CN109329270A - A kind of venous re-transfusion mesenchyme stem cell preserving fluid and preparation method thereof - Google Patents
A kind of venous re-transfusion mesenchyme stem cell preserving fluid and preparation method thereof Download PDFInfo
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- CN109329270A CN109329270A CN201811375272.4A CN201811375272A CN109329270A CN 109329270 A CN109329270 A CN 109329270A CN 201811375272 A CN201811375272 A CN 201811375272A CN 109329270 A CN109329270 A CN 109329270A
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- injection
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Classifications
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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
-
- 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 venous re-transfusion mesenchyme stem cell preserving fluids and preparation method thereof.The preservation liquid includes following components in percentage by weight: autologous plasma lysate mixed liquor 1~5%, human serum albumin 1~5%, VC injection 1~5%, glucose injection 2~3%, Multiple electrolytes injection 23~30%, glucosamine salt injection 23~30% and Amino Acid Compound Injection 23~30%.The preservation liquid that the present invention is prepared is, it can be achieved that the temporary rear direct feedback of refrigeration, reduces cellular damage of the cell of freezing state preservation in the preparation process after recovery and in cell reprocessing process, improve cell utilization rate.
Description
Technical field
The invention belongs to cell preservation technique fields, and in particular to a kind of venous re-transfusion which mescenchymal stem cell preservation
Liquid and preparation method thereof.
Background technique
Stem cell be based on that it is gone back to the nest, migrates, the characteristics such as Proliferation, Differentiation can be applied to treat and repair a variety of damaged cells or
Tissue.Stem cell can secrete cytokine profiles, such as growth factor, chemotactic factor (CF), cell by paracrine action simultaneously
The factor can be combined with the cell receptor of damage, to reach the repair of damaging cells.
Cell therapy is related to the processes such as the preparation, amplification, feedback of cell, and the enforcement place of these usual processes and time are simultaneously
It is inconsistent, and the Quality Control dynamics of cell during transportation is weaker.In cell normal temperature environment, intracellular various enzymes and other biologies
Macromolecular reaction is metabolized vigorous, oxygen demand height.These physiology courses can generate a large amount of oxygen radicals and lipid peroxide, if
It removes not in time, then environment pH and osmotic pressure can be caused to change, cause cell swelling and death, the remote way transport of cell is usually adopted
With freezing and low temperature method.
Freezing state can reduce the metabolic activity of cell to greatest extent, avoid cell metabolism in normal temperature environment and cause
Cellular damage and death, but low-temperature transport needs special preservation medium, keeps cell as long as possible in low temperature environment
Maintenance cell high activity state.Since clinic cell preparation cannot contact animal source component, such as animal sources serum, such as
What guarantees that the safety of cell preparation, stability become the key of cell cryo-conservation, and the higher cost of low-temperature transport.Separately
Outside, cell need to usually be further processed cell before clinical application, increase cell manipulation process, increase cell
Security risk.
The cell that the cell-preservation liquid of existing non-refrigerated state saves is imitated in the Cell viability after the short time and biology
Power etc. is greatly lowered, to limit the treatment of mescenchymal stem cell；Therefore, it is badly in need of a kind of safe effective cell
Save liquid.
Summary of the invention
For above-mentioned deficiency in the prior art, the present invention provides a kind of venous re-transfusion which mescenchymal stem cell preservation
Liquid and preparation method thereof can be effectively solved existing mesenchyme stem cell preserving fluid when saving cell, cause cell living
The problem of rate and biological efficacy are greatly lowered.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:
A kind of venous re-transfusion mesenchyme stem cell preserving fluid, including following components in percentage by weight:
Autologous plasma lysate mixed liquor 1~5%, human serum albumin 1~5%, VC injection 1~5%, glucose injection
Liquid 2~3%, Multiple electrolytes injection 23~30%, glucosamine salt injection 23~30% and Amino Acid Compound Injection
23~30%.
Wherein, Multiple electrolytes injection trade name Bomaili A is purchased from Shanghai Baxter Healthcare Ltd.；It is multiple
Square amino acid 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%, VC injection 1%, glucose injection 3%, is answered at human serum albumin 1%
Square electrolyte injection 30%, glucosamine salt injection 30% and Amino Acid Compound Injection 30%.
Further, the 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, the human serum albumin is the human serum albumin solution that mass fraction is 25%.
Further, the glucose injection is the D-40 glucose injection that mass fraction is 10%
Liquid.
Further, the glucosamine salt injection is Dextrose and Sodium Chloride Inj..
The preparation method of above-mentioned venous re-transfusion mesenchyme stem cell preserving fluid, comprising the following steps:
(1) glucose injection, Multiple electrolytes injection, glucosamine salt injection and amino acid are infused by formula
Liquid is penetrated to be 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, 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 clinic mesenchyme stem cell preserving fluid in the present invention can realize the temporary rear direct feedback of refrigeration, reduce cold
Cellular damage of the cell that jelly state saves in the preparation process after recovery and in cell reprocessing process, improves cell benefit
With rate.
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.
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 venous re-transfusion mesenchyme stem cell preserving fluid, including following components in percentage by weight:
Autologous plasma lysate mixed liquor 5%, 25% human serum albumin 1%, VC injection 1%, 10% low molecule dextrorotation
Sugared acid anhydride glucose injection 3%, Multiple electrolytes injection 30%, Dextrose and Sodium Chloride Inj. 30% and amino acid
Injection 30%.
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 preparation method of above-mentioned venous re-transfusion mesenchyme stem cell preserving fluid, 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, 25% human serum albumin are sequentially added into mixed liquor obtained by step (1) and are recovered in 37 DEG C
Autologous plasma lysate mixed liquor, be uniformly mixed, adjustment pH value be 7.35, osmotic pressure be 290~310mmol/L.
Embodiment 2
A kind of venous re-transfusion mesenchyme stem cell preserving fluid, including following components in percentage by weight:
Autologous plasma lysate mixed liquor 5%, 25% human serum albumin 5%, VC injection 5%, 10% low molecule dextrorotation
Sugared acid anhydride glucose injection 3%, Multiple electrolytes injection 27%, Dextrose and Sodium Chloride Inj. 28% and amino acid
Injection 27%.
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 12% aqueous trehalose and autologous plasma lysate solution according to volume ratio be 1:8'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 preparation method of above-mentioned venous re-transfusion mesenchyme stem cell preserving fluid, 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, 25% human serum albumin are sequentially added into mixed liquor obtained by step (1) and are recovered in 37 DEG C
Autologous plasma lysate mixed liquor, be uniformly mixed, adjustment pH value be 7.35, osmotic pressure be 290~310mmol/L.
Embodiment 3
A kind of venous re-transfusion mesenchyme stem cell preserving fluid, including following components in percentage by weight:
Autologous plasma lysate mixed liquor 1%, 25% human serum albumin 5%, VC injection 1%, 10% low molecule dextrorotation
Sugared acid anhydride glucose injection 3%, Multiple electrolytes injection 30%, Dextrose and Sodium Chloride Inj. 30% and amino acid
Injection 30%.
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 preparation method of above-mentioned venous re-transfusion mesenchyme stem cell preserving fluid, 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, 25% human serum albumin are sequentially added into mixed liquor obtained by step (1) and are recovered in 37 DEG C
Autologous plasma lysate mixed liquor, be uniformly mixed, adjustment pH value be 7.35, osmotic pressure be 290~310mmol/L.
Comparative example
Using normal saline solution as cell-preservation liquid.
Experimental example
The preparation of clinical mescenchymal stem cell
1, the selection good P3-P5 of growth conditions is reached for the good mescenchymal stem cell of growth conditions to cell fusion degree
When to 85-90%, it is prepared into single cell suspension with 0.25% trypsin digestion, is counted.
2, the preservation liquid adjustment cell density being prepared respectively with embodiment 1 and comparative example is 2~5 × 106It is a, packing
Sealing, is then stored in 2~8 DEG C of environment, and monitors the survival rate of cell at any time, and the result is shown in tables 1.
1 cell survival rate of table
Wherein ,-: the agglomerating phenomenon of cell is not found；+: discovery is no more than 2 cell masses；++: 3-5 cell mass of discovery；++
+: more than 5 cell masses of discovery；
From the data in table 1, it can be seen that the preservation liquid being prepared using the method for the present invention saves cell, cell is 2~8
DEG C environment in when saving 32h, remain to maintain Cell viability 90% or more, and the agglomerating rate of cell is extremely low, ensure that cell exists
Safety and validity in treatment clinical course, and when the preservation liquid that comparative example is prepared saves cell, only
After 2h, the survival rate of cell has just been down to 77.08%, and after saving 20h, cell is completely dead, shows only to pass through as a result,
The preservation liquid that can guarantee Cell viability can be just prepared in the formula referred in the method for the present invention.
Claims (7)
1. a kind of venous re-transfusion mesenchyme stem cell preserving fluid, which is characterized in that including following components in percentage by weight:
Autologous plasma lysate mixed liquor 1~5%, human serum albumin 1~5%, VC injection 1~5%, glucose injection 2
~3%, Multiple electrolytes injection 23~30%, glucosamine salt injection 23~30% and Amino Acid Compound Injection 23~
30%.
2. venous re-transfusion mesenchyme stem cell preserving fluid 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 1%, glucose injection 3%, compound electricity
Solve matter injection 30%, glucosamine salt injection 30% and Amino Acid Compound Injection 30%.
3. venous re-transfusion mesenchyme stem cell preserving fluid 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. venous re-transfusion mesenchyme stem cell preserving fluid according to claim 1 or 2, which is characterized in that people's blood
Albumin is the human serum albumin solution that mass fraction is 25%.
5. venous re-transfusion mesenchyme stem cell preserving fluid 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%.
6. venous re-transfusion mesenchyme stem cell preserving fluid according to claim 1 or 2, which is characterized in that the grape
Sugared saline injection is Dextrose and Sodium Chloride Inj..
7. the preparation method of the described in any item venous re-transfusion mesenchyme stem cell preserving fluids of claim 1~6, feature exist
In, comprising the following steps:
(1) formula is pressed by glucose injection, Multiple electrolytes injection, glucosamine salt injection and Amino Acid Compound Injection
It is uniformly mixed；
(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 is uniformly mixed, and adjustment pH value is 7.35~7.45, and osmotic pressure is 290~310mmol/L.
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| WO2021190620A1 (en) * | 2020-03-27 | 2021-09-30 | 上海我武干细胞科技有限公司 | Cell preservation fluid and kit thereof |
| WO2023001147A1 (en) * | 2021-07-19 | 2023-01-26 | 无锡赛比曼生物科技有限公司 | Preservation solution for low-temperature long-term preservation of adipose-derived mesenchymal stem cell |
| WO2023016510A1 (en) * | 2021-08-13 | 2023-02-16 | 无锡赛比曼生物科技有限公司 | Preservation solution for low-temperature preservation of adipose-derived mesenchymal stem cells |
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