Device and method for gradually adjusting glucose concentration of in-vitro embryo culture solution

Abstract

The invention belongs to the technical field of assisted reproduction, and particularly relates to a device and a method for gradually adjusting the glucose concentration of an in-vitro embryo culture solution. The device comprises at least two miniature liquid storage bins, wherein each miniature liquid storage bin is filled with a high-concentration glucose solution in advance; the slow release channel is respectively connected with each micro liquid storage bin and the culture solution main container; the culture solution main container is used for accommodating an embryo and a basal culture medium; the control unit is used for sequentially opening the release ports of the miniature liquid storage bins, so that the high-concentration glucose solution sequentially flows into the culture solution main container through the slow release channels. The technical means of staged closed-loop micro sugar control is introduced into the structure and the using method of the device, so that the accurate adjustment of the sugar concentration of the in-vitro embryo culture solution is realized, the requirements of embryos in different development stages can be effectively met, and the embryo culture quality and the subsequent development potential are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of assisted reproductive technologies, and particularly relates to a device and method for gradually adjusting the glucose concentration of in vitro embryo culture medium. Background Art

[0002] During the in vitro embryo culture process, the glucose concentration requirements of embryos at different developmental stages vary significantly. Specifically: 1. Embryo metabolism during the cleavage stage (the first 3 days) is sensitive to glucose, and a low-glucose environment needs to be maintained to avoid the inhibition of embryonic cell mitosis by high glucose concentration.

[0003] 2. After the blastocyst stage (after the 3rd day), the energy demand of embryos increases significantly, and a high-glucose environment must be provided to support the rapid growth and metabolic needs of blastocysts.

[0004] The prior art mainly adjusts the glucose concentration in the culture medium in the following way: replacing the culture medium stage by stage. Through artificial intervention, at the end of the 3rd day of embryo culture, the embryo is transferred to a new culture medium containing a higher glucose concentration. Defects: The embryo transfer operation will cause a mechanical stress response of the embryo, which may cause embryo damage or metabolic disorders; sudden environmental changes (such as pH, osmotic pressure) may also induce an environmental stress response of the embryo, affecting embryo quality and culture success rate.

[0005] Therefore, there are obvious deficiencies in the prior art, and it is difficult to achieve efficient and convenient glucose concentration regulation while ensuring the quality of embryo culture. There is an urgent need to propose a simpler, more accurate, and non-destructive culture strategy to meet the needs of different developmental stages of embryos, avoid mechanical and environmental stress responses, and improve the success rate and quality of embryo culture. Summary of the Invention

[0006] The purpose of the present invention is to solve the deficiencies existing in the above background art, and provide a device and method for gradually adjusting the glucose concentration of in vitro embryo culture medium.

[0007] The technical solution adopted by the present invention is: a device for gradually adjusting the glucose concentration of in vitro embryo culture medium, the device comprising: At least two micro liquid storage chambers, each of which is pre-filled with a high-concentration glucose solution; A slow-release channel, the slow-release channel is respectively connected to each of the micro liquid storage chambers and the main culture medium container; A main culture medium container for accommodating embryos and a basic culture medium; A control unit, the control unit is used to sequentially open the release ports of each of the micro liquid storage chambers, so that the high-concentration glucose solution flows into the main culture medium container through the slow-release channel in sequence.

[0008] In the above technical solution, the sequential opening of multiple micro-liquid storage chambers can provide different amounts of glucose to the culture medium at different stages, avoiding sudden changes in the embryo environment caused by a one-time addition of a high concentration. At least two (or more) micro-liquid storage chambers facilitate the design of release schemes according to the glucose requirements at different embryo development stages. The control unit can be programmed to set the opening sequence and timing, reducing manual intervention and improving repeatability.

[0009] More preferably, it further includes a regulating valve, which is installed at the release port of each of the micro-liquid storage chambers and is used to control the release of the high-concentration glucose solution; a sensor, which is arranged in the main culture medium container. The sensor is a micro-electrochemical sensor or an optical sensor based on the glucose oxidase reaction and is used to real-time monitor the glucose concentration in the main culture medium container; The control unit controls the opening and closing and the opening degree of the regulating valve according to the glucose concentration in the main culture medium container, so that the glucose concentration of the culture medium reaches the final target concentration threshold.

[0010] In the above technical solution, through real-time sensor detection and valve opening degree adjustment, it can quickly respond to the change in the sugar concentration in the current culture medium, greatly reducing the concentration fluctuation. Once the concentration reaches or exceeds the set threshold, the control unit can automatically command the next operation (such as switching to the next chamber or micro-compensation), reducing the manual judgment error. A lower sugar environment can be maintained in the early stage and moderately increased in the later stage, which conforms to the gradual sugar requirement law of the embryo in vivo environment.

[0011] More preferably, the number of the micro-liquid storage chambers is 2 to 6, and the micro-liquid storage chambers are arranged in a linear arrangement, a circular arrangement or a stacked arrangement, and each micro-liquid storage chamber has a sealing structure to ensure that the internal liquid does not leak before release.

[0012] In the above technical solution, the range of 2 to 6 chambers can not only meet the sugar requirement for staged in vitro embryo culture, but also is not too complex, facilitating design and maintenance. Different arrangement methods are convenient for matching the space layout of the specific experimental platform, and the independent sealing avoids cross-contamination or leakage between chambers. The solution concentration or characteristics of a certain chamber can be replaced according to requirements without changing other chambers, with low maintenance costs.

[0013] More preferably, by controlling the regulating valve at a predetermined opening degree, the flow rate of the glucose solution in the slow-release channel does not exceed 50 μL / hour.

[0014] In the above technical solution, slow release can avoid sudden concentration changes, has a milder impact on the embryo environment, and meets the gradual nutritional requirements of the embryo. Excessive flow rates may cause rapid changes in osmotic pressure, pH, etc.; micro flow rates can keep the characteristics of the culture medium within a safe range. At lower flow rates, the adjustment accuracy of the sensor and valve can more easily play a role to achieve a relatively stable concentration curve.

[0015] More preferably, the total volume of the glucose solution flowing into the main culture medium container does not exceed 1 mL.

[0016] In the above technical solution, the volume of the main culture medium container is usually limited. If too much exogenous liquid is added, it may dilute the basal medium or change its balance; controlling it below 1 mL can maintain the stability of the culture environment. The embryo can tolerate limited changes in body fluids in the in vivo environment, and moderate rather than excessive addition is more in line with physiological requirements. With a small injection volume, combined with the flow rate and time, the actual amount of glucose added can be accurately recorded, facilitating subsequent experimental analysis.

[0017] More preferably, the slow-release channel is composed of a capillary with a small diameter or a catheter made of a hydrophilic material.

[0018] In the above technical solution, the capillary action of the capillary with a small diameter or the surface wettability of the hydrophilic material helps to maintain a low flow rate and reduce a large instantaneous influx. The hydrophilic material can help the liquid to flow smoothly in the channel, avoiding pulsed liquid release caused by uneven flow or air bubbles. The capillary / hydrophilic catheter can be made of medical-grade materials to ensure non-toxicity to the embryo; at the same time, it is easy to achieve independent packaging and sterilization treatment.

[0019] More preferably, the slow-release channel is made of hydrophilic silica gel, polytetrafluoroethylene or microporous membrane material; the length and pore diameter of the slow-release channel are set so that the release rate of glucose reaches a predetermined value.

[0020] More preferably, the glucose concentration of the high-concentration glucose solution is 5 - 50 g / L.

[0021] In the above technical solution, 5 - 50 g / L (about 0.028 - 0.28 M) is sufficient to meet the conventional requirements from low to high during in vitro embryo culture after gradual dilution. The high-concentration stock solution can continuously provide sufficient sugar in the later stage, while avoiding the risk of hypertonicity through micro-release in the early stage. A high concentration means that less volume is required to store enough glucose, which is beneficial for the miniaturized design of the microchamber.

[0022] A method for in vitro embryo culture using the device for gradually adjusting the glucose concentration of in vitro embryo culture medium described above, comprising the following steps: Add basal medium to the main culture medium container and place the embryo; Control the release of high-concentration glucose solution through the slow-release channel, so that the glucose concentration in the main culture solution container gradually rises to the final target concentration threshold.

[0023] In the above technical solution, only the embryo needs to be placed in the main culture solution container containing the basal medium, and then the device is started for automatic glucose control, without frequent manual supplementation. Low glucose can be maintained in the early stage and gradually increased in the later stage according to the needs of embryo development, which conforms to the physiological law and helps to improve the quality of embryo development. Avoid high-concentration impact at one time; also reduce the number of times of opening the culture system and reduce the risk of contamination.

[0024] More preferably, the above steps include: initially only opening the first micro-liquid storage bin to inject high-concentration glucose solution at a low rate to maintain a low concentration to simulate the early environment. After the concentration reaches the first-stage target concentration threshold, close the first micro-liquid storage bin and open the second micro-liquid storage bin to gradually increase the concentration; at each stage, automatically switch to the next stage after reaching the corresponding target concentration threshold to form a stepped release based on the feedback of the glucose concentration in the main culture solution container; until the glucose concentration in the main culture solution container reaches the final target concentration threshold in the last stage and enters the maintenance mode.

[0025] In the above technical solution, a target concentration threshold is set for each stage. With the cooperation of sensor monitoring, it can be automatically switched at the corresponding time without overshoot or shortage. The concentration climbs steadily, and the embryo has enough time to adapt; the final maintenance mode ensures that the concentration no longer fluctuates significantly. Practice shows that different developmental stages have different requirements for sugar concentration, and gradually increasing it can significantly improve the blastocyst formation rate, quality score and subsequent implantation rate.

[0026] The present invention realizes the precise regulation of the sugar concentration in the in vitro embryo culture solution by introducing technical means of phased and closed-loop micro glucose control in the device structure and usage method, can effectively meet the needs of different developmental stages of the embryo, and significantly improve the quality of embryo culture and subsequent developmental potential. Specifically, the following beneficial effects can be achieved: 1. It can realize refined glucose supply in multiple stages and low flow rate, which highly coincides with the actual demand curve of the embryo; 2. The closed-loop control of the concentration sensor and the valve significantly reduces the concentration fluctuation and improves the quality of embryo development; 3. The separate liquid storage of the sub-compartments and the hydrophilic slow-release channel fully consider factors such as volume, osmotic pressure and avoiding contamination while ensuring automation; 4. The stepped release and maintenance mode make the whole culture cycle without frequent manual operation, greatly improving the reliability, reproducibility and experimental efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic structural diagram of a device for gradually regulating the glucose concentration in the in vitro embryo culture solution according to Embodiment 1 of the present invention; Figure 2 This is the flowchart of the method for culturing in vitro embryos using the device for gradually adjusting the glucose concentration of the in vitro embryo culture medium in Embodiment 2 of the present invention; Figure 3 It is a graph showing the change of glucose concentration in the main culture medium container over time.

[0028] In the figure, 100 - micro - liquid storage bin; 200 - slow - release channel; 300 - main culture medium container; 400 - control unit; 500 - regulating valve; 600 - sensor. Specific embodiments

[0029] The following further describes the specific embodiments of the present invention with reference to the accompanying drawings. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] Embodiment 1 As Figure 1 shown, the present invention provides a device for gradually adjusting the glucose concentration of the in vitro embryo culture medium, and the device includes: At least two micro - liquid storage bins 100, each of the micro - liquid storage bins 100 is pre - filled with a high - concentration glucose solution; A slow - release channel 200, the slow - release channel 200 is respectively connected to each of the micro - liquid storage bins 100 and the main culture medium container; A main culture medium container 300, which is used to accommodate embryos and a basal medium; A control unit 400, the control unit 400 is used to sequentially open the discharge ports of each of the micro - liquid storage bins 100, so that the high - concentration glucose solution flows into the main culture medium container 300 through the slow - release channel 200 in sequence.

[0031] In the above - mentioned technical solution, it further includes A regulating valve 500, the regulating valve 500 is installed at the discharge port of each of the micro - liquid storage bins 100, and is used to control the release of the high - concentration glucose solution; A sensor 600, the sensor 600 is arranged in the main culture medium container 300, and the sensor 600 is a micro - electrochemical sensor or an optical sensor based on the glucose oxidase reaction, and is used to monitor the glucose concentration in the main culture medium container 300 in real time; The control unit 400 controls the opening and closing and the opening degree of the regulating valve 500 according to the glucose concentration in the main culture medium container 300, so that the glucose concentration of the culture medium reaches the final target concentration threshold.

[0032] In the above technical solution, the number of the micro liquid storage chambers 100 is 2 to 6, and the micro liquid storage chambers 100 are arranged in a linear arrangement, a circular arrangement or a stacked arrangement, and each micro liquid storage chamber 100 has a sealing structure to ensure that the liquid inside does not leak before being released.

[0033] In the above technical solution, by controlling the regulating valve 500 to be at a predetermined opening degree, the flow rate of the glucose solution in the slow-release channel 200 does not exceed 50 μL / hour.

[0034] In the above technical solution, the total volume of the glucose solution flowing into the main culture solution container 300 does not exceed 1 mL.

[0035] In the above technical solution, the slow-release channel 200 is composed of a capillary with a small diameter or a conduit made of a hydrophilic material.

[0036] In the above technical solution, the slow-release channel 200 is made of hydrophilic silica gel, polytetrafluoroethylene or microporous membrane material; the length and pore diameter of the slow-release channel 200 are set so that the release rate of glucose reaches a predetermined value.

[0037] In the above technical solution, the glucose concentration of the high-concentration glucose solution is 5 to 50 g / L.

[0038] Example 2 As Figure 2 shown, the present invention also provides a method for in vitro embryo culture using the device for gradually adjusting the glucose concentration of in vitro embryo culture medium, including the following steps: S1: Add a basic culture medium into the main culture solution container 300 and place the embryo therein; S2: Control the release of the high-concentration glucose solution through the slow-release channel 200 so that the glucose concentration in the main culture solution container 300 gradually rises to the final target concentration threshold.

[0039] The above steps include: initially only opening the first micro liquid storage chamber to inject the high-concentration glucose solution at a low rate to maintain a low concentration to simulate the early environment. After the concentration reaches the first-stage target concentration threshold, close the first micro liquid storage chamber and open the second micro liquid storage chamber to gradually increase the concentration; at each stage, automatically switch to the next stage after reaching the corresponding target concentration threshold to form a stepped release based on the feedback of the glucose concentration in the main culture solution container 300; until the glucose concentration in the main culture solution container 300 reaches the final target concentration threshold in the last stage and then enters the maintenance mode.

[0040] Combined with Figure 3 the curve of the change of the glucose concentration in the main culture solution container 300 with time shown in, specifically includes the following four stages: 1. Initial Phase Setup: At the start of culture, the first phase begins. The control unit 400 initially opens only the regulating valve 500 connected to the first micro-liquid reservoir, adjusting it to a predetermined opening. The regulating valves 500 of the remaining micro-liquid reservoirs remain closed. The glucose solution in the first micro-liquid reservoir is then slowly released into the main culture medium container 300 at a rate of no more than 50 μL / hour, mixing with the culture medium. At this point, the glucose concentration in the culture medium is maintained at a low level (e.g., approximately 0.5 mM, to simulate the early stage environment of the fallopian tube). This continuous, trace replenishment maintains the required low initial concentration, providing a suitable environment for early embryonic development.

[0041] 2. Concentration Monitoring and Phase Triggering: As the first phase progresses, the sensor 600 continuously monitors changes in the glucose concentration of the culture medium. The control unit 400 compares the real-time concentration with the first-phase target concentration threshold. When the control unit 400 detects that the glucose concentration of the culture medium has risen over time and reached the pre-set first-phase target concentration threshold, it indicates that the glucose concentration has reached the required level for that phase, meeting the triggering conditions for phase switching. At this point, the control unit 400 issues instructions to sequentially execute the following actions: close the regulating valve 500 of the first micro-liquid reservoir (if necessary, this can be maintained at a very small opening to prevent a rapid drop in concentration), and simultaneously open the regulating valve 500 of the second micro-liquid reservoir to the appropriate opening. This initiates the second phase, and glucose solution begins to be released from the second micro-liquid reservoir into the main culture medium container 300. After the second phase is initiated, the glucose concentration of the culture medium will further increase due to the new supply.

[0042] 3. Step-by-step cyclic release: Similarly, during the second stage of operation, the control unit 400 monitors the concentration and gradually increases and approaches the target concentration threshold of the second stage. When the target concentration threshold of the second stage is reached, the control unit 400 triggers the transition to the next stage: close the regulating valve 500 of the second micro liquid storage tank and open the regulating valve 500 of the third micro liquid storage tank to enter the third stage. This cycle is repeated, and multi-stage release control can be performed according to the set number of stages. The start of each stage is automatically triggered by the condition that the culture solution concentration of the previous stage "reaches the threshold", forming a step-by-step release rhythm driven by concentration feedback. The switching process between stages is fast and accurate, and the control unit 400 strictly executes the order to avoid the simultaneous release of large flow rates from multiple micro liquid storage tanks 100, causing concentration to be out of control.

[0043] 4. Termination and Maintenance: When the last stage (e.g., the Nth stage) runs and the glucose concentration in the culture medium reaches the final target concentration threshold, the control unit 400 closes the regulating valve 500 of the last micro-liquid storage bin 100 to end the active release process. At this time, the entire release process is completed. The total volume of the glucose solution added to the main container 300 of the culture medium does not exceed the limit of 1 mL, and the total volume is calculated by cumulatively calculating the flow rate parameter of the glucose solution in the known slow-release channel 200 and the opening duration of the regulating valve 500. In the later stage of cultivation, it enters the monitoring and maintenance mode: The sensor 600 continues to monitor the concentration of the culture medium in real time. If it is found that the concentration decreases due to reasons such as embryo uptake, the control unit 400 can appropriately open the valve in the last stage for micro-compensation to maintain a constant concentration. However, no new liquid storage source is opened. Through the above closed-loop control mechanism, even if there are differences in the glucose consumption rates of embryos, the release can be automatically adjusted to achieve the established concentration curve without manual intervention.

[0044] In the above solution, assume that: The volume of the main container 300 of the culture medium is V, The concentration of the glucose solution in the micro-liquid storage bin 100 used in the current stage is C sol , The release flow rate of the micro-liquid storage bin 100 (the flow rate after opening the regulating valve 500) is F i (t), with the unit such as μL / h, The uptake rate of glucose by the embryo is R(t), and the unit can be regarded as μmol / h, Let the instantaneous glucose concentration in the culture medium be C(t). At a certain moment, if only one micro-liquid storage bin 100 is releasing (with a flow rate of F i (t)), then the change in the glucose content in the main container 300 of the culture medium over time can be described by the following balance equation: ; Among them: represents the total amount of glucose in the main container 300 of the culture medium; F i (t) × C sol represents the total amount of glucose entering the main container 300 of the culture medium per unit time (if the regulating valve 500 is completely closed, then F i (t) = 0); R(t) represents the net consumption rate of glucose by the embryo at this moment (which can be regarded as a quantity at the μmol / h level and needs to be unified in terms of units), Convert it to the change rate of concentration over time: ; Assume that there are a total of N stages, and each stage has a target concentration threshold , and is equipped with a micro-liquid storage chamber 100 (or different regulating valves 500 in the same chamber are set), and its release flow rate can be approximately regarded as a constant F at this stage i , Target concentration in the first stage: ; Target concentration in the second stage: ; ... Target concentration in the Nth stage: ; And there are initial conditions: C(0) = C0, about 0.5 mM, At the beginning of each stage i, Only open the regulating valve 500 corresponding to the micro-liquid storage chamber 100 (the flow rate is F when the opening is constant i ), and the regulating valves 500 of the remaining chambers are closed or kept at a very small opening, The sensor 600 measures C(t) in real time. When it detects that C(t) ≥ (that is, the target concentration threshold of the current stage is reached): close the regulating valve 500 of the current chamber (or adjust it to a very small flow rate to prevent too fast a drop), open the regulating valve 500 of the next stage chamber (if i < N), and enter stage i + 1. This "closed-loop trigger" can be formalized as: ; If the opening duration of the regulating valve 500 in the ith stage is Δt i (which can be recorded by the control unit 400 for the start and end times), and the flow rate of this chamber is basically stable at F i , then the total volume of the glucose solution released into the main culture solution container 300 in this stage is approximately: ; Here, if the unit of F i is μL / h and the unit of Δt i [[ID=...]] is hours, then the unit of V release,i is microliters (μL), and it needs to satisfy: ; To ensure that the final concentration achieves the corresponding gradient, an approximate check can also be carried out from the total amount perspective: The expected concentration span of each stage , where can be regarded as the initial concentration C0, If the consumption by the embryo is not considered, in stage i, in order to increase the concentration in the main container from to , approximately [[ID=...]] of the total amount of glucose needs to be added to the volume V, and the corresponding solution volume is approximately: ; When switching to the Nth stage and detecting : Close all the micro-liquid storage chambers 100, and monitor C(t) regularly or continuously. ; Wherein, can be a tiny threshold value to avoid frequent switching of the regulating valve 500. At this time, the release flow rate can be greatly reduced to meet the requirement of only compensating for consumption without a large increase. In this way, the final concentration can be kept stable while ensuring that the total release amount of the glucose solution does not exceed 1 mL.

[0045] The following exemplarily shows the comparison of key indicators between the control group (the technical solution that does not use the method described in Example 2) and the experimental group (the technical solution that uses the method described in Example 2) under similar experimental conditions, so as to intuitively reflect the advantages brought by using the above technical solution. Among them, the control group adjusts the glucose concentration in the culture solution by replacing the culture medium in stages. In the initial stage of embryo culture (starting from 0 hours), a basic culture solution containing 0.3 mM glucose is used. At the end of the 3rd day of culture (72 hours), the whole embryo is manually transferred to a fresh culture solution, and the glucose concentration of this culture solution is 2.0 mM, so as to provide the glucose concentration required for subsequent development. The volume of the culture solution replaced each time is about 600 - 800 μL, covering the entire internal environment of the culture dish. During the culture process, no automatic sugar supply or sensor closed-loop regulation device is adopted, and the environment is maintained in a static culture system at 37 °C, 5% CO2, and humidity above 95%. It should be emphasized that the following data are only for illustration, and the specific values will vary due to various factors such as experimental design, embryo type and quantity, and culture medium composition. The actual results shall be subject to real experimental measurements, as shown in the following table: Verified by the above experimental data, using the method of this embodiment has the following effects: 1. A smoother glucose concentration curve: The multi-stage micro-release combined with sensor closed-loop monitoring enables the target concentration in each stage to be closer to the set threshold value while avoiding large fluctuations.

[0046] 2. Higher embryo development indicators: Including the early cleavage rate, blastocyst formation rate, and subsequent implantation / survival rate, etc., all have different degrees of improvement in the experimental group, indicating that supplying glucose in stages more meets the physiological needs of embryos in the early, middle, and late stages.

[0047] 3. Reduced manual intervention: Relying on automatic valve switching and sensor feedback, the number of operations and the manual judgment links can be significantly reduced, improving the repeatability and safety of the culture process.

[0048] 4. Meet the volume limit: By recording and calculating the valve opening and opening duration in real time, the total injection volume can be controlled within 1 mL to avoid affecting the embryo environment due to excessive addition.

[0049] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. The content not described in detail in this specification belongs to the prior art well known to those skilled in the art.

Claims

1. An apparatus for gradually adjusting the glucose concentration of in vitro embryo culture medium, characterized in that: The device includes: At least two micro-liquid storage chambers, each of which is pre-filled with a high-concentration glucose solution; A slow-release channel, which is respectively connected to each of the micro-liquid storage chambers and the main culture medium container; A main culture medium container for accommodating embryos and a basic culture medium; A control unit, which is used to sequentially open the release ports of each of the micro-liquid storage chambers, so that the high-concentration glucose solution flows into the main culture medium container through the slow-release channel in sequence.

2. The device for gradually adjusting the glucose concentration of the in vitro embryo culture medium according to claim 1, wherein: It also includes Adjusting valves, which are installed at the release ports of each of the micro-liquid storage chambers and are used to control the release of the high-concentration glucose solution; A sensor, which is arranged in the main culture medium container, and the sensor is a micro-electrochemical sensor or an optical sensor based on the glucose oxidase reaction, and is used to real-time monitor the glucose concentration in the main culture medium container; The control unit controls the opening and closing and the opening degree of the adjusting valve according to the glucose concentration in the main culture medium container, so that the glucose concentration of the culture medium reaches the final target concentration threshold.

3. The device for gradually adjusting the glucose concentration of in vitro embryo culture solution according to claim 1, wherein: The number of the micro-liquid storage chambers is 2 to 6, and the micro-liquid storage chambers are arranged in a linear arrangement, a circular arrangement or a stacked arrangement, and each micro-liquid storage chamber has a sealing structure.

4. The device for gradually adjusting the glucose concentration of an in vitro embryo culture solution according to claim 2, characterized in that: By controlling the adjusting valve at a predetermined opening degree, the flow rate of the glucose solution in the slow-release channel does not exceed 50 μL / hour.

5. The device for gradually adjusting the glucose concentration of in vitro embryo culture medium according to claim 1, characterized in that: The total volume of the glucose solution flowing into the main culture medium container does not exceed 1 mL.

6. The device for gradually adjusting the glucose concentration of in vitro embryo culture medium according to claim 1, characterized in that: The slow-release channel is composed of a capillary with a small diameter or a catheter made of a hydrophilic material.

7. The device for gradually adjusting the glucose concentration of the in vitro embryo culture solution according to claim 6, characterized in that: The slow-release channel is made of hydrophilic silica gel, polytetrafluoroethylene or microporous membrane material; the length and pore diameter of the slow-release channel are set so that the release rate of glucose reaches a predetermined value.

8. The device for gradually adjusting the glucose concentration of in vitro embryo culture solution according to claim 1, characterized in that: The glucose concentration of the high-concentration glucose solution is 5 to 50 g / L.

9. A method for in vitro embryo culture using the device for gradually adjusting the glucose concentration of in vitro embryo culture medium according to claim 1, characterized in that: It includes the following steps: Adding a basic culture medium to the main culture medium container and placing embryos; Controlling the release of the high-concentration glucose solution through the slow-release channel, so that the glucose concentration in the main culture medium container gradually rises to the final target concentration threshold.

10. A method for in vitro embryo culture using the device for gradually adjusting the glucose concentration of in vitro embryo culture medium according to claim 9, characterized in that: The above steps include: initially only opening the first micro-liquid storage chamber to inject the high-concentration glucose solution at a low rate, maintaining a low concentration to simulate the early environment, after the concentration reaches the first-stage target concentration threshold, closing the first micro-liquid storage chamber and opening the second micro-liquid storage chamber, and gradually increasing the concentration; at each stage, it automatically switches to the next stage after reaching the corresponding target concentration threshold, forming a stepped release based on the feedback of the glucose concentration in the main culture medium container; until the glucose concentration in the main culture medium container reaches the final target concentration threshold in the last stage and then enters the maintenance mode.

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