Use of polystyrene microspheres in the preparation of reproductive material simulators

Abstract

The present application relates to the biomedical technology field, especially to the application of polystyrene microspheres in the preparation of reproductive material simulation.The present application researches and finds that the polystyrene microspheres provided by the present application can realize high simulation of different reproductive cell morphological characteristics and surface characteristics, including sperm cells, egg cumulus cell complex, oocytes, zygotes, blastomeres and blastocysts, and the like, to provide a standardized and repeatable cell substitution model for reproductive medical research, compared with the existing simulation materials, has the advantages of convenient material taking, simple operation, good stability and the like, and the preparation method is simple, and the advantages are obvious.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of polystyrene microspheres in the preparation of reproductive material mimics. Background Technology

[0002] The assisted reproductive embryology laboratory is often referred to as the "heart" of a reproductive center. In the assisted reproductive technology process, it plays a crucial role in the in vitro fusion of gametes (oocytes and sperm) to form embryos, the in vitro culture and development of these embryos, and their eventual transfer to the mother's uterus or vitrification and cryopreservation. Compared to human tissues, gametes, and embryonic cells, which are relatively few in number, the unique nature of in vitro embryonic development requires embryologists to possess highly skilled in vitro manipulation techniques to handle individual cells or cell clusters. Currently, the only available resources for embryologists to practice in vitro manipulation techniques are oocytes or embryos with developmental problems. These are severely limited in their availability, have short lifespans, cannot be used immediately, pose ethical risks, and require informed consent from patients. Furthermore, they cannot cover the size changes of oocytes and embryos throughout the entire in vitro development process, making them unsuitable for practicing advanced techniques such as embryo biopsies, and the samples themselves are fragile and easily damaged. This is especially problematic for reproductive centers newly implementing the technology, where the lack of discarded cells or embryos cannot meet the needs of newly trained embryologists for rapid, repeated, and on-demand practice.

[0003] Currently, several models for training in assisted reproductive technologies exist both domestically and internationally, but they generally suffer from significant shortcomings. Some models are prepared using simple materials such as agarose or gelatin, which, while inexpensive, differ considerably in mechanical properties from real oocytes or embryos, resulting in distorted manipulation and hindering effective training. Furthermore, existing microsphere models cannot simulate the mixed states of multiple cells, such as the cumulus-oocyte complex, cleavage embryos, and blastocysts, limiting their application. They also exhibit poor stability and dispersion, easily agglomerating and drying out, making long-term preservation and transportation difficult. Additionally, they readily adsorb non-specific substances like proteins, rendering them unusable in embryo culture media containing proteins and amino acids. Therefore, developing an in vitro manipulation training model that can simulate the physical characteristics of real oocytes and embryos is of significant practical importance. Summary of the Invention

[0004] In view of this, the present invention provides the application of polystyrene microspheres in the preparation of reproductive material mimics.

[0005] This invention provides the application of modified or unmodified polystyrene microspheres in the preparation of reproductive material mimics; the reproductive material includes at least one of sperm, oocyte, cumulus-oocyte complex, embryo, cleavage embryo, and blastocyst.

[0006] This invention reveals that polystyrene microspheres can be used to simulate specific technical operations such as in vitro oocyte processing, in vitro fertilization, gamete transfer, embryo transfer, intracytoplasmic sperm injection (ICSI), embryo biopsy, and embryo freezing and thawing, thereby replacing the corresponding cells. In this invention, the modifiers include at least one of ethylene glycol-polypropylene glycol-polyethylene glycol copolymer and poly(N-isopropylacrylamide) copolymer.

[0007] In this invention, the diameter of the polystyrene microspheres is 1-300 μm. The polystyrene microspheres are regularly spherical or ellipsoidal in shape, and can simulate any one or more cells such as human sperm cells, cumulus-oocyte complex, oocytes, fertilized eggs, cleavage embryos, and blastocysts. The diameter is preferably 1-150 μm; in specific embodiments of this invention, it can be 2-4 μm, 5-25 μm, 100-150 μm, or 120-300 μm.

[0008] In this invention, the polystyrene microspheres can be prepared by microfluidic methods, solvent evaporation methods, phase separation methods, spray drying methods, electrostatic spraying methods, template methods, suspension polymerization methods, or soap-free emulsion polymerization methods. The size and production quantity of the microspheres can be adjusted as needed to meet application requirements. In specific embodiments of this invention, dispersion polymerization and suspension polymerization methods are used to prepare microspheres of different particle sizes. Specifically, dispersion polymerization is used to prepare microspheres of 2-4 μm and 5-25 μm, while suspension polymerization is used to prepare microspheres of 100-150 μm or 120-300 μm.

[0009] Specifically, the method for preparing the polystyrene microspheres includes the steps shown in (1) or (2):

[0010] (1) In an inert atmosphere, polyvinylpyrrolidone, styrene monomer and initiator were mixed in a solvent and reacted at 70°C for 6 hours. After centrifugation and washing, unmodified polystyrene microspheres were obtained.

[0011] (2) Mix and stir polyvinyl alcohol, styrene monomer and initiator in water to obtain monomer droplets; stir the monomer droplets at 85~90℃ for 1.5~2 hours to obtain unmodified polystyrene microspheres.

[0012] In this invention, the inert atmosphere includes nitrogen.

[0013] In this invention, the initiator includes at least one of azobisisobutyronitrile, potassium persulfate, or benzoyl peroxide.

[0014] In this invention, the solvent is water or an alcohol-water mixture.

[0015] The method for preparing polystyrene microspheres provided by the present invention further includes the step of: performing plasma treatment on the surface of the unmodified polystyrene microspheres, and then adding a modifier to modify them to obtain modified polystyrene microspheres.

[0016] This invention, in the preparation of polystyrene microspheres, utilizes methods including but not limited to surface chemical modification and physical treatment to imbue the microspheres with hydrophilicity (contact angle between water droplets and microspheres < 90°), enabling the microspheres to exhibit good biocompatibility in culture media; and solving problems such as poor dispersibility and easy aggregation of microspheres. The physical treatment methods include, but are not limited to, plasma treatment, ion beam radiation, and coating methods. The surface of the microspheres may bear hydrophilic groups such as carboxyl, hydroxyl, and amino groups. The functional monomers providing carboxyl or amino groups may be sodium alginate, aminosilane coupling agents, carboxylating agents, etc. The modified groups on the microsphere surface can be combined with other functional molecules, providing a wider range of applications. For microspheres treated with plasma, the gases used in the modification process mainly include air, oxygen, nitrogen, argon, ammonia, helium, carbon dioxide, etc., or two or more gases mixed in a certain proportion; in a specific embodiment of this invention, air is used.

[0017] Furthermore, the plasma treatment includes: using air as the working gas to perform plasma treatment on the surface of the polystyrene microspheres for 30 minutes.

[0018] Furthermore, the modification includes: adding an aqueous solution containing the modifier and mixing at room temperature for 2 hours.

[0019] The method for preparing polystyrene microspheres as described above in this invention further includes a step of screening the prepared polystyrene microspheres using a sieve.

[0020] Microspheres of different sizes were screened to simulate different germ cells.

[0021] Specifically, spherical or ellipsoidal microspheres C with a particle size of 2-4 μm are used to simulate the head of sperm cells;

[0022] Ellipsoidal microspheres A with a particle size of 5-25 μm were used to simulate granulosa cells in the cumulus cell complex of an oocyte;

[0023] Spherical microspheres B with a particle size of 100-150 μm were used to simulate oocytes;

[0024] Spherical microspheres D with a particle size of 120-300 μm were used to simulate fertilized eggs, cleavage embryos, or blastocyst cells;

[0025] Microspheres A and B were made into composite microspheres to simulate the cumulus-oocyte complex.

[0026] In this invention, the method for preparing the composite microspheres includes:

[0027] Microsphere A was immersed in a hyaluronic acid aqueous solution. Microsphere A was then removed and brought into contact with microsphere B. After standing, the microspheres were passed through a 100-mesh filter and the retained microspheres were collected to obtain composite microspheres that simulate the cumulus cell complex of an oocyte.

[0028] In this invention, the mass-volume concentration of the hyaluronic acid aqueous solution is 1.5-2% w / v, calculated in g / ml; specifically, the mass-volume concentration can be 1.5% w / v, 1.8% w / v, or 2% w / v.

[0029] The composite microspheres prepared in this invention are used to simulate the cumulus-oocyte complex, specifically for simulating oocyte dissection and removal (removing granulosa cells under high magnification to fully expose the oocyte, facilitating observation of oocyte maturity, ICSI procedures, and monitoring fertilization and embryonic development—essential procedures for in vitro fertilization, including enzymatic digestion and mechanical methods, etc., detailed in [link to documentation]). Figure 2 (and other processing tasks.)

[0030] In the processes of in vitro fertilization (IVF), gamete transfer, and intracytoplasmic sperm injection (ICSI), microspheres with diameters of 2-4 μm (sperm cells) and 100-150 μm (oocytes) are required. These microspheres are used to simulate sperm cell treatment (density gradient centrifugation reduces or removes prostaglandins, immune-active cells, antisperm antibodies, bacteria, and debris from the seminal plasma, lowers semen viscosity, and promotes sperm capacitation to improve fertilization capacity) and artificial insemination, in vitro mixed fertilization of sperm cells and oocytes, and ICSI. Figure 3 Procedures such as directly injecting a single active sperm into the egg under high magnification.

[0031] In the processes of embryo biopsy, embryo freezing and thawing, it is necessary to prepare microspheres with a diameter of 120-300 μm to simulate fertilized eggs, cleavage embryos or blastocyst cells, and to simulate operations such as cell transfer between different culture media.

[0032] In this invention, during the preparation of polystyrene microspheres, components including but not limited to polyvinyl alcohol and poly(N-isopropylacrylamide) copolymers are added to give the microspheres an anti-protein adsorption effect, which can prevent the microspheres from adsorbing protein components in the culture medium and affecting the operation.

[0033] This invention primarily develops a simulated microsphere capable of mimicking cells including, but not limited to, human sperm cells, oocyte-cumulus cell complexes, oocytes, fertilized eggs, cleavage embryos, and blastocysts. This microsphere allows for readily available practice. Furthermore, in assisted reproductive technology training centers, discarded eggs and embryos cannot meet the practice needs of trainees. This simulated microsphere enables on-the-spot practice, improving skills during training. Attached Figure Description

[0034] Figure 1 A diagram of a styrene polymerization apparatus, where 1 is a stirrer; 2 is a sealing sleeve; 3 is a thermometer; 4 is a thermometer sleeve; 5 is a condenser; and 6 is a three-necked flask.

[0035] Figure 2 A comparison image of the eggs before and after egg removal;

[0036] Figure 3 This is a diagram of intracytoplasmic sperm injection (ICSI). Detailed Implementation

[0037] This invention provides the application of polystyrene microspheres in the preparation of reproductive material mimics. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0038] Terminology Explanation

[0039] Assisted reproductive technology (ART) refers to techniques that use medical assistance to help infertile couples conceive, including artificial insemination, in-vitro fertilization (IVF), embryo transfer, and related technologies. Specifically, it includes in-vitro fertilization of oocytes, gamete transfer, embryo transfer, intracytoplasmic sperm injection (ICSI), embryo biopsy, and embryo freezing and thawing.

[0040] The assisted reproductive embryo laboratory is the core component determining the success of assisted reproductive technology. Staff there are responsible for sperm and egg processing, fertilization, embryo culture, embryo transfer, vitrification, and thawing. The laboratory environment is designed to closely resemble the human body's internal environment through air purification and control of external interferences such as light and noise. This includes precise control of pH, osmotic pressure, temperature, and humidity to provide the optimal environment for healthy embryo development.

[0041] The cumulus-oocyte complex, also known as the coronal-cumulus complex, is a group of granulosa cells that gather around the oocyte during follicular development and are closely connected to the oocyte through intercellular junctions, forming a complex together.

[0042] Cleavage-stage embryo: also known as a cleavage embryo, refers to an embryo that has developed from a fertilized egg to the third day, containing 6-8 cells.

[0043] Blastocyst stage embryo: also known as blastocyst, is the final stage of early embryo culture in vitro, usually forming on the 5th to 6th day after fertilization. It contains an increased number of embryonic cells, forming a fused blastocoel.

[0044] Vitrification: This involves solidifying germ cells or embryos in a high-concentration cryoprotectant environment at extremely low temperatures, forming an irregular vitrified solid. This preserves the normal molecular and ion distribution found in the liquid state, thus providing protection during vitrification within the cell. When the preserved cells are dehydrated to a certain extent in the cryoprotectant solution, endogenous cytoplasmic macromolecules such as proteins and the cryoprotectant that has permeated into the cell concentrate, thereby protecting the cells during the drastic and rapid cooling process.

[0045] In this document, the terms "including", "comprising", and "having" describe both closed-loop technical solutions consisting of the listed features and open-loop technical solutions that include the listed features.

[0046] In this document, the term “and / or” as used includes any and all combinations of one or more of the related listed items.

[0047] In this document, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0048] In this article, when referring to units for data ranges, if the unit is only followed by the right endpoint, it means that the units for the left and right endpoints are the same. For example, 1~300μm means that the units for the left endpoint "1" and the right endpoint "300" are both μm.

[0049] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0050] The present invention will be further illustrated below with reference to the embodiments:

[0051] Example 1: Preparation of 2-4 μm microspheres (sperm cells) (dispersion polymerization method)

[0052] (1) Weigh anhydrous ethanol and deionized water according to the formula and add them to a 250ml four-necked flask equipped with a condenser. Purge with nitrogen, add polyvinylpyrrolidone, stir until completely dissolved, then add styrene monomer dissolved in azobisisobutyronitrile. Maintain a nitrogen atmosphere and perform a constant temperature polymerization reaction at 70℃ in a water bath with electric stirring for 6 hours to obtain a polystyrene emulsion. Centrifuge the polystyrene emulsion, wash with anhydrous ethanol, and wash with water 3 times.

[0053] (2) Fourier transform infrared spectroscopy (FT-IR) determination involves grinding the dried powder sample, pressing it into a KBr pellet, and measuring it using a Tensor27 Fourier transform infrared spectrometer with a scanning range of 400~4000 cm⁻¹. -1 The particle size and morphology of the microspheres were observed by a TECHAI-12 transmission electron microscope (TEM) with an accelerating voltage of 120 kV, with the sample directly dropped onto a copper grid, and by an S4800 field emission scanning electron microscope (FE-SEM) with an accelerating voltage of 15 kV, with the sample surface sputtered with gold. The particle size and particle size distribution of the dispersed microspheres were determined by a Nano-ZS90 laser particle size analyzer.

[0054] (3) Microspheres of corresponding particle size were screened through 5μm and 25μm pore size sieves to simulate cells of corresponding particle size.

[0055] Example 2: Preparation of 100-300 μm microspheres (oocytes, embryos, cleavage embryos, blastocysts, etc.) (suspension polymerization method)

[0056] (1) Addition: Weigh 0.3g of benzoyl peroxide (BPO) using a balance and place it in a 100mL Erlenmeyer flask. Then, add styrene to the Erlenmeyer flask using a pipette. Gently shake until the BPO is completely dissolved, then add it to a three-necked flask. Next, add 20mL of 1.5% polyvinyl alcohol solution to the three-necked flask (e.g., ...). Figure 1 (As shown in the image), finally rinse the conical flask and graduated cylinder with 130 mL of water and then add the water to the three-necked flask.

[0057] (2) During polymerization, condensate is introduced, stirring is started, and the temperature is raised to 90°C within 25 minutes at a constant speed to begin the polymerization reaction. After one hour of reaction, the dispersed particles in the system become sticky, at which point it is crucial to carefully control the stirring speed. In the later stages of the reaction, the temperature can be raised to the upper limit of the reaction temperature to accelerate the reaction and improve the conversion rate. After 2 hours of reaction, a small amount of particles can be taken with a pipette and observed in a petri dish. If the particles become hard and brittle, the reaction can be terminated.

[0058] (3) After discharging, stop heating and cool the three-necked flask to room temperature with cold water while stirring. Then stop stirring and remove the three-necked flask. Filter with a Buchner funnel and wash several times with hot water. Finally, dry the product, weigh it, and calculate the yield.

[0059] (4) Microspheres of corresponding particle size were screened through sieves with pore sizes of 100μm, 200μm and 300μm to simulate cells of corresponding particle size.

[0060] Example 3

[0061] By comparing the performance differences between polystyrene (PS) microspheres and traditional light agarose microspheres in simulating reproductive structures such as sperm, oocytes, cumulus-oocyte complex (COC), cleavage embryos, and blastocysts, the significant advantages of PS microspheres as germ cell / embryo mimics are verified.

[0062] Seven embryology researchers were randomly selected to conduct preference experiments using the polystyrene microspheres screened in this application and existing waste eggs / embryos and other cells.

[0063] Scoring criteria (1–5 points): 5 points: very close to the handling feel, morphological stability and dispersion of real cells; 4 points: relatively close, basically meeting the needs of operation training; 3 points: fair, with slight aggregation or deformation; 2 points: poor, significantly affecting operation judgment; 1 point: extremely poor, unusable for simulation training.

[0064] Table 1. Scoring Results (Mean Score ± SD)

[0065]

[0066] Note: * indicates p < 0.01.

[0067] Experimental results show that the overall score of the polystyrene microspheres of this invention is significantly higher than that of traditional light agarose microspheres and waste biological samples, and the handling feel is highly similar to that of real cells.

[0068] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of modified or unmodified polystyrene microspheres in the preparation of reproductive material mimics; wherein the reproductive material includes at least one of sperm, oocyte, cumulus-oocyte complex, embryo, cleavage embryo, and blastocyst.

2. The application according to claim 1, characterized in that, The modifiers used in the modification include at least one of ethylene glycol-polypropylene glycol-polyethylene glycol copolymer and poly(N-isopropylacrylamide) copolymer.

3. The application according to claim 1 or 2, characterized in that, The polystyrene microspheres have a diameter of 1~300μm.

4. The application according to any one of claims 1 to 3, characterized in that, The method for preparing the polystyrene microspheres includes the steps shown in (1) or (2): (1) In an inert atmosphere, polyvinylpyrrolidone, styrene monomer and initiator were mixed in a solvent and reacted at 70°C for 6 hours. After centrifugation and washing, unmodified polystyrene microspheres were obtained. (2) Mix and stir polyvinyl alcohol, styrene monomer and initiator in water to obtain monomer droplets; stir the monomer droplets at 85~90℃ for 1.5~2 hours to obtain unmodified polystyrene microspheres.

5. The application according to claim 4, characterized in that, The inert atmosphere includes nitrogen; And / or, the initiator includes at least one of azobisisobutyronitrile, potassium persulfate, or benzoyl peroxide; The solvent is water or an alcohol-water mixture.

6. The application according to claim 4 or 5, characterized in that, Also includes: The steps involve plasma treating the surface of the unmodified polystyrene microspheres, followed by adding a modifier to obtain modified polystyrene microspheres.

7. The application according to claim 6, characterized in that, The plasma treatment includes: using air as the working gas to perform plasma treatment on the surface of the polystyrene microspheres for 30 minutes; The modification includes adding an aqueous solution containing the modifier and mixing at room temperature for 2 hours.

8. The application according to any one of claims 4 to 7, characterized in that, The method for preparing polystyrene microspheres further includes screening the obtained polystyrene microspheres using a sieve. Microspheres C, with a particle size of 2-4 μm, are used to simulate the head of sperm cells; Ellipsoidal microspheres A with a particle size of 5-25 μm were used to simulate granulosa cells in the cumulus cell complex of an oocyte; Spherical microspheres B with a particle size of 100-150 μm were used to simulate oocytes; Spherical microspheres D with a particle size of 120-300 μm were used to simulate fertilized eggs, cleavage embryos, or blastocyst cells; Microspheres A and B were made into composite microspheres to simulate the cumulus-oocyte complex.

9. The application according to claim 5, characterized in that, The method for preparing the composite microspheres includes: Microsphere A was immersed in a hyaluronic acid aqueous solution. Microsphere A was then removed and brought into contact with microsphere B. After standing, the microspheres were passed through a 100-mesh filter and the retained microspheres were collected to obtain composite microspheres that simulate the cumulus cell complex of an oocyte.

10. The application according to claim 9, characterized in that, The mass-volume concentration of the hyaluronic acid aqueous solution is 1.5-2% w / v, calculated in g / ml.

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