CN105794772A - Serum-free cell cryopreservation liquid - Google Patents
Serum-free cell cryopreservation liquid Download PDFInfo
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- CN105794772A CN105794772A CN201610322202.7A CN201610322202A CN105794772A CN 105794772 A CN105794772 A CN 105794772A CN 201610322202 A CN201610322202 A CN 201610322202A CN 105794772 A CN105794772 A CN 105794772A
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- serum
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- stock solution
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- free cell
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention discloses serum-free cell cryopreservation liquid and belongs to the biotechnology field.The serum-free cell cryopreservation liquid is used for solving the problems that existing cryopreservation liquid is inconvenient to use, high in cost and low in cell survival rate.The serum-free cell cryopreservation liquid contains, by volume, 80%-89% of serum-free medium and 11%-20% of a serum and DMSO substitute.The serum and DMSO substitute contains, by volume, 30% of glycerol, 20% of chitosan and 50% of alginic acid.According to the technical scheme, use is convenient, cost is low, and cells are high in survival rate after recovery.
Description
Technical field
The invention belongs to biological technical field, specifically, be a kind of serum-free cell frozen stock solution.
Background technology
Cell cryopreservation and recovery are one of important technologies in cell culture technology, and cell cryopreservation is the one side preserving and cultivating cell
Method.The basic principle of cell cryopreservation and recovery is to freeze slowly to melt soon, so can preserve cell viability to greatest extent.
Having to use for a kind of solution when cells frozen storing liquid is cell cryopreservation, its effect is frozen cell suspension will to be needed in frozen
Liquid, supplies nutrient substance necessary to cell life metabolism, is possible to prevent simultaneously or reduces the freezing ice crystal damaging action to cell.
Freeze-stored cell all uses the method that serum mixes with DMSO, each freeze-stored cell to need first to melt serum in the prior art, and
And cell survival rate after cryopreservation resuscitation is relatively low, particularly some relatively fragile cells, and need to change liquid after recovering,
Use inconvenience；Because animal individual is different, the serum place of production, lot number difference, every quality of lot difference is very big, and its composition can not
Keep consistent；Mycoplasma, virus may be brought in drawing materials into, cell is produced potential impact, the failure of an experiment or experiment may be caused
Result unreliability；Serum cost is high, and every 500ml serum price may be up to 3000-4000 unit；The live body collection of serum is to dynamic
Thing has certain infringement.
Summary of the invention
The present invention seeks to have aimed to provide after a kind of easy to use, cell recovery that survival rate is high, pollution risk is low, low cost
Serum-free cell frozen stock solution.
For realizing above-mentioned technical purpose, the technical solution used in the present invention is as follows:
A kind of serum-free cell frozen stock solution, by volume percent includes the serum-free medium of 80%-89%, the serum of 11%-20% with
DMSO substitute, described serum and DMSO substitute by volume percent include 30% glycerol, the chitosan of 20%, the sea of 50%
Alginic acid.
Use the invention of technique scheme, cells frozen storing liquid do not contain serum, can effectively avoid because animal individual is different,
The serum place of production, lot number are different, and every quality of lot difference is very big, and its composition can not keep consistent, and may bring in drawing materials former
Body, virus, produce potential impact to cell, the situation of the failure of an experiment or experimental result unreliability may be caused to occur, thus
Reduce cost, the reliability reducing the risk polluted, ensureing experimental result；Frozen stock solution preserves at 4 DEG C, plug and play, nothing
Needing to melt, after cell recovery, survival rate is high, and the cell after recovery need not change liquid, can cover with rear direct transfer, convenience
Fast.
Limit further, described serum-free medium include adhesion factor, associated proteins, somatomedin, trace quantity mineral substance element,
Hormone, vitamin.
Limit further, described serum-free medium by volume percent include 20% adhesion factor, the associated proteins of 31%, 9%
Somatomedin, the trace quantity mineral substance element of 3%, the hormone of 9%, 18% vitamin.
Above-mentioned serum-free medium not only contains the whole key elements needed for cell is cultivated, suitably cost-effective than row are possible not only to,
And the survival rate after cell recovery can be improved.
Limiting further, described serum-free medium also includes L-glutaminate.
Limiting further, described associated proteins includes recombinant human insulin, cattle transferrins.
L-glutaminate, recombinant human insulin, the addition of cattle transferrins can improve the survival rate after cell recovery further.
Compared to existing technology, after easy to use, cell recovery, survival rate is high, pollution risk is low, low cost for the present invention.
Detailed description of the invention
In order to make those skilled in the art that the present invention be may be better understood, below in conjunction with embodiment, technical solution of the present invention is entered
One step explanation.
A kind of serum-free cell frozen stock solution, by volume mark includes that the serum-free medium of 80% and the serum of 20% replace with DMSO
For thing.Wherein serum and DMSO substitute by volume percent include 30% glycerol, the chitosan of 20%, the alginic acid of 50%；
Wherein serum-free medium by volume percent include 20% adhesion factor, the associated proteins of 30%, the somatomedin of 9%, 3%
Trace quantity mineral substance element, the hormone of 9%, 18% vitamin, the L-glutaminate of 1%；Wherein by volume mark bag in associated proteins
Include the cattle transferrins of 3% recombinant human insulin and 2%.
Embodiment 1:
First take the logarithm the A549 cell of trophophase, with trypsin, the cell dissociation of monolayer growth is got off, suspension growth thin
Born of the same parents are then moved directly in 15ml centrifuge tube；Then, centrifugal 1000rpm, 5min；Then, trypsin and old cultivation are removed
Liquid, adds appropriate above-mentioned serum-free cell frozen stock solution, blows and beats gently with suction pipe and makes cell arrangement uniform, and counting regulates frozen stock solution
The final densities of middle cell is 5 × 106Individual/ml；Secondly, cell is distributed in cryopreservation tube, often pipe 1.5ml；Finally will freeze
Deposit in the liquid nitrogen that pipe puts into subzero 100 DEG C, preserve 30 days.After 30 days, the cryopreservation tube in liquid nitrogen is taken out, puts into 35 DEG C
Warm water in carry out cell recovery, then examine under a microscope A549 cell state, cell survival number and cell attachment
Situation.
Embodiment 2 to 3:
A549 cell in embodiment 1 is replaced by PC3 cell, MB231 cell respectively, and remaining holding is constant.
Embodiment 4 to 6:
Serum-free cell frozen stock solution in embodiment 1 to 3 is replaced by the frozen stock solution of routine, and remaining keeps constant equally.
Viewed result is shown in table 1.
Table 1
| Embodiment | Survival rate | Adherent rate |
| Embodiment 1 | 95% | 80% |
| Embodiment 2 | 96% | 81% |
| Embodiment 3 | 94% | 85% |
| Embodiment 4 | 78% | 66% |
| Embodiment 5 | 75% | 68% |
| Embodiment 6 | 78% | 66% |
From upper table it can be clearly seen that, use that survival rate after the cell recovery of this serum-free frozen stock solution is high, cell attachment state is good,
Without changing frozen stock solution；And survival rate is much lower, the most adherent compared with many cells after using the cell recovery of conventional cryopreservation liquid, need more
Change frozen stock solution.
A kind of serum-free cell frozen stock solution provided the present invention above is described in detail.The explanation of specific embodiment is simply used
In helping to understand method and the core concept thereof of the present invention.It should be pointed out that, for those skilled in the art,
Under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out some improvement and modification, these improve and modify also to fall
Enter in the protection domain of the claims in the present invention.
Claims (5)
1. a serum-free cell frozen stock solution, it is characterised in that: by volume percent includes the serum-free medium of 80%-89%,
The serum of 11%-20% and DMSO substitute, described serum and DMSO substitute by volume percent include 30% glycerol, 20%
Chitosan, the alginic acid of 50%.
A kind of serum-free cell frozen stock solution the most according to claim 1, it is characterised in that: described serum-free medium includes
Adhesion factor, associated proteins, somatomedin, trace quantity mineral substance element, hormone, vitamin.
A kind of serum-free cell frozen stock solution the most according to claim 2, it is characterised in that: described serum-free medium presses body
Long-pending percent include 20% adhesion factor, the associated proteins of 31%, the somatomedin of 9%, the trace quantity mineral substance element of 3%, the lotus of 9%
You cover, 18% vitamin.
A kind of serum-free cell frozen stock solution the most according to claim 2, it is characterised in that: described serum-free medium also wraps
Include L-glutaminate.
5. according to a kind of serum-free cell frozen stock solution described in claim 2 or 3, it is characterised in that: described associated proteins bag
Include recombinant human insulin, cattle transferrins.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610322202.7A CN105794772A (en) | 2016-05-16 | 2016-05-16 | Serum-free cell cryopreservation liquid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610322202.7A CN105794772A (en) | 2016-05-16 | 2016-05-16 | Serum-free cell cryopreservation liquid |
Publications (1)
| Publication Number | Publication Date |
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| CN105794772A true CN105794772A (en) | 2016-07-27 |
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| CN201610322202.7A Pending CN105794772A (en) | 2016-05-16 | 2016-05-16 | Serum-free cell cryopreservation liquid |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107027743A (en) * | 2017-06-14 | 2017-08-11 | 深圳市泰华细胞工程有限公司 | Cells frozen storing liquid and cell freezing method |
| CN107385517A (en) * | 2017-08-07 | 2017-11-24 | 章毅 | The construction method of mesenchyma stem cell |
| CN109777767A (en) * | 2019-03-09 | 2019-05-21 | 和携科技(北京)有限公司 | A kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to osteoblast differentiation |
| CN109820869A (en) * | 2019-03-09 | 2019-05-31 | 和携科技(北京)有限公司 | A kind of preparation method of source of people umbilical cord mesenchymal stem cells injection that injecting treatment osteoarthritis for lacuna |
| CN109876010A (en) * | 2019-03-09 | 2019-06-14 | 和携科技(北京)有限公司 | It is a kind of for treat burn and scald it is postoperative repair subcutaneous tissue human umbilical cord mesenchymal stem cells secretion factor injection preparation method |
| CN109876011A (en) * | 2019-03-09 | 2019-06-14 | 和携科技(北京)有限公司 | A kind of preparation method of source of people umbilical cord mesenchymal stem cells injection that injecting treatment ischemic femoral head necrosis for lacuna |
| CN109929800A (en) * | 2019-03-09 | 2019-06-25 | 和携科技(北京)有限公司 | A kind of acquisition of stem cell secretion factor and purification process |
| CN109957542A (en) * | 2019-03-09 | 2019-07-02 | 和携科技(北京)有限公司 | A kind of preparation method of the source of people umbilical cord mesenchymal stem cells preparation for conventional antidotal therapy |
| CN110250162A (en) * | 2019-07-05 | 2019-09-20 | 北京太东生物科技有限公司 | Frozen Biological protective agent and its application |
| WO2020259194A1 (en) * | 2019-06-27 | 2020-12-30 | 北京太东生物科技有限公司 | Biological sample cryoprotectant and use thereof |
| CN112674080A (en) * | 2021-01-29 | 2021-04-20 | 华夏源细胞工程集团股份有限公司 | Programmed cooling method for human neural stem cell working cell bank |
| CN114766471A (en) * | 2022-05-31 | 2022-07-22 | 宁波建顺生物科技有限公司 | Cryopreservation liquid and cryopreservation method for long-term storage of stem cells and products thereof |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107027743A (en) * | 2017-06-14 | 2017-08-11 | 深圳市泰华细胞工程有限公司 | Cells frozen storing liquid and cell freezing method |
| CN107027743B (en) * | 2017-06-14 | 2020-12-29 | 沃昕生物科技(深圳)有限公司 | Cell cryopreservation solution and cell cryopreservation method |
| CN107385517A (en) * | 2017-08-07 | 2017-11-24 | 章毅 | The construction method of mesenchyma stem cell |
| CN109820869A (en) * | 2019-03-09 | 2019-05-31 | 和携科技(北京)有限公司 | A kind of preparation method of source of people umbilical cord mesenchymal stem cells injection that injecting treatment osteoarthritis for lacuna |
| CN109876010A (en) * | 2019-03-09 | 2019-06-14 | 和携科技(北京)有限公司 | It is a kind of for treat burn and scald it is postoperative repair subcutaneous tissue human umbilical cord mesenchymal stem cells secretion factor injection preparation method |
| CN109876011A (en) * | 2019-03-09 | 2019-06-14 | 和携科技(北京)有限公司 | A kind of preparation method of source of people umbilical cord mesenchymal stem cells injection that injecting treatment ischemic femoral head necrosis for lacuna |
| CN109929800A (en) * | 2019-03-09 | 2019-06-25 | 和携科技(北京)有限公司 | A kind of acquisition of stem cell secretion factor and purification process |
| CN109957542A (en) * | 2019-03-09 | 2019-07-02 | 和携科技(北京)有限公司 | A kind of preparation method of the source of people umbilical cord mesenchymal stem cells preparation for conventional antidotal therapy |
| CN109777767A (en) * | 2019-03-09 | 2019-05-21 | 和携科技(北京)有限公司 | A kind of promotion abductive approach of the source of people umbilical cord mesenchymal stem cells to osteoblast differentiation |
| WO2020259194A1 (en) * | 2019-06-27 | 2020-12-30 | 北京太东生物科技有限公司 | Biological sample cryoprotectant and use thereof |
| CN110250162A (en) * | 2019-07-05 | 2019-09-20 | 北京太东生物科技有限公司 | Frozen Biological protective agent and its application |
| CN110250162B (en) * | 2019-07-05 | 2021-04-06 | 北京太东生物科技有限公司 | Biological sample cryoprotectant and application thereof |
| CN112674080A (en) * | 2021-01-29 | 2021-04-20 | 华夏源细胞工程集团股份有限公司 | Programmed cooling method for human neural stem cell working cell bank |
| CN114766471A (en) * | 2022-05-31 | 2022-07-22 | 宁波建顺生物科技有限公司 | Cryopreservation liquid and cryopreservation method for long-term storage of stem cells and products thereof |
| CN116636525A (en) * | 2022-05-31 | 2023-08-25 | 宁波建顺生物科技有限公司 | Freezing solution and freezing method for long-term preservation of stem cells and products thereof |
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