CN113069407A - Preparation method of stem cell filtrate and stem cell factor mixed solution - Google Patents
Preparation method of stem cell filtrate and stem cell factor mixed solution Download PDFInfo
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- CN113069407A CN113069407A CN202110227036.3A CN202110227036A CN113069407A CN 113069407 A CN113069407 A CN 113069407A CN 202110227036 A CN202110227036 A CN 202110227036A CN 113069407 A CN113069407 A CN 113069407A
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- 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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Abstract
The invention discloses a preparation method of a stem cell filtrate and a stem cell factor mixed solution in the technical field of cosmetics, which comprises the following steps: the preparation method of the stem cell filtrate and the stem cell factor mixed solution can effectively and continuously improve the skin state fundamentally, achieves the effects of removing acne, acne mark, scar, wrinkle and the like, does not break the skin, is painless, does not have infection risk and side effect in the whole process, can repair the skin radically and resist aging, is simple to operate, does not need any medical quality in the operation process, has moderate price of required instruments, and is suitable for daily use in home.
Description
Technical Field
The invention discloses a preparation method of a stem cell filtrate and a stem cell factor mixed solution, and particularly relates to the technical field of cosmetics.
Background
Skin refers to the tissue that covers the surface of the human body and is in direct contact with the external environment. Has the functions of protection, sensation, secretion, excretion, respiration, etc., and is formed by tightly combining epidermis and dermis. The skin covers the entire body surface and is one of the largest organs of the human body, accounting for about 16% of body weight. In the current society, with the improvement of living standard of people, the demand of people on skin management is more and more urgent, the common skin problems mainly include the problems of acne, dryness, wound scar, pigmentation, wrinkle and the like caused by secretion disorder, severe conditions of work and life, injury, diseases, aging and the like, and the conventional skin care is mainly skin care products, medicines or means of micro-cosmetic and the like at present. Various problems of the skin cannot be fundamentally solved through the forms of cosmetics, medicine application and facial masks, and emerging medical and cosmetic anti-aging means such as a water acupuncture, a thermal magic flower, cell filling and the like have certain defects. The hydro-acupuncture needle directly injects hyaluronic acid into the dermis layer to provide partial nutrient components for the skin, so that the skin is in a fuller and more smooth state, and the beautifying effect is achieved. However, the side effects of pain, red swelling and rash may occur when the water needle is used, and the injected hyaluronic acid is absorbed and metabolized within one to two months, and frequent injection is required to preserve the beauty effect. The principle of the thermal Maji is that high-energy electric waves penetrate through the skin to activate skin collagen and fibroblasts instantly and efficiently, so that the regeneration of collagen is promoted, but the aging is accelerated by sequela. The face pack mainly comprises collagen, autologous adipose-derived stem cells, autologous fat and the like, can achieve a wrinkle removing effect, is easy to generate rejection reaction after injection, and has infection risk. The above-mentioned methods for resisting aging through medical and beauty require special instruments, fixed places such as medical and beauty institutions, etc., and have a certain qualification of operators and a high price compared with general households. Therefore, it is important to develop a method for improving the skin condition from the root without skin breakage, pain, infection risk, side effects, and high cost, which is acceptable to general households and can be used at home daily.
The mesenchymal stem cell is a pluripotent stem cell, has all the commonalities of the stem cell, namely self-renewal and multidirectional differentiation capacity, and can secrete a large number of bioactive factors which can participate in functional links such as immunoregulation, anti-apoptosis and the like, such as stem cell growth factor (SCF), Nerve Growth Factor (NGF), stromal cell derived growth factor (SDF), vascular endothelial cell growth factor (VEGF), exosome and the like, in an autocrine or paracrine mode, so that the local microenvironment can be improved, the migration and differentiation of endogenous stem cells to a wound part can be promoted, and cells with body injury, pathological changes and aging can be further physiologically repaired or replaced.
In addition to bioactive factors secreted by stem cells, the stem cells contain abundant bioactive substances which play an important role in proliferation and differentiation of the stem cells, but the substances are not secreted to the cells in the proliferation and differentiation process, so that the cells are cracked in a proper mode, and mesenchymal stem cell filtrate can be fully extracted, so that on one hand, the problems of immunological rejection reaction and the like caused by direct injection of the mesenchymal stem cells can be avoided, on the other hand, the bioactive substances generated by stem cell cracking can be used for activating the stem cells of an organism, and the cells with body injury, lesion and aging can be physiologically repaired or replaced by the stem cells. In conclusion, the bioactive factor secreted by the mesenchymal stem cell and the bioactive substance in the mesenchymal stem cell have wide application prospects in the fields of disease prevention and treatment, health care and beauty.
The needleless water light is a method for injecting nutrient substances into a skin basal layer in a high-pressure injection mode by adopting a high-pressure vacuum technology, does not need to break the skin, has no pain and almost no recovery period, and is suitable for being used at home by customers due to moderate price of required instruments and simple operation.
At present, the practical method has the following defects:
1. the supernatant obtained by culturing the cells is obtained by concentrating the cell supernatant of several generations, the method is complex and takes long time, and pollution is easily caused in the operation process;
2. at present, products obtained by cell lysis are only resuspended by a culture medium and then simply smeared on the face, and the repairing effect of bioactive substances in the products is not fully exerted;
3. the commonly used medical and aesthetic methods are not only expensive and usually require complicated instrument operation, but also have extremely high requirements on operators and are easy to cause various side effects.
Disclosure of Invention
In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of a stem cell filtrate and a stem cell factor mixed solution comprises the following steps:
step 1: the adopted human umbilical cord source donor has no pathogen (virus, mycoplasma, chlamydia and the like) infection;
step 2: preparing seed cells and product cells of the umbilical cord mesenchymal stem cells by adopting a serum-free and animal-derived-component-free basic culture medium and an additive factor;
and step 3: adopting P4-P6 generation seed cells, carrying out mesenchymal stem cell amplification culture in a culture box, and extracting a culture solution containing cell factors when the fusion degree reaches 100%;
and 4, step 4: placing the culture solution obtained in the step (3) in a centrifuge for centrifugation, transferring the supernatant part into a new sterile centrifuge tube, and storing in a refrigerator at the temperature of 4 ℃;
and 5: adding a proper amount of physiological saline into the culture bottle/dish from which the supernatant is taken out in the step 3, and continuously culturing the cells in the incubator for 24 hours;
step 6: the cells were all suspended by the treatment of step 5. Pumping the suspended cells and physiological saline into a centrifuge tube;
and 7: putting the centrifugal tube filled with the cell suspension in the step 6 into liquid nitrogen for freezing, and then dissolving;
and 8: fully blowing the cell suspension in the centrifugal tube in the step 7 to make organelles adhered to the cell membrane fall off as much as possible;
and step 9: repeating the operations of the step 7 and the step 8 again;
step 10: placing the mixed solution filled with the broken cells in the step 9 in a centrifuge for centrifugation;
step 11: taking out the supernatant in the centrifuge tube in the step 10, and adding the supernatant into the culture solution in the step 3;
step 12: fully mixing the mixed solution obtained in the step 11, and subpackaging the mixture into sealable sterile containers according to the volume of 5ml of each part;
step 13: sealing the container filled with the mixed solution in the step 12, and freezing the container in a refrigerator at the temperature of below 20 ℃ below zero;
step 14: and (4) dissolving the product in the step (13) and then introducing the solution without needle water.
Preferably, the mixture used is a mixture of stem cell factor and cell filtrate that produces stem cell factor.
Preferably, the culture conditions of the culture boxes in the step 3 and the step 5 are both at the temperature of 37 ℃, and CO is2The concentration was 5%.
Preferably, the freezing temperature and the melting temperature of the centrifuge tube filled with the cell suspension obtained in the step 7 are respectively-196 ℃ and 25 ℃.
Preferably, the rotating speed of the centrifuge in the step 4 and the step 10 is 2000rpm, and the time is 10 min.
Compared with the prior art, the cell filtrate obtained after cell disruption and the supernatant obtained by culturing the mesenchymal stem cells are mixed and introduced into human skin in a needleless water light mode, so that cell proliferation and differentiation can be activated, epidermal cell repair is realized, skin state can be effectively and continuously improved fundamentally, acne removal, acne mark removal, scar lightening, wrinkle removal and other effects are achieved, the whole process is free of skin disruption, pain, infection risk and side effects, the skin can be repaired radically, the skin is anti-aging, the operation is simple, no medical quality is needed in the operation process, the price of a needed instrument is moderate, and the device is suitable for daily use in homes.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
a preparation method of a stem cell filtrate and stem cell factor mixed solution comprises the following steps:
step 1: the donor from human umbilical cord used is free of pathogen (virus, mycoplasma, chlamydia, etc.) infection.
Step 2: the preparation of the seed cells and the product cells of the umbilical cord mesenchymal stem cells is carried out by adopting a serum-free and animal-derived-component-free basic culture medium and an additive factor.
And step 3: adopting P4-P6 generation seed cells, at 37 deg.C and 5% CO2The culture box is used for carrying out the amplification culture of the mesenchymal stem cells, and when the fusion degree reaches 80 percent, a culture solution containing the cell factors is extracted.
And 4, step 4: centrifuging the culture solution obtained in step 3 at 2000rpm for 10min, transferring the supernatant into a new sterile centrifuge tube, and storing in a refrigerator at 4 deg.C.
And 5: adding proper amount of normal saline into the culture bottle/dish with the supernatant liquid removed in the step 3, and adding 5% CO at 37 DEG C2The incubator in (1) was continued for 24 h.
Step 6: the cells were all suspended by the treatment of step 5. The suspended cells and saline solution were pumped into the centrifuge tube.
And 7: and (4) putting the centrifuge tube filled with the cell suspension in the step 6 into liquid nitrogen for freezing, and then dissolving.
And 8: and (4) fully blowing the cell suspension in the centrifugal tube in the step (7) to ensure that organelles adhered to the cell membrane fall off as much as possible.
And step 9: and repeating the operations of the step 7 and the step 8 again.
Step 10: the mixture containing the disrupted cells in step 9 was centrifuged at 2000rpm for 10 min.
Step 11: and (4) taking out the supernatant in the centrifugal tube in the step 10, and adding the supernatant into the culture solution in the step 3.
Step 12: and (3) fully mixing the mixed solution obtained in the step (11), and subpackaging the mixture into sealable sterile containers according to the volume of 5ml each.
Step 13: sealing the container filled with the mixed solution in the step 12, and freezing in a refrigerator below-20 ℃.
Step 14: and (4) dissolving the product in the step (13) and then introducing the solution without needle water.
Example 2:
a preparation method of a stem cell filtrate and stem cell factor mixed solution comprises the following steps:
step 1: the donor from human umbilical cord used is free of pathogen (virus, mycoplasma, chlamydia, etc.) infection.
Step 2: the preparation of the seed cells and the product cells of the umbilical cord mesenchymal stem cells is carried out by adopting a serum-free and animal-derived-component-free basic culture medium and an additive factor.
And step 3: adopting P4-P6 generation seed cells, at 37 deg.C and 5% CO2The culture box is used for carrying out the amplification culture of the mesenchymal stem cells, and when the fusion degree reaches 90 percent, a culture solution containing the cell factors is extracted.
And 4, step 4: and (4) centrifuging the culture solution obtained in the step (3) at 2000rpm for 10min, transferring the supernatant part into a new sterile centrifuge tube, and storing the tube in liquid nitrogen.
And 5: adding proper amount of normal saline into the culture bottle/dish with the supernatant liquid removed in the step 3, and adding 5% CO at 37 DEG C2The incubator in (1) was continued for 24 h.
Step 6: the cells were all suspended by the treatment of step 5. The suspended cells and saline solution were pumped into the centrifuge tube.
And 7: and (4) putting the centrifuge tube filled with the cell suspension in the step 6 into liquid nitrogen for freezing, and then dissolving.
And 8: and (4) fully blowing the cell suspension in the centrifugal tube in the step (7) to ensure that organelles adhered to the cell membrane fall off as much as possible.
And step 9: and repeating the operations of the step 7 and the step 8 again.
Step 10: the mixture containing the disrupted cells in step 9 was centrifuged at 2000rpm for 10 min.
Step 11: and (4) taking out the supernatant in the centrifugal tube in the step 10, and adding the supernatant into the culture solution in the step 3.
Step 12: and (3) fully mixing the mixed solution obtained in the step (11), and subpackaging the mixture into sealable sterile containers according to the volume of 5ml each.
Step 13: sealing the container filled with the mixed solution in the step 12, and freezing in a refrigerator below-20 ℃.
Step 14: and (4) dissolving the product in the step (13) and then introducing the solution without needle water.
Example 3:
step 1: the donor from human umbilical cord used is free of pathogen (virus, mycoplasma, chlamydia, etc.) infection.
Step 2: the preparation of the seed cells and the product cells of the umbilical cord mesenchymal stem cells is carried out by adopting a serum-free and animal-derived-component-free basic culture medium and an additive factor.
And step 3: adopting P4-P6 generation seed cells, at 37 deg.C and 5% CO2The culture box is used for carrying out the amplification culture of the mesenchymal stem cells, and when the fusion degree reaches 100 percent, a culture solution containing the cell factors is extracted.
And 4, step 4: centrifuging the culture solution obtained in step 3 at 2000rpm for 10min, transferring the supernatant into a new sterile centrifuge tube, and storing in a refrigerator at 4 deg.C.
And 5: adding proper amount of normal saline into the culture bottle/dish with the supernatant liquid removed in the step 3, and adding 5% CO at 37 DEG C2The incubator in (1) was continued for 24 h.
Step 6: the cells were all suspended by the treatment of step 5. The suspended cells and saline solution were pumped into the centrifuge tube.
And 7: and (4) putting the centrifuge tube filled with the cell suspension in the step 6 into liquid nitrogen for freezing, and then dissolving.
And 8: and (4) fully blowing the cell suspension in the centrifugal tube in the step (7) to ensure that organelles adhered to the cell membrane fall off as much as possible.
And step 9: and repeating the operations of the step 7 and the step 8 again.
Step 10: the mixture containing the disrupted cells in step 9 was centrifuged at 2000rpm for 10 min.
Step 11: and (4) taking out the supernatant in the centrifugal tube in the step 10, and adding the supernatant into the culture solution in the step 3.
Step 12: and (3) fully mixing the mixed solution obtained in the step (11), and subpackaging the mixture into sealable sterile containers according to the volume of 5ml each.
Step 13: sealing the container filled with the mixed solution in the step 12, and freezing in a refrigerator below-20 ℃.
Step 14: and (4) dissolving the product in the step (13) and then introducing the solution without needle water.
Investigation of use effect:
the using method comprises the following steps: the invention selects 10 patients with acne, scars, color spots and wrinkles on the face to treat, one course of treatment is to carry out needleless light introduction for 1 time every day, 5mL of the medicine is used every time, and the medicine is continuously used for 10 days, which is determined according to the specific conditions of the patients.
Among them, female patients with a large amount of acne on their faces showed that the number of facial acne was significantly reduced, pigmentation was significantly improved, and scars were lightened after receiving needleless light.
Among the female patients with herpes on the face, the results show that the herpes on the face is obviously reduced, the healing condition is good and no obvious scar is left after healing after receiving needleless water light introduction.
Among them, the results of male patients with extensive scars on the face showed that almost no scars were left on the sutured sites after receiving the introduction of the needleless water light, except that the zygomatic sites were not completely restored.
In conclusion, the cell filtrate obtained after cell disruption and the supernatant obtained by culturing the mesenchymal stem cells are mixed and introduced into the skin of a human body in a needleless water-light mode, so that the skin state can be effectively and continuously improved fundamentally, the effects of removing acne, fading scars and the like are achieved, the skin is not disrupted, the pain and the infection risk are avoided in the whole process, the skin can be fundamentally repaired, the anti-aging effect is realized, the operation is simple, the price of required instruments is moderate, and the device is suitable for daily use at home.
While the invention has been described above with reference to an embodiment, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the various features of the embodiments disclosed herein may be used in any combination, provided that there is no structural conflict, and the combinations are not exhaustively described in this specification merely for the sake of brevity and conservation of resources. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (5)
1. A preparation method of a stem cell filtrate and stem cell factor mixed solution is characterized in that: the preparation method of the stem cell filtrate and the stem cell factor mixed solution comprises the following steps:
step 1: the adopted human umbilical cord source donor has no pathogen (virus, mycoplasma, chlamydia and the like) infection;
step 2: preparing seed cells and product cells of the umbilical cord mesenchymal stem cells by adopting a serum-free and animal-derived-component-free basic culture medium and an additive factor;
and step 3: adopting P4-P6 generation seed cells, carrying out mesenchymal stem cell amplification culture in a culture box, and extracting a culture solution containing cell factors when the fusion degree reaches 100%;
and 4, step 4: placing the culture solution obtained in the step (3) in a centrifuge for centrifugation, transferring the supernatant part into a new sterile centrifuge tube, and storing in a refrigerator at the temperature of 4 ℃;
and 5: adding a proper amount of physiological saline into the culture bottle/dish from which the supernatant is taken out in the step 3, and continuously culturing the cells in the incubator for 24 hours;
step 6: the cells were all suspended by the treatment of step 5. Pumping the suspended cells and physiological saline into a centrifuge tube;
and 7: putting the centrifugal tube filled with the cell suspension in the step 6 into liquid nitrogen for freezing, and then dissolving;
and 8: fully blowing the cell suspension in the centrifugal tube in the step 7 to make organelles adhered to the cell membrane fall off as much as possible;
and step 9: repeating the operations of the step 7 and the step 8 again;
step 10: placing the mixed solution filled with the broken cells in the step 9 in a centrifuge for centrifugation;
step 11: taking out the supernatant in the centrifuge tube in the step 10, and adding the supernatant into the culture solution in the step 3;
step 12: fully mixing the mixed solution obtained in the step 11, and subpackaging the mixture into sealable sterile containers according to the volume of 5ml of each part;
step 13: sealing the container filled with the mixed solution in the step 12, and freezing the container in a refrigerator at the temperature of below 20 ℃ below zero;
step 14: and (4) dissolving the product in the step (13) and then introducing the solution without needle water.
2. The method of claim 1, wherein the step of preparing the mixture of stem cell filtrate and stem cell factor comprises: the mixture used was stem cell factor and a cell filtrate mixture that produced stem cell factor.
3. The method of claim 1, wherein the step of preparing the mixture of stem cell filtrate and stem cell factor comprises: the culture conditions of the culture boxes in the step 3 and the step 5 are both at the temperature of 37 ℃, and CO is adopted2The concentration was 5%.
4. The method of claim 1, wherein the step of preparing the mixture of stem cell filtrate and stem cell factor comprises: the freezing temperature of the centrifuge tube filled with the cell suspension obtained in the step 7 is-196 ℃, and the melting temperature is 25 ℃.
5. The method of claim 1, wherein the step of preparing the mixture of stem cell filtrate and stem cell factor comprises: the rotating speed of the centrifuge in the step 4 and the step 10 is 2000rpm, and the time is 10 min.
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| CN116392575A (en) * | 2023-03-22 | 2023-07-07 | 广东唯泰生物科技有限公司 | Preparation for treating acne by combining stem cell active ingredients with hyaluronic acid |
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Application publication date: 20210706 |
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