CN109042627A - A kind of people's Amniotic Fluid-derived Mesenchymal Stem Cells transport liquid - Google Patents
A kind of people's Amniotic Fluid-derived Mesenchymal Stem Cells transport liquid Download PDFInfo
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- CN109042627A CN109042627A CN201810983592.1A CN201810983592A CN109042627A CN 109042627 A CN109042627 A CN 109042627A CN 201810983592 A CN201810983592 A CN 201810983592A CN 109042627 A CN109042627 A CN 109042627A
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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
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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
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Abstract
The present invention relates to a kind of people's Amniotic Fluid-derived Mesenchymal Stem Cells to transport liquid.The present invention adds 5 μM of Radix pseudostellariae cyclic peptides A in prior art basis can effectively overcome Cell viability reduction after prior art transport liquid suspension 72h more clearly disadvantageous, the present invention transports formula of liquid are as follows: is uniformly mixed forms by 30% 0.2% aqueous trehalose (PBS solution of trehalose), 25% red blood cell storage solution, 5% 0.2% heparin calcium solution (PBS solution of calciparine) and 40% human serum albumin injection room temperature by mass percentage, and contain 5 μM of Radix pseudostellariae cyclic peptides A.Motility rate when people's Amniotic Fluid-derived Mesenchymal Stem Cells transport 72h not only can be improved in the transport liquid, and does not influence its Multidirectional Differentiation ability.
Description
Technical field
The present invention relates to stem cell fields more particularly to a kind of people's Amniotic Fluid-derived Mesenchymal Stem Cells to transport liquid.
Background technique
Stem cell is a kind of cell with self-renewing and Multidirectional Differentiation ability.Exactly because its regeneration potential and can
Plasticity is widely used in it in regenerative medicine and stem-cell research.Stem cell is many for lacking effective treatment method at present
Disease brings hope, including cerebral apoplexy, Huntington chorea, Alzheimer disease and Parkinson's disease.
Mescenchymal stem cell be from the multipotency stroma cell that is separated with fetal tissue of adult, be defined as sticking at fiber
Cell-like cell.Mescenchymal stem cell has inherent ability of going back to the nest, and can move to injury tissue, play an active part in tissue and repair
It is multiple.Although mescenchymal stem cell has versatility, self-renewal capacity, which gradually decreases, leads to cell division arrest, in vitro table
Reveal limited service life, limits its application in the treatment.Compared with adult tissue, perinatal period tissue-derived mesenchymal
Stem cell has lower mutation risk, and shows excellent cell activity, including bigger differentiation, goes back to the nest and is implanted into effect
Power and lower immunogenicity.The mescenchymal stem cell of separate sources has similar or even identical phenotype, but solely due to it
There is different treatments to imitate for the hereditary feature of spy, transfer ability, separation method, different perinatal period tissue-derived mesenchymal stem cells
Fruit.
There are two types of currently used cell means of transportation, and one kind is non-cultivation conditions low temperature liquid nitrogen transport, and another kind is training
Culture medium transport is filled under the state of supporting.Both of which has certain drawbacks.It wherein can be in specific liquid using liquid nitrogen transport
It is transported after the cell of nitrogen cascade memory amplification quantity, but there are some potential safety problemss, and cost is high, frozen stock solution ingredient is not
It is clear, the cell activity meeting that typically contains the serum of different proportion, can affect to subsequent experimental, while freezing
Decline.And attached cell is transported, single transport cells quantity is lower, and cell culture medium expends excessively, and content liquid
When higher cannot use air transportion mode, while if it is poorly sealed or during transportation exist it is a series of collision if be easy cause
Culture medium is set to leak outside, cell will appear death without culture medium.
Chinese patent CN108432742A discloses a kind of mescenchymal stem cell and is transported at room temperature liquid, and this application overcomes tradition
The drawbacks of mescenchymal stem cell transports, realizes room temperature, amount transport, avoids culture medium in Traditional Transportation mode, liquid nitrogen
A large amount of wastes, effectively reduce cost；And the ingredient of this application transport liquid is simple, it is convenient, pollution-free to prepare, to mescenchymal stem cell
With excellent biological characteristics preservation effect.But 3 kinds of cells (people's amniotic fluid, umbilical cord, placenta derived mesenchymal stem cell) exist
Living cells quantity in cell transport liquid after room temperature 72h significantly reduces, and is not suitable for transporting for long-distance.
Summary of the invention
The present invention is improvements over the prior art, it is desirable to provide a kind of people's Amniotic Fluid-derived Mesenchymal Stem Cells of suitable long-distance transport
Transport liquid.
To achieve the above object, the present invention provides following technical schemes:
A kind of stem cell is transported at room temperature liquid, by mass percentage by 30% 0.2% aqueous trehalose, 25% it is red thin
Born of the same parents' storing liquid, 5% 0.2% heparin calcium solution and 40% human serum albumin injection room temperature are uniformly mixed composition, also contain
Radix pseudostellariae cyclic peptides A.
Preferably, 0.2% aqueous trehalose is formulated by trehalose and PBS buffer solution, and 0.2% is quality volume basis
Content.
Preferably, 0.2% heparin calcium solution is formulated by calciparine and PBS buffer solution, and 0.2% is quality volume basis
Content.
Preferably, Radix pseudostellariae cyclic peptides A concentration is 5 μM.
Above-mentioned stem cell is transported at room temperature application of the liquid in the transport of people's Amniotic Fluid-derived Mesenchymal Stem Cells.
The present invention adds 5 μM of Radix pseudostellariae cyclic peptides A in prior art basis can effectively overcome prior art transport liquid outstanding
Cell viability reduces more clearly disadvantageous after floating 72h, and the present invention transports formula of liquid are as follows: by mass percentage by 30%
0.2% aqueous trehalose (PBS solution of trehalose), 25% red blood cell storage solution, 5% 0.2% heparin calcium solution (heparin
The PBS solution of calcium) and 40% human serum albumin injection room temperature be uniformly mixed composition, and contain 5 μM of Radix pseudostellariae cyclic peptides A.It should
Motility rate when people's Amniotic Fluid-derived Mesenchymal Stem Cells transport 72h not only can be improved in transport liquid, and does not influence its Multidirectional Differentiation ability.
Specific embodiment
One, experimental material
The second trimester amniotic fluid of healthy pregnant women is selected, informed consent form is signed, agrees to be used for scientific experiment.
People's Amniotic Fluid-derived Mesenchymal Stem Cells special culture media is purchased from BI company；Fetal calf serum is purchased from U.S. Thermo company；
DMEM culture medium is purchased from U.S. Gibco company；Red blood cell storage solution is purchased from Beijing Suo Laibao Science and Technology Ltd；Human serum albumin
Injection is purchased from Shanghai Laishi Blood Product Co., Ltd；Stem cell Osteoblast Differentiation culture medium is purchased from PromoCell company；
Neural Differentiation culture medium: the epidermal growth factor of 20 μ g/L and the alkali of 20 μ g/L are added in the DMEM/F12 culture medium containing 2%B27
Property fibroblast growth factor is formulated.
Two, experimental method
1, people's Amniotic Fluid-derived Mesenchymal Stem Cells (hAFSCs) is separately cultured
Amniotic fluid and the PBS containing EDTA are diluted by 1:1,5min is centrifuged with 1200r/min, is added between amniotic fluid after abandoning supernatant
Mesenchymal stem cells special culture media blows and beats mixing repeatedly；Then it is seeded in the coated culture dish of gelatin, is placed in 37 DEG C, volume fraction
For 5%CO2Incubator culture.Cell it is long to 90% or so when digestion count, by 1:2 passage.
2, the preparation of experimental group and cell suspending liquid
The 4th generation cell is taken, is cleaned 3 times with PBS, trypsin digestion and cell is added；It falls off, is added without blood to cell rounding
Clear DMEM culture medium dispels, and is centrifuged 5min under 1200rpm；Supernatant liquid is outwelled, 1000rpm after PBS is resuspended is added and is centrifuged
3min；It abandons supernatant liquid and PBS is added and is resuspended and continue to be centrifuged 3min under 1000rpm；Divide respectively according to following after discarding supernatant liquid
Group is added the corresponding liquid that is transported at room temperature and blows and beats uniformly, and cell concentration is 1 × 106A cell/mL.
Prior art group: liquid is transported at room temperature as the formula of Chinese patent CN108432742A specification embodiment 3, that is, presses raw material
Mass percent meter is by 30% 0.2% aqueous trehalose (PBS solution of trehalose), 25% red blood cell storage solution, 5%
0.2% calciparine (PBS solution of calciparine) and 40% human serum albumin injection room temperature be uniformly mixed composition；
It tests 1 group: adding 5 μM of Radix pseudostellariae cyclic peptides A on the basis of control group transports liquid.
It tests 2 groups: adding 5 μM of Radix pseudostellariae cyclic peptides D on the basis of control group transports liquid.
72h will take 300 μ L to record viable count after cell liquid piping and druming uniformly.
3, the measurement of the front and back transport liquid suspension 72h Osteoblast Differentiation ability
The hAFSCs of 1 group of the experiment 0,72h that suspends in transport liquid is taken, with 5 × 104The density of/mL is inoculated in 24 orifice plates,
For 24 hours after it is paved with bottom hole, it is changed to Osteoblast Differentiation culture medium Fiber differentiation 9d (every 3d replaces a subculture) and collects afterwards carefully
Born of the same parents, RT-PCR detect the relative expression levels of ALP mRNA.
Total serum IgE is extracted using TRIZOL one-step method.3 μ g of total serum IgE is taken, it is complete using RevertAid TM Reverse Transcriptase kit
At reverse transcription.Reaction total volume is 50 μ L, wherein 10 × Taqbuffer 5.0 μ L, MgCl2(25mmol/L) 5.0 μ L, dNTP
The 2 each 5 μ L of 1 μ L, cDNA in the upstream and downstream μ L, ALP and β-actin primer (10 μm of ol/L) of Mix (10mmol/L), Taq enzyme (5U/ μ L)
0.5 μ L supplies distilled water to 50 μ L.Reaction condition are as follows: 95 DEG C of initial denaturation 5min, 94 DEG C of denaturation 40s, 55 DEG C of annealing 60s, 72 DEG C
Extend 60s, 72 DEG C of terminals extend 10min, totally 30 circulation.
5 '-CCAACTCTTTTGTGCCAGAGA-3 ' of ALP upstream primer
5 '-GGCTACATTGGTGTTGAGCTTTT-3 ' of ALP downstream primer
5 '-CATGGGTCAGAAGGATTCCT-3 ' of β-actin upstream primer
5 '-TGATCTGGGTCATCTTCTCG-3 ' of β-actin downstream primer
It after electrophoresis, is scanned and is taken pictures with gel imaging system, analyze the gray value of purpose band, by ALP gray value/β-
The relative expression levels of the ratio calculation ALP mRNA of actin gray value.
4, at the measurement of Neural Differentiation ability before and after transport liquid suspension 72h
The hAFSCs of 1 group of the experiment 0,72h that suspends in transport liquid is taken, with 5 × 104The density of/mL is inoculated in 24 orifice plates,
For 24 hours after it is paved with bottom hole, it is changed to Neural Differentiation culture medium Fiber differentiation 9d (every 3d replaces a subculture) and collects afterwards carefully
Born of the same parents, RT-PCR detect the relative expression levels of nerve cell labelled protein nestin mRNA.
Total serum IgE is extracted using TRIZOL one-step method.3 μ g of total serum IgE is taken, it is complete using RevertAid TM Reverse Transcriptase kit
At reverse transcription.Reaction total volume is 50 μ L, wherein 10 × Taqbuffer 5.0 μ L, MgCl2(25mmol/L) 5.0 μ L, dNTP
2 μ L of Mix (10mmol/L), nestin and the upstream and downstream β-actin primer (10 μm of ol/L) each 5 μ L of 1 μ L, cDNA, Taq enzyme (5U/
μ L) 0.5 μ L, supplies distilled water to 50 μ L.Reaction condition are as follows: 95 DEG C of initial denaturations 5min, 94 DEG C of denaturation 40s, 55 DEG C of annealing 60s,
72 DEG C of extension 60s, 72 DEG C of terminals extend 10min, totally 30 circulation.
5 '-CTCCAAGAATGGAGGCTGTAGGAA-3 ' of nestin upstream primer
5 '-CCTATGAGATGGAGCAGGCAAGA-3 ' of nestin downstream primer
5 '-CATGGGTCAGAAGGATTCCT-3 ' of β-actin upstream primer
5 '-TGATCTGGGTCATCTTCTCG-3 ' of β-actin downstream primer
It after electrophoresis, is scanned and is taken pictures with gel imaging system, analyze the gray value of purpose band, by nestin gray scale
The relative expression levels of value/β-actin gray value ratio calculation nestin mRNA.
5, statistical analysis
It is analyzed using SPSS19.0 software, two independent samples compare using independent samples t test, different comparison among groups
Using one-way analysis of variance, measurement data is indicated with means standard deviation, and P < 0.05 is that difference has significant.
Three, experimental result
1, the influence that transport liquid suspends to cell viability measurement
The results are shown in Table 1.After only testing 1 group of transport liquid suspension 72h, living cells quantity is preceding poor without conspicuousness with suspension
Different, it is minimum that this illustrates that the transport liquid of present invention addition Radix pseudostellariae cyclic peptides A influences hAFSCs motility rate.
Table 1 transports each group viable count before and after liquid suspends
| Suspension 0h | Suspension 72h | |
| Prior art group | 3×105A cell | 2.13×105A cell |
| Test 1 group | 3×105A cell | 2.89×105A cell |
| Test 2 groups | 3×105A cell | 2.25×105A cell |
2, influence of the transport liquid suspension 72h to hAFSCs Osteoblast Differentiation ability
The results are shown in Table 2.1 group of transport liquid suspension 72h is tested to have no significant effect hAFSCs Osteoblast Differentiation ability.
2 ALP mRNA relative expression levels' measurement result of table
| 0h | 72h | |
| Liquid is transported to suspend | 5.84±0.09 | 5.90±0.08 |
Alkaline phosphatase (ALP) is early stage skeletonization mark, is distributed mainly on cell membrane, promotes cell calcification, ALP's quantifies
Detection can reflect the level of differentiation of osteoblast, and activity is higher, illustrate preosteoblast to mature osteoblast differentiation
Be more obvious.The active high expression of ALP is the early sign of osteoblast differentiation maturation, when ALP increased activity, bon e formation increasing
By force, and bone matrix mineralising is promoted to be formed, therefore the activity of ALP is the good finger for reflecting osteoblast differentiation degree and functional status
Mark.
3, influence of the transport liquid suspension 72h to hAFSCs Neural Differentiation ability
The results are shown in Table 3.1 group of transport liquid suspension 72h is tested to have no significant effect hAFSCs Neural Differentiation ability.
3 nestin mRNA relative expression levels' measurement result of table
| 0h | 72h | |
| Liquid is transported to suspend | 8.95±0.08 | 9.26±0.09 |
Nestin (Nestin) is characteristic mark's albumen of neural stem cell, and expression is reflection nerve cell point
The good index of change degree and functional status.
To sum up, the present invention adds 5 μM of Radix pseudostellariae cyclic peptides A in prior art basis can effectively overcome the prior art
Transport liquid suspension 72h after Cell viability reduce it is more clearly disadvantageous, the present invention transport formula of liquid are as follows: by mass percentage by
30% 0.2% aqueous trehalose (PBS solution of trehalose), 25% red blood cell storage solution, 5% 0.2% calciparine (liver
The PBS solution of plain calcium) and 40% human serum albumin injection room temperature be uniformly mixed composition, and contain 5 μM of Radix pseudostellariae cyclic peptides A.
Motility rate when people's Amniotic Fluid-derived Mesenchymal Stem Cells transport 72h not only can be improved in the transport liquid, does not influence its differentiation capability also.
Claims (5)
1. a kind of stem cell is transported at room temperature liquid, by mass percentage by 30% 0.2% aqueous trehalose, 25% red blood cell
Storing liquid, 5% 0.2% heparin calcium solution and 40% human serum albumin injection room temperature are uniformly mixed composition, and feature exists
In: also contain Radix pseudostellariae cyclic peptides A.
2. stem cell according to claim 1 is transported at room temperature liquid, it is characterised in that: 0.2% aqueous trehalose is by sea
Algae sugar and PBS buffer solution are formulated, and 0.2% is quality volumn concentration.
3. stem cell according to claim 1 is transported at room temperature liquid, it is characterised in that: the 0.2% heparin calcium solution is by liver
Plain calcium and PBS buffer solution are formulated, and 0.2% is quality volumn concentration.
4. stem cell according to claim 1 is transported at room temperature liquid, it is characterised in that: Radix pseudostellariae cyclic peptides A concentration is 5 μM.
5. any stem cell of Claims 1 to 4 is transported at room temperature application of the liquid in the transport of people's Amniotic Fluid-derived Mesenchymal Stem Cells.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109938010A (en) * | 2019-03-20 | 2019-06-28 | 江苏瑞思坦生物科技有限公司 | A kind of human adipose mesenchymal stem cells transport liquid and preparation method thereof |
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|---|---|---|---|---|
| US20130267008A1 (en) * | 2010-12-31 | 2013-10-10 | Kyungpook National University Hospital | Freezing medium composition for cryopreserving amniotic fluid-derived stem cells and a method for cryopreserving the same |
| CN106472486A (en) * | 2016-12-26 | 2017-03-08 | 南京佰泰克生物技术有限公司 | A kind of freezing protective agent of maintenance DC CIK cell High Fragmentation power |
| CN106754699A (en) * | 2016-12-22 | 2017-05-31 | 南京佰泰克生物技术有限公司 | The application of miRNA 155 and its inhibitor in terms of DC CIK cell cultures |
| CN108432742A (en) * | 2018-04-09 | 2018-08-24 | 佛山科学技术学院 | A kind of mescenchymal stem cell room temperature transport liquid |
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- 2018-08-27 CN CN201810983592.1A patent/CN109042627B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130267008A1 (en) * | 2010-12-31 | 2013-10-10 | Kyungpook National University Hospital | Freezing medium composition for cryopreserving amniotic fluid-derived stem cells and a method for cryopreserving the same |
| CN106754699A (en) * | 2016-12-22 | 2017-05-31 | 南京佰泰克生物技术有限公司 | The application of miRNA 155 and its inhibitor in terms of DC CIK cell cultures |
| CN106472486A (en) * | 2016-12-26 | 2017-03-08 | 南京佰泰克生物技术有限公司 | A kind of freezing protective agent of maintenance DC CIK cell High Fragmentation power |
| CN108432742A (en) * | 2018-04-09 | 2018-08-24 | 佛山科学技术学院 | A kind of mescenchymal stem cell room temperature transport liquid |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109938010A (en) * | 2019-03-20 | 2019-06-28 | 江苏瑞思坦生物科技有限公司 | A kind of human adipose mesenchymal stem cells transport liquid and preparation method thereof |
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