CN102511471B - Mesenchymal stem cell frozen stock solution and preparation method thereof - Google Patents
Mesenchymal stem cell frozen stock solution and preparation method thereof Download PDFInfo
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- CN102511471B CN102511471B CN 201110419684 CN201110419684A CN102511471B CN 102511471 B CN102511471 B CN 102511471B CN 201110419684 CN201110419684 CN 201110419684 CN 201110419684 A CN201110419684 A CN 201110419684A CN 102511471 B CN102511471 B CN 102511471B
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Abstract
The invention discloses a mesenchymal stem cell frozen stock solution which does not contain animal proteins and DMSO (dimethyl sulfoxide). The frozen stock solution comprises trehaloses, a carbohydrate and electrolyte injection, ATP (adenosine triphosphate) and human serum albumins. The frozen stock solution has the advantages of high recovery rate (90%), no influence on characteristics of stem cells, unchanged cell proliferation, unchanged cell differentiation, easy use, good stability and the like. The invention also discloses a preparation method of the frozen stock solution.
Description
Technical field
The application relates to a kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof, belongs to biomedicine field.
Background technology
Mescenchymal stem cell [mesenchymal stem cells, MSC] is the important member of stem cell family, derives to grow early stage mesoderm and ectoderm.MSC finds in marrow at first, because it has multidirectional differentiation potential, hematopoiesis support and promotes characteristics such as stem cell implantation, immunoregulation and self-replacation to be subjected to people's attention day by day.As mescenchymal stem cell in vivo or under the external specific inductive condition, can be divided into multiple histocytes such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, still have multidirectional differentiation potential after continuous passage cultivation and the freezing preservation, can be used as desirable seed cell and be used for injuries of tissues and organs reparation old and feeble and that pathology causes.
Mescenchymal stem cell has the advantages that to increase in a large number, and the stem cell after the amplification needs sometimes by frozen, in order to using in scientific research and clinical recovery at any time.Main virose dimethyl sulfoxide (DMSO) (DMSO) and the animal blood serum etc. of using in mescenchymal stem cell frozen, DMSO has toxicity inevitably can have certain damage to cell, animal blood serum then has the animal pathogenic contamination of heavy, and this all is that the system of cell has increased operating procedure and uncertain factor.Therefore, seek a kind of alternative DMSO freeze-stored cell, and can be directly used in clinical product and have very important significance.
Trehalose (Trehalose) is with a by two glucose molecules; a; 1; the 1-glycosidic bond constitutes nonreducing sugar; self property is highly stable, and trehalose has magical protective effect to organism, is because trehalose can form the diaphragm of uniqueness at cell surface under severe environmental conditions such as high temperature, high and cold, hyperosmosis and dry dehydration; protected protein matter molecule consistency inactivation effectively, thereby the life process of the body that earns a bare living and biological characteristic.Many adverse circumstances to external world show the species of outstanding degeneration-resistant tolerance, all with in their bodies exist a large amount of trehaloses that direct relation is arranged.With occurring in nature such as other carbohydrate such as sucrose, glucose, all do not possess this function.The functional characteristic that this is unique makes trehalose except can be used as the excellent activity protectant of pharmaceutical grade protein, enzyme, vaccine and other biological goods.
Summary of the invention
The purpose of this invention is to provide a kind of mesenchymal stem cell cryopreserving liquid that does not contain animal sources albumen and DMSO.
Above-mentioned purpose of the present invention is achieved by the following technical solution: a kind of mesenchymal stem cell cryopreserving liquid, it is made up of trehalose, mixed sugar electrolyte injection, ATP and human serum albumins.
In the mesenchymal stem cell cryopreserving liquid in the present invention, the quality that adds trehalose in every 100ml mixed sugar electrolyte solution is 2g ~ 15g, preferred 7.5g; The quality that adds ATP is 0.05 ~ 0.3g, preferred 0.15g; The quality that adds human serum albumins is 0.01 ~ 0.1g, preferred 0.025g.
Another object of the present invention provides a kind of preparation method of above-mentioned mesenchymal stem cell cryopreserving liquid, comprises the steps:
1) takes by weighing trehalose, be dissolved in the mixed sugar electrolyte injection.
2) add ATP and human serum albumins.
3) mixed liquor is used the bacterial filter filtration sterilization, be the mesenchymal stem cell cryopreserving liquid of preparation.
As preferably, the bacterial filter in the step 3 is 0.22
Bacterial filter.
Another object of the present invention provides a kind of using method of above-mentioned mesenchymal stem cell cryopreserving liquid, comprises the steps:
1) gets the mescenchymal stem cell through identifying, add 10%FBS with low sugar DMEM and cultivate amplification cultivation to 10
7Standby during individual cell.
2) the cells frozen storing liquid suspension cell that prepared of the frozen usefulness of mescenchymal stem cell is according to 10
6Individual/ml amount is frozen to-76 ℃ of refrigerators with the programmed cooling box.
In the present invention, get freeze-stored cell after 4 weeks of cell motility rate analysis, 37 ℃ of water-bath recoveries.Get 100
Cell suspension and 100
Platform is expected the dyed blended analysis motility rate of blue dye liquor, and the result shows that the freeze-stored cell motility rate all reaches more than 90%.
Beneficial effect of the present invention
The invention a kind of new mesenchymal stem cell cryopreserving liquid, can be outside cell membrane moment form layer protecting film, the protection cell is not subjected to the infringement of extracellular ice crystal.Have following characteristics: do not contain animal sources albumen and DMSO; High anabiosis rate〉90%, do not influence the characteristic of stem cell, cell proliferation does not change, the cell differentiation does not change; Easy use, good stability.
Embodiment
Embodiment 1
The cryopreserving liquid that contains 200Mm trehalose, 3mM ATP and 0.025% human serum albumins with the preparation of mixed sugar electrolyte solution, get respectively then that umbilical cord mesenchymal stem cells, fat mesenchymal stem cell and the mesenchymal stem cells MSCs of culture identification carry out frozen experiment, the result shows that preserving a month cell motility rate for three kinds of mescenchymal stem cells under-76 ℃ of temperature all can maintain more than 90%.
Embodiment 2: cryopreserving liquid of the present invention with contain DMSO cryopreserving liquid effect contrast
Cryopreserving liquid 1: trehalose 200mM;
ATP 3mM；
Human serum albumins 0.025%;
According to above-mentioned concentration, above-mentioned three kinds of materials are joined in the mixed sugar electrolyte solution preparation cryopreserving liquid 1.
Cryopreserving liquid 2:DMSO volume ratio 10%;
Hyclone volume ratio 10%;
The DMEM culture volume is than 80%;
Mix cryopreserving liquid 2 according to the aforementioned proportion preparation.
Operating procedure:
1) trehalose, ATP, human serum albumins, mixed sugar electrolyte solution are made cryopreserving liquid 1 according to the above ratio; DMSO, hyclone, DMEM medium are made cryopreserving liquid 2 according to the above ratio;
2) use cryopreserving liquid 1 and cryopreserving liquid 2 frozen fat mesenchymal stem cells, umbilical cord mesenchymal stem cells, dental pulp mescenchymal stem cell respectively, in the frozen pipe of the 2ml that packs into, wherein stem cell population is 1 * 10
6/ ml; Each prepares 5 pipes, and the programmed cooling box is frozen to-76 ℃ of low temperature refrigerators;
3) cell recovery: frozen good cell is put rapidly to 37 ℃ of water-baths, it was recovered in 1 ~ 2 minute, expect that with platform blue dyeing carries out cell counting, calculate motility rate.Every group of results averaged is as described in Table 1.
Table 1: mescenchymal stem cell is in the testing result of cryopreserving liquid 1 and cryopreserving liquid 2 freeze-stored cells recovery back cell motility rate
。
As can be seen from Table 1, obtain the effect the same with traditional DMSO cryopreserving liquid during the frozen three kinds of mescenchymal stem cells of novel mesenchymal cell cryopreserving liquid 1, illustrated that it can replace traditional cells frozen storing liquid, become a kind of novel mesenchymal stem cell cryopreserving liquid.
Claims (7)
1. a mesenchymal stem cell cryopreserving liquid is made up of trehalose, mixed sugar electrolyte injection, ATP and human serum albumins; The quality that wherein adds trehalose in every 100ml mixed sugar electrolyte solution is 2g-15g, and the quality that adds ATP is 0.05-0.3g, and the quality that adds human serum albumins is 0.01-0.1g.
2. mesenchymal stem cell cryopreserving liquid as claimed in claim 1, the quality that wherein adds trehalose in every 100ml mixed sugar electrolyte solution is 7.5g.
3. mesenchymal stem cell cryopreserving liquid as claimed in claim 1, the quality that wherein adds ATP in every 100ml mixed sugar electrolyte solution is 0.15g.
4. mesenchymal stem cell cryopreserving liquid as claimed in claim 1, the quality that wherein adds human serum albumins in every 100ml mixed sugar electrolyte solution is 0.025g.
5. the preparation method as each described mesenchymal stem cell cryopreserving liquid of claim 1-4 comprises the steps:
1) takes by weighing trehalose, be dissolved in the mixed sugar electrolyte injection;
2) add ATP and human serum albumins;
3) mixed liquor is used the bacterial filter filtration sterilization, be the mesenchymal stem cell cryopreserving liquid of preparation.
6. method as claimed in claim 5, wherein the bacterial filter in the step 3 is 0.22
Bacterial filter.
7. the using method as each described mesenchymal stem cell cryopreserving liquid of claim 1-4 comprises the steps:
1) gets the mescenchymal stem cell through identifying, add 10%FBS with low sugar DMEM and cultivate amplification cultivation to 10
7Standby during individual cell;
2) the cells frozen storing liquid suspension cell that prepared of the frozen usefulness of mescenchymal stem cell is according to 10
6Individual/ml amount is frozen to-76 ℃ of refrigerators with the programmed cooling box.
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| CN 201110419684 CN102511471B (en) | 2011-12-15 | 2011-12-15 | Mesenchymal stem cell frozen stock solution and preparation method thereof |
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| CN 201110419684 CN102511471B (en) | 2011-12-15 | 2011-12-15 | Mesenchymal stem cell frozen stock solution and preparation method thereof |
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| CN102511471B true CN102511471B (en) | 2013-08-28 |
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| CN104012519B (en) * | 2013-02-28 | 2015-11-18 | 杭州启迪生物科技有限公司 | A kind of fat stem cell-biomaterial composites cryopreserving liquid and uses thereof |
| CN104873542A (en) * | 2015-05-21 | 2015-09-02 | 北京青藤谷禧干细胞科技研究院有限公司 | Umbilical cord mesenchymal stem cell injection as well as preparation method and application thereof |
| CA3022685A1 (en) * | 2016-03-28 | 2017-10-05 | Cook General Biotechnology Llc | Methods of cryopreservation of viable cells, compositions of viable cells and uses thereof |
| CN105961374A (en) * | 2016-07-04 | 2016-09-28 | 深圳市合康生物科技股份有限公司 | Cell cryopreservation fluid |
| BR112019006900A2 (en) | 2016-10-05 | 2019-07-02 | Zoetis Services Llc | lyophilization methods that provide stable dehydrated protozoa for use as potent live vaccines |
| CN108029677A (en) * | 2017-12-26 | 2018-05-15 | 重庆斯德姆生物技术有限公司 | The frozen stock solution and cryopreservation methods of a kind of endothelial progenitor cells |
| CN109042625A (en) * | 2018-08-01 | 2018-12-21 | 广州润虹医药科技股份有限公司 | A kind of frozen stock solution and its application in umbilical cord mesenchymal stem cells freeze |
| CN108739798A (en) * | 2018-08-22 | 2018-11-06 | 北京常箐藤生物科技有限公司 | A kind of cells frozen storing liquid |
| CN109329271A (en) * | 2018-11-19 | 2019-02-15 | 成都清科生物科技有限公司 | A kind of clinical mescenchymal stem cell freezen protective liquid and preparation method thereof |
| CN109362714A (en) * | 2018-12-18 | 2019-02-22 | 广州赛莱拉干细胞科技股份有限公司 | A kind of fat mesenchymal stem cell saves liquid and preparation method and application |
| CN109619088A (en) * | 2018-12-27 | 2019-04-16 | 广州赛莱拉干细胞科技股份有限公司 | A kind of preservation liquid of fat mesenchymal stem cell |
| CN109744227A (en) * | 2018-12-28 | 2019-05-14 | 广州益养生物科技有限公司 | A kind of cells frozen storing liquid and its application |
| CN109792985A (en) * | 2019-02-20 | 2019-05-24 | 华东理工大学 | Cells frozen storing liquid |
| CN111011363B (en) * | 2019-12-16 | 2021-12-17 | 广东唯泰生物科技有限公司 | Mesenchymal stem cell cryopreservation liquid, cryopreservation method, preservation kit and recovery method |
| CN111838138A (en) * | 2020-08-31 | 2020-10-30 | 海南济民博鳌国际医院有限公司 | Stem cell cryopreservation liquid and preparation and use methods thereof |
| CN113367123B (en) * | 2021-06-07 | 2022-05-13 | 南京三生生物技术股份有限公司 | Cell cryopreservation method |
| CN115633675A (en) * | 2021-07-19 | 2023-01-24 | 无锡赛比曼生物科技有限公司 | Preservation solution for preserving adipose-derived mesenchymal stem cells at low temperature for long time |
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