Microfluidic chip for vitrification cryopreservation of oocytes or embryos, preparation method and application thereof

Abstract

The present invention discloses a microfluidic chip for vitrification and cryopreservation of oocytes or embryos, a preparation method and its application, the microfluidic chip comprises a microfluidic channel layer, according to the flow direction of the microfluid, the microfluidic channel layer comprises one or more solution inflow channels, serpentine mixing channels, one or more shunt channels, serpentine channels, cell storage chambers and waste liquid discharge channels connected in sequence; wherein, a first microcolumn array is provided at the outlet of the cell storage chamber, and a second microcolumn array is provided in the channel outside the inlet; a cell channel is connected between the cell storage chamber and the second microcolumn array. The microfluidic chip can realize the integration of vitrification and cryopreservation, rewarming and other operations, reduce the number of cell transfers to avoid contamination, improve processing efficiency, and significantly improve the survival rate of oocytes or embryos after rewarming, which can further promote the development of assisted reproductive technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic chips, and in particular relates to a microfluidic chip for vitrification and cryopreservation of oocytes or embryos, a preparation method of the microfluidic chip, and application of the microfluidic chip in vitrification and cryopreservation of oocytes or embryos. Background Art

[0002] Assisted reproductive technology (ART) refers to the use of medical assistance to help infertile couples become pregnant, such as in vitro fertilization (IVF). It usually requires the freezing and preservation of oocytes or fertilized eggs under appropriate conditions for future use when needed.

[0003] At present, the more conventional methods for cryopreservation of oocytes are slow freezing and vitrification. Among them, vitrification can improve the freezing survival rate of oocytes, so it is currently widely used. At present, the conventional freezing egg process in clinical practice generally has the following steps: ovarian stimulation, egg collection, egg processing and freezing, storage and custody. After egg collection, the dense granulosa cells around the egg must first be separated and removed, and then the cryoprotectant is added in multiple steps through gradient concentrations. After that, the egg loaded with cryoprotectant is moved into the freezing carrier and placed in liquid nitrogen for freezing. In these processes, the eggs need to be transferred continuously, which is not only complicated and cumbersome, but also consumes a lot of manpower and material resources. It will also cause stress damage to the eggs, and it is highly professional and requires professional personnel to operate. At the same time, the oocytes are always in an open environment during the freezing process and are easily contaminated. Therefore, optimizing the freezing egg process is of great significance for the cryopreservation of clinical eggs.

[0004] At present, some schemes have designed some microfluidic cell processing chips or microfluidic-based vitrification preservation devices for the cryopreservation of oocytes. However, the existing microfluidic chips are mainly for the processing of cryoprotectants and cannot cover the entire oocyte cryopreservation / thawing operation process, or the device design is relatively complex and cumbersome, the cost is high, and it is not conducive to promotion. Summary of the invention

[0005] In view of this, the primary purpose of the present invention is to provide a microfluidic chip for vitrification and cryopreservation of oocytes or embryos. The microfluidic chip uses microfluidic technology to achieve continuous linear changes in the concentration of cryoprotectant / thaw solution, reduce the number of cell transfers, realize the integration of addition / removal of cryoprotectant additives and on-chip freezing, realize the automation of vitrification and cryopreservation of oocytes or embryos, reduce labor costs, and improve the survival rate of oocytes / embryos after thaw.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention first provides a microfluidic chip for vitrification and cryopreservation of oocytes or embryos. The microfluidic chip can realize the integrated operation of adding cryoprotectant, vitrification and cryopreservation, thawing and removing cryoprotectant, reduce the number of cell transfers, realize the automation of vitrification and cryopreservation of oocytes or embryos, reduce labor costs, and improve the survival rate of oocytes / embryos after thawing.

[0008] In the present invention, according to the flow direction of the microfluid, the microfluidic channel layer includes one or more solution inflow channels, serpentine mixing channels, one or more diversion channels, serpentine channels, cell storage chambers and waste liquid discharge channels that are sequentially connected;

[0009] Wherein, a first micro-column array is arranged at the outlet of the cell storage chamber, and a second micro-column array is arranged in the channel outside the inlet thereof; a cell channel is connected between the cell storage chamber and the second micro-column array.

[0010] The "plurality" mentioned in this article refers to more than two, which can be set as needed.

[0011] Wherein, the cryopreservative (or rewarming solution) and the buffer solution enter the chip through one or more solution inflow channels, and flow in the chip under the drive of an external device (such as a syringe pump, etc.). After the solution inflow channel converges at the outlet, it is communicated with the entrance of the serpentine mixing channel, wherein the serpentine mixing channel can be provided with more than two as needed, at least two solution inflow channels should be satisfied to correspond to one serpentine mixing channel, or multiple solution inflow channels correspond to one serpentine mixing channel, and the pressure before and after the cell storage chamber is ensured by setting the serpentine mixing channel, thereby reducing the osmotic damage of the solution to the oocyte or embryo. In some specific embodiments of the present invention, the solution inflow channel has two, and the comprehensive design considerations are that the outlets of the solution inflow channel are mutually obtuse and communicated with the entrance of the serpentine mixing channel after convergence.

[0012] Furthermore, the outlet of each serpentine mixing channel is connected to one or more shunt channels to further reduce the osmotic damage of the solution to the oocytes or embryos in the cell storage chamber; at the same time, the provision of multiple shunt channels can also enable a microfluidic chip to store multiple oocytes or embryos at the same time, thereby improving the efficiency of vitrification and cryopreservation of oocytes or embryos. In some specific implementation cases of the present invention, two shunt channels are provided.

[0013] Furthermore, according to the flow direction of the microfluid, each shunt channel is connected in series with a serpentine channel, a cell storage chamber and a waste liquid discharge channel, so as to realize the vitrification and cryopreservation of multiple oocytes or embryos. The outlet of each shunt channel is connected to a section of serpentine channel, and the pressure brought by the serpentine mixing channel is buffered by the setting of the section of serpentine channel, so as to prevent the oocytes or embryos in the cell storage chamber from being damaged by the impact of the solution.

[0014] Furthermore, in the present invention, the shape of the cell storage chamber can be any shape, and the size can be designed according to the specific situation of the oocyte or embryo. Preferably, the inner wall of the cell storage chamber is set to be arc-shaped to avoid damage to the oocyte or embryo. In some specific implementation cases of the present invention, the cell storage chamber is a cylindrical chamber.

[0015] Furthermore, in order to store the oocyte or embryo in the cell storage chamber, a first microcolumn array is provided at the exit of the cell storage chamber, and a second microcolumn array is provided in a section of the passage outside the entrance of the cell storage chamber, wherein the first microcolumn array and the second microcolumn array are composed of a plurality of microcolumns arranged at intervals, and the specific microcolumn interval distance can be designed according to the oocyte or embryo, so as to intercept the oocyte or embryo so that the oocyte or embryo cannot pass through. It is understandable that the shape of the microcolumn can be conventional in the art, such as a regular prism, a cylinder or a triangular prism, etc., but similarly, in order to avoid damage to the oocyte or embryo as much as possible, preferably, a cylindrical microcolumn is used.

[0016] Furthermore, in order to smoothly introduce the oocyte or embryo into the cell storage chamber, a cell channel is connected between the entrance of the cell storage chamber and the second microcolumn array, so that the oocyte or embryo can be smoothly introduced into the cell storage chamber.

[0017] It can be understood that a number of through holes are provided on the surface of the microfluidic channel layer, and these through holes are respectively connected to the solution inlet channel, the cell channel, and the waste liquid discharge channel, so that the microfluidic channel layer can be connected to external equipment to realize the process of vitrification and cryopreservation of oocytes or embryos.

[0018] The second aspect of the present invention provides a method for preparing the microfluidic chip as described above, which specifically comprises the following steps:

[0019] S1. Provide a microfluidic channel layer and a substrate layer.

[0020] Wherein, the microfluidic channel layer can be formed by conventional materials or conventional methods (such as photolithography) in the art. For example, in some specific implementation cases of the present invention, the microfluidic channel layer is made of polydimethylsiloxane (PDMS) by photolithography. It is understandable that the material of the microfluidic channel layer is transparent or translucent, so that the oocyte or embryo contained in the microfluidic chip can be easily observed from the outside.

[0021] In the present invention, the base layer is made of transparent alumina ceramic material, which is due to the fact that transparent alumina ceramic has stronger low temperature resistance and hardness than conventional glass, and can provide a higher heat transfer rate during freezing and thawing.

[0022] S2. Soaking the base layer in (3-mercaptopropyl)trimethoxysilane for 1-5 minutes for surface modification.

[0023] Since the transparent alumina ceramic material is different from the general glass substrate, it needs to be immersed in (3-mercaptopropyl) trimethoxysilane (MPTMS) for 1-5 minutes for surface modification before bonding.

[0024] S3. After cleaning the surface-modified substrate layer, assemble and bond it with the microfluidic channel layer to obtain a microfluidic chip.

[0025] In the present invention, preferably, the surface modified substrate layer is cleaned by high-speed water flow. The substrate layer and the microfluidic channel layer can be bonded by conventional bonding processes in the art, for example, in some specific implementation cases of the present invention, a plasma oxidation bonding process is used.

[0026] The third aspect of the present invention provides the use of the microfluidic chip described above in vitrification cryopreservation of oocytes or embryos.

[0027] A fourth aspect of the present invention provides a method for vitrification cryopreservation of oocytes or embryos based on the microfluidic chip described above, comprising the following steps:

[0028] a: Add cryoprotectant;

[0029] b: Vitrification of oocytes or embryos;

[0030] c: Rewarming of oocytes or embryos after vitrification;

[0031] d: Remove cryoprotectant.

[0032] In the present invention, in step a,

[0033] In the present invention, in step a, the process of adding a cryoprotectant comprises the following steps:

[0034] Connecting the solution inflow channel to a sample injection device, injecting the cryoprotectant and the buffer solution into the microfluidic chip through the sample injection device, and driving the flow of the solution in the chip;

[0035] The injection speeds of the cryoprotectant and the buffer solution are respectively set so that the concentration of the cryoprotectant in the mixed solution increases linearly;

[0036] When the cryoprotectant is completely added, stop injecting the solution; let it stand to ensure that the oocytes or embryos are completely mixed with the cryoprotectant;

[0037] The injection device described here can be any device in the art that can inject cryoprotectants and buffer solutions into the microfluidic chip and drive the cryoprotectants and buffer solutions to flow in the chip. Specific examples include injection pumps, peristaltic pumps, etc., but are not limited to these. These injection devices can control the advancement speed of the cryoprotectants and buffer solutions so as to be flexibly adjusted according to the needs of different cryoprotectant additives and buffer solutions.

[0038] Furthermore, the cryoprotectant additive and buffer solution used may be conventional compositions in the art, for example, the cryoprotectant may be at least one of glycerol, ethylene glycol, 1,2-propylene glycol or DMSO, etc. In some specific implementation cases of the present invention, the formula composition of the cryoprotectant is 15% ethylene glycol + 15% propylene glycol + cell culture medium M2 + 0.5 mol / L trehalose in terms of mass percentage; the buffer solution is cell culture medium M2.

[0039] In the present invention, in step b, the process of vitrification cryopreservation of oocytes or embryos comprises the following steps:

[0040] The microfluidic chip containing oocytes or embryos is placed directly into liquid nitrogen for vitrification.

[0041] In the present invention, in step c and step d, the process of thawing the oocyte or embryo after vitrification and removing the cryoprotectant comprises the following steps:

[0042] Preheat the rewarming solution and buffer solution at 37°C;

[0043] The cryopreserved microfluidic chip was taken out and quickly placed in a 37°C water bath for rewarming;

[0044] Injecting a rewarming solution and a buffer solution into the chip through the solution inflow channel, and setting the injection speed of the rewarming solution and the buffer solution so that the concentration of the rewarming solution in the mixed solution decreases linearly;

[0045] When the rewarming solution is completely added, stop injecting the solution; let it stand to ensure that the oocyte or embryo is completely mixed with the buffer solution;

[0046] After the oocytes or embryos are aspirated, they are placed in a cell culture medium and placed in a cell culture incubator for culture, thereby completing the removal of the cryoprotectant from the oocytes or embryos.

[0047] The rewarming solution and buffer solution used in the present invention can be solutions conventionally used for cryopreservation of oocytes or embryos in the art, without particular limitation. In some specific implementation cases of the present invention, the formula composition of the rewarming solution used is 1 mol / L trehalose + cell culture medium M2 in terms of mass percentage; the buffer solution is cell culture medium M2.

[0048] Beneficial effects of the present invention:

[0049] (1) The microfluidic chip design of the present invention is based on microfluidic technology for precise control of complex fluids at a microscale. The cryoprotectant / rewarming solution and the buffer solution enter the microfluidic channel through different inlets, flow in the channel in a laminar flow manner, and are fully mixed in the serpentine mixing channel by diffusion. The concentration of the cryoprotectant / rewarming solution in the mixed solution is linearly increased / decreased by changing the flow rate of the cryoprotectant / rewarming solution and the buffer solution, thereby reducing the osmotic pressure shock caused by the concentration difference between the cryoprotectant and the intracellular fluid, and the toxic damage of cells in high-concentration cryoprotectants.

[0050] (2) The microfluidic chip for vitrification and cryopreservation of oocytes or embryos of the present invention realizes the integrated automation of addition / removal of cryoprotectants, freezing and recovery. The operation is simple and convenient, and the high precision and stability brought by the equipment are much better than manual operation. During the whole operation, oocytes or embryos are stored in the microfluidic chip in the process to avoid being released from the external environment and prevent contamination by external bacteria. The survival rate of oocytes or embryos cryopreserved by the microfluidic chip in the present invention after rewarming has a significant improvement.

[0051] (3) The microfluidic chip of the present invention has the advantages of controllable cost, simple preparation, and reusability, and has broad prospects for commercialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Schematic diagram of the three-dimensional structure of a microfluidic chip in a preferred embodiment of the present invention.

[0053] Figure 2 for Figure 1 Schematic diagram of the planar structure of the microfluidic chip.

[0054] Figure 3 for Figure 2 A schematic diagram of the enlarged structure of part A in the middle.

[0055] Figure 4 This is an overall physical picture of the microfluidic chip produced in Example 3.

[0056] Figure 5 This is a picture taken under an optical microscope of the oocyte stored in the cell storage chamber 15 in Example 4.

[0057] Figure 6 The optical microscope and fluorescence microscope images of cells alive and dead after vitrification of oocytes by the microfluidic chip in Example 4 and conventional vitrification in the comparative example, wherein: Figure 6 a, 6b are optical microscope and fluorescence microscope images of live and dead cells after conventional vitrification; Figure 6 c, 6d are optical microscope and fluorescence microscope images of live and dead cells after oocyte vitrification using microfluidic chip.

[0058] In the figure: 1-microfluidic channel layer, 11-solution inflow channel, 12-serpentine mixing channel, 13-diversion channel, 14-serpentine channel, 15-cell storage chamber, 151-first microcolumn array, 152-second microcolumn array, 16-cell channel, 17-waste liquid discharge channel; 2-basal layer. DETAILED DESCRIPTION

[0059] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, unless otherwise specified, methods that do not specifically record conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0061] Example 1

[0062] This embodiment provides a microfluidic chip for vitrification and cryopreservation of oocytes or embryos, and its structure is as follows: Figure 1 and Figure 2 As shown in .

[0063] The microfluidic chip includes two layers, the upper layer is a microfluidic channel layer 1, and the lower layer is a substrate layer 2. The microfluidic channel layer 1 and the substrate layer 2 are bonded by a method well known in the art to form the microfluidic chip in this embodiment.

[0064] The microfluidic channel layer 1 includes a solution inflow channel 11, a serpentine mixing channel 12, a diversion channel 13, a serpentine channel 14, a cell storage chamber 15 and a waste liquid discharge channel 17 which are sequentially connected in series according to the flow direction of the microfluid.

[0065] In this embodiment, there are two solution inflow channels 11, whose outlets merge with the inlet of the serpentine mixing channel 12 and are connected with the serpentine mixing channel 12; wherein the two solution inflow channels 11 form a certain angle, specifically, an obtuse angle with each other.

[0066] Furthermore, in this embodiment, there are two diverter channels 13, whose inlets merge with the outlet of the serpentine mixing channel 12 and communicate with the serpentine mixing channel 12. A section of serpentine channel 14 is connected at the outlet of each diverter channel 13, and the outlet of the serpentine channel 14 is connected to the cell storage chamber 15, and the outlet of the cell storage chamber 15 is connected to the inlet of the waste liquid discharge channel 17.

[0067] The cell storage chamber 15 is cylindrical. For details, see Figure 3 A first micro-pillar array 151 is provided at the exit of the cell storage chamber 15. The first micro-pillar array 151 is composed of three cylindrical micro-pillars arranged at intervals, and is used to intercept oocytes or embryos. Furthermore, a second micro-pillar array 152 is provided in the channel connected to the entrance of the cell storage chamber 15. The second micro-pillar array 152 is also composed of three cylindrical micro-pillars arranged at intervals.

[0068] Furthermore, a cell channel 16 is connected between the entrance of the cell storage chamber 15 and the second micro-column array 152 , through which an oocyte or embryo can be introduced into or led out of the microfluidic chip.

[0069] In addition, in this embodiment, a serpentine channel is connected to the outlet of the waste liquid discharge channel 17, the purpose of which is to ensure the front and rear pressures of the cell storage chamber 15 and reduce the osmotic damage of the solution to the oocyte or embryo.

[0070] It can be understood that a number of through holes (not marked in the figure) are provided on the surface of the microfluidic channel layer 1, which are respectively connected to the solution inlet channel 11, the cell channel 16 and the waste liquid discharge channel 17, thereby connecting external equipment with these channels, controlling the flow of microfluids, and the introduction and / or extraction of solutions, oocytes or embryos into and / or out of the microfluidic chip.

[0071] Furthermore, it can be understood that the specific length, width and height design of the microfluidic channel, the spacing distance between the cell storage chamber 15, the first microcolumn array 151, and the second microcolumn array 152, etc. can all be designed according to the actual needs of the oocytes or embryos to be vitrified and cryopreserved, without any special limitations.

[0072] Example 2

[0073] In this embodiment 2, a microfluidic chip for vitrification and cryopreservation of oocytes is provided, and its structure is the same as that in embodiment 1. In this embodiment, the structural parameters of the microfluidic chip are specifically defined so that it can be used for vitrification and cryopreservation of oocytes.

[0074] Specifically, in the microfluidic chip of this embodiment, the width of the overall microfluidic channel is 0.02 mm and the height is 0.15 mm. The cell storage chamber 15 is a cylindrical chamber with a diameter of 0.3 mm. The first microcolumn array 151 is composed of three cylindrical microcolumns with a diameter of 0.07 mm, and the interval between adjacent microcolumns is 0.03 mm. The second microcolumn array 152 is composed of three cylindrical microcolumns with a diameter of 0.05 mm, and the interval between adjacent microcolumns is 0.01-0.02 mm.

[0075] Through the above parameter design, the microfluidic chip can be suitable for vitrification and cryopreservation of oocytes.

[0076] Example 3

[0077] This embodiment provides a process for manufacturing the microfluidic chip in Example 2, and the specific steps are as follows:

[0078] (1) Mask production

[0079] The channel structure of the microfluidic chip was designed and drawn using CAD software, and the mask was prepared using a high-precision plate making machine.

[0080] (2) SU-8 template processing

[0081] Take a 4-inch single-polished silicon wafer for pretreatment and place it in a vacuum oven; take an appropriate amount of SU-8 negative photoresist and spin-coat it on the surface of the silicon wafer to form a layer of about 150 μm thick and then put it in a 95°C oven for soft baking; use a mask template to cling to the silicon wafer with the photoresist spin-coated, and use high-intensity ultraviolet light in the photolithography machine for exposure; then put the silicon wafer in a 95°C oven for hard baking; after baking, use a negative photoresist developer to soak the silicon wafer to remove the unexposed SU-8 photoresist, and after it is completely removed, rinse it with isopropyl alcohol and ultrapure water in turn, and blow dry the surface with nitrogen.

[0082] (3) Fabrication of microchannels and chambers

[0083] Take appropriate amount of PDMS prepolymer and cross-linking agent in a mass ratio of 10:1, mix them and put them in a vacuum box to remove bubbles, cast them on a SU-8 template, and bake them on a 70°C glue baking table for 2 hours; peel off the cured PDMS from the template to obtain the microfluidic channel layer 1 of the chip, and use a punch to punch out the inlets of the solution inflow channel 11, the cell channel 16, and the waste liquid discharge channel 17.

[0084] (4) Assembly and bonding of microfluidic chips

[0085] First, the transparent alumina ceramics were immersed in (3-mercaptopropyl)trimethoxysilane (MPTMS) for 1-5 minutes for surface modification; then, the microfluidic channel layer 1 and the base layer 2 made of 1 mm thick alumina transparent ceramics were cleaned, kept dry and placed in a plasma cleaner for plasma oxidation for 90 seconds. After taking out, the two layers were butt-jointed and pressed, and placed on a 70°C glue drying table for heating for 2 hours to make the two layers fit tightly and completely. The bonded chip was the microfluidic chip used for the subsequent vitrification of oocytes.

[0086] The microfluidic chip structure for oocyte vitrification cryopreservation prepared in this embodiment is as follows: Figure 4 shown.

[0087] Example 4

[0088] This embodiment discloses the steps of freezing, preserving, and thawing oocytes based on the microfluidic chip in Example 3, which are as follows:

[0089] 1. Add cryopreservative

[0090] (1) Sterilize the microfluidic chip by ultraviolet irradiation to ensure the chip environment;

[0091] (2) The syringes containing the cryoprotectant (15% ethylene glycol + 15% propylene glycol + cell culture medium M2 + 0.5 mol / L trehalose) and the buffer solution (cell culture medium M2) are connected to the inlets of the two solution inflow channels 11 of the microfluidic chip through silicone hoses, respectively; wherein the cryoprotectant enters the chip through one of the solution inflow channels 11, and the buffer solution enters the chip through the remaining solution inflow channel 11, and the syringe is automatically pushed by the syringe pump;

[0092] (3) First push the syringe filled with buffer solution to fill the microfluidic channel with buffer solution, and at the same time exhaust the air in the channel to prevent bubbles from damaging the cells;

[0093] (4) connecting the oocyte in the cell culture medium to the inlet of the cell channel 16 through a silicone hose connected to a syringe, and injecting the oocyte into the cell storage chamber 15 through the cell channel 16;

[0094] (5) After the cells are stabilized, the syringes containing the cryoprotectant and the buffer solution are pushed simultaneously, and the cryoprotectant flow rate is controlled to increase slowly from 0 to 10 μL / min, and the buffer solution flow rate is controlled to decrease slowly from 10 μL / min to 0; in the process of changing the flow rate, the cryoprotectant and the buffer solution are completely mixed in the serpentine mixing channel 12, and the content of the cryoprotectant in the mixed solution increases linearly from 0% to 100%;

[0095] (6) When the cryoprotectant content in the mixed solution is 100%, stop pushing the cryoprotectant and let it stand for 0.5 min to allow the cryoprotectant and oocyte to be fully mixed; by linearly increasing the cryoprotectant concentration, it is possible to prevent the sudden concentration difference from causing osmotic shock to the oocyte and reduce the toxic damage caused by the cryoprotectant.

[0096] 2. Vitrification of oocytes

[0097] (1) Turn off the syringe pump and disconnect the syringe from the chip; use a hose to connect and seal the inlet of the solution inflow channel 11 and the outlet of the waste liquid discharge channel 17, and connect the inlets of the two cell channels 16;

[0098] (2) Place the sealed microfluidic chip directly into liquid nitrogen for cryopreservation. Figure 5 As shown in .

[0099] 3. Thawing of oocytes and removal of cryoprotectants

[0100] (1) Preheat the rewarming solution (1 mol / L trehalose + cell culture medium M2) to 37°C in a 37°C water bath;

[0101] (2) Take out the microfluidic chip from liquid nitrogen and place it in a 37°C water bath for 1 min;

[0102] (3) The microfluidic chip is taken out, wiped clean, and then syringes containing a rewarming solution and a buffer solution (cell culture medium M2) are connected to the solution inflow channel 11 through silicone hoses. The syringes are controlled by a syringe pump, wherein the rewarming solution syringe is connected to one of the solution inflow channels 11, and the buffer solution is connected to the remaining solution inflow channel 11;

[0103] (4) First push the syringe containing the rewarming solution to fill the channel with the rewarming solution so that the oocyte is fully exposed to the rewarming solution;

[0104] (5) After the oocytes and the rewarming solution are fully mixed, the syringes containing the rewarming solution and the buffer solution are pushed simultaneously, and the flow rate of the rewarming solution is controlled to slowly decrease from 10 μL / min to 0, and the flow rate of the buffer solution is slowly increased from 0 to 10 μL / min; in the process of changing the flow rate, the rewarming solution and the buffer solution are completely mixed in the serpentine mixing channel, and the content of the rewarming solution in the mixed solution is linearly reduced from 100% to 0%;

[0105] (6) When the buffer solution content reaches 100%, stop pushing the buffer solution and let it stand for 0.5 min to allow the buffer solution, i.e., the cell culture medium M2, to be fully mixed with the oocytes. During this process, the concentration of the rewarming solution decreases linearly, which can prevent the sudden concentration difference from causing osmotic shock to the oocytes, and the cryoprotectant is completely removed.

[0106] (7) Use a syringe connected to a silicone hose to suck out the rewarmed oocytes through the cell channel 16, place them in a cell culture dish containing cell culture medium M2, and place them in a cell culture incubator for recovery and culture.

[0107] Comparative Example

[0108] This comparative example provides a conventional vitrification cryopreservation method for oocytes, and the specific steps are as follows:

[0109] 1. Conventional vitrification of oocytes

[0110] (1) placing the retrieved oocytes in a buffer solution (cell culture medium M2);

[0111] (2) Move the oocytes in the buffer solution to the equilibrated solution (7.5% ethylene glycol + 7.5% propylene glycol + cell culture medium M2) for 5-10 minutes;

[0112] (3) Move the oocytes in the equilibrated solution into the vitrification solution (15% ethylene glycol + 15% propylene glycol + cell culture medium M2 + 0.5 mol / L trehalose) for 1 min;

[0113] (4) The oocytes loaded with cryoprotectant are then quickly transferred to a cryocarrier (such as a freezing straw or cryotop), and the cryocarrier containing the oocytes is placed in liquid nitrogen to complete conventional vitrification cryopreservation of the oocytes.

[0114] 2. Routine rewarming of oocytes

[0115] (1) Prepare rewarming solution (1 mol / L trehalose + cell culture medium M2), dilution solution 1 (0.5 mol / L trehalose + cell culture medium M2), dilution solution 2 (0.25 mol / L trehalose + cell culture medium M2), and buffer solution and heat them to 37°C;

[0116] (2) Take the cryocarrier containing the oocytes out of liquid nitrogen and quickly place it in a 37°C water bath for 10-20 seconds;

[0117] (3) Move the oocytes in the cryocarrier to the rewarming solution and incubate at 37°C for 1 minute;

[0118] (4) Move the oocytes in the rewarming solution to the dilution solution 1 and incubate at 37°C for 3 minutes;

[0119] (5) Move the oocytes in dilution solution 2 to dilution solution 2 and incubate at 37°C for 3 minutes;

[0120] (6) The oocytes in dilution solution 2 are transferred to the buffer solution and placed in a cell culture incubator for culture to complete routine rewarming of the oocytes.

[0121] in, Figure 6 Figure 4 shows the optical microscope and fluorescence microscope images of the cells after vitrification of oocytes by the microfluidic chip in Example 4 and conventional vitrification in the comparative example. Figure 6 It can be clearly seen from the imaging results that the survival rate of oocytes after vitrification and cryopreservation using the microfluidic chip in the embodiment of the present invention is significantly higher than that of conventional vitrification in the comparative example (wherein green represents living cells and red represents dead cells).

[0122] It can be known from the above embodiments and comparative examples that the microfluidic chip in the present invention can realize the integrated operation of adding cryoprotectant, cryopreservation, rewarming and cryoprotectant removal in the vitrification and freezing preservation of oocytes or embryos, and the step is simple compared to conventional vitrification and freezing preservation, and easy to operate; the vitrification and freezing preservation of multiple oocytes or embryos and rewarming etc. can be stored and operated simultaneously, and the cryopreservation and rewarming efficiency of oocytes or embryos are significantly improved. During the whole operation process, oocytes or embryos are always in the microfluidic chip, and the pollution caused by the external environment or personnel operation can be greatly avoided. The survival rate after the rewarming of oocytes or embryos can be significantly improved by the microfluidic chip in the present invention, and the development of assisted reproductive technology can be further promoted.

[0123] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0124] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A microfluidic chip for vitrification and cryopreservation of oocytes or embryos, comprising a microfluidic channel layer, characterized in that: According to the flow direction of the microfluid, the microfluidic channel layer includes two or more solution inflow channels, a serpentine mixing channel, and two or more shunt channels that are connected in sequence, and each shunt channel is connected in series with a serpentine channel, a cell storage chamber, and a waste liquid discharge channel; Wherein, a first micro-column array is arranged at the outlet of the cell storage chamber, and a second micro-column array is arranged in the channel outside the inlet thereof; a cell channel is connected between the inlet of the cell storage chamber and the second micro-column array.

2. The microfluidic chip for vitrification and cryopreservation of oocytes or embryos as claimed in claim 1, characterized in that: The solution inflow channel has two outlets, and the outlets thereof are connected with the inlet of the serpentine mixing channel after merging.

3. The microfluidic chip for vitrification and cryopreservation of oocytes or embryos as claimed in claim 2, characterized in that: The outlets of the solution inflow channels merge at an obtuse angle.

4. The microfluidic chip for vitrification and cryopreservation of oocytes or embryos as claimed in claim 1, characterized in that: There are two diversion channels.

5. The microfluidic chip for vitrification and cryopreservation of oocytes or embryos as claimed in claim 1, characterized in that: The cell storage chamber is a cylindrical chamber.

6. The microfluidic chip for vitrification and cryopreservation of oocytes or embryos as claimed in claim 1, characterized in that: The first micro-column array and the second micro-column array are respectively formed by a plurality of micro-columns arranged at intervals.

7. The microfluidic chip for vitrification and cryopreservation of oocytes or embryos according to claim 1, characterized in that: The outlet of the waste liquid discharge channel is connected with a serpentine channel.

8. A microfluidic chip for vitrification and cryopreservation of oocytes or embryos as described in any one of claims 1 to 7, characterized in that: The microfluidic chip also includes a base layer, and the base layer is made of a transparent alumina ceramic material.

9. A method for preparing the microfluidic chip according to any one of claims 1 to 8, characterized in that: The following steps are involved: providing a microfluidic channel layer and a substrate layer; The substrate layer is immersed in (3-mercaptopropyl)trimethoxysilane for 1-5 minutes for surface modification; The surface-modified substrate layer is cleaned and then assembled and bonded with the microfluidic channel layer to obtain a microfluidic chip.

10. Use of the microfluidic chip according to any one of claims 1 to 8 in vitrification cryopreservation of oocytes or embryos.

11. A method for vitrification cryopreservation of oocytes or embryos based on the microfluidic chip according to any one of claims 1 to 8, characterized in that: The process includes: a: Adding cryoprotectant: injecting cryoprotectant and buffer solution into the microfluidic chip through the solution inflow channel, setting the injection speed of cryoprotectant and buffer solution, and realizing linear addition of cryoprotectant to oocytes or embryos; b: The microfluidic chip containing the oocyte or embryo is directly placed in liquid nitrogen to achieve vitrification and cryopreservation of the oocyte or embryo; c: Take out the cryopreserved microfluidic chip and place it in a 37°C water bath to rewarm the vitrified oocytes or embryos; d: Removal of cryoprotectant: Inject rewarming solution and buffer solution into the chip through the solution inflow channel, set the injection speed of the rewarming solution and buffer solution so that the rewarming solution decreases linearly to achieve linear removal of cryoprotectant, and finally aspirate the oocyte or embryo out of the cell channel of the microfluidic chip.

12. The method according to claim 11, characterized in that In step a, the process of adding a cryoprotectant comprises the following steps: Connecting the solution inflow channel to a sample injection device, injecting the cryoprotectant and the buffer solution into the microfluidic chip through the sample injection device, and driving the flow of the solution in the chip; The injection speeds of the cryoprotectant and the buffer solution are respectively set so that the concentration of the cryoprotectant in the mixed solution increases linearly; When the cryoprotectant is completely added, stop injecting the solution; let it stand to ensure that the oocytes or embryos are completely mixed with the cryoprotectant.

13. The method according to claim 11, characterized in that In step c and step d, the process of thawing the oocyte or embryo after vitrification and removing the cryoprotectant comprises the following steps: Preheat the rewarming solution and buffer solution at 37°C; The cryopreserved microfluidic chip was taken out and quickly placed in a 37°C water bath for rewarming; Injecting a rewarming solution and a buffer solution into the chip through the solution inflow channel, and setting the injection speed of the rewarming solution and the buffer solution so that the concentration of the rewarming solution in the mixed solution decreases linearly; When the buffer solution content in the mixed solution is 100%, stop injecting the solution; let it stand to ensure that the oocyte or embryo is completely mixed with the buffer solution, and complete the removal of the cryoprotectant for the oocyte or embryo; After the oocytes or embryos are aspirated, they are placed in cell culture medium and cultured in a cell culture incubator.

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