CN107475187A - Nutrient solution and a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body - Google Patents
Nutrient solution and a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body Download PDFInfo
- Publication number
- CN107475187A CN107475187A CN201710790115.9A CN201710790115A CN107475187A CN 107475187 A CN107475187 A CN 107475187A CN 201710790115 A CN201710790115 A CN 201710790115A CN 107475187 A CN107475187 A CN 107475187A
- Authority
- CN
- China
- Prior art keywords
- excretion body
- umbilical cord
- stem cells
- mesenchymal stem
- nutrient solution
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/24—Interferons [IFN]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2531/00—Microcarriers
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Rheumatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The production technology of umbilical cord mesenchymal stem cells excretion body is largely obtained the invention discloses a kind of nutrient solution and using the nutrient solution, belongs to biomedicine field.Recombinant interferon-gamma, serum substitute, penicillin and streptomysin are added in nutrient solution of the present invention, compared with being not added with above-mentioned four kinds of components, hence it is evident that improve the content of excretion body in nutrient solution.Recombinant interferon-gamma is especially with the addition of, is compared through parallel test, addition recombinant interferon-gamma causes the amount of excretion body in nutrient solution to increase by more than 40%.The structure of excretion body will not come to harm obtained by handling process of the present invention, so as to keep the high activity of umbilical cord mesenchymal stem cells excretion body, though compensate for the existing precipitation method can it is relatively large acquisition excretion body can but destroy excretion body structure so as to influence the problem of its is active, though while compensate for existing supercentrifugal process can guarantee that excretion body structure be not damaged sample throughput it is small the problem of.The present invention is suitable to commercial Application.
Description
Technical field
The present invention relates to biomedicine field, more particularly to a kind of nutrient solution and largely obtained between umbilical cord using the nutrient solution
The production technology of mesenchymal stem cells excretion body.
Background technology
Stem cell is a kind of cell with the of self-replication capacity and multi-lineage potential characteristic, can be used as therapeutic gram
Grand research and treatment resource, have turned into one of frontier science and technology field for being attracted attention.Mescenchymal stem cell is embryo in a group
The multipotential stem cell with multi-lineage potential in layer source, verified its are present in marrow, liver, spleen, Cheng Renhe
The tissue such as the skeletal muscle of the elderly, peripheral blood, fat, placenta, umbilical cord, bleeding of the umbilicus, skin.
Umbilical cord mesenchymal stem cells are located away from the postnatal discarded tissue of neonate -- umbilical cord.Because of its wide material sources, easily obtain
Take, without moral check dispute, immunogenicity it is low, to advantages such as donor fanout free regions, turned between most Transformation Application prospect
Mesenchymal stem cells.Umbilical cord mesenchymal stem cells can be used for treating diabetes, central nervous system injury, rheumatic arthritis, erythema
The diseases such as lupus.The various biological function that mescenchymal stem cell has clinically has obtained extensive concern, its therapy apparatus
It is made for the focus of stem cell field correlative study in recent years.Excretion body is considered as that mescenchymal stem cell plays paracrine action
Carrier.
Excretion body is that mescenchymal stem cell is discharged into extracellular nanometer in incubation after a series of metabolism of intracellular
Level vesicles.These vesicles have been proved to have and the same or analogous treatment function of stem cell.Compared with stem cell, outside
Secreting body has many advantages, such as stablize it is easy to maintain, without immunogenicity etc..
Traditional umbilical cord mesenchymal stem cells culture is using 2 dimension cultural methods（Planar process）, the area for cultivating face limits
The amplification times of stem cell.In recent years, it is to solve umbilical cord mesenchymal stem cells amplification times using the Maitland culture of microcarrier
A kind of limited feasible means.The excretion body and cell quantity of umbilical cord mesenchymal stem cells are positively related, using microcarrier
While cultivating umbilical cord mesenchymal stem cells, substantial amounts of excretion body can be harvested.
The collection of traditional excretion body has two kinds of supercentrifugation and the precipitation method.Supercentrifugation can ensure excretion body structure
It is not damaged but sample throughput is small, cost is high, it is difficult to realizes requirements of mass production.The precipitation method can be with relatively large extraction excretion
Body, but excretion body structure can be destroyed so as to influence activity, in addition, traditional precipitation method have protein contamination.
The content of the invention
In order to make up the deficiencies in the prior art, solve existing umbilical cord mesenchymal stem cells excretion body production technology yield it is small,
The problem of excretion body is destructurized, purity is not high, largely obtained the invention provides a kind of nutrient solution and using the nutrient solution
The production technology of umbilical cord mesenchymal stem cells excretion body.Culture process of the invention by changing umbilical cord mesenchymal stem cells, and
Irritating cell factor-people's recombinant interferon-gamma is added, suitable bioreactor is selected, optimization culture parameter, obtains
The nutrient solution of excretion body rich content.Then the method for using ultrafiltration, optimizes parameters, realizes the first of large volume excretion body stoste
Step concentration；Low temperature ultracentrifugation is reused, obtains the satisfactory excretion body product of function, form indices.
The technical scheme is that：
It is a kind of be used for largely obtain umbilical cord mesenchymal stem cells excretion bodies production technologies nutrient solution, the nutrient solution by
Recombinant interferon-gamma, serum substitute, penicillin and streptomysin is added in MSCGM-CD mescenchymal stem cell culture mediums to be made.
Preferably, the concentration of recombinant interferon-gamma is 45-55 μ g/L in the nutrient solution, the matter of serum substitute
Amount fraction is 8%-12%, and the concentration of penicillin is 80-120 units/mL, and the concentration of streptomysin is 80-120 units/mL.
The production technology of umbilical cord mesenchymal stem cells excretion body is largely obtained using the nutrient solution, umbilical cord mesenchyma is dry thin
Born of the same parents, which are inoculated in the bioreactor containing Cultispher-G microcarriers and the nutrient solution, carries out amplification cultivation, and culture finishes
Remove umbilical cord mesenchymal stem cells, purified acquisition umbilical cord mesenchymal stem cells excretion body.
Recombinant interferon-gamma is used for the generation for promoting excretion body in nutrient solution used in the present invention.
Preferably, umbilical cord mesenchymal stem cells condition of culture is：Rotating speed 40-50 rpm, PH 7.0-7.2,
36.5-37.5 DEG C, 5% CO2。
Preferably, the density of the Cultispher-G microcarriers is 1.5-2.5g/L.
Preferably, the inoculum density of umbilical cord mesenchymal stem cells is 2-6 × 106Individual/mL.
Preferably, the concentration of recombinant interferon-gamma is 45-55 μ g/L in the nutrient solution, the matter of serum substitute
Amount fraction is 8%-12%, and the concentration of penicillin is 80-120 units/mL, and the concentration of streptomysin is 80-120 units/mL.
Preferably, umbilical cord mesenchymal stem cells are removed by centrifuging.
Further, described purify is specially：Centrifugation gained supernatant is put into milipore filter ultrafiltration, ultrafiltration finishes, and use is micro-
Rice filter filtration sterilization, obtains excretion body crude extract；Excretion body crude extract is centrifuged using Ultracentrifuge, then uses PBS
Packing is resuspended, obtains the umbilical cord mesenchymal stem cells excretion body of purifying.
Further, the milipore filter is 10000NWCO milipore filters.
Beneficial effects of the present invention are：
Recombinant interferon-gamma, serum substitute, penicillin and streptomysin are added in nutrient solution of the present invention, with being not added with above-mentioned four kinds
Component is compared, hence it is evident that improves the content of excretion body in nutrient solution.In addition, parallel test proves, recombinant interferon in nutrient solution
γ, serum substitute, penicillin and the component of streptomysin four complement each other, indispensable；Lack any, umbilical cord mesenchyma is dry thin
The growing state of born of the same parents can substantially be deteriorated.Especially recombinant interferon-gamma, compared through parallel test, addition recombinant interferon-gamma causes
The amount increase by more than 40% of excretion body in nutrient solution.
The structure of excretion body will not come to harm obtained by handling process of the present invention, so as to keep outside umbilical cord mesenchymal stem cells
The high activity of body is secreted, excretion body structure can be destroyed so as to influence it though compensate for the existing precipitation method and relatively large can obtain excretion body
The problem of active, though while it compensate for existing supercentrifugal process and can guarantee that the excretion body structure sample throughput that is not damaged is small
Problem.The present invention is suitable to commercial Application.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, without having to pay creative labor, may be used also
To obtain other accompanying drawings according to these accompanying drawings.
Fig. 1 is growth curve chart of the human umbilical cord mesenchymal stem cells in culture systems of the present invention；
Fig. 2 is the transmission electron microscope photo of present invention gained excretion body；Wherein black arrow points to excretion body.
Embodiment
Embodiment 1
A kind of a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body, including step：
1st, under aseptic condition, umbilical cord 8 obtained by the production operation of moon plane palace is taken fully to 10cm；Puerpera knows umbilical cord processing, and passes through
Cross the paper committee of institute approval.
2nd, under aseptic condition, tried one's best wash clean Cord blood with PBS, peel off umbilical cord outer membrane, arteria umbilicalis, umbilical vein, the China taken
Tong Shi glue.The magnificent Tong Shi glue of acquisition is built up into 1mm × 1mm × 1mm tissue block.It is after PBS 3 times, tissue block is uniform
Be layered in T75 blake bottles, add a small amount of nutrient solution and keep tissue block moistening；Liquid is changed per 5d once.
3rd, passed on after cell density reaches 80%：0.25% 37 DEG C of pancreas enzyme -EDTA digests 2min, after nutrient solution neutralizes,
1000 rpm centrifuge 5 min, add new nutrient solution and are resuspended and with 8 × 104Individual cell/ml density passage is trained to T75
Support in bottle, 37 DEG C, 5 % CO2Under the conditions of cultivate.A nutrient solution is changed within every 2 days, after cell growth to 80-90 % merges
, passage is once.
4th, ware of making even grows to the 3rd generation human umbilical cord mesenchymal stem cells of 80-90% fusions, the digestion of 0.25% pancreas enzyme -EDTA
Afterwards, nutrient solution neutralizes, and 1000rpm centrifugation 5min, fresh medium is resuspended.4 × 10 will be contained6The suspension of individual cell is separately added into
Bioreactor containing Cultispher-G microcarriers（The density of Cultispher-G microcarriers is 2g/L）, add culture
Liquid 200ml, intermittent stirring condition are that 45rpm stirs 2min, stop 13min, so circulate 3h, 37 DEG C, 5% CO2.Inoculation terminates
Afterwards, 500ml nutrient solutions are filled into the final working volumes of 1000ml, adjustment of rotational speed 55rpm.Add nutrient solution 200mL within every 3 days,
To final working volume 1000mL.
Nutrient solution described in step 1-4 is by adding recombinant interferon in MSCGM-CD mescenchymal stem cell culture mediums
γ, serum substitute, penicillin and nutrient solution made from streptomysin；Wherein, the concentration of recombinant interferon-gamma is 50 μ g/L, serum
The mass fraction of substitute is 10%, and the concentration of penicillin is 100 units/mL, and the concentration of streptomysin is 100 units/mL.
Growth curve chart of the human umbilical cord mesenchymal stem cells in nutrient solution of the present invention as shown in figure 1, as shown in Figure 1, this
Invention culture liquid system can promote the growth of stem cell.
5th, step 4 amplification cultivation and the nutrient solution 1000ml after excretion body is secreted, point four centrifuge tube 1000rpm centrifugations
After 10min removes cell, supernatant is put into 10000NWCO milipore filters, admission pressure 480kPa.
6th, PBS is used after the completion of ultrafiltration 3 times, is settled to 20mL, 0.22 micron filter filtration sterilization.Obtain excretion body
Crude extract.
7th, obtained excretion body crude extract Ultracentrifuge is centrifuged 4 DEG C, 100000g centrifugations 70min.With PBS weights
Dispensed after outstanding, minus 80 DEG C of preservations.The transmission electron microscope photo of excretion body is as shown in Fig. 2 wherein black arrow refers to obtained by the present embodiment
To excretion body, excretion body is nanoscale vesicles in Fig. 2.As shown in Figure 2, the method for purification of excretion body can protect in the present embodiment
Hold the structural intergrity of excretion body.
Embodiment 2
A kind of a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body, including step：
1st, under aseptic condition, umbilical cord 8 obtained by the production operation of moon plane palace is taken fully to 10cm；Puerpera knows umbilical cord processing, and passes through
Cross the paper committee of institute approval.
2nd, under aseptic condition, tried one's best wash clean Cord blood with PBS, peel off umbilical cord outer membrane, arteria umbilicalis, umbilical vein, the China taken
Tong Shi glue.The magnificent Tong Shi glue of acquisition is built up into 1mm × 1mm × 1mm tissue block.It is after PBS 3 times, tissue block is uniform
Be layered in T75 blake bottles, add a small amount of nutrient solution and keep tissue block moistening；Liquid is changed per 5d once.
3rd, passed on after cell density reaches 80%：0.3% 37 DEG C of pancreas enzyme -EDTA digests 2min, after nutrient solution neutralizes,
1500 rpm centrifuge 4 min, add new nutrient solution and are resuspended and with 1 × 105Individual cell/ml density passage is cultivated to T75
In bottle, 37 DEG C, 4.7% CO2Under the conditions of cultivate.Change a nutrient solution within every 2 days, after cell growth to 80-90 % merges,
Passage is once.
4th, ware of making even grows to the 3rd generation human umbilical cord mesenchymal stem cells of 80-90% fusions, the digestion of 0.3% pancreas enzyme -EDTA
Afterwards, nutrient solution neutralizes, and 1500rpm centrifugation 4min, fresh medium is resuspended.7 × 10 will be contained6The suspension of individual cell is separately added into
Bioreactor containing Cultispher-G microcarriers（The density of Cultispher-G microcarriers is 1.5g/L）, add training
Nutrient solution 200ml, intermittent stirring condition are that 45rpm stirs 2min, stop 13min, so circulate 3h, 37 DEG C, 4.7% CO2.Inoculation
After end, 500ml nutrient solutions are filled into the final working volumes of 1000ml, adjustment of rotational speed 55rpm.Add nutrient solution within every 3 days
200mL, to final working volume 1000mL.
Nutrient solution described in step 1-4 is by adding recombinant interferon in MSCGM-CD mescenchymal stem cell culture mediums
γ, serum substitute, penicillin and nutrient solution made from streptomysin；Wherein, the concentration of recombinant interferon-gamma is 45 μ g/L, serum
The mass fraction of substitute is 8.5%, and the concentration of penicillin is 85 units/mL, and the concentration of streptomysin is 115 units/mL.
5th, step 4 amplification cultivation and the nutrient solution 1000ml after excretion body is secreted, point four centrifuge tube 1000rpm centrifugations
After 10min removes cell, supernatant is put into 10000NWCO milipore filters, admission pressure 480kPa.
6th, PBS is used after the completion of ultrafiltration 3 times, is settled to 20mL, 0.22 micron filter filtration sterilization.Obtain excretion body
Crude extract.
7th, obtained excretion body crude extract Ultracentrifuge is centrifuged 4 DEG C, 100000g centrifugations 70min.With PBS weights
Dispensed after outstanding, minus 80 DEG C of preservations.
Embodiment 3
A kind of a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body, including step：
1st, under aseptic condition, umbilical cord 8 obtained by the production operation of moon plane palace is taken fully to 10cm；Puerpera knows umbilical cord processing, and passes through
Cross the paper committee of institute approval.
2nd, under aseptic condition, tried one's best wash clean Cord blood with PBS, peel off umbilical cord outer membrane, arteria umbilicalis, umbilical vein, the China taken
Tong Shi glue.The magnificent Tong Shi glue of acquisition is built up into 1mm × 1mm × 1mm tissue block.It is after PBS 3 times, tissue block is uniform
Be layered in T75 blake bottles, add a small amount of nutrient solution and keep tissue block moistening；Liquid is changed per 5d once.
3rd, passed on after cell density reaches 80%：0.20% 37 DEG C of pancreas enzyme -EDTA digests 3min, after nutrient solution neutralizes,
1000 rpm centrifuge 5 min, add new nutrient solution and are resuspended and with 5 × 104Individual cell/ml density passage is trained to T75
Support in bottle, 37 DEG C, 5.2% CO2Under the conditions of cultivate.A nutrient solution is changed within every 2 days, after cell growth to 80-90 % merges
, passage is once.
4th, ware of making even grows to the 3rd generation human umbilical cord mesenchymal stem cells of 80-90% fusions, the digestion of 0.25% pancreas enzyme -EDTA
Afterwards, nutrient solution neutralizes, and 1000rpm centrifugation 5min, fresh medium is resuspended.5 × 10 will be contained6The suspension of individual cell is separately added into
Bioreactor containing Cultispher-G microcarriers（The density of Cultispher-G microcarriers is 2.5g/L）, add training
Nutrient solution 200ml, intermittent stirring condition are that 45rpm stirs 2min, stop 13min, so circulate 3h, 37 DEG C, 5.2% CO2.Inoculation
After end, 500ml nutrient solutions are filled into the final working volumes of 1000ml, adjustment of rotational speed 55rpm.Add nutrient solution within every 3 days
200mL, to final working volume 1000mL.
Nutrient solution described in step 1-4 is by adding recombinant interferon in MSCGM-CD mescenchymal stem cell culture mediums
γ, serum substitute, penicillin and nutrient solution made from streptomysin；Wherein, the concentration of recombinant interferon-gamma is 53 μ g/L, serum
The mass fraction of substitute is 12%, and the concentration of penicillin is 115 units/mL, and the concentration of streptomysin is 85 units/mL.
5th, step 4 amplification cultivation and the nutrient solution 1000ml after excretion body is secreted, point four centrifuge tube 1000rpm centrifugations
After 10min removes cell, supernatant is put into 10000NWCO milipore filters, admission pressure 480kPa.
6th, PBS is used after the completion of ultrafiltration 3 times, is settled to 20mL, 0.22 micron filter filtration sterilization.Obtain excretion body
Crude extract.
7th, obtained excretion body crude extract Ultracentrifuge is centrifuged 4 DEG C, 100000g centrifugations 70min.With PBS weights
Dispensed after outstanding, minus 80 DEG C of preservations.
Claims (10)
- A kind of 1. nutrient solution for being used to largely obtain the production technology of umbilical cord mesenchymal stem cells excretion body, it is characterised in that：Institute Nutrient solution is stated by adding recombinant interferon-gamma, serum substitute, penicillin and chain in MSCGM-CD mescenchymal stem cell culture mediums Mycin is made.
- 2. it is used for the nutrient solution for largely obtaining the production technology of umbilical cord mesenchymal stem cells excretion body as claimed in claim 1, its It is characterised by：The concentration of recombinant interferon-gamma is 45-55 μ g/L in the nutrient solution, and the mass fraction of serum substitute is 8%- 12%, the concentration of penicillin is 80-120 units/mL, and the concentration of streptomysin is 80-120 units/mL.
- 3. largely obtaining the production technology of umbilical cord mesenchymal stem cells excretion body using nutrient solution as claimed in claim 1, it is special Sign is：Umbilical cord mesenchymal stem cells are inoculated in the bioreactor containing Cultispher-G microcarriers and the nutrient solution Amplification cultivation is carried out, culture finishes removal umbilical cord mesenchymal stem cells, purified acquisition umbilical cord mesenchymal stem cells excretion body.
- 4. a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body as claimed in claim 3, it is characterised in that umbilical cord Mescenchymal stem cell condition of culture is：Rotating speed 40-50 rpm, PH 7.0-7.2,36.5-37.5 DEG C, 5%CO2。
- 5. the production technology of umbilical cord mesenchymal stem cells excretion body is largely obtained as described in claim 3 or 4, it is characterised in that： The density of the Cultispher-G microcarriers is 1.5-2.5g/L.
- 6. a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body as claimed in claim 3, it is characterised in that：Umbilical cord The inoculum density of mescenchymal stem cell is 2-6 × 106Individual/mL.
- 7. largely obtaining the production technology of umbilical cord mesenchymal stem cells excretion body as described in any one of claim 3,4,6, it is special Sign is：The concentration of recombinant interferon-gamma is 45-55 μ g/L in the nutrient solution, and the mass fraction of serum substitute is 8%- 12%, the concentration of penicillin is 80-120 units/mL, and the concentration of streptomysin is 80-120 units/mL.
- 8. a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body as claimed in claim 3, it is characterised in that：Pass through Centrifugation removes umbilical cord mesenchymal stem cells.
- 9. a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body as claimed in claim 8, it is characterised in that described Purifying is specially：Centrifugation gained supernatant is put into milipore filter ultrafiltration, ultrafiltration finishes, and using micron filter filtration sterilization, obtains Excretion body crude extract；Excretion body crude extract is centrifuged using Ultracentrifuge, is then resuspended and dispensed with PBS, obtains the umbilical cord of purifying Mescenchymal stem cell excretion body.
- 10. a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body as claimed in claim 9, it is characterised in that：Institute It is 10000NWCO milipore filters to state milipore filter.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710790115.9A CN107475187A (en) | 2017-09-05 | 2017-09-05 | Nutrient solution and a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710790115.9A CN107475187A (en) | 2017-09-05 | 2017-09-05 | Nutrient solution and a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107475187A true CN107475187A (en) | 2017-12-15 |
Family
ID=60604635
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710790115.9A Pending CN107475187A (en) | 2017-09-05 | 2017-09-05 | Nutrient solution and a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107475187A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108633877A (en) * | 2018-05-17 | 2018-10-12 | 广东芙金干细胞再生医学有限公司 | A kind of human umbilical cord mesenchymal stem cells excretion body freeze-dried powder and its method of preparation |
| CN108753697A (en) * | 2018-06-22 | 2018-11-06 | 天津市第三中心医院 | A kind of method that induced multi-potent stem cell excretion body extracts culture medium and preparation method thereof and extracts excretion body using it |
| CN109628392A (en) * | 2019-01-08 | 2019-04-16 | 深圳市第二人民医院 | Large-scale production meets the clinical grade excretion body of GMP specification |
| CN109913408A (en) * | 2019-02-25 | 2019-06-21 | 深圳市第二人民医院 | Large-scale production meets the clinical grade excretion body of GMP specification |
| CN109929799A (en) * | 2019-02-01 | 2019-06-25 | 浙江清华长三角研究院 | Human umbilical cord mesenchymal stem cells excretion body and its preparation method and application |
| CN110669729A (en) * | 2019-11-11 | 2020-01-10 | 广东国科细胞科技有限公司 | Method for preparing mesenchymal stem cell exosome |
| CN113728091A (en) * | 2019-06-10 | 2021-11-30 | 布瑞克斯奥根株式会社 | Composition for promoting production of stem cell-derived exosomes and increasing dryness |
| CN114107186A (en) * | 2021-11-09 | 2022-03-01 | 四川泊麦科技发展股份有限公司 | Preparation process for obtaining umbilical cord mesenchymal stem cell exosomes in low-temperature environment |
| CN115044543A (en) * | 2022-08-17 | 2022-09-13 | 山东卓东生物科技有限公司 | Method for improving activity of aged human body-derived muscle stem cells |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106913583A (en) * | 2017-04-20 | 2017-07-04 | 深圳市赛欧细胞生物科技有限公司 | The preparation method and application of human mesenchymal stem cell source excretion body biologically active agents |
-
2017
- 2017-09-05 CN CN201710790115.9A patent/CN107475187A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106913583A (en) * | 2017-04-20 | 2017-07-04 | 深圳市赛欧细胞生物科技有限公司 | The preparation method and application of human mesenchymal stem cell source excretion body biologically active agents |
Non-Patent Citations (5)
| Title |
|---|
| LIPING SUN等: "exosomes derived from human umbilical cord mesenchymal stem cells preotect against cisplatin-induced ovarian granulose cell stress and apoptosis in vitro", 《SCIENTIFIC REPORTS》 * |
| RICHARD J LOBB等: "optimized exosome isolation protocol for cell culture supernatant and human plasma", 《JOURNAL OF EXTRACELLULAR VESICLES》 * |
| 杨翔宇等: "干扰素γ刺激hUC-MSCs分泌外泌体促进调节性T细胞生成", 《中国药理学通报》 * |
| 程琳等: "间充质干细胞源性外泌体及其在肿瘤治疗中的研究进展", 《现代肿瘤医学》 * |
| 赵贵芳: "脐带间充质干细胞及其来源外泌体修复皮肤损伤的机制研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108633877A (en) * | 2018-05-17 | 2018-10-12 | 广东芙金干细胞再生医学有限公司 | A kind of human umbilical cord mesenchymal stem cells excretion body freeze-dried powder and its method of preparation |
| CN108753697A (en) * | 2018-06-22 | 2018-11-06 | 天津市第三中心医院 | A kind of method that induced multi-potent stem cell excretion body extracts culture medium and preparation method thereof and extracts excretion body using it |
| CN109628392A (en) * | 2019-01-08 | 2019-04-16 | 深圳市第二人民医院 | Large-scale production meets the clinical grade excretion body of GMP specification |
| CN109929799A (en) * | 2019-02-01 | 2019-06-25 | 浙江清华长三角研究院 | Human umbilical cord mesenchymal stem cells excretion body and its preparation method and application |
| CN109929799B (en) * | 2019-02-01 | 2023-02-28 | 浙江清华长三角研究院 | Human umbilical cord mesenchymal stem cell exosome and preparation method and application thereof |
| CN109913408A (en) * | 2019-02-25 | 2019-06-21 | 深圳市第二人民医院 | Large-scale production meets the clinical grade excretion body of GMP specification |
| CN113728091A (en) * | 2019-06-10 | 2021-11-30 | 布瑞克斯奥根株式会社 | Composition for promoting production of stem cell-derived exosomes and increasing dryness |
| CN110669729A (en) * | 2019-11-11 | 2020-01-10 | 广东国科细胞科技有限公司 | Method for preparing mesenchymal stem cell exosome |
| CN110669729B (en) * | 2019-11-11 | 2021-09-07 | 广州赛莱拉干细胞科技股份有限公司 | Method for preparing mesenchymal stem cell exosome |
| CN114107186A (en) * | 2021-11-09 | 2022-03-01 | 四川泊麦科技发展股份有限公司 | Preparation process for obtaining umbilical cord mesenchymal stem cell exosomes in low-temperature environment |
| CN115044543A (en) * | 2022-08-17 | 2022-09-13 | 山东卓东生物科技有限公司 | Method for improving activity of aged human body-derived muscle stem cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107475187A (en) | Nutrient solution and a large amount of production technologies for obtaining umbilical cord mesenchymal stem cells excretion body | |
| CN103667187B (en) | A kind of isolated culture method of human adipose-derived stem cell and the construction method of stem cell bank | |
| CN102127522B (en) | Human umbilical mesenchymal stem cell and preparation method thereof | |
| CN102443566B (en) | Acquisition method of adipose-derived stem cells | |
| CN104726406B (en) | It is a kind of to induce the method that dental pulp Derived from Mesenchymal Stem Cells is nerve cell | |
| CN106754674A (en) | Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN104450611A (en) | Primary separation and culture method of human amniotic mesenchymal stem cells | |
| CN101591644A (en) | The preparation of umbilical cord-mesenchymal stem cells (UC-MSCs) used in clinical therapy and storage | |
| CN106318906A (en) | Method for large-scale culture of human umbilical cord mesenchymal stem cells | |
| CN110564682A (en) | Method for large-scale production of human adipose-derived mesenchymal stem cell exosomes | |
| CN104974984A (en) | Adipose tissue-derived mesenchymal stem cell amplification culture method | |
| CN104651305A (en) | Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells | |
| CN102732586A (en) | Method for culturing mesenchymal stem cell secretin | |
| CN105647860A (en) | Serum-free in-vitro extraction and preparation method of clinical treatment-grade placental decidua basalis-mesenchymal stem cells (PDB-MSCs) | |
| CN111925982A (en) | Human bone marrow mesenchymal stem cell culture process applying three-dimensional cells | |
| CN108179137B (en) | Preparation method of recombinant adeno-associated virus of serum 8 type | |
| WO2020000636A1 (en) | Method for producing recombinant adenovirus | |
| CN106399236A (en) | Culture method for promoting growth of adipose stem cells | |
| CN105695409B (en) | A kind of naked mole oligodendrocyte precursor cells cultural method | |
| CN106479970A (en) | A kind of method of large-scale culture human adipose mesenchymal stem cells | |
| CN104195100A (en) | In-vitro culture method of mammary gland epithelial cells | |
| CN104818243A (en) | Separation method of placenta-derived fetal stem cells | |
| CN104560875A (en) | Method for separating and culturing trigeminal root ganglion neurons of mammal in vitro and application of method | |
| CN112280733B (en) | Amniotic stem cell extraction method | |
| CN102021140A (en) | Method for separating and purifying schwann cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171215 |