CN104762260A - Preparation method and application of adipose-derived mesenchymal stem cells and preparation thereof - Google Patents
Preparation method and application of adipose-derived mesenchymal stem cells and preparation thereof Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, and in particular relates to a preparation method and an application of adipose-derived mesenchymal stem cells and a preparation thereof. The preparation method comprises the following steps: fat is obtained; pretreatment, enzymolysis and centrifugation are carried out; precipitate is collected, such that the adipose-derived mesenchymal stem cells are obtained; the adipose-derived mesenchymal stem cells are cultured; when the adipose-derived mesenchymal stem cells grow to 80% fusion, the adipose-derived mesenchymal stem cells are mixed with EDTA-pancreatin for digestion; digestion is terminated, and centrifugation is carried out; and subculturing is carried out. The method provided by the invention has the following advantages: 1, the purity of cultured stem cells is high; 2, various factors secreted during an adipose-derived mesenchymal stem cell culturing process are recovered, and epidermal cell proliferation can be substantially promoted, which means that epidermal cell renewing and replacement can be accelerated, and a significant anti-aging effect can be achieved.
Description
Technical field
The present invention relates to biological technical field, particularly the preparation method of fat mesenchymal stem cell and preparation thereof and purposes.
Background technology
Fat stem cell (adipose-derived stem cells, ADSCs) is from fatty tissue, be separated a kind of stem cell with multi-lineage potential obtained in recent years.Research finds that ADSCs cell can stablize propagation in vitro and decline rate is low, simultaneously it have draw materials easily, a small amount of tissue can obtain a large amount of stem cell, suitable large scale culturing, to advantages such as body injury are little, and its wide material sources, cylinder storage amount is large, suitable autotransplantation, becomes one of new in recent years study hotspot gradually.Current fat stem cell mainly applies to the beauty industry such as chest enlarge, smoothing wrinkle in adipose tissue transplantation, and it is promoting that the survival rate of transplanting rear adipocyte improves a lot.Another fat stem cell, after external large scale culturing, activates stem cell function, promotes the cytokine such as secretion of VEGF, BFGF, IGF.
Aging be biology As time goes on, spontaneous necessary process, it is complicated spontaneous phenomenon, and show as structure and hypofunction, adaptability and resistibility go down.Essence is the process that the function of body parts tract fails gradually.
In normal physiological processes, human body has a large amount of necrocytosis every day, and simultaneously with the new life of a large amount of cell, to maintain the integrity of various tissue and organ, assurance function is in standard state, and what play cell neogenesis function is various stem cells in body.With advancing age, when the stem cell amount in body reduces, make the death of cell and newborn overbalance, stem cell cannot meet the demand of body to neonatal cell, will cause the going down of tissue organ function, disease occurs, even death.So in time in stem cell amount or activation in added body, dummy senile cell be antidotal important channel to maintain cells in vivo dead with the balance of new life.
Main transplanting plant and the protein induced property hemopoietic stem cell of adopting reaches antidotal object at present.But there is following shortcoming: 1, induce the mainly hemopoietic stem cell obtained, be functionally confined to blood system; 2, cultivate the hemopoietic stem cell purity that obtains at 5-40%, purity is too low.
Summary of the invention
In view of this, the invention provides preparation method and the purposes of a kind of fat mesenchymal stem cell and preparation thereof, the fat mesenchymal stem cell preparation of acquisition, fat mesenchymal stem cell high purity more than 90%, the function of stem cell can better be played, reach anti-ageing object.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of preparation method of fat mesenchymal stem cell, comprise the steps:
Step 1: obtain fat, pre-treatment, enzymolysis, centrifugal, collecting precipitation, obtains fat mesenchymal stem cell;
Step 2: cultivate described fat mesenchymal stem cell, when described fat mesenchymal stem cell grows to 80% fusion, abandons nutrient solution, add PBS cleaning, then add EDTA-trypsin solution digestion, stop digestion, centrifugal, Secondary Culture.
In specific embodiments more of the present invention, pre-treatment described in preparation method's step 1 of fat mesenchymal stem cell for add PBS washing, centrifugal.
Preferably, centrifugal condition is the centrifugal 5min of 1500rpm.
In specific embodiments more of the present invention, enzymolysis described in preparation method's step 1 of fat mesenchymal stem cell is employing 0.5% NTx enzyme, 37 DEG C, 100R digests 50min; The volume ratio of described NTx enzyme and described fat is 0.5 ~ 1:1.
In specific embodiments more of the present invention, cultivate described in preparation method's step 2 of fat mesenchymal stem cell and adopt perfect medium adjustment cell density to be 1 × 10
5cell/mL, adds 1 μ g/mL EGF, in 5%CO
2, 37 DEG C, saturated humidity is cultivate under the condition of 95%; Described perfect medium comprises DMEM/F12 and 10%FBS.
As preferably, clean, repeat 2 times described in step 2 after cultivating 48h with 10mL PBS, the cell suspension getting 10mL adds the 1ug/mL EGF of 100uL, in 5%CO
2, 37 DEG C, saturated humidity is continue under the condition of 95% to cultivate; The suspension of described fat mesenchymal stem cell and the volume ratio of described EGF are 10:0.1.
In specific embodiments more of the present invention, in the preparation method EDTA-trypsin solution of fat mesenchymal stem cell, the concentration of EDTA-pancreatin is 0.25w/v%, and the ratio of the adherent area of described fat mesenchymal stem cell and EDTA-trypsin solution is: every 10cm
2area add 0.5 ~ 1ml EDTA-trypsin solution, the time of described digestion is 1 ~ 2min.
As preferably, stop digestion described in step 2 and be specially and add FBS and stop digestion, the volume ratio of the suspension of FBS and described fat mesenchymal stem cell is 1:10.
As preferably, centrifugal described in step 2 is the centrifugal 5min of 1500rpm/min.
In specific embodiments more of the present invention, Secondary Culture described in the preparation method of fat mesenchymal stem cell is specially and adds perfect medium and EGF, in 5%CO
2, 37 DEG C, saturated humidity is cultivate under the condition of 95%; Described perfect medium comprises DMEM/F12 and 10%FBS; Described passage culture density is 1 × 10
5cell/mL.。
Present invention also offers a kind of preparation method of fat mesenchymal stem cell preparation, comprise the steps:
Step 1: the fat mesenchymal stem cell that the preparation method as described in any one of claim 1 to 6 obtains;
Step 2: by described fat mesenchymal stem cell Secondary Culture to P3 for time, collect supernatant liquor, change nutrient solution;
Step 3: repeating step 2, merges supernatant liquor, ultrafiltration and concentration, freeze-drying, obtains supernatant liquor lyophilized powder;
Step 4: by described fat mesenchymal stem cell Secondary Culture to P3 for time, through trysinization, collect obtain stem cell, with HClO
4solution lyses, centrifugal, collect supernatant liquor;
Step 5: supernatant liquor step 4 obtained neutralizes through alkali lye, centrifugal, collects supernatant liquor;
Step 6: supernatant liquor step 5 obtained mixes with gac, wash, after alkali lye wash-out, adjust ph is neutral, obtains stem cell extract;
Step 7: the stem cell extract that the supernatant liquor lyophilized powder and the step 6 that step 3 are obtained obtain mixes.
As preferably, step 3 ultrafiltration adopts the following particle diameter small molecules of 0.45um, 0.22um membrane filtration ultrafiltration system filter 23 kd.
As preferably, in the supernatant liquor lyophilized powder that step 3 obtains, in every 1mg, the content of protein factor is 0.5 ~ 0.8mg.
As preferably, in step 4, the concentration of pancreatin is 0.25w/v%, and the ratio of the adherent area of described fat mesenchymal stem cell and EDTA-pancreatin is: every 10cm
2area add 0.5 ~ 1ml EDTA-pancreatin.
As preferably, HClO in step 4
4massfraction be 40%, HClO
4the volume ratio of solution and described fat mesenchymal stem cell is 1:5.
As preferably, the number obtaining stem cell in step 4 is 1-1.2 × 10
8individual.
As preferably, condition centrifugal in step 4 is the centrifugal 5min of 1000rpm/min-1500rpm/min.
As preferably, in step 5, alkali lye is KOH solution, and concentration is 0.05mol/mL.
As preferably, condition centrifugal in step 5 is the centrifugal 5min of 1000rpm/min-1500rpm/min.
As preferably, in step 6, the concentration of stem cell extract is 5 ~ 20 μ g/mL.
In specific embodiments more of the present invention, the mass ratio of the stem cell extract that the supernatant liquor lyophilized powder of preparation method's step 3 acquisition of fat mesenchymal stem cell preparation and step 6 obtain is 50 ~ 100:1.
Flash liberation is cultivated, and can obtain 100 supernatant liquor lyophilized powders and 100ml stem cell extract solution; Often prop up lyophilized powder 0.5-2mg, every milliliter of stem cell extract solution is containing stem cell extract 5-20ug, and mix with 1ml stem cell extract solution after dissolving 1ml sterilized water according to 1 supernatant liquor lyophilized powder, the mass ratio obtained is 50-100:1 again.
As preferably, supernatant liquor lyophilized powder step 3 obtained is dissolved in sterilized water, obtained supernatant liquor diluent, and extension rate is 5 ~ 10 times, and the concentration of supernatant liquor diluent is 0.5-2mg/ml; The stem cell extract obtained with step 6 again mixes, the volume ratio of the stem cell extract that supernatant liquor diluent and step 6 obtain is preferably 1:1, and in the stem cell extract that the effective constituent of supernatant liquor diluent and step 6 obtain, the mass ratio of effective constituent is 5 ~ 8:1.
The fat mesenchymal stem cell preparation that the preparation method that present invention also offers above-mentioned fat mesenchymal stem cell preparation obtains.
Present invention also offers the application of above-mentioned fat mesenchymal stem cell preparation in the antidotal medicine of preparation, food, healthcare products, makeup.
The invention provides a kind of preparation method of fat mesenchymal stem cell, comprise the steps: to obtain fat, pre-treatment, enzymolysis, centrifugal, collecting precipitation, obtains fat mesenchymal stem cell; Cultivate described fat mesenchymal stem cell, when described fat mesenchymal stem cell grows to 80% fusion, with the mixture slaking of EDTA-pancreatin, stop digestion, centrifugal, Secondary Culture.Flow process as shown in Figure 1.Compared with prior art, the present invention has following advantage:
1, the stem cell purity obtained is cultivated high;
2, reclaim the oozy all kinds of factor in fat mesenchymal stem cell culturing process, significantly can promote the propagation of epidermic cell, show new life and the replacement that can accelerate epidermic cell, reach significant anti-aging effects.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 shows preparation method's schema of fat mesenchymal stem cell provided by the invention and preparation thereof;
Fig. 2 shows fat mesenchymal stem cell streaming qualification figure.
Embodiment
The invention discloses preparation method and the purposes of fat mesenchymal stem cell and preparation thereof, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In the preparation method of fat mesenchymal stem cell provided by the invention and preparation thereof and purposes, raw materials used and reagent all can be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1 fat mesenchymal stem cell extracts
1. obtain fatty solution, 4 DEG C of low temperature shift are in the super clean bench of laboratory;
2. evenly divided by fatty tissue in 50mL centrifuge tube, often pipe adds 0.5%I Collagenase Type, and after sealing, level is rocked back and forth, and fully mixes.The volume ratio of described NTx enzyme and described fat is 0.5 ~ 1:1.
3. be transferred in Tempeerature-constant air shaking table, 37 DEG C, 100R digests 50min; Add 1mLFBS with 1mL liquid transfer gun head and stop digestion, blow with pasteur pipet even, after centrifuge tube trim, put into whizzer, the centrifugal 5min of 1500rpm/min.
4. sop up supernatant liquor with pasteur pipet, often pipe adds 40mLPBS re-suspended cell, and get 20uL cell suspension cell counter and count, centrifuge tube trim is placed in whizzer, the centrifugal 5min of 1500rpm/min.
5. sop up centrifuged supernatant with pasteur pipet, according to cell counts, adjusting cell density with perfect medium (DMEM/F12+10%FBS) is 1 × 10
5cell/mL, is seeded in the culture dish of 10cm, and every ware adds the 1 μ g/mL EGF of 100 μ L.
6. carry out mark in the culture dish side of covering, be transferred to 5%CO
2, 37 DEG C, saturated humidity is cultivate in the cell culture incubator of 95%.
Embodiment 2 fat mesenchymal stem cell streaming is identified
As shown in Figure 2, from streaming result, fat mesenchymal stem cell surface markers
CD59+CD90+CD45-HLA-DR-is up to 99.7%.
Embodiment 3 fat mesenchymal stem cell is cultivated
After 1.48h, sop up substratum in the obtained ware of embodiment 1 with pasteur pipet, every ware adds 10mL PBS and cleans, and repeats 2 times.
2. inoculate the cell suspension of 10mL in the culture dish of 10cm, every ware adds the 1ug/mLEGF of 100uL.Cross rocks culture dish 5 times, makes cell suspension be uniformly distributed in culture dish, is transferred to 5%CO
2, 37 DEG C, saturated humidity is continue in the cell culture incubator of 95% to cultivate, every day observation of cell state taking pictures.
3. when Growth of Cells to 80% merges, remove nutrient solution, add 10mlPBS cleaning once, add the EDTA-trypsin solution digestion 1-2min of the 0.25w/v% of 4ml.In EDTA-trypsin solution, the concentration of EDTA-pancreatin is 0.25w/v%, and the ratio of the adherent area of described fat mesenchymal stem cell and EDTA-trypsin solution is: every 10cm
2area add 0.5 ~ 1ml EDTA-trypsin solution.
4. add 1mlFBS and stop digestion, draw Digestive system to 15ml centrifuge tube, the centrifugal 5min of 1500rpm/min
5. add perfect medium and be seeded to 10ml culture dish 1-2 ware, every ware adds the 1 μ g/mL EGF of 100 μ L.
6. carry out mark in the culture dish side of covering, be transferred to 5%CO
2, 37 DEG C, saturated humidity is cultivate in the cell culture incubator of 95%.
The preparation of embodiment 4 fat mesenchymal stem cell preparation
1. fat mesenchymal stem cell preparation divides two portions, supernatant liquor lyophilized powder part and stem cell extract part
2. supernatant liquor lyophilized powder part
The obtained fat mesenchymal stem cell of 2.1 examples to be performed 3 be cultured to P3 for time (ware of 15cm can be expanded to the 16-20 ware in P3 generation by 1 ware during inoculation), for three days on end, every day changes liquid, and collects supernatant liquor, can obtain 480-600ml supernatant liquor
2.2 supernatant liquors are through Sartocon slice 200 cross-flow ultrafiltration system thickening filtration, and obtained 100-150ml solution, through obtained 100 lyophilized powders of freeze-drying, often props up containing protein factor composition 0.5-2mg.
3. stem cell extract part
The fat mesenchymal stem cell that the embodiments 3 of 3.1 inoculations, 1 15cm ware are obtained, be cultured to P3 for time, cell can obtain 1-1.2 × 10 through collected by trypsinisation
8individual cell;
3.2 collect the HCLO4 solution lyses that the stem cell obtained with massfraction is 40%, and the centrifugal 5min of 1000rpm/min-1500rpm/min, gets supernatant liquor; HClO
4the volume ratio of solution and described fat mesenchymal stem cell is 1:5.
3.3 supernatant liquors take concentration as the KOH neutralization of 0.05mol/mL, and the centrifugal 5min of 1000rpm/min-1500rpm/min, gets supernatant liquor;
After 3.4 supernatant liquors mix with gac, wash with neutral distilled water, basic solution wash-out, regulate pH value to be neutral, obtain stem cell extract solution 100ml, extract concentrations is 5-20 μ g/ml, is product 1.
4. get 0.5-2mg lyophilized powder and be dissolved in sterilized water 1ml, obtained product 2 (lyophilized powder is the 100 pairs of lyophilized powders obtained in step 2)
Product 1 and product 2 press 1:1 mixing, are fat mesenchymal stem cell preparation provided by the invention.
The volume ratio of the stem cell extract that supernatant liquor diluent and step 6 obtain is preferably 1:1, and in the stem cell extract that the effective constituent of supernatant liquor diluent and step 6 obtain, the mass ratio of effective constituent is 5 ~ 8:1.
Embodiment 5 anti-aging effects is verified
The aging of people is exactly the aging of cell, and by cell cultures, prolongation cell proliferation multiple and passage number are to confirm its function of delaying senility.Proof scheme is as follows:
Get P1 for mouse epidermal cells number ware, centrifugal after digestion, be inoculated in 12 wares (10cm ware, every ware 5*10 respectively
5individual cell).Divide 4 groups, often organize 3 wares.
1 group is left intact, cellar culture;
2 groups are added product 1 in embodiment 4 is 2:1 with the ratio of product 2 in embodiment 4;
3 groups are added product 1 in embodiment 4 is 1:1 with the ratio of product 2 in embodiment 4;
4 groups are added product 1 in embodiment 4 is 1:2 with the ratio of product 2 in embodiment 4.
Respectively 12h, 24h often group get 1 ware cell dissociation counting, result is as shown in table 1:
Table 1 mouse epidermal cells culture experiment result
Can find out in mouse epidermal cells culture experiment, the cell concentration obtained in group 3 is apparently higher than other groups, and P < 0.05, has statistical significance.Visible, fat mesenchymal stem cell preparation provided by the invention can promote the propagation of epidermic cell, shows new life and the replacement that can accelerate epidermic cell, reaches anti-aging effects.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a preparation method for fat mesenchymal stem cell, is characterized in that, comprises the steps:
Step 1: obtain fat, pre-treatment, enzymolysis, centrifugal, collecting precipitation, obtains fat mesenchymal stem cell;
Step 2: cultivate described fat mesenchymal stem cell, when described fat mesenchymal stem cell grows to 80% fusion, abandons nutrient solution, add PBS cleaning, then add EDTA-trypsin solution digestion, stop digestion, centrifugal, Secondary Culture.
2. preparation method according to claim 1, is characterized in that, pre-treatment described in step 1 for add PBS washing, centrifugal.
3. preparation method according to claim 1 and 2, is characterized in that, enzymolysis described in step 1 is employing 0.5% NTx enzyme, 37 DEG C, 100R digests 50min; The volume ratio of described NTx enzyme and described fat is 0.5 ~ 1:1.
4. the preparation method according to any one of claims 1 to 3, is characterized in that, cultivates and adopt perfect medium adjustment cell density to be 1 × 10 described in step 2
5cell/mL, adds 1 μ g/mL EGF, in 5%CO
2, 37 DEG C, saturated humidity is cultivate under the condition of 95%; Described perfect medium comprises DMEM/F12 and 10%FBS.
5. the preparation method according to any one of Claims 1-4, is characterized in that, in described EDTA-trypsin solution, the concentration of EDTA-pancreatin is 0.25w/v%, and the ratio of the adherent area of described fat mesenchymal stem cell and EDTA-trypsin solution is: every 10cm
2area add 0.5 ~ 1ml EDTA-trypsin solution, the time of described digestion is 1 ~ 2min.
6. the preparation method according to any one of claim 1 to 5, is characterized in that, described Secondary Culture is specially and adds perfect medium and EGF, in 5%CO
2, 37 DEG C, saturated humidity is cultivate under the condition of 95%; Described perfect medium comprises DMEM/F12 and 10%FBS, and it is 1 × 10 that described passage cultivates inoculum density
5cell/mL.
7. a preparation method for fat mesenchymal stem cell preparation, is characterized in that, comprises the steps
Step 1: the fat mesenchymal stem cell that the preparation method as described in any one of claim 1 to 6 obtains;
Step 2: by described fat mesenchymal stem cell Secondary Culture to P3 for time, collect supernatant liquor, change nutrient solution;
Step 3: repeating step 2, merges supernatant liquor, ultrafiltration and concentration, freeze-drying, obtains supernatant liquor lyophilized powder;
Step 4: by described fat mesenchymal stem cell Secondary Culture to P3 for time, through trysinization, collect obtain stem cell, with HClO
4solution lyses, centrifugal, collect supernatant liquor;
Step 5: supernatant liquor step 4 obtained neutralizes through alkali lye, centrifugal, collects supernatant liquor;
Step 6: supernatant liquor step 5 obtained mixes with gac, wash, after alkali lye wash-out, adjust ph is neutral, obtains stem cell extract;
Step 7: the stem cell extract that the supernatant liquor lyophilized powder and the step 6 that step 3 are obtained obtain mixes.
8. preparation method according to claim 7, is characterized in that, the mass ratio of the supernatant liquor lyophilized powder that step 3 obtains and the stem cell extract that step 6 obtains is 50 ~ 100:1.
9. the fat mesenchymal stem cell preparation of preparation method's acquisition of fat mesenchymal stem cell preparation according to claim 8.
10. the application of fat mesenchymal stem cell preparation according to claim 9 in the antidotal medicine of preparation, food, healthcare products, makeup.
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