CN101919380B - Improved mesenchyme stem cell protection solution and application thereof - Google Patents
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Abstract
The invention relates to an improved mesenchyme stem cell protection solution as well as application and a preparation method thereof. The protection solution can effectively prolong the activity remaining time of mesenchyme stem cells, reduces preparation cost, and has the advantages of wide raw material source, simple preparation, safe and reliable direct clinical application; and after the mesenchyme stem cells are preserved for 48 hours in the protection solution, the cell activity is still above 90 percent, the cell morphology is normal, and the multiplication capacity and mesenchyme stem cell phenotype characteristics are not influenced.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of improved mesenchyme stem cell protection solution, purposes and preparation method thereof.
Technical background
Mescenchymal stem cell (mesenchymal stem cells, MSCs) be the stem cell that the class in mesoderm source has Multidirectional Differentiation ability, extensively be present in whole body Various Tissues, amplification can be cultivated in vitro, and myocardial cell, neurocyte and adipocyte etc. can be divided under given conditions.Along with Chinese scholars is learned the further investigation of characteristic and function to mesenchymal stem cell biological, can from Various Tissues, isolate mescenchymal stem cell at present.Mescenchymal stem cell has hematopoiesis support and regulates the effects such as immunity, at aspects such as hematopoietic reconstitution, tissue repair, immunotherapies, has wide potential applicability in clinical practice.Mescenchymal stem cell is not expressed MHC II quasi-molecule and costimulating factor B7-1, B7-2, CD40, CD40L etc., seldom expresses MHC I quasi-molecule, so a little less than immunogenicity.Due to effect and the characteristic of above mescenchymal stem cell, in recent years, mescenchymal stem cell is used in the clinical trial and treatment of clinical various diseases, and shows good application prospect.
At present domestic have mechanism that qualification carries out extensive separation, amplification, cultivator mescenchymal stem cell seldom; and the clinical application range of mescenchymal stem cell is very extensive; therefore be badly in need of a kind of mesenchyme stem cell protection solution of research; make mescenchymal stem cell activity in this kind of protection liquid hold time longer; can meet the requirement that is transported to most domestic area and surrounding countries; and can directly apply to human body, safety non-toxic.
Solution medium when at present the domestic mechanism that human mesenchymal stem cell can be provided in a large number normally adopts physiological saline or cell culture medium as clinical mescenchymal stem cell application.If after human mesenchymal stem cell separation, cultivating, increase, to patient, apply immediately, physiological saline is convenient and safe, but the mescenchymal stem cell speed that vigor declines in physiological saline is very fast, with physiological saline, preserve mescenchymal stem cell merely, after 2 hours, cell viability will reduce by 10% left and right, is therefore only applicable to the instant use of mescenchymal stem cell.Existing another Cell protective solutions is using cell culture medium as one of composition, in cell culture medium, contain the required carbohydrate of Growth of Cells metabolism, amino acid, inorganic salt, the materials such as VITAMIN, for maintaining, Premeabilisation of cells is pressed and vigor has good effect, but most domestic cell culture medium is all only for scientific research at present, not as the conventional reagent of clinical application, therefore security and the reliability of direct application cell substratum is uncertain when clinical application human mesenchymal stem cell, and cell culture medium is due to manufacturer, the difference of production method, quality, purity, price is not etc. yet, purity, quality all reasonable import substratum price is more expensive.
Prior art is to use the solution of TC199 serum free medium, human serum albumin and low molecular heparin calcium configuration as the preservation liquid of human mesenchymal stem cell.This technology will use respectively the TC199 that contains 1% human serum albumin and the physiological saline that contains 1% human serum albumin to preserve after human mesenchymal stem cell separation, cultivation, collection, cell concn is 5 * 10
5/ ml, 4 degree were preserved after 8 hours, and the cell viability of physiological saline group is starkly lower than TC199 group; Adjust the concentration of human serum albumin, make respectively in TC199, to contain 0.5%, 1%, 2%, 5% human serum albumin, 1%, 2%, 5% human serum albumin is all useful for keeping the vigor of mescenchymal stem cell; With the TC199 containing 1% human serum albumin, prepare respectively the solution depositary mescenchymal stem cell containing 0.01%EDTA, 75AXaIU/ml low molecular sodium heparin, 75AXaIU/ml low molecular heparin calcium, find that low molecular heparin calcium can effectively prevent mescenchymal stem cell gathering.Prior art is using TC199 substratum, human serum albumin, low molecular heparin calcium as preserving liquid depositary mescenchymal stem cell in a word, stem cell can keep more than 90% vigor 24 hours, cell viability extends gradually and successively decreases in time, and can prevent the adhesion of stem cell and container inner wall.
One of shortcoming of prior art is that the TC199 in mesenchyme stem cell preserving fluid is a kind of cell culture medium, cell culture medium is usually used to carry out cell or tissue culture, not state approval can clinical application in the reagent of human body, complicated, the substratum quality of different manufacturers differs, and import reagent is expensive, and in recent years along with the development in biological reagent field, TC199 has exited substratum market gradually, substituted, so its source is limited by other substratum.In a word, although prior art can keep cell viability 24 hours with TC199 as the effective ingredient of mesenchyme stem cell preserving fluid, from the viewpoint of legitimacy, the reagent cost of reagent source, clinical application, be all unfavorable for that mescenchymal stem cell clinical expansion is used.The solution medium using in this improved mesenchyme stem cell protection solution of the present invention is 5% glucose injection, and it is the reagent that state approval can clinical use, with its alternative TC199, cheap, source is abundant, clinical application is safe and reliable.
The another one weak point of prior art is that it keeps the time of mescenchymal stem cell vigor shorter, mescenchymal stem cell can keep 90% above vigor only 24 hours in the cell-preservation liquid of prior art, and along with time lengthening, cell viability declines, so just mean over 24 hours, cell viability will drop to below 90%, yet most cities and external a plurality of country carry out at home for stem-cell therapy and clinical trial, utilize the existing vehicles, the range of application of mescenchymal stem cell that the vigor of 24 hours has been held time significant limitation.And the present invention improves existing mesenchyme stem cell protection solution; make mescenchymal stem cell in this kind of protection liquid, keep 90% above vigor can maintain 48 hours; with regard to having extended the activity of mescenchymal stem cell, hold time like this; greatly expanded the scope of clinical stem cell widespread use; utilize the existing vehicles, can be transported to most domestic city and outskirts country.
Summary of the invention
The problem existing for prior art, the improved mesenchyme stem cell protection solution of the present invention is to use 5% glucose injection, 1%-5% human serum albumin and low molecular heparin calcium are as effective ingredient, compare with the mesenchyme stem cell protection solution of prior art, the activity that has extended mescenchymal stem cell is held time, reduced the preparation cost of protection liquid, wide material sources, preparation is simple, clinical direct application safety is reliable, mescenchymal stem cell is preserved after 48 hours in this kind of protection liquid, cellular form is normal, do not affect its propagation amplification ability, do not affect mescenchymal stem cell phenotypic characteristic.
The technical solution adopted in the present invention is:
An improved mesenchyme stem cell protection solution is to contain
Human serum albumin injection 1%-5% (volume ratio) and
Low molecular heparin calcium 75AXaIU/ml
5% (mass ratio) glucose injection.
Described human serum albumin injection is the injection liquid containing human serum albumin 10g/50ml.
The preferred concentration of wherein said human serum albumin injection is 1%.
Wherein said mescenchymal stem cell is umbilical cord mesenchymal stem cells.
Described 5% glucose injection is the glucose injection that clinical approval is used.
Described human serum albumin injection is the human serum albumin injection that clinical approval is used.
Described low molecular heparin calcium is the low molecular heparin calcium that clinical approval is used.
Accompanying drawing explanation
Fig. 1: umbilical cord mesenchymal stem cells flow cytometry cell phenotype analytical results
Fig. 2: umbilical cord mesenchymal stem cells is protected the cell phenotype analytical results after liquid is preserved through the present invention
Technique effect
Glucose is that body weight for humans is wanted one of nutritive ingredient and main origin of heat, also be the main energy sources material of cellular metabolism, glucose resolves into carbonic acid gas and water through cellular metabolism, clinical conventional glucose injection is as the supplementary liquid of energy and moisture, and 5% glucose is usually used to dissolved substance as a kind of isotonic solution and is applied to clinical.
Above effect based on glucose; the present invention adopts 5% glucose injection to replace TC199; result shows that mescenchymal stem cell is in this kind of improved Cell protective solutions; in the time of envrionment temperature 2-8 ℃, cell viability more than 90% can maintain 48 hours; cellular form is good; mescenchymal stem cell property preservation is constant, has greatly expanded the range of application of clinical mescenchymal stem cell, can meet the mescenchymal stem cell application needs in multinational many areas.
The present invention adopts 5% conventional clinically glucose injection to substitute TC199; originate abundanter, that cost is cheaper, security is higher, cytoactive is held time is longer; expand the range of application of clinical mescenchymal stem cell, improved the direct applied security of stem cell protection solution and reliability.
The cell-preservation liquid composition using in the present invention is the solvent that clinical approval is used, the human serum albumin that wherein albumin is clinical use, the low molecular heparin calcium that heparin is clinical use, issuable untoward reaction while avoiding mescenchymal stem cell application.
Embodiment
1, the separation of umbilical cord mesenchymal stem cells, cultivation and phenotypic evaluation
(1) umbilical cord is soaked in the aseptic PBS damping fluid (Solarbio company) containing 100IU/L penicillin, 100mg/L Streptomycin sulphate to 10-15 minute.
(2) with the above-mentioned aseptic PBS damping fluid containing penicillin, Streptomycin sulphate, fully rinse umbilical cord, umbilical cord tissue piece is shredded as far as possible, shred into 1mm
3fritter.
(3) tissue shredding is joined to DMEM substratum (Gibco company, the U.S.) washing, under 2000rpm condition centrifugal 10 minutes, abandon supernatant.
(4) pancreatin (Sigma company, the U.S.) is added in DMEM substratum, be made into the Digestive system that final concentration is 0.25% (volume ratio), with Digestive system suspended tissue piece, be placed in 37 ℃ of incubators and digest 30 minutes, centrifugal, abandon supernatant.
(5) add DMEM substratum suspended tissue piece, whole tissue block are inoculated in Tissue Culture Dish, put 37 ℃, 5%CO
2saturated humidity incubator (Thermo company, the U.S.) in cultivate.
(6) tissue block is cultivated full dose after 1 week and is changed DMEM substratum, discards tissue block and non-adherent cell.
(7) after, every 3 days half amounts are changed DMEM substratum, and every day, observation of cell growth conditions, during observation of cell to 80% fusion, with above-mentioned Digestive system peptic cell, went down to posterity by 1: 3.
(8) collecting cell is with CD45, CD90, CD146 (the BD company of FITC mark, the U.S.), the CD105 of PE mark, CD34 (BD company) and HLA-DR (BD company) carry out flow cytometer (Fascalibur, BD company, the U.S.) and detect.
Umbilical cord mesenchymal stem cells flow cytometry cell phenotype analytical results shows: mescenchymal stem cell high expression level CD90,, CD105, CD146, do not express CD45, CD34 and HLA-DR.(as Fig. 1)
2,5% glucose injection (Huaren Pharmaceutical Co., Ltd., Qingdao) and TC199 substratum (Gibco company) impact (table 1) on human mesenchymal stem cell
, purchased from Guangdong Shuanglin Bio-pharmaceutical Co., Ltd, low molecular heparin calcium is purchased from Jiangsu Wanbang Biological Pharmaceutical Co., Ltd. for the human serum albumin injection of the following stated (containing human serum albumin 10g/50ml).
(1) liquid I is preserved in configuration: containing the TC199 substratum of (volume ratio) 1% human serum albumin injection+75AXaIU/ml low molecular heparin calcium
(2) liquid II is preserved in configuration: containing (mass ratio) 5% glucose injection of (volume ratio) 1% human serum albumin injection+75AXaIU/ml low molecular heparin calcium
(3) use respectively above-mentioned two kinds of resuspended human umbilical cord mesenchymal stem cells of Cell protective solutions, adjusting cell concn is 1 * 10
6/ ml, is placed in the cillin bottle of sterilizing, and every 2ml is placed at 4 ℃, preserve 48 hours, during with platform, expect blue dyeing counting mescenchymal stem cell survival rate during respectively at the 12nd, 24,36,48 hours.
Result: 4 ℃ of preservations are after 48 hours in 5% glucose injection group for human umbilical cord mesenchymal stem cells, and cell viability is still more than 90%, and cellular form is constant, and cytolemma is smooth, and cell outline is clear, and cell size is even, cell good dispersion degree, diopter is good; And the human umbilical cord mesenchymal stem cells that is stored in TC199 group is preserved 24 hour cell vigor and is dropped to below 90% under same condition, along with the prolongation of shelf time, cell viability declines obviously, and cellular form becomes and differs in size, cell outline is fuzzy, and it is large that cell volume becomes.
(table 1)
Cell survival rate=(total cellular score-painted cell count)/total cellular score * 100%
3, the selection (table 2) of (volume ratio) 1%-10% human serum albumin injection concentration
(1) liquid I is preserved in configuration: containing 5% glucose injection of 1% human serum albumin injection+75AXaIU/ml low molecular heparin calcium
(2) liquid II is preserved in configuration: containing 5% glucose injection of 5% human serum albumin injection+75AXaIU/ml low molecular heparin calcium
(3) liquid III is preserved in configuration: containing 5% glucose injection of 10% human serum albumin injection+75AXaIU/ml low molecular heparin calcium
Use respectively three kinds of resuspended human umbilical cord mesenchymal stem cells of Cell protective solutions, adjusting cell concn is 1 * 10
6/ ml, is placed in the cillin bottle of sterilizing, and every 2ml is placed in 4 ℃, during respectively at 12,24,48 hours counting mescenchymal stem cell survival rates.
Result: human umbilical cord mesenchymal stem cells is stored in the protection liquid of 5% glucose injection that contains different concns human serum albumin injection+75AXaIU/ml low molecular heparin calcium; human serum albumin injection concentration is respectively 1%, 5%, 10%; result shows that the human serum albumin of 1%-5% concentration is more suitable for maintaining of cell viability and form; not human albumin concentration is higher, and cell viability is better.
(table 2)
Cell survival rate=(total cellular score-painted cell count)/total cellular score * 100%
4, after human umbilical cord mesenchymal stem cells is preserved 48 hours in protection liquid, cell phenotype is identified (Fig. 2)
Human umbilical cord mesenchymal stem cells is (5% glucose injection of take containing 5% human serum albumin injection+75AXaIU/ml low molecular heparin calcium is example) in Cell protective solutions of the present invention; at 4 ℃, preserve after 48 hours, flow cytometer detects cell surface CD45, CD90, CD146, CD105, CD34 and HLA-DR.
Result: 4 ℃ of preservations are after 48 hours in protection liquid of the present invention for human umbilical cord mesenchymal stem cells, and cell phenotype is constant, meets mescenchymal stem cell phenotypic characteristic.(as Fig. 2)
Claims (3)
1. an improved mesenchyme stem cell protection solution, it is characterized in that containing
Human serum albumin injection volume ratio 1%-5%;
Low molecular heparin calcium 75AXaIU/ml; With
Mass ratio 5% glucose injection;
Described human serum albumin injection is the injection liquid containing human serum albumin 10g/50ml; Described mescenchymal stem cell is umbilical cord mesenchymal stem cells.
2. protection liquid as claimed in claim 1, described 5% glucose injection is the glucose injection that clinical approval is used; Described human serum albumin injection is the human serum albumin that clinical approval is used; Described low molecular heparin calcium is the low molecular heparin calcium that clinical approval is used.
3. protection liquid as claimed in claim 1, the volume ratio 1% of described human serum albumin injection.
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| CN102028970A (en) * | 2010-12-27 | 2011-04-27 | 协和干细胞基因工程有限公司 | Stem cell preparation for treating cirrhosis |
| CN102228475A (en) * | 2011-06-09 | 2011-11-02 | 无锡人寰生物医药科技有限公司 | Stem cell compound medicament for preventing and/or treating cirrhosis and preparation method thereof |
| CN102334472B (en) * | 2011-08-02 | 2013-04-10 | 江苏省北科生物科技有限公司 | Umbilical cord preserving fluid and preparation method thereof |
| CN102511471B (en) * | 2011-12-15 | 2013-08-28 | 成都清科生物科技有限公司 | Mesenchymal stem cell frozen stock solution and preparation method thereof |
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