A device and method for constructing a localized cell load model in a three-dimensional scaffold

Abstract

The present invention relates to the field of tissue engineering, and discloses a device and method for constructing a localized cell load model in a three-dimensional scaffold. The device includes a culture container, and a magnet group arranged outside the culture container for forming a non-uniformly distributed magnetic field in the culture container. The method for constructing a localized cell load model in a three-dimensional scaffold using the device includes the following steps: placing a three-dimensional scaffold with a connected porous structure at the bottom of the culture container, and placing the culture container above the lower magnet group; dispersing cells in a cell culture medium containing paramagnetic substances, hydrogel prepolymers and photoinitiators, and adding them to the three-dimensional scaffold; placing the upper magnet group above the culture container; after standing, irradiating the top of the device with an ultraviolet lamp. The device of the present invention can realize the simulation of complex tissue structures, and has universal applicability to different three-dimensional scaffold materials, and can realize the spatial control of the internal cells without damaging the scaffold. The method is simple, efficient and low-cost.

Description

Technical Field

[0001] The present invention relates to the field of tissue engineering, and in particular to a device and method for constructing a localized cell load model in a three-dimensional scaffold. Background Art

[0002] Combining cells with three-dimensional scaffolds, constructing tissues and organs in vitro, and then conducting research and transplantation is a classic method of tissue engineering. Currently, filling cells into scaffolds mainly relies on liquid diffusion and vacuum extraction, and cells are often randomly dispersed in the porous structure of the scaffold. The distribution of cells in actual tissues and organs has a strong regularity. Controlling the partitioning of cells in the scaffold and performing localized loading are of great significance for constructing tissues with real structure and function in vitro. In addition, before implantation in the body, localized loading of cells in the scaffold or localized co-culture of multiple cells to prepare engineered tissues is beneficial to tissue reconstruction and regeneration after implantation.

[0003] For example, many tissues have rich vascular structures. When constructing three-dimensional tissues, a specific distribution of vascular networks is introduced for pre-vascularization, which is beneficial to the survival of tissues after implantation. Skeletal muscle cells are characterized by oriented distribution. By arranging cells in strips in the direction of skeletal muscle orientation, it is beneficial to the cell orientation after tissue implantation. The hair follicles on the surface of the skin are arranged in an array. There are about 30 to 150 hair follicles on 1 square centimeter of skin. Through cell localization technology, hair follicle stem cells are pre-formed into a lattice aggregation, which is beneficial to the regeneration of hair follicles after artificial skin implantation. At present, the main methods for realizing cell localization loading are template method and 3D printing. Both methods have high requirements for equipment and material properties, such as the need for materials to solidify under cell culture conditions and the fast solidification speed. In addition, materials with self-healing properties can also be used to manually splice three-dimensional materials loaded with different cells to obtain engineered tissues with localized loading, but this method has poor controllability and is difficult to scale. In addition, the mechanical properties of materials solidified in a cell culture environment are usually weak and the stability is poor. Traditional water-insoluble biomaterials that have been approved for use, such as polylactic acid-co-glycolic acid (PLGA) and polycaprolactone (PCL), have good mechanical properties and biocompatibility, but there is currently no better method to achieve localized cell loading.

[0004] In addition, the scarcity of seed cells is a bottleneck problem in the development of engineered tissues. Methods such as 3D printing control the distribution of cells by controlling the distribution of materials rather than directly controlling cells. Cells are evenly distributed in three-dimensional materials. During the material molding process, a large number of cells are discarded along with some materials, resulting in huge waste. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a device and method for constructing a localized cell load model in a three-dimensional scaffold. The device of the present invention can simulate complex tissue structures, is universal to different three-dimensional scaffold materials, and can achieve spatial control of cells inside the scaffold without damaging the scaffold. The method is simple, efficient and low-cost.

[0006] The specific technical scheme of the present invention is:

[0007] In a first aspect, the present invention provides a device for constructing a localized cell load model in a three-dimensional scaffold, comprising a culture container and a magnet group for forming a non-uniformly distributed magnetic field in the culture container; the magnet group is arranged outside the culture container.

[0008] The present invention utilizes the magnetic Archimedean effect to construct a localized cell load model in a three-dimensional scaffold. The specific mechanism is as follows: Since the cells have antimagnetism, in the non-uniformly distributed magnetic field in the culture container, the cells will move to the area with weaker magnetic field. After placing a three-dimensional scaffold with a connected porous structure in the culture container as a support, localized cell-dense areas and cell-sparse or blank areas can be formed inside the scaffold to obtain an engineered tissue with regular cell arrangement. In the above manner, different types of magnetic field distributions can be formed in the culture container according to the structure of different tissues by adjusting the number, shape, size and distribution of magnets in the upper and lower magnet groups, and the construction of different tissues can be achieved. Moreover, after the cells are fixed by hydrogel cross-linking or attached to the wall inside the scaffold, a variety of cell localization cultures can be achieved through secondary operations. Therefore, the device of the present invention can realize the simulation of complex tissue structures, and the obtained engineered tissue can be used for in vitro research and can also be used for in vivo repair and regeneration.

[0009] In the device of the present invention, the ferromagnetic material will not come into direct contact with the cell culture environment, and the cells in the culture container will not come into contact with metals and other substances that may have high cytotoxicity. The materials that are in direct contact with the cell culture environment are all commonly used materials for cell culture, thereby avoiding related risks and preventing cells from being damaged; and the use of the device of the present invention to construct engineered tissues is not limited by the type of three-dimensional scaffolds, and spatial control of the cells inside can be achieved without damaging the scaffolds. In addition, compared with the cell localization loading methods such as 3D printing in the prior art, the present invention acts directly on cells and can directly aggregate cells in the target area to reduce cell loss.

[0010] Preferably, the magnet group comprises an upper magnet group and a lower magnet group respectively arranged above and below the culture container; the upper magnet group and the lower magnet group are completely identical, aligned in position, and have the same magnetic field direction.

[0011] Controlling the arrangement of cells inside the three-dimensional scaffold, because the three-dimensional scaffold has a certain thickness, it is difficult to control the vertical magnetic field inside the scaffold to be consistent through a unidirectional magnetic field, and it is difficult to completely clear the cells in the strong magnetic field area. Therefore, when the cells are required to be distributed consistently in the vertical direction, or a cell blank area needs to be formed in the vertical direction, the upper and lower magnet groups can be arranged relative to each other, because the magnetic lines of force between the relative magnetic fields are arranged in parallel, and the magnetic field size is consistent in the vertical direction, so that the cells can be controlled to be distributed consistently in the vertical direction, and a cell blank area can be formed in the vertical direction, thereby obtaining an ideal cell localization load model in the three-dimensional scaffold.

[0012] Preferably, the upper magnet group and the lower magnet group respectively include one or more magnets.

[0013] The magnets in the upper magnet group and the lower magnet group can be solid magnets or hollow magnets with holes, and the shapes include but are not limited to spherical, cylindrical, conical, rectangular or horseshoe.

[0014] Preferably, the upper magnet group and the lower magnet group are composed of a plurality of magnets of the same type, or a plurality of magnets of different types, or a plurality of magnets and one or more non-magnetic spacer materials.

[0015] Non-magnetic spacer materials include but are not limited to silicone, rubber, paper, metal, foam, and plastic products.

[0016] Preferably, the magnets in the upper magnet group and the lower magnet group are permanent magnets, including but not limited to neodymium iron boron magnets, ferrite magnets, rubber magnets, etc.

[0017] Preferably, the difference in magnetic field strength between the maximum magnetic field and the minimum magnetic field in the culture container is not less than 1000 Gs.

[0018] Controlling the difference in magnetic field between different areas in the culture container to be large enough can help speed up cell movement and reduce the sedimentation of cells before they reach the predetermined position.

[0019] In a second aspect, the present invention provides a method for constructing a localized cell loading model in a three-dimensional scaffold using the device, comprising the following steps:

[0020] (1) placing a three-dimensional scaffold with a connected porous structure at the bottom of a culture container, and placing the culture container above a lower magnet group;

[0021] (2) dispersing cells in a cell culture medium containing paramagnetic substances, hydrogel prepolymers, and photoinitiators, and adding them to the three-dimensional scaffold;

[0022] (3) Place the upper magnet assembly above the culture container;

[0023] (4) After standing still, the device is irradiated with ultraviolet light above to complete the construction of the localized cell load model within the three-dimensional scaffold.

[0024] By adding paramagnetic substances, the difference in magnetic susceptibility between cells and the external environment can be increased, which is conducive to the formation of localized distribution of cells under the drive of non-uniform magnetic field. After the cells form a localized distribution under the drive of magnetic field, ultraviolet light is used to irradiate the device above, which can cause photo-crosslinking in the light-transmitting area of ​​the scaffold (which is also the cell-dense area with weaker magnetic field), thereby achieving the fixation of cells in the dense area and obtaining a localized cell load model in the three-dimensional scaffold.

[0025] Preferably, in step (3), the standing time is 30 seconds to 10 minutes.

[0026] A static time that is too long or too short is not conducive to the formation of a localized cell load model. Specifically, when the static time is too short, the cells cannot completely move from the strong magnetic area to the weak magnetic area; when the static time is too long, the cells will settle to the bottom of the three-dimensional scaffold due to gravity.

[0027] Preferably, in step (2), the paramagnetic substance is a clinically approved magnetic resonance contrast agent, including but not limited to gadoxetate disodium, gadobenate dimeglumine, gadopentetate dimeglumine, gadoteric acid dimeglumine, gadoteridol and gadodiamide.

[0028] Preferably, in step (2), the content of the paramagnetic substance in the cell culture medium is 1-500 mmol / L.

[0029] Preferably, in step (2), the hydrogel prepolymer comprises one or more of methacrylated gelatin, methacrylated hyaluronic acid and methacrylated polyethylene glycol. The above materials all have good cell compatibility.

[0030] Preferably, in step (2), the content of the hydrogel prepolymer in the cell culture medium is 1-50 wt %.

[0031] Preferably, in step (2), the photoinitiator includes LAP and / or Iracure 2959.

[0032] The photoinitiators LAP and Iracure 2959 are water-soluble photoinitiators that can be dissolved in cell culture medium and have low cytotoxicity, and are therefore suitable for constructing a localized cell loading model within a three-dimensional scaffold in the present invention.

[0033] Optionally, in step (4), after irradiating the culture container with ultraviolet light, the uncross-linked hydrogel prepolymer in the three-dimensional scaffold is washed away, the cell type, the content of cells in the cell culture medium and the structure of the magnet group are changed, and steps (1) to (4) are repeated to complete the construction of a localized loading model of multiple cells in the three-dimensional scaffold.

[0034] When performing another cell localization load as described above, changing the magnetic field distribution can make the two cells be arranged in different ways. In this way, the simulation of various complex tissue structures can be achieved.

[0035] In a third aspect, the present invention provides a localized cell loading model within a three-dimensional scaffold obtained by the method.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] (1) The device of the present invention does not require the use of self-repairing materials or 3D printing technology for multiple cells. It is universally applicable to different three-dimensional scaffold materials and can achieve spatial control of the cells inside the scaffold without damaging the scaffold. The method is simple, efficient and low-cost.

[0038] (2) Using a magnetic field in a three-dimensional culture system to first localize and aggregate cells and then perform regional cross-linking can help reduce cell loss in the uncross-linked area and improve cell utilization during the operation process;

[0039] (2) The device of the present invention can be used to simulate complex tissue structures and realize the precise construction of in vitro complex tissue models and tissue engineering implant tissues, which has positive significance for the development of tissue engineering regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a structural schematic diagram of the present invention;

[0041] Figure 2 yes Figure 1 Left view of .

[0042] The reference numerals are: culture container 1 , magnet group 2 , upper magnet group 2 - 1 , lower magnet group 2 - 2 , and three-dimensional support 3 . DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with the embodiments.

[0044] Example 1

[0045] like Figure 1 and Figure 2As shown, a device for constructing a localized cell load model in a three-dimensional scaffold includes a culture container 1 and a magnet group 2 arranged outside the culture container 1 for forming a non-uniformly distributed magnetic field in the culture container 1.

[0046] The magnet group 2 includes an upper magnet group 2-1 and a lower magnet group 2-2, which are respectively arranged above and below the culture container 1. The upper magnet group 2-1 and the lower magnet group 2-2 are identical, aligned with each other, and have the same magnetic field direction. The upper magnet group 2-1 and the lower magnet group 2-2 are composed of 4 pieces of 30mm×10mm×1mm neodymium iron boron magnets and 3 pieces of 5mm×10mm×1mm silicone ( Figure 1 and Figure 2 The NdFeB magnet is arranged with a gap between it and the silica gel.

[0047] The culture container 1 is made of silica gel with a thickness of 2 mm and a through hole of 1 cm×1 cm×2 mm. Glass slides are bonded to the inner wall and bottom of the through hole, and a transparent glass slide is covered on top as a lid of the culture container 1 .

[0048] Example 2

[0049] The only difference between this embodiment and embodiment 1 is that, in this embodiment, the upper magnet group 2-1 and the lower magnet group 2-2 are both composed of 5 pieces of 30mm×10mm×1mm NdFeB magnets and 4 pieces of 5mm×10mm×1mm silicone rubber, and the NdFeB magnets and the silicone rubber are arranged at intervals.

[0050] Example 3

[0051] The only difference between this embodiment and embodiment 1 is that in this embodiment, the upper magnet group 2-1 and the lower magnet group 2-2 are both composed of 64 cylindrical magnets with a diameter of 1.5 mm and a height of 20 mm arranged by side adsorption to form an 8×8 array.

[0052] Example 4

[0053] The only difference between this embodiment and embodiment 1 is that in this embodiment, the upper magnet group 2-1 and the lower magnet group 2-2 are both composed of a cylindrical magnet with an outer diameter of 12 mm, an inner diameter of 5 mm, and a height of 5 mm.

[0054] Application Example 1

[0055] The device in Example 2 was used to construct a localized loading model of a single cell in a three-dimensional scaffold. The specific steps are as follows:

[0056] (1) NdFeB magnets and silica gel are arranged at intervals to form an upper magnetic field group 2-1 and a lower magnetic field group 2-2;

[0057] (2) Place a dry PLGA three-dimensional scaffold 3 (10 mm × 10 mm × 2 mm) with a connected porous structure and a flat upper and lower surface at the bottom of the culture container 1, and place the culture container 1 above the lower magnet group 2-2;

[0058] (3) Cells were cultured at 200 × 10 4 The density of cells / mL was dispersed in a cell culture medium containing 30 mM gadopentetate dimeglumine, 10 wt % methacryloyl gelatin and 1 wt % photoinitiator LAP, and was dropped onto the three-dimensional scaffold 3, and the lid of the culture container 1 was covered;

[0059] (4) placing the upper magnet group 2-1 above the culture container 1, opposite to the lower magnet group 2-2, and aligning them through magnetic field attraction;

[0060] (5) After standing for 3 min, irradiate the top of the device with a 405 nm ultraviolet lamp for 30 s to complete the construction of the localized cell loading model within the three-dimensional scaffold.

[0061] Application Example 2

[0062] Using the device in Example 2, a localized loading model of two cells in a three-dimensional scaffold was constructed. The specific steps are as follows:

[0063] (1) NdFeB magnets and silica gel are arranged at intervals to form an upper magnetic field group 2-1 and a lower magnetic field group 2-2;

[0064] (2) Place a dry PLGA three-dimensional scaffold 3 (10 mm × 10 mm × 2 mm) with a connected porous structure and a flat upper and lower surface at the bottom of the culture container 1, and place the culture container 1 above the lower magnet group 2-2;

[0065] (3) Endothelial cells were cultured at 200×10 4 The density of cells / mL was dispersed in a cell culture medium containing 30 mM gadopentetate dimeglumine, 10 wt % methacryloyl gelatin and 1 wt % photoinitiator LAP, and was dropped onto the three-dimensional scaffold 3, and the lid of the culture container 1 was covered;

[0066] (4) placing the upper magnet group 2-1 above the culture container 1, opposite to the lower magnet group 2-2, and aligning them through magnetic field attraction;

[0067] (5) After standing for 3 min, irradiate the device with a 405 nm ultraviolet lamp for 30 s to obtain a single cell localized loading model;

[0068] (6) Soak the single cell localized loading model in 37°C culture medium to wash away the uncross-linked methacryloyl gelatin, and absorb the excess methacryloyl gelatin with filter paper;

[0069] (7) Bone marrow mesenchymal stem cells were cultured at 400×10 4 The density of cells / mL is dispersed in a cell culture medium containing 10wt% methacryloyl gelatin and 1wt% photoinitiator LAP, and is dropped onto the single cell localized load model treated in step (6), and the lid of the culture container 1 is closed;

[0070] (8) Irradiate the device with a 405 nm ultraviolet lamp for 30 seconds to complete the construction of the two cell localized loading models within the three-dimensional scaffold.

[0071] Application Example 3

[0072] Using the device in Example 2, a cell localized load model for simulating thigh muscle tissue was constructed, and the specific steps were as follows:

[0073] (1) NdFeB magnets and silica gel are arranged at intervals to form an upper magnetic field group 2-1 and a lower magnetic field group 2-2;

[0074] (2) Place a dry collagen sponge three-dimensional scaffold 3 (10 mm × 10 mm × 1 mm) with a connected porous structure and a flat upper and lower surface at the bottom of the culture container 1, and place the culture container 1 above the lower magnet group 2-2;

[0075] (3) Bone marrow mesenchymal stem cells were cultured at 200×10 4 The density of cells / mL was dispersed in a cell culture medium containing 30 mM gadopentetate dimeglumine, 10 wt % methacryloyl gelatin and 1 wt % photoinitiator LAP, and was dropped onto the three-dimensional scaffold 3, and the lid of the culture container 1 was covered;

[0076] (4) placing the upper magnet group 2-1 above the culture container 1, opposite to the lower magnet group 2-2, and aligning them through magnetic field attraction;

[0077] (5) After standing for 10 min, the device was irradiated with a 405 nm ultraviolet lamp for 30 s, and then immersed in a 37°C culture medium to wash away the uncross-linked methacryloyl gelatin to obtain a cell localized loading model (5 strips with a width of approximately 1 mm, used to simulate thigh muscle tissue).

[0078] The cell localized loading model obtained in this application example is placed in a culture medium containing induction factors and cultured for 5 days, then taken out and rolled into a cylindrical shape with the stripe pattern direction as the axis, and can be implanted into damaged thigh muscle tissue.

[0079] Application Example 4

[0080] Using the device in Example 3, a cell localized loading model for simulating the hair follicle structure was constructed, and the specific steps were as follows:

[0081] (1) NdFeB magnets and silica gel are arranged at intervals to form an upper magnetic field group 2-1 and a lower magnetic field group 2-2;

[0082] (2) Place a dry collagen chitosan three-dimensional scaffold 3 (10 mm × 10 mm × 2 mm) with a connected porous structure and a flat upper and lower surface at the bottom of the culture container 1, and place the culture container 1 above the lower magnet group 2-2;

[0083] (3) Adipose stem cells and hair follicle stem cells were cultured at a concentration of 100 × 10 4 The density of cells / mL was dispersed in a cell culture medium containing 30 mM gadopentetate dimeglumine, 10 wt % methacryloyl gelatin and 1 wt % photoinitiator LAP, and was dropped onto the three-dimensional scaffold 3, and the lid of the culture container 1 was covered;

[0084] (4) placing the upper magnet group 2-1 above the culture container 1, opposite to the lower magnet group 2-2, and aligning them through magnetic field attraction;

[0085] (5) After standing for 10 min, the device was irradiated with a 405 nm ultraviolet lamp for 30 s, and then immersed in a 37°C culture medium to wash away the uncross-linked methacryloyl gelatin, thereby obtaining a cell localized load model with a lattice distribution (100 lattices with a diameter of approximately 400 μm, used to simulate the structure of skin hair follicles).

[0086] The cell localized loading model obtained in this application example is placed in a culture medium containing hair follicle cell inducing factors and cultured for 7 days before being taken out and implanted into the skin defect site.

[0087] Application Example 5

[0088] Using the device in Example 4, a cell localized loading model for use as a cardiac patch was constructed, and the specific steps were as follows:

[0089] (1) NdFeB magnets and silica gel are arranged at intervals to form an upper magnetic field group 2-1 and a lower magnetic field group 2-2;

[0090] (2) placing a woven poly (L-lactic acid-co-caprolactone) (PLCL) three-dimensional scaffold 3 (8 mm × 8 mm × 1 mm) with a connected porous structure and made of gelatin as a porogen with flat upper and lower surfaces at the bottom of a culture container 1, and placing the culture container 1 above the lower magnet group 2-2;

[0091] (3) Cardiomyocytes and fibroblasts differentiated from induced pluripotent stem cells were cultured at a rate of 100 × 10 cells per cell. 4 The density of cells / mL was dispersed in a cell culture medium containing 30 mM gadopentetate dimeglumine, 10 wt % methacryloyl gelatin and 1 wt % photoinitiator LAP, and was dropped onto the three-dimensional scaffold 3, and the lid of the culture container 1 was covered;

[0092] (4) placing the upper magnet group 2-1 above the culture container 1, opposite to the lower magnet group 2-2, and aligning them through magnetic field attraction;

[0093] (5) After standing for 10 min, the device was irradiated with a 405 nm ultraviolet lamp for 30 s and then immersed in a 37°C culture medium to wash away the uncross-linked methacryloyl gelatin to obtain a cell-localized loading model, i.e., a cardiac patch with cells distributed only in the central area and no cells in the surrounding area.

[0094] Suturing the localized cell loading model obtained in this application example to the site of myocardial infarction can reduce cell loss during the cardiac suturing process of the myocardial patch.

[0095] Application Example 6

[0096] The difference between this application example and application example 1 is that in this application example, the standing time in step (5) is 15 minutes. After observing the localized cell load model in the three-dimensional scaffold finally obtained, it is found that in application example 1, the cell density in the vertical direction of the three-dimensional scaffold is relatively uniform, while in application example 3, the cell density at the bottom of the three-dimensional scaffold is significantly higher than that at the top.

[0097] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0098] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for constructing a localized cell loading model in a three-dimensional scaffold, characterized in that: The following steps are involved: (1) placing a three-dimensional scaffold with a connected porous structure at the bottom of a culture container, and placing the culture container above a lower magnet group; (2) dispersing cells in a cell culture medium containing paramagnetic substances, hydrogel prepolymers, and photoinitiators, and adding them to the three-dimensional scaffold; (3) placing an upper magnet group above the culture container, wherein the upper magnet group and the lower magnet group each include a plurality of magnets and form a non-uniformly distributed magnetic field in the culture container; (4) After standing for 30 seconds to 10 minutes, irradiate with ultraviolet light above the upper magnet group to complete the construction of the localized cell load model in the three-dimensional scaffold.

2. The method according to claim 1, characterized in that The upper magnet group and the lower magnet group are completely identical, their positions are aligned with each other, and their magnetic field directions are the same.

3. The method according to claim 1, characterized in that In step (2), the paramagnetic substance is a clinically approved magnetic resonance contrast agent.

4. The method according to claim 1, characterized in that In step (2), the content of the paramagnetic substance in the cell culture medium is 1-500 mmol / L.

5. The method according to claim 1, characterized in that In step (2), the hydrogel prepolymer includes one or more of methacrylated gelatin, methacrylated hyaluronic acid and methacrylated polyethylene glycol.

6. The method according to claim 1 or 5, characterized in that In step (2), the content of the hydrogel prepolymer in the cell culture medium is 1-50 wt %.

7. The method according to claim 1, characterized in that In step (2), the photoinitiator includes LAP and / or Iracure 2959.

8. The method according to claim 1, characterized in that In step (4), after irradiating the culture container with ultraviolet light, the uncross-linked hydrogel prepolymer in the three-dimensional scaffold is washed away, and one or more of the cell type, the cell content in the cell culture medium, and the structure of the magnet group are replaced, and steps (1) to (4) are repeated to complete the construction of a localized loading model of multiple cells in the three-dimensional scaffold.

9. A localized cell loading model within a three-dimensional scaffold obtained by the method according to any one of claims 1 to 8.

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