CN103081892A - Frozen stock solution used for preserving adult stem cells for long time - Google Patents
Frozen stock solution used for preserving adult stem cells for long time Download PDFInfo
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- CN103081892A CN103081892A CN2011103309583A CN201110330958A CN103081892A CN 103081892 A CN103081892 A CN 103081892A CN 2011103309583 A CN2011103309583 A CN 2011103309583A CN 201110330958 A CN201110330958 A CN 201110330958A CN 103081892 A CN103081892 A CN 103081892A
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- adult stem
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- frozen stock
- stock solution
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Abstract
The invention relates to a frozen stock solution used for preserving adult stem cells for a long time, and a preparation method and an application thereof, specifically for preserving the adult stem cells used for tissue engineering. The frozen stock solution is prepared mainly by using epimedium and a serum substitute (Knock-out serum), can improve capacities for preventing cells from frozen damage and resisting oxidation and apoptosis after the cells are recovered, prevents the cells from contacting with heterologous animal original serum during a frozen stock process and further increases security of the adult stem cells for clinic applications.
Description
Technical field
The invention belongs to the cellular system engineering field, relate to a kind of cryopreserving liquid for the long preservation adult stem cell.
Background technology
Barren wort total chromocor (EF) is the extract of Chinese medicine, can promote propagation, migration and the oxidation resistance of stem cell.Adopt biochip technology to find that EF has remarkable rise effect to cortisone rat growth hormone (GH), growth hormone releasing hormone (GHRH) and all kinds of somatomedin such as insulin-like growth factor binding protein (IGFBP), nerve growth factor (NGF) etc.Find that naturally-aged rat (nephrasthenia syndrome model) EF can make the gene expression rejuvenation of a plurality of tissues, also make GH, the GHRH of senile rat downward and the up-regulated of IGFBP, NGF etc.Adopt the embryo mouse neural stem cell of in-vitro separation to prove that further barrenwort and extract thereof have directly short propagation, anti-apoptotic effect to stem cell.In addition, damage has repair to the Cold exposure mouse tissue antioxidation that report shows barren wort total chromocor.
At present, low temperature is preserved stem cell and is mainly relied on anti frozen liquid dimethyl sulfoxide (DMSO) (DMSO) (SIGMA, D5879)
Protection with serum.And dimethyl sulfoxide (DMSO) has damage to cell, and easily causes stem cell oxidative damage and differentiation potential to reduce in the recovery process.
Summary of the invention
Protect the problems such as composition is single in order to solve in existing cells frozen storing liquid; the present invention uses barren wort total chromocor (Xi'an grass roots biotechnology Co., Ltd; 20100806) be additive; with Knock-out serum (Gibco; 10828028) substitute conventional hyclone; improve anti-apoptosis, oxidation resistance after adult stem cell is recovered, and remove external source animal sources serum to the impact of cell.
The present invention is by the following technical solutions:
The cryopreserving liquid of long preservation adult stem cell of the present invention is taked the following methods preparation, comprises the following steps:
In dimethyl sulfoxide (DMSO): serum: DMEM medium ratio is 1: 2: 7 concentration configure base cryopreserving liquid;
The preparation of barren wort total chromocor freezing liquid: the 1mg barren wort total chromocor is added in the DMEM culture fluid of 10ml, be mixed with the mother liquor of 105ng/ml, then get mother liquor 1ul and be diluted in the above-mentioned basic cryopreserving liquid of 10ml, namely get the cryopreserving liquid that required final concentration is 10ng/ml.
Embodiment
The configuration of basis cryopreserving liquid:
Cryopreserving liquid configuration sequence: first add dimethyl sulfoxide (DMSO) in the DMEM medium, then add Knock-out serum;
Allocation ratio: DMEM medium, dimethyl sulfoxide (DMSO), serum were by 7: 1: 2 basic cryopreserving liquids of proportional arrangement;
The preparation of barren wort total chromocor freezing liquid:
The 1mg barren wort total chromocor is added in the basic cryopreserving liquid of 10ml, be mixed with the mother liquor of 105ng/ml;
Get again mother liquor 1ul and be diluted in the above-mentioned basic cryopreserving liquid of 10ml, namely get the cryopreserving liquid that required final concentration is 10ng/ml.
Cell cryopreservation:
Cell is through 1000rpm, and the centrifugal supernatant of outwelling of 10min uses the cryopreserving liquid that contains barren wort total chromocor resuspended, in the aseptic cell cryopreservation tube of packing into.
Frozen program:
Put into the programmed cell freeze box, put into the liquid nitrogen long preservation after-80 ℃ of placement 12h.
Place 30min under 4 ℃ of conditions, place 2h for-20 ℃, directly put into the liquid nitrogen long preservation after-80 ℃ of placement 16-18h.
Cell recovery:
Cryopreservation tube stirs to dissolving rapidly in 37～40 ℃ of waters after taking out from liquid nitrogen, adds the DMEM medium, and the centrifugal 10min of 1500rp abandons supernatant, puts in 37 ℃, 5%CO2 incubator and cultivates.
Platform is expected blue dyeing counting cytoactive rate (seeing Table 1).
Activity ratio after the recovery of table 1. mesenchymal stem cells MSCs
Claims (2)
1. cryopreserving liquid that is used for the long preservation adult stem cell is characterized in that containing in the cryopreserving liquid of every 100ml basis:
Barren wort total chromocor 10～20ng/ml.
2. cryopreserving liquid that is used for the long preservation adult stem cell, its basic cryopreserving liquid feature are to contain in the cryopreserving liquid of every 100ml basis:
DMEM medium 50%～70%
Dimethyl sulfoxide (DMSO) 10%
Serum is by 40%～20%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201110330958.3A CN103081892B (en) | 2011-10-27 | 2011-10-27 | Frozen stock solution used for preserving adult stem cells for long time and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110330958.3A CN103081892B (en) | 2011-10-27 | 2011-10-27 | Frozen stock solution used for preserving adult stem cells for long time and preparation method thereof |
Publications (2)
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| CN103081892A true CN103081892A (en) | 2013-05-08 |
| CN103081892B CN103081892B (en) | 2014-04-09 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105532649A (en) * | 2016-02-16 | 2016-05-04 | 崔长友 | Freezing medium for long-term storage of adult stem cells and preparing method thereof |
| CN106689117A (en) * | 2017-01-03 | 2017-05-24 | 郑州汉东科技有限公司 | Application and method of isodon excisoides ketone in preparation of cell freezing medium |
| CN106982822A (en) * | 2017-05-24 | 2017-07-28 | 湖南赛诺生物科技股份有限公司 | A kind of porcine islet frozen stock solution and cryopreservation methods |
| CN110771599A (en) * | 2019-11-25 | 2020-02-11 | 深圳科学之门生物工程有限公司 | Stem cell refrigerating fluid and refrigerating method thereof |
| CN115119831A (en) * | 2022-08-03 | 2022-09-30 | 深圳知因细胞生物科技有限公司 | A cryopreservation solution for adult stem cells containing ginsenoside |
| CN115299433A (en) * | 2022-08-03 | 2022-11-08 | 深圳知因细胞生物科技有限公司 | Cryopreservation frozen stock solution for promoting growth of adult stem cells by using saponin and flavone |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105532649A (en) * | 2016-02-16 | 2016-05-04 | 崔长友 | Freezing medium for long-term storage of adult stem cells and preparing method thereof |
| CN105532649B (en) * | 2016-02-16 | 2016-09-21 | 河北佑仁生物科技有限公司 | A kind of frozen stock solution preserved for a long time for adult stem cell and preparation method thereof |
| CN108347915A (en) * | 2016-02-16 | 2018-07-31 | 李建尧 | A kind of frozen stock solution and preparation method thereof for adult stem cell long-term preservation |
| CN106689117A (en) * | 2017-01-03 | 2017-05-24 | 郑州汉东科技有限公司 | Application and method of isodon excisoides ketone in preparation of cell freezing medium |
| CN106982822A (en) * | 2017-05-24 | 2017-07-28 | 湖南赛诺生物科技股份有限公司 | A kind of porcine islet frozen stock solution and cryopreservation methods |
| CN106982822B (en) * | 2017-05-24 | 2018-01-09 | 湖南赛诺生物科技股份有限公司 | A kind of porcine islet frozen stock solution and cryopreservation methods |
| CN110771599A (en) * | 2019-11-25 | 2020-02-11 | 深圳科学之门生物工程有限公司 | Stem cell refrigerating fluid and refrigerating method thereof |
| CN115119831A (en) * | 2022-08-03 | 2022-09-30 | 深圳知因细胞生物科技有限公司 | A cryopreservation solution for adult stem cells containing ginsenoside |
| CN115299433A (en) * | 2022-08-03 | 2022-11-08 | 深圳知因细胞生物科技有限公司 | Cryopreservation frozen stock solution for promoting growth of adult stem cells by using saponin and flavone |
| CN115299433B (en) * | 2022-08-03 | 2023-10-31 | 深圳知因细胞生物科技有限公司 | Adult stem cell low-temperature preservation frozen stock solution for promoting growth by utilizing saponin and flavone |
| CN115119831B (en) * | 2022-08-03 | 2023-11-24 | 深圳知因细胞生物科技有限公司 | Low-temperature preservation solution for adult stem cells containing ginsenoside |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103081892B (en) | 2014-04-09 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| DD01 | Delivery of document by public notice |
Addressee: Beijing QMLC Stem Cell Technology Co., Ltd. Document name: Notification of Publication and of Entering the Substantive Examination Stage of the Application for Invention |
|
| C53 | Correction of patent of invention or patent application | ||
| CB02 | Change of applicant information |
Address after: 100084, C building, Tsinghua Science and technology building, No. 1 Zhongguancun East Road, Beijing, Haidian District, 301 Applicant after: Beijing QMLC Stem Cell Technology Co., Ltd. Address before: 100097 Beijing city Haidian District minzhuang Road No. 3, Tsinghua Science Park building 204 Yuquan Huigu 7 Applicant before: Beijing QMLC Stem Cell Technology Co., Ltd. |
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| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |