CN104877962A - Serum-free adipose-derived stem cell culture medium and preparation method thereof - Google Patents
Serum-free adipose-derived stem cell culture medium and preparation method thereof Download PDFInfo
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Abstract
The invention provides a serum-free adipose-derived stem cell culture medium, which belongs to the technical field of stem cells, and comprises an IMDM (intrinsic mode differentiation) basal 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, tea polyphenol and stem cell growth factor. The serum-free adipose-derived stem cell culture medium provided by the invention does not need to be coated by a culture bottle, and has good cell adherence and morphology and high cell proliferation rate.
Description
Technical field
The invention belongs to stem cells technology field, fat stem cell substratum relating to a kind of serum-free and preparation method thereof.
Background technology
Fat stem cell (adipose-derived stem cells, ADSCs) is from fatty tissue, be separated a kind of stem cell with multi-lineage potential obtained.Research finds that ADSCs cell can stablize propagation in vitro and decline rate is low, has wide material sources simultaneously, and cylinder storage amount is large, draws materials easily, and a small amount of tissue can obtain a large amount of stem cell, and suitable large scale culturing, to advantages such as body injury are little.
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 stem cell serum-free culture mediums at present, the mesenchymal stem cell serum-free culture medium StemPro MSC SFM serum free medium of such as Life Technology company.But existing commercially available serum free medium exists cell attachment difference, the defects such as operation inconvenience, the such as application of Gibco serum free medium needs to carry out culturing bottle bag quilt.In addition, also there is the defects such as cell proliferation is not ideal enough in existing commercially available serum free medium.Chinese patent CN103255103 discloses a kind of fat stem cell substratum of serum-free, this substratum substratum based on DMEM-LG, although with the addition of the components such as heparin, glutamine and leukaemia inhibitory factor, but do not add the nutritive ingredient such as Regular Insulin, albumin, the growing multiplication effect of fat stem cell is not ideal enough.Chinese patent CN102732477 discloses a kind of fat stem cell substratum of serum-free; substratum based on high glycoform DMEM; the concentration of glucose is up to 4500mg/L; add the components such as taurine, reduced glutathion and copper-protein; but do not promote adherent cytokine, exist cell attachment poor, breed defect slowly.
The problems such as therefore, it is poor that prior art exists cell attachment, and cell proliferation rate is not ideal enough.
Summary of the invention
Poor for solving the cell attachment existed in prior art, the dissatisfactory problem of cell proliferation rate, contriver is screened by lot of experiments, obtain a kind of new fats stem cell serum-free culture medium, 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 fat stem cell substratum 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, tea-polyphenol 5 ~ 50 μ g/mL and stem cell factor 5 ~ 30ng/mL.
Adopt the fat stem cell substratum of the serum-free of technique scheme, without the need to adding animal serum, the fast breeding of fat stem cell can be realized, maintaining good stem cell puerilism, there is good cell induction differentiation potential.
In the fat stem cell substratum of serum-free provided by the invention, by the synergy of Fibronectin (fibronectin) and Pp63 glycophosphoproteins (Fetuin), greatly strengthen the adherent performance of fat stem cell; Tea-polyphenol contributes to the puerilism and the cell dryness that maintain fat stem cell, prevents the aging of fat stem cell; Stem cell factor (SCF) then can promote the growing multiplication of fat stem cell.
Preferably, the fat stem cell substratum 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, tea-polyphenol 5 ~ 20 μ g/mL and stem cell factor 5 ~ 15ng/mL.
More preferably, the fat stem cell substratum 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, tea-polyphenol 10 μ g/mL and stem cell factor 10ng/mL.
Preferably, described fibroblast growth factor is Prostatropin (bFGF), described Thr6 PDGF BB is PDGF-bb, described rhIGF-1 is insulin-like growth factor-i (IGF-1), and described transforming growth factor is transforming growth factor-beta (TGF-β).
Preferably, the fat stem cell substratum 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.Those skilled in the art also on basis of the present invention, can add the composition that other can not cause counter productive according to actual needs.
Correspondingly, the present invention also provides the preparation method of the fat stem cell substratum 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, tea polyphenol mother solution and stem cell factor mother liquor, stir, cross film degerming and get final product.
Preferably, described fibroblast growth factor is Prostatropin (bFGF), described Thr6 PDGF BB is PDGF-bb, described rhIGF-1 is insulin-like growth factor-i (IGF-1), and described transforming growth factor is transforming growth factor-beta (TGF-β).
Correspondingly, the present invention also provides the purposes of the fat stem cell substratum of serum-free in fat stem cell is cultivated.
Compared with prior art, the invention has the beneficial effects as follows: the fat stem cell substratum that the invention provides a kind of new serum-free, compare with other serum free mediums containing blood serum medium with traditional, the fat stem cell 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 fat stem cell substratum of obtained serum-free is used for the cultivation of fat stem cell, shows excellent cell performance.
Accompanying drawing explanation
Fig. 1 is containing the impact of the substratum on cell proliferation of different concns stem cell factor.
The form that Fig. 2 P5 fat subsitutes stem cell is cultivated in 3 kinds of substratum.
The growth curve of Fig. 3 fat stem cell in 3 kinds of substratum.
The cell clone quantity of Fig. 4 P3 fat subsitutes stem cell in 3 kinds of substratum.
The surface antigen expression level of the P5 fat subsitutes stem cell that Fig. 5 substratum 1 is cultivated.
The surface antigen expression level of the P5 fat subsitutes stem cell that Fig. 6 substratum 2 is cultivated.
The surface antigen expression level of the P5 fat subsitutes stem cell that Fig. 7 substratum 3 is cultivated.
The adipogenic induction differential stain result figure of the P5 fat subsitutes stem cell of Fig. 83 kinds of culture medium culturing.
The expression of results figure of the adipogenic induction differentiation associated gene FABP4 of P5 fat subsitutes stem cell in inducing culture of Fig. 93 kinds of culture medium culturing.
The Osteoinductive differentiation coloration result figure of the P5 fat subsitutes stem cell of Figure 10 3 kinds of culture medium culturing.
The expression of results figure of the Osteoinductive differentiation genes involved OPN of P5 fat subsitutes stem cell in inducing culture of Figure 11 3 kinds of culture medium culturing.
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 stem cell factor is purchased from Sigma, article No. S7901.In the present invention, substratum 1: the fat stem cell substratum 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 stem cell factor
Contriver carries out the fat stem cell culture medium prescription that lot of experiments screening obtains serum-free provided by the invention, and investigates the concentration impact of stem cell factor further.By P1 for ADSCs with 5000/cm
2density be inoculated in six orifice plates, use the fat stem cell substratum of the serum-free containing different concns stem cell factor respectively (compared with substratum 1, difference is only that the concentration of stem cell factor 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 stem cell factor 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 fat stem cell substratum of concentration on serum-free provided by the invention of stem cell factor has vital impact.
the fat stem cell substratum of embodiment two serum-free
The fat stem cell substratum 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, tea-polyphenol 10 μ g/mL and stem cell factor 10ng/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, tea polyphenol mother solution and stem cell factor 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 tea polyphenol mother solution: get 100mg, dissolves with IMDM basic medium, is made into the mother liquor of 10mg/mL, and-20 degree are preserved.The preparation of stem cell factor 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 fat stem cell substratum of embodiment three serum-free
The fat stem cell substratum of serum-free, comprise IMDM basic medium, also comprise following component and concentration thereof: recombinant human insulin 2mg/mL, recombinant transferrin 2 μ g/mL, vitamins C 20 μ g/mL, human serum albumin 1mg/mL, bFGF 15ng/mL, PDGF-bb 10ng/mL, EGF 15ng/mL, IGF-1 10ng/mL, TGF-β 10ng/mL, fibronectin 1 5ng/mL, Pp63 glycophosphoproteins 2mg/mL, tea-polyphenol 15 μ g/mL and stem cell factor 15ng/mL.
Preparation method: similar with the preparation method in embodiment two.
the fat stem cell substratum of embodiment four serum-free
The fat stem cell substratum of serum-free, comprise IMDM basic medium, also comprise following component and concentration thereof: recombinant human insulin 1mg/mL, recombinant transferrin 1 μ g/mL, vitamins C 10 μ g/mL, human serum albumin 1.5mg/mL, bFGF 20ng/mL, PDGF-bb 15ng/mL, EGF 10ng/mL, IGF-1 15ng/mL, TGF-β 10ng/mL, fibronectin 1 0ng/mL, Pp63 glycophosphoproteins 1mg/mL, tea-polyphenol 15 μ g/mL and stem cell factor 5ng/mL.
Preparation method: similar with the preparation method in embodiment two.
the form that embodiment five fat stem cell is cultivated in the medium
By P1 for ADSCs 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 provided by the invention 1 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 fat stem cell is cultivated in the medium
By P1 for ADSCs 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 fat stem cell is cultivated in the medium
Getting P3 is inoculated in 10cm culture dish for ADSCs 100, 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 fat stem cell 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 fat stem cell.
the adipogenic induction differentiation potential that embodiment nine fat stem cell 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 fat stem cell 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 fat stem cell substratum 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, tea-polyphenol 5 ~ 50 μ g/mL and stem cell factor 5 ~ 30ng/mL.
2. the fat stem cell substratum 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, tea-polyphenol 5 ~ 20 μ g/mL and stem cell factor 5 ~ 15ng/mL.
3. the fat stem cell substratum 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, tea-polyphenol 10 μ g/mL and stem cell factor 10ng/mL.
4. the fat stem cell substratum 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 fat stem cell substratum 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 fat stem cell substratum 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, tea polyphenol mother solution and stem cell factor mother liquor, stir, cross film degerming and get final product.
7. the preparation method of the fat stem cell substratum 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 fat stem cell substratum in fat stem cell is cultivated of serum-free according to any one of claim 1 to 5.
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