CN108812645A - A kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells - Google Patents
A kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells Download PDFInfo
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- CN108812645A CN108812645A CN201810804980.9A CN201810804980A CN108812645A CN 108812645 A CN108812645 A CN 108812645A CN 201810804980 A CN201810804980 A CN 201810804980A CN 108812645 A CN108812645 A CN 108812645A
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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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- 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
Abstract
The invention discloses a kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells, wherein the percentage of ingredient is：Serum-free medium：41.8-51.8%, serum sub：40-50%, dimethyl sulfoxide：8%, the streptomycin sulphate of 200ug/ml：0.1% and additive：0.1%, the present invention also provides a kind of cryopreservation methods of cells frozen storing liquid using everybody umbilical cord mesenchymal stem cells, this method includes：The suspension cell centrifugation of secondary culture is placed in centrifuge tube, by frozen stock solution into centrifuge tube containing cell, cryopreservation tube is moved to, the cryopreservation tube that need to be frozen is placed in the program temperature reduction box equipped with isopropanol, -80 DEG C overnight, and next day moves into liquid nitrogen container.The present invention is able to solve fetal calf serum bring animal derived materials and pathogen contamination problem in conventional cryopreservation liquid, the streptomycin sulphate of addition, antibiotic content can be reduced, murder by poisoning of the antibiotic to everybody umbilical cord mesenchymal stem cells is reduced, cell death is reduced and maintains stem cell properties.
Description
Technical field
The present invention relates to cell culture technology, in particular to a kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells.
Background technique
Human umbilical cord mesenchymal stem cells refer to a kind of multipotential stem cell separated from umbilical cord, the mesenchyma with other sources
Stem cell relatively has an apparent clinical application advantage, such as materials are easy, easily collecting, freezen protective and relatively pure, have compared with
High differentiation potential can be broken up to multiple directions, as osteoblast, cartilage cell, quasi-liver cell, para-insulin cell,
Nerve cell and cardiac muscle cell etc., and the problem of without in terms of ethics.Currently, human umbilical cord mesenchymal stem cells are in clinical field
It is applied and obtains encouraging therapeutic effect.
The seed bank of human umbilical cord mesenchymal stem cells has clinically been widely applied, but there is also limited by conditions of cryopreservation
And the problem of can not be applied on a large scale, it without exogenous pollution is that human umbilical cord mesenchymal stem cells are facing in the frozen storage process of stem cell
Safe and reliable key in bed treatment use, on the one hand due to the fetal calf serum in conventional cryopreservation liquid contain it is various indefinite thin
The intracellular growth factor can be differentiated to form interference to cell, on the other hand carry out clinic using the stem cell of animal blood serum culture and answer
With may cause the cross-infection of pathogen, it is therefore desirable to which the human umbilical cord mesenchymal stem cells serum-free for developing definite ingredients freezes
Liquid storage.
The long period of activity of human umbilical cord mesenchymal stem cells seed bank cell and freeze bad border and have a substantial connection, frozen stock solution it is micro-
Environment has an important influence everybody umbilical cord mesenchymal stem cells, and existing frozen stock solution contains a large amount of antibiotic, exists to cell
Toxic action.
Summary of the invention
It is the deficiency for solving existing conditions of cryopreservation present invention aim to address existing technical assignment of the invention, provides one
The frozen stock solution of kind human umbilical cord mesenchymal stem cells, solves fetal calf serum bring animal derived materials and cause of disease in conventional cryopreservation liquid
Body pollution problem reduces cell death and maintains stem cell properties, and the streptomycin sulphate of addition can reduce antibiotic content, subtract
Murder by poisoning of the small antibiotic to human umbilical cord mesenchymal stem cells increases cell survival rate, promotes cell growth.
The technical solution of offer of the invention is：
A kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells, it is sub- by serum-free medium, serum sub, dimethyl
Sulfone, streptomycin sulphate and additive composition, wherein the percentage of ingredient is：Serum-free medium：41.8-51.8%, serum generation
For object：40-50%, dimethyl sulfoxide：8%, the streptomycin sulphate of 200ug/ml：0.1%, additive：0.1%.
Preferably, it is made of serum-free medium, serum sub, dimethyl sulfoxide, streptomycin sulphate and additive,
Wherein the percentage of ingredient is：Serum-free medium：41.8%, serum sub：50%, dimethyl sulfoxide：8%, 200ug/
The streptomycin sulphate of ml：0.1%, additive：0.1%.
Preferably, it is made of serum-free medium, serum sub, dimethyl sulfoxide, streptomycin sulphate and additive,
Wherein the percentage of ingredient is：Serum-free medium：46.8%, serum sub：45%, dimethyl sulfoxide：8%, 200ug/
The streptomycin sulphate of ml：0.1%, additive：0.1%.
Preferably, it is made of serum-free medium, serum sub, dimethyl sulfoxide, streptomycin sulphate and additive,
Wherein the percentage of ingredient is：Serum-free medium：51.8%, serum sub：40%, dimethyl sulfoxide：8%, 200ug/
The streptomycin sulphate of ml：0.1%, additive：0.1%.
Preferably, described to be added with following components in basic culture solution containing serum free medium, each component is dense eventually
Degree is respectively：1~2g/L of human serum albumins, 5~10mg/L of transferrins, 2~8mg/L of fibronectin, laminin 1
It is~4mg/L, Fe (NO3) 39H2O50g/L, FeSO47H2O417g/L, 1~3 μ g/L of estradiol, 2~5 μ g/L of testosterone, pregnant
1~3 μ g/L of ketone, 39.25~117.74 μ g/L of dexamethasone, 5~10mg/L of insulin, riboflavin 376.36mg/L, coenzyme
A80.96~242.87mg/L, 4.41~6.17mg/L of butanediamine, 1~2mg/L of taurine, 0.61~1.85mg/ of ethylaminoethanol
L, 8.81~26.42mg/L of pyruvic acid, 3.78~7.56 μ g/L of sodium selenate, 292.3~584.6mg/L of L-Glutamine, also wrap
Include 35.74~71.48ng/L of parathyroid hormone, 0.5~2.5 μ g/L of interleukin 2, hydrocortisone 0.36~
1.09mg/L, 10~30mg/L of nonessential amino acid, 1~2mg/L of soybean pancreatin inhibitor, 1 μ g/L of hematopoietin, α-
D-Glucose 3135mg/L, vitamin C 176.13mg/L, NaHCO32.2g/L.
Preferably, the basic culture solution is DMEM/F12.
Preferably, the additive includes μ g/L of platelet derived growth factor AB0.5~2.5, transforming growth factor α
1~4 μ g/L, 0.5~2.5 μ g/L of tumor necrosis factor α, 2~8 μ g/L of vascular endothelial growth factor, epidermal growth factor 4~10
μ g/L, 4~10 μ g/L of basic fibroblast growth factor, 1~5 μ g/L of LIF ELISA, insulin-like growth factor-Ⅱ
μ g/L of I1~5,2~8 μ g/L of stem cell factor composition.
It is a kind of to prepare a kind of freezing for human umbilical cord mesenchymal stem cells described in claim 1-7 any one claim
Method, this method include the following steps：
1) culture dish is rinsed with appropriate PBS culture medium.
2) 0.05% pancreas enzyme -EDTA of the human umbilical cord mesenchymal stem cells after secondary culture is proliferated is digested, the passage
The human umbilical cord mesenchymal stem cells of culture were no more than for five generations, and digestion extremely observes that cell is most of by shuttle shape under inverted microscope
It becomes round and falls off.
3) basal medium is added and terminates digestion.
4) culture dish gently to be blown and beaten, cell is collected, cell suspension is centrifuged after cell screen clothes filter, centrifugal rotational speed 600g,
20 DEG C of centrifugation time 5min.
5) cell count：Be added cell washing solution gently blow and beat be resuspended cell, piping and druming mix, sampling counting, after counting from
Heart washing.
6) it freezes：It inhales and abandons supernatant, stay a small amount of stoste, flick suspension cell with finger.Take out freezing storing box, the frozen stock solution of pre-cooling
And post the cryopreservation tube of label.Frozen stock solution is gently slowly instilled along tube wall, is mixed gently into centrifuge tube containing cell.
7) it dispenses：Every cryopreservation tube 1.0mL is labeled on cryopreservation tube corresponding.
8) cell that need to be frozen is placed in the program temperature reduction box equipped with isopropanol, -80 DEG C overnight, and next day moves into liquid nitrogen
Tank.
Detailed description of the invention
Fig. 1 is the image under the umbilical cord mesenchymal stem cells Electronic Speculum after recovery.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text
Word can be implemented accordingly.
Embodiment 1：
A kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells, it is sub- by serum-free medium, serum sub, dimethyl
Sulfone, streptomycin sulphate and additive composition, wherein the percentage of ingredient is：Serum-free medium：41.8%, serum sub：
50%, dimethyl sulfoxide：8%, the streptomycin sulphate of 200ug/ml：0.1%, additive：0.1%.
Described to be added with following components in basic culture solution containing serum free medium, each component final concentration is respectively：
1~2g/L of human serum albumins, 5~10mg/L of transferrins, 2~8mg/L of fibronectin, laminin 1~4mg/L,
Fe (NO3) 39H2O50g/L, FeSO47H2O417g/L, 1~3 μ g/L of estradiol, 2~5 μ g/L of testosterone, 1~3 μ g/ of progesterone
L, 39.25~117.74 μ g/L of dexamethasone, 5~10mg/L of insulin, riboflavin 376.36mg/L, coacetylase 80.96~
242.87mg/L, 4.41~6.17mg/L of butanediamine, 1~2mg/L of taurine, 0.61~1.85mg/L of ethylaminoethanol, pyruvic acid
It 8.81~26.42mg/L, 3.78~7.56 μ g/L of sodium selenate, 292.3~584.6mg/L of L-Glutamine, further include by first shape
35.74~71.48ng/L of glandular hormone, 0.5~2.5 μ g/L of interleukin 2,0.36~1.09mg/L of hydrocortisone, it is non-must
Need 0~30mg/L of amino acid 1,1~2mg/L of soybean pancreatin inhibitor, 1 μ g/L of hematopoietin, alpha-D-glucose
3135mg/L, vitamin C 176.13mg/L, NaHCO32.2g/L., the basic culture solution is DMEM/F12., described to add
Adding agent includes μ g/L of platelet derived growth factor AB0.5~2.5,1~4 μ g/L of transforming growth factor α, tumor necrosis factor α
0.5~2.5 μ g/L, 2~8 μ g/L of vascular endothelial growth factor, 4~10 μ g/L of epidermal growth factor, basic fibroblast are raw
Long 4~10 μ g/L of the factor, 1~5 μ g/L of LIF ELISA, 1~5 μ g/L of insulin like growth factor-1, stem cell factor 2
~8 μ g/L composition.
The present invention also provides a kind of cryopreservation methods of human umbilical cord mesenchymal stem cells, this method includes the following steps：
1) culture dish is rinsed with appropriate PBS culture medium.
2) 0.05% pancreas enzyme -EDTA of the human umbilical cord mesenchymal stem cells after secondary culture is proliferated is digested, the passage
The human umbilical cord mesenchymal stem cells of culture were no more than for five generations, and digestion extremely observes that cell is most of by shuttle shape under inverted microscope
It becomes round and falls off.
3) basal medium is added and terminates digestion.
4) culture dish gently to be blown and beaten, cell is collected, cell suspension is centrifuged after cell screen clothes filter, centrifugal rotational speed 600g,
20 DEG C of centrifugation time 5min.
5) cell count：Be added cell washing solution gently blow and beat be resuspended cell, piping and druming mix, sampling counting, after counting from
Heart washing.
6) it freezes：It inhales and abandons supernatant, stay a small amount of stoste, flick suspension cell with finger.Take out freezing storing box, the frozen stock solution of pre-cooling
And post the cryopreservation tube of label.Frozen stock solution is gently slowly instilled along tube wall, is mixed gently into centrifuge tube containing cell.
7) it dispenses：Every cryopreservation tube 1.0mL is labeled on cryopreservation tube corresponding.
8) cell that need to be frozen is placed in the program temperature reduction box equipped with isopropanol, -80 DEG C overnight, and next day moves into liquid nitrogen
Tank.
Embodiment 2：
A kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells, it is sub- by serum-free medium, serum sub, dimethyl
Sulfone, streptomycin sulphate and additive composition, wherein the percentage of ingredient is：Serum-free medium：46.8%, serum sub：
45%, dimethyl sulfoxide：8%, the streptomycin sulphate of 200ug/ml：0.1%, additive：0.1%.
Described to be added with following components in basic culture solution containing serum free medium, each component final concentration is respectively：
1~2g/L of human serum albumins, 5~10mg/L of transferrins, 2~8mg/L of fibronectin, laminin 1~4mg/L,
Fe (NO3) 39H2O50g/L, FeSO47H2O417g/L, 1~3 μ g/L of estradiol, 2~5 μ g/L of testosterone, 1~3 μ g/ of progesterone
L, 39.25~117.74 μ g/L of dexamethasone, 5~10mg/L of insulin, riboflavin 376.36mg/L, coacetylase 80.96~
242.87mg/L, 4.41~6.17mg/L of butanediamine, 1~2mg/L of taurine, 0.61~1.85mg/L of ethylaminoethanol, pyruvic acid
It 8.81~26.42mg/L, 3.78~7.56 μ g/L of sodium selenate, 292.3~584.6mg/L of L-Glutamine, further include by first shape
35.74~71.48ng/L of glandular hormone, 0.5~2.5 μ g/L of interleukin 2,0.36~1.09mg/L of hydrocortisone, it is non-must
Need 0~30mg/L of amino acid 1,1~2mg/L of soybean pancreatin inhibitor, 1 μ g/L of hematopoietin, alpha-D-glucose
3135mg/L, vitamin C 176.13mg/L, NaHCO32.2g/L., the basic culture solution is DMEM/F12., described to add
Adding agent includes μ g/L of platelet derived growth factor AB0.5~2.5,1~4 μ g/L of transforming growth factor α, tumor necrosis factor α
0.5~2.5 μ g/L, 2~8 μ g/L of vascular endothelial growth factor, 4~10 μ g/L of epidermal growth factor, basic fibroblast are raw
Long 4~10 μ g/L of the factor, 1~5 μ g/L of LIF ELISA, 1~5 μ g/L of insulin like growth factor-1, stem cell factor 2
~8 μ g/L composition.
The present invention also provides a kind of cryopreservation methods of human umbilical cord mesenchymal stem cells, this method includes the following steps：
1) culture dish is rinsed with appropriate PBS culture medium.
2) 0.05% pancreas enzyme -EDTA of the human umbilical cord mesenchymal stem cells after secondary culture is proliferated is digested, the passage
The human umbilical cord mesenchymal stem cells of culture were no more than for five generations, and digestion extremely observes that cell is most of by shuttle shape under inverted microscope
It becomes round and falls off.
3) basal medium is added and terminates digestion.
4) culture dish gently to be blown and beaten, cell is collected, cell suspension is centrifuged after cell screen clothes filter, centrifugal rotational speed 600g,
20 DEG C of centrifugation time 5min.
5) cell count：Be added cell washing solution gently blow and beat be resuspended cell, piping and druming mix, sampling counting, after counting from
Heart washing.
6) it freezes：It inhales and abandons supernatant, stay a small amount of stoste, flick suspension cell with finger.Take out freezing storing box, the frozen stock solution of pre-cooling
And post the cryopreservation tube of label.Frozen stock solution is gently slowly instilled along tube wall, is mixed gently into centrifuge tube containing cell.
7) it dispenses：Every cryopreservation tube 1.0mL is labeled on cryopreservation tube corresponding.
8) cell that need to be frozen is placed in the program temperature reduction box equipped with isopropanol, -80 DEG C overnight, and next day moves into liquid nitrogen
Tank.
Embodiment 3：
A kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells, it is sub- by serum-free medium, serum sub, dimethyl
Sulfone, streptomycin sulphate and additive composition, wherein the percentage of ingredient is：Serum-free medium：51.8%, serum sub：
40%, dimethyl sulfoxide：8%, the streptomycin sulphate of 200ug/ml：0.1%, additive：0.1%.
Described to be added with following components in basic culture solution containing serum free medium, each component final concentration is respectively：
1~2g/L of human serum albumins, 5~10mg/L of transferrins, 2~8mg/L of fibronectin, laminin 1~4mg/L,
Fe (NO3) 39H2O50g/L, FeSO47H2O417g/L, 1~3 μ g/L of estradiol, 2~5 μ g/L of testosterone, 1~3 μ g/ of progesterone
L, 39.25~117.74 μ g/L of dexamethasone, 5~10mg/L of insulin, riboflavin 376.36mg/L, coacetylase 80.96~
242.87mg/L, 4.41~6.17mg/L of butanediamine, 1~2mg/L of taurine, 0.61~1.85mg/L of ethylaminoethanol, pyruvic acid
It 8.81~26.42mg/L, 3.78~7.56 μ g/L of sodium selenate, 292.3~584.6mg/L of L-Glutamine, further include by first shape
35.74~71.48ng/L of glandular hormone, 0.5~2.5 μ g/L of interleukin 2,0.36~1.09mg/L of hydrocortisone, it is non-must
Need 0~30mg/L of amino acid 1,1~2mg/L of soybean pancreatin inhibitor, 1 μ g/L of hematopoietin, alpha-D-glucose
3135mg/L, vitamin C 176.13mg/L, NaHCO32.2g/L., the basic culture solution is DMEM/F12., described to add
Adding agent includes μ g/L of platelet derived growth factor AB0.5~2.5,1~4 μ g/L of transforming growth factor α, tumor necrosis factor α
0.5~2.5 μ g/L, 2~8 μ g/L of vascular endothelial growth factor, 4~10 μ g/L of epidermal growth factor, basic fibroblast are raw
Long 4~10 μ g/L of the factor, 1~5 μ g/L of LIF ELISA, 1~5 μ g/L of insulin like growth factor-1, stem cell factor 2
~8 μ g/L composition.
The present invention also provides a kind of cryopreservation methods of human umbilical cord mesenchymal stem cells, this method includes the following steps：
1) culture dish is rinsed with appropriate PBS culture medium.
2) 0.05% pancreas enzyme -EDTA of the human umbilical cord mesenchymal stem cells after secondary culture is proliferated is digested, the passage
The human umbilical cord mesenchymal stem cells of culture were no more than for five generations, and digestion extremely observes that cell is most of by shuttle shape under inverted microscope
It becomes round and falls off.
3) basal medium is added and terminates digestion.
4) culture dish gently to be blown and beaten, cell is collected, cell suspension is centrifuged after cell screen clothes filter, centrifugal rotational speed 600g,
20 DEG C of centrifugation time 5min.
5) cell count：Be added cell washing solution gently blow and beat be resuspended cell, piping and druming mix, sampling counting, after counting from
Heart washing.
6) it freezes：It inhales and abandons supernatant, stay a small amount of stoste, flick suspension cell with finger.Take out freezing storing box, the frozen stock solution of pre-cooling
And post the cryopreservation tube of label.Frozen stock solution is gently slowly instilled along tube wall, is mixed gently into centrifuge tube containing cell.
7) it dispenses：Every cryopreservation tube 1.0mL is labeled on cryopreservation tube corresponding.
8) cell that need to be frozen is placed in the program temperature reduction box equipped with isopropanol, -80 DEG C overnight, and next day moves into liquid nitrogen
Tank.
Comparative example：
The cells frozen storing liquid of commercially available human umbilical cord mesenchymal stem cells, by complete culture solution, calf serum, dimethyl sulfoxide,
Wherein the percentage of ingredient is：Complete culture solution：42%, calf serum：50%, dimethyl sulfoxide：8%.
The cryopreservation methods of comparative example human umbilical cord mesenchymal stem cells, this method include the following steps：
1) culture dish is rinsed with appropriate PBS culture medium.
2) 0.05% pancreas enzyme -EDTA of the human umbilical cord mesenchymal stem cells after secondary culture is proliferated is digested, the passage
The human umbilical cord mesenchymal stem cells of culture were no more than for five generations, and digestion extremely observes that cell is most of by shuttle shape under inverted microscope
It becomes round and falls off.
3) basal medium is added and terminates digestion.
4) culture dish gently to be blown and beaten, cell is collected, cell suspension is centrifuged after cell screen clothes filter, centrifugal rotational speed 600g,
20 DEG C of centrifugation time 5min.
5) cell count：Be added cell washing solution gently blow and beat be resuspended cell, piping and druming mix, sampling counting, after counting from
Heart washing.
6) it freezes：It inhales and abandons supernatant, stay a small amount of stoste, flick suspension cell with finger.Take out freezing storing box, the frozen stock solution of pre-cooling
And post the cryopreservation tube of label.Frozen stock solution is gently slowly instilled along tube wall, is mixed gently into centrifuge tube containing cell.
7) it dispenses：Every cryopreservation tube 1.0mL is labeled on cryopreservation tube corresponding.
8) cell that need to be frozen is placed in the program temperature reduction box equipped with isopropanol, -80 DEG C overnight, and next day moves into liquid nitrogen
Tank.
The recovering experiment of the human umbilical cord mesenchymal stem cells frozen：
It is immediately placed in after freeze-stored cell is taken out in 40 DEG C of constant water bath box, makes its rapid fluid resuscitation in 1min, then will
Cell moves into centrifuge tube, and DMEM/F12 culture medium is added, and suction, which is beaten, to be uniformly mixed, and 1000r/min is centrifuged 5min, after abandoning supernatant,
It is washed 2 times with PBS again, cell suspending liquid finally is made with 1mlPBS.
Testing index
(1) motility rate：After Trypan Blue 3-5min of the 50 μ L cell suspending liquids with 50 μ L 0.4%, blood count is used
Plate counting is not dyed to the percentage that blue cell number accounts for total cell number, as Cell viability.
(2) rate of recovery：The cell number collected after freezing accounts for the percentage for freezing preceding total number of cells, the as rate of recovery.
(3) immunizing antigen CD44 detection and integrated value：After cell is dripped piece, CD44 detection is completed in 24 hours.Signal
When colour developing, each sample observes 100 or more cells, be divided by the power of its signal ± ,+, ++ 3 grades, 1,2,4 point is counted respectively,
Calculate total mark value.
Experimental result：
1 embodiment 1-3 of table, comparative example Cell viability and the rate of recovery (%)
| Testing index | Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example |
| Freeze preceding motility rate | 99 | 99 | 99 | 99 |
| Freeze rear motility rate | 91 | 92 | 90 | 81 |
| The rate of recovery | 90 | 93 | 91 | 73 |
2 embodiment 1-3 of table, comparative example cell CD44 integrated value
Detection freezes the variation of rear cellular morphology and freezes the cellular morphology difference between rear each group：
As shown in Figure 1, people's umbilical cord through detecting, in human umbilical cord mesenchymal stem cells serum-free frozen stock solution provided by the invention
Mescenchymal stem cell keeps good adherent characteristic, human umbilical cord mesenchymal stem cells show shuttle shape at fibrous cell's shape
State, while warp is excessively commissioned to train in the case where supporting, cell can keep normal condition.
Analysis of experimental results：
This experiments experiment statistics indicate that, the human umbilical cord mesenchymal stem cells in embodiment 1-3 freeze after motility rate, the rate of recovery
It is significantly greater than the cells frozen storing liquid of common commercially available umbilical cord mesenchymal stem cells with cell CD44 integrated value, wherein embodiment 2
Effect is best, is the most preferred embodiment of this experiment.Everybody navel in the present inventor's umbilical cord mesenchymal stem cells serum-free frozen stock solution
Band mescenchymal stem cell keeps good adherent characteristic, everybody umbilical cord mesenchymal stem cells show the thin at threadiness of shuttle shape
Born of the same parents' form, while warp is excessively commissioned to train in the case where supporting, cell can keep normal condition.
It is obtained from above, the cells frozen storing liquid of everybody umbilical cord mesenchymal stem cells of offer of the invention overcomes animal sources
Property serum contamination defect and and solve the problems, such as cell amplification quantity and maintain stem cell properties：Without serum, avoid
Animal derived pollution in unknown serum composition；It is added to streptomycin sulphate and additive in culture medium, can promote and fill between umbilical cord
The proliferation and maintenance stem cell properties of matter stem cell, increase the survival rate of everybody umbilical cord mesenchymal stem cells, reduce cell toxicant
Property, it is ensured that the quality and safety of cell increase cell survival rate, promote cell growth.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily
Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details.
Claims (8)
1. a kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells, which is characterized in that replaced by serum-free medium, serum
Object, dimethyl sulfoxide, streptomycin sulphate and additive composition, wherein the percentage of ingredient is：Serum-free medium：41.8-
51.8%, serum sub：40-50%, dimethyl sulfoxide：8%, the streptomycin sulphate of 200ug/ml：0.1%, additive：
0.1%.
2. a kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that by no blood
Clear culture solution, serum sub, dimethyl sulfoxide, streptomycin sulphate and additive composition, wherein the percentage of ingredient is：Without blood
Clear culture solution：41.8%, serum sub：50%, dimethyl sulfoxide：8%, the streptomycin sulphate of 200ug/ml：0.1%, add
Add agent：0.1%.
3. a kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that by no blood
Clear culture solution, serum sub, dimethyl sulfoxide, streptomycin sulphate and additive composition, wherein the percentage of ingredient is：Without blood
Clear culture solution：46.8%, serum sub：45%, dimethyl sulfoxide：8%, the streptomycin sulphate of 200ug/ml：0.1%, add
Add agent：0.1%.
4. a kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that by no blood
Clear culture solution, serum sub, dimethyl sulfoxide, streptomycin sulphate and additive composition, wherein the percentage of ingredient is：Without blood
Clear culture solution：51.8%, serum sub：40%, dimethyl sulfoxide：8%, the streptomycin sulphate of 200ug/ml：0.1%, add
Add agent：0.1%.
5. a kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that described to contain
There is serum free medium in basic culture solution added with following components, each component final concentration is respectively：Human serum albumins 1~
2g/L, 5~10mg/L of transferrins, 2~8mg/L of fibronectin, laminin 1~4mg/L, Fe (NO3) 3
9H2O50g/L, FeSO47H2O417g/L, 1~3 μ g/L of estradiol, 2~5 μ g/L of testosterone, 1~3 μ g/L of progesterone, dexamethasone
39.25~117.74 μ g/L, 5~10mg/L of insulin, riboflavin 376.36mg/L, 80.96~242.87mg/L of coacetylase, fourth
4.41~6.17mg/L of diamines, 1~2mg/L of taurine, 0.61~1.85mg/L of ethylaminoethanol, 8.81~26.42mg/ of pyruvic acid
L, 3.78~7.56 μ g/L of sodium selenate, 292.3~584.6mg/L of L-Glutamine, further include parathyroid hormone 35.74~
71.48ng/L, 0.5~2.5 μ g/L of interleukin 2,0.36~1.09mg/L of hydrocortisone, nonessential amino acid 10~
30mg/L, 1~2mg/L of soybean pancreatin inhibitor, 1 μ g/L of hematopoietin, alpha-D-glucose 3135mg/L, vitamin C
176.13mg/L、NaHCO32.2g/L。
6. a kind of human umbilical cord mesenchymal stem cells frozen stock solution according to claim 2, it is characterised in that：The basis training
Nutrient solution is DMEM/F12.
7. a kind of cells frozen storing liquid of human umbilical cord mesenchymal stem cells according to claim 1, which is characterized in that described to add
Adding agent includes μ g/L of platelet derived growth factor AB0.5~2.5,1~4 μ g/L of transforming growth factor α, tumor necrosis factor α
0.5~2.5 μ g/L, 2~8 μ g/L of vascular endothelial growth factor, 4~10 μ g/L of epidermal growth factor, basic fibroblast are raw
Long 4~10 μ g/L of the factor, 1~5 μ g/L of LIF ELISA, 1~5 μ g/L of insulin like growth factor-1, stem cell factor 2
~8 μ g/L composition.
8. a kind of prepare a kind of side of freezing of human umbilical cord mesenchymal stem cells described in claim 1-7 any one claim
Method, which is characterized in that this method includes the following steps：
1) culture dish is rinsed with appropriate PBS culture medium；
2) 0.05% pancreas enzyme -EDTA of the human umbilical cord mesenchymal stem cells after secondary culture is proliferated is digested, the secondary culture
Human umbilical cord mesenchymal stem cells be no more than five generations, digestion to observed under inverted microscope cell largely become from shuttle shape
Circle simultaneously falls off；
3) basal medium is added and terminates digestion；
4) it gently blows and beats culture dish, collects cell, cell suspension is centrifuged after cell screen clothes filter, centrifugal rotational speed 600g, and 20 DEG C
Centrifugation time 5min；
5) cell count：Cell washing solution is added and gently blows and beats resuspension cell, piping and druming mixes, and sampling counts, and is centrifuged and washes after counting
It washs；
6) it freezes：It inhales and abandons supernatant, stay a small amount of stoste, flick suspension cell with finger.Take out pre-cooling freezing storing box, frozen stock solution and
Post the cryopreservation tube of label.Frozen stock solution is gently slowly instilled along tube wall, is mixed gently into centrifuge tube containing cell；
7) it dispenses：Every cryopreservation tube 1.0mL is labeled on cryopreservation tube corresponding；
8) cell that need to be frozen is placed in the program temperature reduction box equipped with isopropanol, -80 DEG C overnight, and next day moves into liquid nitrogen container.
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