CN110840758A - Essence containing umbilical cord mesenchymal stem cell factor and preparation method thereof - Google Patents
Essence containing umbilical cord mesenchymal stem cell factor and preparation method thereof Download PDFInfo
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- 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
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/34—Alcohols
- A61K8/345—Alcohols containing more than one hydroxy group
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- 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
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
- A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
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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
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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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
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
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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
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
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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
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/733—Alginic acid; Salts thereof
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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
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
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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
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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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
- A61Q19/08—Anti-ageing preparations
Abstract
The invention discloses essence containing umbilical cord mesenchymal stem cell factors and a preparation method thereof, and relates to the technical field of skin care products. The essence comprises concentrated solution of umbilical cord mesenchymal stem cell factor, human albumin, dextran, hyaluronic acid, sodium alginate, collagen, glycerol and catechin, and has effects of whitening skin, lightening macula, repairing skin, reducing wrinkle and delaying aging. The preparation method of the essence is simple and rapid, the content and the recovery rate of the mesenchymal stem cell factor in the mesenchymal stem cell factor concentrated solution are obviously improved by optimizing the preparation method, the proliferation capacity of the umbilical cord mesenchymal stem cells is improved, the preparation time of the essence is effectively shortened, and the essence is suitable for large-scale production.
Description
Technical Field
The invention relates to the technical field of skin care products, and particularly relates to an essence containing umbilical cord mesenchymal stem cell factors and a preparation method thereof.
Background
With the improvement of living standard and the development of science and technology, people pay more attention to natural health while paying attention to face care. Conventional cosmetic compositions containing chemical components having adverse effects on the skin, such as preservatives, artificial pigments, perfumes, and mineral oils, have been gradually replaced by cosmetic compositions containing natural raw materials. With the development of the biotechnology field, cosmetics containing bioactive factors are favored in the beauty industry.
Mesenchymal Stem Cells (MSCs) are a type of pluripotent cells derived from mesoderm and having self-replicating ability, and can be differentiated into various functional cells under certain conditions. The source of the mesenchymal stem cells is wide, and the mesenchymal stem cells are separated from tissues such as bone marrow, peripheral blood, embryo, fat, cord blood, umbilical cord and the like. Wherein the culture time of the peripheral blood and the bone marrow mesenchymal stem cells is long, the content is low, and the cell proliferation and differentiation potential is obviously reduced along with the increase of age; the separation efficiency of the cord blood source MSCs is low, and embryos are easy to pollute and have ethical problems. The umbilical cord tissue has the advantages of convenient material acquisition, large cell number, small infection risk and the like, so that the umbilical cord tissue becomes a very potential source of the MSCs. Umbilical cord Wharton jelly mesenchymal stem cells (UW-MSCs) are mesenchymal stem cells derived from umbilical cord Wharton jelly tissues, and like other mesenchymal stem cells, umbilical cord Wharton jelly mesenchymal stem cells can also secrete various cytokines, such as: epidermal growth factor, vascular endothelial growth factor, platelet derived growth factor, and the like. These cytokines can effectively regulate and control the cell signal transmission of the organism and activate the stem cells of the human body, thereby physiologically repairing or replacing the damaged, pathological and aged cells of the organism.
With the continuous development of biotechnology, more and more beauty cosmetics are added with cytokines and active substances, so that the beauty cosmetics have the effects of moisturizing and whitening, and can repair damaged skin and eliminate skin wrinkles. At present, Chinese patent CN106344493A discloses a preparation method of essence containing human mesenchymal stem cell factors, which is a preparation method of essence containing human mesenchymal stem cell factors through the steps of preparation of human mesenchymal stem cell factor freeze-dried powder, preparation of a solvent and mixing. Chinese patent CN105078777A discloses a mesenchymal stem cell secretion factor essence and a preparation method and application thereof, wherein the mesenchymal stem cells are derived from cord blood, the components of the used culture medium are complex, and the preparation method is relatively complex.
Aiming at the problems in the prior art, the invention provides the essence containing the umbilical cord mesenchymal stem cell factor and the preparation method thereof.
Disclosure of Invention
The invention aims to provide essence containing umbilical cord mesenchymal stem cell factors and a preparation method thereof. The proliferation capacity of umbilical cord mesenchymal stem cells, the content and the recovery rate of mesenchymal stem cell factors are improved, and the preparation time of the essence is effectively shortened.
The invention provides an essence containing umbilical cord mesenchymal stem cell factors, which comprises the following components in part by weight: the preparation method comprises the following steps of 50% (v/v) of umbilical cord mesenchymal stem cell factor concentrated solution, 1% -2.5% (w/v) of human albumin, 0.5% -2% (w/v) of glucan, 0.5% -1.4% (w/v) of hyaluronic acid, 0.4% -1.1% (w/v) of sodium alginate, 1% -2.5% (w/v) of collagen, 0.3% -0.9% (v/v) of glycerol and 0.2% -0.5% (w/v) of catechin.
Preferably, the essence comprises: 50% (v/v) of umbilical cord mesenchymal stem cell factor concentrated solution, 1.5% -2.5% (w/v) of human albumin, 1% -2% (w/v) of glucan, 0.8% -1.4% (w/v) of hyaluronic acid, 0.7% -1.1% (w/v) of sodium alginate, 1.4% -2.5% (w/v) of collagen, 0.5% -0.9% (v/v) of glycerol and 0.3% -0.5% (w/v) of catechin.
Further preferably, the essence comprises: the umbilical cord mesenchymal stem cell factor concentrated solution comprises 50% (v/v) of umbilical cord mesenchymal stem cell factor concentrated solution, 1.5% (w/v) of human albumin, 1% (w/v) of dextran, 0.8% (w/v) of hyaluronic acid, 0.7% (w/v) of sodium alginate, 1.4% (w/v) of collagen, 0.5% (v/v) of glycerol and 0.3% (w/v) of catechin.
The invention also provides a preparation method of the essence, which comprises the following steps:
(1) preparing an umbilical cord mesenchymal stem cell factor concentrated solution:
A. separating and culturing umbilical cord Wharton jelly mesenchymal stem cells: soaking human umbilical cord tissue in 75% ethanol, cutting off 1cm from each end of the tissue, washing the rest with normal saline until the tissue is clear, cutting into small segments, washing with normal saline for 3 times, cutting along the venous lumen of the umbilical cord tissue, removing veins, arteries and amnion after tiling, and taking jelly between blood vessels and adventitia, namely: cutting the huatong glue, transferring the huatong glue to a complete culture medium for primary cell culture, and carrying out cell passage when the huatong glue is cultured until 80-90% of fusion;
B. subculturing mesenchymal stem cells: removing the culture solution of the cells to be subcultured in the step A, adding physiological saline to wash the tissue and stick to the wall surface, adding 0.25% of pancreatin, incubating at 37 ℃ for 1-3min, adding a stop solution to stop digestion after the cells become round, quickly shaking, blowing and beating the cells, sucking out cell suspension, adding physiological saline to wash, centrifuging, collecting precipitates, resuspending the precipitates by using a complete culture medium, inoculating the precipitates to the complete culture medium to perform P1 generation culture, and continuously performing subculture when the cells grow to 80% -90% fusion, and subculturing to P6 generation;
C. preparing an umbilical cord mesenchymal stem cell factor concentrated solution: collecting cell culture solution of the P2-P6 generation, filtering and concentrating through a microfiltration membrane and an ultrafiltration membrane, and collecting trapped fluid to obtain umbilical cord mesenchymal stem cell factor concentrated solution;
the complete culture medium comprises: 80% -90% (v/v) of Dayou MSCMB culture medium, 7% -12% (v/v) of Helios serum substitute, 20.5% -2% (w/v) of interleukin, 0.5% -2% (w/v) of sodium citrate and 2% -4% (w/v) of dextran sulfate;
the stop solution comprises α -MEM culture medium 92% -96% (v/v), fetal bovine serum 2% -5% (v/v), thiamine hydrochloride 0.8% -1.5% (w/v) and 4-hydroxyethyl piperazine ethanesulfonic acid 1.2% -1.5% (w/v);
(2) preparation of a diluent: preparing human albumin, dextran, hyaluronic acid, sodium alginate, collagen, glycerol and catechin in proportion with sterile ultrapure water, and shaking for 5-10min to obtain diluent;
(3) preparing an umbilical cord mesenchymal stem cell factor essence: and (3) mixing the concentrated umbilical cord mesenchymal stem cell factor solution obtained in the step (1) with the diluent obtained in the step (2) according to the volume ratio of 1:1, and shaking for 5-10min to obtain the essence containing the umbilical cord mesenchymal stem cell factor.
Preferably, the complete medium comprises: 86-90% (v/v) of Dayou MSCMB culture medium, 7-8% (v/v) of Helios serum substitute, 20.5-1.5% (w/v) of interleukin, 0.5-1.5% (w/v) of sodium citrate and 2-3% (w/v) of dextran sulfate.
Further preferably, said complete medium comprises: 86% (v/v) of Dayou MSCMB culture medium, 8% (v/v) of Helios serum substitute, 21.5% (w/v) of interleukin, 1.5% (w/v) of sodium citrate and 3% (w/v) of dextran sulfate.
Preferably, the stop solution comprises α -MEM culture medium 92% -94% (v/v), fetal bovine serum 3.5% -5% (v/v), thiamine hydrochloride 1.2% -1.5% (w/v) and 4-hydroxyethyl piperazine ethanesulfonic acid 1.3% -1.5% (w/v).
Further preferably, the stop solution comprises α -MEM medium 94% (v/v), fetal bovine serum 3.5% (v/v), thiamine hydrochloride 1.2% (w/v) and 4-hydroxyethyl piperazine ethanesulfonic acid 1.3% (w/v).
Preferably, the diluent comprises: 2-5% (w/v) of human albumin, 1-4% (w/v) of glucan, 1-2.8% (w/v) of hyaluronic acid, 0.8-2.2% (w/v) of sodium alginate, 2-5% (w/v) of collagen, 0.6-1.8% (v/v) of glycerol and 0.4-1% (w/v) of catechin.
Further preferably, the diluent comprises: 3% -5% (w/v) of human albumin, 2% -4% (w/v) of glucan, 1.6% -2.8% (w/v) of hyaluronic acid, 1.4% -2.2% (w/v) of sodium alginate, 2.8% -5% (w/v) of collagen, 1% -1.8% (v/v) of glycerol and 0.6% -1% (w/v) of catechin.
Still more preferably, the diluent comprises: human albumin 3% (w/v), dextran 2% (w/v), hyaluronic acid 1.6% (w/v), sodium alginate 1.4% (w/v), collagen 2.8% (w/v), glycerol 1% (v/v) and catechin 0.6% (w/v).
Preferably, the primary cell culture and the subculture are carried out under the culture conditions of 37 ℃ and 5% CO2The humidity was 95%, and the medium was replaced with fresh medium every 3 days.
Preferably, the seeded cell density in step B is 2-5X 104Per mL; further preferably, the seeded cell density in step B is 3X 104/mL。
Preferably, the ultrafiltration membrane in step C is an ultrafiltration membrane having a molecular weight of 50KD and 3 KD.
Specifically, the preparation method of the umbilical cord mesenchymal stem cell factor essence comprises the following steps:
(1) preparing an umbilical cord mesenchymal stem cell factor concentrated solution:
A. separating and culturing umbilical cord Wharton jelly mesenchymal stem cells: aseptically taking healthy human umbilical cord tissue, soaking in 75% ethanol for 1min, cutting off the two ends of the umbilical cord tissue by 1cm length, washing the rest part with normal saline until the part is clear, cutting into small segments of 2cm, washing with normal saline for 3 times, transferring the umbilical cord tissue to a new culture dish, adding normal saline into the culture dish until the umbilical cord tissue is submerged at 1/2, cutting along the venous lumen of the umbilical cord tissue, flatly laying, removing 1 vein, 2 arteries and amnion, taking jelly between blood vessels and between the blood vessels and adventitia,namely: cutting HUATONG gum to size of 1mm3, transferring to complete culture medium, culturing, placing culture flask in 37 deg.C and 5% CO2And (4) carrying out cell passage when the cells are cultured in an incubator until 80-90% of the cells are fused, and replacing fresh culture medium every 3 days.
B. Subculturing mesenchymal stem cells: removing the culture solution of the cells to be transfected in the step A by suction, adding 10mL of physiological saline into a non-cell culture surface, slightly shaking to wash a tissue pasting wall surface, removing the tissue pasting wall surface, repeating the steps for 2 times, adding 3mL of 0.25% pancreatin, uniformly infiltrating the cell pasting wall surface, incubating for 1min at 37 ℃, adding an equal amount of termination solution to stop digestion after the cells become round, quickly shaking, blowing the cell pasting wall surface by using a 10mL pipette, sucking cell suspension into 2 50mL centrifuge tubes, adding 10mL of physiological saline into each culture bottle, blowing once, converging into 50mL centrifuge tubes, centrifuging at 1200rpm for 6min, removing supernatant, combining precipitates to 1 tube, adding 40mL of physiological saline, centrifuging and washing again, re-suspending the precipitates by using a complete culture medium, inoculating and culturing for P1 generation, paving bottles (T175 bottles) according to the number of the cells, wherein the cell density is 2-5 multiplied by 104Per mL, each bottle volume is 25mL, the culture flask is placed at 37 ℃ with 5% CO2When the culture is carried out in an incubator until 80% -90% of fusion, subculture is continued until P6 generation.
C. Preparing an umbilical cord mesenchymal stem cell factor concentrated solution: collecting cell culture solution of P2-P6 generation, filtering with 0.22 μm filter membrane, concentrating the supernatant of stem cells with 50KD ultrafiltration membrane, passing the concentrated solution with 3KD ultrafiltration membrane, and collecting the trapped fluid, i.e. concentrated solution of umbilical cord mesenchymal stem cell factor;
the complete culture medium comprises: 80% -90% (v/v) of Dayou MSCMB culture medium, 7% -12% (v/v) of Helios serum substitute, 20.5% -2% (w/v) of interleukin, 0.5% -2% (w/v) of sodium citrate and 2% -4% (w/v) of dextran sulfate;
the stop solution comprises α -MEM culture medium 92% -95% (v/v), fetal bovine serum 2% -5% (v/v), thiamine hydrochloride 0.8% -1.5% (w/v) and 4-hydroxyethyl piperazine ethanesulfonic acid 1.2% -1.5% (w/v).
(2) Preparation of a diluent: preparing human albumin, dextran, hyaluronic acid, sodium alginate, collagen, glycerol and catechin with sterile ultrapure water in proportion, placing into a constant temperature oscillator, oscillating for 5-10min, and uniformly oscillating to obtain a diluent;
(3) preparing an umbilical cord mesenchymal stem cell factor essence: mixing the concentrated umbilical cord mesenchymal stem cell factor solution obtained in the step (1) and the diluent obtained in the step (2) according to the volume ratio of 1:1, placing the mixture into a constant-temperature oscillator, oscillating for 5-10min, and oscillating uniformly to obtain the umbilical cord mesenchymal stem cell factor essence.
Compared with the prior art, the invention has the advantages that:
the essence containing the umbilical cord mesenchymal stem cell factor and the preparation method thereof have the effects of whitening and fading spots, repairing skin, reducing wrinkles, delaying senescence and the like. By optimizing the preparation method, the content and the recovery rate of the mesenchymal stem cell factor in the mesenchymal stem cell factor concentrated solution are obviously improved, the proliferation capacity of the umbilical cord mesenchymal stem cells is improved, the preparation time of the essence is effectively shortened, and the method is suitable for large-scale production.
Detailed Description
The following description of the embodiments is only intended to aid in the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
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 examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The various reagents used are, unless otherwise specified, available from conventional commercial sources.
Example 1
An essence containing umbilical cord mesenchymal stem cell factors comprises: the preparation method comprises the following steps of 50% (v/v) of umbilical cord mesenchymal stem cell factor concentrated solution, 1% (w/v) of human albumin, 0.5% (w/v) of glucan, 0.5% (w/v) of hyaluronic acid, 0.4% (w/v) of sodium alginate, 1% (w/v) of collagen, 0.3% (v/v) of glycerol and 0.2% (w/v) of catechin.
The preparation method of the essence comprises the following steps:
(1) preparing an umbilical cord mesenchymal stem cell factor concentrated solution:
A. separating and culturing umbilical cord Wharton jelly mesenchymal stem cells: aseptically taking healthy human umbilical cord tissue, soaking in 75% ethanol for 1min, cutting off the two ends of the umbilical cord tissue by 1cm length respectively, washing the rest part with normal saline until the umbilical cord tissue is clear, cutting into small segments of 2cm, cleaning with normal saline for 3 times, transferring the umbilical cord tissue to a new culture dish, adding normal saline into the culture dish until the umbilical cord tissue is submerged at 1/2, cutting along the venous lumen of the umbilical cord tissue, removing 1 vein, 2 arteries and amnion after tiling, taking jelly between blood vessels and between the blood vessels and adventitia, namely: cutting HUATONG gum into pieces of 1mm3Size, transferred to complete medium for culture, and the flask was placed at 37 ℃ in 5% CO2And (4) carrying out cell passage when the cells are cultured in an incubator until 80-90% of the cells are fused, and replacing fresh culture medium every 3 days.
B. Subculturing mesenchymal stem cells: discarding the culture of the cells to be transfected in step AAdding 10mL of physiological saline into a non-cell culture surface, slightly shaking to wash a tissue wall-attached surface, discarding, repeating for 2 times, adding 3mL of 0.25% pancreatin, uniformly infiltrating the cell wall-attached surface, incubating at 37 ℃ for 1min, adding an equal amount of stop solution after the cells become round to stop digestion, quickly shaking, blowing the cell wall-attached surface by using a 10mL pipette, sucking cell suspension out to 2 50mL centrifuge tubes, adding 10mL of physiological saline into each culture bottle, blowing once, converging into a 50mL centrifuge tube, centrifuging at 1200rpm, centrifuging for 6min, discarding supernatant, combining precipitates to 1 tube, adding 40mL of physiological saline, centrifuging again, washing the precipitates with complete culture medium for re-suspension, inoculating, performing P1-substituted culture, paving bottles (T175 bottles) according to the number of the cells, wherein the cell density is 2 × 104Per mL, each bottle volume is 25mL, the culture flask is placed at 37 ℃ with 5% CO2When the culture is carried out in an incubator until 80% -90% of fusion, subculture is continued until P6 generation.
C. Preparing an umbilical cord mesenchymal stem cell factor concentrated solution: collecting cell culture solution of P2-P6 generation, filtering with 0.22 μm filter membrane, concentrating the supernatant of stem cells with 50KD ultrafiltration membrane, passing the concentrated solution with 3KD ultrafiltration membrane, and collecting the trapped fluid, i.e. concentrated solution of umbilical cord mesenchymal stem cell factor;
wherein the complete medium comprises: 80% (v/v) of Dayou MSCMB culture medium, 12% (v/v) of Helios serum substitute, 22% (w/v) of interleukin, 2% (w/v) of sodium citrate and 4% (w/v) of dextran sulfate;
the stop solution comprises α -MEM culture medium 92%% (v/v), fetal bovine serum 5% (v/v), thiamine hydrochloride 1.5% (w/v) and 4-hydroxyethyl piperazine ethanesulfonic acid 1.5% (w/v).
(2) Preparation of a diluent: preparing human albumin, dextran, hyaluronic acid, sodium alginate, collagen, glycerol and catechin with sterile ultrapure water in proportion, placing into a constant temperature oscillator, oscillating for 5min, and uniformly oscillating to obtain a diluent; the diluent comprises: human albumin 2% (w/v), dextran 1% (w/v), hyaluronic acid 1% (w/v), sodium alginate 0.8% (w/v), collagen 2% (w/v), glycerol 0.6% (v/v) and catechin 0.4% (w/v);
(3) preparing an umbilical cord mesenchymal stem cell factor essence: mixing the concentrated umbilical cord mesenchymal stem cell factor solution obtained in the step (1) and the diluent obtained in the step (2) according to the volume ratio of 1:1, placing the mixture into a constant-temperature oscillator, oscillating for 5min, and oscillating uniformly to obtain the umbilical cord mesenchymal stem cell factor essence.
Example 2
An essence containing umbilical cord mesenchymal stem cell factors comprises: the umbilical cord mesenchymal stem cell factor concentrated solution is 50% (v/v), human albumin 2.5% (w/v), dextran 2% (w/v), hyaluronic acid 1.4% (w/v), sodium alginate 1.1% (w/v), collagen 2.5% (w/v), glycerol 0.9% (v/v) and catechin 0.5% (w/v).
The preparation method of the essence comprises the following steps:
(1) preparing an umbilical cord mesenchymal stem cell factor concentrated solution:
A. separating and culturing umbilical cord Wharton jelly mesenchymal stem cells: aseptically taking healthy human umbilical cord tissue, soaking in 75% ethanol for 1min, cutting off the two ends of the umbilical cord tissue by 1cm length respectively, washing the rest part with normal saline until the umbilical cord tissue is clear, cutting into small segments of 2cm, cleaning with normal saline for 3 times, transferring the umbilical cord tissue to a new culture dish, adding normal saline into the culture dish until the umbilical cord tissue is submerged at 1/2, cutting along the venous lumen of the umbilical cord tissue, removing 1 vein, 2 arteries and amnion after tiling, taking jelly between blood vessels and between the blood vessels and adventitia, namely: cutting HUATONG gum into pieces of 1mm3Size, transferred to complete medium for culture, and the flask was placed at 37 ℃ in 5% CO2And (4) carrying out cell passage when the cells are cultured in an incubator until 80-90% of the cells are fused, and replacing fresh culture medium every 3 days.
B. Subculturing mesenchymal stem cells: removing the culture solution of the cells to be transferred in the step A, adding 10mL of physiological saline into a non-cell culture surface, slightly shaking to wash the tissue wall surface, removing, repeating for 2 times, adding 3mL of 0.25% pancreatin, uniformly infiltrating the cell wall surface, incubating at 37 ℃ for 3min, adding an equal amount of stop solution after the cells become round to stop digestion, quickly shaking, blowing the cell wall surface by using a 10mL pipette, sucking cell suspension into 2 50mL centrifuge tubes, adding 10mL of physiological saline into each culture bottle, and blowing and washingCollecting the obtained product once, centrifuging for 6min at 1200rpm in a 50mL centrifuge tube, discarding supernatant, mixing the precipitate to 1 tube, adding 40mL physiological saline, centrifuging and washing again, re-suspending the precipitate with complete culture medium, inoculating for P1 generation culture, bottling according to cell number (T175 bottle) and cell density of 5 × 104Per mL, each bottle volume is 25mL, the culture flask is placed at 37 ℃ with 5% CO2When the culture is carried out in an incubator until 80% -90% of fusion, subculture is continued until P6 generation.
C. Preparing an umbilical cord mesenchymal stem cell factor concentrated solution: collecting cell culture solution of P2-P6 generation, filtering with 0.22 μm filter membrane, concentrating the supernatant of stem cells with 50KD ultrafiltration membrane, passing the concentrated solution with 3KD ultrafiltration membrane, and collecting the trapped fluid, i.e. concentrated solution of umbilical cord mesenchymal stem cell factor;
wherein the complete medium comprises: 90% (v/v) of Dayou MSCMB culture medium, 7% (v/v) of Helios serum substitute, 20.5% (w/v) of interleukin, 0.5% (w/v) of sodium citrate and 2% (w/v) of dextran sulfate;
the stop solution comprises α -MEM culture medium 96% (v/v), fetal bovine serum 2% (v/v), thiamine hydrochloride 0.8% (w/v) and 4-hydroxyethyl piperazine ethanesulfonic acid 1.2% (w/v).
(2) Preparation of a diluent: preparing human albumin, dextran, hyaluronic acid, sodium alginate, collagen, glycerol and catechin with sterile ultrapure water in proportion, placing into a constant temperature oscillator, oscillating for 10min, and uniformly oscillating to obtain a diluent; the diluent comprises: human albumin 5% (w/v), dextran 4% (w/v), hyaluronic acid 2.8% (w/v), sodium alginate 2.2% (w/v), collagen 5% (w/v), glycerol 1.8% (v/v) and catechin 1% (w/v);
(3) preparing an umbilical cord mesenchymal stem cell factor essence: mixing the concentrated umbilical cord mesenchymal stem cell factor solution obtained in the step (1) and the diluent obtained in the step (2) according to the volume ratio of 1:1, placing the mixture into a constant-temperature oscillator, oscillating for 10min, and oscillating uniformly to obtain the umbilical cord mesenchymal stem cell factor essence.
Example 3
An essence containing umbilical cord mesenchymal stem cell factors comprises: the umbilical cord mesenchymal stem cell factor concentrated solution comprises 50% (v/v) of umbilical cord mesenchymal stem cell factor concentrated solution, 1.5% (w/v) of human albumin, 1% (w/v) of dextran, 0.8% (w/v) of hyaluronic acid, 0.7% (w/v) of sodium alginate, 1.4% (w/v) of collagen, 0.5% (v/v) of glycerol and 0.3% (w/v) of catechin.
The preparation method of the essence comprises the following steps:
(1) preparing an umbilical cord mesenchymal stem cell factor concentrated solution:
A. separating and culturing umbilical cord Wharton jelly mesenchymal stem cells: aseptically taking healthy human umbilical cord tissue, soaking in 75% ethanol for 1min, cutting off the two ends of the umbilical cord tissue by 1cm length respectively, washing the rest part with normal saline until the umbilical cord tissue is clear, cutting into small segments of 2cm, cleaning with normal saline for 3 times, transferring the umbilical cord tissue to a new culture dish, adding normal saline into the culture dish until the umbilical cord tissue is submerged at 1/2, cutting along the venous lumen of the umbilical cord tissue, removing 1 vein, 2 arteries and amnion after tiling, taking jelly between blood vessels and between the blood vessels and adventitia, namely: cutting HUATONG gum into pieces of 1mm3Size, transferred to complete medium for culture, and the flask was placed at 37 ℃ in 5% CO2And (4) carrying out cell passage when the cells are cultured in an incubator until 80-90% of the cells are fused, and replacing fresh culture medium every 3 days.
B. Subculturing mesenchymal stem cells: removing the culture solution of the cells to be transfected in the step A, adding 10mL of physiological saline into a non-cell culture surface, slightly shaking to wash a tissue pasting wall surface, removing the tissue pasting wall surface, repeating the steps for 2 times, adding 3mL of 0.25% pancreatin, uniformly infiltrating the cell pasting wall surface, incubating for 2min at 37 ℃, adding an equal amount of termination solution after the cells become round to stop digestion, quickly shaking, blowing the cell pasting wall surface by using a 10mL pipette, sucking cell suspension into 2 50mL centrifuge tubes, adding 10mL of physiological saline into each culture bottle, blowing once, converging into a 50mL centrifuge tube, centrifuging at 1200rpm for 6min, removing supernatant, combining precipitates to 1 tube, adding 40mL of physiological saline, centrifuging and washing again, re-suspending the precipitates by using complete culture medium, inoculating and culturing for P1 generation, paving bottles (T175 bottles) according to the number of the cells, wherein the cell density is 3 multiplied by 104Per mL, each bottle volume is 25mL, the culture flask is placed at 37 ℃ with 5% CO2When the mixture is cultured in an incubator until 80% -90% of the mixture is fused, continuing to culture the mixtureSubculture was carried out until P6 generation.
C. Preparing an umbilical cord mesenchymal stem cell factor concentrated solution: collecting cell culture solution of P2-P6 generation, filtering with 0.22 μm filter membrane, concentrating the supernatant of stem cells with 50KD ultrafiltration membrane, passing the concentrated solution with 3KD ultrafiltration membrane, and collecting the trapped fluid, i.e. concentrated solution of umbilical cord mesenchymal stem cell factor;
wherein the complete medium comprises: 86% (v/v) of Dayou MSCMB culture medium, 8% (v/v) of Helios serum substitute, 21.5% (w/v) of interleukin, 1.5% (w/v) of sodium citrate and 3% (w/v) of dextran sulfate;
the stop solution comprises α -MEM medium 94% (v/v), fetal bovine serum 3.5% (v/v), thiamine hydrochloride 1.2% (w/v) and 4-hydroxyethyl piperazine ethanesulfonic acid 1.3% (w/v).
(2) Preparation of a diluent: preparing human albumin, dextran, hyaluronic acid, sodium alginate, collagen, glycerol and catechin with sterile ultrapure water in proportion, placing into a constant temperature oscillator, oscillating for 8min, and uniformly oscillating to obtain a diluent; the diluent comprises: human albumin 3% (w/v), dextran 2% (w/v), hyaluronic acid 1.6% (w/v), sodium alginate 1.4% (w/v), collagen 2.8% (w/v), glycerol 1% (v/v) and catechin 0.6% (w/v);
(3) preparing an umbilical cord mesenchymal stem cell factor essence: mixing the concentrated umbilical cord mesenchymal stem cell factor solution obtained in the step (1) and the diluent obtained in the step (2) according to the volume ratio of 1:1, placing the mixture into a constant-temperature oscillator, oscillating for 8min, and oscillating uniformly to obtain the umbilical cord mesenchymal stem cell factor essence.
Example 4
An essence containing umbilical cord mesenchymal stem cell factors comprises: the umbilical cord mesenchymal stem cell factor concentrated solution comprises 50% (v/v) of umbilical cord mesenchymal stem cell factor concentrated solution, 1.5% (w/v) of human albumin, 1% (w/v) of dextran, 0.8% (w/v) of hyaluronic acid, 0.7% (w/v) of sodium alginate, 1.4% (w/v) of collagen, 0.5% (v/v) of glycerol and 0.3% (w/v) of catechin.
The preparation method of the essence comprises the following steps:
(1) preparing an umbilical cord mesenchymal stem cell factor concentrated solution:
A. separating and culturing umbilical cord Wharton jelly mesenchymal stem cells: aseptically taking healthy human umbilical cord tissue, soaking in 75% ethanol for 1min, cutting off the two ends of the umbilical cord tissue by 1cm length respectively, washing the rest part with normal saline until the umbilical cord tissue is clear, cutting into small segments of 2cm, cleaning with normal saline for 3 times, transferring the umbilical cord tissue to a new culture dish, adding normal saline into the culture dish until the umbilical cord tissue is submerged at 1/2, cutting along the venous lumen of the umbilical cord tissue, removing 1 vein, 2 arteries and amnion after tiling, taking jelly between blood vessels and between the blood vessels and adventitia, namely: cutting HUATONG gum into pieces of 1mm3Size, transferred to complete medium for culture, and the flask was placed at 37 ℃ in 5% CO2And (4) carrying out cell passage when the cells are cultured in an incubator until 80-90% of the cells are fused, and replacing fresh culture medium every 3 days.
B. Subculturing mesenchymal stem cells: removing the culture solution of the cells to be transfected in the step A, adding 10mL of physiological saline into a non-cell culture surface, slightly shaking to wash a tissue pasting wall surface, removing the tissue pasting wall surface, repeating the steps for 2 times, adding 3mL of 0.25% pancreatin, uniformly infiltrating the cell pasting wall surface, incubating for 1min at 37 ℃, adding an equal amount of termination solution after the cells become round to stop digestion, quickly shaking, blowing the cell pasting wall surface by using a 10mL pipette, sucking cell suspension into 2 50mL centrifuge tubes, adding 10mL of physiological saline into each culture bottle, blowing once, converging into 50mL centrifuge tubes, centrifuging at 1200rpm for 6min, removing supernatant, combining precipitates to 1 tube, adding 40mL of physiological saline, centrifuging and washing again, re-suspending the precipitates by using complete culture medium, inoculating and culturing for P1 generation, paving bottles (T175 bottles) according to the number of the cells, wherein the cell density is 2 multiplied by 104Per mL, each bottle volume is 25mL, the culture flask is placed at 37 ℃ with 5% CO2When the culture is carried out in an incubator until 80% -90% of fusion, subculture is continued until P6 generation.
C. Preparing an umbilical cord mesenchymal stem cell factor concentrated solution: collecting cell culture solution of P2-P6 generation, filtering with 0.22 μm filter membrane, concentrating the supernatant of stem cells with 50KD ultrafiltration membrane, passing the concentrated solution with 3KD ultrafiltration membrane, and collecting the trapped fluid, i.e. concentrated solution of umbilical cord mesenchymal stem cell factor;
wherein the complete medium comprises: 86% (v/v) of Dayou MSCMB culture medium, 8% (v/v) of Helios serum substitute, 21.5% (w/v) of interleukin, 1.5% (w/v) of sodium citrate and 3% (w/v) of dextran sulfate;
the stop solution comprises α -MEM culture medium 92% (v/v), fetal bovine serum 5% (v/v), thiamine hydrochloride 1.5% (w/v) and 4-hydroxyethyl piperazine ethanesulfonic acid 1.5% (w/v).
(2) Preparation of a diluent: preparing human albumin, dextran, hyaluronic acid, sodium alginate, collagen, glycerol and catechin with sterile ultrapure water in proportion, placing into a constant temperature oscillator, oscillating for 6min, and uniformly oscillating to obtain a diluent; the diluent comprises: human albumin 3% (w/v), dextran 2% (w/v), hyaluronic acid 1.6% (w/v), sodium alginate 1.4% (w/v), collagen 2.8% (w/v), glycerol 1% (v/v) and catechin 0.6% (w/v);
(3) preparing an umbilical cord mesenchymal stem cell factor essence: mixing the concentrated umbilical cord mesenchymal stem cell factor solution obtained in the step (1) and the diluent obtained in the step (2) according to the volume ratio of 1:1, placing the mixture into a constant-temperature oscillator, oscillating for 6min, and oscillating uniformly to obtain the umbilical cord mesenchymal stem cell factor essence.
Example 5
An essence containing umbilical cord mesenchymal stem cell factors comprises: the umbilical cord mesenchymal stem cell factor concentrated solution is 50% (v/v), human albumin 2.5% (w/v), dextran 2% (w/v), hyaluronic acid 1.4% (w/v), sodium alginate 1.1% (w/v), collagen 2.5% (w/v), glycerol 0.9% (v/v) and catechin 0.5% (w/v).
The preparation method of the essence comprises the following steps:
(1) preparing an umbilical cord mesenchymal stem cell factor concentrated solution:
A. separating and culturing umbilical cord Wharton jelly mesenchymal stem cells: aseptically taking healthy human umbilical cord tissue, soaking in 75% ethanol for 1min, cutting off length of 1cm from both ends of umbilical cord tissue, washing the rest part with normal saline to clear, cutting into 2cm small segments, cleaning with normal saline for 3 times, transferring umbilical cord tissue to a new culture dish, adding normal saline into the culture dishCutting along the vein cavity of the umbilical cord tissue to submerge the umbilical cord tissue 1/2, removing 1 vein, 2 arteries and amnion after tiling, and taking jelly between blood vessels and between the blood vessels and the adventitia, namely: cutting HUATONG gum into pieces of 1mm3Size, transferred to complete medium for culture, and the flask was placed at 37 ℃ in 5% CO2And (4) carrying out cell passage when the cells are cultured in an incubator until 80-90% of the cells are fused, and replacing fresh culture medium every 3 days.
B. Subculturing mesenchymal stem cells: removing the culture solution of the cells to be transfected in the step A, adding 10mL of physiological saline into a non-cell culture surface, slightly shaking to wash a tissue pasting wall surface, removing the tissue pasting wall surface, repeating the steps for 2 times, adding 3mL of 0.25% pancreatin, uniformly infiltrating the cell pasting wall surface, incubating for 3min at 37 ℃, adding an equal amount of termination solution after the cells become round to stop digestion, quickly shaking, blowing the cell pasting wall surface by using a 10mL pipette, sucking cell suspension into 2 50mL centrifuge tubes, adding 10mL of physiological saline into each culture bottle, blowing once, converging into 50mL centrifuge tubes, centrifuging at 1200rpm for 6min, removing supernatant, combining precipitates to 1 tube, adding 40mL of physiological saline, centrifuging and washing again, re-suspending the precipitates by using complete culture medium, inoculating and culturing for P1 generation, paving bottles (T175 bottles) according to the number of the cells, wherein the cell density is 5 multiplied by 104Per mL, each bottle volume is 25mL, the culture flask is placed at 37 ℃ with 5% CO2When the culture is carried out in an incubator until 80% -90% of fusion, subculture is continued until P6 generation.
C. Preparing an umbilical cord mesenchymal stem cell factor concentrated solution: collecting cell culture solution of P2-P6 generation, filtering with 0.22 μm filter membrane, concentrating the supernatant of stem cells with 50KD ultrafiltration membrane, passing the concentrated solution with 3KD ultrafiltration membrane, and collecting the trapped fluid, i.e. concentrated solution of umbilical cord mesenchymal stem cell factor;
wherein the complete medium comprises: 90% (v/v) of Dayou MSCMB culture medium, 7% (v/v) of Helios serum substitute, 20.5% (w/v) of interleukin, 0.5% (w/v) of sodium citrate and 2% (w/v) of dextran sulfate;
the stop solution comprises α -MEM medium 94% (v/v), fetal bovine serum 3.5% (v/v), thiamine hydrochloride 1.2% (w/v) and 4-hydroxyethyl piperazine ethanesulfonic acid 1.3% (w/v).
(2) Preparation of a diluent: preparing human albumin, dextran, hyaluronic acid, sodium alginate, collagen, glycerol and catechin with sterile ultrapure water in proportion, placing into a constant temperature oscillator, oscillating for 9min, and uniformly oscillating to obtain a diluent; the diluent comprises: human albumin 5% (w/v), dextran 4% (w/v), hyaluronic acid 2.8% (w/v), sodium alginate 2.2% (w/v), collagen 5% (w/v), glycerol 1.8% (v/v) and catechin 1% (w/v);
(3) preparing an umbilical cord mesenchymal stem cell factor essence: mixing the concentrated umbilical cord mesenchymal stem cell factor solution obtained in the step (1) and the diluent obtained in the step (2) according to the volume ratio of 1:1, placing the mixture into a constant-temperature oscillator, oscillating for 9min, and oscillating uniformly to obtain the umbilical cord mesenchymal stem cell factor essence.
Comparative example 1
The difference from example 3 is only that, the essence containing the umbilical cord mesenchymal stem cell factor comprises: the umbilical cord mesenchymal stem cell factor concentrated solution comprises 50% (v/v) of umbilical cord mesenchymal stem cell factor concentrated solution, 3% (w/v) of human albumin, 0.3% (w/v) of glucan, 1.5% (w/v) of hyaluronic acid, 0.3% (w/v) of sodium alginate, 3% (w/v) of collagen, 0.2% (v/v) of glycerol and 1% (w/v) of catechin.
Comparative example 2
The difference from example 3 is only that, the essence containing the umbilical cord mesenchymal stem cell factor comprises: the preparation method comprises the following steps of 50% (v/v) of umbilical cord mesenchymal stem cell factor concentrated solution, 0.5% (w/v) of human albumin, 2.5% (w/v) of glucan, 0.3% (w/v) of hyaluronic acid, 1.5% (w/v) of sodium alginate, 0.5% (w/v) of collagen, 1% (v/v) of glycerol and 0.1% (w/v) of catechin.
Comparative example 3
The only difference from example 3 is that the complete medium comprises: 75% (v/v) of Dayou MSCMB culture medium, 15% (v/v) of Helios serum substitute, 20.3% (w/v) of interleukin, 2.7% (w/v) of sodium citrate and 7% (w/v) of dextran sulfate.
Comparative example 4
The difference from example 3 is only that the stop solution comprises α -MEM medium 90% (v/v), fetal bovine serum 6% (v/v), thiamine hydrochloride 0.5% (w/v) and 4-hydroxyethyl piperazine ethanesulfonic acid 3.5% (w/v).
First, detection of cytokine concentration variation
The contents and recovery rates of epidermal growth factor, vascular endothelial growth factor and platelet-derived growth factor before and after concentration of umbilical cord mesenchymal stem cells in examples 1 to 5 and comparative examples 3 to 4 were measured using an ELASIA kit, and table 1 was obtained:
TABLE 1 cytokine content and recovery variation
As can be seen from Table 1, the cell supernatant in this application was concentrated by about 40-fold by filtration using an ultrafiltration membrane, and the cytokine concentrations and recovery rates in examples 1 to 5 were high, and the recovery rates for epidermal growth factor, vascular epidermal growth factor and platelet-derived growth factor were in the ranges of 97.72-98.12%, 96.60-97.47% and 98.05-99.30%, respectively, wherein the cytokine concentration and recovery rate in example 3 were the highest, and the cytokine concentrations and recovery rates in comparative examples 3 to 4 were lower than those in example 3. The results show that the optimization of the preparation method in the application obviously improves the content and recovery rate of the mesenchymal stem cell factor in the mesenchymal stem cell factor concentrated solution.
Second, essence effect test
35 subjects with wrinkles, spots, dull skin or loose skin were found, and randomly divided into 7 groups of 5 persons, and the essences of examples 1 to 5 and comparative examples 1 to 2 were tried, respectively, continuously used for two months, and the skin condition of the subjects was observed and scored according to Table 2, to obtain Table 3:
TABLE 2 Scoring basis
| Facial condition | Fractional interval (points) |
| Facial wrinkle and mottle/dark and rough skin | 1-3 |
| Facial wrinkles, mottle/skin whitening and smoothing | 4-6 |
| The facial wrinkles and color spots become light, and the skin becomes white and smooth | 7-9 |
| The face has no wrinkle, color spot/skin whitening and smoothing effects | 10 |
Table 3 serum efficacy test scores
| Examples of the invention | Fraction (point) |
| Example 1 | 8.4 |
| Example 2 | 8.5 |
| Example 3 | 9.2 |
| Example 4 | 8.7 |
| Example 5 | 8.7 |
| Comparative example 1 | 5.5 |
| Comparative example 2 | 5.7 |
Note: the data in the table are mean values.
As can be seen from Table 3, the scores of the examples 1-5 are higher and are all more than 8, wherein the score of the example 3 is the highest and is 9.2, and the scores of the comparative examples 1-2 are lower and are respectively 5.5 and 5.7, which indicates that the essence in the protection range of the application has the effects of whitening and lightening spots, repairing skin, reducing wrinkles, delaying aging and the like, and the effect is influenced by changing the proportion of the essence.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The essence containing the umbilical cord mesenchymal stem cell factor is characterized by comprising the following components in parts by weight: the preparation method comprises the following steps of 50% (v/v) of umbilical cord mesenchymal stem cell factor concentrated solution, 1% -2.5% (w/v) of human albumin, 0.5% -2% (w/v) of glucan, 0.5% -1.4% (w/v) of hyaluronic acid, 0.4% -1.1% (w/v) of sodium alginate, 1% -2.5% (w/v) of collagen, 0.3% -0.9% (v/v) of glycerol and 0.2% -0.5% (w/v) of catechin.
2. The essence of claim 1, wherein the essence comprises: 50% (v/v) of umbilical cord mesenchymal stem cell factor concentrated solution, 1.5% -2.5% (w/v) of human albumin, 1% -2% (w/v) of glucan, 0.8% -1.4% (w/v) of hyaluronic acid, 0.7% -1.1% (w/v) of sodium alginate, 1.4% -2.5% (w/v) of collagen, 0.5% -0.9% (v/v) of glycerol and 0.3% -0.5% (w/v) of catechin.
3. A method for preparing the essence according to any one of claims 1 to 2, wherein the method comprises the following steps:
(1) preparing an umbilical cord mesenchymal stem cell factor concentrated solution:
A. separating and culturing umbilical cord Wharton jelly mesenchymal stem cells: soaking human umbilical cord tissue in 75% ethanol, cutting off 1cm from each end of the tissue, washing the rest with normal saline until the tissue is clear, cutting into small segments, washing with normal saline for 3 times, cutting along the venous lumen of the umbilical cord tissue, removing veins, arteries and amnion after tiling, and taking jelly between blood vessels and adventitia, namely: cutting the huatong glue, transferring the huatong glue to a complete culture medium for primary cell culture, and carrying out cell passage when the huatong glue is cultured until 80-90% of fusion;
B. subculturing mesenchymal stem cells: removing the culture solution of the cells to be subcultured in the step A, adding physiological saline to wash the tissue and stick to the wall surface, adding 0.25% of pancreatin, incubating at 37 ℃ for 1-3min, adding a stop solution to stop digestion after the cells become round, quickly shaking, blowing and beating the cells, sucking out cell suspension, adding physiological saline to wash, centrifuging, collecting precipitates, resuspending the precipitates by using a complete culture medium, inoculating the precipitates to the complete culture medium to perform P1 generation culture, and continuously performing subculture when the cells grow to 80% -90% fusion, and subculturing to P6 generation;
C. preparing an umbilical cord mesenchymal stem cell factor concentrated solution: collecting cell culture solution of the P2-P6 generation, filtering and concentrating through a microfiltration membrane and an ultrafiltration membrane, and collecting trapped fluid to obtain umbilical cord mesenchymal stem cell factor concentrated solution;
the complete culture medium comprises: 80% -90% (v/v) of Dayou MSCMB culture medium, 7% -12% (v/v) of Helios serum substitute, 20.5% -2% (w/v) of interleukin, 0.5% -2% (w/v) of sodium citrate and 2% -4% (w/v) of dextran sulfate;
the stop solution comprises α -MEM culture medium 92% -96% (v/v), fetal bovine serum 2% -5% (v/v), thiamine hydrochloride 0.8% -1.5% (w/v) and 4-hydroxyethyl piperazine ethanesulfonic acid 1.2% -1.5% (w/v);
(2) preparation of a diluent: preparing human albumin, dextran, hyaluronic acid, sodium alginate, collagen, glycerol and catechin in proportion with sterile ultrapure water, and shaking for 5-10min to obtain diluent;
(3) preparing an umbilical cord mesenchymal stem cell factor essence: and (3) mixing the concentrated umbilical cord mesenchymal stem cell factor solution obtained in the step (1) with the diluent obtained in the step (2) according to the volume ratio of 1:1, and shaking for 5-10min to obtain the essence containing the umbilical cord mesenchymal stem cell factor.
4. The method of claim 3, wherein the complete medium comprises: 86-90% (v/v) of Dayou MSCMB culture medium, 7-8% (v/v) of Helios serum substitute, 20.5-1.5% (w/v) of interleukin, 0.5-1.5% (w/v) of sodium citrate and 2-3% (w/v) of dextran sulfate.
5. The method according to claim 3, wherein the stop solution comprises α -MEM medium 92% -94% (v/v), fetal bovine serum 3.5% -5% (v/v), thiamine hydrochloride 1.2% -1.5% (w/v), and 4-hydroxyethylpiperazine ethanesulfonic acid 1.3% -1.5% (w/v).
6. The method of claim 3, wherein the diluent comprises: 2-5% (w/v) of human albumin, 1-4% (w/v) of glucan, 1-2.8% (w/v) of hyaluronic acid, 0.8-2.2% (w/v) of sodium alginate, 2-5% (w/v) of collagen, 0.6-1.8% (v/v) of glycerol and 0.4-1% (w/v) of catechin.
7. The method of claim 6, wherein the diluent comprises: 3% -5% (w/v) of human albumin, 2% -4% (w/v) of glucan, 1.6% -2.8% (w/v) of hyaluronic acid, 1.4% -2.2% (w/v) of sodium alginate, 2.8% -5% (w/v) of collagen, 1% -1.8% (v/v) of glycerol and 0.6% -1% (w/v) of catechin.
8. The method according to claim 3, wherein the primary cell culture and the subculture are carried out under the culture conditions of 37 ℃ and 5% CO2The humidity was 95%, and the medium was replaced with fresh medium every 3 days.
9. The method according to claim 3, wherein the seeded cell density in the step B is 2 to 5X 104/mL。
10. The method according to claim 3, wherein the ultrafiltration membrane in step C is an ultrafiltration membrane having a molecular weight of 50KD and 3 KD.
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