Three-dimensional culture system and culture method of sheep embryonic stem cells
By using a three-dimensional culture system of polylactic acid-glycolic acid copolymer nanofibers and a specific culture medium, the problems of low expansion efficiency and poor directional differentiation induction of sheep embryonic stem cells were solved, achieving efficient cell expansion and differentiation.
Patent Information
- Application Number
- CN202511143839.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-15
- Publication Date
- 2025-11-18
AI Technical Summary
Traditional two-dimensional culture methods result in low expansion efficiency of sheep embryonic stem cells and poor induction of directed differentiation, making it difficult to meet the needs of practical applications.
Biodegradable polylactic acid-glycolic acid copolymer nanofibers were used as a three-dimensional culture scaffold, combined with specific concentrations of DMEM/F12 medium and growth factors such as leukemia inhibitory factor and bone morphogenetic protein-4 for three-dimensional culture. The growth and differentiation of cells in the three-dimensional environment were controlled by adding differentiation-inducing factors such as dexamethasone and sodium β-glycerophosphate.
It significantly improves the expansion efficiency of sheep embryonic stem cells, increasing the expansion rate by 3-5 times in 7 days, while greatly enhancing the directional differentiation induction effect and significantly improving the differentiation rate.
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of animal cell engineering, and particularly relates to a three-dimensional culture system and culture method of sheep embryonic stem cells. BACKGROUND
[0002] Sheep embryonic stem cells have the potential of self-renewal and multi-directional differentiation, and have important application value in livestock breeding, animal disease model construction and tissue engineering research. The traditional two-dimensional culture method is to culture sheep embryonic stem cells on a flat carrier such as a culture dish, but this culture method has many disadvantages.
[0003] The two-dimensional culture environment is quite different from the growth microenvironment of sheep embryonic stem cells in vivo, and cannot simulate the three-dimensional spatial structure and cell-cell interaction of cells in vivo, resulting in low cell expansion efficiency. It usually takes a long time to obtain a sufficient number of cells, which is difficult to meet the needs of practical applications. At the same time, under the two-dimensional culture condition, the differentiation direction of cells is difficult to control accurately, the differentiation induction effect is poor, the differentiation rate is low, and the function of differentiated cells may also be affected.
[0004] Therefore, it has become an urgent problem in the field to develop a culture system and method that can improve the expansion efficiency and directional differentiation induction effect of sheep embryonic stem cells. SUMMARY
[0005] The present application aims to provide a three-dimensional culture system and culture method of sheep embryonic stem cells. The three-dimensional culture system can effectively solve the problems of low expansion efficiency and poor directional differentiation induction effect of traditional two-dimensional culture of sheep embryonic stem cells.
[0006] A three-dimensional culture system of sheep embryonic stem cells comprises a three-dimensional culture scaffold and a culture medium. The material of the three-dimensional culture scaffold is a degradable polylactic acid-glycolic acid copolymer nanofiber material, the pore size of which is 50-150 nm, and the fiber spacing is 20-80 μm. The culture medium comprises a DMEM / F12 basic medium and growth factors, and the growth factors comprise leukemia inhibitory factor and bone morphogenetic protein-4. In the culture medium, the concentration of leukemia inhibitory factor is 1000-2000 IU / mL, and the concentration of bone morphogenetic protein-4 is 10-50 ng / mL.
[0007] Alternatively, in the degradable polylactic acid-glycolic acid copolymer nanofiber material, the molar ratio of lactic acid to glycolic acid is 7:3.
[0008] Alternatively, the culture medium comprises 10% fetal bovine serum, 100 units / mL penicillin and 100 μg / mL streptomycin by volume percentage of the culture medium.
[0009] Optionally, the culture medium further comprises 10 -8 -10 -7 mol / L dexamethasone and 5-10 mmol / L sodium beta-glycerophosphate.
[0010] Optionally, the culture medium further comprises 0.5-1 mmol / L 3-isobutyl-1-methylxanthine and 5-10 μg / mL insulin.
[0011] Optionally, the ovine embryonic stem cells are derived from ovine embryos at a gestation period of 40-50 days.
[0012] A culture method of the three-dimensional culture system described above, comprising the following steps:
[0013] S101: inoculating the ovine embryonic stem cells on the three-dimensional culture scaffold at an inoculation density of 1×10 4 -5×10 4 / cm 2 ;
[0014] S102: placing the three-dimensional culture scaffold after inoculation in the culture medium and culturing in a 37℃, 5% CO2 incubator for 3-5 days for proliferation;
[0015] S103: after proliferation, adding corresponding differentiation inducers in the culture medium according to the required differentiation direction and continuing to culture in a 37℃, 5% CO2 incubator.
[0016] Optionally, in S103, the differentiation inducers are 10 -8 -10 -7 mol / L dexamethasone and 5-10 mmol / L sodium beta-glycerophosphate, and the culture is continued in a 37℃, 5% CO2 incubator for 21-28 days.
[0017] Optionally, in S103, the differentiation inducers are 0.5-1 mmol / L 3-isobutyl-1-methylxanthine and 5-10 μg / mL insulin, and the culture is continued in a 37℃, 5% CO2 incubator for 14-21 days.
[0018] Optionally, an inverted microscope is used to regularly observe the growth and differentiation of the cells in the three-dimensional culture scaffold.
[0019] Advantages
[0020] The three-dimensional culture system of the present application can significantly improve the expansion efficiency of ovine embryonic stem cells, with an expansion multiple of 3-5 times compared with two-dimensional culture in 7 days; and greatly improves the effect of directional differentiation induction. DETAILED DESCRIPTION
[0021] An embodiment of the present application provides a three-dimensional culture system of sheep embryonic stem cells, which comprises a three-dimensional culture support and a culture medium; the material of the three-dimensional culture support is degradable polylactic acid-glycolic acid copolymer (PLGA) nanofiber material, the pore size of which is 50-150 nm, and the fiber spacing is 20-80 μm; the culture medium comprises DMEM / F12 basic medium and growth factors, and the growth factors comprise leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP-4); in the culture medium, the concentration of the leukemia inhibitory factor is 1000-2000 IU / mL, and the concentration of the bone morphogenetic protein-4 is 10-50 ng / mL. The three-dimensional culture system can significantly improve the amplification efficiency of the sheep embryonic stem cells, and the amplification multiple in 7 days is increased by 3-5 times compared with two-dimensional culture; and the effect of directional differentiation induction is greatly improved.
[0022] In some embodiments, in the degradable polylactic acid-glycolic acid copolymer nanofiber material, the molar ratio of lactic acid and glycolic acid is 7:3. The ratio of lactic acid and glycolic acid can make the three-dimensional culture support have a more suitable degradation rate and mechanical property, and be more conducive to the adhesion, growth and proliferation of the sheep embryonic stem cells, and improve the stability and efficiency of cell culture.
[0023] In some embodiments, the culture medium comprises 10% fetal bovine serum, 100 units / mL penicillin and 100 μg / mL streptomycin by volume percentage of the culture medium. The fetal bovine serum can provide rich nutrients for cells, the penicillin and streptomycin mixture can effectively prevent bacterial contamination in the cell culture process, and guarantee the normal growth of cells, cooperate with the three-dimensional culture support, and jointly promote cell growth, and further improve the overall performance of the culture system.
[0024] In some embodiments, the culture medium further comprises 10 -8 -10 -7 mol / L dexamethasone and 5-10 mmol / L β-glycerophosphate sodium. At this time, the three-dimensional culture system can induce osteogenic differentiation.
[0025] In some embodiments, the culture medium further comprises 0.5-1 mmol / L 3-isobutyl-1-methyl xanthine and 5-10 μg / mL insulin. At this time, adipogenic differentiation can be induced.
[0026] In some embodiments, the sheep embryonic stem cells are derived from sheep embryos in a gestation period of 40-50 days. The stem cells with high activity are more likely to grow and differentiate in the suitable three-dimensional culture system, fully exert the advantages of the system, and improve the amplification and differentiation efficiency.
[0027] The application further provides a culture method of the three-dimensional culture system of the sheep embryonic stem cell.
[0028] S101: inoculate the sheep embryonic stem cell on the three-dimensional culture support with a density of 1×10 4 -5×10 4 cm 2 -2 ;
[0029] S102: place the inoculated three-dimensional culture support in a culture medium and culture in a 37℃, 5% CO2 incubator for 3-5 days for proliferation.
[0030] S103: after the proliferation, add a corresponding differentiation inducer in the culture medium according to the required differentiation direction and continue to culture in the 37℃, 5% CO2 incubator.
[0031] Optionally, in S103, the differentiation inducer is 10 -8 -10 -7 mol / L dexamethasone and 5-10 mmol / L sodium beta-glycerophosphate, and the culture in the 37℃, 5% CO2 incubator is continued for 21-28 days.
[0032] Optionally, in S103, the differentiation inducer is 0.5-1 mmol / L 3-isobutyl-1-methyl xanthine and 5-10 μg / mL insulin, and the culture in the 37℃, 5% CO2 incubator is continued for 14-21 days.
[0033] Optionally, an inverted microscope is used to regularly observe the growth and differentiation of the cell in the three-dimensional culture support.
[0034] Optionally, S101 further comprises: selecting a sheep embryo with a gestation period of 40-50 days, obtaining the sheep embryonic stem cell by using a tissue block culture method or an enzyme digestion method, and purifying and identifying the sheep embryonic stem cell. Further, the identification of the sheep embryonic stem cell is performed by using flow cytometry to detect the expression of stem cell surface markers such as Oct4 and Nanog.
[0035] Embodiment 1
[0036] The culture method of the three-dimensional culture system of the sheep embryonic stem cell in this embodiment is as follows:
[0037] S01: Obtaining of sheep embryonic stem cells: Healthy ewes were selected, and sheep embryos were obtained by surgery when the ewes were about 40 days pregnant. The embryo tissue was cut into small pieces of about 1 mm3, inoculated into culture dishes, and cultured in a 37°C, 5% CO2 incubator. After the cells migrated out of the tissue pieces, they were purified and cultured. Flow cytometry was used to detect the expression of stem cell surface markers Oct4 and Nanog. The results showed that the positive rate of Oct4 was 95%, and the positive rate of Nanog was 93%, indicating that the obtained cells were high-purity sheep embryonic stem cells.
[0038] S02: Preparation of three-dimensional culture scaffold: A PLGA three-dimensional culture scaffold was prepared using electrospinning technology, with a molar ratio of lactic acid to glycolic acid of 7:3, a pore size of 100 μm, and a fiber spacing of 50 μm.
[0039] S03: Preparation of culture medium: DMEM / F12 was used as the base medium, and 10% fetal bovine serum, 100 units / mL penicillin, 100 μg / mL streptomycin, 1500 IU / mL LIF, and 30 ng / mL BMP-4 were added. The adipogenic differentiation medium was prepared by adding 0.8 mmol / L 3-isobutyl-1-methyl xanthine, 8 μg / mL insulin, and 80 μmol / L indomethacin. The osteogenic differentiation medium was prepared by adding 5×10 -8 mol / L dexamethasone and 8 mmol / L β-glycerophosphate sodium.
[0040] S04: Cell inoculation and culture: The purified sheep embryonic stem cells were inoculated onto the three-dimensional culture scaffold at a density of 3×10 4 cells / cm2, and cultured in the initial expansion medium in a 37°C, 5% CO2 incubator. The medium was changed every 24 hours, and the cells were cultured for 4 days. Then, the adipogenic differentiation medium and the osteogenic differentiation medium were used, and the cells were cultured for 18 days and 25 days, respectively.
[0041] Test:
[0042] (1) Expansion ratio test: After 7 days of culture, the cells in the three-dimensional scaffold were collected, and the cell count method was used to calculate the number of cells. Compared with the initial inoculation number, the expansion ratio was obtained. The results showed that the expansion ratio was 6.2 times. The test method was trypsin digestion to collect cells, and a hemocytometer was used for counting.
[0043] (2) Adipogenic differentiation induction rate test: After the adipogenic differentiation culture ended, oil red O staining method was used to observe and count the proportion of positive cells (cells containing lipid droplets) under a microscope. The results showed that the adipogenic differentiation induction rate was 88%. The test method was the conventional oil red O staining procedure, and 5 random fields were counted.
[0044] (3) Osteogenic differentiation marker test: After the osteogenic differentiation culture, the content of osteocalcin in the cell culture solution was detected by ELISA method, and the alkaline phosphatase activity was detected by colorimetric method. The results showed that the content of osteocalcin was 150 ng / mL, and the alkaline phosphatase activity was 250 U / L. The test method was strictly operated according to the corresponding kit instructions.
[0045] (4) Calcium nodule formation test: After the osteogenic differentiation culture, the formation of calcium nodule was observed under a microscope by alizarin red staining method. The results showed that a large number of obvious calcium nodules were visible.
[0046] Comparative Example 1
[0047] In Comparative Example 1, the traditional two-dimensional culture method was used: the same sheep embryonic stem cells as in Example 1 were inoculated in a culture dish, and the conventional two-dimensional culture method was used, and the culture medium was the same as in Example 1.
[0048] Test:
[0049] (1) Expansion fold test: After 7 days of culture, the same method as in Example 1 was used for testing, and the results showed that the expansion fold was 1.8 times.
[0050] (2) Adipogenic differentiation induction rate test: The same adipogenic differentiation medium and test method as in Example 1 were used, and the results showed that the adipogenic differentiation induction rate was 52%.
[0051] (3) Osteogenic differentiation marker test: The same osteogenic differentiation medium and test method as in Example 1 were used, and the results showed that the content of osteocalcin was 65 ng / mL, and the alkaline phosphatase activity was 120 U / L.
[0052] (4) Calcium nodule formation test: The same test method as in Example 1 was used, and the results showed that only a small amount of scattered calcium nodules were present.
[0053] From Example 1 and Comparative Example 1, it can be seen that the culture method of the present application can significantly improve the expansion efficiency of sheep embryonic stem cells, and greatly improve the effect of directional differentiation induction.
[0054] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.
[0055] For those skilled in the art, according to the idea of the present application, there will be changes in specific embodiments and application scope. In summary, the content of the specification should not be understood as a limitation of the present application.
Claims
1. A three-dimensional culture system for sheep embryonic stem cells, characterized in that, The invention includes a three-dimensional culture scaffold and a culture medium. The three-dimensional culture scaffold is made of biodegradable polylactic acid-glycolic acid copolymer nanofiber material with a pore size of 50-150 nm and a fiber spacing of 20-80 μm. The culture medium includes DMEM / F12 basal medium and growth factors, including leukemia inhibitory factor and bone morphogenetic protein-4. In the culture medium, the concentration of leukemia inhibitory factor is 1000-2000 IU / mL, and the concentration of bone morphogenetic protein-4 is 10-50 ng / mL.
2. The three-dimensional culture system for sheep embryonic stem cells as described in claim 1, characterized in that, In the biodegradable polylactic acid-glycolic acid copolymer nanofiber material, the molar ratio of lactic acid to glycolic acid is 7:
3.
3. The three-dimensional culture system of sheep embryonic stem cells as described in claim 1, characterized in that, The culture medium comprises 10% fetal bovine serum, 100 units / mL of penicillin, and 100 μg / mL of streptomycin, by volume.
4. The three-dimensional culture system of sheep embryonic stem cells as described in any one of claims 1-3, characterized in that, The culture medium also includes 10 -8 -10 -7 mol / L dexamethasone and 5-10 mmol / L sodium β-glycerophosphate.
5. The three-dimensional culture system of sheep embryonic stem cells as described in any one of claims 1-3, characterized in that, The culture medium also includes 0.5-1 mmol / L of 3-isobutyl-1-methylxanthine and 5-10 μg / mL of insulin.
6. The three-dimensional culture system of sheep embryonic stem cells as described in any one of claims 1-3, characterized in that, The sheep embryonic stem cells are derived from sheep embryos that are 40-50 days old.
7. A method for culturing sheep embryonic stem cells in a three-dimensional culture system according to any one of claims 1-6, characterized in that, Includes the following steps: S101: The sheep embryonic stem cells are seeded onto the three-dimensional culture scaffold at a density of 1×10⁻⁶. 4 -5×10 4 pcs / cm 2 ; S102: Place the inoculated three-dimensional culture scaffold in the culture medium and culture it in an incubator at 37°C and 5% CO2 for 3-5 days for proliferation; S103: After proliferation, according to the desired differentiation direction, add the corresponding differentiation inducing factor to the culture medium and continue to culture in an incubator at 37°C and 5% CO2.
8. The method for culturing sheep embryonic stem cells in a three-dimensional culture system as described in claim 7, characterized in that, In S103, the differentiation-inducing factor is 10. -8 -10 -7 The solution was incubated with dexamethasone at 0.1 mol / L and sodium β-glycerophosphate at 5-10 mmol / L for 21-28 days in an incubator at 37°C and 5% CO2.
9. The method for culturing sheep embryonic stem cells in a three-dimensional culture system as described in claim 7, characterized in that, In S103, the differentiation-inducing factors are 0.5-1 mmol / L of 3-isobutyl-1-methylxanthine and 5-10 μg / mL of insulin, and the mixture is cultured in an incubator at 37°C and 5% CO2 for 14-21 days.
10. The method for culturing sheep embryonic stem cells in a three-dimensional culture system as described in any one of claims 7-9, characterized in that, The growth and differentiation of cells within the three-dimensional culture scaffold were observed periodically using an inverted microscope.