CN107245474A - A kind of mankind's inductive pluripotent stem cells isolated culture method without feeder layer serum-free - Google Patents
A kind of mankind's inductive pluripotent stem cells isolated culture method without feeder layer serum-free Download PDFInfo
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- 230000001939 inductive effect Effects 0.000 title claims abstract description 19
- 210000001778 pluripotent stem cell Anatomy 0.000 title claims abstract description 18
- 238000012136 culture method Methods 0.000 title claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 73
- 238000000034 method Methods 0.000 claims abstract description 18
- 230000004069 differentiation Effects 0.000 claims description 16
- 238000001514 detection method Methods 0.000 claims description 14
- 230000008672 reprogramming Effects 0.000 claims description 14
- 238000002474 experimental method Methods 0.000 claims description 11
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 210000002242 embryoid body Anatomy 0.000 claims description 7
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 6
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- 206010043276 Teratoma Diseases 0.000 claims description 5
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- 238000000338 in vitro Methods 0.000 claims description 4
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- 108091023040 Transcription factor Proteins 0.000 claims description 3
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- 239000003797 essential amino acid Substances 0.000 claims description 3
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- 238000007901 in situ hybridization Methods 0.000 claims description 3
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- 238000012360 testing method Methods 0.000 claims description 2
- 230000002159 abnormal effect Effects 0.000 claims 1
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Abstract
The present invention relates to a kind of mankind's inductive pluripotent stem cells isolated culture method without feeder layer serum-free.This method does not need feeder cells and serum, medium component is clearly, simple to operate, can efficiently and stably separate mankind's iPS cells, its self-renewing and versatility are maintained, is that the final mankind's iPS cell separation cultivating systems for setting up clinical practice rank lay good experimental basis.
Description
Technical field
The present invention relates to cell engineering field, specifically a kind of mankind's inductive pluripotent without feeder layer serum-free is dry thin
Born of the same parents' isolated culture method.
Background technology
Now, in inductive pluripotent stem cells (induced pluripotent the stem cells, iPS generally used
Cells) in separation and cultivating system, feeder cells and serum are largely dependent upon, they for iPS cells from
I updates and the maintenance of versatility plays an important role (Zhou et al., 2015).
Application of the feeder cells in multipotential cell culture is long-standing, early in teratocarcinoma cell (embryonic
Carcinoma cells, ECC) in vitro culture when begun to using lose mitotic capabilities mouse embryo fibroblast it is thin
Born of the same parents (mouse embryo fibroblasts, MEFs) are as feeder cells, and always in mouse, hESC
In continue to use (Zwaka and Thomson, 2005).Conventional Mouse feeder confluent monolayer cells include two kinds at present：MEFs and STO are thin
Born of the same parents.Feeder cells are acted on of both may being played on multipotential cell self-renewing and versatility is maintained, and one is to provide
Various cell factors, two are to provide and stick face, such as the direct contact of feeder cells and multipotential cell or feeder layer are thin
Contact of the extracellular matrix that born of the same parents provide with multipotential cell.In mouse embryo stem cell and hESC's incubation
In, it is already possible to feeder cells are not needed, corresponding cell factor is only added.But for iPS cells, this is asked
Topic be not yet completely resolved, individually add LIF ELISA (Leukemia Inhibitory Factor, LIF) or
Basic fibroblast growth factor (basic fibroblast growth factor, bFGF) is unable to sertoli cell reprogramming
Completion, iPS cells self-renewing and pluripotent state can not be maintained.
The a variety of cell factors secreted by feeder cells are have now been discovered, as exploitation without feeder layer culture body
The candidate of system, such as some of FGF superfamilies and TGF superfamilies member (Rao et al., 2005).On the other hand, section
Scholar also directly suppresses the differentiation of self of embryonic stem cell, such as Wnt in the material for actively finding the layer secretion of some non-breeding
The activator BIO of signal can maintain mouse and the self-renewing of hESC, and the searching and application of these materials will
Make embryonic stem cell to clinical practice huge step.Meanwhile, some scientists abandon MEC, and select
Body cell from the mankind is as feeder layer (Du et al., 2015), still, and this purifying to iPS cells brings some fiber crops
Tired, potential safety hazard can not be completely eliminated.
In addition to the dependence to feeder cells, iPS cells occur spontaneous point during separation and culture, easily
Change, this serum being likely due in culture medium contains various growth factors and hormone, can induce iPS cells
Differentiation, meanwhile, the influence that the serum of different batches is cultivated cell reprogramming efficiency and iPS cells is widely different, so
Free serum culture is imperative.Separation iPS cells are generally just using serum replacement (Knockout Serum at present
Replacement) serum in original culture systems is substituted.Regrettably in serum replacement, still containing heterologous clear
Albumen, and specific composition and its content due to commercial relations it is still not very clear.
It is to come from other animals just because of feeder cells and serum, its composition is extremely complex, composition is not clear, Er Qiepin
Matter is unstable, causes iPS cells cultivating systems now not only to there is potential safety hazard, it is difficult to large-scale culture iPS
Cells supplies clinical practice, and also brings many troubles to the explanation of experimental result.
In a word, the system that mankind's inductive pluripotent stem cells (iPS cells) are separately cultured is largely dependent upon feeding
Confluent monolayer cells and serum are supported, this system that is separately cultured has potential safety hazard, it is difficult to which large-scale culture iPS cells are answered for clinical
With.
Therefore, for the improvement that iPS cells are separately cultured system, the present invention develops no feeder cells, serum-free
Isolated culture method.
The content of the invention
For defect present in prior art, the present invention will set up a kind of specific chemical components and without feeder layer and blood
Clear mankind's iPS cells isolated culture methods, this method does not need feeder cells and serum, and medium component clearly, is grasped
Make simple, can efficiently and stably separate mankind iPS cells, maintain its self-renewing and versatility, be that final foundation is clinical
The mankind iPS cells of application level are separately cultured system and lay good experimental basis.
To achieve the above objectives, the present invention is adopted the technical scheme that：
A kind of reprogramming culture medium without feeder layer serum-free, including：
DMEM/F12 basal mediums (11330-032, Gibco), 47.75mL；N-2Supplement(17502-048,
Gibco),0.5mL；B-27Supplement(17504-044,Gibco),1mL；MEM Non-essential amino acid
solution(11140-050,Gibco),0.25mL；Gluamax-I(100X)(35050-061,Gibco),0.25mL；
Beta-Mercaptoethnol(21985-023,Gibco),90.9μL；100ng/ml bFGF,159.1μL.
A kind of mankind's inductive pluripotent stem cells culture medium without feeder layer serum-free, including：TeSRTM-E8TM Kit
For hESC/hiPSC Maintenance (article No.s：05940, STEMCELL Technologies).
A kind of mankind's inductive pluripotent stem cells isolated culture method without feeder layer serum-free, comprises the following steps：
1st, human peripheral blood mononuclear cells are separated, and are expanded in vitro；
2nd, episomol carriers are built, tetra- kinds of transcription factors of OCT-4, NANOG, KLF-4 and l-MYC are included；
3rd, by episomol carriers electrotransfection to human peripheral blood mononuclear cells；
4th, the human peripheral blood mononuclear cells after the reprogramming inoculation of medium electrotransfection without feeder layer serum-free, training
After supporting 20 days, cell reprogramming efficiency detection is carried out, satisfactory mankind's inductive pluripotent stem cells are filtered out；
The 5th, the satisfactory mankind's inductive pluripotent stem cells filtered out are transferred to the mankind of no feeder layer serum-free
Cultivated in inductive pluripotent stem cells culture medium 14-21 days, picking and passage then are carried out to iPS cells samples colony；
6th, iPS cells are identified and specific expression gene detection, and carries out vitro differentiation experiment and differentiation in vivo
Experiment.
On the basis of above-mentioned technical proposal, in step 1, immunomagnetic beads human peripheral blood mononuclear cells are utilized.
On the basis of above-mentioned technical proposal, in step 4, cell reprogramming efficiency is carried out using alkaline phosphatase staining method
Detection.
It is described to identify the caryogram identification for including iPS cells and multipotency in step 6 on the basis of above-mentioned technical proposal
Property identification.
On the basis of above-mentioned technical proposal, caryogram identification is carried out using fluorescence in-situ hybridization method, every 10 generations to iPS
Cells caryogram is identified.
On the basis of above-mentioned technical proposal, alkaline phosphatase staining, immunohistochemistry, monster neoplasia, plan are utilized
Idiosome (EB) breaks up and the method for real-time quantitative PCR (Polymerase Chain Reaction) carries out versatility identification.
On the basis of above-mentioned technical proposal, in step 6, detect that iPS cells are special by immunofluorescence and RT-PCR
The gene of expression, the gene of specifically expressing includes OCT-4, NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81.
On the basis of above-mentioned technical proposal, in step 6, the vitro differentiation experiment utilizes spontaneous point of embryoid body (EB)
Change, noble cells of the detection from ectoderm, mesoderm and entoderm.
On the basis of above-mentioned technical proposal, in step 6, the differentiation in vivo experiment utilizes teratoma and histotomy side
The noble cells of ectoderm, mesoderm and entoderm is derived from method detection teratoma.
The invention has the advantages that：
The mankind iPScells without feeder layer and serum that the present invention sets up a kind of specific chemical components is separately cultured body
System, this system can need not be used as feeder cells by the use of MEC (MEFs), it is not required that serum
As additive, its medium component is clearly, simple to operate, efficiently and stably can separate and cultivate mankind's iPS cells, maintain it
Self-renewing and versatility, are that the final mankind's iPS cell separation cultivating systems for setting up clinical practice rank lay good examination
Test basis.
Brief description of the drawings
The present invention has drawings described below：
The method flow schematic diagram of Fig. 1 present invention.
Embodiment
The present invention is described in further details below in conjunction with accompanying drawing and specific implementation case.
A kind of reprogramming culture medium without feeder layer serum-free, including：
DMEM/F12 basal mediums (11330-032, Gibco), 47.75mL；N-2Supplement(17502-048,
Gibco),0.5mL；B-27Supplement(17504-044,Gibco),1mL；MEM Non-essential amino acid
solution(11140-050,Gibco),0.25mL；Gluamax-I(100X)(35050-061,Gibco),0.25mL；
Beta-Mercaptoethnol(21985-023,Gibco),90.9μL；100ng/ml bFGF,159.1μL.
A kind of mankind's inductive pluripotent stem cells culture medium without feeder layer serum-free, including：TeSRTM-E8TM Kit
For hESC/hiPSC Maintenance (article No.s：05940, STEMCELL Technologies).
As shown in figure 1, a kind of mankind's inductive pluripotent stem cells isolated culture method without feeder layer serum-free, including such as
Lower step：
1st, human peripheral blood mononuclear cells are separated, and are expanded in vitro；
2nd, episomol carriers are built, tetra- kinds of transcription factors of OCT-4, NANOG, KLF-4 and l-MYC are included；
3rd, by episomol carriers electrotransfection to human peripheral blood mononuclear cells；
4th, the human peripheral blood mononuclear cells after the reprogramming inoculation of medium electrotransfection without feeder layer serum-free, training
After supporting 20 days, cell reprogramming efficiency detection is carried out, satisfactory mankind's inductive pluripotent stem cells are filtered out；
The 5th, the satisfactory mankind's inductive pluripotent stem cells filtered out are transferred to the mankind of no feeder layer serum-free
Cultivated in inductive pluripotent stem cells culture medium 14-21 days, picking and passage then are carried out to iPS cells samples colony；
6th, iPS cells are identified and specific expression gene detection, and carries out vitro differentiation experiment and differentiation in vivo
Experiment.
On the basis of above-mentioned technical proposal, in step 1, immunomagnetic beads human peripheral blood mononuclear cells are utilized.
On the basis of above-mentioned technical proposal, in step 4, cell reprogramming efficiency is carried out using alkaline phosphatase staining method
Detection.
It is described to identify the caryogram identification for including iPS cells and multipotency in step 6 on the basis of above-mentioned technical proposal
Property identification.
On the basis of above-mentioned technical proposal, caryogram identification is carried out using fluorescence in-situ hybridization method, every 10 generations to iPS
Cells caryogram is identified.
On the basis of above-mentioned technical proposal, alkaline phosphatase staining, immunohistochemistry, monster neoplasia, plan are utilized
Idiosome (EB) breaks up and the method for real-time quantitative PCR (Polymerase Chain Reaction) carries out versatility identification.
On the basis of above-mentioned technical proposal, in step 6, detect that iPS cells are special by immunofluorescence and RT-PCR
The gene of expression, the gene of specifically expressing includes OCT-4, NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81.
On the basis of above-mentioned technical proposal, in step 6, the vitro differentiation experiment utilizes spontaneous point of embryoid body (EB)
Change, noble cells of the detection from ectoderm, mesoderm and entoderm.
On the basis of above-mentioned technical proposal, in step 6, the differentiation in vivo experiment utilizes teratoma and histotomy side
The noble cells of ectoderm, mesoderm and entoderm is derived from method detection teratoma.
The content not being described in detail in this specification belongs to prior art known to professional and technical personnel in the field.
Claims (10)
1. a kind of reprogramming culture medium without feeder layer serum-free, it is characterised in that including：
DMEM/F12 basal mediums, 47.75mL；N-2Supplement,0.5mL；B-27Supplement,1mL；MEM
Non-essential amino acid solution,0.25mL；Gluamax-I,0.25mL；Beta-
Mercaptoethnol,90.9μL；100ng/ml bFGF,159.1μL.
2. a kind of mankind's inductive pluripotent stem cells isolated culture method without feeder layer serum-free, using above-mentioned culture medium, bag
Include following steps：
(1) human peripheral blood mononuclear cells are separated, and are expanded in vitro；
(2) episomol carriers are built, tetra- kinds of transcription factors of OCT-4, NANOG, KLF-4 and l-MYC are included；
(3) by episomol carriers electrotransfection to human peripheral blood mononuclear cells；
(4) human peripheral blood mononuclear cells after the reprogramming inoculation of medium electrotransfection without feeder layer serum-free, culture
After 20 days, cell reprogramming efficiency detection is carried out, satisfactory mankind's inductive pluripotent stem cells are filtered out；
(5) mankind that the satisfactory mankind's inductive pluripotent stem cells filtered out are transferred into no feeder layer serum-free induce
Property multipotential stem cell culture medium in cultivate 14-21 days, then to iPS cells samples colony progress picking and passage；
(6) iPS cells are identified and specific expression gene detection, and carries out vitro differentiation experiment and differentiation in vivo examination
Test.
3. method as claimed in claim 2, it is characterised in that in the step (1), using the immunomagnetic beads mankind outside
All blood monocytes.
4. method as claimed in claim 2, it is characterised in that in the step (4), is carried out using alkaline phosphatase staining method
Cell reprogramming efficiency is detected.
5. method as claimed in claim 2, it is characterised in that identification described in step (6) includes iPS cells caryogram mirror
The identification of fixed and versatility.
6. caryogram identification as claimed in claim 5, it is characterised in that carry out caryogram identification using fluorescence in-situ hybridization method,
IPS cells caryogram is identified every 10 generations.
7. versatility identification as claimed in claim 5, it is characterised in that using alkaline phosphatase staining, immunohistochemistry,
The method of monster neoplasia, embryoid body differentiation and real-time quantitative PCR carries out versatility identification.
8. method as claimed in claim 2, it is characterised in that in the step (6), is detected by immunofluorescence and RT-PCR
The gene of iPS cells specifically expressings, the gene of specifically expressing includes OCT-4, NANOG, SSEA-3, SSEA-4, TRA-1-60
And TRA-1-81.
9. method as claimed in claim 2, it is characterised in that in the step (6), the vitro differentiation experiment utilizes embryoid
The Spontaneous Differentiation of body, noble cells of the detection from ectoderm, mesoderm and entoderm.
10. method as claimed in claim 2, it is characterised in that in the step (6), the differentiation in vivo experiment utilizes abnormal
The noble cells of ectoderm, mesoderm and entoderm is derived from tire knurl and tissue section method detection teratoma.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108085299A (en) * | 2017-12-28 | 2018-05-29 | 安徽中盛溯源生物科技有限公司 | A kind of efficient induced multi-potent stem cell reprogramming method of blood cell |
| CN113403262A (en) * | 2021-06-23 | 2021-09-17 | 昆明理工大学 | Layer-free culture method for stem cells of cynomolgus monkeys |
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| CN103003416A (en) * | 2010-06-15 | 2013-03-27 | 细胞动力学国际有限公司 | Generation of induced pluripotent stem cells from small volumes of peripheral blood |
| CN103667349A (en) * | 2013-11-15 | 2014-03-26 | 安徽农业大学 | Method for efficiently acquiring inductive pluripotent stem cells (iPSCs) |
| CN103756969A (en) * | 2014-01-02 | 2014-04-30 | 中国医学科学院血液病医院（血液学研究所） | Method for establishing iPS |
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| CN113403262A (en) * | 2021-06-23 | 2021-09-17 | 昆明理工大学 | Layer-free culture method for stem cells of cynomolgus monkeys |
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