In-situ stress loading device and method for living cells in three-dimensional matrix material

Abstract

The invention discloses an in-situ stress loading device and method for living cells in a three-dimensional matrix material. The device comprises a tannic acid-N-isopropylacrylamide micro actuator, embedding a tannic acid-N-isopropylacrylamide micro actuator into the three-dimensional matrix hydrogel wrapping a large number of cells to prepare a micro hydrogel sample for in-situ stress loading; the tannic acid-N-isopropylacrylamide micro actuator is in a single-layer strip shape, a double-layer strip shape, a bent triangular shape or a notch circular ring shape in the three-dimensional matrix hydrogel, and the tannic acid-N-isopropylacrylamide micro actuator is deformed by controlling the temperature change so as to apply stress to cells around the tannic acid-N-isopropylacrylamide micro actuator. According to the living cell in-situ stress loading device and method in the three-dimensional matrix material, the tannic acid content can be used for quantitatively adjusting the actuating rate and mechanical property, and a repeated reversible driving effect is achieved; and the actuator does not need any external power supply or lead, so that the disturbance of the matrix hydrogel structure caused by the lead is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of long-time continuous imaging of cells under external forces, and particularly to a device and method for in-situ stress loading of living cells in a three-dimensional matrix material. Background Art

[0002] The changes in the behavior of living cells under mechanical stimuli are of great significance to biology and medicine. Cell behaviors such as cell growth, proliferation, apoptosis, and migration are all affected by mechanical factors. To study the regulation law of force signals on cell behaviors, overall stretching and compression or other forms of external force loading are often applied to the extracellular matrix material (such as hydrogel), so that the overall deformation of the matrix material drives the cells inside the matrix material to be stressed and deformed.

[0003] On the one hand, such methods will make the volume of the external force loading device relatively large, significantly larger than the sample size of the matrix material. On the other hand, it will cause the stress in the internal area of the matrix to be gradually attenuated in the direction towards the central area, making it difficult to apply a relatively significant stress to the cells at a specific local target position. In addition, the current cell stress loading device also requires a special observation and imaging device and cannot be adapted to a general small-stage incubator. Summary of the Invention

[0004] To solve the above problems, the present invention provides a device and method for in-situ stress loading of living cells in a three-dimensional matrix material, which can perform close-range stress loading on cells with a relatively delicate small-volume structure and without cumbersome peripheral devices.

[0005] To achieve the above object, the present invention provides a device for in-situ stress loading of living cells in a three-dimensional matrix material, including a tannic acid-N-isopropylacrylamide microactuator. The tannic acid-N-isopropylacrylamide microactuator is embedded in a three-dimensional matrix hydrogel containing a large number of cells to prepare a microhydrogel sample for in-situ stress loading;

[0006] The tannic acid-N-isopropylacrylamide microactuator is in a single-layer strip shape, a double-layer strip shape, a curved triangular shape, or a notched circular ring shape in the three-dimensional matrix hydrogel;

[0007] The environmental temperature is controlled to rise above the critical transition temperature of the tannic acid-N-isopropylacrylamide microactuator, so that the tannic acid-N-isopropylacrylamide microactuator deforms, thereby applying stress to the surrounding cells through the three-dimensional matrix hydrogel.

[0008] Preferably, the preparation of the tannic acid-N-isopropylacrylamide microactuator includes:

[0009] S1. Accurately measure 1% 1,4 - dioxane and 4% deionized water and place them in a beaker. After stirring evenly, set aside. Then, add 1 - 5% N - isopropylacrylamide and 1 - 2.5% tannic acid to the beaker in sequence. After fully dissolving, add 3 - 7% N,N'-methylenebisacrylamide and 3 - 7% 2 - hydroxy - 4′-(2 - hydroxyethoxy)-2 - methylpropiophenone powder to obtain a pre - polymerization solution;

[0010] S2. Inject the pre - polymerization solution into a mold, and prepare a gel through cross - linking reaction under ultraviolet light irradiation. After taking out the gel, soak it with deionized water to make the gel fully swell and remove unreacted substances, obtaining a tannic acid - N - isopropylacrylamide micro - actuator.

[0011] Preferably, by regulating the relative ratio of the amounts of substance of tannic acid and N - isopropylacrylamide, the strength of the tannic acid - N - isopropylacrylamide micro - actuator is ≥1 kPa, and the deformable range is ≥200 μm.

[0012] Preferably, in step S2, the distance between the ultraviolet lamp and the surface of the mold is 1 cm, and the polymerization temperature is 26°C.

[0013] Preferably, the micro - hydrogel sample is placed in an in - situ incubator on the stage for cultivation, and the cultivation temperature is set at 37°C; during the process of heating from room temperature to 37°C, the tannic acid - N - isopropylacrylamide micro - actuator shrinks and deforms, and the deformation process of the tannic acid - N - isopropylacrylamide micro - actuator and the stretching or contraction of cells can be observed in real - time through the objective lens below the in - situ incubator on the stage through the heat - insulating transparent film.

[0014] Preferably, the curved triangular tannic acid - N - isopropylacrylamide micro - actuator is formed by three strip - shaped tannic acid - N - isopropylacrylamide micro - actuators enclosing a curved - edge triangular closed area as a whole.

[0015] Preferably, the notched circular - ring - shaped tannic acid - N - isopropylacrylamide micro - actuator is formed by a strip - shaped tannic acid - N - isopropylacrylamide micro - actuator enclosing a circumference with a notch on one side.

[0016] Preferably, the critical transition temperature of the tannic acid - N - isopropylacrylamide micro - actuator is 32 - 33°C.

[0017] A method for in - situ stress loading of living cells in a three - dimensional matrix material is carried out through the above - mentioned device for in - situ stress loading of living cells in a three - dimensional matrix material.

[0018] Preferably, the mechanical properties of the three-dimensional matrix hydrogel are directly measured by a rotational rheometer; according to the mechanical parameter values of the tannic acid-N-isopropylacrylamide microactuator, the stress distributions within the current tannic acid-N-isopropylacrylamide microactuator and the matrix hydrogel are calculated through finite element simulation, and then the stress on the cells is obtained.

[0019] The present invention adopts the above-mentioned in-situ stress loading device and method for living cells in a three-dimensional matrix material, and has the following beneficial effects:

[0020] (1) The content of tannic acid in the tannic acid-N-isopropylacrylamide microactuator can quantitatively regulate its actuation rate and mechanical properties, and has a repeated and reversible driving effect;

[0021] (2) The tannic acid-N-isopropylacrylamide microactuator does not require any external power supply and has no wires, greatly avoiding the disturbance of the matrix hydrogel structure caused by wires;

[0022] (3) It can cause cells to migrate and grow along the direction of its contraction through the volume contraction of the actuator, and it is regulated by the geometric and mechanical characteristics of the microactuator; moreover, the expression level of intracellular F-actin related to migration can be regulated by the force loading parameters.

[0023] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0024] Figure 1 is the preparation process of the tannic acid-N-isopropylacrylamide microactuator in Embodiment 1 of the present invention;

[0025] Figure 2 is the schematic diagram of the embedding structure of the tannic acid-N-isopropylacrylamide microactuator in Embodiment 1 of the present invention;

[0026] Figure 3 is the deformation schematic diagram of the strip-shaped tannic acid-N-isopropylacrylamide microactuator in Embodiment 3 of the present invention;

[0027] Figure 4 is the deformation schematic diagram of the curved triangular tannic acid-N-isopropylacrylamide microactuator in Embodiment 4 of the present invention;

[0028] Figure 5 is the deformation schematic diagram of the notched circular-ring-shaped tannic acid-N-isopropylacrylamide microactuator in Embodiment 5 of the present invention;

[0029] Figure 6 is the deformation schematic diagram of the double-layer strip-shaped tannic acid-N-isopropylacrylamide microactuator in Embodiment 6 of the present invention;

[0030] Figure 7 Schematic structural diagram of the in-situ stress loading device of the present invention;

[0031] Figure 8 Schematic diagram of the 3D printing device of the present invention;

[0032] Figure 9 Complete deformation process of the TA-NIPAM gel strip during the test;

[0033] Figure 10 Graph of the change in the distance between points A and B of the TA-NIPAM actuator over time during the test;

[0034] Figure 11 Duration of deformation (shrinkage) of actuators with different TA contents during the test;

[0035] Figure 12 Morphology of the cells around the upper end of the side of the TA-NIPAM actuator after being loaded with stress after 24 hours of 3D culture.

[0036] Reference numerals

[0037] 1. Three-dimensional matrix hydrogel; 2. TA-NIPAM micro actuator; 3. Cells; 4. In-situ culture device for the stage; 5. Objective lens; 6. Print head; 7. Top-fixed frame; 8. Flexible soft film;

[0038] 21. First strip-shaped actuator; 22. Second strip-shaped actuator; 23. Third strip-shaped actuator; 24. Notched ring-shaped actuator; 25. Fourth strip-shaped actuator; 26. Fifth strip-shaped actuator; 41. Heat-insulating transparent film. Detailed implementation manners

[0039] In order to make the purpose, technical solutions and advantages of the embodiments disclosed in the present invention clearer and more understandable, the following further elaborates on the embodiments of the present invention in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not used to limit the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts fall within the scope of protection of this application. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions throughout.

[0040] It should be noted that the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or server that includes a series of steps or units does not necessarily have to be limited to those clearly listed steps or units, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0041] Like reference numerals and letters refer to like items in the following figures. Therefore, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

[0042] Example 1

[0043] The tannic acid-N-isopropylacrylamide microactuator has a preparation process as Figure 1 shown and includes:

[0044] (1) Preparation of the molding die during the experiment: The molding die consists of upper and lower layers of quartz glass plates and a polytetrafluoroethylene frame in the middle. The thickness of the polytetrafluoroethylene frame is 0.5 mm, and the inner frame size is 30 mm × 30 mm. The mold is fixed with clips around it, and a small opening is made on one side of the polytetrafluoroethylene frame for convenient injection of the pre-solution. Quartz glass with good light transmittance should be selected to achieve excellent photocrosslinking effect. In this example, quartz glass with a thickness of 1 mm and an ultraviolet light transmittance of 92% is used.

[0045] (2) Preparation process of the polymerization precursor solution: Use a pipette to accurately measure 0.2 mL of 1,4-dioxane and 0.8 mL of deionized water into a 10 mL beaker, stir evenly and set aside.

[0046] Add 226 mg of N-isopropylacrylamide (N-IPAM) and 12 mg of tannic acid (TA) to the beaker in sequence. After fully dissolving, add 5 mg of BIS and 5 mg of Irg2959 to obtain the polymerization precursor solution.

[0047] Among them, N-isopropylacrylamide is the main monomer for crosslinking. Tannic acid has strong ultraviolet light absorption ability and can play a role in improving the crosslinking strength. N,N'-methylenebisacrylamide (BIS, chemical formula C 12 H 20 N2O2) is used as a chemical crosslinking agent, and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irg2959, chemical formula C 12 H 16 O4) is used as a photoinitiator.

[0048] (3) Crosslinking process and treatment of the actuator sample: Use a syringe to inject the polymerization precursor solution into the mold and crosslink it under ultraviolet light (wavelength 365 nm, power 20 W) for 3 minutes. Note that the ultraviolet lamp is 1 cm away from the surface of the mold, and keep the polymerization temperature at about 26 °C.

[0049] After crosslinking is completed, the mold is removed, and the transparent gel is taken out. The crosslinked gel is soaked in deionized water (or distilled water). After soaking for 1 hour, it is rinsed twice and the water source is changed to clean water, and then changed every 4 hours. The sample is soaked for a total of 2 days. The purpose is to fully swell the gel and remove unreacted substances. The prepared gel actuator is stored in deionized water for standby and named tannic acid-N-isopropylacrylamide (TA-NIPAM) microactuator.

[0050] (4) After the experiment, the operator thoroughly washes hands and face.

[0051] The steps for constructing an in-situ stress loading device for living cells in a three-dimensional matrix material are as follows:

[0052] (1) Prepare the TA-NIPAM microactuator using the above steps, and characterize the material properties and mechanical properties of the TA-NIPAM microactuator. The mechanical property parameters and force-displacement curves of the TA-NIPAM microactuator can be measured by a soft material mechanical testing machine / biomechanical testing machine.

[0053] (2) Judge whether the strength of the TA-NIPAM microactuator reaches above 1 kPa; judge whether the deformable range of the TA-NIPAM microactuator exceeds 200 μm. If the above requirements are met, proceed to step (3). If the above requirements are not met, repeat step (1) and change the relative proportion of the amounts of substance of tannic acid and N-isopropylacrylamide to meet the above requirements.

[0054] (3) Use a slow-crosslinking hydrogel (complete crosslinking time is between 20 and 30 minutes) for three-dimensional cell culture. Cut the prepared TA-NIPAM microactuator into strips with dimensions of 20 mm × 5 mm × 0.5 mm, and embed it at a specific position (such as Figure 2 shown) inside the three-dimensional matrix hydrogel 1 wrapping a large number of cells 3, and perform external stress loading on the internal cells in a "close-range" non-direct contact manner. After the TA-NIPAM microactuator absorbs water, its thickness swells to more than 2 times, and the swelling growth in length and width is 20%-40% of the original.

[0055] (4) Place the micro-hydrogel sample wrapping the TA-NIPAM microactuator and cells in step (3) in an in-situ incubator on the stage for culture.

[0056] Example 2

[0057] A method for using an in-situ stress loading device for living cells in a three-dimensional matrix material includes the following steps:

[0058] (1) The TA-NIPAM microactuator 2 is in the shape of a strip with dimensions of 20 mm × 5 mm × 0.5 mm. After the TA-NIPAM microactuator absorbs water, its thickness swells to more than twice the original, and the swelling growth in length and width is 20%-40% of the original. As Figure 3 shown. During the process of heating the strip-shaped TA-NIPAM microactuator from room temperature to 37°C, shrinkage deformation occurs, causing the cells 3 on both sides of the TA-NIPAM microactuator to be stretched at the two ends of the strip, while the cells near the middle part of the strip are compressed.

[0059] (2) A microhydrogel sample is prepared using the strip-shaped TA-NIPAM microactuator. As Figure 7 shown, the three-dimensional matrix hydrogel 1 encapsulating the cells and the TA-NIPAM microactuator inside it are placed together on the stage in-situ incubator 4. The stage in-situ incubator can provide an accurate temperature and gas environment suitable for cell growth (the concentrations of carbon dioxide and oxygen can be set). The deformation process of the TA-NIPAM microactuator and the stretching or contraction of the cells beside it can be observed in real time through the objective lens 5 below the stage in-situ incubator through the heat-insulating transparent film 41. The cells used in this example are C2C12 cells.

[0060] (3) The mechanical properties of the three-dimensional matrix hydrogel are measured using a rotational rheometer; based on the mechanical parameter values of the TA-NIPAM microactuator, the stress distribution within the current TA-NIPAM microactuator and the matrix hydrogel is calculated through finite element simulation, and then the stress on the cells is obtained.

[0061] Example Three

[0062] A method for using a device for in-situ stress loading on living cells in a three-dimensional matrix material is the same as that in Example Two, except that the TA-NIPAM microactuator used is in the shape of a curved triangle, as Figure 4 shown.

[0063] The curved triangle-shaped TA-NIPAM microactuator is formed by the first strip-shaped actuator 21, the second strip-shaped actuator 22, and the third strip-shaped actuator 23, which together enclose a curved triangle closed area. When all three actuators contract, the entire curved triangle contracts inward, and the cells inside this area will be squeezed. The first strip-shaped actuator, the second strip-shaped actuator, and the third strip-shaped actuator are all in the shape of a strip with dimensions of 8 mm × 2 mm × 0.5 mm.

[0064] Example Four

[0065] A method for using a device for in-situ stress loading on living cells in a three-dimensional matrix material is the same as that in Example Two, except that the TA-NIPAM microactuator used is in the shape of a notched ring, asFigure 5 as shown

[0066] The notched ring actuator 24 is formed by surrounding a strip actuator of 20 mm×5 mm×0.5 mm, characterized by having a notch 241 on the circumference, and contracting at 37°C to extrude the cells therein through the matrix hydrogel.

[0067] Example Five

[0068] A method of using a live cell in-situ stress loading device in a three-dimensional matrix material is the same as that in Example Two, except that the TA-NIPAM micro actuator used is composed of a double-layer strip actuator of 10 mm×2.5 mm×0.5 mm, including a fourth strip actuator 25 and a fifth strip actuator 26, as Figure 6 shown. The fourth strip actuator 25 and the fifth strip actuator 26 have different degrees of contraction, so that the cells on both sides are subjected to different stresses.

[0069] Example Six

[0070] The hydrogel matrix encapsulating the cells is printed layer by layer in a 3D printing manner in a fixed area. The TA-NIPAM micro actuator 2 is placed or embedded at a specific position inside the three-dimensional matrix hydrogel, and the print head 6 will print the hydrogel matrix layer encapsulating the cells layer by layer around the TA-NIPAM micro actuator. As Figure 8 shown, the top fixing frame 7 is connected above the print head.

[0071] The entire printing process can be completed inside the stage incubator 4 (with the upper cover removed), and a foldable flexible soft film 8 is used to cover this working space. Since the stage incubator can provide closed-loop temperature control and humidity control, with a constant temperature of 37°C and a humidity control of about 60%-80%, the temperature and humidity of the cells during the printing process will be better than in the conventional situation, which is used to improve the problem of reduced cell viability caused by long-term printing.

[0072] Experimental Test

[0073] (1) Test on the single-drive response effect of the TA-NIPAM actuator

[0074] The prepared TA-NIPAM gel is cut into strips with a size of 20 mm×5 mm×0.5 mm. After being fully swollen in deionized water at 20°C, it is transferred to water at 50°C. In the experiment, the TA-NIPAM gel strip changes from a flat state to a curled state.

[0075] At both ends of the TA-NIPAM gel strip in the swelling equilibrium state, they are respectively marked as point A and point B. When the straight-line distance between A and B is 20 mm, it is defined as the initial deformation state. Immerse the gel strip in deionized water at 50 °C, observe and record the relationship between the distance between points A and B changing with time. When points A and B approximately coincide due to the bending of the gel strip, stop recording, and define this moment as the end time of deformation. At this moment, the distance is approximately 0.

[0076] During the test, the deformation process of the TA-NIPAM gel strip is as Figure 9 shown. When the gel strip is in an aqueous solution above the critical transition temperature (LCST, about 32 - 33 °C), it slowly loses water, and while the volume shrinks, it generates a bending deformation, and the strip actuator quickly curls into a ring shape.

[0077] (2) Reversible driving performance test of the TA-NIPAM actuator with repeated deformation

[0078] Cut the prepared TA-NIPAM gel into strips with dimensions of 20 mm × 5 mm × 0.5 mm. Transfer the sample that has reached swelling equilibrium at 20 °C into deionized water at 50 °C, observe its deformation process, and record the time consumed during the process when the distance between points A and B of the gel strip shortens from 20 mm to approximately 0 mm.

[0079] When the two ends of the gel strip just start to contact, immediately transfer it into water at 20 °C, observe and record the time taken for the gel to absorb water and expand and return to the state before deformation. After the gel reaches swelling equilibrium, put it into water at 50 °C again for actuation response. Repeat this experiment three times to obtain a curve graph showing the change of the distance between points A and B of the TA-NIPAM actuator with time as Figure 10 shown.

[0080] It can be seen that the actuation deformation (contraction) process of the gel is significantly faster than the recovery (unfolding) process. The time consumed for deformation (contraction) is about 10 s, showing the characteristics of rapid response and fast actuation.

[0081] The time consumed for the first recovery process is 4.5 min. As the number of cycles increases, the deformation time also slightly increases. When it comes to the third cycle, the time taken for the distance between the two ends of the gel strip to return to the flat state is 5.5 min, and it is only flat to a certain extent. It is difficult for the gel to truly return to a completely flat state in a short time. After waiting for 3 - 5 times the time, it will eventually return to the initial state of the experiment.

[0082] There is no obvious difference in the three actuation and recovery processes of the TA-NIPAM gel actuator, indicating that the repeated driving effect of the actuator is good and it can be reused repeatedly. Through experimental tests, when sufficient time is provided for the actuator to recover after deformation, its deformation recovery cycle can reach more than fifty times, showing a good service life.

[0083] (III) Tannic acid (TA) is a natural plant polyphenol that is easily available in nature, so it is inexpensive, non-toxic, harmless, and biocompatible. In addition, tannic acid has a strong ability to absorb ultraviolet light and can enhance the effect of ultraviolet light cross-linking. Therefore, the TA content will affect the cross-linking effect and response performance of the gel actuator. In order to explore the effect of TA content on the temperature response rate and mechanical properties of TA-NIPAM actuators, a series of actuators with different TA contents were prepared, and the duration of their deformation (contraction) was tested. The results are as follows: Figure 11 shown.

[0084] The deformation time of the TA-NIPAM actuator shows a trend of first decreasing and then increasing with the increase of TA content. The increase of tannic acid content increases the cross-linking density. When the temperature is higher than LCST, the response speed is faster, which is reflected in the shortening of the time taken for the distance between points A and B of the gel strip to decrease from 20mm to 0mm. When the tannic acid content increases further, the pores of the gel under microscopic conditions are further reduced, which affects the water loss rate, which is reflected in the long actuation time and reduced response rate under macroscopic conditions.

[0085] (IV) Stress loading experiment of TA-NIPAM microactuator on cells

[0086] After the TA-NIPAM microactuator was cut into 5mm×2mm×0.5mm strips and embedded into the dextran hydrogel that encapsulated the cells, it was found through experiments that it had no effect on the activity of C2C12 cells and could apply local stress to the cells, causing the cells to stretch in an aligned manner, such as Figure 12 shown.

[0087] The cell culture environment temperature is 37°C, which is higher than the critical transition temperature of the actuator, 32-33°C. The actuator will produce the characteristic of volume contraction. The two ends of the strip actuator contract toward the middle, driving the slow gel to contract toward the middle, and the cells wrapped in the dextran gel are then driven to stretch and grow. After 24 hours of cell culture, the growth condition is still good, and the number of cells has increased significantly compared to the initial time, and the body shape has been significantly elongated, and the closer to the edge of the actuator, the more obvious it is.

[0088] Figure 12 The image shows a microscopic image of the part near the upper end of the actuator. The black strip in the center of the image is the TA-NIPAM microactuator. The actuator is covered with cells on both sides. It can be observed that the cells on the left and right sides of the actuator have different degrees of elongation (more obvious on the left). From the local magnified image, it can be seen that the cells are in the shape of narrow strips, while the cells cultured for 24 hours are more spherical when no stress is applied, without such significant elongation, which proves that the stress applied by the microactuator can change the cell growth morphology.

[0089] The present invention relates to a device and method for in-situ stress loading of living cells in a three-dimensional matrix material. The content of tannic acid in the actuator can quantitatively regulate its actuation rate and mechanical properties and has a repeated and reversible driving effect; the TA-NIPAM microactuator does not require any external power supply and has no wires, greatly avoiding the disturbance of the matrix hydrogel structure caused by wires; it can cause cells to migrate and grow along the direction of its contraction through the volume contraction of the actuator, and it is regulated by the geometric and mechanical characteristics of the microactuator.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A device for in-situ stress loading of living cells in a three-dimensional matrix material, characterized in that: It includes a tannic acid-N-isopropylacrylamide microactuator, and the tannic acid-N-isopropylacrylamide microactuator is embedded in a three-dimensional matrix hydrogel wrapping a large number of cells to prepare a microhydrogel sample for in-situ stress loading; The tannic acid-N-isopropylacrylamide microactuator is in the form of a single-layer strip, a double-layer strip, a curved triangle, or a notched circular ring in the three-dimensional matrix hydrogel; The environmental temperature is controlled to rise above the critical transition temperature of the tannic acid-N-isopropylacrylamide microactuator, so that the tannic acid-N-isopropylacrylamide microactuator deforms, thereby applying stress to the surrounding cells through the three-dimensional matrix hydrogel.

2. The in-situ stress loading device for living cells in a three-dimensional matrix material according to claim 1, characterized in that, The preparation of the tannic acid-N-isopropylacrylamide microactuator includes: S1. Accurately measure 1% 1,4-dioxane and 4% deionized water and place them in a beaker. After stirring evenly, set aside. Then, add 1-5% N-isopropylacrylamide and 1-2.5% tannic acid to the beaker in sequence. After fully dissolving, add 3-7% N,N'-methylenebisacrylamide and 3-7% 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone powder to obtain a pre-polymerization solution; S2. Inject the pre-polymerization solution into a mold, and carry out a cross-linking reaction under ultraviolet light irradiation to prepare a gel; after taking out the gel, soak it in deionized water to make the gel fully swell and remove unreacted substances, thereby obtaining the tannic acid-N-isopropylacrylamide microactuator.

3. The in-situ stress loading device for living cells in a three-dimensional matrix material according to claim 2, characterized in that: By regulating the relative proportion of the amounts of substance of tannic acid and N-isopropylacrylamide, the strength of the tannic acid-N-isopropylacrylamide microactuator is ≥1 kPa, and the deformable range is ≥200 μm.

4. A device for in-situ stress loading of living cells in a three-dimensional matrix material according to claim 2, characterized in that: In step S2, the distance between the ultraviolet lamp and the surface of the mold is 1 cm, and the polymerization temperature is 26 °C.

5. A device for in-situ stress loading of living cells in a three-dimensional matrix material according to claim 1, characterized in that: The microhydrogel sample is placed in an in-situ incubator on the stage for cultivation, and the cultivation temperature is set at 37 °C; during the process of heating from room temperature to 37 °C, the tannic acid-N-isopropylacrylamide microactuator shrinks and deforms, and the deformation process of the tannic acid-N-isopropylacrylamide microactuator and the stretching or contraction of the cells can be observed in real time through the objective lens under the in-situ incubator on the stage through the heat-insulating transparent film.

6. The in-situ stress loading device for living cells in a three-dimensional matrix material according to claim 1, characterized in that: The curved triangular tannic acid-N-isopropylacrylamide microactuator is formed by three strip-shaped tannic acid-N-isopropylacrylamide microactuators enclosing a closed area with a curved triangular shape as a whole.

7. A device for in-situ stress loading of living cells in a three-dimensional matrix material according to claim 1, characterized in that: The notched circular-ring-shaped tannic acid-N-isopropylacrylamide microactuator is formed by a strip-shaped tannic acid-N-isopropylacrylamide microactuator enclosing a circumference with a notch on one side.

8. A device for in-situ stress loading of living cells in a three-dimensional matrix material according to claim 1, characterized in that: The critical transition temperature of the tannic acid-N-isopropylacrylamide microactuator is 32-33 °C.

9. A method for in-situ stress loading of living cells in a three-dimensional matrix material, characterized in that: It is carried out by using the device for in-situ stress loading of living cells in a three-dimensional matrix material according to any one of claims 1 to 7.

10. A method for in-situ stress loading of living cells in a three-dimensional matrix material according to claim 9, characterized in that: The mechanical properties of the three-dimensional matrix hydrogel are directly measured by a rotational rheometer; according to the mechanical parameter values of the tannic acid-N-isopropylacrylamide microactuator, the stress distribution in the current tannic acid-N-isopropylacrylamide microactuator and the matrix hydrogel is calculated by finite element simulation, and then the stress received by the cells is obtained.

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