Detachable cell sheet transfer printing device and cell sheet transfer printing method

Abstract

The invention discloses a detachable cell sheet transfer printing device and a cell sheet transfer printing method. The detachable cell sheet transfer printing device comprises a support, a hydrogel cavity and a connecting column, the bottom surface of the supporter is detachably connected with a heating film; the hydrogel cavity is clamped at the lower end of the supporter; thermal response hydrogel is injected into the hydrogel cavity; after the heating film is electrified, the volume of the thermal response hydrogel can be shrunk; the connecting column is in threaded connection with the lower end of the hydrogel cavity; and a PDMS adsorption sheet is fixed at the lower end of the connecting column to adsorb a cell sheet. The process of capturing and releasing objects by cephalopods is simulated, the volume of the thermal response hydrogel is changed by charging and discharging of the heating film, and a pressure difference is generated in the transfer printing device through contraction and expansion of the volume of the thermal response hydrogel, so that adsorption and release of the cell sheet are realized; large-scale, rapid and repeatable transfer printing can be carried out on thin and fragile cell sheets, and the detachable connection mode facilitates disassembly, replacement and assembly of the transfer printing device.

Description

Technical Field

[0001] This invention relates to the field of cell membrane transfer technology, and more specifically to a detachable cell membrane transfer device and a cell membrane transfer method. Background Technology

[0002] For a long time, the shortage of donor organs and transplant rejection have been two major challenges in the field of organ transplantation. The emergence of tissue engineering has provided a new approach to solving these problems. Tissue engineering, based on the principles of cell biology, materials science, and bioengineering, constructs bioactive tissue analogs in vitro to replace, repair, improve, or regenerate damaged or diseased tissues and organs in the human body. Skin, as the largest organ in the human body, has a complex structure and diverse functions. Located on the outermost layer of the body, it is easily damaged by external factors such as mechanical forces, temperature, and chemicals, leading to impaired skin structural integrity and loss of normal function. Furthermore, chronic underlying diseases such as metabolic disorders and vascular dysfunction can also cause skin trauma. Therefore, utilizing tissue engineering techniques to repair, regenerate, and reconstruct the function of skin defects is of great significance.

[0003] Cell sheets have been shown to replicate the physiological activity of original tissues and, due to cell-cell interactions and the presence of the extracellular matrix, exhibit better therapeutic effects than single cells. Cell sheet transfer technology is an important technique in tissue engineering. It involves culturing cells into cell sheets with specific structures and functions, and then transplanting them to damaged tissue sites to promote tissue repair and regeneration. This technology not only addresses the donor shortage problem in traditional organ transplantation but also effectively reduces the incidence of transplant rejection, providing a new approach for the treatment of tissue injuries such as skin defects.

[0004] However, cell membrane materials are thin and fragile, making transportation and assembly during the cell membrane transfer process a significant challenge. One method for cell membrane transfer involves adhering the cell membrane to a polymer carrier and then removing the carrier via chemical or thermal treatment. This process cannot be repeated and suffers from problems such as long transfer times and high experimental costs.

[0005] To meet the growing demand for cell sheet-based applications, future technological developments must improve existing technologies to enable large-scale, rapid, and reproducible cell sheet transfer. Summary of the Invention

[0006] In view of this, the present invention provides a detachable cell membrane transfer device and a cell membrane transfer method, which simulates the process of cephalopods capturing and releasing objects. It utilizes the discharge of a heated membrane to cause a change in the volume of a thermoresponsive hydrogel. The contraction and expansion of the thermoresponsive hydrogel volume generates a pressure difference within the transfer device, thereby achieving the adsorption and release of cell membranes. This enables large-scale, rapid, and repeatable transfer of thin and fragile cell membranes, and the detachable connection method facilitates the disassembly, replacement, and assembly of the transfer device.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A detachable cell membrane transfer device includes a support, a hydrogel cavity, and a connecting column;

[0009] A heating membrane is detachably connected to the bottom surface of the support; the hydrogel cavity is snapped into the lower end of the support; the hydrogel cavity is filled with thermoresponsive hydrogel, and the thermoresponsive hydrogel can shrink in volume when the heating membrane is energized; the upper end of the connecting post is threadedly connected to the lower end of the hydrogel cavity; a PDMS suction sheet is fixed to the lower end of the connecting post to adsorb cell membrane sheets.

[0010] The beneficial effects of the technical solution of this invention are that when the heating membrane is energized, the volume of the thermally responsive hydrogel shrinks, and the shrinkage of the thermally responsive hydrogel creates a pressure difference within the transfer device. Under the action of the pressure difference, the PDMS absorbent sheet will adsorb cell membrane sheets. By controlling the temperature of the heating membrane, the shrinkage and expansion of the thermally responsive hydrogel volume can be controlled, thereby controlling the adsorption and release of cell membrane sheets. The support, hydrogel cavity and connecting column are connected in a detachable manner, which facilitates the disassembly, replacement and assembly of the transfer device.

[0011] Preferably, the support includes a support plate and a support rod; the lower end of the support rod is vertically fixed to the upper surface of the support plate; the heating film is bonded to the lower surface of the support plate with double-sided adhesive. The support rod can be held by hand, connected to a robotic arm or a three-axis platform, and can adapt to different cell membrane transfer scenarios. The bonding and fixing method between the support plate and the heating film makes installation and removal convenient and the heating film easy to replace.

[0012] Preferably, the heating film is manufactured on a copper / polyimide film; the surface of the heating film away from the support plate is plated with a copper layer; the heating film is connected to a DC power supply via a wire. The conductive coating enables the heating film to conduct electricity, and the discharge of the heating film allows for the thermal response of the hydrogel volume to shrink and expand.

[0013] Preferably, the hydrogel cavity has multiple locking blocks fixed to its circumferential surface; the support plate has multiple locking slots circumferentially formed; and the multiple locking blocks are correspondingly engaged in the multiple locking slots. The locking blocks and slots achieve the engagement between the hydrogel cavity and the support plate, making installation and disassembly convenient, quick, and efficient.

[0014] Preferably, the connecting column is a hollow column, and an electrospun film is bonded to the inner wall of the middle part of the hollow column to divide its inner cavity into an upper chamber and a lower chamber; the upper inner wall of the hollow column has an internal thread, and the lower outer wall of the hydrogel cavity has an external thread that engages with the internal thread; the inner cavity of the hydrogel cavity is connected to the upper chamber. The electrospun film has selective permeability, allowing air molecules to pass freely, but exhibits a significant blocking effect on water molecules. When the volume of the thermally responsive hydrogel changes, the pressure change in the two chambers connecting the hollow column and the hydrogel cavity prevents water molecules released by the phase transition of the thermally responsive hydrogel from passing through the electrospun film into the connecting column, and at the same time prevents water molecules in the cell membrane from passing through the electrospun film into the hydrogel cavity when adsorbing cell membranes.

[0015] Preferably, an annular boss is fixed at the lower end of the hollow column; the PDMS absorbent sheet is interference-fitted with the annular boss. This interference fit ensures a tight seal between the PDMS absorbent sheet and the hollow column, preventing pressure leakage due to pressure differences caused by thermal response hydrogel volume changes, thus ensuring effective adsorption of the cell membrane.

[0016] Preferably, the PDMS absorbent sheet has multiple adsorption pores. When the PDMS absorbent sheet comes into contact with the cell membrane sheet, the volume of the thermally responsive hydrogel changes, generating a pressure difference. The pressure is used to adsorb the cell membrane sheet through the adsorption pores, thus forming a sealed structure between the cell membrane sheet and the PDMS absorbent sheet.

[0017] Preferably, the PDMS absorbent sheet is prepared by curing a prepolymer and a curing agent, followed by ethanol immersion, ultraviolet sterilization, and drying. By using a mold to produce PDMS absorbent sheets of different sizes, pore numbers, pore sizes, and thicknesses, the transfer of cell membrane sheets of different sizes can be achieved.

[0018] Preferably, the thermoresponsive hydrogel is polymerized from NIPAAm, crosslinking agent BIS, and photoinitiator under ultraviolet light. The thermoresponsive hydrogel undergoes a phase transition above the critical solution temperature (LCST), changing from hydrophilic to hydrophobic, resulting in a rapid change in its volume. When the temperature is below the LCST, it reverts to hydrophilicity. This characteristic allows for the formation of a sealed structure when the transfer device contacts the cell membrane. The volume change of the thermoresponsive hydrogel within the hydrogel cavity due to temperature variations causes a corresponding change in air pressure within the sealed structure. By controlling the temperature of the heating membrane, the hydrogel's contraction and expansion are controlled, thereby controlling the adsorption and release of the cell membrane.

[0019] The present invention also provides a cell membrane transfer method, employing a detachable cell membrane transfer device from the above-mentioned technical solution, comprising the following steps:

[0020] S1. Inject the thermo-responsive hydrogel into the hydrogel cavity and irradiate it with ultraviolet light to polymerize the thermo-responsive hydrogel.

[0021] S2. Remove the culture medium from the cell membrane sheet, and bring the PDMS absorbent sheet into contact with the surface of the cell membrane sheet;

[0022] S3. When the heating membrane is energized, the volume of the thermally responsive hydrogel shrinks, and the PDMS absorbent sheet adsorbs the cell membrane under the pressure difference.

[0023] S4. After the cell membrane sheet is transferred to the predetermined position, the heating membrane is de-energized, and after the volume of the thermally responsive hydrogel recovers, the PDMS absorbent sheet separates from the cell membrane sheet, completing the transfer.

[0024] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a detachable cell membrane transfer device and a cell membrane transfer method. By adjusting the temperature change of the heating membrane, the pressure difference inside the transfer device is changed, thereby completing the adsorption and transfer of cell membranes. The detachable connection method can adapt to the needs of different transfer scenarios. The electrospun membrane is used to reduce the interaction between substances in the cell membrane and the thermally responsive hydrogel. The materials are simple and readily available, low in cost, and highly efficient. The miniaturization of the transfer device is achieved, making it suitable for transfer in various scenarios. It has the advantages of simple and reliable structure, good biological functionality, and easy control. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the transfer device structure provided by the present invention;

[0027] Figure 2 This is an exploded view of the transfer device provided by the present invention;

[0028] Figure 3 This is a schematic diagram of the cell membrane transfer process provided by the present invention.

[0029] in,

[0030] 1-Supporter; 11-Support rod; 12-Support plate; 13-Heating film; 14-Card slot; 2-Hydrogel cavity; 21-Card block; 3-Connecting column; 31-Electrospun film; 32-PDMS suction sheet; 33-Adsorption pore; 4-Cell membrane sheet. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] See appendix Figure 1 and 2According to an embodiment of the present invention, a detachable cell membrane transfer device includes a support 1, a hydrogel cavity 2, and a connecting column 3; a heating film 13 is detachably connected to the bottom surface of the support 1; the hydrogel cavity 2 is snapped into the lower end of the support 1; the support 1 includes a support plate 12 and a support rod 11; the lower end of the support rod 11 is vertically fixed to the middle of the upper surface of the support plate 12, and the support rod 11 can be held by hand or connected to a robotic arm or a triaxial platform to meet different transfer scenarios; the hydrogel cavity 2 is snapped into the lower end of the support 1; the hydrogel cavity 2 is injected with thermally responsive hydrogel, and the heating film 13 can be energized to... The hydrogel shrinks in volume in response to heat; multiple locking blocks 21 are fixed on the circumferential surface of the hydrogel cavity 21; multiple slots 14 are opened circumferentially on the support plate 12; multiple locking blocks 21 are locked into the multiple slots 14 one by one; the detachable connection between the hydrogel cavity 2 and the support 1 is achieved by the cooperation of the locking blocks 21 and the slots 14; in this embodiment, the heating film 13 is bonded to the lower surface of the support plate 12 with 3M double-sided adhesive, the connection method is simple and reliable, and it is convenient to replace the heating film 13; the upper end of the hydrogel cavity 21 is covered by the support plate 12, and the support plate 12 is used as the top plate of the hydrogel cavity 21. The upper end of the connecting column 3 is threaded to the lower end of the hydrogel cavity 2; a PDMS suction plate 32 is fixed to the lower end of the connecting column 3 to adsorb the cell membrane sheet 4; the connecting column 3 is a hollow column, and an electrospun film 31 is bonded to the inner wall of the middle part of the hollow column to divide its inner cavity into an upper chamber and a lower chamber; the inner wall of the upper end of the hollow column has an internal thread, and the outer wall of the lower end of the hydrogel cavity 2 has an external thread that engages with the internal thread; the inner cavity of the hydrogel cavity 2 is connected to the upper chamber. The electrospun film 32 has selective barrier properties, allowing air molecules to pass through while blocking water molecules; the hydrogel cavity 2 is filled with thermally responsive hydrogel. The PDMS suction plate 32 has multiple adsorption pores 33 to adsorb the cell membrane sheet 4.

[0034] In this embodiment, the threaded connection between the hollow column and the hydrogel cavity ensures a sealing effect between them; an annular boss is fixed at the lower end of the hollow column; the PDMS suction pad 32 is interference-fitted with the annular boss. This interference fit ensures a sealing effect between the PDMS suction pad 32 and the annular boss.

[0035] Specifically, such as Figure 2 As shown, the PDMS absorbent pad has a bowl-shaped structure, and its inner wall is connected to the outer wall of the annular boss by a cyanoacrylate adhesive, so that the inner wall of the PDMS absorbent pad and the outer wall of the annular boss are sealed.

[0036] To further optimize the above technical solution, the support plate 12 and the support rod 11 are designed using 3D modeling software and then integrally formed using FDM 3D printing.

[0037] To further optimize the above technical solution, the heating film 13 is manufactured on a copper / polyimide film; a copper layer is plated on the side of the heating film 13 away from the support plate 12; the heating film 13 is connected to a DC power supply via wires. The support is simple and convenient to manufacture, and the materials are readily available and inexpensive.

[0038] In this embodiment, the hydrogel cavity is designed using 3D modeling software and then formed using SLA photopolymerization 3D printing. The printing consumable uses a biocompatible photosensitive resin. After printing, post-processing is required, such as ultrasonic cleaning of the hydrogel cavity in an isopropanol solution, followed by immersion in anhydrous ethanol to remove residual isopropanol and resin. After cleaning, it is dried in an oven, and then the hydrogel cavity is completely polymerized using ultraviolet light. The thermoresponsive hydrogel is prepared by NIPAAm and the crosslinking agent BIS, and a photoinitiator is added. The resulting solution is polymerized by irradiation with an ultraviolet lamp, which shapes the thermoresponsive hydrogel and improves its mechanical properties. This thermoresponsive hydrogel undergoes a phase transition above its critical solution temperature (LCST), changing from hydrophilic to hydrophobic, resulting in a dramatic volume change. Below the LCST, it reverts to hydrophilicity. This property allows it to form a sealed structure when the transfer device contacts a cell membrane. The volume change of the hydrogel within the cavity due to temperature variations causes a corresponding change in pressure within the sealed structure. This pressure difference and adsorption pores are used to adsorb the cell membrane. By controlling the temperature of the heating membrane, the thermoresponsive hydrogel's volume can be controlled to contract and expand, thereby controlling the adsorption and release of the cell membrane.

[0039] To prepare the thermoresponsive hydrogel, NIPAAm and N,N′-methylenebisacrylamide were dissolved in distilled water at 25°C and stirred on a magnetic stirrer for 15 min. The photoinitiator LAP phenyl-2,4,6-trimethylbenzoyl lithium phosphinate was added to the stirred NIPAAm solution, and then the solution was stirred on a magnetic stirrer for 30 min in the dark. The resulting solution was injected into the hydrogel cavity and polymerized by irradiation with a UV lamp for 10 min to obtain the thermoresponsive hydrogel.

[0040] In other specific embodiments, the hollow column and the hydrogel cavity are prepared in the same way. The electrospun film is prepared by applying a high-voltage electrostatic field, which causes the polymer solution to form an ultrafine fiber film under the action of the electric field force. It has selective permeability, allowing air molecules to pass freely, but exhibits a significant blocking effect on water molecules. When the volume of the hydrogel changes in response to heat, the pressure change in the two chambers of the hollow column and the hydrogel cavity prevents water molecules released by the hydrogel phase transition from passing through the film, and also prevents water molecules adsorbed in the cell membrane from passing through the film and entering the hydrogel cavity during adsorption.

[0041] To further optimize the above technical solution, PDMS absorbent sheet 32 ​​is prepared by curing prepolymer and curing agent, and then sterilized and dried by ethanol immersion and ultraviolet sterilization.

[0042] The preparation of PDMS absorbent sheets involves two processes: surface treatment and preparation. Specifically, the prepolymer and curing agent are weighed using a weighing balance, stirred evenly in a 10:1 ratio, and then poured into a mold printed using SLA photopolymerization 3D printing. The mold is then placed in a vacuum chamber, vacuumed, and left for 20 minutes to eliminate air bubbles. After that, it is dried and cured in a 60°C oven for six hours. The cured PDMS absorbent sheets are then sterilized by first soaking them in 75% ethanol, then irradiating them with a UV lamp for 30 minutes, rinsing them three times with phosphate-buffered saline, and then soaking them in Pluronic F-127 solution for three hours. After soaking, the PDMS absorbent sheets are placed in an incubator to dry completely, giving the PDMS absorbent sheets anti-adhesion properties and protecting cell membranes from damage.

[0043] Example 2

[0044] According to a cell membrane transfer method of this embodiment, using a detachable cell membrane transfer device as described in Embodiment 1, the method includes the following steps:

[0045] S1. Inject the thermo-responsive hydrogel into the hydrogel cavity 2 and irradiate it with ultraviolet light to polymerize the thermo-responsive hydrogel;

[0046] S2. Remove the culture medium from cell membrane sheet 4, so that PDMS absorbent sheet 32 ​​comes into contact with the surface of cell membrane sheet 4;

[0047] S3. When the heating membrane 13 is energized, the thermally responsive hydrogel shrinks in volume, and the PDMS absorbent sheet 32 ​​adsorbs the cell membrane sheet 4 under the pressure difference.

[0048] S4. After the cell membrane sheet 4 is transferred to the predetermined position, the heating membrane 13 is de-energized. After the volume of the thermally responsive hydrogel recovers, the PDMS suction sheet 32 ​​separates from the cell membrane sheet 4, completing the transfer.

[0049] In this embodiment, to prevent cell membrane contamination, the support 1, hydrogel cavity 2, and connecting column 3 need to be sterilized under ultraviolet light for 30 minutes before the transfer begins.

[0050] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0051] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A detachable cell membrane transfer device, characterized in that, It includes a support (1), a hydrogel cavity (2), and a connecting column (3); The support (1) is detachably connected to a heating membrane (13) on its bottom surface; the hydrogel cavity (2) is snapped into the lower end of the support (1); the hydrogel cavity (2) is filled with a thermo-responsive hydrogel and the thermo-responsive hydrogel can shrink in volume when the heating membrane (13) is energized; the upper end of the connecting post (3) is threadedly connected to the lower end of the hydrogel cavity (2); a PDMS suction sheet (32) is fixed to the lower end of the connecting post (3) to adsorb cell membrane sheets (4).

2. The detachable cell membrane transfer device according to claim 1, characterized in that, The support (1) includes a support plate (12) and a support rod (11); the lower end of the support rod (11) is vertically fixed to the upper surface of the support plate (12); the heating film (13) is bonded to the lower surface of the support plate (12) by double-sided adhesive.

3. The detachable cell membrane transfer device according to claim 2, characterized in that, The heating film (13) is manufactured on a copper / polyimide film; the surface of the heating film (13) away from the support plate (12) is plated with a copper layer; the heating film (13) is connected to a DC power supply via a wire.

4. The detachable cell membrane transfer device according to claim 3, characterized in that, The hydrogel cavity (2) has multiple locking blocks (21) fixed on its circumferential surface; the support plate (12) has multiple slots (14) in its circumferential direction; the multiple locking blocks (21) are locked into the multiple slots (14) one by one.

5. A detachable cell membrane transfer device according to claim 4, characterized in that, The connecting column (3) is a hollow column, and an electrospun film (31) is bonded to the inner wall of the middle part of the hollow column to divide its inner cavity into an upper chamber and a lower chamber; the inner wall of the upper end of the hollow column is provided with an internal thread, and the outer wall of the lower end of the hydrogel cavity (2) has an external thread that engages with the internal thread; the inner cavity of the hydrogel cavity (2) is connected to the upper chamber.

6. The detachable cell membrane transfer device according to claim 5, characterized in that, The lower end of the hollow column is fixed with an annular boss; the PDMS suction plate (32) is interference-fitted with the annular boss.

7. The detachable cell membrane transfer device according to claim 1, characterized in that, The PDMS absorbent sheet (32) has multiple adsorption holes (33).

8. The detachable cell membrane transfer device according to claim 1, characterized in that, The PDMS absorbent sheet (32) is prepared by curing a prepolymer and a curing agent, and then sterilized and dried by ethanol immersion and ultraviolet sterilization.

9. A detachable cell membrane transfer device according to claim 1, characterized in that, The thermoresponsive hydrogel is polymerized from NIPAAm, crosslinking agent BIS, and photoinitiator under ultraviolet light.

10. A method for transferring cell membrane sheets, characterized in that, The detachable cell membrane transfer device according to any one of claims 1 to 9 includes the following steps: S1. Inject the thermo-responsive hydrogel into the hydrogel cavity (2) and irradiate it with ultraviolet light to polymerize the thermo-responsive hydrogel; S2. Remove the culture medium from the cell membrane sheet (4) and bring the PDMS suction sheet (32) into contact with the surface of the cell membrane sheet (4); S3. When the heating membrane (13) is energized, the thermally responsive hydrogel shrinks in volume, and the PDMS absorbent sheet (32) adsorbs the cell membrane sheet (4) under the pressure difference. S4. After the cell membrane sheet (4) is transferred to the predetermined position, the heating membrane (13) is de-energized. After the volume of the thermally responsive hydrogel is restored, the PDMS absorbent sheet (32) separates from the cell membrane sheet (4), thus completing the transfer.

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