Preparation method and application of hydrogel core-shell tube

Abstract

The present invention relates to a preparation method and application of a hydrogel core-shell tube, comprising the following steps: injecting a core layer photocurable gel solution into the inner channel of an inner and outer double-layer coaxial needle, and injecting a shell layer photocurable gel solution into the outer channel of the inner and outer double-layer coaxial needle, wherein the inner channel is sleeved within the outer channel; simultaneously extruding the core layer photocurable gel solution and the shell layer photocurable gel solution into a collecting liquid, cross-linking the shell layer photocurable gel solution, and obtaining a preliminarily cross-linked core-shell tube; placing the preliminarily cross-linked core-shell tube in a light environment, cross-linking the core layer photocurable gel solution, and further cross-linking the shell layer photocurable gel solution, thereby obtaining a hydrogel core-shell tube. The hydrogel core-shell tube prepared by the present invention has obvious core-shell stratification, and when the hydrogel core-shell tube is applied to cell culture, cell partitioning can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a preparation method and application of a hydrogel core-shell tube. Background Art

[0002] Conventional two-dimensional (2D) cell culture techniques struggle to replicate the true physiological conditions of living tissues, such as the three-dimensional (3D) structure, cellular heterogeneity, and cell-matrix interactions. Mixing cells into a hydrogel precursor solution and cross-linking them is a simple method for achieving 3D cell culture, but it lacks the ability to control cell compartments, hinders oxygen and nutrient delivery, and compromises cell metabolism. Summary of the Invention

[0003] Based on this, it is necessary to provide a method for preparing hydrogel core-shell tubes that can achieve cell partitioning.

[0004] To achieve the above object, the present invention provides a technical solution:

[0005] A method for preparing a hydrogel core-shell tube comprises the following steps:

[0006] Injecting a core layer photocurable gel solution into the inner channel of the inner and outer double-layer coaxial needle, and injecting a shell layer photocurable gel solution into the outer channel of the inner and outer double-layer coaxial needle, wherein the inner channel is sheathed within the outer channel;

[0007] extruding the core layer photocurable gel solution and the shell layer photocurable gel solution simultaneously into a collection solution, and crosslinking the shell layer photocurable gel solution to obtain a preliminarily crosslinked core-shell tube;

[0008] The preliminarily cross-linked core-shell tube is placed in a light environment, the core layer photocurable gel solution is cross-linked, and the shell layer photocurable gel solution is further cross-linked to obtain the hydrogel core-shell tube.

[0009] Preferably, the core layer photocurable gel solution includes a GelMA solution.

[0010] Preferably, the steps of preparing the GelMA solution include:

[0011] GelMA, a photoinitiator and a CaCl2 solution are stirred and dissolved to obtain the GelMA solution.

[0012] Preferably, the shell layer photocurable gel solution comprises a GelMA / alginate solution.

[0013] Preferably, the steps of preparing the GelMA / alginate solution include:

[0014] adding sodium alginate into a NaCl solution to swell the alginate to obtain a swollen sodium alginate solution;

[0015] GelMA and a photoinitiator are added to the swollen sodium alginate solution, and the mixture is stirred and dissolved to obtain the GelMA / alginate solution.

[0016] Preferably, the inner and outer double-layer coaxial needle includes an inner needle and an outer needle, the inner needle is inserted into the outer needle, the inner diameter of the inner needle forms the inner channel, and the inner diameter of the outer needle and the outer diameter of the inner needle form the outer channel.

[0017] Preferably, the outer diameter of the inner needle is 1.28 mm, the inner diameter of the inner needle is 0.84 mm, the outer diameter of the outer needle is 1.81 mm, and the inner diameter of the outer needle is 1.65 mm.

[0018] Preferably, in the step of placing the preliminarily cross-linked core-shell tubes under a light irradiation environment, the wavelength of the light is 400-410 nm, and the intensity of the light is 1-1.5 μW / cm 2 .

[0019] Preferably, the injection speed of the core layer photocurable gel solution is 10 to 20 mL / h, and the injection speed of the shell layer photocurable gel solution is 10 to 40 mL / h.

[0020] The present invention also provides an application of the preparation method of the hydrogel core-shell tube as described above in cell culture.

[0021] Beneficial effects of the present invention:

[0022] The present invention injects a core layer photocurable gel solution and a shell layer photocurable gel solution into the inner and outer channels of the inner and outer double-layer coaxial needles respectively, first extrudes the core layer photocurable gel solution and the shell layer photocurable gel solution into a collecting liquid, crosslinks the shell layer photocurable gel solution to obtain a preliminarily crosslinked core-shell tube, and a preliminarily crosslinked reaction occurs due to the crosslinking of the shell layer photocurable gel solution. The tube wall of the preliminarily crosslinked core-shell tube consists of a crosslinked shell layer photocurable gel solution and an uncrosslinked shell layer photocurable gel solution. The core layer photocurable gel solution is placed in the preliminarily crosslinked core-shell tube, and the core-shell tube is further placed under light conditions for a photocuring reaction to obtain a hydrogel core-shell tube with obvious core-shell stratification. The hydrogel core-shell tube is applied to cell culture to achieve cell zoning control. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the preparation method of hydrogel core-shell tubes;

[0024] Figure 2 Schematic diagram of the cross section of the hydrogel core-shell tube;

[0025] Figure 3This is a magnified microscope diagram of the hydrogel core-shell tube;

[0026] Figure 4 A magnified microscope diagram of the cross section of the hydrogel core-shell tube;

[0027] Figure 5 When the flow rate of the core layer photocurable gel solution is 10 mL / h, the total diameter, core layer diameter and shell layer thickness of the hydrogel core-shell tube change with the flow rate of the shell layer photocurable gel solution;

[0028] Figure 6 When the flow rate of the core layer photocurable gel solution is 15 mL / h, the total diameter, core layer diameter and shell layer thickness of the hydrogel core-shell tube change with the flow rate of the shell layer photocurable gel solution;

[0029] Figure 7 When the flow rate of the core layer photocurable gel solution is 20 mL / h, the total diameter, core layer diameter and shell layer thickness of the hydrogel core-shell tube change with the flow rate of the shell layer photocurable gel solution;

[0030] Figure 8 This is a microscopic magnified schematic diagram of the hydrogel core-shell tube when the flow rate of the core layer photocurable gel solution is 10 mL / h and the flow rate of the shell layer photocurable gel solution is 40 mL / h;

[0031] Figure 9 This is a microscopic magnified schematic diagram of the hydrogel core-shell tube when the flow rate of the core layer photocurable gel solution is 15 mL / h and the flow rate of the shell layer photocurable gel solution is 20 mL / h;

[0032] Figure 10 This is a microscopic magnified schematic diagram of the hydrogel core-shell tube when the flow rate of the core layer photocurable gel solution is 20 mL / h and the flow rate of the shell layer photocurable gel solution is 10 mL / h;

[0033] Figure 11 Fluorescence microscopy image of hydrogel shell-core tube encapsulating cells;

[0034] Figure 12 Fluorescence microscopy image of a cross-section of a hydrogel shell-core tube encapsulating cells.

[0035] Among them, 100. Inner channel; 200. Outer channel; 300. Inner needle; 400. Outer needle; 500. Hydrogel core-shell tube; 510. Nuclear layer; 520. Shell layer; 530. Cell A; 540. Cell B; 600. Collection fluid. DETAILED DESCRIPTION

[0036] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific embodiments.

[0037] In the examples, the test methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0038] A method for preparing a hydrogel core-shell tube 500 comprises the following steps:

[0039] S100. Preparation of nuclear layer photocurable gel solution

[0040] Specifically, the core layer photocurable gel solution includes a GelMA solution. The steps for preparing the GelMA solution are as follows:

[0041] Add GelMA and a photoinitiator to a 100 mM CaCl2 solution, heat to 35-39°C, and stir on a magnetic stirrer for 30 minutes to fully dissolve. This yields a GelMA solution with a GelMA concentration of 80-100 g / L and a photoinitiator concentration of 2-5 g / L.

[0042] S200. Preparation of Shell Photocurable Gel Solution

[0043] Specifically, the shell layer photocurable gel solution includes a GelMA / alginate solution. The steps for preparing the GelMA / alginate solution are as follows:

[0044] Sodium alginate is added into physiological saline (NaCl solution) with a concentration of 0.85-0.95% to swell, and the swollen sodium alginate solution is obtained after swelling for 12-16 hours.

[0045] GelMA and a photoinitiator were added to the swollen sodium alginate solution, and the mixture was heated to 35-39° C. and stirred on a magnetic stirrer for 30 minutes to fully dissolve the mixture, thereby obtaining a GelMA / alginate solution.

[0046] The mass concentration of GelMA is 80-100 g / L, the mass concentration of the photoinitiator is 2-5 g / L, and the mass concentration of sodium alginate is 9-10 g / L.

[0047] More specifically, the photoinitiator is LAP.

[0048] S300. Preparation of hydrogel core-shell tube 500

[0049] like Figure 1As shown, the inner and outer double-layer coaxial needle includes an inner needle 300 and an outer needle 400. The inner needle 300 is inserted into the outer needle 400. The inner diameter of the inner needle 300 forms an inner channel 100. The inner diameter of the outer needle 400 and the outer diameter of the inner needle 300 form an outer channel 200. The outer diameter of the inner needle 300 is 1.28 mm, the inner diameter of the inner needle 300 is 0.84 mm, the outer diameter of the outer needle 400 is 1.81 mm, and the inner diameter of the outer needle 400 is 1.65 mm. The core layer photocurable gel solution and the shell layer photocurable gel solution are respectively loaded into syringes and connected to the inner channel 100 and outer channel 200 of the inner and outer double-layer coaxial needles through a flexible tube. The core layer photocurable gel solution is injected into the inner channel 100 of the inner and outer double-layer coaxial needles through the syringe, and the shell layer photocurable gel solution is injected into the outer channel 200 of the inner and outer double-layer coaxial needles through the syringe. The inner channel 100 is arranged inside the outer channel 200. The injection rate of the core layer photocurable gel solution is 10 to 20 mL / h, and the injection rate of the shell layer photocurable gel solution is 10 to 40 mL / h.

[0050] Specifically, when injecting with two syringes, the inner needle 300 and the outer needle 400 of the inner and outer double-layer coaxial needle are fixed vertically on the collection liquid 600, and the front ends of the needle nozzles of the inner needle 300 and the outer needle 400 just touch the liquid surface of the collection liquid 600. Preferably, the collection liquid 600 includes a CaCl2 solution.

[0051] The syringe pumps are used to independently adjust the flow rates of each solution, so that the core layer 510 solution and the shell layer 520 solution flow out through the inner and outer channels 200 of the coaxial needle respectively and are injected into the collection solution 600. After the two solutions flow out of the needle nozzle, the sodium alginate contacts the calcium ions in the collection solution 600 and the core layer 510 to form ionic crosslinks. The calcium ions in the collection solution 600 and the core layer photocurable gel solution are 2+ Rapidly cross-linking the sodium alginate in the photocurable gel solution of the outer core layer to obtain a preliminarily cross-linked shell-core tube;

[0052] The preliminarily cross-linked core-shell tube is placed in a light environment, the core layer photocurable gel solution is cross-linked, and the shell layer photocurable gel solution is further cross-linked to obtain a hydrogel core-shell tube 500. Specifically, the wavelength of the light is 405 nm, and the intensity of the light is 1.2 μW / cm 2 .

[0053] The present invention also provides an application of the preparation method of any one of the above hydrogel core-shell tubes 500 in cell culture.

[0054] Specifically, the specific steps of its application are as follows:

[0055] When preparing the core layer photocurable gel solution, target cells A530 were added and mixed evenly. Then, the shell layer photocurable gel solution was prepared and cells B540 were added to prepare the hydrogel core-shell tube 500 to achieve the partitioning of cells A530 and cells B540. More specifically, the cell concentration of cells A530 was 2.0-3.0×10 5 The cell concentration of B540 cells is 2.0-3.0×10 5 .

[0056] More specifically, if Figure 2 As shown, the hydrogel core-shell tube 500 includes a core layer 510 and a shell layer 520. The core layer 510 is formed by photocuring a core layer photocurable gel solution (i.e., GelMA, CaCl2 and cell A530), and the shell layer 520 is formed by curing a shell layer photocurable gel solution (i.e., GelMA, sodium alginate and cell B540).

[0057] Example 1

[0058] S100. Preparation of nuclear layer photocurable gel solution

[0059] The core layer photocurable gel solution includes a GelMA solution.

[0060] GelMA, photoinitiator LAP, and CaCl2 were added to a solution, heated to 37°C, and stirred on a magnetic stirrer for 30 minutes to fully dissolve, thereby obtaining a GelMA solution. The mass concentration of GelMA was 80 g / L, and the mass concentration of photoinitiator LAP was 5 g / L.

[0061] S200. Preparation of Shell Photocurable Gel Solution

[0062] The shell layer photocurable gel solution includes GelMA / alginate solution.

[0063] Sodium alginate was added to a 0.9% saline solution (NaCl solution) to swell for 12 hours to obtain a swollen sodium alginate solution;

[0064] GelMA and photoinitiator LAP were added to the swollen sodium alginate solution, and the mixture was heated to 37° C. and stirred on a magnetic stirrer for 30 min to fully dissolve the mixture, thereby obtaining a GelMA / alginate solution.

[0065] Among them, the mass concentration of GelMA is 80 g / L, the mass concentration of photoinitiator LAP is 5 g / L, and the mass concentration of sodium alginate is 10 g / L.

[0066] S300. Preparation of hydrogel core-shell tubes

[0067] The core layer photocurable gel solution and the shell layer photocurable gel solution are respectively loaded into syringes, and connected to the inner channel and outer channel of the inner and outer double-layer coaxial needles through a hose. The core layer photocurable gel solution is injected into the inner channel of the inner and outer double-layer coaxial needles through the syringe, and the shell layer photocurable gel solution is injected into the outer channel of the inner and outer double-layer coaxial needles through the syringe, and the inner channel is arranged inside the outer channel; the injection rate of the core layer photocurable gel solution is 10 mL / h, and the injection rate of the shell layer photocurable gel solution is 15 mL / h.

[0068] Specifically, the inner and outer double-layer coaxial needle includes an inner needle and an outer needle. The inner needle is inserted into the outer needle, the inner needle is connected to the inner channel, and the outer needle is connected to the outer channel. The outer diameter of the inner needle is 1.28 mm, the inner diameter of the inner needle is 0.84 mm, the outer diameter of the outer needle is 1.81 mm, and the inner diameter of the outer needle is 1.65 mm.

[0069] When injecting with two syringes, the inner and outer needles of the inner and outer double-layer coaxial needles are fixed vertically on the collecting liquid CaCl2 solution, and the front ends of the needle nozzles of the inner and outer needles just touch the liquid surface of the collecting liquid.

[0070] The preliminarily cross-linked core-shell tube was placed under a blue light illumination environment, the core layer photocurable gel solution was cross-linked, and the shell layer photocurable gel solution was further cross-linked to obtain a hydrogel core-shell tube. Specifically, the wavelength of the light was 405nm and the intensity of the light was 1.2μW / cm 2 The structure of the hydrogel tubes was observed under an optical microscope and their dimensions were measured using ImageJ.

[0071] like Figures 3-4 As shown, the preparation method of the hydrogel core-shell tube provided by the embodiment of the present invention can prepare a hydrogel tube with obvious core-shell stratification, wherein: Figure 3 A magnified microscopic image of the hydrogel core-shell tube is shown; scale bar: 500 μm; Figure 4 A magnified microscopic image of a cross section of a hydrogel core-shell tube is shown; scale bar: 500 μm. The total diameter of the hydrogel core-shell tube is 1682 ± 59 μm, while the core diameter is 1042 ± 37 μm.

[0072] Example 2

[0073] By adjusting the flow rates of the core and shell layers, hydrogel core-shell tubes with varying diameters can be prepared. The injection rate for the core layer photocurable gel solution was 10-20 mL / h, while the injection rate for the shell layer photocurable gel solution was 10-40 mL / h. Only one flow rate was varied at a time, while the other parameters remained constant. The hydrogel tube structure was observed under an optical microscope, and its dimensions were measured using ImageJ.

[0074] Figures 5-7The total diameter, core diameter and shell thickness of the hydrogel core-shell tubes are shown as a function of flow rate. Figures 5-7 As shown in the figure, when the core layer flow velocity remains constant, as the shell layer flow velocity increases, the total diameter and shell thickness of the hydrogel core-shell tube gradually increase, while the core layer diameter does not show an obvious change pattern.

[0075] Figures 8-10 A microscopic diagram showing the hydrogel core-shell tubes prepared with different core and shell flow rates. Figures 8-10 Scale bar in the figure: 500 μm.

[0076] Example 3

[0077] Mouse fibroblast L929 cells were collected and the L929 cell membrane was made red and green fluorescent with DiI and DiO fluorescent dyes, respectively. L929-DiI and L929-DiO were mixed evenly with the nuclear layer photocurable gel solution and the shell layer photocurable gel solution, respectively, to prepare a cell concentration of 2.5×10 5 / mL cell suspension. Heterogeneous cell-loaded hydrogel core-shell tubes were prepared according to the method and steps in Example 1. After collection, the cells were placed in a 6-well plate, and cell culture medium was added. The cells were cultured in a 37°C, 5% CO2 incubator. After 5 days, the cell growth and morphology were observed under a fluorescence microscope.

[0078] Figure 11 Fluorescence microscopy images of hydrogel core-shell tubes encapsulating cells are shown; Figure 11 The scale bar in the figure is 500 μm. Figure 12 shows a cross-sectional fluorescence microscopy image of a hydrogel core-shell tube encapsulating cells; Figure 12 Scale bar in the figure: 500 μm.

[0079] like Figures 11-12 As shown, L929-DiI and L929-DiO are wrapped in the core layer and shell layer of the hydrogel tube in different regions. The core layer wraps L929 stained with red fluorescence, and the shell layer wraps L929 stained with green fluorescence.

[0080] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. All equivalent structural transformations made using the contents of the present invention's description and drawings, or direct / indirect applications in other related technical fields, within the scope of the present invention are included in the patent protection scope of the present invention.

Claims

1. A method for preparing a hydrogel core-shell tube, characterized in that: Including steps: Injecting a core layer photocurable gel solution into the inner channel of the inner and outer double-layer coaxial needle, and injecting a shell layer photocurable gel solution into the outer channel of the inner and outer double-layer coaxial needle, wherein the inner channel is sheathed within the outer channel; The core layer photocurable gel solution and the shell layer photocurable gel solution are simultaneously extruded into a collecting solution, wherein the injection rate of the core layer photocurable gel solution is 10-20 mL / h, and the injection rate of the shell layer photocurable gel solution is 10-40 mL / h; the collecting solution is a CaCl2 solution; the shell layer photocurable gel solution is cross-linked to obtain a preliminarily cross-linked core-shell tube; placing the preliminarily cross-linked core-shell tube under light irradiation, cross-linking the core layer photocurable gel solution, and further cross-linking the shell layer photocurable gel solution, thereby obtaining the hydrogel core-shell tube; The core layer photocurable gel solution is a GelMA solution, and the preparation steps of the GelMA solution include: adding GelMA and a photoinitiator to a 100mM CaCl2 solution, heating the solution to 35-39°C, and stirring to dissolve the solution to obtain the GelMA solution; The shell layer photocurable gel solution is a GelMA / alginate solution. The preparation steps of the GelMA / alginate solution include: adding sodium alginate to a NaCl solution to swell, thereby obtaining a swollen sodium alginate solution; adding GelMA and a photoinitiator to the swollen sodium alginate solution, heating the solution to 35-39° C., and stirring to dissolve the solution, thereby obtaining the GelMA / alginate solution.

2. The method for preparing the hydrogel core-shell tube according to claim 1, wherein The inner and outer double-layer coaxial needle includes an inner needle and an outer needle. The inner needle is inserted into the outer needle. The inner diameter of the inner needle forms the inner channel. The inner diameter of the outer needle and the outer diameter of the inner needle form the outer channel.

3. The method for preparing the hydrogel core-shell tube according to claim 2, wherein: The outer diameter of the inner needle is 1.28 mm, the inner diameter of the inner needle is 0.84 mm, the outer diameter of the outer needle is 1.81 mm, and the inner diameter of the outer needle is 1.65 mm.

4. The method for preparing the hydrogel core-shell tube according to claim 1, wherein In the step of placing the preliminarily cross-linked core-shell tubes under a light irradiation environment, the wavelength of the light is 400-410 nm, and the intensity of the light is 1.0-1.5 μW / cm 2 .

5. Use of the method for preparing the hydrogel core-shell tube according to any one of claims 1 to 4 in cell culture, characterized in that: The application is to prepare a nuclear layer photocurable gel solution, add target cells A and mix evenly, prepare a shell layer photocurable gel solution, add cells B and mix evenly, prepare a hydrogel shell-core tube, and realize the partitioning of cells A and cells B.

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