CN106692196A - Method for preparing autologous beautifying micro-needle preparation and application thereof - Google Patents
Method for preparing autologous beautifying micro-needle preparation and application thereof Download PDFInfo
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
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Abstract
The invention belongs to the technical field of cosmetic mini-plastic surgery and specifically discloses a method for preparing autologous beautifying micro-needle preparation and application thereof. The preparation of the micro-needle preparation comprises the following steps: separating autologous platelet-rich plasma and peripheral blood mononuclear cells; culturing by using an autologous activated cell factor solution and a cell culture medium; replacing a fresh autologous activated cell factor solution and a cell culture medium the next day; collecting circulating fibrocytes, endothelial progenitor cells and immune cells on the fifth day, resuspending by using the autologous platelet-rich plasma and autologous plasma, thereby obtaining the autologous beautifying micro-needle preparation. Compared with the prior art, the method disclosed by the invention has the advantages that a composition of the circulating fibrocytes, endothelial progenitor cells, immune cells and platelet-rich plasma is used for cosmetic preparations, the circulating fibrocytes are capable of reconstructing skin tissues, secreting collagens and eliminating wrinkles; the endothelial progenitor cells can achieve the effects of local skin microcirculation and improving the skin nutrition state; the immune cells can achieve the effects of swallowing and taking away metabolic products; and the combination of the endothelial progenitor cells and the immune cells can achieve the effects of repairing spotted dull skin.
Description
Technical field
The invention belongs to beauty micro-shaping technical field, specifically a kind of preparation method of autologous beauty micropin preparation
And its application.
Technical background
Facial anti-aging is the more and more popular problem of current aesthetic surgery.With the development of life science and entering for medical science
Step, makes some progress in terms of prevention and anti-aging.Most directly embodiment is the table in wrinkle and color spot for facial aging
Now go up, smoothing wrinkle and nti-freckle turn into the topic that people increasingly pay close attention to.
With the improvement of people ' s living standards, people have requirement higher to spiritual life level, to makings profile
It is required that, to beautiful requirement more and more higher.Face rejuvenation is the aging for delaying by various methods and resisting skin of face, makes face
Portion keeps even recovering rejuvenation appearance.Microneedle injection technology is minimally invasive, painless because of its, and recovers the fast and good advantage of effect, receives
The favor of numerous people seeking beauties is arrived.
Fibroblast is the main cell in dermal tissue, with very strong division, multiplication capacity, additionally it is possible to secrete glue
The extracellular matrix proteins such as original and Porcine HGF, the stromatin of secretion can Methodistic reconstruction skin histology, it is extensive
The globality of multiple its original, to smoothing wrinkle and desalination scar, the slickness for increasing skin has good facilitation.Endothelial progenitor cells
Promote the formation of capilary, improve blood supply and the nutritional status of skin, while immunocyte can be assisted to take away metabolite again, it is right
Color spot and dim heavy skin have improvement result.Immunocyte can swallow metabolite, and can secrete abundant interleukins,
TGF and all kinds of growth factors etc., including TGF-β, EGF, FGF, IL-2 etc., can promote cell with repairing for organizing
It is multiple, moreover it is possible to eliminate pigmentation, promote wound healing, preventing from scar.And autologous PRP contains abundant blood platelet and various types of cells
The factor, can speed up the repair of fibroblast, vascular endothelial cell to skin histology.
But existing platelet rich plasma（PRP）Deng cosmetic formulation, the effect of its smoothing wrinkle is not obvious enough, particularly checks colors
The fading effect of spot is poor.Therefore, the smoothing wrinkle and desalination color spot effect of cosmetic formulation are further improved, is to need to solve at present
Problem.
The content of the invention
To solve problem of the prior art and defect, present invention aim at a kind of effective autologous beauty micropin system of offer
Agent and preparation method thereof, so as to reach effective smoothing wrinkle and desalination color spot, improves the effect of skin quality.
To achieve the above object, autologous beauty micropin preparation provided by the present invention, comprising autologous peripheral blood source
PRP, PMNC（PBMCs）Circulation fibroblast, endothelial progenitor cells and the immunocyte obtained by culture,
And autologous plasma.
To achieve the above object, a kind of preparation method of autologous beauty micropin preparation, comprises the following steps：
Step 1）：Centrifuging autologous peripheral blood, obtains autologous plasma layer and cellular layer；
Step 2）：Autologous plasma layer keeps its unactivated state by obtaining platelet rich plasma after centrifugal concentrating, and to blood plasma
Low-temperature storage is carried out with platelet rich plasma.
Step 3）：Cellular layer further separates acquisition human peripheral blood single nucleus cell；
Step 4）：The human peripheral blood single nucleus cell of separation is placed in the cell culture medium containing activation cytokines solution,
Carry out Fiber differentiation；
Step 5）：Culture 12-36h, is collected by centrifugation cell, resuspended with the fresh cell culture medium containing activation cytokines solution
Cell, continues to cultivate；
Step 6）：Culture collects cell to 100-140h, with autologous plasma re-suspended cell and adds the rich platelet blood of preservation
Slurry, that is, obtain this autologous beauty micropin preparation.
Preferably, in the step 1, following steps are specifically included：Collection autologous peripheral venous blood, 300 ~ 500 g, room
Temperature centrifugation 10 ~ 20 minutes, after centrifugation terminates, collects plasma layer and is put into new centrifuge tube using pasteur pipet.
Preferably, in the step 2, will containing the g of hematoblastic autologous plasma 1000 ~ 2000, room temperature centrifugation 10 ~
20 minutes.Upper serum is collected into new centrifuge tube, is separated with lower sediment（Retain bottom 3-5ml serum during separation to exist
Do not taken out in pipe）.Remaining 3-5ml blood plasma must concentrate PRP with pellet platelets are resuspended in pipe.
Preferably, in the step 3, with physiological saline two-fold dilution lower floor haemocyte, add and contain lymphocyte separation medium
Centrifuge tube in, 500 ~ 800 g are centrifuged 15 ~ 25 minutes, draw tunica albuginea layer, with brine 2 times, that is, obtain outer
All blood mononuclear cell PBMCs.
Preferably, in the step 4, culture medium based on cell culture medium.
Preferably, in the step 4, the density of the PBMCs of resuspended rear initial incubation is 0.5 ~ 2.0 × 106/ml。
Preferably, in the step 4, activating cell growth factor solution compound method is to add in every ml cell culture mediums
Enter 30 μ l activating cell growth factors.
Preferably, in the step 4, activation cytokines are basic fibroblast growth factor, vascular endothelial growth
The factor and stem cell factor.
Preferably, in the step 6, collecting cell mainly includes circulation fibroblast, and endothelial progenitor cells are immunized thin
Born of the same parents.
Additionally, preservation and application method present invention also offers the beauty micropin preparation, including：This beauty micropin system
The storage temperature of agent is 2-8 DEG C.This beauty micropin preparation can apply to cure U.S. product, using in skin surface, dosage
It is 0.1-3ml/cm2。
The advantage of the invention is that：
（1）The present invention uses autogenous cell and PRP, and preparation method is simple, and with low cost, security is higher.
（2）Using activation cytokines solution treatment cell, Fiber differentiation circulation fibroblast is to skin for the present invention
The reconstruction of tissue, recovers its globality, and can secrete collagen, recovers skin elasticity, plays filling and the effect dispelled；
（3）Using activation cytokines solution treatment cell, Fiber differentiation endothelial progenitor cells can be played and promote skin the present invention
Local microcirculation, improves skin-nourishing state, while immunocyte can be assisted to take away metabolite again, to dim heavy, color spot flesh
Skin problem has good effect.
（4）The present invention using activation cytokines solution treatment cell, Fiber differentiation immunocyte, immunocyte can
Phagocytosis metabolite, and abundant interleukins, TGF and all kinds of growth factors etc., including TGF- can be secreted
β, EGF, FGF, IL-2 etc., can promote the reparation of cell and tissue, moreover it is possible to eliminate pigmentation, promote wound healing, not stay
Scar.
（5）Autologous PRP contains abundant cell factor, can speed up fibroblast, vascular endothelial cell to skin group
The repair knitted.
（6）The present invention uses these four materials as beauty micropin preparation, the interaction between them, further lifting
Skin histology reconstruction, smoothing wrinkle, desalination pigment, improve the functions such as the colour of skin.
Brief description of the drawings
Accompanying drawing 1：For the PMNC picture that example 1 is separate；
Accompanying drawing 2：It is the culture of example 2 and 3 to the 3rd day and the cell picture of the 5th day；
Accompanying drawing 3：It is the cell streaming phenotypic analysis result of example 3；
Accompanying drawing 4：For the subject of example 4 uses the skin photo before this beauty micropin preparation；
Accompanying drawing 5：For the subject of example 4 uses the skin photo after this beauty micropin preparation 18 days；
Specific embodiment
The present invention is illustrated with example below, but the present invention is not intended to be limited thereto.All unreceipted specific bars in Examples below
The experimental technique of part is the operating instruction execution that the method for observing a usual practice and producer provide.
Example 1
This example 1 is to separate autologous PRP and PMNC PBMCs：
Detection in peripheral blood of patients underwent 20ml aseptically is gathered with 20ml syringes, liquaemin anti-freezing is used.By the periphery of anti-freezing
Blood is transferred in 50ml centrifuge tubes, 400g, is centrifuged 15 minutes, after centrifugation terminates, is collected plasma layer using pasteur pipet and is transferred to
New 15ml centrifuge tubes, draw on cellular layer 1-2 millimeters.Hematoblastic autologous plasma 1500g, 15 minutes will be contained.
Upper plasma is collected into new 15ml centrifuge tubes, is separated with lower sediment（Reservation bottom 3-5ml blood plasma is in pipe during separation
Do not take out）.Remaining 3-5ml blood plasma must concentrate PRP with pellet platelets are resuspended in pipe.Will concentration PRP and remaining blood plasma be placed in-
80 DEG C of preservations；With physiological saline two-fold dilution lower floor haemocyte, human lymphocyte separating liquid is with dilute blood according to 1:2 ratio
Add in centrifuge tube, 680g/ minute, be centrifuged 20 minutes, draw tunica albuginea layer, with brine 2 times, rotating speed is respectively
500g/ minutes, 410g/ minutes, it is centrifuged 7 minutes, obtains PMNC（PBMCs）.
Example 2
This example 2 is induced fibroblast, endothelial progenitor cells and immunocyte in vitro：
The PBMCs activating cell initial mediums that will be separate（Contain activation cytokines solution）It is resuspended, control cell density
1 × 106Cells/ml, during cell moved into T75 blake bottles, shakes up, be positioned over saturated humidity in CO2 incubators, 37 DEG C, 5%
CO2 concentration cultures.
After culture 2 days, 500g/ minutes, it is centrifuged 6 minutes, cell is collected, by cell precipitation activating cell initial medium
（Contain activation cytokines solution）It is resuspended, and be transferred in T75 blake bottles, shaking up, saturation is wet in being positioned over CO2 incubators
Degree, 37 DEG C, the continuation culture of 5%CO2 concentration.
Example 3
Example 3 is to carry out quality testing to the cell of above-mentioned culture
Morphology, purity, cell phenotype detection, cell quantity, motility rate, aseptic, endotoxin are carried out to the culture cell of the 3rd day
Deng.Concrete operations are comprised the following steps：Take 0.5ml cell liquid to be counted using cell counter, Trypan Blue meter Cell viability,
Flow cytomery cell phenotype（CD45, CD3, CD56, CD34, CD31, CD42b, vimentin（Vimentin））；Centrifugation
Cell supernatant is collected, using Mei Liai full automatic microorganism detecting system detection bacteriums and fungi, the examination of LONZA detection of mycoplasma
Agent box detects common mycoplasma, TAL detection endotoxin.After testing, at the 3rd day, cell quantity is 2.72 × 107cells（n
=20）, Trypan Blue meter Cell viability reaches 92.6%（n=20）, cell phenotype detection CD34+ cells reach 0.8%, wherein interior
Skin progenitor cells account for 84.7%, and circulation fibroblast accounts for 20.8%, and the ratio of immunocyte is 59.2%（n=20）, it is full-automatic
Microbiological analysis instrument detection bacterium, fungi are feminine gender（n=20）, mycoplasma pneumoniae testing result is feminine gender（n=20）, endotoxin
Content≤0.5EU/ml（n=20）.
Example 4
This example 4 is with autologous plasma re-suspended cell and adds autologous PRP, prepares this beauty micropin preparation.
After culture 5 days, 500g/ minutes, it is centrifuged 6 minutes, collects cell；The cell brine 2 times that will be collected,
500g/ minutes, it is centrifuged 6 minutes, abandoning supernatant.
By 37 DEG C of rapid autologous plasma 900g for thawing, it is centrifuged 10 minutes, supernatant is what is prepared for micropin preparation
Autologous plasma；The cell that will have been washed with the autologous plasma is resuspended, constant volume to 9ml, and the PRP that will be frozen is placed in 37 DEG C of water-baths
Recovery, takes the PRP after 3ml recoveries and is added in cell suspension, and shaking table is transferred into 2 5ml cryopreservation tubes after vibrating 20 minutes
In, every pipe 5ml, this is this beauty micropin preparation.Remaining preparation is transferred to 2ml cell cryopreservation tube interior sealings, and -20 DEG C preserve 6
Keep sample within individual month to be checked.
Example 5
This example is with subjects recruitment trial edition micropin preparation.
20 Chinese women subjects at age 30-60 Sui are recruited, trial edition beauty micropin preparation reports to every subject
The whole research process of this beauty micropin preparation, and sign Informed Consent Form.
Microneedle injection this beauty micropin preparation 9-10ml is used to 20 subject's faces, according to subject's face situation,
Assess each injection location dosage；Wherein each injection 0.2- in 0.5-1.5ml, tear ditch position or so is respectively injected at decree line position or so
0.3-1.2ml is respectively injected at 1ml, puppet line position or so, and nasion injection location 0.1-0.5ml, whole skin of face inject 3-
6ml.Continuous Observation one month, period does not use other skin-protection products, normal activity to note sun-proof.As For the illustrated example, Fig. 3 is
Using preceding skin photo, Fig. 4 is, using the skin photo after latter month of this beauty micropin preparation, by contrast, to note
After penetrating this beauty micropin preparation, skin becomes pale bright and clean, and microgroove is largely reduced.
Claims (10)
1. a kind of preparation method of autologous beauty micropin preparation, comprises the following steps：
Step 1）：Centrifuging autologous peripheral blood, obtains autologous plasma layer and cellular layer；
Step 2）：Autologous plasma layer keeps its unactivated state by obtaining platelet rich plasma after centrifugal concentrating, and to blood plasma
Low-temperature storage is carried out with platelet rich plasma；
Step 3）：Cellular layer further separates acquisition human peripheral blood single nucleus cell；
Step 4）：The human peripheral blood single nucleus cell of separation is placed in the cell culture medium containing activation cytokines solution,
Carry out Fiber differentiation；
Step 5）：Culture 12-36h, is collected by centrifugation cell, resuspended with the fresh cell culture medium containing activation cytokines solution
Cell, continues to cultivate；
Step 6）：Culture collects cell to 100-140h, with autologous plasma re-suspended cell and adds the rich platelet blood of preservation
Slurry, that is, obtain this autologous beauty micropin preparation.
2. according to right 1 autologous beauty micropin preparation preparation method, it is characterised in that the step 1）Specifically include：
Collection autologous peripheral venous blood, 300-500g, room temperature are centrifuged 10-20 minutes.
3. according to right 1 autologous beauty micropin preparation preparation method, it is characterised in that the step 1）In, centrifugation knot
Shu Hou, collects plasma layer and is put into new centrifuge tube, for red cell volume reaches in the platelet rich plasma for making preparation using pasteur pipet
To minimum, draw on cellular layer 0.5-3 millimeters.
4. according to right 1 autologous beauty micropin preparation preparation method, it is characterised in that the step 2）In, blood will be contained
The autologous plasma 1000-2000g of platelet, room temperature is centrifuged 10-20 minutes；Upper plasma is collected into new centrifuge tube, with
Layer precipitate and separate, retains bottom 3-5ml blood plasma and is not taken out in pipe during separation, remaining 3-5ml blood plasma sinks with blood platelet in pipe
Shallow lake is resuspended must to concentrate platelet rich plasma.
5. according to right 1 autologous beauty micropin preparation preparation method, it is characterised in that the step 2）In, will concentrate
The low temperature environment that concentration platelet rich plasma is placed in less than -80 DEG C with remaining blood plasma is preserved.
6. according to right 1 autologous beauty micropin preparation preparation method, it is characterised in that the step 3）In, use physiology
Salt solution two-fold dilution lower floor haemocyte, adds in the centrifuge tube containing lymphocyte separation medium, and 500-800g is centrifuged 15-25 minutes,
Tunica albuginea layer is drawn, with brine, that is, PMNC is obtained.
7. according to right 1 autologous beauty micropin preparation preparation method, it is characterised in that the step 4）In, cell training
Support culture medium based on base；After re-suspended cell, the density of initial incubation PMNC is 0.5-2 × 106cells/
ml；Activating cell growth factor solution compound method is that 30 μ l activating cell growth factors are added in every milliliter of cell culture medium；
The activating cell growth factor be basic fibroblast growth factor, VEGF and stem cell growth because
Son.
8. according to right 1 autologous beauty micropin preparation preparation method, it is characterised in that the step 6）In, use 10-
14ml autologous plasmas and the resuspended fibroblast of platelet rich plasma, endothelial progenitor cells and immunocyte, obtain beauty micropin system
Agent；The temperature that the cosmetic formulation is preserved is 2-8 DEG C.
9. the autologous beauty micropin preparation that prepared by the preparation method of the autologous beauty micropin preparation of claim 1-8 any one, should
For curing U.S. product.
10. application method according to claim 9, it is characterised in that the dosage of the beauty micropin preparation is
0.1-3ml/cm2。
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108815114A (en) * | 2018-07-13 | 2018-11-16 | 深圳市润科生物科技有限公司 | A kind of beauty injection and preparation method thereof |
| CN109288868A (en) * | 2018-10-31 | 2019-02-01 | 广州市天河诺亚生物工程有限公司 | M-PRP store method, preparation method and applications |
| CN110484492A (en) * | 2019-08-27 | 2019-11-22 | 广州准优生物科技有限公司 | Endothelial progenitor cells culture preparation, culture solution and endothelial progenitor cells isolated culture method |
| CN111110623A (en) * | 2019-06-28 | 2020-05-08 | 青岛华澳现代医疗美容有限公司 | Self-repairing small needle nutrient solution formula |
| CN111214431A (en) * | 2020-02-28 | 2020-06-02 | 深圳市芙丽嘉生物科技有限公司 | Composition for promoting skin recovery and application thereof |
| CN112553158A (en) * | 2020-12-14 | 2021-03-26 | 北京博奥体质宝健康科技有限公司 | Extraction and preparation process of multi-functional repaired cell MDMC |
| CN113057965A (en) * | 2021-03-31 | 2021-07-02 | 河北康腾生物科技有限公司 | Activating and rejuvenating beauty liquid and preparation method and application thereof |
| CN114042086A (en) * | 2021-11-10 | 2022-02-15 | 张江平 | Autologous venous blood cell activation therapy |
| CN114983926A (en) * | 2022-07-01 | 2022-09-02 | 南方医科大学南方医院 | Soluble and detachable microneedle for promoting skin and hair regeneration and preparation method thereof |
| CN115125202A (en) * | 2022-07-08 | 2022-09-30 | 上海揽微赛尔生物科技有限公司 | Autologous blood cell factor and exosome serum and preparation method thereof |
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| CN104173252A (en) * | 2014-07-29 | 2014-12-03 | 蔡贤芬 | Biological beautifying preparation containing autologous stroma cells |
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| CN104173252A (en) * | 2014-07-29 | 2014-12-03 | 蔡贤芬 | Biological beautifying preparation containing autologous stroma cells |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108815114A (en) * | 2018-07-13 | 2018-11-16 | 深圳市润科生物科技有限公司 | A kind of beauty injection and preparation method thereof |
| CN108815114B (en) * | 2018-07-13 | 2021-06-15 | 深圳市润科生物科技有限公司 | Injection for beauty treatment and its preparation method |
| CN109288868A (en) * | 2018-10-31 | 2019-02-01 | 广州市天河诺亚生物工程有限公司 | M-PRP store method, preparation method and applications |
| CN111110623A (en) * | 2019-06-28 | 2020-05-08 | 青岛华澳现代医疗美容有限公司 | Self-repairing small needle nutrient solution formula |
| CN110484492A (en) * | 2019-08-27 | 2019-11-22 | 广州准优生物科技有限公司 | Endothelial progenitor cells culture preparation, culture solution and endothelial progenitor cells isolated culture method |
| CN111214431A (en) * | 2020-02-28 | 2020-06-02 | 深圳市芙丽嘉生物科技有限公司 | Composition for promoting skin recovery and application thereof |
| CN112553158A (en) * | 2020-12-14 | 2021-03-26 | 北京博奥体质宝健康科技有限公司 | Extraction and preparation process of multi-functional repaired cell MDMC |
| CN113057965A (en) * | 2021-03-31 | 2021-07-02 | 河北康腾生物科技有限公司 | Activating and rejuvenating beauty liquid and preparation method and application thereof |
| CN113057965B (en) * | 2021-03-31 | 2023-07-04 | 河北康腾生物科技有限公司 | Activating and rejuvenating beauty liquid and preparation method and application thereof |
| CN114042086A (en) * | 2021-11-10 | 2022-02-15 | 张江平 | Autologous venous blood cell activation therapy |
| CN114983926A (en) * | 2022-07-01 | 2022-09-02 | 南方医科大学南方医院 | Soluble and detachable microneedle for promoting skin and hair regeneration and preparation method thereof |
| CN114983926B (en) * | 2022-07-01 | 2024-01-26 | 南方医科大学南方医院 | Detachable soluble microneedle for promoting skin and hair regeneration and preparation method thereof |
| CN115125202A (en) * | 2022-07-08 | 2022-09-30 | 上海揽微赛尔生物科技有限公司 | Autologous blood cell factor and exosome serum and preparation method thereof |
| CN115125202B (en) * | 2022-07-08 | 2023-08-04 | 上海揽微赛尔生物科技有限公司 | Autologous blood cell factor and exosome serum and preparation method thereof |
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