Efficient serum-free culture medium for in-vitro culture of poultry primordial germ cells as well as preparation method and application of efficient serum-free culture medium

Abstract

The invention provides an efficient serum-free culture medium for in-vitro culture of poultry primordial germ cells as well as a preparation method and application of the efficient serum-free culture medium, and belongs to the technical field of biology. The culture medium comprises a calcium-free Knockout DMEM (Dulbecco Modified Eagle Medium), glucose, a B-27 supplement, a glutamine substitute, non-essential amino acid, an EmryoMax nucleoside mixture, beta-mercaptoethanol, ovalbumin, sodium pyruvate, CaCl2, heparin sodium, Activin A, FGF2 (Fibroblast Growth Factor 2), ovotransferrin, vitamin B12 and folic acid. According to the method, serum does not need to be used, the culture process is remarkably simplified, the pollution risk is reduced, cell apoptosis is synergistically inhibited by optimizing a culture medium formula and introducing a multi-channel inhibitor combination, the cell proliferation rate is remarkably increased, and the pluripotency of primordial germ cells is maintained.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture, and particularly relates to a high-efficiency serum-free medium for in vitro culture of avian primordial germ cells, a preparation method thereof, and an application thereof. Background Art

[0002] Avian primordial germ cells (PGCs), as precursor cells of germ stem cells, have pluripotency and reproductive characteristics, and are important tools for constructing transgenic avian models and protecting species genetic resources. In the early stage of avian embryo development, PGCs originate from the germinal crescent area in the extra-embryonic region, migrate to the gonad through the blood circulation, and differentiate into gametes. Their unique migration characteristics provide an ideal way for transgenic technology and germplasm resource preservation. However, the in vitro culture of PGCs still faces many challenges.

[0003] In the prior art, the in vitro culture system of avian PGCs mainly relies on a medium containing fetal bovine serum (FBS). For example, based on KO-DMEM, FBS, chicken serum, growth factors (such as bFGF, LIF), and various auxiliary components are added. Such a system has significant defects: the serum components are complex and there are large differences between batches, which are prone to introducing exogenous contamination (such as viruses, mycoplasmas). At the same time, the proteins in the serum will interfere with the transgenic operation mediated by cationic liposomes, resulting in low transfection efficiency. In addition, when cultured using the traditional culture system, the culture period is long, and after long-term culture, the expression of pluripotency markers such as DAZL and CVH in PGCs decreases, and the migration ability weakens.

[0004] In recent years, the development of serum-free media has become a research hotspot. However, existing serum-free systems mostly rely on single signaling pathway inhibitors (such as Rock inhibitors) or cytokines, and it is difficult to effectively inhibit cell apoptosis and differentiation, resulting in insufficient proliferation ability and short survival time of PGCs. For example, some studies have tried to add insulin, transferrin, etc. to replace serum, but still cannot fully simulate the supporting effect of serum, and the cost remains high. In addition, the synergistic effect of the FGF2 concentration (4 - 10 ng / mL) and Activin A (20 - 25 ng / mL) in the existing formulations is insufficient, resulting in an imbalance between proliferation and differentiation, which is not conducive to large-scale production. Therefore, it is imperative to develop a high-efficiency serum-free medium for in vitro culture of avian primordial germ cells. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a high-efficiency serum-free medium for in vitro culture of avian primordial germ cells, a preparation method thereof, and an application thereof. The present invention optimizes the medium formula, introduces a combination of multi-pathway inhibitors, synergistically inhibits cell apoptosis, promotes cell proliferation, and maintains the pluripotency of primordial germ cells.

[0006] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] The present invention provides a high-efficiency serum-free medium for in vitro culture of avian primordial germ cells. The medium uses calcium-free Knockout DMEM as the basal medium and further includes components with the following final concentrations: 10-15 mM glucose, 0.5-1.5× B-27 supplement, 1-3 mM glutamine substitute, 0.5-1.5× non-essential amino acids, 0.5-1.5× EmbryoMax nucleoside mixture, 0.01-0.2 mM β-mercaptoethanol, 0.1%-0.3% ovalbumin, 1-1.5 mM sodium pyruvate, 0.05-0.2 mM CaCl2, 0.001%-0.02% sodium heparin, 15-35 ng / mL Activin A, 30-50 ng / mL FGF2, 5-10 μg / mL ovotransferrin, 1-10 μg / L vitamin B12, 1-10 μg / L folic acid.

[0008] Preferably, the medium uses calcium-free Knockout DMEM as the basal medium and further includes components with the following final concentrations: 11-14 mM glucose, 0.7-1.2× B-27 supplement, 1.5-2.5 mM glutamine substitute, 0.7-1.2× non-essential amino acids, 0.7-1.2× EmbryoMax nucleoside mixture, 0.05-0.15 mM β-mercaptoethanol, 0.15%-0.25% ovalbumin, 1.1-1.3 mM sodium pyruvate, 0.1-0.17 mM CaCl2, 0.005%-0.015% sodium heparin, 20-30 ng / mL Activin A, 35-45 ng / mL FGF2, 7-9 μg / mL ovotransferrin, 3-8 μg / L vitamin B12, 3-8 μg / L folic acid.

[0009] More preferably, the medium uses calcium-free Knockout DMEM as the basal medium and further includes components with the following final concentrations: 12 mM glucose, 1× B-27 supplement, 2.0 mM glutamine substitute, 1× non-essential amino acids, 1× EmbryoMax nucleoside mixture, 0.1 mM β-mercaptoethanol, 0.2% ovalbumin, 1.2 mM sodium pyruvate, 0.15 mM CaCl2, 0.01% sodium heparin, 25 ng / mL Activin A, 40 ng / mL FGF2, 8 μg / mL ovotransferrin, 5 μg / L vitamin B12, 4 μg / L folic acid.

[0010] The present invention provides a preparation method of the above-mentioned high-efficiency serum-free medium, including the following steps:

[0011] (1) Glucose and CaCl₂ were added to calcium-free Knockout DMEM medium, and the osmotic pressure was adjusted to 250 - 260 mOsm / kg to obtain Solution 1;

[0012] (2) B-27 supplement, glutamine substitute, non-essential amino acids, EmbryoMax nucleoside mixture, β-mercaptoethanol, ovalbumin, sodium pyruvate, sodium heparin, vitamin B₁₂, and folic acid were added to Solution 1 and mixed evenly to obtain Solution 2;

[0013] (3) Pre-dissolved ovotransferrin, Activin A, and FGF2 were added to Solution 2, and the mixture was filtered and sterilized to obtain a highly efficient serum-free medium.

[0014] Preferably, the solution used for the pre-dissolved ovotransferrin in step (3) is PBS with a pH of 7.0 - 7.6.

[0015] Preferably, the concentration of the pre-dissolved ovotransferrin is 0.05 - 0.2 mg / mL.

[0016] Preferably, before adding Activin A and FGF2 to Solution 2, it also includes dissolving Activin A and FGF2. The concentration of Activin A after dissolution is 0.5 - 2 μg / mL, and the concentration of FGF2 after dissolution is 0.5 - 2 μg / mL.

[0017] Preferably, the solution used for dissolution is calcium-free Knockout DMEM medium.

[0018] The present invention also provides the application of the above-mentioned highly efficient serum-free medium or the highly efficient serum-free medium obtained according to the above-mentioned preparation method in promoting the proliferation of avian primordial germ cells.

[0019] Preferably, the avian is a chicken.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a highly efficient serum-free medium for in vitro culture of avian primordial germ cells, its preparation method and application. In the present invention, serum is not required, which significantly simplifies the culture process and reduces the risk of contamination. By optimizing the medium formula and introducing a multi-pathway inhibitor combination to synergistically inhibit apoptosis, the present invention significantly improves the cell proliferation rate and maintains the pluripotency of primordial germ cells. It provides a reliable technical basis for the in vitro culture of avian primordial germ cells and is of great significance for the protection of endangered avian species. Description of the Drawings

[0021] Figure 1 Cell viability of chicken primordial germ cells cultured in different media. Detailed implementation mode

[0022] The present invention provides a highly efficient serum-free medium for in vitro culture of avian primordial germ cells. The medium uses calcium-free Knockout DMEM as the basal medium and further includes components with the following final concentrations: 10-15 mM glucose, preferably 11-14 mM, and more preferably 12 mM;

[0023] 0.5-1.5× B-27 supplement, preferably 0.7-1.2×, and more preferably 1×;

[0024] 1-3 mM glutamine substitute, preferably 1.5-2.5 mM, and more preferably 2.0 mM;

[0025] 0.5-1.5× non-essential amino acids, preferably 0.7-1.2×, and more preferably 1×;

[0026] 0.5-1.5× EmbryoMax nucleoside mixture, preferably 0.7-1.2×, and more preferably 1×;

[0027] 0.01-0.2 mM β-mercaptoethanol, preferably 0.05-0.15 mM, and more preferably 0.1 mM;

[0028] 0.1%-0.3% ovalbumin, preferably 0.15%-0.25%, and more preferably 0.2%;

[0029] 1-1.5 mM sodium pyruvate, preferably 1.1-1.3 mM, and more preferably 1.2 mM;

[0030] 0.05-0.2 mM CaCl2, preferably 0.1-0.17 mM, and more preferably 0.15 mM;

[0031] 0.001%-0.02% sodium heparin, preferably 0.005%-0.015%, and more preferably 0.01%;

[0032] 15-35 ng / mL Activin A, preferably 20-30 ng / mL, and more preferably 25 ng / mL;

[0033] 30-50 ng / mL FGF2, preferably 35-45 ng / mL, and more preferably 40 ng / mL;

[0034] 5-10 μg / mL ovotransferrin, preferably 7-9 μg / mL, and more preferably 8 μg / mL;

[0035] 1 - 10 μg / L of vitamin B12, preferably 3 - 8 μg / L, more preferably 5 μg / L;

[0036] 1 - 10 μg / L of folic acid, preferably 3 - 8 μg / L, more preferably 4 μg / L.

[0037] In the present invention, calcium - free Knockout DMEM as the basal medium provides a flexible and controllable culture environment for avian primordial germ cells, and can precisely regulate the calcium level during the culture of avian primordial germ cells;

[0038] Glucose is the main energy source, providing energy for avian primordial germ cells through glycolysis and oxidative phosphorylation;

[0039] B - 27 supplement provides antioxidants, hormones and trace elements for avian primordial germ cells, supporting cell survival and differentiation;

[0040] Glutamine substitute provides a nitrogen source for avian primordial germ cells and participates in energy metabolism. Since the glutamine substitute is a stable precursor of glutamine, it can effectively avoid the problem of easy degradation of traditional glutamine;

[0041] The addition of non - essential amino acids can reduce the metabolic burden of avian primordial germ cells and promote proliferation;

[0042] EmbryoMax nucleoside mixture can provide purine and pyrimidine nucleosides, promote DNA / RNA synthesis, and support the growth of avian primordial germ cells;

[0043] β - mercaptoethanol can maintain the reduced state of glutathione in avian primordial germ cells and prevent oxidative stress;

[0044] Ovalbumin, as a carrier protein, transports lipophilic substances and mimics the function of serum;

[0045] Sodium pyruvate, as an alternative energy source, can enter the tricarboxylic acid cycle to supply energy, and can maintain the survival of avian primordial germ cells especially when glucose is insufficient;

[0046] CaCl2 provides calcium ions, precisely regulates the calcium level during the culture of avian primordial germ cells, and participates in signal transduction, cell adhesion and enzyme activity regulation;

[0047] Heparin sodium can prevent the medium from coagulating and promote the binding of avian primordial germ cells to growth factors;

[0048] Activin A is a member of the TGF - β superfamily, promoting mesoderm differentiation and maintaining the pluripotency of avian primordial germ cells;

[0049] FGF2 can stimulate cell proliferation and migration, and maintain the undifferentiated state of avian primordial germ cells;

[0050] Ovotransferrin can bind and transport iron ions to avoid the toxicity of free iron and provide iron elements for avian primordial germ cells;

[0051] Vitamin B12 participates in the methylation reaction, promoting DNA synthesis and erythropoiesis;

[0052] Folic acid participates in nucleotide synthesis, amino acid metabolism, and DNA methylation.

[0053] In the present invention, all components jointly maintain the nutritional, energy, and signaling environment required for the growth, proliferation, and differentiation of avian primordial germ cells.

[0054] The present invention provides a method for preparing the described high-efficiency serum-free medium, comprising the following steps:

[0055] (1) Glucose and CaCl2 are added to a calcium-free Knockout DMEM medium, and the osmotic pressure is adjusted to 250 - 260 mOsm / kg to obtain Solution 1;

[0056] (2) A B-27 supplement, a glutamine substitute, non-essential amino acids, an EmbryoMax nucleoside mixture, β-mercaptoethanol, ovalbumin, sodium pyruvate, sodium heparin, vitamin B12, and folic acid are added to Solution 1 and mixed evenly to obtain Solution 2;

[0057] (3) Pre-dissolved ovotransferrin, Activin A, and FGF2 are added to Solution 2, and the mixture is filtered and sterilized to obtain the high-efficiency serum-free medium.

[0058] In the present invention, the solution used for the pre-dissolved ovotransferrin in step (3) is PBS with a pH of 7.0 - 7.6, preferably 7.2 - 7.5, and more preferably 7.4; the concentration of the pre-dissolved ovotransferrin is 0.05 - 0.2 mg / mL, preferably 0.08 - 0.15 mg / mL, and more preferably 0.1 mg / mL.

[0059] In the present invention, before adding Activin A and FGF2 to Solution 2, it further includes dissolving Activin A and FGF2. The Activin A is dissolved by using calcium-free Knockout DMEM medium to dissolve Activin A, obtaining an Activin A solution with a concentration of 0.5 - 2 μg / mL. The concentration of Activin A is preferably 0.7 - 1.5 μg / mL, and more preferably 1 μg / mL. The FGF2 is dissolved by using calcium-free Knockout DMEM medium to dissolve FGF2, obtaining an FGF2 solution with a concentration of 0.5 - 2 μg / mL. The concentration of the FGF2 solution is preferably 0.7 - 1.5 μg / mL, and more preferably 1 μg / mL.

[0060] In the present invention, due to the low contents of CaCl2, β-mercaptoethanol, heparin sodium, vitamin B12, and folic acid, to ensure the accuracy of the concentration, they need to be dissolved and then added to the calcium-free Knockout DMEM medium. Among them, the prepared concentration of CaCl2 is 1 M, the concentration of β-mercaptoethanol is 1 M, the concentration of heparin sodium is 10 mg / mL, the concentration of vitamin B12 is 5 mg / L, and the concentration of folic acid is 4 mg / L.

[0061] In the present invention, the filtration and sterilization use a filter membrane with a pore size of 0.1 - 0.45 μm, preferably 0.22 μm.

[0062] The present invention also provides the application of the described high-efficiency serum-free medium or the high-efficiency serum-free medium obtained according to the described preparation method in promoting the proliferation of avian primordial germ cells.

[0063] In the present invention, the avian is a chicken.

[0064] The following combines examples to elaborate in detail on the technical solutions provided by the present invention, but they cannot be understood as limiting the protection scope of the present invention.

[0065] Preparation of Activin A solution: Dilute the purchased Activin A (10 μg) from Wokaway (Beijing) Biotechnology Co., Ltd. with 10 mL of calcium-free Knockout DMEM medium to obtain an Activin A solution with a concentration of 1 μg / mL, and store it at -80 °C for standby.

[0066] Preparation of FGF2 solution: Dilute the purchased FGF2 (10 μg) from Andy Biotechnology (Shanghai) Co., Ltd. with 10 mL of calcium-free Knockout DMEM medium to obtain an FGF2 solution with a concentration of 1 μg / mL, and store it at -80 °C for standby.

[0067] Preparation of ovotransferrin solution: Dissolve the ovotransferrin (5 mg) purchased from MedChemExpress in PBS with a pH of 7.4 to a concentration of 0.1 mg / mL for standby.

[0068] Preparation of 1 M CaCl₂ solution: Weigh 11.10 g of anhydrous CaCl₂ and dissolve it in 100 mL of sterile water to obtain a 1 M CaCl₂ solution.

[0069] Preparation of 1 M β-mercaptoethanol solution: Measure 7 μL of β-mercaptoethanol and dissolve it in 100 mL of sterile water to obtain a 1 M β-mercaptoethanol solution.

[0070] Preparation of 10 mg / mL heparin sodium solution: Dissolve the heparin sodium (10 mg) purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. in 1 mL of sterile water to obtain a 10 mg / mL heparin sodium solution.

[0071] Preparation of 5 mg / L vitamin B₁₂ solution: Weigh 0.5 mg of vitamin B₁₂ and dissolve it in 100 mL of sterile water to obtain a 5 mg / L vitamin B₁₂ solution.

[0072] Preparation of 4 mg / L folic acid solution: Weigh 0.4 mg of folic acid and dissolve it in 100 mL of sterile water to obtain a 4 mg / L folic acid solution.

[0073] Calcium-free Knockout DMEM medium, B-27 supplement, glutamine substitute, and non-essential amino acids were purchased from Thermo Fisher Scientific; EmbryoMax nucleoside mixture was purchased from MilliporeSigma; glucose, CaCl₂, β-mercaptoethanol, ovalbumin, sodium pyruvate (100 mM), vitamin B₁₂, and folic acid were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0074] Activin A, FGF2, ovotransferrin, CaCl₂, heparin sodium, vitamin B₁₂, and folic acid used in the subsequent examples were all obtained in the above manner.

[0075] Example 1

[0076] Preparation of a highly efficient serum-free medium for in vitro culture of avian primordial germ cells

[0077] Add 50 mL of calcium-free Knockout DMEM medium, 0.216 g of glucose, and 15 μL of CaCl₂ into a 100 mL centrifuge tube. Measure the current osmotic pressure using an osmometer. After adjusting the osmotic pressure to 250 - 260 mOsm / kg with calcium-free Knockout DMEM medium, add 1 mL of B-27 supplement, 1 mL of glutamine substitute, 1 mL of non-essential amino acids, 1 mL of EmbryoMax nucleoside mixture, 10 μL of β-mercaptoethanol, 0.2 g of ovalbumin, 1.2 mL of sodium pyruvate, 1 mL of sodium heparin, 100 μL of vitamin B₁₂, 100 μL of folic acid, 8 mL of ovotransferrin, 2.5 mL of Activin A, and 4 mL of FGF2. Make up the volume to 100 mL with calcium-free Knockout DMEM medium. Filter and sterilize the prepared medium through a 0.22 μm filter membrane to obtain a highly efficient serum-free medium, and store it sealed in a 4°C refrigerator.

[0078] Example 2

[0079] Preparation of a Highly Efficient Serum-Free Medium for in Vitro Culture of Avian Primordial Germ Cells

[0080] Add 50 mL of calcium-free Knockout DMEM medium, 0.180 g of glucose, and 5 μL of CaCl₂ into a 100 mL centrifuge tube. Measure the current osmotic pressure using an osmometer. After adjusting the osmotic pressure to 250 - 260 mOsm / kg with calcium-free Knockout DMEM medium, add 0.5 mL of B-27 supplement, 0.5 mL of glutamine substitute, 0.5 mL of non-essential amino acids, 0.5 mL of EmbryoMax nucleoside mixture, 1 μL of β-mercaptoethanol, 0.1 g of ovalbumin, 1 mL of sodium pyruvate, 0.1 mL of sodium heparin, 20 μL of vitamin B₁₂, 25 μL of folic acid, 5 mL of ovotransferrin, 1.5 mL of Activin A, and 3 mL of FGF2. Make up the volume to 100 mL with calcium-free Knockout DMEM medium. Filter and sterilize the prepared medium through a 0.22 μm filter membrane to obtain a highly efficient serum-free medium, and store it sealed in a 4°C refrigerator.

[0081] Example 3

[0082] Preparation of a Highly Efficient Serum-Free Medium for in Vitro Culture of Avian Primordial Germ Cells

[0083] Add 50 mL of calcium-free Knockout DMEM medium, 0.270 g of glucose, and 20 μL of CaCl2 into a 100 mL centrifuge tube. Measure the current osmotic pressure using an osmometer. After adjusting the osmotic pressure to 250 - 260 mOsm / kg with calcium-free Knockout DMEM medium, add 1.5 mL of B-27 supplement, 1.5 mL of glutamine substitute, 1.5 mL of non-essential amino acids, 1.5 mL of EmbryoMax nucleoside mixture, 20 μL of β-mercaptoethanol, 0.3 g of ovalbumin, 1.5 mL of sodium pyruvate, 2 mL of heparin sodium (prepared from 2 vials of 10 mg heparin sodium), 200 μL of vitamin B12, 250 μL of folic acid, 10 mL of ovotransferrin, 3.5 mL of Activin A, and 5 mL of FGF2. Make up the volume to 100 mL with calcium-free Knockout DMEM medium. Filter and sterilize the prepared medium through a 0.22 μm filter membrane to obtain a high-efficiency serum-free medium, and store it sealed in a 4°C refrigerator.

[0084] Control Example 1

[0085] Preparation of a High-Efficiency Serum-Free Medium for in Vitro Culture of Avian Primordial Germ Cells

[0086] Add 50 mL of calcium-free Knockout DMEM medium, 0.090 g of glucose, and 3 μL of CaCl2 into a 100 mL centrifuge tube. Measure the current osmotic pressure using an osmometer. After adjusting the osmotic pressure to 250 - 260 mOsm / kg with calcium-free Knockout DMEM medium, add 0.3 mL of B-27 supplement, 0.25 mL of glutamine substitute, 0.3 mL of non-essential amino acids, 0.3 mL of EmbryoMax nucleoside mixture, 1 μL of β-mercaptoethanol, 0.05 g of ovalbumin, 0.5 mL of sodium pyruvate, 0.05 mL of heparin sodium, 10 μL of vitamin B12, 13 μL of folic acid, 2.5 mL of ovotransferrin, 1 mL of Activin A, and 2 mL of FGF2. Make up the volume to 100 mL with calcium-free Knockout DMEM medium. Filter and sterilize the prepared medium through a 0.22 μm filter membrane to obtain a high-efficiency serum-free medium, and store it sealed in a 4°C refrigerator.

[0087] Control Example 2

[0088] Preparation of a High-Efficiency Serum-Free Medium for in Vitro Culture of Avian Primordial Germ Cells

[0089] Add 50 mL of calcium-free Knockout DMEM medium, 0.360 g of glucose, and 30 μL of CaCl2 into a 100 mL centrifuge tube. Measure the current osmotic pressure using an osmometer. After adjusting the osmotic pressure to 250 - 260 mOsm / kg with calcium-free Knockout DMEM medium, add 2 mL of B-27 supplement, 2 mL of glutamine substitute, 2 mL of non-essential amino acids, 2 mL of EmbryoMax nucleoside mixture, 30 μL of β-mercaptoethanol, 0.4 g of ovalbumin, 2 mL of sodium pyruvate, 3 mL of heparin sodium (prepared from 3 vials of 10 mg heparin sodium), 300 μL of vitamin B12, 300 μL of folic acid, 15 mL of ovotransferrin, 4 mL of Activin A, and 6 mL of FGF2. Make up the volume to 100 mL with calcium-free Knockout DMEM medium. Filter and sterilize the prepared medium through a 0.22 μm filter membrane to obtain a high-efficiency serum-free medium, and store it sealed in a 4°C refrigerator.

[0090] Comparative Example 3

[0091] Prepare a high-efficiency serum-free medium for in vitro culture of avian primordial germ cells in the same manner as in Example 1, except that the medium does not contain ovotransferrin, vitamin B12, and folic acid, but adds chicken serum with a final concentration of 0.2%.

[0092] Comparative Example 4

[0093] Prepare a high-efficiency serum-free medium for in vitro culture of avian primordial germ cells in the same manner as in Example 1, except that the medium does not contain ovotransferrin, but adds chicken serum with a final concentration of 0.2%.

[0094] Comparative Example 5

[0095] Prepare a high-efficiency serum-free medium for in vitro culture of avian primordial germ cells in the same manner as in Example 1, except that the medium does not contain ovotransferrin and vitamin B12, but adds chicken serum with a final concentration of 0.2%.

[0096] Comparative Example 6

[0097] Prepare a high-efficiency serum-free medium for in vitro culture of avian primordial germ cells in the same manner as in Example 1, except that the medium does not contain ovotransferrin and folic acid, but adds chicken serum with a final concentration of 0.2%.

[0098] Comparative Example 7

[0099] Prepare a high-efficiency serum-free medium for in vitro culture of avian primordial germ cells in the same manner as in Example 1, except that vitamin B12 in the medium is replaced with vitamin C.

[0100] Comparative Example 8

[0101] The high-efficiency serum-free medium for in vitro culture of avian primordial germ cells was prepared in the same manner as in Example 1, except that the medium did not contain vitamin B12 and folic acid.

[0102] Comparative Example 9

[0103] The high-efficiency serum-free medium for in vitro culture of avian primordial germ cells was prepared in the same manner as in Example 1, except that the medium did not contain ovotransferrin and folic acid.

[0104] Comparative Example 10

[0105] The high-efficiency serum-free medium for in vitro culture of avian primordial germ cells was prepared in the same manner as in Example 1, except that the medium did not contain ovotransferrin and vitamin B12.

[0106] Comparative Example 11

[0107] The high-efficiency serum-free medium for in vitro culture of avian primordial germ cells was prepared in the same manner as in Example 1, except that vitamin B12 in the medium was replaced with mecobalamin.

[0108] Comparative Example 12

[0109] The high-efficiency serum-free medium for in vitro culture of avian primordial germ cells was prepared in the same manner as in Example 1, except that folic acid in the medium was replaced with choline.

[0110] Comparative Example 13

[0111] The high-efficiency serum-free medium for in vitro culture of avian primordial germ cells was prepared in the same manner as in Example 1, except that the concentration of ovotransferrin in the medium was adjusted to 15 μg / mL.

[0112] Comparative Example 14

[0113] The high-efficiency serum-free medium for in vitro culture of avian primordial germ cells was prepared in the same manner as in Example 1, except that the concentration of vitamin B12 in the medium was adjusted to 15 μg / mL.

[0114] Comparative Example 15

[0115] The high-efficiency serum-free medium for in vitro culture of avian primordial germ cells was prepared in the same manner as in Example 1, except that the concentration of folic acid in the medium was adjusted to 15 μg / mL.

[0116] Experimental Example

[0117] The culture media obtained from Examples 1 to 3 and Comparative Examples 1 to 14 were used as the culture solution for chicken primordial germ cells. The hatching eggs were hatched at a temperature of 8°C, a relative humidity of 60%, and an incubation time of 7 days. The eggshells were disinfected, and after opening the hatching eggs, they were placed in a petri dish. The gonads were isolated under a stereomicroscope and placed in a 1.5 mL centrifuge tube containing 100 μL of phosphate buffer. 0.25% trypsin digestion solution was added to the centrifuge tube and digested at 37°C for 20 min. The gonads were completely digested into single cells by repeatedly pipetting, and an equal volume of the culture solution was added to terminate the digestion. Then, it was centrifuged at 2500 rpm for 3 min to obtain chicken primordial germ cells. The chicken primordial germ cells were transferred into a 24-well cell culture plate at a density of 50 cells per microliter of the culture medium and cultured in an incubator at 37°C and 5% CO2 for 4 days. After culturing for 12 h, the culture solution was completely replaced, and the culture was continued until the 4th day. The cell concentration and proliferation rate were calculated, and the results are shown in Table 1. At the same time, the morphology of chicken primordial germ cells was observed under a microscope.

[0118] Proliferation rate = (cell concentration - initial cell density added) / initial cell density added

[0119] Among them, the initial cell density added was 50 cells / μL, that is, 5×10 4 cells / mL.

[0120] The cell viability was detected: Using a cell counting kit (CCK-8), the cells cultured for 4 days were inoculated into a 96-well plate and cultured for another 24 h. Then, the cells were processed according to the instructions. CCK-8 (10 μL) was added to each well and incubated for 1.5 h, and then the cell viability was detected. The absorbance was measured at 450 nm using a Microplate reader. The cell viability was calculated according to the following formula: Cell viability = (control OD - treated OD) / (control OD - blank OD) × 100%. The results are shown in Figure 1 .

[0121] Table 1 Cell concentration and proliferation rate

[0122]

[0123]

[0124] As can be seen from Table 1, the culture medium of the present invention can significantly promote the proliferation efficiency of chicken primordial germ cells in vitro. Moreover, by observing the morphology of chicken primordial germ cells under a microscope, it was found that the chicken primordial germ cells cultured with the culture media of Examples 1 to 3 and those except Comparative Example 2 were all round, with bright edges and relatively large volumes, and it was observable that the cells were connected in pairs or in strings, indicating that the culture medium of the present invention is helpful for the proliferation of chicken primordial germ cells in vitro and maintains the pluripotency of primordial germ cells. However, the primordial germ cells cultured with the culture medium of Comparative Example 2 showed an increase in volume, and some cells showed a blurred cell boundary, indicating that when the component concentration in the culture medium used is high, it will affect the cell morphology of primordial germ cells and thus affect the pluripotency of primordial germ cells.

[0125] By comparing Example 1 with Comparative Examples 3 to 6, it can be seen that the addition of ovotransferrin, vitamin B12 and folic acid has an important impact on the proliferation of primordial germ cells and the maintenance of their pluripotency. And by analyzing the results of Comparative Examples 7 to 15, it can be known that whether a certain component is replaced, omitted or the dosage of a certain component is changed, it will lead to a decrease in the proliferation rate of primordial germ cells. Thus, the present invention significantly improves the cell proliferation rate and maintains the pluripotency of primordial germ cells by optimizing the culture medium formula and introducing a multi-pathway inhibitor combination to synergistically inhibit apoptosis.

[0126] As Figure 1 can be seen, the chicken primordial germ cells cultured with the culture medium of the present invention have good cell viability.

[0127] By comparing Example 1 with the comparative examples, it can be seen that whether the concentration of each component in the culture medium component is changed, or one or more components in the culture medium are replaced, or the concentration of a certain component in the culture medium is changed, it will all affect the proliferation of primordial germ cells. The culture medium of the present invention can significantly increase the cell proliferation rate and maintain the pluripotency of primordial germ cells.

[0128] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A high-efficiency serum-free medium for in vitro culture of avian primordial germ cells, characterized in that: The culture medium is based on calcium-free Knockout DMEM and also includes the following components at final concentrations: 10-15 mM glucose, 0.5-1.5×B-27 supplement, 1-3 mM glutamine substitute, 0.5-1.5×non-essential amino acids, 0.5-1.5×EmbryoMax nucleoside mixture, 0.01-0.2 mMβ-mercaptoethanol, 0.1%-0.3% ovalbumin, 1-1.5 mM sodium pyruvate, 0.05-0.2 mM CaCl2, 0.001%-0.02% heparin sodium, 15-35 ng / mL ActivinA, 30-50 ng / mL FGF2, 5-10 μg / mL ovotransferrin, 1-10 μg / L vitamin B12, and 1-10 μg / L folic acid.

2. The high-efficiency serum-free medium according to claim 1, characterized in that The culture medium is based on calcium-free Knockout DMEM and also includes the following components at final concentrations: 11-14 mM glucose, 0.7-1.2×B-27 supplement, 1.5-2.5 mM glutamine substitute, 0.7-1.2×non-essential amino acids, 0.7-1.2×EmbryoMax nucleoside mixture, 0.05-0.15 mMβ-mercaptoethanol, 0.15%-0.25% ovalbumin, 1.1-1.3 mM sodium pyruvate, 0.1-0.17 mM CaCl2, 0.005%-0.015% heparin sodium, 20-30 ng / mL Activin A, 35-45 ng / mL FGF2, 7-9 μg / mL ovotransferrin, 3-8 μg / L vitamin B12, and 3-8 μg / L folic acid.

3. The high-efficiency serum-free medium according to claim 2, characterized in that The culture medium is based on calcium-free Knockout DMEM and also includes the following components at final concentrations: 12 mM glucose, 1× B-27 supplement, 2.0 mM glutamine substitute, 1× non-essential amino acid, 1× EmbryoMax nucleoside mixture, 0.1 mM β-mercaptoethanol, 0.2% ovalbumin, 1.2 mM sodium pyruvate, 0.15 mM CaCl2, 0.01% sodium heparin, 25 ng / mL Activin A, 40 ng / mL FGF2, 8 μg / mL ovotransferrin, 5 μg / L vitamin B12, and 4 μg / L folic acid.

4. The method for preparing a high-efficiency serum-free medium according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Glucose and CaCl2 were added to the calcium-free knockout DMEM medium, and the osmotic pressure was adjusted to 250-260 mOsm / kg to obtain solution 1; (2) adding B-27 supplement, glutamine substitute, non-essential amino acids, EmbryoMax nucleoside mixture, β-mercaptoethanol, ovalbumin, sodium pyruvate, sodium heparin, vitamin B12, and folic acid to solution 1, and mixing well to obtain solution 2; (3) Pre-dissolved ovotransferrin, Activin A and FGF2 were added to solution 2, and the mixture was sterilized by filtration to obtain a high-efficiency serum-free culture medium.

5. The preparation method according to claim 4, characterized in that: The solution used for the pre-dissolved ovotransferrin in step (3) is PBS with a pH of 7.0 to 7.

6.

6. The preparation method according to claim 5, characterized in that: The concentration of the pre-dissolved ovotransferrin is 0.05-0.2 mg / mL.

7. The preparation method according to claim 4, characterized in that: Before adding Activin A and FGF2 to Solution 2, Activin A and FGF2 are dissolved. The concentration of Activin A after dissolution is 0.5-2 μg / mL, and the concentration of FGF2 after dissolution is 0.5-2 μg / mL.

8. The preparation method according to claim 7, characterized in that: The solution used for the dissolution is a calcium-free Knockout DMEM culture medium.

9. Use of the high-efficiency serum-free medium according to any one of claims 1 to 3 or the high-efficiency serum-free medium obtained according to the preparation method according to any one of claims 4 to 8 in promoting the proliferation of avian primordial germ cells.

10. The use according to claim 9, characterized in that: The poultry is chicken.

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