CN104304233B - The method of a kind of cryopreserving liquid and uses thereof and preservation umbilical cord mesenchymal stem cells - Google Patents
The method of a kind of cryopreserving liquid and uses thereof and preservation umbilical cord mesenchymal stem cells Download PDFInfo
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- CN104304233B CN104304233B CN201410459778.9A CN201410459778A CN104304233B CN 104304233 B CN104304233 B CN 104304233B CN 201410459778 A CN201410459778 A CN 201410459778A CN 104304233 B CN104304233 B CN 104304233B
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Abstract
The invention discloses and a kind ofly the invention provides a kind of cryopreserving liquid for preservation stem cell, this cryopreserving liquid is mixed to get by dimethyl sulfoxide (DMSO) and growth medium, described growth medium contains basal medium and additive, and described additive contains the antibody of human vessel endothelium growth factor resisting, the antibody of anti-human ErbB1, the antibody of anti-human epidermal growth factor acceptor 2 and guanosine-5-triphosphoric acid trisodium salt.Present invention also offers the purposes of described growth medium in preservation umbilical cord mesenchymal stem cells.Present invention also offers a kind of method of preservation umbilical cord mesenchymal stem cells, the method comprises: be suspended in by umbilical cord mesenchymal stem cells in cryopreserving liquid as above, then preservation in the temperature of subzero 200 DEG C to subzero 70 DEG C.By technique scheme, the present invention can promote the umbilical cord mesenchymal stem cells amplification ability in vitro after preservation significantly.
Description
Technical field
The present invention relates to technical field of cell biology, particularly, relate to a kind of method for the cryopreserving liquid of preservation stem cell, the purposes of this cryopreserving liquid and preservation umbilical cord mesenchymal stem cells.
Background technology
Stem cell is that a class has not breaking up or PD cell of the of self-replication capacity and multi-lineage potential.Under certain condition, it can be divided into several functions cell.Potentiality of development according to stem cell is divided three classes: myeloid-lymphoid stem cell, multipotential stem cell and unipotent stem cell.
Mescenchymal stem cell, with a kind of multipotential stem cell, is the important member of stem cell line, derives from and grows early stage mesoderm and ectoderm.Mescenchymal stem cell has the features such as multi-lineage potential, hematopoiesis support and the implantation of promotion stem cell, immunoregulation and self-replacation because of it and day by day receives the concern of people.If mescenchymal stem cell is in vivo or under external specific inductive condition, the Various Tissues cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium can be divided into, still there is multi-lineage potential after continuous passage cultivation and freezen protective, can be used as the injuries of tissues and organs reparation that desirable seed cell causes for old and feeble and pathology.Mescenchymal stem cell, due to its wide material sources, is easy to be separated and cultivates, and has stronger differentiation potential and can the advantage such as autoplastic transplantation, has higher clinical value.
Umbilical cord mesenchymal stem cells refers to a kind of multipotential stem cell be present in neonatal umbilical cord tissue.Isolated umbilical cord mesenchymal stem cells from people's umbilical cord, its cell content and multiplication capacity are all better than mesenchymal stem cells MSCs, and immunogenicity is lower than mesenchymal stem cells MSCs, have in addition and draw materials conveniently, without advantages such as ethics disputes, therefore there is in mescenchymal stem cell higher clinical value.
In practical clinical, the sufficient amount of umbilical cord mesenchymal stem cells is the necessary factor ensureing stem-cell therapy.The necessary ways of the quantity being increase umbilical cord mesenchymal stem cells that in vitro umbilical cord mesenchymal stem cells increased.But current umbilical cord mesenchymal stem cells can only go down to posterity 15-20 generation in vitro, and go down to posterity within latter more than 3 days, just can reach more than 80% degrees of fusion, also there will be after these external 15 generations of going down to posterity and lose the phenomenon of differentiation potential.Therefore, there is the defect of amplification ability in the method for existing cultivation umbilical cord mesenchymal stem cells.In addition, umbilical cord mesenchymal stem cells is after frozen, and amplification ability there will be decline.
Summary of the invention
The object of the invention is to overcome umbilical cord mesenchymal stem cells after frozen, amplification ability there will be the defect of decline, provides a kind of method for the cryopreserving liquid of preservation stem cell, the purposes of this cryopreserving liquid and preservation umbilical cord mesenchymal stem cells.
The present inventor finds, by adding specific antibody and ATP in basal medium, the amplification ability of umbilical cord mesenchymal stem cells can be improved significantly, and, also specific antibody and ATP is added in cryopreserving liquid, can improve significantly frozen after the amplification ability of umbilical cord mesenchymal stem cells, resulting in the present invention.
On the one hand, the invention provides a kind of cryopreserving liquid for preservation stem cell, this cryopreserving liquid is mixed to get by dimethyl sulfoxide (DMSO) and growth medium, and the volume ratio of dimethyl sulfoxide (DMSO) and described growth medium is (0.05-0.4): 1; Described growth medium contains basal medium and additive, and described additive contains the antibody of human vessel endothelium growth factor resisting, the antibody of anti-human ErbB1, the antibody of anti-human epidermal growth factor acceptor 2 and guanosine-5-triphosphoric acid trisodium salt.
On the other hand, present invention also offers a kind of method of preservation umbilical cord mesenchymal stem cells, the method comprises: be suspended in by umbilical cord mesenchymal stem cells in cryopreserving liquid as above, then preservation in the temperature of subzero 200 DEG C to subzero 70 DEG C.
Again on the one hand, present invention also offers the purposes of cryopreserving liquid as above in preservation umbilical cord mesenchymal stem cells.
By technique scheme, the present invention can promote significantly frozen after umbilical cord mesenchymal stem cells amplification ability in vitro.Such as, the subculture in vitro separately number of the umbilical cord mesenchymal stem cells after frozen can be brought up to 25-40 generation by the present invention, and can reach the degrees of fusion of more than 80% in 40-60 hour after going down to posterity, and also there will not be the phenomenon of losing differentiation potential after these external 20 generations of going down to posterity.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
Fig. 1 is the growth curve chart of the umbilical cord mesenchymal stem cells grown in growth medium different in testing example 1.In Fig. 1,1 and 2 represent that preparation embodiment 1 and 2,3-6 represent preparation comparative example 1-4 respectively, and abscissa represents growth time, and ordinate represents that 550nm place measures each hole absorption photometric value (D value).
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
In the present invention, when not doing contrary explanation, the volumetric quantities of the liquid of use is the numerical value at 20 DEG C; The volumetric quantities of the gas used is the numerical value of 20 DEG C and 1 normal atmosphere pressures.
The invention provides a kind of cryopreserving liquid for preservation stem cell, this cryopreserving liquid is mixed to get by dimethyl sulfoxide (DMSO) and growth medium, and the volume ratio of dimethyl sulfoxide (DMSO) and described growth medium is (0.05-0.4): 1; Described growth medium contains basal medium and additive, and described additive contains the antibody of human vessel endothelium growth factor resisting, the antibody of anti-human ErbB1, the antibody of anti-human epidermal growth factor acceptor 2 and guanosine-5-triphosphoric acid trisodium salt.
According to cryopreserving liquid provided by the invention, wherein, human vascular endothelial growth factor (humanvascularendothelialgrowthfactor, hVEGF) refer to that gene accession number in NCBI (NCBIGeneID) is the translation product of the gene of 7422, its official's full name (OfficialFullName) is VEGF-A (vascularendothelialgrowthfactorA, VEGFA), its common another name also comprises VPF, VEGF or MVCD1.
According to cryopreserving liquid provided by the invention, wherein, human epidermal growth factor acceptor 1 (humanepidermalgrowthfactorreceptor1, HER1) refer to that gene accession number in NCBI (NCBIGeneID) is the translation product of the gene of 1956, its official's full name (OfficialFullName) is EGF-R ELISA (epidermalgrowthfactorreceptor, EGFR), its common another name also comprises EGFR, ERBB, mENA, ERBB1 or PIG61.
According to cryopreserving liquid provided by the invention, wherein, ErbB-2 (humanepidermalgrowthfactorreceptor2, HER2) refer to that gene accession number in NCBI (NCBIGeneID) is the translation product of the gene of 2064, its official's full name (OfficialFullName) is v-erb-b2 bird EBL viral oncogene autoploid 2 (v-erb-b2avianerythroblasticleukemiaviraloncogenehomolog2), its common another name also comprises NEU, NGL, TKR1, CD340, HER-2, MLN19 or HER-2/neu.
According to cryopreserving liquid provided by the invention, wherein, guanosine-5-triphosphoric acid trisodium salt (guanosine-5'-triphosphatetrisodiumsalt) refers to the compound shown in formula (1), and its No. CAS is 36051-31-7.
According to cryopreserving liquid provided by the invention, wherein, the content of the antibody of described human vessel endothelium growth factor resisting can be 0.01-0.2ng/mL, the content of the antibody of described anti-human ErbB1 can be 0.01-0.1ng/mL, and the content of the antibody of described anti-human epidermal growth factor acceptor 2 can be 0.05-0.4ng/mL; The content of guanosine-5-triphosphoric acid trisodium salt can be 0.2-4ng/mL.
According to cryopreserving liquid provided by the invention, wherein, according to the preferred embodiment of the present invention, the content of the antibody of human vessel endothelium growth factor resisting is 0.05-0.1ng/mL, the content of the antibody of anti-human ErbB1 is 0.03-0.08ng/mL, and the content of the antibody of anti-human epidermal growth factor acceptor 2 is 0.1-0.2ng/mL; The content of guanosine-5-triphosphoric acid trisodium salt is 0.5-2ng/mL.In this preferred embodiment, growth medium of the present invention can strengthen the expanding effect to umbilical cord mesenchymal stem cells further.
According to cryopreserving liquid provided by the invention, wherein, as long as the antibody of described human vessel endothelium growth factor resisting can be combined with human vascular endothelial growth factor specifically and hinder the enforcement of its biological function; As long as the antibody of described anti-human ErbB1 can be combined with human epidermal growth factor acceptor 1 specifically and hinder the enforcement of its biological function; As long as the antibody of described anti-human epidermal growth factor acceptor 2 can be combined with ErbB-2 specifically and hinder the enforcement of its biological function.Above-mentioned antibody is by being commercially available.According to the preferred embodiment of the present invention, the antibody of described human vessel endothelium growth factor resisting is bevacizumab; The antibody of described anti-human ErbB1 is Cetuximab, and the antibody of described anti-human epidermal growth factor acceptor 2 is Herceptin.
According to cryopreserving liquid provided by the invention, wherein, the English of described bevacizumab is called Bevacizumab, and commodity are called Arastin (Avastin); The English of described Cetuximab is called Cetuximab, and commodity are called Erbitux (Erbitux); Described Herceptin English Trastuzumab by name, commodity are called Trastuzumab (Herceptin).
According to cryopreserving liquid provided by the invention, wherein, the not special requirement of described basal medium, mammiferous medium can be cultivated for conventional the various of use, such as, described basal medium includes but not limited at least one in DMEM medium, MEM medium, IMEM medium and RPMI1640 medium.Above-mentioned basal medium can be commercially available, such as, can buy from Gibco or Hyclone company and obtain.Such as, according to the goods catalogue of Gibco, the article number of the article number of DMEM medium to be the article number of 11965, MEM medium be 11095, IMEM medium is the article number of 21700, RPMI1640 medium is 11875.
According to cryopreserving liquid provided by the invention, wherein, growth medium of the present invention can, for the medium containing serum, also can be not containing the medium of serum.In order to accelerate the adherent of umbilical cord mesenchymal stem cells, preferably, the serum of described growth medium also containing 5-20 volume %.Wherein, described serum can comprise at least one in hyclone, NBCS and calf serum, is more preferably hyclone.
According to cryopreserving liquid provided by the invention, wherein, the preparation method of above-mentioned cryopreserving liquid of the present invention can comprise: dimethyl sulfoxide (DMSO), basal medium are mixed with described additive.
Present invention also offers the purposes of cryopreserving liquid as above in preservation umbilical cord mesenchymal stem cells.
Present invention also offers a kind of method of preservation umbilical cord mesenchymal stem cells, the method comprises: be suspended in by umbilical cord mesenchymal stem cells in cryopreserving liquid as above, then preservation in the temperature of subzero 200 DEG C to subzero 70 DEG C.
According to the method for preservation umbilical cord mesenchymal stem cells provided by the invention, wherein, relative to the cryopreserving liquid of every mL, the consumption of umbilical cord mesenchymal stem cells can be (1-5) × 10
7individual.
According to the method for preservation umbilical cord mesenchymal stem cells provided by the invention, wherein, frozen rate of temperature fall used can be 0.5-2 DEG C/min.Above-mentioned rate of temperature fall can be realized by programmed cooling instrument.
Wherein, the umbilical cord mesenchymal stem cells be under preservation state continues after can recovering to cultivate.The method of recovery can comprise: the water-bath 1-2 minute umbilical cord mesenchymal stem cells be under preservation state being placed in 35-38 DEG C.
Wherein, temperature and the CO of cultivation is continued after recovery
2concentration can be the selection of umbilical cord mesenchymal stem cells field routine; Such as, the temperature of cultivation can be 36-38 DEG C; The CO cultivated
2concentration can be 4-6 volume %.Continuing after recovery to cultivate the medium used can be growth medium as above.
Wherein, in the present invention, growth medium as above can promote that umbilical cord mesenchymal stem cells increases quickly, and correspondingly shorten and go down to posterity the cycle, therefore under preferable case, the cycle of going down to posterity of cultivation is 40-60 hour.
Wherein, in the present invention, growth medium as above can promote that umbilical cord mesenchymal stem cells extends passage number, and under preferable case, preferably, the algebraically gone down to posterity of cultivation is 25-40 generation.
Wherein, described umbilical cord mesenchymal stem cells can be obtained by tissue block adherent cultivation or homogenate collagenase digestion.
Below, the present invention is further described by embodiment.
Preparation embodiment 1
At DMEM medium (purchased from Gibco, article number is 11965) in add hyclone (purchased from Gibco), bevacizumab (purchased from Roche Holding Ag), Cetuximab (purchased from Merck & Co., Inc.), Herceptin (purchased from Roche Holding Ag) and guanosine-5-triphosphoric acid trisodium salt (purchased from Sigma, identical below); The amount of adding makes final concentration to be: hyclone 10 volume %, bevacizumab 0.08ng/mL, Cetuximab 0.06ng/mL, Herceptin 0.15ng/mL and guanosine-5-triphosphoric acid trisodium salt 1ng/mL; Obtain growth medium.Growth medium and dimethyl sulfoxide (DMSO) (purchased from Invitrogen, article No. D8370, identical below), according to volume ratio 1:0.1 mixing, are obtained the cryopreserving liquid that this prepares embodiment.
Preparation embodiment 2
Hyclone (purchased from Gibco), bevacizumab (purchased from Roche Holding Ag), Cetuximab (purchased from Merck & Co., Inc.), Herceptin (purchased from Roche Holding Ag) and guanosine-5-triphosphoric acid trisodium salt is added in DMEM medium (purchased from Gibco, article number is 11965); The amount of adding makes final concentration to be: hyclone 10 volume %, bevacizumab 0.01ng/mL, Cetuximab 0.1ng/mL, Herceptin 0.05ng/mL and guanosine-5-triphosphoric acid trisodium salt 300 μ g/mL; Obtain growth medium.Growth medium and dimethyl sulfoxide (DMSO) are mixed according to volume ratio 1:0.4, obtains the cryopreserving liquid that this prepares embodiment.
Preparation comparative example 1
At DMEM medium (purchased from Gibco, article number is 11965) in add hyclone (purchased from Gibco), Cetuximab (purchased from Merck & Co., Inc.), Herceptin (purchased from Roche Holding Ag) and guanosine-5-triphosphoric acid trisodium salt (purchased from Sigma, identical below); The amount of adding makes final concentration to be: hyclone 10 volume %, Cetuximab 0.06ng/mL, Herceptin 0.15ng/mL and guanosine-5-triphosphoric acid trisodium salt 1ng/mL; Obtain growth medium.Growth medium and dimethyl sulfoxide (DMSO) are mixed according to volume ratio 1:0.1, obtains the cryopreserving liquid that this prepares comparative example.
Preparation comparative example 2
At DMEM medium (purchased from Gibco, article number is 11965) in add hyclone (purchased from Gibco), bevacizumab (purchased from Roche Holding Ag), Herceptin (purchased from Roche Holding Ag) and guanosine-5-triphosphoric acid trisodium salt (purchased from Sigma, identical below); The amount of adding makes final concentration to be: hyclone 10 volume %, bevacizumab 0.08ng/mL, Herceptin 0.15ng/mL and guanosine-5-triphosphoric acid trisodium salt 1ng/mL; Obtain growth medium.Growth medium and dimethyl sulfoxide (DMSO) are mixed according to volume ratio 1:0.1, obtains the cryopreserving liquid that this prepares comparative example.
Preparation comparative example 3
At DMEM medium (purchased from Gibco, article number is 11965) in add hyclone (purchased from Gibco), bevacizumab (purchased from Roche Holding Ag), Cetuximab (purchased from Merck & Co., Inc.) and guanosine-5-triphosphoric acid trisodium salt (purchased from Sigma, identical below); The amount of adding makes final concentration to be: hyclone 10 volume %, bevacizumab 0.08ng/mL, Cetuximab 0.06ng/mL and guanosine-5-triphosphoric acid trisodium salt 1ng/mL; Obtain growth medium.Growth medium and dimethyl sulfoxide (DMSO) are mixed according to volume ratio 1:0.1, obtains the cryopreserving liquid that this prepares comparative example.
Preparation comparative example 4
Hyclone (purchased from Gibco), bevacizumab (purchased from Roche Holding Ag), Cetuximab (purchased from Merck & Co., Inc.) and Herceptin (purchased from Roche Holding Ag) is added in DMEM medium (purchased from Gibco, article number is 11965); The amount of adding makes final concentration to be: hyclone 10 volume %, bevacizumab 0.08ng/mL, Cetuximab 0.06ng/mL and Herceptin 0.15ng/mL; Obtain growth medium.Growth medium and dimethyl sulfoxide (DMSO) are mixed according to volume ratio 1:0.1, obtains the cryopreserving liquid that this prepares comparative example.
Testing example 1
After caesarean birth fetal birth, intercept umbilical cord, peel off amnion, peel off Wal Tong Shi glue (Wharton'sjelly), shred into 0.5-1.5mm
3tissue block, add type Ⅳ collagenase (10g/L), after putting 37 DEG C of constant-temperature shaking incubators vibration digestion 3h, filter with 80 mesh filter screens, by the cell suspension centrifuge washing of filtration.Cell is mixed, with 1.0 × 10 with serum free medium (purchased from Cambrex, UItraCULTURE)
5/ cm
2inoculum density be inoculated in T-75cm
2in blake bottle, be placed in 37 DEG C, saturated humidity, 5 volume % CO
2be cultured to the degrees of fusion of 80% in incubator, obtain 1st generation umbilical cord mesenchymal stem cells.
Adhere-wall culture obtained above had in the blake bottle of 1st generation umbilical cord mesenchymal stem cells and add 2.5g/L trypsinization, and be passaged in Tissue Culture Plate in the ratio of 1:3, the growth medium used that goes down to posterity is the growth medium in preparation embodiment 1.When reaching for the 6th generation and be cultured to the degrees of fusion of 80%, use 2.5g/L trypsin digestion cell, 200 × g centrifugal collecting cell, the cryopreserving liquid suspension cell obtained with preparation embodiment 1-2 and preparation comparative example 1-4.Relative to the cryopreserving liquid of every mL, the consumption of umbilical cord mesenchymal stem cells is 2 × 10
7individual.The cell that cryopreserving liquid suspends is placed in programmed cooling instrument, is cooled to subzero 80 DEG C with the rate of temperature fall of 1 DEG C/min, is then placed in liquid nitrogen and preserves.
Preserve and recover after 10 days, the cell of preservation is placed in the water-bath 5 minutes of 37 DEG C, then the growth medium added in preparation embodiment 1 suspends, the cryopreserving liquid of relatively every mL, the addition of growth medium is 10mL, 200 × g centrifugal collecting cell, then the growth medium added in preparation embodiment 1 suspends, adjustment cell concentration to 1 × 10
4individual/mL, incubation growth to 80% degrees of fusion, then use tetrazolium bromide (MTT) method to detect cytoactive and draw the growth curve of cell, concrete operations comprise: suspended by the umbilical cord mesenchymal stem cells enzymolysis growing to 80% degrees of fusion, adjustment cell concentration to 1 × 10
4individual/mL, adds 24 orifice plates, every hole 800 μ L, be placed in 37 DEG C, saturated humidity, 5 volume % CO
2cultivate in incubator, in 5 days, a 24 orifice plates are taken out every 8 hours, after every hole adds the MTT solution (5g/L) of 80 μ L, 4h is placed at 37 DEG C, the dimethyl sulfoxide (DMSO) (DMSO) of 600 μ L is added in every hole, each hole absorption photometric value (D value) is measured at 550nm place by microplate reader, the relative populations of cell is represented with D value, draw growth curve, result as shown in Figure 1, in Fig. 1, 1 and 2 represent preparation embodiment 1 and 2, 3-6 represents preparation comparative example 1-4 respectively, abscissa represents growth time, ordinate represents that 550nm place measures each hole absorption photometric value (D value).
Can find out according to growth curve, the cryopreserving liquid that obtains of preparation embodiment 1 and 2 preserve umbilical cord mesenchymal stem cells can grow to plateau before 56 hours.But the umbilical cord mesenchymal stem cells in the growth medium that preparation comparative example 1-4 obtains can only can grow to plateau after 72 hours.This shows, cryopreserving liquid of the present invention can effectively accelerate frozen after the growth rate of umbilical cord mesenchymal stem cells.
Testing example 2
After caesarean birth fetal birth, intercept umbilical cord, peel off amnion, peel off Wal Tong Shi glue (Wharton'sjelly), shred into 0.5-1.5mm
3tissue block, add type Ⅳ collagenase (10g/L), after putting 37 DEG C of constant-temperature shaking incubators vibration digestion 3h, filter with 80 mesh filter screens, by the cell suspension centrifuge washing of filtration.Cell is mixed, with 1.0 × 10 with serum free medium (purchased from Cambrex, UItraCULTURE)
5/ cm
2inoculum density be inoculated in T-75cm
2in blake bottle, be placed in 37 DEG C, saturated humidity, 5 volume % CO
2be cultured to the degrees of fusion of 80% in incubator, obtain 1st generation umbilical cord mesenchymal stem cells.
Adhere-wall culture obtained above had in the blake bottle of 1st generation umbilical cord mesenchymal stem cells and add 2.5g/L trypsinization, and be passaged in Tissue Culture Plate in the ratio of 1:3, the growth medium used that goes down to posterity is the growth medium in preparation embodiment 1.When reaching for the 6th generation and be cultured to the degrees of fusion of 80%, use 2.5g/L trypsin digestion cell, 200 × g centrifugal collecting cell, the cryopreserving liquid suspension cell obtained with preparation embodiment 1-2.Relative to the cryopreserving liquid of every mL, the consumption of umbilical cord mesenchymal stem cells is 2 × 10
7individual.The cell that cryopreserving liquid suspends is placed in programmed cooling instrument, is cooled to subzero 80 DEG C with the rate of temperature fall of 1 DEG C/min, is then placed in liquid nitrogen and preserves.
Preserve and recover after 10 days, the cell of preservation is placed in the water-bath 5 minutes of 37 DEG C, then the growth medium added in preparation embodiment 1 suspends, the cryopreserving liquid of relatively every mL, the addition of growth medium is 10mL, 200 × g centrifugal collecting cell, then the growth medium added in preparation embodiment 1 suspends, adjustment cell concentration to 1 × 10
4individual/mL, incubation growth to 80% degrees of fusion, go down to posterity in the ratio of 1:3, the 1st generation umbilical cord mesenchymal stem cells gone down to posterity after obtaining cryopreservation resuscitation, after carrying out and so forth being passaged to and obtaining the 25th generation umbilical cord mesenchymal stem cells, to the 25th generation umbilical cord mesenchymal stem cells carry out stem cell surface mark mensuration, the detection to osteoblast differentiation potential, the detection to Adipocyte Differentiation potential, the detection to quasi-liver cell differentiation potential, the detection to myoblast differentiation potential, the detection of differentiating into nerve cells potential and the detection to hematopoetic cell differentiation potential.Concrete detection method comprises: stem cell surface mark mensuration, the detection to osteoblast differentiation potential and the detection to Adipocyte Differentiation potential are according to document (Wang Juan etc., human umbilical cord mesenchymal stem cells in-vitro separation, Isolation and characterization, Ji'nan University's journal, 30th volume, 2009) method in is carried out; Detection to quasi-liver cell differentiation potential is carried out according to document (bear China etc., cryopreserved human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells differentiation-inducing, Chinese Tissue Engineering Study and clinical rehabilitation, the 7th phase in 2007); To myoblast differentiation potential detection according to document (Liu Dai, mesenchymal stem cells derived from human umbilical blood to the in vitro study of myoblast differentiation, master thesis, 2009) carry out; The detection of differentiating into nerve cells potential is carried out according to document (Sun Hongtao etc., umbilical cord mesenchymal stem cells separation, qualification and Neural Differentiation, Chinese neurosurgery related disease research magazine, in April, 2008); Detection to hematopoetic cell differentiation potential is carried out according to document (Hu Kaimeng etc., the research that human umbilical cord mesenchymal stem cells breaks up to hematopoietic cell direction, Ph.D. Dissertation, 2012).
The result of above-mentioned detection shows, being separated the cryopreserving liquid using preparation embodiment 1-2 to obtain after the Umbilical cord blood mesenchymal stem cells cultivated reached for the 6th generation carries out frozen, after recovery, carried out for 25 generations when going down to posterity, also there is the specific surfaces mark of stem cell, to osteoblast differentiation potential, to Adipocyte Differentiation potential, to quasi-liver cell differentiation potential, to myoblast differentiation potential, differentiating into nerve cells potential and to hematopoetic cell differentiation potential.
As can be seen here, the subculture in vitro separately number of the umbilical cord mesenchymal stem cells after frozen can improve by the present invention, and the growth rate after going down to posterity is accelerated, this is external repeatedly go down to posterity after also there will not be the phenomenon of loss differentiation potential.
Below the preferred embodiment of the present invention is described in detail by reference to the accompanying drawings; but; the present invention is not limited to the detail in above-mentioned embodiment; within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible combination.
In addition, also can be combined between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. for a cryopreserving liquid for preservation stem cell, this cryopreserving liquid is mixed to get by dimethyl sulfoxide (DMSO) and growth medium, and the volume ratio of dimethyl sulfoxide (DMSO) and described growth medium is (0.05-0.4): 1; Described growth medium contains basal medium and additive, it is characterized in that: described additive contains the antibody of human vessel endothelium growth factor resisting, the antibody of anti-human ErbB1, the antibody of anti-human epidermal growth factor acceptor 2 and guanosine-5-triphosphoric acid trisodium salt.
2. cryopreserving liquid according to claim 1, it is characterized in that: the content of the antibody of human vessel endothelium growth factor resisting is 0.01-0.2ng/mL, the content of the antibody of anti-human ErbB1 is 0.01-0.1ng/mL, and the content of the antibody of anti-human epidermal growth factor acceptor 2 is 0.05-0.4ng/mL; The content of guanosine-5-triphosphoric acid trisodium salt is 0.2-4ng/mL.
3. cryopreserving liquid according to claim 2, it is characterized in that: the content of the antibody of human vessel endothelium growth factor resisting is 0.05-0.1ng/mL, the content of the antibody of anti-human ErbB1 is 0.03-0.08ng/mL, and the content of the antibody of anti-human epidermal growth factor acceptor 2 is 0.1-0.2ng/mL; The content of guanosine-5-triphosphoric acid trisodium salt is 0.5-2ng/mL.
4. according to the cryopreserving liquid in claim 1-3 described in any, it is characterized in that: the antibody of described human vessel endothelium growth factor resisting is bevacizumab; The antibody of described anti-human ErbB1 is Cetuximab, and the antibody of described anti-human epidermal growth factor acceptor 2 is Herceptin.
5. according to cryopreserving liquid according to claim 1, it is characterized in that: described basal medium comprises at least one in DMEM medium, MEM medium, IMEM medium and RPMI1640 medium.
6. cryopreserving liquid according to claim 1, is characterized in that: the serum of described growth medium also containing 5-20 volume %.
7. the purposes of the cryopreserving liquid in claim 1-6 described in any one in preservation umbilical cord mesenchymal stem cells.
8. a method for preservation umbilical cord mesenchymal stem cells, is characterized in that: the method comprises: be suspended in by umbilical cord mesenchymal stem cells in the cryopreserving liquid in claim 1-6 described in any one, then preservation in the temperature of subzero 200 DEG C to subzero 70 DEG C.
9. method according to claim 8, is characterized in that: relative to the cryopreserving liquid of every mL, and the consumption of umbilical cord mesenchymal stem cells is (1-5) × 10
7individual.
10. method according to claim 8, is characterized in that: frozen rate of temperature fall used is 0.5-2 DEG C/min.
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| 培养基对兔骨髓间充质干细胞扩增与分化的影响;须珏华等;《中国组织工程研究与临床康复》;20070121;第11卷(第3期);第468-470页 * |
| 无血清培养基体外分离培养人胶质瘤干细胞与鉴定;袁鲁等;《中国组织工程研究与临床康复》;20090302(第10期);第1896-1900 * |
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