CN106386783A - A mesenchymal stem cell cryoprotectant - Google Patents
A mesenchymal stem cell cryoprotectant Download PDFInfo
- Publication number
- CN106386783A CN106386783A CN201610483019.5A CN201610483019A CN106386783A CN 106386783 A CN106386783 A CN 106386783A CN 201610483019 A CN201610483019 A CN 201610483019A CN 106386783 A CN106386783 A CN 106386783A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- mesenchymal stem
- growth factor
- human albumin
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A mesenchymal stem cell cryoprotectant is disclosed. Raw materials of the cryoprotectant includes, by volume, 65.5-75.4% of 1640 culture liquid, 9.8-10.1% of dimethyl sulfoxide, 9.8-11.2% of human albumin, 9.9-10.8% of hydroxyethyl starch and 0.5-1.5% of a fibroblast growth factor. The cryoprotectant is more suitable for cryopreservation of mesenchymal stem cells, the anchoring rate of the mesenchymal stem cells after resuscitation reaches 90% or above, dead cells are few, and the quantity of cells obtained after culturing for 7 days is 2*10<7> per flask, thus greatly increasing the quantity of cells. During cell phenotype detection, CD44<+> is 99% or above and CD105<+> is 99% or above.
Description
Technical field
The present invention relates to frozen stock solution technical field, more particularly, to a kind of mesenchymal stem cell cryopreserving liquid.
Background technology
The using method of stem cell cryopreserving liquid is at present：After cell culture reaches certain density, need to freeze
It is saved in the activity keeping cell in liquid nitrogen, can normally cultivate after in the future using recovery, therefore exist
Need when frozen to add the injury fall completely with to cell that a certain proportion of frozen stock solution keeps cell when frozen
Extremely minimum.
Cells frozen storing liquid of the prior art is often 9 by hyclone and dimethyl sulfoxide by volume：1 mixes
Conjunction obtains, and due to using zoogenous hyclone, leading to mix inhuman source material in cells frozen storing liquid, makes
Become residual or pollute, and existing cells frozen storing liquid stem cell in stem cell follow-up recovery incubation
Adherent rate be 80% about, dead cell is more, and the final cell number of acquisition is 1.2 × 107About/bottle, become
This is also higher.
Content of the invention
The technical problem being existed based on background technology, the present invention proposes a kind of mesenchymal stem cell cryopreserving liquid,
It is more suitable for the frozen of mescenchymal stem cell, after mescenchymal stem cell is recovered again, adherent rate reaches more than 90%,
Dead cell is less, and culture harvests the cell number obtaining after 7 days be 2 × 107/ bottle, substantially increases the number of cell
Amount；When detecting cell phenotype simultaneously, CD44+Reach more than 99%, CD105+Reach more than 99%.
A kind of mesenchymal stem cell cryopreserving liquid proposed by the present invention, its raw material is included by percent by volume：1640
Culture fluid 65.5～75.4%, dimethyl sulfoxide 9.8～10.1%, human albumin 9.8～11.2%, ethoxy
Starch 9.9～10.8%, fibroblast growth factor 0.5～1.5%.
In specific embodiment, the percent by volume of RPMI-1640 can for 65.5%, 66%, 66.5%,
67%th, 67.5%, 68%, 68.5%, 69%, 69.5%, 70%, 70.5%, 71%, 71.5%, 72%,
72.5%th, 73%, 73.5%, 74%, 74.5%, 75%, 75.4%, the percent by volume of dimethyl sulfoxide can
Think 9.8%, 9.9%, 10.0%, 10.1%, the percent by volume of human albumin can for 9.8%, 9.9%,
10%th, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%,
11%th, 11.1%, 11.2%, the percent by volume of hetastarch can for 9.9%, 10%, 10.1%, 10.2%,
10.3%th, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, the volume hundred of fibroblast growth factor
Point ratio can for 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%,
1.5%.
Preferably, its raw material is included by percent by volume：RPMI-1640 68～72%, dimethyl sulfoxide 9.9～
10.0%, human albumin 10～11%, hetastarch 10.0～10.2%, fibroblast growth factor
0.8～1.2%.
Preferably, human albumin, hetastarch, the weight volume ratio of fibroblast growth factor are 10.2：
10：1.1.
Preferably, human albumin, hetastarch, the weight volume ratio of fibroblast growth factor are 10.8：
10.1：1.
Preferably, human albumin, hetastarch, the weight volume ratio of fibroblast growth factor are 10.5：
10.2：0.9.
Preferably, its raw material is included by percent by volume：RPMI-1640 69%, dimethyl sulfoxide 10%,
Human albumin 10%, hetastarch 10%, desmocyte growth factor-21 %.
The present invention adopts RPMI-1640, human albumin, hetastarch, fibroblast growth factor
Cooperate, mescenchymal stem cell can be nourished, promote mescenchymal stem cell to be in semidormancy state, fill between making
The energy expenditure of matter stem cell maintains relatively low level, promotes mescenchymal stem cell to keep cell in liquid nitrogen
Activity, be allowed to recovery after can normally be cultivated；Wherein RPMI-1640, human albumin, hydroxyl
Hydroxyethyl starch provides nutritional labeling for frozen mescenchymal stem cell, and human albumin, hetastarch and two
Methyl sulfoxide cooperates and can reduce intracellular aqueous fusion point, maintains intraor extracellular osmotic balance, becomes fiber finer
The intracellular growth factor can make mescenchymal stem cell slowly be repaired in frozen period, maintains cytoactive.To sum up
Described, the present invention is more suitable for the frozen of mescenchymal stem cell compared to frozen stock solution of the prior art.
Carry out the frozen of mescenchymal stem cell using the present invention, adherent rate after mescenchymal stem cell is recovered again
Reach more than 90%, dead cell is less, culture harvests the cell number obtaining after 7 days be 2 × 107/ bottle, significantly
Improve the quantity of cell；When detecting cell phenotype simultaneously, CD44+Reach more than 99%, CD105+Reach
More than 99%.
Brief description
Fig. 1 is that the embodiment of the present invention 5 gained mesenchymal stem cell cryopreserving liquid carries out cell jelly with existing frozen stock solution
Deposit, recover, recovery stem cell adherent rate comparison diagram.
Fig. 2 is that the embodiment of the present invention 5 gained mesenchymal stem cell cryopreserving liquid carries out cell jelly with existing frozen stock solution
Deposit, recover, recovery Stem Cell Activity comparison diagram.
Fig. 3 is that the embodiment of the present invention 5 gained mesenchymal stem cell cryopreserving liquid carries out cell jelly with existing frozen stock solution
Deposit, recover, recovery stem Cell Phenotypic detection figure.
Specific embodiment
Below, by specific embodiment, technical scheme is described in detail.
Embodiment 1
A kind of mesenchymal stem cell cryopreserving liquid proposed by the present invention, its raw material is included by percent by volume：1640
Culture fluid 67.6%, dimethyl sulfoxide 9.8%, human albumin 11.2%, hetastarch 9.9%, becomes fine
Dimension growth factor-21 .5%.
Embodiment 2
A kind of mesenchymal stem cell cryopreserving liquid proposed by the present invention, its raw material is included by percent by volume：1640
Culture fluid 68.8%, dimethyl sulfoxide 10.1%, human albumin 9.8%, hetastarch 10.8%, becomes fine
Dimension cell growth factor 0.5%.
Embodiment 3
A kind of mesenchymal stem cell cryopreserving liquid proposed by the present invention, its raw material is included by percent by volume：1640
Culture fluid 67.9%, dimethyl sulfoxide 9.9%, human albumin 11%, hetastarch 10%, becomes fiber
Growth factor-21 .2%.
Embodiment 4
A kind of mesenchymal stem cell cryopreserving liquid proposed by the present invention, its raw material is included by percent by volume：1640
Culture fluid 69%, dimethyl sulfoxide 10%, human albumin 10%, hetastarch 10.2%, becomes fiber
Cell growth factor 0.8%.
Embodiment 5
A kind of mesenchymal stem cell cryopreserving liquid proposed by the present invention, its raw material is included by percent by volume：1640
Culture fluid 69%, dimethyl sulfoxide 10%, human albumin 10%, hetastarch 10%, becomes fiber finer
The intracellular growth factor 1%.
Embodiment 5 gained mesenchymal stem cell cryopreserving liquid is carried out cell cryopreservation and recovery cell is detected,
Concrete operations are as follows：
1st, by embodiment 5 gained mesenchymal stem cell cryopreserving liquid add to stem cell resuspended after, be saved in cryopreservation tube
In, 5 × 106/ pipe, the mode of programmed cooling is frozen, transfers in liquid nitrogen pipe and preserve after 24h；
2nd, take out a cryopreservation tube containing freeze-stored cell, after 37 DEG C of water-baths are redissolved, be added in culture bottle, then plus
Enter 30mL stem cell special culture media, place 37 °C of O2Cultivate in incubator；
3rd, culture fluid in culture bottle, the adherent situation of observation of cell are changed within 2nd；
4th, pass within 4th, detect cell number and activity；
5th, attached cell, detection cell number and activity are harvested within 7th, and stem Cell Phenotypic.
As Figure 1-3, Fig. 1 is recovery stem cell adherent rate comparison diagram to its result, and Fig. 2 is recovery stem cell
Active comparison diagram, Fig. 3 detects figure for recovery stem Cell Phenotypic.From Fig. 1 and Fig. 2：Using the present invention
Embodiment 5 gained mesenchymal stem cell cryopreserving liquid carry out frozen, recovery after, the activity of cell and adherent rate remote
Far above existing frozen stock solution, its adherent rate and activity all can reach more than 90%；And as shown in Figure 3：Adopt
With the embodiment of the present invention 5 gained mesenchymal stem cell cryopreserving liquid carry out frozen, recovery after, in cell phenotype
CD44+Reach more than 99%, CD105+Reach more than 99%.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention not office
Be limited to this, any those familiar with the art the invention discloses technical scope in, according to this
The technical scheme of invention and its inventive concept in addition equivalent or change, all should cover the protection in the present invention
Within the scope of.
Claims (6)
1. a kind of mesenchymal stem cell cryopreserving liquid is it is characterised in that its raw material is included by percent by volume：1640
Culture fluid 65.5～75.4%, dimethyl sulfoxide 9.8～10.1%, human albumin 9.8～11.2%, ethoxy
Starch 9.9～10.8%, fibroblast growth factor 0.5～1.5%.
2. according to claim 1 mesenchymal stem cell cryopreserving liquid it is characterised in that its raw material press volume
Percentage ratio includes：RPMI-1640 68～72%, dimethyl sulfoxide 9.9～10.0%, human albumin 10～
11%, hetastarch 10.0～10.2%, fibroblast growth factor 0.8～1.2%.
3. according to claim 1 mesenchymal stem cell cryopreserving liquid it is characterised in that human albumin,
Hetastarch, the weight volume ratio of fibroblast growth factor are 10.2：10：1.1.
4. according to claim 1 mesenchymal stem cell cryopreserving liquid it is characterised in that human albumin,
Hetastarch, the weight volume ratio of fibroblast growth factor are 10.8：10.1：1.
5. according to claim 1 mesenchymal stem cell cryopreserving liquid it is characterised in that human albumin,
Hetastarch, the weight volume ratio of fibroblast growth factor are 10.5：10.2：0.9.
6. according to claim 1 mesenchymal stem cell cryopreserving liquid it is characterised in that its raw material press volume
Percentage ratio includes：RPMI-1640 69%, dimethyl sulfoxide 10%, human albumin 10%, and ethoxy forms sediment
Powder 10%, desmocyte growth factor-21 %.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610483019.5A CN106386783A (en) | 2016-06-24 | 2016-06-24 | A mesenchymal stem cell cryoprotectant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610483019.5A CN106386783A (en) | 2016-06-24 | 2016-06-24 | A mesenchymal stem cell cryoprotectant |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106386783A true CN106386783A (en) | 2017-02-15 |
Family
ID=58005904
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610483019.5A Pending CN106386783A (en) | 2016-06-24 | 2016-06-24 | A mesenchymal stem cell cryoprotectant |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106386783A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106465710A (en) * | 2016-08-19 | 2017-03-01 | 杭州易文赛生物技术有限公司 | Fatty tissue frozen stock solution and fatty tissue cryopreservation methods |
| 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 |
| CN107258769A (en) * | 2017-06-30 | 2017-10-20 | 苏州北开生化设备有限公司 | A kind of cryopreservation methods of the Vero cells of high viability |
| CN107683849A (en) * | 2017-10-12 | 2018-02-13 | 北京臻惠康生物科技有限公司 | A kind of mesenchymal stem cell cryopreserving liquid, preparation method and its application method of directly intravenous applications |
| CN107996559A (en) * | 2018-01-03 | 2018-05-08 | 吉林省拓华生物科技有限公司 | A kind of neural stem cell frozen stock solution and its application method |
| CN109090100A (en) * | 2018-08-27 | 2018-12-28 | 深圳市浊安认证生物技术有限公司 | A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method |
| CN109845726A (en) * | 2019-02-27 | 2019-06-07 | 李冲杰 | A kind of mescenchymal stem cell Cryopreservation protective agent |
| CN112450205A (en) * | 2020-11-04 | 2021-03-09 | 深圳盛皓生物科技有限公司 | Mesenchymal stem cell storage solution |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2016
- 2016-06-24 CN CN201610483019.5A patent/CN106386783A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106465710A (en) * | 2016-08-19 | 2017-03-01 | 杭州易文赛生物技术有限公司 | Fatty tissue frozen stock solution and fatty tissue cryopreservation methods |
| CN106465710B (en) * | 2016-08-19 | 2020-04-14 | 杭州易文赛生物技术有限公司 | Adipose tissue cryopreservation liquid and adipose tissue cryopreservation method |
| 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 |
| CN107258769A (en) * | 2017-06-30 | 2017-10-20 | 苏州北开生化设备有限公司 | A kind of cryopreservation methods of the Vero cells of high viability |
| CN107683849A (en) * | 2017-10-12 | 2018-02-13 | 北京臻惠康生物科技有限公司 | A kind of mesenchymal stem cell cryopreserving liquid, preparation method and its application method of directly intravenous applications |
| CN107996559A (en) * | 2018-01-03 | 2018-05-08 | 吉林省拓华生物科技有限公司 | A kind of neural stem cell frozen stock solution and its application method |
| CN107996559B (en) * | 2018-01-03 | 2021-03-16 | 吉林省拓华生物科技有限公司 | Neural stem cell cryopreservation liquid and using method thereof |
| CN109090100A (en) * | 2018-08-27 | 2018-12-28 | 深圳市浊安认证生物技术有限公司 | A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method |
| CN109845726A (en) * | 2019-02-27 | 2019-06-07 | 李冲杰 | A kind of mescenchymal stem cell Cryopreservation protective agent |
| CN112450205A (en) * | 2020-11-04 | 2021-03-09 | 深圳盛皓生物科技有限公司 | Mesenchymal stem cell storage solution |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106386783A (en) | A mesenchymal stem cell cryoprotectant | |
| IL273159B2 (en) | Culture medium composition, and method for culturing cell or tissue using said composition | |
| PH12015501465A1 (en) | Culturing of human embryonic stem cells at the air-liquid interface for differentiation into pancreatic endocrine cells | |
| RU2016121409A (en) | SUSPENDING AND CLUSTERING OF HUMAN PLURIPOTENT STEM CELLS WITH THE PURPOSE OF THEIR DIFFERENTIATION IN PANCREATIC ENDOCRINE CELLS | |
| CN106465710A (en) | Fatty tissue frozen stock solution and fatty tissue cryopreservation methods | |
| CN101474426B (en) | Vascular matrix for removing cells in vascular tissue and preparation method thereof | |
| CN103849576B (en) | One strain has the recombinant Saccharomyces cerevisiae bacterial strain of stress tolerance | |
| EP3202894A1 (en) | Method for producing three-dimensional cell aggregates | |
| CN104719285B (en) | Ovary room-temperature extender and store method | |
| CN104472474A (en) | Human adipose tissue-derived stromal cell frozen stock solution | |
| CN106011054A (en) | Method for differentiation of murine hippocampal neural stem cells into chondrocytes | |
| CN103773734B (en) | There is the preparation method of the cell sheet engineering that bone occurs to differentiation and blood vessel | |
| Welter et al. | Assessing adipogenic potential of mesenchymal stem cells: a rapid three‐dimensional culture screening technique | |
| WO2011011660A3 (en) | Drain down and re-feed of microcarrier bioreactor | |
| Tisdell et al. | The cost of production of giant clam seed Tridacna gigas | |
| KR20150085244A (en) | Microalgae Harvest Apparatus | |
| CN103305418A (en) | Stem cell isolated culturing device | |
| CN204937970U (en) | Store the holding vessel of MBS raw material | |
| CN105670990A (en) | Preparation method and application of tissue engineering material for promoting directional differentiation of mesenchymal stem cells | |
| CN203417494U (en) | Perfume storage tank | |
| CN106508891A (en) | Construction method of kidney cell line of Schizothorax taliensis | |
| CN202643694U (en) | Anti-fog culture dish | |
| WO2019075166A3 (en) | Hollow microcarrier for shear-free culture of adherent cells in bioreactors | |
| Kopecká et al. | Storage of Pitching Yeast in Brewery | |
| CN106804341A (en) | A kind of flue-cured tobacco transplanting tobacco seedling growth cup cover and its application method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170215 |
|
| RJ01 | Rejection of invention patent application after publication |