Special culture dish for embryo vitrification unfreezing and use method thereof

Abstract

The invention relates to the technical field of assisted reproduction, and discloses a culture dish special for embryo vitrification thawing. The culture dish is prepared by compounding a medical-grade cycloolefin copolymer and boron nitride nanosheets, and the heat conductivity coefficient is not lower than 0.75 W / (m.K). A groove with the depth of 1-2mm and the volume of 100-200mu L is formed in the bottom of the dish body and is used for accommodating unfreezing liquid. By means of the high-thermal-conductivity material and the microstructure design, the problems that a traditional culture dish is poor in thermal conductivity, unstable in temperature, large in reagent dosage and difficult in operation standardization are solved, and rapid, stable and low-cost embryo vitrification unfreezing operation is achieved.

Description

Technical Field

[0001] This invention relates to the field of assisted reproductive technology, and more specifically, to a special culture dish for thawing and thawing embryos in glass and a method for performing standardized thawing operations using the culture dish. Background Technology

[0002] Vitrification and thawing are core technologies for embryo preservation in modern assisted reproductive technology. Currently, ordinary plastic culture dishes are routinely used for thawing in clinical practice, which has the following inherent drawbacks: Poor thermal conductivity: The low thermal conductivity of plastics (approximately 0.1-0.2 W / (m·K)) leads to unstable thawing fluid temperature, unsatisfactory rewarming rate, and easy damage to the embryo due to the re-formation of ice crystals.

[0003] High reagent and time costs: Large droplets (500-1000μL) need to be prepared and often covered with mineral oil to prevent evaporation, resulting in serious reagent waste; and thawing dishes need to be prepared and equilibrated a day in advance, making the process cumbersome.

[0004] Standardizing procedures is difficult: large temperature fluctuations and droplet size and position depend on the operator's experience, resulting in significant differences in resuscitation effects between different batches and poor consistency in clinical outcomes.

[0005] Therefore, there is an urgent need for a thawing device and method that combines excellent thermal conductivity, significant cost savings, and standardized operation to improve embryo recovery efficiency and clinical pregnancy success rate. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing technologies and provide a low-cost, high-thermal-conductivity glass culture dish for thawing embryos and its standardized usage method. Through systematic innovation in material formulation, structural design, and operational procedures, it achieves dual savings in reagents and time, ultimately leading to excellent clinical outcomes.

[0007] In a first aspect, to achieve the above objectives, the present invention adopts the following technical solution: a special culture dish for embryo glass thawing, comprising a dish body and a dish lid made of a high thermal conductivity composite material, wherein the composite material comprises a medical-grade cyclic olefin copolymer matrix and boron nitride (BN) nanosheet filler uniformly dispersed therein; the bottom of the dish body is provided with a groove for containing thawing fluid; the depth of the groove is 1~2 mm and the volume is 100~200 μL; the bottom of the dish body is flush with its edge and fits snugly against the table surface during use.

[0008] Preferably, as an improvement, the volume percentage of the boron nitride nanosheets is 2% to 5%.

[0009] Preferably, as an improvement, the volume percentage of the boron nitride nanosheets is preferably 4%.

[0010] Preferably, as an improvement, the thermal conductivity of the composite material is not less than 0.75 W / (m·K).

[0011] Preferably, as an improvement, the bottom outer side of the dish body is provided with a plurality of un-perforated alignment grooves; the top outer side of the dish lid is provided with alignment protrusions that match the position and size of the alignment grooves.

[0012] Preferably, as an improvement, the sidewall edge of the dish body is provided with an arc-shaped notch, and the sidewall edge of the dish lid is provided with an arc-shaped protrusion that matches the arc-shaped notch. The arc-shaped notch is used to temporarily embed the embryo freezing carrier rod.

[0013] Preferably, as an improvement, an operation time indicator is provided next to the groove.

[0014] Secondly, the present invention also provides a method for thawing embryos using the aforementioned embryo vitrification thawing culture dish, comprising the following steps: S1. After preheating the culture dish to 37°C, place the pre-equilibrium thawing solution into the groove; S2. After removing the embryo cryopreservation carrier from the low-temperature storage environment, quickly transfer it to the pre-equilibrium thawing solution. Transfer the pre-equilibrium thawing solution droplets sequentially in three positions, with the time controlled within 1 minute. S3. Transfer the thawed and cleaned embryos to a culture medium for post-thaw culture.

[0015] Preferably, as an improvement, the total volume of the pre-equilibrated thawing solution used in the entire culture dish does not exceed 200 μL.

[0016] The core working principle of this invention is as follows: Regarding the materials, highly thermally conductive BN nanosheets are uniformly dispersed in a medical-grade COC matrix to form a three-dimensional thermally conductive network, which significantly improves the overall thermal conductivity of the culture dish and enables rapid and uniform heat transfer.

[0017] Regarding the structure, a precisely sized groove is formed at the bottom of the dish to pre-place the required thawing droplets. This design significantly reduces the amount of reagents used (no mineral oil required) and utilizes the dish's own high thermal conductivity to ensure that small droplets can be stably maintained at 37℃±0.5℃ on the work surface for a long time.

[0018] In addition, the combination of laser engraving time indicators and color-coded zoning marks transforms the complex thawing process into a standardized operation that follows the instructions, reducing reliance on personnel experience and ensuring consistency and repeatability of operations.

[0019] Compared with the prior art, the beneficial effects of the present invention include: 1. The thermal conductivity of the petri dish remained stable at approximately 0.75 W / (m·K). Under room temperature (22℃) conditions, within 10 minutes of removing the dish from the table after preheating, the core temperature fluctuation of the liquid inside the dish was less than ±0.5℃, which is far superior to ±2-3℃ for ordinary petri dishes.

[0020] 2. Reagent consumption is reduced by more than 40%; no overnight equilibration is required, and it can be used immediately; standardized procedures reduce the average operation time by about 25%.

[0021] 3. Droplets in all functional areas are pre-positioned through grooves, eliminating concerns about droplet evaporation and cross-contamination inherent in traditional methods. 4. Significantly improves clinical outcomes. Based on large-scale clinical retrospective analysis, the use of the present invention can significantly improve the hCG positivity rate, clinical pregnancy rate and live birth rate, while significantly reducing the miscarriage rate, especially at the blastocyst stage.

[0022] 5. The bottom of the dish is flush with its edge, ensuring a seamless fit with the desktop during use and enhancing heat conduction. This avoids the problem of poor heat conduction caused by the protruding edge and concave bottom in existing technologies, which prevents complete contact with the desktop. Attached Figure Description

[0023] Figure 1 These are top and bottom views of the lid of the embryo glass thawing culture dish described in Embodiment 1 of the present invention.

[0024] Figure 2 These are top and bottom views of the culture dish for defrosting embryos in glass as described in Embodiment 1 of the present invention.

[0025] Figure 3 for Figure 2 Cross-sectional view along the AA direction.

[0026] Figure 4 This is a schematic diagram of the structure of a traditional petri dish (items 2-4 in the diagram are schematic representations of thawing fluid and are not structural features).

[0027] The reference numerals in the accompanying drawings include: 1. Cover of the dish, 101. Alignment protrusion, 102. Slot, 2. Dish body, 201. Groove, 202. Flange alignment groove, 203. Arc-shaped protrusion, 3. Arc-shaped notch. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will describe in detail the embryo glass thawing culture dish and its application, in conjunction with the accompanying drawings and specific embodiments.

[0029] Example 1: Structure and preparation of the culture dish of the present invention This embodiment describes in detail a specific structure and preparation method of the embryo glass thawing culture dish described in this invention.

[0030] like Figure 1 and Figure 2 As shown, the culture dish includes a dish body 2 (made of a high thermal conductivity composite material through precision injection molding) Figure 2 ) and the matching lid 1 ( Figure 1 The high thermal conductivity composite material uses medical-grade cyclic olefin copolymer (COC) as the matrix and boron nitride (BN) nanosheets as the thermally conductive filler. The BN nanosheets are uniformly dispersed in the COC matrix, with a volume percentage of 2% to 5%. In a preferred embodiment, the volume percentage of BN nanosheets is 4%. This ratio has been experimentally verified to significantly improve the thermal conductivity of the material while maintaining good injection molding processability, optical transparency, and cost controllability. The composite material prepared thereby has a stable thermal conductivity of not less than 0.75 W / (m·K), which is approximately 4 to 5 times that of ordinary plastic petri dishes.

[0031] Combination Figure 3 As shown, a circular groove 201 for containing thawing solution is provided in the central area of ​​the bottom of the petri dish 2. The groove 201 has a depth of 1~2 mm and a volume of 100~200 μL. This groove 201 design replaces the traditional method of directly adding a large number of droplets to the bottom of the petri dish, realizing the pre-positioning and quantitative control of reagents. The amount of reagent used per droplet is reduced by more than 60% compared to the traditional 500-1000 μL, and the droplet can be kept stable without covering it with mineral oil.

[0032] To further enhance operational convenience and stability, the bottom outer side of the dish body 2 is provided with three un-perforated micro-alignment grooves (approximately 0.2 mm deep), arranged in an equilateral triangle. Correspondingly, the top outer side of the dish lid 1 is provided with three micro-alignment protrusions 101 that match the position and size of the alignment grooves. When the dish lid 1 is closed, the alignment grooves and protrusions 101 on adjacent dish bodies 2 and dish lid 1 fit together when multiple culture dishes are stacked, helping to maintain the relative stability of the culture dishes during movement or stacking and preventing slippage. At the same time, the bottom of the dish body 1 is flush with its edge, allowing it to fit snugly against the table surface during use, enhancing heat conduction.

[0033] Similar to conventional petri dishes, the petri dish of the present invention has corresponding annular buckles on the outer side of the dish body 2 and the inner side of the dish lid 1. The annular buckles are a combination of flange 202 and groove 102.

[0034] On the adjacent sidewalls of the dish body 2 and the dish lid 1, there are correspondingly positioned arc-shaped protrusions 3 and arc-shaped notches 4. The arc-shaped notches 4 are used to temporarily embed the embryo freezing carrier rod. This design allows the operator to stably place the carrier rod on the edge of the culture dish during the intervals of embryo transfer, avoiding contamination or temperature fluctuations caused by the carrier rod contacting the work surface.

[0035] Each groove 201 is equipped with a clear operation time indicator (e.g., "Step 1: 1 min") through laser engraving technology. Different function droplet areas (such as balancing area, defrosting area, and cleaning area) can also be distinguished by different colored backgrounds to achieve "convenient" operation guidance and reduce reliance on personnel experience.

[0036] The preparation process includes the following steps: Raw material premixing and drying: Accurately weigh the dried medical-grade COC particles and BN nanosheets at a volume ratio of 4%.

[0037] Melt blending and granulation: The mixture is fed into a high-speed mixer for thorough premixing, and then melt-blended, extruded, cooled and granulated by a twin-screw extruder at 200~240℃ to obtain a high thermal conductivity composite masterbatch.

[0038] Injection molding: The composite masterbatch is fed into a precision injection molding machine, and injection molding is performed at a barrel temperature of 240-260℃ and a mold temperature of 60-80℃ to obtain a culture dish preform (including dish body 2 and dish lid 1).

[0039] Post-processing and labeling: After cleaning the molded preform, operation time indicators and color-coded zones are engraved at designated locations using a laser. Because the COC material itself has suitable surface energy, the inner surface of the culture dish after molding does not require additional hydrophilic coating treatment to meet the droplet molding requirements.

[0040] Performance verification: The culture dishes prepared according to this embodiment were tested: Thermal conductivity: The thermal conductivity was tested using the heat flow method and was approximately 0.75 W / (m·K).

[0041] Temperature stability: At room temperature (22℃), the culture dish was preheated for 5 minutes on a 37℃ hot plate and then moved to a regular work surface. The center temperature of a 150μL water droplet inside the dish was monitored using a high-precision temperature probe. The results showed that the temperature fluctuation range within 10 minutes was less than ±0.5℃, demonstrating excellent heat retention.

[0042] Biocompatibility: The extract was tested for cytotoxicity according to relevant medical device standards, and the results met the requirements for use in embryo culture.

[0043] Example 2: Vitrification and thawing of human blastocysts under standard procedures This embodiment demonstrates the application of the petri dish and accompanying standard procedure of Embodiment 1 of the present invention for routine glass thawing, including the following steps: Preheat the culture dish at 37°C for 5 minutes. Within 1 second of removing the blastocyst cryopreservation carrier from liquid nitrogen, immerse it in the preheated 1.0M sucrose thawing solution on the dish. Then, strictly follow the time markings engraved on the dish (e.g., "Step 1: 1min") to sequentially transfer it to the subsequent thawing and washing areas. The entire process takes about 1 minute. Finally, transfer it to the culture medium droplet for incubation.

[0044] Comparative example: using, for example Figure 4 The process of thawing human blastocysts in a traditional culture dish is shown below: Transfer to the No. 1 defrosting fluid zone, changing the location every minute, for a total of 3 minutes.

[0045] Move to the No. 2 defrosting fluid area, changing the location every 1-2 minutes, for a total of 5 minutes.

[0046] Transfer to thawing solution area 3, rinse briefly, and then transfer to culture medium for post-recovery culture.

[0047] Comparison of clinical outcomes (based on a retrospective analysis of 9,145 cryo-thaw transplant cycles): Overall outcome: Compared with the conventional method group (5136 cycles), the invention group (1925 cycles) had significantly higher hCG positivity rate (64.2% vs 52.2%, P<0.001), clinical pregnancy rate (57.9% vs 46.2%, P<0.001), live birth rate (48.6% vs 37.2%, P<0.001), and significantly lower miscarriage rate (16.1% vs 19.5%, P=0.003).

[0048] Blastocyst stage-specific analysis: In blastocyst transfer cycles, compared with the conventional method group (3295 cycles), the live birth rate of the group of this invention (1346 cycles) was increased by 13.2% (58.5% [788 / 1346] vs 45.3% [1491 / 3295]), and the miscarriage rate was reduced by 5.4% (13.7% [125 / 913] vs 19.1% [351 / 1842]).

[0049] Conclusion: By providing a stable thermal environment and standardized operation, the present invention significantly improves the live birth rate of high-value blastocysts after thawing and reduces the risk of miscarriage while saving costs.

[0050] Example 3: Application of the Ultra-Fast Recovery Process This embodiment demonstrates the application of the culture dish of the present invention in an extremely simplified ultra-fast thawing scheme, further reflecting its technical versatility.

[0051] Petri dish and process design: Using the petri dish from Example 1, only two droplets were prepared on it: droplet 1 (1.0M sucrose thawing solution, 100-150 μL) and droplet 2 (basal culture washing solution, 100-150 μL). A separate conventional petri dish was prepared with 3 drops of culture solution for final washing.

[0052] Ultra-fast operating procedure: After preheating the culture dish, immerse the blastocyst carrier rod in droplet 1 and hold for 1 minute; quickly transfer to droplet 2 and rinse for 10-15 seconds; then transfer to 3 drops of culture medium in a conventional dish and rinse for about 15 seconds each, for a total time of about 2 minutes. Then place it in a culture drop and incubate at 37°C in a 6% CO2 incubator.

[0053] Clinical results: In 112 thawing cycles using this ultra-rapid protocol, the hCG positivity rate was 62.5% (70 / 112), and the clinical pregnancy rate was 57.1% (64 / 112). These results were significantly better than the corresponding indicators (52.2% and 46.2%) in the conventional resuscitation group mentioned in the background technique.

[0054] Conclusion: Even with the "two-step" ultra-rapid approach, relying on the superior thermal conductivity and temperature maintenance capabilities of the culture dish of this invention, the resuscitation time can be drastically reduced from the traditional 10 minutes to approximately 2 minutes, and the reagent usage can be minimized (only 2 droplets are required). Clinical pregnancy outcomes superior to those of traditional methods can still be obtained. This fully demonstrates the technical versatility and strong reliability of the culture dish of this invention, providing a more efficient, economical, and superior new solution for embryo thawing.

Claims

1. A glass culture dish specifically for thawing frozen embryos, characterized in that, The device includes a dish body and a dish lid made of a high thermal conductivity composite material, the composite material comprising a medical-grade cyclic olefin copolymer matrix and boron nitride (BN) nanosheet filler uniformly dispersed therein; the bottom of the dish body is provided with a groove for containing thawing fluid; the groove has a depth of 1~2 mm and a volume of 100~200 μL; the bottom of the dish body is flush with its edge and fits snugly against the table surface during use.

2. The embryo glass thawing culture dish according to claim 1, characterized in that, The volume percentage of the boron nitride nanosheets is 2% to 5%.

3. The embryo glass thawing culture dish according to claim 1, characterized in that, The volume fraction of the boron nitride nanosheets is preferably 4%.

4. A glass culture dish for thawing embryos according to any one of claims 1 to 3, characterized in that, The thermal conductivity of the composite material is not less than 0.75 W / (m·K).

5. The embryo glass thawing culture dish according to claim 1, characterized in that, The bottom outer side of the dish body is provided with multiple un-perforated alignment grooves; the top outer side of the dish lid is provided with alignment protrusions that match the position and size of the alignment grooves.

6. The embryo glass thawing culture dish according to claim 1, characterized in that, The sidewall edge of the dish body is provided with an arc-shaped notch, and the sidewall edge of the dish lid is provided with an arc-shaped protrusion that matches the arc-shaped notch. The arc-shaped notch is used to temporarily embed the embryo freezing carrier rod.

7. The embryo glass thawing culture dish according to claim 1, characterized in that, An operation time indicator is provided next to the groove.

8. A method for vitrifying embryos using a special culture dish for vitrification of embryos as described in any one of claims 1-4 and 5-6, characterized in that, Includes the following steps: S1. After preheating the culture dish to 37°C, place the pre-equilibrium thawing solution into the groove; S2. After removing the embryo cryopreservation carrier from the low-temperature storage environment, quickly transfer it to the pre-equilibrium thawing solution. Transfer the pre-equilibrium thawing solution droplets sequentially in three positions, with the time controlled within 1 minute. S3. Transfer the thawed and cleaned embryos to a culture medium for post-thaw culture.

9. The method according to claim 8, characterized in that, The total volume of pre-equilibrated thawing solution used in the entire culture dish should not exceed 200 μL.

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