CN111888459A - Lyophilized powder containing mesenchymal stem cell secretory factor and solvent thereof - Google Patents
Lyophilized powder containing mesenchymal stem cell secretory factor and solvent thereof Download PDFInfo
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- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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Abstract
The invention relates to the technical field of biology, and discloses a lyophilized powder containing mesenchymal stem cell secretion factors, which is prepared by freeze-drying human embryo mesenchymal stem cell secretion, and comprises the following components: mannitol, oligopeptide-1, oligopeptide-3, oligopeptide-5, disodium hydrogen phosphate and sodium dihydrogen phosphate; also discloses a solvent, and the components of the solvent comprise water, Hamamelis virginiana extract, butanediol, hydrolyzed sclerotium rolfsii gum, beta-glucan, nicotinamide, purslane extract, sodium hyaluronate, dipotassium glycyrrhizinate and ethylhexylglycerin. The freeze-dried powder with high activity and anti-aging effect is obtained in a low-cost and original ecological mode, the freeze-dried powder has great use value in the aspects of improving skin repairing force and resisting aging, and the provided solvent can efficiently, quickly and completely dissolve the freeze-dried powder, so that guarantee is provided for efficient exertion of the activity effect of the freeze-dried powder.
Description
Technical Field
The invention relates to the technical field of biology, in particular to lyophilized powder containing mesenchymal stem cell secretory factors and a solvent thereof.
Background
Mesenchymal Stem Cells (MSCs) are adult stem cells with self-renewal, proliferation and multidirectional differentiation potential, can stimulate the growth and repair of tissues, enhance the regeneration capacity of the tissues, influence the immunoregulation, have very wide application prospects in the field of cell therapy, can secrete active factors in the culture process, and have the functions of immunoregulation, nutrition support and the like.
The conventional mesenchymal stem cell secretory factors are utilized in a freeze-dried powder form, but at present, the most widely utilized mesenchymal stem cells mainly adopt goat fetal tissues and human umbilical cord tissues, the adopted raw materials are different, and secretory active ingredients obtained by preparation are different, so that at present, no freeze-dried powder with high activity and anti-aging effect of oligopeptides is obtained by the secretory factors of the mesenchymal stem cells of any tissue in the market.
The solvent is used for dissolving the freeze-dried powder, but the existing solvent has poor solubility to the freeze-dried powder and cannot achieve the effect of quick and complete dissolution.
Disclosure of Invention
Aiming at the defects in the prior art, the technical problem to be solved by the invention is to provide the freeze-dried powder containing the mesenchymal stem cell secretion factors, the freeze-dried powder is prepared by freeze-drying human embryo mesenchymal stem cell secretion, and the components of the freeze-dried powder comprise: mannitol, oligopeptide-1, oligopeptide-3, oligopeptide-5, disodium hydrogen phosphate and sodium dihydrogen phosphate.
Further, the freeze-dried powder is prepared by the following steps:
(1) obtaining mesenchymal stem cells: separating and purifying mesenchymal stem cells from human embryonic tissues;
(2) culturing mesenchymal stem cells: taking human embryo mesenchymal stem cells with good growth condition, and carrying out primary culture and subculture by using a fresh serum-free culture medium without antibiotics;
(3) extracting a supernatant: collecting cell culture supernatant of 3-4 generations, filtering the supernatant by using a sterile filter with the diameter of 0.22 mu m, and taking filtrate to obtain supernatant containing human embryo mesenchymal stem cell secretion;
(4) freeze drying and centrifuging: and (3) adding a freeze-drying agent containing the components including mannitol, disodium hydrogen phosphate and sodium dihydrogen phosphate into the supernatant obtained in the step (3), filtering the mixture by using a 0.22-micron filter membrane, subpackaging the mixture, pre-freezing the mixture for 24 hours at a low temperature, freeze-drying the mixture in an ultralow temperature freeze-dryer, centrifuging the mixture for 12-24 hours, and obtaining the freeze-dried powder containing the mesenchymal stem cell secretory factors after the freeze-drying is finished.
Further, in the step 4, the pre-freezing temperature is-30 ℃, the freeze-drying temperature is-80 to-60 ℃, and the centrifugal speed is 1800 rpm.
The invention also provides an application of the lyophilized powder containing the mesenchymal stem cell secretory factor, and the specific scheme is as follows: the application of lyophilized powder containing mesenchymal stem cell secretion factor in preparing antiaging medicine is provided.
The invention also provides a solvent for dissolving the freeze-dried powder, and the components of the solvent comprise water, Hamamelis virginiana (HAMAMELIS VIRGINIANA) extract, butanediol, SCLEROTIUM rolfsii (scleritum rolfsii) gum, beta-glucan, nicotinamide, purslane (PORTULACA OLERACEA) extract, sodium hyaluronate, dipotassium glycyrrhizinate and ethylhexylglycerin.
The solvent is prepared by mixing 69% of water, 1.2% of Hamamelis virginiana (HAMAMELIS VIRGINIANA) extract, 4% of butanediol, 3.6% of SCLEROTIUM rolfsii (scleroticum ROLFSSII) gum, 1.2% of beta-glucan, 2.5% of nicotinamide, 6% of purslane (Portulaca OLERACEA) extract, 3.3% of sodium hyaluronate, 6.8% of dipotassium glycyrrhizinate and 2.4% of ethylhexyl glycerol.
Further, the v/w ratio of the solvent for dissolving the freeze-dried powder to the freeze-dried powder is 1: 25.
Compared with the prior art, the invention has the beneficial effects that: the invention obtains the secretion factors such as oligopeptide-1, oligopeptide-3, oligopeptide-5 and the like by culturing human embryo mesenchymal stem cells, and prepares the supernatant containing the secretion factors into lyophilized powder by lyophilization, wherein the oligopeptide-1, the oligopeptide-3 and the oligopeptide-5 are cell growth peptides, which can promote the growth of epidermal keratinocytes, repair damaged epidermal cells and accelerate the metabolism of cells, and have the function of anti-aging.
Furthermore, the solvent is prepared by adopting a special preparation process, can quickly and completely dissolve the freeze-dried powder, has a good dissolving effect, and is beneficial to fully exerting the effect of the active factors of the freeze-dried powder.
Detailed Description
The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention and to clearly and unequivocally define the scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention provides a freeze-dried powder containing mesenchymal stem cell secretion factors, which is prepared by freeze-drying human embryo mesenchymal stem cell secretion and comprises the following components: mannitol, oligopeptide-1, oligopeptide-3, oligopeptide-5, disodium hydrogen phosphate and sodium dihydrogen phosphate.
In the preferred embodiment, the lyophilized powder is prepared by the following steps:
(1) obtaining mesenchymal stem cells: separating and purifying mesenchymal stem cells from human embryonic tissues;
(2) culturing mesenchymal stem cells: taking human embryo mesenchymal stem cells with good growth condition, and carrying out primary culture and subculture by using a fresh serum-free culture medium without antibiotics;
(3) extracting a supernatant: collecting cell culture supernatant of 3-4 generations, filtering the supernatant by using a sterile filter with the diameter of 0.22 mu m, and taking filtrate to obtain supernatant containing human embryo mesenchymal stem cell secretion;
(4) freeze drying and centrifuging: and (3) adding a freeze-drying agent containing the components including mannitol, disodium hydrogen phosphate and sodium dihydrogen phosphate into the supernatant obtained in the step (3), filtering the mixture by using a 0.22-micron filter membrane, subpackaging the mixture, pre-freezing the mixture for 24 hours at a low temperature, freeze-drying the mixture in an ultralow temperature freeze-dryer, centrifuging the mixture for 12-24 hours, and obtaining the freeze-dried powder containing the mesenchymal stem cell secretory factors after the freeze-drying is finished.
Preferably, in step 4, the prefreezing temperature is-30 ℃, the freeze-drying temperature is-80, and the centrifugation speed is 1800 rpm.
Mannitol, disodium hydrogen phosphate and sodium dihydrogen phosphate are cryoprotectants, which are effective in maintaining the activity of secreted factors.
It should be noted that the culture technology of the mesenchymal stem cells is the prior art, and the culture steps of the mesenchymal stem cells of the invention are as follows: 201910104124.7 the specific steps are carried out for cell culture.
The invention provides an application of lyophilized powder containing mesenchymal stem cell secretory factors, which comprises the following specific steps: the active factor oligopeptide-1 secreted by the mesenchymal stem cells can promote the growth of epidermal keratinocytes, repair damaged epidermal cells and accelerate the metabolism of cells, and after the oligopeptides specifically repair the keratinocytes, the immunocompetence and resistance of the skin can be improved, so that the skin can recover the healthy and active state, and the senescence of the cells is delayed; the active factor oligopeptide-5 secreted by the mesenchymal cells can quickly heal wound skin and renew skin cell composition, can also promote the growth of skin capillaries, promote cell metabolism, promote the synthesis of elastic fibers and collagen, increase the elasticity of the skin and make the skin glowing; the active factor oligopeptide-3 secreted by the mesenchymal cells has the effects of repairing skin epidermal tissues, resisting aging, fading color spots, homogenizing skin color and repairing wrinkles.
The invention also provides a solvent for dissolving the freeze-dried powder, and the components of the solvent comprise water, Hamamelis virginiana (HAMAMELIS VIRGINIANA) extract, butanediol, SCLEROTIUM rolfsii (scleritum rolfsii) gum, beta-glucan, nicotinamide, purslane (PORTULACA OLERACEA) extract, sodium hyaluronate, dipotassium glycyrrhizinate and ethylhexylglycerin.
In the preferred embodiment, the solvent is prepared by mixing 69% of water, 1.2% of Hamamelis virginiana (HAMAMELIS VIRGINIANA) extract, 4% of butanediol, 3.6% of SCLEROTIUM rolfsii (scleroticum ROLFSSII) gum, 1.2% of beta-glucan, 2.5% of nicotinamide, 6% of purslane (Portulaca OLERACEA) extract, 3.3% of sodium hyaluronate, 6.8% of dipotassium glycyrrhizinate and 2.4% of ethylhexyl glycerol.
It can be understood that the freeze-dried powder and the solvent need to be matched for use, and the solvent has the functions of dissolving the freeze-dried powder and enhancing the activity of active factors in the freeze-dried powder; in the preferred embodiment, the v/w ratio of the solvent to the lyophilized powder is 1:25, for example, 50mg of lyophilized powder is dissolved in 2ml of solvent.
To further illustrate the high dissolution efficiency of the vehicle, the following is illustrated by 5 groups of dissolution experiments, the results of which are shown in table 1, table 1 records the complete dissolution time and dissolution amount of the lyophilized powder:
group A: mixing 50mg of freeze-dried powder with 2ml of solvent for dissolving; group B: mixing 25mg of freeze-dried powder with 1ml of solvent for dissolving; group C: mixing 75mg of freeze-dried powder with 3ml of solvent for dissolving; group D: mixing 50mg of freeze-dried powder with 2ml of clear water for dissolving; group E: mixing 50mg of freeze-dried powder with 2ml of normal saline for dissolving; during the experiment, the solvent is added into the freeze-dried powder and is shaken up.
TABLE 1 dissolution of lyophilized powder
| Whether or not to dissolve completely | Dissolution time (approximation) | Amount of dissolution (approximation) | |
| Group A | Is that | 2s | 100％ |
| Group B | Is that | 2s | 100％ |
| Group C | Is that | 3s | 100％ |
| Group D | Whether or not | >10min | 50％ |
| Group E | Whether or not | >10min | 60％ |
The experiments show that the freeze-dried powder can be efficiently, quickly and completely dissolved under the action of the special solvent, so that the solvent can provide guarantee for the efficient exertion of the activity of the freeze-dried powder.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (7)
1. The freeze-dried powder containing the mesenchymal stem cell secretion factors is characterized by being prepared by freeze-drying human embryo mesenchymal stem cell secretion, and comprises the following components: mannitol, oligopeptide-1, oligopeptide-3, oligopeptide-5, disodium hydrogen phosphate and sodium dihydrogen phosphate.
2. The lyophilized powder containing mesenchymal stem cell secretory factors of claim 1, which is prepared by the following steps:
(1) obtaining mesenchymal stem cells: separating and purifying mesenchymal stem cells from human embryonic tissues;
(2) culturing mesenchymal stem cells: taking human embryo mesenchymal stem cells with good growth condition, and carrying out primary culture and subculture by using a fresh serum-free culture medium without antibiotics;
(3) extracting a supernatant: collecting cell culture supernatant of 3-4 generations, filtering the supernatant by using a sterile filter with the diameter of 0.22 mu m, and taking filtrate to obtain supernatant containing human embryo mesenchymal stem cell secretion;
(4) freeze drying and centrifuging: and (3) adding a freeze-drying agent containing the components including mannitol, disodium hydrogen phosphate and sodium dihydrogen phosphate into the supernatant obtained in the step (3), filtering the mixture by using a 0.22-micron filter membrane, subpackaging the mixture, pre-freezing the mixture for 24 hours at a low temperature, freeze-drying the mixture in an ultralow temperature freeze-dryer, centrifuging the mixture for 12-24 hours, and obtaining the freeze-dried powder containing the mesenchymal stem cell secretory factors after the freeze-drying is finished.
3. The lyophilized powder containing mesenchymal stem cell secretory factors according to claim 2, wherein: in the step 4, the pre-freezing temperature is-30 ℃, the freeze-drying temperature is-80 to-60 ℃, and the centrifugal speed is 1800 rpm.
4. The use of the lyophilized powder containing mesenchymal stem cell secretory factors as defined in claims 1 to 3 for preparing an anti-aging medicament.
5. A vehicle for dissolving the lyophilized powder of any one of claims 1-3, wherein the vehicle comprises water, Hamamelis virginiana (HAMAMELIS VIRGINIANA) extract, butylene glycol, sclerotium rolfsii (sclerotium rolfssii) gum, beta-glucan, niacinamide, Portulaca OLERACEA (Portulaca OLERACEA) extract, sodium hyaluronate, dipotassium glycyrrhizinate, and ethylhexylglycerin.
6. The vehicle according to claim 5, wherein: the solvent is prepared by mixing 69% of water, 1.2% of Hamamelis virginiana (HAMAMELIS VIRGINIANA) extract, 4% of butanediol, 3.6% of SCLEROTIUM rolfsii (scleritum rolfsii) gum, 1.2% of beta-glucan, 2.5% of nicotinamide, 6% of purslane (Portulaca OLERACEA) extract, 3.3% of sodium hyaluronate, 6.8% of dipotassium glycyrrhizinate and 2.4% of ethylhexyl glycerol.
7. The vehicle of claim 5, wherein the v/w ratio of the vehicle for dissolving the lyophilized powder to the lyophilized powder is 1: 25.
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Cited By (4)
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| CN113797151A (en) * | 2021-10-09 | 2021-12-17 | 四川伍衍生物科技有限公司 | Repairing gel containing mesenchymal stem cell exosomes and preparation method thereof |
| CN115025035A (en) * | 2022-07-11 | 2022-09-09 | 睿博斯(北京)生物科技有限公司 | Freeze-dried powder, preparation method and mask |
| CN115737501A (en) * | 2022-11-16 | 2023-03-07 | 珂蓝(重庆)生物医药科技有限责任公司 | Composition for rapidly repairing basement membrane band and reconstructing collagen and application |
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| CN115737501A (en) * | 2022-11-16 | 2023-03-07 | 珂蓝(重庆)生物医药科技有限责任公司 | Composition for rapidly repairing basement membrane band and reconstructing collagen and application |
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