CN104877963A - Serum-free human umbilical cord mesenchymal stem cell culture medium and preparation method thereof - Google Patents
Serum-free human umbilical cord mesenchymal stem cell culture medium and preparation method thereof Download PDFInfo
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Abstract
The invention provides a serum-free human umbilical cord mesenchymal stem cell culture medium, which belongs to the technical field of stem cells, and comprises an IMDM (intrinsic mode division multiplexing) basic culture medium and the following components: recombinant human insulin, recombinant transferrin, vitamin C, human serum albumin, fibroblast growth factor, platelet derived growth factor, epidermal growth factor, insulin-like growth factor, transforming growth factor, fibronectin, fetuin and beta-cell element. The serum-free human umbilical cord mesenchymal stem cell culture medium provided by the invention does not need culture bottle coating, and has good cell adherence and morphology and high cell proliferation rate.
Description
Technical field
The invention belongs to stem cells technology field, relate to Human umbilical cord mesenchymal stem cell culture medium of a kind of serum-free and preparation method thereof.
Background technology
Human umbilical cord mesenchymal stem cells is derived from umbilical cord Hua Ershi and leads to glue, it is a kind of adult stem cell with obvious plasticity-and multi-lineage potential, under the adjustment of different somatomedin, the different tissues cell type deriving from 3 germinal layers can be divided into, as adipocyte, scleroblast, hepatic lineage, cardiac-like muscle cell, neurogliocyte, neuronal cell and insulin-like cell etc.Except this, human umbilical cord mesenchymal stem cells does not express ajor histocompatibility II class antigen, trace expression ajor histocompatibility I class antigen, immunogenicity is low, transplant rejection effect is little, and there is immunomodulatory effect, the nutritional factor of the human umbilical cord mesenchymal stem cells secretion after transplanting, has the effects such as Angiogensis, promoting growth of cell, anti-inflammatory and anti-fibrosis.
Traditional stem cell media containing animal serum brings multiple unfavorable factor to the production and scientific research of stem cell.Serum can provide hormone, somatomedin, transfer protein and other nutritive substance needed for growing multiplication for stem cell, but there is shortcomings in it: differences between batches are larger, source is unstable, need to verify work in a large number, expensive, use inconvenience, composition is indefinite, there is cytostatic composition, be unfavorable for the object such as vaccine and monoclonal antibody product separation purifying, easily by virus and mycoplasma infection etc.Serum free medium is on the basis of basic medium, adds the blood serum substituting composition of definite ingredients, can meet the fostering requirement of stem cell, effectively can avoid again the many unfavorable factors because using serum to bring.Therefore, developing serum free medium is the essential condition that stem cell moves towards clinical application.
More existing commercially available mesenchymal stem cell serum-free culture mediums at present, the mesenchymal stem cell serum-free culture medium StemPro SFM of such as Life Technology.But there is the defect such as cell attachment difference, operation inconvenience in existing commercially available serum free medium.The such as application of Gibco serum free medium, Stem CELL serum free medium needs to carry out culturing bottle bag quilt.In addition, the cell proliferation rate of these culture medium culturing is not ideal enough.
Chinese patent CN 102433302 discloses a kind of serum free medium, this substratum substratum based on DMEM/F12, but this invention substratum only has a kind of anchoring factor, and cell attachment performance is not ideal enough.Chinese patent application CN 104164404 discloses a kind of people's umbilical cord mesenchymal stem cells substratum, this substratum substratum based on a-MEM, and the cytokine of interpolation and other components reach 29 kinds, and component is very complicated.
The problems such as therefore, it is poor that prior art exists cell attachment, and component is too complicated, and cell proliferation rate is not ideal enough.
Summary of the invention
Poor for solving the cell attachment existed in prior art, component is too complicated, the problems such as cell proliferation rate is not ideal enough, contriver is screened by lot of experiments, obtain a kind of Human umbilical cord mesenchymal stem cell culture medium of new serum-free, this substratum is without the need to carrying out culturing bottle bag quilt, easy and simple to handle, well, cell proliferation rate is high for cell attachment and form.
Object of the present invention will be further described in detail below reflect and description.
The invention provides a kind of Human umbilical cord mesenchymal stem cell culture medium of serum-free, comprise IMDM basic medium, also comprise following component and concentration thereof: recombinant human insulin 0.5 ~ 5mg/mL, recombinant transferrin 1 ~ 5 μ g/mL, vitamins C 5 ~ 50 μ g/mL, human serum albumin 0.5 ~ 5mg/mL, FGF5 ~ 50ng/mL, Thr6 PDGF BB 5 ~ 50ng/mL, Urogastron 5 ~ 50ng/mL, rhIGF-1 5 ~ 50ng/mL, transforming growth factor 5 ~ 50ng/mL, fibronectin 5 ~ 50ng/mL, Pp63 glycophosphoproteins 0.5 ~ 5mg/mL and β cytokine 5 ~ 50 μ g/mL.
Adopt the Human umbilical cord mesenchymal stem cell culture medium of the serum-free of technique scheme, without the need to adding animal serum, the fast breeding of human umbilical cord mesenchymal stem cells can be realized, maintaining good stem cell puerilism, there is good cell induction differentiation potential.
In the Human umbilical cord mesenchymal stem cell culture medium of serum-free provided by the invention, by the synergy of Fibronectin (fibronectin) and Pp63 glycophosphoproteins (Fetuin), greatly strengthen the adherent performance of human umbilical cord mesenchymal stem cells; β cytokine (betacellulin) contributes to the stem cell properties maintaining human umbilical cord mesenchymal stem cells, and prevents cell senescence.
Preferably, the Human umbilical cord mesenchymal stem cell culture medium of serum-free provided by the invention, comprise IMDM basic medium, also comprise following component and concentration thereof: recombinant human insulin 0.5 ~ 2mg/mL, recombinant transferrin 1 ~ 4 μ g/mL, vitamins C 5 ~ 30 μ g/mL, human serum albumin 0.5 ~ 2mg/mL, FGF5 ~ 20ng/mL, Thr6 PDGF BB 5 ~ 20ng/mL, Urogastron 5 ~ 20ng/mL, rhIGF-1 5 ~ 20ng/mL, transforming growth factor 5 ~ 20ng/mL, fibronectin 5 ~ 20ng/mL, Pp63 glycophosphoproteins 0.5 ~ 3mg/mL and β cytokine 5 ~ 20 μ g/mL.
More preferably, the Human umbilical cord mesenchymal stem cell culture medium of serum-free provided by the invention, comprise IMDM basic medium, also comprise following component and concentration thereof: recombinant human insulin 1mg/mL, recombinant transferrin 2.5 μ g/mL, vitamins C 16.1 μ g/mL, human serum albumin 1mg/mL, Fibroblast growth factor-10 ng/mL, Thr6 PDGF BB 10ng/mL, Urogastron 10ng/mL, type-1 insulin like growth factor 0ng/mL, TGF-1 0ng/mL, fibronectin 1 0ng/mL, Pp63 glycophosphoproteins 1mg/mL and β cytokine 10 μ g/mL.
Preferably, described fibroblast growth factor is Prostatropin, described Thr6 PDGF BB is PDGF-bb, and described rhIGF-1 is insulin-like growth factor-i, and described transforming growth factor is transforming growth factor-beta.
Preferably, the Human umbilical cord mesenchymal stem cell culture medium of serum-free provided by the invention, also comprises the non-essential amino acid of 1% volume percent, such as, purchased from the nonessential amino acid of Sai Ye bio tech ltd, Guangzhou, and article No. NEAA-10201-100.
Correspondingly, the present invention also provides the preparation method of the Human umbilical cord mesenchymal stem cell culture medium of serum-free, comprise the steps: in IMDM basic medium, recombinant human insulin's mother liquor is added by described concentration, recombinant transferrin mother liquor, vitamins C mother liquor, human serum albumin, fibroblast growth factor mother liquor, Thr6 PDGF BB mother liquor, Urogastron mother liquor, rhIGF-1 mother liquor, transforming growth factor mother liquor, fibronectin mother liquor, Pp63 glycophosphoproteins mother liquor and β cytokine mother liquor, stir, cross film degerming and get final product.
Preferably, described fibroblast growth factor is Prostatropin, described Thr6 PDGF BB is PDGF-bb, and described rhIGF-1 is insulin-like growth factor-i, and described transforming growth factor is transforming growth factor-beta.
Correspondingly, the present invention also provides the purposes of the Human umbilical cord mesenchymal stem cell culture medium of serum-free in human umbilical cord mesenchymal stem cells is cultivated.
Compared with prior art, the invention has the beneficial effects as follows: the Human umbilical cord mesenchymal stem cell culture medium that the invention provides a kind of new serum-free, compare with other serum free mediums containing blood serum medium with traditional, the human umbilical cord mesenchymal stem cells of culture medium culturing provided by the invention can keep good stem cell pedomorphism, has better cell proliferation rate, cell dryness and cell induction differentiation potential.Preparation method provided by the invention is simple, and the Human umbilical cord mesenchymal stem cell culture medium of obtained serum-free is used for the cultivation of human umbilical cord mesenchymal stem cells, shows excellent cell performance.
Accompanying drawing explanation
Fig. 1 is containing the impact of the substratum on cell proliferation of different concns β cytokine.
The form that Fig. 2 P5 cultivates in 3 kinds of substratum for human umbilical cord mesenchymal stem cells.
The growth curve of Fig. 3 human umbilical cord mesenchymal stem cells in 3 kinds of substratum.
Fig. 4 P3 is for the cell clone quantity of human umbilical cord mesenchymal stem cells in 3 kinds of substratum.
The P5 that Fig. 5 substratum 1 is cultivated is for the surface antigen expression level of human umbilical cord mesenchymal stem cells.
The P5 that Fig. 6 substratum 2 is cultivated is for the surface antigen expression level of human umbilical cord mesenchymal stem cells.
The P5 that Fig. 7 substratum 3 is cultivated is for the surface antigen expression level of human umbilical cord mesenchymal stem cells.
The P5 of Fig. 83 kinds of culture medium culturing is for the adipogenic induction differential stain result figure of human umbilical cord mesenchymal stem cells.
The P5 of Fig. 93 kinds of culture medium culturing is for the expression of results figure of the adipogenic induction differentiation associated gene FABP4 of human umbilical cord mesenchymal stem cells in inducing culture.
The P5 of Figure 10 3 kinds of culture medium culturing is for the Osteoinductive differentiation coloration result figure of human umbilical cord mesenchymal stem cells.
The P5 of Figure 11 3 kinds of culture medium culturing is for the expression of results figure of the Osteoinductive differentiation genes involved OPN of human umbilical cord mesenchymal stem cells in inducing culture.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further details.
Each component in the present invention is conventional commercial product, and such as β cytokine is purchased from Sigma, article No. B3670.In the present invention, substratum 1: the Human umbilical cord mesenchymal stem cell culture medium of the serum-free that the embodiment of the present invention two provides; Substratum 2:Life Tecnology company StemPro MSC SFM serum free medium, article No. A10332-01; The FBS of substratum 3:IMDM basic medium+10% volume percent.
embodiment one is containing the impact of the substratum on cell proliferation of different concns β cytokine
Contriver carries out the Human umbilical cord mesenchymal stem cell culture medium formula that lot of experiments screening obtains serum-free provided by the invention, and investigates the concentration impact of β cytokine further.By P1 for HUMSC with 5000/cm
2density be inoculated in six orifice plates, use the Human umbilical cord mesenchymal stem cell culture medium of the serum-free containing different concns β cytokine respectively (compared with substratum 1, difference is only that the concentration of β cytokine is different) cultivate, cell was used 0.25% trysinization in every 3 days, collecting cell number of computations, and with 5000/cm
2density inoculation culture again, until P5 generation, according to the cell harvesting quantity of each generation, draw cell proliferation curve, result is as shown in Figure 1.
As can be seen from Figure 1, when the concentration of β cytokine is 10ng/mL, cell proliferation rate is the highest, when concentration is 5ng/mL and 20ng/mL, also there is higher cell proliferation rate, but when concentration reaches 30ng/mL, not only do not improve cell proliferation rate, reduce cell proliferation rate on the contrary.Therefore, the Human umbilical cord mesenchymal stem cell culture medium of concentration on serum-free provided by the invention of β cytokine has vital impact.
the Human umbilical cord mesenchymal stem cell culture medium of embodiment two serum-free
The Human umbilical cord mesenchymal stem cell culture medium of serum-free, comprise IMDM basic medium, also comprise following component and concentration thereof: recombinant human insulin 1mg/mL, recombinant transferrin 2.5 μ g/mL, vitamins C 16.1 μ g/mL, human serum albumin 1mg/mL, bFGF 10ng/mL, PDGF-bb 10ng/mL, EGF 10ng/mL, IGF-1 10ng/mL, TGF-β 10ng/mL, fibronectin 1 0ng/mL, Pp63 glycophosphoproteins 1mg/mL and β cytokine 10 μ g/mL.
Preparation method: in IMDM basic medium, recombinant human insulin's mother liquor, recombinant transferrin mother liquor, vitamins C mother liquor, human serum albumin, bFGF mother liquor, PDGF-bb mother liquor, EGF mother liquor, IGF-1 mother liquor, TGF-β mother liquor, fibronectin mother liquor, Pp63 glycophosphoproteins mother liquor and β cytokine mother liquor is added by described concentration, stir, mistake film is degerming and get final product.
The preparation of recombinant human insulin's mother liquor: get 1g, uses 0.01mol/L dissolving with hydrochloric acid, is made into the mother liquor of 100mg/mL, and-20 degree are preserved.The preparation of recombinant transferrin mother liquor: get 10mg, dissolves with IMDM basic medium, is made into the mother liquor of 2.5mg/mL, and-20 degree are preserved.The preparation of vitamins C mother liquor: get 10g, dissolves with IMDM basic medium, is made into the mother liquor of 16.1mg/mL, and-20 degree are preserved.The preparation of bFGF mother liquor: get 10mg, dissolves with IMDM basic medium, is made into the mother liquor of 1mg/mL, and-20 degree are preserved.The preparation of PDGF-bb mother liquor: get 10mg, dissolves with IMDM basic medium, is made into the mother liquor of 1mg/mL, and-20 degree are preserved.The preparation of EGF mother liquor: get 10mg, dissolves with IMDM basic medium, is made into the mother liquor of 1mg/mL, and-20 degree are preserved.The preparation of IGF-1 mother liquor: get 10mg, dissolves with IMDM basic medium, is made into the mother liquor of 1mg/mL, and-20 degree are preserved.The preparation of TGF-β mother liquor: get 10mg, dissolves with IMDM basic medium, is made into the mother liquor of 1mg/mL, and-20 degree are preserved.The preparation of fibronectin mother liquor: get 10mg, dissolves with IMDM basic medium, is made into the mother liquor of 1mg/mL, and-20 degree are preserved.The preparation of Fetuin mother liquor: get 1g, dissolves with IMDM basic medium, is made into the mother liquor of 100mg/mL, and-20 degree are preserved.The preparation of β cytokine mother liquor: get 100 μ g, dissolves with IMDM basic medium, is made into the mother liquor of 10mg/mL, and-20 degree are preserved.
the Human umbilical cord mesenchymal stem cell culture medium of embodiment three serum-free
The Human umbilical cord mesenchymal stem cell culture medium of serum-free, comprise IMDM basic medium, also comprise following component and concentration thereof: recombinant human insulin 1mg/mL, recombinant transferrin 2 μ g/mL, vitamins C 15 μ g/mL, human serum albumin 1.5mg/mL, bFGF 15ng/mL, PDGF-bb 10ng/mL, EGF 10ng/mL, IGF-1 15ng/mL, TGF-β 10ng/mL, fibronectin 1 5ng/mL, Pp63 glycophosphoproteins 3mg/mL and β cytokine 15ng/mL.
Preparation method: similar with the preparation method in embodiment two.
the Human umbilical cord mesenchymal stem cell culture medium of embodiment four serum-free
The Human umbilical cord mesenchymal stem cell culture medium of serum-free, comprise IMDM basic medium, also comprise following component and concentration thereof: recombinant human insulin 2mg/mL, recombinant transferrin 1 μ g/mL, vitamins C 10 μ g/mL, human serum albumin 2mg/mL, bFGF 15ng/mL, PDGF-bb 15ng/mL, EGF 15ng/mL, IGF-1 10ng/mL, TGF-β 10ng/mL, fibronectin 1 0ng/mL, Pp63 glycophosphoproteins 2mg/mL and β cytokine 5ng/mL.
Preparation method: similar with the preparation method in embodiment two.
the form that embodiment five human umbilical cord mesenchymal stem cells is cultivated in the medium
By P1 for HUMSC with 5000/cm
2density be inoculated in culturing bottle, cultivate with substratum 1, substratum 2 and substratum 3 respectively, when cell confluency degree reaches 85%, carry out Secondary Culture, until P5 generation, basis of microscopic observation cellular form, result is as shown in Figure 2.As can be seen from Figure 2, substratum institute provided by the invention cultured cells form is more homogeneous, in fusiformis, maintain good stem cell puerilism, and the cellular form of other culture medium culturing differs, and presents ageing state.
the propagation that embodiment six human umbilical cord mesenchymal stem cells is cultivated in the medium
By P1 for HUMSC with 5000/cm
2density be inoculated in six orifice plates, cultivate with substratum 1, substratum 2 and substratum 3 respectively, cell 0.25% trysinization got off in every 3 days, collecting cell number of computations, and with 5000/cm
2density inoculation culture again, until P5 generation, according to the cell harvesting quantity of each generation, draw cell proliferation curve, result is as shown in Figure 3.As can be seen from Figure 3, substratum provided by the invention 1 cultured cells multiplication rate is higher.
the clonal expansion that embodiment seven human umbilical cord mesenchymal stem cells is cultivated in the medium
Getting P3 is inoculated in 10cm culture dish for HUMSC100, add 10mL substratum 1, substratum 2 and substratum 3 respectively to cultivate, within every 3 days, half amount changes liquid once, when contained by maximum cell clone, cell number is more than 200, dye by Viola crystallina, naked eyes calculate the cell clone quantity in culture dish, and result as shown in Figure 4.As can be seen from Figure 4, substratum provided by the invention 1 cultured cells clone is more.
the surface antigen that embodiment eight human umbilical cord mesenchymal stem cells is cultivated in the medium is expressed
Use the P5 of substratum 1, substratum 2 and substratum 3 cultivation for cell in Example five respectively, the expression level of application flow cytometry cell-surface antigens CD59, CD45, CD90, HLA-DR, result as shown in Figures 5 to 7.From Fig. 5 to Fig. 7, substratum 1, substratum 2 and substratum 3 cultured cells surface antigen all meet the surface antigen requirement of human umbilical cord mesenchymal stem cells.
the adipogenic induction differentiation potential that embodiment nine human umbilical cord mesenchymal stem cells is cultivated in the medium
Use the P5 of substratum 1, substratum 2 and substratum 3 cultivation for cell in Example five respectively, with 5000/cm
2density be inoculated in six orifice plates, and add corresponding substratum 1, substratum 2 and substratum 3 and cultivate, when cell confluency degree reaches 80%, discard substratum, and add adipogenic induction division culture medium (purchased from Life Tecnology company, article No. A10070-01), within every 3 days, change liquid once, after cultivating 3 weeks, carry out into fat qualification with oil red O, result as shown in Figure 8.Meanwhile, identify the expression level of stearoblast genes involved FABP4 with qPCR, result as shown in Figure 9.From Fig. 8 and Fig. 9, substratum provided by the invention 1 cultured cells adipogenic induction differentiation potential is better.
the Osteoinductive differentiation potential that embodiment ten human umbilical cord mesenchymal stem cells is cultivated in the medium
Use the P5 of substratum 1, substratum 2 and substratum 3 cultivation for cell in Example five respectively, with 5000/cm
2density be inoculated in six orifice plates, and add corresponding substratum 1, substratum 2 and substratum 3 and cultivate, when cell confluency degree reaches 80%, discard substratum, and add Osteoblast Differentiation substratum (purchased from Life Tecnology company, article No. A10072-01), within every 3 days, change liquid once, after cultivating 4 weeks, carry out skeletonization qualification by sodium alizarinsulfonate, result as shown in Figure 10.Meanwhile, identify the expression level of scleroblast genes involved OPN with qPCR, result as shown in figure 11.From Figure 10 and Figure 11, substratum provided by the invention 1 cultured cells Osteoinductive differentiation potential is better.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.
Claims (8)
1. the Human umbilical cord mesenchymal stem cell culture medium of a serum-free, it is characterized in that: comprise IMDM basic medium, also comprise following component and concentration thereof: recombinant human insulin 0.5 ~ 5mg/mL, recombinant transferrin 1 ~ 5 μ g/mL, vitamins C 5 ~ 50 μ g/mL, human serum albumin 0.5 ~ 5mg/mL, FGF5 ~ 50ng/mL, Thr6 PDGF BB 5 ~ 50ng/mL, Urogastron 5 ~ 50ng/mL, rhIGF-1 5 ~ 50ng/mL, transforming growth factor 5 ~ 50ng/mL, fibronectin 5 ~ 50ng/mL, Pp63 glycophosphoproteins 0.5 ~ 5mg/mL and β cytokine 5 ~ 50 μ g/mL.
2. the Human umbilical cord mesenchymal stem cell culture medium of serum-free according to claim 1, it is characterized in that: comprise IMDM basic medium, also comprise following component and concentration thereof: recombinant human insulin 0.5 ~ 2mg/mL, recombinant transferrin 1 ~ 4 μ g/mL, vitamins C 5 ~ 30 μ g/mL, human serum albumin 0.5 ~ 2mg/mL, FGF5 ~ 20ng/mL, Thr6 PDGF BB 5 ~ 20ng/mL, Urogastron 5 ~ 20ng/mL, rhIGF-1 5 ~ 20ng/mL, transforming growth factor 5 ~ 20ng/mL, fibronectin 5 ~ 20ng/mL, Pp63 glycophosphoproteins 0.5 ~ 3mg/mL and β cytokine 5 ~ 20 μ g/mL.
3. the Human umbilical cord mesenchymal stem cell culture medium of serum-free according to claim 2, it is characterized in that: comprise IMDM basic medium, also comprise following component and concentration thereof: recombinant human insulin 1mg/mL, recombinant transferrin 2.5 μ g/mL, vitamins C 16.1 μ g/mL, human serum albumin 1mg/mL, Fibroblast growth factor-10 ng/mL, Thr6 PDGF BB 10ng/mL, Urogastron 10ng/mL, type-1 insulin like growth factor 0ng/mL, TGF-1 0ng/mL, fibronectin 1 0ng/mL, Pp63 glycophosphoproteins 1mg/mL and β cytokine 10 μ g/mL.
4. the Human umbilical cord mesenchymal stem cell culture medium of serum-free according to any one of claim 1 to 3, it is characterized in that: described fibroblast growth factor is Prostatropin, described Thr6 PDGF BB is PDGF-bb, described rhIGF-1 is insulin-like growth factor-i, and described transforming growth factor is transforming growth factor-beta.
5. the Human umbilical cord mesenchymal stem cell culture medium of serum-free according to any one of claim 1 to 3, is characterized in that: the non-essential amino acid also comprising 1% volume percent.
6. the preparation method of the Human umbilical cord mesenchymal stem cell culture medium of serum-free according to claim 1, it is characterized in that: comprise the steps: in IMDM basic medium, recombinant human insulin's mother liquor is added by described concentration, recombinant transferrin mother liquor, vitamins C mother liquor, human serum albumin, fibroblast growth factor mother liquor, Thr6 PDGF BB mother liquor, Urogastron mother liquor, rhIGF-1 mother liquor, transforming growth factor mother liquor, fibronectin mother liquor, Pp63 glycophosphoproteins mother liquor and β cytokine mother liquor, stir, cross film degerming and get final product.
7. the preparation method of the Human umbilical cord mesenchymal stem cell culture medium of serum-free according to claim 6, it is characterized in that: described fibroblast growth factor is Prostatropin, described Thr6 PDGF BB is PDGF-bb, described rhIGF-1 is insulin-like growth factor-i, and described transforming growth factor is transforming growth factor-beta.
8. the purposes of Human umbilical cord mesenchymal stem cell culture medium in human umbilical cord mesenchymal stem cells is cultivated of serum-free according to any one of claim 1 to 5.
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