CN109988747A - A kind of separation amplification method of adipose-derived fat stem cell and its application - Google Patents
A kind of separation amplification method of adipose-derived fat stem cell and its application Download PDFInfo
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Abstract
The present invention provides a kind of separation amplification methods of adipose-derived fat stem cell, it is chosen including adipose tissue, enzyme digestion obtains from adipose tissue fat stem cell, amplification fat stem cell, the separation amplification method cell amplification rate of fat stem cell provided by the invention is fast, in defined cell proliferation algebra, and available concentration is 1 × 106Cell/ml fat stem cell, and the fat injection liquid that a kind of application fat stem cell is used for chest enlarge is provided, when can effectively solve simple fat injection, the complication such as liquefaction of fat, necrosis of appearance.
Description
Technical field
The present invention relates to a kind of separation amplification methods of adipose-derived fat stem cell, and apply the fat stem cell
Fat injection liquid is prepared for chest enlarge.
Background technique
Being constantly progressive and develop with society, people in enriching oneself while for oneself appearance increasingly
Concern, however, some women are innately or with advancing age, mastatrophy becomes smaller or relaxes sagging, therefore passes through shaping hand
Art carries out chest enlarge and reforms into women ideal selection in the heart.
Chest enlarge, also referred to as enlarges the bosom or breast augmentation, is one kind that women is moulded to promote glamour for thorax shape
Behavior.Common breast enlarging method has following several: food chest enlarge, i.e., by adjusting dietary, borrows the nutrients in vegetables
Matter achievees the purpose that chest enlarge；Chest enlarge is moved, breast development is stimulated by daily exercise；Drug chest enlarge takes drugs carry out chest enlarge；
Prosthese chest enlarge achievees the purpose that chest enlarge to products such as chest filling gels；Autologous fat injection chest enlarge is obtained by liposuction method
Autologous adipose tissue carries out chest enlarge by injection.
First three chest enlarge mode, can complete, but effect varies with each individual, difference is larger at home.Latter two chest enlarge mode needs
It is completed in cosmetic surgery hospital.Prosthese chest enlarge is that silicone is placed in gap under pectoralis major by notch.This method damage location
Greatly, the convalescence after Miles operation is longer, and the several years need to separately be replaced, and the psychological financial burden of patient is heavier, it is postoperative need to long-term massaging breasts,
Permanent protection breast is not by wound etc..Autologous fat injection chest enlarge is easy to appear the complication such as liquefaction of fat, necrosis, causes
Adverse consequences.
In conclusion method used at present all safe and effective cannot fundamentally solve the problems, such as proprietary chest enlarge.
Stem cell be it is a kind of there is self-renewing, the multipotential cell of Multidirectional Differentiation, have regenerate various histoorgans and
The potential function of human body, medical field are known as " general-purpose cell ".Wherein fat stem cell (adipos derived Mesnchymal
Stem cells, ADSCs) from adipose tissue, compared to other stem cells, ADSCs has the significant excellent of following five aspect
Gesture: (one) abundance, materials are easy.(2) it is easy to expand, is not easy aging.It is demonstrated experimentally that repeatedly cell growth is fast after passage
Degree can continue to keep stability structurally and functionally also without trend is slowed down.(3) have the function of paracrine, promotion blood can be secreted
The correlation factor that pipe generates promotes revascularization.(4) there is lower immunogenicity.(5) ADSCs enters through hematological system
After human body, have the function of improving whole body well, the flexibility and tolerance, adjusting body of body can be improved significantly
Endocrine Levels and improve nervous system to the domination function of body.
The basic skills of fractionation of fatty stem cell is from adipose tissue at present: firstly, to adipose tissue sample obtained
Product are washed, and haemocyte is removed；It is crushed, digests secondly, carrying out fat, remove other heteroproteose cells；Again, it removes indigested
Tissue, obtains the filtrate of fatty stem cell；Finally, centrifugation obtain fat stem cell, can be used for it is subsequent culture, passage and
It freezes.
The technical problem that the basic skills is faced includes: that fetal calf serum cannot be used to carry out cell amplification, so that subculture
Growth rate is slow, can not obtain the fat stem cell for needing quantity in defined algebra.
Summary of the invention
The object of the present invention is to provide a kind of separation amplification methods of adipose-derived fat stem cell to solve above-mentioned training
In the method for supporting cell amplification rate it is slow, in defined cell proliferation algebra, be unable to get the fat stem cell etc. for needing quantity
Technical problem, and the fat injection liquid that a kind of application fat stem cell is used for chest enlarge is provided.
The technical proposal adopted by the invention to solve the above technical problems is that: a kind of point of adipose-derived fat stem cell
From amplification method, include the following steps:
(1) adipose tissue is chosen: aseptically extracting 20~30ml of adipose tissue from femoribus internus or abdomen；
(2) enzyme digestion is from obtaining fat stem cell in adipose tissue: under superclean bench gnotobasis, in the fat of extraction
In tissue, 1~5mg/mL I-type collagen enzyme of two volumes is added, digestive juice is centrifuged by 37 degree of lower digestion 1h at 800 turns
Under, it is centrifuged 5 minutes, abandons supernatant, cell is resuspended with the DMEM/F12 culture medium containing EGF, bFGF and PRP；
(3) it expands fat stem cell: alkaline cell growth factor EGF, bFGF being added in DMEM/F12 culture medium and be rich in blood
The blood plasma of platelet, cultivates fat stem cell in 35~37 DEG C, 1~5% carbon dioxide incubator of concentration, and the alkaline cell is raw
The concentration of the long factor are as follows: EGF unit of activity is 50~200U/mL, and bFGF unit of activity is 100~500U/mL, described to be rich in blood
The blood plasma of platelet is the platelet concentrate that self whole blood obtains after being centrifuged, is added into DMEM/F12 culture medium, blood
Starching ultimate density is 5~20%.
Based on above technical scheme, the separation amplification method of fat stem cell provided by the invention, a kind of preferably method,
Include the following steps: under 1) aseptic condition, extracts adipose tissue from femoribus internus or abdomen, 5mg/ is added in example 1:2 by volume
The I-type collagen enzyme of mL, 37 degree of lower digestion 1h are centrifuged 5 minutes by digestive juice under 800 turns of centrifugations, abandon supernatant；
2) cell is resuspended with the DMEM/F12 culture medium containing EGF, bFGF and PRP, under 37 degree, 5% gas concentration lwevel,
It is cultivated in incubator 8 days, wherein EGF unit of activity is 50U/mL, and bFGF unit of activity is 100U/mL, and the concentration of PRP is 10%.
The present invention also provides a kind of applications of the fat stem cell of above method preparation, by the fat stem cell of amplification and newly
Fresh adipose tissue mixes, and is prepared into fat injection liquid, and final concentration of the 1 of fat stem cell in the fat injection liquid being prepared into ×
106Cell/ml.
It should be noted that blood plasma (PRP), the epidermis that autologous platelet rich is added in the present invention in basal medium are thin
The intracellular growth factor (EGF) and basic fibroblast growth factor (bFGF) are rich in blood for the amplification of fat stem cell self
The blood plasma of platelet growth factor rich in, i.e. platelet derived growth factor, transforminggrowthfactor-β1 and β 2, para-insulin
Growth factor, vascular endothelial growth factor, nerve growth factor, epithelial cell growth factor etc..Growth factor is added simultaneously,
EGF and bFGF will preferably expand fat stem cell.
The separation amplification method cell amplification rate of fat stem cell provided by the invention is fast, in defined cell proliferation generation
In number, available concentration is 1 × 106Cell/ml fat stem cell, and a kind of application fat stem cell is provided for rich
The fat injection liquid of chest, when can effectively solve simple fat injection, the complication such as liquefaction of fat, necrosis of appearance, fat is done thin
Born of the same parents injection after, cytokine profiles can be generated, as vascular endothelial growth factor, hepatocyte growth factor, insulin-like growth because
Son, platelet derived growth factor and transforming growth factor etc., these growth factors not only can promote revascularization, and can improve new
The microenvironment of adipose tissue is injected, guarantees the nutrition supply of new injection adipose tissue, survives it, complete the weight of chest enlarge and chest
Modeling.It is natural, mellow and full with chest enlarge effect, the features such as having no toxic side effect.Using autologous fat stem cell chest enlarge, it is only necessary to disposable to move
Plant the effect of can reach chest enlarge.The method wound is small, and burden is light, without obvious notch, safely and effectively；Extracting autologous fat simultaneously
While as chest enlarge material, can also reduce weight body beautification.
Detailed description of the invention
Fig. 1 shows the screening curve figures of optimum N GF concentration；
Fig. 2 expression, the screening curve figure of best bFGF concentration；
Fig. 3 is indicated, under identical condition of culture, the fat stem cell growth curve of different culture medium culture.
Specific embodiment
1 material and method
1.1 material
20~the 30ml of adipose tissue for extracting abdomen or femoribus internus is provided by Xi'an Li Zun cosmetic surgery hospital；I-type collagen,
EGF, bFGF are purchased from sigma；DMEM/F12, FBS are purchased from Gibco；Cell counting Kit has purchased from Xi'an He Te biotechnology
Limit company；The serum free medium of fat stem cell is purchased from STEMCELL；PRP is the blood platelet that self whole blood obtains after being centrifuged
Concentrate.
1.2 are analyzed with EXCEL statistical software, carry out variance analysis, and as p < 0.05, difference has statistical significance.
Embodiment 1: fat stem cell is separately cultured.In operating room, abdomen or femoribus internus 20~30ml of fat are taken, it will
It is stored in sterile saline, with cold chain transportation to laboratory.On the hundred-grade super-clean workbench in the laboratory GMP, in rouge
In fat tissue, 5mg/mL I-type collagen is added by the volume ratio of 1:2, digests 1h under 37 degree, 800 turns of digestive juice is centrifuged
5 minutes, supernatant is abandoned, cell is resuspended with the DMEM/F12 culture medium containing EGF, bFGF and PRP, is added to T25 cell culture
In bottle, cultivate in 37 degree, 5% carbon dioxide incubator to P1 generation.
Embodiment 2: experiment is divided into five groups, A group (DMEM/F12+ 10%PRP+10U/mL EGF), B group (DMEM/F12+
10%PRP+20U/mL EGF), C group (DMEM/F12+ 10%PRP+50U/mL EGF), D group (DMEM/F12+ 10%PRP
+ 100U/mL EGF), E group (DMEM/F12+ 10%PRP+200U/mL EGF).
It by P1 for cell, after centrifugation, is resuspended with the DMEM/F12 culture medium containing 10%PRP, adjusts density to 5 × 104
A/mL is inoculated in 5 piece of 24 orifice plate with 500 holes μ L/.According to grouping, in corresponding hole, the EGF of various concentration is added, often
24 holes of group.5 piece of 24 orifice plate is then put into 37 degree, is cultivated in 5% carbon dioxide incubator, and in 1 d, 2 d, 3 d, 4
D, 5 d, 6 d, 7 d, 8d, totally 8 time points, each time point take the cell in 3 holes in each group to carry out cell count.
Experimental result shows that C group cell proliferation rate is most fast.The optium concentration for showing EGF is 50U/mL.
Embodiment 3: experiment is divided into five groups, A group (DMEM/F12+ 10%PRP+20U/mL bFGF), B group (DMEM/
F12+ 10%PRP+50U/mL bFGF), C group (DMEM/F12+ 10%PRP+100U/mL bFGF), D group (DMEM/F12+
10%PRP+200U/mL bFGF), E group (DMEM/F12+ 10%PRP+500U/mL bFGF).
It by P1 for cell, after centrifugation, is resuspended with the DMEM/F12 culture medium containing 10%PRP, adjusts density to 5 × 104
A/mL is inoculated in 5 piece of 24 orifice plate with 500 holes μ L/.According to grouping, in corresponding hole, the bFGF of various concentration is added, often
24 holes of group.5 piece of 24 orifice plate is then put into 37 degree, is cultivated in 5% carbon dioxide incubator, and in 1 d, 2 d, 3 d, 4
D, 5 d, 6 d, 7 d, 8d, totally 8 time points, each time point take the cell in 3 holes in each group to carry out cell count.
Experimental result shows that C group cell proliferation rate is most fast.The optium concentration for showing bFGF is 100U/mL.
Embodiment 4: experiment is divided into three groups, A group (serum free medium of fat stem cell), B group (DMEM/F12+10%
FBS), C group (DMEM/F12+ 10%PRP+50U/mL EGF, 100U/mL bFGF).
P1 is divided into three parts, after centrifugation for cell, according to grouping situation, is resuspended with corresponding culture medium, adjusts density to 5
×104A/mL is inoculated in 3 piece of 24 orifice plate with 500 holes μ L/, every group of 24 holes.3 piece of 24 orifice plate is then put into 37 degree, 5%
It is cultivated in carbon dioxide incubator, and in 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8d, totally 8 time points, each
Time point takes the cell in 3 holes in each group to carry out cell count.
It is the results show that three groups of cells start to be proliferated and enter increased logarithmic phase after culture 2d.Wherein B group is in culture 3
Cell Proliferation is slightly fast between ~ 6d, and A group and C the group cell Proliferation between cultivating 3~6d are slightly slow, but after three groups of cell culture 7d, without aobvious
It writes sex differernce (p < 0.01).
Cell growth curve experiment confirms, using C group (DMEM/F12+ 10%PRP+50U/mL EGF, 100U/mL
BFGF) culture medium can effectively facilitate the proliferation of cell, growth rate and A group (serum free medium of fat stem cell), B group
(DMEM/F12+10%FBS) quite.A group is the serum free medium for the fat stem cell that STEMCELL company develops, and causes to make
Valence is excessively high, and can not promote the use of a large area.Contain fetal calf serum in B group culture medium, in the fat stem cell training being transfused to people
In supporting, it is not recommended that use allogeneic serum.Cell growth curve the experimental results showed that, in the DMEM/F12 culture medium containing 10%PRP
Middle addition 50U/mL EGF, 100U/mL bFGF, carry out fat stem cell culture, and effect is similar to fetal calf serum is added.
Embodiment 5: using containing 50U/mL EGF, 100U/mL bFGF, 10%PRP DMEM/F12 culture medium to fat
Stem cell carries out amplification cultivation.Aseptically, adipose tissue and fat stem cell are mixed, makes the concentration of fat stem cell
Reach 1 × 106Cell/ml.According to udder shape, injection point is chosen respectively at breast bottom, middle part and top.Using self
Fat stem cell chest enlarge, it is only necessary to the effect of disposable transplanting can reach chest enlarge.The method wound is small, and burden is light, without obvious notch,
Safely and effectively.
The present invention relates to acquisition 20~30ml of volunteer's autologous fat, are used for fractionation of fatty stem cell, with the aforedescribed process
Expand fat stem cell.Volunteer's autologous fat 200~250ml of cell is acquired again, it is uniformly mixed with fat stem cell,
The final concentration of fat stem cell is set to reach 1 × 106Cell/ml.According to udder shape, distinguish at breast bottom, middle part and top
Choose injection point.
After mixing from the fat of the fat stem cell and extraction that separate in adipose tissue, injection chest enlarge is carried out.Can have
When effect solves simple fat injection, the complication such as liquefaction of fat, necrosis of appearance.After fat stem cell injection, it can generate a variety of
Cell factor, as vascular endothelial growth factor, hepatocyte growth factor, insulin-like growth factor, platelet derived growth factor and
Transforming growth factor etc., these growth factors not only can promote revascularization, and can improve the micro-loop of new injection adipose tissue
Border guarantees the nutrition supply of new injection adipose tissue, survives it, complete chest enlarge and the remodeling of chest.Certainly with chest enlarge effect
So, mellow and full, the features such as having no toxic side effect.Using autologous fat stem cell chest enlarge, it is only necessary to disposable transplanting can reach chest enlarge it
Effect.The method wound is small, and burden is light, without obvious notch, safely and effectively；Autologous fat is being extracted as the same of chest enlarge material simultaneously
When, can also reduce weight body beautification.
Claims (4)
1. a kind of separation amplification method of adipose-derived fat stem cell, which comprises the steps of:
Adipose tissue is chosen: aseptically extracting 20~30ml of adipose tissue from femoribus internus or abdomen；
Enzyme digestion is from obtaining fat stem cell in adipose tissue: under superclean bench gnotobasis, at fatty group of extraction
In knitting, 1~5mg/mL I-type collagen enzyme of two volumes is added, digestive juice is centrifuged by 37 degree of lower digestion 1h at 800 turns
Under, it is centrifuged 5 minutes, abandons supernatant, cell is resuspended with the DMEM/F12 culture medium containing EGF, bFGF and PRP；
It expands fat stem cell: epithelical cell growth factor (EGF), basic fibroblast being added in DMEM/F12 culture medium
Growth factor (bFGF) and blood plasma (PRP) rich in blood platelet, are trained in 35~37 DEG C, 1~5% carbon dioxide incubator of concentration
Support fat stem cell, the concentration of the alkaline cell growth factor are as follows: EGF unit of activity is 50~200U/mL, bFGF vigor list
Position is 100~500U/mL, and the blood plasma rich in blood platelet is the platelet concentrate that self whole blood obtains after being centrifuged, will
It is added in DMEM/F12 culture medium, and blood plasma ultimate density is 5~20%.
2. a kind of separation amplification method of adipose-derived fat stem cell, which comprises the steps of: 1) sterile item
Under part, adipose tissue is extracted from femoribus internus or abdomen, the I-type collagen enzyme of the 5mg/mL of example 1:2 addition by volume, 37 degree
Lower digestion 1h is centrifuged 5 minutes by digestive juice under 800 turns of centrifugations, abandons supernatant；
2) cell is resuspended with the DMEM/F12 culture medium containing EGF, bFGF and PRP, under 37 degree, 5% gas concentration lwevel,
It is cultivated in incubator 8 days, wherein EGF unit of activity is 50U/mL, and bFGF unit of activity is 100U/mL, and the concentration of PRP is 10%.
3. the application of any one claim fat stem cell obtained expanded, feature exist according to claim 1~2
In the fat stem cell of amplification and fresh fat tissue are mixed, fat injection liquid is prepared into.
4. the application of the fat stem cell of the amplification according to made from claim 3, which is characterized in that described to be prepared into fat injection
Liquid, final concentration of the 1 × 10 of fat stem cell6Cell/ml.
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