CN101210232A - Mesenchyme stem cell preserving fluid and use thereof - Google Patents
Mesenchyme stem cell preserving fluid and use thereof Download PDFInfo
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Abstract
The invention discloses a preservation solution for mesenchymal stem cells and application thereof. The cell preservation solution contains human albumin and heparin as the main components, and other auxiliary reagents such as human cytokine, phosphate ion, metal ions or monosaccharide are contained in a buffer solution for preserving human mesenchymal stem cells. The preservation solution can keep high survival rate of human mesenchymal stem cells in transportation process, reduce adhesion between cells and between the cell and the inner wall of a container, and reduce the possible occurrence of cell mass embolism in blood vessel while clinically infusing human mesenchymal stem cells. The mesenchymal stem cells can be maintained in a state of single-cell suspension at an environment temperature of 4 to 15 DEG C for 24 h, thus greatly enlarging the clinic application range of the human mesenchymal stem cells. The components used in the solution accord with the clinic application, and can meet the requirement for clinic use of the human mesenchymal stem cells.
Description
Technical field
The present invention relates to biological technical field, especially a kind of mesenchyme stem cell preserving fluid and uses thereof.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs) be to derive to grow an early stage mesoderm and an ectodermic class multipotential stem cell, in marrow, find at first, be subjected to people's attention day by day: 1) hematopoiesis support and short the implantation because of it has following characteristics.As the main cellular constituent of hematopoieticmicroenviron-ment---the ancestral cells of stromal cell lines, MSCs realizes the finely regulating to hematopoiesis, the growth of hematopoiesis support stem/progenitor cells by direct effect, secretory cell epimatrix and the various kinds of cell factor of cell pair cell.Can promote the implantation of hematopoietic stem/progenitor cells with the hemopoietic stem cell co-transplantation, for improving the umbilical cord blood transplantation success ratio, it is significant to widen the umbilical cord blood transplantation scope of application.2) immunoregulation.MSCs expresses the MHC-I quasi-molecule, do not express CD80, CD86, collaborative stimulation molecule such as HLA-DR, externally suppress mixed lymphocyte reacion, inducing immune tolerance in the body for the graft versus host disease (GVH disease) that causes behind prevention and the treatment recessive allele hematopoietic cell transplantation, induces the tolerance of organ transplantation immunity that application promise in clinical practice is arranged.3) gene therapy.Because MSCs amplification in vitro, multiplication capacity are strong, can be used as the stem cell platform of genetic manipulation, very tempting prospect is arranged in gene therapy.4) multidirectional differentiation potential.In vivo/can be divided into multiple histocytes such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium under the outer specific inductive condition, still have multidirectional differentiation potential after continuous passage cultivation and the freezing preservation, can be used as the ideal seed cell and be used for organizational project.
Mescenchymal stem cell MSCs is an attached cell, is prepared into suspension and is easy to assemble agglomerating later on, is easy to take place the cell mass vascular embolization by blood infusion MSCs when doing experimentation on animals, causes the death of laboratory animal." primary mesenchymal stem cells " injection liquid phase clinical study that is used for leukemia treating of China's development at present.The umbilical cord mesenchyma stem cell of country's stem cell line systems engineering important component part is also set up in Tianjin.Mescenchymal stem cell acceleration from now on is trend of the times in clinical application, is very important so study a kind of effective mesenchyme stem cell preserving fluid.Can make present human mesenchymal stem cell can only be at home among a small circle application extension to the whole nation, and even surrounding countries.And can carry out at present that people MSCs separates and the unit of large scale culturing seldom, and horse back infusion after need finishing dealing with cell, in case after the placement several hrs, it is agglomerating a large amount of gatherings of MSCs to occur, bring danger for patient's infusion, make the scope that to carry out the MSCs clinical application be very limited.It is agglomerating that present most laboratory generally uses diethylamine tetraacethyl (EDTA) to suppress the mescenchymal stem cell gathering, EDTA (clinical in claiming edetate) is a kind of sequestrant, clinical disodium salt or calcium disodium salt with edetic acid, edetate has teratogenesis in experimentation on animals, though zinc can resist this effect, but what we preserved is mescenchymal stem cell, for caution's sake preferably without edetate.We find that heparin also can be used for suppressing mescenchymal stem cell and assemble agglomerating.Heparin is present clinical anticoagulant commonly used, and the heparin of clinical usefulness is generally heparinates such as heparin sodium, calciparine, Lithium heparinate, low molecular sodium heparin and low molecular heparin calcium.But Low molecular heparin and unfractionated heparin ratio have long half time, the advantage that biological activity is high, and hemorrhage side effect is few, can monitor coagulation indexes during with Low molecular heparin, relatively safety.The bioavailability height of low molecular heparin calcium, the anti thrombotic action time is 24h, and bleeding tendency is seldom arranged.So selecting for use low molecular heparin calcium to suppress MSCs in experiment assembles agglomerating.
It is general with containing the high viability that serum keeps cell to preserve cell at present, but also be not fit to the human serum that clinical infusion uses at present, and the serum composition complexity, quality is unexpectedly controlled, nutritive ingredient is more relatively, and the gathering that can increase MSCs in the high viability that keeps cell is agglomerating.And the albumin of suitable concn also can keep the high viability of cell within a certain period of time.Clinical widely used albumin preparation is also arranged at present.In experiment and clinical application, also there is the people singly to use albumin to keep the high viability of cell.Up to the present find no the people and in mesenchyme stem cell preserving fluid, use albumin and heparin simultaneously.
Summary of the invention
Technical problem to be solved by this invention is, a kind of mesenchyme stem cell preserving fluid and uses thereof is provided, can make the maintenance high viability of human mesenchymal stem cell in transportation, reduced between the cell and the sticking of cell and container inner wall, occurred the possibility of cell mass embolism when reducing clinical infusion human mesenchymal stem cell in the blood vessel.
In order to address the above problem, the technical solution used in the present invention is: a kind of human mesenchymal stem cell is preserved liquid, comprises 0.01%-20% albumin, 1-5000AXaIU/ml heparin and solution medium.
Described albumin is the human serum albumin that clinical approval is used.
Described heparin is the heparinate that clinical approval is used.
Described heparinate is a kind of in low molecular heparin calcium or the low molecular sodium heparin.
Described solution medium is no bacterium, virus, mycoplasma contamination, the liquid medium of endotoxin content＜5EU/ml.
Described solution medium mainly contains serum free medium or the phosphate buffered saline buffer of physiological saline, TC199.
Aforesaid human mesenchymal stem cell is preserved liquid, is used to preserve the mescenchymal stem cell that the source comprises all or part of histoorgan of marrow, blood, fat, umbilical cord, placenta, amniotic fluid, skin, liver, muscle, blood vessel, nerve, lung, kidney, brain, miscarriage embryo/fetus.
Beneficial effect of the present invention: mesenchyme stem cell preserving fluid of the present invention, contain human albumin and heparin as main component, other auxiliary reagents such as human cytokine, phosphate anion, metal ion or monose etc. are included in the buffered soln that depositary's mescenchymal stem cell adopted together.Above-mentioned cell-preservation liquid can make the maintenance high viability of human mesenchymal stem cell in transportation, has reduced between the cell and the sticking of cell and container inner wall, and occurs the possibility of cell mass embolism when reducing clinical infusion human mesenchymal stem cell in the blood vessel.It is main state that cell-preservation liquid of the present invention can make mescenchymal stem cell keep single cell suspension in the time of envrionment temperature 4-15 ℃ in 24 hours, the mescenchymal stem cell that this cell-preservation liquid is preserved can be sent to all parts of the country and surrounding countries by the modern transportation instrument, increased the scope of clinical end user's mescenchymal stem cell greatly, used composition is the composition that meets clinical use, can satisfy the needs of clinical end user's mescenchymal stem cell.
Description of drawings
Fig. 1 is the streaming result that the immunophenotype of umbilical cord mesenchymal stem cells is analyzed;
Fig. 2 is that artificial the setting judges that MSC assembles the standard of situation, is divided into level Four according to the weight of aggregation extent :+, ++, +++, ++ ++;
Fig. 3 respectively organized the gathering situation under the different preservation conditions after 24 hours; Be respectively the image of EDTA+1% albumin group, low molecular heparin calcium+1% albumin group and 1% albumin group aggregation extent from left to right.
Fig. 4 to be mescenchymal stem cell preserve in preserving liquid streaming result that immunophenotype is analyzed after 24 hours, on be 0 hour, is 24 hours down.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
Human mesenchymal stem cell of the present invention is preserved liquid, comprises 0.01%-20% albumin, 1-5000AXaIU/ml heparin and solution medium.Described albumin is the human serum albumin that clinical approval is used.
Described heparin is the heparinate that clinical approval is used.Described heparinate is a kind of in low molecular heparin calcium or the low molecular sodium heparin.Described solution medium is no bacterium, virus, mycoplasma contamination, the liquid medium of endotoxin content＜5EU/ml.Described solution medium mainly contains serum free medium or the phosphate buffered saline buffer of physiological saline, TC199.
Aforesaid human mesenchymal stem cell is preserved liquid, is used to preserve the mescenchymal stem cell that the source comprises all or part of histoorgan of marrow, blood, fat, umbilical cord, placenta, amniotic fluid, skin, liver, muscle, blood vessel, nerve, lung, kidney, brain, miscarriage embryo/fetus.
Solution medium in the mesenchyme stem cell preserving fluid can be used physiological saline, serum free medium etc.In experimenting, we find out that with mescenchymal stem cell at physiological saline, albumin, preserve above 6 hours later survival rates in the preservation liquid that low molecular heparin calcium is configured to and be lower than serum free medium, albumin, the survival rate in the preservation liquid that low molecular heparin calcium is configured at TC199.And the solution medium of the medullary cell that is used for when at present the TC199 serum free medium often is used as clinical bone marrow transplantation suspending.So we select for use the TC199 serum free medium as the solution medium of preserving in the liquid.
Anticoagulin Xa international unit/milliliter is expressed as AXaIU/ml.
Separation and the cultivation and the phenotypic evaluation of embodiment 1 people's umbilical cord cell
(1) external, people's umbilical cord is washed repeatedly by phosphoric acid buffer (PBS), remove residual blood, shred to about 1mm
3Fritter, add the collagenase of raw material weight 0.1%, 37 ℃ of digestion 45 minutes; (2) add raw material weight 0.125% pancreatin (Sigma, USA) 37 ℃ of digestion is 45 minutes, stirs with rotor in digestive process, adds the effect of an amount of serum termination pancreatin; (3) Digestive system screen filtration is successively used 100 orders and 200 purpose strainer filterings, removes indigested tissue; (4) Digestive system after will filtering dilutes with phosphoric acid buffer, and Digestive system and phosphoric acid buffer liquid proportional are 1: 10 ratio; (5) centrifugal, obtain cell, the rotating speed of whizzer is the centrifugal 15min of 1500rpm/min, obtains cell; (6) cell is by 1～2 * 10
1/ cm
2Be inoculated in the plastic culture bottle, (Sigma, USA), (Gibco BRL, substratum USA) are put 37 ℃, 5%CO to 5% foetal calf serum (FCS) with containing the DMEM-LG/F12 substratum
2Incubator is cultivated, and changes liquid after 4-5 days, discards non-adherent cell, and half amount was changed liquid in later every 3-4 days.Treat that cell 80% merges, 0.25% trysinization was gone down to posterity by 1: 3.
The conventional digestion of attached cell back is with CD45, CD34, CD31 and the CD90 of FITC mark, the CD29 of PE mark, CD44, CD73, CD105, HLA-DR antibody and corresponding homotype contrasting marking cell thereof, flow cytometer (FACA Calibur type, USA) detection.Above fluorescent-labeled antibody is all available from BD company (USA).
The streaming immunophenotype detected result of umbilical cord mesenchymal stem cells shows: umbilical cord mesenchymal stem cells high expression level CD90, CD29, CD44, CD73 and CD105, do not express CD45, CD34, CD31 and HLA-DR.(Fig. 1)
The solution medium that embodiment 2 preserves in the liquid and preparation method thereof is selected
1.1 operation steps
1.1.1 contain the TC199 and the preparation that contains 1% human serum albumin physiological saline of 1% human serum albumin.
1.1.2 with the TC199 that contains 1% human serum albumin with contain 1% human serum albumin physiological saline and prepare umbilical cord mesenchymal stem cells suspension, 5 * 10 respectively
5/ ml/ml.Packing 6 pipes (preserving 3 parallel pipes of liquid for every kind), the 2.5ml/ pipe.
1.1.3 the umbilical cord mesenchymal stem cells suspension is transferred to respectively in the cillin bottle, places 4 ℃, after the preservation in 8 hours, platform is expected blue dyeing counting cell survival rate.
The result judges: dead cell can be expected that orchid catches look by platform, visible navy blue cell under the mirror, and viable cell is not colored, and is the water white transparency shape under the mirror.
The result:
| The TC199 group | The physiological saline group | |
| The counting cells number | 300 | 300 |
| Painted total cellular score | 19±2 | 167±16 |
| Cell survival rate % | 93.7±0.7 | 44.3±5.3 |
The selection of embodiment 3 human serum albumin concentration
1.1 operation steps
1.1.1 contain the preparation of 5%, 2%, 1% and 0.5% human serum albumin TC199.
1.1.2 preparation umbilical cord mesenchymal stem cells suspension, 5 * 10
5/ ml/ml.Packing 12 pipes (preserving 3 parallel pipes of liquid for every kind), the 2.5ml/ pipe.
1.1.3 800rpm, after 5min is centrifugal, supernatant discarded.Add the various preservation liquid that 2.5ml prepares respectively.
1.1.4 transfer to respectively behind the cell mixing in 12 cillin bottles, place 4 ℃, after the preservation in 8 hours, platform is expected blue dyeing counting cell survival rate.
The result judges: dead cell can be expected that orchid catches look by platform, visible navy blue cell under the mirror, and viable cell is not colored, and is the water white transparency shape under the mirror.
The result:
1.1 operation steps
1.1.1 prepare respectively with the TC199 that contains 1% human serum albumin and to contain 0.01%EDTA, 75AXaIU/ml heparin sodium, the preservation liquid of 75AXaIU/ml low molecular heparin calcium.
1.1.2 preparation umbilical cord mesenchymal stem cells suspension, 5 * 10
5/ ml.Packing 9 pipes (preserving 3 parallel pipes of liquid for every kind), the 2.5ml/ pipe.
1.1.3 800rpm, after 5min is centrifugal, supernatant discarded.Add the various preservation liquid that 2.5ml prepares respectively.
1.1.4 transfer to respectively behind the cell mixing in 9 cillin bottles, respectively at 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, the 24 hours time points of preserving, sampling and measuring cell survival rate.
1.1.5 the result judges: dead cell can be expected that orchid catches look by platform, visible navy blue cell under the mirror, and viable cell is not colored, and is the water white transparency shape under the mirror.
The result:
| 0.01％EDTA | The 75AXaIU/ml heparin sodium | The 75AXaIU/ml low |
|
| 0 hour | 100％ | 100％ | 100％ |
| 2 hours | 91.30％ | 93.30％ | 98.41％ |
| 4 hours | 84.57％ | 91.54％ | 97.65％ |
| 6 hours | 75.19％ | 87.36％ | 95.19％ |
| 8 hours | 69.67％ | 83.49％ | 94.3％ |
| 24 hours | 57.58％ | 74.71％ | 90.57％ |
Embodiment 5 EDTA, low molecular heparin calcium influences the umbilical cord mesenchymal stem cells accumulative
1.1 operation steps
1.1.1 prepare respectively with the TC199 that contains 1% human serum albumin and to contain 0.01%EDTA, the preservation liquid of 75AXaIU/ml low molecular heparin calcium.
1.1.2 preparation umbilical cord mesenchymal stem cells suspension, 5 * 10
5/ ml.Packing 9 pipes (preserving 3 parallel pipes of liquid for every kind), the 2.5ml/ pipe.
1.1.3 800rpm, after 5min is centrifugal, supernatant discarded.Add the various preservation liquid that 2.5ml prepares respectively.
1.1.4 transfer to respectively behind the cell mixing in 6 cillin bottles, respectively at 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, the 24 hours time points of preserving, microscope is according to imaging under 4 times of object lens.
1.1.5 the result judges: judge the gathering situation according to standard (Fig. 2).With set wherein a certain aggregation extent and just be corresponding+number number when suitable, as: ++; Before being written as between certain two aggregation extent ++ number number/back one has added number, as :+/ ++.
Result: EDTA+1% albumin group: ++, low molecular heparin calcium+1% albumin group :-/+, 1% albumin group: +++/ ++ ++.(Fig. 3)
Embodiment 6 mescenchymal stem cells are preserved the analysis of immunophenotype after 24 hours in preserving liquid
Preserve cell at preservation liquid and get 1 * 10 respectively after 0 hour and 24 hours
6Individual cell, with the CD45 of the method indicia band FITC mark that the professional was familiar with, the CD73 of PE mark, CD105 and HLA-DR streaming antibody and corresponding homotype contrasting marking antibody thereof, (FACA Calibur type USA) detects flow cytometer.Above fluorescent-labeled antibody is all available from BD company (USA).
Flow cytometer detects to be preserved before the liquid preservation respectively and the cellular immunization phenotype of preservation after 24 hours.Showed cell high expression level CD105, CD73 do not express hematopoietic cell phenotype HLA-DR, CD45 as a result, and immunophenotype does not have considerable change.Streaming figure sees (Fig. 4)
The effect of embodiment 7 preservation liquid with the mescenchymal stem cell that keeps derived from bone marrow
The mescenchymal stem cell of derived from bone marrow is digested, counts with the method that the professional knows, prepare mesenchymal stem cells MSCs suspension, 5 * 10 with the TC199 that contains 1% human serum albumin and 75AXaIU/ml low molecular heparin calcium
5/ ml.Transfer to respectively behind the cell mixing in 6 cillin bottles, respectively at 0 hour, 2 hours, 6 hours, 12 hours, 18 hours, the 24 hours time points of preserving, microscope is according to imaging under 4 times of object lens.Judge the gathering situation according to standard (Fig. 2).
The result:
| Time (hour) | 0 | 2 | 6 | 12 | 18 | 24 |
| State of aggregation | - | - | - | - | -/+ | -/+ |
The cell-preservation liquid of the present invention preparation can keep the mescenchymal stem cell in various sources, can make the maintenance high viability of mescenchymal stem cell in transportation, has reduced between the cell and the sticking of cell and container inner wall.Can satisfy the needs of clinical end user's mescenchymal stem cell, increase the scope of clinical end user's mescenchymal stem cell greatly.
Claims (7)
1. a human mesenchymal stem cell is preserved liquid, it is characterized in that, comprises 0.01%-20% albumin, 15000AXaIU/ml heparin and solution medium.
2. human mesenchymal stem cell according to claim 1 is preserved liquid, it is characterized in that, described albumin is the human serum albumin that clinical approval is used.
3. human mesenchymal stem cell according to claim 1 is preserved liquid, it is characterized in that, described heparin is the heparinate that clinical approval is used.
4. human mesenchymal stem cell according to claim 3 is preserved liquid, it is characterized in that, described heparinate is a kind of in low molecular heparin calcium or the low molecular sodium heparin.
5. human mesenchymal stem cell according to claim 1 is preserved liquid, it is characterized in that described solution medium is no bacterium, virus, mycoplasma contamination, the liquid medium of endotoxin content＜5EU/ml.
6. human mesenchymal stem cell according to claim 5 is preserved liquid, it is characterized in that described solution medium mainly contains serum free medium or the phosphate buffered saline buffer of physiological saline, TC199.
7. human mesenchymal stem cell as claimed in claim 1 is preserved liquid, is used to preserve the mescenchymal stem cell that the source comprises all or part of histoorgan of marrow, blood, fat, umbilical cord, placenta, amniotic fluid, skin, liver, muscle, blood vessel, nerve, lung, kidney, brain, miscarriage embryo/fetus.
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