 |
| Group B | 0.1 | 0.9 |
| Group C | 0.15 | 1.2 |
| Group D | 0.2 | 0.7 |
Data analysis shows that:
by comparing the exosome extraction rates of the treatment groups, it was found that the exosome extraction rate of group C (0.15% trypsin concentration) was highest relative to the control group, reaching 1.2 times.
Exosome extraction rate of group B (0.1% trypsin concentration) was also higher, reaching 0.9 fold.
Exosome extraction rates for group a (0.05% trypsin concentration) and group D (0.2% trypsin concentration) were relatively low, 0.8-fold and 0.7-fold, respectively.
Thus, from these data, it can be initially deduced that the addition of 0.15% trypsin digest was the optimal concentration during extraction of exosomes in adipose tissue.
Further, in the above technical solution, the specific method for separating stem cells from a culture medium by a molecular biological method includes:
firstly, adding cells in a culture medium into a centrifuge for centrifugation, collecting supernatant, and re-suspending the supernatant with the culture medium to prepare a cell suspension;
secondly, adding the cell suspension into a sample cell of a flow cytometer, and adding an anti-CD 3 marker for identifying and marking stem cells;
and thirdly, starting a flow cytometer, sequentially passing the cells in the sample cell through a cell separation channel, and carrying out cell separation according to the expression condition of the cell surface markers to obtain separated stem cells.
Further, in the above technical scheme, the specific steps of placing the stem cells in a culture medium to induce the stem cells to release exosomes after the stem cells are expanded include:
Firstly, amplifying stem cells, separating the stem cells from a culture medium by a molecular biological method, repeating for a plurality of times, and improving the purity of the stem cells;
Secondly, placing the stem cells in a proper culture medium, and placing the stem cells under proper temperature and humidity conditions to promote the growth and proliferation of the stem cells;
Thirdly, adding cytokines, growth factors and medicines into the culture medium to induce the stem cells to release exosomes;
the proper temperature and humidity conditions are sterile and nontoxic environment with the temperature of 36-37 ℃ and the humidity of 70-90%, and the content ratio of air to carbon dioxide in the environment is 19:1.
Further, in the above technical solution, the exosome extraction assembly includes a housing 10, a fluid tank 20, an anti-splashing assembly 30, a liquid flow assembly 40, and a temperature control assembly 50, the housing 10 has a square structure, the fluid tank 20 is fixed at the central position inside the housing 10, the fluid is placed inside the fluid tank 20, and the stem cells enter the fluid to be chromatographically filtered through the movement of the fluid; a splash guard assembly 30 is fixed to the top of the fluid tank 20, the splash guard assembly 30 being used to guard against splashing of fluid inside the fluid tank 20; the two sides of the top of the fluid tank 20 are provided with liquid flow components 40, and the liquid flow components 40 are opposite to the two sides of the fluid tank 20 and are used for applying pressure to the fluid in the fluid tank 20 so as to enable the fluid to move in the fluid tank 20 from top to bottom; water molecule filtering membranes are fixed at the two sides of the fluid box 20 and only can pass water molecules; films with different pore sizes are arranged between the water molecule filtering films at the two sides of the fluid box 20 from top to bottom, and the purity of the exosome is improved by absorbing the fluid after the fluid flows through the films from top to bottom; a temperature control assembly 50 is arranged between the housing 10 and the fluid tank 20, and the temperature control assembly 50 is used for controlling the temperature of the fluid in the fluid tank 20 so as to reduce the release of cell substances;
The top center and the bottom center of the fluid tank 20 are provided with communicating pipes which are in through connection with the top center and the bottom center of the fluid tank 20; wherein, the communicating pipe on one side is connected with the deionized water barrel, and the communicating pipe on the other side is used for outputting purified exosome solution fluid;
the two sides of the communicating pipe far away from the fluid tank 20 are provided with fluid pumps, the two sides close to the fluid tank 20 are provided with electromagnetic valves, the electromagnetic valves are used for controlling the flow of fluid, and the fluid pumps are used for conveying the fluid;
The housing 10 is made of transparent toughened glass.
When the device is used, the electromagnetic valve on the communicating pipe connecting the fluid tank 20 and the deionized water barrel is opened, so that fluid enters the fluid tank 20, stem cells are added into the fluid tank 20, the liquid flow component 40 pumps compressed air into the side edge position of the fluid tank 20, fluid in the fluid tank 20 is driven to move from top to bottom through the fluidity of the fluid, substances in the fluid are separated through chromatography, the separated communicating pipe passing through the bottom is led out, purified cells are subjected to further substance separation through density gradient centrifugation, and a final exosome is obtained.
Further, in the above-mentioned technical solution, the liquid flow assembly 40 includes a fixed shell 41, an air duct 42, an air suction valve 43, a compression cylinder 44, a nozzle 45 and a filter assembly 46, wherein the fixed shell 41 is fixed at the top end of the inner wall of the housing 10, and corresponds to the positions of two sides of the fluid tank 20; one end of the air pipe 42 is connected with the top of the fixed shell 41 in a sealing way, and the other end of the air pipe extends out of the shell 10 and is used for sucking air into the shell 10; a compression cylinder 44 is fixed in the fixed shell 41, and the compression cylinder 44 corresponds to the position of the air pipe 42 and is used for compressing air sucked by the air pipe 42; one end of the nozzle 45 is connected with the fixed shell 41 in a penetrating way, and the other end of the nozzle penetrates into the fluid tank 20, so that air compressed by the compression cylinder 44 is pumped into the fluid tank 20; an air suction valve 43 is fixed on the air pipe 42, an air outlet valve 47 is fixed on the nozzle 45, and the air suction valve 43 and the air outlet valve 47 are used for controlling air inlet and outlet inside the fixed shell 41;
after the suction valve 43 and the air outlet valve 47 are closed, the inside of the fixed housing 41 is sealed for rapidly compressing the gas;
The air duct 42 is formed by combining two funnels into a whole, and the cross-sectional area of the two ends is larger than that of the middle part, so that a pressure difference is formed; the area of the air duct 42 with a small middle cross-sectional area is provided with a filter assembly 46, and the filter assembly 46 is used for sterilizing and filtering the air entering the liquid flow assembly 40;
the filter assembly 46 is an air cleaner for filtering contaminants in the air;
a pressure sensor is fixed to the inner wall of the fixed case 41 for monitoring pressure change inside the fixed case 41.
When the chromatographic separation device is used, the air pipe 42 sucks air into the fixed shell 41, the air suction valve 43 is closed, the air in the fixed shell 41 is compressed by the compression cylinder 44, the air outlet valve 47 is opened, the compressed air is pumped into the two side positions of the fluid box 20, the fluid in the fluid box 20 is driven to move from top to bottom by the fluidity of the fluid, and substances in the fluid are separated by chromatography.
Further, in the above technical solution, the splash-preventing assembly 30 is a grid structure, and glass balls are fixed in each grid of the grid structure, and the diameter of each glass ball is the same as the length of each grid;
Around the splash guard assembly 30 is an inwardly curved rounded corner structure for re-reflecting water droplets that splash around the splash guard assembly 30 to the interior of the fluid tank 20.
Further, in the above technical scheme, the pore diameters of the films with different pore diameters are changed from top to bottom to 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 500nm and 300nm, so as to purify exosomes layer by layer.
Further, in the above-mentioned technical solution, the temperature control assembly 50 includes a flow pipe, the liquid nitrogen circulates in the flow pipe, and the temperature in the fluid tank 20 is controlled between-10 ℃ and 4 ℃ by adjusting the distance between the flow pipe and the fluid tank 20.
Further, in the above technical solution, the fluid placed inside the fluid tank 20 is phosphate buffer.
When in use, the sample is separated into single cells by means of trypsin hydrolysis and centrifugation, and the stem cells are separated out by stem cell factors; after the stem cells are amplified, placing the stem cells in a culture medium to induce the stem cells to release exosomes; the stem cells are added into the exosome extraction component, the electromagnetic valve on the communicating pipe connecting the fluid tank 20 and the deionized water barrel is opened, so that fluid enters the fluid tank 20, the stem cells are added into the fluid tank 20, the air pipe 42 sucks air into the fixed shell 41, the air suction valve 43 is closed, the compression cylinder 44 compresses the air in the fixed shell 41, the air outlet valve 47 is opened, the compressed air is pumped into the two side positions of the fluid tank 20, the fluid in the fluid tank 20 is driven to move from top to bottom through the fluidity of the fluid, substances in the fluid are separated through chromatography, the separated communicating pipe passing through the bottom is led out, and the purified cells are subjected to further substance separation through density gradient centrifugation, so that the final exosome is obtained.
Specifically, the principle of the invention is as follows: when in use, the sample is separated into single cells by means of trypsin hydrolysis and centrifugation, and the stem cells are separated out by stem cell factors; placing the stem cells in a culture medium to induce the stem cells to release exosomes after the stem cells are amplified; the stem cells are added into the exosome extraction component, the electromagnetic valve on the communicating pipe connecting the fluid tank 20 and the deionized water barrel is opened, so that fluid enters the fluid tank 20, the stem cells are added into the fluid tank 20, the air pipe 42 sucks air into the fixed shell 41, the air suction valve 43 is closed, the air in the fixed shell 41 is compressed by the compression cylinder 44, the air outlet valve 47 is opened, the compressed air is pumped into the two side positions of the fluid tank 20, the fluid in the fluid tank 20 is driven to move from top to bottom by the flowability of the fluid, substances in the fluid are separated by chromatography, the separated communicating pipe passing through the bottom is led out, and the purified cells are further separated by density gradient centrifugation, so that the final exosome is obtained.
Claims (10)
1. A method for extracting stem cell exosomes, comprising the steps of:
S10: separating a sample into single cells by means of trypsin hydrolysis and centrifugation, and separating stem cells by stem cell factors;
s20: placing the stem cells in a culture medium to induce the stem cells to release exosomes after the stem cells are amplified;
s30: adding the stem cells into an exosome extraction component, and purifying by low-temperature chromatography, so that exosomes are fully extracted from a culture medium and separated from cell substances under the condition of not affecting the intracellular substances;
S40: and (3) separating further substances from the purified exosomes by density gradient centrifugation to obtain the final exosomes.
2. The method for extracting stem cell exosomes according to claim 1, wherein the specific steps of separating the sample into individual cells by trypsin hydrolysis and centrifugation, and separating the stem cells by stem cell factor comprise:
firstly, sampling tissues and blood, and adding trypsin into the samples for hydrolysis;
adding the hydrolyzed sample into a sample layer of a centrifuge tube;
thirdly, adding centrifugal liquid into a centrifugal liquid layer of the centrifugal tube, and fixing the centrifugal tube on the centrifugal machine for rotary separation;
A fourth step of sucking the separated single cells onto a medium by a pipette;
fifthly, adding stem cell factors to the culture medium to promote proliferation and differentiation of the stem cells;
sixthly, separating the stem cells from the culture medium by a molecular biological method;
The centrifugal liquid is physiological saline, the rotating speed of the centrifugal machine is 1000-3000rpm, and the centrifugal time is 5-30min;
The specific steps of sampling from tissues and blood and adding trypsin to the sample for hydrolysis comprise:
Firstly, preparing required tissue and blood samples, and dissolving and diluting trypsin powder according to the preparation concentration;
secondly, processing the sample;
Thirdly, adding the treated sample into digestion liquid containing trypsin with proper concentration, and gently shaking the culture dish to ensure that the sample is fully and uniformly mixed with the trypsin;
fourthly, carrying out hydrolysis treatment on the sample added with the trypsin at the temperature of 37 ℃;
fifth, after hydrolysis reaches the expected degree, placing the culture dish in a temperature below 20 ℃, and stopping trypsin activity by changing the temperature to prevent excessive hydrolysis;
the specific steps of adding the treated sample into digestion liquid containing trypsin with proper concentration, and gently shaking the culture dish to ensure that the sample is fully and uniformly mixed with the trypsin are as follows:
and designing a quantitative control experiment, and determining the optimal concentration according to an experimental result.
3. The method for extracting stem cell exosomes according to claim 2, wherein the specific method for separating stem cells from the culture medium by molecular biology method comprises:
firstly, adding cells in a culture medium into a centrifuge for centrifugation, collecting supernatant, and re-suspending the supernatant with the culture medium to prepare a cell suspension;
Secondly, adding the cell suspension into a sample cell of a flow cytometer, and adding an anti-CD 3 marker for identifying and marking stem cells;
and thirdly, starting a flow cytometer, sequentially passing the cells in the sample cell through a cell separation channel, and carrying out cell separation according to the expression condition of the cell surface markers to obtain separated stem cells.
4. A method for extracting exosomes from stem cells according to claim 3, wherein the specific step of placing the stem cells in a culture medium to induce release of exosomes after expansion of the stem cells comprises:
Firstly, amplifying stem cells, separating the stem cells from a culture medium by a molecular biological method, repeating for a plurality of times, and improving the purity of the stem cells;
Secondly, placing the stem cells in a proper culture medium, and placing the stem cells under proper temperature and humidity conditions to promote the growth and proliferation of the stem cells;
Thirdly, adding cytokines, growth factors and medicines into the culture medium to induce the stem cells to release exosomes;
the proper temperature and humidity conditions are sterile and nontoxic environment with the temperature of 36-37 ℃ and the humidity of 70-90%, and the content ratio of air to carbon dioxide in the environment is 19:1.
5. The stem cell exosome extraction method according to claim 4, wherein the exosome extraction assembly comprises a housing (10), a fluid tank (20), an anti-splash assembly (30), a liquid flow assembly (40) and a temperature control assembly (50), the housing (10) has a square structure, the fluid tank (20) is fixed at an inner central position of the housing (10), a fluid is placed in the fluid tank (20), and the stem cells enter the fluid for chromatographic filtration through movement of the fluid; the top of the fluid tank (20) is fixedly provided with the anti-splashing component (30), and the anti-splashing component (30) is used for preventing fluid inside the fluid tank (20) from splashing; the liquid flow components (40) are arranged on two sides of the top of the fluid tank (20), the liquid flow components (40) are opposite to two sides of the fluid tank (20), and are used for applying pressure to fluid in the fluid tank (20) so that the fluid moves in the fluid tank (20) from top to bottom; water molecule filtering membranes are fixed at the two sides of the fluid box (20), and only water molecules can pass through the water molecule filtering membranes; films with different pore sizes are arranged between water molecule filtering films at two sides of the fluid box (20) from top to bottom, and the purity of the exosome is improved by adsorbing the fluid after the fluid flows through the films from top to bottom; the temperature control assembly (50) is arranged between the shell (10) and the fluid tank (20), and the temperature control assembly (50) is used for controlling the temperature of fluid in the fluid tank (20) so as to reduce the release of cell substances;
Communicating pipes are arranged at the top center and the bottom center of the fluid tank (20), and are in through connection with the top center and the bottom center of the fluid tank (20); wherein, the communicating pipe on one side is connected with the deionized water barrel, and the communicating pipe on the other side is used for outputting the purified exosome solution fluid;
the two sides of the communicating pipe, which are far away from the fluid tank (20), are provided with fluid pumps, the two sides, which are close to the fluid tank (20), are provided with electromagnetic valves, the electromagnetic valves are used for controlling the flow of fluid, and the fluid pumps are used for conveying the fluid;
the shell (10) is made of transparent toughened glass.
6. The stem cell exosome extraction method according to claim 5, wherein the liquid flow assembly (40) comprises a fixed shell (41), an air pipe (42), an air suction valve (43), a compression cylinder (44), a nozzle (45) and a filtering assembly (46), wherein the fixed shell (41) is fixed at the top end of the inner wall of the shell (10) and corresponds to two side positions of the fluid tank (20); one end of the air pipe (42) is connected with the top of the fixed shell (41) in a sealing way, and the other end of the air pipe extends out of the shell (10) and is used for sucking air into the shell (10); the compression cylinder (44) is fixed in the fixed shell (41), and the compression cylinder (44) corresponds to the position of the air pipe (42) and is used for compressing air sucked by the air pipe (42); one end of the nozzle (45) is connected with the fixed shell (41) in a penetrating way, and the other end of the nozzle penetrates into the fluid tank (20) and is used for pumping air compressed by the compression cylinder (44) into the fluid tank (20); the air suction valve (43) is fixed on the air pipe (42), the air outlet valve (47) is fixed on the nozzle (45), and the air suction valve (43) and the air outlet valve (47) are used for controlling air inlet and outlet in the fixed shell (41);
After the air suction valve (43) and the air outlet valve (47) are closed, the inside of the fixed shell (41) is sealed for rapidly compressing air;
The air pipe (42) is in a shape of two funnels combined into an integrated structure, and the cross-sectional area of two ends is larger than that of the middle part, so that pressure difference is formed; the area with small middle cross-sectional area of the air pipe (42) is provided with the filtering component (46), and the filtering component (46) is used for sterilizing and filtering air entering the liquid flow component (40);
The filter assembly (46) is an air purifier for filtering contaminants in air;
a pressure sensor is fixed on the inner wall of the fixed shell (41) and is used for monitoring the pressure change inside the fixed shell (41).
7. The stem cell exosome extraction method according to claim 6, wherein the splash prevention assembly (30) is a grid structure, and glass spheres are fixed in each grid of the grid structure, and the diameter of each glass sphere is the same as the length of each grid;
the periphery of the splash-proof assembly (30) is provided with an inward-bending round corner structure, and the round corner structure is used for reflecting water drops splashed on the periphery of the splash-proof assembly (30) to the inside of the fluid tank (20).
8. The method according to claim 7, wherein the membranes with different pore sizes have pore diameters varying from top to bottom to 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 500nm, 300nm for purifying the exosomes layer by layer.
9. The method according to claim 8, wherein the temperature control assembly (50) comprises a flow tube, through which liquid nitrogen circulates, and wherein the temperature inside the fluid tank (20) is controlled between-10 ℃ and 4 ℃ by adjusting the distance between the flow tube and the fluid tank (20).
10. The method according to claim 9, wherein the fluid placed inside the fluid tank (20) is phosphate buffer.
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