Method for preparing and vitrification of oocyte / embryo hydrogel microspheres

Abstract

The present application relates to the field of oocyte / embryo vitrification, and particularly relates to a method for preparing and vitrification of oocyte / embryo-loaded hydrogel microspheres. The present application first prepares oocyte-loaded cell microspheres encapsulated by sodium alginate hydrogel through a microfluidic chip, then places the collected cell-loaded microspheres in a freezing carrier, then places the freezing carrier loaded with the microspheres in a vitrification solution for CPA loading, and finally puts the freezing carrier loaded with the microspheres into liquid nitrogen for freezing preservation. The microfluidic chip used in the present application can stably generate oocyte-loaded sodium alginate microspheres with high dispersity and uniformity, the microsphere empty package rate and oocyte loss rate are low, and the survival rate and later development of the oocytes are good; the oocyte vitrification preservation technology can effectively reduce the concentration of the protective agent and the loading time, reduce the toxic effect and osmotic damage to the cells, and improve the survival rate and development rate of the oocytes.

Description

Technical Field

[0001] This invention relates to the field of oocyte / embryo vitrification technology, and in particular to the preparation and vitrification preservation method of oocyte / embryo-loaded hydrogel microspheres. Background Technology

[0002] Cryopreservation of oocytes / embryos is not only used for human fertility preservation but also provides technical support for protecting species resources and saving endangered species. It plays an important role in basic research, the application of genetic preservation models, and clinical applications. Cryopreservation techniques for oocytes / embryos are generally divided into slow freezing and vitrification. Slow freezing involves programmed cooling of pre-treated oocytes / embryos. During this cooling process, ice crystal formation can damage the cells, leading to unsatisfactory cryopreservation results. This method has now been superseded by vitrification. Vitrification involves loading cells with a high concentration of cryoprotective agents (CPA) before freezing, followed by a rapid transition from a liquid to a glassy state at an extremely high cooling rate. During this stage, no ice crystals are formed, reducing intracellular ice damage.

[0003] Currently, the most commonly used vitrification technique in clinical practice is the Cryotop method proposed by Kuwayama et al. Although the survival rate of oocytes / embryos after cryopreservation and thawing is as high as 90-97%, the high concentrations of cryoprotectants used can be toxic to cells, causing DNA damage and reducing their developmental capacity. Rapid changes in osmotic pressure inside and outside the cell during the equilibration phase and warming process can easily cause osmotic damage, potentially leading to oocyte atrophy and deformation, thus affecting post-freezing survival and development. Furthermore, contact between the citrate granulation tissue (CPA) and oocytes / embryos increases intracellular calcium ions, inducing premature exocytosis of cortical granules, resulting in hardening of the zona pellucida and affecting sperm penetration and fertilization. Therefore, novel vitrification methods for oocytes / embryos are needed to improve post-freezing cell developmental capacity.

[0004] Hydrogels are three-dimensional network structures composed of hydrophilic polymers interacting through covalent bonds or intermolecular forces. They can absorb large amounts of water or biological fluids and exhibit good biocompatibility. The water in hydrogels can be classified into three types: free water, intermediate water, and bound water, with bound water maintaining its liquid state and fluidity even below -100°C. Due to their unique structure and chemical composition, hydrogels possess a certain anti-icing ability. Based on this special property, the cryopreservation of hydrogels as cell carriers has attracted great interest from researchers in related fields. Sarbani et al. used a mixed hydrogel prepared from silk, carrageenan, and gelatin to encapsulate osteoblasts for cryopreservation; the cells maintained high differentiation and proliferation capabilities after thawing. Paweena et al. studied the effect of encapsulating cat ovarian tissue with a biodegradable fibrinogen hydrogel on its cryopreservation resistance; the morphology and number of follicles in the ovarian cortex were higher in the frozen group than in the unencapsulated group. These studies on cryopreservation of cells or tissues encapsulated with hydrogels are gradually emerging in the field of cryopreservation; however, there are currently no reports of hydrogels being used to encapsulate oocytes.

[0005] Sodium alginate is a collective term for polysaccharides produced by brown algae or bacteria, which can form stable hydrogels under the action of millimolecular concentrations of calcium or other divalent cations. This gelling property can encapsulate cells under physiological conditions and ensure uniform distribution of cells throughout the matrix. The unique three-dimensional network structure of sodium alginate hydrogels restricts ice crystal growth. Using sodium alginate hydrogels to encapsulate cells or tissues for cryopreservation can improve the survival rate and developmental capacity of cells or tissues after rewarming.

[0006] When sodium alginate hydrogels are used to encapsulate cells for cryopreservation, the cell-carrying microspheres must possess good biocompatibility, homogeneity, and freeze resistance. During cryopreservation, poor homogeneity or a fragile hydrogel shell may affect the CPA (cell-peptide permeation) process, leading to reduced CPA loading efficiency. After immersion in liquid nitrogen, an uneven hydrogel shell, under thermal stress, may crack at weak points, damaging the cells and affecting cryopreservation efficacy. Therefore, issues regarding encapsulation materials, encapsulation methods, and shell shape and size remain to be addressed when using sodium alginate hydrogel microspheres to encapsulate cells.

[0007] Currently, methods for preparing sodium alginate hydrogel microspheres include manual preparation and automated preparation assisted by electrical equipment. Manual preparation typically involves placing a droplet of a mixed solution containing sodium alginate and cells onto a mesh placed upside down over a calcium chloride solution, then rapidly shaking or gently tapping to allow the droplet to fall into a crosslinking bath; alternatively, the mixed solution containing sodium alginate and cells can be directly dripped into the crosslinking bath from the end of a pipette to generate microspheres. While this method does not require any instruments, the operation of shaking the droplet through the mesh or drawing the cell-containing sodium alginate solution with a pipette requires a certain liquid volume, typically a minimum of 2.5 μL. Manual operation cannot precisely control the morphology and size of the generated hydrogel microspheres, resulting in microspheres that are too large or too small, or with uneven size distribution. These factors may affect the effectiveness of cell encapsulation and cryopreservation, as well as post-freeze survival and development. Automated preparation of sodium alginate hydrogel microspheres by electrical equipment includes coaxial airflow and electrostatic spraying methods. The coaxial airflow method uses a coaxial airflow to droplets from the tip of a needle into a cross-linking bath, producing microspheres as small as approximately 400 μm, with a typically large particle size distribution. The droplet generation process of the electrostatic spraying method is similar to that of the coaxial airflow method; however, it can produce microspheres smaller than 200 μm with a smaller size distribution. While these automated methods can produce hydrogel microspheres with relatively uniform particle size, the electrical equipment used may cause some damage to cells during operation. Therefore, there is a need to research and develop methods that are both gentle to operate and can stably and uniformly generate cell-loaded hydrogel microspheres. Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to provide a method for preparing and vitrifying hydrogel microspheres loaded with oocytes / embryos. The microfluidic chip used in this invention can stably generate sodium alginate microspheres loaded with oocytes that exhibit high dispersibility and uniformity, with low microsphere empty package rate and oocyte loss rate, and good oocyte survival and subsequent development. The oocyte vitrification cryopreservation technology effectively reduces the concentration of cryoprotectant and the loading time, minimizing toxicity and osmotic damage to cells, and improving oocyte survival and development rates.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] The first objective of this invention is to provide a method for preparing oocyte / embryo-loaded hydrogel microspheres, comprising the following steps:

[0011] (S1) Disperse oocytes in sodium alginate solution to obtain sodium alginate solution containing oocytes;

[0012] (S2) The sodium alginate solution containing oocytes obtained in step (S1), mineral oil, and oil emulsifier are pumped into a microfluidic chip. The sodium alginate solution containing oocytes and mineral oil form a first droplet in the first junction area of ​​the microfluidic chip. The first droplet and oil emulsifier are mixed in the second junction area of ​​the microfluidic chip and further cross-linked in the S-shaped cross-linking channel of the microfluidic chip to form oocyte / embryo hydrogel microspheres.

[0013] In one embodiment of the present invention, in step (S1), the mass concentration of the sodium alginate solution is 0.5% to 1.5%; preferably, the mass concentration of the sodium alginate solution is 1%.

[0014] The ratio of oocytes to sodium alginate solution is 30-35 oocytes: 800 μL.

[0015] In one embodiment of the present invention, in step (S2), the microfluidic chip is composed of a top layer, a distribution layer, a channel layer and a bottom layer, which are connected sequentially from top to bottom and are provided with an outlet and an inlet.

[0016] The channel layer has a three-channel structure, which includes a first channel where sodium alginate solution containing oocytes meets mineral oil, a second channel where the first droplet meets oil emulsifier, and an S-shaped cross-linking channel.

[0017] The first channel is connected to the inlet of the sodium alginate solution containing oocytes, the inlet of mineral oil, and the inlet of the second channel, respectively; a throat is provided on the side near the inlet of the second channel where the sodium alginate solution containing oocytes and mineral oil meet;

[0018] The second channel is connected to the outlet of the first channel, the inlet of the oil emulsifier, and the inlet of the S-type crosslinking channel, respectively;

[0019] The outlet of the S-shaped crosslinking channel is connected to the outlet of the top layer.

[0020] In one embodiment of the present invention, the microfluidic chip is a flow-focusing microfluidic chip made of PMMA material.

[0021] The microfluidic system mainly consists of a liquid pumping zone, a microsphere generation zone, and a collection zone. It controls the generation rate and particle size of microspheres by adjusting the flow rates of each phase solution. The microfluidic system achieves uniform, high-throughput preparation of cell-loaded microspheres by microencapsulating cells using microfluidic chip technology.

[0022] In one embodiment of the present invention, the channel layer is 150 mm long and 60 mm wide, and the total length of the S-shaped cross-linked channel is 10 mm × 40 mm, wherein the throat is 300 μm long and 120 μm wide.

[0023] In one embodiment of the present invention, in step (S2), the oil emulsifier is a mixture of anhydrous calcium chloride, mineral oil and Span 80;

[0024] The pumping rates of sodium alginate solution containing oocytes, mineral oil, and oil emulsifier were 2 μL / min, 20 μL / min to 40 μL / min, and 20 μL / min to 40 μL / min, respectively.

[0025] In one embodiment of the invention, the mineral oil and the oil emulsifier are pumped at the same rate.

[0026] The second objective of this invention is to provide a hydrogel microsphere carrying oocytes / embryos prepared by the above method, wherein the microsphere has a particle size of 100–320 μm;

[0027] Preferably, the microspheres have a particle size of 262 μm.

[0028] A third objective of this invention is to provide a method for vitrification preservation of oocyte / embryo-loaded hydrogel microspheres, comprising the following steps:

[0029] (A1) Place oocyte / embryo hydrogel microspheres in a cryocarrier to obtain a cryocarrier loaded with microspheres;

[0030] (A2) The cryocarrier carrying microspheres prepared in step (A1) is placed in a vitrification solution for CPA loading;

[0031] (A3) After step (A2) is completed, the cryogenic carrier containing the microspheres is placed in liquid nitrogen for cryopreservation;

[0032] Before use, the hydrogel microspheres carrying oocytes / embryos are subjected to rewarming treatment.

[0033] In one embodiment of the present invention, in step (A1), the freezing carrier is an 80-mesh metal screen made of 304 stainless steel.

[0034] In one embodiment of the present invention, in step (A2), the vitrification solution is a mixture of DMSO, EG and trehalose.

[0035] In one embodiment of the present invention, in step (A2), the vitrification solution contains 10% DMSO by mass, 10% EG by mass, and 0.5M trehalose by mass.

[0036] In one embodiment of the present invention, in step (A2), the time for CPA loading is 4 to 12 minutes;

[0037] Preferably, the CPA loading process takes 8 minutes.

[0038] In one embodiment of the present invention, in step (A3), during the rewarming process, the cryogenic carrier carrying the microspheres is placed in a preheated rewarming solution for rewarming, and then transferred to a dilution and release solution for dilution and release;

[0039] The diluted release solution is a mixed solution of trehalose and sodium citrate, and the dilution and release process is carried out simultaneously.

[0040] In one embodiment of the present invention, the rewarming process is specifically as follows:

[0041] Before use (when oocytes need to be rewarmed), use tweezers to remove the metal sieve from the liquid nitrogen and quickly place it into the preheated 37°C rewarming solution. After 1 minute, transfer it to the dilution and release solution for 3 minutes. During this time, shake the sieve to allow the sodium alginate microspheres to fall into the solution. The outer sodium alginate hydrogel is decrosslinked by the sodium citrate in the dilution and release solution, and then the oocytes are released from the inside of the sodium alginate hydrogel into the solution. After 3 minutes, use a mouth aspirator to wash the released oocytes three times in BS solution, and finally blow them into M2 solution for recovery.

[0042] This invention first prepares sodium alginate hydrogel-encapsulated oocyte-loaded microspheres using a microfluidic system. The collected microspheres are then placed in a cryocarrier, followed by CPA loading in a vitrification solution. Finally, the cryocarrier is immersed entirely in liquid nitrogen for cryopreservation. The rewarming and release procedure can be a one-step, two-step, or three-step method, preferably a two-step method involving rewarming followed by simultaneous dilution and release.

[0043] The microfluidic chip used in this invention can stably generate sodium alginate microspheres carrying oocytes with high dispersion and uniformity. The microsphere empty rate and oocyte loss rate are low, and the oocyte survival rate and subsequent development are good.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The present invention provides a method for vitrification preservation of oocytes encapsulated in sodium alginate hydrogel. Compared with traditional oocyte vitrification freezing technology, this method can effectively reduce the concentration of the protective agent and the loading time, thereby reducing the toxicity and osmotic damage to the oocytes. Furthermore, the sodium alginate hydrogel used in this method not only has good biocompatibility but also a unique three-dimensional network structure. When encapsulating oocytes for vitrification, it can limit ice crystal growth, exhibit good freeze resistance, and reduce freezing damage to the oocytes. The encapsulated oocytes can also be easily released during thawing. Using the method provided by this invention can improve the survival rate and development rate of oocytes, representing a novel and relatively mild oocyte vitrification freezing technology.

[0046] (2) The present invention provides a method for preparing cell-carrying microspheres encapsulated with sodium alginate hydrogel. This method utilizes a highly controllable microfluidic chip, and the microfluidic system built upon this chip can generate cell-carrying microspheres of different sizes as needed. This microfluidic system solves the problems of low generation efficiency and uneven size of cell-carrying microspheres in cell encapsulation. By adjusting the solution concentration and flow rate, different size requirements for the microspheres can be met. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the microfluidic chip structure in Example 3. Labels in the diagram: 1. Top layer; 2. Distribution layer; 3. Channel layer; 4. Bottom layer.

[0048] Figure 2 This is a schematic diagram of the throat structure of the chip in Example 4. Wherein: (a) no throat; (b) throat width 150μm; (c) throat length 300μm, width 120μm.

[0049] Figure 3 This shows the formation of droplets in the throat in Example 4.

[0050] Figure 4 The three cross-linking structures of the microfluidic chip in Example 5 are shown. (a) External cross-linking chip; (b) Dual-channel internal cross-linking chip; (c) Three-channel internal cross-linking chip; Labels in the figure: 5. Sodium alginate inlet; 6. Mineral oil inlet; 7. Oil emulsifier inlet; 8. First junction; 9. Second junction; 10. S-shaped cross-linking channel; 11. Outlet; 12. Sodium alginate channel; 13. Mineral oil channel; 14. Oil emulsifier channel; 15. First droplet channel; 16. Mixing channel.

[0051] Figure 5 The diagram shows the microsphere size distribution in Example 5. (a) Microsphere size distribution of the externally cross-linked chip; (b) Microsphere size distribution of the dual-channel internally cross-linked chip; (c) Microsphere size distribution of the three-channel internally cross-linked chip.

[0052] Figure 6 This is a schematic diagram of the preparation of sodium alginate hydrogel microspheres carrying oocytes using the droplet microfluidic system in Example 7.

[0053] Figure 7 This shows the survival and development of oocytes encapsulated and released by sodium alginate hydrogel in Example 7.

[0054] Figure 8 The survival and in vitro development of oocytes under different vitrification protocols in Example 8 are shown. Letters (a-d) indicate significant differences (P<0.05) in the Tukey multiple range test.

[0055] Figure 9 This section describes the oocyte survival and in vitro development under different oocyte-bearing hydrogel microsphere sizes in Example 9. Letters (a-d) indicate significant differences (P<0.05) in the Tukey multiple range test.

[0056] Figure 10 This section describes the oocyte survival and in vitro development capabilities at different concentrations of protective agents in Example 10. Letters (a-d) indicate significant differences (P<0.05) in the Tukey multiple range test.

[0057] Figure 11 This section describes the oocyte survival and in vitro development under different protective agent loading durations in Example 11. Letters (a-d) indicate significant differences (P<0.05) in the Tukey multiple range test.

[0058] Figure 12 The survival and in vitro development of oocytes under different rewarming and release procedures in Example 12 are shown. Letters (a-d) indicate significant differences (P<0.05) in the Tukey new complex range test. Detailed Implementation

[0059] This invention provides a method for preparing oocyte / embryo-loaded hydrogel microspheres, comprising the following steps:

[0060] (S1) Disperse oocytes in sodium alginate solution to obtain sodium alginate solution containing oocytes;

[0061] (S2) The sodium alginate solution containing oocytes obtained in step (S1), mineral oil, and oil emulsifier are pumped into a microfluidic chip. The sodium alginate solution containing oocytes and mineral oil form a first droplet in the first junction area of ​​the microfluidic chip. The first droplet and oil emulsifier are mixed in the second junction area of ​​the microfluidic chip and further cross-linked in the S-shaped cross-linking channel of the microfluidic chip to form oocyte / embryo hydrogel microspheres.

[0062] In one embodiment of the present invention, in step (S1), the mass concentration of the sodium alginate solution is 0.5% to 1.5%; preferably, the mass concentration of the sodium alginate solution is 1%.

[0063] The ratio of oocytes to sodium alginate solution is 30-35 oocytes: 800 μL.

[0064] In one embodiment of the present invention, in step (S2), the microfluidic chip is composed of a top layer, a distribution layer, a channel layer and a bottom layer, which are connected sequentially from top to bottom and are provided with an outlet and an inlet.

[0065] The channel layer has a three-channel structure, which includes a first channel where sodium alginate solution containing oocytes meets mineral oil, a second channel where the first droplet meets oil emulsifier, and an S-shaped cross-linking channel.

[0066] The first channel is connected to the inlet of the sodium alginate solution containing oocytes, the inlet of mineral oil, and the inlet of the second channel, respectively; a throat is provided on the side near the inlet of the second channel where the sodium alginate solution containing oocytes and mineral oil meet;

[0067] The second channel is connected to the outlet of the first channel, the inlet of the oil emulsifier, and the inlet of the S-type crosslinking channel, respectively;

[0068] The outlet of the S-shaped crosslinking channel is connected to the outlet of the top layer.

[0069] In one embodiment of the present invention, the microfluidic chip is a flow-focusing microfluidic chip made of PMMA material.

[0070] The microfluidic system mainly consists of a liquid pumping zone, a microsphere generation zone, and a collection zone. It controls the generation rate and particle size of microspheres by adjusting the flow rates of each phase solution. The microfluidic system achieves uniform, high-throughput preparation of cell-loaded microspheres by microencapsulating cells using microfluidic chip technology.

[0071] In one embodiment of the present invention, the channel layer is 150 mm long and 60 mm wide, and the total length of the S-shaped cross-linked channel is 10 mm × 40 mm, wherein the throat is 300 μm long and 120 μm wide.

[0072] In one embodiment of the present invention, in step (S2), the oil emulsifier is a mixture of anhydrous calcium chloride, mineral oil and Span 80;

[0073] The pumping rates of sodium alginate solution containing oocytes, mineral oil, and oil emulsifier were 2 μL / min, 20 μL / min to 40 μL / min, and 20 μL / min to 40 μL / min, respectively.

[0074] In one embodiment of the invention, the mineral oil and the oil emulsifier are pumped at the same rate.

[0075] This invention provides a hydrogel microsphere carrying oocytes / embryos prepared by the above method, wherein the microsphere has a particle size of 100-320 μm;

[0076] Preferably, the microspheres have a particle size of 262 μm.

[0077] This invention provides a method for vitrification preservation of oocyte / embryo-loaded hydrogel microspheres, comprising the following steps:

[0078] (A1) Place oocyte / embryo hydrogel microspheres in a cryocarrier to obtain a cryocarrier loaded with microspheres;

[0079] (A2) The cryocarrier carrying microspheres prepared in step (A1) is placed in a vitrification solution for CPA loading;

[0080] (A3) After step (A2) is completed, the cryogenic carrier containing the microspheres is placed in liquid nitrogen for cryopreservation;

[0081] Before use, the hydrogel microspheres carrying oocytes / embryos are subjected to rewarming treatment.

[0082] In one embodiment of the present invention, in step (A1), the freezing carrier is an 80-mesh metal screen made of 304 stainless steel.

[0083] In one embodiment of the present invention, in step (A2), the vitrification solution is a mixture of DMSO, EG and trehalose.

[0084] In one embodiment of the present invention, in step (A2), the vitrification solution contains 10% DMSO by mass, 10% EG by mass, and 0.5M trehalose by mass.

[0085] In one embodiment of the present invention, in step (A2), the time for CPA loading is 4 to 12 minutes;

[0086] Preferably, the CPA loading process takes 8 minutes.

[0087] In one embodiment of the present invention, in step (A3), during the rewarming process, the cryogenic carrier carrying the microspheres is placed in a preheated rewarming solution for rewarming, and then transferred to a dilution and release solution for dilution and release;

[0088] The diluted release solution is a mixed solution of trehalose and sodium citrate, and the dilution and release process is carried out simultaneously.

[0089] In one embodiment of the present invention, the rewarming process is specifically as follows:

[0090] Before use (when oocytes need to be rewarmed), use tweezers to remove the metal sieve from the liquid nitrogen and quickly place it into the preheated 37°C rewarming solution. After 1 minute, transfer it to the dilution and release solution for 3 minutes. During this time, shake the sieve to allow the sodium alginate microspheres to fall into the solution. The outer sodium alginate hydrogel is decrosslinked by the sodium citrate in the dilution and release solution, and then the oocytes are released from the inside of the sodium alginate hydrogel into the solution. After 3 minutes, use a mouth aspirator to wash the released oocytes three times in BS solution, and finally blow them into M2 solution for recovery.

[0091] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0092] Unless otherwise specified, all reagents used in the following embodiments are commercially available, and all detection methods and techniques used are conventional detection methods and techniques in the art; unless a special chip is used, the detailed structure of the microfluidic chip is the same as that of the prior art microfluidic chip.

[0093] Example 1

[0094] This embodiment provides the main reagents and their preparation methods.

[0095] (1) Basic solution (BS): Pipette 40mL TCM 199 into a 50mL centrifuge tube, add 10mL fetal bovine serum, mix well to prepare a basic solution containing 20% ​​fetal bovine serum;

[0096] (2) Sodium alginate solution: Weigh 50mg, 100mg, 150mg, and 200mg using a balance and place them into 15mL centrifuge tubes respectively. Add 100mg of D-mannitol to each centrifuge tube, then add BS solution to make up to 10mL. Refrigerate and let stand overnight to allow the sodium alginate to dissolve completely. Finally, prepare sodium alginate solutions of four concentrations: 0.5%, 1%, 1.5%, and 2%.

[0097] (3) Egg collection solution: Take an appropriate amount of M2 culture medium, add 1% penicillin and streptomycin to prepare an egg collection solution containing 100 IU / mL penicillin and streptomycin.

[0098] (4) In vitro culture medium for oocytes: Take an appropriate amount of KSOM culture medium, add 1% penicillin and streptomycin, and prepare an in vitro culture medium for oocytes containing 100 IU / mL of penicillin and streptomycin.

[0099] (5) Calcium chloride solution: Weigh 166.47 mg of anhydrous calcium chloride powder into a centrifuge tube, add 10 mL of BS to dissolve it completely, and the final concentration is 0.15 M; this solution is used for the cross-linking of sodium alginate;

[0100] (6) Sodium citrate solution: Weigh 220.56 mg of trisodium citrate crystals into a centrifuge tube, add 10 mL of BS to prepare a 0.075 M sodium citrate solution. This solution is used for decrosslinking sodium alginate hydrogels;

[0101] (7) Oil emulsifier: Weigh 2.1g of anhydrous calcium chloride into a 15mL centrifuge tube, add 3mL of PBS, shake well, and bring the volume to a final concentration of 0.7g / mL calcium chloride solution. Add 9mL of mineral oil and 0.6mL of Span 80 to the calcium chloride solution, inverting the tube repeatedly until the oil and water are mixed into an emulsion. Then place the centrifuge tube in an ultrasonic cleaner and run it in ultrasonic mode for 5 minutes. The final solution is an oil emulsifier containing 0.175g / mL calcium chloride.

[0102] (8) Oil phase liquid: Measure 10 mL of mineral oil and 0.2 mL of Span 80, and mix them evenly at room temperature;

[0103] (9) Cytochalasin B solution: Take 1 mg of cytochalasin B, add 1 mL of M2 culture medium, dissolve it completely, take 20 μL into a centrifuge tube, add 5 mL of M2 culture medium, mix well to prepare a 5 μg / mL cytochalasin B solution.

[0104] (10) Strontium chloride solution: Weigh 266.6 mg of strontium chloride, add 1 mL of M2, and dissolve it completely to prepare a 1 μmol / μL strontium chloride stock solution;

[0105] (11) Activation solution: Take 10 μL of strontium chloride stock solution, 1 mL of M2 and 5 μL of CB stock solution, mix well and set aside. The final activation solution contains 5 μg / mL of cytochalasin B and 10 μg / mL of strontium chloride.

[0106] (12) Cryoprotectant: Weigh 1.7115g of trehalose and pour it into a centrifuge tube. Add 1.5mL of EG and 1.5mL of DMSO, and then add BS to bring the volume to 10mL. The final cryoprotectant concentration is 15% DMSO + 15% EG + 0.5M trehalose;

[0107] (13) Equilibrium solution (ES): DMSO + EG + BS; In ES, the mass concentration of DMSO is 7.5% and the mass concentration of EG is 7.5%;

[0108] (14) Vitrification solution 1 (VS1): DMSO + EG + trehalose + BS; In VS1, the mass concentration of DMSO is 7.5%, the mass concentration of EG is 7.5%, and the concentration of trehalose is 1M;

[0109] (15) Glass transition solution 2 (VS2): DMSO + EG + trehalose + BS; In VS2, the mass concentration of DMSO is 8.75%, the mass concentration of EG is 8.75%, and the concentration of trehalose is 0.5M;

[0110] (16) Glass transition solution 3 (VS3): DMSO + EG + trehalose + BS; In VS3, the mass concentration of DMSO is 10%, the mass concentration of EG is 10%, and the concentration of trehalose is 0.5M;

[0111] (17) Vitrification solution 4 (VS4): DMSO + EG + trehalose + BS; In VS4, the mass concentration of DMSO is 12.5%, the mass concentration of EG is 12.5%, and the concentration of trehalose is 0.5M;

[0112] (18) Vitrification solution 5 (VS5): DMSO + EG + trehalose + BS; In VS5, the mass concentration of DMSO is 15%, the mass concentration of EG is 15%, and the concentration of trehalose is 0.5M;

[0113] (19) Rewarm solution (TS): Trehalose + BS; The concentration of trehalose in TS is 1M;

[0114] (20) Dilution solution (DS): Trehalose + BS; The concentration of trehalose in DS is 0.5M;

[0115] (21) Dilution release solution: trehalose + sodium citrate + BS; in the dilution release solution, the concentration of trehalose is 0.5M and the concentration of sodium citrate is 0.075M;

[0116] (22) Rewarming release solution: trehalose + sodium citrate + BS; the concentration of trehalose in the rewarming release solution is 0.75M and the concentration of sodium citrate is 0.075M.

[0117] Example 2

[0118] This embodiment provides a method for obtaining and processing oocytes.

[0119] (1) Seven-week-old ICR SPF mice were selected. After they were familiar with and adapted to the environment for one week, 0.2 mL of pregnant mare serum gonadotropin (PMSG) was injected into the peritoneum. 48 hours later, 0.2 mL of human chorionic gonadotropin (HCG) was injected into the peritoneum to stimulate the mouse ovaries and induce a large number of follicles to mature and ovulate.

[0120] (2) Within 15.5 hours after HCG injection, mice were quickly euthanized by cervical dislocation. The abdominal skin was disinfected with 75% ethanol, and the skin was cut open with scissors to expose the peritoneum. The peritoneum was then cut open with another pair of sterile ophthalmic scissors, and the internal organs were turned upwards to expose the uterus, fallopian tubes, and ovaries. The upper part of the uterus was grasped with forceps and lifted, and the excess fat pad was removed. The connection between the fallopian tubes and the uterus was cut, and the fusion of the fallopian tubes and ovaries was removed.

[0121] (3) Rinse the fallopian tubes three times in PBS, then transfer them into preheated M2 oocyte collection solution. Under stereoscopic vision, locate the enlarged area at the junction of the fallopian tube and ovary, and use a 1mL syringe to puncture the ampulla of the fallopian tube. Oocyte-cumulus cell complex (COC) can be seen flowing out.

[0122] (4) Use a pipette to transfer COC to a 50 μL drop of hyaluronidase solution, and repeatedly pipette with a 10 μL pipette for 4 minutes. Once the granulosa cells are loosened, immediately transfer them to M2 culture medium to avoid prolonged exposure to the digestive enzymes, which could damage the oocytes. Then wash the naked oocytes four times in M2 culture medium droplets to thoroughly remove the hyaluronidase.

[0123] (5) Prepare two 50 mL M2 droplets in a 35 mm culture dish. Select oocytes with intact morphology, uniform cytoplasm, and smooth polar body surfaces and place them into the M2 culture medium droplets. The number of oocytes in each droplet should be controlled to be 10-20. Finally, seal the droplets with 2 mL of embryonic mineral oil and place them in an incubator for later use.

[0124] Example 3

[0125] This embodiment provides the design and fabrication of a microfluidic chip.

[0126] The microfluidic chip for preparing sodium alginate hydrogel microspheres consists of four parts: top layer 1, distribution layer 2, channel layer 3, and bottom layer 4. The structure and order of each layer are as follows: Figure 1 As shown. Top layer 1 has inlets and outlets for each phase solution. The solution is pumped in from the inlet, enters the distribution layer 2 for uniform dispersion, then converges to form droplets in the channel layer 3, and finally flows out from the outlet of top layer 1. The thickness of top layer 1 and bottom layer 4 (PMMA board) is 0.5 mm, and the thickness of distribution layer 2 and channel layer 3 is 0.3 mm. The fabrication process of the PMMA microfluidic chip is as follows:

[0127] (1) Drawing of the structure of each layer of the microfluidic chip

[0128] Design the overall structure of the microfluidic chip according to the required functions, and draw the geometry of each layer of the chip in AutoCAD 2018.

[0129] (2) Laser engraving machine processing

[0130] Double-sided tape is applied to the substrate containing channel layer 3 and continuous phase distribution layer 2, and the substrate is placed in the operating chamber of the laser engraving machine. CAD drawings are uploaded to the laser engraving machine's software, printing parameters are set, and the CAD graphics are engraved onto the PMMA substrate. This process is repeated to complete the fabrication of each layer of the chip.

[0131] (3) Cleaning of each layer of chips

[0132] After engraving, place each layer of the chip into an ultrasonic cleaner filled with deionized water and clean it for 20 minutes in ultrasonic mode to remove residual dust and powder from the chip. After cleaning, wipe each layer of the chip dry and allow it to air dry at room temperature.

[0133] (4) Chip bonding and bonding

[0134] After cleaning and drying, each layer of the chip is bonded together, ensuring that all structural positions on the chip are aligned. The bonded chips are then placed in a vacuum bonding machine with a vacuum bonding time of 4 minutes. After bonding, the chips are placed in a degassing chamber with a degassing time of 10 minutes.

[0135] (5) Component connection

[0136] Anti-collision stickers were affixed to the solution inlet and outlet of the microfluidic chip. Steel needles of appropriate size were inserted into each inlet and outlet according to experimental requirements. UV adhesive was then applied, and the chips were cured under light for 2 hours. During the experiment, the chip was connected to other instruments and equipment via silicone tubing to form a complete microfluidic chip system.

[0137] Example 4

[0138] The Influence of Different Throat Structures on Droplet Generation in Microfluidic Chips

[0139] To evaluate the impact of the structure at the two-phase flow intersection, i.e., the chip throat, on droplet formation, this invention designed three different types and sizes of throats, as shown in the schematic diagram below. Figure 2 As shown, some parameters were designed as follows: (a) no throat; (b) a relatively wide throat structure, 150 μm wide; (c) a throat length of 300 μm and a width of 120 μm. The flow rate of the dispersed phase 1% sodium alginate solution was set at 2 μL / min, and the flow rate of the continuous phase mineral oil was set at 20 μL / min. The droplet formation in the throat of the microfluidic chip was observed using a microscope.

[0140] See results. Figure 3 In microfluidic chips without a throat structure, droplets are difficult to form within the channel; in microfluidic chips with a relatively wide throat structure of 150 μm, droplets still cannot be stably generated in the throat, the dispersed phase solution continues to tail, and the formed droplets have different particle sizes and uneven size distribution; in microfluidic chips with a throat structure of 300 μm in length and 120 μm in width, droplets can be generated stably and uniformly in the throat region.

[0141] Example 5

[0142] The Influence of Different Crosslinking Structures on the Formation of Sodium Alginate Hydrogel Microspheres in Microfluidic Chips

[0143] This invention designs such as Figure 4 The three microfluidic chip structures shown are (a) as follows: Figure 4As shown in (a), the externally cross-linked chip is 80 mm long and 40 mm wide. A sodium alginate solution containing oocytes enters the chip through sodium alginate inlet 5 and flows through sodium alginate channel 12 to the junction. Mineral oil enters the chip through mineral oil inlet 6 and flows through mineral oil channel 13 to the first junction 8. The two meet at the throat, where they are sheared to form droplets, which then flow out through outlet 11 and into the calcium chloride solution via the silicone tube to cross-link and form oocyte / embryo-loaded hydrogel microspheres. (b) As shown in (b) Figure 4 As shown in b, this is a dual-channel internal cross-linking chip, 125 mm long and 50 mm wide. The S-shaped channel on the right is the droplet cross-linking region, with a total length of 12 × 30 mm. Sodium alginate solution containing oocytes and oil emulsifier are pumped in from sodium alginate inlet 5 and oil emulsifier inlet 7, respectively, and enter the chip channel through sodium alginate channel 12 and oil emulsifier channel 14, respectively. They converge at the throat of the first confluence 8, shearing to form spherical droplets while simultaneously entering the S-shaped cross-linking channel 10. Sodium alginate directly cross-links with calcium chloride in the oil emulsifier to form hydrogel microspheres. Finally, the cross-linked oocyte / embryo-carrying hydrogel microspheres flow from outlet 11 into a collection dish containing PBS; (c) as Figure 4 As shown in Figure c, this is a three-channel internal cross-linked chip (i.e., the channel layer has a three-channel structure, including a first channel (sodium alginate channel 12, mineral oil channel 13, first junction 8) where the sodium alginate solution containing oocytes meets mineral oil, a second channel (first droplet channel 15, oil emulsifier channel 14, and second junction 9) where the first droplet meets the oil emulsifier, and an S-shaped cross-linked channel 10; the first channel is connected to the sodium alginate solution inlet 5, the mineral oil inlet 6, and the inlet of the second channel respectively; a throat is provided on the side of the outlet of the first channel near the sodium alginate solution inlet 5; the second channel is connected to the outlet of the first channel, the oil emulsifier inlet 7, and the inlet of the S-shaped cross-linked channel 10 respectively; the outlet of the S-shaped cross-linked channel 10 is connected to the bottom layer), the chip is 150mm long and 60mm wide, and the total length of the S-shaped cross-linked channel 10 is 10×40mm. Sodium alginate solution containing oocytes enters through sodium alginate inlet 5, and mineral oil enters through mineral oil inlet 6. The two solutions converge at the first confluence port 8 through sodium alginate channel 12 and mineral oil channel 13, respectively, where they are sheared to form the first droplet. Oil emulsifier enters through oil emulsifier inlet 7, flows along oil emulsifier channel 14, and converges with the first droplet flowing along the first droplet channel 15 at the second confluence port 9. Then, in the right-side S-shaped cross-linking channel 10, the first droplet, after contacting the oil emulsifier, further cross-links into the S-shaped cross-linking channel 10 through mixing channel 16 to form oocyte / embryo-loaded hydrogel microspheres. The microspheres flow out through outlet 11 into a collection dish containing PBS for collection.

[0144] The experimental setup included a 1% sodium alginate solution as the dispersed phase at a flow rate of 2 μL / min. The flow rates for both the mineral oil and oil emulsifier phases were 20 μL / min. Sodium alginate hydrogel microspheres generated by the three microfluidic chips were collected, and their morphology was observed and photographed under an inverted microscope. Finally, the particle size of each group of microspheres was measured using measurement tools in ImageJ.

[0145] See results. Figure 5 Different cross-linking methods and chip structures can significantly affect the particle size and uniformity of microspheres. The average particle size of microspheres generated by externally cross-linked microfluidic chips was 208.29 μm, with a coefficient of variation as high as 29.73%, indicating that the sodium alginate hydrogel microspheres generated by externally cross-linked chips had a wide particle size distribution and poor uniformity. Microspheres generated by dual-channel and three-channel internally cross-linked microfluidic chips had more uniform particle sizes, with average particle sizes of 263.74 μm and 262.76 μm, respectively. This suggests that internally cross-linked chips are more conducive to maintaining the uniformity of microsphere particle size.

[0146] Taking into account the influence of microfluidic chip geometry and channel parameters on droplet and microsphere generation, a three-channel microfluidic chip is preferred in the following embodiments.

[0147] Example 6

[0148] Effects of solution concentration and flow rate on microsphere formation in microfluidic chips

[0149] A three-channel microfluidic chip was used, with sodium alginate solution as the continuous phase at concentrations of 0.5%, 1%, and 1.5%, and a fixed flow rate of 2 μL / min. The oil phase consisted of mineral oil with 0.1% Span 80 added, with flow rates set at 20 μL / min, 30 μL / min, and 40 μL / min. The flow rate of the oil emulsifier phase was the same as that of the oil phase. Nine experiments were conducted at three different flow rate ratios for each of the three groups of sodium alginate concentrations to analyze the microsphere formation effect and particle size, and to determine the optimal formation parameters.

[0150] Table 1 Comparison of average particle size and coefficient of variation of microspheres at different flow rates and concentrations.

[0151]

[0152] As shown in Table 1, at a flow rate ratio of 1:10, the 0.5% sodium alginate solution produced the largest microspheres, while the 1% sodium alginate group had the lowest coefficient of variation. With increasing sodium alginate solution concentration, the average microsphere size gradually decreased. Overall, the 1% group consistently produced highly uniform microspheres at all three flow rate ratios, and the microsphere size could be effectively controlled by adjusting the flow rate of the continuous phase.

[0153] Therefore, the optimal parameters were selected as 1% sodium alginate solution concentration and 2 μL / min dispersed phase flow rate, and these are the preferred parameters for the following examples.

[0154] Example 7

[0155] Evaluation of the effectiveness of microfluidic chip in preparing sodium alginate hydrogel microspheres carrying oocytes

[0156] (1) Construction of microfluidic system

[0157] A schematic diagram of the device for fabricating sodium alginate hydrogel microspheres loaded with oocytes using a microfluidic chip is shown below. Figure 6 As shown, the system mainly consists of a liquid pumping zone, a microsphere generation zone, and a collection zone. The speed and particle size of microsphere generation can be controlled by adjusting the flow rate of each phase solution.

[0158] (2) Fabrication of sodium alginate hydrogel microspheres carrying oocytes using microfluidic chips

[0159] Using a pipette, 30-35 oocytes were blown into a 1% sodium alginate solution. After the oocytes were fully dispersed, a 1 mL syringe was used to draw up the sodium alginate solution containing the oocytes. The syringe was then fixed to the syringe pump and connected to the dispersion phase inlet of the microfluidic chip via a silicone tube, with a flow rate set to 2 μL / min. Mineral oil and oil emulsifier were then drawn up separately using 5 mL syringes, and after being fixed and connected, flow rates were set to 20 μL / min, 30 μL / min, and 40 μL / min, respectively. The syringe pump was turned on, and after the flow rates of each phase solution stabilized, uniformly formed droplets could be observed at the chip throat. The droplets continued to flow through the junction of the mineral oil and oil emulsifier towards the S-shaped cross-linking zone, where they cross-linked into sodium alginate microspheres. After pumping was complete, the sodium alginate microspheres could be collected in a collection dish containing PBS solution connected to the chip outlet.

[0160] (3) Determination of the survival and development of oocytes encapsulated in sodium alginate hydrogel

[0161] Microspheres were collected from the collection dish, and sodium citrate solution was added to decrosslink the sodium alginate hydrogel, releasing oocytes. The oocytes were then washed three times in M2 solution. The survival rate of the encapsulated and released oocytes was observed, and parthenogenetic activation was performed on the surviving oocytes and fresh oocytes. The specific steps are as follows:

[0162] 1) First, prepare two 50 μL KSOM droplets in a culture dish. Add 2 mL of embryos along the edge of the culture dish and seal with mineral oil. Place the dish in an incubator and equilibrate overnight. Then, take a new culture dish, prepare a 50 μL activation solution droplet, cover the embryos with mineral oil, and place the dish in an incubator to equilibrate for 3.5 h.

[0163] 2) After equilibration, transfer the oocytes into an activation solution droplet and place them in an incubator for activation culture for 4 hours. After 4 hours, remove the oocytes and wash them three times in M2 solution. Place the washed oocytes in a KSOM droplet and continue culturing in an incubator.

[0164] 3) Observe the activation status of oocytes after 6 hours, with the expulsion of polar bodies as the marker of activation success. Thereafter, observe the oocyte status every 24 hours; observe the cleavage status of oocytes after 48 hours, select cleaved cells, change the medium, and continue culturing; observe cell development after 96 hours and calculate the blastocyst rate.

[0165] The methods for calculating survival rate, cleavage rate, and blastocyst rate are as follows:

[0166] Survival rate (%) = Number of surviving cells / Total number of oocytes × 100%

[0167] Cleavage rate (%) = Number of cleavages / Number of surviving eggs × 100%

[0168] Blastocyst rate (%) = Number of blastocysts / Number of cleavages × 100%

[0169] Table 2 Comparison of empty cell rate and oocyte loss rate of sodium alginate hydrogel microspheres at different flow rates

[0170]

[0171] As shown in Table 2 and Figure 7 As shown, the results indicate that at high flow rates, the microspheres have a higher empty encapsulation rate and poor encapsulation effect, and a higher oocyte loss rate. As the flow rate decreases, the empty encapsulation rate and oocyte loss rate of the microspheres decrease accordingly. Encapsulating oocytes with microfluidic chips and then releasing the oocytes did not significantly affect the oocyte survival rate and subsequent developmental ability compared with the fresh group.

[0172] Example 8

[0173] Evaluation of the effectiveness of sodium alginate hydrogel encapsulation for oocyte vitrification cryopreservation

[0174] The Cryotop method, the low-concentration CPA-hydrogel encapsulation method, and the low-concentration CPA-Cryotop method were used for comparative verification. The specific steps are as follows:

[0175] (1) Cryotop group: During the freezing process, 10 oocytes were taken from each group. Using a pipette, the oocytes were transferred from the culture medium to a 400 μL droplet of ES solution for equilibration for 15 min, and then transferred to 400 μL of VS5 solution for 1 min. After CPA loading, the oocytes were quickly blown onto Cryotop carriers, with 2-3 oocytes placed on each carrier. The Cryotop carriers carrying the oocytes were then placed directly into liquid nitrogen. After the liquid nitrogen stabilized, a plastic shell was put on top, and the whole unit was immersed in liquid nitrogen for freezing for 1 h. During the thawing process, after freezing for 1 h, the Cryotop carrier was removed from the liquid nitrogen, the plastic shell was removed, and the tip of the carrier was quickly immersed in 800 μL of preheated 37°C TS solution. The oocytes were gently shaken to detach from the carrier into the solution. After 1 min, the oocytes were transferred to 800 μL of DS solution for dilution for 5 min. Finally, the oocytes were washed 3 times in BS solution and then transferred to M2 solution.

[0176] (2) Low-concentration CPA-sodium alginate hydrogel encapsulation group: During the encapsulation process, 30-35 oocytes were mixed with 800 μL of 1% sodium alginate solution and evenly distributed in the solution. A 1 mL syringe was used to draw up the sodium alginate and oocyte mixture. The syringe, a syringe containing oil emulsifier, and a syringe containing mineral oil were respectively fixed to an injection pump, with flow rates set at 2 μL / min, 30 μL / min, and 30 μL / min, respectively. The syringe was connected to the microfluidic chip through a 0.5 mm inner diameter silicone tube, and the pump was turned on. The overall pumping time was approximately 5-8 min. The prepared oocyte-loaded sodium alginate hydrogel microspheres were collected from the recovery dish, transferred to a metal mesh carrier, and washed three times with PBS. During the freezing process, a metal sieve containing microspheres was directly immersed in 1 mL of VS3 for CPA loading for 10 minutes. After CPA loading, excess liquid was absorbed from under the metal sieve using absorbent paper, and then the metal sieve was immersed in liquid nitrogen for freezing. During the thawing process, after 1 hour of freezing, the metal sieve was removed from the liquid nitrogen with tweezers and quickly placed into a preheated 37°C thawing solution. After 1 minute, it was transferred to a dilution and release solution for 3 minutes. During this time, the sieve was shaken to allow the sodium alginate microspheres to fall into the solution. The outer sodium alginate hydrogel was decrosslinked by the sodium citrate in the dilution and release solution, and then the oocytes were released from the inside of the sodium alginate hydrogel into the solution. After 3 minutes, the released oocytes were washed three times in BS solution using a mouthpiece and finally blown into M2 solution for recovery.

[0177] (3) Low concentration CPA-Cryotop group: All experimental procedures were the same as those of the Cyotop group, except that the vitrification solution was replaced with VS3, which was the same as that of the sodium alginate hydrogel encapsulation group.

[0178] All oocytes collected from each experimental group were allowed to recover in an incubator for 1 hour, and the number of surviving cells was counted.

[0179] Survival rates were calculated, followed by in vitro parthenogenetic activation to determine oocyte cleavage and blastocyst rates. All experiments were repeated three times.

[0180] like Figure 8 As shown, the survival and development rates of oocytes encapsulated in sodium alginate hydrogel and vitrified in low-concentration preservative VS3 were not significantly different from those obtained by the Cryotop method in high-concentration preservative VS5, indicating that encapsulation with sodium alginate hydrogel can reduce the concentration of preservative required for oocyte vitrification.

[0181] Example 9

[0182] Evaluation of the vitrification cryopreservation effect of oocyte-loaded hydrogel microspheres of different particle sizes

[0183] The particle size of sodium alginate microspheres carrying oocytes was varied by controlling the flow rates of mineral oil and oil emulsifier in a microfluidic chip, and cryopreservation and thawing experiments were conducted. The flow rate of the 1% sodium alginate solution containing oocytes was fixed at 2 μL / min; the flow rates of mineral oil and oil emulsifier were the same, set at 20 μL / min, 30 μL / min, and 40 μL / min. The average particle sizes of the sodium alginate microspheres generated at flow rate ratios of 10, 15, and 20 were 262 μm, 193 μm, and 156 μm, respectively. The oocyte-carrying microspheres generated at different flow rate ratios were subjected to cryopreservation and thawing, following the same experimental procedure as step (2) in Example 8, and the survival and subsequent development of the oocytes were statistically analyzed.

[0184] like Figure 9 As shown, when the microsphere size was 262 μm, there were no significant differences in oocyte survival rate (93.16%), cleavage rate (71.78%), and blastocyst rate (21.06%) compared to the 193 μm group (92.48%), cleavage rate (70.80%), and blastocyst rate (20.42%). The oocyte survival rate and cleavage rate in the 156 μm group were only 68.26% and 59.44%, respectively, significantly lower than the control group and the other two groups. Therefore, controlling the average particle size of the hydrogel microspheres can regulate the permeation rate of the protective agent, thereby reducing permeation damage to oocytes.

[0185] Example 10

[0186] Effects of different cryoprotectant concentrations on vitrification cryopreservation of oocyte-loaded hydrogel microspheres

[0187] Four different concentrations of cryoprotectants, namely VS1, VS2, VS3, and VS4, were selected to determine the optimal concentration of cryoprotectant after encapsulating oocytes with sodium alginate hydrogel. For each group, oocyte-loaded microspheres with a preferred particle size of approximately 262 μm were prepared and collected using a microfluidic chip. The oocyte-loaded microspheres were placed in a metal sieve and then immersed in 1 mL of each of the four different concentrations of cryoprotectant for 10 min. Subsequent cryopreservation, thawing, and release procedures were the same as step (2) in Example 8.

[0188] like Figure 10 As shown, the survival rate and development rate of oocytes increased with increasing protectant concentration, but the increase was not monotonous. When the protectant concentration was increased to 12.5% ​​EG + 12.5% ​​DMSO + 0.5M trehalose (VS4), the survival rate, cleavage rate, and blastocyst rate of oocytes in the VS4 group were not significantly different from those in the VS3 group, but they decreased slightly, indicating that there is an optimal balance point between sodium alginate hydrogel and protectant concentration.

[0189] Example 11

[0190] Effects of different cryoprotectant loading times on vitrification cryopreservation of oocyte-loaded hydrogel microspheres

[0191] Sodium alginate hydrogel microspheres carrying oocytes, prepared using a microfluidic chip, were loaded into VS3 cryoprotectant for 4 min, 8 min, and 12 min, respectively. After loading, excess cryoprotectant was removed with absorbent paper, and the cryopreservation and thawing process was continued. Other operating steps were the same as step (2) in Example 8.

[0192] like Figure 11 As shown, the oocyte survival rate in the 4-min group was much lower than that in other groups, at only 23.99%. There was no significant difference in cell survival rate between the 8-min group (91.98%) and the 12-min group (92.28%), but the cell cleavage rate (75.84%) and blastocyst rate (23.86%) in the 8-min group were significantly higher than those in the 12-min group (61.88% and 15.70%, respectively).

[0193] Example 12

[0194] Effects of different rewarming and release procedures on the cryopreservation and rewarming of oocyte-loaded hydrogel microspheres

[0195] Three rewarming and oocyte release steps were designed. The cryopreservation and preservation steps for each group involved loading 262 μm 1% sodium alginate microspheres with VS3 cryopreservation agent in a single step for 8 minutes, followed by freezing in liquid nitrogen for 1 hour. The specific steps are as follows:

[0196] (1) One-step method: The rewarming and release processes are carried out simultaneously. After the metal sieve is removed from the liquid nitrogen, it is placed directly into the rewarming and release solution, and the microspheres are gently touched with tweezers to detach from the inner wall into the solution. After 3 minutes of rewarming and release, the oocytes are aspirated into the BS using a mouth pipette.

[0197] (2) Two-step method: dilution and release are carried out simultaneously. First, the microspheres are immersed in 1M trehalose solution for 1 min to warm them. Then, the microspheres are transferred to a mixed solution of 0.5M trehalose and 0.075M sodium citrate for 3 min to dilute and release them. Finally, the oocytes are transferred to BS.

[0198] (3) Three-step method: rewarming, dilution, and release are performed separately. After freezing for 1 hour, the metal sieve containing cell-carrying microspheres is removed from liquid nitrogen and first immersed in a 1M trehalose solution at 37℃ for 1 minute for rewarming. Then, it is transferred to a 0.5M trehalose solution for dilution for 3 minutes. Finally, the rewarmed sodium alginate microspheres are shaken off into a 0.075M sodium citrate solution to decrosslink for 3 minutes, releasing the oocytes. Under stereoscopic visualization, the oocytes are transferred into the BS using a mouth pipette.

[0199] After being transferred to BS, oocytes from each group were washed three times sequentially, then transferred to M2 droplets and incubated for 2 hours. The number of surviving oocytes was then counted, and the survival rate was calculated. The surviving oocytes were then parthenogenetically activated, and the number of cleavage cells and cells developing to the blastocyst stage were counted.

[0200] like Figure 12 As shown, comparing the effects of three different rewarming and release methods on the survival and development rate of oocytes after vitrification and rewarming, the two-step method of rewarming first and then simultaneously performing dilution and release had significantly higher survival rate, cleavage rate and blastocyst rate than the one-step method of rewarming and releasing simultaneously and the three-step method of rewarming, dilution and release separately.

[0201] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A method for the preparation of oocyte / embryo hydrogel microspheres, characterized by, The method comprises the following steps: (S1) dispersing the oocyte in a sodium alginate solution to obtain a sodium alginate solution containing the oocyte; (S2) pumping the sodium alginate solution containing the oocyte obtained in step (S1), mineral oil and oil emulsifier into a microfluidic chip, the sodium alginate solution containing the oocyte first forms a first droplet with the mineral oil, and the first droplet is mixed with the oil emulsifier to further crosslink to form an oocyte / embryo hydrogel microsphere; In step (S2), the microfluidic chip comprises, from top to bottom, a top layer, a distribution layer, a channel layer and a bottom layer which are sequentially connected and are respectively provided with an outlet and an inlet; the channel layer is a three-channel structure, the three-channel structure comprises a first channel for the intersection of the sodium alginate solution containing the oocyte and the mineral oil, a second channel for the intersection of the first droplet and the oil emulsifier, and an S-shaped crosslinking channel for the crosslinking of the first droplet and the oil emulsifier; the first channel is connected with the inlet of the sodium alginate solution containing the oocyte, the inlet of the mineral oil and the inlet of the second channel respectively; the intersection of the sodium alginate solution containing the oocyte and the mineral oil is provided with a throat near one side of the inlet of the second channel; the second channel is connected with the outlet of the first channel, the inlet of the oil emulsifier and the inlet of the S-shaped crosslinking channel respectively; the outlet of the S-shaped crosslinking channel is connected with the outlet of the top layer; the pumping speeds of the sodium alginate solution containing the oocyte, the mineral oil and the oil emulsifier are 2 μL / min, 20 μL / min-40 μL / min and 20 μL / min-40 μL / min respectively.

2. The method for preparing oocyte / embryo-loaded hydrogel microspheres according to claim 1, characterized in that, In step (S1), the mass concentration of the sodium alginate solution is 0.5%-1.5%; the dosage ratio of the oocyte to the sodium alginate solution is 30-35:800 μL.

3. The method of claim 1, wherein the oocyte / embryo hydrogel microspheres are prepared by the steps of: In step (S2), the oil emulsifier is a mixture of anhydrous calcium chloride, mineral oil and Span 80.

4. The oocyte / embryo hydrogel microspheres prepared by the method of any one of claims 1 to 3, characterized in that, The particle size of the microspheres is 100 μm-320 μm.

5. A method of vitrification of oocyte / embryo hydrogel microspheres as claimed in claim 4, wherein, The method comprises the following steps: (A1) placing the oocyte / embryo hydrogel microspheres in a freezing carrier to obtain a freezing carrier loaded with the microspheres; (A2) placing the freezing carrier loaded with the microspheres prepared in step (A1) in a vitrification solution for CPA loading; (A3) after step (A2) is completed, placing the freezing carrier loaded with the microspheres in liquid nitrogen for freezing preservation; Before use, the oocyte / embryo hydrogel microspheres are subjected to a rewarming treatment.

6. The vitrification method of oocyte / embryo hydrogel microspheres according to claim 5, wherein, In step (A1), the freezing carrier is a 80-mesh metal screen made of 304 stainless steel.

7. The vitrification method of oocyte / embryo hydrogel microspheres according to claim 5, wherein, In step (A2), the vitrification solution is a mixture of DMSO, EG and trehalose; In the CPA loading process, the time is 4 min-12 min.

8. The vitrification method of oocyte / embryo hydrogel microspheres according to claim 5, wherein, In step (A3), in the rewarming process, the freezing carrier loaded with the microspheres is placed in a preheated rewarming solution for rewarming, and then transferred to a dilution release solution for dilution release; wherein the dilution release solution is a mixed solution of trehalose and sodium citrate, and the dilution release process is performed synchronously.

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