CN110438071A - People's umbilical cord China Tong Shi glue mesenchymal stem cells and preparation method thereof - Google Patents
People's umbilical cord China Tong Shi glue mesenchymal stem cells and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 26
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- 230000029087 digestion Effects 0.000 claims abstract description 42
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- 229940009444 amphotericin Drugs 0.000 description 2
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- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
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- 230000008014 freezing Effects 0.000 description 1
- CEAZRRDELHUEMR-UHFFFAOYSA-N gentamicin Chemical class O1C(C(C)NC)CCC(N)C1OC1C(O)C(OC2C(C(NC)C(C)(O)CO2)O)C(N)CC1N CEAZRRDELHUEMR-UHFFFAOYSA-N 0.000 description 1
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- 230000030097 organ senescence Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- CWERGRDVMFNCDR-UHFFFAOYSA-M thioglycolate(1-) Chemical compound [O-]C(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-M 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- 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
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- 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
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- Bioinformatics & Cheminformatics (AREA)
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Abstract
The present invention provides a kind of people's umbilical cord China Tong Shi glue mesenchymal stem cells and preparation method thereof, is related to stem cell and regenerative medicine field.The preparation method, comprising the following steps: umbilical cord China Tong Shi glue Antigen repairing: isolated umbilical cord China Tong Shi glue tissue is cut to tissue pieces；It digests for the first time: taking the tissue pieces to mix with tissue digestion liquid and carry out first time digestion, be centrifuged after digestion, remove supernatant, retain precipitating, be added and be resuspended containing dual anti-complete medium, obtain suspension；Second of digestion: suspension being dispensed and is cultivated, and is carried out second with trypsase and is digested, is centrifuged after digestion, and removal supernatant retains precipitating, is added and is resuspended containing dual anti-complete medium, obtain umbilical cord mesenchymal stem cells suspension；Culture: the umbilical cord mesenchymal stem cells suspension is dispensed, and continues culture to get people's umbilical cord China Tong Shi mescenchymal stem cell.Preparation method recovery rate of the invention is high, and purity is high, activity are good.
Description
Technical field
The present invention relates to stem cell and regenerative medicine field, more particularly to people's umbilical cord China Tong Shi glue mesenchymal stem cells and
Preparation method.
Background technique
Mescenchymal stem cell is the important member in stem cell line, with self-renewing, Multidirectional Differentiation and increment energy
The strong functional character of power.Deriving from extensively for mescenchymal stem cell, is isolated in the tissue such as marrow, umbilical cord and muscle at present
Mescenchymal stem cell.Wherein, marrow is the tissue that earliest discovery can extract mescenchymal stem cell, but from myeloid tissue
It is big to extract mescenchymal stem cell difficulty, and there may be ethics problems.
Umbilical cord mesenchymal stem cells are rich in people's umbilical cord China Tong Shi glue tissue, the functional spy of institute with mescenchymal stem cell
Ethics problem is also not present in sign, abundance.Umbilical cord mesenchymal stem cells can be used as treatment tissue organ senescence and lesion
Ideal seed cell can be saved to make the possible period of want or need, and umbilical cord mesenchymal stem cells are increasingly valued by people.
Traditional umbilical cord mesenchymal stem cells extract and store method, the pick-up rate of cell umbilical cord mesenchymal stem cells, pure
Degree is all lower, is difficult to meet studies and clinical application.
Summary of the invention
Based on this, it is necessary to low, pure for the existing method recovery rate for obtaining people's umbilical cord China Tong Shi glue mesenchymal stem cells
Low problem is spent, a kind of preparation method of people's umbilical cord China Tong Shi glue mesenchymal stem cells is provided, people's navel is extracted from umbilical cord tissue
Recovery rate with China's Tong Shi glue mesenchymal stem cells is high, and purity is high, activity are good.
A kind of preparation method of people's umbilical cord China Tong Shi mescenchymal stem cell, comprising the following steps:
Umbilical cord China Tong Shi glue Antigen repairing: isolated umbilical cord China Tong Shi glue tissue is cut to 1~2mm3Tissue pieces；
It digests for the first time: taking the tissue pieces to mix with tissue digestion liquid and carry out first time digestion, be centrifuged, go after digestion
Except supernatant, retain precipitating, is added and is resuspended containing dual anti-complete medium, obtain suspension；The tissue digestion liquid packet
Include collagenase type I and III Collagenase Type；
Second of digestion: suspension being dispensed and is cultivated, and is carried out second with trypsase and is digested, is centrifuged after digestion, in removal
Clear liquid retains precipitating, is added and is resuspended containing dual anti-complete medium, obtains umbilical cord mesenchymal stem cells suspension；
Culture: the umbilical cord mesenchymal stem cells suspension is dispensed, and it is dry to get people's umbilical cord China Tong Shi mesenchyma to continue culture
Cell.
Above-mentioned preparation method digests umbilical cord tissue using two steps, can more fully digest umbilical cord tissue, maximum limitation
Acquisition umbilical cord mesenchymal stem cells, obtained people's umbilical cord China Tong Shi mescenchymal stem cell yield is high, purity is high, activity are good, has
It is clinical conducive to being applied to.
In one of the embodiments, in the umbilical cord China Tong Shi glue Antigen repairing, umbilical cord China Tong Shi glue tissue is separated
The step of are as follows: the blood for cleaning umbilical cord tissue is cleaned up with organization protection's liquid, vein and artery is removed, isolates stick-slip
Clear gum object, i.e. umbilical cord China Tong Shi glue；Organization protection's liquid be include 0.5wt%~1.5wt% gentamicin and
The physiological saline of 0.5wt%~1.5wt% amphotericin B.
In one of the embodiments, in the first time digestion step, the time of digestion is 2~3min, 3 after digestion
It is centrifuged at~5 DEG C, centrifugal rotational speed is 800~1100rpm, and centrifugation time is 4~6min.
The concentration of collagenase type I is 0.7~1.1g/mL, III in the tissue digestion liquid in one of the embodiments,
The concentration of Collagenase Type is 0.4~0.8g/mL, and solvent is PBS solution；The additive amount of the tissue digestion liquid be and the tissue
Fragment is isometric.Preferably, collagenase type I and the concentration of type III clostridiopetidase A ratio are 3:2.
Dual anti-in the complete medium is 0.5~1.0 μ g/mL gentamicin and 0.5 in one of the embodiments,
~1.0 μ g/mL amphotericin Bs further include 8wt%~12wt% cell factor in the complete medium.Preferably, training completely
The content for supporting cell factor in base is 10wt%.
In one of the embodiments, in second of digestion step, suspension is dispensed into T75 culture bottle, in temperature
Degree is 37 ± 0.5 DEG C, CO2Concentration is to be cultivated under 5% ± 0.5% environment, after cell growth is fused to 60%~70%,
It adds trypsase and carries out second of digestion.
The tissue pieces are when unused in one of the embodiments, packing to cryopreservation tube, and cell cryopreservation is added
Liquid, program are placed in liquid nitrogen after being cooled to -80~-90 DEG C and save.
People's umbilical cord China Tong Shi mescenchymal stem cell is when unused in one of the embodiments, and packing is to freezing
Pipe, is added cells frozen storing liquid, and program is placed in liquid nitrogen after being cooled to -80~-90 DEG C and saves.
The ingredient of the frozen stock solution in one of the embodiments, are as follows: glycerol, mannitol and containing dual anti-DMEM, it is sweet
Oil, mannosan and the mass ratio containing dual anti-DMEM are 1:(2~3): (6~7).Preferably, glycerol, mannosan and contain
The mass ratio for having dual anti-DMEM is 2:5:13.Contain DMSO in traditional frozen stock solution, when applying to clinic after recovery, DMSO is residual
Staying will cause patient under consideration and the symptoms such as Nausea and vomiting occurs, frozen stock solution of the invention can to avoid the generation of above-mentioned side effect,
And mannitol, which is added, to provide nutritional ingredient for cell culture.Cells frozen storing liquid is placed at 4 DEG C and saves.
One aspect of the present invention also provides a kind of people's umbilical cord China Tong Shi mescenchymal stem cell that above-mentioned preparation method obtains.The people
Umbilical cord China Tong Shi mescenchymal stem cell has the advantages that purity is high, activity are high, is advantageously applied to clinic.
Compared with prior art, the invention has the following advantages:
Of the invention digests umbilical cord tissue using two steps, can more fully digest umbilical cord tissue, maximum limitation obtains
Umbilical cord mesenchymal stem cells are obtained, obtained people's umbilical cord China Tong Shi mescenchymal stem cell yield is high, with high purity, activity is good, is conducive to
Applied to clinic.
Detailed description of the invention
Fig. 1 is the flow chart of 1 method of embodiment；
Fig. 2 is flow cytometer detection result figure；
Fig. 3 be using embodiment 1 the obtained P0 of method for umbilical cord mesenchymal stem cells culture 5 days photo；
Fig. 4 be using comparative example 1 the obtained P0 of method for umbilical cord mesenchymal stem cells culture 5 days photo；
Fig. 5 be using comparative example 2 the obtained P0 of method for umbilical cord mesenchymal stem cells culture 5 days photo；
Fig. 6 be using embodiment 1 the obtained P3 of method for umbilical cord mesenchymal stem cells culture 3 days photo；
Fig. 7 be using comparative example 1 the obtained P3 of method for umbilical cord mesenchymal stem cells culture 3 days photo；
Fig. 8 be using comparative example 2 the obtained P3 of method for umbilical cord mesenchymal stem cells culture 3 days photo.
Specific embodiment
It to facilitate the understanding of the present invention, below will be to invention is more fully described.The present invention can be with many not
With form realize, however it is not limited to embodiment described herein.On the contrary, purpose of providing these embodiments is makes to this
The understanding of the disclosure of invention is more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
Embodiment 1
A kind of preparation method of people's umbilical cord China Tong Shi mescenchymal stem cell, as shown in Figure 1, comprising the following steps:
1) Preservation in sterile condition: umbilical cord tissue being placed in organization protection's liquid and carries out Preservation in sterile condition, organization protection's liquid be containing
The physiological saline of 1wt% gentamicin and 1wt% amphotericin B；Extract the group for impregnating umbilical cord tissue respectively with 2 syringes
Protection each 5mL of liquid is knitted, thioglycollate solution medium is injected separately into, improves in Martin's broth；
2) blood: the sterile umbilical cord tissue of 15~18cm of clip is handled, rinses liquid remained on surface with organization protection's liquid,
The length for being cut into 2~3cm is put into the culture dish that diameter is 10cm, 75% alcohol being added in culture dish, and immersion 1~
After 2min, umbilical cord tissue taking-up is placed in the culture dish equipped with clean organization protection's liquid, the blood vessel in umbilical cord tissue is squeezed,
Until organization protection's liquid cleans up the blood inside blood vessel；
3) it separates umbilical cord China Tong Shi glue: with 1 vein and 2 arteries of aseptic nipper removal umbilical cord, isolating stick-slip
Clear gum object, the clear gum object are China's Tong Shi glue, and the width of magnificent Tong Shi glue is about 2~3mm, weighs about 5g, and 2.5g China is led to
Family name's glue is transferred in 50mL centrifuge tube, is shredded to 2~3mm3Tissue pieces；
4) it stores China's Tong Shi glue: when non-immediate use China's Tong Shi glue, being loaded into cryopreservation tube and freeze, every 2mL freezes
2.5g China Tong Shi glue, 1mL cells frozen storing liquid are added in pipe, is cooled to -80~-90 DEG C by programmed cooling instrument, then be placed in liquid
It is stored in nitrogen；Wherein, the component of cells frozen storing liquid are as follows: glycerol, mannitol and contain dual anti-DMEM, wherein glycerol, sweet dew are poly-
Sugared and mass ratio containing dual anti-DMEM is 2:5:13；
5) it digests for the first time: taking the magnificent Tong Shi glue tissue pieces of 2g in 50mL centrifuge tube, continue to shred to 1~2mm3's
Fragment is added and is centrifuged 5min, centrifugal rotational speed after digestion with the isometric tissue digestion liquid of above-mentioned tissue pieces, 2~3min of digestion
For 1000rpm, temperature is 4 DEG C, removes supernatant, retains precipitating, and addition 20mL contains dual anti-complete medium and is resuspended,
Obtain suspension；Wherein, tissue digestion liquid is molten for the PBS of the collagenase type I comprising 0.9g/mL and the type III clostridiopetidase A of 0.6g/mL
Liquid includes 5 μ g/mL gentamicins, 10 μ g/mL amphotericin Bs and 10wt% cell factor in complete medium；
6) it digests for second: suspension being dispensed into T75 culture bottle, P0 generation is denoted as, is placed in 37 DEG C, 5%CO2Incubator
A culture solution is changed in middle culture for 5 days, when P0 for cell fusion to 60%~70% when, with 1.5mL trypsin digestion 2-
3min is centrifuged 5min, centrifugal rotational speed 1000rpm after digestion, temperature is 4 DEG C, removes supernatant, retains precipitating, 10mL is added
It is resuspended containing dual anti-complete medium, obtains cell suspension, dispensed into T75 culture bottle, be labeled as P1 generation, continue to cultivate,
The present embodiment culture is to P3 generation；
7) cell cryopreservation: by P1 in cell suspension and the cells frozen storing liquid cryopreservation tube of 1:4 addition by volume, pass through program
Cooling instrument is cooled to -80~-90 DEG C, then is placed in liquid nitrogen and freezes；The ingredient of frozen stock solution are as follows: glycerol, mannitol and containing double
Anti- DMEM, glycerol, mannosan and the mass ratio containing dual anti-DMEM are 2:5:13.
Embodiment 2
A kind of preparation method of people's umbilical cord China Tong Shi mescenchymal stem cell, the difference from embodiment 1 is that, tissue digestion liquid
The concentration of middle collagenase type I is 0.7g/mL, and the concentration of type III clostridiopetidase A is 0.8g/mL.
Embodiment 3
A kind of preparation method of people's umbilical cord China Tong Shi mescenchymal stem cell, the difference from embodiment 1 is that, tissue digestion liquid
The concentration of middle collagenase type I is 1.1g/mL, and the concentration of type III clostridiopetidase A is 0.4g/mL.
Comparative example 1
A kind of preparation method of people's umbilical cord China Tong Shi mescenchymal stem cell, the difference from embodiment 1 is that, tissue digestion liquid
For the PBS solution of the collagenase type I comprising 1g/mL.
Comparative example 2
A kind of preparation method of people's umbilical cord China Tong Shi mescenchymal stem cell, the difference from embodiment 1 is that, tissue digestion liquid
For the PBS solution of the type III clostridiopetidase A comprising 1.5g/mL.
Comparative example 3
A kind of preparation method of people's umbilical cord China Tong Shi mescenchymal stem cell, the difference from embodiment 1 is that, tissue digestion liquid
For the PBS solution of the II Collagenase Type comprising 1.5g/mL, the DNA hydrolase of 1g/mL.
Experimental example
The cell that Example 1 obtains carries out flow cytometer detection, as a result as shown in Figure 2；
Cell and P3 after being commissioned to train feeding 5 days using inverted microscope to the P0 that embodiment and comparative example obtains are commissioned to train feeding 3 days
Cell afterwards is taken pictures, and photo is as can be seen from figures 6 to 8；Cell count, count results such as 1 institute of table are carried out using Cell count
Show:
1 people's umbilical cord China Tong Shi mescenchymal stem cell count table of table
As seen from Table 1, compared to comparative example, the umbilical cord China Tong Shi glue mesenchymal stem cells being obtained by the present invention are adopted
Quantity it is higher, show higher using method recovery rate of the invention, obtained cell proliferation rate is fast, and activity is good.Especially
Tissue digestion liquid in the embodiment of the present invention 1 is used cooperatively using collagenase type I and type III clostridiopetidase A, obtained cell quantity
It is 1.5~2.75 times of comparative example, shows that its growth rate is most fast, activity is best.
It can be determined that umbilical cord China Tong Shi glue mesenchymal stem cells from the flow cytometer detection in Fig. 7.It can be seen that from Fig. 1~6
The purity for adopting the umbilical cord China Tong Shi glue mesenchymal stem cells being obtained by the present invention is higher, P1 for when the uniformity and growth
Situation is preferable, and wherein the cell growth state of embodiment 1 is best, purity highest.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of preparation method of people's umbilical cord China Tong Shi mescenchymal stem cell, which comprises the following steps:
Umbilical cord China Tong Shi glue Antigen repairing: isolated umbilical cord China Tong Shi glue tissue is cut to 1~2mm3Tissue pieces；
It digests for the first time: taking the tissue pieces to mix with tissue digestion liquid and carry out first time digestion, be centrifuged after digestion, in removal
Clear liquid retains precipitating, is added and is resuspended containing dual anti-complete medium, obtains suspension；The tissue digestion liquid includes I type
Clostridiopetidase A and III Collagenase Type；
Second of digestion: suspension being dispensed and is cultivated, and is carried out second with trypsase and is digested, is centrifuged after digestion, removal supernatant,
Retain precipitating, is added and is resuspended containing dual anti-complete medium, obtain umbilical cord mesenchymal stem cells suspension；
Culture: the umbilical cord mesenchymal stem cells suspension is dispensed, and it is dry thin to get people's umbilical cord China Tong Shi mesenchyma to continue culture
Born of the same parents.
2. the preparation method of people's umbilical cord according to claim 1 China Tong Shi mescenchymal stem cell, which is characterized in that the navel
With the step of in China's Tong Shi glue Antigen repairing, separating umbilical cord China Tong Shi glue tissue are as follows: umbilical cord group will be cleaned with organization protection's liquid
The blood knitted cleans up, and removes vein and artery, isolates the clear gum object of stick-slip, i.e. umbilical cord China Tong Shi glue；Described group
Knitting protection liquid is the physiological saline for including 0.5wt%~1.5wt% gentamicin and 0.5wt%~1.5wt% amphotericin B.
3. the preparation method of people's umbilical cord according to claim 1 China Tong Shi mescenchymal stem cell, which is characterized in that described
In digestion step, time of digestion is 2~3min, is centrifuged at 3~5 DEG C after digestion, centrifugal rotational speed is 800~
1100rpm, centrifugation time are 4~6min.
4. the preparation method of people's umbilical cord according to claim 1 China Tong Shi mescenchymal stem cell, which is characterized in that described group
The concentration for knitting collagenase type I in digestive juice is 0.7~1.1g/mL, and the concentration of type III clostridiopetidase A is 0.4~0.8g/mL, solvent
For PBS solution；The additive amount of the tissue digestion liquid is isometric with the tissue pieces.
5. the preparation method of people's umbilical cord according to claim 1 China Tong Shi mescenchymal stem cell, which is characterized in that described complete
Dual anti-in full culture medium is 0.5~1.0 μ g/mL gentamicin and 0.5~1.0 μ g/mL amphotericin B, the complete culture
It further include 8wt%~12wt% cell factor in base.
6. the preparation method of people's umbilical cord according to claim 1 China Tong Shi mescenchymal stem cell, which is characterized in that described
In secondary digestion step, suspension is dispensed into T75 culture bottle, is 37 ± 0.5 DEG C in temperature, CO2Concentration is 5% ± 0.5%
It is cultivated under environment, after cell growth is fused to 60%~70%, adds trypsase and carry out second of digestion.
7. the preparation method of people's umbilical cord according to claim 1 China Tong Shi mescenchymal stem cell, which is characterized in that described group
Fragment is knitted when unused, cells frozen storing liquid is added in packing to cryopreservation tube, and program is placed in liquid nitrogen after being cooled to -80~-90 DEG C
Middle preservation.
8. the preparation method of people's umbilical cord according to claim 1 China Tong Shi mescenchymal stem cell, which is characterized in that the people
For umbilical cord China Tong Shi mescenchymal stem cell when unused, cells frozen storing liquid is added to cryopreservation tube in packing, and program is cooled to -80~-
It is placed in liquid nitrogen and saves after 90 DEG C.
9. the preparation method of people's umbilical cord according to claim 7 or 8 China Tong Shi mescenchymal stem cell, which is characterized in that institute
State the ingredient of frozen stock solution are as follows: glycerol, mannitol and containing dual anti-DMEM and contain dual anti-DMEM's at glycerol, mannosan
Mass ratio is 1:(2~3): (6~7).
10. the people's umbilical cord China Tong Shi mescenchymal stem cell obtained by preparation method according to any one of claims 1 to 9.
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