CN113528433A

CN113528433A - Umbilical cord mesenchymal stem cell exosome based on oxygen concentration gradient and preparation method and application thereof

Info

Publication number: CN113528433A
Application number: CN202110884894.5A
Authority: CN
Inventors: 罗彦军; 常国辉
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-08-03
Filing date: 2021-08-03
Publication date: 2021-10-22

Abstract

The invention discloses an umbilical cord mesenchymal stem cell exosome based on oxygen concentration gradient and a preparation method and application thereof. The invention provides a preparation method of an umbilical cord mesenchymal stem cell exosome, which comprises the following steps: placing the umbilical cord mesenchymal stem cells under a series of concentration gradients of oxygen concentration from low to high to culture in sequence; collecting culture supernatant, and separating and purifying to obtain the umbilical cord mesenchymal stem cell exosome. Based on an oxygen concentration gradient culture technology, the proliferation capacity of umbilical cord mesenchymal stem cells and the potential of stimulating and secreting functional exosomes are stimulated to the utmost extent, a supernatant stock solution rich in 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, asiaticoside with an anti-inflammatory and moisturizing effect, hyaluronic acid and the like are added, the multiple repairing and nursing effects are achieved, and the microenvironment for repairing striae gravidarum is improved.

Description

Umbilical cord mesenchymal stem cell exosome based on oxygen concentration gradient and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an umbilical cord mesenchymal stem cell exosome based on an oxygen concentration gradient, and a preparation method and application thereof.

Background

The umbilical cord mesenchymal stem cells are derived from a mucosal tissue, namely umbilical cord Wharton's jelly tissue. Compared with stem cells derived from tissues such as bone marrow, fat, dental pulp and the like, the human umbilical cord tissue is waste after delivery of pregnant women, has the advantages of low cost, easy access and the like, and has the advantages of high cell unicity, low immunogenicity and the like in later-stage preparation and application; multiple studies show that umbilical cord mesenchymal stem cells can secrete a large amount of cytokines, act on damaged parts of organisms through a paracrine mechanism, promote the function repair of tissue cells, show extremely potential clinical curative effects on exploratory clinical treatment of various diseases, and are also widely applied to repair of skin injuries such as burns, frostbites, ultraviolet radiation injuries and the like and skin health care.

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.

The exosome is regarded as a specific secretory membrane bubble at present, and 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.

The formation reasons of the striae gravidarum are many, the influence of hormones in the gestation period of women is one of the main factors, meanwhile, along with the growth of a fetus during pregnancy, the abdomen of a pregnant woman swells to cause the elastic fibers and the collagen fibers of the skin to be damaged or broken, so that the skin of the abdomen becomes thinner and thinner, and pink or purple wavy fine lines with different widths and different lengths appear, and after delivery, the fine lines gradually disappear to leave white or silvery glossy scar lines, namely the striae gravidarum. Stretch marks appear mainly on the anterior wall of the abdomen, and may also appear on the inside and outside of the thighs, buttocks, chest, back waist, arms, and the like. The first time of pregnancy and parturient has the most obvious performance. Once the striae gravidarum appears, the striae gravidarum cannot disappear, and the later period is accompanied by skin looseness, breast drop and abdominal fat accumulation, so that the postpartum physical state and physical and psychological health of women are seriously affected.

Disclosure of Invention

The invention aims to provide an umbilical cord mesenchymal 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 umbilical cord mesenchymal stem cell exosomes.

The preparation method of the umbilical cord mesenchymal stem cell exosome comprises the following steps:

(I) placing the umbilical cord mesenchymal stem cells under a series of concentration gradients of oxygen concentration from low to high to culture in sequence; the series of concentration gradients of the oxygen concentration from low to high covers low oxygen and high oxygen; the low oxygen refers to an oxygen concentration significantly lower than 20.9% (atmospheric standard oxygen content), and the high oxygen refers to an oxygen concentration significantly higher than 20.9% (atmospheric standard oxygen content).

(II) collecting the umbilical cord mesenchymal stem cells cultured in the step (I) or culture supernatant of the umbilical cord mesenchymal stem cells after passage, and separating and purifying to obtain the umbilical cord mesenchymal stem cell exosome.

Further, the series of concentration gradients of the oxygen concentration from low to high may specifically refer to the oxygen concentration from 4% to 32%.

In a specific embodiment of the present invention, the series of concentration gradients of the oxygen concentration from low to high is set as follows: 4%, 8%, 16%, 24%, 28% and 32%.

In the invention, the oxygen concentration is volume percentage.

Further, in the step (I), the umbilical cord mesenchymal stem cells may be cultured for 10-14h (e.g. 12h) at each concentration gradient.

Further, in the step (I), when the umbilical cord mesenchymal stem cells are cultured under each oxygen concentration gradient, CO is generated₂May be present in a concentration of 5% by volume. Except thatO₂And CO₂In addition, the rest gas is nitrogen.

Further, in the step (I), the culturing temperature of the umbilical cord mesenchymal stem cells may be 37 ℃ for each oxygen concentration gradient.

Further, in the step (I), in each oxygen concentration gradient, the culture medium adopted in culturing the umbilical cord mesenchymal stem cells is a serum-free stem cell culture medium.

In a specific embodiment of the present invention, in step (I), the culture medium used for culturing the umbilical cord mesenchymal stem cells is TheraPEAK at each oxygen concentration gradient^TM MSCGM-CD^TMMesenchymal Stem Cell Medium, chemical Defined (Chinese name: TheraPEAK)^TM MSCGM-CD^TMHuman mesenchymal stem cell culture medium), product coding: YS000471, supplier: LONZA corporation.

Further, in step (I), before the umbilical cord mesenchymal stem cells are placed in a series of concentration gradients from low to high oxygen concentration for sequential culture, the method may further comprise the following steps: the umbilical cord mesenchymal stem cells are placed in a serum-free stem cell culture medium for culture for 8h, and then washed by PBS (such as twice).

Further, in the step (II), the umbilical cord mesenchymal stem cell exosome may be isolated and purified according to a method comprising the following steps: after collecting the culture supernatant of the umbilical cord mesenchymal 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 umbilical cord mesenchymal 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 ℃.

Furthermore, the method also comprises the steps of suspending the umbilical cord mesenchymal stem cell exosome precipitate in a PBS solution, and filtering by using 0.45 μm and 022 μm filters, wherein the obtained filtrate is a stock solution rich in umbilical cord mesenchymal stem cell exosomes.

In a second aspect, the present invention claims umbilical cord mesenchymal stem cell exosomes or umbilical cord mesenchymal stem cell exosome-rich stock solution prepared by the method of the first aspect.

In a third aspect, the invention claims a repair care solution for repairing postpartum striae gravidarum of a pregnant woman.

The repair care solution for repairing the postpartum striae gravidarum of the pregnant woman, which is claimed by the invention, can be composed of a solution B and a solution C which are respectively and independently packaged, and can also be formed by mixing the solution B and the solution C in equal volume.

The solution B is a stock solution rich in umbilical cord mesenchymal stem cell exosomes as described in the second aspect.

The composition of the solution C is as follows: 0.92 percent of asiaticoside, 0.26 percent of hyaluronic acid, 0.12 percent of glycerin, 0.15 percent of olive oil and the balance of normal saline.

In a fourth aspect, the invention claims any of the following applications:

use of P1, an umbilical cord mesenchymal stem cell exosome or a dope enriched in umbilical cord mesenchymal stem cell exosomes as described in the second aspect hereinbefore in the preparation of a repair solution as described in the third aspect hereinbefore.

P2, and the application of the repairing care solution in the third aspect in repairing the postpartum striae gravidarum of the pregnant woman.

Use of P3, an umbilical cord mesenchymal stem cell exosome or a dope enriched in umbilical cord mesenchymal stem cell exosomes as described hereinbefore in the second aspect or a repair care solution as described hereinbefore in the third aspect for tissue repair.

In a fifth aspect, the invention claims any of the following methods:

method A, a method for improving the proliferation capacity of umbilical cord mesenchymal stem cells, comprising the step (I) in the first aspect, to obtain umbilical cord mesenchymal stem cells with improved proliferation capacity.

The proliferation capacity of the umbilical cord mesenchymal stem cells cultured in the step (I) is stronger than that of the umbilical cord mesenchymal stem cells cultured under the condition of normal oxygen concentration (20.9 percent) (except for different oxygen concentrations, the other conditions are the same).

Method B, a method of repairing stretch marks, comprising the following step B1 or step B2:

b1: smearing 2-3 ml of the solution B to the striae gravidarum of the abdomen, and gently patting and massaging uniformly; taking 2-3 ml of the liquid C, and gently massaging the abdomen until the liquid B and the liquid C are completely absorbed;

b2: the solution B and the solution C are mixed in the palm in equal volumes, and then dipped by a low-temperature probe of a physical low-temperature introduction device (an appropriate amount is enough to ensure that the probe is wet), and gently massaged on the abdomen until the solution is completely absorbed.

The method C comprises the step (I) in the first aspect, and the tissue repair capacity of the exosomes secreted by the umbilical cord mesenchymal stem cells cultured in the step (I) is improved.

The tissue repair capacity of the exosome secreted by the umbilical cord mesenchymal stem cell cultured in the step (I) is stronger than that of the exosome secreted by the umbilical cord mesenchymal stem cell cultured under the condition of normal oxygen concentration (20.9 percent) (except for different oxygen concentrations, the other conditions are the same).

In the invention, the umbilical cord mesenchymal stem cells can be commercial umbilical cord mesenchymal stem cells and also can be umbilical cord mesenchymal stem cells separated from an isolated placenta. The umbilical cord mesenchymal stem cells can be human umbilical cord mesenchymal stem cells.

According to the invention, the human umbilical cord mesenchymal stem cells are separated and extracted, the striae gravidarum repair stock solution rich in umbilical cord mesenchymal stem cell exosomes is prepared, and the repair care solution for repairing the postpartum striae gravidarum of the pregnant woman is prepared by adding the protection solution with the moisturizing and anti-inflammatory effects. The product can remove macromolecules which are difficult to absorb by enriching active factors, RNA (ribonucleic acid), enzyme and other active substances secreted to cell culture supernatant in the growth process of stem cells and filtering through a 0.22 mu m filter membrane, effectively promote the regeneration of dermal fibroblast and the recovery of physiological function, and achieve the double effects of repairing and moistening by matching with components with inflammation diminishing, moisture retention and lubrication in a substrate solution, thereby effectively improving the repairing effect of striae gravidarum.

Based on an oxygen concentration gradient culture technology, the proliferation capacity of umbilical cord mesenchymal stem cells and the potential of stimulating and secreting functional exosomes are stimulated to the utmost extent, a supernatant stock solution rich in 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, asiaticoside with an anti-inflammatory and moisturizing effect, hyaluronic acid and the like are added, the multiple repairing and nursing effects are achieved, and the microenvironment for repairing striae gravidarum is improved.

Drawings

Fig. 1 is a growth curve of umbilical cord mesenchymal stem cells under different oxygen concentration conditions.

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 preparation method and application of umbilical cord mesenchymal stem cell exosome based on oxygen concentration gradient

Culture of umbilical cord mesenchymal stem cells

Collecting in vitro umbilical cord of newborn produced by full-term cesarean section, cutting into 2-3cm tissue pieces with high-pressure sterilization surgical scissors, cleaning, peeling off Wharton's jelly under aseptic condition, and cutting to 1mm²Uniformly spreading small tissue blocks of 75cm²Placing in a culture flask at 37 deg.C and 5% CO₂The cell culture box is used for culturing, 15ml of complete culture medium is added into 8-24hs to cover the tissue block, the solution is changed after 132hs culture, the solution is changed every 48-72hs, and the cell fusion degree reaches 7At 0-80%, remove the tissue mass to another new 75cm²And (3) supplementing a culture medium to 15ml in a culture bottle, continuously observing the growth condition that new cells gradually spread outwards from the periphery of the tissue block by 24-48hs, carrying out passage when the cell growth fusion degree reaches about 70% -80%, recording as P1, carrying out continuous passage, obtaining cells from P2 generation to P5 generation for secondary adherent growth, and storing the cells as umbilical cord mesenchymal stem cells for experiments.

Second, test grouping

Transferring the P3 generation umbilical cord mesenchymal stem cells with good growth state, wherein the number is 5 × 10⁶Cell culture flasks passaged 175 cm square, using commercial serum-free stem cell medium (English name: TherAPAK)^TM MSCGM-CD^TMMesenchymal Stem Cell Medium, chemical Defined, Chinese name: therapeak^TM MSCGM-CD^TMHuman mesenchymal stem cell culture medium, product code: YS000471, supplier: LONZA corporation), incubated for 8 hours, the cells were washed twice with PBS, and the cells were randomly divided into two groups. 1. 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. 2. Oxygen concentration normal treatment group: 20.9% O₂，5％CO₂The culture temperature is 37 ℃, and the culture time is 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 used^TM MSCGM-CD^TMMesenchymal Stem Cell Medium, chemical Defined, Chinese name: therapeak^TMMSCGM-CD^TMHuman mesenchymal stem cell culture medium, product code: YS000471, supplier: LONZA corporation).

Third, CCK-8(Cell Counting Kit 8) method for detecting Cell proliferation

(1) Respectively inoculating the umbilical cord mesenchymal stem cells of the oxygen concentration gradient treatment group and the oxygen concentration normal treatment group in the second step into a 96-hole 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 umbilical cord mesenchymal 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.

Fourthly, stock solution rich in exosomes derived from umbilical cord mesenchymal stem cells is prepared

Transferring the umbilical cord mesenchymal stem cells of the oxygen concentration gradient group with good growth state in the second step, wherein the number of the umbilical cord mesenchymal stem cells is 5 multiplied by 10⁶Cell culture flasks subcultured to 175 cm square, commercial serum-free Stem cell Medium (English name: TheraPEAK)^TM MSCGM-CD^TMMesenchymal Stem Cell Medium, chemical Defined, Chinese name: therapeak^TM MSCGM-CD^TMHuman mesenchymal stem cell culture medium, product code: YS000471, supplier: LONZA corporation), and collecting a culture supernatant when the degree of fusion of cell growth reaches 80% to 90%; centrifuging at 4 deg.C for 10min, and centrifuging at 2000g to remove intact cells and dead cell debris; obtaining a supernatant A; filtering the supernatant A with 0.45 μm and 0.22 μm filters to obtain a stock solution B rich in exosomes; the solution B is rich in active factors, and needs to be stored at a low temperature of 4 ℃.

(1) Preparing base solution C (first preparing hyaluronic acid, glycerol, olive oil, normal saline into mixed solution, adding asiaticoside, and making into solution C)

Wherein the concentration of each substance is the final concentration in the solution C. CAS number of asiaticoside: 16830-15-2, supplier: dingrui chemical (Shanghai) Co., Ltd.

(2) Application method

The method I comprises the following steps: taking out the liquid B stored at low temperature, extruding 2-3 ml of the liquid B to the palm of the hand, smearing the liquid B to the striae gravidarum of the abdomen, and gently patting and massaging the liquid B uniformly; squeeze out appropriate amount of solution C (equal volume to solution B), massage gently on abdomen until B, C is completely absorbed.

Method II: or used in combination with physical low temperature introducing device (Plabeau Spectrum Lebo low temperature plasma skin care instrument, model: PB-S1, supplier: Korean (L.) Spectrum Labao PLABIO CO, LTD.); mixing the solutions B and C at palm, dipping with low temperature probe (proper amount, ensuring probe wetting), and gently massaging at abdomen until completely absorbed.

Fifth, use case

According to the method I in the step four (2), the number and the coverage area of the striae gravidarum larger than 2cm are obviously reduced after the striae gravidarum is continuously used for 1 time every day for 1 month, 2 months and 3 months, wherein after the striae gravidarum is used for 3 months, the exosome repair effect derived from the oxygen concentration gradient group is more obvious, and detailed results are shown in a table 1. Meanwhile, the striae gravidarum becomes light in color, the torn lines become light, and the abdomen becomes tight.

According to the method II in the step four (2), after the instrument is matched with the instrument to conduct (liquid B + liquid C) for 1 time every week and continuously used for 1 month, 2 months and 3 months, similar change rules are displayed on the repairing efficacy of the striae gravidarum, the related indexes are improved by 2-3 percentage points, and the repairing and nursing effect of the umbilical cord mesenchymal stem cell exosomes can be more efficiently exerted by matching with the instrument to conduct.

TABLE 1 analysis of effect of oxygen concentration change stimulating umbilical cord mesenchymal stem cells to generate exosome to repair striae gravidarum

Note: the reduction ratio of the striae gravidarum number to the striae gravidarum number before use (number of striae gravidarum before use-number of striae gravidarum after use)/number of striae gravidarum before use. Stretch mark coverage area is reduced from before use by the ratio (stretch mark area before use-stretch mark area after use)/stretch mark 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

1. A preparation method of umbilical cord mesenchymal stem cell exosomes comprises the following steps:

(I) placing the umbilical cord mesenchymal stem cells under a series of concentration gradients of oxygen concentration from low to high to culture in sequence; the series of concentration gradients of the oxygen concentration from low to high covers 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;

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 concentration gradient, the culture time is 10-14h when the umbilical cord mesenchymal stem cells are cultured; and/or

In the step (I), CO is added when the umbilical cord mesenchymal stem cells are cultured under each concentration gradient₂The concentration of (A) is 5%; and/or

In the step (I), under each concentration gradient, the culture temperature is 37 ℃ when the umbilical cord mesenchymal stem cells are cultured; and/or

In the step (I), the culture medium adopted when the umbilical cord mesenchymal stem cells are cultured under each concentration gradient is a serum-free stem cell culture medium.

5. The method according to any one of claims 1-4, wherein: in the step (I), before the umbilical cord mesenchymal 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 umbilical cord mesenchymal 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 umbilical cord mesenchymal stem cell exosome is obtained by separation and purification according to the method comprising the following steps: after collecting the culture supernatant of the umbilical cord mesenchymal 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 umbilical cord mesenchymal 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 umbilical cord mesenchymal stem cell exosome precipitate in a PBS solution, and filtering by using 0.45-micrometer and 0.22-micrometer filters in sequence, wherein the obtained filtrate is a stock solution rich in umbilical cord mesenchymal stem cell exosomes.

7. Umbilical cord mesenchymal stem cell exosomes or umbilical cord mesenchymal stem cell exosome-rich stock solution prepared by the method of any one of claims 1-6.

8. A repairing care solution for repairing striae gravidarum consists of a solution B and a solution C which are respectively and independently packaged, or is formed by mixing the solution B and the solution C in equal volumes;

the liquid B is a stock solution rich in umbilical cord mesenchymal stem cell exosomes of claim 7;

9. Any of the following applications:

use of P1, the umbilical cord mesenchymal stem cell exosomes of claim 7 or a stock solution enriched in umbilical cord mesenchymal stem cell exosomes in the preparation of the repair care solution of claim 8;

the use of P2 or the repair solution of claim 8 for repairing stretch marks;

p3, the umbilical cord mesenchymal stem cell exosome of claim 7 or stock solution rich in umbilical cord mesenchymal stem cell exosome or the use of the repair care solution of claim 8 in tissue repair.

10. Any one of the following methods:

method A, a method for increasing the proliferation capacity of umbilical cord mesenchymal stem cells, comprising the step (I) of any one of claims 1 to 5, obtaining umbilical cord mesenchymal stem cells with increased proliferation capacity;

b1: applying 2-3 ml of the solution B in the claim 8 to the abdominal striae gravidarum, and massaging uniformly; taking 2-3 ml of the liquid C in claim 8, massaging the abdomen until the liquid B and the liquid C are completely absorbed;

b2: mixing the solution B and the solution C in the claim 8 in equal volume, dipping with a low temperature probe of a physical low temperature introduction device, and massaging the abdomen until the solution is completely absorbed;

method C, a method for improving the tissue repair capacity of exosomes of umbilical cord mesenchymal stem cells, comprising the step (I) of any one of claims 1 to 5, wherein the tissue repair capacity of exosomes secreted by umbilical cord mesenchymal stem cells cultured in the step (I) is improved.