Film delay partition type culture dish bottom glucose slow release structure and use method

Abstract

The invention belongs to the technical field of assisted reproduction, and particularly relates to a film delay partition type culture dish bottom glucose slow release structure and a use method. Comprising a culture dish, a glucose slow-release structure in the culture dish and a thin film covering the glucose slow-release structure, the glucose slow-release structure is fixed to the bottom of the culture dish, the thin film covers the glucose slow-release structure, a partition is formed between the thin film and the bottom of the culture dish, and the thin film is fixed to the bottom of the culture dish. The thin film is made of a biodegradable high polymer material with a delayed degradation characteristic, and is used for blocking the release of glucose in the glucose slow-release structure in the early stage of embryo culture and starting to generate hydrolysis-triggered swelling dissolution after being placed in an in-vitro culture solution environment for a preset time; sealing partition of the glucose slow-release structure is removed, and glucose in the glucose slow-release structure is released into the culture solution, so that pollution and mechanical stress risks are reduced.

Description

Technical Field

[0001] This invention belongs to the field of assisted reproductive technology, specifically relating to a thin-film delayed-release glucose slow-release structure at the bottom of a culture dish and its usage. Background Technology

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

[0003] 2. Blastocyst stage (after day 3): The embryo's energy requirements increase significantly, and a high concentration of glucose must be provided to support the rapid growth and metabolic needs of the blastocyst.

[0004] Current technologies primarily adjust glucose concentration in the culture medium through staged medium replacement. This involves manually transferring the embryo to a new culture medium with a higher glucose concentration at the end of day 3 of embryo culture. However, this process has drawbacks: embryo transfer can cause mechanical stress, potentially leading to embryo damage or metabolic disorders; sudden environmental changes (such as pH or osmotic pressure) can also induce environmental stress in the embryo, affecting embryo quality and culture success rate.

[0005] Therefore, existing technologies have significant shortcomings, making it difficult to achieve efficient and convenient glucose concentration control while ensuring embryo culture quality. There is an urgent need to propose simpler, more precise, and non-destructive culture methods to meet the needs of different embryo developmental stages, avoid mechanical and environmental stress responses, and improve the success rate and quality of embryo culture. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the aforementioned background technology and provide a thin-film delayed-release glucose slow-release structure at the bottom of a culture dish and its usage method.

[0007] The technical solution adopted in this invention is: a film-coated delayed-release glucose slow-release structure at the bottom of a culture dish, comprising a culture dish, a glucose slow-release structure inside the culture dish, and a film covering the glucose slow-release structure. The glucose slow-release structure is fixed to the bottom of the culture dish, and the film covers the glucose slow-release structure. The film is sealed and adhered to the bottom of the culture dish along the periphery of the glucose slow-release structure, forming a sealed cavity covering the glucose slow-release structure. Initially, it is isolated from the external culture medium. The film is made of a biodegradable polymer material with delayed degradation characteristics. The film is used to block the release of glucose from the glucose slow-release structure in the early stage of embryo culture. After being placed in the in vitro culture medium environment for a predetermined time, it begins to undergo hydrolysis-triggered swelling and dissolution, releasing the sealed barrier of the glucose slow-release structure and releasing the glucose from the glucose slow-release structure into the culture medium, thereby achieving controlled release of glucose concentration in the culture medium.

[0008] The aforementioned technical solution employs a delayed-release design in its overall structure, enabling precise temporal control of glucose concentration in the embryo culture medium. This avoids the toxic effects that may arise from excessively high glucose concentrations in the early stages of embryonic development and ensures the safety of the embryo during its sensitive developmental period. The nutritional supply is matched to the embryonic developmental needs, thereby improving the quality of the embryo culture environment. Ultimately, the overall quality of embryo culture is significantly enhanced.

[0009] Preferably, the film is a blend of polylactic-co-glycolic acid (PLA) and polyethylene glycol (PEG). The PLA-PEG blend film exhibits a degradation process in the in vitro environment characterized by the initial formation of pores followed by induced main chain hydrolysis. This staged degradation mechanism provides a more precise delayed release time window because the initial formation of pores effectively postpones main chain breakage and glucose release. Furthermore, the blend material exhibits high stability and good biocompatibility, and will not prematurely disintegrate or generate harmful byproducts during the delayed release period. This ensures the reliability of the system operation and guarantees its friendliness and harmlessness to the embryo culture environment.

[0010] Preferably, the film is made of hydroxypropyl methylcellulose. Hydroxypropyl methylcellulose has excellent hydrophilicity and pH-responsive properties. When the pH of the culture medium becomes acidic, the material swells or degrades, thereby triggering the release of internal glucose. Using the change in culture medium acidity as a triggering condition makes the timing of glucose release more specific and predictable. Through this pH-responsive mechanism, nutrient release can be better matched with the metabolic stage of the embryo, further improving the predictability and control precision of the release time.

[0011] Preferably, the glucose sustained-release structure includes at least one glucose-containing inner layer and a biodegradable material outer layer covering the inner layer. The biodegradable material outer layer includes a matrix with multiple micropores and at least one sealing material layer for sealing the pores of the multiple micropores.

[0012] Preferably, the matrix is ​​made of one or more of polylactic acid, polylactic acid-glycolic acid copolymer, polyglycolic acid, chitosan, gelatin, carboxymethyl cellulose or their crosslinked derivatives, and pores are formed in the matrix by adding a pore-forming agent or a foaming process during manufacturing; the porous or microporous morphology gradually transforms from a closed-cell structure in the dry state to an interconnected pore network after absorbing water and swelling.

[0013] Preferably, the sealing material layer is a swellable hydrophilic polymer or a hydrolyzable hydrophobic polymer, which is sealed on the pore surface.

[0014] In the above technical solution, the glucose sustained-release structure adopts a multi-layer design, with the outer controlled-release layer and the inner sugar storage layer working synergistically to achieve a unique release behavior. The outer membrane maintains a closed pore structure in the initial stage, effectively preventing the leakage of sugar from the inner layer during the delay period, thus providing a longer release delay. When the delay time ends, the outer pore structure changes from closed to open, and the glucose stored in the inner layer is rapidly released. This design, combining outer layer sustained release with inner layer rapid release, possesses both a longer delay and instantaneous release regulation capability, flexibly meeting the nutritional needs of different embryonic developmental stages.

[0015] Preferably, the thickness of the film is in the range of 10 to 100 micrometers, and its coverage area is 50% to 100% of the bottom area of ​​the culture dish. The film has a microporous structure with an initial porosity of no more than 20%. The film is light-transmitting, and its transmittance in the wavelength range of 400 to 700 nm is no less than 70%.

[0016] In the above technical solution, by adjusting the thickness, coverage, and optical properties of the film, this invention ensures that controlled glucose release is achieved without affecting micromanipulation and observation during embryo culture. The film is optimized to conform to the embryo culture environment while maintaining high transparency and suitable thickness, ensuring clear and unobstructed monitoring of embryo morphology under a microscope. Researchers will not be disturbed during microinjection, embryo transfer, and other procedures, ensuring the smooth execution of routine embryo manipulation processes. Therefore, this design provides delayed controlled release while simultaneously addressing the convenience of experimental operations and the need for embryo development monitoring.

[0017] A method for in vitro embryo culture using the aforementioned film-delayed septum glucose slow-release structure at the bottom of a culture dish includes the following steps: placing a human embryo and culture medium together into a culture dish containing the glucose slow-release structure; starting culture; and 48–72 hours after the start of culture, the glucose slow-release structure automatically begins to release glucose into the culture medium under the delayed release effect of the film, with the film undergoing hydrolysis or swelling and dissolution triggered by the culture environment to release glucose.

[0018] Preferably, when the film is a blend of polylactic acid-glycolic acid copolymer and polyethylene glycol, the mass ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 55:45, the mass percentage of polyethylene glycol in the film is 5-10%, and the film thickness is 20-50 µm. In the culture medium environment, polyethylene glycol dissolves and forms interconnected micropores inside the film within the first 12-24 hours. Subsequently, the culture medium permeates into the polylactic acid-glycolic acid copolymer matrix through the micropores, initiating a hydrolysis reaction of its main chain ester bonds. Through this hydrolysis, the autocatalytically formed lactic acid and glycolic acid lower the local pH of the film and accelerate chain breakage, causing the film to reach a porosity of 10-20% or higher within 48-72 hours after the start of culture and begin to degrade and disintegrate, thereby releasing glucose from the glucose slow-release structure into the culture medium. Pre-dissolution of PEG is used to induce the hydrolysis of PLGA, and local autocatalytic degradation is achieved through the acidity generated by PLGA itself. After PEG dissolves at a predetermined time, it forms initial pores, leading to a decrease in the local pH environment. This lower pH further accelerates the breakage of the PLGA backbone. Through this chain-reaction degradation mechanism, pore formation and sugar release are strictly controlled within a precise time window. This mechanism enables timed release as designed without external intervention, giving the system a highly predictable automatic release characteristic. Simultaneously, the controllability and reliability of the controlled-release process are significantly improved.

[0019] Preferably, when the membrane is made of hydroxypropyl methylcellulose, the timing of membrane swelling is determined by monitoring whether the pH of the culture medium reaches a threshold. The swelling and disintegration time range is 60–72 hours after the start of culture. Monitoring is performed using either a phenol red indicator or a miniature optical non-invasive sensor, avoiding direct probe insertion into the embryo region. The release trigger point of the hydroxypropyl methylcellulose membrane is determined by applying a pH indicator and monitoring method. By integrating a pH indicator into the culture system, the system provides a clear signal when the environmental acidity reaches the trigger threshold, allowing the operator to know the start of membrane release without interfering with embryo culture. This intelligent monitoring mechanism improves the accuracy of the system's perception of the release timing and allows the operator to obtain timely information about the system status without affecting the embryo culture environment. Therefore, the accuracy and practicality of release control are greatly improved, ensuring that nutrient supply can more precisely match the developmental needs of the embryo.

[0020] Compared with the prior art, the beneficial effects of the present invention are: 1. Timing-based control of glucose concentration in culture medium: By integrating a sustained-release structure and a delayed-release film at the bottom of the culture dish, the timing and rate of glucose release can be precisely controlled, ensuring that the embryo receives a suitable nutritional environment at different developmental stages. For example, early embryos are in a low-glucose environment, which helps reduce the potential toxicity of glucose metabolism byproducts to the embryo; after 48–72 hours of culture, glucose levels can be automatically increased to meet the higher energy requirements of the blastocyst stage.

[0021] 2. Reduced manual medium changes and operational interference: Traditional methods require changing the culture medium or adding extra nutrients during embryo culture, a cumbersome process that may disrupt the stability of the culture environment. Using the structure of this invention, there is no need to open the culture dish midway to add glucose or change the culture medium, avoiding external interference and contamination risks, and reducing the likelihood of embryos being damaged due to handling or environmental fluctuations.

[0022] 3. Maintaining a stable culture environment: The slow-release system of this invention gradually releases nutrients within a closed culture dish, avoiding fluctuations in temperature, pH, and gas environment caused by frequent opening and closing of the incubator. The high light transmittance of the film ensures the microscopic observation and illumination requirements during the culture process, and both the film and the slow-release material are biodegradable and biocompatible, and will not adversely affect the normal development of the embryo.

[0023] 4. Improved Culture Efficiency and Success Rate: By automatically adjusting the culture medium composition, this invention reduces manual operation steps and increases the automation level of the culture process. This is expected to improve the efficiency of embryo culture and reduce the workload of personnel. Simultaneously, by creating a dynamic culture environment that better meets physiological needs, the quality of embryo development and the success rate of in vitro culture are expected to improve, providing stronger support for in vitro fertilization and embryo transfer. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of a thin-film delayed-release glucose slow-release structure at the bottom of a culture dish according to the present invention; Figure 2 This is a schematic diagram of the glucose sustained-release structure; Figure 3 Microscopic images of embryos in the control group (conventional culture, without sustained release) at T1=24h; Figure 4 Microscopic images of embryos in the sustained-release group (those with a thin-film delayed-release glucose-releasing structure at the bottom of a culture dish) at T1=24h; Figure 5 Microscopic images of embryos in the control group (conventional culture, without sustained release) at T2=72h; Figure 6Microscopic images of embryos in the sustained-release group (those with a thin-film delayed-release glucose-releasing structure at the bottom of a culture dish) at T2=72h; Figure 7 Microscopic images of embryos in the control group (conventional culture, without sustained release) at T3=120h; Figure 8 Microscopic images of embryos in the sustained-release group (those with a thin-film delayed-release septum bottom glucose sustained-release structure) at T3=120h.

[0025] Wherein, 1-culture dish; 2-film; 3-glucose sustained-release structure (3.1-inner layer of glucose; 3.2-outer layer of biodegradable material; 3.3-matrix; 3.4-sealing material layer). Detailed Implementation

[0026] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, 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.

[0027] Example 1 like Figure 1 As shown, a film-delayed isolation type culture dish bottom glucose slow-release structure includes a culture dish 1, a glucose slow-release structure 3 inside the culture dish, and a film 2 covering the glucose slow-release structure. The glucose slow-release structure 3 is fixed to the bottom of the culture dish 1. The film 2 covers the glucose slow-release structure 3 and forms an isolation with the bottom of the culture dish 1. The film 2 is sealed and adhered to the bottom of the culture dish 1 along the periphery of the glucose slow-release structure 3, forming a closed cavity covering the glucose slow-release structure 3. Initially, it is isolated from the external culture medium. The film 2 is made of a biodegradable polymer material with delayed degradation characteristics. The film 2 is used to block the release of glucose from the glucose slow-release structure 3 in the early stage of embryo culture. After being placed in the in vitro culture medium environment for a predetermined time, it begins to undergo hydrolysis-triggered swelling and dissolution, releasing the sealed isolation of the glucose slow-release structure 3 and releasing the glucose from the glucose slow-release structure 3 into the culture medium, so as to achieve controlled release of glucose concentration in the culture medium.

[0028] In one optional embodiment, film 2 is a film formed by blending polylactic acid-glycolic acid copolymer (PLGA) with polyethylene glycol. By mass percentage: 80-85% polylactic acid-glycolic acid copolymer (PLGA, lactic acid:glycolic acid = 55:45), 5-10% polyethylene glycol (PEG), and 10-15% glucose.

[0029] In another optional embodiment, film 2 is a film made of hydroxypropyl methylcellulose. By mass percentage: 95-100% hydroxypropyl methylcellulose (HPMC), 0-5% glycerol, and 5-10% glucose.

[0030] In the above technical solution, the thickness of the film 2 ranges from 10 to 100 micrometers, and its coverage area is 50% to 100% of the bottom area of ​​the culture dish 1. The film 2 has a microporous structure with an initial porosity of no more than 20%. The film 2 is light-transmitting, and its transmittance in the wavelength range of 400 to 700 nm is no less than 70%. The film 2 itself does not contain exudable sugars.

[0031] In one alternative embodiment, the glucose sustained-release structure 3 is fixed to the bottom of the culture dish 1 in any of the following ways to form an initial airtight barrier together with the film 2: (i) Mechanical locking: An annular groove or step is set at the bottom of the culture dish 1, the grape slow-release structure 3 is placed in the groove and fixed by a retaining ring; the film 2 is laid on top of the slow-release structure, and an inert elastic sealing ring is added between its periphery and the bottom of the dish to press and seal it. (ii) Hot pressing / welding edge sealing: After surface activation, a continuous edge sealing is formed between the periphery of the film and the bottom of the dish by means of local low temperature hot pressing, ultrasonic / laser plastic welding, etc.; (iii) Adhesion sealing: Apply medical-grade adhesive between the periphery of the film and the bottom of the dish and allow it to cure completely to form a ring seal, and the adhesive layer does not extend to the culture area where the embryo is in direct contact.

[0032] The above methods all achieve initial sealing and isolation of the sustained-release structure between the film 2 and the bottom of the culture dish 1, blocking glucose release at the beginning of the culture stage. Subsequently, the film opens within the trigger time window to connect the pores, thus meeting the delayed release and time-controlled release requirements of the present invention.

[0033] After assembly, it is preferable to use electrochemical impedance spectroscopy (EIS) and fluorescence ratio method (FBI) to confirm the sealing and opening time; the transmittance in the visible light band (400–700nm) should not be less than 70%; during the incubation process, the pH should be maintained at 7.2–7.4, and there should be no visible particles / film debris.

[0034] like Figure 2 As shown, the glucose sustained-release structure 3 includes at least one glucose-containing inner layer 3.1 and a biodegradable material outer layer 3.2 covering the inner layer. The biodegradable material outer layer 3.2 includes a matrix 3.3 with multiple micropores and at least one sealing material layer 3.4 for sealing the pores of the multiple micropores.

[0035] In the above technical solution, the matrix is ​​made of one or more of polylactic acid, polylactic acid-glycolic acid copolymer, polyglycolic acid, chitosan, gelatin, carboxymethyl cellulose or their crosslinked derivatives, and pores are formed in the matrix by adding a pore-forming agent or a foaming process during manufacturing.

[0036] In the above technical solution, the porous or microporous morphology gradually transforms from a closed-cell structure in a dry state to a connected pore network after absorbing water and swelling.

[0037] In the above technical solution, the sealing material layer is a swellable hydrophilic polymer or a hydrolyzable hydrophobic polymer, which is sealed on the pore surface.

[0038] In the above technical solution, the shape of the glucose sustained-release structure is one of microspheres, microcapsules, gel blocks, or membranes.

[0039] Example 2 A method for using the aforementioned film-delayed septum glucose slow-release structure at the bottom of a culture dish includes the following steps: placing a human embryo and culture medium together in a culture dish containing the glucose slow-release structure; initiating culture; 48–72 hours after the start of culture, the glucose slow-release structure automatically begins to release glucose into the culture medium under the delayed-release effect of the film, the film undergoing hydrolysis or swelling and dissolution triggered by the culture environment to release glucose; when the film is a film formed by blending polylactic acid-glycolic acid copolymer and polyethylene glycol, wherein the mass ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 55:45. The polyethylene glycol content in the film is 5-10% by mass, and the film thickness is 20-50 µm. In the culture medium environment, polyethylene glycol dissolves and forms interconnected micropores inside the film within the first 12-24 hours. Subsequently, the culture medium permeates into the polylactic acid-glycolic acid copolymer matrix through the micropores, initiating a hydrolysis reaction of its main chain ester bonds. Through this hydrolysis, the lactic acid and glycolic acid formed by autocatalysis lower the local pH of the film and accelerate chain breakage, so that the film reaches a porosity of more than 10-20% within 48-72 hours after the start of culture and begins to degrade and disintegrate, thereby releasing glucose from the glucose sustained-release structure into the culture medium.

[0040] Example 3 A method for using the aforementioned film-delayed septum glucose slow-release structure at the bottom of a culture dish includes the following steps: placing a human embryo and culture medium together in a culture dish containing the glucose slow-release structure; initiating culture; 48–72 hours after the start of culture, the glucose slow-release structure automatically begins to release glucose into the culture medium under the delayed-release effect of the film, and the film undergoes hydrolysis or swelling and dissolution triggered by the culture environment to release glucose; when the film is made of hydroxypropyl methylcellulose, the timing of film swelling triggering is determined by monitoring whether the pH of the culture medium reaches a threshold, and the film swelling and disintegration time range is 60–72 hours after the start of culture, and the monitoring method is one of phenol red indicator or micro-optical non-invasive sensor, and the probe should be avoided from directly entering the area where the embryo is located.

[0041] In Examples 2 and 3 above, the outer layer of the biodegradable material with glucose sustained-release structure maintains its structure unchanged from the start of embryo culture to the first time threshold T1; the outer layer of the biodegradable material swells, degrades or peels off during the period from the first time threshold T1 to the second time threshold T2, and the glucose-containing inner layer begins to release glucose into the culture medium until after the second time threshold T2, when the glucose concentration in the culture medium gradually rises to the target concentration required for the blastocyst stage.

[0042] The culture medium is a single-step medium, or other media with buffering or adaptation mechanisms for the low glucose requirement during cleavage and the high glucose requirement during blastocyst stage can be selected.

[0043] The sealing material layer keeps the pores in a non-connected state before the first time threshold T1, and begins to swell, degrade or peel off after reaching the first time threshold T1, so that the pores gradually connect and the glucose-containing inner layer begins to release glucose.

[0044] The first time threshold T1 is set as a critical time threshold before the end of the embryonic cleavage stage, and the second time threshold T2 is set as a critical time threshold before or at the beginning of blastocyst formation, so that the sugar concentration required during the blastocyst stage gradually reaches and is maintained within the target range in the culture medium.

[0045] The outer layer of the biodegradable material is further incorporated with a buffer that can be gradually released at 37°C and pH 7.2-7.4 to maintain the pH of the culture medium stable within the range of 7.2-7.4 during the swelling, degradation or peeling of the outer layer.

[0046] The glucose sustained-release structure in this embodiment employs a triple system: a sugar-containing inner layer, a biodegradable porous outer layer, and a sealing material layer. During preparation, a high concentration of glucose is first uniformly embedded in the inner layer. The outer layer uses biocompatible materials such as polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), or chitosan, forming dry closed pores through a pore-forming agent or foaming process. Subsequently, a swellable or hydrolyzable sealing material layer is applied to the pore openings, creating a dense barrier state initially after culture medium addition. During the cleavage stage (days 0-3 of culture), water penetration is slow, and glucose diffuses at a very low rate, maintaining a low glucose level in the culture medium. This perfectly matches the early embryonic physiological needs of relying on pyruvate / lactic acid metabolism and avoiding high glucose levels.

[0047] When cultured to the set time threshold T1 (approximately day 2.5-3, when the embryo enters the morula stage), the sealing material layer begins to swell or hydrolyze, and the outer closed pores gradually transform into an interconnected network as they absorb water and swell. Thus, the glucose release pathway switches from "diffusion-restricted" to "through diffusion," but the release rate is still regulated by both the outer layer thickness and the network size to avoid explosive release. Subsequently, in the T1-T2 stage (approximately day 3-5), the outer polyester continues to hydrolyze, and chitosan undergoes enzymatic hydrolysis, continuously increasing the network density. The inner sugar source is released stably with near-zero-order kinetics, and the glucose concentration in the culture medium gradually increases from approximately 0.2-0.3 mM (millimoles / L) to the 1-2 mM (millimoles / L) required for blastocyst formation, reaching a plateau around T2.

[0048] During this process, acidic intermediates such as lactic acid and glycolic acid produced by polyester degradation are neutralized by pre-doped buffers and chitosan degradation products, stabilizing the culture medium pH at 7.2-7.4 and preventing localized acidification that could damage the embryo. In the later stages of blastocyst expansion (≥T2), the inner layer glucose is almost completely released, and the outer layer material degrades to safe small molecules, naturally decreasing the release rate. The glucose concentration in the culture medium is maintained at the blastocyst maintenance level without becoming excessive. Through this time-controlled degradation and diffusion coupling mechanism, this invention achieves sequential energy supply from low glucose in the cleavage stage to high glucose in the blastocyst stage with a single application, eliminating the need for medium changes, reducing mechanical stress and contamination risks, significantly improving blastocyst formation rate and subsequent implantation potential, and demonstrating outstanding clinical application value.

[0049] To achieve the functional effect of "keeping the pores in a non-connected state before the first time threshold T1 and starting to swell, degrade, or peel off after T1" in the above scheme, the sealing material needs to have good environmental responsiveness and time delay controllability, which can be controlled from three aspects: material selection, structural design, and preparation process. First, the sealing material layer should be a polymer with a delayed response to the cumulative hydration effect of temperature or time, preferably a hydrophilic polymer with controllable hydration behavior or slow degradation properties, such as gelatin, hydroxypropyl methylcellulose (HPMC), polyvinyl alcohol (PVA), cross-linked chitosan, or their complexes. These materials exhibit predictable water absorption and swelling rates or hydrolysis initiation times at 37°C and physiological pH (approximately 7.2–7.4).

[0050] Secondly, to ensure that the sealed pores are physically disconnected before T1, the density and barrier properties of the sealing material layer can be improved in the following ways: A uniform, crack-free sealing film is formed at the pore openings of the matrix using solution casting or spraying methods. Moderate cross-linking of the sealing material (e.g., using glutaraldehyde, ionic cross-linking agents, or UV curing) can slow down its water absorption and degradation rate. Control the coating thickness between 10 and 100 μm to avoid premature cracking due to excessive thinness, and to avoid affecting complete swelling in the later stage due to excessive thickness.

[0051] Furthermore, to ensure that the swelling or degradation process can be initiated after T1, the following parameters can be adjusted: By pre-setting the molecular weight, cross-linking degree, and hydrophilicity parameters of the sealing material layer, it can achieve a glass transition (Tg drops below body temperature) after a specific hydration time (such as about 48–60 hours), changing from a glassy state to a highly elastic state, thereby rapidly absorbing water and swelling. Alternatively, a double-layered sealing material layer can be used, with the outer layer being a rapidly hydrolytic hydrophilic polymer (such as PVA) and the inner layer being a slow-release shedding structure. The outer layer degrades first, and then the inner layer is exposed, allowing the entire sealing material layer to naturally disintegrate around time T1.

[0052] Therefore, in practical implementation, by optimizing the composition (polymer type, molecular weight, degree of cross-linking), structural thickness, and construction process (coating uniformity, curing method) of the sealing material, it can maintain its density and effectively block pore connectivity at the beginning of the culture stage. It can also gradually hydrate, loosen, degrade, or peel off in a sheet-like manner under the influence of the receptor fluid environment before and after T1, thereby achieving time-series regulation of the glucose release pathway and providing dynamic nutritional support for embryonic development.

[0053] The preparation process of the glucose sustained-release structure in this embodiment includes: using a gelatable natural or synthetic polymer as a carrier, mixing an aqueous glucose solution with the carrier and gelling it in a low-temperature environment to obtain a glucose-containing inner layer with controlled particle size, and drying the glucose-containing inner layer to form a sugar-containing core; selecting a biodegradable hydrophobic or amphoteric polymer, dissolving or dispersing it together with a pore-forming agent to form a coating liquid, covering the surface of the sugar-containing core by spraying or dipping, and obtaining an outer layer with multiple microporous structures after curing and removal of the pore-forming agent; using a hydrophilic polymer material that can absorb water and swell or controllably degrade under aqueous conditions, preparing a coating liquid and applying it to the pore surface of the outer layer to form an initial sealing material layer; and performing chemical or physical cross-linking treatment on the microspheres after forming the sealing material layer.

[0054] Specifically, it includes: Inner layer preparation: A high-concentration glucose solution (30% by mass) was mixed with a gelatin solution (10% by mass), stirred evenly at room temperature, and then dropped into liquid nitrogen to form gel microspheres. The diameter of the microspheres was controlled at 200–300 μm, and the sugar-containing core was obtained after freeze-drying.

[0055] Porous outer layer coating: Polylactic acid (PLA) is dissolved in chloroform (10% by mass), and 10% (by mass) sodium chloride is added as a pore-forming agent. The mixture is then coated onto the surface of the dried core using a spray coating method and cured by rotation to form a closed-cell outer layer.

[0056] Sealing material coating: Hydroxypropyl methylcellulose (HPMC, K4M grade) was selected as the sealing material and dissolved in an ethanol / water (30:70) mixture to prepare a 3% mass concentration solution. The solution was uniformly formed on the pore surface of the outer layer by electrostatic spraying, with a film thickness of about 30 μm. Then it was dried at 45℃ for 1 hour.

[0057] Crosslinking treatment: Glutaraldehyde vapor crosslinking treatment for 10 minutes was used to improve the density and initial stability of the sealing material layer.

[0058] Experiment 1 As shown in Tables 1-1 and 1-2, the control group followed the traditional staged medium change pathway: at D3 / approximately 72h, the embryos were transferred from low-glucose droplets to pre-equilibrated high-glucose droplets to meet the energy requirements of the blastocyst stage and serve as a baseline control with the sustained-release group; at T1=24h, the morphology and rhythm of both groups were basically consistent, and both were still in a low-glucose state, indicating that the membrane effectively "delayed the cutoff" in the early stage and the control group had not yet undergone medium change; at T2=72h, the control group showed a characteristic of instantaneous "upward jump" from a low pH level due to medium change (accompanied by a possible slight pH transition), while the sustained-release group was in the preset trigger window of 48–72h and began releasing. The sugar content reflects the fundamental difference between the two pathways in terms of sugar supply methods and environmental disturbances. At T3=120h, both groups reached the blastocyst stage. The control group experienced a decline from its high level after the medium change due to continuous consumption, while the sustained-release group could smoothly increase and maintain the glucose in the culture medium at the target plateau of about 1–2mM during the blastocyst stage without changing the medium, and it also helped maintain a stable pH of 7.2–7.4 throughout the process. Based on this, we can conclude that compared with the traditional D3 medium change, the present invention can achieve the technical effects of timely (T1→T2) triggering, stable (T2→T3) controlled release, and less environmental fluctuation without reducing the morphological quality of the embryo.

[0059] Table 1-1 Morphological Validation Table of Control Group Based on Embryonic Development Images Table 1-2 Morphological Validation Table of the Sustained-Release Group Based on Embryonic Development Images Experiment 2 Table 2 records glucose concentration, pH, temperature, and morphology side-by-side on the same time axis for the experimental group (culture dish) and the control group (traditional medium change). The control group follows the traditional staged medium change procedure: initially using a low-glucose medium (target glucose ≈ 0.25 mM), and at D3 / ≈ 72 h, transferring the embryos from the original low-glucose droplets to a pre-equilibrated high-glucose droplet (target ≈ 1.5 mM) in the incubator to meet the energy requirements of the blastocyst stage. Both groups share the environmental parameters of constant temperature incubation at 37℃ and stable pH of the culture system at 7.2–7.4. The unified monitoring / recording time points are 0, 24, 48, 60, 72 (before medium change), 72 (10–15 min after medium change), 84, 96, and 120 h, corresponding to each item in the table. At each time point, 1 µL of glucose was taken from a 20 µL droplet in the oil for quantitative analysis (GOx-POD colorimetric method or HPLC-RID; the volume was replenished after sampling to maintain a constant volume). pH and temperature were recorded simultaneously, and a brief morphological score was performed (e.g., 2–4 c, 8 c / mulberry, blastocyst). At the 72-hour media change point, the "before" value was recorded first, and then recorded again 5–10 minutes after the change to capture the concentration "jump" and slight pH jump caused by the media change. Thereafter, no artificial intervention was performed for 72–120 hours, allowing the concentration to decrease slowly as the embryo was consumed, thus forming a positive comparison with the curve of the sustained-release group, which smoothly increased to 1–2 mM after T1 and maintained a plateau with a more stable pH.

[0060] It can be seen that: Release rate: The concentration in the experimental group increased nearly linearly over 48–96 h (example slope ~0.026 mM·h). -1 This corresponds to the near-zero-order release that is jointly controlled by the outer layer thickness and pore size after the transition from "closed pore to interconnected pore network". Concentration dynamic control: The experimental group remained stable at a plateau of 1.2–1.7 mM for 72–120 h; Stability: The experimental group maintained a constant pH of 7.2–7.4 and a constant temperature throughout the process, and was less affected by manual solution changes; while the control group showed a sawtooth pattern of "sudden increase in concentration followed by decrease as it was consumed" after a solution change at 72 hours.

[0061] These features validate the technical effectiveness of the present invention: "48–72h initiation and release, 1–2mM target platform, and reduced fluid changes to maintain a stable environment".

[0062] Table 2. Glucose Concentration + Environmental Parameters + Speciation / Comparison Table Experiment 3 Control group: Standard dishes (no film, 37℃, pH 7.2–7.4, high glucose on day 3); Experimental group: In a simulated system isotonic with the culture medium (37℃, pH 7.2–7.4), in-situ membrane parameters were measured on dishes with a film. T1-EIS(h): The electrochemical impedance of the thin film at 50% of the baseline (the inflection point where the interconnected pores appear, reflecting "on"); PEG dissolution leader (%): The percentage of PEG dissolved in the film at 12h and 24h, used to verify the two-stage triggering logic of "PEG dissolution first → micropores → main chain hydrolysis"; Blocking index FBI (the faster the decrease, the more likely it is to be activated): using 10kDa fluorescent dextran as a neutral tracer, the rate of decrease in the fluorescence ratio of the upper / lower chambers of the membrane was measured (without involving the embryo or directly measuring glucose). The theoretical basis for the trigger window: In the first 12–24 hours, PEG dissolves to form micropores in the PLGA-PEG blend membrane, followed by hydrolysis of the PLGA main chain and autocatalytic acidification to accelerate degradation, making it permeable and starting to release at 48–72 hours; HPMC membrane is pH-triggered, and the swelling and disintegration window is 60–72 hours.

[0063] Transmittance: Sample thickness, spectrophotometer model, scanning step; Porosity: Mercury pressure / Image method / Mass-volume method and sampling location; T1-EIS: Electrode arrangement, frequency sweep range, 50% baseline definition; FBI formula (upper / lower chamber fluorescence ratio), sample size; PEG leaching: sampling volume, analytical method (HPLC / TOC), calibration curve.

[0064] Table 3 Engineering Trigger Platform Data Both T1-EIS and FBI should fall within the invention setting window (approximately 48–72 hours for PLGA-PEG and approximately 60–72 hours for HPMC); a transmittance of ≥70% ensures ease of microscopic observation and operation.

[0065] Table 4 Material By-products and Compatibility Tables 3 and 4 show that the membrane opens on time within the T1 window, the embryo enters the high metabolic phase as scheduled during the blastocyst stage with less fluctuation, the pH remains stable at 7.2–7.4 throughout the process, and the operational burden and risk of contamination are lower; this confirms that the present invention achieves timely, stable and convenient glucose concentration regulation without reducing the quality of embryo development.

[0066] Structural level: The experimental group transitioned from isolation to connectivity within the T1 window on time, while the conventional method achieved connectivity from 0h, requiring only chemical medium replacement on D3 without "timed opening"; the "delayed isolation + timed release" has been confirmed from an engineering perspective. Safety / environmental level (Table 4): The experimental group exhibited low byproduct load, stable pH (7.2–7.4), and no debris, while the control group had a "zero material" baseline; the comparison shows that the invention achieves controllable degradation without disrupting the culture steady state. In direct comparison with the conventional method (control group: high glucose replacement on D3), Table 3 proves that it "opens on time," and Table 4 proves that it is "stable and safe even when open"; after cross-validation with Experiment 1 (morphology) and Experiment 2 (glucose dynamics), it can be concluded that this film-film delayed isolation bottom-release structure can form and maintain the target glucose platform during the blastocyst stage without mid-term medium replacement, while maintaining a stable culture environment, achieving efficient and convenient glucose concentration control.

[0067] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Contents not described in detail in this specification belong to prior art known to those skilled in the art.

Claims

1. A thin film delayed partitioned petri dish bottom glucose release structure comprising a petri dish, characterized by: The application also discloses a glucose slow-release structure in a culture dish and a film covering the glucose slow-release structure, wherein the glucose slow-release structure is fixed to the bottom of the culture dish, the film covers the glucose slow-release structure, and the film is sealed to the bottom of the culture dish along the periphery of the glucose slow-release structure to form a sealed cavity covering the glucose slow-release structure and isolating the glucose slow-release structure from the outside culture solution in an initial state; the film is made of a biodegradable polymer material with delayed degradation characteristics, and is used for blocking the release of glucose in the glucose slow-release structure in the initial stage of embryo culture and for starting hydrolysis-triggered swelling and dissolution after being placed in an in-vitro culture solution environment for a predetermined time to remove the sealing of the glucose slow-release structure and release the glucose in the glucose slow-release structure into the culture solution to realize controlled release of the glucose concentration in the culture solution.

2. The thin film delayed partitioned petri dish bottom glucose release structure of claim 1, wherein: The film is a film formed by blending polylactic acid-glycolic acid copolymer and polyethylene glycol.

3. The thin film delayed partitioned petri dish bottom glucose release structure of claim 1, wherein: The film is a film made of hydroxypropyl methyl cellulose.

4. The thin film delayed partitioned petri dish bottom glucose release structure of claim 1, wherein: The glucose slow-release structure comprises at least one inner layer containing glucose and a biodegradable material outer layer covering the inner layer, and the biodegradable material outer layer comprises a substrate with a plurality of micropores and at least one layer of sealing material layer for sealing the pores.

5. The thin film delayed partitioned petri dish bottom glucose release structure of claim 4, wherein: The substrate is made of a mixture of one or more of polylactic acid, polylactic acid-glycolic acid copolymer, polyglycolic acid, chitosan, gelatin, carboxymethyl cellulose or a cross-linked derivative thereof, and the pores are formed in the substrate by adding a porogen or a foaming process during manufacturing; the plurality of micropores are transformed from a closed pore structure in a dry state to a connected pore network after water absorption and swelling.

6. The thin film delayed partitioned petri dish bottom glucose release structure of claim 4, wherein: The sealing material layer is a swellable hydrophilic polymer or a hydrolysable hydrophobic polymer, and is sealed to the surface of the pores.

7. The thin film delayed partitioned petri dish bottom glucose release structure of claim 1, wherein: The thickness of the film ranges from 10 to 100 microns, the covering area of the film ranges from 50% to 100% of the bottom area of the culture dish, the film has a microporous structure, the initial porosity of the film is not higher than 20%, the film has light transmittance, and the light transmittance of the film in the wavelength range of 400-700 nm is not lower than 70%.

8. A method of using the glucose release delaying structure of the bottom of the delayed contact culture dish of claim 1, characterized by: The application also discloses a method for controlling the glucose concentration in an in-vitro culture solution, which comprises the following steps: The human embryo is placed in the culture dish containing the glucose slow-release structure together with the culture solution, and the culture is started; 48-72 hours after the culture is started, the glucose slow-release structure starts to release glucose into the culture solution under the delayed control of the film, and the film is triggered to hydrolyze or swell and dissolve in the culture environment to release the glucose.

9. The method of claim 8, wherein the thin film delayed partitioned petri dish bottom glucose release structure is used for: When the film is a film formed by blending polylactic-glycolic acid copolymer and polyethylene glycol, wherein the mass ratio of lactic acid to glycolic acid in the polylactic-glycolic acid copolymer is 55:45, the mass percentage of polyethylene glycol in the film is 5-10%, and the film thickness is 20-50 µm; in the culture medium environment, polyethylene glycol is dissolved out and forms interconnected micropores inside the film within the first 12-24 hours, and then the culture medium penetrates into the polylactic-glycolic acid copolymer matrix through the micropores, triggering the hydrolysis reaction of the ester bond in the main chain of the polylactic-glycolic acid copolymer, and through the hydrolysis, the lactic acid and glycolic acid formed by autocatalysis reduce the local pH of the film and accelerate the chain scission, so that the film reaches a porosity of more than 10-20% within 48-72 hours after the start of the culture and begins to degrade and disintegrate, thereby releasing the glucose in the glucose slow-release structure into the culture medium.

10. The method of claim 8, wherein the thin film delayed partitioned petri dish bottom glucose release structure is used. When the film is a film made of hydroxypropyl methyl cellulose, the swelling time of the film is in the range of 60-72 hours after the start of the culture by monitoring whether the pH of the culture medium reaches a threshold value to determine the timing of film swelling, and the monitoring method is one of using phenol red indicator or a miniature optical non-invasive sensor.

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