CN111407717A - Stem cell factor freeze-dried powder and preparation method and application thereof - Google Patents
Stem cell factor freeze-dried powder and preparation method and application thereof Download PDFInfo
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- CN111407717A CN111407717A CN202010228082.0A CN202010228082A CN111407717A CN 111407717 A CN111407717 A CN 111407717A CN 202010228082 A CN202010228082 A CN 202010228082A CN 111407717 A CN111407717 A CN 111407717A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/0216—Solid or semisolid forms
- A61K8/022—Powders; Compacted Powders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
- A61K8/982—Reproductive organs; Embryos, Eggs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
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Abstract
The invention discloses stem cell factor freeze-dried powder which is prepared by the following method: (1) centrifuging the stem cell factor-rich solution to remove cell debris and other large granular impurities; (2) adding G10-G50 sephadex into the solution obtained after centrifugation, and soaking for 1-12 h, wherein inorganic salt molecules can enter the meshes of the gel, and proteins and polypeptides such as cytokines are excluded from the meshes of the gel due to overlarge molecular weight; filtering with screen mesh to remove gel after swelling with adsorbed inorganic salt solution to obtain stem cell factor concentrated solution, and freeze drying to obtain lyophilized powder which can be used as raw material for preparing cosmetics and cosmetics. The stem cell factor freeze-dried powder is added with the glucan gel filtration, so that a large amount of inorganic salt components can be removed, and the stem cell factor freeze-dried powder has a concentration effect; the freeze-drying can greatly prolong the effective period of the raw materials, reduce the storage difficulty and is more beneficial to realizing large-scale application.
Description
Technical Field
The invention relates to stem cell factor freeze-dried powder, a preparation method thereof and application thereof in preparing cosmetics and cosmetics.
Background
The umbilical cord Mesenchymal Stem Cells (MSCs) are multifunctional Stem Cells existing in umbilical cord tissues of newborns, have high differentiation potential, can be differentiated into various tissue Cells, and have wide clinical application prospects. Scientific data indicate that stem cells decrease both quantitatively and qualitatively as humans age, a key factor in human aging. The increase in survival time and replication times leads to the accumulation of false signals at the gene level within the stem cells, which means that the stem cell characteristics of these cells are impaired even if these cells are available and expansion is achieved. The umbilical cord mesenchymal stem cells obtained from the umbilical cord tissues have the advantages of good cell content and proliferation capacity, low immunogenicity, convenient material acquisition, no ethical dispute and the like.
The umbilical cord mesenchymal stem cells can secrete a large number of growth factors in the growth process, such as Epidermal Growth Factor (EGF), (HGF), PDGF and the like, the factors have obvious effects on anti-aging beauty, and the umbilical cord mesenchymal stem cells have extremely wide application prospects in the field of biological beauty. In the prior art, a plurality of studies on using mesenchymal stem cell factor-rich solution as a biological beauty raw material exist, but the factor is applied simply in the market at present, and after stem cell culture supernatant is obtained, cell fragments are removed only through one-step centrifugation. In addition, the stem cell factor has poor stability in solution, so that the activity is easy to decline and lose efficacy, and the stem cell factor is not beneficial to being used as a biological beauty raw material.
Disclosure of Invention
Aiming at the prior art, the invention provides stem cell factor freeze-dried powder, a preparation method thereof and application thereof in preparing cosmetics and cosmetics.
The invention is realized by the following technical scheme:
a preparation method of stem cell factor freeze-dried powder comprises the following steps:
(1) centrifuging the stem cell factor-rich solution to remove cell debris and other large granular impurities;
(2) adding G10-G50 sephadex (the specification of G10-G50 is suitable for small molecules with the molecular weight less than 5000, such as G10, G15, G25 and G50; the adding amount of the sephadex is related to the concentration of inorganic salt), soaking for 1-12 h, wherein inorganic salt molecules can enter gel meshes, and proteins and polypeptides such as cytokines are excluded from the gel meshes due to overlarge molecular weight; and then filtering with a screen to remove gel after swelling with the adsorbed inorganic salt solution to obtain a dry cell factor concentrated solution, subpackaging the dry cell factor concentrated solution into penicillin bottles with 2-5 ml of each vial, putting the vials into a freeze dryer for freeze drying to obtain freeze-dried powder, sealing the freeze-dried powder with a gland, and storing the freeze-dried powder at-20 ℃.
In the step (1), the centrifugal force during centrifugation is 500-5000 g, preferably 3000 g.
In the step (1), the centrifugation time is 5-20 min, preferably 10 min.
In the step (1), the stem cell factor-rich solution is selected from mesenchymal stem cell culture supernatants, namely: when the growth confluence of the mesenchymal stem cells reaches 70-100% (preferably 90%), collecting the culture supernatant, which is the prior art.
In the step (2), the final concentration of the G10-G50 glucan gel is 4-50G/m L.
In the step (2), the aperture of the screen is 8-100 meshes, and preferably 40 meshes.
The stem cell factor freeze-dried powder prepared by the method is used as a raw material for preparing cosmetics and cosmetics.
The stem cell factor freeze-dried powder provided by the invention is added with two steps of sephadex filtration and freeze-drying on the basis of removing cell debris by centrifugation. The sephadex filtration has two functions, namely removing a large amount of inorganic salt components and concentrating the stem cell factor. The freeze-dried powder obtained by the freeze-drying step can greatly prolong the validity period of the raw materials, reduce the storage difficulty and is more beneficial to realizing large-scale application.
The method takes a stem cell factor-rich solution as an initial raw material, and comprises the steps of centrifugation, sephadex filtration and freeze-drying. The purpose of centrifugation is to remove insoluble particles such as cell debris or other large particulate impurities, and to remove components that cannot be absorbed by skin follicles. Because the factor-rich solution of the stem cells contains a large amount of inorganic salt and trace micromolecule components and has potential risks of dehydration, mutagenesis and the like on epidermal cells such as squamous epithelial cells and the like, the invention adopts gel filtration chromatography to remove micromolecule substances, has high removal efficiency and high yield of target substances.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
Example 1 preparation of Stem cell factor lyophilized powder
The method comprises the following steps:
(1) placing 35ml of the stem cell factor-rich solution into a 50ml centrifuge tube, centrifuging, and centrifuging for 10min at a centrifugal force of 3000g to remove cell debris and other large-particle impurities;
the stem cell factor-rich solution is derived from mesenchymal stem cell culture supernatant, namely the culture supernatant is collected when the growth confluency of the mesenchymal stem cells reaches 90%;
(2) adding Sephadex G-10 into the solution obtained after centrifugation according to the concentration of 4g/100ml, and soaking for 10h, wherein inorganic salt molecules can enter into gel meshes, and proteins and polypeptides such as cell factors are excluded from the gel meshes due to overlarge molecular weight; and filtering with a 40-mesh screen to remove gel after swelling with the adsorbed inorganic salt solution to obtain a dry cell factor concentrated solution, subpackaging the dry cell factor concentrated solution into penicillin bottles with 2-5 ml of each vial, putting the vials into a freeze dryer for freeze drying to obtain freeze-dried powder, sealing the freeze-dried powder with a gland, and storing the freeze-dried powder at-20 ℃.
Example 2 preparation of Stem cell factor lyophilized powder
The method comprises the following steps:
(1) placing 35ml of the stem cell factor-rich solution into a 50ml centrifuge tube, centrifuging, and centrifuging for 20min at the centrifugal force of 500g to remove cell debris and other large-particle impurities;
the stem cell factor-rich solution is derived from mesenchymal stem cell culture supernatant, namely the culture supernatant is collected when the growth confluency of the mesenchymal stem cells reaches 70%;
(2) adding Sephadex G-15 into the solution obtained after centrifugation according to the concentration of 50g/100ml, and soaking for 1h, wherein inorganic salt molecules can enter into gel meshes, and proteins and polypeptides such as cell factors are excluded from the gel meshes due to overlarge molecular weight; and filtering with an 8-mesh screen to remove gel after swelling with the adsorbed inorganic salt solution to obtain a dry cell factor concentrated solution, subpackaging the dry cell factor concentrated solution into penicillin bottles with 2-5 ml of each vial, freeze-drying the vials in a freeze dryer to obtain freeze-dried powder, sealing the freeze-dried powder with a gland, and storing the freeze-dried powder at-20 ℃.
Example 3 preparation of Stem cell factor lyophilized powder
The method comprises the following steps:
(1) placing 35ml of the stem cell factor-rich solution into a 50ml centrifuge tube, centrifuging, and centrifuging for 5min at the centrifugal force of 5000g to remove cell debris and other large-particle impurities;
the stem cell factor-rich solution is derived from mesenchymal stem cell culture supernatant, namely the culture supernatant is collected when the growth confluency of the mesenchymal stem cells reaches 80%;
(2) adding Sephadex G-50 into the solution obtained after centrifugation according to the concentration of 25g/100ml, and soaking for 12h, wherein inorganic salt molecules can enter into gel meshes, and proteins and polypeptides such as cell factors are excluded from the gel meshes due to overlarge molecular weight; and then filtering with a 100-mesh screen to remove gel after swelling with the adsorbed inorganic salt solution to obtain a dry cell factor concentrated solution, subpackaging the dry cell factor concentrated solution into penicillin bottles with 2-5 ml of each vial, putting the vials into a freeze dryer for freeze drying to obtain freeze-dried powder, sealing the freeze-dried powder with a gland, and storing the freeze-dried powder at-20 ℃.
Example 4 preparation of Stem cell factor lyophilized powder
The method comprises the following steps:
(1) placing 35ml of the stem cell factor-rich solution into a 50ml centrifuge tube, centrifuging, and centrifuging for 15min at a centrifugal force of 2000g to remove cell debris and other large-particle impurities;
the stem cell factor-rich solution is derived from mesenchymal stem cell culture supernatant, namely the culture supernatant is collected when the growth confluency of the mesenchymal stem cells reaches 90%;
(2) adding Sephadex G-25 into the solution obtained after centrifugation according to the concentration of 30g/100ml, and soaking for 8h, wherein inorganic salt molecules can enter into gel meshes, and proteins and polypeptides such as cell factors are excluded from the gel meshes due to overlarge molecular weight; and filtering with a 60-mesh screen to remove gel after swelling with the adsorbed inorganic salt solution to obtain a dry cell factor concentrated solution, subpackaging the dry cell factor concentrated solution into penicillin bottles with 2-5 ml of each vial, freeze-drying the vials in a freeze dryer to obtain freeze-dried powder, sealing the freeze-dried powder with a gland, and storing the freeze-dried powder at-20 ℃.
Example 5 preparation of Stem cell factor lyophilized powder
The method comprises the following steps:
(1) placing 35ml of the stem cell factor-rich solution into a 50ml centrifuge tube, centrifuging, and centrifuging for 10min at the centrifugal force of 1000g to remove cell debris and other large-particle impurities;
the stem cell factor-rich solution is derived from mesenchymal stem cell culture supernatant, namely the culture supernatant is collected when the growth confluence of the mesenchymal stem cells reaches 95%;
(2) adding Sephadex G-25 into the solution obtained after centrifugation according to the concentration of 4g/100ml, and soaking for 6h, wherein inorganic salt molecules can enter into gel meshes, and proteins and polypeptides such as cell factors are excluded from the gel meshes due to overlarge molecular weight; and filtering with a 80-mesh screen to remove gel after swelling with the adsorbed inorganic salt solution to obtain a dry cell factor concentrated solution, subpackaging the dry cell factor concentrated solution into penicillin bottles with 2-5 ml of each vial, freeze-drying the vials in a freeze dryer to obtain freeze-dried powder, sealing the freeze-dried powder with a gland, and storing the freeze-dried powder at-20 ℃.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Claims (10)
1. A preparation method of stem cell factor freeze-dried powder is characterized by comprising the following steps: comprises the following steps
(1) Centrifuging the stem cell factor-rich solution to remove cell debris and other large granular impurities;
(2) adding G10-G50 sephadex into the solution obtained after centrifugation, and soaking for 1-12 h, wherein inorganic salt molecules can enter the meshes of the gel, and proteins and polypeptides such as cytokines are excluded from the meshes of the gel due to overlarge molecular weight; and filtering with a screen to remove the gel swollen by the adsorbed inorganic salt solution to obtain a stem cell factor concentrated solution, and freeze-drying to obtain the freeze-dried powder.
2. The method for preparing the stem cell factor freeze-dried powder according to claim 1, which is characterized in that: in the step (1), the centrifugal force during centrifugation is 500-5000 g.
3. The method for preparing the stem cell factor freeze-dried powder according to claim 1, which is characterized in that: in the step (1), the centrifugation time is 5-20 min.
4. The method for preparing the stem cell factor freeze-dried powder according to claim 1, which is characterized in that: in the step (1), the solution rich in the stem cells is selected from mesenchymal stem cell culture supernatant.
5. The method for preparing the stem cell factor freeze-dried powder according to claim 4, which is characterized in that: the stem cell factor-rich solution is prepared by the following method: and collecting the culture supernatant when the growth confluency of the mesenchymal stem cells reaches 70-100%.
6. The method for preparing the stem cell factor freeze-dried powder according to claim 1, which is characterized in that: in the step (2), the G10-G50 glucan gel is selected from G10, G15, G25 and G50.
7. The method for preparing the stem cell factor freeze-dried powder according to claim 1, wherein in the step (2), the final concentration of the G10-G50 sephadex is 4-50G/m L.
8. The method for preparing the stem cell factor freeze-dried powder according to claim 1, which is characterized in that: in the step (2), the aperture of the screen is 8-100 meshes.
9. The stem cell factor freeze-dried powder prepared by the preparation method of the stem cell factor freeze-dried powder of any one of claims 1 to 8.
10. The use of the lyophilized powder of stem cell factor of claim 9 in the preparation of cosmetics and toiletries.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114042150A (en) * | 2021-12-06 | 2022-02-15 | 陕西中鸿科瑞再生医学研究院有限公司 | Oral stem cell factor compound and application thereof |
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2020
- 2020-03-27 CN CN202010228082.0A patent/CN111407717A/en active Pending
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| WO1991001334A1 (en) * | 1989-07-19 | 1991-02-07 | Sloan-Kettering Institute For Cancer Research | A purified, human autoregulatory cytokine, compositions containing same and uses thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114042150A (en) * | 2021-12-06 | 2022-02-15 | 陕西中鸿科瑞再生医学研究院有限公司 | Oral stem cell factor compound and application thereof |
| CN114042150B (en) * | 2021-12-06 | 2023-09-26 | 陕西中鸿科瑞再生医学研究院有限公司 | Oral stem cell factor compound and application thereof |
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Application publication date: 20200714 |