CN105838675A - Hematopoietic stem cell serum-free culture medium - Google Patents

Hematopoietic stem cell serum-free culture medium Download PDF

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Publication number
CN105838675A
CN105838675A CN201610398475.XA CN201610398475A CN105838675A CN 105838675 A CN105838675 A CN 105838675A CN 201610398475 A CN201610398475 A CN 201610398475A CN 105838675 A CN105838675 A CN 105838675A
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stem cell
concentration
candidate stem
free medium
serum free
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张严冬
谢海涛
李相鲁
孟建军
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Guangdong Wanhai Cell Biotechnology Co Ltd
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Guangdong Wanhai Cell Biotechnology Co Ltd
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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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2306—Interleukin-6 (IL-6)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/305—Growth hormone [GH], aka. somatotropin

Abstract

The invention aims at providing a hematopoietic stem cell serum-free culture medium, which comprises the following ingredients of cell factor combinations, DMEM culture liquid, insulin, transferrin, BMP-4, glutathione, FEG-2 and growth hormone, wherein the cell factor combinations include IL-3, IL-6, SCF and FL; the concentration of the IL-3 is 1 to 20ng/ml; the concentration of the IL-6 is 1 to 100ng/ml; the concentration of the SCF is 1 to 100ng/ml; the concentration of the FL is 1 to 100ng/mL. Through developing the serum-free culture medium and changing the adding sequence of the haemopoietic factor, the technical defects of the hematopoietic stem cell serum-free culture medium in the prior art are overcome.

Description

A kind of candidate stem cell serum free medium
Technical field
The present invention relates to biological field, particularly relate to a kind of candidate stem cell serum free medium.
Background technology
Candidate stem cell plays key effect in body fluid circulatory: on the one hand, and in body fluid, various haemocytes are all by hematopoiesis Stem cell differentiates；On the other hand, the haemocyte in body fluid updates and supplements and also relies on the increment of candidate stem cell and divide Change.Along with the rise of personalized regeneration medicine technology, candidate stem cell plays in terms of anti-aging and disease treatment and becomes more and more important Effect be clinically used for now the treatment that diabetes, tumour, disease of immune system and ischemic tissue are downright bad, candidate stem cell Transplant the supportive treatment after being also used for tumor chemoradiotherapy, to improve oncotherapy effect, reduce recurrence rate.
And how candidate stem cell cultivation realizes rapid amplifying and does not affect the key that its function is candidate stem cell cultivation, But it is not mature enough to cultivate candidate stem cell technology in prior art, show that the growth rate of cell is slow and passage number relatively Low.
Existing stem cell media with the addition of hyclone (FBS) and the growth factor of animal origin.But these The branch that becomes of animal origin causes the most potential danger clinically: immunogene and toxalbumin that (1) is contained can excite immunity , there is immunological rejection in reaction, such as, in vitro culture, with the cell of animal origin Neu5GC antibody, is transplanted and the most afterwards can Produce serious immunological rejection.The cell of animal-based components amplification can cause serious allergy anti-the most afterwards Should and immunological rejection；(2) danger of microbiological contamination stem cell is added, including virus and bacterium infection, protein Virus and unknown zoonosis；It is unfavorable for clinical practice and the basic research of stem cell.
The increment differentiation of candidate stem cell, maturation process during fetal growth are considerably complicated, depend on the regulation of various Hemopoietic factor, its The effect that middle LSF plays is the most crucial.III type tyrosine kinase receptors ligand (flt3-ligand FL) is nearest A kind of cell factor regulating hematopoiesis in early days found for several years, primary bioactivity shows as stimulating the increasing of candidate stem cell in early days Value and differentiation.Interleukin Ⅲ (Interleukin3 IL-3) is conducive to the growth of hematopoietic cell and activity to maintain, but right Candidate stem cell increment differentiation in early days has depression effect.
By research and development serum free medium, the addition sequence of change Hemopoietic factor, solve current candidate stem cell culture medium The drawback of prior art.
Summary of the invention
It is an object of the invention to provide the candidate stem cell culture medium of a kind of serum-free.
For realizing the purpose of the present invention, the invention provides techniques below scheme:
A kind of candidate stem cell serum free medium, composition includes, combination of cytokines, DMEM nutrient solution, insulin, turns Ferritin, BMP-4, glutathione, FGF-2 and growth hormone.
Described combination of cytokines includes that IL-3, IL-6, SCF and FL, concentration are respectively as follows: IL-3 1-20ng/ml；IL-6 1-100ng/ml；SCF 1-100ng/ml；FL 1-100ng/ml.
The concentration of transferrins is 0.05-0.15 μ g/ml；The concentration of BMP-4 is 0.05-0.15ng/ml；Described polypeptide is dense Degree is 100-300 μ g/ml；It is characterized in that described FGF-2 concentration is 5-15ng/ml；Described growth hormone concentration is 1-20ng/ ml。
Insulin concentration is 5-15 μ g/ml.
Described DMEM nutrient solution is high glycoform DMEM nutrient solution, and concentration of glucose is 3000mg/L-4500mg/L.
Described DMEM nutrient solution includes following components: anhydrous calcium chloride, anhydrous cupric sulfate, nine water ferric nitrates, seven water sulphur Acid ferrous iron, potassium chloride, magnesium chloride, anhydrous magnesium sulfate, sodium chloride, AMSP, disodium hydrogen phosphate, white vitriol, L-arginine hydrochloride, CYSTINE hydrochloride, Glu, glycine, L-Histidine hydrochloride, ILE, L- Leucine, L lysine HCL, L-Methionine, hypoxanthine, L-phenylalanine, Serine, L-threonine, L-the third ammonia Acid, L-asparagine, D-Glucose, ASPARTIC ACID, L-cysteine hydrochloride, Pidolidone, L-PROLINE, L-look Propylhomoserin, TYR, Valine, linoleic acid, lipoic acid, phenol red, vitamin B12,1,4-butanediamine dihydrochloride, pyruvic acid Sodium, biotin, D-VB5 calcium, Choline Chloride, thymidine, folic acid, i-inositol, niacinamide, pyridoxal hydrochloride, hydrochloric acid pyrrole are trembled pure, core Flavine, thiamine hydrochloride.
The concentration of the described each component of DMEM nutrient solution is:
A kind of using method of candidate stem cell serum free medium, cultivate candidate stem cell process as follows: a, preparation Good DMEM nutrient solution adds insulin, transferrins, BMP-4, glutathione, FGF-2 and growth hormone by concentration be fabricated to Nutrient solution；B, cultivating in 24 orifice plates, every hole 1ml nutrient solution, the candidate stem cell needed for inoculation inoculation, inoculum density is 1×105Cells/ml, in 37 DEG C, 5%CO2Full saturated humidity under cultivate；C, cultivation add IL-3+IL-6+SCF in first week； Within 7th day, it is simultaneously introduced FL until 4th week terminates；14d rises and disables IL-3.
Supplementary notes
FL is (flt3-ligand) III type tyrosine kinase receptors ligand；
IL-3 is Interleukin3 interleukin Ⅲ；
IL-6 is Interleukin6 interleukin-6；
SCF is stem cell factor stem cell factor；
BMP-4 is Bone Morphogenetic Protein bone morphogenetic protein 4；
DMEM is (Dulbeco ' s Modified Eagle Medium) Dole primary section minimum essential medium；
FGF-2 is Fibroblast Growth Factor 2 FGF2；
FBS is hyclone；
Cell number/TCS × 100% that apoptosis rate=apoptosis is positive.
Beneficial effect: the present invention is by being not added with hyclone in research and development serum free medium, i.e. culture medium, thus avoids Issuable immunological rejection in clinical practice, decreases the risk of microbiological contamination；By changing Hemopoietic factor Addition sequence, can weaken FL inhibitory action value-added to candidate stem cell getting up early, improve candidate stem cell and breed in vitro point The activity changed, reduces apoptosis rate.
Detailed description of the invention
High glycoform DMEM nutrient solution is prepared according to form 1
Table 1 high glycoform DMEM formula table
A, preparation candidate stem cell nutrient solution: in DMEM nutrient solution by concentration add insulin, transferrins, BMP-4, Glutathione, FGF-2 and growth hormone are fabricated to candidate stem cell nutrient solution；
B, addition cell factor: experiment is divided into five groups, and first group of control group being to be not added with IL-3, second group for being not added with FL Control group, the 3rd group is that four kinds of cell factors are simultaneously introduced, in no particular order order, the 4th group be candidate stem cell of the present invention training Breeding method, the 5th group is blank experiment, and four kinds of cell factors are all not added with, and concrete condition is as follows:
First group of IL-6+SCF+FL
Second group of IL-3+IL-6+SCF
3rd group of IL-3+IL-6+SCF+FL
Cultivate first week for 4th group and add IL-3+IL-6+SCF；Within 7th day, it is simultaneously introduced FL until 4th week terminates；14d Rise and disable IL-3；
Wherein the concentration of cell factor is IL-3 5ng/ml, IL-6 50ng/ml, SCF 50ng/ml, FL 50ng/ml；
C, inoculation candidate stem cell: culture experiment is carried out in 24 orifice plates, every hole 1ml nutrient solution, the AC133 that inoculation purifies + cell, inoculum density is 1 × 105cells/ml, in 37 DEG C, cultivate under the full saturated humidity of 5%CO2.Start and the cultivating Within 7 days, 14 days, 21 days, 28 days, calculate TCS respectively.
Result statistics and analysis:
AC133+ cell is amplification times under different cytokines combination of stimulation
Interpretation of result: cell amplification aspect, in addition to the 4th group, other combination all cultivate 21d time amplification times reach The highest, occur subsequently declining, and be both less than the 4th group of amplification times when 21d, the highest amplification times of the 4th group is for cultivating 28d Time (9847.2 times).Illustrate that the present invention combines expanding effect best.
AC133+ cell Apoptosis situation under different cytokines combination of stimulation compares (%)
Interpretation of result: result two: in terms of Apoptosis, all reaches the highest in cultivation to 14d, has declined.The The highest apoptosis rate of four groups isCombine less than other, illustrate that the present invention combines apoptosis rate minimum.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention is not limited thereto, Any those of ordinary skill in the art in the technical scope that the invention discloses, the change that can readily occur in or replacement, all answer Contain within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with scope of the claims.

Claims (8)

1. a candidate stem cell serum free medium, it is characterised in that: composition includes, combination of cytokines, DMEM nutrient solution, Insulin, transferrins, BMP-4, glutathione, FGF-2 and growth hormone.
A kind of candidate stem cell serum free medium the most according to claim 1, it is characterised in that: described cell factor group Conjunction includes that IL-3, IL-6, SCF and FL, concentration are respectively as follows: IL-3 1-20ng/ml；IL-6 1-100ng/ml；SCF 1- 100ng/ml；FL 1-100ng/ml.
A kind of candidate stem cell serum free medium the most according to claim 1, its feature exists: the concentration of transferrins is 0.05-0.15μg/ml；The concentration of BMP-4 is 0.05-0.15ng/ml；Described peptide concentration is 100-300 μ g/ml；Its feature It is that described FGF-2 concentration is 5-15ng/ml；Described growth hormone concentration is 1-20ng/ml.
A kind of candidate stem cell serum free medium the most according to claim 1, it is characterised in that: insulin concentration is 5- 15μg/ml。
A kind of candidate stem cell serum free medium the most according to claim 1, it is characterised in that: described DMEM nutrient solution For high glycoform DMEM nutrient solution, concentration of glucose is 3000mg/L-4500mg/L.
A kind of candidate stem cell serum free medium the most according to claim 1, it is characterised in that: described DMEM cultivates Liquid includes following components: anhydrous calcium chloride, anhydrous cupric sulfate, nine water ferric nitrates, ferrous sulfate heptahydrate, potassium chloride, magnesium chloride, nothing Water magnesium sulfate, sodium chloride, AMSP, disodium hydrogen phosphate, white vitriol, L-arginine hydrochloride, CYSTINE Hydrochloride, Glu, glycine, L-Histidine hydrochloride, ILE, L-Leu, L lysine HCL, L- Methionine, hypoxanthine, L-phenylalanine, Serine, L-threonine, ALANINE, L-asparagine, D-Glucose, ASPARTIC ACID, L-cysteine hydrochloride, Pidolidone, L-PROLINE, L-Trp, TYR, Valine, Asia Oleic acid, lipoic acid, phenol red, vitamin B12,1,4-butanediamine dihydrochloride, Sodium Pyruvate, biotin, D-VB5 calcium, chlorination Choline, thymidine, folic acid, i-inositol, niacinamide, pyridoxal hydrochloride, hydrochloric acid pyrrole are trembled pure, riboflavin, thiamine hydrochloride.
A kind of candidate stem cell serum free medium the most according to claim 1, it is characterised in that: described DMEM cultivates The concentration of each component of liquid is:
8. the using method of the candidate stem cell serum free medium described in an any one of claim 1-7, it is characterised in that: Cultivate candidate stem cell process as follows: a, in preparing DMEM nutrient solution by concentration add insulin, transferrins, BMP-4, Glutathione, FGF-2 and growth hormone are fabricated to nutrient solution；B, cultivate in 24 orifice plates, every hole 1ml nutrient solution, inoculation Candidate stem cell needed for inoculation, inoculum density is 1 × 105Cells/ml, in 37 DEG C, 5%CO2Full saturated humidity under cultivate； C, cultivation add IL-3+IL-6+SCF in first week；Within 7th day, it is simultaneously introduced FL until 4th week terminates；14d rises and disables IL-3.
CN201610398475.XA 2016-06-07 2016-06-07 Hematopoietic stem cell serum-free culture medium Pending CN105838675A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220241A (en) * 2017-12-28 2018-06-29 安徽中盛溯源生物科技有限公司 A kind of erythroid progenitor cells serum free medium and its application method
CN108265028A (en) * 2016-12-30 2018-07-10 联亘生物科技（上海）有限公司 For the cultivating system of amplifying candidate stem cell in vitro
CN109593717A (en) * 2018-12-29 2019-04-09 广州和能生物科技有限公司 A kind of stem cell serum-free culture medium
CN110894490A (en) * 2018-09-13 2020-03-20 李陶 Umbilical cord hematopoietic stem cell in-vitro amplification culture system and umbilical cord hematopoietic stem cell in-vitro amplification method
CN113151172A (en) * 2021-05-14 2021-07-23 郑州优倍得生物科技有限公司 Culture medium for amplifying umbilical cord blood hematopoietic stem cells

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CN104711226A (en) * 2015-04-09 2015-06-17 广州赛莱拉干细胞科技股份有限公司 Preparation method of placenta hematopoietic stem cells

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CN103805562A (en) * 2014-01-13 2014-05-21 章毅 Serum-free medium for culturing placenta mesenchymal stem cells
CN104711226A (en) * 2015-04-09 2015-06-17 广州赛莱拉干细胞科技股份有限公司 Preparation method of placenta hematopoietic stem cells

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108265028A (en) * 2016-12-30 2018-07-10 联亘生物科技（上海）有限公司 For the cultivating system of amplifying candidate stem cell in vitro
CN108220241A (en) * 2017-12-28 2018-06-29 安徽中盛溯源生物科技有限公司 A kind of erythroid progenitor cells serum free medium and its application method
CN108220241B (en) * 2017-12-28 2021-02-09 安徽中盛溯源生物科技有限公司 Erythrocyte progenitor cell serum-free medium and use method thereof
CN110894490A (en) * 2018-09-13 2020-03-20 李陶 Umbilical cord hematopoietic stem cell in-vitro amplification culture system and umbilical cord hematopoietic stem cell in-vitro amplification method
CN109593717A (en) * 2018-12-29 2019-04-09 广州和能生物科技有限公司 A kind of stem cell serum-free culture medium
CN113151172A (en) * 2021-05-14 2021-07-23 郑州优倍得生物科技有限公司 Culture medium for amplifying umbilical cord blood hematopoietic stem cells

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