CN109097326A - A kind of method and its application preparing mescenchymal stem cell excretion body - Google Patents
A kind of method and its application preparing mescenchymal stem cell excretion body Download PDFInfo
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Abstract
The present invention provides a kind of methods for preparing mescenchymal stem cell excretion body, comprising: cleaning human umbilical tissue surface blood removes epidermis and vascular tissue, shreds after taking out huatong plastic cleaning, inoculated and cultured；Secondary culture；Second generation umbilical cord mesenchymal stem cells inoculated and cultured；The cell stimulation factor for promoting the secretion of excretion body is added；The first cell supernatant is sucked out when cell grows to 90% degrees of fusion for separating excretion body；First cell supernatant is centrifuged removal cell fragment, collects the second cell supernatant；Second cell supernatant is subjected to first time ultrafiltration concentration, excessive moisture is removed, obtains the first concentrate；First concentrate is carried out second to be concentrated by ultrafiltration, excessive moisture is removed, obtains the second concentrate；Second concentrate is subjected to first time ultracentrifugation, supernatant is taken to carry out second of ultracentrifugation, supernatant is removed, is precipitated as the excretion body of stem cell secretion；Physiological saline cleaning is added, centrifugation removes supernatant, obtains excretion body precipitating.
Description
Technical field
The invention belongs to stem cells and regenerative medicine field, and in particular to a kind of side for preparing mescenchymal stem cell excretion body
Method and its application.
Background technique
Excretion body (Exosome) is by intracellular vesicles structure release to a kind of extracellular film property vesica, diameter about 30-
150nm, cell can all secrete excretion body under normal physiological status, be believed by protein in vesica and RNA in iuntercellular
Number conduction and iuntercellular mutually regulate and control in play an important role, and this signal transduction and mutual regulatory mechanism complexity, especially
Between tumour cell, whether the excretion body of tumor cell secretion is to immunosupress, tumor cell migration, the increasing in tumor tissues
Grow and be impregnated with facilitation etc., it not yet makes clear completely now.
Excretion body is studied there are the separation and Extraction that a bottleneck problem urgently to be resolved is exactly excretion body, and existing method includes
Ultracentrifugation, immunological magnetic bead sorting etc..Ultracentrifugation is the most popular method for separating excretion body, but ultracentrifugation needs to configure
Ultracentrifuge, this equipment belongs to special equipment, expensive, and using complexity, and disengaging time is long, and fractional dose is few, often
It is secondary to separate a small amount of liquid, the centrifugation time of a few hours is needed, separative efficiency is low, limits some middle-size and small-size laboratories pair
The research of excretion body and the batch production of excretion body, do not have the possibility of batch production.Immunological magnetic bead sorting method is to select CD69 anti-
The magnetic bead of body connection is connected on excretion body in such a way that antibody antigen combines, then is adsorbed on excretion body point by high-intensity magnetic field
It selects on instrument, so that excretion body be separated from liquid.For immunological magnetic bead sorting method there is also reagent consumptive material valuableness, sorting amount is few
The shortcomings that, thus seek new method for separating can low-cost, high-volume separation excretion body become the emphasis studied at present.
In addition, the mescenchymal stem cell excretion body secretory volume normally cultivated is few, content is extremely low in cell culture supernatant,
If be not concentrated in advance, the cell culture supernatant bulky that required excretion body needs is obtained, separation is difficult.Although by low
The available a large amount of excretion bodies of the specific process processing cell such as oxygen, hungry culture and cytokine protein, but cell state
Impaired, the mescenchymal stem cell after collecting excretion body is no longer available for other treatment.
Summary of the invention
The purpose of the present invention is for mescenchymal stem cell excretion bulk concentration is low, ultracentrifugation is complicated for operation, extraction efficiency
The technical problems such as low provide a kind of preparation method of mescenchymal stem cell excretion body, the mescenchymal stem cell excretion of preparation
Bulk concentration is high, and ultracentrifugation is easy to operate, and extraction efficiency is high.
For this purpose, the present invention provides a kind of preparation methods of mescenchymal stem cell excretion body, comprising the following steps:
Step 1: cleaning human umbilical tissue surface blood removes epidermis and vascular tissue, shreds after taking out huatong plastic cleaning,
Using tissue adherent method inoculated and cultured；
Step 2: carrying out secondary culture when primary cell length to 70% degrees of fusion；Select the good second generation of upgrowth situation
Umbilical cord mesenchymal stem cells inoculated and cultured；
Step 3: the cell stimulation factor for promoting the secretion of excretion body being added in cell cultivation process, promotes cell production
It is secreted with excretion body；
Step 4: the first cell supernatant being sucked out when cell grows to 90% degrees of fusion for separating excretion body；
Step 5: first cell supernatant being centrifuged removal cell fragment, collects the second cell supernatant；
Step 6: second cell supernatant being subjected to first time ultrafiltration concentration, excessive moisture is removed, it is dense to obtain first
Contracting liquid；
Step 7: first concentrate being carried out second and is concentrated by ultrafiltration, excessive moisture is removed, obtains the second concentrate；
Step 8: the second concentrate being subjected to first time ultracentrifugation, removes tiny cell fragment；
Step 9: taking supernatant to carry out second of ultracentrifugation, remove supernatant, be precipitated as the excretion body of stem cell secretion；
Step 10: physiological saline cleaning is added, ultracentrifugation removes supernatant, obtains excretion body precipitating.
As a preferred embodiment, the cell stimulation factor include vitamin C, INF- γ (interferon),
HGF (hepatocyte growth factor), bFGF (basic fibroblast growth factor).It is highly preferred that the cell stimulation factor by
The group of following concentration is grouped as: vitamin C 50ng/ml, INF- γ 25ng/ml, HGF10ng/ml, bFGF5ng/ml.
As a preferred embodiment, centrifugal speed is 3000 revs/min in the step 5, centrifugation time is extremely
It is 20 minutes few.
As a preferred embodiment, in the step 6, ultrafiltration membrane aperture is 3KD, centrifugal speed is 4500 turns/
Minute, centrifugation time is at least 30 minutes.
As a preferred embodiment, second of condition being concentrated by ultrafiltration and first time ultrafiltration are dense in the step 7
The condition of contracting is identical.
As a preferred embodiment, the volume of second concentrate is the second cell conditioned medium liquid in the step 7
Long-pending 20%.
As a preferred embodiment, ultracentrifugal speed is 10000 revs/min, centrifugation in the step 8
Time is at least 1 hour.
As a preferred embodiment, in the step 9 and step 10, ultracentrifugal speed is 100000 turns/
Minute, centrifugation time is at least 1 hour.
As a preferred embodiment, method of the invention further includes that excretion body precipitating is resuspended with physiological saline, it is dilute
It is interpreted as excretion liquid solution.
As a preferred embodiment, method of the invention further includes that excretion body precipitating is resuspended with physiological saline, it is dilute
After being interpreted as excretion liquid solution, freeze-dried powder is made into after human serum albumins, trehalose, mannitol is added to excretion liquid solution.It is preferred that
Ground, which includes based on concentration be the excretion liquid solution percent by volume of 10 μ g/ml be 2% human serum albumins, end
The mannitol that trehalose that concentration is 0.2M, mass-volume concentration are 6%.
The present invention also provides a kind of cosmetics comprising utilizes excretion body made from the above method.
In preparation method of the invention, by adding excretion body stimulating factor, it is dry thin that umbilical cord mesenchyma can be effectively facilitated
Born of the same parents largely discharge excretion body in normal incubation, and do not influence umbilical cord mesenchymal stem cells normal growth process；Pass through
It in conjunction with ultrafiltration and supercentrifugation, is first concentrated using super filter tube, excessive moisture in cell culture supernatant is removed, to excretion body
It is concentrated, the ultracentrifugal volume of excretion body can be effectively reduced, improve excretion bulk concentration in solution, improve separative efficiency,
Save disengaging time and cost.In addition, in excretion body freeze-dried powder disclosed by the invention formula, by adding human serum albumins
The structure of protein and microRNA and activity stabilized can be effectively kept in excretion body with trehalose.
Detailed description of the invention
Fig. 1 is the comparing result of total protein concentration in the supernatant of concentration front and back.
Fig. 2 is the form of excretion body of the invention under a scanning electron microscope.
Fig. 3 is the particle diameter distribution range of isolated excretion body.
Specific embodiment
Technical solution of the present invention is described in further detail combined with specific embodiments below, but the present invention is not limited to
Lower embodiment.
As unspecified, reagent used in the present invention is available reagent, can be obtained by commercial channel.
Injection is prepared according to the following steps:
1, hospital acquires mature caesarean birth fetal cord tissue, signs client's informed consent form before acquiring, be organized in 4 DEG C it is cold
It transports laboratory under hiding gnotobasis to, uses organization protection's liquid protection umbilical cord tissue biological activity, tissue guarantor in transportational process
The amphotericin B for protecting gentamicin sulphate and 5 μ g/ml that liquid adds 25 μ g/ml by physiological saline is prepared, it is ensured that in transportational process
Without bacterium and fungal contamination；
2, umbilical cord tissue surface blood is cleaned with organization protection's liquid in laboratory, removes epidermis and vascular tissue, take out China
1-2mm is shredded into after logical glue cleaning3Volume fragment is inoculated in T75 culture bottle using tissue adherent method and is cultivated, uses serum-free
And the proliferated culture medium culture of definite ingredients；
3, primary cell is long to 70% degrees of fusion progress secondary culture through culture in 14 days；
4, the good second generation umbilical cord mesenchymal stem cells of upgrowth situation are selected, by 10000/cm2Density is inoculated in healthy and free from worry
In T175 culture bottle, 30ml serum-free proliferated culture medium is added and normally cultivates；
5, the cell stimulation factor for promoting the secretion of excretion body, including vitamin C are added in cell cultivation process
(50ng/ml, by the final concentration in cell culture fluid), INF- γ (25ng/ml), HGF (10ng/ml), bFGF (5ng/
Ml), promote cell production and the secretion of excretion body；
6, supernatant is sucked out when cell grows to 90% degrees of fusion after cultivating 3-4 days for separating excretion body, mesenchyma is dry
The normal secondary culture of cell determines culture generation according to cell growth state；
7,3000 revs/min of cell supernatant centrifugations, the 20 minutes removal cell fragments collected collect supernatant；
8, cell supernatant is added in 15ml super filter tube and is concentrated, and ultrafiltration membrane aperture 3KD, 4500 revs/min are centrifuged 30 minutes,
Remove excessive moisture；
9, it collects liquid in super filter tube and removal excessive moisture is concentrated again, concentration final volume is the 20% of initial volume, is made
With total protein concentration (the result is shown in Figure 1) in BCA (bicinchoninic acid) method measurement supernatant, forefathers are concentrated as the result is shown
Total protein concentration is (1.43 ± 0.21) μ g/ml in umbilical cord mesenchymal stem cells free serum culture supernatant, after liquid concentration, on
Total protein concentration is (6.8 ± 0.34) μ g/ml in clear liquid；
10, concentrated liquid ultracentrifugation, 10000 revs/min of centrifugations remove tiny cell fragment in 1 hour, take supernatant again
Secondary 100000 revs/min are centrifuged 1 hour, remove supernatant, are precipitated as the excretion body of stem cell secretion；
11, it is primary that the cleaning of 10ml physiological saline is added, 100000 revs/min are centrifuged 1 hour, remove supernatant；
12, excretion body precipitating is resuspended in 1ml physiological saline, and BCA (bicinchoninic acid) method measures excretion body precipitating
Middle total protein concentration, scanning electron microscope shooting excretion volume morphing (result is shown in Fig. 2), with Nanosight nano-particle size analysis
Instrument carries out particle distribution range (result is shown in Fig. 3) analysis, the excretion body protein concentration extracted as the result is shown to isolated excretion body
For (1.21 ± 0.15) mg/ml；
13, according to BCA method measurement total protein concentration using physiological saline to excretion body be diluted for 10 μ g/ml it is molten
Liquid；
14, excretion liquid solution be added human serum albumins (the total volume meter volume fraction based on excretion liquid solution be 2%),
Trehalose (the final concentration of 0.2M of total volume meter based on excretion liquid solution), the mannitol (total volume meter based on excretion liquid solution
Quality percent by volume be 6%) after be made into freeze-dried powder long-term preservation (in 3ml cillin bottle fill 1ml liquid freeze-drying)；
15, excretion body freeze-dried powder for skin of face maintain, in use, excretion body freeze-dried powder be added 1.5ml solvent (go from
Sub- water+0.5wt% hyaluronic acid) be completely dissolved after use.
Compliance test result:
It is sterilized after choosing 20 subject's facial cleansings with 75% (v/v) medicinal alcohol face, by dissolved excretion body
Solution is uniformly applied to face, is gently rolled 5 minutes up and down with needle roller, without cleaning, makes weekly respectively using once sooner or later daily
With 2 times, it is used continuously 4 weeks.CBS-807 skin analysis system detection skin hair is used behind the 0th day, 7 days, 14 days, 21 days, 28 days
Hole size, skin elasticity, the colour of skin, wrinkle etc..As a result as shown in the following Table 1, subject can see skin after 1 week and improve,
Skin improves obvious after being used continuously 4 weeks.
Table 1
| 0 day | 7 days | 14 days | 21 days | 28 days | |
| Pore | ++++ | +++ | +++ | ++ | ++ |
| Elasticity | ++ | +++ | ++++ | ++++ | +++++ |
| Wrinkle | +++ | +++ | ++ | ++ | + |
| The colour of skin | ++ | ++ | +++ | +++ | ++++ |
| Skin shine | ++ | ++ | +++ | +++ | ++++ |
Claims (10)
1. a kind of method for preparing mescenchymal stem cell excretion body, it is characterised in that the following steps are included:
Step 1: cleaning human umbilical tissue surface blood removes epidermis and vascular tissue, shreds, uses after taking out huatong plastic cleaning
Organize adherent method inoculated and cultured；
Step 2: carrying out secondary culture when primary cell length to 70% degrees of fusion；Select the good second generation umbilical cord of upgrowth situation
Mescenchymal stem cell inoculated and cultured；
Step 3: the cell stimulation factor for promoting the secretion of excretion body being added in cell cultivation process, promotes cell production and outer
Secrete body secretion；
Step 4: the first cell supernatant being sucked out when cell grows to 90% degrees of fusion for separating excretion body；
Step 5: first cell supernatant being centrifuged removal cell fragment, collects the second cell supernatant；
Step 6: second cell supernatant being subjected to first time ultrafiltration concentration, excessive moisture is removed, obtains the first concentrate；
Step 7: first concentrate being carried out second and is concentrated by ultrafiltration, excessive moisture is removed, obtains the second concentrate；
Step 8: second concentrate being subjected to first time ultracentrifugation, removes tiny cell fragment；
Step 9: taking supernatant to carry out second of ultracentrifugation, remove supernatant, be precipitated as the excretion body of stem cell secretion；
Step 10: physiological saline cleaning is added, centrifugation removes supernatant, obtains excretion body precipitating.
2. the method according to claim 1, wherein the cell stimulation factor include vitamin C, INF- γ,
HGF and bFGF.
3. centrifugal speed is 3000 revs/min the method according to claim 1, wherein in the step 5, from
The heart time is at least 20 minutes.
4. the method according to claim 1, wherein ultrafiltration membrane aperture is 3KD, centrifugal speed in the step 6
It is 4500 revs/min, centrifugation time is at least 30 minutes.
5. the method according to claim 1, wherein the volume of second concentrate is institute in the step 7
State the 20% of the second cell supernatant volume.
6. the method according to claim 1, wherein in the step 8, ultracentrifugal speed is 10000 turns/
Minute, centrifugation time is at least 1 hour.
7. the method according to claim 1, wherein in the step 9 and step 10, ultracentrifugal speed is
100000 revs/min, centrifugation time is at least 1 hour.
8. the method according to claim 1, wherein further include that excretion body precipitating is resuspended with physiological saline,
It is diluted to excretion liquid solution.
9. the method according to claim 1, wherein further include that excretion body precipitating is resuspended with physiological saline,
After being diluted to excretion liquid solution, freeze-drying is made into after human serum albumins, trehalose, mannitol is added to the excretion liquid solution
Powder.
10. a kind of cosmetics, it is characterised in that including excretion body made from the described in any item methods of claims 1 to 10.
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| CN109943533A (en) * | 2019-03-29 | 2019-06-28 | 上海交通大学医学院附属第九人民医院 | A kind of method preparing fat stem cell excretion body, fat stem cell excretion body and its application |
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Application publication date: 20181228 |