CN112535692B - Preparation method of stem cell factor repair liquid capable of promoting wound healing - Google Patents
Preparation method of stem cell factor repair liquid capable of promoting wound healing Download PDFInfo
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- A61K38/18—Growth factors; Growth regulators
- A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
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- A61K38/18—Growth factors; Growth regulators
- A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
- A61K38/1866—Vascular endothelial growth factor [VEGF]
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- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/02—Atmosphere, e.g. low oxygen conditions
Abstract
The invention relates to a preparation method of stem cell factor repair liquid capable of promoting wound healing, which comprises the following steps: s1: providing a stem cell to be cultured; s2: pre-culturing stem cells under conditions suitable for growth of the stem cells to obtain a first culture; s3: continuously culturing the first culture under the condition of low-oxygen culture to obtain a second culture solution; s4: separating the second culture solution, thereby obtaining a culture supernatant; wherein the hypoxia condition refers to 0.1-5% of oxygen by volume, and N 2 、CO 2 And O 2 Is defined as the total volume. The method adopts the hypoxia technology to culture the stem cells, and can enable the stem cells to secrete more bioactive factors, thereby better promoting wound healing.
Description
Technical Field
The invention relates to the field of biological product preparation, in particular to a preparation method of stem cell factor repair liquid capable of promoting wound healing.
Background
The skin refers to the tissue covered outside the body, is the largest organ of the human body, and mainly plays the roles of protecting the body, perspiration, feeling cold and hot, pressure and the like. The skin covers the whole body and protects various tissues and organs in the body from physical, mechanical, chemical and pathogenic microorganisms, so the skin is a protective barrier for our body, but the skin is very fragile, various reasons are often left in life to leave wounds, some wounds automatically heal within three to five days, but some people find own wounds difficult to heal, and if careless, the wounds are infected by bacteria, the wound healing is affected, and even suppuration, infection and even life threatening are caused.
The causes of difficult wound healing are numerous, such as: age, nutritional status, systemic disease, etc., especially those elderly and suffering from chronic diseases such as diabetes, once there is a wound on the limb, conventional drugs have little effect on such wounds.
Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) are one type of adult stem cells, whose repair and regeneration in vivo have been preliminarily confirmed. Its key role in wound healing includes 2 aspects: firstly, directly participating in reconstruction of damaged tissues, mainly comprising proliferation, migration, homing, differentiation and the like of cells; secondly, the cell is indirectly involved in the repair and reconstruction of tissue injury in a mode of regulating sexual function cells, and mainly relates to the secretion of a large amount of bioactive molecules in a paracrine mode after stem cells are activated so as to promote the functions of other repair cells. Mesenchymal stem cells secrete various growth factors during culture, such as Stem Cell Factor (SCF), epidermal Growth Factor (EGF), vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor (TGF- β), nerve Growth Factor (NGF), insulin-like growth factor (IGF), etc. The growth factors secreted by the stem cells can promote proliferation and differentiation of various cells, and new cells are generated to replace aged and damaged cells; promote cell synthesis and secretion of extracellular matrix components, and provide a good nutritional environment. In addition, the secretion factors of the stem cells also contain various extracellular matrix proteins, so that the skin quality can be improved, and particularly, the skin scar and the scar can be repaired well.
However, most of the current stem cell culture is carried out under the condition of 21% oxygen concentration, so that the secretion amount of stem cell factors is low, and the requirement of wound healing cannot be met.
Disclosure of Invention
The invention aims to provide a preparation method of stem cell factor repair liquid for promoting wound healing, which uses low-oxygen culture conditions, and the prepared repair liquid can effectively promote wound healing and has better effect.
In a first aspect of the present invention, there is provided a method for preparing a stem cell factor repair liquid, comprising the steps of:
s1: providing a stem cell to be cultured;
s2: pre-culturing stem cells under conditions suitable for growth of the stem cells to obtain a first culture;
s3: continuously culturing the first culture under the condition of low-oxygen culture to obtain a second culture solution;
s4: separating the second culture solution, thereby obtaining a culture supernatant;
wherein the hypoxia condition refers to 0.1-5% of oxygen by volume, and N 2 、CO 2 And O 2 Is defined as the total volume.
In another preferred embodiment, the hypoxic culture conditions are 0.2% or less of oxygen by volume < 2%, preferably 0.5-1.5%, more preferably 1%, N 2 、CO 2 And O 2 Is defined as the total volume.
In another preferred embodiment, the culture conditions for the preculture are 15 to 25% by volume of oxygen, preferably 20 to 21% by volume, in N 2 、CO 2 And O 2 Is defined as the total volume.
In another preferred embodiment, the conditions for the hypoxic culture are 5% CO 2 、1％O 2 、94％N 2 In N 2 、CO 2 And O 2 Is defined as the total volume.
In another preferred embodiment, the culture temperature of the preculture is 25 to 40 ℃, preferably 35 to 38 ℃, more preferably 37 ℃.
In another preferred embodiment, the low oxygen conditions are used at a cultivation temperature of 25-40deg.C, preferably 35-38deg.C, more preferably 37deg.C.
In another preferred embodiment, the preculture conditions are: the stem cells were exposed to 5% CO at 37℃ 2 、21％O 2 And 74% N 2 Culturing in a conventional wet environment with concentration.
In another preferred embodiment, the cultivation time for the preculture is 2.5 to 10 days, preferably 3 to 8 days, more preferably 4 to 6 days.
In another preferred embodiment, in step s2, the commercially available stem cells are subcultured for at least 2 or 3 passages, such as 3-7 passages (e.g., 3, 4, 5, 6, or 7 passages).
In another preferred embodiment, the period of time for the low oxygen cultivation is 1 to 48 hours, preferably 12 to 36 hours, more preferably 24 hours.
In another preferred embodiment, the stem cells are human umbilical cord or adipose mesenchymal stem cells.
In another preferred embodiment, the preparation method further comprises the following steps:
s5: and mixing the stabilizer with the obtained culture supernatant according to the volume ratio of 1:1-1:10 to obtain the stem cell factor repairing liquid.
In another preferred embodiment, the stabilizer is a mixture of pharmaceutically acceptable glycerol and sterile distilled water.
In another preferred embodiment, the volume ratio of the pharmaceutically acceptable glycerol to the sterile distilled water is 1:1 to 1:5.
In another preferred example, the prepared stem cell factor repair liquid is stored in a medical refrigerator at 4 ℃.
In another preferred embodiment, the culture supernatant is a culture supernatant of passaged stem cells P3-P7.
In another preferred embodiment, the culture supernatant is a culture supernatant of passaged stem cells P4-P6.
In another preferred embodiment, the passaged stem cells are P6 human umbilical cord mesenchymal stem cells.
In another preferred embodiment, the passaged stem cells are P4 adipose-derived mesenchymal stem cells.
In another preferred embodiment, the preculture and the hypoxic culture each employ a medium selected from the group consisting of: serum-free medium, phenol red-free medium, or a combination thereof.
In another preferred embodiment, the second culture broth is separated using a 0.22um filter.
In another preferred embodiment, the positive proportion of positive markers CD73, CD90 and CD105 (based on the total number of stem cells) of said passaged stem cells is greater than 90%.
In another preferred embodiment, the positive proportion of positive markers CD73, CD90 and CD105 (based on the total number of stem cells) of said passaged stem cells is greater than 95%.
In another preferred embodiment, the positive proportion of negative markers CD31, CD34, CD45 and HLA-DR of the passaged stem cells is less than 5% (based on the total number of stem cells).
In another preferred embodiment, the positive proportion of negative markers CD31, CD34, CD45 and HLA-DR of the passaged stem cells is less than 2% (based on the total number of stem cells).
In another preferred embodiment, the culture supernatant comprises:
in another preferred embodiment, the stem cell factor repair liquid is mainly used for repairing ischemic injury wounds.
In a second aspect of the present invention, there is provided a stem cell culture supernatant comprising:
in another preferred embodiment, the culture supernatant is prepared by the method for preparing the stem cell factor repair liquid of the first aspect.
In a third aspect of the invention, there is provided an article of manufacture comprising the culture supernatant of the second aspect, and a pharmaceutically acceptable carrier.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
Figure 1 shows the effect of a stem cell factor repair fluid prepared using the method of the present invention on a wound that is not effective after treatment with amoxicillin for two weeks or more.
FIG. 2 shows the results of comparing mesenchymal stem cell factor secretion under hypoxic and normoxic conditions.
Detailed Description
The present inventors have made extensive and intensive studies, and for the first time, unexpectedly developed a method for preparing a stem cell factor repair liquid for promoting wound healing, which uses a specific culture method for culturing umbilical cord or adipose stem cells by means of normal oxygen pre-culture in combination with hypoxia culture, thereby unexpectedly enabling the cultured stem cells to secrete more beneficial bioactive factors. Experiments show that the cytokine repairing liquid prepared by the method can heal wounds more effectively and more rapidly. The present invention has been completed on the basis of this finding.
Preparation method of stem cell factor repair liquid
In the invention, the preparation method of the stem cell factor repair liquid comprises the following steps:
s1: providing a stem cell to be cultured;
s2: pre-culturing stem cells under conditions suitable for growth of the stem cells to obtain a first culture;
s3: continuously culturing the first culture under the condition of low-oxygen culture to obtain a second culture solution;
s4: separating the second culture solution, thereby obtaining a culture supernatant;
wherein the hypoxia condition refers to 0.1-5% of oxygen by volume, and N 2 、CO 2 And O 2 Is defined as the total volume.
Preferably, the hypoxic culture conditions are 0.2% or less of oxygen by volume < 2%, preferably 0.5-1.5%, more preferably 1%, N 2 、CO 2 And O 2 Is defined as the total volume.
Preferably, the culture conditions for the preculture are those in which the volume percentage of oxygen is 15 to 25%, preferably 20 to 21%, in N 2 、CO 2 And O 2 Is defined as the total volume.
Preferably, the culture temperature of the preculture and the hypoxic condition is 25-40deg.C, preferably 35-38deg.C, more preferably 37deg.C.
Preferably, the conditions for the hypoxic culture are 5% CO 2 、1％O 2 、94％N 2 In N 2 、CO 2 And O 2 Is defined as the total volume.
Preferably, the preculture conditions are: the stem cells were exposed to 5% CO at 37℃ 2 、21％O 2 And 74% N 2 Culturing in a conventional wet environment with concentration.
Preferably, the cultivation time for the preculture is 2.5 to 10 days, preferably 3 to 8 days, more preferably 4 to 6 days.
Preferably, the time for the low-oxygen cultivation is 1 to 48 hours, preferably 12 to 36 hours, more preferably 24 hours.
Preferably, the preparation method further comprises the following steps:
and mixing the stabilizer with the obtained culture supernatant according to the volume ratio of 1:1-1:10 to obtain the stem cell factor repairing liquid.
Preferably, the stabilizer is a mixture of medicinal glycerol and sterile distilled water, and preferably, the volume ratio of the medicinal glycerol to the sterile distilled water is 1:1-1:5.
Preferably, the prepared stem cell factor repair liquid is stored in a medical refrigerator at 4 ℃.
Preferably, the stem cells are human umbilical cord or adipose mesenchymal stem cells.
Preferably, the culture supernatant is a culture supernatant of passage stem cells P3-P7, preferably a culture supernatant of P4-P6, more preferably a culture supernatant of P6 human umbilical cord mesenchymal stem cells or P4 adipose mesenchymal stem cells.
Preferably, the positive proportion of positive markers CD73, CD90 and CD105 (based on total stem cells) of said passaged stem cells is greater than 90%, preferably greater than 95%; the positive proportion of the negative markers CD31, CD34, CD45 and HLA-DR is less than 5%, preferably less than 2%.
Preferably, the method of the invention employs a medium selected from the group consisting of: serum-free medium, phenol red-free medium, or a combination thereof.
Preferably, the second culture broth is separated with a 0.22um filter.
Culture supernatant
The culture supernatant is prepared by the preparation method of the stem cell factor repair liquid.
Preferably, the culture supernatant (stem cell factor repair stock solution) comprises:
preferably, the amount of VEGF in the culture supernatant is 1800-2000pg/ml.
Preferably, the amount of IGF-1 in the culture supernatant is 350-450pg/ml.
Preferably, the amount of HGF in the culture supernatant is 2500-3000pg/ml.
Preferably, the stem cell factor repair liquid is mainly used for repairing ischemic injury wounds, and preferably, ineffective wounds after treatment by conventional OTC anti-inflammatory drugs (such as penicillin anti-inflammatory drugs).
Article of manufacture
The preparation of the invention comprises the culture supernatant as described above and a pharmaceutically acceptable carrier.
"pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, and the like), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, and the like), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, and the like), emulsifiers (e.g.
) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.
Preferably the preparation of the invention comprises a culture supernatant and a stabilizing agent.
Preferably, the stabilizer is a mixture of medicinal glycerol and sterile distilled water, and preferably, the volume ratio of the medicinal glycerol to the sterile distilled water is 1:1-1:5.
Hypoxia culture
The low-oxygen culture according to the present invention means a culture under normoxic culture conditions (i.e., oxygen content of about the oxygen content of air) for a certain period of time (e.g., 3 to 4 days), followed by a low-oxygen culture (i.e., oxygen less than normoxic culture) for a certain period of time (e.g., 24 hours) before collecting culture supernatant.
Compared with the prior art, the invention has the following beneficial effects:
1. the preparation method of the invention adopts the hypoxia technology to culture umbilical cord or fat stem cells, and can enable the stem cells to secrete more bioactive factors, thereby being capable of promoting wound healing more effectively.
2. The preparation method adopts the serum-free culture medium to culture the umbilical cord or the adipose-derived stem cells, is safe and reliable, and can not introduce heterologous pathogenic microorganisms and heterologous proteins or even cause immune rejection reaction like the traditional animal serum culture method.
3. The stem cell factor repairing liquid prepared by the method can rapidly relieve local inflammation and promote the establishment of collateral circulation, thereby playing remarkable roles of easing pain and promoting wound healing.
4. The method of the invention does not contain cells, particulate matters or microorganisms, and is safe and reliable.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Example 1 preparation of umbilical cord Stem cell factor repair liquid
1. Preparation of umbilical cord stem cells
1. Placing umbilical cord tissue of healthy person with four detected infectious diseases and other viruses and microorganisms and negative in an ultra clean bench, cutting into 1cm segments, repeatedly cleaning umbilical cord with sterile physiological saline for several times, washing umbilical cord blood, cutting the outer wall of umbilical cord with surgical scissors, carefully removing three blood vessels with forceps, taking Wharton's jelly tissue with forceps into a culture dish, cutting into finer fragments, adding appropriate amount of serum-free and phenol-free red complete culture medium into the culture dish, and adding conventional CO 2 In a cell incubator.
2. After cell culture for 72 hours, the supernatant was aspirated, fresh serum-free phenol red-free complete medium was added, and CO was then added 2 Half of the medium was then changed every two days in the incubator until 85% of the dish bottom was seen to be full of cells. TrypLE with Gibco whenever 85% of cells were in the dish TM The Select digest digests the cells, and the stem cells are resuspended in fresh serum-free medium and inoculated into a cell culture flask. Thus passaged to P6 umbilical cord stem cells.
3. Culturing umbilical cord mesenchymal stem cells to P6, taking out a proper amount of cells, labeling the stem cells with antibodies, and detecting the expression level of stem cell markers on a flow cytometer.
The detection result shows that the positive proportion of the positive markers CD73, CD90 and CD105 of the mesenchymal stem cells is more than or equal to 95 percent, and the positive proportion of the negative markers CD31, CD34, CD45 and HLA-DR of the mesenchymal stem cells is less than 2 percent.
2. Preparation of umbilical cord Stem cell culture supernatant
Umbilical cord stem cells of P6 were used as a source of stem cell factor repair fluid. Cells were first incubated at 37℃with 5% carbon dioxide, 21% oxygen and 74% N 2 (volume percent) in a conventional humid environment, 24 hours before harvesting, the cells were transferred to a special culture environment containing 1% oxygen, after 24 hours the whole cell culture supernatant (stem cell factor repair stock solution) was collected and stored in a medical refrigerator at 4 ℃ for subsequent preparation.
3. Filtration of umbilical cord stem cell supernatant
After the supernatant is accumulated to a certain volume, the supernatant is taken out from the refrigerator, put into an ultra-clean workbench, filtered by a 0.2 mu m sterile filter and recycled into a new sterile bottle for standby.
4. Preparation of the stabilizer
In an ultra clean bench, medical glycerol and sterile distilled water are mixed uniformly in a volume ratio of 1:1 (if necessary, a sterile filter of 0.2um can be used for filtration).
5. Preparation of umbilical cord stem cell factor repairing liquid
In an ultra clean bench, the stabilizer and the filtered supernatant are mixed uniformly according to the volume ratio of 1:6, and are packaged into 1ml sterile dropping bottles, and are placed in a medical refrigerator of 4 degrees.
Example 2 preparation of adipose-derived mesenchymal Stem cell factor repair liquid
1. Preparation of adipose-derived mesenchymal Stem cells
The same procedure as in example 1 was employed except that the umbilical cord tissue was replaced with fat tissue obtained by liposuction or liposuction. And (5) subculturing to obtain the P4 adipose-derived stem cells.
2. Preparation of adipose-derived mesenchymal Stem cell culture supernatant
Fat stem cells of P4 were used as a source of stem cell factor repair fluid. Cells always had 5% carbon dioxide, 21% oxygen and 74% N at 37℃ 2 The cells were transferred to a special culture environment containing 1% oxygen for 24 hours before harvesting, and after 24 hours the whole cell culture supernatant was collected and stored in a medical refrigerator at 4 ℃ for subsequent preparation.
3. Filtration of adipose-derived mesenchymal stem cell supernatant
After the supernatant is accumulated to a certain volume, the supernatant is taken out from the refrigerator and put into an ultra-clean workbench, filtered by a 0.2um sterile filter, and recovered into a new sterile bottle for standby.
4. Preparation of the stabilizer
In an ultra clean bench, medical glycerol and sterile distilled water are mixed uniformly in a volume ratio of 1:1 (if necessary, a sterile filter of 0.2um can be used for filtration).
5. Preparation of adipose-derived mesenchymal stem cell factor repair liquid
In an ultra clean bench, the stabilizer and the filtered supernatant are mixed uniformly according to the volume ratio of 1:6, and are packaged into 1ml sterile dropping bottles, and are placed in a medical refrigerator of 4 degrees.
EXAMPLE 3 different conditions of mesenchymal Stem cell factor secretion
Experimental method
1. Hypoxic conditions (5% CO) 2 、1％O 2 、94％N 2 ) Group:
umbilical cord stem cells of P6 were used as a source of stem cell factor repair fluid. Cells were first incubated at 37℃with 5% carbon dioxide, 21% oxygen and 74% N 2 (volume percent) in a conventional humid environment, 24 hours prior to harvest, the cells were transferred to a special culture environment containing 1% oxygen, and after 24 hours the whole cell culture supernatant (stem cell factor repair stock) was collected. Culture of umbilical cord stem cells and collection of supernatant can be seen in example 1.
2. Normoxic conditions (5% CO) 2 、21％O 2 、74％N 2 ) Group:
materials and media are in the same "hypoxic conditions" group. Umbilical cord stem cells of P6 were used as a source of stem cell factor repair fluid. Cells were kept at 37℃C, 5% carbon dioxide, 21% oxygen and 74% N 2 (volume percent) in a conventional humid environment, and directly collecting the whole cell culture supernatant.
Experimental results
For the secretion of mesenchymal stem cell factor in hypoxia and normoxic condition, the results are shown in table 1 and fig. 2 using a commercially available ELISA assay kit.
TABLE 1
Experimental results show that compared with normoxic culture conditions, the mesenchymal stem cell factor secretion can be remarkably improved by performing hypoxia culture for a short time before collecting culture supernatant. The rise of VEGF, IGF-1, HGF and KGF is most remarkable, and the rise is 68-149%.
EXAMPLE 4 healing Effect on ineffective wounds after conventional OTC drug treatment
The experimental object: 4 volunteers, who had wounds in the extremities or feet and who were not effective half a year after amoxicillin treatment.
The using method comprises the following steps: after the wound is cleaned, the stem cell factor repairing liquid prepared in the example 1 is uniformly dripped on the affected part every 4 hours, and amoxicillin is coated on the affected part for continuous use for 2 weeks.
Test results: the wound of 4 subjects suffering from the wound healed, and the comparison of the front and back of 1 subject using the stem cell factor repair liquid prepared by the present invention is shown in fig. 1.
The result shows that the stem cell factor repair liquid prepared by the method can obviously promote the healing of ineffective wounds after the treatment of conventional OTC medicaments (such as amoxicillin).
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (19)
1. The preparation method of the stem cell factor repair liquid is characterized by comprising the following steps of:
s1: providing a stem cell to be cultured;
s2: pre-culturing stem cells under conditions suitable for growth of the stem cells to obtain a first culture, wherein the stem cells are subcultured for 6 generations;
s3: continuously culturing the first culture under the condition of low-oxygen culture to obtain a second culture solution; and
s4: separating the second culture solution, thereby obtaining a culture supernatant;
wherein the culture condition of the preculture is that the volume percentage of oxygen is 20-21%, and N2 and CO are used 2 And O 2 Is based on the total volume of the container; the culture temperature of the pre-culture is 25-40 ℃; the culture time of the preculture is 2.5-10 days;
wherein the hypoxia condition is that the volume percentage of oxygen is 0.5-1.5%, and N2 and CO are used 2 And O 2 Is based on the total volume of the container; the time of the hypoxia culture is 24-36 h; the culture temperature of the low-oxygen condition is 25-40 ℃;
wherein the stem cells are human umbilical cord stem cells;
the preculture and the hypoxia culture are both carried out by adopting a serum-free phenol red-free culture medium;
the culture supernatant comprises:
2. the method according to claim 1, wherein the low-oxygen culture condition is 1% by volume of oxygen, N 2 、CO 2 And O 2 Is defined as the total volume.
3. The method according to claim 2, wherein the conditions for the low-oxygen culture are 5% CO 2 、1％O 2 、94％N 2 In N 2 、CO 2 And O 2 Is defined as the total volume.
4. A method according to any one of claims 1 to 3, wherein the low oxygen conditions are at a temperature of 35 to 38 ℃.
5. The method of claim 1, wherein the period of time for the hypoxic culture is 24 hours.
6. The method according to claim 1, wherein the low oxygen condition is a culture temperature of 37 ℃.
7. The method according to claim 1, wherein the culture temperature of the preculture is 35 to 38 ℃.
8. The method of any one of claims 1 and 6-7, wherein the pre-culture conditions are: the stem cells were exposed to 5% CO at 37℃ 2 、21％O 2 And a 74% N2 concentration in a conventional humid environment.
9. The method of claim 1, wherein the pre-incubation time is 3-8 days.
10. The method of claim 1, wherein the pre-incubation time is 4-6 days.
11. The method according to claim 1, wherein the conditions for the low-oxygen culture are 5% CO 2 、1％O 2 94% N2, in N2, CO 2 And O 2 The preculture conditions are: the stem cells were exposed to 5% CO at 37℃ 2 、21％O 2 And a 74% N2 concentration in a conventional humid environment.
12. The method of manufacturing as claimed in claim 1, further comprising the steps of:
s5: and mixing the stabilizer with the obtained culture supernatant according to the volume ratio of 1:1-1:10 to obtain the stem cell factor repairing liquid.
13. The method of claim 12, wherein the stabilizer is a mixture of pharmaceutically acceptable glycerol and sterile distilled water.
14. The method of claim 13, wherein the volume ratio of the pharmaceutically acceptable glycerol to the sterile distilled water is from 1:1 to 1:5.
15. The method of claim 1, wherein the second culture broth is separated using a 0.22um filter.
16. The method of claim 1, wherein the positive proportion of positive markers CD73, CD90 and CD105 of the passaged stem cells is greater than 95% based on the total number of stem cells.
17. The method of claim 1, wherein the negative markers CD31, CD34, CD45 and HLA-DR of the passaged stem cells have a positive proportion of less than 2% based on the total number of stem cells.
18. Use of a stem cell factor repair liquid prepared by the preparation method of any one of claims 1 to 17 in the preparation of an agent for repairing an ischemic injury wound.
19. The method of claim 1, wherein the method further comprises: mixing the culture supernatant with a pharmaceutically acceptable carrier, thereby producing a product comprising the culture supernatant and the pharmaceutically acceptable carrier.
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