CN106577637A - Preservation method of human amniotic mesenchymal stem cells - Google Patents
Preservation method of human amniotic mesenchymal stem cells Download PDFInfo
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- CN106577637A CN106577637A CN201710052311.6A CN201710052311A CN106577637A CN 106577637 A CN106577637 A CN 106577637A CN 201710052311 A CN201710052311 A CN 201710052311A CN 106577637 A CN106577637 A CN 106577637A
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- mesenchymal stem
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- amnion mesenchymal
- human amnion
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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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Abstract
The invention relates to a preservation method of human amniotic mesenchymal stem cells, and belongs to the biotechnology field. A human amniotic mesenchymal stem cell preservation solution is prepared from 12-15 g of sodium cirate (C6H5Na3O7.2H2O), 3-6 g of citric acid (C6H8O7.H2O), 12-16 g of glucose (C6H12O6.H2O), 10 ml of human plasma, 10 g of human albumin and 990 ml of water. The preservation method has the advantages that the human amniotic mesenchymal stem cell preservation solution does not contain animal serum and has the high safety; experiments prove that the human amniotic mesenchymal stem cells still can express following three kinds of mesenchymal stem cell membrane molecules including a human leukocyte differentiation antigen CD73, a human leukocyte differentiation antigen CD90 and a human leukocyte differentiation antigen CD105 after being preserved in the preservation solution for 24 hours at 4 DEG C.
Description
Technical field
The present invention relates to a kind of store method of human amnion mesenchymal stem cell, belongs to biological technical field.
Background technology
People amnion stem cell has height self, propagation, implantable and polyphyly differentiation capability, and allogene is individual to be moved
Without immunological rejection and oncogenicity risk after plant.And, the placenta that people's amnion source of human stem cell is discarded after parturient childbirth is applied to
Clinic will not bring Medical Ethics to dispute on.Amnion stem cell have obvious Colony forming ability, multiplication capacity, embryo's dryness,
Immunoregulation capability and without excellent biological characteristicses such as allotransplantation rejection and oncogenicity, can be utilized for many diseases
Cell therapy, the reparation of injuries of tissues and organs with rebuild, be the preferable seed cell resource of regenerative medicine field, with wide
Potential applicability in clinical practice.
In the last few years, it is substantial amounts of research show, amnion stem cell, both with to same germinal layer different type cell differentiation
Multi-lineage potential, there is the interdepartmental ability across differentiation of germinal layers again, shows good plasticity.Such as, originate from mesoblastic cell
Can break up to mesoblastemas such as skeletonization, cartilage, fat, also can be thin to the endoderm cells such as pancreatic cell, liver cell, nerve
The ectoderm cells such as born of the same parents break up.
People's amnion stem cell not only has significant self-renewal capacity and interdepartmental across a germinal layer polyphyly differentiation potential, and relatively
Embryonic stem cell and other adult stem cells, its aboundresources is easily obtained, and without dispute of ethic, immunogenicity is low, without tumorigenesis wind
Danger, also has a powerful paracrine or autocrine work(of the multiple biological activities factor such as secretory immune inhibiting factor, neurotrophic factor
Energy.These characteristics impart it and face the correlative regeneration medical domain such as organizational project, cell therapy, gene therapy is immeasurable
Bed using value.
The content of the invention
First purpose of the present invention is to provide human amnion mesenchymal stem cell and preserves liquid.
Second object of the present invention is to provide the store method of human amnion mesenchymal stem cell.
For achieving the above object, the present invention is employed the following technical solutions：
Human amnion mesenchymal stem cell preserves liquid, is made up of following compositions：
Sodium citrate (C6H5Na3O7·2H2O) 12-15g, citric acid (C6H8O7·H2O) 3-6g, glucose (C6H12O6·
H2O) 12-16g, human plasma 10ml, human serum albumin 10g, water 990ml.
The human amnion mesenchymal stem cell preserves liquid, is preferably made up of following compositions：
Sodium citrate (C6H5Na3O7·2H2O) 13-14g, citric acid (C6H8O7·H2O) 4-5g, glucose (C6H12O6·
H2O) 14-15g, human plasma 10ml, human serum albumin 10g, water 990ml.
The human amnion mesenchymal stem cell preserves liquid, is most preferably made up of following compositions：
Sodium citrate (C6H5Na3O7·2H2O) 13.2g, citric acid (C6H8O7·H2O) 4.8g, glucose (C6H12O6·
H2O) 14.7g, human plasma 10ml, human serum albumin 10g and water 990ml.
The human plasma behaviour AB blood plasma.
It is 7.2-7.4 that the human amnion mesenchymal stem cell preserves the pH value of liquid.
The store method of human amnion mesenchymal stem cell, comprises the steps：Human amnion mesenchymal stem cell is stored in
Above-mentioned human amnion mesenchymal stem cell is preserved in liquid.
Content of the human amnion mesenchymal stem cell in the preservation liquid is (2-3) × 108Individual cell/L.
The temperature of the preservation is 2-8 DEG C, preferably 4 DEG C.
The store method of human amnion mesenchymal stem cell：By human amnion mesenchymal stem cell in the material group by following proportioning
Into solution in preserve：Sodium citrate (C6H5Na3O7·2H2O) 13.2g, citric acid (C6H8O7·H2O) 4.8g, glucose
(C6H12O6·H2O) 14.7g, people AB blood plasma 10ml, human serum albumin 10g and water 990ml.
The human amnion mesenchymal stem cell is expressed as follows three kinds of mescenchymal stem cell membrane molecules：HL breaks up
Antigens c D73, HL's differentiation antigen CD90 and HL differentiation antigen CD105.It is not expressed as follows two kinds of cell membranes point
Son：HL's differentiation antigen CD45 and human leucocyte antigen (HLA) HLA-DR.
Preserve in liquid in said temperature and human amnion mesenchymal stem cell, it can be 12-24 hours that the time of the preservation is,
Such as 24 hours.
It is an advantage of the invention that：The human amnion mesenchymal stem cell that the present invention is provided preserves liquid and does not contain animal blood serum, has
High security.It is demonstrated experimentally that human amnion mesenchymal stem cell is preserved 24 hours in above-mentioned preservation liquid at 4 DEG C, still it is expressed as follows
Three kinds of mescenchymal stem cell membrane molecules：HL differentiation antigen CD73, HL's differentiation antigen CD90 and people are thin in vain
Born of the same parents differentiation antigen CD105.
The present invention is elaborated with reference to the accompanying drawings and detailed description, not limitation of the invention, it is all according to
The equivalent of any this area carried out according to disclosure file, belongs to protection scope of the present invention.
Description of the drawings
Figure 1A is situation when human amnion mesenchymal stem cell is just adherent
Figure 1B starts situation when growing for human amnion mesenchymal stem cell
Fig. 1 C are the situation after human amnion mesenchymal stem cell propagation
Fig. 2A is expression of the human amnion mesenchymal stem cell by flow cytomery HL differentiation antigen CD73
Situation
Fig. 2 B are expression of the human amnion mesenchymal stem cell by flow cytomery HL differentiation antigen CD90
Situation
Fig. 2 C are expression of the human amnion mesenchymal stem cell by flow cytomery HL differentiation antigen CD105
Situation
Fig. 2 D are expression of the human amnion mesenchymal stem cell by flow cytomery HL differentiation antigen CD45
Situation
Fig. 2 E are expression feelings of the human amnion mesenchymal stem cell by flow cytomery human leucocyte antigen (HLA) HLA-DR
Condition
Fig. 3 A be human amnion mesenchymal stem cell human amnion mesenchymal stem cell preserve liquid in 4 DEG C preserve 24 hours after lead to
Overflow-type cell instrument detects the expression of HL differentiation antigen CD73
Fig. 3 B be human amnion mesenchymal stem cell human amnion mesenchymal stem cell preserve liquid in 4 DEG C preserve 24 hours after lead to
Overflow-type cell instrument detects the expression of HL differentiation antigen CD90
Fig. 3 C be human amnion mesenchymal stem cell human amnion mesenchymal stem cell preserve liquid in 4 DEG C preserve 24 hours after lead to
Overflow-type cell instrument detects the expression of HL differentiation antigen CD105
Fig. 3 D be human amnion mesenchymal stem cell human amnion mesenchymal stem cell preserve liquid in 4 DEG C preserve 24 hours after lead to
Overflow-type cell instrument detects the expression of HL differentiation antigen CD45
Fig. 3 E be human amnion mesenchymal stem cell human amnion mesenchymal stem cell preserve liquid in 4 DEG C preserve 24 hours after lead to
Overflow-type cell instrument detects the expression of human leucocyte antigen (HLA) HLA-DR
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, commercially obtain.
α-MEM culture mediums are the product of Gibco companies, and its production code member is：11900024；People AB blood plasma is red purchased from Beijing
Cross Blood Center；Injection human serum albumin (Chinese medicines quasi-word S20030043).
Human amnion mesenchymal stem cell special culture media：Add people AB blood plasma and people in α-MEM (Gibco, USA) culture medium
Blood albumin, concentration of the people AB blood plasma in the human amnion mesenchymal stem cell special culture media is that 10% (volume basis are dense
Degree), concentration of the human serum albumin in the human amnion mesenchymal stem cell special culture media is 1% (concentration expressed in percentage by volume), pH
It is worth for 7.2-7.4.
Embodiment 1, human amnion mesenchymal stem cell preserves liquid
First, prescription
2nd, preparation method
The sodium citrate of recipe quantity, citric acid, glucose, people's AB blood plasma, human serum albumin are sequentially added in water, is stirred
Dissolving, mixes, and sterilizing is obtained final product.
Embodiment 2, human amnion mesenchymal stem cell is separately cultured
1st, cell separation
(1) amnion takes from mature Cesarean esction health puerpera, 6～12h process after collection.
(2) amnion of a diameter of 6cm × 6cm is cleaned with 0.9% sodium chloride solution, is shredded to about 1mm × 1mm × 1mm,
The clostridiopetidase As of Jing 0.1% and 0.125% 37 DEG C of digestion 30-60min of pancreatin, with α-MEM (Gibco, USA) 40ml is diluted to.
(3) aforesaid liquid is successively filtered with 100 mesh and 200 mesh filter screens, removes indigested tissue.
(4) filter after celliferous liquid be placed in 50ml centrifuge tubes, be placed on after trim in refrigerated centrifuge, with from
Mental and physical efforts 300Xg, 4 ± 2 DEG C of temperature is centrifuged 8 minutes.
(5) gently take out centrifuge tube after shutting down to be placed on rack for test tube, centrifuge tube content is divided into two parts, and upper strata is collagen
Enzyme, pancreatin and α-MEM culture mediums, lower floor is tissue pieces and cell mixture.
(6) upper strata clostridiopetidase A, pancreatin and α-MEM culture mediums are suctioned out.
(7) separated people's amnion mononuclearcell is washed with 2000r/min centrifugation 10min after the dilution of α-MEM culture mediums
It is secondary.
2nd, cell culture
Detached people's amnion mononuclearcell is pressed into 1 × 106Individual cell/cm2It is inoculated in 75cm2Plastic culture bottle, is incubated at
37 DEG C, 5%CO2Incubator, nutrient solution is human amnion mesenchymal stem cell special culture media.Liquid is changed after 24 hours, non-adherent is abandoned
Cell, changed liquid per 2～3 days later；Treat that human amnion mesenchymal stem cell grows into 70～80% fusions, use 0.25% pancreatin
(Sigma, USA) digests (1～2 minute), by 0.8 × 104～1.0 × 104Individual cell/cm2Pass on.Per 2～3 days with the people sheep
Pass on once in intermembranous mesenchymal stem cells special culture media, make cell concentration maintain 5 × 105～10 × 105Individual cell/mL.
Every time the condition of Secondary Culture is 37 DEG C, 5%CO2.Passing for 5 generations obtains human amnion mesenchymal stem cell.
Situations of the Figure 1A for human amnion mesenchymal stem cell when just adherent is form in tiny circle.
Figure 1B starts situation when growing for human amnion mesenchymal stem cell, and after 48 hours, cell starts propagation, Xiang Changsuo
Shape changes.
Fig. 1 C are the situation after human amnion mesenchymal stem cell propagation, and cell starts to breed what formation was differed in size after one week
Cell colony.
Embodiment 3：FCM analysis
First, method
The human amnion mesenchymal stem cell that embodiment 2 is obtained is logical with anti-human CD73 monoclonal antibodies (BioLegend, USA)
Overflow-type cell instrument detects the expression of HL differentiation antigen CD73, with anti-human CD90 monoclonal antibodies
The expression that (BioLegend, USA) passes through flow cytomery HL differentiation antigen CD90, it is mono- with anti-human CD105
The expression that clonal antibody (BioLegend, USA) passes through flow cytomery HL differentiation antigen CD105, uses anti-
The expression feelings that people's CD45 monoclonal antibodies (BioLegend, USA) passes through flow cytomery HL differentiation antigen CD45
Condition, with anti-human HLA HLA-DR monoclonal antibodies (BioLegend, USA) flow cytometer (model is passed through
FACSCalibur342975, BD company) detection human leucocyte antigen (HLA) HLA-DR expression.
2nd, result
As a result as shown in Fig. 2A, Fig. 2 B, Fig. 2 C, Fig. 2 D and Fig. 2 E, show that human amnion mesenchymal stem cell expression people is white
Cell differentiation antigen CD73, HL's differentiation antigen CD90 and HL differentiation antigen CD105.HL point is not expressed
Change antigen CD4 5 and human leucocyte antigen (HLA) HLA-DR.Flow cytomery result shows the pure of the human amnion mesenchymal stem cell
Degree reaches 94.3-95.2%.
Embodiment 4：The preservation of human amnion mesenchymal stem cell
First, material：
1st, the human amnion mesenchymal stem cell that embodiment 2 is obtained
2nd, human amnion mesenchymal stem cell preserves liquid, prepares by embodiment 1
2nd, method：
The human amnion mesenchymal stem cell that embodiment 2 is obtained adds human amnion mesenchymal stem cell prepared by embodiment 1
In preserving liquid, the pH value of the preservation liquid is 7.2-7.4.Human amnion mesenchymal stem cell is protected in the human amnion mesenchymal stem cell
The content of liquid storage is (2-3) × 108Individual cell/L.
The human amnion mesenchymal stem cell for accessing human amnion mesenchymal stem cell is preserved into liquid to be fitted in haemocyte storage bag,
Haemocyte storage bag is cut off with high-frequency thermocompressor, is preserved 24 hours at 4 DEG C.
Following five kinds of HL's cell membranes of the human amnion mesenchymal stem cell after being preserved with flow cytomery resist
Original molecule expression：HL differentiation antigen CD73, HL differentiation antigen CD90, HL's differentiation antigen
CD105, HL's differentiation antigen CD45 and human leucocyte antigen (HLA) HLA-DR.
3rd, result：
As a result as shown in Fig. 3 A to Fig. 3 E, human amnion mesenchymal stem cell preserves liquid in above-mentioned human amnion mesenchymal stem cell
In preserve 24 hours at 4 DEG C, still express HL differentiation antigen CD73, HL's differentiation antigen CD90 and HL point
Change antigens c D105.HL's differentiation antigen CD45 and human leucocyte antigen (HLA) HLA-DR is not expressed.
Claims (10)
1. human amnion mesenchymal stem cell preserves liquid, it is characterised in that be made up of following compositions：
Sodium citrate (C6H5Na3O7·2H2O) 12-15g, citric acid (C6H8O7·H2O) 3-6g, glucose (C6H12O6·H2O)
12-16g, human plasma 10ml, human serum albumin 10g, water 990ml.
2. human amnion mesenchymal stem cell according to claim 1 preserves liquid, it is characterised in that the human amnion mesenchymal
Stem cell preserving fluid is made up of following compositions：
Sodium citrate (C6H5Na3O7·2H2O) 13-14g, citric acid (C6H8O7·H2O) 4-5g, glucose (C6H12O6·H2O)
14-15g, human plasma 10ml, human serum albumin 10g, water 990ml.
3. human amnion mesenchymal stem cell according to claim 2 preserves liquid, it is characterised in that the human amnion mesenchymal
Stem cell preserving fluid is made up of following compositions：
Sodium citrate (C6H5Na3O7·2H2O) 13.2g, citric acid (C6H8O7·H2O) 4.8g, glucose (C6H12O6·H2O)
14.7g, human plasma 10ml, human serum albumin 10g and water 990ml.
4. the human amnion mesenchymal stem cell according to any one of claims 1 to 3 preserves liquid, it is characterised in that：Institute
State human plasma behaviour AB blood plasma.
5. human amnion mesenchymal stem cell according to claim 4 preserves liquid, it is characterised in that：The human amnion mesenchymal
The pH value of stem cell preserving fluid is 7.2-7.4.
6. the store method of human amnion mesenchymal stem cell, it is characterised in that：Human amnion mesenchymal stem cell is stored in into right
It is required that the human amnion mesenchymal stem cell any one of 1 to 5 is preserved in liquid.
7. the store method of human amnion mesenchymal stem cell according to claim 6, it is characterised in that：Between people's amnion
Content of the mesenchymal stem cells in the preservation liquid is 2 × 108-3×108Individual cell/L.
8. the store method of the human amnion mesenchymal stem cell according to claim 6 or 7, it is characterised in that：The preservation
Temperature be 2-8 DEG C.
9. the store method of human amnion mesenchymal stem cell according to claim 8, it is characterised in that：The temperature of the preservation
Spend for 4 DEG C.
10. the store method of human amnion mesenchymal stem cell, it is characterised in that：By human amnion mesenchymal stem cell by matching somebody with somebody as follows
Preserve in the solution of the material composition of ratio：Sodium citrate (C6H5Na3O7·2H2O) 13.2g, citric acid (C6H8O7·H2O)4.8g、
Glucose (C6H12O6·H2O) 14.7g, people AB blood plasma 10ml, human serum albumin 10g and water 990ml；Fill between everybody amnion
The content of matter stem cell is 2 × 108-3×108Individual cell/L；The time of preservation is 12-24 hours.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108124853A (en) * | 2017-12-29 | 2018-06-08 | 天津普瑞赛尔生物科技有限公司 | A kind of store method of mescenchymal stem cell |
| CN112841170A (en) * | 2021-01-09 | 2021-05-28 | 于威 | Preservation solution and preservation method for maintaining activity of amniotic mesenchymal stem cells |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108124853A (en) * | 2017-12-29 | 2018-06-08 | 天津普瑞赛尔生物科技有限公司 | A kind of store method of mescenchymal stem cell |
| CN112841170A (en) * | 2021-01-09 | 2021-05-28 | 于威 | Preservation solution and preservation method for maintaining activity of amniotic mesenchymal stem cells |
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Application publication date: 20170426 |