CN108315300A - A kind of cell factor composition cultivates the application in candidate stem cell in vitro - Google Patents
A kind of cell factor composition cultivates the application in candidate stem cell in vitro Download PDFInfo
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Abstract
Cultivate the application in candidate stem cell in vitro the invention discloses a kind of cell factor composition, belong to bioengineering and biomedicine technical field, the cell factor composition includes mainly translation growth factor-β, stem cell factor, interleukin-1 beta, platelet derived growth factor, differentiation inhibiting factor and interleukin-6, and prepared for blood serum medium using cell factor composition, the preparation method of the culture medium includes：(1) culture substrate layer is prepared, (2) prepare magnetic antigen, and (3) prepare sterile culture base fluid；The amplification method of culture medium in vitro culture candidate stem cell includes：(1) enrichment of candidate stem cell, the amplification in vitro and harvest of (2) candidate stem cell, the Magneto separate of (3) candidate stem cell.In short, the present invention can effectively improve the in-vitro multiplication rate of candidate stem cell, there is positive effect in drug research and clinical treatment.
Description
Technical field
The invention belongs to bioengineering and biomedicine technical field, and in particular to a kind of cell factor composition is in vitro
Cultivate the application in candidate stem cell.
Background technology
Hematopoietic stem cell transplantation has become one of the important means of clinical treatment blood disease and malignant tumour at present, due to
Derived from cord blood is abundant, acquisition is convenient, immunogenicity is weak, has more advantages compared with marrow and peripheral blood transplanting.But hematopoiesis in bleeding of the umbilicus
Ancestral cells content is relatively low, it is difficult to meet adult and the needs of the larger patient of weight, hematopoietic reconstitution postpones after transplanting, it is difficult to receive
To satisfied curative effect, its application in clinic has seriously been limited.How foot expeditiously to be obtained by the method for amplification in vitro
The candidate stem cell of amount is to meet clinical needs, it has also become a urgent problem to be solved.Previously researches show that：Traditional cell
The amplification in vitro system of combinations of factors also accelerates the process of its differentiation and maturation while promoting hematopoietic stem/progenitor proliferation,
The self-renewing of hematopoietic stem/progenitor and the potential of hematopoietic reconstitution are lost, is unable to maintain that long term hematopoietic；With to hematopoiesis micro-loop
The understanding for regulating and controlling the molecular mechanism of hemopoietic stem cell proliferation and self-renewing in border provides newly for amplifying candidate stem cell in vitro
Thinking.In vivo, hematopoieticmicroenviron-ment maintains candidate stem cell by iuntercellular close contact and all kinds of hematopoietic regulation factors
The potential of self-renewing and polyphyly differentiation.Therefore, the microenvironment for simulating hematopoietic stem cell growth becomes and improves amplification in vitro effect
One of rate important method.
Publication No. CN103509101A's discloses a kind of cell factor of amplification umbilical cord blood hematopoietic stem cell, the cell
The factor is obtained by following preparation method comprising following steps：(1) processing is for stroma cell；(2) it obtains immortalizing thin
Born of the same parents；(3) the fetal liver cell continuous passage for filtering out (2) step obtains cell line；(4) total serum IgE is obtained；(5) reverse transcription at
cDNA；(6) gene microarray analysis is done；(7) it chooses in the fetal mouse liver cell that can stimulate umbilical cord blood hematopoietic stem cell in-vitro multiplication
The gene order of height expression；(8) (7) are retrieved into nucleotide sequence；(9) 293T cells are obtained；(10) it is opposite that 293T cells are obtained
The albumen answered；(11) albumen that (10) purify is added to containing SCF, TPO, FLT3L, in the StemSpan culture mediums of PTN, is tested
Card can promote the in-vitro multiplication of umbilical cord blood hematopoietic stem cell.But used by the patent it is traditional blood serum medium, due to
For serum origin in heterogenous animal, ingredient is unknown, and quality is difficult to control, and there is virus infection, immunological rejection equivalent risk.Also,
The proliferation times of candidate stem cell are not high, low to the differentiation inhibiting rate of candidate stem cell, and there is the hematopoiesis to Successful amplification
The defect that stem cell is not readily separated with other interference cells.
Invention content
For the above technical problem, the present invention provides a kind of cell factor composition and cultivates in candidate stem cell in vitro
Using.
The technical scheme is that：A kind of cell factor composition cultivates the application in candidate stem cell, institute in vitro
It is with weight ratio by main component and auxiliary element for 10-20 to state cell factor composition:1-3 is formed；
The main component includes in percentage by weight：11.3-15.2% translation growth factor-βs, 17.6-
22.4% stem cell factor, 9.1-14.4% interleukin-1 ' beta 's, 5.8-8.6% platelet derived growth factors, 4.7-7.6% point
Change inhibiting factor, surplus is interleukin-6；The platelet derived growth factor in the Colony cultivation of candidate stem cell to having orientation
The effect for breaking up megakaryoblast, can be improved the proliferation of candidate stem cell with other combination of cytokines in vitro in amplification cultivation
Rate and amplification times；The stem cell factor is the stimulating factor of highly important early stage candidate stem cell；The monocyte
Chemotactic factor (CF) 1 can promote the expression of the scavenger receptor of mononuclear macrophage；The differentiation inhibiting factor can inhibit Hematopoietic Stem
Cell further breaks up.
The auxiliary element includes in percentage by weight：9.8-14.1% insulin-like growth factors 1,11.5-
The prostaglandin mediated some growth factor of 12.7% inflammatory cytokine, 7.2-10.1% hypoxia inducible factors, 0.8-1.3%,
7.8-9.1% eosinophil chemokine, 14.8-20.2% cell activating agents, 6.7-8.4% micromolecular inhibitors,
Surplus behaviour galactose agglutinin 1；The insulin-like growth factor 1 can promote the proliferation rate of candidate stem cell；It is described thermophilic
Eosinophil chemotactic factor (CF) and inflammatory cytokine can carry out proliferation of the candidate stem cell in amplification procedure and differentiation micro-
See regulation and control；The prostaglandin mediated some growth factor, which has, promotes candidate stem cell to divide and may also function as certain cell
Protective effect；The hypoxia inducible factor can ensure that the candidate stem cell after division amplification can adapt to being transplanted in human body
Low-oxygen environment；The cell activating agent can activate candidate stem cell, promote its division；The micromolecular inhibitor can press down
Manufacture hemocytoblast is broken up；Differentiation of the 1 controllable candidate stem cell of the human galactose agglutinin after being transplanted to human body
Direction.
Further, the micromolecular inhibitor is the micromolecular inhibitor based on SPOP protein structures, the SPOP eggs
In white amino acid sequence such as sequence table shown in SEQ ID NO.1：
Further, the cell factor composition is used to manufacture the culture medium of in vitro culture candidate stem cell.
Further, the culture medium includes in percentage by weight：It cultivates on the bases 10.2-12.3%DMEM/F12
Base, 6.8-7.9% cell factor compositions, 5.6-6.7% nanometer magnetic beads, 5.1-6.4% vascular endothelial cells adhesion factor,
4.6-5.7%PBS buffer solutions, 0.3-0.8%rhVEGF, 0.5-1.0% multi-vitamins, 0.08-0.56% trace elements
Close object, 0.7-1.2% tyrosine, 0.8-1.3% peptides, 1.1-1.7%3D Poly L-lactic acid, 0.5-0.6% lecithin,
0.1-0.3% small active peptides, 0.05-0.10% tea polyphenols, 1.0-2.0% human hemoglobins, 3.3-4.1% water-setting collagens
Protein adhesive, 3.3-4.1% glacial acetic acid, 3.4-4.5% human placental extracts, 0.1-0.2% superoxide dismutases, 0.1-
0.3% polyunsaturated fatty acid, 0.01-0.03% prednisones, 0.01-0.03% progesterone, 0.2-0.3% Trenaxmines, surplus
For ultra-pure water；The polyunsaturated fatty acid is used to safeguard the structure and function of cell membrane；The prednisone has anti-inflammatory and anti-
Allergy acts on；Active skull cap components containing Cord blood development in the human placental extract, simulate the internal hair of Cord blood
Microenvironment is educated, the amplification rate of candidate stem cell is greatly improved；The small active peptides can be directly entered into the cell with original shape,
Abundant nutriment is provided for candidate stem cell；The superoxide dismutase can eliminate candidate stem cell in fission process
In itself generate free radical；The Trenaxmine can be to inhibiting the differentiation of candidate stem cell to play certain booster action.
Further, the multi-vitamins are by vitamin A, vitamin B6, adenine phosphate, vitamin E with quality
Than being 1:1:2:2 compositions；The multi-vitamins can provide nutrition for candidate stem cell, improve the work of candidate stem cell
Property.
Further, the trace element compound is selenium yeast, zinc sulfate, two carboxyethyl germanium sesquioxide germanium, chlorination
Any one or a few in calcium, iron chloride；The selenium yeast can provide trace elements of selenium for candidate stem cell, promote Hematopoietic Stem
The division of cell.
Further, the preparation method of the culture medium includes the following steps：
(1) culture substrate layer is prepared：The water-setting collagen protein adhesive is dissolved in the glacial acetic acid, and molten by what is obtained
It solves liquid and utilizes 0.22 μm of membrane filtration, obtain water-setting collagen protein adhesive lysate；Then again that the water-setting collagen protein adhesive is molten
Liquid is solved with 100l/ μ cm2Dosage be fitted into culture roller bottle in, then will the culture roller bottle sealing after be put into constant temperature incubator,
Temperature be 40-45 DEG C and rotating speed be 6-12r/h under the conditions of adherent incubation 2h, obtain culture substrate layer, for use；Water-setting collagen
Glue, as culture substrate layer, has at fast compared with rate, mechanical strength is good, degradation time is longer, immune row compared to collagen
The advantages that different reaction is weaker.
(2) magnetic antigen is prepared：The nanometer magnetic bead activates 60min by EDC/NHSS modes, obtains activation nano magnetic
Pearl, then the activation nanometer magnetic bead, vascular endothelial cell adhesion factor, PBS buffer solution are mixed according to aforementioned proportion, with
30-60r/min mechanical agitations 30-40min so that activation nanometer magnetic bead is specifically tied with vascular endothelial cell adhesion factor
It closes, later, the rhVEGF is added, in 37 DEG C of CO24-6h is cultivated in incubator, activates vascular endothelial cell adhesion factor,
Magnetic antigen is obtained, for carrying out specific adhesion with candidate stem cell；
(3) sterile culture base fluid is prepared：By the cell factor composition, multi-vitamins, trace element compound, junket
Propylhomoserin, peptide, 3D Poly L-lactic acid, lecithin, small active peptides, tea polyphenols, human hemoglobin, human placental extract,
Recombination human source vascular endothelial growth factor, superoxide dismutase, polyunsaturated fatty acid, prednisone, progesterone, Trenaxmine with
Aforementioned proportion is added in the DMEM/F12 basal mediums, and is dissolved using the ultra-pure water, and 0.22 μ is finally utilized
M membrane filtration degermings, obtain sterile culture base fluid；The sterile culture base fluid and the magnetic antigen are added to containing again
It states in the culture roller bottle of culture substrate layer to get to culture medium.
Further, included the following steps using the amplification method of the culture medium in vitro culture candidate stem cell：
(1) enrichment of candidate stem cell：Aseptic collection umbilical cord blood 40-50ml, using Ficoll density gradients from
The sedimentation of heart method detaches mononuclearcell in the Cord blood；
(2) amplification in vitro and harvest of candidate stem cell：The nucleus is added to 1 × 1010cells/L concentration
In culture roller bottle containing the culture medium, it is placed in 37 DEG C, 5%CO2Incubator in, culture roller bottle in 6-12r/h rotating speeds
Lower adhere-wall culture 4-6 days, changes the liquid once every three days, and 3-5 is for people's umbilical cord candidate stem cell for harvest, and then adding volume ratio is
1:The mixed enzyme solution of 1 0.25% trypsase and 0.1% clostridiopetidase A digests 30-60min, and vortex mixer is recycled to carry out
5-10min is shaken, stripping is adsorbed on the cell film layer of culture roller bottle wall, obtains band passage candidate stem cell；
(3) Magneto separate of candidate stem cell：By the passage candidate stem cell by vortex magnetic separator screening, discards and do not glue
Attached cell obtains the passage candidate stem cell mutually adhered to magnetic antigen.
Compared with prior art, beneficial effects of the present invention are：
(1) the present invention provides the specific cell factor compositions of component, wherein in main component it is platelet-derived because
Son is thin with other in amplification cultivation in vitro to playing the role of directed differentiation megakaryoblast in the Colony cultivation of candidate stem cell
Intracellular cytokine combines the proliferation rate and amplification times that candidate stem cell can be improved；Stem cell factor is highly important early stage Hematopoietic Stem
The stimulating factor of cell；Translation growth factor-β can promote the expression of the scavenger receptor of mononuclear macrophage；Differentiation inhibits
The factor can inhibit candidate stem cell further to break up.Insulin-like growth factor 1 in auxiliary element can promote Hematopoietic Stem
The proliferation rate of cell；Eosinophil chemokine and inflammatory cytokine can be to increasing of the candidate stem cell in amplification procedure
It grows and breaks up and carry out microcosmic regulation and control；The prostaglandin mediated some growth factor, which has, promotes candidate stem cell to divide and may also function as
Certain cytoprotection；Hypoxia inducible factor can ensure that the candidate stem cell after division amplification is being transplanted to energy in human body
Enough adapt to low-oxygen environment；Cell activating agent can activate candidate stem cell, promote its division；Micromolecular inhibitor can inhibit
Candidate stem cell is broken up；Differentiation direction of the 1 controllable candidate stem cell of human galactose agglutinin after being transplanted to human body.
(2) present invention prepares serum free medium using cell factor composition, avoids since serum origin is in xenogenesis
Animal, ingredient is unknown, and quality is difficult to control, and there is virus infection, immunological rejection equivalent risk.Wherein, in culture medium it is more not
Saturated fatty acid is used to safeguard the structure and function of cell membrane；Prednisone has anti-inflammatory and anti-allergic effects；Human placental extract
In containing Cord blood development active skull cap components, simulate the internal development microenvironment of Cord blood, be greatly improved Hematopoietic Stem
The amplification rate of cell；Small active peptides can be directly entered into the cell with original shape, and abundant nutrients is provided for candidate stem cell
Matter；Superoxide dismutase can eliminate the candidate stem cell free radical that itself is generated in fission process；Trenaxmine can be right
The differentiation of candidate stem cell is inhibited to play certain booster action.
(3) the present invention also provides a kind of preparation methods of culture medium, prepare and cultivate first with water-setting collagen protein adhesive
Substrate layer, water-setting collagen protein adhesive compared to collagen as culture substrate layer, have at fast compared with rate, mechanical strength is good,
The advantages that longer, the immune rejection of degradation time is weaker.Secondly, by nanometer magnetic bead and vascular endothelial cell adhesion factor into
Row combine, and utilize rhVEGF to activate vascular endothelial cell adhesion factor, obtain magnetic antigen, for candidate stem cell into
Row specific adhesion.
(4) the present invention also provides the amplification of candidate stem cell and separation methods, wherein is passed using vortex magnetic separator screening
For candidate stem cell, nonadherent cell is discarded, the passage candidate stem cell mutually adhered to magnetic antigen is obtained, can efficiently carry
The separation purity of high candidate stem cell is implanted into human body in addition, sticking to be incorporated in the candidate stem cell after magnetic antigen binding
Interior, can targeting control according to external magnetic control, it is gone back to the nest, and improves the utilization rate of candidate stem cell.
In short, present invention proportioning is reasonable, the in-vitro multiplication rate of candidate stem cell can be effectively improved, while also can be improved and making
Hemocytoblast inhibit after utilization rate, in drug research and clinical treatment have positive effect, and with it is good society and
Economic benefit.
Specific implementation mode
With reference to specific embodiment, present invention be described in more detail, but embodiment does not limit the scope of the invention,
The scope of the present invention is determined with core content according to claims.
Embodiment 1
A kind of cell factor composition cultivates the application in candidate stem cell in vitro, the cell factor composition be by
Main component and auxiliary element are with weight ratio for 10:1 composition；
The main component includes in percentage by weight：11.3% translation growth factor-β, 17.6% stem cell
The factor, 9.1% interleukin-1 ' beta ', 5.8% platelet derived growth factor, 4.7% differentiation inhibiting factor, surplus are situated between for leucocyte
Element 6；The platelet derived growth factor in the Colony cultivation of candidate stem cell to playing the role of directed differentiation megakaryoblast, in body
The proliferation rate and amplification times of candidate stem cell can be improved in outer amplification cultivation with other combination of cytokines；The stem cell because
Son is the stimulating factor of highly important early stage candidate stem cell；The translation growth factor-β can promote monokaryon macrophage thin
The expression of the scavenger receptor of born of the same parents；The differentiation inhibiting factor can inhibit candidate stem cell further to break up.
The auxiliary element includes in percentage by weight：9.8% insulin-like growth factor 1,11.5% inflammation are thin
Intracellular cytokine, 7.2% hypoxia inducible factor, the 0.8% prostaglandin mediated some growth factor, 7.8% eosinophil chemotactic
The factor, 14.8% cell activating agent, 6.7% micromolecular inhibitor, surplus behaviour galactose agglutinin 1；The para-insulin
Growth factor 1 can promote the proliferation rate of candidate stem cell；The eosinophil chemokine and inflammatory cytokine can
Microcosmic regulation and control are carried out to proliferation of the candidate stem cell in amplification procedure and differentiation；The prostaglandin mediated some growth factor
It is divided with promotion candidate stem cell and may also function as certain cytoprotection；The hypoxia inducible factor can ensure point
It splits the candidate stem cell after amplification and can adapt to low-oxygen environment being transplanted in human body；The cell activating agent can be activated and be made
Hemocytoblast promotes its division；The micromolecular inhibitor can inhibit candidate stem cell to be broken up；The human galactose is solidifying
Differentiation direction of the plain 1 controllable candidate stem cell of collection after being transplanted to human body.Wherein, the micromolecular inhibitor is to be based on SPOP
The micromolecular inhibitor of protein structure, in the amino acid sequence such as sequence table of the SPOP albumen shown in SEQ ID NO.1.
The cell factor composition is used to manufacture the culture medium of in vitro culture candidate stem cell.Wherein, the culture medium
Include in percentage by weight：10.2%DMEM/F12 basal mediums, 6.8% cell factor composition, 5.6% nano magnetic
Pearl, 5.1% vascular endothelial cell adhesion factor, 4.6%PBS buffer solutions, 0.3%rhVEGF, 0.5% multi-vitamins,
0.08% trace element compound, 0.7% tyrosine, 0.8% peptide, 1.1%3D Poly L-lactic acid, 0.5% lecithin,
0.1% small active peptides, 0.05% tea polyphenols, 1.0% human hemoglobin, 3.3% water-setting collagen protein adhesive, 3.3% ice vinegar
Acid, 3.4% human placental extract, 0.1-0.2% superoxide dismutases, 0.1% polyunsaturated fatty acid, 0.01% bold and vigorous Buddhist nun
Pine, 0.01% progesterone, 0.2% Trenaxmine, surplus is ultra-pure water；The polyunsaturated fatty acid is used to safeguard the knot of cell membrane
Structure and function；The prednisone has anti-inflammatory and anti-allergic effects；Day containing Cord blood development in the human placental extract
Right active constituent simulates the internal development microenvironment of Cord blood, is greatly improved the amplification rate of candidate stem cell；Described small point
Sub- active peptide can be directly entered into the cell with original shape, and abundant nutriment is provided for candidate stem cell；The superoxides discrimination
The candidate stem cell free radical that itself is generated in fission process can be eliminated by changing enzyme；The Trenaxmine can be to inhibiting Hematopoietic Stem
Certain booster action is played in the differentiation of cell.Wherein, the multi-vitamins are by vitamin A, vitamin B6, vitamin
B4, vitamin E are with mass ratio for 1:1:2:2 compositions；The multi-vitamins can provide nutrition for candidate stem cell, improve
The activity of candidate stem cell.Wherein, the multi-vitamins are by vitamin A, vitamin B6, adenine phosphate, vitamin E with matter
Amount is than being 1:1:2:2 compositions；The multi-vitamins can provide nutrition for candidate stem cell, improve the work of candidate stem cell
Property.Wherein, the trace element compound is zinc sulfate.
The preparation method of the culture medium includes the following steps：
(1) culture substrate layer is prepared：The water-setting collagen protein adhesive is dissolved in the glacial acetic acid, and molten by what is obtained
It solves liquid and utilizes 0.22 μm of membrane filtration, obtain water-setting collagen protein adhesive lysate；Then again that the water-setting collagen protein adhesive is molten
Liquid is solved with 100l/ μ cm2Dosage be fitted into culture roller bottle in, then will the culture roller bottle sealing after be put into constant temperature incubator,
Temperature be 40 DEG C and rotating speed be 6r/h under the conditions of adherent incubation 2h, obtain culture substrate layer, for use；Water-setting collagen protein adhesive is compared
In collagen as culture substrate layer, have at fast compared with rate, mechanical strength is good, degradation time is longer, immune rejection
The advantages that weaker.
(2) magnetic antigen is prepared：The nanometer magnetic bead activates 60min by EDC/NHSS modes, obtains activation nano magnetic
Pearl, then the activation nanometer magnetic bead, vascular endothelial cell adhesion factor, PBS buffer solution are mixed according to aforementioned proportion, with
30r/min mechanical agitations 30min so that activation nanometer magnetic bead carries out specific bond with vascular endothelial cell adhesion factor, later,
The rhVEGF is added, in 37 DEG C of CO24h is cultivated in incubator, activates vascular endothelial cell adhesion factor, is obtained magnetic anti-
Original, for carrying out specific adhesion with candidate stem cell；
(3) sterile culture base fluid is prepared：By the cell factor composition, multi-vitamins, trace element compound, junket
Propylhomoserin, peptide, 3D Poly L-lactic acid, lecithin, small active peptides, tea polyphenols, human hemoglobin, human placental extract,
Recombination human source vascular endothelial growth factor, superoxide dismutase, polyunsaturated fatty acid, prednisone, progesterone, Trenaxmine with
Aforementioned proportion is added in the DMEM/F12 basal mediums, and is dissolved using the ultra-pure water, and 0.22 μ is finally utilized
M membrane filtration degermings, obtain sterile culture base fluid；The sterile culture base fluid and the magnetic antigen are added to containing again
It states in the culture roller bottle of culture substrate layer to get to culture medium.
Amplification method using the culture medium in vitro culture candidate stem cell includes the following steps：
(1) enrichment of candidate stem cell：Aseptic collection umbilical cord blood 40ml, using Ficoll density-gradient centrifugation methods
It settles, detach mononuclearcell in the Cord blood；
(2) amplification in vitro and harvest of candidate stem cell：By the nucleus with 1 × 1010Cells/L concentration, which is added to, to be contained
Have in the culture roller bottle of the culture medium, is placed in 37 DEG C, 5%CO2Incubator in, culture roller bottle it is adherent under 6r/h rotating speeds
Culture 4 days, changes the liquid once every three days, harvests 3 generation people's umbilical cord candidate stem cells, and it is 1 then to add volume ratio:1
The mixed enzyme solution of 0.25% trypsase and 0.1% clostridiopetidase A digests 30min, and vortex mixer is recycled to carry out concussion 5min,
Stripping is adsorbed on the cell film layer of culture roller bottle wall, obtains band passage candidate stem cell；
(3) Magneto separate of candidate stem cell：By the passage candidate stem cell by vortex magnetic separator screening, discards and do not glue
Attached cell obtains the passage candidate stem cell mutually adhered to magnetic antigen.
Embodiment 2
A kind of cell factor composition cultivates the application in candidate stem cell in vitro, the cell factor composition be by
Main component and auxiliary element are with weight ratio for 15:2 compositions；
The main component includes in percentage by weight：It is 13.25% translation growth factor-β, 20.0% dry thin
Intracellular cytokine, 11.75% interleukin-1 ' beta ', 7.2% platelet derived growth factor, 6.15% differentiation inhibiting factor, surplus are white thin
Born of the same parents' interleukin 6；The platelet derived growth factor to playing the role of directed differentiation megakaryoblast in the Colony cultivation of candidate stem cell,
The proliferation rate and amplification times of candidate stem cell can be improved in amplification cultivation with other combination of cytokines in vitro；It is described dry thin
Intracellular cytokine is the stimulating factor of highly important early stage candidate stem cell；The translation growth factor-β can promote monokaryon huge
The expression of the scavenger receptor of phagocyte；The differentiation inhibiting factor can inhibit candidate stem cell further to break up.
The auxiliary element includes in percentage by weight：11.95% insulin-like growth factor 1,12.1% inflammation
Cell factor, 8.65% hypoxia inducible factor, the 1.05% prostaglandin mediated some growth factor, 8.45% eosinophil
Chemotactic factor (CF), 17.5% cell activating agent, 7.55% micromolecular inhibitor, surplus behaviour galactose agglutinin 1；The class pancreas
Island element growth factor 1 can promote the proliferation rate of candidate stem cell；The eosinophil chemokine and inflammatory cell because
Son can carry out microcosmic regulation and control to proliferation of the candidate stem cell in amplification procedure and differentiation；The prostaglandin mediated some growth
The factor, which has, promotes candidate stem cell to divide and may also function as certain cytoprotection；The hypoxia inducible factor can be protected
Candidate stem cell after card division amplification can adapt to low-oxygen environment being transplanted in human body；The cell activating agent can swash
Candidate stem cell living, promotes its division；The micromolecular inhibitor can inhibit candidate stem cell to be broken up；People's gala
Differentiation direction of the 1 controllable candidate stem cell of sugared agglutinin after being transplanted to human body.Wherein, the micromolecular inhibitor is to be based on
The micromolecular inhibitor of SPOP protein structures, in the amino acid sequence such as sequence table of the SPOP albumen shown in SEQ ID NO.1.
The cell factor composition is used to manufacture the culture medium of in vitro culture candidate stem cell.Wherein, the culture medium
Include in percentage by weight：11.25%DMEM/F12 basal mediums, 7.35% cell factor composition, 6.15% are received
Rice magnetic bead, 5.75% vascular endothelial cell adhesion factor, 5.15%PBS buffer solutions, 0.55%rhVEGF, 0.75% compound dimension life
Element, 0.32% trace element compound, 0.95% tyrosine, 1.1% peptide, 1.4%3D Poly L-lactic acid, 0.55% lecithin
Fat, 0.2% small active peptides, 0.07% tea polyphenols, 1.5% human hemoglobin, 3.7% water-setting collagen protein adhesive, 3.7% ice
Acetic acid, 4.0% human placental extract, 0.15% superoxide dismutase, 0.2% polyunsaturated fatty acid, 0.02% prednisone,
0.02% progesterone, 0.2-0.3% Trenaxmines, surplus are ultra-pure water；The polyunsaturated fatty acid is for safeguarding cell membrane
Structure and function；The prednisone has anti-inflammatory and anti-allergic effects；Contain Cord blood development in the human placental extract
Active skull cap components simulate the internal development microenvironment of Cord blood, are greatly improved the amplification rate of candidate stem cell；It is described small
Molecular activity peptide can be directly entered into the cell with original shape, and abundant nutriment is provided for candidate stem cell；The superoxides
Mutase can eliminate the candidate stem cell free radical that itself is generated in fission process；The Trenaxmine can be to inhibiting hematopoiesis
Certain booster action is played in the differentiation of stem cell.Wherein, the multi-vitamins are by vitamin A, vitamin B6, vitamin
B4, vitamin E are with mass ratio for 1:1:2:2 compositions；The multi-vitamins can provide nutrition for candidate stem cell, improve
The activity of candidate stem cell.Wherein, the multi-vitamins are by vitamin A, vitamin B6, adenine phosphate, vitamin E with matter
Amount is than being 1:1:2:2 compositions；The multi-vitamins can provide nutrition for candidate stem cell, improve the work of candidate stem cell
Property.Wherein, the trace element compound is selenium yeast.
The preparation method of the culture medium includes the following steps：
(1) culture substrate layer is prepared：The water-setting collagen protein adhesive is dissolved in the glacial acetic acid, and molten by what is obtained
It solves liquid and utilizes 0.22 μm of membrane filtration, obtain water-setting collagen protein adhesive lysate；Then again that the water-setting collagen protein adhesive is molten
Liquid is solved with 100l/ μ cm2Dosage be fitted into culture roller bottle in, then will the culture roller bottle sealing after be put into constant temperature incubator,
Temperature be 43 DEG C and rotating speed be 10r/h under the conditions of adherent incubation 2h, obtain culture substrate layer, for use；Water-setting collagen protein adhesive phase
Compared with collagen as culture substrate layer, have at fast compared with rate, mechanical strength is good, degradation time is longer, immune rejection is anti-
Should be weaker the advantages that.
(2) magnetic antigen is prepared：The nanometer magnetic bead activates 60min by EDC/NHSS modes, obtains activation nano magnetic
Pearl, then the activation nanometer magnetic bead, vascular endothelial cell adhesion factor, PBS buffer solution are mixed according to aforementioned proportion, with
45r/min mechanical agitations 35min so that activation nanometer magnetic bead carries out specific bond with vascular endothelial cell adhesion factor, later,
The rhVEGF is added, in 37 DEG C of CO25h is cultivated in incubator, activates vascular endothelial cell adhesion factor, is obtained magnetic anti-
Original, for carrying out specific adhesion with candidate stem cell；
(3) sterile culture base fluid is prepared：By the cell factor composition, multi-vitamins, trace element compound, junket
Propylhomoserin, peptide, 3D Poly L-lactic acid, lecithin, small active peptides, tea polyphenols, human hemoglobin, human placental extract,
Recombination human source vascular endothelial growth factor, superoxide dismutase, polyunsaturated fatty acid, prednisone, progesterone, Trenaxmine with
Aforementioned proportion is added in the DMEM/F12 basal mediums, and is dissolved using the ultra-pure water, and 0.22 μ is finally utilized
M membrane filtration degermings, obtain sterile culture base fluid；The sterile culture base fluid and the magnetic antigen are added to containing again
It states in the culture roller bottle of culture substrate layer to get to culture medium.
Amplification method using the culture medium in vitro culture candidate stem cell includes the following steps：
(1) enrichment of candidate stem cell：Aseptic collection umbilical cord blood 45ml, using Ficoll density-gradient centrifugation methods
It settles, detach mononuclearcell in the Cord blood；
(2) amplification in vitro and harvest of candidate stem cell：By the nucleus with 1 × 1010Cells/L concentration, which is added to, to be contained
Have in the culture roller bottle of the culture medium, is placed in 37 DEG C, 5%CO2Incubator in, culture roller bottle pasted under 10r/h rotating speeds
Wall culture 5 days, changes the liquid once every three days, harvests 4 generation people's umbilical cord candidate stem cells, and it is 1 then to add volume ratio:1
The mixed enzyme solution of 0.25% trypsase and 0.1% clostridiopetidase A digests 45min, and vortex mixer is recycled to carry out concussion 7min,
Stripping is adsorbed on the cell film layer of culture roller bottle wall, obtains band passage candidate stem cell；
(3) Magneto separate of candidate stem cell：By the passage candidate stem cell by vortex magnetic separator screening, discards and do not glue
Attached cell obtains the passage candidate stem cell mutually adhered to magnetic antigen.
Embodiment 3
A kind of cell factor composition cultivates the application in candidate stem cell in vitro, the cell factor composition be by
Main component and auxiliary element are with weight ratio for 20:3 compositions；
The main component includes in percentage by weight：15.2% translation growth factor-β, 22.4% stem cell
The factor, 14.4% interleukin-1 ' beta ', 8.6% platelet derived growth factor, 7.6% differentiation inhibiting factor, surplus are situated between for leucocyte
Element 6；The platelet derived growth factor in the Colony cultivation of candidate stem cell to playing the role of directed differentiation megakaryoblast, in body
The proliferation rate and amplification times of candidate stem cell can be improved in outer amplification cultivation with other combination of cytokines；The stem cell because
Son is the stimulating factor of highly important early stage candidate stem cell；The translation growth factor-β can promote monokaryon macrophage thin
The expression of the scavenger receptor of born of the same parents；The differentiation inhibiting factor can inhibit candidate stem cell further to break up.
The auxiliary element includes in percentage by weight：14.1% insulin-like growth factor 1,12.7% inflammation are thin
Intracellular cytokine, 10.1% hypoxia inducible factor, the 1.3% prostaglandin mediated some growth factor, 9.1% eosinophil chemotactic
The factor, 20.2% cell activating agent, 8.4% micromolecular inhibitor, surplus behaviour galactose agglutinin 1；The para-insulin
Growth factor 1 can promote the proliferation rate of candidate stem cell；The eosinophil chemokine and inflammatory cytokine can
Microcosmic regulation and control are carried out to proliferation of the candidate stem cell in amplification procedure and differentiation；The prostaglandin mediated some growth factor
It is divided with promotion candidate stem cell and may also function as certain cytoprotection；The hypoxia inducible factor can ensure point
It splits the candidate stem cell after amplification and can adapt to low-oxygen environment being transplanted in human body；The cell activating agent can be activated and be made
Hemocytoblast promotes its division；The micromolecular inhibitor can inhibit candidate stem cell to be broken up；The human galactose is solidifying
Differentiation direction of the plain 1 controllable candidate stem cell of collection after being transplanted to human body.Wherein, the micromolecular inhibitor is to be based on SPOP
The micromolecular inhibitor of protein structure, in the amino acid sequence such as sequence table of the SPOP albumen shown in SEQ ID NO.1.
The cell factor composition is used to manufacture the culture medium of in vitro culture candidate stem cell.Wherein, the culture medium
Include in percentage by weight：12.3%DMEM/F12 basal mediums, 7.9% cell factor composition, 6.7% nano magnetic
Pearl, 6.4% vascular endothelial cell adhesion factor, 5.7%PBS buffer solutions, 0.8%rhVEGF, 1.0% multi-vitamins,
0.56% trace element compound, 1.2% tyrosine, 1.3% peptide, 1.7%3D Poly L-lactic acid, 0.6% lecithin,
0.3% small active peptides, 0.10% tea polyphenols, 2.0% human hemoglobin, 4.1% water-setting collagen protein adhesive, 4.1% ice vinegar
Acid, 4.5% human placental extract, 0.2% superoxide dismutase, 0.3% polyunsaturated fatty acid, 0.03% prednisone,
0.03% progesterone, 0.3% Trenaxmine, surplus are ultra-pure water；The polyunsaturated fatty acid is used to safeguard the structure of cell membrane
And function；The prednisone has anti-inflammatory and anti-allergic effects；Contain the natural of Cord blood development in the human placental extract
Active constituent simulates the internal development microenvironment of Cord blood, is greatly improved the amplification rate of candidate stem cell；The small molecule
Active peptide can be directly entered into the cell with original shape, and abundant nutriment is provided for candidate stem cell；The superoxide dismutase
Enzyme can eliminate the candidate stem cell free radical that itself is generated in fission process；The Trenaxmine can be to inhibiting Hematopoietic Stem thin
Certain booster action is played in the differentiation of born of the same parents.Wherein, the multi-vitamins be by vitamin A, vitamin B6, adenine phosphate,
Vitamin E is with mass ratio for 1:1:2:2 compositions；The multi-vitamins can provide nutrition for candidate stem cell, and raising is made
The activity of hemocytoblast.Wherein, the multi-vitamins are by vitamin A, vitamin B6, adenine phosphate, vitamin E with quality
Than being 1:1:2:2 compositions；The multi-vitamins can provide nutrition for candidate stem cell, improve the work of candidate stem cell
Property.Wherein, the trace element compound is two carboxyethyl germanium sesquioxide germanium.
The preparation method of the culture medium includes the following steps：
(1) culture substrate layer is prepared：The water-setting collagen protein adhesive is dissolved in the glacial acetic acid, and molten by what is obtained
It solves liquid and utilizes 0.22 μm of membrane filtration, obtain water-setting collagen protein adhesive lysate；Then again that the water-setting collagen protein adhesive is molten
Liquid is solved with 100l/ μ cm2Dosage be fitted into culture roller bottle in, then will the culture roller bottle sealing after be put into constant temperature incubator,
Temperature be 45 DEG C and rotating speed be 12r/h under the conditions of adherent incubation 2h, obtain culture substrate layer, for use；Water-setting collagen protein adhesive phase
Compared with collagen as culture substrate layer, have at fast compared with rate, mechanical strength is good, degradation time is longer, immune rejection is anti-
Should be weaker the advantages that.
(2) magnetic antigen is prepared：The nanometer magnetic bead activates 60min by EDC/NHSS modes, obtains activation nano magnetic
Pearl, then the activation nanometer magnetic bead, vascular endothelial cell adhesion factor, PBS buffer solution are mixed according to aforementioned proportion, with
60r/min mechanical agitations 40min so that activation nanometer magnetic bead carries out specific bond with vascular endothelial cell adhesion factor, later,
The rhVEGF is added, in 37 DEG C of CO26h is cultivated in incubator, activates vascular endothelial cell adhesion factor, is obtained magnetic anti-
Original, for carrying out specific adhesion with candidate stem cell；
(3) sterile culture base fluid is prepared：By the cell factor composition, multi-vitamins, trace element compound, junket
Propylhomoserin, peptide, 3D Poly L-lactic acid, lecithin, small active peptides, tea polyphenols, human hemoglobin, human placental extract,
Recombination human source vascular endothelial growth factor, superoxide dismutase, polyunsaturated fatty acid, prednisone, progesterone, Trenaxmine with
Aforementioned proportion is added in the DMEM/F12 basal mediums, and is dissolved using the ultra-pure water, and 0.22 μ is finally utilized
M membrane filtration degermings, obtain sterile culture base fluid；The sterile culture base fluid and the magnetic antigen are added to containing again
It states in the culture roller bottle of culture substrate layer to get to culture medium.
Amplification method using the culture medium in vitro culture candidate stem cell includes the following steps：
(1) enrichment of candidate stem cell：Aseptic collection umbilical cord blood 50ml, using Ficoll density-gradient centrifugation methods
It settles, detach mononuclearcell in the Cord blood；
(2) amplification in vitro and harvest of candidate stem cell：By the nucleus with 1 × 1010Cells/L concentration, which is added to, to be contained
Have in the culture roller bottle of the culture medium, is placed in 37 DEG C, 5%CO2Incubator in, culture roller bottle pasted under 12r/h rotating speeds
Wall culture 6 days, changes the liquid once every three days, harvests 5 generation people's umbilical cord candidate stem cells, and it is 1 then to add volume ratio:1
The mixed enzyme solution of 0.25% trypsase and 0.1% clostridiopetidase A digests 60min, and vortex mixer is recycled to be shaken
10min, stripping are adsorbed on the cell film layer of culture roller bottle wall, obtain band passage candidate stem cell；
(3) Magneto separate of candidate stem cell：By the passage candidate stem cell by vortex magnetic separator screening, discards and do not glue
Attached cell obtains the passage candidate stem cell mutually adhered to magnetic antigen.
Interpretation of result
Using the embodiment of the present invention 1-3 as experimental group 1-3, routine culture group is contrast groups, utilizes cell colony technical bid
Accurate and correction of typist's errors formula counts Cord blood amplifying candidate stem cell in vitro, and is carried out using SPSS10.0 statistical softwares
Statistical disposition, measurement data withIt indicates, comparison among groups variance uses paired t-test, is difference with P ＜ 0.05, when statistics
Between be 4 weeks, statistical result is shown in Table 1.
1 umbilical cord blood hematopoietic stem cell amplification in vitro multiple of table (N=5)
As can be seen from the above table, experimental group 1-3 is all remarkably higher than control group in Each point in time, when by the 4th week, Cord blood
Hematopoietic stem cell expansion is up to 84.65-87.86 times, and control group merely adds 8.56 times.
Although the present invention is described and illustrated with reference to its specific embodiment, it will be appreciated by those skilled in the art that
It can be variously modified without departing from the spirit and scope of the present invention, changed and replaced.Therefore, of the invention
It is intended to the scope limitation only by following claims and these claims should be as broadly as possible explained in rational degree.
Sequence table
<110>Dalian Jin Ma health industry Development Co., Ltd
<120>A kind of cell factor composition cultivates the application in candidate stem cell in vitro
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 374
<212> PRT
<213> A
<220>
<221> LIPID
<222> (1)..(374)
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Met Ser Arg Val Pro Ser Pro Pro Pro Pro Ala Glu Met Ser Ser Gly
1 5 10 15
Pro Val Ala Glu Ser Trp Cys Tyr Thr Gln Ile Lys Val Val Lys Phe
20 25 30
Ser Tyr Met Trp Thr Ile Asn Asn Phe Ser Phe Cys Arg Glu Glu Met
35 40 45
Gly Glu Val Ile Lys Ser Ser Thr Phe Ser Ser Gly Ala Asn Asp Lys
50 55 60
Leu Lys Trp Cys Leu Arg Val Asn Pro Lys Gly Leu Asp Glu Glu Ser
65 70 75 80
Lys Asp Tyr Leu Ser Leu Tyr Leu Leu Leu Val Ser Cys Pro Lys Ser
85 90 95
Glu Val Arg Ala Lys Phe Lys Phe Ser Ile Leu Asn Ala Lys Gly Glu
100 105 110
Glu Thr Lys Ala Met Glu Ser Gln Arg Ala Tyr Arg Phe Val Gln Gly
115 120 125
Lys Asp Trp Gly Phe Lys Lys Phe Ile Arg Arg Asp Phe Leu Leu Asp
130 135 140
Glu Ala Asn Gly Leu Leu Pro Asp Asp Lys Leu Thr Leu Phe Cys Glu
145 150 155 160
Val Ser Val Val Gln Asp Ser Val Asn Ile Ser Gly Gln Asn Thr Met
165 170 175
Asn Met Val Lys Val Pro Glu Cys Arg Leu Ala Asp Glu Leu Gly Gly
180 185 190
Leu Trp Glu Asn Ser Arg Phe Thr Asp Cys Cys Leu Cys Val Ala Gly
195 200 205
Gln Glu Phe Gln Ala His Lys Ala Ile Leu Ala Ala Arg Ser Pro Val
210 215 220
Phe Ser Ala Met Phe Glu His Glu Met Glu Glu Ser Lys Lys Asn Arg
225 230 235 240
Val Glu Ile Asn Asp Val Glu Pro Glu Val Phe Lys Glu Met Met Cys
245 250 255
Phe Ile Tyr Thr Gly Lys Ala Pro Asn Leu Asp Lys Met Ala Asp Asp
260 265 270
Leu Leu Ala Ala Ala Asp Lys Tyr Ala Leu Glu Arg Leu Lys Val Met
275 280 285
Cys Glu Asp Ala Leu Cys Ser Asn Leu Ser Val Glu Asn Ala Ala Glu
290 295 300
Ile Leu Ile Leu Ala Asp Leu His Ser Ala Asp Gln Leu Lys Thr Gln
305 310 315 320
Ala Val Asp Phe Ile Asn Tyr His Ala Ser Asp Val Leu Glu Thr Ser
325 330 335
Gly Trp Lys Ser Met Val Val Ser His Pro His Leu Val Ala Glu Ala
340 345 350
Tyr Arg Ser Leu Ala Ser Ala Gln Cys Pro Phe Leu Gly Pro Pro Arg
355 360 365
Lys Arg Leu Lys Gln Ser
370
Claims (8)
1. a kind of cell factor composition cultivates the application in candidate stem cell in vitro, which is characterized in that the cell factor
Composition is with weight ratio by main component and auxiliary element for 10-20:1-3 is formed；
The main component includes in percentage by weight：11.3-15.2% translation growth factor-βs, 17.6-22.4%
Stem cell factor, 9.1-14.4% interleukin-1 ' beta 's, 5.8-8.6% platelet derived growth factors, 4.7-7.6% differentiation inhibit
The factor, surplus are interleukin-6；
The auxiliary element includes in percentage by weight：9.8-14.1% insulin-like growth factors 1,11.5-12.7%
The prostaglandin mediated some growth factor of inflammatory cytokine, 7.2-10.1% hypoxia inducible factors, 0.8-1.3%, 7.8-
9.1% eosinophil chemokine, 14.8-20.2% cell activating agents, 6.7-8.4% micromolecular inhibitors, surplus
For people's galactose agglutinin 1.
2. a kind of cell factor composition as described in claim 1 cultivates the application in candidate stem cell, feature in vitro
It is, the cell factor composition is used to manufacture the culture medium of in vitro culture candidate stem cell.
3. a kind of cell factor composition as claimed in claim 2 cultivates the application in candidate stem cell, feature in vitro
It is, the culture medium includes in percentage by weight：10.2-12.3%DMEM/F12 basal mediums, 6.8-7.9% are thin
Intracellular cytokine composition, 5.6-6.7% nanometer magnetic beads, 5.1-6.4% vascular endothelial cells adhesion factor, 4.6-5.7%PBS bufferings
Liquid, 0.3-0.8%rhVEGF, 0.5-1.0% multi-vitamins, 0.08-0.56% trace element compounds, 0.7-1.2% junket
Propylhomoserin, 0.8-1.3% peptides, 1.1-1.7%3D Poly L-lactic acid, 0.5-0.6% lecithin, 0.1-0.3% small molecules are lived
Property peptide, 0.05-0.10% tea polyphenols, 1.0-2.0% human hemoglobins, 3.3-4.1% water-setting collagens protein adhesive, 3.3-4.1%
Glacial acetic acid, 3.4-4.5% human placental extracts, 0.1-0.2% superoxide dismutases, 0.1-0.3% polyunsaturated fatty acids,
0.01-0.03% prednisones, 0.01-0.03% progesterone, 0.2-0.3% Trenaxmines, surplus are ultra-pure water.
4. a kind of cell factor composition as claimed in claim 3 cultivates the application in candidate stem cell, feature in vitro
It is, the multi-vitamins are with mass ratio by vitamin A, vitamin B6, adenine phosphate, vitamin E for 1:1:2:2 compositions
's.
5. a kind of cell factor composition as claimed in claim 3 cultivates the application in candidate stem cell, feature in vitro
It is, the multi-vitamins are with mass ratio by vitamin B6, adenine phosphate, VitAVitE for 1:2:1:2 compositions
's.
6. a kind of cell factor composition as claimed in claim 3 cultivates the application in candidate stem cell, feature in vitro
It is, the trace element compound is arbitrary in selenium yeast, zinc sulfate, two carboxyethyl germanium sesquioxide germanium, calcium chloride, iron chloride
It is one or more of.
7. a kind of cell factor composition as claimed in claim 3 cultivates the application in candidate stem cell, feature in vitro
It is, the preparation method of the culture medium includes the following steps：
(1) culture substrate layer is prepared：The lysate that the water-setting collagen protein adhesive is dissolved in the glacial acetic acid, and will obtained
Using 0.22 μm of membrane filtration, water-setting collagen protein adhesive lysate is obtained；Then again by the water-setting collagen protein adhesive lysate
With 100l/ μ cm2Dosage be fitted into culture roller bottle in, then will the culture roller bottle sealing after be put into constant temperature incubator, in temperature
Adherent incubation 2h under the conditions of being 6-12r/h for 40-45 DEG C and rotating speed, obtains culture substrate layer, for use；
(2) magnetic antigen is prepared：The nanometer magnetic bead activates 60min by EDC/NHSS modes, obtains activation nanometer magnetic bead, then
The activation nanometer magnetic bead, vascular endothelial cell adhesion factor, PBS buffer solution are mixed according to aforementioned proportion, with 30-
60r/min mechanical agitations 30-40min so that activation nanometer magnetic bead carries out specific bond with vascular endothelial cell adhesion factor, it
Afterwards, the rhVEGF is added, in 37 DEG C of CO24-6h is cultivated in incubator, is activated vascular endothelial cell adhesion factor, is obtained magnetic
Property antigen, be used for and candidate stem cell carry out specific adhesion；
(3) sterile culture base fluid is prepared：By the cell factor composition, multi-vitamins, trace element compound, junket ammonia
Acid, peptide, 3D Poly L-lactic acid, lecithin, small active peptides, tea polyphenols, human hemoglobin, human placental extract, again
Group people source vascular endothelial growth factor, superoxide dismutase, polyunsaturated fatty acid, prednisone, progesterone, it is more than Trenaxmine
The ratio of stating is added in the DMEM/F12 basal mediums, and is dissolved using the ultra-pure water, finally utilizes 0.22 μm
Membrane filtration degerming obtains sterile culture base fluid；The sterile culture base fluid and the magnetic antigen are added to containing again
It states in the culture roller bottle of culture substrate layer to get to culture medium.
8. a kind of cell factor composition as claimed in claim 7 cultivates the application in candidate stem cell, feature in vitro
It is, the amplification method using the culture medium in vitro culture candidate stem cell includes the following steps：
(1) enrichment of candidate stem cell：Aseptic collection umbilical cord blood 40-50ml, using Ficoll density-gradient centrifugation methods
It settles, detach mononuclearcell in the Cord blood；
(2) amplification in vitro and harvest of candidate stem cell：By the nucleus with 1 × 1010Cells/L concentration is added to containing
In the culture roller bottle for stating culture medium, it is placed in 37 DEG C, 5%CO2Incubator in, culture roller bottle it is adherent under 6-12r/h rotating speeds
Culture 4-6 days, changes the liquid once every three days, and for harvest 3-5 for people's umbilical cord candidate stem cell, it is 1 then to add volume ratio:1
The mixed enzyme solution of 0.25% trypsase and 0.1% clostridiopetidase A digests 30-60min, and vortex mixer is recycled to carry out concussion 5-
10min, stripping are adsorbed on the cell film layer of culture roller bottle wall, obtain band passage candidate stem cell；
(3) Magneto separate of candidate stem cell：By the passage candidate stem cell by vortex magnetic separator screening, discard nonadherent
Cell obtains the passage candidate stem cell mutually adhered to magnetic antigen.
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| EP4089164A4 (en) * | 2020-01-06 | 2024-03-06 | Scm Lifescience Co Ltd | Composition for reinforcing function of stem cell |
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