CN108823160A - A kind of umbilical cord mesenchymal stem cells primary culture medium and its primary culture method - Google Patents
A kind of umbilical cord mesenchymal stem cells primary culture medium and its primary culture method Download PDFInfo
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- CN108823160A CN108823160A CN201810824245.4A CN201810824245A CN108823160A CN 108823160 A CN108823160 A CN 108823160A CN 201810824245 A CN201810824245 A CN 201810824245A CN 108823160 A CN108823160 A CN 108823160A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Abstract
The present invention relates to technical field of stem cell culture, in particular to a kind of umbilical cord mesenchymal stem cells primary culture medium and its primary culture method.The primary culture medium includes tranexamic acid, G-CSF, EGF and basal medium；The concentration of each component is in umbilical cord mesenchymal stem cells primary culture medium：Tranexamic acid：500~15000mg/L；G-CSF：10~50ng/L；EGF：5~30ng/mL；Basal medium：It supplies.Tranexamic acid, G-CSF, EGF are added into basal medium by the present invention, can be shortened the originally culture time, and effect is better than conventional medium and adds the culture medium of monofactor；There can be higher clinical safety simultaneously to avoid the introducing of heterologous substance.
Description
Technical field
The present invention relates to technical field of stem cell culture, in particular to a kind of umbilical cord mesenchymal stem cells primary culture medium and
Its primary culture method.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is with height self-renewal capacity and multidirectional
The multipotential stem cell of differentiation potential is widely present in marrow, fat, amniotic fluid, placenta, Cord blood and umbilical cord tissue.Umbilical cord
Mescenchymal stem cell (UC-MSCs) is present in umbilical cord Wall and leads in glue (Wharton's jelly) and tissues surrounding vascular
A kind of stem cell, it is from a wealth of sources, convenient for materials, it is easily isolated amplification in vitro.
Umbilical cord mesenchymal stem cells are more more advantageous than the MSCs from embryo, marrow, fat and its hetero-organization.It is first
First, umbilical cord source is not by dispute of ethic, and cost is relatively low；Secondly, hUC-MSCs will not cause teratoma, and there is suppression cancer
Property, since hUC-MSCs also has low immunogenicity, immunological regulation, matrix support, paracrine, migration and gene stability, therefore
With good clinical treatment potential.The common primary separation method of umbilical cord has：Tissue block adherent cultivation, umbilical cord are homogenized collagen
Enzyme digestion and improvement 3 kinds of methods of collagenase digestion.
Existing conventional cultivation technique uses external source serum (such as fetal calf serum) although promoting umbilical cord mesenchymal stem cells proliferation
Proliferation purpose can be reached, originally culture is time-consuming longer, though and its composition of external source serum is most of it is known that but there are also one
Part is unclear, and serum composition and content it is often different with the gender of blood supply animal, age, physiological condition and nutritional condition and
It is different.
The external rapid amplifying side for being used for umbilical cord mesenchymal stem cells therefore, it is necessary to seek a kind of alternative fetal calf serum
Method.
Summary of the invention
In view of this, the present invention provides a kind of umbilical cord mesenchymal stem cells primary culture medium and its primary culture methods.
Tranexamic acid, G-CSF, EGF are added into basal medium by the present invention, can be shortened the originally culture time, and effect is better than conventional training
It supports base and adds the culture medium of monofactor；There can be higher clinical safety simultaneously to avoid the introducing of heterologous substance.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of umbilical cord mesenchymal stem cells primary culture mediums, including tranexamic acid, G-CSF, EGF and base
Basal culture medium；The concentration of each component is in umbilical cord mesenchymal stem cells primary culture medium：
Tranexamic acid：500~15000mg/L；
G-CSF：10~50ng/L；
EGF：5~30ng/mL；
Basal medium：It supplies.
Preferably, the concentration of each component is in umbilical cord mesenchymal stem cells primary culture medium：
Tranexamic acid：500~10000mg/L；
G-CSF：10~20ng/L；
EGF：10~20ng/mL；
Basal medium：It supplies.
Preferably, the concentration of each component is in umbilical cord mesenchymal stem cells primary culture medium：
Tranexamic acid：10000mg/L；
G-CSF：10~20ng/L；
EGF：20ng/mL；
Basal medium：It supplies.
It is highly preferred that the concentration of each component is in umbilical cord mesenchymal stem cells primary culture medium：
Tranexamic acid：10000mg/L；
G-CSF：10ng/L；
EGF：20ng/mL；
Basal medium：It supplies.
It is highly preferred that the concentration of each component is in umbilical cord mesenchymal stem cells primary culture medium：
Tranexamic acid：10000mg/L；
G-CSF：20ng/L；
EGF：20ng/mL；
Basal medium：It supplies.
Preferably, basal medium is DMEM/F12 culture medium.
The present invention also provides a kind of umbilical cord mesenchymal stem cells primary culture methods, include the following steps：
Umbilical cord outer membrane, vein and artery are removed, magnificent Tong Shi glue is obtained；
Magnificent Tong Shi glue is shredded, the magnificent Tong Shi glue shredded is inoculated in above-mentioned umbilical cord mesenchymal stem cells primary culture medium,
Carry out originally culture.
Preferably, shredding magnificent Tong Shi glue to 1~2mm3。
Preferably, inoculum density is 2.5~3mL/15cm ware.
Preferably, the condition of originally culture is 37 DEG C, 5%CO2。
The present invention provides a kind of umbilical cord mesenchymal stem cells primary culture medium and its primary culture methods.The originally culture
Base includes tranexamic acid, G-CSF, EGF and basal medium；The concentration of each component in umbilical cord mesenchymal stem cells primary culture medium
For：Tranexamic acid：500~15000mg/L；G-CSF：10~50ng/L；EGF：5~30ng/mL；Basal medium：It supplies.This
Inventing the technical effect that has is：
Tranexamic acid, G-CSF, EGF are added into basal medium by the present invention, can be shortened the originally culture time, and effect is good
In conventional medium and the culture medium of addition monofactor；
The mixture of tranexamic acid, G-CSF, EGF are replaced fetal calf serum by the present invention, can drawing to avoid heterologous substance
Enter, there is higher clinical safety.
Detailed description of the invention
Fig. 1 shows the skeletonization effect of umbilical cord mesenchymal stem cells；
Fig. 2 shows the expression of umbilical cord mesenchymal stem cells surface marker.
Specific embodiment
The invention discloses a kind of umbilical cord mesenchymal stem cells primary culture medium and its primary culture method, art technologies
Personnel can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar replacements and changing
Move apparent to those skilled in the art, they are considered as being included in the present invention.It method of the invention and answers
With being described by preferred embodiment, related personnel can obviously not depart from the content of present invention, in spirit and scope
To method described herein and application is modified or appropriate changes and combinations, carrys out implementation and application the technology of the present invention.
Term is explained：
Umbilical cord：It is the cord structures of fetal period connection parent and fetus, it is outer by amnion, include 2 arteria umbilicalis, 1
Umbilical vein contains special embryo's mucoid connective tissue-Wal Tong Shi glue (Whartonps Jelly) between arteriovenous.
Human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells, hUC-
MSCs)：The stroma cell isolated from Wal Tong Shi glue is a kind of fibroblast-like cells with multinomial differentiation potential, in
It is isolated and cultured for the first time by McElreavery within 1991.HUC-MSCs ratio is from marrow, fat and its hetero-organization
MSCs is more advantageous, firstly, umbilical cord source is by dispute of ethic, and cost is relatively low；Secondly, hUC-MSCs will not cause it is abnormal
Tire tumor, and have press down it is carcinous, since hUC-MSCs also has low immunogenicity, immunological regulation, matrix support, paracrine, moves
Shifting and gene stability, therefore there is good clinical treatment potential.
DMEM/F12 culture medium：F12 medium component is abundant, containing various trace elements and DMEM with 1：1 combines, and claims
It is relatively abundant to be contained using F12 as the basis of exploitation serum-free formula for DMEM/F12 culture medium (DME/F12medium)
Ingredient and DMEM contain higher concentration nutritional ingredient be advantage.The culture medium be suitable for serum content it is lower under the conditions of lactation
Animal cell culture.
Tranexamic acid, English name：Tranexamic Acid, chemical name：To aminomethyl cyclohexane formic acid, it is trans--
4- aminomethyl-naphthenic acid.Tranexamic acid can inhibit the effect of fibrinolysin, to show hemostasis, antiallergic action, disappear
Scorching effect.It is mainly used for various bleedings caused by acute or chronic, limitation or systemic hyperfibrinolysis.
Granulocyte colony stimulating factor (G-CSF) is a kind of glycoprotein, contains 174 amino acid, and molecular weight is about
20000.G-CSF mainly acts on proliferation, differentiation and the activation of neutrophil series (lineage) hematopoietic cell.Recombined human grain
Granulomacrophage colony stimulating factor (rhGM-CSF) acts on hematopoietic progenitor cells, it is promoted to be proliferated and break up, important work
With being stimulation grain, mononuclear macrophage maturation, mature cell is promoted to discharge to peripheral blood, and macrophage can be promoted and bite acidity
The multiple functions of cell.
Epithelical cell growth factor (EGF) also known as people oligopeptides -1 are a kind of intracorporal active materials of people, by 53 amino
The active peptides of composition reach repairing hyperplasia skin table by stimulating the tyrosine phosphorylation of epidermal growth factor receptor
Confluent monolayer cells, it is stated that excellent curative effect is possessed to injured, impaired epidermis skin.Its biggest characteristic is that the proliferation of cell can be promoted
Differentiation, to replace aging and dead cell with newborn cell.EGF can also stop blooding, and have and accelerate skin and mucous membrane wound
The effect of recover from injury conjunction, anti-inflammatory analgesic, prevents ulcer.The stability of EGF is fabulous, is not easy flowing of scattering, energy and people at normal temperature
Internal various enzymes form good coordinating effect.Initial EGF is mainly employed for medical domain, is primarily to facilitate defect table
The reparation and regeneration of skin, such as treatment burn, scald.
Agents useful for same or instrument in umbilical cord mesenchymal stem cells primary culture medium and its primary culture method provided by the invention
Device is available on the market.
Below with reference to embodiment, the present invention is further explained：
Embodiment 1, mixture ratio
Mixture working concentration：
(1) tranexamic acid working concentration is 500mg/mL, 10000mg/mL；
(2) G-CSF working concentration is 10ng/mL, 20ng/mL；
(3) EGF working concentration is 10ng/mL, 20ng/mL.
Prepare two kinds of culture mediums：
Experimental group：It is named as A group, is DMEM/F12+ mixture；
Control group：It is named as B group, is DMEM/F12+10%FBS.
Specifically it is grouped as follows：
Each component concentration proportioning in 1 A group culture medium of table
Embodiment 2, observation compare primary cell and climb out of situation
(1) umbilical cord outer membrane, 1 vein and 2 arteries are removed, magnificent Tong Shi glue is obtained；
(2) magnificent Tong Shi glue is shredded to 1~2mm3, 3ml/15cm ware inoculation China's Tong Shi glue；
(3) with two class culture medium culture China Tong Shi glue in embodiment 1；Condition of culture is：37 DEG C, 5%CO2It is trained in incubator
It supports, changes the liquid once every three days.
(4) it observes within every 3 days, compares and climb out of cell situation.
It is as shown in the table to climb out of cell situation：
2 each group of table climbs out of cell situation
| Number of days | 3d | 6d | 9d | 12d |
| A1 | 0CFU | 1CFU | 3CFU | 5CFU |
| A2 | 0CFU | 1CFU | 5CFU | 7CFU |
| A3 | 1CFU | 3CFU | 6CFU | 8CFU |
| A4 | 1CFU | 2CFU | 5CFU | 9CFU |
| A5 | 1CFU | 3CFU | 7CFU | 10CFU |
| A6 | 3CFU | 6CFU | 15CFU | 16CFU |
| A7 | 1CFU | 4CFU | 12CFU | 13CFU |
| A8 | 2CFU | 6CFU | 16CFU | 17CFU |
| B | 0CFU | 1CFU | 5CFU | 9CFU |
Thus table is as it can be seen that in the case where being added to mixture (A6/A8), primary umbilical cord mesenchymal stem cells growth rate
It is substantially better than the culture medium of addition FBS.
Embodiment 3, AB group differentiation capability compare
Formula the 3rd generation cell of A8, B is taken, by 2.5 × 104Cell/mL concentration is inoculated in be trained containing DMEM/F12+20%FBS
Support base 24 orifice plates in, until cell 80% merge when, be changed to containing 1nmol/L dexamethasone, 10mg/L insulin,
The culture solution of 0.5mmol/L 3-isobutyl-1-methylxanthine, 100 μm of ol/L Indomethacins, changes weekly liquid 2 times, 3 Zhou Houyong
Paraformaldehyde is fixed, oil red O stain.Coloration result is shown in Fig. 1.It can be seen that being clearly colored fat drips, it was demonstrated that A8 group of the present invention is cultivated
The umbilical cord mesenchymal stem cells arrived have into rouge differentiation capability.
Embodiment 4, Immunophenotyping analysis
Formula the 3rd generation cell of A8, B group is taken, is cleaned 3 times after digestion, cell suspension is made, antibody, flow cytometer is added
Test and analyze the expression of CD73, CD90, CD105, CD11b, CD19, CD34, CD45, HLA-DR.Test result is shown in Table 3,
Fig. 2.
The expression of 3 umbilical cord mesenchymal stem cells surface marker of table
Above-mentioned testing result shows that the cell is umbilical cord mesenchymal stem cells.
Embodiment 5, Piglet s colibacillosis
Experimental group：A8 group；
Control group：It is named as C group, is DMEM/F12+ monofactor.
Each component concentration proportioning in 4 test group of table and control group
(1) umbilical cord outer membrane, 1 vein and 2 arteries are removed, magnificent Tong Shi glue is obtained；
(2) magnificent Tong Shi glue is shredded to 1~2mm3, 2.5~3ml/15cm ware inoculation China's Tong Shi glue；
(3) with above-mentioned two classes culture medium culture China Tong Shi glue；
(4) it observes within every 3 days, compares and climb out of cell situation.
It is as shown in the table to climb out of cell situation：
5 each group of table climbs out of cell situation
Add the primary umbilical cord mesenchymal stem cells growth rate of mixture culture medium A 8 bright it can be seen from the above results
The aobvious conventional medium better than addition monofactor.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of umbilical cord mesenchymal stem cells primary culture medium, which is characterized in that including tranexamic acid, G-CSF, EGF and basis
Culture medium；The concentration of each component is in the umbilical cord mesenchymal stem cells primary culture medium：
Tranexamic acid：500~15000mg/L；
G-CSF：10~50ng/L；
EGF：5~30ng/mL；
Basal medium：It supplies.
2. umbilical cord mesenchymal stem cells primary culture medium according to claim 1, which is characterized in that the umbilical cord mesenchyma
The concentration of each component is in stem cell primary culture medium：
Tranexamic acid：500~10000mg/L；
G-CSF：10~20ng/L；
EGF：10~20ng/mL；
Basal medium：It supplies.
3. umbilical cord mesenchymal stem cells primary culture medium according to claim 1, which is characterized in that the umbilical cord mesenchyma
The concentration of each component is in stem cell primary culture medium：
Tranexamic acid：10000mg/L；
G-CSF：10~20ng/L；
EGF：20ng/mL；
Basal medium：It supplies.
4. umbilical cord mesenchymal stem cells primary culture medium according to claim 1, which is characterized in that the umbilical cord mesenchyma
The concentration of each component is in stem cell primary culture medium：
Tranexamic acid：10000mg/L；
G-CSF：10ng/L；
EGF：20ng/mL；
Basal medium：It supplies.
5. umbilical cord mesenchymal stem cells primary culture medium according to claim 1, which is characterized in that the umbilical cord mesenchyma
The concentration of each component is in stem cell primary culture medium：
Tranexamic acid：10000mg/L；
G-CSF：20ng/L；
EGF：20ng/mL；
Basal medium：It supplies.
6. umbilical cord mesenchymal stem cells primary culture medium according to any one of claim 1 to 5, which is characterized in that institute
Stating basal medium is DMEM/F12 culture medium.
7. a kind of umbilical cord mesenchymal stem cells primary culture method, which is characterized in that include the following steps：
Umbilical cord outer membrane, vein and artery are removed, magnificent Tong Shi glue is obtained；
Magnificent Tong Shi glue is shredded, it is dry thin that the magnificent Tong Shi glue shredded is inoculated in umbilical cord mesenchyma described in any one of claims 1 to 6
In born of the same parents' primary culture medium, originally culture is carried out.
8. primary culture method according to claim 7, which is characterized in that described to shred magnificent Tong Shi glue to 1~2mm3。
9. primary culture method according to claim 7, which is characterized in that the inoculum density is 2.5~3mL/15cm
Ware.
10. primary culture method according to any one of claims 7 to 9, which is characterized in that the item of the originally culture
Part is 37 DEG C, 5%CO2。
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