Improved blastocyst culture solution as well as preparation method and application thereof

Abstract

The invention provides an improved blastocyst culture solution as well as a preparation method and application thereof, and relates to the field of embryonic cell in-vitro culture. Based on a formula of a conventional blastocyst culture solution, colony stimulating factor-1 (CSF-1) and a transforming growth factor-alpha (TGF-alpha) are added, and the colony stimulating factor-1 and the transforming growth factor-alpha are dissolved in the conventional culture solution according to the concentration of 0.02-0.06 mmol / L and the concentration of 0.01-0.05 mmol / L; the obtained improved blastocyst culture solution can improve the mulberry embryo formation rate or blastocyst formation rate in in-vitro culture, promote the development of low-quality embryos and improve the mulberry embryo quality or blastocyst quality, and can be used for preparing medicines for promoting embryo implantation or subsequent growth and development.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro culture of embryonic cells, relates to blastocyst culture medium, and specifically relates to an improved blastocyst culture medium, a preparation method thereof, and an application thereof. Background Art

[0002] The emergence of in vitro fertilization-embryo transfer (IVF-ET) technology provides an opportunity for infertile couples to have healthy children. However, data shows that embryos of some couples will experience repeated pregnancy loss after in vitro culture and transfer, and the incidence rate is about 2% - 5% and shows an upward trend in recent years. Common causes of repeated pregnancy loss include abnormal embryo development, maternal anatomical abnormalities, maternal endocrine abnormalities, maternal metabolic abnormalities, etc., and 40% - 60% of cases still cannot find clear pathogenic factors. One of the common causes of abnormal embryo development is low embryo quality. Among them, compared with young couples, older couples have a higher probability of obtaining low-quality embryos, and low-quality embryos will lead to abnormal subsequent development and further cause repeated pregnancy loss.

[0003] Research shows that the human maternal reproductive tract can secrete growth factors, which exist in oviduct fluid and uterine fluid, and embryos can express their receptors. Therefore, in vivo, the development of embryos is regulated by growth factors secreted by the maternal reproductive tract. In addition, many growth factors have been proven to affect the development speed of embryos in vitro, embryo quality, the number of blastocyst cells, embryo metabolism, and embryo cell apoptosis.

[0004] Colony stimulating factor is a group of cytokines that can selectively stimulate the proliferation of hematopoietic progenitor cells in vivo and in vitro, and differentiate to form a colony of cells of a certain lineage, named colony stimulating factor (CSF). Colony stimulating factor can bind to the receptor protein on the surface of hematopoietic stem cells, activate the intracellular signal pathway, promote cell proliferation, and differentiate into specific types of blood cells. According to the different target cells it acts on, CSF can be divided into four types: granulocyte and macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), macrophage CSF (M-CSF), and multi-CSF (usually called IL-3). Among them, macrophage CSF (M-CSF) is also called colony stimulating factor-1 (CSF-1), and was initially discovered in serum, urine or other body fluids. The action of CSF-1 is mediated by the high-affinity receptor tyrosine kinase (CSF-1R) encoded by the c-fms proto-oncogene. It regulates the survival, proliferation, and differentiation of mononuclear macrophages, and also regulates the proliferation and differentiation of cells in the female reproductive tract.

[0005] Transforming growth factor (TGF) refers to two types of polypeptide growth factors, transforming growth factor-α and transforming growth factor-β. Transforming growth factor-α is produced by macrophages, brain cells, and epidermal cells and can induce epithelial development. Transforming growth factor-β has three subtypes, namely TGF-β1, TGF-β2, and TGF-β3. Transforming growth factor-α is a small molecule polypeptide. It binds to the epidermal growth factor receptor (EGFR) on the cell surface, activates the tyrosine kinase activity in the receptor, and thus initiates signal transduction, leading to various biochemical reactions. Among them, the intracellular calcium concentration increases, glycolysis and protein synthesis are enhanced, the expression of some genes (including the epidermal growth factor receptor) is promoted, and ultimately cell proliferation is promoted.

[0006] The number of blastocyst cells in mice lacking colony-stimulating factor-1 (CSF-1) is reduced, while the apoptosis level of blastocyst cells in mice lacking transforming growth factor-α (TGF-α) is higher compared to wild-type mice. CSF-1 increases the number of trophoblast cells. TGF-α acts on the receptors in the inner cell mass cells and local polarized trophoblast cells, and adding TGF-α to the culture medium can reduce the apoptosis of mouse blastocyst cells cultured in vitro.

[0007] The prior art US20020028509A1 discloses that at least two growth factors selected from the group consisting of hepatocyte growth factor, also known as HGF, transforming growth factor α, also known as TGF-α, granulocyte-macrophage colony-stimulating factor, also known as GM-CSF, epidermal growth factor, also known as EGF and / or HB-EGF, growth and differentiation factor, also known as GDF, such as gdf-9, insulin-like growth factor, also known as IGF, such as IGF-1 and / or IGF-2 can be added to an in vitro cell culture composition. The disadvantage of this technology is that the specific combination of growth factors is not clear and the effect of promoting embryo growth is poor.

[0008] For the embryos of patients with recurrent pregnancy loss during in vitro culture, the current reproductive center uses the same culture medium as that for conventional embryos without special treatment. These embryos often show poor blastocyst quality and low blastocyst rate during in vitro culture, and most of them will be directly discarded. Therefore, there is an urgent need for a blastocyst culture medium specifically for the embryos of patients with recurrent pregnancy loss to optimize the in vitro culture environment of poor-quality embryos, improve their developmental potential, and thus improve the reproductive outcomes of such patients, which is of great significance to couples eager to have children. Summary of the Invention

[0009] In view of the problems existing in the prior art, the present invention provides an improved blastocyst culture medium, a preparation method thereof, and an application thereof. Based on the formula of a conventional blastocyst culture medium, colony stimulating factor-1 (CSF-1) and transforming growth factor-α (TGF-α) are added, and colony stimulating factor-1 and transforming growth factor-α are dissolved in the conventional culture medium at concentrations of 0.02-0.06 mmol / L and 0.01-0.05 mmol / L respectively. The preparation method is simple. The obtained improved blastocyst culture medium can improve the morula formation rate or blastocyst formation rate in in vitro culture, promote the development of low-quality embryos, and improve the quality of morulas or blastocysts, and can be used for preparing drugs to promote embryo implantation or subsequent growth and development.

[0010] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0011] On the one hand, the present invention provides an improved blastocyst culture medium which simultaneously contains colony stimulating factor-1 and transforming growth factor-α, and the concentration of colony stimulating factor-1 is 0.02-0.06 mmol / L, and the concentration of transforming growth factor-α is 0.01-0.05 mmol / L.

[0012] Preferably, the concentration of colony stimulating factor-1 is 0.03-0.05 mmol / L, and the concentration of transforming growth factor-α is 0.02-0.04 mmol / L.

[0013] Preferably, the concentration of colony stimulating factor-1 is 0.04 mmol / L, and the concentration of transforming growth factor-α is 0.03 mmol / L.

[0014] On the other hand, the present invention provides a preparation method of the above-mentioned improved blastocyst culture medium, which includes the following steps:

[0015] S1. Prepare a conventional blastocyst culture medium;

[0016] S2. Dissolve colony stimulating factor-1 and transforming growth factor-α in the conventional blastocyst culture medium prepared in step S1;

[0017] S3. Detect and maintain the osmotic pressure and pH value of the culture medium obtained in step S2;

[0018] S4. Filter and sterilize the solution obtained in step S3 through a filter membrane to obtain the improved blastocyst culture medium.

[0019] Preferably, the components of the conventional blastocyst culture medium in step S1 include: glutamic acid 0.08 - 0.13 mmol / L, aspartic acid 0.08 - 0.13 mmol / L, L-histidine hydrochloride 0.17 - 0.23 mmol / L, L-arginine hydrochloride 0.58 - 0.63 mmol / L, isoleucine 0.37 - 0.42 mmol / L, leucine 0.37 - 0.42 mmol / L, methionine 0.08 - 0.12 mmol / L, phenylalanine 0.18 - 0.23 mmol / L, alanine 0.08 - 0.13 mmol / L, glycine 0.08 - 0.13 mmol / L, proline 0.08 - 0.13 mmol / L, serine 0.08 - 0.13 mmol / L, asparagine 0.08 - 0.13 mmol / L, valine 0.38 - 0.42 mmol / L, L-lysine hydrochloride 0.38 - 0.42 mmol / L, threonine 0.38 - 0.42 mmol / L, cystine 0.08 - 0.13 mmol / L, tryptophan 0.48 - 0.53 mmol / L, tyrosine 0.17 - 0.22 mmol / L, taurine 0.08 - 0.13 mmol / L, alanyl-glutamine 0.90 - 1.10 mmol / L, sodium chloride 95.60 - 100.60 mmol / L, potassium chloride 5.30 - 5.80 mmol / L, magnesium sulfate 0.90 - 1.20 mmol / L, potassium dihydrogen phosphate 0.23 - 0.27 mmol / L, calcium chloride 1.60 - 1.90 mmol / L, anhydrous glucose 3.11 - 3.17 mmol / L, sodium pyruvate 0.08 - 0.13 mmol / L, sodium bicarbonate 23.00 - 27.00 mmol / L, sodium lactate 5.85 - 5.86 mmol / L, choline chloride 0.0052 - 0.0072 mmol / L, calcium pantothenate 0.0011 - 0.0021 mmol / L, folic acid 0.0016 - 0.0023 mmol / L, nicotinamide 0.0077 - 0.0082 mmol / L, pyridoxal hydrochloride 0.0045 - 0.0049 mmol / L, riboflavin 0.0001 -

[0020] 0.0003 mmol / L, thiamine hydrochloride 0.0020 - 0.0030 mmol / L, inositol 0.0106 - 0.0111 mmol / L, gentamicin sulfate 8 - 12 mg / L, and human albumin 4 - 6 g / L.

[0021] Preferably, the components of the conventional blastocyst culture medium include: glutamic acid 0.08 - 0.10 mmol / L, aspartic acid 0.08 - 0.10 mmol / L, L-histidine hydrochloride 0.17 - 0.19 mmol / L, L-arginine hydrochloride 0.58 - 0.61 mmol / L, isoleucine 0.37 - 0.39 mmol / L, leucine 0.37 - 0.39 mmol / L, methionine 0.08 - 0.10 mmol / L, phenylalanine 0.18 - 0.20 mmol / L, alanine 0.08 - 0.10 mmol / L, glycine 0.08 - 0.10 mmol / L, proline 0.08 - 0.10 mmol / L, serine 0.08 - 0.10 mmol / L, asparagine 0.08 - 0.10 mmol / L, valine 0.38 - 0.40 mmol / L, L-lysine hydrochloride 0.38 - 0.40 mmol / L, threonine 0.38 - 0.40 mmol / L, cystine 0.08 - 0.10 mmol / L, tryptophan 0.48 - 0.51 mmol / L, tyrosine 0.17 - 0.20 mmol / L, taurine 0.08 - 0.11 mmol / L, L-alanyl-L-glutamine 1.00 - 1.10 mmol / L, sodium chloride 95.60 - 98.60 mmol / L, potassium chloride 5.30 - 5.60 mmol / L, magnesium sulfate 0.90 - 1.10 mmol / L, potassium dihydrogen phosphate 0.24 - 0.26 mmol / L, calcium chloride 1.70 - 1.80 mmol / L, anhydrous glucose 3.13 - 3.15 mmol / L, sodium pyruvate 0.09 - 0.12 mmol / L, sodium bicarbonate 24.00 - 26.00 mmol / L, sodium lactate 5.85 - 5.86 mmol / L, choline chloride 0.0052 - 0.0062 mmol / L, calcium pantothenate 0.0013 - 0.0019 mmol / L, folic acid 0.0018 - 0.0021 mmol / L, nicotinamide 0.0079 - 0.0081 mmol / L, pyridoxal hydrochloride 0.0046 - 0.0048 mmol / L, riboflavin 0.0001 - 0.0003 mmol / L, thiamine hydrochloride 0.0022 - 0.0028 mmol / L, inositol 0.0107 - 0.0110 mmol / L, gentamicin sulfate 9 - 11 mg / L, and human serum albumin 4.5 - 5.5 g / L.

[0022] Specifically, the components of the conventional blastocyst culture medium in step S1 include: glutamic acid 0.09 mmol / L, aspartic acid 0.09 mmol / L, L-histidine hydrochloride 0.18 mmol / L, L-arginine hydrochloride

[0023] 0.60 mmol / L, isoleucine 0.38 mmol / L, leucine 0.38 mmol / L, methionine

[0024] 0.09 mmol / L, phenylalanine 0.19 mmol / L, alanine 0.09 mmol / L, glycine

[0025] 0.09 mmol / L, proline 0.09 mmol / L, serine 0.09 mmol / L, asparagine

[0026] 0.09 mmol / L, valine 0.39 mmol / L, lysine hydrochloride 0.39 mmol / L, threonine 0.39 mmol / L, cystine 0.09 mmol / L, tryptophan 0.49 mmol / L, tyrosine 0.18 mmol / L, taurine 0.10 mmol / L, alanyl glutamine 1.00 mmol / L, sodium chloride 97.60 mmol / L, potassium chloride 5.44 mmol / L, magnesium sulfate 1.00 mmol / L, potassium dihydrogen phosphate 0.25 mmol / L, calcium chloride 1.75 mmol / L, anhydrous glucose 3.15 mmol / L, sodium pyruvate 0.10 mmol / L, sodium bicarbonate

[0027] 25.00 mmol / L, sodium lactate 5.85 mmol / L, choline chloride 0.0060 mmol / L, calcium pantothenate 0.0015 mmol / L, folic acid 0.0020 mmol / L, nicotinamide 0.0080 mmol / L, pyridoxal hydrochloride

[0028] 0.0047 mmol / L, riboflavin 0.0002 mmol / L, thiamine hydrochloride 0.0025 mmol / L, inositol 0.0108 mmol / L, gentamicin sulfate 10 mg / L and human albumin 5 g / L.

[0029] Preferably, in step S1, each of the above components is weighed sequentially and dissolved in water for injection, and the principle of adding solids first and then liquids is followed during the dissolution process.

[0030] Preferably, the water for injection is filtered through a 0.1 - 0.3 μM filter membrane, and the endotoxin is detected to be less than 0.5 EU / ml.

[0031] Preferably, the water for injection is filtered through a 0.22 μM filter membrane.

[0032] Preferably, in step S3, the osmotic pressure is maintained at 265 - 295 mOsm / Kg.

[0033] Preferably, in step S3, the pH value is maintained at 7.8 - 8.2.

[0034] Preferably, step S4 includes filtering and sterilizing the solution obtained in step S3 through a 0.22 μm filter membrane.

[0035] On the other hand, the present invention provides the application of the above-mentioned improved blastocyst culture medium or the above-mentioned preparation method in the in vitro culture of embryonic cells.

[0036] Preferably, the embryonic cells include morulae and blastocysts.

[0037] Preferably, the application is selected from one or more of the following applications:

[0038] 1) Application in the preparation of drugs for increasing the formation rate of morulae or blastocysts;

[0039] 2) Application in the preparation of drugs for promoting the development of low-quality embryos;

[0040] 3) Application in the preparation of drugs for improving the quality of morulae or blastocysts;

[0041] 4) Application in the preparation of drugs for promoting embryo implantation or subsequent growth and development.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The improved blastocyst culture medium disclosed by the present invention only needs to add colony-stimulating factor-1 and transforming growth factor-α on the basis of a conventional blastocyst culture medium, and the preparation method is simple;

[0044] 2. The improved blastocyst culture medium disclosed by the present invention can increase the formation rate of morulae or blastocysts, promote the development of low-quality embryos, improve the quality of morulae or blastocysts in in vitro culture, and can be used for the preparation of drugs for promoting embryo implantation or subsequent growth and development. Detailed implementation mode

[0045] Example 1: Preparation of blastocyst culture medium

[0046] Preparation method:

[0047] 1) Prepare a conventional blastocyst culture medium: Weigh each component in turn and dissolve it in injection-grade water. During the dissolution process, follow the principle of adding solids first and then liquids; the injection-grade water is filtered through a 0.22 μM filter membrane, and the endotoxin is detected to be <0.5 EU / ml; the prepared culture medium; the components of the conventional blastocyst culture medium are glutamic acid

[0048] 0.09 mmol / L of aspartic acid, 0.09 mmol / L of histidine hydrochloride, 0.18 mmol / L of arginine hydrochloride, 0.60 mmol / L of isoleucine, 0.38 mmol / L of leucine, 0.09 mmol / L of methionine, 0.19 mmol / L of phenylalanine, 0.09 mmol / L of alanine, 0.09 mmol / L of glycine, 0.09 mmol / L of proline, 0.09 mmol / L of serine, 0.09 mmol / L of asparagine, 0.39 mmol / L of valine, 0.39 mmol / L of lysine hydrochloride, 0.39 mmol / L of threonine, 0.09 mmol / L of cystine, 0.49 mmol / L of tryptophan, 0.18 mmol / L of tyrosine, 0.10 mmol / L of taurine, 1.00 mmol / L of alanyl glutamine, 97.60 mmol / L of sodium chloride, 5.44 mmol / L of potassium chloride, 1.00 mmol / L of magnesium sulfate, 0.25 mmol / L of potassium dihydrogen phosphate, 1.75 mmol / L of calcium chloride, 3.15 mmol / L of anhydrous glucose, 0.10 mmol / L of sodium pyruvate, 25.00 mmol / L of sodium bicarbonate, 5.85 mmol / L of sodium lactate, 0.0060 mmol / L of choline chloride, 0.0015 mmol / L of calcium pantothenate, 0.0020 mmol / L of folic acid, 0.0080 mmol / L of nicotinamide, 0.0047 mmol / L of pyridoxal hydrochloride, 0.0002 mmol / L of riboflavin, 0.0025 mmol / L of thiamine hydrochloride, 0.0108 mmol / L of inositol, 10 mg / L of gentamicin sulfate, and 5 g / L of human albumin.

[0049] 2) Detect and record the osmotic pressure and pH value of the culture medium obtained in step 1; the osmotic pressure is maintained at 280 mOsm / Kg, and the pH value is maintained at 8.0;

[0050] 3) Dissolve colony-stimulating factor-1 (purchased from Sigma, catalog number M9170) and transforming growth factor-α (purchased from MCE, catalog number HY-P77857) in the conventional blastocyst culture medium prepared in step 2) at concentrations of 0.02 mmol / L and 0.05 mmol / L, respectively;

[0051] 4) Filter and sterilize the solution obtained in step 3) through a 0.22 μm filter membrane to obtain a modified blastocyst culture medium.

[0052] Example 2: Preparation of blastocyst culture medium

[0053] Prepare the modified blastocyst culture medium according to the method of Example 1, except that the concentrations of colony-stimulating factor-1 and transforming growth factor-α are 0.04 mmol / L and 0.03 mmol / L, respectively.

[0054] Example 3: Preparation of Blastocyst Culture Medium

[0055] Prepare the improved blastocyst culture medium according to the method of Example 1, except that the concentrations of colony-stimulating factor-1 and transforming growth factor-α are 0.06 mmol / L and 0.01 mmol / L, respectively.

[0056] Comparative Example 1: Preparation of Blastocyst Culture Medium

[0057] Prepare the improved blastocyst culture medium according to the method of Example 1, except that the concentration of colony-stimulating factor-1 is 0.02 mmol / L and transforming growth factor-α is not added.

[0058] Comparative Example 2: Preparation of Blastocyst Culture Medium

[0059] Prepare the improved blastocyst culture medium according to the method of Example 1, except that the concentration of colony-stimulating factor-1 is 0.04 mmol / L and transforming growth factor-α is not added.

[0060] Comparative Example 3: Preparation of Blastocyst Culture Medium

[0061] Prepare the improved blastocyst culture medium according to the method of Example 1, except that the concentration of colony-stimulating factor-1 is 0.06 mmol / L and transforming growth factor-α is not added.

[0062] Comparative Example 4: Preparation of Blastocyst Culture Medium

[0063] Prepare the improved blastocyst culture medium according to the method of Example 1, except that the concentration of transforming growth factor-α is 0.05 mmol / L and colony-stimulating factor-1 is not added.

[0064] Comparative Example 5: Preparation of Blastocyst Culture Medium

[0065] Prepare the improved blastocyst culture medium according to the method of Example 1, except that the concentration of transforming growth factor-α is 0.03 mmol / L and colony-stimulating factor-1 is not added.

[0066] Comparative Example 6: Preparation of Blastocyst Culture Medium

[0067] Prepare the improved blastocyst culture medium according to the method of Example 1, except that the concentration of transforming growth factor-α is 0.01 mmol / L and colony-stimulating factor-1 is not added.

[0068] Comparative Example 7: Preparation of Blastocyst Culture Medium

[0069] Prepare the improved blastocyst culture medium according to the method of Example 1, except that colony-stimulating factor-1 is replaced with granulocyte-macrophage colony-stimulating factor (GM-CSF, purchased from Sigma, catalog number G0792), and the concentration is 0.02 mmol / L.

[0070] Comparative Example 8: Preparation of blastocyst culture medium

[0071] Prepare the improved blastocyst culture medium according to the method of Example 1, except that colony-stimulating factor-1 is replaced with granulocyte-macrophage colony-stimulating factor (GM-CSF), and the concentration is 0.04 mmol / L.

[0072] Comparative Example 9: Preparation of blastocyst culture medium

[0073] Prepare the improved blastocyst culture medium according to the method of Example 1, except that colony-stimulating factor-1 is replaced with granulocyte-macrophage colony-stimulating factor (GM-CSF), and the concentration is 0.06 mmol / L.

[0074] Effect Example: Evaluation of the effect of blastocyst medium on improving the embryo development rate of aged mice

[0075] Instructions for storing blastocyst culture medium:

[0076] Store the blastocyst culture medium in an environment of 2°C - 8°C; the blastocyst culture medium is a single-use product and cannot be reused; in order to avoid contamination, use the product with aseptic technique; if the product changes color, becomes turbid or shows signs of microbial contamination, do not use it.

[0077] Instructions for using blastocyst culture medium:

[0078] 1) After culturing the embryos to the 3rd day, they need to be transferred to the blastocyst culture medium.

[0079] 2) In the afternoon of the 2nd day, take a 60 mm culture dish, use a sterile pipette tip pre-rinsed with the blastocyst culture medium, prepare 6 30 μL droplets in a single culture dish, and immediately cover them with paraffin oil.

[0080] 3) Immediately after preparation, place the culture dish in an incubator with 6% carbon dioxide concentration and 37°C. Gently remove the culture dish lid and lean it against the edge of the culture dish for medium equilibration.

[0081] 4) In the morning of the 3rd day, transfer the embryos into the prepared blastocyst culture medium and place them in an incubator with 6% carbon dioxide concentration and 37°C for culture.

[0082] The blastocyst culture media of Examples 1-3 and Comparative Examples 1-9 were evaluated for their effects. Nine-month-old (aged mouse model) C57BL / 6J female mice were selected as experimental subjects, and all animal experiments were conducted in accordance with the guidelines of the Animal Ethics Committee. For the ovulation induction experiment, oocytes were obtained from 9-month-old (aged) mice. At 05:00 pm on the first day, pregnant mare serum gonadotropin (PMSG) 10 IU was injected intraperitoneally. After 48 hours, human chorionic gonadotropin (HCG) 10 IU was injected intraperitoneally. Fourteen hours after the injection of HCG, the mice were dissected and the cumulus-oocyte complexes were obtained from the ampulla of the fallopian tube. Epididymal sperm from 8-week-old wild-type male mice were used for conventional in vitro fertilization. The grouping and results of the blastocyst medium are shown in Table 1. Three parallel experiments were set up for each group, and the blastocyst rates of embryos in different groups were statistically analyzed.

[0083] Table 1 Effects of Different Groupings

[0084]

[0085]

[0086] Note: The same letter annotation in the same column indicates that there is no significant difference between the data (P>0.05), and different letter annotations in the same column indicate that there is a significant difference between the data (P<0.05).

[0087] Further analysis of the results: The blastocyst rates of Examples 1-3 were significantly higher than those of the control group, indicating that compared with the conventional blastocyst culture medium, the blastocyst culture medium supplemented with CSF-1 and TGF-α could increase the blastocyst rate of embryos. When comparing Example 1 (blastocyst rate 93.30%) with the aged control group (blastocyst rate 48.95%), the blastocyst rate of Example 1 increased and the difference was significant (p<0.001). When comparing Example 2 (blastocyst rate 96.65%) with the aged control group (blastocyst rate 48.95%), the blastocyst rate of Example 2 increased and the difference was significant (p<0.001). When comparing Example 3 (blastocyst rate 93.85%) with the aged control group (blastocyst rate 48.95%), the blastocyst rate of Example 3 increased and the difference was significant (p<0.001).

[0088] When comparing Comparative Examples 1-6 with the aged control group (blastocyst rate 48.95%), the addition of either CSF-1 or TGF-α alone had a certain promoting effect, but the simultaneous addition of CSF-1 and TGF-α (Examples 1-3) had a significant synergistic effect. When comparing Comparative Examples 7-9 with Examples 1-3, it was found that if CSF-1 was replaced with GM-CSF, the blastocyst rate was significantly lower than that of the combination of CSF-1 and TGF-α, indicating that the combination of CSF-1 and TGF-α in the present invention cannot be easily replaced. The blastocyst culture medium promoted the development of low-quality embryos in aged mice by adding colony-stimulating factor-1 and transforming growth factor-α, and increased the blastocyst rate of mouse embryos.

[0089] In addition, 8-week-old (low-age control group) and 9-month-old (high-age mouse model group) C57BL / 6J female mice were selected as experimental subjects. Epididymal sperm of 8-week-old wild-type male mice were taken for conventional in vitro fertilization. All animal experiments were conducted in accordance with the guidelines of the Animal Ethics Committee. The blastocyst cells were stained by binding of a DNA staining reagent to the cell nucleus. The markers expressed by the inner cell mass cells were stained by immunofluorescence staining. The total number of blastocyst cells and the number of inner cell mass cells in 20 blastocysts each in the low-age control group (using conventional blastocyst culture medium), high-age control group (using conventional blastocyst culture medium), and high-age experimental group (using the improved blastocyst medium in Example 2) were counted under a fluorescence microscope. The statistical results are shown in Table 2. According to industry standards, the total number of well-developed blastocyst cells ≥ 50, and the number of inner cell mass cells ≥ 12. As shown in Table 2, the blastocysts in the low-age control group had good quality, with the average number of inner cell mass cells being 16.1 ± 1.75 and the average total cell number being 53.1 ± 3.67; the blastocysts in the high-age control group had poor quality, with the average number of inner cell mass cells being 8.3 ± 1.34 and the average total cell number being 22.95 ± 5.47; the average number of inner cell mass cells in the high-age experimental group was 11.95 ± 2.15, and the average total cell number was 44.5 ± 7.62. The development of the inner cell mass and trophectoderm of the blastocysts in the high-age experimental group was better than that in the high-age control group and was close to that of the good blastocysts in the low-age control group.

[0090] Table 2 Blastocyst quality of different groups

[0091]

[0092] The above results indicate that the blastocyst culture medium promoted the development of low-quality embryos by adding colony-stimulating factor-1 and transforming growth factor-α, improving the blastocyst rate (see Table 1) and blastocyst quality (see Table 2) of high-age mouse embryos, thus facilitating their implantation and subsequent growth and development, and can be used for the preparation of related drugs.

[0093] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A modified blastocyst culture medium, characterized in that: The improved blastocyst culture medium contains colony stimulating factor-1 and transforming growth factor-α at the same time, and the concentration of colony stimulating factor-1 is 0.02-0.06 mmol / L, and the concentration of transforming growth factor-α is 0.01-0.05 mmol / L.

2. The improved blastocyst culture medium according to claim 1, characterized in that: The concentration of colony stimulating factor-1 is 0.03-0.05 mmol / L, and the concentration of transforming growth factor-α is 0.02-0.04 mmol / L.

3. The improved blastocyst culture medium according to claim 2, characterized in that: The concentration of colony stimulating factor-1 is 0.04 mmol / L, and the concentration of transforming growth factor-α is 0.03 mmol / L.

4. The method for preparing the improved blastocyst culture medium according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Prepare conventional blastocyst culture medium; S2, dissolving colony stimulating factor-1 and transforming growth factor-α in the conventional blastocyst culture medium prepared in step S1; S3, detecting and maintaining the osmotic pressure and pH value of the culture solution obtained in step S2; S4. Filter and sterilize the solution obtained in step S3 through a filter membrane to obtain a modified blastocyst culture medium.

5. The preparation method according to claim 4, characterized in that: The components of the conventional blastocyst culture medium in step S1 include: glutamic acid 0.08-0.13mmol / L, aspartic acid 0.08-0.13mmol / L, histidine hydrochloride 0.17-0.23mmol / L, arginine hydrochloride 0.58-0.63mmol / L, isoleucine 0.37-0.42mmol / L, leucine 0.37-0.42mmol / L, methionine 0.08-0.12mmol / L, phenylalanine 0.18-0.23mmol / L, alanine 0.08-0.13mmol / L, glycine 0.08- 0.13mmol / L, proline 0.08-0.13mmol / L, serine 0.08-0.13mmol / L, asparagine 0.08-0.13mmol / L, valine 0.38-0.42mmol / L, lysine hydrochloride 0.38-0.42mmol / L, threonine 0.38-0.42mmol / L, cystine 0.08-0.13mmol / L, tryptophan 0.48-0.53mmol / L, tyrosine 0.17-0.22mmol / L, taurine 0.08-0.13mmol / L, alanine Glutamine 0.90-1.10mmol / L, sodium chloride 95.60-100.60mmol / L, potassium chloride 5.30-5.80mmol / L, magnesium sulfate 0.90-1.20mmol / L, potassium dihydrogen phosphate 0.23-0.27mmol / L, calcium chloride 1.60-1.90mmol / L, anhydrous glucose 3.11-3.17mmol / L, sodium pyruvate 0.08-0.13mmol / L, sodium bicarbonate 23.00-27.00mmol / L, sodium lactate 5.85-5.86mmol / L, choline chloride 0 .0052-0.0072mmol / L, calcium pantothenate 0.0011-0.0021mmol / L, folic acid 0.0016-0.0023mmol / L, nicotinamide 0.0077-0.0082mmol / L, pyridoxal hydrochloride 0.0045-0.0049mmol / L, riboflavin 0.0001-0.0003mmol / L, thiamine hydrochloride 0.0020-0.0030mmol / L, inositol 0.0106-0.0111mmol / L, gentamicin sulfate 8-12mg / L and human serum albumin 4-6g / L.

6. The preparation method according to claim 5, characterized in that: The components of the conventional blastocyst culture medium include: glutamic acid 0.08-0.10mmol / L, aspartic acid 0.08-0.10mmol / L, histidine hydrochloride 0.17-0.19mmol / L, arginine hydrochloride 0.58-0.61mmol / L, isoleucine 0.37-0.39mmol / L, leucine 0.37-0.39mmol / L, methionine 0.08-0.10mmol / L, phenylalanine 0.18-0.20mmol / L, alanine 0.08-0.10mmol / L, glycine 0.08-0.10mmol / L mmol / L, proline 0.08-0.10mmol / L, serine 0.08-0.10mmol / L, asparagine 0.08-0.10mmol / L, valine 0.38-0.40mmol / L, lysine hydrochloride 0.38-0.40mmol / L, threonine 0.38-0.40mmol / L, cystine 0.08-0.10mmol / L, tryptophan 0.48-0.51mmol / L, tyrosine 0.17-0.20mmol / L, taurine 0.08-0.11mmol / L, alanyl-glutamine 1.00-1.10mmol / L, sodium chloride 95.60-98.60mmol / L, potassium chloride 5.30-5.60mmol / L, magnesium sulfate 0.90-1.10mmol / L, potassium dihydrogen phosphate 0.24-0.26mmol / L, calcium chloride 1.70-1.80mmol / L, anhydrous glucose 3.13-3.15mmol / L, sodium pyruvate 0.09-0.12mmol / L, sodium bicarbonate 24.00-26.00mmol / L, sodium lactate 5.85-5.86mmol / L, choline chloride 0.0052mmol / L -0.0062mmol / L, calcium pantothenate 0.0013-0.0019mmol / L, folic acid 0.0018-0.0021mmol / L, niacinamide 0.0079-0.0081mmol / L, pyridoxal hydrochloride 0.0046-0.0048mmol / L, riboflavin 0.0001-0.0003mmol / L, thiamine hydrochloride 0.0022-0.0028mmol / L, inositol 0.0107-0.0110mmol / L, gentamicin sulfate 9-11mg / L and human serum albumin 4.5-5.5g / L.

7. The preparation method according to claim 6, characterized in that: The components of the conventional blastocyst culture medium in step S1 include: glutamic acid 0.09mmol / L, aspartic acid 0.09mmol / L, histidine hydrochloride 0.18mmol / L, arginine hydrochloride 0.60mmol / L, isoleucine 0.38mmol / L, leucine 0.38mmol / L, methionine 0.09mmol / L, phenylalanine 0.19mmol / L, alanine 0.09mmol / L, glycine 0.09mmol / L, proline 0.09mmol / L, serine 0.09mmol / L, asparagine 0.09mmol / L, valine 0.39mmol / L, lysine hydrochloride 0.39mmol / L, threonine 0.39mmol / L, cystine 0.09mmol / L, tryptophan 0.49mmol / L, tyrosine 0.18mmol / L, taurine 0.10mmol / L, alanyl-glutamine 1.00mmol / L, sodium chloride 97.60mmol / L, potassium chloride 5.44mmol / L, magnesium sulfate 1.00mmol / L, potassium dihydrogen phosphate 0.25mmol / L, calcium chloride 1.75mmol / L, anhydrous glucose 3.15mmol / L, sodium pyruvate 0.10mmol / L, sodium bicarbonate 25.00mmol / L, sodium lactate 5.85mmol / L, choline chloride 0.0060mmol / L, calcium pantothenate 0.0015mmol / L, folic acid 0.0020mmol / L, nicotinamide 0.0080mmol / L, pyridoxal hydrochloride 0.0047mmol / L, riboflavin 0.0002mmol / L, thiamine hydrochloride 0.0025mmol / L, inositol 0.0108mmol / L, gentamicin sulfate 10mg / L and human serum albumin 5g / L.

8. The preparation method according to claim 4, characterized in that: The step S1 comprises weighing the components of the conventional blastocyst culture solution in sequence and dissolving them in injection-grade water, wherein the principle of solid first and liquid second is followed during the dissolution process.

9. The preparation method according to claim 8, characterized in that: The injection-grade water is filtered through a 0.1-0.3 μM filter membrane, and the endotoxin is detected to be less than 0.5 EU / ml.

10. The preparation method according to claim 9, characterized in that: The injection-grade water was filtered through a 0.22 μM filter membrane.

11. The preparation method according to claim 4, characterized in that: The osmotic pressure in step S3 is maintained at 265-295 mOsm / Kg.

12. The preparation method according to claim 4, characterized in that: The pH value in step S3 is maintained at 7.8-8.

2.

13. The preparation method according to claim 4, characterized in that: The step S4 comprises filtering the solution obtained in step S3 through a 0.22 μm filter membrane for sterilization.

14. Use of the improved blastocyst culture medium according to any one of claims 1 to 3 or the preparation method according to any one of claims 4 to 13 in in vitro culture of embryonic cells.

15. The use according to claim 14, characterized in that: The embryonic cells include morula and blastocyst.

16. The use according to claim 14, characterized in that The application is selected from one or more of the following applications: 1) Application in the preparation of drugs for improving the morula formation rate or blastocyst formation rate; 2) Application in the preparation of drugs for promoting the development of low-quality embryos; 3) Application in the preparation of drugs for improving the quality of morula or blastocyst; 4) Application in the preparation of drugs for promoting embryo implantation or subsequent growth and development.

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