CN115478047A - Extraction method and application of umbilical cord stem cell exosome - Google Patents

Extraction method and application of umbilical cord stem cell exosome Download PDF

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CN115478047A
CN115478047A CN202211265720.1A CN202211265720A CN115478047A CN 115478047 A CN115478047 A CN 115478047A CN 202211265720 A CN202211265720 A CN 202211265720A CN 115478047 A CN115478047 A CN 115478047A
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exosome
exosomes
umbilical cord
stem cell
extracting
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李本金
郝建华
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Beijing North American Institute Of Anti Aging Medicine
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Beijing North American Institute Of Anti Aging Medicine
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Abstract

The invention relates to the technical field of biomedicine, in particular to an extraction method and application of an umbilical cord stem cell exosome. The serum-free culture medium is adopted for culture, the defect of animal-derived serum pollution is overcome, the problems of cell amplification quantity and stem cell characteristic maintenance are solved, serum is not contained, the influence of unknown serum components on cell culture and animal-derived pollution are avoided, cell proliferation is promoted, the stem cell characteristic is maintained, and PEG is adopted for crosslinking into a net shape and is attached to exosomes; carry out the exosome protection through adding sucrose solution, avoided the exosome to suffer destruction at the ultracentrifugation in-process to stabilize the exosome, reduce ultracentrifugation's mechanical damage and lead to the exosome to break the piece that brings, improve the purity of exosome extract product, whole operation step easy operation, it is consuming time shorter, and the stem cell exosome purity of extracting is high, be fit for the mass and draw, the stem cell exosome who makes can be at the skin aging problem of solving the people as active ingredient.

Description

Extraction method and application of umbilical cord stem cell exosome
Technical Field
The invention relates to the technical field of biomedicine, in particular to an extraction method and application of an umbilical cord stem cell exosome.
Background
The stem cell exosome is a cell secretion from stem cells, and the exosome is a tiny vesicle (30-150 nm wide) secreted by the stem cells, is provided with bioactive chemical substances such as protein, mRNA and microRNA of the stem cells, and has some functions of the stem cells. The exosome accords with the definition of the nanoparticle, the stem cell exosome can penetrate through a blood brain barrier, is a key substance for transferring the intermediate information content between the stem cell and other cells, and is favorable for finishing the function of the stem cell. They resemble intercellular "communication soldiers" that can transfer DNA, RNA or proteins between cells, thereby affecting the function of recipient cells, and exosomes are being used to treat diseases ranging from heart disease to respiratory disease in the stem cell and regenerative medicine fields.
The stem cell exosome has similar biological performance with the stem cell, but the stem cell exosome is safer, more stable and more efficient, the exosome does not respond to the damaged microenvironment, has stronger and complex signal molecule transporting and regulating capacity, and has the capacity of changing extracellular matrix, changing transcriptome and proteome of receptor cells, and regulating apoptosis, growth, proliferation and differentiation pathways. Exosomes contain a variety of biological factors that have been found to induce migration and proliferation of fibroblasts and collagen synthesis; has important biological functions of reducing apoptosis, relieving inflammatory reaction, promoting angiogenesis, inhibiting fibrosis, improving tissue repair potential and the like, and has good application prospect in the aspects of tissue injury repair, chronic wound healing, wound scar formation inhibition, aging resistance and the like. And the high-purity stem cell exosome is obtained on the premise of developing application research. At present, the traditional essence in the market is mainly prepared by exogenous substances such as plant component extraction or fine chemical manufacturing process, and although the traditional essence can produce transient improvement effect on skin, the traditional essence does not fundamentally change the aging and repairing problems of the skin.
Disclosure of Invention
Aiming at the defects of the background technology, the invention provides an extraction method and application of an umbilical cord stem cell exosome.
On one hand, the technical scheme adopted by the invention is as follows: the method for extracting the exosomes of the umbilical cord stem cells is characterized by comprising the following steps of:
s1, extracting an exosome stock solution in a constant-temperature sterile environment by adopting a serum-free culture medium;
s2, mixing the exosome stock solution with PEG to enable the concentration of the PEG to be 1.6-2.5%, and incubating for 1-3 hours to obtain a stable exosome crosslinking system;
s3, adding an exosome extracting solution to obtain an exosome ultracentrifugation system;
s4, carrying out density gradient centrifugation collection on the obtained exosomes to obtain high-purity exosomes;
s5, freezing and storing, and refrigerating the cultured stem cell exosomes at a low temperature.
Preferably, the serum-free medium in S1 comprises DMEM-F12 medium, platelet-derived factor 1-50ng/mL, basic fibroblast growth factor 20-50ng/mL aloe extract 10-50ng/mL ginseng extract 10-50ng/mL, cistanche deserticola extract 10-50ng/mL.
Preferably, S1 is specifically to collect the culture supernatant of the stem cells, remove cell debris and impurities to obtain an exosome stock solution, and remove the cell debris by centrifugation for 8-10 minutes under the relative centrifugal force of 200-300 g.
Preferably, the molecular weight of PEG in S2 is 4000-6000, and the incubation temperature is controlled at 2-6 ℃.
Preferably, S3 is in particular added to 45% wt of the sucrose solution until the mass fraction of sucrose in the exosome ultracentrifugation system is 10-15%.
Preferably, S4 is centrifuged for 1-2h at 110000g and 2-6 ℃ under the centrifugation condition.
Preferably, the S5 is stored in a frozen state, and the extracted stem cell exosomes are preserved in an environment of-80 ℃.
In another aspect, the invention provides an application of the umbilical cord stem cell exosome in a cosmetic preparation.
In another aspect, the present invention provides an anti-aging cosmetic product comprising the umbilical cord stem cell exosome extracted by the above method.
Has the advantages that: compared with the prior art, the invention adopts serum-free culture medium for culture, overcomes the defect of animal-derived serum pollution and solves the problems of cell amplification quantity and stem cell characteristic maintenance, does not contain serum, avoids the influence of unknown serum components on cell culture and animal-derived pollution, promotes cell proliferation and maintains stem cell characteristics, adopts PEG (polyethylene glycol) for crosslinking into a net shape, and is attached to exosomes; carry out the exosome protection through adding sucrose solution, ultracentrifugation enrichment and purification exosome on this basis, avoided exosome to suffer destruction in ultracentrifugation in-process, thereby stabilize exosome, reduce ultracentrifugation's mechanical damage and lead to the exosome to break the piece that brings, improve the purity of exosome extract product, whole operation steps easy operation, it is consuming time shorter, and the stem cell exosome purity of extracting is high, be fit for the mass and draw, the stem cell exosome who makes is as active ingredient, can be in the ageing problem of skin of solving the people.
Detailed Description
The present invention will be described in detail with reference to specific embodiments in order to make those skilled in the art better understand the technical solutions of the present invention. The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the industrial raw materials (reagents and raw materials are selected according to the situation) are all conventional industrial raw materials which are sold in the market if not specified; the processing and manufacturing methods (detection, test, preparation method and the like are selected according to the situation) are conventional methods unless otherwise specified.
Example 1 extraction of umbilical cord mesenchymal Stem cells
Taking out umbilical cord from the preservation solution, physically crushing the umbilical cord, removing the outer sides of two ends of the umbilical cord ligation, cutting the middle section into small sections with the length of 1-2 cm, repeatedly flushing the small sections with normal saline, splitting open, removing arteries and veins, repeatedly flushing with normal saline to remove blood and blood clots, soaking in 10 times of double-resistance solution, and shearing with scissors to 0.5-2 mm 3 Obtaining umbilical cord tissue fragments; sequentially digesting the umbilical cord tissue fragments by adopting a type I collagenase solution, an EDTA solution and trypsin, wherein the digestion conditions in each time are as follows: the temperature is 36-38 ℃, the time is 20-30 min, after digestion is ended, the mixture is washed twice by physiological saline, a 100-micron mesh screen is used for filtering tissue residues, filtrate containing cells is collected, and mesenchymal stem cells obtained by centrifugation are washed; inoculating umbilical cord mesenchymal stem cells into a serum-free culture medium, wherein the serum-free culture medium comprises a DMEM-F12 culture medium, platelet-derived factors 15ng/mL, basic fibroblast growth factors 35ng/mL, aloe extracts 35ng/mL, ginseng extracts 10ng/mL, and cistanche extracts 40ng/mL,incubating for 15-25h in an incubator at 37 ℃, 5% by volume of carbon dioxide and 6% by volume of oxygen, replacing a new culture medium, carrying out subculture according to the proportion of 1 2 The test results are shown in table 1 below.
TABLE 1
Algebra Number of surviving cells Multiple of increase Survival rate
P1 2.47×10 6 117.8 96.7％
P2 3.24×10 6 154.6 97.1
P3 1.78×10 6 84.9 95.8
Example 2 extraction of umbilical cord stem cell exosomes
Centrifuging the culture medium after the cell culture in the embodiment 1 to remove impurities, collecting the supernatant of the stem cell culture solution, removing cell fragments and impurities to obtain an exosome stock solution, and preferably centrifuging the culture medium for 8 to 10 minutes under the relative centrifugal force of 200g to 300g to remove the cell fragments to obtain the exosome stock solution; mixing the exosome stock solution with PEG (polyethylene glycol) with the concentration of 1.6-2.5% and the molecular weight of 4000-6000, and incubating for 1-3 hours at the temperature of 2-6 ℃ to obtain a stable exosome crosslinking system; adding 45 percent by weight of sucrose solution until the mass fraction of sucrose in the system is 10-15 percent, centrifuging at the low temperature of 110000g for 1-2h under the centrifugation condition of 2-6 ℃ to form a density layer which is continuously distributed from low to high, and collecting exosomes in a sample in the density range of 1.1-1.15 g/ml by density gradient centrifugation to obtain high-purity exosomes; when the frozen storage is performed, the storage is required to be performed at-80 ℃.
Example 3 exosome-containing lyophilized powder
And adding mannitol (1-15%) into the exosome prepared in the embodiment 2, uniformly mixing, filtering (0.22 mu m), standing for 10 hours at-80 ℃, and freeze-drying under the vacuum degree of 10Pa to obtain the human mesenchymal stem cell source exosome freeze-dried powder.
And (3) stability test: the obtained cosmetic preparation is stored at-20 deg.C. After six months, no significant change was observed.
And (3) testing the effectiveness: 60 female test volunteers aged 30-50 years were screened, and the subjects were divided into 3 groups of 20 persons each according to skin conditions, and the evaluation results after completion of one treatment course (30 days) using the product of example 3 are shown in Table 2.
TABLE 2
Figure BDA0003893041920000051
Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.

Claims (9)

1. An extraction method of an umbilical cord stem cell exosome is characterized by comprising the following steps:
s1, extracting an exosome stock solution in a constant-temperature sterile environment by adopting a serum-free culture medium;
s2, mixing the exosome stock solution with PEG to ensure that the concentration of the PEG is between 1.6 and 2.5 percent, and incubating for 1 to 3 hours to obtain a stable exosome crosslinking system;
s3, adding an exosome extracting solution to obtain an exosome ultracentrifugation system;
s4, carrying out density gradient centrifugal collection on the obtained exosomes to obtain high-purity exosomes;
s5, freezing and storing, and refrigerating the cultured stem cell exosomes at a low temperature.
2. The method for extracting exosomes from umbilical cord stem cells according to claim 1, wherein the serum-free culture medium in S1 comprises DMEM-F12 culture medium, platelet-derived factor 1-50ng/mL, basic fibroblast growth factor 20-50ng/mL aloe extract 10-50ng/mL ginseng extract 10-50ng/mL, cistanche deserticola extract 10-50ng/mL.
3. The method for extracting the exosomes from the umbilical cord stem cells according to claim 1, wherein S1 is specifically used for collecting culture solution supernatant of the stem cells, removing cell debris and impurities to obtain an exosome stock solution, and preferably removing the cell debris by centrifugation at a relative centrifugal force of 200 g-300 g for 8-10 minutes.
4. The method for extracting the exosomes of the umbilical cord stem cells according to claim 1, wherein the molecular weight of PEG in S2 is 4000-6000, and the incubation temperature is controlled to be 2-6 ℃.
5. The method for extracting exosomes from umbilical cord stem cells according to claim 1, characterized in that S3 is concretely by adding 45% by weight of sucrose solution until the mass fraction of sucrose in the exosome ultracentrifugation system is 10-15%.
6. The method for extracting the exosomes from the umbilical cord stem cells according to claim 1, which is characterized in that S4 is centrifuged for 1-2 hours at a low temperature of 110000g and at a temperature of 2-6 ℃.
7. The method for extracting exosomes from umbilical cord stem cells according to claim 1, wherein the extracted exosomes from stem cells are stored in an environment of-80 ℃ when S5 is stored in a frozen state.
8. Use of an umbilical cord stem cell exosome extracted by the method of any one of claims 1-7 in a cosmetic preparation.
9. An anti-aging cosmetic product characterized by: comprising umbilical cord stem cell exosomes extracted by the method of any of claims 1-7.
CN202211265720.1A 2022-10-17 2022-10-17 Extraction method and application of umbilical cord stem cell exosome Pending CN115478047A (en)

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