CN109329271A - A kind of clinical mescenchymal stem cell freezen protective liquid and preparation method thereof - Google Patents
A kind of clinical mescenchymal stem cell freezen protective liquid and preparation method thereof Download PDFInfo
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- CN109329271A CN109329271A CN201811375580.7A CN201811375580A CN109329271A CN 109329271 A CN109329271 A CN 109329271A CN 201811375580 A CN201811375580 A CN 201811375580A CN 109329271 A CN109329271 A CN 109329271A
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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 kind of clinical mescenchymal stem cell freezen protective liquid and preparation method thereof.The preservation liquid includes following components in percentage by weight: autologous plasma lysate mixed liquor 1~5%, human serum albumin 1~2%, VC injection 0~1%, glucose injection 5~10%, class electrolyte solution 20~30%, glucosamine salt injection 20~30%, Amino Acid Compound Injection 20~30%, dimethyl sulfoxide 2~5% and trehalose 2.5~5%.The freezen protective liquid that the present invention is prepared, it can be used in carrying out profound hypothermia long term storage to cell, long distance transportation can be achieved, to expand the dispatching range of cell, realize the teletherapy of stem cell, and cell after recovery can direct feedback, reduce many and diverse operation bring security risk.
Description
Technical field
The invention belongs to cell preservation technique fields, and in particular to it is a kind of it is clinical with mescenchymal stem cell freezen protective liquid and
Preparation method.
Background technique
Cell therapy is related to the processes such as the preparation, amplification, feedback of cell, in general, the enforcement place of these processes and time
Not consistent, so that cell be needed to transport to destination, the Quality Control dynamics in cellular transport processes is weaker.Cell normal temperature environment
In, intracellular various enzymes and the reaction of other large biological molecules, metabolism is vigorous, and aerobic height, energy consumption is high.These physiology courses can generate
A large amount of oxygen radicals and lipid peroxide can cause environment pH and osmotic pressure to change, cause cell molten if removing not in time
It is swollen and dead, excessive cell culture medium and the required oxygen of a large amount of metabolism are provided simultaneously due to inconvenient, and the remote way of cell is transported logical
Frequently with freezing and low temperature method.
The preserving type of cell mainly have cryogenic freezing save and it is stored refrigerated, freezen protective can reduce to greatest extent
The metabolic activities of cell save cell activity.
Chilled save of usual cell needs special cryoprotector, reduces damage of the cell in frozen storage process, existing
There is cryoprotector mainly to have a dimethyl sulfoxide, low concentration freezes that effect is poor, and high concentration has certain toxic action to cell,
Especially under normal temperature state, dimethyl sulfoxide has biggish toxic action to cell.Therefore through dimethyl sulfoxide low temperature cold
Dimethyl sulfoxide should be removed immediately after freezing the cell recovery saved, to reduce the damage under room temperature to cell, guarantee that cell is living
Rate.
But cell, after resuscitation, activity and state are in relatively fragile state, easily cause the damage of cell,
To reduce effective cell amount, the biopotency of cell, which is also likely to occur, to be greatly lowered, and it is dry to thus greatly reduce mesenchyma
The clinical effectiveness of cell.
It is badly in need of a kind of stem cell freezen protective liquid, under the premise of meeting long-distance transportation, it is multiple is able to maintain cell freezing
High activity after Soviet Union, and cell can be applied directly after recovery.
Summary of the invention
For above-mentioned deficiency in the prior art, the present invention provide it is a kind of it is clinical with mescenchymal stem cell freezen protective liquid and
Preparation method can be effectively solved existing freezen protective liquid after saving to cell, and cell can be made to exist and lived
Property is low, the problem of not can be used directly after recovery.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:
A kind of clinical mescenchymal stem cell freezen protective liquid, including following components in percentage by weight:
Autologous plasma lysate mixed liquor 1~5%, human serum albumin 1~2%, VC injection 0~1%, glucose injection
Liquid 5~10%, class electrolyte solution 20~30%, glucosamine salt injection 20~30%, Amino Acid Compound Injection 20~
30%, dimethyl sulfoxide 2~5% and trehalose 2.5~5%.
Wherein, Amino Acid Compound Injection trade name 18AA is purchased from Chengdu Brilliant Pharmaceutical Co., Ltd..
Further, including following components in percentage by weight:
Autologous plasma lysate mixed liquor 5%, human serum albumin 1%, VC injection 0.5%, glucose injection 5%,
Class electrolyte solution 24%, glucosamine salt injection 28%, Amino Acid Compound Injection 27%, dimethyl sulfoxide 5% and sea
Algae sugar 4.5%.
Further, autologous plasma lysate mixed liquor the preparation method comprises the following steps:
(1) blood plasma is acquired, is then centrifuged 8~10min in 4000~5000r/min, draws upper plasma, and make tunica albuginea layer
It mixes with blood plasma, is saved backup in -80~70 DEG C；
(2) in 35~37 DEG C of recovery step (1) products therefroms, it then is centrifuged 10~15min in 3800~4000r/min,
Supernatant is collected, autologous plasma lysate is made；
(3) aqueous trehalose that mass fraction is 10~15% is mixed with autologous plasma lysate solution, the seaweed
The volume ratio of sugar juice and autologous plasma lysate solution is 1:5~10, autologous plasma lysate mixed liquor is made, and in -80
~70 DEG C save backup.
Further, glucose injection is the D-40 glucose injection that mass fraction is 10%.
Further, class electrolyte solution is Multiple electrolytes injection, and trade name Bomaili A is purchased from Shanghai hundred
Special medical supplies Co., Ltd.
Further, glucosamine salt injection is Dextrose and Sodium Chloride Inj..
Further, the concentration of trehalose is 0.35mol/L.
The above-mentioned clinical preparation method for using mescenchymal stem cell freezen protective liquid, which comprises the following steps:
(1) formula is pressed by glucose injection, class electrolyte solution, glucosamine salt injection and Amino Acid Compound Injection
It is uniformly mixed；
(2) VC injection, human serum albumin are sequentially added into mixed liquor obtained by step (1) and are recovered in 37~40 DEG C
Autologous plasma lysate mixed liquor, add dimethyl sulfoxide and trehalose, be uniformly mixed.
The invention has the benefit that
1, contain platelet derived growth factor, transforming growth factor, epidermal growth factor, pancreas islet in autologous plasma lysate
Plain like growth factor, vascular endothelial growth factor etc. can provide preferable living environment for cell.
2, human serum albumin can be used as stabilizer and protective agent, and be the important component of cell culture medium, have to cell
There is stronger protective effect, the growth of cell can be promoted, extends the time of cell survival；Increase blood volume and maintains infiltration
Pressure, the main dynamic equilibrium for adjusting moisture between tissue and blood vessel, and can be used as nitrogen source is tissue with nutrient.
3, vitamin C, amino acid are added in the present invention, can effectively slow down permeability and damage stable eucaryotic cell structure, resist
The effects of oxidation, can help cell to maintain the activity of various peroxidase, reduce cellular damage.
4, the freezen protective liquid that is prepared of the present invention, can be used in carrying out profound hypothermia long term storage to cell, it can be achieved that
Long distance transportation realizes the teletherapy of stem cell, and cell can after recovery to expand the dispatching range of cell
Direct feedback reduces many and diverse operation bring security risk.
5, the present invention uses autologous plasma lysate, and non-animal derived serum composition can preferably maintain cell state, pole
Big reduces external source infection risk, and trehalose ingredient is added, and has helped to improve the work of the blood platelet of cryo-conservation state
Property, more mild living environment is provided for the preservation of stem cell.
6, the present invention saves liquid under the premise of guaranteeing cell state, when greatly extending the preservation after prepared by cell
Effect.
Detailed description of the invention
Adherent growth ability test map when Fig. 1 is cell cryopreservation one month；Wherein, Fig. 1 a is 1 cell of embodiment jelly
2nd day adherent growth picture after recovery is inoculated with when depositing one month；Recovery inoculation when Fig. 1 b is 1 cell cryopreservation of embodiment one month
4th day adherent growth picture afterwards；2nd day adherent growth figure after recovery is inoculated with when Fig. 1 c is comparative example cell cryopreservation one month
Piece；4th day adherent growth picture after recovery is inoculated with when Fig. 1 d is comparative example cell cryopreservation one month；
Adherent growth ability test map when Fig. 2 is cell cryopreservation three months；Wherein, Fig. 2 a is 1 cell of embodiment jelly
2nd day adherent growth picture after recovery is inoculated with when depositing three months；Recovery inoculation when Fig. 2 b is 1 cell cryopreservation of embodiment three months
4th day adherent growth picture afterwards；2nd day adherent growth figure after recovery is inoculated with when Fig. 2 c is comparative example cell cryopreservation three months
Piece；4th day adherent growth picture after recovery is inoculated with when Fig. 2 d is comparative example cell cryopreservation three months；
Adherent growth ability test map when Fig. 3 is cell cryopreservation six months；Wherein, Fig. 3 a is 1 cell of embodiment jelly
2nd day adherent growth picture after recovery is inoculated with when depositing six months；Recovery inoculation when Fig. 3 b is 1 cell cryopreservation of embodiment six months
4th day adherent growth picture afterwards；2nd day adherent growth figure after recovery is inoculated with when Fig. 3 c is comparative example cell cryopreservation six months
Piece；4th day adherent growth picture after recovery is inoculated with when Fig. 3 d is comparative example cell cryopreservation six months；
Adherent growth ability test map when Fig. 4 is cell cryopreservation 12 months；Wherein, Fig. 4 a is 1 cell of embodiment
2nd day adherent growth picture after recovery is inoculated with when freezing 12 months；It is multiple when Fig. 4 b is 1 cell cryopreservation of embodiment 12 months
4th day adherent growth picture after Soviet Union's inoculation；2nd day patch after recovery is inoculated with when Fig. 4 c is comparative example cell cryopreservation 12 months
Wall grows picture；4th day adherent growth picture after recovery is inoculated with when Fig. 4 d is comparative example cell cryopreservation 12 months.
Specific embodiment
A specific embodiment of the invention is described below, in order to facilitate understanding by those skilled in the art this hair
It is bright, it should be apparent that the present invention is not limited to the ranges of specific embodiment, for those skilled in the art,
As long as various change is in the spirit and scope of the present invention that the attached claims limit and determine, these variations are aobvious and easy
See, all are using the innovation and creation of present inventive concept in the column of protection.
Embodiment 1
A kind of clinical mescenchymal stem cell freezen protective liquid, including following components in percentage by weight:
Autologous plasma lysate mixed liquor 5%, human serum albumin 1%, VC injection 0.5%, 10% low molecule dextrose
Acid anhydride glucose injection 5%, Multiple electrolytes injection 24%, Dextrose and Sodium Chloride Inj. 28%, amino acid injection
Liquid 27%, dimethyl sulfoxide 5% and trehalose 4.5%.
Wherein, autologous plasma lysate mixed liquor the preparation method comprises the following steps:
(1) acquisition donor peripheral blood 16mL completes blood plasma preparation in EDTA anticoagulant blood-collecting pipe in acquisition 2h；
(2) it is centrifuged 8min in 4000r/min, draws upper plasma and gently blows and beats tunica albuginea layer, so that tunica albuginea layer and blood plasma are mixed
It is even, blood plasma and tunica albuginea layer are transferred in cryopreservation tube, are temporarily stored into -80 ° of ultra low temperature freezers overnight；
(3) blood plasma that recovery step (2) is prepared under conditions of 37 DEG C is centrifuged 15min in 3800r/min, will be upper
It is transferred to the preparation for being used for clinical mesenchyme stem cell preserving fluid in new centrifuge tube clearly, discards centrifugation, derives from body blood
Starch lysate；
(4) by mass fraction be 10% aqueous trehalose and autologous plasma lysate solution according to volume ratio be 1:10's
Ratio is uniformly mixed, and autologous plasma lysate mixed liquor is made, then dispenses and is stored in spare in -80 DEG C of environment.
The above-mentioned clinical preparation method for using mescenchymal stem cell freezen protective liquid, comprising the following steps:
(1) formula is pressed by 10% D-40 glucose injection, Multiple electrolytes injection, glucose chlorination
Sodium injection and Amino Acid Compound Injection are uniformly mixed；
(2) VC injection, human serum albumin and oneself to recover in 37 DEG C are sequentially added into mixed liquor obtained by step (1)
Body blood plasma lysate mixed liquor, adds dimethyl sulfoxide and trehalose, is uniformly mixed, is then no less than in 2-8 DEG C of pre-cooling
30min can be used.
Embodiment 2
A kind of clinical mescenchymal stem cell freezen protective liquid, including following components in percentage by weight:
Autologous plasma lysate mixed liquor 1%, human serum albumin 2%, 10% D-40 glucose injection
10%, Multiple electrolytes injection 30%, Dextrose and Sodium Chloride Inj. 20%, Amino Acid Compound Injection 30%, dimethyl
Sulfoxide 2% and trehalose 5%.
Wherein, autologous plasma lysate mixed liquor the preparation method comprises the following steps:
(1) acquisition donor peripheral blood 16mL completes blood plasma preparation in EDTA anticoagulant blood-collecting pipe in acquisition 2h；
(2) it is centrifuged 8min in 4000r/min, draws upper plasma and gently blows and beats tunica albuginea layer, so that tunica albuginea layer and blood plasma are mixed
It is even, blood plasma and tunica albuginea layer are transferred in cryopreservation tube, are temporarily stored into -80 ° of ultra low temperature freezers overnight；
(3) blood plasma that recovery step (2) is prepared under conditions of 37 DEG C is centrifuged 15min in 3800r/min, will be upper
It is transferred to the preparation for being used for clinical mesenchyme stem cell preserving fluid in new centrifuge tube clearly, discards centrifugation, derives from body blood
Starch lysate；
(4) by mass fraction be 10% aqueous trehalose and autologous plasma lysate solution according to volume ratio be 1:10's
Ratio is uniformly mixed, and autologous plasma lysate mixed liquor is made, then dispenses and is stored in spare in -80 DEG C of environment.
The above-mentioned clinical preparation method for using mescenchymal stem cell freezen protective liquid, comprising the following steps:
(1) formula is pressed by 10% D-40 glucose injection, Multiple electrolytes injection, glucose chlorination
Sodium injection and Amino Acid Compound Injection are uniformly mixed；
(2) autologous plasma for sequentially adding human serum albumin into mixed liquor obtained by step (1) and recovering in 37 DEG C is split
Object mixed liquor is solved, dimethyl sulfoxide and trehalose are added, is uniformly mixed, being then no less than 30min in 2-8 DEG C of pre-cooling can make
With.
Embodiment 3
A kind of clinical mescenchymal stem cell freezen protective liquid, including following components in percentage by weight:
Autologous plasma lysate mixed liquor 5%, VC injection 1%, glucose injection 5%, is answered at human serum albumin 2%
Square electrolyte injection 23.5%, glucosamine salt injection 25%, Amino Acid Compound Injection 30%, dimethyl sulfoxide 4% with
And trehalose 4.5%.
Wherein, autologous plasma lysate mixed liquor the preparation method comprises the following steps:
(1) acquisition donor peripheral blood 16mL completes blood plasma preparation in EDTA anticoagulant blood-collecting pipe in acquisition 2h；
(2) it is centrifuged 8min in 4000r/min, draws upper plasma and gently blows and beats tunica albuginea layer, so that tunica albuginea layer and blood plasma are mixed
It is even, blood plasma and tunica albuginea layer are transferred in cryopreservation tube, are temporarily stored into -80 ° of ultra low temperature freezers overnight；
(3) blood plasma that recovery step (2) is prepared under conditions of 37 DEG C is centrifuged 15min in 3800r/min, will be upper
It is transferred to the preparation for being used for clinical mesenchyme stem cell preserving fluid in new centrifuge tube clearly, discards centrifugation, derives from body blood
Starch lysate；
(4) by mass fraction be 15% aqueous trehalose and autologous plasma lysate solution according to volume ratio be 1:5's
Ratio is uniformly mixed, and autologous plasma lysate mixed liquor is made, then dispenses and is stored in spare in -80 DEG C of environment.
The above-mentioned clinical preparation method for using mescenchymal stem cell freezen protective liquid, comprising the following steps:
(1) formula is pressed by 10% D-40 glucose injection, Multiple electrolytes injection, glucose chlorination
Sodium injection and Amino Acid Compound Injection are uniformly mixed；
(2) VC injection, human serum albumin and oneself to recover in 37 DEG C are sequentially added into mixed liquor obtained by step (1)
Body blood plasma lysate mixed liquor, adds dimethyl sulfoxide and trehalose, is uniformly mixed, is then no less than in 2-8 DEG C of pre-cooling
30min can be used.
Comparative example
Cell culture medium and dimethyl sulfoxide are mixed according to the ratio that volume ratio is 1:9, are then 0.22 μm with aperture
Filter filtering after, 10% cells frozen storing liquid is prepared, can make after at least 30min is pre-chilled in 2-8 DEG C of refrigerator
With.
Experimental example
1, the clinical preparation for using placenta source mescenchymal stem cell
Select P3-P5 for placenta source mescenchymal stem cell, cell fusion degree reaches 85-90% degrees of fusion, and digestion is prepared into
Single cell suspension.
2, adjustment cell density is 0.5~2 × 107Then a/mL is prepared using embodiment 1 and comparative example respectively
Freezen protective liquid freezen protective is carried out to cell suspension, and when freezing 1 month, 3 months, 6 months and 12 months, in 37
~40 DEG C of progress water-bath recoveries, are counted with Invitrogen company full-automatic cell calculating instrument Countess, count 3 respectively
Secondary to be averaged, the average motility rate value of cell during detection freezes, the result is shown in tables 1；Then again to freezing 1 month, 3 months, 6
The cell recovered at a month and 12 months is cultivated, and second day and third day carry out observation of taking pictures after recovery inoculation,
The result is shown in Figure 1~4, the cell that this preservation liquid is frozen it can be seen from cell picture exist compared to the cell that comparative example freezes
Inoculated and cultured is able to maintain higher motility rate after cell recovery, and retains higher proliferation rate.
Cell survival rate during table 1 freezes
| Freeze time (moon) | 1 motility rate of embodiment | Comparative example motility rate |
| 1 | 95.32% | 87.02% |
| 3 | 94.88% | 89.21% |
| 6 | 95.37% | 84.55% |
| 12 | 94.09% | 80.67% |
3, it after freezing, recovers in 37~40 DEG C to cell, the cell after recovery is temporarily stored into 2-8 degrees Celsius of environment
In, respectively at 0h, 2h, 4h, 6h monitors Cell viability, is carried out with Invitrogen company full-automatic cell calculating instrument Countess
It counts, counts 3 times be averaged respectively, the result is shown in tables 2.
The survival rate of cell after table 2 is recovered
From the data in table 2, it can be seen that carrying out freezen protective to cell using the freezen protective liquid that the present invention is prepared, cell exists
2h is still able to maintain 90% or more Cell viability after recovery, and 80% or more Cell viability is able to maintain that in 4h, is thus illustrated, warp
It crosses the cell that the freezing liquid that is prepared of the present invention saves, in the very short time after recovery, can have greater activity, so that
Cell can be applied directly after recovery.
Claims (8)
1. a kind of clinical mescenchymal stem cell freezen protective liquid, which is characterized in that including following components in percentage by weight:
Autologous plasma lysate mixed liquor 1~5%, human serum albumin 1~2%, VC injection 0~1%, glucose injection 5
~10%, class electrolyte solution 20~30%, glucosamine salt injection 20~30%, Amino Acid Compound Injection 20~30%,
Dimethyl sulfoxide 2~5% and trehalose 2.5~5%.
2. clinical mescenchymal stem cell freezen protective liquid according to claim 1, which is characterized in that including following weight
The component of percentage:
Autologous plasma lysate mixed liquor 5%, human serum albumin 1%, VC injection 0.5%, glucose injection 5%, class electricity
Electrolyte solution 24%, glucosamine salt injection 28%, Amino Acid Compound Injection 27%, dimethyl sulfoxide 5% and trehalose
4.5%.
3. clinical mescenchymal stem cell freezen protective liquid according to claim 1 or 2, which is characterized in that described self
Blood plasma lysate mixed liquor the preparation method comprises the following steps:
(1) blood plasma is acquired, is then centrifuged 8~10min in 4000~5000r/min, draws upper plasma, and make tunica albuginea layer and blood
Slurry mixes, and saves backup in -80~70 DEG C；
(2) in 35~37 DEG C of recovery step (1) products therefroms, it then is centrifuged 10~15min in 3800~4000r/min, is collected
Autologous plasma lysate is made in supernatant；
(3) aqueous trehalose that mass fraction is 10~15% is mixed with autologous plasma lysate solution, the trehalose is molten
The volume ratio of liquid and autologous plasma lysate solution is 1:5~10, autologous plasma lysate mixed liquor is made, and in -80~70
It DEG C saves backup.
4. clinical mescenchymal stem cell freezen protective liquid according to claim 1 or 2, which is characterized in that the grape
Sugared injection is the D-40 glucose injection that mass fraction is 10%.
5. clinical mescenchymal stem cell freezen protective liquid according to claim 1 or 2, which is characterized in that the class electricity
Electrolyte solution is Multiple electrolytes injection.
6. clinical mescenchymal stem cell freezen protective liquid according to claim 1 or 2, which is characterized in that the grape
Sugared saline injection is Dextrose and Sodium Chloride Inj..
7. clinical mescenchymal stem cell freezen protective liquid according to claim 1 or 2, which is characterized in that the seaweed
The concentration of sugar is 0.35mol/L.
8. the described in any item clinical preparation methods for using mescenchymal stem cell freezen protective liquid of claim 1~7, feature exist
In, comprising the following steps:
(1) glucose injection, class electrolyte solution, glucosamine salt injection and Amino Acid Compound Injection are mixed by formula
Uniformly；
(2) VC injection, human serum albumin and oneself to recover in 37~40 DEG C are sequentially added into mixed liquor obtained by step (1)
Body blood plasma lysate mixed liquor, adds dimethyl sulfoxide and trehalose, is uniformly mixed.
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