Microfluidic device for magnetic functionalized cell hydrogel microsphere immobilization and cryopreservation

Abstract

The application discloses a microfluidic device for magnetic functionalized cell hydrogel microsphere fixation and cryopreservation, and belongs to the technical field of cell cryopreservation. The microfluidic device comprises a substrate layer provided with a cavity with a recess, the bottom of the cavity is provided with a receiving part, and a magnetic part is arranged in the receiving part; according to the microfluid flow direction, the microfluid channel layer comprises two inlet channels, a serpentine mixing channel, a perfusion chamber and an outlet channel which are sequentially communicated; the bottom of the perfusion chamber is provided with a plurality of recesses, and the recesses are used for fixing the magnetic functionalized cell hydrogel microspheres to be cryopreserved; and the microfluidic device further comprises a top layer and a sealing cover used for sealing the perfusion chamber. The microfluidic device can well realize the fixation and cryopreservation of the magnetic functionalized cell hydrogel microspheres, and can conveniently realize the loading or removal of cells, improve the operation convenience and the cell survival rate.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell cryopreservation, and particularly relates to a microfluidic device for magnetic functionalization of cell hydrogel microspheres and cryopreservation. BACKGROUND

[0002] Cell cryopreservation is a key technology in biological sample banks, regenerative medicine and cell therapy, and its goal is to maintain the activity and function of cells in a super-low temperature environment for a long time. As the current mainstream technology, vitrification cryopreservation rapidly cools cells to a glass state, thereby avoiding the formation of ice crystals. However, the traditional vitrification cryopreservation method relies on high concentrations (usually 4-6M) of cryoprotective agents (CPA). These CPAs can effectively inhibit the generation of ice crystals, but they also have significant cytotoxicity, and during the loading and removal process, they can cause strong osmotic stress, resulting in a dramatic change in cell volume, ultimately causing cell membrane damage, organelle dysfunction, and even cell death, which severely restricts the survival rate of cells after cryopreservation.

[0003] In order to overcome the osmotic shock caused by the one-step or multi-step addition of CPA in the traditional method, microfluidic technology has emerged. This technology can achieve precise and gradual control of the concentration of CPA, allowing cells to gradually adapt to a continuously changing concentration gradient, thereby minimizing osmotic damage and improving cell survival rate. Although microfluidic technology has shown great potential in CPA loading control, it still faces a series of key technical bottlenecks and defects that have not been overcome in practical applications, especially in conventional laboratory environments. Specifically, (1) insufficient physical protection of cells in microfluidic processes, which are prone to mechanical damage: in microfluidic channels, cells are directly exposed to fluid shear force. Especially during solution exchange and sample recovery, collisions, friction between cells and channel walls, and fluid suction can cause irreversible mechanical damage to the fragile cell membrane and internal structure. Especially for some cells with large volume and sensitive to mechanical stress (such as oocytes), this design without physical buffering will reduce their survival rate in the microfluidic system. (2) Low efficiency of cell loading and recovery: due to the design of the inlet and outlet of traditional microfluidic chips, which is not optimized for efficient and non-destructive loading and recovery of single or small amounts of valuable cells (such as oocytes); cells are prone to block the microchannel during loading, and it is also difficult to recover the treated cells from the chip completely and efficiently after the experiment, resulting in low recovery rate.

[0004] In general, existing microfluidic cryopreservation schemes have deficiencies in convenience, reliability and repeatability in actual operation. SUMMARY

[0005] Therefore, the primary purpose of the present application is to provide a microfluidic device for fixing and cryopreserving magnetically functionalized cell hydrogel microspheres, which facilitates the fixing and cryopreserving of magnetically functionalized cell hydrogel microspheres, cooperatively solves the problems of complicated operation and low cell survival rate, and promotes the development of cell freezing technology.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] One aspect of the present application discloses a microfluidic device for fixing and cryopreserving magnetically functionalized cell hydrogel microspheres, comprising:

[0008] a substrate layer provided with a recessed chamber, the bottom of the chamber is provided with a receiving part, and the receiving part is provided with a magnetic member;

[0009] a microfluidic channel layer, according to the microfluidic flow direction, the microfluidic channel layer comprises two inlet channels, a serpentine mixing channel, a perfusion chamber and an outlet channel which are sequentially communicated; the bottom of the perfusion chamber is provided with a plurality of recesses, and the recesses are used for fixing magnetically functionalized cell hydrogel microspheres to be cryopreserved;

[0010] a top layer provided with an inlet through hole corresponding to the inlet channel, an outlet through hole corresponding to the outlet channel, and a hollow part corresponding to the perfusion chamber;

[0011] and a sealing cover, the sealing cover is matched with the hollow part, and is used for sealing the perfusion chamber;

[0012] Wherein, the microfluidic channel layer and the top layer are sequentially bonded and received in the chamber, and the perfusion chamber corresponds to the receiving part.

[0013] Another aspect of the present application discloses a method for fixing and cryopreserving magnetically functionalized cell hydrogel microspheres, which is realized based on the microfluidic device described in the present application, and the method comprises the following steps:

[0014] providing the microfluidic device described in the present application;

[0015] pouring cell culture medium into the microfluidic device with the sealing cover, so that the cell culture medium fills the entire perfusion chamber and the recesses, and is discharged through the outlet channel;

[0016] opening the sealing cover, placing the prepared magnetically functionalized cell hydrogel microspheres in the recesses, and then tightly closing the sealing cover;

[0017] The perfusion chamber is perfused with a cryoprotective agent and a cell culture medium through two inlet channels respectively, one of which perfuses the cryoprotective agent and the other of which perfuses the cell culture medium, and linear addition of the cryoprotective agent is realized by adjusting the injection speed of the cryoprotective agent and the cell culture medium; after the cryoprotective agent addition is completed, the injection of the solution is stopped, and the inlet channel and the outlet channel are sealed;

[0018] The microfluidic device containing the magnetic functionalized cell hydrogel microspheres is cryopreserved.

[0019] The beneficial effects of the present application are:

[0020] The microfluidic device of the present application realizes convenient operation of cell cryopreservation and improves the survival rate of cells by optimizing the structure of the substrate layer, the microfluidic channel layer and the top layer. Specifically, a chamber is arranged in the substrate layer, and a magnetic part mounting space is arranged at the bottom of the chamber, and a plurality of recesses are arranged in the perfusion chamber of the microfluidic channel layer, the perfusion chamber corresponds to the magnetic part, and the size of the magnetic functionalized cell hydrogel microspheres to be cryopreserved is similar to that of the recesses, so that the magnetic functionalized cell hydrogel microspheres to be cryopreserved can be well fixed in the recesses under the cooperation of the recesses and the magnetic part, which is not only convenient to operate, but also facilitates related research during the cryopreservation process. In addition, a sealing cover that can be quickly opened and closed is arranged on the top layer, thereby solving the problem of difficult cell loading and recovery in the existing microfluidic device, and realizing lossless and efficient recovery of encapsulated cells. In this closed and controllable microfluidic environment, the chip can accurately generate a stable cryoprotective agent concentration gradient, so that the cells in the microspheres can complete progressive osmotic adaptation under mild conditions, thereby minimizing osmotic stress damage.

[0021] Based on the microfluidic device in the present application, the present application further proposes a cryopreservation method, which encapsulates cells by using magnetic hydrogel microspheres, provides comprehensive physical protection for the cells, and introduces biocompatible magnetic nanoparticles to endow the cells with magnetic response characteristics, so that it is possible to easily, quickly and losslessly transfer, position and recover a large number of encapsulated cells by using an external magnetic field, and the fluid shear damage to individual fragile cells caused by traditional pipetting operation is avoided, thereby laying a solid foundation for realizing high-throughput parallel processing.

[0022] In general, the magnetic hydrogel encapsulation strategy is combined with a specially designed device in the present application, thereby constructing an efficient, mild and simple-to-operate cell cryopreservation platform, which solves the problems of complicated operation and low cell survival rate in traditional technologies. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Fig. 1 is a schematic diagram of the overall structure of the microfluidic device 100 in a preferred embodiment of the present application.

[0024] Figure 2 For Figure 1 Structural exploded view of the microfluidic device 100.

[0025] Figure 3 For Figure 2 Structural view of the substrate layer 10.

[0026] Figure 4 For Figure 2 Structural view of the microfluidic channel layer 20.

[0027] Figure 5 For Figure 2 Structural view of the top layer 30.

[0028] Figure 6 For Figure 2 Structural view of the sealing cover 40.

[0029] In the figure: 10-substrate layer, 11-chamber, 12-receiving part; 20-microfluidic channel layer, 21-inlet channel, 22-snake-shaped mixing channel, 23-perfusion chamber, 24-outlet channel, 25-recess; 30-top layer, 31-inlet through hole, 32-outlet through hole, 33-hollow part; 40-sealing cover, 41- boss. DETAILED DESCRIPTION

[0030] The present application will be further illustrated below with specific examples. It should be noted that the following specific examples are for illustrative purposes only, and do not limit the scope of the present application in any way.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0032] In addition, unless otherwise specified, the methods without specifically recorded conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial channels.

[0033] Example 1 Microfluidic device

[0034] A microfluidic device for magnetic functionalized cell hydrogel microsphere fixation and cryopreservation is provided in this embodiment, and the structure of the microfluidic device is as shown in Figure 1 and Figure 2 The microfluidic device includes a substrate layer 10, a microfluidic channel layer 20, a top layer 30, and a sealing cover 40, wherein the microfluidic channel layer 20 and the top layer 30 are sequentially bonded and placed in the substrate layer 10.

[0035] Referring to Figure 3 , the substrate layer 10 is provided with a recessed cavity 11, which is used to accommodate the microfluidic channel layer 20 and the top layer 30, so the sum of the thicknesses of the microfluidic channel layer 20 and the top layer 30 is the same as the depth of the cavity 11. A receiving part 12 is provided at the bottom of the cavity 11, which is a groove for placing a magnetic part. In this application, the magnetic part refers to a magnet or a magnet, or any other component with magnetism.

[0036] Referring to Figure 4 , the microfluidic channel layer 20 is configured as a channel for microfluidic flow, which includes two inlet channels 21, a serpentine mixing channel 22, a perfusion chamber 23 and an outlet channel 24 according to the flow direction of the microfluidic flow. Specifically, the two inlet channels 21 communicate with the inlet of the serpentine mixing channel 22 after converging at the outlet; the outlet of the serpentine mixing channel 22 communicates with the inlet of the perfusion chamber 23, and the outlet of the perfusion chamber 23 communicates with the inlet of the outlet channel 24; as a preferred example, the outlets of the inlet channels 21 converge at a certain angle, and more preferably, the outlets of the inlet channels 21 converge at an obtuse angle; wherein the arrangement of the serpentine mixing channel 22 can ensure that the cryoprotective agent and the cell culture medium can be fully mixed and uniform before reaching the perfusion chamber 23; the number of bends of the serpentine mixing channel 22 can be designed according to needs, without particular limitation. Further, a plurality of recesses 25 are provided at the bottom of the perfusion chamber 23, wherein "a plurality of" refers to one or more than one, for example, one, two, three or four, etc., and the specific number can be set according to needs. These recesses 25 are used to fix the magnetically functionalized cell hydrogel microspheres, and therefore, preferably, the size of the recesses 25 is adapted to the microspheres. In this application, the perfusion chamber 23 corresponds to the receiving part 12, so that the magnetically functionalized cell hydrogel microspheres located in the recesses 25 are better fixed.

[0037] Referring to Figure 5 , a plurality of through holes are provided on the top layer 30, which are located corresponding to the positions of the inlet channels 21 and the outlet channels 24 in the microfluidic channel layer 20, specifically, the inlet through hole 31 and the outlet through hole 32, the position of the inlet through hole 31 corresponds to the position of the inlet channel 21, and the position of the outlet through hole 32 corresponds to the position of the outlet channel 24. A hollow part 33 is provided on the top layer 30, which is located corresponding to the position of the perfusion chamber 23, so as to facilitate the placement and removal of the magnetically functionalized cell hydrogel microspheres in the perfusion chamber 23.

[0038] Further, the sealing cover 40 is configured to cover the hollow part 33 of the top layer, thereby achieving the sealing of the perfusion chamber 23. Specifically, the structure can be designed as needed, as long as the purpose of sealing the perfusion chamber 23 can be achieved. In the present embodiment, please refer to Figure 5 and Figure 6 , the hollow part 33 is configured as a recessed step, wherein the middle part of the step platform is hollow, and the hollow part corresponds to the position of the perfusion chamber 23. The lower surface of the sealing cover 40 is provided with a boss 41, which is matched with the hollow part of the step platform. The whole sealing cover 40 is embedded in the step platform, and the boss 41 fills the hollow part, thereby achieving the sealing of the perfusion chamber 23. Further, as a preferred embodiment, a groove is formed around the side wall of the boss 41, and a sealing ring is sleeved in the groove, thereby achieving better sealing effect.

[0039] For the material of the microfluidic device in the present application, a material with good biocompatibility and transparency known in the art or independently developed can be selected. As a preferred example, in the present embodiment, the material of the microfluidic channel layer 20 and the top layer 30 is PDMS (polydimethylsiloxane), which has the characteristics of transparency, elasticity, chemical inertness and good biocompatibility. The bonding method of the PDMS chip is various, simple to operate, and can realize reversible bonding by simple physical action, or irreversible bonding by chemical modification method, so that multiple same microchannel fluid layers can be completed quickly and conveniently. Further, in the present application, the substrate layer 10 and the sealing cover 40 also use a material with good biocompatibility and high hardness, thereby achieving better sealing. As a preferred example, PMMA (polymethyl methacrylate) material is used.

[0040] In addition, for the specific size of the microfluidic chip (such as the length, width and height of the microfluidic channel, the depth of the perfusion chamber and the size of the recess, etc.), corresponding optimization design can be made according to the actual cells to be cryopreserved, and those skilled in the art have such ability, so there is no special limitation.

[0041] Example 2: Fabrication of microfluidic device

[0042] In the present embodiment, a specific example of the fabrication of a microfluidic device is provided. In the microfluidic device, the width of the microfluidic channel in the microfluidic channel layer 20 is 0.4 mm, and the height is 0.4 mm; the length of the perfusion chamber 23 is 8 mm, the width is 4 mm, and the depth is 1 mm; the diameter of the inlet microcolumn of the inlet channel 21 is 2 mm. In the substrate layer 10, the length of the chamber 11 is 50 mm, the width is 20 mm, and the depth is 3 mm; the length of the receiving part 12 is 10 mm, the width is 10 mm, and the depth is 2 mm. Among them, the microfluidic channel layer 20 and the top layer 30 are made by PDMS inverse molding, and the mold is carved from PMMA.

[0043] The specific manufacturing process is as follows:

[0044] 2.1 Substrate layer 10

[0045] The substrate layer 10 is directly carved from PMMA, with a 3mm deep cavity 11 for placing the entire microfluidic chip. A groove-shaped receiving part 12 is excavated at the bottom of the cavity 11, with dimensions of 10mm x 10mm x 2mm, for placing a magnet.

[0046] 2.2 Microfluidic chip (microfluidic channel layer 20 and top layer 30)

[0047] 2.2.1 PMMA mold processing

[0048] (1) Microfluidic channel layer 20 mold carving: first select a PMMA plate of appropriate size and thickness, carve a microfluidic channel of corresponding size, with a channel design height of 0.4mm and a perfusion chamber 23 height of 0.4mm. There are four protruding blocks on the perfusion chamber 23, with the same size as the designed hydrogel microsphere recess 25, with dimensions of 0.6mm x 0.6mm x 0.2mm. After reverse molding, the corresponding perfusion chamber 23 and recess 25 positions are formed, with a mold overall depth design of 1.6mm. At the same time, two microcolumns with a diameter of 2mm are designed as the inlet of the inlet channel 21 and the perfusion chamber 23, with a height design of 1.4mm and a mold overall depth design of 1.4mm. After the PMMA mold is completed, it is ready for use.

[0049] (2) Top layer 30 mold processing: the size of the top layer 30 is designed to be 50mm long and 20mm wide. The upper mold thickness is 1.6mm thick, with two different size rectangular grooves in the center. The upper part of the rectangular groove is 14mm long, 12mm wide, and 0.6mm deep. The lower part of the rectangular groove is 8mm long, 4mm wide, and 1mm thick, which is just the same size as the position of the perfusion chamber 23 in the microfluidic channel layer 20, and just penetrates through the entire upper layer of the chip.

[0050] 2.2.2 Microfluidic chip manufacturing

[0051] The pre-polymer and curing agent of PDMS are mixed in a mass ratio of 10:1, poured into the two templates of the upper and lower layers of the chip (PMMA mold made in 2.2.1), and placed in a desiccator to extract vacuum to remove bubbles. Then put it in a 70℃ oven for 2 hours to make the PDMS fully cured. The cured PDMS layer is carefully peeled off from the PMMA mold, and the chip microfluidic channel layer 20 and top layer 30 are cut out with a scalpel.

[0052] The obtained microfluidic channel layer 20 and top layer 30 are bonded to form a complete microfluidic chip. In this embodiment, the oxygen plasma bonding technology is used, and the specific steps are as follows: the upper and lower chip surfaces of PDMS are treated in a plasma cleaning instrument for 90 seconds. By changing the surface properties of the contact piece, both sides have strong hydrophilic effect, and after mutual contact, O-Si-O covalent bond is formed, thereby realizing irreversible bonding. The channel depth and width of the finally obtained microfluidic chip are both 0.4 mm, and the size of the perfusion chamber 23 formed is 8 mm long, 4 mm wide, and 1.4 mm deep.

[0053] 2.3 Sealing cover 40

[0054] The sealing cover 40 is integrally formed by engraving from polymethyl methacrylate (PMMA), and the main structure includes a boss 41 (size: 8 mm long x 4 mm wide x 0.6 mm deep) matched with the perfusion chamber 23. The outer edge of the boss 41 is provided with an annular sealing groove (14 mm long x 12 mm wide x 0.6 mm deep), which is buckled with the edge of the perfusion chamber 23 to realize the airtight sealing of the perfusion chamber 23.

[0055] In the operation of the microfluidic device in this application, only the sealing cover 40 needs to be opened, thereby realizing the convenience of cell loading and recovery, avoiding complex pipeline connection and cell suspension injection operation, and the risk of cell blockage caused thereby. After the treatment is completed, the encapsulated cells can be recovered efficiently and non-destructively by opening the sealing cover 40 and using a magnetic field. In this closed and controllable microfluidic environment, a stable cryoprotective agent concentration gradient can be accurately generated, so that the encapsulated cells can complete progressive osmotic adaptation under mild conditions, realize the integration and automation of linear addition / removal of cryoprotective agents, freezing and recovery, and the permeability of cryoprotective agents to cryopreserved cells.

[0056] Example 3 Cryopreservation of cells

[0057] In this embodiment, oocytes are taken as examples to illustrate the method for cryopreservation of cells by the microfluidic device in this application. The preparation of magnetically functionalized oocyte hydrogel microspheres can refer to the literature Tian C, Shen L, Gong C, et al. Microencapsulation and nanowarming enables vitrification cryopreservation of mouse preantral follicles [J]. Nature Communications [2025-11-07]. DOI: 10.1038 / s41467-022-34549-2.

[0058] The formula of the cryoprotectant used is 15% ethylene glycol + 15% propylene glycol + cell culture medium M2 + 0.5 mol / L trehalose, and the buffer solution used is cell culture medium M2.

[0059] The specific steps are as follows:

[0060] (1) Place the magnet in the accommodation part 12 of the substrate layer 10, and then place the bonded double-layer microfluidic chip (including the microfluidic channel layer 20 and the top layer 30) above. Prepare the magnetic oocyte hydrogel microspheres, open the sealing cover 40, and use the pipette gun to inject the microspheres into the recess 25 at the bottom of the perfusion chamber 23. Due to the action of the magnetic field below, the microspheres can be well fixed in the recess 25. Then, the sealing cover 40 is covered on the perfusion chamber 23 to seal the perfusion chamber 23, which is convenient for subsequent operation. Before starting, the microfluidic device with the sealed cover 40 needs to be perfused with cell culture medium, so that the culture medium fills the entire perfusion chamber 23 and the recess 25, and the air in the device is exhausted to prevent the influence of air bubbles in the subsequent experiment.

[0061] (2) Two micro-injection pumps are respectively filled with isotonic solution (M2 medium) and hypertonic solution (cryoprotectant). When the isotonic solution is introduced, the cell volume in the microspheres can be used as the initial volume of the cells. When the hypertonic solution is gradually introduced, the cell volume in the microspheres changes due to the increase of osmotic pressure. The volume change data of the cells in the microspheres can be dynamically monitored and recorded by a high-speed camera. Specifically, open the injection pump controlling the isotonic solution, adjust the rate to 15 μL / min, and introduce the isotonic solution for 3 minutes. Then, open the injection pump controlling the hypertonic solution, gradually adjust the flow rate ratio of the isotonic solution and the hypertonic solution, and control the total flow rate at 15 μL / min. Until the flow rate of the hypertonic solution reaches 15 μL / min and the flow rate of the isotonic solution decreases to 0 μL / min, the whole process of perfusing the cryoprotectant lasts for 5 min. After the process of loading the cryoprotectant into the oocyte microspheres is completed, the volume change of the oocytes is recorded by the high-speed camera, and the membrane permeability of the oocytes can be studied.

[0062] (3) After the perfusion of the cryoprotectant is completed, remove all the hoses of the injection pumps, and seal and fix the inlet through hole 31 and the outlet through hole 32 of the top layer 30 using adhesive tape. After sealing and fixing, the whole microfluidic device can be placed in liquid nitrogen for cryopreservation of oocytes.

[0063] (4) After the freezing and preservation of the mature oocyte, the chip is taken out of the liquid nitrogen and placed in a 37°C water bath for thawing. After thawing, the tape used for fixation and sealing is removed, and the syringe pump is connected to the chip, and the process of oocyte cryoprotectant removal can be carried out. Then the chip is taken out, the upper cover is opened, the oocyte microspheres in the trap are taken out with a pipette, and the oocyte hydrogel microspheres are taken out and placed in sodium citrate for decrosslinking. After decrosslinking, the oocytes are taken out and placed in a cell incubator for culture, completing the cryoprotectant removal and decrosslinking of the oocytes or embryos, and then the next step of research is carried out.

[0064] The application introduces a magnetic material hydrogel to package cells, and cooperates with a corresponding microfluidic device to achieve good fixation of cells to be frozen, convenient cell loading and removal, improved cell recovery efficiency, and guaranteed cell survival rate.

[0065] It can be understood that the oocyte is taken as an example in the embodiments of the application, but the cell cryopreservation strategy in the application is applicable to any cell having a cryopreservation requirement in the art, including but not limited to oocytes, embryos, red blood cells, stem cells, etc. For different frozen cells, those skilled in the art can adjust the size of the microfluidic device according to the needs, and those skilled in the art have such ability, so there is no special limitation.

[0066] It should be noted that the application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and embodiments having the same technical idea and playing the same role and effect within the scope of the technical solutions of the application are all included in the technical scope of the application. In addition, within the scope of the main idea of the application, various modifications that can be thought of by those skilled in the art are applied to the embodiments, and other ways constructed by combining part of the constituent elements in the embodiments are also included in the scope of the application.

Claims

1. A microfluidic device, characterized in that, The microfluidic device comprises: a substrate layer provided with a concave chamber, the bottom of the chamber is provided with a receiving part, and the receiving part is provided with a magnetic member; a microfluidic channel layer, according to the microfluidic flow direction, the microfluidic channel layer comprises two inlet channels, a serpentine mixing channel, a perfusion chamber and an outlet channel which are sequentially communicated; the bottom of the perfusion chamber is provided with a plurality of recesses for fixing the magnetic functionalized cell hydrogel microspheres to be cryopreserved; a top layer provided with an inlet through hole corresponding to the inlet channel, an outlet through hole corresponding to the outlet channel, and a hollow part corresponding to the perfusion chamber; and a sealing cover which is matched with the hollow part and used for sealing the perfusion chamber. The microfluidic channel layer and the top layer are sequentially bonded and received in the chamber, and the perfusion chamber corresponds to the receiving part.

2. The microfluidic device of claim 1, wherein, The material of the microfluidic channel layer and the top layer is PDMS; and / or the material of the substrate layer and the sealing cover is PMMA.

3. The microfluidic device of claim 1, wherein, The outlets of the inlet channels are connected to the inlet of the serpentine mixing channel.

4. The microfluidic device of claim 3, wherein, The outlets of the inlet channels are connected at an obtuse angle.

5. The microfluidic device of claim 1, wherein, The sum of the thicknesses of the microfluidic channel layer and the top layer is consistent with the depth of the chamber in the substrate layer.

6. The microfluidic device of claim 1, wherein, The diameter of the inlet microcolumn of the inlet channel is 2 mm.

7. The microfluidic device of claim 1, wherein, The inner surface of the sealing cover is provided with a boss corresponding to the hollow part, and a groove is formed around the side wall of the boss, and a sealing ring is sleeved in the groove.

8. A method for the immobilization and cryopreservation of magnetically functionalized cell hydrogel microspheres, based on the microfluidic device of any one of claims 1-7, characterized in that, The method comprises the following steps: providing the microfluidic device according to any one of claims 1-7; injecting cell culture medium into the microfluidic device with the sealing cover, so that the cell culture medium fills the entire perfusion chamber and the recesses, and is discharged through the outlet channel; opening the sealing cover, placing the prepared magnetic functionalized cell hydrogel microspheres in the recesses, and then tightly closing the sealing cover; injecting cryoprotective agent and cell culture medium into the perfusion chamber through the two inlet channels, and adjusting the injection speed of the cryoprotective agent and the cell culture medium to realize linear addition of the cryoprotective agent; after the addition of the cryoprotective agent is completed, stop injecting the solution, and seal the inlet channel and the outlet channel; freezing the microfluidic device containing the magnetic functionalized cell hydrogel microspheres.

9. The method of claim 8, wherein, The method further comprises the steps of thawing and removing the cryoprotective agent.

10. The method of claim 9, wherein, The thawing and cryoprotective agent removal comprises the following steps: preheating the thawing solution and the buffer solution at 37°C; taking out the cryopreserved microfluidic device and quickly placing it in a 37°C water bath for thawing; injecting the thawing solution and the buffer solution into the perfusion chamber through the two inlet channels, and adjusting the injection speed of the thawing solution and the buffer solution to linearly reduce the concentration of the thawing solution in the mixed solution; when the addition of the thawing solution is completed, stop injecting the solution; opening the sealing cover of the perfusion chamber, taking out the magnetic functionalized cell hydrogel microspheres and performing decrosslinking; after decrosslinking, taking out the cryopreserved cells and placing them in a cell incubator for culture, completing the removal of the cryoprotective agent and decrosslinking of the cells.

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