Sterilizable clear heat-cut sealer and method of making same

Abstract

The application discloses a sterilizable transparent hot cutting seal and a preparation method thereof. The seal comprises a base, an electric heating wire, a top cover and a power supply; the electric heating wire is linear at both ends and is a sealing part in the middle; the base is made of transparent material and is provided with a groove track matched with the electric heating wire; the top cover is made of flexible transparent material; and the electric heating wire is connected with the power supply during use. The molds of the base and the top cover are constructed by using a 3D printing method, and transparent high-temperature-resistant polymers are poured in culture dishes and other containers; the base and the top cover are obtained after solidification; the electric heating wire is vertically placed in the groove track of the base and is connected with the power supply; and the base and the top cover are fixed, so that the transparent hot cutting seal is finally formed. The application solves the reliable sealing problem of a water-containing porous membrane and has wide application prospects in the fields of cell packaging, tissue engineering, medical health, food and the like.

Description

Technical Field

[0001] This invention relates to a sterilizable transparent thermal cutting sealer and its preparation method, belonging to the field of materials processing technology. Background Technology

[0002] The fabrication of various cell encapsulation devices relies heavily on sealing processes. For example, in the fabrication of islet encapsulation devices, porous membranes are typically encapsulated into small pouches. After the islets are infused into these pouches, the pores in the membrane become filled with liquid, making the sealing process particularly challenging. Therefore, developing a technology capable of reliably sealing aqueous porous membranes is crucial. Otherwise, sealing defects can lead to cell invasion, thereby reducing or even eliminating the immunoprotective efficacy of the encapsulation device. Furthermore, sealing defects can also cause cell escape, posing a risk of carcinogenesis when encapsulating stem cell-derived islets.

[0003] Generally, cell encapsulation devices can be sealed using solvent-based adhesives, solvent-free polymer melts, UV adhesives, and heat sealers. For example, a nylon 6 (PA6) / formic acid solution has been used to seal islet encapsulation devices based on PA6 nanofiber membranes, but formic acid is toxic and may impair the function of the encapsulated islets. To avoid the use of organic solvents, the scientific community has developed a solvent-free underwater adhesive based on low-modulus polyester containing catechol groups and coumarins. However, its adhesive strength (0.7 MPa) is lower than that of most membrane materials, such as PA6 nanofiber tubes (6.0 MPa). Furthermore, the adhesive must have a certain degree of fluidity before curing, making precise bonding very difficult.

[0004] Another approach involves creating a hydrophobic port during the fabrication of the encapsulation device, and then sealing the port with a UV-curable adhesive after cell loading. The port is typically non-porous and small in diameter, thus achieving a relatively reliable seal. However, this strategy is only suitable for loading cells dispersed in buffer solutions, not for loading cell-loaded scaffolds. Furthermore, commercial heat sealers have been used to encapsulate porous encapsulation devices. However, the sealed ends obtained by this method are typically rough and have sharp edges, inevitably leading to irritation and tissue adhesion after implantation. Additionally, commercial heat sealers struggle to achieve reliable seals when encapsulating water-loaded cell-loaded devices.

[0005] Patent No. 202080048213.5 discloses a polymer fiber having a main chain with a positively charged component including zwitterionic parts. In order to encapsulate pancreatic islet cells, the nanofiber tube is encapsulated by a manual pulse sealer, which may lead to an inability to control the degree of fiber melting, and the high temperature around the heating wire may cause irreversible damage to the islets.

[0006] Patent No. 202111222197.X discloses a silk protein-based underwater adhesive and its preparation method. In order to make the adhesive flowable, the patent dissolves degummed silk in an inorganic salt / anhydrous formic acid solution. However, formic acid is toxic and may impair the function of the encapsulated cells.

[0007] While the aforementioned existing technologies have achieved the sealing of water-containing porous membranes, research on issues such as cell biocompatibility and sealing surface morphology is limited, and they have not yet fully met the practical application requirements of cell encapsulation. This invention aims to overcome these and other deficiencies in the field. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a heat-cutting sealer with sterilization function and high visibility.

[0009] To address the aforementioned problems, this invention provides a sterilizable transparent thermal cutting sealer, comprising a base, an electric heating wire, a top cover, and a power source; the electric heating wire has linear ends and a sealing portion in the middle; the base is made of transparent material and has grooves that mate with the electric heating wire; the top cover is made of flexible transparent material, and the electric heating wire is connected to the power source during use.

[0010] Preferably, the width of the groove track is 0.1-10mm and the depth is 0.1-20mm.

[0011] Preferably, the thickness of the base is 0.1-20 cm, and the area is 0.3-1000 cm². 2 .

[0012] Preferably, the top cover has a bendability of 1°-180°, a thickness of 0.01-20cm, and an area of ​​0.3-1000cm². 2 .

[0013] Preferably, the edge of the top cover is provided with a notch that is the same or similar in shape to the sealing part of the electric heating wire, so as to reduce the temperature diffusion area during the sealing process.

[0014] Preferably, the heating wire has a heat resistance temperature of 50-3000℃ and is made of nickel-chromium alloy, iron-chromium-aluminum alloy, tungsten, or molybdenum; the heating wire has a thickness of 0.01-1mm, a length of 1-50cm, and a sealing width of 1-10mm; the power supply has a rated power greater than 2.5W and a rated current greater than 0.5A.

[0015] The base, heating wire, and top cover can be sterilized at high temperature and used in a sterile environment. The heat sealing process can be observed in real time with the naked eye or a microscope. The resulting sealing surface is smooth, the sealing strength is high, the temperature diffusion area during the sealing process is small, and it has no significant impact on the internal cells and other components.

[0016] Preferably, the base and top cover have a transparency of 1%-100% and a heat resistance temperature of 50-3000℃; the base and top cover are made of any one of the following: polydimethylsiloxane, polylactic acid, polyurethane, polyamide, polyvinyl chloride, epoxy resin, phenolic resin, vulcanized rubber, silicone, styrene, olefins, dienes, vinyl chloride, urethanes, esters, amides, organofluorine compounds, organosilicon compounds, and ethylene thermoplastic elastomers.

[0017] The present invention also provides a method for preparing the above-mentioned sterilizable transparent thermal cutting sealer: a mold for the base and the top cover is constructed by 3D printing, and a transparent high-temperature resistant polymer is poured into a container such as a petri dish. After curing, the base and the top cover are obtained. An electric heating wire is vertically placed in the groove track of the base and connected to a power source to fix the base and the top cover, thus forming the transparent thermal cutting sealer.

[0018] Preferably, the preparation method includes the following steps:

[0019] Step 1: Prepare a groove mold with a thickness similar to that of the electric heating wire using 3D printing. This mold will be used to prepare the base and top cover, which will be placed vertically in the petri dish.

[0020] Step 2: Mix the PDMS polymer and crosslinking agent, pour the mixture into a petri dish containing a grooved mold and an empty petri dish, remove air bubbles under vacuum, and place it in an oven to cure, thus preparing the base and top cover respectively.

[0021] Step 3: Remove the cured PDMS base and top cover;

[0022] Step 4: Bend the material of the electric heating wire and place it into the groove of the PDMS base. Use a punch to cut a notch on the edge of the top cover to form a notch with the same or similar shape as the sealing part. Fix the top cover and the base with a long tail clip to obtain the transparent sterilizable heat-cutting sealer.

[0023] Preferably, the consumable material for 3D printing is at least one of polylactic acid, polyvinyl alcohol, polyurethane, polyamide, acrylonitrile-butadiene-styrene terpolymer, polyethylene terephthalate-1,4-cyclohexanediol ester, photosensitive resin, and metal powder, and the 3D printer used is a fused deposition modeling 3D printer, a photopolymerization 3D printer, or a laser sintering 3D printer.

[0024] Preferably, the culture dish is circular, triangular, quadrilateral, pentagonal, or hexagonal in shape, with a thickness of 0.1-20 cm and an area of ​​0.3-1000 cm². 2 The material is glass, metal or polymer.

[0025] Preferably, the mass ratio of the PDMS polymer to the crosslinking agent is 10:0.1 to 0.1:10, the oven temperature is 20-500℃, and the curing time is 2-72h.

[0026] This invention provides an autoclavable transparent thermal liner (ATCC) and its preparation method. ATCC offers a reliable solution for encapsulating water-bearing cell-carrying devices. The sterilizable transparent thermal liner prepared by this invention can be used with porous membranes of any thermoplastic material, and is particularly suitable for reliably sealing water-bearing porous membranes, showing broad application prospects in cell encapsulation, tissue engineering, medical and health fields, and food.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) Compared with similar commercial sealers, the sterilizable transparent thermal cutting sealer and its preparation method of the present invention use transparent PDMS instead of high-temperature resistant polytetrafluoroethylene or glass cloth and base. During the sealing process, the melting state of the sealing film can be observed, and the heating wire can be stopped when the sealing state reaches the optimal point, thus improving the sealing stability. Moreover, PDMS has good heat resistance, is non-toxic and environmentally friendly, and ensures the long-term use of the sealer. The entire preparation process is simple and low-cost. The prepared transparent thermal cutter can be sterilized by high-temperature steam and has broad application prospects in the biomedical field.

[0029] (2) In the first, second and third steps of this invention, the grooved PDMS base is prepared by using a 3D printer to prepare a mold. 3D printing can achieve high precision and complexity in mold preparation, and the base can be customized and diversified to meet the usage needs of different scenarios.

[0030] (3) In the fourth step of this invention, the heating wire is placed vertically in the groove of the base. The heating wire extends beyond the base to provide space for the diffusion of moisture and heat, thereby improving the efficiency of moisture removal when sealing the water-containing porous membrane and reducing the transfer of heat on the film during the heating process. In addition, the melted part of the film will be cut off by the pressure of the heating wire and the top cover, and a smooth seal is achieved after cooling.

[0031] (4) In the fourth step of this invention, a notch is formed by cutting the edge of the top cover with a punch. The top cover with the notch is more conducive to the diffusion of moisture and heat during the heating process, which is beneficial to the encapsulation of the water-containing porous membrane and reduces the impact of heat accumulation on cell activity.

[0032] (5) During the sealing strength test, the porous membrane broke before the seal, indicating that the sealing strength was higher than the porous membrane strength. Cell proliferation and cell viability staining tests showed that the seal had no effect on cell viability. Attached Figure Description

[0033] Figure 1 For the design of ATTC, a) Schematic diagram of ATCC structure; b) Thermogravimetric curve of PDMS; c) Temperature change of nichrome resistance wire under 20 cycles of heating (60 seconds on, 60 seconds off, voltage 5V, heating element length 12.5cm); df) Schematic diagram including: d) A nanofiber tube placed on top of the heating wire; e) The nanofiber tube is cut into two independent parts after melting; f) Sealed nanofiber tube; gk) Image of the sealed PA6 nanofiber tube; g) Digital image; hk) Scanning electron microscope image;

[0034] Figure 2 Digital images of ATCC: a) PDMS cap; b) PDMS base plate; c) Assembled ATCC; d) Heating wire; e) ATCC placed in a sterilization bag for autoclaving; fh) Sealing process.

[0035] Figure 3 To optimize the ATTC encapsulation process, a) Schematic diagram of the ATCC sealing process of the nanofiber membrane; b) Schematic diagram of the sample preparation and tensile testing process; c, d) Microscopic images of the sealing area at different sealing heights: c) 2.5 mm, d) 0.5 mm; ef) Effect of sealing height on sealing: e) Stress-strain curve, f) Tensile strength; g, h) Sample fracture at: g) sealing area, h) other locations, tensile test images when the sealing height is 0.0 mm and 0.5 mm; ik) Effect of heating wire length on sealing: i) Heating time-temperature curve, j) Heating temperature equilibrium time and sealing time, k) Tensile strength; lm) Effect of sealing time on sealing: l) Tensile strength, m) Tensile strain; Unless otherwise stated, the heating wire length and sealing height will remain constant at 18.5 cm and 0.5 mm, respectively, and the symbols on the bar chart indicate comparisons with unlabeled samples;

[0036] Figure 4 To optimize ATTC sealing conditions; a) Images of the sealed end at different sealing heights. bd) Effect of sealing height on sealing: b) tensile strain, c) statistics of fracture locations, d) Effect of sealing width; e) Effect of heating wire length on sealing, e) Stress-strain curve, f) tensile strain, g) statistics of fracture locations, h) Sealing width, i) Burnt sealing caused when heating wire length is 12.5cm; jl) Effect of sealing time on sealing: j) stress-strain curve, k) statistics of fracture locations, l) Sealing width;

[0037] Figure 5Customization and diversification for ATTC; a) Schematic diagram of the process for preparing PDMS substrates of arbitrary shapes; bo) Generation of seals with arbitrary shapes; bi) Arc seals with different diameters and curvatures; j, k) Trapezoidal seals; l, m) Triangular seals; n, o) Serrated seals; pu) ATTC seals of different polymer nanofiber tubes; p, q) Digital images; r) Tensile strength; s) Stress-strain curves; t) Tensile strain; u) Statistical analysis of fracture locations.

[0038] Figure 6 ATTC sealing of hydrous nanofiber tubes; a) Schematic diagram of ATTC sealing of hydrous nanofiber membrane, where the gap between the base and the top cover allows the liquid to evaporate rapidly during heating, thus achieving melt bonding; be) Image of ATTC sealing of hydrous nanofiber membrane, b) Melting of hydrous porous membrane seal during heating, c) Melting observed within 0.7 seconds, d) Complete melting within 9.3 seconds, e) Scanning electron microscope image of the sealing area; fi) Effect of water content of hydrous nanofiber membrane on sealing, f) Stress-strain curve, g) Tensile strength, h) Tensile strain, i) Sealing time; jm) Bursting test, j) Schematic diagram of bursting test, k) Bursting of hydrous nanofiber tube sealed at one end in the unsealed area, l) Pressure change over time, m) Bursting pressure;

[0039] Figure 7 Biocompatibility testing of the cell encapsulation device prepared for ATTC. a) Schematic diagram of the cell encapsulation device preparation; b) Temperature of the device surface after sealing, captured by infrared thermal imaging; c) Distance between the 60°C boundary line and one end of the seal; d, e) Live / dead fluorescence images of NIH / 3T3 cells inside the sealed nanofiber tubes; f) Metabolic activity of encapsulated cells in nanofiber tubes with different pore sizes sealed by ATTC; g) Cell escape statistics (day 14) from encapsulation devices with different pore sizes.

[0040] Figure 8 Cell escape experiment results in nanofiber devices with different pore sizes sealed with ATTC; a1) Fluorescence image of the outer surface of the device, ac) Day 2, df) Day 7, gi) Day 10, jl) Day 14. mo) NIH3T3 / GFP cells in the device after 14 days of culture;

[0041] Figure 9 This is a schematic diagram of the structure of the transparent thermal cutting sealer provided by the present invention. Detailed Implementation

[0042] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0043] like Figure 9As shown, this invention provides a sterilizable transparent thermal cutting sealer, which includes a base 1, an electric heating wire 2, a top cover 3, and a power source; the two ends of the electric heating wire 2 are linear, and the middle is the sealing part; the base 1 is made of transparent material, and the base 1 is provided with grooves and tracks that cooperate with the electric heating wire 2; the top cover 3 is made of flexible transparent material, and the electric heating wire 2 is connected to the power source during use.

[0044] The width of the groove track is 0.1-10mm, and the depth is 0.1-20mm.

[0045] The base 1 can be circular, triangular, quadrilateral, pentagonal, hexagonal, or other shapes, with a thickness of 0.1-20 cm and an area of ​​0.3-1000 cm². 2 .

[0046] The top cover 3 has a bendability of 1°-180°, a thickness of 0.01-20cm, and an area of ​​0.3-1000cm². 2 The top cover can be circular, triangular, quadrilateral, pentagonal, hexagonal, or other shapes.

[0047] The edge of the top cover 3 is provided with a notch that is the same as or similar in shape to the sealing part of the electric heating wire 2, so as to reduce the temperature diffusion area during the sealing process. The edge shape includes straight line, arc, semi-circle, triangle, trapezoid, zigzag or other shapes.

[0048] The shape of the sealing part of the electric heating wire 2 can be arc-shaped, semi-circular, triangular, trapezoidal, zigzag, or other shapes; the heat resistance temperature of the electric heating wire 2 is 50-3000℃, and the material is nickel-chromium alloy, iron-chromium-aluminum alloy, tungsten, or molybdenum; the thickness of the electric heating wire 2 is 0.01-1mm, the length is 1-50cm, and the width of the sealing part is 1-10mm; the rated power of the power supply is greater than 2.5W, and the rated current is greater than 0.5A.

[0049] The base 1 and top cover 3 have a transparency of 1%-100% and a heat resistance temperature of 50-3000℃; the base 1 and top cover 3 are made of any one of the following: polydimethylsiloxane, polylactic acid, polyurethane, polyamide, polyvinyl chloride, epoxy resin, phenolic resin, vulcanized rubber, silicone, styrene, olefin, diene, vinyl chloride, urethane, ester, amide, organofluorine, organosilicon, and ethylene thermoplastic elastomers.

[0050] Materials and Methods:

[0051] Polycaprolactam (Nylon 6, N823205) was purchased from Macklin; polyvinylidene fluoride (PVDF, BD148542) from Bide Pharmatech; thermoplastic polyurethane (TPU, BTE-75A) from Evermore Chemical Industry; 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, 90106D), formic acid (FA, 73553A), N,N-dimethylformamide (DMF, 76259B), and acetone (Ace, 75902G) from Adamas; polydimethylsiloxane elastomer (PDMS, Sylgard 184) from Dow Corning; fibrinogen (F8051) from Solarbio; thrombin (ST1699) from Beyotime; and gelatin (1288485) from Sigma-Aldrich. All reagents were not further purified before use.

[0052] Example 1

[0053] ATCC Fabrication: A template for the PDMS substrate was fabricated using PLA 3D printing. Before casting, the template was placed vertically on a petri dish (35 mm in diameter). Approximately 10 g of liquid PDMS (10:1 weight ratio) was then poured into the petri dish, degassed, and heated overnight at 50°C. By removing the printed template and petri dish, a custom-grooved PDMS base (11 mm thick, 35 mm in diameter) was obtained. The length and thickness of the printed template were maintained at 31 mm and 0.5 mm, respectively. To achieve sealing heights ranging from 0.0 mm to 3.0 mm, the height of the printed template was set from 5 mm to 2 mm. Furthermore, to verify the generation of arbitrary-shaped seals by ATTC, templates with the same shape as the heating wire were printed. A circular PDMS cover (1.8 mm thick, 35 mm in diameter) was obtained by pouring approximately 1.6 g of liquid PDMS into a petri dish (35 mm in diameter) and creating curved edges using a 5 mm circular punch. To manufacture heating wires of arbitrary shapes and varying lengths, commercial nichrome resistance wire (5 mm wide, 0.2 mm thick) was cut to lengths ranging from 12.5 cm to 30.5 cm, bent into the desired shape using pliers, and then embedded into grooves in the PDMS substrate. Finally, the PDMS substrate and PDMS cover were assembled together using tail clips, while the two ends of the heating wire were connected to a switch and a power supply (rated voltage 5V, rated current 40A).

[0054] Example 2

[0055] ATTC Sealing: First, attach a nanofiber tube to the top of the heating wire, then apply pressure to secure it by pressing the cap with tweezers. Turn on the power to begin sealing and stop when the nanofibers are observed to have completely melted. To create a stable thermally bonded seal, maintain pressure until the molten polymer cools and solidifies.

[0056] Example 3

[0057] Electrospinning: Nanofiber tubes were prepared using a custom-designed electrospinning apparatus, including an injection pump, a moving platform, a rotating bar collector, and a high-voltage power supply (Dongwen High Voltage, DW-P303-1ACH2). The diameter of the nanofiber tubes was controlled by the diameter of the rotating bar collector. A thin layer of 80% sucrose syrup was coated onto the bar before electrospinning. The nanofiber tubes were removed by immersion in water, washed five times, and dried in an oven at 50°C. To generate PA6 nanofiber membranes with pore sizes of 4.08 μm, 1.06 μm, and 0.35 μm, electrospinning was performed using solutions of 0.2 g / mL PA6 / HFIP, 0.2 g / mL PA6 / (HFIP / FA 8:2), and 0.15 g / mL PA6 / (HFIP / FA 8:2). To prepare PVDF and TPU nanofiber tubes, electrospinning was performed using 0.12 g / mL PVDF / (DMF / Ace 1:2) and 0.22 g / mL TPU / DMF solutions. During electrospinning, the needle size, collecting rod diameter, collecting distance, collecting rod rotation speed, and moving platform speed remained constant at 18 G, 3.2 mm, 8 cm, 150 rpm, and 500 mm / min, respectively. The electrospinning parameters for nanofiber tubes with different pore sizes are shown in Table 1.

[0058] Table 1

[0059]

[0060] Example 4

[0061] Characterization of nanofiber tubes: The pore size of the nanofiber tubes was measured using a pore size analyzer (PMI, CFP-1100AI). Sealing quality was imaged using a scanning electron microscope (Hitachi, FLEX 1000). The sealing process was imaged using a stereomicroscope (AOSVI, T2-HD208C) and an infrared thermal imager (FLIR, T630SC). Sealing mechanical properties were measured using a tensile testing machine (DARONG, YG(B)026G). The moving speed and clamping distance were 50 mm / min and 20 mm, respectively. Specifically, straight-sealed nanofiber tubes (1.5 cm in length, 3.2 mm in diameter, and 160 μm in wall thickness) were cut from both sides using scissors. Bursting pressure was measured using a self-made electronic pressure gauge. Specifically, one end of a dried and moist PA6 nanofiber tube (3.5 cm in length, 3.2 mm in diameter, and 160 μm in wall thickness) was sealed with ATTC, and the other end was mounted on a polypropylene tube (11 cm in length, 3 mm in diameter, and 0.5 mm in thickness) via PDMS. A three-way valve connected the syringe, the polypropylene tube, and a digital pressure gauge. During testing, a 6% pink alginate solution was injected into the sealed nanofiber tube at a rate of 3 mL / min using a syringe pump.

[0062] Example 5

[0063] Cell escape assay: NIH3T3 / GFP mouse fibroblasts were dispersed at a density of 1 million / mL in a solution containing 10 mg / mL fibrinogen, 0.25 units / mL thrombin, and 50 mg / mL gelatin / saline. Then, 60 μL of the cell-matrix suspension was pipetted into one-end-sealed PA6 tubes (1.5 cm long, 3.2 mm in diameter). The other end of the cell-filled PA6 tubes was sealed with ATTC, and imaging was performed on days 2, 7, 10, and 14 using an inverted fluorescence microscope (Cossim FRD-6C). Cell metabolic activity was measured on days 2, 4, and 7 using a cell counting kit-8 (CCK-8) according to the manufacturer's instructions (Adamas). All samples were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The medium was changed every other day. The culture environment was 37°C and a 5 vol% CO2 atmosphere.

[0064] Example 6

[0065] Cell viability assay: To investigate the effect of the sealing process on cell viability, NIH3T3 mouse fibroblasts were dispersed at a density of 1 million / mL in a solution containing 10% gelatin / saline. Then, 60 μL of the cell-matrix suspension was pipetted into a PA6 tube sealed at one end. The other end of the PA6 tube containing cells was sealed with ATTC. After two days of culture, the middle of the PA6 tube was sealed again with ATTC. Live / dead cell staining was performed according to the manufacturer's instructions (Beyotime). After cutting open the sealed PA6 tube from both sides, the cells in the apparatus were subjected to fluorescence imaging.

[0066] Example 7

[0067] Statistical Analysis: All statistical analyses were performed using Origin 2022b. All bar charts represent the mean and standard deviation. All data were processed using analysis of variance (ANOVA). For three or more groups, Bonferroni multiple comparisons were used, and for two groups of quantitative data, t-tests were used. Statistical significance was expressed as ns, *, **, and *** when p-values ​​were greater than 0.05, less than 0.05, less than 0.01, and less than 0.001, respectively.

[0068] Related experiments in Examples 1-7

[0069] I. Design and Manufacturing of ATCC

[0070] The ATTC consists of a custom-designed base, top cover, and heating wire. Figure 1 (a) Both the base and the top cover are made of PDMS ( Figure 2 (a and b) are fixed together by clips. Figure 2 (c) The heating wire is made from a modified commercial nickel-chromium alloy. Figure 2 (d). It's important to note that the top cap is very thin (approximately 1.8mm thick), allowing for flexible opening and closing. It's worth noting that PDMS was chosen primarily for three reasons. First, PDMS is transparent, which facilitates visualization of the sealing process and ensures proper sealing. Second, PDMS can withstand temperatures up to 635°C (…). Figure 1 (b) Therefore, ATTC can perform high-temperature sterilization. Figure 2 (e). Third, the highest temperature of the heating wire during the repeated cycle is 505.6℃ ( Figure 1 c) The temperature is much lower than the decomposition temperature of PDMS, so the PDMS substrate and top cover can maintain a stable structure during repeated sealing processes.

[0071] Specifically, the heating wire is placed vertically in the groove of the PDMS base. Figure 1 a, Figure 2(b, c). This design ensures a narrow heating width (approximately 0.2 mm), and the invention can generate a smooth sealing surface through the cutting process. Figure 1 (df). The encapsulation process involves several steps. First, the nanofibers in contact with the heating wire will rapidly melt ( Figure 1 (d). Next, the nanofiber tube will be cut into two segments ( Figure 1 (e, f). Then, after heating is stopped, the molten areas will solidify and bond together at room temperature. It is important to note that throughout the process, pressure should be applied to the sealing area using tweezers to achieve rapid melting and cutting, as well as sufficient contact during bonding. It is worth noting that this invention assumes that ATTC can be applied to encapsulate hydrous porous membranes when the heating element is above the groove depth of the PDMS substrate. This creates a gap between the substrate and the top cap, allowing for rapid liquid evaporation during heating, thus achieving melt bonding after the moisture disappears.

[0072] To verify the feasibility of ATTC, this invention first uses a dried PA6 nanofiber tube (3.2 mm in diameter, 160 μm in wall thickness) with an arc-shaped seal for sealing. Figure 1 g, Figure 2 (fh). It is worth noting that the melting process can be completed within 7 seconds. Figure 2 (f, g), and the sealed tube can be easily separated into two parts ( Figure 2 (h). Even better, the entire sealing end is very smooth (h). Figure 1 (hj). Furthermore, a distinct gradient melting zone can be observed at the sealed end. Figure 1 (k) indicates that the melting of the nanofiber membrane exists not only in the heating element region, but also extends to a region a certain distance away from the heating wire due to heat conduction, which is particularly important for reliable sealing based on the ATTC heat-cutting sealing method.

[0073] II. Optimization of ATCC Sealing Process

[0074] Based on the ATTC sealing principle, this invention considers the heating wire height, sealing temperature, and heating time to be the key parameters determining the encapsulation performance. Figure 3 (a) To facilitate quantification of encapsulation strength, this invention uses a fixed voltage of 5V to linearly seal the PA6 nanofiber tube, and then cuts the PA6 tube sealed at one end from both sides of its length for tensile testing. Figure 3 (b) To avoid the influence of temperature on the sealing quality, the sealing process is carried out at room temperature (24°C). Similar to arc sealing, straight sealing can also be completed quickly, and the two parts can be easily separated after bonding.

[0075] First, this invention optimizes the heating wire height by fixing the length of the heating wire to 12.5 cm. When the heating wire height is equal to or greater than 1.5 mm, insufficient contact between the top and bottom occurs during the curing process of the PA6 nanofiber tube, resulting in incomplete closure of the sealing area. Figure 3 c in the middle Figure 4 (a) In contrast, a better sealing morphology was observed when the sealing height was less than 1.5 mm. Figure 3 d, Figure 4 (a) Tensile test results show that a higher heating wire height leads to lower tensile strength and a larger strength variation ( Figure 3 (e, f) shorter tensile strain ( Figure 4 (b) and more breaks appear at the seal ( Figure 3 g, Figure 4 (c) Conversely, when the heating wire height is 0.5 mm, the tensile strength is 7.98 ± 0.32 MPa, which is similar to that of the original PA6 nanofiber tube. Figure 3 (e, f). Furthermore, the tensile strain is significantly increased ( Figure 4 (b) The fracture mainly occurs in the unsealed area rather than the sealed end. Figure 3 h, Figure 4 (c) indicates that the mechanical properties of the sealed end are even superior to those of the original PA6 nanofiber membrane. However, when the sealing height is further reduced to 0 mm, the tensile strength and tensile strain decrease significantly. This is attributed to the heating element being completely hidden in the groove of the PDMS base, resulting in poor heat conduction, which can be verified by the narrower sealing width. Figure 4 (d). In summary, the results of this invention demonstrate that a heating wire height of 0.5 mm, similar to the tube wall thickness, can achieve excellent sealing quality.

[0076] Next, this invention investigated the effect of heating temperature on sealing quality while maintaining the heating wire height at 0.5 mm. It is noteworthy that the heating temperature was adjusted by changing the length of the heating wire. Specifically, when the length of the heating element was reduced from 30.5 cm to 12.5 cm, the equilibrium temperature decreased from 221.7 ± 4.0 °C to 511.0 ± 1.1 °C. Figure 3 (i). Accordingly, the balancing time was reduced from 70.0 seconds to 37.6 seconds, while the sealing time was reduced from 62.3 ± 4.0 seconds to 2.9 ± 0.5 seconds. Figure 3 The shorter heating wire (j) than the equilibrium time indicates that the actual sealing temperature is much lower than the equilibrium temperature. Overall, a shorter heating wire results in better sealing quality. Figure 3 k, Figure 4 (e.g.), this is due to the larger sealing width ( Figure 4(h). However, when the length of the heating element is 12.5 cm, the present invention observed scorching at the sealing end (h). Figure 4 The result in (i) indicates that the heating temperature is too high for the dried PA6 nanofiber tubes. Therefore, this invention selected a heating element length of 18.5 cm for further optimization.

[0077] In theory, better sealing quality can be achieved by increasing the heating time, due to the sufficient diffusion of the molten polymer and a larger sealing width. Figure 4 Specifically, when the heating time increased from 6 s to 10 s, the tensile strength and tensile strain increased from 3.04 ± 0.53 MPa to 8.46 ± 0.63 MPa, respectively. Figure 3 The value of l increased from 0.03±0.01 mm / mm to 1.26±0.31 mm / mm. Figure 3 (m). Notably, when the sealing time was 10 s, the tensile strength and tensile strain were the same as the original PA6 nanofiber tube, indicating that ATTC can achieve a seal with excellent quality. However, when the sealing time was further increased to 30 seconds, the seal quality deteriorated. This was attributed to the flexible PDMS cap failing to provide sufficient pressure to the sealing end when the sealing width was too large, although this issue could be addressed by using a flat plate instead of tweezers to apply pressure if a wider sealing end was required.

[0078] III. ATTC Customization and Diversification

[0079] Based on the above optimization results, it is clear that the shape of the heating wire directly determines the shape of the sealing end. To verify ATTC's ability to generate seals of arbitrary shapes, this invention uses 3D printing technology to create templates of various shapes. Figure 5 (a) Therefore, the present invention produces a PDMS base plate that closely fits various shapes through a casting process, thereby enabling the stable installation of nickel-chromium wire heating wires that are adapted to various shapes.

[0080] Except for the straight sealing end ( Figure 3 In addition to (d), the present invention can also easily prepare arc-shaped sealing ends ( Figure 5 (In the middle of the bg). For example, ATTC can seal nanofiber tubes with diameters from 3.2 mm to 9.0 mm ( Figure 5 (The middle section is a stencil material), which is advantageous for loading cells when fabricating larger diameter encapsulation devices. As expected, the curvature of the arc can also be adjusted. Figure 5 (in Chinese). It is worth noting that a smooth sealing end is expected to help reduce irritation and immune responses. Furthermore, sealing ends of any shape, such as trapezoidal, triangular, and serrated, can be easily achieved. Figure 5 injo).

[0081] Since ATTC sealing is based on melt bonding, this invention anticipates that ATTC will be applicable to sealing of all thermoplastic polymer-based membrane materials. Therefore, this invention uses polyvinylidene fluoride (PVDF) and thermoplastic polyurethane (TPU) to fabricate nanofiber tubes with a diameter of 3.2 mm to test the sealing capability of ATTC. Similar to PA6 nanofiber tubes, PVDF and TPU nanofiber tubes can produce smooth, curved seals. Figure 5 (p, q). Furthermore, this invention quantifies the sealing quality through tensile testing. The results show that the tensile strength and tensile strain of the PVDF and TPU nanofiber tubes are the same as those of the original material (p, q). Figure 5 (middle rt). Furthermore, the fractures in the tensile tests all occurred on the porous membrane ( Figure 5 (u), which indicates that ATTC is suitable for encapsulating a variety of thermoplastic film materials.

[0082] IV. ATCC Encapsulation of Aqueous Porous Membranes

[0083] The porous membrane of the cell encapsulation device becomes wetted by the solution after cell loading, which poses a significant challenge to the subsequent sealing process. Therefore, reliable encapsulation of aqueous porous membrane materials is crucial for the fabrication of cell encapsulation devices. This invention assumes that the heating zone of the ATTC is narrow and that there is a gap between the PDMS base and the top cover to allow for liquid evaporation. Figure 6 (a) ATTC can encapsulate aqueous nanofiber membranes. To verify ATTC's ability to package aqueous nanofiber membranes, this invention prepared moistened PA6 nanofiber tubes by immersing dried nanofiber tubes in water for sealing tests. Unlike previous optimizations for dry membrane materials, the sealing height was set to 1 mm to allow for sufficient liquid evaporation. Furthermore, the length of the heating wire was set to 12.5 cm to accelerate the melting process. During encapsulation, moisture evaporated immediately upon energization, and water vapor was observed passing through the PDMS top cover within 0.7 s. Figure 6 (b, c). As expected, the sealing time for the hydrous porous membrane material is longer than that for the dry membrane material. In particular, when the water content of the nanofiber tubes is 212.7%, it takes 9.3 seconds for the heated area to dry and melt. Figure 6 (d). Even better, a smooth seal can be obtained when encapsulating hydrous nanofiber tubes. Figure 6 (e).

[0084] To investigate whether the moisture content of hydrated nanofiber tubes affects sealing quality, this invention controls the moisture content by measuring the absorption time of the paper towel. As expected, the moisture in the nanofiber tubes negatively impacts sealing quality. Specifically, when the moisture content increases from 99.2% ± 14.4% to 346.3% ± 39.0%, the tensile strength and tensile strain decrease from 5.4 ± 1.0 MPa to 2.5 ± 1.3 MPa and from 0.08 ± 0.04 mm / mm to 0.01 ± 0.01 mm / mm, respectively. Figure 6 The value (fh) is lower than that of encapsulated dry nanofiber tubes. This should be attributed to the continuous movement of moisture towards the sealing area due to capillary effects, resulting in a narrower seal width (fh). Figure 6 (e). Correspondingly, the sealing time increased from 4.5±0.7s to 9.8±1.7s. Figure 6 The sealing time is longer than that of dry nanofiber tubes, indicating that moisture evaporation takes time. Therefore, in order to better encapsulate water-containing porous membrane materials, the moisture around the sealing area should be sufficiently absorbed.

[0085] To further quantify whether the seal of the hydrous nanofiber tubes was sufficient for practical applications, the burst pressure was measured using an electronic pressure gauge. Specifically, one end of a dry and hydrous PA6 nanofiber tube was sealed with ATTC, while the other end was connected to a polypropylene tube via PDMS for connection to the pressure gauge. During the test, a 6% pink alginate solution was injected into the nanofiber tube using a syringe pump. Figure 6 (j). It is worth noting that the burst occurred in the middle of the fiber tube rather than at the sealed end. Figure 6 (kPa). Furthermore, the burst pressures of the dry encapsulation device (313.9 ± 17.8 kPa) and the aqueous encapsulation device (313.2 ± 19.3 kPa) are similar, indicating that the sealing end has a stronger burst pressure than the membrane material itself. Therefore, this invention suggests that ATTC can be used as a reliable method for encapsulating aqueous cell encapsulation devices.

[0086] V. Evaluation of ATCC biocompatibility by encapsulating cell-loaded porous membranes

[0087] To evaluate the suitability of ATTC for encapsulating cell-carrying devices, this invention dispersed 60 μL (cell density of 1 million / mL) of NIH3T3 or NIH3T3 / GFP cell suspension in fibrinogen / gelatin hydrogel, and loaded it into a nanofiber tube (3.2 mm in diameter and 25 mm in length) sealed at one end, then sealed the other end with an arc-shaped ATTC. Figure 7 (a). Furthermore, to reduce heat transfer throughout the packaging device, a top cover with curved edges is fabricated using perforation. Figure 2(a). Because the heat generated by ATTC during the sealing process not only aids in thermal welding but also affects the survival rate of the loaded cells, the surface temperature of the encapsulation device was measured immediately after sealing using an infrared thermal imager. Figure 7 (b) The results showed that when the length of the heating wire increased from 12.5 cm to 24.5 cm, the distance between the 60°C boundary line and the sealing end increased from 3.37 ± 0.35 mm to 5.54 ± 0.29 mm. Figure 7 c) indicates that a faster encapsulation process can reduce heat transfer from the heating wire along the length of the encapsulation device by a higher sealing temperature. Therefore, a heating wire of 12.5 cm in length was used in further quantitative evaluation.

[0088] To better investigate the impact of the encapsulation process on cell survival, NIH3T3 cell suspension was loaded into nanofiber tubes and cultured in an incubator for 48 hours to promote cell attachment; then, cell viability was quantitatively analyzed using a live / dead staining method. The results showed that the cell viability in the main area of ​​the device was 95.51% ± 2.25%. Figure 7 In sections d and e), although most cells died in the sealed area due to high temperature, the distance between the dead cell boundary and the sealed end was 3.45 ± 0.24 mm. Figure 7 (d) is relatively short. It is worth noting that in actual cell encapsulation devices, the cells are not placed in the sealing area to achieve a reliable seal without damaging the encapsulated cells. Therefore, this invention suggests that ATTC can be used to prepare cell encapsulation devices.

[0089] To further evaluate the biocompatibility of ATTC, this invention prepared nanofiber tubes with different pore sizes (0.35 μm, 1.06 μm, and 4.08 μm) using electrospinning technology for better comparison. In this case, NIH3T3 / GFP cells were encapsulated to evaluate cell proliferation and escape. CCK-8 results showed that cells could proliferate continuously for up to 7 days in a sealed device. Figure 7 (f) indicates the cell compatibility of the ATTC encapsulation process with the manufacturing process of the encapsulation device. To confirm the reliability of the seal, the present invention monitored cell escape using a fluorescence microscope daily for up to 14 days. Starting from day 10, cells began to be detected on the outer surface of the encapsulation devices with pore sizes of 1.06 μm and 4.08 μm. In contrast, no cell escape was detected for the encapsulation device with a pore size of 0.35 μm. Figure 7 g, Figure 8 (al) confirmed that ATTC can generate a reliable seal with no cell leakage. On day 14, the present invention opened these devices and found that the cells were still viable in the devices. Figure 8The result (in the text) indicates that ATTC is highly compatible with cells during the fabrication of the cell encapsulation device.

[0090] This invention provides a transparent, sterilizable ATCC for reliably encapsulating hydrous porous membranes with smooth, arbitrary shapes. Notably, the transparent PDMS base and top cap allow for high-temperature, high-pressure sterilization and better control of sealing quality through in-situ visualization. Furthermore, the vertically embedded heating wire enables the sealing edges to achieve arbitrary shapes. Through system optimization, the ATCC achieves a robust seal with the same tensile strength of 8.46 ± 0.63 MPa as the original porous membrane. More importantly, the ATCC reliably encapsulates hydrous porous membranes, with a burst pressure (313.16 ± 19.34 kPa) similar to that of dry-sealed porous membranes. Moreover, cell survival, proliferation, and escape tests validated the ATCC encapsulation process's high compatibility with the fabrication of cell encapsulation devices. In conclusion, the ATCC provides a novel option for encapsulating hydrous cell-loaded devices.

Claims

1. A sterilizable transparent heat-cutting sealer, characterized in that, The device includes a base (1), an electric heating wire (2), a top cover (3), and a power supply. The electric heating wire (2) has two linear ends and a sealing section in the middle. The base (1) is made of transparent material and has grooves that cooperate with the electric heating wire (2). The top cover (3) is made of flexible transparent material and has notches on its edge that are the same or similar in shape to the sealing section of the electric heating wire (2) to reduce the area of ​​temperature diffusion during the sealing process. When in use, the electric heating wire (2) is connected to the power supply and is placed vertically in the grooves of the base (1). The electric heating wire (2) extends beyond the base (1) to provide space for the diffusion of moisture and heat. This sealer is used for sealing water-containing porous membranes.

2. The sterilizable transparent heat-cutting sealer as described in claim 1, characterized in that, The width of the groove track is 0.1-10mm, and the depth is 0.1-20mm.

3. The sterilizable transparent heat-cutting sealer as described in claim 1, characterized in that, The thickness of the base (1) is 0.1-20cm, and the area is 0.3-1000cm². 2 .

4. The sterilizable transparent heat-cutting sealer as described in claim 1, characterized in that, The top cover (3) has a bendability of 1°-180°, a thickness of 0.01-20cm, and an area of ​​0.3-1000cm². 2 .

5. The sterilizable transparent heat-cutting sealer as described in claim 1, characterized in that, The electric heating wire (2) has a heat resistance temperature of 50-3000℃ and is made of nickel-chromium alloy, iron-chromium-aluminum alloy, tungsten or molybdenum. The electric heating wire (2) has a thickness of 0.01-1mm, a length of 1-50cm, and a sealing width of 1-10mm. The power supply has a rated power greater than 2.5W and a rated current greater than 0.5A.

6. The sterilizable transparent heat-cutting sealer as described in claim 1, characterized in that, The base (1) and top cover (3) have a transparency of 1%-100% and a heat resistance temperature of 50-3000℃; the base (1) and top cover (3) are made of any one of polydimethylsiloxane, polylactic acid, polyurethane, polyamide, polyvinyl chloride, epoxy resin, phenolic resin, and vulcanized rubber.

7. The method for preparing the sterilizable transparent thermal cutting sealer according to any one of claims 1-6, characterized in that, The molds for the base and top cover are constructed using 3D printing. A transparent high-temperature resistant polymer is poured into a petri dish and cured to obtain the base and top cover. An electric heating wire is vertically placed in the groove track of the base and connected to a power source to fix the base and top cover, ultimately forming a transparent thermal cutting sealer.

8. The method for preparing the sterilizable transparent thermal cutting sealer as described in claim 7, characterized in that, Includes the following steps: Step 1: Prepare a groove mold with a thickness similar to that of the electric heating wire using 3D printing. This mold will be used to prepare the base and top cover, which will be placed vertically in the petri dish. Step 2: Mix the PDMS polymer and crosslinking agent, pour the mixture into a petri dish containing a grooved mold and an empty petri dish, remove air bubbles under vacuum, and place it in an oven to cure, thus preparing the base and top cover respectively. Step 3: Remove the cured PDMS base and top cover; Step 4: Bend the material of the electric heating wire and place it into the groove of the PDMS base. Use a punch to cut a notch on the edge of the top cover to form a notch with the same or similar shape as the sealing part. Fix the top cover and the base with a long tail clip to obtain the sterilizable transparent heat-cutting sealer.

9. The method for preparing the sterilizable transparent thermal cutting sealer as described in claim 8, characterized in that, The consumables for 3D printing are at least one of polylactic acid, polyvinyl alcohol, polyurethane, polyamide, acrylonitrile-butadiene-styrene terpolymer, polyethylene terephthalate-1,4-cyclohexanediol ester, and metal powder. The 3D printing equipment used is a fused deposition modeling 3D printer, a photopolymerization 3D printer, or a laser sintering 3D printer.

10. The method for preparing the sterilizable transparent thermal cutting sealer as described in claim 8, characterized in that, The grooved mold-containing petri dishes and empty petri dishes are both circular, triangular, quadrilateral, pentagonal, or hexagonal in shape, with a thickness of 0.1-20 cm and an area of ​​0.3-1000 cm². 2 The material is glass, metal or polymer.

11. The method for preparing the sterilizable transparent thermal cutting sealer as described in claim 8, characterized in that, The mass ratio of the PDMS polymer to the crosslinking agent is 10:0.1~0.1:10, the oven temperature is 20-500℃, and the curing time is 2-72h.

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