Pancreatic cell culture hydrogel microfiber with core-shell structure and preparation method thereof
The preparation of hydrogel microfibers of nucleocapsular structures through microfluidic control technology has solved the problem of insufficient bionic pancreatic structure simulation in the prior art, and achieved low-cost and efficient pancreatic tissue simulation and cell interaction, which is suitable for tissue engineering and diabetes research.
Patent Information
- Application Number
- CN202510437415.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-08-22
AI Technical Summary
Bionic pancreas constructed in the prior art usually focus only on single cell-like functions or simple mixed plate cultures, failing to simulate the bionic structure.
Microfluidic control technology was used to prepare pancreatic cell culture hydrogel microfibers with core-shell structures. The laminar flow state was formed by adjusting the flow rate of internal and external phase solutions, and secondary cross-linking was performed using ion cross-linking and light irradiation to prepare hydrogel microfibers carrying pancreatic islet β cells and pancreatic stellate cells.
It has achieved better simulation of pancreatic tissue, provided a three-dimensional bionic microenvironment, promoted cell-to-cell interactions, was cheap and easy to operate, and was suitable for tissue engineering and exploration of diabetes pathogenesis.
Smart Images
Figure CN120519984A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedical engineering, and in particular relates to pancreatic cell culture hydrogel microfibers with a core-shell structure and a preparation method thereof. Background Art
[0002] With the improvement of quality of life and changes in lifestyle habits, the incidence of pancreatic diseases has continued to rise, causing a serious social burden. To better understand pancreatic function during disease progression, constructing a biomimetic pancreas is crucial in tissue engineering, but also extremely challenging.
[0003] However, in the prior art, the constructed bionic pancreas usually only focuses on the function of a single type of cells, or simply mixes two types of cells and cultures them on a plate, failing to achieve the simulation of bionic structures. To this end, the present invention proposes pancreatic cell culture hydrogel microfibers with a core-shell structure and a preparation method. Summary of the Invention
[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide pancreatic cell culture hydrogel microfibers with a core-shell structure and a preparation method, which solves the problems in the prior art.
[0005] The purpose of the present invention can be achieved through the following technical solutions:
[0006] A method for preparing pancreatic cell culture hydrogel microfibers with a core-shell structure comprises the following steps:
[0007] The photoinitiator is dissolved in the inner phase cross-linked water-soluble polymer solution, and after filtering and sterilization, pancreatic beta cells are added to obtain an inner phase hydrogel cell prepolymer solution, which is then passed into the inner phase tube of the microfluidic device;
[0008] The outer phase cross-linked water-soluble polymer solution is filtered and sterilized, and then pancreatic stellate cells are added to obtain an outer phase hydrogel cell prepolymer solution, which is then passed into the outer phase tube of the microfluidic device;
[0009] By adjusting the flow rates of the internal and external phase solutions, the liquids form a mutually immiscible laminar flow state, and the external phase solution is brought into contact with the ion-crosslinked aqueous phase solution for ion crosslinking; then secondary crosslinking is performed by blue light irradiation to obtain the product.
[0010] Furthermore, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.
[0011] Furthermore, the mass fraction of the photoinitiator is 0.25%.
[0012] Furthermore, the internal phase cross-linked water-soluble polymer solution includes: deionized water and methacryloylated hyaluronic acid, and the mass fraction of the methacryloylated hyaluronic acid is 2%.
[0013] Furthermore, the external phase cross-linked water-soluble polymer solution includes: deionized water and sodium alginate, and the mass fraction of the sodium alginate is 1.5%.
[0014] Furthermore, the ion-crosslinked aqueous solution is a calcium chloride solution, and the mass concentration of the calcium chloride solution is 2-5%.
[0015] Furthermore, the ratio of the number of the pancreatic islet β cells to the number of the pancreatic stellate cells is 1:1.
[0016] Furthermore, the inner diameter of the inner phase tube is 200-300 μm; the inner diameter of the outer phase tube is 600-750 μm; and the diameter of the hydrogel microfiber is 250-350 μm.
[0017] The pancreatic cell culture hydrogel microfibers with a core-shell structure are prepared using the above-mentioned preparation method.
[0018] Application of the pancreatic cell culture hydrogel microfibers with core-shell structure in simulating pancreatic tissue.
[0019] Beneficial effects of the present invention:
[0020] 1. The present invention prepares hydrogel microfibers based on microfluidic technology, which is low-cost and easy to operate. The inner and outer diameters of the microfibers can be adjusted by adjusting the microfluidic parameters, and stable mass production can be achieved.
[0021] 2. The present invention prepares hydrogel microfibers with a core-shell double-layer structure containing pancreatic β cells and pancreatic stellate cells based on microfluidic technology, which is conducive to better in vitro simulation of pancreatic tissue and exploration of cell-cell interactions.
[0022] 3. The core-shell structured hydrogel microfibers prepared by the present invention have good biocompatibility. Their structure provides a three-dimensional biomimetic microenvironment for the growth of pancreatic β cells, which is conducive to the formation of pseudo-islet cell spheres, simulating the natural islet structure and improving the function of islet cells. At the same time, the outer pancreatic stellate cells are added to simulate the distribution of two cells in the natural pancreas, which is expected to be used as an ideal cell interaction model in the fields of tissue engineering and exploration of the pathogenesis of diabetes. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.
[0024] Figure 1 Schematic diagram of the preparation of the core-shell structured pancreatic cell three-dimensional culture hydrogel microfibers of the present invention;
[0025] Figure 2 This is a physical picture of the hydrogel microfiber of the present invention;
[0026] Figure 3 is a graph showing the relationship between the diameter parameter of the hydrogel microfiber of the present invention and the flow rate of the solution;
[0027] Figure 4 The pancreatic β cells in the present invention form a pseudo-islet image;
[0028] Figure 5 This is a diagram of the interaction between pancreatic β cells and pancreatic stellate cells of the present invention;
[0029] Figure 6 This is a schematic diagram of the evaluation of the pancreatic β-cell and pancreatic stellate cell drug of the present invention;
[0030] In the figure: 1-internal phase liquid inlet device; 2-external phase liquid inlet device; 3-internal phase capillary; 4-observation tube; 5-external phase capillary; 6-405nm blue light source; 7-collecting device. DETAILED DESCRIPTION
[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.
[0032] A method for preparing pancreatic cell culture hydrogel microfibers with a core-shell structure comprises the following steps:
[0033] S1, dissolving a photoinitiator with a mass fraction of 0.25% in an inner phase cross-linked water-soluble polymer solution, filtering and sterilizing, adding pancreatic β cells to obtain an inner phase hydrogel cell prepolymer solution, and passing it into the inner phase tube of the microfluidic device;
[0034] S2, filtering and sterilizing the external phase cross-linked water-soluble polymer solution, then adding pancreatic stellate cells to obtain an external phase hydrogel cell prepolymer solution, and passing it into the external phase tube of the microfluidic device;
[0035] S3, by adjusting the flow rate of the internal and external phase solutions, the liquids form a mutually immiscible laminar state, and the collection port of the microfluidic device is placed in the ionically cross-linked aqueous phase solution. After the external phase solution contacts the ionically cross-linked aqueous phase solution at the collection point, the ionically cross-linked components in the external phase solution are ionically cross-linked with the collection solution to form preliminary hydrogel microfibers; then, 405nm blue light is irradiated to make the photoinitiator produce free radicals, which initiates the cross-linking and polymerization of the internal phase hydrogel cell pre-polymer solution, and finally obtains pancreatic cell culture hydrogel microfibers with a core-shell structure.
[0036] In S1, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt.
[0037] In S1, the internal phase cross-linked water-soluble polymer solution includes: deionized water and methacryloylated hyaluronic acid, and the mass fraction of the methacryloylated hyaluronic acid is 2%.
[0038] In S2, the external phase cross-linked water-soluble polymer solution includes: deionized water and sodium alginate, and the mass fraction of the sodium alginate is 1.5%.
[0039] In S3, the ion-crosslinked aqueous solution is a calcium chloride solution, and the mass concentration of the calcium chloride solution is 2-5%.
[0040] The ratio of the number of the pancreatic islet β cells to the number of the pancreatic stellate cells is 1:1.
[0041] In S1, the inner diameter of the inner phase tube is 200-300 μm; in S2, the inner diameter of the outer phase tube is 600-750 μm; in S3, the diameter of the finally prepared hydrogel microfibers is 250-350 μm.
[0042] The specific solution is introduced below through the following embodiments;
[0043] Example 1
[0044] like Figure 1 As shown, this embodiment introduces the construction of a microfluidic two-phase core-shell hydrogel microfiber generation device, including the following contents:
[0045] 1) Fabrication of the inner capillary tube 3, the outer capillary tube 5, and the observation tube 4;
[0046] A glass capillary with an outer diameter of 1000 μm and an inner diameter of 580 μm was stretched by a tube puller until the tip was needle-shaped. The outlet was then polished with 1500-grit sandpaper to make it smooth. The inner diameter of the tip was 200-300 μm. This capillary served as the inner phase tube of the microfluidic two-phase core-shell hydrogel microfiber generation device, i.e., inner phase capillary 3.
[0047] Select another glass capillary with an outer diameter of 1000 μm and an inner diameter of 750 μm, cut it to a suitable length, and polish both ends to serve as the external phase capillary 5;
[0048] A glass capillary with an outer diameter of 1500 μm and an inner diameter of 1100 μm is cut and polished at both ends to serve as the observation tube 4 in the generating device.
[0049] Finally, all the treated capillaries were immersed in ethanol solution and ultrasonically cleaned for 5-10 minutes. After cleaning, they were blown dry with nitrogen or allowed to dry naturally for later use.
[0050] 2) assembly of the generating device;
[0051] A glass slide is selected as the carrier of the microfluidic core-shell hydrogel microfiber generation device. First, use quick-drying glue to fix the observation tube 4 in the center of the glass slide. Then, the inner phase capillary 3 is nested in the outer phase capillary 5, ensuring that the tip of the inner phase capillary 3 is inserted into the outer phase capillary 5 and aligned with the central axis of the two. The inner and outer capillaries are fixed to the glass slide with quick-drying glue, and the opening at the junction of the observation tube 4 and the outer phase capillary 5 is closed. After that, take a flat-head needle, carve a groove at the bottom that fits the outer diameter of the glass capillary, and fix it vertically on the glass slide, located at the junction of the opening of the inner phase capillary 3 and the observation tube 4. Finally, use quick-drying glue to seal around the base of the needle to ensure that it is stable for subsequent use.
[0052] Example 2
[0053] like Figure 1 As shown, this embodiment introduces a process for preparing pancreatic cell culture hydrogel microfibers with a core-shell structure, which includes the following steps:
[0054] Step 1: Add 0.15g of sodium alginate to 10ml of deionized water and stir to fully dissolve it; dissolve 0.2g of methacryloylated hyaluronic acid (HAMA) and 0.025g of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) in 10ml of deionized water and stir to dissolve in the dark. Filter the two solutions using a 0.22μm filter membrane for sterilization. Then, mix the HAMA solution with the cultured pancreatic β cells (10 7 The sodium alginate solution was mixed with pancreatic stellate cells (10 7 / ml) to prepare the external phase hydrogel cell prepolymerization solution.
[0055] Step 2: The inner phase hydrogel cell prepolymer solution is passed into the inlet of the inner phase capillary 3 of the microfluidic device through the inner phase liquid inlet device 1, and the outer phase hydrogel cell prepolymer solution is passed into the opening of the outer phase capillary 5 through the outer phase liquid inlet device 2. By adjusting the flow rate of the inner and outer phase solutions, the liquids are formed into a mutually immiscible laminar state. At the outlet of the microfluidic device, after the outer phase solution contacts the collection liquid (calcium chloride solution) in the collection device 7, the sodium alginate in the solution is cross-linked by the calcium ions in the collection liquid to initially form hydrogel microfibers, which are then irradiated by a 405nm blue light source 6 for secondary cross-linking, so that the inner phase HAMA is solidified, and finally a core-shell hydrogel microfiber containing pancreatic β cells and pancreatic stellate cells is formed.
[0056] The collected microfibers were washed with sterile water to remove unreacted components, then transferred to a culture dish and incubated in a 37°C incubator containing 5% carbon dioxide. The medium was changed every other day to maintain cell viability.
[0057] Example 3
[0058] In this example, relevant experiments were conducted to verify the performance of the hydrogel microfibers prepared in Example 2.
[0059] 1) In order to observe the continuity and microstructure of hydrogel microfibers, e.g. Figure 2 As shown, they were characterized using an optical microscope to clearly observe that the collected microfibers maintained the core-shell structure and uniformity of diameter; Figure 2 (a) is the process of hydrogel microfiber formation. Figure 2 (b) is a microfiber light microscope image of the core-shell structure without cells. Figure 2 (c) is a microfiber optical microscope image of the core-shell structure of the cell. Figure 2 (d) is a scanning electron microscope image of core-shell structured microfibers; it can be seen that by freeze-drying the microfibers and then observing them using a scanning electron microscope, the microfibers have a dense shell structure and a loose core structure.
[0060] 2) By adjusting the parameters of the microfluidic system, the inner diameter, outer diameter and shell thickness of the hydrogel microfiber can be precisely controlled, such as Figure 3 As shown; Figure 3 (a) is the relationship between the flow rate of the microfluidic internal phase and the inner diameter of the microfiber. Figure 3 (b) is the relationship between the microfluidic external phase flow rate and the microfiber inner diameter. Figure 3 (c) is the relationship between the microfluidic internal phase flow rate and the microfiber outer diameter. Figure 3 (d) is the relationship between the microfluidic external phase flow rate and the microfiber outer diameter. Figure 3 (e) is the relationship between the microfluidic internal phase flow rate and the microfiber shell thickness. Figure 3 (f) in the figure is the relationship between the microfluidic external phase flow rate and the microfiber shell thickness; it can be seen that: the inner diameter of the microfiber is proportional to the flow rate of the internal phase solution and inversely proportional to the flow rate of the external phase solution; the outer diameter has no obvious correlation with the internal phase flow rate and is proportional to the external phase flow rate; the shell thickness is inversely proportional to the inward flow velocity and directly proportional to the external phase flow velocity.
[0061] 3) The pancreatic β cell-loaded hydrogel microfibers were cultured to observe the effects of the culture time, such as Figure 4 As shown, Figure 4 (a) is a fluorescence image of pseudo-islet cytoskeleton staining. Figure 4(b) shows the GSIS function results of pseudo-islets compared with 2D culture; it can be seen that pancreatic β cells form pseudo-islets with spherical structures in the core. The glucose-stimulated insulin secretion (GSIS) experiment confirms that these three-dimensionally cultured pseudo-islets have better insulin secretion function than traditional flat plate culture.
[0062] 4) Staining of frozen sections of microfibrils and extraction of RNA from co-cultured pancreatic β cells for real-time quantitative fluorescence PCR detection, e.g. Figure 5 shown; among them, Figure 5 (a) is the TUNEL staining image after co-culture. Figure 5 (b) is the result of Caspase3 mRNA expression. Figure 5 (c) in the figure is the result of Caspase 8 mRNA expression; it can be seen that activated pancreatic stellate cells promote the apoptosis of pancreatic β cells.
[0063] 5) Treat the co-cultured cells with a glucagon-like peptide 1 (GLP-1) receptor agonist, such as Figure 6 As shown, it can be seen that Exenatide (Exendin-4), a representative drug of GLP-1 receptor agonists, can improve pancreatic β-cell function.
[0064] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
[0065] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.
Claims
1. A method for preparing pancreatic cell culture hydrogel microfibers with a core-shell structure, characterized in that: The following steps are involved: The photoinitiator is dissolved in the inner phase cross-linked water-soluble polymer solution, and after filtering and sterilization, pancreatic beta cells are added to obtain an inner phase hydrogel cell prepolymer solution, which is then passed into the inner phase tube of the microfluidic device; The outer phase cross-linked water-soluble polymer solution is filtered and sterilized, and then pancreatic stellate cells are added to obtain an outer phase hydrogel cell prepolymer solution, which is then passed into the outer phase tube of the microfluidic device; By adjusting the flow rates of the internal and external phase solutions, the liquids form a mutually immiscible laminar flow state, and the external phase solution is brought into contact with the ion-crosslinked aqueous phase solution for ion crosslinking; then secondary crosslinking is performed by blue light irradiation to obtain the product.
2. The method for preparing pancreatic cell culture hydrogel microfibers with a core-shell structure according to claim 1, characterized in that: The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.
3. The method for preparing pancreatic cell culture hydrogel microfibers with a core-shell structure according to claim 1 or 2, characterized in that: The mass fraction of the photoinitiator is 0.25%.
4. The method for preparing pancreatic cell culture hydrogel microfibers with a core-shell structure according to claim 1, characterized in that: The internal phase cross-linked water-soluble polymer solution includes: deionized water and methacryloylated hyaluronic acid, and the mass fraction of the methacryloylated hyaluronic acid is 2%.
5. The method for preparing pancreatic cell culture hydrogel microfibers with a core-shell structure according to claim 1, characterized in that: The external phase cross-linked water-soluble polymer solution includes: deionized water and sodium alginate, and the mass fraction of the sodium alginate is 1.5%.
6. The method for preparing pancreatic cell culture hydrogel microfibers with a core-shell structure according to claim 1, characterized in that: The ion-crosslinked aqueous solution is a calcium chloride solution, and the mass concentration of the calcium chloride solution is 2-5%.
7. The method for preparing pancreatic cell culture hydrogel microfibers with a core-shell structure according to claim 1, characterized in that: The ratio of the number of the pancreatic islet β cells to the number of the pancreatic stellate cells is 1:
1.
8. The method for preparing pancreatic cell culture hydrogel microfibers with a core-shell structure according to claim 1, characterized in that: The inner diameter of the inner phase tube is 200-300 μm; the inner diameter of the outer phase tube is 600-750 μm; and the diameter of the hydrogel microfiber is 250-350 μm.
9. A pancreatic cell culture hydrogel microfiber having a core-shell structure, characterized in that: It is prepared using the preparation method according to any one of claims 1 to 8.
10. Use of the pancreatic cell culture hydrogel microfiber with a core-shell structure according to claim 9 in simulating pancreatic tissue.