CN106417252A - Novel cell cryopreservation reagent - Google Patents
Novel cell cryopreservation reagent Download PDFInfo
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- CN106417252A CN106417252A CN201610851438.XA CN201610851438A CN106417252A CN 106417252 A CN106417252 A CN 106417252A CN 201610851438 A CN201610851438 A CN 201610851438A CN 106417252 A CN106417252 A CN 106417252A
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- cell
- reagent
- recombinant human
- frozen
- cell cryopreservation
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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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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a novel cell cryopreservation reagent which comprises, 100ng of recombinant human epidermal growth factors, 500ng of recombinant human basic fibroblast growth factors, 300ng of recombinant human hepatocyte growth factors, 0.5ml of DMSO (dimethyl sulfoxide), 2ml of recombinant human serum albumin and 8ml of DMEM (Dulbecco's modified eagle medium). The novel cell cryopreservation reagent has the advantages that the novel cell cryopreservation reagent is free from animal components and safe and reliable to use, components are completely definite, quality control is facilitated, the reagent is ready to use and can be directly used for cell resuspension after cell counting, the reagent is more convenient to operate without additional preparation, applicable cell types are unlimited, and cells can be maintained in long-term safe storage in a -196 DEG C liquid nitrogen environment.
Description
Technical field
The present invention relates to a kind of cell cryopreservation reagent, and in particular to a kind of new cell cryopreservation reagent, belong to biological skill
Art field.
Background technology
Cell cryopreservation using frozen reagent have containing serum and two class of serum-free, as serum has complicated component, quality
Unstable, expensive the shortcomings of, so serum-free Cryopreservation Technology becomes the trend of development.The present invention is primarily directed to serum-free
The research that Cryopreservation Technology is carried out.
Existing cell cryopreservation reagent, which is primarily present following both sides problem：
First, contain animal component, using dangerous；
When the 2nd, using, matching while using, inconvenient operation is needed, and is easily polluted.
Content of the invention
For solving the deficiencies in the prior art, it is an object of the invention to provide a kind of new cell cryopreservation reagent, which is not
Animal component is not only contained, and is instant, after cell counting, you can directly cell is carried out using this reagent resuspended.
In order to realize above-mentioned target, the present invention is adopted the following technical scheme that：
A kind of new cell cryopreservation reagent, it is characterised in that each constituent and consumption are respectively：
The invention has benefit that：
(1) animal component is not contained, safe and reliable；
(2) composition is completely clear and definite, is easy to quality control；
(3) instant, after cell counting, you can directly carry out cell using this reagent resuspended, more convenient to operate, need not
Extra preparation；
(4) cell type being suitable for is not limited；
(5) cell long-term safety under -196 DEG C of liquid nitrogen environments can be maintained to preserve.
Specific embodiment
Make specific introduction to the present invention below in conjunction with specific embodiment.
First, prepare each related reagent
The constituent of 1 each group of table and consumption
Existing cell cryopreservation reagent, its formula is generally hyclone collocation DMEM culture medium, while adding a certain amount of
DMSO, the frozen reagent of this formula contains animal component, and needs matching while using, much asks to actually used bringing
Topic.
And in the present invention, we replace hyclone from recombinant human serum albumin, and by recombinant human serum albumin
With DMEM culture medium according to 2:8 volume ratio mixing, additionally, for the activity for preferably keeping freeze-stored cell, we are through repeatedly
Checking, finally have selected recombinant human epidermal growth factor, recombination human basic fibroblast growth factor and human hepatocyte growth
The factor is the most suitable factor concentration of checking, We conducted the various combination of said components as complementary element, to verify
Specific frozen effect.
2nd, preparation method
Prepare DMEM culture medium (purchased from Life company of the U.S.), recombinant human serum albumin (unit is biological purchased from Wuhan standing grain),
DMSO (purchased from Sigma Co., USA), recombinant human epidermal growth factor (purchased from Peprotech company of the U.S.), recombination human basic
Fibroblast growth factor (purchased from Peprotech company of the U.S.), recombinant human hepatocyte growth factor are (purchased from the U.S.
Peprotech company).
The preparation of related reagent is carried out in hundred grades of aseptic operating platform.Recombinant human epidermal growth factor, recombination human basic
Fibroblast growth factor, the preparation of recombinant human hepatocyte growth factor are operated according to the description of producer.
3rd, using method
After cell counting, directly carry out cell using this reagent resuspended, density be6-5×106/ ml's, i.e. 1.8ml
Load 1ml cell suspension in cryopreservation tube.
4th, frozen effect
Tested from Hela cell.Enough Hela cells are collected, with aforementioned different component formulated in combination
It is resuspended that frozen reagent carries out cell, will meet density requirements (3 × 106/ ml) cell load cryopreservation tube.Dropped using program gradient
Wen Yi is lowered the temperature to cryopreservation tube, proceeds to -196 DEG C of liquid nitrogen environments afterwards.After frozen a period of time, cell recovery is carried out, observation
The recovery situation of each group cell, and calculate the recovery survival rate (abbreviation anabiosis rate) of each group cell.
1st, the morphological observation of Hela cell
Frozen one week, then recover, apply phase contrast microscope the survival of each group cell and differentiation situation after recovery is observed,
Culture calculated cloning efficiency after 3 days.
It is observed that the survival of each group cell and differentiation situation are shown in Table 2.
The survival of 2 each group cell of table and differentiation situation
| Survival condition | Differentiation situation | |
| 1st group | Difference | There is differentiation |
| 2nd group | Well | There is not differentiation |
| 3rd group | Well | There is not differentiation |
| 4th group | Well | There is not differentiation |
| 5th group | Well | There is not differentiation |
| 6th group | Well | There is not differentiation |
| 7th group | Well | There is not differentiation |
| 8th group | Well | There is not differentiation |
| 9th group | Well | There is not differentiation |
It is computed, the cloning efficiency of each group cell is shown in Table 3.
The cloning efficiency of 3 each group cell of table
2nd, the characteristic measurement of Hela cell
Frozen one week, then recover, determine the characteristic of Hela cell.
Measurement result shows：
(1) Hela cell maintains very high proliferation activity.
(2), there is no great change in Hela cell form stable.
3rd, the frozen timing of the safety of Hela cell
Frozen one week, one month, 2 years, then recover, calculate the anabiosis rate of each group cell.
Result of calculation is shown in Table 4.
The anabiosis rate of 4 each group cell of table
As shown in Table 4：
(1) frozen one week, the frozen effect of the 3rd group of frozen reagent was best, and cell recovery rate is up to 95%, and the 1st group of jelly
The cell recovery rate for depositing reagent is only 50%；
(2) frozen one month, or the frozen effect of the 3rd group of frozen reagent is best, cell recovery rate was up to 95%, and the
The cell recovery rate of 1 group of frozen reagent is only 40%；
(3) frozen 2 years, or the frozen effect of the 3rd group of frozen reagent is best, cell recovery rate was up to 93%, and the 1st
The cell recovery rate for organizing frozen reagent is only 30%.
Above result of the test explanation：The cell cryopreservation reagent of the present invention can maintain cell long under -196 DEG C of liquid nitrogen environments
Phase safe storage.
The cell cryopreservation reagent of the present invention, by adding three kinds of recombinant growth factors (recombinant human epidermal growth factor, restructuring
Human alkaline fibroblast growth factor, recombinant human hepatocyte growth factor) and recombinant human serum albumin, use can be broken away from
Ox blood serum is carrying out cell cryopreservation, and three kinds of recombinant growth factors of interpolation compensate for being taken with recombinant human serum albumin merely well
For the relevant issues that Ox blood serum brings.
5th, the cell type being suitable for
From PBMC cell, MSC cell, CIK cell, NK cell, 293 cells, HUVEC cell, fibroblast, people ES
Cell, people iPS cell amount to 9 kinds of different types of cells to be tested.
From the 3rd group of best frozen reagent of frozen effect.
Using being tested with the 4th step identical test method.
Result of the test is shown in Table 5.
Table 5 is for the frozen effect of different types of cell cryopreservation reagent
| Cell Name | Cell recovery rate after one week |
| PBMC cell | 90% |
| MSC cell | 98% |
| CIK cell | 96% |
| NK cell | 90% |
| 293 cells | 97% |
| HUVEC cell | 89% |
| Fibroblast | 96% |
| People's ES cell | 85% |
| People's iPS cell | 87% |
As shown in Table 5：For different types of cell, frozen one week using the cell cryopreservation reagent of the present invention, cell is multiple
What Soviet Union's rate was minimum can also reach 85%.
Above result of the test explanation：The cell type that the cell cryopreservation reagent of the present invention is suitable for is not limited.
It should be noted that above-described embodiment the invention is not limited in any way, all employing equivalents or equivalent change
The technical scheme obtained by the mode that changes, all falls within protection scope of the present invention.
Claims (1)
1. a kind of new cell cryopreservation reagent, it is characterised in that each constituent and consumption are respectively：
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106900695A (en) * | 2017-04-01 | 2017-06-30 | 北京焕生汇生物技术研究院 | A kind of mankind's inductive pluripotent stem cells are special to freeze reagent and cryopreservation methods |
| CN107006453A (en) * | 2017-06-07 | 2017-08-04 | 湖南惠益森细胞基因工程有限公司 | A kind of umbilical cord mesenchymal stem cells frozen stock solution and its application method |
| CN107034195A (en) * | 2017-05-18 | 2017-08-11 | 北京焕生汇生物技术研究院 | A kind of mankind's inductive pluripotent stem cells special recovery reagent and method for resuscitation |
| CN109479871A (en) * | 2017-09-12 | 2019-03-19 | 深圳华云生物科技发展有限公司 | Frozen stock solution, preparation method, application and China's Tong Shi glue tissue cryopreservation methods |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152488A (en) * | 2014-08-15 | 2014-11-19 | 济南干细胞再生与转化医学研究院 | Construction method of human nerve stem cell bank |
-
2016
- 2016-09-26 CN CN201610851438.XA patent/CN106417252A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152488A (en) * | 2014-08-15 | 2014-11-19 | 济南干细胞再生与转化医学研究院 | Construction method of human nerve stem cell bank |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106900695A (en) * | 2017-04-01 | 2017-06-30 | 北京焕生汇生物技术研究院 | A kind of mankind's inductive pluripotent stem cells are special to freeze reagent and cryopreservation methods |
| CN107034195A (en) * | 2017-05-18 | 2017-08-11 | 北京焕生汇生物技术研究院 | A kind of mankind's inductive pluripotent stem cells special recovery reagent and method for resuscitation |
| CN107034195B (en) * | 2017-05-18 | 2021-02-09 | 焕生汇生物基因技术(北京)有限公司 | Resuscitation reagent special for human induced pluripotent stem cells and resuscitation method |
| CN107006453A (en) * | 2017-06-07 | 2017-08-04 | 湖南惠益森细胞基因工程有限公司 | A kind of umbilical cord mesenchymal stem cells frozen stock solution and its application method |
| CN109479871A (en) * | 2017-09-12 | 2019-03-19 | 深圳华云生物科技发展有限公司 | Frozen stock solution, preparation method, application and China's Tong Shi glue tissue cryopreservation methods |
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Application publication date: 20170222 |