CN105394026B - A kind of candidate stem cell freezes new method - Google Patents
A kind of candidate stem cell freezes new method Download PDFInfo
- Publication number
- CN105394026B CN105394026B CN201510732004.3A CN201510732004A CN105394026B CN 105394026 B CN105394026 B CN 105394026B CN 201510732004 A CN201510732004 A CN 201510732004A CN 105394026 B CN105394026 B CN 105394026B
- Authority
- CN
- China
- Prior art keywords
- stem cell
- candidate stem
- protective agent
- reagent
- bone marrow
- 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.)
- Active
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
-
- 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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Abstract
New method is frozen the invention discloses a kind of candidate stem cell, the first step takes candidate stem cell protective agent, candidate stem cell suspension or bone marrow fluid, and volume ratio is 12:6；Second step, candidate stem cell suspension or bone marrow fluid are well mixed with candidate stem cell protective agent, and then emptying air, sealing is put into 80 DEG C of refrigerators and preserved 12 hours；3rd step, takes out above-mentioned candidate stem cell suspension or bone marrow fluid and candidate stem cell protective agent mixed liquor, then adds candidate stem cell protective agent, and the volume ratio of candidate stem cell protective agent and mixed liquor is 1：3, emptying air, sealing again is put into 80 DEG C of refrigerator preservations；Cryopreservation methods of the present invention are simple and easy to operate, freeze effect more preferable.
Description
Technical field
The present invention relates to a kind of candidate stem cell cryopreservation methods.
Background technology
Candidate stem cell is the adult stem cell in hematological system, is a heterogeneous colony, with it is long-term self more
New ability and the potential for being divided into all kinds of mature blood cells.It is that research history is most long and a most deep class is thin into soma
Born of the same parents, to study of various stem cell, including tumor stem cell, with great importance.The mature cell life-span in hematological system
It is extremely short, therefore in people in life, candidate stem cell need according to the in good time supplemental blood system of the psychological need of body each
Mature cell component.Simultaneously under the stress situations such as damage, inflammation, candidate stem cell also plays regulation and maintains internal blood
The role of the physiological equilibrium of each cellular component of system.
Candidate stem cell clinically applies extremely wide, and it is to ensure HSCT success that candidate stem cell, which freezes,
One of key technology, therefore its to freeze mode particularly important.Clinic candidate stem cell has frozen the program control drop of -196 DEG C of liquid nitrogen at present
Temperature is preserved and -80 DEG C of non-Programmed freezings are preserved, and the former can preserve for a long time and cellular damage is few, can typically preserve 23~25 years, but
Complex operation, equipment are expensive, have cell agglutination phenomenon after defrosting, the latter can preserve stem cell 1~2 year, its operation relative ease,
Expense is low, but preservation cell stage and cytoactive are limited.
A kind of stem cell cryopreserving method easy to operate, quick, with low cost and longer the holding time is explored, for clinic
The storage of candidate stem cell, using and HSCT success it is significant.In the freezing of candidate stem cell, make
Freeze agent and method is extremely important.The protective agent applied to -80 DEG C of Cord blood stem cells has dimethyl sulfoxide (DMSO) at present
(calling DMSO in the following text), CP-1, HES, RPMI1640 culture mediums, human serum albumin etc., different protectant formula and make
Larger with method divergence, the preservation effect and holding time to stem cell are there is also difference, and the survival rate that majority preserves cells is
80% or so, stem cell is difficult to ensure that safely, and some cryopreservation methods also have complex operation shortcoming.
The content of the invention
It is an object of the invention to provide a kind of freezing and storing method of candidate stem cell, easy to operate, preservation cost
Low, cell survival rate is higher.
In order to realize the above object technical scheme is as follows：Candidate stem cell freezes new method, and the first step takes
Candidate stem cell protective agent, candidate stem cell suspension or bone marrow fluid, volume ratio is 1-2:6；Second step, by candidate stem cell suspension
Or bone marrow fluid is well mixed with candidate stem cell protective agent, then emptying air, sealing is put into -80 DEG C of refrigerators and preserved 12 hours；
3rd step, takes out above-mentioned candidate stem cell suspension or bone marrow fluid and candidate stem cell protective agent mixed liquor, then adds and make
The volume ratio of hemocytoblast protective agent, candidate stem cell protective agent and mixed liquor is 1：3, emptying air, sealing again is put into -80
DEG C refrigerator is preserved；
Described candidate stem cell protective agent is made up of reagent A and reagent B, and reagent A and reagent B volume ratio are 2：3；
The reagent A is made up of dimethyl sulfoxide (DMSO), the aseptic culture mediums of RPMI 1640, HES, and solvent is physiology salt
Water, dimethyl sulfoxide (DMSO), the aseptic culture mediums of RPMI 1640, the mass percent of HES are respectively 30%, 5%, 15%；
The reagent B is people's albumin solution, and the mass percent of human serum albumin is 10%.
More preferred technical scheme, the body of described candidate stem cell protective agent, candidate stem cell suspension or bone marrow fluid
Product is than being 1.5:6.
Unit with upper volume is ml.
The volume ratio of candidate stem cell protective agent and mixed liquor is 1 in wherein the 3rd step：3, mixed liquor refers to second step hematopoiesis
Stem cell suspension or bone marrow fluid are well mixed obtained mixed liquor with candidate stem cell protective agent.
Dimethyl sulfoxide (DMSO) (DMSO) has good, the miscible with water characteristic of highly polar, higher boiling, heat endurance, can be dissolved in second
Most of organic matters such as alcohol, propyl alcohol, benzene and chloroform, are described as " alembroth ".
Compared with prior art, beneficial effects of the present invention：First, candidate stem cell protective agent is added in two times to be made
Hemocytoblast suspension or bone marrow fluid, are found through experiments that granulocyte-macrophage colony-producing units CFU-GM is slightly higher and conventional freeze
Deposit method, but the detection of cell viability, CD34+ cell recoveries is apparently higher than conventional method preservation effect；Second, the present invention
Store method easier can preserve candidate stem cell under conditions of -80 DEG C of non-Programmed freezings, preserve simple and convenient, be clinical
The storage application and HSCT of peripheral blood hematopoietic stem cells provide Reliable guarantee.
Embodiment
The invention will be further described below.
Embodiment：Candidate stem cell freezes new method, and the first step takes candidate stem cell protective agent, candidate stem cell suspension
Or bone marrow fluid, volume ratio is 1.5:6；Second step, candidate stem cell suspension or bone marrow fluid are mixed with candidate stem cell protective agent
Uniformly, then emptying air, sealing are put into -80 DEG C of refrigerators and preserved 12 hours；3rd step, takes out above-mentioned candidate stem cell suspension
Or bone marrow fluid and candidate stem cell protective agent mixed liquor, then add candidate stem cell protective agent, candidate stem cell protective agent
Volume ratio with mixed liquor is 1：3, emptying air, sealing again is put into -80 DEG C of refrigerator preservations；Candidate stem cell protective agent is by trying
Agent A and reagent B compositions, reagent A and reagent B volume ratio are 2：3；Reagent A is by dimethyl sulfoxide (DMSO), the sterile cultures of RPMI 1640
Base, HES composition, solvent is physiological saline, dimethyl sulfoxide (DMSO), the aseptic culture mediums of RPMI 1640, HES
Mass percent is respectively 30%, 5%, 15%；Reagent B is people's albumin solution, and the mass percent of human serum albumin is
10%.
Candidate stem cell protective agent 150ml+250ml is prepared as described above, prepares 600ml (A groups)+600ml (B groups)
+ 600ml (C groups) candidate stem cell suspension, takes above-mentioned 150ml protective agents to be well mixed with 600ml (C groups) candidate stem cell, point
10 hermetic bag preservations are respectively charged into 10 parts, tests and uses as C groups.
Experiment one is divided into 3 groups i.e. A, B, C, and every group is respectively adopted 10 hermetic bags preservations, using technical solution of the present invention and
Traditional scheme that freezes is sampled detection in different time points, and every group of data are averaged, and (i.e. every group of data are
Take the average value of 10 data), it is as a result as follows.
Cryopreservation methods of the present invention are compared as follows table with traditional candidate stem cell cryopreservation methods actual effect, and wherein tradition freezes
Method refers to that -196 DEG C of liquid nitrogen Programmed freezings are preserved and -80 DEG C of non-Programmed freezings are preserved, traditional cryopreservation methods (including protective agent is matched somebody with somebody
Than) belong to existing known technology, it is not described in detail.But numerical value slightly has not in the proportioning of traditional protection agent each information paper
Together, in order to preferably be tested, traditional cryopreservation methods (i.e. -196 DEG C of liquid nitrogen Programmed freezings of tradition are preserved) are adopted in tests below
Dimethyl sulfoxide (DMSO), HES, the mass percent of human serum albumin are 10%, 6%, 4% in protective agent；Protective agent
Volume ratio with candidate stem cell suspension is 1:4.The protection that traditional cryopreservation methods (the non-Programmed freezing of -80 DEG C of tradition is preserved) use
Dimethyl sulfoxide (DMSO) in agent, 1640, anticoagulant for storage of whole blood I mass percent be 10%, 5%, 4%, protective agent and candidate stem cell
The volume ratio of suspension is 1:4.
Table 1 is the detection (%) of different time points cell viability during freezing
Table 2 is the detection (every 10 of different time points granulocyte-macrophage colony-producing units CFU-GM during freezing
Ten thousand cells)
Table 3 is the detection (%) of different time points CD34+ cells (candidate stem cell) during freezing
3 groups of experiments more than are it can be found that cytoactive, the granulocyte macrophage of the stem cell that the method for the present invention freezes
Cell colony generation unit CFU-GM and CD34+ cell recoveries are slightly better than traditional cryopreservation methods.
Claims (2)
1. a kind of candidate stem cell freezes new method, it is characterised in that：The first step, takes candidate stem cell protective agent, Hematopoietic Stem thin
Born of the same parents' suspension or bone marrow fluid, volume ratio are 1-2:6；Second step, candidate stem cell suspension or bone marrow fluid are protected with candidate stem cell
Agent is well mixed, and then emptying air, sealing is put into -80 DEG C of refrigerators and preserved 12 hours；3rd step, takes out above-mentioned Hematopoietic Stem thin
Born of the same parents' suspension or bone marrow fluid and candidate stem cell protective agent mixed liquor, then add candidate stem cell protective agent, candidate stem cell
The volume ratio of protective agent and mixed liquor is 1：3, emptying air, sealing again is put into -80 DEG C of refrigerator preservations；
Described candidate stem cell protective agent is made up of reagent A and reagent B, and reagent A and reagent B volume ratio are 2：3；
The reagent A is made up of dimethyl sulfoxide (DMSO), the aseptic culture mediums of RPMI 1640, HES, and solvent is physiological saline,
Dimethyl sulfoxide (DMSO), the aseptic culture mediums of RPMI 1640, the mass percent of HES are respectively 30%, 5%, 15%；
The reagent B is people's albumin solution, and the mass percent of human serum albumin is 10%.
2. candidate stem cell according to claim 1 freezes new method, it is characterised in that：The first step, candidate stem cell is protected
The volume ratio for protecting agent, candidate stem cell suspension or bone marrow fluid is 1.5:6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510732004.3A CN105394026B (en) | 2015-10-31 | 2015-10-31 | A kind of candidate stem cell freezes new method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510732004.3A CN105394026B (en) | 2015-10-31 | 2015-10-31 | A kind of candidate stem cell freezes new method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105394026A CN105394026A (en) | 2016-03-16 |
| CN105394026B true CN105394026B (en) | 2017-09-29 |
Family
ID=55460079
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510732004.3A Active CN105394026B (en) | 2015-10-31 | 2015-10-31 | A kind of candidate stem cell freezes new method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105394026B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107396920A (en) * | 2017-08-31 | 2017-11-28 | 重庆金时代生物技术有限公司 | Store method during a kind of transport of candidate stem cell |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3022685A1 (en) * | 2016-03-28 | 2017-10-05 | Cook General Biotechnology Llc | Methods of cryopreservation of viable cells, compositions of viable cells and uses thereof |
| CN105941389A (en) * | 2016-05-03 | 2016-09-21 | 上海安集协康生物技术股份有限公司 | Animal derived serum-free cell freezing medium |
| CN108925548A (en) * | 2017-05-24 | 2018-12-04 | 西比曼生物科技（香港）有限公司 | A kind of freeze-stored cell preparation and cell recovery mode |
| CN107094753A (en) * | 2017-05-31 | 2017-08-29 | 东莞市保莱生物科技有限公司 | A kind of candidate stem cell frozen stock solution and candidate stem cell cryopreservation methods |
| CN107232182A (en) * | 2017-06-30 | 2017-10-10 | 陈印平 | A kind of mesenchymal stem cells MSCs cell cryopreservation agent |
| CN107156111A (en) * | 2017-06-30 | 2017-09-15 | 陈印平 | A kind of candidate stem cell cell cryopreservation agent |
| CN107853290A (en) * | 2017-12-13 | 2018-03-30 | 朱郎平 | A kind of candidate stem cell freezes new method |
| CN108157351A (en) * | 2017-12-25 | 2018-06-15 | 朱郎平 | A kind of candidate stem cell cryopreservation methods |
| CN113564121A (en) * | 2021-08-10 | 2021-10-29 | 合肥滴碧云生物科技有限公司 | Hematopoietic stem cell cryopreservation method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5580714A (en) * | 1995-03-08 | 1996-12-03 | Celox Laboratories, Inc. | Cryopreservation solution |
| CN103583511B (en) * | 2012-09-03 | 2015-07-29 | 四川新生命干细胞科技股份有限公司 | A kind of mesenchymal stem cell cryopreserving liquid and parenteral solution |
| CN103548814B (en) * | 2013-11-07 | 2015-04-08 | 中国人民解放军第三军医大学第一附属医院 | Non-programmable-controlled cooling cryopreservation method and protection agent for hematopoietic stem cells at minus 80DEG C |
-
2015
- 2015-10-31 CN CN201510732004.3A patent/CN105394026B/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107396920A (en) * | 2017-08-31 | 2017-11-28 | 重庆金时代生物技术有限公司 | Store method during a kind of transport of candidate stem cell |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105394026A (en) | 2016-03-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105394026B (en) | A kind of candidate stem cell freezes new method | |
| CN108207930A (en) | A kind of cocktail type cryoprotector and its application | |
| Mazur | Principles of cryobiology | |
| CN103548814B (en) | Non-programmable-controlled cooling cryopreservation method and protection agent for hematopoietic stem cells at minus 80DEG C | |
| Liu et al. | Comparing normothermic machine perfusion preservation with different perfusates on porcine livers from donors after circulatory death | |
| Pegg | The relevance of ice crystal formation for the cryopreservation of tissues and organs | |
| RU2396748C2 (en) | Medium for storage of cells | |
| CN104026118A (en) | Immunization cell frozen stock solution, preparation method and application | |
| CA2719806A1 (en) | Method, system, and apparatus for hypothermic collection, storage, transport and banking of birth tissue | |
| JP2018518965A (en) | Cryopreservation method of tumor infiltrating lymphocytes | |
| Johnson et al. | PAS‐G supports platelet reconstitution after cryopreservation in the absence of plasma | |
| Clarke et al. | Improved post-thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular-like cryopreservation solution | |
| CN105941389A (en) | Animal derived serum-free cell freezing medium | |
| CN102144630B (en) | Adding liquid for storing platelets under liquid low temperature condition and application thereof | |
| CN110839615A (en) | Cryopreservation liquid and preservation method for oocyte | |
| CN104222069A (en) | Erythroid progenitor cell cryopreservation liquid and application thereof | |
| Stylianou et al. | Novel cryoprotectant significantly improves the post-thaw recovery and quality of HSC from CB | |
| CN103004751A (en) | Hematopoietic stem cell cryopreserving method and protective agent | |
| US9005886B2 (en) | Method for cryopreservation of human spermatozoa free from seminal plasma using a fast and simple aseptic vitrification-devitrification process; portable kit for carrying out the method; and use of the same for treatment of disorders related to reproductive failures | |
| CN108157351A (en) | A kind of candidate stem cell cryopreservation methods | |
| Hubel et al. | Cryopreservation of cord blood after liquid storage | |
| JP6329468B2 (en) | Vitrification cryopreservation method of fibroblasts | |
| CN107853290A (en) | A kind of candidate stem cell freezes new method | |
| CN105613484B (en) | Megakaryoblast frozen stock solution and its application | |
| Pereira | Principles of cryopreservation and applicabilities in intestinal organoids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Tian Liping Inventor before: Qin Ling |
|
| CB03 | Change of inventor or designer information | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20170811 Address after: 100096 Beijing City, Huilongguan Changping District small town north of the village Applicant after: Beijing army joint Stem Cell Biotechnology Co., Ltd. Address before: 225300 Jiangsu city of Taizhou province China pharmaceutical Road East, Xinyang City Road on the north side of G25 7 storey building on the west side Applicant before: JIANGSU SAIERT BIOTECHNOLOGY CO., LTD. |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |