CN108056095A - A kind of fat mesenchymal stem cell transport protection liquid and its application - Google Patents
A kind of fat mesenchymal stem cell transport protection liquid and its application Download PDFInfo
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- CN108056095A CN108056095A CN201711466774.3A CN201711466774A CN108056095A CN 108056095 A CN108056095 A CN 108056095A CN 201711466774 A CN201711466774 A CN 201711466774A CN 108056095 A CN108056095 A CN 108056095A
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- mesenchymal stem
- stem cell
- protection liquid
- transport protection
- fat mesenchymal
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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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
Abstract
The present invention relates to biological technical fields, provide a kind of stem cell transport protection liquid, and in particular, to a kind of fat mesenchymal stem cell transport protection liquid of suitable long distance transportation.Stem cell of the present invention transport protection liquid is made of the μ g/mL streptomysins of 10%~30%DMSO (dimethyl sulfoxide (DMSO)), 50 μ g/mL~150,50%~80%DMEM, 8%~20%HES (hydroxyethyl starch).It is an advantage of the invention that the stability and cytoactive of cell can be kept, convenient for preservation and long distance transportation.
Description
Technical field
The invention belongs to biological technical fields, are related to a kind of stem cell transport protection liquid, and in particular, to one kind is suitble to remote
The fat mesenchymal stem cell transport protection liquid of distance transport.
Background technology
Fat mesenchymal stem cell (Adipose-derived Mesenchymal Stem Cells, ADSCs) is source
In the stem cell of adipose tissue, as other adult stem cells there is the ability of self-renewing and Multidirectional Differentiation, lured suitable
Osteoblast, adipocyte, cartilage cell etc. can be divided under the conditions of leading, is had for regenerative medicine and cell therapy research
There is important value.
Mesenchymal stem cell (bone marrow mesenchymal stem cells, BMSCs) is because of its skeletonization energy
Power, it has also become generally acknowledged bone seeding cell source.But mesenchymal stem cell is because extraction is not easy, marrow extracted amount is limited, is carried
It fetches low reason, causes its application often limited.And fat mesenchymal stem cell (adipose-derived
Mesenchymal stem cells, ADSCs) in extraction process to small for bulk damage, and can be to be abandoned in liposuction fat
Fat suspensions of tissues is important sources, therefore has become hot spot to the research of fat mesenchymal stem cell.Pass through research in recent years
It was found that mescenchymal stem cell can be separated by fat it is a large amount of obtain, and can to osteoblast, chondroblast, muscle cell,
The directional inductions such as cardiac muscle cell, adipocyte convert, and are with a wide range of applications in terms of organizational project and cell therapy, inhale
The concern of more and more researchers is drawn.
2001, Zuk etc. was separated from human fat tissue and is obtained the cell with Multidirectional Differentiation ability, according to domestic and international
Result of study is referred to as fat mesenchymal stem cell.It is proved with vitro study in body, this cell, which has, is divided into fat
The ability of the various kinds of cell such as fat cell, cartilage cell, osteoblast, sarcoblast, vascular endothelial cell and nerve cell, has
Good potential applicability in clinical practice.However, in the treatment use of clinical practice, fat mesenchymal stem cell need to be in stringent GMP
Clinical treatment can just be entered after laboratory treatment, can not realize instant infusion, transition period need to inevitably use can quilt
The protection liquid of clinical infusion is preserved.When the suitable culture environment of cell detachment enters in protection liquid, new environment necessarily makes
Cell generates adaptive change, to reach preferable therapeutic effect, it is necessary to assure the stability and cell of stem cell in protection liquid
Activity, particularly during long distance transportation.
The research of related human adipose mesenchymal stem cells transport protection liquid is very few at present, and therefore, there is an urgent need to develop one kind
Fat mesenchymal stem cell transport protection liquid, realizes that fat mesenchymal stem cell can keep the steady of cell in long distance transportation
Qualitative and cytoactive so that stem cell can effectively be applied.
The content of the invention
It is an object of the invention to provide a kind of fat mesenchymal stem cell transport protection liquid, can solve it is existing fat between
The shortcomings of mesenchymal stem cells transport protection liquid can not keep cell stability and cytoactive in long distance transportation.
One aspect of the present invention provides a kind of fat mesenchymal stem cell transport protection liquid, which does carefully
Born of the same parents transport protection liquid by the μ g/mL streptomysins of 10%~30%DMSO (dimethyl sulfoxide (DMSO)), 50 μ g/mL~150,50%~80%
DMEM (culture medium containing various amino acid and glucose), 8%~20%HES (hydroxyethyl starch) are formed.
In another preference, in the fat mesenchymal stem cell transport protection liquid concentration of DMSO for 10%~
20%；In a particular embodiment, the concentration of the DMSO is 20%.
In another preference, the concentration of the fat mesenchymal stem cell transport protection liquid streptomycin is 100 μ g/mL
~150 μ g/mL；In a particular embodiment, the concentration of the streptomysin is 100 μ g/mL.
In another preference, in the fat mesenchymal stem cell transport protection liquid concentration of DMEM for 70%~
80%；In a particular embodiment, the concentration of the DMEM is 70%.
In another preference, the concentration of HES is 10%~20% in the fat mesenchymal stem cell transport protection liquid；
In a particular embodiment, the concentration of the HES is 12%.
In another preference, the fat mesenchymal stem cell is to adjust to be added to fat mesenchymal and do in a manner of resuspension
In cell transport protection liquid, the concentration of human adipose mesenchymal stem cells is 6 × 10 in transport protection liquid5~7 × 105A/ml.Tool
Body, comprise the following steps：It takes the logarithm the 3rd fat subsitutes mescenchymal stem cell (ADMSCs) in growth period, 1000r/min centrifugations
5min abandons supernatant, adds in the transport protection liquid that 4 DEG C of precoolings freeze, blows and beats cell precipitation, cell is made uniformly to divide in liquid is protected
Cloth, adjustment viable cell concentrations are 6 × 105~7 × 105A/ml, dispenses and is preserved into cryopreservation tube.
Preferably, present invention additionally comprises cryopreservation tube is placed in -20 DEG C~-80 DEG C freezing storing box to preserve；It is preferred that be placed in-
It is preserved in 80 DEG C of freezing storing box.
In another preference, the assay method of the viable cell concentrations comprises the following steps：Take a little cell suspension by
9：1 and 0.4% trypan blue solution mixing, the dead cell for being dyed to blueness and the viable count of anti-dye are counted under microscope.
The present invention in a specific embodiment, additionally provides the preparation method of the human adipose mesenchymal stem cells, including
Following steps：
(1) 5~10g of block adipose tissue is collected under aseptic condition, is immersed in the DMEM containing antibiotic.Remove naked eyes
Visible vascular tissue rinses 3 times to remove haemocyte and other impurities repeatedly with the DMEM containing antibiotic, and sterile scissors will
Tissue block shreds as far as possible.
(2) adipose tissue is placed in 0.1% (mass concentration) type i collagen enzyme of double volume, in 37 DEG C of constant water bath box
Vibration digestion 60min, 1500r/min centrifugation 10min, abandons supernatant and umbrella organisations, adds in the complete culture solution ((hydroxyl containing 12%HES
Hydroxyethyl starch), 100 μ g/mL streptomysins, 70%DMEM) into single cell suspension, the filtering of 200 mesh cell sieves connects piping and druming cell precipitation
Kind is in T25 blake bottles, 37 DEG C, 5%CO2It is cultivated in constant incubator.
(3) liquid is changed after 3d for the first time, changes liquid 1 time every 2d later.When cell fusion is up to 70%~80%, with 0.25%
(mass concentration) pancreatin digests, and collects cell, and 1000r/min centrifuges 5min, and PBS is washed 3 times (1000r/min × 10min), centrifuges
Terminate, supernatant is abandoned in suction, obtains human adipose mesenchymal stem cells.
The present invention in a particular embodiment, additionally provides the fat mesenchymal stem cell transport protection liquid and is freezing
Deposit the application in human adipose mesenchymal stem cells and fat mesenchymal stem cell transport.
Advantageous effect of the invention
The transport protection liquid of different component of the present invention carries out food preservation test to the human adipose mesenchymal stem cells of identical source,
The survival rate of each processing group human adipose mesenchymal stem cells is counted after handling at the same time, the results show that the fortune of the present invention
Defeated protection liquid has higher Cell viability.It follows that human adipose mesenchymal stem cells transport protection liquid provided by the invention
The stability and cytoactive of cell can be improved, convenient for preservation and long distance transportation, use is safer, has great clinic
Value.
Description of the drawings
Fig. 1 show stem cell transport protection liquid treated the Cell viability of different formulations.
Specific embodiment
Embodiment of the present invention is described in detail in the following with reference to the drawings and specific embodiments, but art technology
Personnel will be understood that the following example is merely to illustrate the present invention, and should not be construed as the limitation of the present invention.In following embodiments
Experimental method, unless otherwise specified, be according to normal condition or manufacturer suggestion condition carry out.In following embodiments
Material, reagent used etc. are commercially or the conventional products of acquisition purchased in market unless otherwise specified.
1 fat mesenchymal stem cell of the present invention of embodiment transport protection liquid
(1) it is formulated
Fat mesenchymal stem cell transport protection liquid by 10%~30%DMSO (dimethyl sulfoxide (DMSO)), 50 μ g/mL~
150 μ g/mL streptomysins, 50%~80%DMEM (culture medium containing various amino acid and glucose), 8%~20%HES (hydroxyl second
Base starch) composition.
(2) preparation method
In ratio in formula, streptomysin is dissolved and is uniformly mixed with DMEM and HES, DMSO is added, is placed on ice water
To nearly 0 DEG C of frozen stock solution temperature in bath.
The preparation method of 2 human adipose mesenchymal stem cells of embodiment
The preparation method of the human adipose mesenchymal stem cells, comprises the following steps：
(1) 5~10g of block adipose tissue is collected under aseptic condition, is immersed in the DMEM containing antibiotic.Remove naked eyes
Visible vascular tissue rinses 3 times to remove haemocyte and other impurities repeatedly with the DMEM containing antibiotic, and sterile scissors will
Tissue block shreds as far as possible.
(2) adipose tissue is placed in 0.1% (mass concentration) type i collagen enzyme of double volume, in 37 DEG C of constant water bath box
Vibration digestion 60min, 1500r/min centrifugation 10min, abandons supernatant and umbrella organisations, adds in the complete culture solution ((hydroxyl containing 12%HES
Hydroxyethyl starch), 100 μ g/mL streptomysins, 70%DMEM) into single cell suspension, the filtering of 200 mesh cell sieves connects piping and druming cell precipitation
Kind is in T25 blake bottles, 37 DEG C, 5%CO2It is cultivated in constant incubator.
(3) liquid is changed after 3d for the first time, changes liquid 1 time every 2d later.When cell fusion is up to 70%~80%, with 0.25%
(mass concentration) pancreatin digests, and collects cell, and 1000r/min centrifuges 5min, and PBS is washed 3 times (1000r/min × 10min), centrifuges
Terminate, supernatant is abandoned in suction, obtains human adipose mesenchymal stem cells.
The hybrid mode of 3 fat mesenchymal stem cell of embodiment and protection liquid
The fat mesenchymal stem cell is to adjust to be added to fat mesenchymal stem cell transport protection liquid in a manner of resuspension
In, the concentration of human adipose mesenchymal stem cells is 6 × 10 in transport protection liquid5~7 × 105A/ml.Specifically, including following step
Suddenly：It takes the logarithm the 3rd fat subsitutes mescenchymal stem cell (ADMSCs) in growth period, 1000r/min centrifugation 5min abandon supernatant, add in 4
The transport protection liquid that DEG C precooling freezes, blows and beats cell precipitation, cell is made to be uniformly distributed in liquid protect, and adjusting viable cell concentrations is
6×105~7 × 105A/ml, dispenses and is preserved into cryopreservation tube.
4 human adipose mesenchymal stem cells of embodiment preserve viability experiment
(1) Blend grouping as shown in table 1 carries out cell and preserves viability experiment.
Table 1 is formulated
| A groups | 30%DMSO, 50 μ g/mL streptomysins, 80%DMEM, 8%HES |
| B groups | 10%DMSO, 100 μ g/mL streptomysins, 50%DMEM, 20%HES |
| C groups | 20%DMSO, 100 μ g/mL streptomysins, 70%DMEM, 12%HES |
| D groups | 30%DMSO, 80 μ g/mL streptomysins, 50%DMEM, 16%HES |
(2) test method
It takes the logarithm the 3rd fat subsitutes mescenchymal stem cell (ADMSCs) in growth period, is separately added into isometric A-D tetra- and assembles
The stem cell transport protection liquid of side.It after gently piping and druming uniformly dispenses, is placed in -80 DEG C of freezing storing box 3 days, takes out calculating cell and live
Rate.
Cell viability assay method is as follows：A little cell suspension is taken by 9：1 and 0.4% trypan blue solution mixing, it is placed in aobvious
Under micro mirror, viable count and dead cell number, Cell viability=viable count/total cell number * 100% are calculated.
The results are shown in Figure 1, and C group of formula (20%DMSO, 100 μ g/mL streptomysins, 70%DMEM, 12%HES) cell is lived
Rate highest, better than other groups.It follows that the human adipose mesenchymal stem cells of the present invention have higher Cell viability, can protect
The vigor and stability of cell are demonstrate,proved, human adipose mesenchymal stem cells is enable effectively to be applied.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that root
According to all introductions having disclosed, these embodiments can be carry out various modifications and replaced, these change the present invention's
Within protection domain.The four corner of the present invention is provided by appended claims and its any equivalent.
Claims (10)
1. a kind of fat mesenchymal stem cell transport protection liquid, which is characterized in that by 10%~30%DMSO (dimethyl sulfoxide (DMSO)),
The μ g/mL streptomysins of 50 μ g/mL~150,50%~80%DMEM, 8%~20%HES (hydroxyethyl starch) compositions.
2. fat mesenchymal stem cell transport protection liquid according to claim 1, which is characterized in that the fat mesenchymal
Stem cell is to adjust to be added in fat mesenchymal stem cell transport protection liquid in a manner of resuspension, in transport protection liquid between people's fat
The concentration of mesenchymal stem cells is 6 × 105~7 × 105A/ml.
3. fat mesenchymal stem cell transport protection liquid according to claim 1 or 2, which is characterized in that the DMSO's
Concentration is 10%~20%；Preferably, the concentration of the DMSO is 20%.
4. fat mesenchymal stem cell transport protection liquid according to claim 1 or 2, which is characterized in that the streptomysin
Concentration be the μ g/mL of 100 μ g/mL~150；Preferably, the concentration of the streptomysin is 100 μ g/mL.
5. fat mesenchymal stem cell transport protection liquid according to claim 1 or 2, which is characterized in that the DMEM's
Concentration is 70%~80%；Preferably, the concentration of the DMEM is 70%.
6. fat mesenchymal stem cell transport protection liquid according to claim 1 or 2, which is characterized in that the HES's is dense
It spends for 10%~20%；Preferably, the concentration of the HES is 12%.
7. fat mesenchymal stem cell transport protection liquid according to claim 2, which is characterized in that the fat mesenchymal
Stem cell is to adjust to be added in fat mesenchymal stem cell transport protection liquid in a manner of resuspension, is comprised the following steps：It takes the logarithm
The 3rd fat subsitutes mescenchymal stem cell (ADMSCs) in growth period, 1000r/min centrifugation 5min, abandons supernatant, adds in 4 DEG C of precoolings
The transport protection liquid deposited, blows and beats cell precipitation, cell is made to be uniformly distributed in liquid is protected, adjustment viable cell concentrations are 6 × 105~
7×105A/ml, dispenses and is preserved into cryopreservation tube.
8. fat mesenchymal stem cell transport protection liquid according to claim 7, which is characterized in that the adjustment living cells
Concentration comprises the following steps：A little cell suspension is taken by 9：1 and 0.4% trypan blue solution mixing, it counts under microscope and is dyed to
The dead cell of blueness and the viable count of anti-dye.
9. fat mesenchymal stem cell according to claim 7 transport protection liquid, which is characterized in that still further comprise by
Cryopreservation tube is placed in -20 DEG C~-80 DEG C freezing storing box and preserves；Preferably, it is placed in -80 DEG C of freezing storing box and preserves.
10. claim 1-9 any one of them fat mesenchymal stem cell transport protection liquid is done in cryopreserved human fat mesenchymal
Application in cell and fat mesenchymal stem cell transport.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753712A (en) * | 2018-07-04 | 2018-11-06 | 成都清科生物科技有限公司 | A kind of fat stem cell extracting method |
| CN108849857A (en) * | 2018-07-20 | 2018-11-23 | 吉林济惠生物科技有限公司 | A kind of transport protection liquid of umbilical cord mesenchymal stem cells |
| CN109266604A (en) * | 2018-10-10 | 2019-01-25 | 浙江神雁精准医疗科技有限公司 | Composition and preparation method thereof comprising Stem Cell Activity substance |
| CN109757469A (en) * | 2019-03-20 | 2019-05-17 | 江苏瑞思坦生物科技有限公司 | A kind of adipose tissue transport liquid and preparation method |
| CN110074096A (en) * | 2019-05-28 | 2019-08-02 | 苏州博特龙免疫技术有限公司 | A kind of serum-free cell frozen stock solution and its preparation method and application |
| CN111418579A (en) * | 2020-04-13 | 2020-07-17 | 广东华夏健康生命科学有限公司 | Preservation method of adipose tissues, preservation solution of adipose tissues and preparation method of preservation solution |
| CN112425603A (en) * | 2020-11-10 | 2021-03-02 | 郑州贝贝生物科技有限公司 | Transportation and preservation solution for adipose-derived stem cells |
| CN113498779A (en) * | 2021-04-22 | 2021-10-15 | 重庆医科大学附属儿童医院 | Reagent and method for transporting cells |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753712A (en) * | 2018-07-04 | 2018-11-06 | 成都清科生物科技有限公司 | A kind of fat stem cell extracting method |
| CN108753712B (en) * | 2018-07-04 | 2022-11-01 | 成都清科生物科技有限公司 | Method for extracting adipose-derived stem cells |
| CN108849857A (en) * | 2018-07-20 | 2018-11-23 | 吉林济惠生物科技有限公司 | A kind of transport protection liquid of umbilical cord mesenchymal stem cells |
| CN109266604A (en) * | 2018-10-10 | 2019-01-25 | 浙江神雁精准医疗科技有限公司 | Composition and preparation method thereof comprising Stem Cell Activity substance |
| CN109757469A (en) * | 2019-03-20 | 2019-05-17 | 江苏瑞思坦生物科技有限公司 | A kind of adipose tissue transport liquid and preparation method |
| CN110074096A (en) * | 2019-05-28 | 2019-08-02 | 苏州博特龙免疫技术有限公司 | A kind of serum-free cell frozen stock solution and its preparation method and application |
| CN110074096B (en) * | 2019-05-28 | 2020-05-15 | 苏州博特龙免疫技术有限公司 | Serum-free cell cryopreservation liquid and preparation method and application thereof |
| CN111418579A (en) * | 2020-04-13 | 2020-07-17 | 广东华夏健康生命科学有限公司 | Preservation method of adipose tissues, preservation solution of adipose tissues and preparation method of preservation solution |
| CN111418579B (en) * | 2020-04-13 | 2021-05-18 | 广东华夏健康生命科学有限公司 | Preservation method of adipose tissues, preservation solution of adipose tissues and preparation method of preservation solution |
| CN112425603A (en) * | 2020-11-10 | 2021-03-02 | 郑州贝贝生物科技有限公司 | Transportation and preservation solution for adipose-derived stem cells |
| CN113498779A (en) * | 2021-04-22 | 2021-10-15 | 重庆医科大学附属儿童医院 | Reagent and method for transporting cells |
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