CN106109496B - Human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method - Google Patents
Human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method Download PDFInfo
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Abstract
The present invention relates to human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation methods, the preparation method of the human umbilical cord mesenchymal stem cells extract freeze-drying powder is the following steps are included: obtain the primary stem cell of the higher human umbilical cord mesenchymal of purity using density-gradient centrifugation method, and carried out secondary culture, supernatant and trehalose are mixed and made into freeze-dried powder by the cell culture supernatant for collecting for the 3rd~18 generation.The resulting human umbilical cord mesenchymal stem cells extract freeze-drying powder of the present invention has good appearance character；Freeze-dried powder is redissolved, dissolution is very fast, and dissolution is complete；Zoopery proves that its irritation is small, highly-safe, using reliable.In addition, freeze-dried powder of the present invention can effectively save the various biologically active cell factors of human umbilical cord mesenchymal stem cells culture supernatant, the ultraviolet damage of skin can defend, promote skin collagen synthesis, have effects that whitening, delay senescence.
Description
Technical field
The present invention relates to stem cell fields, and in particular to human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation side
Method.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is that a kind of have self-renewing, proliferation and more
To the adult stem cell of differentiation potential, tissue growth and reparation can be stimulated, enhances the power of regeneration of tissue, influences immunological regulation,
There is extremely wide application prospect in cell therapy field.MSCs can be from marrow, Cord blood, fat, synovial membrane, placenta, connective
The positions such as tissue obtain.Currently, research shows that derived from umbilical cord MSCs with the MSCs in other sources compared with it is from a wealth of sources, adopt
Collection is convenient, Proliferation, Differentiation ability is strong, immunogenicity is weak, without ethics problem many advantages, such as, led in organizational project, genetic engineering etc.
Domain is of increasing concern.
However, the abundance of MSCs in the tissue is extremely low, therefore, in vitro culture is to realize must walking for MSCs application value
Suddenly, studies have shown that the MSCs from fresh separated is different, after cultured and amplified in vitro, biological characteristics can occur very big cell
Variation, and as the extension of incubation time, the ability of cell differentiation are gradually lost, this has with environment locating for the cell of inside and outside
It closes, intracorporal microenvironment composition is considerably complicated, it is impossible to these extrinsic factor institute moulds of stable temperature, pH value and nutritional ingredient
Quasi-.How the short time obtains a large amount of good MSCs of functional status, maintains the reset condition of MSCs, and the control differentiation side MSCs
It is also difficult point to the hot spot for being current research.In addition, more and more evidences show no matter to be injected intravenously or locally injecting,
After the MSCs of in vitro culture is entered in vivo, only minimal amount of cells survival, and the cell of this minority survival is big by secretion
The small-molecule substance of amount, for example, cell factor, growth factor, chemotactic factor (CF) etc. and play a role.
Stem cell extract generally refer to stem cell medium supernatant and (or) cell pyrolysis liquid, on cell culture fluid
Clear liquid contains a large amount of protein, polypeptide and cell factor with multiple biological activities, they participate in cyto-architectural maintenance
Motion information exchange and tissue repair and regeneration, there is preferable anti-light aging, anti-oxidant, crease-resistant, skin whitening, wound to be cured
Conjunction, cell repair and other effects.Therefore, as the application value of stem cell extract has been to be concerned by more and more people, how to obtain
It obtains largely, stem cell extract stable and controllable for quality becomes the problem of people endeavour research.Chinese patent application
201510033971.0 disclosing the preparation side of human umbilical cord mesenchymal stem cells culture supernatant active factors and cell pyrolysis liquid
Method, product and application, the preparation method carry out the sorting of healthy human umbilical cord mesenchymal stem cells using magnetic lossless method for separating
It extracts, then carries out secondary culture, whole cell culture supernatants and cell pyrolysis liquid between collecting 2~10 instead of, most afterwards through low
The freeze-dried powder of cell culture supernatant and cell pyrolysis liquid is prepared in warm vacuum, which is effectively saved in the form of freeze-dried powder
Various biologically active cytokine mixtures in human mesenchymal stem cell culture supernatant are human mesenchymal stem cell
The industrial application of culture supernatant is laid a good foundation.
Summary of the invention
In order to solve the problems existing in the prior art, high, bioactivity that the purpose of the present invention is to provide a kind of product purities
High human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method, the preparation method easy to operate 00 is complete effectively, energy
Enough realize that the industrialization of human umbilical cord mesenchymal stem cells extract freeze-drying powder utilizes.
The present invention is by following technical solution to be attained in that
A kind of preparation method of human umbilical cord mesenchymal stem cells extract freeze-drying powder, comprising the following steps:
(1) primary separation, purifying and the culture of stem cell: separating umbilical cord jelly of Wharton from Freshman umbilical cord, and digestive juice disappears
Change, be filtered with 150um cell filter, collects filtrate, filtrate is placed under the conditions of 1200r/min and is centrifuged 5min, in abandoning
Clearly, it is washed cell precipitation 2~3 times with balanced salt solution, then cell is resuspended with the balanced salt solution of 2 times of volumes, utilization is close
Gradient centrifugation purifying cells suspension is spent, the primary stem cell of umbilical cord mesenchyma is obtained, the primary stem cell of umbilical cord mesenchyma is inoculated in
Adhere-wall culture is carried out in serum free medium containing 100U/ml penicillin and 100g/ml streptomysin；
(2) acquisition of stem cell extract: to the primary stem cell of umbilical cord mesenchyma close to 90% fusion, replace it is fresh not
Antibiotic serum free medium, and secondary culture is carried out, the cell culture supernatant in the 3rd~18 generation is collected, by supernatant
It is filtered with 0.22 μm of sterilizing filter, takes filtrate to get umbilical cord mesenchymal stem cells extract；
(3) preparation of stem cell extract freeze-drying powder: umbilical cord mesenchymal stem cells extract and freeze drying protectant are mixed
Afterwards, cold then using water for injection regulatory protein concentration to 45 ± 0.5 μ g/ml with 0.22 μm of sterilizing filter filtration sterilization
It is lyophilized dry to get human umbilical cord mesenchymal stem cells extract freeze-drying powder；
The freeze drying protectant is trehalose, and the dosage of freeze drying protectant is 5%wt umbilical cord mesenchymal stem cells extract；
The freeze-drying specifically: pre-freeze 12h in -80 DEG C of ultra low temperature freezers is subsequently placed in vacuum freeze drier
In, -30 DEG C of 1~2h of holding are 1.0 × 10 in vacuum degree-2~1.5 × 10-210~12h is lyophilized under the conditions of -5 DEG C by mbar, protects
Hold vacuum degree, continue at 20 DEG C 2~4h of freeze-drying to get.
Serum free medium (SYL-SF) of the present invention is commercial product, public by the advantageous scientific and technological limited development in Beijing three
Independent research is taken charge of, the basal medium of the SYL-SF is α-MEM, wherein addition albumin, transferrins, insulin, epidermis
The blood serum substitutings ingredient such as growth factor, fibroblast growth factor, hydrocortisone, lipid, without any animal-derived sera and
Antibiotic.
Further, the density-gradient centrifugation method in the step (1) specifically: cell suspension is slowly added into and is equipped with
In the centrifuge tube of isometric separating liquid, 4 DEG C, 2000r/min is centrifuged 15~20min, collects intermediate tunica albuginea layer, adds into tunica albuginea layer
Enter isometric phosphate buffer solution, 4 DEG C, 1000r/min is centrifuged 5~15min, abandons supernatant, adds isometric phosphoric acid
Salt buffer solution, 4 DEG C, 800r/min is centrifuged 5~15min, abandons supernatant, takes precipitating to get the primary stem cell of umbilical cord mesenchyma.
Preferably, the density-gradient centrifugation method in the step (1) specifically: by cell suspension be slowly added into equipped with etc.
In the centrifuge tube of volume separating liquid, 4 DEG C, 2000r/min is centrifuged 20min, collects intermediate tunica albuginea layer, the bodies such as addition into tunica albuginea layer
Long-pending phosphate buffer solution, 4 DEG C, 1000r/min is centrifuged 10min, abandons supernatant, it is molten to add isometric phosphate-buffered
Liquid, 4 DEG C, 800r/min is centrifuged 10min, abandons supernatant, takes precipitating to get the primary stem cell of umbilical cord mesenchyma.
Further, the separating liquid is ficoll-Sodium Amidotrizoate solution that concentration is 1.073g/mL.
Further, in the step (1) digestive juice digestion specifically: by 1:3 volume ratio be added digestive juice, 37 DEG C
Digest 3h, digestive juice be the phosphate buffer solution containing clostridiopetidase A II, hyaluronidase and EDTA, wherein clostridiopetidase A II, thoroughly
The quality percent by volume final concentration of bright matter acid enzyme and EDTA in phosphate buffer solution is respectively 0.12%, 0.06%,
0.02%.
A kind of human umbilical cord mesenchymal stem cells extract freeze-drying powder, is prepared by above-mentioned preparation method.
Using trehalose, as freeze drying protectant, freeze-dried powder is made in human umbilical cord mesenchymal stem cells extract by the present invention,
The freeze-dried powder has good appearance character, and freeze-dried powder is redissolved, and dissolution is very fast, and dissolution is complete.Zoopery card
Its bright irritation is small, highly-safe, using reliable.The freeze-dried powder can preferably keep human umbilical cord mesenchymal stem cells culture
The bioactivity of supernatant cell factor.It is proved through test cell line, freeze-dried powder of the present invention is remarkably improved application on human skin into fiber finer
The vigor of born of the same parents, and cell SOD activity is significantly increased, ultraviolet light is effective against to human fibroblasts oxidative damage.Through animal
Test proves that freeze-dried powder of the present invention is remarkably improved nude mice skin elasticity and moisture content, and promotes nude mice skin Type I collagen egg
The synthesis of white and III collagen type, improves the content of collagen in skin.
Compared with prior art, present invention has an advantage that
(1) present invention uses density-gradient centrifugation method purifying cells suspension, and it is former to obtain the higher human umbilical cord mesenchymal of purity
For stem cell, the primary stem cell of isolated human umbilical cord mesenchymal cultivate in vitro in have that vigor is high, differentiation capability is good, expands
Several high features are doubled, to realize that the industrial application of human umbilical cord mesenchymal stem cells extract is laid a good foundation.
(2) using trehalose as freeze drying protectant freeze-dried powder is made in human umbilical cord mesenchymal stem cells extract by the present invention,
The bioactivity of human umbilical cord mesenchymal stem cells extract can be preferably kept, it is not degradable in prolonged storage, and make
With conveniently.
Specific embodiment
The present invention is further described below by way of specific embodiment, but the present invention is not limited only to following embodiment.
The separation of 1 human umbilical cord mesenchymal stem cells of embodiment, purifying, culture and identification
(1) human umbilical cord mesenchymal stem cells separation, purifying and culture
It, will be fresh using the balanced salt solution containing 100U/ml penicillin and 100g/ml streptomysin in sterile condition
The umbilical cord of acquisition rinses 3 times repeatedly, and umbilical cord outer membrane and artery and vein are removed after cleaning up, and takes between blood vessel, blood vessel and outer membrane
Between jelly (umbilical cord jelly of Wharton) and be cut into 1mm3Digestive juice is added by the volume ratio of 1:3 in the tissue block of size, and 37 DEG C disappear
Change 3h, wherein digestive juice be the phosphate buffer solution containing clostridiopetidase A II, hyaluronidase and EDTA, and clostridiopetidase A II,
The quality percent by volume final concentration of hyaluronidase and EDTA in phosphate buffer solution is respectively 0.12%, 0.06%,
0.02%.Then it is filtered with 150um cell filter, collects filtrate, filtrate is placed under the conditions of 1200r/min and is centrifuged
5min abandons supernatant, is washed cell precipitation 3 times with balanced salt solution, then cell is resuspended with the balanced salt solution of 2 times of volumes,
Obtain cell suspension.Cell suspension is slowly added into molten equipped with ficoll-Sodium Amidotrizoate that isometric concentration is 1.073g/mL
In the centrifuge tube of liquid, 4 DEG C, 2000r/min is centrifuged 20min, collects intermediate tunica albuginea layer, isometric phosphoric acid is added into tunica albuginea layer
Salt buffer solution, 4 DEG C, 1000r/min is centrifuged 10min, abandons supernatant, adds isometric phosphate buffer solution, and 4 DEG C,
800r/min is centrifuged 10min, abandons supernatant, takes precipitating to get the primary stem cell of umbilical cord mesenchyma.Umbilical cord mesenchyma is primary dry thin
Born of the same parents are inoculated in the serum free medium containing 100U/ml penicillin and 100U/ml streptomysin, in 37 DEG C, 5%CO2Incubator
Middle carry out adhere-wall culture.
(2) human umbilical cord mesenchymal stem cells are identified
By flow cytometry human umbilical cord mesenchymal stem cells surface marker, detected using Flow Cytometry glimmering
Signal antibody.According to antigen-antibody combination principle, the thin of corresponding antigens is carried to known with the antibody that specific fluorescent element marks
Born of the same parents dye.The cell dyed through fluorescein labelled antibody can be identified by flow cytometer, and be taken according to known cell
The intensity of the fluorescein of band carries out qualitative, quantitative analysis to labelled antibody.
The result shows that human umbilical cord mesenchymal stem cells do not express hematopoietic cell marker such as: CD45, CD34, CD14,
CD11b (is negative), and the cells and characteristic of stem surface antigen such as CD105, CD109, CD73, CD90 is positive and (is shown in Table 1), show through
Density-gradient centrifugation method purifying cells suspension obtains the primary stem cell of the higher human umbilical cord mesenchymal of purity.
The testing result of 1 human umbilical cord mesenchymal stem cells surface marker of table
| Testing index | Expression rate | Conclusion |
| CD105 | 99.7% | It is positive |
| CD109 | 99.4% | It is positive |
| CD73 | 99.8% | It is positive |
| CD90 | 99.6% | It is positive |
| CD45 | 0.1% | It is negative |
| CD34 | 0.5% | It is negative |
| CD14 | 0.2% | It is negative |
| CD11b | 0.4% | It is negative |
The preparation of 2 human umbilical cord mesenchymal stem cells extract freeze-drying powder of embodiment
(1) it the acquisition of the extract of human umbilical cord mesenchymal stem cells: to umbilical cord mesenchymal stem cells close to 90% fusion, goes
It except culture solution, is cleaned 3 times with phosphate buffer solution, the pancreatin digestive juice that mass fraction is 0.25%, 37 DEG C of digestion is added
10min is added the isometric not antibiotic serum free medium of digestive juice and terminates digestion, is centrifuged under the conditions of 1200r/min
5min abandons supernatant, obtains cell, and cell is resuspended using serum free medium, carries out passage inoculation according to the ratio of 1:3, is placed in
37 DEG C, 5%CO2It is cultivated in incubator, the culture solution of replacement in every 2 days melts to umbilical cord mesenchymal stem cells close to 80%
It closes, discards culture solution, replace fresh serum free medium and continue 72h, collect supernatant, and continue passage training with same method
It supports.
(2) the extract freeze-drying powder preparation of human umbilical cord mesenchymal stem cells: merge the cells and supernatant in the 3rd~18 generation
Trehalose is added in liquid, and the dosage of the trehalose is 5%wt cell culture supernatant, mixes, with 0.22 μm of sterilizing filter
Filtration sterilization then using water for injection regulatory protein concentration to 45 ± 0.5 μ g/ml, then is placed in -80 DEG C of ultra low temperature freezers pre-
Freeze 12h, then be placed in vacuum freeze drier, -30 DEG C of holding 2h, is 1.0 × 10 in vacuum degree-2Mbar freezes under the conditions of -5 DEG C
Dry 12h, keeps vacuum degree, continues to be lyophilized 2h at 20 DEG C to get human umbilical cord mesenchymal stem cells extract freeze-drying powder.
Embodiment 3 prepares human umbilical cord mesenchymal stem cells extract freeze-drying powder using different freeze drying protectants and compares
Respectively using the trehalose in mannitol, Lactis Anhydrous, the dextran replacement present invention as freeze drying protectant, according to
Freeze-dried powder is made in human umbilical cord mesenchymal stem cells extract by the technique of embodiment 2, and freeze-dried powder obtained and embodiment 2 are made
The freeze-dried powder obtained carries out the results are shown in Table 2 than table.
Table 2 prepares human umbilical cord mesenchymal stem cells extract freeze-drying powder physical behavior using different freeze drying protectants and compares
| Group | Appearance | It redissolves dissolution time (s) | The clarity of solution after redissolution | pH |
| Embodiment 2 | Dense powder shape | 4 | It clarifies, is transparent | 6.5~7.5 |
| Mannitol group | Part atrophy is powdered | 8 | It clarifies, is transparent | 6.5~7.5 |
| Lactis Anhydrous group | Block | 12 | Generate opalescence, muddiness | 6.0~7.0 |
| Dextran group | Block | 15 | It clarifies, is transparent | 6.0~7.0 |
As seen from the above table, it is lyophilized using trehalose as human umbilical cord mesenchymal stem cells extract made from freeze drying protectant
Powder has good appearance character；Freeze-dried powder is redissolved, dissolution is very fast, and dissolution is complete.
4 human umbilical cord mesenchymal stem cells extract freeze-drying powder safety evaluatio of embodiment
(1) acute toxicity test of Mouse oral human umbilical cord mesenchymal stem cells extract freeze-drying powder
10 kunming mices (half male and half female, average weight 23.5g) are taken, embodiment 2 is given to stomach-filling of mouse and makes
The dose of 10 times of the freeze-dried powder mark concentration obtained, observes its toxic reaction, when not causing animal dead, then no longer carries out multiple doses
The acute oral toxicity test of amount.The results show that any adverse reaction does not occur in mouse, there is death without animal, show
Human umbilical cord mesenchymal stem cells extract freeze-drying powder produced by the present invention is highly-safe.
(2) rabbit smears the skin irritation test of human umbilical cord mesenchymal stem cells extract freeze-drying powder
It takes 10 Japan large ear rabbits (half male and half female, weight 2kg or so), for 24 hours with depilatory agent to medication area (back before experiment
Portion two sides) carry out depilation processing, each 3cm × 3cm of unhairing range or so.Baoding is carried out to rabbit, rabbit left dorsal is positioned
For check plot, right positioner is trial zone, and physiological saline 0.5ml is given in check plot, and trial zone is between people's umbilical cord made from embodiment 2
Mesenchymal stem cells extract freeze-drying powder redissolved with physiological saline after solution 0.5ml, add gauze covering protection, use immobilization with adhesive tape.
Wipe tested material afterwards for 24 hours, warm water cleaning observes 30min, 2h, 6h, and whether there is or not erythema and oedema situation for 12h coating part.As a result it shows
Show, trial zone and check plot show that human umbilical cord mesenchymal stem cells produced by the present invention mention without there is erythema and oedema situation
Take object freeze-dried powder small to skin irritation.
5 human umbilical cord mesenchymal stem cells extract freeze-drying powder of embodiment damages ultraviolet induction human skin fibroblasts
Protective effect
1. experimental material: human umbilical cord mesenchymal stem cells extract freeze-drying powder made from embodiment 2, described in embodiment 3
Respectively using the trehalose in mannitol, Lactis Anhydrous, the dextran replacement present invention as people's umbilical cord made from freeze drying protectant
Mescenchymal stem cell extract freeze-drying powder, human skin fibroblasts (Guangzhou Ji Niou Biotechnology Co., Ltd).
2. experimental method: the human skin fibroblasts in logarithmic growth phase being inoculated in 96 orifice plates, to cell monolayer
After adherent (for 24 hours), culture solution is abandoned, 200 μ lPBS are added in every hole, use 30J/cm respectively2UVA irradiation, blank control group aluminium
Foil lid is lived, and irradiates 2h daily, Continuous irradiation be randomly divided into after 4 days blank control group, model group, 2 groups of embodiment, mannitol group,
Lactis Anhydrous group and dextran group.Wherein, blank control group and model group give isometric physiological saline, and 2 groups of embodiment
Give human umbilical cord mesenchymal stem cells extract freeze-drying powder made from embodiment 2, final protein concentration is respectively 40 μ g/ml, sweet
Dew alcohol group, Lactis Anhydrous group and dextran group are given described in embodiment 3 with mannitol, Lactis Anhydrous and dextran respectively
As human umbilical cord mesenchymal stem cells extract freeze-drying powder made of freeze drying protectant, final protein concentration is 40 μ g/ml,.
After 48h, 20 μ lMTT (5mg/ml) are added to every hole, continue to abandon supernatant after cultivating 4h, every hole adds 150 μ lDMSO to dissolve, room temperature vibration
10min is swung, crystallization is dissolved, microplate reader detects light absorption (OD) value at each hole 492nm, then calculates cell viability, cell
Vigor (%)=experimental group absorbance value/blank control group absorbance value × 100%, and to the superoxide dismutase of cell
(SOD) activity is detected, and the results are shown in Table 3.
3. experimental result
Influence of the 3 human umbilical cord mesenchymal stem cells extract freeze-drying powder of table to human skin fibroblasts vigor
Note: compared with blank control group,**P < 0.01；***P < 0.001；Compared with model group,##P < 0.01.
As seen from the above table, using trehalose as human umbilical cord mesenchymal stem cells extract freeze-drying powder made from freeze drying protectant
Be remarkably improved the vigor of human skin fibroblasts, and significantly increase cell SOD activity, be effective against ultraviolet light to people at
Fibrocyte oxidative damage shows that human umbilical cord mesenchymal stem cells extract freeze-drying powder made from embodiment 2 can effective depositary
The various biologically active cell factors of umbilical cord mesenchymal stem cells culture supernatant, and with mannitol, Lactis Anhydrous and
Dextran cannot effectively save the activity of human umbilical cord mesenchymal stem cells extract as freeze drying protectant, especially with nothing
The effect of water and milk sugar group is worst.
The influence that 6 human umbilical cord mesenchymal stem cells extract freeze-drying powder of embodiment synthesizes skin collagen
1. experimental method: taking BALB/C nude mice 40 of 6 week old, half male and half female is provided by Guangdong Province's animal experimental center.
It is divided into 2 groups of embodiment, mannitol group, Lactis Anhydrous group and dextran group, every group 10.Respectively in each group nude mice skin of back
The right half of embodiment 2, described in embodiment 3 using mannitol, Lactis Anhydrous and dextran as freeze drying protectant system of smearing
At human umbilical cord mesenchymal stem cells extract freeze-drying powder redissolved with physiological saline after aqueous solution, the skin of back of left one side of something is then
Physiological saline is coated as control, 200 μ l is smeared every time, is tested after 3 times/day, 6 weeks using special skin elasticity (water content)
Instrument measures the elasticity and water content of nude mice skin of back.The big of elasticity is evaluated according to the situation that skin restores in a short time
Small, elasticity number 1 is maximum, i.e. skin 100% restores to the original state.Nude mice is put to death after experiment, removes the skin group at nude mice back
It knits, left and right sides respectively takes 0.1g, using the Type I collagen and III Collagen Type VI transcriptional level in RT-PCR detection tissue.It the results are shown in Table 4 Hes
Table 5.
2. experimental result
Influence of the 4 human umbilical cord mesenchymal stem cells extract freeze-drying powder of table to nude mice skin elasticity and moisture
Note: compared with the control group,*P < 0.05.
Influence of the 5 human umbilical cord mesenchymal stem cells extract freeze-drying powder of table to nude mice skin collagen transcriptional level
Note: compared with the control group,**P < 0.01.
As upper table 4 and 5 it is found that freezing using trehalose as human umbilical cord mesenchymal stem cells extract made from freeze drying protectant
Dry powder is remarkably improved nude mice skin elasticity and moisture content, and promotes nude mice skin Type I collagen albumen and III collagen type
Synthesis improves the content of collagen in skin.Show human umbilical cord mesenchymal stem cells extract freeze-drying powder made from embodiment 2
The various biologically active cell factors of human umbilical cord mesenchymal stem cells culture supernatant can be effectively saved, and with sweet dew
Alcohol, Lactis Anhydrous and dextran cannot effectively save human umbilical cord mesenchymal stem cells extract as freeze drying protectant
Activity, it is especially worst with the effect of Lactis Anhydrous group.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Claims (5)
1. a kind of preparation method of human umbilical cord mesenchymal stem cells extract freeze-drying powder, it is characterised in that the following steps are included:
(1) primary separation, purifying and the culture of stem cell: separating umbilical cord jelly of Wharton from Freshman umbilical cord, and digestive juice digestion is used
150um cell filter is filtered, and collects filtrate, filtrate is placed under the conditions of 1200r/min and is centrifuged 5min, abandons supernatant, is used
Balanced salt solution washs cell precipitation 2~3 times, then cell is resuspended with the balanced salt solution of 2 times of volumes, utilizes density gradient
Centrifugal process purifying cells suspension obtains the primary stem cell of umbilical cord mesenchyma, by the primary stem cell of umbilical cord mesenchyma be inoculated in containing
Adhere-wall culture is carried out in the serum free medium of 100U/ml penicillin and 100g/ml streptomysin；
(2) it the acquisition of stem cell extract: to the primary stem cell of umbilical cord mesenchyma close to 90% fusion, replaces fresh being free of and resists
The serum free medium of raw element, and secondary culture is carried out, the cell culture supernatant in the 3rd~18 generation is collected, supernatant is used
0.22 μm of sterilizing filter filtering, takes filtrate to get umbilical cord mesenchymal stem cells extract；
(3) it the preparation of stem cell extract freeze-drying powder: after umbilical cord mesenchymal stem cells extract and freeze drying protectant are mixed, uses
0.22 μm of sterilizing filter filtration sterilization, then using water for injection regulatory protein concentration to 45 ± 0.5 μ g/ml, freezing is dry
It is dry to get human umbilical cord mesenchymal stem cells extract freeze-drying powder；
The freeze drying protectant is trehalose, and the dosage of freeze drying protectant is 5%wt umbilical cord mesenchymal stem cells extract；
The freeze-drying specifically: pre-freeze 12h in -80 DEG C of ultra low temperature freezers is subsequently placed in vacuum freeze drier, -30
DEG C keep 1~2h, vacuum degree be 1.0 × 10-2~1.5 × 10-210~12h is lyophilized under the conditions of -5 DEG C by mbar, keeps vacuum
Degree, continue at 20 DEG C 2~4h of freeze-drying to get；
Density-gradient centrifugation method in the step (1) specifically: cell suspension is slowly added into equipped with isometric separating liquid
Centrifuge tube in, 4 DEG C, 2000r/min is centrifuged 15~20min, collects intermediate tunica albuginea layer, isometric phosphorus is added into tunica albuginea layer
Hydrochlorate buffer solution, 4 DEG C, 1000r/min is centrifuged 5~15min, abandons supernatant, adds isometric phosphate buffer solution, and 4
DEG C, 800r/min is centrifuged 5~15min, abandons supernatant, takes precipitating to get the primary stem cell of umbilical cord mesenchyma.
2. the preparation method of human umbilical cord mesenchymal stem cells extract freeze-drying powder according to claim 1, which is characterized in that
Density-gradient centrifugation method in the step (1) specifically: by cell suspension be slowly added into equipped with isometric separating liquid from
In heart pipe, 4 DEG C, 2000r/min is centrifuged 20min, collects intermediate tunica albuginea layer, isometric phosphate-buffered is added into tunica albuginea layer
Solution, 4 DEG C, 1000r/min is centrifuged 10min, abandons supernatant, adds isometric phosphate buffer solution, and 4 DEG C, 800r/min
It is centrifuged 10min, abandons supernatant, takes precipitating to get the primary stem cell of umbilical cord mesenchyma.
3. the preparation method of human umbilical cord mesenchymal stem cells extract freeze-drying powder according to claim 1 or 2, feature exist
In the separating liquid is ficoll-Sodium Amidotrizoate solution that concentration is 1.073g/mL.
4. the preparation method of human umbilical cord mesenchymal stem cells extract freeze-drying powder according to claim 1, which is characterized in that
In the step (1) digestive juice digestion specifically: by 1:3 volume ratio be added digestive juice, 37 DEG C of digestion 3h, digestive juice for containing
There are the phosphate buffer solution of clostridiopetidase A II, hyaluronidase and EDTA, wherein clostridiopetidase A II, hyaluronidase and EDTA exist
Quality percent by volume final concentration in phosphate buffer solution is respectively 0.12%, 0.06%, 0.02%.
5. human umbilical cord mesenchymal stem cells extract freeze-drying powder, which is characterized in that system according to any one of claims 1 to 4
Preparation Method obtains.
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