Hydrogel microfiber loaded with pancreatic alpha and beta cells, preparation method and application thereof

Abstract

The present invention belongs to the field of biomedical engineering technology, and specifically relates to a hydrogel microfiber carrying pancreatic islet α and β cells, a preparation method and an application thereof; the preparation method comprises: dissolving a photoinitiator in a cross-linked water-soluble polymer solution, filtering and sterilizing, adding pancreatic islet α and β cells to obtain a hydrogel cell prepolymer solution, passing the hydrogel cell prepolymer solution into an inner phase tube, passing an ionically cross-linked aqueous phase solution into an outer phase tube, and adjusting the flow rates of the solutions in the inner and outer phase tubes so that the liquids form a mutually immiscible laminar flow state; in a microfluidic device, after the inner phase solution contacts the outer phase solution, the ionically cross-linked components in the inner phase solution undergo ionically cross-linking with the outer phase solution to preliminarily form hydrogel microfibers, which are then secondary cross-linked by 405nm blue light irradiation to ultimately form hydrogel microfibers carrying pancreatic islet α and β cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering, and in particular relates to a hydrogel microfiber carrying pancreatic islet α and β cells, a preparation method and an application thereof. Background Art

[0002] Type 1 diabetes is a serious chronic disease that poses a major public health problem worldwide. Traditional treatment strategies are highly dependent on daily subcutaneous injections of insulin, while the rise of islet transplantation has brought new solutions to the treatment of type 1 diabetes by restoring endogenous insulin secretion. However, direct transplantation of islets is limited by the shortage of donor sources and can also cause immune rejection, resulting in the inability of the graft to survive in the body for a long time. Therefore, researchers have developed biocompatible hydrogels to encapsulate pancreatic beta cells for immune isolation. However, existing hydrogels tend to encapsulate individual beta cells and fail to simulate natural islet components. Based on this, in the present invention, we designed and invented a porous microcarrier carrying three-dimensional pancreatic beta cell aggregates based on microfluidic technology for use in tissue engineering and diabetes treatment. Summary of the Invention

[0003] In response to the shortcomings of the existing technology, the present invention provides a method for preparing porous microcarriers carrying three-dimensional pancreatic beta cell aggregates. Based on microfluidic technology, hydrogel microfibers are formed through secondary cross-linking, which not only simulates natural pancreatic islet components but also ensures material exchange within the hydrogel and the release of pancreatic cell hormones, thereby being used for tissue engineering research and diabetes treatment.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] A method for preparing pancreatic α and β cell hydrogel microfibers, the method specifically comprising: dissolving a photoinitiator with a mass fraction of 0.5% in a cross-linked water-soluble polymer solution, filtering and sterilizing, and adding 4×10 6 pancreatic α cells and 1.4×10 7 The hydrogel cell prepolymer solution is obtained by passing the hydrogel cell prepolymer solution into the inner phase tube, and the ion-crosslinked aqueous phase solution is passed into the outer phase tube. By adjusting the flow rates of the inner and outer phase solutions, the liquids form a mutually immiscible laminar state; in the microfluidic device, after the inner phase solution contacts the outer phase solution, the ion-crosslinking components in the inner phase solution undergo ion-crosslinking with the outer phase solution to initially form hydrogel microfibers, which are then secondary crosslinked by blue light irradiation to finally form hydrogel microfibers carrying pancreatic islet α and β cells.

[0006] Preferably, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt.

[0007] Preferably, the cross-linked water-soluble polymer solution comprises deionized water, sodium alginate and methacrylated hyaluronic acid, wherein the mass fraction of the sodium alginate is 1.5% and the mass fraction of the methacrylated hyaluronic acid is 2%.

[0008] Preferably, the ion-crosslinked aqueous solution is a calcium chloride solution, and the concentration of the calcium chloride solution is 2-5%.

[0009] Preferably, the ratio of pancreatic α cells to pancreatic β cells is 2:7.

[0010] Preferably, the inner phase tube and the outer phase tube are both glass capillaries, the inner diameter of the inner phase tube is 250-300 μm, and the inner diameter of the outer phase tube is 700-750 μm.

[0011] Preferably, a hydrogel microfiber carrying pancreatic α and β cells is prepared by the method described above.

[0012] Preferably, the pancreatic islet α and β cell-carrying hydrogel microfibers have a diameter of 250-300 μm.

[0013] Preferably, the pancreatic islet α and β cell-carrying hydrogel microfibers are used in the preparation of drugs for treating diabetes.

[0014] Beneficial effects of the present invention:

[0015] (1) The present invention prepares hydrogel microfibers based on microfluidic technology, which is low-cost and easy to operate. The size of the microfibers can be adjusted by adjusting the microfluidic parameters, and stable mass production can be achieved.

[0016] (2) The present invention prepares pancreatic islet α and β cell-loaded hydrogel microfibers based on microfluidic technology, and adds pancreatic islet α and β cells according to the ratio of natural pancreatic islet cell components, which is beneficial to cell-cell interaction and better simulates pancreatic islet tissue in vitro.

[0017] (3) The pancreatic α and β cell-loaded hydrogel microfibers prepared by the present invention have good biocompatibility. Their structure provides support for cell growth and also has certain pores to ensure the diffusion efficiency of oxygen and nutrients and the release of hormones secreted by pancreatic cells. They are expected to be used as ideal pancreatic organoids in the fields of tissue engineering and diabetes treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present disclosure 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.

[0019] Figure 1Schematic diagram of the preparation of hydrogel microfibers carrying pancreatic α and β cells according to an embodiment of the present invention; wherein: 1. Inner phase liquid inlet device; 2. Outer phase liquid inlet device; 3. 405nm blue light source; 4. Inner phase capillary; 5. Observation tube; 6. Outer phase capillary;

[0020] Figure 2 Figure 1 shows the preparation of hydrogel microfibers carrying pancreatic α and β cells according to an embodiment of the present invention; Figure a shows the hydrogel microfiber generation process, Figure b shows the relationship between the hydrogel microfiber diameter and the flow rate of the inner phase solution, and Figure c shows the relationship between the hydrogel microfiber diameter and the flow rate of the outer phase solution;

[0021] Figure 3 Figure 1 is a diagram showing the general morphology of hydrogel microfibers according to an embodiment of the present invention; Figure a shows the general morphology of microfibers collected in a dish, and Figure b is a light microscopy image of the hydrogel microfibers;

[0022] Figure 4 : Electron microscope images of hydrogel microfibers according to an embodiment of the present invention; Figure a is an electron microscope image of the hydrogel microfiber as a whole, and Figure b is a partial magnified cross-section of the hydrogel microfiber;

[0023] Figure 5 Schematic diagram of the hydrogel culture and proliferation of pancreatic α and β cells according to the present invention; Figure a is a bright field fluorescence image of hydrogel microfibers and calcein-characterized living cells at 1, 4, and 7 days of culture, and Figure b is a statistical graph of the fluorescence intensity of living cells.

[0024] Figure 6 Schematic diagram of the permeability of the hydrogel carrying pancreatic α and β cells according to the present invention; Figure a is a graph showing the change in fluorescence intensity in the culture medium after FITC-BSA is encapsulated into the microfiber hydrogel, and Figure b is a statistical graph of the fluorescence intensity of FITC-BSA in the hydrogel microfibers.

[0025] Figure 7 Figure 3 is a diagram illustrating the therapeutic effect of pancreatic α and β cell hydrogel transplantation in diabetic mice according to the present invention; Figure a is a blood glucose change curve of the diabetic control group and the pancreatic α and β cell hydrogel group, Figure b is an insulin immunohistochemical staining image of the transplant removed 42 days after the experiment, with the asterisk indicating the insulin-positive area, and Figure c is a glucagon immunohistochemical staining image of the transplant removed 42 days after the experiment, with the asterisk indicating the glucagon-positive area. DETAILED DESCRIPTION

[0026] 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.

[0027] The present invention provides a method for preparing hydrogel microfibers loaded with pancreatic α- and β-cells. The method comprises an internal phase comprising a hydrogel cell prepolymer solution containing a cross-linked water-soluble polymer solution, a photoinitiator, and pancreatic α- and β-cells; and an external phase comprising an ionically cross-linked aqueous solution. The internal phase solution is first ionically cross-linked within a microfluidic chip to form a preliminary microfiber structure, followed by secondary cross-linking via irradiation to ultimately form the hydrogel microfibers.

[0028] (1) Construction of a microfluidic two-phase hydrogel microfiber generation device

[0029] Use a tube puller to pull a glass capillary with an outer diameter of 1000 μm and an inner diameter of 580 μm, and sandpaper to polish it into a tip capillary with an inner diameter of 250-300 μm, which serves as the inner phase tube of the microfluidic two-phase hydrogel microfiber generation device; take a 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 make it smooth as the outer phase tube of the two-phase hydrogel microfiber generation device; take another glass capillary with an outer diameter of 1500 μm and an inner diameter of 1100 μm, cut it to a suitable length, polish both ends to make it smooth, and use it as the observation tube of the hydrogel microfiber generation device; soak each capillary in ethanol solution, ultrasonically clean it for 5-10 minutes, blow dry it with nitrogen or air dry it at room temperature before use;

[0030] A glass slide was used as the carrier for the microfluidic hydrogel microfiber generation device. First, the observation tube was fixed to the center of the glass slide with quick-drying glue. The inner and outer phase tubes were then nested within the observation tube, with the inner tube's tip inserted into the outer tube, aligning their central axes. The inner and outer phase tubes were fixed to the glass slide with quick-drying glue, and the opening at the junction of the observation tube and outer phase tube was sealed with quick-drying glue. A flat-tipped needle was cut into the bottom with a groove appropriate to the outer diameter of the glass capillary. This needle was fixed perpendicular to the glass slide at the opening of the inner phase tube and the junction of the inner phase tube and observation tube. The base of the needle was sealed with quick-drying glue for ready use.

[0031] Example 1

[0032] Dissolve 0.15g of sodium alginate in 10ml of deionized water, heat and stir at 60℃, add 0.2g of methacryloylated hyaluronic acid (HAMA) and 0.025g of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) to the above sodium alginate solution, stir and dissolve in the dark. Sterilize the solution by filtering with a 0.22μm filter membrane and mix with cultured pancreatic α and β cells at a ratio of 2:7 (pancreatic α cells 4×10 6 pancreatic β cells 1.4×10 72g of calcium chloride solid was dissolved in 100ml of pure water and sterilized by filtration using a 0.22μm filter membrane to obtain an external phase ion crosslinked aqueous phase solution.

[0033] The hydrogel cell prepolymer solution is introduced into the inlet of the inner phase tube of the microfluidic device, and the calcium chloride solution is introduced into the opening of the outer phase tube. By adjusting the flow rate of the inner and outer phase solutions, the liquids form a mutually immiscible laminar flow state. In the microfluidic device, after the inner phase solution contacts the outer phase solution, the sodium alginate in the inner phase solution is first cross-linked by the calcium ions in the outer phase solution, initially forming hydrogel microfibers. Then, secondary cross-linking is carried out by blue light irradiation, allowing the HAMA to solidify, and finally forming hydrogel microfibers carrying pancreatic α and β cells. The collected microfibers are washed with sterile pure water to remove unreacted components, then transferred to a culture dish, added with DMEM complete medium, and cultured in an incubator at 37°C containing 5% carbon dioxide. The medium is changed every other day to maintain cell activity.

[0034] By adjusting the parameters of the microfluidic system, the diameter of the hydrogel microfibers can be precisely controlled, such as Figure 2 The microfiber diameter is proportional to the flow rate of the inner phase solution and inversely proportional to the flow rate of the outer phase solution.

[0035] In order to observe the continuity and microstructure of hydrogel microfibers, they were characterized using optical microscopy, e.g. Figure 3 As shown in Figure 2, it is clearly observed that the collected microfibers maintain complete continuity and uniform diameter. The microfibers were freeze-dried and then observed using a scanning electron microscope. Figure 4 The dense structure of the microfibers can be seen, and further magnification reveals uniform pores, which ensure the exchange of oxygen and nutrients and the release of hormones secreted by cells encapsulated in the microfibers. The hydrogel microfibers carrying pancreatic α and β cells were cultured to observe the changes in fluorescence intensity over time through calcein staining, showing that the cells within the hydrogel microfibers maintained a state of continuous growth, indicating that the hydrogel microfibers have good biocompatibility and the ability to maintain cell survival and proliferation. After the hydrogel microfibers carrying pancreatic α and β cells were transplanted into diabetic mice, they had the effect of lowering the blood sugar of the mice.

[0036] 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.

[0037] 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 hydrogel microfibers carrying pancreatic α and β cells, characterized in that: The method is specifically as follows: dissolving a photoinitiator with a mass fraction of 0.25% in a cross-linked water-soluble polymer solution, filtering and sterilizing, and adding 4×10 6 pancreatic α cells and 1.4×10 7 The hydrogel cell prepolymer solution is obtained by adding β cells, the hydrogel cell prepolymer solution is passed into the inner phase tube, and the ion cross-linked aqueous phase solution is passed into the outer phase tube, and the flow rates of the inner and outer phase solutions are adjusted to make the liquids form a mutually immiscible laminar flow state; In the microfluidic device, after the inner phase solution contacts the outer phase solution, the ionic crosslinking components in the inner phase solution undergo ionic crosslinking with the outer phase solution to initially form hydrogel microfibers, which are then secondary crosslinked by 405nm blue light irradiation to ultimately form hydrogel microfibers carrying pancreatic α and β cells; the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate; the crosslinked water-soluble polymer solution components include deionized water, sodium alginate and methacryloylated hyaluronic acid, the mass fraction of the sodium alginate is 1.5%, and the mass fraction of the methacryloylated hyaluronic acid is 2%; the ion-crosslinked aqueous phase solution is a calcium chloride solution, and the concentration of the calcium chloride solution is 2-5%.

2. The method for preparing pancreatic α and β cell-carrying hydrogel microfibers according to claim 1, characterized in that: The inner phase tube and the outer phase tube are both glass capillaries. The inner diameter of the inner phase tube is 250-300 μm, and the inner diameter of the outer phase tube is 700-750 μm.

3. A pancreatic islet α and β cell-carrying hydrogel microfiber prepared by the preparation method of pancreatic islet α and β cell-carrying hydrogel microfiber according to any one of claims 1-2.

4. The hydrogel microfiber carrying pancreatic α and β cells according to claim 3, characterized in that: The diameter of the hydrogel microfiber carrying pancreatic islet α and β cells is 250-300 μm.

5. Use of the pancreatic islet α and β cell-carrying hydrogel microfiber according to any one of claims 3 to 4 in the preparation of a drug for treating diabetes.

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