Millimeter-sized calcium alginate gel spheres having an open macroporous structure and a method for preparing the same

Abstract

The present application provides a kind of millimeter calcium alginate gel ball with open macroporous structure and its preparation method.The base material of the gel ball is calcium alginate, the gel ball has micron-sized pore structure that is interconnected inside, and the micron-sized pore structure that is interconnected inside the gel ball is open on the surface of the gel ball, the average pore size of the micron-sized pore structure inside the gel ball is 120-160 μm, and the particle size of the gel ball is 2-5 mm when it reaches swelling equilibrium in water.The present application can solve the problem that it is difficult to obtain open macroporous structure when preparing calcium alginate gel ball in the prior art, and realize the preparation of calcium alginate gel ball with open and interconnected macroporous structure and millimeter small size at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to millimeter-scale calcium alginate gel spheres with an open macroporous structure and a preparation method thereof. Background Art

[0002] The growth of tissues and cells in the body is based on the in vivo environment. Traditional two-dimensional cell culture can only provide each cell with a balanced supply of nutrients. Cell morphology differs significantly from that in vivo and cannot simulate in vivo differentiation and gene expression. As an emerging in vitro cell culture technology, three-dimensional cell culture provides cells with a three-dimensional growth space, promoting their growth, proliferation, and migration. The cell morphology is also closer to that of natural growth. Three-dimensional cell culture technology combines the advantages of in vitro two-dimensional cell culture and in vivo models to form a convenient and efficient research system. It can also provide an important platform for drug screening, disease treatment, and tissue engineering.

[0003] With the continuous development of biomaterials, an increasing number of scaffold materials are being used for in vitro three-dimensional cell culture. Scaffold materials used for 3D cell culture include hydrogels, fibers, porous structures, and 3D-printed scaffolds. Traditional macroporous scaffolds are generally bulky. While large pores can enhance nutrient delivery, their bulky structure results in excessively long delivery distances. Therefore, some researchers have combined macroporous structures with microspheres to develop macroporous gel spheres. Macroporous gel spheres have a high specific surface area, which facilitates cell growth, proliferation, differentiation, and expression. They enhance nutrient and oxygen delivery to cells and facilitate the timely removal of metabolic waste. Their open and interconnected pores also promote cell-cell and cell-extracellular matrix (ECM) interactions. Consequently, macroporous gel spheres have garnered widespread attention. In 3D cell culture, the pore size, porosity, and pore connectivity of macroporous gel spheres significantly influence cell adhesion, growth, migration, proliferation, differentiation, and protein expression.

[0004] Sodium alginate (NaAlg) is an anionic polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G), which can form alginate hydrogels with multivalent cations. Alginate hydrogels are widely used as cell microcarrier materials due to their good biocompatibility, biodegradability and bioadhesion. The preparation of cell-loaded alginate gel balls can be divided into pre-loading cell method and post-loading cell method. The pre-loading cell method requires stirring the cell suspension to mix it with the target solution. The cells will be subjected to fluid shear force. During the preparation process, the cells may also come into contact with slightly toxic reagents, surfactants, etc., and sometimes ultraviolet curing is required. These factors will cause certain damage to the cells. The post-loading cell method can avoid these adverse effects and load the cells into the interior of the gel ball without loss. The post-loading cell method requires that the outside of the alginate gel ball have large pores to facilitate the entry of cells into the interior of the gel ball. However, in the process of preparing calcium alginate gel balls, due to the interaction between alginate and Ca 2+ The cross-linking speed is too fast, and it is difficult to obtain open pores. Currently, there are still great challenges in preparing calcium alginate gel spheres with open interconnected macroporous structures.

[0005] For example, Quraishi et al. prepared a calcium alginate scaffold by exposing an aqueous solution of alginate containing lignin and calcium carbonate to carbon dioxide bubbles under low pressure. After the bubbles stopped forming, the solution was transferred to an ethanol solution for supercritical drying, resulting in a calcium alginate scaffold (see Quraishi S., Martins M., Barros AA, et al. L. Novel non-cytotoxic alginate–lignin hybrid aerogels as scaffolds for tissue engineering [J]. The Journal of Supercritical Fluids, 2015, 105: 1-8). Although this scaffold has structural and morphological properties suitable for tissue engineering applications, its overall structure is relatively large, reaching a size of 10 mm, and its pore structure is mostly composed of pores around 1 μm. The pore size is too small, making it difficult for cells to adhere to the scaffold. Summary of the Invention

[0006] In view of the problem that it is difficult to obtain an open macroporous structure when preparing calcium alginate gel balls in the prior art, the present invention provides millimeter-sized calcium alginate gel balls with an open macroporous structure and a preparation method thereof, so as to achieve the preparation of alginate gel balls with both an open interconnected macroporous structure and a millimeter-sized small size.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0008] The invention relates to a millimeter-scale calcium alginate gel ball with an open macroporous structure. The matrix material of the gel ball is calcium alginate. The interior of the gel ball has a micron-scale pore structure that is interconnected, and the micron-scale pore structure that is interconnected inside the gel ball is opened on the surface of the gel ball. The average pore size of the micron-scale pore structure inside the gel ball is 120 to 160 μm. The particle size of the gel ball when it reaches swelling equilibrium in water is 2 to 5 mm.

[0009] In the technical solution of the above-mentioned millimeter-scale calcium alginate gel sphere with an open macroporous structure, the average pore diameter of the pore structure on the surface of the gel sphere is 150-200 μm, and further, the porosity of the pore structure on the surface of the gel sphere is 20%-40%.

[0010] Furthermore, in the technical solution of the above-mentioned millimeter-sized calcium alginate gel spheres with an open macroporous structure, the porosity of the pore structure inside the gel spheres is 50% to 70%.

[0011] The present invention also provides a method for preparing the above-mentioned millimeter-sized calcium alginate gel spheres with an open macroporous structure, comprising the following steps:

[0012] (1) dissolving sodium alginate, a porogen, and a water-soluble anionic surfactant in water, and homogenizing and emulsifying the resulting mixture to incorporate bubbles into the mixture to obtain a gel precursor solution; in the gel precursor solution, the concentration of sodium alginate is 1.5 wt% to 2 wt%, the concentration of the porogen is 1 wt% to 2 wt%, and the concentration of the water-soluble anionic surfactant is 0.05 wt% to 0.15 wt%; the porogen is sodium bicarbonate or potassium bicarbonate;

[0013] Dissolving a water-soluble calcium salt and glacial acetic acid in water to obtain a coagulation bath solution, wherein the concentration of the water-soluble calcium salt in the coagulation bath solution is 5 wt% to 15 wt%, and the concentration of the glacial acetic acid is 5 wt% to 15 wt%;

[0014] (2) dripping a gel precursor into a coagulation bath solution through an injection needle, wherein the sodium alginate in the gel precursor droplets dripping into the coagulation bath solution undergoes a cross-linking reaction with calcium ions, and simultaneously, the porogen in the gel precursor droplets dripping into the coagulation bath solution reacts with glacial acetic acid to generate CO2 gas in the gel precursor droplets, thereby causing the bubbles in the gel precursor droplets to be connected. After the gel precursor droplets are cross-linked and solidified, solid-liquid separation is performed, unreacted water-soluble calcium salts and glacial acetic acid in the obtained solid phase are removed by washing, and freeze-drying is performed to obtain millimeter-sized calcium alginate gel spheres having interconnected macropores inside.

[0015] (3) Immersing the gel balls obtained in step (2) in an etchant aqueous solution, etching under stirring, so that the etchant is complexed with the calcium ions in the calcium alginate on the surface of the gel balls to form an open-pore structure on the surface of the gel balls, immediately replacing the etchant aqueous solution with water to terminate the reaction between the etchant and the calcium ions, separating the solid and the liquid, washing and drying the obtained solid phase, and obtaining the product; the etchant is potassium citrate or sodium citrate, and the concentration of the etchant aqueous solution is 10 to 15 g / L.

[0016] In step (3) of the technical solution of the above preparation method, the etching time is preferably controlled to be 3 to 10 minutes.

[0017] In step (2) of the technical solution of the above preparation method, the gel precursor solution is preferably dripped into the coagulation bath solution through an injection needle with an inner diameter of 0.5 to 1 mm.

[0018] In step (2) of the technical solution of the above preparation method, the gel precursor solution is dripped into the coagulation bath solution through the injection needle at a flow rate of 15 to 25 mL / h by the injection pump.

[0019] In step (2) of the technical solution of the above preparation method, the cross-linking time should ensure that the gel precursor droplets are completely cross-linked and solidified, and the cross-linking time can usually be controlled to be 10 to 15 hours.

[0020] In step (1) of the technical solution of the above preparation method, the feasible water-soluble anionic surfactant can be sodium lauryl sulfate or other water-soluble anionic surfactants.

[0021] The design concept of the present invention is mainly as follows:

[0022] The present invention provides a new strategy for preparing millimeter-sized calcium alginate (CaAlg) gel spheres with an open macroporous structure. First, sodium alginate (NaAlg), a porogen, and a water-soluble anionic surfactant are dissolved in water. Then, gas is added to the resulting mixture using a homogeneous emulsification method to form a mixed solution containing bubbles, namely, a gel precursor. Due to the presence of the water-soluble anionic surfactant and the relatively high viscosity of NaAlg, the bubbles in the gel precursor can exist stably. After the gel precursor is dropped into a coagulation bath solution containing a water-soluble calcium salt and glacial acetic acid, the NaAlg in the gel precursor droplets reacts with the CaAlg to form a mixed solution containing bubbles. 2+A cross-linking reaction occurs, causing the gel precursor droplets to gradually solidify. At the same time, the porogen reacts with glacial acetic acid to produce a large amount of CO2 gas. The generation of CO2 gas can promote the rapid nucleation and growth of bubbles inside the gel precursor droplets, and cause the bubbles in the gel precursor droplets to become connected. After the cross-linking is complete, millimeter-sized CaAlg gel balls with interconnected macropores inside (macroporous CaAlg gel balls) are obtained. The macroporous CaAlg gel balls are immersed in an etchant aqueous solution. During the immersion process, the anions of the etchant will compete with the alginate for Ca 2+ The CaAlg gel is transformed into a sol by complexing the etchant, the concentration of the etchant solution and the etching time. 2+ The complexation reaction mainly occurs on the surface of the macroporous CaAlg gel spheres, and then an open pore structure is formed on the surface of the macroporous CaAlg gel spheres. That is, the pore structure inside the macroporous CaAlg gel spheres that are interconnected are opened on the surface of the gel spheres, thereby obtaining millimeter-scale CaAlg gel spheres with an open macroporous structure.

[0023] Compared with the prior art, the technical solution of the present invention produces the following beneficial technical effects:

[0024] 1. The present invention provides a millimeter-scale calcium alginate gel ball with an open macroporous structure, wherein the interior of the gel ball has a micron-scale pore structure that is interconnected, and the micron-scale pore structure that is interconnected inside the gel ball is opened on the surface of the gel ball. The particle size of the gel ball when it reaches swelling equilibrium in water is 2 to 5 mm. When the gel ball of the present invention is used for in vitro 3D cell culture, this interconnected open pore structure and relatively small gel ball size are conducive to cell adsorption and uniform dispersion. When the cell suspension is added dropwise to the gel ball, the cells can easily diffuse into the interior of the gel ball and disperse evenly through the open pores that are interconnected, which is conducive to the material exchange between the cells and the external environment. It is a macroporous gel ball scaffold with good biocompatibility and convenient for cell transfer and culture. Compared with the existing calcium alginate porous scaffold with large size and small pore structure, the reduction in the size of the gel ball of the present invention is conducive to shortening the transmission distance of the material. The increase in the pores inside and on the surface of the gel ball of the present invention (the average pore size is above 100 μm) is more conducive to cell adsorption into the interior of the gel ball.

[0025] 2. The present invention has confirmed through cell adsorption experiments that, compared with gel balls with interconnected macroporous structures inside but no open pore structure on the surface, the millimeter-sized calcium alginate gel balls with open macroporous structures described in the present invention can more evenly adsorb HepG2 cells.

[0026] 3. The present invention demonstrated through HepG2 cell activity and proliferation experiments that the millimeter-sized calcium alginate gel spheres with open macroporous structures provide a favorable environment for cell growth and proliferation, with cells growing in clusters within the spheres. After 24, 48, and 72 hours of culture, the activity of HepG2 cells was above 95%. The cell count after 48 and 72 hours of culture was 1.4 and 2.3 times that of the 24-hour culture, respectively, ensuring normal cell growth and proliferation.

[0027] 4. The present invention confirms through in vitro secretion experiments of HepG2 cells that the millimeter-sized calcium alginate gel spheres with an open macroporous structure of the present invention can maintain the functional expression of cells.

[0028] 5. The present invention also provides a method for preparing the above-mentioned millimeter-sized calcium alginate gel balls with open macroporous structure. The method comprises the following steps: adding air bubbles into the gel precursor solution, and forming an interconnected macroporous structure in the cross-linked and solidified gel balls by using an etchant and glacial acetic acid during the cross-linking process of sodium alginate and calcium ions, thereby obtaining macroporous calcium alginate gel balls. On this basis, the etchant is combined with the calcium ions in the surface layer of the macroporous calcium alginate gel balls to form an open pore structure on the surface of the macroporous calcium alginate gel balls. This solves the problem in the prior art caused by the cross-linking of sodium alginate and calcium ions. 2+ The cross-linking speed is too fast, making it difficult to obtain open pores. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a scanning electron microscope image of the macroporous CaAlg gel spheres prepared in step (2) of Example 1.

[0030] Figure 2 These are optical photographs of macroporous CaAlg gel spheres prepared in four groups of experiments in Example 2.

[0031] Figure 3 These are optical photographs of the four gel precursor solutions in Example 3 after being placed for different periods of time.

[0032] Figure 4 These are optical photographs of macroporous CaAlg gel spheres prepared in four groups of experiments in Example 3.

[0033] Figure 5 3 is a scanning electron microscope image of the cross section of the macroporous CaAlg gel spheres prepared in the four groups of experiments in Example 3.

[0034] Figure 6 This is an optical photograph of macroporous CaAlg gel spheres prepared by four groups of experiments in Example 4.

[0035] Figure 7 3 is a scanning electron microscope image of the macroporous CaAlg gel spheres prepared in the four groups of experiments in Example 4.

[0036] Figure 8 This is an optical photograph of the macroporous CaAlg gel sphere during the etching process in Example 5.

[0037] Figure 9 3 and 4 are scanning electron micrographs of the open-pore CaAlg gel spheres obtained by etching for different times in Example 5.

[0038] Figure 10 3 is a scanning electron microscope image of the open-pore CaAlg gel spheres prepared in each group of experiments when potassium citrate was used as the etchant in Example 5.

[0039] Figure 11 These are the fluorescence images of the macroporous CaAlg gel spheres (a, b) and open-pore CaAlg gel spheres (c, d) after adsorption of HepG2 cells in Example 6, where a and c are bright field images and b and d are fluorescence field images; the scale bar is 500 μm.

[0040] Figure 12 This is the result of observing the activity of HepG2 cells using the live-dead cell staining method in Example 7. In the figure, UGS represents macroporous CaAlg gel spheres, and OGS represents open-pore CaAlg gel spheres.

[0041] Figure 13 Figures a and b show the cell activity test results of HepG2 cells cultured with open-pore CaAlg gel spheres in Example 7 for 24, 48, and 72 hours, as well as the cell proliferation results of HepG2 cells cultured with macroporous CaAlg gel spheres and open-pore CaAlg gel spheres for 24, 48, and 72 hours, respectively. In the figures, General porous represents macroporous CaAlg gel spheres, and Open porous represents open-pore CaAlg gel spheres.

[0042] Figure 14 Figures a and b show the OD value and urea concentration of urea in the supernatant after HepG2 cells were cultured for different time periods using macroporous CaAlg gel spheres and open-porous CaAlg gel spheres, respectively, in Example 8. In the figures, General porous represents macroporous CaAlg gel spheres, and Open porous represents open-porous CaAlg gel spheres.

[0043] Figure 15 Figures a and b show the OD values ​​and CYP450 concentrations in the supernatant of HepG2 cells cultured for different periods of time using macroporous CaAlg gel spheres and open-porous CaAlg gel spheres, respectively, in Example 8. In the figures, "General porous" represents macroporous CaAlg gel spheres, and "Open porous" represents open-porous CaAlg gel spheres. DETAILED DESCRIPTION

[0044] The following examples further illustrate the millimeter-sized calcium alginate gel spheres with an open macroporous structure and the preparation method thereof provided by the present invention. These examples are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art may make some non-essential improvements and adjustments to the present invention based on the above-mentioned invention content, which still fall within the scope of protection of the present invention.

[0045] Example 1

[0046] In this example, millimeter-sized calcium alginate (CaAlg) gel spheres with an open macroporous structure were prepared by the following steps:

[0047] (1) Sodium alginate (NaAlg) was dissolved in deionized water, and then sodium bicarbonate (NaHCO3) and sodium dodecyl sulfate (SDS) were added and stirred until NaHCO3 and SDS were completely dissolved. The resulting mixture was homogenized and emulsified in a homogenizer at a speed of 19000 rpm in an air atmosphere for 30 seconds to incorporate bubbles into the mixture to obtain a gel precursor solution; in the gel precursor solution, the concentration of NaAlg was 1.5wt%, the concentration of NaHCO3 was 1.5wt%, and the concentration of SDS was 0.1wt%.

[0048] Calcium chloride and glacial acetic acid (HAc) were dissolved in deionized water to obtain a coagulation bath solution, wherein the concentration of calcium chloride in the coagulation bath solution was 10 wt %, and the concentration of HAc was 10 wt %.

[0049] (2) The gel precursor solution was transferred to a 20 mL syringe with a flat needle of 0.52 mm (type 21G) in inner diameter. The syringe was pushed vertically downward by a syringe pump at a flow rate of 20 mL / h. The gel precursor solution naturally dripped into the 150 mL coagulation bath solution through the needle of the syringe. The NaAlg in the formed gel precursor droplets reacted with the Ca in the coagulation bath. 2+ A cross-linking reaction occurs, and at the same time, the NaHCO3 in the gel precursor droplets reacts with the HAc in the coagulation bath to produce a large amount of CO2. The produced CO2 promotes the rapid nucleation and growth of bubbles in the gel precursor droplets, causing the bubbles in the gel precursor droplets to connect. After 12 hours of cross-linking reaction, the gel precursor droplets solidify and the solid-liquid is separated. The resulting solid phase is washed 2 to 3 times with deionized water to remove unreacted calcium chloride and HAc, and freeze-dried to obtain millimeter-scale CaAlg gel spheres with interconnected macropores inside (referred to as macroporous CaAlg gel spheres).

[0050] (3) According to the ratio of adding 4g macroporous CaAlg gel balls to 1L potassium citrate aqueous solution, the macroporous CaAlg gel balls were added to the potassium citrate aqueous solution with a concentration of 15g / L and etched at a stirring speed of 20rpm for 5min. During the etching process, the citrate ions compete with the alginate ions for the Ca2+ The complexation of citrate and Ca 2+ After complexation, the outer layer of the macroporous CaAlg gel spheres is converted into a sol state, and an open-pore structure is formed on the surface of the gel spheres. After the etching is completed, the potassium citrate solution is immediately absorbed and a large amount of deionized water is added to terminate the reaction. The solid-liquid separation is performed, and the resulting solid phase is washed with deionized water 2 to 3 times and dried to obtain millimeter-sized CaAlg gel spheres with an open macroporous structure (referred to as open-pore CaAlg gel spheres).

[0051] The scanning electron microscope image of the macroporous CaAlg gel spheres prepared in step (2) of this embodiment is as follows: Figure 1 As shown, Figures a1 and a2 show the whole gel ball and the cut gel ball. Figure 1 The scale in the table is 1 mm. Figure 1 It can be seen that the macroporous CaAlg gel spheres prepared in step (2) have interconnected pores in their interior, and the pore sizes are relatively uniform, but no pores are formed on their surface. This is because the CaAlg 2+ During the diffusion process, it first contacts the outer layer of the gel precursor droplet and undergoes a rapid cross-linking reaction to generate CaAlg. The CaAlg formed on the outer layer of the gel precursor droplet encapsulates the CO2 bubbles generated by the reaction of NaHCO3 and HAc inside, making it impossible to form a pore structure on the surface of the gel ball, and it is impossible to obtain millimeter-scale CaAlg gel balls with an open macroporous structure.

[0052] The scanning electron microscope image of the open-pore CaAlg gel sphere prepared in step (3) of this embodiment is as follows: Figure 10 As shown in Figure c3, it can be seen from the figure that after the etching operation in step (3), a relatively uniformly distributed open pore structure is formed on the surface of the gel ball, and a millimeter-scale CaAlg gel ball with an open macroporous structure is obtained.

[0053] Example 2

[0054] In this embodiment, syringe needles with different inner diameters were used to prepare millimeter-sized CaAlg gel spheres with an open macroporous structure.

[0055] In this embodiment, a total of 4 groups of experiments were set up. The operations of each group of experiments were basically the same as those in Example 1, except that the inner diameters of the flat-blade needles used in step (2) of each experimental group were 0.26 mm (type 25G), 0.42 mm (type 22G), 0.52 mm (type 21G) and 0.61 mm (type 20G), respectively.

[0056] Figure 2are optical photos of the millimeter-scale CaAlg gel spheres with interconnected macropores (referred to as macroporous CaAlg gel spheres) prepared in the four groups of experiments, wherein the inner diameters of the flat-end needles corresponding to the photos a-d are 0.26 mm, 0.42 mm, 0.52 mm and 0.61 mm, respectively, and the scale in the photos is 2 mm. Figure 2 It can be seen that as the inner diameter of the needle increases, the size of the macroporous CaAlg gel spheres also increases; when the size of the macroporous CaAlg gel spheres is small, the outer layer of the cross-linked CaAlg gel is continuously extruded and deformed due to the continuous generation of CO2 bubbles inside, thus showing a non-spherical shape.

[0057] The macroporous CaAlg gel spheres prepared in the four groups of experiments were dispersed in deionized water, and the diameters thereof were measured and the coefficient of variation (CV) values of the diameters were calculated. When the inner diameters of the needles were 0.26 mm, 0.42 mm, 0.52 mm and 0.61 mm, respectively, the diameters of the prepared macroporous CaAlg gel spheres were 1.61 mm, 1.92 mm, 2.08 mm and 2.42 mm, respectively, and the CV values of the diameters were 7.7%, 6.7%, 3.1% and 4.2%, respectively. When the inner diameters of the needles were 0.52 mm and 0.61 mm, the sphericity of the prepared macroporous CaAlg gel spheres was better, and the CV values were relatively lower. Accordingly, a needle with an inner diameter of at least 0.42 mm can be selected for preparation, and the specific inner diameter of the needle can be determined according to the actual application requirements, for example, the inner diameter of the needle can generally be selected to be between 0.5 mm and 1 mm.

[0058] Example 3

[0059] In this example, millimeter-scale CaAlg gel spheres with open macroporous structures were prepared using gel precursor solutions with different concentrations of NaAlg.

[0060] In this example, four groups of experiments were set up, and the operations of the groups of experiments were basically the same as those in Example 1, except that the concentrations of NaAlg in the gel precursor solutions used in the groups of experiments were different. In the gel precursor solutions used in the four groups of experiments, the concentration of NaHCO3 was 1.5 wt%, the concentration of SDS was 0.1 wt%, and the concentration of NaAlg was 0.5 wt%, 1.0 wt%, 1.5 wt% and 2.0 wt%, respectively.

[0061] The four prepared gel precursor solutions were left to stand for different times, and the changes in their states with the standing time were observed, and the results are shown in Figure 3 Figure 3 ​It can be seen that when the concentration of NaAlg in the gel precursor is 0.5wt%, as the standing time increases, the bubbles in the gel precursor quickly dissipate and stratify with the solution, and the gel precursor has obvious stratification after standing for less than 5 minutes; when the concentration of NaAlg in the gel precursor is 1wt% and 1.5wt%, the gel precursor begins to stratify after standing for 10 minutes and 20 minutes respectively; when the concentration of NaAlg in the gel precursor is 2wt%, the gel precursor does not stratify after standing for 20 minutes. The increase in the stability of the gel precursor is conducive to the uniform distribution of the number of bubble templates in the gel precursor droplets dripped into the coagulation bath solution, thereby increasing the consistency of the internal pore structure of the different gel spheres finally prepared.

[0062] The optical photographs of the millimeter-sized CaAlg gel spheres with interconnected macropores (abbreviated as macroporous CaAlg gel spheres) prepared in the four experimental groups of this embodiment are shown in FIG. Figure 4 As shown, Figures a to d correspond to the experimental groups with NaAlg concentrations of 0.5wt%, 1.0wt%, 1.5wt% and 2.0wt% in the gel precursor solution, and the scale in the figure is 2mm. Figure 4 It can be seen that when the concentration of NaAlg in the gel precursor is 0.5wt%, the texture of the prepared macroporous CaAlg gel spheres is similar to plastic, and the overall shape is milky white and irregular. This is because when the concentration of NaAlg is low, it is more porous than CaAlg. 2+ The cross-linked gel has low strength and is easily deformed by internal bubbles. When the NaAlg concentration in the gel precursor solution is between 1 and 2 wt%, the prepared macroporous CaAlg gel spheres are essentially white spherical. The prepared macroporous CaAlg gel spheres were dispersed in deionized water, and their sizes were measured and their diameter CV values ​​were calculated. When the NaAlg concentrations in the gel precursor solution were 0.5 wt%, 1.0 wt%, 1.5 wt%, and 2.0 wt%, the diameters of the prepared macroporous CaAlg gel spheres were 2.15 mm, 2.01 mm, 2.12 mm, and 2.08 mm, respectively, with diameter CV values ​​of 9.7%, 5.4%, 4.4%, and 4.4%, respectively. As the NaAlg concentration in the gel precursor solution increased, the diameter CV values ​​of the macroporous CaAlg gel spheres gradually decreased, and the size distribution became more uniform.

[0063] The scanning electron microscope images of the cross-sections of the macroporous CaAlg gel spheres prepared in the four groups of experiments in this example are as follows: Figure 5 As shown in Figures a to d, the graphs correspond to the experimental groups with NaAlg concentrations of 0.5wt%, 1.0wt%, 1.5wt% and 2.0wt% in the gel precursor solution, and the scale bar in the graph is 200μm. Figure 5It can be seen that when the NaAlg concentration in the gel precursor solution is 0.5wt% and 1.0wt%, large-volume pores appear inside the macroporous CaAlg gel spheres, which is caused by the coalescence and coarsening of bubbles. When the NaAlg concentration in the gel precursor solution is 1.5wt% and 2.0wt%, the pores inside the macroporous CaAlg gel spheres are evenly distributed and interconnected.

[0064] The porosity, pore size, and CV values ​​of porosity and pore size of the macroporous CaAlg gel spheres prepared in the four experimental experiments of this embodiment were calculated. When the NaAlg concentration in the gel precursor solution was 0.5wt%, 1.0wt%, 1.5wt%, and 2.0wt%, the porosity of the macroporous CaAlg gel spheres was 77.54%, 67.21%, 63.11%, and 61.08%, respectively. The CV values ​​of porosity were 10.8%, 5.1%, 2.7%, and 2.4%, respectively. The average pore size of the macroporous CaAlg gel spheres was 240.64μm, 162.27μm, 143.67μm, and 128.34μm, respectively. The CV values ​​of pore size were 14.3%, 19.6%, 21.7%, and 17.5%, respectively. The higher the NaAlg concentration in the gel precursor solution, the smaller the difference in porosity of the macroporous CaAlg gel spheres. The CV values ​​of the pore sizes of the macroporous CaAlg gel spheres prepared in the four groups of experiments were distributed between 10% and 20%, indicating that the pore sizes inside the macroporous CaAlg gel spheres are mixed, rather than a single large or small pore size, which is beneficial to cell growth and differentiation.

[0065] From the above, it can be seen that, considering the morphology and pore structure of the gel spheres and other factors, the concentration of NaAlg in the gel precursor solution is preferably 1.5 wt % to 2 wt %.

[0066] Example 4

[0067] In this embodiment, millimeter-sized CaAlg gel spheres with an open macroporous structure were prepared using gel precursor solutions with different NaHCO3 concentrations.

[0068] This example provides four sets of experiments. The procedures for each set of experiments are essentially the same as those in Example 1, except that the concentration of NaHCO3 in the gel precursor solution used in each set of experiments is different. In the gel precursor solution used in the four sets of experiments, the concentration of NaAlg is 1.5wt%, the concentration of SDS is 0.1wt%, and the concentration of NaHCO3 is 0.5wt%, 1.0wt%, 1.5wt%, and 2.0wt%, respectively.

[0069] The optical photographs of the millimeter-sized CaAlg gel spheres with interconnected macropores (abbreviated as macroporous CaAlg gel spheres) prepared in the four experimental groups of this embodiment are shown in FIG. Figure 6The a~d figures in the figure correspond to the experimental groups with NaHCO3 concentration of 0.5wt%, 1.0wt%, 1.5wt% and 2.0wt% in the gel precursor solution, and the scale in the figure is 2mm. The prepared macroporous CaAlg gel spheres were dispersed in deionized water, the diameter of the macroporous CaAlg gel spheres was measured and the CV value of the diameter was calculated. When the NaHCO3 concentration in the gel precursor solution was 0.5wt%, 1.0wt%, 1.5wt% and 2.0wt%, the diameter of the prepared macroporous CaAlg gel spheres was 1.81mm, 2.19mm, 2.12mm and 2.29mm respectively, and the CV value of the diameter was 3.0%, 3.5%, 4.2% and 4.3% respectively. With the increase of the concentration of NaHCO3, the size of the macroporous CaAlg gel spheres gradually increased and the surface gradually became uneven, and many protrusions were increased, because the amount of gas bubbles gradually increased, and the outer layer of crosslinked CaAlg was squeezed.

[0070] The scanning electron microscope images of the cross section of the macroporous CaAlg gel spheres prepared in the four groups of experiments of the present embodiment are shown in Figure 7 The a~d figures in the figure correspond to the experimental groups with NaHCO3 concentration of 0.5wt%, 1.0wt%, 1.5wt% and 2.0wt% in the gel precursor solution, and the scale in the figure is 2mm. The prepared macroporous CaAlg gel spheres were dispersed in deionized water, the diameter of the macroporous CaAlg gel spheres was measured and the CV value of the diameter was calculated. When the NaHCO3 concentration in the gel precursor solution was 0.5wt%, 1.0wt%, 1.5wt% and 2.0wt%, the diameter of the prepared macroporous CaAlg gel spheres was 1.81mm, 2.19mm, 2.12mm and 2.29mm respectively, and the CV value of the diameter was 3.0%, 3.5%, 4.2% and 4.3% respectively. With the increase of the concentration of NaHCO3, the size of the macroporous CaAlg gel spheres gradually increased and the surface gradually became uneven, and many protrusions were increased, because the amount of gas bubbles gradually increased, and the outer layer of crosslinked CaAlg was squeezed. Figure 7 It can be seen from

[0071] The porosity, pore size, and CV values of the porosity and pore size of the macroporous CaAlg gel spheres prepared in the four groups of experiments of the present embodiment were calculated. When the NaHCO3 concentration in the gel precursor solution was 0.5wt%, 1.0wt%, 1.5wt% and 2.0wt%, the porosity of the macroporous CaAlg gel spheres was 49.40%, 54.69%, 63.11% and 67.89% respectively, and the porosity increased with the increase of the concentration of NaHCO3. It can be seen that increasing the concentration of NaHCO3 can provide more nucleation sites for the generation of CO2 bubbles. The CV value of the porosity was 2.6%, 3.5%, 2.7% and 5.5% respectively, indicating that the pore structure inside the macroporous CaAlg gel spheres was relatively uniform. The average pore size of the macroporous CaAlg gel spheres was 144.19μm, 133.22μm, 143.66μm and 142.65μm respectively, and the CV value of the pore size was 12.0%, 13.4%, 21.2% and 21.2% respectively. The gradual increase of the CV value of the pore size indicates that increasing the concentration of NaHCO3 will increase the disorder of bubble growth.

[0072] Example 5

[0073] In this embodiment, etchants of different types and concentrations are used to etch the macroporous CaAlg gel spheres.

[0074] (1) The operation is the same as step (1) of Example 1.

[0075] (2) The same operation as step (2) of Example 1 was performed to prepare millimeter-sized CaAlg gel spheres with interconnected macropores (referred to as macroporous CaAlg gel spheres).

[0076] (3) The macroporous CaAlg gel spheres were added to the etchant aqueous solution at a ratio of 4 g per 1 L of etchant aqueous solution and etched for 120 min at a stirring speed of 20 rpm. After the etching was completed, the etchant aqueous solution was immediately removed and a large amount of deionized water was added to terminate the reaction. The solid-liquid separation was performed and the resulting gel spheres were washed with deionized water 2 to 3 times to obtain open-pore CaAlg gel spheres. During the etching process, photos were taken at intervals to record the surface morphology changes of the macroporous CaAlg gel spheres.

[0077] The concentrations of the etchants used and their aqueous solutions are: sodium citrate (33.50 g / L), sodium bicarbonate (9.57 g / L), sodium acetate (9.35 g / L), sodium chloride (6.66 g / L), sodium hydroxide (4.56 g / L), potassium citrate (36.96 g / L), ammonium citrate (27.71 g / L), and ferric citrate (27.90 g / L).

[0078] Figure 8 This is an optical photograph of the macroporous CaAlg gel sphere during the etching process. The scale in the figure is 1 mm. Figure 8 It can be seen that sodium bicarbonate, sodium acetate and sodium chloride have no chemical effect on the macroporous CaAlg gel spheres; the macroporous CaAlg gel spheres gradually begin to dissolve and release some encapsulated bubbles after soaking in sodium hydroxide aqueous solution for 60 minutes, and most of the macroporous CaAlg gel spheres are etched after soaking for 120 minutes; the ferric citrate aqueous solution has a certain swelling effect on the macroporous CaAlg gel spheres, but no obvious etching occurs; the sodium citrate, potassium citrate and ammonium citrate aqueous solutions have an etching effect on the macroporous CaAlg gel spheres, but the etching rates are different, with ammonium citrate being the fastest, followed by sodium citrate, and potassium citrate being the slowest.

[0079] (4) Adjust the concentration of the etchant aqueous solution in step (3), and repeat the operation of step (3). The concentration of the etchant aqueous solution used is: sodium citrate (6.7 g / L), sodium bicarbonate (9.57 g / L), sodium acetate (9.35 g / L), sodium chloride (6.66 g / L), sodium hydroxide (4.56 g / L), potassium citrate (7.39 g / L), ammonium citrate (5.54 g / L), and ferric citrate (27.90 g / L).

[0080] The scanning electron microscope test results of the open-pored CaAlg gel spheres obtained by etching for different times are shown in Figs. 8a-8h. Figure 9 Figure 9 In Figs. 8a-8h, the corresponding etchants are sodium citrate, sodium bicarbonate, sodium acetate, sodium chloride, sodium hydroxide, potassium citrate, ammonium citrate, and ferric citrate, and the corresponding etching times are 20 min, 120 min, 120 min, 120 min, 120 min, 40 min, 20 min, and 120 min, respectively. The scale in the figure is 1 mm. As can be seen from Figs. 8a-8h, Figure 9 sodium bicarbonate, sodium acetate, and sodium chloride have basically no etching effect on the macroporous CaAlg gel spheres; ferric citrate has a weak etching effect on the macroporous CaAlg gel spheres, and uneven etching occurs after 120 min of immersion; the macroporous CaAlg gel spheres are etched after 20 min of immersion in sodium citrate and ammonium citrate aqueous solutions, and the etching of sodium citrate is more uniform; the macroporous CaAlg gel spheres are etched after 40 min of immersion in potassium citrate aqueous solution, and many holes can be observed on the surface thereof. In general, the etching effects of sodium citrate and potassium citrate are relatively better.

[0081] (5) Repeat the operation of step (3) using potassium citrate as the etchant. A total of 3 groups of experiments are set, and the details are as follows:

[0082] In experimental group ①, the concentrations of potassium citrate are 30 g / L, 35 g / L, 40 g / L, 45 g / L, and 50 g / L, and the etching time is 5 min. The scanning electron microscope images of the open-pored CaAlg gel spheres prepared are shown in Figs. 9a1-9a5. Figure 10

[0083] In experimental group ②, the concentrations of potassium citrate are 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1.0 g / L, and the immersion time is 120 min. The scanning electron microscope images of the open-pored CaAlg gel spheres prepared are shown in Figs. 9b1-9b5. Figure 10

[0084] ​​​Experimental group ③, the potassium citrate concentrations were 5g / L, 10g / L, 15g / L, 20g / L and 25g / L, and the immersion time was 120min. The scanning electron microscopy images of the prepared open-pore CaAlg gel spheres are shown in Figure 3. Figure 10 As shown in Figure c1 to c5.

[0085] Depend on Figure 10 As can be seen, in experimental group ①, under the conditions of high concentration and short etching time, the surface of the open-pore CaAlg gel spheres prepared was covered with open pores, but the structure of the open-pore CaAlg gel spheres collapsed, which was mainly caused by the high etchant concentration. In experimental group ②, under the conditions of low concentration and long etching time, the surface of the open-pore CaAlg gel spheres prepared did not show a large area of ​​open pore structure and showed shrinkage. This is because the etching time was too long, and the etchant gradually diffused into the interior of the gel spheres, dissolving the gel with a low degree of internal cross-linking. In experimental group ③, when the potassium citrate concentration was 10-15 g / L, the open-pore CaAlg gel spheres prepared by etching for 5 minutes had a relatively uniform distribution of open macropores on the surface, and the spheres did not collapse.

[0086] In this step, the porosity of the open pores on the surface of the open-pored CaAlg gel spheres prepared under the conditions of potassium citrate concentrations of 10 g / L and 15 g / L was 26.41% and 37.73%, respectively, and the average pore size of the open pores on the surface of the open-pored CaAlg gel spheres was 159.50 μm and 194.37 μm, respectively.

[0087] Example 6

[0088] In this example, the ability of the millimeter-sized CaAlg gel spheres with an open macroporous structure prepared in Example 1 (referred to as open-pore CaAlg gel spheres) to adsorb cells was tested.

[0089] HepG2 cells in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension with a cell density of 5 × 10 6 / mL. Soak the gel balls in PBS buffer to fully swell, and sterilize with ultraviolet light. After sterilization, discard the PBS buffer and soak the gel balls with complete culture medium. Spread the gel balls soaked in complete culture medium on a 35mm culture dish, add 1.5mL of cell suspension, and after observing that the cells are absorbed into the gel balls, transfer the cell-loaded gel balls to a new culture dish, add 4% paraformaldehyde solution, fix at room temperature for 20 minutes, discard the paraformaldehyde solution, and wash with PBS buffer to remove the residual paraformaldehyde solution. Then use 10μg·mL -1The cells were stained with 4',6-diamidino-2-phenylindole (DAPI) solution at room temperature in the dark for 5 minutes, washed with PBS buffer, and the cell distribution was observed under a fluorescence microscope. The open-pore CaAlg gel spheres and macroporous CaAlg gel spheres prepared in Example 1 were used as the experimental group and the control group, respectively.

[0090] The adsorption capacity of gel balls to cells and the distribution of cells in gel balls are crucial for cell culture. It is necessary for cells to be adsorbed into the interior of the gel balls and to be dispersed as evenly as possible. After the gel balls adsorb the cell suspension for 30 minutes, transfer it to a new culture dish and stain the cells with DAPI solution. Blue fluorescence can be observed under a fluorescence microscope, such as Figure 11 As shown, Figures a and b are fluorescence images of HepG2 cells after adsorption by macroporous CaAlg gel spheres, and Figures c and d are fluorescence images of HepG2 cells after adsorption by open-pore CaAlg gel spheres. Figures a and c are bright field images, and Figures b and d are fluorescence field images. The scale bar in the figure is 500 μm. Figure 11 As can be seen, the open-pore CaAlg gel spheres are extremely transparent after being immersed in PBS buffer, and the complex pores inside them can be vaguely seen under bright field light. The blue fluorescence representing cells is almost invisible in the macroporous CaAlg gel spheres, while many HepG2 cells are adsorbed inside the open-pore CaAlg gel spheres and evenly dispersed within the pores of the gel spheres. This shows that the present invention effectively improves the gel spheres' ability to adsorb cells by etching open, interconnected macropores.

[0091] Example 7

[0092] In this example, the activity and proliferation of HepG2 cells in the millimeter-sized CaAlg gel spheres with open macroporous structure prepared in Example 1 (referred to as open-pore CaAlg gel spheres) were tested.

[0093] (1) ① HepG2 cells in the logarithmic growth phase were digested with trypsin to prepare a single cell suspension with a cell density of 5×10 6 After sterilization, the gel spheres (the macroporous CaAlg gel spheres and the open-pore CaAlg gel spheres prepared in Example 1) were immersed in a complete culture medium solution. A 35 mm culture dish was pre-filled with the gel spheres, and 1.5 mL of the cell suspension was added. After observing that the gel spheres were absorbed by the cells, the cell-loaded gel spheres were transferred to a new culture dish, and 1 mL of fresh complete culture medium was added. The dish was then incubated in a 5% CO2, 37°C constant temperature cell culture incubator.

[0094] ②After 24h, 48h and 72h of culture, the original culture medium was discarded, and the cells were washed with PBS buffer for 2-3 times. Then 1 mL of cell double staining working solution (Calcein-AM: 2 μM, PI: 4.5 μM) was added, and the cells were stained at room temperature for 15 min in the dark.

[0095] ③After the staining was completed, the staining solution was discarded, and the cells were washed with PBS buffer. Then the cells were observed under a fluorescence microscope to determine the survival of the cells, and the results are shown in Figure 12

[0096] As shown in Figure 12 , neither green fluorescence representing living cells nor red fluorescence representing dead cells was observed in the macroporous CaAlg gel spheres, which indicated that it was difficult for the macroporous CaAlg gel spheres to adsorb HepG2 cells from the beginning. The HepG2 cells in the open-pore CaAlg gel spheres showed strong green fluorescence after 24h, 48h and 72h of culture. After 48h and 72h of culture, a small number of dead cells appeared in the interior of the gel spheres, and the number of living cells gradually increased and the cell morphology gradually aggregated with the increase of the culture time, which was completely inconsistent with the morphology in 2D adherent culture. It was indicated that the open-pore CaAlg gel spheres provided by the application were suitable for use as a carrier for 3D cell culture in vitro.

[0097] (2) ①HepG2 cells in the logarithmic growth phase were trypsinized to prepare a single cell suspension, and the cell density was 5×10 6

[0098] ②After 24h, 48h and 72h of culture, the original culture medium was discarded, and 1 mL of complete culture medium containing 10% CCK-8 solution was added (experimental group). The complete culture medium containing 10% CCK-8 solution was set as a control group, and was incubated at 37°C for 2h together with the experimental group.

[0099] ③After the incubation was completed, the supernatant in the culture dish was collected into a 96-well plate, and the OD value at 450 nm was determined by using an enzyme marker. Six parallel experiments were set for each group, and the final OD value of each group was the average value of the six parallel experiments.

[0100] After the HepG2 cells were cultured in the open-pore CaAlg gel spheres for 24h, 48h and 72h, the activity of the HepG2 cells was all above 95%, as shown in Figure 13 ​​As shown in Figure a, it shows that open-pore CaAlg gel spheres are suitable as carriers for cell culture. The cell proliferation of HepG2 cells after culturing with macroporous CaAlg gel spheres and open-pore CaAlg gel spheres for 24h, 48h and 72h respectively is shown in Figure a. Figure 13 As shown in Figure b, after culturing HepG2 cells with open-pore CaAlg gel spheres for 48 and 72 hours, the OD values ​​were 1.4 and 2.3 times higher than those after culturing for 24 hours. In contrast, after culturing HepG2 cells with macroporous CaAlg gel spheres for 24, 48, and 72 hours, the OD values ​​remained essentially unchanged, demonstrating that the open-pore CaAlg gel spheres of the present invention can maintain conditions for cell growth and proliferation, ensuring the diffusion supply of cellular nutrients.

[0101] Example 8

[0102] In this example, a specific secretion experiment of HepG2 cells was conducted in the millimeter-sized CaAlg gel spheres with open macroporous structures (hereinafter referred to as open-pore CaAlg gel spheres) prepared in Example 1. The macroporous CaAlg gel spheres prepared in step (2) of Example 1 were used as a control group, and the open-pore CaAlg gel spheres prepared in step (3) of Example 1 were used as an experimental group. The secretion concentrations of the two groups at different incubation times after cell adsorption were studied.

[0103] (1) Detection of urea concentration secreted by cells using double antibody sandwich ELISA

[0104] HepG2 cells in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension with a cell density of 5 × 10 6 After sterilization, soak the gel spheres in complete culture medium. Pre-spread the gel spheres in a 35 mm culture dish, then add 1.5 mL of the cell suspension. Once the gel spheres are observed to be absorbed by the cells, transfer the cell-loaded gel spheres to a new 35 mm culture dish and incubate the cells in a 5% CO2, 37°C incubator. After 24, 48, and 72 hours of incubation, transfer the gel spheres to a sterile centrifuge tube, centrifuge at 2000 rpm for 20 minutes, and collect the supernatant. Add 50 μL of urea standard and the collected supernatant to the pre-coated plate, then add 100 μL of HPR-labeled detection antibody, seal the plate, and incubate at room temperature for 60 minutes. Tap and wash the plate five times, then add 50 μL of chromogen A and chromogen B, seal the plate, and incubate at 37°C in the dark for 15 minutes. Finally, add 50 μL of acidic solution to terminate the reaction. Within 15 minutes, measure the absorbance at 450 nm using a microplate reader. Four parallel experiments were set up, and the absorbance value of each group was the average value of the four parallel experiments.

[0105] Figure 14Figures (a) and (b) show the OD values ​​and urea concentrations in the supernatant of HepG2 cells cultured for different time periods using macroporous and open-pore CaAlg gel spheres, respectively. The urea concentration in the supernatant showed a negative correlation with increasing culture time, which may be due to the rapid degradation of urea or the complexity of maintaining and expressing hepatocyte function.

[0106] (2) Detection of cytochrome P450 (CYP450) concentration in cells using competitive ELISA

[0107] HepG2 cells in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension with a cell density of 5 × 10 6 / mL. After sterilization, soak the gel balls in complete culture medium solution. Pre-cover the 35mm culture dish with gel balls, then add 1.5mL of cell suspension. After observing that the gel balls are absorbed by the cells, transfer the cell-loaded gel balls to a new 35mm culture dish to culture the cells. Place the gel balls in a 5% CO2 and 37℃ constant temperature cell culture incubator for culturing. After culturing for 24h, 48h and 72h, discard the original culture medium and add PBS buffer containing 5mol / L citric acid. After dissolving overnight, transfer the mixture to a sterile centrifuge tube, centrifuge at 2000rpm for 20min, and discard the supernatant. Add 100μL of cell lysis buffer to the precipitate, react at 4℃ for 30min, centrifuge at 2000rpm for 20min, and collect the supernatant. Add 50 μL of CYP450 standard and collected supernatant to the pre-coated plate. Then, add 50 μL of biotinylated antigen working solution, seal the plate, and incubate at 37°C for 30 minutes. Tap the plate and wash it five times before adding 50 μL of avidin-HPR working solution. Seal the plate and incubate at 37°C for 30 minutes. Next, tap the plate and wash it five times before adding 50 μL of color developer A and 50 μL of color developer B. Seal the plate and incubate at 37°C in the dark for 10 minutes. Finally, add 50 μL of acidic solution to terminate the reaction. Measure the absorbance at 450 nm using a microplate reader within 10 minutes. Set up four parallel experiments, and the absorbance value for each group is the average of the four parallel experiments.

[0108] Figure 15 Figures a and b show the OD values ​​and CYP450 concentrations in the supernatant of HepG2 cells cultured for different time periods using macroporous CaAlg gel spheres and open-pore CaAlg gel spheres, respectively. The concentration of CYP450 in the macroporous CaAlg gel spheres was always lower than the lowest concentration of the CYP450 standard, and its specific concentration could not be calculated using the fitting curve. Figure 15In panel b, the expression "none" is indicated by "NA." The concentration of CYP450 in the open-pore CaAlg gel spheres gradually increased with culture time, indicating that the functional expression of HepG2 cells was maintained. These experimental results demonstrate that the physiological function of HepG2 cells is enhanced in open-pore CaAlg gel spheres, making them suitable as a scaffold for cell culture.

Claims

1. Millimeter-sized calcium alginate gel spheres with open macroporous structure, characterized in that: The matrix material of the gel ball is calcium alginate. The interior of the gel ball has an interconnected micron-scale pore structure, and the interconnected micron-scale pore structure inside the gel ball opens on the surface of the gel ball. The average pore size of the micron-scale pore structure inside the gel ball is 120-160 μm, and the porosity of the pore structure inside the gel ball is 50%-70%. The average pore size of the pore structure on the surface of the gel ball is 150-200 μm, and the porosity of the pore structure on the surface of the gel ball is 20%-40%. The particle size of the gel ball when it reaches swelling equilibrium in water is 2-5 mm. The gel ball is prepared by the following method: (1) Sodium alginate, a porogen, and a water-soluble anionic surfactant are dissolved in water, and the resulting mixture is homogenized and emulsified to incorporate bubbles into the mixture to obtain a gel precursor solution; in the gel precursor solution, the concentration of sodium alginate is 1.5 wt% to 2 wt%, the concentration of the porogen is 1 wt% to 2 wt%, and the concentration of the water-soluble anionic surfactant is 0.05 wt% to 0.15 wt%; the porogen is sodium bicarbonate or potassium bicarbonate; Dissolving a water-soluble calcium salt and glacial acetic acid in water to obtain a coagulation bath solution, wherein the concentration of the water-soluble calcium salt in the coagulation bath solution is 5 wt% to 15 wt%, and the concentration of the glacial acetic acid is 5 wt% to 15 wt%; (2) dripping the gel precursor into the coagulation bath solution through an injection needle, the sodium alginate in the gel precursor droplets dripping into the coagulation bath solution reacts with calcium ions to undergo a cross-linking reaction, and at the same time, the porogen in the gel precursor droplets dripping into the coagulation bath solution reacts with glacial acetic acid to generate CO2 gas in the gel precursor droplets, causing the bubbles in the gel precursor droplets to connect, cross-linking until the gel precursor droplets solidify, solid-liquid separation, washing to remove unreacted water-soluble calcium salts and glacial acetic acid in the obtained solid phase, and freeze-drying to obtain millimeter-scale calcium alginate gel spheres with interconnected macropores inside; (3) Immersing the gel spheres obtained in step (2) in an etchant aqueous solution, etching the spheres under stirring, so that the etchant is complexed with calcium ions in the calcium alginate on the surface of the gel spheres to form an open-pore structure on the surface of the gel spheres, immediately replacing the etchant aqueous solution with water to terminate the reaction between the etchant and the calcium ions, separating the solid and the liquid, washing and drying the obtained solid phase, and obtaining millimeter-sized calcium alginate gel spheres with an open macroporous structure; the etchant is potassium citrate or sodium citrate, and the concentration of the etchant aqueous solution is 10-15 g / L.

2. The millimeter-sized calcium alginate gel sphere with an open macroporous structure according to claim 1, characterized in that: In step (3), the etching time is controlled to be 3 to 10 minutes.

3. The millimeter-sized calcium alginate gel sphere with an open macroporous structure according to claim 1, characterized in that: In step (2), the gel precursor solution is dripped into the coagulation bath solution through an injection needle with an inner diameter of 0.5~1 mm.

4. The millimeter-sized calcium alginate gel sphere with an open macroporous structure according to any one of claims 1 to 3, characterized in that: In step (2), the gel precursor solution is dripped into the coagulation bath solution through the injection needle at a flow rate of 15-25 mL / h by the injection pump.

5. The millimeter-sized calcium alginate gel sphere with an open macroporous structure according to any one of claims 1 to 3, characterized in that: In step (2), the cross-linking time is controlled to be 10~15 h.

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