CN1566333A - Method for cryopreservation of cell - Google Patents
Method for cryopreservation of cell Download PDFInfo
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- CN1566333A CN1566333A CN 03148397 CN03148397A CN1566333A CN 1566333 A CN1566333 A CN 1566333A CN 03148397 CN03148397 CN 03148397 CN 03148397 A CN03148397 A CN 03148397A CN 1566333 A CN1566333 A CN 1566333A
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Abstract
A method for cryopreservation of cell, wherein unliquefied sodium alginate - polylysine - sodium alginate microcapsule and refrigeration protective material (glycerine or dimethyl sulphoxide) are combined to proceed cell refrigeration.
Description
Technical field
The invention belongs to the biomass cells technical field, be specifically related to a kind of method of freezing preservation cell.
Background technology
The microencapsulated cell technology has been used to treat the various diseases that cause owing to cell injury, and by pancreas islet treatment diabetes, bag is by the hyperthyroidism of parathyroid gland cell therapy as bag, and bag is by liver cell treatment liver failure, and bag is by genetically engineered cell treatment cancer.The cellular transplantation therapy diabetes are achieved success.Microencapsulation pancreas islet treatment people's diabetes also obtain certain progress.The microencapsulated cell technology be applied to clinical after, it is another problem that need solve that long-term frozen is preserved.It is frozen that Sun etc. studies have shown that the frozen effect of microencapsulation pancreas islet is better than not microencapsulation.Find behind the freezing microencapsulated hepatocyte such as Chang the to thaw hepatocellular secreting function in back and ordinary method and indifference, this illustrates microencapsulation technology pair cell frozenly has a provide protection.The micro-capsule of application such as Sun and Chang is the Lalgine-poly-lysine-alginate microcapsule of liquefaction.The center of this micro-capsule is a liquid seaweed acid sodium, and outer bread is by the film of one deck poly-lysine and sodium alginate composition.Someone thinks that the permeability of this micro-capsule is good, helps the discharge of cell growth and emiocytosis product, but also the someone thinks that the micro-capsule of its permeability and not liquefaction does not have difference.The anti-mechanical force of micro-capsule of liquefaction and impermeabilisation are pressed ability, easily break.When carrying out cell freezing, the ice crystal that the outer liquid of micro-capsule forms can damage microcapsule membrane, and in addition, the micro-capsule of liquefaction is because its center is liquid, and the height that water content does not more liquefy easily forms ice crystal in the micro-capsule when therefore freezing, and pair cell causes damage.And the micro-capsule that does not liquefy, because its inside is solid, so good stability is difficult for breaking.Anti-mechanical force and impermeabilisation pressure energy power are strong, have reduced the infringement of ice crystal to film.In addition, interior moisture content is few, and ice crystal is few, so the infringement of pair cell is little.
Summary of the invention
The micro-capsule that the present invention uses not liquefaction carries out the cell freezing preservation, forms the damage that pair cell causes to avoid ice crystal, keeps the membranous structure of micro-capsule simultaneously again, guarantees to have immune buffer action after the transplanting in the body.Its method is: will treat that at first refrigerated cell and sodium alginate soln are mixed, and form drop through high-voltage pulse static droplet generator, the drop that forms is splashed into CaCl
2In the solution.Calcium chloride and sodium alginate generation gelatinization reaction form the glue pearl, handle with physiological saline, poly-lysine, physiological saline, sodium alginate, physiological saline successively then.Final formation diameter is the micro-capsule of 50-1000 μ m.Different with the micro-capsule of liquefaction, at last without Trisodium Citrate liquefaction, so that the center of micro-capsule is a solid.Then the micro-capsule of making is mixed with the cryoprotection liquid phase.The composition of frozen solution is: 10% glycerine or 10% DMSO, 30% foetal calf serum, 60% substratum.At last mixed solution is put into frozen pipe, seal.Keep flat in-20 ℃ the refrigerator.The purpose that keeps flat is to prevent that the micro-capsule deposition is agglomerating, the cryoprotectant skewness.Put into-70 ℃ after 2 hours, take out after 12 hours, directly drop into-196 ℃ in preservation.The invention is characterized in the micro-capsule that application is not liquefied, because its inside is solid, so good stability is difficult for breaking; Anti-mechanical force and impermeabilisation pressure energy power are strong, have reduced the infringement of film to ice crystal.In addition, interior moisture content is few, and ice crystal is few, so the infringement of pair cell is little.Therefore recovery back micro-capsule percentage of head rice height, cellular form and active good can be directly used in the body and transplant.
Embodiment
The making of embodiment 1 micro-capsule
Collect HK-2 cell (people's renal cells system), adding 1.5% sodium alginate soln to cell concn is 1 * 10
6Individual/ml.Cell suspension is through high-voltage pulse static droplet generator, and the drop of formation splashes into 2.0% CaCl
2In the solution, soaked 10 minutes, finally form the glue pearl.The glue pearl is put into physiological saline clean, add 0.05% poly-lysine then, reacted 6 minutes, physiological saline cleans, and 0.03% sodium alginate soaked 6 minutes, and physiological saline cleans.The microscopically micrometer is observed, and the diameter of micro-capsule is 500 μ m.Average each micro-capsule contains 100 cells.
Embodiment 2 micro-capsules freezing
1 milliliter of micro-capsule is mixed in the frozen pipe of packing into 1 milliliter of frozen storing liquid.For avoiding micro-capsule to pile up, kept flat in-20 ℃ 2 hours, put into then-70 ℃ 12 hours, take out after 12 hours, put in-196 ℃ of liquid nitrogen and preserve.
After 1 week, 37 ℃ thaw, and with the 55mM Trisodium Citrate micro-capsule that breaks, discharge cell.The back cell motility rate that thaws is 85%.Illustrate frozen respond well.
Claims (6)
1 the present invention is the method that a kind of cell freezing is preserved.It is characterized in that cell envelope in micro-capsule, add cryoprotectant, follow procedure is lowered the temperature.
Said micro-capsule in 2 claims 1, but it is characterized in that forming by gel material, metal ion and polyamino acid.
Said micro-capsule in 3 claims 1 is characterized in that diameter can be between 50～1000 microns.
But said gel material is a sodium alginate in 4 claims 2, and metal ion is a calcium ion, and polyamino acid is a poly-lysine.
Said cryoprotectant in 5 claims 1 is characterized in that it can being glycerine, also can be dimethyl sulfoxide (DMSO).
Said cooling process in 6 claims 1, it is characterized in that-20 ℃ 2 hours ,-70 ℃ 12 hours, go into-196 ℃ of liquid nitrogen at last and preserve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03148397 CN1566333A (en) | 2003-07-02 | 2003-07-02 | Method for cryopreservation of cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03148397 CN1566333A (en) | 2003-07-02 | 2003-07-02 | Method for cryopreservation of cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1566333A true CN1566333A (en) | 2005-01-19 |
Family
ID=34472275
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03148397 Pending CN1566333A (en) | 2003-07-02 | 2003-07-02 | Method for cryopreservation of cell |
Country Status (1)
| Country | Link |
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| CN (1) | CN1566333A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103648507A (en) * | 2010-12-30 | 2014-03-19 | 人类起源公司 | Methods for cryopreserving and encapsulating cells |
| CN105746493A (en) * | 2016-03-29 | 2016-07-13 | 深圳爱生再生医学科技有限公司 | Frozen-preserving and thawing method for microencapsulation immune cells |
| CN108990964A (en) * | 2018-08-09 | 2018-12-14 | 华东理工大学 | Cells frozen storing liquid |
-
2003
- 2003-07-02 CN CN 03148397 patent/CN1566333A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103648507A (en) * | 2010-12-30 | 2014-03-19 | 人类起源公司 | Methods for cryopreserving and encapsulating cells |
| CN105746493A (en) * | 2016-03-29 | 2016-07-13 | 深圳爱生再生医学科技有限公司 | Frozen-preserving and thawing method for microencapsulation immune cells |
| CN108990964A (en) * | 2018-08-09 | 2018-12-14 | 华东理工大学 | Cells frozen storing liquid |
| CN108990964B (en) * | 2018-08-09 | 2022-01-28 | 华东理工大学 | Cell cryopreservation liquid |
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
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