CN105713880A - Serum-free culture medium for hematopoietic stem cell in vitro expansion culture and application thereof - Google Patents
Serum-free culture medium for hematopoietic stem cell in vitro expansion culture and application thereof Download PDFInfo
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Abstract
The invention discloses a serum-free culture medium for hematopoietic stem cell in vitro expansion culture and a preparation method thereof. The culture medium for in vitro expansion of the hematopoietic stem cell is free from introducing exogenous gene expression, and the genome stability of the original stem cell is unchanged, the tumorigenic risk is avoided, and the stem cell transplantation safety problem caused by feeder layer cell and other exogenous cell mixing is not involved; through the combined application of various cytokines, a proper culture condition and properexpansion time are selected to overcome the defect that the expansion number is insufficient, the number is effectively expanded, and the quality is maintained at the same time, namely, the effect of maintaining hematopoietic reconstitution capacity through the expansion of the early hematopoietic progenitor cell and reservation of the HSC is achieved.
Description
Technical field
The invention belongs to the research field of cytobiology, molecular biology and medical domain, relate to the serum-free medium that a kind of hematopoietic stem cell population is cultivated, more particularly, it relates to be used for serum-free medium and the application thereof that hematopoietic stem cell population is cultivated.
Background technology
Hematopoietic stem cell (hematopoieticstemcell, HSC) refers to that internal not yet to reach maturity and be responsible for generating all be respectively the most original cell mass of hemocyte.HSC is versatile stem cell, is medically called " omnipotent cell ".Hematopoietic stem cell has height self-renewal capacity and multiple differentiation potential, can produce the hemocyte of all maturations, such as erythrocyte, leukocyte, platelet and lymphocyte etc..HSC is a kind of heterogeneity, immature hemopoietic forebody cell, can rebuild whole hemopoietic system in life process, has to B cell and two kinds of approach differentiation of granulocyte, and has the function maintaining long term hematopoietic.In recent years, HSC is applied to clinical treatment Malignancy, solid tumor, marrow failure disease and some congenital diseases etc. more and more, the hematopathy of department of pediatrics, tumor, heredopathia treatment in also show certain application prospect.During clinical discovery HSCT the quantity of hematopoietic stem cell with transplant after the reconstruction of hemopoietic and immunologic function have substantial connection, and the infusion of the hematopoietic stem cell of heavy dose is beneficial to donor hematopoietic stem cell by internal implantation.So when HSC is used for treating, being also faced with a severe problem: there is no abundant HSC for transplanting.
It is a good solution that HSC carries out amplification in vitro cultivation, is also the technology of a great challenge simultaneously, based on following several reasons: while 1. the amplification in vitro of HSC requires to keep lasting self-renewal capacity, also stop it to break up in amplification；But, the understanding about the physiological mechanisms of the self renewal and prevention HSC amplification that regulate HSC is limited；2. the quantity of expanding stem cells and the success rate of transplanting are closely related.
Summary of the invention
It is an object of the present invention to provide a kind of serum-free medium for cultivating cell.
Serum-free medium for cultivating cell provided by the invention includes recombinant human insulin, recombined human transferrins, recombinant human serum albumin, beta-mercaptoethanol, ethanolamine, cholesterol, linoleic plus oleic acid.
In above-mentioned serum-free medium, described recombinant human insulin, described recombined human transferrins and described recombinant human serum albumin refer both to be undertaken what vivoexpression obtained by genetic engineering means, are able to prepared by repetitive manual.
In above-mentioned serum-free medium,
The mass ratio of described recombinant human insulin, described recombined human transferrins, described recombinant human serum albumin, described beta-mercaptoethanol, described ethanolamine, described cholesterol, described linoleic acid and described oleic acid is 5-30:20-80:50-300:10-100:1-10:1-10:0.1-5:1.5.
In above-mentioned serum-free medium,
The mass ratio of described recombinant human insulin, described recombined human transferrins, described recombinant human serum albumin, described beta-mercaptoethanol, described ethanolamine, described cholesterol, described linoleic acid and described oleic acid is 10:50:100:40:5:5:1:1.5.
In above-mentioned serum-free medium,
Described culture medium also includes basal medium, L-glutaminate, stem cell factor, interleukin Ⅲ, interleukin-6, thrombopoietin, erythropoietin, granulocyte-macrophage colony stimutaing factor and granulocyte colony-stimulating factor；
Described basal medium is Cell Basal Medium；Described Cell Basal Medium is specially IMDM basal medium.
Described culture medium is by described IMDM basal medium, described recombinant human insulin, described recombined human transferrins, described recombinant human serum albumin, described beta-mercaptoethanol, described ethanolamine, described cholesterol, described linoleic acid, described oleic acid, described L-glutaminate, described stem cell factor, described interleukin Ⅲ, described interleukin-6, described thrombopoietin, described erythropoietin, described granulocyte-macrophage colony stimutaing factor becomes with described G-CSF group.Wherein, described L-glutaminate adds before cell faces cultivation；Described stem cell factor, described interleukin Ⅲ, described interleukin-6, described thrombopoietin, described erythropoietin, described granulocyte-macrophage colony stimutaing factor and described granulocyte colony-stimulating factor all add in the process that cell is cultivated.
In above-mentioned serum-free medium, the pH of described culture medium is 6.8-7.2.
In above-mentioned serum-free medium,
Described recombinant human insulin concentration in described serum-free medium is 5-30mg/L；
Described recombined human transferrins concentration in described serum-free medium is 20-80mg/L；
Described recombinant human serum albumin concentration in described serum-free medium is 50-300mg/L
Described beta-mercaptoethanol concentration in described serum-free medium is 10-100mg/L；
Described ethanolamine concentration in described serum-free medium is 1-10mg/L；
Described cholesterol concentration in described serum-free medium is 1-10mg/L；
Described linoleic acid concentration in described serum-free medium is 0.1-5mg/L；
Described oleic acid concentration in described serum-free medium is 0.1-5mg/L；
Described L-glutaminate concentration in described serum-free medium is 100-800mg/L
Described stem cell factor concentration in described serum-free medium is 10-100ng/mL；
Described interleukin Ⅲ concentration in described serum-free medium is 10-100ng/mL；
Described interleukin-6 concentration in described serum-free medium is 10-100ng/mL；
Described thrombopoietin concentration in described serum-free medium is 1-10U/ml；
Described erythropoietin concentration in described serum-free medium is 1-10U/ml；
Described granulocyte-macrophage colony stimutaing factor concentration in described serum-free medium is 10-50ng/ml；
Described granulocyte colony-stimulating factor concentration in described serum-free medium is 10-50ng/ml.
In above-mentioned serum-free medium,
Described recombinant human insulin concentration in described serum-free medium is 10mg/L；
Described recombined human transferrins concentration in described serum-free medium is 50mg/L；
Described recombinant human serum albumin concentration in described serum-free medium is 100mg/L；
Described beta-mercaptoethanol concentration in described serum-free medium is 40mg/L；
Described ethanolamine concentration in described serum-free medium is 5mg/L；
Described cholesterol concentration in described serum-free medium is 5mg/L；
Described linoleic acid concentration in described serum-free medium is 1mg/L；
Described oleic acid concentration in described serum-free medium is 1.5mg/L；
Described L-glutaminate concentration in described serum-free medium is 584mg/L；
Described stem cell factor concentration in described serum-free medium is 50ng/mL；
Described interleukin Ⅲ concentration in described serum-free medium is 20ng/mL；
Described interleukin-6 concentration in described serum-free medium is 20ng/mL；
Described thrombopoietin concentration in described serum-free medium is 4U/ml；
Described erythropoietin concentration in described serum-free medium is 3U/ml；
Described granulocyte-macrophage colony stimutaing factor concentration in described serum-free medium is 14ng/ml；
Described granulocyte colony-stimulating factor concentration in described serum-free medium is 20ng/ml.
The serum-free medium of the present invention is in concrete use, first by 1000mlIMDM basic culture solution (Gibco31980030), recombinant human insulin's (Recombinant protein expression, LIFE company), (yeast is recombinant expressed for recombined human transferrins, Invitria company), recombinant human serum albumin (Invitria company, Cellastim), beta-mercaptoethanol (Invitrogen, 21985-023), ethanolamine (Sigma, E0135), cholesterol (Sigma, C1231), linoleic acid (Sigma, and oleic acid (Sigma L1012), O1383) mixing, obtain hematopoietic stem cell IMDM cultivating system 1；Face in the cultivation above-mentioned hematopoietic stem cell IMDM cultivating system of forward direction at cell and add L-glutaminate, obtain hematopoietic stem cell IMDM cultivating system 2；Again immunocyte to be cultivated is added in above-mentioned hematopoietic stem cell IMDM cultivating system 2, and in the process that cell is cultivated, add stem cell factor (SCF), interleukin Ⅲ (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), erythropoietin (EPO), granulocyte-macrophage colony stimutaing factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), obtain hematopoietic stem cell serum-free medium.
It is a further object to provide the new application of above-mentioned culture medium.
The invention provides the application in cell expansion ex vivo is cultivated of the above-mentioned culture medium.
Last purpose of the present invention is to provide the cultural method of a kind of cell.
The cultural method of cell provided by the invention comprises the steps: with above-mentioned culture medium culturing cell.
In above-mentioned culture medium or above-mentioned application or said method, described cell is hematopoietic stem cell, and described hematopoietic stem cell is specially CD34+Cell.
Compared with traditional hematopoietic stem cell expansion method, the serum-free medium of the amplifying candidate stem cell in vitro of the present invention has following several main feature and advantage: first, do not introduce expression of exogenous genes, therefore do not change the Genome stability of former stem cell, without tumorigenesis risk；Second, it is not related to the exogenous cells such as feeder layer cells and mixes and the stem cell transplantation safety problem that causes.
The serum-free medium of the amplifying candidate stem cell in vitro of the present invention passes through use in conjunction cytokine profiles, select suitable condition of culture and proliferation time, the shortcoming overcoming amplification lazy weight, effectively expand its quantity, it is maintained with its quality, namely reaches retain HSC and expand early stage hemopoietic progenitor cell to maintain the effect of hematopoietic reconstitution ability.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
The product of stem cell factor (SCF) in following embodiment, interleukin Ⅲ (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), erythropoietin (EPO), granulocyte-macrophage colony stimutaing factor (GM-CSF) and PeproTech company of granulocyte colony-stimulating factor (G-CSF) the Jun Shi U.S..
Semisolid culturemedium in following embodiment is the culture medium obtained after being mixed with IMDM culture fluid according to volume ratio 1:2 by the methylated cellulose aqueous solution that mass fraction is 2.7%.Wherein, IMDM culture fluid is the culture fluid that will obtain after following cytokine IL-3, SCF, IL-6, GM-CSF, G-CSF, EPO, TPO and IMDM basic culture solution (Gibco31980030) mixing；The concentration of IL-3, SCF, IL-6, GM-CSF, G-CSF, EPO, TPO in IMDM culture fluid respectively IL-320ng/ml, SCF50ng/ml, IL-620ng/ml, GM-CSF14ng/ml, G-CSF20ng/ml, EPO3U/ml, TPO4U/ml.
Embodiment 1, hematopoietic stem cell serum-free medium preparation
By 1000mlIMDM basic culture solution (Gibco31980030), recombinant human insulin's (Recombinant protein expression, LIFE company), (yeast is recombinant expressed for recombined human transferrins, Invitria company), recombinant human serum albumin (Invitria company, Cellastim), beta-mercaptoethanol (Invitrogen, 21985-023), ethanolamine (Sigma, E0135), cholesterol (Sigma, C1231), linoleic acid (Sigma, and oleic acid (Sigma L1012), O1383) mixing, obtain hematopoietic stem cell IMDM cultivating system 1.
Face in the cultivation above-mentioned hematopoietic stem cell IMDM cultivating system of forward direction at cell and add L-glutaminate, obtain hematopoietic stem cell IMDM cultivating system 1；The process that cell is cultivated adds stem cell factor (SCF), interleukin Ⅲ (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), erythropoietin (EPO), granulocyte-macrophage colony stimutaing factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) again in above-mentioned hematopoietic stem cell IMDM cultivating system 2, obtains hematopoietic stem cell serum-free medium.
Wherein, each solute component and the concentration in hematopoietic stem cell serum-free medium thereof are respectively as follows: recombinant human insulin 10mg/L, recombined human transferrins 50mg/L, recombinant human serum albumin 0.1g/L, beta-mercaptoethanol 40mg/L, ethanolamine 5mg/L, cholesterol 5mg/L, linoleic acid 1.0mg/L, oleic acid 1.5mg/L, L-glutaminate 584mg/L, stem cell factor (SCF) 50ng/mL, interleukin Ⅲ (IL-3) 20ng/mL, interleukin-6 (IL-6) 20ng/mL, thrombopoietin (TPO) 4U/ml, erythropoietin (EPO) 3U/ml, granulocyte-macrophage colony stimutaing factor (GM-CSF) 14ng/ml, granulocyte colony-stimulating factor (G-CSF) 20ng/ml.
The application in cultivating hematopoietic stem cell of embodiment 2, hematopoietic stem cell serum-free medium
One, the separation of the collection of Cord blood and mononuclearcell
Gathering the Cord blood that Placenta Hominis is given birth to, every part of umbilical blood is taken from maternity & child care center's (requiring that anemia of pregnant woman is without obstetrics, internal medicine complication and other hematologic disease), aseptically extracts Cord blood, injects sterile blood sampling bag and is placed on 4 DEG C of preservations.It is easily separated in all sample standard deviations 12h after acquisition, adopts Ficoll lymphocyte separation medium density gradient centrifugation compatriot's Cord blood.Specifically comprise the following steps that
1, the centrifugal 30min of Cord blood 2000r/min that will gather.Careful draw in the middle of tunica albuginea layer, adds 5 times and fully inhales with the IMDM culture fluid of upper volume and beat, wash, 1500r/min is centrifuged 10min, abandons supernatant.Repeat aforesaid operations, wash twice altogether, after final centrifugation, quickly abandon supernatant, make the IMDM cell suspension of 1mL.
2, Mei Tian Ni company MACS magnetic bead sorting system is used to separate CD34 from IMDM cell suspension+Cell, it is thus achieved that cell quantity is 0.38 × 106Individual CD34+Cell, parallel type cell instrument detection CD34+The purity of cell colony is 93.3%.
Two, the In vitro culture of hematopoietic stem cell
1, experiment packet
Step one is separated the CD34 obtained+Cell is inoculated in following culture fluid respectively, divides three groups and carries out suspension culture.
(1) blank group
Hyclone (GIBCO, 10099141) and the IMDM basic culture solution adding L-glutaminate are mixed, obtains the IMDM culture medium of 10% (volume fraction) hyclone；By CD34+Cell is inoculated in the IMDM culture medium of 10% (volume fraction) hyclone, makes CD34+Cell concentration in cultivating system is 1 × 106/ ml, obtains blank group cultivating system.
(2) serum-free group
By L-glutaminate and the hematopoietic stem cell IMDM cultivating system mixing in embodiment 1, obtain cultivating system 1, by CD34+Cell is inoculated in cultivating system 1, obtain cultivating system 2, again following cytokine is joined in cultivating system 2: stem cell factor (SCF), interleukin Ⅲ (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), erythropoietin (EPO), granulocyte-macrophage colony stimutaing factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), make CD34+Cell, L-glutaminate, stem cell factor (SCF), interleukin Ⅲ (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), erythropoietin (EPO), granulocyte-macrophage colony stimutaing factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) the concentration respectively CD34 in cultivating system 2+Cell 1 × 106/ ml, L-glutaminate 584mg/L, stem cell factor (SCF) 50ng/mL, interleukin Ⅲ (IL-3) 20ng/mL, interleukin-6 (IL-6) 20ng/mL, thrombopoietin (TPO) 4U/ml, erythropoietin (EPO) 3U/ml, granulocyte-macrophage colony stimutaing factor (GM-CSF) 14ng/ml, granulocyte colony-stimulating factor (G-CSF) 20ng/ml, obtain serum-free group cultivating system.
(3) there is serum group
Hyclone (GIBCO, 10099141) and the IMDM basic culture solution adding L-glutaminate are mixed, obtains the IMDM culture medium of 10% (volume fraction) hyclone；By CD34+Cell is inoculated in the hematopoietic stem cell culture medium of 10% (volume fraction) hyclone, obtain cultivating system 3, again following cytokine is joined in cultivating system 3: stem cell factor (SCF), interleukin Ⅲ (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), erythropoietin (EPO), granulocyte-macrophage colony stimutaing factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), make CD34+Cell, stem cell factor (SCF), interleukin Ⅲ (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), erythropoietin (EPO), granulocyte-macrophage colony stimutaing factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) the concentration respectively CD34 in cultivating system 2+Cell 1 × 106/ ml, L-glutaminate 584mg/L, stem cell factor (SCF) 50ng/mL, interleukin Ⅲ (IL-3) 20ng/mL, interleukin-6 (IL-6) 20ng/mL, thrombopoietin (TPO) 4U/ml, erythropoietin (EPO) 3U/ml, granulocyte-macrophage colony stimutaing factor (GM-CSF) 14ng/ml, granulocyte colony-stimulating factor (G-CSF) 20ng/ml, obtain there is serum group cultivating system.
2, In vitro culture
Taking above-mentioned each group of cultivating system (blank group cultivating system, serum-free group cultivating system and have serum group cultivating system) lml respectively, be placed in 24 hole flat-bottomed plates, often group multiple cropping three hole, is placed in 37 DEG C, 5%CO2Under concentration, cultivating in saturated humidity incubator, cultivate 7d, 14d, 21d respectively, half amount is changed liquid and adds corresponding cytokine weekly, co-culture three weeks, respectively obtain the blank group cultivating system after cultivation, serum-free group cultivating system and have serum group cultivating system.
Three, the detection of cell after cultivating
1, microorganism detection
To blank group cultivating system, the serum-free group cultivating system after cultivating and serum group cultivating system is had to carry out microorganism detection (antibacterial, mycoplasma and bacterial endotoxin) according to " Chinese Pharmacopoeia " (version annex Ⅻ in 2010).Result shows: blank group cultivating system, serum-free group cultivating system after cultivation and have the equal asepsis growth of serum group cultivating system, without mycoplasma contamination with without bacterial endotoxin.
Above-mentioned Sterility Test Methods is as follows: take THIOGLYCOLLIC ACID salt broth, improvement Martin's culture medium, selective medium, nutrient broth medium, nutrient agar and each 10 parts of improvement Martin's agar culture medium at random, inoculation 0.5ml cell suspension, put 35 DEG C of cultivations for 5 parts, putting 25 DEG C for 5 parts to cultivate 14 days, every day, observation had asepsis growth.
Above-mentioned mycoplasma inspection method is as follows: be inoculated in by cell culture supernatant in indicator cells (free of contamination Vero cell) culture fluid, dibenzamide fluorochrome is adopted to dye, fluorescence microscopy Microscopic observation is merely the nucleus of cell and presents yellow-green fluorescence, it was shown that grow without mycoplasma.
Above-mentioned Bacterial Endotoxin Test checks according to gel method.
2, umbilical blood CD34+The change of total cellular score
Adopt flow cytometer detect cultivation respectively after blank group cultivating system, serum-free group cultivating system and have CD34 in serum group cultivating system+Cell content.
Testing result is as shown in table 1, from table 1 it follows that along with the increase of cultivated days, blank group CD34+Total cellular score declines rapidly, with serum-free group, have serum group compared with have significant difference；But serum-free group is compared with having serum group, expand CD34+Cell effect is close, there was no significant difference.Wherein, the CD34 of serum-free group+Cell proportion has begun to when cultivating 7 days rise, and continues to rise, reached peak by 14 days when 10 days, continues to cultivate by 21 days, CD34+Cell proportion is gradually reduced, but level before remaining above cultivation.
Table 1, there is serum-free amplification umbilical blood CD34+Total cellular score (104Individual/ml) comparison
3, cell phenotype analysis
The CD34 separated+Cell is suspension growth in the medium, and propagation rapidly, forms the granulocyte colony group of CFU sample, over time prolongation, cell colony is on the increase expansion.
The antibody (CD29, CD34 and CD45) using FITC labelling detects cell surface marker, flow cytometer is adopted to carry out cell phenotype analysis, collect 10000 cells to detect every time, carry out data analysis with CellQuest software (U.S. company BD).Result shows: cell expresses CD34 and CD45, does not express CD29.
4, cell survival rate
The cell suspension and the trypan blue fuel that take a serum-free group cultivating system mix, and drop in the counting chamber of blood cell counting plate, stand 2min, and basis of microscopic observation counts, and does 10 parallel tests.Cell survival rate is bright and non-staining for living cells, dye blueness for dead cell.Calculate cell survival rate.Cell survival rate computing formula is number of viable cells/(number of viable cells+dead cell number) × 100%.
Result shows: the cell survival rate of Trypan Blue detection serum-free group is 93.9%.
5, Colony forming ability detection
Respectively by the blank group cultivating system after above-mentioned cultivation 10d, serum-free group cultivating system with there is the CD34 in serum group cultivating system+Cell is inoculated in semisolid culturemedium and detects Colony forming ability.Specifically comprise the following steps that
Take 1 × 104Cell after individual each group of cultivation is inoculated in 24 well culture plates respectively, and every hole adds 0.5ml semisolid culturemedium, and often group sets 3 multiple holes.It is placed in 37 DEG C, 5%CO2Under concentration, after saturated humidity incubator cultivates 14d, carry out colony count with phase contrast microscope, be designated as a colony containing the cell cluster having more than 50 cells.Adopt counting method of blood cell counting mix-colony forming unit (CFU-mix).
Testing result is as shown in table 2: as can be seen from the table, and along with the increase of cultivated days, blank group umbilical blood CFU-mix productivity declines rapidly, with serum-free group, have serum group compared with have significant difference；But serum-free group is compared with having serum group, the colony productivity often organized weekly is all on a declining curve, and umbilical blood CFU-mix productivity effect is close, and two group differences are little.
Table 2, have serum-free amplification umbilical blood CFU-mix productivity (individual/2 × 105Cell) comparison
Claims (10)
1., for cultivating a serum-free medium for cell, described culture medium includes recombinant human insulin, recombined human transferrins, recombinant human serum albumin, beta-mercaptoethanol, ethanolamine, cholesterol, linoleic plus oleic acid.
2. serum-free medium according to claim 1, it is characterised in that:
The mass ratio of described recombinant human insulin, described recombined human transferrins, described recombinant human serum albumin, described beta-mercaptoethanol, described ethanolamine, described cholesterol, described linoleic acid and described oleic acid is 5-30:20-80:50-300:10-100:1-10:1-10:0.1-5:1.5.
3. serum-free medium according to claim 1 and 2, it is characterised in that: the mass ratio of described recombinant human insulin, described recombined human transferrins, described recombinant human serum albumin, described beta-mercaptoethanol, described ethanolamine, described cholesterol, described linoleic acid and described oleic acid is 10:50:100:40:5:5:1:1.5.
4. according to described serum-free medium arbitrary in claim 1-3, it is characterised in that:
Described culture medium also includes basal medium, L-glutaminate, stem cell factor, interleukin Ⅲ, interleukin-6, thrombopoietin, erythropoietin, granulocyte-macrophage colony stimutaing factor and granulocyte colony-stimulating factor；
Described L-glutaminate adds before cell faces cultivation；
Described stem cell factor, described interleukin Ⅲ, described interleukin-6, described thrombopoietin, described erythropoietin, described granulocyte-macrophage colony stimutaing factor and described granulocyte colony-stimulating factor all add in the process that cell is cultivated.
5. according to described serum-free medium arbitrary in claim 1-4, it is characterised in that:
The pH of described culture medium is 6.8-7.2；
Described basal medium is Cell Basal Medium；Described Cell Basal Medium is specially IMDM basal medium.
6. according to described serum-free medium arbitrary in claim 1-5, it is characterised in that:
Described recombinant human insulin concentration in described serum-free medium is 5-30mg/L；
Described recombined human transferrins concentration in described serum-free medium is 20-80mg/L；
Described recombinant human serum albumin concentration in described serum-free medium is 50-300mg/L
Described beta-mercaptoethanol concentration in described serum-free medium is 10-100mg/L；
Described ethanolamine concentration in described serum-free medium is 1-10mg/L；
Described cholesterol concentration in described serum-free medium is 1-10mg/L；
Described linoleic acid concentration in described serum-free medium is 0.1-5mg/L；
Described oleic acid concentration in described serum-free medium is 0.1-5mg/L；
Described L-glutaminate concentration in described serum-free medium is 100-800mg/L
Described stem cell factor concentration in described serum-free medium is 10-100ng/mL；
Described interleukin Ⅲ concentration in described serum-free medium is 10-100ng/mL；
Described interleukin-6 concentration in described serum-free medium is 10-100ng/mL；
Described thrombopoietin concentration in described serum-free medium is 1-10U/ml；
Described erythropoietin concentration in described serum-free medium is 1-10U/ml；
Described granulocyte-macrophage colony stimutaing factor concentration in described serum-free medium is 10-50ng/ml；
Described granulocyte colony-stimulating factor concentration in described serum-free medium is 10-50ng/ml.
7. according to described serum-free medium arbitrary in claim 1-6, it is characterised in that:
Described recombinant human insulin concentration in described serum-free medium is 10mg/L；
Described recombined human transferrins concentration in described serum-free medium is 50mg/L；
Described recombinant human serum albumin concentration in described serum-free medium is 100mg/L；
Described beta-mercaptoethanol concentration in described serum-free medium is 40mg/L；
Described ethanolamine concentration in described serum-free medium is 5mg/L；
Described cholesterol concentration in described serum-free medium is 5mg/L；
Described linoleic acid concentration in described serum-free medium is 1mg/L；
Described oleic acid concentration in described serum-free medium is 1.5mg/L；
Described L-glutaminate concentration in described serum-free medium is 584mg/L；
Described stem cell factor concentration in described serum-free medium is 50ng/mL；
Described interleukin Ⅲ concentration in described serum-free medium is 20ng/mL；
Described interleukin-6 concentration in described serum-free medium is 20ng/mL；
Described thrombopoietin concentration in described serum-free medium is 4U/ml；
Described erythropoietin concentration in described serum-free medium is 3U/ml；
Described granulocyte-macrophage colony stimutaing factor concentration in described serum-free medium is 14ng/ml；
Described granulocyte colony-stimulating factor concentration in described serum-free medium is 20ng/ml.
8. arbitrary described culture medium application in cell expansion ex vivo is cultivated in claim 1-7.
9. a cultural method for cell, comprises the steps: with described culture medium culturing cell arbitrary in claim 1-7.
10. the application according to described culture medium arbitrary in claim 1-7 or claim 8 or the method described in claim 9, it is characterised in that: described cell is hematopoietic stem cell.
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