CN109536441A - A kind of culture medium and preparation method thereof of fatty stem cell secretion object - Google Patents
A kind of culture medium and preparation method thereof of fatty stem cell secretion object Download PDFInfo
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- CN109536441A CN109536441A CN201811464152.1A CN201811464152A CN109536441A CN 109536441 A CN109536441 A CN 109536441A CN 201811464152 A CN201811464152 A CN 201811464152A CN 109536441 A CN109536441 A CN 109536441A
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Abstract
The invention discloses a kind of preparation methods of the culture medium of fatty stem cell secretion object comprising following steps: 1) takes the fat stem cell of the third generation with 1 × 106It is a to be inoculated in the coated Tissue Culture Dish of gelatin, supernatant liquid is discarded after cell is adherent, PBS is washed three times；2) base culture base 48h is added, is filtered after being centrifuged, obtains the culture medium of fatty stem cell secretion object.Culture medium of the invention overcomes traditional fatty stem cell that will now match and is applied to the cell therapy test unhandy various drawbacks of process, it combines fatty stem cell secretion object with culture medium, realize preservation it is convenient, can in batches using, without temporarily now matching, avoiding Liquid nitrogen storage to need recovery etc. in due course, and can effectively reduce contaminated possibility when cell during test shifts, be conducive to the application in practice in production.
Description
Technical field
The present invention relates to bioengineering field, in particular to a kind of culture medium and preparation method thereof.
Background technique
Fat stem cell (adipose-derived stem cells, ADSCs) is that a kind of multipotency is dry thin in adipose tissue
Born of the same parents, rich content are easily isolated, and donor site is easily accepted by patients, the mescenchymal stem cell pursued in recent years as heat, again
It has had received widespread attention and has applied in raw medicine and clinical position.Research shows that ADSCs can enhance cell Proliferation, migration
Vigor, improve damaged cell vitality, antagonism Apoptosis.
Existing research shows that fat stem cell have the function of promote union of wounded skin, can also with delay skin aging,
Clinically fat stem cell is introduced into cell therapy and has obtained certain effect, however because cell therapy to cell consumption amount very
Greatly, and scholar even clearly proposes that ADSCs has potential carcinogenicity, injection cell is not optimal method.Therefore
How preferably to play the use of fat stem cell clinically is current problem to be solved.
Human skin fibroblasts play very crucial effect in skin biology, can not only promote the surface of a wound
Healing, and important role is also play during keeping the integrality and rejuvenation of skin.Studies have shown that ADSCs
Have the function of powerful paracrine, can produce a variety of growth factors, activation closes on cell or tissue and generates corresponding biology
Effect, has obvious action in terms of anti-aging, such as can promote the secretion of fibroblast proliferation and collagen, make intradermal
Collagen content increases, a large amount of new extracellular matrix components of secretion, so that original skin corium elastic fibers break is repaired, weight
Build, repair skin texture, reach dermis of skin thicken, skin elasticity increase, so as to improve skin texture.
How to have under the premise of avoiding potential risk brought by direct injection fat stem cell by fat stem cell
Effect improves the activity of skin fibroblasts, is conducive to skin regeneration, becomes a big problem of urgent need to resolve.
Summary of the invention
It is an object of the invention in view of the above shortcomings of the prior art, provide a kind of training of fatty stem cell secretion object
The preparation method of base is supported, the culture medium obtained has the substitution effect of cell culture medium, is also beneficial to skin regeneration, avoids straight
A possibility that connecing potential risk brought by injection fat stem cell, increasing clinical application.
The technical solution used in the present invention is: a kind of preparation method of the culture medium of fatty stem cell secretion object,
Include the following steps:
1) take the fat stem cell of the third generation with 1 × 106It is a to be inoculated in the coated Tissue Culture Dish of gelatin, it is adherent to cell
After discard supernatant liquid, PBS is washed three times；
2) base culture base 48h is added, is filtered after being centrifuged, obtains the culture medium of fatty stem cell secretion object.
As a further improvement of the foregoing solution, basal medium described in step 2) is by mass percentage by 10%
Fetal calf serum and 90% DMEM composition.
As a further improvement of the foregoing solution, the centrifugal speed of centrifugation described in step 2) is 4000rpm, centrifugation time
For 10min.
As a further improvement of the foregoing solution, it is filtered into described in step 2) and is filtered through 0.22 μm.
The object of the invention is also to provide a kind of culture mediums of fatty stem cell secretion object, by making as described above
Preparation Method preparation gained.
The beneficial effects of the present invention are: culture medium of the invention overcomes and traditional will now be applied to fatty stem cell
Cell therapy tests the unhandy various drawbacks of process, and fatty stem cell secretion object is combined with culture medium, realizes
Save it is convenient, can in batches using, without temporarily now matching, avoiding Liquid nitrogen storage to need recovery etc. in due course, and can effectively reduce examination
Contaminated possibility when cell shifts during testing is conducive to the application in practice in production.
Detailed description of the invention
Fig. 1 is the human adipose-derived stem cell of the embodiment of the present invention 2 into rouge Osteoblast Differentiation result figure, and wherein Figure 1A is into rouge point
Change figure, Figure 1B is Osteoblast Differentiation figure；
Fig. 2 is the cellular morphology figure of the human skin fibroblasts of the embodiment of the present invention 4, and wherein Fig. 2A is ordinary culture medium
Skin fibroblasts aspect graph after cultivating 72h, Fig. 2 B are the skin fibroblasts shape that 72h is co-cultured with fat stem cell
State figure, Fig. 2 C are the skin fibroblasts aspect graph that 72h is co-cultured with fat stem cell cultivation of cervical secretions base.
Fig. 3 is the beta galactosidase coloration result figure of the human skin fibroblasts of the embodiment of the present invention 4, wherein Fig. 3 A
For the result figure of ordinary culture medium group, Fig. 3 B is the skin fibroblasts beta galactose glycosides that 72h is co-cultured with fat stem cell
Enzyme dyeing result figure, Fig. 3 C are the coloration result figure of fat stem cell cultivation of cervical secretions base culture group；
Fig. 4 is each resulting cell beta galactosidase staining cell number of three kinds of training methods in the embodiment of the present invention 4
Comparison result.
Specific embodiment
The present invention is specifically described below with reference to embodiment, in order to technical field personnel to of the invention
Understand.It is necessary to it is emphasized that embodiment is only intended to, the present invention will be further described herein, should not be understood as to this
The limitation of invention protection scope, fields person skilled in the art, the non-intrinsically safe that the present invention is made according to foregoing invention content
The modifications and adaptations of property, should still fall within protection scope of the present invention.Mentioned raw materials following simultaneously are unspecified, are
Commercial product；The processing step or preparation method not referred in detail be processing step known to a person skilled in the art or
Preparation method.
Embodiment 1: separation and culture human adipose-derived stem cell
Be separately cultured people ADSCs: fat carries out the volunteer of liposuction from beauty hospital.Following procedure is sterile
Under the conditions of operate: take liposuction obtain people's abdominal adipose tissue, be added PBS solution cleaning, 2000rpm be centrifuged 5min, removal
Upper layer fat drips, take underlying adipose tissue, are added 0.1% I-type collagen enzyme solutions of the preheating newly prepared in equal volume, and 37 DEG C
60min is digested, until seeming more smooth, is centrifuged, carefully removes upper layer grease and lower layer from top to bottom with pipette
Collagenase solution, then be added PBS solution be resuspended precipitating, dispel, by it is postdigestive tissue with 200 mesh cell strainers filtering,
1200rpm is centrifuged 10min, abandons supernatant, the resuspension of its complete medium (DMEM/F12 containing 10% fetal calf serum) is added, then connect
Kind carries out cell secondary culture in culture plate.
Embodiment 2: the Multidirectional Differentiation identification of human adipose-derived stem cell
Human adipose-derived stem cell when 1 cell secondary culture of Example to third time passes on, is seeded to six with equal densities
In wherein three holes of orifice plate, label 1,2,3, wherein 1,2 liang of hole is experimental group, 3 holes are control group, when cell grows to 80%
When converging, remove culture medium, then wash cell three times with PBS.
It will be added in 1 hole test group at rouge induction culture medium；2 hole test groups are added in Osteoblast Differentiation induced medium
In；It will be added in complete medium in 3 hole control groups.
Culture medium replacement completely in every 3~4 days.After induction 5 days, observes and pass through oil red O when generating a large amount of greases in 1 hole
Staining evaluation fat generative capacity passes through Alizarin red staining when observing that apparent a large amount of calcium tubercles occur in 2 holes after inducing 7 days
Measure osteogenic ability.It observes and takes pictures under an optical microscope, obtain differentiated result figure as shown in Figure 1, wherein Figure 1A is 1 hole
Breaking up at rouge for test group is schemed, and Figure 1B is the Osteoblast Differentiation figure of 2 hole test groups.
The results show that the human adipose-derived stem cell taken is external evoked to carry out into rouge and Osteoblast Differentiation.
Embodiment 3: the preparation method of the culture medium of fatty stem cell secretion object
1) take the fat stem cell of the third generation with 1 × 106A Tissue Culture Dish for being inoculated in the coated 100mm of gelatin, to
After cell is adherent, supernatant liquid is discarded, PBS is washed three times；
2) be added 7ml base culture base 48h, the basal medium by mass percentage by 10% fetal calf serum
DMEM with 90% is formed, and after 4000rpm is centrifuged 10min and 0.22 μm of filtering, obtains the culture of fatty stem cell secretion object
Base.
Embodiment 4: human adipose-derived stem cell secretion is to the fibroblastic effect of ageing skin
Experimental procedure；
1) culture of aging human skin fibroblasts: the human skin fibroblasts cultivated are cell line, reach 45
In generation, is hereinafter, be naturally-aged cell.Its used complete medium is the DMEM/F12 containing 10% fetal calf serum, is incubated at
37 DEG C, 5%CO2In incubator, ordinary culture medium group is obtained, skin fibroblasts aspect graph is as shown in Figure 2 A；
2) co-cultivation of human skin fibroblasts and fat stem cell:
By amniotic fluid stem cell 1 × 103A small interior for being placed in 0.4 μm of aperture Transwell plate, each cell are added
250ul complete medium is added 5 × 104500 μ are added in the lower layer for co-culturing plate, every hole in a aging human skin fibroblasts
L culture medium cultivates 72h, obtain with fat stem cell co-cultivation group, skin fibroblasts aspect graph is as shown in Figure 2 B；
3) culture medium of human skin fibroblasts and fatty stem cell secretion object co-cultures:
After the digestion centrifugation of aging human skin fibroblasts, cell precipitation is resuspended, by every hole 5 × 104A cell inoculation
In 24 orifice plates, 37 DEG C, 5%CO2Culture, after cell is adherent, every hole is changed to the fatty dry of the preparation of the embodiment of the present invention 3
The culture medium of cell secreta is cultivated 72h, is obtained and fat stem cell secretion co-cultivation group, skin fibroblasts shape
State figure is as shown in Figure 2 C；
4) β of the human skin fibroblasts of three kinds of training method cultures-galactoside enzyme dyeing:
Absorb respectively it is above-mentioned 1)~3) the kind resulting cell culture medium of training method, washed once, be added appropriate with PBS
SA- β-gal dyes fixer, and the fixed 15min of room temperature absorbs cell fixer, PBS washing is added three times, each 3min.It absorbs
PBS, is added appropriate cell dyeing working solution, and 37 DEG C of overnight incubations are observed under an optical microscope.It is in pale blue under senile cell mirror
To navy blue, percentage of the sample positive rate with positive sample in total number of samples is indicated.
The beta galactosidase coloration result figure of three groups of human skin fibroblasts is as shown in figure 3, wherein Fig. 3 A is general
The result figure of logical culture medium group, Fig. 3 B are to dye with the skin fibroblasts beta galactosidase of fat stem cell co-cultivation group
Result figure, Fig. 3 C are the skin fibroblasts beta galactosidase coloration result with fat stem cell secretion co-cultivation group
Figure.
The comparison result of the beta galactosidase staining cell number of three groups of human skin fibroblasts is as shown in Figure 4.
The culture medium of fatty stem cell secretion object prepared by the present invention contains growth factor, and experimental result shows that it can
To effectively improve the vigor of the human skin fibroblasts of aging, senile cell number is reduced, and its effect is no less than and fat
Stem cell co-cultivation group, it was demonstrated that it has the function of delay skin aging, can be applied to cosmetics and skincare product industry, reach and repair
Multiple skin texture, increases skin elasticity, so as to improve the purpose of skin texture.
Above-described embodiment is the preferred embodiment of the present invention, all with similar technique of the invention and made equivalence changes,
It should belong to protection category of the invention.
Claims (5)
1. a kind of preparation method of the culture medium of fatty stem cell secretion object, it is characterised in that include the following steps:
1) take the fat stem cell of the third generation with 1 × 106It is a to be inoculated in the coated Tissue Culture Dish of gelatin, it is abandoned after cell is adherent
Supernatant liquid is removed, PBS is washed three times；
2) base culture base 48h is added, is filtered after being centrifuged, obtains the culture medium of fatty stem cell secretion object.
2. a kind of preparation method of fatty stem cell secretion object according to claim 1, it is characterised in that: in step 2)
The basal medium is made of 10% fetal calf serum and 90% DMEM by mass percentage.
3. a kind of preparation method of fatty stem cell secretion object according to claim 1, it is characterised in that: in step 2)
The centrifugal speed of the centrifugation is 4000rpm, centrifugation time 10min.
4. a kind of preparation method of fatty stem cell secretion object according to claim 1, it is characterised in that: in step 2)
Described be filtered into is filtered through 0.22 μm.
5. a kind of culture medium of fatty stem cell secretion object is prepared by the described in any item preparation methods of Claims 1 to 4
Gained.
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Citations (5)
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| CN105820997A (en) * | 2015-01-06 | 2016-08-03 | 陈礼明 | Adipose-derived stem cell active ingredient and beauty preparation containing active ingredient |
| CN108865990A (en) * | 2018-07-25 | 2018-11-23 | 广州赛莱拉干细胞科技股份有限公司 | A kind of biological agent and preparation method thereof for skin injury reparation |
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2018
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| CN101461772A (en) * | 2009-01-07 | 2009-06-24 | 天津欧瑞生物科技有限公司 | Method for preparing stem cell secretion factor for beauty treatment and skin-protection |
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| CN105820997A (en) * | 2015-01-06 | 2016-08-03 | 陈礼明 | Adipose-derived stem cell active ingredient and beauty preparation containing active ingredient |
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