Method for evaluating adhesion capacity of adherent cells through supergravity

Abstract

The invention discloses a method for evaluating the adhesion capacity of adherent cells through supergravity. The method comprises the following steps: patterning adherent cells to be detected in a container into an annular structure; fixing the container on a rotating platform, and keeping the center of the annular structure concentric with the rotating center of the rotating platform; the rotating platform is rotated to apply supergravity, and the cell adhesion capability is evaluated in a manner of applying the supergravity. Compared with the prior art, the method has the advantages of high precision, high flux and low cost, avoids interference of biochemical reagents, is suitable for drug screening, biological material development and clinical detection, and remarkably improves the efficiency and reliability of cell mechanics research.

Description

Technical Field

[0001] The present invention belongs to the field of tissue engineering and biomanufacturing technology under biomedical engineering, and particularly relates to a method for evaluating the adhesion ability of adherent cells through hypergravity. Background Art

[0002] Hypergravity is an artificially created environment exceeding Earth's gravity, typically applied to cells using equipment such as centrifuges. This environment not only provides mechanical stimulation distinct from conventional gravity but can also simulate specific physiological or pathological conditions. Hypergravity can affect cell proliferation and differentiation, stimulating cell proliferation or altering its differentiation state. This approach can simulate cell behavior under specific physiological or pathological conditions, such as promoting cell proliferation and differentiation in tissue engineering. Furthermore, studies have shown that hypergravity can influence cellular gene expression patterns. Under hypergravity, cells regulate a range of genes related to stress response, cell cycle, metabolism, and differentiation, reflecting their adaptive mechanisms in different physical environments. Hypergravity provides a unique experimental platform for studying cellular responses to external mechanical stimuli. It can simulate specific physical stress environments within the human body, such as the gravitational changes experienced by the skeletal system and the mechanical stresses experienced by tissues like the heart, providing technical support for space biology.

[0003] Cell adhesion is a core biological process underlying the interaction between cells and the extracellular matrix (ECM), other cells, or artificial surfaces, regulating cell migration, proliferation, differentiation, and tissue homeostasis. In basic research, adhesion strength directly reflects the mechanical properties of cell-substrate interactions and is a key parameter for studying mechanisms such as cancer metastasis, immune responses, and stem cell differentiation. Cell adhesion is also an important cellular assessment tool, particularly for myogenic cell lines (e.g., C2C12 skeletal muscle stem cells), where it is crucial for assessing stem cell transformation and regeneration. In applied fields, accurate assessment of cell adhesion strength is crucial for drug screening (e.g., anti-tumor and anti-inflammatory drugs), biomaterial development (e.g., implants and tissue engineering scaffolds), and clinical diagnostics (e.g., capturing circulating tumor cells). However, existing technologies, such as atomic force microscopy (AFM), which directly measures the adhesion force of single cells to substrates with a probe and can achieve piconewton (pN) accuracy, are prohibitively expensive (typically exceeding $500,000) and require specialized operators, limiting their widespread use in general laboratories. Furthermore, throughput is extremely low, measuring only a single cell at a time. Statistical analysis requires hours to days, making it difficult to meet the demands of high-throughput drug screening. Atomic force microscopy (AFM) technology requires high cell viability; probe contact can damage the cell membrane. Furthermore, cells must be securely fixed to the cantilever, placing stringent demands on cell viability and morphological stability. While the spinning dish method (such as the one described in "A novel mode of cell detachment from fibrillar fibronectin matrix under shear") cleverly utilizes centrifugal force to induce cell detachment, it is low-cost but suffers from fundamental flaws. Its centrifugal force calculations suffer from large errors, and the random distribution of cells leads to variations in the centrifugal radius (R). Cells within the same dish experience centrifugal forces that can vary several times (e.g., between cells at the edge and the center), resulting in high data dispersion. Furthermore, this method fails to account for intercellular biomechanics and lacks a single control variable for measuring cell-matrix adhesion. Furthermore, it relies on manual counting of detached cells under a microscope, resulting in low efficiency and subjectivity, making automation difficult.

[0004] In summary, the aforementioned methods all suffer from an imbalance in the "precision-cost-throughput" triangle. High-precision methods (such as AFM) sacrifice throughput and cost, while low-cost solutions (such as the spinning plate method) cannot meet research needs due to precision limitations. Therefore, a cell adhesion strength assessment method that combines high precision, high throughput, low cost, and ease of use is urgently needed to meet the diverse needs of biomedical research, drug development, and clinical testing. Summary of the Invention

[0005] To solve the problems existing in the prior art, the present invention provides a method for evaluating the adhesion strength of adherent cells using hypergravity. The culture method is simple and easy to operate, has low additional cost, and can evaluate cell adhesion ability with high throughput.

[0006] The method provided by the present invention for evaluating the adhesion ability of adherent cells by hypergravity breaks through the inability of traditional cell adhesion measurement methods to achieve both simplicity and ease of operation, low additional cost, and accurate and simple evaluation of the adhesion ability of adherent cells, and has great commercial and scientific value.

[0007] A method for evaluating the adhesion ability of adherent cells by hypergravity comprises: patterning the adherent cells to be tested in a container into a ring structure; fixing the container on a rotating platform, keeping the center of the ring structure concentric with the rotation center of the rotating platform; rotating the rotating platform to apply hypergravity, and evaluating the cell adhesion ability by applying hypergravity.

[0008] The present invention ensures the uniformity of centrifugal force by patterning the circular cells by keeping the center of the annular structure concentric with the center of rotation of the rotating platform. Various forms of adhesion ability tests can be implemented. For example, the adhesion ability of different cells can be measured at a fixed rotation speed; or different rotation speeds under specific detachment states can be detected for different cells; both qualitative and quantitative detection can be achieved. Preferably, when performing quantitative detection, after the application of supergravity is completed, microscopic images before and after centrifugation are collected, and the de-adhesion rate is calculated by counting the number of cells before and after centrifugation, thereby evaluating the adhesion ability of the adherent cells. When counting, manual identification and counting can be performed using a microscope, or an automatic cell recognition method based on microscope images can be used, such as the "A cell recognition and counting method, device and computer storage medium based on Faster-RCNN" mentioned in the patent document with publication number 116758072A.

[0009] Preferably, a three-jaw chuck is used to fix the container on the rotating platform while keeping the center of the annular structure concentric with the rotation center of the rotating platform.

[0010] Preferably, the container is sucked and fixed on the rotating platform by negative pressure, while the center of the annular structure is kept concentric with the rotation center of the rotating platform.

[0011] As a further preferred embodiment, the container is fixed on the rotating platform by using a three-jaw chuck and negative pressure, while keeping the center of the annular structure concentric with the rotation center of the rotating platform, thereby further ensuring the concentricity of the culture dish and the rotation center during centrifugation.

[0012] Preferably, the container is a culture dish, and further, the container is a circular culture dish of 5 mm to 90 mm.

[0013] A method for evaluating the adhesion ability of adherent cells by hypergravity comprises: patterning the adherent cells into a multi-layer coaxial circular ring structure through a photosensitive hydrogel protective layer; fixing a culture dish on a rotating platform by vacuum negative pressure, wherein the platform is provided with a three-jaw chuck and a central suction hole to ensure concentric positioning; controlling the platform speed and centrifugation time by a single-chip microcomputer, applying hypergravity, and evaluating the cell adhesion ability by applying hypergravity; collecting microscopic images before and after centrifugation, calculating the de-adhesion rate by counting the number of cells before and after centrifugation, and then evaluating the adhesion ability of the adherent cells.

[0014] Furthermore, a method for evaluating the adhesion ability of adherent cells by hypergravity includes: patterning the adherent cells in a circular culture dish into a multi-layer fine ring pattern through a biolithographic hydrogel protective layer, fixing the culture dish on a rotating platform by generating vacuum negative pressure through a vacuum pump, and providing three positioning claws symmetrically distributed at 120° intervals on the edge of the rotating platform, which cooperate with the suction hole in the center of the turntable to accurately and concentrically fix the culture dish. The platform speed and centrifugation time are controlled by a single-chip microcomputer, and microscopic images before and after centrifugation are collected. The de-adhesion rate is calculated by counting the number of cells before and after centrifugation, and the adhesion ability of the adherent cells is evaluated. The microscopic images before and after centrifugation are collected, and the de-adhesion rate is calculated by counting the number of cells before and after centrifugation, and the adhesion ability of the adherent cells is evaluated. The microscopic images before and after centrifugation are collected, and the de-adhesion rate is calculated by counting the number of cells before and after centrifugation, and the adhesion ability of the adherent cells is evaluated.

[0015] The vacuum pump has an air extraction rate of ≥60 L / min for a circular culture dish with a diameter of 5 mm to 90 mm.

[0016] The adherent cells are cells that grow attached to the wall during cell culture. The above-mentioned evaluation method can be applied to the adhesion ability evaluation of normal adherent cells, and can also be applied to the application of hypergravity to adherent cells.

[0017] Preferably, the hydrogel is a photosensitive bio-hydrogel modified with methacrylic groups. Further preferably, the hydrogel contains Gelma90 hydrogel with a photoinitiator concentration of 0.25% wt and a hydrogel concentration of 7.5% wt and a high degree of methacrylation substitution (degree of substitution greater than 85%).

[0018] Preferably, no light absorber is added to the hydrogel to ensure sufficient crosslinking. Preferably, the hydrogel is sterilized using a syringe filter before being added to the culture vessel. Further preferably, the hydrogel solution is filtered using a 0.22 μl syringe filter.

[0019] Preferably, the adherent cells are thawed frozen cells or subcultured and digested cells.

[0020] Preferably, the culture container is a commercial round cell culture dish with a diameter of 5 mm to 90 mm.

[0021] As a preference, the cell growth density of the adherent cells is the best when the entire bottom of the culture dish is covered. As a further preference, the cell growth density of the adherent cells is 2×10 4 to 5×10 4 cells / mL.

[0022] Preferably, the surface of the rotating platform is flat, and a 1mm diameter suction hole is built into the center.

[0023] Preferably, during testing, the vacuum negative pressure suction hole built into the rotating platform should be facing the center of the culture dish.

[0024] Preferably, the rotating platform is equipped with a three-jaw chuck structure, which uses a three-point positioning principle to ensure the coaxial center of the culture dish. As a further preferred embodiment, taking the 35mm culture dish in this experimental example as an example, its inner diameter is 34.8mm, forming a 0.2mm interference fit with the culture dish. The inner wall of the three-jaw chuck is designed with a micro-serrated texture to increase the static friction coefficient with the edge of the culture dish (μ≥0.5).

[0025] Preferably, the microcontroller controls the rotating platform to rotate at a speed of 500-8000 rpm, depending on the cell type and the assay intensity, and the centrifugation time is 1-10 minutes.

[0026] Preferably, the cell microscopic images before and after centrifugation should be analyzed for cell number, diameter, area-to-perimeter, and aspect ratio to comprehensively evaluate adhesion ability.

[0027] The circular ring structure pattern can be obtained by the method in the patent application of the inventor with application number 2025103095302 (A method for obtaining patterned adherent cells).

[0028] Preferably, the method for patterning the adherent cells to be tested in the container into a ring structure is as follows:

[0029] Step 1: Add the hydrogel solution to the adherent cell culture vessel, with the volume of the solution covering the bottom of the culture dish by 2 mm or more;

[0030] Step 2: Place the mask with the ring pattern under the culture container and perform photolithography;

[0031] Step 3: After solidifying the hydrogel, add PBS buffer to the culture dish to remove the unsolidified hydrogel;

[0032] Step 4: Add trypsin to the culture vessel to complete the digestion reaction and remove cells not protected by the solidified hydrogel;

[0033] Step 5: After digestion is complete, collagenase type II is added to degrade the solidified hydrogel;

[0034] Step 6: After the hydrogel is completely degraded, the upper suspension is discarded to obtain adherent cells with a circular pattern.

[0035] Preferably, the annular structure is a single ring or multiple concentric rings. Using an annular structure with multiple rings can simultaneously apply different centrifugal forces to the cells, further improving the detection efficiency.

[0036] As a specific preference, a method for evaluating the adhesion ability of adherent cells by hypergravity comprises the following steps.

[0037] Step 1: Sterilize the hydrogel solution and add it to the adherent cell culture vessel in advance. Add the solution according to the size of the culture dish, and the volume should be 2mm above the bottom of the culture dish.

[0038] Step 2: Place the ultra-fine ring pattern mask under the culture dish and use a 405nm wavelength light source to illuminate the photolithography. As a preferred method, the exposure intensity is 20mw / cm 2 , exposure time is 60s;

[0039] Step 3: After the hydrogel solidifies, add 2 ml of 37°C PBS buffer to the culture dish, pipette and swirl several times, and then aspirate the suspension.

[0040] Step 4: Add 2 ml of trypsin to the culture dish and place it in the incubator for 5 minutes to complete the digestion reaction, so that the cells are digested and suspended in the area not protected by the solidified hydrogel.

[0041] Step 5: After digestion is complete, add 2 ml of high-glucose medium to terminate the digestion reaction. Repeatedly pipette and evenly shake the mixture more than ten times. Discard the supernatant and add 5 ml of 2 U / ml collagenase type II. Observe the degradation of the hydrogel under a microscope.

[0042] Step 6: After the hydrogel is completely degraded, the upper suspension is discarded to obtain adherent cells in a very fine ring pattern.

[0043] Step 7: Place the circular culture dish on the rotating platform, align the center of rotation with the center of the culture dish, adjust the clamping position of the three-jaw chuck, and ensure that the center of the culture dish and the center of the turntable platform (suction hole) are coaxial.

[0044] Step 8: Start the vacuum pump to ensure that the culture dish is accurately and firmly adsorbed on the rotary platform

[0045] Step 9: Cover the protective cover, adjust the rotation parameters including speed and centrifugal time through the microcontroller, and start the motor to rotate the platform.

[0046] Step 10: After the rotation is completed, turn off the rotating motor first, then turn off the vacuum pump to cancel the adsorption, remove the culture dish, take pictures and observe, and count the cells.

[0047] The present invention provides a method for evaluating the adhesion ability of adherent cells by hypergravity, which for the first time quantitatively utilizes the spinning dish method to accurately apply hypergravity, thereby achieving adhesion evaluation of adherent cells and expanding existing cell mechanics application methods.

[0048] The present invention also provides a device for evaluating the adhesion ability of adherent cells by hypergravity, comprising:

[0049] A rotating platform is provided with claws and vacuum suction holes. During the test, the container containing the adherent cells to be tested is placed on the rotating platform;

[0050] A motor driving the rotating platform;

[0051] A vacuum pump connected to the vacuum negative pressure suction hole.

[0052] As an option, it also includes:

[0053] A controller that controls the motor speed;

[0054] A microscope for capturing images of adherent cells to be tested before and after centrifugation;

[0055] An image analysis module is used to analyze the image.

[0056] The controller may be a computer, a single chip microcomputer, or other types of controllers.

[0057] The image analysis module may employ a computer including corresponding software codes.

[0058] More specifically, a device for evaluating the adhesion ability of adherent cells under hypergravity comprises:

[0059] (a) A rotating platform equipped with claws and vacuum suction holes;

[0060] (b) Vacuum pump and single chip microcomputer control system;

[0061] (c) Motor that drives the rotation

[0062] (d) Microscope and image analysis module, used to acquire and process cell images before and after centrifugation.

[0063] The vacuum pump of the rotating platform has an air extraction rate of ≥60 L / min and is suitable for circular culture dishes with a diameter of 5 mm to 90 mm.

[0064] The rotation speed range of the high gravity is 500-8000 rpm, and the centrifugation time is 1-10 minutes.

[0065] In response to the defects of the existing technology, the present invention innovatively proposes a method for evaluating cell adhesion ability through hypergravity. Vacuum adsorption fixation and a three-jaw chuck are used to eliminate the displacement of the culture dish. The circular cell patterning ensures the uniformity of centrifugal force and greatly eliminates the influence of biological forces between cells. The integrated image recognition algorithm automatically identifies the number of cells and automatically and accurately identifies the status of cells before and after hypergravity, comprehensively evaluating the adhesion ability of adherent cells.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] (1) The method of the present invention for evaluating the adhesion ability of adherent cells by hypergravity is a complementary method to the existing spinning disk method. By arranging cells in a ring and combining micropatterning technology, the magnitude of hypergravity can be quantitatively controlled and the influence of lateral interactions between cells in the traditional spinning disk method can be significantly reduced, thus making up for the accuracy defects of the existing method. The method is simple and easy to operate, and has low additional cost, and has great commercial value.

[0068] (2) The method of the present invention for evaluating the adhesion ability of adherent cells by hypergravity does not use biochemical reagents such as cell detachment reagents to de-adhere the cells, which greatly ensures the stability of the adherent cells and avoids the influence of the biological factor of the reagent on the biochemical level of the cells. The cells after the test are still in a healthy and stable state.

[0069] (3) The present method for assessing adherent cell adhesion using hypergravity allows for the simultaneous assessment of a larger number of cells. Compared to atomic force microscopy, this method offers higher throughput and is particularly valuable for large-scale assessments requiring less stringent precision. Compared to the traditional spinning plate method, this method offers higher precision, fundamentally overcoming the significant drawbacks of the original method.

[0070] (4) The method of the present invention for evaluating the adhesion ability of adherent cells by hypergravity can be used in a culture process and can also be used as a means of quantitatively applying hypergravity to adherent cells. It has a wide range of applications and great commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 This is a conceptual diagram of the device in an embodiment of the present invention; 1 is a 35mm culture dish, in which cells have been patterned into ring-shaped cells according to the method described in the present invention. 2 is a rotating platform with a three-jaw chuck, 3 is a rotating shaft, 4 is a vacuum adsorption hole, 5 is a protective cover, 6 is a vacuum pump, and 7 is a rotating motor.

[0072] Figure 2 This is a schematic diagram of the effect of evaluating the adhesion ability of adherent cells according to an embodiment of the present invention; wherein, Figure 2 A is a diagram showing the initial adherent growth of cells in a culture dish according to an embodiment of the present invention. Figure 2 B is a 4x microscope image of cells forming a circular pattern in a culture dish according to an embodiment of the present invention. The transparent portion mixed with gray shadows in the image is the hydrogel protective layer. As shown in the figure, the adherent cells protected by the circular hydrogel were not digested by trypsin. Figure 2 C is a 4x microscope image of cells forming a ring pattern in a culture dish according to an embodiment of the present invention. The image is an image of adherent cells after collagenase degradation, showing that the adherent cells finally formed a ring pattern. Figure 2 D is a 4x microscope image of the cells after hypergravity loading in an embodiment of the present invention. Figure 3 This is a statistical graph showing the results of evaluating the adhesion ability of old, middle-aged, and young generations of adherent cells in an embodiment of the present invention. DETAILED DESCRIPTION

[0073] To further clarify the objectives, technical solutions, and advantages of the present invention, the present invention is further described in detail below with reference to the following examples. Unless otherwise specified, the equipment and reagents used in each example and test example are commercially available. The specific examples described herein are intended only to illustrate the present invention and are not intended to limit the present invention. To characterize the evaluation results of the present invention, three generations of fibroblasts (elderly, middle-aged, and young) were used for the experiments.

[0074] The method uses photosensitive hydrogel patterning technology to arrange adherent cells into a single or multi-layer coaxial ring structure, eliminating the effects of uneven centrifugal force distribution and cell-cell interactions. A vacuum pressure combined with a three-jaw chuck secures the culture dish to ensure concentric positioning of the rotating platform and the dish. A single-chip microcomputer controls the rotation speed and centrifugation time, precisely applying hypergravity to each cell cycle. Microscopic images are collected before and after centrifugation, and the detachment rate is calculated by counting the number of cells before and after centrifugation, thereby evaluating the adhesion capacity of the adherent cells.

[0075] The supporting devices used in this embodiment are as follows Figure 1 As shown, the apparatus comprises a rotating platform 1, a rotating shaft 3, a protective cover 5, a vacuum pump 6, a rotating motor 7, a single-chip microcomputer control system, a light source mask assembly, and a microscopic image analysis module. The rotating platform 1 is provided with vacuum adsorption holes 4. A commercially available spin coater can be used, with a three-jaw chuck installed on the rotating platform. The culture vessel used is a commercially available round cell culture dish with a diameter of 5 mm to 90 mm.

[0076] like Figure 1 As shown, a method for evaluating the adhesion ability of adherent cells by hypergravity comprises the following steps:

[0077] 1. Sterilize the GELMA90 hydrogel solution (the concentration of the photoinitiator LAP is 0.25% wt, the hydrogel concentration is 7.5% wt, and the degree of substitution is 90%) using a 0.22 ul syringe filter.

[0078] 2. Take a culture dish containing adherent cells (using human oral epithelial fibroblasts as an example) and observe the growth and cell density under a microscope.

[0079] 3. The density of adherent cells is 5×10 4 cells / mL, discard the supernatant and add sterilized hydrogel solution until the level is 2mm-3mm above the bottom of the dish.

[0080] 4. Place the culture dish on a mask with a set pattern (such as a circular structure in this embodiment) and use a surface curing light source to cure the film. Set the exposure intensity to 20mw / cm 2 , the exposure time is 60s. The role of the photocuring process is to fix the hydrogel structure and prevent it from deformation or degradation in subsequent processes.

[0081] 5. Add 2 ml of 37°C PBS buffer to the solidified culture dish, pipette thoroughly, and then aspirate the supernatant to remove the unsolidified hydrogel.

[0082] 6. Add 2 ml of trypsin to the culture dish and incubate it in a carbon dioxide incubator at 37°C and 5% carbon dioxide concentration for 5 minutes to allow trypsin to digest the cells.

[0083] 7. After digestion is complete, add 2ml of high-glucose medium to terminate digestion, pipette, and discard the supernatant to remove cells not solidified by the hydrogel. Add 5ml of 2U / ml collagenase type II. After observing the hydrogel degradation under a microscope, discard the supernatant to obtain patterned adherent cells.

[0084] 8. After the above steps, the following can be collected in the culture dish: Figure 2 C shows the ring-shaped cells.

[0085] 9. Place the round culture dish 1 Figure 1 On the rotating platform 2 shown, the rotation center, i.e., the vacuum suction port, is aligned with the center of the culture dish. The position of the three-jaw chuck's jaws is adjusted. Using the three-point positioning principle, the radial constraint force of the three-jaw chuck ensures that the center of the culture dish and the center of the turntable platform (suction hole) are coaxial.

[0086] 10. Start the vacuum pump 6 to ensure that the culture dish is firmly adsorbed on the rotating platform 2.

[0087] 11. Cover the protective cover and adjust the rotation parameters through the single chip microcomputer. The shaft 3 drives the rotating platform 2 to rotate at a speed of 4000 rpm for 4 minutes.

[0088] 12. After the rotation is completed, turn off the rotating motor 7 first, then turn off the vacuum pump 6 to cancel the adsorption, remove the culture dish and observe it under a microscope, collect microscopic images before and after centrifugation, calculate the de-adhesion rate by counting the number of cells before and after centrifugation, and then evaluate the adhesion ability of the adherent cells.

[0089] Evaluation results:

[0090] After the above operation, if Figure 2 In middle D, it can be seen that there is a significant difference in the number of cells before and after rotation, and the cell morphology has changed significantly due to the influence of hypergravity, specifically the color of the cell nucleus has darkened and the overall shape has become round. Figure 3 It is known that the adhesion capacity of adherent cells is greater in young, middle-aged, and elderly individuals. The results of the example show that the number of de-adhesions in elderly individuals is significantly higher, while the number of de-adhesions in young and middle-aged individuals is lower, with a significant gradient difference. This demonstrates the effectiveness of this method for evaluating the adhesion capacity of adherent cells using hypergravity.

[0091] The above-described embodiments are only preferred implementations of the present invention. It should be noted that the above-described embodiments are illustrative and cannot be understood as limiting the present invention. A person skilled in the art can make several changes, modifications, substitutions and variations without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for evaluating the adhesion ability of adherent cells by hypergravity, characterized in that: include: Patterning the adherent cells to be tested in the container into a ring structure; fixing the container on a rotating platform, keeping the center of the ring structure concentric with the rotation center of the rotating platform; The rotating platform was rotated to apply hypergravity, and the cell adhesion ability was evaluated by applying hypergravity.

2. The method for evaluating the adhesion ability of adherent cells by hypergravity according to claim 1, wherein: After the application of hypergravity, microscopic images before and after centrifugation were collected, and the de-adhesion rate was calculated by counting the number of cells before and after centrifugation to evaluate the adhesion ability of adherent cells.

3. The method for evaluating the adhesion ability of adherent cells by hypergravity according to claim 1, wherein: The container is a culture dish, which is fixed on the rotating platform by a three-jaw chuck, while keeping the center of the annular structure concentric with the rotation center of the rotating platform.

4. The method for evaluating the adhesion ability of adherent cells by hypergravity according to claim 1 or 3, characterized in that: The container is a culture dish, which is fixed on the rotating platform by suction through negative pressure, while keeping the center of the annular structure concentric with the rotation center of the rotating platform.

5. The method for evaluating the adhesion ability of adherent cells by hypergravity according to claim 4, wherein: The culture dish is a circular culture dish with a diameter of 5 mm to 90 mm; the negative pressure is provided by a vacuum pump with a pumping rate of ≥60 L / min.

6. The method for evaluating the adhesion ability of adherent cells by hypergravity according to claim 1, wherein: The rotation speed range of the high gravity is 500-8000 rpm, and the centrifugation time is 1-10 minutes.

7. The method for evaluating the adhesion ability of adherent cells by hypergravity according to claim 1, wherein: The method for patterning the adherent cells to be tested into a ring structure in a container is as follows: Step 1: Add the hydrogel solution to the adherent cell culture vessel, with the volume of the solution covering the bottom of the culture dish by 2 mm or more; Step 2: Place the mask with the ring pattern under the culture container and perform photolithography; Step 3: After solidifying the hydrogel, add PBS buffer to the culture dish to remove the unsolidified hydrogel; Step 4: Add trypsin to the culture vessel to complete the digestion reaction and remove cells not protected by the solidified hydrogel; Step 5: After digestion is complete, collagenase type II is added to degrade the solidified hydrogel; Step 6: After the hydrogel is completely degraded, the upper suspension is discarded to obtain adherent cells with a circular pattern.

8. The method for evaluating the adhesion ability of adherent cells by hypergravity according to claim 1, wherein: The annular structure is a single ring or a plurality of concentric rings.

9. A device for evaluating the adhesion ability of adherent cells by hypergravity, characterized in that: include: A rotating platform is provided with claws and vacuum suction holes. During the test, the container containing the adherent cells to be tested is placed on the rotating platform; A motor driving the rotating platform; A vacuum pump connected to the vacuum negative pressure suction hole.

10. The device for evaluating the adhesion ability of adherent cells by hypergravity according to claim 9, characterized in that: Also includes: A controller that controls the motor speed; A microscope for capturing images of adherent cells to be tested before and after centrifugation; An image analysis module is used to analyze the image.

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