CN112972493A - Preparation method and application of anti-aging agent - Google Patents
Preparation method and application of anti-aging agent Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- 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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Abstract
The invention discloses a preparation method and application of an anti-aging agent, wherein the anti-aging agent is prepared by taking healthy newborn umbilical cords as raw materials according to the following steps: a) extracting mesenchymal stem cells; b) culturing the mesenchymal stem cells in a serum-free culture medium until the fusion rate of the mesenchymal stem cells reaches 40-50 percent; c) adding a glucose solution with the final concentration of 25-55 mmol/L into the monolayer cells to prepare a mixed solution; d) and (e) continuing culturing the mixed solution for 46-50 hours to prepare a preparation culture medium, and concentrating and purifying the culture medium.
Description
Technical Field
The invention relates to the field of stem cell anti-aging, in particular to a preparation method and application of an anti-aging agent.
Background
With the increasing prominence of the aging problem of the population, the anti-aging field becomes the focus of global attention. The skin is the largest organ of the human body and plays roles in protecting, feeling, regulating body temperature, secretion, excretion, immunity and the like, but with the increase of age, the skin can generate degeneration, for example, the skin becomes thin, the water content is reduced, the elasticity is reduced, the skin atrophy and the wrinkle are generated, and then the clinical symptoms such as the wrinkle, the senile plaque and the like are generated, so that people can be aware of the beginning of aging, the complex psychological and physiological influence is generated on the life and the work of people, and the psychological problems such as anxiety, depression, self-inferior and the like can be caused. Therefore, anti-aging treatments, particularly for the skin, are one of the focuses of research, and it is desired to improve and enhance the quality of life by anti-aging treatments.
Skin aging is a long and complex evolutionary process. Intrinsic factors are, for example, that with increasing age, cell telomeres are progressively shortened, while oxidative damage caused by aerobic cell metabolism is progressively increased. The specific manifestations are thinning of epidermis layer and dermis layer of skin, thinning of blood vessel wall, reduction and dysfunction of fibroblast in dermis layer, and gradual reduction of synthesis speed of collagen fiber. Extrinsic factors include lifestyle, nutritional status, environmental factors, and the like. Among them, photodamage (photodamage) caused by ultraviolet rays in sunlight is a major environmental factor causing skin aging. With the improvement of living standard and the rapid development of modern biology and medicine, especially the emergence of stem cell technology, the physiological function of skin can be finally changed fundamentally by activating skin cells, promoting the proliferation and differentiation of skin stem cells and repairing or replacing aged skin cells.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the existing defects and provide a preparation method and application of an anti-aging agent, thereby solving the problems.
In order to achieve the above object, the present invention provides a preparation method and an application of an anti-aging agent according to the following technical scheme, wherein the anti-aging agent is prepared from a healthy newborn umbilical cord as a raw material according to the following steps:
a) extracting mesenchymal stem cells;
b) culturing the mesenchymal stem cells in a serum culture medium until the mesenchymal stem cells are fused to 40-50% of a monolayer of cells;
c) adding a glucose solution with the concentration of 25-55 mmol/L into the monolayer cells to prepare a target culture medium;
d) continuously culturing the target culture medium for 46-50 hours to prepare a preparation culture medium;
e) concentrating and purifying the preparation medium;
in the step a), taking a fresh healthy umbilical cord of a full-term fetus, washing blood stains, stripping and shearing into small pieces by using normal saline, uniformly coating, slowly adding 10-15 mL of serum-free culture medium, culturing in an incubator, culturing primary cells for 7-10 days, then replacing the serum-free culture medium, culturing until the cells are fused to form umbilical cord mesenchymal stem cells with the concentration of more than 70%, removing the serum-free culture medium, adding a proper amount of trypsin-sodium Ethylenediaminetetraacetate (EDTA) for digestion for l minutes to make the umbilical cord mesenchymal stem cells shrink and separate from the wall of the flask, adding the culture supernatant removed previously, slightly blowing by using a pipette, transferring the suspension of the umbilical cord mesenchymal stem cells into a 50mL centrifuge tube, centrifuging, removing the supernatant, adding the serum-free culture medium again to resuspend the umbilical cord mesenchymal stem cells, counting, and carrying out subculture according to the method;
in the step b), serum containing 5-10% of amino acid is added into the 3 rd generation cells of the umbilical cord mesenchymal stem cells to be cultured to 40-50% of monolayer cells;
in the step c), adding a glucose solution into the monolayer cells, wherein the concentration of glucose is 25-55 mmol/L, and preparing a target culture medium;
in the step d), after the target culture medium is continuously cultured for 46-52 hours, filtering by using a filter to prepare a preparation culture medium;
in the step e), the preparation culture medium is dried, concentrated and purified.
Preferably, the physiological saline is penicillin and streptomycin with the concentration of 50-100 ug/mL.
Preferably, the culture conditions in the incubator are 37 ℃, 5% by volume of carbon dioxide and 95% saturated humidity.
Preferably, the mass ratio of the trypsin to the sodium Ethylene Diamine Tetracetate (EDTA) is 0.1-0.2.
Preferably, the filter mesh is 0.2 wn.
Compared with the prior art, the invention has the beneficial effects that: the umbilical cord mesenchymal stem cell conditioned medium contains a large amount of growth factors required by cells, can effectively reduce the generation of R0S in the cells and delay the senescence of the cells.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a preparation method of an anti-aging agent and mesenchymal stem cell fusion degree data applied thereto;
fig. 2 is a flow chart of a preparation method of an anti-aging agent and a preparation process of mesenchymal stem cells using the same.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without any creative work based on the embodiments of the present invention belong to the protection scope of the present invention.
The invention provides a technical scheme that: the preparation method and the application of the anti-aging agent are characterized in that the anti-aging agent takes healthy newborn umbilical cords as raw materials and is prepared according to the following steps:
a) extracting mesenchymal stem cells;
b) culturing the mesenchymal stem cells in a serum culture medium until the mesenchymal stem cells are fused to 40-50% of a monolayer of cells;
c) adding a glucose solution with the concentration of 25-55 mmol/L into the monolayer cells to prepare a target culture medium;
d) continuously culturing the target culture medium for 46-50 hours to prepare a preparation culture medium;
e) concentrating and purifying the preparation medium;
in the step a), taking a fresh healthy umbilical cord of a full-term fetus, washing blood stains, stripping and shearing into small pieces by using normal saline, uniformly coating, slowly adding 10-15 mL of serum-free culture medium, culturing in an incubator, culturing primary cells for 7-10 days, then replacing the serum-free culture medium, culturing until the cells are fused to form umbilical cord mesenchymal stem cells with the concentration of more than 70%, removing the serum-free culture medium, adding a proper amount of trypsin-sodium Ethylenediaminetetraacetate (EDTA) for digestion for l minutes to make the umbilical cord mesenchymal stem cells shrink and separate from the wall of the flask, adding the culture supernatant removed previously, slightly blowing by using a pipette, transferring the suspension of the umbilical cord mesenchymal stem cells into a 50mL centrifuge tube, centrifuging, removing the supernatant, adding the serum-free culture medium again to resuspend the umbilical cord mesenchymal stem cells, counting, and carrying out subculture according to the method;
in the step b), serum containing 5-10% of amino acid is added into the 3 rd generation cells of the umbilical cord mesenchymal stem cells to be cultured to 40-50% of monolayer cells;
in the step c), adding a glucose solution into the monolayer cells, wherein the concentration of glucose is 25-55 mmol/L, and preparing a target culture medium;
in the step d), after the target culture medium is continuously cultured for 46-52 hours, filtering by using a filter to prepare a preparation culture medium;
in the step e), the preparation culture medium is dried, concentrated and purified.
The physiological saline is penicillin and streptomycin with the concentration of 50-100 ug/mL.
The culture conditions in the incubator are that the temperature is 37 ℃, the volume fraction of carbon dioxide is 5 percent, and the saturation humidity is 95 percent.
The mass ratio of the trypsin to the ethylenediaminetetraacetic acid (EDTA) is 0.1-0.2.
The filter mesh number is 0.2 wn.
Example 1: operating in an ultraclean workbench, taking fresh healthy umbilical cord of a full-term fetus, washing with 60ug/mL physiological saline to remove blood stain, cutting into small pieces, uniformly coating, slowly adding 10mL serum-free culture medium, culturing in an incubator with the temperature of 37 ℃, the volume fraction of carbon dioxide of 5% and the saturation humidity of 95%, culturing primary cells for 7 days, replacing the serum-free culture medium, culturing until the cells are fused to 70%, removing the serum-free culture medium, adding trypsin-Ethylene Diamine Tetraacetic Acid (EDTA) with the mass proportion of 0.13% to digest lmin, shrinking and separating umbilical cord mesenchymal stem cells from the bottle wall, adding the previously removed culture supernatant, gently blowing by using a pipette, transferring the umbilical cord mesenchymal stem cell suspension into a 50mL centrifuge tube, centrifuging for 8min at the centrifugation speed of 1000rpm, removing the supernatant, adding the serum-free complete culture medium again to resuspend the umbilical cord mesenchymal stem cells, counting, and subculturing to 3 rd generation cells according to the method;
culturing the 3 rd generation cells of umbilical cord mesenchymal stem cells in serum containing 8% amino acids to a 40-50% monolayer of cells, adding a glucose solution to a final concentration of 25Lmmol/L glucose, and continuing the culturing for 48 hours. Centrifuging for 5 minutes at a centrifugation speed of 2000rpm, collecting culture supernatant, namely conditioned medium, as umbilical cord mesenchymal stem cells, filtering by using a filter with a mesh number of 0.25wn, drying, concentrating by 10 times, and grinding a target culture solution, namely an anti-aging agent into powder, and adding the powder as an active ingredient into skin care water for improving the aging problem of skin.
Example 2: operating in an ultraclean workbench, taking fresh healthy umbilical cord of a term fetus, washing with 80ug/mL physiological saline to remove blood stain, cutting into small pieces, uniformly coating, slowly adding 10mL serum-free culture medium, culturing in an incubator with the temperature of 37 ℃, the volume fraction of carbon dioxide of 5% and the saturation humidity of 95%, culturing primary cells for 10 days, replacing the serum-free culture medium, culturing until the cells are 70% fused, removing the serum-free culture medium, adding trypsin-sodium Ethylenediaminetetraacetate (EDTA) with the mass ratio of 0.15% for digestion for 1.5min, shrinking the umbilical cord mesenchymal stem cells to separate from the bottle wall, adding the previously removed culture supernatant, gently blowing and beating by using a pipette, transferring the umbilical cord mesenchymal stem cell suspension into a 50mL centrifuge tube, centrifuging for 9min at the centrifugation speed of 1070rpm, removing the supernatant, adding again the serum-free complete culture medium to resuspend the umbilical cord mesenchymal stem cells, counting, and subculturing to 3 rd generation cells according to the method;
culturing the 3 rd generation cells of umbilical cord mesenchymal stem cells in serum containing 10% amino acids to a 40-50% monolayer of cells, adding a glucose solution to a final concentration of 45mmol/L glucose, and continuing the culturing for 50 hours. Centrifuging for 8 minutes at a centrifuging speed of 2080rpm, collecting culture supernatant, namely conditioned medium, as umbilical cord mesenchymal stem cells, filtering by using a filter with a mesh number of 0.2wn, drying, concentrating by 12 times, and grinding a target culture solution, namely an anti-aging solution into powder, and adding the powder as an active ingredient into eye cream for improving the eye skin problem.
Example 3: operating in an ultraclean workbench, taking fresh healthy umbilical cord of a term fetus, washing with 100ug/mL physiological saline to remove blood stain, cutting into small pieces, uniformly coating, slowly adding 10mL serum-free culture medium, culturing in an incubator with the temperature of 37 ℃, the volume fraction of carbon dioxide of 5% and the saturation humidity of 95%, culturing primary cells for 8 days, replacing the serum-free culture medium, culturing until the cells are fused to 70%, removing the serum-free culture medium, adding trypsin-Ethylene Diamine Tetraacetic Acid (EDTA) with the mass proportion of 0.18% to digest lmin, shrinking and separating umbilical cord mesenchymal stem cells from the bottle wall, adding the previously removed culture supernatant, gently blowing and beating by using a pipette, transferring the suspension of umbilical cord mesenchymal stem cells into a 50mL centrifuge tube, centrifuging for 9min at the centrifugation speed of 950rpm, removing the supernatant, adding again the serum-free complete culture medium to resuspend the umbilical cord mesenchymal stem cells, counting, and subculturing to 3 rd generation cells according to the method;
culturing the 3 rd generation cells of umbilical cord mesenchymal stem cells in serum containing 8% amino acids to a 40-50% monolayer of cells, adding a glucose solution to a final concentration of 48mmol/L glucose, and continuing the culturing for 52 hours. Centrifuging for 6 minutes at 2250rpm, collecting culture supernatant as conditioned medium, filtering with a filter having a mesh of 0.15wn as umbilical cord mesenchymal stem cells, oven drying, concentrating by 15 times, and grinding the target culture solution as anti-aging agent, which can be added into hand cream as active ingredient for improving skin problems of hands.
Finally, it should be further noted that the above examples and descriptions are not limited to the above embodiments, and technical features of the present invention that are not described may be implemented by or using the prior art, and are not described herein again; the above embodiments and drawings are only for illustrating the technical solutions of the present invention and not for limiting the present invention, and the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that changes, modifications, additions or substitutions within the spirit and scope of the present invention may be made by those skilled in the art without departing from the spirit of the present invention, and the present invention should also fall within the scope of the claims of the present invention, and the above embodiments are only given as an arbitrary example of one currently used embodiment.
Claims (5)
1. The preparation method and the application of the anti-aging agent are characterized in that the anti-aging agent takes healthy newborn umbilical cords as raw materials and is prepared according to the following steps:
a) extracting mesenchymal stem cells;
b) culturing the mesenchymal stem cells in a serum culture medium until the mesenchymal stem cells are fused to 40-50% of a monolayer of cells;
c) adding a glucose solution with the concentration of 25-55 mmol/L into the monolayer cells to prepare a target culture medium;
d) continuously culturing the target culture medium for 46-50 hours to prepare a preparation culture medium;
e) concentrating and purifying the preparation medium;
in the step a), the fresh full-term healthy fetal umbilical cord is taken, the bloodstain is washed by normal saline, stripped and cut into small pieces, evenly coated, 10-15 mL of serum-free culture medium is slowly added, culturing in an incubator, culturing the primary cells for 7-10 days, replacing serum-free culture solution, culturing until the cells are fused to more than 70% to form umbilical cord mesenchymal stem cells, removing the serum-free culture solution, adding a proper amount of trypsin-sodium Ethylene Diamine Tetracetate (EDTA) for digestion for l min to make the umbilical cord mesenchymal stem cells shrink and separate from the bottle wall, adding the culture supernatant removed previously, gently blowing and beating by using a pipette, transferring the umbilical cord mesenchymal stem cell suspension into a 50mL centrifuge tube, centrifuging, removing supernatant, adding a serum-free culture medium again to resuspend the umbilical cord mesenchymal stem cells, counting, and subculturing according to the method;
in the step b), serum containing 5-10% of amino acid is added into the 3 rd generation cells of the umbilical cord mesenchymal stem cells to be cultured to 40-50% of monolayer cells;
in the step c), adding a glucose solution into the monolayer cells, wherein the concentration of glucose is 25-55 mmol/L, and preparing a target culture medium;
in the step d), after the target culture medium is continuously cultured for 46-52 hours, filtering by using a filter to prepare a preparation culture medium;
in the step e), the preparation culture medium is dried, concentrated and purified.
2. The method for preparing anti-aging agent and the application thereof according to claim 1, wherein the physiological saline is penicillin and streptomycin with the concentration of 50-100 ug/mL.
3. The method for preparing anti-aging agent and the use thereof according to claim 1, wherein the culture conditions in the incubator are 37 ℃, 5% volume fraction of carbon dioxide and 95% saturation humidity.
4. The method for preparing an anti-aging agent and the application thereof according to claim 1, wherein the mass ratio of trypsin to sodium Ethylene Diamine Tetracetate (EDTA) is 0.1-0.2.
5. The method for preparing an anti-aging agent and the application thereof according to claim 1, wherein the mesh number of the filter is 0.15-0.25 wn.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114159374A (en) * | 2021-12-10 | 2022-03-11 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method, product and application of skin anti-aging mesenchymal stem cell exosome composition |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130012771A (en) * | 2011-07-26 | 2013-02-05 | 주식회사 바이오에프디엔씨 | Non-antibiotic culture solution of mesenchymal stem cell |
| US20150299650A1 (en) * | 2014-04-18 | 2015-10-22 | Growgene Biotech Inc. | Use of stem cell conditioned medium to inhibit oxidation for anti-aging skin |
| CN106361771A (en) * | 2016-11-08 | 2017-02-01 | 北京恒峰铭成生物科技有限公司 | High-glucose activated mesenchymal stem cell injection and application thereof for diabetic drugs |
| WO2017096611A1 (en) * | 2015-12-11 | 2017-06-15 | 郭镭 | Method for separating and culturing mesenchymal stem cells from wharton's jelly tissue of umbilical cord |
| CN106967679A (en) * | 2017-03-23 | 2017-07-21 | 北京恒峰铭成生物科技有限公司 | A kind of preparation method and applications of anti-senility liquid |
| CN107090431A (en) * | 2017-03-31 | 2017-08-25 | 北京恒峰铭成生物科技有限公司 | A kind of active mesenchymal stem cell injection for being used to be transfused diabetes |
| US20180280441A1 (en) * | 2015-04-03 | 2018-10-04 | Stemmedicare Co., Ltd. | Method for mass producing proteins in mesenchymal stem cells |
-
2019
- 2019-12-12 CN CN201911270731.7A patent/CN112972493A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130012771A (en) * | 2011-07-26 | 2013-02-05 | 주식회사 바이오에프디엔씨 | Non-antibiotic culture solution of mesenchymal stem cell |
| US20150299650A1 (en) * | 2014-04-18 | 2015-10-22 | Growgene Biotech Inc. | Use of stem cell conditioned medium to inhibit oxidation for anti-aging skin |
| US20180280441A1 (en) * | 2015-04-03 | 2018-10-04 | Stemmedicare Co., Ltd. | Method for mass producing proteins in mesenchymal stem cells |
| WO2017096611A1 (en) * | 2015-12-11 | 2017-06-15 | 郭镭 | Method for separating and culturing mesenchymal stem cells from wharton's jelly tissue of umbilical cord |
| CN106361771A (en) * | 2016-11-08 | 2017-02-01 | 北京恒峰铭成生物科技有限公司 | High-glucose activated mesenchymal stem cell injection and application thereof for diabetic drugs |
| CN106967679A (en) * | 2017-03-23 | 2017-07-21 | 北京恒峰铭成生物科技有限公司 | A kind of preparation method and applications of anti-senility liquid |
| CN107090431A (en) * | 2017-03-31 | 2017-08-25 | 北京恒峰铭成生物科技有限公司 | A kind of active mesenchymal stem cell injection for being used to be transfused diabetes |
Non-Patent Citations (3)
| Title |
|---|
| YOON-JIN KIM ET AL.: "Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin", 《BIOCHEM BIOPHYS RES COMMUN》, vol. 493, no. 2, pages 1102 - 1108, XP055583511, DOI: 10.1016/j.bbrc.2017.09.056 * |
| 王帅帅 等: "人脐带血间充质干细胞条件培养基的制备及其生物学特性", 中国医药指南, vol. 11, no. 13, pages 426 - 427 * |
| 郑桂纯 等: "不同来源间充质干细胞条件培养基对内源性衰老细胞作用的比较", 《中国组织工程研究》, vol. 23, no. 21, pages 3357 - 3363 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114159374A (en) * | 2021-12-10 | 2022-03-11 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method, product and application of skin anti-aging mesenchymal stem cell exosome composition |
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