CN108157351A - A kind of candidate stem cell cryopreservation methods - Google Patents
A kind of candidate stem cell cryopreservation methods Download PDFInfo
- Publication number
- CN108157351A CN108157351A CN201711420682.1A CN201711420682A CN108157351A CN 108157351 A CN108157351 A CN 108157351A CN 201711420682 A CN201711420682 A CN 201711420682A CN 108157351 A CN108157351 A CN 108157351A
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- China
- Prior art keywords
- stem cell
- sealing container
- candidate stem
- syringe
- reagent
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- 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.)
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Classifications
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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
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
-
- 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
Abstract
The invention discloses a kind of candidate stem cell cryopreservation methods, including rubber stopper, multiple hollow rubber balls and sealing container；An opening is provided at the top of sealing container, rubber stopper is provided in opening, multiple hollow rubber balls are set to sealed container interior, and are connected between each hollow rubber ball, and the diameter of hollow rubber ball is more than or equal to 2cm；The first step, prepares the syringe after a disinfection, and the pin hole of syringe is inserted into rubber stopper, is exhausted the air in sealing container using syringe；Second step, takes candidate stem cell protective agent, candidate stem cell suspension or bone marrow fluid, and volume ratio is 12:6；Candidate stem cell suspension or bone marrow fluid with candidate stem cell protective agent are uniformly mixed, are injected into sealing container by syringe, gently rock sealing container 23 minutes；Third walks, and the air that prior high-temperature process is crossed is injected into sealing container using syringe.
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 group, have it is long-term self more
New ability and the potential for being divided into all kinds of mature blood cells.It is research history longest and one kind the most deep is thin into soma
Born of the same parents to study of various stem cell, including tumor stem cell, have great importance.The mature cell service life in hematological system
It is extremely short, therefore in people in life, candidate stem cell needs each according to the in due course supplemental blood system of the psychological need of body
Mature cell component.Simultaneously under the stress situations such as damage, inflammation, candidate stem cell also plays adjusting and maintains internal blood
The role of the physiological equilibrium of each cellular component of system.Candidate stem cell is clinically using extremely wide, candidate stem cell jelly
It is to ensure one of successful key technology of hematopoietic stem cell transplantation to deposit, thus its to freeze mode particularly important.Clinic Hematopoietic Stem at present
Cell cryopreservation has -196 DEG C of liquid nitrogen Programmed freezings preservations and -80 DEG C of non-Programmed freezings to preserve, the former can preserve for a long time and cell damages
Wound is few, can generally preserve 23~25 years, but it is complicated for operation, equipment is expensive, there is cell agglutination phenomenon after defrosting, the latter can preserve dry
Cell 1~2 year, operation relative ease, expense are low, but preservation cell stage and cell activity are limited.Explore a kind of operation letter
Just stem cell cryopreserving method, quick, of low cost and longer the holding time, storage, application for clinical candidate stem cell
And hematopoietic stem cell transplantation is successfully of great significance.In the freezing of candidate stem cell, use freeze agent and method is non-
It is often important.There are dimethyl sulfoxide (DMSO) (calling DMSO in the following text), CP-1, hydroxyl second applied to the protective agent of -80 DEG C of Cord blood stem cells at present
Base starch, RPMI1640 culture mediums, human serum albumin etc., different protectant formulas and application method differ greatly, to dry
The preservation effect of cell is with the holding time there is also difference, and the survival rate that majority preserves cell is 80% or so, stem cell safety
It is difficult to ensure that, also there are shortcomings complicated for operation for some cryopreservation methods.
Invention content
The purpose of the present invention is to provide a kind of freezing and storing method of candidate stem cell, cell survival rate higher, this hairs
It is bright to overcome traditional technology prejudice.
In order to achieve the goal above, technical scheme is as follows：Candidate stem cell cryopreservation methods, it is characterised in that：
Including rubber stopper, multiple hollow rubber balls and sealing container；
An opening is provided at the top of sealing container, rubber stopper is provided in opening, multiple hollow rubber balls are set to close
It seals inside container, and is connected between each hollow rubber ball, the diameter of hollow rubber ball is more than or equal to 2cm；Hollow rubber ball
Surface sets multiple holes；
The first step prepares the syringe after a disinfection, and the pin hole of syringe is inserted into rubber stopper, will be sealed using syringe
Air in container exhausts；
Second step takes candidate stem cell protective agent, candidate stem cell suspension or bone marrow fluid, volume ratio 1-2:6；By hematopoiesis
Stem cell suspension or bone marrow fluid are uniformly mixed with candidate stem cell protective agent, are injected into sealing container by syringe, gently
Rock sealing container 2-3 minutes；
Third walks, and the air that prior high-temperature process is crossed is injected into sealing container using syringe so that sealing container
Interior pressure is between 2.0-3.0 atmospheric pressure；Then sealing container is put into -80 DEG C of refrigerators to preserve 12 hours；
The candidate stem cell protective agent is made of reagent A and reagent B, and the volume ratio of reagent A and reagent B are 2：3；
The reagent A is made of dimethyl sulfoxide (DMSO), RPMI1640 aseptic culture mediums, hydroxyethyl starch, and solvent is physiology salt
Water, dimethyl sulfoxide (DMSO), RPMI1640 aseptic culture mediums, hydroxyethyl starch mass percent be 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 solution, the body of the 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.
Dimethyl sulfoxide (DMSO) (DMSO) has highly polar, higher boiling, the characteristic that thermal stability is good, miscible with water, can be dissolved in second
Most of organic matters such as alcohol, propyl alcohol, benzene and chloroform, are known as " alembroth ".
Compared with prior art, beneficial effects of the present invention：The method of disinfecting air is filled with using sealing container, is led to
It crosses experiment and finds that granulocyte-macrophage colony-producing units CFU-GM, cell viability, the detection of CD34+ cell recoveries are apparent
Higher than conventional method preservation effect, reason is existing candidate stem cell protective agent in refrigerating process compared to candidate stem cell
What is freezed for suspension hurries up, and about the cell in Millisecond, candidate stem cell suspension, which also compares to be active in, is slower than protection
It is easily reduced during agent freezing with protective agent collision friction so as to cause cell viability, CD34+ and CFU-GM numerical value drop
Low, the present invention overcomes technology prejudice, and the air of high-temperature sterilization (after cooling) is injected sealing container and a part is filled in
In hollow rubber ball, when candidate stem cell suspension and protective agent enter in sealing container, air can enter candidate stem cell
In suspension and protective agent, although candidate stem cell is slower than the speed of protective agent freezing due to being full of in this way in refrigerating process
A large amount of air can be very good the frictional impact between protection candidate stem cell and protective agent.
Description of the drawings
Fig. 1 is the structure diagram of sealing container.
Wherein 1 is rubber stopper；2 be hollow rubber ball；3 be sealing container.
Specific embodiment
The invention will be further described below in conjunction with the accompanying drawings.
Embodiment：Candidate stem cell freezes new method, including rubber stopper, multiple hollow rubber balls and sealing container；
An opening is provided at the top of sealing container, rubber stopper is provided in opening, multiple hollow rubber balls are set to close
It seals inside container, and is connected between each hollow rubber ball, the diameter of hollow rubber ball is equal to 2cm；
The first step prepares the syringe after a disinfection, and the pin hole of syringe is inserted into rubber stopper, will be sealed using syringe
Air in container exhausts；
Second step takes candidate stem cell protective agent, candidate stem cell suspension or bone marrow fluid, volume ratio 1:4；By Hematopoietic Stem
Cell suspension or bone marrow fluid are uniformly mixed with candidate stem cell protective agent, are injected into sealing container by syringe, gently
Rock sealing container 3 minutes；
Third walks, and the air that prior high-temperature process is crossed is injected into sealing container using syringe so that sealing container
Interior pressure is between 2.8 atmospheric pressure；Then sealing container is put into -80 DEG C of refrigerators to preserve 12 hours；
The candidate stem cell protective agent is made of reagent A and reagent B, and the volume ratio of reagent A and reagent B are 2：3；Institute
It states reagent A to be made of dimethyl sulfoxide (DMSO), RPMI1640 aseptic culture mediums, hydroxyethyl starch, solvent is physiological saline, and dimethyl is sub-
Sulfone, RPMI1640 aseptic culture mediums, hydroxyethyl starch mass percent be respectively 30%, 5%, 15%；The reagent B is people
Albumin solution, 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 uniformly mixed with 600ml (C groups) candidate stem cell, point
10 hermetic bags are respectively charged into 10 parts to preserve, and are tested and are used as C groups.
Experiment one is divided into 3 groups i.e. A, B, C, and every group is respectively adopted 10 hermetic bags and preserves, 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 that (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 -196 DEG C of liquid nitrogen Programmed freezings preservations and -80 DEG C of non-Programmed freezings preserve, and traditional cryopreservation methods (are matched including protective agent
Than) belong to existing known technology, it is not described in detail.But numerical value is slightly not in each information paper of proportioning of traditional protection agent
Together, in order to preferably be tested, traditional cryopreservation methods (i.e. -196 DEG C of liquid nitrogen Programmed freezings of tradition preserve) are adopted in tests below
Dimethyl sulfoxide (DMSO), hydroxyethyl starch, 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 preserves) use
Dimethyl sulfoxide (DMSO) in agent, 1640, the mass percent of anticoagulant for storage of whole blood I be 10%, 5%, 4%, protective agent and candidate stem cell
The volume ratio of suspension is 1:4.
A kind of device frozen for candidate stem cell, including rubber stopper, multiple hollow rubber balls and sealing container；
An opening is provided at the top of sealing container, rubber stopper is provided in opening, multiple hollow rubber balls are set to close
It seals inside container, and is connected between each hollow rubber ball, the diameter of hollow rubber ball is more than or equal to 2cm；Hollow rubber ball
Surface multiple holes are set.
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
By above 3 groups of experiments it can be found that the cell activity of stem cell, granulocyte macrophage 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 (1)
1. a kind of candidate stem cell cryopreservation methods, it is characterised in that：Hold including rubber stopper, multiple hollow rubber balls and sealing
Device；
An opening is provided at the top of sealing container, rubber stopper is provided in opening, multiple hollow rubber balls, which are set to sealing, to be held
It inside device, and is connected between each hollow rubber ball, the diameter of hollow rubber ball is more than or equal to 2cm；Hollow rubber ball surface
Multiple holes are set；
The first step prepares the syringe after a disinfection, and the pin hole of syringe is inserted into rubber stopper, using syringe by sealing container
Interior air exhausts；
Second step takes candidate stem cell protective agent, candidate stem cell suspension or bone marrow fluid, volume ratio 1-2:6；Hematopoietic Stem is thin
Born of the same parents' suspension or bone marrow fluid are uniformly mixed with candidate stem cell protective agent, are injected into sealing container by syringe, shaking gently
Shake sealing container 2-3 minutes；
Third walks, and the air that prior high-temperature process is crossed is injected into sealing container using syringe so that in sealing container
Pressure is between 2.0-3.0 atmospheric pressure；Then sealing container is put into -80 DEG C of refrigerators to preserve 12 hours；
The candidate stem cell protective agent is made of reagent A and reagent B, and the volume ratio of reagent A and reagent B are 2：3；
The reagent A is made of dimethyl sulfoxide (DMSO), RPMI1640 aseptic culture mediums, hydroxyethyl starch, and solvent is physiological saline, and two
Methyl sulfoxide, RPMI1640 aseptic culture mediums, hydroxyethyl starch mass percent be respectively 30%, 5%, 15%；
The reagent B is people's albumin solution, and the mass percent of human serum albumin is 10%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711420682.1A CN108157351A (en) | 2017-12-25 | 2017-12-25 | A kind of candidate stem cell cryopreservation methods |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711420682.1A CN108157351A (en) | 2017-12-25 | 2017-12-25 | A kind of candidate stem cell cryopreservation methods |
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| Publication Number | Publication Date |
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| CN108157351A true CN108157351A (en) | 2018-06-15 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201711420682.1A Pending CN108157351A (en) | 2017-12-25 | 2017-12-25 | A kind of candidate stem cell cryopreservation methods |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109845726A (en) * | 2019-02-27 | 2019-06-07 | 李冲杰 | A kind of mescenchymal stem cell Cryopreservation protective agent |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1625684A (en) * | 2002-01-30 | 2005-06-08 | 弗劳恩霍弗应用技术研究院 | Sample support for the cryoconservation of biological samples |
| CA2760175A1 (en) * | 2009-04-29 | 2010-11-04 | Biolife Solutions, Inc. | Apparatuses and compositions for cryopreservation of cellular monolayers |
| CN105394026A (en) * | 2015-10-31 | 2016-03-16 | 江苏赛尔特生物技术有限公司 | New method for cryopreserving hematopoietic stem cells |
-
2017
- 2017-12-25 CN CN201711420682.1A patent/CN108157351A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1625684A (en) * | 2002-01-30 | 2005-06-08 | 弗劳恩霍弗应用技术研究院 | Sample support for the cryoconservation of biological samples |
| CA2760175A1 (en) * | 2009-04-29 | 2010-11-04 | Biolife Solutions, Inc. | Apparatuses and compositions for cryopreservation of cellular monolayers |
| CN105394026A (en) * | 2015-10-31 | 2016-03-16 | 江苏赛尔特生物技术有限公司 | New method for cryopreserving hematopoietic stem cells |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109845726A (en) * | 2019-02-27 | 2019-06-07 | 李冲杰 | A kind of mescenchymal stem cell Cryopreservation protective agent |
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Application publication date: 20180615 |