CN117158408A

CN117158408A - Preparation and vitrification preservation method of oocyte/embryo-loaded hydrogel microsphere

Info

Publication number: CN117158408A
Application number: CN202310985067.4A
Authority: CN
Inventors: 周新丽; 张宇琪; 林春燕
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2023-08-07
Filing date: 2023-08-07
Publication date: 2023-12-05

Abstract

The invention relates to the technical field of oocyte/embryo vitrification freezing, in particular to a preparation method and a vitrification preservation method of an oocyte/embryo hydrogel microsphere. Firstly preparing cell-carrying microspheres of sodium alginate hydrogel encapsulated oocytes through a microfluidic chip, then placing the collected cell-carrying microspheres into a frozen carrier, then placing the frozen carrier loaded with the microspheres into a vitrification solution for CPA loading, and finally, integrally putting the frozen carrier loaded with the microspheres into liquid nitrogen for freezing preservation. The micro-fluidic chip applied in the invention can stably generate the oocyte-loaded sodium alginate microsphere with high dispersibility and uniformity, the empty microsphere inclusion rate and the oocyte loss rate are low, and the survival rate and the later development condition of the oocyte are good; the oocyte vitrification cryopreservation technology can effectively reduce the concentration and loading time of the protective agent, reduce the toxic effect and osmotic damage to cells, and improve the survival rate and the development rate of oocytes.

Description

Preparation and vitrification preservation method of oocyte/embryo-loaded hydrogel microsphere

Technical Field

The invention relates to the technical field of oocyte/embryo vitrification freezing, in particular to a preparation method and a vitrification preservation method of an oocyte/embryo hydrogel microsphere.

Background

The cryopreservation of oocytes/embryos can be used for the fertility preservation of human beings, can provide technical guarantees for protecting species resources and saving endangered species, and has important roles in basic research, application of genetic preservation models and clinical application. Oocyte/embryo cryopreservation techniques are generally classified into slow freezing and vitrification freezing. The slow freezing is to perform program cooling on the pretreated oocyte/embryo, and the formation of ice crystals in the cooling process can damage cells, so that the freezing effect is not ideal, and the method is replaced by a vitrification freezing technology at present. Vitrification refers to the loading of cells with a high concentration of cryoprotectant (Cryoprotective agents, CPA) prior to freezing, followed by a rapid transition of the liquid state to the glassy phase at an extremely high cooling rate, which is free of ice crystal formation and reduces intracellular ice damage.

The vitrification freezing technique commonly used in clinical practice is still the Cryotop method proposed by Kuwayama et al. Although the survival rate of oocytes/embryos after freezing and re-warming is as high as 90-97%, the high concentration of protective agent used can have toxic effect on cells, and cause DNA damage of the oocytes/embryos to reduce the development capacity of the oocytes/embryos. The too fast osmotic pressure change inside and outside the cells in the balancing stage and the heating process is extremely easy to cause the osmotic damage of the cells, and the oocytes can be possibly atrophic and deformed, thereby influencing the survival rate and the development rate of the cells after freezing. In addition, contact of CPA with the oocyte/embryo causes an increase in intracellular calcium ions, which induces premature exocytosis of cortical particles, resulting in stiffening of the zona pellucida of the oocyte, affecting sperm penetration and fertilization. Thus, a novel method of preserving oocytes/embryos by vitrification is needed to improve the post-freezing development of cells.

The hydrogel is a three-dimensional network structure substance formed by interaction of hydrophilic polymers through covalent bonds or intermolecular forces, can absorb a large amount of water or biological liquid, and has good biocompatibility. The water in hydrogels can be divided into three categories: free water, intermediate water and bound water, wherein the bound water remains liquid and fluid at temperatures below-100 ℃. Because of the special structure and chemical composition of the hydrogel, the hydrogel has certain ice inhibition capability. Based on this particular property, cryopreservation studies of hydrogels as cell carriers have attracted considerable interest to researchers in the relevant field. The mixed hydrogel prepared by using silk, carrageenan and gelatin and Sarbani and the like encapsulates osteoblasts for freezing preservation, and the cells after rewarming still keep high differentiation and proliferation capability. Paweena et al studied the effect of biodegradable fibrinogen gel coating cat ovarian tissue on its resistance to freezing, and the morphology and number of follicles in the frozen ovarian cortex were higher than those in the unencapsulated group. The cryopreservation studies of these hydrogel-encapsulated cells or tissues have been gradually pursued in the field of cryopreservation, however, no hydrogel has been reported to be applied to the encapsulation of oocytes.

Sodium alginate is a generic term for a polysaccharide produced by brown algae or bacteria that forms stable hydrogels under the action of millimoles concentrations of calcium or other divalent cations. This gelling property encapsulates the cells under physiological conditions and allows for a uniform distribution of cells throughout the matrix. The unique three-dimensional network structure of the sodium alginate hydrogel has the effect of limiting the growth of ice crystals, and the sodium alginate hydrogel is used for encapsulating cells or tissues for low-temperature preservation, so that the survival rate and the development capability of the cells or tissues after rewarming can be improved.

When the sodium alginate hydrogel encapsulated cells are used for low-temperature preservation, the cell-carrying microspheres are required to have good biocompatibility, uniformity and freezing resistance. During the low temperature preservation process, the poor uniformity or the fragile hydrogel shell can influence the process of the CPA to permeate the cell-loaded hydrogel microspheres, so that the CPA loading efficiency is reduced; after liquid nitrogen is added, the uneven hydrogel shell may form cracks at weak parts under the action of heat stress, so that damage is caused to cells, and the freezing effect of the cells is affected. Therefore, when the sodium alginate hydrogel microsphere is used for encapsulating cells, the problems of an encapsulating material, an encapsulating method, the shape and the size of a shell and the like are still to be solved.

At present, the preparation method of the sodium alginate hydrogel microsphere comprises manual preparation and automatic preparation under the assistance of electrical equipment. Wherein, the manual preparation is generally to place the mixed solution drop containing sodium alginate and cells on a net and invert the mixed solution drop above the calcium chloride solution, and then shake or tap the mixed solution drop rapidly to make the mixed solution drop fall into a crosslinking bath; or a mixed solution containing sodium alginate and cells is directly dropped into a crosslinking bath from the pipette tip to thereby produce microspheres. Although the method does not need any instrument, the liquid drops are shaken off through a grid, or the operation of sucking the sodium alginate solution containing cells by a liquid-transfering gun has certain requirements on the liquid quantity of the liquid drops, the minimum liquid quantity is usually 2.5 mu L, the shape and the size of the generated hydrogel microspheres cannot be precisely controlled by manual operation, the sizes of the microspheres are too large or too small, the size distribution is uneven, and the factors may influence the effects of cell encapsulation and freezing and the survival and development conditions after freezing. The electric automatization preparation of the sodium alginate hydrogel microsphere comprises a coaxial air flow method, an electrostatic spraying method and the like. The coaxial gas flow method is to drop the liquid drop on the needle point at the outlet into the crosslinking bath by using coaxial gas flow, and can produce microspheres with a minimum size of about 400 μm, and the particle size distribution is usually larger. The droplet generation process of the electrostatic spraying method is similar to that of the coaxial air flow method, and the electrostatic spraying method can produce microspheres with smaller size distribution and smaller size distribution, wherein the size of the microspheres is smaller than 200 mu m. Although the automatic methods can generate hydrogel microspheres with uniform particle size, the electrical equipment may cause certain damage to cells in the use process, so that research and development of a method which is mild in operation and can stably and uniformly generate the cell-loaded hydrogel microspheres are required.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a preparation method and a vitrification preservation method of oocyte/embryo-loaded hydrogel microspheres. The micro-fluidic chip applied in the invention can stably generate the oocyte-loaded sodium alginate microsphere with high dispersibility and uniformity, the empty microsphere inclusion rate and the oocyte loss rate are low, and the survival rate and the later development condition of the oocyte are good; the oocyte vitrification cryopreservation technology can effectively reduce the concentration and loading time of the protective agent, reduce the toxic effect and osmotic damage to cells, and improve the survival rate and the development rate of oocytes.

The aim of the invention can be achieved by the following technical scheme:

the first object of the present invention is to provide a method for preparing oocyte/embryo-loaded hydrogel microspheres, which comprises the following steps:

(S1) dispersing oocytes in a sodium alginate solution to obtain a sodium alginate solution containing oocytes;

and (S2) pumping the sodium alginate solution containing the oocyte, the mineral oil and the oil emulsifier obtained in the step (S1) into a microfluidic chip, wherein the sodium alginate solution containing the oocyte and the mineral oil form a first liquid drop in a first intersection region of the microfluidic chip, the first liquid drop and the oil emulsifier are mixed in a second intersection region of the microfluidic chip, and further cross-linking is performed in an S-shaped cross-linking channel of the microfluidic chip to form the oocyte/embryo-carrying hydrogel microsphere.

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

The dosage ratio of the oocyte to the sodium alginate solution is 30-35: 800 mul.

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

the channel layer is of a three-channel structure, and the three-channel structure comprises a first channel in which sodium alginate solution containing oocytes is intersected with mineral oil, a second channel in which first liquid drops are intersected with an oil emulsifier, and an S-shaped crosslinking channel;

the first channel is respectively connected with an inlet of sodium alginate solution containing oocytes, an inlet of mineral oil and an inlet of the second channel; a throat part is arranged at one side of the junction of sodium alginate solution containing oocytes and mineral oil, which is close to the inlet of the second channel;

the second channel is respectively connected with the outlet of the first channel, the inlet of the oil emulsifier and the inlet of the S-shaped crosslinking channel;

the outlet of the S-shaped cross-linking channel is connected with the outlet of the top layer.

In one embodiment of the present invention, the microfluidic chip is a flow focusing type microfluidic chip made of PMMA material.

The microfluidic system mainly comprises a liquid pumping area, a microsphere generation area and a collection area, and the generation speed and the particle size of the microspheres can be controlled by adjusting the flow rate of each phase of solution. The micro-fluidic system realizes uniform and high-flux preparation of the cell-carrying microspheres by using micro-fluidic chip technology to encapsulate cells.

In one embodiment of the invention, the channel layer is 150mm long by 60mm wide and the S-shaped cross-linked channels have a total length of 10mm by 40mm, wherein the throat is 300 μm long by 120 μm wide.

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;

the pumping speed of sodium alginate solution, mineral oil and oil emulsifier containing oocyte is 2 mu L/min, 20 mu L/min-40 mu L/min and 20 mu L/min-40 mu L/min in sequence.

In one embodiment of the invention, the pumping rates of the mineral oil and oil emulsifier are the same.

A second object of the present invention is to provide an oocyte/embryo-carrying hydrogel microsphere prepared by the above method, the microsphere having a particle size of 100 to 320 μm;

Preferably, the particle size of the microspheres is 262 μm.

The third object of the present invention is to provide a vitrification preservation method of oocyte/embryo-loaded hydrogel microspheres, comprising the following steps:

(A1) Placing the oocyte/embryo-carrying hydrogel microspheres in a frozen carrier to obtain a frozen carrier carrying the microspheres;

(A2) The frozen carrier carrying the microspheres prepared in the step (A1) is placed in vitrification solution for CPA loading;

(A3) After the step (A2) is finished, placing the frozen carrier loaded with the microspheres into liquid nitrogen for freezing preservation;

before use, the oocyte/embryo-carrying hydrogel microsphere is subjected to rewarming treatment.

In one embodiment of the present invention, in the step (A1), the freezing support is a 304 stainless steel 80 mesh metal screen.

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

In one embodiment of the present invention, in step (A2), the glass transition solution has a DMSO mass fraction of 10%, an EG mass fraction of 10%, and a trehalose concentration of 0.5M.

In one embodiment of the present invention, in step (A2), the duration of CPA loading is 4-12 min;

Preferably, the time during CPA loading is 8 minutes.

In one embodiment of the present invention, in the step (A3), during the re-heating process, the frozen carrier loaded with the microspheres is placed in a preheated re-heating solution for re-heating, and then transferred to a diluted release solution for dilution release;

wherein the dilution releasing solution is a mixed solution of trehalose and sodium citrate, and the dilution releasing process is synchronously carried out.

In one embodiment of the present invention, the re-warming process is specifically as follows:

before use (when the oocyte needs to be rewarmed), taking out the metal screen from the liquid nitrogen by using tweezers, rapidly putting the metal screen into a preheated rewarming solution at 37 ℃, transferring the preheated solution into a dilution release solution for dilution and release for 3min after 1min, shaking the screen during the period to enable sodium alginate microspheres to fall into the solution, enabling external sodium alginate hydrogel to be decrosslinked by sodium citrate in the dilution release solution, and then releasing the oocyte into the solution from the inside of the sodium alginate hydrogel; after 3min, the released oocytes were washed sequentially 3 times in BS solution using an oral aspirator, and finally blown into M2 solution for recovery.

Firstly preparing cell-carrying microspheres of sodium alginate hydrogel encapsulated oocytes through a microfluidic system, then placing the collected cell-carrying microspheres in a frozen carrier, then placing the frozen carrier carrying the microspheres in a vitrification solution for CPA loading, and finally, integrally putting the frozen carrier carrying the microspheres in liquid nitrogen for freezing preservation. The rewarming release procedure can be a one-step, two-step or three-step procedure, preferably a two-step procedure in which the rewarming is followed by simultaneous dilution and release procedures.

The micro-fluidic chip applied in the invention can stably generate the oocyte-loaded sodium alginate microsphere with high dispersibility and uniformity, the microsphere empty package rate and the oocyte loss rate are low, and the survival rate and the later development condition of the oocyte are good.

Compared with the prior art, the invention has the following beneficial effects:

(1) Compared with the traditional oocyte vitrification freezing technology, the vitrification preservation method for the sodium alginate hydrogel encapsulated oocyte can effectively reduce the concentration and loading time of the protective agent and reduce the toxic effect and osmotic damage to the oocyte; secondly, the sodium alginate hydrogel used in the method has good biocompatibility and a unique three-dimensional network structure, and when the encapsulated oocyte is used for vitrification cryopreservation, the growth of ice crystals can be limited, the freezing resistance is good, and the freezing damage of the oocyte is reduced. The encapsulated oocyte can also be easily released during rewarming. The method provided by the invention can improve the survival rate and the development rate of the oocyte, and is a novel milder oocyte vitrification freezing technology.

(2) According to the preparation method of the cell-loaded microsphere of the sodium alginate hydrogel encapsulated oocyte, provided by the invention, the microfluidic chip with high controllability is adopted, and the microfluidic system built based on the chip can generate cell-loaded microspheres with different sizes according to requirements. The microfluidic system solves the problems of low generation efficiency, uneven size and the like of cell-loaded microspheres in cell encapsulation, and can meet the requirements of different sizes of microspheres by adjusting the concentration and the flow rate of a solution.

Drawings

Fig. 1 is a schematic view of the structure of each layer of the microfluidic chip in example 3. Reference numerals in the drawings: 1. a top layer; 2. a distribution layer; 3. a channel layer; 4. a bottom layer.

FIG. 2 is a schematic diagram of the throat structure of the chip in example 4. Wherein: (a) no throat; (b) throat width 150 μm; (c) throat length 300 μm and width 120. Mu.m.

FIG. 3 shows the formation of droplets in the throat of example 4.

Fig. 4 shows three cross-linked structures of the microfluidic chip in example 5. Wherein: (a) an externally crosslinked chip; (b) a dual channel internal cross-linked chip; (c) three-way internal crosslinking chips; reference numerals in the drawings: 5. a sodium alginate inlet; 6. a mineral oil inlet; 7. an oil emulsifier inlet; 8. a first junction; 9. a second junction; s-type cross-linking channel; 11. an outlet; 12. sodium alginate flow channels; 13. a mineral oil flow passage; 14. an oil emulsifier flow channel; 15. a first droplet flow path; 16. and a mixing runner.

FIG. 5 is a graph showing the particle size distribution of microspheres in example 5. Wherein: (a) an external cross-linked chip microsphere particle size distribution; (b) dual channel internal cross-linked chip microsphere particle size distribution; (c) three-channel internal cross-linked chip microsphere particle size distribution.

Fig. 6 is a schematic diagram of the preparation of oocyte-loaded sodium alginate hydrogel microspheres by the droplet microfluidic system in example 7.

FIG. 7 shows the survival and development of oocytes released by encapsulation with sodium alginate hydrogel in example 7.

FIG. 8 shows the survival and in vitro development of oocytes in different vitrification protocols in example 8. Wherein: letters (a-d) represent significant differences in Tukey new complex polar differences (P < 0.05).

FIG. 9 shows the survival and in vitro development of oocytes under different particle sizes of oocyte-loaded hydrogel microspheres in example 9. Wherein: letters (a-d) represent significant differences in Tukey new complex polar differences (P < 0.05).

FIG. 10 shows the viability and in vitro viability of oocytes at different concentrations of protective agent in example 10. Wherein: letters (a-d) represent significant differences in Tukey new complex polar differences (P < 0.05).

FIG. 11 shows the survival and in vitro development of oocytes under different loading periods of protective agents in example 11. Wherein: letters (a-d) represent significant differences in Tukey new complex polar differences (P < 0.05).

FIG. 12 shows the survival and in vitro development of oocytes in the various rewarming and release procedures of example 12. Wherein: letters (a-d) represent significant differences in Tukey new complex polar differences (P < 0.05).

Detailed Description

The invention provides a preparation method of oocyte/embryo-loaded hydrogel microspheres, which comprises the following steps:

The invention provides an oocyte-carrying/embryo hydrogel microsphere prepared by the method, wherein the particle size of the microsphere is 100-320 mu m;

preferably, the particle size of the microspheres is 262 μm.

The invention provides a vitrification preservation method of oocyte/embryo hydrogel microspheres, which comprises the following steps:

preferably, the time during CPA loading is 8 minutes.

The invention will now be described in detail with reference to the drawings and specific examples.

In the following examples, unless specified otherwise, all reagents used were commercial reagents and all detection means and methods used were conventional in the art; if no special chip exists, the detail structure of the microfluidic chip is the same as that of the microfluidic chip in the prior art.

Example 1

The present examples provide major reagents and methods of formulating the same.

(1) Base Solution (BS): sucking 40mL of TCM 199 in a 50mL centrifuge tube, adding 10mL of fetal bovine serum, and uniformly mixing to prepare a base solution containing 20% of fetal bovine serum;

(2) Sodium alginate solution: weighing 50mg, 100mg, 150mg and 200mg by a balance, respectively placing into a 15mL centrifuge tube, adding 100mg of D-mannitol into each centrifuge tube, adding a BS solution to a constant volume of 10mL, refrigerating and standing overnight to fully dissolve sodium alginate. Finally preparing sodium alginate solution with four concentrations of 0.5%, 1%, 1.5% and 2%;

(3) Egg picking operation liquid: taking a proper amount of M2 culture solution, adding 1% of streptomycin, and preparing an egg picking operation solution containing 100IU/mL of double antibody;

(4) Oocyte in vitro culture solution: taking a proper amount of KSOM culture solution, adding 1% of streptomycin, and preparing an oocyte in-vitro culture solution containing 100IU/mL of double antibody;

(5) Calcium chloride solution: 166.47mg of anhydrous calcium chloride powder is weighed into a centrifuge tube, 10mL of BS is added to be fully dissolved, and the final concentration is 0.15M; the solution is used for crosslinking sodium alginate;

(6) Sodium citrate solution: 220.56mg of trisodium citrate crystals are weighed into a centrifuge tube and 10mL of BS is added to prepare a 0.075M sodium citrate solution. The solution is used for the decrosslinking of sodium alginate hydrogel;

(7) Oil emulsifier: 2.1g of anhydrous calcium chloride is weighed into a 15mL centrifuge tube, 3mL of PBS is added, shaking is carried out, and the volume of the calcium chloride solution is fixed to be 0.7 g/mL. 9mL of mineral oil and 0.6mL of span 80 were aspirated and placed in a calcium chloride solution, turned upside down until the oil and water mixed to an emulsion. And then the centrifuge tube is put into an ultrasonic cleaner, and the centrifuge tube is placed in an ultrasonic mode for 5min. An oil emulsifier eventually formulated to contain 0.175g/mL calcium chloride;

(8) Oil phase liquid: weighing 10mL of mineral oil and 0.2mL of span 80, and uniformly mixing at normal temperature;

(9) Cytochalasin B solution: 1mg of cytochalasin B is taken, 1mL of M2 culture solution is added, 20 mu L of the culture solution is taken to be placed in a centrifuge tube after the culture solution is fully dissolved, then 5mL of M2 culture solution is added, and the mixture is uniformly mixed to prepare 5 mu g/mL of cytochalasin B solution;

(10) Strontium chloride solution: 266.6mg of strontium chloride is weighed, 1mL of M2 is added, and 1 mu mol/mu L of strontium chloride stock solution is prepared after full dissolution;

(11) Activating solution: 10. Mu.L of strontium chloride stock solution, 1mL of M2 and 5. Mu.L of CB stock solution were taken and mixed well for later use. The concentration of cytochalasin B in the final activation solution is 5 mug/mL, and the concentration of strontium chloride is 10 mug/mL;

(12) Low temperature protective agent: 1.7115g of trehalose was weighed and poured into a centrifuge tube, EG 1.5mL and DMSO1.5 mL were added, and BS was added to a constant volume of 10mL. The final protectant concentration was 15% dmso+15% eg+0.5m trehalose;

(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%;

(14) Vitrification solution 1 (VS 1): dmso+eg+trehalose+bs; in VS1, the DMSO mass concentration is 7.5%, the EG mass concentration is 7.5%, and the trehalose concentration is 1M;

(15) Vitrification solution 2 (VS 2): dmso+eg+trehalose+bs; in VS2, the DMSO mass concentration was 8.75%, the EG mass concentration was 8.75%, and the trehalose concentration was 0.5M;

(16) Vitrification solution 3 (VS 3): dmso+eg+trehalose+bs; in VS3, the DMSO mass concentration was 10%, the EG mass concentration was 10%, and the trehalose concentration was 0.5M;

(17) Vitrification solution 4 (VS 4): dmso+eg+trehalose+bs; in VS4, the DMSO mass concentration is 12.5%, the EG mass concentration is 12.5%, and the trehalose concentration is 0.5M;

(18) Vitrification solution 5 (VS 5): dmso+eg+trehalose+bs; in VS5, the DMSO mass concentration is 15%, the EG mass concentration is 15%, and the trehalose concentration is 0.5M;

(19) Rewarming solution (TS): trehalose+bs; in TS, the concentration of trehalose is 1M;

(20) Dilution Solution (DS): trehalose+bs; in DS, the concentration of trehalose is 0.5M;

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

(22) Rewarming the release solution: trehalose + sodium citrate + BS; the solution was released by rewarming with trehalose at a concentration of 0.75M and sodium citrate at a concentration of 0.075M.

Example 2

The present embodiments provide methods for oocyte retrieval and processing.

(1) Selecting an ICR (ICR) SPF-class mouse with 7 weeks of age, and after the ICR is familiar with the adaptation environment for one week, injecting 0.2mL of Pregnant Mare Serum Gonadotropin (PMSG) into the abdominal cavity, injecting 0.2mL of Human Chorionic Gonadotropin (HCG) into the abdominal cavity after 48 hours to stimulate the ovary of the mouse, and inducing the follicle to mature greatly and ovulate;

(2) Mice were quickly sacrificed by cervical dislocation within 15.5 hours after HCG injection, abdominal skin was sterilized with 75% ethanol, the peritoneum was exposed by shearing the skin with scissors, the peritoneum was sheared with another sterile ophthalmology scissors, the viscera turned up, and uterus, fallopian tubes and ovaries were exposed. The upper part of the uterus is clamped by forceps to lift the uterus, redundant fat pads are removed, the connection between the oviduct and the uterus is cut off, and the oviduct and ovary combination body is taken out;

(3) The oviduct is put into PBS for rinsing for 3 times, and then is transferred into preheated M2 egg picking liquid. Under the field of view of the stereo-mirror, finding an expansion part at the joint of the oviduct and the ovary, and using a 1mL syringe to scratch the ampulla of the oviduct, so that the oocyte and the cumulus cell complex (COC) can flow out;

(4) COC was transferred to 50. Mu.L of hyaluronidase droplets using an aspirator and repeatedly blown for 4min with a 10. Mu.L pipette. And the granulosa cells are immediately transferred into an M2 culture solution after loosening, so that the damage to oocytes caused by overlong time in digestive enzymes is avoided. Then cleaning the naked eggs in M2 culture solution drops for 4 times in sequence, and thoroughly removing hyaluronidase;

(5) Two 50mL M2 liquid drops are manufactured in a 35mm culture dish, oocytes with complete morphology, uniform cytoplasm and smooth surface of the polar body are selected and put into M2 culture solution micro-drops, and the number of the oocytes in each micro-drop is controlled to be 10-20. Finally, 2mL embryos are sealed by mineral oil and placed in an incubator for standby.

Example 3

The present embodiment provides the design and fabrication of microfluidic chips.

The micro-fluidic chip main body for preparing the sodium alginate hydrogel microsphere consists of a top layer 1, a distribution layer 2, a channel layer 3 and a bottom layer 4. The structure and sequence of the layers are shown in figure 1. The top layer 1 is provided with an inlet and an outlet for each phase of solution, the solution is pumped in from the inlet, enters the distribution layer 2 and is uniformly dispersed, then the solution is converged in the channel layer 3 to form liquid drops, and finally, the liquid drops flow out from the outlet of the top layer 1. Wherein the thickness of the top layer 1 and the bottom layer 4PMMA plates is 0.5mm, and the thickness of the distribution layer 2 and the channel layer 3 is 0.3mm. The PMMA microfluidic chip has the following manufacturing flow:

(1) Drawing each layer of structure of microfluidic chip

The overall structure of the microfluidic chip is designed according to the required functions, and the geometric figures of the chips of each layer are respectively drawn in Auto CAD 2018.

(2) Laser engraving machine processing

And (3) attaching double-sided adhesive tape to the plates of the channel layer 3 and the continuous phase distribution layer 2, and putting the plates into an operation bin of a laser engraving machine. And uploading the CAD drawing to matched software of a laser engraving machine, setting printing parameters, engraving the CAD graph on the PMMA plate, and sequentially completing the manufacture of each layer of the chip.

(3) Cleaning each layer of chip

And placing the engraved chips in an ultrasonic cleaner containing deionized water, and cleaning for 20min in an ultrasonic mode to remove residual dust and powder on the chips. After cleaning, each layer of chip is wiped dry, and the chips are left to dry at natural temperature.

(4) Chip bonding

And each layer of chip after washing and drying is laminated layer by layer, so that the corresponding structure positions of the chips are ensured. And placing the bonded chips into a vacuum bonding machine, setting the vacuum bonding time to be 4min, and placing the bonded chips into a bubble removal bin, wherein the bubble removal time is set to be 10min.

(5) Component connection

And (3) attaching an anti-collision patch at the solution inlet and outlet of the microfluidic chip, selecting steel needles with proper sizes to be inserted into the inlets and outlets according to experimental requirements, dripping UV (ultraviolet) glue, and carrying out light curing for 2 hours. In the experiment, the chip is connected with other instruments and equipment through a silica gel tube to form a complete micro-fluidic chip system.

Example 4

Influence of different throat structures of microfluidic chip on droplet generation

In order to evaluate the influence of the two-phase flow intersection structure, namely the chip throat on the generation of liquid drops, three different types and sizes of throats are designed, the schematic diagram of the throat structure is shown in fig. 2, and part of parameters are designed as follows: (a) no throat; (b) a wider throat structure 150 μm wide; (c) throat length 300 μm and width 120. Mu.m. The flow rate of the 1% sodium alginate solution of the disperse phase was set to 2. Mu.L/min, and the flow rate of the mineral oil of the continuous phase was set to 20. Mu.L/min. And observing the generation condition of liquid drops at the throat of the microfluidic chip through a microscope.

As a result, referring to fig. 3, droplets in a microfluidic chip channel without a throat structure are difficult to form; in a microfluidic chip with a wider throat structure of 150 mu m, liquid drops still cannot be stably generated at the throat, a dispersed phase solution is continuously trailing, and the formed liquid drops have different particle sizes and uneven size distribution; in a throat structured microfluidic chip 300 μm long and 120 μm wide, droplets can be stably and uniformly generated in the throat region.

Example 5

Influence of different cross-linked structures of microfluidic chip on generation effect of sodium alginate hydrogel microsphere

The invention designs 3 micro-fluidic chips with structures shown in fig. 4, wherein (a) is an external cross-linked chip with the length of 80mm and the width of 40mm shown in fig. 4 a; sodium alginate solution containing oocytes enters the chip from the sodium alginate inlet 5 and enters the junction through the sodium alginate runner 12; mineral oil enters the chip from the mineral oil inlet 6, enters the first intersection 8 through the mineral oil flow passage 13, and is intersected at the throat part, liquid drops are sheared and generated, and then flows out from the outlet 11 and flows into calcium chloride solution along the silica gel pipe to be crosslinked to generate oocyte-carrying/embryo hydrogel microspheres; (b) As shown in FIG. 4b, the chip is a two-channel internal crosslinking chip, the chip is 125mm long and 50mm wide, the S-shaped channel on the right side is a droplet crosslinking area, and the total length is 12×30mm. Sodium alginate solution and oil emulsifier containing oocytes are pumped in from a sodium alginate inlet 5 and an oil emulsifier inlet 7 respectively, enter a chip channel through a sodium alginate runner 12 and an oil emulsifier runner 14 respectively, are intersected at the throat part of a first intersection 8, enter an S-shaped crosslinking channel 10 while being sheared to form spherical liquid drops, calcium chloride in sodium alginate contact oil emulsifier is directly crosslinked into hydrogel microspheres, and finally the crosslinked oocyte-carrying/embryo hydrogel microspheres flow into a collecting dish containing PBS from an outlet 11; (c) As shown in fig. 4c, the three-channel internal crosslinking chip (i.e. the channel layer is of a three-channel structure, the three-channel structure comprises a first channel (sodium alginate channel 12, mineral oil channel 13, first intersection 8) for intersecting sodium alginate solution containing oocyte with mineral oil, a second channel (first droplet channel 15, oil emulsifier channel 14 and second intersection 9) for intersecting first droplet with oil emulsifier, and an S-shaped crosslinking channel 10, wherein the first channel is respectively connected with an inlet 5 of sodium alginate solution, an inlet 6 of mineral oil and an inlet of the second channel, one side of an outlet of the first channel, which is close to the inlet 5 of sodium alginate solution, is provided with a throat, the second channel is respectively connected with an outlet of the first channel, an inlet 7 of oil emulsifier and an inlet of the S-shaped crosslinking channel 10, the outlet of the S-shaped crosslinking channel 10 is connected with a bottom layer, the chip has a length of 150mm, a width of 60mm, and the total length of the S-shaped crosslinking channel 10 is 10×40mm. Sodium alginate solution containing oocytes enters through a sodium alginate inlet 5, mineral oil enters through a mineral oil inlet 6, and the sodium alginate solution and the mineral oil respectively meet at a first intersection 8 through a sodium alginate runner 12 and a mineral oil runner 13, and first liquid drops are generated by shearing; the oil emulsifier enters through the oil emulsifier inlet 7, enters along the oil emulsifier flow channel 14, and is intersected with the first liquid drop flowing along the first liquid drop flow channel 15 at the second intersection 9, then in the right S-shaped cross-linking channel 10, the first liquid drop further enters into the S-shaped cross-linking channel 10 through the mixing flow channel 16 after being contacted with the oil emulsifier to be cross-linked into oocyte-carrying/embryo hydrogel microspheres, and the microspheres flow out to a collecting dish containing PBS through the outlet 11 for collection.

The experiment set the disperse phase as 1% sodium alginate solution and the flow rate as 2. Mu.L/min. The flow rates of the mineral oil phase and the oil emulsifier phase were 20. Mu.L/min. And respectively collecting sodium alginate hydrogel microspheres generated by 3 microfluidic chips, observing the morphology of the microspheres under an inverted microscope, photographing and recording, and finally measuring the particle size of each group of microspheres by using a measuring tool in Image J.

As a result, referring to fig. 5, different crosslinking modes and chip structures have a great influence on the particle size and uniformity of the microspheres. The average particle size of the microspheres generated by the external crosslinking microfluidic chip is 208.29 mu m, the coefficient of variation is up to 29.73%, which indicates that the microsphere size distribution range of the sodium alginate hydrogel microspheres generated by the external crosslinking chip is wide and the uniformity is poor; the microsphere particle sizes generated by the two-channel and three-channel internal crosslinking microfluidic chips are more uniform, and the average particle sizes of the microspheres generated by the two-channel and three-channel internal crosslinking microfluidic chips are 263.74 mu m and 262.76 mu m respectively, which shows that the internal crosslinking chips are more beneficial to maintaining the uniformity of the particle sizes of the microspheres.

The influence of the geometry of the microfluidic chip and channel parameters on the generation effect of the liquid drops and the microspheres is comprehensively considered, and a three-channel microfluidic chip is preferred in the following embodiments.

Example 6

Influence of solution concentration and flow velocity of microfluidic chip on microsphere generation

And a three-channel microfluidic chip is used, the continuous phase is sodium alginate solution, the concentration is 0.5 percent, 1 percent and 1.5 percent respectively, and the flow rate of the solution is fixed to be 2 mu L/min. The oil phase was mineral oil with 0.1% span 80 added, and the flow rates were set at 20. Mu.L/min, 30. Mu.L/min, and 40. Mu.L/min, respectively. The oil emulsifier phase flow rate is the same as the oil phase. 3 groups of sodium alginate with different concentrations are subjected to 3 different flow speed ratio experiments, 9 groups are added, the microsphere generation effect and the particle size are analyzed, and the optimal generation parameters are determined.

TABLE 1 average particle size and coefficient of variation of microspheres at different flow rates and concentrations

As shown in table 1, at a flow rate ratio of 1:10, the microsphere particle size generated by the 0.5% sodium alginate solution is the largest, and the variation coefficient of the 1% sodium alginate group is the lowest. As the concentration of the sodium alginate solution increases, the average particle size of the microspheres gradually decreases. Overall, the 1% group stably produced microspheres with higher uniformity at all 3 flow rate ratios, and the size of the microspheres can be effectively controlled by controlling the flow rate of the continuous phase.

Therefore, the concentration of the sodium alginate solution was 1%, the flow rate of the dispersed phase was 2. Mu.L/min, and the following examples were all given as the optimum parameters.

Example 7

Evaluation of effect of preparing oocyte-loaded sodium alginate hydrogel microspheres by microfluidic chip

(1) Construction of microfluidic systems

The schematic diagram of a device for preparing the oocyte-loaded sodium alginate hydrogel microsphere by the microfluidic chip is shown in fig. 6, the system mainly comprises a liquid pumping area, a microsphere generating area and a collecting area, and the speed and the particle size of microsphere generation can be controlled by adjusting the flow rate of each phase of solution.

(2) Preparation of oocyte-loaded sodium alginate hydrogel microsphere by microfluidic chip

Blowing 30-35 oocytes into 1% sodium alginate solution by using an oral aspirator, sucking the sodium alginate solution containing the oocytes by using a 1mL syringe after the oocytes are fully dispersed, fixing the syringe on a syringe pump, connecting the syringe with a dispersion phase inlet of a microfluidic chip by using a silicone tube, and setting the flow rate to be 2 mu L/min. The mineral oil and the oil emulsifier are respectively sucked by a 5mL syringe, and the flow rates are respectively set to be 20 mu L/min, 30 mu L/min and 40 mu L/min after the mineral oil and the oil emulsifier are fixedly connected. After the flow rate of each phase of solution is stable, the uniformly formed liquid drops can be observed at the throat of the chip. The droplets continue to flow to the S-shaped crosslinking zone through the junction of the mineral oil and the oil emulsifier where they crosslink into sodium alginate microspheres. After the pumping is completed, the sodium alginate microspheres can be collected in a collection dish containing PBS solution connected with the outlet of the chip.

(3) Determination of survival and development of oocytes released by encapsulation of sodium alginate hydrogel

The microspheres were collected from the collection dish, and then sodium alginate hydrogel was decrosslinked by adding sodium citrate solution to release oocytes, and the oocytes were washed 3 times in M2 solution. Observing the survival condition of the oocytes after the encapsulation and release, and carrying out parthenogenetic activation on the surviving oocytes and the fresh group of oocytes, wherein the specific steps are as follows:

1) Two 50. Mu.L droplets of KSOM were first made in a petri dish, 2mL of mineral oil-sealed layer for embryos was added along the edge of the petri dish, and placed in an incubator for equilibration overnight. Then, a new culture dish was taken, 50. Mu.L of droplets of the activating solution were prepared, and the embryos were covered with mineral oil and placed in an incubator for equilibration for 3.5 hours.

2) After the balance is well, transferring the oocyte into an activating solution drop, and then placing the activating solution drop into an incubator for activating culture for 4 hours. After 4h, oocytes were retrieved and washed 3 times in M2 solution. Placing the cleaned oocyte in KSOM liquid drops, and placing the KSOM liquid drops in an incubator for continuous culture.

3) After 6 hours, the activation condition of the oocyte is observed, and the polar body is discharged to serve as an activation result sign. Observing the oocyte every 24 hours thereafter; observing the oozing condition of the oocyte after 48 hours, selecting oozing cell replacement liquid for continuous culture; after 96 hours, the cell development was observed, and the blastocyst rate was calculated.

The calculation method of the survival rate, the cleavage rate and the blastula rate comprises the following steps:

survival (%) = survival number/total number of oocytes x 100%

Cleavage rate (%) =number of cleavage/number of survival×100%

Blastula rate (%) = blastula number/number of cleavage x 100%

TABLE 2 comparison of blank Rate of sodium alginate hydrogel microspheres with oocyte loss Rate at different flow Rate ratios

As shown in table 2 and fig. 7, the results show that the empty package rate of the microsphere is higher and the encapsulation effect is poor under the condition of high flow rate, and meanwhile, the higher oocyte loss rate can occur, and the empty package rate and the oocyte loss rate of the microsphere are reduced along with the reduction of the flow rate; the micro-fluidic chip is used for encapsulating the oocyte, and then the oocyte is released, so that the survival rate and the subsequent development capacity of the oocyte are not significantly different from those of a fresh group.

Example 8

Evaluation of vitrification cryopreservation effect of sodium alginate hydrogel encapsulated oocyte

And (3) respectively adopting a Cryotop method, a low-concentration CPA-hydrogel encapsulation method and a low-concentration CPA-Cryotop method for comparison verification. The method comprises the following specific steps:

(1) Cryotop group: during the freezing process, 10 oocytes were taken per group, and the oocytes were transferred from the culture broth into 400. Mu.L of ES droplets using an oral aspirator for 15min of equilibration, and then transferred into 400. Mu.L of VS5 for 1min of infiltration. After CPA loading is completed, the oocytes are quickly blown onto Cryotop carrier rods, and 2-3 oocytes are placed on each carrier rod. And then the Cryotop carrier rod carrying the oocytes is directly placed into liquid nitrogen, a plastic shell is sleeved after the liquid nitrogen is stabilized, and the whole carrier rod is put into the liquid nitrogen for freezing for 1h. And (3) in the re-warming process, freezing for 1h, taking the Cryotop carrier out of liquid nitrogen, taking off the plastic shell, quickly immersing the front end of the carrier rod into 800 mu L TS of which the temperature is 37 ℃ preheated in advance, slightly shaking to enable the oocyte to be separated from the carrier rod into solution, transferring the oocyte into 800 mu L DS, and diluting for 5min after 1min. Finally, the oocytes were washed 3 times in BS and transferred to M2 solution.

(2) Low concentration CPA-sodium alginate hydrogel encapsulation group: and in the encapsulation process, 30-35 oocytes are taken from each group, mixed with 800 mu L of 1% sodium alginate solution and uniformly distributed in the solution. The mixed solution of sodium alginate and oocyte was sucked by using a 1mL syringe, and the syringe, the syringe containing the oil emulsifier and the syringe containing the mineral oil were fixed on a syringe pump, respectively, with the flow rates of 2. Mu.L/min, 30. Mu.L/min, and 30. Mu.L/min, respectively. The syringe and the micro-fluidic chip are connected through a silica gel tube with the inner diameter of 0.5mm, and pumping is started, so that the whole duration is about 5-8 min. And collecting the prepared oocyte-carrying sodium alginate hydrogel microspheres from a recovery dish, transferring the oocyte-carrying sodium alginate hydrogel microspheres into a metal screen carrier, and washing the oocyte-carrying sodium alginate hydrogel microspheres with PBS for three times. And in the freezing process, directly immersing a metal screen containing microspheres into VS3 containing 1mL for CPA loading for 10min, absorbing excessive liquid below the metal screen by using water absorbing paper after CPA loading, and then throwing the metal screen into liquid nitrogen for freezing. And in the rewarming process, after freezing for 1h, taking out the metal screen from the liquid nitrogen by using tweezers, rapidly putting the metal screen into a preheated rewarming solution at 37 ℃, transferring the preheated solution into a dilution release solution for dilution and release for 3min after 1min, shaking the screen during the period to enable sodium alginate microspheres to fall into the solution, and decrosslinking the sodium citrate in the dilution release solution by using external sodium alginate hydrogel, so that oocytes are released into the solution from the inside of the sodium alginate hydrogel. After 3min, the released oocytes were washed sequentially 3 times in BS solution using an oral aspirator, and finally blown into M2 solution for recovery.

(3) Low concentration CPA-Cryotop group: all experimental procedures were identical to the Cryotop group, with only the vitrification solution being changed to VS3, which was identical to the sodium alginate hydrogel encapsulation group.

The oocytes finally collected in each experimental group are recovered for 1h in an incubator, and the number of surviving cells is counted

And calculating the survival rate, and then performing in vitro parthenogenesis activation operation to calculate the cleavage rate and blastula rate of the oocyte. All experiments were repeated 3 times.

As shown in fig. 8, the survival rate and the development rate of the sodium alginate hydrogel encapsulated oocyte in the low-concentration protective agent VS3 in vitrification preservation are not significantly different from the result of the Cryotop method in the high-concentration protective agent VS5 in vitrification preservation, which indicates that encapsulation of the sodium alginate hydrogel can reduce the concentration of the protective agent required for vitrification of the oocyte.

Example 9

Evaluation of vitrification cryopreservation effect of oocyte-carrying hydrogel microspheres with different particle sizes

The particle size of the oocyte-loaded sodium alginate microspheres is changed by controlling the flow rates of mineral oil and oil emulsifier in the microfluidic chip, and freezing and rewarming experiments are carried out respectively. The flow rate of the 1% sodium alginate solution containing oocytes is fixed to be 2 mu L/min; the mineral oil and the oil emulsifier have the same flow rate, and are set to 20 mu L/min, 30 mu L/min and 40 mu L/min. The average particle diameters of the sodium alginate microspheres produced at flow rate ratios of 10, 15 and 20 were 262 μm, 193 μm and 156 μm, respectively. Freezing and rewarming the oocyte-carrying microspheres generated under different flow velocity ratios respectively, and counting the survival and subsequent development conditions of the oocytes in the experimental procedure in the step (2) in the embodiment 8.

As shown in FIG. 9, when the particle size of the microspheres was 262 μm, there was no significant difference in the survival rate of oocytes (93.16%), the cleavage rate (71.78%), the blastocyst rate (21.06%) and the survival rate of 193 μm group (92.48%), the cleavage rate (70.80%), and the blastocyst rate (20.42%). The oocyte viability and cleavage rate for the 156 μm group were only 68.26% and 59.44%, much lower than for the control group and the other two groups. Therefore, the control of the average particle size of the hydrogel microspheres can regulate the permeation rate of the protective agent, thereby reducing the permeation damage of the oocyte.

Example 10

Influence of different protective agent concentrations on vitrification cryopreservation of oocyte-loaded hydrogel microspheres

Four different concentrations of cryoprotectants, namely VS1, VS2, VS3 and VS4, were selected to determine the optimum concentration of cryoprotectant after encapsulating the oocyte with sodium alginate hydrogel. The oocyte-carrying microspheres with the particle size of 262 mu m are prepared and collected by a microfluidic chip, and are respectively immersed into four low-temperature protective agents with different concentrations in 1mL after being placed in a metal screen, and the loading time is 10min. The subsequent freezing, rewarming and releasing procedures were the same as in step (2) of example 8.

As shown in FIG. 10, the survival and development rates of oocytes increased with increasing concentration of the protective agent, but did not show a monotonic increase. When the concentration of the protective agent was increased to 12.5% EG+12.5% DMSO+0.5M trehalose (VS 4), the survival rate, cleavage rate and blastula rate of the VS4 group oocytes were not significantly different from those of the VS3 group, but were slightly decreased, indicating that there was an optimal balance point between the sodium alginate hydrogel and the protective agent concentration.

Example 11

Influence of different loading time lengths of protective agent on vitrification cryopreservation of oocyte-loaded hydrogel microspheres

And loading the oocyte-loaded sodium alginate hydrogel microsphere prepared by the microfluidic chip into a low-temperature protective agent of VS3 for 4min, 8min and 12min respectively. After loading, the water absorbing paper absorbs the superfluous protective agent, and the freezing and rewarming operation is continued, and other operation steps are the same as the step (2) in the embodiment 8.

As shown in FIG. 11, the 4min group had much lower oocyte viability than the other groups, which was only 23.99%; the cell viability of the 8min group (91.98%) was not significantly different from that of the 12min group (92.28%), but the cell burst rate (75.84%) and blastula rate (23.86%) were significantly higher than those of the 12min group (61.88% and 15.70%).

Example 12

Influence of different rewarming release procedures on freezing and rewarming of oocyte-loaded hydrogel microspheres

3 groups of restoration and oocyte release steps are designed, the protective agent loading and freezing steps of each group of oocyte-loaded microspheres are all 262 mu m of 1% sodium alginate microspheres, the VS3 protective agent is loaded in one step, the duration is 8min, and the oocyte-loaded microspheres are put into liquid nitrogen and frozen for 1h. The method comprises the following specific steps:

(1) The one-step method comprises the following steps: the rewarming and releasing processes are performed simultaneously. After the metal screen is taken out of the liquid nitrogen, the metal screen is directly put into a rewarming release solution, and tweezers are used for lightly touching the screen to enable the microspheres to fall off from the inner wall into the solution. After 3min of rewarming release, the oocytes were aspirated into BS using an oral aspirator.

(2) The two-step method comprises the following steps: dilution and release are performed simultaneously. The microspheres are immersed in a 1M trehalose solution for rewarming for 1min, then the microspheres are moved to a mixed solution of 0.5M trehalose and 0.075M sodium citrate for dilution and release for 3min, and then the oocytes are moved to BS.

(3) The three-step method comprises the following steps: the rewarming, dilution and release are respectively carried out. Taking out the frozen metal screen with the cell-carrying microspheres from liquid nitrogen, immersing the metal screen into a trehalose solution with the temperature of 37 ℃ and the temperature of 1M for 1min, transferring the metal screen into a trehalose solution with the temperature of 0.5M for 3min, shaking the sodium alginate microspheres subjected to the temperature recovery out of the sodium citrate solution with the temperature of 0.075M for 3min, and releasing oocytes. The oocyte was transferred into the BS under the field of view of the stereoscopic vision, with the use of an aspirator.

After transferring each group of oocytes into BS, washing for 3 times in sequence, transferring into M2 liquid drops, culturing in an incubator for 2 hours, counting the survival number of the oocytes, and calculating the survival rate. And then parthenogenetic activation is carried out on the surviving oocytes, and the number of the blastomeres and the cells which develop to the blastula stage is counted.

As shown in fig. 12, compared with the influence of three different rewarming and releasing modes on the survival rate and the development rate of the oocyte after vitrification and rewarming, the survival rate, the cleavage rate and the blastocyst rate of the two-step method of the procedure of rewarming and then simultaneously diluting and releasing are remarkably higher Yu Fuwen than those of the one-step method of the procedure of releasing and the three-step method of the procedure of rewarming, diluting and releasing respectively.

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the explanation of the present invention, should make improvements and modifications without departing from the scope of the present invention.

Claims

1. The preparation method of the oocyte/embryo-loaded hydrogel microsphere is characterized by comprising the following steps of:

and (S2) pumping the sodium alginate solution containing the oocyte, the mineral oil and the oil emulsifier obtained in the step (S1) into a microfluidic chip, wherein the sodium alginate solution containing the oocyte and the mineral oil form first liquid drops, and the first liquid drops are mixed with the oil emulsifier and then are further crosslinked to form the oocyte-carrying/embryo hydrogel microspheres.

2. The method for preparing oocyte/embryo-loaded hydrogel microspheres according to claim 1, wherein in the step (S1), the mass concentration of the sodium alginate solution is 0.5% to 1.5%;

3. The method for preparing oocyte/embryo-loaded hydrogel microspheres according to claim 1, wherein in the step (S2), the microfluidic chip is a top layer, a distribution layer, a channel layer and a bottom layer which are sequentially connected from top to bottom and are provided with an outlet and an inlet, respectively; the channel layer is of a three-channel structure, and the three-channel structure comprises a first channel for converging sodium alginate solution containing oocytes with mineral oil, a second channel for converging first liquid drops with an oil emulsifier, and an S-shaped crosslinking channel for crosslinking the first liquid drops with the oil emulsifier.

4. A method of preparing an oocyte/embryo-loaded hydrogel microsphere according to claim 3, wherein the first channel is connected to the inlet of sodium alginate solution containing oocytes, the inlet of mineral oil and the inlet of the second channel, respectively; a throat part is arranged at one side of the junction of sodium alginate solution containing oocytes and mineral oil, which is close to the inlet of the second channel;

5. The method of claim 1, wherein in the step (S2), the oil emulsifier is a mixture of anhydrous calcium chloride, mineral oil and span 80;

6. An oocyte-loaded/embryo hydrogel microsphere prepared by the method according to any one of claims 1 to 5, wherein the particle size of the microsphere is 100 μm to 320 μm.

7. A method for vitrification preservation of oocyte/embryo-loaded hydrogel microspheres as set forth in claim 6, comprising the steps of:

8. The method for vitrification preservation of oocyte/embryo-loaded hydrogel microspheres according to claim 7, wherein in step (A1), the frozen carrier is a 304 stainless steel 80 mesh metal screen.

9. The method for vitrification preservation of oocyte/embryo-loaded hydrogel microspheres according to claim 7, wherein in step (A2), the vitrification solution is a mixture of DMSO, EG and trehalose;

in the CPA loading process, the time is 4-12 min.

10. The method for vitrification preservation of oocyte/embryo hydrogel microspheres according to claim 7, wherein in the step (A3), the frozen carrier loaded with the microspheres is put into a preheated rewarming solution for rewarming and then transferred to a diluted release solution for dilution release;