Embryo culture solution as well as preparation method and application thereof

Abstract

The invention discloses an embryo culture solution as well as a preparation method and application thereof, and belongs to the technical field of assisted reproduction. The technical problem to be solved is to reduce the ammonium ion concentration in the use process of the cell culture fluid. According to the technical scheme, the embryo culture solution is characterized by comprising sodium chloride, potassium chloride, monopotassium phosphate, calcium chloride, magnesium sulfate, sodium bicarbonate, a sodium lactate solution, sodium pyruvate, glucose, edetate disodium, gentamicin sulfate, human serum albumin, taurine, alanyl glutamine, non-essential amino acid and essential amino acid. The total concentration of the alanyl glutamine, the non-essential amino acid and the essential amino acid is 0.3-0.7 g / L; the mass ratio of the alanyl glutamine to the non-essential amino acid to the essential amino acid is (0.05 to 0.2) to (0.03 to 0.05) to (0.2 to 0.45); the mass ratio of the human serum albumin to the alanyl glutamine is (3-5): (0.05-0.2).

Description

Technical Field

[0001] This invention provides an embryo culture medium, its preparation method, and its application, belonging to the field of assisted reproductive technology. Background Technology

[0002] For understanding the technical content of this invention: Amino acids in embryo culture medium play a crucial role in embryonic development; however, amino acid metabolism produces ammonia, which is toxic to the embryo. Ammonia produced in embryo culture comes from two sources: the embryo's own metabolism and the spontaneous deamination of amino acids in the culture medium. The ammonia produced in the culture medium is protonated, accumulating into ammonium ions, which are toxic to the embryo. Normally, this accumulation is rare in the dynamically balanced fallopian tubes and uterine cavity. Experiments have shown that the ammonium ion concentration in the fallopian tubes is zero. The accumulation of ammonium ions in the embryo culture medium has been proven to have adverse effects on embryonic development and differentiation. When the ammonium ion concentration reaches 300 μmol / L (0.3 mM), it can also affect embryonic development after implantation. Furthermore, ammonium ions affect embryonic metabolism, intracellular pH regulation, and gene expression.

[0003] Relevant patent documents retrieved: This document, published in China (CN108251355A) on July 6, 2018, discloses a one-step embryo culture medium. The composition used to prepare this one-step embryo culture medium comprises (±)-α-lipoic acid and N-acetyl-L-cysteine; the molar ratio of (±)-α-lipoic acid to N-acetyl-L-cysteine ​​is 0.055-0.098:0.702-0.858. However, when this embryo culture medium is used to culture mouse embryos, the ammonium ion concentration in the culture medium after 96 hours of culture is excessively high, reaching 0.325 mM, exceeding 0.3 mM, which will affect the development of the embryo after transplantation.

[0004] Relevant non-patent literature retrieved: The journal or book title is *China Pharmaceutical Industry*, and the document title is "Analysis of Factors Affecting Ammonium Ion Content in Fluids Used in Human Assisted Reproductive Technology," Volume 33, Issue 12, published on June 20, 2024. This document discloses that animal experiments have shown that ammonia has adverse effects on embryonic and fetal development, reducing implantation rates and inhibiting fetal growth in a concentration-dependent manner. In human embryo studies, increased ammonium ion content in ART culture media after in vitro fertilization (IVF) has been found to reduce blastocyst development rates, inhibit human embryo metabolism, and alter gene expression. In this study, storing embryo culture media at 4°C resulted in an increase in ammonium ion content over time; storing embryo culture media at 37°C allowed for the rapid accumulation of large amounts of ammonia.

[0005] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Embryo culture medium can generate large amounts of ammonia during storage and use, affecting embryo development. Evidence for this includes the finding in the study "Analysis of Factors Affecting Ammonium Ion Content in Fluids Used in Human Assisted Reproductive Technology," which states that storing embryo culture medium at 4°C increases the ammonium ion content over time; storing it at 37°C can also lead to a rapid accumulation of ammonia. When the embryo culture medium described in CN108251355A was used to culture mouse embryos, the ammonium ion concentration in the culture medium was excessively high after 96 hours of culture. Summary of the Invention

[0006] The purpose of this invention is to provide: An embryo culture medium, and related technologies thereof, to solve technical problems such as reducing ammonium ion concentration during the use of cell culture medium, or a combination thereof.

[0007] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0008] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0009] The definition of the standard chemical term can be found in the reference "Animal Cell Culture Technology: Publisher: Sun Yat-sen University Press; Publication Date: June 2018; Edition: 3rd Edition; General Requirements for Human and Mouse Pluripotent Stem Cells, ISO 24603, International Standard Recommendation".

[0010] Unless otherwise stated, conventional methods within the scope of the art, such as ammonium ion concentration detection methods, shall be used.

[0011] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0012] As used in this article, "amino acid" refers to an organic compound containing an amino group (-NH2) and a carboxyl group (-COOH), which is the basic building block of proteins. There are 20 standard amino acids in living organisms, which are linked by peptide bonds to form polypeptide chains, which then fold into proteins with specific functions.

[0013] The term "human serum albumin" as used in this article refers to: human serum albumin is a plasma protein synthesized by the liver and is one of the most abundant proteins in blood plasma. It plays an important role in maintaining plasma osmotic pressure, transporting various substances (such as hormones, drugs, and fatty acids), and regulating acid-base balance.

[0014] The term "embryonic cell" as used in this article refers to cells that develop in the early stages of embryonic development and have the ability to differentiate into various cell types. Embryonic cells mainly include fertilized egg cells, cleavage-stage cells, and blastocyst-stage cells.

[0015] The term "non-essential amino acids" used in this article refers to: alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, and serine.

[0016] The term "essential amino acids" as used in this article refers to: arginine, cystine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine.

[0017] In a first aspect, the present invention provides: an embryo culture medium comprising: sodium chloride, potassium chloride, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, sodium bicarbonate, sodium lactate solution, sodium pyruvate, glucose, disodium edetate, gentamicin sulfate, human serum albumin, taurine, alanylglutamine, non-essential amino acids, and essential amino acids; wherein the total concentration of alanylglutamine, non-essential amino acids, and essential amino acids is 0.3-0.7 g / L; and the mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids is (0.05-0.2):(0.03-0.05):(0.2-0.45).

[0018] Among them, the technical characteristics of alanine glutamine, the total concentration of non-essential amino acids and essential amino acids are selected from 0.3-0.7 g / L.

[0019] The preferred total concentration of alanine glutamine, non-essential amino acids, and essential amino acids is 0.3343-0.4291 g / L.

[0020] The preferred concentrations of alanylglutamine, non-essential amino acids, and essential amino acids are: 0.3343 g / L, 0.34 g / L, 0.35 g / L, 0.36 g / L, 0.37 g / L, 0.38 g / L, 0.39 g / L, 0.40 g / L, 0.41 g / L, 0.42 g / L, and 0.4291 g / L.

[0021] The preferred technical feature is that the total concentration of alanine glutamine, non-essential amino acids, and essential amino acids is 0.4291 g / L.

[0022] The technical feature is that the mass ratio of alanine glutamine, non-essential amino acids and essential amino acids is selected from (0.05-0.2): (0.03-0.05): (0.2-0.45).

[0023] The preferred mass ratio of alanine glutamine, non-essential amino acids, and essential amino acids is (0.05-0.11):(0.03-0.041):(0.24-0.28).

[0024] The preferred mass ratio of alanine glutamine, non-essential amino acids, and essential amino acids is (0.0547-0.1086): (0.0355-0.041): (0.2441-0.2795).

[0025] The preferred mass ratio of alanine glutamine, non-essential amino acids, and essential amino acids is 0.1086:0.041:0.2795.

[0026] The technical characteristic of the embryo culture medium is selected from the following dosage relationships: 4.5-6.5 g / L sodium chloride, 0.3-0.6 g / L potassium chloride, 0.02-0.05 g / L potassium dihydrogen phosphate, 0.1-0.4 g / L calcium chloride, 0.1-0.4 g / L magnesium sulfate, 2-2.5 g / L sodium bicarbonate, 1.5-2.5 g / L sodium lactate solution, and 0.01-0.05 g / L pyruvate. Sodium, 0.1-0.3 g / L glucose, 0.002-0.005 g / L disodium edetate, 0.01-0.05 g / L gentamicin sulfate, 3-5 g / L human serum albumin, 0.005-0.008 g / L taurine, 0.05-0.2 g / L alanylglutamine, 0.03-0.05 g / L non-essential amino acids, and 0.2-0.3 g / L essential amino acids.

[0027] The preferred dosage relationships for the embryo culture medium are as follows: 4.5-5.6 g / L sodium chloride, 0.30-0.42 g / L potassium chloride, 0.02-0.04 g / L potassium dihydrogen phosphate, 0.15-0.27 g / L calcium chloride, 0.16-0.25 g / L magnesium sulfate, 2.00-2.11 g / L sodium bicarbonate, 1.5-2.0 g / L sodium lactate solution, and 0.01-0.04 g / L sodium pyruvate. 0.10-0.18 g / L glucose, 0.002-0.004 g / L disodium edetate, 0.01-0.012 g / L gentamicin sulfate, 3.00-3.13 g / L human serum albumin, 0.005-0.0065 g / L taurine, 0.05-0.11 g / L alanylglutamine, 0.03-0.045 g / L non-essential amino acids, and 0.24-0.28 g / L essential amino acids.

[0028] The optimal ratio of the amounts of embryo culture medium used in this technical feature is as follows: 4.5546-5.5518 g / L sodium chloride, 0.3100-0.4100 g / L potassium chloride, 0.0240-0.0340 g / L potassium dihydrogen phosphate, 0.1522-0.2646 g / L calcium chloride, 0.1674-0.2465 g / L magnesium sulfate, 2.0125-2.1003 g / L sodium bicarbonate, 1.5717-1.9617 g / L sodium lactate solution, and 0.0114-0.0352 g / L sodium chloride. Sodium pyruvate (g / L), glucose (0.1128-0.1802g / L), disodium edetate (0.0021-0.0037g / L), gentamicin sulfate (0.0100-0.0111g / L), human serum albumin (3.0147-3.1247g / L), taurine (0.005-0.0063g / L), alanylglutamine (0.0547-0.1086g / L), non-essential amino acids (0.0355-0.041g / L), and essential amino acids (0.2441-0.2795g / L).

[0029] The preferred ratio of the amounts of embryo culture medium is as follows: 5.5518 g / L sodium chloride, 0.41 g / L potassium chloride, 0.0340 g / L potassium dihydrogen phosphate, 0.2646 g / L calcium chloride, 0.2465 g / L magnesium sulfate, 2.1003 g / L sodium bicarbonate, 1.9617 g / L sodium lactate solution, 0.0352 g / L sodium pyruvate, 0.1802 g / L glucose, 0.0037 g / L disodium edetate, 0.01 g / L gentamicin sulfate, 3.0147 g / L human serum albumin, 0.0063 g / L taurine, 0.1086 g / L alanylglutamine, 0.041 g / L non-essential amino acids, and 0.2795 g / L essential amino acids.

[0030] Among them, the dosage relationship of the non-essential amino acids in the technical feature is selected from: 0.004-0.006 g / L alanine, 0.006-0.008 g / L asparagine, 0.005-0.008 g / L aspartic acid, 0.006-0.008 g / L glutamic acid, 0.003-0.005 g / L glycine, 0.005-0.008 g / L proline, and 0.005-0.008 g / L serine.

[0031] The preferred mass ratio of alanine glutamine, non-essential amino acids, and essential amino acids is as follows: 0.0045-0.006 g / L alanine, 0.006-0.0075 g / L asparagine, 0.0067-0.008 g / L aspartic acid, 0.006-0.0074 g / L glutamic acid, 0.0038-0.005 g / L glycine, 0.0058-0.008 g / L proline, and 0.0053-0.008 g / L serine.

[0032] The preferred ratio of non-essential amino acids in the technical features is as follows: 0.0045 g / L alanine, 0.0075 g / L asparagine, 0.0067 g / L aspartic acid, 0.0074 g / L glutamic acid, 0.0038 g / L glycine, 0.0058 g / L proline, and 0.0053 g / L serine.

[0033] The essential amino acids used in the technical features are selected from the following amounts: 0.06-0.08 g / L arginine, 0.01-0.03 g / L cysteine, 0.02-0.04 g / L histidine, 0.02-0.04 g / L isoleucine, 0.02-0.04 g / L leucine, 0.03-0.05 g / L lysine, 0.007-0.009 g / L methionine, 0.01-0.03 g / L phenylalanine, 0.02-0.04 g / L threonine, 0.005-0.007 g / L tryptophan, 0.01-0.03 g / L tyrosine, and 0.02-0.04 g / L valine.

[0034] The optimal ratio of essential amino acids for the technical features is as follows: 0.0632 g / L arginine, 0.0120 g / L cysteine, 0.0210 g / L histidine, 0.0262 g / L isoleucine, 0.0262 g / L leucine, 0.0365 g / L lysine, 0.0075 g / L methionine, 0.0165 g / L phenylalanine, 0.0238 g / L threonine, 0.0051 g / L tryptophan, 0.0181 g / L tyrosine, and 0.0234 g / L valine.

[0035] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred solution: the total concentration of alanylglutamine, non-essential amino acids, and essential amino acids. This technical solution, having already addressed the technical problem of "reducing the ammonium ion concentration during cell culture medium use," further addresses the technical problem of "further reducing the ammonium ion concentration during cell culture medium use."

[0036] The second preferred option is the mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids. This technical solution, building upon the existing solution to "reducing the ammonium ion concentration during cell culture medium use," further addresses the technical problem of "further reducing the ammonium ion concentration during cell culture medium use."

[0037] The third priority solution: the relationship between the amount of embryo culture medium used. This technical solution, having already solved the technical problem of "reducing the ammonium ion concentration during the use of cell culture medium", further addresses the technical problem of "further reducing the ammonium ion concentration during the use of cell culture medium".

[0038] Secondly, the present invention provides a method for preparing an embryo culture medium, comprising the following steps: dissolving sodium chloride, potassium chloride, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, sodium bicarbonate, sodium lactate solution, sodium pyruvate, glucose, disodium edetate, gentamicin sulfate, human serum albumin, taurine, alanine glutamine, non-essential amino acids and essential amino acids in water until the solution is clear and transparent.

[0039] Thirdly, the present invention provides the application of the above-mentioned embryo culture medium in the preparation of assisted reproductive products.

[0040] Fourthly, a product for assisted reproduction.

[0041] Among them, products with technical features include assisted reproductive technology.

[0042] Among them, the products with technical features for assisted reproduction are selected from: assisted reproduction kits.

[0043] Among them, the assisted reproductive technology kits are selected from: in vitro embryo preservation kits and embryo culture kits.

[0044] Embodiments 1-3 of this invention at least support the protection scope of claim 1.

[0045] Regarding the claim 1: The technical feature “total concentration of alanylglutamine, non-essential amino acids and essential amino acids 0.3-0.7 g / L” is derived from the common feature “total concentration of alanylglutamine, non-essential amino acids and essential amino acids” summarized from the aforementioned explanation and / or the corresponding technical features in Examples 1-3, which are 0.4291 g / L, 0.3343 g / L, and 0.6584 g / L. Therefore, those skilled in the art can reasonably presume that the technical features alanylglutamine, the total concentration of non-essential and essential amino acids, the subordinate concepts of the total concentration of alanylglutamine, non-essential and essential amino acids, the technical means that are substantially equivalent to the total concentration of alanylglutamine, non-essential and essential amino acids, and the technical means that can replace the total concentration of alanylglutamine, non-essential and essential amino acids based on the existing level of technology and common knowledge should all fall within the protection scope of claim 1. For example, replacing the total concentration of alanylglutamine, non-essential and essential amino acids with the total molar concentration of alanylglutamine, non-essential and essential amino acids, or the total volume concentration of alanylglutamine, non-essential and essential amino acids while keeping other technical features unchanged still falls within the protection scope of claim 1 of this invention.

[0046] The technical feature “the mass ratio of alanylglutamine, non-essential amino acids and essential amino acids is (0.05-0.2):(0.03-0.05):(0.2-0.45)” is derived from the corresponding technical features in the foregoing explanation and / or Examples 1-3, such as the mass ratios of alanylglutamine, non-essential amino acids and essential amino acids of 0.1086:0.041:0.2795, 0.0547:0.0355:0.2441, 0.1907:0.0497:0.418, etc., which are summarized from the common feature “mass ratio of alanylglutamine, non-essential amino acids and essential amino acids”. Therefore, those skilled in the art can reasonably presume that the mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids, the subordinate concept of the mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids, the technical means that are substantially equivalent to the mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids, and the technical means that can replace the mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids based on the existing level of technology and within the scope of conventional technical means and common knowledge, should all fall within the protection scope of claim 1. For example, if the mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids is replaced with the molar ratio of alanylglutamine, non-essential amino acids, and essential amino acids, or the volume ratio of alanylglutamine, non-essential amino acids, and essential amino acids while other technical features remain unchanged, it still falls within the protection scope of claim 1 of this invention.

[0047] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: 1. Compared with the prior art, the present invention has better technical effects in reducing the concentration of ammonium ions during the use of cell culture medium.

[0048] According to experimental tests, this invention reduces the ammonium ion concentration in the cell culture medium during use from the existing technology's concentrations of 0.1 mM for 24h, 0.112 mM for 48h, 0.236 mM for 72h, and 0.325 mM for 96h to below 0.064 mM for 24h, 0.082 mM for 48h, 0.168 mM for 72h, and 0.259 mM for 96h.

[0049] Furthermore, based on the present invention: 1. Based on the comparison of Example 1 and Comparative Examples 1-3, the present invention employs a combination of specific technical means, including the total concentration of alanylglutamine, non-essential amino acids, and essential amino acids; a specific mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids; and a specific mass ratio of human serum albumin to alanylglutamine in the embryo culture medium. This combination achieves a new technical effect by reducing the ammonium ion concentration during the use of the cell culture medium. The combined technical effect is superior to the sum of the effects of each individual technical means. Detailed Implementation

[0050] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0051] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0052] The human serum albumin of this invention was purchased from Shenzhen Weiguang Biological Products Co., Ltd.

[0053] Example 1 The bill of materials for Example 1 is shown in Table 1.

[0054] Table 1

[0055] In Example 1, the mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids was 0.1086:0.041:0.2795.

[0056] In Example 1, the total concentration of alanine glutamine, non-essential amino acids, and essential amino acids was 0.4291 g / L.

[0057] In Example 1, the mass ratio of human serum albumin to alanine glutamine was 3:0.1086.

[0058] Preparation method of Example 1: (1) Preparation of non-essential amino acid stock solution (200×): Digital display heated magnetic stirrer (hereinafter referred to as "stirrer"), turn on, temperature set to 25℃, speed 300 r / min, use these parameters for stirring throughout. Add 50 mL of water for injection to a 100 mL beaker, add the rotor and place it on the stirrer. Weigh the 7 non-essential amino acids sequentially according to the dosage relationship in Example 1 using a balance and add them to the beaker. After the sample is fully dissolved until clear and transparent, transfer it to a 100 mL volumetric flask. Wash the beaker with water for injection, and transfer the rinsing solution to the volumetric flask. Repeat step 4 at least 3 times and then make up to volume.

[0059] (2) Preparation of essential amino acid stock solution (100×): Turn on the digital display heated magnetic stirrer (hereinafter referred to as the "stirrer"), set the temperature to 25℃ and the rotation speed to 300 r / min, and use these parameters for stirring throughout the process. Add about 50 mL of injection solution to a 100 mL beaker and place the rotor on the stirrer. Weigh the essential amino acids (excluding cystine and tyrosine) sequentially according to the dosage relationship in Example 1 using a balance and add them to the beaker. After the sample is fully dissolved until clear and transparent, transfer it to a 100 mL volumetric flask. Rinse the beaker with an appropriate amount of water for injection, and transfer the rinsing solution to the volumetric flask. Repeat step 4 at least 3 times and then make up to volume.

[0060] (3) Prepare the disodium edetate stock solution (1000×): Turn on the digital display heated magnetic stirrer (hereinafter referred to as the "stirrer"), set the temperature to 25℃ and the rotation speed to 300 r / min, and use these parameters for stirring throughout the process. Add 50 mL of injection solution to a 100 mL beaker and place the rotor on the stirrer. Weigh the disodium edetate into the beaker using a balance according to the dosage relationship in Example 1. After the sample is fully dissolved until clear and transparent, transfer it to a 100 mL volumetric flask. Rinse the beaker from step 2 with an appropriate amount of water for injection, and transfer the rinsing solution to the volumetric flask. Repeat step 4 at least 3 times and then make up to volume.

[0061] (4) Preparation of taurine stock solution (200×): Turn on the digital display heated magnetic stirrer (hereinafter referred to as the "stirrer"), set the temperature to 25℃ and the rotation speed to 300 r / min, and use these parameters for stirring throughout the process. Add 50 mL of injection solution to a 100 mL beaker and place the rotor on the stirrer. Weigh the taurine according to the dosage relationship in Example 1 using a balance and add it to the beaker. After the sample is fully dissolved until clear and transparent, transfer it to a 100 mL volumetric flask. Rinse the beaker with water for injection, and transfer the rinsing solution to the volumetric flask. Repeat step 4 at least 3 times and then make up to volume.

[0062] (5) Prepare embryo culture medium (1L): 1) Digital display heated magnetic stirrer (hereinafter referred to as "stirrer"), turn on, temperature set to 25℃, speed 300 r / min, use these parameters for stirring throughout. Add 400 mL of injection water to a 1000 mL beaker and put it into the rotor.

[0063] 2) Take 5.5518 g of sodium chloride, accurately weigh it using a ten-thousandth balance, and slowly and carefully add it to the above 1000 mL beaker. Stir until clear and free of solid particles to form mixture A.

[0064] 3) Weigh 0.0120 g of cystine and 0.0181 g of tyrosine using a balance. Slowly and carefully add them to mixture A, stir at 300 r / min for 10 min, and sonicate for about 60 min. Stir with a glass rod for 2-3 min every 20 minutes until clear and free of solid particles, forming mixture B.

[0065] 4) Place a 25 mL empty beaker in a balance and zero it. Take 1 / 20 of the weight of the non-essential amino acid mother liquor. The balance should be set to a weighing error of ±1%. Slowly and carefully add the mixture B and rinse the beaker with water for injection at least 3 times. Add the mixture B to form the mixture C.

[0066] 5) Place a 25 mL empty beaker in a balance and zero it. Take 1 / 10 of the weight of the essential amino acid stock solution. The balance should be set to ±1% of the original weight. Slowly and carefully add the mixture C and rinse the beaker with water for injection at least 3 times. Add the mixture C to form the mixture D.

[0067] 6) Take 0.4100 g of potassium chloride, weigh it using a ten-thousandth balance, and slowly and carefully add it to mixture D. Stir until it is clear and free of solid particles to form mixture E.

[0068] 7) Take 0.2465 g of magnesium sulfate, weigh it using a ten-thousandth balance, and slowly and carefully add it to mixture E. Stir until it is clear and free of solid particles to form mixture F.

[0069] 8) Take 0.0340 g of potassium dihydrogen phosphate, weigh it using a ten-thousandth balance, and slowly and carefully add it to the mixture F. Stir until it is clear and free of solid particles to form the mixture G.

[0070] 9) Place a 25 mL empty beaker in a balance and zero it. Take 0.2646 g of calcium chloride and weigh it using a ten-thousandth balance. Add water for injection until all the calcium chloride is fully dissolved. Slowly and carefully add the fully dissolved calcium chloride to the mixture G. Rinse the beaker with water for injection at least 3 times for any residual calcium chloride. Add the mixture G and stir until clear and free of solid particles to form the mixture H.

[0071] 10) Weigh 0.0352 g of sodium pyruvate using a ten-thousandth balance, slowly and carefully add it to mixture H, and stir until clear and free of solid particles to form mixture I.

[0072] 11) Take 0.1802 g of anhydrous glucose, weigh it using a ten-thousandth balance, and slowly and carefully add it to mixture I. Stir until clear and free of solid particles to form mixture J.

[0073] 12) Take 0.2172 g of alanine glutamine, weigh it using a ten-thousandth balance, and slowly and carefully add it to the mixture J. Stir until it is clear and free of solid particles to form the mixture K.

[0074] 13) Weigh 0.0100 g of gentamicin sulfate using a 1 / 10,000 balance, and slowly and carefully add it to the mixture K. Stir until clear and free of solid particles, forming a mixture L.

[0075] 14) Place a 25 mL empty beaker in a balance and zero it. Take 1 / 100 of the weight of the disodium edetate mother solution, and weigh it with a balance of ±1%. Slowly and carefully add the mixture L, and rinse the beaker with water for injection to form the mixture M.

[0076] 15) Place a 25 mL empty beaker in the balance and zero it. Take 1 / 20 of the weight of the taurine mother liquor. The balance should be set to a weighing error of ±1%. Slowly and carefully add the mixture M and rinse the beaker with water for injection to form the mixture N.

[0077] 16) Place a 25 mL empty beaker in a zero balance, weigh 1.9617 g of sodium lactate solution into the beaker, and weigh it precisely using a ten-thousandth balance. Slowly and carefully add the mixture N, rinse the beaker with water for injection at least 3 times to remove any residual sodium lactate solution, add the mixture N, and stir until clear to form mixture O.

[0078] 17) Add water for injection to about 600 mL, weigh 2.1003 g of sodium bicarbonate using a ten-thousandth balance, and slowly and carefully add it to mixture O. Stir until clear and free of solid particles to form mixture P.

[0079] 18) Place a 100 mL empty beaker in a zero position on a balance, weigh 26.1675 g of human serum albumin into the beaker, weigh it accurately, slowly and carefully add the mixture P, rinse the beaker three times with water for injection to remove any residual human serum albumin, add the mixture P, stir until clear, and form the mixture Q.

[0080] 19) Slowly pour the mixture Q from the 1000 mL beaker into the 1000 mL volumetric flask, rinse the beaker three times with water for injection, pour it into the 1000 mL volumetric flask, and bring the volume to 1000 mL to form the mixture S.

[0081] 20) Calibration of the osmolality meter: Perform two-point calibration using purified water and a 300 mOsm / kg sodium chloride standard solution. The retest of the 300 mOsm / kg sodium chloride standard solution should yield 298-302 mOsm / kg. The S value of the mixed solution should be 260-280 mOsm / kg.

[0082] After passing the osmotic pressure test, it proceeds to aseptic filtration and filling.

[0083] Example 2 The bill of materials for Example 2 is shown in Table 2.

[0084] Table 2

[0085] In Example 2, the mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids was 0.0547:0.0355:0.2441.

[0086] In Example 2, the total concentration of alanine glutamine, non-essential amino acids, and essential amino acids was 0.3343 g / L.

[0087] In Example 2, the mass ratio of human serum albumin to alanine glutamine was 3:0.0547.

[0088] Preparation method: The preparation was carried out according to the material usage in Table 2, using the preparation method of Example 1.

[0089] Example 3 The bill of materials for Example 3 is shown in Table 3.

[0090] Table 3

[0091] In Example 3, the mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids was 0.1907:0.0497:0.418.

[0092] In Example 3, the total concentration of alanine glutamine, non-essential amino acids, and essential amino acids was 0.6584 g / L.

[0093] In Example 3, the mass ratio of human serum albumin to alanine glutamine was 5:0.1907.

[0094] Preparation method: The preparation was carried out according to the material usage in Table 3, using the preparation method of Example 1.

[0095] Comparative Example 1 The bill of materials for Comparative Example 1 is shown in Table 4.

[0096] Table 4

[0097] In Comparative Example 1, the mass ratio of alanine glutamine, non-essential amino acids, and essential amino acids was 0.2503:0.0280:0.2795.

[0098] In Comparative Example 1, the total concentration of alanine glutamine, non-essential amino acids, and essential amino acids was 0.5578 g / L.

[0099] In Comparative Example 1, the mass ratio of human serum albumin to alanine glutamine was 3:0.2503.

[0100] Comparative Example 1 was prepared according to the preparation method of Example 1, using the same dosage relationship as Comparative Example 1.

[0101] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that the concentration of human serum albumin is 5.5124 g / L.

[0102] In Comparative Example 2, the mass ratio of human serum albumin to alanine glutamine was 5.5:0.1086.

[0103] Comparative Example 3 The embryo culture medium of Example 1 in Chinese Patent CN108251355A has all range values ​​taken as the minimum value.

[0104] Example 1: Embryo Development Experiment (1) Experimental materials Sample group: Examples 1-3 and Comparative Examples 1-3; Negative control: Blastocyst culture medium (manufacturer: Nanjing Youer Biotechnology Development Co., Ltd.); Positive control: Endotoxin (manufacturer: Xiamen Limulus Amebocyte Lysate Reagent Biotechnology Co., Ltd.).

[0105] SPF grade B6D2F1 mice, sex: half male and half female; age: females 35-41 days old, males 49-55 days old.

[0106] Reagents: M2 culture medium (SIGMA, batch number: 0000310849).

[0107] Culture oil (Dongyun Medical Technology Co., Ltd., batch number: 990224002).

[0108] PMSG: Pregnant mare serum gonadotropin (Nanjing Aibei Biotechnology Co., Ltd., batch number: 2410A).

[0109] HCG: Human chorionic gonadotropin (Nanjing Aibei Biotechnology Co., Ltd., batch number: 2409A).

[0110] Ready-to-use hyaluronidase (Nanjing Aibei Biotechnology Co., Ltd., batch number: 2403A).

[0111] (2) Experimental methods According to the experimental groups, before culturing embryos, 50 μL of negative control solution, positive control solution and sample group (Example 1-3 groups and Comparative Example 1-3 groups) liquid droplets were prepared in cell culture dishes, with 5 droplets in each group. The droplets were covered with culture oil and pre-equilibrated for more than 30 minutes in an incubator at 37°C and 5% CO2 saturated humidity for embryo pretreatment and culture.

[0112] After the sample group was prepared, liquid droplets were prepared directly from the sample. For the negative control group, liquid droplets were prepared from blastocyst culture medium. For the positive control group, 120 EU of endotoxin was dissolved in M16 culture medium to prepare liquid droplets at a concentration of 120 EU / mL. All prepared liquid droplets were clear.

[0113] Female mice were injected intraperitoneally with 10 IU of PMSG per mouse, and 48 hours later, they were injected intraperitoneally with 10 IU of HCG per mouse. On the day of the HCG injection, the female mice were housed with male mice of the same strain in a 1:1 ratio overnight.

[0114] 18-22 hours after HCG injection, female mice were euthanized by cervical dislocation, and the oviducts and part of the uterus were removed. The ampulla of the oviduct was torn open with microforceps to obtain the oocyte cluster. The granulosa cells were removed by digestion with hyaluronidase preheated to 37°C, and the cells were washed clean with M2 culture medium.

[0115] The microdroplet culture method was used, and the collected mouse embryos were randomly divided into 8 groups, with no less than 50 embryos in each group; Treatment of experimental group mouse embryos: The experimental group embryos were placed in equilibrated experimental sample droplets without any other treatment and cultured in an incubator at 37°C and 5% CO2 saturated humidity for 96 hours.

[0116] Embryos from the negative and positive control groups were placed in equilibrated M16 culture medium without any other treatment and cultured for 96 hours in an incubator at 37°C and 5% CO2 saturated humidity.

[0117] A well-developed blastocyst has a fully expanded blastocoel, a moderately sized inner cell mass, and tightly connected, uniformly sized trophoblast cells. A poorly developed blastocyst has a small blastocoel, a small or absent inner cell mass, and sparse trophoblast cells.

[0118] Blastocyst formation rate = (Number of well-developed blastocysts / Total number of blastocysts) × 100%.

[0119] The experimental results of blastocyst formation rate are shown in Table 5.

[0120] Table 5

[0121] (3) Changes in ammonium ion concentration During the 96-hour incubation period, changes in ammonium ion concentration were detected at 0h, 24h, 48h, 72h, and 96h.

[0122] The amount of ammonium salts produced by a single mouse embryo at each developmental stage was determined using ultra-micro fluorescence technology. This technique is a miniaturization of traditional biochemical analysis, using a quantitative fluorescence microscope as a micro-fluorometer.

[0123] The following assay methods and reagents were used to analyze ammonium salts:

[0124] The reagent composition was as follows: 0.24 mM NADH, 0.75 mM α-ketoglutarate, 0.63 mM ADP, 14.15 mM sodium bicarbonate, and 3 U / ml glutamate dehydrogenase, dissolved in 157 mM triethanolamine buffer, pH 8.0. A calibration curve was run for each experiment using 0–0.5 mM ammonium chloride. The mean coefficient of determination for the calibration curves was 0.996.

[0125] By taking out a culture medium sample and reacting it with the above reagents (reaction ratio: 1:3, when ammonium salt (NH4) + In the presence of ), NADH is oxidized to NAD. + The fluorescence intensity decreases as NADH is consumed, and the ammonium salt concentration can be calculated by measuring the fluorescence change.

[0126] The ammonium salt concentration was calculated using the calibration curve of 0-0.5 mM ammonium chloride, and the experimental results are shown in Table 6.

[0127] Table 6

[0128] Verification of technical effectiveness and / or analysis of technical problem solving The blastocyst formation rate in the positive control group was significantly lower than that in the negative control group, at only 0%.

[0129] The blastocyst formation rate of the negative control group and the groups in Examples 1-3 was ≥94%.

[0130] Although the blastocyst formation rate of Comparative Examples 1-3 reached 90%, their ammonium ion concentration was much higher than that of Examples 1-3, exceeding 0.3 mM, which would affect the development of the embryos after transplantation.

[0131] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An embryo culture medium, comprising: The ingredients include sodium chloride, potassium chloride, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, sodium bicarbonate, sodium lactate solution, sodium pyruvate, glucose, disodium edetate, gentamicin sulfate, human serum albumin, taurine, alanylglutamine, non-essential amino acids, and essential amino acids; the total concentration of alanylglutamine, non-essential amino acids, and essential amino acids is 0.3-0.7 g / L; the mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids is (0.05-0.2):(0.03-0.05):(0.2-0.45); and the mass ratio of human serum albumin to alanylglutamine is (3-5):(0.05-0.2).

2. The embryo culture medium according to claim 1, characterized in that, The total concentration of alanylglutamine, non-essential amino acids, and essential amino acids is further preferably: 0.3343-0.4291 g / L.

3. The embryo culture medium according to claim 1, characterized in that, The mass ratio of alanylglutamine, non-essential amino acids, and essential amino acids is (0.0547-0.1086): (0.0355-0.041): (0.2441-0.2795).

4. The embryo culture medium according to any one of claims 1-3, characterized in that, The components include the following concentrations: 4.5-6.5 g / L sodium chloride, 0.3-0.6 g / L potassium chloride, 0.02-0.05 g / L potassium dihydrogen phosphate, 0.1-0.4 g / L calcium chloride, 0.1-0.4 g / L magnesium sulfate, 2-2.5 g / L sodium bicarbonate, 1.5-2.5 g / L sodium lactate solution, 0.01-0.05 g / L sodium pyruvate, 0.1-0.3 g / L glucose, 0.002-0.005 g / L disodium edetate, 0.01-0.05 g / L gentamicin sulfate, 3-5 g / L human serum albumin, 0.005-0.008 g / L taurine, 0.05-0.2 g / L alanylglutamine, 0.03-0.05 g / L non-essential amino acids, and 0.2-0.3 g / L essential amino acids.

5. The embryo culture medium according to claim 4, characterized in that, include: 4.5-5.6 g / L sodium chloride, 0.30-0.42 g / L potassium chloride, 0.02-0.04 g / L potassium dihydrogen phosphate, 0.15-0.27 g / L calcium chloride, 0.16-0.25 g / L magnesium sulfate, 2.00-2.11 g / L sodium bicarbonate, 1.5-2.0 g / L sodium lactate solution, 0.01-0.04 g / L sodium pyruvate, 0.10-0.18 g / L glucose, 0.002-0.004g / L disodium edetate, 0.01-0.012g / L gentamicin sulfate, 3.00-3.13g / L human serum albumin, 0.005-0.0065g / L taurine, 0.05-0.11g / L alanylglutamine, 0.03-0.045g / L non-essential amino acids and 0.24-0.28g / L essential amino acids.

6. The embryo culture medium according to claim 1, characterized in that, The non-essential amino acids include: 0.004-0.006 g / L alanine, 0.006-0.008 g / L asparagine, 0.005-0.008 g / L aspartic acid, 0.006-0.008 g / L glutamic acid, 0.003-0.005 g / L glycine, 0.005-0.008 g / L proline, and 0.005-0.008 g / L serine; the essential amino acids include: 0.06-0.08 g / L arginine, 0.01-0.03 g / L glutamic acid, and 0.005-0.008 g / L serine. 0.02-0.04 g / L cystine, 0.02-0.04 g / L histidine, 0.02-0.04 g / L isoleucine, 0.02-0.04 g / L leucine, 0.03-0.05 g / L lysine, 0.007-0.009 g / L methionine, 0.01-0.03 g / L phenylalanine, 0.02-0.04 g / L threonine, 0.005-0.007 g / L tryptophan, 0.01-0.03 g / L tyrosine, 0.02-0.04 g / L valine.

7. The embryo culture medium according to claim 1, characterized in that, The mass ratio of human serum albumin to alanine glutamine is 3:(0.0547-0.1086).

8. The method for preparing the embryo culture medium according to any one of claims 1-7, characterized in that, Includes the following steps: Sodium chloride, potassium chloride, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, sodium bicarbonate, sodium lactate solution, sodium pyruvate, glucose, disodium edetate, gentamicin sulfate, human serum albumin, taurine, alanine glutamine, non-essential amino acids and essential amino acids are dissolved in water until clear and transparent.

9. The use of the embryo culture medium according to any one of claims 1-7 in the preparation of assisted reproductive products.

10. A product for assisted reproduction, characterized in that, Includes the embryo culture medium according to any one of claims 1-7.

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