CN101971799A - Freezing storage protective solution for human umbilical Wharton's jelly tissue block - Google Patents
Freezing storage protective solution for human umbilical Wharton's jelly tissue block Download PDFInfo
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- CN101971799A CN101971799A CN2010105287315A CN201010528731A CN101971799A CN 101971799 A CN101971799 A CN 101971799A CN 2010105287315 A CN2010105287315 A CN 2010105287315A CN 201010528731 A CN201010528731 A CN 201010528731A CN 101971799 A CN101971799 A CN 101971799A
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Abstract
The invention discloses freezing storage protective solution for a human umbilical Wharton's jelly tissue block. 1 liter of the freezing storage protective solution comprises 0.8 to 1.2 mol of glycol, 0.4 to 0.6 mol of dimethyl sulfoxide, 0.1 to 0.3 mol of cane sugar, 100 to 200 milliliters of cord blood autoserum and the balance of culture solution DMEM/F12. In the freezing storage protective solution, the cord blood autoserum and the culture solution DMEM/F12 serve as base solution to provide nutrient for the tissue block, so osmotic equilibrium inside and outside cells is guaranteed, the cell environment in the tissue block is guaranteed to be similar to the in vivo environment, and the freezing storage effect is good; the low-concentration glycol and dimethyl sulfoxide serve as a permeability freezing protective agent, so the toxicity of the freezing storage protective solution to the cells is reduced; and the cane sugar serves as a non-permeability freezing protective agent, so the viscosity of the solution is increased and the osmotic pressure inside and outside cell membranes is relieved when the cells are frozen and stored. The human umbilical Wharton's jelly tissue block can be directly frozen and stored by the freezing storage protective solution, so the freezing storage effect is good, the cell damage is little, and high-quality human umbilical mesenchymal stem cells can be provided for clinic.
Description
Technical field
The present invention relates to a kind of frozen protection liquid of people's umbilical cord China Tong Shi glue piece of tissue.
Background technology
Cell in people's umbilical cord China Tong Shi glue (Wharton ' s Jelly) has can self and the feature of stem cell such as multidirectional differentiation, multiplication capacity be strong, and we are referred to as umbilical cord mesenchymal stem cells (huMSCs).Mescenchymal stem cell is the important member of stem cell family, is a kind of stem cell of reduced immunogenicity, have hematopoiesis support and promote characteristics such as stem cell is implanted, regulation and control immunity, and be to be used for the desirable seed cell that injuries of tissues and organs old and feeble and that pathology causes is repaired.Umbilical cord mesenchymal stem cells easily obtains, and has all characteristics of mescenchymal stem cell because of its wide material sources, is subjected to people's attention day by day, and increasing people begins to store umbilical cord mesenchymal stem cells and thinks the possible period of want or need in the future.The prescription of frozen protection liquid and frozen operation scheme directly influence the quality of the umbilical cord mesenchymal stem cells of storage, and be most important to cell transplantation effect in the future.Cell or tissue freezing preserved several critical links: the selection of cryoprotector and combination, the proportioning of various cryoprotectors, frozen step, frozen temperature and recovery temperature.
At present; the traditional cell freezing method of the many employings of the frozen method of umbilical cord mesenchymal stem cells, cell freezing mainly adopts the frozen protection liquid that contains the permeability cryoprotector, and effect is to reduce the cell freezing point; reduce the formation of ice crystal, alleviate cells injury in the refrigerating process.Generally; people select the single frozen stem cell of permeability cryoprotector dimethyl sulfoxide (DMSO); the recovery of frozen back has the residual of frozen protection liquid; clinical usefulness contains the residual stem cell of frozen protection liquid of dimethyl sulfoxide (DMSO); higher toxic and side effect is arranged, easily cause symptoms such as nauseating, vomiting, stomachache.
Summary of the invention
The objective of the invention is to adopt a kind of frozen protection liquid of forming by basal liquid, permeability cryoprotector, impermeability cryoprotector that directly people's umbilical cord China Tong Shi glue piece of tissue is carried out the long-term safety stored frozen; frozen effective; cellular damage is few; frozen protection liquid is residual low; toxic and side effect is little, and human body is not had injury.
In order to achieve the above object; the frozen protection liquid of a kind of people's umbilical cord China's Tong Shi glue piece of tissue of the present invention comprises in the volume of 1L: ethylene glycol 0.8-1.2mol, dimethyl sulfoxide (DMSO) 0.4-0.6mol, sucrose 0.1-0.3mol, be culture fluid DMEM/F12 from body bleeding of the umbilicus serum 100-200ml, surplus.
Further; the frozen protection liquid of a kind of people's umbilical cord China's Tong Shi glue piece of tissue preferably comprises in the volume of 1L: ethylene glycol 0.9-1.1mol, dimethyl sulfoxide (DMSO) 0.4-0.5mol, sucrose 0.1-0.2mol, be culture fluid DMEM/F12 from body bleeding of the umbilicus serum 100-150ml, surplus.
The present invention adopts from body bleeding of the umbilicus serum, culture fluid DMEM/F12 as basal liquid, can guarantee the osmotic equilibrium of cell, especially adopted come from same individuality provide nutrient as basal liquid for piece of tissue from body bleeding of the umbilicus serum, be simple and easy to from body bleeding of the umbilicus serum, clinical easy realization, do not have the adding of outer derived components, guaranteed that to greatest extent the cellular environment in the piece of tissue is consistent with internal milieu, frozen effective in the frozen process; Adopt ethylene glycol, dimethyl sulfoxide (DMSO) as the permeability cryoprotector, can permeate through cell membranes be penetrated in the cell, reduce the formation of ice crystal in cell when freezing, its concentration all is on the lower level, has reduced the toxicity of frozen protection liquid to greatest extent; Adopt sucrose as the impermeability cryoprotector; its low price; comparatively common; molecular weight very much not porous in cell; a part seldom just can increase the viscosity of solution greatly, makes cell dehydration, the inside and outside osmotic pressure of cell membrane when alleviating cell cryopreservation; the ice crystal that reduces cell when freezing forms, and to stem cell nontoxic.
The present invention changes the frozen protection liquid that tradition contains single cryoprotector; employing is to the direct frozen frozen protection liquid that contains multiple cryoprotector of people's umbilical cord China Tong Shi glue piece of tissue; frozen effective; cellular damage is few; it is intracellular residual in piece of tissue recovery back to reduce frozen protection liquid to greatest extent; toxic and side effect is little, and human body is not had injury, can be for the umbilical cord mesenchymal stem cells of better quality is provided clinically.
Embodiment
To the detailed description of the invention, but protection scope of the present invention is not limited to the scope that embodiment lists by following examples.
Example one
The culture fluid DMEM/F12 that gets 500ml adds that the filter with the 0.22um filter membrane filters back 4 ℃ of precoolings behind the abundant mixing of sucrose 0.1mol; add successively on the ice bath 4 ℃ of precoolings from body bleeding of the umbilicus serum 100ml; the ethylene glycol 0.8mol of 4 ℃ of precoolings; the dimethyl sulfoxide (DMSO) 0.4mol of 4 ℃ of precoolings adds culture fluid DMEM/F12 to 1L and promptly is the frozen protection liquid of people's umbilical cord China's Tong Shi glue piece of tissue.
Example two
Get the culture fluid DMEM/F12 of 500ml; filter with the 0.22um filter membrane behind the abundant mixing of adding sucrose 0.3mol filters back 4 ℃ of precoolings; add successively on the ice bath 4 ℃ of precoolings from body bleeding of the umbilicus serum 200ml; the ethylene glycol 1.2mol of 4 ℃ of precoolings; the dimethyl sulfoxide (DMSO) 0.6mol of 4 ℃ of precoolings adds culture fluid DMEM/F12 to 1L and promptly is the frozen protection liquid of people's umbilical cord China's Tong Shi glue piece of tissue.
Example three
Get the culture fluid DMEM/F12 of 500ml; filter with the 0.22um filter membrane behind the abundant mixing of adding sucrose 0.2mol filters back 4 ℃ of precoolings; add successively on the ice bath 4 ℃ of precoolings from body bleeding of the umbilicus serum 150ml; the ethylene glycol 1mol of 4 ℃ of precoolings; the dimethyl sulfoxide (DMSO) 0.5mol of 4 ℃ of precoolings adds culture fluid DMEM/F12 to 1L and promptly is the frozen protection liquid of people's umbilical cord China's Tong Shi glue piece of tissue.
Claims (2)
1. the frozen protection liquid of people's umbilical cord China's Tong Shi glue piece of tissue comprises in the volume of 1L:
Ethylene glycol 0.8-1.2mol
Dimethyl sulfoxide (DMSO) 0.4-0.6mol
From body bleeding of the umbilicus serum 100-200ml
Sucrose 0.1-0.3mol
Surplus is culture fluid DMEM/F12.
2. require the frozen protection liquid of 1 described a kind of people's umbilical cord China's Tong Shi glue piece of tissue according to the obligee, preferably comprise in the volume of 1L:
Ethylene glycol 0.9-1.1mol
Dimethyl sulfoxide (DMSO) 0.4-0.5mol
From body bleeding of the umbilicus serum 100-150ml
Sucrose 0.1-0.2mol
Surplus is culture fluid DMEM/F12.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105287315A CN101971799A (en) | 2010-11-03 | 2010-11-03 | Freezing storage protective solution for human umbilical Wharton's jelly tissue block |
| CN2011103434210A CN102428910A (en) | 2010-11-03 | 2011-11-03 | Protecting liquid for freezing and storing human umbilical cord Wharton jelly tissue block |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105287315A CN101971799A (en) | 2010-11-03 | 2010-11-03 | Freezing storage protective solution for human umbilical Wharton's jelly tissue block |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101971799A true CN101971799A (en) | 2011-02-16 |
Family
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Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105287315A Pending CN101971799A (en) | 2010-11-03 | 2010-11-03 | Freezing storage protective solution for human umbilical Wharton's jelly tissue block |
| CN2011103434210A Pending CN102428910A (en) | 2010-11-03 | 2011-11-03 | Protecting liquid for freezing and storing human umbilical cord Wharton jelly tissue block |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011103434210A Pending CN102428910A (en) | 2010-11-03 | 2011-11-03 | Protecting liquid for freezing and storing human umbilical cord Wharton jelly tissue block |
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| CN (2) | CN101971799A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103518702A (en) * | 2012-07-02 | 2014-01-22 | 复旦大学 | Cell cryopreservation method |
| CN104472473A (en) * | 2014-11-20 | 2015-04-01 | 黑龙江天晴干细胞股份有限公司 | Cryopreservation method of umbilical cord tissue blocks |
| CN107173382A (en) * | 2017-07-14 | 2017-09-19 | 重庆市畜牧科学院 | Porcine fetus fibroblastses frozen stock solution |
| WO2018095323A1 (en) * | 2016-11-22 | 2018-05-31 | 华东理工大学 | Application of living cell sensor in cryopreservation of plant cells |
| CN111345285A (en) * | 2020-04-24 | 2020-06-30 | 湖南南华爱世普林生物技术有限公司 | Umbilical cord mesenchymal stem cell cryopreservation liquid and method without immunogenicity |
| CN112075416A (en) * | 2020-09-25 | 2020-12-15 | 山东省齐鲁干细胞工程有限公司 | Cryopreservation and recovery method of umbilical cord mesenchymal stem cell tissue |
Families Citing this family (1)
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| CN109392891B (en) * | 2018-10-26 | 2020-10-16 | 银丰生物工程集团有限公司 | Method for cryopreserving human umbilical cord tissues according to structural hierarchy system and application |
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| CN101003791A (en) * | 2007-01-11 | 2007-07-25 | 山东省农业科学院畜牧兽医研究所 | Refrigeration method for preserving embryo and oocyte by vitrification of glasscapillary |
| CN100459861C (en) * | 2007-06-18 | 2009-02-11 | 北京锦绣大地农业股份有限公司 | Livestock embryo vitrifying freeze process on metal surface |
| CN101338299A (en) * | 2008-07-29 | 2009-01-07 | 山东省农业科学院畜牧兽医研究所 | Vitrification freezing and storing method of a little somatic cells for cloning domestic animal |
| CN101861857B (en) * | 2008-11-24 | 2012-11-07 | 浙江大学 | Preservation method of ruminant mammary gland epithelial cells |
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2010
- 2010-11-03 CN CN2010105287315A patent/CN101971799A/en active Pending
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2011
- 2011-11-03 CN CN2011103434210A patent/CN102428910A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103518702A (en) * | 2012-07-02 | 2014-01-22 | 复旦大学 | Cell cryopreservation method |
| CN104472473A (en) * | 2014-11-20 | 2015-04-01 | 黑龙江天晴干细胞股份有限公司 | Cryopreservation method of umbilical cord tissue blocks |
| CN104472473B (en) * | 2014-11-20 | 2016-03-16 | 黑龙江天晴干细胞股份有限公司 | Umbilical cord tissue block cryopreservation methods |
| WO2018095323A1 (en) * | 2016-11-22 | 2018-05-31 | 华东理工大学 | Application of living cell sensor in cryopreservation of plant cells |
| US11291200B2 (en) | 2016-11-22 | 2022-04-05 | East China University Of Science And Technology | Application of viable cell monitor to cryopreservation of plant cells |
| CN107173382A (en) * | 2017-07-14 | 2017-09-19 | 重庆市畜牧科学院 | Porcine fetus fibroblastses frozen stock solution |
| CN107173382B (en) * | 2017-07-14 | 2020-12-01 | 重庆市畜牧科学院 | Frozen stock solution of pig fetal fibroblast |
| CN111345285A (en) * | 2020-04-24 | 2020-06-30 | 湖南南华爱世普林生物技术有限公司 | Umbilical cord mesenchymal stem cell cryopreservation liquid and method without immunogenicity |
| CN112075416A (en) * | 2020-09-25 | 2020-12-15 | 山东省齐鲁干细胞工程有限公司 | Cryopreservation and recovery method of umbilical cord mesenchymal stem cell tissue |
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| Publication number | Publication date |
|---|---|
| CN102428910A (en) | 2012-05-02 |
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Open date: 20110216 |