CN106109496A - 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
- Publication number
- CN106109496A CN106109496A CN201610529860.3A CN201610529860A CN106109496A CN 106109496 A CN106109496 A CN 106109496A CN 201610529860 A CN201610529860 A CN 201610529860A CN 106109496 A CN106109496 A CN 106109496A
- Authority
- CN
- China
- Prior art keywords
- umbilical cord
- stem cells
- mesenchymal stem
- cord mesenchymal
- freeze
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method, the preparation method of described human umbilical cord mesenchymal stem cells extract freeze-drying powder comprises the following steps: use density－gradient centrifuga－tion method to obtain the purity primary stem cell of higher human umbilical cord mesenchymal, and carried out Secondary Culture, collect the cell culture supernatant in the 3rd～18 generations, supernatant and trehalose are mixed and made into lyophilized powder.The human umbilical cord mesenchymal stem cells extract freeze-drying powder of gained of the present invention has good appearance character；Lyophilized powder is redissolved, dissolves very fast, dissolve completely；Zoopery proves that its zest is little, and safety is high, uses reliable.Additionally, lyophilized powder of the present invention can have bioactive cytokine by the various of depositary's umbilical cord mesenchymal stem cells culture supernatant effectively, skin ultraviolet can be defendd to damage, promote skin collagen synthesis, there is effect of whitening, slow down aging.
Description
Technical field
The present invention relates to stem cell field, be specifically related to human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation side
Method.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is that a class has self renewal, propagation and many
To the adult stem cell of differentiation potential, tissue growth and reparation can be stimulated, strengthen the regeneration capacity of tissue, affect immunomodulating,
The most wide application prospect is had in cell therapy field.MSCs can be from bone marrow, Cord blood, fat, synovial membrane, Placenta Hominis, connective
The positions such as tissue obtain.At present, research shows that the MSCs coming from umbilical cord has wide material sources compared with the MSCs that other are originated, adopts
Collection is convenient, proliferation and differentiation ability is strong, immunogenicity is weak, without plurality of advantages such as ethical issuess, at the neck such as organizational project, genetic engineering
Territory is of increasing concern.
But, MSCs abundance in the tissue is extremely low, and therefore, In vitro culture is to realize must walking of MSCs using value
Suddenly, research shows, different from the MSCs of fresh separated, and cell is after cultured and amplified in vitro, and its biological characteristics can occur the biggest
Change, and along with the prolongation of incubation time, the ability of cell differentiation is gradually lost, and this has with the environment residing for the cell of inside and outside
Closing, internal microenvironment composition is considerably complicated, it is impossible to these extrinsic factor institute moulds of stable temperature, pH value and nutritional labeling
Intend.How the short time obtains the MSCs that a large amount of functional statuses are good, maintains the initial condition of MSCs, and controls MSCs differentiation side
Also it is difficult point to the focus being current research.It addition, increasing evidence shows, no matter intravenous injection or local injection,
The MSCs of In vitro culture enter into internal after, the most minimal amount of cells survival, and the cell of this minority survival is big by secretion
Amount small-molecule substance, such as, cytokine, somatomedin, chemotactic factor etc. and play a role.
Stem cell extract generally refer to stem cell medium supernatant and (or) cell pyrolysis liquid, on its cell culture fluid
Clear liquid contains and substantial amounts of has multiple bioactive protein, polypeptide and cytokine, and they participate in cyto-architectural maintenance
Movable information exchange and tissue repair and regeneration, have preferable anti-light aging, antioxidation, crease-resistant, skin whitening, wound more
The effects such as conjunction, cytothesis.Therefore, along with the using value of stem cell extract is increasingly paid close attention to by people, how to obtain
Obtaining substantial amounts of, stem cell extract stable and controllable for quality becomes the problem that people endeavour research.Chinese patent application
201510033971.0 disclose human umbilical cord mesenchymal stem cells culture supernatant active factors and the preparation side of cell pyrolysis liquid
Method, product and application, this preparation method uses magnetic lossless method for separating to carry out the sorting of Healthy People umbilical cord mesenchymal stem cells
Extract, then carry out Secondary Culture, collect 2～10 instead of between all cell culture supernatants and cell pyrolysis liquid, after through low
Temperature vacuum prepares the lyophilized powder of cell culture supernatant and cell pyrolysis liquid, and this invention preserves effectively with lyophilized powder form
Various in human mesenchymal stem cell culture supernatant have bioactive cytokine mixture, for human mesenchymal stem cell
The commercial application of culture supernatant is laid a good foundation.
Summary of the invention
For solving the problem that prior art exists, it is an object of the invention to provide a kind of product purity high, biological activity
High human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method, described preparation method simple to operate 00 is complete effectively, energy
The industrialization enough realizing human umbilical cord mesenchymal stem cells extract freeze-drying powder utilizes.
The present invention is by following technical solution to be attained in that
The preparation method of a kind of human umbilical cord mesenchymal stem cells extract freeze-drying powder, comprises the following steps:
(1) primary separation, purification and the cultivation of stem cell: separating umbilical cord jelly of Wharton from Freshman umbilical cord, Digestive system disappears
Changing, filter with 150um cell filter, collect filtrate, under the conditions of filtrate is placed in 1200r/min, centrifugal 5min, abandons
Clearly, precipitate 2～3 times with balanced salt solution washed cell, then with the balanced salt solution re-suspended cell of 2 times of volumes, utilize close
Degree gradient centrifugation purifying cells suspension, obtains the primary stem cell of umbilical cord mesenchyma, is inoculated in by primary for umbilical cord mesenchyma stem cell
Serum-free medium containing 100U/ml penicillin and 100g/ml streptomycin carries out adhere-wall culture；
(2) acquisition of stem cell extract: treat the primary stem cell of umbilical cord mesenchyma close to 90% merge, change fresh not
Serum-free medium containing antibiotic, and carry out Secondary Culture, collect the cell culture supernatant in the 3rd～18 generations, by supernatant
Filter with the sterilizing filter of 0.22 μm, take filtrate, obtain umbilical cord mesenchymal stem cells extract；
(3) preparation of stem cell extract freeze-drying powder: umbilical cord mesenchymal stem cells extract is mixed with freeze drying protectant
After, with the sterilizing filter filtration sterilization of 0.22 μm, then use water for injection regulation protein concentration is to 45 ± 0.5 μ g/ml, cold
Lyophilizing is dry, obtains human umbilical cord mesenchymal stem cells extract freeze-drying powder；
Described freeze drying protectant is trehalose, and the consumption of freeze drying protectant is 5%wt umbilical cord mesenchymal stem cells extract；
Described lyophilization, particularly as follows: pre-freeze 12h in-80 DEG C of ultra cold storage freezers, is subsequently placed in vacuum freeze drier
In ,-30 DEG C keep 1～2h, are 1.0 × 10 in vacuum-2～1.5 × 10-2Mbar, lyophilizing 10～12h under the conditions of-5 DEG C, protect
Hold vacuum, continue lyophilizing 2～4h at 20 DEG C, to obtain final product.
Serum-free medium of the present invention (SYL-SF) is commercially available prod, public by Beijing three favourable science and technology limited development
Department's independent research, the basal medium of described SYL-SF is α-MEM, wherein adds albumin, transferrins, insulin, epidermis
The blood serum substituting compositions such as somatomedin, fibroblast growth factor, hydrocortisone, lipid, without any animal-derived sera and
Antibiotic.
Further, the density－gradient centrifuga－tion method in described step (1) particularly as follows: cell suspension is slowly added into equipped with
In the centrifuge tube of equal-volume separation liquid, 4 DEG C, 2000r/min is centrifuged 15～20min, collects middle tunica albuginea layer, adds in tunica albuginea layer
Entering isopyknic phosphate buffered solution, 4 DEG C, 1000r/min is centrifuged 5～15min, abandons supernatant, adds isopyknic phosphoric acid
Salt buffer solution, 4 DEG C, 800r/min is centrifuged 5～15min, abandons supernatant, takes precipitation, obtains the primary stem cell of umbilical cord mesenchyma.
Preferably, the density－gradient centrifuga－tion method in described step (1) particularly as follows: cell suspension is slowly added into equipped with etc.
In the centrifuge tube of volume integral chaotropic, 4 DEG C, 2000r/min is centrifuged 20min, collect in the middle of tunica albuginea layer, the body such as addition in tunica albuginea layer
Long-pending phosphate buffered solution, 4 DEG C, 1000r/min is centrifuged 10min, abandons supernatant, adds isopyknic phosphate-buffered molten
Liquid, 4 DEG C, 800r/min is centrifuged 10min, abandons supernatant, takes precipitation, obtains the primary stem cell of umbilical cord mesenchyma.
Further, described separation liquid be concentration be the ficoll-sodium amidotrizoate solution of 1.073g/mL.
Further, the Digestive system in described step (1) digests particularly as follows: press the volume ratio addition Digestive system of 1:3,37 DEG C
Digestion 3h, Digestive system is the phosphate buffered solution containing collagenase II, hyaluronidase and EDTA, wherein, collagenase II, thoroughly
Bright matter acid enzyme and the EDTA quality percent by volume final concentration in phosphate buffered 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.
The present invention uses trehalose as freeze drying protectant, and human umbilical cord mesenchymal stem cells extract is made lyophilized powder,
Described lyophilized powder has good appearance character, is redissolved by lyophilized powder, dissolves very fast, dissolves completely.Zoopery is demonstrate,proved
Its zest bright is little, and safety is high, uses reliable.Described lyophilized powder can preferably keep human umbilical cord mesenchymal stem cells to cultivate
The biological activity of supernatant cytokine.Proving through test cell line, lyophilized powder of the present invention is remarkably improved application on human skin and becomes fiber finer
The vigor of born of the same parents, and it is obviously enhanced cell SOD activity, it is effective against ultraviolet to human fibroblasts oxidative damage.Through animal
Test proves, lyophilized powder of the present invention is remarkably improved nude mice skin elasticity and moisture, and promotes nude mice skin NTx egg
White and III collagen type synthesizes, and improves the content of collagen protein in skin.
Compared with prior art, present invention have an advantage that
(1) present invention uses density－gradient centrifuga－tion method purifying cells suspension, and the human umbilical cord mesenchymal obtaining purity higher is former
For stem cell, the primary stem cell of human umbilical cord mesenchymal of isolated has in cultivating in vitro that vigor is high, differentiation capability is good, expands
Double several high feature, lay a good foundation for realizing the commercial application of human umbilical cord mesenchymal stem cells extract.
(2) present invention is using trehalose as freeze drying protectant, and human umbilical cord mesenchymal stem cells extract is made lyophilized powder,
Can preferably keep the biological activity of human umbilical cord mesenchymal stem cells extract, the most degradable in prolonged storage, and make
With convenient.
Detailed description of the invention
Further describe the present invention below by way of detailed description of the invention, but the present invention is not limited only to following example.
Embodiment 1 human umbilical cord mesenchymal stem cells separation, purification, cultivate and identify
(1) human umbilical cord mesenchymal stem cells separation, purification and cultivation
In aseptic condition, use the balanced salt solution containing 100U/ml penicillin and 100g/ml streptomycin by fresh
The umbilical cord gathered rinses 3 times repeatedly, removes umbilical cord adventitia and artery and vein after cleaning up, and takes between blood vessel, blood vessel and adventitia
Between jelly (umbilical cord jelly of Wharton) and be cut into 1mm3The piece of tissue of size, adds Digestive system by the volume ratio of 1:3, and 37 DEG C disappear
Changing 3h, wherein, Digestive system is the phosphate buffered solution containing collagenase II, hyaluronidase and EDTA, and collagenase II,
Hyaluronidase and the EDTA quality percent by volume final concentration in phosphate buffered solution is respectively 0.12%, 0.06%,
0.02%.Then filter with 150um cell filter, collect filtrate, centrifugal under the conditions of filtrate is placed in 1200r/min
5min, abandons supernatant, precipitates 3 times with balanced salt solution washed cell, then with the balanced salt solution re-suspended cell of 2 times of volumes,
Obtain cell suspension.Cell suspension is slowly added into the ficoll-sodium amidotrizoate equipped with isopyknic concentration is 1.073g/mL molten
In the centrifuge tube of liquid, 4 DEG C, 2000r/min is centrifuged 20min, collects middle tunica albuginea layer, adds isopyknic phosphoric acid in tunica albuginea layer
Salt buffer solution, 4 DEG C, 1000r/min is centrifuged 10min, abandons supernatant, adds isopyknic phosphate buffered solution, 4 DEG C,
800r/min is centrifuged 10min, abandons supernatant, takes precipitation, obtains the primary stem cell of umbilical cord mesenchyma.By primary for umbilical cord mesenchyma dry thin
Born of the same parents are inoculated in the serum-free medium containing 100U/ml penicillin and 100U/ml streptomycin, in 37 DEG C, 5%CO2Incubator
In carry out adhere-wall culture.
(2) human umbilical cord mesenchymal stem cells is identified
By flow cytometry human umbilical cord mesenchymal stem cells surface marker, utilize Flow Cytometry detection glimmering
Signal antibody.According to antigen-antibody combination principle, carry the thin of corresponding antigens with the antibody of specific fluorescent element labelling to known
Born of the same parents dye.The cell dyeed through fluorescein labelled antibody by flow cytometer identification, and can be taken according to known cell
The intensity of the fluorescein of band, carries out qualitative to traget antibody, quantitative analysis.
Result shows, human umbilical cord mesenchymal stem cells does not express hematopoietic cell mark such as: CD45, CD34, CD14,
CD11b (is negative), and the cells and characteristic of stem surface antigens such as CD105, CD109, CD73, CD90 are positive (being shown in Table 1), show through
Density－gradient centrifuga－tion method purifying cells suspension, obtains the purity primary stem cell of higher human umbilical cord mesenchymal.
The testing result of table 1 human umbilical cord mesenchymal stem cells surface marker
| Testing index | Expression rate | Conclusion |
| CD105 | 99.7% | Positive |
| CD109 | 99.4% | Positive |
| CD73 | 99.8% | Positive |
| CD90 | 99.6% | Positive |
| CD45 | 0.1% | Negative |
| CD34 | 0.5% | Negative |
| CD14 | 0.2% | Negative |
| CD11b | 0.4% | Negative |
Prepared by embodiment 2 human umbilical cord mesenchymal stem cells extract freeze-drying powder
(1) acquisition of the extract of human umbilical cord mesenchymal stem cells: treat that umbilical cord mesenchymal stem cells merges close to 90%, go
Except culture fluid, clean 3 times by phosphate buffered solution, add the trypsinization liquid that mass fraction is 0.25%, 37 DEG C of digestion
10min, adds the isopyknic serum-free medium without antibiotic of Digestive system and terminates digestion, centrifugal under the conditions of 1200r/min
5min, abandons supernatant, it is thus achieved that cell, uses serum-free medium re-suspended cell, carries out passing on inoculation according to the ratio of 1:3, be placed in
37 DEG C, 5%CO2Incubator is cultivated, within every 2 days, changes a culture fluid, treat that umbilical cord mesenchymal stem cells melts close to 80%
Close, discard culture fluid, change fresh serum-free medium and continue 72h, collect supernatant, and continue to pass on training with same method
Support.
(2) prepared by the extract freeze-drying powder of human umbilical cord mesenchymal stem cells: merge the cells and supernatant in the 3rd～18 generations
Liquid, adds trehalose, and the consumption of described trehalose is 5%wt cell culture supernatant, and mixing, with the sterilizing filter of 0.22 μm
Filtration sterilization, then use water for injection regulation protein concentration is to 45 ± 0.5 μ g/ml, then is placed in-80 DEG C of ultra cold storage freezers pre-
Freezing 12h, then be placed in vacuum freeze drier ,-30 DEG C keep 2h, are 1.0 × 10 in vacuum-2Mbar, freezes under the conditions of-5 DEG C
Dry 12h, keeps vacuum, continues lyophilizing 2h at 20 DEG C, obtains human umbilical cord mesenchymal stem cells extract freeze-drying powder.
Embodiment 3 uses different freeze drying protectant to prepare human umbilical cord mesenchymal stem cells extract freeze-drying powder to compare
Trehalose in the present invention is replaced as freeze drying protectant respectively using mannitol, Lactis Anhydrous, dextran, according to
Human umbilical cord mesenchymal stem cells extract is made lyophilized powder by the technique of embodiment 2, is made with embodiment 2 by the lyophilized powder obtained
The lyophilized powder obtained carries out ratio table, the results are shown in Table 2.
Table 2 uses different freeze drying protectant to prepare human umbilical cord mesenchymal stem cells extract freeze-drying powder physical behavior to compare
| Group | Outward appearance | Redissolution dissolution time (s) | The clarity of solution after redissolution | pH |
| Embodiment 2 | Dense powder shape | 4 | Clarification, transparent | 6.5～7.5 |
| Mannitol group | Part atrophy powder | 8 | Clarification, transparent | 6.5～7.5 |
| Lactis Anhydrous group | Block | 12 | Produce opalescence, muddiness | 6.0～7.0 |
| Dextran group | Block | 15 | Clarification, transparent | 6.0～7.0 |
As seen from the above table, the human umbilical cord mesenchymal stem cells extract lyophilizing that trehalose prepares is used as freeze drying protectant
Powder has good appearance character；Lyophilized powder is redissolved, dissolves very fast, dissolve completely.
Embodiment 4 human umbilical cord mesenchymal stem cells extract freeze-drying powder safety evaluatio
(1) acute toxicity test of Mouse oral human umbilical cord mesenchymal stem cells extract freeze-drying powder
Take 10 kunming mices (male and female half and half, average weight is 23.5g), give embodiment 2 to gavage of mice and make
The lyophilized powder 10 times obtained indicates the dose of concentration, observes its toxic reaction, when not causing animal dead, the most no longer carries out multiple doses
The acute oral toxicity test of amount.Result shows, any untoward reaction does not occur in mice, does not has animal death occur, shows
The human umbilical cord mesenchymal stem cells extract freeze-drying powder safety that the present invention prepares is high.
(2) rabbit smears the irritation test of human umbilical cord mesenchymal stem cells extract freeze-drying powder
Taking 10 Japan large ear rabbits (male and female half and half, about body weight 2kg), before experiment, 24h depilatory is to the medication district (back of the body
Both sides, portion) carry out depilation process, each 3cm × 3cm in unhairing scope left and right.Rabbit is carried out Baoding, rabbit left dorsal is positioned
For check plot, right positioner is trial zone, and check plot is to normal saline 0.5ml, between trial zone is to the prepared people's umbilical cord of embodiment 2
Mesenchymal stem cells extract freeze-drying powder solution 0.5ml after normal saline redissolution, adds gauze covering protection, uses immobilization with adhesive tape.
Wiping tested material, warm water cleaning after 24h, observe 30min, 2h, 6h, 12h coating part is with or without erythema and edema situation.Result shows
Showing, trial zone and check plot all without there is erythema and edema situation, show that the human umbilical cord mesenchymal stem cells that the present invention prepares carries
Take thing lyophilized powder little to skin irritation.
Ultraviolet induction human skin fibroblast is damaged by embodiment 5 human umbilical cord mesenchymal stem cells extract freeze-drying powder
Protective effect
1. experiment material: described in the prepared human umbilical cord mesenchymal stem cells extract freeze-drying powder of embodiment 2, embodiment 3
People's umbilical cord that the trehalose in the present invention prepares is replaced as freeze drying protectant respectively using mannitol, Lactis Anhydrous, dextran
Mescenchymal stem cell extract freeze-drying powder, human skin fibroblast (Ji Niou bio tech ltd, Guangzhou).
2. experimental technique: the human skin fibroblast being in exponential phase is inoculated in 96 orifice plates, treats cell monolayer
After adherent (24h), abandoning culture fluid, every hole adds 200 μ lPBS, uses 30J/cm respectively2UVA irradiate, blank group aluminum
Foil lid live, irradiate 2h every day, Continuous irradiation be randomly divided into after 4 days blank group, model group, embodiment 2 groups, mannitol group,
Lactis Anhydrous group and dextran group.Wherein, blank group and model group give isopyknic normal saline, embodiment 2 groups
Giving the human umbilical cord mesenchymal stem cells extract freeze-drying powder that embodiment 2 prepares, final protein concentration is respectively 40 μ g/ml, sweet
Dew alcohol group, Lactis Anhydrous group and dextran group give respectively described in embodiment 3 with mannitol, Lactis Anhydrous and dextran
The human umbilical cord mesenchymal stem cells extract freeze-drying powder made as freeze drying protectant, final protein concentration is 40 μ g/ml,.
After 48h, adding 20 μ lMTT (5mg/ml) to every hole, abandon supernatant after continuing to cultivate 4h, every hole adds 150 μ lDMSO and dissolves, and room temperature is shaken
Swinging 10min, make crystallization dissolve, microplate reader detects the light at each hole 492nm and absorbs (OD) value, then calculates cell viability, cell
Vigor (%)=experimental group absorbance value/blank group absorbance value × 100%, and the superoxide dismutase to cell
(SOD) activity detects, and the results are shown in Table 3.
3. experimental result
The impact on human skin fibroblast vigor of the table 3 human umbilical cord mesenchymal stem cells extract freeze-drying powder
Note: compare with blank group,**P ＜ 0.01；***P ＜ 0.001；Compare with model group,##P ＜ 0.01.
As seen from the above table, the human umbilical cord mesenchymal stem cells extract freeze-drying powder prepared using trehalose as freeze drying protectant
It is remarkably improved the vigor of human skin fibroblast, and is obviously enhanced cell SOD activity, be effective against ultraviolet and people is become
Fibrocyte oxidative damage, shows that human umbilical cord mesenchymal stem cells extract freeze-drying powder that embodiment 2 prepares can effective depositary
Umbilical cord mesenchymal stem cells culture supernatant various have a bioactive cytokine, and with mannitol, Lactis Anhydrous and
Dextran as freeze drying protectant all can not the activity of depositary's umbilical cord mesenchymal stem cells extract effectively, especially with nothing
The effect of water and milk sugar group is worst.
The impact that skin collagen is synthesized by embodiment 6 human umbilical cord mesenchymal stem cells extract freeze-drying powder
1. experimental technique: take the BALB/C nude mice 40 of 6 week old, male and female half and half, Guangdong Province's animal experimental center provide.
It is divided into embodiment 2 groups, mannitol group, Lactis Anhydrous group and dextran group, often group 10.Respectively at each group of nude mice skin of back
Right one side of something smear described in embodiment 2, embodiment 3 using mannitol, Lactis Anhydrous and dextran as freeze drying protectant system
The human umbilical cord mesenchymal stem cells extract freeze-drying powder become aqueous solution after normal saline redissolution, the skin of back of left one side of something is then
Coat normal saline as comparison, smear 200 μ l every time, 3 times/day, after 6 weeks, use special skin elasticity (water content) to test
Elasticity and the water content of nude mice skin of back measured by instrument.The situation recovered at short notice according to skin evaluates the big of elasticity
Little, elasticity number 1 is maximum, i.e. skin 100% restores to the original state.Experiment puts to death nude mice after terminating, and peels off the skin group at nude mice back
Knitting, left and right sides respectively takes 0.1g, uses the NTx in RT-PCR detection tissue and III Collagen Type VI transcriptional level.The results are shown in Table 4 Hes
Table 5.
2. experimental result
Table 4 human umbilical cord mesenchymal stem cells extract freeze-drying powder is the elastic and impact of moisture on nude mice skin
Note: compare with matched group,*P ＜ 0.05.
The impact on nude mice skin collagen protein transcriptional level of the table 5 human umbilical cord mesenchymal stem cells extract freeze-drying powder
Note: compare with matched group,**P ＜ 0.01.
From upper table 4 and 5, freeze using the human umbilical cord mesenchymal stem cells extract that trehalose prepares as freeze drying protectant
Dry powder is remarkably improved nude mice skin elasticity and moisture, and promotes nude mice skin NTx albumen and III collagen type
Synthesis, improves the content of collagen protein in skin.Show the human umbilical cord mesenchymal stem cells extract freeze-drying powder that embodiment 2 prepares
Effectively can have bioactive cytokine by the various of depositary's umbilical cord mesenchymal stem cells culture supernatant, and with manna
Alcohol, Lactis Anhydrous and dextran all can not depositary's umbilical cord mesenchymal stem cells extracts effectively as freeze drying protectant
Activity, the especially effect with Lactis Anhydrous group are worst.
Below it is only the preferred embodiment of the present invention, it is noted that it is right that above-mentioned preferred implementation is not construed as
The restriction of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, it is also possible to make some improvements and modifications, these change
Enter and retouch and also should be regarded as protection scope of the present invention.
Claims (6)
1. the preparation method of a human umbilical cord mesenchymal stem cells extract freeze-drying powder, it is characterised in that comprise the following steps:
(1) primary separation, purification and the cultivation of stem cell: separate umbilical cord jelly of Wharton from Freshman umbilical cord, Digestive system digests, and uses
150um cell filter filters, and collects filtrate, and under the conditions of filtrate is placed in 1200r/min, centrifugal 5min, abandons supernatant, uses
Balanced salt solution washed cell precipitates 2～3 times, then with the balanced salt solution re-suspended cell of 2 times of volumes, utilizes density gradient
Centrifuging purifying cells suspension, obtains the primary stem cell of umbilical cord mesenchyma, primary for umbilical cord mesenchyma stem cell is inoculated in containing
The serum-free medium of 100U/ml penicillin and 100g/ml streptomycin carries out adhere-wall culture；
(2) acquisition of stem cell extract: treat that the primary stem cell of umbilical cord mesenchyma is merged close to 90%, changes fresh not containing and resists
The serum-free medium of raw element, and carry out Secondary Culture, collect the cell culture supernatant in the 3rd～18 generations, supernatant is used
The sterilizing filter of 0.22 μm filters, and takes filtrate, obtains umbilical cord mesenchymal stem cells extract；
(3) preparation of stem cell extract freeze-drying powder: after being mixed with freeze drying protectant by umbilical cord mesenchymal stem cells extract, uses
The sterilizing filter filtration sterilization of 0.22 μm, then uses water for injection regulation protein concentration to 45 ± 0.5 μ g/ml, and freezing is dry
Dry, obtain human umbilical cord mesenchymal stem cells extract freeze-drying powder；
Described freeze drying protectant is trehalose, and the consumption of freeze drying protectant is 5%wt umbilical cord mesenchymal stem cells extract；
Described lyophilization, particularly as follows: pre-freeze 12h in-80 DEG C of ultra cold storage freezers, is subsequently placed in vacuum freeze drier ,-30
DEG C keep 1～2h, be 1.0 × 10 in vacuum-2～1.5 × 10-2Mbar, lyophilizing 10～12h under the conditions of-5 DEG C, keep vacuum
Degree, continues lyophilizing 2～4h at 20 DEG C, to obtain final product.
The preparation method of human umbilical cord mesenchymal stem cells extract freeze-drying powder the most according to claim 1, it is characterised in that
Density－gradient centrifuga－tion method in described step (1) particularly as follows: cell suspension is slowly added into equipped with equal-volume separation liquid from
In heart pipe, 4 DEG C, 2000r/min is centrifuged 15～20min, collects middle tunica albuginea layer, adds isopyknic phosphate in tunica albuginea layer
Buffer solution, 4 DEG C, 1000r/min is centrifuged 5～15min, abandons supernatant, adds isopyknic phosphate buffered solution, 4 DEG C,
800r/min is centrifuged 5～15min, abandons supernatant, takes precipitation, obtains the primary stem cell of umbilical cord mesenchyma.
The preparation method of human umbilical cord mesenchymal stem cells extract freeze-drying powder the most according to claim 2, it is characterised in that
Density－gradient centrifuga－tion method in described step (1) particularly as follows: cell suspension is slowly added into equipped with equal-volume separation liquid from
In heart pipe, 4 DEG C, 2000r/min is centrifuged 20min, collects middle tunica albuginea layer, adds isopyknic flat phosphate and delay in tunica albuginea layer
Dissolved liquid, 4 DEG C, 1000r/min is centrifuged 10min, abandons supernatant, adds isopyknic phosphate buffered solution, 4 DEG C, 800r/
Min is centrifuged 10min, abandons supernatant, takes precipitation, obtains the primary stem cell of umbilical cord mesenchyma.
4., according to the preparation method of the human umbilical cord mesenchymal stem cells extract freeze-drying powder described in Claims 2 or 3, its feature exists
In, described separation liquid be concentration be the ficoll-sodium amidotrizoate solution of 1.073g/mL.
The preparation method of human umbilical cord mesenchymal stem cells extract freeze-drying powder the most according to claim 1, it is characterised in that
Digestive system digestion in described step (1) is particularly as follows: the volume ratio pressing 1:3 adds Digestive system, and 37 DEG C of digestion 3h, Digestive system is for containing
Having the phosphate buffered solution of collagenase II, hyaluronidase and EDTA, wherein, collagenase II, hyaluronidase and EDTA exist
Quality percent by volume final concentration in phosphate buffered solution is respectively 0.12%, 0.06%, 0.02%.
6. human umbilical cord mesenchymal stem cells extract freeze-drying powder, it is characterised in that according to the arbitrary described preparation of claim 1-5
Method obtains.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610529860.3A CN106109496B (en) | 2016-07-06 | 2016-07-06 | Human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610529860.3A CN106109496B (en) | 2016-07-06 | 2016-07-06 | Human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106109496A true CN106109496A (en) | 2016-11-16 |
| CN106109496B CN106109496B (en) | 2019-11-05 |
Family
ID=57282833
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610529860.3A Active CN106109496B (en) | 2016-07-06 | 2016-07-06 | Human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106109496B (en) |
Cited By (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367389A (en) * | 2016-11-15 | 2017-02-01 | 东莞自然衡健康科技有限公司 | Preparation method and application of human umbilical cord mesenchymal stem cell factors |
| CN107260570A (en) * | 2017-06-14 | 2017-10-20 | 南京仯素生物科技有限公司 | A kind of mescenchymal stem cell factor composition and its production and use |
| CN108159078A (en) * | 2018-01-26 | 2018-06-15 | 深圳市新仑生物科技有限公司 | A kind of Porcine HGF freeze-dried powder, preparation method and application |
| CN108186682A (en) * | 2018-02-01 | 2018-06-22 | 伯仕利生物科技发展（盐城）有限公司 | A kind of preparation method of placenta mesenchyma stem cell freeze-dried powder |
| CN108309921A (en) * | 2017-12-21 | 2018-07-24 | 云南舜喜再生医学工程有限公司 | A kind of method for preparing freeze-dried powder rich in cell factor |
| CN108888634A (en) * | 2018-09-06 | 2018-11-27 | 广州苿莱生物科技有限公司 | The preparation method and application of hair follicle stem cells extract freeze-drying powder |
| CN109106728A (en) * | 2018-09-06 | 2019-01-01 | 广州苿莱生物科技有限公司 | The preparation method and application of human placenta stem cell extract freeze-drying powder |
| CN109453200A (en) * | 2018-11-29 | 2019-03-12 | 云南研灵生物科技有限公司 | The preparation method of mostly tissue-derived mescenchymal stem cell factor lytic freeze-dried powder |
| CN110179826A (en) * | 2019-05-29 | 2019-08-30 | 武汉五州润达生物医药科技有限公司 | Human umbilical cord mesenchymal stem cells Derived Stem Cells factor microcapsule bubble preparation and preparation method |
| CN110448572A (en) * | 2019-08-04 | 2019-11-15 | 丰泽康生物医药（深圳）有限公司 | A kind of preparation method of umbilical cord mesenchymal stem cells active matter and the compound of cord blood stem cell active matter |
| WO2020095592A1 (en) * | 2018-11-07 | 2020-05-14 | 剛士 田邊 | Pharmaceutical composition and cosmetic composition |
| CN111351930A (en) * | 2020-02-20 | 2020-06-30 | 泛肽生物科技(浙江)有限公司 | Quality control product of active human lymphocyte surface antigen and preparation method thereof |
| CN112695013A (en) * | 2021-01-15 | 2021-04-23 | 苏州京赛诺生物科技有限公司 | Conditioned medium, freeze-dried powder, preparation method and application |
| CN113133447A (en) * | 2021-05-20 | 2021-07-20 | 郑州优倍得生物科技有限公司 | Cryopreservation liquid and cryopreservation method for umbilical cord mesenchymal stem cells |
| JP2021120398A (en) * | 2018-11-07 | 2021-08-19 | 剛士 田邊 | Pharmaceutical composition and cosmetic composition |
| CN113521019A (en) * | 2021-07-27 | 2021-10-22 | 吉林大学第一医院 | Mesenchymal stem cell supernatant freeze-dried preparation and preparation method thereof |
| CN113583950A (en) * | 2021-08-06 | 2021-11-02 | 合肥滴碧云生物科技有限公司 | Method for preparing stem cell active factor and application thereof |
| CN114262684A (en) * | 2021-12-07 | 2022-04-01 | 上海泽充生物技术有限公司 | Method for extracting supernatant of stem cells |
| CN114469997A (en) * | 2022-03-18 | 2022-05-13 | 北京岳淘生物科技有限公司 | Application of umbilical cord stem cell freeze-dried powder in preparation of medicines and cosmetics |
| CN114990059A (en) * | 2022-06-30 | 2022-09-02 | 阿谷巴(杭州)生物科技发展有限公司 | Preparation method of umbilical cord mesenchymal stem cell freeze-dried powder |
| CN115181172A (en) * | 2022-07-12 | 2022-10-14 | 广东唯泰生物科技有限公司 | Cell factor for removing freckles and whitening skin and preparation method and application thereof |
| CN116004530A (en) * | 2023-02-09 | 2023-04-25 | 广东省科玮智丽生物医药有限公司 | Freeze-dried powder extracted from umbilical cord mesenchymal stem cells and preparation method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090081171A1 (en) * | 2006-08-11 | 2009-03-26 | Yu-Show Fu | Cell system for alleviating syndromes of Parkinson's disease in a mammal |
| CN101525594A (en) * | 2009-04-17 | 2009-09-09 | 中国医学科学院血液学研究所 | Complete medium with low serum concentration for cultivating mesenchymal stem cells and method for cultivating mesenchymal stem cells using same |
| CN104523753A (en) * | 2015-01-22 | 2015-04-22 | 中国科学院广州生物医药与健康研究院 | Preparation method, product and application of human umbilical cord mesenchymal stem cell cultural supernatant active factor and cell lysis buffer |
| CN105543313A (en) * | 2015-12-29 | 2016-05-04 | 四川新生命干细胞科技股份有限公司 | Human-derived mesenchymal stem cell factor, and preparation method and application thereof |
-
2016
- 2016-07-06 CN CN201610529860.3A patent/CN106109496B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090081171A1 (en) * | 2006-08-11 | 2009-03-26 | Yu-Show Fu | Cell system for alleviating syndromes of Parkinson's disease in a mammal |
| CN101525594A (en) * | 2009-04-17 | 2009-09-09 | 中国医学科学院血液学研究所 | Complete medium with low serum concentration for cultivating mesenchymal stem cells and method for cultivating mesenchymal stem cells using same |
| CN104523753A (en) * | 2015-01-22 | 2015-04-22 | 中国科学院广州生物医药与健康研究院 | Preparation method, product and application of human umbilical cord mesenchymal stem cell cultural supernatant active factor and cell lysis buffer |
| CN105543313A (en) * | 2015-12-29 | 2016-05-04 | 四川新生命干细胞科技股份有限公司 | Human-derived mesenchymal stem cell factor, and preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| KOCH,TG ET AL.: "Isolation of mesenchymal stem cells from equine umbilical cord blood", 《BMC BIOTECHNOLOGY》 * |
| 洪小杨等: ""人脐带血间充质干细胞体外分离培养研究"", 《中国误诊学杂志》 * |
| 范菊莉等: ""冷冻干燥法保存人骨髓间充质干细胞的实验初探"", 《世界科技研究与发展》 * |
| 陆士新: "《干细胞与肿瘤》", 31 July 2009, 中国协和医科大学出版社 * |
Cited By (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367389A (en) * | 2016-11-15 | 2017-02-01 | 东莞自然衡健康科技有限公司 | Preparation method and application of human umbilical cord mesenchymal stem cell factors |
| CN107260570B (en) * | 2017-06-14 | 2020-07-31 | 南京仯素生物科技有限公司 | Mesenchymal stem cell factor composition and preparation method and application thereof |
| CN107260570A (en) * | 2017-06-14 | 2017-10-20 | 南京仯素生物科技有限公司 | A kind of mescenchymal stem cell factor composition and its production and use |
| CN108309921A (en) * | 2017-12-21 | 2018-07-24 | 云南舜喜再生医学工程有限公司 | A kind of method for preparing freeze-dried powder rich in cell factor |
| CN108159078A (en) * | 2018-01-26 | 2018-06-15 | 深圳市新仑生物科技有限公司 | A kind of Porcine HGF freeze-dried powder, preparation method and application |
| CN108186682A (en) * | 2018-02-01 | 2018-06-22 | 伯仕利生物科技发展（盐城）有限公司 | A kind of preparation method of placenta mesenchyma stem cell freeze-dried powder |
| CN108888634A (en) * | 2018-09-06 | 2018-11-27 | 广州苿莱生物科技有限公司 | The preparation method and application of hair follicle stem cells extract freeze-drying powder |
| CN109106728A (en) * | 2018-09-06 | 2019-01-01 | 广州苿莱生物科技有限公司 | The preparation method and application of human placenta stem cell extract freeze-drying powder |
| US12036245B2 (en) | 2018-11-07 | 2024-07-16 | I Peace, Inc. | Pharmaceutical composition and cosmetic composition |
| JP7518513B2 (en) | 2018-11-07 | 2024-07-18 | 剛士 田邊 | Pharmaceutical and cosmetic compositions |
| JP2021120398A (en) * | 2018-11-07 | 2021-08-19 | 剛士 田邊 | Pharmaceutical composition and cosmetic composition |
| WO2020095592A1 (en) * | 2018-11-07 | 2020-05-14 | 剛士 田邊 | Pharmaceutical composition and cosmetic composition |
| CN109453200A (en) * | 2018-11-29 | 2019-03-12 | 云南研灵生物科技有限公司 | The preparation method of mostly tissue-derived mescenchymal stem cell factor lytic freeze-dried powder |
| CN110179826A (en) * | 2019-05-29 | 2019-08-30 | 武汉五州润达生物医药科技有限公司 | Human umbilical cord mesenchymal stem cells Derived Stem Cells factor microcapsule bubble preparation and preparation method |
| CN110448572A (en) * | 2019-08-04 | 2019-11-15 | 丰泽康生物医药（深圳）有限公司 | A kind of preparation method of umbilical cord mesenchymal stem cells active matter and the compound of cord blood stem cell active matter |
| CN111351930A (en) * | 2020-02-20 | 2020-06-30 | 泛肽生物科技(浙江)有限公司 | Quality control product of active human lymphocyte surface antigen and preparation method thereof |
| CN112695013A (en) * | 2021-01-15 | 2021-04-23 | 苏州京赛诺生物科技有限公司 | Conditioned medium, freeze-dried powder, preparation method and application |
| CN113133447A (en) * | 2021-05-20 | 2021-07-20 | 郑州优倍得生物科技有限公司 | Cryopreservation liquid and cryopreservation method for umbilical cord mesenchymal stem cells |
| CN113521019B (en) * | 2021-07-27 | 2023-08-18 | 吉林大学第一医院 | Mesenchymal stem cell supernatant freeze-dried preparation and preparation method thereof |
| CN113521019A (en) * | 2021-07-27 | 2021-10-22 | 吉林大学第一医院 | Mesenchymal stem cell supernatant freeze-dried preparation and preparation method thereof |
| CN113583950A (en) * | 2021-08-06 | 2021-11-02 | 合肥滴碧云生物科技有限公司 | Method for preparing stem cell active factor and application thereof |
| CN114262684A (en) * | 2021-12-07 | 2022-04-01 | 上海泽充生物技术有限公司 | Method for extracting supernatant of stem cells |
| CN114469997A (en) * | 2022-03-18 | 2022-05-13 | 北京岳淘生物科技有限公司 | Application of umbilical cord stem cell freeze-dried powder in preparation of medicines and cosmetics |
| CN114469997B (en) * | 2022-03-18 | 2023-11-07 | 陕西三八妇乐科技股份有限公司 | Application of umbilical cord stem cell freeze-dried powder in preparation of medicines |
| CN114990059A (en) * | 2022-06-30 | 2022-09-02 | 阿谷巴(杭州)生物科技发展有限公司 | Preparation method of umbilical cord mesenchymal stem cell freeze-dried powder |
| CN114990059B (en) * | 2022-06-30 | 2024-04-26 | 阿谷巴(杭州)生物科技发展有限公司 | Preparation method of freeze-dried powder of umbilical mesenchymal stem cells |
| CN115181172A (en) * | 2022-07-12 | 2022-10-14 | 广东唯泰生物科技有限公司 | Cell factor for removing freckles and whitening skin and preparation method and application thereof |
| CN116004530A (en) * | 2023-02-09 | 2023-04-25 | 广东省科玮智丽生物医药有限公司 | Freeze-dried powder extracted from umbilical cord mesenchymal stem cells and preparation method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106109496B (en) | 2019-11-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106109496B (en) | Human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method | |
| CN101757691B (en) | Preparation method of tissue engineering cornea | |
| CN101890050B (en) | Human umbilical cordmesenchymal stem cell-derived exosome and application thereof | |
| CN108823156A (en) | For the clinical grade human umbilical cord mesenchymal stem cells composite factor of reparation and the preparation method of freeze-dried powder | |
| CN106754674A (en) | Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN108486047B (en) | Medical dressing of stem cell extract and preparation method thereof | |
| CN107073037A (en) | Composition comprising mescenchymal stem cell hydrogel and preparation method thereof | |
| KR20100065338A (en) | Method for extracting mesenchymal stem cell from human or animal embryo and for extracting the secretion product thereof | |
| CN108159078A (en) | A kind of Porcine HGF freeze-dried powder, preparation method and application | |
| CN104622902B (en) | It is a kind of for treating the stem cell medicine of liver fibrosis | |
| CN103881971B (en) | Culture medium for culturing and/or amplifying mesenchymal stem cells and culture method thereof | |
| CN104726406A (en) | Method for inducing dental pulp mesenchymal stem cells to be differentiated into nerve cells | |
| WO2008002063A1 (en) | Soft tissue filler composition comprising autologous dermis-derived cell culture product and hyaluronic acid | |
| CN113304103A (en) | Preparation method and application of human mesenchymal stem cell supernatant dry powder gel | |
| CN109893541B (en) | Application of exosome derived from menstrual blood stem cells in preparation of medicine for treating intrauterine adhesion | |
| CN104523753A (en) | Preparation method, product and application of human umbilical cord mesenchymal stem cell cultural supernatant active factor and cell lysis buffer | |
| CN102965337A (en) | Method for separating and extracting human subcutaneous adipose-derived mesenchymal stem cells and special culture medium for extraction | |
| CN108186682A (en) | A kind of preparation method of placenta mesenchyma stem cell freeze-dried powder | |
| CN107349220A (en) | A kind of preparation comprising fibroblast excretion body and application thereof | |
| CN106701670A (en) | Methods for enhancing bioactive factor secretion capacity of mesenchymal stem cells and extracting active factors in culture solution | |
| CN108795852A (en) | A kind of preparation method, product and its application of people's myoblast excretion body | |
| CN102161981B (en) | Method for jointly inducing bone marrow mesenchymal stem cells into sweat gland cells by recombinant protein | |
| AU2014396937A1 (en) | Method for manufacturing induced pluripotent stem cells from adipose-derived mesenchymal stem cells and induced pluripotent stem cells manufactured by same method | |
| CN103468744B (en) | VEGF165 gene modified hair follicle stem cells and preparation method thereof | |
| CN115125192A (en) | Bone marrow supernatant and application thereof in cell culture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230630 Address after: Room 101, No. 39 Baoying Avenue, Huangpu District, Guangzhou City, Guangdong Province, 510700, self-made unit 102 Patentee after: Guangdong Huasang Lixi Biotechnology Co.,Ltd. Address before: Room 301 and 401, No. 39 Baoying Avenue, Guangzhou Free Trade Zone, Guangdong Province, 510070 Patentee before: GUANGDONG COOWAY BIOTECHNOLOGY Co.,Ltd. |
|
| TR01 | Transfer of patent right |