CN108823160B - Umbilical cord mesenchymal stem cell primary culture medium and primary culture method thereof - Google Patents
Umbilical cord mesenchymal stem cell primary culture medium and primary culture method thereof Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention relates to the technical field of stem cell culture, in particular to a primary culture medium of umbilical cord mesenchymal stem cells and a primary culture method thereof. The primary culture medium comprises tranexamic acid, G-CSF, EGF and a basal culture medium; the concentration of each component in the umbilical cord mesenchymal stem cell primary culture medium is as follows: tranexamic acid: 500-15000 mg/L; G-CSF: 10-50 ng/L; EGF: 5-30 ng/mL; basic culture medium: and (4) complementing. According to the invention, tranexamic acid, G-CSF and EGF are added into a basic culture medium, so that the primary culture time can be shortened, and the effect is better than that of a conventional culture medium and a culture medium added with a single factor; meanwhile, the introduction of heterologous substances can be avoided, and the clinical safety is higher.
Description
Technical Field
The invention relates to the technical field of stem cell culture, in particular to a primary culture medium of umbilical cord mesenchymal stem cells and a primary culture method thereof.
Background
Mesenchymal Stem Cells (MSCs) are pluripotent stem cells with high self-renewal capacity and multipotentiality to multipotentiality, and widely present in bone marrow, fat, amniotic fluid, placenta, umbilical cord blood, and umbilical cord tissue. Umbilical cord mesenchymal stem cells (UC-MSCs) are stem cells existing in Wharton's jelly and perivascular tissues, have wide sources, are convenient to obtain materials, and are easy to separate and amplify in vitro.
Umbilical cord mesenchymal stem cells are more advantageous than MSCs derived from embryos, bone marrow, fat, and other tissues. Firstly, the umbilical cord source is not disputed by ethics, and the cost is lower; secondly, the hUC-MSCs do not cause teratoma and have cancer suppression, and the hUC-MSCs also have low immunogenicity, immunoregulation, stroma support, paracrine, migration and gene stability, so the hUC-MSCs have good clinical treatment potential. Common methods for primary isolation of umbilical cords are: tissue block adherent culture method, umbilical cord homogenate collagenase digestion method and improved collagenase digestion method 3 methods.
Although the conventional culture technology can achieve the purpose of proliferation by adopting exogenous serum (such as fetal calf serum) to promote the proliferation of umbilical cord mesenchymal stem cells, the primary culture takes a long time, most of the components of the exogenous serum are known, but a part of the exogenous serum is not clear, and the serum composition and the content are different according to the sex, age, physiological condition and nutritional condition of a blood-supply animal.
Therefore, a method for rapidly expanding umbilical cord mesenchymal stem cells in vitro instead of fetal bovine serum needs to be searched.
Disclosure of Invention
In view of this, the invention provides an umbilical cord mesenchymal stem cell primary culture medium and a primary culture method thereof. According to the invention, tranexamic acid, G-CSF and EGF are added into a basic culture medium, so that the primary culture time can be shortened, and the effect is better than that of a conventional culture medium and a culture medium added with a single factor; meanwhile, the introduction of heterologous substances can be avoided, and the clinical safety is higher.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primary culture medium of umbilical cord mesenchymal stem cells, which comprises tranexamic acid, G-CSF, EGF and a basal culture medium; the concentration of each component in the umbilical cord mesenchymal stem cell primary culture medium is as follows:
tranexamic acid: 500-15000 mg/L;
G-CSF：10～50ng/L；
EGF：5～30ng/mL；
basic culture medium: and (4) complementing.
Preferably, the concentration of each component in the umbilical cord mesenchymal stem cell primary culture medium is as follows:
tranexamic acid: 500-10000 mg/L;
G-CSF：10～20ng/L；
EGF：10～20ng/mL；
basic culture medium: and (4) complementing.
Preferably, the concentration of each component in the umbilical cord mesenchymal stem cell primary culture medium is as follows:
tranexamic acid: 10000 mg/L;
G-CSF：10～20ng/L；
EGF：20ng/mL；
basic culture medium: and (4) complementing.
More preferably, the concentration of each component in the umbilical cord mesenchymal stem cell primary culture medium is as follows:
tranexamic acid: 10000 mg/L;
G-CSF：10ng/L；
EGF：20ng/mL；
basic culture medium: and (4) complementing.
More preferably, the concentration of each component in the umbilical cord mesenchymal stem cell primary culture medium is as follows:
tranexamic acid: 10000 mg/L;
G-CSF：20ng/L；
EGF：20ng/mL；
basic culture medium: and (4) complementing.
Preferably, the basal medium is DMEM/F12 medium.
The invention also provides a primary culture method of the umbilical cord mesenchymal stem cells, which comprises the following steps:
stripping the umbilical cord adventitia, the vein and the artery to obtain Wharton's jelly;
shearing the Wharton jelly, inoculating the sheared Wharton jelly to the primary culture medium of the umbilical cord mesenchymal stem cells, and carrying out primary culture.
Preferably, the gordonia gum is sheared to 1-2 mm3。
Preferably, the inoculation density is 2.5-3 mL/15cm dish.
Preferably, the primary culture is carried out at 37 ℃ and 5% CO2。
The invention provides a primary culture medium and a primary culture method of umbilical cord mesenchymal stem cells. The primary culture medium comprises tranexamic acid, G-CSF, EGF and a basal culture medium; the concentration of each component in the umbilical cord mesenchymal stem cell primary culture medium is as follows: tranexamic acid: 500-15000 mg/L; G-CSF: 10-50 ng/L; EGF: 5-30 ng/mL; basic culture medium: and (4) complementing. The invention has the technical effects that:
according to the invention, tranexamic acid, G-CSF and EGF are added into a basic culture medium, so that the primary culture time can be shortened, and the effect is better than that of a conventional culture medium and a culture medium added with a single factor;
the invention replaces the fetal calf serum with the mixture of tranexamic acid, G-CSF and EGF, can avoid the introduction of heterologous substances and has higher clinical safety.
Drawings
FIG. 1 shows the osteogenic effect of umbilical cord mesenchymal stem cells;
FIG. 2 shows the expression of surface markers of umbilical cord mesenchymal stem cells.
Detailed Description
The invention discloses a primary culture medium of umbilical cord mesenchymal stem cells and a primary culture method thereof, and a person skilled in the art can realize the culture by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Interpretation of terms:
umbilical cord: the cord-shaped structure is connected with a mother body and a fetus in a fetus period, the exterior of the cord-shaped structure is coated with amnion, 2 umbilical arteries and 1 umbilical vein are contained, and special embryo mucus-like connective tissue-Wharton Jelly (Whartonps Jelly) is contained between the arteriovenous.
Human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells, hUC-MSCs): stromal cells isolated from Wallace's jelly, a fibroblast-like cell with a diverse differentiation potential, were first isolated and cultured by McElreaavery in 1991. The hUC-MSCs are more advantageous than MSCs derived from bone marrow, fat and other tissues, firstly, the umbilical cord source is not subject to ethical dispute and the cost is lower; secondly, the hUC-MSCs do not cause teratoma and have cancer suppression, and the hUC-MSCs also have low immunogenicity, immunoregulation, stroma support, paracrine, migration and gene stability, so the hUC-MSCs have good clinical treatment potential.
DMEM/F12 medium: the F12medium is rich in components and contains various trace elements, and DMEM is added in a proportion of 1: 1 combination, known as DMEM/F12 medium (DME/F12medium), was used as a basis for the development of serum-free formulations, taking advantage of the relatively rich content of F12 and the relatively high concentration of nutrients in DMEM. The culture medium is suitable for mammalian cell culture under the condition of low serum content.
Tranexamic acid, english name: tranxamic Acid, chemical name: p-aminomethyl-cyclohexanecarboxylic acid, trans-4-aminomethyl-cyclohexanecarboxylic acid. Tranexamic acid can inhibit the action of plasmin, thereby showing hemostatic, antiallergic and anti-inflammatory effects. It is mainly used for various bleedings caused by acute or chronic, localized or systemic hyperfibrino lysis.
Granulocyte colony stimulating factor (G-CSF) is a glycoprotein comprising 174 amino acids and having a molecular weight of about 20000. G-CSF acts mainly on the proliferation, differentiation and activation of hematopoietic cells of the neutrophil lineage (lineage). The recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) acts on hemopoietic progenitor cells to promote the proliferation and differentiation of the hemopoietic progenitor cells, and has the important functions of stimulating the maturation of granulocyte and monocyte macrophages, promoting the release of mature cells to peripheral blood and promoting multiple functions of the macrophages and the phagocytic acid cells.
Epidermal Growth Factor (EGF), also known as human oligopeptide-1, is an active substance in human body, and an active polypeptide consisting of 53 amino groups can repair hyperplastic skin surface cells by stimulating tyrosine phosphorylation of epidermal growth factor receptors, so that the therapeutic effect on injured and damaged epidermal skin is said to be excellent. The main characteristic is that it can promote the proliferation and differentiation of cells, so that the new cells can replace the aged and dead cells. EGF also stops bleeding, and has effects of accelerating wound healing of skin and mucosa, diminishing inflammation, relieving pain, and preventing ulcer. The EGF has excellent stability, is not easy to lose and flow at normal temperature, and can form good coordination effect with various enzymes in human bodies. The original EGF is mainly used in the medical field, and is mainly used for promoting the repair and regeneration of damaged epidermis, such as treating burns, scalds and the like.
The umbilical cord mesenchymal stem cell primary culture medium and the reagent or instrument used in the primary culture method thereof provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 mixture ratio
Working concentration of the mixture:
(1) the working concentration of tranexamic acid is 500mg/mL and 10000 mg/mL;
(2) the working concentration of G-CSF is 10ng/mL and 20 ng/mL;
(3) EGF working concentration is 10ng/mL and 20 ng/mL.
Two media were prepared:
experimental groups: named group A, is DMEM/F12+ mixture;
control group: designated as group B, DMEM/F12+ 10% FBS.
The specific grouping is as follows:
TABLE 1 concentration ratio of each component in group A Medium
Example 2 Observation and comparison of Primary cell climbing-out
(1) Stripping the umbilical cord adventitia, 1 vein and 2 arteries to obtain Wharton's jelly;
(2) shearing the Wharton's jelly to 1-2 mm3Inoculating Wharton's jelly in a dish of 3ml/15 cm;
(3) culturing the Wharton jelly by using the two culture mediums in the example 1; the culture conditions were: 37 ℃ and 5% CO2Cultured in an incubator every timeThe liquid is changed once in three days.
(4) Observed every 3 days, the cell climbing-out condition was compared.
The cell climbing-out is shown in the following table:
TABLE 2 cell climbing-out for each group
| 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 |
As can be seen from the table, the proliferation rate of the primary umbilical cord mesenchymal stem cells is obviously superior to that of the FBS-added culture medium under the condition that the mixture (A6/A8) is added.
Example 3 comparison of AB Components in ability to differentiate
Taking the 3 rd generation cells of the formulas A8 and B according to the ratio of 2.5 × 104The cells/mL are inoculated in a 24-well plate containing DMEM/F12+ 20% FBS medium, when the cells are fused at 80%, the cells are replaced by a culture solution containing 1nmol/L dexamethasone, 10mg/L insulin, 0.5 mmol/L3-isobutyl-1-methylxanthine and 100 mu mol/L indomethacin, the solution is replaced 2 times per week, and after 3 weeks, the cells are fixed by paraformaldehyde and stained by oil red O. The staining results are shown in FIG. 1. Clearly stained lipid droplets can be seen, and the umbilical cord mesenchymal stem cells cultured by the group A8 are proved to have adipogenic differentiation capacity.
Example 4 cellular immunophenotyping
Taking 3 rd generation cells of the formula A8 and the formula B, digesting, cleaning for 3 times, preparing cell suspension, adding an antibody, and detecting and analyzing the expression conditions of CD73, CD90, CD105, CD11B, CD19, CD34, CD45 and HLA-DR by a flow cytometer. The test results are shown in table 3 and fig. 2.
TABLE 3 expression of surface markers of umbilical cord mesenchymal stem cells
The detection result shows that the cell is the umbilical cord mesenchymal stem cell.
Example 5 comparative test of Effect
Experimental groups: group A8;
control group: named group C, is DMEM/F12+ single factor.
TABLE 4 concentration ratios of the components in the test group and the control group
(1) Stripping the umbilical cord adventitia, 1 vein and 2 arteries to obtain Wharton's jelly;
(2) shearing the Wharton's jelly to 1-2 mm3Inoculating Wharton's jelly in a dish of 2.5-3 ml/15 cm;
(3) culturing Wharton's jelly with the above two culture mediums;
(4) observed every 3 days, the cell climbing-out condition was compared.
The cell climbing-out is shown in the following table:
TABLE 5 cell climbing-out of each group
As can be seen from the above results, the proliferation rate of the primary umbilical cord mesenchymal stem cells added with the mixture culture medium A8 is obviously superior to that of the conventional culture medium added with a single factor.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. The primary culture medium of the umbilical cord mesenchymal stem cells is characterized by consisting of tranexamic acid, G-CSF, EGF and DMEM/F12 culture medium; the concentration of each component in the umbilical cord mesenchymal stem cell primary culture medium is as follows:
tranexamic acid: 10000 mg/L;
G-CSF：20ng/L；
EGF：20ng/mL；
DMEM/F12 medium: complementing;
or the concentration of each component in the umbilical cord mesenchymal stem cell primary culture medium is as follows:
tranexamic acid: 10000 mg/L;
G-CSF：20ng/L；
EGF：10ng/mL；
DMEM/F12 medium: and (4) complementing.
2. A primary culture method of umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
stripping the umbilical cord adventitia, the vein and the artery to obtain Wharton's jelly;
shearing Wharton's jelly, and inoculating the sheared Wharton's jelly in the umbilical cord mesenchymal stem cell primary culture medium of claim 1 for primary culture.
3. The primary culture method of claim 2, wherein the size of the pieces of Wharton's jelly is cut to 1-2 mm3。
4. The primary culture method of claim 2, wherein the seeding density is 2.5-3 mL/15cm dish.
5. The primary culture method according to any of claims 2 to 4, characterized in that the conditions of the primary culture are 37 ℃ and 5% CO2。
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