WO2023245812A1 - Cell sheet clamping device, use method, and preparation method for neurorestorative material - Google Patents

Abstract

A cell sheet clamping device, a use method, and a preparation method for a neurorestorative material. The cell sheet clamping device comprises a frame, a fixed plate being fixedly arranged on the frame. A first clamping member is adjustably arranged on the frame and cooperates with the fixed plate so as to clamp one end of a cell sheet. A movable plate is movably arranged on the frame. A second clamping member is adjustably arranged on the frame and cooperates with the movable plate so as to clamp the other end of the cell sheet. The movable plate can move relatively close to or far away from the fixed plate in a preset plane, and when the movable plate moves in the preset plane, the second clamping member moves synchronously. One end of a cell sheet is clamped by the cooperated first clamping member and fixed plate, while the other end of the cell sheet is clamped by the cooperated second clamping member and movable plate; the second clamping member and the movable plate are synchronously adjusted such that the cell sheet has a natural length; and then the positions of the fixed plate and the movable plate are relatively fixed and cells are cultured for a preset time, so that the cell sheet spontaneously rolls up into a cylindrical shape to form a neurorestorative material.

Description

Cell film clamping device, method of use and preparation method of nerve repair material Technical field

The invention relates to the technical field of cell tissue engineering, and in particular to a cell film clamping device, a method of use and a preparation method of nerve repair materials.

Background technique

Nerve repair materials are scaffold-type nerve repair materials composed of biodegradable synthetic polymers and natural biological materials. However, scaffold-type nerve repair materials have disadvantages such as difficulty in controlling the degradation rate, local inflammatory reaction, excessive internal guidance space of the material causing axon dispersion and inhibiting nerve function recovery, and intratubular collapse and compression during nerve regeneration.

Contents of the invention

The purpose of the present invention is to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a cell film clamping device that can prepare self-assembled nerve repair materials with better effects.

The invention also provides a method of using the cell film clamping device.

The invention also provides a method for preparing nerve repair materials.

The cell film clamping device according to the first embodiment of the present invention includes:

frame;

a fixed plate, fixedly provided on the frame and used to carry the part of the cell membrane;

The first clamping part is adjustably provided on the frame, and the first clamping part can be relatively close to or relatively far away from the fixed plate in the longitudinal direction, so that the first clamping part can pass through the fixed plate. The fixed plate clamps one end of the cell membrane in a cooperative manner;

A movable plate, movably provided on the frame, for carrying other parts of the cell membrane;

The second clamping part is adjustably provided on the frame. The second clamping part can be relatively close to or relatively far away from the movable plate in the longitudinal direction, so that the second clamping part can pass through the movable plate. The movable plate clamps the other end of the cell membrane in a cooperative manner; and

The movable plate can be relatively close to or relatively far away from the fixed plate in a preset plane, and when the movable plate moves in the preset plane, the second clamping part moves synchronously, so that the first clamp The holding part and the second holding part can adjust the unfolded state of the cell film after respectively holding the cell film.

The cell film clamping device according to the first embodiment of the present invention has at least the following beneficial effects: the first clamping part cooperates with the fixed plate to clamp one end of the cell membrane, and the second clamping part cooperates with the movable plate to clamp At the other end of the cell film, the second clamping part and the movable plate are synchronously adjusted so that the cell film is at a natural length, and then the positions of the fixed plate and the movable plate are relatively fixed and the cell film is cultured for a preset time, so that the cell film It spontaneously shrinks into a cylindrical shape to form a nerve repair material. This scaffold-free nerve repair material has the advantages of low immune rejection and can reconstruct the mechanical and structural properties of natural tissue.

According to the cell film clamping device according to the first embodiment of the present invention, the frame includes a top plate, a support part, a connector and a slide assembly, and the bottom end of the support part is connected to the fixed plate, so The top end of the support part is used to support the top plate, the first clamping part is provided on the top plate, the bottom end of the connecting piece is connected to the movable plate, and the top plate is connected to the slider assembly. The slider assembly is slidably provided on the top plate, and the second clamping portion is provided on the slider assembly.

According to the cell film clamping device according to the first embodiment of the present invention, the bottom end of the connecting member and the movable plate are connected through a profile connection;

And/or the bottom end of the support part and the fixing plate are connected through profile connection.

According to the cell film clamping device according to the first embodiment of the present invention, the top surface of the top plate is provided with a first guide rail, the bottom surface of the top plate is provided with a second guide rail, and the slider assembly includes a locking member , a first slide block and a second slide block, the first slide block is slidably disposed on the first guide rail, the second slide block is slidably disposed on the second guide rail, and the locking member Adjustably penetrated through the first slide block and the second slide block, so that the slide block assembly can be locked on the top plate when the locking member is adjusted to a preset state.

According to the cell film clamping device according to the first embodiment of the present invention, the locking member is covered with an elastic component, and the elastic component is arranged between the first slider and the second slider. time to provide locking preload.

According to the cell film clamping device according to the first embodiment of the present invention, a third guide rail is provided between the first guide rail and the second guide rail, and the top end of the third guide rail is connected to the first guide rail The bottom end of the third guide rail is connected to the top end of the second guide rail. A first protrusion and a second protrusion are provided on the top surface of the second slider. The first protrusion The raised portion is slidably disposed on the third guide rail, the second protruding portion is slidably disposed on the second guide rail, the locking member is passed through the first protruding portion, and the elastic component is disposed on the second guide rail. The first raised portion.

According to the cell film clamping device according to the first embodiment of the present invention, the fixing plate is provided with a first silica gel protrusion, and the bottom end of the first clamping portion is provided with a second silica gel protrusion. The first silicone protrusion and the second silicone protrusion cooperate with each other to clamp one end of the cell film;

And/or the movable plate is provided with a third silicone protrusion, the bottom end of the second clamping part is provided with a fourth silicone protrusion, the third silicone protrusion and the fourth silicone protrusion are mutually exclusive. Fit the other end for clamping the cell membrane.

The method of using the cell film clamping device according to the second embodiment of the present invention includes:

The cell film clamping device used includes: a frame, a fixed plate, a first clamping part, a movable plate and a second clamping part, wherein the second clamping part is slidably arranged on the machine through the slider assembly. Frame, the sliding assembly includes a locking piece;

The method of using the cell membrane clamping device includes the following steps:

The rack is placed in a petri dish containing a preset amount of culture fluid, and the culture fluid at least covers the first silica gel protrusion of the fixed plate and the third silica gel protrusion of the movable plate;

Transfer the cell film to the culture dish, use a pipette to absorb the culture medium and drop it so that the cell film spreads in the culture medium;

Use a pipette to blow the unfolded cell film to the first silica gel protrusion, adjust the first clamping part accordingly, control the clamping force to clamp the edge of the cell film, and then blow the other side of the cell film to the first silica gel protrusion. There are three silicone protrusions, and the second clamping part is adjusted accordingly to control the clamping force to clamp the other edge of the cell membrane;

Loosen the locking piece and adjust the slider assembly to simultaneously drive the second clamping part and the movable plate to move, so that the cell film is at its natural length and naturally suspended in the culture medium;

Lock the locking member to fix the relative positions of the first silicone protrusion and the third silicone protrusion.

The method of using the cell film clamping device according to the second embodiment of the present invention has at least the following beneficial effects: a cylindrical self-assembled nerve repair material is prepared by clamping and utilizing the shrinkage characteristics of the cell film. The scaffold-free type The nerve repair material has the advantages of low immune rejection, can reconstruct the mechanical and structural properties of natural tissue, and also forms an ordered extracellular matrix through clamping and self-shrinkage after cell membrane peeling. The effect of guiding nerve regeneration in the target direction is more significant, and the preparation cycle is shorter than that of other scaffold-free nerve repair materials based on cell films.

The preparation method of nerve repair materials according to the third embodiment of the present invention includes the following steps: culturing and preparing cell sheets; using the cell clamping device described in the first aspect of the present invention to clamp the cell sheets, and culturing to obtain the nerve repair materials.

According to the preparation method of nerve repair materials according to the third embodiment of the present invention, the culture conditions are 3 to 5 days at 35-39°C and 3%-7% CO ₂ concentration.

According to the preparation method of nerve repair materials according to the embodiment of the third aspect of the present invention, the present invention does not limit the specifications of the culture dish; preferably, a ring-shaped material can be pasted on the culture dish, and the size of the ring-shaped material can be determined according to The specifications of the culture dish are adjusted, and the ring-shaped material is prepared by using at least one of polymer hydrogel or silica gel.

According to the method for preparing nerve repair materials according to the embodiment of the third aspect of the present invention, the cells include: at least one of NHDF fibroblasts, 3T3 fibroblasts, Schwann cells and other cells that can be used to prepare nerve repair materials. .

The preparation method of nerve repair materials according to the third embodiment of the present invention has at least the following beneficial effects:

In the non-patent document "Cardiomyocyte Film Sensor for Marine Toxin Detection", it is recorded that a clip is used to fix the cardiomyocyte film, but the other end is in a free state, forming a cantilever structure. The spontaneous contraction movement of the cardiomyocytes will cause the film to However, the method in this document is only used to characterize the beating state of cells through the spontaneous contraction movement of cardiomyocytes and cannot be used to prepare cylindrical materials.

In the present invention, by clamping and fixing the cell film, the cell film spontaneously shrinks into a cylindrical shape to form a nerve repair material. Specifically, the cell membrane is clamped at both ends to shrink into a cylindrical shape due to the constraints of the two fixed points.

The method used in the present invention is simple to operate, and makes full use of the self-shrinking characteristics of the cell membrane after peeling off, so that the cell membrane can self-assemble to form an extracellular matrix with an orderly structure, and then form a nerve repair material. The effect of extracellular matrix with well-organized structure in guiding nerve regeneration in the target direction is more significant. Moreover, the preparation cycle is shorter than that of other scaffold-free nerve repair materials prepared based on cell films.

The repair material according to the embodiment of the fourth aspect of the present invention is prepared by the preparation method of the nerve repair material described in the third aspect of the present invention.

The nerve repair material according to the fourth embodiment of the present invention has at least the following beneficial effects: the present invention prepares a cylindrical self-assembled nerve repair material by clamping and utilizing the shrinkage characteristics of cell films. The material has other advantages based on The scaffold-free nerve repair material prepared from cell films has the advantages of low immune rejection and the ability to reconstruct the mechanical and structural properties of natural tissue. Moreover, due to the self-shrinkage after clamping and cell film peeling, it also forms a structurally arranged structure. The orderly extracellular matrix has a more significant effect of guiding nerve regeneration in the target direction.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of the drawings

The present invention will be further described below in conjunction with the accompanying drawings and examples;

Figure 1 is a schematic structural diagram of an embodiment of the present invention;

Figure 2 is an exploded view of the top plate and slider assembly in the embodiment of the present invention;

Figure 3 is an internal schematic diagram of A in Figure 1;

Figure 4 is a schematic flow chart for preparing tissue engineering self-assembled nerve repair materials based on the shrinkage characteristics of cell films in an embodiment of the present invention;

Figure 5 is a rendering of the preparation of tissue engineering self-assembled nerve repair materials based on the shrinkage properties of NHDF fibroblast films in the embodiment of the present invention;

Figure 6 is a diagram showing the effect of natural suspension culture of NHDF fibroblast cell films without clamping in Comparative Example 1.

Reference signs:

Fixed plate 100, first silicone protrusion 110, first clamping part 200, second silicone protrusion 210, movable plate 300, third silicone protrusion 310, second clamping part 400, fourth silicone protrusion 410, Top plate 510, first guide rail 511, second guide rail 512, third guide rail 513, support part 520, first slide block 531, second slide block 532, first protruding part 5321, second protruding part 5322, locking Component 533, elastic component 534, connecting component 540.

Detailed ways

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the drawings are exemplary and are only used to explain the present invention and cannot be understood as limiting the present invention.

In the description of the present invention, it should be understood that orientation descriptions, such as up, down, front, back, left, right, etc., are based on the orientation or position relationships shown in the drawings and are only In order to facilitate the description of the present invention and simplify the description, it is not intended to indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore should not be construed as a limitation of the present invention.

In the description of the present invention, several means one or more, plural means at least two, greater than, less than, more than, etc. are understood to exclude the original number, and above, below, within, etc. are understood to include the original number. If there is a description of first and second, it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of indicated technical features or implicitly indicating the order of indicated technical features. relation.

In the description of the present invention, unless otherwise explicitly limited, words such as setting, installation, and connection should be understood in a broad sense. Those skilled in the art can reasonably determine the specific meaning of the above words in the present invention in combination with the specific content of the technical solution.

Peripheral nerves and central nervous systems are damaged due to diseases, accidents, etc., and the limited ability of nerve tissue to self-regenerate may lead to loss of nerve function and the inability to control target muscles to complete specific responses. When the injured nerve gap is larger than 5mm, it is difficult to directly suture the nerve gap of peripheral nerve damage through neurosorture to achieve therapeutic effect. Treatment options such as autologous nerve transplantation and nerve repair material transplantation are needed to repair the nerve damage. Although autologous nerve transplantation can achieve better nerve repair effects, its shortcomings are also very significant. The amount of donor nerve tissue available in the human body is limited, the size does not match the damaged nerve tissue, and there is a risk of neuroma, loss of function, and scarring at the donor site, leading to secondary physical damage and reducing the patient's quality of life after treatment. . Therefore, it is of great significance to develop and prepare artificial nerve repair materials with good transplantation and repair functions.

At present, nerve repair materials are mainly designed and developed through tissue engineering technology, combined with engineering, materials science and cell biology. Most FDA-approved nerve repair materials are scaffold-type nerve repair materials composed of biodegradable synthetic polymers and natural biological materials. Scaffold-type nerve repair materials have shortcomings such as too fast or too slow degradation rate, local inflammatory reaction, excessive internal guidance space of the material causing axon dispersion and inhibiting nerve function recovery, and intratubular collapse and compression during nerve regeneration. In order to optimize the internal guidance space of the material, it has been proposed to use hydrogels containing related matrices to fill the interior of the material. This can prevent collapse and compression, and can also simulate the extracellular matrix microenvironment to promote nerve repair. However, this optimization method requires studying the filler composition and filling density, which is a cumbersome process, and the different degradation rates of the filler and the scaffold will affect nerve repair. Scaffold-free nerve repair materials are mainly composed of cell products such as extracellular matrix. The raw materials used in this method can be produced from the host's stem cells, which can minimize immune rejection reactions. Moreover, the nerve repair materials prepared through cell film technology can better reconstruct the mechanical and structural properties of natural tissues compared with scaffolds. There is no need to simulate the microenvironment provided by the extracellular matrix, and the extracellular matrix can be directly used as the main body of the repair material.

The difficulty in using cell films to prepare nerve repair materials is to form the films into a cylindrical shape. At present, the method of directly rolling the film or mandrel-assisted rolling is mainly used to shape the cell film from a membrane into a cylindrical shape. However, this method is complex to operate, and it takes a long time to fuse the gaps between the cell films after being rolled into a cylindrical shape. If the roll is rolled into a cylindrical shape with the help of a mandrel, there will be disadvantages such as inconvenience in extracting the mandrel and the formation of a large cavity after the mandrel is extracted. If the cell film rolling method is used to prepare nerve repair materials, the lack of an orderly arrangement of extracellular matrix in the structure will affect the effect of guiding nerve directional repair, and a longer culture period is required.

Referring to Figures 1 to 3, the cell film clamping device according to the first embodiment of the present invention is used in the preparation of self-assembled nerve repair materials in tissue engineering. The cell film clamping device includes a frame, a fixing plate 100, a first The clamping part 200, the movable plate 300 and the second clamping part 400.

The frame serves as a bearing platform, and the fixed plate 100, the first clamping part 200, the movable plate 300 and the second clamping part 400 are all arranged on the frame. The fixed plate 100 is fixedly arranged, and is used to carry the part of the cell film; the first clamping part 200 is adjustably arranged on the frame, and the first clamping part 200 can be relatively close to or relatively far away from the fixed plate 100 in the longitudinal direction. , so that the first clamping part 200 can clamp one end of the cell film by cooperating with the fixed plate 100; the movable plate 300 is movably provided on the frame and is used to carry other parts of the cell film; the second clamping The second clamping part 400 is adjustably disposed on the frame, and the second clamping part 400 can be relatively close to or relatively far away from the movable plate 300 in the longitudinal direction, so that the second clamping part 400 can clamp the cell film by cooperating with the movable plate 300 and the movable plate 300 can be relatively close to or relatively far away from the fixed plate 100 in the preset plane, and when the movable plate 300 moves in the preset plane, the second clamping part 400 moves synchronously, so that the first clamping part 200 and the second clamping part 400 can adjust the unfolded state of the cell membrane after clamping the cell membrane respectively. The first clamping part 200 and the fixed plate 100 cooperate to clamp one end of the cell film, and the second clamping part 400 and the movable plate 300 cooperate to clamp the other end of the cell membrane. By synchronously adjusting the second clamping part 400 and the movable plate 300 so that the cell film is at a natural length, and then the positions of the fixed plate 100 and the movable plate 300 are relatively fixed and the cells are cultured for a preset time, so that the cell film spontaneously shrinks into a cylindrical shape to form a nerve repair material. This scaffold-free nerve Repair materials have the advantages of low immune rejection and the ability to reconstruct the mechanical and structural properties of natural tissue. This method is simple to operate, and makes full use of the self-shrinking properties of cell membranes after peeling off, so that the cell membranes can self-assemble to form an ordered extracellular matrix, thereby forming nerve repair materials. The effect of extracellular matrix with well-organized structure in guiding nerve regeneration in the target direction is more significant.

It can be understood that the first clamping part 200 can be adjusted up and down above the fixed plate 100 and cooperates with the fixed plate 100 , and the second clamping part 400 can be adjusted up and down above the movable plate 300 and cooperates with the movable plate 300 . The plate 300 is configured so as to be able to clamp two positions of the cell membrane through the two clamping ends. Based on the spontaneous shrinkage characteristics, the cell membrane will shrink based on the two clamping ends, and finally form a cell membrane based on the two clamping ends. For a cylindrical organizational structure whose connection line is the axis, preferably the first clamping part 200 and the second clamping part 400 are both screws, and the adjustment effect is achieved by rotating the screws.

In some embodiments of the present invention, specifically referring to Figure 1, the rack includes a top plate 510, a support part 520, a connector 540 and a slider assembly. The bottom end of the support part 520 is connected to the fixed plate 100, and the top end of the support part 520 is connected with On the supporting top plate 510, the first clamping part 200 is provided on the top plate 510, the bottom end of the connector 540 is connected to the movable plate 300, the top plate 510 is connected to the slider assembly, the slider assembly is slidably provided on the top plate 510, and the second clamp The holding portion 400 is provided on the slider assembly. It can be understood that the support part 520 is arranged vertically as a force-bearing foundation, the top plate 510 is fixedly connected to the support part 520 and is arranged horizontally, the top plate 510 serves as the installation foundation, and the first clamping part 200 and the slider assembly are both arranged on the top plate 510. In the embodiment in which the first clamping part 200 is a screw, the first clamping part 200 is inserted into the threaded hole of the top plate 510, and the threaded hole is arranged vertically so that the first clamping part 200 can only be adjusted longitudinally. In the embodiment where the slider assembly is slidably disposed on the top plate 510, and the second clamping part 400 is a screw, the second clamping part 400 is inserted into the threaded hole of the slider assembly, and the threaded hole is arranged vertically. The second clamping part 400 can only be adjusted longitudinally. At the same time, the movable plate 300 is connected to the slider assembly through the connector 540, so that the second clamping part 400 and the movable plate 300 move synchronously when the slider assembly is slidingly adjusted, thereby achieving tensioning. The effect of cell membranes. Preferably, both the supporting part 520 and the connecting piece 540 are configured as plate-shaped members and exist as side plates in the rack to ensure the stability of the rack.

Preferably, specifically referring to FIG. 3 , the bottom end of the connecting member 540 is connected to the movable plate 300 through a profile connection, and the bottom end of the support portion 520 is connected to the fixed plate 100 through a profile connection. The connection through profile connection can avoid the contact between the metal and the culture medium when using metal screws to connect, which will affect the cell film. It ensures that only non-toxic materials such as PC (polycarbonate) and silica gel are in direct contact with the culture medium.

In some embodiments of the present invention, specifically referring to Figure 2, the top surface of the top plate 510 is provided with a first guide rail 511, and the bottom surface of the top plate 510 is provided with a second guide rail 512. The slider assembly includes a locking member 533, a first slider 531 and the second slider 532, the first slider 531 is slidably disposed on the first guide rail 511, the second slider 532 is slidably disposed on the second guide rail 512, and the locking member 533 is adjustably disposed on the first guide rail 511. The sliding block 531 and the second sliding block 532 enable the sliding block assembly to be locked on the top plate 510 when the locking member 533 is adjusted to the preset state. It can be understood that the position adjustment of the second clamping part 400 is achieved by sliding the first slider 531. Since the locking member 533 is passed through the first slider 531 and the second slider 532, the first slider 531 is slid. When the second slide block 532 is required to be locked, the first slide block 531 and the second slide block 532 are brought relatively close to each other through the locking member 533, and locking is achieved through the static friction force of the relative close proximity. Preferably, the locking member 533 is a threaded fastener, and the second slider 532 is processed with a threaded hole. By rotating the locking member 533, the second slider 532 can be adjusted relatively close to and relatively away from the first slider 531, thereby Correspondingly loosen and lock the first slider 531.

Preferably, the locking member 533 is covered with an elastic component 534, and the elastic component 534 is disposed between the first slide block 531 and the second slide block 532 to provide a locking pre-tightening force. Specifically, the elastic component 534 is a spring, which can enhance the locking effect of the locking member 533 on the slider assembly and maintain the stability of the device when it is not locked.

Preferably, with specific reference to Figure 2, a third guide rail 513 is provided between the first guide rail 511 and the second guide rail 512. The top end of the third guide rail 513 is connected to the bottom end of the first guide rail 511, and the bottom end of the third guide rail 513 is connected to the third guide rail 511. The top of the second guide rail 512 and the top surface of the second slider 532 are provided with a first protruding portion 5321 and a second protruding portion 5322. The first protruding portion 5321 is slidably disposed on the third guide rail 513, and the second protruding portion 5322 is slidably disposed on the second guide rail 512 , the locking member 533 is disposed on the first protruding portion 5321 , and the elastic component 534 is disposed on the first protruding portion 5321 . In some embodiments, the fixed plate 100 and the movable plate 300 are spaced apart along the length direction of the frame, and the movable plate 300 is adjustable along the length direction. Then the first guide rail 511 , the second guide rail 512 and the third guide rail 513 are all parallel to each other. It is arranged in the length direction, and the first guide rail 511, the second guide rail 512 and the third guide rail 513 can be processed and formed with the top plate 510 based on the process of processing grooves. The specific structural shapes of the first slider 531 and the second slider 532 are set as The slider structure is adapted to the first guide rail 511, the second guide rail 512 and the third guide rail 513 to ensure normal sliding. Therefore, the first protruding part 5321 is located in the middle of the top surface of the second slider 532. There are two second protruding parts 5322 symmetrically arranged on both sides of the first protruding part 5321. The second guide rail 512 and the third guide rail 513 Correspondingly, the first protruding part 5321 serves as the installation basis for the elastic component 534 and the second clamping part 400 to ensure that the adjustment of the second clamping part 400, the adjustment of the slider assembly and the adjustment of the locking part 533 can be more efficient. Reasonable and do not interfere with each other.

In some embodiments of the present invention, the fixing plate 100 is provided with a first silicone protrusion 110, the bottom end of the first clamping portion 200 is provided with a second silicone protrusion 210, the first silicone protrusion 110 and the second silicone protrusion The protrusions 210 cooperate with each other to clamp one end of the cell membrane; the movable plate 300 is provided with a third silica gel protrusion 310, and the bottom end of the second clamping part 400 is provided with a fourth silica gel protrusion 410. The protrusion 310 and the fourth silicone protrusion 410 cooperate with each other to clamp the other end of the cell membrane. It can be understood that the mutual cooperation of the first silicone protrusion 110 and the second silicone protrusion 210 serves as a clamping end, and the mutual cooperation of the third silicone protrusion 310 and the fourth silicone protrusion 410 serves as another clamping end. end, using two clamping ends to clamp the cell film, so that the cell film can shrink at its natural length and finally form a cylindrical shape, in which the first silica gel protrusion 110, the second silica gel protrusion 210, and the third silica gel protrusion The protrusions 310 and the fourth silicone protrusion 410 are both made of silicone material, and the connector 540, the support part 520, the fixed plate 100 and the movable plate 300 that can contact the culture medium are all made of PC material (polycarbonate). Effectively avoid the impact on the cell membrane and ensure the effect of cell culture.

Referring to Figures 1 to 3, the method of using the cell film clamping device according to the second embodiment of the present invention is shown. The method of using the cell film clamping device can be the method of using the cell film clamping device according to the first embodiment of the present invention. , the cell film clamping device used includes: a frame, a fixed plate 100, a first clamping part 200, a movable plate 300 and a second clamping part 400, where the second clamping part 400 is slidably arranged on the machine through a slider assembly. Frame, sliding assembly includes locking piece 533;

The method of using the cell membrane clamping device includes the following steps:

Place the rack in a petri dish containing a preset amount of culture solution, and the culture solution at least covers the first silicone protrusion 110 of the fixed plate 100 and the third silicone protrusion 310 of the movable plate 300;

Transfer the cell film to the culture dish, use a pipette to absorb the culture medium and drop it so that the cell film spreads in the culture medium;

Use a pipette to blow the unfolded cell film to the first silica gel protrusion 110, adjust the first clamping part 200 accordingly, control the clamping force to clamp the edge of the cell film, and then blow the other side of the cell film to the third silica gel protrusion 310, and adjust the second clamping portion 400 accordingly to control the clamping force to clamp the other edge of the cell film;

Loosen the locking member 533 and adjust the slider assembly to simultaneously drive the second clamping part 400 and the movable plate 300 to move, so that the cell film is at its natural length and naturally suspended in the culture medium;

Lock the locking member 533 to fix the relative positions of the first silicone protrusion 110 and the third silicone protrusion 310 .

A cylindrical self-assembled nerve repair material is prepared by clamping and utilizing the shrinkage properties of cell films. This scaffold-free nerve repair material has the advantages of low immune rejection and can reconstruct the mechanical and structural properties of natural tissue. Moreover, through clamping and self-shrinkage after peeling off the cell film, a structurally ordered extracellular matrix is formed, which has a more significant effect of guiding nerve regeneration in the target direction, and the preparation cycle is shorter than other scaffold-free types based on cell film preparation. Preparation cycle of nerve repair materials.

Specific usage methods include:

Cells are cultured routinely;

Passage the cells and inoculate them on a 35mm temperature culture dish or a temperature culture dish with a ring-shaped material with a square interior of 35*35mm. Inoculate 1 million cells and place them in a 37°C, 5% CO2 incubator for 7 days;

Place the 35mm temperature culture dish with the cell film cultured on it in a 20°C incubator for 1h-2h to peel off the cell film;

Move the slider assembly to the minimum lateral length of the device, and lock the locking piece 533 to keep the slider assembly at the shortest lateral length;

Place the rack in a 10cm petri dish containing a certain amount of culture solution. The culture solution has just covered the silicone protrusions of the fixed plate 100 and the movable plate 300;

Transfer the peeled cell film to a 10cm culture dish, use a pipette to absorb the culture medium, and drop the culture medium drop by drop to allow the cell film to spread in the culture medium;

Use a pipette to slowly blow the unfolded cell film onto one of the silicone protrusions in the device, and then unscrew the corresponding first clamping part 200 or second clamping part 400 to form two silicone protrusions up and down. Contact, clamp the edge of the cell membrane, and clamp the other side of the cell membrane in the same way;

Loosen the locking piece 533 and slowly move the slider assembly so that the cell film is at its natural length and naturally suspended in the culture medium. Then lock the locking piece 533 to fix the lateral length of the clamping device;

Place the entire device with the cell film clamped in a 37°C, 5% CO2 incubator and culture it for 3-5 days;

The cells can be NHDF fibroblasts, 3T3 fibroblasts, and other cells; further, the cell film can be prepared from a single type of cell, multiple types of cells, a single type of cell composite degradable material, and a multiple type of cell type composite degradable material. Specifically, the cell film can be an NHDF fibroblast film, a 3T3 fibroblast film, an NHDF fibroblast film co-cultured with Schwann cells or a 3T3 fibroblast film co-cultured with Schwann cells, as well as a single type of cell. Cell films made of composite degradable materials and various cell-like composite degradable materials;

Further, the culture dish is a 35*35mm square ring-shaped material made of at least one of polymer hydrogel or silica gel.

Referring to Figures 1 to 3, a method for preparing nerve repair materials according to a third embodiment of the present invention, specifically a method for preparing tissue engineering self-assembled nerve repair materials from NHDF fibroblast cell films, is shown in Figure 4 for a flow chart.

Specific preparation methods of nerve repair materials include:

Add 2 ml of fetal bovine serum to a 35 mm temperature culture dish (purchased from UpCell) and coat at 37°C overnight. NHDF fibroblasts were cultured according to the instructions specified in the instructions, digested with trypsin, and set aside. Aspirate the fetal bovine serum in the temperature culture dish, inoculate NHDF fibroblasts on the temperature culture dish pre-coated with fetal bovine serum, inoculate 1 million cells, and place them in a 37°C, 5% _CO2 incubator using low sugar culture The culture medium was cultured for 7 days, and the culture medium was changed every 2 days.

Move the slider assembly to the minimum lateral length of the device, and lock the locking piece 533 to keep the device at the shortest lateral length; place the rack in a 10cm petri dish containing a certain amount of high-sugar culture solution, and the high-sugar culture solution has just disappeared Through the silicone protrusions of the fixed plate 100 and the movable plate 300; the high-glucose culture medium includes 89% DMEM (dulbecco's modified eagle medium) high-glucose basic medium, 10% fetal bovine serum, and 1% double antibody.

Place the 35mm temperature culture dish cultured with NHDF cell film in a 20°C incubator for 1h-2h, and use the pipetting method in the patent document CN202010601331.6 (a liver cell in vitro co-culture system and its construction method and application). Gently pipet the edge of the culture dish to peel off the NHDF cell film. Transfer the stripped NHDF cell film to the culture dish, use a pipette to absorb the high-sugar culture medium, and drop the high-sugar culture medium drop by drop, so that the NHDF cell film expands in the high-glucose culture medium; transfer the expanded NHDF cell film, Use a pipette to slowly blow onto one of the silicone protrusions in the device, and then unscrew the corresponding first clamping part 200 or second clamping part 400 so that the upper and lower silicone protrusions are in contact to clamp the NHDF cells edge of the membrane and clamp the other side of the NHDF cell membrane in the same manner.

Loosen the locking member 533 and slowly slide the slider assembly so that the NHDF cell membrane is at its natural length and naturally suspended in the high-sugar culture medium. Then lock the locking member 533 to fix the lateral length of the clamping device.

Place the entire device with the NHDF cell film clamped in a 37°C, 5% _CO2 incubator for 3-5 days, and record the state of the NHDF cell film on one day (Figure 5); finally, tissue engineering self-assembly is obtained Type nerve repair materials, proving that cylindrical self-assembled nerve repair materials can be prepared by clamping and utilizing the shrinkage properties of cell films.

Comparative Example 1NHDF fibroblast cell film natural suspension culture without clamping

Use the NHDF cell film peeled off in the embodiment, transfer it to a 10cm culture dish containing high sugar culture solution, use a pipette to absorb the high sugar culture solution, and drop the culture solution drop by drop, so that the NHDF cell film is in the high sugar culture medium. The culture medium was expanded and naturally suspended in the high-sugar culture medium. The culture dish was placed in a 37°C, 5% CO ₂ incubator and cultured for 7 days. The status of the NHDF cell membrane was recorded on days 1, 3, and 7 respectively. The results are shown in Figure 6. This shows that the suspension culture of NHDF cell films without clamping cannot form tissue engineering self-assembled nerve repair materials. It only shrinks into a thick round cake-shaped tissue, indicating that the suspension culture of NHDF cell films without clamping cannot form tissue engineering. The self-assembling nerve repair material only shrinks itself into a thick round cake-shaped tissue.

The technical features of the above-described embodiments can be combined in any way. To simplify the description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, all possible combinations can be used. It should be considered to be within the scope described in this manual.

The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the technical field, various modifications can be made without departing from the purpose of the present invention. kind of change.

Claims (11)

1. A cell film clamping device, characterized by including:

   frame;

   a fixed plate, fixedly provided on the frame and used to carry the part of the cell membrane;

   The first clamping part is adjustably provided on the frame, and the first clamping part can be relatively close to or relatively far away from the fixed plate in the longitudinal direction, so that the first clamping part can pass through the fixed plate. The fixed plate clamps one end of the cell membrane in a cooperative manner;

   A movable plate, movably provided on the frame, for carrying other parts of the cell membrane;

   The second clamping part is adjustably provided on the frame. The second clamping part can be relatively close to or relatively far away from the movable plate in the longitudinal direction, so that the second clamping part can pass through the movable plate. The movable plate clamps the other end of the cell membrane in a cooperative manner; and

   The movable plate can be relatively close to or relatively far away from the fixed plate in a preset plane, and when the movable plate moves in the preset plane, the second clamping part moves synchronously, so that the first clamp The holding part and the second holding part can adjust the unfolded state of the cell film after respectively holding the cell film.
2. The cell film clamping device according to claim 1, characterized in that: the frame includes a top plate, a support part, a connecting piece and a slide assembly, the bottom end of the support part is connected to the fixed plate, and the The top end of the support part is used to support the top plate, the first clamping part is provided on the top plate, the bottom end of the connecting piece is connected to the movable plate, and the top plate is connected to the slider assembly. The slider assembly is slidably provided on the top plate, and the second clamping portion is provided on the slider assembly.
3. The cell film clamping device according to claim 2, characterized in that: the bottom end of the connecting piece and the movable plate are connected through a profile connection;

   And/or the bottom end of the support part and the fixing plate are connected through profile connection.
4. The cell film clamping device according to claim 2, wherein the top surface of the top plate is provided with a first guide rail, the bottom surface of the top plate is provided with a second guide rail, and the slider assembly includes a locking member, A first slide block and a second slide block, the first slide block is slidably disposed on the first guide rail, the second slide block is slidably disposed on the second guide rail, and the locking member can Adjustably penetrated through the first slide block and the second slide block, so that the slide block assembly can be locked on the top plate when the locking member is adjusted to a preset state.
5. The cell film clamping device according to claim 4, characterized in that: the locking member is covered with an elastic component, and the elastic component is arranged between the first slider and the second slider. To provide locking pre-tightening force.
6. The cell film clamping device according to claim 5, characterized in that: a third guide rail is provided between the first guide rail and the second guide rail, and the top end of the third guide rail is connected to the top of the first guide rail. The bottom end of the third guide rail is connected to the top end of the second guide rail. A first protrusion and a second protrusion are provided on the top surface of the second slider. The first protrusion The second protruding part is slidably disposed on the third guide rail, the second protruding part is slidably disposed on the second guide rail, the locking member is passed through the first protruding part, and the elastic component is disposed on the The first bulge.
7. The cell film clamping device according to any one of claims 1 to 6, characterized in that: the fixing plate is provided with a first silica gel protrusion, and the bottom end of the first clamping part is provided with a second silica gel Protrusions, the first silicone protrusion and the second silicone protrusion cooperate with each other to clamp one end of the cell membrane;

   And/or the movable plate is provided with a third silicone protrusion, the bottom end of the second clamping part is provided with a fourth silicone protrusion, the third silicone protrusion and the fourth silicone protrusion are mutually exclusive. Fit the other end for clamping the cell membrane.
8. A method of using a cell film clamping device, which is characterized by:

   The cell film clamping device used includes: a frame, a fixed plate, a first clamping part, a movable plate and a second clamping part, wherein the second clamping part is slidably arranged on the frame through a slider assembly, The sliding assembly includes a locking piece;

   The method of using the cell membrane clamping device includes the following steps:

   The rack is placed in a petri dish containing a preset amount of culture fluid, and the culture fluid at least covers the first silica gel protrusion of the fixed plate and the third silica gel protrusion of the movable plate;

   Transfer the cell film to the culture dish, use a pipette to absorb the culture medium and drop it so that the cell film spreads in the culture medium;

   Use a pipette to blow the unfolded cell film to the first silica gel protrusion, adjust the first clamping part accordingly, control the clamping force to clamp the edge of the cell film, and then blow the other side of the cell film to the first silica gel protrusion. There are three silicone protrusions, and the second clamping part is adjusted accordingly to control the clamping force to clamp the other edge of the cell membrane;

   Loosen the locking piece and adjust the slider assembly to simultaneously drive the second clamping part and the movable plate to move, so that the cell film is at its natural length and naturally suspended in the culture medium;

   Lock the locking member to fix the relative positions of the first silicone protrusion and the third silicone protrusion.
9. A method for preparing nerve repair materials, which is characterized in that it includes the following steps: cultivating and preparing cell films; using the cell film clamping device according to any one of claims 1 to 7 for clamping, and cultivating to obtain the nerve Repair materials.
10. The method for preparing nerve repair materials according to claim 9, wherein the cells include: at least one of NHDF fibroblasts, 3T3 fibroblasts, and Schwann cells.
11. A nerve repair material, characterized in that it is prepared by the preparation method described in claim 9.

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