CN109988744A - Human pluripotent stem cells culture medium and its preparation method and application - Google Patents
Human pluripotent stem cells culture medium and its preparation method and application Download PDFInfo
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- 239000006143 cell culture medium Substances 0.000 claims abstract description 36
- 239000003112 inhibitor Substances 0.000 claims abstract description 36
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- PMYDPQQPEAYXKD-UHFFFAOYSA-N 3-hydroxy-n-naphthalen-2-ylnaphthalene-2-carboxamide Chemical compound C1=CC=CC2=CC(NC(=O)C3=CC4=CC=CC=C4C=C3O)=CC=C21 PMYDPQQPEAYXKD-UHFFFAOYSA-N 0.000 claims abstract description 27
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
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Abstract
The invention discloses a kind of human pluripotent stem cells culture mediums and its preparation method and application, wherein, the cell culture medium contains DMEM culture medium, F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin-like growth factor, bFGF, transferrins and transforming growth factor β signal pathway inhibitor.It realizes without containing animal source component, and enough nutriments and stable living environment can be provided for culture cell, while preparation method step is simple, raw material is easy to get, and the cell generation cultivated is more, proliferative capacity is strong, the good effect of state.
Description
Technical field
The present invention relates to culture medium fields, and in particular, to human pluripotent stem cells culture medium and its preparation method and application.
Background technique
Stem cell is a kind of cell for referring to self-renewing, dividing and be divided into other cell populations.From stem cell
In development from the point of view of the locating stage, stem cell can be divided into embryonic stem cell (embryonic stem ceLL, ES cell) and
Adult stem cell (aduLt stem ceLL).It is divided into three classes according to the potentiality of development of stem cell: myeloid-lymphoid stem cell
(totipotent stem cell, TSC), multipotential stem cell (pluripotent stem cell) and unipotent stem cell
(unipotent stem cell) (specially energy stem cell).Embryonic stem cell is a kind of multipotential stem cell (pLuripotent stem
CeLL), the tissue and organ that can break up 200 multiple types cell of adult body, form body, can be divided into various kinds of cell
It, and can be with infinite multiplication with the stem cell of the potential of tissue.Evans and Kaufman in 1981 is from mouse inner cell mass
(inner ceLL mass, ICM) isolates embryonic stem cell, and ES cell research later becomes the hot spot of scientists from all over the world's research,
Occur (1981 like the mushrooms after rain around the research that stem cell directional is divided into other cell types in recent years；Nature 292
(5819):154-6).Thomson is successfully separated, cultivates hESC within 1998, is the physianthropy application of stem cell
Provide possible (1998；Science 282 (5391): 1145-7).It stretches in the mountain of Kyoto Univ Japan in 2006 and more etc. takes the lead in
Report the research (2006 of iPS cell；CeLL 126 (4): 663-76), they are by the mouse skin to terminal differentiation at fibre
It ties up and imports four kinds of transcription factor OSKM (Oct4, Sox2, KLf4, c-Myc) in cell, the cell Cheng Gan of terminal differentiation can be induced
Hereafter there are various manufacture iPS cells in cell state, i.e. iPS cell (induced pLuripotent stem ceLL)
Technology Ways.And in mountain doctor also because cell reprogramming field outstanding contribution, obtain Nobel's physiology in 2012 or
Medicine.The multipotential stem cell of artificial induction has differentiation capability similar with embryonic stem cell, epigenetic modification and cell
Other features such as form, and from a wealth of sources, amoral dispute of ethic provide to do thin field of biology and clinical regenerative medicine
New research direction.
Stem cell has the potentiality of a variety of differentiation potentials and self-renewing and infinite multiplication, and can grow in vitro
Phase saves.These features of stem cell provide good cell origin for scientific research and medical application.Stem cell master at present
It studies and specifically includes that 1) drug development and drug screening, external evoked stem cell directional are divided into certain kinds with application direction
Various drugs are added after type cell, research drug is saved numerous studies cost, answered for clinic to the toxicity of specific organization or cell
With offer mass data.2) cell therapy, hepatoblast can be broken up for stem cell directional (notification number is by having been reported
The patent of CN102666853A) and cardiac muscle cell's patent of CN103602633A and CN104293730A (notification number be) etc. other
The cell of type provides the cell origin for largely having physiological function for cell therapy；3) regenerative medicine field, conventional regeneration
Medicine such as organ transplant are by donor person organ transplant to receptor body, and this transplanting means can lead to the problem of very much, are such as exempted from
Epidemic disease rejection and infection etc..Being divided by the stem cell directional of external patient artificial induction oneself mature has physiology function
The organ of energy can efficiently solve these negative effects, provide new method for regenerative medicine.
Currently, often contain animal source component in culture medium used in the incubation of multipotential stem cell, especially in
The culture medium of entoderm precursor, for example use MEF (mouse embryonic fibroblasts, Mouse Embryonic
FibrobLast) the animal blood serum in trophocyte and culture medium.Although above-mentioned culture medium can guarantee stem cell and its lure
The cell for leading differentiation has excellent self-renewal capacity, but due to introducing a large amount of animal derived substances in culture medium, to dry
The cell self-renewal and atomization of cell and its induction differentiation bring uncertain factor, increase error；Simultaneously may
Introduce animal derived pollution, such as mycoplasma contamination；In addition, can also induce immune response during cell therapy.
Summary of the invention
For the above-mentioned prior art, it is an object of the invention to overcome in the incubation of multipotential stem cell in the prior art
Often contain animal source component in the culture medium used, to occur error in atomization or introduce animal derived pollution
The problems such as, to provide a kind of without containing animal source component, and enough nutriments and surely can be provided for culture cell
Fixed living environment, while preparation method step is simple, raw material is easy to get, and the cell generation cultivated is more, proliferative capacity is strong, state
Good human pluripotent stem cells culture medium and its preparation method and application.
To achieve the goals above, the present invention provides a kind of human pluripotent stem cells culture mediums, wherein people's multipotency is dry
Cell culture medium contain DMEM culture medium, F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin-like growth factor,
BFGF, transferrins and transforming growth factor β signal pathway inhibitor.
The present invention also provides a kind of preparation methods of human pluripotent stem cells culture medium, wherein the preparation method includes:
1) by DMEM culture medium, F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin-like growth factor, bFGF,
Transferrins and the mixing of transforming growth factor β signal pathway inhibitor, are made mixture M 1；
2) mixture M 1 is adjusted into pH and osmotic pressure, obtains mixture M 2；
3) mixture M 2 is subjected to sterilization treatment, cell culture medium is made.
The present invention also provides a kind of human pluripotent stem cells culture mediums to break up in culture human pluripotent stem cells to various kinds of cell
In application, wherein the human pluripotent stem cells culture medium is as described above.
Through the above technical solutions, cell culture medium provided by the invention passes through DMEM culture medium and F12 culture medium, selenic acid
Sodium, sodium bicarbonate, vitamin C, insulin-like growth factor, bFGF, transferrins and transforming growth factor β signal pathway inhibitor
Synergistic effect with to culture cell enough nutriments and stable living environment are provided so that culture cell have it is excellent
Self-renewal capacity.Animal source component is not used in the culture medium simultaneously, and then has effectively evaded due to animal derived object
The case where self-renewing of confrontation culture cell and atomization bring uncertain factor and pollute to culture cell
Generation, and can be avoided completely during cell therapy induce be immunoreacted generation.In addition, the cell culture medium
Preparation method step is simple, raw material is easy to get, be free of serum, dedicated for hESC and induce multi-potent stem cell
Without the culture and amplification under the conditions of feeder cells.The culture medium can keep undifferentiated, the multipotency state of hESC and hiPSCs.Training
The removal for supporting animal blood serum in base can avoid interference of the animal component to human pluripotent stem cell culture, simplified culture mistake significantly
Journey increases the confidence level of experimental result.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the aspect graph of human pluripotent stem cell when expanding for 4 generation in application examples 1；
Fig. 2 is the aspect graph of human pluripotent stem cell when expanding for 15 generation in application examples 1；
Fig. 3 is the increment curve graph that 15 generations are expanded in application examples 1；
Fig. 4 be versatility protein marker Sox2, Oct4 that the human pluripotent stem cell after 15 generations is expanded in application examples 1,
The identification of Nanog, TRA1-81；
Fig. 5 is identification of the human pluripotent stem cell to various kinds of cell differentiation capability after expanding for 15 generations in application examples 1；
Fig. 6-Fig. 8 is the identification of the human pluripotent stem cell differentiating into nerve cells ability after expanding for 15 generations in application examples 1；
Wherein, using Nestin and Sox2 to mark in Fig. 6 is neural stem cell, and it is neuronal cell that Tuji label is used in Fig. 7,
It is neuronal cell that Fig. 8, which uses Map2 label,；
Fig. 9 is the identification of the human pluripotent stem cell myocardiac differentiation ability after expanding for 15 generations in application examples 1；
Figure 10 is mirror of the human pluripotent stem cell after expanding for 15 generations in application examples 1 to vascular endothelial cell differentiation capability
It is fixed.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
The present invention provides a kind of human pluripotent stem cells culture mediums, wherein the human pluripotent stem cells culture medium contains
DMEM culture medium, F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin-like growth factor, bFGF, transferrins and turn
Change grouth factor beta signal pathway inhibitor.
Cell culture medium provided by the invention is given birth to by DMEM culture medium and F12 culture medium, sodium selenate, sodium bicarbonate, dimension
Plain C, insulin-like growth factor, bFGF, transferrins and transforming growth factor β signal pathway inhibitor synergistic effect with to training
Feeding cell provides enough nutriments and stable living environment, so that culture cell has excellent self-renewal capacity.
Animal source component is not used in the culture medium simultaneously, so effectively evaded due to animal derived substance to culture cell from
I updates and generation the case where atomization brings uncertain factor and pollutes to culture cell, and can be
It avoids inducing the generation being immunoreacted completely during cell therapy.In addition, the preparation method step letter of the cell culture medium
Single, raw material is easy to get, and is free of serum, dedicated for hESC and induces multi-potent stem cell in no feeder cells condition
Under culture and amplification.The culture medium can keep undifferentiated, the multipotency state of hESC and hiPSCs.Animal blood serum in culture medium
Removal can avoid interference of the animal component to human pluripotent stem cell culture significantly, and simplified culture process increases experimental result
Confidence level.
The content of above-mentioned raw materials can select in a wide range, for example, in a kind of preferred embodiment of the invention
In, in order to enable cell can have more stable culture environment, the DMEM culture medium and the F12 during the cultivation process
The volume ratio of the content of culture medium is 1:1-4；And on the basis of the total amount of the cell culture medium, the ascorbic concentration
For 1-100 μ g/mL, the concentration of the insulin-like growth factor is 1-100ng/mL, and the concentration of the bFGF is 1-200ng/mL,
The concentration of the transforming growth factor β signal pathway inhibitor is 1-50-ng/mL, and the concentration of the sodium selenate is 1-100 μ g/
L, the concentration of the sodium bicarbonate are 100-1000mg/L, and the concentration of the transferrins is 1-50 μ g/mL.
In further preferred embodiment, the ascorbic concentration is 20-100 μ g/mL, the insulin growth
The concentration of the factor is 5-100ng/mL, and the concentration of the bFGF is 5-150ng/mL, the transforming growth factor β signal path suppression
The concentration of preparation is 1-20-ng/mL, and the concentration of the sodium selenate is 1-50 μ g/L, and the concentration of the sodium bicarbonate is 200-
700mg/L, the concentration of the transferrins are 5-50 μ g/mL.
In a kind of more preferably embodiment, in order to guarantee nutritional sufficiency in cell cultivation process and environment is more stable
Etc. demands, the ascorbic concentration be 50-100 μ g/mL, the concentration of the insulin-like growth factor is 20-60ng/mL, institute
The concentration for stating bFGF is 50-100ng/mL, and the concentration of the transforming growth factor β signal pathway inhibitor is 5-10-ng/mL,
The concentration of the sodium selenate is 2-30 μ g/L, and the concentration of the sodium bicarbonate is 400-600mg/L, the concentration of the transferrins
For 10-30 μ g/mL.
Certainly, transforming growth factor β signal pathway inhibitor here can be selected from skilled artisans appreciate that
With the type used, for example, in a kind of more preferably embodiment, in order to make it have better regulating effect, described turn
Change grouth factor beta signal pathway inhibitor and is selected from the transforming growth factor β signal pathway inhibitor that the trade mark is SB431542.
Certainly, the conditions such as specific pH value of cell culture medium here can select in a wide range, for example, in this hair
In a kind of bright preferred embodiment, in order to further ensure the stability of culture environment, the pH value of the cell culture medium
For 7.3-8.
Similarly, in another preferred embodiment, the osmotic pressure of the cell culture medium is 320-360mOsm/kg.
The present invention also provides a kind of preparation methods of human pluripotent stem cells culture medium, wherein the preparation method includes:
1) by DMEM culture medium, F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin-like growth factor, bFGF,
Transferrins and the mixing of transforming growth factor β signal pathway inhibitor, are made mixture M 1；
2) mixture M 1 is adjusted into pH and osmotic pressure, obtains mixture M 2；
3) mixture M 2 is subjected to sterilization treatment, cell culture medium is made.
The dosage of above-mentioned raw materials can select in a wide range, for example, in a kind of preferred embodiment of the invention
In, in order to enable cell can have more stable culture environment, the DMEM culture medium and the F12 during the cultivation process
The volume ratio of the dosage of culture medium is 1:1-4；And on the basis of the total amount of the cell culture medium, the ascorbic concentration
For 1-100 μ g/mL, the concentration of the insulin-like growth factor is 1-100ng/mL, and the concentration of the bFGF is 1-200ng/mL,
The concentration of the transforming growth factor β signal pathway inhibitor is 1-50ng/mL, and the concentration of the sodium selenate is 1-100 μ g/L,
The concentration of the sodium bicarbonate is 100-1000mg/L, and the concentration of the transferrins is 1-50 μ g/mL.
In further preferred embodiment, the ascorbic concentration is 20-100 μ g/mL, the insulin growth
The concentration of the factor is 5-100n g/mL, and the concentration of the bFGF is 5-150ng/mL, the transforming growth factor β signal path
The concentration of inhibitor is 1-20ng/mL, and the concentration of the sodium selenate is 1-50 μ g/L, and the concentration of the sodium bicarbonate is 200-
700mg/L, the concentration of the transferrins are 5-50 μ g/mL.
In a kind of more preferably embodiment, in order to guarantee nutritional sufficiency in cell cultivation process and environment is more stable
Etc. demands, the ascorbic concentration be 50-100 μ g/mL, the concentration of the insulin-like growth factor is 20-60n g/mL,
The concentration of the bFGF is 50-100ng/mL, and the concentration of the transforming growth factor β signal pathway inhibitor is 5-10ng/mL,
The concentration of the sodium selenate is 2-30 μ g/L, and the concentration of the sodium bicarbonate is 400-600mg/L, the concentration of the transferrins
For 10-30 μ g/mL.
Similarly, in a kind of preferred embodiment, the transforming growth factor β signal pathway inhibitor is selected from the trade mark
The transforming growth factor β signal pathway inhibitor of SB431542.
Adjust in step 2) pH operation can using skilled artisans appreciate that by the way of operated, example
Such as, it can be adjusted using acid-base modifier is added, in a preferred embodiment of the invention, be added in step 2)
Enter alkali and adjusts pH value.
Here alkali can be selected from skilled artisans appreciate that and the type that uses, for example, one kind is more highly preferred to
Embodiment in, the alkali be selected from sodium hydroxide and/or potassium hydroxide.
Certainly, the pH value after adjusting can select in a wide range, for example, in a kind of preferred embodiment, in order to
It is further ensured that the stability of culture environment, the pH value of mixture M 2 are 7.3-8.
In further preferred embodiment, the osmotic pressure of mixture M 2 is 320-360mOsm/kg.
In step 3) sterilization treatment can using those skilled in the art institute it will be appreciated that by the way of operate, for example,
In a preferred embodiment of the invention, sterilization treatment is selected from ray sterilizing, hot air sterilization, moist heat sterilization in step 3)
At least one of with filtration sterilization.
In further preferred embodiment, sterilization treatment uses moist heat sterilization.
More preferably in embodiment, sterilization treatment is to be gone out by the membrane filtration with 0.1-0.3 μ m diameter micropore
Bacterium.
The present invention also provides a kind of human pluripotent stem cells culture mediums to break up in culture human pluripotent stem cells to various kinds of cell
In application, wherein the human pluripotent stem cells culture medium is as described above.
The present invention will be described in detail by way of examples below.In following embodiment, DMEM culture is Thermo
The commercially available product of Fisher Scientific company, F12 culture medium are the commercially available of Thermo Fisher Scientific company
Product, vitamin C are the commercially available product of Sigma ALdrich company, and vascular endothelial growth factor is R&D Biosystems company
Commercially available product, transforming growth factor β signal pathway inhibitor are the commercially available product that the Sigma ALdrich company trade mark is SB431542,
Transferrins is the commercially available product that the Sigma ALdrich company trade mark is T0665.
Embodiment 1
1) at 25 DEG C, by DMEM culture medium, F12 culture medium, vitamin C, sodium selenate, sodium bicarbonate, insulin growth
The factor, bFGF and the mixing of transforming growth factor β signal pathway inhibitor sufficiently obtain mixture M 1；Wherein, DMEM culture medium with
The volume ratio of F12 culture medium is 1:2, and ascorbic concentration is 50 μ g/mL, and the concentration of insulin-like growth factor is 50ng/mL,
The concentration of bFGF is 100ng/mL, and the concentration of transferrins is 25 μ g/mL, transforming growth factor β signal pathway inhibitor it is dense
Degree is 25ng/mL, and the concentration of sodium selenate is 20 μ g/L, and the concentration of sodium bicarbonate is 500mg/L.
2) it after sodium hydroxide being added into above-mentioned mixture M 1 so that pH is adjusted to 7.4, adds sodium chloride and adjusts infiltration
It is depressed into 340mOsm/kg, mixture M 2 is made；
3) said mixture M2 is sterilized by the membrane filtration with 0.2 μ m diameter micropore and cell culture medium A1 is made.
Embodiment 2
1) at 25 DEG C, by DMEM culture medium, F12 culture medium, vitamin C, sodium selenate, sodium bicarbonate, insulin growth
The factor, bFGF and the mixing of transforming growth factor β signal pathway inhibitor sufficiently obtain mixture M 1；Wherein, DMEM culture medium with
The volume ratio of F12 culture medium is 1:1, and ascorbic concentration is 2 μ g/mL, and the concentration of insulin-like growth factor is 4ng/mL, is turned
The concentration of ferritin is that the concentration of 5 μ g/mL, bFGF is 5ng/mL, and the concentration of transforming growth factor β signal pathway inhibitor is
3ng/mL, the concentration of sodium selenate are 2 μ g/L, and the concentration of sodium bicarbonate is 200mg/L.
2) it after sodium hydroxide being added into above-mentioned mixture M 1 so that pH is adjusted to 7.4, adds sodium chloride and adjusts infiltration
It is depressed into 340mOsm/kg, mixture M 2 is made；
3) said mixture M2 is sterilized by the membrane filtration with 0.2 μ m diameter micropore and cell culture medium A2 is made.
Embodiment 3
1) at 25 DEG C, by DMEM culture medium, F12 culture medium, vitamin C, sodium selenate, sodium bicarbonate, insulin growth
The factor, bFGF and the mixing of transforming growth factor β signal pathway inhibitor sufficiently obtain mixture M 1；Wherein, DMEM culture medium with
The volume ratio of F12 culture medium is 1:4, and ascorbic concentration is 90 μ g/mL, and the concentration of insulin-like growth factor is 95ng/mL,
The concentration that the concentration of transferrins is 50 μ g/mL, bFGF is 200ng/mL, transforming growth factor β signal pathway inhibitor it is dense
Degree is 45ng/mL, and the concentration of sodium selenate is 50 μ g/L, and the concentration of sodium bicarbonate is 600mg/L.
2) it after sodium hydroxide being added into said mixture W1 so that pH is adjusted to 7.4, adds sodium chloride and adjusts infiltration
It is depressed into 340mOsm/kg, mixture M 2 is made；
3) said mixture W3 is sterilized by the membrane filtration with 0.2 μ m diameter micropore and cell culture medium A3 is made.
Comparative example 1
It carries out according to the method for embodiment 1, unlike, vitamin C is not used in step 1), cell culture medium is made
D1。
Comparative example 2
It carries out according to the method for embodiment 1, unlike, insulin-like growth factor is not used in step 1), cell is made
Culture medium D2.
Comparative example 3
It carries out according to the method for embodiment 1, unlike, bFGF is not used in step 1), cell culture medium D3 is made.
Comparative example 4
It carries out according to the method for embodiment 1, unlike, the suppression of transforming growth factor β signal path is not used in step 1)
Cell culture medium D4 is made in preparation.
Comparative example 5
It carries out according to the method for embodiment 1, unlike, sodium selenate is not used in step 1), cell culture medium D5 is made.
Comparative example 6
If the method for embodiment 1 carries out, unlike, it is added without bFGF and transforming growth factor β signal pathway inhibitor,
Cell culture medium D6 is made.
Application examples 1
1) culture dish is coated with MatrigeL matrigel and (certainly, connects mucoprotein Vitronection using glass
Also can) 2h；Then it recovers in 37 DEG C of water-bath the human pluripotent stem cell frozen, and the cell inoculation to above-mentioned A1 is cultivated
In base, in 37 DEG C, 5%CO2Under cultivated, and culture medium is replaced daily, until proliferation of pluripotent stem cells is to 80% fusion
Spend (confLuency) when, then with the EDTA of 0.5mmol/L (PH=8.0, osmotic pressure (OsmoLarity)=340mOsm) into
Row had digestive transfer culture is to keep the state of human pluripotent stem cells cell mass.When needing unicellular passage, using Trypsin enzyme (when
Right TrypLE Express enzyme can also be with) digestion, and the Rock inhibitor (work that the trade mark is Y-27632 is added into culture medium
Concentration is 10 μm of ol/L, improves the survival rate of cell) culture is for 24 hours.
2) culture medium is removed, with PBS buffer solution cleaning 3 times, then with the EDTA of 0.5mmol/L (PH=8.0, infiltration
Press (OsmoLarity)=340mOsm) digestion 5min, EDTA is sucked, A1 culture medium is added and gently blows and beats 3 to 5 times, to keep people
The state of multipotential stem cell cell mass reaches new use MatrigeL matrigel or Vitronectin with area ratio 1:8 and is coated with
Culture dish in, be added cell culture medium A1 in 37 DEG C, 5%CO2Lower culture, replaces culture medium daily, when cell fusion degree reaches
When to 80%, cell B1 is obtained.
Detect example 1
Firstly, EDTA (PH=8.0, osmotic pressure (OsmoLarity)=340mOsm) digestion with 0.5mmol/L is above-mentioned thin
Born of the same parents B1 counts to cell mass state, takes a part passage (expanding) down.Passage 15 times.The result is shown in Figure 1 is to Fig. 3.
Wherein: Fig. 1-2 is aspect graph of the human pluripotent stem cell after the culture medium culture.Fig. 1 is the 4th generation of amplification, Fig. 2
It is the 15th generation of amplification, Fig. 3 is the growth curve that human pluripotent stem cell expands 15 generations with the culture medium culture.
Similarly, by application examples 1 and detect example 1 method it is found that culture medium A 2 and A3 also can be stable culture people
Class multipotential stem cell.
It is operated according to the method for application examples 1 and detection example 1, unlike, cell culture medium is replaced with into D1- respectively
D6, the results show that cell culture medium D1-D6 can not successfully cultivate human pluripotent stem cell.
Meanwhile it is as shown in figure 5, dry thin for mankind's multipotency after provided 15 generation of cell culture medium culture through the invention
Born of the same parents (H1) and the trophocyte (TBL) of directed differentiation, mesendoderm group cell (ME), nerve group cell (NPC) express pedigree
Special gene, it was demonstrated that under this cultivating system of cell culture medium provided by the invention, human pluripotent stem cell is maintain
The ability broken up to various kinds of cell.
As Figure 6-Figure 8, dry thin for mankind's multipotency after provided 15 generation of cell culture medium culture through the invention
The identification of born of the same parents' differentiating into nerve cells ability, wherein using Nestin and Sox2 to mark in Fig. 6 is neural stem cell, in Fig. 7
Using Tuji label is neuronal cell, and it is neuronal cell that Fig. 8, which uses Map2 label,.
As shown in figure 9, for the human pluripotent stem cell after 15 generation of cell culture medium culture provided by through the invention to
The identification of Cardiomyocyte Differentiation ability, wherein α-actinin and cTnT marks cardiac muscle cell, the visible Mature myocardium of fluorescent staining
The texture of cell-specific.
As shown in Figure 10, for the human pluripotent stem cell after 15 generation of cell culture medium culture provided by through the invention to
The identification of vascular endothelial cell differentiation capability, wherein CD31 label vascular endothelial cell, it can self-organizing shape in 3D culture systems
At capillary network.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (10)
1. a kind of human pluripotent stem cells culture medium, which is characterized in that the human pluripotent stem cells culture medium contain DMEM culture medium,
F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin-like growth factor, bFGF, transferrins and transforming growth factor β
Signal pathway inhibitor.
2. cell culture medium according to claim 1, wherein the content of the DMEM culture medium and the F12 culture medium
Volume ratio be 1:1-4；And
On the basis of the total amount of the cell culture medium, the ascorbic concentration is 1-100 μ g/mL, the insulin growth
The concentration of the factor is 1-100ng/mL, and the concentration of the bFGF is 1-200ng/mL, the transforming growth factor β signal path suppression
The concentration of preparation is 1-50ng/mL, and the concentration of the sodium selenate is 1-100 μ g/L, and the concentration of the sodium bicarbonate is 100-
1000mg/L, the concentration of the transferrins are 1-50 μ g/mL；
Preferably, the ascorbic concentration is 20-100 μ g/mL, and the concentration of the insulin-like growth factor is 5-100ng/
The concentration of mL, the bFGF are 5-150ng/mL, and the concentration of the transforming growth factor β signal pathway inhibitor is 1-20ng/
ML, the concentration of the sodium selenate are 1-50 μ g/L, and the concentration of the sodium bicarbonate is 200-700mg/L, the transferrins
Concentration is 5-50 μ g/mL；
It is further preferable that the ascorbic concentration is 50-100 μ g/mL, the concentration of the insulin-like growth factor is 20-
The concentration of 60ng/mL, the bFGF are 50-100ng/mL, and the concentration of the transforming growth factor β signal pathway inhibitor is 5-
10ng/mL, the concentration of the sodium selenate are 2-30 μ g/L, and the concentration of the sodium bicarbonate is 400-600mg/L, described to turn iron egg
White concentration is 10-30 μ g/mL.
3. cell culture medium according to claim 1 or 2, wherein the transforming growth factor β signal pathway inhibitor choosing
The transforming growth factor β signal pathway inhibitor for being SB431542 from the trade mark.
4. cell culture medium according to claim 1 or 2, wherein the pH value of the cell culture medium is 7.3-8；
Preferably, the osmotic pressure of the cell culture medium is 320-360mOsm/kg.
5. a kind of preparation method of human pluripotent stem cells culture medium, which is characterized in that the preparation method includes:
1) by DMEM culture medium, F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin-like growth factor, bFGF, turn iron
Albumen and the mixing of transforming growth factor β signal pathway inhibitor, are made mixture M 1；
2) mixture M 1 is adjusted into pH and osmotic pressure, obtains mixture M 2；
3) mixture M 2 is subjected to sterilization treatment, human pluripotent stem cells culture medium is made.
6. preparation method according to claim 5, wherein the dosage of the DMEM culture medium and the F12 culture medium
Volume ratio is 1:1-4；And
On the basis of the total amount of the cell culture medium, the ascorbic concentration is 1-100 μ g/mL, the insulin growth
The concentration of the factor is 1-100ng/mL, and the concentration of the bFGF is 1-200ng/mL, the transforming growth factor β signal path suppression
The concentration of preparation is 1-50ng/mL, and the concentration of the sodium selenate is 1-100 μ g/L, and the concentration of the sodium bicarbonate is 100-
1000mg/L, the concentration of the transferrins are 1-50 μ g/mL；
Preferably, the ascorbic concentration is 20-100 μ g/mL, and the concentration of the insulin-like growth factor is 5-100ng/
The concentration of mL, the bFGF are 5-150ng/mL, and the concentration of the transforming growth factor β signal pathway inhibitor is 1-20ng/
ML, the concentration of the sodium selenate are 1-50 μ g/L, and the concentration of the sodium bicarbonate is 200-700mg/L, the transferrins
Concentration is 5-50 μ g/mL；
It is further preferable that the ascorbic concentration is 50-100 μ g/mL, the concentration of the insulin-like growth factor is 20-
The concentration of 60ng/mL, the bFGF are 50-100ng/mL, and the concentration of the transforming growth factor β signal pathway inhibitor is 5-
10ng/mL, the concentration of the sodium selenate are 2-30 μ g/L, and the concentration of the sodium bicarbonate is 400-600mg/L, described to turn iron egg
White concentration is 10-30 μ g/mL.
7. preparation method according to claim 5 or 6, wherein the transforming growth factor β signal pathway inhibitor is selected from
The trade mark is the transforming growth factor β signal pathway inhibitor of SB431542.
8. preparation method according to claim 5 or 6, wherein adjust pH value in step 2) for alkali is added；
Preferably, the alkali is selected from sodium hydroxide and/or potassium hydroxide；
It is further preferable that the pH value of mixture M 2 is 7.3-8；
It is further preferred that the osmotic pressure of mixture M 2 is 320-360mOsm/kg.
9. preparation method according to claim 5 or 6, wherein sterilization treatment is selected from ray sterilizing, xeothermic goes out in step 3)
At least one of bacterium, moist heat sterilization and filtration sterilization；
Preferably, sterilization treatment uses moist heat sterilization；
It is further preferable that sterilization treatment is to be sterilized by the membrane filtration with 0.1-0.3 μ m diameter micropore.
10. a kind of application of human pluripotent stem cells culture medium in culture human pluripotent stem cells into various kinds of cell differentiation, feature
It is, the human pluripotent stem cells culture medium is any in claim 5-9 as described in any one of claim 1-4 or such as
Preparation method described in one is made.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111019896A (en) * | 2019-12-27 | 2020-04-17 | 浙江大学 | Nucleus pulposus progenitor cell culture medium and preparation method and application thereof |
| CN111057677A (en) * | 2019-12-27 | 2020-04-24 | 浙江大学 | Chondroprogenitor cell culture medium and preparation method and application thereof |
| CN113416709A (en) * | 2021-06-21 | 2021-09-21 | 香港再生医学有限公司 | Method, culture medium and system for promoting iPSC to differentiate into peripheral neuron cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952374A (en) * | 2014-05-07 | 2014-07-30 | 北京赛贝生物技术有限公司 | Human pluripotent stem cell culture medium and culture method of human pluripotent stem cells |
| CN104928231A (en) * | 2015-06-05 | 2015-09-23 | 芜湖医诺生物技术有限公司 | Cell culture medium, and preparation method and application thereof |
| CN104928230A (en) * | 2015-06-05 | 2015-09-23 | 芜湖医诺生物技术有限公司 | Vascular endothelial cell culture method |
-
2019
- 2019-03-01 CN CN201910155177.1A patent/CN109988744A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103952374A (en) * | 2014-05-07 | 2014-07-30 | 北京赛贝生物技术有限公司 | Human pluripotent stem cell culture medium and culture method of human pluripotent stem cells |
| CN104928231A (en) * | 2015-06-05 | 2015-09-23 | 芜湖医诺生物技术有限公司 | Cell culture medium, and preparation method and application thereof |
| CN104928230A (en) * | 2015-06-05 | 2015-09-23 | 芜湖医诺生物技术有限公司 | Vascular endothelial cell culture method |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111019896A (en) * | 2019-12-27 | 2020-04-17 | 浙江大学 | Nucleus pulposus progenitor cell culture medium and preparation method and application thereof |
| CN111057677A (en) * | 2019-12-27 | 2020-04-24 | 浙江大学 | Chondroprogenitor cell culture medium and preparation method and application thereof |
| WO2021129030A1 (en) * | 2019-12-27 | 2021-07-01 | 浙江大学 | Nucleus pulposus progenitor cell culture medium, preparation method therefor and use thereof |
| CN111057677B (en) * | 2019-12-27 | 2021-08-03 | 浙江大学 | Chondroprogenitor cell culture medium and preparation method and application thereof |
| CN113416709A (en) * | 2021-06-21 | 2021-09-21 | 香港再生医学有限公司 | Method, culture medium and system for promoting iPSC to differentiate into peripheral neuron cells |
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