CN113621569A - Autologous adipose-derived stem cell exosome based on oxygen concentration gradient and preparation method and application thereof - Google Patents
Autologous adipose-derived stem cell exosome based on oxygen concentration gradient and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an autologous adipose-derived stem cell exosome based on an oxygen concentration gradient, and a preparation method and application thereof. The invention provides a preparation method of an adipose-derived stem cell exosome, which comprises the following steps: placing the adipose-derived stem cells under a series of concentration gradients of which the oxygen concentration is from low to high to culture in sequence; collecting culture supernatant, separating and purifying to obtain the adipose-derived stem cell exosome. Based on an oxygen concentration gradient culture technology, the proliferation capacity of adipose-derived stem cells is stimulated to the utmost extent and the potential of functional exosomes are stimulated and secreted, a supernatant stock solution rich in the stem cell exosomes is separated through centrifugal concentration and nano-membrane filtration, nutrient components and active factors for promoting cell growth in the stem cell culture supernatant are retained to the maximum extent, other components which are difficult to absorb, such as macromolecular proteins and cell fragments, are removed, the scalp hair regeneration and repair micro-environment is improved effectively.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to an autologous adipose-derived stem cell exosome based on an oxygen concentration gradient and a preparation method and application thereof.
Background
The hair is composed of two parts of hair shaft and hair root. The part extending out of the skin is called hair shaft, and the part embedded in the skin is called hair root. Hair loss includes natural androgenic hair loss, burn hair loss, traumatic hair loss, iatrogenic hair loss, and the like. There are many methods of repairing hair defects: scalp expansion, scalp reduction, scalp strip free transplantation, flap method, and autologous hair transplantation method. The hair loss can be repaired by the repairing methods, but the hair transplant is large, so the defects of unnatural hair direction and density distribution after the operation, large damage of the affected area, low survival rate, influence on the appearance and the like are caused.
Exosomes (exosomes) are micro vesicles secreted by various cells under normal or pathological conditions, have the diameter of 30-150nm, are mainly derived from a multivesicular body formed by invagination of intracellular lysosomal microparticles, are released into an extracellular matrix in a budding-like manner after the outer membrane of the multivesicular body is fused with a cell membrane, and have a lipid bilayer membrane structure. Exosomes contain complex RNA and proteins, and play important roles in immune response, antigen presentation, cell differentiation, intercellular information transfer, tumor growth and invasion, tissue homeostasis control, and the like. Compared with stem cells, the stem cell exosome is more stable and safer, is more convenient to store and better controls the quality, and is expected to become a new treatment method.
Disclosure of Invention
The invention aims to provide an autologous adipose-derived stem cell exosome based on an oxygen concentration gradient, and a preparation method and application thereof.
In a first aspect, the invention claims a preparation method of an adipose-derived stem cell exosome.
The preparation method of the adipose-derived stem cell exosome comprises the following steps:
(I) placing the adipose-derived stem cells under a series of concentration gradients of which the oxygen concentration is from low to high to culture in sequence; the series of concentration gradients of the oxygen concentration from low to high comprises low oxygen and high oxygen; the low oxygen refers to an oxygen concentration lower than 20.9% (air standard oxygen content), and the high oxygen refers to an oxygen concentration higher than 20.9% (air standard oxygen content).
And (II) collecting the adipose-derived stem cells cultured in the step (I) or culture supernatant of the adipose-derived stem cells after passage, and separating and purifying to obtain the adipose-derived stem cell exosomes.
Wherein, the series of concentration gradients of the oxygen concentration from low to high refers to the oxygen concentration from 4% to 32%.
In the invention, the series of concentration gradients of the oxygen concentration from low to high is specifically set as follows: 4%, 8%, 16%, 24%, 28% and 32%.
In the present invention, the oxygen concentration refers to volume percentage.
In the step (I), when the adipose-derived stem cells are cultured under each oxygen concentration gradient, the culture time can be 10-14h (such as 12 h); CO 22The concentration of (A) can be 5% (volume percentage); the culture temperature can be 37 ℃; the adopted culture medium can be serum-free stem cell culture medium. In a particular embodiment of the invention, said serum-free stem cell culture medium is in particular TherAPAKTMMSCGM-CDTMMesenchymal Stem Cell Medium, chemical Defined (Chinese name: TheraPEAK)TMMSCGM-CDTMHuman mesenchymal stem cell culture medium), product coding: YS000471, supplier: LONZA corporation.
In step (I), before the adipose-derived stem cells are sequentially cultured in a series of concentration gradients from low to high oxygen concentration, the method may further include the following steps: and (3) placing the adipose-derived stem cells in a serum-free stem cell culture medium for culturing for 8 h. Followed by another PBS wash (e.g., two washes).
In the step (II), the adipose-derived stem cell exosomes can be isolated and purified according to a method comprising the following steps: after collecting the culture supernatant of the adipose-derived stem cells, firstly removing intact cells and dead cell debris through low-speed centrifugation, and then carrying out high-speed centrifugation on the centrifuged supernatant to obtain an adipose-derived stem cell exosome precipitate; the low-speed centrifugation is carried out for 8-10min at the temperature of 4 ℃ and 2000 g; the high-speed centrifugation is performed for 90min at 100000g at 4 ℃.
Further, the method can also comprise the step of suspending the adipose-derived stem cell exosome sediment in a PBS solution, and then sequentially filtering the sediment by using 0.45-micron and 0.22-micron filters, wherein the obtained filtrate is a stock solution rich in the adipose-derived stem cell exosomes.
In a second aspect, the present invention claims an adipose stem cell exosome or an adipose stem cell exosome-rich stock solution prepared by the method described in the first aspect.
In a third aspect, the invention claims a hair growth promoting injection.
The hair growth promoting injection claimed by the invention is prepared from the stock solution rich in the adipose-derived stem cell exosome and the normal saline for injection; the hair growth promoting injection at least contains 1 × 10 of the components per milliliter5Exosomes produced by individual adipose-derived stem cells (i.e. exosomes prepared according to the method described in the first aspect above).
Furthermore, the hair growth promoting injection contains 1 × 10 of the total weight of the hair growth promoting injection per milliliter5Exosomes produced by individual adipose-derived stem cells (i.e. exosomes prepared according to the method described in the first aspect above).
In a fourth aspect, the invention claims any of the following applications:
use of P1, an adipose stem cell exosome or an adipose stem cell exosome-rich stock solution as described in the second aspect, in the preparation of a hair growth promoting injection as described in the third aspect.
Use of P2, an adipose stem cell exosome or an adipose stem cell exosome-rich stock solution as described hereinbefore in the second aspect or a hair-growth injection as described hereinbefore in the third aspect for hair growth (i.e. hair regeneration).
Use of P3, an adipose stem cell exosome or an adipose stem cell exosome-rich stock solution as described in the second aspect or a hair-growing injection as described in the third aspect for tissue repair.
In a fifth aspect, the invention claims any of the following methods:
method A, a method for increasing the proliferation potency of adipose-derived stem cells, comprising the step (I) of the method of the first aspect, to obtain adipose-derived stem cells with increased proliferation potency.
The proliferation capacity of the adipose-derived stem cells cultured in the step (I) is stronger than that of the adipose-derived stem cells cultured under the condition of normal oxygen concentration (20.9%), except that the oxygen concentration is different and the other conditions are the same.
Method B, a method for improving tissue repair capacity of adipose-derived stem cells, comprising the step (I) of the method of the first aspect, wherein the tissue repair capacity of exosomes secreted by the adipose-derived stem cells cultured in the step (I) is improved.
The tissue repair capacity of the exosome secreted by the adipose-derived stem cell cultured in the step (I) is stronger than that of the exosome secreted by the adipose-derived stem cell cultured under the condition of normal oxygen concentration (20.9%) (the other conditions are the same except for different oxygen concentrations).
In the present invention, the adipose-derived stem cells may be commercially available adipose-derived stem cells, or adipose-derived stem cells isolated from adipose tissue ex vivo.
In the present invention, the adipose-derived stem cells may be human adipose-derived stem cells.
In the present invention, the adipose-derived stem cells are preferably autologous adipose-derived stem cells.
The autologous adipose-derived stem cell exosome prepared by the method can effectively utilize autologous tissues, and has the advantages of easiness in operation, no immunogenic reaction risk, high activity, obvious effect, low cost, strong operability and the like. The exosome is an exosome derived from autologous adipose-derived stem cells, and is used for regulating and restoring the functions of hair follicle stem cells and realizing hair regeneration. The method has the advantages of convenient material acquisition, convenient use, safety, reliability and obvious effect.
Based on an oxygen concentration gradient culture technology, the proliferation capacity of adipose-derived stem cells is stimulated to the utmost extent and the potential of functional exosomes are stimulated and secreted, a supernatant stock solution rich in the stem cell exosomes is separated through centrifugal concentration and nano-membrane filtration, nutrient components and active factors for promoting cell growth in the stem cell culture supernatant are retained to the maximum extent, other components which are difficult to absorb, such as macromolecular proteins and cell fragments, are removed, the scalp hair regeneration and repair micro-environment is improved effectively.
Drawings
FIG. 1 is a graph showing the growth curve of autologous adipose-derived stem cells under different oxygen concentrations.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 adipose-derived stem cell exosomes based on oxygen concentration gradient and preparation method and application thereof
Selection of adipose tissue
Autologous fat was selected for the experiment in this example. 150 ml of aseptically collected autologous adipose tissues of the abdomen or the hip in vitro are quickly put into PBS buffer solution containing 0.01M gentamicin, and the pH value is pH7.2.
Preparation of adipose-derived stem cells
1. Placing autologous adipose tissue into a tissue culture dish under GMP conditions, removing macroscopic blood vessels and fiber parts in the fat by using surgical scissors and forceps, washing for 3 times by using PBS buffer solution, removing fat on the upper layer of the fat and blood liquid on the bottom layer, and obtaining pure fat on the middle layer.
2. Cutting adipose tissue to about 1mm by tissue scissors3The left and right tissue pieces were transferred to a T175 flask, and DMEM/F12 medium containing 10% (by volume) fetal bovine serum was added and incubated in a 37 ℃ constant temperature cell incubator.
3. Changing the liquid every 5 days, and obtaining primary adipose-derived stem cells (P0 generation) when the cell fusion degree reaches 20 percent, wherein the cell fusion degree is determined according to the following steps of 1: 3 subculture, culturing to 5 th generation (P1-P5 generation), and storing as experimental autologous adipose-derived stem cells.
Third, test grouping
Transplanting the good-growth-state P3 autologous adipose-derived stem cells, wherein the number of the autologous adipose-derived stem cells is 1 × 105Passage 175 cm square cell culture flasks, trade markPurified serum-free stem cell culture medium (TherAPAK)TMMSCGM-CDTMMesenchymal Stem Cell Medium, chemical Defined (Chinese name: TheraPEAK)TMMSCGM-CDTMHuman mesenchymal stem cell culture medium), product coding: YS000471, supplier: LONZA corporation) for 8 hours, the cells were washed twice with PBS, and the cells were randomly divided into two groups.
Oxygen concentration gradient treatment group: 4% of O2，5％CO2Culturing at 37 deg.C for 12 hs; 8% of O2，5％CO2Culturing at 37 deg.C for 12 hs; 16% O2，5％CO2Culturing at 37 deg.C for 12 hs; 24% O2，5％CO2Culturing at 37 deg.C for 12 hs; 28% O2，5％CO2Culturing at 37 deg.C for 12 hs; 32% O2，5％CO2And culturing at 37 ℃ for 12 hs.
Oxygen concentration normal treatment group: 20.9% O2，5％CO2And culturing at 37 ℃ for 72 hs. Nitrogen equilibration if necessary (i.e. other than O)2And CO2In addition, the remaining gas was nitrogen).
Both groups were cultured at 72 hs. Wherein, the gas content is volume percentage content. When the above two groups of cells were cultured, the above commercial serum-free stem cell culture medium (English name: TherAPAK) was still usedTMMSCGM-CDTMMesenchymal Stem Cell Medium, chemical Defined, Chinese name: therapeakTMMSCGM-CDTMHuman mesenchymal stem cell culture medium, product code: YS000471, supplier: LONZA corporation).
Fourth, CCK-8(CellCounting Kit 8) method detects cell proliferation
1. Respectively inoculating the autologous adipose-derived stem cells of the three oxygen concentration gradient treatment group and the oxygen concentration normal treatment group to a 96-well cell culture plate, wherein the cell concentration is 2 multiplied by 104100 μ l/well, in 8 duplicate wells per group, seven groups were inoculated in sequence, from 1 to 7.
2. The culture was continued for 7 days, and during the Cell culture, a CCK-8 solution (Cell Counting Kit 8, cat # 96992, product of Sigma-Aldrich, supplier: Merck, USA) was added to 1 group of Cell culture media at fixed time points daily at 10. mu.l/well, and after incubation for 2.5hs, OD was measured at a wavelength of 450 nm.
3. The results show that: the proliferation trends of the autologous adipose-derived stem cells of the oxygen concentration gradient group and the oxygen concentration normal group are both in a typical S-shaped stem cell growth curve, and the proliferation capacity of the stem cells of the oxygen concentration gradient group is obviously higher than that of the stem cells of the oxygen concentration normal group, which is shown in figure 1. The extreme stress factors can effectively stimulate the proliferation potential of the stem cells to be fully exerted.
Fifth, application of stock solution and hair-growing injection for preparing exosome rich in autologous adipose-derived stem cells
1. Preparation of exosome stock solution rich in autologous adipose-derived stem cells
Transplanting the autologous adipose-derived stem cells with good growth state obtained by the step of three oxygen concentration gradient treatment, wherein the number of the autologous adipose-derived stem cells is 1 multiplied by 105Cell culture flasks subcultured to 175 cm square, using commercial serum-free Stem cell Medium (TheraPEAK)TMMSCGM-CDTMMesenchymal Stem Cell Medium, chemical Defined (Chinese name: TheraPEAK)TMMSCGM-CDTMHuman mesenchymal stem cell culture medium), product coding: YS000471, supplier: LONZA corporation), when the fusion degree of the cell growth reaches 80% -90%, the cell reaches 5X 106Collecting culture supernatant; centrifuging at 4 ℃ for 10min, and centrifuging at 2000g to remove intact cells and dead cell debris to obtain adipose-derived stem cell supernatant; filtering the supernatant with 0.45 μm and 0.22 μm filters to obtain stock solution rich in autologous adipose-derived stem cell exosome, storing at 4 deg.C in refrigerator due to the stock solution being rich in active factors, and storing at low temperature.
2. Preparation of hair-growing injection
Diluting the autologous adipose-derived stem cell exosome stock solution obtained in the step 1 by using normal saline for injection under aseptic conditions to prepare a hair growth injection, wherein each milliliter of the hair growth injection contains 1 multiplied by 105And (3) exosomes produced by the autologous adipose-derived stem cells.
3. Application method
Adopts a special hair-growing micro-needle injector (Haifeel-S)TMNumber: 10034073610321 supplier: beijing east century trade limited), will grow hairThe injection is injected into scalp at a dose of 20 μ l/part once a week, and 4-8 times is a treatment course according to alopecia range and degree, and the treatment course is 2-3 treatment courses per year.
The results show that: after 2 courses of treatment, alopecia is obviously improved, hair growth is gradually increased, and the exosome from the oxygen concentration gradient group has a more obvious hair growth effect, and detailed results are shown in table 1. The activation of hair follicle cells by various cytokines after treatment, as well as the maintenance of this activation state and the improvement of the overall scalp condition, are revealed to have an effective promoting effect on the growth of head hair.
TABLE 1 analysis of the effect of stimulating the production of exosomes from autologous adipose-derived stem cells to promote hair regeneration in the head by oxygen concentration variation
Note: the ratio of increase in hair number compared to before use (number of hairs after use-number of hairs before use)/number of hairs before use. The ratio of the head hair defect area to the reduction before use (head hair defect area before use-head hair defect area after use)/head hair defect area before use.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A preparation method of an adipose-derived stem cell exosome comprises the following steps:
(I) placing the adipose-derived stem cells under a series of concentration gradients of which the oxygen concentration is from low to high to culture in sequence; the series of concentration gradients of the oxygen concentration from low to high comprises low oxygen and high oxygen; the low oxygen refers to the oxygen concentration being lower than 20.9 percent, and the high oxygen refers to the oxygen concentration being higher than 20.9 percent;
and (II) collecting the adipose-derived stem cells cultured in the step (I) or culture supernatant of the adipose-derived stem cells after passage, and separating and purifying to obtain the adipose-derived stem cell exosomes.
2. The method of claim 1, wherein: the series of concentration gradients of the oxygen concentration from low to high refers to the oxygen concentration from 4% to 32%.
3. The method of claim 2, wherein: the series of concentration gradients of the oxygen concentration from low to high is set as follows: 4%, 8%, 16%, 24%, 28% and 32%.
4. A method according to any one of claims 1-3, characterized in that: in the step (I), under each oxygen concentration gradient, the culture time is 10-14h when the adipose-derived stem cells are cultured; and/or
In the step (I), when the adipose-derived stem cells are cultured under each oxygen concentration gradient, CO is added2The concentration of (A) is 5%; and/or
In the step (I), under each oxygen concentration gradient, the culture temperature is 37 ℃ when the adipose-derived stem cells are cultured; and/or
In the step (I), the adopted culture medium is a serum-free stem cell culture medium when the adipose-derived stem cells are cultured under each oxygen concentration gradient.
5. The method according to any one of claims 1-4, wherein: in the step (I), before the adipose-derived stem cells are placed in a series of concentration gradients from low to high in oxygen concentration for culture, the method further comprises the following steps: and (3) placing the adipose-derived stem cells in a serum-free stem cell culture medium for culturing for 8 h.
6. The method according to any one of claims 1-5, wherein: in the step (II), the adipose-derived stem cell exosomes are obtained by separation and purification according to a method comprising the following steps: after collecting the culture supernatant of the adipose-derived stem cells, firstly removing intact cells and dead cell debris through low-speed centrifugation, and then carrying out high-speed centrifugation on the centrifuged supernatant to obtain an adipose-derived stem cell exosome precipitate; the low-speed centrifugation is carried out for 8-10min at the temperature of 4 ℃ and 2000 g; the high-speed centrifugation is performed for 90min at the temperature of 4 ℃ at 100000 g;
further, the method comprises the steps of suspending the adipose-derived stem cell exosome sediment in a PBS solution, and filtering by using 0.45-micron and 0.22-micron filters sequentially to obtain filtrate which is stock solution rich in the adipose-derived stem cell exosomes.
7. Adipose-derived stem cell exosomes or adipose-derived stem cell exosome-rich stock solutions prepared by the method of any one of claims 1 to 6.
8. A hair growth promoting injection, which is prepared from the stock solution rich in the adipose-derived stem cell exosome of claim 7 and normal saline for injection; the hair growth promoting injection at least contains 1 × 10 of the components per milliliter5Exosomes produced by individual adipose-derived stem cells;
furthermore, the hair growth promoting injection contains 1 × 10 of the total weight of the hair growth promoting injection per milliliter5Exosomes produced by individual adipose-derived stem cells.
9. Any of the following applications:
use of P1, the adipose stem cell exosomes of claim 7 or a stock solution rich in adipose stem cell exosomes for preparing the hair growth promoting injection of claim 8;
p2, the adipose stem cell exosome of claim 7 or a stock solution rich in adipose stem cell exosomes or the use of the hair growth promoting injection of claim 8 in hair growth;
p3, the adipose stem cell exosome of claim 7 or stock solution rich in adipose stem cell exosome or the hair growth injection of claim 8 for use in tissue repair.
10. Any one of the following methods:
method A, a method for increasing the proliferative capacity of adipose-derived stem cells, comprising the step (I) of any one of claims 1 to 5, to obtain adipose-derived stem cells with increased proliferative capacity;
method B, a method for improving tissue repair ability of adipose-derived stem cells, comprising the step (I) of any one of claims 1 to 5, wherein the tissue repair ability of the exosomes secreted by the adipose-derived stem cells cultured in the step (I) is improved.
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| CN115778892A (en) * | 2022-12-30 | 2023-03-14 | 广州赛莱拉生物基因工程有限公司 | Scalp care composition and scalp care product containing (animal) umbilical cord extract |
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Application publication date: 20211109 |