CN106635957A - Stem cell culture medium and preparation method thereof - Google Patents
Stem cell culture medium and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of cell culture mediums, and particularly relates to a stem cell culture medium and a preparation method thereof. The stem cell culture medium provided by the invention comprises a basic culture medium and an additive, wherein counted by final concentration, the additive is prepared from 1 to 3mg/mL human serum albumin, 8 to 13mug/mL transferrin, 0.12 to 0.15mug/mL glucagon, 2.5 to 3mug/mL activated carbon, 13 to 18ng/mL fibroblast growth factor, 7 to 11ng/mL epidermal growth factor, 3 to 6 muM linoleic acid, 5 to 9mug/mL quercetin, 20 to 25mug/mL platycodin, 2 to 5mg/mL laminine, 3 to 7mug/mL adenosine deaminase and 1.2 to 1.6muM myristic acid. By adopting the stem cell culture medium provided by the invention, stem cells can be amplified rapidly, and the culture time of the stem cells is shortened greatly.
Description
Technical field
The invention belongs to cell culture medium technical field, and in particular to a kind of stem cell media and preparation method thereof.
Background technology
Stem cell can not survive in simple basal medium, and some trace nutrients are must provide in cell culture
Matter and growth factor can just enable cell grow and maintain growth conditions.Therefore the basal medium for adopting is often required to add blood
Clearly, such as horse serum or hyclone；Because the extremely complex mixture that serum is made up of many biomolecule of different sizes,
It can provide growth factor, hormone, associated proteins required when cell is cultivated in vitro, and provide protective effect；And this
Mitogenic factor is also rich in body serum, also beneficial to clone and original cuiture.But the drawbacks of blood serum medium has certain,
The alloplasm in animal blood serum cannot be evaded, if the cell cultivated using animal blood serum culture medium, can there is human body different
Source body repels and conflicts.Especially during stem cell culture, the protein of a large amount of complicated components in serum gives cell training
Foster standardization brings difficulty, wherein complicated albumen and multiple cell factor, can cause to be easy for the dry thin of differentiation in itself
Born of the same parents bear various diverse cell phenotypes, and produce various diverse cell differentiation trend, also give cell culture table
Repeatability up to separation and purification of products and result brings very big difficulty.
Chinese patent application CN105087480A discloses a kind of serum-free stem cell media, including basal medium and
The recruitment factor being added in the basal medium, recruitment factor includes sodium selenite, transferrins, bovine serum albumin(BSA), pancreas
Island element, FTN and hydrocortisone.Chinese patent application CN104357379A discloses a kind of stem cell media, bag
Containing following components：Water-soluble non-polyelectrolyte macromolecule, inorganic salts, vitamin, amino acid, glucose, transferrins, pancreas islet
Element or para-insulin function replacement product such as (IGF-1/2), fibroblast growth factor.
At present, the stem cell media without serum is had been developed for, but, there is cell in existing stem cell media
The slow-footed problem of propagation.
The content of the invention
For the deficiencies in the prior art, it is an object of the invention to provide a kind of stem cell media and preparation method thereof.
The stem cell media energy rapid amplifying stem cell that the present invention is provided, substantially reduces the time of stem cell culture.
The technical scheme is that：
A kind of stem cell media, including basal medium and additive, the composition of the additive is with final concentration, bag
Include：Human serum albumin 1-3mg/mL, transferrins 8-13 μ g/mL, hyperglycemic factor 0.12-0.15 μ g/mL, activated carbon 2.5-3 μ
G/mL, desmocyte growth factor-21 3-18ng/mL, EGF 7-11ng/mL, 3-6 μM of linoleic acid, Quercetin 5-9
μ g/mL, Platycodin D 20-25 μ g/mL, laminine 2-5mg/mL, adenosine deaminase 3-7 μ g/mL, myristic acid 1.2-1.6
μM。
Further, the stem cell media includes basal medium and additive, and the composition of the additive is with end
Densimeter, including：Human serum albumin 2mg/mL, the μ g/mL of transferrins 10, the μ g/mL of hyperglycemic factor 0.14, the μ g/ of activated carbon 2.8
ML, desmocyte growth factor-21 5ng/mL, EGF 9ng/mL, 5 μM of linoleic acid, the μ g/mL of Quercetin 6, balloonflower root soap
Glycosides D23 μ g/mL, laminine 4mg/mL, the μ g/mL of adenosine deaminase 5,1.4 μM of myristic acid.
Further, the basal medium is DMEM or F12 culture mediums.
In addition, present invention also offers the preparation method of the stem cell media, comprises the following steps：
S1 adds human serum albumin, transferrins, hyperglycemic factor, Desmocyte growth factor in basal medium
Son, EGF, linoleic acid, Quercetin, Platycodin D, laminine and myristic acid, stir 25-35 minutes, add
Adenosine deaminase, continues to stir 30-40 minutes, adds activated carbon, stirs, and stands 2-4 hours, obtains mixture；
S2 adds mass fraction for the sodium carbonate liquor of 6-8%, the pH of regulation culture medium in step S1 gained mixture
To 7.2-7.4, sterilising temp is 118 DEG C -123 DEG C, and sterilization time is 16-20 minutes, is obtained final product.
Preferably, step S1 is stirred 30 minutes.
Preferably, step S1 continues to stir 36 minutes.
Preferably, step S1 stands 3 hours.
Preferably, step S2 adds the sodium carbonate liquor that mass fraction is 7% in step S1 gained mixture.
Preferably, step S2 adjusts the pH to 7.3 of culture medium.
Preferably, the step S2 sterilising temp is 121 DEG C.
Preferably, the step S2 sterilization time is 18 minutes.
The stem cell media that the present invention is provided includes basal medium and additive, and the additive of addition includes that human blood is white
Albumen, transferrins, hyperglycemic factor and activated carbon etc..In the composition of the various culture mediums of the present invention, basal medium energy
The existence of stem cell and minimum physiological activity are enough provided.In the present invention, transferrins can adjust the generation of internal ferro element
Thank, be transferred to ferro element by the TfR of cell surface intracellular；Meanwhile, it can be with micro- with other
With reference to so as to adjust the growing multiplication and functional expression of stem cell.In the present invention, human serum albumin is mainly used as osmotic pressure tune
Section agent, the carrier of growth factor, stabilizer and free radical scavenger and nutritional agents etc..In the present invention, hyperglycemic factor
Glycometabolism, lipid metabolism of stem cell etc. are participated in, the propagation and functional expression of stem cell can be adjusted.Make in culture medium of the present invention
Activated carbon, can adsorb the noxious material of metabolism in stem cell growth, but, activated carbon is harmful in absorption culture medium
While material, composition that also can be in Absorption Growth Auto-regulator and other culture mediums, inventor is when using activated carbon, it is considered to
To the dual character of activated carbon, Jing many experiments, the proportioning of suitable activated carbon and growth regulatory substance in culture medium is obtained so as to
Can preferably play a role.In the present invention, fibroblast growth factor and EGF are dry mainly as maintenance
Recruitment factor needed for cell injuring model existence, propagation and differentiation.In the present invention, linoleic acid can provide cell membrane synthesis
Required lipid.In the present invention, laminine can promote the adhesion of stem cell, and its adherent growth.In the present invention, gland
Guanosine deaminase can speed up cell metabolism.In the present invention, myristic acid can promote the growth of stem cell.
Research discovery, adds Platycodin D and Quercetin, both composition energy and other compositions in stem cell media
Interphase interaction, the amplification rate for making stem cell is further improved.Stem cell media of the present invention collocation is reasonable, respectively into
Mutually coordinated, collective effect between point, improves expansion of stem cells speed, substantially reduces the time of stem cell culture.
Compared with prior art, the stem cell media that the present invention is provided has the advantage that：
(1) present invention provide stem cell media energy rapid amplifying stem cell, substantially reduce stem cell culture when
Between.
(2) stem cell media that the present invention is provided does not contain serum, eliminates the pathogen contamination of animal origin.
(3) the present invention is to provide a kind of non-animal derived property composition, the stem cell media of composition determination, can effectively protect
The quality stability of card product batches, is conducive to the standardization of products.
Specific embodiment
Below by way of the description of specific embodiment, the invention will be further described, but this is not the limit to the present invention
System, those skilled in the art's basic thought of the invention, various modifications may be made or improves, but without departing from this
The basic thought of invention, within the scope of the present invention.
DMEM culture mediums are purchased from Sigma companies in the present invention, and F12 culture mediums are purchased from Sigma companies, and Quercetin is commercially available
Biological Co., Ltd of shaking is composed from Shanghai,.
Embodiment 1, a kind of stem cell media
The stem cell media includes basal medium and additive, the composition of the additive with final concentration, such as
Shown in table 1.
Table 1：The final concentration of the additive being added in basal medium
| Additive | Final concentration |
| Human serum albumin | 1mg/mL |
| Transferrins | 8μg/mL |
| Hyperglycemic factor | 0.12μg/mL |
| Activated carbon | 2.5μg/mL |
| Fibroblast growth factor | 13ng/mL |
| EGF | 7ng/mL |
| Linoleic acid | 3μM |
| Quercetin | 5μg/mL |
| Platycodin D | 20μg/mL |
| Laminine | 2mg/mL |
| Adenosine deaminase | 3μg/mL |
| Myristic acid | 1.2μM |
The basal medium is DMEM culture mediums.
Preparation method：
S1 adds human serum albumin, transferrins, hyperglycemic factor, Desmocyte growth factor in basal medium
Son, EGF, linoleic acid, Quercetin, Platycodin D, laminine and myristic acid, stir 25 minutes, add gland
Guanosine deaminase, continues to stir 30 minutes, adds activated carbon, stirs, and stands 2 hours, obtains mixture；
S2 to step S1 gained mixture in add mass fraction be 6% sodium carbonate liquor, adjust culture medium pH to
7.2, sterilising temp is 118 DEG C, and sterilization time is 16 minutes, is obtained final product.
Embodiment 2, a kind of stem cell media
The stem cell media includes basal medium and additive, the composition of the additive with final concentration, such as
Shown in table 2.
Table 2：The final concentration of the additive being added in basal medium
| Additive | Final concentration |
| Human serum albumin | 3mg/mL |
| Transferrins | 13μg/mL |
| Hyperglycemic factor | 0.15μg/mL |
| Activated carbon | 3μg/mL |
| Fibroblast growth factor | 18ng/mL |
| EGF | 11ng/mL |
| Linoleic acid | 6μM |
| Quercetin | 9μg/mL |
| Platycodin D | 25μg/mL |
| Laminine | 5mg/mL |
| Adenosine deaminase | 7μg/mL |
| Myristic acid | 1.6μM |
The basal medium is F12 culture mediums.
Preparation method：
S1 adds human serum albumin, transferrins, hyperglycemic factor, Desmocyte growth factor in basal medium
Son, EGF, linoleic acid, Quercetin, Platycodin D, laminine and myristic acid, stir 35 minutes, add gland
Guanosine deaminase, continues to stir 40 minutes, adds activated carbon, stirs, and stands 4 hours, obtains mixture；
S2 to step S1 gained mixture in add mass fraction be 8% sodium carbonate liquor, adjust culture medium pH to
7.4, sterilising temp is 123 DEG C, and sterilization time is 20 minutes, is obtained final product.
Embodiment 3, a kind of stem cell media
The stem cell media includes basal medium and additive, the composition of the additive with final concentration, such as
Shown in table 3.
Table 3：The final concentration of the additive being added in basal medium
| Additive | Final concentration |
| Human serum albumin | 2mg/mL |
| Transferrins | 10μg/mL |
| Hyperglycemic factor | 0.14μg/mL |
| Activated carbon | 2.8μg/mL |
| Fibroblast growth factor | 15ng/mL |
| EGF | 9ng/mL |
| Linoleic acid | 5μM |
| Quercetin | 6μg/mL |
| Platycodin D | 23μg/mL |
| Laminine | 4mg/mL |
| Adenosine deaminase | 5μg/mL |
| Myristic acid | 1.4μM |
The basal medium is F12 culture mediums.
Preparation method：
S1 adds human serum albumin, transferrins, hyperglycemic factor, Desmocyte growth factor in basal medium
Son, EGF, linoleic acid, Quercetin, Platycodin D, laminine and myristic acid, stir 30 minutes, add gland
Guanosine deaminase, continues to stir 36 minutes, adds activated carbon, stirs, and stands 3 hours, obtains mixture；
S2 to step S1 gained mixture in add mass fraction be 7% sodium carbonate liquor, adjust culture medium pH to
7.3, sterilising temp is 121 DEG C, and sterilization time is 18 minutes, is obtained final product.
Comparative example 1, a kind of stem cell media
The stem cell media includes basal medium and additive, the composition of the additive with final concentration, such as
Shown in table 4.
Table 4：The final concentration of the additive being added in basal medium
The basal medium is F12 culture mediums.
Preparation method is similar to Example 3.
It is that Platycodin D is replaced with into ginsenoside with the difference of embodiment 3.
Comparative example 2, a kind of stem cell media
The stem cell media includes basal medium and additive, the composition of the additive with final concentration, such as
Shown in table 5.
Table 5：The final concentration of the additive being added in basal medium
The basal medium is F12 culture mediums.
Preparation method is similar to Example 3.
It is that Quercetin is replaced with into baicalein with the difference of embodiment 3.
Comparative example 3, a kind of stem cell media
The stem cell media includes basal medium and additive, the composition of the additive with final concentration, such as
Shown in table 6.
Table 6：The final concentration of the additive being added in basal medium
| Additive | Final concentration |
| Human serum albumin | 2mg/mL |
| Transferrins | 10μg/mL |
| Hyperglycemic factor | 1.47μg/mL |
| Activated carbon | 1.47μg/mL |
| Fibroblast growth factor | 15ng/mL |
| EGF | 9ng/mL |
| Linoleic acid | 5μM |
| Quercetin | 6μg/mL |
| Platycodin D | 23μg/mL |
| Laminine | 4mg/mL |
| Adenosine deaminase | 5μg/mL |
| Myristic acid | 1.4μM |
The basal medium is F12 culture mediums.
Preparation method is similar to Example 3.
It is that the final concentration of hyperglycemic factor and activated carbon is adjusted to into 1.47 μ g/mL with the difference of embodiment 3.
The impact of test example one, stem cell media of the present invention to stem cell growth
1st, subjects：Embodiment of the present invention 1-3 gained stem cell media, and the gained stem cell training of comparative example 1-3
Foster base.
2nd, test method：
Deep fat tissue 20mL is drawn from stern top with aseptic needle tubing, is moved it in 1h in centrifuge tube, add etc.
800r/min is centrifuged 10 minutes after amount PBS, fully vibration, 3 times is rinsed repeatedly and adds equivalent 15g/L NTx enzyme afterwards,
Fully vibrate 30 minutes in 37 DEG C of water-baths.800r/min be centrifuged 10 minutes after supernatant discarded.Add after lower confluent monolayer cells are suspended with PBS
Enter in 37 DEG C of water-baths of 16mmol/L NH4Cl 10min with splitting erythrocyte.After 800r/min is centrifuged 10 minutes, then rinsed with PBS
3 times.Finally, cell is used respectively invention embodiment 1-3 gained stem cell media, and comparative example 1-3 gained
Stem cell media is moved in blake bottle after suspending, 37 DEG C, 50mL/L CO2It is incubated in incubator.Start in culture, and the 7th
My god, 14 days, 21 days and when 28 days, TCS is calculated respectively.As a result it is as shown in table 7.
Table 7：Stem cell amplification times in different culture media
| Culture medium | 7 days | 14 days | 21 days | 28 days |
| Embodiment 1 | 156.3±24.5 | 1371.6±48.5 | 6567.2±42.4 | 11236.1±77.3 |
| Embodiment 2 | 157.8±25.6 | 1370.6±42.8 | 6464.7±55.4 | 11238.5±81.3 |
| Embodiment 3 | 161.9±25.3 | 1374.5±41.7 | 6472.6±58.3 | 11244.2±83.3 |
| Comparative example 1 | 51.2±14.7 | 742.3±26.8 | 3825.6±45.1 | 6132.8±52.5 |
| Comparative example 2 | 52.7±15.2 | 752.1±25.7 | 3886.2±43.5 | 6242.9±50.6 |
| Comparative example 3 | 52.5±14.5 | 756.4±23.2 | 3873.5±44.8 | 6253.4±51.2 |
As can be seen from Table 7, in identical incubation time, it is grown in the gained stem cell culture of embodiment of the present invention 1-3
The amplification times of cell in base, hence it is evident that more than the amplification times for being grown in cell in comparative example 1-3 gained stem cell media.This
Illustrate, the expanding effect of stem cell media of the present invention is good.
Claims (10)
1. a kind of stem cell media, it is characterised in that including basal medium and additive, the composition of the additive is with end
Densimeter, including：Human serum albumin 1-3mg/mL, transferrins 8-13 μ g/mL, hyperglycemic factor 0.12-0.15 μ g/mL are living
Property charcoal 2.5-3 μ g/mL, desmocyte growth factor-21 3-18ng/mL, EGF 7-11ng/mL, linoleic acid 3-6 μ
M, Quercetin 5-9 μ g/mL, Platycodin D 20-25 μ g/mL, laminine 2-5mg/mL, adenosine deaminase 3-7 μ g/mL, Pork and beans
Cool sour 1.2-1.6 μM.
2. stem cell media as claimed in claim 1, it is characterised in that described to add including basal medium and additive
Plus the composition of agent is with final concentration, including：Human serum albumin 2mg/mL, the μ g/mL of transferrins 10, the μ g/ of hyperglycemic factor 0.14
ML, the μ g/mL of activated carbon 2.8, desmocyte growth factor-21 5ng/mL, EGF 9ng/mL, 5 μM of linoleic acid, quercitrin
Element 6 μ g/mL, the μ g/mL of Platycodin D 23, laminine 4mg/mL, the μ g/mL of adenosine deaminase 5,1.4 μM of myristic acid.
3. stem cell media as claimed in claim 1 or 2, it is characterised in that the basal medium is that DMEM or F12 is trained
Foster base.
4. the preparation method of the stem cell media as described in claim 1-3 is arbitrary, it is characterised in that comprise the following steps：
S1 adds human serum albumin, transferrins, hyperglycemic factor, fibroblast growth factor, table in basal medium
Skin growth factor, linoleic acid, Quercetin, Platycodin D, laminine and myristic acid, stir 25-35 minutes, add adenosine
Deaminase, continues to stir 30-40 minutes, adds activated carbon, stirs, and stands 2-4 hours, obtains mixture；
S2 to step S1 gained mixture in add mass fraction for 6-8% sodium carbonate liquor, adjust culture medium pH to
7.2-7.4, sterilising temp is 118 DEG C -123 DEG C, and sterilization time is 16-20 minutes, is obtained final product.
5. the preparation method of stem cell media as claimed in claim 4, it is characterised in that step S1 stirs 30 points
Clock.
6. the preparation method of stem cell media as claimed in claim 4, it is characterised in that step S1 continues to stir 36
Minute.
7. the preparation method of stem cell media as claimed in claim 4, it is characterised in that step S2 is to step S1 institute
Obtain the sodium carbonate liquor for adding that mass fraction is 7% in mixture.
8. the preparation method of stem cell media as claimed in claim 4, it is characterised in that step S2 adjusts culture medium
PH to 7.3.
9. the preparation method of stem cell media as claimed in claim 4, it is characterised in that the step S2 sterilising temp is
121℃。
10. the preparation method of stem cell media as claimed in claim 4, it is characterised in that the step S2 sterilization time
For 18 minutes.
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| CN201611209708.3A CN106635957A (en) | 2016-12-23 | 2016-12-23 | Stem cell culture medium and preparation method thereof |
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| CN201611209708.3A CN106635957A (en) | 2016-12-23 | 2016-12-23 | Stem cell culture medium and preparation method thereof |
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Cited By (3)
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| CN108913647A (en) * | 2018-08-08 | 2018-11-30 | 新乡医学院 | A kind of stem cell media and preparation method thereof |
| CN111733130A (en) * | 2020-06-15 | 2020-10-02 | 湖南科诺康美生命科技有限公司 | Culture method for inducing amplification of DC-CIK |
| CN111944752A (en) * | 2020-08-28 | 2020-11-17 | 广州同康生物科技有限公司 | Skeletal muscle stem cell serum-free medium and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913647A (en) * | 2018-08-08 | 2018-11-30 | 新乡医学院 | A kind of stem cell media and preparation method thereof |
| CN108913647B (en) * | 2018-08-08 | 2021-06-04 | 新乡医学院 | Stem cell culture medium and preparation method thereof |
| CN111733130A (en) * | 2020-06-15 | 2020-10-02 | 湖南科诺康美生命科技有限公司 | Culture method for inducing amplification of DC-CIK |
| CN111944752A (en) * | 2020-08-28 | 2020-11-17 | 广州同康生物科技有限公司 | Skeletal muscle stem cell serum-free medium and preparation method thereof |
| CN111944752B (en) * | 2020-08-28 | 2021-09-03 | 广州同康生物科技有限公司 | Skeletal muscle stem cell serum-free medium and preparation method thereof |
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