CN107771782A - A kind of mesenchyme stem cell protection solution and application thereof - Google Patents
A kind of mesenchyme stem cell protection solution and application thereof Download PDFInfo
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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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
The invention provides a kind of mesenchyme stem cell protection solution and application thereof.Protection liquid provided by the invention is made up of parts by weight for 0.2 1 parts of AB types umbilical cord blood, 8 15 parts of hemoprotein, 48 parts of myoglobins, HEPES and 500 1500 of 5 10 parts part of the aqueous solution.The nutrient provided by the invention that abundance is provided for mescenchymal stem cell, maintains its survival rate, extends its holding time.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of mesenchyme stem cell protection solution and application thereof.
Background technology
Mescenchymal stem cell (MSCs) is to belong to mesoblastic a kind of multipotential stem cell, has powerful multiplication capacity and more
To differentiation potential, MSCs is distributed in the Various Tissues such as marrow, liver, muscle, brain, skin and organ, inside suitably, outer shroud
The mesoblastemas such as hematopoietic cell, bone, cartilage, fat and muscle can be not only divided under border, in specific environment can be with
Transdifferentiationof is entoderm and ectodermic cell, and MSCs is because having Multidirectional Differentiation and itself renewal, low immunogenicity and being immunized
Inhibitory action is adjusted, there is very wide application in the field of organizational project, organ transplant and treatment immunologically mediated disease
Prospect.
MSCs had once been considered as once the main function for treating injury disease because having stronger propagation and differentiation capability
Mechanism, the research of reparations of the MSCs to structural organization defect comparative maturity, can successfully repair bone, cartilage and tendon etc.
The defect of tissue, and the effect of satisfactory has been achieved in the application of clinical tissue engineering, and to baroque organ
Repair and the research of the lasting existing disease of the cause of disease is still in the exploratory stage.
MSCs is in clinical transplantation treatment, it is impossible to which, by the stem cell direct infusion of fresh separated in human body, it needs elder generation
Certain time in the preservation liquid (such as 0.9% sodium chloride injection) that can be transfused is stored in, then is transfused in internal.It is clinical at present normal
Preservation liquid has 0.9% sodium chloride injection, 5% glucose injection, 1% human serum albumin liquid etc., but MSCs is at these
Preserving activity in liquid can decline, and influence the therapeutic effect of clinic.
The content of the invention
In order to solve the problems such as the holding time of MSCs in the prior art is short, activity rate is low, the invention provides an inter-species
Mesenchymal stem cells protect liquid, and the activity that the protection liquid extends mescenchymal stem cell is held time, reduces its pollution rate, improve it
Motility rate and vigor, make it to preserve activity within 10 days normal.
Concrete technical scheme of the present invention is as follows:
The invention provides a kind of mesenchyme stem cell protection solution, AB type of the protection liquid by parts by weight for 0.2-1 parts
Umbilical cord blood, the hemoprotein of 8-15 parts, 4-8 parts myoglobins, HEPES and 500-1500 part aqueous solution groups of 5-10 parts
Into.
Further to improve, protection liquid is by AB type umbilical cord blood of the parts by weight for 0.5 part, 11 parts of ferroheme egg
In vain, 7 portions of myoglobins, HEPES and 1000 of 7 parts part of aqueous solution composition.
The present invention in liquid is protected by adding AB types umbilical cord blood, hemoprotein, myoglobins, HEPES and water
Solution, the nutrient of abundance is provided for mescenchymal stem cell, improves its survival rate.
Further to improve, protection liquid also includes parts by weight for the sodium citrate of 1-5 parts and the Bomaili A of 5-15 parts.
The mixed electrolyte that the present invention is formed by adding sodium citrate and Bomaili A in liquid is protected, fills between can improving
The vigor of matter stem cell.
Further to improve, protection liquid also includes carrotene, the rosmanol and 5- of 2-8 parts that parts by weight are 1-5 parts
10 parts of green-tea extract.
The present invention is anti-oxidant by adding the joint that carrotene, rosmanol and green-tea extract form in liquid is protected
Agent, the proliferation activity of cell during the preservation of mescenchymal stem cell, can be maintained.
Preferably, the extracting method of green-tea extract comprises the following steps:
1) after being 15% by tea-drying to water content, cut out to 1cm2, obtain dry tealeaves；
2) the drying tealeaves obtained by step 1) is positioned in steam box and steams 30min, obtain softening tealeaves；
3) the softening tealeaves obtained by step 2) is positioned in round-bottomed flask, adds the water extraction 30min of 8 times of amounts, filter,
Residue repeats above step 3 times, merges each secondary filtrate；
4) merging filtrate obtained by step 3) is concentrated, dries to powder, produce.
The present invention prepares green-tea extract by above method, can improve the content of polyphenoils in extract, Jin Erti
Protective capability of the height protection liquid to mescenchymal stem cell.
Further to improve, protection liquid also includes Tobramycin, the UDP-3-O- (R- of 1-5 parts that parts by weight are 0.2-1 parts
Hydroxyl myristoyl) -2-Acetamido-2-deoxy-D-glucose deacetylase -1 (LpxC-1) and 0.2-1 parts teicoplanin.
The mixing antibiotic that the present invention is formed by adding Tobramycin, LpxC-1 and teicoplanin in liquid is protected, is reduced
The contamination rate of mescenchymal stem cell.
It is -80 DEG C to protect operating temperature of the liquid when preserving mescenchymal stem cell.
Present invention also offers a kind of preparation method of mesenchyme stem cell protection solution, this method comprises the following steps:
S1:1000 parts of aqueous solution are taken, 11 parts of hemoprotein is added to the aqueous solution of stirring with 5s/d speed
In, continue to stir 25min with 400r/min speed after being added dropwise, filtered with biofilter, it is water-soluble to obtain human serum albumin
Liquid；
S2:0.5 part of AB types umbilical cord blood, 7 parts of myoglobins and 7 parts of HEPES are positioned over to the people of S1 acquisitions
In the blood albumin aqueous solution, sufficiently stir under the conditions of 15 DEG C, filtered with biofilter；
S3:Mixed solution obtained by step S2 is down to 0 DEG C with ice-water bath, produced.
The present invention provides a kind of mesenchyme stem cell protection solution, and the protection liquid can be used for preserving mescenchymal stem cell, extend
The activity of mescenchymal stem cell is held time.
Beneficial effects of the present invention are as follows:Mesenchyme stem cell protection solution provided by the invention can reduce its contamination rate, and
Slow down its rate of metabolism, survival rate reaches 96.87 after being kept for three days, and adherent rate reaches 98.33, preserves activity after 1920h
Rate is 67.5, and the activity for farthest extending mescenchymal stem cell is held time.
Embodiment
Embodiment 1
A kind of mesenchyme stem cell protection solution, AB type umbilical cord blood, 8 part of ferroheme egg of the protection liquid by 0.2 part
In vain, 4 parts of myoglobins, HEPES and 500 of 5 parts part of aqueous solution composition.
Embodiment 2
A kind of mesenchyme stem cell protection solution, AB type umbilical cord blood, 11 part of ferroheme egg of the protection liquid by 0.5 part
White 7 parts of myoglobins, HEPES and 1000 of 7 parts part of aqueous solution composition.
Embodiment 3
A kind of mesenchyme stem cell protection solution, AB type umbilical cord blood, 15 part of hemoprotein of the protection liquid by 1 part
8 parts of myoglobins, HEPES and 1500 of 10 parts part of aqueous solution composition.
Embodiment 4
A kind of mesenchyme stem cell protection solution, AB type umbilical cord blood, 11 part of ferroheme egg of the protection liquid by 0.5 part
White 7 parts of myoglobins and 7 parts of HEPES compositions.
The preparation method of the mesenchyme stem cell protection solution comprises the following steps:
S1:1000 parts of aqueous solution are taken, 11 parts of hemoprotein is added to the aqueous solution of stirring with 5s/d speed
In, continue to stir 25min with 400r/min speed after being added dropwise, filtered with biofilter, it is water-soluble to obtain human serum albumin
Liquid；
S2:0.5 part of AB types umbilical cord blood, 7 parts of myoglobins and HEPES are positioned over to the white egg of people's blood of S1 acquisitions
In white water solution, sufficiently stir, filtered with biofilter；
S3:Mixed solution obtained by step S2 is down to 0 DEG C with ice-water bath, produced.
Embodiment 5
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
1 part of sodium citrate and 5 parts of Bomaili A.
Embodiment 6
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
3 parts of sodium citrate and 10 parts of Bomaili A.
Embodiment 7
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
5 parts of sodium citrate and 15 parts of Bomaili A.
Embodiment 8
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
5 parts of sodium citrate and 15 parts of Bomaili A.
The preparation method of the protection liquid comprises the following steps:
S1:1000 parts of aqueous solution are taken, 11 parts of hemoprotein is added to the aqueous solution of stirring with 5s/d speed
In, continue to stir 25min with 400r/min speed after being added dropwise, filtered with biofilter, it is water-soluble to obtain human serum albumin
Liquid；
S2:0.5 part of AB types umbilical cord blood and 7 parts of HEPES are positioned over to the human serum albumin aqueous solution of S1 acquisitions
In, sufficiently stir, filtered with biofilter, obtain and tentatively protect liquid；
S3:5 parts of sodium citrate and 15 parts of vigorous arteries and veins power are dispersed in the preliminary protection liquid of step S2 acquisitions, used
Biofilter filters；
S4:Mixed solution obtained by step S3 is down to 0 DEG C with ice-water bath, produced.
Embodiment 9
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
1 part of carrotene, 2 parts of rosmanol and 5 parts of green-tea extract.
Embodiment 10
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
3 parts of carrotene, 5 parts of rosmanol and 8 parts of green-tea extract.
Embodiment 11
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
5 parts of carrotene, 8 parts of rosmanol and 10 parts of green-tea extract.
Embodiment 12
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
5 parts of carrotene, 8 parts of rosmanol and 10 parts of green-tea extract.
Wherein, the extracting method of green-tea extract comprises the following steps:
1) after being 15% by tea-drying to water content, cut out to 1cm2, obtain dry tealeaves；
2) the drying tealeaves obtained by step 1) is positioned in steam box and steams 30min, obtain softening tealeaves；
3) the softening tealeaves obtained by step 2) is positioned in round-bottomed flask, adds the water extraction 30min of 8 times of amounts, filter,
Residue repeats above step 3 times, merges each secondary filtrate；
4) merging filtrate obtained by step 3) is concentrated, dries to powder, produce.
Embodiment 13
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
5 parts of carrotene, 8 parts of rosmanol and 10 parts of green-tea extract.
Wherein, the preparation method of the protection liquid comprises the following steps:
S1:1000 parts of aqueous solution are taken, 11 parts of hemoprotein is added to the aqueous solution of stirring with 5s/d speed
In, continue to stir 25min with 400r/min speed after being added dropwise, filtered with biofilter, it is water-soluble to obtain human serum albumin
Liquid；
S2:0.5 part of AB types umbilical cord blood and 7 parts of HEPES are positioned over to the human serum albumin aqueous solution of S1 acquisitions
In, sufficiently stir, obtain and tentatively protect liquid；
S3:5 parts of carrotene, 8 parts of rosmanol and 10 parts of green-tea extract are dispersed in into step S2 to obtain
Preliminary protection liquid in；
S4:Mixed solution obtained by step S3 is down to 0 DEG C with ice-water bath, produced.
Embodiment 14
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
0.2 part of Tobramycin, LpxC-1 and 0.2 of 1 part part of teicoplanin.
Embodiment 15
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
0.6 part of Tobramycin, LpxC-1 and 0.6 of 3 parts part of teicoplanin.
Embodiment 16
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
1 part of Tobramycin, LpxC-1 and 1 of 5 parts part of teicoplanin.
Embodiment 17
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 2, includes the group of following parts by weight
Point:
0.6 part of Tobramycin, LpxC-1 and 0.6 of 3 parts part of teicoplanin.
Wherein, the preparation method of the protection liquid comprises the following steps:
S1:1000 parts of aqueous solution are taken, 11 parts of hemoprotein is added to the aqueous solution of stirring with 5s/d speed
In, continue to stir 25min with 400r/min speed after being added dropwise, filtered with biofilter, it is water-soluble to obtain human serum albumin
Liquid；
S2:0.5 part of AB types umbilical cord blood and 7 parts of HEPES are positioned over to the human serum albumin aqueous solution of S1 acquisitions
In, sufficiently stir, filtered with biofilter, obtain and tentatively protect liquid；
S3:By 0.6 part of Tobramycin, LpxC-1 and 0.6 of 3 parts part of teicoplanin be dispersed in that step S2 obtains just
Stir to being uniformly dissolved in step protection liquid, filtered with biofilter；
S4:Mixed solution obtained by step S3 is down to 0 DEG C with ice-water bath, produced.
Reference examples 1
It is in place of a kind of mesenchyme stem cell protection solution, with the difference of embodiment 1, adds 1 part of hyclone.
Reference examples 2
It is in place of a kind of mesenchyme stem cell protection solution, with the difference of embodiment 1, instead of with phosphate-buffered liquid calcium
HEPES。
Reference examples 3
It is in place of a kind of mesenchyme stem cell protection solution, with the difference of embodiment 1, deletes myoglobins.
Reference examples 4
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 6, adds 2 parts of ammonium chloride.
Reference examples 5
A kind of mesenchyme stem cell protection solution, is with the difference of embodiment 6, and citric acid is instead of with potassium citrate
Sodium.
Reference examples 6
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 6, deletes Bomaili A.
Reference examples 7
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 10, adds 1 part of superoxides discrimination
Change enzyme.
Reference examples 8
A kind of mesenchyme stem cell protection solution, is with the difference of embodiment 10, and carrot is instead of with vitamin C
Element.
Reference examples 9
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 10, deletes green-tea extract.
Reference examples 10
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 15, adds 0.2 part of chloramphenicol.
Reference examples 11
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 15, instead of with gentamicin sulphate
Tobramycin.
Reference examples 12
A kind of mesenchyme stem cell protection solution, it is with the difference of embodiment 15, deletes LpxC-1.
The survival experiment of test example 1
Mescenchymal stem cell is divided into 6 groups, it is immersed in respectively in embodiment 1-4 and reference examples 1-3 preservation liquid, put
Preserved 3 days in -80 DEG C of refrigerator, the quantity and growing state of the mescenchymal stem cell in the different protection liquid of mtt assay detection, knot
Fruit is as shown in table 1；
Wherein, after survival rate (%)=preservation cell quantity/cell original amount × 100%.
The different survival assays result for preserving the mescenchymal stem cell that liquid preserves of table 1
As shown in Table 1, the survival rate for the mescenchymal stem cell being stored in embodiment 1-4 protection liquid is above reference examples
1-3, and the preservation effect of embodiment 2 is better than embodiment 1 and embodiment 3, it was demonstrated that when protection liquid contains parts by weight for 0.5 part
When AB types umbilical cord blood, 11 parts of hemoprotein, 7 parts of myoglobins, HEPES and 1000 of 7 parts part of aqueous solution, between pair
The protecting effect of mesenchymal stem cells is best, and the preservation effect of embodiment 4 is better than embodiment 1-3, it was demonstrated that provided by the invention
The protection liquid that the preparation method of mesenchymal stem cells protection liquid is prepared is more preferable to the protecting effect of mescenchymal stem cell.
The adherent rate of test example 2 is tested
Mescenchymal stem cell is preserved with embodiment 5-8 and reference examples 4-6 protection liquid respectively, respectively 24,48,96,240
Enter test procedure with 480h, adjust cell density, be seeded in plate, after it grows 48h naturally, not adherent thin of removal
Born of the same parents, digested with 0.25m/v% trypsase, calculate adherent mescenchymal stem cell, draw adherent rate, as a result such as table 2
It is shown.
The different adherent rate result of the test for preserving the mescenchymal stem cell that liquid preserves of table 2
As shown in Table 2, embodiment 5-8 protection liquid in mescenchymal stem cell adherent rate apparently higher than reference examples 4-6, and
The adherent rate for the mescenchymal stem cell that the protection liquid of embodiment 6 preserves is higher than embodiment 5 and embodiment 7, it was demonstrated that when protection liquid contains
When to have parts by weight be 3 parts of sodium citrate and 10 parts of Bomaili A, the adherent rate effect of mescenchymal stem cell is best, fills
Matter Stem Cell Activity is best, and the adherent rate for the mescenchymal stem cell that the protection liquid of embodiment 8 preserves is higher than embodiment 6, it was demonstrated that
Protection liquid energy prepared by method provided by the invention improves the adherent rate of the mescenchymal stem cell after preserving.
The proliferation activity of test example 3 is tested
Mescenchymal stem cell is preserved with embodiment 9-11 and reference examples 7-9 protection liquid respectively, then will be filled respectively between each group
Matter stem cell is with 1 × 106Individual/T75 bottles inoculation, adds complete medium culture, then be inoculated in 96 holes with every 5000 cells in hole
In plate, 200 μ L L-DMEM is inoculated with per hole, 200 μ LPBS are added in the collar aperture of 96 orifice plate outermost one, after adherent growth, control group after
It is continuous to use complete medium culture, detected after 24h, often hole is separately added into the 5g/L μ L of MTT solution 20, and in incubator 37
Under the conditions of DEG C after lucifuge culture 4h, supernatant is removed, the μ L of dimethyl sulphoxide solution 150 are added per hole, is determined with micropore board detector
Absorbance (A value) of each hole at 570nm wavelength, calculate the average value and its proliferation activity of the hole absorbance of each group 10, knot
Fruit is shown in Table 3.
The proliferation activity for the mescenchymal stem cell that the different preservation liquid of table 3 preserves tests result
Wherein, F=9.789, P=0.000；
As shown in Table 3, the proliferation activity for the mescenchymal stem cell that the protection liquid that embodiment 10-11 is provided preserves has notable
Property, and the protection liquid that reference examples 7-9 is provided to the proliferation activity of mescenchymal stem cell without conspicuousness, and the protection liquid of embodiment 10
The proliferation activity highest of the mescenchymal stem cell of holding, it was demonstrated that when the carrotene of 3 parts of addition, 5 in mesenchyme stem cell protection solution
When the rosmanol and 8 parts of green-tea extract of part, the proliferation activity highest of mescenchymal stem cell, any composition is improved or reduced
Content can all reduce its holding mescenchymal stem cell proliferation activity.
The pollution rate of test example 4 is tested
Mescenchymal stem cell is divided into 6 groups, it is immersed in embodiment 14-16 and reference examples 10-12 protection liquid respectively
In, preserved 12 days under the conditions of being -80 DEG C in temperature, cell will be detected with Flow cytometry after the cell dissociation processing of preservation
The quantity of pollution, and pollution rate is calculated, as a result as shown in table 4.
The different pollution rate result of the test for preserving the mescenchymal stem cell that liquid preserves of table 4
As shown in Table 4, the pollution rate for the mescenchymal stem cell that embodiment 14-16 preservation liquid preserves is substantially less than reference examples
The preservation liquid of 10-12 groups, and the pollution rate for preserving the mescenchymal stem cell in liquid of embodiment 15 is minimum, antipollution effect is most
It is good, after protecting the composition in the Tobramycin in liquid, LpxC-1 and teicoplanin to lack one or replaced by other composition, fill
The pollution rate of matter stem cell greatly promotes, it was demonstrated that when protect in liquid containing 0.6 part of Tobramycin, LpxC-1 and 0.6 of 3 parts part
During teicoplanin, protect the contamination resistance of liquid most strong.
Claims (9)
- A kind of 1. mesenchyme stem cell protection solution, it is characterised in that AB type navel of the protection liquid by parts by weight for 0.2-1 parts With blood blood plasma, the hemoprotein of 8-15 parts, the myoglobins of 4-8 parts, 5-10 parts HEPES and 500-1500 parts the aqueous solution Composition.
- 2. the mesenchyme stem cell protection solution described in claim 1, it is characterised in that the protection liquid is 0.5 by parts by weight The AB types umbilical cord blood, 11 parts of hemoprotein, 7 parts of myoglobins, HEPES and 1000 of 7 parts part of the aqueous solution of part Composition.
- 3. mesenchyme stem cell protection solution as claimed in claim 1, it is characterised in that the protection liquid also includes parts by weight The Bomaili A of sodium citrate and 5-15 parts for 1-5 parts.
- 4. mesenchyme stem cell protection solution as claimed in claim 1, it is characterised in that the protection liquid also includes parts by weight For the green-tea extract of the carrotene of 1-5 parts, the rosmanol of 2-8 parts and 5-10 parts.
- 5. mesenchyme stem cell protection solution as claimed in claim 4, body characteristicses are, the extracting method of the green-tea extract Comprise the following steps:1) after being 15% by tea-drying to water content, cut out to 1cm2, obtain dry tealeaves；2) the drying tealeaves obtained by step 1) is positioned in steam box and steams 30min, obtain softening tealeaves；3) the softening tealeaves obtained by step 2) is positioned in round-bottomed flask, adds the water extraction 30min of 8 times of amounts, filtering, residue Repeat above step 3 times, merge each secondary filtrate；4) merging filtrate obtained by step 3) is concentrated, dries to powder, produce.
- 6. mesenchyme stem cell protection solution as claimed in claim 1, it is characterised in that the protection liquid also includes parts by weight For the Tobramycin of 0.2-1 parts, the teicoplanin of LpxC-1 and 0.2-1 parts of 1-5 parts.
- 7. mesenchyme stem cell protection solution as claimed in claim 1, it is characterised in that the protection liquid is done in preservation mesenchyma Operating temperature during cell is -80 DEG C.
- 8. a kind of preparation method of mesenchyme stem cell protection solution, it is characterised in that the preparation method comprises the following steps:S1:1000 parts of aqueous solution are taken, 11 parts of hemoprotein are added in the aqueous solution of stirring with 5s/d speed, drop Add and continue to stir 25min with 400r/min speed after finishing, filtered with biofilter, obtain the human serum albumin aqueous solution；S2:People's blood that 0.5 part of AB types umbilical cord blood, 7 parts of myoglobins and 7 parts of HEPES are positioned over to S1 acquisitions is white In protein solution, sufficiently stir, filtered with biofilter；S3:Mixed solution obtained by step S2 is down to 0 DEG C with ice-water bath, produced.
- 9. any described protection liquid of claim 1-7 is preparing the application in being used to preserve the device of mescenchymal stem cell.
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| CN108441549A (en) * | 2018-04-02 | 2018-08-24 | 南京千年健干细胞基因工程有限公司 | A kind of identification mesenchymal stem cell based on circular rna freezes the gene detecting kit of time |
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