CN113957040A - Adipose-derived stem cell growth factor extract and preparation method and application thereof - Google Patents
Adipose-derived stem cell growth factor extract and preparation method and application thereof Download PDFInfo
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- CN113957040A CN113957040A CN202010698271.4A CN202010698271A CN113957040A CN 113957040 A CN113957040 A CN 113957040A CN 202010698271 A CN202010698271 A CN 202010698271A CN 113957040 A CN113957040 A CN 113957040A
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Abstract
The invention discloses a method for preparing an adipose-derived stem cell growth factor, which comprises the steps of separating adipose-derived stem cells, culturing the adipose-derived stem cells and culturing the adipose-derived stem cell growth factor. The adipose-derived stem cell growth factor can be used for repairing skin aging, and particularly can be used for promoting the expression of hyaluronic acid, elastin, collagen and/or reticulin in the skin, stimulating the cell proliferation of a using area, stimulating the cell proliferation of the skin and secreting nutritional factors of the using area. The adipose-derived stem cell growth factor can be used for preparing cosmetics or medical beauty products.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to an adipose-derived stem cell growth factor extract, and a preparation method and application thereof.
Background
Stem cells are a cell population with self-renewal, high proliferation and multidirectional differentiation capacity, and can be divided into embryonic stem cells and adult stem cells according to different sequences appearing in the ontogeny process, wherein the embryonic stem cells can be differentiated into stem cells with specific functions, such as skin stem cells which can be differentiated into various skin cells, hematopoietic stem cells which can be differentiated into erythrocytes, leukocytes, platelets and the like. An adult stem cell is an undifferentiated stem cell extracted from an already differentiated tissue, which cell is capable of self-renewal and, under certain conditions, of differentiating into cells of that tissue. They are primarily used to maintain homeostasis of cell function. With the development of research, the types of tissues in which stem cells exist are found to be more and more extensive, including bone marrow, peripheral blood, skin, fat and the like. Adult stem cells have a limited proliferative capacity compared to embryonic stem cells. Embryonic stem cells are pluripotent in terms of differentiation potential, and can form a variety of cells, whereas adult stem cells can differentiate into only a specific cell type. However, the results of the study demonstrate the potential of stem cells to be plastic and differentiate into other cell types.
At present, pluripotent stem cells have great potential in the fields of tissue engineering and gene therapy, and are generally divided into embryonic stem cells and adult stem cells, but the adult stem cells are qualitatively compared with the embryonic stem cells, and have no immunological rejection and some theoretical problems.
Adipose-derived stem cells (ADSCs) are adult stem cells derived from adipose tissue, are static undifferentiated stem cells, and have strong in-vitro amplification and self-proliferation capabilities. Has repairing function on tissue cells, and can promote cell regeneration. Because adipose tissues are easy to obtain, patients have little pain and sufficient supply, and can take materials repeatedly, the adipose-derived stem cells have huge potential in clinical treatment no matter what reason is controversial.
The traditional autologous fat transplantation refers to sucking redundant fat from waist, abdomen, thigh and other parts, centrifuging and purifying, selecting fat cell particles, and filling/wrinkle removing/shaping the face by using an injection technology. However, the survival rate of fat transplantation is low (generally reported to be 30%), and sometimes a "small number of times" approach is needed and overkill is needed. The wide use of the surgical method is limited due to unstable therapeutic effects. ZUK et al, 2001, isolated fibroblast-like cells from adipose tissue, similar in morphology to bone marrow stem cells, called impacted-derived stem cells (ADSCs), that yielded an average of 2x10 per 300ml of adipose tissue8-6X108Such cells can be proliferated well and a large number of cells having differentiation ability can be obtained. The adipose-derived stem cells are cultured in vitro and then injected into human bodies, so that the tissues and organs can be renewed and repaired, and the adipose-derived stem cells can spontaneously move to a wound area and repair the wound. Cell therapy must be performed by injecting cells into a subject by subcutaneous injection. However, this therapy is invasive and carries some risks, and will cause many traumatic wounds that may affect the appearance of the skin. In addition, since the safety of live cells should be strictly evaluated before injection due to the use of live cells in the therapy, since live cells should be derived from human stem cells, the application of conventional cell therapy of injecting into a human body has many limitations.
Disclosure of Invention
Adipose stem cells secrete a large number of growth factors during therapy, which promote cell growth, repair, and supplement nutrition. Therefore, in order to overcome the defects of the conventional cell therapy, the invention provides a method for preparing the adipose-derived stem cell growth factor. The above problems can be solved by preparing a growth factor cultured in adipose-derived stem cells into a cosmetic or medical cosmetic.
The technical scheme of the invention is as follows:
a method for preparing an adipose-derived stem cell growth factor extract, comprising the steps of:
(1) extracting adipose-derived stem cells from in vitro adipose tissues to obtain primary adipose-derived stem cells;
(2) inoculating the primary adipose-derived stem cells into a serum-containing culture medium through a cell sieve for culture to obtain 4-6 generations of adipose-derived stem cells;
(3) when the cell confluence rate is 70-95%, replacing a protein-free culture medium, culturing under the condition of 5% carbon dioxide hypoxia, and centrifuging to obtain a supernatant which is an adipose-derived stem cell growth factor extract. The extract can be further prepared into a freeze-dried powder product form.
As a preferred embodiment, step (1) is specifically: cleaning and carrying out enzymolysis on the isolated adipose tissue containing the adipose-derived stem cells, terminating enzymolysis digestion by using a culture medium, and then carrying out resuspension and centrifugal separation to obtain the primary adipose-derived stem cells.
In a preferred embodiment, the enzymolysis is carried out by using pancreatin and/or collagenase at 35-38 ℃ for 20-60 min.
Further preferably, the volume ratio of the pancreatin and/or collagenase to the tissue is 1: 3-5.
In a preferred embodiment, the primary adipose-derived stem cells obtained in step (2) are screened by a 50-100 μm cell sieve, and then inoculated into MEM (minimum essential medium) for culture.
As a preferred embodiment, the time of the hypoxic condition culture in the step (3) is 3-4 d. The stem cells are cultured in a hypoxic environment, and the secretion amount of growth factors is increased.
The adipose stem cell growth factor extract prepared by the method comprises Hepatocyte Growth Factor (HGF), Platelet Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF), transforming growth factor beta (TGF-beta), basic fibroblast growth factor (bFGF), indoleamine 2, 3-dioxygenase (I DO), interleukin (IL-10), Keratinocyte Growth Factor (KGF), Vascular Endothelial Growth Factor (VEGF), insulin-like growth factor (IGF-1) and the like.
The cell growth factor is a multifunctional strong cell factor and plays an important role in promoting the metabolism of fibroblasts and the formation of collagen. The cell growth factor can promote the growth and the propagation of skin tissues, regulates and controls the division, the propagation and the growth and the differentiation of skin epithelium, endothelium and stromal cells by combining with a cell surface specific receptor, promotes the cell metabolism and enhances the oxidation effect; can promote the rapid growth and reproduction of cells related to skin injury, and regulate the synthesis, secretion and decomposition of intercellular matrix; can promote the regeneration of stratum corneum cells, accelerate the repair of stratum corneum and matrix layers of the skin and promote the growth of skin cells of a human body; can enhance the synthesis and cell metabolism of skin cells, delay skin cell aging, promote repair and growth of epidermal cells, and make skin smooth and plump.
Wherein, (1) Hepatocyte Growth Factor (HGF) can promote the generation, survival and regeneration of histiocytes, inhibit apoptosis, regulate the synthesis and inflammatory reaction of collagen fibers, and play an important role in promoting wound healing and preventing and treating tissue fibrosis.
(2) The Epidermal Growth Factor (EGF) can promote the synthesis of DNA, RNA and hydroxyproline in the process of repairing skin wound tissues, induce the mature epidermal cells of induced differentiation to be reversed into epidermal stem cells, accelerate the generation of wound granulation tissues and the proliferation of epithelial cells, thereby shortening the healing time of the wound and improving the quality of wound repair.
(3) Fibroblast Growth Factor (FGF) promotes endothelial cell migration and smooth muscle cell proliferation, but does not allow smooth muscle cell migration. Can promote neovascularization and repair damaged endothelial cells.
(4) Keratinocyte Growth Factor (KGF) is capable of specifically stimulating physiological processes such as metabolism of epithelial cells, including regeneration, differentiation, migration, etc.
(5) Vascular endothelial cell growth factor (VEGF) is a highly specific vascular endothelial cell growth factor, and has the effects of promoting vascular permeability increase, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, vascularization and the like, so that the wound can be quickly healed.
(6) Insulin-like growth factors (IGFs) mediate the stimulation of growth hormones, regulating tissue growth and development, and play important roles in the regulation of muscle volume and strength, maintenance of body constituents, and nutrient metabolism.
Based on the functional effect of the cell growth factor, the invention also comprises the application of the adipose-derived stem cell growth factor obtained by the method in the preparation of the composition with the function of repairing skin aging.
It is another object of the present invention to provide a cosmetic or cosmeceutical containing the adipose stem cell growth factor extract. The cosmetic or medical cosmetic containing the adipose-derived stem cell growth factor is topically applied to a human body, and can promote the expression of hyaluronic acid, elastin, collagen or reticulin in the skin, stimulate the proliferation of cells in an administration area and secrete trophic factors, thereby providing an excellent effect of repairing skin aging.
As a preferred embodiment, when the cosmetic or cosmeceutical article of the present invention is applied to the skin surface in the form of external application, a transdermal polypeptide-type substance, which carries a growth factor into the dermis layer of the skin through the epidermis but is harmless to the skin, may be further added.
In the present invention, the cosmetics or medical cosmetics containing the adipose-derived stem cell growth factor may be lyophilized powder, emulsion, cream, gel, paste, spray or solution.
In the invention, phosphate buffer solution, penetration enhancer laurocapram, isosorbide dimethyl ether, thiaketone, inositol and the like can be added into the freeze-dried powder, and polypeptides for promoting growth factors to enter skin can also be added.
The cosmetic or cosmeceutical product of the present invention may also be prepared in the form of food for swallowing or drinking, such as health food or cosmetic drink. The medical cosmetic product of the invention can be prepared and used in the form of oral administration or subcutaneous injection.
In the present invention, since various growth factors contained in adipose stem cells are used to directly utilize the effective components contained therein to provide desired effects, the amount of the adipose stem cell growth factors can be more easily controlled, unlike conventional stem cell therapy methods. The adipose stem cell growth factor provides a desired effect by stimulating active ingredients of living cells in the body.
Drawings
FIG. 1 is a flow chart of a method for preparing lyophilized powder of cell growth factor;
fig. 2 is a photograph comparing the improvement of skin of volunteers after applying the freeze-dried powder of the present invention for one month.
Fig. 3-5 are photographs comparing skin improvement of volunteers after being smeared with the freeze-dried powder of the invention for ten days.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood 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.
Example 1
Preparation of adipose-derived stem cell growth factor extract
Referring to fig. 1, the preparation method of the cell growth factor extract of this example includes:
sterilizing a container containing 25ml of adipose tissues, cleaning the adipose tissues by using normal saline, standing for layering, removing the upper layer, repeatedly cleaning until the liquid at the bottom layer is clear, and taking the clean adipose tissues at the lower layer for later use.
5ml of 0.25% collagenase was added to the adipose tissue at 37 ℃ and 400g/h and digested for 30 min.
Then, 5ml of serum-containing MEM medium was added to terminate the digestion, the cells neutralized by the digestion were placed in a centrifuge tube, centrifuged at 1800rpm for 10min, the supernatant was removed, the cells in the lower layer were collected, the cells were blown with 5ml of 10% fetal bovine serum MEM medium to disperse them uniformly, and then they were sieved through a 100 μm cell sieve to obtain a primary adipose-derived stem cell pellet.
Adding 10ml of MEM (MEM) culture medium containing 10% fetal calf serum into the obtained primary adipose tissue stem cells, inoculating the primary adipose tissue stem cells into a T150 culture bottle, putting the culture bottle into an incubator, and culturing for 4 days at 37 ℃ in a 5% carbon dioxide humid environment;
when the confluence rate is 95%, digesting with 3ml of 0.1% pancreatin for 3min, centrifuging at 1200rpm for 5min, removing the supernatant, and collecting the lower layer cells to obtain the first generation adipose-derived stem cells. The cells were blown up in 45ml of 10% fetal bovine serum MEM medium and passaged into 3T 150 flasks as second generation adipose stem cells. And carrying out cell passage at the ratio of 1:3 in each passage until the third generation of the adipose-derived stem cells are obtained. And collecting the third generation adipose-derived stem cells, uniformly blowing and beating the third generation adipose-derived stem cells by using cell freezing solution, and freezing and storing the third generation adipose-derived stem cells according to the cell amount of 200 ten thousand per cell. And (4) freezing and storing the purified adipose-derived stem cells in liquid nitrogen to obtain the seed cells. When the cell viability reaches 95% through detection, the flow detection marker is qualified. The detection results are as follows:
| surface sign | Sample 1 | Sample 2 | Detection standard |
| CD44 | 96.20 | 97.21 | ≥95％ |
| CD105 | 98.23 | 98.15 | ≥95％ |
| CD34 | 0.19 | 0.14 | ≤2％ |
| CD45 | 0.90 | 0.81 | ≤2％ |
As can be seen from the above table, the expression of the positive markers of CD105 and CD44 is more than or equal to 95 percent and the expression of the positive markers of CD34 and CD45 is less than or equal to 2 percent through flow detection.
Taking out a frozen adipose-derived stem cell, resuscitating the cell in a water bath at 37 ℃, adding 10ml of physiological saline, centrifuging for 5min at 1200rpm, removing supernatant, collecting lower layer cells, blowing the cells with 15ml of MEM (minimum medium) of 10% fetal calf serum, passaging the cells into a T150 culture bottle, and culturing for 72h at 37 ℃ in a 5% carbon dioxide humid environment until the confluence rate is 95%; removing the culture medium from the culture flask, and washing the flask with physiological saline for 3 times; adding DMEM/F12 culture medium into a culture bottle, culturing for 72h at 37 ℃ in a 5% carbon dioxide hypoxic environment to obtain a mixture containing cell growth factors; collecting the mixture, centrifuging at 10000rpm for 5min, and collecting supernatant as the extract containing growth factor.
Detection of growth factor concentration in supernatant with ELISA kit: HGF hepatocyte growth factor; PDGF platelet-derived growth factor; EGF epidermal growth factor; TGF- β transforming growth factor β; bFGF basic fibroblast growth factor; i DO indoleamine 2, 3-dioxygenase; IL-10 interleukin; KGF keratinocyte growth factor; VEGF vascular endothelial cell growth factor; and (4) freezing and storing the IGF-1 insulin-like growth factor at-20 ℃ after the IGF-1 insulin-like growth factor concentration is detected to be qualified.
The table below shows the lowest concentration of growth factor below which the test failed.
The detection results are as follows:
example 2
Lyophilized powder of adipose-derived stem cell growth factor
Freeze-drying the supernatant containing growth factor at vacuum degree of 0.2mbar and temperature of-30 deg.C for 40H to obtain sterile lyophilized powder, wherein each lyophilized powder is 0.13 mg.
Before use, the lyophilized powder is dissolved by solvent, and then the microcrystals are introduced into skin, so that the growth factors can easily permeate into the skin, and the expression of hyaluronic acid, elastin, collagen or reticular protein in the skin is promoted.
The lyophilized powder of the adipose stem cell growth factor of the present invention may be used at various administration frequencies, for example, 2 pieces at a time, once every two weeks, according to the needs of the subject. The dosage can be adjusted according to the skin aging degree of the subject, and can be increased by 2-5 times.
FIG. 2 shows that volunteers use 2 lyophilized powder of the present invention each time, once every two weeks. After one month, freckles were significantly reduced. Fig. 3-5 show that the volunteers use the freeze-dried powder once and 10 days after 2 times of use, the facial spots are reduced, and the fine lines of the eyes are greatly improved.
In addition, in order to compare the using effect with the common growth factor freeze-dried powder product, 20 cases of test persons are selected for comparison test, and the common facial mask group uses commercially available common growth factor freeze-dried powder; the experimental group used the growth factor lyophilized powder of the present invention. The common growth factor is usually a recombinant artificially synthesized unilateral EGF growth factor, which is obtained by obtaining DNA fragments through artificial genetic engineering recombination and then obtaining the EGF growth factor through a bacterial fermentation mode. After 1 month, the user was asked about the skin condition, including aspects of skin color, gloss, fine lines, etc. The results are given in the following table:
| total number of cases | Has obvious improvement | No obvious abnormality | High efficiency | |
| Common growth factor freeze-dried powder | 20 | 15 | 15 | 50％ |
| Experimental group | 20 | 25 | 5 | 85％ |
The above test data shows that the skin improvement effective rate of the growth factor freeze-dried powder prepared by the invention for a user is 35% higher than that of the common growth factor freeze-dried powder for the user, which indicates that the growth factor freeze-dried powder prepared by the method of the invention can effectively improve the skin condition compared with the common growth factor freeze-dried powder.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for preparing an adipose-derived stem cell growth factor extract, which comprises the following steps:
(1) extracting adipose-derived stem cells from in vitro adipose tissues to obtain primary adipose-derived stem cells;
(2) inoculating the primary adipose-derived stem cells into a serum-containing culture medium through a cell sieve for culture to obtain 4-6 generations of adipose-derived stem cells;
(3) when the cell confluence rate is 70-95%, replacing a protein-free culture medium, culturing under a low-oxygen condition, and centrifuging to obtain a supernatant which is an adipose-derived stem cell growth factor extract.
2. The method according to claim 1, wherein step (1) is specifically: cleaning and carrying out enzymolysis on the isolated adipose tissue containing the adipose-derived stem cells, terminating enzymolysis digestion by using a culture medium, and then carrying out resuspension and centrifugal separation to obtain the primary adipose-derived stem cells.
3. The method of claim 2, wherein the enzymatic hydrolysis is carried out with pancreatin and/or collagenase at 35-38 ℃ for 20-60 min.
4. The method of claim 3, wherein the pancreatin and/or collagenase is present in a volume ratio to the tissue of 1: 3-5.
5. The method according to claim 1, wherein the primary adipose stem cells obtained in step (2) are cultured in MEM through a cell sieve of 50-100 μm.
6. The method according to claim 1, wherein the time for the hypoxic condition culture in step (3) is 3-4 d.
7. The adipose-derived stem cell growth factor extract prepared by the method of claims 1 to 6, wherein the adipose-derived stem cell growth factor extract comprises hepatocyte growth factor, platelet-derived growth factor, epidermal growth factor, transforming growth factor beta, basic fibroblast growth factor, indoleamine 2, 3-dioxygenase, interleukin, keratinocyte growth factor, vascular endothelial growth factor and insulin-like growth factor.
8. Use of the adipose stem cell growth factor extract according to claim 7 in the preparation of a composition having the function of repairing skin aging.
9. A cosmetic or cosmeceutical comprising the adipose stem cell growth factor extract according to claim 7.
10. The cosmetic or medical cosmetic product according to claim 9, further comprising a transdermal polypeptide.
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| CN114540295A (en) * | 2022-03-15 | 2022-05-27 | 梁浩楠 | Culture medium for culturing mesenchymal stem cells |
| CN114948998A (en) * | 2022-06-01 | 2022-08-30 | 复旦大学附属中山医院 | Polysaccharide biomedical colloidal fluid rich in human adipose-derived mesenchymal stem cell factor compound and preparation method and application thereof |
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| CN114540295A (en) * | 2022-03-15 | 2022-05-27 | 梁浩楠 | Culture medium for culturing mesenchymal stem cells |
| CN114948998A (en) * | 2022-06-01 | 2022-08-30 | 复旦大学附属中山医院 | Polysaccharide biomedical colloidal fluid rich in human adipose-derived mesenchymal stem cell factor compound and preparation method and application thereof |
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