CN105284787A - Preservation reagent for mesenchymal stem cells and application thereof - Google Patents
Preservation reagent for mesenchymal stem cells and application thereof Download PDFInfo
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Abstract
The invention provides a preservation reagent for mesenchymal stem cells and application of the preservation reagent to preserving the mesenchymal stem cells. The preservation reagent is prepared from 80-100 mmol/L glucose, 40-50 mmol/L mannitol, 24-28 mmol/L dipotassium hydrogen phosphate, 12-16 mmol/L potassium dihydrogen phosphate, 3-4 mmol/L adenine, 1-1.5 mmol/L inosine, 14-18 mmol/L potassium citrate, 35-45 mmol/L ammonium chloride, 75-80 mmol/L sodium chloride and cellular antioxidants. Due to the adoption of the preservation reagent for the mesenchymal stem cells, basic life metabolism of the cells is maintained through glucose, potassium citrate and other few nutrients, and meanwhile no large number of rich nutrients are added so that fast metabolism and proliferation of the cells can be avoided; by means of further assistance of adenine, inosine and mannitol, the shapes and the characters of the cells are maintained, the purposes of protecting cell membranes and enzyme activity are achieved, and the cell activity does not degrade in the preservation process; finally, oxidization damage of the cells is inhibited through the cellular antioxidants, and compared with existing common preservation liquid, the cells have better activity and quality after being preserved.
Description
Technical field
The invention belongs to cell-preservation liquid technical field, be specifically related to a kind of preservation reagent and application thereof of mesenchymal stem cells MSCs.
Background technology
Stem-cell therapy is after being cultivated in vitro by the stem cell with continuous self-reproduction ability and multidirectionalization potential, then is implanted into the impaired or defective tissue of patient, thus realizes supplementing and repairing damaged tissue.And human marrow mesenchymal stem cell can be realized at present to be separated and the cell of large-scale culture is little, after cell chulture completes, also need that Phenotypic examination is carried out to cell simultaneously, cell shape is analyzed etc., afterwards cell is kept in conserving liquid, carries out infusion more when needed.Carry out in the stage of preserving after human marrow mesenchymal stem cell is cultivated, the basic vital metabolic that should maintain cell, to prevent its death, avoids its metabolism amount excessive and a large amount of shapes variations of continuation a large amount of propagation generation simultaneously again.So in cell preservation, all adopt 0.9WT% sodium chloride injection, 5WT% glucose injection, Multiple electrolytes injection (PlasmalyteA), 1 ~ 5WT% human serum albumin solution etc. to carry out as the preservation medium of cell.
But in the process that cell is preserved, the conserving liquid of above-mentioned MSCs is adopted to preserve, when needing the holding time shorter, the character of (within 4h) cell can also maintain more normal level with change, and the degree that cytoactive reduces also significantly can not reduce the quality for the treatment of.If but the time of preserving upper to increase further, when preserving slightly for a long time, first cell there is phenotype mutation, actively to reduce, assemble the tendency such as agglomerating and Character change more obvious, these all can reduce the effect of qualitative effects treatment of cell; And above-mentioned conserving liquid itself does not have the ability alleviating or reduce problem in these preservation processes, after preserving, cytoactive reduces greatly, slowly turns on dormancy or apoptosis.
Summary of the invention
Object of the invention process is to overcome existing defect, provides a kind of and can maintain its cellular metabolism state and active preservation reagent in mesenchymal stem cells MSCs is preserved.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
A preservation reagent for mesenchymal stem cells MSCs, comprises the glucose of 0 ~ 100mmol/L, the mannitol of 40 ~ 50mmol/L, the dipotassium hydrogen phosphate of 24 ~ 28mmol/L, the potassium dihydrogen phosphate of 12 ~ 16mmol/L, the adenine of 3 ~ 4mmol/L, the inosine of 1 ~ 1.5mmol/L, 14 ~ 18mmol/L potassium citrate, the ammonium chloride of 35 ~ 45mmol/L, the sodium chloride of 75 ~ 80mmol/L and cellular antioxidants.
The preservation reagent of above-mentioned mesenchymal stem cells MSCs of the present invention, maintains the basic vital metabolic of cell by glucose, a few nutrition of potassium citrate, does not add a large amount of abundant nutrition simultaneously and avoids its a large amount of tachymetabolism and propagation; And assist adenine, inosine, mannitol to maintain form and the proterties of cell self further, the object of Cell protection film and enzymic activity, makes cytoactive in the process of preservation not degenerate; Finally with the oxidative damage of cellular anti-oxidant agent inhibit cell, compare existing common conserving liquid, after preserving, cell has better active and quality.
On the basis of the preservation reagent of above-mentioned mesenchymal stem cells MSCs of the present invention, the present invention also propose further its mesenchymal stem cells MSCs preserve in application.Adopt above-mentioned preservation reagent to preserve mesenchymal stem cells MSCs, the energy generating mechanism of Cell protection in preservation, makes it maintain normal configuration and function; And the cell metabolization rate that as far as possible slows down, reduce energy ezpenditure and supplying energy material, the form simultaneously also maintaining cell suppresses and breaks up and damage, and after comparing existing common conserving liquid preservation, cell has better activity and form.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Example of the present invention proposes a kind of preservation reagent of mesenchymal stem cells MSCs, and this preservation agent formulations includes the glucose of 80 ~ 100mmol/L, the mannitol of 40 ~ 50mmol/L, the dipotassium hydrogen phosphate of 24 ~ 28mmol/L, the potassium dihydrogen phosphate of 12 ~ 16mmol/L, the adenine of 3 ~ 4mmol/L, the inosine of 1 ~ 1.5mmol/L, 14 ~ 18mmol/L potassium citrate, the ammonium chloride of 35 ~ 45mmol/L, the sodium chloride of 75 ~ 80mmol/L and cellular antioxidants.
The preservation reagent of above-mentioned mesenchymal stem cells MSCs of the present invention, the while of being metabolic activity basic based on maintenance cell, being the energy generating mechanism of Cell protection on the one hand, making it maintain normal configuration and function; Two is the cell metabolization rate that as far as possible slow down, and reduces energy ezpenditure and supplying energy material; And auxiliary reparation composition, reduces the cellular damage in preservation process.
Wherein, cellular antioxidants relatively in important complementarity cellular damage reparation and suppress engineering composition, energy scavenging free radicals and the material suppressing free radical activity, the formation of free radical can be prevented, or prevent them to be combined with other cellular elements after free radical is formed, thus free radical is stoped to cause aging and the oxidative damage of cell.In force, above-mentioned cellular oxidation agent can adopt one or more in superoxide dismutase (SOD), glutathione peroxidase, vitamin C, vitamin E and reduced glutathione.
Certainly, most preferably superoxide dismutase (SOD), be indispensable, the important free scavenger of oxygen of human body relatively, it is using the free radical of instability as catalytic substrate.This enzyme is important for the survival of aerobic cell, it catalysis can remove superoxide radical, the enzyme of to be also find so far unique with free radical be substrate, thus biometric safeguard machine interior free yl produce with the dynamic equilibrium of removing in play extremely important effect.Compare cellular antioxidants in other base, its direct scavenging activated oxygen, in effect definitely, stronger to suppressing the specific aim of oxidative cell damage.
In addition; glutathione peroxidase is the peroxide breakdown enzyme extensively existed in body; for activated centre with selenocystein structure; GSSG can be become by catalysis GSH; poisonous peroxide is made to be reduced into nontoxic hydroxy compounds; thus the structure of Cell protection film and function be not by interference and the infringement of peroxide, mainly for the injury repair in membrane structure.Other several, vitamin C, vitamin E and reduced glutathione are all the biotic components with reproducibility, can slow down the oxidative damage of the oxidation material causing cellular oxidation to damage.
Further in the process implemented, because the type of above-mentioned cellular antioxidants is not identical with composition, superoxide dismutase and glutathione peroxidase belong to protein enzyme, vitamin C, vitamin E belong to reproducibility vitamin, reduced glutathione is oligopeptides, add according to the amount be applicable to separately respectively in the process of adding, there is not unified standardized amount (enzyme measures in the mode such as mol, quality with unit of activity U metering, chemical composition) each other.Such as, in enforcement superoxide dismutase in preservation reagent, to control addition 35000 ~ 45000U/L more moderate; Glutathione peroxidase its be peroxide substrate catalyzing enzyme, compare the amount of peroxides of generation, control the concentration range of interpolation 20000 ~ 25000U/L; Both vitamin C and vitamin E add 3 ~ 10mmol/L, when there being above-mentioned enzyme collaborate to use, adopt low amounts to add in the above range; Simultaneously, because reduced glutathione self is also polypeptide, addition is many likely can carry out destructive metabolism as C, N source, also differentiation can may be produced by inducing cell, control low amounts 1 ~ 1.5mmol/L, when being that above-mentioned multiple antioxidant uses jointly, low amounts can be adopted under above-mentioned scope to add.
When cell oxidative damage is well alleviated, namely remaining composition is in the employing that the energy generating mechanism of above-mentioned Cell protection makes it maintain normal configuration and function and slow down cell metabolization rate supplying energy material carry out.Such as, wherein glucose as the basic life energy; Potassium citrate and sodium chloride maintain the normal operation of sodium potassium pump (Na+-K+-ATPase) on erythrocyte membrane; Adenine is used for maintaining and recovering ATP, DPG level, can also stablize stem cell morphology; Inosine and phosphate are form buffer system on the one hand, maintain environment pH, can also play the object of Cell protection film and enzymic activity simultaneously; Mannitol and sodium chloride can maintain the crystal and colloidal substance that are applicable to cells survival, keep suitable extracellular osmotic pressure, the membrane structure of stabilized cell.As can be seen from above-mentioned selection, there is not a large amount of multiple nutrients, in preservation, control cell metabolization rate remain at low levels.
Under the conception of above-mentioned basic function, mesenchymal stem cells MSCs own belongs to the stem cell than being easier to break up, if so preservation reagent self is provided with the composition that inducing cell phenotype changes, cell just probably can be caused in the process of preserving to produce differentiation; Do not add in conjunction with in this case the composition that cell factor etc. has inducing effect, and with inosine, mannitol and sodium chloride stabilizing cell membrane Structure and form in external environment, can more well T suppression cell character mutation or break up.
Meanwhile, preserving reagent in use, is all be placed in Conventional spatial by cell under very most of situation, instead of gnotobasis, so be necessary to add the antibiotic agent suppressing living contaminants further; Based in this case to the situation that mesenchymal stem cells MSCs is preserved, preserving in reagent the chloramphenicol adopting interpolation 5 ~ 10mmol/L, the antibiotic of other types compared by chloramphenicol, and it belongs to biocidal property broad-spectrum antibiotic.Be adopt based on its broad spectrum activity on the one hand in the present invention, do not reach good fungistatic effect to avoid narrow spectrum antibiotic antibacterial type very few; On the other hand, the type of its mainly biocidal property, instead of bactericidal type; Can avoid substantially like this producing the metabolism of mesenchymal stem cells MSCs self killing and wounding.
Meanwhile, above-mentioned preservation reagent of the present invention is in the process of preparation, although most composition is all water miscible (vitamin E belongs to fat-soluble) substantially, simultaneously chloramphenicol dissolves slow and solvability is very low in cold water; So the conserving liquid of this case configuration process in the composition except the enzyme of cellular antioxidants all can have been added after, overall possibility can not directly be dissolved completely, but suspension jelly, can adopt suitably be heated to suspension present more homogeneous after carry out packing and high-temperature sterilization process; And after completion of the sterilization cycle, finally add superoxide dismutase (SOD) and glutathione peroxidase again, cause deactivation in follow-up sterilization treatment to avoid adding in advance.Simultaneously, after configuration, immediately mentioned reagent may might not be carried out cell preservation, and superoxide dismutase (SOD) and glutathione peroxidase may self may or easily be degraded or inactivation in the process of long-term storage, preserve stored frozen after can configuring in the manner described above so long-term; Or after having configured, temporarily do not add superoxide dismutase (SOD) and glutathione peroxidase these two kinds, and add between use.
Adopt the preservation reagent of above-mentioned mesenchymal stem cells MSCs of the present invention, maintain the basic vital metabolic of cell by glucose, a few nutrition of potassium citrate, do not add a large amount of abundant nutrition simultaneously and avoid its a large amount of tachymetabolism and propagation; And assist adenine, inosine, mannitol to maintain form and the proterties of cell self further, the object of Cell protection film and enzymic activity, makes cytoactive in the process of preservation not degenerate; Last with the oxidative damage of cellular anti-oxidant agent inhibit cell, maintain activity and the quality of cell as much as possible.
On the basis of the preservation reagent of above-mentioned mesenchymal stem cells MSCs of the present invention, the present invention also propose further its mesenchymal stem cells MSCs preserve in application, above-mentioned preservation reagent is adopted to preserve mesenchymal stem cells MSCs, the energy generating mechanism of Cell protection in preservation, makes it maintain normal configuration and function; And the cell metabolization rate that as far as possible slows down, reduce energy ezpenditure and supplying energy material, the form simultaneously also maintaining cell suppresses and breaks up and damage, and after comparing existing common conserving liquid preservation, cell has better activity and form.
Implement to be easier to the understanding of those skilled in the art for making being prepared in during mesenchymal stem cells MSCs is preserved of above-mentioned preservation reagent of the present invention and implement reference, highlight the quality effect that the present invention preserves reagent simultaneously, be illustrated below by way of specific embodiment.
Embodiment 1
S10, configures mesenchymal stem cells MSCs of the present invention and preserves reagent, according to following concentration of preserving agent formulations, calculates according to the gauge of configuration 5L volume and obtains each composition:
Glucose 90mmol/L, mannitol 45mmol/L, dipotassium hydrogen phosphate 25mmol/L, potassium dihydrogen phosphate 14mmol/L, adenine 3.5mmol/L, inosine 1.3mmol/L, potassium citrate 16mmol/L, ammonium chloride 40mmol/L, sodium chloride 77mmol/L;
And, chloramphenicol 7mmol/L, superoxide dismutase 40000U/L, vitamin C 3mmol/L.
Then the mesenchymal stem cells MSCs configuring this case in accordance with the following steps preserves reagent:
Mentioned component 3L water for injection outside chloramphenicol and superoxide dismutase is dissolved and makes main liquid;
Then being equipped with in the bottle of chloramphenicol of purchase is injected with water and after rocking 1 ~ 2min, be transferred in the main liquid of above-mentioned 3L with syringe; Certainly, chloramphenicol is added in main liquid and can repeats several times, is transferred in main liquid after the bottle water for injection that chloramphenicol is housed simultaneously repeatedly cleans;
Main liquid water-bath is heated slightly, to main liquid be suspended be reduced to solution comparatively homogeneous after carry out sterilization treatment, preserve in ice bath after sterilizing; By the time after ice bath enough time, finally add superoxide dismutase again, after dissolving, be settled to 5L, continue ice bath and save backup.(certainly in this enforcement, what antioxidant adopted only has superoxide dismutase a kind of, if also arranged in pairs or groups in force have the glutathione peroxidase of pyrosol inactivation, then the add step same with SOD can be adopted to add)
S20, obtains mesenchymal stem cells MSCs:
The marrow specimen being separated for primary MSCs and cultivating is collected, anticoagulant heparin when getting people's operation on hip joint or leg bone open operation;
Every part of marrow specimen is added in the centrifugal 30min of 2300rpm on the percoll parting liquid that density is 1.073g/ml in the ratio of 1:2, get middle level monocyte, add sterilization PBS buffer solution, the centrifugal 10min of 1000rpm, wash 3 times, the cell obtained after abandoning supernatant is MSCs.
Gained MSCs counts under an optical microscope, is adjusted to 1 × 10
7/ blake bottle (25ml) density is used for cell primary and cultivates, and the mode of cultivation adopts serum free culture system system to cultivate, and concrete medium is for containing 10% hyclone, hydrocortisone (10
-6mol/L), the low sugar DMEM (L-DMEM) of penicillin (100U/ml) and streptomycin (100mg/ml).
S30, MSCs go down to posterity and amplification in vitro:
When the Growth of Cells of step S10 is to when almost merging completely, incline old medium, and with PBS liquid rinsing 2 ~ 3 times, incline PBS liquid, add the trypsase (including 0.02%EDTA) of 0.25%, addition is as the criterion with the cell at the bottom of making trypsase can cover completely bottle.
Observe under blake bottle being placed in inverted microscope, see that kytoplasm bounces back, after space between cells increases, the low sugar DMEM added immediately containing 10% hyclone cultivates termination digestion, suction pipe order is blown and beaten at the bottom of bottle gently, under inverted microscope after the completely de-wall of observation of cell, go down to posterity in 1:2 ~ 1:3 ratio and be inoculated in 25ml blake bottle (medium adopts identical with step S20), be positioned over 37 DEG C, 5%CO
2, 95% humidity incubator in cultivate, until Growth of Cells is to when almost merging completely, again go down to posterity; Every day is observation of cell form and growing state under inverted microscope.
S40, when being completed in the cell chulture of step S20 to P3 generation, the mesenchymal stem cells MSCs obtaining P3 generation is used for food preservation test: after 0.25% trypsinization, cell suspension carries out cell counting, and about 1 × 10
6individual/m1, with every hole 1 × 10
4individually be inoculated in 96 well culture plates, in CO
2after incubator 37 DEG C of quiescent culture 30h, after sucked away culture fluid, add the existing conserving liquid of 3ml (0.9WT% sodium chloride injection, 5WT% glucose injection, Multiple electrolytes injection (PlasmalyteA), 1WT% human serum albumin solution or 5WT% human serum albumin solution are in contrast) and conserving liquid of the present invention after washing 2 times with corresponding conserving liquid, establish 3 identical multiple holes for each group; Activity rating is carried out respectively after placing 4h, 8h, 12h, 24h in 4 DEG C after changing liquid.
In the mode of the activity rating of the cell after above-mentioned steps S40 preserves with different conserving liquid, first detect the cell survival rate after preserving, method adopts trypan blue staining to detect:
MSCs after preserving with different conserving liquid (i.e. existing several conserving liquid and) is centrifugal through corresponding holding time point, l000rpm, 5min supernatant discarded; PBS re-suspended cell precipitates, and fully mix, the 0.4% trypan blu e dye liquor getting 18 μ l cell suspensions and 2 μ l fully mixes; Get appropriate suspension to be added drop-wise in blood counting chamber counting chamber, add cover glass, basis of microscopic observation; Living cell counting and dead cell respectively, counting process completes in 3min, and uncoloured cell is living cells, and dying blue cell is dead cell.
Cell survival rate (%)=viable count/total cell number × 100%.
| Sodium chloride injection | Glucose injection | Human albumin solution | Multiple electrolytes injection | The present invention | |
| 4h | 72.37±10.32％ | 78.54±9.64％ | 85.62±8.45％ | 75.24±8.67％ | 90.52±11.69％ |
| 8h | 43.70±10.28％ | 51.12±6.47％ | 60.42±7.97％ | 52.84±6.12％ | 81.26±12.91％ |
| 12h | 21.94±4.34％ | 23.65±5.65％ | 30.36±6.45％ | 25.13±6.25％ | 59.88±10.30％ |
| 24h | 4～6％ | 4～7％ | 8.21±3.51％ | 4～7％ | 27.70±5.29％ |
The result of calculation of the cell survival rate after 4h is preserved from the various conserving liquid of upper table, can find out except physiological saline (sodium chloride), after other several conserving liquid preservations, cell survival rate difference is little, although survival rate of the present invention is slightly higher, amplitude difference is but very not large.Reason is that the time of preservation is 4h, also cannot have the effect of very large embodiment because the time is shorter on the one hand.After the holding time continues to extend, the reduction Amplitude Ratio of the Apoptosis number after the preservation of usual conserving liquid is comparatively large, and the cell survival rate after last 24h after the preservation of routine preservation liquid is less than 10%, and this case obviously wants high.The quality of each conserving liquid can be embodied after extending the holding time, first conventional human albumin solution is the living matter as comparing balance, so higher than the cell survival rate after the preservation of conserving liquid in other base, and in the present invention, have a few seed amino acid and glucose jointly to sustain life, nutrition is more balanced, can also have relatively high survival rate after long-time preservation.
On the basis that above-mentioned basic survival rate detects, continue to carry out adherence rate mensuration further, filling dry between marrow is a kind of adherent type cell, its survival an index be exactly cell can adherent, propagation, namely the difference of therefore filling dry doubling wall performance between marrow reflect the difference of cell viability; Detect adherent rate to be used for reflecting the Lymphocytic phenotype degree of its survivaling cell and the activity of cell:
The P3 of above-mentioned cultivation is made cell suspension for after cell dissociation with PBS, with 2 × l0
6under/group is kept at corresponding preservation medium and storage temperature; Temporally put and preserve after 4h, 8h terminate respectively, centrifugal l000rpm, 5min, abandon supernatant; With normal perfect medium, rinsing cell is once gently; Use perfect medium re-suspended cell again, often group is with 2 × 10
4/ hole is inoculated in 24 orifice plates, is positioned in cell culture incubator and cultivates 24h; After cell attachment, micropipettor removes culture fluid, with PBS rinsing 2 times gently, removes non-attached cell; PBS rinsing 1 ~ 2 time gently to be fixed after 20min by 70% ethanol; Crystal violet dye liquor dyeing 15min (100 μ l, did not have bottom orifice plate); Deionized water rinsing 2 ~ 3 times gently, removes background color as far as possible; Be placed in basis of microscopic observation, count, take pictures; Adherence rate=attached cell sum/inoculating cell number × l00%.
| Sodium chloride injection | Glucose injection | Human albumin solution | Multiple electrolytes injection | The present invention | |
| 4h | 55.23±9.53％ | 48.22±7.58％ | 46.65±7.89％ | 51.22±11.80％ | 72.83±13.62％ |
| 8h | 25.62±4.21％ | 23.67±4.85％ | 15.68±2.54％ | 23.21±3.97％ | 54.97±17.61％ |
The Structure Comparison of the adherence rate after preserving above-mentioned preservation 4h can be found out, the contrast of adherent rate is obvious, the damage that Multiple electrolytes injection compares cell in the conserving liquid of existing routine or the situation causing its cell phenotype to change less, comparing in other base may be larger, and the cell comparing conserving liquid of the present invention preservation should be that adherent ability is weakened to half adherent degree, so the difference of structure can embody on the one hand at this.And when saved between continue to extend to 8h after, data more can embody the above results, after 8h, common conserving liquid substantially cell attachment performance is more weak, continue time expand to 12h, 24h again row detect substantially can predict adherent about 10%, to contrast embody difference when, continuations detection is not carried out in this case.Illustrate conserving liquid of the present invention maintain cell activity, prevent Apoptosis and turn in the problem of dormancy have obvious advantage.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. the preservation reagent of a mesenchymal stem cells MSCs, it is characterized in that, comprise the glucose of 0 ~ 100mmol/L, the mannitol of 40 ~ 50mmol/L, the dipotassium hydrogen phosphate of 24 ~ 28mmol/L, the potassium dihydrogen phosphate of 12 ~ 16mmol/L, the adenine of 3 ~ 4mmol/L, the inosine of 1 ~ 1.5mmol/L, 14 ~ 18mmol/L potassium citrate, the ammonium chloride of 35 ~ 45mmol/L, the sodium chloride of 75 ~ 80mmol/L and cellular antioxidants.
2. the preservation reagent of mesenchymal stem cells MSCs as claimed in claim 1, it is characterized in that, described cellular antioxidants comprises at least one in superoxide dismutase, glutathione peroxidase, vitamin C, vitamin E and reduced glutathione.
3. the preservation reagent of mesenchymal stem cells MSCs as claimed in claim 2, it is characterized in that, in described cellular antioxidants, superoxide dismutase concentration is 35000 ~ 45000U/L;
And/or described cellular antioxidants Glutathione Peroxidase concentration is 20000 ~ 25000U/L;
And/or vitamin C concentration is 3 ~ 10mmol/L in described cellular antioxidants;
And/or Vitamin E levels is 3 ~ 10mmol/L in described cellular antioxidants;
And/or reduced glutathione concentration is 1 ~ 1.5mmol/L in described cellular antioxidants.
4. the preservation reagent of the mesenchymal stem cells MSCs as described in any one of claims 1 to 3, is characterized in that, the preservation reagent of described mesenchymal stem cells MSCs also includes antibiotic.
5. the preservation reagent of mesenchymal stem cells MSCs as claimed in claim 4, it is characterized in that, described antibiotic is the chloramphenicol of 5 ~ 10mmol/L.
6. the application of the mesenchymal stem cells MSCs conserving liquid as described in any one of claim 1 to 5 in mesenchymal stem cells MSCs is preserved.
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