CN113943700A - Method for culturing stem cells by using bovine serum exosomes - Google Patents
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Abstract
The invention discloses a method for culturing stem cells by using bovine serum exosomes, which comprises the following steps: adding a predetermined amount of bovine serum into the stem cell culture medium, and additionally adding bovine serum exosomes with a predetermined concentration. The method for culturing the stem cells by using the bovine serum exosomes optimizes the culture conditions of the stem cells and can improve the growth state of the stem cells. Under the condition that the adding amount of the bovine serum is not required to be increased, the content of bovine serum exosomes can be increased, and more active ingredients and nutrients are provided for the growth of stem cells. Meanwhile, impurity factors such as excessive grease, hormone, antibiotics and the like are not introduced, and the influence on stem cells is reduced. In addition, by additionally adding the bovine serum exosome, bovine serum differences caused by factors such as different bovine production places, different bovine serum separation processes, different bovine serum batches and the like can be reduced, and stable culture of stem cells is facilitated.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for culturing stem cells by using bovine serum exosomes.
Background
Mesenchymal Stem Cells (MSCs) are adult Stem Cells, which are present in tissues such as umbilical cord, bone marrow, fat, and cord blood and have strong self-renewal and multi-embryonic-layer differentiation abilities. Research shows that the mesenchymal stem cells have obvious curative effect on immunoregulation and promotion of tissue repair, and are widely used for clinical research and treatment of various major diseases such as rheumatoid arthritis, lupus erythematosus, graft-versus-host disease, hepatic fibrosis and the like. The umbilical cord mesenchymal stem cells are extracted from the umbilical cord of the newborn after full-term delivery and umbilical cord rupture, have the advantages of rich sources, no harm to the body, strong proliferation capacity, multi-embryo layer differentiation capacity, low immunogenicity and the like, and are accepted and applied by more and more professionals.
When the umbilical cord stem cells are cultured, a DMEM medium is generally selected as a basic medium, and then bovine serum (5% -15% different) with a certain concentration is added. In the existing culture method, when the adding proportion of bovine serum is too small, the growth and the adherent state of cells are not good. When the concentration of the bovine serum is increased, the content of impurity micromolecule substances such as oil, hormone, antibiotics and the like contained in the bovine serum is also increased, on one hand, the specific content and the function of the impurity micromolecule are uncertain, on the other hand, the oil can change the spectrum of cell metabolites and can cause the increase of complement content, the hormone can accelerate the differentiation of stem cells, and the factors are not beneficial to the culture of the cells. In addition, these impurity factors can mask the function of the active ingredients in the bovine serum. And the factors of different cattle producing areas, different bovine serum separation processes, different bovine serum batches and the like can cause the difference between different bovine serums, and the unstable factors are not beneficial to the stable culture of stem cells.
That is, the conventional bovine serum used for stem cell culture has the following defects: (1) when the concentration of the bovine serum is low, the growth and the adherence of cells are not good; when the concentration of the bovine serum is high, more serum impurity factors can be brought in, and the cell culture is influenced; (2) the specific components of different bovine serums have large differences, which is not beneficial to the stable culture of stem cells.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for culturing stem cells by using bovine serum exosomes, which optimizes the culture conditions of the stem cells and can improve the growth state of the stem cells.
The purpose of the invention is realized by adopting the following technical scheme:
a method for culturing stem cells by using bovine serum exosomes comprises the following steps: adding a predetermined amount of bovine serum into the stem cell culture medium, and additionally adding bovine serum exosomes with a predetermined concentration.
Further, the adding amount of the bovine serum is 3-10% of the using amount of the stem cell culture medium; the concentration of the additionally added bovine serum exosomes in the stem cell culture medium was 1.0 × 108～1.0×109particals/mL。
Further, the bovine serum exosome is an exosome extracted by using a kit.
Further, the bovine serum is fetal bovine serum, and the bovine serum exosome is fetal bovine serum exosome.
Further, the stem cell is a mesenchymal stem cell, preferably an umbilical cord mesenchymal stem cell.
Further, stem cell culture was performed by a tissue culture method.
Further, additionally adding bovine serum exosome with preset concentration in the primary culture of the stem cells; or, additionally adding bovine serum exosome with preset concentration in the subculture of the stem cells.
Further, the method comprises the following steps: inoculating the tissue block in a culture container, adding stem cell culture solution into the culture container, and additionally adding bovine serum exosome, wherein the concentration of the additionally added bovine serum exosome in the stem cell culture solution is 1.0 multiplied by 108～1.0×109particles/mL; then the culture container is placed in an incubator for culture, and bovine serum exosome with the same concentration is additionally added into the culture solution every time the culture solution is replaced in the culture process.
Further, the tissue block is umbilical cord Wharton's jelly; the stem cell culture solution is DMEM/F12 phenol red-free culture solution containing 5% serum; the culture container is a culture dish, and the culture dish is placed at 37 ℃ and 5% CO2The incubator is used for static culture, and the culture process is replaced every 48 to 72 hoursAnd (3) adding bovine serum exosome with the same concentration into the culture solution every time the culture solution is changed.
Further, the culture process is subcultured every 48 to 72 hours.
Compared with the prior art, the invention has the beneficial effects that:
the method for culturing the stem cells by using the bovine serum exosomes optimizes the culture conditions of the stem cells and can improve the growth state of the stem cells. Under the condition that the adding amount of the bovine serum is not required to be increased, the content of bovine serum exosomes can be increased, and more active ingredients and nutrients are provided for the growth of stem cells. Meanwhile, impurity factors such as excessive grease, hormone, antibiotics and the like are not introduced, and the influence on stem cells is reduced. In addition, by additionally adding the bovine serum exosome, bovine serum differences caused by factors such as different bovine production places, different bovine serum separation processes, different bovine serum batches and the like can be reduced, and stable culture of stem cells is facilitated.
Drawings
Fig. 1 is an electron microscope morphology of bovine serum exosomes extracted by the kit provided by the embodiment of the present invention and an electron microscope morphology of exosomes extracted by the UC method;
FIG. 2 is a diagram showing a distribution of bovine serum exosomes extracted from the kit according to the embodiment of the present invention;
FIG. 3 is a diagram showing the growth state of primary cells after 13 days of culture according to the method of example 1;
FIG. 4 is a diagram showing the growth state of primary cells after 13 days of culture according to the method provided in example 2 of the present invention;
FIG. 5 is a diagram showing the growth state of cells after subculture for 3 days according to the culture method provided in example 3 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict. The raw materials, equipments and the like used in the following examples are commercially available unless otherwise specified.
A method for culturing stem cells by using bovine serum exosomes comprises the following steps: adding a predetermined amount of bovine serum into the stem cell culture medium, and additionally adding bovine serum exosomes with a predetermined concentration. Wherein, the bovine serum is preferably fetal bovine serum, and the bovine serum exosome is preferably fetal bovine serum exosome; the stem cell is a mesenchymal stem cell, preferably an umbilical cord mesenchymal stem cell. For the cell culture, a tissue culture method may be employed.
The serum exosome is a core active component in serum, has the diameter of about 40-100nm, is rich in a phospholipid bilayer membrane structure the same as a cell membrane, contains specific protein, cell factors, growth factors and the like on the surface, wraps protein, lipid and miRNA segments, and can be used for positioning a cell injury area through a biological membrane barrier. The bovine serum exosome of the embodiment of the invention is derived from bovine serum, other impurities such as hormone which can adversely affect the growth of cells in the serum are removed, and the bovine serum exosome as an active ingredient is additionally added into a basal medium of stem cells, and the invention has the following advantages:
1. when the fetal calf is born by cow caesarean section, the fresh fetal calf whole blood obtained by heart closed puncture blood collection is collected after natural chromatography and centrifugation, and the light yellow transparent supernatant liquid with blood cells, fibrin and other components removed is treated by a multi-stage high-position flow membrane filtration technology and a three-stage terminal microfiltration method. The exosome is derived from fetal calf serum, the commercialization degree is high, commercial serum does not contain virus components, the serum extraction process is stable, and the quality is stable.
2. Exosomes are derived from the blood of fetal cows. Fetal bovine blood contains various cell sources, including umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, hematopoietic stem cells, neural stem cells, liver stem cells and other cells which have high activity, are in different developmental stages, are derived from different germ layers and have a complex functional state. Since the exosomes of fetal bovine serum are derived from the above-mentioned cells, the exosomes in fetal bovine serum have biological activities necessary for development in association with the above-mentioned cells.
3. Bovine serum is a necessary additive in the stem cell culture process, can be used for supplementing the defect that the nutrition of a culture medium is insufficient to maintain the growth of stem cells, and a main active ingredient exosome extracted from the bovine serum can be used as a nutritional additive in the stem cell culture process.
Furthermore, the adding amount of the bovine serum is 3-10% of the using amount of the stem cell culture medium; the concentration of the additionally added bovine serum exosomes in the stem cell culture medium was 1.0 × 108～1.0×109particles/mL. The invention can increase the content of bovine serum exosome without increasing the adding amount of bovine serum, provides more active ingredients and nutrients for stem cell growth, and when the final concentration is 1.0 multiplied by 108～1.0×109When the particles/mL serum exosome is used, the stem cell migration rate is high, the growth state is optimal, the cell saturation is high, and the refractive index is better.
Further, the bovine serum exosome is an exosome extracted by using a kit. In the production process of bovine serum, a part of vesicles with the size of more than 100nm are removed when viruses are removed, exosome components with the size of 40-100nm can be detected in the final serum product, and the exosome concentration in the serum product is detected to be about 1 multiplied by 108particles/mL. The exosomes are separated from serum by an exosome extraction kit (umibi-UR 52101), the kit adopts a chromatographic purification method, compared with a traditional exosome extraction gold standard ultra-separation (UC) method, large-scale extraction can be performed (extraction of 500ml samples can be realized once, each batch of the UC method can only process dozens of ml samples), the method is not limited by expensive instruments of the ultra-separation method, and the purified exosomes have clean backgrounds (as shown in figure 1, the form of the serum exosomes under an electron microscope (A: extraction of the kit; B: extraction of the UC method)). As shown in FIG. 2, the particle size distribution of bovine serum exosomes was measured using a nano-flow meter.
Further, the stem cell culture method provided by the embodiment of the invention comprises the following steps: inoculating the tissue block in a culture container, adding stem cell culture solution into the culture container, adding bovine serum exosome, and addingThe concentration of bovine serum exosomes in stem cell culture solution is 1.0 × 108～1.0×109particles/mL; then the culture container is placed in an incubator for culture, and bovine serum exosome with the same concentration is additionally added into the culture solution every time the culture solution is replaced in the culture process.
Further, the tissue mass is umbilical cord Wharton's jelly; the stem cell culture solution is DMEM/F12 phenol red-free culture solution containing 5% serum; the culture container is a culture dish, and the culture dish is placed at 37 ℃ and 5% CO2The culture box is used for static culture, the culture solution is replaced every 48 to 72 hours in the culture process, and bovine serum exosome with the same concentration is additionally added into the culture solution when the culture solution is replaced every time.
More specifically, the umbilical artery and umbilical vein of the umbilical cord are removed simultaneously; uniformly shearing the peeled Wharton's jelly; resuspending the minced Wharton jelly with MSCs culture medium (DMEM-F12 phenol red-free culture medium containing 5% serum), inoculating to a culture dish, adding culture medium (DMEM-F12 phenol red-free culture medium containing 5% fetal calf serum), submerging Wharton jelly on the surface of the culture medium, and adding into different culture dishes to final concentrations of 1.0 × 107～1.0×109Fetal bovine serum exosomes of particles/mL, the culture dish was placed at 37 ℃ in 5% CO2And (5) standing and culturing in an incubator. The culture process 2-3d, the culture medium is changed once, and the serum exosome with the same concentration is added into the culture medium every time the culture medium is changed.
The method provided by the embodiment of the invention is suitable for primary culture and subculture, and specifically comprises the following steps: additionally adding bovine serum exosomes with preset concentration into primary culture of stem cells; or, adding bovine serum exosome with preset concentration in the subculture of the stem cells, preferably, carrying out subculture every 48-72 hours during the culture process.
Example 1
Simultaneously removing umbilical artery and umbilical vein of umbilical cord; uniformly shearing the peeled Wharton's jelly; resuspending the minced Wharton's jelly in MSCs medium (DMEM-F12 phenol red-free medium containing 5% serum), inoculating to a petri dish, adding culture medium (DMEM-F12 phenol red-free medium containing 5% fetal calf serum), and culturingImmersing the Huatong glue on the surface of the base liquid, and adding the Huatong glue into different culture dishes respectively to the final concentration of 1.0 multiplied by 107、1.0×108、1.0×109Fetal bovine serum exosomes of particles/mL, the culture dish was placed at 37 ℃ in 5% CO2And (5) standing and culturing in an incubator. The culture process is carried out for 48h, the culture medium is replaced once, and serum exosomes with the same concentration are added into the culture medium every time the culture medium is replaced. Wharton's jelly was cultured in conditioned medium containing bovine serum exosomes at different concentrations, the medium was changed every 48h, and the growth of primary cells after 13 days was as shown in FIG. 3.
From fig. 3, it can be seen that bovine serum exosomes were added to the stem cell culture solution, and the stem cells grew faster and in better condition. Adding the mixture to a final concentration of 1.0X 107When bovine serum exosomes are particles/mL, the migration rate of stem cells is accelerated, but the cell morphology is fine; adding the mixture to a final concentration of 1.0X 108particles/mL and 1.0X 109When the bovine serum exosomes of particles/mL are used, the stem cells have high migration rate, the optimal growth state, high cell saturation and better refractive index.
Example 2
Simultaneously removing umbilical artery and umbilical vein of umbilical cord; uniformly shearing the peeled Wharton's jelly; resuspending the minced Wharton jelly with MSCs culture medium (DMEM-F12 phenol red-free culture medium containing 5% serum), inoculating to a culture dish, adding culture medium (DMEM-F12 phenol red-free culture medium containing 5% fetal calf serum), submerging Wharton jelly on the surface of the culture medium, and adding into different culture dishes to final concentrations of 1.0 × 107、1.0×108、1.0×109Fetal bovine serum exosomes of particles/mL, the culture dish was placed at 37 ℃ in 5% CO2And (5) standing and culturing in an incubator. The culture process is carried out for 72h, the culture medium is replaced once, and serum exosomes with the same concentration are added into the culture medium every time the culture medium is replaced. The Wharton jelly was cultured in conditioned medium containing bovine serum exosomes at different concentrations, the medium was changed every 72h, and the growth of primary cells after 13 days was as shown in FIG. 4.
From fig. 4, it can be seen that bovine serum exosomes were added to the stem cell culture solution, and the stem cells grew faster and in better condition. Adding into the final productConcentration 1.0X 107When bovine serum exosomes are particles/mL, the migration rate of stem cells is accelerated, but the cell morphology is fine; adding the mixture to a final concentration of 1.0X 108particles/mL and 1.0X 109When the bovine serum exosomes of particles/mL are used, the stem cells have high migration rate, the optimal growth state, high cell saturation and better refractive index.
Example 3
Simultaneously removing umbilical artery and umbilical vein of umbilical cord; uniformly shearing the peeled Wharton's jelly; resuspending the minced Wharton jelly with MSCs culture medium (DMEM-F12 phenol red-free culture medium containing 5% serum), inoculating to a culture dish, adding culture medium (DMEM-F12 phenol red-free culture medium containing 5% fetal calf serum), submerging Wharton jelly on the surface of the culture medium, and adding into different culture dishes to final concentrations of 1.0 × 107、1.0×108、1.0×109Fetal bovine serum exosomes of particles/mL, the culture dish was placed at 37 ℃ in 5% CO2And (5) standing and culturing in an incubator. The culture process was carried out for 48h for one subculture, and the subculture medium was still a conditioned medium containing exosomes of different concentrations (each the same as the medium in the primary culture). The growth of the cells after 3 passages is shown in FIG. 5.
As can be seen from FIG. 5, in the case of subculture, a final concentration of 1.0X 10 was added to the primary medium and the subculture medium8particles/mL and 1.0X 109When the bovine serum exosomes of particles/mL are used, the stem cells are in the best growth state, the saturation is high, and the refractive index is better.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (10)
1. A method for culturing stem cells by using bovine serum exosomes is characterized by comprising the following steps: adding a predetermined amount of bovine serum into the stem cell culture medium, and additionally adding bovine serum exosomes with a predetermined concentration.
2. The method for culturing the bovine serum exosomes according to claim 1, wherein the adding amount of the bovine serum is 3-10% of the using amount of the stem cell culture medium; the concentration of the additionally added bovine serum exosomes in the stem cell culture medium was 1.0 × 108～1.0×109particals/mL。
3. The method for culturing the bovine serum exosomes according to claim 1, wherein the bovine serum exosomes are exosomes extracted by a kit.
4. The method of claim 1, wherein the bovine serum is fetal bovine serum and the bovine serum exosomes are fetal bovine serum exosomes.
5. Method of bovine serum exosomes for stem cell culture according to claim 1, characterized in that the stem cells are mesenchymal stem cells, preferably umbilical cord mesenchymal stem cells.
6. The method for culturing stem cells using bovine serum exosomes according to claim 1, wherein the stem cell culture is performed by a tissue culture method.
7. The method for culturing bovine serum exosomes according to claim 1, wherein bovine serum exosomes with preset concentration is additionally added in the primary culture of stem cells; or, additionally adding bovine serum exosome with preset concentration in the subculture of the stem cells.
8. The method of using bovine serum exosomes according to claim 1 for stem cell culture, comprising the steps of: inoculating the tissue block in a culture container, adding stem cell culture solution into the culture container, and adding additional bovine serum exosome into the culture containerThe concentration of the stem cell culture solution is 1.0 x 108～1.0×109particles/mL; then the culture container is placed in an incubator for culture, and bovine serum exosome with the same concentration is additionally added into the culture solution every time the culture solution is replaced in the culture process.
9. The method of culturing bovine serum exosomes according to claim 8, wherein the tissue block is umbilical cord Wharton's jelly; the stem cell culture solution is DMEM/F12 phenol red-free culture solution containing 5% serum; the culture container is a culture dish, and the culture dish is placed at 37 ℃ and 5% CO2The culture box is used for static culture, the culture solution is replaced every 48 to 72 hours in the culture process, and bovine serum exosome with the same concentration is additionally added into the culture solution when the culture solution is replaced every time.
10. A method of bovine serum exosomes according to claim 8 or 9 for stem cell culture, wherein the culture process is sub-cultured every 48-72 hours.
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