A method for in vitro rotational culture of embryos

Abstract

The application belongs to the technical field of biology and particularly relates to a method for in-vitro rotation culture of embryos. The method places the embryos in embryo culture solution for culture, wherein the embryo culture solution comprises fresh rat serum, antibiotics, ascorbic acid or derivatives thereof, pyruvic acid or salts thereof, glutamine, buffer salt and optionally glucose; the embryo culture solution does not contain human umbilical cord blood serum; the use of human umbilical cord blood serum is avoided, so that the embryo rotation culture system can be quickly popularized; and further limitation is made to the use of fresh rat serum, adjustment of oxygen concentration at different periods, which can improve the proportion of embryos with continued blood flow in the yolk sac blood vessels at the E7.5+3 day period, realize the rapid blood flow in the yolk sac blood vessels of mouse embryos at the E7.5+5 day period, and complete the in-vitro culture process of mouse embryos from E7.5 to E7.5+6 day.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a method for in vitro rotation culture of embryos. Background Technology

[0002] Understanding the developmental processes of tissue and organ formation is a fundamental question in developmental biology. In mammals, this process occurs after embryo implantation in the uterus, making observation and manipulation relatively difficult. Therefore, the process from gastrulation to organogenesis (heart, lungs, etc.) remains incompletely understood and difficult to intervene artificially. Currently, in vitro culture of mouse embryos is limited to the period from E7.5 to E11. E11 is considered late organogenesis, where many organs, such as the mouse lungs, have only completed one branch on each side, and organs like the liver and kidneys are still in the primordial stage. However, mouse embryos at E12.5 and E13.5 have already passed the organogenesis stage and are considered fetal. The mouse lungs have completed the structure of one branch on the left and four branches on the right, the liver has hematopoietic function, and the kidneys are visible to the naked eye on both sides. The basic shapes of other organs are not significantly different from those of organs in later stages. Therefore, culturing mouse embryos from E7.5 to E12.3 and even E13.5 is more conducive to studying the early organogenesis and developmental processes of mouse embryos in vitro.

[0003] Traditional in vitro mouse embryo culture systems (from E6.5 to E8.5), while having some application value in embryonic development research, also have some significant drawbacks. For example, they lack data on organ development (heart, lungs, etc.). Traditional static culture systems typically only provide a two-dimensional culture environment, failing to fully simulate the three-dimensional development of the embryo under natural maternal physiological conditions. Oxygen and nutrients in the culture medium may be unevenly distributed in a static environment, leading to unbalanced nutrient acquisition by the embryo. Furthermore, in a static culture environment, embryos may settle at the bottom of the culture flask due to gravity, potentially causing morphological abnormalities or physical damage, ultimately resulting in a lower embryonic development rate.

[0004] As research progresses, novel culture methods such as rotating culture systems can support embryos to achieve better in vitro developmental capabilities. In 2021, the Jacob H. Hanna laboratory in Israel optimized the embryo rotating culture system and published their findings in Nature. Mouse embryos were cultured from the E7.5 stage for four days further, eventually reaching the E11 stage. The article describes how the gas pressure in the culture flask was adjusted by changing the pressure, and the number of expelled bubbles was controlled by adjusting the vent valve. Using a culture medium consisting of 25% DMEM, 50% frozen rat serum, and 25% human umbilical cord blood serum, E7.5 embryos were cultured in vitro, yielding E11 embryos with a developmental efficiency of 75%.

[0005] However, Hanna's laboratory's rotary culture device regulates the number of bubbles expelled by adjusting pressure and the vent valve, making it impossible to calculate the volume of gas passing through the culture flask. This operation is cumbersome, and whether pressure affects embryonic development requires further verification. Furthermore, the culture medium contains human umbilical cord blood serum, which must be obtained from mothers who have given birth vaginally without diabetes or infectious diseases. Each mother can only obtain a maximum of 30-50 mL of umbilical cord blood serum, making it extremely difficult to obtain and hindering the widespread use of the rotary embryo culture system. In the article, mouse embryos were only cultured to the E11 stage, with a culture efficiency of only about 75%. Summary of the Invention

[0006] The first aspect of the present invention is to provide a method for in vitro rotation culture of embryos.

[0007] The second aspect of the present invention aims to provide the application of embryos obtained by the culture method of the first aspect of the present invention.

[0008] A third aspect of the present invention is to provide an embryo culture medium.

[0009] The fourth aspect of this invention aims to provide a whole embryo in vitro rotation culture system.

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

[0011] In a first aspect, the present invention provides a method for in vitro rotational culture of embryos, wherein an embryo is placed in an embryo culture medium for culture, the embryo culture medium comprising fresh rat serum, antibiotics, ascorbic acid or a derivative thereof, pyruvate or a salt thereof, glutamine, buffer salts, and optionally glucose; the embryo culture medium does not contain human umbilical cord blood serum.

[0012] In some embodiments, the antibiotic comprises at least one of amphotericin B, nystatin, gentamicin, tetracycline, erythromycin, penicillin, and streptomycin; more particularly penicillin and streptomycin.

[0013] In some embodiments, the ascorbic acid or its derivative comprises at least one selected from ascorbic acid, calcium ascorbate, magnesium ascorbate, zinc ascorbate, potassium ascorbate, sodium ascorbate, dehydroascorbic acid, L-threonic acid, L-xylitolic acid, L-lythreonic acid, L-ascorbic acid monostearate, L-ascorbic acid dipalmitoate, L-ascorbic acid 6-hexadecanoic acid compound, L-ascorbic acid 2-phosphide, L-ascorbic acid 3-phosphide, and L-ascorbic acid 2-sulfate; further comprising ascorbic acid (vitamin C).

[0014] In some embodiments, the pyruvate or its salt is sodium pyruvate.

[0015] In some embodiments, the glutamine is L-glutamine; more specifically, it is GlutaMax.

[0016] In some embodiments, the buffer salt comprises at least one of phosphate, Tris, and HEPES; more specifically, HEPES.

[0017] In some embodiments, a mixed gas containing oxygen and carbon dioxide is introduced into the culture system at a gas flow rate of 12.5-200 mL / min.

[0018] In some embodiments, the gas flow rate is 12.5-60 mL / min; further, 25-60 mL / min; and even further, 45-55 mL / min.

[0019] In some embodiments, the gas pressure in the culture system is 0.1-15 psi; more specifically, 0.1-13 psi.

[0020] In some embodiments, the embryo culture medium includes embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, and optionally embryo culture medium 4, embryo culture medium 5 and / or embryo culture medium 6;

[0021] The embryo culture medium 1 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine and buffer salts, wherein the concentration of ascorbic acid or its derivatives in the embryo culture medium 1 is P μg / mL;

[0022] The embryo culture medium 2 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine, buffer salts and glucose, wherein the concentration of ascorbic acid or its derivatives in the embryo culture medium 2 is P+300 μg / mL and the concentration of glucose in the embryo culture medium 2 is N mg / mL.

[0023] The embryo culture medium 3 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine, buffer salts and glucose. The concentration of ascorbic acid or its derivatives in the embryo culture medium 3 is P+700 μg / mL, and the concentration of glucose in the embryo culture medium 3 is N+0.5 mg / mL.

[0024] The embryo culture medium 4 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine, buffer salts and glucose, wherein the concentration of ascorbic acid or its derivatives in the embryo culture medium 4 is P+700 μg / mL and the concentration of glucose in the embryo culture medium 4 is N+1 mg / mL.

[0025] The embryo culture medium 5 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine, buffer salts and glucose, wherein the concentration of ascorbic acid or its derivatives in the embryo culture medium 5 is P+700 μg / mL and the concentration of glucose in the embryo culture medium 5 is N+1.5 mg / mL.

[0026] The embryo culture medium 6 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine, buffer salts and glucose, wherein the concentration of ascorbic acid or its derivatives in the embryo culture medium 6 is P+700 μg / mL and the concentration of glucose in the embryo culture medium 6 is N+2 mg / mL.

[0027] The value of N is 2-4; further, it is 2.5-3.5; even further, it is 2.9-3.1;

[0028] The value of P is 25-200; further, it is 50-150; and even further, it is 95-105.

[0029] In some embodiments, the embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 do not contain human umbilical cord blood serum.

[0030] In some embodiments, the antibiotics in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 are each independently selected from at least one of amphotericin B, nystatin, gentamicin, tetracycline, erythromycin, penicillin and streptomycin; more specifically penicillin and streptomycin.

[0031] In some embodiments, the ascorbic acid or its derivative in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5, and embryo culture medium 6 is each independently selected from at least one of ascorbic acid, calcium ascorbate, magnesium ascorbate, zinc ascorbate, potassium ascorbate, sodium ascorbate, dehydroascorbic acid, L-threonic acid, L-xylitolic acid, L-lythreonic acid, L-ascorbic acid monostearate, L-ascorbic acid dipalmitoate, L-ascorbic acid 6-hexadecanoic acid compound, L-ascorbic acid 2-phosphoside, L-ascorbic acid 3-phosphoside, and L-ascorbic acid 2-sulfate; further, it is ascorbic acid (vitamin C).

[0032] In some embodiments, the pyruvate or its salt in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is sodium pyruvate.

[0033] In some embodiments, the glutamine in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is L-glutamine; more specifically, it is GlutaMax.

[0034] In some embodiments, the buffer salts in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 are each independently selected from at least one of phosphate, Tris, and HEPES; more specifically, HEPES.

[0035] In some embodiments, the concentration of the antibiotic in the embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is independently 0.2%-2% by volume; further 0.5%-1.5%; and even further 0.9%-1.1%.

[0036] In some embodiments, the concentration of the buffer salt in each of the embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is independently 3-11 mM; further, 4-7 mM; and even further, 5-6 mM.

[0037] In some embodiments, the concentration of pyruvate or its salt in the embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is independently 0.5-2 mM; further 0.6-1.4 mM; and even further 0.9-1.1 mM.

[0038] In some embodiments, the concentration of glutamine in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is independently 1-3 mM; further 1.2-2.8 mM; and even further 1.8-2.2 mM.

[0039] In some embodiments, the in vitro embryo rotation culture method includes the following steps:

[0040] 1) Place the embryos in embryo culture medium 1 for the first culture;

[0041] 2) Embryos cultured for the first time are placed in embryo culture medium 2 for a second culture;

[0042] 3) The embryos that have undergone the second culture are placed in embryo culture medium 3 for a third culture.

[0043] In some embodiments, the in vitro embryo rotation culture method includes the following steps:

[0044] 4) The embryos that have undergone the third culture are placed in embryo culture medium 4 for a fourth culture.

[0045] In some embodiments, the in vitro embryo rotation culture method includes the following steps:

[0046] 5) Embryos that have undergone the fourth culture are placed in embryo culture medium 5 for a fifth culture;

[0047] 6) Embryos cultured for the fifth time are placed in embryo culture medium 6 for a sixth culture.

[0048] In some embodiments, before the embryos that have undergone the fifth culture are placed in the embryo culture medium 6 for the sixth culture, the following steps are also included: separating the yolk sac and amnion, while retaining the placental cone portion; preferably, this is done when the blood in the yolk sac is not flowing, and only the blood of the embryo itself is flowing.

[0049] In some embodiments, each of the first, second, third, fourth, fifth, and sixth culture systems is independently introduced with a mixed gas containing oxygen and carbon dioxide at a gas flow rate of 12.5-200 mL / min.

[0050] In some embodiments, the gas flow rate is 12.5-60 mL / min; further, 25-60 mL / min; and even further, 45-55 mL / min.

[0051] In some embodiments, the gas pressure in the first, second, third, fourth, fifth, and sixth culture systems is independently 0.1-15 psi; more specifically, 0.1-13 psi.

[0052] In some embodiments, the carbon dioxide content in the first, second, third, fourth, fifth, and sixth culture systems is independently 3%-7% by volume; further, it is 4%-6%.

[0053] In some embodiments, the temperatures for the first, second, third, fourth, fifth, and sixth cultures are each independently 33-40°C; more specifically, 36-38°C.

[0054] In some embodiments, the rotation speeds of the first, second, third, fourth, fifth, and sixth cultures are each independently 15-50 rpm; further, 20-40 rpm; and even further, 25-35 rpm.

[0055] In some implementations, the time for the first, second, third, fourth, fifth, and sixth cultivations is independently 18-30 hours; further, 20-28 hours; and even further, 22-26 hours.

[0056] In some embodiments, the oxygen content in the first culture system is M% by volume; and / or

[0057] The oxygen content in the second culture system, by volume percentage, is (M+8)%; and / or

[0058] The oxygen content in the third culture system, expressed as a volume percentage, is (M+25)%; and / or

[0059] The oxygen content in the fourth and fifth culture systems, by volume percentage, was independently (M+85)%; and / or

[0060] The oxygen content in the sixth culture system, expressed as a volume percentage, was (M+55)%.

[0061] The value of M is 4-8; further, it is 4.5-5.5; and even further, it is 4.8-5.2.

[0062] In some embodiments, the temperature of embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is 33-40°C; more specifically, it is 36-38°C.

[0063] In some embodiments, the in vitro embryo culture is performed in a whole embryo in vitro rotation culture system;

[0064] The whole embryo in vitro rotation culture system includes:

[0065] A gas mixing device includes a gas source, a gas mixer, a gas pipeline, and a gas flow and quality controller, wherein the gas source and the gas mixer are connected through the gas pipeline, and the gas flow and quality controller is disposed on the gas pipeline; and a culture device includes an incubator and a culture assembly disposed within the chamber of the incubator, wherein the culture assembly includes a turntable and culture flasks disposed on the turntable, the turntable includes a gas channel, the gas channel including a gas inlet, a culture flask interface, and a gas outlet that are interconnected, the gas inlet being connected to the gas mixer, the culture flask interface being connected to the culture flask, the gas outlet being configured to discharge waste gas, and the culture flask being configured to contain culture medium.

[0066] In some implementations, each culture involves placing the embryo and embryo culture medium (embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5, or embryo culture medium 6) in a culture flask.

[0067] In some embodiments, each culture is first placed in a culture flask and preheated to 33-40°C (preferably 36-38°C) before the embryos are transferred in.

[0068] In some embodiments, the gas source includes one or more of a nitrogen source, an oxygen source, and a carbon dioxide source.

[0069] In some embodiments, when there are multiple gas sources, the gas sources are connected to the gas mixer through corresponding gas pipelines.

[0070] In some embodiments, the gas mixing device further includes a gas detection component connected to the gas mixer to detect the proportion of gas in the gas mixer.

[0071] In some embodiments, the gas detection assembly includes an oxygen detector and / or a carbon dioxide detector.

[0072] In some embodiments, the gas mixing device further includes a filter that is in communication with the gas mixer and is located downstream of the gas mixer.

[0073] In some embodiments, the whole embryo in vitro rotation culture system further includes:

[0074] A humidity control device is disposed between the filter and the gas inlet.

[0075] In some embodiments, the humidity regulating device includes a distilled water container.

[0076] In some embodiments, the whole embryo in vitro rotation culture system further includes:

[0077] A pressure control device includes a gas cylinder, a first gas pipeline, a second gas pipeline, and a gas pressure controller. The gas cylinder is located downstream of the turntable. The inlet of the gas cylinder is connected to the gas outlet through the first gas pipeline. The outlet of the gas cylinder is connected to the second gas pipeline. The gas pressure controller is configured to control the pressure inside the gas cylinder. The valve of the gas pressure controller is located on the second gas pipeline, and the opening degree of the valve is adjustable.

[0078] In some embodiments, the whole embryo in vitro rotation culture system further includes:

[0079] A temperature control device configured to regulate the temperature of the chamber of the incubator.

[0080] In some embodiments, the temperature control device includes a constant temperature drying oven and a thermometer, wherein the incubator is the constant temperature drying oven, and the thermometer is configured to detect the temperature of the constant temperature drying oven.

[0081] In some embodiments, the embryo is derived from non-human mammals (e.g., non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), livestock (e.g., horses, cattle, sheep, pigs, rabbits)).

[0082] In some embodiments, the embryo is derived from rodents; further, from mice; and even further, from mice.

[0083] In some embodiments, the embryo is an E5.5-E12.5 embryo; more specifically, an E7-E8 embryo.

[0084] In some embodiments, the fresh rat serum refers to serum that has been in vivo from the rat for no more than 6 hours before use.

[0085] In some embodiments, the fresh rat serum is prepared as follows: rat blood is centrifuged for the first time, the obtained serum is fixed with fibrinogen, then centrifuged for the second time, the obtained serum is inactivated with complement system, and finally filtered.

[0086] In some embodiments, the first centrifugation is performed at 2000-6000 rpm for 6-20 min; further, at 3000-5000 rpm for 8-13 min.

[0087] In some embodiments, the method for fixing fibrinogen involves placing the product at 0-4°C for 1-2 hours.

[0088] In some embodiments, the conditions for the second centrifugation are 2000-6000 rpm for 3-10 min; further, 3000-5000 rpm for 4-7 min.

[0089] In some embodiments, the method for inactivating the complement system is to incubate at 50-65°C for 20-40 min; further, to incubate at 56°C for 30 min.

[0090] In some implementations, a 0.22 μm filter is used for filtration.

[0091] A second aspect of the invention provides the use of embryos obtained by the culture method of the first aspect of the invention in any one of a1)-a7):

[0092] a1) Preparation of embryo models;

[0093] a2) Embryo research;

[0094] a3) Screening for key genes causing embryonic developmental defects;

[0095] a4) Investigate the effects of knockout or overexpression libraries on embryonic development;

[0096] a5) Testing drug safety;

[0097] a6) Preparation of tissues and / or organs (such as eyes, bones, lungs, heart, intestines, liver, kidneys, head, placenta, amnion, etc.);

[0098] a7) Prepare a product, said product being used in any one of a1) to a6).

[0099] In some implementations, the product is a reagent kit or reagent kit.

[0100] A third aspect of the present invention provides an embryo culture medium, wherein the embryo culture medium is the embryo culture medium of the first aspect of the present invention.

[0101] In some embodiments, the embryo culture medium also includes an instruction manual describing a method for in vitro rotational culture of embryos according to a first aspect of the present invention.

[0102] A fourth aspect of the present invention provides a whole embryo in vitro rotation culture system, wherein the whole embryo in vitro rotation culture system is the same as the whole embryo in vitro rotation culture system in the first aspect of the present invention.

[0103] Compared with the prior art, this application includes at least the following beneficial effects:

[0104] An in vitro rotation culture method for embryos involves culturing embryos in an embryo culture medium containing fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine, buffer salts, and optionally glucose; the embryo culture medium does not contain human umbilical cord blood serum; this avoids the use of human umbilical cord blood serum, allowing for the rapid popularization of the embryo rotation culture system; and further limiting the use of fresh rat serum can increase the proportion of embryos with continued blood flow in the yolk sac vessels at the E10.5 stage.

[0105] Furthermore, by using fresh rat serum and adjusting the concentrations of vitamin C and oxygen at different stages, the proportion of embryos with continued blood flow in the yolk sac vessels at E7.5+3 days was increased, and rapid blood flow in the yolk sac vessels of mouse embryos at E7.5+5 days was achieved.

[0106] The in vitro rotation culture method for embryos provided by this invention improves the culture efficiency from E7.5 to E11.5 to 100%, and completes the in vitro culture process from E7.5 to E7.5+6 days. By E7.5+6 days, the eyes of mouse embryos are basically fully developed, the femur, patella, tibia and fibula of the limbs are basically developed, the paws are paddle-like, and the external ear is formed. In contrast, the in vitro culture efficiency of Hanna's laboratory from E7.5 to E7.5+4 days is about 75%, and only the in vitro culture process of E7.5+4 days is achieved. Attached Figure Description

[0107] Figure 1 A structural diagram of a whole embryo in vitro rotation culture system according to an embodiment of this application is shown.

[0108] Figure 2 A physical diagram of a whole embryo in vitro rotation culture system according to an embodiment of this application is shown.

[0109] Figure 3 This study demonstrates that cryopreserved rat serum causes embryonic developmental arrest.

[0110] Figure 4 The proportion of embryos that continued to have blood flow in their yolk sac vessels at the E7.5+3 day stage (E10.5 stage) under different flow rates / embryo culture media is shown.

[0111] Figure 5 The proportion of embryos that developed to the E7.5+4 day stage (E11.5 stage) under different pressures is shown.

[0112] Figure 6 Bright field images of embryos at various stages of in vitro culture, from E7.5 to E7.5+6 days, are shown. Detailed Implementation

[0113] The technical solution of this application will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely for explaining this application and not for limiting it. Furthermore, it should be noted that, for ease of description, only the parts relevant to this application are shown in the accompanying drawings, not all of them.

[0114] This application defines certain directional terms. Unless otherwise stated, the directional terms used, such as "up," "down," "left," "right," "inner," and "outer," are used for ease of understanding and therefore do not constitute a limitation on the scope of protection of this application.

[0115] In this application, unless otherwise expressly specified and limited, "above" or "below" the second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of the second feature includes the first feature being directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature includes the first feature being directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0116] In the description of this application, unless otherwise expressly specified and limited, the terms "connected," "linked," and "fixed" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0117] Figure 1 A schematic diagram of a whole embryo in vitro rotation culture system is shown. Figure 1 As shown, the whole embryo in vitro rotation culture system includes electronic devices, gas mixing devices, culture devices, pressure control devices, and temperature control devices.

[0118] For example, the electronic device may be a computer, tablet, mobile phone, etc. The electronic device may include a memory and a processor. The memory stores programs (also known as embryonic host software), such as programs controlling gas mixing devices, culture devices, pressure control devices, and / or temperature control devices. The processor is configured to execute the programs in the memory. Furthermore, the electronic device may also include a display screen configured to display information, such as the proportions of various gases in the gas mixing device, the gas flow rate supplied to the culture device, the rotational speed of the turntable in the culture device, and the pressure of the culture flasks in the culture device.

[0119] The gas mixing device includes one or more gas sources. For example, the gas mixing device may include three gas sources: a nitrogen source, an oxygen source, and a carbon dioxide source. It should be noted that in other embodiments, there may be only one gas source, i.e., only one of the nitrogen, oxygen, and carbon dioxide sources; or there may be two gas sources, i.e., any two of the nitrogen, oxygen, and carbon dioxide sources.

[0120] The gas mixing device also includes a gas mixer. Gas from a gas source is mixed in the gas mixer. Specifically, the gas source and the gas mixer are connected via a corresponding gas pipeline. For example, in... Figure 1 In this system, a nitrogen gas source is connected to the gas mixer via a nitrogen gas pipeline, an oxygen gas source is connected to the gas mixer via an oxygen gas pipeline, and a carbon dioxide gas source is connected to the gas mixer via a carbon dioxide gas pipeline. For example, the gas mixer can be a three-in-one gas mixer, model MR2403-03-1 / 8PTFE, manufactured by Tianjin Jisite Instrument Co., Ltd.

[0121] The gas mixing device also includes a gas flow and quality controller disposed on the gas pipeline. For example, in... Figure 1 The image shows three gas flow quality controllers: a nitrogen gas flow quality controller, an oxygen gas flow quality controller, and a carbon dioxide gas flow quality controller. Specifically, the nitrogen gas flow quality controller is installed on the nitrogen gas pipeline to control the flow rate of nitrogen, the oxygen gas flow quality controller is installed on the oxygen gas pipeline to control the flow rate of oxygen, and the carbon dioxide gas flow quality controller is installed on the carbon dioxide gas pipeline to control the flow rate of carbon dioxide.

[0122] The nitrogen gas flow quality controller, oxygen gas flow quality controller, and carbon dioxide gas flow quality controller can be coupled to an electronic device, thereby controlling the flow rate of each gas flow quality controller through a program in the electronic device.

[0123] For example, the gas flow quality controller can be a gas flow quality controller with product model G300C-200-P-24-1 manufactured by Beijing Spentech Technology Co., Ltd. For example, the flow range of the gas flow quality controller can be 0-200 mL / min.

[0124] By controlling the flow rates of various gas flow quality controllers, different proportions of mixed gases can be obtained in the gas mixer. For example, the proportion of carbon dioxide can be 5%, and the oxygen concentration can be set electronically via the embryo host computer software and monitored in real time by an oxygen concentration detector, with adjustments made in stages as the embryo culture time changes. For example, if it is desired to increase the proportion of oxygen in the mixed gas, the flow rate of the oxygen gas flow quality controller can be increased, or the flow rates of the nitrogen gas flow quality controller and the carbon dioxide gas flow quality controller can be decreased.

[0125] The gas mixing device also includes a gas detection component connected to the gas mixer to detect the proportion of gas in the gas mixer. For example, the gas detection component may include an oxygen detector and / or a carbon dioxide detector.

[0126] For example, the oxygen detector is the ST8100A model manufactured by CIMA, with a measurement range of 0-100% volume. The carbon dioxide detector is the GDAP-V20 model manufactured by Shanghai Huigan Co., Ltd., with a measurement range of 0-20% volume.

[0127] The gas mixing device may also include a filter, such as a 0.22 μm filter, which is connected to the gas mixer and located downstream of the gas mixer, so that the gas in the gas mixer can be filtered by the filter before entering the culture device, thus avoiding impurities in the gas from affecting the culture process.

[0128] Figure 2 This is a physical diagram of a whole embryo in vitro rotational culture system. The culture device includes an incubator and a culture assembly. The incubator may include a chamber, in which the culture assembly is disposed. The culture assembly includes a turntable and culture flasks disposed on the turntable. The turntable may be connected to a drive component (e.g., a motor) to rotate under drive. A gas channel is provided on the turntable, which may include a gas inlet, a culture flask interface, and a gas outlet that are interconnected. The gas inlet is connected to a gas mixer to receive gas from the gas mixer, the culture flask interface is connected to a culture flask to deliver gas into the culture flask and to discharge gas from the culture flask, and the gas outlet is configured to discharge waste gas. Gas from the gas mixer enters the gas channel from the gas inlet, then enters the culture flask through the culture flask interface, then returns to the gas channel from the culture flask interface, and finally exits from the gas outlet.

[0129] The culture flask is set up to contain a culture medium, such as rat serum.

[0130] For example, the motor's rotational speed ranges from 0 to 200 rpm / min. The volume of the culture flasks ranges from 10 to 100 mL. The turntable can have 20 culture flask ports, for example, 10 on the left and 10 on the right side of the turntable.

[0131] The motor can be coupled to an electronic device, so that the program in the electronic device can control the speed of the motor, which in turn controls the speed of the turntable, and ultimately controls the speed of the culture flask.

[0132] The pressure control device includes a gas cylinder, a first gas pipeline, a second gas pipeline, and a gas pressure controller. The gas cylinder is located downstream of the turntable. The inlet of the gas cylinder is connected to the gas outlet of the gas channel via the first gas pipeline, and the outlet of the gas cylinder is connected to the second gas pipeline for venting waste gas. The gas pressure controller is used to control the gas pressure inside the gas cylinder. Specifically, the gas pressure controller includes a valve located on the second gas pipeline. By adjusting the opening degree of the valve, the pressure of the gas cylinder can be controlled, thereby controlling the pressure inside the culture flask.

[0133] For example, the gas pressure controller may be a gas pressure controller with model number GT201-015-2 manufactured by Beijing Spentech Technology Co., Ltd., whose gas pressure detection range is 0-15 psi.

[0134] Optionally, the pressure control device also includes a pressure gauge connected to the gas cylinder to detect the gas pressure in the cylinder. For example, the pressure gauge could be a high-precision digital display pressure gauge, model AZ8230, manufactured by Hengxin Industrial Co., Ltd., with a gas pressure detection range of 0-30 psi.

[0135] The temperature control device is configured to regulate the temperature of the chamber in the incubator. For example, the temperature control device includes a constant-temperature drying oven and a thermometer, wherein the incubator can be a constant-temperature drying oven, and the thermometer (e.g., a mercury thermometer) is used to detect the temperature of the constant-temperature drying oven.

[0136] For example, the constant temperature drying oven can be a product model DHP-9052 manufactured by Shanghai Yiheng Scientific Instruments Co., Ltd., with a power of 250W and a temperature control range of 30-65℃.

[0137] In addition, a humidity control device, such as a distilled water container, can be installed between the filter and the gas inlet of the turntable. The gas filtered by the filter is first passed into the distilled water and then into the gas inlet, thereby humidifying the gas and preventing the flow of dry gas from causing the culture medium in the culture bottle to evaporate, which would lead to a change in the osmotic pressure of the culture medium.

[0138] During the cultivation process, the total gas flow rate and the ratio of the three gases in the culture bottle, the rotation speed of the turntable, and the gas pressure in the culture bottle are controlled by electronic devices. At the same time, oxygen detectors, carbon dioxide detectors, gas pressure controllers, and mercury thermometers are used to monitor the system in real time to ensure stable operation.

[0139] During the operation of the whole embryo in vitro rotational culture system, nitrogen, carbon dioxide, and oxygen are mixed in a certain proportion in a gas mixer after passing through corresponding gas mass flow controllers. For example, carbon dioxide accounts for 5% of the total volume, and the oxygen concentration is adjusted in stages according to the embryo culture time. The flow rate of the mixed gas ranges from 0-200 mL / min, and is adjusted according to the embryo culture efficiency. The gas is then filtered and enters a constant temperature drying oven to ensure that the gas entering the culture flask does not contaminate the embryo culture medium. The temperature of the constant temperature drying oven is set at 37℃ for mouse embryo in vitro culture. The gas then flows into distilled water for humidification to prevent the flow of dry gas from causing evaporation of the culture medium in the culture flask, which would alter the osmotic pressure of the culture medium. The mixed gas then enters the culture flask, with the rotor speed at 20-30 rpm / min. The pressure in the culture flask ranges from 0.1-15 psi, regulated by valves and monitored in real time by a gas pressure controller. Finally, the waste gas is discharged into the environment through a second gas pipeline.

[0140] The preparation method of fresh rat serum involved in the following examples is as follows: Male rats weighing approximately 300g were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. and starved for 18 hours in advance, but were allowed to drink water normally. The starved rats were placed in a transparent box containing isoflurane, with the isoflurane concentration maintained at 2.5-4.0%. After the rats were mildly anesthetized, force was applied to the tail of the rats until they did not struggle. Then, the abdomen of the rats was disinfected with 75% alcohol. A V-shaped incision was made along the midline of the abdomen from bottom to top with scissors to expose the abdomen. Then, the adipose tissue above the abdominal aorta was torn to the left and right with two forceps to expose the abdominal aorta. A 21G needle was attached to a 20 mL syringe. Then, the needle was inserted into the abdominal aorta at a 15° angle with the incision point facing downwards. The rat blood was slowly drawn into the syringe. Approximately 12 mL of rat blood could be obtained from each 300g rat. Transfer rat blood from the syringe to a 15 mL centrifuge tube pre-placed on ice, then centrifuge at 4000 rpm for 10 min in a pre-chilled 4°C centrifuge. After centrifugation, gently aspirate the serum from the 15 mL centrifuge tube, avoiding the aspiration of red blood cells, and transfer it to a new 15 mL centrifuge tube. 5-7 mL of rat serum can be obtained from each 300g rat. Place the rat serum in a 4°C freezer for 1-2 h to fix fibrinogen. Squeeze the coagulated fibrinogen with forceps, then centrifuge at 4000 rpm for 5 min in a pre-chilled 4°C centrifuge. Transfer the supernatant serum to a new 15 mL centrifuge tube and incubate in 56°C water for 30 min to inactivate the complement system. The obtained rat serum is filtered through a 0.22 μm filter to avoid using hemolyzed rat serum, and the rat serum should be used on the same day it is drawn (within 6 hours).

[0141] The preparation method of E7.5 embryos involved in the following examples is as follows: Female C57BL / 6 mice approximately 8 weeks old, purchased from Shanghai Southern Model Biotechnology Co., Ltd., were mated with male C57BL / 6 mice aged 8-12 weeks. The mating was checked the morning after mating; the presence of vaginal plugs indicated successful fertilization, and this day was defined as day E0.5 of the embryo. When the mouse embryos developed to the E7.5 stage, the mice were euthanized by cervical dislocation, the abdomen was opened, and the uterus was removed and placed in a dissection fluid. The dissection fluid was a mixed culture medium consisting of DMEM (GIBCO 11880):FBS (GIBCO, 10099141) = 1:3 (volume ratio). The dissection fluid was filtered through a 0.22 μm filter. The dissection fluid was equilibrated in a 37°C incubator for at least 1 hour beforehand. Using forceps, carefully peel away the uterine wall to expose the tunica vaginalis. Hold the forceps on both sides of the tunica vaginalis with your left hand, and use the forceps in your right hand to carefully lift along the middle line of the tunica vaginalis from the posterior end to the posterior end, being careful to only lift the upper 1 / 3 of the tunica vaginalis to avoid damaging the embryo in the middle. Then, use both forceps to grasp the two sides of the tunica vaginalis and tear it open to the left and right to expose the embryo. Then, carefully lift the embryo out of the tunica vaginalis with the forceps. Using two sets of 1mL syringes with 30G needles (BD, 305106), carefully detach the yolk sac from the embryo to avoid damaging the embryo. Embryo dissection is performed under an optical microscope equipped with a 37℃ heating plate, and each operation lasts no more than 30 minutes.

[0142] Example 1: Whole Embryo In Vitro Rotation Culture System

[0143] like Figure 1 As shown, the whole embryo in vitro rotation culture system includes electronic devices, gas mixing devices, culture devices, pressure control devices, and temperature control devices.

[0144] In this embodiment, the electronic device is a computer. The computer is electrically connected to the gas mixing device, the culture device, the pressure control device, and the temperature control device, thereby controlling the operation of each device.

[0145] In this embodiment, the gas mixing device includes a nitrogen source, an oxygen source, a carbon dioxide source, a nitrogen gas pipeline, an oxygen gas pipeline, a carbon dioxide gas pipeline, a nitrogen gas flow quality controller, an oxygen gas flow quality controller, a carbon dioxide gas flow quality controller, a gas mixer, an oxygen detector, a carbon dioxide detector, and a filter.

[0146] Specifically, the gas mixer is a three-in-one gas mixer. A nitrogen gas source is connected to the gas mixer via a nitrogen gas pipeline, and a nitrogen gas flow quality controller is installed on the nitrogen gas pipeline to control the nitrogen flow rate. An oxygen gas source is connected to the gas mixer via an oxygen gas pipeline, and an oxygen gas flow quality controller is installed on the oxygen gas pipeline to control the oxygen flow rate. A carbon dioxide gas source is connected to the gas mixer via a carbon dioxide gas pipeline, and a carbon dioxide gas flow quality controller is installed on the carbon dioxide gas pipeline to control the carbon dioxide flow rate. Oxygen and carbon dioxide detectors are placed in the gas mixer to detect the concentrations of oxygen and carbon dioxide, respectively. A filter is installed downstream of the gas mixer; the gas in the gas mixer is filtered through the filter before being delivered to the cultivation device.

[0147] The nitrogen, oxygen, and carbon dioxide gas flow quality controllers are all electrically connected to a computer so that the computer can control each gas flow quality controller.

[0148] In this embodiment, the culture apparatus includes an incubator and a culture assembly. The incubator is a constant temperature drying oven. The culture assembly is placed inside the constant temperature drying incubator. The culture assembly includes a motor, a turntable, and culture flasks. The motor is electrically connected to a computer and the turntable; the computer controls the motor's rotation, which in turn drives the turntable to rotate. The turntable can rotate around its own axis, and culture flasks can be installed on both the left and right sides of the turntable. The turntable has a gas channel inside, which includes a gas inlet, a culture flask interface, and a gas outlet that are interconnected. The gas inlet is connected to a gas mixer to receive gas from the gas mixer, the culture flask interface is connected to a culture flask to deliver gas into the culture flask and to discharge gas from the culture flask, and the gas outlet is configured to discharge waste gas.

[0149] Gas from the gas mixer passes through a filter and a distilled water container before entering the gas channel through the gas inlet. It then enters the culture flask through the culture flask interface, returns to the gas channel through the culture flask interface, and finally exits through the gas outlet.

[0150] In this embodiment, the pressure control device includes a gas cylinder, a first gas pipeline, a second gas pipeline, and a gas pressure controller. The gas cylinder is located downstream of the turntable. The inlet of the gas cylinder is connected to the gas outlet of the gas channel via the first gas pipeline, and the outlet of the gas cylinder is connected to the second gas pipeline for venting waste gas. The gas pressure controller is used to control the gas pressure inside the gas cylinder. Specifically, the gas pressure controller includes a valve located on the second gas pipeline. By adjusting the opening degree of the valve, the pressure of the gas cylinder can be controlled, thereby controlling the pressure inside the culture flask. The gas pressure controller is electrically connected to a computer so that the computer can control the gas pressure controller.

[0151] In this embodiment, the temperature control device includes a constant temperature drying oven and a thermometer. The constant temperature drying oven serves as an incubator, and the thermometer is used to detect the temperature inside the incubator. The constant temperature drying oven is electrically connected to a computer so that the computer can control the temperature of the drying oven.

[0152] Example 2: Effect of embryo culture medium on in vitro rotation culture of embryos

[0153] a1) A method for in vitro rotation culture of embryos, using the whole embryo in vitro rotation culture system described in Example 1 to culture E7.5 embryos, specifically as follows: Take 2 mL of embryo culture medium 1 (embryo culture medium 1 consists of the following components: 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), 100 μg / mL vitamin C (Sigma, A5960 and fresh rat serum (pH=7.2-7.4) were added to a 10 mL culture flask (Duran) in the whole embryo in vitro rotational culture system of Example 1. The flask was then placed on a rotational culture plate and preheated at 37°C for 1 h. Five to six E7.5 embryos were transferred to a culture flask containing 2 mL of embryo culture medium 1 using a pipette. The flasks were cultured at 37°C, 5% oxygen, 5% carbon dioxide, 0.1 psi (0.1 pounds per square inch), a gas flow rate of 50 mL / min, and a flask rotation speed of 30 rpm. After 24 hours of culture (in the embodiments of this invention, "XX" in "after XX hours of culture" is calculated from the time the E7.5 embryos are transferred to a culture flask containing 2 mL of embryo culture medium 1, E7.5 + 1 day), three embryos per group are transferred to a culture flask containing 2 mL of embryo culture medium 2 (embryo culture medium 2 consists of the following components: 3 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 400 μg / mL vitamin C (Sigma, A5960 and fresh rat serum (pH=7.2-7.4) were cultured in 10 mL Duran flasks (preheated at 37°C for 1 h on a rotating culture plate before transfer) at 37°C, 13% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate, and 30 rpm flask rotation.After 48 hours of culture (E7.5 + 2 days), two embryos per group were transferred to embryo culture medium 3 containing 2 mL of embryo culture medium. Embryo culture medium 3 consisted of the following components: 3.5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma). A5960) and fresh rat serum (pH=7.2-7.4) were cultured in 10 mL Duran flasks (preheated at 37°C for 1 h on a rotating culture plate before transfer) at 37°C, 30% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and flask rotation speed of 30 rpm. After 72 hours of culture (E7.5 + 3 days), one embryo per group was transferred to embryo culture medium 4 containing 2.5 mL of embryo culture medium (embryo culture medium 4 consisted of the following components: 4 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured for 24 h at 37°C, 90% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and flask rotation speed of 30 rpm.After culturing for 96 hours (E7.5 + 4 days), one embryo per group was transferred to embryo culture medium 5 containing 2.5 mL of embryo culture medium (embryo culture medium 5 consisted of the following components: 4.5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), 800 μg / mL of glucose. The culture was prepared in 10 mL Duran flasks containing vitamin C (Sigma, A5960) and fresh rat serum (pH 7.2-7.4) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured for 24 h at 37°C, 90% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate, and flask rotation speed of 30 rpm. After 120 hours of culture (E7.5 + 5 days), one embryo per group was transferred to embryo culture medium 6 containing 2.5 mL of embryo culture medium 6 (embryo culture medium 6 consists of the following components: 5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured for 24 h at 37°C, 60% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and flask rotation speed of 30 rpm.

[0154] a2) A method for in vitro rotation culture of embryos, the same as a1), except that the fresh rat serum in embryo culture media 1, 2, and 3 is replaced with a mixture of DMEM and fresh rat serum, wherein the volume ratio of DMEM to fresh rat serum is 1:3.

[0155] a3) A method for in vitro rotational culture of embryos, the same as a1), except that the fresh rat serum in embryo culture media 1 and 2 is replaced with cryopreserved rat serum (the preparation method of cryopreserved rat serum is as follows: the fresh rat serum obtained by "squeezing the coagulated fibrinogen with tweezers and then centrifuging it at 4000 rpm / min for 5 min in a centrifuge pre-cooled at 4℃" is immediately placed in a -80℃ freezer for 3 weeks and used within one month. When using, it is incubated in water at 56℃ for 30 min to inactivate the complement system, and then filtered through a 0.22μm filter to obtain usable cryopreserved rat serum).

[0156] When embryos are cultured to E7.5 + 3 days, they absorb nutrients through the yolk sac cells, which then transport these nutrients to the yolk sac blood vessels and circulate them into the embryo, promoting development. However, due to the in vitro culture process, blood flow in the yolk sac gradually ceases at this stage, preventing the blood vessels from continuing to deliver nutrients to the embryo and causing developmental arrest. Therefore, observing whether blood flow continues in the yolk sac at E10.5 is used as a criterion for assessing whether the embryo can continue developing.

[0157] In embryos at stage E10.5 (a1)-a3), the proportion of embryos with continued blood flow in the yolk sac vessels is as follows: Figure 3 , 4 As shown: in a1), the proportion of embryos with continued blood flow in the yolk sac vessels at E10.5 was 94.12%; while in a2), the proportion was only 63.64%. Therefore, the embryonic development effect of the whole rat fresh serum combination was better than that of the DMEM:fresh rat serum = 1:3 combination. In a3), the embryos underwent developmental arrest at E9.5 and could not complete the embryonic turning process. Figure 3 The rightmost "E7.5+2Day" shows the developmental status of the embryos whose yolk sacs were removed at E7.5 + 2 Day. Therefore, the embryos with the frozen rat serum combination were arrested at the E9.5 stage and could not continue to develop. In summary, the embryo culture medium containing fresh rat serum (i.e., a1) showed the best embryo development effect.

[0158] Example 3: Effect of gas flow rate on in vitro rotation culture of embryos

[0159] b1) A method for in vitro rotation culture of embryos, using the whole embryo in vitro rotation culture system of Example 1 to culture E7.5 embryos, as follows:

[0160] b11) Take 2 mL of embryo culture medium 1 (embryo culture medium 1 consists of the following components: 1% (v / v) penicillin-streptomycin (Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), 100 μg / mL vitamin C (Sigma, A5960), and fresh rat serum, pH=7.2-7.4) and add it to a 10 mL culture flask (Duran) in the whole embryo in vitro rotational culture system of Example 1. Then place it on a rotational culture plate and preheat at 37°C for 1 h. Use a pipette to transfer 5-6 E7.5 embryos to a culture flask containing 2 mL of embryo culture medium 1 and incubate at 37°C, 5% oxygen, 5% carbon dioxide, 0.1 Incubate at a gas flow rate of 50 mL / min and a bottle rotation speed of 30 rpm;

[0161] After culturing for 24 hours (E7.5 + 1 day), 3 embryos per group were transferred to embryo culture medium 2 containing 2 mL of each medium. Embryo culture medium 2 consisted of the following components: 3 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 400 μg / mL vitamin C (Sigma). A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured at 37°C, 13% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate, and 30 rpm flask rotation.

[0162] After culturing for 48 hours (E7.5 + 2 days), two embryos per group were transferred to embryo culture medium 3 containing 2 mL of embryo culture medium. Embryo culture medium 3 consisted of the following components: 3.5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma). 10 mL culture flasks (Duran) containing A5960 and fresh rat serum (pH=7.2-7.4) (preheated at 37°C for 1 h on a rotating culture plate before transfer) were incubated for 24 h at 37°C, 30% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate, and flask rotation speed of 30 rpm.

[0163] After culturing b14 for 72 hours (E7.5 + 3 days), one embryo per group was transferred to a culture medium containing 2.5 mL of embryo culture medium 4 (embryo culture medium 4 consists of the following components: 4 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured for 24 h at 37°C, 90% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and flask rotation speed of 30 rpm.

[0164] After culturing b15 for 96 hours (E7.5 + 4 days), one embryo per group was transferred to embryo culture medium 5 containing 2.5 mL of embryo culture medium (embryo culture medium 5 consisted of the following components: 4.5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured for 24 h at 37°C, 90% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and flask rotation speed of 30 rpm.

[0165] After culturing b16 for 120 hours (E7.5 + 5 days), one embryo per group was transferred to embryo culture medium 6 containing 2.5 mL of embryo culture medium 6 (embryo culture medium 6 consists of the following components: 5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured for 24 h at 37°C, 60% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and flask rotation speed of 30 rpm.

[0166] b2) A method for in vitro rotation culture of embryos, the same as b1), except that 50 mL / min in b11), b12), b13), b14), b15), b16) is replaced with 12.5 mL / min.

[0167] b3) A method for in vitro rotation culture of embryos, the same as b1), except that 50 mL / min in b11), b12), b13), b14), b15), b16) is replaced with 25 mL / min.

[0168] b4) A method for in vitro rotation culture of embryos, the same as b1), except that 50 mL / min in b11), b12), b13), b14), b15), b16) is replaced with 100 mL / min.

[0169] b5) A method for in vitro rotation culture of embryos, the same as b1), except that 50 mL / min in b11), b12), b13), b14), b15), b16) is replaced with 200 mL / min.

[0170] In the E10.5 stage of embryos (b1)-b5), the proportion of embryos with continued blood flow in the yolk sac vessels is as follows: Figure 4 As shown: in b1), the proportion of embryos with continued blood flow in the yolk sac vessels at E10.5 was 94.12%; in b2), 66.67%; in b3), 75%; in b4), 64.71%; and in b5), 42.86%. The presence or absence of blood flow in the yolk sac at E10.5 in in vitro cultured embryos can be used as a criterion for judging whether the embryo continues to develop. Therefore, when mouse embryos are cultured with a mixed gas flow rate of 50 mL / min, the embryonic development effect is better than other gas flow rate combinations.

[0171] Example 4: Effect of gas pressure on in vitro rotation culture of embryos

[0172] c1) A method for in vitro rotation culture of embryos, using the whole embryo in vitro rotation culture system of Example 1 to culture E7.5 embryos, as follows:

[0173] c11) Take 2 mL of embryo culture medium 1 (embryo culture medium 1 consists of the following components: 1% (v / v) penicillin-streptomycin (Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), 100 μg / mL vitamin C (Sigma, A5960), and fresh rat serum, pH=7.2-7.4) and add it to a 10 mL culture flask (Duran) in the whole embryo in vitro rotational culture system of Example 1. Then place it on a rotational culture plate and preheat at 37°C for 1 h. Use a pipette to transfer 5-6 E7.5 embryos to a culture flask containing 2 mL of embryo culture medium 1 and incubate at 37°C, 5% oxygen, 5% carbon dioxide, 0.1 Incubate at a gas flow rate of 50 mL / min and a bottle rotation speed of 30 rpm;

[0174] After culturing c12 for 24 hours (E7.5 + 1 day), three embryos per group were transferred to embryo culture medium 2 containing 2 mL of each medium. Embryo culture medium 2 consisted of the following components: 3 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 400 μg / mL vitamin C (Sigma). A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured at 37°C, 13% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate, and 30 rpm flask rotation.

[0175] After culturing c13 for 48 hours (E7.5 + 2 days), two embryos per group were transferred to embryo culture medium 3 containing 2 mL of embryo culture medium 3 (embryo culture medium 3 consists of the following components: 3.5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured at 37°C, 30% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate, and 30 rpm flask rotation.

[0176] After culturing c14 for 72 hours (E7.5 + 3 days), one embryo per group was transferred to a culture medium containing 2.5 mL of embryo culture medium 4 (embryo culture medium 4 consisted of the following components: 4 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured for 24 h at 37°C, 90% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and flask rotation speed of 30 rpm.

[0177] After culturing c15 for 96 hours (E7.5 + 4 days), one embryo per group was transferred to a culture medium containing 2.5 mL of embryo culture medium 5 (composed of the following components: 4.5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured for 24 h at 37°C, 90% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and flask rotation speed of 30 rpm.

[0178] After culturing c16 embryos for 120 hours (E7.5 + 5 days), one embryo per group was transferred to embryo culture medium 6 containing 2.5 mL of embryo culture medium 6 (embryo culture medium 6 consists of the following components: 5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured for 24 h at 37°C, 60% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and flask rotation speed of 30 rpm.

[0179] c2) A method for in vitro rotation culture of embryos, the same as c1), except that 0.1 psi in c11), c12), c13), c14), c15), and c16) is replaced with 6.5 psi.

[0180] c3) A method for in vitro rotation culture of embryos, the same as c1), except that 0.1 psi in c11), c12), c13), c14), c15), and c16) is replaced with 13 psi.

[0181] The efficiency of c1)-c3) from E7.5 to the E7.5+4Day stage (E11.5 period) is as follows: Figure 5 As shown: c1)-c3) The efficiency of culturing embryos from E7.5 to E7.5+4Day (E11.5 stage) is 100%. That is, the gas pressure between 0.1psi and 13psi does not affect the efficiency of culturing embryos from E7.5 to E7.5+4Day (E11.5 stage). Considering that embryo culture requires changing the culture medium every 24 hours, if the embryos are cultured at a pressure of 13psi, it would take a long time to increase the gas pressure in the culture bottle after changing the medium, which is cumbersome. Therefore, 0.1psi represents a pressure slightly higher than atmospheric pressure generated by gas flow. After changing the medium, it immediately reaches 0.1psi, which is simple to operate and more conducive to in vitro embryo culture.

[0182] Example 5: A method for in vitro rotational culture of embryos

[0183] A method for in vitro rotation culture of embryos, using the whole embryo in vitro rotation culture system of Example 1 to culture E7.5 embryos, is as follows:

[0184] Take 2 mL of embryo culture medium 1 (embryo culture medium 1 consists of the following components: 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 100 μg / mL vitamin C (Sigma, A5960 and fresh rat serum (pH 7.2-7.4) were added to a 10 mL culture flask (Duran) in the whole embryo in vitro rotational culture system of Example 1. The flask was then placed on a rotational culture plate and preheated at 37°C for 1 h. Five to six E7.5 embryos were transferred to a culture flask containing 2 mL of embryo culture medium 1 using a pipette. The flasks were cultured at 37°C, 5% oxygen, 5% carbon dioxide, 0.1 psi, a gas flow rate of 50 mL / min, and a flask rotation speed of 30 rpm.

[0185] After 24 hours of culture (E7.5 + 1 day), three embryos per group were transferred to embryo culture medium 2 containing 2 mL of each medium. Embryo culture medium 2 consisted of the following components: 3 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 400 μg / mL vitamin C (Sigma). A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured at 37°C, 13% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate, and 30 rpm flask rotation.

[0186] After 48 hours of culture (E7.5 + 2 days), two embryos per group were transferred to embryo culture medium 3 containing 2 mL of embryo culture medium. Embryo culture medium 3 consisted of the following components: 3.5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma). A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured at 37°C, 30% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate, and 30 rpm flask rotation.

[0187] After 72 hours of culture (E7.5 + 3 days), one embryo per group was transferred to embryo culture medium 4 containing 2 mL of embryo culture medium (embryo culture medium 4 consisted of the following components: 4 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured at 37°C, 90% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and 30 rpm flask rotation.

[0188] After culturing for 96 hours (E7.5 + 4 days), one embryo per group was transferred to embryo culture medium 5 containing 2.5 mL of embryo culture medium (embryo culture medium 5 consisted of the following components: 4.5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM sodium pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured at 37°C, 90% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and 30 rpm flask rotation.

[0189] After 120 hours of culture (E7.5 + 5 days), when the blood flow from the yolk sac and umbilical cord ceases and only the embryo's own blood is flowing, the yolk sac and amnion of the in vitro cultured embryos are detached, preserving the placental cone. One embryo per group is then transferred to a culture medium containing 2.5 mL of embryo culture medium 6 (embryo culture medium 6 consists of the following components: 5 mg / mL glucose (Gibco, A2494001), 1% (v / v) penicillin-streptomycin (penicillin-streptomycin bispecific antibody, Gibco; 15140122), 5.5 mM HEPES (GIBCO, 15630130), 1 mM pyruvate (Gbico, 11360039), 2 mM glutamax (Gbico, 35050038), and 800 μg / mL vitamin C (Sigma, ...). A5960) and fresh rat serum (pH=7.2-7.4) were placed in 10 mL culture flasks (Duran) (preheated at 37°C for 1 h on a rotating culture plate before transfer) and cultured for 24 h at 37°C, 60% oxygen, 5% carbon dioxide, 0.1 psi, 50 mL / min gas flow rate and flask rotation speed of 30 rpm.

[0190] During the culture process, images were taken daily using an Olympus imaging system (SZX16 microscope, DP71 camera), and the results are as follows. Figure 6 As shown: By E7.5 + 6 days, the mouse embryos' eyes are basically fully developed, the femurs, patellae, tibias, and fibulae of the limbs are basically formed, the paws are paddle-like, and the external ear has formed. Figure 6 In this context, "Intact yolk sac" refers to an embryo at the corresponding stage where the yolk sac has not yet been separated, while "Dissected yolk sac" refers to an embryo at the corresponding stage where the yolk sac has been separated, used to illustrate the developmental status of the corresponding embryo.

[0191] Although this application has been described in detail above with general descriptions, specific embodiments, and experiments, some modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of this application fall within the scope of protection claimed in this application.

Claims

1. A method for in vitro rotation culture of embryos, wherein the embryos are placed in an embryo culture medium for culture; The embryo culture medium includes embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, and optionally embryo culture medium 4, embryo culture medium 5 and / or embryo culture medium 6. None of the embryo culture media 1, embryo culture media 2, embryo culture media 3, embryo culture media 4, embryo culture media 5 and embryo culture media 6 contain human umbilical cord blood serum; The embryo culture medium 1 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine and buffer salts, wherein the concentration of ascorbic acid or its derivatives in the embryo culture medium 1 is P μg / mL; The embryo culture medium 2 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine, buffer salts and glucose. The concentration of ascorbic acid or its derivatives in the embryo culture medium 2 is P+300μg / mL, and the concentration of glucose in the embryo culture medium 2 is N mg / mL. The embryo culture medium 3 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine, buffer salts and glucose. The concentration of ascorbic acid or its derivatives in the embryo culture medium 3 is P+700μg / mL, and the concentration of glucose in the embryo culture medium 3 is N+0.5mg / mL. The embryo culture medium 4 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine, buffer salts and glucose. The concentration of ascorbic acid or its derivatives in the embryo culture medium 4 is P+700μg / mL, and the concentration of glucose in the embryo culture medium 4 is N+1mg / mL. The embryo culture medium 5 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine, buffer salts and glucose, wherein the concentration of ascorbic acid or its derivatives in the embryo culture medium 5 is P+700μg / mL and the concentration of glucose in the embryo culture medium 5 is N+1.5mg / mL. The embryo culture medium 6 contains fresh rat serum, antibiotics, ascorbic acid or its derivatives, pyruvate or its salts, glutamine, buffer salts and glucose, wherein the concentration of ascorbic acid or its derivatives in the embryo culture medium 6 is P+700μg / mL and the concentration of glucose in the embryo culture medium 6 is N+2mg / mL. The value of N is 2.5-3.5; The value of P is 50-150; The ascorbic acid or its derivative in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 are each independently selected from at least one of ascorbic acid, calcium ascorbate, magnesium ascorbate, zinc ascorbate, potassium ascorbate, sodium ascorbate, L-ascorbic acid 2-phosphate, and L-ascorbic acid 3-phosphate; The in vitro rotation culture method for embryos includes the following steps: 1) Place the embryos in embryo culture medium 1 for the first culture; 2) Embryos cultured for the first time are placed in embryo culture medium 2 for a second culture; 3) The embryos that have undergone the second culture are placed in embryo culture medium 3 for a third culture; In each of the first, second, and third culture systems, a mixed gas containing oxygen and carbon dioxide is independently introduced at a gas flow rate of 25-60 mL / min. The oxygen content in the first culture system was M% by volume. The oxygen content in the second culture system was (M+8)% by volume. The oxygen content in the system during the third culture was (M+25)% by volume. The value of M is 4.5-5.5; The gas pressure in the first, second, and third culture systems is independently 0.1-15 psi; The time for the first, second, and third cultures is 20-28 hours each. The embryos were derived from mice; The embryos are E7-E8 embryos.

2. The method according to claim 1, characterized in that, The value of N is 2.9-3.1; and / or The P is 95-105; and / or In each of the first, second, and third culture systems, a mixed gas is independently introduced at a gas flow rate of 45-55 mL / min; and / or The value of M is 4.8-5.2; and / or The gas pressure in each of the first, second, and third culture systems is independently 0.1-13 psi; and / or The duration of the first, second, and third culture processes is independently 22-26 hours; and / or The embryos were derived from mice.

3. The method according to claim 2, characterized in that, The antibiotics in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5, and embryo culture medium 6 are each independently selected from at least one of amphotericin B, nystatin, gentamicin, tetracycline, erythromycin, penicillin, and streptomycin; and / or The ascorbic acid or its derivative in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is ascorbic acid; and / or The buffer salts in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5, and embryo culture medium 6 are each independently selected from at least one of phosphate, Tris, and HEPES; and / or The pyruvate or its salt in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is sodium pyruvate; and / or The concentration of the antibiotic in each of the embryo culture media 1, embryo culture media 2, embryo culture media 3, embryo culture media 4, embryo culture media 5, and embryo culture media 6 is independently 0.2%-2% by volume; and / or The concentration of pyruvate or its salt in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is independently 0.5-2 mM; and / or The concentration of glutamine in each of the embryo culture media 1, embryo culture media 2, embryo culture media 3, embryo culture media 4, embryo culture media 5, and embryo culture media 6 is independently 1-3 mM; and / or The concentration of the buffer salt in each of the embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is independently 3-11 mM.

4. The method according to claim 3, characterized in that, The antibiotics in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5, and embryo culture medium 6 are penicillin and streptomycin; and / or The buffer salts in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5, and embryo culture medium 6 are HEPES; and / or The concentration of the antibiotic in each of the embryo culture media 1, embryo culture media 2, embryo culture media 3, embryo culture media 4, embryo culture media 5, and embryo culture media 6 is independently 0.5%-1.5% by volume; and / or The concentration of pyruvate or its salt in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is independently 0.6-1.4 mM; and / or The concentration of glutamine in each of the embryo culture media 1, embryo culture media 2, embryo culture media 3, embryo culture media 4, embryo culture media 5, and embryo culture media 6 is independently 1.2-2.8 mM; and / or The concentration of the buffer salt in each of the embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is independently 4-7 mM.

5. The method according to claim 4, characterized in that, The concentration of the antibiotic in each of the embryo culture media 1, embryo culture media 2, embryo culture media 3, embryo culture media 4, embryo culture media 5, and embryo culture media 6 is independently 0.9%-1.1% by volume; and / or The concentration of pyruvate or its salt in embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is independently 0.9-1.1 mM; and / or The concentration of glutamine in each of the embryo culture media 1, embryo culture media 2, embryo culture media 3, embryo culture media 4, embryo culture media 5, and embryo culture media 6 is independently 1.8-2.2 mM; and / or The concentration of the buffer salt in each of the embryo culture medium 1, embryo culture medium 2, embryo culture medium 3, embryo culture medium 4, embryo culture medium 5 and embryo culture medium 6 is independently 5-6 mM.

6. The method according to any one of claims 1-5, characterized in that, The in vitro rotation culture method for embryos includes the following steps: 4) Embryos that have undergone the third culture are placed in embryo culture medium 4 for a fourth culture; Optionally, the in vitro rotation culture method for embryos includes the following steps: 5) Embryos that have undergone the fourth culture are placed in embryo culture medium 5 for a fifth culture; 6) Embryos cultured for the fifth time were placed in embryo culture medium 6 for a sixth culture; In each of the fourth, fifth, and sixth culture systems, a mixed gas containing oxygen and carbon dioxide is independently introduced at a gas flow rate of 25-60 mL / min. The oxygen content in the fourth and fifth culture systems, by volume percentage, was independently (M+85)% each. The oxygen content in the sixth culture system, expressed as a volume percentage, was (M+55)%. The gas pressure in the fourth, fifth, and sixth culture systems is independently 0.1-15 psi; The time for the fourth, fifth, and sixth culture cycles is 20-28 hours each.

7. The method according to claim 6, characterized in that, In each of the fourth, fifth, and sixth culture systems, a mixed gas is independently introduced at a gas flow rate of 45-55 mL / min; and / or The gas pressure in the fourth, fifth, and sixth culture systems is independently 0.1-13 psi; and / or The time for the fourth, fifth, and sixth culture cycles is 22-26 hours each.

8. The method according to claim 6, characterized in that, The carbon dioxide content in each of the first, second, third, fourth, fifth, and sixth culture systems is independently 3%-7% by volume; and / or The temperatures for the first, second, third, fourth, fifth, and sixth cultures are each independently 33-40℃; and / or The rotation speed for the first, second, third, fourth, fifth, and sixth cultures is independently 15-50 rpm.

9. The method according to claim 8, characterized in that, The carbon dioxide content in each of the first, second, third, fourth, fifth, and sixth culture systems is independently 4%-6% by volume; and / or The temperatures for the first, second, third, fourth, fifth, and sixth cultures are each independently 36-38℃; and / or The rotation speed for the first, second, third, fourth, fifth, and sixth cultures is independently 20-40 rpm.

10. The method according to claim 9, characterized in that, The rotation speed for the first, second, third, fourth, fifth, and sixth cultures is independently 25-35 rpm.

11. The method according to any one of claims 1-5 and 7-10, characterized in that, The in vitro rotation culture of embryos is performed in a whole embryo in vitro rotation culture system; The whole embryo in vitro rotation culture system includes: A gas mixing device includes a gas source, a gas mixer, a gas pipeline, and a gas flow and quality controller. The gas source and the gas mixer are connected through the gas pipeline, and the gas flow and quality controller is disposed on the gas pipeline. A culture apparatus includes an incubator and a culture assembly disposed within a chamber of the incubator. The culture assembly includes a turntable and a culture flask disposed on the turntable. The turntable includes a gas channel, which includes a gas inlet, a culture flask interface, and a gas outlet that are interconnected. The gas inlet is connected to a gas mixer, the culture flask interface is connected to the culture flask, the gas outlet is configured to discharge waste gas, and the culture flask is configured to contain embryo culture medium.

12. The method according to claim 11, characterized in that, The gas source includes one or more of nitrogen, oxygen, and carbon dioxide; and / or The gas mixing device further includes a gas detection component, which is connected to the gas mixer to detect the proportion of gas in the gas mixer; and / or The whole embryo in vitro rotation culture system also includes: A pressure control device includes a gas cylinder, a first gas pipeline, a second gas pipeline, and a gas pressure controller. The gas cylinder is located downstream of the turntable. The inlet of the gas cylinder is connected to the gas outlet via the first gas pipeline. The outlet of the gas cylinder is connected to the second gas pipeline. The gas pressure controller is configured to control the pressure inside the gas cylinder. A valve of the gas pressure controller is located on the second gas pipeline, and the opening degree of the valve is adjustable; and / or The whole embryo in vitro rotation culture system also includes: A temperature control device is configured to regulate the temperature of the chamber of the incubator.

13. The method according to claim 12, characterized in that, When there are multiple gas sources, each gas source is connected to the gas mixer through a corresponding gas pipeline; and / or The gas detection assembly includes an oxygen detector and / or a carbon dioxide detector.

14. The method according to claim 6, characterized in that, The in vitro rotation culture of embryos is performed in a whole embryo in vitro rotation culture system; The whole embryo in vitro rotation culture system includes: A gas mixing device includes a gas source, a gas mixer, a gas pipeline, and a gas flow and quality controller. The gas source and the gas mixer are connected through the gas pipeline, and the gas flow and quality controller is disposed on the gas pipeline. A culture apparatus includes an incubator and a culture assembly disposed within a chamber of the incubator. The culture assembly includes a turntable and a culture flask disposed on the turntable. The turntable includes a gas channel, which includes a gas inlet, a culture flask interface, and a gas outlet that are interconnected. The gas inlet is connected to a gas mixer, the culture flask interface is connected to the culture flask, the gas outlet is configured to discharge waste gas, and the culture flask is configured to contain embryo culture medium.

15. The method according to claim 14, characterized in that, The gas source includes one or more of nitrogen, oxygen, and carbon dioxide; and / or The gas mixing device further includes a gas detection component, which is connected to the gas mixer to detect the proportion of gas in the gas mixer; and / or The whole embryo in vitro rotation culture system also includes: A pressure control device includes a gas cylinder, a first gas pipeline, a second gas pipeline, and a gas pressure controller. The gas cylinder is located downstream of the turntable. The inlet of the gas cylinder is connected to the gas outlet via the first gas pipeline. The outlet of the gas cylinder is connected to the second gas pipeline. The gas pressure controller is configured to control the pressure inside the gas cylinder. A valve of the gas pressure controller is located on the second gas pipeline, and the opening degree of the valve is adjustable; and / or The whole embryo in vitro rotation culture system also includes: A temperature control device configured to regulate the temperature of the chamber of the incubator.

16. The method according to claim 15, characterized in that, When there are multiple gas sources, each gas source is connected to the gas mixer through a corresponding gas pipeline; and / or The gas detection assembly includes an oxygen detector and / or a carbon dioxide detector.

17. The use of the embryo obtained by the culture method according to any one of claims 1-16 in any one of a1)-a7): a1) Preparation of embryo models; a2) Embryo research; a3) Screening for key genes causing embryonic developmental defects; a4) Investigate the effects of knockout or overexpression libraries on embryonic development; a5) Testing drug safety; a6) Preparation of tissues and / or organs; a7) Prepare a product, said product being used in any one of a1) to a6); The tissue and / or organ is the eye or bone.

18. An embryo culture medium, wherein the embryo culture medium is the embryo culture medium according to any one of claims 1-16.

19. The embryo culture medium according to claim 18, characterized in that, The embryo culture medium also includes an instruction manual, which describes the in vitro rotation culture method for embryos according to any one of claims 1-16.

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