CN101919380A - Improved mesenchyme stem cell protection solution and application thereof - Google Patents
Improved mesenchyme stem cell protection solution and application thereof Download PDFInfo
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- CN101919380A CN101919380A CN2010102464248A CN201010246424A CN101919380A CN 101919380 A CN101919380 A CN 101919380A CN 2010102464248 A CN2010102464248 A CN 2010102464248A CN 201010246424 A CN201010246424 A CN 201010246424A CN 101919380 A CN101919380 A CN 101919380A
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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 capability, extensively be present in the multiple tissue of whole body, can be at cultured and amplified in vitro, and can be divided into cardiac muscle cell, nerve cell and adipocyte etc. under given conditions.Along with the further investigation of Chinese scholars, can from multiple tissue, isolate mescenchymal stem cell at present to mesenchymal stem cell biological characteristic and function.Mescenchymal stem cell has effects such as hematopoiesis support and adjusting immunity, has broad clinical application prospect at aspects such as hematopoietic reconstitution, tissue repair, immunization therapies.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 the immunogenicity.Because the effect and the characteristic of above mescenchymal stem cell, in recent years, mescenchymal stem cell is used in the clinical testing and treatment of clinical multiple disease, and shows good prospects for application.
At present domestic have mechanism that qualification carries out extensive separation, amplification, cultivation human mesenchymal 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 protection liquid hold time longer; can satisfy the requirement that is transported to domestic most of area and surrounding countries; and can directly apply to human body, safety non-toxic.
Solution medium when the at present domestic mechanism that human mesenchymal stem cell can be provided in a large number normally adopts physiological saline or cell culture medium to use as clinical mescenchymal stem cell.If after the human mesenchymal stem cell separation, cultivating, increase, use to patient immediately, physiological saline is convenient and safe, but the mescenchymal stem cell speed that vigor descends in physiological saline is very fast, preserve mescenchymal stem cell with physiological saline merely, cell viability will reduce about 10% after 2 hours, therefore only is applicable to the instant use of mescenchymal stem cell.Existing another cytoprotection liquid is as one of composition with cell culture medium; contain the required carbohydrate of cell growth metabolism in the cell culture medium; amino acid; mineral salt; materials such as vitamin; Premeabilisation of cells is pressed and vigor has better action for keeping; but present domestic most of cell culture medium all is only to use for scientific research; not as the conventional reagent of clinical practice; therefore the safety and the reliability of direct application cell medium is uncertain the clinical practice human mesenchymal stem cell time; and cell culture medium is because manufacturer; the difference of production method; quality; purity; price does not wait yet, purity; the all reasonable import medium of quality price is more expensive.
Prior art is to use the preservation liquid of the solution of TC199 serum free medium, human serum albumin and the configuration of LMWHs calcium as human mesenchymal stem cell.This technology is used the physiological saline preservation that contains the TC199 of 1% human serum albumins and contain 1% human serum albumins respectively after human mesenchymal stem cell is separated, cultivates, collects, and cell concentration is 5 * 10
5/ ml, 4 degree were preserved after 8 hours, and the cell viability of physiological saline group is starkly lower than the TC199 group; Adjust the concentration of human serum albumin, make respectively and contain 0.5%, 1%, 2%, 5% human serum albumin among the TC199,1%, 2%, 5% human serum albumin all is useful for the vigor that keeps mescenchymal stem cell; Prepare the solution depositary mescenchymal stem cell that contains 0.01%EDTA, 75AXaIU/ml low molecular sodium heparin, 75AXaIU/ml LMWHs calcium respectively with the TC199 that contains 1% human serum albumin, find that LMWHs calcium can effectively prevent the mescenchymal stem cell gathering.Prior art is as preserving liquid depositary mescenchymal stem cell with TC199 medium, human serum albumin, LMWHs calcium in a word, stem cell can keep the vigor 24 hours more than 90%, cell viability prolongs gradually in time and successively decreases, and can prevent the adhesion of stem cell and container inner wall.
One of shortcoming of prior art is that the TC199 in the mesenchyme stem cell preserving fluid is a kind of cell culture medium, cell culture medium is used to carry out cell and tissue culture usually, be not state approval can clinical practice in the reagent of human body, the composition complexity, the medium quality of different manufacturers differs, and import reagent costs an arm and a leg, and in recent years along with the continuous development in biological reagent field, TC199 has withdrawed from medium market gradually, substituted by other medium, so its source is limited.In a word, although prior art can keep cell viability 24 hours with TC199 as the effective ingredient of mesenchyme stem cell preserving fluid, consider all to be unfavorable for that from legitimacy, the reagent cost aspect of reagent source, clinical practice the mescenchymal stem cell clinical expansion uses.The solution medium that uses 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, and is with its alternative TC199, cheap, the source is abundant, clinical practice 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 descends, so just mean above 24 hours, cell viability will drop to below 90%, yet the most at home cities of stem-cell therapy and clinical testing and external a plurality of country carry out, utilize the existing vehicles, the range of application of mescenchymal stem cell that 24 hours vigor has been held time significant limitation.And will having mesenchyme stem cell protection solution now, the present invention improves; make mescenchymal stem cell in this kind protection liquid, keep 90% above vigor can keep 48 hours; hold time with regard to the activity that has prolonged mescenchymal stem cell like this; enlarged the scope of clinical stem cell extensive use greatly; utilize the existing vehicles, can be transported to domestic most of city and surrounding countries.
Summary of the invention
Problem at the prior art existence; the improved mesenchyme stem cell protection solution of the present invention is to use 5% glucose injection; 1%-5% human serum albumin and LMWHs calcium are as effective ingredient; compare with the mesenchyme stem cell protection solution of prior art; the activity that has prolonged 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 48 hours in this kind protection liquid after, cellular morphology is normal; do not influence its propagation amplification ability; do not influence the mescenchymal stem cell phenotypic characteristic.
The technical solution adopted in the present invention is:
A kind of improved mesenchyme stem cell protection solution is to contain
Human serum albumin parenteral solution 1%-5% (volume ratio) and
LMWHs calcium 75AXaIU/ml
5% (mass ratio) glucose injection.
Described human serum albumin parenteral solution is the parenteral solution that contains human serum albumin 10g/50ml.
The preferred concentration of wherein said human serum albumin parenteral solution is 1%.
Wherein said mescenchymal stem cell is a umbilical cord mesenchymal stem cells.
Described 5% glucose injection is the glucose injection that clinical approval is used.
Described human serum albumin parenteral solution is the human serum albumin parenteral solution that clinical approval is used.
Described LMWHs calcium is the LMWHs calcium that clinical approval is used.
Description of drawings
Fig. 1: umbilical cord mesenchymal stem cells flow cytometry cell phenotype analysis result
Fig. 2: the cell phenotype analysis result of umbilical cord mesenchymal stem cells after the present invention protects the liquid preservation
Technique effect
Glucose is that body weight for humans is wanted one of nutritional labeling and main origin of heat, it also is the main energy sources material of cell metabolism, carbon dioxide and water are resolved in the metabolism of glucose process cell, clinical glucose injection liquid commonly used is as the additional 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 liquid to replace TC199; the result shows that a mesenchymal stem cells is in the improved Cell protective solutions of this kind; cell viability can kept more than 90% 48 hours when 2-8 ℃ of environment temperature; the cell form is good; between the mesenchymal stem cells property preservation constant, greatly enlarged the range of application of clinical mesenchymal stem cells, can satisfy multinational many areas between mesenchymal stem cells use needs.
The present invention adopts clinical 5% glucose injection liquid commonly used to substitute TC199; originate abundanter, cost is cheaper, security is higher, active to keep the time longer for cell; enlarge the range of application of clinical mesenchymal stem cells, improved the direct applied security of stem cell protection solution and reliability.
Employed cell-preservation liquid composition is the solvent that clinical approval is used among the present invention, and wherein albumin is the human serum albumin of clinical use, and heparin is the Low-molecular-weight Heparins Calcium of clinical use, the bad reaction that may produce when avoiding a mesenchymal stem cells to use.
Embodiment
1, the separation of umbilical cord mesenchymal stem cells, cultivation and phenotypic evaluation
(1) umbilical cord was soaked 10-15 minute in containing the aseptic PBS buffer solution (Solarbio company) of 100IU/L penicillin, 100mg/L streptomycin.
(2) fully wash umbilical cord with the above-mentioned aseptic PBS buffer solution that contains penicillin, streptomycin, the umbilical cord tissue piece is shredded as far as possible, shred into 1mm
3Fritter.
(3) tissue that will shred joins DMEM medium (Gibco company, the U.S.) washing, under the 2000rpm condition centrifugal 10 minutes, abandons supernatant.
(4) pancreatin (Sigma company, the U.S.) is added in the DMEM medium, be made into the digestive juice that final concentration is 0.25% (volume ratio),, place 37 ℃ of incubator digestion 30 minutes with digestive juice suspended tissue piece, centrifugal, abandon supernatant.
(5) add DMEM medium suspended tissue piece, whole piece of tissue are inoculated in the Tissue Culture Dish, put 37 ℃, 5%CO
2Saturated humidity incubator (Thermo company, the U.S.) in cultivate.
(6) full dose was changed the DMEM medium after piece of tissue cultivated for 1 week, discarded piece of tissue and non-adherent cell.
(7) per 3 days half amounts are changed the DMEM medium after, and every day, the observation of cell growth conditions when observation of cell to 80% merges, with above-mentioned digestive juice vitellophag, went down to posterity by 1: 3.
(8) collecting cell is with CD45, CD90, the CD146 (BD company, the U.S.) of FITC mark, and 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 analysis result shows: mescenchymal stem cell high expressed 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 medium (Gibco company) are to the influence (table 1) of human mesenchymal stem cell
LMWHs calcium is available from Jiangsu Wanbang Biological Pharmaceutical Co., Ltd. available from Guangdong Shuanglin Bio-pharmaceutical Co., Ltd for the human serum albumin parenteral solution of the following stated (containing human serum albumin 10g/50ml).
(1) liquid I is preserved in configuration: the TC199 medium that contains (volume ratio) 1% human serum albumin parenteral solution+75AXaIU/ml LMWHs calcium
(2) liquid II is preserved in configuration: (mass ratio) 5% glucose injection that contains (volume ratio) 1% human serum albumin parenteral solution+75AXaIU/ml LMWHs calcium
(3) use above-mentioned two kinds of resuspended human umbilical cord mesenchymal stem cells of cytoprotection liquid respectively, adjusting cell concentration is 1 * 10
6/ ml places the cillin bottle of sterilization, and every 2ml places under 4 ℃, preserved 48 hours, during expect blue dyeing counting mescenchymal stem cell survival rate with platform during respectively at the 12nd, 24,36,48 hour.
The result: 4 ℃ of preservations are after 48 hours in 5% glucose injection group for human umbilical cord mesenchymal stem cells, and still more than 90%, cellular morphology is constant for cell viability, and cell membrane is smooth, and cell outline is clear, and the cell size is even, the cell good dispersion degree, and 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 promptly dropped to below 90% under same condition, along with the prolongation of holding time, cell viability descends obviously, and cellular morphology becomes and differs in size, cell outline is fuzzy, and it is big that cell volume becomes.
(table 1)
Cell survival rate=(total cellular score-painted cell number)/total cellular score * 100%
3, the selection (table 2) of (volume ratio) 1%-10% human serum albumin parenteral solution concentration
(1) liquid I is preserved in configuration: 5% glucose injection that contains 1% human serum albumin parenteral solution+75AXaIU/ml LMWHs calcium
(2) liquid II is preserved in configuration: 5% glucose injection that contains 5% human serum albumin parenteral solution+75AXaIU/ml LMWHs calcium
(3) liquid III is preserved in configuration: 5% glucose injection that contains 10% human serum albumin parenteral solution+75AXaIU/ml LMWHs calcium
Use three kinds of resuspended human umbilical cord mesenchymal stem cells of cytoprotection liquid respectively, adjusting cell concentration is 1 * 10
6/ ml places the cillin bottle of sterilization, and every 2ml places 4 ℃, during respectively at 12,24,48 hours counting mescenchymal stem cell survival rates.
The result: human umbilical cord mesenchymal stem cells is stored in the protection liquid of 5% glucose injection that contains variable concentrations human serum albumin parenteral solution+75AXaIU/ml LMWHs calcium; human serum albumin parenteral solution concentration is respectively 1%, 5%, 10%; the result shows that the human serum albumin of 1%-5% concentration is more suitable for keeping of cell viability and form; be not that human albumin concentration is high more, cell viability is good more.
(table 2)
Cell survival rate=(total cellular score-painted cell number)/total cellular score * 100%
4, cell phenotype was identified (Fig. 2) after human umbilical cord mesenchymal stem cells was preserved 48 hours in protection liquid
Human umbilical cord mesenchymal stem cells is (is example with 5% glucose injection that contains 5% human serum albumin parenteral solution+75AXaIU/ml LMWHs calcium) in cytoprotection liquid of the present invention; preservation is after 48 hours down at 4 ℃, and flow cytometer detects cell surface CD45, CD90, CD146, CD105, CD34 and HLA-DR.
The 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 the mescenchymal stem cell phenotypic characteristic.(as Fig. 2)
Claims (4)
1. an improved mesenchyme stem cell protection solution is characterized in that it being to contain
Human serum albumin parenteral solution volume than 1%-5% and
LMWHs calcium 75AXaIU/ml
Mass ratio 5% glucose injection.
Described human serum albumin parenteral solution is the parenteral solution that contains human serum albumin 10g/50ml.
2. protection liquid as claimed in claim 1, the preferred concentration of described human serum albumin parenteral solution are volume ratio 1%.
3. protection liquid as claimed in claim 1, described mescenchymal stem cell are umbilical cord mesenchymal stem cells.
4. as the arbitrary described protection liquid of claim 13, described 5% glucose injection is the glucose injection that clinical approval is used; Described human serum albumin parenteral solution is the human serum albumin that clinical approval is used; Described LMWHs calcium is the LMWHs calcium that clinical approval is used.
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Cited By (19)
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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 |
| CN102334472A (en) * | 2011-08-02 | 2012-02-01 | 江苏省北科生物科技有限公司 | Umbilical cord preserving fluid and preparation method thereof |
| CN102487939A (en) * | 2011-12-29 | 2012-06-13 | 上海润生生物技术有限公司 | Mesenchymal stem cell stock solution |
| CN102511471A (en) * | 2011-12-15 | 2012-06-27 | 成都清科生物科技有限公司 | Mesenchymal stem cell frozen stock solution and preparation method thereof |
| CN102920735A (en) * | 2012-11-14 | 2013-02-13 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection, preparation method and application thereof in preparing medicine for treating diabetes |
| WO2014075592A1 (en) * | 2012-11-14 | 2014-05-22 | 贾在美 | Mesenchymal stem cell injection and preparation method thereof and use in preparing drugs for treating dilated cardiomyopathy in children |
| CN105802905A (en) * | 2016-04-21 | 2016-07-27 | 天津普瑞赛尔生物科技有限公司 | Kit for collecting, separating and amplifying dental pulp stem cells and using method thereof |
| CN106177918A (en) * | 2016-09-30 | 2016-12-07 | 广州赛莱拉干细胞科技股份有限公司 | A kind of mesenchymal stem cell injection and its preparation method and application |
| CN106237313A (en) * | 2016-09-30 | 2016-12-21 | 广州赛莱拉干细胞科技股份有限公司 | A kind of umbilical cord mesenchymal stem cells injection and its preparation method and application |
| CN106361771A (en) * | 2016-11-08 | 2017-02-01 | 北京恒峰铭成生物科技有限公司 | High-glucose activated mesenchymal stem cell injection and application thereof for diabetic drugs |
| CN106554940A (en) * | 2015-09-24 | 2017-04-05 | 苏州爱乐桢医疗器械有限公司 | A kind of fat mesenchymal stem cell transport protection liquid |
| CN106922648A (en) * | 2017-02-22 | 2017-07-07 | 海南新生命干细胞医疗有限公司 | A kind of mescenchymal stem cell cryopreservation solution and preparation method thereof |
| CN107912420A (en) * | 2017-10-27 | 2018-04-17 | 北京协科医药科技有限公司 | A kind of cell preservation method, preserve liquid and preserve liquid and preparation method thereof |
| CN108432742A (en) * | 2018-04-09 | 2018-08-24 | 佛山科学技术学院 | A kind of mescenchymal stem cell room temperature transport liquid |
| CN109876012A (en) * | 2019-03-21 | 2019-06-14 | 北京隆祺生物科技有限公司 | A kind of Pharmaceutical composition and its application in promotion skin wound healing |
| CN110622956A (en) * | 2019-09-27 | 2019-12-31 | 广州南医大生物工程有限公司 | Umbilical cord mesenchymal stem cell preservation solution |
| CN112522190A (en) * | 2020-12-08 | 2021-03-19 | 山东省齐鲁干细胞工程有限公司 | High-survival-rate storage and transportation method for umbilical cord tissues |
| CN113287603A (en) * | 2021-06-18 | 2021-08-24 | 吉林省拓华生物科技有限公司 | Biological sample preservation solution and preparation method and application thereof |
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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 |
| CN102334472A (en) * | 2011-08-02 | 2012-02-01 | 江苏省北科生物科技有限公司 | Umbilical cord preserving fluid 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 |
| CN102511471A (en) * | 2011-12-15 | 2012-06-27 | 成都清科生物科技有限公司 | Mesenchymal stem cell frozen stock solution 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 |
| CN102487939A (en) * | 2011-12-29 | 2012-06-13 | 上海润生生物技术有限公司 | Mesenchymal stem cell stock solution |
| CN102920735A (en) * | 2012-11-14 | 2013-02-13 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection, preparation method and application thereof in preparing medicine for treating diabetes |
| WO2014075592A1 (en) * | 2012-11-14 | 2014-05-22 | 贾在美 | Mesenchymal stem cell injection and preparation method thereof and use in preparing drugs for treating dilated cardiomyopathy in children |
| WO2014075589A1 (en) * | 2012-11-14 | 2014-05-22 | 贾在美 | Mesenchymal stem cell injection, preparation method thereof, and application thereof in preparing diabetes drug |
| CN106554940A (en) * | 2015-09-24 | 2017-04-05 | 苏州爱乐桢医疗器械有限公司 | A kind of fat mesenchymal stem cell transport protection liquid |
| CN105802905A (en) * | 2016-04-21 | 2016-07-27 | 天津普瑞赛尔生物科技有限公司 | Kit for collecting, separating and amplifying dental pulp stem cells and using method thereof |
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| CN106361771A (en) * | 2016-11-08 | 2017-02-01 | 北京恒峰铭成生物科技有限公司 | High-glucose activated mesenchymal stem cell injection and application thereof for diabetic drugs |
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| CN107912420A (en) * | 2017-10-27 | 2018-04-17 | 北京协科医药科技有限公司 | A kind of cell preservation method, preserve liquid and preserve liquid and preparation method thereof |
| CN108432742A (en) * | 2018-04-09 | 2018-08-24 | 佛山科学技术学院 | A kind of mescenchymal stem cell room temperature transport liquid |
| CN109876012A (en) * | 2019-03-21 | 2019-06-14 | 北京隆祺生物科技有限公司 | A kind of Pharmaceutical composition and its application in promotion skin wound healing |
| CN110622956A (en) * | 2019-09-27 | 2019-12-31 | 广州南医大生物工程有限公司 | Umbilical cord mesenchymal stem cell preservation solution |
| CN112522190A (en) * | 2020-12-08 | 2021-03-19 | 山东省齐鲁干细胞工程有限公司 | High-survival-rate storage and transportation method for umbilical cord tissues |
| CN113287603A (en) * | 2021-06-18 | 2021-08-24 | 吉林省拓华生物科技有限公司 | Biological sample preservation solution and preparation method and application thereof |
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