CN113046307A - Preparation method and application of iPSC (induced pluripotent stem cell) source stem cell exosome - Google Patents
Preparation method and application of iPSC (induced pluripotent stem cell) source stem cell exosome Download PDFInfo
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Abstract
The invention belongs to the technical field of stem cell exosomes, and particularly relates to a preparation method of an iPSC (induced pluripotent stem cell) source stem cell exosome, which comprises the following specific steps: s1: stem cell culture: obtaining stem cell tissue, dividing the stem cell tissue into small particles, and culturing the small particles of the stem cell tissue in a culture medium without exosome serum; s2: obtaining a supernatant through multistage centrifugation: diluting the upper layer of the culture medium and the cultured granular stem cell tissue in the step S1, placing the diluent in a centrifuge for 2-4 times of centrifugation, and obtaining supernatant liquid by each time of centrifugation; s3: separating and filtering the supernatant to obtain exosomes: separating the supernatant finally obtained in the step S2 to obtain exosomes; s4: purifying the exosomes. Through multi-stage centrifugal treatment, the obtained exosome has high purity; the purification of the exosome is further improved through the operation of purifying the exosome; the preparation steps are simple, and the efficiency is high.
Description
Technical Field
The invention relates to the technical field of stem cell exosomes, in particular to a preparation method and application of an iPSC source stem cell exosome.
Background
The iPSC-induced pluripotent stem cells are all called induced pluripotent stem cells and are obtained by artificially inducing non-pluripotent cells to express a specific gene. iPSCs and natural pluripotent stem cells share similarities in many respects, such as expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling, embryoid body formation, teratoma formation, formation of different chimeras, and differentiation potential.
Exosomes are small membrane vesicles (30-150nm) containing complex RNAs and proteins; in 1983, exosomes were first found in sheep reticulocytes, which were named "exosomes" by Johnstone in 1987.
The exosome has wide application, can be used for treating various diseases, has small side effect, reduces the rejection reaction of human bodies, has lower exosome purity prepared by the existing exosome preparation scheme, and influences the using effect.
Disclosure of Invention
The invention aims to provide a preparation method of an iPSC source stem cell exosome and application thereof, and aims to solve the problems that the exosome prepared by the existing exosome preparation scheme in the background technology has low purity and the using effect is influenced.
In order to achieve the purpose, the invention provides the following technical scheme: an iPSC source stem cell exosome preparation method comprises the following specific steps:
s1: stem cell culture: obtaining stem cell tissue, dividing the stem cell tissue into small particles, culturing the small particle stem cell tissue in a culture medium without exosome serum, simultaneously filling excessive oxygen into the culture medium, keeping the temperature of the culture medium at 25 ℃, and shaking the culture medium every 30min during the culture process to enable the oxygen to be dissolved into the culture medium;
s2: obtaining a supernatant through multistage centrifugation: diluting the upper layer of the culture medium and the cultured granular stem cell tissue in the step S1, placing the diluent in a centrifuge for 2-4 times of centrifugation, and obtaining supernatant liquid by each time of centrifugation;
s3: separating and filtering the supernatant to obtain exosomes: separating the supernatant finally obtained in the step S2 to obtain exosomes;
s4: purifying the exosomes: diluting the exosome obtained in the step S3, performing centrifugal separation again to obtain a supernatant, and drying the supernatant to obtain the purified exosome.
Preferably, the stem cell tissue in step S1 is adipose stem cell tissue, and the adipose stem cell tissue is collected from the inside of the fat at the neck, face, arm, back, waist, hip, abdomen, thigh, and calf.
Preferably, the tissue size of the small granular stem cells is 2mm by 1mm in a rectangular parallelepiped shape.
Preferably, in step S1, the pre-tissue culture state of the small granular stem cells is 2-4 generations, and the tissue culture time of the small granular stem cells is 4-5 days.
Preferably, the rotation speed of the centrifuge in the step S2 is 800-.
Preferably, the centrifugation in step S2 is performed in such a manner that the rotation speed of the centrifuge is gradually increased from slow to fast, the increasing time is 10min, and the centrifuge is kept in a stable speed after the increasing.
Preferably, the operation of separating and obtaining exosomes in step S3 is as follows: the centrifuged supernatant is removed, filtered through an ultrafiltration membrane, pressurized from one side so that the liquid and exosomes pass through the ultrafiltration membrane, and the small granular stem cell tissue and impurities are blocked at one side.
An application of an iPSC source stem cell exosome is disclosed, wherein the iPSC source stem cell exosome is used for repairing skin, treating alopecia and early screening cancers.
Compared with the prior art, the invention has the beneficial effects that:
1) through multi-stage centrifugal treatment, the obtained exosome has high purity;
2) the purification of the exosome is further improved through the operation of purifying the exosome;
3) the preparation steps are simple, and the efficiency is high.
Drawings
FIG. 1 is a flow chart of the preparation process of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it is to be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention.
Example 1:
referring to fig. 1, the present invention provides a technical solution: an iPSC source stem cell exosome preparation method comprises the following specific steps:
s1: stem cell culture: the stem cell tissue is obtained and divided into small granular stem cell tissues, the size of the small granular stem cell tissues is 2mm x 1mm in a cuboid shape, the stem cell tissues are adipose stem cell tissues, the adipose stem cell tissues are collected from the fat at the neck, the face, the arm, the back, the waist, the hip, the abdomen, the thigh and the calf, the safety value of the liposuction amount of one liposuction operation is 3000ml, and if the obesity degree is too high, multiple times of suction is needed to achieve the final effect. About 3 percent. There are 2-5 million stem cells in 40 grams of adipose tissue. Is 2500 times more than that in bone marrow, and can obtain at least 16-40 hundred million stem cells per 40 g of adipose tissue in three successive generations of culture. The autologous mesenchymal stem cells can be used for the stem cell transfusion of the donor. Because the cells are autologous cells, immune rejection reactions such as graft-versus-host disease and the like are avoided. The stem cell reinfusion can be used for beauty treatment, health care, and treatment and prevention of some indications. Placing small granular stem cell tissues in a culture medium without exosome serum for culture, wherein the state of the small granular stem cell tissues before culture is 2 generations, the culture time of the small granular stem cell tissues is 4 days, meanwhile, excess oxygen is filled into the culture medium, the culture medium is kept at 25 ℃, in the culture process, the culture medium is shaken every 30min, so that the oxygen is dissolved into the culture medium, an oxygen pump is adopted to fill the oxygen into the culture medium, the oxygen enters the culture medium to accelerate the respiration of the stem cell tissues, the generation rate of exosomes can be improved, the stem cell tissues are better dissolved into the culture medium in a shaking mode, the stem cell tissues are cultured at a proper temperature through heat preservation measures, and the exosomes can be released more quickly;
s2: obtaining a supernatant through multistage centrifugation: diluting the upper layer of the culture medium and the cultured granular stem cell tissue in the step S1, placing the diluent in a centrifuge for 2 times of centrifugation, wherein the rotation speed of the centrifuge is 800r/min, the centrifugation time is 20-80min each time, the centrifugation mode is that the rotation speed of the centrifuge is gradually increased from slow to fast, the increasing time is 10min, the centrifuge is kept in a stable speed for centrifugal operation after increasing, and supernatant is obtained by each centrifugation;
s3: separating and filtering the supernatant to obtain exosomes: and (4) separating the supernatant finally obtained in the step S2 to obtain exosomes, wherein the specific mode of the operation of separating and obtaining exosomes is as follows: removing the centrifuged supernatant, filtering the supernatant through an ultrafiltration membrane, and pressurizing from one side to enable the liquid and the exosomes to pass through the ultrafiltration membrane, so that the granular stem cell tissues and impurities are blocked at one side;
s4: purifying the exosomes: diluting the exosome obtained in the step S3, performing centrifugal separation again to obtain a supernatant, and drying the supernatant to obtain the purified exosome.
An application of an iPSC source stem cell exosome is disclosed, wherein the iPSC source stem cell exosome is used for repairing skin, treating alopecia and early screening cancers. Extracellular nicotinamide phosphoribosyl phosphate transfer mediated by exosomes can reverse mouse senescence and prolong life. The exosome secreted by the human dermal fibroblast can better repair the skin damage caused by photoaging. The sustained release of extracellular vesicles derived from the Dermal Papilla (DP) from the injected microgel can promote hair growth.
Example 2:
referring to fig. 1, the present invention provides a technical solution: an iPSC source stem cell exosome preparation method comprises the following specific steps:
s1: stem cell culture: the stem cell tissue is obtained and divided into small granular stem cell tissues, the size of the small granular stem cell tissues is 2mm x 1mm in a cuboid shape, the stem cell tissues are adipose stem cell tissues, the adipose stem cell tissues are collected from the fat at the neck, the face, the arm, the back, the waist, the hip, the abdomen, the thigh and the calf, the safety value of the liposuction amount of one liposuction operation is 3000ml, and if the obesity degree is too high, multiple times of suction is needed to achieve the final effect. About 3 percent. There are 2-5 million stem cells in 40 grams of adipose tissue. Is 2500 times more than that in bone marrow, and can obtain at least 16-40 hundred million stem cells per 40 g of adipose tissue in three successive generations of culture. The autologous mesenchymal stem cells can be used for the stem cell transfusion of the donor. Because the cells are autologous cells, immune rejection reactions such as graft-versus-host disease and the like are avoided. The stem cell reinfusion can be used for beauty treatment, health care, and treatment and prevention of some indications. Placing small granular stem cell tissues in a culture medium without exosome serum for culture, wherein the state of the small granular stem cell tissues before culture is 3 generations, the culture time of the small granular stem cell tissues is 4.5 days, meanwhile, excess oxygen is filled into the culture medium, the culture medium is kept at 25 ℃, in the culture process, the culture medium is shaken every 30min to ensure that the oxygen is dissolved into the culture medium, an oxygen pump is adopted to fill the oxygen into the culture medium, the oxygen enters the culture medium to accelerate the respiration of the stem cell tissues, the generation rate of exosomes can be improved, the stem cell tissues are better dissolved into the culture medium in a shaking mode, the stem cell tissues are cultured at a proper temperature through heat preservation measures, and the exosomes can be released more quickly;
s2: obtaining a supernatant through multistage centrifugation: diluting the upper layer of the culture medium and the cultured granular stem cell tissue in the step S1, placing the diluent in a centrifuge for 3 times of centrifugation, wherein the rotation speed of the centrifuge is 900r/min, the centrifugation time is 20-80min each time, the centrifugation mode is that the rotation speed of the centrifuge is gradually increased from slow to fast, the increasing time is 10min, the centrifuge is kept in a stable speed for centrifugal operation after increasing, and supernatant is obtained by each centrifugation;
s3: separating and filtering the supernatant to obtain exosomes: and (4) separating the supernatant finally obtained in the step S2 to obtain exosomes, wherein the specific mode of the operation of separating and obtaining exosomes is as follows: removing the centrifuged supernatant, filtering the supernatant through an ultrafiltration membrane, and pressurizing from one side to enable the liquid and the exosomes to pass through the ultrafiltration membrane, so that the granular stem cell tissues and impurities are blocked at one side;
s4: purifying the exosomes: diluting the exosome obtained in the step S3, performing centrifugal separation again to obtain a supernatant, and drying the supernatant to obtain the purified exosome.
An application of an iPSC source stem cell exosome is disclosed, wherein the iPSC source stem cell exosome is used for repairing skin, treating alopecia and early screening cancers. Extracellular nicotinamide phosphoribosyl phosphate transfer mediated by exosomes can reverse mouse senescence and prolong life. The exosome secreted by the human dermal fibroblast can better repair the skin damage caused by photoaging. The sustained release of extracellular vesicles derived from the Dermal Papilla (DP) from the injected microgel can promote hair growth.
Example 3:
referring to fig. 1, the present invention provides a technical solution: an iPSC source stem cell exosome preparation method comprises the following specific steps:
s1: stem cell culture: the stem cell tissue is obtained and divided into small granular stem cell tissues, the size of the small granular stem cell tissues is 2mm x 1mm in a cuboid shape, the stem cell tissues are adipose stem cell tissues, the adipose stem cell tissues are collected from the fat at the neck, the face, the arm, the back, the waist, the hip, the abdomen, the thigh and the calf, the safety value of the liposuction amount of one liposuction operation is 3000ml, and if the obesity degree is too high, multiple times of suction is needed to achieve the final effect. About 3 percent. There are 2-5 million stem cells in 40 grams of adipose tissue. Is 2500 times more than that in bone marrow, and can obtain at least 16-40 hundred million stem cells per 40 g of adipose tissue in three successive generations of culture. The autologous mesenchymal stem cells can be used for the stem cell transfusion of the donor. Because the cells are autologous cells, immune rejection reactions such as graft-versus-host disease and the like are avoided. The stem cell reinfusion can be used for beauty treatment, health care, and treatment and prevention of some indications. Placing small granular stem cell tissues in a culture medium without exosome serum for culture, wherein the state of the small granular stem cell tissues before culture is 4 generations, the culture time of the small granular stem cell tissues is 5 days, meanwhile, excess oxygen is filled into the culture medium, the culture medium is kept at 25 ℃, in the culture process, the culture medium is shaken every 30min, so that the oxygen is dissolved into the culture medium, an oxygen pump is adopted to fill the oxygen into the culture medium, the oxygen enters the culture medium to accelerate the respiration of the stem cell tissues, the generation rate of exosomes can be improved, the stem cell tissues are better dissolved into the culture medium in a shaking mode, the stem cell tissues are cultured at a proper temperature through heat preservation measures, and the exosomes can be released more quickly;
s2: obtaining a supernatant through multistage centrifugation: diluting the upper layer of the culture medium and the cultured granular stem cell tissue in the step S1, placing the diluent in a centrifuge for 4 times of centrifugation, wherein the rotation speed of the centrifuge is 1000r/min, the centrifugation time is 20-80min each time, the centrifugation mode is that the rotation speed of the centrifuge is gradually increased from slow to fast, the increasing time is 10min, the centrifuge is kept in a stable speed for centrifugal operation after increasing, and supernatant is obtained by each centrifugation;
s3: separating and filtering the supernatant to obtain exosomes: and (4) separating the supernatant finally obtained in the step S2 to obtain exosomes, wherein the specific mode of the operation of separating and obtaining exosomes is as follows: removing the centrifuged supernatant, filtering the supernatant through an ultrafiltration membrane, and pressurizing from one side to enable the liquid and the exosomes to pass through the ultrafiltration membrane, so that the granular stem cell tissues and impurities are blocked at one side;
s4: purifying the exosomes: diluting the exosome obtained in the step S3, performing centrifugal separation again to obtain a supernatant, and drying the supernatant to obtain the purified exosome.
An application of an iPSC source stem cell exosome is disclosed, wherein the iPSC source stem cell exosome is used for repairing skin, treating alopecia and early screening cancers. Extracellular nicotinamide phosphoribosyl phosphate transfer mediated by exosomes can reverse mouse senescence and prolong life. The exosome secreted by the human dermal fibroblast can better repair the skin damage caused by photoaging. The sustained release of extracellular vesicles derived from the Dermal Papilla (DP) from the injected microgel can promote hair growth.
While there have been shown and described the fundamental principles and essential features of the invention and advantages thereof, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof; the present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein, and any reference signs in the claims are not intended to be construed as limiting the claim concerned.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A preparation method of an iPSC source stem cell exosome is characterized by comprising the following steps: the method for preparing the exosome of the iPSC source stem cell comprises the following specific steps:
s1: stem cell culture: obtaining stem cell tissue, dividing the stem cell tissue into small particles, culturing the small particle stem cell tissue in a culture medium without exosome serum, simultaneously filling excessive oxygen into the culture medium, keeping the temperature of the culture medium at 25 ℃, and shaking the culture medium every 30min during the culture process to enable the oxygen to be dissolved into the culture medium;
s2: obtaining a supernatant through multistage centrifugation: diluting the upper layer of the culture medium and the cultured granular stem cell tissue in the step S1, placing the diluent in a centrifuge for 2-4 times of centrifugation, and obtaining supernatant liquid by each time of centrifugation;
s3: separating and filtering the supernatant to obtain exosomes: separating the supernatant finally obtained in the step S2 to obtain exosomes;
s4: purifying the exosomes: diluting the exosome obtained in the step S3, performing centrifugal separation again to obtain a supernatant, and drying the supernatant to obtain the purified exosome.
2. The method for preparing an iPSC-derived stem cell exosome according to claim 1, which is characterized in that: the stem cell tissue in step S1 is adipose stem cell tissue, and the adipose stem cell tissue is collected from the inside of fat at the neck, face, arm, back, waist, hip, abdomen, thigh, and calf.
3. The method for preparing an iPSC-derived stem cell exosome according to claim 1, which is characterized in that: the small granular stem cell tissue size is 2mm by 1mm cuboid.
4. The method for preparing an iPSC-derived stem cell exosome according to claim 1, which is characterized in that: the pre-tissue culture state of the small granular stem cells in the step S1 is 2-4 generations, and the tissue culture time of the small granular stem cells is 4-5 days.
5. The method for preparing an iPSC-derived stem cell exosome according to claim 1, which is characterized in that: the rotation speed of the centrifuge in the step S2 is 800-1000r/min, and the centrifugation time is 20-80min each time.
6. The method for preparing an iPSC-derived stem cell exosome according to claim 5, which is characterized in that: the centrifugation mode in the step S2 is that the rotation speed of the centrifuge is gradually increased from slow to fast, the increasing time is 10min, and the centrifuge operation is kept at a stable speed after the increasing time.
7. The method for preparing an iPSC-derived stem cell exosome according to claim 1, which is characterized in that: the specific way of the step S3 of separating and obtaining exosomes is as follows: the centrifuged supernatant is removed, filtered through an ultrafiltration membrane, pressurized from one side so that the liquid and exosomes pass through the ultrafiltration membrane, and the small granular stem cell tissue and impurities are blocked at one side.
8. The application of an iPSC source stem cell exosome is characterized in that: the iPSC source stem cell exosome is used for repairing skin, treating alopecia and early screening cancers.
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