CN106561632A - Freezing preservation solution used for adipose-derived stem cells as well as preparation method for freezing preservation solution and culture medium - Google Patents
Freezing preservation solution used for adipose-derived stem cells as well as preparation method for freezing preservation solution and culture medium Download PDFInfo
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- CN106561632A CN106561632A CN201610938658.6A CN201610938658A CN106561632A CN 106561632 A CN106561632 A CN 106561632A CN 201610938658 A CN201610938658 A CN 201610938658A CN 106561632 A CN106561632 A CN 106561632A
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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
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Abstract
The invention discloses a freezing preservation solution used for adipose-derived stem cells as well as a preparation method for the freezing preservation solution and a culture medium. The culture medium comprises the following components in parts by weight: 7200-7300 parts of inorganic salt composite, 980-1000 parts of amino acid complex, 40-45 parts of vitamin composite, 1000-1100 parts of glucose, 100-110 parts of sodium pyruvate, 60-75 parts of butyric acid, 80-110 parts of sodium butyrate, 0.15-0.22 part of ferric citrate and 1,000,000 parts of water. The freezing preservation solution has the beneficial effects that: the culture medium has excellent freezing preservation effect in the freezing preservation solution which is compounded by a serum substitute component KSR and dimethyl sulfoxide, and is used for the adipose-derived stem cells.
Description
Technical field
The present invention relates to frozen stock solution field, more particularly to a kind of frozen stock solution for fat stem cell and preparation method thereof with
And culture medium.
Background technology
Bone marrow stem cell, umbilical cord stem cells and fat stem cell (adipose-derived stem cells, ADSCs) are
The more several stem cell of Recent study.Bone marrow stem cell is as not easy to drawing materials, amplification are relatively slow, for weak and gerontal patient
The problems such as inapplicable and ethics, make troubles to clinical practice.Umbilical cord mesenchymal stem cells are originated compared with bone marrow stem cell
Wide, immunogenicity is low, almost without immunological rejection.Fat stem cell is isolated from fatty tissue in recent years
A kind of stem cell with multi-lineage potential, it has many good qualities compared with bone marrow stem cell, umbilical cord stem cells, and research finds
ADSCs cells stably can be bred in vitro and decline rate is low, while obtaining a large amount of dry by having easy, a small amount of tissue of drawing materials
Cell, suitable large-scale culture, growth is uniform, it is little to body injury the advantages of, and its wide material sources, cylinder storage amount it is big,
Suitable autotransplantation, security performance are high, and the foundation in fat stem cell storehouse is increasingly becoming one of study hotspot new in recent years.
The Chinese patent of Publication No. CN102550542A discloses a kind of serum-free frozen stock solution for fat stem cell
And the foundation in fat stem cell storehouse.The serum-free frozen stock solution can be used in the frozen of fat stem cell, and frozen effect is better than containing
The frozen effect of serum frozen stock solution, it is to avoid the risk that may be present in Clinical practice of the frozen stock solution containing serum.
Based on the further investigation to the serum-free frozen stock solution, although inventors have realized that the serum-free frozen stock solution have it is good
Good frozen effect, but the serum-free frozen stock solution is only to provide the specific serum-free frozen stock solution that a kind of KSR is prepared, not
Further the serum-free medium in serum-free frozen stock solution is furtherd investigate.For this purpose, the present inventor is to serum-free frozen stock solution
In serum-free medium further studied, make the serum-free medium and blood serum substituting component KSR and dimethyl sub-
Frozen effect after sulfone compounding is further lifted.
The content of the invention
It is an object of the invention to provide a kind of culture medium, the culture medium is sub- with blood serum substituting component KSR and dimethyl
The frozen stock solution for fat stem cell that sulfone compounding is obtained has excellent frozen effect.
The present invention above-mentioned technical purpose technical scheme is that：
A kind of culture medium, including the component of following parts by weight：
By adopting above-mentioned technical proposal, inorganic salt composite can adjust Premeabilisation of cells pressure, the activity for adjusting enzyme and
Solution acid alkalinity.Amino acid complex is the necessary raw material of cell synthetic protein.Vitamin complex is to maintain cell life
The bioactive substance of activity, can form prothetic group or coenzyme in cell body.Glucose is the main energy of cell activities
Amount source.Sodium Pyruvate can Energy supply material as an alternative.In the case of glucose deficiency, cell can also metabolism acetone acid
Sodium obtains energy.Butanoic acid and sodium butyrate constitute the pH buffer systems of solution, maintain the pH balances of solution.Ferric citrate can be thin
Born of the same parents provide ferrum element, and the transferrinss in alternative serum play biological effect.Containing sub- selenium in the composition of serum substitute KSR
Sour sodium and Monohydrated selenium dioxide, while including containing barium compound, stanniferous inorganic compound, silver-containing inorganic compound, ammonium-containing compound and containing
Calcium compounds.Toxic gas can be discharged when KSR contact acids or bleaching liquid.And on the one hand the culture medium is able to maintain that pH puts down
Weighing apparatus, on the other hand has the reducing agents such as Sodium Pyruvate again, and the danger of poison is discharged so as to reduce KSR.PH balances can also be tieed up simultaneously
Hold stablizing for ammonium-containing compound.And the lifting that can stably bring frozen effect of system.Therefore, the jelly for being obtained using the culture medium
Liquid storage improves its frozen effect from many aspects.
The present invention is further arranged to：According to weight portion, also including phenol red 9-10 parts.
By adopting above-mentioned technical proposal, phenol red is a kind of deep red crystalline powder, is practically insoluble in ether and chloroform, molten
It is in peony in alkali hydroxide or alkali carbonate solution, it is stable in the air.It is phenol red can be as acid-base indicator, compared with phenolphthalein spirit
It is quick, meta-acid, scope 6.8-8.0, by yellow to red.
The present invention is further arranged to：According to weight portion, also including fibronectin 0.1-1 part.
The adherent growth of cell can be promoted by adopting above-mentioned technical proposal, fibronectin, while being also important
The differentiation factor of mitogen and maintenance cell normal function, the breeding and differentiation important role to cell.The present inventor
It was unexpectedly observed that the compound use of fibronectin and ferric citrate can be mutually promoted, strengthen the frozen effect of frozen stock solution.
The present invention is further arranged to：According to weight portion, also including hormone complex 5-25 parts, the hormone complex bag
Insulin, glucagon and progesterone are included, according to weight ratio, the insulin: glucagon: progesterone=3-6: 3-5: 1.
By adopting above-mentioned technical proposal, hormone complex is stimulating cellular growth, the important substance for maintaining cell function.
Insulin is even more a kind of requisite hormone of cell.And glucagon can be coordinated with insulin, concentration of glucose is maintained
It is stable, promote the growth of cell.Progesterone also known as Progesterone, are a kind of natural progestogens secreted by corpus luteum.By insulin,
Glucagon and progesterone compound use, strengthen the facilitation to cell.
The present invention is further arranged to：According to weight portion, also including 50000 parts of antibiotic, the antibiotic is penicillin
Or streptomycin.
By adopting above-mentioned technical proposal, the use of antibiotic can avoid the culture of cell, in frozen and resuscitation process
Disturbed by miscellaneous bacteria, affected result of the test.And penicillin and streptomycin belong to common antibiotics, cost is relatively low, antibacterial effect
Fruit is significantly.
The present invention is further arranged to：The inorganic salt composite includes anhydrous calcium chloride, nickel sulfate, potassium chloride, anhydrous
Magnesium sulfate, Sodium Chloride, zinc sulfate, according to weight ratio, the anhydrous calcium chloride: nickel sulfate: potassium chloride: anhydrous magnesium sulfate: chlorination
Sodium: zinc sulfate=53: 4: 80: 20: 1280: 9.
By adopting above-mentioned technical proposal, the presence of plurality of inorganic salt fully can adjust the ionic equilibrium of intraor extracellular and
Solution acid alkalinity, the growth for cell provide suitable growing environment.
The present invention is further arranged to：The vitamin complex includes calcium pantothenate, (2-Hydroxyethyl)trimethylammonium bitartrate, Folic Acid, inositol, cigarette
Amide, riboflavin, thiamine hydrochloride, Pyridoxine Hydrochloride, vitamin A, vitamin C and Vitamin E, according to weight ratio, the pantothenic acid
Calcium: (2-Hydroxyethyl)trimethylammonium bitartrate: Folic Acid: inositol: nicotiamide: riboflavin: thiamine hydrochloride: Pyridoxine Hydrochloride: vitamin A: vitamin C and dimension
Raw element E=2: 3.6: 2: 3.6: 2: 0.2: 2: 2: 1.1: 2: 1.
Various different prothetic groups or auxiliary can be formed in cell body by adopting above-mentioned technical proposal, multivitamin
Enzyme, so as to participate in different vital movements, maintains the normal activities of cell.
The present invention is further arranged to：The amino acid complex includes L-Arginine Hydrochloride, L- hydrochloric acid cystine, sweet ammonia
Acid, L- histidine monohydrochlorides, L-Isoleucine, L-Leu, L-Lysine Hydrochloride, METHIONINE, L-phenylalanine, L- silk ammonia
Acid, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, according to weight ratio, L-Arginine Hydrochloride: L- hydrochloric acid cystine:
Glycine: L- histidine monohydrochlorides: L-Isoleucine: L-Leu: L-Lysine Hydrochloride: METHIONINE: L-phenylalanine: L-
Serine: L-Threonine: L-Tryptophan: L-Tyrosine: L-Valine=84: 63: 30: 42: 105: 105: 146: 30: 66: 42:
95∶16∶72∶94。
By adopting above-mentioned technical proposal, as the necessary raw material of cell synthetic protein, there is provided several amino acids can
Meet raw materials requirement during cell synthetic protein, it is to avoid cell cannot synthesize the protein of needs because of lack of material, from
And affect the vital movement of cell.
Another goal of the invention of the present invention is to provide a kind of frozen stock solution for fat stem cell, including following component：
Dimethyl sulfoxide, blood serum substituting component KSR and above-mentioned culture medium, the volume fraction of the dimethyl sulfoxide is 8-20%, described
The volume fraction of blood serum substituting component KSR is 70-85%, and the volume fraction of the culture medium is 5-15%.
Compound with blood serum substituting component KSR and dimethyl sulfoxide, strengthen and use by adopting above-mentioned technical proposal, culture medium
In the frozen effect of the frozen stock solution of fat stem cell.
It is to provide a kind of preparation method of the frozen stock solution for fat stem cell that the present invention also has a goal of the invention, including
Following preparation process：
Step1：According to weight portion, inorganic salt composite 7200-7300 parts, amino acid complex 980-1000 parts, dimension life are weighed
Plain complex 40-45 parts, glucose 1000-1100 parts, Sodium Pyruvate 100-110 parts, butanoic acid 60-75 parts, sodium butyrate 80-110
Part, ferric citrate 0.15-0.22 parts, 1000000 parts of water, phenol red 9-10 parts, fibronectin 0.1-1 part, hormone complex 5-
25 parts, 50000 parts of antibiotic, are mixed and stirred for uniformly, obtaining culture medium；
Step2：According to volume fraction, the dimethyl sulfoxide of 8-20%, blood serum substituting component KSR of 70-85% and 5- are measured
15% culture medium, is mixed and stirred for uniform.
Fully can be mixed with blood serum substituting component KSR and dimethyl sulfoxide by adopting above-mentioned technical proposal, culture medium
Close, each component interphase interaction is mutually promoted, and strengthens the frozen effect of the frozen stock solution for fat stem cell.
In sum, the invention has the advantages that：
1st, the compound use of fibronectin and ferric citrate can be mutually promoted, and strengthen the frozen effect of frozen stock solution and preserve effect
Really；
2nd, the presence of sodium butyrate not only constitutes pH buffer systems with butanoic acid, and maintenance system pH's stablizes, while can also be with Fructus Citri Limoniae
Sour ferrum collective effect, promotes protein expression.Vitamin C can affect the release of the iron ion in ferric citrate, control iron ion
Concentration, so as to the expression for also further affecting albumen；
3rd, component of the present invention by the culture medium in improvement frozen stock solution, and content optimization design is carried out, so that culture medium exists
When compounding with blood serum substituting component KSR and dimethyl sulfoxide, the frozen effect of the frozen stock solution for fat stem cell can be strengthened.
Specific embodiment
This specific embodiment is only explanation of the invention, and which is not limitation of the present invention, people in the art
Member can make the modification for not having creative contribution after this specification is read as needed to the present embodiment, but as long as at this
All protected by Patent Law in the right of invention.
In embodiment one to five, inorganic salt composite include anhydrous calcium chloride, nickel sulfate, potassium chloride, anhydrous magnesium sulfate,
Sodium Chloride, zinc sulfate, according to weight ratio, anhydrous calcium chloride: nickel sulfate: potassium chloride: anhydrous magnesium sulfate: Sodium Chloride: zinc sulfate=
53∶4∶80∶20∶1280∶9.Amino acid complex includes L-Arginine Hydrochloride, L- hydrochloric acid cystine, glycine, L- hydrochloric acid group ammonia
Acid, L-Isoleucine, L-Leu, L-Lysine Hydrochloride, METHIONINE, L-phenylalanine, L-Serine, L-Threonine,
L-Tryptophan, L-Tyrosine, L-Valine, according to weight ratio, L-Arginine Hydrochloride: L- hydrochloric acid cystine: glycine: L- hydrochloric acid
Histidine: L-Isoleucine: L-Leu: L-Lysine Hydrochloride: METHIONINE: L-phenylalanine: L-Serine: L- Soviet Unions ammonia
Acid: L-Tryptophan: L-Tyrosine: L-Valine=84: 63: 30: 42: 105: 105: 146: 30: 66: 42: 95: 16: 72: 94.
Vitamin complex includes that calcium pantothenate, (2-Hydroxyethyl)trimethylammonium bitartrate, Folic Acid, inositol, nicotiamide, riboflavin, thiamine hydrochloride, hydrochloric acid pyrrole are more
Pungent, vitamin A, vitamin C and Vitamin E, according to weight ratio, calcium pantothenate: (2-Hydroxyethyl)trimethylammonium bitartrate: Folic Acid: inositol: nicotiamide: core
Flavin: thiamine hydrochloride: Pyridoxine Hydrochloride: vitamin A: vitamin C and Vitamin E=2: 3.6: 2: 3.6: 2: 0.2: 2: 2: 1.1:
2∶1。
Embodiment one
A kind of preparation method of the frozen stock solution for fat stem cell, including following preparation process：
Step1：According to weight portion, 7200 parts of inorganic salt composite, 990 parts of amino acid complex, vitamin complex 40 are weighed
Part, 1000 parts of glucose, 110 parts of Sodium Pyruvate, 68 parts of butanoic acid, 80 parts of sodium butyrate, 0.15 part of ferric citrate, 1000000 parts of water,
Phenol red 9 parts, 1 part of fibronectin, 25 parts of hormone complex, 50000 parts of penicillin, be mixed and stirred for uniform, obtain culture medium；
Step2：According to volume fraction, the culture of 8% dimethyl sulfoxide, 77% blood serum substituting component KSR and 15% is measured
Base, is mixed and stirred for uniform.
Wherein hormone complex includes insulin, glucagon and progesterone, according to weight ratio, the insulin: pancreas is high
Blood glucose element: progesterone=3: 3: 1.
Embodiment two
A kind of preparation method of the frozen stock solution for fat stem cell, including following preparation process：
Step1：According to weight portion, 7230 parts of inorganic salt composite, 980 parts of amino acid complex, vitamin complex 41 are weighed
Part, 1030 parts of glucose, 107 parts of Sodium Pyruvate, 60 parts of butanoic acid, 85 parts of sodium butyrate, 0.22 part of ferric citrate, 1000000 parts of water,
Phenol red 9.2 parts, 0.8 part of fibronectin, 20 parts of hormone complex, 50000 parts of streptomycin, be mixed and stirred for uniform, trained
Foster base；
Step2：According to volume fraction, the culture of 14% dimethyl sulfoxide, 74% blood serum substituting component KSR and 12% is measured
Base, is mixed and stirred for uniform.
Wherein hormone complex includes insulin, glucagon and progesterone, according to weight ratio, the insulin: pancreas is high
Blood glucose element: progesterone=3: 5: 1.
Embodiment three
A kind of preparation method of the frozen stock solution for fat stem cell, including following preparation process：
Step1：According to weight portion, 7260 parts of inorganic salt composite, 985 parts of amino acid complex, vitamin complex 43 are weighed
Part, 1060 parts of glucose, 105 parts of Sodium Pyruvate, 65 parts of butanoic acid, 90 parts of sodium butyrate, 0.18 part of ferric citrate, 1000000 parts of water,
Phenol red 9.4 parts, 0.6 part of fibronectin, 15 parts of hormone complex, 50000 parts of penicillin, be mixed and stirred for uniform, trained
Foster base；
Step2：According to volume fraction, the culture of 20% dimethyl sulfoxide, 70% blood serum substituting component KSR and 10% is measured
Base, is mixed and stirred for uniform.
Wherein hormone complex includes insulin, glucagon and progesterone, according to weight ratio, the insulin: pancreas is high
Blood glucose element: progesterone=4: 3: 1.
Example IV
A kind of preparation method of the frozen stock solution for fat stem cell, including following preparation process：
Step1：According to weight portion, 7280 parts of inorganic salt composite, 995 parts of amino acid complex, vitamin complex 42 are weighed
Part, 1080 parts of glucose, 103 parts of Sodium Pyruvate, 70 parts of butanoic acid, 95 parts of sodium butyrate, 0.2 part of ferric citrate, 1000000 parts of water,
Phenol red 9.6 parts, 0.4 part of fibronectin, 10 parts of hormone complex, 50000 parts of streptomycin, be mixed and stirred for uniform, trained
Foster base；
Step2：According to volume fraction, the culture of 10% dimethyl sulfoxide, 85% blood serum substituting component KSR and 5% is measured
Base, is mixed and stirred for uniform.
Wherein hormone complex includes insulin, glucagon and progesterone, according to weight ratio, the insulin: pancreas is high
Blood glucose element: progesterone=6: 4: 1.
Embodiment five
A kind of preparation method of the frozen stock solution for fat stem cell, including following preparation process：
Step1：According to weight portion, 7300 parts of inorganic salt composite, 1000 parts of amino acid complex, vitamin complex 45 are weighed
Part, 1100 parts of glucose, 100 parts of Sodium Pyruvate, 75 parts of butanoic acid, 100 parts of sodium butyrate, 0.16 part of ferric citrate, water 1000000
Part, phenol red 10 parts, 0.1 part of fibronectin, 5 parts of hormone complex, 50000 parts of penicillin, be mixed and stirred for uniform, trained
Foster base；
Step2：According to volume fraction, the culture of 17% dimethyl sulfoxide, 75% blood serum substituting component KSR and 8% is measured
Base, is mixed and stirred for uniform.
Wherein hormone complex includes insulin, glucagon and progesterone, according to weight ratio, the insulin: pancreas is high
Blood glucose element: progesterone=6: 5: 1.
Frozen test
Carry out the separation of fat stem cell, culture, freeze with reference to embodiment 1-4 of the Chinese patent of Publication No. CN102550542A
Deposit, recover.Wherein, carry out fat stem cell it is frozen when, be respectively adopted according to embodiment one to five prepare frozen stock solution carry out
It is frozen.
With reference to the survival rate of the fat stem cell of 5 pairs of recoveries of embodiment of the Chinese patent of Publication No. CN102550542A
It is measured and records.
The survival rate log of the fat stem cell of 1 embodiment of table, one to five frozen rear recovery
| Group | Recover within frozen 1 month | Recover within frozen 3 months | Recover within frozen 6 months |
| Embodiment one | 96.4% | 96.4% | 96.7% |
| Embodiment two | 96.8% | 96.6% | 96.5% |
| Embodiment three | 97.1% | 97.0% | 96.8% |
| Example IV | 96.7% | 96.8% | 96.6% |
| Embodiment five | 96.5% | 96.5% | 96.5% |
| Meansigma methodss | 96.7% | 96.66% | 96.62% |
As known from Table 1, the cell cryopreservation cell survival rate meansigma methodss of 1 month, 3 months and 6 months be respectively 96.7%,
96.66% and 96.62%.It can be seen that, the frozen effect of frozen stock solution of the present invention is affected less by the frozen time, and frozen performance is steady
It is fixed, frozen excellent effect.And in embodiment one to five, the survival rate highest of embodiment three, it is seen that using according to embodiment three
The frozen stock solution of preparation is optimum to the frozen effect of cell.
Food preservation test
The frozen stock solution prepared according to embodiment one to five is respectively taken, in depositing in an aseptic refrigerator of cleaning, refrigerator is controlled
Temperature be -20 DEG C, storage was taken out and returns to room temperature after one month.
The separation of fat stem cell, training are carried out with reference to embodiment 1-4 of the Chinese patent of Publication No. CN102550542A
Foster, frozen, recovery.Wherein, carry out fat stem cell it is frozen when, be respectively adopted according to one to five preparation of embodiment, pass through -20
DEG C storage one month after take out and return to room temperature frozen stock solution carry out it is frozen.
Embodiments of the invention one to five are distinguished with reference to the embodiment 5 of the Chinese patent of Publication No. CN102550542A
The survival rate of the fat stem cell recovered is determined and is recorded.
The survival rate log of the fat stem cell of 2 embodiment of table, one to five frozen rear recovery (deposit through -20 DEG C by frozen stock solution
Put a process in month)
| Group | Recover within frozen 1 month | Recover within frozen 3 months | Recover within frozen 6 months |
| Embodiment one | 96.4% | 96.4% | 96.7% |
| Embodiment two | 96.6% | 96.5% | 96.5% |
| Embodiment three | 97.1% | 97.0% | 96.8% |
| Example IV | 96.7% | 96.7% | 96.4% |
| Embodiment five | 96.5% | 96.4% | 96.3% |
| Meansigma methodss | 96.66% | 96.6% | 96.54% |
The survival rate change log of the fat stem cell of 3 embodiment of table, one to five frozen rear recovery (deposit through -20 DEG C by frozen stock solution
Put the survival rate after processing for month and deduct the survival rate after frozen stock solution is processed without -20 DEG C of storages for month)
| Group | Recover within frozen 1 month | Recover within frozen 3 months | Recover within frozen 6 months |
| Embodiment one | 0 | 0 | 0% |
| Embodiment two | - 0.2% | - 0.1% | 0 |
| Embodiment three | 0 | 0 | 0% |
| Example IV | 0 | - 0.1% | - 0.2% |
| Embodiment five | 0% | - 0.1% | - 0.2% |
| Meansigma methodss | - 0.04% | - 0.06% | - 0.08% |
As known from Table 3, after -20 DEG C of storages are processed for one month, the frozen effect of frozen stock solution hardly changes, it is seen that this
Invention is with good preservation effect.
Comparative example one
As a comparison case one is carried out from the group 1 in the embodiment 11 of the Chinese patent of Publication No. CN102550542A.
Comparative example two
From removal ferric citrate unlike embodiment three, other are identical with embodiment three.
Comparative example three
From removal fibronectin unlike embodiment three, other are identical with embodiment three.
Comparative example four
From ferric citrate and fibronectin are simultaneously removed unlike embodiment three, other are identical with embodiment three.
Comparative example five
From unlike embodiment three, according to weight portion, butanoic acid is 55 parts, 78 parts of sodium butyrate, ferric citrate is 0.1 part, fine adhesion
Albumen is 0.05 part, and other are identical with embodiment three.
Comparative example six
From unlike embodiment three, according to weight portion, butanoic acid is 80 parts, 113 parts of sodium butyrate, ferric citrate is 0.25 part, fine viscous
Even albumen is 1.1 parts, and other are identical with embodiment three.
Embodiment three and comparative example one to six are measured and are recorded with reference to frozen test.
The survival rate log of the fat stem cell of recovery after 4 embodiment three of table and comparative example one to six are frozen
| Group | Recover within frozen 1 month | Recover within frozen 3 months | Recover within frozen 6 months |
| Embodiment three | 97.1% | 97.0% | 96.8% |
| Comparative example one | 94.8 | 93.6 | 92.3 |
| Comparative example two | 96.1 | 95.8 | 95.3 |
| Comparative example three | 95.6 | 95.3 | 94.3 |
| Comparative example four | 95.1 | 94.8 | 93.6 |
| Comparative example five | 96.7 | 96.6 | 96.4 |
| Comparative example six | 96.5 | 96.2 | 96.0 |
Can draw to draw a conclusion from table 4：
Comparative example three and comparative example one understand, the survival rate of 1 month, 3 months and 6 months of embodiment three is above contrast
Example one.It can be seen that the frozen effect of the frozen stock solution of the present invention is better than comparative example one.
Comparative example three and comparative example two to four understand, the survival rate of 1 month, 3 months and 6 months of embodiment three is equal
Higher than comparative example two and three, and the survival rate of 1 month of comparative example four, 3 months and 6 months is below comparative example two and three.By
This is visible, and ferric citrate and fibronectin can each strengthen frozen effect of the invention.And when ferric citrate and fibre are viscous
When even albumen is used simultaneously, can occur to interact between ferric citrate and fibronectin, strengthen the frozen effect of the present invention
Really.
Comparative example three and comparative example five to six understand, the survival rate of 1 month, 3 months and 6 months of embodiment three is equal
Higher than comparative example five to six.As can be seen here, when the consumption of butanoic acid, sodium butyrate, ferric citrate and fibronectin is higher than or low
During corresponding amounts of components in embodiment three, the frozen effect of frozen stock solution can decline.
Embodiment three and comparative example one to six are tested and recorded with reference to food preservation test.
Survival rate log (the frozen stock solution of the fat stem cell of recovery after 5 embodiment three of table and comparative example one to six are frozen
Process within one month through -20 DEG C of storages)
| Group | Recover within frozen 1 month | Recover within frozen 3 months | Recover within frozen 6 months |
| Embodiment three | 97.1% | 97.0% | 96.8% |
| Comparative example one | 94.3% | 92.4% | 90.6% |
| Comparative example two | 95.6% | 95% | 94.2% |
| Comparative example three | 94.7% | 94.1% | 92.5% |
| Comparative example four | 94.1% | 93.5% | 91.7% |
| Comparative example five | 96.6% | 96.5% | 96.3% |
| Comparative example six | 96.5% | 96.1% | 96.0% |
The survival rate change log (frozen stock solution of the fat stem cell of recovery after 6 embodiment three of table and comparative example one to six are frozen
Survival rate after -20 DEG C of storages are processed for one month deducts the survival after frozen stock solution is processed without -20 DEG C of storages for month
Rate)
| Group | Recover within frozen 1 month | Recover within frozen 3 months | Recover within frozen 6 months |
| Embodiment three | 0 | 0 | 0 |
| Comparative example one | - 0.5% | - 1.2% | - 1.7% |
| Comparative example two | - 0.5% | - 0.8% | - 1.1% |
| Comparative example three | - 0.9% | - 1.2% | - 1.8% |
| Comparative example four | - 1% | - 1.3% | - 1.9% |
| Comparative example five | - 0.1% | - 0.1% | - 0.1% |
| Comparative example six | 0 | - 0.1% | 0 |
Can draw to draw a conclusion from table 6：
Comparative example three and comparative example one understand, in frozen stock solution after -20 DEG C of storages are processed for one month, the 1 of embodiment three
There is no significant change in the survival rate of individual month, 3 months and 6 months, and the survival of 1 month of comparative example one, 3 months and 6 months
Rate changing value is far above embodiment three.It can be seen that the preservation effect of the frozen stock solution of the present invention is better than comparative example one.
Comparative example three and comparative example two to four understand, in frozen stock solution after -20 DEG C of storages are processed for one month, implement
The survival rate changing value of 1 month, 3 months and 6 months of example three is below comparative example two and three, and 1 month of comparative example four, 3
The survival rate changing value of individual month and 6 months is higher than comparative example two.The survival rate of 1 month, 3 months and 6 months of comparative example three becomes
Change value is close with the survival rate changing value of 1 month, 3 months and 6 months of comparative example four.As can be seen here, ferric citrate is to this
Bright preservation effect affects less, and fibronectin can strengthen the preservation effect of the present invention.But, when ferric citrate and fibre
When Fibronectin is used simultaneously, can occur to interact between ferric citrate and fibronectin, strengthen the preservation of the present invention
Effect.
Comparative example three and comparative example five to six understand, the survival rate of 1 month, 3 months and 6 months of embodiment three becomes
The change value and comparative example survival rate changing value of five to six 1 months, 3 months and 6 months differs less.As can be seen here, when butanoic acid, fourth
When the consumption of sour sodium, ferric citrate and fibronectin is higher or lower than corresponding amounts of components in embodiment three, frozen stock solution
Preservation effect significant change will not occur.
Claims (10)
1. a kind of culture medium, is characterized in that：Including the component of following parts by weight：
Inorganic salt composite 7200-7300 parts
Amino acid complex 980-1000 parts
Vitamin complex 40-45 parts
Glucose 1000-1100 parts
Sodium Pyruvate 100-110 parts
Butanoic acid 60-75 parts
Sodium butyrate 80-110 parts
Ferric citrate 0.15-0.22 parts
1000000 parts of water.
2. culture medium according to claim 1, is characterized in that：According to weight portion, also including phenol red 9-10 parts.
3. culture medium according to claim 2, is characterized in that：According to weight portion, also including fibronectin 0.1-1 part.
4. culture medium according to claim 3, is characterized in that：According to weight portion, also including hormone complex 5-25 parts, institute
Stating hormone complex includes insulin, glucagon and progesterone, according to weight ratio, the insulin：Glucagon：Progesterone
=3-6：3-5：1.
5. culture medium according to claim 4, is characterized in that：It is according to weight portion, also including 50000 parts of antibiotic, described
Antibiotic is penicillin or streptomycin.
6. culture medium according to claim 5, is characterized in that：The inorganic salt composite includes anhydrous calcium chloride, sulphuric acid
Nickel, potassium chloride, anhydrous magnesium sulfate, Sodium Chloride, zinc sulfate, according to weight ratio, the anhydrous calcium chloride：Nickel sulfate：Potassium chloride：Nothing
Water magnesium sulfate：Sodium Chloride：Zinc sulfate=53:4:80:20:1280:9.
7. culture medium according to claim 5, is characterized in that：The vitamin complex includes calcium pantothenate, tartaric acid gallbladder
Alkali, Folic Acid, inositol, nicotiamide, riboflavin, thiamine hydrochloride, Pyridoxine Hydrochloride, vitamin A, vitamin C and Vitamin E, according to
Weight ratio, the calcium pantothenate：(2-Hydroxyethyl)trimethylammonium bitartrate：Folic Acid：Inositol：Nicotiamide：Riboflavin：Thiamine hydrochloride：Pyridoxine Hydrochloride：Dimension life
Plain A：Vitamin C and Vitamin E=2:3.6:2:3.6:2:0.2:2:2:1.1:2:1.
8. culture medium according to claim 5, is characterized in that：The amino acid complex includes L-Arginine Hydrochloride, L-
Hydrochloric acid cystine, glycine, L- histidine monohydrochlorides, L-Isoleucine, L-Leu, L-Lysine Hydrochloride, METHIONINE, L-
Phenylalanine, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, according to weight ratio, L- hydrochloric acid essence ammonia
Acid：L- hydrochloric acid cystine：Glycine：L- histidine monohydrochlorides：L-Isoleucine：L-Leu：L-Lysine Hydrochloride：L- first sulfur ammonia
Acid：L-phenylalanine：L-Serine：L-Threonine：L-Tryptophan：L-Tyrosine：L-Valine=84:63:30:42:105:
105:146:30:66:42:95:16:72:94。
9. a kind of frozen stock solution for fat stem cell, is characterized in that：Including following component：Dimethyl sulfoxide, blood serum substituting group
Divide KSR and the culture medium as described in claim 5-8 any one, the volume fraction of the dimethyl sulfoxide is 8-20%, described
The volume fraction of blood serum substituting component KSR is 70-85%, and the volume fraction of the culture medium is 5-15%.
10. a kind of preparation method of the frozen stock solution for being used for fat stem cell as claimed in claim 9, is characterized in that：Including such as
Lower preparation process：
Step1：According to weight portion, inorganic salt composite 7200-7300 parts, amino acid complex 980-1000 parts, dimension life are weighed
Plain complex 40-45 parts, glucose 1000-1100 parts, Sodium Pyruvate 100-110 parts, butanoic acid 60-75 parts, sodium butyrate 80-110
Part, ferric citrate 0.15-0.22 parts, 1000000 parts of water, phenol red 9-10 parts, fibronectin 0.1-1 part, hormone complex 5-
25 parts, 50000 parts of antibiotic, are mixed and stirred for uniformly, obtaining culture medium；
Step2：According to volume fraction, the dimethyl sulfoxide of 8-20%, blood serum substituting component KSR of 70-85% and 5-15% are measured
Culture medium, is mixed and stirred for uniform.
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| CN109769799A (en) * | 2019-03-20 | 2019-05-21 | 江苏瑞思坦生物科技有限公司 | A kind of human fat tissue saves liquid and preparation method thereof |
| CN114698630A (en) * | 2022-06-06 | 2022-07-05 | 天津百恩生物科技有限公司 | Placenta tissue preserving fluid and preparation method and application thereof |
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