A method for measuring intercellular adhesion in ultra-low field magnetic fields

Abstract

This invention discloses an ultra-low field magnetic field method for measuring intercellular adhesion force. It utilizes magnetic borosilicates as probes to measure cell-cell adhesion force in an ultra-low field. The magnetic borosilicate probes used in this invention are characterized by easy mass production, high remanence, and biocompatibility. This invention enables high-throughput, high-sensitivity measurement of intercellular adhesion force. The metastatic ability of tumor cells can be determined by comparing the magnitude of intercellular adhesion forces. This invention simulates the in vivo environment to construct an intercellular adhesion system; the magnetic probes used do not interfere with intercellular adhesion, and the measured values ​​are closer to those in the real environment.

Description

Technical Field

[0001] This invention relates to a method for measuring intercellular adhesion force in ultra-low field using magnetosomes as probes, belonging to the field of biomaterials and medical nanomaterials technology. Background Technology

[0002] Intercellular adhesion plays a crucial role in tissue morphology and homeostasis. Many physiological processes and cellular functions are related to intercellular adhesion. For example, during tumor metastasis, tumor cells reduce their adhesion to neighboring cells and escape the primary site; subsequently, tumor cells enter the bloodstream and metastasize, adhering to the endothelial cells of blood vessel walls in distant tissues and then invading the blood vessels; finally, they colonize distant tissues to form secondary tumors. Therefore, analyzing intercellular adhesion forces can enable the diagnosis of diseases such as tumors (e.g., tumor metastatic ability).

[0003] Traditional methods for measuring intercellular adhesion forces mainly include atomic force microscopy (AFM) and magnetic tweezers. These techniques are single-cell measurements, requiring hundreds or thousands of measurements to obtain statistically significant data, making the process cumbersome. Ultra-low field force spectroscopy (FIRMS), on the other hand, can analyze tens of thousands of cells in a single measurement. Its detection principle is primarily based on the remanent magnetic signals generated by magnetic probes or cells, bacteria, and biomolecules labeled with magnetic probes. Ultra-low field magnetic probes possessing high remanence, good dispersibility, and low cytotoxicity are key to FIRMS applications in the biological field. Currently used ultra-low field magnetic probes are all artificially synthesized. Because they are prepared in an oil phase, water conversion is required when applying them to cell systems, a complex process. Furthermore, the use of organic ligands significantly increases the cytotoxicity of the magnetic probes. Summary of the Invention

[0004] The purpose of this invention is to provide a method for measuring ultra-low field intercellular adhesion using magnetosomes as probes. This method achieves high-throughput measurement of intercellular adhesion at the pN level by labeling cells with magnetosome probes, making it of great application potential in biomedical fields such as tumor metastasis diagnosis.

[0005] Firstly, this invention protects an ultra-low field measurement method for cell-cell adhesion force using a magnetosome as a probe. Currently, there is no existing method for measuring cell-cell adhesion force in ultra-low field measurement technology.

[0006] The method for measuring cell-cell adhesion force using a magnetic bobbin as a probe in an ultra-low field includes the following steps:

[0007] (1) Stable adhesion of lower layer cells to the substrate: Cells were seeded in a fibronectin-modified substrate and cultured until a monolayer fusion state was obtained to obtain lower layer cells that were stably adhered to the substrate.

[0008] (2) Magnetic probe labeling of upper layer cells: upper layer cells are co-incubated with magnetosome probes to obtain magnetically probe-labeled cells;

[0009] (3) Construction of intercellular adhesion system: The cells labeled with magnetic probe in step (2) were seeded on the lower layer of cells that formed stable adhesion with the substrate in step (1) and cultured and magnetized.

[0010] (4) Measurement of intercellular adhesion force: The intercellular adhesion system obtained in step (3) was subjected to external force interference, causing the upper layer cells to separate from the lower layer cells. The remanent magnetic signal change spectrum during the dissociation process was recorded by an ultra-low field magnetometer.

[0011] Among them, the applied external force corresponding to the decrease of half of the remanent magnetic signal is defined as the intercellular adhesion force.

[0012] In step (1) of the above method, the substrate material is polydimethylsilane.

[0013] The base can specifically be a cuboid, more specifically a cuboid with a length, width, and height of 1.4cm, 0.6cm, and 0.2cm, respectively;

[0014] The surface of the substrate is provided with a groove (the groove is the site for fibronectin modification and cell culture), and the groove can be a square depression with a side length of 0.5cm and a depth of 0.1cm.

[0015] The fibronectin-modified substrate was prepared by the following method:

[0016] The substrate was incubated with fibronectin, the fibronectin solution was removed, and the fibronectin-modified substrate was washed with complete cell culture medium to obtain the final product.

[0017] The concentration of fibronectin can be 20–50 μg / mL, specifically 50 μg / mL;

[0018] The incubation temperature can be 4℃-37℃, and the time can be 2-12h, specifically 4℃ for 12h or 37℃ for 2h.

[0019] In step (1), the lower layer cells can specifically be human umbilical vein endothelial cells (HUVECs);

[0020] The cell culture temperature can be 35-37℃, and the time can be 10-24h, specifically 12h.

[0021] In step (2), the upper layer cells are cancer cells, specifically any one of human breast cancer cells MDA-MB-231, MCF-7, and MCF-10A.

[0022] The magnetosomes are derived from magnetotactic bacteria, specifically magnetotactic spirobacter AMB-1.

[0023] The magnetosomes are prepared by a method including the following steps: taking a sample of magnetotactic bacteria, sonicating it in PBS buffer, using a magnet to adsorb the broken cells, separating the precipitate, and thus obtaining the magnetosomes;

[0024] The concentration of the PBS buffer can be 10 mM; the ratio of magnetotactic bacteria cells to PBS buffer is 1:10 (w / v);

[0025] Ultrasonic fragmentation in an ice bath;

[0026] The ultrasonic power can be 300W, and the breaking time can be 30 minutes.

[0027] This further includes repeated ultrasonic cleaning of the resulting magnetic particles.

[0028] After purification, the concentration of bacterial contaminants in the magnetosomes was less than 0.1 μg / mL.

[0029] The obtained magnetic particles have an average particle size of 48.7±8.9 nm, exhibiting good size uniformity and dispersibility.

[0030] The ratio of cells to magnetosome probes can be 1×10⁻⁶. 6 Individual: 20-500μg, specifically 1×10 6 100μg;

[0031] The co-incubation temperature can be 35-37℃, and the time can be 2-12 hours, specifically 4 hours.

[0032] In step (3), the ratio of the number of upper layer cells to lower layer cells can be 0.3 to 2:1.

[0033] The culture time for the upper and lower cell layers can be 30 min to 1 h, specifically 30 min.

[0034] The magnetization is performed using a magnet with a strength of 0.5-1T, specifically 1T. The magnetization distance is 1.5-2cm, specifically 2cm. The magnetization time is 1-5min, specifically 2min.

[0035] In this invention, the method of obtaining the remanent magnetization signal change spectrum recorded by the ultra-low field magnetometer through external force interference, and the definition of the force corresponding to the decrease of half of the remanent magnetization signal as intercellular adhesion force, are known methods in the art.

[0036] In this invention, the applied external force is centrifugal force, shear force, etc.

[0037] The applied external force is centrifugal force. The magnitude of the centrifugal force on the cell is calculated based on the centrifugal speed.

[0038] The formula for calculating centrifugal force is: F = (ρ cell –ρ medium )·V cell ·ω 2 ·r, where F is the centrifugal force, ρ cell It is the average cell density (~1.07 kg·m³). -3 ), ρ medium It is the average density of the cell culture medium (~1 kg·m³). -3 V cell The average cell volume is (~525 μm) 3 ω is the centrifugal angular velocity, and r is the distance between the sample and the center of rotation (0.045m).

[0039] Specifically, in step (4), the external force is centrifugal force, and the intensity of the centrifugal force is 12pN-163pN, specifically 12pN, 18pN, 26pN, 36pN, 46pN, 59pN, 88pN, 104pN, 123pN, 142pN and 163pN;

[0040] In this invention, the remanent magnetic signal of the intracellular magnetic probe can be stably maintained for at least 36 hours without the application of external force.

[0041] In this invention, the intercellular adhesion force is mainly obtained by analyzing the changes in remanent magnetic signals caused by the dissociation of upper and lower cells labeled by magnetic probes.

[0042] Secondly, this invention protects the ultra-low field application of magnetosome probes.

[0043] No applications of magnetosomes in ultra-low fields have been reported in the prior art. The magnetosomes in this invention have multiple applications in ultra-low field magnetometers. First, magnetosomes can provide significant remanent magnetization signals in ultra-low fields, thus enabling their use in ultra-low field magnetic imaging, targeted therapy, and visual tracking. Second, magnetosomes can achieve stable, high signal-to-noise ratio magnetic labeling of cells, thus serving as ultra-low field magnetic probes for measuring cell-cell adhesion. The magnetosomes in this invention are synthesized by magnetotactic bacteria, not artificially, and are easily mass-produced. The magnetosomes synthesized by magnetotactic bacteria are encapsulated by a natural phospholipid membrane, exhibiting good biocompatibility and high remanence while significantly reducing the aggregation of magnetic probes.

[0044] The present invention has the following advantages:

[0045] 1. This invention enables high-throughput and high-sensitivity measurement of intercellular adhesion forces.

[0046] 2. This invention can determine the metastatic ability of tumor cells by comparing the magnitude of intercellular adhesion forces.

[0047] 3. The magnetic boson probe used in this invention has the characteristics of easy mass production, high remanence, and biocompatibility.

[0048] 4. This invention simulates the in vivo environment to construct an intercellular adhesion system. The magnetic probe used does not interfere with intercellular adhesion, and the measured values ​​are closer to the real environment. Attached Figure Description

[0049] Figure 1 TEM characterization of the ultra-low field force spectrum magnetosome probe prepared in Example 1 of this invention.

[0050] Figure 2 TEM characterization of the outer film of the ultra-low field force spectrum magnetosome probe prepared in Example 1 of the present invention.

[0051] Figure 3 TEM characterization of cells labeled with ultra-low field force spectrum magnetosome probes prepared in Example 3 of this invention.

[0052] Figure 4 The cell-labeled remanent magnetic signal of the ultra-low field force spectrum magnetic probe magnetosome prepared in Example 3 of this invention.

[0053] Figure 5 This is a characterization of the cell labeling time stability of the ultra-low field force spectrum magnetic probe magnetosomes prepared in Example 3 of the present invention.

[0054] Figure 6 Cytotoxicity characterization of the ultra-low field force spectrum magnetic probe magnetosomes prepared in Example 3 of this invention.

[0055] Figure 7 This is a schematic diagram of the measurement of intercellular adhesion force in ultra-low field force spectrum in Embodiment 4 of the present invention.

[0056] Figure 8 The force-dependent remanent magnetization spectrum and quantified intercellular adhesion force between HUVEC cells and MDA-MB-231 cells as measured by FIRMS in Example 4 of this invention are shown below. Figure 8 (a) is the force-dependent remanence spectrum. Figure 8 (b) represents the quantified intercellular adhesion force.

[0057] Figure 9 This is a remanent magnetization spectrum showing the adhesion force between the lower layer cells labeled with magnetosomes and the substrate in Embodiment 4 of the present invention.

[0058] Figure 10This refers to the intercellular adhesion between HUVEC cells and three different types of tumor cells (MDA-MB-231, MCF-7, and MCF-10A) as measured by FIRMS in Example 5 of this invention. Detailed Implementation

[0059] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0061] Example 1: Fabrication of ultra-low field magnetic probe magnets

[0062] AMB-1 bacterial samples were collected and PBS buffer (10 mM) was added until the bacterial-to-PBS buffer ratio was 1:10 (w / v). The samples were then sonicated at 300 W for 30 min in an ice bath (3 s working time, 5 s interval). The lysed cells were then adsorbed onto a magnet for 12 h to separate the precipitated magnetosomes, which were resuspended in PBS buffer (1:1, w / v). The resuspended samples were repeatedly sonicated in an ice bath at ultrasonic powers of 250 W, 200 W, 150 W, 100 W, and 80 W for 15 min (3 s working time, 5 s interval). After each washing, the magnetosomes were adsorbed onto a magnet for 15 min and then resuspended in PBS buffer (1:1, w / v). The final resuspended sample was collected and stored at 4 °C to obtain the ultra-low field magnetosome probe.

[0063] TEM images of the obtained ultra-low field magnetosome probe are shown below. Figure 1 The magnetic bosons have an average particle size of 48.7 ± 8.9 nm, exhibiting good size uniformity and dispersibility. The obtained ultra-low field magnetic boson probes are encapsulated by a natural phospholipid film, and the core-film structure of the ultra-low field magnetic boson probes is as follows: Figure 2 As shown, the film thickness is 4 nm.

[0064] Example 2: Stable adhesion between cells and substrate

[0065] The substrate (a cuboid made of polydimethylsilane, with dimensions of 1.4 cm x 0.6 cm x 0.2 cm, and a square depression of 0.1 cm on each side, with a side length of 0.5 cm) was incubated with fibronectin (50 μg / mL) for 4 h. The fibronectin solution was then removed, and the substrate was washed three times with complete cell culture medium. Then, 5 × 10⁻⁶ cells / mL were added to the substrate. 4 Personal umbilical vein endothelial cells (HUVECs) were seeded on a fibronectin-modified substrate and cultured in a cell culture incubator at 37°C for 12 hours until they fused into a monolayer of cells for later use.

[0066] Example 3: Cellular Magnetosome Probe Labeling

[0067] 1×10 6 One MDA-MB-231 cell line was seeded in a 6-well cell culture plate and cultured at 37°C for 16 h. The culture medium was then removed, and the cells were washed three times with PBS buffer. 2 mL of serum-free medium containing magnetosomes (50 μg / mL) was added, and the cells were cultured for 4 h. The culture medium was then aspirated, and the cells were washed five times with PBS buffer. The magnetosome-labeled cells were then digested with trypsin for 2 min, and digestion was terminated by adding complete culture medium. The cells were collected, centrifuged at 1200 rpm for 3 min, and the cell pellet was redispersed in complete culture medium for later use.

[0068] TEM images of the obtained cells labeled with magnetosome probes are shown below. Figure 3 As shown, the magnetosome probe is internalized into intracellular vesicles such as endosomes or lysosomes. The intracellular labeling remanent magnetic signal of the magnetosome probe is as follows: Figure 4 As shown. The temporal stability of the intracellular labeling remanent magnetization signal of the magnetosome is as follows. Figure 5 As shown, the remanent magnetic signal of magnetosomes within the cells did not change significantly within 36 hours. To verify whether the obtained magnetosome probes affected cell viability, a 24-hour cytotoxicity assessment was performed, as follows: Figure 6 As shown, within a concentration range of 100 μg / mL, the obtained magnetosome probes had no significant effect on the activity of MCF-7 cells.

[0069] Example 4: Measurement of intercellular adhesion force

[0070] like Figure 7 The schematic diagram of intercellular adhesion force measurement shows that 5×10 4 Personal umbilical vein endothelial cells (HUVECs) were seeded on a fibronectin-modified substrate and cultured for 12 hours until they merged into a monolayer. Then, 5 × 10⁻⁶ cells were added to the substrate. 4Human breast cancer cells (MDA-MB-231) labeled with magnetosome probes were incubated in a cell culture incubator for 30 min. Then, the cells were magnetized with a 1T magnet at a distance of 2 cm for 2 min, and the initial remanent magnetization signal was measured by FIRMS. Subsequently, centrifugation was used to dissociate the upper layer of magnetosome-labeled cells from the lower layer. As the centrifugal force gradually increased, the number of dissociated upper layer magnetosome-labeled cells increased, leading to a continuous decrease in the remanent magnetization signal. The magnitude of the intercellular adhesion force can be obtained from the FIRMS-recorded remanent magnetization signal versus centrifugal force spectrum. The formula for calculating the centrifugal force on the cells is: F = (ρ cell –ρ medium )·V cell ·ω 2 ·r, where F is the centrifugal force, ρ cell It is the average cell density (~1.07 kg·m³). -3 ), ρ medium It is the average density of the cell culture medium (~1 kg·m³). -3 V cell The average cell volume is (~525 μm) 3 ω is the centrifugal angular velocity, and r is the distance between the sample and the center of rotation (0.045 m). The relative adhesion force between cells is defined as the centrifugal force corresponding to a 50% reduction in remanence signal. The remanence spectra and relative adhesion forces between human breast cancer cells (MDA-MB-231) and human umbilical vein endothelial cells (HUVECs) are shown below. Figure 8 a and Figure 8 As shown in b.

[0071] To verify that the change in remanent magnetization signal was due to dissociation between upper and lower cells labeled by the magnetosome probe, rather than dissociation between lower cells and the substrate, 5×10 4 MCF-7 cells labeled with magnetosome probes were seeded on a fibronectin-modified substrate. After culturing for 12 hours to form a cell monolayer, the cells were magnetized for 2 minutes at a distance of 2 cm using a 1T magnet. The initial remanent magnetization signal was measured by FIRMS, and the change in remanent magnetization signal with gradually increasing centrifugal force was recorded. Figure 9 As shown, as the centrifugal force gradually increased from 12 pN to 163 pN, the remanent magnetic signal between the MCF-7 cells labeled with the magnetosome probe and the substrate did not change significantly. This indicates that the adhesion between the cells and the substrate is stable within the 163 pN force range, and the magnetosome probe stably labels cells and exhibits good mechanical stability. Based on these results, it can be inferred that the decrease in remanent magnetic signal with centrifugal force in the intercellular adhesion force measurement system is due to the dissociation between the upper and lower cells, meaning that intercellular adhesion force can be measured using the FIRMS method.

[0072] Example 5: Expanding FIRMS technology for identifying tumor cell metastatic potential

[0073] According to the method for determining the intercellular adhesion between human breast cancer cells (MDA-MB-231) and human umbilical vein endothelial cells (HUVECs) using FIRMS in Example 4 of this invention, the intercellular adhesion between three different types of invasive tumor cells (MDA-MB-231, MCF-7, and MCF-10A) and HUVECs was measured. Since both internal and external invasion during tumor metastasis involve interaction with endothelial cells, the metastatic potential of tumor cells can be determined by the magnitude of the adhesion force between tumor cells and endothelial cells.

[0074] like Figure 10 As shown, the intercellular adhesion between MDA-MB-231, MCF-7, and MCF-10A cells and HUVECs decreased sequentially. This result is consistent with the known invasiveness of MDA-MB-231, MCF-7, and MCF-10A cells (the invasiveness of MDA-MB-231, MCF-7, and MCF-10A cells decreases sequentially), indicating that intercellular adhesion can be measured by FIRMS to identify the metastatic potential of tumor cells.

[0075] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for measuring cell-cell adhesion force in an ultra-low field, comprising the following steps: (1) Stable adhesion of lower layer cells to substrate: Cells were seeded in a fibronectin-modified substrate and cultured until a monolayer fusion state was obtained to obtain lower layer cells that were stably adhered to the substrate. (2) Magnetic probe labeling of upper layer cells: upper layer cells are co-incubated with magnetosome probes to obtain magnetically probe-labeled cells; (3) Construction of intercellular adhesion system: The cells labeled with magnetic probe in step (2) were seeded on the lower layer of cells that formed stable adhesion with the substrate in step (1) and cultured and magnetized; (4) Measurement of intercellular adhesion force: The intercellular adhesion system obtained by external force interference in step (3) causes the upper layer cells to separate from the lower layer cells, and the remanent magnetic signal change spectrum of the dissociation process is recorded by an ultra-low field magnetometer. in, The force corresponding to a decrease of half in remanence signal is defined as the intercellular adhesion force; In step (1), the substrate material is polydimethylsilane; The base is a cuboid; The surface of the substrate is provided with a groove; The fibronectin-modified substrate was prepared by the following method: The substrate was incubated with fibronectin, the fibronectin solution was removed, and the fibronectin-modified substrate was washed with complete cell culture medium to obtain the final product. The concentration of the fibronectin is 20–50 μg / mL; The incubation temperature is 4℃-37℃, and the time is 2h-12h; The cell culture temperature is 35-37℃, and the time is 10-24 hours; In step (2), the upper layer of cells are cancer cells. The magnetosomes are derived from magnetotactic bacteria; The magnetosomes are prepared by a method including the following steps: taking a sample of magnetotactic bacteria, sonicating it in PBS buffer, using a magnet to adsorb the lysed cells, separating the precipitate, and thus obtaining the magnetosomes; The ratio of cells to magnetosome probes is 1×10⁻⁶. 6 Individuals: 20-500 μg; The co-incubation temperature is 35-37℃, and the time is 2-12 hours; In step (3), the ratio of the number of upper layer cells to the number of lower layer cells is 0.3 to 2:1; The culture time for the upper and lower cell layers is 30 min to 1 h; The magnetization is performed using a magnet; In step (4), the external force is centrifugal force, and the intensity of the centrifugal force is 12pN-163pN.

2. The method according to claim 1, characterized in that: The strength of the magnet is 0.5-1T, the magnetization distance is 1.5-2cm, and the magnetization time is 1-5 min.

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