Gradient magnetic field device for targeted transplantation of magnetized cells, and application thereof

Abstract

A gradient magnetic field device for targeted transplantation of magnetized cells, and an application thereof. A magnetic pole pair, a magnet yoke and an electromagnetic coil are used to constitute an electromagnet; the magnetic pole pair consists of a convex circular cylindrical surface small magnetic pole and a concave circular cylindrical surface large magnetic pole, which are coaxial and directly face each other; a gradient magnetic field is formed in a gap region of the magnetic pole pair, and magnetized cells are subjected to magnetic attraction due to the gradient of the magnetic field, thereby realizing targeted attraction of the magnetized cells; moreover, a magnetic field gradient value at any point in a gradient magnetic field space is in direct proportion to the total current of the electromagnetic coil, such that the magnetic attraction applied to the magnetized cells is adjusted by means of adjusting the current.

Description

Gradient magnetic field device for magnetized cell targeted transplantation and its application Technical Field

[0001] The present invention relates to the field of magnetic field technology, in particular to a gradient magnetic field device for magnetized cell targeted transplantation and its application. Background Art

[0002] Regenerative medicine has always been a hot topic in medical research. Cell transplantation is an important research direction in regenerative medicine. It has the function of replacing and supplementing locally defective cells, and is expected to treat diseases caused by the loss of non-dividing cells (such as neurons and other cells). The transplantation of some special cells, such as mesenchymal stem cells (MSC) and neural stem cells (NSC), also has the advantages of differentiation (such as the transformation of hematopoietic stem cells into different blood cells), anti-inflammatory (such as neural stem cells secreting cytokines, promoting the polarization of brain glial cells M1 into M2, alleviating local inflammation, etc.), and low immunogenicity. At present, several types of cell transplantation treatments have achieved good results in clinical practice, bringing good news to patients with diseases that are difficult to respond to simple surgical treatment and chemotherapy.

[0003] There are two main methods of cell transplantation: in situ injection and vascular infusion. In situ injection has high cell transplantation efficiency, but it requires the use of in situ puncture and other means, which is relatively traumatic. The impact on important parts and tissues of the central nervous system cannot be ignored, and may ultimately lead to poor prognosis. Compared with in situ injection, vascular infusion can avoid important parts and tissues and achieve systemic cell transplantation. Vascular infusion is further divided into arterial puncture and venous puncture. Arterial puncture is more dangerous and difficult to operate, so it is less used; veins are superficial, and the puncture operation is safe and easy. Therefore, intravenous infusion is the preferred cell transplantation method in experimental research and clinical operations, in addition to in situ injection, such as hematopoietic stem cell transplantation in clinical practice.

[0004] Although intravenous infusion is safe and easy, after cells enter the systemic blood circulation system through the veins, they are often intercepted by the capillaries of organs such as the lungs, liver, spleen, and kidneys, making it difficult for them to reach and remain in the target transplant site. In addition, after being diluted by the circulating blood, the cell concentration decreases, further reducing the transplantation efficiency. Therefore, how to modify and label cells and improve the targeting and efficiency of cell transplantation has become an urgent problem to be solved for intravenous infusion of transplanted cells. Previous studies have modified and labeled cells in various ways (such as embedding corresponding ligands or receptors into the cell membrane, specifically binding to the target area to enhance its targeting, etc.), but some of these methods will change the characteristics of the cells themselves and affect the normal physiological activities of the cells; or they are more cumbersome and have a long processing cycle, which reduces the vitality of the cells and makes it difficult to mass produce and clinically transform them.

[0005] The application of superparamagnetic nanoparticles (SPIONs) offers new insights into cell modification and labeling. SPIONs are iron oxides (such as Fe2O3 and Fe3O4) with small (nanoscale) particle sizes, allowing direct cellular uptake and high biocompatibility. SPIONs can also be surface-coated or loaded into the pores of materials for cellular uptake. SPIONs have low toxicity and do not alter normal cellular function even at high doses. Their powerful magnetic responsiveness and superparamagnetism, when attracted by an external magnetic field, enable labeled cells to acquire magnetic response and targeted migration capabilities, significantly improving the efficiency of targeted cell transplantation.

[0006] For exogenous magnetic fields, permanent magnets are commonly used in domestic and foreign studies, such as the SCI paper "High-efficiency magnetic targeting of mesenchymal stem cells in spinal cord injury" in the International Journal of Nanomedicine. In this paper, two types of permanent magnets were used: (1) flat neodymium magnets with geometric dimensions of 13×7×2mm, surface magnetic induction intensity Bs=0.35T, residual magnetic field Br=1.2T, and the described magnetic field line distribution (see Figure 4-1 provided in the paper for details); (2) stepped neodymium magnets, with one step added to the center of the first neodymium magnet. The step geometric dimensions are 4×7×2mm, and other parameters remain unchanged. The magnetic field line distribution (see Figure 4-2 provided in the paper for details) is 4-4.5mm from the neodymium magnet to the spinal cord. It can be seen that the magnetic induction intensity generated by it is uncontrollable, the spatial distribution of magnetic field lines is difficult to describe, and the maximum magnetic induction intensity of the magnet surface is limited (Bs=0.35T), making it difficult to estimate and control the magnetic response and targeted migration of magnetically labeled cells, and therefore, it is difficult to achieve satisfactory transplantation and treatment effects. To achieve better magnetic targeting transplantation effects, it is necessary to increase the magnetic induction intensity of the exogenous magnetic field and improve the spatial distribution of magnetic lines of force, or increase the magnetic targeting attraction time.

[0007] A few previous studies have used electromagnets to provide an external magnetic field, but these devices often provide a dispersed magnetic field, targeting a wide range of sites and lacking precision, making the results difficult to quantify and translate clinically. Other studies have used electromagnets to provide an external magnetic field for magnetic drugs, but these are not suitable for cell transplantation.

[0008] In summary, the use of exogenous magnetic fields, such as permanent magnets or electromagnets, in research on magnetized cell-targeted transplantation has hindered the development and clinical translation of magnetically targeted cell transplantation experiments due to issues such as the uncontrollable magnetic induction intensity of permanent magnets, the poor description of the spatial distribution of magnetic field lines, and the limited maximum magnetic induction intensity on the magnet surface. Furthermore, the dispersion of the magnetic field of electromagnets, the large targeting range, and the lack of precision have hindered the development of magnetically targeted cell transplantation experiments. Currently, there are no reports on the design of electromagnet devices specifically for magnetically targeted cell transplantation.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide a gradient magnetic field device for magnetized cell targeted transplantation and its application to solve the problems existing in the above-mentioned prior art.

[0011] To achieve the above-mentioned object, the present invention provides the following solution: a gradient magnetic field device for magnetized cell targeted transplantation, comprising:

[0012] A magnetic pole pair, the magnetic pole pair comprising a small magnetic pole and a large magnetic pole, the top surface of the small magnetic pole being arranged in a convex arc cylindrical surface, the end surface of the large magnetic pole facing the small magnetic pole being arranged in a concave arc cylindrical surface, and a gradient magnetic field being formed in the space facing the small magnetic pole and the large magnetic pole;

[0013] A magnetic yoke, wherein the magnetic yoke includes a second magnetic yoke rod, and the two ends of the second magnetic yoke rod are respectively connected to a first magnetic yoke rod and a third magnetic yoke rod, the end of the first magnetic yoke rod away from the second magnetic yoke rod is connected to the large magnetic pole, and the end of the third magnetic yoke rod away from the second magnetic yoke rod is connected to the small magnetic pole, and the small magnetic pole, the large magnetic pole, the first magnetic yoke rod, the second magnetic yoke rod, the third magnetic yoke rod, and the space between the magnetic pole pair together constitute a closed magnetic circuit;

[0014] An electromagnetic coil, comprising four parallel coils, the coils being sleeved on the second magnetic yoke rod and used to provide magnetic potential to the magnetic circuit;

[0015] The bottom plate has a top surface connected to the yoke rod 1 and the yoke rod 3 via four bolts, and the bottom surfaces of the bottom plate, the yoke rod 1, and the yoke rod 3 are equipped with four feet.

[0016] Preferably, the radius of the convex arc cylinder of the small magnetic pole is 2 mm, and the height is 4 mm. The small magnetic pole is arranged in a quadrangular pyramid, and the top surface of the quadrangular pyramid is a convex arc cylinder with an axis perpendicular to the horizontal plane. The left and right sides of the cone are tangent to the convex arc cylinder, and the upper and lower sides of the cone are inclined planes; the radius of the concave arc cylinder of the large magnetic pole is 45 mm, the chord width is 84.57 mm, and the height is 30 mm. The appearance dimensions of the large magnetic pole are a rectangular parallelepiped with a length of 135 mm, a width of 88 mm, and a height of 30 mm.

[0017] Preferably, the convex arc cylindrical surface of the small magnetic pole and the concave arc cylindrical surface of the large magnetic pole are coaxial and arranged opposite each other, and their axes are perpendicular to the horizontal plane.

[0018] Preferably, the bottom surface of the small magnetic pole and the bottom surface of the large magnetic pole are both rectangles with a width of 88 mm and a height of 30 mm.

[0019] Preferably, the cross-sections of the first magnetic yoke rod, the second magnetic yoke rod, and the third magnetic yoke rod are all rectangular with a height of 30 mm.

[0020] Preferably, the number of turns of the coil is 1164, the wire diameter is 0.49 mm, the inner hole of the coil is a rectangular through hole, and the size of the rectangular through hole is 63 mm long*30 mm wide*40 mm deep.

[0021] Preferably, there is a gap between the small magnetic pole and the arc cylinder facing the large magnetic pole, and a working area is selected in the gap. The magnetic lines of force in the working area are projected as a fan in the horizontal plane and as an isosceles trapezoid in the vertical plane where the radius line of the arc cylinder is located.

[0022] Preferably, the material of the small magnetic pole, the large magnetic pole, the first magnetic yoke rod, the second magnetic yoke rod, and the third magnetic yoke rod are all electrical pure iron.

[0023] The present invention also provides an application of a gradient magnetic field device for targeted transplantation of magnetized cells. By energizing the electromagnetic coil, a magnetic potential is generated, and a gradient magnetic field is generated between the magnetic pole pairs. In this gradient magnetic field, the magnetized cells are subjected to a magnetic force directed toward the top of the small magnetic pole. By adjusting the current of the electromagnetic coil, the targeted migration speed of the magnetized cells can be adjusted, so that the target point of the targeted migration of the magnetized cells can be more clearly described and the migration speed can be controlled.

[0024] The present invention discloses the following technical effects:

[0025] The present invention generates a magnetic potential by energizing an electromagnetic coil, and the magnetic potential can be adjusted by adjusting the current of the electromagnetic coil. The geometric shape, size and position of the magnetic pole pair are used to determine the distribution of magnetic lines of force between the magnetic pole pairs. The projection on the horizontal plane is fan-shaped, and the projection on the vertical plane where the radius line of the arc cylinder is located is an isosceles trapezoid. Such a spatial magnetic field is a gradient magnetic field. At the same time, a mathematical function of the gradient magnetic field is established. Magnetized cells in the gradient magnetic field are subjected to a magnetic force directed toward the top of the small magnetic pole. The magnetized cells overcome their own gravity and the resistance of the liquid environment in which they are located, migrate toward the top of the small magnetic pole, and finally gather in an area near the top of the small magnetic pole, achieving targeted migration. The speed of the targeted migration of the magnetized cells is positively correlated with the current of the electromagnetic coil. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] FIG1 is a schematic structural diagram of the present invention;

[0028] FIG2 is a diagram showing the power supply and circuit principles of the present invention;

[0029] FIG3 is a projection of the magnetic lines of force of the present invention on a horizontal plane;

[0030] FIG4 is a projection of the magnetic lines of force of the present invention on the vertical plane where the radius line of the arc cylinder is located;

[0031] FIG5 shows the isosceles trapezoidal parameters of the magnetic lines of force projected on the vertical plane where the radius line of the arc cylinder is located;

[0032] FIG6 is a magnetic induction intensity curve of the working area of ​​the present invention (electromagnetic coil I=1A);

[0033] FIG7 is a magnetic field gradient curve of the working area of ​​the present invention (electromagnetic coil I=1A);

[0034] FIG8 is a diagram of detecting cell viability using CCK-8 of the present invention;

[0035] FIG9 is a Perls staining of the present invention after formaldehyde fixation;

[0036] FIG10 shows the effect of the present invention on attracting magnetized cells in vitro (photographed under a microscope);

[0037] FIG11 shows the effect of attracting magnetized cells in vitro (liquid phase bottle method) of the present invention;

[0038] FIG12 is a comparison of the states of suspended magnetized HUVECs before and after attraction according to the present invention;

[0039] FIG13 shows the present invention attracting magnetic cells in nude mice (targeting the left cerebral hemisphere);

[0040] FIG14 is a fluorescence imaging diagram of the present invention showing the in vivo attraction effect;

[0041] FIG15 is a fluorescence imaging diagram of the present invention showing the in vivo attraction effect of different working currents;

[0042] FIG16 is a fluorescence imaging diagram of the present invention showing the in vivo attraction effect of groups with different attraction time lengths;

[0043] Among them, 1. Small magnetic pole; 2. Large magnetic pole; 3. Magnetic yoke rod 1; 4. Magnetic yoke rod 2; 5. Magnetic yoke rod 3; 6. Electromagnetic coil; 7. Base plate; 8. Foot. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0045] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0046] 1-7 , the present invention provides a gradient magnetic field device for magnetized cell targeted transplantation, comprising: a magnetic pole pair, the magnetic pole pair comprising a small magnetic pole 1 and a large magnetic pole 2, the top surface of the small magnetic pole 1 being a convex arc cylindrical surface, the end surface of the large magnetic pole 2 facing the small magnetic pole 1 being a concave arc cylindrical surface, and the space facing the small magnetic pole 1 and the large magnetic pole 2 forming a gradient magnetic field; a magnetic yoke, the magnetic yoke comprising a second magnetic yoke rod 4, the two ends of the second magnetic yoke rod 4 being respectively connected to a first magnetic yoke rod 3 and a third magnetic yoke rod 5, the end of the first magnetic yoke rod 3 away from the second magnetic yoke rod 4 being connected to the large magnetic pole 2, One end of the yoke rod three 5 away from the yoke rod two 4 is connected to the small magnetic pole 1, and the small magnetic pole 1, the large magnetic pole 2, the yoke rod one 3, the yoke rod two 4, the yoke rod three 5 and the space between the magnetic pole pairs together constitute a closed magnetic circuit; the electromagnetic coil, the electromagnetic coil includes four parallel coils 6, the coils 6 are sleeved on the yoke rod two 4, and the coils 6 are used to provide magnetic potential to the magnetic circuit; the bottom plate 7, the top surface of the bottom plate 7 is connected to the yoke rod one 3 and the yoke rod three 5 by four bolts, and the bottom surfaces of the bottom plate 7, the yoke rod one 3, and the yoke rod three 5 are installed with four feet 8.

[0047] Further optimization plan, the radius of the convex arc cylinder of the small magnetic pole 1 is 2mm, the height is 4mm, the appearance of the small magnetic pole 1 is a horizontal quadrangular pyramid, the height of the quadrangular pyramid is 152mm, the top surface of the cone is a convex arc cylinder with the axis perpendicular to the horizontal plane, the left and right sides of the cone are tangent to the convex arc cylinder, and the upper and lower sides of the cone are inclined planes; the radius of the concave arc cylinder of the large magnetic pole 2 is 45mm, the chord width is 84.57mm, and the height is 30mm. The appearance size of the large magnetic pole 1 is a rectangular parallelepiped with a length of 135mm, a width of 88mm, and a height of 30mm.

[0048] In a further optimization, the convex arc cylinder of the small magnetic pole 1 and the concave arc cylinder of the large magnetic pole 2 are coaxial and face-to-face, with their axes perpendicular to the horizontal plane. Coaxial means the axes of the two arc cylinders coincide, and facing means the two radii passing through the center points of the two arc cylinders lie on the same straight line.

[0049] To further optimize the solution, the opposite side of the convex arc cylinder of the small magnetic pole 1 is a rectangle, and the opposite side of the concave arc cylinder of the large magnetic pole 2 is also a rectangle. Both rectangles are 88mm wide and 30mm high.

[0050] To further optimize the solution, the cross-sections of yoke rod 1 3, yoke rod 2 4, and yoke rod 3 5 are all rectangular with a height of 30 mm. After assembly, the top and bottom surfaces of the iron parts are aligned at the joints to reduce the escape of magnetic lines of force at the joints.

[0051] A further optimization scheme has been developed, with coil 6 having 1164 turns and a wire diameter of 0.49mm. The inner hole of coil 6 is a rectangular through-hole with dimensions of 63mm long, 30mm wide, and 40mm deep. Under natural cooling conditions, when the total current of the electromagnetic coil is less than 3.2A, the coil temperature rise is less than 50°C. Since "total current × number of turns per coil = magnetic potential," the magnetic potential in the magnetic circuit can be adjusted by adjusting the total current.

[0052] A further optimization scheme involves creating a gap between the arc-shaped cylinders facing the small magnetic pole 1 and the large magnetic pole 2. A working area is selected within this gap. The magnetic field lines within this working area are projected as a sector on the horizontal plane and an isosceles trapezoid on the vertical plane corresponding to the radius of the arc-shaped cylinder. This allows for a mathematical function to describe the magnetic induction intensity and magnetic field gradient within the working area.

[0053] To further optimize the solution, the small magnetic pole 1, large magnetic pole 2, yoke rod 1 3, yoke rod 2 4, and yoke rod 3 5 are all made of electrically pure iron. When there is no magnetic potential in the magnetic circuit, the residual magnetism is low, allowing the magnetic induction intensity and magnetic field gradient to vary with the coil current, thereby controlling the magnetic attraction force on the magnetized cells in the working area.

[0054] Due to the geometry, size, and position of the magnetic pole pairs, a gradient magnetic field, described by a mathematical function, is formed in the gap between the pole pairs. This magnetic field gradient causes the paramagnetic particles to experience a magnetic force directed toward the tip of the small magnetic pole. The electromagnetic coil consists of four coils 6 connected in parallel. The enameled copper wire of all coils 6 is wound in the same direction and is powered by an adjustable DC regulated power supply with parameters of 0-32V and 0-5A. During use, due to the magnetic saturation characteristics of electrical pure iron, when the current I is less than 2.0A, the magnetic induction intensity is approximately proportional to the current; when I is greater than or equal to 2.0A, the magnetic induction intensity at the center of the arc cylindrical surface of the small magnetic pole 1 begins to saturate; when I is equal to or equal to 3.5A, the arc surface of the small magnetic pole 1 tends to magnetic saturation. At this time, the magnetic induction intensity at the center of the arc surface of the small magnetic pole 1 is 0.556T (the surface magnetic induction intensity of the neodymium magnet used in the paper, ABC Magnet Ltd, Prague, Czech, is Bs=0.35T). Therefore, when using, the current should not exceed 3.2A, otherwise the magnetic induction intensity will no longer increase due to saturation, but the coil resistance heating power will increase with I 2 The increase causes the coil temperature to rise too much.

[0055] To further optimize the solution, the working area distance can be increased or decreased as needed. The small magnetic pole 1 remains unchanged, and only the radius R = 45 ± δ mm and the length of the concave arc cylinder of the large magnetic pole 2 need to be changed. All other parameters, including the shape, size, and position of the magnetic pole pair, remain unchanged. After the design modification, a new large magnetic pole 2 is fabricated from electrical pure iron and replaced. The modified gradient magnetic field maintains a fan-shaped distribution of magnetic field lines when projected horizontally, and an isosceles trapezoidal shape when projected vertically along the radius of the circular arc cylinder. However, the height of the trapezoid, H, is set to 43 ± δ mm, and the function describing the magnetic field gradient changes accordingly.

[0056] To further optimize the present invention, to increase the saturation magnetic flux density, soft magnetic materials with a higher saturation magnetic flux density than electrical pure iron, such as silicon steel sheets and Permalloy, can be used. If silicon steel sheets are used, magnetic saturation will not occur at the center of the convex arc cylindrical surface of the small magnetic pole 1 when the magnetic flux density on the surface of the small magnetic pole 1 is less than 1.5T.

[0057] The present invention also provides an application of a gradient magnetic field device for magnetized cell targeted transplantation, wherein a magnetic potential is generated by energizing an electromagnetic coil, a magnetic circuit is formed by a magnetic yoke, a magnetic pole pair, and the space between the magnetic pole pairs, and the magnetic pole pairs are designed to have specific geometric dimensions, shapes, and installation positions, so that a describable gradient magnetic field is formed in the space between the magnetic pole pairs; in the gradient magnetic field, an area with a relatively regular distribution of magnetic lines of force is selected as a working area, and at any point in the working area, the magnetic field gradient G x The size is proportional to the current I of the electromagnetic coil and is a function of the distance x from the axis of the convex arc cylinder. Indicates that the direction of the gradient magnetic field points to the axis of the convex arc cylinder, and the paramagnetic particles are subjected to a magnetic force F = M·G in the gradient magnetic field. x In the equation, M is the degree to which the particle is magnetized, G x It is the magnetic field gradient at the location of the particle; the magnetic field gradient, that is, the magnetic force F exerted on the magnetized cell, is controlled by controlling the current of the electromagnetic coil, so that the target point of the magnetized cell's targeted migration can be clearly described and the migration speed can be controlled.

[0058] Referring to Figure 3-5, the magnetic field in the working area, the projection of the magnetic lines of force on the horizontal plane is fan-shaped, and the projection of the magnetic lines of force on the vertical plane where the radius line of the arc cylinder is located isosceles trapezoidal.

[0059] The magnetic induction intensity on the center surface of the small pole arc cylinder was measured using a "VICTOR 862A digital display Teslameter", and the results are shown in Table 1.

[0060] Table 1: Magnetic induction intensity at the center surface of the small pole arc cylinder

[0061] Note [1]: Rate of change n =(average n+1 -average value n ) / (Current n+1 - Current n ).

[0062] Data analysis in Table 1: When the current is less than 2.0A, the magnetic induction intensity is approximately proportional to the current; when I is greater than or equal to 2.0A, the magnetic induction intensity at the center of the small magnetic pole arc cylinder begins to saturate; when I is equal to or equal to 3.5A, the small magnetic pole arc cylinder tends to magnetic saturation, at which time the magnetic induction intensity at the center surface of the small magnetic pole arc cylinder is 0.556T (the magnetic induction intensity Bs of the neodymium magnet surface used in the paper is 0.35T of ABC Magnet Ltd, Prague, Czech Republic).

[0063] Take any point in the working area that is xmm away from the axis of the small magnetic pole arc cylinder as the research object

[0064] When the current I is less than 2.0A, the following equation can be approximately considered to be true: r =0.207I (1)

[0065] In formula (1)

[0066] B r : Magnetic induction intensity at the center surface of the convex arc cylinder (x = r), unit T

[0067] I: total input current of electromagnet, unit A

[0068] The projection of the magnetic lines of force on the horizontal plane of the working area is approximately fan-shaped (Figure 3), and the projection on the vertical plane where the radius line of the arc surface is located is approximately an isosceles trapezoid (Figure 5). The area S of the arc cylinder through which the original magnetic lines of force pass at a distance xmm from the axis of the arc cylinder is obtained. x Relationship with x. S x =(θ·x)·h

[0069] In formula (2)

[0070] θ: fan angle of horizontal projection, unit rad

[0071] h2: The height of the concave arc cylinder of the large magnetic pole is 30mm

[0072] h1: The height of the convex arc cylinder of the small magnetic pole is 4mm

[0073] R: The radius of the concave arc cylinder is 45mm

[0074] r: radius of the convex arc cylinder 2mm

[0075] Since "the magnetic flux through any closed surface is 0", we can get "the magnetic flux of the convex arc cylinder = the magnetic flux of the original magnetic field line on the arc cylinder with radius x", so B r ·S r =B x ·S x(3)

[0076] In formula (3), the area S of the convex arc cylinder is r =r·θ·h1

[0077] The solution of the combined equations (2) and (3) is

[0078] In formula (4), the fraction is a dimensionless factor. Substituting the numerical value, we get

[0079] Formula (1)B r =0.207I Substitute, we get

[0080] In formula (5):

[0081] x: distance from the axis of the small magnetic pole arc cylinder, unit: mm

[0082] I: total input current of electromagnetic coil, unit A

[0083] To B x Take the derivative of x and get the magnetic field gradient G x G x =d(B x ) / dx

[0084] Use Excel software to perform numerical analysis on equations (5) and (6), setting current I = 1A, and we get Table 2 and Figures 6 and 7 are obtained through numerical analysis.

[0085] It can be seen that as x increases, the magnetic field gradient G x It quickly becomes smaller, and the negative sign represents that the direction of the magnetic field gradient is opposite to the x direction, that is, pointing to the axis of the small magnetic pole arc cylinder; x increases from 2 to 10mm. The magnetic field gradient G x Reduced to 2.4% (see Table 2, G2 = -0.1347, G 10 =-0.0032, G 10 / G2=2.4%).

[0086] Table 2: Spatial distribution of magnetic induction intensity and magnetic field gradient (I = 1A)

[0087] Application Example: Magnetized cells were first cultured, and then the present invention was used to verify the effect of targeted attraction of magnetized cells in nude mice. The application example was divided into seven experiments in chronological order: Experiment 1, determining the concentration of SPIONs used; Experiment 2, SPION uptake by target cells; Experiment 3, the effect of the present invention on attracting magnetized cells in vitro (culture dish method); Experiment 4, the effect of the present invention on attracting magnetized cells in vitro (liquid phase flask method); Experiment 5, the effect of the present invention on attracting magnetized cells in vivo; Experiment 6, the optimal operating current of the present invention; Experiment 7, the optimal attraction duration of the present invention. The following describes these seven experiments in detail.

[0088] Experiment 1: Determine the concentration of SPIONs used

[0089] Purpose and Materials: This invention provides an external magnetic field for magnetized cell targeted transplantation experiments or clinical trials, so preparing magnetized cells is a necessary preparatory step. With the goal of promoting vascular repair through targeted cell transplantation after craniocerebral trauma, human umbilical vein endothelial cells (HUVECs) with the ability to form new blood vessels, commonly used in angiogenesis experiments, were selected. These cells were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.; SPION solution (particle size 30-50 nm, concentration 5 mg / mL, purchased from Aladdin Biochemical Technology Co., Ltd.); HUVEC-specific culture medium; trypsin; PBS; and a Cell Counting Kit-8 (CCK-8) kit (Biyuntian).

[0090] Procedure: SPION was added to HUVEC culture medium at final concentrations of 0, 10, 20, 40, 60, 80, 100, and 120 μg / mL, and HUVEC was incubated for 24 hours. Since the amount of SPION solution used was small, the dilution of the culture medium by the solute (water) was negligible. HUVEC cell viability (absorbance at 450 nm) was measured using a CCK-8 kit on a microplate reader.

[0091] Results and Conclusion: As shown in Figure 8, HUVEC were labeled with SPION at different concentrations for 24 h, and cell viability was detected by CCK-8. It can be seen that when HUVEC were incubated with SPION at a concentration higher than 60 μg / mL, cell viability would decrease, affecting the success rate of transplantation. Therefore, a SPION concentration of 60 μg / mL was selected to continue.

[0092] Experiment 2: SPION uptake by target cells

[0093] Objective and Materials: After determining the SPION concentration, it is necessary to verify that the cells have taken up the SPIONs, which is necessary for the cells to possess magnetic properties and be able to migrate in response to an external magnetic field. Materials used are the same as those in Experiment 1, including 4% formaldehyde solution and Perls' Prussian Blue staining kit (Biyuntian).

[0094] Procedure: Incubate HUVECs with HUVEC-specific culture medium containing 60 μg / mL SPION for 24 hours, rinse three times with PBS, and detect using the Perls assay kit.

[0095] Results and Conclusion: As shown in Figure 9, after formaldehyde fixation, Perls staining showed that the SPIONs in HUVEC cells appeared blue (scale bar = 50 μm). After rinsing with PBS, the SPIONs that had not entered the HUVEC cells were basically removed. The blue shadows co-labeled outside the nucleus and cytoplasm were the SPIONs taken up by HUVEC, indicating that HUVECs successfully took up SPIONs, making them magnetized HUVECs, which can be targeted and migrated under the attraction of a permanent magnet or an exogenous magnetic field such as the present invention.

[0096] Experiment 3: Effect of the present invention on attracting magnetized cells in vitro (culture dish method)

[0097] Objective and Materials: To verify that magnetized HUVEC can achieve targeted migration and aggregation on a horizontal plane in the external magnetic field provided by the present invention. Materials are the same as those in Experiment 1, including lentivirus carrying a green fluorescent protein (GFP) tag.

[0098] The steps are as follows: HUVECs are infected with lentivirus to carry GFP, becoming HUVEC-GFP (HG), which facilitates microscopic observation and confirmation of cell activity; HG is incubated with SPIONs to convert the HG into magnetized HG; trypsin digestion and centrifugation are performed, and the magnetized HG is resuspended in PBS and placed in a 3.5 mm circular culture dish, which is placed in the working area of ​​the present invention, with one side of the liquid phase bottle against the tip of the small magnetic pole, and the working current is 2A, and the magnetization is carried out for 3 minutes;

[0099] Results and conclusion: As shown in Figure 10, the in vitro attraction effect of magnetized cells (under the microscope) was as follows: the device current was 2A, the attraction (power-on) time was 3 min, the cells were HUVECs incubated with Fe3O4 (60 μg / mL 30-50 nm) for 24 h and carrying GFP fluorescent labels, the cell number was 1.2×10^6, scale bar=50 μm, and under the bright field of the microscope, the magnetized HG was uniformly dispersed before attraction, and after attraction, the magnetized HG gathered in the lower left quadrant near the small magnetic pole; under the green fluorescence channel, the cells all showed bright, non-quenchable GFP signals, proving that the modification of SPION and the attraction of the present invention had no significant effect on the activity of HG cells.

[0100] Experiment 4: Effect of the present invention on attracting magnetized cells in vitro (liquid phase bottle method)

[0101] Objective and Materials: To verify that magnetized HUVECs can achieve targeted migration and aggregation in three-dimensional space in the external magnetic field provided by the present invention. Materials are the same as those in Experiment 1, including a cell membrane red fluorescence staining kit (DiI, Bio-Tech).

[0102] Steps: As shown in Figure 11, a photograph of the effect of in vitro attraction of magnetized cells (liquid phase bottle method) was taken. The cells were stained bright red using a DiI kit; trypsin digestion, centrifugation, and magnetized HUVECs were resuspended in PBS and placed in a 1.5 mL liquid phase bottle. The bottle was placed in the working area of ​​the present invention with one side of the liquid phase bottle against the tip of the small magnetic pole. The working current was 2A and the attraction was carried out for 3 minutes.

[0103] Results and Conclusion: As shown in Figure 12, magnetized HUVECs were uniformly dispersed and suspended in PBS before attraction. After attraction, they aggregated on the flask wall in the attraction area of ​​the small magnetic tip. Under the influence of the magnetic force, the red cells shifted from a uniformly dispersed state to aggregate on the flask wall in contact with the small magnetic tip, likely maintaining their aggregated form by overcoming gravity through intercellular adhesion.

[0104] Experiment 5: Effect of the present invention on attracting magnetized cells in vivo

[0105] Objectives and Materials: The most important function of this invention is to achieve targeted transplantation of magnetized cells. To verify this function, in vivo application is crucial. Five-week-old healthy male BALB / C nude mice (weighing 20.7 ± 0.8 g) were randomly selected. Other materials were the same as those in Experiment 4.

[0106] Steps: As shown in Figure 13, magnetized cell targeted transplantation uses a gradient magnetic field device to attract magnetic cells in nude mice, targeting the left cerebral hemisphere, and HUVEC is treated with Fe3O4 (60μg / mL 30~50nm) were incubated for 24h, stained with a cell membrane red fluorescent staining kit (DiI), digested with trypsin, centrifuged, resuspended in PBS, and prepared into a cell suspension with a concentration of 1×10^6 cells / mL. The cells were kept on ice to maintain activity and injected into nude mice via one side of the tail vein. The injection volume for each mouse was 400μl. After the tail vein injection, the nude mice were randomly divided into 3 groups (Group I, Group II, and Group III), with n≥6. Group I was a non-attraction group. The nude mice were anesthetized with isoflurane and no attraction was performed. After 30min, fluorescence imaging was performed under a small animal living imager. Group II was an attraction group. The nude mice were anesthetized with isoflurane. The target area for targeted transplantation was placed within the working area of ​​the present invention, and the small magnetic pole was against the center of the target area. During the actual operation, the left brain was selected as the target area. The present invention used a current of 2A and attraction for 30min. After the end of attraction, fluorescence imaging was performed under a small animal living imager immediately. Group III was a post-attraction waiting group. The previous steps were the same as those of Group II, but after the end of attraction, fluorescence imaging was performed after waiting for 60min.

[0107] Results and Conclusions: As shown in Figure 14, fluorescence imaging shows the in vivo attraction effect. The device current is 2A and the magnetic induction intensity on the surface of the small magnetic pole is 0.414T (refer to formula (5) x = 2 mm, I = 2A), attraction time 30 min; A. Magnetized HUVEC cell suspension, concentration 1 × 10^6 cells / mL; B. Group I, injected with magnetized HUVEC, not attracted in the present device; C. Group II, injected with magnetized HUVEC, attracted in the present device for 30 min; D. Group III, 60 min after stopping attraction, compared with Group I, Group II showed a higher fluorescence signal in the left brain of nude mice, indicating that the present invention effectively attracted the magnetized cells in vivo and targeted them to accumulate in the target area; Group I showed a stronger signal in the lungs, which is consistent with the view in previous studies that intravenously infused cells mainly accumulate in the lungs; Group III still had a high fluorescence signal in the target area, indicating that the cells did not move with the blood flow and were successfully transplanted in the target area.

[0108] Experiment 6: Optimal working current of the present invention

[0109] Objective and Materials: After confirming that the present invention has a targeted attraction effect on magnetized cells in vivo, it is necessary to verify the effects of the working current and attraction duration on the attraction effect. First, verify the effect of current and find the optimal working current; materials are the same as those in Experiment 5;

[0110] Procedure: Magnetized HUVECs were prepared as in Experiment 4. Nude mice were randomly divided into five groups (n ≥ 6). After injection of magnetized HUVECs, the mice were attracted with currents of 0.5 A, 1 A, 1.5 A, 2 A, and 3 A for 30 min, respectively. The maximum magnetic induction intensity in each group varied approximately arithmetic progression. Fluorescence imaging was performed immediately after the attraction period using a small animal in vivo imaging system.

[0111] Results and Conclusions: As shown in Figure 15, fluorescence imaging demonstrates the in vivo attraction effect at different operating currents, with an attraction time of 30 minutes. The numerical values ​​below the image represent the current and the measured maximum magnetic induction intensity (see Table 1). Color Scale: Min = (3±1) × 10^7, Max = (6±1) × 10^9. The optimal attraction effect was achieved at 2A. At 3A, the attraction effect was less noticeable than at 2A. Furthermore, the coil of the present invention generates increased heat, resulting in a significant temperature rise, making it unsuitable for prolonged use. Therefore, 2A is the optimal operating current.

[0112] Experiment 7: Optimal attraction duration of the present invention

[0113] Objective and Materials: Next, we will verify the effect of attraction duration and find the optimal attraction duration. The materials are the same as those in Experiment 5.

[0114] Procedure: Preparation of magnetized HUVEC was the same as in Experiment 4. Nude mice were randomly divided into four groups (n ≥ 6). After injection of magnetized HUVEC, the HUVECs were attracted at an optimal working current of 2 A for 5, 10, 30, and 60 minutes, respectively. Fluorescence imaging was performed immediately after the attraction.

[0115] Results and Conclusion: As shown in Figure 16, fluorescence imaging demonstrates the in vivo aspiration effect in groups with different aspiration durations. The operating current was 2A. The numerical value below the image indicates the aspiration duration for that group. Color Scale: Min = (3±1) × 10^7, Max = (6±1) × 10^9. The optimal aspiration effect was achieved at 30 minutes. However, at 60 minutes, the nude mice underwent prolonged anesthesia, leading to a decrease in body temperature, which may have affected their health and cell transplantation efficacy. Therefore, 30 minutes was the optimal aspiration duration.

[0116] In summary, through experiments three to seven, the present invention can attract magnetized cells in vitro in a horizontal plane, three-dimensional space, and in vivo, so that they can be targeted and migrated to the target area, thereby realizing targeted transplantation of magnetized cells; and, through experiment five, the effectiveness of the present invention in attracting magnetized cells in vivo was verified; through experiment six, when the working current is ≤2A, the working current of the present invention is positively correlated with the in vivo attraction effect, and the optimal current is 2A; through experiment seven, when the attraction time is ≤30min, the attraction time of the present invention is positively correlated with the in vivo attraction effect, and the optimal time is 30min.

[0117] In the description of the present invention, it should be understood that the terms "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside" and "outside" and the like to indicate orientations or positional relationships are based on the orientations or positional relationships shown in the accompanying drawings and are only for the convenience of describing the present invention, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0118] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A gradient magnetic field device for magnetized cell targeted transplantation, characterized in that: include: A magnetic pole pair, the magnetic pole pair comprising a small magnetic pole (1) and a large magnetic pole (2), the top surface of the small magnetic pole (1) being arranged in the form of a convex arc cylinder, the end surface of the large magnetic pole (2) facing the small magnetic pole (1) being arranged in the form of a concave arc cylinder, and the space facing the small magnetic pole (1) and the large magnetic pole (2) forming a gradient magnetic field; A magnetic yoke, the magnetic yoke comprising a second magnetic yoke rod (4), the two ends of the second magnetic yoke rod (4) being connected to a first magnetic yoke rod (3) and a third magnetic yoke rod (5) respectively, the end of the first magnetic yoke rod (3) away from the second magnetic yoke rod (4) being connected to the large magnetic pole (2), the end of the third magnetic yoke rod (5) away from the second magnetic yoke rod (4) being connected to the small magnetic pole (1), the small magnetic pole (1), the large magnetic pole (2), the first magnetic yoke rod (3), the second magnetic yoke rod (4), the third magnetic yoke rod (5) and the space between the magnetic pole pairs together forming a closed magnetic circuit; An electromagnetic coil, the electromagnetic coil comprising four coils (6) connected in parallel, the coil (6) being sleeved on the second magnetic yoke rod (4), the coil (6) being used to provide a magnetic potential to the magnetic circuit; A bottom plate (7), the top surface of the bottom plate (7) being connected to the yoke rod one (3) and the yoke rod three (5) via a plurality of bolts, and the bottom surfaces of the bottom plate (7), the yoke rod one (3) and the yoke rod three (5) are provided with four footrests (8).

2. The gradient magnetic field device for magnetized cell targeted transplantation according to claim 1, characterized in that: The radius of the convex arc cylinder of the small magnetic pole (1) is 2 mm, and the height is 4 mm. The small magnetic pole (1) is arranged in a quadrangular pyramid, and the top surface of the quadrangular pyramid is a convex arc cylinder with an axis perpendicular to the horizontal plane. The left and right side surfaces of the cone are tangent to the convex arc cylinder, and the upper and lower side surfaces of the cone are inclined planes; the radius of the concave arc cylinder of the large magnetic pole (2) is 45 mm, the chord width is 84.57 mm, and the height is 30 mm. The appearance size of the large magnetic pole (1) is a rectangular parallelepiped with a length of 135 mm, a width of 88 mm, and a height of 30 mm.

3. The gradient magnetic field device for magnetized cell targeted transplantation according to claim 2, characterized in that: The convex arc cylindrical surface of the small magnetic pole (1) and the concave arc cylindrical surface of the large magnetic pole (2) are coaxial and arranged opposite to each other, and their axes are perpendicular to the horizontal plane.

4. The gradient magnetic field device for magnetized cell targeted transplantation according to claim 1, characterized in that: The bottom surface of the small magnetic pole (1) and the bottom surface of the large magnetic pole (2) are both rectangular with a width of 88 mm and a height of 30 mm.

5. The gradient magnetic field device for magnetized cell targeted transplantation according to claim 1, characterized in that: The cross-sections of the yoke rod one (3), the yoke rod two (4), and the yoke rod three (5) are all rectangular with a height of 30 mm.

6. The gradient magnetic field device for magnetized cell targeted transplantation according to claim 1, characterized in that: The number of turns of the coil (6) is 1164, the wire diameter is 0.49 mm, the inner hole of the coil (6) is a rectangular through hole, and the size of the rectangular through hole is 63 mm long*30 mm wide*40 mm deep.

7. The gradient magnetic field device for magnetized cell targeted transplantation according to claim 1, characterized in that: There is a gap between the small magnetic pole (1) and the arc cylinder directly facing the large magnetic pole (2), and a working area is selected in the gap. The magnetic lines of force in the working area are projected into a sector shape on the horizontal plane and into an isosceles trapezoid on the vertical plane where the radius line of the arc cylinder is located.

8. The gradient magnetic field device for magnetized cell targeted transplantation according to claim 1, characterized in that: The material of the small magnetic pole (1), the large magnetic pole (2), the yoke rod one (3), the yoke rod two (4), and the yoke rod three (5) is all electrical pure iron.

9. An application of the gradient magnetic field device for magnetized cell targeted transplantation according to any one of claims 1 to 8, characterized in that: By energizing the electromagnetic coil, a magnetic potential is generated, and a gradient magnetic field is generated between the magnetic pole pairs. In this gradient magnetic field, the magnetized cells are subjected to a magnetic force directed toward the top of the small magnetic pole (1), and the targeted migration speed of the magnetized cells can be adjusted by adjusting the current of the electromagnetic coil, so that the target point of the targeted migration of the magnetized cells can be more clearly described and the migration speed can be controlled.

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